Inducible Cardiac-specific Pyruvate Dehydrogenase E1α Deficiency Sensitizes Heart To Ischemic Insults

Ischemic heart disease is a condition where the heart muscle is weakened and the left ventricle is usually enlarged and dilated. This condition can result from coronary artery stenosis or occlusion or chronic myocardial ischemia. Myocardial ischemia is also a metabolic disease.Ischemia inhibits the oxidative metabolism of both free fatty acids and glucose due to the restriction of oxygen and nutrients; however, glucose transport and glycolytic ATP production are increased as an adaptation to the dramatic switch from aerobic to anaerobic metabolism. Thus, the switch between fatty acid and glucose metabolism can be exploited for the treatment of myocardial ischemia.PDHc, a multi-enzyme complex located in the mitochondrial matrix,plays a key role in aerobic energy metabolism. This multienzyme complex occupies a central crossroad of glycolysis and the tricarboxylic acid cycle by catalyzing the oxidative decarboxylation of pyruvate to form acetyl Co A. There is altered cardiac glucose in various pathophysiological states consequently causing PDHc to be altered. Because of the importance of PDH in cell energy metabolism and the high mortality in PDH knockdown mice, the biological effects of conventional cardiac PDH lack of good research on animal models. Moreover, the associated signal transduction mechanisms are lacking depth discussion under ischemia condition.AMP-activated protein kinase(AMP-activated protein kinase, AMPK)enzyme is one of the most important regulator of intracellular stress response, the activation and deactivation is mainly affected by the level of intracellular energy regulation. AMPK activation enhance the activity of ATP synthesis system while reducing the consumption of ATP. Our previous work demonstrated that ischemic myocardium AMPK activation can regulate energy substrate metabolism in ischemic myocardium,increase glucose utilization by affecting glucose transporter GLUT4 membrane translocation medium. Also, our previous work also found that the phosphorylation of AMPK activation in ischemic myocardium depends on its upstream kinase LKB1 and Sestrin2.Both PDH and the AMPK signaling pathway exert cardioprotective effects on the myocardium when it is subjected to stress. However, the mechanism by which PDH and AMPK activation modulate cardiac metabolism and execute cardioprotection remains elusive. Therefore, the present study, we constructed an inducible cardiac specific PDH E1αknockout mice(inducible cardiac-specific PDH E1α knockout, PDH E1αi CKO), discussed the deficiency of the cardiac specific PDH E1α onmyocardial ischemic injury, and analysis of relevant signaling mechanisms.Aim:(1) To determine the role of PDH in regulating cardiac function during ischemia stress;(2) To confirm PDH regulation of myocardial glucose metabolism during ischemia;(3) To determine the regulation between PDH and AMPK and the mechenism of the regulation.Method: Construction of PDH E1α i CKO mice, with Cre mice as controls, establish myocardial ischemia, myocardial tissues were collected to detect the expression of the correlation signal, analyze systolic and diastolic function at different time points after ischemia. Working-Heart Perfusion System was used to detect myocardial glucose and fatty acid metabolism, glycolysis and glucose uptake.Result:(1) Cardiac-specific deletion of the Pdha1 gene was generated by breeding PDH E1α flox/flox mice bred with transgenic male mice that carried an autosomally integrated Cre gene driven by the cardiac-specific alpha-myosin heavy chain promoter(αMHC). Genotyping mice by PCR analysis, the resulting progeny were Cre ERT2-PDHflox/flox mice. Tamoxifen was injected(0.08 mg/g, i.p. 5 days) to induce cardiac PDH E1α knockoutin vivo, generated inducible cardiac-specific PDH E1α knockout(PDH E1α i CKO) mice.(2) Compared to Cre ERT2 mice, PDH E1α i CKO mice gradually increased myocardial hypertrophy phenotype with age. To 4 months of age, the ratio of heart weight / body weight in PDH E1α i CKO mice significantly increased; TRITC-WGA staining confirmed PDH E1α i CKO mouse cardiomyocyte hypertrophy; Mac-2 immunohistochemical staining,Masson staining and metalloproteinase-9(MMP-9) and interleukin-6(IL-6) RT-PCR confirmed the increased fibrosis and inflammation in PDH E1α i CKO mice. Moreover, compared with Cre ERT2 mice, PDH E1αi CKO mice increased mortality significantly by shorter survival period.(3) Consistent with the results of myocardial fibrosis, using echocardiography confirmed: PDH E1α knockout leading to cardiac systolic and diastolic dysfunction, PDH E1α i CKO mice left ventricular inner diameter(LVID d) during diastole increased and ejection fraction(EF%) reduced compared to Cre ERT2 mice. And we found, the myocardial basal metabolic rate of glucose oxidation in mice significantly reduced after PDH E1α knockout. These results confirm the important role of PDH in maintaining cardiac function and metabolism.(4) To clarify the biological consequences in PDH E1α i CKO mice and control mice, the in vivo model of myocardial ischemia were established. The results showed, PDH E1α knockout significantlyincreased the extent of myocardial ischemia and myocardial infarct size.After using PDH specific agonist dichloroacetate(DCA) can effectively increase cardiac PDH activity, effectively reducing the extent of myocardial injury. Cardioprotective effect of DCA was completely blocked in PDH E1α i CKO.(5) The results further confirmed that the use of DCA can effectively increase the glucose transporter(GLUT4) membrane translocation during myocardial ischemia, promoting ischemic myocardial glucose uptake.And DCA activate cardiac PDH can effectively optimize energy substrate metabolism, increase glucose utilization and inhibit fatty acid oxidation.(6) More importantly, our results showed that, using PDH specific agonists DCA treatment can effectively enhance AMPK phosphorylation level during ischemia. Conversely, DCA regulatory role in energy metabolism were inhibited in AMPK KD mice. The results suggest that the biological effects of cardiac PDH may be mediated by AMPK activity.(7) A direct relationship between the regulation of cardiac PDH and AMPK was found by Co-IP. We first found after cardiac specific PDH E1α knockout, complex subunit interactions of Sestrin2 and PDH E2/3significantly enhanced, in turn weakened the formation Sestrin2-LKB1 complex, resulting the inhibition of AMPK activity in PDH E1α i CKO mice during myocardial ischemia.Conclusion: This is the first study using cardiac specific PDH E1αinducible knockout mice confirmed that PDH is a key factor to maintain myocardial energy metabolism and myocardial systolic and diastolic function, activation of PDH can effectively adjust the ischemic myocardium substrate metabolism. We first discovered the existence of a direct relationship between the regulation of cardiac PDH and AMPK,PDH deletions may lead to inactivation of AMPK and significantly increased myocardial ischemic injury.