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Protective effects of metoprolol and ascorbic acid during the development of diabetic cardiomyopathy Saran, Varun Vivashan 2012

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Protective Effects of Metoprolol and Ascorbic Acid During the Development of Diabetic Cardiomyopathy by Varun Vivashan Saran B.Sc., The University of British Columbia, 2008  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in The Faculty of Graduate Studies (Pathology and Laboratory Medicine)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2012       © Varun Vivashan Saran, 2012     Abstract   The	existence	of	a	heart	muscle	disorder	specific	to	Diabetes	Mellitus	(DM)  has	been	proposed,	termed,	Diabetic	Cardiomyopathy	(DCM).		DCM	is	defined	as	the presence	of	an	early	asymptomatic	diastolic	dysfunction	that	eventually	progresses to	overt	systolic	dysfunction	in	the	absence	of	ischemic	or	valvular	heart	disease. Metabolic	impairment	and	increased	oxidative	stress	have	been	highlighted	as causes.		The	β‐blocker	metoprolol	is	known	to	improve	function	in	diabetic	rat hearts,	possibly	through	amelioration	of	the	sequelae	associated	with	oxidative stress,	without	lowering	oxidative	stress.		It	is	unclear	if	lowering	oxidative	stress	in concert	with	metoprolol	treatment	would	improve	function	further.		Ascorbic	Acid (AA)	is	a	potent	antioxidant	and	has	been	shown	to	improve	function	in	the	diabetic rat	heart.  Hypothesis: We	propose	that	metabolic	changes	that	occur	during	diabetes	elevate oxidative	stress,	leading	to	protein	damage,	signaling	changes,	cell	death	and	other sequelea;	the	eventual	sum	of	these	changes	is	an	impairment	of	function.		Treatment of	either	the	sequelae	of	oxidative	stress	or	oxidative	stress	directly	will	be	beneficial but	treatment	of	both	will	improve	function	further.    To	accomplish	our	study	we	induced	DM	in	male	Wistar	rats	using	60	mg/kg  streptozotocin	and	treated	them	with	metoprolol	at	15	mg/kg/day	via	osmotic        ii   pump	and/or	AA	at	1000	mg/kg/day	via	drinking	water.		In	order	to	study	the	effect of	treatment	on	the	development	of	dysfunction	we	studied	a	time	point	before	and after	development	of	dysfunction	(5	and	7	weeks,	respectively).		Blood	was	collected to	assess	the	severity	of	diabetes	and	echocardiography	performed	to	assess	in	vivo heart	function.		At	termination,	ex	vivo	heart	function	and	substrate	use	were measured	by	working	heart	perfusion.		Tissue	was	collected	for	measurements	of metabolite	levels	and	oxidative	protein	damage.   Function	significantly	worsened	in	association	with	metabolism	and  oxidative	damage.		Both	drugs	improved	function,	while	only	AA	reduced	oxidative damage.		Combined	treatment	led	to	improvement	in	function	more	pronounced then	single	treatment.	Our	β‐blocker	and	antioxidant	treatment	strategy	focuses	on oxidative	stress,	and	not	on	diabetes	specifically,	thus	it	may	prove	useful	in	other disease	where	oxidative	stress	contributes	to	pathology.           iii     Preface   Ethics	approval	for	this	study	was	attained	from	the	Animal	Care	Committee  at	the	University	of	British	Columbia.		Study	1	was	listed	under	the	certificate	titled: Modulation	of	cardiac	metabolism	by	metoprolol	in	the	diabetic	heart	(#A06‐0420). Study	2	was	listed	under	the	certificate	titled:	Malonyl	Co‐A‐independent	regulation of	carnitine	palmitoyltransferase‐1	by	B	adrenoceptor	signaling	in	the	heart	(#A07‐ 0730).        iv    Table of Contents   Abstract	....................................................................................................................................................	ii  Preface	.....................................................................................................................................................	iv  Table	of	Contents...................................................................................................................................	v  List	of	Tables	.......................................................................................................................................	viii  List	of	Figures	.......................................................................................................................................	ix  List	of	Schemes	.......................................................................................................................................	x  List	of	Abbreviations	..........................................................................................................................	xi  Acknowledgements	..........................................................................................................................	xiii  CHAPTER	1	 Introduction	............................................................................................................	1  1.1.	Diabetes	Mellitus	..........................................................................................	1  1.2.	Complications	During	Diabetes	..............................................................	2  1.3.	Diabetic	Cardiomyopathy	.........................................................................	3  1.4.	Causes	of	Diabetic	Cardiomyopathy	.....................................................	5  				1.4.1.	Calcium	Handling	Abnormalities	...................................................	6  				1.4.2.	Cell	Death	and	Fibrosis	......................................................................	6  				1.4.3.	Metabolism	and	Oxidative	Stress	..................................................	8  								1.4.3.1.	Fuel	Usage	........................................................................................	8  								1.4.3.2.	Lipid	Accumulation	......................................................................	9  								1.4.3.3.	Hyperglycemia	.............................................................................	11  1.5.	Selected	Treatment	Strategies	..............................................................	14  				1.5.1.	β‐blockers	..............................................................................................	14  								1.5.1.1.	β‐blockers	and	Diabetic	Cardiomyopathy	.......................	15  				1.5.2.	Ascorbic	Acid	........................................................................................	17  1.6.	Hypothesis	and	Study	Objectives	........................................................	19  CHAPTER	2	 Methods	...................................................................................................................	22  2.1.	Animals/	Treatment	Groups	..................................................................	22  2.2.	Echocardiography	......................................................................................	24  2.3.	Function	and	Fuel	Usage	.........................................................................	24  				2.3.1.	Perfusion	Conditions	.........................................................................	24        v           CHAPTER	3                 CHAPTER	4                      				2.3.2.	Function	..................................................................................................	25 				2.3.3.	Fuel	Usage	..............................................................................................	25 2.4.	Metabolism	....................................................................................................	27 				2.4.1.	Tissue	Triglyceride	Assay	...............................................................	27 				2.4.2.	Tissue	Glycogen	...................................................................................	28 2.5.	Oxidative	Stress	Assessment,	Oxyblot	...............................................	28 2.6.	Statistics	..........................................................................................................	30 Results	......................................................................................................................	32 3.1.	General	Characteristics	and	Plasma	Parameters	..........................	32 				3.1.1.	Study	1	.....................................................................................................	32 				3.1.2.	Study	2	.....................................................................................................	33 3.2.	In	vivo	Cardiac	Function	of	Diabetic	Rats	........................................	34 				3.2.1.	Study	1	.....................................................................................................	34 				3.2.2.	Study	2	.....................................................................................................	34 3.3.	Ex	vivo	Cardiac	Function	of	Diabetic	Rats	.......................................	35 				3.3.1.	Study	1	.....................................................................................................	35 				3.3.2.	Study	2	.....................................................................................................	36 3.4.	Substrate	Oxidation	and	Metabolite	Content	.................................	37 				3.4.1.	Study	1	.....................................................................................................	37 				3.4.2.	Study	2	.....................................................................................................	38 3.5.	Oxidative	Protein	Damage	......................................................................	39 				3.5.1.	Study	1	.....................................................................................................	39 				3.5.2.	Study	2	.....................................................................................................	40 Discussion...............................................................................................................	52 4.1.	Overview	of	Study	......................................................................................	52 4.2.	General	Physical	Characteristics	–	Body	Weight	and	Heart Weight	are	More	Perturbed	with	Disease	Progression	.............	53 4.3.	Plasma	Triglyceride	and	Cholesterol	–	Levels	and Persistence	of	Disturbance	with	Disease	Progression	..............	54 				4.3.1.	Triglycerides	.........................................................................................	54 				4.3.2.	Cholesterol	.............................................................................................	56 4.4.	Heart	Function	–	Relationship	to	Disease	Progression	..............	57 4.5.	Heart	Metabolism	–	Metabolic	Alterations	Worsen	with Disease	Progression	..................................................................................	61 				4.5.1.	Palmitate	Oxidation	...........................................................................	61 				4.5.2.	Glucose	Oxidation	...............................................................................	62 4.6.	Oxidative	Protein	Damage	in	Diabetic	Hearts	Worsen	with Disease	Progression	..................................................................................	64 4.7.	Summary	........................................................................................................	65 				4.7.1.	Progression	of	Diabetes	from	5	to	7	weeks	–	An Important	Time	Point	in	the	Development	of	Cardiac Dysfunction	..........................................................................................	65 				4.7.2.	Benefits	of	β‐blocker	Therapy	Supplemented	with Antioxidants	........................................................................................	66     vi    				4.7.3	Hypothesis	and	Conclusion	.............................................................	67  4.8.	Importance	of	the	Study	..........................................................................	69  4.9.	Future	Directions	........................................................................................	69   WORKS	CITED	.....................................................................................................................................	73          vii    List of Tables Table	1 Table	2 Table	3 Table	4        General	Characteristics	.....................................................................................	41 Plasma	Parameters	.............................................................................................	42 Functional	Parameters	as	Measured	by	Echocardiography	.............	43 Functional	Parameters	as	Measured	by	Working	Heart Perfusion	.................................................................................................................	44        viii    List of Figures Figure	1 Figure	2 Figure	3 Figure	4 Figure	5 Figure	6 Figure	7    Cardiac	Output	as	Measured	by	Echocardiography	.............................	45 Mechanical	Function	as	Measured	by	Working	Heart	Perfusion	...	46 Palmitate	and	Glucose	Oxidation	Rates	.....................................................	47 Glycogen	and	Triglyceride	Content	in	Cardiac	Tissue	........................	48 Oxidative	Protein	Damage,	Control	vs.	Diabetic	....................................	49 Oxidative	Protein	Damage,	Study	1	.............................................................	50 Oxidative	Protein	Damage,	Study	2	.............................................................	52      ix    List of Schemes Scheme	1 Scheme	2 Scheme	3       Outline	of	Overall	Hypothesis	........................................................................	21 Timeline	of	Study	................................................................................................	31 Outline	of	Findings	.............................................................................................	72        x    List of Abbreviations  AA AGE(s) Akt AMP ATP BSA cAMP CDM CHO COMET CVD DAG DCM DM DNA DNP EDTA EDV EF ESV FS GlcNAc GLUT4 HbA1c HBP HEPES LPL LVEDD LVEDV LVESD LVESV MOPS NADH(NAD+) NADPH NCX PKA PKC PKC-β2 PLB PMCA   Ascorbic	Acid Advanced	Glycation	End	Products Protein	Kinase	B Adenosine	Monophosphate Adenosine	Triphosphate Bovine	Serum	Albumin Cyclic	Adenosine	Monophosphate Centre	for	Disease	Modeling Chinese	Hamster	Ovary	Cells Carvedilol	or	Metoprolol	European	Trial Cardiovascular	Disease Diacylglycerol Diabetic	Cardiomyopathy Diabetes	Mellitus Deoxyribonucleic	Acid Dinitrophenol Ethylenediaminetetraacetic	Acid End	Diastolic	Volume Ejection	Fraction End	Systolic	Volume Fractional	Shortening N‐Acetylglucosamine Glucost	Transporter	4 Glycated	Hemoglobin Hexosamine	Biosynthetic	Pathway 4‐(2‐Hydroxyethyl)‐1‐Piperazineethanesulfonic	Acid Lipoprotein	Lipase Left	Ventricular	End	Diastolic	Diameter Left	Ventricular	End	Diastolic	Volume Left	Ventricular	End	Systolic	Diameter Left	Ventricular	End	Systolic	Volume 3‐(N‐Morpholino)‐Propanesulfonic	Acid Nicotinamide	Adenine	Dinucleotide Nicotinamide	Adenine	Dinucleotide	Phosphate Sodium	Calcium	Exchanger Protein	Kinase	A Protein	Kinase	C Protein	Kinase	C	β2 Phospholamban Plasma	Membrane	Ca2+	ATPase     xi   RAAS Rac1 RAGE ROS RPP RyR SEM SERCA STZ T1DM T2DM TUNEL TWEEN UDP-GlcNAc v/v VLDL    Renin‐Angiotensin‐Aldosterone‐System Rho	Family,	Small	GTP	Binding	Protein AGE	Receptor Reactive	Oxygen	Species Rate	Pressure	Product Ryanodine	Receptor Standard	Error	of	the	Mean Sarco/Endoplasmic	Reticulum	Ca2+‐ATPase Streptozotocin Type	2	Diabetes	Mellitus Type	2	Diabetes	Mellitus Terminal	Deoxynucleotidyl	Transferase	(dUTP)	Nick	End Labeling Polyoxyethylenesorbitan Uridine	Diphosphate	N‐Acetylglucosamine Volume	by	Volume Very	Low	Density	Lipoprotein      xii  Introduction       Acknowledgements I	would	like	to	thank	first	and	foremost	my	supervisors	John	H.	McNeill	and  Dr.	Michael	F.	Allard.		Throughout	my	degree	Dr.	McNeill	has	been	a	constant	source of	knowledge,	experience	and	stability.		The	pragmatism	and	professionalism	he conducts	himself	with	will	serve	as	something	for	me	to	strive	towards	in	all	my future	endeavors.		I’ve	worked	for	Dr.	Allard	for	a	number	of	years	and	throughout that	time	he	has	been	a	constant	source	of	guidance,	support.		It	has	been	an invaluable	experience	working	under	someone	with	so	much	energy	and	so	much drive.		I	could	not	have	asked	for	a	better	set	of	supervisors	and	it	was	an	honor working	under	them	both.   I	would	also	like	to	thank	Dr.	Vijay	Sharma.		Dr.	Sharma	produced	the	rich  background	of	data	this	work	is	based	on	and	conceived	the	original	concept	for	this thesis.		Furthermore,	he	was	a	constant	source	of	intellectual	guidance	during	the completing	of	experiments	and	writing	of	this	report.   I	also	owe	special	thanks	to	Mr.	Rich	Wambolt,	Dr.	Allard’s	lab	manager,	for  providing	the	immense	technical	expertise	required	for	working	heart	perfusion, and	helping	me	organize	the	study.		Also	from	the	Allard	lab,	I	would	like	to	thank Nathan	Wong	for	collecting	food	and	water	measurements	when	I	was	unable	to.		To Ms.	Violet	Yuen	I	would	also	like	to	say	thanks	for	handling	all	of	the	plasma	analysis and	being	a	constant	source	of	technical	knowledge.		Also	from	the	McNeill	lab	I would	like	to	thank	Sally	Mustafa	and	Linda	Tran	for	assisting	with	the	plasma analysis.        xiii  Introduction     I	would	also	like	to	acknowledge	the	time	and	hard	work	committed	to	this  project	by	the	Genetically	Engineered	Models	(GEM)	facility	staff;	Claire	Smits, Tatjana	Bozin,	Lubos	Bohunek	and	Lynne	Carter.		They	were	involved	in	every aspect	related	to	the	experimental	animals.		They	performed	all	the	necessary surgeries,	all	of	the	echocardiography	and	they	helped	ensure	that	all	procedures were	ethical	and	correct.		Furthermore	they	assisted	me	by	collecting	blood	samples and	caring	for	the	animals	when	I	was	not	available.		Without	their	guidance	I	would have	been	lost.   I	would	like	to	thank	my	supervisory	committee,	Dr.	John	Hill	(committee  chair)	and	Dr.	Greg	Bondy	for	their	guidance	and	support	during	my	journey.		I would	also	like	to	extend	a	thank	you	to	the	Department	of	Pathology	program advisor,	Dr.	Hayden	Pritchard.		He	was	always	available	when	needed	and	I	greatly appreciated	that	he	placed	a	high	priority	on	the	students	needs.   Finally,	I	would	like	to	thank	the	Canadian	Institute	of	Health	Research	for  not	only	providing	funding	for	the	project	but	also	for	providing	a	stipend	through the	Fredrick	Banting	and	Charles	Best	Canada	Graduate	Scholorships	Master’s Award.        xiv  Introduction    1. Introduction  1.1. Diabetes Mellitus Diabetes	mellitus	(diabetes)	is	a	condition	of	chronically	elevated	blood glucose	levels.		It	is	clinically	defined	as	an	8‐hour	fasting	plasma	glucose	greater then	7.0	mM,	a	non‐fasting	plasma	glucose	of	greater	then	11.1	mM	or	a	2‐hour plasma	glucose	of	greater	then	11.1	mM	on	an	oral	glucose	tolerance	test	1.		The current	world	wide	prevalence	is	estimated	at	285	million	people,	and	the	number is	fast	rising,	with	some	estimating	an	increase	in	confirmed	sufferers	to	438	million by	the	year	20302.		Most	sufferers	today	are	found	in	the	developed	world,	however, the	greatest	increases	in	prevalence	are	expected	to	be	in	developing	countries	in Asia	and	Africa3. Diabetes	is	a	disease	of	damaged	insulin	signaling.		Insulin	is	a	peptide hormone	produced	by	β‐cells	located	in	the	islets	of	Langerhans	in	the	pancreas,	and it	is	used	to	regulate	blood	glucose	content.		Diabetes	develops	either	through failure	of	β‐cells	to	secrete	enough	insulin,	or	through	failure	of	cells	to	detect insulin.		Regardless	of	the	specific	early	cause,	β‐cell	death,	loss	of	insulin	sensitivity and	hyperglycemia	will	develop	in	most	cases4‐6		.		However,	diabetes	affects	more then	just	glucose	homeostasis,	protein	and	lipid	metabolism	are	also	severely disturbed7.        1  Introduction    Type	1	diabetes	is	caused	by	the	destruction	of	insulin	secreting	β‐cells.		The  presence	of	antibodies	targeting	insulin	or	other	antigens	associated	with	islet	cells indicate	that	the	effector	mechanism	behind	β‐cell	death	is	often	autoimmunity, however,	the	underlying	cause	is	a	combination	of	genetic	or	epigenetic susceptibility	and	environmental	stimuli	8. Type	2	diabetes	is	caused	by	a	reduction	in	sensitivity	to	insulin,	termed: insulin	resistance.		Development	of	insulin	resistance	is	due	to	a	combination	of;	a genetic	predisposition,	often	indicated	by	family	history	or	race/ethnicity;	and	an unhealthy	lifestyle,	usually	low	physical	activity,	excess	caloric	intake	and	obesity9. Of	the	two	major	types	of	diabetes	mellitus,	Type	1	diabetes	sufferers comprise	about	5‐10%	of	the	total	diabetic	population	and	Type	2	diabetics comprise	almost	the	entire	remainder	2	.		A	third	common	form	of	diabetes,	known as	gestational	diabetes,	affects	about	3.7%	of	non‐aboriginal	and	8‐18%	of aboriginal	women	during	pregnancy.		The	presence	of	gestational	diabetes	increases the	likelihood	that	the	mother	will	later	develop	Type	2	diabetes	10.  1.2. Complications during Diabetes Diabetes	mellitus	is	a	disease	that	causes	severe	homeostatic	disturbances	in the	blood,	thus	deleterious	effects	can	be	found	in	many	different	body	tissues.		For instance,	the	effects	of	diabetes	on	the	microvasculature	and	peripheral	nervous system	are:	suspected	to	be	the	leading	cause	of	new	cases	of	blindness	in	adults between	20‐74	years	of	age;	are	the	leading	cause	of	kidney	failure,	accounting	for 44%	of	all	new	cases	in	2008;	and	are	the	leading	cause	of	non‐traumatic	lower	limb       2  Introduction   amputation11.		Diabetes	also	increases	the	risk	for	macrovascular	disease,	including development	of	atherosclerosis	and	a	150‐400%	increase	in	the	likelihood	of stroke12,	13	.		In	addition,	diabetes	has	a	powerful	association	with	cardiovascular disease	(CVD).		CVD	is	the	primary	cause	of	death	in	both	Type	1	and	Type	2	diabetic patients	and	is	the	single	largest	component	of	health	care	expenses	associated	with diabetes12.   1.3. Diabetic Cardiomyopathy   In	1972	Rubler	et	al.	first	proposed	the	presence	of	a	diabetes	specific  cardiomyopathy	in	four	patients	who	developed	cardiac	hypertrophy,	interstitial fibrosis	and	eventually	heart	failure	without	any	discernable	cause14.		In	1974 Kannel	et	al.	demonstrated,	in	the	landmark	Framingham	Heart	Study,	that	diabetes is	an	independent	risk	factor	for	the	development	of	heart	failure.		Furthermore, they	showed	that	diabetic	men	displayed	a	greater	then	two‐fold,	and	women,	a	five‐ fold	increase	in	risk15.		Today	there	is	much	evidence	describing	the	increased cardiovascular	disease	risk	to	diabetic	patients,	however,	the	existence	of	a cardiomyopathy	which	develops	independent	of	hypertension,	valvular	and congenital	heart	disease	or	coronary	artery	disease	is	still	somewhat	controversial in	the	eyes	of	some	clinicians16.		In	addition,	clinical	societies	have	often	not recognized	it	as	a	stand‐alone	entity,	only	Type	1	diabetes	was	mentioned	as	a	cause of	secondary	cardiomyopathy	in	the	2006	American	Heart	Association	Classification of	Cardiomyopathies,	and	a	similar	assertion	was	made	in	the	2008	European Society	of	Cardiology	scientific	statement17,	18	.		Sharma	et	al.	point	out	that	diabetic       3  Introduction   cardiomyopathy	(DCM)	often	leads	to	heart	failure	when	it	is	combined	with ischemic	heart	disease	or	hypertension,	thus	the	presence	of	DCM	may	not	be obvious	and	other	complications	may	be	highlighted	19.		Similarly,	Maisch	argues that	the	problem	may	lay	with	the	definition	of	DCM.		He	points	out	that	in	a	clinical setting	DCM	will	be	classified	using	functional	measurements,	thus	DCM	may	be classified	as	a	non‐ischemic	or	ischemic	cardiomyopathy	or,	‘heart	failure	with normal	ejection	fraction’,	or	‘heart	failure	with	reduced	ejection	fraction’	20.		Maisch goes	on	to	suggest	a	common	definition	for	DCM	‐	“distinct	entity	characterized	by the	presence	of	abnormal	myocardial	performance	or	structure	in	the	absence	of epicardial	coronary	artery	disease,	hypertension,	and	significant	valvular	disease”, which	is	inline	with	accepted	definition	or	cardiomyopathy	in	general20.		A commonly	reported	characteristic	of	DCM,	in	both	young	Type	1	and	60%	of	well controlled	normotensive	Type	2	diabetics,	is	the	development	of	diastolic dysfunction,	defined	as	“early	diastolic	filling,	prolongation	of	isovolumetric relaxation,	and	increased	atrial	filling”	21‐23		.		Thus,	others	extend	the	definition	of DCM	to	include	an	early	phase	indicated	by	an	asymptomatic	diastolic	dysfunction, which	later	progresses	to	overt	systolic	dysfunction	and	eventual	heart	failure24,	25	.   Several	diabetic	animal	models	have	been	used	to	study	the	progression	of  DCM.		Akita	mice	are	a	genetic	model	of	Type	1	diabetes,	they	possess	a	single	base pair	mutation	which	causes	misfolding	of	proinsulin,	endoplasmic	reticulum	stress and	β‐cell	death	leading	to	hyperglycemia26.		The	Akita	mouse	can	also	display diastolic	dysfunction	in	absence	of	systolic	dysfunction,	similar	to	the	early	phase	of DCM	in	humans	27.		The	ob/ob	mouse	model	of	diabetes	shows	a	deficiency	in	the        4  Introduction   hormone	leptin,	as	a	result	animals	become	obese	and	eventually	develop	Type	2 diabetes28.		Diastolic	dysfunction	has	also	been	reported	in	these	animals29.		The streptozotocin	(STZ)	induced	model	of	Type	1	diabetes	has	been	used	in	over	7600 scientific	publications,	making	it	the	most	commonly	used	model	of	diabetes,	and the	second	most	commonly	used	animal	model	in	research.		In	this	model,	diabetes is	induced	using	STZ,	a	glucose	moiety	produced	by	the	bacterium	Streptomyces griseus,	that	is	toxic	to	insulin	producing	β‐cells	in	the	pancreas30.		The	STZ	model displays	DCM,	with	an	initial	asymptomatic	diastolic	dysfunction	followed	by development	of	an	overt	systolic	dysfunction.		Importantly,	this	model	does	not develop	hypertension,	or	atherosclerosis,	showing	that	DCM	can	occur	in	the absence	of	any	other	cardiovascular	complication31‐35				.  1.4. Causes of Diabetic Cardiomyopathy A	variety	of	molecular	and	structural	causes	have	been	implicated	in	the development	of	DCM.		They	range	from	abnormalities	in	calcium	handling, structural	changes	within	the	heart,	development	of	neuropathy,	hormonal abnormalities	and	finally	metabolic	changes	and	oxidative	stress36.				When considering	the	diversity	of	causes	of	DCM,	it	is	important	to	remember	that	all	the discussed	causes	converge	on	two	effector	mechanisms,	either	reduced	ventricular compliance	or	reduced	ventricular	contractility19.           5  Introduction    1.4.1.   Calcium Handling Abnormalities  Disturbances	in	calcium	handling	are	a	hallmark	of	DCM.		Calcium	is	essential  for	coupling	of	excitation	and	contraction,	thus,	precise	control	of	calcium	levels	is essential	for	normal	function.			There	are	numerous	studies	in	animal	models	that show	reductions	in	expression	and	activity	in	all	calcium	transporters	that	are involved	in	excitation	and	contraction	coupling.		These	include,	sarcoplasmic reticulum	Ca2+‐ATPase	(SERCA),	Na+/Ca2+	exchanger	(NCX),	ryanodine	receptor (RyR),	and	plasma	membrane	Ca2+‐ATPase	(PMCA)	36.		Some	of	the	observed changes	in	activity	are	due	to	Protein	kinase	C,	which	is	activated	during	diabetes and	heart	failure,	and	is	known	to	phosphorylate	a	number	of	enzymes	that	are involved	in	cellular	calcium	handling	37‐39		.   The	effect	diabetes	has	on	the	SERCA	homologue	SERCA‐2a	and	its	inhibitor  phospholamban	(PLB),	seem	to	be	particularly	important.		Protein	and	mRNA	levels of	both	proteins	are	reduced	during	diabetes,	and	depression	in	SERCA	activity	is known	to	lead	to	calcium	overload	in	the	cytosol	and	impaired	contraction40,	41	. Furthermore,	overexpression	of	SERCA	in	diabetic	rodent	models	can	lead	to normalization	of	function42.   1.4.2.   Cell Death and Fibrosis  Sharma	et	al.	have	demonstrated	development	of	a	pro‐apoptotic	signaling  state	in	the	diabetic	heart34.		In	addition,	myocyte	apoptosis	and	necrosis	in	the diabetic	heart	are	often	reported	in	literature43.		Cell	death	has	an	important	impact on	the	heart	due	to	its	limited	regenerative	capacity.		According	to	Cai	et	al.	cell       6  Introduction   death	will	lead	to	“loss	of	contractile	units,	conduction	disturbances,	compensatory hypertrophy	of	myocardial	cells	and	fibrosis”		43‐45		.		The	molecular	basis	behind increased	cell	death	centers	around	metabolic	disturbances	and	the	resultant oxidative	stress,	as	well	as	inflammation43. Although	the	presence	of	increased	apoptosis	in	the	diabetic	heart	is	difficult to	doubt,	the	reported	rates	might	not	be	as	high	as	initially	thought.		Many	studies use	Terminal	deoxynucleotidyl	transferase	dUTP	Nick	End	Labeling	(TUNEL)	or detection	of	activated	caspase‐3	to	assess	apoptosis46,	47	.		However,	TUNEL	is	not completely	specific	for	apoptosis,	as	it	will	generate	a	positive	signal	in	cells	that	are undergoing	DNA	repair	48.		Also,	levels	of	activated	caspase‐3	are	not	always	an accurate	indicator	of	apoptosis.		Sharma	et	al.	have	demonstrated	that	cleaved caspase‐3	can	be	sequestered	and	deactivated	by	caveolins34.   Development	of	myocardial	fibrosis,	is	another	key	characteristic	observed  during	DCM	and	is	known	to	be	at	least	partially	due	to	replacement	fibrosis following	necrotic	or	apoptotic	cell	death36.		There	is	also	a	relationship	between fibrosis	and	diastolic	dysfunction,	as	demonstrated	in	both	Type	2	diabetic	rodents and	in	human	Type	1	diabetics	with	asymptomatic	diastolic	dysfunction49,	50	. Activation	of	Protein	Kinase	C	β2	(PKC‐β2)	and	the	Renin	Angiotensin‐ Aldosterone	System	(RAAS)	are	both	involved	in	development	of	fibrosis	during diabetes.		PKC‐β2	is	a	mediator	of	fibrosis	and	its	activity	is	known	to	increase during	hyperglycemia.		Furthermore	its	expression	is	increased	in	rodent	models	of STZ	induced	diabetes51.		Overexpression	of	PKC‐β2	in	animal	models	causes	cardiac hypertrophy,	fibrosis	and	left	ventricular	dysfunction52.		The	RAAS	is	also	activated        7  Introduction   during	diabetes,	and	is	thought	to	contribute	to	myocyte	necrosis	and	development of	fibrosis	in	the	heart36.   1.4.3.  Metabolism and Oxidative Stress  1.4.3.1. Fuel Usage   In	the	normal	heart	60‐80%	of	ATP	is	derived	from	oxidation	of	fatty	acids,  and	20‐40%	from	oxidation	of	carbohydrates,	during	diabetes	fuel	usage	is	severely disturbed,	with	a	near	complete	loss	of	carbohydrate	oxidation	and	an	acceleration in	fatty	acid	oxidation53‐55		.			Changes	in	substrate	uptake	and	reciprocal	regulation of	fatty	acid	and	glucose	oxidation	are	blamed	for	the	metabolic	disturbances	in	the diabetic	heart. GLUT4	is	the	primary	inducible	glucose	transporter	in	the	adult	heart. GLUT4	is	normally	sequestered	into	intracellular	vesicles,	and	upon	stimulation	by insulin	or	contraction	it	is	translocated	to	the	sarcolemma56.		During	diabetes	there is	a	reduction	in	glucose	transport	into	cardiac	myocytes,	this	is	primarily	due	to	a reduction	in	total	expression	of	GLUT4	as	well	as	reduction	in	translocation	of	the remaining	protein53. Lipids	are	supplied	to	tissue	from	lipoprotein	such	as	chylomicrons	and	Very Low	Density	Lipoprotein	(VLDL)	that	contain	esterified	fatty	acids	collected	from the	gut	and	the	liver,	respectively;	or	from	free	non‐esterified	fatty	acids	that	are bound	to	albumin.		Fatty	acids	from	chylomicrons	and	VLDL,	are	greater	in	molar concentration	by	approximately	10‐fold	compared	to	albumin	bound	fatty	acids57. Albumin	bound	fatty	acids	can	be	taken	up	by	mycotyes	directly	(via	transporters       8  Introduction   on	the	myocyte	surface),	while	lipoprotein	bound	fatty	acid	must	first	be	released	by the	enzyme	lipoprotein	lipase	(LPL)	found	on	the	vascular	luminal	surface	of endothelial	cells.		During	diabetes,	levels	of	lipoproteins	and	albumin	bound	fatty acid	increase,	as	does	LPL	activity	at	it’s	functional	site	in	the	vasculature,	thus,	fatty acid	uptake	into	cardiomyocytes	is	increased58‐60		. Fatty	acid	oxidation	and	carbohydrate	oxidation	are	reciprocally	regulated, thus,	rates	of	glucose	metabolism	are	further	inhibited	by	accelerated	rates	of	fatty acid	oxidation,	this	effect	is	referred	to	as	the	Randle	cycle60.		The	Randle	cycle	is mediated	through	changes	in	ratios	of	NADH/NAD+,	acetyl‐CoA/free	CoA	and	citrate levels.		Accelerated	fatty	acid	oxidation	increases	NADH/NAD+	ratio,	and	acetyl‐ CoA/free	CoA	ratios,	both	of	these	actions	inhibit	the	pyruvate	dehydrogenase complex,	reducing	flux	through	the	tricarboxylic	acid	cycle	and	preventing	full oxidation	of	glucose60.		Furthermore,	citrate	levels	are	also	increased	by	fatty	acid oxidation,	citrate	inhibits	phosphofructokinase‐1,	a	key	rate	controlling	step	in glycolysis61.  1.4.3.2. Lipid Accumulation     Increased	lipid	uptake	and	oxidation	by	the	diabetic	heart	has	several  deleterious	consequences.		Accelerated	fatty	acid	uptake	decreases	the	diabetic heart’s	oxygen	efficiency,	the	ex	vivo	oxygen	requirements	for	Type	1	and	Type	2 diabetic	rodent	hearts	are	increased	by	57%	and	85%,	respectively.		A	similar	effect is	observed	when	fatty	acid	levels	that	the	heart	is	exposed	to	are	increased,	the        9  Introduction   same	group	reported	a	15%	increase	in	control	hearts	when	they	were	perfused with	a	high	versus	low	concentration	of	fat62. Another	consequence	of	increased	lipid	uptake	and	oxidation	is	the development	of	lipotoxicity,	or	cellular	dysfunction	and	death	associated	with	lipid accumulation63.		A	substantial	amount	of	evidence	has	emerged	linking	excess	lipid supply	and	accumulation	in	myocytes	with	cardiomyopathy,	both	in	humans	with impaired	metabolic	regulation	and	in	animal	models	of	diabetes	and	other	metabolic disorders63‐65		.		Certain	fatty	acids	have	been	shown	to	induce	apoptosis	in	several different	cell	types,	including	cardiomyocytes66.		Often	referred	to	as	‘palmitate induced	apoptosis’,	the	effect	seems	to	be	most	pronounced	with	long	chain saturated	fatty	acids,	such	as	palmitate	(C16:0)	and	stearate	(C18:0),	shorter	chain lengths	and	unsaturated	fatty	acids	do	not	seem	to	cause	lipotoxicity67,	68	.		The	key effector	mechanism	in	palmitate‐induced	apoptosis	is	believed	to	be	production	of ceramide	and	increased	oxidiatve	stress.		Ceramides	are	a	lipid	signaling	molecule that	are	involved	in	propagating	pro‐apoptotic	signaling	and	are	synthesized	from saturated	fatty	acids	like	palmitate69.		Furthermore,	cell	permeable	ceramide analogues	generate	the	same	effect	as	palmitate	and	inihibiton	of	ceramide synthesis	often	prevents	apoptosis	in	the	presence	of	palmitate70.	However, ceramide	production	is	not	essential	for	palmitate	induced	apoptosis	in	all	cell types,	as	isolated	cardiomyocytes	from	chick	embryos,	Chinese	Hamster	Ovaries (CHO)	and	H4IIE	liver	cells	undergo	apoptosis	via	a	ceramide‐independent pathway71‐73		.		On	the	other	hand,	oxidative	stress	appears	to	be	an	essential mediator	of	palmitate‐induced	cell	death,	as	generation	of	reactive	oxygen	and        10  Introduction   nitrogen	species	increases	during	lipid	accumulation	in	a	variety	of	cell	types	and apoptosis	can	be	prevented	using	agents	that	scavenge	reactive	species73‐76			.  1.4.3.3. Hyperglycemia   Similar	to	excess	lipid	accumulation,	excess	glucose	load	during	diabetes	can  cause	significant	cellular	damage	and	eventually	lead	to	myocyte	death, hyperglycemia’s	toxic	effects	are	referred	to	as,	glucotoxicity77.		The	clinical relevance	of	hyperglycemia	is	well	recognized.		For	every	1%	increase	in	levels	of glycated	hemoglobin,	(HbA1c,	glucose	permanently	modifies	a	portion	of	total hemoglobin	upon	long	term	exposure,	greater	glucose	concentrations	yield modification	of	more	hemoglobin)	there	is	an	8%	increase	in	risk	of	heart	failure78. Furthermore,	elevated	plasma	glucose,	even	without	diabetes,	is	a	predictor	for	the development	of	cardiovascular	disease79.		Although	there	are	many	factors	and processes	involved	in	the	pathologic	effects	of	glucotoxicity,	the	primary	effector mechanisms	revolve	around	protein	glycation,	formation	of	reactive	oxygen	species and	glucose	flux	through	alternate	pathways25,	77	. Glycation,	or	‘non‐enzymatic	glycosylation’,	is	a	posttranslational modification	where	a	carbohydrate	group	is	added	to	a	protein,	lipid	or	nucleic	acid molecule.		Glycation	can	affect	the	activity	of	proteins,	such	as	p53;	a	transcription factor	known	to	regulate	cell	death80.		Increased	glycosylation	of	p53	in	isolated myocytes	exposed	to	hyperglycemia	has	been	demonstrated	to	increase	its	activity, leading	eventually	to	activation	of	the	local	RAAS,	production	of	angiotensin	II	and increase	in	ratio	of	pro‐/anti‐apoptotic	proteins77.		Glycation	is	also	responsible	for        11  Introduction   the	production	of	Advanced	Glycation	End	products	(AGEs).		AGEs	are	proteins, lipids	or	nucleic	acids	that	have	been	modified	as	a	result	of	increases	in	oxidative stress	and	excess	carbohydrates81.		AGE	formation	is	deleterious	not	only	because proteins	are	structurally	modified,	but	also	because	they	can	activate	pro‐ inflammatory	pathways	when	they	bind	their	receptors,	RAGE.		Furthermore, formation	of	the	AGE‐RAGE	complex	can	activate	the	NADPH	oxidase	complex, leading	to	further	reactive	oxygen	species	(ROS)	production82,	83	. NADHP	oxidase	mediated	ROS	production	is	also	dependent	on	several	other signaling	molecules	and	proteins	such	as,	diacylglycerol	(DAG),	angiotensin	II	and Rac184,	85	.		During	hyperglycemia	de	novo	DAG	synthesis	has	been	demonstrated	to increase.		DAG	is	a	second	messenger	signaling	molecule	that	is	a	physiological activator	of	Protein	Kinase	C	(PKC),	activated	PKC	is	known	to	stimulate	NADPH oxidase	activity82,	84	.			Angiotensin	II	is	an	effector	molecule	produced	by	the	RAAS. During	diabetes	the	RAAS	is	locally	activated,	the	resultant	angiotensin	II	stimulates NADPH	oxidase	activity36,	85	.		Rac	is	a	small	guanosine	triphosphate	binding	protein that	is	a	member	of	the	NADPH	oxidase	complex	and	is	essential	for	its	formation, Rac1	is	the	major	isoform	in	the	cardiomyocyte86,	87	.		Rac1	was	recently demonstrated	to	be	essential	for	the	development	of	hyperglycemia	induced apoptosis	in	cardiomyocytes,	and	this	role	was	demonstrated	to	be	mediated through	NADPH	oxidase88. Glucotoxicity	can	also	result	from	diversion	of	glucose	from	the	oxidative pathway	into	alternate	metabolic	pathways19,	53	.	During	diabetes,	glucose	uptake	is significantly	reduced	but	not	completely	abolished.		However,	due	to	inhibition	of        12  Introduction   glucose	oxidation	and	glycolysis	caused	by	fatty	acid	oxidation,	the	small	portion	of glucose	that	enters	the	cell	can	be	shunted	towards	the	polyol	or	hexosamine biosynthetic	pathways	89. The	polyol	pathway	consists	of	an	NADPH	requiring	reduction	of	glucose	to sorbitol	via	aldose	reductase	and	an	oxidation	of	sorbitol	to	fructose	via	sorbitol dehydrogenase90,	91	.		During	diabetes,	excess	glucose	enters	this	pathway	and stimulates	aldose	reductase	but	not	sorbitol	dehydrogenase,	thus	excess	flux through	the	polyol	pathway	leads	to	reduction	of	the	NADPH/NADP+	ratio.		NADPH is	required	to	produce	glutathione,	a	major	endogenous	antioxidant,	thus	polyol	flux can	weaken	cellular	antioxidant	defenses90. Under	physiologic	conditions	the	Hexosamine	Biosynthetic	Pathway’s	(HBP) role	is	to	act	as	a	fuel	sensor,	and	to	help	partition	fuels	to	the	appropriate	storage sites	within	the	body92.		Carbohydrates	enter	this	pathway	as	fructose‐6‐phosphate, immediately	before	the	rate	controlling	phosphofructokinase‐1‐mediated	step	in glycolysis.		The	final	product	of	the	HBP	is	UDP‐N‐acetylglucosamine	(UDP‐GlcNAc), this	then	serves	as	a	substrate	for	O‐GlcNAc	transferase,	which	then	attaches	the GlcNAc	moiety	to	specific	sites	on	target	proteins93.		The	HBP	is	implicated	in	a number	of	cellular	processes,	including	intracellular	signaling,	modification	of protein	degradation	and	modulating	protein‐protein	interaction93,	94	.		During diabetes,	flux	through	the	HBP	is	increased,	possibly	due	to	inhibition	of	PFK‐1	via the	Randle	cycle	and	accelerated	fatty	acid	oxidation19.		Recently,	Rajamani	et	al. demonstrated	increased	GlcNAc	tagging	of	the	pro‐apoptotic	Bad	protein	in	myocyte        13  Introduction   during	hyperglycemia,	furthermore,	GlcNAc	tagging	prevented	interaction	of	Bad and	the	apoptosis	inhibitor,	BCl‐295.   1.5. Selected Treatment Strategies 1.5.1.  β-Blockers  β‐adrenergic	receptors	are	a	class	of	G‐protein‐linked	receptors	(β‐receptors) that	accept	catecholamines,	such	as	epinephrine	and	norepinephrine,	and	signal	as part	of	the	sympathetic	nervous	system.		All	three	subtypes	of	β‐receptors	are	found in	the	heart,	β1,	β2,	and	β3,	however,	β1	is	the	most	abundant	and	has	the	most powerful	effect	on	contractile	function96‐98		.		Outcomes	of	β	receptor	signaling	are dependent	on	the	specific	heterotrimeric	G	proteins	they	couple	to;	β1	and	β2	can couple	to	stimulatory	Gs	proteins	while	β2	and	β3	can	couple	to	the	inhibitory	Gi protein.		Signaling	through	Gs	leads	to	activation	of	adenylyl	cyclase	triggering increases	in	cAMP	levels	and	activation	of	Protein	Kinase	A	(PKA),	while	signaling through	Gi	inhibits	adenylyl	cyclase.		PKA	phosphorylates	several	different sarcolemmal	proteins	including	L‐type	Ca2+	channels	and	phospholamban,	these actions	enhance	calcium	influx	into	the	myocyte	and	calcium	uptake	into	the sarcoplasmic	reticulum.		Thus	the	net	effect	of	Gs	signaling	is	enhanced contraction99,	100	. β‐adrenergic	receptor	antagonists	(β‐blockers),	are	a	class	of	drugs	that	bind and	block	the	action	of	one	particular,	or	several,	β‐receptors,	thus,	they	block sympathetic	signaling	and	have	acute	negative	inotropic	and	chronotropic	effects101.        14  Introduction   Due	to	their	suppression	of	contracton,	β‐blockers	were	originally	considered dangerous	for	heart	failure	sufferers102‐104		.		However,	it	is	now	known	that	with chronic	treatment	they	improve	cardiac	function,	and	reduce	morbidity	and mortality	in	heart	failure	patients.		Consequently,	their	use	is	currently	strongly supported	by	clinical	guidelines	and	by	consensus105‐107		. β‐blockers	are	not	created	equal	in	terms	of	receptor	specificities	and	chemical properties.		Currently	only	three	β‐blockers,	bisprolol,	metoprolol	and	carvedilol, have	been	approved	for	patients	undergoing	heart	failure.		Metoprolol	and	bisprolol are	β1	selective	inverse	agonist,	meaning	that	they	bind	and	block	the	β1	receptor (and	β2	at	high	doses)	but	also	reduce	receptor	signaling	below	basal	levels108. Carvedilol	is	a	nonselective	β	blocker	which	also	displays	antagonism	for	the	α1 receptor.		Carvedilol	is	also	known	to	have	clinically	relevant	antioxidant	and vasodilating	properties109.  1.5.1.1. β-Blockers and Diabetic Cardiomyopathy The	mechanism	of	β‐blockers	therapeutic	effect	during	heart	failure	is thought	to	center	around	their	ability	to	mitigate	excessive	adrenergic	drive,	helping normalize	impaired	calcium	handling110.		Increased	adrenergic	signaling	has	also been	demonstrated	during	DCM,	as	has	the	associated	reduction	in	β1	receptor sensitivity	and	expression,	and	impaired	calcium	handling36,	111,	112		.		Thus,	long term	β‐blocker	therapy	may	help	to	ameliorate	some	of	the	dysfunction	observed during	DCM.		In	a	series	of	studies	by	Sharma	et	al.	the	effect	of	metoprolol	on	STZ        15  Introduction   diabetic	rats	was	examined19,	33,	34,	112,	113.	Metoprolol	in	this	model	was demonstrated	to	ameliorate	functional	impairments	in	isolated	perfused	working hearts,	improving	reduced	hydraulic	power,	rate	pressure	product	and	cardiac output33.		Independent	to	its	functional	effects,	metoprolol	also	partially	ameliorated metabolic	disturbances	during	diabetes,	with	Sharma	et	al.	reporting	reductions	in fatty	acid	oxidation	and	secondary	increases	in	glucose	oxidation33,	34.		Finally, metoprolol	also	appeared	to	switch	the	diabetic	heart	away	from	an	activity	pattern promoting	activation	of	the	pro‐apoptotic	Bad	and	inhibition	of	anti‐apoptotic	BCl‐2, to	inhibition	of	Bad	and	activation	of	BCl‐2,	while	not	reducing	oxidative	DNA damage.		Thus	metoprolol	improved	function	and	reduced	the	sequela	of	diabetes and	oxidative	stress	without	reducing	oxidative	stress34. However,	the	concept	of	treating	diabetics	with	β‐blockers	is	somewhat controversial.		Much	of	the	controversy	centers	around	the	belief	that	β‐blockers will	reduce	awareness	of	symptoms	of	hypoglycemia,	however,	there	is	direct scientific	evidence	from	studies	in	human	diabetics	and	normals	refuting	this claim114‐116		.		Another	perceived	negative	consequence	of	β‐blockade,	and metoprolol	in	particular,	is	that	it	may	promote	development	of	new	cases	of diabetes.		This	belief	stems	largely	from	retrospective	analysis	of	data	from	the Carvedilol	Or	Metoprolol	European	Trial	(COMET),	where	it	was	found	that metoprolol	tartrate	was	associated	with	an	increase	in	new	onset	diabetes	of	10.1% compared	to	8.7%	for	carvedilol117.		However,	there	has	been	much	criticism	of these	findings,	as	the	actual	comparison	being	made	is	to	carvedilol	treated	patients, thus	there	is	no	proof	that	metoprolol	actually	triggers	new	onset	diabetes.        16  Introduction   Furthermore,	an	unreccomended	low	dose	of	the	non‐clinically	proven	metoprolol tartrate	(50	mg,	twice	daily;	quick	release	formulation)	was	used	instead	of	the more	effective	metoprolol	succinate	(slow	release	formulation)	118.		Comments	from cardiologist	Dr.	John	McMurray	summarize	these	results	best,	“a	proven	dose	of carvedilol	is	clearly	superior	to	a	non‐recommended,	low	dose	of	a	short	acting formulation	of	metoprolol…”119.   1.5.2.  Ascorbic Acid  There	are	many	sources	of	increased	oxidative	stress	during	diabetes, including	altered	fuel	usage,	accumulation	of	lipids	and	hyperglycemia.		This increased	oxidative	stress,	as	discussed	above,	is	thought	to	contribute	to	the contractile	dysfunction	observed	during	DM.		The	cardioprotective	effects	of	a number	of	antioxidant	molecules	have	been	evaluated,	including,	β‐carotene, vitamin	E	and	Ascorbic	Acid	(AA).		To	date,	there	have	been	many	encouraging findings	in	epidemiological	studies.		High	β‐carotene	intake	has	been	shown	to reduce	cardiovascular	risk	in	the	Nurses	Health	Study,	reduce	cardiovascular mortality	and	myocardial	infarction	in	the	elderly	in	the	Massachusetts	Health	Care Panel	Study,	and	reduce	cardiovascular	risk	in	the	Health	Professionals	Follow	Up Study120‐122		.		Vitamin	E	has	been	associated	with	reduced	cardiovascular	death	and risk	in	the	Nurses	Health	and	Health	Professional	Follow	Up	Study120‐122.		AA	is among	one	of	the	most	common	antioxidants	available	and	it	has	proven	effective	in reducing	cardiovascular	risk	in	both	the	National	Health	and	Nutrition	Examination Survey	and	the	Eastern	Finland	Study120,	123,	124.       17  Introduction    Basic	scientific	studies	have	also	provided	evidence	for	a	therapeutic	role	for  antioxidants.		In	a	study	by	Qin	et	al.	it	was	demonstrated	that	a	combination treatment	of	AA	and	vitamin	E	was	able	to	reduce	oxidative	stress,	increase	BCl‐2 expression	and	lower	Caspase	3	activity	in	rabbit	hearts	post	myocardial	infarction 125.		Furthermore,	the	ability	of	the	β‐blocker	carvedilol	to	reduce	infarct	size	is  matched	by	SB	211475,	a	metabolite	of	carvedilol	without	any	adrenergic	receptor blocking	ability,	but	possessing	its	antioxidant	strength126.		Finally,	Dai	et	al.	were able	to	show	that	oral	AA	intake	could	lead	to	partial	amelioration	of	myocardial dysfunction,	including	filling	rates,	in	a	dose	dependent	manner,	in	the	STZ	diabetic rat.		Furthermore,	they	demonstrated	that	AA	was	able	to	lower	elevated	plasma triglycerides,	cholesterol	and	free	fatty	acid	levels,	again	in	a	dose	dependent manner127. In	sharp	contrast	to	the	findings	in	support	of	antioxidant	therapy	in epidemiological	and	basic	science	studies	lies	the	largely	negative	data	from	clinical trials128,	129.		Generally	speaking,	clinical	trials	have	not	been	able	to	demonstrate	a clear	therapeutic	relationship	between	antioxidants	and	cardiovascular	disease, however,	several	methodological	aspects	are	outlined	by	Ye	et	al.	and	Steinhubl	et al.	that	may	explain	the	discrepancy.		First,	clinical	trials	often	select	antioxidants based	on	ease	of	availability	and	deliverability.		For	example,	in	clinical	studies	a synthetic	vitamin	E	is	often	used,	whereas	natural	vitamin	E	consists	of	8	different forms	with	differing	properties.		Next,	the	duration	of	study	for	a	clinical	trial	is often	shorter	then	an	observational	study,	running	only	5	years	or	so,	whereas observational	studies	could	span	decades.		Finally,	the	study	population	selected        18  Introduction   during	clinical	trials	is	often	older	and	has	preexisting	disease,	therefore	treatment periods	are	much	shorter,	for	example:	2	years	of	antioxidant	therapy	after	40	years of	oxidative	stress.		In	comparison,	in	animal	studies	treatment	often	begins	before overt	disease	is	present	and	prospective	studies	often	begin	when	subjects	are younger	and	healthier.		Although	clinical	trials	provide	greater	control	of	variables then	epidemiological	studies,	due	to	their	shortcomings,	their	findings	in	regard	to antioxidant	therapy	are	at	best	inconclusive	128,	129	.   1.6. Hypothesis and Study Objectives Primary	Hypothesis	(see	Scheme	1):  We	propose	that	metabolic	changes	that	occur	during	diabetes	elevate oxidative	stress,	leading	to	protein	damage,	signaling	changes,	cell	death	and	other sequelea;	the	eventual	sum	of	these	changes	is	an	impairment	of	function.		Treatment of	either	the	sequelae	of	oxidative	stress	or	oxidative	stress	directly	will	be	beneficial but	treatment	of	both	will	improve	function	further.    We	will	address	our	hypothesis	in	two	ways.		First,	we	will	observe	the  development	of	metabolic	impairment,	oxidative	damage	and	DCM	by	studying	a time	point	before	(referred	to	as	Study	1)	and	a	time	point	after	(Study	2) development	of	overt	cardiac	dysfunction.		Second,	we	will	use	two	drugs	that	are known	to	ameliorate	functional	impairment,	metoprolol	and	ascorbic	acid,	to	study        19  Introduction   how	they	affect	oxidative	damage	and	function	from	Study	1	to	Study	2.		Thus	our hypothesis	can	be	broken	down	into	three	sub‐hypotheses:  1. Disturbances	in	metabolism	will	appear	before	the	development	of	overt dysfunction,	while	changes	in	oxidative	protein	damage	will	appear	most prominent	at	the	point	of	dysfunction. 2. Both	metoprolol	and	ascorbic	acid	will	improve	cardiac	function,	however,	only ascorbic	acid	will	reduce	oxidative	stress. 3. Combined	metoprolol	and	ascorbic	acid	treatment	will	improve	function further	then	single	treatment.  We	feel	that	our	approach	will	provide	us	with	new	insights	into	DCM.		By assessing	changes	in	metabolism,	oxidative	damage	and	function	from	Study	1	to Study	2,	we	will	be	able	to	clarify	the	sequence	of	events	involved	in	the development	of	cardiac	dysfunction.			Furthermore,	by	assessing	the	effect	of	our drug	treatments,	we	will	be	able	to	report	whether	reduction	of	oxidative	stress will	supplement	the	beneficial	effects	observed	with	metoprolol.		Finally,	our study	should	provide	insight	into	the	mechanism	of	action	of	metoprolol	and ascorbic	acid.        20  Introduction          21  Methods    2. Methods  2.1. Animals/ Treatment Groups   Animals	were	cared	for	in	accordance	with	the	guidelines	of	the	Canadian  Council	on	Animal	Care.		The	Animal	Care	Committee	at	the	University	of	British Columbia,	Office	of	Research	Services,	approved	the	protocol	for	animal	care.		All animal	experiments	were	conducted	at	the	Genetically	Engineered	Models	Facility located	at	the	James	Hogg	Research	Centre.		Animals	were	divided	into	two	study groups,	titled	Study	1	and	Study	2.		For	both	studies,	weight	matched	(200‐220	g) Male	Wistar	rats	were	used.		Animals	from	Study	1	were	purchased	from	Charles River	(St.	Constant,	Quebec),	while	Study	2	animals	were	obtained	from	the	Centre for	Disease	Modeling	(CDM,	University	of	British	Columbia,	Vancouver,	British Colombia).		The	strain	of	animal	purchased	from	both	sources	was	Wistar,	and animals	were	all	male	with	the	same	approximate	age	at	the	time	of	study. Furthermore,	the	CDM	sourced	animals	used	in	Study	2	display	similar	cardiac functional	and	metabolic	characteristics	during	diabetes	similar	to	rats	from	Charles River	and	elsewhere33,	130,	131		.		Unless	noted,	all	animals	were	allowed	ad	libitum access	to	standard	rat	chow	and	water. For	Study	1,	animals	were	given	one	week	after	arrival	to	acclimatize	and were	then	randomly	divided	into	either	diabetic	or	control	groups.		Diabetes	was induced	by	a	single	intravenous	injection	of	streptozotocin	(STZ)	at	60	mg/kg	body        22  Methods   weight	(60	mg/ml	STZ	in	a	sterile	saline,	0.9%	NaCl	w/v	solution,	delivered	at	1	μl/g body	weight)	into	the	caudal	vein.		Control	animals	were	injected	with	sterile	saline only.		Blood	was	collected	at	one‐week	post	STZ	and	at	termination;	all	animals	were fasted	for	5	hours	before	collection	to	allow	stabilization	of	plasma	insulin	levels. Plasma	glucose	and	insulin	were	measured	at	one‐week	post	STZ	to	ensure induction	of	diabetes	(this	time	point	will	hereafter	be	known	as	‘induction	of diabetes’).		Similar	measurements	were	also	made	at	termination.		One‐week	post induction	of	diabetes	(Week	2	in	Scheme	2),	the	control	and	diabetic	animals	(C,	D) were	divided	into	metoprolol	treated	(CM,	DM),	ascorbic	acid	treated	(CA,	DA),	and metoprolol	with	ascorbic	acid	treated	(CMA,	DMA).		Metoprolol	was	administered via	subcutaneous	Alzet	2ML4	osmotic	pumps,	from	the	Durect	Corporation (Cupertino,	California),	at	a	dose	of	15	mg/kg/day,	respectively.		Ascorbic	acid	was delivered	at	a	dose	of	1000	mg/kg/day	in	the	drinking	water.		β‐blocker	and ascorbic	acid	treatment	lasted	for	a	total	of	four	weeks.		Animals	were	terminated, and	their	hearts	collected	and	perfused	at	five	week	post‐induction	of	diabetes,	a period	during	which	metabolic	abnormalities	have	occurred,	but	overt	cardiac dysfunction	is	reportedly	not	yet	evident	130	.		A	final	blood	collection	from	the	chest cavity	was	made	after	excision	of	the	heart;	urine	was	also	collected	from	the bladder	(Scheme	2). Study	2	was	identical,	with	the	following	exceptions:	Alzet	2006	osmotic pumps	were	used,	treatment	lasted	six	weeks	and	termination	occurred	seven weeks	post‐induction	of	diabetes,	a	period	during	which	both	metabolic abnormalities	and	cardiac	dysfunction	are	reportedly	evident	130	(Scheme	2).        23  Methods    2.2. Echocardiography Heart	function	was	measured	in	vivo	by	echocardiography	using	the	VEVO	770	High Resolution	Imaging	System	with	a	RMV	716	probe,	all	from	Visual	Sonics	(Toronto, Ontario).		All	animals	were	anesthetized	with	2	%	isoflurane.		Left	ventricular	end diastolic	and	end	systolic	diameters	(LVEDD	and	LVESD,	respectively)	were measured.		Calculations	were	automatically	generated	by	the	manufacturer’s software	using	the	following	formulas:		Fractional	Shortening	(FS,	%)	=	[(LVEDD	– LVESD)/LVEDD]	×100%,	Left	Ventricular	End	Diastolic	Volume	(LVEDV,	μl)	= [7.0/(2.4	+	LVEDD)]	x	(LVEDD)3	x	1000,	Left	Ventricular	End	Systolic	Volume (LVESV,	μl)	=	same	as	previous	except	using	LVESD	in	place	of	LVEDD,	ejection fraction	(EF,	%)	=	[(LVEDV	–	LVESV)/LVEDV]	×100%.		All	values	represent	an average	of	a	minimum	of	three	measurements	from	each	animal.		Values	generated for	each	animal	were	then	combined	to	produce	a	value	for	its	treatment	group.  2.3. Function and Fuel Usage 2.3.1.   Perfusion Conditions   Cardiac	function	and	metabolism	were	measured	as	previously	described	132‐  134	.		At	termination,	animals	were	anesthetized	by	4%	isofluorane	anesthesia	and  the	hearts	were	isolated	and	perfused	as	working	heart	preparations	using	a modified	Krebs‐Henseleit	solution	(perfusion	buffer),	supplemented	with	substrates at	physiologically	relevant	concentrations.		Perfusion	buffer	consisted	of	118	mM NaCl,	4.7	mM	KCl,	1.2	mM	KH2PO4,	1.2	mM	MgSO4,	2	mM	CaCl2,	5.5	mM	glucose,	0.5        24  Methods   mM	lactate,	20	U/ml	insulin,	and	0.6	mM	palmitate	bound	to	3%	BSA	132.		Hearts were	perfused	in	working	heart	mode	for	30	minutes,	during	this	time	metabolic and	functional	measurements	were	made	every	6	minutes.		After	completion	of perfusion,	hearts	were	freeze	clamped,	weighed,	and	stored	at	‐80°C	for	further analysis.   2.3.2.   Function  In	order	to	measure	heart	rate	and	peak	systolic	pressure,	a	pressure  transducer,	from	Viggo‐Spectramed	(Oxnard,	California),	was	inserted	into	the afterload	line.		Cardiac	output	and	aortic	flow	were	measured	using	external	flow probes	attached	to	the	preload	and	aortic	outflow	lines;	probes	were	purchased from	Transonic	Systems	Inc.	(Ithaca,	New	York).		From	these	measurements,	cardiac output,	coronary	flow	(cardiac	output	–	aortic	outflow),	rate	pressure	product (heart	rate	x	peak	systolic	pressure)	and	hydraulic	work	(cardiac	output	x	peak systolic	pressure)	were	calculated135	.   2.3.3.  Fuel Usage  In	all	animals	from	Study	1,	and	selected	animals	from	Study	2,	the	rates	of glucose	oxidation	and	palmitate	oxidation	were	quantified	by	measuring	14CO2	and 3H O	produced	by	oxidation	of	[14C]glucose	and	[3H]palmitate,	respectively.		In	the 2  remainder	of	the	hearts	from	Study	2,	glycolysis	and	palmitate	oxidation	rates	were quantified	by	measuring	3H2O,	and	14CO2,	produced	by	oxidation	of	[3H]glucose	and        25  Methods   [14C]	palmitate,	respectively.		All	radioisotope	labeled	glucose	and	palmitate	were purchased	from	Perkin‐Elmer	(Woodbridge,	Ontario). In	order	to	accurately	measure	3H2O	released	into	the	perfusion	buffer	it must	first	be	separated	from	the	[3H]glucose	or	[3H]palmitate	present.		To	separate 3H O	we	loaded	200	μl	of	collected	perfusion	buffer	samples	into	a	cap‐less	500	μl 2  centrifuge	tube	and	placed	this	inside	of	a	larger	7	ml	scintillation	vial	containing 500	μl	of	water.		After	sealing	the	scintillation	vial,	we	allowed	the	3H2O	to	evaporate from	the	smaller	tube	at	60C	for	24	hours.		Samples	were	then	moved	to	a refrigerator	and	incubated	at	4C	to	allow	evaporated	3H2O	to	re‐condense	into	the larger	outer	tube.		After	24	hours,	the	cap‐less	tubes	were	removed	and	the radioactivity	of	recovered	water	within	the	7	ml	scintillation	tube	was	measured using	a	Beckman	LS6500	Liquid	Scintillation	Counter	(Mississauga,	Ontario).	All samples	were	run	in	duplicate	and	control	tubes	loaded	with	known	amounts	of 3H O	were	also	run	in	order	to	measure	efficiency	of	3H O	collection.		3H O	was 2 2 2  purchased	from	Perkin‐Elmer	(Woodbridge,	Ontario). In	order	to	accurately	measure	oxidation	of	14C	containing	substrates,	we must	account	for	both	the	14CO2	released	into	the	atmosphere	and	the	14CO2 converted	to	H14CO3‐	that	was	released	into	the	perfusion	buffer.		This	procedure has	been	previously	described133	.		Briefly,	14CO2	was	captured	by	bubbling	gas produced	inside	of	the	sealed	working	heart	perfusion	rig	through	a	solution	of	the strong	base,	10‐X	hyamine	hydroxide	(1	M	methylbensethonium	hydroxide	in methanol),	hyamine	samples	were	then	collected	and	stored.		H14CO3‐	was	extracted from	the	perfusion	buffer	by	converting	it	to	14CO2	using	9N	H2SO4,	it	was	then        26  Methods   captured	onto	filter	paper	soaked	with	10‐X	hyamine	hydroxide.		The	radioactivity of	the	10‐X	hyamine	samples	and	hyamine	soaked	filter	paper	was	measured	using	a Beckman	LS6500	Liquid	Scintillation	Counter	(Mississauga,	Ontario).  2.4. Metabolism   Plasma	glucose	concentrations	were	measured	using	the	Beckman	Glucose  Analyzer	II.		Plasma	insulin	was	measured	using	the	radioimmunoassay	kit	from Linco	supplied	by	Cedarlane	(Burlington,	Ontario).		Plasma	cholesterol,	and triglycerides	were	determined	by	colorimetric	assay	kits	available	from	Sigma	(St. Louis,	Missouri).		Whole	blood	ketone	levels	were	measured	using	the	CardioChek analyzer	from	Polymer	Technology	Systems	(Indianapolis,	Indiana).   2.4.1.   Tissue Triglyceride Assay  Myocardial	triglyceride	content	was	measured	in	30‐40	mg	of	tissue,	as  previously	described	136	.		Briefly,	tissue	was	powdered	in	liquid	nitrogen	cooled mortar	and	pestle,	then	transferred	to	glass	test	tubes	with	3	ml	of	a	2:1 chloroform:methanol	(v/v).		After	a	1	hour	room	temperature	incubation	mounted on	a	shaking	platform,	0.6	ml	of	0.05%	H2SO4	was	added	and	tubes	were	left overnight	at	4C	to	separate	liquid	phases.	The	lower	liquid	phase	was	collected,	1 ml	of	1%	Triton‐X100	in	chloroform	(v/v)	was	added	and	the	samples	were	dried under	N2	gas	at	45C.		Samples	were	then	reconstituted	in	500	μl	PBS	and	assayed using	a	colorimetric	triglyceride	kit	purchased	from	Caymen	Chemical	supplied	by        27  Methods   Cedarlane	(Burlington,	Ontario).   2.4.2.   Tissue Glycogen  Myocardial	glycogen	content	was	measured	in	95‐110	mg	of	powdered	tissue  as	previously	described	137	.		Briefly,	tissue	was	boiled	in	0.3	ml	of	30%	KOH	for	1 hour	in	pre‐weighed	Corex®	test	tubes	topped	with	glass	marbles,	tubes	were manufactured	by	Corning	and	supplied	by	VWR	International	(Edmonton,	Alberta). Samples	were	allowed	to	cool,	then	0.2	ml	of	Na2SO4	and	2	ml	of	absolute	ethanol were	added	to	each	tube.		Tubes	were	left	at	‐20C	over	night	for	ethanol precipitation	of	released	glycogen.		The	following	day,	samples	were	spun	at	3500	× g,	the	supernatant	which	contained	free	glucose,	was	discarded.		The	pellet	was washed	in	66%	ethanol	and	then	boiled	in	1	ml	of	2N	H2SO4	for	3	hours	to	hydrolyze glycogen	to	glucose.		After	cooling,	0.5	ml	of	a	1M	MOPS	buffer	was	added	and samples	were	individually	brought	to	a	pH	of	6.8‐7	using	NaOH.		The	Corex®	tubes were	weighed	to	determine	the	end	sample	dilution	volume.		Finally,	samples	were assayed	using	a	colorimetric	glucose	assay	kit	purchased	from	Caymen	Chemical supplied	by	Cedarlane	(Burlington,	Ontario).   2.5. Oxidative Stress Assessment, Oxyblot   Increased	oxidative	stress	has	been	shown	to	cause	the	introduction	of  carbonyl	groups	into	protein,	therefore	oxidative	stress	was	assessed	by	measuring changes	in	the	number	of	carbonyl	groups	present	in	whole	heart	homogenates.		For        28  Methods   homogenization,	30‐40	mg	of	powdered	heart	tissue	was	weighed	into	liquid nitrogen	cooled	tubes	and	0.5	ml	of	cold	total	protein	extraction	buffer	plus	β‐ mercaptoethanol,	(total	protein	extraction	buffer:	20	mM	HEPES,	1	mM ethylenediamine	tetraacetic	acid	(EDTA),	250	mM	sucrose,	100	mM	sodium pyrophosphate,	10	mM	sodium	orthovanadate,	100	mM	sodium	fluoride,	5	μl/ml protease	inhibitor	cocktail	from	Sigma‐Aldrich	(St.	Louis,	Missouri)	and	2%	(v/v)	β ‐mercaptoethanol)	was	added.		Tissue	was	then	homogenized	using	an	Ultra‐Turrax TR‐10	tissue	homogenizer,	from	Rose	Scientific	Ltd.	(Edmonton,	Alberta),	for	2	x	5 second	bursts.		Tissue	homogenates	were	then	spun	at	500	g	in	order	to	separate soluble	proteins	from	membrane	components	and	nuclei,	the	supernatant	was	then collected	for	further	analysis. Homogenates	were	analyzed	using	the	OxyBlot	kit	from	Millipore	supplied	by Cedarlane	(Burlington,	Ontario).		This	kit	was	used	to	label	carbonyl	groups	with	a 2,4‐dinitrophenylhydrazone	(DNP)	tag.		After	tagging	samples	were	blotted	directly onto	nitrocellulose	membranes. After	the	membranes	had	fully	absorbed	samples,	they	were	blocked	for	1 hour	with	blocking	buffer	(2.5%	bovine	serum	albumin	in	tris‐buffered	saline	plus 0.1%	polyoxyethylenesorbitan,	(TWEEN))	and	were	incubated	at	room	temperature for	two	hours	with	primary	anti‐DNP	antibody	that	was	supplied	with	the	kit. Membranes	were	then	washed	with	tris‐buffered	saline	plus	TWEEN	(2	rinses	and	3 x	5	minute	washes),	incubated	for	1	hour	at	room	temperature	with	a	supplied horse	radish	peroxidase	tagged	secondary	antibody	followed	by	another	set	of washes.		Detection	of	blotted	proteins	was	accomplished	using	the	Super	Signal       29  Methods   West	Femto	Maximum	Sensitivity	substrate,	from	Pierce	Biotechnology	supplied	by Fisher	Scientific	(Ottawa,	Ontario).		Images	were	taken	using	the	ChemigeniusQ Image	Analyzer,	purchased	from	Geneflow	(Alexandria,	Virginia),	densitometry analysis	was	conducted	using	the	software	program	ImageJ,	from	the	National Institutes	of	Health	(Bethesda,	Maryland).  2.6. Statistics   Values	are	expressed	as	means	±	Standard	Error	of	the	Mean	(SEM).		When  the	means	of	more	then	two	groups	was	compared,	the	One‐Way	Analysis	of Variance	technique	was	used,	with	Bonferroni	post‐hoc	analysis.		When	the	means of	two	groups	were	compared	a	Student’s	t‐test	was	performed.		For	all	analysis	a pre‐adjustment	α	level	of	0.05	was	chosen.		All	analysis	was	performed	using	Prism 5	software	from	GraphPad	Software	Inc.	(La	Jolla,	California).		The	Statistical Consulting	and	Research	Laboratory	reviewed	and	approved	the	statistical	analysis performed	for	this	study138.        30  Methods    Study 1 Timeline     Metoprolol pump implant and ascorbic acid treatment  Saline or STZ  Perfusion and termina on  Diabetes induced  Study 2 Timeline  Normal Diabe c  ‐1  0  2  1  3  Weeks  4  5  6  7  8  Scheme 2. Outline of treatments for Study 1 and Study 2. For both studies, animals were allowed to acclima ze for 1 week, a er which they were separated into control and diabe c groups. Diabe c animals were injected with streptozotocin (STZ, 60 mg/kg body weight), and control animals with saline. Diabetes was confirmed by measuring blood glucose levels 1 week post injec on. 1 week post diabetes confirma on, osmo c pumps containing saline or metoprolol (15 mg/kg/day) were implanted. At the same me ascorbic acid treatment (1000 mg/kg/day, in drinking water) was ini ated in selected animals. For Study 1, perfusion and termina on were performed 5 weeks post diabetes confirma on, for Study 2, this occurred 7 weeks post diabetes confirma on.        31  Results  3. Results 3.1. General Characteristics and Plasma Parameters 3.1.1.   Study 1  General	physical	characteristics,	nutrient	intake	and	a	list	of	plasma  parameter	values	during	Study	1	can	be	found	in	Table	1	and	Table	2.		Body	weight was	significantly	reduced	in	diabetic	animals	and	was	unaffected	by	any	drug treatments.		Heart	weight	was	unchanged	in	diabetic	animals,	or	by	any	treatment. Diabetic	animals	were	also	observed	to	consume	2‐fold	more	food	and	4‐fold	more water	than	the	untreated	control	animals	(Table	1).   Decreased	plasma	insulin	and	elevated	glucose	levels	were	observed	in  diabetic	rats,	indicating	a	perturbation	of	glucose	homeostasis,	as	expected. Treatment	of	diabetic	rats	with	metoprolol	or	ascorbic	acid	had	no	effect	on	these parameters.		Plasma	triglyceride	levels	were	also	significantly	disturbed	during diabetes.		Metoprolol,	but	not	ascorbic	acid,	treatment	partially	ameliorated	changes in	triglyceride	content	in	diabetic	animals.		Plasma	cholesterol	levels	were unaffected	by	diabetes	or	treatment.		Plasma	ketone	levels	appeared	to	double	in	all diabetic	groups,	however,	a	full	statistical	analysis	of	plasma	ketone	levels	was	not possible	because	of	the	lack	of	a	sufficient	number	of	animals	in	the	control untreated	group	(Table	2).   32  Results  3.1.2.  Study 2  General	physical	characteristics,	nutrient	intake	and	a	list	of	plasma parameter	values	during	Study	2	can	be	found	in	Table	1	and	Table	2.		All	diabetic animals	experienced	significantly	reduced	body	and	heart	weights	compared	to untreated	control	animals.		All	diabetic	animals	consumed	a	half‐fold	more	food	and four‐fold	more	water	than	untreated	controls,	both	observed	changes	were significant	(Table	1). Glucose	homeostasis	was	severely	disturbed	in	all	diabetic	groups;	insulin levels	were	significantly	reduced	and	an	associated	rise	in	plasma	glucose	was observed.	Treatment	had	no	effect.		Plasma	triglyceride	and	ketone	levels	were significantly	increased	during	diabetes.	Treatment	with	metoprolol	had	no	effects	in control	or	diabetic	animals,	however,	ascorbic	acid	caused	reductions	in	plasma triglyceride	content	in	both	control	and	diabetic	animals	that	did	not	achieve statistical	significance.		Plasma	ketone	levels	were	not	altered	by	treatment.		Plasma cholesterol	levels	were	significantly	increased	in	diabetic	animals	compared	to controls.		Metoprolol	alone	had	no	effect	on	plasma	cholesterol	levels,	however, ascorbic	acid	with	or	without	metoprolol,	lowered	cholesterol	levels	to	a	point where	they	were	no	longer	significantly	different	from	the	untreated	control	(Table 2).   33  Results  3.2. In vivo Cardiac Function of Diabetic Rats 3.2.1.   Study 1  Cardiac	function	was	assessed	in	vivo	by	echocardiography;	volume	and  function	measurements	observed	during	Study	1	can	be	found	in	Table	3	and	Figure 1A.		Heart	rate,	in	vivo,	was	significantly	reduced	in	all	diabetic	hearts	compared	to controls.	Metoprolol	had	a	significant	negative	chronotropic	effect	in	control	heart, and	when	combined	with	diabetes,	produced	a	significant	and	nearly	additive reduction	in	metoprolol	treated	diabetic	heart	rates.			Ascorbic	acid	did	not	have	an effect	on	heart	rates.		Ejection	Fraction	(EF),	Fractional	Shortening	(FS),	End	Systolic and	Diastolic	Volume	(ESV	and	EDV)	were	not	significantly	altered	in	diabetic animals	compared	to	controls.		Treatment	had	little	effect	on	EF,	FS	and	ESV,	except for	the	combined	metoprolol	and	ascorbic	acid	treatment,	which	significantly reduced	EF	and	FS,	and	significantly	raised	ESV	in	diabetic	hearts.		EDV	was significantly	increased	in	all	metoprolol	treated	groups	compared	to	controls. Stroke	volume	was	not	significantly	modified	by	diabetes;	metoprolol	delivered alone	did	significantly	raise	stroke	volume	in	both	control	and	diabetic	hearts, however		(Table	3).		Cardiac	output	was	not	significantly	affected	by	diabetes	or	any treatment	(Figure	1).   3.2.2.   Study 2  Volume	and	function	measurements	observed	during	Study	2	can	be	found	in  Table	3	and	Figure	1B.		Heart	rate	was	significantly	reduced	in	all	diabetic	animals  34  Results  and	in	metoprolol	treated	control	animals.		Diabetic	animals	treated	with metoprolol	experienced	a	further	reduction	in	heart	rate,	however	this	effect	was not	statistically	significant.		Ascorbic	acid	had	no	effect	on	heart	rates.		EF	and	FS were	significantly	reduced	in	all	diabetic	animals	compared	to	untreated	controls, except	for	EF	in	diabetics		treated	with	ascorbic	acid	alone.		ESV	and	EDV	were	both increased	by	diabetes,	but	neither	change	was	significant.		Treatment	with metoprolol	caused	a	significant	increase	in	ESV	in	control	animals,	and	in	ESV	and EDV	in	diabetic	animals.		Stroke	volume	was	unaffected	by	diabetes	or	treatment (Table	3).		Cardiac	output	was	lower	in	all	diabetic	animals;	however,	this	change was	only	significant	in	animals	treated	with	metoprolol	alone	(Figure	1).   3.3. Ex vivo Cardiac Function of Diabetic Rats 3.3.1.   Study 1  In	order	to	assess	changes	in	cardiac	performance	independent	of	whole  body	effects	of	both	diabetes	and	treatment,	function	was	also	measured	ex	vivo during	working	heart	perfusion.		Functional	parameters	observed	during	Study	1 can	be	found	in	Table	4	and	Figure	2A	and	2B.		Heart	rate	measured	ex	vivo demonstrated	that	diabetes	had	a	significant	negative	chronotropic	effect. Metoprolol	treatment	demonstrated	a	significant	positive	chronotropic	effect	when it	was	given	alone	in	control	hearts,	and	raised	diabetic	heart	rates	in	treated	groups to	a	point	where	they	were	no	longer	significantly	different	from	controls.	Ascorbic acid	had	no	significant	effect	except	that	it	appeared	to	somewhat	blunt  35  Results  metoprolol’s	increase	of	heart	rate,	as	demonstrated	in	the	control	dual	treated group.		There	were	no	significant	changes	in	peak	systolic	pressure,	rate	pressure product,	and	hydraulic	work	caused	by	diabetes	or	treatment	(Table	4).		Coronary flow	and	cardiac	output	were	not	significantly	affected	by	diabetes	(Figure	2A,B). Treatment	with	ascorbic	acid	did	significantly	increase	coronary	flow	rates	in diabetic	hearts	when	administered	in	combination	with	metoprolol,	and	in	control hearts	with	or	without	metoprolol	(Figure	2A).   3.3.2.   Study 2  Functional	parameters	observed	during	Study	2	can	be	found	in	Table	4	and  Figure	2C	and	2D.		Diabetic	hearts	experienced	a	significant	reduction	in	heart	rates compared	to	the	untreated	control.		Metoprolol,	when	administered	alone, significantly	raised	rates	above	the	untreated	diabetic;	these	rates	were	also	no longer	significantly	different	from	controls.		Treatment	of	diabetic	animals	with ascorbic	acid	alone	had	no	effect	on	heart	rates,	and	when	given	with	metoprolol, seemed	to	remove	metoprolol’s	normalizing	effect.		There	were	no	significant treatment	effects	on	control	heart	rates.		Peak	systolic	pressure	was	unchanged	by diabetes	or	treatment.		Rate	Pressure	Product	(RPP)	was	significantly	reduced	by diabetes.		Treatment	with	metoprolol	alone	significantly	increased	RPP	compared	to untreated	diabetics	and	ascorbic	acid	alone	partially	ameliorated	RPP,	raising	it	to	a level	that	was	not	significantly	different	from	control.		However,	both	these correcting	effects	disappeared	when	metoprolol	and	ascorbic	acid	treatments	were given	in	the	same	animal.		Cardiac	work	and	coronary	flow	were	significantly  36  Results  reduced	during	diabetes.		Treatment	of	diabetics	with	metoprolol	alone	had	no effect,	however,	ascorbic	acid	with	or	without	metoprolol,	caused	increases	in	both parameters	substantial	enough	that	they	were	no	longer	different	from	control (Table	4	and	Figure	2C).		Cardiac	output	was	significantly	reduced	during	diabetes. Metoprolol	alone	had	no	significant	effect.		Cardiac	output	of	ascorbic	acid‐treated diabetic	animals	was	not	statistically	different	from	those	of	control	animals indicating	a	partial	amelioration	of	diabetes‐induced	reductions	in	cardiac	output. When	both	drugs	were	combined	cardiac	output	was	significantly	increased compared	to	the	untreated	diabetic	group	(Figure	2D).   3.4. Substrate Oxidation and Metabolite Content 3.4.1.   Study 1  Oxidation	rates	of	exogenous,	radiolabeled,	palmitate	and	glucose	measured  ex	vivo	during	working	heart	perfusion	are	shown	in	Figures	3A	and	3B.		Palmitate oxidation	rates	were	significantly	accelerated	by	20%	in	diabetic	hearts	compared to	controls.		All	treatments	significantly	lowered	palmitate	oxidation	rates	in diabetic	hearts	and	rates	in	all	diabetic	treated	groups	were	not	significantly different	from	control	(Figure	3A).		Oxidation	of	exogenous	glucose	was	reduced	to nearly	undetectable	levels	in	diabetic	hearts.	Although	treatment	with	metoprolol	or ascorbic	acid	individually	did	not	alter	glucose	oxidation	rates,	combined	treatment did	cause	a	noticeable,	but	not	significant,	increase.	Treatment	had	no	effect	on exogenous	glucose	oxidation	in	control	hearts	(Figure	3B).  37  Results    Tissue	glycogen	content	was	measured	in	hearts	flash	frozen	after	working  heart	perfusion,	data	for	Study	1	is	charted	in	Figure	4A.		Glycogen	content	was higher	in	all	diabetic	hearts,	however,	changes	were	most	pronounced	and	only significant	in	the	dual	metoprolol	and	ascorbic	acid	treated	group.		Treatment	had no	effect	on	control	hearts	(Figure	4A).   Tissue	triglycerides	were	extracted	and	measured	in	hearts	flash	frozen	after  working	heart	perfusion,	data	for	Study	1	is	charted	in	Figure	4B.		Cardiac triglyceride	content	was	not	significantly	modified	by	diabetes	or	any	treatment (Figure	4B).   3.4.2.  Study 2  Oxidation	rates	of	exogenous,	radiolabeled,	palmitate	and	glucose	for	Study	2 are	displayed	in	Figure	3C	and	3D.		Palmitate	oxidation	rates	were	significantly increased	by	70%	in	diabetic	compared	to	controls	animals.		Treatment	with metoprolol	caused	a	non‐significant	reduction	in	diabetic	hearts	only	while	ascorbic acid	had	no	effect	on	control	or	diabetic	animals.		Dual	treatment	significantly reduced	rates	of	palmitate	oxidation	in	diabetic	animals	(Figure	3C).		Glucose oxidation	rates	were	significantly	lowered	to	almost	undetectable	levels	in	diabetic hearts.		Treatment	with	metoprolol	or	ascorbic	acid	had	no	significant	effects	on glucose	oxidation	in	control	or	diabetic	hearts	(Figure	3D). Tissue	glycogen	content	for	Study	2	is	displayed	in	Figure	4C.		Tissue glycogen	content	was	significantly	increased	in	diabetic	hearts	compared	to untreated	controls.		Metoprolol	treatment	appeared	to	raise	glycogen	levels	in  38  Results  diabetic	hearts	over	the	untreated	diabetic	group,	however	changes	were	not significant.		Ascorbic	acid	administered	alone	or	with	metoprolol	had	no	effect	on glycogen	levels	in	diabetic	hearts.		Control	hearts	were	unaffected	by	treatment (Figure	4C). Tissue	triglyceride	content	for	hearts	from	Study	2	is	displayed	in	Figure	4D. Tissue	triglyceride	levels	appeared	higher	in	the	untreated	diabetics	compared	to controls,	however	changes	were	not	significant.		Treatment	did	not	have	any significant	effects	(Figure	4D).   3.5. Oxidative Protein Damage 3.5.1.   Study 1  Oxidative	protein	damage	as	measured	by	OxyBlot	analysis	for	hearts	from  Study	1	can	be	found	in	Figures	5	and	6.		There	was	a	60%	rise	in	oxidative	protein damage	in	diabetic	hearts	during	Study	1,	however	this	change	was	not	significant (Figure	5). The	effect	of	treatment	on	oxidative	protein	damage	for	hearts	from	Study	1 can	be	found	in	Figure	6A	and	6B.		Separate	charts	are	displayed	for	control	and diabetic	hearts	in	order	to	illustrate	that	the	analyses	for	these	two	groups	were completed	on	separate	immunoblots.		Comparisons	can,	therefore,	only	be	made within	the	control	treatments	or	diabetic	treatments,	but	not	across	control	and diabetic	treatments.		In	control	hearts,	metoprolol	treatment	caused	increases	in oxidative	protein	damage,	while	ascorbic	acid	did	not	appear	to	have	any	effect.		In  39  Results  diabetic	hearts	only	metoprolol	alone	caused	increased	damage	while	both	ascorbic acid	treated	hearts	caused	reductions.		However,	none	of	these	treatment	effects were	significant	(Figure	6A,B).   3.5.2.  Study 2  During	Study	2	there	was	a	significant,	100%	rise	in	oxidative	protein damage	in	diabetic	hearts	compared	to	control	hearts	(Figure	5).   The	effect	of	treatment	on	oxidative	protein	damage	for	hearts	from	Study	2  can	be	found	in	Figure	7A	and	7B.		In	control	hearts	metoprolol	treatment	did	not appear	to	have	an	effect,	however,	ascorbic	acid	with	or	without	metoprolol significantly	lowered	oxidative	protein	damage	(Figure	7A).		In	diabetic	hearts,	an identical	pattern	was	observed,	however	changes	were	not	significant	(Figure	7B).     40  Results  Table 1 - General characteristics   Control untreated C  STZ untreated D   Control metoprolol CM   STZ metoprolol DM   Control ascorbic acid CA  STZ ascorbic acid DA  Control metoprolol +	ascorbic acid CMA  STZ metoprolol +	ascorbic acid DMA  Study	1 N Body	Weight	(g) Heart	Weight	(g) Food	(g/day) Water	(ml/day)  10  8  10  10  10  10  10  11  476±15  385±9a  488±11  377±18	(9)a  475±12  380±20	(8)a  467±11  356±9a  1.75±0.04  1.91±0.07  1.68±0.06	(9)  1.77±0.05  33±3  1.67±0.07 54±1a  32±1  47±2  30±1  1.62±0.05	(8) 58±7a  49±1  223±10a  47±1  181±15a  35±2  1.76±0.05 30±1  1.57±0.03 52±5	(10)a  194±12a  34±2  183±17	(10)a  Study	2 N Body	Weight	(g) Heart	Weight	(g) Food	(g/day) Water	(ml/day)  10  12  10  12  4  8  8  6  447±11  259±21a  480±18  283±16a  412±16  286±12a  423±9  277±37a  1.93±0.08  1.47±0.07a  1.94±0.06  1.43±0.05a  1.77±0.12  1.61±0.06a  1.78±0.09  1.37±0.10a  31±1  42±2	(14)a  34±3  41±2a  27±0  46±1a  27±1  37±3  42±1  160±8	(14)a  41±2  149±10a  26±0  161±9a  30±1  137±8a   Table 1 -	Values	are	means	±	SEM	(N	number,	if	different	from	above,	is	noted	in	brackets).		Diabetes	was	induced	(D,	DM,	DA, DMA)	with	a	60	mg/kg	streptozotocin	(STZ)	injection	into	the	caudal	vein,	control	animals	received	equivalent	volumes	of	saline. Two	weeks	post	STZ,	metoprolol	treatment	(15	mg/	kg/	day;	CM,	CMA,	DM,	DMA),	and	ascorbic	acid	treatment	(1000	mg/kg/day; CA,	CMA,	DA,	DMA)	were	started.	Study	1	and	Study	2	animals	were	terminated	6	and	8	weeks	after	STZ	injection,	respectively.		All values	 were	 measured	 at	 or	 near	 termination,	 food	 and	 water	 measurements	 are	 averages	 over	 the	 last	 two	 weeks	 before termination.	 	 Statistical	 analysis	 was	 by	 1‐Way	 ANOVA	 with	 Bonferroni	 post	 hoc	 test.	 a=p<0.05	 vs.	 untreated	 control	 (C). b=p<0.05	vs.	untreated	diabetic	(D).       41  Results  Table 2 – Plasma parameters   Control untreated C  STZ untreated D   Control metoprolol CM   STZ metoprolol DM   Control ascorbic acid CA  STZ ascorbic acid DA  Control metoprolol +	ascorbic acid CMA  STZ metoprolol +	ascorbic acid DMA  Study	1 N Insulin	(ng/ml) Glucose	(mM) Triglyceride	(mM) Cholesterol	(mM) Ketones	(mM)  10  8  10  1.20±0.21  0.28±0.05a  0.97±0.14  7.3±0.3	(6)  23.1±1.2	(5)a  1.35±0.16  10  10  10  10  11  0.28±0.03a  1.31±0.29  0.26±0.04a  7.3±0.2	(6)  0.32±0.07	(8)a	 1.17±0.19 22.6±1.3	(5)a	 7.8±0.4  22.2±0.8	(8)a  7.1±0.3  20.4±1.7a  2.63±0.31a  1.70±0.16  1.77±0.30  1.46±0.14  2.47±0.39a  1.18±0.17  2.04±0.33  2.18±0.11  2.35±0.14  2.00±0.09  2.33±0.24  1.88±0.09  2.49±0.20  1.98±0.07  2.85±0.34  0.43±0.05	(2)  1.26±0.40	(4)  0.65±0.07	(6)  0.98±0.16	(5)  0.65±0.15	(8)  1.34±0.27	(8)  0.59±0.03	(8)  1.34±0.23	(8)  Study	2 N Insulin	(ng/ml) Glucose	(mM) Triglyceride	(mM) Cholesterol	(mM) Ketones	(mM)  10  12  10  12  4  8  8  6  1.28±0.13  0.34±0.09a  1.85±0.35  0.39±0.14a  1.26±0.15  0.30±0.12a  1.35±0.16  0.31±0.16a  9.1±0.4  22.2±1.6a  9.3±0.5  23.0±1.6a  11.0±0.6  22.9±1.4a  9.6±0.5  22.6±2.7a  1.31±0.09  3.49±0.26a  1.42±0.14  3.34±0.32a  1.29±0.20  3.17±0.40a  1.16±0.12  2.73±0.42a  1.92±0.08  3.72±0.41a  1.73±0.08  3.22±0.33a  1.70±0.03  0.54±0.04	(8)  2.37±0.30a  0.56±0.04  2.11±0.30a  0.69±0.09  3.01±0.27 1.93±0.43a  1.83±0.08 0.63±0.04  2.81±0.24 2.42±0.44a   Table 2 -	Values	are	means	±	SEM	(N	number,	if	different	from	above,	is	noted	in	brackets).		Diabetes	was	induced	(D,	DM,	DA, DMA)	with	a	60	mg/kg	streptozotocin	(STZ)	injection	into	the	caudal	vein,	control	animals	received	equivalent	volumes	of	saline. Two	weeks	post	STZ,	metoprolol	treatment	(15	mg/	kg/	day;	CM,	CMA,	DM,	DMA),	and	ascorbic	acid	treatment	(1000	mg/kg/day; CA,	CMA,	DA,	DMA)	were	started.	Study	1	and	Study	2	animals	were	terminated	6	and	8	weeks	after	STZ	injection,	respectively.		All values	 were	 measured	 at	 or	 near	 termination,	 food	 and	 water	 measurements	 are	 averages	 over	 the	 last	 two	 weeks	 before termination.	 	 Statistical	 analysis	 was	 by	 1‐Way	 ANOVA	 with	 Bonferroni	 post	 hoc	 test.	 a=p<0.05	 vs.	 untreated	 control	 (C). b=p<0.05	vs.	untreated	diabetic	(D). 42  Results  Table 3 - Functional parameters as measured by echocardiography   Control untreated C  STZ untreated D   Control STZ metoprolol metoprolol CM DM    Control ascorbic acid CA  STZ ascorbic acid DA  Control STZ metoprolol	 metoprolol +	ascorbic	 +	ascorbic acid acid CMA DMA  Study	1 N Heart	Rate	(BPM) Ejection	Fraction	(%) Fractional	Shortening	(%) End	Systolic	Volume	µl End	Diastolic	Volume	µl Stroke	Volume	µl  10  9  10  9  10  8  10  11  387±9  323±9a  331±8a  281±9a	b  380±10  301±12a  317±8a  272±8a	b  73.4±2.0  69.1±3.0  73.0±1.4  70.8±2.1  73.4±1.4  66.2±1.6  69.8±1.6  65.0±1.6a  44.3±1.8  40.9±2.5  44.0±1.2  42.2±1.8  44.2±1.3  38.3±1.3  41.1±1.3  37.4±1.2a  84±12  120±15  106±8  90±7  128±10  305±23  377±21  391±15a  123±14 411±25a  338±11  374±15  110±11 361±24a  220±13  257±14  284±10a  288±14a  248±8  246±6  251±16  257±7  141±11a 398±16a  Study	2 N Heart	Rate	(BPM) Ejection	Fraction	(%) Fractional	Shortening	(%) End	Systolic	Volume	µl End	Diastolic	Volume	µl Stroke	Volume	µl  10  13  10  12  4  8  8  6  359±12  287±12a  293±8a  261±8a  338±19  293±14a  295±8a  258±22a  73.0±1.6  66.8±0.5a  68.7±1.0  64.7±1.1a  66.9±1.9  70.6±2.3  64.7±2.1a  43.7±1.4  38.4±0.4a  41.9±1.9  36.9±1.7a  81±8  37.0±0.9a  38.6±1.5  112±6  40.2±0.8 125±12a  66.3±1.2 38.1±1.0a  129±8a  110±3  112±6  107±12  116±13  300±19  335±16  395±31  362±15a  334±13  332±12  358±23  325±28  219±15  224±10  270±19  233±9  224±14  220±8  251±16  209±17   Table 3 - Values	are	means	±	SEM	(N	number,	if	different	from	above,	is	noted	in	brackets).		Animals	were	anesthetised	with	2% isoflurane	during	the	measurement	period.		STZ	was	delivered	at	60	mg/kg	by	IV	injection,	metoprolol	at	15	mg/kg/day	by osmotic	pump	and	ascorbic	acid	at	1000	mg/kg/day	in	drinking	water.		Statistical	analysis	was	by	1‐Way	ANOVA	with	Bonferroni post	hoc	test.	a=p<0.05	vs.	untreated	control	(C).		b=p<0.05	vs.	untreated	diabetic	(D). 43  Results    Table 4 - Functional parameters as measured during working heart perfusion   Control untreated C  STZ untreated D   Control STZ metoprolol metoprolol CM DM    Control ascorbic acid CA  STZ ascorbic acid DA  Control STZ metoprolol	 metoprolol +	ascorbic	 +	ascorbic acid acid CMA DMA  Study	1 N Heart	Rate	(BPM)  10  7  10  9  10  8  10  11  251±6  227±5a  275±6a  245±7  253±4  226±5a  264±4  245±3  108±2  112±2  104±3  109±4  110±1  115±2  109±1  110±1  Peak	Systolic	Pressure	(mm Hg) Rate	Pressure	Product	(BPM x	mm	Hg/	1000) Cardiac	Work	(ml	x	mm Hg/min	x	1000)  27.0±0.8  25.3±0.4  28.5±0.8  26.5±0.7  28.0±0.5  25.9±0.5  28.7±0.5  27.0±0.5  84.9±2.6  84.3±2.4  81.5±4.1  80.3±5.4  94.9±1.9  95.1±4.4  95.8±1.8  89.8±2.0  N  10  12  10  12  4  8  8  6  234±4  202±2a  239±3  223±5b  240±4  209±7a  260±12  210±6a  119±2  111±4  122±3  113±3  120±2  118±2  112±5  116±2  27.8±0.8  22.4±0.8a  29.2±0.6  25.2±0.7b  28.8±0.6  24.6±1.0  28.8±0.5  24.3±1.0a  104.8±2.7  76.8±5.2a  106.7±5.1  84.5±3.7a  100.7±3.7  87.7±2.9  94.9±5.0  92.6±3.4  Study	2 Heart	Rate	(BPM) Peak	Systolic	Pressure	(mm Hg) Rate	Pressure	Product	(BPM x	mm	Hg/	1000) Cardiac	Work	(ml	x	mm Hg/min	x	1000)   Table 4 -	Values	are	averages	of	measurements	taken	over	the	perfusion	period	±	SEM.		Hearts	were	perfused	in	working	heart mode	for	30	minutes	with	perfusion	buffer	containing	118	mM	NaCl,	4.7	mM	KCl,	1.2	mM	KH2PO4,	1.2	mM	MgSO4,	2	mM	CaCl2,	5.5 mM	glucose,	0.5	mM	lactate,	20	µU/ml	insulin,	and	0.6	mM	palmitate	bound	to	3%	BSA.		All	animals	received	the	same	perfusion buffer,	and	no	treatments	were	delivered	during	perfusion.	Statistical	analysis	was	by	1‐Way	ANOVA	with	Bonferroni	post	hoc test.	a=p<0.05	vs.	untreated	control	(C).		b=p<0.05	vs.	untreated	diabetic	(D).  44  Results    45  Results         46  Results        47  +  +  +  +  + DMA  +  CMA  +  DA  +  +  CA  DMA  +  DM  CMA  +  CM  DA  +  D  µmole triglyceride/ g dry heart weight µmole triglyceride/ g dry heart weight  0  C  +  CA  DM  CM  D  +  N=6  +  N=7  +  N=8  +  N=4  5  N=5  C  10  + +  N=7  15  N=6  Ascorbic acid  D  20  N=5  +  0  N=3  +  N=5  +  N=5  +  N=7  Metoprolol  N=3  +  N=5  STZ  N=5  0  N=6  100  N=7  200  N=7  a  N=7  a  5  N=7  300  aC  10  N=7  a  15  N=7  400  20  N=7  N=8  N=8  N=4  N=4  N=4  0  B Study 2 – Cardiac triglyceride Study 1 – Cardiac triglyceride  100  N=4  Study 1 – Cardiac glycogen  200  N=4  Study 2 – Cardiac glycogen  a,b  N=5  µmole glucose/ g dry heart weight  A  300  N=8  µmole glucose/ g dry heart weight  Results  Figure 4 – Cardiac	glycogen	(A)	and	cardiac	triglyceride	(B)	content	of	hearts	from	Study	1, and	Study	2	(C	&	D,	respectively).	Value	are	group	means,	error	bars	represent	SEM.	Statistical analysis	was	by	1‐Way	ANOVA	with	Bonferroni	post	hoc	test.	a=p<0.05	vs.	untreated	control (C).		b=p<0.05	vs.	untreated	diabetic	(D).        48  Results        49  Results        50  Results  51  Discussion  4. Discussion  4.1. Overview of Study The	purpose	of	this	study	was	to	investigate	the	hypothesis	(See	Scheme	3 for	outline	of	hypothesis	and	findings):  We	propose	that	metabolic	changes	that	occur	during	diabetes	elevate oxidative	stress,	leading	to	protein	damage,	signaling	changes,	cell	death	and	other sequelea;	the	eventual	sum	of	these	changes	is	an	impairment	of	function.		Treatment of	either	the	sequelae	of	oxidative	stress	or	oxidative	stress	directly	will	be	beneficial but	treatment	of	both	will	improve	function	further.    We	approached	our	question	in	two	ways.		First,	we	chose	to	investigate  contributions	of	potentially	relevant	factors	in	the	development	of	diabetic	cardiac dysfunction	by	comparing	metabolic,	functional	and	oxidative	stress	parameters measured	before	overt	dysfunction	(Study	1	–	Diabetes	week	5)	and	after	the development	of	overt	cardiac	dysfunction	(Study	2	–	Diabetes	week	7).		We	were able	to	show	that	cardiac	function	significantly	worsened	in	concert	with	increases in	metabolic	disturbance	and	oxidative	protein	damage.   Second,	we	set	out	to	determine	if	metoprolol’s	beneficial	effects	could	be  supplemented	by	reduction	of	oxidative	stress	by	ascorbic	acid.		We	observed	that    52  Discussion  both	drugs	improved	cardiac	function	and	had	metabolic	effects,	while	only	ascorbic acid	appeared	to	reduce	oxidative	protein	damage.		When	metoprolol	and	ascorbic acid	were	combined	the	observed	improvement	in	function	was	more	powerful	then the	drugs	alone.   4.2. General Physical Characteristics – Body Weight and Heart Weight are More Perturbed with Disease Progression   Diabetic	animals	from	Study	1	displayed	significant	reductions	in	mass	as  compared	to	controls	(Table	1).		Weight	loss	in	diabetic	animals	is	usually	due	to loss	of	diaphaseal	bone	due	to	disturbances	in	calcium	homeostasis	and	loss	of muscle,	adipose	tissue	and	liver	due	to	insulinopenia	with	tissue	loss	evident	within 1	week	following	onset	of	diabetes139,	140	.		The	observed	mass	gap	doubled	by	the time	Study	2	was	terminated	2	weeks	later.		This	finding	is	not	novel,	others	have also	shown	that	the	mass	gap	increases	as	diabetes	progresses130,	141	.   Heart	weights	during	Study	1	were	not	significantly	lower	in	diabetics	as  compared	to	controls	(Table	1).	However,	in	Study	2,	diabetic	heart	weights	had dropped	significantly	by	about	25%.		Hoit	et	al.	reported	that	heart	weights	began	to trend	lower	at	8	weeks	after	induction	of	diabetes,	a	time	point	which	is	shortly after	Study	2130.		We	also	observed	that	our	treatments	had	no	effect	on	heart weight,	however,	Hanada	et	al.	and	Sharma	et	al.	previously	reported	that metoprolol	treatment	causes	reductions	in	heart	weight	(Table	1).		These	studies differed	from	ours	in	several	ways.		Firstly,	metoprolol	was	used	to	reverse    53  Discussion  isoproterenol	induced	cardiac	hypertrophy	in	Hanada’s	study.		Heart	weights	in treated	animals	were	returned	to	normal	and	not	below.		Secondly,	in	Sharma’s study,	a	much	higher	dose	of	metoprolol	was	used	(75	mg/kg/day)	and	treatment was	delivered	by	daily	intraperitoneal	injection.	This	high	but	transient	dose	likely has	different	effects	and	is	not	fully	comparable	to	our	lower	(15	mg/kg/day)	and more	constant	dosing.   4.3. Plasma Triglyceride and Cholesterol – Levels and Persistence of Disturbance Worsen with Disease Progression 4.3.1.   Triglycerides  We	observed	an	increase	in	plasma	triglyceride	of	94%	in	Study	1	diabetic  rats	and	160%	in	Study	2	(Table	2).		These	results	are	in	line	with	observations	in literature	that	also	show	rises	in	diabetic	animals	at	more	advanced	time	points	33, 127,	141,	142	.  Metoprolol	treatment	reduced	plasma	triglyceride	levels	in	diabetics	in	Study 1	but	had	no	effect	during	Study	2,	indicating	that	disturbances	in	triglyceride	levels not	only	become	more	pronounced,	but	they	also	become	more	resistant	to treatment	(Table	2).		Results	for	Study1,	but	not	Study	2,	are	in	keeping	with	similar findings	in	the	literature.	Sharma	et	al.	performed	their	research	in	an	identical model	to	ours	at	a	time	point	in	between	Study	1	and	Study	2	and	found	an approximate	40%	rise	in	diabetic	triglyceride	content	and	that	metoprolol normalized	this	rise33.		Results	are	confirmed	by	others	who	have	found	that	STZ   54  Discussion  treated	Sprague‐Dawley	rats	at	the	same	time	point	show	a	70%	increase	in	plasma triglyceride	that	was	corrected	by	metoprolol131.		However,	results	from	Study	2	are not	corroborated	by	results	of	Olbrich	et	al.	who	preformed	a	long‐term	study	on metoprolol	treated	STZ	rats.		They	found	that	metoprolol	still	had	a	corrective	effect, but	only	noted	a	10%	increase	in	triglyceride	levels	at	their	6	month	time	point, much	lower	then	observed	in	Study	2143.		This	finding	likely	indicates	that	their animals	were	not	as	metabolically	perturbed	and	thus	their	metabolic	phenotype was	at	a	point	that	could	be	rescued,	similar	to	those	in	Study	1.		The	changing	effect of	metoprolol	in	Study	1	versus	Study	2,	likely	reflects	the	increase	in	severity	of metabolic	disturbances	over	the	short	span	of	time.   Ascorbic	acid	administered	alone	did	not	produce	significant	reductions	in  plasma	triglyceride	content,	although	values	trended	lower	then	the	untreated diabetics	(Table	2).		Dai	et	al.	performed	a	similar	study	to	ours	and	found	that	at	a time	point	equivalent	to	Study	2,	diabetic	triglyceride	levels	were	4‐fold	higher	than control	levels	and	were	significantly	reduced	using	ascorbic	acid,	with	the	reduction occurring	in	a	dose	dependent	manner.		Importantly,	the	highest	dose	was equivalent	to	that	used	in	this	study127.		It	is	unclear	why	Dai	found	such	high triglyceride	values	in	diabetics,	their	model	was	nearly	identical	to	ours,	differing only	slightly	in	the	STZ	dose	(55	vs.	60	mg/kg	for	us).		The	improved	performance	of ascorbic	acid	in	their	hands	can	likely	be	explained	by	the	fact	that	they	began treatment	3	days	post	STZ	administration,	whereas	it	was	delayed	2	weeks	in	our study.    55  Discussion    Clinically,	metoprolol	has	been	shown	to	increase	triglyceride	levels	in	non‐  diabetic	hypertensive	patients	and	in	type	2	diabetic	patients	144‐146	.		Ascorbic	acid has	been	shown	by	some	to	reduce	triglyceride	levels	in	type	2	diabetics147.   4.3.2.   Cholesterol  Plasma	cholesterol	content	in	diabetics	was	significantly	elevated	in	Study	2  but	not	in	Study	1	(Table	2).		Akula	et	al.	also	observed	an	increase	in	cholesterol levels	in	Sprague‐Dawley	rats	as	diabetes	progressed.		The	degree	of	increase	was also	similar,	with	a	doubling	in	levels	observed	from	week	4	to	week	8.	Cholesteral increased	in	a	similar	manner	from	week	5	to	week	7	in	the	present	study	(Study	1 and	Study	2)	141. Metoprolol	had	no	effect	on	plasma	cholesterol	levels	in	our	study,	and	in two	similar	studies	in	Wistar	and	Sprague‐Dawley	rats	with	STZ	induced	diabetes (Table	2)	33,	131	. Ascorbic	acid	partially	ameliorated	increased	plasma	cholesterol	levels during	Study	2.		Dai	et	al.	also	observed	ascorbic	acid’s	cholesterol	lowering	effects, with	600	and	1000	mg/kg/day	lowering	levels	in	a	dose	dependent	manner127. Ascorbic	acid’s	cholesterol	lowering	abilities	are	shared	by	other	potent antioxidants,	such	as	alpha	lipoic	acid,	coenzyme	Q10	and	resveratrol148‐150	.		It	is interesting	to	note	that	each	of	these	agents	also	increase	plasma	ascorbic	acid levels.	Although	this	finding	raises	the	possibility	of	a	deeper	role	for	this	vitamin, the	effect	may	simply	reflect	replenishment	of	the	body’s	antioxidant	reserves	as levels	of	other	non‐enzymatic	antioxidants	also	increase150.    56  Discussion  In	humans,	plasma	cholesterol	is	not	modified	by	metoprolol	in	type	2 diabetic	patients,	but	is	reduced	by	ascorbic	acid146,	147	.   4.4. Heart Function – Relationship to Disease Progression   Heart	function	in	diabetic	rat	hearts	compared	to	controls	decreased  significantly	from	Study	1	to	Study	2.		Echocardiography	and	isolated	working	heart perfusion	revealed	virtually	no	changes	to	any	diabetic	functional	measurements compared	to	controls	during	Study	1	(Table	3,4,	Figure	1A,	2A,	2B).		In	contrast, diabetic	hearts	in	Study	2	showed	reductions	in	fractional	shortening	and	ejection fraction	as	measured	be	echocardiography,	and	rate	pressure	product,	cardiac	work, coronary	flow	and	cardiac	output	as	measured	by	working	heart	perfusion	(Table 3,4	and	Figure	1B,	2C,	2D).		These	findings	are	consistent	with	the	work	of	several others	who	have	demonstrated	cardiac	dysfunction	in	STZ	treated	animals	near	the 7	week	time	point	of	Study	2	33,	127,	130	.   Use	of	non‐invasive	and	invasive	methods	to	study	cardiac	function  highlights	the	fact	that	disturbed	ex	vivo	heart	function	does	not	always	correlate with	in	vivo	dysfunction.		Study	2	showed	reductions	in	cardiac	output	as	measured by	working	heart	perfusion,	but	not	as	measured	by	echocardiography	(Figure	2D and	1B,	respectively).		This	discrepancy	has	been	previously	reported.		Akita	mice, which	display	a	genetic	form	of	Type	1	diabetes,	showed	reduced	cardiac	output	as measured	by	echocardiography	at	54	weeks	of	age,	while	reductions	in	cardiac    57  Discussion  power	and	left	ventricular	developed	pressure	measured	by	working	heart perfusion	becomes	evident	at	24	weeks26.		In	studies	using	STZ	treated	rats	similar to	that	in	the	current	study,	cardiac	dysfunction	as	measured	by	echocardiography, only	subtly	appeared	as	reductions	in	filling	rate	at	8	weeks	and	changes	in	chamber volume	only	appeared	at	12	weeks130,	141	.		In	contrast,	studies	using	working	heart perfusion	showed	differences	by	6	weeks	of	diabetes	in	STZ	treated	rats	33,	127	. There	are	several	explanations	for	the	discrepancy	between	in	vivo	and	ex vivo	function.		First,	in	vivo	cardiac	output,	and	other	related	functional	parameters, are	largely	dependent	on	factors	affecting	venous	return	and	not	by	the	heart	itself, thus	contractile	problems	could	actually	be	masked	by	changes	in	mean	systemic pressure,	vascular	compliance	and	blood	volume151.		Second,	during echocardiography	animals	are	anesthetized,	and	the	hearts	unchallenged,	thus functional	problems	might	not	be	obvious.		This	is	supported	by	the	fact	that	when isoproterenol	was	used	to	increase	heart	rate	in	diabetic	rats,	differences	in	function as	measured	by	echocardiography,	became	evident	as	early	as	5	weeks	of	diabetes, whereas	unchallenged	hearts	showed	no	changes	at	5	weeks	and	only	subtle changes	in	filling	rate	at	8	weeks130.		Third,	there	is	the	possibility	that	worsened function	observed	on	the	working	heart	apparatus	may	be	a	result	of	poor	recovery after	an	ischemic	period	that	exists	between	the	removal	of	the	heart	from	the	chest cavity	and	the	mounting	of	the	heart	on	the	perfusion	apparatus.		However,	this	is unlikely	as	there	is	considerable	evidence	that	hearts	from	STZ	treated	animals actually	recover	better	then	control	hearts	after	an	ischemic	period152‐154		,	as	long as	the	duration	is	short	and	diabetes	is	not	too	severe155.		Finally,	there	are    58  Discussion  important	differences	in	the	conditions	in	which	the	heart	operates	during	perfusion and	echocardiography.		During	echocardiography	diabetic	hearts	are	exposed	to	3‐ fold	higher	glucose	concentrations,	4‐fold	lower	insulin,	2.5‐fold	more	triglycerides and	a	more	then	4‐fold	increase	in	ketone	levels	compared	to	control	hearts,	with accompanying	neurohormonal	signals	attempting	to	ensure	proper	tissue	perfusion (Table	2).		This	is	important	as	changes	in	substrate	availability	and	hormone	levels are	known	to	cause	functional	modifications,	thus	in	vivo	perfusion	conditions	may have	partially	normalized	function	in	the	diabetic	heart134,	156	.		In	contrast,	during working	heart	perfusion	all	substrate	and	hormone	levels	are	identical,	highlighting intrinsic	differences	in	the	hearts	themselves.   Metoprolol	and	ascorbic	acid	improved	ex	vivo	but	not	in	vivo	function	in  diabetic	hearts	during	Study	2.		When	metoprolol	was	given	alone	it	was	able	to significantly	improve	rate	pressure	product;	ascorbic	acid	alone	modified	rate pressure	product,	cardiac	(hydraulic)	work,	coronary	flow	and	cardiac	output	to	a point	where	they	were	no	longer	significantly	different	from	controls.		Dual metoprolol	and	ascorbic	acid	treatment	did	the	same	with	hydraulic	work	and coronary	flow,	but	significantly	improved	cardiac	output	above	the	untreated diabetic	group	(Figure	1B,	2C,	2D	and	Table	4). It	should	be	noted	that	our	results	with	metoprolol	are	less	pronounced	then those	reported	in	the	literature.		Sharma	et	al	observed	a	significant	increase	in cardiac	output	and	hydraulic	work	in	metoprolol	treated	diabetic	rats.		However,	in Sharma’s	study	function	was	found	to	be	far	worse	in	the	untreated	diabetic	than	in the	present	study.		Where	we	observed	a	25%	reduction	in	cardiac	output,	they    59  Discussion  showed	a	60%	reduction	(Figure	2D).		Metoprolol	in	their	hands	only	improved	the treated	animals	to	a	level	that	was	very	close	to	our	untreated	diabetic	animals33. Thus	it	is	possible	that	the	reason	they	saw	a	greater	effect	with	metoprolol	was because	cardiac	function	was	disturbed	to	a	greater	extent. The	improvements	in	function	we	observed	with	ascorbic	acid	are	similar	to previously	reported	results.		Dai	et	al.	noted	that	ascorbic	acid	improved	left ventricular	developed	pressure	and	left	ventricular	end	diastolic	pressure	in	STZ treated	rats127.		Other	researchers	have	also	shown	that	results	observed	with ascorbic	acid	can	be	extended	to	other	antioxidants;	Koksoy	et	al.	noted	similar improvements	in	pressure	related	parameters	after	treatment	with	sodium	selanate and	omega‐3	fish	oil	supplemented	with	vitamin	E157. The	additive	functional	effects	observed	with	dual	treatment	seem	to	imply that	metoprolol	and	ascorbic	acid	did	not	directly	act	on	the	same	site.		Sharma	et	al. have	proposed	that	metoprolol’s	functional	improvement	effects	are,	at	least partially,	mediated	through	normalization	of	signaling,	sequestration	of	cell	death mediators	and	reduction	in	myocardial	fibrosis,	but	not	reduction	of	oxidative	stress 34,	112	.		Positive	effects	observed	with	antioxidants,	on	the	other	hand,	are	thought	to  be	regulated	through	reduction	in	oxidative	damage	to	proteins157.       60  Discussion  4.5. Heart Metabolism – Metabolic Alterations Worsen with Disease Progression 4.5.1.   Palmitate Oxidation  Oxidation	of	exogenous	palmitate	is	increased	by	20%	in	diabetic	hearts  compared	to	controls	during	Study	1	and	by	70%	above	controls	by	the	end	of	Study 2	(Figure	3A,	3C).		Thus,	it	appears	that	there	was	a	large	metabolic	shift	that	occurs during	the	period	between	Study	1	and	Study	2.		Literature	values	for	exogenous palmitate	oxidation	at	early	and	late	time	points	show	increases	in	diabetic	hearts that	are	between	rates	observed	in	Study	1	and	Study	2.		However,	the	degree	of disturbance	in	palmitate	oxidation	does	not	correlate	consistently	with	time	point. Kewalramani	et	al.	and		Ghosh	et	al.	both	used	Wistar	rats	treated	with	55	mg/kg and	both	measured	palmitate	oxidation	rates	at	4	days.	Kewalramani	observed	a 100%,	while	Ghosh	found	only	a	40%	increase142,	158	.		Workload	is	identical	in	both cases,	and	substrate	concentrations	are	similar.		Thus,	there	is	no	clear	explanation why	consistent	palmitate	oxidation	values	were	not	observed.		One	possibility	is that	hearts	with	lower	measured	substrate	oxidation	rates	were	actually metabolizing	significant	amounts	of	endogenous	triglyceride	stores	and	since	only the	exogenous	palmitate	is	labeled	with	isotope,	oxidation	of	endogenous	fuels would	be	invisible. Treatment	with	metoprolol	and	ascorbic	acid	completely	normalized palmitate	oxidation	rates	in	Study	1	(Figure	3A).		In	Study	2,	oxidation	rates	were more	resistant	to	change.		Although	metoprolol	consistently	caused	a	20%	decrease in	treated	diabetic	groups	the	change	was	not	significant	(Figure	3C).		This	is	in   61  Discussion  contrast	to	the	work	of	Sharma	et	al.	who	showed	that	metoprolol	treatment	could lead	to	an	approximate	one‐half	reduction	in	rates.		However,	it	is	important	to	note that	their	goal	was	to	model	in	vivo	oxidation	rates,	so	they	did	not	provide	diabetic hearts	with	insulin	during	perfusion,	and	provided	controls	with	5‐fold	the	insulin provided	in	our	study.		As	a	result	their	palmitate	oxidation	rates	in	diabetic	hearts were	400%	higher	then	their	controls.	Perfusion	conditions	combined	with	their higher,	75	mg/kg/day	dosage,	delivered	via	intraperitoneal	injection,	might	explain the	larger	effect	they	observed	with	metoprolol.		Ascorbic	acid	produced	no detectable	effect	on	palmitate	oxidation	rates	in	our	study,	and	no	literature	data was	found	on	the	effect	of	ascorbic	acid	on	fatty	acid	oxidation	during	diabetes33.		In animals	receiving	dual	treatment	there	was	a	significant	30%	reduction	in	rates, thus	it	appears	that	ascorbic	acid	some	how	sensitized	hearts	to	the	effects	of metoprolol.   4.5.2.   Glucose Oxidation  Glucose	oxidation	in	diabetic	hearts	were	lowered	to	nearly	undetectable  levels	in	both	studies	(Figure	3B,	3D).		Changes	in	glucose	oxidation	rates	are	known to	begin	early	and	correspond	with	the	induction	of	diabetes.		Ghosh	et	al.	showed that	at	4	days	post	STZ	injection,	animals	had	50%	reduced	glucose	oxidation	rates compared	to	controls	and	by	6	weeks	showed	oxidation	rates	similar	to	those observed	in	Study	1	and	Study	2158.		Glucose	oxidation	is	reduced	in	diabetes partially	because	of	inhibition	of	pyruvate	dehydrogenase,	a	key	regulator	of	glucose oxidation,	caused	by	increased	fatty	acid	oxidation	rates	and	partially	because	of    62  Discussion  reduced	substrate	availability	due	to	reduced	glucose	transporter	4	(GLUT	4) expression159. During	Study	1	and	Study	2,	treatment	with	metoprolol	or	ascorbic	acid, individiually	or	in	combination,	showed	no	significnat	beneficial	effects.		However, dual	treatment	in	Study	1,	and	all	treatments	in	Study	2	did	show	small	increases	in oxidation	rates.	Interestingly,	the	degree	of	increase	correlates	well	with	increases in	cardiac	glycogen	levels,	with	the	largest	increases	observed	in	groups	with	the most	glycogen	(Figure	3B,	3D	and	4A,	4C).		Although	our	study	did	not	include	a comprehensive	analysis	of	cardiac	glucose	use,	increased	oxidation	and	increased glycogen	levels	might	indicate	increased	glucose	uptake.		Sharma	et	al.	have	shown that	metoprolol	can	cause	a	small	but	significant	stimulation	of	glucose	oxidation, however	they	did	not	observe	changes	in	tissue	glycogen	levels33.		Of	note,	other antioxidants	are	known	to	improve	glucose	uptake.		Resveratrol,	a	potent antidoxidant,	has	been	shown	to	improve	glucose	uptake,	by	increasing	GLUT	4 translocation	in	Sprague‐Dawley	rats	made	diabetic	with	65	mg/kg	of	STZ.		In addition,	the	antioxidant	alpha–tocopherol	has	been	demonstrated	to	improve glucose	uptake	in	diaphragm	muscle	from	STZ	treated	rats160.		Further	studies	are required	to	confirm	whether	our	treatments	caused	real	increases	in	glucose	uptake and	oxidation.       63  Discussion  4.6. Oxidative Protein Damage in Diabetic Hearts Worsen with Disease Progression Oxidative	cardiac	protein	damage	was	increased	by	about	65%	in	diabetics from	Study	1,	although	these	changes	are	not	significant.		In	Study	2,	oxidative damage	in	diabetics	had	increased	by	over	95%	compared	to	controls	and	the changes	were	statistically	significant	(Figure	5).		The	cause	of	the	increased oxidative	protein	damage	may	be	due	to	increases	in	levels	of	ceramide,	which	is	a key	mediator	of	lipotoxicity,	oxidative	stress	and	cell	death161.		Increased	oxidative damage	could	also	be	due	to	increased	glycotoxicity,	or	directed	flow	of	glucose catabolism	through	the	polyol	pathway,	formation	of	advanced	glycation	end products	leading	to	reactive	oxygen	species	production,	and	increased	flux	through the	hexosamine	biosynthetic	pathway91,	162,	163		. During	Study	2,	when	oxidative	stress	is	most	prominent,	there	is	a	large	and significant	reduction	in	oxidative	protein	damage	associated	with	ascorbic	acid treatment	in	control	hearts,	but	a	lesser	and	non‐significant	reduction	in	diabetic hearts	(Figure	7).		Although	it	appears	that	ascorbic	acid,	with	or	without metoprolol,	has	an	approximately	one‐half	lesser	effect	in	diabetic	animals	(80	and 95%	reductions	in	controls	versus	45	and	55%	in	diabetics,	respectively),	it	is important	to	note	that	oxidative	damage	in	diabetics	is	doubled	at	this	time	point. Thus,	in	terms	of	absolute	degree	of	reduction,	ascorbic	acid	has	a	similar	effect	on both	control	and	diabetic	hearts.		Ascorbic	acid’s	reduction	of	oxidative	stress	could partially	be	due	to	its	chemical	antioxidant	properties,	but	also	could	be	due	to	its    64  Discussion  ability	to	inhibit	aldose	reductase,	the	rate	limiting	enzyme	in	the	reactive	oxygen species	generating	polyol	pathway164. In	contrast	to	Study	1,	during	Study	2	all	groups	treated	with	metoprolol tended	to	show	slightly	lower	oxidative	protein	damage	then	their	respective counterparts	(Figure	7).		Although	metoprolol	is	not	known	to	have	biologically relevant	antioxidant	activity,	its	ability	to	reduce	cell	death	signaling	increased during	diabetes	may	improve	cellular	stability	and	reduce	reactive	species generation34.		Further	experiments	examining	the	dose	dependency	of	oxidative damage	reductions	using	metoprolol	and	ascorbic	acid	would	be	useful	to	confirm our	findings	and	proposed	mechanisms.   4.7. Summary (see Scheme 3) 4.7.1.  Progression of Diabetes from 5 to 7 Weeks - An  Important Time Point in the Development of Cardiac Dysfunction   In	the	progression	of	diabetes,	as	demonstrated	by	differences	between  Study	1	and	Study	2,	a	number	of	important	changes	occurred.		First,	diabetic animals	experienced	greater	reductions	in	body	weight	and	began	to	develop reductions	in	heart	weight.		Second,	fatty	acid	oxidation	in	diabetic	animals compared	to	controls	increases	from	a	mild	20%	disturbance	to	a	pronounced 100%	increase.		Furthermore,	these	changes	actually	become	more	persistent,	as metoprolol	and	ascorbic	acid	lost	their	beneficial	effects.		Third,	more	pronounced functional	impairments	developed	ex	vivo	then	in	vivo,	indicating	that	intrinsic    65  Discussion  changes	occur	in	the	myocardium	over	this	critical	time	point	and	that	they	can	be masked	in	vivo.		Finally,	oxidative	protein	damage	in	diabetic	hearts	worsens	from 65%	over	controls	to	95%	over	controls.		Thus	it	appears	that	physical,	metabolic, functional	characteristics	in	the	diabetic	heart	all	worsen	significantly	over	a relatively	short	2‐week	period,	a	period	over	which	oxidative	protein	damage	also shows	large	changes.   4.7.2.  Benefits of β-Blocker Therapy Supplemented with  Antioxidants. Although	we	observed	two	time	points	during	our	study,	Study	2	is	most	useful in	assessing	the	effects	of	dual	therapy	on	metabolism,	function	and	oxidative damage	because	it	is	here	that	overt	dysfunction	developed.		During	Study	2 metoprolol	alone	had	no	significant	effects	on	metabolism	or	oxidative	damage	but did	have	some	effect	on	function.		Furthermore,	values	from	metoprolol	treated diabetic	animals	often	trended	more	towards	controls.		Ascorbic	acid	was	not observed	to	have	an	effect	on	metabolism	except	for	increasing	the	effect	of metoprolol	on	palmitate	oxidation,	also	it	did	cause	reductions	in	oxidative	protein damage	and	improved	function	to	a	point	where	treated	animals	were	not significantly	different	from	controls.		When	both	treatments	were	combined function	was	further	(and	significantly)	improved	as	compared	to	diabetic untreated	hearts.       66  Discussion  4.7.3.  Hypothesis and Conclusions  The	following	conclusions	were	made	in	regards	to	the	three	sub‐hypotheses that	we	addressed	during	our	study:  1. Disturbances	in	metabolism	will	appear	before	the	development	of	overt dysfunction,	while	changes	in	oxidative	protein	damage	will	appear	most prominent	at	the	point	of	dysfunction.      During	our	study,	we	were	able	to	demonstrate	that	metabolic	disturbances  do	in	fact	appear	before	development	of	overt	cardiac	dysfunction,	however,	they	do become	more	prominent	once	dysfunction	has	set	in.		Changes	in	oxidative	protein damage	appear	before	dysfunction,	but	do	become	much	more	pronounced	after development	of	cardiac	dysfunction.		Thus,	we	were	able	to	provide	support	for	this hypothesis.  2. Both	metoprolol	and	ascorbic	acid	will	improve	cardiac	function,	however,	only ascorbic	acid	will	reduce	oxidative	stress.  In	our	hands	metoprolol	and	ascorbic	acid	improved	functional	parameters in	the	diabetic	heart,	with	ascorbic	acid	having	the	more	pronounced	effect. Ascorbic	acid,	but	not	metoprolol,	was	able	to	lower	oxidative	stress	(although changes	were	pronounced	they	were	only	significant	in	control	hearts).		We	feel there	is	strong	support	for	this	hypothesis.    67  Discussion   3. Combined	metoprolol	and	ascorbic	acid	treatment	will	improve	function further	then	single	treatment.  There	is	strong	support	for	this	hypothesis.		When	the	two	drugs	were combined,	several	functional	parameters	were	raised	to	a	point	that	they	were	not different	from	controls	and,	in	the	case	of	cardiac	output,	significantly	improved above	diabetics.  Our	overall	hypothesis	was:    We	propose	that	metabolic	changes	that	occur	during	diabetes	elevate  oxidative	stress,	leading	to	protein	damage,	signaling	changes,	cell	death	and	other sequelea;	the	eventual	sum	of	these	changes	is	an	impairment	of	function.		Treatment of	either	the	sequelae	of	oxidative	stress	or	oxidative	stress	directly	will	be	beneficial but	treatment	of	both	will	improve	function	further.    We	were	able	to	show	that	metabolism	became	significantly	worse	at	a	time  point	that	was	associated	with	impaired	function,	we	were	also	able	to	show increased	oxidative	protein	damage,	a	possible	link	between	function	and metabolism.		Although	it	should	be	noted	we	have	not	conclusively	shown	a	cause and	effect	relationship	between	the	two.		Finally,	we	were	able	to	show	that	by    68  Discussion  treating	both	oxidative	stress	and	it’s	consequences,	function	was	improved.		Thus, we	feel	we	have	supported	our	hypothesis.   4.8. Importance of the Study   The	results	of	this	study	are	important	because	they	demonstrate	the  effectiveness	of	a	novel	treatment	strategy	targeting	both	the	signaling	changes,	cell death	and	other	sequela	(by	metoprolol)	and	oxidative	stress	(by	ascorbic	acid)	that are	associated	with	STZ‐induced	diabetic	cardiomyopathy.		This	strategy	is especially	helpful	because	it	targets	oxidative	stress	and	its	effects	without interfering	with	oxidative	lipid	metabolism,	avoiding	problems	associated	with	lipid accumulation.		Furthermore,	our	results	may	provide	insight	into	the	superior therapeutic	effects	of	β‐blockers	that	incorporate	antioxidant	properties,	such	as carvedilol.		Our	β‐blocker	and	antioxidant	treatment	strategy’s	focus	is	on	oxidative stress,	and	not	on	diabetes	specifically,	thus	it	may	prove	helpful	in	other	disease where	metabolic	disturbances	contribute	to	oxidative	stress,	such	as	heart	failure.   4.9. 	Future Directions   The	present	study	sheds	light	onto	the	progression	of	diabetic	cardiovascular  disease,	and	especially	diabetes‐induced	cardiac	dysfunction.	However,	there	are	a number	of	important	questions	that	remain.		The	most	obvious	issue	is	to	clarify	the role	of	metabolism	in	the	development	of	cardiovascular	dysfunction.		To	that	end, one	could	attempt	to	modify	metabolism	further	then	was	achieved	in	our	study	by    69  Discussion  using	higher	doses	of	metoprolol,	or	using	other	agents	that	are	known	to	inhibit long	chain	fatty	acid	oxidation,	such	as	etomoxir,	however,	one	would	have	to	also lower	lipid	uptake	in	order	to	prevent	lipid	accumulation165.		Furthermore,	it	would be	useful	to	perform	a	full	assessment	of	expression	and	activation	states	of	key metabolic	flux	regulating	proteins	such	as;	hexokinase	and	phosphofructokinase‐1, which	control	glycolytic	flux;	pyruvate	dehydrogenase,	which	controls	rate	of	flux through	the	citric	acid	cycle;	and	carnitine	palmitoyltransferase‐155.		Assessment	of glucose	uptake	capacity	in	untreated	and	treated	diabetic	hearts	by	measurement	of subcellular	localization	and	total	expression	of	GLUT	4	could	also	prove	useful, particularly	in	explaining	some	of	ascorbic	acids	effects	on	glucose	metabolism. Another	important	area	to	investigate	would	be	to	assess	the	contribution	of lipotoxicity	and	glucotoxicity	in	the	development	of	oxidative	stress.		This	would include	quantitation	of	ceramide	levels	and	measuring	the	flux	through	oxidative stress	causing	pathways,	such	as	the	polyol	pathway,	and	the	hexosamine biosynthetic	pathways.		Further	study	of	the	polyol	pathway	in	particular,	by measuring	expression	and	activity	of	aldose	reductase,	could	shed	further	light	onto the	mechanism	of	action	of	ascorbic	acid,	as	it	is	an	inhibitor	of	that	key	enzyme164. Recently,	metoprolol	was	shown	to	move	the	diabetic	heart	from	a	pro‐	to anti‐apoptotic	state.		This	was	accomplished	by	shifting	signaling	from	protein‐ kinase‐A	to	protein‐kinase‐B	signaling,	and	through	sequesteration	of	activated caspase‐3	by	caveolins34.		It	would	be	useful	to	investigate	ascorbic	acid’s	effect	on apoptotic	signaling	within	diabetic	heart	as	reduction	of	oxidative	damage	may result	in	reduced	stimulus	for	a	pro‐apoptotic	state.    70  Discussion  Our	experimental	model	of	type‐1	diabetes	provided	a	clear	look	at	cardiac dysfunction	in	a	hyperglycemic	model	without	complications	such	as	hypertension. 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