The long term goal is a comprehensive understanding of the regulation of cardiac excitation-contraction coupling (ECC) and contractile force, particularly with respect to cellular Ca regulation, which is central in regulating physiological function in the heart (both electrical & mechanical). Indeed, altered myocytes Ca transport has been implicated in hypertrophy, heart failure and arrhythmias. This project period focuses on: 1) fundamental aspects of ECC, 2) regulation of Na/Ca exchange (NCX) and 3) dynamic regulation of calmodulin (CaM) in ventricular myocytes. Studies will use mainly isolated adult ventricular myocytes under voltage clamp, with confocal imaging & dynamic fluorescence measurements of [Ca]i, [Na]i, [CaM] and [Ca-CaM], complemented by some biochemical/ molecular work. Genetically altered mice will also be used to test specific mechanisms.
Aim 1 will address important fundamental ECC questions such as: A. What dictates ECC gain, single Ca channel current amplitude (iCa) or number of open channels (NP0)? B. How many Ca channels must open physiologically to activate local ECC? C. Whether gradedness of ECC as a function of ICa is due exclusively to the number of junctions firing. D. Whether the shut-off of SR Ca release depends on local [Ca] inside or outside the sarcoplasmic reticulum (SR). E. What role does SR Ca reloading (vs. ryanodine receptor recovery) play in restitution of ECC in heart. The results will help us understand better how Ca release is controlled in both the normal heart and during pathology (e.g. heart failure & arrhythmias). NCX is the main pathway to extrude the Ca that enters the myocyte at each beat, and determines diastolic tone and carries arrhythmogenic current.
Aim 2 addresses currently critical NCX questions as to: A. how NCX is activated and deactivated in the cellular environment, B. what the true stoichiometry of transport is (and how local [Ca]i and [Na]i gradients can complicate this), and C. whether NCX plays an important modulatory role on ECC.
Aim 3 focuses on CaM, which is a major mediator of many Ca-dependent signaling pathways in heart, but is understudied.
The aims assess: A. the total and free [CaM] in myocytes, its localization & binding kinetics in the myocyte (at different [Ca]i), and B. dynamic changes in [Ca-CaM] and its binding to targets in the myocyte during normal Ca transients (e.g. as a function of frequency & amplitude). Different fluorescent CaM variants and dual GFP Ca-CaM sensors (which alter fluorescence resonance energy transfer or FRET upon binding) will be used. This work will greatly increase fundamental understanding of key cardiac Ca transport systems in the intact cellular environment under physiological conditions.
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