Background-Despite increased secondary cardiovascular events in patients with ischemic cardiomyopathy (ICM), the expression of innate cardiac protective molecules in the hearts of patients with ICM is incompletely characterized. Therefore, we used a nonbiased RNAseq approach to determine whether differences in cardiac protective molecules occur with ICM. Methods and Results-RNAseq analysis of human control and ICM left ventricular samples demonstrated a significant decrease in KCNJ11 expression with ICM. KCNJ11 encodes the Kir6.2 subunit of the cardioprotective K-ATP channel. Using wild-type mice and kcnj11-deficient (kcnj11-null) mice, we examined the effect of kcnj11 expression on cardiac function during ischemia-reperfusion injury. Reactive oxygen species generation increased in kcnj11-null hearts above that found in wild-type mice hearts after ischemia-reperfusion injury. Continuous left ventricular pressure measurement during ischemia and reperfusion demonstrated a more compromised diastolic function in kcnj11-null compared with wild-type mice during reperfusion. Analysis of key calcium-regulating proteins revealed significant differences in kcnj11-null mice. Despite impaired relaxation, kcnj11-null hearts increased phospholamban Ser16 phosphorylation, a modification that results in the dissociation of phospholamban from sarcoendoplasmic reticulum Ca2+, thereby increasing sarcoendoplasmic reticulum Ca2+-mediated calcium reuptake. However, kcnj11-null mice also had increased 3-nitrotyrosine modification of the sarcoendoplasmic reticulum Ca2+-ATPase, a modification that irreversibly impairs sarcoendoplasmic reticulum Ca2+ function, thereby contributing to diastolic dysfunction. Conclusions-KCNJ11 expression is decreased in human ICM. Lack of kcnj11 expression increases peroxynitrite-mediated modification of the key calcium-handling protein sarcoendoplasmic reticulum Ca2+-ATPase after myocardial ischemia-reperfusion injury, contributing to impaired diastolic function. These data suggest a mechanism for ischemia-induced diastolic dysfunction in patients with ICM.