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.

Nitrosative stress drives heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.

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.

p38γ and p38δ regulate postnatal cardiac metabolism through glycogen synthase 1.

During the first weeks of postnatal heart development, cardiomyocytes undergo a major adaptive metabolic shift from glycolytic energy production to fatty acid oxidation. This metabolic change is contemporaneous to the up-regulation and activation of the p38γ and p38δ stress-activated protein kinases in the heart. We demonstrate that p38γ/δ contribute to the early postnatal cardiac metabolic switch through inhibitory phosphorylation of glycogen synthase 1 (GYS1) and glycogen metabolism inactivation. Premature induction of p38γ/δ activation in cardiomyocytes of newborn mice results in an early GYS1 phosphorylation and inhibition of cardiac glycogen production, triggering an early metabolic shift that induces a deficit in cardiomyocyte fuel supply, leading to whole-body metabolic deregulation and maladaptive cardiac pathogenesis. Notably, the adverse effects of forced premature cardiac p38γ/δ activation in neonate mice are prevented by maternal diet supplementation of fatty acids during pregnancy and lactation. These results suggest that diet interventions have a potential for treating human cardiac genetic diseases that affect heart metabolism.

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.

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.

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.

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.

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.

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.

An ultrasensitive biosensor for high-resolution kinase activity imaging in awake mice.

Protein kinases control nearly every facet of cellular function. These key signaling nodes integrate diverse pathway inputs to regulate complex physiological processes, and aberrant kinase signaling is linked to numerous pathologies. While fluorescent protein-based biosensors have revolutionized the study of kinase signaling by allowing direct, spatiotemporally precise kinase activity measurements in living cells, powerful new molecular tools capable of robustly tracking kinase activity dynamics across diverse experimental contexts are needed to fully dissect the role of kinase signaling in physiology and disease. Here, we report the development of an ultrasensitive, second-generation excitation-ratiometric protein kinase A (PKA) activity reporter (ExRai-AKAR2), obtained via high-throughput linker library screening, that enables sensitive and rapid monitoring of live-cell PKA activity across multiple fluorescence detection modalities, including plate reading, cell sorting and one- or two-photon imaging. Notably, in vivo visual cortex imaging in awake mice reveals highly dynamic neuronal PKA activity rapidly recruited by forced locomotion.

Nampt Potentiates Antioxidant Defense in Diabetic Cardiomyopathy.

[Figure: see text].

CaMKII Serine 280 O-GlcNAcylation Links Diabetic Hyperglycemia to Proarrhythmia.

[Figure: see text].

COVID-19 and Cardiovascular Disease: From Bench to Bedside.

A pandemic of historic impact, coronavirus disease 2019 (COVID-19) has potential consequences on the cardiovascular health of millions of people who survive infection worldwide. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), the etiologic agent of COVID-19, can infect the heart, vascular tissues, and circulating cells through ACE2 (angiotensin-converting enzyme 2), the host cell receptor for the viral spike protein. Acute cardiac injury is a common extrapulmonary manifestation of COVID-19 with potential chronic consequences. This update provides a review of the clinical manifestations of cardiovascular involvement, potential direct SARS-CoV-2 and indirect immune response mechanisms impacting the cardiovascular system, and implications for the management of patients after recovery from acute COVID-19 infection.

Serine biosynthesis as a novel therapeutic target for dilated cardiomyopathy.

AIMS: Genetic dilated cardiomyopathy (DCM) is a leading cause of heart failure. Despite significant progress in understanding the genetic aetiologies of DCM, the molecular mechanisms underlying the pathogenesis of familial DCM remain unknown, translating to a lack of disease-specific therapies. The discovery of novel targets for the treatment of DCM was sought using phenotypic sceening assays in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) that recapitulate the disease phenotypes in vitro. METHODS AND RESULTS: Using patient-specific iPSCs carrying a pathogenic TNNT2 gene mutation (p.R183W) and CRISPR-based genome editing, a faithful DCM model in vitro was developed. An unbiased phenotypic screening in TNNT2 mutant iPSC-derived cardiomyocytes (iPSC-CMs) with small molecule kinase inhibitors (SMKIs) was performed to identify novel therapeutic targets. Two SMKIs, Gö 6976 and SB 203580, were discovered whose combinatorial treatment rescued contractile dysfunction in DCM iPSC-CMs carrying gene mutations of various ontologies (TNNT2, TTN, LMNA, PLN, TPM1, LAMA2). The combinatorial SMKI treatment upregulated the expression of genes that encode serine, glycine, and one-carbon metabolism enzymes and significantly increased the intracellular levels of glucose-derived serine and glycine in DCM iPSC-CMs. Furthermore, the treatment rescued the mitochondrial respiration defects and increased the levels of the tricarboxylic acid cycle metabolites and ATP in DCM iPSC-CMs. Finally, the rescue of the DCM phenotypes was mediated by the activating transcription factor 4 (ATF4) and its downstream effector genes, phosphoglycerate dehydrogenase (PHGDH), which encodes a critical enzyme of the serine biosynthesis pathway, and Tribbles 3 (TRIB3), a pseudokinase with pleiotropic cellular functions. CONCLUSIONS: A phenotypic screening platform using DCM iPSC-CMs was established for therapeutic target discovery. A combination of SMKIs ameliorated contractile and metabolic dysfunction in DCM iPSC-CMs mediated via the ATF4-dependent serine biosynthesis pathway. Together, these findings suggest that modulation of serine biosynthesis signalling may represent a novel genotype-agnostic therapeutic strategy for genetic DCM.

Reversing mitochondrial defects in aged hearts: role of mitochondrial calpain activation.

Aging chronically increases endoplasmic reticulum (ER) stress that contributes to mitochondrial dysfunction. Activation of calpain 1 (CPN1) impairs mitochondrial function during acute ER stress. We proposed that aging-induced ER stress led to mitochondrial dysfunction by activating CPN1. We posit that attenuation of the ER stress or direct inhibition of CPN1 in aged hearts can decrease cardiac injury during ischemia-reperfusion by improving mitochondrial function. Male young (3 mo) and aged mice (24 mo) were used in the present study, and 4-phenylbutyrate (4-PBA) was used to decrease the ER stress in aged mice. Subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) were isolated. Chronic 4-PBA treatment for 2 wk decreased CPN1 activation as shown by the decreased cleavage of spectrin in cytosol and apoptosis inducing factor (AIF) and the α1 subunit of pyruvate dehydrogenase (PDH) in mitochondria. Treatment improved oxidative phosphorylation in 24-mo-old SSM and IFM at baseline compared with vehicle. When 4-PBA-treated 24-mo-old hearts were subjected to ischemia-reperfusion, infarct size was decreased. These results support that attenuation of the ER stress decreased cardiac injury in aged hearts by improving mitochondrial function before ischemia. To challenge the role of CPN1 as an effector of the ER stress, aged mice were treated with MDL-28170 (MDL, an inhibitor of calpain 1). MDL treatment improved mitochondrial function in aged SSM and IFM. MDL-treated 24-mo-old hearts sustained less cardiac injury following ischemia-reperfusion. These results support that age-induced ER stress augments cardiac injury during ischemia-reperfusion by impairing mitochondrial function through activation of CPN1.

Catalpol alleviates myocardial ischemia reperfusion injury by activating the Nrf2/HO-1 signaling pathway.

PURPOSE: Myocardial ischemia/reperfusion injury (MI/RI) is a major problem in the clinical treatment of ischemic cardiomyopathy, and its specific underlying mechanisms are complicated and still unclear. A number of studies have indicated that the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxidase-1(HO-1) signaling pathway might serve as an important target for the management of MI/RI. Catalpol is a kind of iridoid glucoside that has been found to exhibit diverse anti-inflammatory and antioxidant properties. This study was aimed at investigating the role of Catalpol in targeting MI/RI and its related mechanisms in an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro and a preclinical ischemia/reperfusion (I/R) model. METHODS: This study using both in vitro and in vivo models investigated the possible role and underlying mechanisms used by Catalpol for modulating of MI/RI. The potential effects of Catalpol on the viability of cardiomyocytes were measured by cell counting kit-8 (CCK-8) assays. The phenotypes of myocardial injury, oxidative stress and inflammation markers were measured by western blot, immunofluorescence, enzyme-linked immunosorbent assay (ELISA) etc. Nrf2/HO-1 signaling pathway was detected by immunofluorescence and western blot analysis. RESULTS: We found that Catalpol significantly suppressed the process of MI/RI and protected OGD/R-treated cardiomyocytes by inhibiting the various markers of inflammation and suppressing oxidative stress. Additionally, mechanistically it was also demonstrated that Catalpol could effectively activate Nrf2/HO-1 signaling pathway to suppress the damage caused by inflammation and oxidative stress in MI/RI. CONCLUSION: In summary, the findings suggest that Catalpol exerted significant cardioprotective effects following myocardial ischemia, possibly through the activation of the Nrf2/HO-1 signaling pathway.

GRK5 Controls SAP97-Dependent Cardiotoxic ß1 Adrenergic Receptor-CaMKII Signaling in Heart Failure.

RATIONALE: Cardiotoxic ß1 adrenergic receptor (ß1AR)-CaMKII (calmodulin-dependent kinase II) signaling is a major and critical feature associated with development of heart failure. SAP97 (synapse-associated protein 97) is a multifunctional scaffold protein that binds directly to the C-terminus of ß1AR and organizes a receptor signalosome. OBJECTIVE: We aim to elucidate the dynamics of ß1AR-SAP97 signalosome and its potential role in chronic cardiotoxic ß1AR-CaMKII signaling that contributes to development of heart failure. METHODS AND RESULTS: The integrity of cardiac ß1AR-SAP97 complex was examined in heart failure. Cardiac-specific deletion of SAP97 was developed to examine ß1AR signaling in aging mice, after chronic adrenergic stimulation, and in pressure overload hypertrophic heart failure. We show that the ß1AR-SAP97 signaling complex is reduced in heart failure. Cardiac-specific deletion of SAP97 yields an aging-dependent cardiomyopathy and exacerbates cardiac dysfunction induced by chronic adrenergic stimulation and pressure overload, which are associated with elevated CaMKII activity. Loss of SAP97 promotes PKA (protein kinase A)-dependent association of ß1AR with arrestin2 and CaMKII and turns on an Epac (exchange protein directly activated by cAMP)-dependent activation of CaMKII, which drives detrimental functional and structural remodeling in myocardium. Moreover, we have identified that GRK5 (G-protein receptor kinase-5) is necessary to promote agonist-induced dissociation of SAP97 from ß1AR. Cardiac deletion of GRK5 prevents adrenergic-induced dissociation of ß1AR-SAP97 complex and increases in CaMKII activity in hearts. CONCLUSIONS: These data reveal a critical role of SAP97 in maintaining the integrity of cardiac ß1AR signaling and a detrimental cardiac GRK5-CaMKII axis that can be potentially targeted in heart failure therapy. Graphical Abstract: A graphical abstract is available for this article.

Atrial Myocyte NLRP3/CaMKII Nexus Forms a Substrate for Postoperative Atrial Fibrillation.

RATIONALE: Postoperative atrial fibrillation (POAF) is a common and troublesome complication of cardiac surgery. POAF is generally believed to occur when postoperative triggers act on a preexisting vulnerable substrate, but the underlying cellular and molecular mechanisms are largely unknown. OBJECTIVE: To identify cellular POAF mechanisms in right atrial samples from patients without a history of atrial fibrillation undergoing open-heart surgery. METHODS AND RESULTS: Multicellular action potentials, membrane ion-currents (perforated patch-clamp), or simultaneous membrane-current (ruptured patch-clamp) and [Ca2+]i-recordings in atrial cardiomyocytes, along with protein-expression levels in tissue homogenates or cardiomyocytes, were assessed in 265 atrial samples from patients without or with POAF. No indices of electrical, profibrotic, or connexin remodeling were noted in POAF, but Ca2+-transient amplitude was smaller, although spontaneous sarcoplasmic reticulum (SR) Ca2+-release events and L-type Ca2+-current alternans occurred more frequently. CaMKII (Ca2+/calmodulin-dependent protein kinase-II) protein-expression, CaMKII-dependent phosphorylation of the cardiac RyR2 (ryanodine-receptor channel type-2), and RyR2 single-channel open-probability were significantly increased in POAF. SR Ca2+-content was unchanged in POAF despite greater SR Ca2+-leak, with a trend towards increased SR Ca2+-ATPase activity. Patients with POAF also showed stronger expression of activated components of the NLRP3 (NACHT, LRR, and PYD domains-containing protein-3)-inflammasome system in atrial whole-tissue homogenates and cardiomyocytes. Acute application of interleukin-1ß caused NLRP3-signaling activation and CaMKII-dependent RyR2/phospholamban hyperphosphorylation in an immortalized mouse atrial cardiomyocyte cell-line (HL-1-cardiomyocytes) and enhanced spontaneous SR Ca2+-release events in both POAF cardiomyocytes and HL-1-cardiomyocytes. Computational modeling showed that RyR2 dysfunction and increased SR Ca2+-uptake are sufficient to reproduce the Ca2+-handling phenotype and indicated an increased risk of proarrhythmic delayed afterdepolarizations in POAF subjects in response to interleukin-1ß. CONCLUSIONS: Preexisting Ca2+-handling abnormalities and activation of NLRP3-inflammasome/CaMKII signaling are evident in atrial cardiomyocytes from patients who subsequently develop POAF. These molecular substrates sensitize cardiomyocytes to spontaneous Ca2+-releases and arrhythmogenic afterdepolarizations, particularly upon exposure to inflammatory mediators. Our data reveal a potential cellular and molecular substrate for this important clinical problem.

Hyperglycemia Acutely Increases Cytosolic Reactive Oxygen Species via O-linked GlcNAcylation and CaMKII Activation in Mouse Ventricular Myocytes.

RATIONALE: Diabetes mellitus is a complex, multisystem disease, affecting large populations worldwide. Chronic CaMKII (Ca2+/calmodulin-dependent kinase II) activation may occur in diabetes mellitus and be arrhythmogenic. Diabetic hyperglycemia was shown to activate CaMKII by (1) O-linked attachment of N-acetylglucosamine (O-GlcNAc) at S280 leading to arrhythmia and (2) a reactive oxygen species (ROS)-mediated oxidation of CaMKII that can increase postinfarction mortality. OBJECTIVE: To test whether high extracellular glucose (Hi-Glu) promotes ventricular myocyte ROS generation and the role played by CaMKII. METHODS AND RESULTS: We tested how extracellular Hi-Glu influences ROS production in adult ventricular myocytes, using DCF (2',7'-dichlorodihydrofluorescein diacetate) and genetically targeted Grx-roGFP2 redox sensors. Hi-Glu (30 mmol/L) significantly increased the rate of ROS generation-an effect prevented in myocytes pretreated with CaMKII inhibitor KN-93 or from either global or cardiac-specific CaMKIIδ KO (knockout) mice. CaMKII KO or inhibition also prevented Hi-Glu-induced sarcoplasmic reticulum Ca2+ release events (Ca2+ sparks). Thus, CaMKII activation is required for Hi-Glu-induced ROS generation and sarcoplasmic reticulum Ca2+ leak in cardiomyocytes. To test the involvement of O-GlcNAc-CaMKII pathway, we inhibited GlcNAcylation removal by Thiamet G (ThmG), which mimicked the Hi-Glu-induced ROS production. Conversely, inhibition of GlcNAcylation (OSMI-1 [(αR)-α-[[(1,2-dihydro-2-oxo-6-quinolinyl)sulfonyl]amino]-N-(2-furanylmethyl)-2-methoxy-N-(2-thienylmethyl)-benzeneacetamide]) prevented ROS induction in response to either Hi-Glu or ThmG. Moreover, in a CRSPR-based knock-in mouse in which the functional GlcNAcylation site on CaMKIIδ was ablated (S280A), neither Hi-Glu nor ThmG induced myocyte ROS generation. So CaMKIIδ-S280 is required for the Hi-Glu-induced (and GlcNAc dependent) ROS production. To identify the ROS source(s), we used different inhibitors of NOX (NADPH oxidase) 2 (Gp91ds-tat peptide), NOX4 (GKT137831), mitochondrial ROS (MitoTempo), and NOS (NO synthase) pathway inhibitors (L-NAME, L-NIO, and L-NPA). Only NOX2 inhibition or KO prevented Hi-Glu/ThmG-induced ROS generation. CONCLUSIONS: Diabetic hyperglycemia induces acute cardiac myocyte ROS production by NOX2 that requires O-GlcNAcylation of CaMKIIδ at S280. This novel ROS induction may exacerbate pathological consequences of diabetic hyperglycemia.