Hypertrophic cardiomyopathy (HCM) is an inherited cardiac disease characterized by an increased left ventricular mass in the absence of another cause for hypertrophy. This disease provides a model to study the process of cardiac hypertrophy. In approximately 15% of affected families, the disease gene encodes a beta myosin heavy chain (BMHC) gene with a missense mutation. We previously identified 32 distinct mutations in the BMHC gene and mapped them onto the 3D structure of the head of skeletal muscle myosin. The families evaluated at NIH are referred to this tertiary care facility for severe disease. From a group of 320 such families more than 60 mapped to the Beta Myosin gene. The mutations cluster in 4 regions, suggesting different types of interference in the actomyosin cross-bridge kinetics as a function of mutation location. Subsequent large scale sequencing of many more patients by the Seidnman group has filled in the regions between these clusters. However, the patients that we studied had severe disease and perhaps this is why the clustering was so evident. We have studied the mechanical properties of extracted mutant myosins and muscle fibers expressing these myosins, to analyze the pathophysiology at a molecular level. One of the clusters of mutations has led us to the discovery of mutations in the myosin light chains which cause a variant of HCM characterized by an obstruction in the middle of the left ventricle. Through a series of arguments, the association of the myosin light chain mutations with the rare subtype of HCM led us to hypothesize the importance of the """"""""stretch-activation response"""""""" to the function of the normal heart. The stretch-activation response in Drosophila flight muscle has been previously shown to be distorted by a mutation in the """"""""regulatory"""""""" myosin light chain (RLC), resulting in flightless flies whose wings do not beat properly. We have developed transgenic mice expressing the mutant myosin """"""""essential"""""""" light chain (ELC), from a patient with cardiac hypertrophy. The hearts from these mice also do not beat properly. That is, there is a shift of the frequency of maximum power output to a rate beyond the physiologic range, with consequent loss of oscillatory power. We have cloned a human enzyme that phosphorylates the RLC and and identified a genetic mutation in a small family. This mutant enzyme has been expressed and shown to have an increased maximum velocity compared to the normal enzyme. We have performed mechanical studies on isolated muscle fibers treated with this cloned enzyme and demonstrated a change in the stretch-activation response. We are continuing to use muscle fibers from normal and transgenic mice to study the basic biophysical response of muscle fibers to light chain phosphorylation. One of the observations from our recent temperature dependency studies evaluating cross-bridge kinetics of fast and slow muscle fibers has generated an intriquing hypothesis. That is, that cardiac muscle fibers may be able to drive the cross-bridge kinetics in reverse and produce the equivalent of new ATP. This is based on the observation of a Q10 of 2 for the force producing state of fast muscle myosin and a corresponding Q10 of 20 for cardiac myosin. This disparity suggests an isolated force producing state in fast myosin but a back linked force producing step in cardiac myosin. If elastic forces in the oscillating heart are timed correctly then portions of the heart that are stretched by contracting portions could conserve energy rather then produce heat with the net energy saving equivalent to new energy production. We are presently developing single molecule instrumentation that will allow the direct test of this hypothesis. We have published a manuscript (Biophys J. 2007 Apr 15;92(8):2865-74. Epub 2007 Jan 26.) that takes advantage of an observation we made in our basic single muscle fiber studies. Under rare conditions, a ploot of tension vs. temperature of fast skeletal muscle fibers is sigmoidal, with an inflection point. Therefore tension production can be represented as a simple 2 state reaction. This has allowed, for the first time, the calculation of the backwards and forward reaction rates of myosin force production as well as the enthalpy and entropy of the reaction. The forward and backward reactions are Arrhenious and anti-Arrhenious respectively, suggesting that force production involves a localized unfolding and the backwards reaction is inhibited by an entropic barrier (refolding). These findings are consistent with previous crystallographic findings of Myosin VI, published by the Houdusse laboratory. Our 2009 publication in PNAS compares the exponentials extracted from the temperature induced myosin force production (involving no movement) to movement induced force production. This highlights the buffering of tension by an ensemble of crossbridges that have released inorganic phosphate but not yet cycled through the strong attachment phase. Both of these papers add to our basic understanding of muscle contraction. Most recently, a review of our previous genetic screens has identified a naturally occurring mutation in a new cardiac kinase that phosphorylates both RLC and Myosin Binding Protein-C. This mutation causes severe disease in patients. The vector for a Knock-in mouse has been constructed and is now being sequenced before insertion into a stem cell for mouse production. The wild type and mutant form of this kinase is being expressed in a Baculoviral system to be used together with our previously expressed MLCK-2. Such studies will be carried out in single fiber studies, as we have done previously.
|Davis, Julien S; Epstein, Neal D (2009) Mechanistic role of movement and strain sensitivity in muscle contraction. Proc Natl Acad Sci U S A 106:6140-5|