As a VA anesthesiologist and physiologist I study protection against myocardial infarction, a significant medical problem in aging Veterans. In Ischemic Preconditioning (IPC), for example, several shorter ischemic periods before sustained ischemia/reperfusion (IR) attenuate infarction. Various triggering mechanisms have been described for IPC, including nitric oxide (NO) and superoxide formation. Genetic predisposition, however, may be an important confounding factor when trying to transfer it into clinical practice;while some patients may benefit others do not. Profound differences in ischemic tolerance exist not just among but also within certain species. For example, Dahl Salt Sensitive (SS) rats are more susceptible to IR injury than Brown Norways (BN), making them an ideal model to study the genotype of diseases phenotypically similar to African-American patients. In a unique chromosomal substitution (consomic) model constructed at the Medical College of Wisconsin introgression of BN chromosome 6 into SS renders the resulting SS6BN consomic more IR resistant while narrowing the genetic difference to a single chromosome. This offers an excellent starting point to study the genetic basis of cardioprotective strategies like IPC. Although NO has been implicated in decreasing infarct size in BN vs SS without IPC, it is yet unknown if SS can respond to IPC and if resistance to IPC is related to differences in endothelial NO synthase (eNOS) activity possibly modulated by the DNA-binding protein inhibitor Id2 and the peroxisome proliferator-activated receptor 3 (PPAR3). eNOS can produce NO or superoxide. Both can modulate mitochondrial function, which in turn can function as a trigger and effector of IPC. Thus, my overall hypothesis is that failure/success of cardioprotection by IPC is mediated by genes regulating NO production and mitochondrial function. I therefore propose to use this consomic model to study two specific aims: I) Determine if a different genetic background is responsible for differential cellular and mitochondrial protection by IPC, and II) if eNOS and/or its upstream modulators Id2 and PPAR3 are candidate genes responsible for this differential protection. Four hypotheses are tested: 1) Genes on BN chromosome 6 are necessary for IPC as evidenced by better cardiac function and less infarction in BN &SS6BN vs SS. 2) Genes on BN chromosome 6 are necessary for IPC as evidenced by more efficient mitochondrial function in BN &SS6BN vs SS. 3) Protection of cellular and mitochondrial function by IPC in intact hearts depends on the signaling pathway Id2->PPAR3->eNOS->NO modulated by genes on rat chromosome 6. 4) Differential protection of cellular and mitochondrial function in isolated cardiomyocytes depends on NO availability. Approaches for 1 &2: Various cardiac and mitochondrial functions, NO production and infarct size are measured in intact, beating hearts to assess quantity and quality of genetically determined protection by IPC. Approaches for 3 &4: Additional beating heart experiments are conducted in the presence of NOS inhibitors, an NO-donor, or a PPAR3 agonist or antagonist, and differences in Id2, PPAR3, and eNOS expression and NO levels and localization are determined. In addition, different mitochondrial functions in the absence or presence of NO are measured in isolated myocytes to determine the role and origin of NO in mediating differential cellular and mitochondrial protection by IPC in this genetic model. Genetic tools are essential to study the role of genetic predisposition. Correlating functional outcomes, infarct size, and mitochondrial functions with IPC, NO levels, protein expressions and genotype will allow us to define the signaling pathway and role of NO and mitochondrial function in cardioprotection and to delineate the subcellular and mitochondrial phenotypes associated with the cardioprotective genotype variation. This CDA marks the indispensable basis for further investigations on the role of genetics in cardioprotection and will be a critical milestone to achieve investigative independence in cardiovascular research at the VA.
As a VA anesthesiologist and cardiac physiologist I study protective strategies against myocardial infarction, a significant medical problem in Veterans. One such strategy is "Preconditioning". Genetic predisposition, however, may be a confounding factor when trying to transfer its use into clinical practice. Profound differences in tolerance to ischemia exist among and even within certain species. Utilizing a unique genetic rat model it could be shown that transfer of one chromosome from a more resistant strain into a more sensitive strain renders the resulting "consomic" more resistant against infarction while narrowing the genetic difference to a single chromosome compared to the sensitive parental strain. This offers an excellent starting point to study the genetic basis and mechanisms of cardioprotection, from intact hearts to isolated mitochondria. This proposal is the indispensable basis for me to further investigate the role of genetics in cardioprotection and represents a critical milestone to achieve independence in cardiovascular research in the VA system.