CCBM: Calcium Cycling and Regulation of the Cardiac AP

Dilated cardiomyopathy (DCM) is the most common form of primary cardiac muscle disease, with prevalence estimated at 36.5 cases per 100,0002. DCM is characterized by ventricular dilatation, decreased myocardial contractility and cardiac output, and increased risk of sudden cardiac death3-5. Ventricular myocytes isolated from dilated cardiomyopathic hearts ("failing hearts") exhibit changes in expression levels of proteins involved in repolarization of the action potential (AP) and intracellular calcium (Ca 2+ ) cycling. These changes are accompanied by reduction of junctional sarcoplasmic reticulum (JSR) Ca 2+ concentration6-8, peak intracellular Ca 2+ transient amplitude9-11, slowed diastolic Ca 2+ extrusion11,12 and prolongation of AP duration13,14. We have previously formulated a "minimal" computational model of the failing canine ventricular myocyte that incorporates experimental data on down-regulation of potassium (K + ) currents and the SR Ca 2+ -ATPase, and up-regulation of the Na + -Ca 2+ exchanger11,15. This model is able to qualitatively reconstruct changes in AP and Ca 2+ transient morphology observed in failing myocytes. Model simulations predict that down-regulation of the SR Ca 2+ -ATPase and up-regulation of the Na + -Ca 2+ exchanger reduces JSR Ca 2+ level, JSR Ca 2+ release and the magnitude of Ca 2+ -dependent inactivation of L-type Ca 2+ current (I Ca,L ). On depolarization, I Ca ,L in canine (and human) ventricular myocytes exhibits an initial peak, which declines with time to generate a maintained inward current during the plateau phase14. The models predict that decreased Ca 2+ -dependent inactivation leads to an increase in magnitude of this maintained component of I Ca ,L . during the plateau phase, thereby prolonging AP duration. This modeling result has led us to hypothesize that the maintained component of I Ca ,L , active during the plateau phase of the AP, is increased in heart failure. Further, we hypothesize that JSR Ca 2+ level, through effects on JSR Ca 2+ release and Ca 2+ -dependent inactivation of I Ca ,L , is an important modulator of AP duration under a range of conditions producing changes in JSR Ca 2+ level . The general goal of the proposed research is to test this hypothesis by means of experiments coupled with computational modeling. Specific Aims of this four year proposal are:

Aim 1: Develop computational models of voltage-dependent activation, and voltage- and Ca 2+ -dependent inactivation of I Ca ,L based on experimental measurements in both normal and failing canine ventricular myocytes. Test the hypothesis that Ca 2+ -dependent inactivation of I Ca ,L is the dominant inactivation mechanism over sub-second time scales.

Aim 2: Develop integrative models of normal and failing canine ventricular myocytes based on the I Ca ,L model of Aim 1, and incorporating local-control of JSR Ca 2+ release. Existing common pool models of the cardiac myocyte exhibit all-or-none Ca 2+ release rather than the graded release measured experimentally. APs simulated using these models become unstable when the balance between voltage- and Ca 2+ -dependent I Ca ,L inactivation is adjusted to match experimental data. We hypothesize that an integrative model of the myocyte incorporating emerging principles of local-control of JSR Ca 2+ release will not exhibit these instabilities, and will be necessary for quantifying the relationship between JSR Ca 2+ load and release, Ca 2+ -dependent inactivation, and AP morphology to be investigated in Aim 3. Aim 2 will develop such a model and test this hypothesis.

Aim 3: Use the models developed in Aims 1 and 2, in conjunction with electrophysiological experiments, to test the hypothesis that JSR Ca 2+ level and Ca 2+ release are important modulators of I Ca ,L magnitude and AP duration.

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