Titin is a 3000 kD protein found only in heart and skeletal muscle. Studies will be conducted to characterize the molecular properties of the cardiac isoform of this protein. The cDNA sequence of rabbit cardiac titin will be extended from our recently isolated clones coding for a section near the Z line. A random primed cDNA expression library will be further probed with polyclonal and monoclonal antibodies which primarily bind to titin in the I band region of the sarcomere (most published sequence to date is internal and associated with the A band region of titin). Alternatively oligo-nucleotides based on amino acid sequence from isolated peptides from the titin I band region will be used to screen for titin clones. New clones will be sequenced and differences between cardiac and skeletal muscle titin cDNA will be explored using PCR techniques. Attempts will be made to relate any sequence variations found to the known differences in resting tension between these tissues. Preliminary results indicate a marked effect of protein kinase A on the rest tension of skinned cardiomyocytes. We will test the hypothesis that titin, C protein, or TnI phosphorylation state alters rest tension by selective exchange of C protein or TnI have differing phosphorylation states into skinned cells. The role of the kinase domain in titin will be examined, and its ability to autophosphorylate and/or phosphorylate other cardiac contractile proteins determined. Studies will be conducted to identify which peptides in titin are phosphorylated (either in vivo or by autophosphorylation). The phosphorylated peptide (s) will be isolated and sequenced and their location in the sarcomere determined using antibody staining of myofibrils or skinned myocytes prior to light and electron microscopy. Different purified kinases (protein kinase A, protein kinase C, casein kinase II) will be tested for their ability to phosphorylate purified bovine cardiac titin and titin in skinned cardiac cells using gamma 32p labelled ATP. The phosphorylation pattern as determined by 2D peptide maps will also be compared to those obtained after treatment of intact cardiomyocytes with beta-agonist or phosphatase inhibitors. The possible role of titin in beta-agonist mediated changes in rest tension merits further study since adrenergic regulation is an important means of altering cardiac function in response to exercise or ischemia.
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