H9c2 Rat Cardiomyocytes Research Articles

MiR-15b-5p REGULATES THE NLRP3 INFLAMMASOME SIGNAL THROUGH TARGETING SIRT3 TO REGULATE HYPOXIA/REOXYGENATION-INDUCED CARDIOMYOCYTE PYROPTOSIS PROCESS.

Hypoxia/reoxygenation (H/R) induces pyroptosis in the setting of acute myocardial infarction (AMI). Previous studies have shown that the expression of the miR-15 family is stimulated in myocardial ischemia-reperfusion injury or H/R-induced cardiomyocyte injury, and miR-15 is a promoter of cardiac ischemia-reperfusion or H/R injury. However, whether miR-15b-5p regulates H/R injury and cardiomyocyte pyroptosis and its mechanism still need to be further clarified. Bioinformatics analysis elicited that SIRT3 was the downstream regulatory target gene of miR-15b-5p. SIRT3 has been shown to participate in the regulation of pyroptosis by negatively regulating the NLRP3 inflammasome pathway. Therefore, we hypothesized that miR-15b-5p targets SIRT3 and activated the NLRP3 inflammasome pathway to promote H/R-induced cardiomyocyte pyroptosis. We first show that H/R increases miR-15b-5p in rat cardiomyocytes H9C2. Next, we tested the effects of inhibition of miR-15b-5p or overexpression of SIRT3. We found that miR-15b-5p downregulation or SIRT3 overexpression could reverse the H/R-induced pyroptosis. Furthermore, silencing SIRT3 antagonized the protective effect of miR-15b-5p downregulation on H9C2 cells. NLRP3 inhibitor MCC950 annulled the previously mentioned antagonistic effect of silencing SIRT3 on the protection of miR-15b-5p downregulation against pyroptosis. We then used a rat AMI model to analyze myocardial infarction area by triphenyl tetrazolium chloride staining and assess serum cardiac troponin T level by ELISA and found that miR-15b-5p silencing reduced AMI injury in rats. Collectively, these results suggest that miR-15b-5p increase H/R-induced pyroptosis in cardiomyocytes by targeting SIRT3 and activating the NLRP3 inflammasome.

PFKM inhibits doxorubicin-induced cardiotoxicity by enhancing oxidative phosphorylation and glycolysis

Heart failure (HF) is a global pandemic which affects about 26 million people. PFKM (Phosphofructokinase, Muscle), catalyzing the phosphorylation of fructose-6-phosphate, plays a very important role in cardiovascular diseases. However, the effect of PFKM in glycolysis and HF remains to be elucidated. H9c2 rat cardiomyocyte cells were treated with doxorubicin (DOX) to establish injury models, and the cell viability, apoptosis and glycolysis were measured. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting were used for gene expression. DOX treatment significantly inhibited PFKM expression in H9c2 cells. Overexpression of PFKM inhibited DOX-induced cell apoptosis and DOX-decreased glycolysis and oxidative phosphorylation (OXPHOS), while silencing PFKM promoted cell apoptosis and inhibited glycolysis and OXPHOS in H9c2 cells. Moreover, PFKM regulated DOX-mediated cell viability and apoptosis through glycolysis pathway. Mechanism study showed that histone deacetylase 1 (HDAC1) inhibited H3K27ac-induced transcription of PFKM in DOX-treated cells and regulated glycolysis. PFKM could inhibit DOX-induced cardiotoxicity by enhancing OXPHOS and glycolysis, which might benefit us in developing novel therapeutics for prevention or treatment of HF.

Retraction Statement.

Retraction Statement.

Evaluating the protective effects of Panax bipinnatifidus Seem. extracts on hypoxia/reoxygenation-subjected cardiomyocytes

The search for new products to treat ischemic heart diseases has interested scientists for years as it is the main cause of cardiovascular disease mortality. In this study, we evaluated the protective effects of Panaxbipinnatifidus Seem. extracts (PSE) on hypoxia/reoxygenation (HR)-treated rat H9C2 cardiomyocytes. Rat H9C2 cardiomyocytes were either grown under normal condition or subjected to HR conditions. NecroX-5 (10 μM) was used as positive control for cardiac protective effect. The PSE (5-500 μg/ml) was employed to culture media at the onset of reoxygenation. Cell viability and mitochondrial function were tested using a Cell Counting Kit-8 and suitable fluorescence kits. The obtained data shows that HR conditions dramatically induced H9C2 cell death (p<0.05). Treatment of PSE (5-62.5 μg/ml) effectively reduced HR injuries in a dose-dependent manner. Similar to the NecroX-5 group, ginseng extract-treated cells had higher viability levels compared to that of the HR-subjected cells (p<0.05). In addition, supplementing PSE at the onset of reoxygenation strongly prevented the collapse of mitochondrial membrane potential and reduced mitochondrial oxidative stress (p<0.05). This study suggested the total saponin extracts from Panax bipinnatifidus Seem. exerts cardioprotective properties against HR damage.

Protective Effects of Amauroderma rugosum on Doxorubicin-Induced Cardiotoxicity through Suppressing Oxidative Stress, Mitochondrial Dysfunction, Apoptosis, and Activating Akt/mTOR and Nrf2/HO-1 Signaling Pathways.

Clinical outcomes for doxorubicin (Dox) are limited by its cardiotoxicity but a combination of Dox and agents with cardioprotective activities is an effective strategy to improve its therapeutic outcome. Natural products provide abundant resources to search for novel cardioprotective agents. Ganoderma lucidum (GL) is the most well-known edible mushroom within the Ganodermataceae family. It is commonly used in traditional Chinese medicine or as a healthcare product. Amauroderma rugosum (AR) is another genus of mushroom from the Ganodermataceae family, but its pharmacological activity and medicinal value have rarely been reported. In the present study, the cardioprotective effects of the AR water extract against Dox-induced cardiotoxicity were studied in vitro and in vivo. Results showed that both the AR and GL extracts could potentiate the anticancer effect of Dox. The AR extract significantly decreased the oxidative stress, mitochondrial dysfunction, and apoptosis seen in Dox-treated H9c2 rat cardiomyocytes. However, knockdown of Nrf2 by siRNA abolished the protective effects of AR in these cells. In addition, Dox upregulated the expression of proapoptotic proteins and downregulated the Akt/mTOR and Nrf2/HO-1 signaling pathways, and these effects could be reversed by the AR extract. Consistently, the AR extract significantly prolonged survival time, reversed weight loss, and reduced cardiac dysfunction in Dox-treated mice. In addition, oxidative stress and apoptosis were suppressed, while Nrf2 and HO-1 expressions were elevated in the heart tissues of Dox-treated mice after treatment with the AR extract. However, the GL extract had less cardioprotective effect against Dox in both the cell and animal models. In conclusion, the AR water extract demonstrated a remarkable cardioprotective effect against Dox-induced cardiotoxicity. One of the possible mechanisms for this effect was the upregulation of the mTOR/Akt and Nrf2/HO-1-dependent pathways, which may reduce oxidative stress, mitochondrial dysfunction, and cardiomyocyte apoptosis. These findings suggested that AR may be beneficial for the heart, especially in patients receiving Dox-based chemotherapy.

Pretreatment with Panaxatriol Saponin Attenuates Mitochondrial Apoptosis and Oxidative Stress to Facilitate Treatment of Myocardial Ischemia-Reperfusion Injury via the Regulation of Keap1/Nrf2 Activity.

Myocardial ischemia-reperfusion injury (MIRI) is a type of severe injury to the ischemic myocardium that can occur following recovery of blood flow, and for which, there is no effective treatment. Panaxatriol saponin (PTS), a major active component of P. notoginseng, has been used clinically to treat ischemia-related encephalopathy due to its antioxidant activity, but its effect on ischemic cardiomyopathy and underlying mechanism of action is still unclear. This study was performed to investigate the protective effect of PTS against MIRI and explore the potential underlying mechanisms. Hydrogen peroxide (H2O2) was used to stimulate cardiomyocytes, to mimic MIRI in vitro. Cell viability was tested using the CCK-8 method. The antioxidant activity of PTS in the H9c2 rat cardiomyocyte cell line was examined using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). The levels of superoxide dismutase-1 (SOD1), SOD2, and heme oxygenase (HO-1) were determined by Western blotting and/or immunofluorescence. The antiapoptotic effect of PTS was determined. In addition, mitochondrial permeability transition pore (mPTP) opening and mitochondrial membrane potential (ΔΨm) changes were assessed. Changes in Keap1/Nrf2 activation were evaluated by Western blotting analysis, molecular docking, and immunoprecipitation. An in vivo MIRI model was established in rats, and the myocardial infarct size was measured by 2,3,5-triphenyltetrazolium chloride (TTC) staining. Myocardial enzyme activities were determined by ELISA or biochemical analyses. Furthermore, changes in Nrf2 activation were evaluated, and the regulatory effect of PTS on cardiomyocyte apoptosis was examined using the Nrf2 blocker, ML385. The results showed that PTS ameliorated the cardiomyocyte injury induced by H2O2, characterized by increased cell viability, decreased reactive oxygen species (ROS) production, and promotion of SOD1, SOD2, and HO1 expression. PTS inhibited cardiomyocyte apoptosis in vivo and in vitro. PTS also reduced mPTP opening and stabilized ΔΨm in H9c2 cells. Molecular docking and immunoprecipitation study revealed that PTS can disrupt Keap1/Nrf2 interaction by directly blocking the binding site of Nrf2 in the Keap1 protein. In vivo, PTS decreased the area of myocardial infarction and attenuated pathological damage in ischemia-reperfusion (I/R) rats. In addition, the activities of myocardial injury markers were decreased by PTS. Finally, PTS regulated nuclear translocation of Nrf2, and ML385 blocked the therapeutic effect of PTS in vivo and in vitro. These results suggested that PTS has therapeutic potential for MIRI by targeting Keap1/Nrf2 activity.

Evaluation of mitochondrial oxidative toxicity in mammalian cardiomyocytes by determining the highly reproducible and reliable increase in mitochondrial superoxides after exposure to therapeutic drugs

Mitochondria are important cytoplasmic elements present in eukaryotic cells, and are involved in converting energy to ATP through oxidative phosphorylation. Mitochondria are vulnerable to reactive oxygen species (ROS), thereby making it imperative to evaluate the toxicity. However, existing methods that evaluate mitochondrial toxicity in cardiomyocytes are limited. In the current study, we aimed to determine a mitochondrial biomarker that measures the toxicity of mitochondria, and subsequently suggest an efficient evaluation system for evaluating mitochondrial-specific oxidative toxicity. To achieve this, AC16 human cardiomyocytes, H9C2 rat cardiomyocytes were exposed to acetaminophen (AP), amiodarone hydrochloride (AMD), doxorubicin hydrochloride (Dox), valproic acid sodium salt (Val), and (Z)-4-hydroxytamoxifen (4-OHT). Mitochondrial oxidative stress was determined by staining the drug-treated cells with MitoSOX™ red fluorescence dye, followed by imaging with a fluorescence microscope. All working concentrations of Dox showed increased levels of red fluorescence in AC16 and H9C2 cells, whereas exposure to Val did not alter the red fluorescence level of both cells. Considering our results, increased MitoSOX™ subsequent to drug exposure is a highly reproducible and reliable method to measure the mitochondrial-specific oxidative toxicity. These results indicate that a screening system using MitoSOX™ has the potential to be applied as a reliable biomarker for determining mitochondrial oxidative toxicity in new drug development.

Patchouli alcohol protects against myocardial ischaemia–reperfusion injury by regulating the Notch1/Hes1 pathway

Context Patchouli alcohol (PA) has protective effects on cerebral ischaemia/reperfusion (I/R) injury, but its efficacy on myocardial ischaemia-reperfusion (MI/R) has yet to be addressed. Objective To examine the therapeutic effect of PA on myocardial ischaemia-reperfusion (I/R) injury. Materials and methods C57BL/6 male mice were randomly divided into sham, MI/R, MI/R + PA-10, MI/R + PA-20 and MI/R + PA-40 groups. In vivo MI/R model was established by ligating the anterior descending coronary artery of the heart. In vitro stimulated IR cell model was constructed by using the rat cardiomyocyte H9C2 cell line. Mice in the treatment groups were intraperitoneally injected with PA (10, 20, 40 mg/kg) for 30 days then subjected to surgery, and cells in the experimental group were pre-treated with PA (1, 10 or 100 μmol/L). After treatment, mouse heart function, myocardial injury markers, myocardial infarction and Notch1/Hes1 expression, endoplasmic reticulum stress markers, and apoptosis-related proteins were determined. Results In vivo, PA treatment improved hemodynamic parameter changes and myocardial enzymes, increased the left ventricular ejection fraction and left ventricular fractional shortening, reduced the left ventricular end-systolic diameter and inhibited CK-MB, cTnI and cTnT levels. In addition, PA attenuated myocardial tissue damage and apoptosis. PA treatment elevated Notch1, NICD and Hes1 levels and suppressed the levels of ATF4, p-PERK/PERK, and cleaved caspase-3/caspase-3 in vitro and in vivo. Discussion and conclusion PA protects against MI/R, possibly by modulating ER stress, apoptosis and the Notch1/Hes1 signalling pathways. These findings indicate that PA may be a promising candidate for treating ischaemic heart diseases.

Establishment of a platform for measuring mitochondrial oxygen consumption rate for cardiac mitochondrial toxicity.

The heart has an abundance of mitochondria since cardiac muscles require copious amounts of energy for providing continuous blood through the circulatory system, thereby implying that myocardial function is largely reliant on mitochondrial energy. Thus, cardiomyocytes are susceptible to mitochondrial dysfunction and are likely targets of mitochondrial toxic drugs. Various methods have been developed to evaluate mitochondrial toxicity by evaluating toxicological mechanisms, but an optimized and standardized assay for cardiomyocytes remains unmet. We have therefore attempted to standardize the evaluation system for determining cardiac mitochondrial toxicity, using AC16 human and H9C2 rat cardiomyocytes. Three clinically administered drugs (acetaminophen, amiodarone, and valproic acid) and two anticancer drugs (doxorubicin and tamoxifen) which are reported to have mitochondrial effects, were applied in this study. The oxygen consumption rate (OCR), which directly reflects mitochondrial function, and changes in mRNA levels of mitochondrial respiratory complex I to complex V, were analyzed. Our results reveal that exposure to all five drugs results in a concentration-dependent decrease in the basal and maximal levels of OCR in AC16 cells and H9C2 cells. In particular, marked reduction in the OCR was observed after treatment with doxorubicin. The reduction in OCR after exposure to mitochondrial toxic drugs was found to be associated with reduced mRNA expression in the mitochondrial respiratory complexes, suggesting that the cardiac mitochondrial toxicity of drugs is majorly due to dysfunction of mitochondrial respiration. Based on the results of this study, we established and standardized a protocol to measure OCR in cardiomyocytes. We expect that this standardized evaluation system for mitochondrial toxicity can be applied as basic data for establishing a screening platform to evaluate cardiac mitochondrial toxicity of drugs, during the developmental stage of new drug discovery.

Dexmedetomidine protects H9C2 rat cardiomyocytes against hypoxia/reoxygenation injury by regulating the long non-coding RNA colon cancer-associated transcript 1/microRNA-8063/Wnt/β-catenin axis

ABSTRACT Dexmedetomidine (Dex) protects the heart from ischemia/reperfusion (I/R) injury. The differential expression of long non-coding RNAs (lncRNAs) is associated with myocardial injury, but whether the lncRNA colon cancer-associated transcript 1 (CCAT1) is associated with Dex-mediated myocardial protection remains unclear. In this study, a hypoxia/reoxygenation (H/R) H9C2 model was established to simulate the in vitro characteristics of I/R. CCAT1 and microRNA (miR)-8063 expression levels in H/R H9C2 cells pretreated with Dex were determined via quantitative reverse transcription-polymerase chain reaction. The survival and apoptotic rates of H9C2 cells were determined via cell counting kit-8 and flow cytometry assays. Wnt3a, Wnt5a, and β-catenin protein levels were measured via western blotting. Luciferase and RNA immunoprecipitation assays were used to explore the binding relationship between miR-8063 and CCAT1. Dex pretreatment increased H/R H9C2 cell viability and CCAT1 expression, while decreasing the cell apoptosis and Wnt3a, Wnt5a, and β-catenin protein levels. Knockdown of CCAT1 abolished the protective effects of Dex on H/R H9C2 cells, and the downregulation of miR-8063 expression eliminated the effect of CCAT1 knockdown. These results revealed that CCAT1, a sponge for miR-8063, is involved in Dex-mediated H9C2 cell H/R injury by negatively targeting miR-8063 and inactivating the Wnt/β-catenin pathway. Dex protects H9C2 cells from H/R impairment by regulating the lncRNA CCAT1/miR-8063/Wnt/β-catenin axis.

Momordica. charantia-Derived Extracellular Vesicles-Like Nanovesicles Protect Cardiomyocytes Against Radiation Injury via Attenuating DNA Damage and Mitochondria Dysfunction.

Thoracic radiotherapy patients have higher risks of developing radiation-induced heart disease (RIHD). Ionizing radiation generates excessive reactive oxygens species (ROS) causing oxidative stress, while Momordica. charantia and its extract have antioxidant activity. Plant-derived extracellular vesicles (EVs) is emerging as novel therapeutic agent. Therefore, we explored the protective effects of Momordica. charantia-derived EVs-like nanovesicles (MCELNs) against RIHD. Using density gradient centrifugation, we successfully isolated MCELNs with similar shape, size, and markers as EVs. Confocal imaging revealed that rat cardiomyocytes H9C2 cells internalized PKH67 labeled MCELNs time-dependently. In vitro assay identified that MCELNs promoted cell proliferation, suppressed cell apoptosis, and alleviated the DNA damage in irradiated (16 Gy, X-ray) H9C2 cells. Moreover, elevated mitochondria ROS in irradiated H9C2 cells were scavenged by MCELNs, protecting mitochondria function with re-balanced mitochondria membrane potential. Furthermore, the phosphorylation of ROS-related proteins was recovered with increased ratios of p-AKT/AKT and p-ERK/ERK in MCELNs treated irradiated H9C2 cells. Last, intraperitoneal administration of MCELNs mitigated myocardial injury and fibrosis in a thoracic radiation mice model. Our data demonstrated the potential protective effects of MCELNs against RIHD. The MCELNs shed light on preventive regime development for radiation-related toxicity.

An Olive Oil Mill Wastewater Extract Improves Chemotherapeutic Activity Against Breast Cancer Cells While Protecting From Cardiotoxicity.

Cardiovascular toxicity in cancer patients receiving chemotherapy remains one of the most undesirable side effects, limiting the choice of the most efficient therapeutic regimen, including combinations of different anticancer agents. Anthracyclines (doxorubicin) and antimetabolites (5-fluorouracil (5-FU), capecitabine) are among the most known agents used in breast cancer and other neoplasms and are associated with cardiotoxic effects. Extra-virgin olive oil (EVOO) is rich in polyphenols endowed with antioxidant cardioprotective activities. Olive mill wastewater (OMWW), a waste product generated by EVOO processing, has been reported to be enriched in polyphenols. In this study, we investigated the activities of polyphenol-rich extract from OMWW, A009, in cooperation with chemotherapy on two breast cancer cell lines, namely, BT459 and MDA-MB-231, in a cardio-oncology perspective. The effects of A009 on cardiac cells were also investigated with and without chemotherapeutic agents. Cell viability was determined on BT459 and MDA-MB-231 (i.e., breast cancer cells) and H9C2 (i.e., rat cardiomyocytes) cells, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A spheroids assay was used as a 3D in vitro model on BT459 and MDA-MB-231 cells. For in vivo studies, the murine sponge assay of angiogenesis was used as a model of breast cancer-associated vascularization. The embryo of Danio rerio (zebrafish) was used to detect the cardioprotective activities of the OMWW. We found that the A009 extract exhibited antiangiogenic activities induced by breast cancer cell supernatants and increased T-cell recruitment in vivo. The combination of the OMWW extracts with doxorubicin or 5-FU limited BT459 and MDA-MB-231 cell viability and the diameter of 3D spheroids, while mitigating their toxic effects on the rat H9C2 cardiomyocytes. Cardioprotective effects were observed by the combination of OMWW extracts with doxorubicin in zebrafish embryos. Finally, in human cardio myocytes, we observed 5-FU-induced upregulation of the inflammatory, senescence-associated cytokine IL6 and p16 genes, which expression was reduced by OMWW treatment. Our study demonstrates that the polyphenol-rich purified OMWW extract A009 combined with cancer chemotherapy could represent a potential candidate for cardiovascular protection in breast cancer patients, while increasing the effects of breast cancer chemotherapy.

Micropatterned conductive elastomer patch based on poly (glycerol sebacate)-graphene for cardiac tissue repair

Preparing a micropatterned elastomer film with characteristics that can simulate the mechanical properties, anisotropy, and electroactivity of natural myocardial tissues is crucial in cardiac tissue engineering after myocardial infarction (MI). Therefore, in this study, we developed several elastomeric films with a surface micropattern based on poly (glycerol sebacate) (PGS) and graphene (Gr). These films have sufficient mechanical strength (0.6 ± 0.1–3.2 ± 0.08 MPa) to withstand heartbeats, and the micropatterned structure also satisfies the natural myocardium anisotropy in the transverse and vertical. Moreover, Gr makes these films conductive (up to 5.80 × 10−7 S m−1), which is necessary for the conduction of electrical signals between cardiomyocytes and the cardiac tissue. Furthermore, they have good cytocompatibility and can promote cell proliferation in H9c2 rat cardiomyocyte cell lines. In vivo test results indicate that these films have good biocompatibility. Notably, a film with 1 wt% Gr content (PGS–Gr1) significantly affects the recovery of myocardial function in rats after MI. This film effectively decreased the infarct size and degree of myocardial fibrosis and reduced collagen deposition. Echocardiographic evaluation showed that after treatment with this film, the left ventricular internal dimension (LVID) in systole and LVID in diastole of rats exhibited a significant downward trend, whereas the fractional shortening and ejection fraction were significantly increased compared with the control group. These data indicate that this electroactive micropatterned anisotropic elastomer film can be applied in cardiac tissue engineering.

Minocycline relieves myocardial ischemia-reperfusion injury in rats by inhibiting inflammation, oxidative stress and apoptosis.

Myocardial ischemia-reperfusion (I/R) injury (MIRI) is an important cause of irreversible injury to the myocardium in patients with acute myocardial infarction. The purpose of this study was to investigate the effects of minocycline (MC) on inflammation, oxidative stress and apoptosis of myocardial tissues. We used rats to establish MIRI model by ligating coronary arteries. The structure and function of rat myocardium were determined by 2, 3, 5-triphenyl tetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining and echocardiography. In addition, we detected the expression of inflammatory factors, antioxidant enzymes and apoptosis-related molecules in rats by enzyme-linked immunosorbent assay (ELISA), immunohistochemical (IHC) staining and reverse transcription-polymerase chain reaction (RT-PCR) to determine the effect of MC on inflammation, oxidative stress and apoptosis in I/R rats. Finally, we studied the effect of MC stimulation on the viability of rat cardiomyocytes (H9c2 cells) in vitro. After I/R, the heart function of rats decreased, and the structure of myocardium was destroyed. The levels of inflammation and oxidative stress in I/R rats also increased significantly, manifested by increased inflammatory factors and decreased antioxidant enzymes in serum and myocardial tissue. After treatment of I/R rats with MC, the structure and function of rat myocardium improved significantly, and MC reduced inflammation and oxidative stress levels in rats, thus inhibiting the apoptosis of cardiomyocytes. MC also improved the viability of H9c2 cells in vitro. MC reduced inflammation and oxidative stress levels in MIRI rat model or H9c2 cells, thus inhibiting cardiomyocyte apoptosis. Therefore, MC has potential application prospects for the treatment of MIRI.

Protective effect of resveratrol on rat cardiomyocyte H9C2 cells injured by hypoxia/reoxygenation by regulating mitochondrial autophagy PTEN-induced putative kinase protein 1/Parkinson disease protein 2 signaling pathway.

To investigate the protective effect of resveratrol on cardiomyocytes after hypoxia/ reoxygenation intervention based on PTEN-induced putative kinase protein 1/Parkinson disease protein 2 (PINK1/PARKIN) signaling pathway. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide was used to detect the effect of resveratrol on the viability of H9C2 cells; the hypoxia/ reoxygenation (H/R) model was established in tri-gas incubator; 2', 7'-Dichlorofluorescin diacetate staining was used to measure the content of reactive oxygen species (ROS); the changes of mitochondrial membrane potential was determined by 5,5',6,6'-Tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide staining; the changes of mitochondrial respiratory chain complex activity was evaluated by enzyme activity kits; flow cytometry was used to detect the ratio of apoptotic cells; transmission electron microscope was used to observe the ultrastructure of H9C2 cells; Western blot was used to detect the protein changes of mitochondrial 20 kDa outer membrane protein (TOM20), translocase of inner mitochondrial membrane 23 (TIM23), presenilins associated rhomboid-like protein (PARL), PINK1, PARKIN and mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), phosphotyrosine independent ligand for the Lck SH2 domain of 62 kDa (P62), microtubule-associated protein 1 light chain 3 beta (LC3B); the mRNA levels of PINK1 and PARKIN was detected by quantitative polymerase chain reaction; immunoprecipitation assay was used to detect the interaction between PARKIN and Ubiquitin. Resveratrol could inhibit the proliferation of H9C2 cells in a time- and concentration- dependent manner; however, pretreatment with low cytotoxic resveratrol could reduce the H/R-induced increase in cellular ROS levels, alleviate the loss of mitochondrial membrane potential induced by H/R, inhibit H/R-induced apoptosis of H9C2 cells, and protect the mitochondrial structure and respiratory chain of H9C2 cells from H/R damage. Resveratrol could further increase the levels of p62, PINK1, PARKIN protein, the expression of PINK1, PARKIN mRNA and the ratio of LC3BⅡ/LC3BⅠin H/R-induced H9C2 cells, inhibit the interaction between PARKIN and Ubiquitin in H/R-induced H9C2 cells, and further reduce the expression of TOM20,TIM23, PARL, Mfn1 and Mfn2 protein in H/R-induced H9C2 cells. The effect of resveratrol is consistent with that of autophagy activator on H/R-induced H9C2 cells. Resveratrol can protect H9C2 cells from H/R injury, which may be related to resveratrol promoting mitochondrial autophagy by activating PINK1/PARKIN signaling pathway.

Smilax glabra Roxb. flavonoids protect against pathological cardiac hypertrophy by inhibiting the Raf/MEK/ERK pathway: In vivo and in vitro studies

Ethnopharmacological relevanceSmilax glabra Roxb., the dry rhizome of Sarsaparilla, which is also known as Tu fuling (TFL) in China, is a well-known traditional CHINESE medicine that is widely used for detoxication, relieving dampness and as a diuretic. We have previously shown that the extracted TFL flavonoids (designated TFLF) possess anti-cardiac hypertrophy effects in vitro. However, the anti-cardiac hypertrophy effects of TFLF in vivo and the underlying mechanisms remain to be elucidated. Aim of the studyTo reveal the underlying therapeutic mechanism of TFLF on cardiac hypertrophy by using transverse aortic constriction (TAC) model and cellular assays in vitro. Material & methodsCardiac hypertrophy was replicated by TAC surgery in rats or by isoprenaline treatment of rat H9C2 myocardial cells in vitro. Cardiac structure and function were evaluated by echocardiographic and hemodynamic examinations in vivo and histological analysis of tissues ex vivo. Biochemical kits and quantitative PCR were used to analyze markers of cardiac hypertrophy. Expression and phosphorylation of key proteins in the Raf/MEK/ERK pathway were quantified by Western blotting. We further confirmed our findings in H9C2 rat cardiomyocytes treated with isoprenaline and the ERK inhibitor in vitro. ResultsTFLF attenuated cardiac hypertrophy and fibrosis and improved cardiac dysfunction in TAC rats. TFLF treatment induced a strong reduction in serum NT-proBNP levels. Cardiac hypertrophy marker gene (ANP, BNP and β-MHC) expression and the phosphorylation levels of c-Raf and ERK1/2 were decreased by TFLF treatment. TFLF also protected H9C2 cells from isoprenaline-induced hypertrophy in vitro via a similar molecular mechanism as that observed in the rat heart. Moreover, pretreatment with TRLF and the ERK inhibitor further inhibited the mRNA overexpression of hypertrophic genes in vitro. ConclusionsTFLFs may protect against pathological cardiac hypertrophy via negative regulation of the Raf/MEK/ERK pathway. Thus, TFLFs are implicated as a potential pharmacological agent for treating cardiac hypertrophy in clinical practice.

Direct cardio-protection of Dapagliflozin against obesity-related cardiomyopathy via NHE1/MAPK signaling.

Obesity is an important independent risk factor for cardiovascular diseases, remaining an important health concern worldwide. Evidence shows that saturated fatty acid-induced inflammation in cardiomyocytes contributes to obesity-related cardiomyopathy. Dapagliflozin (Dapa), a selective SGLT2 inhibitor, exerts a favorable preventive activity in heart failure. In this study, we investigated the protective effect of Dapa against cardiomyopathy caused by high fat diet-induced obesity in vitro and in vivo. Cultured rat cardiomyocyte H9c2 cells were pretreated with Dapa (1, 2.5 μM) for 1.5 h, followed by treatment with palmitic acid (PA, 200 μM) for 24 h. We showed that Dapa pretreatment concentration-dependently attenuated PA-induced cell hypertrophy, fibrosis and apoptosis. Transcriptome analysis revealed that inhibition of PA-activated MAPK/AP-1 pathway contributed to the protective effect of Dapa in H9c2 cells, and this was confirmed by anti-p-cJUN fluorescence staining assay. Using surface plasmon resonance analysis we found the direct binding of Dapa with NHE1. Gain and loss of function experiments further demonstrated the role of NHE1 in the protection of Dapa. In vivo experiments were conducted in mice fed a high fat diet for 5 months. The mice were administered Dapa (1 mg·kg-1·d-1, i.g.) in the last 2 months. Dapa administration significantly reduced the body weight and improved the serum lipid profiles. Dapa administration also alleviated HFD-induced cardiac dysfunction and cardiac aberrant remodeling via inhibiting MAPK/AP-1 pathway and ameliorating cardiac inflammation. In conclusion, Dapa exerts a direct protective effect against saturated fatty acid-induced cardiomyocyte injury in addition to the lowering effect on serum lipids. The protective effect results from negative regulating MAPK/AP-1 pathway in a NHE1-dependent way. The current study highlights the potential of clinical use of Dapa in the prevention of obesity-related cardiac dysfunction.

Maltol mitigates cisplatin‐evoked cardiotoxicity via inhibiting the PI3K/Akt signaling pathway in rodents in vivo and in vitro

Our current research aims to evaluate the efficiency of a flavor enhancer, maltol (produced by heating ginseng) against cisplatin-evoked cardiotoxicity by establishing cisplatin-induced heart injury in vivo and H9C2 rat cardiomyocyte model. The cisplatin-treated mice at 3mg/kg for four times on the 7th, 9th, 11th and 13th day, and in them appeared a serious cardiac damage accompanied with the increase in indicators of heart damage. Multiple exposure of 3mg/kg for four times of cisplatin increased cardiac cells apoptosis with increased expression of Bax and cleaved-caspase 3, and decreased expression of Bcl-2. Interestingly, supplement of maltol at doses of 50 and 100 mg/kg for 15 days significantly suppressed the cardiac disturbance. In cultured H9C2 cells, maltol enhanced PI3K/Akt expression level during cisplatin treatment, and reduced cisplatin-induced apoptosis. Notably, inhibition of PI3K/Akt by LY294002 and HY-10249A lessened the efficacy of maltol. In mice, maltol apparently induced PI3K/Akt in heart tissues and protected against cisplatin-induced cardiotoxicity. In conclusion, maltol exerted the protective effects against cisplatin-induced cardiotoxicity, at least partially by inhibiting the activation of PI3K/Akt signaling pathways in cardiomyocytes, to ease oxidative stress, and alleviate reactive oxygen species-mediated apoptosis.

Mitochondrial ROS-Mediated Metabolic and Cytotoxic Effects of Isoproterenol on Cardiomyocytes Are p53-Dependent and Reversed by Curcumin.

Acute β-adrenergic stimulation contributes to heart failure. Here, we investigated the role of p53 in isoproterenol (ISO)-mediated metabolic and oxidative stress effects on cardiomyocytes and explored the direct protective effects offered by the antioxidant nutraceutical curcumin. Differentiated H9C2 rat cardiomyocytes treated with ISO were assayed for glucose uptake, lactate release, and mitochondrial reactive oxygen species (ROS) generation. Survival was assessed by sulforhodamine B assays. Cardiomyocytes showed significantly decreased glucose uptake and lactate release, as well as increased cellular toxicity by ISO treatment. This was accompanied by marked dose-dependent increases of mitochondria-derived ROS. Scavenging with N-acetyl-L-cysteine (NAC) effectively lowered ROS levels, which completely recovered glycolytic metabolism and survival suppressed by ISO. Mechanistically, ISO reduced extracellular-signal-regulated kinase (ERK) activation, whereas it upregulated p53 expression in an ROS-dependent manner. Silencing of p53 with siRNA blocked the ability of ISO to stimulate mitochondrial ROS and suppress glucose uptake, and partially recovered cell survival. Finally, curcumin completely reversed the metabolic and ROS-stimulating effects of ISO. Furthermore, curcumin improved survival of cardiomyocytes exposed to ISO. Thus, ISO suppresses cardiomyocyte glycolytic metabolism and survival by stimulating mitochondrial ROS in a p53-dependent manner. Furthermore, curcumin can efficiently rescue cardiomyocytes from these adverse effects.

CTRP3 alleviates cardiac ischemia/reperfusion injury via LAMP1/JIP2/JNK signaling pathway.

Background: C1q/tumor necrosis factor-related protein 3 (CTRP3) has been reported to be a crucial regulator in myocardial infarction. Nevertheless, the potential molecular mechanism of CTRP3 in ischemia/reperfusion (I/R) injury remains largely unclear.Methods: The cell model of myocardial I/R injury was established by oxygen-glucose deprivation/reoxygenation (OGD/R) of rat cardiomyocyte H9C2. Expression of CTRP3 and lysosomal-associated membrane protein 1 (LAMP1) was detected in H9C2 cells treated with oxygen-glucose deprivation/reoxygenation (OGD/R). H9C2 cells were transfected with overexpression plasmids of CTRP3 (pcDNA-CTRP3) and LAMP1 (pcDNA-LAMP1), or CTRP3 small interfering RNA (si-CTRP3) or/and pcDNA-LAMP1, and cell proliferation, apoptosis and oxidative stress were testified. Co-IP assay was performed to validate the relationship among CTRP3, LAMP1 and JIP2. The role of CTRP3 and LAMP1 in JIP2/JNK pathway was evaluated with Western blot assay. Furthermore, in vivo myocardial I/R injury model was constructed to investigate the effect of CTRP3.Results: Overexpression of CTRP3 and LAMP1 both significantly promoted cell proliferation, inhibited apoptosis and the production of reactive oxygen species (ROS), malondialdehyde (MAD) and cardiac troponin (cTn-I), while silencing CTRP3 exerted the opposite effects, and LAMP1 overexpression reversed the effect of silencing CTRP3 on the aspects above. CTRP3 interacted with LAMP1, and both CTRP3 and LAMP1 bound with JIP2. SP600125 (JNK inhibitor) could restore the effects of CTRP3 or LAMP1 overexpression on the expression of JIP2 and phosphorylated-JNK (p-JNK), proliferation and apoptosis. Moreover, overexpression of CTRP3 improved cardiac I/R injury in vivo.Conclusion: CTRP3 alleviates cardiac I/R injury by elevating LAMP1 and activating JIP2/JNK signaling pathway, which may serve as a potential therapeutic target for I/R injury.