ABSTRACT
Hypertension is a major cause of morbidity and mortality in patients with hypertrophic cardiomyopathy (HCM), suggesting a potential role for mechanics in HCM pathogenesis. Here, we developed an in vitro physiological model to investigate how mechanics acts together with HCM-linked myosin binding protein C (MYBPC3) mutations to trigger disease. Micro-heart muscles (µHM) were engineered from induced pluripotent stem cell (iPSC)-derived cardiomyocytes bearing MYBPC3+/- mutations and challenged to contract against substrates of different elasticity. µHMs that worked against substrates with stiffness at or exceeding the stiffness of healthy adult heart muscle exhibited several hallmarks of HCM, including cellular hypertrophy, impaired contractile energetics, and maladaptive calcium handling. Remarkably, we discovered changes in troponin C and T localization in MYBPC3+/- µHM that were entirely absent in 2D culture. Pharmacologic studies suggested that excessive Ca2+ intake through membrane-embedded channels underlie the observed electrophysiological abnormalities. These results illustrate the power of physiologically relevant engineered tissue models to study inherited disease with iPSC technology.
ABSTRACT
Hypertrophic cardiomyopathy is the most common cause of sudden death in the young. Because the disease exhibits variable penetrance, there are likely nongenetic factors that contribute to the manifestation of the disease phenotype. Clinically, hypertension is a major cause of morbidity and mortality in patients with HCM, suggesting a potential synergistic role for the sarcomeric mutations associated with HCM and mechanical stress on the heart. We developed an in vitro physiological model to investigate how the afterload that the heart muscle works against during contraction acts together with HCM-linked MYBPC3 mutations to trigger a disease phenotype. Micro-heart muscle arrays (µHM) were engineered from iPSC-derived cardiomyocytes bearing MYBPC3 loss-of-function mutations and challenged to contract against mechanical resistance with substrates stiffnesses ranging from the of embryonic hearts (0.4 kPa) up to the stiffness of fibrotic adult hearts (114 kPa). Whereas MYBPC3 +/- iPSC-cardiomyocytes showed little signs of disease pathology in standard 2D culture, µHMs that included components of afterload revealed several hallmarks of HCM, including cellular hypertrophy, impaired contractile energetics, and maladaptive calcium handling. Remarkably, we discovered changes in troponin C and T localization in the MYBPC3 +/- µHM that were entirely absent in 2D culture. Pharmacologic studies suggested that excessive Ca 2+ intake through membrane-embedded channels, rather than sarcoplasmic reticulum Ca 2+ ATPase (SERCA) dysfunction or Ca 2+ buffering at myofilaments underlie the observed electrophysiological abnormalities. These results illustrate the power of physiologically relevant engineered tissue models to study inherited disease mechanisms with iPSC technology.
ABSTRACT
INTRODUCTION: In clinical and animal studies, Hypertrophic Cardiomyopathy (HCM) shares many similarities with non-inherited cardiac hypertrophy induced by pressure overload (hypertension). This suggests a potential role for mechanical stress in priming tissues with mutation-induced changes in the sarcomere to develop phenotypes associated with HCM, including hypercontractility and aberrant calcium handling. Here, we tested the hypothesis that heterozygous loss of function of Myosin Binding Protein C (MYBCP3 +/- , mutations in which account for almost 50% of inherited HCM) combines with environmental stiffness to drive HCM phenotypes. METHODS: We differentiated isogenic control (WTC) and MYBPC3 +/- iPSC into cardiomyocytes using small molecule manipulation of Wnt signaling, and then purified them using lactate media. The purified cardiomyocytes were seeded into "dog bone" shaped stencil molds to form micro-heart muscle arrays (µHM). To mimic changes in myocardial stiffness stemming from pressure overload, we varied the rigidity of the substrates µHM contract against. Stiffness levels ranged from those corresponding to fetal (5 kPa), healthy (15 kPa), pre-fibrotic (30 kPa) to fibrotic (65 kPa) myocardium. Substrates were embedded with a thin layer of fluorescent beads to track contractile force, and parent iPSC were engineered to express the genetic calcium indicator, GCaMP6f. High speed video microscopy and image analysis were used to quantify calcium handling and contractility of µHM. RESULTS: Substrate rigidity triggered physiological adaptation for both genotypes. However, MYBPC3 +/- µHM showed a lower tolerance to substrate stiffness with the peak traction on 15 kPa, while WTC µHM had peak traction on 30 kPa. MYBPC3 +/- µHM exhibited hypercontractility, which was exaggerated by substrate rigidity. MYBPC3 +/- µHM hypercontractility was associated with longer rise times for calcium uptake and force development, along with higher overall Ca2+ intake. CONCLUSION: We found MYBPC3 +/- mutations cause iPSC-µHM to exhibit hypercontractility, and also a lower tolerance for mechanical stiffness. Understanding how genetics work in combination with mechanical stiffness to trigger and/or exacerbate pathophysiology may lead to more effective therapies for HCM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at (10.1007/s12195-021-00684-x).
ABSTRACT
Cell encapsulating scaffolds are necessary for the study of cellular mechanosensing of cultured cells. However, conventional scaffolds used for loading cells in bulk generally fail at low compressive strain, while hydrogels designed for high toughness and strain resistance are generally unsuitable for cell encapsulation. Here we describe an alginate/gelatin methacryloyl interpenetrating network with multiple crosslinking modes that is robust to compressive strains greater than 70%, highly biocompatible, enzymatically degradable and able to effectively transfer strain to encapsulated cells. In future studies, this gel formula may allow researchers to probe cellular mechanosensing in bulk at levels of compressive strain previously difficult to investigate.
ABSTRACT
Mechanical loading plays a critical role in cardiac pathophysiology. Engineered heart tissues derived from human induced pluripotent stem cells (iPSCs) allow rigorous investigations of the molecular and pathophysiological consequences of mechanical cues. However, many engineered heart muscle models have complex fabrication processes and require large cell numbers, making it difficult to use them together with iPSC-derived cardiomyocytes to study the influence of mechanical loading on pharmacology and genotype-phenotype relationships. To address this challenge, simple and scalable iPSC-derived micro-heart-muscle arrays (µHM) have been developed. "Dog-bone-shaped" molds define the boundary conditions for tissue formation. Here, we extend the µHM model by forming these tissues on elastomeric substrates with stiffnesses spanning from 5 to 30 kPa. Tissue assembly was achieved by covalently grafting fibronectin to the substrate. Compared to µHM formed on plastic, elastomer-grafted µHM exhibited a similar gross morphology, sarcomere assembly, and tissue alignment. When these tissues were formed on substrates with different elasticity, we observed marked shifts in contractility. Increased contractility was correlated with increases in calcium flux and a slight increase in cell size. This afterload-enhanced µHM system enables mechanical control of µHM and real-time tissue traction force microscopy for cardiac physiology measurements, providing a dynamic tool for studying pathophysiology and pharmacology.