Human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) allow investigations in a human cardiac model system, but disorganized mechanics and immaturity of hPSC-CMs on standard two-dimensional surfaces have been hurdles. Here, we developed a platform of micron-scale cardiac muscle bundles to control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips on two-dimensional elastomer substrates with far greater throughput than single cell methods. By defining geometry and workload in this reductionist platform, we show that myofibrillar alignment and auxotonic contractions at physiologic workload drive maturation of contractile function, calcium handling, and electrophysiology. Using transcriptomics, reporter hPSC-CMs, and quantitative immunofluorescence, these cardiac muscle bundles can be used to parse orthogonal cues in early development, including contractile force, calcium load, and metabolic signals. Additionally, the resultant organized biomechanics facilitates automated extraction of contractile kinetics from brightfield microscopy imaging, increasing the accessibility, reproducibility, and throughput of pharmacologic testing and cardiomyopathy disease modeling.
Human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) allow novel investigations of human cardiac disease, but disorganized mechanics and immaturity of hPSC-CMs on two-dimensional (2D) surfaces have been hurdles for efficient and reproducible study of these cells. Here, we developed a platform of micron-scale 2D cardiac tissues (M2DCTs) to precisely control biomechanics in arrays of thousands of purified, independently contracting cardiac muscle strips in 2D. By defining geometry and workload in M2DCTs in this reductionist platform that does not incorporate other cell types, we show that myofibrillar alignment and auxotonic contractions at physiologic workload critically drive maturation of cardiac contractile function, calcium handling, and electrophysiology. Additionally, the organized biomechanics in this system facilitates rapid and automated extraction of contractile kinetic parameters from brightfield microscopy images, increasing the reproducibility and throughput of pharmacologic testing. Finally, we show that M2DCTs enable precise and efficient dissection of contractile kinetics in cardiomyopathy disease models.
Introduction: Genetic hypertrophic cardiomyopathy (HCM) is an autosomal dominant inherited condition primarily due to pathogenic variants in sarcomere genes, often with marked clinical variability. We hypothesized that this variability may, in part, be due to additive effects from low penetrance sarcomere variants that would otherwise be dismissed due to high population prevalence. Methods: Low-penetrance HCM-associated sarcomere gene variants were identified by meeting a threshold for enrichment in HCM (ascertained from the Sarcomeric Human Cardiomyopathy Registry, SHaRe) versus the general population (ascertained from the Genome Aggregation Database, gnomAD), defined by an odds ratio (OR) >5, and a population prevalence greater than the most common autosomal dominant pathogenic HCM variant, MYBPC3 R502W (4x10 -5 ). Clinical variables and time-event analyses were performed from SHaRe . Results: A total of 507 unique sarcomere gene variants were present in 6384 individuals with genetic testing. Ten putative low-penetrance variants were identified in the genes MYBPC3 (N=2), TNNT2 (N=1), TNNI3 (N=2), and MYH7 (N=5) with a combined OR of 12.7 (range 5.8 - 28.4). These variants had a population prevalence of 4.02x10 -5 to 3.5x10 -4 . Family members of low-penetrance variant carriers were less likely to have HCM (35/171, 20%) than those of MYBPC3 R502W carriers (44/113, 39%, p<0.005). Age of diagnosis was older in patients with isolated low-penetrance variants than those with pathogenic variants (42 ± 19 vs 37 ± 18, p=0.005). Composite adverse events were less likely in patients with isolated low-penetrance variants than typical pathogenic sarcomere variants, but the risk was additive with both present (see Figure). Conclusions: A subset of low-penetrance sarcomere gene variants are tolerated in the general population at higher than expected proportions for HCM and may exert an additive pathogenic effect. These findings support an oligogenic risk model of HCM.
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