Differential 02 Sensing in Right and Left hearts: Crosstalk with Ca-Signaling

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DESCRIPTION (provided by applicant): Acute oxygen deprivation leads to an array of events in the myocardium that include ischemic infarction, arrhythmia, cardiomyopathy, and heart failure. Intrinsic cardioprotection during hypoxia occurs by decreasing oxygen demand through alternative energy handling and Ca2+ signaling. However, the exact mechanisms by which the left and right hearts sense O2 and activate such protective mechanisms at the time of injury are mostly unknown. While current cardio-protective therapies mainly prevent reoccurrence or progression of disease post myocardial infarction, they fail to combat cell death during hypoxic injury. Moreover, the left and right ventricles differ in their response to injury. Although, current therapies improve left ventricular function, right heart pathologies are not effectively treated. Our long-term objective is to determine differential mechanisms of O2 sensing and protection during hypoxia in left and right hearts to help develop specific therapies against early phase hypoxic injury. Our main hypothesis is that anoxia/hypoxia differentially alters calcium signaling in right versus left ventricles. This hypothesis is based on the following observations: 1) acute anoxia leads to instant loss of electrically evoked calcium transients in single isolated ventriclular myocytes 2) the Ca content of the sarcoplamic reticulum (SR) in response to application of 10mM caffeine solutions decreases secondary to anoxia in a reversible manner 3) The timeline of SR suppression and recovery is different between right and left ventricles 4) It has been shown that loss of oxygen-sensor Hypoxia Inducible Factor-la (HIF-1a) gene alters Ca-signaling in the myocardium and we have found higher baseline mRNA levels of this protein in the right ventricle. Based on these observations, the focus of this proposal is to: 1. Characterize membrane currents and calcium signaling in right versus left ventricular myocytes in response to anoxia/hypoxia. We will expose isolated cardiomyocytes to anoxia/hypoxia and monitor i) current magnitude and kinetics of K, Na, Ca ii) magnitude of Ca release and uptake. 2. Determine mechanisms of differential SR regulation in right and left ventricles during hypoxia. Here we will examine the role of reactive oxygen species and activated kinases (PKA, Ca/Calmodulin Kinase II) on i) redox regulation of ionic currents and SR Ca release/uptake ii) regulation of SR Ca release/uptake by ryanodine receptor (RyR)/SERCA-Phospholamban phosphorylation 3. Determine the effect of transient HIF-1a overexpression on calcium signaling in right and left ventricles. We will overexpress HIF-1a gene in cardiomyocytes using adenoviral vectors and evaluate the i) magnitude and kinetics of calcium currents and calcium release/uptake ii) degree of RyR and SERCA-2A expression.