Functional Assessment of Cardiac Responses of Adult Zebrafish (Danio rerio) to Acute and Chronic Temperature Change Using High-Resolution Echocardiography

The zebrafish (Danio rerio) is an important organism as a model for understanding vertebrate cardiovascular development. However, little is known about adult ZF cardiac function and how contractile function changes to cope with fluctuations in ambient temperature. The goals of this study were to: 1) determine if high resolution echocardiography (HRE) in the presence of reduced cardiodepressant anesthetics could be used to accurately investigate the structural and functional properties of the ZF heart and 2) if the effect of ambient temperature changes both acutely and chronically could be determined non-invasively using HRE in vivo. Heart rate (HR) appears to be the critical factor in modifying cardiac output (CO) with ambient temperature fluctuation as it increases from 78 ± 5.9 bpm at 18°C to 162 ± 9.7 bpm at 28°C regardless of acclimation state (cold acclimated CA– 18°C; warm acclimated WA– 28°C). Stroke volume (SV) is highest when the ambient temperature matches the acclimation temperature, though this difference did not constitute a significant effect (CA 1.17 ± 0.15 μL at 18°C vs 1.06 ± 0.14 μl at 28°C; WA 1.10 ± 0.13 μL at 18°C vs 1.12 ± 0.12 μl at 28°C). The isovolumetric contraction time (IVCT) was significantly shorter in CA fish at 18°C. The CA group showed improved systolic function at 18°C in comparison to the WA group with significant increases in both ejection fraction and fractional shortening and decreases in IVCT. The decreased early peak (E) velocity and early peak velocity / atrial peak velocity (E/A) ratio in the CA group are likely associated with increased reliance on atrial contraction for ventricular filling

Zebrafish as a Model of Mammalian Cardiac Function: Optically Mapping the Interplay of Temperature and Rate on Voltage and Calcium Dynamics

The zebrafish (Danio rerio) heart is a viable model of mammalian cardiovascular function due to similarities in heart rate, ultrastructure, and action potential morphology. Zebrafish are able to tolerate a wide range of naturally occurring temperatures through altering chronotropic and inotropic properties of the heart. Optical mapping of cannulated zebrafish hearts can be used to assess the effect of temperature on excitation-contraction (EC) coupling and to explore the mechanisms underlying voltage (Vm) and calcium (Ca2+) transients. Applicability of zebrafish as a model of mammalian cardiac physiology should be understood in the context of numerous subtle differences in structure, ion channel expression, and Ca2+ handling. In contrast to mammalian systems, Ca2+ release from the sarcoplasmic reticulum (SR) plays a relatively small role in activating the contractile apparatus in teleosts, which may contribute to differences in restitution. The contractile function of the zebrafish heart is closely tied to extracellular Ca2+ which enters cardiomyocytes through L-type Ca2+ channel (LTCC), T-type Ca2+ channel (TTCC), and the sodium-calcium exchanger (NCX). Novel data found that despite large temperature effects on heart rate, Vm, and Ca2+ durations, the relationship between Vm and Ca2+ signals was only minimally altered in the face of acute temperature change. This suggests that zebrafish Vm and Ca2+ kinetics are largely rate-independent. In comparison to mammalian systems, zebrafish Ca2+ cycling is inherently more dependent on transsarcolemmal Ca2+ transport and less reliant on SR Ca2+ release. However, the compensatory actions of various components of the Ca2+ cycling machinery of the zebrafish cardiomyocytes, allow for maintenance of EC coupling over a wide range of environmental temperatures

Thermodynamic characterization of hypertrophic cardiomyopathy associated troponin C mutations

Hypertrophic Cardiomyopathy (HCM) is the leading cause of sudden cardiac death in young adults under the age of 35; a devastating disease that is not yet well understood. To date, greater than 1000 HCM-associated mutations have been found in genes that encode mostly sarcomeric proteins. Familial Hypertrophic Cardiomyopathy (FHC) is the heritable form of HCM. The overlying phenotype of FHC is thought to be derived from an increase in calcium (Ca2+) sensitivity of contraction and impaired relaxation of the myocardium. Dilated Cardiomyopathy (DCM) associated mutations are thought to have the opposite functional effect. This study focuses on cardiac troponin C (cTnC) a component of the cardiac troponin complex where binding of Ca2+ acts as the regulatory switch, leading to a series of conformational changes that culminate in muscle contraction. This project explores Ca2+ binding by focusing on the proximal-most unit of the contractile apparatus. The interaction of Ca2+ with the regulatory domain of cTnC is studied through isothermal titration calorimetry in conjunction with Molecular Dynamics simulations to understand structural and functional changes in the N-terminal region of cTnC. Initially, we established a workflow by exploring the functional consequences of sequence variations in coordinating Ca2+ binding and the genetic control of paralog expression in response to environmental temperature change in zebrafish. We then focused on a series of FHC-associated mutations (A8V, L29Q, A31S, and C84Y), as well as an engineered Ca2+ sensitizing mutation (L48Q), and a DCM-associated mutation (Q50R). The effects of temperature in modulating the Ca2+-cTnC interaction was also studied in these mutants. We further explored the role of cellularly abundant magnesium (Mg2+) which also interacts with cTnC and may modulate the Ca2+ coordinating capabilities of this contractile protein. Lastly, the role of Mg2+ binding to the mutants of interest, under normal cellular condition and in energy depleted states was explored to better understand the etiology of FHC and provide biomedical and physiological insight into potential treatments for this disease

Familial Hypertrophic Cardiomyopathy Related Cardiac Troponin C L29Q Mutation Alters Length-Dependent Activation and Functional Effects of Phosphomimetic Troponin I*

The Ca2+ binding properties of the FHC-associated cardiac troponin C (cTnC) mutation L29Q were examined in isolated cTnC, troponin complexes, reconstituted thin filament preparations, and skinned cardiomyocytes. While higher Ca2+ binding affinity was apparent for the L29Q mutant in isolated cTnC, this phenomenon was not observed in the cTn complex. At the level of the thin filament in the presence of phosphomimetic TnI, L29Q cTnC further reduced the Ca2+ affinity by 27% in the steady-state measurement and increased the Ca2+ dissociation rate by 20% in the kinetic studies. Molecular dynamics simulations suggest that L29Q destabilizes the conformation of cNTnC in the presence of phosphomimetic cTnI and potentially modulates the Ca2+ sensitivity due to the changes of the opening/closing equilibrium of cNTnC. In the skinned cardiomyocyte preparation, L29Q cTnC increased Ca2+ sensitivity in a highly sarcomere length (SL)-dependent manner. The well-established reduction of Ca2+ sensitivity by phosphomimetic cTnI was diminished by 68% in the presence of the mutation and it also depressed the SL-dependent increase in myofilament Ca2+ sensitivity. This might result from its modified interaction with cTnI which altered the feedback effects of cross-bridges on the L29Q cTnC-cTnI-Tm complex. This study demonstrates that the L29Q mutation alters the contractility and the functional effects of the phosphomimetic cTnI in both thin filament and single skinned cardiomyocytes and importantly that this effect is highly sarcomere length dependent

Investigating the Utility of Adult Zebrafish Ex Vivo Whole Hearts to Pharmacologically Screen hERG Channel Activator Compounds

There is significant interest in the potential utility of small molecule activator compounds to mitigate cardiac arrhythmia caused by loss-of-function of hERG1a voltage-gated potassium channels. Zebrafish (Danio rerio) have been proposed as a cost effective, high throughput drug-screening model to identify compounds that cause hERG1a dysfunction. However, there are no reports on the effects of hERG1a activator compounds in zebrafish, and consequently on the utility of the model to screen for potential gain-of-function therapeutics. Here, we examined the effects of hERG1a blocker, and type 1 and type 2 activator, compounds on isolated zkcnh6a (zERG3) channels in the Xenopus laevis oocyte heterologous expression system, as well as action potentials recorded from ex vivo adult zebrafish whole hearts using optical mapping. Our functional data from isolated zkcnh6a channels show that these channels respond to hERG1a channel blockers (dofetilide and terfenadine), and type 1 (RPR260243) and type 2 (NS1643, PD-118057) hERG1a activator compounds, in a similar manner to hKCNH2a channels, with minor differences largely accounted for by subtly different biophysical properties in the two channels. In ex vivo zebrafish whole hearts, two of the three hERG1a activators examined caused abbreviation of the APD, while hERG1a blockers caused APD prolongation. These data represent, to our knowledge, the first pharmacological characterization of isolated zkcnh6a channels and the first assessment of hERG enhancing therapeutics in zebrafish. Our findings suggest that the zebrafish ex vivo whole heart model serves as a valuable tool in the screening of hKCNH2a blocker and activator compounds

Physiological phenotyping of the adult zebrafish heart

The zebrafish has proven to be an excellent organism for manipulation of its genome from a long history of transcript down-regulation using morpholino oligimers to more recent genome editing tools such as CRISPR-Cas9. Early forward and reverse genetic screens significantly benefited from the transparency of zebrafish embryos, allowing cardiac development as a function of genetics to be directly observed. However, gradual loss of transparency with subsequent maturation limited many of these approaches to the first several days post-fertilization. As many genes are developmentally regulated, the immature phenotype is not entirely indicative of that of the mature zebrafish. For accurate phenotyping, subsequent developmental stages including full maturation must also be considered. In adult zebrafish, cardiac function can now be studied in great detail due both to the size of the hearts as well as recent technological improvements. Because of their small size, zebrafish are particularly amenable to high frequency echocardiography for detailed functional recordings. Although relatively small, the hearts are easily excised and contractile parameters can be measured from whole hearts, heart slices, individual cardiomyocytes and even single myofibrils. Similarly, electrical activity can also be measured using a variety of techniques, including in vivo and ex vivo electrocardiograms, optical mapping and traditional microelectrode techniques. In this report, the major advantages and technical considerations of these physiological tools are discussed.The grant support to GFT from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canada Foundation for Innovation and the Canada Research Chairs programs are gratefully acknowledged

Effects of the mutant L29Q cTnC and SD cTnI determined by Molecular Dynamics simulations.

<p>(A) MDS-based model for the interaction of the cardiac specific N-terminal extension of cTnI peptide1–32 (green; PDB: 2JPW) with the pseudo-phosphorylated Asp23/24 indicated. The N-terminus of L29Q cTnC is shown in red and cTnI switch peptide147–163 is shown in blue (PDB: 1MXL) with the L29Q mutation indicated. Electrostatic surface of the N-terminus of L29Q cTnC was generated in pyMOL using APBS with 50% transparency (red, negatively charged; blue, positively charged; white, neutral). This is at t = 100 ns of simulation. (B) WT cNTnC (white) is superimposed with L29Q cNTnC (red) with its cTnI switch peptide bound to its central hydrophobic cavity at t = 100 ns simulation. (cyan: cTnI switch peptide bound within WT cNTnC; dark blue: cTnI switch peptide bound within L29Q cNTnC). The pseudo-phosphorylated cTnI is omitted from this snapshot for clarity. The Ca²⁺ ion bound in site II is shown as a black sphere. (C) shows the largest movement caused by the L29Q mutation in the presence of the pseudo-phosphorylated cTnI, which is the re-orientation of helix A and helix B of cNTnC accompanied by a decrease of interhelical angle of 13°.</p