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Background: Natural compounds offer promising targets for cardioprotection, which could lead to enhanced clinical outcomes. We aimed to determine the cardioprotective effects of Fruitflow®, a water-soluble tomato extract known for its anti-platelet effects in doxorubicin-induced toxicity in rat cardiomyoblast cell line pathological alteration in heart tissue of high fat-fed Wistar Albino rats., Methods: The cardioprotective effect of Fruitflow® was investigated using H9c2 (2-1) cells (rat cardiomyoblast cell line) and high-fat diet-fed Wistar Albino rats. We evaluated morphological changes, cell proliferation, cell migration, antioxidant activity, cell cycle progression, and mitochondrial membrane potential after the Fruitflow® treatment in the Doxorubicin-injured H9c2 (2-1) cell line. We studied lipid profiles, inflammation, oxidative stress, and cardiac function regulatory enzyme activity in the rat model., Results: Fruitflow® dose-dependently stimulated cell proliferation and migration in Doxorubicin-injured H9c2 (2-1) cells, potentially promoting cardiac regeneration and supporting tissue repair. Fruitflow® modulated the cell cycle, improved mitochondrial function, and reduced oxidative stress. Furthermore, it significantly improved lipid profiles and enzyme activities and reduced inflammation and oxidative stress in high-fat-fed rats. Fruitflow® also modulated the expression of genes involved in cardiac remodeling, mitochondrial biogenesis, inflammation, and vascular function., Conclusion: Our findings suggest Fruitflow® may have cardioprotective effects, making it a potential treatment option for cardiac ailments. Larger-scale clinical trials were recommended further to determine the efficacy and safety of Fruitflow® as a potential therapeutic agent for cardiac diseases, potentially in combination with other cardioprotective medications., Competing Interests: Declaration of Competing Interest Prof. Asim K Duttaroy holds a patent related to the technology used in this study. Prof. Asim K Duttaroy is a member of the scientific advisory board of Provexis, PLC, which advocates for policies related to the research topic “The cardioprotective effects of Fruitflow® against Doxorubicin-induced toxicity in rat cardiomyoblast cells H9c2 (2−1) and high-fat diet-induced dyslipidemia and pathological alteration in cardiac tissue of Wistar Albino rats,”. All the authors were kept blinded before the completion of the study and data analysis. All other authors declare no conflict of interest, (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Cardiovascular disease is one of the leading causes of death worldwide and has a high prevalence. Insulin-like growth factor-II receptor α (IGF-IIRα) acts as a stress-inducible negative regulator. This study focused on the substantial impact of heightened expression of IGF-IIRα in cardiac myoblasts and its association with the exacerbation of cardiac dysfunction. Using lipopolysaccharide (LPS)-induced H9c2 cardiac myoblasts as a model for sepsis, we aimed to elucidate the molecular interactions between IGF-IIRα and LPS in exacerbating cardiac injury. Our findings demonstrated a synergistic induction of cardiac inflammation and hypertrophy by LPS stimulation and IGF-IIRα overexpression, leading to decreased cell survival. Excessive calcineurin activity, triggered by this combined condition, was identified as a key factor exacerbating the negative effects on cell survival. Cellular changes such as cell enlargement, disrupted actin filaments, and upregulation of hypertrophy-related and inflammation-related proteins contributed to the overall hypertrophic and inflammatory responses. Overexpression of IGF-IIRα also exacerbated apoptosis induced by LPS in H9c2 cardiac myoblasts. Inhibiting calcineurin in LPS-treated H9c2 cardiac myoblasts with IGF-IIRα overexpression effectively reversed the detrimental effects, reducing cell damage and mitigating apoptosis-related cardiac mechanisms. Our study suggests that under sepsis-like conditions in the heart with IGF-IIRα overexpression, hyperactivation of calcineurin worsens cardiac damage. Suppressing IGF-IIRα and calcineurin expression could be a potential intervention to alleviate the impact of the illness and improve cardiac function., (© 2024 Wiley Periodicals LLC.)

Tyrosine kinase inhibitors (TKIs) offer targeted therapy for cancers but can cause severe cardiotoxicities. Determining their dose‑dependent impact on cardiac function is required to optimize therapy and minimize adverse effects. The dose‑dependent cardiotoxic effects of two TKIs, imatinib and ponatinib, were assessed in vitro using H9c2 cardiomyoblasts and in vivo using zebrafish embryos. In vitro , H9c2 cardiomyocyte viability, apoptosis, size, and surface area were evaluated to assess the impact on cellular health. In vivo , zebrafish embryos were analyzed for heart rate, blood flow velocity, and morphological malformations to determine functional and structural changes. Additionally, reverse transcription‑quantitative PCR (RT‑qPCR) was employed to measure the gene expression of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), established markers of cardiac injury. This comprehensive approach, utilizing both in vitro and in vivo models alongside functional and molecular analyses, provides a robust assessment of the potential cardiotoxic effects. TKI exposure decreased viability and surface area in H9c2 cells in a dose‑dependent manner. Similarly, zebrafish embryos exposed to TKIs exhibited dose‑dependent heart malformation. Both TKIs upregulated ANP and BNP expression, indicating heart injury. The present study demonstrated dose‑dependent cardiotoxic effects of imatinib and ponatinib in H9c2 cells and zebrafish models. These findings emphasize the importance of tailoring TKI dosage to minimize cardiac risks while maintaining therapeutic efficacy. Future research should explore the underlying mechanisms and potential mitigation strategies of TKI‑induced cardiotoxicities.

Carboxylic ionophores are polyether antibiotics used in production animals as feed additives, with a wide range of benefits. However, ionophore toxicosis often occurs as a result of food mixing errors or extra-label use and primarily targets the cardiac and skeletal muscles of livestock. The ultrastructural changes induced by 48 hours of exposure to 0.1 μM monensin, salinomycin, and lasalocid in cardiac (H9c2) and skeletal (L6) myoblasts in vitro were investigated using transmission electron microscopy and scanning electron microscopy. Ionophore exposure resulted in condensed mitochondria, dilated Golgi apparatus, and cytoplasmic vacuolization which appeared as indentations on the myoblast surface. Ultrastructurally, it appears that both apoptotic and necrotic myoblasts were present after exposure to the ionophores. Apoptotic myoblasts contained condensed chromatin and apoptotic bodies budding from their surface. Necrotic myoblasts had disrupted plasma membranes and damaged cytoplasmic organelles. Of the three ionophores, monensin induced the most alterations in myoblasts of both cell lines., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Henn et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Oxidative stress plays a pivotal role in the development of diabetic cardiomyopathy (DCM). Previous studies have revealed that inhibition of mitochondrial fission suppressed oxidative stress and alleviated mitochondrial dysfunction and cardiac dysfunction in diabetic mice. However, no research has confirmed whether mitochondria fission accentuates hyperglycemia-induced cardiomyoblast oxidative stress through regulating fatty acid oxidation (FAO). We used H9c2 cardiomyoblasts exposed to high glucose (HG) 33 mM to simulate DCM in vitro. Excessive mitochondrial fission, poor cell viability, and lipid accumulation were observed in hyperglycemia-induced H9c2 cardiomyoblasts. Also, the cells were led to oxidative stress injury, lower adenosine triphosphate (ATP) levels, and apoptosis. Dynamin-related protein 1 (Drp1) short interfering RNA (siRNA) decreased targeted marker expression, inhibited mitochondrial fragmentation and lipid accumulation, suppressed oxidative stress, reduced cardiomyoblast apoptosis, and improved cell viability and ATP levels in HG-exposed H9c2 cardiomyoblasts, but not in carnitine palmitoyltransferase 1 (CPT1) inhibitor etomoxir treatment cells. We also found subcellular localization of CPT1 on the mitochondrial membrane, FAO, and levels of nicotinamide adenine dinucleotide phosphate (NADPH) were suppressed after exposure to HG treatment, whereas Drp1 siRNA normalized mitochondrial CPT1, FAO, and NADPH. However, the blockade of FAO with etomoxir abolished the above effects of Drp1 siRNA in hyperglycemia-induced H9c2 cardiomyoblasts. The preservation of mitochondrial function through the Drp1/CPT1/FAO pathway is the potential mechanism of inhibited mitochondria fission in attenuating oxidative stress injury of hyperglycemia-induced H9c2 cardiomyoblasts., (© 2024 The Author(s). Cell Biology International published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.)

Ethnopharmacological Relevance: Astragali Radix (AR) is a popular traditional Chinese medicine that has been used for more than 2000 years. It is a well-known tonic for weak people with chronic diseases, such as heart failure and cerebral ischemia. Previous studies have reported that AR could support the "weak heart" of cancer patients who suffered from doxorubicin (DOX)-induced cardiotoxicity (DIC). However, the underlying mechanism remains unclear., Aim of the Study: This study aimed to uncover the critical pathways and molecular determinants for AR against DIC by fully characterizing the network-based relationship., Materials and Methods: We integrated ultra-high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) profiling, database and literature searching, and the human protein-protein interactome to discover the specific network module associated with AR against DIC. To validate the network-based findings, a low-dose, long-term DIC mouse model and rat cardiomyoblast H9c2 cells were employed. The levels of potential key metabolites and proteins in hearts and cells were quantified by the LC-MS/MS targeted analysis and western blotting, respectively., Results: We constructed one of the most comprehensive AR component-target network described to date, which included 730 interactions connecting 64 unique components and 359 unique targets. Relying on the network-based evaluation, we identified fatty acid metabolism as a putative critical pathway and peroxisome proliferator-activated receptors (PPARα and PPARγ) as potential molecular determinants. We then confirmed that DOX caused the accumulation of fatty acids in the mouse failing heart, while AR promoted fatty acid metabolism and preserved heart function. By inhibiting PPARγ in H9c2 cells, we further found that AR could alleviate DIC by activating PPARγ to maintain fatty acid homeostasis., Conclusions: Our findings imply that AR is a promising drug candidate that treats DIC by maintaining fatty acid homeostasis. More importantly, the network-based method developed here could facilitate the mechanism discovery of AR therapy and help catalyze innovation in its clinical application., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Tyrosine kinase inhibitors (TKIs) are associated with cardiac toxicity, which may be caused by mitochondrial toxicity. The underlying mechanisms are currently unclear and require further investigation. In the present study, we aimed to investigate in more detail the role of the enzyme complexes of the electron transfer system (ETS), mitochondrial oxidative stress, and mechanisms of cell death in cardiac toxicity associated with imatinib and sorafenib. Cardiac myoblast H9c2 cells were exposed to imatinib and sorafenib (1 to 100 µM) for 24 h. Permeabilized rat cardiac fibers were treated with both drugs for 15 min. H9c2 cells exposed to sorafenib for 24 h showed a higher membrane toxicity and ATP depletion in the presence of galactose (favoring mitochondrial metabolism) compared to glucose (favoring glycolysis) but not when exposed to imatinib. Both TKIs resulted in a higher dissipation of the mitochondrial membrane potential in galactose compared to glucose media. Imatinib inhibited Complex I (CI)- and CIII- linked respiration under both conditions. Sorafenib impaired CI-, CII-, and CIII-linked respiration in H9c2 cells cultured with glucose, whereas it inhibited all ETS complexes with galactose. In permeabilized rat cardiac myofibers, acute exposure to imatinib and sorafenib decreased CI- and CIV-linked respiration in the presence of the drugs. Electron microscopy showed enlarged mitochondria with disorganized cristae. In addition, both TKIs caused mitochondrial superoxide accumulation and decreased the cellular GSH pool. Both TKIs induced caspase 3/7 activation, suggesting apoptosis as a mechanism of cell death. Imatinib and sorafenib impaired the function of cardiac mitochondria in isolated rat cardiac fibers and in H9c2 cells at plasma concentrations reached in humans. Both imatinib and sorafenib impaired the function of enzyme complexes of the ETS, which was associated with mitochondrial ROS accumulation and cell death by apoptosis.

Ethnopharmacological Relevance: Solanum nigrum, commonly known as Makoi or black shade has been traditionally used in Asian countries and other regions of world to treat liver disorders, diarrhoea, inflammatory conditions, chronic skin ailments (psoriasis and ringworm), fever, hydrophobia, painful periods, eye diseases, etc. It has been observed that S. nigrum contains substances, like steroidal saponins, total alkaloid, steroid alkaloid, and glycoprotein, which show anti-tumor activity. However; there is no scientific evidence of the efficacy of S. nigrum in the treatment of cardiac hypertrophy., Aim: To investigate the ability of S. nigrum to attenuate Angiotensin II - induced cardiac hypertrophy and improve cardiac function through the suppression of protein kinase PKC-ζ and Mel-18-IGF-IIR signaling leading to the restoration of HSF2 desumolyation., Materials and Methods: Cardiomyoblast cells (H9c2) were challenged with 100 nM Angiotensin-II (AngII) for 24 h and were then treated with different concentration of S.nigrum or Calphostin C for 24 h. The hypertrophic effect in cardiomyoblast cells were determined by immunofluorescence staining and the modulations in hypertrophic protein marker along with Protein Kinase C-ζ, MEL18, HSF2, and Insulin like growth factor II (IGFIIR), markers were analyzed by western blotting. In vivo experiments were performed using 12 week old male Wistar Kyoto rats (WKY) and Spontaneously hypertensive rats (SHR) separated into five groups. [1]Control WKY, [2] WKY -100 mg/kg of S.nigrum treatment, [3] SHR, [4] SHR-100 mg/kg of S.nigrum treatment, [5] SHR-300 mg/kg of S.nigrum treatment. S. nigrum was administered intraperitoneally for 8 week time interval., Results: Western blotting results indicate that S. nigrum significantly attenuates AngII induced cardiac hypertrophy. Furthermore, actin staining confirmed the ability of S. nigrum to ameliorate AngII induced cardiac hypertrophy. Moreover, S. nigrum administration suppressed the hypertrophic signaling mediators like Protein Kinase C-ζ, Mel-18, and IGFIIR in a dose-dependent manner and HSF2 activation (restore deSUMOlyation) that leads to downregulation of IGF-IIR expression. Additionally in vivo experiments demonstrate the reduced heart sizes of S. nigrum treated SHRs rats when compared to control WKY rats., Conclusion: Collectively, the data reveals the cardioprotective effect of S. nigrum inhibiting PKC-ζ with alleviated IGF IIR level in the heart that profoundly remits cardiac hypertrophy for hypertension-induced heart failure., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Abstract: Glucagon-like peptide (GLP)-1(7-36), a major active form of GLP-1 hormone, is rapidly cleaved by dipeptidyl peptidase-4 to generate a truncated metabolite, GLP-1(9-36) which has a low affinity for GLP-1 receptor (GLP-1R). GLP-1(7-36) has been shown to have protective effects on cardiovascular system through GLP-1R-dependent pathway. Nevertheless, the cardioprotective effects of GLP-1(9-36) have not fully understood. The present study investigated the effects of GLP-1(9-36), including its underlying mechanisms against oxidative stress and apoptosis in H9c2 cells. Here, we reported that GLP-1(9-36) protects H9c2 cardiomyoblasts from hydrogen peroxide (H2O2)-induced oxidative stress by promoting the synthesis of antioxidant enzymes, glutathione peroxidase-1, catalase, and heme oxygenase-1. In addition, treatment with GLP-1(9-36) suppressed H2O2-induced apoptosis by attenuating caspase-3 activity and upregulating antiapoptotic proteins, Bcl-2 and Bcl-xL. These protective effects of GLP-1(9-36) are attenuated by blockade of PI3K-mediated Akt phosphorylation and prevention of nitric oxide synthase-induced nitric oxide production. Thus, GLP-1(9-36) represents the potential therapeutic target for prevention of oxidative stress and apoptosis in the heart via PI3K/Akt/nitric oxide synthase signaling pathway., Competing Interests: The authors report no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The aim of this study was to examine the effects of lipid emulsions on carnitine palmitoyltransferase I (CPT-I), carnitine acylcarnitine translocase (CACT), carnitine palmitoyltransferase II (CPT-II), and the mitochondrial dysfunctions induced by toxic doses of local anesthetics in H9c2 rat cardiomyoblasts. The effects of local anesthetics and lipid emulsions on the activities of CPT-I, CACT, and CPT-II, and concentrations of local anesthetics were examined. The effects of lipid emulsions, N-acetyl-L-cysteine (NAC), and mitotempo on the bupivacaine-induced changes in cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and intracellular calcium levels were examined. CACT, without significantly altering CPT-I and CPT-II, was inhibited by toxic concentration of local anesthetics. The levobupivacaine- and bupivacaine-induced inhibition of CACT was attenuated by all concentrations of lipid emulsion, whereas the ropivacaine-induced inhibition of CACT was attenuated by medium and high concentrations of lipid emulsion. The concentration of levobupivacaine was slightly attenuated by lipid emulsion. The bupivacaine-induced increase of ROS and calcium and the bupivacaine-induced decrease of MMP were attenuated by ROS scavengers NAC and mitotempo, and the lipid emulsion. Collectively, these results suggested that the lipid emulsion attenuated the levobupivacaine-induced inhibition of CACT, probably through the lipid emulsion-mediated sequestration of levobupivacaine.

Although numerous studies have demonstrated the biological and multifaceted nature of dimethyl sulfoxide (DMSO) across different in vitro models, the direct effect of "non-toxic" low DMSO doses on cardiac and cancer cells has not been clearly explored. In the present study, H9c2 cardiomyoblasts and MCF-7 breast cancer cells were treated with varying concentrations of DMSO (0.001-3.7%) for 6 days. Here, DMSO doses < 0.5% enhanced the cardiomyoblasts respiratory control ratio and cellular viability relative to the control cells. However, 3.7% DMSO exposure enhanced the rate of apoptosis, which was driven by mitochondrial dysfunction and oxidative stress in the cardiomyoblasts. Additionally, in the cancer cells, DMSO (≥0.009) led to a reduction in the cell's maximal respiratory capacity and ATP-linked respiration and turnover. As a result, the reduced bioenergetics accelerated ROS production whilst increasing early and late apoptosis in these cells. Surprisingly, 0.001% DMSO exposure led to a significant increase in the cancer cells proliferative activity. The latter, therefore, suggests that the use of DMSO, as a solvent or therapeutic compound, should be applied with caution in the cancer cells. Paradoxically, in the cardiomyoblasts, the application of DMSO (≤0.5%) demonstrated no cytotoxic or overt therapeutic benefits.

Doxorubicin (Doxo) is a widely used antineoplastic drug which often induces cardiomyopathy, leading to congestive heart failure through the intramyocardial production of reactive oxygen species (ROS). Icariin (Ica) is a flavonoid isolated from Epimedii Herba (Berberidaceae). Some reports on the pharmacological activity of Ica explained its antioxidant and cardioprotective effects. The aim of our study was to assess the protective activities of Ica against Doxo-detrimental effects on rat heart-tissue derived embryonic cardiac myoblasts (H9c2 cells) and to identify, at least in part, the molecular mechanisms involved. Our results showed that pretreatment of H9c2 cells with 1 μM and 5 μM of Ica, prior to Doxo exposure, resulted in an improvement in cell viability, a reduction in ROS generation, the prevention of mitochondrial dysfunction and mPTP opening. Furthermore, for the first time, we identified one feasible molecular mechanism through which Ica could exerts its cardioprotective effects. Indeed, our data showed a significant reduction in Caveolin-1(Cav-1) expression levels and a specific inhibitory effect on phosphodiesterase 5 (PDE5a) activity, improving mitochondrial function compared to Doxo-treated cells. Besides, Ica significantly prevented apoptotic cell death and downregulated the main pro-autophagic marker Beclin-1 and LC3 lipidation rate, restoring physiological levels of activation of the protective autophagic process. These results suggest that Ica might have beneficial cardioprotective effects in attenuating cardiotoxicity in patients requiring anthracycline chemotherapy through the inhibition of oxidative stress and, in particular, through the modulation of Cav-1 expression levels and the involvement of PDE5a activity, thereby leading to cardiac cell survival.

Background: Acylcarnitine is an intermediate product of fatty acid oxidation. It is reported to be closely associated with the occurrence of diabetic cardiomyopathy (DCM). However, the mechanism of acylcarnitine affecting myocardial disorders is yet to be explored. This current research explores the different chain lengths of acylcarnitines as biomarkers for the early diagnosis of DCM and the mechanism of acylcarnitines for the development of DCM in-vitro., Methods: In a retrospective non-interventional study, 50 simple type 2 diabetes mellitus patients and 50 DCM patients were recruited. Plasma samples from both groups were analyzed by high throughput metabolomics and cluster heat map using mass spectrometry. Principal component analysis was used to compare the changes occurring in the studied 25 acylcarnitines. Multivariable binary logistic regression was used to analyze the odds ratio of each group for factors and the 95% confidence interval in DCM. Myristoylcarnitine (C14) exogenous intervention was given to H9c2 cells to verify the expression of lipid metabolism-related protein, inflammation-related protein expression, apoptosis-related protein expression, and cardiomyocyte hypertrophy and fibrosis-related protein expression., Results: Factor 1 (C14, lauroylcarnitine, tetradecanoyldiacylcarnitine, 3-hydroxyl-tetradecanoylcarnitine, arachidic carnitine, octadecanoylcarnitine, 3-hydroxypalmitoleylcarnitine) and factor 4 (octanoylcarnitine, hexanoylcarnitine, decanoylcarnitine) were positively correlated with the risk of DCM. Exogenous C14 supplementation to cardiomyocytes led to increased lipid deposition in cardiomyocytes along with the obstacles in adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) signaling pathways and affecting fatty acid oxidation. This further caused myocardial lipotoxicity, ultimately leading to cardiomyocyte hypertrophy, fibrotic remodeling, and increased apoptosis. However, this effect was mitigated by the AMPK agonist acadesine., Conclusions: The increased plasma levels in medium and long-chain acylcarnitine extracted from factors 1 and 4 are closely related to the risk of DCM, indicating that these factors can be an important tool for DCM risk assessment. C14 supplementation associated lipid accumulation by inhibiting the AMPK/ACC/CPT1 signaling pathway, aggravated myocardial lipotoxicity, increased apoptosis apart from cardiomyocyte hypertrophy and fibrosis were alleviated by the acadesine., (© 2021. The Author(s).)

Objectives: Considering the complementary nature of signalling mechanisms and the therapeutic effects of nebivolol, a β1-adrenoreceptor antagonist, and valsartan, an angiotensin receptor blocker (ARB), here we aimed to investigate whether nebivolol/valsartan combination would complement the cardioprotective effects of nebivolol on angiotensin II (ANG II)-induced pathology in H9c2 cardiomyoblasts., Methods: H9c2 cardiomyoblasts were used to investigate the protective effects of nebivolol and nebivolol and valsartan combination against ANG II-induced pathology. Reactive oxygen species (ROS) generation was determined by 2',7'-dichlorofluorescein diacetate (DCFDA) and MitoSOX Red staining. Real-time PCR and immunoblotting were employed to quantify the changes in mRNA and protein expression levels, respectively., Key Findings: Our data revealed that pretreatment with nebivolol and nebivolol/valsartan combination significantly reduced ANG II-induced oxidative stress and mTORC1 signalling. Concurrently, ANG II-induced activation of inflammatory cytokines and fetal gene expressions were significantly suppressed by nebivolol and nebivolol/valsartan combination. Pretreatment with nebivolol and nebivolol/valsartan combination alleviated ANG II-induced impairment of mitochondrial biogenesis by restoring the gene expression levels of PGC-1α, TFAM, NRF-1 and SIRT3. Our data further show that nebivolol and nebivolol/valsartan combination mediated up-regulation in mitochondrial biogenesis is accompanied by decrease in ANG II-stimulated mitochondrial ROS generation as well as increase in expression of mitochondrial fusion genes MFN2 and OPA1, indicative of improved mitochondrial dynamics., Summary: These findings suggest that both nebivolol and nebivolol/valsartan combination exert protective effects on ANG II-induced mitochondrial dysfunction by alleviating its biogenesis and dynamics. Moreover, addition of valsartan to nebivolol do not produce any additive effects compared with nebivolol alone on ANG II-induced cardiac pathology., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Royal Pharmaceutical Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Pathological cardiac hypertrophy is associated with many diseases including hypertension. Recent studies have identified important roles for microRNAs (miRNAs) in many cardiac pathophysiological processes, including the regulation of cardiomyocyte hypertrophy. However, the role of miR-145-5p in the cardiac setting is still unclear. In this study, H9C2 cells were overexpressed with microRNA-145-5p, and then treated with Ang-II for 24 h, to study the effect of miR-145-5p on Ang-II-induced myocardial hypertrophy in vitro. Results showed that Ang-II treatment down-regulated miR-145-5p expression were revered after miR-145-5p overexpression. Based on results of bioinformatics algorithms, paxillin was predicted as a candidate target gene of miR-145-5p, luciferase activity assay revealed that the luciferase activity of cells was substantial downregulated the following co-transfection with wild paxillin 3'UTR and miR-145-5p compared to that in scramble control, while the inhibitory effect of miR-145-5p was abolished after transfection of mutant paxillin 3'UTR. Additionally, overexpression of miR-145-5p markedly inhibited activation of Rac-1/ JNK /c-jun/ NFATc3 and ANP expression and induced SIRT1 expression in Ang-II treated H9c2 cells. Jointly, our study suggested that miR-145-5p inhibited cardiac hypertrophy by targeting paxillin and through modulating Rac-1/ JNK /c-jun/ NFATc3/ ANP / Sirt1 signaling, therefore proving novel downstream molecular pathway of miR-145-5p in cardiac hypertrophy., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Benomyl, benzimidazole group pesticide, has been prohibited in Europe and USA since 2003 due to its toxic effects and it has been still determined as food and environmental contaminant. In the present study, the toxic effect mechanisms of benomyl were evaluated in rat cardiomyoblast (H9c2) cells. Cytotoxicity was determined by MTT and NRU assay and, oxidative stress potential was evaluated by reactive oxygen species (ROS) production and glutathione levels. DNA damage was assessed by alkaline comet assay. Relative expressions of apoptosis related genes were evaluated; furthermore, NF-κB and JNK protein levels were determined. At 4 μM concentration (at which cell viability was >70%), benomyl increased 2-fold of ROS production level and 2-fold of apoptosis as well as DNA damage. Benomyl down-regulated miR21, TNF-α and Akt1 ≥ 48.75 and ≥ 97.90; respectively. PTEN, JNK and NF-κB expressions were upregulated. The dramatic changes in JNK and NF-κB expression levels were not observed in protein levels. These findings showed the oxidative stress related DNA damage and apoptosis in cardiomyoblast cells exposed to benomyl. However, further mechanistic and in vivo studies are needed to understand the cardiotoxic effects of benomyl and benzimidazol fungucides., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Myocardial ischemia is the main reason for ischemic heart diseases. Antioxidant treatment is considered as a possible approach to prevent myocardial ischemia injury, because oxidative stress is a key factor triggering it. This study was to investigate the protective effects of 3,5-dimethoxy-4-hydroxy myricanol (DHM) against oxidative stress-induced cytotoxicity on H9c2 cells and further explore its mechanisms. The oxidative stress and inflammatory response markers were detected by H2DCFDA fluorescent measurement, enzyme-linked immunosorbent assay (ELISA), real-time PCR and Western blot. Results showed DHM exerted inhibitory effects against H9c2 cell damage. Furthermore, DHM decreased oxidative stress in H9c2 cells through up-regulating protein expression of heme oxygenase-1 (HO-1) and nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Moreover, DHM inhibited inflammatory responses through down-regulating the protein expression of mitogen-activated protein kinases (MAPKs) and nuclear factor kappa B (NF-κB). DHM exerted protective activities against oxidative stress-induced cell damage, at least through decreasing oxidative stress and inhibiting inflammatory responses, indicating that DHM have the potential to be developed as therapeutic agents for the treatment of myocardial ischemia.

Arsenic trioxide (ATO) is among the first-line chemotherapeutic drugs for treating acute promyelocytic leukemia patients, but its clinical use is hampered due to cardiotoxicity. The present investigation unveils the mechanism underlying ATO-induced oxidative stress that promotes calcineurin (a ubiquitous Ca 2+ /calmodulin-dependent serine/threonine phosphatase expressed only during sustained Ca 2+ elevation) expression, inflammatory cytokine release and apoptosis in H9c2 cardiomyoblasts, and its possible modulation with phloretin (PHL, an antioxidant polyphenol present in apple peel). ATO caused Ca 2+ overload resulting in elevated expression of calcineurin and its downstream transcriptional effector NFATc causing the release of cytokines such as IL-2, IL-6, MCP-1, IFN-γ, and TNF-α in H9c2 cardiomyoblast. There was a visible increase in the nuclear fraction of NF-κB and ROS-mediated apoptotic cell death. The expression levels of cardiac-specific genes (troponin, desmin, and caveolin-3) and genes of the apoptotic signaling pathway (BCL-2, BAX, IGF1, AKT, ERK1, -2, RAF1, and JNK) in response to ATO and PHL were studied. The putative binding mode and the potential ligand-target interactions of PHL with calcineurin using docking software (Autodock and iGEMDOCKv2) showed the high binding affinity of PHL to calcineurin. PHL co-treatment significantly reduced Ca 2+ influx and normalized the expression of calcineurin, NFATc, NF-κB, and other cytokines. PHL co-treatment resulted in activation of BCL-2, IGF1, AKT, RAF1, ERK1, and ERK2 and inhibition of BAX and JNK. Overall, these results revealed that PHL has a protective effect against ATO-induced apoptosis and we propose calcineurin as a druggable target for the interaction of PHL in ATO cardiotoxicity in H9c2 cells.

Rutin is a flavonoid with antioxidant property. It has been shown to exert cardioprotection against cardiomyocyte hypertrophy. However, studies regarding its antihypertrophic property are still lacking, whether it demonstrates similar antihypertrophic effect to its metabolite, quercetin. Hence, this study aimed to investigate the effects of both flavonoids on oxidative stress and mitogen-activated protein kinase (MAPK) pathway in H9c2 cardiomyocytes that were exposed to angiotensin II (Ang II) to induce hypertrophy. Cardiomyocytes were exposed to Ang II (600 nM) with or without quercetin (331 μM) or rutin (50 μM) for 24 h. A group given vehicle served as the control. The concentration of the flavonoids was chosen based on the reported effective concentration to reduce cell hypertrophy or cardiac injury in H9c2 cells. Exposure to Ang II increased cell surface area, intracellular superoxide anion level, NADPH oxidase and inducible nitric oxide synthase activities, and reduced cellular superoxide dismutase activity and nitrite level, which were similarly reversed by both rutin and quercetin. Rutin had no significant effects on phosphorylated proteins of extracellular signal-related kinases (ERK1/2) and p38 but downregulated phosphorylated c-Jun N-terminal kinases (JNK1/2), which were induced by Ang II. Quercetin, on the other hand, had significantly downregulated the phosphorylated proteins of ERK1/2, p38, and JNK1/2. The quercetin inhibitory effect on JNK1/2 was stronger than the rutin. In conclusion, both flavonoids afford similar protective effects against Ang II-induced cardiomyocyte hypertrophy, but they differently modulate MAPK pathway.

Amlodipine-induced toxicity has detrimental effects on cardiac cells. The aim of this study was to examine the effect of lipid emulsion on decreased H9c2 rat cardiomyoblast viability induced by amlodipine toxicity. The effects of amlodipine, lipid emulsion, LY 294002, and glibenclamide, either alone or in combination, on cell viability and count, apoptosis, and expression of cleaved caspase-3 and -8, and Bax were examined. LY 294002 and glibenclamide partially reversed lipid emulsion-mediated attenuation of decreased cell viability and count induced by amlodipine. Amlodipine increased caspase-3 and -8 expression, but it did not alter Bax expression. LY 294002 and glibenclamide reversed lipid emulsion-mediated inhibition of cleaved caspase-3 and -8 expression induced by amlodipine. Lipid emulsion inhibited early and late apoptosis induced by amlodipine. LY 294002 and glibenclamide inhibited lipid emulsion-mediated inhibition of late apoptosis induced by amlodipine, but they did not significantly alter lipid emulsion-mediated inhibition of early apoptosis induced by amlodipine. Lipid emulsion decreased amlodipine-induced TUNEL-positive cells. These results suggest that lipid emulsion inhibits late apoptosis induced by amlodipine at toxic dose via the activation of phosphoinositide-3 kinase and ATP-sensitive potassium channels in the extrinsic apoptotic pathway.

Kirenol (KRL) is a biologically active substance extracted from Herba Siegesbeckiae. This natural type of diterpenoid has been widely adopted for its important anti-inflammatory and anti-rheumatic properties. Despite several studies claiming the benefits of KRL, its cardiac effects have not yet been clarified. Cardiotoxicity remains a key concern associated with the long-term administration of doxorubicin (DOX). The generation of reactive oxygen species (ROS) causes oxidative stress, significantly contributing to DOX-induced cardiac damage. The purpose of the current study is to investigate the cardio-protective effects of KRL against apoptosis in H9c2 cells induced by DOX. The analysis of cellular apoptosis was performed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining assay and measuring the modulation in the expression levels of proteins involved in apoptosis and Nrf2 signaling, the oxidative stress markers. Furthermore, Western blotting was used to determine cell survival. KRL treatment, with Nrf2 upregulation and activation, accompanied by activation of PI3K/AKT, could prevent the administration of DOX to induce cardiac oxidative stress, remodeling, and other effects. Additionally, the diterpenoid enhanced the activation of Bcl2 and Bcl-xL, while suppressing apoptosis marker proteins. As a result, KRL is considered a potential agent against hypertrophy resulting from cardiac deterioration. The study results show that KRL not only activates the IGF-IR-dependent p-PI3K/p-AKT and Nrf2 signaling pathway, but also suppresses caspase-dependent apoptosis.

As an important complication of diabetes mellitus, diabetic cardiomyopathy (DCM) is thought to arise as a result of insulin resistance (IR) in cardiomyocytes. Improving IR in cardiomyocytes may therefore be a way to treat DCM. A recently discovered myokine, irisin, has been shown to be significantly associated with increased insulin sensitivity both in clinical and pre-clinical studies of diabetes mellitus. Based on previously research, we hypothesized that irisin may be a potential candidate for increasing the insulin sensitivity of cardiomyocytes. The aim of the present study was to examine the ability of irisin to affect IR induced by treatment of rat cardiomyocyte H9c2 cells with palmitic acid (PA) and to explore its underlying mechanism. Differentiated H9c2 cells were treated with 500 μM PA, 200 ng/mL irisin, and 500 μM PA + 200 ng/mL irisin with or without 100 nM rapamycin (RAP) for 24 h. We found that coincubation with 200 ng/mL irisin for 24 h significantly increased insulin-stimulated glucose consumption compared to the 500 μM PA group alone. Additionally, coincubation with irisin significantly alleviated the degree of autophagy compared to the 500 μM PA group alone as evidenced by monodansylcadaverine (MDC) fluorescence, the LC3II/LC3I protein levels ratio, and the protein levels of Atg5 and Atg7. Coincubation with irisin increased the levels of PI3Kp110α, pAkt and Akt compared to the 500 μM PA group alone. All these effects of irisin were reversed by RAP. Our results indicate that irisin improves IR in H9c2 cells, possibly in part by inhibiting autophagy through activating the PI3K/Akt pathway., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Inorganic nitrate or nitrite supplementation has been reported to demonstrate positive outcomes in rodent models of obesity and diabetes as well as in type 2 diabetic humans and even included in clinical trials pertaining to cardiovascular diseases in the recent decade. However, there are contrasting data regarding the useful and toxic effects of the anions. The primary scope of this study was to analyze the beneficial/detrimental alterations in redox status, mitochondrial dynamics and function, and cellular fitness in cardiomyoblasts inflicted by nitrite under hyperglycemic conditions compared with normoglycemia. Nitrite supplementation in H9c2 myoblasts under high glucose diminishes the Bcl-x L expression and mitochondrial ROS levels without significant initiation of cell death or decline in total ROS levels. Concomitantly, there are tendencies towards lowering of mitochondrial membrane potential, but without noteworthy changes in mitochondrial biogenesis and respiration. The study also revealed that under high glucose stress, nitrite may alter mitochondrial dynamics by Drp1 activation possibly via Akt1-Pim1 axis. Moreover, the study revealed differential effects of Drp1 silencing and/or nitrite under the above glycemic conditions. Overall, the study warrants more research regarding the effects of nitrite therapy in cardiac cells exposed to hyperglycemia., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Dengue virus (DENV) infection elevates intracellular Ca 2+ concentration ([Ca 2+ ] i ), but it is unknown whether Ca 2+ and calmodulin (CaM) are involved in DENV infection. We conducted immunofluorescence and western blot experiments and measured [Ca 2+ ] i examining the effects of DENV infection and drugs that alter Ca 2+ /CaM functions on CaM translocation, DENV2 infection, protein expression, virus-inducible STAT2 protein abundance, and CREB phosphorylation in H9c2 cells. DENV infection increased CaM expression, its nuclear translocation and NS3 and E viral proteins expression and colocalization in a manner that could be blocked by the ryanodine receptor antagonist dantrolene. DENV infection also increased CREB phosphorylation, an effect inhibited by either dantrolene or the CaM inhibitor W7. Dantrolene substantially hindered infection as assessed by focus assays in Vero cells. These results suggest that Ca 2+ and CaM play an important role in DENV infection of cardiac cells and that dantrolene may protect against severe DENV cardiac morbidity., (Copyright © 2020 Elsevier Inc. All rights reserved.)

6-Bromoindirubin-3'-oxime (6BIO) is a novel small molecule that exerts positive effects on several age-related alterations. However, the anti-aging effects of 6BIO on the aging heart remain unknown. Herein, we aim to investigate the effects of 6BIO on the myocardium and its underlying mechanism in vivo and vitro. Following 6BIO treatment, an increased p53 contents, a reduced p16 and β-gal levels, and attenuation of cardiac fibrosis were observed, suggesting 6BIO retarded aging of cardiomyocytes. As observed, 6BIO reduced p62 contents, elevated the levels of Beclin-1 and the ratio of LC3II/I, indicating the induction of autophagy, while the reduction of the accumulation of ROS indicated 6BIO alleviated oxidative stress. In addition, 6BIO treatment inhibited both GSK3β signaling and mTOR signaling. 6BIO might be a promising agent for preventing myocardium from aging.

Micrurus surinamensis (Cuvier, 1817), popularly known as aquatic coral snake, has a broad geographic distribution in the Rainforest of South America. The purpose of this study was to investigate the cytotoxic effect caused by M. surinamensis venom in H9c2 cardiomyoblast cells and to identify protein components involved in cardiotoxic processes. Venom cardiotoxic potential is evidenced by cell viability reduction in a concentration-dependent manner. We have purified one of venom components responsible for this effect after three chromatographic steps: a cytotoxic 23.461 kDa protein, as determined by mass spectrometry. A 19-residue sequence (DCPSGWSSYEGSCYNFFQR) of the purified protein was deduced by MS/MS and exhibited high homology with N-terminal region of C-type lectin from snake venoms. This protein was named Ms-CTL. Morphologically, H9c2 incubation with Ms-CTL led to a significant cellular retraction and formation of cellular aggregates, as observed by microscopy phase-contrast images. Our results indicate that M. surinamensis venom is highly toxic to H9c2 cardiomyoblast cell and less or not cytotoxic to other cell lines, such as HaCat, VERO and U373. Results presented herein will help understanding the mechanisms that underlie cellular damage and tissue destruction, being useful in the development of alternative therapies against these coral snake bites., Competing Interests: Declaration of competing interest The authors report no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The clinical application of doxorubicin, one of the most effective anticancer drugs, has been limited due to its adverse effects, including cardiotoxicity. One of the hallmarks of doxorubicin-induced cytotoxicity is mitochondrial dysfunction. Despite intensive research over recent decades, there are no effective approaches for alleviating doxorubicin-induced cytotoxicity. Melatonin, a natural hormone that is primarily secreted by the pineal gland, is emerging as a promising adjuvant that protects against doxorubicin-induced cytotoxicity owing to its pharmaceutical effect of preserving mitochondrial integrity. However, the underlying mechanisms are far from completely understood. Here, we provide novel evidence that treatment of H9c2 cardiomyoblasts with doxorubicin strongly induced AMP-activated protein kinase α2 (AMPKα2), which translocated to mitochondria and interfered with their function and integrity, ultimately leading to cellular apoptosis. These phenomena were significantly blocked by melatonin treatment. The levels of AMPKα2 in murine hearts were tightly associated with cardiotoxicity in the context of doxorubicin and melatonin treatment. Therefore, our study suggests that the maintenance of mitochondrial integrity is a key factor in reducing doxorubicin-induced cytotoxicity and indicates that AMPKα2 may serve as a novel target in the design of cytoprotective combination therapies that include doxorubicin.

Natural products black cumin- Nigella sativa ( N. sativa ) and wild garlic- Allium ursinum (AU) are known for their potential role in reducing cardiovascular risk factors, including antracycline chemotherapy. Therefore, this study investigates the effect of N. sativa and AU water and methanolic extracts in a cellular model of doxorubicin (doxo)-induced cardiotoxicity. The extracts were characterized using Ultraviolet-visible (UV-VIS) spectroscopy, Fourier-transform infrared (FT-IR) spectroscopy, Liquid Chromatography coupled with Mass Spectrometry (LC-MS) and Gas Chromatography coupled with Mass Spectrometry (GC-MS) techniques. Antioxidant activity was evaluated on H9c2 cells. Cytosolic and mitochondrial reactive oxygen species (ROS) release was evaluated using 2',7'-dichlorofluorescin-diacetate (DHCF-DA) and mitochondria-targeted superoxide indicator (MitoSOX red), respectively. Mitochondrial membrane depolarization was evaluated by flow cytometry. LC-MS analysis identified 12 and 10 phenolic compounds in NSS and AU extracts, respectively, with flavonols as predominant compounds. FT-IR analysis identified the presence of carbohydrates, amino acids and lipids in both plants. GC-MS identified the sulfur compounds in the AU water extract. N. sativa seeds (NSS) methanolic extract had the highest antioxidant activity reducing both intracellular and mitochondrial ROS release. All extracts (excepting AU methanolic extract) preserved H9c2 cells viability. None of the investigated plants affected the mitochondrial membrane depolarization. N. sativa and AU are important sources of bioactive compounds with increased antioxidant activities, requiring different extraction solvents to obtain the pharmacological effects.

Recent works reported the relevance of cellular exosomes in the evolution of different pathologies. However, most of these studies focused on the ability of exosomes to convey mi-RNA from cell to cell. The level of knowledge concerning the transport of lipid mediators by these nanovesicles is more than fragmented. The role of lipid mediators in the inflammatory signaling is fairly well described, in particular concerning the derivatives of the arachidonic acid (AA), called eicosanoïds or lipid mediators. The aim of the present work was to study the transport of these lipids within the extracellular vesicles of rat bone marrow mesenchymal stem cells (BM-MSC) and the cardiomyoblast cell line H9c2. We were able to characterize, for the first time, complete profiles of oxilipins within these nanovesicles. We studied also the impact on these profiles, of the polyunsaturated fatty acids (PUFAs) know to be precursors of the inflammatory signaling molecules (AA, eicosapentaenoic acid EPA and Docosahexaenoic acid DHA), at physiological concentrations. By growing the progenitor cells under PUFAs supplementation, we provide a comprehensive assessment of the beneficial effect of ω-3 PUFA therapy. Actually, our results tend to support the resolving role of the inflammation that stromal cell-derived extracellular vesicles can have within the cardiac microenvironment., (Copyright © 2020 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

In this study, we used ICI 182 780 (ICI), an estrogen receptor (ER) antagonist, to investigate the estrogenic activity of Danshen, and to further explored whether Danshen extract can block Leu27IGF-II-induced hypertrophy in H9c2 cardiomyoblast cells. We first used an IGF-II analog Leu27IGF-II, which specifically activates IGF2R signaling cascades and induces H9c2 cardiomyoblast cell hypertrophy. However, Danshen extract completely inhibited Leu27IGF-II-induced cell size increase, ANP and BNP hypertrophic marker expression, and IGF2R induction. We also observed that Danshen extract inhibited calcineurin protein expression and NFAT3 nuclear translocation, leading to suppression of Leu27IGF-II-induced cardiac hypertrophy. Moreover, the anti-Leu27IGF-II-IGF2R signaling effect of Danshen was totally reversed by ICI, which suggest the cardio protective effect of Danshen is mediated through estrogen receptors. Our study suggests that, Danshen exerts estrogenic activity, and thus, it could be used as a selective ER modulator in IGFIIR induced hypertrophy model., (© 2020 Wiley Periodicals, Inc.)

Doxorubicin (DOX) is an anticancer drug widely used in oncology, especially for breast cancer. The main limitation of DOX treatment is its cardiotoxicity due to the cumulative dose. Clinically, DOX-induced cardiomyopathy develops as a progressive heart failure caused by a progressive cardiomyocyte's death. For long, the oxidative stress induced by DOX was considered as the main toxic mechanism responsible for heart damage, but it is now controverted, and other processes are investigated to develop cardioprotective strategies. Previously, we studied DOX-induced cardiotoxicity and dexrazoxane (DEX), the only cardioprotective compound authorized by the FDA, by 1 H-NMR metabonomics in H9C2 cells. We observed an increased succinate secretion in the extracellular fluid of DEX-exposed cardiomyocytes, a finding that led us to the hypothesis of a possible protective role of this agonist of the GPR91 receptor. The objective of the present work was to study the effect of succinate (SUC) and cis- epoxysuccinate ( cis -ES), two agonists of the GPR91 receptor, on DOX-induced cardiotoxicity to H9C2 cells. To this purpose, several toxicity parameters, including cell viability, oxidative stress and apoptosis, as well as the GPR91 expression, were measured to assess the effects of DEX, SUC and cis -ES either alone or in combination with DOX in H9C2 cells. A 1 H-NMR-based metabonomic study was carried out on cellular fluids collected after 24 h to highlight the metabolic changes induced by those protective compounds. Moreover, the effects of each agonist given either alone or in combination with DOX were evaluated on MCF-7 breast cancer cells. GPR91 expression was confirmed in H9C2 cells, while no expression was found in MCF-7 cells. Under such experimental conditions, both SUC and cis -ES decreased partially the cellular mortality, the oxidative stress and the apoptosis induced by DOX. The SUC protective effect was similar to the DEX effect, but the protective effect of cis -ES was higher on oxidative stress and apoptosis. In addition, the metabonomics findings pointed out several metabolic pathways involved in the cardioprotective effects of both GPR91 agonists: the stimulation of aerobic metabolism with glucose as the main fuel, redox balance and phospholipids synthesis. Finally, none of the GPR91 agonists jeopardized the pharmacological effects of DOX on MCF-7 breast cancer cells.

Oxidative stress is widely accepted as a key factor of doxorubicin (Doxo)‑induced cardiotoxicity. There is evidence to indicate that nitrosative stress is involved in this process, and that Doxo interacts by amplifying cell damage. Mitochondrial connexin 43 (mitoCx43) can confer cardioprotective effects through the reduction of mitochondrial reactive oxygen species production during Doxo‑induced cardiotoxicity. The present study aimed to evaluate the involvement of mitoCx43 in Doxo‑induced nitrosative stress. Rat H9c2 cardiomyoblasts were treated with Doxo in the absence or presence of radicicol, an inhibitor of Hsp90, the molecular chaperone involved in Cx43 translocation to the mitochondria that underlies its role in cardioprotection. FACS analysis and RT‑qPCR revealed that Doxo increased superoxide dismutase, and catalase gene and protein expression. As shown by hypodiploid nuclei and confirmed by western blot analysis, Doxo increased caspase 9 expression and reduced procaspase 3 levels, which induced cell death. Moreover, a significant increase in the activation of the NF‑κB signaling pathway was observed. It is well known that the increased expression of inducible nitric oxide synthase results in nitric oxide overproduction, which then rapidly reacts with hydrogen peroxide or superoxide generated by the mitochondria, to form highly reactive and harmful peroxynitrite, which ultimately induces nitrotyrosine formation. Herein, these interactions were confirmed and increased effects were observed in the presence of radicicol. On the whole, the data of the present study indicate that an interplay between oxidative and nitrosative stress is involved in Doxo‑induced cardiotoxicity, and that both aspects are responsible for the induction of apoptosis. Furthermore, it is demonstrated that the mechanisms that further increase mitochondrial superoxide generation (e.g., the inhibition of Cx43 translocation into the mitochondria) significantly accelerate the occurrence of cell death.

Macrolides are clinically well-established class of antibiotics. Macrolides induce cardiotoxicity by blocking ether-a-go-go -related gene (ERG) potassium channels in cardiac myocytes. The aim of this study was to compare the effects of erythromycin, clarithromycin and azithromycin on cell viability and expression of ERG1 gene in H9c2 cells. Cell viability and ERG1 gene expression of H9c2 cells in 3 different concentrations, 1, 10 and 25 µg/ml, after 48 and 72 h were determined by MTT test and Real time-PCR method respectively. After 48 h, the growth of H9c2 cells treated with erythromycin, clarithromycin and Azithromycin (except two doses) were inhibited significantly compared to control group (p<0.05). All three groups of antibiotics showed toxic effects on cells after 72 h in all concentrations. Azithromycin-inhibiting effects were significantly higher than two other groups after 72 h of treatment. The expression of ERG1 gene increased in all three groups of antibiotics by increasing the concentration and duration of treatment. Azithromycin had the most pronounced effect on ERG1 expression in 48 and 72 h. This study indicated that these macrolides affect ERG1 expression due to their potential cardiac adverse effects. Further investigations are required to understand the exact mechanism of cardiotoxicity associated with macrolides., Competing Interests: The authors declare that they have no conflict of interest., (© Georg Thieme Verlag KG Stuttgart · New York.)

Ciguatera fish poisoning is a frequently reported non-bacterial food-borne illness related to the consumption of seafood contaminated with ciguatoxins, and possibly maitotoxins. These toxins are synthesized by marine dinoflagellate species of Gambierdiscus and Fukuyoa genera, and their abundance is a matter of great concern due to their adverse effects to aquatic life and human health. The present study aims to assess the sensitivity of rat cardiomyoblast H9c2(2-1) cells to Gambierdiscus toxic compounds using concentration- and time-dependent sulforhodamine B (SRB) colorimetric assays. Low concentrations of Gambierdiscus extracts (corresponding to 1.3-2.3 cells mL -1 ) induced a concentration-dependent response. Specificity in time-dependent response of H9c2(2-1) cells was demonstrated for G. excentricus after a 180 min exposure compared to both G. cf. belizeanus and G. silvae species, with EC 50s obtained after 720 and 360 min, respectively. The sensitivity of H9c2(2-1) cells to dinoflagellate toxic compounds was also tested with other genera from benthic (Coolia malayensis, Ostreopsis cf. ovata, Prorocentrum hoffmannianum and P. lima) and planktonic (Amphidinium carterae and Lingulodinium polyedrum) habitats. Amphidinium, Coolia and Lingulodinium data did not present any concentration-response relationships, and EC 50 values could only be obtained after 720 and 1440 min of exposure to both Prorocentrum species and O. cf. ovata, respectively. This study demonstrated that the H9c2(2-1) SRB assay represents a promising and sensitive tool for the detection of Gambierdiscus toxic compounds present in water samples, particularly of G. excentricus at very low cell abundances., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The mammalian target of rapamycin (mTOR) signaling pathway efficiently regulates the energy state of cells and maintains tissue homeostasis. Dysregulation of the mTOR pathway has been implicated in several human diseases. Rapamycin is a specific inhibitor of mTOR and pharmacological inhibition of mTOR with rapamycin promote cardiac cell generation from the differentiation of mouse and human embryonic stem cells. These studies strongly implicate a role of sustained mTOR activity in the differentiating functions of embryonic stem cells; however, they do not directly address the required effect for sustained mTOR activity in human cardiac progenitor cells. In the present study, we evaluated the effect of mTOR inhibition by rapamycin on the cellular function of human cardiac progenitor cells and discovered that treatment with rapamycin markedly attenuated replicative cell senescence in human cardiac progenitor cells (hCPCs) and promoted their cellular functions. Furthermore, rapamycin not only inhibited mTOR signaling but also influenced signaling pathways, including STAT3 and PIM1, in hCPCs. Therefore, these data reveal a crucial function for rapamycin in senescent hCPCs and provide clinical strategies based on chronic mTOR activity.

Myocardial ischemia/reperfusion (I/R) injury is a major complication of reperfusion therapy in myocardial infarction. Ischemic myocardium produces a variety of peptides. We recently identified PDRPS7 as a novel peptide in cardiomyocytes that can be induced by hypoxia. However, the role of PDRPS7 is unknown. Here, we investigated the effects of PDRPS7 on hypoxia/reoxygenation (H/R)-induced injury in rat cardiomyoblast H9c2 cells and NRCMs. We found that PDRPS7 improved cell survival and attenuated lactate dehydrogenase leakage following H/R in H9c2 cells and NRCMs. PDRPS7 also alleviated H/R-induced pulsation reduction in NRCMs. Moreover, H/R-induced cell apoptosis was decreased in the presence of PDRPS7. H/R-induced reactive oxygen species generation was reduced by PDRPS7; in addition, PDRPS7 did not impact H 2 O 2 -induced cell injury. Signaling analysis demonstrated that H/R increased the phosphorylation levels of JNKs, ERKs, and p38 mitogen-activated protein kinases. However, PDRPS7 only attenuated H/R-induced JNK phosphorylation, but not phosphorylation of ERKs and p38. PDRPS7 protected cardiomyocytes from apoptosis by inhibiting JNK phosphorylation and c-Jun phosphorylation pathways, markedly upregulating anti-apoptotic Bcl-2 expression and inhibiting that of pro-apoptotic Bax and cleaved caspase-3. Importantly, pharmacological activation of JNKs diminished the protective effect of PDRPS7 in terms of cell survival against H/R stimulation. In summary, our study identified PDRPS7 as a novel cardioprotective peptide against H/R challenge and this action was mediated, at least in part, through inactivation of JNKs., (© 2020 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.)

Cruzain, an essential cysteine protease of the parasitic protozoan, Trypanosoma cruzi , is an important drug target for Chagas disease. We describe here a new series of reversible but time-dependent inhibitors of cruzain, composed of a dipeptide scaffold appended to vinyl heterocycles meant to provide replacements for the irreversible reactive "warheads" of vinyl sulfone inactivators of cruzain. Peptidomimetic vinyl heterocyclic inhibitors (PVHIs) containing Cbz-Phe-Phe/homoPhe scaffolds with vinyl-2-pyrimidine, vinyl-2-pyridine, and vinyl-2-( N -methyl)-pyridine groups conferred reversible, time-dependent inhibition of cruzain ( K i * = 0.1-0.4 μM). These cruzain inhibitors exhibited moderate to excellent selectivity versus human cathepsins B, L, and S and showed no apparent toxicity to human cells but were effective in cell cultures of Trypanosoma brucei brucei (EC 50 = 1-15 μM) and eliminated T. cruzi in infected murine cardiomyoblasts (EC 50 = 5-8 μM). PVHIs represent a new class of cruzain inhibitors that could progress to viable candidate compounds to treat Chagas disease and human sleeping sickness.

Objective: Heart dysfunctions are the major complications of trastuzumab in patients with Human Epidermal growth factor Receptor-2 (HER2)-positive breast cancers., Methods: In this study, the cytotoxicity of trastuzumab on H9c2 cardiomyoblasts was demonstrated, and the proteome changes of cells were investigated by a tandem mass tagging quantitative approach. The Differentially Abundant Proteins (DAPs) were identified and functionally enriched., Results: We determined that carvedilol, a non-selective beta-blocker, could effectively inhibit trastuzumab toxicity when administrated in a proper dose and at the same time. The proteomics analysis of carvedilol co-treated cardiomyoblasts showed complete or partial reversion in expressional levels of trastuzumab-induced DAPs., Conclusion: Downregulation of proteins involved in the translation biological process is one of the most important changes induced by trastuzumab and reversed by carvedilol. These findings provide novel insights to develop new strategies for the cardiotoxicity of trastuzumab., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Phytoestrogens exert protective effects on the cardiovascular system through mechanisms that have yet to be clearly demonstrated. The aim of this study was to evaluate the protective effects exerted by genistein on cardiomyoblasts (H9C2) against oxidative stress, nitric oxide (NO) release, viability, proliferation/migration and mitochondrial function. H9C2 cultured in physiological or peroxidative conditions, were treated with genistein in the absence or presence of estrogen receptors (ERs), G protein‑​coupled‑estrogenic‑receptors (GPER), Akt, extracellular‑​signal‑regulated kinases 1/2 (ERK1/2) and p38 mitogen activated protein kinase (p38MAPK) blockers. Cell viability, proliferation, migration, mitochondrial membrane potential, mitochondrial oxygen consumption and oxidant/antioxidant system, were measured by specific assays. Western blot assay was used for the analysis of NO synthase (NOS) subtypes' and expression and activation of various kinases. In all experiments 17β‑estradiol was used for comparison. The results showed that phytoestrogens and estrogens can increase cell viability, proliferation/migration and improve mitochondrial membrane potential and oxygen consumption of H9C2. Furthermore, NO release was modulated by genistein and 17β‑estradiol. These effects were reduced or abolished by the pre‑treatment with ERs, GPER, Akt, ERK1/2 and p38MAPK blockers. Finally, a reduction of reactive oxygen species production and an increase of glutathione content was found in response to the two agents. In H9C2 cultured in physiological conditions, genistein induced endothelial NOS‑dependent NO production through the involvement of estrogenic receptors and by the modulation of intracellular signalling related to Akt, ERK1/2, and p38MAPK. Moreover, estrogens and phytoestrogens protected H9C2 against oxidative stress by reducing inducible NOS expression and through the modulation of the antioxidant system and mitochondrial functioning.

ω-3 fish oil fat emulsions contain a considerable quantity of unsaturated carbon-carbon double bonds, which undergo lipid peroxidation to yield low-dose aldehydes. These aldehydes may stimulate the production of antioxidant enzymes, thereby mitigating myocardial oxidative damage. This study aims to (1) verify the cardioprotective effect of ω-3 fish oil fat emulsion in vivo and in vitro, and (2) determine whether aldehyde stress is a protective mechanism. For modeling purposes, we pretreated rats with 2 ml/kg of a 10% ω-3 fish oil fat emulsion for 5 days in order to generate a sufficient aldehyde stress response to trigger the production of antioxidant enzymes, and we obtained similar response with H9C2 cells that were pretreated with a 0.5% ω-3 fish oil fat emulsion for 24 h. ω-3 fish oil fat emulsion pretreatment in vivo reduced the myocardial infarct size, decreased the incidence of arrhythmias, and promoted the recovery of cardiac function after myocardial ischemia/reperfusion injury. Once the expression of nuclear factor E2-related factor 2 (Nrf2) was silenced in H9C2 cells, aldehydes no longer produced enough antioxidant enzymes to reverse the oxidative damage caused by tert-butyl hydroperoxide (TBHP). Our results demonstrated that ω-3 fish oil fat emulsion enhanced the inhibition of oxidation and production of free radicals, and alleviated myocardial oxidative injury via activation of the Nrf2 signaling pathway., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

A key action of current aquatic environmental sciences is the determination of concentration-response assessments to quantitatively measure the risk of pollutants, and fish lethal tests are still a regulatory requirement for assessing the environmental risk of both new and existing substances in the aquatic compartment. However, animal health and welfare aspects, as well as the time and resources required to support fish lethal testing, stimulate research for alternative in vitro methods, and cell-based assays are considered as one such alternative. The first goal of the present study was to compile existing fish short-term toxicity and cell toxicity data of pesticides through a scientific literature search, and relate in vivo and in vitro results to identify sensitive cell models, mammalian-, fish- or arthropod-derived. Discovering cell models which are sensitive is of great importance, since the risk of false negatives, believed to be the main limitation of cell-based assays as an alternative to fish tests, may decrease. As to the second goal, this study also determined and compared cell toxicity results for 12 pesticides using rat cardiomyoblast H9c2(2-1) cells with the corresponding fish LC 50,96h (50% lethal concentration at 96 h of exposure) values collected in literature. On the whole, the in vivo/in vitro ratio was obtained for 50 pesticides belonging to 23 groups, and presented only 27% of positive results, thus confirming the low sensitivity of cell-based assays in relation to fish lethal data for pesticides. In general, cell-based assays still do not seem to be an alternative to the regulatory short-term fish assay for pesticides, making further studies necessary., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Hyperkalemia is often associated with cardiac dysfunction. In this study an earthworm extract (dilong) was prepared from dried Pheretima aspergillum powder and its effect against high-KCl challenge was determined in H9c2 cardiomyoblast cells. H9c2 cells pre-treated with dilong (31.25, 62.5, 125, and 250 mg/mL) for 24 hours, where challenged with different doses of KCl treatment for 3 hours to determine the protective mechanisms of dilong against cardiac fibrosis. High-KCl administration induced mitochondrial injury and elevated the levels of pro-apoptotic proteins. The mediators of fibrosis such as ERK, uPA, SP1, and CTGF were also found to be upregulated in high-KCl condition. However, dilong treatment enhanced IGF1R/PI3k/Akt activation which is associated with cell survival. In addition, dilong also reversed high-KCl induced cardiac fibrosis related events in H9c2 cells and displayed a strong cardio-protective effect. Therefore, dilong is a potential agent to overcome cardiac events associated with high-KCl toxicity., (© 2019 Wiley Periodicals, Inc.)

Cell-based therapies represent a very promising strategy to repair and regenerate the injured heart to prevent progression to heart failure. To date, these therapies have had limited success due to a lack of survival and retention of the infused cells. Therefore, it is important to increase our understanding of the biology of these cells and utilize this information to enhance their survival and function in the injured heart. Mitochondria are critical for progenitor cell function and survival. Here, we demonstrate the importance of mitochondrial autophagy, or mitophagy, in the differentiation process in adult cardiac progenitor cells (CPCs). We found that mitophagy was rapidly induced upon initiation of differentiation in CPCs. We also found that mitophagy was mediated by mitophagy receptors, rather than the PINK1-PRKN/PARKIN pathway. Mitophagy mediated by BNIP3L/NIX and FUNDC1 was not involved in regulating progenitor cell fate determination, mitochondrial biogenesis, or reprogramming. Instead, mitophagy facilitated the CPCs to undergo proper mitochondrial network reorganization during differentiation. Abrogating BNIP3L- and FUNDC1-mediated mitophagy during differentiation led to sustained mitochondrial fission and formation of donut-shaped impaired mitochondria. It also resulted in increased susceptibility to cell death and failure to survive the infarcted heart. Finally, aging is associated with accumulation of mitochondrial DNA (mtDNA) damage in cells and we found that acquiring mtDNA mutations selectively disrupted the differentiation-activated mitophagy program in CPCs. These findings demonstrate the importance of BNIP3L- and FUNDC1-mediated mitophagy as a critical regulator of mitochondrial network formation during differentiation, as well as the consequences of accumulating mtDNA mutations. Abbreviations : Baf: bafilomycin A 1 ; BCL2L13: BCL2 like 13; BNIP3: BCL2 interacting protein 3; BNIP3L: BCL2 interacting protein 3 like; CPCs: cardiac progenitor cells; DM: differentiation media; DNM1L: dynamin 1 like; EPCs: endothelial progenitor cells; FCCP: carbonyl cyanide- 4 -(trifluoromethoxy)phenylhydrazone; FUNDC1: FUN14 domain containing 1; HSCs: hematopoietic stem cells; MAP1LC3B/LC3: microtubule-associated protein 1 light chain 3 beta; MFN1/2: mitofusin 1/2; MSCs: mesenchymal stem cells; mtDNA: mitochondrial DNA; OXPHOS: oxidative phosphorylation; PPARGC1A: PPARG coactivator 1 alpha; PHB2: prohibitin 2; POLG: DNA polymerase gamma, catalytic subunit; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TMRM: tetramethylrhodamine methyl ester.

Endocrine disrupters are strictly associated to cancer and several cardiovascular risk factors. Bisphenol A (BPA) is an endocrine disrupter commonly used in the manufacturing of plastics based on polycarbonate, polyvinyl chloride and resins. Our study aims to investigate whether BPA may cause pro-oxidative and pro-inflammatory effects on cardiomyoblasts, thus exacerbating the Doxorubicin (DOXO)-induced cardiotoxicity phenomena. We tested the metabolic effects of BPA at low doses analyzing its affections on the intracellular calcium uptake, oxidative stress, lipid peroxidation and production of nitric oxide and interleukins. Co-incubation of BPA and DOXO significantly reduced the cardiomyoblast viability, compared to only DOXO exposure cells. The mechanisms underlying these effects are based on the stimulation of the intracellular calcium accumulation and lipid peroxidation. Notably, BPA increase the production of pro-inflammatory interleukins involved in cardiovascular diseases as well as in DOXO-Induced cardiotoxicity phenomena. This study provides a rationale for translational studies in the field of cardio-oncology., (Copyright © 2019. Published by Elsevier B.V.)

Recurrent cardiotoxicity limits the clinical application of doxorubicin (DOX); however the detailed molecular mechanism of DOX cardiotoxicity remains unclear. In the current study, we found that a natural product extracted from Illicium verum, isodunnianol (IDN), mitigates DOX-induced cardiotoxicity by regulating autophagy and apoptosis both in vitro and in vivo. DOX suppressed protective autophagy and induced apoptosis in H9C2 cardiac myoblasts. Additionally, IDN demonstrated up-regulated autophagy and reduced apoptosis through the activation of the AMPK-ULK1 pathway. In addition, the beneficial effects of IDN on DOX which induced myocardial injury were dependent on AMPK and ULK1 phosphorylation. Similar results were also observed in a DOX-induced cardiotoxicity rat model. The combination of IDN and DOX resulted in decreased apoptosis and inflammatory myocardial fibrosis compared to the DOX mono-treatment group. In summary, our findings provide novel insights into the prevention of DOX-related toxicity by isodunnianol, a food source natural product, warranting further investigation.

Lipid emulsion (LE) therapy has been used to reduce overdose of bupivacaine (BPV)-induced cardiotoxicity. The TWIK-related potassium channel-1 (TREK-1) is inhibited by BPV and activated by polyunsaturated fatty acids, which are the main component in LE. These pharmacological properties inspired us to investigate whether the TREK-1 channel is associated with cell viability of H9c2 cardiomyoblasts affected by BPV and LE. Consistent with previous studies, BPV-induced cell death was reduced by LE treatment. The reduction in the TREK-1 expression level by BPV was alleviated by LE. The BPV cytotoxicity highly decreased in TREK-1 overexpressed cells but was the opposite in TREK-1 knocked-down cells. TREK-1 channel activators and inhibitors increased and decreased cell viability, respectively. BPV-induced depolarization of the plasma and mitochondrial membrane potential and increase in intracellular Ca 2+ level were blocked by LE treatment. BPV-induced depolarization of membrane potential was reduced in TREK-1 overexpressed cells, indicating that TREK-1 channels mediate setting the resting membrane potentials as a background K + channel in H9c2 cells. These results show that TREK-1 activity is involved in the BPV cytotoxicity and the antagonistic effect of LE in H9c2 cells and suggest that TREK-1 could be a target for action of BPV and LE., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Diabetic cardiomyopathy (DCM) is one of the chief diabetes mellitus complications. Inflammation factors may be one reason for the damage from DM. The purpose of this research is to study the potential protective effects of puerarin on DM and the possible mechanisms of action related to NF-κB signal pathway. Following administration of puerarin to the disease model rat, several changes were observed including the changes of serum biochemical index, improved diastolic dysfunction, and enhanced endogenous antioxidant enzymes activities, further NF-κB signaling activation. Puerarin showed cardio-protective effects on DCM by inhibiting inflammation, and it might be a potential candidate for the treatment of DCM.

Cigarette smoking causes a vast array of diseases including cardiovascular diseases. Our laboratory focuses on investigating cigarette smoke (CS)-induced cardiovascular malfunction and the responsible mechanisms utilizing the model, c-kit-positive cardiac stem cells (CSCs). The main objective of our study is to investigate whether CS extracts (CSEs) cause impairment of CSC functions via oxidative damage. We hypothesized that CSE, via oxidative modifications of CSC proteins and antioxidant enzymes, can modulate CSC functions and these modifications can be attenuated by ascorbate treatment. Our specific aims are (1) to investigate CSE-induced oxidative modification of CSC proteins via carbonylation, and prevention by ascorbic acid; (2) to investigate CSE-induced oxidative modification of antioxidant enzymes and ascorbic acid-mediated modulations; and (3) to investigate CSE-induced changes in CSC functions and protection by ascorbic acid. CSCs were cultured, and the aqueous extracts of CSE were prepared. CSE-induced modulations of CSC viability, oxidative modification of proteins, and antioxidant enzyme activities were detected using standard assays including Apostain, bromodeoxyuridine, and Oxiblot. CSE caused oxidative modification of CSC proteins, changed antioxidant enzyme levels, attenuated CSC proliferation, and accelerated CSC apoptosis. Ascorbic acid prevented CSE-induced CSC malfunctions, and ascorbic acid therapy might be useful in smoker CSC recipients and to condition CSCs prior to the transplant in the future. Cardiac stem cell therapy is currently undergoing in clinical trials.

Scope: High fat (HF)-diet-induced insulin resistance is a major contributor to the pathogenesis of cardiovascular diseases. However, the molecular mechanisms that regulate cardiac insulin signaling are not fully understood. The regulatory role of tafazzin in the hearts of HF-diet-fed mice is investigated., Methods and Results: Mice are fed a HF diet or low fat (LF) diet for up to 24 weeks. After 24 weeks, it is found that HF-diet-induced cardiac dysfunction is linked to overexpression of the mitochondrial protein tafazzin. Increased tafazzin promotes mitochondrial fission and impairs insulin signaling, which is mediated by dynamin-related protein 1 (Drp-1) translocation from the cytosol to the mitochondria. Furthermore, knockdown of tafazzin with siRNA inhibits palmitic-acid-induced mitochondrial fission and restores insulin sensitivity. Moreover, miR-125b-5p as an upstream regulator targeting tafazzin is identified and palmitate-induced insulin resistance further rescued., Conclusion: In HF-diet-fed mouse hearts, increased tafazzin contributes to insulin resistance via mediating Drp-1 translocation to the mitochondria, and a small non-coding RNA, miR-125b-5p, at least partially regulates this signaling pathway and alleviates insulin resistance., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)