The focus of the project is on the effects of SR [Ca] upon excitation-contraction coupling (ECC). The gain of ECC and fractional SR Ca release during a cardiac twitch depend very steeply on SR Ca content. It has also been suggested that an increased diastolic SR Ca leak (and consequent unloading of the SR during diastole), along with the SR Ca pump fluxes, is a major factor in reducing contractility in heart failure (HF). However, the critical factors which limits the SR Ca content ([Ca]SRT) and free intra-SR ([Ca]SR) are still somewhat controversial. In addition the work suggesting increased diastolic SR Ca leak (Jleak) in HF has only been measured at the single channel in lipid bilayers, and has not been assessed in an intact cellular setting. The overall hypothesis is that Jleak, along with alterations in SR Ca pump flux (Jpump), affect the SR [Ca] and that changes in these take place physiologically and pharmacologically, thus affecting ECC.
Specific Aim 1 is to determine what limits [Ca]SR in intact myocytes during diastole. We had reported that the maximal diastolic SR Ca load approached the thermodynamic limit of the SR Ca pump, and that the diastolic leak of Ca from the SR was very small compared to SR Ca-pump flux. In contrast, it has been demonstrated that blocking Jleak in intact cells can dramatically increase [Ca]SRT. We hypothesize that this dichotomy has a rational explanation and that the leak is very steeply dependent on [Ca]SRT. To clarify this we will measure the forward and reverse SR Ca Pump fluxes (Jpumpf & Jpumpr or"""""""" backflux"""""""") and Jleak in intact cells as a function of [Ca]SRT. These experiments will allow us to quantitatively assess how Jpumpr vs Jleak balance Jpumpf during diastole.
Specific Aim 2 is to measure [Ca]SR directly in intact myocytes during diastole. We also have some promising new preliminary results using for the first time an intra-SR free Ca indicator in intact ventricular myocytes. We therefore have the unique ability to assess changes in [Ca]SR in intact cells. This is especially important because [Ca]SR (not [Ca]SRT) is what governs: a) intra SR buffering, b) the driving force for SR Ca release, c) single RyR conductance and d) is what thermodynamically limits SR Ca uptake. Overall these experiments using noveel methods and protocols will provide new mechanistic and quantitative understanding about how [Ca]SRT and release are regulated in intact ventricular myocytes. ? ?
