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PubMed2003

Physiological coupling of donor and host cardiomyocytes after cellular transplantation.

Cellular transplantation has emerged as a potential approach to treat diseased hearts. Although cell transplantation can affect global heart function, it is not known if this results directly via functional integration of donor myocytes or indirectly via enhanced revascularization and/or altered postinjury remodeling. To determine the degree to which donor cardiomyocytes are able to functionally integrate with the host myocardium, fetal transgenic cardiomyocytes expressing enhanced green fluorescent protein were transplanted into the hearts of nontransgenic adult mice. Two-photon molecular excitation laser scanning microscopy was then used to simultaneously image cellular calcium transients in donor and host cells within the intact recipient hearts. Calcium transients in the donor cardiomyocytes were synchronous with and had kinetics indistinguishable from those of neighboring host cardiomyocytes. These results strongly suggest that donor cardiomyocytes functionally couple with host cardiomyocytes and support the notion that transplanted cardiomyocytes can form a functional syncytium with the host myocardium.

Animals↗
PubMed2003

Spontaneous cardiomyocyte differentiation from adipose tissue stroma cells.

Cardiomyocyte regeneration is limited in adult life. Thus, the identification of a putative source of cardiomyocyte progenitors is of great interest to provide a usable model in vitro and new perspective in regenerative therapy. As adipose tissues were recently demonstrated to contain pluripotent stem cells, the emergence of cardiomyocyte phenotype from adipose-derived cells was investigated. We demonstrated that rare beating cells with cardiomyocyte features could be identified after culture of adipose stroma cells without addition of 5-azacytidine. The cardiomyocyte phenotype was first identified by morphological observation, confirmed with expression of specific cardiac markers, immunocytochemistry staining, and ultrastructural analysis, revealing the presence of ventricle- and atrial-like cells. Electrophysiological studies performed on early culture revealed a pacemaker activity of the cells. Finally, functional studies showed that adrenergic agonist stimulated the beating rate whereas cholinergic agonist decreased it. Taken together, this study demonstrated that functional cardiomyocyte-like cells could be directly obtained from adipose tissue. According to the large amount of this tissue in adult mammal, it could represent a useful source of cardiomyocyte progenitors.

Adipose Tissue↗
PubMed2004

Expression of mutant p193 and p53 permits cardiomyocyte cell cycle reentry after myocardial infarction in transgenic mice.

Previous studies have demonstrated that expression of p193 and p53 mutants with dominant-interfering activities renders embryonic stem cell-derived cardiomyocytes responsive to the growth promoting activities of the E1A viral oncoproteins. In this study, the effects of p53 and p193 antagonization on cardiomyocyte cell cycle activity in normal and infarcted hearts were examined. Transgenic mice expressing the p193 and/or the p53 dominant-interfering mutants in the heart were generated. Transgene expression had no effect on cardiomyocyte cell cycle activity in uninjured adult hearts. In contrast expression of either transgene resulted in a marked induction of cardiomyocyte cell cycle activity at the infarct border zone at 4 weeks after permanent coronary artery occlusion. Expression of the p193 dominant-interfering mutant was also associated with an induction of cardiomyocyte DNA synthesis in the interventricular septa of infarcted hearts. A concomitant and marked reduction in hypertrophic cardiomyocyte growth was observed in the septa of hearts expressing the p193 dominant-interfering transgene, suggesting that cell cycle activation might partially counteract the adverse ventricular remodeling that occurs after infarction. Collectively these data suggest that antagonization of p193 and p53 activity relaxes the otherwise stringent regulation of cardiomyocyte cell cycle reentry in the injured adult heart.

Amino Acid Substitution↗
PubMed1994

Hypoxia induces apoptosis with enhanced expression of Fas antigen messenger RNA in cultured neonatal rat cardiomyocytes.

We examined whether apoptosis occurs in cardiomyocytes by hypoxia in vitro. Neonatal rat cardiomyocytes and nonmyocytes were cultured in 95% N2-5% CO2 atmosphere to produce hypoxic conditions. DNA fragmentation into integer multiples of the internucleosomal DNA length was observed in cardiomyocytes as early as 12 hours, whereas nonmyocytes did not show fragmentation of DNA up to 72 hours. DNA fragmentation of cardiomyocytes induced by hypoxia was also confirmed by nick-end labeling in situ. Messenger RNA for Fas antigen, a mediator of apoptotic cell death, was expressed in both cardiomyocytes and nonmyocytes as revealed by Northern blotting and in situ hybridization. In hypoxic condition, Fas messenger RNA levels in cardiomyocytes were upregulated by twofold over controls, whereas those of nonmyocytes were downregulated. These results indicate that cardiomyocyte death by hypoxia can occur via apoptosis and that Fas antigen may be associated with the mechanism of this apoptotic process.

Animals↗
PubMed1996

Regulation of rDNA transcription during endothelin-1-induced hypertrophy of neonatal cardiomyocytes. Hyperphosphorylation of upstream binding factor, an rDNA transcription factor.

Treatment of cultured neonatal cardiomyocytes with endothelin-1 and phorbol 12-myristate 13-acetate (PMA) results in cardiomyocyte hypertrophy. However, the signal transduction pathways involved in this process are poorly understood. Because increased ribosome biogenesis is a requisite for hypertrophy, we sought to (1) confirm the hypothesis that these two hypertrophic agents did indeed induce rRNA synthesis and (2) examine the mechanism through which this induction was accomplished. In this study, hypertrophy of contraction-arrested neonatal cardiomyocytes induced by treatment with either endothelin-1 or PMA was associated with increased rDNA transcription. Western blots demonstrated that the enhanced rates of rDNA transcription were not mediated by increased amounts of either RNA polymerase I or upstream binding factor (UBF), an rDNA transcription factor. However, immunoprecipitation of [32P] orthophosphate-labeled UBF from hypertrophying neonatal cardiomyocytes suggested that the increased rate of rDNA transcription may be due to the hyperphosphorylation of UBF, which would increase the activity of UBF. The increase in UBF phosphorylation occurred within 3 to 6 hours after exposure to either agent, was maximal at 12 hours, and was sustained for at least the first 24 hours of exposure. Phosphoamino acid analysis of UBF immunoprecipitated from control and treated cardiomyocytes demonstrated that UBF was phosphorylated exclusively on serine residues. Our previous studies have shown that the cellular UBF content increased in adrenergic- and contraction-induced models of cardiac hypertrophy. This study with endothelin-1 and PMA demonstrates that the modulation of UBF phosphorylation is an additional pathway by which ribosome biogenesis may be regulated in neonatal cardiomyocytes. These results support the hypothesis that UBF is an important regulatory factor during the initiation and maintenance of the accelerated rate of rDNA transcription observed during neonatal cardiomyocyte hypertrophy mediated by both phorbol esters and endothelin-1.

Animals↗
PubMed1999

Establishment of beta-adrenergic modulation of L-type Ca2+ current in the early stages of cardiomyocyte development.

beta-Adrenergic modulation of the L-type Ca2+ current (ICaL) was characterized for different developmental stages in murine embryonic stem cell-derived cardiomyocytes using the whole-cell patch-clamp technique at 37 degreesC. Cardiomyocytes first appeared in embryonic stem cell-derived embryoid bodies grown for 7 days (7d). ICaL was insensitive to isoproterenol, forskolin, and 8-bromo-cAMP in very early developmental stage (VEDS) cardiomyocytes (from 7+1d to 7+2d) but highly stimulated by these substances in late developmental stage (LDS) cardiomyocytes (from 7+9d to 7+12d), indicating that all signaling cascade components became functionally coupled during development. In early developmental stage (EDS) cells (from 7+3d to 7+5d), the stimulatory response to forskolin and 8-bromo-cAMP was relatively weak. The forskolin effect was strongly augmented by ATP-gamma-S. At this stage, basal ICaL was stimulated by the nonselective phosphodiesterase (PDE) inhibitor isobutylmethylxanthine, by PDE inhibitors selective for the PDE II, III, and IV isoforms, as well as by the phosphatase inhibitor okadaic acid. Stimulation of ICaL by the catalytic subunit of the cAMP-dependent protein kinase A (PKA) was found to be similar (about 3 times) throughout development and in adult mouse ventricular cardiomyocytes, indicating that no structural changes of the Ca2+ channel related to phosphorylation occurred during development. ICaL was stimulated by isoproterenol in the presence of a PKA inhibitor and GTP-gamma-S in LDS but not VEDS cardiomyocytes, suggesting the development of a membrane-delimited stimulatory pathway mediated through the stimulatory GTP binding protein, Gs. We conclude that uncoupling and/or low expression of Gs protein accounted for the ICaL insensitivity to beta-adrenergic stimulation in VEDS cardiomyocytes. Furthermore, in EDS cells at the 7+4d stage, the reduced beta-adrenergic response is due, at least in part, to high intrinsic PDE and phosphatase activities.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

A mammalian myocardial cell-free system to study cell cycle reentry in terminally differentiated cardiomyocytes.

Cardiomyocytes withdraw from the cell cycle in the early neonatal period, rendering the adult heart incapable to regenerate after injury. In the present study, we report the establishment of a cell-free system to investigate the control of cell cycle reentry in mammalian ventricular cardiomyocyte nuclei and to specifically address the question of whether nuclei from terminally differentiated cardiomyocytes can be stimulated to reenter S phase when incubated with extracts from S-phase cells. Immobilized cardiomyocyte nuclei were incubated with nuclei and cytoplasmic extract of synchronized H9c2 muscle cells or cardiac nonmyocytes. Ongoing DNA synthesis was monitored by biotin-16-dUTP incorporation as well as proliferating cell nuclear antigen expression and localization. Nuclei and cytoplasmic extract from S-phase H9c2 cells but not from H9c2 myotubes induced DNA synthesis in 92% of neonatal cardiomyocyte nuclei. Coincubation in the presence of cycloheximide indicated that de novo translation is required for the reinduction of S phase. Similar results were obtained with adult cardiomyocyte nuclei. When coincubated with both cytoplasmic extract and nuclei or nuclear extracts of S-phase cells, >70% of adult cardiomyocyte nuclei underwent DNA synthesis. In conclusion, these results demonstrate that postmitotic ventricular myocyte nuclei are responsive to stimuli derived from S-phase cells and can thus bypass the cell cycle block. This cell-free system now makes it feasible to analyze the molecular requirements for the release of the cell cycle block and will help to engineer strategies for regenerative growth in cardiac muscle.

Animals↗
PubMed2001

Coexpression of mutant p53 and p193 renders embryonic stem cell-derived cardiomyocytes responsive to the growth-promoting activities of adenoviral E1A.

Expression of adenoviral E1A in cardiomyocytes results in the activation of DNA synthesis followed by apoptosis. In contrast, expression of simian virus 40 large T antigen induces sustained cardiomyocyte proliferation. Previous studies have shown that T antigen binds to 2 proapoptotic proteins in cardiomyocytes, namely the p53 tumor suppressor and p193 (a new member of the BH3-only proapoptosis subfamily). Structure-function analyses identified a p193 C-terminal truncation mutant that encodes prosurvival activity. This mutant was used to test the role of p193 in E1A-induced cardiomyocyte apoptosis. E1A induced apoptosis in cardiomyocytes derived from differentiating embryonic stem cells. Expression of the prosurvival p193 mutant alone or a mutant p53 alone did not block E1A-induced apoptosis. In contrast, combinatorial expression of mutant p193 and mutant p53 blocked E1A-induced apoptosis, resulting in a proliferative response indistinguishable from that seen with T antigen. These results confirm the hypothesis that there are 2 proapoptotic pathways, encoded by p53 and p193, respectively, which restrict cardiomyocyte cell cycle activity in differentiating embryonic stem cell cultures. Furthermore, these results explain in molecular terms the phenotypic differences of E1A versus T-antigen gene transfer in cardiomyocytes.

3T3 Cells↗
PubMed2001

Cardiomyocyte apoptotic cell death in arterial hypertension: mechanisms and potential management.

Hypertensive heart disease is a progressive condition in which the compensatory left ventricular hypertrophy that maintains cardiac output leads to myocardial remodeling, characterized by fibrosis, insufficient vascularization, and alterations in cardiomyocytes, including contractile disturbances, changes in gene expression, and decrease in the number of cells. Structural abnormalities in the myocardial wall accelerate the development of diastolic and systolic dysfunction, resulting in heart failure. Many observations point to the apoptotic cell death of cardiomyocytes as a relevant factor in the transition from compensatory hypertrophy to pump failure in experimental and human hypertension. Potential inducers of cardiomyocyte apoptosis in overloaded hearts include extrinsic factors, such as mechanical forces, neurohormonal activation, oxidative stress, hypoxia, and cytokines. Some lines of evidence indicate that angiotensin II and the overstretching of cardiomyocytes are originally involved in the triggering of apoptosis in hypertension, whereas other factors are being investigated. Furthermore, intracellular changes, such as downregulation of survival proteins or activation of death proteins, seem to play an important role. The assumption that the apoptosis of cardiomyocytes worsens hypertensive heart disease prognosis brings forth new approaches to avoid or slow the transition to pump failure. In this respect, experimental data indicate that currently used antihypertensive drugs interfere with cardiomyocyte apoptosis. Moreover, the knowledge of intracellular apoptotic processes in cardiomyocytes provides novel therapeutic strategies to be added to the multimodal approach in the prevention of heart failure.

Angiotensin II↗
PubMed2004

Expression of glutamate transporters in rat cardiomyocytes and their localization in the T-tubular system.

Glutamate and aspartate play important roles in the intermediary metabolism of the myocardium and have been shown to improve cardiac recovery after hypoxia or ischemia. Limited data are available about the expression of glutamate transporters that are involved in the uptake of glutamate and aspartate in cardiomyocytes. In this study, non-radioactive in situ hybridization (ISH) using complementary RNA probes was applied to detect the glutamate transporters GLT1 variant (GLT1v) and EAAC1 mRNA in rat cardiomyocytes. The transporter proteins were demonstrated by Western blotting and immunocytochemistry using affinity-purified antibodies against transporter peptides. ISH and immunocytochemistry showed that both glutamate transporters are coexpressed in cardiomyocytes. The ISH labeling indicates the distribution of transporter mRNA throughout the cytoplasm of cardiomyocytes. GLT1v and EAAC1 proteins, which showed in Western blots a molecular mass of approximately 60 kD, are strongly enriched and colocalized in the transverse (T)-tubular system of cardiomyocytes. These results may indicate that glutamate/aspartate uptake into cardiomyocytes could be mediated by the high-affinity transporters GLT1v and EAAC1. A high efficiency of glutamate/aspartate transport into cardiomyocytes could be achieved by their localization in the T-tubular system, which consists of tubular invaginations of the sarcolemma extending deep into the cell.

Amino Acid Transport System X-AG↗
PubMed2002

Autologous cardiomyocyte transplantation in an ovine myocardial infarction model.

BACKGROUND: To evaluate the possibility of autologous cardiomyocyte transplantation, we transplanted cultured autologous cells into an infarct region developed by coronary artery ligation in an ovine model. MATERIALS AND METHODS: A chronic heart failure model with a considerable portion of myocardial infarction was created in sheep using sequential ligation of the homonymous artery and its diagonal branch. Autologous cardiomyocytes were cultured and isolated from the right ventricular infundibulum. After a predetermined interval (one animal for two months and the other for three months), the two animals were reanesthetized and a suspension of cultured autologous vetricular cells in 0.3 ml of culture medium (1.2 x 10(7) cells) was injected into the center of three out of the four sites in the infarcted area using a tuberculin syringe. The same amount of culture medium was injected with an identical procedure into the center of the remaining site, as control. The animals were kept alive for a further month, and then sacrificed for postmortem heart examinations. Light microscopic analysis and immunohistochemical study for myoglobin were done. RESULTS AND CONCLUSIONS: On postmortem gross examination, well-demarcated thin-walled anteroseptal infarcts with chamber enlargement were clearly seen in specimens from the two animals. Microscopic analysis showed homogenous fibrosis throughout the infarcted areas. In both animals, one of the three sites of cardiomycyte injection showed an islet of cardiomyocytes in the mid-myocardium, while none were observed in the control site of either animal. A layer of cardiomyocytes was observed in subendocardial regions, as it was in the control areas. In conclusion, cardiomyocyte transplantation into the infarct regions developed by coronary artery ligation in an ovine model was achieved with only limited success. An understanding of why only 33% of cardiomyocyte-injection sites demonstrated viable cardiomyocytes, in the form of tiny cell islets, remains to be elucidated.

Animals↗
PubMedDate not supplied

Cardiomyocyte transplantation in a porcine myocardial infarction model.

Transplantation of cardiomyocytes into the heart is a potential treatment for replacing damaged cardiac muscle. To investigate the feasibility and efficiency of this technique, either a cardiac-derived cell line (HL-1 cells), or normal fetal or neonatal pig cardiomyocytes were grafted into a porcine model of myocardial infarction. The myocardial infarction was created by the placement of an embolization coil in the distal portion of the left anterior descending artery in Yorkshire pigs (n = 9). Four to 5 wk after creation of an infarct, the three preparations of cardiomyocytes were grafted, at 1 x 10(6) cells/20 microL into normal and into the middle of the infarcted myocardium. The hearts were harvested and processed for histologic examinations 4 to 5 wk after the cell grafts. Histologic evaluation of the graft sites demonstrated that HL-1 cells and fetal pig cardiomyocytes formed stable grafts within the normal myocardium without any detrimental effect including arrhythmia. In addition, a marked increase in angiogenesis was observed both within the grafts and adjacent host myocardium. Electron microscopy studies demonstrated that fetal pig cardiomyocytes and the host myocardial cells were coupled with adherens-type junctions and gap junctions. Histologic examination of graft sites from infarct tissue failed to show the presence of grafted HL-1 cells, fetal, or neonatal pig cardiomyocytes. Cardiomyocyte transplantation may provide the potential means for cell-mediated gene therapy for introduction of therapeutic molecules into the heart.

Animals↗
PubMed1991

Contractile response to sympathetic innervation in neonatal ventricular cardiomyocytes of the spontaneously hypertensive rat.

Differences in cardiac development between spontaneously hypertensive rats (SHR) and their normotensive Wistar Kyoto (WKY) controls are observed before the onset of hypertension. To determine whether intrinsic differences in myocardium or autonomic neurons might be responsible for these observations, we studied primary cultures of isolated, never previously innervated ventricular cardiomyocytes from neonatal rats of both strains, and sympathetic innervation was produced by addition of neurons from thoracolumbar sympathetic ganglia. Both same-strain and cross-strain innervation were compared. Amplitude and frequency of contraction were measured from video images of spontaneously contracting cells by on-line video motion analysis. Sympathetic innervation improved contractile function by 61% in SHR cardiomyocytes (p less than 0.001), an effect qualitatively similar to that previously reported for WKY cardiomyocytes. Contractile function of SHR cardiomyocytes cultured without sympathetic explants was 25% less than that of WKY cells (p less than 0.005), but the response of SHR cardiomyocytes to sympathetic innervation was twice as great as that of WKY myocytes (p less than 0.01). Cross-strain innervation experiments showed that sympathetic neurons from both strains were equally effective; interstrain differences were confined to the cardiomyocytes. Interstrain differences in cardiomyocyte contractile function and contractile response to sympathetic innervation are present before the onset of hypertension, and may in part account for alterations in cardiac function observed in prehypertensive SHR.

Animals↗
PubMed2004

Sequential myofibrillar breakdown accompanies mitotic division of mammalian cardiomyocytes.

The contractile tissue of the heart is composed of individual cardiomyocytes. During mammalian embryonic development, heart growth is achieved by cell division while at the same time the heart is already exerting its essential pumping activity. There is still some debate whether the proliferative activity is carried out by a less differentiated, stem cell-like type of cardiomyocytes or whether embryonic cardiomyocytes are able to perform both of these completely different dynamic tasks, contraction and cell division. Our analysis of triple-stained specimen of cultured embryonic cardiomyocytes and of whole mount preparations of embryonic mouse hearts by confocal microscopy revealed that differentiated cardiomyocytes are indeed able to proliferate. However, to go through cell division, a disassembly of the contractile elements, the myofibrils, has to take place. This disassembly occurs in two steps with Z-disk and thin (actin)-filament-associated proteins getting disassembled before disassembly of the M-bands and the thick (myosin) filaments happens. After cytokinesis reassembly of the myofibrillar proteins to their mature cross-striated pattern can be seen. Another interesting observation was that the cell-cell contacts remain seemingly intact during division, probably reflecting the requirement of intact integration sites of the individual cells in the contractile tissue. Our results suggest that embryonic cardiomyocytes have developed an interesting strategy to deal with their major cytoskeletal elements, the myofibrils, during mitosis. The complex disassembly-reassembly process might also provide a mechanistic explanation, why cardiomyocytes cede to divide postnatally.

Actinin↗
PubMed2006

Re-programming of newt cardiomyocytes is induced by tissue regeneration.

Newt hearts are able to repair substantial cardiac injuries without functional impairment, whereas mammalian hearts cannot regenerate. The cellular and molecular mechanisms that control the regenerative capacity of the newt heart are unknown. Here, we show that the ability of newt cardiomyocytes to regenerate cardiac injuries correlates with their ability to transdifferentiate into different cell types. Mechanical injury of the heart led to a severe reduction of sarcomeric proteins in the myocardium, indicating a partial de-differentiation of adult newt cardiomyocytes during regeneration. Newt cardiomyocytes implanted into regenerating limbs lost their cardiac phenotype and acquired skeletal muscle or chondrocyte fates. Reprogramming of cardiomyocytes depended on contact with the limb blastema because cardiomyocytes implanted into intact, non-regenerating limbs or cultured in vitro retained their original identity. We reason that signals from the limb blastema led to de-differentiation of cardiomyocytes, cell proliferation and re-differentiation into specialized cells and propose that the ability of cardiomyocytes to transdifferentiate into different cell types reflects the cellular program that enables heart regeneration.

Animals↗
PubMed1999

Role of ATP-dependent K(+) channels in the electrical excitability of early embryonic stem cell-derived cardiomyocytes.

Single, murine embryonic stem cell-derived early stage cardiomyocytes dissociated from embryoid bodies expressed two inward rectifier K(+) channels, I(K1) and the ATP dependent K(+) current. I(K1) exhibited low density in early stage cardiomyocytes, but increased significantly in late stage cells. In contrast, the ATP dependent K(+) current was expressed at similar densities in early and late stage cardiomyocytes. This current was found to be involved in the determination of the membrane potential, since glibenclamide depolarized early cardiomyocytes and exerted a positive chronotropic effect. Some cardiomyocytes displayed a bursting behavior of action potentials, characterized by alternating periods with and without action potentials. During the phases without action potentials, the membrane potential was hyperpolarized, indicating the involvement of K(+) channels in the generation of this bursting behavior. Extracellular recording techniques were applied to spontaneously contracting areas of whole embryoid bodies. In 20% of these bursting behavior similar to that seen in the single cells was observed. In regularly beating embryoid bodies, bursting could be induced by reduction of substrates from the extracellular medium as well as by superfusion with the positive chronotropic agents Bay K 8644 or isoproterenol. Perfusion with substrate-reduced medium induced bursting behavior after a short latency, isoproterenol and Bay K 8644 resulted in a positive chronotropic response followed by bursting behavior with longer latencies. The spontaneous bursting was blocked by glibenclamide. These experimental results suggest that intermittent activation of ATP dependent K(+) channels underlies the bursting behavior observed in single cardiomyocytes and in the whole embryoid body. Conditions of metabolic stress lead to the rhythmic suppression of action potential generation. Our data indicate that ATP dependent K(+) channels play a prominent role in the cellular excitability of early cardiomyocytes.

2,4-Dinitrophenol↗
PubMed2000

Insulin-like growth factor I inhibits cardiomyocyte apoptosis and the underlying signal transduction pathways.

The effects of insulin-like growth factor I (IGF-I) on cardiomyocyte apoptosis induced by hypoxia in cultured neonatal rat cardiomyocyte were investigated. Primary neonatal rat cardiomyocytes were cultured in 95% N2-5% CO2 to imitate the in vivo hypoxic condition. Electron microscopic observation revealed a series of typical morphological changes characteristic of apoptosis in cardiomyocytes under the hypoxic condition. DNA gel electrophoresis showed DNA laddering in an ischemic duration-dependent manner. The hypoxia-induced cardiomyocyte apoptosis was also evidenced by flow cytometry and TUNEL assay. DNA gel electrophoresis showed that IGF-I in a dose range of 10(-9)-10(-7) mol/l could significantly prevent the hypoxia-induced cardiomyocyte apoptosis. The protective effects of IGF-I against hypoxia-induced apoptosis could also be verified by flow cytometry and TUNEL assay. A tyrosine kinase inhibitor (genistein), a MAPK inhibitor (PD-098059) and a P13 kinase inhibitor (wortmannin) could also suppress the antiapoptotic effects of IGF-I. These results suggest that IGF-I can directly alleviate the hypoxia-induced cardiomyocyte apoptosis and that the three kinase routes mentioned above may be involved in its signaling pathways.

Androstadienes↗
PubMed2006

Bone morphogenetic protein-4 enhances cardiomyocyte differentiation of cynomolgus monkey ESCs in knockout serum replacement medium.

Despite extensive research in the differentiation of rodent ESCs into cardiomyocytes, there have been few studies of this process in primates. In this study, we examined the role of bone morphogenic protein-4 (BMP-4) to induce cardiomyocyte differentiation of cynomolgus monkey ESCs. To study the role of BMP-4, EBs were formed and cultured in Knockout Serum Replacement (KSR) medium containing BMP-4 for 8 days and subsequently seeded in gelatin-coated dishes for 20 days. It was found that ESCs differentiated into cardiomyocytes upon stimulation with BMP-4 in KSR medium, which resulted in a large fraction of beating EBs ( approximately 16%) and the upregulation of cardiac-specific proteins in a dose and time-dependent manner. In contrast, the addition of BMP-4 in FBS-containing medium resulted in a lower fraction of beating EBs ( approximately 6%). BMP-4 acted principally between mesendodermal and mesoderm progenitors and subsequently enhanced their expression. Ultrastructural observation revealed that beating EBs contained mature cardiomyocytes with sarcomeric structures. In addition, immunostaining, reverse transcription-polymerase chain reaction, and Western blotting for cardiac markers confirmed the increased differentiation of cardiomyocytes in these cultures. Moreover, electrophysiological studies demonstrated that the differentiated cardiomyocytes were electrically activated. These findings may be useful in developing effective culture conditions to differentiate cynomolgus monkey ESCs into cardiomyocytes for studying developmental biology and for regenerative medicine.

Animals↗