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PubMed2005

Functional role of phosphodiesterase 3 in cardiomyocyte apoptosis: implication in heart failure.

BACKGROUND: Myocyte apoptosis plays an important role in pathological cardiac remodeling and the progression of heart failure. cAMP signaling is crucial in the regulation of myocyte apoptosis and cardiac remodeling. Multiple cAMP-hydrolyzing phosphodiesterases (PDEs), such as PDE3 and PDE4, coexist in cardiomyocytes and elicit differential temporal/spatial regulation of cAMP signaling. However, the role of PDE3 and PDE4 in the regulation of cardiomyocyte apoptosis remains unclear. Although chronic treatment with PDE3 inhibitors increases mortality in patients with heart failure, the contribution of PDE3 expression/activity in heart failure is not well known. METHODS AND RESULTS: In this study we report that PDE3A expression and activity were significantly reduced in human failing hearts as well as mouse hearts with chronic pressure overload. In primary cultured cardiomyocytes, chronic inhibition of PDE3 but not PDE4 activity by pharmacological agents or adenovirus-delivered antisense PDE3A promoted cardiomyocyte apoptosis. Both angiotensin II (Ang II) and the beta-adrenergic receptor agonist isoproterenol selectively induced a sustained downregulation of PDE3A expression and induced cardiomyocyte apoptosis. Restoring PDE3A via adenovirus-delivered expression of wild-type PDE3A1 completely blocked Ang II- and isoproterenol-induced cardiomyocyte apoptosis, suggesting the critical role of PDE3A reduction in cardiomyocyte apoptosis. Moreover, we defined a crucial role for inducible cAMP early repressor expression in PDE3A reduction-mediated cardiomyocyte apoptosis. CONCLUSIONS: Our results suggest that PDE3A reduction and consequent inducible cAMP early repressor induction are critical events in Ang II- and isoproterenol-induced cardiomyocyte apoptosis and may contribute to the development of heart failure. Drugs that maintain PDE3A function may represent an attractive therapeutic approach to treat heart failure.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1999

Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts.

BACKGROUND: Cardiomyocyte grafting augments myocyte numbers in the heart. We investigated (1) how developmental stage influences graft survival; (2) whether acutely necrotic or healing cardiac lesions support grafts; and (3) the differentiation and integration of cardiomyocyte grafts in injured hearts. METHODS AND RESULTS: Cardiomyocytes from fetal, neonatal, or adult inbred rats were grafted into normal myocardium, acutely cryoinjured myocardium, or granulation tissue (6 days after injury). Adult cardiomyocytes did not survive under any conditions. In contrast, fetal and neonatal cardiomyocytes formed viable grafts under all conditions. Time-course studies with neonatal cardiomyocytes showed that the grafts recapitulated many aspects of normal development. The adherens junction protein N-cadherin was distributed circumferentially at day 1 but began to organize into intercalated disk-like structures by day 6. The gap junction protein connexin43 followed a similar but delayed pattern relative to N-cadherin. From 2 to 8 weeks, there was progressive hypertrophy and the formation of mature intercalated disks. In some hearts, graft cells formed adherens and gap junctions with host cardiomyocytes, suggesting electromechanical coupling. More commonly, however, grafts were separated from the host myocardium by scar tissue. Gap and adherens junctions formed between neonatal and adult cardiomyocytes in coculture, as evidenced by dye transfer and localization of cadherin and connexin43 at intercellular junctions. CONCLUSIONS: Grafted fetal and neonatal cardiomyocytes form new, mature myocardium with the capacity to couple with injured host myocardium. Optimal repair, however, may require reducing the isolation of the graft by the intervening scar tissue.

Aging↗
PubMed1996

Intracellular Ca2+ modulation by angiotensin II and endothelin-1 in cardiomyocytes and fibroblasts from hypertrophied hearts of spontaneously hypertensive rats.

The vasoactive peptides angiotensin II (Ang II) and endothelin-1 (ET-1) have been implicated in cardiac hypertrophy. This study investigates Ang II and ET-1 effects on intracellular free calcium concentration and the receptor subtype through which agonist-induced calcium responses are mediated in isolated cardiomyocytes and fibroblasts from hypertrophied hearts of spontaneously hypertensive rats (SHR). We measured intracellular free calcium concentration by fura 2 methodology and determined receptor status by radioligand binding assays. Ang II (10(-12) to 10(-7) mol/L) had no effect on cardiomyocyte calcium levels in control Wistar-Kyoto rats but significantly increased (P < .01) intracellular free calcium concentration in a dose-dependent manner in cardiomyocytes from SHR. Ang II total and specific binding were increased (P < .05) in SHR cardiomyocytes. Calcium responses elicited by 10(-7) to 10(-5) mol/L Ang II were significantly reduced (P < .01) in SHR fibroblasts despite no significant change in Ang II receptor density. The angiotensin type 1 receptor blocker losartan (1 mumol/L) blocked Ang II-stimulated calcium transients, whereas the angiotensin type 2 receptor blocker PD 123319 had no effect. ET-1- and sarafotoxin S6c-induced calcium responses in cardiomyocytes and fibroblasts were not different between hypertensive and control groups. In conclusion, Ang II and ET-1 elicit distinct and differential responses in a cell-specific manner in cardiomyocytes and fibroblasts from hypertrophied hearts of SHR. Whereas Ang II-mediated effects, which are elicited via angiotensin type 1 receptors, are detectable in cardiomyocytes from SHR, responses to Ang II are blunted in fibroblasts from SHR, and ET-1-related actions are similar in cells from both rat groups. Stimulation of cardiomyocytes by Ang II in hypertrophied hearts associated with pressure overload in genetic hypertension suggests that Ang II could modulate the function of cardiomyocytes of SHR but not those of Wistar-Kyoto rats, whereas cardiac actions of ET-1 do not change with the development of hypertension.

Angiotensin II↗
PubMed2002

Identification of IEX-1 as a biomechanically controlled nuclear factor-kappaB target gene that inhibits cardiomyocyte hypertrophy.

Biomechanical strain is a stimulus for cardiomyocyte hypertrophy and heart failure, but the underlying molecular mechanisms remain incompletely understood. Using an in vivo murine model of pressure overload and an in vitro model of mechanical stimulation of primary cardiomyocytes, we identified iex-1 as a gene activated during the early response of cardiomyocytes to hypertrophic stimuli and as a gene product that inhibits hypertrophy without affecting cardiomyocyte viability. On stimulation of cardiomyocytes, iex-1 mRNA and protein expression increased and translocation of the gene product to the cardiomyocyte nucleus occurred. iex-1 has previously been proposed as a mediator of NF-kappaB-dependent cell survival and growth in tumor cells. Here, we demonstrate that the biomechanical induction of iex-1 in cardiomyocytes was NF-kappaB-dependent, as overexpression of the NF-kappaB inhibitor IkappaBalpha completely inhibited strain-mediated iex-1 mRNA accumulation. The functional role of iex-1 was investigated by overexpressing wild-type iex-1 with replication-defective adenoviral gene transfer. Overexpression of iex-1 abolished cardiomyocyte hypertrophy by mechanical strain, phenylephrine, or endothelin-1 at levels that did not affect cell viability. These studies identify iex-1 as a biomechanical stress-inducible and NF-kappaB-dependent gene in cardiac muscle cells during the acute phase of hypertrophy with negative growth regulatory effects that may counterbalance early hypertrophic responses in activated cardiomyocytes.

Adenoviridae↗
PubMed2003

Critical role of cyclin D1 nuclear import in cardiomyocyte proliferation.

Mammalian cardiomyocytes irreversibly lose their capacity to proliferate soon after birth, yet the underlying mechanisms have been unclear. Cyclin D1 and its partner, cyclin-dependent kinase 4 (CDK4), are important for promoting the G1-to-S phase progression via phosphorylation of the retinoblastoma (Rb) protein. Mitogenic stimulation induces hypertrophic cell growth and upregulates expression of cyclin D1 in postmitotic cardiomyocytes. In the present study, we show that, in neonatal rat cardiomyocytes, D-type cyclins and CDK4 were predominantly cytoplasmic, whereas Rb remained in an underphosphorylated state. Ectopically expressed cyclin D1 localized in the nucleus of fetal but not neonatal cardiomyocytes. To target cyclin D1 to the nucleus efficiently, we constructed a variant of cyclin D1 (D1NLS), which directly linked to nuclear localization signals (NLSs). Coinfection of recombinant adenoviruses expressing D1NLS and CDK4 induced Rb phosphorylation and CDK2 kinase activity. Furthermore, D1NLS/CDK4 was sufficient to promote the reentry into the cell cycle, leading to cell division. The number of cardiomyocytes coinfected with these viruses increased 3-fold 5 days after infection. Finally, D1NLS/CDK4 promoted cell cycle reentry of cardiomyocytes in adult hearts injected with these viruses, evaluated by the expression of Ki-67, which is expressed in proliferating cells in all phases of the cell cycle, and BrdU incorporation. Thus, postmitotic cardiomyocytes have the potential to proliferate provided that cyclin D1/CDK4 accumulate in the nucleus, and the prevention of their nuclear import plays a critical role as a physical barrier to prevent cardiomyocyte proliferation. Our results provide new insights into the development of therapeutics strategies to induce regeneration of cardiomyocytes. The full text of this article is available at http://www.circresaha.org.

Active Transport, Cell Nucleus↗
PubMed2004

Preinduced molecular chaperones in the endoplasmic reticulum protect cardiomyocytes from lethal injury.

Although molecular chaperones in the endoplasmic reticulum (ER) are known to be involved in folding and assembly of glycosylated proteins, it is unclear whether preinduced ER chaperones can protect cardiomyocytes from lethal injury. In this study we used tunicamycin, an inhibitor of N-linked glycosylation in the ER, to preinduce ER chaperones in H9c2 cardiomyocytes and we tested the cytoprotective role of preinduced ER chaperones in the cardiomyocytes. Expression of GRP78 at both protein and mRNA levels was markedly increased in cardiomyocytes pretreated with tunicamycin, when compared to non-treatment controls. Following prolonged ATP depletion or oxidative stress, which was used to simulate cardiac ischemia and reperfusion injury, respectively, the release of lactate dehydrogenase (LDH) from tunicamycin-pretreated cardiomyocytes was significantly lower than from non-pretreated cardiomycocytes. Tunicamycin-pretreated cardiomyocytes showed significantly higher Ca2+ release into cytoplasm than controls when treated with both caffeine and thapsigargin, indicating higher storage of Ca2+ in the ER. After oxidative stress, cytosolic Ca2+ levels were maintained relatively stable in tunicamycin-pretreated cardiomyocytes, when compared to control cardiomyocytes. These observations suggest that preinduced ER chaperones protect cardiomyocytes from lethal injury, at least in part, by preventing an increase in cytosolic Ca2+.

Animals↗
PubMed2006

Changes in important apoptosis-related molecules in the endothelin-1-induced hypertrophied cardiomyocytes: effect of the pretreatment with eicosapentaenoic Acid.

Human heart failure is preceded by a process called cardiac remodeling, in which heart chambers progressively enlarge and contractile function deteriorates. Programmed cell death (apoptosis) of cardiac muscle cells has been identified as an essential process in the progression to heart failure. The execution of the apoptotic program entails complex interactions between and execution of multiple molecular subprograms. Endothelin (ET)-1, a potent vasoconstrictor peptide, is synthesized and secreted by cardiomyocytes and induces hypertrophy of cardiomyocytes. The cardiovascular benefit of fish oil containing eicosapentaenoic acid (EPA) in humans and experimental animals was reported. Recently, we found that ET-1-induced cardiomyocytic remodeling could be prevented by pretreatment with EPA. The aim of the present study is to investigate whether there would be any alteration in the expression of important apoptosis-related molecules in ET-1-administered hypertrophied cardiomyocytes. We also sought to determine, if there are alterations in apoptotic molecules, what type of role for EPA would then exist. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control, the ET-1 (0.1 nM)-treated group, and the ET-1 group pretreated with EPA (10 microM). Twenty-four hours after the treatment, the gene expressions of three important molecules related to apoptosis (caspase-3, Bax, and Bcl-2) in three experimental groups were evaluated by real-time polymerase chain reaction. The present study could not demonstrate any significant or representative alteration in any of the above three apoptosis-related important markers in either ET-1-induced hypertrophied cardiomyocytes with or without EPA pretreatment. The present study would at least be able to exclude the involvement of some representative molecules related to apoptosis in ET-1-induced hypertrophied cardiomyocytes. In addition, the present study demonstrates that the antihypertrophic effect of EPA to ET-1-administered cardiomyocytes appears not to modulate the apoptosis signaling cascade.

Animals↗
PubMed2001

Expression of HNK1 epitope by the cardiomyocytes of the early embryonic chick: in situ and in vitro studies.

Monoclonal antibody HNK1 reacts with a carbohydrate epitope in cell surface glycoproteins and glycolipids. During development, in various species the HNK1 epitopes are expressed in migrating neural crest cells and in the developing conduction cardiomyocytes. The conduction system is generally thought to be developed from cardiomyocytes, but some investigators have hypothesized that it is derived from the neural crest because conduction myocytes express neural antigens, including HNK1. Using immunohistochemistry, we examined the spatiotemporal expression of HNK1 in early chick cardiogenesis (stages 4 to 18) and whether cultured precardiac mesoderm does or does not express HNK1 as well as sarcomeric myosin (MF20). HNK1 was first expressed in the premyocardium at stage 8. At stage 10, HNK1-positive cardiomyocytes were scattered along the straight heart tube. By stage 18, HNK1-positive cardiomyocytes had become restricted to the atrium and sinus venosus. Atrioventricular cushion mesenchyme also expressed an HNK1 epitope. Immunostaining of HNK1 and MF20 in cultured precardiac mesoderm showed that there are at least three types of cells: 1) cardiomyocytes without HNK1 expression, 2) cells possessing both HNK1- and MF20-immunoreactivity, and 3) mesenchymal cells with HNK1. Immunogold electron microscopy showed that cardiomyocytes containing sparsely distributed myofibrils associated with the Z-band react with anti-HNK1 antibody. Our observations showed a direct evidence for the first time that the precardiac mesoderm generates HNK1-positive cardiomyocytes with morphological features similar to those of conduction cardiomyocytes.

Animals↗
PubMed2007

Role of endothelin-1, sodium hydrogen exchanger-1 and mitogen activated protein kinase (MAPK) activation in glucose-induced cardiomyocyte hypertrophy.

BACKGROUND: Cardiac hypertrophy is a key structural feature of diabetic cardiomyopathy. Previous studies have shown that diabetes-induced endothelin-1 (ET-1) and sodium hydrogen exchanger-1 (NHE-1) mediate structural and functional deficits in the heart. In order to gain a mechanistic understanding of the role of ET-1 and NHE-1 in cardiomyocyte hypertrophy, we have utilized an in vitro endothelial-myocyte co-culture system to reveal cellular interactions that may arbitrate cardiomyocyte deficits in diabetes. METHODS AND RESULTS: Rat ventricular cardiomyocytes were cultured in high glucose levels, which caused cellular hypertrophy. Hypertrophic markers, atrial natruritic peptide (ANP) and angiotensinogen (Agt), as well as inducible nitric oxide synthase (iNOS) were upregulated by high glucose. Treatment of cells with ET antagonist bosentan and NHE-1 inhibitor cariporide prevented glucose-induced cardiomyocyte hypertrophy and expression of ANP, Agt, and iNOS. Bosentan and cariporide treatment of cardiomyocytes co-cultured with endothelial cells produced a more pronounced normalization of glucose-induced changes as compared to cardiomyocyte cultured alone. To further explore the signaling mechanisms involved, we investigated the mitogen activated protein kinase (MAPK) pathway and its cross-interaction with signaling proteins known to be altered in diabetes. Our results indicate that MAPK activation is associated with cardiomyocyte hypertrophy and is inhibited by bosentan, cariporide, as well as protein kinase C inhibiton. Furthermore, MAPK activation was found to be upstream of the transcription factors, nuclear factor-kappaB and activating protein-1. CONCLUSION: These results demonstrate that ET-1 and NHE-1 may mediate cardiomyocyte hypertrophy via MAPK activation and provide an insight into the pathogenesis of diabetic cardiomyopathy.

Animals↗
PubMed1993

Infection with the avian polyomavirus, BFDV, selectively affects myofibril structure in embryonic chick ventricle cardiomyocytes.

Embryonic cardiomyocytes can both beat and divide. They assemble cardiac muscle-specific proteins into sarcomeric myofibrils and contract. In addition, they periodically synthesize DNA, complete mitosis, disassemble sarcomeric myofibrils in the area of the mitotic spindle, assemble cytoplasmic isoform-specific proteins into a cleavage furrow contractile ring, undergo cytokinesis, and then reform sarcomeric myofibrils in daughter cells. Little is known about how embryonic cardiomyocytes disassemble their myofibrils as they traverse the cell cycle and divide. In the present study, beating embryonic avian ventricular cardiomyocytes in primary culture were stimulated to initiate DNA synthesis without subsequent mitosis or cytokinesis by infection with the lytic avian polyomavirus, Budgerigar Fledgling Disease Virus (BFDV). Within 48 hours, infected, adherent cardiomyocytes disassemble most of their sarcomeric myofibrils, retaining cardiac myosin only in thin myofibrils with disrupted sarcomeric periodicity and in amorphous nonfibrillar pools. By 72 hours, infected cardiomyocytes contain no myofibrils and no longer react with antibodies to cardiac myosin. In contrast, infected cardiomyocytes continue to display cytoplasmic myosin localized in stress-fiber-like-structures in adherent cells, or in disrupted fibers and dispersed pools in detaching cells. Infected cardiomyocytes also continue to display interphase-like arrays of polymerized microtubules, even when rounded-up just prior to lysis. These results suggest that polyomavirus infection may provide a useful model system for further study of the regulation of myofibrils disassembly in embryonic cardiomyocytes.

Animals↗
PubMed1996

Activation of ecto-5'-nucleotidase by protein kinase C attenuates irreversible cellular injury due to hypoxia and reoxygenation in rat cardiomyocytes.

Adenosine, synthesized by ecto-5'-nucleotidase, is cardioprotective against ischemia and reperfusion injury. We have previously reported that activation of protein kinase C increases ecto-5'-nucleotidase activity of the rat cardiomyocytes, raising the possibility that activation of protein kinase C protects cardiomyocytes from the irreversible cellular injury via activation of ecto-5'-nucleotidase. To test this hypothesis, cardiomyocytes were isolated from adult male Wistar rats and suspended in modified HEPES-Tyrode buffer solution. The cardiomyocytes were incubated with and without exposure to methoxamine (1 x 10(-6) mol/l) or phorbol 12-myristate 13-acetate (PMA. 1 x 10(-8) mol/l). Ecto-5'-nucleotidase activity increased 15 min after the onset of an exposure to either methoxamine or PMA. Adenosine release during hypoxia and reperfusion was augmented in the methoxamine- and PMA-pretreated cardiomyocytes compared with the untreated cardiomyocytes, which was inhibited by alpha, beta-methyleneadenosine 5'-diphosphate (AOPCP), an inhibitor of ecto-5'-nucleotidase. Irreversible cellular injury assessed by the extent of release of lactate dehydrogenase and the trypan blue exclusion test following 60 min of hypoxia and 60 min of reoxygenation was attenuated in the methoxamine- and PMA-pretreated cardiomyocytes compared with the untreated group, which was also blunted by AOPCP and 8-sulfophenyltheophylline, an adenosine receptor antagonist. An adenosine A1 receptor agonist, N6-cyclohexyladenosine, restored the cardioprotection under the treatment with PMA and AOPCP. We conclude that activation of ecto-5'-nucleotidase via protein kinase C contributes to the attenuation of the irreversible injury of the rat cardiomyocytes due to hypoxia and reoxygenation.

5'-Nucleotidase↗
PubMed1997

Retinoic acid accelerates embryonic stem cell-derived cardiac differentiation and enhances development of ventricular cardiomyocytes.

Pluripotent embryonic stem (ES) cells spontaneously differentiate via embryo-like aggregates into cardiomyocytes of pacemaker-, atrium- and ventricle-like type, which can be distinguished by their specific patterns of action potentials. It has been shown that retinoic acid (RA) treatment during ES cell differentiation increases the number of cardiomyocytes in a time- and concentration-dependent manner. In order to test the effect of RA on cardiomyocyte differentiation and specialization into ventricle-like cardiomyocytes, we studied gene expression of beta-galactosidase driven by the ventricular myosin light chain-2 (MLC-2v) promoter as an indicator for ventricular differentiation. Clones containing the stably integrated expression vector pGNA/MLC-2.1 were selected, which revealed an increase of beta-galactosidase activity in cardiomyocytes of embryoid bodies at day 7 + 16. RA, both, in the all-trans and in the 9-cis configuration resulted in a significant acceleration of cardiomyocyte differentiation and a transient increase of beta-galactosidase activity. To test whether this acceleration of cardiac differentiation and RA-induced increase of the MLC-2v promotor/beta-galactosidase activity reflects an increase of cardiac- and ventricle-specific gene expression, a semi-quantitative RT-PCR analysis was performed for alpha-cardiac myosin heavy chain (alpha-MHC) and MLC-2v genes. It was shown that both 10(-8) M and 10(-9) M RA resulted in an increased level of alpha-cardiac MHC and MLC-2v mRNA in embryoid bodies in early, but not in terminal developmental stages. This led us to the conclusion that the RA-induced accelerated expression of cardiac-specific genes results in an enhanced development of ventricular cardiomyocytes. An increased number of ventricle-like cells after RA treatment was also found by patch-clamp analysis. The number of cardiomyocytes with Purkinje- and ventricle-like properties was shown to be increased by RA, whereas the number of pacemaker- and atrium-like cells was reduced and early pacemaker cells were not quantitatively affected.

Alitretinoin↗
PubMed1996

Exposure to the n-3 polyunsaturated fatty acid docosahexaenoic acid impairs alpha 1-adrenoceptor-mediated contractile responses and inositol phosphate formation in rat cardiomyocytes.

The beneficial effects of n-3 polyunsaturated fatty acids of fish oil in the prevention of fatal arrhythmias in myocardial ischemia were suggested to be at least in part mediated by a modulation of dihydropyridine-sensitive L-type calcium channels. As cardiac alpha 1-adrenoceptor stimulation has been suggested to have no significant effect on L-type calcium channels, the aim of this study using cultured neonatal rat cardiomyocytes was to investigate whether chronic n-3 polyunsaturated fatty acid exposure may have an influence on alpha 1-adrenoceptor-induced positive inotropic effects and induction of arrhythmias. Pretreatment of the rat cardiomyocytes for 3 days in the presence of the n-3 polyunsaturated fish oil-derived fatty acid docosahexaenoic acid (60 mumol/l) markedly decreased alpha 1-adrenoceptor-stimulated increase in contraction velocity and induction of arrhythmias. The increase in contraction velocity of the cardiomyocytes induced by the beta-adrenoceptor agonist isoprenaline was also markedly reduced by the n-3 fatty acid pretreatment. Basal contractile amplitude and spontaneous beating frequency of the cardiomyocytes were not significantly altered by the docosahexaenoic acid exposure. The pretreatment of the rat cardiomyocytes for 3 days in the presence of docosahexaenoic acid (60 mumol/l) decreased alpha 1-adrenoceptor-stimulated formation of the calcium-mobilizing second messenger IP3 and its metabolites IP2 and IP1 by 55%. The depression of IP3 formation by docosahexaenoic acid treatment was not mediated by a decreased uptake of myo-inositol into the cardiomyocytes nor by a decreased synthesis of phosphatidylinositol bisphosphate (PIP2), the substrate of phospholipase C. The level of glycerol-3-phosphate, an important substrate of the phosphoinositide cycle, was unaltered by the docosahexaenoic acid pretreatment. Receptor binding studies revealed that the dissociation constant and maximal binding capacity of the alpha 1-adrenoceptor antagonist (3H)prazosin was unchanged by the n-3 polyunsaturated fatty acid exposure. Beta-Adrenoceptor-and forskolin-stimulated adenylyl cyclase activities were not diminished by the docosahexaenoic acid pretreatment. Chronic exposure of the cardiomyocytes to the n-6 polyunsaturated fatty acid arachidonic acid (60 mumol/l) did neither significantly alter alpha 1-adrenoceptor-induced inositol phosphate formation nor alpha 1-adrenoceptor-stimulated increase in contraction velocity. The results presented show that chronic n-3 polyunsaturated fatty acid pretreatment of rat cardiomyocytes leads to a marked impairment of alpha 1-adrenoceptor-induced positive inotropic effects and induction of arrhythmias concomitant with a n-3 fatty acid-induced decrease in IP3 formation. This derangement of the phosphoinositide pathway by chronic n-3 fatty acid exposure may, thus, contribute to the beneficial effects of fish oil-derived fatty acids in the prevention of fatal arrhythmias in myocardial ischemia.

Adenylyl Cyclases↗
PubMed1999

Repolarization abnormalities in cardiomyocytes of dogs with chronic heart failure: role of sustained inward current.

We previously showed that a canine model of chronic heart failure (HF) produced by multiple coronary microembolizations manifests ventricular arrhythmias similar to those observed in patients with chronic HF. In the present study, we used single canine cardiomyocytes isolated from the left ventricle (LV) of normal dogs (n = 13) and dogs with HF (n = 15) to examine the cellular substrate of these arrhythmias. Action potentials (APs) and ion currents were measured by perforated and whole cell patch clamp, respectively. We found prolonged APs and alterations of AP duration resulting in early afterdepolarizations (EADs) at the low pacing rates of 0.5 Hz and 0.2 Hz. Na+ channel blockers saxitoxin (STX, 100 nM) and lidocaine (90 microM) reduced AP duration dispersion and abolished EADs in HF cardiomyocytes. The steady-state current (Iss)-voltage relation, in the voltage range from -25 mV to 25 mV analogous to the AP plateau level, was significantly shifted inward in HF cardiomyocytes. STX and lidocaine shifted the Iss-voltage relationship in an outward direction. The shifts produced by both drugs was significantly greater in cardiomyocytes of dogs with HF, indicating an increase in inward current. In the experimental configuration in which K+ currents were blocked, the density of the steady-state Ca2+ current (ICa) was found to decrease in HF cardiomyocytes by approximately 33%. In contrast, the density of the steady-state Na+ current (INa) significantly (P < 0.01) increased in HF cardiomyocytes (0.17 +/- 0.06 pA/pF) compared with normal cells (0.08 +/- 0.02 pA/pF). The relative contribution of INa to the net inward current was greater in HF cardiomyocytes, as evident from the increased ratio of INa/ICa (from 0.22 to 0.68). These observation support a hypothesis that anomalous repolarization of HF cardiomyocytes is due, at least in part, to an increased steady-state inward Na+ current.

Animals↗
PubMed2006

Angiotensin II stimulates apoptosis via TGF-beta1 signaling in ventricular cardiomyocytes of rat.

Elevations in angiotensin II (AngII) and transforming growth factor (TGF-beta1) levels are often found under conditions leading to progression of heart failure. From several studies, it is evident that AngII enhances TGF-beta1 expression via activator protein 1 (AP-1) activation, and that this pathway is involved in hypertrophic growth of the heart muscle and in the development of cardiac fibrosis. We now continued characterization of the signaling pathway stimulated by AngII in ventricular cardiomyocytes of rat and analyzed if the enhancement of TGF-beta1 expression by AngII may also contribute to apoptosis induction, which is another predictor of heart failure progression. Stimulation of cardiomyocytes with 100 nM AngII for 2 h activated the transcription factors AP-1 and GATA by 68.6+/-23.9 or 70.7+/-9.8%. Induction of both factors was mediated by p38 mitogen-activated protein kinase (MAPK) because it was totally blocked using a specific inhibitor of the kinase (SB202190). When GATA was inhibited by transformation of cardiomyocytes with decoy oligonucleotides, AngII could not enhance TGF-beta1 expression. This inhibition was observed on the mRNA level in real-time polymerase chain reaction and on the protein level in Western blots. As a consequence, upon AngII stimulation for 24 h, release of TGF-beta1 from cardiomyocytes was also reduced from 240.5+/-50.4 to 130.5+/-22.1% (p<0.05). In contrast to the early induction of GATA and AP-1, the transcription factor similar to mothers against decapentaplegic homolog (SMAD) was induced by AngII after 24 h. This stimulation was dependent on TGF-beta1 because it was blocked by antibodies specific for TGF-beta1. Twenty-four hours after AngII addition, the number of apoptotic cardiomyocytes increased by 6.5+/-1.2%, and this apoptosis induction was blocked when SMAD activity was inhibited by transformation of cardiomyocytes with SMAD decoy oligonucleotides. In conclusion, the transcription factors AP-1 and GATA are activated by p38 MAPK upon AngII stimulation, and both are needed to enhance TGF-beta1 expression in ventricular cardiomyocytes. TGF-beta1 acts in an autocrine loop on the cells to induce apoptosis via SMAD signaling. Thus, the often-found correlation between AngII, TGF-beta1, AP-1, and SMAD in pathogenesis of heart disease reflects the proapoptotic signaling pathway induced by AngII in cardiomyocytes.

Angiotensin II↗
PubMed2002

Characteristics of death of neonatal rat cardiomyocytes following hypoxia or hypoxia-reoxygenation: the association of apoptosis and cell membrane disintegrity.

Irreversibly injured cardiomyocytes are positive for terminal deoxynucleotidyl transferase nick end-labeling (TUNEL), making it controversial as to whether TUNEL-positive cardiomyocytes induced by hypoxia-reoxygenation are apoptotic (secondarily necrotic) or primarily necrotic. We investigated the relationship between plasma membrane integrity and DNA fragmentation in hypoxic-reoxygenated cardiomyocytes. Cardiomyocytes were prepared from neonatal rat heart and exposed to hypoxia. The plasma membrane integrity was assessed by propidium iodide (PI) staining. The mode of DNA fragmentation was assessed by TUNEL and in situ polymerase chain reaction ligation assay. Furthermore, caspase-3 activity was measured in hypoxic-reoxygenated cardiomyocytes. Reoxygenation for 24 h after 3-8 h of hypoxia increased TUNEL positivity. However, the appearance of PI-positivity preceded that of TUNEL at various time points following reoxygenation. In contrast, TUNEL-positive but PI-negative cells were rarely found. In the hypoxic-reoxygenated cells, caspase-3 activity was increased, and PI- and TUNEL-positive cardiomyocytes possessed a sufficient number of double-strand DNA breaks with single-base 3'-OH terminals. In cardiomyocytes subjected to hypoxia-reoxygenation, the appearance of TUNEL positivity was delayed in comparison to membrane disintegrity, but in these cells caspase-3 has been activated and the mode of DNA fragmentation was apoptosis-specific. Thus, hypoxia-reoxygenation induces apoptosis associated with cell membrane disintegrity in cardiomyocytes.

Animals↗
PubMed2005

Differential roles of 3H-1,2-dithiole-3-thione-induced glutathione, glutathione S-transferase and aldose reductase in protecting against 4-hydroxy-2-nonenal toxicity in cultured cardiomyocytes.

4-hydroxy-2-nonenal (HNE) plays an important role in the pathogenesis of cardiac disorders. While conjugation with glutathione (GSH) catalyzed by GSH S-transferase (GST) has been suggested to be a major detoxification mechanism for HNE in target cells, whether chemically upregulated cellular GSH and GST afford protection against HNE toxicity in cardiac cells has not been investigated. In addition, the differential roles of chemically induced GSH and GST as well as other cellular factors in detoxifying HNE in cardiomyocytes are unclear. In this study, we have characterized the induction of GSH and GST by 3H-1,2-dithiole-3-thione (D3T) and the protective effects of the D3T-elevated cellular defenses on HNE-mediated toxicity in rat H9C2 cardiomyocytes. Treatment of cardiomyocytes with D3T resulted in a significant induction of both GSH and GST as well as the mRNA expression of gamma-glutamylcysteine ligase catalytic subunit and GSTA. Both GSH and GST remained elevated for at least 72 h after removal of D3T from the culture media. Treatment of cells with HNE led to a significant decrease in cell viability and an increased formation of HNE-protein adducts. Pretreatment of cells with D3T dramatically protected against HNE-mediated cytotoxicity and protein-adduct formation. HNE treatment caused a significant decrease in cellular GSH level, which preceded the loss of cell viability. Either depletion of cellular GSH by buthionine sulfoximine (BSO) or inhibition of GST by sulfasalazine markedly sensitized the cells to HNE toxicity. Co-treatment of cardiomyocytes with BSO was found to completely block the D3T-mediated GSH elevation, which however failed to reverse the cytoprotective effects of D3T, suggesting that other cellular factor(s) might be involved in D3T cytotprotection. In this regard, D3T was shown to induce cellular aldose reductase (AR). Surprisingly, inhibition of AR by sorbinil failed to potentiate HNE toxicity in cardiomyocytes. In contrast, sorbinil dramatically augmented HNE cytotoxicity in cells with GSH depletion induced by BSO. Similarly, in BSO-treated cells, D3T cytoprotection was also largely reversed by sorbinil, indicating that AR played a significant role in detoxifying HNE only under the condition of GSH depletion in cardiomyocytes. Taken together, this study demonstrates that D3T can induce GSH, GST, and AR in cardiomyocytes, and that the above cellular factors appear to play differential roles in detoxification of HNE in cardiomyocytes.

Aldehyde Reductase↗
PubMed2005

Granulocyte colony-stimulating factor treatment enhances the efficacy of cellular cardiomyoplasty with transplantation of embryonic stem cell-derived cardiomyocytes in infarcted myocardium.

We tested the hypothesis that granulocyte colony-stimulating factor (G-CSF) administration would enhance the efficacy of cellular cardiomyoplasty with embryonic stem (ES) cell-derived cardiomyocytes in infarcted myocardium. Three weeks after myocardial infarction by cryoinjury, Sprague-Dawley rats were randomized to receive either an injection of medium, ES cell-derived cardiomyocyte transplantation, G-CSF administration, or a combination of G-CSF administration and ES cell-derived cardiomyocyte transplantation. Eight weeks after treatment, the cardiac tissue formation, neovascularization, and apoptotic activity in the infarct regions were evaluated by histology and immunohistochemistry. The left ventricular (LV) dimensions and function of the treated heart were evaluated by echocardiography. Transplanted ES cell-derived cardiomyocytes survived and participated in the myocardial regeneration in the infarcted heart. A combination of G-CSF treatment and ES cell-derived cardiomyocyte transplantation significantly promoted angiogenesis and reduced the infarct area and cell apoptosis in the infarcted myocardium compared with ES cell-derived cardiomyocyte transplantation alone. The combination therapy also attenuated LV dilation, as compared with ES cell-derived cardiomyocyte transplantation alone. G-CSF treatment can enhance the efficacy of cellular cardiomyoplasty by ES cell-derived cardiomyocyte transplantation to treat myocardial infarction.

Animals↗