H9c2 Myoblasts Research Articles

Pyruvate dehydrogenase kinase 1 controls triacylglycerol hydrolysis in cardiomyocytes.

Pyruvate dehydrogenase kinase (PDK) 1 is one of four isozymes that inhibit the oxidative decarboxylation of pyruvate to acetyl-CoA via pyruvate dehydrogenase. PDK activity is elevated in fasting or starvation conditions to conserve carbohydrate reserves. PDK has also been shown to increase mitochondrial fatty acid utilization. In cardiomyocytes, metabolic flexibility is crucial for the fulfillment of high energy requirements. The PDK1 isoform is abundant in cardiomyocytes, but its specific contribution to cardiomyocyte metabolism is unclear. Here we show that PDK1 regulates cardiomyocyte fuel preference by mediating triacylglycerol turnover in differentiated H9c2 myoblasts using lentiviral shRNA to knockdown Pdk1 . Somewhat surprisingly, PDK1 loss did not affect overall PDH activity, basal glycolysis, or glucose oxidation revealed by oxygen consumption rate experiments and 13 C 6 glucose labelling. On the other hand, we observed decreased triacylglycerol turnover in H9c2 cells with PDK1 knockdown, which was accompanied by decreased mitochondrial fatty acid utilization following nutrient deprivation. 13 C 16 palmitate tracing of uniformly labelled acyl chains revealed minimal acyl chain shuffling within triacylglycerol, indicating that the triacylglycerol hydrolysis, and not re-esterification, was dysfunctional in PDK1 suppressed cells. Importantly, PDK1 loss did not significantly impact the cellular lipidome or triacylglycerol accumulation following palmitic acid treatment, suggesting that effects of PDK1 on lipid metabolism were specific to the nutrient-deprived state. We validated that PDK1 loss decreased triacylglycerol turnover in Pdk1 knockout mice. Together, these findings implicate a novel role for PDK1 in lipid metabolism in cardiomyocytes, independent of its canonical roles in glucose metabolism.

Abstract Tu081: T-tubule microdomains promote protective mitophagy in failing hearts

Heart failure (HF) with preserved ejection fraction (HFpEF) represents about 50% of all HF incidence, yet with limited therapeutic options. Although poorly understood, a key pathophysiology of HFpEF includes mitochondrial dysfunction and increased oxidative stress. We recently identified that abnormal remodeling of the cardiomyocyte t-tubule microdomains organized by the scaffolding protein cardiac bridging integrator 1 (cBIN1) contributes to HFpEF progression in db/db mice with type 2 diabetes, which can be rescued by cBIN1 gene therapy. In db/db mice, cBIN1 gene therapy (2x10 12 vg/kg) rescues HFpEF by normalizing t-tubule microdomains. With transmission electron microscopy imaging, here we report that t-tubule cBIN1-microdomains are in close proximity to mitochondria and can correct abnormal mitochondrial accumulation in db/db cardiomyocytes (Figure 1A-B). Oroboros and seahorse analysis indicates that reduced mitochondrial calcium retention, elevated oxidative stress, and impaired oxygen consumption in diabetic cardiomyocytes can be normalized by cBIN1 therapy (Figure 1C-E). Furthermore, proteomics analysis indicates accumulation of mitochondrial proteins in db/db cardiomyocytes can be reversed by the therapy. cBIN1-normalized mitochondrial number, proteomics, and function in db/db cardiomyocytes are associated with activation of LC3 but not changes in PGC1a, indicating increased mitophagy by the therapy. Mechanistic exploration in rat myoblast H9C2 cells indicates that FCCP stress induces mitophagy as evidenced by elevated LC3II/I ratio, which is further accumulated with autophagic flux blockade with chloroquine. Knockdown of BIN1 results in further accumulation of LC3II/I in FCCP-stressed cells (Figure 1F with quantification in the bar graph below), which is abolished by chloroquine. These data indicate that cBIN1 promotes protective mitophagy in diabetic failing hearts, suggesting its therapeutic potential for HFpEF in general.

In vitro characterization of Trypanosoma cruzi infection dynamics in skeletal and cardiac myotubes models suggests a potential cell-to-cell transmission in mediating cardiac pathology.

Chagas disease predominantly affects the heart, esophagus, and colon in its chronic phase. However, the precise infection mechanisms of the causal agent Trypanosoma cruzi in these tissue types remain incompletely understood. This study investigated T. cruzi infection dynamics in skeletal (SM) and cardiac myotubes (CM) differentiated from H9c2(2-1) myoblasts (control). SM and CM were generated using 1% fetal bovine serum (FBS) without or with retinoic acid, respectively. Initial invasion efficiencies and numbers of released parasites were equivalent between undifferentiated and differentiated cells (~0.3-0.6%). Concomitantly, parasite motility patterns were similar across cell lines. However, CM demonstrated significantly higher infection kinetics over time, reaching 13.26% infected cells versus 3.12% for SM and 3.70% for myoblasts at later stages. Cellular automata modeling suggested an enhanced role for cell-to-cell transmission in driving the heightened parasitism observed in CM. The increased late-stage susceptibility of CM, potentially mediated by cell-to-cell transfer mechanisms of the parasite, aligns with reported clinical tropism patterns. The myotube infection models provide novel insights into Chagas disease pathogenesis that are not fully attainable through in vivo examination alone. Expanding knowledge in this area could aid therapeutic development for this neglected illness.

Abstract 2038: Combination therapy leads to decreased viability in cardiomyblasts cells

Abstract Anti-cancer therapies (ACT) have been extremely effective in improving survival in cancer patients; however, they have also been known to cause cardiovascular diseases (CVD) in the same patients. Published research shows that ACTs are toxic to cardiomyocytes through decreased viability. However, they have mainly focused on drugs that are phasing out of clinical practice while newly generated tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs) have rarely been studied, despite their increase in popularity for treating multiple cancers as monotherapies and combination therapies. Additionally, most of the concentrations tested tend to be higher than the concentration that directly interacts with cardiomyocytes, therefore overestimating its effects. The goal of this project is to elucidate whether newly generated TKIs and ICIs, both by itself and in combination, lead to decreased viability in cells. We tested three TKIs (Axitinib, Cabozantinib, and Lenvatinib) and two ICIs (Nivolumab and Pembrolizumab), both solely and together, on rat ventricular myoblasts (H9c2) cells using clinically relevant concentrations by calculating the unbound serum drug concentrations found in cancer patients. After 24 hours, we tested cell viability using the cell counting kit-8 (CCK-8) assay. After 24 hours, neither TKIs nor ICIs by themselves caused decreased viability compared to the control group. However, there were significant decreases in viability when treated with combination of the two, with more than 50% depletion in the Lenvatinib/Pembrolizumab group. Our study indicates although TKIs and ICIs by themselves do not effect viability, they have significant effects after 24 hours when the drugs are combined, even at clinically relevant concentrations. We plan to further characterize whether these drug combinations lead to cardiomyocyte death and through which pathway. Additionally, our lab has successfully differentiated and validated human-induced pluripotent ventricular myocytes (hiPSC-CMs) and testing whether these monotherapies and combination therapies effect viability and functionality on human-induced pluripotent ventricular myocytes (hiPSC-CMs) is currently in progress. Citation Format: Ashley N. Greenlee, Ethan J. Schwendeman, Sakima A. Smith. Combination therapy leads to decreased viability in cardiomyblasts cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2038.

Effects of different cryopreservation parameters on the differences between trypan blue and fluorescent SYTO 13/GelRed assays

Post-thaw cell viability assessment is very important in cryopreservation because it is the main assessment method used to optimize cryopreservation protocols for each cell type; hence, having standardized accurate, quick, and reliable assays for post-thaw cell viability measurements is of utmost importance. The trypan blue exclusion assay and nucleic-acid-binding fluorescence-based assays are two different methods for cell viability assessment. Both assays identify cells with damaged membranes by whether they let a compound enter the cell. In this study, these two assays are compared in the context of cryopreservation and the impacts of important cryopreservation parameters on the differences in measurements are investigated. H9c2 myoblasts were cryopreserved with different freezing protocols. Cell membrane integrities were measured immediately after thaw as well as after cryoprotectant removal by a hemocytometer-based trypan blue dye exclusion assay and a dual fluorometric SYTO 13/GelRed assay; and the results were compared. This study quantifies how (i) the absence or presence of different cryoprotectants, (ii) different cell–cryoprotectant incubation conditions, and (iii) the presence or removal of cryoprotectants after thaw affect the differences between these two viability assays.

Systematic cryopreservation study of cardiac myoblasts in suspension.

H9c2 myoblasts are a cell line derived from embryonic rat heart tissue and demonstrate the ability to differentiate to cardiac myotubes upon reduction of the serum concentration (from 10% to 1%) and addition of all-trans retinoic acid in the growth medium. H9c2 cells are increasingly being used as an easy-to-culture proxy for some functions of cardiomyocytes. The cryobiology of cardiac cells including H9c2 myoblasts has not been studied as extensively as that of some cell types. Consequently, it is important to characterize the cryobiological response and systematically develop well-optimized cryopreservation protocols for H9c2 cells to have optimal and consistent viability and functionality after thaw for high quality studies with this cell type. In this work, an interrupted slow cooling protocol (graded freezing) was applied to characterize H9c2 response throughout the cooling profile. Important factors that affect the cell response were examined, and final protocols that provided the highest post-thaw viability are reported. One protocol uses the common cryoprotectant dimethyl sulfoxide combined with hydroxyethyl starch, which will be suitable for applications in which the presence of dimethyl sulfoxide is not an issue; and the other protocol uses glycerol as a substitute when there is a desire to avoid dimethyl sulfoxide. Both protocols achieved comparable post-thaw viabilities (higher than 80%) based on SYTO 13/GelRed flow cytometry results. H9c2 cells cryopreserved by either protocol showed ability to differentiate to cardiac myotubes comparable to fresh (unfrozen) H9c2 cells, and their differentiation to cardiac myotubes was confirmed with i) change in cell morphology, ii) expression of cardiac marker troponin I, and iii) increase in mitochondrial mass.

Random cellulose acetate nanofibers: a breakthrough for cultivated meat production.

Overcoming the challenge of creating thick, tissue-resembling muscle constructs is paramount in the field of cultivated meat production. This study investigates the remarkable potential of random cellulose acetate nanofibers (CAN) as a transformative scaffold for muscle tissue engineering (MTE), specifically in the context of cultivated meat applications. Through a comparative analysis between random and aligned CAN, utilizing C2C12 and H9c2 myoblasts, we unveil the unparalleled capabilities of random CAN in facilitating muscle differentiation, independent of differentiation media, by exploiting the YAP/TAZ-related mechanotransduction pathway. In addition, we have successfully developed a novel process for stacking cell-loaded CAN sheets, enabling the production of a three-dimensional meat product. C2C12 and H9c2 loaded CAN sheets were stacked (up to four layers) to form a ~300-400 μm thick tissue 2 cm in length, organized in a mesh of uniaxial aligned cells. To further demonstrate the effectiveness of this methodology for cultivated meat purposes, we have generated thick and viable constructs using chicken muscle satellite cells (cSCs) and random CAN. This groundbreaking discovery offers a cost-effective and biomimetic solution for cultivating and differentiating muscle cells, forging a crucial link between tissue engineering and the pursuit of sustainable and affordable cultivated meat production.

Mitochondrial succinate transport as a novel therapeutic target for cardiac ischemia/reperfusion injury

Abstract Background The accumulation of the mitochondrial metabolite succinate during ischemia and its rapid oxidation by succinate dehydrogenase (SDH) upon reperfusion drives ischemia/reperfusion injury (IRI) in myocardial infarction. Succinate oxidation leads to the production of reactive oxygen species (ROS) by reverse electron transport (RET) at mitochondrial complex I, orchestrating a cascade of events leading to cardiomyocyte death. Modulating succinate metabolism, such as SDH inhibition, is a promising therapeutic strategy. During ischemia, the mitochondria produced succinate is transported into the cytosol, enabling the accumulation of a large succinate pool. On reperfusion, the succinate is transported back into mitochondria and oxidised by SDH, thus we hypothesised that targeting succinate mitochondrial transport could be a novel therapeutic target to prevent cardiac IRI. Purpose To assess the role of succinate mitochondrial transport as a novel therapeutic target to prevent cardiac IRI. Methods Isolated heart mitochondria were used to assess mitochondrial respiration by Oroboros, RET-ROS production by Amplex Red and butylmalonate delivery. Cultured H9C2 myoblasts and isolated primary adult cardiomyocytes from C57BL/6J mice were used to assess butylmalonate uptake and delivery from esters, as well as their impact on succinate accumulation and oxidation in vitro. LAD ligation and reperfusion in C57BL/6J mice was used as an in vivo MI model. Metabolite levels were measured by LC-MS/MS. Results Butylmalonate, an inhibitor of the mitochondrial dicarboxylate carrier (DIC), blocks succinate-dependent respiration and RET-ROS production in isolated mitochondria. However, butylmalonate entry into cells was inefficient and does not impact infarct size after IRI in vivo. We developed novel butylmalonate esters to improve butylmalonate delivery to cardiomyocytes in vivo. We found that diacetoxymethyl butylmalonate (DAB) effectively delivered butylmalonate and could prevent succinate accumulation and oxidation in vitro. DAB could also prevent succinate-mediated respiration and RET-ROS at lower concentrations than butylmalonate alone. Finally, DAB reduced acute infarct size in vivo when administered at reperfusion. Conclusions Blocking succinate transport back into mitochondria at reperfusion prevents the damage in IRI by preventing the oxidation of succinate by SDH and downstream RET-ROS production. Therefore, targeting the mitochondrial transport of succinate may be a promising novel strategy in preventing cardiac IRI.

Abstract 15483: Mitoquinone Pretreatment Mitigates Dox-induced Systolic Dysfunction in Isolated Rat Hearts

Background: Doxorubicin (DOX) is a common and effective chemotherapeutic agent that can exert cardiotoxicity by inducing oxidative stress in mitochondria, compromising mitochondrial function, and inducing apoptosis. Currently, there is no treatment that targets this mechanism. We previously found that pretreatment with higher concentrations of mitoquinone (MQ), a mitochondrial antioxidant, exerts cardioprotection against DOX in myoblast H9C2 cells. Hypothesis: We hypothesize that MQ pretreatment would preserve cardiac function against DOX in isolated rat hearts. Methods: Cardiac parameters (heart rate (HR) and left ventricular end systolic pressure (LVESP)) were monitored before and after 60 min infusion of DOX, MQ, or DOX with MQ pretreatment (10-15 min) in a Langendorff preparation of male SD rat hearts. TUNEL assay was conducted on heart tissue slides to evaluate apoptosis. The ratio between the final and initial cardiac parameters was calculated and compared among experimental groups. Results: Higher dose DOX (25 μM, n=5) induced a higher reduction in the ratios of LVESP, to 39±5% than lower dose DOX infusion (20 μM, n=3; 74±3%). DOX had no effects on HR. A 60-minute perfusion of higher doses of MQ (1-5 μM, n=7) unexpectedly reduced the ratios of LVESP and HR to 73±12% and 66±5%, respectively. Higher dose MQ (1-2.5 μM, n=6) pretreatment showed similar improvement of LVESP ratio (68±9%) to lower dose MQ (0.25-0.5 μM, n=6; 71±11%). TUNEL assay showed that higher dose DOX caused a higher rate of apoptosis (number of TUNEL-positive nuclei) (326.3±6.4. n=3) than lower dose (214±29.5, n=3). Pretreatment with higher dose MQ prior to DOX 25 μM infusion resulted in a notable reduction of apoptosis (60.3±5, n=6) compared to DOX alone, while lower dose pretreatment reduced apoptosis to 70.5± 0.5 (n=4). Conclusion: This study suggests that infusion of DOX into the heart acutely attenuated cardiac systolic function accompanied with higher apoptotic cell damage. Although prolonged administration of higher doses of MQ suppressed cardiac function, MQ pretreatment for 10-15 minutes mitigated DOX-induced systolic dysfunction and reduced cardiomyocyte apoptosis. In summary, MQ pretreatment may mitigate DOX-induced cardiac damage.

Pimobendan prevents cardiac dysfunction, mitigates cardiac mitochondrial dysfunction, and preserves myocyte ultrastructure in a rat model of mitral regurgitation

BackgroundPimobendan has been proven to delay the onset of congestive heart failure (CHF) in dogs with mitral regurgitation (MR); however, molecular underlying mechanisms have not been fully elucidated. This study aimed to investigate (1) the effects of pimobendan on cardiac function, cardiac mitochondrial quality and morphology, and cardiac ultrastructure in a rat model of chronic MR and (2) the direct effect of pimobendan on intracellular reactive oxygen species (ROS) production in cardiac cells. MR was surgically induced in 20 Sprague-Dawley rats, and sham procedures were performed on 10 rats. Eight weeks post-surgery, the MR rats were randomly divided into two groups: the MR group and the MR + pimobendan group. Pimobendan (0.15 mg/kg) was administered twice a day via oral gavage for 4 weeks, whereas the sham and MR groups received equivalent volumes of drinking water. Echocardiography was performed at baseline (8 weeks post-surgery) and at the end of the study (4 weeks after treatment). At the end of the study protocol, all rats were euthanized, and their hearts were immediately collected, weighed, and used for transmission electron microscopy and mitochondrial quality assessments. To evaluate the role of pimobendan on intracellular ROS production, preventive or scavenging properties were tested with H2O2-induced ROS generation in rat cardiac myoblasts (H9c2).ResultsPimobendan preserved cardiac functions and structure in MR rats. In addition, pimobendan significantly improved mitochondrial quality by attenuating ROS production and depolarization (P < 0.05). The cardiac ultrastructure and mitochondrial morphology were significantly preserved in the MR + pimobendan group. In addition, pimobendan appeared to play as a ROS scavenger, but not as a ROS preventer, in H2O2-induced ROS production in H9c2 cells.ConclusionsPimobendan demonstrated cardioprotective effects on cardiac function and ultrastructure by preserving mitochondrial quality and acted as an ROS scavenger in a rat model of MR.

The retinoic acid response is a minor component of the cardiac phenotype in H9c2 myoblast differentiation

The H9c2 myoblast cell line, isolated from the left ventricular tissue of rat, is currently used in vitro as a mimetic for skeletal and cardiac muscle due to its biochemical, morphological, and electrical/hormonal signaling properties. During culture, H9c2 cells acquire a myotube phenotype, where a critical component is the inclusion of retinoic acid (RA). The results from some authors on H9c2 suggested that thousands of genes respond to RA stimuli, while others report hundreds of genes responding to RA over different cell types. In this article, using a more appropriate experimental design, we first confirm the H9c2 cardiac phenotype with and without RA and report transcriptomic and physiological changes regarding calcium handling, bioenergetics, and other biological concepts. Interestingly, of the 2360 genes showing a transcriptional change, 622 genes were statistically associated with the RA response. Of these genes, only 305 were RA-specific, and the rest also showed a culture-time component. Thus, the major expression changes (from 74 to 87%) were indeed due to culture conditions over time. Unexpectedly, only a few components of the retinol pathway in KEGG responded to RA. Our results show the role of RA in the H9c2 cultures impacting the interpretation using H9c2 as an in vitro model.

Potentilla reptans L. preconditioning regulates H19 and MIAT long noncoding RNAs in H9C2 myoblasts Ischemia/Reperfusion model

The present study aimed to evaluate the effect of the ethyl acetate fraction of P. reptans root (PEF) preconditioning on expressions of lncRNAs H19 and MIAT in H9C2 myoblasts I/R injury.H9C2 cells were treated with different concentrations ranging from (10–400 µg/ml) of PEF for 24 h, followed by simulation of I/R condition. For I/R experiments, H9C2 cells were subjected with the oxygen and glucose deprivation for 2 h.H9C2 cell viability was significantly enhanced by PEF preconditioning under I/R condition in a concentration-dependent manner up to 200 µg/ml as a EC50. The PEF significantly diminished the expression of lncRNA MIAT and rate of apoptosis against the I/R group. In addition, PEF pretreated before stimulation I/R condition increased H19 expression compared to the normal PEF group with no statistically significant differences between groups. Hence, the results suggest that PEF can protect cardiomyocytes during hypoxia-induced myocardial cell injury by targeting specific involved genes.

Indoxyl sulfate induces left ventricular hypertrophy via the AhR-FGF23-FGFR4 signaling pathway.

Patients with chronic kidney disease (CKD) have a high risk of left ventricular hypertrophy (LVH). Fibroblast growth factor 23 (FGF23) and indoxyl sulfate (IS) are associated with LVH in patients with CKD, but the interactions between these molecules remain unknown. We investigated whether IS contributes to LVH associated with FGF23 in cultured cardiomyocytes and CKD mice. In cultured rat cardiac myoblast H9c2 cells incubated with IS, mRNA levels of the LVH markers atrial natriuretic factor, brain natriuretic peptide, and β-myosin heavy chain were significantly upregulated. Levels of mRNA of the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3), which regulates FGF23 O-glycosylation, and FGF23 were also upregulated in H9c2 cells. Intact FGF23 protein expression and fibroblast growth factor receptor 4 (FGFR4) phosphorylation were increased in cell lysates by IS administration. In C57BL/6J mice with heminephrectomy, IS promoted LVH, whereas the inhibition of FGFR4 significantly reduced heart weight and left ventricular wall thickness in IS-treated groups. While there was no significant difference in serum FGF23 concentrations, cardiac FGF23 protein expression was markedly increased in IS-injected mice. GALNT3, hypoxia-inducible factor 1 alpha, and FGF23 protein expression was induced in H9c2 cells by IS treatment and suppressed by the inhibition of Aryl hydrocarbon receptor which is the receptor for IS. This study suggests that IS increases FGF23 protein expression via an increase in GALNT3 and hypoxia-inducible factor 1 alpha expression, and activates FGF23-FGFR4 signaling in cardiomyocytes, leading to LVH.

Highly Efficient Cardiac Differentiation and Maintenance by Thrombin-Coagulated Fibrin Hydrogels Enriched with Decellularized Porcine Heart Extracellular Matrix.

Biochemical and biophysical properties instruct cardiac tissue morphogenesis. Here, we are reporting on a blend of cardiac decellularized extracellular matrix (dECM) from porcine ventricular tissue and fibrinogen that is suitable for investigations employing an in vitro 3D cardiac cell culture model. Rapid and specific coagulation with thrombin facilitates the gentle inclusion of cells while avoiding sedimentation during formation of the dECM-fibrin composite. Our investigations revealed enhanced cardiogenic differentiation in the H9c2 myoblast cells when using the system in a co-culture with Nor-10 fibroblasts. Further enhancement of differentiation efficiency was achieved by 3D embedding of rat neonatal cardiomyocytes in the 3D system. Calcium imaging and analysis of beating motion both indicate that the dECM-fibrin composite significantly enhances recovery, frequency, synchrony, and the maintenance of spontaneous beating, as compared to various controls including Matrigel, pure fibrin and collagen I as well as a fibrin-collagen I blend.

Doxorubicin induces cardiomyocyte death owing to the accumulation of dysfunctional mitochondria by inhibiting the autophagy fusion process

Doxorubicin (Dox), an anthracycline antibiotic, is an anticancer drug that inhibits DNA replication and cellular metabolic processes in cancer cells with high proliferative potential. However, Dox causes severe side effects, including myocardial damage and heart failure, but the molecular mechanism underlying Dox-induced myocardial injury remains uncertain. In the present study, we evaluated the effects of Dox on the mitochondrial quality control system and regulation of mitochondrial respiration and autophagy in an in vitro rat myoblast H9c2 cell culture model using western blotting, immunohistochemistry, the Seahorse XF24 system, and flow cytometry. Our results showed that Dox did not impair the initiation of autophagic flux or the functions of lysosomes; however, Dox affected the mitochondrial quality control system, leading to a fission-dominant morphology and impaired regulation of mitochondrial respiration, thereby increasing oxidative stress and inhibited progression of autophagy, particularly the fusion of autophagosomes with lysosomes. This inhibition caused a significant decrease in the formation of autolysosomes and was responsible for the accumulation of dysfunctional mitochondria and subsequent increase in oxidative stress, eventually leading to increased myocardial cell death.

Ginsenoside Rb1 from Panax notoginseng Suppressed TNF-α-Induced Matrix Metalloproteinase-9 via the Suppression of Double-Strand RNA-Dependent Protein Kinase (PKR)/NF-κB Pathway.

Chronic inflammation is commonly accompanied by the stimulation of matrix metalloproteinases (MMPs) production and the degradation of the extracellular matrix. The overexpression of MMP-9 (Gelatinase B) highly participates in the progression of pathetic cardiac remodeling and liver cancer metastasis. Panax notoginseng (Burkill) F. H. Chen (Sanqi), a widely used traditional Chinese medicinal herb, shows myocardial protective and anti-tumor effects. In this study, we examined the inhibitory effect of different PNG extracts on tumor necrosis factor (TNF)-α-induced MMP-9 expression in cardiac myoblast H9c2 cells. Using a bioassay-guided fractionation scheme, the most active extract was fractionated by silica gel column chromatography and high-performance liquid chromatography until an active compound was obtained. The compound was identified as Ginsenoside Rb1 by nuclear magnetic resonance. Ginsenoside Rb1 inhibited TNF-α-induced MMP-9 production in both H9c2 and liver carcinoma HepG-2 cells. Interestingly, it did not affect the MMP-2 (Gelatinase A) level and the cell proliferation of the two cell lines. The inhibitory effects of Ginsenoside Rb1 may be due to its modulation of double-strand RNA-dependent protein kinase and nuclear factor kappa B signaling pathways. The results reveal the potential use of Ginsenoside Rb1 for the treatment of inflammatory and MMP-9-related cardiac remodeling and metastasis of hepatocellular carcinomas.

Disruption of mitochondrial bioenergetics, calcium retention capacity and cell viability caused by D-2-hydroxyglutaric acid in the heart

Accumulation of D-2-hydroxyglutaric acid (D-2-HG) is the biochemical hallmark of D-2-hydroxyglutaric aciduria type I and, particularly, of D-2-hydroxyglutaric aciduria type II (D2HGA2). D2HGA2 is a metabolic inherited disease caused by gain-of-function mutations in the gene isocitrate dehydrogenase 2. It is clinically characterized by neurological abnormalities and a severe cardiomyopathy whose pathogenesis is still poorly established. The present work investigated the potential cardiotoxicity D-2-HG, by studying its in vitro effects on a large spectrum of bioenergetics parameters in heart of young rats and in cultivated H9c2 cardiac myoblasts. D-2-HG impaired cellular respiration in purified mitochondrial preparations and crude homogenates from heart of young rats, as well as in digitonin-permeabilized H9c2 cells. ATP production and the activities of cytochrome c oxidase (complex IV), alpha-ketoglutarate dehydrogenase, citrate synthase and creatine kinase were also inhibited by D-2-HG, whereas the activities of complexes I, II and II-III of the respiratory chain, glutamate, succinate and malate dehydrogenases were not altered. We also found that this organic acid compromised mitochondrial Ca2+ retention capacity in heart mitochondrial preparations and H9c2 myoblasts. Finally, D-2-HG reduced the viability of H9c2 cardiac myoblasts, as determined by the MTT test and by propidium iodide incorporation. Noteworthy, L-2-hydroxyglutaric acid did not change some of these measurements (complex IV and creatine kinase activities) in heart preparations, indicating a selective inhibitory effect of the enantiomer D. In conclusion, it is presumed that D-2-HG-disrupts mitochondrial bioenergetics and Ca2+ retention capacity, which may be involved in the cardiomyopathy commonly observed in D2HGA2.

Abstract 12041: Tyrosine Kinase Inhibitors Leads to Sodium Channel Dysfunction in the Heart

Tyrosine Kinase Inhibitors (TKIs) are used by tens of thousands of patients and are efficacious for treating renal cell carcinoma . However, TKIs have been reported to have numerous off-target effects, including arrhythmias and heart failure. Many patients who receive TKIs are over 60 years of age and have existing cardiac diseases, thus increasing their risk of cardiotoxicity. However, the exact mechanism to how these TKIs cause cardiotoxicity remains unclear. Our research goal is to understand how TKIs lead to cardiotoxicity by learning what proteins are being targeted and what are potential upstream regulators so we can introduce possible treatments that can combat these effects. Recent literature has stated that the TKI Sorafenib led to increased reactive oxygen species (ROS) production and Calcium (Ca 2+ )/Calmodulin dependent kinase II (CaMKII) activity in rat ventricular cardiomyocytes. ROS can activate CaMKII, and increased CaMKII activity can lead to hyperphosphorylation of the voltage-gated sodium channel, leading to pathophysiological enhancement of late sodium current (I Na,L ). Increased activity of CaMKII and I Na,L can be seen in arrhythmias and heart failure. We hypothesize that TKIs lead to an increase enhancement of I Na,L due to an increase in ROS production and increased CaMKII activity. To test this hypothesis, we treated H9c2 myoblasts with Pazopanib (n=3) or DMSO (n=3) and wild-type mice with Pazopanib (n=5) or DMSO (n=5). After treatment, we assessed cytosolic and mitochondrial ROS production in vitro and functional affects, CaMKII activity, and I Na,L current in vivo . Results showed that Pazopanib led to a significant increase in ROS production in H9c2 cells and more arrhythmogenic events in WT mice. While CaMKII activity or I Na,L expression was not significantly changed between the two groups, with electrophysiology studies Pazopanib did lead to alterations in amplitude, peak current, ultra-slow inactivation, and recovery. In conclusion, Pazopanib leads to cardiovascular dysfunction and this could be due to an increase in ROS production and altered sodium channels. Our study indicates a potential mechanism as to how TKI treatment leads to cardiotoxicity in patients.

Impaired autophagy and mitochondrial dynamics are involved in Sorafenib-induced cardiomyocyte apoptosis

Sorafenib (Sor), a novel multi-target anticancer drug also induces severe toxicity in heart, while the mechanism of its cardiotoxicity remains to be fully elucidated. Dysregulation of autophagy and mitochondrial dynamics imbalance have been implicated in cardiomyocyte death. The aim of this study is to test the hypothesis that Sor disrupts autophagy and mitochondrial dynamics, thereby aggravating Sor-induced oxidative stress damage to cardiomyocytes. Our results revealed that Sor (≥ 5 μM) concentration- and time-dependently reduced cell viability and induced apoptosis in H9c2 myoblasts. Sor treatment promoted intracellular reactive oxygen species (ROS) generation, and subsequent Ca2+ overload as well as apoptosis, which were abolished by the ROS scavenger MPG. Sor inhibited the basal autophagy activity of cells, as supported by the fact that ERK1/2 inhibition-dependent decreases of autophagosomes and autolysosomes, and p62 accumulation in a concentration- and time-dependent manner. Improving autophagy with rapamycin abrogated Sor-induced ROS and Ca2+ overloads, and cell apoptosis. Furthermore, Sor compromised mitochondrial morphology and caused excessive mitochondrial fragmentation in cells. The imbalance of mitochondrial dynamics was attributed to ROS-mediated CaMKII overactivity, and increased phosphorylation of dynamin-related protein 1 (phosph-Drp1). Suppression of CaMKII with KN-93 or mitochondrial fission with mitochondrial division inhibitor-1 (Mdivi-1) attenuated Sor-induced ROS and Ca2+ overloads as well as apoptosis. In conclusion, these results provide the first evidence that impairments in autophagy and mitochondrial dynamics are involved in Sor-induced cardiomyocyte apoptosis. The present study may provide a potential strategy for preventing or reducing cardiotoxicity of Sor.

BRD4 Silencing Protects Angiotensin II-Induced Cardiac Hypertrophy by Inhibiting TLR4/NF-κB and Activating Nrf2-HO-1 Pathways.

Background Heart failure is a critical health problem worldwide, and cardiac hypertrophy is an important characteristic of heart failure. Bromodomain-containing protein 4 (BRD4) is involved in various cellular processes, including cardiac hypertrophy. This study aimed to investigate the mechanism underlying the effects of BRD4 on cardiac hypertrophy. Methods Rat myoblast H9c2 cells were treated with angiotensin II (Ang II) to increase the mRNA and protein expressions of BRD4. BRD4 was silenced by small interfering RNA (siRNA) in H9c2 cells. Proteins involved in Nrf2-HO-1 pathway were determined by Western blot. Results Our data suggest that BRD4 silencing attenuated Ang II, increased the percentage of TUNEL + cells and caspase-3 activity, increased oxidative stress, and increased the expression and content of pro-inflammatory cytokines. Mechanistically, we found that BRD4 silencing enhanced the protein expressions of Nrf2 and HO-1 and inhibited the TLR4 and phosphorylation of NF-kappa B in Ang II-stimulated H9c2 cells. TLR4 overexpression attenuated cardioprotection against Ang II by BRD4 silencing, including cardiac hypertrophy, oxidative stress, and inflammatory cytokine production. Additionally, TLR4 overexpression attenuated an increase in Nrf2 and HO-1 proteins and decreased phosphorylated NF-kappa B in H9c2 cells. Conclusion Our results speculate that the BRD4/TLR4 axis might be a promising strategy for treating cardiovascular diseases with cardiac hypertrophy, including HF.