In the last decade alone fish hearts have become an increasingly popular model for studying heart function. Although fish hearts contain a single atrium and ventricle and present a fundamentally different cardiovascular system when compared to mammalian models, there are many developmental, structural, and functional commonalities between the two vertebrate classes. The goldfish heart, specifically, has remarkably similar electrical properties to that of humans’. For example, the heart rate, action potential morphology, and Ca2+ transient kinetics and dynamics of adult goldfish closely parallel those of humans, even more so than mice. In nearly all vertebrate species, direct input from the autonomic nervous system tightly controls cardiac contractility and excitability. Although there is an abundant amount of research on the autonomic control of cardiac contractility and excitability in numerous mammalian species, the characterization of pathophysiological mechanisms is still difficult to obtain for humans specifically. This is in part due to humans having strikingly dissimilar AP characteristics and electrocardiographic morphology in comparison to commonly used animal models such as mice, rats, and rabbits. Fish, on the other hand, are the largest and most diverse group of vertebrates, and as such, their autonomic nervous system regulation can often deviate from the classical vertebrate models used to study autonomic control of cardiac contractility and excitability. Ventricular APs, electrocardiograms, and Ca2+ transients recorded from the goldfish intact heart showed perfusion with either 100 nM isoproterenol (sympathetic agonist) or 5 µM carbamylcholine (parasympathetic agonist), was enough to stimulate the sympathetic branch or parasympathetic branch, respectively. Interestingly, our results indicate stimulation of the goldfish autonomic nervous system by these commonly used agonists resulted in a corresponding change in cardiac dromotropism, chronotropism, ionotropism, and lusitropism in a similar manner observed in humans. The data obtained from our experiments have led us to propose the goldfish heart as an excellent model for performing physiological experiments at the intact-heart level. Moreover, its shared ionic and electrical similarities with larger mammals open a new avenue for goldfish hearts to be used as a model to study human physiology.

Cardiac calsequestrin (Casq2) is a Ca²⁺ binding protein inside the lumen of the sarcoplasmic reticulum (SR) that binds Ca²⁺ and regulates the gating of the Ca²⁺ channel called the ryanodine receptor (RyR2). The role of Casq2 during cardiac contraction is still unclear. We hypothesize Casq2 serves as a lumenal Ca²⁺ sensor that increases RyR2 open probability as a function of [C²⁺] inside the SR. To better understand how Casq2 affects RyR2 open probability (Po), single channel electrophysiology experiments were performed using transgenic SR microsomes with (Casq2WT) and without (Casq2KO) Casq2. Increasing lumenal [Ca²⁺] from 2μM to 2mM produced a significant increase in Po and a decrease in the amplitude of the current in Casq2WT microsomes; though, it is uncertain whether this effect was due to a lumenal regulation by Casq2 or a cytosolic regulation by a feed-through activation. Increasing lumenal [Mg²⁺], a RyR2 cytosolic inhibitor, from 0 mM to 3 mM also produced an increase in RyR2 Po. An increase in lumenal Tb³⁺ [nM] activated RyR2s from Casq2WT but not from Casq2KO microsomes without modifications in the RyR2 current, indicating no feed-through activation. These results support the idea that Casq2 is involved in the lumenal regulation of RyR2.

T-wave alternans are alternating beat-to-beat changes in the morphology of the electrocardiographic T-wave. Despite being the only indicator of ventricular fibrillation leading to sudden cardiac death (SCD), the molecular mechanisms of alternans remain unclear. At the cellular level, the beat-to-beat changes in the T-wave are paralleled with beat-to-beat changes in the electrical repolarization duration (action potential duration (APD) alternans) and in the myoplasmic Ca2+ levels (Ca2+ alternans). The study presented in this thesis aimed to understand how T-wave alternans are generated by assessing how the two cardiac properties, electrical (measured as the action potential) and contractile (Ca2+ dynamics, measured as Ca2+ transients), change under conditions favoring alternans. Research has pointed to the mishandling of Ca2+ as the source of T-wave alternans. However, the precise cause of this mishandling has not been established. In this thesis, we assessed three variables that modulate the handling of intracellular Ca2+: heart rate (HR), temperature, and autonomic nervous system (ANS) regulation. Studying these variables will allow us to determine what mishandling occurs in the presence of Ca2+ alternans. Tachycardia, or increased HRs, is a condition that favors the genesis of T-wave alternans. Thus, HR was used to induce alternans and to study what changes occur in the cardiac properties as the HR is increased. Since temperature dramatically affects the handling of intracellular Ca2+ content, we also assessed how temperature modulates Ca2+ alternans. In addition, the role of the ANS on Ca2+ alternans was studied because it is the body’s primary method of changing the HR and it also modulates Ca2+ cycling that may affect alternans. We hypothesized that insufficient Ca2+ re-uptake by the sarcoplasmic reticulum (Ca2+ storage sites inside the cell) during increased HRs, hypothermia, or ANS dysfunction produces Ca2+ alternans. Consequently, Ca2+ alternans cause beat-to-beat alternations in the AP repolarization leading to T-wave alternans. The major conclusion from this thesis is that increases in the HR and colder temperatures (conditions favoring alternans) primarily affect the Ca2+ transient relaxation. These results point to the importance of the SR re-uptake mechanisms in the genesis of Ca2+ alternans. The ANS, in particular the sympathetic nervous system, is capable of reducing Ca2+ alternans by improving the re-sequestering of intracellular Ca2+ into the SR. Thus, targeting SR-Ca2+ reuptake may be a possible intervention method in the treatment of T-wave alternans before SCD occurs.

The Na+/Ca2+ exchanger (NCX1) plays an important role in defining cardiac function by regulating the contractility and excitability of the heart. The aim of my research was to study the effect of impairing NCX1 with various LiCl concentrations (Na+ replaced by Li+), known NCX blockers KBR-7943 and SEA0400 in wild-type intact mouse hearts. In addition to this, cardiac-specific NCX1 KO mice were used to further evaluate intracellular Ca2+ dynamics and the electrical properties of the heart. We hypothesized that impairment of the exchanger would lead to an increase of Ca2+ in the cytosol and consequently an accumulation of Ca2+in the sarcoplasmic reticulum (SR). This accumulation will result in a larger release of Ca2+from the SR. In addition to this, NCX1 impairment will lead to a change in the membrane potential during the repolarization phase of epicardial action potentials. Experiments performed in this dissertation were by the use of a modified version of the pulsed local-field fluorescent microscopy (PLFFM), where hearts were loaded with Ca2+ sensitive fluorescent dyes (rhod-2, AM and mag-fluo-4, AM) or the potentiometric dye (DI-8ANEPPS). Microelectrode impalement for epicardial action potential recordings were also used in this dissertation for simultaneous recordings of intracellular Ca2+ dynamics and left ventricular transmural electrocardiographic recordings. To conclude, impairment of the NCX1 alters intracellular Ca2+ dynamics and changes the electrical properties of the heart.

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