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Updated: Aug 7, 2025
Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes
Published on: December 16, 2022
cMyBP-C ablation in human engineered cardiac tissue causes progressive Ca2+-handling abnormalities
Willem J De Lange1, Emily T Farrell1, Jonathan J Hernandez1
1Departments of Pediatrics, School of Medicine and Public Health, University of Wisconsin-Madison , Madison, WI, USA.
Insights
Truncation mutations in cardiac myosin binding protein C (cMyBP-C) cause hypertrophic cardiomyopathy (HCM). This study shows cMyBP-C deficiency leads to progressive contractile dysfunction, initially hypercontractile then hypocontractile with impaired relaxation.
Area of Science:
- Cardiovascular Biology
- Genetics
- Biomedical Engineering
Background:
- Truncation mutations in cardiac myosin binding protein C (cMyBP-C) are a leading genetic cause of hypertrophic cardiomyopathy (HCM).
- Understanding the functional consequences of cMyBP-C mutations is crucial for developing targeted therapies for HCM.
- Existing models do not fully recapitulate the progressive nature of HCM phenotypes associated with cMyBP-C deficiency.
Purpose of the Study:
- To investigate the functional impact of heterozygous and homozygous cMyBP-C deficiency on cardiomyocyte contractility and Ca2+-handling.
- To model progressive hypertrophic cardiomyopathy using CRISPR-Cas9 gene editing in human induced pluripotent stem cells (iPSCs).
- To elucidate the molecular mechanisms underlying cMyBP-C related cardiac dysfunction.
Main Methods:
- CRISPR-Cas9 gene editing to create isogenic human iPSC lines with heterozygous (cMyBP-C+/-) and homozygous (cMyBP-C-/-) MYBPC3 frame-shift mutations.
- Generation of cardiac micropatterns and engineered cardiac tissue constructs (ECTs) from patient-derived cardiomyocytes.
- Comprehensive characterization of contractile function, Ca2+-handling, Ca2+-sensitivity, and gene expression (RNA-seq).
Main Results:
- Heterozygous cMyBP-C+/- ECTs exhibited haploinsufficiency and progressive hypocontractility with impaired relaxation.
- Homozygous cMyBP-C-/- micropatterns showed increased initial strain, while ECTs developed severe contractile depression and slower Ca2+-release over time.
- RNA-seq revealed differential expression of hypertrophic, sarcomeric, Ca2+-handling, and metabolic genes in cMyBP-C deficient models.
- Phenotype severity correlated with cMyBP-C levels, with homozygous deficiency showing more severe and earlier onset dysfunction.
Conclusions:
- cMyBP-C haploinsufficiency and ablation result in a progressive cardiac phenotype, transitioning from hypercontractility to hypocontractility with impaired relaxation.
- Calcium handling abnormalities are central to the observed contractile dysfunction in cMyBP-C deficient cardiomyocytes.
- These findings provide critical insights into the pathophysiology of HCM caused by MYBPC3 mutations and highlight the utility of engineered cardiac tissues for disease modeling.
Abstract:
Truncation mutations in cardiac myosin binding protein C (cMyBP-C) are common causes of hypertrophic cardiomyopathy (HCM). Heterozygous carriers present with classical HCM, while homozygous carriers present with early onset HCM that rapidly progress to heart failure. We used CRISPR-Cas9 to introduce heterozygous (cMyBP-C+/-) and homozygous (cMyBP-C-/-) frame-shift mutations into MYBPC3 in human iPSCs. Cardiomyocytes derived from these isogenic lines were used to generate cardiac micropatterns and engineered cardiac tissue constructs (ECTs) that were characterized for contractile function, Ca2+-handling, and Ca2+-sensitivity. While heterozygous frame shifts did not alter cMyBP-C protein levels in 2-D cardiomyocytes, cMyBP-C+/- ECTs were haploinsufficient. cMyBP-C-/- cardiac micropatterns produced increased strain with normal Ca2+-handling. After 2 wk of culture in ECT, contractile function was similar between the three genotypes; however, Ca2+-release was slower in the setting of reduced or absent cMyBP-C. At 6 wk in ECT culture, the Ca2+-handling abnormalities became more pronounced in both cMyBP-C+/- and cMyBP-C-/- ECTs, and force production became severely depressed in cMyBP-C-/- ECTs. RNA-seq analysis revealed enrichment of differentially expressed hypertrophic, sarcomeric, Ca2+-handling, and metabolic genes in cMyBP-C+/- and cMyBP-C-/- ECTs. Our data suggest a progressive phenotype caused by cMyBP-C haploinsufficiency and ablation that initially is hypercontractile, but progresses to hypocontractility with impaired relaxation. The severity of the phenotype correlates with the amount of cMyBP-C present, with more severe earlier phenotypes observed in cMyBP-C-/- than cMyBP-C+/- ECTs. We propose that while the primary effect of cMyBP-C haploinsufficiency or ablation may relate to myosin crossbridge orientation, the observed contractile phenotype is Ca2+-mediated.
