In order to investigate the effects of aconitine on [Ca2+] oscillation patterns in cultured myocytes of neonatal rats, fluorescent Ca2+ indicator Fluo-4 NW and laser scanning confocal microscope (LSCM) were used to detect the real-time changes of [Ca2+] oscillation patterns in the cultured myocytes before and after aconitine (1.0 micromol/L) incubation or antiarrhythmic peptide (AAP) and aconitine co-incubation. The results showed under control conditions, [Ca2+] oscillations were irregular but relatively stable, occasionally accompanied by small calcium sparks. After incubation of the cultures with aconitine, high frequency [Ca2+] oscillations emerged in both nuclear and cytoplasmic regions, whereas typical calcium sparks disappeared and the average [Ca2+] in the cytoplasm of the cardiomyocyte did not change significantly. In AAP-treated cultures, intracellular [Ca2+] oscillation also changed, with periodic frequency, increased amplitudes and prolonged duration of calcium sparks. These patterns were not altered significantly by subsequent aconitine incubation. The basal value of [Ca2+] in nuclear region was higher than that in the cytoplasmic region. In the presence or absence of drugs, the [Ca2+] oscillated synchronously in both the nuclear and cytoplasmic regions of the same cardiomyocyte. It was concluded that although oscillating strenuously at high frequency, the average [Ca2+] in the cytoplasm of cardiomyocyte did not change significantly after aconitine incubation, compared to the controls. The observations indicate that aconitine induces the changes in [Ca2+] oscillation frequency other than the Ca2+ overload.
Aconitine/*pharmacology ; Animals, Newborn ; Calcium Signaling/*drug effects ; Cells, Cultured ; Myocytes, Cardiac/cytology ; Myocytes, Cardiac/*metabolism ; Rats, Sprague-Dawley

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

In developed countries, in which people have nutrient-rich diets, convenient environments, and access to numerous medications, the disease paradigm has changed. Nowadays, heart failure is one of the major causes of death. In spite of this, the therapeutic efficacies of medications are generally unsatisfactory. Although whole heart transplantation is ideal for younger patients with heart failure, many patients are deemed to be unsuitable for this type of surgery due to complications and/or age. The need for therapeutic alternatives to heart transplantation is great. Regenerative therapy is a strong option. For this purpose, several cell sources have been investigated, including intrinsic adult stem or progenitor cells and extrinsic pluripotent stem cells. Most intrinsic stem cells seem to contribute to a regenerative environment via paracrine factors and/or angiogenesis, whereas extrinsic pluripotent stem cells are unlimited sources of cardiomyocytes. In this review, we summarize the various strategies for using regenerative cardiomyocytes including our recent progressions: non-genetic approaches for the purification of cardiomyocytes and efficient transplantation. We expect that use of intrinsic and extrinsic stem cells in combination will enhance therapeutic effectiveness.
Animals ; Embryonic Stem Cells/cytology ; Humans ; Myocardium/*cytology/*metabolism ; Myocytes, Cardiac/*cytology ; *Regeneration ; Stem Cell Transplantation ; Tissue Engineering

Cardiomyocyte apoptosis leads to the functional incapacitation of myocardial plasmodium and plays an important role in the pathogenesis of heart failure transformed from compensable cardiac hypertrophy. Mitochondria are the main source of apoptosis-inducing molecule of various cells, and the role of caspartate-specific cysteinyl proteinase (caspase)-dependent mechanism has generally been accepted in the cardiomyocyte apoptosis. However, the significance of caspase-independent apoptosis-inducing factor (AIF) mechanism is not yet understood. The purpose of this study was to evaluate hypoxia-reperfusion-induced alterations of AIF mRNA and protein expressions in hypertrophic cardiomyocytes. Cardiomyocyte hypertrophy was produced by angiotensin II (0.1 mumol/L). The cells were cultured under the condition of hypoxia (95% N2 and 5% CO2; the O2 partial pressure was lower than 5 mmHg) for 8 h or 12 h (named as H8h and H12h groups, respectively), and then exposed to normal culture environment (named as H8h/R and H12h/R groups, respectively). Apoptosis was detected with Hoechst 33258 staining. The AIF mRNA and protein expressions were detected by RT-PCR and Western blot and quantified by gel scanning. The results were as follows: (1) The level of AIF mRNA expression was 0.29+/-0.08 (optical density, relative value) in the control group (hypertrophic cardiomyocytes cultured in normal environment). Compared with that in the control group, the levels of AIF mRNA expression were significantly higher in the groups of H8h and H12h (0.52+/-0.04 and 0.85+/-0.10), indicating that this effect was time-dependent. A further increase of AIF mRNA expression was observed in the groups of H8h/R (1.09+/-0.12) and H12h/R (1.41+/-0.23). (2) The level of AIF protein expression was 0.29+/-0.04 in the control group. Compared with that in the control group, the levels of AIF protein expression were significantly higher in the groups of H8h and H12h (2.07+/-0.15 and 3.12+/-0.19). The AIF protein expression was increased further in the groups of H8h/R (4.57+/-0.25) and H12h/R (5.71+/-0.27). The nuclear translocation of AIF protein was obvious only in the groups of H8h/R and H12h/R. (3) The expressions of AIF mRNA and protein were almost completely inhibited by AIF siRNA transfection. The siRNA transfection also reduced the apoptosis of hypertrophic cardiomyocytes in the groups of H8h/R and H12h/R but not in the groups of H8h and H12h. The apoptosis rate was significantly reduced by both AIF siRNA transfection and Ac-DEVD-cmk, an inhibitor of caspase-3. This reduction induced by two factors was more evident than that by one factor. (4) AIF nuclear translocation induced by hypoxia-reperfusion was not affected by inhibition of the activity of caspase-3. These data suggest that AIF plays a pivotal role in the apoptosis of hypertrophic cardiomyocytes induced by hypoxia-reperfusion.
Apoptosis ; Apoptosis Inducing Factor ; metabolism ; Cardiomegaly ; Cell Hypoxia ; Myocytes, Cardiac ; cytology ; Reperfusion Injury

Animals ; Cell Proliferation ; Cells, Cultured ; Coxsackievirus Infections ; metabolism ; Enterovirus B, Human ; Myocytes, Cardiac ; cytology ; metabolism ; virology ; Osteopontin ; metabolism ; Rats

We investigated the effect of phenylephrine (PE)- and isoproterenol (ISO)-induced cardiac hypertrophy on subcellular localization and expression of caveolin-3 and STAT3 in H9c2 cardiomyoblast cells. Caveolin-3 localization to plasma membrane was attenuated and localization of caveolin-3 to caveolae in the plasma membrane was 24.3% reduced by the catecholamine-induced hypertrophy. STAT3 and phospho-STAT3 were up-regulated but verapamil and cyclosporin A synergistically decreased the STAT3 and phospho-STAT3 levels in PE- and ISO-induced hypertrophic cells. Both expression and activation of STAT3 were increased in the nucleus by the hypertrophy. Immunofluorescence analysis revealed that the catecholamine-induced hypertrophy promoted nuclear localization of pY705-STAT3. Of interest, phosphorylation of pS727-STAT3 in mitochondria was significantly reduced by catecholamine-induced hypertrophy. In addition, mitochondrial complexes II and III were greatly down-regulated in the hypertrophic cells. Our data suggest that the alterations in nuclear and mitochondrial activation of STAT3 and caveolae localization of caveolin-3 are related to the development of the catecholamine-induced cardiac hypertrophy.
Animals ; Catecholamines/*pharmacology ; Caveolae/*metabolism ; Caveolin 3/*metabolism ; Cell Line ; Hypertrophy/metabolism ; Mitochondria/*metabolism ; Myocardium/cytology/*pathology ; Myocytes, Cardiac/cytology/*drug effects/metabolism ; Rats ; STAT3 Transcription Factor/*metabolism

We investigated the effects of simulated ischemia on intracellular Cl- concentration ([Cl-]i) in guinea pig ventricular myocardial cells and possible role of the [Cl-]i on the ischemia/reperfusion-induced arrhythmias in perfused rat hearts. Our results provided direct evidence that the [Cl-]i in ventricular muscle was increased under ischemic conditions, which suggested that activation of the Cl-(-)HCO3- exchanger by ischemia would partially contribute to the elevation of [Cl-]i. Application of stilbene derivatives or lowering Cl- concentration in perfusion solution delayed the onset of ischemia-induced deterioration in action potentials, pHi, [Cl-]i, and suppressed the incidence of ischemia/reperfusion-induced arrhythmias. The conclusion was made to emphasize the important role of intracellular Cl- homeostasis in cardiac physiology and pathogenesis of myocardial ischemia/reperfusion injury.
Action Potentials ; Animals ; Arrhythmias, Cardiac ; etiology ; metabolism ; Chlorides ; metabolism ; Guinea Pigs ; Hydrogen-Ion Concentration ; Microelectrodes ; Myocardial Reperfusion Injury ; complications ; metabolism ; Myocytes, Cardiac ; metabolism ; Papillary Muscles ; cytology

AIMTo examine the effects of pHi on [Ca2+]i, cell length and Ca2+ sensitivity of myofilaments in rat myocyte.

METHODSWe used microscopic spectral imaging approach to monitor simultaneously [Ca2+]i, pHi and cell length with fluorescent indicator indo-1 and SNARF-1 in isolated single rat myocyte.

RESULTSExposure of cell to 20 mmol/L Sodium Propionate induced an intracellular acidosis which increased slightly systolic and diastolic [Ca2+]i, decreased the cell shortening (CS) and Ca2+ sensitivity of myofilament (P < 0.01). Exposure of cell to 15 mmol/L NH4Cl induced an intracellular alkalosis which decreased systolic and diastolic [Ca2+]i, increased the cell shortening and Ca2+ sensitivity of myofilament (P < 0.01).

CONCLUSIONSIn the early time of acidosis, [Ca2+]i, as well as cell length increases respectively. While alkalosis, [Ca2+]i and cell length decreases respectively. The effect of acidosis and alkalosis on Ca2+ sensitivity presents non-linear relationship, i.e. the effect of acidosis on sensitivity, caused by pHi change, is less than that of alkalosis.

Animals ; Calcium ; metabolism ; Cell Size ; Hydrogen-Ion Concentration ; Male ; Myocardium ; cytology ; Myocytes, Cardiac ; metabolism ; Rats ; Rats, Wistar

AIMFast 2-dimension scanning and line-scanning of confocal imaging were employed for measurement of cardiac Ca2+ transients, and the advantages and disadvantages about these two scannings were discussed.

METHODSSingle adult SD rat cardiac myocytes were made freshly and loaded with fluo4-AM. Intracellular Ca2+ was imaging by the LSMS10 META system. The Ca2+ transients were evoked by electrical field stimulation from an electronic stimulator which was triggered to work synchronically with the confocal imaging system.

RESULTSFast 2-dimension scanning showed the global Ca2+ signal clearly, which would be more helpful especially in monitoring a cell of Ca2+ overload or in other pathological conditions. And the images could be packaged into a vivid animation, which showed the process of Ca2+ transients and cell contraction visually and virtually. Line-scanning showed the Ca2+ transients in good temporal and spacial resolutions along the long axis of the cell. And the dynamic shortening of the cell length could be used for indicating the contraction of the cell. Data from line-scanning would be helpful for drawing some more exact conclusions.

CONCLUSIONIn general, fast 2-dimension scanning and line-scanning could work reciprocally to show a more perfect picture of the intracellular Ca2+ transients in cardiac myocytes.

Animals ; Calcium ; analysis ; metabolism ; Calcium Signaling ; physiology ; Female ; Male ; Microscopy, Confocal ; methods ; Myocytes, Cardiac ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

AIMTo observe the effects of adenosine on intracellular calcium concentration ([Ca2+]i) level in guinea pig ventricular myocytes and to define the possible mechanisms involved.

METHODSThe effects of adenosine on [Ca2+]i were investigated in guinea pig ventricular myocytes. [Ca2+]i was detected by laser confocal microscopy and represented by relative fluorescent intensity ((FI-FI0)/FI0, %, FIo: control, FI: administration of drugs).

RESULTS(1) Adenosine (10, 50, 100 micromol/L) reduced [Ca2+]i of ventricular myocytes in both normal Tyrode's solution and Ca(2+) -free Tyrode's solution in a concentration-dependent manner. (2) Tyrode's solution containing 30 mmol/L KCl (high K+ Tyrode's solution) induced [Ca2+]i elevation in ventricular myocytes, while adenosine (10, 50, 100 micromol/L) markedly inhibited the increase in [Ca2+]i induced by KCl. (3) Pretreatment with DPCPX (1 micromol/L) significantly reduced the effects of adenosine (100 micromol/L) in high K+ Tyrode's solution. The effects of adenosine (100 micromol/L) on [Ca2+]i in high K+ Tyrode's solution were also partially attenuated by pretreatment with L-NAME (1 mmol/L). (4) Adenosine (100 micromol/L) markedly inhibited the low concentration of ryanodine-induced [Ca2+]i increase in Ca(2+) -free Tyrode's solution. (5) When the propagating waves of elevated [Ca2+]i (Ca2+ waves) were produced by increasing extracellular Ca2+ concentration from 1 mmol/L to 10 mmol/L, adenosine (100 micromol/L) could block the propagating waves of elevated [Ca2+]i, reduce the frequency and duration of propagating waves, and reduce [Ca2+]i as well.

CONCLUSIONAdenosine may reduce the [Ca2+]i in isolated guinea pig ventricular myocytes via inhibiting Ca2+ influx and alleviating Ca2+ release from sarcoplasmic reticulum(SR). The reduction of Ca2+ influx might be due to the inhibition of voltage-dependent Ca2+ channel via adenosine A1 receptor, and NO might be involved in this process.

Adenosine ; pharmacology ; Animals ; Calcium ; metabolism ; Cells, Cultured ; Guinea Pigs ; Heart Ventricles ; cytology ; Myocytes, Cardiac ; drug effects ; metabolism