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PubMed2003

Transdifferentiation of blood-derived human adult endothelial progenitor cells into functionally active cardiomyocytes.

BACKGROUND: Further to promoting angiogenesis, cell therapy may be an approach for cardiac regeneration. Recent studies suggest that progenitor cells can transdifferentiate into other lineages. However, the transdifferentiation potential of endothelial progenitor cells (EPCs) is unknown. METHODS AND RESULTS: EPCs were obtained from peripheral blood mononuclear cells of healthy adults or coronary artery disease (CAD) patients by cultivating with endothelial cell medium and growth factors. After 3 days, >95% of adherent cells were functionally and phenotypically EPCs. Diacetylated LDL-labeled EPCs were then cocultivated with rat cardiomyocytes for 6 days, resulting in significant increases of EPC cell length and size to a cardiomyocyte-like morphology. Biochemically, 9.94+/-1.39% and 5.04+/-1.09% of EPCs from healthy adults (n=15) or CAD patients (n=14, P<0.01 versus healthy adults), respectively, expressed alpha-sarcomeric actinin as measured by flow cytometry. Immunocytochemistry showed that human EPCs expressed alpha-sarcomeric actinin, cardiac troponin I (both with partial sarcomeric organization), atrial natriuretic peptide, and myocyte enhancer factor 2. Fluo 4 imaging demonstrated calcium transients synchronized with adjacent rat cardiomyocytes in transdifferentiated human EPCs. Single-cell microinjection of Lucifer yellow and calcein-AM labeling of cardiomyocytes demonstrated gap junctional communication between 51+/-7% of EPCs (16 hours after labeling, n=4) and cardiomyocytes. EPC transdifferentiation into cardiomyocytes was not observed with conditioned medium but in coculture with paraformaldehyde-fixed cardiomyocytes. CONCLUSIONS: EPCs from healthy volunteers and CAD patients can transdifferentiate in vitro into functionally active cardiomyocytes when cocultivated with rat cardiomyocytes. Cell-to-cell contact but not cellular fusion mediates EPC transdifferentiation. The therapeutic use of autologous EPCs may aid cardiomyocyte regeneration in patients with ischemic heart disease.

Adult↗
PubMed1999

Formation of cell junctions between grafted and host cardiomyocytes at the border zone of rat myocardial infarction.

BACKGROUND: Cardiomyocyte transplantation is an innovative strategy for the treatment of heart failure after myocardial infarction. Cell junctions show diverse temporal polarization toward intercalated disks during postnatal development and exhibit altered distribution in diseased hearts. To elucidate the formation of cell junctions between grafted and host cardiomyocytes at the border zone of myocardial infarction, the 3D distribution of cell junctions was examined using immunohistochemistry and confocal microscopy. METHODS AND RESULTS: Neonatal cardiomyocytes obtained from 3-day-old rats by collagenase digestion and Percoll density centrifugation were injected into the border zones of infarction sites 10 days after coronary ligation in adult rats. At 4 to 14 days after transplantation, hearts were harvested and processed by immunohistochemistry. Antibodies against connexin43, desmoplakin, and cadherin were used to analyze the distribution of gap junctions, desmosomes, and adherens junctions, respectively. Grafted cardiomyocytes were identified by immunohistochemistry for alpha-smooth muscle actin. Grafted cardiomyocytes tended to align parallel to the host cardiomyocytes. Connexin43, desmoplakin, and cadherin were localized between grafted cardiomyocytes themselves and between grafted and host cardiomyocytes. Semiquantitative analysis revealed that all junctions showed increasing polarization to longitudinal cell termini, especially at the border of grafted and host cardiomyocytes, as time advanced from 4 to 7 days after transplantation. CONCLUSIONS: These findings indicate that grafted cardiomyocytes foster electrical pathways with host counterparts through the gap junction and suggest that the environment in infarcted hearts could influence the localization of gap junctions, desmosomes, and adherens junctions.

Animals↗
PubMed2005

Cardiomyocyte cell cycle activation ameliorates fibrosis in the atrium.

MHC-TGFcys33ser transgenic mice have elevated levels of active transforming growth factor (TGF)-beta1 in the myocardium. Previous studies have shown that these animals develop atrial, but not ventricular, fibrosis. Here we show that atrial fibrosis was accompanied with cardiomyocyte apoptosis. Although similar levels of cardiomyocyte apoptosis were present in the right and left atria of MHC-TGFcys33ser hearts, the extent of fibrosis was more pronounced in the right atrium. Thus, additional factors influence the degree of atrial fibrosis in this model. Tritiated thymidine incorporation studies revealed cardiomyocyte cell cycle activity in left atrial cardiomyocytes, but not in right atrial cardiomyocytes. These observations suggested that cardiomyocyte cell cycle activation ameliorated the severity of atrial fibrosis. To directly test this hypothesis, MHC-TGFcys33ser mice were crossed with MHC-cycD2 mice (which have constitutive cardiomyocyte cell cycle activity in the right atrium). Mice inheriting both transgenes exhibited right atrial cardiomyocyte cell cycle activity and a concomitant reduction in the severity of right atrial fibrosis, despite the presence of a similar level of cardiomyocyte apoptosis as was observed in mice inheriting the MHC-TGFcys33ser transgene alone. These data support the notion that cardiomyocyte cell cycle induction can antagonize fibrosis in the myocardium.

Animals↗
PubMed1996

In vivo survival and function of transplanted rat cardiomyocytes.

Recent studies have demonstrated the feasibility of transplanting fetal mouse cardiomyocytes into the hearts of adult syngeneic mice. However, the function of the transplanted cardiomyocytes and their capacity to survive in fibrous connective tissue were not assessed. In the present study, we evaluated the viability and contractility of transplanted fetal and neonatal rat cardiomyocytes in the connective tissue of the adult rat hindlimb. Purified fetal or neonatal rat cardiomyocytes were cultured. These cells contained sarcomeres, formed junctions composed of desmosomes and fascia adherens, and contracted regularly and spontaneously. A fetal or neonatal cardiomyocyte suspension was injected into the subcutaneous tissue of adult rat hindlimbs. Cyclosporin A (5 mg/kg) was administered subcutaneously daily for the 3-month duration of the study, at which time the animals were killed. The transplanted cardiomyocytes formed 'tissue' in vivo that increased in size for the first 2 weeks and remained the same size at the third week. The tissue derived from the transplanted fetal cardiomyocytes contracted spontaneously at a rate of 73 +/- 12 bpm, and that from the neonatal cardiomyocytes contracted at a rate of 43 +/- 21 bpm. The electrocardiogram was similar to that seen in myocardium with an idioventricular rhythm. Histologically, the tissue appeared to be cardiac muscle with sarcomeres. Angiogenesis occurred in the cardiomyocyte graft. In summary, a cell suspension of cultured fetal and neonatal rat cardiomyocytes transplanted into the adult rat hindlimb formed contractile cardiac tissue in the subcutaneous connective tissue.

Animals↗
PubMed1995

Stable fetal cardiomyocyte grafts in the hearts of dystrophic mice and dogs.

This report documents the formation of stable fetal cardiomyocyte grafts in the myocardium of dystrophic dogs. Preliminary experiments established that the dystrophin gene product could be used to follow the fate of engrafted cardiomyocytes in dystrophic mdx mice. Importantly, ultrastructural analyses revealed the presence of intercalated discs consisting of fascia adherens, desmosomes, and gap junctions at the donor-host cardiomyocyte border. To determine if isolated cardiomyocytes could form stable intracardiac grafts in a larger species, preparations of dissociated fetal canine cardiomyocytes were delivered into the hearts of adult CXMD (canine X-linked muscular dystrophy) dogs. CXMD dogs, like Duchenne muscular dystrophy patients and mdx mice, fail to express dystrophin in both cardiac and skeletal muscle. Engrafted fetal cardiomyocytes, identified by virtue of dystrophin immunoreactivity, were observed to be tightly juxtaposed with host cardiomyocytes as long as 10 wk after engraftment, the latest date analyzed. Confocal laser scanning microscopy revealed the presence of connexin43, a major constituent of the gap junction, at the donor-host cardiomyocyte border. The presence of intracardiac grafts was not associated with arrhythmogenesis in the CXMD model. These results indicate that fetal cardiomyocyte grafting can successfully augment cardiomyocyte number in larger animals.

Animals↗
PubMed2005

Effect of intrauterine growth restriction on the number of cardiomyocytes in rat hearts.

Epidemiologic studies have linked intrauterine growth restriction (IUGR) with an increased incidence of cardiovascular disease later in life; reduced cardiomyocyte number in IUGR hearts may underlie such prenatal programming. Our aim was to examine the effect of IUGR, as a result of maternal protein restriction, on the number of cardiomyocytes in the rat heart at birth. Rats were fed either a low-protein diet (LPD) or a normal-protein diet (NPD) during pregnancy. At birth, the offspring were killed and the hearts were immersion-fixed. The number of cardiomyocyte nuclei in the hearts were stereologically determined using an optical disector-fractionator approach. In some litters, cardiomyocytes were enzymatically isolated from freshly excised hearts and the proportion of binucleated cells was determined. Taking into account the number of binucleated cells, the nuclear counts were adjusted to estimate total cardiomyocyte number. Birth weight and heart weight were significantly reduced in the LPD offspring. This was accompanied by a significant reduction in the number of cardiomyocytes per heart in the LPD offspring compared with the NPD offspring (1.18 +/- 0.05 x 10(7) and 1.41 +/- 0.06 x 10(7), respectively; p = 0.001). The number of binucleated cardiomyocytes was low (approximately 3%) and equal in both groups. In conclusion, IUGR as a result of maternal protein restriction leads to a reduction in the number of cardiomyocytes per heart. As cardiomyocyte proliferation is rare after birth, it is plausible that this reduction in cardiomyocytes may lead to compromised cardiac function later in life.

Animals↗
PubMed2006

Apoptosis of cardiomyocytes in myocarditis.

Apoptosis of cardiomyocytes has been reported to be involved in the pathogenesis of heart failure of different aetiologies. The purpose of this study is to assess the role and extent of apoptosis of cardiomyocytes in active myocarditis. Endomyocardial samples from the right ventricle of 22 patients with active myocarditis were compared with 25 traffic accident victims without a history of cardiovascular disease. Twenty-two patients fulfilled the histopathologic Dallas criteria for myocarditis. The TUNEL method and immunostaining for active caspase 3 were used for the detection of apoptosis. Immunohistochemical methods were used for the evaluation of regulators of apoptosis (p53, Bcl-2) and evaluation of interstitial cells (macrophages, T and B lymphocytes). Apoptosis of cardiomyocytes (TUNEL-positive and anti-caspase 3-positive cardiomyocytes), which was not p53-dependent, was present in 0.3 to 0.4% (0.3% by TUNEL method and 0.4% by immunostaining for active caspase 3) of cardiomyocytes in active myocarditis, whereas only few apoptotic cardiomyocytes (0.0006 +/- 0.002% TUNEL-positive cardiomyocytes and 0.001 +/- 0.002% active caspase 3-positive cardiomyocytes) were found in the control group (P = 0.001). Apoptotic (TUNEL-positive and active caspase 3-positive) cardiomyocytes were found in small clusters. An increased expression of Bcl-2 was found in active myocarditis compared to the controls (P < 0.01), yet Bcl-2 failed to protect myocytes from apoptosis. We provide evidence of apoptosis of cardiomyocytes in active myocarditis, which may be involved in the development of heart failure.

Apoptosis↗
PubMed2006

Effect of eicosapentaenoic acid on the different endothelin system components in endothelin-1-induced hypertrophied cardiomyocytes.

The cardiovascular benefit of fish oil, including eicosapentaenoic acid (EPA), in humans and experimental animals has been reported. The role of endothelin-1 (ET-1) in cardiac hypertrophy is well known. Endothelin-1 stimulates prepro-ET-1 mRNA expression in cardiomyocytes, and the autocrine/paracrine system of ET-1 is important for cardiomyocyte hypertrophy. Although many studies link EPA to cardiac protection, the effect of EPA on cardiac hypertrophy has yet to be clarified. Recently, we demonstrated that ET-1-induced cardiomyocytic change could be prevented by pretreatment with EPA. The present study investigated the changes of different components of the ET system at the mRNA level in ET-1-administered cardiomyocytes, and examined the effect of EPA pretreatment. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats, cultured in Dulbecco's modified Eagle's medium and Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into 3 groups: control group, ET-1-treated (0.1 nM) group, and ET-1-treated group pretreated with EPA (10 microM). Twenty-four hours after treatment, the gene expressions of different components of the endothelin system in three experimental groups were evaluated by real-time polymerase chain reaction. Prepro-ET-1 mRNA expression was 53% upregulated in ET-1-induced hypertrophied cardiomyocytes and suppressed in the EPA-pretreated group. Endothelin-converting enzyme-1 (ECE-1) was also increased in ET-1-administered cardiomyocytes by 42% compared with the control group and was reversed in the EPA-pretreated group. The two receptors of ET system, ET(A) and ET(B), tended to be increased in the ET-1-treated group, but no statistical significance was seen among study groups. Endothelin-1 increased prepro-ET-1 and ECE-1 mRNA expression in hypertrophied-neonatal cardiomyocytes, and this was reversed with EPA pretreatment. Thus, EPA may play a crucial role in the regression of ET-1-induced cardiomyocyte hypertrophy, partly through the suppression of ET-1 and ECE-1 expression.

Animals↗
PubMed2006

[Electrophysiological effects of hyperpolarization activated cyclic nucleotide gated channel 4 overexpression in neonatal cardiomyocytes mediated by recombinant adenovirus].

OBJECTIVE: To explore the electrophysiological effects of hyperpolarization activated cyclic nucleotide gated channel 4 (HCN4) overexpression in rat neonatal cardiomyocytes mediated by recombinant adenovirus. METHODS: Ventricular cardiomyocytes were obtained from 20 neonatal rats. Recombinant adenovirus carrying HCN4 gene, AdHCN4, a dominant isoform of hyperpolarization activated cyclic nucleotide gated cation channel gene in cardiac transduction system, was constructed and used to transfect the neonatal rat ventricular cardiomyocytes. Untransfected cardiomyocytes were used as controls. RT-PCR and immunofluorescence cytochemistry were used to detect the HCN4 mRNA and protein expression. The electrophysiological characteristics of infected cardiomyocytes were studied by patch clamp. RESULTS: The mRNA and protein expression levels of HCN4 in AdHCN4 infected cardiomyocytes were both markedly higher than those of the control cardiomyocytes. AdHCN4 caused a significant increase in The spontaneous rate in the transfected cardiomyocytes was 92.5 + 7.4 bpm, significantly higher than that of the control cells (68.9 + 6.2 bpm, P < 0.05). Patch clamp experiments showed that the pacemaker current density in the AdHCN4 infected cardiomyocytes was 115.7 + 7.8 pA/pF, significantly higher than that of the untransfected cells (7.2 + 0.6 pA/pF, P < 0.05). CONCLUSION: Overexpression of HCN4 can enhance the autorhythmicity of neonatal cardiomyocytes and significantly increase the spontaneous beat rate. HCN4 channel gene may represent a candidate gene in gene therapy for bradycardia diseases.

Action Potentials↗
PubMed2006

The cardiomyocyte cell cycle.

Many forms of cardiac disease are characterized by cardiomyocyte death due to necrosis, apoptosis and/or oncosis. Recently, the notion of promoting cardiac regeneration as a means to replace damaged heart tissue has engendered considerable interest. One approach to accomplish heart muscle regeneration entails promoting cardiomyocyte cell cycle activity in the surviving myocardium. Genetically modified mice have provided useful model systems to test the efficacy of specific pathways to promote cardiomyocyte proliferation in normal and diseased hearts. For example, expression of a heart-restricted dominant interfering version of p193 (an E3 ubiquitin ligase also known as Cul7) resulted in an induction of cardiomyocyte cell cycle activity at the infarct border zone and ventricular septum 4 weeks after permanent coronary artery occlusion. A concomitant reduction in hypertrophic cardiomyocyte growth was also observed in this model, suggesting that cell cycle activation partially counteracted the adverse ventricular remodelling that occurs post-infarction. In other studies, targeted expression of cyclin D2 promoted cardiomyocyte cell cycle activity in adult hearts. The level of cardiomyocyte cell cycle activity increased after myocardial infarction, ultimately resulting in a marked increase in cardiomyocyte number and a concomitant regression of infarct size. Collectively, these data suggest that modulation of cardiomyocyte cell cycle activity can be exploited to promote regenerative growth in injured hearts.

Animals↗
PubMed2004

Cardiomyocyte hyperplasia after plasmid-mediated vascular endothelial growth factor gene transfer in pigs with chronic myocardial ischemia.

BACKGROUND: For over 40 years it has been proposed that cardiomyocyte hyperplasia may occur in hypertrophic human hearts. While this implies that heart myocytes can undergo cytokinesis, evidence of conventional cell division has been exceptionally reported. Recently, we found that gene transfer of vascular endothelial growth factor (VEGF) displays a mitogenic effect on adult cardiomyocytes. In the present study we searched for cardiomyocyte hyperplasia as evidence of VEGF-induced cardiomyocyte cytokinesis. METHODS: Three weeks after implanting an Ameroid constrictor at the origin of the left circumflex artery, 16 pigs were randomized to receive 10 direct intramyocardial injections of 3.8 mg of plasmid encoding for VEGF (pVEGF) or empty plasmid. Five weeks later, hearts were weighed, myocyte diameter was measured in tissue sections, and myocyte length and nuclei number were studied in isolated myocytes. A resting echocardiogram was performed immediately before reoperation and before sacrifice to evaluate global and regional left ventricular function. Investigators were blinded to the study groups and nature of the injectate until the end of data analysis. RESULTS: No heart weight differences existed between groups. However, in the ischemic myocardium, pVEGF-treated hearts had 22% more cardiomyocytes per unit volume and exhibited significantly more oligonucleated (1 or 2 nuclei) cardiomyocytes than hearts receiving empty plasmid. CONCLUSIONS: In pigs with chronic myocardial ischemia, VEGF gene transfer induced cardiomyocyte cytokinesis, as revealed by cardiomyocyte hyperplasia. Our finding extends the previously reported mitogenic effect of VEGF on adult cardiomyocytes and supports the hypothesis that VEGF may have a therapeutic role in diseases characterized by myocardial cell loss.

Animals↗
PubMed1996

Mild hypoxia induces hypertrophy of cultured neonatal rat cardiomyocytes: a possible endogenous endothelin-1-mediated mechanism.

Hypoxic or ischemic stresses on cardiomyocytes may cause a variety of compensatory responses including cell hypertrophy. In this study, we examined whether hypoxia induces hypertrophy of cardiomyocytes in vitro and whether hypoxia-induced hypertrophy is inhibited by an endothelin A receptor antagonist (BQ123). Neonatal rat cardiomyocytes were cultured in 10% O2/85% N2/5% CO2 or 95% N2/5% CO2 to produce a mild or severe hypoxic condition, respectively. Cardiomyocytes exposed to severe hypoxia revealed degenerative morphological changes and a decrease of cell number, suggesting the toxicity of severe hypoxia on cardiomyocytes. In contrast, cardiomyocytes with mild hypoxia developed hypertrophy; cell surface area of cardiomyocytes as evaluated by an image analyser system increased by 1.6-fold over control after 48 h. [3H]leucine incorporation into the cells was significantly increased by mild hypoxia but decreased by severe hypoxia, mRNA level of skeletal alpha-actin, a genetic marker of cardiac hypertrophy, up-regulated after 6-24 h by mild hypoxia. A transient increase of preproET-1 mRNA and a time-dependent increase of ET-1 protein in the culture medium were also observed in cardiomyocytes exposed to mild hypoxia. BQ123 partially inhibited either hypoxia-induced [3H]leucine incorporation or skeletal alpha-actin mRNA in a dose-dependent manner. These data suggest that mild hypoxia induces hypertrophy of cardiomyocytes and that activation of endogenous ET-1 may, at least in part, mediate this hypertrophic responses as an autocrine/ paracrine factor.

Actins↗
PubMedDate not supplied

Temperature regulates the arrhythmogenic activity of pulmonary vein cardiomyocytes.

Temperature plays an important role in the electrophysiology of cardiomyocytes. Pulmonary veins (PVs) are known to initiate paroxysmal atrial fibrillation. The effects of temperature on the arrhythmogenic activity of rabbit single PV and atrial cardiomyocytes were assessed using the whole-cell clamp technique. PV cardiomyocytes had different beating rates at low (22-25 degrees C), normal (38-39 degrees C) and high (40-41 degrees C) temperatures (0.9 +/- 0.1, 3.2 +/- 0.4, 6.4 +/- 0.6 Hz, respectively; p < 0.001). There were different action potential durations and incidences of delayed afterdepolarization in PV cardiomyocytes with pacemaker activity (31, 59, 63%; p < 0.05), PV cardiomyocytes without pacemaker activity (16, 47, 60%; p < 0.001), and atrial myocytes (0, 0, 21%; p < 0.05). However, oscillatory afterpotentials were only found in PV cardiomyocytes with pacemaker activity at normal (50%) or high (68%) temperatures, but not at low temperatures (p < 0.001). Both PV and atrial cardiomyocytes had larger transient inward currents and inward rectified currents at high temperatures. Additionally, PV cardiomyocytes with and without pacemaker activity had larger pacemaker currents at higher temperatures. This study demonstrated that PV cardiomyocytes have an increase in arrhythmogenic activity at high temperatures because of enhanced automaticity, induced triggered activity, or shortening of action potential duration.

Action Potentials↗
PubMed2006

Postconditioning attenuates cardiomyocyte apoptosis via inhibition of JNK and p38 mitogen-activated protein kinase signaling pathways.

A sequence of intermittent interruptions of oxygen supply (i.e., postconditioning, Postcon) at reoxygenation reduces oxidant-induced cardiomyocyte loss. This study tested the hypothesis that prevention of cardiomyocyte apoptosis by Postcon is mediated by mitogen-activated protein kinases pathways. Primary cultured neonatal rat cardiomyocytes were exposed to 3 h hypoxia followed by 6 h of reoxygenation. Cardiomyocytes were postconditioned by three cycles each of 5 min reoxygenation and 5 min hypoxia after prolonged hypoxia. Relative to hypoxia alone, reoxygenation stimulated expression of JNKs and p38 kinases, corresponding to increased activity of JNKs (phospho-c-Jun) and p38 (phospho-ATF2). The level of TNFalpha in cell lysates, activity of cytosolic caspases-8, -3, expression of Bax and the number of apoptotic cardiomyocytes were increased while expression of Bcl-2 was decreased with reoxygenation. Consistent with an attenuation in generation of superoxide anions detected by lucigenin-enhanced chemiluminescence at early period of reoxygenation, treatment of cardiomyocytes with Postcon further reduced expression and activity of JNKs and p38 kinases, level of TNFalpha, the frequency of apoptotic cells and expression of Bax. However, the inhibitory effects of Postcon on these changes were lost when its application was delayed by 5 min after the start of reoxygenation. Addition of a JNK/p38 stimulator, anisomycin into cardiomyocytes at the beginning of reoxygenation eliminated protection by Postcon. These data suggest that 1) hypoxia/reoxygenation elicits cardiomyocyte apoptosis in conjunction with expression and activation of JNK and p38 kinases, release of TNFalpha, activation of caspases, and an increase in imbalance of pro-/anti-apoptotic proteins; 2) Postcon attenuates cardiomyocyte apoptosis, potentially mediated by inhibiting JNKs/p-38 signaling pathways and reducing TNFalpha release and caspase expression.

Animals↗
PubMed2002

Inhibition of tumor necrosis factor-alpha-dependent cardiomyocyte apoptosis by metallothionein.

Myocardial cell death is an important cellular event of heart failure. Tumor necrosis factor-alpha (TNF) accumulates in the failing heart and causes myocyte apoptosis, but the mechanism of this action is unclear. This study was undertaken to examine the relationship between TNF-induced cardiomyocyte apoptosis and activation of p38 mitogen-activated protein kinase (MAPK) through oxidative stress. Primary cultures of neonatal cardiomyocytes isolated from transgenic mouse hearts that overexpress metallothionein (MT) as well as cardiomyocytes isolated from wild-type mice were used. The treatment of wildtype cardiomyocytes with TNF at 10 ng/mL induced apoptosis, as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and confirmed by Annexin V-fluorescein isothiocyanate binding. The apoptotic effect of TNF was significantly inhibited in the MT-overexpressing cardiomyocytes. Corresponding to the apoptotic effect, TNF at 10 ng/mL caused rapid phosphorylation of p38 MAPK in wild-type cardiomyocytes. The activation of p38 MAPK was further confirmed by an in vivo experiment treating the mice with TNF and measuring p38 MAPK activity using an immune complex kinase assay. The activation of p38 MAPK was not observed in the MT-overexpressing cardiomyocytes either in vitro or in vivo. Importantly, TNF-induced accumulation of reactive oxygen species was dramatically reduced in the MT-overexpressing cardiomyocytes as determined by a carboxy-H(2)-DCFDA staining method. This study thus suggests that p38 MAPK activation is likely involved in TNFinduced cardiomyocyte apoptosis, which is also related to reactive oxygen species accumulation.

Animals↗
PubMed1996

Human pediatric and adult ventricular cardiomyocytes in culture: assessment of phenotypic changes with passaging.

OBJECTIVES: The purpose of this study was to assess morphologically and biochemically the phenotypic changes which occur in vitro with passaging of human pediatric and adult ventricular cardiomyocytes. METHODS: Human ventricular cardiomyocytes from 3 children (1 to 2 years of age) and an adult patient (65 years of age) undergoing open heart surgery and an adult heart transplant patient (55 years of age) were isolated, cultured, purified, and passaged. Growth curves and 3H-thymidine uptake studies were performed. Characterization of the cells was done by light microscopy, transmission electron microscopy, immunofluorescent staining for myoglobin, CK-MB, and cardiac-specific troponin I isoform, human ventricular myosin heavy chain (HVMHC) and light chain 1 (HVMLC1), Northern blot analysis of HVMHC, and CK-MB activity and mass measurements. Passage 3 cardiomyocyte and pediatric myocardial phospholipids were analysed by gas chromatography. RESULTS: Pediatric cells were smaller (P < 0.01) and divided faster (P < 0.001, ANOCOVA) than adult cells. The cardiomyocytes showed phenotypic changes in primary culture with essentially complete loss of sarcomeres by 10 days and a gradual loss of myofilaments with passaging. The cells were identified as cardiomyocytes by immunohistochemistry for myoglobin, CK-MB, cardiac-specific troponin I isoform, HVMHC and HVMLC1, and by Northern blot analysis for the 3'-end of HVMHC mRNA. The composition of phospholipid fatty acids in the cultured pediatric cells was similar to that found in the pediatric myocardium. CK-MB activity and mass could be measured in the cardiomyocytes. The adult cardiomyocytes were more difficult to maintain than the pediatric cells which could be cultured for as long as 6 months. CONCLUSIONS: Primary cultures of human pediatric and adult partially differentiated ventricular cardiomyocytes can be passaged. Although rapid disorganization of the myofibrils occurs, the non-contractile cells can be identified as cardiomyocytes by morphological appearance, immunofluorescent staining, Northern blot analysis for HVMHC, and CK-MB activity.

Actin Cytoskeleton↗
PubMed2006

Jagged1 protein enhances the differentiation of mesenchymal stem cells into cardiomyocytes.

BACKGROUND: Mesenchymal stem cells (MSCs) can differentiate into cardiomyocytes if an appropriate cellular environment is provided. Notch signals exchanged between neighboring cells through the Notch receptor can eventually dictate cell differentiation. In our study, we show that MSC differentiation into cardiomyocytes is dependent on the Notch signal. METHODS: We created a myocardial infarction model in rat by coronary ligation, administered direct intramyocardial injection of DAPI-labeled MSC immediately, and observed the differentiation of MSCs after 14 days by immunofluorescence staining against troponin T. We cultured MSCs and cardiomyocytes in four ways, respectively, in vitro. (1) MSCs cocultured with cardiomyocytes obtained from neonatal rat ventricles in a ratio of 1:10. (2) The two types of cells were cultured in two chambers separated by a semipermeable membrane as indirect coculture group. (3) Notch receptor-soluble jagged1 protein was added to indirect coculture group. (4) Both jagged1 protein and gamma-secretase inhibitor-DAPT were added to indirect coculture group. Two weeks later, we observed the differentiation percentage, respectively, by immunofluorescence staining. RESULTS: We found the differentiation of MSCs which were close to cardiomyocytes in vivo. The differentiation percentage of the four cell culture group was 30.13+/-2.16%, 12.52+/-1.18%, 26.33+/-2.20%, and 13.08+/-1.15%. CONCLUSIONS: MSCs can differentiate into cardiomyocytes in vitro and in vivo if a cardiomyocyte microenvironment is provided. 2. Cell-to-cell interaction is very important for the differentiation of MSCs into cardiomyocytes. 3. Jagged1 protein can activate Notch signal and enhance the differentiation of MSC into cardiomyocyte, while the effect can be inhibited by DAPT.

Animals↗
PubMed2004

Growth hormone releasing peptide (ghrelin) is synthesized and secreted by cardiomyocytes.

OBJECTIVE: Ghrelin, the endogenous ligand of growth hormone secretagogue receptor (GHS-R), acts on the pituitary and the hypothalamus to stimulate the release of growth hormone (GH) and promotes appetite and adiposity. It has also been reported to increase myocardial contractility, induce vasodilation, and protect against myocardial-infarction-induced heart failure. Though principally gastric in origin, it is also produced by other tissues. This work investigated whether cardiomyocytes synthesize and secrete ghrelin, and how its production in these cells responds to stress and exogenous apoptotic agents. METHODS: Ghrelin and its receptor expression was studied by RT-PCR, immunohistochemistry, and competitive binding studies in mouse adult cardiomyocyte cell line HL-1, and primary cultured human cardiomyocytes. Ghrelin accumulation in cardiomyocyte culture medium was measured by radioimmunoassay. Viability and apoptosis assays were carried on by MTT and Hoechst dye vital staining, respectively. RESULTS: RT-PCR showed that HL-1 cells produce mRNAs for both ghrelin and GHS-R, and that GHS-R1a is expressed in human cardiomyocytes; and competitive binding studies using (125)I-labelled ghrelin showed efficient constitutive expression of GHS-R at the surface of HL-1 cells. Immunohistochemistry confirmed the presence of ghrelin in the cytoplasm of HL-1 cells and of isolated human cardiomyocytes in primary culture. Radioimmunoassay showed that ghrelin was secreted by HL-1 cells and human cardiomyocytes into the culture medium. Ghrelin did not modify the viability of HL-1 cells subjected to 12-h starvation, but did protect against the apoptosis inducer cytosine arabinoside (AraC). Finally, production of ghrelin mRNA in HL-1 cardiomyocytes was reduced by AraC but increased if exposure to AraC was preceded by GH treatment. CONCLUSIONS: Ghrelin is synthesized and secreted by isolated murine and human cardiomyocytes, probably with paracrine/autocrine effects, and may be involved in protecting these cells from apoptosis.

Animals↗