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PubMed1999

Nitric oxide induces apoptotic death of cardiomyocytes via a cyclic-GMP-dependent pathway.

Recently, we have reported that excess amounts of nitric oxide (NO) produced by inducible NO synthase are involved in the development of myocardial damage in rats with induced myocarditis. However, there remain many problems to be solved concerning its mechanism of action. In this study, we examined whether NO induces apoptotic cell death in cardiomyocytes. Cultured neonatal rat cardiomyocytes were exposed to S-nitroso-N-acetylpenicillamine (SNAP) and (+/-)-E-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine (NOR 3), as NO donors, or 8-bromo-cyclic GMP (cGMP), an analog of cGMP which functions as a second messenger in cells stimulated by NO. DNA fragmentation was confirmed by electron microscopy, by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method, and by agarose gel electrophoresis. Exogenously supplied SNAP or NOR 3 induced cardiomyocyte apoptosis in a dose- and time-dependent manner. Cardiomyocytes exposed to SNAP displayed typical features of apoptosis as demonstrated by electron microscopy. Treatment of the cells with 8-bromo-cGMP also induced apoptosis. In cardiomyocytes, SNAP-induced apoptosis was completely blocked by a PKG inhibitor (KT5823) and by a soluble guanylate cyclase inhibitor (ODQ) and was suppressed by hemoglobin and was completely blocked by ZVAD-FMK, a caspase inhibitor. These results show that NO-mediated apoptosis of cardiomyocytes is cGMP dependent and that caspases are involved in this process.

Animals↗
PubMed1996

Endotoxin and cytokines induce direct cardiodepressive effects in mammalian cardiomyocytes via induction of nitric oxide synthase.

In patients with septic shock or inflammatory cardiac diseases like myocarditis myocardial contractility is depressed. These patients have elevated circulating levels of bacterial endotoxins (lipopolysaccharides, LPS) and pro-inflammatory cytokines like interleukin-1 beta (IL-alpha 1 beta) or tumor necrosis factor-alpha (TNF-alpha). It is not clear, whether LPS and/or cytokines have direct inotropic effects on cardiomyocytes and whether these effects are mediated via the L-arginine-nitric oxide synthase (NOS) pathway as demonstrated in vascular smooth muscle cells. Therefore, we examined the direct effects of LPS. IL-1 beta and TNF-alpha on contractility and cGMP content in isolated guinea-pig ventricular cardiomyocytes. Furthermore, the influence of the NOS inhibitor NG-nitro-L-arginine (L-NNA) and dexamethasone on these effects was studied as well as inducible NOS (iNOS) protein expression. LPS (1000 ng/ml), IL-1 beta (25 ng/ml) and TNF-alpha (100 ng/ml) decreased contractility by 48%, 55% and 65% and augmented cGMP content by 135%, 88% or 70% after long-term treatment (18 h) in cardiomyocytes, without altering contractility or cGMP content after short-term treatment (30 min). These effects were blocked by L-NNA (100 microM) and dexamethasone (3 microM). Furthermore iNOS protein was expressed in LPS- and cytokine-treated cardiomyocytes. These findings demonstrate that LPS. IL-1 beta and TNF-alpha have direct negative inotropic effects on cardiomyocytes, which are accompanied by an increase in cGMP content. These effects are mediated via de novo synthesis of a myocardial iNOS. The direct negative inotropic effects of endotoxins and cytokines on cardiomyocytes may in part contribute to the contractile dysfunction observed in patients with septic shock or inflammatory cardiac diseases.

Animals↗
PubMed1997

Differential protein expression and subcellular distribution of TGFbeta1, beta2 and beta3 in cardiomyocytes during pressure overload-induced hypertrophy.

The transforming growth factor beta (TGFbeta) superfamily plays an important role in the myocardial response to hypertrophy. We have investigated the protein expression of TGFbeta1, beta2 and beta3 in left ventricular tissue, and determined their subcellular distribution in myocytes by immunoblotting and immunocytochemistry during the development of left ventricular hypertrophy (LVH), using isoform specific antibodies to TGFbeta1, beta2 and beta3. LVH was produced in rats by aortic constriction (AC) and LV tissue was obtained at days (d)0, 1, 3, 7, 14, 21 and 42 following operation. Compared with age matched sham-operated controls (SH), TGFbeta1 levels in LV tissue of AC rats increased significantly from d1-d14 (P<0.03) concomitant with the adaptive growth of LV tissue. In contrast, TGFbeta3 levels decreased in LV tissue of AC rats from d3 post-operation (significant from d14-d42, P<0.03). No significant difference in TGFbeta2 levels were observed from SH and AC rats after operation. Antibodies to TGFbeta1 stained intercalated disks, sarcolemmal membranes and cytoplasm, but not nuclei, of cardiomyocytes on LV sections from untreated and SH rats. However, a trans-localisation of TGFbeta1 to the nuclei of cardiomyocytes was observed in AC hearts. Antibodies to TGFbeta3 stained T tubules, cytoplasm and the nuclei of cardiomyocytes from untreated and SH rats. However, by d7 post-AC operation, TGFbeta3 expression was lost rapidly from nuclei of cardiomyocytes followed by a reduction in total TGFbeta3 immunofluorescence in myocytes. Antibodies to TGFbeta2 stained sarcolemmal membranes of cardiomyocytes from both SH and AC rats without significant difference between groups. Thus, the differential pattern of protein expression and subcellular distribution of TGFbeta1, beta2 and beta3 in myocytes during the development of LVH suggests that these molecules play different roles in the response of cardiomyocytes to LVH.

Animals↗
PubMed1998

Angiotensin II potentiates DNA synthesis in AT-1 transformed cardiomyocytes.

Angiotensin II has been shown to be mitogenic in various cell types. In cultured neonatal cardiomyocytes, we have demonstrated that angiotensin II causes hypertrophy, not hyperplasia. However, fetal or neonatal cardiomyocytes exhibit limited proliferation in primary culture, and are mitotically less potent. In order to determine whether angiotensin II is simply a hypertrophic or hyperplastic growth factor for mitotically-potent cardiomyocytes, we analysed [3H]-thymidine uptake and cell cycle-regulated gene expression using SV40 large T-transformed AT-1 cardiomyocytes. Angiotensin II, alone and in combination with other growth factors, increased [3H]-thymidine uptake in a dose-dependent manner. The mRNA expression of G1 cyclins (Cyclin C, D1, D2, D3) and histone H1-kinase activity by CDK2 increased 6 h after angiotensin II stimulation. Western blot analysis revealed cyclin B1 expression after 18 h , which peaked at 30 h. Histone H1-kinase activity by cdc2 was also increased by angiotensin II, and peaked at 24-36 h, indicating that these changes were cell cycle dependent. Double immunofluorescent photography showed that AT-1 cells incorporated BrdU, and expressed cdc2 by angiotensin II stimulation. [3H]-thymidine and BrdU uptake were blocked by losartan, but not by PD123319. In contrast with neonatal cardiomyocytes, angiotensin II potentiated DNA synthesis and induced cell cycle regulated gene expression in AT-1 cardiomyocytes, and this activity was mediated by the angiotensin II type-1 receptor.

Angiotensin II↗
PubMed2001

Cardiomyocyte grafting for cardiac repair: graft cell death and anti-death strategies.

M. Zhang, D. Methot, V. Poppa, Y. Fujio, K. Walsh and C. E. Murry. Cardiomyocyte Grafting for Cardiac Repair: Graft Cell Death and Anti-Death Strategies. Journal of Molecular and Cellular Cardiology (2001) 33, 907-921. Recent studies indicate that cardiomyocyte grafting forms new myocardium in injured hearts. It is unknown, however, whether physiologically significant amounts of new myocardium can be generated. Pilot experiments showed that death of grafted rat neonatal cardiomyocytes limited formation of new myocardium after acute cryoinjury. Time-course studies showed that, at 30 min after grafting, only 1.8(+/-0.4)% of graft cells were TUNEL-positive. At 1 day, however, TUNEL indices increased to 32.1(+/-3.5)% and remained high at 4 days, averaging 9.8(+/-3.8)%. By 7 days, TUNEL decreased to 1.0(+/-0.2)%. Electron microscopy revealed that dead cells had features of both irreversible ischemic injury and apoptosis. To test whether ischemia contributed to poor graft survival, grafts were placed into vascularized 2-week-old cardiac granulation tissue or normal myocardium. TUNEL indices were reduced by 53% and 86%, respectively. Adenoviral infection of graft cells with the cytoprotective kinase Akt, or constitutively active Akt, reduced TUNEL indices by 31% and 40%, respectively, compared to beta -gal-transfected controls. Neither treatment reached statistical significance compared to untreated controls, however. Heat shock reduced cardiomyocyte death in vitro in response to serum deprivation, glucose depletion, and viral activation of the Fas death pathway. When cardiomyocytes were heat shocked prior to grafting, graft cell death in vivo was reduced by 54% at day 1. Therefore, high levels of cardiomyocyte death occur for at least 4 days after grafting into injured hearts, in large part due to ischemia. Death can be limited by activating the Akt pathway and even more effectively by heat shock prior to transplantation.

Adenoviridae↗
PubMed2001

Functional abrogation of p53 is required for T-Ag induced proliferation in cardiomyocytes.

Targeted expression of the SV40 large T-antigen oncoprotein (T-Ag) induces cardiomyocyte proliferation in the atria and ventricles of transgenic mice. Previous studies have identified the p53 tumor suppressor, p107 (a homologue of the retinoblastoma tumor suppressor), and p193 (a novel BH3 only proapoptosis protein) as prominent TAg binding proteins in cardiomyocyte cell lines derived from these transgenic mice. To further explore the significance of these protein-protein interactions in the regulation of cardiomyocyte proliferation, a transgene comprising the human atrial natriuretic factor (ANF) promoter and sequences encoding a mutant T-Ag lacking the p53 binding domain was generated. Repeated micro-injection of this DNA gave rise to genetically mosaic animals with minimal transgene content, suggesting that widespread cardiac expression of mutant T-Ag was deleterious. This notion was supported by the observation that the transgene was selectively lost from the cardiac myocytes (but not the cardiac fibroblasts) in the mosaic animals. Crosses between the mosaic mice and animals expressing a cardiac restricted dominant negative p53 resulted in transgene transmission with ensuing overt cardiac tumorigenesis. Transfection of the mutant T-Ag in embryonic stem (ES) cell-derived cardiomyocytes resulted in wide-spread cell death with characteristics typical of apoptosis. Co-transfection with a dominant negative p53 transgene rescued mutant TAg-induced cell death in the ES-derived cardiomyocyte cultures, resulting in a marked proliferative response similar to that seen in vivo with the rescued transgenic mouse study. These results indicate that T-Ag expression in the absence of p53 functional abrogation results in cardiomyocyte death.

Animals↗
PubMed2002

Involvement of reactive oxygen species-mediated NF-kappa B activation in TNF-alpha-induced cardiomyocyte hypertrophy.

We examined the intracellular signaling mechanism for tumor necrosis factor-alpha (TNF-alpha)-induced cardiac hypertrophy in isolated rat neonatal cardiomyocytes. TNF-alpha enhanced the expression of a kappa B-dependent reporter gene construct in a dose-dependent manner, which was transiently transfected in cardiomyocytes. Electrophoretic mobility shift assay demonstrated that TNF-alpha induced nuclear factor- kappa B (NF-kappa B)-specific DNA binding. Cultured cardiomyocytes were infected with a recombinant adenoviral vector expressing a degradation-resistant mutant of I kappa B alpha (AdI kappa B alpha 32/36A). The I kappa B alpha mutant suppressed NF-kappa B activation induced by TNF- alpha. In cardiomyocytes infected with AdI kappa B alpha 32/36A, TNF-alpha-induced hypertrophic responses, including increases in cell size, protein synthesis and atrial natriuretic factor production and enhancement of sarcomeric organization, were remarkably attenuated compared to the cells infected with an adenovirus expressing bacterial beta-galactosidase. Using a reactive oxygen species (ROS)-sensitive fluorescent dye, 2', 7'-dichlorofluorescin, we observed an increase in fluorescent signal in cardiomyocytes over time, upon addition of TNF-alpha. Preincubation of n-acetyl cysteine (NAC), an antioxidant, prior to TNF-alpha treatment, abolished TNF-alpha -induced ROS generation. NAC abolished TNF-alpha-induced NF-kappa B activation and hypertrophic responses. These findings indicated that TNF-alpha-induced cardiomyocyte hypertrophy is mediated through NF-kappa B activation via the generation of ROS.

Animals↗
PubMed2002

Differential regulation of the cardiac sodium calcium exchanger promoter in adult and neonatal cardiomyocytes by Nkx2.5 and serum response factor.

Nkx2.5 and serum response factor (SRF) are critically important transcription factors in cardiac morphogenesis. They are also widely expressed in adult cardiomyocytes, but there is little data to indicate their possible role in adult cardiac cells. In this paper we demonstrate that the interaction of Nkx2.5 and SRF in cardiac-specific gene regulation is different between neonatal and adult cardiomyocytes. Our experimental model utilizes transient transfection and adenovirus mediated gene transfer of the proximal promoter fragment of the cardiac isoform of the sodium-calcium exchanger gene (NCX1). This promoter construct (NCX184) contains a single Nkx2.5-response element (NKE) and a single serum response element (CArG). In rat neonatal cardiomyocytes NCX184 activity is substantially induced with Nkx2.5 or SRF and additively with both. Mutagenesis of these NKE and CArG elements demonstrated the specificity of the interactions, which was confirmed with gel retardation analysis of cardiac ventricular tissue. In contrast, in adult cardiomyocytes, co-infection of Nkx2.5 and SRF adenovirus vectors showed Nkx2.5 induction but SRF did not have additive effects on NCX1 promoter regulation. As opposed to NCX1, the proximal atrial natriuretic factor (ANF) promoter was regulated identically in response to SRF and Nkx2.5 in both adult and neonatal cardiomyocytes. These results show that Nkx2.5-SRF interactions are capable of producing different transcriptional responses in adult versus neonatal cardiomyocytes, implying important differences in NCX1 promoter tertiary complex formation dependent on developmental stage.

Animals↗
PubMed1996

Cardiogel: a biosynthetic extracellular matrix for cardiomyocyte culture.

Tissue-cultured neonatal cardiomyocytes can be successfully maintained in culture on a variety of extracellular matrix components such as laminin, fibronectin, and interstitial collagens (Types I and III). In vivo, however, cardiomyocytes (as well as many other cells) exist in a highly complex extracellular matrix environment composed of, in addition to the above three components, other proteins, proteoglycans, and growth factors. We have developed a procedure for culturing cardiomyocytes on a naturally occurring complete extracellular matrix, Cardiogel. This substrate, synthesized by cardiac fibroblasts, contains laminin, fibronectin. Types I and III collagen, and proteoglycans. When compared to cardiomyocytes grown on laminin alone or fibronectin alone, Candiogel-supported cardiomyocytes adhere more rapidly after plating, exhibit spontaneous contractility earlier, undergo cytoskeletal and myofibrillar differentiation earlier, and grow larger than their counterparts. We suggest that their superior growth characteristics reflect the synergistic effect of numerous extracellular matrix components signals in Cardiogel transduced by the cardiomyocyte cytoskeletal elements.

Animals↗
PubMed2002

Effect of ginsenoside-Rb1 on cardiomyocyte apoptosis after ischemia and reperfusion in rats.

The effect of ginsenoside Rb1 on cardiomyocyte apoptosis after ischemia (30 min) and reperfusion (6 h) in rats was observed. The ischemia/reperfusion heart model was established by ligating left anterior descending branch of coronary artery in Wistar rats. The apoptotic cardiomyocytes were examined under transmission electron microscopy and counted by in situ nick end labeling (TUNEL) method and light microscopy. Results showed that (1) The apoptotic cardiomyocytes were found in ischemic regions in the ischemia/reperfusion group, but not in the sham-operating group under transmission electron microscopy; (2) The number of apoptotic cells were 134.45 +/- 45.61/field in the ischemia/reperfusion group, 0/field in the sham-operating group and 51.65 +/- 13.71/field in the ginsenoside Rb1-treated group. The differences were significant among the three groups (P < 0.01). It was concluded that myocardial ischemia-reperfusion could induce cardiomyocyte apoptosis, and ginsenoside Rb1 could significantly inhibit cardiomyocyte apoptosis induced by ischemia-reperfusion in rats, indicating that ginsenoside Rb1 could inhibit cardiomyocyte apoptosis induced by ischemia-reperfusion, thus alleviating ischemia-reperfusion injury.

Animals↗
PubMed1997

Neuropeptide Y as a stimulator of Na+ -dependent Ca2+ efflux from freshly isolated adult rat cardiomyocytes.

Several physiological stimuli cause a rise in intracellular Ca2+ concentration ([Ca2+]i) in cardiomyocytes. This increased [Ca2+]i must be restored to physiological resting level to ensure response to further stimuli. In the present study, we examined the effect of neuropeptide Y (NPY), which is secreted from certain adrenergic or nonadrenergic neurons, on Ca2+ efflux from freshly isolated, quiescent adult rat cardiomyocytes. The isolated cardiomyocytes were preloaded with 45CaCl2 for 1 h. Then, the fractional release of 45Ca2+ from the cells was measured. NPY stimulated the efflux of 45Ca2+ from isolated adult rat cardiomyocytes in a concentration-dependent manner (10(-8) M to 10(-6) M). NPY (10(-6) M)-induced Ca2+ efflux was 2.0 +/- 0.16% of the total cellular content. The 45Ca2+ efflux from the cells was also stimulated by Y1 receptor agonist, [Leu31, Pro34]NPY, but not by Y2 receptor agonist, NPY13-36. The effect of NPY was inhibited by a peptide NPY inhibitor, NPY18-36 and a non-peptide NPY inhibitor, benextramine to a similar extent. From these results, it is conceivable that the effect of NPY on Ca2+ efflux from cardiomyocytes is mediated through Y1 receptors. It was also observed that NPY caused a rise in [Ca2+]i to almost 150 nM. NPY-stimulated 45Ca2+ efflux was not affected by removal of extracellular Ca2+, but was dependent on the presence of extracellular Na+. Moreover, NPY caused a 22Na+ influx into the cells of about 1.6-fold over the basal value which was inhibited by amiloride and 5-(N,N-dimethyl)-amiloride, known Na+/Ca2+ exchange inhibitors. In addition, isoproterenol also caused 45Ca2+ efflux from the cells and which was enhanced by the addition of NPY. These results suggest that NPY stimulates extracellular Na+-dependent 45Ca2+ efflux from freshly isolated adult rat cardiomyocytes, probably through its stimulatory effect on plasma membrane Y1 receptors with which NPY may couple during Na+/Ca2+ exchange.

Adenosine Triphosphatases↗
PubMed2004

Urotensin-II-mediated cardiomyocyte hypertrophy: effect of receptor antagonism and role of inflammatory mediators.

Urotensin-II (U-II), the most potent mammalian vasoconstrictor identified, and its receptor, UT, exhibits increased expression in cardiac tissue and plasma in congestive heart failure (CHF) patients. Cardiomyocyte hypertrophy is primarily responsible for increased myocardial mass associated with cardiac injury. Neurohumoral factors such as angiotensin-II, endothelin-1, catecholamines, and inflammatory cytokines are thought to mediate this response. U-II shares similar biological activities with other hypertrophic G(q)-coupled receptor ligands such as angiotensin-II and endothelin-1, but a role for U-II in cardiomyocyte hypertrophy has not been characterized. The hypothesis of the current study was that U-II, acting through its G(q)-coupled receptor UT plays a hypertrophic role in cardiac hypertrophic remodeling. We report that adenoviral upregulation of the UT receptor "unmasked" U-II-induced hypertrophy in H9c2 cardiomyocytes, with a threshold response of 202+/-8 binding sites/cell. U-II was equally as efficacious as phenylephrine in inducing hypertrophy, measured by a reporter assay (EC(50) 0.7+/-0.2 nM) and [(3)H]-leucine incorporation (EC(50) 150+/-40 nM). A competitive peptidic UT receptor antagonist, BIM-23127, inhibited U-II-induced hypertrophy ( K(B) 34+/-6 nM). U-II did not affect cell proliferation or apoptosis, indicating that U-II is more hypertrophic than apoptotic or hyperplastic in cardiomyocytes. U-II (10 nM) stimulated interleukin-6 release in UT-expressing cardiomyocytes (4.6-fold at 6 h). Finally, in a rat heart failure model, cardiac ventricular mRNA expression of U-II, UT receptor, interleukin-6, and interleukin-1-beta is increased time-dependently following myocardial injury. These results indicate that U-II might play a role in cardiac remodeling associated with CHF by stimulation of cardiomyocyte hypertrophy via UT, and through upregulation of inflammatory cytokines. As such, UT antagonism may represent a novel therapeutic target for the clinical management of heart failure.

Animals↗
PubMed2003

Measurements of the mechanical properties of contracted collagen gels populated with rat fibroblasts or cardiomyocytes.

In this paper the mechanical properties of contracted collagen gels populated with rat fibroblasts or cardiomyocytes were investigated by means of uniaxial tensile testing. Rat type I collagen-Dulbecco's modified Eagle's medium (DMEM) gels (each 2 ml in volume, 0.5 mg/ml collagen concentration) populated with different numbers of rat fibroblasts or cardiomyocytes were made in 31 x 17-mm wells cut in silicone rubber located in a 100-mm diameter plastic dish. Identically treated gels were incubated for 4 days floating in DMEM and then were subjected to uniaxial tensile testing. Rapid contraction occurred within the first 3 days for both the fibroblast and cardiomyocyte gels, but the cardiomyocyte gels consistently contracted to smaller sizes than the fibroblast gels for each number of cells used. The tension-strain curve of the contracted collagen gels demonstrated exponential behavior in the low stress region, followed by a linear section, and finally a maximum tension point, giving the ultimate strength of the gel tested. The cardiomyocyte gels had higher tension-strain curves than the fibroblast gels for each number of cells used. The tension relaxation and cyclic creep phenomena were observed in both kinds of gels, and these phenomena coincide with prior observations in collagen gels contracted by human fibroblasts. This experiment shows that type I collagen gels can be significantly contracted by rat fibroblasts or cardiomyocytes so as to achieve a certain mechanical strength. The contracted collagen structures made in these experiments have potential for developing tissue-engineered structures for cardiac muscle studies.

Animals↗
PubMed2006

Hypoxic postconditioning enhances the survival and inhibits apoptosis of cardiomyocytes following reoxygenation: role of peroxynitrite formation.

OBJECTIVES: Our previous study has shown that slow or "controlled" reperfusion for the ischemic heart reduces cardiomyocyte injury and myocardial infarction, while the mechanisms involved are largely unclear. In this study, we tested the hypothesis that enhancement of survival and prevention of apoptosis in hypoxic/reoxygenated cardiomyocytes by hypoxic postconditioning (HPC) are associated with the reduction in peroxynitrite (ONOO(-)) formation induced by hypoxia/reoxygenation (H/R). METHODS: Isolated adult rat cardiomyocytes were exposed to 2 h of hypoxia followed by 3 h of reoxygenation. After 2 h of hypoxia the cardiomyocytes were either abruptly reperfused with pre-oxygenized culture medium or postconditioned by two cycles of 5 min of brief reoxygenation and 5 min of re-hypoxia followed by 160 min of abrupt reoxygenation. RESULTS: H/R resulted in severe injury in cardiomyocytes as evidenced by decreased cell viability, increased LDH leakage in the culture medium, increased apoptotic index (P values all less than 0.01 vs. normoxia control group) and DNA ladder formation, which could be significantly attenuated by HPC treatment applied before the abrupt reoxygenation (P < 0.05 vs. H/R group). In addition, H/R induced a significant increase in ONOO(-) formation as determined by nitrotyrosine content in cardiomyocytes (P < 0.01 vs. normoxia control). Treatment with the potent ONOO(-) scavenger uric acid (UA) at reoxygenation significantly decreased ONOO(-) production and protected myocytes against H/R injury, whereas the same treatment with UA could not further enhance myocyte survival in HPC group (P > 0.05 vs. HPC alone). Statistical analysis showed that cell viability closely correlated inversely with myocyte ONOO(-) formation (P < 0.01). CONCLUSION: These data demonstrate that hypoxic postconditioning protects myocytes against apoptosis following reoxygenation and enhances myocytes survival, which is partly attributable to the reduced ONOO(-) formation following reoxygenation.

Animals↗
PubMed2005

Role of mitogen-activated protein kinase pathway in reactive oxygen species-mediated endothelin-1-induced beta-myosin heavy chain gene expression and cardiomyocyte hypertrophy.

Endothelin-1 (ET-1) has been found to increase cardiac beta-myosin heavy chain (beta-MyHC) gene expression and induce hypertrophy in cardiomyocytes. ET-1 has been demonstrated to increase intracellular reactive oxygen species (ROS) in cardiomyocytes. The exact molecular mechanism by which ROS regulate ET-1-induced beta-MyHC gene expression and hypertrophy in cardiomyocytes, however, has not yet been fully described. We aim to elucidate the molecular regulatory mechanism of ROS on ET-1-induced beta-MyHC gene expression and hypertrophic signaling in neonatal rat cardiomyocytes. Following stimulation with ET-1, cultured neonatal rat cardiomyocytes were examined for 3H-leucine incorporation and beta-MyHC promoter activities. The effects of antioxidant pretreatment on ET-1-induced cardiac hypertrophy and mitogen-activated protein kinase (MAPKs) phosphorylation were studied to elucidate the redox-sensitive pathway in cardiomyocyte hypertrophy and beta-MyHC gene expression. ET-1 increased 3H-leucine incorporation and beta-MyHC promoter activities, which were blocked by the specific ET(A) receptor antagonist BQ-485. Antioxidants significantly reduced ET-1-induced 3H-leucine incorporation, beta-MyHC gene promoter activities and MAPK (extracellular signal-regulated kinase, p38, and c-Jun NH2 -terminal kinase) phosphorylation. Both PD98059 and SB203580 inhibited ET-1-increased 3H-leucine incorporation and beta-MyHC promoter activities. Co-transfection of the dominant negative mutant of Ras, Raf, and MEK1 decreased the ET-1-induced beta-MyHC promoter activities, suggesting that the Ras-Raf-MAPK pathway is required for ET-1 action. Truncation analysis of the beta-MyHC gene promoter showed that the activator protein-2 (AP-2)/specificity protein-1 (SP-1) binding site(s) were(was) important cis-element(s) in ET-1-induced beta-MyHC gene expression. Moreover, ET-1-induced AP-2 and SP-1 binding activities were also inhibited by antioxidant. These data demonstrate the involvement of ROS in ET-1-induced hypertrophic responses and beta-MyHC expression. ROS mediate ET-1-induced activation of MAPK pathways, which culminates in hypertrophic responses and beta-MyHC expression.

Animals↗
PubMed2002

Increased cardiomyocyte apoptosis following ischemia and reperfusion in diet-induced hypercholesterolemia: relation to Bcl-2 and Bax proteins and caspase-3 activity.

It has been reported that apoptosis is a significant contributor to myocardial cell death as a result of reperfusion injury. However, whether the extent of cardiomyocyte apoptosis following ischemia and reperfusion varies in different pathophysiological backgrounds is still uncertain. In this study, we examined whether hypercholesterolemia increases the extent of myocardial reperfusion injury by aggravating cardiomyocyte apoptosis and the effects of hypercholesterolemia on the expression of Bcl-2 and Bax proteins and the activation of caspase-3. Twenty-eight male New Zealand white rabbits were fed standard chow (control, n = 14) or chow supplemented with 10% cholesterol (hypercholesterolemic, n = 14) for 8 wk. Anesthetized rabbits were then subjected to 30 min of left circumflex artery occlusion followed by 4 h of reperfusion. Apoptosis was identified as "DNA ladders" by gel electrophoresis and confirmed histologically using the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay. The infarct size (% of risk region) was significantly greater in hypercholesterolemic rabbits than in controls (39 +/- 6 vs. 23 +/- 2%, P = 0.02). Very few TUNEL-positive cardiomyocytes could be identified in the nonischemic regions in both groups, consistent with an absence of DNA laddering. In contrast, TUNEL-positive cardiomyocytes were significantly displayed in the ischemic, nonnecrotic myocardium, and DNA ladder occurred in all animals. The percentage of TUNEL-positive cardiomyocytes in the ischemic nonnecrotic myocardium was significantly higher in hypercholesterolemic rabbits compared with controls (40 +/- 5 vs. 17 +/- 11%, P < 0.001). Western blot analysis showed that, in the nonischemic myocardium, hypercholesterolemic rabbits exhibited an approximately 50% increase in the expression of Bcl-2 (P < 0.05), but not Bax, than control rabbits. However, compared with controls, hypercholesterolemic rabbits exhibited a more pronounced decrease in the expression of Bcl-2 (42 +/- 4 vs. 26 +/- 2%, P < 0.01) and a similar extent of increase in the expression of Bax in the ischemic myocardium. Furthermore, hypercholesterolemic rabbits were associated with a markedly increased activation of caspase-3 within the ischemic myocardium compared to control rabbits. This study demonstrates that although hypercholesterolemia is associated with an increased myocardial Bcl-2/Bax ratio at baseline, it still significantly exacerbates cardiac reperfusion injury, not only by increasing the infarct size but also by increasing the extent of cardiomyocyte apoptosis.

Animals↗
PubMed1984

Characterization of purine nucleotide metabolism in primary rat cardiomyocyte cultures.

Primary rat cardiomyocyte cultures were utilized as a model for the study of purine nucleotide metabolism in the heart muscle, especially in connection with the mechanisms operating for the conservation of adenine nucleotides. The cultures exhibited capacity to produce purine nucleotides from nonpurine molecules (de novo synthesis), as well as from preformed purines (salvage synthesis). The conversion of adenosine to AMP, catalyzed by adenosine kinase, appears to be the most important physiological salvage pathway of adenine nucleotide synthesis in the cardiomyocytes. The study of the metabolic fate of IMP formed from [14C]formate or [14C]hypoxanthine and that of AMP formed from [14C]adenine or [14C]adenosine revealed that in the cardiomyocyte the main flow in the nucleotide interconversion pathways is from IMP to AMP, whereas the flux from AMP to IMP appeared to be markedly slower. Following synthesis from labeled precursors by either de novo or salvage pathways, most of the radioactivity in purine nucleotides accumulated in adenine nucleotides, and only a small proportion of it resided in IMP. The results suggest that the main pathway of AMP degradation in the cardiomyocyte proceeds through adenosine rather than through IMP. About 90% of the total radioactivity in purines effluxed from the cells during de novo synthesis from [14C]formate or following prelabeling of adenine nucleotides with [14C]adenine were found to reside in hypoxanthine. The activities in cell extracts of AMP 5'-nucleotidase and IMP 5'-nucleotidase, which catalyze nucleotide degradation, and of AMP deaminase, a key enzyme in the purine nucleotide cycle, were low. The nucleotidase activity resembles, and that of the AMP deaminase contrasts the respective enzyme activities in extracts of cultured skeletal-muscle myotubes. The results indicate that in the cardiomyocyte, in contrast to the myotube, the main mechanism operating for conservation of nucleotides is prompt phosphorylation of AMP, rather than operation of the purine nucleotide cycle. The primary cardiomyocyte cultures are a plausible model for the study of purine nucleotide metabolism in the heart muscle.

5'-Nucleotidase↗
PubMed1991

In vivo regulation of mitochondrial respiration in cardiomyocytes: specific restrictions for intracellular diffusion of ADP.

Relative diffusivities of ADP and creatine in cardiomyocytes were studied. The isolated rat cardiomyocytes were lysed with saponin (40 micrograms/ml) to perforate or completely disrupt sarcolemma that was evidenced by leakage of 80-100% lactate dehydrogenase. In these cardiomyocytes mitochondria were used as 'enzymatic probes' to determine the average local concentration of substrates exerting acceptor control of respiration--ADP or creatine (the latter activates respiration via mitochondrial creatine kinase reaction)--when their concentrations in the surrounding medium were changed. The kinetic parameters for ADP and creatine in control of respiration of saponin-treated cardiomyocytes were compared with those determined in isolated mitochondria and skinned cardiac fibers. The apparent Km for creatine (at 0.2 mM ATP) was very close and in a range of 6.0-6.9 mM in all systems studied, showing the absence of diffusion difficulties for this substrate. On the contrary, the apparent Km for ADP increased from 18 +/- 1 microM for isolated mitochondria to 250 +/- 59 microM for cardiomyocytes with the lysed sarcolemma and to 264 +/- 57 microM for skinned fibers. This elevation of Km was not eliminated by inhibition of myokinase with diadenosine pentaphosphate. When 25 mM creatine was present, the apparent Km for ADP decreased to 36 +/- 6 microM. These data are taken to indicate specific restrictions of diffusion of ADP most probably due to its interaction with intermediate binding sites in cardiomyocytes. The important role of phosphocreatine-creatine kinase system of energy transport is to overcome the restrictions in regulation of energy fluxes due to decreased diffusivity of ADP.

Adenosine Diphosphate↗