Abstract
Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super-relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned hiPSC-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super-relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.
Significance Statement Heart disease is the leading cause of death worldwide, and hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, affecting over 1 in 200 people. Mutations in myosin, the motor protein responsible for contraction of the heart, are a common cause of HCM but have diverse effects on the biomechanics of the myosin protein. We demonstrate that complex biomechanical effects of mutations associated with heart disease can be effectively studied and understood using a multi-scale experimental and computational modeling approach. This work confirmed an important role for disruption of the super-relaxed state for one particular HCM mutation, and our approach can be extended to aid in the development of new targeted therapies for patients with different mutations.
Competing Interest Statement
JAS is co-founder and on the Scientific Advisory Board of Cytokinetics, Inc., a company developing small molecule therapeutics for treatment of hypertrophic cardiomyopathy. DB has also consulted for Cytokinetics, Inc.
Footnotes
Competing Interest Statement: JAS is co-founder and on the Scientific Advisory Board of Cytokinetics, Inc., a company developing small molecule therapeutics for treatment of hypertrophic cardiomyopathy. DB has also consulted for Cytokinetics, Inc.
Classification: Major classification: Biological Sciences; Minor classification: Biochemistry; Biophysics and Computational Biology; Cell Biology;
