Comparative analyses of myosin from control human hearts an from individuals with idiopathic dilated cardiomyopathy (IDC) revealed a significant decrease in the content of the regulatory light chain (LC2) in IDC heart in which actomyosin ATPase activity and filament assembly were correspondingly altered. These studies with contractile proteins will be extended to evaluate the state of Ca++-regulation during IDC using myofibirls and purified myosin and regulated actin. Based on initial observations, it appears that Ca++- regulation was totally suppressed during IDC because of a possible proteolysis of the TnT subunit of troponin. The presence or absence of TnT in IDC tissues will be established by Western blots and radioimmunoassays of myofibrils and troponin. In addition, paracrystals of relaxing factor and regulated actin will be analyzed by immunoelectron microscopy using gold coupled anti-TnT antibody to provide further evidence for the absence of TnT. Functional measurements to achieve the same goal will include MgATPase and in vitro motility assays using myosin and regulated actin from both tissues: it is expected that proteins from IDC samples will be calcium insensitive due to the absence of TnT. The reduction in LC2 content and possibly that of TnT in IDC hearts was linked to an active protease. Experiments are designed to obtain a highly purified protease by a combination of cation-exchange, gel- exclusion and hydrophobic chromatography. At the same time it will e identified by gel autography and electroblotted onto nitrocellulose. The protease band will be excised to raise antibodies against it, to determine its amino acid sequence and from the sequence oligonucleotides generated to be used as specific CDNA probes. The purified protease will be characterized by sedimentation equilibrium ultracentrifugation to confirm its homogeneity, its secondary structure determined by circular dichroism spectra and its distribution in cardiac myocytes delineated by immunoelectron microscopy and by immunofluorescence. The characteriza- tion of the protease will further involve establishing its specificity, using human heart, gizzard and scallop myosin LC2, clarifying its divalent cation requirements, its Ph profile, the effect of specific inhibitors on activity, and calculating its V/m and K/m with respect to human heart LC2. Finally the protease gene will be isolated with complementary CDNA clones to examine the expression of the gene in myopathic and control tissues, in order to differentiate between transcriptional and post-transcriptional mechanisms for increased protease activity. By comparing the levels of the protease in control and pathologic samples by Western blotting and immunofluorescent staining of thin sections, it will be possible to determine if muscle contains latent forms of the protease that are activated in the disease state.
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