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171.
The proliferation, structural differentiation, and capacity of association of human ES cell-derived cardiomyocytes were assessed in culture and in extracardiac graft tissues. Embryoid body (EB) outgrowths having cardiomyocytes, and their transplants in mice retroperitoneum or renal subcapsular region were analyzed mainly by immunochemistry. During the culture of EB outgrowths, colonies of cardiomyocytes grew in size exhibiting synchronized beatings. Subcellular structures of those cardiomyocytes involved in the contraction, hormone production, and intercellular integration differentiated with distinct immunoreactivity for constituent proteins/peptides. Judging from PCNA staining, proliferation potential was maintained in part for more than 70 days. In teratoma tissues on post-transplantation Day 7, cardiomyocytes maintained their integration with connexin 43 and cadherin at their junctions. They partly exhibited strong PCNA reactivity. On Day 28, large part of the cardiomyocytes lost their association, dispersing among non-cardiac cells without discernible cadherin reactivity. Proliferation potential was generally low irrespective of their tissue diversity. From these results, structural differentiation and active proliferation of human ES cell-derived cardiomyocytes occurred in vitro, maintaining their association. When developed in extracardiac tissues, however, the cardiomyocytes showed low proliferation potential and reduced cellular integration. This leads to the proposal that some procedure will be necessary to accelerate or maintain the proliferation of cardiomyocytes in vivo. 相似文献
172.
de Jonge HW Dekkers DH Houtsmuller AB Sharma HS Lamers JM 《Cell biochemistry and biophysics》2007,47(1):21-32
Numerous neurohumoral factors such as endothelin (ET)-1 and angiotensin (Ang) II as well as the stretch stimulus act concertedly
in the in vivo overloaded heart in inducing hypertrophy and failure. The primary culture of rat neonatal cardiomyocytes is
the only in vitro model that allows the comparative analysis of growth responses and signaling events in response to different
stimuli. In the present study, we examined stretched rat cardiomyocytes grown on flexible bottomed cultured plates for hypertrophic
growth responses (protein synthesis, protein/DNA ratio, and cell volume), F-actin filaments rearrangement (by confocal, laser
scanning microscopy), and for signaling events (activation of phospholipase C [PLC-β, protein kinase C [PKC], mitogenactivated
protein [MAP] kinases] and compared these responses with ET-1 (10−8
M)-stimulated cells. Cyclic stretch for 48 h induced hypertrophic growth in cardiomyocytes indicated by increases in the rate
of protein synthesis, cell volume, and diameter, which were less pronounced in comparison to stimulation by ET-1. During cyclic
stretch, we observed disoriented F-actin, particularly stress-fibers whereas during ET-1 stimulation, F-actins rearranged
clearly in alignment with sarcomeres and fibers. The upstream part of signaling by cyclic stretch did not follow the PLCβ-PKC
cascade, which, in contrast, was strongly activated during ET-1 stimulation. Cyclic stretch and, to greater extent, ET-1 stimulated
downstream signaling through ERK, p38 MAP kinase, and JNK pathways, but the, involvement of tyrosine kinase and PI3 kinase-Akt
signaling during cyclic stretch could not be proven. Taken together, our results demonstrate that both cyclic stretch and
ET-1 induce hypertrophic responses in cardiomyocytes with different effects on organization of F-actin stress fibers in case
of stretch. Furthermore, on the short-term basis, cyclical stretch, unlike ET-1, mediates its hypertrophic response not through
activation of PLC-β and PKC but more likely through integrin-linked pathways, which both lead to downstream activation of
the MAP kinase family. 相似文献
173.
目的探讨膜片钳实验中豚鼠、大鼠心肌细胞急性分离过程中的观察指标和影响因素,并进行膜片钳记录。方法使用改进的Langendorff恒流灌注法分离心肌细胞和全细胞膜片钳记录钾通道电流。结果分离即刻,存活细胞可达80%以上,复钙后约40%~50%呈静息状态,余出现自发性收缩,存活细胞能够进行膜片钳封接,于室温KB液中可成活8 h以上。结论使用恒流灌注法,观测灌注压的变化,可以较客观的掌握酶解消化的程度,利于获取生理状态良好的心肌细胞。且细胞能够进行膜片钳实验。 相似文献
174.
175.
Dan Liu Ming He Bo Yi Wu-hua Guo Ai-ling Que Ji-xiang Zhang 《The international journal of biochemistry & cell biology》2009,41(11):2315-2322
Although anoxic preconditioning (APC) in the myocardium has been investigated for many years, its physiological mechanism is still not completely understood. Increasing evidence indicates that transiently increased resistance to ischemic damage following APC is dependent on de novo proteins synthesis. However, the key effector pathway(s) associated with APC still remains unclear. The proto-oncogene Pim kinase belongs to a serine/threoine protein kinase family, consists of Pim-1, Pim-2 and Pim-3 and has been implicated in stimulating cell growth and inhibiting cell apoptosis. Therefore we assumed that Pim-3 expression might be aberrantly induced in cardiomyocytes that were subjected to anoxia/reoxygenation (A/R) injury and that Pim-3 might also contribute to cardio-protection after APC. To address this hypothesis, we cloned a Pim-3 expression vector, transfected it into rat cardiomyocytes, and examined Pim-3 expression in rat cardiomyocytes that were subjected to A/R injury. Moreover, we studied the role of three major MAPK pathways, e.g. p38 MAPK, JNK, and ERK1/2, in order to evaluate the molecular mechanism underlying Pim-3 up-regulation and A/R induced cardiomyocyte injury. Our experiments showed that APC induced an up-regulation of Pim-3 and the transfection of Pim-3 gene into the cardiomyocytes attenuated A/R injury. The inhibition of p38 MAPK by SB203580 abolished both the Pim-3 up-regulation and the cardio-protection provided by APC. Overall, these results suggest that APC could act to protect the heart from A/R injury with cooperation from the proto-oncogene Pim-3; in addition, it up-regulates Pim-3 expression through a p38 MAPK signaling pathway. 相似文献
176.
Jae-Hong Ko Marwa Ahmed Ibrahim Won Sun Park Nari Kim Inja Lim Jin Han 《Biochemical and biophysical research communications》2009,389(1):74-79
Large-conductance Ca2+-activated K+ (BKCa) channels are widely distributed in cellular membranes of various tissues, but have not previously been found in cardiomyocytes. In this study, we cloned a gene encoding the mouse cardiac BKCa channel α-subunit (mCardBKa). Sequence analysis of the cDNA revealed an open reading frame encoding 1154 amino acids. Another cDNA variant, identical in amino acid sequence, was also identified by sequence analysis. The nucleotide sequences of the two mCardBKa cDNAs, type 1 (mCardBKa1) and type 2 (mCardBKa2), differed by three nucleotide insertions and one nucleotide substitution in the N-terminal sequence. The amino acid sequence demonstrated that mCardBKa was a unique BKCa channel α-subunit in mouse cardiomyocytes, with amino acids 41-1153 being identical to calcium-activated potassium channel SLO1 and amino acids 1-40 corresponding to BKCa channel subfamily M alpha member 1. These findings suggest that a unique BKCa channel α-subunit is expressed in mouse cardiomyocytes. 相似文献
177.
Shenfu injection (the major components of which are ginsenosides compound, extract of Panax ginseng shown to have antioxidant
properties) is a well-known important Chinese traditional medicine used for the treatment of various diseases especial for
cardiac diseases. The precise mechanism of the biological actions of this plant is not fully understood, in order to elucidate
the protection of cardiomyocytes. The aim of the present study was to investigate the effect of Shenfu injection on hypoxia/reoxygenation
(H/R)-induced apoptosis and the expression of bcl-2 and caspase-3 in cultured neonatal rat cardiomyocytes in vitro. Ventricular
myocytes were isolated from neonatal rat hearts and were exposed to 4 h of hypoxia followed by 16 h of reoxygenation. The
results indicated that treatment with different doses of Shenfu injection protected cardiacmyocyte cultures from hypoxia/reoxygenation-induced
apoptosis. Caspase-3 activation was decreased in hypoxic/reoxygenationed cardiomyocytes co-treated with Shenfu injection when
compared to hypoxia/reoxygenation alone treated cultures. Expression of the Bcl-2 proteins was increased in Shenfu injection-treated
cardiomyocytes subjected to hypoxia/reoxygenation. In conclusion, ginsenosides compound has obviously protective effects on
cardiacmyocytes against apoptosis induced by hypoxia/reoxygenation injury, whose mechanisms probably involve the inhibition
of down-regulation of Bcl-2 protein levels and sequential activation of caspase-3. 相似文献
178.
In researches of ketone bodies, D-3-hydroxybutyrate (D-3HB) is usually the major one which has been investigated; in contrast, little attention has been paid to L-3-hydroxybutyrate (L-3HB), because of its presence in trace amounts, its dubious metabolism, and a lack of knowledge about its sources. In the present study we determined the distributions of enantiomers of 3-hydroxybutyrate (3HB) in rat brain, liver, heart, and kidney homogenates, and we found the heart homogenate contained an enriched amount of L-3HB (37.67 microM/mg protein) which generated a significant ratio of 66/34 (D/L). The ratio was altered to be 87/13 in the diabetic rat heart homogenate. We subsequently found this changed ratio of D/L-3HB may contribute to reduce glucose utilization in cardiomyocytes. Glucose utilization by cardiomyocytes with 5 mM of D-3HB was decreased to 61% of the control, but no interference was observed when D-3HB was replaced with L-3HB, suggesting L-3HB is not utilized for the energy fuel as other ketone bodies are. In addition, the reduced glucose utilization caused by D-3HB gradually recovered in a dose-dependent manner with administration of additional L-3HB. The results gave the necessity of taking L-3HB together with D-3HB into account with regard to glucose utilization, and L-3HB may be a helpful substrate for improving inhibited cardiac pyruvate oxidation caused by hyperketonemia. 相似文献
179.
Devillard L Vandroux D Tissier C Dumont L Borgeot J Rochette L Athias P 《Molecular and cellular biochemistry》2008,307(1-2):149-157
Before transplantation, the heart graft is preserved by the use of cold storage in order to limit ischemia-reperfusion stress.
However, sustained exposure to low temperature may induce myocardial ultrastructural damage, particularly microtubules (MT)
disruption. Previous data suggested that tubulin-binding agents are able to attenuate cold-induced cytoskeleton alterations.
Thus, the aim of the present work was to study the influence of docetaxel (DX, a tubulin-binding taxane) on the effects of
deep hypothermia (4°C) and of simulated cold ischemia-reperfusion on the MT network and oxidative stress of cardiomyocyte
(CM) in monolayer cultures prepared from newborn rat ventricles. The MT network was explored by immunocytochemistry and Western-blotting,
the cell stress by tetrazolium dye assay (MTT) and lactate dehydrogenase (LDH) release, and the superoxide production by the
dihydroethidium probe (DHE). The MT assembly remained stable after 4 and 8 h of hypothermia. Tubulin acetylation was promoted
in CM subjected to 4-h hypothermia. Low temperature reduced the mitochondrial function and increased the basal LDH release.
The cold ischemia during 4 and 8 h preserved MT network. Docetaxel promoted MT polymerization and tubulin acetylation in basal
and in cold conditions. This drug decreased the release of LDH induced by cold ischemia. Moreover, hypothermia (4 h) significantly
raised the anion superoxide production. Docetaxel decreased this oxidative stress in the control CM and in CM submitted to
4 h of hypothermia. These data demonstrated that stabilizing MT with DX exerted a protective effect on CM subjected to hypothermia
and to cold ischemia-reperfusion. Tubulin-ligands should be thus considered to improve the tolerance of the heart graft toward
stressing conservative conditions. 相似文献
180.
As the signals required for cardiomyocyte differentiation and functional regulation are complex and only partly understood, the mechanisms prompting the differentiation and specification of pluripotential embryonic stem (ES) cells into cardiomyocytes remain unclear. We hypothesized that a combined technology system, cocultured with a visceral endoderm (VE) - like cell line, END-2, and added cytokine BMP-2, would induce high percentage conversion of murine ES-D3 cell line into cardiomyocytes, and derived cardiomyocytes in this system would exhibit more mature characteristics. It was observed that 92% (P<0.01) ES cell-derived aggregates in this system exhibited rhythmic contractions, and the contractile areas were greater. By contrast, in ES cells cultured alone, on the feeder layer of END-2 cells, or with added BMP-2, the total percentage of beating aggregates was 19, 69 (P<0.01) and 44% (P<0.01), respectively. All the rhythmically contractile cells derived from ES cells expressed cardiac-specific proteins for troponin T. Among them, the combined system resulted in significantly increased cardiac-specific genes (NKx2.5, alpha-MHC). Transmission electron microscopy (TEM) revealed varying degrees of myofibrillar organization, and the combined system resulted in a more mature phenotype such as Z bands, nascent intercalated discs and gap junctions. Before shifting to the cardiomyocyte phenotype, this system could accelerate apoptosis of the cell population (P<0.01). The inductive efficacy of this system can provide an opportunity to facilitate cardiomyocyte differentiation of ES cells. The inducible effects of this system may depend on increasing cardiac-specific gene expression and the induction of apoptosis in cells that are not committed to cardiac differentiation. 相似文献