Multiscale Dissection of HCM Phenotype: From iPSC-Derived Cardiomyocytes to Organ-Level Insights

Introduction: Hypertrophic cardiomyopathy (HCM) is a genetic disorder characterized by excessive cardiac myocyte contractility, resulting in hypertrophy and myocardial fibrosis that can culminate in heart failure and sudden death. Central to the pathology is the alteration of cardiomyocyte contractile mechanics, particularly how myosin's super-relaxed (SRX) and disordered-relaxed (DRX) states contribute to disease manifestation. The novel drug Mavacamten, which targets myosin's function, offers a therapeutic approach to dampen the hypercontractility underlying HCM.

Our study leverages induced pluripotent stem cell (iPSC) models and intricate computational simulations to dissect the effects of the H251N β-myosin mutation, representative of HCM pathology. We explore the mutation's repercussions from molecular changes to cellular mechanics, and integrate insights into patients' organ-scale cardiac structure and function. This correlation aims to connect subcellular molecular dynamics to large-scale organ remodeling and clinical presentations, reinforcing the potential of precision medicine in managing HCM.

With meticulous cross-validation between in vitro assessments and clinical outcomes, specifically in patients who have undergone procedures such as alcohol septal ablation, our approach seeks to bridge the gap between laboratory findings and patient experiences. This study promises to enhance the understanding of HCM's complexity and bolster Mavacamten's role in personalized treatment strategies.

Materials and

Methods: To model hypertrophic cardiomyopathy (HCM) at the cellular level, we harnessed iPSC-derived cardiomyocytes with the H251N β-myosin mutation. We probed their biomechanical properties and sarcomere structure robustly using traction force microscopy (TFM), tailoring substrate stiffness to emulate the biomechanical environment from healthy to fibrotic myocardium. Further, Mant-ATP assays allowed us to delve into the distribution between myosin's super-relaxed (SRX) and disordered-relaxed (DRX) states, an essential aspect of HCM's cellular physiology.

Our study ambitiously spans from these foundational cellular insights to an organ-level understanding through patient-specific computational modeling. Integrating data from late gadolinium-enhanced magnetic resonance imaging (LGE MRI), we gleaned a temporal view of myocardial structure, pre and post-intervention from HCM patients who underwent alcohol septal ablation. This longitudinal perspective revealed the evolution of cardiac fibrosis and hypertrophy, providing snapshots of myocardial architecture across crucial post-procedural milestones at one and six months.

Linking single-cell analyses with clinical imaging marks a significant leap toward personalized medicine, offering a detailed, patient-tailored map of HCM progression. The juxtaposition of in-vitro cellular behavior against the backdrop of patient-specific, in-vivo cardiac remodeling underscores the promise of targeted therapies in correcting the maladaptive responses observed in HCM. Our multi-tiered approach fosters a comprehensive understanding pivotal for innovative interventions.

Results, Conclusions, and Discussions: Our traction force microscopy investigations into iPSC-derived cardiomyocytes with the H251N β-myosin mutation uncovered decreased relaxation velocities reminiscent of the diastolic dysfunction seen in hypertrophic cardiomyopathy (HCM). Subsequent treatment of these mutant cells with Mavacamten led to a significant decrease in contractile force, highlighting the drug's potential to temper the hypercontractility characteristic of HCM.

Further examination using Mant-ATP assays showed that untreated cardiomyocytes exhibited a 38% basal occupancy in the super-relaxed (SRX) state, which increased to 53% following Mavacamten administration. The H251N mutation altered this drug response, with SRX state occupancy rocketing from 24% to 66%. This pronounced shift towards the SRX state suggests that Mavacamten may effectively mitigate the hypercontractile HCM phenotype by promoting an energy-conserving configuration of myosin.

Morphological analyses of patient hearts (n=3) using our segmentation protocols affirmed notable hypertrophy and fibrosis. By simulating cardiac contraction mechanics and integrating these data into kinetic crossbridge models of the H251N mutation, we aim to align these simulations with the actual cellular forces observed. These refined models will eventually be incorporated into a comprehensive organ-scale computational framework, allowing us to predict the impact of Mavacamten on cardiac function in a patient-specific context.

Expectations are that our multiscale models, augmented with clinical data, will substantiate Mavacamten's therapeutic potential to ameliorate the structural and mechanical distortions in HCM. Our cellular data not only validate the drug's mechanism as perturbative to unhealthy cardiomyocyte mechanoenergetics but also as a facilitator of myocardial stress relief, thus reducing fibrotic remodeling.

Future research will involve engineered heart tissue constructs to delve deeper into the effects of the H251N mutation and Mavacamten on cardiomyocytes and fibroblasts together, unraveling the multifactorial mechanisms of fibrosis. We also plan to extend our in vitro and in silico investigations to additional myosin mutations. These endeavors will better elucidate the heterogeneity underlying HCM's pathophysiology and could pioneer precise, mutation-targeted therapies—a testament to the transformative potential of multiscale analysis in precision medicine.

Acknowledgements (Optional): NIH NHLBI K99/R00 HL153679 Pathway to Independence
NIH NIGMS RM1 GM131981 Early Stage Investigator Development