Punicalagin attenuates isoproterenol-induced myocardial infarction through nuclear factor erythroid 2-related factor 2/silent information regulator transcript-1-mediated inhibition of inflammation and cardiac stress markers in experimental animal models.

Myocardial infarction (MI) is a significant global health issue and the leading cause of death. Myocardial infarction (MI) is characterized by events such as damage to heart cells and stress generated by inflammation. Punicalagin (PCN), a naturally occurring bioactive compound found in pomegranates, exhibits a diverse array of pharmacological effects against many disorders. This study aimed to assess the preventive impact of PCN, with its potential anti-inflammatory and antioxidant properties, on myocardial injury caused by isoproterenol (ISO) in rats and elucidate the possible underlying mechanisms. Experimental rats were randomly categorized into four groups: control group (fed a regular diet for 15 days), PCN group (orally administered PCN at 50 mg/kg body weight (b.w.) for 15 days), ISO group (subcutaneously administered ISO (85 mg/kg b.w.) on days 14 and 15 to induce MI), and PCN+ISO group (orally preadministered PCN (50 mg/kg b.w.) for 15 days and administered ISO (85 mg/kg b.w.) on days 14 and 15). The rat cardiac tissue was then investigated for cardiac marker, oxidative stress marker, and inflammatory marker expression levels. PCN prevented ISO-induced myocardial injury, suppressing the levels of creatine kinase-myocardial band, C-reactive protein, homocysteine, cardiac troponin T, and cardiac troponin I in the rats. Moreover, PCN treatment reversed (P<0.01) the ISO-induced increase in blood pressure, attenuated lipid peroxidation markers, and depleted both enzymatic and nonenzymatic markers in the rats. Additionally, PCN inhibited (P<0.01) ISO-induced overexpression of oxidative stress markers (p-38, p-c-Jun N-terminal kinase, and p-extracellular signal-regulated kinase 1), inflammatory markers (nuclear factor-kappa B, tumor necrosis factor-alpha, and interleukin-6), and matrix metalloproteinases and decreased the levels (P<0.01) of apoptosis proteins in the rats. Nuclear factor erythroid 2-related factor 2/silent information regulator transcript-1 (Nrf2/Sirt1) is a major cellular defense protein that regulates and scavenges oxidative toxic substances through apoptosis. Therefore, overexpression of Nrf2/Sirt1 to inhibit inflammation and oxidative stress is considered a novel target for preventing MI. PCN also significantly enhanced the expression of Nrf2/Sirt1 in ISO-induced rats. Histopathological analyses of cardiac tissue revealed that PCN treatment exhibited a protective effect on the heart tissue, mitigating damage. These findings show that by activating the Nrf2/Sirt1 pathway, PCN regulates oxidative stress, inflammation, and apoptosis, hence providing protection against ISO-induced myocardial ischemia.

Knockdown of SCN5A alters metabolic-associated genes and aggravates hypertrophy in the cardiomyoblast.

SCN5A mutations have been reported to cause various cardiomyopathies in humans. Most of the SCN5A mutations causes loss of function and thereby, alters the overall cellular function. Therefore, to understand the loss of SCN5A function in cardiomyocytes, we have knocked down the SCN5A gene (SCN5A-KD) in H9c2 cells and explored the cell phenotype and molecular behaviors in the presence and absence of isoproterenol (ISO), an adrenergic receptor agonist that induces cardiac hypertrophy. Expression of several genes related to hypertrophy, inflammation, fibrosis, and energy metabolism pathways were evaluated. It was found that the mRNA expression of hypertrophy-related gene, brain (B-type) natriuretic peptide (BNP) was significantly increased in SCN5A-KD cells as compared to 'control' H9c2 cells. There was a further increase in the mRNA expressions of BNP and ßMHC in SCN5A-KD cells after ISO treatment compared to their respective controls. Pro-inflammatory cytokine, tumor necrosis factor-alpha expression was significantly increased in 'SCN5A-KD' H9c2 cells. Further, metabolism-related genes like glucose transporter type 4, cluster of differentiation 36, peroxisome proliferator-activated receptor alpha, and peroxisome proliferator-activated receptor-gamma were significantly elevated in the SCN5A-KD cells as compared to the control cells. Upregulation of these metabolic genes is associated with increased ATP production. The study revealed that SCN5A knock-down causes alteration of gene expression related to cardiac hypertrophy, inflammation, and energy metabolism pathways, which may promote cardiac remodelling and cardiomyopathy.

LncRNA GAS5 restrains ISO-induced cardiac fibrosis by modulating mir-217 regulation of SIRT1.

Considering the effect of SIRT1 on improving myocardial fibrosis and GAS5 inhibiting occurrence and development of myocardial fibrosis at the cellular level, the aim of the present study was to investigate whether LncRNA GAS5 could attenuate cardiac fibrosis through regulating mir-217/SIRT1, and whether the NLRP3 inflammasome activation was involved in this process. Isoprenaline (ISO) was given subcutaneously to the male C57BL/6 mice to induce myocardial fibrosis and the AAV9 vectors were randomly injected into the left ventricle of each mouse to overexpress GAS5. Primary myocardial fibroblasts (MCFs) derived from neonatal C57BL/6 mice and TGF-ß1 were used to induce fibrosis. And the GAS5 overexpressed MCFs were treated with mir-217 mimics and mir-217 inhibitor respectively. Then the assays of expression levels of NLRP3, Caspase-1, IL-1ß and SIRT1 were conducted. The findings indicated that the overexpression of GAS5 reduced the expression levels of collagen, NLRP3, Capase-1, IL-1ß and SIRT1 in ISO treated mice and TGF-ß1 treated MCFs. However, this effect was significantly weakened after mir-217 overexpression, but was further enhanced after knockdown of mir-217. mir-217 down-regulates the expression of SIRT1, leading to increased activation of the NLRP3 inflammasome and subsequent pyroptosis. LncRNA GAS5 alleviates cardiac fibrosis induced via regulating mir-217/SIRT1 pathway.

Cardioprotective Potential of Cymbopogon citratus Essential Oil against Isoproterenol-induced Cardiomyocyte Hypertrophy: Possible Involvement of NLRP3 Inflammasome and Oxidative Phosphorylation Complex Subunits.

OBJECTIVE: Cymbopogon citratus (DC.) Stapf is a medicinal and edible herb that is widely used for the treatment of gastric, nervous and hypertensive disorders. In this study, we investigated the cardioprotective effects and mechanisms of the essential oil, the main active ingredient of Cymbopogon citratus, on isoproterenol (ISO)-induced cardiomyocyte hypertrophy. METHODS: The compositions of Cymbopogon citratus essential oil (CCEO) were determined by gas chromatography-mass spectrometry. Cardiomyocytes were pretreated with 16.9 µg/L CCEO for 1 h followed by 10 µmol/L ISO for 24 h. Cardiac hypertrophy-related indicators and NLRP3 inflammasome expression were evaluated. Subsequently, transcriptome sequencing (RNA-seq) and target verification were used to further explore the underlying mechanism. RESULTS: Our results showed that the CCEO mainly included citronellal (45.66%), geraniol (23.32%), and citronellol (10.37%). CCEO inhibited ISO-induced increases in cell surface area and protein content, as well as the upregulation of fetal gene expression. Moreover, CCEO inhibited ISO-induced NLRP3 inflammasome expression, as evidenced by decreased lactate dehydrogenase content and downregulated mRNA levels of NLRP3, ASC, CASP1, GSDMD, and IL-1ß, as well as reduced protein levels of NLRP3, ASC, pro-caspase-1, caspase-1 (p20), GSDMD-FL, GSDMD-N, and pro-IL-1ß. The RNA-seq results showed that CCEO inhibited the increase in the mRNA levels of 26 oxidative phosphorylation complex subunits in ISO-treated cardiomyocytes. Our further experiments confirmed that CCEO suppressed ISO-induced upregulation of mt-Nd1, Sdhd, mt-Cytb, Uqcrq, and mt-Atp6 but had no obvious effects on mt-Col expression. CONCLUSION: CCEO inhibits ISO-induced cardiomyocyte hypertrophy through the suppression of NLRP3 inflammasome expression and the regulation of several oxidative phosphorylation complex subunits.

Real-Time cAMP Dynamics in Live Cells Using the Fluorescent cAMP Difference Detector In Situ.

cAMP Difference Detector In Situ (cADDis) is a novel biosensor that allows for the continuous measurement of cAMP levels in living cells. The biosensor is created from a circularly permuted fluorescent protein linked to the hinge region of Epac2. This creates a single fluorophore biosensor that displays either increased or decreased fluorescence upon binding of cAMP. The biosensor exists in red and green upward versions, as well as green downward versions, and several red and green versions targeted to subcellular locations. To illustrate the effectiveness of the biosensor, the green downward version, which decreases in fluorescence upon cAMP binding, was used. Two protocols using this sensor are demonstrated: one utilizing a 96-well plate reading spectrophotometer compatible with high-throughput screening and another utilizing single-cell imaging on a fluorescent microscope. On the plate reader, HEK-293 cells cultured in 96-well plates were stimulated with 10 µM forskolin or 10 nM isoproterenol, which induced rapid and large decreases in fluorescence in the green downward version. The biosensor was used to measure cAMP levels in individual human airway smooth muscle (HASM) cells monitored under a fluorescent microscope. The green downward biosensor displayed similar responses to populations of cells when stimulated with forskolin or isoproterenol. This single-cell assay allows visualization of the biosensor location at 20x and 40x magnification. Thus, this cAMP biosensor is sensitive and flexible, allowing real-time measurement of cAMP in both immortalized and primary cells, and with single cells or populations of cells. These attributes make cADDis a valuable tool for studying cAMP signaling dynamics in living cells.

The protective effect of Macrostemonoside T from Allium macrostemon Bunge against Isoproterenol-Induced myocardial injury via the PI3K/Akt/mTOR signaling pathway.

Myocardial injury (MI) signifies a pathological aspect of cardiovascular diseases (CVDs) such as coronary artery disease, diabetic cardiomyopathy, and myocarditis. Macrostemonoside T (MST) has been isolated from Allium macrostemon Bunge (AMB), a key traditional Chinese medicine (TCM) used for treating chest stuffiness and pains. Although MST has demonstrated considerable antioxidant activity in vitro, its protective effect against MI remains unexplored. To investigate MST's effects in both in vivo and in vitro models of isoproterenol (ISO)-induced MI and elucidate its underlying molecular mechanisms. This study established an ISO-induced MI model in rats and assessed H9c2 cytotoxicity to examine MST's impact on MI. Various assays, including histopathological staining, TUNEL staining, immunohistochemical staining, DCFH-DA staining, JC-1 staining, ELISA technique, and Western blot (WB), were utilized to explore the potential molecular mechanisms of MI protection. In vivo experiments demonstrated that ISO caused myocardial fiber disorders, elevated cardiac enzyme levels, and apoptosis. However, pretreatment with MST significantly mitigated these detrimental changes. In vitro experiments revealed that MST boosted antioxidant enzyme levels and suppressed malondialdehyde (MDA) production in H9c2 cells. Concurrently, MST inhibited ISO-induced reactive oxygen species (ROS) production and mitigated the decline in mitochondrial membrane potential, thereby reducing the apoptosis rate. Moreover, pretreatment with MST elevated the expression levels of p-PI3K, p-Akt, and p-mTOR, indicating activation of the PI3K/Akt/mTOR signaling pathway and consequent protection against MI. MST attenuated ISO-induced MI in rats by impeding apoptosis through activation of the PI3K/Akt/mTOR signaling pathway. This study presents potential avenues for the development of precursor drugs for CVDs.

AKAP12 Upregulation Associates With PDE8A to Accelerate Cardiac Dysfunction.

BACKGROUND: In heart failure, signaling downstream the ß2-adrenergic receptor is critical. Sympathetic stimulation of ß2-adrenergic receptor alters cAMP (cyclic adenosine 3',5'-monophosphate) and triggers PKA (protein kinase A)-dependent phosphorylation of proteins that regulate cardiac function. cAMP levels are regulated in part by PDEs (phosphodiesterases). Several AKAPs (A kinase anchoring proteins) regulate cardiac function and are proposed as targets for precise pharmacology. AKAP12 is expressed in the heart and has been reported to directly bind ß2-adrenergic receptor, PKA, and PDE4D. However, its roles in cardiac function are unclear. METHODS: cAMP accumulation in real time downstream of the ß2-adrenergic receptor was detected for 60 minutes in live cells using the luciferase-based biosensor (GloSensor) in AC16 human-derived cardiomyocyte cell lines overexpressing AKAP12 versus controls. Cardiomyocyte intracellular calcium and contractility were studied in adult primary cardiomyocytes from male and female mice overexpressing cardiac AKAP12 (AKAP12OX) and wild-type littermates post acute treatment with 100-nM isoproterenol (ISO). Systolic cardiac function was assessed in mice after 14 days of subcutaneous ISO administration (60 mg/kg per day). AKAP12 gene and protein expression levels were evaluated in left ventricular samples from patients with end-stage heart failure. RESULTS: AKAP12 upregulation significantly reduced total intracellular cAMP levels in AC16 cells through PDE8. Adult primary cardiomyocytes from AKAP12OX mice had significantly reduced contractility and impaired calcium handling in response to ISO, which was reversed in the presence of the selective PDE8 inhibitor (PF-04957325). AKAP12OX mice had deteriorated systolic cardiac function and enlarged left ventricles. Patients with end-stage heart failure had upregulated gene and protein levels of AKAP12. CONCLUSIONS: AKAP12 upregulation in cardiac tissue is associated with accelerated cardiac dysfunction through the AKAP12-PDE8 axis.

Overexpressing of the GIPC1 protects against pathological cardiac remodelling.

OBJECTIVE: Pathological cardiac remodelling, including cardiac hypertrophy and fibrosis, is a key pathological process in the development of heart failure. However, effective therapeutic approaches are limited. The ß-adrenergic receptors are pivotal signalling molecules in regulating cardiac function. G-alpha interacting protein (GAIP)-interacting protein, C-terminus 1 (GIPC1) is a multifunctional scaffold protein that directly binds to the C-terminus of ß1-adrenergic receptor (ß1-adrenergic receptor). However, little is known about its roles in heart function. Therefore, we investigated the role of GIPC1 in cardiac remodelling and its underlying molecular mechanisms. METHODS: Pathological cardiac remodelling in mice was established via intraperitoneal injection of isoprenaline for 14 d or transverse aortic constriction surgery for 8 weeks. Myh6-driving cardiomyocyte-specific GIPC1 conditional knockout (GIPC1 cKO) mice and adeno-associated virus 9 (AAV9)-mediated GIPC1 overexpression mice were used. The effect of GIPC1 on cardiac remodelling was assessed using echocardiographic, histological, and biochemical analyses. RESULTS: GIPC1 expression was consistently reduced in the cardiac remodelling model. GIPC1 cKO mice exhibited spontaneous abnormalities, including cardiac hypertrophy, fibrosis, and systolic dysfunction. In contrast, AAV9-mediated GIPC1 overexpression in the heart attenuated isoproterenol-induced pathological cardiac remodelling in mice. Mechanistically, GIPC1 interacted with the ß1-adrenergic receptor and stabilised its expression by preventing its ubiquitination and degradation, maintaining the balance of ß1-adrenergic receptor/ß2-adrenergic receptor, and inhibiting hyperactivation of the mitogen-activated protein kinase signalling pathway. CONCLUSIONS: These results suggested that GIPC1 plays a cardioprotective role and is a promising therapeutic target for the treatment of cardiac remodelling and heart failure.

Arglabin: A mediator of inflammasome modulated and independent myocardial injury (PARA-AMI study).

BACKGROUND: Arglabin is a plant alkaloid (sesquiterpene lactone) that is used as an anticancer drug. It has potential anti-diabetic and anti-atherogenic effects. PURPOSE: Arglabin has drawn particular attention because of its therapeutic effects as an anti-inflammatory agent in multiple diseases. Since arglabin inhibits Epidermal Growth Factor Receptor (EGFR) tyrosine kinase, concerns for cardiotoxic effects are valid. The present study was designed to investigate the protective effects of arglabin on the myocardium. STUDY DESIGN: This study was designed to evaluate the effect of arglabin on the myocardium in an experimental model of myocardial necrosis in rats. Different doses of arglabin (2.5, 5, and 10 µg/kg) were investigated as pre-treatment for 21 days in the isoproterenol (ISO) model of myocardial necrosis groups and per se groups. METHODS: On the 22nd day, hemodynamic, histopathological, electron microscopy, oxidative stress markers, inflammatory mediators, apoptotic markers, inflammasome mediators, and Western blot analysis were performed to evaluate the effects of arglabin. RESULTS: Arglabin pre-treatment showed improvement in hemodynamic parameters and histopathological findings at low doses in isoproterenol-induced myocardial necrosis model of rats. Arglabin administration altered myocardial structure and modulated myocardial function via activation of NFκB/MAPK pathway that led to myocardial injury with an increase in dose. CONCLUSION: Arglabin imparted partial cardio-protection via an inflammasome-dependent pathway and mediated injury through the inflammasome-independent pathway.

Human amniotic MSCs-mediated anti-inflammation of CD206hiIL-10hi macrophages alleviates isoproterenol-induced ventricular remodeling in mice.

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammation which makes them suitable for the treatment of various diseases. OBJECTIVE: This study aimed to explore the therapeutic effect and molecular mechanism of hAMSCs in ventricular remodeling (VR). METHODS: hAMSCs were characterized by a series of experiments such as flow cytometric analysis, immunofluorescence, differentiative induction and tumorigenicity. Mouse VR model was induced by isoproterenol (ISO) peritoneally, and the therapeutic effects and the potential mechanisms of hAMSCs transplantation were evaluated by echocardiography, carboxy fluorescein diacetate succinimidyl ester (CFSE) labeled cell tracing, histochemistry, qRT-PCR and western blot analysis. The co-culturing experiments were carried out for further exploring the mechanisms of hAMSCs-derived conditioned medium (CM) on macrophage polarization and fibroblast fibrosis in vitro. RESULTS: hAMSCs transplantation significantly alleviated ISO-induced VR including cardiac hypertrophy and fibrosis with the improvements of cardiac functions. CFSE labeled hAMSCs kept an undifferentiated state in heart, indicating that hAMSCs-mediated the improvement of ISO-induced VR might be related to their paracrine effects. hAMSCs markedly inhibited ISO-induced inflammation and fibrosis, seen as the increase of M2 macrophage infiltration and the expressions of CD206 and IL-10, and the decreases of CD86, iNOS, COL3 and αSMA expressions in heart, suggesting that hAMSCs transplantation promoted the polarization of M2 macrophages and inhibited the polarization of M1 macrophages. Mechanically, hAMSCs-derived CM significantly increased the expressions of CD206, IL-10, Arg-1 and reduced the expressions of iNOS and IL-6 in RAW264.7 macrophages in vitro. Interestingly, RAW264.7-CM remarkably promoted the expressions of anti-inflammatory factors such as IL-10, IDO, and COX2 in hAMSCs. Furthermore, the CM derived from hAMSCs pretreated with RAW264.7-CM markedly inhibited the expressions of fibrogenesis genes such as αSMA and COL3 in 3T3 cells. CONCLUSION: Our results demonstrated that hAMSCs effectively alleviated ISO-induced cardiac hypertrophy and fibrosis, and improved the cardiac functions in mice, and the underlying mechanisms might be related to inhibiting the inflammation and fibrosis during the ventricular remodeling through promoting the polarization of CD206hiIL-10hi macrophages in heart tissues. Our study strongly suggested that by taking the advantages of the potent immunosuppressive and anti-inflammatory effects, hAMSCs may provide an alternative therapeutic approach for prevention and treatment of VR clinically.

Mycn ameliorates cardiac hypertrophy-induced heart failure in mice by mediating the USP2/JUP/Akt/ß-catenin cascade.

BACKGROUND: Pathological cardiac hypertrophy is associated with cardiac dysfunction and is a key risk factor for heart failure and even sudden death. This study investigates the function of Mycn in cardiac hypertrophy and explores the interacting molecules. METHODS: A mouse model of cardiac hypertrophy was induced by isoproterenol (ISO). The cardiac dysfunction was assessed by the heart weight-to-body weight ratio (HW/BW), echocardiography assessment, pathological staining, biomarker detection, and cell apoptosis. Transcriptome alteration in cardiac hypertrophy was analyzed by bioinformatics analysis. Gain- or loss-of-function studies of MYCN proto-oncogene (Mycn), ubiquitin specific peptidase 2 (USP2), and junction plakoglobin (JUP) were performed. The biological functions of Mycn were further examined in ISO-treated cardiomyocytes. The molecular interactions were verified by luciferase assay or immunoprecipitation assays. RESULTS: Mycn was poorly expressed in ISO-treated mice, and its upregulation reduced HW/BW, cell surface area, oxidative stress, and inflammation while improving cardiac function of mice. It also reduced apoptosis of cardiomyocytes in mice and those in vitro induced by ISO. Mycn bound to the USP2 promoter to activate its transcription. USP2 overexpression exerted similar myocardial protective functions. It stabilized JUP protein by deubiquitination modification, which blocked the Akt/ß-catenin pathway. Knockdown of JUP restored phosphorylation of Akt and ß-catenin protein level, which negated the protective effects of USP2. CONCLUSION: This study demonstrates that Mycn activates USP2 transcription, which mediates ubiquitination and protein stabilization of JUP, thus inactivating the Akt/ß-catenin axis and alleviating cardiac hypertrophy-induced heart failure.

OTUD1 promotes isoprenaline- and myocardial infarction-induced heart failure by targeting PDE5A in cardiomyocytes.

Heart failure represents a major cause of death worldwide. Recent research has emphasized the potential role of protein ubiquitination/deubiquitination protein modification in cardiac pathology. Here, we investigate the role of the ovarian tumor deubiquitinase 1 (OTUD1) in isoprenaline (ISO)- and myocardial infarction (MI)-induced heart failure and its molecular mechanism. OTUD1 protein levels were raised markedly in murine cardiomyocytes after MI and ISO treatment. OTUD1 deficiency attenuated myocardial hypertrophy and cardiac dysfunction induced by ISO infusion or MI operation. In vitro, OTUD1 knockdown in neonatal rat ventricular myocytes (NRVMs) attenuated ISO-induced injuries, while OTUD1 overexpression aggravated the pathological changes. Mechanistically, LC-MS/MS and Co-IP studies showed that OTUD1 bound directly to the GAF1 and PDEase domains of PDE5A. OTUD1 was found to reverse K48 ubiquitin chain in PDE5A through cysteine at position 320 of OTUD1, preventing its proteasomal degradation. PDE5A could inactivates the cGMP-PKG-SERCA2a signaling axis which dysregulate the calcium handling in cardiomyocytes, and leading to the cardiomyocyte injuries. In conclusion, OTUD1 promotes heart failure by deubiquitinating and stabilizing PDE5A in cardiomyocytes. These findings have identified PDE5A as a new target of OTUD1 and emphasize the potential of OTUD1 as a target for treating heart failure.

Regulation of Na+-K+-ATPase leads to disturbances of isoproterenol-induced cardiac dysfunction via interference of Ca2+-dependent cardiac metabolism.

Reductions in Na+-K+-ATPase (NKA) activity and expression are often observed in the progress of various reason-induced heart failure (HF). However, NKA α1 mutation or knockdown cannot cause spontaneous heart disease. Whether the abnormal NKA α1 directly contributes to HF pathogenesis remains unknown. Here, we challenge NKA α1+/- mice with isoproterenol to evaluate the role of NKA α1 haploinsufficiency in isoproterenol (ISO)-induced cardiac dysfunction. Genetic knockdown of NKA α1 accelerated ISO-induced cardiac cell hypertrophy, heart fibrosis, and dysfunction. Further studies revealed decreased Krebs cycle, fatty acid oxidation, and mitochondrial OXPHOS in the hearts of NKA α1+/- mice challenged with ISO. In ISO-treated conditions, inhibition of NKA elevated cytosolic Na+, further reduced mitochondrial Ca2+ via mNCE, and then finally down-regulated cardiac cell energy metabolism. In addition, a supplement of DRm217 alleviated ISO-induced heart dysfunction, mitigated cardiac remodeling, and improved cytosolic Na+ and Ca2+ elevation and mitochondrial Ca2+ depression in the NKA α1+/- mouse model. The findings suggest that targeting NKA and mitochondria Ca2+ could be a promising strategy in the treatment of heart disease.

Adenosine monophosphate-regulated protein kinase inhibition modulates electrophysiological characteristics and calcium homeostasis of rabbit right ventricular outflow tract.

BACKGROUND: Metabolic stress predisposes to ventricular arrhythmias and sudden cardiac death. Right ventricular outflow tract (RVOT) is the common origin of ventricular arrhythmias. Adenosine monophosphate-regulated protein kinase (AMPK) activation is an important compensatory mechanism for cardiac remodeling during metabolic stress. OBJECTIVES: The purpose of this study was to access whether AMPK inhibition would modulate RVOT electrophysiology, calcium (Ca2+ ) regulation, and RVOT arrhythmogenesis or not. METHODS: Conventional microelectrodes were used to record electrical activity before and after compound C (10 µM, an AMPK inhibitor) in isoproterenol (1 µM)-treated rabbit RVOT tissue preparations under electrical pacing. Whole-cell patch-clamp and confocal microscopic examinations were performed in baseline and compound C-treated rabbit RVOT cardiomyocytes to investigate ionic currents and intracellular Ca2+ transients in isolated rabbit RVOT cardiomyocytes. RESULTS: Compound C decreased RVOT contractility, and reversed isoproterenol increased RVOT contractility. Compound C decreased the incidence, rate, and duration of isoproterenol-induced RVOT burst firing under rapid pacing. Compared to baseline, compound C-treated RVOT cardiomyocytes had a longer action potential duration, smaller intracellular Ca2+ transients, late sodium (Na+ ), peak L-type Ca2+ current density, Na+ -Ca2+ exchanger, transient outward potassium (K+ ) current, and rapid and slow delayed rectifier K+ currents. CONCLUSION: AMPK inhibition modulates RVOT electrophysiological characteristics and Ca2+ homeostasis, contributing to lower RVOT arrhythmogenic activity. Accordingly, AMPK inhibition might potentially reduce ventricular tachyarrhythmias.

Chitosan-sodium alginate-polyethylene glycol-Ally isothiocyante nanocomposites ameliorates isoproterenol-induced myocardial infarction in rats.

Myocardial infarction (MI) is a common type of ischemic heart disease that affects millions of people worldwide. In recent times, nanotechnology has become a very promising field with immense applications. The current exploration was conducted to synthesize the chitosan-sodium alginate-polyethylene glycol-Ally isothiocyanate nanocomposites (CSP-AIso-NCs) and evaluate their beneficial roles against the isoproterenol (ISO)-induced MI in rats. The CSP-AIso-NCs were prepared and characterized by several characterization techniques. The MI was initiated in the rats by the administration of 85 mg/kg of ISO for 2 days and treated with 10 and 20 mg/kg of CSP-AIso-NCs for 1 month. The changes in heart weight and bodyweight were measured. The cardiac function markers were assessed with echocardiography. The lipid profiles, Na+, K+, and Ca2+ ions, cardiac biomarkers, antioxidant parameters, and inflammatory cytokines were assessed using corresponding assay kits. The histopathological study was done on the heart tissues. The UV spectral analysis revealed the maximum peak at 208 nm, which confirms the formation of CSP-AIso-NCs. The FT-IR analysis revealed the occurrence of different functional groups, and the crystallinity of the CSP-AIso-NCs was proved by the XRD analysis. DLS analysis indicated the size of the CSP-AIso-NCs at 146.50 nm. The CSP-AIso-NCs treatment increased the bodyweight and decreased the HW/BW ratio in the MI rats. The status of lipids was reduced, and HDL was elevated in the CSP-AIso-NCs administered to MI rats. CSP-AIso-NCs decreased the LVEDs, LVEDd, and NT-proBNP and increased the LVEF level. The oxidative stress markers were decreased, and the antioxidants were increased by the CSP-AIso-NCs treatment in the MI rats. The Na+ and Ca+ ions were reduced, and the K+ ions were increased by the CSP-AIso-NCs. The interleukin-1ß and tumor necrosis factor-α were also depleted, and Nrf-2 was improved in the CSP-AIso-NCs administered to MI rats. The histological study revealed the ameliorative effects of CSP-AIso-NCs. Overall, our outcomes revealed that the CSP-AIso-NCs are effective against the ISO-induced MI rats. Hence, it could be a hopeful therapeutic nanomedicine for MI treatment.

Dapagliflozin Suppresses Isoprenaline-Induced Cardiac Hypertrophy Through Inhibition of Mitochondrial Fission.

ABSTRACT: Dapagliflozin (DAPA) is a novel oral hypoglycemic agent, and there is increasing evidence that DAPA has a protective effect against cardiovascular disease. The study aimed to investigate how DAPA inhibits cardiac hypertrophy and explore its potential mechanisms. By continuously infusing isoprenaline (ISO) for 2 weeks using a subcutaneous osmotic pump, a cardiac hypertrophic model was established in male C57BL/6 mice. On day 14 after surgery, echocardiography showed that left ventricle mass (LV mass), interventricular septum, left ventricle posterior wall diastole, and left ventricular posterior wall systole were significantly increased, and ejection fraction was decreased compared with control mice. Masson and Wheat Germ Agglutinin staining indicated enhanced myocardial fibrosis and cell morphology compared with control mice. Importantly, these effects were inhibited by DAPA treatment in ISO-induced mice. In H9c2 cells and neonatal rat cardiomyocytes, we found that mitochondrial fragmentation and mitochondrial oxidative stress were significantly augmented in the ISO-induced group. However, DAPA rescued the cardiac hypertrophy in ISO-induced H9c2 cells and neonatal rat cardiomyocytes. Mechanistically, we found that DAPA restored the PIM1 activity in ISO-induced H9c2 cells and subsequent increase in dynamin-associated protein 1 (Drp1) phosphorylation at S616 and decrease in Drp1 phosphorylation at S637 in ISO-induced cells. We found that DAPA mitigated ISO-induced cardiac hypertrophy by suppressing Drp1-mediated mitochondrial fission in a PIM1-dependent fashion.

The Detrimental Effect of Pre-Treatment with Ivermectin on Myocardial Ischemia.

INTRODUCTION: Ivermectin (IVM) is a broad-spectrum anti-parasitic agent with potential antibacterial, antiviral, and anti-cancer effects. There are limited studies on the effects of IVM on cardiovascular diseases, so the present study sought to determine the effects of pre-treatment with IVM on myocardial ischemia in both ex vivo and in vivo. METHODS: In the ex vivo part, two groups of control and treated rats with IVM (0.2 mg/kg) were examined for cardiac function and arrhythmias by isolated heart perfusion. In the in vivo part, four groups, namely, control, IVM, Iso (MI), and Iso + IVM 0.2 mg/kg were used. Subcutaneous injection of isoproterenol (100 mg/kg/day) for 2 consecutive days was used for the induction of myocardial infarction (MI) in male Wistar rats. Then electrocardiogram, hemodynamic factors, cardiac hypertrophy, and malondialdehyde (MDA) levels were investigated. RESULTS: The ex vivo results showed that administration of IVM induces cardiac arrhythmia and decreases the left ventricular maximal rate of pressure increase (contractility) and maximal rate of pressure decline (relaxation). The isoproterenol-induced MI model used as an in vivo model showed that cardiac hypertrophy were increased with no improvement in the hemodynamic and electrocardiogram pattern in the IVM-treated group in comparison to MI (Iso) group. However, the MDA level was lower in the IVM-treated group. CONCLUSION: IVM pre-treatment demonstrates detrimental effects in cardiac ischemia through exacerbation of cardiac arrhythmia, myocardial dysfunction, and increased cardiac hypertrophy. Therefore, the use of IVM in ischemic heart patients should be done with great caution.

Isoproterenol induces MD2 activation by ß-AR-cAMP-PKA-ROS signalling axis in cardiomyocytes and macrophages drives inflammatory heart failure.

Cardiac inflammation contributes to heart failure (HF) induced by isoproterenol (ISO) through activating ß-adrenergic receptors (ß-AR). Recent evidence shows that myeloid differentiation factor 2 (MD2), a key protein in endotoxin-induced inflammation, mediates inflammatory heart diseases. In this study, we investigated the role of MD2 in ISO-ß-AR-induced heart injuries and HF. Mice were infused with ISO (30 mg·kg-1·d-1) via osmotic mini-pumps for 2 weeks. We showed that MD2 in cardiomyocytes and cardiac macrophages was significantly increased and activated in the heart tissues of ISO-challenged mice. Either MD2 knockout or administration of MD2 inhibitor L6H21 (10 mg/kg every 2 days, i.g.) could prevent mouse hearts from ISO-induced inflammation, remodelling and dysfunction. Bone marrow transplantation study revealed that both cardiomyocyte MD2 and bone marrow-derived macrophage MD2 contributed to ISO-induced cardiac inflammation and injuries. In ISO-treated H9c2 cardiomyocyte-like cells, neonatal rat primary cardiomyocytes and primary mouse peritoneal macrophages, MD2 knockout or pre-treatment with L6H21 (10 µM) alleviated ISO-induced inflammatory responses, and the conditioned medium from ISO-challenged macrophages promoted the hypertrophy and fibrosis in cardiomyocytes and fibroblasts. We demonstrated that ISO induced MD2 activation in cardiomyocytes via ß1-AR-cAMP-PKA-ROS signalling axis, and induced inflammatory responses in macrophages via ß2-AR-cAMP-PKA-ROS axis. This study identifies MD2 as a key inflammatory mediator and a promising therapeutic target for ISO-induced heart failure.

Empagliflozin protects against isoprenaline-induced fibrosis in rat heart through modulation of TGF-ß/SMAD pathway.

AIM: Cardiac fibrosis is characterized by excessive accumulation of fibrous tissue, particularly collagens, in the myocardium. Accumulated fibrous tissue renders myocardium stiffer and reduces its contractility. Empagliflozin is an oral hypoglycemic agent with extra-diabetic functional profile toward maintaining cardiac functions. The present study aimed to examine protective effect of empagliflozin against an in-vivo model of cardiac fibrosis induced by isoprenaline and targeting TGF-ß/SMAD signaling as a possible pathway responsible for such effect. MAIN METHODS: Sixty animals were divided into six groups; the first was normal, and the second was treated with isoprenaline only (5 mg/kg/day I.P.) as a control. The third received pirfenidone (500 mg/kg/day P.O.), and the remaining groups received graded doses (5, 10, 20 mg/kg respectively) of empagliflozin for 14 days before fibrosis induction by isoprenaline (5 mg/kg/day) for 30 days. KEY FINDINGS: Isoprenaline increased cardiac enzymes, and cardiac tissues revealed elevated concentrations of transforming growth factor ß (TGF-ß1), monocyte chemoattractant protein 1 (MCP-1), tumor necrosis factor α (TNF-α), and c-jun N-terminal kinase (JNK) proteins. Expression of nuclear factor kappa B (NF-κB), alpha smooth muscle actin (α-SMA), collagens, suppressor of mothers against decapentaplegic (SMADs), connective tissue growth factor (CTGF), and fibronectin was upregulated. Empagliflozin improved the histological picture of heart tissue in comparison to fibrosis developed in controls, and protected against fibrosis through significant modulation of all mentioned parameters' concentrations and expressions. SIGNIFICANCE: Empagliflozin demonstrated a promising protective approach against biological model of cardiac fibrosis through an anti-fibrotic effect through targeting TGF-ß signaling pathways.

Unlocking the miRNA-34a-5p/TGF-ß and HMGB1/PI3K/Akt/mTOR crosstalk participate in the enhanced cardiac protection of liraglutide against isoproterenol-induced acute myocardial injury rat model.

Liraglutide (LIRA), a drug used to treat type 2 diabetes mellitus that belongs to the glucagon-like peptide-1 class, has recently drawn attention for its potential cardioprotective properties because of its anti-oxidative and anti-inflammatory properties. This current investigation was designed to assess the impact of LIRA on myocardial injury induced by isoproterenol (ISO). The experiment included 24 male Wistar rats in total, and they were divided into four groups: Control, LIRA (200 µg/kg/12 hrs., S.C.), ISO (85 mg/kg, S.C.), and ISO + LIRA. To assess the results, various biochemical and histopathological analyses were carried out. The findings showed elevated serum enzyme levels, a sign of cardiac injury. ISO-treated rats showed an upregulation of oxidative stress and inflammatory biomarkers like MDA, MPO, nitrites, NADPH oxidase, TNF-α, IL-1ß, IL-6, 8-Hydroxyguanosine (8-OHdG), and TGF-ß, as well as altered gene expressions like TLR-1 and miRNA-34a-5p. According to western blotting analysis, protein levels of AKT, PI3K, and mTOR were obviously enhanced. Additionally, ISO-treated samples showed altered tissue morphology, elevated caspase 3, and decreased Bcl2 concentrations. The levels of these dysregulated parameters were significantly normalized by LIRA therapy, demonstrating its cardioprotective function against ISO-induced myocardial injury in rats. This protective mechanism was linked to anti-inflammatory properties, redox balance restoration, and modulation of the miRNA-34a-5p/TGF-ß pathway.