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Investigation of mechanisms underlying altered alpha-adrenergic receptor-induced contractile responses in the streptozotocin-diabetic rat heart Guo, Tianhai

Abstract

Diabetic cardiomyopathy is one of the major chronic complications in diabetes mellitus. Alterations in the α₁-adrenergic receptor (α₁-AR)-induced positive inotropic effect (PIE) have been shown in the diabetic heart, but the results have not been consistent. The molecular signaling mechanisms underlying the α₁-AR-induced PIE in the heart, though still under debate, have been suggested to be related to two protein kinases, protein kinase C (PKC) and Rho kinase, both of which consist of several isoforms. Thus it was hypothesized that specific PKC and/or Rho Kinase isoforms play a role in the altered α₁-AR-induced PIE in the diabetic heart. The effects of chronic streptozotocin-induced diabetes on the basal contractile function and the α₁-AR-induced PIE, as well as the associated changes in four PKC isoforms (α, β₂, δ and є) and two Rho kinase isoforms (ROCK 1 and ROCK 2) in the rat heart were investigated. Three cardiac contractile parameters, left ventricular developed pressure (LVDP), maximal rate of contraction (+dP/dt) and relaxation (-dP/dt), were measured using the isolated Langendorff-perfused isovolumic heart model. In the absence of adrenergic stimulation, all three contractile parameters were attenuated in hearts from 6∼7 week and 12∼15 week diabetic rats. The selective α₁-AR agonist, phenylephrine (PE), produced greater maximal increase (Rmax) values for LVDP and -dP/dt in both 6∼7 week and 12∼15 week diabetic hearts compared to age-matched controls. It also produced greater pD₂ (-log [ED₅₀]) values for +dP/dt in both 6∼7 week and 12∼15 week diabetic hearts, and greater PD₂ values for LVDP and -dP/dt in 12∼15 week diabetic hearts compared to age-matched controls. In the presence of the non-isoform-selective PKC inhibitor, chelerythrine (CE), the increase in all three contractile parameters in response to PE was partially suppressedin both diabetic and control hearts, and the increase to PE in LVDP and -dP/dt was not different in diabetic and control hearts. The non-isoform-selective Rho kinase inhibitors, Y-27632 and H1152, had no effect on the α₁-AR-induced PIE in either diabetic or control hearts. Western immunoblotting showed that in the absence of adrenergic stimulation, the basal levels of PKCδ and PKCε in the particulate fraction of 12~15 week diabetic hearts were increased compared to control, without any change in the soluble fraction. There was no change in the subcellular distribution of PKCα, PKCβ₂, ROCK 1 or ROCK 2 in diabetic hearts compared to control. PE produced a significant increase in levels of PKCδ and PKCε in the particulate fraction of both 12∼15 week diabetic and control hearts, but without a corresponding decrease in the soluble fraction. The increase in particulate PKCδ over its own basal levels in diabetic hearts was significantly greater than control, whereas the increase in particulate PKCє over its own basal levels in diabetic and control hearts was not different. In the presence of CE, the PE-induced increase in levels of PKCδ and PKCε in the particulate fraction of both diabetic and control hearts was completely suppressed. PE had no effect on the subcellular distribution of PKCα, PKCβ₂, ROCK 1 or ROCK 2 in either diabetic or control hearts. Activation of the renin-angiotensin system (RAS) has been suggested to contribute to diabetic cardiomyopathy. It has been shown that in isolated cardiomyocytes from diabetic rats, PKCε translocated from the soluble to the particulate fraction, while treatment with the angiotensin II type 1 receptor antagonist, L-158,809, normalized the alteration in PKCε. Thus it was hypothesized that treatment with this antagonist would improve the attenuated basal contractile function and normalize the enhanced α₁-AR-induced PIE, as well as the associated changes in PKC isoforms in thediabetic heart. The results showed that treatment with L-158,809 significantly improved the basal contractile function of 12-week diabetic hearts. However, it did not normalize the enhanced α₁-AR-induced PIE. This antagonist also had no effect on the basal levels of PKCδ and PKCε in the particulate fraction of diabetic hearts, nor did it affect the PEinduced changes in these two PKC isozymes in either diabetic or control hearts. The results from the present study suggest a role for PKCδ and/or PKCε in the PIE to α₁-AR stimulation in the heart, and that PKCδ may contribute to the enhanced α₁-AR-induced PIE in the diabetic heart. These two PKC isoforms appear to be activated under basal conditions in the diabetic heart. The present study does not support a role for Rho kinase in the α₁-AR-induced PIE in the heart or in diabetic cardiomyopathy. The activation of RAS contributes to cardiac contractile dysfunction in diabetes. However, this study does not support an involvement of PKC in this process.

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