Molecular mechanisms of contraction in striated muscles, and the mechanisms of hypertrophic and dilated cardiomyopathy (HCM, DCM).

To study the molecular mechanisms of contraction, it is essential that force can be measured, hence "skinned" single muscle fibers are used, in which the plasma membrane is chemically removed. To learn the function of individual amino acid residues in a contractile protein, it is necessary to replace it with a recombinant protein. We developed a system, in which the thin filament of cardiac fibers is selectively removed by gelsolin, and reconstituted by actin monomers, followed by regulatory proteins tropomyosin (Tpm) and troponin (Tn). Because this procedure does not require any adverse conditions (extreme ionic strength, pH, temperature, etc), the reproducibility of active tension is 104±3%; similarly, the reproducibility of other contractile parameters are excellent. This technique has numerous applications. One can study the actin-myosin interaction without Tpm/Tn; we found that Tpm/Tn increases force/cross-bridge by 50% indicating that the actin-Tpm interaction allosterically activates the actin-myosin interaction. This technique has also been used to study mutant proteins to establish the structure-function relationships. It was further used to learn molecular pathogenesis based on thin filament proteins (actin, Tpm, Tn) that are known to cause HCM and DCM in humans. The technique makes it possible to obtain information on early events of pathogenesis, ie, before complex signaling cascades ensue. In Tpm mutations that cause HCM, we found that tension at pCa 8 increases from 10% (WT) to 30% to cause diastolic problem, whereas in Tpm mutations that cause DCM, tension at pCa 8 and pCa 4.5 both decrease to cause systolic problem.

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