Inducible, Heterozygous Ryr2 Knockout Mice Reveal A Key Role For Sr Ca2+Release In Cardiac Metabolism

BackgroundHeart failure is a multi-faceted condition characterized by numerous functional lesions including contractile dysfunction and metabolic inflexibility. The cardiac ryanodine receptor (RYR2) Ca2+-release channel plays an essential role in excitation contraction and its expression and/or activity is reduced by up to 50% in multiple models of heart disease including both hypertensive and diabetic cardiomyopathies. Given that Ca2+ release is known to stimulate oxidative energy metabolism, RYR2 is poised to have an additional role in pacing cardiomyocyte metabolism. To test which aspects of heart failure may be downstream of RYR2 dysfunction, we have developed a model whereby RYR2 protein is reduced to a stable 50% level in adult cardiomyocytes. The overall objective of our research is to test whether a 50% reduction in RYR2 function can drive the progression of cardiac pathology and metabolic dysfunction.MethodsWe generated mice with a conditional, haploinsufficiency for RYR2 function by crossing mice with only one floxed Ryr2 allele with a cardiac specific, tamoxifen-inducible Cre deleter mouse line.ResultsThis deletion led to a stable 50% decrease in Ryr2 mRNA and RYR2 protein levels within one month after tamoxifen injection. Despite decreased RYR2 levels, no significant changes were observed in cardiac output measured by echocardiography or in the contractility of isolated cardiomyocytes. Only minor decreases in cardiac output were observed during ex vivo perfusion of isolated working hearts. However, observed a significant decrease in glucose oxidation during working heart perfusion despite no significant reductions in ATP levels or in the oxidation of other metabolic substrates. Metabolomic analyses provided evidence for significant decreases in key TCA cycle intermediates but not products of non-oxidative glycolysis. Targeted biochemical analysis revealed further mechanistic details linking partial RYR2 ablation to metabolic changes. Collectively these results indicate that a chronic and stable 50% decrease in RYR2, even without alterations in basal heart function, is sufficient to alter cardiac cellular metabolism and impede the use of glucose as a substrate for oxidative ATP metabolism.ConclusionThese results provide evidence that RYR2 is critically involved in stimulating oxidative metabolism in cardiomyocytes. Furthermore, these findings show that RYR2 dysfunction is able to disrupt cardiac metabolism regardless of its effect on excitation-contraction, which suggests a role for RYR2 dysfunction in mediating cardiac dysfunction in heart disease. BackgroundHeart failure is a multi-faceted condition characterized by numerous functional lesions including contractile dysfunction and metabolic inflexibility. The cardiac ryanodine receptor (RYR2) Ca2+-release channel plays an essential role in excitation contraction and its expression and/or activity is reduced by up to 50% in multiple models of heart disease including both hypertensive and diabetic cardiomyopathies. Given that Ca2+ release is known to stimulate oxidative energy metabolism, RYR2 is poised to have an additional role in pacing cardiomyocyte metabolism. To test which aspects of heart failure may be downstream of RYR2 dysfunction, we have developed a model whereby RYR2 protein is reduced to a stable 50% level in adult cardiomyocytes. The overall objective of our research is to test whether a 50% reduction in RYR2 function can drive the progression of cardiac pathology and metabolic dysfunction. Heart failure is a multi-faceted condition characterized by numerous functional lesions including contractile dysfunction and metabolic inflexibility. The cardiac ryanodine receptor (RYR2) Ca2+-release channel plays an essential role in excitation contraction and its expression and/or activity is reduced by up to 50% in multiple models of heart disease including both hypertensive and diabetic cardiomyopathies. Given that Ca2+ release is known to stimulate oxidative energy metabolism, RYR2 is poised to have an additional role in pacing cardiomyocyte metabolism. To test which aspects of heart failure may be downstream of RYR2 dysfunction, we have developed a model whereby RYR2 protein is reduced to a stable 50% level in adult cardiomyocytes. The overall objective of our research is to test whether a 50% reduction in RYR2 function can drive the progression of cardiac pathology and metabolic dysfunction. MethodsWe generated mice with a conditional, haploinsufficiency for RYR2 function by crossing mice with only one floxed Ryr2 allele with a cardiac specific, tamoxifen-inducible Cre deleter mouse line. We generated mice with a conditional, haploinsufficiency for RYR2 function by crossing mice with only one floxed Ryr2 allele with a cardiac specific, tamoxifen-inducible Cre deleter mouse line. ResultsThis deletion led to a stable 50% decrease in Ryr2 mRNA and RYR2 protein levels within one month after tamoxifen injection. Despite decreased RYR2 levels, no significant changes were observed in cardiac output measured by echocardiography or in the contractility of isolated cardiomyocytes. Only minor decreases in cardiac output were observed during ex vivo perfusion of isolated working hearts. However, observed a significant decrease in glucose oxidation during working heart perfusion despite no significant reductions in ATP levels or in the oxidation of other metabolic substrates. Metabolomic analyses provided evidence for significant decreases in key TCA cycle intermediates but not products of non-oxidative glycolysis. Targeted biochemical analysis revealed further mechanistic details linking partial RYR2 ablation to metabolic changes. Collectively these results indicate that a chronic and stable 50% decrease in RYR2, even without alterations in basal heart function, is sufficient to alter cardiac cellular metabolism and impede the use of glucose as a substrate for oxidative ATP metabolism. This deletion led to a stable 50% decrease in Ryr2 mRNA and RYR2 protein levels within one month after tamoxifen injection. Despite decreased RYR2 levels, no significant changes were observed in cardiac output measured by echocardiography or in the contractility of isolated cardiomyocytes. Only minor decreases in cardiac output were observed during ex vivo perfusion of isolated working hearts. However, observed a significant decrease in glucose oxidation during working heart perfusion despite no significant reductions in ATP levels or in the oxidation of other metabolic substrates. Metabolomic analyses provided evidence for significant decreases in key TCA cycle intermediates but not products of non-oxidative glycolysis. Targeted biochemical analysis revealed further mechanistic details linking partial RYR2 ablation to metabolic changes. Collectively these results indicate that a chronic and stable 50% decrease in RYR2, even without alterations in basal heart function, is sufficient to alter cardiac cellular metabolism and impede the use of glucose as a substrate for oxidative ATP metabolism. ConclusionThese results provide evidence that RYR2 is critically involved in stimulating oxidative metabolism in cardiomyocytes. Furthermore, these findings show that RYR2 dysfunction is able to disrupt cardiac metabolism regardless of its effect on excitation-contraction, which suggests a role for RYR2 dysfunction in mediating cardiac dysfunction in heart disease. These results provide evidence that RYR2 is critically involved in stimulating oxidative metabolism in cardiomyocytes. Furthermore, these findings show that RYR2 dysfunction is able to disrupt cardiac metabolism regardless of its effect on excitation-contraction, which suggests a role for RYR2 dysfunction in mediating cardiac dysfunction in heart disease.