To explore mechanisms by which SGLT2 inhibitors protect diabetic hearts from heart failure, we examined the effect of empagliflozin (Empa) on the ultrastructure of cardiomyocytes in the noninfarcted region of the diabetic heart after myocardial infarction (MI). OLETF, a rat model of type 2 diabetes, and its nondiabetic control, LETO, received a sham operation or left coronary artery ligation 12 h before tissue sampling. Tissues were sampled from the posterior ventricle (i.e., the remote noninfarcted region in rats with MI). The number of mitochondria was larger and small mitochondria were more prevalent in OLETF than in LETO. Fis1 expression level was higher in OLETF than in LETO, while phospho‐Ser637‐Drp1, total Drp1, Mfn1/2, and OPA1 levels were comparable. MI further reduced the size of mitochondria with increased Drp1‐Ser616 phosphorylation in OLETF. The number of autophagic vacuoles was unchanged after MI in LETO but was decreased in OLETF. Lipid droplets in cardiomyocytes and tissue triglycerides were increased in OLETF. Empa administration (10 mg/kg per day) reduced blood glucose and triglycerides and paradoxically increased lipid droplets in cardiomyocytes in OLETF. Empa suppressed Fis1 upregulation, increased Bnip3 expression, and prevented reduction in both mitochondrial size and autophagic vacuole number after MI in OLETF. Together with the results of our parallel study showing upregulation of SOD2 and catalase by Empa, the results indicate that Empa normalizes the size and number of mitochondria in diabetic hearts and that diabetes‐induced excessive reduction in mitochondrial size after MI was prevented by Empa via suppression of ROS and restoration of autophagy.

BackgroundDiabetes mellitus (DM) is associated with an increased risk of ischemic heart disease and of adverse outcomes following myocardial infarction (MI). Here we assessed the role of endoplasmic reticulum (ER) stress in ventricular dysfunction and outcomes after MI in type 2 DM (T2DM).Methodology and Principal FindingsIn hearts of OLETF, a rat model of T2DM, at 25∼30 weeks of age, GRP78 and GRP94, markers of ER stress, were increased and sarcoplasmic reticulum calcium ATPase (SERCA)2a protein was reduced by 35% compared with those in LETO, a non-diabetic control. SERCA2a mRNA levels were similar, but SERCA2a protein was more ubiquitinated in OLETF than in LETO. Left ventricular (LV) end-diastolic elastance (Eed) was higher in OLETF than in LETO (53.9±5.2 vs. 20.2±5.6 mmHg/µl), whereas LV end-systolic elastance and positive inotropic responses to β-adrenergic stimulation were similar in OLETF and LETO. 4-Phenylbutyric acid (4-PBA), an ER stress modulator, suppressed both GRP up-regulation and SERCA2a ubiquitination and normalized SERCA2a protein level and Eed in OLETF. Sodium tauroursodeoxycholic acid, a structurally different ER stress modulator, also restored SERCA2a protein level in OLETF. Though LV dysfunction was modest, mortality within 48 h after coronary occlusion was markedly higher in OLETF than in LETO (61.3% vs. 7.7%). Telemetric recording showed that rapid progression of heart failure was responsible for the high mortality rate in OLETF. ER stress modulators failed to reduce the mortality rate after MI in OLETF.ConclusionsER stress reduces SERCA2a protein via its augmented ubiquitination and degradation, leading to LV diastolic dysfunction in T2DM. Even at a stage without systolic LV dysfunction, susceptibility to lethal heart failure after infarction is markedly increased, which cannot be explained by ER stress or change in myocardial response to sympathetic nerve activation.

Excess cellular iron increases reactive oxygen species (ROS) production and causes cellular damage. Mitochondria are the major site of iron metabolism and ROS production; however, few studies have investigated the role of mitochondrial iron in the development of cardiac disorders, such as ischemic heart disease or cardiomyopathy (CM). We observe increased mitochondrial iron in mice after ischemia/reperfusion (I/R) and in human hearts with ischemic CM, and hypothesize that decreasing mitochondrial iron protects against I/R damage and the development of CM. Reducing mitochondrial iron genetically through cardiac-specific overexpression of a mitochondrial iron export protein or pharmacologically using a mitochondria-permeable iron chelator protects mice against I/R injury. Furthermore, decreasing mitochondrial iron protects the murine hearts in a model of spontaneous CM with mitochondrial iron accumulation. Reduced mitochondrial ROS that is independent of alterations in the electron transport chain's ROS producing capacity contributes to the protective effects. Overall, our findings suggest that mitochondrial iron contributes to cardiac ischemic damage, and may be a novel therapeutic target against ischemic heart disease.

Multiple cardioprotective signal pathways that are activated by ischaemic preconditioning (IPC) and those by IPC mimetics converge on mitochondria. Recent studies have shown that pools of Akt, protein kinase C-ε, extracellular-regulated kinases, glycogen synthase kinase-3beta (GSK-3beta), and hexokinases (HK) I and II, are localized in mitochondria in addition to their pools in the cytosol. Accumulating evidence indicates that such 'mitochondrial protein kinases' receive signals from cytosolic molecules and enhance tolerance of myocytes to injury. Proteomic analyses suggest that these kinases form complexes with each other and with subunit proteins of the mitochondrial permeability transition pore (mPTP). Functional relationships between the protein kinases in mitochondria have not been fully clarified, but GSK-3beta and HKs appear to be at the end of the signal pathways and directly responsible for inhibition of opening of the mPTP and, thus, for myocyte protection from necrosis. In this review, recent findings supporting roles of mitochondrial protein kinases in protection from myocardial necrosis after ischaemia/reperfusion are summarized and discussed.

The mechanism by which SGLT2 inhibitors reduce cardiac events in diabetic patients remains unclear. Here, we examined the effects of an SGLT2 inhibitor on the acute survival rate after myocardial infarction (MI) in an animal model of type 2 diabetes mellitus (DM) and the possible involvement of modification of cardiac metabolomes and antioxidative proteins. MI was induced in DM Otsuka Long-Evans Tokushima Fatty (OLETF) rats and Long-Evans Tokushima Otsuka (LETO) control rats. Treatment with empagliflozin (10 mg/kg per day, 14 days) before MI reduced blood glucose and increased blood and myocardial b-hydroxybutyrate (bOHB) levels in OLETF. Survival rate at 48 hours after MI was significantly lower in OLETF rats than in LETO rats (40% vs. 84%), and empagliflozin significantly improved the survival rate in OLETF rats to 70%, although the sizes of MI were comparable. Patterns of metabolomes and gene expression in the noninfarcted myocardium of OLETF rats were consistent with increased fatty acid oxidation and decreased glucose oxidation. The patterns were modified by empagliflozin, suggesting both increased glucose oxidation and ketone utilization in OLETF rats. Empagliflozin prevented reduction of ATP level in the noninfarcted myocardium after MI and significantly increased myocardial levels of Sirt3 and superoxide dismutase 2 in OLETF rats. Administration of bOHB partially mimicked the effects of empagliflozin in OLETF rats. The results suggest that empagliflozin prevents DM-induced increase in post-MI mortality, possibly by protective modification of cardiac energy metabolism and antioxidant proteins.