Animals ; Apoptosis ; Cardiomegaly ; metabolism ; pathology ; Glycogen Synthase Kinase 3 ; metabolism ; Mice ; Mice, Transgenic ; Myocytes, Cardiac ; cytology

OBJECTIVETo investigate the degree of injury to microtubule of myocardium at early post-hypoxia stage.

METHODSCardiomyocytes from Wistar rats were isolated and cultured, and they were then divided into normal control and hypoxia groups. The distribution and morphological changes in microtubules were observed with laser confocal microscopy and scanning electron microscope at 10, 20, 30 post-hypoxia minutes (PHM) and 1 post-hypoxia hour (PHH). Then the fluorescence intensity of alpha-microtubule was detected with RT-PCR, the morphology of microtubule was observed, and the expression of dissociative alpha-microtubule was determined by Western blot.

RESULTSCompared with normal control group, the bead-like structure of the microtubule in hypoxia group disappeared at 10 PHM, but no obvious change was observed in the distribution and number of microtubules. Despite the disappearance of bead-like structure of the microtubule, the microtubule derangement and loss of microtubule at the edge of cell were observed at 20 PHM. The fragmentation, derangement of texture, and loss of regularity in cardiomyocytes were observed at 30 PHM and 1 PHH. The fluorescence intensity of alpha-microtubule in hypoxia group was evidently decreased than that in normal group in a time-dependent manner. The expression of dissociative alpha-microtubule in hypoxia group at 10 PHM (46,644 +/- 145) was obviously higher than that in normal group (13,357 +/- 98, P < 0.01), and its increase was maintained with elapse of time.

CONCLUSIONMicrotubule injury to cardiomyocytes occurs at early stage of post-hypoxia, with destruction of its structure and distribution.

Animals ; Cell Hypoxia ; Cells, Cultured ; Microtubule-Associated Proteins ; biosynthesis ; Microtubules ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Rats ; Rats, Wistar

AIMThe observation of the relationship between the level of NO detected by ESR in the blood and the myocardial apoptosis and function caused by the recurrent, reversible myocardial ischemia/reperfusion injury.

METHODSFifteen New Zealand white rabbits were randomly divided into three groups (n = 5): (1) control group, (2) L-Arg group, (3) L-NNA group. The rabbits were anesthetized with intravenous pentobarbital. A suture ligature was passed around the left anterior descending coronary artery (LAD), so it could be snare occluded and reperfused. The LAD was occluded for 10 min three times, the first and second occlusions were followed by 10 min of reflow, after the third occlusion, the reperfusion was 120 min.

RESULTSIn all groups dp/dt(max) began to decrease at 5 min after the first ischemia. But compared with control group at 5 min after first reperfusion: in L-Arg group NO and apoptosis level were elevated but dp/dt(max) decreased significantly. In L-NNA group NO and apoptosis decreased significantly, dp/dt(max) improved significantly.

CONCLUSIONThe fact that the level of NO and apoptosis elevated suggested that they had taken part in the process of myocardial stunning.

Animals ; Apoptosis ; Female ; Male ; Myocardial Reperfusion Injury ; metabolism ; pathology ; physiopathology ; Myocytes, Cardiac ; metabolism ; Nitric Oxide ; metabolism ; Rabbits

OBJECTIVETo observe the alterations of taurine transport, taurine transporter (TAUT) and cysteine sulfinate decarboxylase (CSD) mRNA in the calcification of myocardial cells in vitro.

METHODS3H-taurine measured the amount of taurine uptake. TAUT and CSD mRNA consents were measured using competitive quantitative RT-PCR in cultured and calcified myocardial cells.

RESULTSIn calcification of myocardial cells, taurine concentration was decreased by 27% (P < 0.05), taurine uptake was markedly reduced, Vmax reduced by 39% (P < 0.01), there were no statistical significance of Km values between the two groups. TAUT mRNA decreased by 45% (P < 0.01), but CSD mRNA increased by 25% (P < 0.05).

CONCLUSIONSThe data suggest that there were impediment of taurine transport in calcification of myocardial cells, as TAUT mRNA level was decreased, but CSD mRNA concentration was improved.

Animals ; Biological Transport ; Calcinosis ; metabolism ; pathology ; Calcium ; metabolism ; Carboxy-Lyases ; metabolism ; Cells, Cultured ; Myocytes, Cardiac ; metabolism ; pathology ; RNA, Messenger ; metabolism ; Rats ; Taurine ; biosynthesis ; genetics ; metabolism

OBJECTIVETo investigate the influence of hypoxia induced microtubule damage on the opening of mitochondrial permeable transition pore (MPTP)of cardiac myocytes and on the decrease of respiratory function in rat.

METHODSPrimary cultured myocardial cells from 30 neonatal rats were randomized as normoxic group (A), hypoxia group (B), normoxia with microtubule destabilizing agent group (C, with treatment of 8 micromol/L colchicines for 30 minutes before normoxia), and hypoxia with microtubule stabilizing agent group (D, with treatment of 10 micromol/L taxol for 30 minutes before hypoxia). beta-tubulin immunofluorescence ,the opening of mitochondria permeability transition pore, and the mitochondrial inner membrane potential were detected at 0.5, 1, 3, 6 and 12 post-treatment hours (PTH), and the mitochondrial respiratory function was determined by MTT method. The changes in these indices were also determined in A group at the corresponding time-points.

RESULTSObvious damage of polymerized microtubule, opening of MPTP, mitochondrial inner membrane potential loss and decrease of myocardial respiratory activity were observed in both group B and C at 0.5 PTH, and they became more and more serious afterwards. However, the changes in the above indices in D group were much better than those in B group (P < 0.05 or 0.01), and no difference was found between D (92.8 +/- 4.0)% and C [(100.0 +/- 0.0) %, P > 0.05] groups.

CONCLUSIONHypoxia played a role in the myocardial microtubule damage as well as in the opening of MPTP. Moreover, hypoxia could also impair the mitochondrial respiratory function. Microtubule destabilizing agent could reproduce well the process of hypoxia induced microtubule damage, while the stabilizing agent exerted protective effect by improving the transition of mitochondrial permeability and the mitochondria respiratory function.

Animals ; Cell Hypoxia ; Cells, Cultured ; Hypoxia ; metabolism ; pathology ; Membrane Potential, Mitochondrial ; Microtubules ; pathology ; Mitochondria, Heart ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo evaluate the in vitro anti-hypertrophic effect of total Glycosides of Ranunculus Japonius (TGRJ).

METHODSNeonatal rat cardiomyocytes were cultured and hypertrophy was induced by administrating isoproterenol (ISO, 10 µmol/L) or angiotensin 2 (Ang 2, 1 µmol/L) for 48 hours. In the treatment groups, cells were pretreated with TGRJ (0.3 g/L) for 30 minutes prior to hypertrophic stimuli. The anti-hypertrophic effects of TGRJ were examined by measuring cell size, total protein content, and protein synthesis. Intracellular free Ca(2+) concentration ([Ca(2+)]i) was evaluated using fluorescence dye Fura-2/AM. Sacroplasmic/endoplasmic reticulum Ca(2+) ATPase 2a (SERCA2a), atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and beta-myosin heavy chain (β-MHC) protein expression levels were measured by Western blotting . SERCA2a activity was assayed by p-nitrophenal phosphate disodium salt hexahydrate method.

RESULTSIncreased cell size, total protein content, and protein synthesis following ISO or Ang 2 stimulation were significantly inhibited by pretreatment with TGRJ (all P<0.05). This anti-hypertrophic effect of TGRJ was confirmed by its suppressing effect on elevated expression of the three hypertrophic related genetic markers, ANP, BNP, and β-MHC. In addition, TGRJ inhibited ISO or Ang 2 induced up-regulation of [Ca(2+)]i under chronic but not acute conditions. And ISO or Ang 2 induced down-regulation of SERCA2a expression and activity was also effectively rectified by TGRJ pretreatment.

CONCLUSIONSThe results of present study suggested that TGRJ could prevent ISO or Ang 2 induced cardiac hypertrophy through improving chronic [Ca(2+)]i disorder, might via normalizing SERCA2a expression and activity.

Animals ; Animals, Newborn ; Calcium ; metabolism ; Cells, Cultured ; Glycosides ; analysis ; pharmacology ; Myocytes, Cardiac ; drug effects ; metabolism ; pathology ; Ranunculus ; chemistry ; Rats

OBJECTIVE: To observe whether necroptosis was happened in high glucose (HG) - induced primary cardiomyocytes injury and to investigate the likely mechanism. METHODS: The primary cultured cardiomyocytes were divided into 4 groups (n=9): control group (the cardiomyocytes were incubated with 5.5 mmol/L glucose for 48 h), HG group (the cardiomyocytes were incubated with 30 mmol/L glucose for 48 h), HG + necrostatin-1 (Nec-1) group (the cardiomyocytes was co-incubated with necroptosis inhibitor Nec-1 at 100 μmol/L and HG for 48 h) and hypertonic pressure group (HPG, the cardiomyocytes was co-incubated with 5.5 mmol/L glucose and 24.5 mmol/L mannitol for 48 h). Cell viability was measured by MTT method, reactive oxygen species (ROS) generation was measured by DHE staining. The levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1β (IL-1β) were tested by ELISA method. The mRNA and protein expressions of necroptosis related genes receptor interacting serine/threonine protein kinase 1 (RIP1), RIP3, mixed lineage kinase domain-like protein (MLKL) were tested by quantitative real-time PCR and Western blot. RESULTS: The results showed HG intervention decreased cardiomyocytes viability, increased ROS generation, up-regulated the levels of TNF-α, IL-6 and IL-1β, increased RIP1, RIP3, MLKL expressions at mRNA and protein levels. Nec-1 treatment attenuated HG-induced increased cardiomyocytes viability, reduced ROS generation, down-regulated the levels of TNF-α, IL-6 and IL-1β, decreased RIP1, RIP3, MLKL expressions at mRNA and protein levels. CONCLUSION Necroptosis was happened in high glucose-induced primary cardiomyocytes injury. Inhibition of necroptosis can reduce high glucose-induced cardiomyocytes damage, may be related to inhibition of oxidative stress and depression of inflammative factors releasing.
Apoptosis ; Cells, Cultured ; Cytokines ; metabolism ; Glucose ; adverse effects ; Humans ; Myocytes, Cardiac ; cytology ; pathology ; Necrosis ; Oxidative Stress ; Reactive Oxygen Species ; metabolism

To elucidate the mechanism of arrhythmia in healed myocardial infarction (HMI), the changes of action potential duration (APD), transient outward potassium current (Ito), delayed rectifier potassium current (IK) and inward rectifier potassium current (IK1) of left ventricular myocytes in non-infarcted zone of HMI were investigated. Rabbits were randomly assigned into two groups: HMI group, in which animals were subjected to thoracotomy and ligation of the circumflex coronary and sham-operated group, in which rabbits underwent thoracotomy but no conorary ligation. 3 months after the operation, the whole myocyte patch clamp technique was used to record APD, Ito, IK, and IK1 of ventricular myocytes in non-infarcted zone. Our results showed that the membrane capacitance was larger in HMI group than in sham-operated group. Action potential duration was significantly lengthened in HMI group and early afterdepolarization (EAD) appeared in HMI group. The densities of Ito, I(K, tail), and IK1 were reduced significantly in HMI group, from 6.72 +/- 0.42 pA/pF, 1.54 +/- 0.13 pA/pF and 25.6 +/- 2.6 pA/pF in sham-operated group to 4.03 +/- 0.33 pA/pF, 1.14 +/- 0.11 pA/pF and 17.6 +/- 2.3 pA/pF, respectively. It is concluded that the reduced densities of Ito, I(K, tail) and IK1 in ventricular myocytes of non-infarcted zone in HMI were responsible for the prolongation of APD and the presentation of EAD which played important roles in the development of malignant arrhythmia in HMI.
Action Potentials ; Animals ; Arrhythmias, Cardiac ; etiology ; Female ; Heart Ventricles ; metabolism ; Male ; Myocardial Infarction ; complications ; metabolism ; pathology ; Myocytes, Cardiac ; cytology ; Patch-Clamp Techniques ; Potassium Channels ; metabolism ; Rabbits

OBJECTIVETo investigate the effect of angiotensin II (AngII) type 2 (AT2) receptors on pressure overload-induced inflammatory cytokine secretion in adult rat hypertrophied cadiomyocytes.

METHODSRat models of left ventricular hypertrophy induced by pressure overload was established by placing a band around the abdominal aortic of the rats, from which the hypertrophied cadiomyocytes were isolated and purified 8 weeks later. The isolated cardiomyocytes were treated with AngII plus losartan or AngII plus PD123319, and 36 h after the treatments, the expression levels of tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and IL-6 in the supernatant were detected using radioimmunoassay.

RESULTSAngII induced TNF-alpha and IL-1beta secretion from the hypertrophied cardiomyocyets, and pretreatment of the cells with PD123319, but not losartan, decreased their secretion. IL-6 level was not detected in the supernatant.

CONCLUSIONAngII-induced the expression of inflammatory cytokines in adult rat hypertrophied cardiomyocytes is mediated mainly by AT2, not by AT1 receptors.

Animals ; Cells, Cultured ; Cytokines ; secretion ; Hypertrophy ; metabolism ; pathology ; Inflammation Mediators ; metabolism ; Myocytes, Cardiac ; metabolism ; pathology ; Rats ; Rats, Sprague-Dawley ; Receptor, Angiotensin, Type 2 ; metabolism

OBJECTIVETo explore the relationship between expression of Fas, bcl-2 and apoptosis of cardiomyocytes in myocardial ischemia in rats.

METHODSMyocardial ischemia was experimentally induced by ligating the left coronary artery. The rate were killed from 10 minutes to 7 days after the operation. Apoptotic myocardial cells were detected by TUNEL method. The expression of Fas and bcl-2 was studied by ABC immunohistochemistry.

RESULTSCardiomyocytic apoptosis appeared from 3 to 36 hours after ischemia. This phenomenon however could not be detected by TUNEL method 7 days after ischemia. The expression of Fas could be detected by ABC immunohistochemistry from 3 hours to 7 days after ischemia, and the expression of bcl-2 from 10 minutes to 7 days. Cardiomyocytic apoptosis and Fas / bcl-2 expression appeared in different regions of myocardium: apoptosis in the ischemic regions, while Fas and bcl-2 expression in regions without obvious ischemia.

CONCLUSIONIn rats, Fas and bcl-2 may not directly regulate apoptosis of cardiomyocytes in case of myocardial ischemia.

Animals ; Apoptosis ; Male ; Myocardial Infarction ; metabolism ; pathology ; Myocytes, Cardiac ; metabolism ; pathology ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Rats, Sprague-Dawley ; fas Receptor ; metabolism