The long term goal is a comprehensive understanding of the regulation of cardiac contractile force, particularly with respect to cellular Ca regulation, which is central in regulating physiological function in the heart (both electrical and mechanical). Thus it is crucial to understand fundamental aspects of [Ca]i regulation in detail. Indeed, altered myocyte Ca transport has been implicated in hypertrophy, heart failure and arrhythmias). This project focuses on 1) interactions between dihydropyridine receptor (DHPR) and ryanodine receptors (RyR), 2) Ca-Calmodulin dynamics in intact cells, 3) the mechanism of frequency-dependent acceleration of relaxation and 4) some key excitation-contraction (EC) coupling questions made more addressable by recent transgenic or knockout (KO) mice. 1. Two manifestations of functional linkage between DHPR and RyR will be explored. First, studies will examine how the Ca channel agonist Bay K 8644 activates resting Ca release in ventricular myocytes (Ca sparks) independent of Ca influx. These will include studies comparing effects of Bay K 8644 of Ca sparks and Ca current. Second, studies will examine the effects of small peptides from the cardiac DHPR on Ca spark frequency and E-C coupling in voltage clamped ventricular myocytes. Finally, the interaction of these peptides with the Bay K 8644 effect will be studied. These will help to understand how these 2 proteins which are critical in E-C coupling interact in the intact ventricular myocyte. 2. How [Ca-Calmodulin] changes dynamically during the cardiac cycle will be measured. Calmodulin is a ubiquitous second messenger, but little is known about dynamic fluctuations of Ca-calmodulin in the intact ventricular myocyte. This will be examined using new fluorescence resonance energy transfer probes, based on modified green fluorescent proteins linked by calmodulin biding peptides. This will provide fundamental new data about this system. 3. The mechanism of frequency-dependent acceleration of relaxation and [Ca]i decline in cardiac muscle (independent of beta-adrenergic agonists) is unknown, but is important in diastolic filling at high heart rate. Detailed studies will evaluate the role of phospholamban, CaMKII and SR Ca-ATPase isoforms in mediating this important fundamental effect. 4. The importance of intra-SR free [Ca] (vs total SR Ca load) in the regulation of SR Ca release will be determined using mice overexpressing calsequestrin (and/or lacking phospholamban), [Ca]i measurement and voltage clamp. It will also be determined if overexpression of Na/Ca exchange in mice where SR function depressed, can a) rescue hypertrophic phenotype and b) create Ca flux balance like in human ventricle. 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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