Molecular Signaling to Preserve Mitochondrial Integrity against Ischemic Stress in the Heart: Rescue or Remove Mitochondria in Danger

Cardiovascular diseases are one of the leading causes of death and global health problems worldwide, and ischemic heart disease is the most common cause of heart failure (HF). The heart is a high-energy demanding organ, and myocardial energy reserves are limited. Mitochondria are the powerhouses of the cell, but under stress conditions, they become damaged, release necrotic and apoptotic factors, and contribute to cell death. Loss of cardiomyocytes plays a significant role in ischemic heart disease. In response to stress, protective signaling pathways are activated to limit mitochondrial deterioration and protect the heart. To prevent mitochondrial death pathways, damaged mitochondria are removed by mitochondrial autophagy (mitophagy). Mitochondrial quality control mediated by mitophagy is functionally linked to mitochondrial dynamics. This review provides a current understanding of the signaling mechanisms by which the integrity of mitochondria is preserved in the heart against ischemic stress.

Low Intrinsic Aerobic Capacity Limits Recovery Response to Hindlimb Ischemia

Introduction: In this study, we determined the influence of intrinsic exercise capacity on the vascular adaptive responses to hind limb ischemia. High Capacity Running, HCR; Low Capacity Running, LCR, rats were used to assess intrinsic aerobic capacity effects on adaptive responses to ischemia.Methods: Muscle samples from both ischemic and non-ischemic limb in both strains were compared, histologically for the muscle-capillary relationship, and functionally using microspheres to track blood flow and muscle stimulation to test fatigability. PCR was used to identify the differences in gene expression between the phenotypes following occlusive ischemia.Results: Prior to ligation, there were not significant differences between the phenotypes in the exhaustion time with high frequency pacing. Following ligation, LCR decreased significantly in the exhaustion time compare with HCRs (437 ± 47 vs. 824 ± 56, p < 0.001). The immediate decrease in flow was significantly more severe in LCRs than HCRs (52.5 vs. 37.8%, p < 0.001). VEGF, eNOS, and ANG2 (but not ANG1) gene expression were decreased in LCRs vs. HCRs before occlusion, and increased significantly in LCRs 14D after occlusion, but not in HCRs. LCR capillary density (CD) was significantly lower at all time points after occlusion (LCR 7D = 564.76 ± 40.5, LCR 14D = 507.48 ± 54.2, both p < 0.05 vs. HCR for respective time point). NCAF increased significantly in HCR and LCR in response to ischemia.Summary: These results suggest that LCR confers increased risk for ischemic injury and is subject to delayed and less effective adaptive response to ischemic stress.

Protective mitochondrial fission induced by stress-responsive protein GJA1-20k

The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.

Empagliflozin Restores Cardiac Metabolic Flexibility in Diet-induced Obese C57BL6/J Mice

BackgroundSodium-glucose co-transporter type 2 (SGLT2) inhibitor therapy to treat type 2 diabetes unexpectedly reduced all-cause mortality and hospitalization due to heart failure in several large-scale clinical trials, and has since been shown to produce similar cardiovascular disease-protective effects in patients without diabetes. How SGLT2 inhibitor therapy improves cardiovascular disease outcomes remains incompletely understood. Metabolic flexibility refers to the ability of a cell or organ to adjust its use of metabolic substrates, such as glucose or fatty acids, in response to physiological or pathophysiological conditions, and is a feature of a healthy heart that may be lost during diabetic cardiomyopathy and in the failing heart. While several studies have addressed metabolic changes in hearts in response to SGLT2 inhibitor therapy, none have specifically assessed metabolic flexibility in an in vivo system. We therefore undertook the described studies to determine the effects of SGLT2 inhibitor therapy on cardiac metabolic flexibility in vivo in obese, insulin resistant mice.MethodsDiet-induced obese mice were treated with the SGLT2 inhibitor empagliflozin (EMPA; 10 mg/kg/d) for four weeks prior to study and compared with untreated obese and lean controls. We assessed changes in body weight and composition, plasma metabolites in response to fasting/re-feeding, cardiac hypertrophy by echocardiography, the response to ischemic stress following coronary artery ligation, as well as cardiac-specific rates of relative glucose and fatty acid utilization using a [U13C]-glucose infusion during fasting and hyperinsulinemic euglycemic clamp.ResultsEMPA-treated mice presented with reduced cardiac hypertrophy and protection from ischemic stress compared with obese controls. In the fasted state, relative rates of cardiac glucose and fatty acid utilization were similar in control and EMPA-treated mice. During the hyperinsulinemic euglycemic clamp, rates of cardiac glucose utilization and metabolic flexibility were reduced in obese compared with lean mice, and EMPA-treatment partially restored both features. ConclusionsSGLT2 inhibitor therapy restored cardiac metabolic flexibility in obese, insulin resistant mice, and was associated with reduced cardiac hypertrophy and protection from ischemia. These observations suggest that the cardiovascular disease-protective effects of SGLT2 inhibitors may in part be explained by beneficial effects on cardiac metabolic substrate selection.

Differential Association of 4E-BP2-Interacting Proteins Is Related to Selective Delayed Neuronal Death after Ischemia

Cerebral ischemia induces an inhibition of protein synthesis and causes cell death and neuronal deficits. These deleterious effects do not occur in resilient areas of the brain, where protein synthesis is restored. In cellular stress conditions, as brain ischemia, translational repressors named eukaryotic initiation factor (eIF) 4E-binding proteins (4E-BPs) specifically bind to eIF4E and are critical in the translational control. We previously described that 4E-BP2 protein, highly expressed in brain, can be a molecular target for the control of cell death or survival in the reperfusion after ischemia in an animal model of transient cerebral ischemia. Since these previous studies showed that phosphorylation would not be the regulation that controls the binding of 4E-BP2 to eIF4E under ischemic stress, we decided to investigate the differential detection of 4E-BP2-interacting proteins in two brain regions with different vulnerability to ischemia-reperfusion (IR) in this animal model, to discover new potential 4E-BP2 modulators and biomarkers of cerebral ischemia. For this purpose, 4E-BP2 immunoprecipitates from the resistant cortical region and the vulnerable hippocampal cornu ammonis 1 (CA1) region were analyzed by two-dimensional (2-D) fluorescence difference in gel electrophoresis (DIGE), and after a biological variation analysis, 4E-BP2-interacting proteins were identified by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry. Interestingly, among the 4E-BP2-interacting proteins identified, heat shock 70 kDa protein-8 (HSC70), dihydropyrimidinase-related protein-2 (DRP2), enolase-1, ubiquitin carboxyl-terminal hydrolase isozyme-L1 (UCHL1), adenylate kinase isoenzyme-1 (ADK1), nucleoside diphosphate kinase-A (NDKA), and Rho GDP-dissociation inhibitor-1 (Rho-GDI), were of notable interest, showing significant differences in their association with 4E-BP2 between resistant and vulnerable regions to ischemic stress. Our data contributes to the first characterization of the 4E-BP2 interactome, increasing the knowledge in the molecular basis of the protection and vulnerability of the ischemic regions and opens the way to detect new biomarkers and therapeutic targets for diagnosis and treatment of cerebral ischemia.

An integrated comparative physiology and molecular approach pinpoints mediators of breath-hold capacity in dolphins

Ischemic events, such as ischemic heart disease and ischemic stroke, are the number one cause of death globally. Ischemia prevents blood, carrying essential nutrients and oxygen, from reaching the tissues leading to cell death, tissue death, and eventual organ failure. While humans are relatively intolerant to these ischemic events, other species, such as marine mammals, have evolved remarkable tolerance to chronic ischemia/reperfusion during diving. Here we capitalized on the unique adaptations of bottlenose dolphins (Tursiops truncatus) as a comparative model of ischemic stress and hypoxia tolerance to identify molecular features associated with breath-holding. Using RNA-Seq we observed time-dependent upregulation of the arachidonate 5-lipoxygenase (ALOX5) gene during breath-holding. Consistent with the RNA-Seq data, we also observed increased ALOX5 enzymatic activity in the serum of dolphins undergoing breath-holds. ALOX5 has previously been shown to be activated during hypoxia in rodent models, and its metabolites, leukotrienes, induce vasoconstriction. The upregulation of ALOX5 occurred within the estimated aerobic dive limit of the species, suggesting that ALOX5 enzymatic activity may promote tolerance to ischemic stress through sustained vasoconstriction in dolphins during diving. These observations pinpoint a potential molecular mechanism by which dolphins, and perhaps other marine mammals, have adapted to the prolonged breath-holds associated with diving.