Murine HL-1 Cardiomyocytes Research Articles

Cardiomyocyte-targeting exosomes from sulforaphane-treated fibroblasts affords cardioprotection in infarcted rats

BackgroundExosomes (EXOs), tiny extracellular vesicles that facilitate cell–cell communication, are being explored as a heart failure treatment, although the features of the cell source restrict their efficacy. Fibroblasts the most prevalent non-myocyte heart cells, release poor cardioprotective EXOs. A noninvasive method for manufacturing fibroblast-derived exosomes (F-EXOs) that target cardiomyocytes and slow cardiac remodeling is expected. As a cardioprotective isothiocyanate, sulforaphane (SFN)-induced F-EXOs (SFN-F-EXOs) should recapitulate its anti-remodeling properties.MethodsExosomes from low-dose SFN (3 μM/7 days)-treated NIH/3T3 murine cells were examined for number, size, and protein composition. Fluorescence microscopy, RT-qPCR, and western blot assessed cell size, oxidative stress, AcH4 levels, hypertrophic gene expression, and caspase-3 activation in angiotensin II (AngII)-stressed HL-1 murine cardiomyocytes 12 h-treated with various EXOs. The uptake of fluorescently-labeled EXOs was also measured in cardiomyocytes. The cardiac function of infarcted male Wistar rats intramyocardially injected with different EXOs (1·1012) was examined by echocardiography. Left ventricular infarct size, hypertrophy, and capillary density were measured.ResultsSustained treatment of NIH/3T3 with non-toxic SFN concentration significantly enhances the release of CD81 + EXOs rich in TSG101 (Tumor susceptibility gene 101) and Hsp70 (Heat Shock Protein 70), and containing maspin, an endogenous histone deacetylase 1 inhibitor. SFN-F-EXOs counteract angiotensin II (AngII)-induced hypertrophy and apoptosis in murine HL-1 cardiomyocytes enhancing SERCA2a (sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a) levels more effectively than F-EXOs. In stressed cardiomyocytes, SFN-F-EXOs boost AcH4 levels by 30% (p < 0.05) and significantly reduce oxidative stress more than F-EXOs. Fluorescence microscopy showed that mouse cardiomyocytes take in SFN-F-EXOs ~ threefold more than F-EXOs. Compared to vehicle-injected infarcted hearts, SFN-F-EXOs reduce hypertrophy, scar size, and improve contractility.ConclusionsLong-term low-dose SFN treatment of fibroblasts enhances the release of anti-remodeling cardiomyocyte-targeted F-EXOs, which effectively prevent the onset of HF. The proposed method opens a new avenue for large-scale production of cardioprotective exosomes for clinical application using allogeneic fibroblasts.

Hdac1-deficiency affects the cell cycle axis Cdc25-Cdk1 causing impaired G2/M phase progression and reduced cardiomyocyte proliferation in zebrafish

Zebrafish have the ability to fully regenerate their hearts after injury since cardiomyocytes subsequently dedifferentiate, re-enter cell cycle, and proliferate to replace damaged myocardial tissue. Recent research identified the reactivation of dormant developmental pathways during cardiac regeneration in adult zebrafish, suggesting pro-proliferative pathways important for developmental heart growth to be also critical for regenerative heart growth after injury.Histone deacetylase 1 (Hdac1) was recently shown to control both, embryonic as well as adult regenerative cardiomyocyte proliferation in the zebrafish model. Nevertheless, regulatory pathways controlled by Hdac1 are not defined yet. By analyzing RNA-seq-derived transcriptional profiles of the Hdac1-deficient zebrafish mutant baldrian, we here identified DNA damage response (DDR) pathways activated in baldrian mutant embryos. Surprisingly, although the DDR signaling pathway was transcriptionally activated, we found the complete loss of protein expression of the known DDR effector and cell cycle inhibitor p21. Consequently, we observed an upregulation of the p21-downstream target Cdk2, implying elevated G1/S phase transition in Hdac1-deficient zebrafish hearts. Remarkably, Cdk1, another p21-but also Cdc25-downstream target was downregulated. Here, we found the significant downregulation of Cdc25 protein expression, explaining reduced Cdk1 levels and suggesting impaired G2/M phase progression in Hdac1-deficient zebrafish embryos. To finally prove defective cell cycle progression due to Hdac1 loss, we conducted Cytometer-based cell cycle analyses in HDAC1-deficient murine HL-1 cardiomyocytes and indeed found impaired G2/M phase transition resulting in defective cardiomyocyte proliferation.In conclusion, our results suggest a critical role of Hdac1 in maintaining both, regular G1/S and G2/M phase transition in cardiomyocytes by controlling the expression of essential cell cycle regulators such as p21 and Cdc25.

Chronic insulin resistance deteriorates cardiac mitochondrial quality control in a mouse model of SEC-NAFLD-IR

Abstract Background Insulin resistance and nonalcoholic fatty liver disease (NAFLD) both relate to cardiovascular mortality. Using a mouse model of chronic lipid overload and secondary-NAFLD-induced insulin resistance (SEC-NAFLD-IR), we recently deciphered that SEC-NAFLD-IR already at young age provoked myocardial lipotoxicity with reduced mitochondrial efficiency and increased vulnerability to cardiac ischemia. However, long-term consequences of SEC-NAFLD-IR remain elusive. Purpose Here we aimed to elucidate the impact of long-term SEC-NAFLD-IR on multiple mitochondrial quality control (mQC) mechanisms in the heart and its consequences for cardiac function. Methods We studied 36 SEC-NAFLD-IR mice (72-week-old). For mechanistic experiments, we applied palmitate-induced insulin resistant murine HL-1 cells. Cardiac mitochondrial dynamics were measured via quantification of mitochondrial morphology and expression of mitochondrial fusion and fission factors (Opa1, Drp1, Fis1, Mfn 1 &amp; 2). Mitophagy level was evaluated via immunofluorescence and protein expression of key mitophagy-related genes (Parkin, NIX, LC3). Mitochondrial biogenesis and mass were examined via quantitation of PGC-1α expression, mtDNA and citrate synthase activity. Results 72-week-old SEC-NAFLD-IR mice exhibited 21% (p=0.001) and 32% (p&amp;lt;0.001) higher body weight and heart weight compared with controls. Along with elevated oxidative stress, hepatic lipid accumulation and inflammation, 6h-fasted SEC-NAFLD-IR mice were characterized by increased plasma glucose, insulin and cholesterol. SEC-NAFLD-IR mice displayed a cardiac phenotype with 21% higher left ventricular mass (normalized to body weight, p&amp;lt;0.001) and 6% lower ejection fraction compared to controls (73.5% SEM 0.90 vs 69.4% SEM 1.65, p=0.04). We found several advantageous mQC mechanisms suppressed in aged SEC-NAFLD-IR mice including long form OPA1-mediated mitochondrial fusion, Parkin- and NIX-mediated mitophagy. Likewise, mitochondrial biogenesis was suppressed in the aged insulin-resistant heart, which was connected to a 65% downregulation of PGC-1α1 expression (p=0.01). Interestingly, downregulation of cardiac PGC-1α1 in aged SEC-NAFLD-IR mice coincided with upregulation of PARIS, indicating the crucial participation of the Parkin/PARIS pathway in mQC of the insulin-resistant heart. In addition, induction of insulin resistance in murine HL-1 cardiomyocytes also led to increased mitochondrial fragmentation and decreased PGC-1α1 expression. Conclusion This study demonstrated that regulation of mitochondrial network and turnover is hampered by SEC-NAFLD-IR in the hearts of aged mice, which may contribute to hypertrophy and cardiac dysfunction in insulin resistance. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Collaborative Research Centre 1116 (German Research Foundation)

Cardiac-specific overexpression of Claudin-5 exerts protection against myocardial ischemia and reperfusion injury

Claudin-5 has recently attracted increasing attention by its potential as a novel treatment target in the early stage of heart failure. However, whether Claudin-5 produces beneficial effects on myocardial ischemia and reperfusion (IR) injury has not been elucidated yet. In this study, we identified reduced levels of Claudin-5 in the hearts of mice subjected to acute myocardial IR injury and murine HL-1 cardiomyocytes subjected to hypoxia and reoxygenation (HR). We then constructed cardiac-specific Cldn5-overexpressing mice using an adeno-associated virus (AAV9) vector and demonstrated that Cldn5 overexpression ameliorated cardiac dysfunction and myocardial damage in mice subjected to myocardial IR injury. Moreover, Cldn5 overexpression attenuated myocardial oxidative stress (DHE and protein levels of Nrf2, HO-1, and NQO1), inflammatory response (levels of MPO, F4/80, Ly6C, and circulating inflammatory cells), mitochondrial dysfunction (protein levels of PGC-1α, NRF1, and TFAM), endoplasmic reticulum stress (protein levels of GRP78, ATF6, and CHOP and p-PERK), energy metabolism disorder (p-AMPK and ACC), and apoptosis (TUNEL assay and protein levels of Bax and Bcl2) in mice subjected to myocardial IR. Next, we generated Cldn5 knockdown cells by lentiviral shRNA and observed that Cldn5 knockdown inhibited cell viability and affected the expression or activation of these IR-related signalings in HL-1 cardiomyocytes subjected to HR. Mechanistically, SIRT1 was proved to be involved in regulating the expression of Claudin-5 by co-immunoprecipitation analysis and Sirt1 knockdown experiments. Our data demonstrated that targeting Claudin-5 may represent a promising approach for preventing and treating acute myocardial IR injury.

IL-10 and TGF-β Increase Connexin-43 Expression and Membrane Potential of HL-1 Cardiomyocytes Coupled with RAW 264.7 Macrophages.

Cardiac resident macrophages facilitate electrical conduction by interacting with cardiomyocytes via connexin-43 (Cx43) hemichannels. Cx43 is critical for impulse propagation and coordination between muscle contractions. Cardiomyocyte electrophysiology can be altered when coupled with noncardiomyocyte cell types such as M2c tissue-resident macrophages. Using cocultures of murine HL-1 cardiomyocytes and RAW 264.7 macrophages, we examined the hypothesis that cytokine signals, TGF-β1 and IL-10, upregulate Cx43 expression at points of contact between the two cell types. These cytokine signals maintain the macrophages in an M2c anti-inflammatory phenotype, mimicking cardiac resident macrophages. The electrophysiology of cardiomyocytes was examined using di-8-ANEPPS potentiometric dye, which reflects a change in membrane potential. Greater fluorescence intensity of di-8-ANEPPS occurred in areas where macrophages interacted with cardiomyocytes. Suppressor of cytokine signaling 3 (SOCS3) peptide mimetic downregulated fluorescence of this membrane potentiometric stain. Cx43 expression in cocultures was confirmed by fluorescence microscopy and flow cytometry. Confocal images of these interactions demonstrate the Cx43 hemichannel linkages between the cardiomyocytes and macrophages. These results suggest that TGF-β1 and IL-10 upregulate Cx43 hemichannels, thus enhancing macrophage-cardiomyocyte coupling, raising the cellular resting membrane potential and leading to a more excitatory cardiomyocyte.

PINCH1 knockout aggravates myocardial infarction in mice via mediating the NF‑κB signaling pathway

Myocardial infarction (MI), the leading cause of death among patients with cardiovascular diseases, is characterized by acute cardiac muscle injury due to severe impairment of the coronary blood supply, which may lead to cardiogenic shock and cardiac arrest. Particularly interesting new cysteine histidine rich 1 (PINCH1) protein, a key component of the integrin signaling pathway, interacts with several proteins and serves a vital role in numerous cellular processes, including cytoskeleton remodeling, cell proliferation and cell migration. To investigate the role of PINCH1 in heart injury in the present study, PINCH1 was knocked out in the myocardial tissue of mice (age, 18 weeks) to induce MI. In addition, cell viability, migration and apoptosis, as well as the expression levels of NF-κB-associated proteins were determined in murine HL1 cardiomyocytes with a conditional PINCH1 shRNA using Cell Counting Kit-8, Transwell, flow cytometry and western blot assays, respectively. Furthermore, the cardiac expansion and myocardial fibrosis in PINCH1 knockout mice was investigated in vivo by performing morphological and histological examinations. Additionally, the murine ventricular myocardial ultrastructure was evaluated using an electron microscope, and the cardiomyocyte apoptotic rate and expression levels of NF-κB-related proteins were determined using TUNEL and western blot assays, respectively. The results showed that the apoptotic rate in the in vivo PINCH1 knockdown group was significantly increased. In addition, the protein expression levels of NF-κB signaling pathway-related proteins, including NF-κB, myeloid differentiation factor 88, TNF-α and caspase-3, were significantly increased in the in vivo PINCH1 knockdown group compared with the wild-type group, but the protein expression of MMP2 and MMP9 were the opposite. Overall, the in vitro and in vivo results revealed that PINCH1 knockout in mice significantly aggravated MI via the NF-κB signaling pathway.

Effective Accentuation of Voltage-Gated Sodium Current Caused by Apocynin (4'-Hydroxy-3'-methoxyacetophenone), a Known NADPH-Oxidase Inhibitor.

Apocynin (aPO, 4′-Hydroxy-3′-methoxyacetophenone) is a cell-permeable, anti-inflammatory phenolic compound that acts as an inhibitor of NADPH-dependent oxidase (NOX). However, the mechanisms through which aPO can interact directly with plasmalemmal ionic channels to perturb the amplitude or gating of ionic currents in excitable cells remain incompletely understood. Herein, we aimed to investigate any modifications of aPO on ionic currents in pituitary GH3 cells or murine HL-1 cardiomyocytes. In whole-cell current recordings, GH3-cell exposure to aPO effectively stimulated the peak and late components of voltage-gated Na+ current (INa) with different potencies. The EC50 value of aPO required for its differential increase in peak or late INa in GH3 cells was estimated to be 13.2 or 2.8 μM, respectively, whereas the KD value required for its retardation in the slow component of current inactivation was 3.4 μM. The current–voltage relation of INa was shifted slightly to more negative potential during cell exposure to aPO (10 μM); however, the steady-state inactivation curve of the current was shifted in a rightward direction in its presence. Recovery of peak INa inactivation was increased in the presence of 10 μM aPO. In continued presence of aPO, further application of rufinamide or ranolazine attenuated aPO-stimulated INa. In methylglyoxal- or superoxide dismutase-treated cells, the stimulatory effect of aPO on peak INa remained effective. By using upright isosceles-triangular ramp pulse of varying duration, the amplitude of persistent INa measured at low or high threshold was enhanced by the aPO presence, along with increased hysteretic strength appearing at low or high threshold. The addition of aPO (10 μM) mildly inhibited the amplitude of erg-mediated K+ current. Likewise, in HL-1 murine cardiomyocytes, the aPO presence increased the peak amplitude of INa as well as decreased the inactivation or deactivation rate of the current, and further addition of ranolazine or esaxerenone attenuated aPO-accentuated INa. Altogether, this study provides a distinctive yet unidentified finding that, despite its effectiveness in suppressing NOX activity, aPO may directly and concertedly perturb the amplitude, gating and voltage-dependent hysteresis of INa in electrically excitable cells. The interaction of aPO with ionic currents may, at least in part, contribute to the underlying mechanisms through which it affects neuroendocrine, endocrine or cardiac function.

Myeloperoxidase-Derived 2-Chlorohexadecanal Is Generated in Mouse Heart during Endotoxemia and Induces Modification of Distinct Cardiomyocyte Protein Subsets In Vitro.

Sepsis is a major cause of mortality in critically ill patients and associated with cardiac dysfunction, a complication linked to immunological and metabolic aberrations. Cardiac neutrophil infiltration and subsequent release of myeloperoxidase (MPO) leads to the formation of the oxidant hypochlorous acid (HOCl) that is able to chemically modify plasmalogens (ether-phospholipids) abundantly present in the heart. This reaction gives rise to the formation of reactive lipid species including aldehydes and chlorinated fatty acids. During the present study, we tested whether endotoxemia increases MPO-dependent lipid oxidation/modification in the mouse heart. In hearts of lipopolysaccharide-injected mice, we observed significantly higher infiltration of MPO-positive cells, increased fatty acid content, and formation of 2-chlorohexadecanal (2-ClHDA), an MPO-derived plasmalogen modification product. Using murine HL-1 cardiomyocytes as in vitro model, we show that exogenously added HOCl attacks the cellular plasmalogen pool and gives rise to the formation of 2-ClHDA. Addition of 2-ClHDA to HL-1 cardiomyocytes resulted in conversion to 2-chlorohexadecanoic acid and 2-chlorohexadecanol, indicating fatty aldehyde dehydrogenase-mediated redox metabolism. However, a recovery of only 40% indicated the formation of non-extractable (protein) adducts. To identify protein targets, we used a clickable alkynyl analog, 2-chlorohexadec-15-yn-1-al (2-ClHDyA). After Huisgen 1,3-dipolar cycloaddition of 5-tetramethylrhodamine azide (N3-TAMRA) and two dimensional-gel electrophoresis (2D-GE), we were able to identify 51 proteins that form adducts with 2-ClHDyA. Gene ontology enrichment analyses revealed an overrepresentation of heat shock and chaperone, energy metabolism, and cytoskeletal proteins as major targets. Our observations in a murine endotoxemia model demonstrate formation of HOCl-modified lipids in the heart, while pathway analysis in vitro revealed that the chlorinated aldehyde targets specific protein subsets, which are central to cardiac function.

Mitoxantrone impairs proteasome activity and prompts early energetic and proteomic changes in HL-1 cardiomyocytes at clinically relevant concentrations.

Mitoxantrone (MTX) is used to treat several types of cancers and to improve neurological disability in multiple sclerosis. Unfortunately, cardiotoxicity is a severe and common adverse effect in MTX-treated patients. Herein, we aimed to study early and late mechanisms of MTX-induced cardiotoxicity using murine HL-1 cardiomyocytes. Cells were exposed to MTX (0.1, 1 or 10µM) during short (2, 4, 6, or 12h) or longer incubation periods (24 or 48h). At earlier time points, (6 and 12h) cytotoxicity was already observed for 1 and 10µM MTX. Proteomic analysis of total protein extracts found 14 proteins with higher expression and 26 with lower expression in the cells exposed for 12h to MTX (pH gradients 4-7 and 6-11). Of note, the expression of the regulatory protein 14-3-3 protein epsilon was increased by a factor of two and three, after exposure to 1 and 10µM MTX, respectively. At earlier time-points, 10µM MTX increased intracellular ATP levels, while decreasing media lactate levels. At later stages (24 and 48h), MTX-induced cytotoxicity was concentration and time-dependent, according to the MTT reduction and lactate dehydrogenase leakage assays, while caspase-9, -8 and -3 activities increased at 24h. Regarding cellular redox status, total glutathione increased in 1µM MTX (24h), and that increase was dependent on gamma-glutamylcysteine synthetase activity. Meanwhile, for both 1 and 10µM MTX, oxidized glutathione was significantly higher than control at 48h. Moreover, MTX was able to significantly decrease proteasomal chymotrypsin-like activity in a concentration and time-independent manner. In summary, MTX significantly altered proteomic, energetic and oxidative stress homeostasis in cardiomyocytes at clinically relevant concentrations and our data clearly demonstrate that MTX causes early cardiotoxicity that needs further study.

Evidence for Effective Inhibitory Actions on Hyperpolarization-Activated Cation Current Caused by Ganoderma Triterpenoids, the Main Active Constitutents of Ganoderma Spores.

The triterpenoid fraction of Ganoderma (Ganoderma triterpenoids, GTs) has been increasingly demonstrated to provide effective antioxidant, neuroprotective or cardioprotective activities. However, whether GTs is capable of perturbing the transmembrane ionic currents existing in electrically excitable cells is not thoroughly investigated. In this study, an attempt was made to study whether GTs could modify hyperpolarization-activated cation currents (Ih) in pituitary tumor (GH3) cells and in HL-1 atrial cardiomyocytes. In whole-cell current recordings, the addition of GTs produced a dose-dependent reduction in the amplitude of Ih in GH3 cells with an IC50 value of 11.7 µg/mL, in combination with a lengthening in activation time constant of the current. GTs (10 µg/mL) also caused a conceivable shift in the steady-state activation curve of Ih along the voltage axis to a more negative potential by approximately 11 mV. Subsequent addition of neither 8-cyclopentyl-1,3-dipropylxanthine nor 8-(p-sulfophenyl)theophylline, still in the presence of GTs, could attenuate GTs-mediated inhibition of Ih. In current-clamp voltage recordings, GTs diminished the firing frequency of spontaneous action potentials in GH3 cells, and it also decreased the amplitude of sag potential in response to hyperpolarizing current stimuli. In murine HL-1 cardiomyocytes, the GTs addition also suppressed the amplitude of Ih effectively. In DPCPX (1 µM)-treated HL-1 cells, the inhibitory effect of GTs on Ih remained efficacious. Collectively, the inhibition of Ih caused by GTs is independent of its possible binding to adenosine receptors and it might have profound influence in electrical behaviors of different types of electrically excitable cells (e.g., pituitary and heart cells) if similar in vitro or in vivo findings occur.

ADAMTS13 Deficiency Shortens the Life Span of Mice With Experimental Diabetes.

In patients with diabetes, impaired activity of ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats, member 13), the plasma metalloprotease that cleaves highly thrombogenic von Willebrand factor multimers, is a major risk factor of cardiovascular events. Here, using Adamts13-/- mice made diabetic by streptozotocin, we investigated the impact of the lack of ADAMTS13 on the development of diabetes-associated end-organ complications. Adamts13-/- mice experienced a shorter life span than their diabetic wild-type littermates. It was surprising that animal death was not related to the occurrence of detectable thrombotic events. The lack of ADAMTS13 drastically increased the propensity for ventricular arrhythmias during dobutamine-induced stress in diabetic mice. Cardiomyocytes of diabetic Adamts13-/- mice exhibited an aberrant distribution of the ventricular gap junction connexin 43 and increased phosphorylation of Ca2+/calmodulin-dependent kinase II (CaMKII), and with the consequent CaMKII-induced disturbance in Ca2+ handling, which underlie propensity for arrhythmia. In vitro, thrombospondin 1 (TSP1) promoted, in a paracrine manner, CaMKII phosphorylation in murine HL-1 cardiomyocytes, and ADAMTS13 acted to inhibit TSP1-induced CaMKII activation. In conclusion, the deficiency of ADAMTS13 may underlie the onset of lethal arrhythmias in diabetes through increased CaMKII phosphorylation in cardiomyocytes. Our findings disclose a novel function for ADAMTS13 beyond its antithrombotic activity.

Activation of voltage-gated sodium current and inhibition of erg-mediated potassium current caused by telmisartan, an antagonist of angiotensin II type-1 receptor, in HL-1 atrial cardiomyocytes.

Telmisartan (TEL) is a non-peptide blocker of angiotensin II type-1 (AT1 ) receptor. However, the mechanisms through which this drug interacts directly with ion currents in hearts remain largely unclear. Herein, we aim to investigate the effects of TEL the on ionic currents and membrane potential of murine HL-1 cardiomyocytes. In whole-cell recordings, addition of TEL stimulated the peak and late components of voltage-gated Na+ currents (INa ) with different potencies. The EC50 values required to achieve the stimulatory effect of this drug on peak and late INa were 0.2 and 1.2μmol/L, respectively, and the current-voltage relationship of peak INa shifted toward less-depolarized potentials during exposure to TEL. Telmisartan not only increased peak INa but also prolonged the inactivation time course of late INa . Amiodarone (Amio) or ranolazine (Ran), but not angiotensin II, could reverse TEL-mediated effects. The drug enhanced the recovery rate of INa inactivation and exerted an inhibitory effect on erg-mediated K+ and L-type Ca2+ currents. In whole-cell current-clamp recordings, addition of the drug resulted in prolongation of the duration of action potentials (APs) in a dose-dependent manner in HL-1 cells; Amio or Ran could reverse this increase in AP durations. Telmisartan-mediated prolongation of AP was attenuated in KCNH2 siRNA-transfected HL-1 cells. In cultured smooth muscle cells of the human coronary artery, TEL enhanced INa amplitudes and slowed current inactivation. Stimulation by TEL of INa in HL-1 cells did not simply increase current magnitude but altered current kinetics, thereby suggesting state-dependent activation. Telmisartan may have greater affinity to the open/inactivated state than to the resting state residing in NaV channels. Collectively, TEL-mediated stimulation of INa and inhibition of IK(erg) could be an important ionic mechanism underlying the increased cell excitability of HL-1 cells; these actions, however, cannot be entirely explained by its blockade of AT1 receptor.

H2S and homocysteine control a novel feedback regulation of cystathionine beta synthase and cystathionine gamma lyase in cardiomyocytes

Hydrogen sulfide (H2S), a cardioprotective gas, is endogenously produced from homocysteine by cystathionine beta synthase (CBS) and cystathionine gamma lyase (CSE) enzymes. However, effect of H2S or homocysteine on CBS and CSE expression, and cross-talk between CBS and CSE are unclear. We hypothesize that homocysteine and H2S regulate CBS and CSE expressions in a dose dependent manner in cardiomyocytes, and CBS deficiency induces cardiac CSE expression. To test the hypothesis, we treated murine atrial HL1 cardiomyocytes with increasing doses of homocysteine or Na2S/GYY4137, a H2S donor, and measured the levels of CBS and CSE. We found that homocysteine upregulates CSE but downregulates CBS whereas Na2S/GYY4137 downregulates CSE but upregulates CBS in a dose-dependent manner. Moreover, the Na2S-treatment downregulates specificity protein-1 (SP1), an inducer for CSE, and upregulates miR-133a that targets SP1 and inhibits cardiomyocytes hypertrophy. Conversely, in the homocysteine-treated cardiomyocytes, CBS and miR-133a were downregulated and hypertrophy was induced. In vivo studies using CBS+/−, a model for hyperhomocysteinemia, and sibling CBS+/+ control mice revealed that deficiency of CBS upregulates cardiac CSE, plausibly by inducing SP1. In conclusion, we revealed a novel mechanism for H2S-mediated regulation of homocysteine metabolism in cardiomyocytes, and a negative feedback regulation between CBS and CSE in the heart.

Dietary cyanidin 3-glucoside from purple corn ameliorates doxorubicin-induced cardiotoxicity in mice

Background and aimsAnthracyclines are effective anticancer drugs that have improved prognosis of hundred thousand cancer patients worldwide and are currently the most common chemotherapeutic agents used for the treatment of blood, breast, ovarian and lung cancers. However, their use is limited because of a cumulative dose-dependent and irreversible cardiotoxicity that can cause progressive cardiomyopathy and congestive heart failure. Aim of the present study was to determine the cardioprotective activity of a dietary source of cyanidin 3-glucoside (C3G), such as purple corn, against doxorubicin (DOX)-induced cardiotoxicity in mice. Methods and resultsIn vitro studies on murine HL-1 cardiomyocytes showed that pretreatment with both pure C3G and purple corn extract improved survival upon DOX treatment. However, C3G and purple corn extract did not affect the cytotoxic effect of DOX on human cancer cell lines. We then validated in vivo the protective role of a C3G-enriched diet against DOX-induced cardiotoxicity by comparing the effect of dietary consumption of corn isogenic lines with high levels of anthocyanins (purple corn – Red diet – RD) or without anthocyanins (yellow corn – Yellow diet – YD) incorporated in standard rodent diets. Results showed that mice fed RD survived longer than mice fed YD upon injection of a toxic amount of DOX. In addition, ultrastructural analysis of hearts from mice fed RD showed reduced histopathological alterations. ConclusionDietary intake of C3G from purple corn protects mice against DOX-induced cardiotoxicity.

Abstract 132: A Novel Hydrogen Sulfide (H 2 S) Prodrug, SG1002, Protects Against Myocardial Oxidative Damage and Hypertrophic Signaling via Induction of Cystathionine &amp;#914;-Synthase (CBS) and Antioxidant Proteins

Background: Endogenously produced H 2 S is critical for cardiovascular homeostasis. Therapeutic strategies aimed at increasing H 2 S levels have proven cardioprotective in models of acute myocardial infarction and heart failure (HF). The present study was under taken to investigate the effects of a novel H 2 S prodrug, SG1002, on stress induced hypertrophic signaling in murine HL1 cardiomyocytes. Methods: HL1 cells were maintained either in serum starvation (1%) or serum containing (10%) media followed by treatment either with SG1002 or H 2 O 2 , or endothelin-1 (ET-1)/phenylephrine (Phe) or in combination. Treated cells were analyzed for specific experimental needs. Results: SG1002 significantly increased cellular levels of the H 2 S producing enzyme, CBS, as well as production of H 2 S and nitrosothiol in HL1 cells cultured both in serum starvation or serum containing media. SG1002 significantly inhibited H 2 O 2 and ET-1/Phe induced oxidative stress in both culture media as measured by advanced protein oxidation products and MDA levels. Expression of ANP and BNP were markedly attenuated by SG1002 treatment. Cells cultured in media supplemented with serum containing H 2 O 2 /(ET-1 or Phe) or in 1% serum exhibited decreased levels of CBS, SOD1, and catalase. When the HL1 cells were coincubated with SG1002 cellular damage from oxidative stress was significantly attenuated accompanied by an increase in CBS expression. Conclusion: Our data clearly demonstrate that SG1002 attenuates myocardial cellular damage via increasing antioxidant proteins. SG1002 directly increases H 2 S levels and upregulates CBS. Studies are currently underway to evaluate the clinical utility of SG1002 in HF.

The cytoprotective capacity of processed human cardiac extracellular matrix.

Freshly isolated human cardiac extracellular matrix sheets (cECM) have been shown to support stem cell proliferation and tissue-specific lineage commitment. We now developed a protocol for standardized production of durable, bio-functional hcECM microparticles and corresponding hydrogel, and tested its cytoprotective effects on contractile cells subjected to ischemia-like conditions. Human ventricular myocardium was decellularized by a 3-step protocol, including Tris/EDTA, SDS and serum incubation (cECM). Following snap-freezing and lyophilization, microparticles were created and characterized by laser diffraction, dynamic image analysis (DIA), and mass spectrometry. Moreover, cECM hydrogel was produced by pepsin digestion. Baseline cell-support characteristics were determined using murine HL-1 cardiomyocytes, and the cytoprotective effects of ECM products were tested under hypoxia and glucose/serum deprivation. In cECM, glycoproteins (thrombospondin 1, fibronectin, collagens and nidogen-1) and proteoglycans (dermatopontin, lumican and mimecan) were preserved, but residual intracellular and blood-borne proteins were also detected. The median particle feret diameter was 66μm (15-157μm) by laser diffraction, and 57μm (20-182μm) by DIA with crystal violet staining. HL-1 cells displayed enhanced metabolic activity (39±12%, P<0.05) and proliferation (16±3%, P<0.05) when grown on cECM microparticles in normoxia. During simulated ischemia, cECM microparticles exerted distinct cytoprotective effects (MTS conversion, 240±32%; BrdU uptake, 45±14%; LDH release, -72±7%; P<0.01, each). When cECM microparticles were solubilized to form a hydrogel, the cytoprotective effect was initially abolished. However, modifying the preparation process (pepsin digestion at pH 2 and 25°C, 1mg/ml final cECM concentration) restored the cytoprotective cECM activity. Extracellular matrix from human myocardium can be processed to yield standardized durable microparticles that exert specific cytoprotective effects on cardiomyocyte-like cells. The use of processed cECM may help to optimize future clinical-grade myocardial tissue engineering approaches.

Phosphoinositide-3-kinase/akt - dependent signaling is required for maintenance of [Ca2+]i,ICa, and Ca2+ transients in HL-1 cardiomyocytes

The phosphoinositide 3-kinases (PI3K/Akt) dependent signaling pathway plays an important role in cardiac function, specifically cardiac contractility. We have reported that sepsis decreases myocardial Akt activation, which correlates with cardiac dysfunction in sepsis. We also reported that preventing sepsis induced changes in myocardial Akt activation ameliorates cardiovascular dysfunction. In this study we investigated the role of PI3K/Akt on cardiomyocyte function by examining the role of PI3K/Akt-dependent signaling on [Ca2+]i, Ca2+ transients and membrane Ca2+ current, ICa, in cultured murine HL-1 cardiomyocytes. LY294002 (1–20 μM), a specific PI3K inhibitor, dramatically decreased HL-1 [Ca2+]i, Ca2+ transients and ICa. We also examined the effect of PI3K isoform specific inhibitors, i.e. α (PI3-kinase α inhibitor 2; 2–8 nM); β (TGX-221; 100 nM) and γ (AS-252424; 100 nM), to determine the contribution of specific isoforms to HL-1 [Ca2+]i regulation. Pharmacologic inhibition of each of the individual PI3K isoforms significantly decreased [Ca2+]i, and inhibited Ca2+ transients. Triciribine (1–20 μM), which inhibits AKT downstream of the PI3K pathway, also inhibited [Ca2+]i, and Ca2+ transients and ICa. We conclude that the PI3K/Akt pathway is required for normal maintenance of [Ca2+]i in HL-1 cardiomyocytes. Thus, myocardial PI3K/Akt-PKB signaling sustains [Ca2+]i required for excitation-contraction coupling in cardiomyoctyes.

The actions of mdivi-1, an inhibitor of mitochondrial fission, on rapidly activating delayed-rectifier K+ current and membrane potential in HL-1 murine atrial cardiomyocytes

Mdivi-1 is an inhibitor of dynamin related protein 1- (drp1)-mediated mitochondrial fission. However, the mechanisms through which this compound interacts directly with ion currents in heart cells remain unknown. In this study, its effects on ion currents and membrane potential in murine HL-1 cardiomyocytes were investigated. In whole-cell recordings, the addition of mdivi-1 decreased the amplitude of tail current (Itail) for the rapidly activating delayed-rectifier K+ current (IKr) in a concentration-dependent manner with an IC50 value at 11.6μM, a value that resembles the inhibition requirement for mitochondrial division. It shifted the activation curve of Itail to depolarized voltages with no change in the gating charge. However, mdivi-1 did not alter the rate of recovery from current inactivation. In cell-attached configuration, mdivi-1 inside the pipette suppressed the activity of acetylcholine-activated K+ channels without modifying the single-channel conductance. Mdivi-1 (30μM) slightly depressed the peak amplitude of Na+ current with no change in the overall current–voltage relationship. Under current-clamp recordings, addition of mdivi-1 resulted in prolongation for the duration of action potentials (APs) and to increase the firing of spontaneous APs in HL-1 cells. Similarly, in pituitary GH3 cells, mdivi-1 was effective in directly suppressing the amplitude of ether-à-go-go-related gene-mediated K+ current. Therefore, the lengthening of AP duration and increased firing of APs caused by mdivi-1 can be primarily explained by its inhibition of these K+ channels enriched in heart cells. The observed effects of mdivi-1 on ion currents were direct and not associated with its inhibition of mitochondrial division.

Characterisation of electrophysiological conduction in cardiomyocyte co-cultures using co-occurrence analysis

Cardiac arrhythmias are disturbances of the electrical conduction pattern in the heart with severe clinical implications. The damage of existing cells or the transplantation of foreign cells may disturb functional conduction pathways and may increase the risk of arrhythmias. Although these conduction disturbances are easily accessible with the human eye, there is no algorithmic method to extract quantitative features that quickly portray the conduction pattern. Here, we show that co-occurrence analysis, a well-established method for feature recognition in texture analysis, provides insightful quantitative information about the uniformity and the homogeneity of an excitation wave. As a first proof-of-principle, we illustrate the potential of co-occurrence analysis by means of conduction patterns of cardiomyocyte–fibroblast co-cultures, generated both in vitro and in silico. To characterise signal propagation in vitro, we perform a conduction analysis of co-cultured murine HL-1 cardiomyocytes and murine 3T3 fibroblasts using microelectrode arrays. To characterise signal propagation in silico, we establish a conduction analysis of co-cultured electrically active, conductive cardiomyocytes and non-conductive fibroblasts using the finite element method. Our results demonstrate that co-occurrence analysis is a powerful tool to create purity–conduction relationships and to quickly quantify conduction patterns in terms of co-occurrence energy and contrast. We anticipate this first preliminary study to be a starting point for more sophisticated analyses of different co-culture systems. In particular, in view of stem cell therapies, we expect co-occurrence analysis to provide valuable quantitative insight into the integration of foreign cells into a functional host system.

Gata4 and Sp1 regulate expression of the erythropoietin receptor in cardiomyocytes

Experimental studies indicate significant cardioprotective effects of recombinant erythropoietin (Epo) by binding to the Epo receptor (EpoR) and by inducing various molecular mechanisms, including activation of Gata4, a transcription factor that induces anti-apoptotic genes. However, specific molecular mechanisms of EpoR regulation in cardiomyocytes are unknown. We identified a 774 bp regulatory domain in the EpoR 5′ flanking region by reporter gene assays in murine HL-1 cardiomyocytes. The binding sites for Gata and Sp transcription factors both significantly contributed to EpoR promoter activity. DNA-binding studies (EMSA and ChIP assays) identified Gata4 and Sp1 as EpoR promoter-binding proteins in HL1 cardiomyocytes. Although Sp1 alone stimulates EpoR only slightly, forced expression of Gata4 significantly induced EpoR mRNA expression. In addition, knockdown of Gata4 (but also of Sp1) resulted in a significant decrease of EpoR transcript levels in HL-1 cardiomyocytes. Cumulative in vitro data suggest that function of the Sp1 site is essential for the Gata4-mediated transcription. In vivo, analysis of transgenic mice expressing an inducible small-hairpin RNA against Gata4 confirmed suppression of EpoR expression in the heart. Treating mice with high-dose doxorubicin not only resulted in Gata4 protein depletion, but also down-regulated EpoR, followed by up-regulation of EpoR transcripts when Gata4 levels recovered. In conclusion, we identified Gata4 as novel regulator of EpoR transcription in cardiomyocytes. In models of cardiac injury, down-regulation of Gata4 or Sp1 may limit the accessibility of the EpoR for binding of erythropoiesis-stimulating agents (ESA). Thereby our data underline the essential role of Gata4 in mediating cardioprotective effects.