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.

EPAC1 and 2 inhibit K+ currents via PLC/PKC and NOS/PKG pathways in rat ventricular cardiomyocytes.

The exchange protein directly activated by cAMP (EPAC) has been implicated in cardiac proarrhythmic signaling pathways including spontaneous diastolic Ca2+ leak from sarcoplasmic reticulum and increased action potential duration (APD) in isolated ventricular cardiomyocytes. The action potential (AP) lengthening following acute EPAC activation is mainly due to a decrease of repolarizing steady-state K+ current (IKSS) but the mechanisms involved remain unknown. This study aimed to assess the role of EPAC1 and EPAC2 in the decrease of IKSS and to investigate the underlying signaling pathways. AP and K+ currents were recorded with the whole cell configuration of the patch-clamp technique in freshly isolated rat ventricular myocytes. EPAC1 and EPAC2 were pharmacologically activated with 8-(4-chlorophenylthio)-2'-O-methyl-cAMP acetoxymethyl ester (8-CPTAM, 10 µmol/L) and inhibited with R-Ce3F4 and ESI-05, respectively. Inhibition of EPAC1 and EPAC2 significantly decreased the effect of 8-CPTAM on APD and IKSS showing that both EPAC isoforms are involved in these effects. Unexpectedly, calmodulin-dependent protein kinase II (CaMKII) inhibition by AIP or KN-93, and Ca2+ chelation by intracellular BAPTA, did not impact the response to 8-CPTAM. However, inhibition of PLC/PKC and nitric oxide synthase (NOS)/PKG pathways partially prevents the 8-CPTAM-dependent decrease of IKSS. Finally, the cumulative inhibition of PKC and PKG blocked the 8-CPTAM effect, suggesting that these two actors work along parallel pathways to regulate IKSS upon EPAC activation. On the basis of such findings, we propose that EPAC1 and EPAC2 are involved in APD lengthening by inhibiting a K+ current via both PLC/PKC and NOS/PKG pathways. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy.NEW & NOTEWORHTY Exchange protein directly activated by cAMP (EPAC) proteins modulate ventricular electrophysiology at the cellular level. Both EPAC1 and EPAC2 isoforms participate in this effect. Mechanistically, PLC/PKC and nitric oxide synthase (NO)/PKG pathways are involved in regulating K+ repolarizing current whereas the well-known downstream effector of EPAC, calmodulin-dependent protein kinase II (CaMKII), does not participate. This may have pathological implications since EPAC is upregulated in diseases such as cardiac hypertrophy. Thus, EPAC inhibition may be a new approach to prevent arrhythmias under pathological conditions.

Impaired Cardiac AMPK (5'-Adenosine Monophosphate-Activated Protein Kinase) and Ca2+-Handling, and Action Potential Duration Heterogeneity in Ibrutinib-Induced Ventricular Arrhythmia Vulnerability.

BACKGROUND: We recently demonstrated that acute administration of ibrutinib, a Bruton's tyrosine kinase inhibitor used in chemotherapy for blood malignancies, increases ventricular arrhythmia (VA) vulnerability. A pathway of ibrutinib-induced vulnerability to VA that can be modulated for cardioprotection remains unclear. METHODS AND RESULTS: The effects of ibrutinib on cardiac electrical activity and Ca2+ dynamics were investigated in Langendorff-perfused hearts using optical mapping. We also conducted Western blotting analysis to evaluate the impact of ibrutinib on various regulatory and Ca2+-handling proteins in rat cardiac tissues. Treatment with ibrutinib (10 mg/kg per day) for 4 weeks was associated with an increased VA inducibility (72.2%±6.3% versus 38.9±7.0% in controls, P<0.002) and shorter action potential durations during pacing at various frequencies (P<0.05). Ibrutinib also decreased heart rate thresholds for beat-to-beat duration alternans of the cardiac action potential (P<0.05). Significant changes in myocardial Ca2+ transients included lower amplitude alternans ratios (P<0.05), longer times-to-peak (P<0.05), and greater spontaneous intracellular Ca2+ elevations (P<0.01). We also found lower abundance and phosphorylation of myocardial AMPK (5'-adenosine monophosphate-activated protein kinase), indicating reduced AMPK activity in hearts after ibrutinib treatment. An acute treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside ameliorated abnormalities in action potential and Ca2+ dynamics, and significantly reduced VA inducibility (37.1%±13.4% versus 72.2%±6.3% in the absence of 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside, P<0.05) in hearts from ibrutinib-treated rats. CONCLUSIONS: VA vulnerability inflicted by ibrutinib may be mediated in part by an impairment of myocardial AMPK activity. Pharmacological activation of AMPK may be a protective strategy against ibrutinib-induced cardiotoxicity.

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.

Marein Alleviates Doxorubicin-Induced Cardiotoxicity through FAK/AKT Pathway Modulation while Potentiating its Anticancer Activity.

Doxorubicin (DOX) is an effective anticancer agent, yet its clinical utility is hampered by dose-dependent cardiotoxicity. This study explores the cardioprotective potential of Marein (Mar) against DOX-induced cardiac injury and elucidates underlying molecular mechanisms. Neonatal rat cardiomyocytes (NRCMs) and murine models were employed to assess the impact of Mar on DOX-induced cardiotoxicity (DIC). In vitro, cell viability, oxidative stress were evaluated. In vivo, a chronic injection method was employed to induce a DIC mouse model, followed by eight weeks of Mar treatment. Cardiac function, histopathology, and markers of cardiotoxicity were assessed. In vitro, Mar treatment demonstrated significant cardioprotective effects in vivo, as evidenced by improved cardiac function and reduced indicators of cardiac damage. Mechanistically, Mar reduced inflammation, oxidative stress, and apoptosis in cardiomyocytes, potentially via activation of the Focal Adhesion Kinase (FAK)/AKT pathway. Mar also exhibited an anti-ferroptosis effect. Interestingly, Mar did not compromise DOX's efficacy in cancer cells, suggesting a dual benefit in onco-cardiology. Molecular docking studies suggested a potential interaction between Mar and FAK. This study demonstrates Mar's potential as a mitigator of DOX-induced cardiotoxicity, offering a translational perspective on its clinical application. By activating the FAK/AKT pathway, Mar exerts protective effects against DOX-induced cardiomyocyte damage, highlighting its promise in onco-cardiology. Further research is warranted to validate these findings and advance Mar as a potential adjunctive therapy in cancer treatment.

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.

miR-652-3p Suppressed the Protective Effects of Isoflurane Against Myocardial Injury in Hypoxia/Reoxygenation by Targeting ISL1.

This research is concentrated on investigating the role and mechanism of miR-652-3p in the protective effects of isoflurane (ISO) against myocardial ischemia-reperfusion (I/R) injury. H9c2 cells underwent pretreatment with varying concentrations of ISO, and subsequently, a hypoxia/reoxygenation (H/R) model was constructed. The levels of miR-652-3p, ISL LIM homeobox 1 (ISL1), and inflammatory cytokines interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-α) were evaluated through reverse transcription polymerase chain reaction (RT-qPCR). Enzyme-linked immunosorbent assay was employed to investigate concentrations of myocardial injury markers, such as creatine kinase-MB (CK-MB) and cardiac troponin I (cTnI). Cell counting kit-8 was used to evaluate cell viability, while flow cytometry was utilized to measure apoptosis. Additionally, a dual luciferase reporter assay was conducted to validate the targeting relationship between ISL1 and miR-652-3p. Herein, we confirmed that the level of miR-652-3p was gradually increased with prolonged hypoxia; nevertheless, this increase was suppressed by ISO pretreatment (P < 0.05). Additionally, ISO pretreatment prevented the decrease in cell viability, increase in apoptosis, and overproduction of IL-6, TNF-α, CK-MB, and cTnI induced by H/R (P < 0.05). However, the inhibitory effects of ISO were counteracted by the increased levels of miR-652-3p (P < 0.05). ISL1 is a potential target of miR-652-3p. H/R induction suppressed ISL1 levels compared to the control, but ISO treatment increased its expression (P < 0.05). Overexpression of ISL1 inhibited the elimination of the protective effect of ISO on myocardial damage induced by the elevation of miR-652-3p (P < 0.05). The findings of this research confirm that miR-652-3p attenuated the protective effect of ISO on cardiomyocytes in myocardial ischemia by targeting ISL1.

Inhibition of TBL1 cleavage alleviates doxorubicin-induced cardiomyocytes death by regulating the Wnt/ß-catenin signal pathway.

AIMS: Doxorubicin (DOX) is a widely used anthracycline anticancer agent; however, its irreversible effects on the heart can result in DOX-induced cardiotoxicity (DICT) after cancer treatment. Unfortunately, the pathophysiology of DICT has not yet been fully elucidated, and there are no effective strategies for its prevention or treatment. In this investigation, the novel role of transducin beta-like protein 1 (TBL1) in developing and regulating DICT was explored. METHODS AND RESULTS: We observed a reduction in TBL1 protein expression levels as well as cleavage events in the transplanted cardiac tissues of patients diagnosed with Dilated Cardiomyopathy and DICT. It was revealed that DOX selectively induces TBL1 cleavage at caspase-3 preferred sites-D125, D136, and D215. Interestingly, overexpression of the uncleaved TBL1 mutant (TBL1uclv) variant reduced apoptosis, effectively preventing DOX-induced cell death. We confirmed that cleaved TBL1 cannot form a complex with ß-catenin. As a result, Wnt reporter activity and Wnt target gene expression collectively indicate a decrease in Wnt/ß-catenin signalling, leading to DICT progression. Furthermore, the cleaved TBL1 triggered DOX-induced abnormal electrophysiological features and disrupted calcium homeostasis. However, these effects were improved in TBL1uclv-overexpressing human-induced pluripotent stem cell-derived cardiomyocytes. Finally, in a DICT mouse model, TBL1uclv overexpression inhibited the DICT-induced reduction of cardiac contractility and collagen accumulation, ultimately protecting cardiomyocytes from cell death. CONCLUSION: Our findings reveal that the inhibition of TBL1 cleavage not only mitigates apoptosis but also enhances cardiomyocyte function, even in the context of DOX administration. Consequently, this study's results suggest that inhibiting TBL1 cleavage may be a novel strategy to ameliorate DICT.

Empagliflozin inhibits increased Na influx in atrial cardiomyocytes of patients with HFpEF.

AIMS: Heart failure with preserved ejection fraction (HFpEF) causes substantial morbidity and mortality. Importantly, atrial remodelling and atrial fibrillation are frequently observed in HFpEF. Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have recently been shown to improve clinical outcomes in HFpEF, and post-hoc analyses suggest atrial anti-arrhythmic effects. We tested if isolated human atrial cardiomyocytes from patients with HFpEF exhibit an increased Na influx, which is known to cause atrial arrhythmias, and if that is responsive to treatment with the SGTL2i empagliflozin. METHODS AND RESULTS: Cardiomyocytes were isolated from atrial biopsies of 124 patients (82 with HFpEF) undergoing elective cardiac surgery. Na influx was measured with the Na-dye Asante Natrium Green-2 AM (ANG-2). Compared to patients without heart failure (NF), Na influx was doubled in HFpEF patients (NF vs. HFpEF: 0.21 ± 0.02 vs. 0.38 ± 0.04 mmol/L/min (N = 7 vs. 18); P = 0.0078). Moreover, late INa (measured via whole-cell patch clamp) was significantly increased in HFpEF compared to NF. Western blot and HDAC4 pulldown assay indicated a significant increase in CaMKII expression, CaMKII autophosphorylation, CaMKII activity, and CaMKII-dependent NaV1.5 phosphorylation in HFpEF compared to NF, whereas NaV1.5 protein and mRNA abundance remained unchanged. Consistently, increased Na influx was significantly reduced by treatment not only with the CaMKII inhibitor autocamtide-2-related inhibitory peptide (AIP), late INa inhibitor tetrodotoxin (TTX) but also with sodium/hydrogen exchanger 1 (NHE1) inhibitor cariporide. Importantly, empagliflozin abolished both increased Na influx and late INa in HFpEF. Multivariate linear regression analysis, adjusting for important clinical confounders, revealed HFpEF to be an independent predictor for changes in Na handling in atrial cardiomyocytes. CONCLUSION: We show for the first time increased Na influx in human atrial cardiomyocytes from HFpEF patients, partly due to increased late INa and enhanced NHE1-mediated Na influx. Empagliflozin inhibits Na influx and late INa, which could contribute to anti-arrhythmic effects in patients with HFpEF.

Gene therapy with phosphodiesterases 2A and 4B ameliorates heart failure and arrhythmias by improving subcellular cAMP compartmentation.

AIMS: Gene therapy with cardiac phosphodiesterases (PDEs), such as phosphodiesterase 4B (PDE4B), has recently been described to effectively prevent heart failure (HF) in mice. However, exact molecular mechanisms of its beneficial effects, apart from general lowering of cardiomyocyte cyclic adenosine monophosphate (cAMP) levels, have not been elucidated. Here, we studied whether gene therapy with two types of PDEs, namely PDE2A and PDE4B, can prevent pressure-overload-induced HF in mice by acting on and restoring altered cAMP compartmentation in distinct subcellular microdomains. METHODS AND RESULTS: HF was induced by transverse aortic constriction followed by tail-vein injection of adeno-associated-virus type 9 vectors to overexpress PDE2A3, PDE4B3, or luciferase for 8 weeks. Heart morphology and function was assessed by echocardiography and histology which showed that PDE2A and especially PDE4B gene therapy could attenuate cardiac hypertrophy, fibrosis, and decline of contractile function. Live cell imaging using targeted cAMP biosensors showed that PDE overexpression restored altered cAMP compartmentation in microdomains associated with ryanodine receptor type 2 (RyR2) and caveolin-rich plasma membrane. This was accompanied by ameliorated caveolin-3 decline after PDE2A3 overexpression, reduced RyR2 phosphorylation in PDE4B3 overexpressing hearts, and antiarrhythmic effects of both PDEs measured under isoproterenol stimulation in single cells. Strong association of overexpressed PDE4B but not PDE2A with RyR2 microdomain could prevent calcium leak and arrhythmias in human-induced pluripotent stem-derived cardiomyocytes with the A2254V mutation in RyR2 causing catecholaminergic polymorphic ventricular tachycardia. CONCLUSION: Our data indicate that gene therapy with phosphodiesterases can prevent HF including associated cardiac remodelling and arrhythmias by restoring altered cAMP compartmentation in functionally relevant subcellular microdomains.

SIRT6 in Regulation of Mitochondrial Damage and Associated Cardiac Dysfunctions: A Possible Therapeutic Target for CVDs.

Cardiovascular diseases (CVDs) can be described as a global health emergency imploring possible prevention strategies. Although the pathogenesis of CVDs has been extensively studied, the role of mitochondrial dysfunction in CVD development has yet to be investigated. Diabetic cardiomyopathy, ischemic-reperfusion injury, and heart failure are some of the CVDs resulting from mitochondrial dysfunction Recent evidence from the research states that any dysfunction of mitochondria has an impact on metabolic alteration, eventually causes the death of a healthy cell and therefore, progressively directing to the predisposition of disease. Cardiovascular research investigating the targets that both protect and treat mitochondrial damage will help reduce the risk and increase the quality of life of patients suffering from various CVDs. One such target, i.e., nuclear sirtuin SIRT6 is strongly associated with cardiac function. However, the link between mitochondrial dysfunction and SIRT6 concerning cardiovascular pathologies remains poorly understood. Although the Role of SIRT6 in skeletal muscles and cardiomyocytes through mitochondrial regulation has been well understood, its specific role in mitochondrial maintenance in cardiomyocytes is poorly determined. The review aims to explore the domain-specific function of SIRT6 in cardiomyocytes and is an effort to know how SIRT6, mitochondria, and CVDs are related.

Mst4, a novel cardiac STRIPAK complex-associated kinase, regulates cardiomyocyte growth and survival and is upregulated in human cardiomyopathy.

Myocardial failure is associated with adverse remodeling, including loss of cardiomyocytes, hypertrophy, and alterations in cell-cell contacts. Striatin-interacting phosphatase and kinase (STRIPAK) complexes and their mammalian STE20-like kinase 4 (Mst4) have been linked to development of different diseases. The role and targets of Mst4 in cardiomyocytes have not been investigated yet. Multitissue immunoblot experiments show highly enriched Mst4 expression in rodent hearts. Analyses of human biopsy samples from patients suffering from dilated cardiomyopathy revealed that Mst4 is upregulated (5- to 8-fold p < 0.001) compared with nonfailing controls. Increased abundance of Mst4 could also be detected in mouse models of cardiomyopathy. We confirmed that Mst4 interacts with STRIPAK components in neonatal rat ventricular cardiomyocytes, indicating that STRIPAK is present in the heart. Immunofluorescence stainings and molecular interaction studies revealed that Mst4 is localized to the intercalated disc and interacts with several intercalated disc proteins. Overexpression of Mst4 in cardiomyocytes results in hypertrophy compared with controls. In adult rat cardiomyocytes, Mst4 overexpression increases cellular and sarcomeric fractional shortening (p < 0.05), indicating enhanced contractility. Overexpression of Mst4 also inhibits apoptosis shown by reduction of cleaved caspase3 (-69%, p < 0.0001), caspase7 (-80%, p < 0.0001), and cleaved Parp1 (-27%, p < 0.001). To elucidate potential Mst4 targets, we performed phosphoproteomics analyses in neonatal rat cardiomyocytes after Mst4 overexpression and inhibition. The results revealed target candidates of Mst4 at the intercalated disc. We identified Mst4 as a novel cardiac kinase that is upregulated in cardiomyopathy-regulating cardiomyocyte growth and survival.

N -N-Butyl Haloperidol Iodide Mitigates Myocardial Ischemia/Reperfusion Injury Through Activation of SIRT1-Nrf2 Signaling Loop.

ABSTRACT: N -n-butyl haloperidol iodide (F 2 ), a derivative of haloperidol developed by our group, exhibits potent antioxidative properties and confers protection against cardiac ischemia/reperfusion (I/R) injury. The protective mechanisms by which F 2 ameliorates I/R injury remain obscure. The activation of nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor transactivating many antioxidative genes, also attenuates I/R-induced myocardial damage. The present study investigated whether the cardioprotective effect of F 2 depends on Nrf2 using a mouse heart I/R model. F 2 (0.1, 0.2 or 0.4 mg/kg) or vehicle was intravenously injected to mice 5 minutes before reperfusion. Systemic administration of 0.4 mg/kg F 2 led to a significant reduction in I/R injury, which was accompanied by enhanced activation of Nrf2 signaling. The cardioprotection conferred by F 2 was largely abrogated in Nrf2-deficient mice. Importantly, we found F 2 -induced activation of Nrf2 is silent information regulator of transcription 1 (SIRT1)-dependent, as pharmacologically inhibiting SIRT1 by the specific inhibitor EX527 blocked Nrf2 activation. Moreover, F 2 -upregulated expression of SIRT1 was also Nrf2-dependent, as Nrf2 deficiency inhibited SIRT1 upregulation. These results indicate that SIRT1-Nrf2 signaling loop activation is indispensable for the protective effect of F 2 against myocardial I/R injury and may provide new insights for the treatment of ischemic heart disease.

Pyruvate Kinase M2: A Potential Regulator of Cardiac Injury Through Glycolytic and Non-glycolytic Pathways.

ABSTRACT: Adult animals are unable to regenerate heart cells due to postnatal cardiomyocyte cycle arrest, leading to higher mortality rates in cardiomyopathy. However, reprogramming of energy metabolism in cardiomyocytes provides a new perspective on the contribution of glycolysis to repair, regeneration, and fibrosis after cardiac injury. Pyruvate kinase (PK) is a key enzyme in the glycolysis process. This review focuses on the glycolysis function of PKM2, although PKM1 and PKM2 both play significant roles in the process after cardiac injury. PKM2 exists in both low-activity dimer and high-activity tetramer forms. PKM2 dimers promote aerobic glycolysis but have low catalytic activity, leading to the accumulation of glycolytic intermediates. These intermediates enter the pentose phosphate pathway to promote cardiomyocyte proliferation and heart regeneration. Additionally, they activate adenosine triphosphate (ATP)-sensitive K + (K ATP ) channels, protecting the heart against ischemic damage. PKM2 tetramers function similar to PKM1 in glycolysis, promoting pyruvate oxidation and subsequently ATP generation to protect the heart from ischemic damage. They also activate KDM5 through the accumulation of αKG, thereby promoting cardiomyocyte proliferation and cardiac regeneration. Apart from glycolysis, PKM2 interacts with transcription factors like Jmjd4, RAC1, ß-catenin, and hypoxia-inducible factor (HIF)-1α, playing various roles in homeostasis maintenance, remodeling, survival regulation, and neovascularization promotion. However, PKM2 has also been implicated in promoting cardiac fibrosis through mechanisms like sirtuin (SIRT) 3 deletion, TG2 expression enhancement, and activation of transforming growth factor-ß1 (TGF-ß1)/Smad2/3 and Jak2/Stat3 signals. Overall, PKM2 shows promising potential as a therapeutic target for promoting cardiomyocyte proliferation and cardiac regeneration and addressing cardiac fibrosis after injury.

A Non-Apoptotic Pattern of Caspase-9/Caspase-3 Activation During Differentiation of Human Embryonic Stem Cells into Cardiomyocytes.

In vitro studies have demonstrated that the differentiation of embryonic stem cells (ESCs) into cardiomyocytes requires activation of caspases through the mitochondrial pathway. These studies have relied on synthetic substrates for activity measurements, which can be misleading due to potential none-specific hydrolysis of these substrates by proteases other than caspases. Hence, caspase-9 and caspase-3 activation are investigated during the differentiation of human ESCs (hESCs) by directly assessing caspase-9 and -3 cleavage. Western blot reveals the presence of the cleaved caspase-9 prior to and during the differentiation of human ESCs (hESCs) into cardiomyocytes at early stages, which diminishes as the differentiation progresses, without cleavage and activation of endogenous procaspase-3. Activation of exogenous procaspase-3 by endogenous caspase-9 and subsequent cleavage of chromogenic caspase-3 substrate i.e. DEVD-pNA during the course of differentiation confirmes that endogenous caspase-9 has the potency to recognize and activate procaspase-3, but for reasons that are unknown to us fails to do so. These observations suggest the existence of distinct mechanisms of caspase regulation in differentiation as compared to apoptosis. Bioinformatics analysis suggests the presence of caspase-9 regulators, which may influence proteolytic function under specific conditions.

Inhibition of cyclin-dependent kinase 7 mitigates doxorubicin cardiotoxicity and enhances anticancer efficacy.

AIMS: The anthracycline family of anticancer agents such as doxorubicin (DOX) can induce apoptotic death of cardiomyocytes and cause cardiotoxicity. We previously reported that DOX-induced apoptosis is accompanied by cardiomyocyte cell cycle re-entry. Cell cycle progression requires cyclin-dependent kinase 7 (CDK7)-mediated activation of downstream cell cycle CDKs. This study aims to determine whether CDK7 can be targeted for cardioprotection during anthracycline chemotherapy. METHODS AND RESULTS: DOX exposure induced CDK7 activation in mouse heart and isolated cardiomyocytes. Cardiac-specific ablation of Cdk7 attenuated DOX-induced cardiac dysfunction and fibrosis. Treatment with the covalent CDK7 inhibitor THZ1 also protected against DOX-induced cardiomyopathy and apoptosis. DOX treatment induced activation of the proapoptotic CDK2-FOXO1-Bim axis in a CDK7-dependent manner. In response to DOX, endogenous CDK7 directly bound and phosphorylated CDK2 at Thr160 in cardiomyocytes, leading to full CDK2 kinase activation. Importantly, inhibition of CDK7 further suppressed tumour growth when used in combination with DOX in an immunocompetent mouse model of breast cancer. CONCLUSION: Activation of CDK7 is necessary for DOX-induced cardiomyocyte apoptosis and cardiomyopathy. Our findings uncover a novel proapoptotic role for CDK7 in cardiomyocytes. Moreover, this study suggests that inhibition of CDK7 attenuates DOX-induced cardiotoxicity but augments the anticancer efficacy of DOX. Therefore, combined administration of CDK7 inhibitor and DOX may exhibit diminished cardiotoxicity but superior anticancer activity.

Protective Effect of CD137 Deficiency Against Postinfarction Cardiac Fibrosis and Adverse Cardiac Remodeling by ERK1/2 Signaling Pathways.

ABSTRACT: Myocardial fibrosis, a common complication of myocardial infarction (MI), is characterized by excessive collagen deposition and can result in impaired cardiac function. The specific role of CD137 in the development of post-MI myocardial fibrosis remains unclear. Thus, this study aimed to elucidate the effects of CD137 signaling using CD137 knockout mice and in vitro experiments. CD137 expression levels progressively increased in the heart after MI, particularly in myofibroblast, which play a key role in fibrosis. Remarkably, CD137 knockout mice exhibited improved cardiac function and reduced fibrosis compared with wild-type mice at day 28 post-MI. The use of Masson's trichrome and picrosirius red staining demonstrated a reduction in the infarct area and collagen volume fraction in CD137 knockout mice. Furthermore, the expression of alpha-smooth muscle actin and collagen I, key markers of fibrosis, was decreased in heart tissues lacking CD137. In vitro experiments supported these findings because CD137 depletion attenuated cardiac fibroblast differentiation, and migration, and collagen I synthesis. In addition, the administration of CD137L recombinant protein further promoted alpha-smooth muscle actin expression and collagen I synthesis, suggesting a profibrotic effect. Notably, the application of an inhibitor targeting the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway attenuated the profibrotic effects of CD137L. To conclude, this study provides evidence that CD137 plays a significant role in promoting myocardial fibrosis after MI. Inhibition of CD137 signaling pathways may hold therapeutic potential for mitigating pathological cardiac remodeling and improving post-MI cardiac function.

Cardiac monoamine oxidase-A inhibition protects against catecholamine-induced ventricular arrhythmias via enhanced diastolic calcium control.

AIMS: A mechanistic link between depression and risk of arrhythmias could be attributed to altered catecholamine metabolism in the heart. Monoamine oxidase-A (MAO-A), a key enzyme involved in catecholamine metabolism and longstanding antidepressant target, is highly expressed in the myocardium. The present study aimed to elucidate the functional significance and underlying mechanisms of cardiac MAO-A in arrhythmogenesis. METHODS AND RESULTS: Analysis of the TriNetX database revealed that depressed patients treated with MAO inhibitors had a lower risk of arrhythmias compared with those treated with selective serotonin reuptake inhibitors. This effect was phenocopied in mice with cardiomyocyte-specific MAO-A deficiency (cMAO-Adef), which showed a significant reduction in both incidence and duration of catecholamine stress-induced ventricular tachycardia compared with wild-type mice. Additionally, cMAO-Adef cardiomyocytes exhibited altered Ca2+ handling under catecholamine stimulation, with increased diastolic Ca2+ reuptake, reduced diastolic Ca2+ leak, and diminished systolic Ca2+ release. Mechanistically, cMAO-Adef hearts had reduced catecholamine levels under sympathetic stress, along with reduced levels of reactive oxygen species and protein carbonylation, leading to decreased oxidation of Type II PKA and CaMKII. These changes potentiated phospholamban (PLB) phosphorylation, thereby enhancing diastolic Ca2+ reuptake, while reducing ryanodine receptor 2 (RyR2) phosphorylation to decrease diastolic Ca2+ leak. Consequently, cMAO-Adef hearts exhibited lower diastolic Ca2+ levels and fewer arrhythmogenic Ca2+ waves during sympathetic overstimulation. CONCLUSION: Cardiac MAO-A inhibition exerts an anti-arrhythmic effect by enhancing diastolic Ca2+ handling under catecholamine stress.