H9c2 Cardiomyocytes Research Articles

Exogenous 8-hydroxydeoxyguanosine attenuates doxorubicin-induced cardiotoxicity by decreasing pyroptosis in H9c2 cardiomyocytes

Doxorubicin (DOX), which is widely used in cancer treatment, can induce cardiomyopathy. One of the main mechanisms whereby DOX induces cardiotoxicity involves pyroptosis through the NLR family pyrin domain containing 3 (NLRP3) inflammasome and gasdermin D (GSDMD). Increased NAPDH oxidase (NOX) and oxidative stress trigger pyroptosis. Exogenous 8-hydroxydeoxyguanosine (8-OHdG) decreases reactive oxygen species (ROS) production by inactivating NOX. Here, we examined whether 8-OHdG treatment can attenuate DOX-induced pyroptosis in H9c2 cardiomyocytes. Exposure to DOX increased the peroxidative glutathione redox status and NOX1/2/4, toll-like receptor (TLR)2/4, and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) expression, while an additional 8-OHdG treatment attenuated these effects. Furthermore, DOX induced higher expression of NLRP3 inflammasome components, including NLRP3, apoptosis-associated speck-like protein containing a c-terminal caspase recruitment domain (ASC), and pro-caspase-1. Moreover, it increased caspase-1 activity, a marker of pyroptosis, and interleukin (IL)-1β expression. All these effects were attenuated by 8-OHdG treatment. In addition, the expression of the cardiotoxicity markers, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was increased by DOX, whereas the increase of ANP and BNP induced by DOX treatment was reversed by 8-OHdG. In conclusion, exogenous 8-OHdG attenuated DOX-induced pyroptosis by decreasing the expression of NOX1/2/3, TLR2/4, and NF-κB. Thus, 8-OHdG may attenuate DOX-induced cardiotoxicity through the inhibition of pyroptosis.

Cardiac-specific ITCH overexpression ameliorates septic cardiomyopathy via inhibition of the NF-κB signaling pathway

Septic cardiomyopathy is a common complication of septic shock and organ dysfunction. ITCH is a HECT (homologous to the E6-AP carboxyl-terminus)-type ubiquitin E3 ligase that plays a critical role in inflammatory suppression. Herein, we focused on the interaction between ITCH and key regulators of nuclear factor-κB (NF-κB), such as tumor necrosis factor receptor-associated factor 6 (TRAF6) and transforming growth factor-β activated kinase 1 (TAK1), and examined the impact of ITCH on the development of septic cardiomyopathy. In H9C2 cardiomyocytes, ITCH protein expression decreased in response to lipopolysaccharide (LPS) and tumor necrosis factor alpha (TNFα). The protein interactions of ITCH with TRAF6 and TAK1 were confirmed by immunoprecipitation in vitro and in vivo . Based on overexpression and knockdown studies of ITCH in H9C2 cardiomyocytes, ITCH regulates the phosphorylation of NF-κB and subsequent interleukin 6 ( IL-6 ) expression in response to LPS and TNFα stimulation. LPS was intraperitoneally injected into transgenic mice with cardiac-specific overexpression of ITCH (ITCH-Tg) and wild-type (WT) mice. Compared with WT mice, phosphorylation of NF-κB and subsequent IL-6 expression were inhibited in ITCH-Tg mice. Cardiac systolic dysfunction after LPS administration was ameliorated in ITCH-Tg mice, and the survival rate was higher in ITCH-Tg mice than in WT mice. ITCH interacts with TRAF6 and TAK1 in cardiomyocytes and improves cardiac function and survival rates in septic cardiomyopathy by suppressing the NF-κB pathway. • ITCH interacted with TRAF6 and TAK1 in cardiomyocytes. • ITCH modulates activation of NFκB in cardiomyocytes. • Cardiac overexpression of ITCH preserves cardiac function in septic cardiomyopathy. • Cardiac overexpression of ITCH improves survival rate in septic cardiomyopathy. • ITCH plays a protective role in the development of septic cardiomyopathy.

Hyperpolarised magnetic resonance spectroscopy of α-[1-13C]-ketoisocaproic acid reveals reamination of branched chain ketoacids in H9c2 cardiomyocytes in the presence of ketone bodies but not L-glutamate, and this reamination is increased in the presence of the SGLT-2 inhibitor Empagliflozin

Hyperpolarised magnetic resonance spectroscopy of α-[1-13C]-ketoisocaproic acid reveals reamination of branched chain ketoacids in H9c2 cardiomyocytes in the presence of ketone bodies but not L-glutamate, and this reamination is increased in the presence of the SGLT-2 inhibitor Empagliflozin

Diverse lignans with protective effect against hypoxia/oxidative injuries to H9c2 cells from Syringa pinnatifolia Hemsl.

A bioactivity-guided fractionation on the phenolic fractions from the peeled stems of Syringa pinnatifolia Hemsl., one of representative Mongolian folk medicine in China, led to the isolation and structural determination of 11 undescribed lignans and 12 known ones. These lignans cover diverse types, among them syringanones A and B represent an unprecedented carbon skeleton (proposed syringanane) and alashanenol A possesses a rare bicyclo [3.3.1]nonadienemethanol core. Their structures were established by extensive spectroscopic data analysis, X-ray diffraction, and quantum chemical calculations. All isolates were evaluated for their cardioprotective activities on H9c2 cardiomyocytes in vitro. The results showed that five lignans exhibited the protective effects against hypoxia-induced injury at the concentrations of 1.2–40 μM and six lignans exhibited anti-oxidative stress injury at 10–40 μM. These findings account to some extend for the traditional therapeutic effects of S. pinnatifolia for the treatment of ischemic heart diseases in clinic.

Protective efficacy of Shenge San on mitochondria in H9c2 cardiomyocytes.

To investigate whether peroxisome proliferator-activated receptor γ-coactivator-1α/nuclear respiratory factor 1 (PGC-1α/NRF1) activity can protect mitochondrial function in the setting of cardiac hypertrophy and improve cardiomyocyte energy metabolism. Cardiac hypertrophy was modeled in H9c2 cells treated with isoproterenol (ISO) to assess the effects of Shenge San (, SGS) on cell viability and mitochondrial membrane potential. We assessed mitochondrial complex mRNA levels and mitochondrial oxidative phosphorylation factor mRNA and protein levels. Compared with the 100 μM ISO group, cell size was significantly decreased in the 0.3 mg/mL SGS and 20 μM ZLN005 (PGC-1α activator) groups ( < 0.01). Compared with the SGS (0.3) +ISO group, we observed lower phosphorylated adenosine monophosphate-activated kinase (AMPK) protein levels in the ISO and ZLN005+SGS+ISO groups ( < 0.01). Compared with the compound C group, SGS significantly increased PGC-1α expression in ISO-induced cardiac hypertrophy cells ( < 0.01), and this was inhibited by compound C pretreatment ( < 0.05). Compared with the ISO group, the mitochondrial red-green fluorescence ratio increased in the 0.3 mg/mL SGS group ( < 0.05). mRNA levels of cytochrome c oxidase subunit 1 (CO1) in the ISO and compound C groups were lower than those in control group ( 0.01), and the mRNA levels of CO1 and ATP8 were significantly lower in the ISO and compound C groups versus control ( 0.01). Compared with the SGS (0.3) +ISO group, ATP synthetase subunit 8 (ATP8) mRNA was significantly decreased in the ISO group ( < 0.01) and compound C+SGS+ISO group ( < 0.05). Compared with the SGS (0.3) +ISO group, NRF1 mRNA levels were significantly decreased ( < 0.05) in the ISO and compound C+SGS+ISO groups. SGS can attenuate ISO-induced cardiomyocyte hypertrophy, restore the decrease in mitochondrial membrane potential, and upregulate PGC-1α/NRF1 levels. Notably, these effects can be blocked by AMPK inhibitor-compound C.

A novel peptide HSP-17 ameliorates oxidative stress injury and apoptosis in H9c2 cardiomyocytes by activating the PI3K/Akt pathway.

Oxidative stress and cell apoptosis play pivotal roles in the pathogenesis of doxorubicin (DOX)-induced myocardial injury. Heat shock protein-derived peptide (HSP-17) is a peptide which is low-expressed in DOX treated mouse heart tissue. It has high bioactivity and interspecies sequence consistency, and is predicted to have myocardial protective effect. Firstly, we added 1 µM DOX to H9c2 cell culture medium for 24 hours to construct the myocardial cytotoxicity model. Then we detected the effect of HSP-17 on DOX induced H9c2 cardiomyocyte injury by measuring cell viability and lactate dehydrogenase (LDH) level. In addition, reactive oxygen species (ROS) and tetraethylbenzimidazolylcarbocyanine iodide kits are used to evaluate the effect of the HSP-17 peptide on DOX-induced oxidative stress injury to cardiomyocytes, and the detection of apoptosis related proteins and flow cytometry were applied to detect the level of apoptosis. Furthermore, the protein expression levels [phosphorylated Akt (p-Akt) and phosphorylated PI3K (p-PI3K)] of the PI3K/Akt pathway were also detected by western blotting. We found that the HSP-17 peptide can increase cell viability, protect mitochondrial potential, reduce LDH levels, and reduce ROS and cardiomyocyte apoptosis. In addition, we also observed that HSP-17 upregulated the expression level of p-Akt, and LY294002, a typical inhibitor of PI3K/Akt, was found to eliminate the protective roles of HSP-17. In conclusion, this study demonstrated that the HSP-17 peptide protected H9c2 cells against oxidative stress and apoptosis via PI3K/Akt pathway activation, which provides a new idea for the treatment of DOX-induced myocardial injury.

Ameliorative effects of Danshensu from the functional food Salvia miltiorrhiza against arsenic trioxide-induced cardiac toxicity in vivo and in vitro: Involvement of inhibiting the AKT/IKK/NF-κB signaling pathway

• Cardiotoxicity of arsenic trioxide limits its clinical utility. • Danshensu has anti-oxidant, anti-inflammatory, and anti-apoptotic activities. • Danshensu shows the protective effects on arsenic trioxide-induced cardiotoxicity. • Protective mechanisms are related to the inhibition of AKT/IKK/NF-κB pathway. Cardiotoxicity is a serious side effect of arsenic trioxide (ATO) which hinders its clinical use in cancer chemotherapy. Danshensu (DSS) has potential applications in the food and healthcare industry to promote cardiovascular health. The experiment aims to investigate the effects of DSS against ATO-induced cardiotoxicity and its possible mechanism. Our results demonstrated that DSS improved cardiac dysfunction, alleviated histopathological damage, enhanced antioxidant activity, attenuated cardiomyocyte apoptosis and suppressed inflammation. Further studies showed that DSS prevented ATO-caused activation of NF-κB by inhibiting the phosphorylation of AKT and IKK, degradation of IκBα, and nuclear translocation of NF-κB. In H9c2 cardiomyocytes, DSS significantly increased cell survival rate and inhibited ATO-mediated expression of p-AKT, p-IKKα/β, p-IκBα, and NF-κB. Notably, the efficacy of DSS was synergistically increased by AKT inhibitor GSK690693. To sum up, DSS effectively ameliorated ATO-induced cardiac toxicity in vivo and in vitro , which may be achieved through inhibition of the AKT/IKK/NF-κB pathway.

Gender Differences in the Correlations Between Immune Cells and Organ Damage Indexes of Acute Myocardial Infarction Patients.

There are clear gender differences in the pathological process and outcome in acute myocardial infarction (AMI) patients but inflammatory responses remain clarified. Here, we aimed to analyse the correlations between inflammatory cells and organ injury parameters in AMI patients and compared between male and female groups. A total of 603 AMI patients who underwent percutaneous coronary intervention (PCI) within 24 hours of the onset were analysed retrospectively. Basic information and hematological parameters detected 6 hours before the PCI were collected, neutrophil-to-lymphocyte ratio (NLR) and monocyte-to-lymphocyte ratio (MLR) were calculated. Renal, liver function indicators, and myocardial enzymes were measured. Left ventricular ejection fraction (EF) and fractional shortening (FS) on days 5-7 after PCI were obtained. Western blot was performed to detect iNOS, eNOS and nNOS expression in H9C2 rat cardiomyocytes treated with IL-6 with and without estrogen and testosterone. WBC, NEU, MON, MLR, CK, ALT and CREA of male patients were significantly higher than females, but FS was lower in females. NEU, MON and MLR were positively correlated with CK, CK-MB, AST, and ALT in males, whereas LYM were correlated with these parameters in female. NEU and NLR were inversely correlated with EF or FS only in female. Estrogen and testosterone reduced IL-6-induced iNOS protein expression in H9C2 cardiomyocytes, estrogen enhanced IL-6-induced nNOS protein expression. NEU, MON, MLR in male AMI patients, and LYM in female patients were associated with organ injury parameters. Estrogen regulation of nitric oxide pathway may mediate the protective effects in female.

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.

Metallothionein-2A Protects Cardiomyocytes from Hypoxia/reper-Fusion through Inhibiting p38.

The reperfusion of coronary artery blood supply is often accompanied by myocardial hypoxia/reperfusion (H/R) injury, and induced cardiomyocytes apoptosis. The activation of p38 can induce apoptosis, thereby aggravating the myocardial H/R injury. Metallothionein-2A (MT2A) has the functions of anti-apoptosis and protective effect through p38. However, it is not clear that MT2A may protect cardiomyocytes from H/R injury through p38 signaling pathway. Here, we constructed an H/R model for H9c2 cardiomyocytes to explore the protective effect of MT2A on cardiomyocytes apoptosis during the process of H/R through p38 signal pathway. The results revealed that both endogenously overexpressed MT2A and exogenously added MT2A can inhibit the active expression of p-p38 and cleaved caspase-3 under H/R. Based on our results, H/R induced cardiomyocytes apoptosis and activation of p38. And, MT2A can inhibit the active expression of caspase-3 and p38. We found that MT2A can protect cardiomyocytes apoptosis from H/R injury through p38 signaling pathway.

Hesperetin ameliorates ischemia/hypoxia-induced myocardium injury via inhibition of oxidative stress, apoptosis, and regulation of Ca2+ homeostasis.

Ischemia/hypoxia (I/H)-induced myocardial injury has a large burden worldwide. Hesperetin (HSP) has a cardioprotective effect, but the molecular mechanism underlying this is not clearly established. Here, we focused on the protective mechanisms of HSP against I/H-induced myocardium injury. H9c2 cardiomyocytes were challenged with CoCl2 for 22 h to imitate hypoxia after treatment groups received HSP for 4h. The viability of H9c2 cardiomyocytes was evaluated, and cardiac function indices, reactive oxygen species, apoptosis, mitochondrial membrane potential (MMP), and intracellular Ca2+ concentration ([Ca2+ ]i ) were measured. L-type Ca2+ current (ICa-L ), myocardial contraction, and Ca2+ transients in isolated ventricular myocytes were also recorded. We found that HSP significantly increased the cell viability, and MMP while significantly decreasing cardiac impairment, oxidative stress, apoptosis, and [Ca2+ ]i caused by CoCl2 . Furthermore, HSP markedly attenuated ICa-L , myocardial contraction, and Ca2+ transients in a concentration-dependent manner. Our findings suggest a protective mechanism of HSP on I/H-induced myocardium injury by restoring oxidative balance, inhibiting apoptosis, improving mitochondrial function, and reducing Ca2+ influx via L-type Ca2+ channels (LTCCs). These data provide a new direction for HSP applied research as a LTCC inhibitor against I/H-induced myocardium injury.

Resveratrol alleviated 5-FU-induced cardiotoxicity by attenuating GPX4 dependent ferroptosis

The clinical use of 5-fluorouracil (5-FU), a potent antitumor agent, was limited by severe cardiotoxic effects. The present study was aimed to investigate the protective effects of resveratrol (Res) on 5-FU-induced cardiotoxicity and to explore its potential mechanisms.The cardiotoxicity model was intraperitoneal injection of 5-FU at the dose of 30 mg/kg for 7 consecutive days. Plasma enzymes activities, cardiac tissues were assessed after treatment with Res for 3 weeks. Ferrostatin-1 (Fer-1) was used as ferroptosis inhibitor. In H9c2 cardiomyocyte cells, cell viability, generation of reactive oxygen species (ROS), mitochondrial activity and cellular Fe2+ levels were measured. Western-blot assay was performed to evaluate the protein level of ferroptosis in vitro and in vivo. In the mice model, Res reduced 5-FU-induced cardiomyocyte injury (ferroptosis, myofibrillar loss and vacuolization). In addition, increased serum creatine kinase (CK), lactate dehydrogenase (LDH), malonaldehyde (MDA) and Fe2+ activity and decreased activities of glutathione (GSH) were observed in 5-FU group. These changes were prevented by treatment with Res. In H9c2 cardiomyocyte cells, Res increased the cell viability and attenuated cell ferroptosis as measured by DCFH-DA, TMRE and Calcein AM staining. In addition, 5-FU induced a reduction in GPX4, FTH1, Nrf2 and NQO1 and activation of TfR and P53 compared with the control group. However, Res effectively inhibited the changes in ferroptosis associated proteins in vitro and in vivo. Res possessed the cardioprotective potential against 5-FU induced cardiotoxicity. Moreover, Res attenuates 5-FU-induced cardiotoxicity via inhibiting GPX4 dependent ferroptosis.

Myosin 1b Participated in the Modulation of Hypoxia/Reoxygenation-Caused H9c2 Cell Apoptosis and Autophagy.

Myocardial ischemia/reperfusion (I/R) injury seriously threats the health and life of patients with ischemia heart disease. Herein, we probed the potential influence of myosin 1b (myo1b) on hypoxia/reoxygenation- (H/R-) stimulated cardiomyocyte H9c2 cell apoptosis and autophagy. After H/R stimulation, the myo1b mRNA level in H9c2 cells was tested via qRT-PCR. Myo1b overexpression plasmid (OE-myo1b) and small interfering RNA (siRNA) targeting myo1b (si-myo1b) were transfected into H9c2 cells to alter myo1b expression in H9c2 cells. Following H/R stimulation and/or OE-myo1b (or si-myo1b) transfection, H9c2 cell apoptosis, proliferation, and autophagy were detected, respectively. We found that H/R stimulation reduced the mRNA level of myo1b in H9c2 cells and resulted in H9c2 cell apoptosis, proliferation inhibition, and autophagy. Overexpression of myo1b reversed the H/R-resulted H9c2 cell apoptosis, proliferation inhibition, and autophagy. Silence of myo1b had opposite effects, which promoted H9c2 cell apoptosis, reduced cell proliferation, and accelerated cell autophagy. Taken together, Myo1b took part in the modulation of H/R-stimulated cardiomyocyte apoptosis and autophagy, which might be serve as a potential endogenous target for prevention and therapy of I/R injury.

Astragalus polysaccharides alleviates cardiac hypertrophy in diabetic cardiomyopathy via inhibiting the BMP10-mediated signaling pathway

BackgroundCardiac hypertrophy can lead to cardiac dysfunction and is closely associated with mortality in diabetic cardiomyopathy (DCM). Astragalus polysaccharides (APS) is the main component extracted from Astragalus membranaceus (Fisch.) Bunge (AM), which exhibits anti-hypertrophic effects on cardiomyocytes in various diseases. However, whether APS exerts anti-hypertrophic effects in DCM remains unclear. PurposeTo investigate whether APS can attenuate cardiac hypertrophy in DCM and exert anti-hypertrophic effects by inhibiting the bone morphogenetic protein 10 (BMP10) pathway. MethodsThe anti-hypertrophic effects of APS were studied in high-glucose (HG)-stimulated H9c2 cardiomyocytes and streptozotocin (STZ)-induced DCM rats. BMP10 siRNA was used to inhibit BMP10 expression in H9c2 cardiomyocytes. Cardiac function was assessed by echocardiography. Cardiac hypertrophy was evaluated using heart weight/body weight (HW/BW), RT-PCR, hematoxylin-eosin (HE), and rhodamine phalloidin staining. Changes in hypertrophic components, including BMP10 and downstream factors, were measured using western blotting. ResultsIn vitro, HG treatment increased the relative cell surface area of H9c2 cardiomyocytes, whereas BMP10 siRNA transfection or APS treatment alleviated the increase induced by HG. APS treatment improved the general condition, increased cardiac function, and decreased the HW/BW ratio, ANP mRNA level, and cardiomyocyte cross-sectional area of DCM rats in vivo. Molecular experiments demonstrated that APS downregulated the levels of the pro-hypertrophic protein BMP10 and its downstream proteins ALK3, BMPRII, and p-Smad1/5/8 without affecting the level of total Smad1/5/8. ConclusionsOur study demonstrates that APS can alleviate cardiac hypertrophy and protect against DCM by inhibiting activation of the BMP10 pathway. APS is a promising candidate for DCM treatment.

Labdane conjugates protect cardiomyocytes from doxorubicin-induced cardiotoxicity.

The cardiovascular side effects associated with doxorubicin (DOX), a wide spectrum anticancer drug, have limited its clinical application. Therefore, to explore novel strategies with cardioprotective effects, a series of new labdane conjugates were prepared (6a-6cand8a-8d) from the natural diterpene labdanodiol (1). These hybrid compounds contain anti-inflammatory privileged structures such as naphthalimide, naphthoquinone, and furanonaphthoquinone. Biological activity of these conjugates against DOX-induced cardiotoxicity was tested in vitro and the potential molecular mechanisms of protective effects were explored in H9c2 cardiomyocytes. Three compounds 6c, 8a, and 8b significantly improved cardiomyocyte survival, via inhibition of reactive oxygen species-mediated mitogen-activated protein kinasesignaling pathways (extracellular signal-regulated kinase and c-Jun N-terminal kinase) and autophagy mediated by Akt activation. Some structure-activity relationships were outlined, and the best activity was achieved with the labdane-furonaphthoquinone conjugate 8a having anN-cyclohexyl substituent. The findings of this study pave the way for further investigations to obtain more compounds with potential cardioprotective activity.

Dietary Nε-(carboxymethyl) lysine affects cardiac glucose metabolism and myocardial remodeling in mice.

Myocardial remodeling is a key factor in the progression of cardiovascular disease to the end stage. In addition to myocardial infarction or stress overload, dietary factors have recently been considered associated with myocardial remodeling. Nε-(carboxymethyl)lysine (CML) is a representative foodborne toxic product, which can be ingested via daily diet. Therefore, there is a marked need to explore the effects of dietary CML on the myocardium. To explore the effects of dietary CML (dCML) on the heart. C57 BL/6 mice were divided into a control group and a dCML group. The control group and the dCML group were respectively fed a normal diet or diet supplemented with CML for 20 wk. Body weight and blood glucose were recorded every 4 wk. 18F-fluorodeoxyglucose (FDG) was used to trace the glucose uptake in mouse myocardium, followed by visualizing with micro-positron emission tomography (PET). Myocardial remodeling and glucose metabolism were also detected. In vitro, H9C2 cardiomyocytes were added to exogenous CML and cultured for 24 h. The effects of exogenous CML on glucose metabolism, collagen I expression, hypertrophy, and apoptosis of cardiomyocytes were analyzed. Our results suggest that the levels of fasting blood glucose, fasting insulin, and serum CML were significantly increased after 20 wk of dCML. Micro-PET showed that 18F-FDG accumulated more in the myocardium of the dCML group than in the control group. Histological staining revealed that dCML could lead to myocardial fibrosis and hypertrophy. The indexes of myocardial fibrosis, apoptosis, and hypertrophy were also increased in the dCML group, whereas the activities of glucose metabolism-related pathways and citrate synthase (CS) were significantly inhibited. In cardiomyocytes, collagen I expression and cellular size were significantly increased after the addition of exogenous CML. CML significantly promoted cellular hypertrophy and apoptosis, while pathways involved in glucose metabolism and level of Cs mRNA were significantly inhibited. This study reveals that dCML alters myocardial glucose metabolism and promotes myocardial remodeling.

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes via the Whole-Cell Patch-Clamp Technique.

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ion channels, voltage-gated potassium (Kv) channels are closely associated with some serious heart diseases, such as drug-induced myocardial damage and myocardial infarction. In the present study, the whole-cell patch-clamp technique was employed to determine the effects of 1.5 mM 4-aminopyridine (4-AP, a broad-spectrum potassium channel inhibitor) and aconitine (AC, 25 µM, 50 µM, 100 µM, and 200 µM) on the Kv channel current (IKv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the IKv by about 54%, while the inhibitory effect of AC on the IKv showed a dose-dependent trend (no effect for 25 µM, 30% inhibitory rate for 50 µM, 46% inhibitory rate for 100 µM and 54% inhibitory rate for 200 µM). Due to the characteristics of higher sensitivity and precision, this technique will promote the exploration of cardiotoxicity and the pharmacological effects of ethnomedicine targeting ion channels.

Voltage-Dependent Potassium Current Recording on H9c2 Cardiomyocytes &lt;em&gt;via&lt;/em&gt; the Whole-Cell Patch-Clamp Technique

Potassium channels on the myocardial cell membrane play an important role in the regulation of cell electrophysiological activities. Being one of the main ion channels, voltage-gated potassium (Kv) channels are closely associated with some serious heart diseases, such as drug-induced myocardial damage and myocardial infarction. In the present study, the whole-cell patch-clamp technique was employed to determine the effects of 1.5 mM 4-aminopyridine (4-AP, a broad-spectrum potassium channel inhibitor) and aconitine (AC, 25 µM, 50 µM, 100 µM, and 200 µM) on the Kv channel current (IKv) in H9c2 cardiomyocytes. It was found that 4-AP inhibited the IKv by about 54%, while the inhibitory effect of AC on the IKv showed a dose-dependent trend (no effect for 25 µM, 30% inhibitory rate for 50 µM, 46% inhibitory rate for 100 µM and 54% inhibitory rate for 200 µM). Due to the characteristics of higher sensitivity and precision, this technique will promote the exploration of cardiotoxicity and the pharmacological effects of ethnomedicine targeting ion channels.

Abstract 12842: Adipose Tissue Derived Ceramides Regulate the Myocardial Redox State and Predict Cardiovascular Outcomes

Introduction: Ceramides biosynthesis is dysregulated in obesity, yet their role as mediators of obesity-induced myocardial dysfunction is unclear. Objective: We investigate the causal role of AT-derived ceramides in regulating the myocardial redox state and explore their prognostic value in patients with CVD. Methods: In 880 cardiac surgery patients, 20 sphingolipids were measured in plasma, subcutaneous-, thoracic- and epicardial-AT biopsies and their secretomes. Myocardial redox state was measured and the contribution of NOXs, uncoupled eNOS and mitochondrial oxidases to O 2 .- production quantified. Median follow-up was 8.3 years. Genetic variants were identified to predict CVD outcomes using cis-Mendelian Randomisation. The underlying mechanisms were explored using differentiated H9c2 cardiomyocytes in vitro and human myocardial tissue ex vivo . Results: C16:0-ceramide (CerC16) was the most abundant ceramide secreted from AT (p&lt;0.0001). Patients with high plasma CerC16 and C16:0-glucosylceramide (GlcC16) had significantly increased myocardial O 2 .- production (vs low/int.) (A) . Patients with rs112572487 (B) , an intronic SNP in UGCG , had significantly increased plasma GlcC16 (C) and myocardial NOX-derived O 2 .- (D) vs those without. Exogenous CerC16 (20nM) induced O 2 .- production in H9c2 cardiomyocytes and human myocardial tissue (E) , an effect prevented by the UGCG inhibitor D-PDMP. Importantly, high plasma GlcC16 led to a higher risk of cardiac death and/or heart failure (HR=2.128 [1.101, 4.115], p=0.025) vs low/int. levels, a relationship also seen with rs112572487 (F) . rs112572487 also led to an increased risk of new, post-operative atrial fibrillation in hospital (HR=1.805 [1.188, 2.742], p=0.006). Conclusions: We demonstrate for the first time, that AT-derived ceramides are causally related with dysregulated myocardial redox signalling and adverse cardiovascular disease outcomes in patients with advanced atherosclerosis.

Abstract 12308: Presenilin-2 Regulates Mitophagy via Promoting Intracellular Translocation of Parkin to Mitochondria

Introduction: Presenilin-2 (PS2) is a component of the γ-secretase complex, intracellular protease. Variant in PSEN2 , the causative genes of Alzheimer's disease, are also implicated in the development of dilated cardiomyopathy. Hypoxia increases PSEN2 expression 10-fold over normal, and PSEN2 knockout (PS2KO) cells exhibit significantly lower ATP production. However, the mechanism of the mitochondrial dysfunction has not been fully elucidated. Hypothesis: PS2 regulates mitochondrial selective autophagy (mitophagy) by promoting the translocation of Parkin to mitochondria. Methods: Expression of autophagy and mitophagy markers was examined using cultured rat cardiomyocytes (H9c2) silenced for PSEN2 and PSEN1 , exposed to 10 μM carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a mitochondrial uncoupler used to induce mitophagy. Results: Mitochondrial respiration of PS2KO-H9c2 was showed reduced (maximal respiration capacity, 68.4 ± 27.0 pmol/min/cells) compared to PS1KO-H9c2 (147.6 ± 27.2 pmol/min/cells) or normal cells (276.6 ± 11.9 pmol/min/cells) (P&lt;0.001). When mitophagy-dye modified with perylene-3,4-dicarboxylic anhydride was used, mitophagy detection rates were significantly increased by the addition of FCCP to normal H9c2 (34% increased, P=0.041) and PS1KO-H9c2 (46% increased, P=0.035), but there was no change with PS2KO-H9c2 (25% decreased, P=0.232). When FCCP was added to PS2KO-H9c2, Parkin was not only enhanced in whole cell lysate of PS2KO-H9c2, but also increased even at mRNA level; however, in the isolated mitochondrial fraction, Parkin was significantly reduced, and PINK1 and phosphorylated ubiquitin (serine 65) were also attenuated. In Immunofluorescent staining confirmed that Parkin was not co-localized with mitochondria in PS2KO-H9c2, implicating PS2 in promoting the transfer of Parkin from the cytoplasm to mitochondria. As mitochondrial depolarization causes PINK1 stabilization needed for Parkin recruitment, we interrogated mitochondrial membrane potential measurement using Rhodamine-123 and examined expression of OPA1 isoforms; no significant results were obtained. Conclusions: PS2 contributes to the intracellular transport of Parkin to regulate mitophagy.