Regulation of K+ in cellular and extracellular compartments is of central importance to volume regulation, fluid transport, the resting membrane potential and electrical activity in excitable cells. In heart, extracellular [K+] in narrow clefts between cells and in T-tubules is not always in diffusional equilibrium with external K+ ([K+]o ) and can be depleted or accumulated and thereby differ from [K+]o during a wide range of physiological/pathological conditions. The central goal of the project is to elucidate the consequences of extracellular K+ accumulation ([K+]a) on cardiac electrical activity in physiological and pathological conditions. To do so, we developed new K+ sensitive fluorescent probes that can be anchored to plasma membranes and upon binding to K+ produce a fluorescence change that reports the local [K+]. We can calibrate the probe, measure rapid (1 ms) changes in [K+]a in ventricular myocytes and Langendorff hearts.
The Specific Aims are:
Aim 1 : Improve our new K+ sensitive probes consisting of: i) hydrophobic groups to anchor the probe to lipid bilayers, ii) hydrophilic groups to dissolve and prevent the probe from diffusing across the membrane into cells, iii) a fluorescent dye that senses iv) the binding of K+ to a K+- selective organic crown ether ring and exhibits a fluorescence change as a function of [K+] in the range of 1-50 mM.
Aim 2 : To test the hypothesis that different K+ currents contribute differently to [K+]a under different physiological conditions( ?? heartrate, ?? 2-adrenergic activity) and metabolic states. [K+]a responses will be compared during pharmacological interventions of channels responsible for K+ efflux (IK1, It,o, IK,slow, IKr, IKs, IKATP) and during manipulations of K+ re-uptake via the Na/K pumps. [K+]a will be compared in atrial and ventricular myocytes to test the effects of differences in APDs, K+ currents and T-tubules. In perfused hearts, APs and [K+]a will be simultaneously mapped, and changes in [K+]a will be measured on a beat-to-beat basis.
Aim 3 : To test the hypothesis that in pathological conditions, the rise of [K+]a can lead to regions of myocardium with an increase in excitability and a greater propensity to focal activity or premature beats that initiate arrhythmias. Simultaneous optical mapping of APs and [K+]a in perfused hearts will be used to measure APs and beat-to- beat changes in [K+]a transients to elucidate the role of [K+]a during arrhythmias elicited by ischemia/reperfusion applied either globally (zero flow) or locally via an LAD ligation. [K+]a elevation will be correlated with coronary vessels, fiber orientation and the origins of premature impulses. Such a project will develop K+ sensitive dyes for applications to cardiac electrophysiology and for the first time characterize [K+]a on a beat-to-beat basis. Dual optical mapping of [K+]a and APs will determine the changes in [K+]a that occur during physiological and pathological interventions, will validate methods to reduce [K+]a to offer new targets to treat cardiac diseases.
Extracellular K+ accumulation in T-tubules and narrow invaginations of heart muscle has been implicated as a determinant of normal cardiac physiology and pathology. However, [K+] concentrations in narrow regions that are not in diffusional equilibrium with the surrounding aqueous medium have never been measured directly. The project will synthesize new K+ indicator probes, measure [K+] in t-tubules, and interstitial spaces and characterize local [K+] accumulation in normal physiological conditions and various pathologies. The role of K+ accumulation may lead to a new understanding of mechanisms that enhanced the likelihood of arrhythmias, provide novel approaches to reduce defibrillation threshold, reduce the recurrence of VF after defibrillation, improve the success rate of defibrillation past 5 min of fibrillation and may lead to new therapeutic targets for the treatment of ischemic heart disease.
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