Biology subjects

Saswata S Sarkar

Publications and source records attributed to Saswata S Sarkar.

3 recordsLinked to original sources

bioRxiv2016

Mutations in the catalytic domain of human β-cardiac myosin that cause early onset hypertrophic cardiomyopathy significantly increase the fundamental parameters that determine ensemble force and velocity

Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. In infants it can be particularly severe and it is the leading cause of sudden cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in {beta}-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two mutations that cause early-onset HCM, D239N and H251N, affect the mechanical output of human {beta}-cardiac myosin at the molecular level. We observe extremely large increases (25% - 95%) in the actin gliding velocity, single molecule intrinsic force, and ATPase activity of the two mutant myosin motors compared to wild type myosin. In contrast to previous studies of HCM-causing mutations in human {beta}-cardiac myosin, these mutations were striking in that they caused changes in biomechanical parameters that were both greater in magnitude and more uniformly consistent with a hyper-contractile phenotype. In addition, S1-S2 binding studies revealed a significant decrease in affinity of the H251N motor for S2, suggesting that this mutation may further increase hyper-contractility by releasing active motors from a sequestered state. This report shows, for the first time, a clear and significant gain in function for all tested molecular biomechanical parameters due to HCM mutations in human {beta}-cardiac myosin.

Biophysics↗

bioRxiv2016

Biophysical properties of human β-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hyper-contractility, and therapies are aimed toward controlling the hyperactive physiology. {beta}-cardiac myosin comprises ~40 percent of genetic mutations associated with HCM and the converter domain of myosin is a hot spot for HCM-causing mutations, but the underlying primary effects of these mutations on myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human {beta}-cardiac myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations R719W, R723G and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant myosins are decreased compared to wild type, and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15 percent faster for R719W and R723G myosin, while there is no change in velocity for G741R. ATPase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to wild type. These results indicate that the net biomechanical properties of human {beta}-cardiac myosin carrying these converter domain mutations are very similar to wild type or even slightly hypo-contractile, leading us to consider an alternative mechanism for the clinically observed hyper-contractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of myosin heads participating in force production.\n\nOne Sentence Summary: Biophysical analysis of human {beta} -cardiac myosin with converter domain HCM mutations reveal minimal changes compared to wild type myosin to slightly hypo-contractile profiles in ensemble force production, in contrast to the clinically observed hyper-contractility, leading to a speculative alternative mechanism involving availability of myosin heads participating in the sarcomere.

Biophysics↗

bioRxiv2016

Beyond the myosin mesa: a potential unifying hypothesis on the underlying molecular basis of hyper-contractility caused by a majority of hypertrophic cardiomyopathy mutations

AbstractHypertrophic cardiomyopathy (HCM), the most commonly occurring inherited cardiovascular disease, is primarily caused by mutations in human {beta}-cardiac myosin and myosin binding protein-C. It has been thought that such mutations in myosin increase the intrinsic force of the motor, its velocity of contraction, or its ATPase activity, giving rise to hyper-contractility. We hypothesize that while these parameters are mildly affected by most myosin HCM-causing mutations, a major effect of a majority of myosin HCM mutations is likely to involve an increase in the number of myosin heads that are functionally accessible (Na) for interaction with actin in the sarcomere. We consider a model involving three types of interactions involving the myosin mesa and the converter domain of the myosin motor that hold myosin heads in a sequestered state, likely to be released in a graded manner as the demands on the heart increase: 1) the two myosin heads binding to one another, 2) one head binding to its own coiled-coil tail, and 3) the other head binding to myosin binding protein-C. In addition there is clear evidence of interaction between the coiled-coil tail of myosin and myosin binding protein-C. Experimentally, here we focus on myosin head binding to its own coiled-coil tail and to myosin binding protein-C. We show that phosphorylation of the myosin regulatory light chain and myosin binding protein-C weaken these respective associations, consistent with known enhancements of sarcomere function by these phosphorylations. We show that these interactions are weakened as a result of myosin HCM mutations, in a manner consistent with our structural model. Our data suggests a potential unifying hypothesis for the molecular basis of hyper-contractility caused by human hypertrophic cardiomyopathy myosin mutations, whereby the mutations give rise to an increase in the number of myosin heads that are functionally accessible for interaction with actin in the sarcomere, causing the hyper-contractility observed clinically.

Biochemistry↗