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PubMed2005

Toll-like receptor 2 regulates interleukin-1beta-dependent cardiomyocyte hypertrophy triggered by Trypanosoma cruzi.

Trypanosoma cruzi, the intracellular protozoan parasite that causes Chagasic cardiomyopathy, elicits a robust hypertrophic response in isolated cardiomyocytes. Previous studies established that T. cruzi-elicited cardiomyocyte hypertrophy is mediated by interleukin-1beta produced by infected cardiomyocyte cultures. Here, we define key upstream signaling events leading to cardiomyocyte hypertrophy in response to T. cruzi infection, to be dependent on Toll-like receptor 2 and NF-kappaB. Furthermore, we demonstrate that cardiomyocyte hypertrophy, which is initiated by live infective T. cruzi trypomastigotes or stimulation of isolated myocytes with secreted/released trypomastigote molecules, is a common outcome of the cardiomyocyte recognition of pathogen-associated molecular patterns by intrinsic Toll-like receptors. This study is the first to link pathogen recognition by intrinsic Toll-like receptors to cardiomyocyte hypertrophy.

Animals↗
PubMed2004

Morphofunctional integration between skeletal myoblasts and adult cardiomyocytes in coculture is favored by direct cell-cell contacts and relaxin treatment.

The success of cellular cardiomyoplasty, a novel therapy for the repair of postischemic myocardium, depends on the anatomical integration of the engrafted cells with the resident cardiomyocytes. Our aim was to investigate the interaction between undifferentiated mouse skeletal myoblasts (C2C12 cells) and adult rat ventricular cardiomyocytes in an in vitro coculture model. Connexin43 (Cx43) expression, Lucifer yellow microinjection, Ca2+ transient propagation, and electrophysiological analysis demonstrated that myoblasts and cardiomyocytes were coupled by functional gap junctions. We also showed that cardiomyocytes upregulated gap junctional communication and expression of Cx43 in myoblasts. This effect required direct cell-to-cell contact between the two cell types and was potentiated by treatment with relaxin, a cardiotropic hormone with potential effects on cardiac development. Analysis of the gating properties of gap junctions by dual cell patch clamping showed that the copresence of cardiomyocytes in the cultures significantly increased the transjunctional current and conductance between myoblasts. Relaxin enhanced this effect in both the myoblast-myoblast and myoblast-cardiomyocyte cell pairs, likely acting not only on gap junction formation but also on the electrical properties of the preexisting channels. Our findings suggest that myoblasts and cardiomyocytes interact actively through gap junctions and that relaxin potentiates the intercellular coupling. A potential role for gap junctional communication in favoring the intercellular exchange of regulatory molecules, including Ca2+, in the modulation of myoblast differentiation is discussed.

Animals↗
PubMed1996

Mechanical stimulation of organogenic cardiomyocyte growth in vitro.

Adherent cultures of neonatal rat cardiomyocytes were subjected to progressive, unidirectional lengthening for 2-4 days in serum-containing medium. This mechanical stretch (25% increase in initial length each day) simulates the eccentric mechanical load placed on in vivo heart cells by increases in postnatal blood pressure and volume. The in vitro mechanical stimuli initiated a number of morphological alterations in the confluent cardiomyocyte population which were similar to those occurring during in vivo heart growth. These include cardiomyocyte organization into parallel arrays of rod-shaped cells, increased cardiomyocyte binucleation, and cardiomyocyte hypertrophy by longitudinal cell growth. Stretch stimulated DNA synthesis in the noncardiomyocyte population but not in the cardiomyocytes. Myosin heavy chain (MHC) content increased 62% over 4 days of stretch and included increased accumulation of both fetal beta-MHC and adult alpha-MHC isoforms. This new model of stretch-induced cardiomyocyte hypertrophy may assist in examining some of the complex mechanogenic growth processes that occur in the rapidly enlarging neonatal heart.

Animals↗
PubMed2004

Sucrose-induced cardiomyocyte dysfunction is both preventable and reversible with clinically relevant treatments.

We recently identified cardiomyocyte dysfunction in the early stage of type 2 diabetes (i.e., diet-induced insulin resistance). The present investigation was designed to determine whether a variety of clinically relevant interventions are sufficient to prevent and reverse cardiomyocyte dysfunction in sucrose (SU)-fed insulin-resistant rats. Subsets of animals were allowed to exercise (free access to wheel attached to cage) or were treated with bezafibrate in drinking water to determine whether these interventions would prevent the adverse effects of SU feeding on cardiomyocyte function. After 6-8 wk on diet and treatment, animals were surgically prepared to assess whole body insulin sensitivity (intravenous glucose tolerance test), and isolated ventricular myocyte mechanics were evaluated (video edge recording). SU feeding produced hyperinsulinemia and hypertriglyceridemia, with euglycemia, and induced characteristic whole body insulin resistance. Both exercise and bezafibrate treatment prevented these metabolic abnormalities. Ventricular myocyte shortening and relengthening were slower in SU-fed rats (42-63%) compared with starch (ST)-fed controls, and exercise or bezafibrate completely prevented cardiomyocyte dysfunction in SU-fed rats. In separate cohorts of animals, after 5 wk of SU feeding, animals were either switched back to an ST diet or given menhaden oil for an additional 7-9 wk to determine whether the cardiomyocyte dysfunction was reversible. Both interventions have previously been shown to have favorable metabolic effects, and both improved myocyte mechanics, but only the ST diet reversed all indications of cardiomyocyte dysfunction induced by SU feeding. Thus phenotypic changes in cardiomyocyte mechanics associated with early stages of type 2 diabetes were found to be both preventable and reversible with clinically relevant treatments, suggesting that the cellular processes contributing to this dysfunction are modifiable.

Animals↗
PubMed2005

Metabolic effects of insulin on cardiomyocytes from control and diabetic db/db mouse hearts.

Diabetic db/db mice exhibit profound insulin resistance in vivo, but the specific degree of cardiac insensitivity to insulin has not been assessed. Therefore, the effect of insulin on cardiomyocytes from db/db hearts was assessed by measuring two metabolic responses (deoxyglucose uptake and fatty acid oxidation) and the phosphorylation of two enzymes in the insulin-signaling cascade [Akt and AMP-activated protein kinase (AMPK)]. Maximal insulin-stimulated deoxyglucose transport was reduced to 58 and 40% of control in cardiomyocytes from db/db mice at two ages (6 and 12 wk). Insulin-stimulated deoxyglucose uptake was also reduced in myocytes from transgenic db/db mice overexpressing the insulin-sensitive glucose transporter (db/db-hGLUT4). Treatment of db/db mice for 1 wk with an insulin-sensitizing peroxisome proliferator-activated receptor-gamma agonist (COOH) completely normalized insulin-stimulated deoxyglucose uptake. Insulin had no direct effect on palmitate oxidation by either control or db/db cardiomyocytes, but the combination of insulin and glucose reduced palmitate oxidation, likely an indirect effect secondary to increased glucose uptake. Insulin had no effect on AMPK phosphorylation from either control or db/db cardiomyocytes. Insulin increased the phosphorylation of Akt in all cardiomyocyte preparations (control, db/db, COOH-treated db/db) to the same extent. Thus insulin has selective metabolic actions in mouse cardiomyocytes; deoxyglucose uptake and Akt phosphorylation are increased, but fatty acid oxidation and AMPK phosphorylation are unchanged. Insulin resistance in db/db cardiomyocytes is manifested by reduced insulin-stimulated deoxyglucose uptake.

Animals↗
PubMed2005

Clusterin: a protective mediator for ischemic cardiomyocytes?

We examined the relationship between clusterin and activated complement in human heart infarction and evaluated the effect of this protein on ischemic rat neonatal cardiomyoblasts (H9c2) and isolated adult ventricular rat cardiomyocytes as in vitro models of acute myocardial infarction. Clusterin protects cells by inhibiting complement and colocalizes with complement on jeopardized human cardiomyocytes after infarction. The distribution of clusterin and complement factor C3d was evaluated in the infarcted human heart. We also analyzed the protein expression of clusterin in ischemic H9c2 cells. The binding of endogenous and purified human clusterin on H9c2 cells was analyzed by flow cytometry. Furthermore, the effect of clusterin on the viability of ischemically challenged H9c2 cells and isolated adult ventricular rat cardiomyocytes was analyzed. In human myocardial infarcts, clusterin was found on scattered, morphologically viable cardiomyocytes within the infarcted area that were negative for complement. In H9c2 cells, clusterin was rapidly expressed after ischemia. Its expression was reduced after reperfusion. Clusterin bound to single annexin V-positive or annexin V and propidium iodide-positive H9c2 cells. Clusterin inhibited ischemia-induced death in H9c2 cells as well as in isolated adult ventricular rat cardiomyocytes in the absence of complement. We conclude that ischemia induces the upregulation of clusterin in ischemically challenged, but viable, cardiomyocytes. Our data suggest that clusterin protects cardiomyocytes against ischemic cell death via a complement-independent pathway.

Animals↗
PubMed2004

Mechanism of cardiac depression after trauma-hemorrhage: increased cardiomyocyte IL-6 and effect of sex steroids on IL-6 regulation and cardiac function.

A prolonged depression of cardiovascular function occurs in males after trauma-hemorrhagic shock (T-H). Although a correlation between increased circulatory IL-6 levels and poor outcome has been reported after T-H, it remains unknown whether T-H increases IL-6 levels locally in cardiomyocytes and whether there is a correlation between altered cardiac function and local IL-6 production after T-H. T-H was induced in normal, castrated (2 wk before T-H), and 17beta-estradiol (E2)-treated (0.5 mg sc, 1 wk before T-H) adult male rats. At 2 h after T-H or sham operation, cardiac output, heart rate, mean arterial pressure, positive and negative first derivative of pressure (+/-dP/dt), stroke volume, and total peripheral resistance were determined. Cardiomyocytes were isolated and divided into two parts: one was used for measurements of intracellular IL-6 levels using fluorescein-activated cell sorting, and the other was used to isolate RNA to determine IL-6 gene expression by quantitative real-time PCR. In addition, cardiac IL-6 protein levels were measured in freshly isolated hearts by Western blotting. Cardiac output, stroke volume, +dP/dt, -dP/dt, and total peripheral resistance were markedly altered after T-H. These parameters, except -dP/dt, improved significantly in the castrated group; however, all these parameters were restored in E2-treated males. Cardiomyocyte IL-6 mRNA expression and intracellular IL-6 production increased after T-H. Cardiac IL-6 protein levels increased after T-H in freshly isolated heart. Castration and E2 treatment attenuated cardiomyocyte intracellular IL-6 levels and cardiac IL-6 protein levels after T-H; however, only E2 treatment attenuated cardiomyocyte IL-6 gene expression. Thus there is an inverse correlation between cardiomyocyte IL-6 levels and cardiac function after T-H. The salutary effects of E2 on cardiac function after T-H may be due in part to decreased IL-6 synthesis in cardiomyocytes.

Animals↗
PubMed2003

Hexarelin protects rat cardiomyocytes from angiotensin II-induced apoptosis in vitro.

Loss of cardiomyocytes by apoptosis is proposed to cause heart failure. Angiotensin II (ANG II), an important neurohormonal factor during heart failure, can induce cardiomyocyte apoptosis. Inasmuch as hexarelin has been reported to have protective effects in this process, we examined whether hexarelin can prevent cardiomyocytes from ANG II-induced cell death. Cultured cardiomyocytes from neonatal rats were stimulated with ANG II. Apoptosis was evaluated using fluorescence microscopy, TdT-mediated dUTP nick-end labeling (TUNEL) method, flow cytometry, DNA laddering, and analysis of cell viability by (3,4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). It was found that incubation with 0.1 micromol/l ANG II for 48 h increased cardiomyocyte apoptosis. Administration of 0.1 micromol/l hexarelin significantly decreased this ANG II-induced apoptosis and DNA fragmentation and increased myocyte viability. To further investigate the underlying mechanisms, caspase-3 activity assay and mRNA expression of Bax, Bcl-2, and growth hormone secretagogue receptor (GHS-R; the supposed hexarelin binding site) were examined. GHS-R mRNA was abundantly expressed in cardiomyocytes and was upregulated after administration of hexarelin. These results suggest that hexarelin abates cardiomyocytes from ANG II-induced apoptosis possibly via inhibiting the increased caspase-3 activity and Bax expression induced by ANG II and by increasing the expression of Bcl-2, which is depressed by ANG II. Whether the upregulated expression of GHS-R induced by hexarelin is associated with this antiapoptotic effect deserves further investigation.

Angiotensin II↗
PubMed1997

Assessment of cardiomyocyte DNA synthesis in normal and injured adult mouse hearts.

Cardiomyocyte DNA synthesis was examined in normal and injured adult mouse hearts. In preliminary studied DNA synthesis was monitored by [3H]thymidine incorporation, followed by autoradiographic analysis of dispersed cell preparations. No synthetic cells were identified when 20,000 ventricular cardiomyocytes from normal adult hearts were examined. A high throughput assay was developed to establish the actual labeling index for the adult mouse heart. The assay utilized [3H]thymidine incorporation in transgenic mice which expressed a nuclear-localized beta-galactosidase (beta-Gal) reporter gene exclusively in cardiac myocytes. Cardiomyocyte DNA synthesis was evidenced by colocalization of beta-Gal activity and silver grains in autoradiograms of histological sections. Examination of 180,000 ventricular cardiomyocyte nuclei from normal adult transgenic mice identified a single synthetic nucleus, suggesting a maximum labeling index of 0.0005%. Cardiomyocyte DNA synthesis was next examined in hearts injured by focal cauterization of the left ventricular free wall. Only three synthetic nuclei were identified when 36,000 cardiomyocyte nuclei in the perinecrotic zone of the injured heart were examined. No additional synthetic nuclei were identified when 180,000 nuclei in regions distal to the necrotic zone were examined. These data confirm that cardiomyocyte DNA synthesis in the adult mouse heart is extremely rare and provide baseline data for analyses in genetically modified animals.

Animals↗
PubMed2003

Extracellular signal-regulated kinase and phosphoinositol-3 kinase mediate IGF-1 induced proliferation of fetal sheep cardiomyocytes.

Growth of the fetal heart involves cardiomyocyte enlargement, division, and maturation. Insulin-like growth factor-1 (IGF-1) is implicated in many aspects of growth and is likely to be important in developmental heart growth. IGF-1 stimulates the IGF-1 receptor (IGF1R) and downstream signaling pathways, including extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3K). We hypothesized that IGF-1 stimulates cardiomyocyte proliferation and enlargement through stimulation of the ERK cascade and stimulates cardiomyocyte differentiation through the PI3K cascade. In vivo administration of Long R3 IGF-1 (LR3 IGF-1) did not stimulate cardiomyocyte hypertrophy but led to a decreased percentage of cells that were binucleated in vivo. In culture, LR3 IGF-1 increased myocyte bromodeoxyuridine (BrdU) uptake by three- to five-fold. The blockade of either ERK or PI3K signaling (by UO-126 or LY-294002, respectively) completely abolished BrdU uptake stimulated by LR3 IGF-1. LR3 IGF-1 did not increase footprint area, but as expected, phenylephrine stimulated an increase in binucleated cardiomyocyte size. We conclude that 1) IGF-1 through IGF1R stimulates cardiomyocyte division in vivo; hyperplastic growth is the most likely explanation of IGF-1 stimulated heart growth in vivo; 2) IGF-1 through IGF1R does not stimulate binucleation in vitro or in vivo; 3) IGF-1 through IGF1R does not stimulate hypertrophy either in vivo or in vitro; and 4) IGF-1 through IGF1R requires both ERK and PI3K signaling for proliferation of near-term fetal sheep cardiomyocytes in vitro.

Animals↗
PubMed2001

A novel pharmacological action of ET-1 to prevent the cytotoxicity of doxorubicin in cardiomyocytes.

We previously reported that cardiomyocytes produce endothelin (ET)-1 and that the tissue level of ET-1 markedly increased in failing hearts in rats with chronic heart failure. Because the level of plasma ET-1 also increased progressively in patients with breast cancer who received doxorubicin (Dox; Adriamycin), which possesses cardiotoxicity, we hypothesized that ET-1 plays a role in the pathophysiology of cardiomyocytes injured by Dox. In this study, we investigated the effect of ET-1 on the cytotoxicity of Dox in primary cultured neonatal rat cardiomyocytes. The results showed that ET-1 effectively attenuated Dox-induced acute cardiomyocyte cytotoxicity (24-h incubation with Dox) evaluated by in vitro cell toxicity assay [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay and lactate dehydrogenase release]. The cytoprotective effect of ET-1 was mediated via ET(A) receptors, because pretreatment with the ET(A)-receptor antagonist BQ123 completely suppressed the cytoprotective effect of ET-1, whereas the ET(B)-receptor antagonist BQ788 did not. The cytoprotective effect of ET-1 was abolished by pretreatment with cycloheximide or staurosporine. These results suggest that a protein molecule(s), which is synthesized de novo by the stimulation of protein kinase pathway, is involved in the cytoprotective effect of ET-1. ET-1 increased the expression of an endogenous antioxidant, manganese superoxide dismutase (Mn-SOD), in the cardiomyocytes, as demonstrated by a Western blotting analysis. Pretreatment with an antisense oligodeoxyribonucleotide of Mn-SOD markedly attenuated the cytoprotective effect of ET-1 on the Dox-induced cytotoxicity. However, under conditions of prolonged incubation with Dox (48 h), ET-1 did not affect Dox-induced cardiomyocyte cytotoxicity in culture. These results suggest that ET-1 prevents the early phase of Dox-induced cytotoxicity via the upregulation of the antioxidant Mn-SOD through ET(A) receptors in cultured cardiomyocytes.

Animals↗
PubMed2006

Slow diffusion of K+ in the T tubules of rat cardiomyocytes.

Cardiomyocyte contractility is regulated by the extracellular K(+) concentration ([K(+)](o)). Potassium dynamics in the T tubules during the excitation-contraction cycle depends on the diffusion rate of K(+), but this rate is not known. Detubulation of rat cardiomyocytes was induced by osmotic shock using formamide, which separated the surface membrane from the T tubules. Changes in current and membrane potential in voltage-clamped (-80 mV) and current-clamped control and detubulated cardiomyocytes were compared during rapid switches between 5.4 and 8.1 mM [K(+)](o), and the results were simulated in a mathematical model. In the voltage-clamp experiments, the current changed significantly slower in control than in detubulated cardiomyocytes during the switch from 5.4 to 8.1 mM [K(+)](o), as indicated by the times to achieve 25, 50, 90, and 95% of the new steady-state current [control (ms) t(25) = 98 +/- 12, t(50) = 206 +/- 20, t(90) = 570 +/- 72, t(95) = 666 +/- 92; detubulated t(25) = 61 +/- 11, t(50) = 142 +/- 17, t(90) = 352 +/- 52, t(95) = 420 +/- 69]. These time points were not significantly different either during the 8.1 to 5.4 mM [K(+)](o) switch or in current-clamped cardiomyocytes switching from 5.4 to 8.1 mM [K(+)](o). Mathematical simulation of the difference current between control and detubulated cardiomyocytes gave a t-tubular diffusion rate for K(+) of approximately 85 mum(2)/s. We conclude that the diffusion of K(+) in the T tubules is so slow that they constitute a functional compartment. This might play a key role in local regulation of the action potential, and thus in the regulation of cardiomyocyte contractility.

Action Potentials↗
PubMed2006

Nox2-containing NADPH oxidase and Akt activation play a key role in angiotensin II-induced cardiomyocyte hypertrophy.

Angiotensin II (ANG II) has profound effects on the development and progression of pathological cardiac hypertrophy; however, the intracellular signaling mechanisms are not fully understood. In this study, we used genetic tools to test the hypothesis that increased formation of superoxide (O2-*) radicals from a Rac1-regulated Nox2-containing NADPH oxidase is a key upstream mediator of ANG II-induced activation of serine-threonine kinase Akt, and that this signaling cascade plays a crucial role in ANG II-dependent cardiomyocyte hypertrophy. ANG II caused a significant time-dependent increase in Rac1 activation and O2-* production in primary neonatal rat cardiomyocytes, and these responses were abolished by adenoviral (Ad)-mediated expression of a dominant-negative Rac1 (AdN17Rac1) or cytoplasmic Cu/ZnSOD (AdCu/ZnSOD). Moreover, both AdN17Rac1 and AdCu/ZnSOD significantly attenuated ANG II-stimulated increases in cardiomyocyte size. Quantitative real-time PCR analysis demonstrated that Nox2 is the homolog expressed at highest levels in primary neonatal cardiomyocytes, and small interference RNA (siRNA) directed against it selectively decreased Nox2 expression by >95% and abolished both ANG II-induced O2-* generation and cardiomyocyte hypertrophy. Finally, ANG II caused a time-dependent increase in Akt activity via activation of AT(1) receptors, and this response was abolished by Ad-mediated expression of cytosolic human O2-* dismutase (AdCu/ZnSOD). Furthermore, pretreatment of cardiomyocytes with dominant-negative Akt (AdDNAkt) abolished ANG II-induced cellular hypertrophy. These findings suggest that O2-* generated by a Nox2-containing NADPH oxidase is a central mediator of ANG II-induced Akt activation and cardiomyocyte hypertrophy, and that dysregulation of this signaling cascade may play an important role in cardiac hypertrophy.

Adenoviridae↗
PubMed2003

Bone marrow-derived cardiomyocytes are present in adult human heart: A study of gender-mismatched bone marrow transplantation patients.

BACKGROUND: Recent studies have identified cardiomyocytes of extracardiac origin in transplanted human hearts, but the exact origin of these myocyte progenitors is currently unknown. METHODS AND RESULTS: Hearts of female subjects (n=4) who had undergone sex-mismatched bone marrow transplantation (BMT) were recovered at autopsy and analyzed for the presence of Y chromosome-positive cardiomyocytes. Four female gender-matched BMT subjects served as controls. Fluorescence in situ hybridization (FISH) for the Y chromosome was performed on paraffin-embedded sections to identify cells of bone marrow origin with concomitant immunofluorescent labeling for alpha-sarcomeric actin to identify cardiomyocytes. A total of 160 000 cardiomyocyte nuclei were analyzed approximating 20 000 nuclei per patient. The mean percentage of Y chromosome-positive cardiomyocytes in patients with sex-mismatched BMT was 0.23+/-0.06%. Not a single Y chromosome-positive cardiomyocyte was identified in any of the control patients. Immunofluorescent costaining for laminin and chromosomal ploidy analysis with FISH showed no evidence of either pseudonuclei or cell fusion in any of the chimeric cardiac myocytes identified. CONCLUSIONS: These data establish for the first time human bone marrow as a source of extracardiac progenitor cells capable of de novo cardiomyocyte formation.

Actins↗
PubMed2004

Aerobic fitness is associated with cardiomyocyte contractile capacity and endothelial function in exercise training and detraining.

BACKGROUND: Physical fitness and level of regular exercise are closely related to cardiovascular health. A regimen of regular intensity-controlled treadmill exercise was implemented and withdrawn to identify cellular mechanisms associated with exercise capacity and maximal oxygen uptake (VO2max). METHODS AND RESULTS: Time-dependent associations between cardiomyocyte dimensions, contractile capacity, and VO2max were assessed in adult rats after high-level intensity-controlled treadmill running for 2, 4, 8, and 13 weeks and detraining for 2 and 4 weeks. With training, cardiomyocyte length, relaxation, shortening, Ca2+ decay, and estimated cell volume correlated with increased VO2max (r=0.92, -0.92, 0.88, -0.84, 0.73; P<0.01). Multiple regression analysis identified cell length, relaxation, and Ca2+ decay as the main explanatory variables for VO2max (R2=0.87, P<0.02). When training stopped, exercise-gained VO2max decreased 50% within 2 weeks and stabilized at 5% above sedentary controls after 4 weeks. Cardiomyocyte size regressed in parallel with VO2max and remained (9%) above sedentary after 4 weeks, whereas cardiomyocyte shortening, contraction/relaxation- and Ca2+-transient time courses, and endothelium-dependent vasorelaxation regressed completely within 2 to 4 weeks of detraining. Cardiomyocyte length, estimated cell volume, width, shortening, and Ca2+ decay and endothelium-dependent arterial relaxation all correlated with VO2max (r=0.85, 0.84, 0.75, 0.63, -0.54, -0.37; P<0.01). Multiple regression identified cardiomyocyte length and vasorelaxation as the main determinants for regressed VO2max during detraining (R2=0.76, P=0.02). CONCLUSIONS: Cardiovascular adaptation to regular exercise is highly dynamic. On detraining, most of the exercise-gained aerobic fitness acquired over 2 to 3 months is lost within 2 to 4 weeks. The close association between cardiomyocyte dimensions, contractile capacity, arterial relaxation, and aerobic fitness suggests cellular mechanisms underlying these changes.

Acetylcholine↗
PubMed2005

Adenovirus-mediated overexpression of diacylglycerol kinase-zeta inhibits endothelin-1-induced cardiomyocyte hypertrophy.

BACKGROUND: Diacylglycerol (DAG) is a lipid second messenger that transiently accumulates in cells stimulated by endothelin-1 (ET-1) and other Galphaq protein-coupled receptor agonists. Diacylglycerol kinase (DGK) is thought to be an enzyme that controls the cellular levels of DAG by converting it to phosphatidic acid; however, the functional role of DGK has not been examined in cardiomyocytes. Because DGK inactivates DAG, a strong activator of protein kinase C (PKC), we hypothesized that DGK inhibited ET-1-induced activation of a DAG-PKC signaling cascade and subsequent cardiomyocyte hypertrophy. METHODS AND RESULTS: Real-time reverse transcription-polymerase chain reaction demonstrated a significant increase of DGK-zeta mRNA by ET-1 in cardiomyocytes. To determine the functional role of DGK-zeta, we overexpressed DGK-zeta in cardiomyocytes using a recombinant adenovirus encoding rat DGK-zeta (Ad-DGKzeta). ET-1-induced translocation of PKC-epsilon was blocked by Ad-DGKzeta (P<0.01). Ad-DGKzeta also inhibited ET-1-induced activation of extracellular signal-regulated kinase (P<0.01). Luciferase reporter assay revealed that ET-1-mediated increase of activator protein-1 (AP1) DNA-binding activity was significantly inhibited by DGK-zeta (P<0.01). In cardiomyocytes transfected with DGK-zeta, ET-1 failed to cause gene induction of atrial natriuretic factor, increases in [3H]-leucine uptake, and increases in cardiomyocyte surface area. CONCLUSIONS: We demonstrated for the first time that DGK-zeta blocked ET-1-induced activation of the PKC-epsilon-ERK-AP1 signaling pathway, atrial natriuretic factor gene induction, and resultant cardiomyocyte hypertrophy. DGK-zeta might act as a negative regulator of hypertrophic program in response to ET-1, possibly by controlling cellular DAG levels.

Adenoviridae↗
PubMed2000

Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart.

BACKGROUND: IGF-1 has been shown to protect myocardium against death in animal models of infarct and ischemia-reperfusion injury. In the present study, we investigated the role of the IGF-1-regulated protein kinase Akt in cardiac myocyte survival in vitro and in vivo. METHODS AND RESULTS: IGF-1 promoted survival of cultured cardiomyocytes under conditions of serum deprivation in a dose-dependent manner but had no effect on cardiac fibroblast survival. The cytoprotective effect of IGF-1 on cardiomyocytes was abrogated by the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin. Wortmannin had no effect on cardiomyocyte viability in the absence of IGF-1. IGF-1-mediated cytoprotection correlated with the wortmannin-sensitive induction of Akt protein kinase activity. To examine the functional consequences of Akt activation in cardiomyocyte survival, replication-defective adenoviral constructs expressing wild-type, dominant-negative, and constitutively active Akt genes were constructed. Transduction of dominant-negative Akt blocked IGF-1-induced survival but had no effect on cardiomyocyte survival in the absence of IGF-1. In contrast, transduction of wild-type Akt enhanced cardiomyocyte survival at subsaturating levels of IGF-1, whereas constitutively active Akt protected cardiomyocytes from apoptosis in the absence of IGF-1. After transduction into the mouse heart in vivo, constitutively active Akt protected against myocyte apoptosis in response to ischemia-reperfusion injury. CONCLUSIONS: These data are the first documentation that Akt functions to promote cellular survival in vivo, and they indicate that the activation of this pathway may be useful in promoting myocyte survival in the diseased heart.

Animals↗
PubMed2002

Dual actions of the Galpha(q) agonist Pasteurella multocida toxin to promote cardiomyocyte hypertrophy and enhance apoptosis susceptibility.

Previous attempts to delineate the consequences of Galpha (q) activation in cardiomyocytes relied largely on molecular strategies in cultures or transgenic mice. Modest levels of wild-type Galpha(q) overexpression induce stable cardiac hypertrophy, whereas intense Galpha(q) stimulation induces cardiomyocyte apoptosis. The precise mechanism(s) whereby traditional targets of Galpha (q) subunits that induce hypertrophy also trigger cardiomyocyte apoptosis is not obvious and is explored with recombinant Pasteurella multocida toxin (rPMT, a Galpha(q) agonist). Cells cultured with rPMT display cardiomyocyte enlargement, sarcomeric organization, and increased atrial natriuretic factor expression in association with activation of phospholipase C, novel protein kinase C (PKC) isoforms, extracellular signal-regulated protein kinase (ERK), and (to a lesser extent) JNK/p38-MAPK. rPMT stimulates the ERK cascade via epidermal growth factor (EGF) receptor transactivation in cardiac fibroblasts, but EGF receptor transactivation plays no role in ERK activation in cardiomyocytes. Surprisingly, rPMT (or novel PKC isoform activation by PMA) decreases basal Akt phosphorylation; rPMT prevents Akt phosphorylation by EGF or IGF-1 and functionally augments cardiomyocyte apoptosis in response to H2O2. These results identify a Galpha(q)-PKC pathway that represses basal Akt phosphorylation and impairs Akt stimulation by survival factors. Because inhibition of Akt enhances cardiomyocyte susceptibility to apoptosis, this pathway is predicted to contribute to the transition from hypertrophy to cardiac decompensation and could be targeted for therapy in heart failure.

Animals↗