MLP-deficient human pluripotent stem cell derived cardiomyocytes develop hypertrophic cardiomyopathy and heart failure phenotypes due to abnormal calcium handling

Abstract

Muscle LIM protein (MLP, CSRP3 ) is a key regulator of striated muscle function, and its mutations can lead to both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, due to lack of human models, mechanisms underlining the pathogenesis of MLP defects remain unclear. In this study, we generated a knockout MLP/CSRP3 human embryonic stem cell (hESC) H9 cell line using CRISPR/Cas9 mediated gene disruption. CSRP3 disruption had no impact on the cardiac differentiation of H9 cells and led to confirmed MLP deficiency in hESC-derived cardiomyocytes (ESC-CMs). MLP-deficient hESC-CMs were found to develop phenotypic features of HCM early after differentiation, such as enlarged cell size, multinucleation, and disorganized sarcomeric ultrastructure. Cellular phenotypes of MLP-deficient hESC-CMs subsequently progressed to mimic heart failure (HF) by 30 days post differentiation, including exhibiting mitochondrial damage, increased ROS generation, and impaired Ca 2+ handling. Pharmaceutical treatment with beta agonist, such as isoproterenol, was found to accelerate the manifestation of HCM and HF, consistent with transgenic animal models of MLP deficiency. Furthermore, restoration of Ca 2+ homeostasis by verapamil prevented the development of HCM and HF phenotypes, suggesting that elevated intracellular Ca 2+ concentration is a central mechanism for pathogenesis of MLP deficiency. In summary, MLP-deficient hESC-CMs recapitulate the pathogenesis of HCM and its progression toward HF, providing an important human model for investigation of CSRP3/MLP-associated disease pathogenesis. More importantly, correction of the autonomous dysfunction of Ca 2+ handling was found to be an effective method for treating the in vitro development of cardiomyopathy disease phenotype.

RAD-Deficient Human Cardiomyocytes Develop Hypertrophic Cardiomyopathy Phenotypes Due to Calcium Dysregulation

“…Firstly, maturity is critical for studying the function of hESC-CMs. Genetically modified hPSCs have been commonly used to study the gene function in cardiomyocytes, indicating that these cells are excellent models for cardiovascular research (Lan et al, 2013;Fogarty et al, 2017;Mosqueira et al, 2018;Li et al, 2019). In this study, we found that RRAD −/− cardiomyocytes were more prone to hypertrophic phenotype, whereas, WT cardiomyocytes were also exhibited a certain phenotype such as low frequencies of myofibrillar disarray, multi-nucleation under the same culture conditions.…”

“…Results as shown in Figure 3C, although "circadian entrainment" and "axon guidance" rank first and second in GO analysis of 15 significantly different pathways, they were involved in circadian rhythm control of organism (Golombek and Rosenstein, 2010) and neurological disorders (Van Battum et al, 2015), respectively. We focused on the third "calcium signal pathway" for it is closely related to the physiological function of RRAD gene and it has been confirmed as the central mechanism of cardiac hypertrophy in previous studies (Lan et al, 2013;Dewenter et al, 2017;Li et al, 2019). Taken together, these findings suggested that RAD is involved in the regulation of biological processes and is closely related to the regulation of calcium ion function.…”

“…The protocol of both H9 and H9-RRAD −/− GCaMP reporter cell lines were generated as previously reported (Li et al, 2019).…”

“…Homeostasis of calcium activity is crucial for cardiac excitationcontraction (EC) coupling and electrophysiological properties, early disruption of which, however, can lead to various dysfunctions such as cardiac hypertrophy and arrhythmia (Bers, 2008;Monteiro da Rocha et al, 2016). To explore the possible effects of RAD deficiency on calcium regulation, we generated GCaMP reporter cells as previous reported (Li et al, 2019) and assessed calcium transient of cardiomyocytes in both WT and KO lines at days 20, 30, and 40 post-induction. Results as shown in Figures 4A,B, cardiomyocytes derived from KO line showed smaller amplitude and significantly altered calcium transient which may be associated with arrhythmia-like voltage waveforms compared to control cells.…”

“…It acts as a calcium channel regulator by interacting with cardiac L-type Ca 2+ channels (LTCC), which play a fundamental role in normal heart (Finlin et al, 2003). Previous evidence has shown that elevated intracellular Ca 2+ and abnormal calcium regulation are the central mechanism for inducing cardiac hypertrophy (Lan et al, 2013;Monteiro da Rocha et al, 2016;Dewenter et al, 2017;Li et al, 2019). Studies on mice indicated that deficiency of RAD function in cardiomyocytes, which can lead to an increased L-type Ca 2+ current (I Ca−L ) via upregulation of LTCC expression in the plasma membrane (Yada et al, 2007), significantly increased stress-induced cardiac hypertrophy and remodeling in vitro (Chang et al, 2007) and cardiac fibrosis in vivo (Zhang et al, 2011).…”