UBC Theses and Dissertations
Mechanisms of cardiac pacemaking and temperature-dependent depression of cardiac electrical excitation in the zebrafish (Danio rerio) Marchant, James
The physiology of ectotherms is profoundly affected by the environmental temperature which governs the rate of physiological processes. Cardiac function, an essential function of all vertebrates, is no exception: during warming heart rate tracks temperature before declining at temperatures beyond maximum optimal temperature, ultimately collapsing with further warming. In fishes, like other vertebrates, intrinsic heart rate is set by pacemaker cells located in the sino-atrial node that spontaneously generate action potentials. At the cellular level, temperature-dependent deterioration of pacemaking mechanisms may contribute to the decline of cardiac function. Nevertheless, the mechanisms of pacemaking and their temperature-dependent deterioration remain elusive. Hence, I explored cardiac pacemaking mechanisms in zebrafish, as well as their relative thermal performance and limits. I validated blebbistatin as an effective excitation-contraction uncoupling agent that did not modify the cardiac action potential properties, thus providing an essential methodology for future cardiac pacemaking research enabling the direct recording of intracellular electrical activity of pacemaker cells. Using electrocardiograms, I confirmed that cardiac pacemaking involves two major mechanisms. Pharmacological blockade of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels with zatebradine reduced heart rate by up to 60%, suggesting HCN channels play the major role in cardiac pacemaking. Likewise, sarcoplasmic reticulum (SR) calcium cycling was pharmacologically blocked using ryanodine and thapsigargin to block ryanodine receptors and SERCA pumps, respectively, which reduced heart rate by ~40%, suggesting the SR plays a secondarily important role in pacemaking. However, the combination of these pharmacological interventions did not completely stop the heartbeat, suggesting that either mammalian pharmacological agents are less effective in producing total Hcn block in zebrafish, perhaps due to isoform specificity. HCN4, the major HCN channel involved in mammalian pacemaking, was knocked out using CRISPR to explore its role in zebrafish cardiac pacemaking. Heart rate did not differ significantly between mutant and control fish at any test temperature, including fish treated with inhibitors of HCN channels or SR calcium cycling. Thus, alternative Hcn channels compensated for the knockout of Hcn4, presumably contributing to a higher thermal tolerance. In addition, mutant fish had a higher upper thermal tolerance than control fish when SR calcium cycling was inhibited.
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