The onset of cardiac sympathetic innervation is accompanied by developmental changes in cardiac ion channel function. Of particular importance is the evolution of a negative shift in activation voltage of the ventricular pacemaker current, I-f, identified in a previous collaborative study by Projects 8 and 9. I-f is of interest (1) because of its central role in the control of heart rate, which is rapid at birth and slows to less than half its original rate by adolescence, and (2) because it plays a role in rate and rhythm control in instances where disease induces a positive shift in activation of the current. The proposed aims of Project 9 derive from our earlier studies of developmental changes in I-f, as well as three observations made during the current funding period: 1) The developmental shift in I-f is partly reproduced by culturing neonatal ventricular cells with sympathetic nerves or in the sustained presence of neuropeptide Y (NPY) and norepinephrine. 2) I-f activates at less negative voltages in adult ventricular myocytes from mice lacking NPY than in cells from wild type, supporting a developmental role for NPY. 3) Over-expressed HCN2, a molecular correlate of I-f, activates at less negative voltages in neonatal than in adult rat ventricular myocytes, suggesting the existence of an age-specific factor influencing the gating of individual HCN isoforms. In this renewal application, Project 9 has four specific aims, targeted to the following questions: 1) What are the components of the NPY-dependent signaling cascade regulating I-f developmentally? 2) What additional signaling cascade(s) contributes to I-f maturation? 3) What is the molecular basis for the maturational change in I-f? 4) What are the functional consequences of HCN over-expression? We will employ single cells and cell cultures of neonatal and adult ventricular myocytes of rat and mouse, the latter to take advantage of knockout animals to elucidate the relevant signaling cascades. Both native I-f and over-expressed HCN currents will be studied. These studies are relevant to both regulation of normal cardiac developmental by sympathetic innervation and disease states involving abnormal innervation and associated arrhythmias. Further, by studying over-expression of pacemaker channel genes in myocytes rather than heterologous systems, Project 9 provides an innovative approach that uses the power of molecular biology while preserving the physiologic context essential to the ultimate elucidation of how these channels function, and can be controlled, in vivo.
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