TNI ISOFORM SWITCHING AND CALCIUM ION CONTROL IN THE HEART

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The calcium-based regulation of myofilament activity is a crucial determinant in excitation-contraction coupling. It is broadly seen that myofilament isoform switching during normal development and in pathology leads to marked changes in response of this system to cytosolic [CA2+]i. Troponin I (TnI) isoform switching has been implicated as a central player in the differential response of the neonatal versus adult heart to inotropi agents that mediate decreased Ca2+ sensitivity of the contractile apparatus The fetal heart expresses the slow-twitch skeletal muscle isoform of TnI (TnIs). After birth, the adult cardiac isoform, TnIc, becomes the exclusiv TnI isoform present in the heart. The PI has investigated the molecular mechanisms mediating slow TnI isoform expression in the heart by cloning the human TnIx gene and identifying multiple transcriptional elements involved in skeletal versus cardiac muscle-specific gene expression and has found chamber-specific differences in transgene reporter genes into adult myocardium. In vivo gene expression has been examined by analysis of transgenic mice and direct injection of reporter genes into adult myocardium. The investigator will now apply these in vivo gene transfer approaches to examine the molecular mechanisms regulating TnI isoform switching in the heart and assess the consequences of TnI isoform switching to calcium-based myofilament function. (i) New lines of transgenic mice will be analyzed to assess the role of individual transcriptional elements to temporal and spatial gene regulation in the heart; (ii) Transient transfection of primary myocytes, gel mobility shift assays, and DNA footprinting studies will be used to identify factors regulating the TnIs gene in the neonatal versus adult heart; (iii) Null mutations will be introduced into the mouse cardiac and slow skeletal muscle TnI isoform genes to assess the consequences of these knock out mutations on the differentiation and Ca2+ activation of myocytes derived from embryonic stem cells; (iv) To address the functional importance of previously identified TnI interaction and phosphorylation domains, expression vectors containing chimerical cDNA constructs between the TnIc and TnIs isoforms will be expressed in adult rat hearts along with a marker plasmid by direct DNA injection. Myocytes will be prepared from the injected hearts, and the effect of the hybrid TnI proteins on calcium based myofilament regulation will be evaluated. This is an integrative study using in vitro and in vivo approaches to examine the molecular mechanisms controlling thin filament protein gene expression and the physiological consequences of TnI isoform switching in the maturing heart.