Muscle LIM Protein Force-Sensing Mediates Sarcomeric Biomechanical Signaling in Human Familial Hypertrophic Cardiomyopathy

Abstract

Background: Familial hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease and is typically caused by mutations in genes encoding sarcomeric proteins that regulate cardiac contractility. HCM manifestations include left ventricular hypertrophy and heart failure, arrythmias, and sudden cardiac death. How dysregulated sarcomeric force production is sensed and leads to pathological remodeling remains poorly understood in HCM, thereby inhibiting the efficient development of new therapeutics. Methods: Our discovery was based on insights from a severe phenotype of an individual with HCM and a second genetic alteration in a sarcomeric mechanosensing protein. We derived cardiomyocytes from patient-specific induced pluripotent stem cells (iPSC-CMs) and developed robust engineered heart tissues (EHTs) by seeding iPSC-CMs into a laser-cut scaffold possessing native cardiac fiber alignment in order to study human cardiac mechanobiology at both cellular and tissue levels. Coupled with computational modeling for muscle contraction and rescue of disease phenotype via gene editing and pharmacological interventions, we have identified a new mechanotransduction pathway in HCM, shown to be essential in modulating the phenotypic expression of HCM in five families bearing distinct sarcomeric mutations. Results: Enhanced actomyosin crossbridge formation caused by sarcomeric mutations in cardiac myosin heavy chain ( MYH7 ) led to increased force generation, which when coupled with slower twitch relaxation, destabilized the muscle LIM protein (MLP) stretch-sensing complex at the Z-disc. Subsequent reduction in the sarcomeric MLP level caused disinhibition of calcineurin-nuclear factor of activated T-cells (NFAT) signaling, which promoted cardiac hypertrophy. We demonstrate that the common MLP-W4R variant is an important modifier, exacerbating the phenotypic expression of HCM, but alone may not be a disease-causing mutation. By mitigating enhanced actomyosin crossbridge formation through either genetic or pharmacological means, we alleviated stress at the Z-disc, preventing the development of hypertrophy associated with sarcomeric mutations. Conclusions: Our studies have uncovered a novel biomechanical mechanism through which dysregulated sarcomeric force production is sensed and leads to pathological signaling, remodeling, and hypertrophic responses. Together, these establish the foundation for developing innovative mechanism-based treatments for HCM that stabilize the Z-disc MLP mechanosensory complex.

Myosin Heavy Chain Converter Domain Mutations Drive Early-Stage Changes in Extracellular Matrix Dynamics in Hypertrophic Cardiomyopathy

“…Genetic testing can be utilized for screening family members who may be at risk for developing HCM, however mutation type is not currently able to predict the risk of SCD or guide clinical management. However, genetic testing can be clinically important in those patients with greater than 1 sarcomeric gene mutation and HCM phenotype, as they may have more severe disease ( Kelly and Semsarian, 2009 ; Girolami et al, 2010 ; Maron et al, 2012 ; Weissler-Snir et al, 2017 ; Riaz et al, 2022 ). Girolami et al described a HCM patient with MYH7 R723C, E1455X, and MYBPC3 E165D who underwent cardiac transplantation at the age of 43.…”

Signaling network model of cardiomyocyte morphological changes in familial cardiomyopathy

“…To validate a potential drug combination predicted by the model, we conducted an experiment on recently reported patient-specific iPSC-derived cardiomyocytes (iPSC-CMs) harboring two heterozygous missense mutations, one in β-myosin heavy chain 7 (MYH7-R723C), which expressed in sarcomere of cardiomyocytes and the other one in muscle LIM protein (MLP) expressed on Z-disc of sarcomeres 65 . This patient with double mutant (MLP-W4R;MYH7-R723C) was diagnosed with HCM phenotype at the age of 1.3 years 65 and iPSC-derived CMs from this patient exhibited significantly elevated expression of HCM markers including ANF and BNP mRNA levels and enlarged cell area phenotype 65 . Among drug targets predicted by the model in the HCM context (Fig 5A), we selected combination inhibition of ERK1/2 and PI3K-AKT pathways for experimental validation for their efficacy as well as feasibility with existing compounds.…”

“…This patient with double mutant (MLP-W4R;MYH7-R723C) was diagnosed with HCM phenotype at the age of 1.3 years 65 and iPSC-derived CMs from this patient exhibited significantly elevated expression of HCM markers including ANF and BNP mRNA levels and enlarged cell area phenotype 65 Fig. 6a shows iPSC-CMs after all four treatments.…”

“…To validate a potential drug combination predicted by the model, we conducted an experiment on recently reported patient-specific iPSC-derived cardiomyocytes (iPSC-CMs) harboring two heterozygous missense mutations, one in β-myosin heavy chain 7 (MYH7-R723C), which expressed in sarcomere of cardiomyocytes and the other one in muscle LIM protein (MLP) expressed on Z-disc of sarcomeres 65 .…”

“…Detailed information of human iPSCs were reported 65 . Briefly, under Yale University Institutional Review Board's approval, MLP-W4R;MYH7-R723C iPSC lines were generated by infecting peripheral blood mononuclear cells (PBMC) with Sendai virus particles encoding human transcription factors of SOX2, OCT4, KLF4, and c-MYC.…”