Isoliquiritigenin attenuates myocardial ischemia reperfusion through autophagy activation mediated by AMPK/mTOR/ULK1 signaling.

BACKGROUND: Ischemia reperfusion (IR) causes impaired myocardial function, and autophagy activation ameliorates myocardial IR injury. Isoliquiritigenin (ISO) has been found to protect myocardial tissues via AMPK, with exerting anti-tumor property through autophagy activation. This study aims to investigate ISO capacity to attenuate myocardial IR through autophagy activation mediated by AMPK/mTOR/ULK1 signaling. METHODS: ISO effects were explored by SD rats and H9c2 cells. IR rats and IR-induced H9c2 cell models were established by ligating left anterior descending (LAD) coronary artery and hypoxia/re-oxygenation, respectively, followed by low, medium and high dosages of ISO intervention (Rats: 10, 20, and 40 mg/kg; H9c2 cells: 1, 10, and 100 µmol/L). Myocardial tissue injury in rats was assessed by myocardial function-related index, HE staining, Masson trichrome staining, TTC staining, and ELISA. Autophagy of H9c2 cells was detected by transmission electron microscopy (TEM) and immunofluorescence. Autophagy-related and AMPK/mTOR/ULK1 pathway-related protein expressions were detected with western blot. RESULTS: ISO treatment caused myocardial function improvement, and inhibition of myocardial inflammatory infiltration, fibrosis, infarct area, oxidative stress, CK-MB, cTnI, and cTnT expression in IR rats. In IR-modeled H9c2 cells, ISO treatment lowered apoptosis rate and activated autophagy and LC3 fluorescence expression. In vivo and in vitro, ISO intervention exhibited enhanced Beclin1, LC3II/LC3I, and p-AMPK/AMPK levels, whereas inhibited P62, p-mTOR/mTOR and p-ULK1(S757)/ULK1 protein expression, activating autophagy and protecting myocardial tissues from IR injury. CONCLUSION: ISO treatment may induce autophagy by regulating AMPK/mTOR/ULK1 signaling, thereby improving myocardial IR injury, as a potential candidate for treatment of myocardial IR injury.

Checkpoint Kinase 1 Stimulates Endogenous Cardiomyocyte Renewal and Cardiac Repair by Binding to Pyruvate Kinase Isoform M2 C-Domain and Activating Cardiac Metabolic Reprogramming in a Porcine Model of Myocardial Ischemia/Reperfusion Injury.

BACKGROUND: The regenerative capacity of the adult mammalian hearts is limited. Numerous studies have explored mechanisms of adult cardiomyocyte cell-cycle withdrawal. This translational study evaluated the effects and underlying mechanism of rhCHK1 (recombinant human checkpoint kinase 1) on the survival and proliferation of cardiomyocyte and myocardial repair after ischemia/reperfusion injury in swine. METHODS AND RESULTS: Intramyocardial injection of rhCHK1 protein (1 mg/kg) encapsulated in hydrogel stimulated cardiomyocyte proliferation and reduced cardiac inflammation response at 3 days after ischemia/reperfusion injury, improved cardiac function and attenuated ventricular remodeling, and reduced the infarct area at 28 days after ischemia/reperfusion injury. Mechanistically, multiomics sequencing analysis demonstrated enrichment of glycolysis and mTOR (mammalian target of rapamycin) pathways after rhCHK1 treatment. Co-Immunoprecipitation (Co-IP) experiments and protein docking prediction showed that CHK1 (checkpoint kinase 1) directly bound to and activated the Serine 37 (S37) and Tyrosine 105 (Y105) sites of PKM2 (pyruvate kinase isoform M2) to promote metabolic reprogramming. We further constructed plasmids that knocked out different CHK1 and PKM2 amino acid domains and transfected them into Human Embryonic Kidney 293T (HEK293T) cells for CO-IP experiments. Results showed that the 1-265 domain of CHK1 directly binds to the 157-400 amino acids of PKM2. Furthermore, hiPSC-CM (human iPS cell-derived cardiomyocyte) in vitro and in vivo experiments both demonstrated that CHK1 stimulated cardiomyocytes renewal and cardiac repair by activating PKM2 C-domain-mediated cardiac metabolic reprogramming. CONCLUSIONS: This study demonstrates that the 1-265 amino acid domain of CHK1 binds to the 157-400 domain of PKM2 and activates PKM2-mediated metabolic reprogramming to promote cardiomyocyte proliferation and myocardial repair after ischemia/reperfusion injury in adult pigs.

Y4 RNA fragments from cardiosphere-derived cells ameliorate diabetic myocardial ischemia‒reperfusion injury by inhibiting protein kinase C ß-mediated macrophage polarization.

The specific pathophysiological pathways through which diabetes exacerbates myocardial ischemia/reperfusion (I/R) injury remain unclear; however, dysregulation of immune and inflammatory cells, potentially driven by abnormalities in their number and function due to diabetes, may play a significant role. In the present investigation, we simulated myocardial I/R injury by inducing ischemia through ligation of the left anterior descending coronary artery in mice for 40 min, followed by reperfusion for 24 h. Previous studies have indicated that protein kinase Cß (PKCß) is upregulated under hyperglycemic conditions and is implicated in the development of various diabetic complications. The Y4 RNA fragment is identified as the predominant small RNA component present in the extracellular vesicles of cardio sphere-derived cells (CDCs), exhibiting notable anti-inflammatory properties in the contexts of myocardial infarction and cardiac hypertrophy. Our investigation revealed that the administration of Y4 RNA into the ventricular cavity of db/db mice following myocardial I/R injury markedly enhanced cardiac function. Furthermore, Y4 RNA was observed to facilitate M2 macrophage polarization and interleukin-10 secretion through the suppression of PKCß activation. The mechanism by which Y4 RNA affects PKCß by regulating macrophage activation within the inflammatory environment involves the inhibition of ERK1/2 phosphorylation In our study, the role of PKCß in regulating macrophage polarization during myocardial I/R injury was investigated through the use of PKCß knockout mice. Our findings indicate that PKCß plays a crucial role in modulating the inflammatory response associated with macrophage activation in db/db mice experiencing myocardial I/R, with a notable exacerbation of this response observed upon significant upregulation of PKCß expression. In vitro studies further elucidated the protective mechanism by which Y4 RNA modulates the PKCß/ERK1/2 signaling pathway to induce M2 macrophage activation. Overall, our findings suggest that Y4 RNA plays an anti-inflammatory role in diabetic I/R injury, suggesting a novel therapeutic approach for managing myocardial I/R injury in diabetic individuals.

Sevoflurane postconditioning attenuates cardiomyocytes hypoxia/reoxygenation injury via PI3K/AKT pathway mediated HIF-1α to regulate the mitochondrial dynamic balance.

BACKGROUND: Myocardial ischemia-reperfusion injury (I/RI) is a major cause of perioperative cardiac-related adverse events and death. Studies have shown that sevoflurane postconditioning (SpostC), which attenuates I/R injury and exerts cardioprotective effects, regulates mitochondrial dynamic balance via HIF-1α, but the exact mechanism is unknown. This study investigates whether the PI3K/AKT pathway in SpostC regulates mitochondrial dynamic balance by mediating HIF-1α, thereby exerting myocardial protective effects. METHODS: The H9C2 cardiomyocytes were cultured to establish the hypoxia-reoxygenation (H/R) model and randomly divided into 4 groups: Control group, H/R group, sevoflurane postconditioning (H/R + SpostC) group and PI3K/AKT blocker (H/R + SpostC + LY) group. Cell survival rate was determined by CCK-8; Apoptosis rate was determined by flow cytometry; mitochondrial membrane potential was evaluated by Mito Tracker™ Red; mRNA expression levels of AKT, HIF-1α, Opa1and Drp1 were detected by quantitative real-time polymerase chain reaction (qRT-PCR); Western Blot assay was used to detect the protein expression levels of AKT, phosphorylated AKT (p-AKT), HIF-1α, Opa1 and Drp1. RESULTS: Compared with the H/R group, the survival rate of cardiomyocytes in the H/R + SpostC group increased, the apoptosis rate decreased and the mitochondrial membrane potential increased. qRT-PCR showed that the mRNA expression of HIF-1α and Opa1 were higher in the H/R + SpostC group compared with the H/R group, whereas the transcription level of Drp1 was lower in the H/R + SpostC group. In the H/R + SpostC + LY group, the mRNA expression of HIF-1α was lower than the H/R + SpostC group. There was no difference in the expression of Opa1 mRNA between the H/R group and the H/R + SpostC + LY group. WB assay results showed that compared with the H/R group, the protein expression levels of HIF-1α, Opa1, P-AKT were increased and Drp1 protein expression levels were decreased in the H/R + SpostC group. HIF-1α, P-AKT protein expression levels were decreased in the H/R + SpostC + LY group compared to the H/R + SpostC group. CONCLUSION: SpostC mediates HIF-1α-regulated mitochondrial fission and fusion-related protein expression to maintain mitochondrial dynamic balance by activating the PI3K/AKT pathway and increasing AKT phosphorylation, thereby attenuating myocardial I/R injury.

miR-30a-5p attenuates hypoxia/reoxygenation-induced cardiomyocyte apoptosis by regulating PTEN protein expression and activating PI3K/Akt signaling pathway.

BACKGROUND: This study was designed to investigate the mechanism by which miR-30a-5p mediates cardiomyocyte apoptosis after acute myocardial infarction (AMI) induced by hypoxia/reoxygenation (H/R). METHODS: Differentially expressed miRNAs were analyzed by RNA high-throughput sequencing in acute myocardial infarction (ST-elevation myocardial infarction) patients versus healthy individuals (controls). The H/R model was used to assess the regulatory mechanism of miRNAs in AMI. Lentivirus-associated vectors were used to overexpress or knock down miR-30a-5p in cellular models. The pathological mechanisms of miR-30a-5p regulating the development of acute myocardial infarction were serially explored by qPCR, bioinformatics, target gene prediction, dual luciferase, enzyme-linked immunosorbent assays (ELISAs) and Western blotting. RESULTS: The results showed that the expression of miR-30a-5p was significantly increased in AMI patients and H9C2 cells. Hypoxia decreased cardiomyocyte survival over time, and reoxygenation further reduced cell survival. Bax and Phosphatase and tensin homolog (PTEN)were suppressed, while Bcl-2 was upregulated. Additionally, miR-30a-5p specifically targeted the PTEN gene. According to the GO and KEGG analyses, miR-30a-5p may participate in apoptosis by interacting with PTEN. The miR-30a-5p mimic decreased the expression of apoptosis-related proteins and the levels of the proinflammatory markers IL-1ß, IL-6, and TNF-α by activating the PTEN/PI3K/Akt signaling pathway. Conversely, anti-miR-30a-5p treatment attenuated these effects. Additionally, silencing PTEN and anti-miR-30a-5p had opposite effects on H/R-induced cell apoptosis. CONCLUSIONS: miR-30a-5p plays a crucial role in cardiomyocyte apoptosis after hypoxia-induced acute myocardial infarction. Our findings provide translational evidence that miR-30a-5p is a novel potential therapeutic target for AMI.

Depleting inositol pyrophosphate 5-InsP7 protected the heart against ischaemia-reperfusion injury by elevating plasma adiponectin.

AIMS: Adiponectin is an adipocyte-derived circulating protein that exerts cardiovascular and metabolic protection. Due to the futile degradation of endogenous adiponectin and the challenges of exogenous administration, regulatory mechanisms of adiponectin biosynthesis are of significant pharmacological interest. METHODS AND RESULTS: Here, we report that 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) generated by inositol hexakisphosphate kinase 1 (IP6K1) governed circulating adiponectin levels via thiol-mediated protein quality control in the secretory pathway. IP6K1 bound to adiponectin and DsbA-L and generated 5-InsP7 to stabilize adiponectin/ERp44 and DsbA-L/Ero1-Lα interactions, driving adiponectin intracellular degradation. Depleting 5-InsP7 by either IP6K1 deletion or pharmacological inhibition blocked intracellular adiponectin degradation. Whole-body and adipocyte-specific deletion of IP6K1 boosted plasma adiponectin levels, especially its high molecular weight forms, and activated AMPK-mediated protection against myocardial ischaemia-reperfusion injury. Pharmacological inhibition of 5-InsP7 biosynthesis in wild-type but not adiponectin knockout mice attenuated myocardial ischaemia-reperfusion injury. CONCLUSION: Our findings revealed that 5-InsP7 is a physiological regulator of adiponectin biosynthesis that is amenable to pharmacological intervention for cardioprotection.

Selective cyclooxygenase-2 inhibition protects against myocardial damage in experimental acute ischemia

BACKGROUND: Acute myocardial infarction is associated with tissue inflammation. Early coronary reperfusion clearly improves the outcome but may help propagate the inflammatory response and enhance tissue damage. Cyclooxygenase-2 is an enzyme that catalyzes the initial step in the formation of inflammatory prostaglandins from arachidonic acid. Cyclooxygenase-2 levels are increased when ischemic cardiac events occur. The overall function of COX-2 in the inflammatory process generated by myocardial ischemic damage has not yet been elucidated. GOAL: The objective of this study was to determine whether a selective cyclooxygenase-2 inhibitor (rofecoxib) could alter the evolution of acute myocardial infarction after reperfusion. METHODS AND RESULTS: This study was performed with 48 mongrel dogs divided into two groups: controls and those treated with the drug. All animals were prepared for left anterior descending coronary artery occlusion. The dogs then underwent 180 minutes of coronary occlusion, followed by 30 minutes of reperfusion. Blood samples were collected from the venous sinus immediately before coronary occlusion and after 30 minutes of reperfusion for measurements of CPK-MB, CPK-MBm and troponin I. During the experiment we observed the mean blood pressure, heart rate and coronary flow. The coronary flow and heart rate did not change, but in the control group, there was blood pressure instability, in addition to maximal levels of CPK-MB post-infarction. The same results were observed for CPK-MBm and troponin I. CONCLUSION: In a canine model of myocardial ischemia-reperfusion, selective inhibition of Cyclooxygenase-2 with rofecoxib was not associated with early detrimental effects on the hemodynamic profile or the gross extent of infarction; in fact, it may be beneficial by limiting cell necrosis.