TITINS ROLE IN MODULATING CARDIAC PASSIVE TENSION

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Titin is a 3000 kD protein found in heart and skeletal muscle, and it is believed to be largely responsible for the resting tension of cells from these tissues. Studies will be conducted to determine if differences in resting tension between cardiomyocytes from different species are due to altered expression of titin isoforms or altered cassette expression from the single titin gene. These studies will employ slot blots with probes corresponding to the central I-band portion of titin. The cDNA sequence for the region including and flanking the PEVK region of the major isoforms in rat and dog cardiac muscle will be determined. The frequency of the titin N2A and N2B type messages will be estimated by isolation and sequencing multiple clones from rat and dog cDNA libraries using the N2B sequence common in all cardiac titins observed to date. Specific antibodies and quantitative immuno-fluorescence of tissue sections from these species will be used to estimate the protein proportion of isoforms of the N2A and N2B isoform classes. A similar approach will be used to examine titin isoform expression in different strains of rats (including those with spontaneous hypertension- SHR) and in hypertrophied dog heart after chronic pacing. The binding of expressed titin fragments containing the PEVK region to actin will be determined by co-sedimentation assays and by surface plasmon resonance. Such fragments will also be tested for effects on skinned single cardiomyocytes. The possible modulation of rest tension by changes in phosphorylation state will be determined by measuring the effects of several kinases (protein kinase A, protein kinase C, casein kinase II) on resting tension of skinned rat myocytes. Different purified kinases (protein kinase A, protein kinase C, casein kinase II) will be tested for their ability to phosphorylate titin in skinned cardiac cells using gamma 32P labeled ATP, and this phosphorylation related to any mechanical changes in the cells. The phosphorylated peptide(s) will be isolated and sequenced and their location in the sarcomere mapped. The phosphorylation patterns as determined by 2D peptide maps will also be compared to those obtained after treatment of intact cardiomyocytes with beta-agonist or phosphatase inhibitors. Phosphorylation status of hypertrophied versus normal rat and dog heart will be assessed using similar methods. The possible modulation of rest tension either by altering isoform expression or by changing titin's phosphorylation state may be important mechanisms for changing cardiac function in vivo. Titin changes may be related to the hypertrophic response that commonly is brought about by high blood pressure and thus is a frequent problem in human health.