Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na+) channels and sarcoplasmic reticulum calcium (Ca2+) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na+ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.

Background: Molecular targeted chemotherapies have been shown to significantly improve the outcomes of patients who have cancer, but they often cause cardiovascular side effects that limit their use and impair patients’ quality of life. Cardiac dysfunction induced by these therapies, especially trastuzumab, shows a distinct cardiotoxic clinical phenotype in comparison to the cardiotoxicity induced by conventional chemotherapies. Methods: We used the human induced pluripotent stem cell–derived cardiomyocyte (iPSC-CM) platform to determine the underlying cellular mechanisms in trastuzumab-induced cardiac dysfunction. We assessed the effects of trastuzumab on structural and functional properties in iPSC-CMs from healthy individuals and performed RNA-sequencing to further examine the effect of trastuzumab on iPSC-CMs. We also generated human induced pluripotent stem cells from patients receiving trastuzumab and examined whether patients’ phenotype could be recapitulated in vitro by using patient-specific iPSC-CMs. Results: We found that clinically relevant doses of trastuzumab significantly impaired the contractile and calcium-handling properties of iPSC-CMs without inducing cardiomyocyte death or sarcomeric disorganization. RNA-sequencing and subsequent functional analysis revealed mitochondrial dysfunction and altered the cardiac energy metabolism pathway as primary causes of trastuzumab-induced cardiotoxic phenotype. Human iPSC-CMs generated from patients who received trastuzumab and experienced severe cardiac dysfunction were more vulnerable to trastuzumab treatment than iPSC-CMs generated from patients who did not experience cardiac dysfunction following trastuzumab therapy. It is important to note that metabolic modulation with AMP-activated protein kinase activators could avert the adverse effects induced by trastuzumab. Conclusions: Our results indicate that alterations in cellular metabolic pathways in cardiomyocytes could be a key mechanism underlying the development of cardiac dysfunction following trastuzumab therapy; therefore, targeting the altered metabolism may be a promising therapeutic approach for trastuzumab-induced cardiac dysfunction.

Muscular dystrophies (MDs) comprise a group of degenerative muscle disorders characterized by progressive muscle wasting and often premature death. The primary defect common to most MDs involves disruption of the dystrophin-glycoprotein complex (DGC). This leads to sarcolemmal instability and Ca²⁺ influx, inducing cellular necrosis. Here we have shown that the dystrophic phenotype observed in δ-sarcoglycan–null (Sgcd^–/–) mice and dystrophin mutant mdx mice is dramatically improved by skeletal muscle–specific overexpression of sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA1). Rates of myofiber central nucleation, tissue fibrosis, and serum creatine kinase levels were dramatically reduced in Sgcd^–/– and mdx mice with the SERCA1 transgene, which also rescued the loss of exercise capacity in Sgcd^–/– mice. Adeno-associated virus–SERCA2a (AAV-SERCA2a) gene therapy in the gastrocnemius muscle of Sgcd^–/– mice mitigated dystrophic disease. SERCA1 overexpression reversed a defect in sarcoplasmic reticulum Ca²⁺ reuptake that characterizes dystrophic myofibers and reduced total cytosolic Ca²⁺. Further, SERCA1 overexpression almost completely rescued the dystrophic phenotype in a mouse model of MD driven solely by Ca²⁺ influx. Mitochondria isolated from the muscle of SERCA1-Sgcd^–/– mice were no longer swollen and calpain activation was reduced, suggesting protection from Ca²⁺-driven necrosis. Our results suggest a novel therapeutic approach using SERCA1 to abrogate the altered intracellular Ca²⁺ levels that underlie most forms of MD.

Background: The progression toward low-cost and rapid next-generation sequencing has uncovered a multitude of variants of uncertain significance (VUS) in both patients and asymptomatic “healthy” individuals. A VUS is a rare or novel variant for which disease pathogenicity has not been conclusively demonstrated or excluded, and thus cannot be definitively annotated. VUS, therefore, pose critical clinical interpretation and risk-assessment challenges, and new methods are urgently needed to better characterize their pathogenicity. Methods: To address this challenge and showcase the uncertainty surrounding genomic variant interpretation, we recruited a “healthy” asymptomatic individual, lacking cardiac-disease clinical history, carrying a hypertrophic cardiomyopathy (HCM)-associated genetic variant (NM_000258.2:c.170C>A, NP_000249.1:p.Ala57Asp) in the sarcomeric gene MYL3 , reported by the ClinVar database to be “likely pathogenic.” Human-induced pluripotent stem cells (iPSCs) were derived from the heterozygous VUS MYL3 _(170C>A) carrier, and their genome was edited using CRISPR/Cas9 to generate 4 isogenic iPSC lines: (1) corrected “healthy” control; (2) homozygous VUS MYL3 _{(170C>A )} ; (3) heterozygous frameshift mutation MYL3 ^{(170C>A/fs )} ; and (4) known heterozygous MYL3 pathogenic mutation (NM_000258.2:c.170C>G), at the same nucleotide position as VUS MYL3 _(170C>A) , lines. Extensive assays including measurements of gene expression, sarcomere structure, cell size, contractility, action potentials, and calcium handling were performed on the isogenic iPSC-derived cardiomyocytes (iPSC-CMs). Results: The heterozygous VUS MYL3 _(170C>A) -iPSC-CMs did not show an HCM phenotype at the gene expression, morphology, or functional levels. Furthermore, genome-edited homozygous VUS MYL3 _(170C>A) - and frameshift mutation MYL3 ^(170C>A/fs) -iPSC-CMs lines were also asymptomatic, supporting a benign assessment for this particular MYL3 variant. Further assessment of the pathogenic nature of a genome-edited isogenic line carrying a known pathogenic MYL3 mutation, MYL3 _(170C>G) , and a carrier-specific iPSC-CMs line, carrying a MYBPC3 _(961G>A) HCM variant, demonstrated the ability of this combined platform to provide both pathogenic and benign assessments. Conclusions: Our study illustrates the ability of clustered regularly interspaced short palindromic repeats/Cas9 genome-editing of carrier-specific iPSCs to elucidate both benign and pathogenic HCM functional phenotypes in a carrier-specific manner in a dish. As such, this platform represents a promising VUS risk-assessment tool that can be used for assessing HCM-associated VUS specifically, and VUS in general, and thus significantly contribute to the arsenal of precision medicine tools available in this emerging field.