Treated H9c2 Cardiomyocytes Research Articles

Construction of a Near-Infrared Fluorescent Probe for Dynamic Monitoring and Early Diagnosis of Heart Failure.

Cardiac hypertrophy characterized by abnormal cardiomyocyte viscosity is a typical sign of heart failure (HF) with vital importance for early diagnosis. However, current biochemical and imaging diagnostic methods are unable to detect this subclinical manifestation. In this work, we developed a series of NIR-I fluorescence probes for detecting myocardial viscosity based on the pyridazinone scaffold. The probes showed weak fluorescence due to free intramolecular rotation under low-viscosity conditions, while they displayed strong fluorescence with limited intramolecular rotation in response to a high-viscosity environment. Among them, CarVis2 exhibited higher stability and photobleaching resistance than commercial dyes. Its specific response to viscosity was not influenced by the pH and biological species. Furthermore, CarVis2 showed rapid and accurate responses to the viscosity of isoproterenol (ISO)-treated H9C2 cardiomyocytes with good biocompatibility. More importantly, CarVis2 demonstrated excellent sensitivity in monitoring myocardial viscosity variation in HF mice in vivo, potentially enabling earlier noninvasive identification of myocardial abnormalities compared to traditional clinical imaging and biomarkers. These findings revealed that CarVis2 can serve as a powerful tool to monitor myocardial viscosity, providing the potential to advance insights into a pathophysiological mechanism and offering a new reference strategy for early visual diagnosis of HF.

Oxidative stress, cardiomyocytes premature senescence and contractile dysfunction in in vitro and in vivo experimental models of Chagas disease

AimsThe relationship between redox imbalance and cardiovascular senescence in infectious myocarditis is unknown. Thus, the aim of this study was to investigate whether cardiomyocytes parasitism, oxidative stress and contractile dysfunction can be correlated to senescence-associated β-galactosidase (SA-β-Gal) activity in Trypanosoma cruzi-infection in vitro and in vivo. MethodsUninfected, T. cruzi-infected untreated and benznidazole (BZN)-treated H9c2 cardiomyocytes and rats were investigated. Parasitological, prooxidant, antioxidant, microstructural, and senescence-associated markers were quantified in vitro and in vivo. ResultsT. cruzi infection triggered intense cardiomyocytes parasitism in vitro and in vivo, which was accompanied by reactive oxygen species (ROS) upregulation, lipids, proteins and DNA oxidation in cardiomyocytes and cardiac tissue. Oxidative stress was parallel to microstructural cell damage (e.g., increased cardiac toponin I levels) and contractile dysfunction in cardiomyocytes in vitro and in vivo, whose severity accompanied a premature cellular senescence-like phenotype revealed by increased senescence-associated β-galactosidase (SA-β-Gal) activity and DNA oxidation (8-OHdG). Cellular parasitism (e.g., infection rate and parasite load), myocarditis and T. cruzi-induced prooxidant responses were attenuated by early BZN administration to interrupt the progression of T. cruzi infection, protecting against SA-β-gal-based premature cellular senescence, microstructural damage and contractile deterioration in cardiomyocytes from T. cruzi-infected animals. ConclusionOur findings indicated that cell parasitism, redox imbalance and contractile dysfunction were correlated to SA-β-Gal-based cardiomyocytes premature senescence in acute T. cruzi infection. Therefore, in addition to controlling parasitism, inflammation and oxidative stress; inhibiting cardiomyocytes premature senescence should be further investigated as an additional target of specific Chagas disease therapeutics.

β3 adrenoceptor agonist mirabegron protects against right ventricular remodeling and drives Drp1 inhibition.

The right ventricular (RV) function determines the prognosis of patients with pulmonary hypertension (PH). Metabolic disorders have been observed in the RV myocardium in PH. Activation of the β3 adrenoceptor improves cardiac function and restores cardiac metabolic efficiency in rodents with heart failure; however, its role in the RV remains uncertain. Experimental PH was induced by monocrotaline (MCT) in rats. Mirabegron, a selective β3 adrenoceptor agonist, was given to MCT rats daily from the day after MCT injection at the dose of 10 mg/kg. In vivo echocardiography and RV catheterization were performed to assess RV hemodynamics, structure, and function. RV fibrosis and hypertrophy were assessed by Sirius Red (SR) and wheat germ agglutinin (WGA) staining respectively. Western blotting was performed to examine the markers of RV fibrosis and hypertrophy, as well as the levels of the key molecules and their phosphorylated forms. The molecular changes were confirmed in the cardiac hypertrophy model of angiotensin II (Ang II) treated H9c2 cardiomyocytes using western blotting. The overloaded RV had increased β3 adrenoceptor expression, which was further increased by mirabegron. Mirabegron reduced RV pressure and reduced RV structural and functional deterioration in MCT rats. Mirabegron decreased cardiac fibrosis and hypertrophy in the overloaded RV. Mirabegron suppressed dynaminrelated protein 1 (Drp1) and promoted AMP-activated protein kinase (AMPK) signaling in the overloaded RV and Ang II treated cardiomyocytes. The β3 adrenoceptor agonist mirabegron reduced RV hypertrophy and fibrosis in PH rats. The treatment effect involved Drp1 inhibition and AMPK activation.

Roles of transient receptor potential channel 6 in glucose-induced cardiomyocyte injury.

BACKGROUNDDiabetic cardiomyopathy (DCM) is a serious complication of end-stage diabetes that presents symptoms such as cardiac hypertrophy and heart failure. The transient receptor potential channel 6 (TRPC6) protein is a very important selective calcium channel that is closely related to the development of various cardiomyopathies.AIMTo explore whether TRPC6 affects cardiomyocyte apoptosis and proliferation inhibition in DCM.METHODSWe compared cardiac function and myocardial pathological changes in wild-type mice and mice injected with streptozotocin (STZ), in addition to comparing the expression of TRPC6 and P-calmodulin-dependent protein kinase II (P-CaMKII) in them. At the same time, we treated H9C2 cardiomyocytes with high glucose and then evaluated the effects of addition of SAR, a TRPC6 inhibitor, and KN-93, a CaMKII inhibitor, to such H9C2 cells in a high-glucose environment.RESULTSWe found that STZ-treated mice had DCM, decreased cardiac function, necrotic cardiomyocytes, and limited proliferation. Western blot and immunofluorescence were used to detect the expression levels of various appropriate proteins in the myocardial tissue of mice and H9C2 cells. Compared to those in the control group, the expression levels of the apoptosis-related proteins cleaved caspase 3 and Bax were significantly higher in the experimental group, while the expression of the proliferation-related proteins proliferating cell nuclear antigen (PCNA) and CyclinD1 was significantly lower. In vivo and in vitro, the expression of TRPC6 and P-CaMKII increased in a high-glucose environment. However, addition of inhibitors to H9C2 cells in a high-glucose environment resulted in alleviation of both apoptosis and proliferation inhibition.CONCLUSIONThe inhibition of apoptosis and proliferation of cardiomyocytes in a high-glucose environment may be closely related to activation of the TRPC6/P-CaMKII pathway.

Melatonin Attenuates Ischemia/Reperfusion-Induced Oxidative Stress by Activating Mitochondrial Fusion in Cardiomyocytes.

Myocardial ischemia/reperfusion (I/R) injury can stimulate mitochondrial reactive oxygen species production. Optic atrophy 1- (OPA1-) induced mitochondrial fusion is an endogenous antioxidative mechanism that preserves the mitochondrial function. In our study, we investigated whether melatonin augments OPA1-dependent mitochondrial fusion and thus maintains redox balance during myocardial I/R injury. In hypoxia/reoxygenation- (H/R-) treated H9C2 cardiomyocytes, melatonin treatment upregulated OPA1 mRNA and protein expression, thereby enhancing mitochondrial fusion. Melatonin also suppressed apoptosis in H/R-treated cardiomyocytes, as evidenced by increased cell viability, diminished caspase-3 activity, and reduced Troponin T secretion; however, silencing OPA1 abolished these effects. H/R treatment augmented mitochondrial ROS production and repressed antioxidative molecule levels, while melatonin reversed these changes in an OPA1-dependent manner. Melatonin also inhibited mitochondrial permeability transition pore opening and maintained the mitochondrial membrane potential, but OPA1 silencing prevented these outcomes. These results illustrate that melatonin administration alleviates cardiomyocyte I/R injury by activating OPA1-induced mitochondrial fusion and inhibiting mitochondrial oxidative stress.

HSP70 alleviates sepsis-induced cardiomyopathy by attenuating mitochondrial dysfunction-initiated NLRP3 inflammasome-mediated pyroptosis in cardiomyocytes.

Sepsis-induced cardiomyopathy (SIC) is an identified serious complication of sepsis that is associated with adverse outcomes and high mortality. Heat shock proteins (HSPs) have been implicated in suppressing septic inflammation. The aim of this study was to investigate whether HSP70 can attenuate cellular mitochondrial dysfunction, exuberated inflammation and inflammasome-mediated pyroptosis for SIC intervention. Mice with cecal ligation plus perforation (CLP) and lipopolysaccharide (LPS)-treated H9C2 cardiomyocytes were used as models of SIC. The mouse survival rate, gross profile, cardiac function, pathological changes and mitochondrial function were observed by photography, echocardiography, hematoxylin-eosin staining and transmission electron microscopy. In addition, cell proliferation and the levels of cardiac troponin I (cTnI), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were determined by Cell Counting Kit-8, crystal violet staining and enzyme-linked immunosorbent assay. Moreover, mitochondrial membrane potential was assessed by immunofluorescence staining, and dynamin-related protein 1 and pyroptosis-related molecules [nucleotide-binding domain, leucine-rich-repeat containing family pyrin domain-containing 3 (NLRP3), caspase-1, gasdermin-D (GSDMD), gasdermin-D N-terminal (GSDMD-N)] were measured by western blotting, immunoprecipitation and immunoblotting. Finally, hsp70.1 knockout mice with CLP were used to verify the effects of HSP70 on SIC and the underlying mechanism. Models of SIC were successfully established, as reduced consciousness and activity with liparotrichia in CLP mice were observed, and the survival rate and cardiac ejection fraction (EF) were decreased; conversely, the levels of cTnI, TNF-α and IL-1β and myocardial tissue damage were increased in CLP mice. In addition, LPS stimulation resulted in a reduction in cell viability, mitochondrial destabilization and activation of NLRP3-mediated pyroptosis molecules in vitro. HSP70 treatment improved myocardial tissue damage, survival rate and cardiac dysfunction caused by CLP. Additionally, HSP70 intervention reversed LPS-induced mitochondrial destabilization, inhibited activation of the NLRP3 inflammasome, caspase-1, GSDMD and GSDMD-N, and decreased pyroptosis. Finally, knockout of hsp70.1 mice with CLP aggravated cardiac dysfunction and upregulated NLRP3 inflammasome activity, and exogenous HSP70 significantly rescued these changes. It was further confirmed that HSP70 plays a protective role in SIC by attenuating mitochondrial dysfunction and inactivating pyroptotic molecules. Our study demonstrated that mitochondrial destabilization and NLRP3 inflammasome activation-mediated pyroptosis are attributed to SIC. Interestingly, HSP70 ameliorates sepsis-induced myocardial dysfunction by improving mitochondrial dysfunction and inhibiting the activation of NLRP3 inflammasome-mediated pyroptosis, and such a result may provide approaches for novel therapies for SIC.

Pinocembrin Protects Cardiomyocytes Against Isoproterenol-Induced Hypertrophy

Cardiac hypertrophy is characterized by an increase in myocardial cell volume and extracellular matrix production. Persistent cardiac hypertrophy can cause dilated cardiomyopathy, heart failure, and even death. Pinocembrin (5,7-dihydroxyflavanone) is a type of flavonoid, extracted from propolis, that has antimicrobial, antioxidant, antiinflammatory, and anticancer properties. The results of the present study showed that pretreatment of isoproterenol (ISO)-treated H9c2 cardiomyocytes with pinocembrin reduced the messenger RNA levels of hypertrophic markers, including atrial natriuretic factor and βeta-myosin heavy chain, and inflammatory cytokines, such as tumor necrosis factor-α, interleukin-6, interleukin-1β, and interferon-γ, and also inhibited p65 phosphorylation and nuclear factor-kappa B (NF-κB) translocation. In addition, the activity of IκBα, an inhibitor of NF-κB, was increased while that of caspase-3 was reduced under these conditions. These results indicate that pinocembrin may inhibit ISO-induced myocardial hypertrophy by attenuating the NF-κB signaling pathway.

Catalase Mediates the Inhibitory Actions of PPARδ against Angiotensin II-Triggered Hypertrophy in H9c2 Cardiomyocytes.

Hypertrophy of myocytes has been implicated in cardiac dysfunctions affecting wall stress and patterns of gene expression. However, molecular targets potentially preventing cardiac hypertrophy have not been fully elucidated. In the present study, we demonstrate that upregulation of catalase by peroxisome proliferator-activated receptor δ (PPARδ) is involved in the anti-hypertrophic activity of PPARδ in angiotensin II (Ang II)-treated H9c2 cardiomyocytes. Activation of PPARδ by a specific ligand GW501516 significantly inhibited Ang II-induced hypertrophy and the generation of reactive oxygen species (ROS) in H9c2 cardiomyocytes. These effects of GW501516 were almost completely abolished in cells stably expressing small hairpin (sh)RNA targeting PPARδ, indicating that PPARδ mediates these effects. Significant concentration and time-dependent increases in catalase at both mRNA and protein levels were observed in GW501516-treated H9c2 cardiomyocytes. In addition, GW501516-activated PPARδ significantly enhanced catalase promoter activity and protein expression, even in the presence of Ang II. GW501516-activated PPARδ also inhibited the expression of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), which are both marker proteins for hypertrophy. The effects of GW501516 on the expression of ANP and BNP were reversed by 3-amino-1,2,4-triazole (3-AT), a catalase inhibitor. Inhibition or downregulation of catalase by 3-AT or small interfering (si)RNA, respectively, abrogated the effects of PPARδ on Ang II-induced hypertrophy and ROS generation, indicating that these effects of PPARδ are mediated through catalase induction. Furthermore, GW501516-activated PPARδ exerted catalase-dependent inhibitory effects on Ang II-induced hypertrophy by blocking p38 mitogen-activated protein kinase. Taken together, these results indicate that the anti-hypertrophic activity of PPARδ may be achieved, at least in part, by sequestering ROS through fine-tuning the expression of catalase in cardiomyocytes.

MicroRNA-21 mediates the protective effects of salidroside against hypoxia/reoxygenation-induced myocardial oxidative stress and inflammatory response.

Myocardial ischemia-reperfusion (I/R) injury is the oxidative stress and inflammatory response that occurs when a tissue is reperfused following a prolonged period of ischemic injury. Growing evidence has demonstrated that microRNAs (miRs) are essential in the development of myocardial I/R injury. Salidroside, a phenylpropanoid glycoside isolated from a traditional Chinese medicinal plant, Rhodiola rosea, possesses multiple pharmacological functions and protects against myocardial I/R injury in vitro and in vivo. However, the role of miRs in the cardioprotective effects of salidroside against myocardial I/R injury has not been studied, to the best of our knowledge. In the present study, the role of miR21 in the underlying mechanism of salidroside-induced protection against oxidative stress and inflammatory injuries in hypoxia/reoxygenation (H/R)-treated H9c2 cardiomyocytes was determined. The cell viability was assessed with an MTT assay. Lactate dehydrogenase (LDH) release, caspase-3 activity, malondialdehyde (MDA) level, superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities were determined by commercial kits. Cell apoptosis was measured by flow cytometry. Intracellular reactive oxygen species (ROS) generation was monitored by DCFH-DA. The miR-21 level was quantified by reverse transcription-quantitative (RT-q)PCR. The interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α levels were measured by RT-qPCR and ELISA. The results showed that salidroside pretreatment significantly increased cell viability and decreased the release of LDH, accompanied by an increase in miR-21 expression in H/R-treated H9c2 cells and a miR-21 inhibitor reversed these effects. In addition, the miR-21 inhibitor also abrogated the inhibition of salidroside on H/R-induced increases in apoptosis and caspase-3 activity in H9c2 cells. Salidroside mitigated H/R-induced oxidative stress as illustrated by the downregulation of ROS generation and MDA level and increased the activities of the antioxidant enzymes, SOD and GSH-Px, all of which were abrogated in cells transfected with the miR-21 inhibitor. Salidroside induced a decrease in the expression and levels of the pro-inflammatory cytokines, IL-6, IL-1β and TNF-α, which were prevented by the miR-21 inhibitor. Together, these results provide evidence of the beneficial effects of salidroside against myocardial I/R injury by reducing myocardial oxidative stress and inflammation which are enhanced by increasing miR-21 expression.

CD47 deficiency protects cardiomyocytes against hypoxia/reoxygenation injury by rescuing autophagic clearance.

To assess the effect of cluster of differentiation(CD47) downregulation on autophagy in hypoxia/reoxygenation(H/R)‑treated H9c2 cardiomyocytes. H9c2cells were maintained in normoxic conditions (95% air, 5%CO2, 37˚C) without CD47antibodies, Si‑CD47 or chloroquine(CQ) treatment; H9c2cells in the H/R group were subjected to 24h of hypoxia (1%O2, 94%N2, 5%CO2, 37˚C) followed by 12h of reoxygenation (95% air, 5%CO2, 37˚C). All assays were controlled, triplicated and repeated on three separately initiated cultures. The biochemical parameters in the medium supernatant were measured to evaluate the oxidative stress in cardiomyocytes. The AnnexinV‑fluorescein isothiocyanate assay was used to detect the apoptotic rate in the H9c2cells. Transmission electron microscope, immunofluorescent staining and western blot analysis were performed to detect the effect of the CD47 antibody on autophagic flux in H/R‑treated H9c2 cardiomyocytes. The cardiomyocytic oxidative stress and apoptotic rate decreased and autophagic clearance increased after CD47 downregulation. H/R triggered cell autophagy, autophagosome accumulation and apoptosis in H9c2 cell lines. However, these effects can be attenuated by CD47 downregulation. This study demonstrates its clinical implications in ischemia/reperfusion injury treatment.

Curcumin attenuates palmitic acid-induced cell apoptosis by inhibiting endoplasmic reticulum stress in H9C2 cardiomyocytes.

Diabetic cardiomyopathy is mediated by multiple molecular mechanisms including endoplasmic reticulum (ER) stress. Curcumin, a phenolic compound, has cytoprotective properties, but its potential protective action against diabetic cardiomyopathy and the related molecular mechanisms are not fully elucidated. In this study, we evaluated the effects of curcumin on cell viability and apoptosis in palmitic acid (PA)-treated H9C2 cardiomyocytes and investigated the signaling pathways involved. Treatment with PA reduced cell viability, induced apoptosis, enhanced apoptosis-related protein expression (Caspase 3 and BCL-2 associated X protein (BAX)), and activated ER stress marker protein expression (glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP)). Curcumin attenuated PA-induced reduction in cell viability and activation of apoptosis, Caspase 3 activity, BAX, CHOP, and GRP78 expression. 4-Phenylbutyric acid (4-PBA) attenuated the PA-induced effects on cell viability and apoptosis, similar to curcumin. Both curcumin and 4-PBA also attenuated PA-induced increase in ER stress protein (CHOP and GRP78) expression. Curcumin also protected against cytotoxicity, apoptosis, and ER stress induced by thapsigargin. These findings indicate that PA triggers apoptosis in H9C2 cells via ER stress pathways and curcumin protects against this phenomenon.

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy

Assessing the role of extracellular vesicles in renin-angiotensin system signalling in cardiomyocyte hypertrophy

Ginkgetin aglycone attenuates the apoptosis and inflammation response through nuclear factor-kB signaling pathway in ischemic-reperfusion injury.

Acute myocardial infarction (AMI) is one of the most threaten disease in the world. Ginkgetin aglycone (GA) was a new kind of Ginkgo biloba, involved in various diseases, including kidney injury and acute pancreatitis. However, the function of GA in AMI remains unknown. The aim of the study was to investigate the characteristics and function of GA in ischemic-reperfusion injury. H2 O 2 - and CoCl 2 -treated H9C2 cells were used to analyze the function of GA in vitro. Caspase 3, interleukin-6 (IL-6), and tumor necrosis factor-α were detected to evaluate the apoptosis and inflammation response. Rat AMI was performed to elucidate the function in vivo. We found that GA could reduce the apoptosis and improved cell survival of H2 O 2 -treated H9C2 cardiomyocytes and CoCl 2 -treated H9C2 cells. GA attenuated CoCl 2 -induced inflammatory response and the level of cleaved caspase 33, suggesting that GA could alleviate the cell apoptosis. GA improved the cardiac function and attenuated the inflammatory cell infiltration in vivo. We also found that nuclear factor-kB signaling pathway, which was activated under hypoxia environment, was also suppressed in the GA-treated group. We verified the function and mechanism of GA and provide evidence that GA may play a vital role in ischemic-reperfusion injury, and understanding the precise role of GA will undoubtedly shed new light on the clinical treatment.

Salidroside mitigates hypoxia/reoxygenation injury by alleviating endoplasmic reticulum stress‑induced apoptosis in H9c2 cardiomyocytes.

Endoplasmic reticulum (ER) stress-induced apoptosis serves a crucial role in the development of myocardial ischemia/reperfusion (I/R) injury. Salidroside is a phenylpropanoid glycoside isolated from Rhodiola rosea L., which is a plant often used in traditional Chinese medicine. It possesses multiple pharmacological actions and protects against myocardial I/R injury in vitro and in vivo. However, it is not yet clear whether ER stress or ER stress-induced apoptosis contributes to the cardioprotective effects of salidroside against myocardial I/R injury. Hence, hypoxia/reoxygenation (H/R)-treated H9c2 cardiomyocytes were used in the current study to mimic myocardium I/R injury in vivo. It was hypothesized that salidroside alleviates ER stress and ER stress-induced apoptosis, thereby reducing H/R injury in H9c2 cells. The results demonstrated that salidroside attenuated H/R-induced H9c2 cardiomyocyte injury, as cell viability was increased, lactate dehydrogenase release was decreased, morphological changes in apoptotic cells were ameliorated and the apoptosis ratio was reduced compared with the H/R group. ER stress was reversed, indicated by the downregulation of glucose regulated protein 78 and C/EBP homologous protein following pretreatment with salidroside. In addition, salidroside attenuated ER stress-induced apoptosis, as the expression of cleaved caspase-12 and pro-apoptotic protein Bcl-2 associated X protein and activity of caspase-3 was decreased, while the expression of anti-apoptotic protein Bcl-2 was increased following pretreatment with salidroside. Furthermore, the results indicated that salidroside decreases the activation of the ER stress-associated signaling pathway, as the expression of phosphorylated protein kinase RNA (PKR)-like ER kinase (p-PERK) and phosphorylated inositol-requiring enzyme-1α (p-IRE1α) proteins were decreased following pretreatment with salidroside. These results demonstrate that salidroside protects against H/R injury via regulation of the PERK and IRE1α pathways, resulting in alleviation of ER stress or ER stress-induced apoptosis in H9c2 cardiomyocytes.

Pharmacological evaluation of novel PKR inhibitor indirubin-3-hydrazone in-vitro in cardiac myocytes and in-vivo in wistar rats

AimsDouble stranded protein kinase R cellular response is associated with various stress signals such as nutrients, endoplasmic stress, cytokines and mechanical stress. Increased PKR activity has been observed under diabetic and cardiovascular disease conditions. Most of the currently available PKR inhibitors are non-specific and have other effects as well. Thus, the aim of the present study was to examine the effect of novel PKR inhibitor indirubin-3-hydrazone (IHZ) in cultured rat H9C2 cardiomyocytes and wistar rats. Materials and methodsPKR expression was determined by Q-PCR, immunofluorescence and immunoblotting. The expression of different gene markers for apoptosis was measured by RT-PCR. Apoptosis and oxidative stress were determined by flow cytometry. KEY FINDINGS: High glucose (HG) treated H9C2 cardiomyocytes and high fructose (HF) treated wistar rats developed a significant increase in PKR expression. A significant increase in apoptosis and generation of reactive oxygen species was also observed in HG treated H9C2 cells and HF treated rats. Reduced vacuole formation and prominent nuclei were also observed in high glucose treated cells. Cardiac hypertrophy and increased fibrosis were observed in HF treated rats. All these effects of HG and HF were attenuated by novel PKR inhibitor, indirubin-3-hydrazone. SignificanceOur results indicate IHZ as an effective inhibitor of PKR in vitro and in-vivo, thus it may prove very useful in blocking the multiple harmful effects of PKR.

P-Coumaric acid mediated protection of H9c2 cells from Doxorubicin-induced cardiotoxicity: Involvement of augmented Nrf2 and autophagy

Doxorubicin (Dox) is a widely administered chemotherapeutic drug and incidences of cardiotoxicity associated with its administration have been of general concern. Extensive research proposes several mechanisms as a cause of Dox induced cardiotoxicity. However, none of these studies have been able to suggest a find one, cure all antidote for the same. To this end, several studies involving plant based compounds or natural products have gained acclaim for their ability to address at least one factor contributing to drug induced pathogenesis. We had previously reported that p-coumaric (pCA) has a protective effect on Dox induced oxidative stress in rat-derived cardiomyoblasts. In this study we investigated the effects of pCA on the regulation of Nrf-2, mitochondrial viability, autophagy and apoptosis in Doxorubicin treated H9c2 cardiomyocytes. ROS induced mitochondrial stress, changes in mitochondrial membrane potential, loss of membrane integrity; nuclear damage as single/double stranded breaks, autophagy and the effects of pre and co-treatment of pCA on Nrf-2 mediated signaling was evaluated by various approaches. The effect of pCA on drug uptake was evaluated through confocal Raman Spectroscopy. We find that nuclear translocation of Nrf-2 is prominently marked by protein-specific antibody conjugated fluorophore in Dox treated cells especially. Cell survival is mediated to a certain extent by the expression of the anti-apoptotic BCl2 in pCA treated cells. However, mRNA levels of autophagy related (Atg) genes suggest that autophagy plays a decisive role in deciding cellular fate. Caspase-3 activation is also observed in pCA treated cells which suggest an alternative function of caspase-3 in pCA mediated cell survival. Expression of antioxidant enzymes confirm the oxidative stress induced by Dox treatment in cells and the modulation of cell redox homeostasis through treatment with pCA.

MiR-30c Mediates Upregulation of Cdc42 and Pak1 in Diabetic Cardiomyopathy.

Cardiac hypertrophy and myocardial fibrosis significantly contribute to the pathogenesis of diabetic cardiomyopathy (DCM). Altered expression of several genes and their regulation by microRNAs has been reported in hypertrophied failing hearts. This study aims to examine the role of Cdc42, Pak1, and miR-30c in the pathogenesis of cardiac hypertrophy in DCM. DCM was induced in Wistar rats by low-dose streptozotocin-high-fat diet for 12 weeks. Cardiac expression of Cdc42, Pak1 and miR-30c, and hypertrophy markers (ANP and β-MHC) was studied in DCM vs control rats and in high-glucose (HG)-treated H9c2 cardiomyocytes. Diabetic rats showed cardiomyocyte hypertrophy, increased heart-to-body weight ratio, and an increased expression of ANP and β-MHC. Cardiac expression of Cdc42 and Pak1 genes was increased in diabetic hearts and in HG-treated cardiomyocytes. miR-30c was identified to target Cdc42 and Pak1 genes, and cardiac miR-30c expression was found to be decreased in DCM rats, patients with DCM, and in HG-treated cardiomyocytes. miR-30c overexpression decreased Cdc42 and Pak1 genes and attenuated HG-induced cardiomyocyte hypertrophy, whereas miR-30c inhibition increased Cdc42 and Pak1 gene expression and myocyte hypertrophy in HG-treated cardiomyocytes. Downregulation of miR-30c mediates prohypertrophic effects of hyperglycemia in DCM by upregulation of Cdc42 and Pak1 genes.

Cardiomyocyte mitochondrial respiration is reduced by receptor for advanced glycation end-product signaling in a ceramide-dependent manner.

Cigarette smoke exposure is associated with an increased risk of cardiovascular complications. The role of advanced glycation end products (AGEs) is already well established in numerous comorbidities, including cardiomyopathy. Given the role of AGEs and their receptor, RAGE, in activating inflammatory pathways, we sought to determine whether ceramides could be a mediator of RAGE-induced altered heart mitochondrial function. Using an in vitro model, we treated H9C2 cardiomyocytes with the AGE carboxy-methyllysine before mitochondrial respiration assessment. We discovered that mitochondrial respiration was significantly impaired in AGE-treated cells, but not when cotreated with myriocin, an inhibitor of de novo ceramide biosynthesis. Moreover, we exposed wild-type and RAGE knockout mice to secondhand cigarette smoke and found reduced mitochondrial respiration in the left ventricular myocardium from wild-type mice, but RAGE knockout mice were protected from this effect. Finally, conditional overexpression of RAGE in the lungs of transgenic mice elicited a robust increase in left ventricular ceramides in the absence of smoke exposure. Taken together, these findings suggest a RAGE-ceramide axis as an important contributor to AGE-mediated disrupted cardiomyocyte mitochondrial function.

NLRP3 gene silencing ameliorates diabetic cardiomyopathy in a type 2 diabetes rat model.

BackgroundNucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome is associated with metabolic disorder and cell death, which are important triggers in diabetic cardiomyopathy (DCM). We aimed to explore whether NLRP3 inflammasome activation contributes to DCM and the mechanism involved.MethodsType 2 diabetic rat model was induced by high fat diet and low dose streptozotocin. The characteristics of type 2 DCM were evaluated by metabolic tests, echocardiography and histopathology. Gene silencing therapy was used to investigate the role of NLRP3 in the pathogenesis of DCM. High glucose treated H9c2 cardiomyocytes were used to determine the mechanism by which NLRP3 modulated the DCM. The cell death in vitro was detected by TUNEL and EthD-III staining. TXNIP-siRNA and pharmacological inhibitors of ROS and NF-kB were used to explore the mechanism of NLRP3 inflammasome activation.ResultsDiabetic rats showed severe metabolic disorder, cardiac inflammation, cell death, disorganized ultrastructure, fibrosis and excessive activation of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), pro-caspase-1, activated caspase-1 and mature interleukin-1β (IL-1β). Evidence for pyroptosis was found in vivo, and the caspase-1 dependent pyroptosis was found in vitro. Silencing of NLRP3 in vivo did not attenuate systemic metabolic disturbances. However, NLRP3 gene silencing therapy ameliorated cardiac inflammation, pyroptosis, fibrosis and cardiac function. Silencing of NLRP3 in H9c2 cardiomyocytes suppressed pyroptosis under high glucose. ROS inhibition markedly decreased nuclear factor-kB (NF-kB) phosphorylation, thioredoxin interacting/inhibiting protein (TXNIP), NLRP3 inflammasome, and mature IL-1β in high glucose treated H9c2 cells. Inhibition of NF-kB reduced the activation of NLRP3 inflammasome. TXNIP-siRNA decreased the activation of caspase-1 and IL-1β.ConclusionNLRP3 inflammasome contributed to the development of DCM. NF-κB and TXNIP mediated the ROS-induced caspase-1 and IL-1β activation, which are the effectors of NLRP3 inflammasome. NLRP3 gene silencing may exert a protective effect on DCM.

Abstract 57: Impaired Aldehyde Dehydrogenase 2 Activity Contributes To Inhibition Of Mitochondrial Respiration In Hyperglycemic Rats

Aldehyde dehydrogenase (ALDH) 2 is a mitochondrial isozyme of the heart involved in the metabolism of toxic aldehydes produced from oxidative stress. A decrease in ALDH2 levels and activity was reported in the hearts of experimental hyperglycemia along with an increase in 4-hydroxy-2-nonenal (4HNE). 4HNE is produced in the mitochondria upon lipid peroxidation and known to form adduct with mitochondrial proteins. We hypothesize that reduced ALDH2 activity impairs mitochondrial respiration due to 4HNE adducts formation, ultimately resulting in cardiac damage in hyperglycemia. A single dose (65 mg/kg; i.p.) of streptozotocin in rats resulted in hyperglycemia with a blood glucose level of 443 ± 9 mg/dl versus 121 ± 7 mg/dl in control animals, p<0.0001. N=7-11 in both groups. After 6 months of hyperglycemia, heart function was recorded and the rats were sacrificed. Increases in cardiomyocyte cross sectional area (446 ± 32 μm 2 Vs 221 ± 10 μm 2 ; p<0.0001) indicated cardiac hypertrophy in diabetic rats. Both diastolic and systolic dysfunctions were recorded with diabetic rats compared to controls. For instance, % fractional shortening was lower in diabetic group versus control; (p<0.0001). Most importantly, myocardial ALDH2 activity and levels were reduced and immunostaining for 4HNE protein adducts was increased in diabetic hearts compared to controls. The mitochondrial oxygen consumption rate (OCR), an index of mitochondrial respiration, was decreased in mitochondria isolated from diabetic hearts compared to controls (p<0.0001). To check the direct effect of 4HNE and ALDH2 inhibition, we treated H9C2 cardiomyocytes with 4HNE and disufiram, an ALDH inhibitor, respectively, which reduced mitochondrial respiration (p<0.0001) and cell viability (p<0.0001) along with reduced ALDH2 activity and increased 4HNE adducts formation. We conclude that chronic hyperglycemia-induced 4HNE accumulation due to low ALDH2 activity in the heart leads to defective mitochondrial respiration and cardiac damage and dysfunction.