Cardiomyopathy is a disease of myocardium categorized into three major forms, hypertrophic (HCM), dilated (DCM) and restrictive cardiomyopathy (RCM), which has recently been demonstrated to be a monogenic disease due to mutations in various proteins expressed in cardiomyocytes. Mutations in HCM and RCM typically increase the myofilament sensitivity to cytoplasmic Ca²⁺, leading to systolic hyperfunction and diastolic dysfunction. In contrast, mutations in DCM typically decrease the myofilament sensitivity to cytoplasmic Ca²⁺ and/or force generation/transmission, leading to systolic dysfunction. Creation of genetically-manipulated transgenic and knock-in animals expressing mutant proteins exogenously and endogenously, respectively, in their hearts provides valuable animal models to discover the molecular and cellular mechanisms for pathogenesis and promising therapeutic strategy in vivo. Recently, cardiomyocytes have been differentiated from patient’s induced pluripotent stem cells as a model of inherited cardiomyopathies in vitro. In this review, we provide overview of experimental models of cardiomyopathies with a focus on revealed molecular and cellular pathogenic mechanisms and potential therapeutics.

Cardiomyopathies are categorized, based on ventricular morphology and function, into three major forms, hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), restrictive cardiomyopathy (RCM)[1]. HCM is characterized by increased left ventricular (LV) wall thickness, cardiomyocyte disarray, increased myocardial fibrosis and impaired LV diastolic function with normal or increased LV systolic function[2-4]. DCM is characterized by LV dilatation and systolic dysfunction, frequently resulting in heart failure, arrhythmias and sudden death, with heart transplantation being the most effective treatment for survival at end stage because of no effective therapeutic drugs[5]. RCM is an uncommon form of cardiomyopathy, characterized by restrictive filling of LV and/or right ventricle despite normal or near-normal wall thickness and systolic function[6,7].

Following the uncovering of a gene mutation in β-myosin heavy chain (β-MyHC) of familial HCM patients at 1990[8], a large number of mutations in the genes encoding sarcomere proteins in cardiac muscle have been found to cause HCM, DCM and RCM[9]. Many animal models have been created to discover the functional consequences of these mutations and molecular mechanisms for the pathogenesis of cardiomyopathies in vivo, which should be critical for advancement of diagnosis and therapy. Recently, premature cardiomyocytes have been created from induced pluripotent stem cells (iPSC) of patients with inherited cardiomyopathies as a novel disease model in vitro. This review summarizes the recent advances in our understanding about molecular pathogenic mechanisms and potential therapeutic strategy brought about from these experimental models.

HCM, characterized by unexplained LV wall thickening and diastolic dysfunction, has an overall prevalence of 200 per 100000 individuals[10]. It is known that LV systolic function is not impaired but rather increased in HCM patients[2]. Structural remodeling involving hypertrophic growth of LV is believed to be caused by enhanced protein synthesis in cardiomyocytes leading to hyperplasia of myofibrils and thus cardiomyocyte enlargement. The purpose of current therapy for HCM is to improve diastolic dysfunction indirectly through suppressing systolic function using β-blockers, Ca²⁺ channel blockers or Na⁺ channel blockers[11-13].

Human HCM is a monogenic disorder, which is caused by several hundred distinct mutations in many genes found in patients and families with HCM[14,15]. The causal genes for HCM include those encoding cardiac myosin-binding protein C (MYBPC3), β-MyHC (MYH7), cardiac troponin C (TNNC1), cardiac troponin I (TNNI3), cardiac troponin T (TNNT2), cardiac actin (ACTC), α-tropomyosin (TPM1), regulatory myosin light chain, essential myosin light chain and titin/connectin. Mutations in these genes account for approximately 65% of all HCM cases[16], indicating that HCM is a disease of sarcomeric protein genes. The total number of mutations in each genes increase depending on the gene size, so that any one of mutations in two large genes encoding MYH7 and MYBPC3 are identified in about 50% of cases while mutations in other genes only account for less than 20% of cases[16].

Soon after discovery of these mutations in sarcomeric proteins, extensive studies have been started to understand the pathogenic mechanisms by exploring the effects of mutations on the in vitro sarcomeric function as well as the in vivo global structure and function of the heart using genetically modified animal models. In vitro studies revealed that HCM-linked mutations in thin filament-associated regulatory proteins, including TNNT2, consistently increase the myofilament sensitivity to cytoplasmic Ca²⁺ and thus probably impair diastolic function through a malfunction in the troponin-tropomyosin regulatory system[17-26]. Animal models of human HCM with mutations in cardiac troponin T[17,19,20,22,24], TNNI3[21,23] and TPM1[18,25,26] demonstrated that increased cardiac myofilament Ca²⁺ sensitivity is a root cause that initiates molecular cascades involving pathological cardiac remodeling in HCM. These findings indicate that reversal of the increased myofilament Ca²⁺ sensitivity toward normal levels is a promising definitive therapeutic strategy for HCM. At present, however, there exists no drugs that decrease the myofilament Ca²⁺ sensitivity through directly acting on the thin filament regulatory system, making it worthwhile to develop novel drugs “Ca²⁺ desensitizers”. Epigallocatechin gallate, a major polyphenol in green tea, is a potential lead compound for Ca²⁺ desensitizers, which has been demonstrated to decrease the myofilament Ca²⁺ sensitivity in membrane-permeabilized cardiac muscle fibers through binding to a C-terminal lobe region of TNNC1[27]. Poor absorption from the intestine and permeability into cells, however, may be serious problems to be solved. Another potential lead compound is blebbistatin, which has also been demonstrated to decrease the myofilament Ca²⁺ sensitivity in membrane-permeabilized cardiac muscle fibers through inhibiting the interaction between actin and myosin and prevent arrhythmia induced by Ca²⁺ sensitizer[28]. Crossing transgenic mice harboring HCM-linked sarcomeric mutation with transgenic mice harboring DCM-linked sarcomeric mutation conferring decreased myofilament Ca²⁺ sensitivity was found to normalize overall myofilament Ca²⁺ sensitivity and prevent cardiac deterioration[29,30], supporting the idea that Ca²⁺ desensitizer might be beneficial for HCM patients affected by mutations in sarcomeric protein genes.

HCM-causing mutations that increase the myofilament sensitivity to cytoplasmic Ca²⁺ also alter the regulation of intracellular Ca²⁺ level, which could activate hypertrophic response and failure in the myocardium[31]. Cardiomyocytes isolated from experimental mouse models of HCM show abnormal intracellular Ca²⁺ handling, including increased diastolic Ca²⁺ associated with decreased Ca²⁺ store in the sarcoplasmic reticulum (SR), and dysregulation of intracellular Ca²⁺ precede hypertrophic remodeling of the heart[32,33]. The voltage-dependent L-type Ca²⁺ channel inhibitor, diltiazem, restored the normal intracellular Ca²⁺ handling and suppressed cardiac hypertrophy in young mice with HCM-causing myosin R403Q mutation[33], indicating that pharmacologic interventions targeting early key intracellular events caused by abnormal intracellular Ca²⁺ regulation could prevent disease development.

DCM is characterized by progressive LV dilatation and systolic dysfunction, being the most common indication for cardiac transplantation[5]. Many mutations in various genes encoding sarcomeric proteins, cytoskeletal proteins, nuclear envelope proteins and sarcolemmal membrane proteins have been shown to be linked to approximately 25%-30% of the DCM cases[34-39]. Cardiomyocyte hypertrophy and fibrosis, but not cardiomyocyte disarray, are commonly observed as in the case of HCM[36]. DCM is frequently accompanying with abnormal cardiac conduction system, arrhythmias and sudden death probably due to pathophysiological myocardial remodeling and severe fibrosis. Underlying molecular mechanisms include diminished force generation/transmission, altered energy metabolism, and impaired intracellular calcium handling in cardiomyocytes[3]. The purpose of current standard therapy for DCM is to prevent the progression of myocardial remodeling and systolic dysfunction by a combination of cardioprotective drugs, including β-adrenergic receptor blockers, vasodilators (angiotensin converting enzyme inhibitors or angiotensin II receptor blockers), aldosterone antagonists and diuretics[40].

In contrast to HCM-causing mutations, DCM-causing mutations in TPM1[41] and TNNT2 consistently decrease the myofilament sensitivity to cytoplasmic Ca²⁺ and thus impair systolic function through a malfunction in the troponin-tropomyosin regulatory system[42,43]. A mouse model of DCM caused by the deletion mutation ΔK210 in TNNT2 demonstrated that lessened cardiac myofilament Ca²⁺ sensitivity is a root cause that initiates molecular cascades involving pathological cardiac remodeling in DCM[44]. This mouse model developed an early-onset severe LV dilation with high incidence of sudden death despite showing no heart failure symptoms, resembling the phenotypes of a human family of DCM patients with this mutation[35]. These findings indicate that reversal of the decreased myofilament Ca²⁺ sensitivity toward normal levels is a promising definitive therapeutic strategy for DCM linked to sarcomeric regulatory protein gene mutations. Early intervention with a Ca²⁺ sensitizer, pimobendan, had remarkable effects of preventing cardiac remodeling, systolic dysfunction and sudden death in this DCM model mouse[44]. However, it remains to be determined whether pimobendan has also therapeutic effects on DCM mice with this mutation after developing decompensated, end-stage heart failure. It may be worth noting that combination therapy with pimobendan and β-blocker has provided beneficial effects in DCM patients with severe heart failure[45,46].

Cardiomyocyte contraction is evoked by Ca²⁺, which is rapidly released into cytoplasm from SR upon sarcolemmal depolarization. Cytoplasmic Ca²⁺ is rapidly returned to a low level during diastole by reuptake into SR through SR Ca²⁺pump (SERCA2a). Myocardial expression of SERCA2a is down-regulated in the patients with end-stage congestive heart failure[47,48], resulting in a decrease in the rate of Ca²⁺ reuptake by SR[49-51]. Myocardial expression of SERCA2a was also confirmed to be markedly decreased in a mouse model of DCM[52]. In a pressure-overload heart failure model of rats, transfection of adenovirus expression vector carrying SERCA2a cDNA into the heart normalized the hemodynamic parameters, including LV end-systolic pressure, maximum rates of LV pressure increase and decrease, and isovolumic relaxation rate[53]. Another study using a pressure-overload model of rats demonstrated that adenoviral transfection of SERCA2a during heart failure reversed the LV dilation and improved the myocardial energy metabolism and survival[54]. SERCA2a gene transfer also improved the contractile function of cardiomyocytes taken from patients with heart failure by increasing the rates of contraction and relaxation, decreasing and increasing the cytoplasmic Ca²⁺ at diastole and systole, respectively, and normalizing the frequency dependence of force generation[55]. Taken together, these studies suggest that enhancement of SERCA2a expression in cardiomyocytes may serve as potential therapeutic strategy for DCM patients.

RCM is characterized by increased stiffness of ventricular chambers, with wall thickness and systolic function usually being within normal limits. The reduction in myocardial compliance results in an abnormally large increase in early diastolic ventricular pressure against small increment in volume and an abrupt termination of filling. Most individuals with RCM develop heart failure and die within a few years[56]. Several reports suggest clinical and genetic overlaps between RCM and HCM[56-58]. RCM is rare, and its genetic etiology has just started to be explored. To date, RCM-linked mutations are found in sarcomere protein genes, including TNNI3, TNNT2, MYH7 and ACTC[58-61].

Like sarcomeric gene mutations in other types of cardiomyopathy, RCM-causing sarcomeric gene mutations alter myofilament sensitivity to cytoplasmic Ca²⁺ through a malfunction in the troponin-tropomyosin regulatory system. Membrane-permeabilized cardiac muscle fibers prepared from transgenic mouse model of RCM are more sensitive to Ca²⁺ and show more force at low Ca²⁺ levels than those from transgenic mice overexpressing wild-type proteins[62]. This is consistent with the findings from earlier in vitro studies in which recombinant RCM-causing mutant proteins are exchanged into membrane-permeabilized cardiac muscle fibers[63-65]. Kobayashi et al[66] demonstrated that the increase in myofilament Ca²⁺ sensitivity was caused by increased affinity of troponin C for Ca²⁺ in the thin filament. Thus, the myofilament hypersensitivity to cytoplasmic Ca²⁺ is a common feature that RCM-causing mutations share with HCM-causing mutations. In vitro experiments using membrane-permeabilized cardiac muscle fibers reconstituted with recombinant mutant proteins revealed that RCM-causing mutations give much greater Ca²⁺ sensitivity to the myofilament compared with HCM-causing mutations[62,63]. Consistent with these in vitro reconstitution experiments, membrane-permeabilized cardiac muscle fibers prepared from transgenic mice expressing RCM-causing TNNI3 R145W mutant showed a much larger increase in the Ca²⁺ sensitivity of ATPase activity and force generation compared with those from transgenic mice expressing HCM-causing TNNI3 R145G mutant[62,67]. Crossing transgenic mice expressing RCM-causing TNNI3 R193H mutant with transgenic mice expressing N-terminal truncated TNNI3, known to decrease myofilament Ca²⁺ sensitivity, corrected the impaired relaxation in R193H RCM transgenic mice[68], supporting the idea that myofilament Ca²⁺ desensitizer could also be beneficial to treat RCM caused by sarcomeric protein gene mutations. Design of new compounds that exert lusitropic action on the heart directly through decreasing the myofilament Ca²⁺ sensitivity is an innovative and exciting challenge to overcome RCM as well as HCM.

Although the contribution of gene-manipulated animal models to the understanding of inherited cardiomyopathies in in vivo system has been enormous, small animals have significantly different intrinsic properties in the heart from human, including faster heart rate, shorter plateau phase in the action potential of ventricles, and much higher ratio of α/β-MyHC isoforms in ventricles. Intact cardiomyocytes are difficult to obtain from healthy parson and even from cardiomyopathy patients. The iPSC technology may offer a unique opportunity for creating disease-specific models directly from human patients with monogenic disease to investigate underlying mechanisms and carry out drug screening in human cardiomyocytes, though only in vitro[69,70]. Premature but self-beating cells like cardiomyocytes have been shown to be differentiated from human iPSC[71,72]. Patient-specific iPSC-derived cardiomyocytes have been created for HCM-causing missense mutation R663H in MYH7[73]. These iPSC-derived cardiomyocytes developed cellular hypertrophy and arrhythmia at the single cell level accompanying irregular Ca²⁺ cycling and elevation in resting cytoplasmic Ca²⁺ level. Further, pharmacological inhibition of Ca²⁺ entry with L-type Ca²⁺ channel blockers verapamil, nifedipine and diltiazem prevented development of cellular hypertrophy and electrophysiological abnormality. It is somewhat surprising that these numerous aspects of HCM phenotype can be reproduced in an in vitro cultured system without any neurohormornal stimulation, since these phenotypes are thought to develop as a long-term consequence of adaptation or compensation in vivo to an abnormal contractile function conferred by the mutation in a motor protein encoded in MYH7. The results of this study on patient-specific iPSC-derived cardiomyocytes, however, clearly show that iPSC-derived cardiomyocytes are a useful platform to elucidate molecular and cellular pathogenic mechanisms underlying inherited HCM and to identify novel therapies for this disease.

iPSC-derived cardiomyocytes from a three-generation family of DCM patients affected by a missense mutation R173W in TNNT2 have been shown to exhibit a lessened force generation capability, one of the common root causes for DCM, with impaired Ca²⁺ handling and abnormal distribution of Z-band α-actinin but no abnormalities in electrophysiological properties and cell size[74]. β1-selective adrenergic receptor blocker metoprolol improved the sarcomeric disorganization judged by α-actinin distribution, and over-expression of SERCA2a improved contractile function and Ca²⁺ handling. These findings demonstrated that cardiomyocytes differentiated from iPSCs of DCM patients recapitulated the disease phenotype to some extent and could be used as an in vitro experimental model to explore molecular and cellular pathogenic mechanisms underlying inherited DCM and to carry out drug screening for this disease.

Abnormal sensitivity to cytoplasmic Ca²⁺ or force generation/transmission of cardiac myofilament, which is incurred as a direct functional consequence of mutations in genes encoding proteins in cardiomyocytes, is the primary root cause that initiates subsequent molecular and cellular events leading to pathological remodeling in inherited cardiomyopathies. HCM/RCM-causing mutations usually heighten the myofilament sensitivity to cytoplasmic Ca²⁺ or force generation, whereas DCM-causing mutations lessen the myofilament sensitivity to cytoplasmic Ca²⁺ or force generation/transmission. Therefore, reversal of the altered myofilament Ca²⁺ sensitivity or force generation/transmission capability toward normal levels should be a promising definitive therapeutic strategy to prevent or even reverse the progression of the disease in inherited cardiomyopathies (Figure 1). Further studies using gene-manipulated animal models and patient’s iPSC-derived cardiomyocytes briefly summarized in this review are important to develop novel therapeutic drugs for inherited cardiomyopathy patients.

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Figure 1 Essentials of pathogenic mechanisms in inherited cardiomyopathies and potential definitive drug therapies. HCM: Hypertrophic cardiomyopathy; DCM: Dilated cardiomyopathy; RCM: Restrictive cardiomyopathy.