Hook, line and sinker: adult zebrafish offer a valid model to study mammalian cardiac contractile mechanics

On a beat-to-beat basis, cardiac contractile function is driven by a multifaceted and dynamic process that is regulated by both intrinsic (e.g. mechanical loading) and extrinsic (e.g. neuro-hormonal) factors (de Tombe et al. 2010). Specifically, the level of contractile activation of adult mammalian cardiomyocytes is modulated by the magnitude of the Ca²⁺ transient, the dynamic activation–relaxation kinetic response of the sarcomere to activator Ca²⁺ and the responsiveness of the myofilament to Ca²⁺, the last of which is dependent on sarcomere length and is a primary mediator of the Frank–Starling response. While transgenic mouse models have provided important insight into the molecular mechanisms underlying cardiac contractile function in health and disease, they are associated with high cost and relatively long generation times. Recently, the zebrafish has emerged as a promising model for the study of cardiac structure–function relationships, due to short generation times, ease of genetic manipulation and low cost. While multiple studies have characterized zebrafish cardiac electrophysiology, Ca²⁺ dynamics and myofilament mechanical function, it remains unclear whether the cardiac contractile structure–function relationship of adult zebrafish is comparable to that of the mammalian system.

In a recent issue of The Journal of Physiology, Dvornikov et al. (2014) sought to determine whether adult zebrafish cardiac contractile physiology and structure–function relationship are comparable to that of the mammalian system. Using a novel carbon rod-based approach to study the contractile function of single, enzymatically isolated zebrafish ventricular myocytes, Dvornikov et al. (2014) used a sequential approach to characterize cardiac contractile mechanics. Specifically, the following two modes of contraction were studied to characterize the extrinsic and intrinsic factors regulating force production: (i) electrically stimulated twitches in mechanically loaded membrane-intact myocytes; and (ii) Ca²⁺-activated contractions in chemically permeabilized cells. The authors demonstrated that in the absence of change in intracellular Ca²⁺, twitch force increased in response to sarcomere stretch, with maximal Ca²⁺-saturated force increasing with sarcomere length. Furthermore, contractility was shown to respond to increases in extracellular Ca²⁺ concentrations in a dose-dependent manner. In chemically permeabilized cells, steady-state force development increased sigmoidally as a function of activating [Ca²⁺]. These findings suggest that the Frank–Starling response and its cellular correlate, myofilament length-dependent activation, are an intrinsic property and critical mediator of contractile function of both mammalian and zebrafish cardiomyocytes. Using a rapid solution-switching technique, sarcomere Ca²⁺ activation–relaxation and force redevelopment kinetics were examined to determine the dynamic response of the sarcomere to activator Ca²⁺. It was revealed that the kinetic characteristics of single zebrafish myofibrils were similar to those of mammalian myofibrils. Taken together, these findings provide evidence that the regulation of contractile function and structure–function relationships in the zebrafish heart are similar to those of the mammalian heart, suggesting that the zebrafish represents a valid model system for the study of mammalian cardiac contractile mechanics.

The use of novel carbon fibre-based approaches, as used by Dvornikov et al. (2014), has allowed for unprecedented study of active and passive cardiomyocyte force–length and force–Ca²⁺ relationships. Isolated intact cardiomyocytes have the benefit of being independent of connective tissue and endothelium, allowing for the isolation of contractile properties and assessment of subcellular mechanisms to a greater degree compared with intact tissue preparations. The sequential approach used in the study by Dvornikov et al. (2014) allowed the authors to comprehensively assess zebrafish ventricular cardiomyocyte contractile physiology and the regulation of twitch force production. As force development is intimately dependent on the magnitude of the Ca²⁺ transient and sarcomere length, the finding of twitch force modulation by extracellular [Ca²⁺] and sarcomere length in the isolated intact zebrafish myocyte is important for model validation and future translational application. Specifically, the robust myofilament length-dependent activation exhibited by zebrafish ventricular myocytes suggests that this model may help to identify the cellular basis of this phenomenon. Mammalian myofilament length-dependent activation has been extensively studied, but the mechanisms responsible remain unclear, with multiple integrated pathways, including interfilament lattice spacing and post-translational modification of sarcomeric proteins, thought to contribute. Recent work suggests that the length dependence of thin filament Ca²⁺ co-operativity, which reflects the increasing steepness of force development as a function of [Ca²⁺], may contribute to myofilament length-dependent activation in mammalian cardiomyocytes (Farman et al. 2010). However, the absence of this finding in the study by Dvornikov et al. (2014) and in previous work in rodents (Dobesh et al. 2002) highlights the unresolved complexity of the mechanisms underlying this phenomenon. Notwithstanding, the relative ease of genetic manipulation in zebrafish and the conservation of the structure of critical domains of several sarcomeric proteins suggest that the zebrafish will be invaluable for future studies assessing the molecular basis of length-dependent activation and resolving the disparate findings that exist between studies.

While the study by Dvornikov et al. (2014) focused on the characterization of zebrafish ventricular cardiomyocyte contractile function, the integration of isolated cardiomyocytes and whole-heart function in this model system may be limited. The intrinsic mechanical properties of isolated cardiomyocytes are directly responsible for many of the active and passive (e.g. contraction and relaxation) properties of the whole heart. Arguments against the utility of the zebrafish as a model to study human-related health and disease include its ectothermal regulation of body temperature and two-chamber heart morphology. Furthermore, the absence of the t-tubular system and limited contribution of the sarcoplasmic reticulum to Ca²⁺ homeostasis in ventricular cardiomyocytes (Verkerk & Remme, 2012), both of which are key components of excitation–contraction coupling in mammalian cardiomyocytes, suggest that significant mechanistic differences exist between mammalian and zebrafish cardiomyocytes in the control of cytosolic [Ca²⁺] and cardiac contractility. This is further supported by the absence of the slow force response in the study by Dvornikov et al. (2014), which, in addition to the Frank–Starling response, is an important regulator of cardiac contractility. Ultimately, these findings may preclude the use of zebrafish myocardium for integrative assessments of contractile mechanics and cardiac reserve, the latter of which is important to characterize cardiomyocyte responsiveness to physiological and pathological stress stimuli. The zebrafish may therefore be better suited to the study of isolated components of excitation–contraction coupling and the contractile apparatus, with the study by Dvornikov et al. (2014) providing support for this assertion.

Although the focus of the study by Dvornikov et al. (2014) is the validation of the zebrafish as a model for mammalian contractile mechanics, the zebrafish has considerable value as a model for several areas of cardiovascular interest. The similarities between mammalian and zebrafish action potential properties in ventricular cardiomyocytes have made the zebrafish an ideal model for the study of ion channel mutations and the functional effects of electrophysiological abnormalities (Brette et al. 2008). Furthermore, the closed-loop zebrafish cardiovascular system makes for a useful model for cardiovascular developmental biology because it includes similar steps to mammals, with early progenitor cell specification, differentiation and migration, followed by formation of the myocardium and valves and development of the conduction system and pacemaker. Abnormal development may also be modelled, allowing for the study of congenital heart disease, including structural and electrophysiological defects. The majority of congenital heart diseases have a genetic basis, but the role of genetic regulation in cardiovascular developmental disease remains incompletely understood. As the zebrafish genome has been sequenced fully and is publicly available, it is commonly used for both forward and reverse genetic screens, contributing to the identification of candidate genes and single nucleotide polymorphisms (Arnaout et al. 2007) which may underlie cardiovascular developmental abnormalities. However, the aforementioned two-chamber physiology as well as a lack of pulmonary circulation limits the ability of zebrafish to model integrated cardiovascular development completely.

The zebrafish model has also been used to examine molecular mechanisms underlying the pathological progression of numerous human cardiac diseases, most notably intrinsic disorders of the myocardium, including cardiomyopathies. Intrinsic cardiomyopathies are caused by familial and spontaneous mutations in sarcomeric contractile proteins, ion channels, signalling proteins, etc. It should be noted, however, that mutations in sarcomeric contractile proteins are the most prominent cause of intrinsic cardiomyopathies. These mutations disrupt normal sarcomeric protein organization, cardiomyocyte stretch sensing and excitation–contraction coupling, ultimately leading to progressive decompensation of cardiac contractile function as well as enhanced susceptibility to malignant cardiac arrhythmias. The use of transgenic zebrafish models therefore allows for the manipulation of molecular components of the contractile apparatus and contributes to a better understanding of the genetic factors and molecular modifications that underlie human cardiac disease. For example, mybpc3 gene knockdown in the zebrafish was shown to result in structural, functional and electrophysiological abnormalities characteristic of hypertrophic cardiomyopathy and has been used to study the susceptibility of the hypertrophied heart to Ca²⁺ and voltage alternans (Chen et al. 2013). Ultimately, this will allow for the development of novel therapies, including targeted gene delivery, which may eventually complement or replace current pharmacotherapies. In this context, the zebrafish may prove to be a useful model.

In conclusion, Dvornikov et al. (2014) have used a novel carbon fibre-based approach to show that the zebrafish offers a valid model system for the study of mammalian cardiac contractile physiology and cellular structure–function relationships. The zebrafish offers a promising and advantageous model that will contribute significantly to knowledge about the molecular mechanisms of mammalian cardiac contractile regulation, offering insight into the functional consequences of genetic manipulation and therapeutic strategies in human health and disease.

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Competing interests

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