Heart disease with arrhythmia is the primary cause of death in the Western world and there is data to suggest that mechanoelectric feedback plays a role in the development of these arrhythmias. One source of this transduction may be mechanosensitive ion channels that have been identified in a number of cardiocytes. These channels have a different pharmacology from traditional voltage sensitive channels, and understanding their properties may lead tot he development of new therapies and therapeutic agents. The proposed experiments will clarify the behavior of mechanosensitive channels in heart cells at the level of single channels and whole cells using acutely isolated cardiocytes from rats and chicks. At present, only one paper exists on voltage clamp experiments on stretched cardiac cells. Consequently there is almost no information on such fundamental questions as the existence of currents, their magnitude as a function of stretch, their ionic selectivity, the number types of channels or transporters involved in such currents, their pharmacology,a nd their variability throughout different regions of the heart. Although single channel records of mechanosensitive ion channels have been made from heart cells, it is unknown which, if any, of the channels seen under patch clamp conditions are physiologically active. Techniques will be developed to stretch cells with minimum damage using probes attached to piezoelectric manipulators. Mean strain and uniformity will be measured from both cell length, membrane markers and from sarcomere length using video microscopy. A patch clamp pipette attached tot he cell near the fixed pipette will follow the motion of the cell to avoid local strain. The currents will be characterized by permeation (conductance and selectivity), gating (using steady state and transient changes in length and voltage), response to pharmacological agents, temperature and variability with respect to anatomical origin. Stretch dependent changes in the action potential will be recorded under current clamp conditions. To correlate the effects of stretch with changes in intracellular Ca2+ levels, fluorescence microscopy with Ca2+ indicators will be done during defined stretch protocols. Single channel patch clamp studies will be used to characterize the coupling of stress in the cortical region of the cells to channel activation. Stimuli will include transient and steady state mechanical stress, voltage and pharmacological agents. To measure the patch geometry and mechanical properties that are necessary to characterize the stimulus, a pressure servo and video microscopy of the patch combined with image fitting routines will be combined to estimate the mean patch stress and strain.
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