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 have identified 32 distinct mutations in the BMHC gene and mapped them onto the 3D structure of the head of skeletal myosin. The mutations cluster in 4 regions, suggesting different types of interference in the actomyosin cross-bridge kinetics as a function of mutation location. 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 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 the 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.? This year we have published a manuscript in Biophys J., that takes advantage of an observation we made in our basic single muscle fiber studies. Under rare conditions, a plot 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 finding are consistent with new crystallographic findings of Myosin VI, published by the Houdusse laboratory. A recently submitted followup manuscript 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.? In parallel studies, our generation of a transgenic mouse with a null allele of the cardiac MLCK gene has yielded new insights into the importance of the spatial gradient of myosin light chain phosphorylation in the heart, first reported by our laboratory in Cell 2001. Further studies of the effect of MLCK are continuing on patients with hypertrophic or dilated cardiomyopathy, who harbor the common allele or variant of this gene, These African- American individuals are being studied with a new offline analysis package of phase tagged Magnetic Resonance Imaging that allows the evaluation of cardiac dyssynchrony.
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