Apoptosis ; Inhibitor of Apoptosis Proteins ; Myocytes, Cardiac ; cytology

Adult Stem Cells ; Myocytes, Cardiac ; cytology

Cell Culture Techniques ; Humans ; Myocytes, Cardiac ; cytology ; Stem Cells ; cytology

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

To approach the method of isolation of tolerant human atrial myocytes, single myocytes were isolated by modified procedure of enzymatic dissociation with protease (type XXIV) and collagenase (type V). L-type calcium channel current (I(Ca-L)), sodium current (I(Na)), transient outward potassium current (I(to1)), and inward rectifier potassium current (I(K1)) in isolated atrial myocytes were recorded by using whole-cell patch-clamp techniques. Single cardiocytes isolated by this method were smooth, well-striated and rod-shaped. The yields of recordable myocytes, which viable and calcium-tolerant for electrophysiological studies, were 50%-60% of the total isolated cells. Compared with other isolation methods, this method was simple and steady, but with yield of a great number of qualified myocytes. The currents recorded in these cells were functional and active. Our research suggests that the myocytes isolated by the described method in this paper have normal electrophysiological function and are appropriate for patch-clamp experiments.
Cell Separation ; methods ; Humans ; Myocardium ; cytology ; Myocytes, Cardiac ; cytology ; Patch-Clamp Techniques

OBJECTIVETo generate a P19-alphaMHC-EGFP reporter line and induce cardiomyocyte differentiation of this reporter line.

METHODSThe P19 cells were transfected with palphaMHC-EGFP, a P19-alphaMHC-EGFP reporter line was obtained after G418 selection and limited dilution of recombinant clones. The reporter line was induced to differentiate into cardiomyocytes which would beat and express green fluorescent protein. A comparison of cardiomyocyte differentiation rate and cTnI expression amount between the reporter line and the untransfected P19 cells was also performed. The ultrastructure was observed under transmission electron microscope.

RESULTSThe ultrastructure characteristics indicated cardiomyocytes-like changes on induction day 10. The beating cardiomyocytes which express GFP appear in the seventh induction day. The cardiomyocyte differentiation rate and cTnI expression amount of P19-alphaMHC-EGFP reporter line were similar as those in untransfected P19 cells (P > 0.05).

CONCLUSIONThe P19-alphaMHC-EGFP reporter line is of great benefit for identifying and purifying cardiomyocytes from undifferentiated P19 cells without influencing the differentiation of P19 cells. This feature makes P19-alphaMHC-EGFP reporter line a promising cell source for clinical cardiomyocyte replacement therapy.

Animals ; Cell Culture Techniques ; Cell Differentiation ; Cell Line ; Mice ; Myocytes, Cardiac ; cytology ; Stem Cells ; cytology ; Transfection

Animals ; Humans ; Models, Animal ; Myocytes, Cardiac ; cytology ; Stem Cells ; cytology ; Tissue Engineering

OBJECTIVETo establish the integrated discrete multiple organ cell culture (IdMOC) system.

METHODSRat primary cell of hepatocyte, nephrocyte, cardiomyocytes, alveolar macrophage, dermal fibroblasts were isolated by collagenase digestion, separation of bronchial lavage, two-step digestion method and cultured respectively, with monolayer culture. To establish the integrated discrete multiple organ cell culture (IdMOC) system, glass slides of five different cells were used to the same dish with 10% FBS DMEM medium cultured 7d, using MTT comparison primary cells cultured alone and cocultured when growth.

RESULTSEstablished rat hepatocytes, renal cell, cardiomyocyte, alveolar macrophages, dermal fibroblasts separation method was stable, cell separation survival rate was about 90.0%. Hepatocytes separation survival rate 90.3% ,renal cell separation survival rate 91.9%, cardiomyocyte separation survival rate 93.0% and beating rate indifference curve among 3d-15d, alveolar macrophages cell separation survival rate 90.8%, dermal fibroblasts cell separation survival rate 92.7%. Five primary cells multiple organ cells coculture showed cocultured cell growth proliferation well, cultured alone and cocultured cells growth curve basic coincide.

CONCLUSIONEstablished rat multiple organ cell co-culture is successful.

Animals ; Cell Culture Techniques ; methods ; Epithelial Cells ; cytology ; Hepatocytes ; cytology ; Macrophages, Alveolar ; cytology ; Myocytes, Cardiac ; cytology ; Rats ; Rats, Sprague-Dawley

UNLABELLEDEmbryonic germ (EG) cells are pluripotent cells derived from primordial germ cells (PGCs) of gonads, gonadal ridges and mesenteries, analogies of fetuses,with the ability to undergo both highly self-renewal and multiple differentiation. These cells in vitro can differentiate into derivatives of all three embryonic germ layers when transferred to an in vitro environment and have the ability to form any fully differentiated cells of the body. The aim of this study is to investigate the potentiality of human EG cells differentiation into cardiomyocytes. Inducing human EG cells with the method of murine ES cells differentiation into cardiomyocytes, supplemented with 0.75%-1% DMSO, 20% NBS, 10(-7) mM RA and 20% cardiomyocytes conditioned medium.

RESULTS20 heart-like (rhythmic beating cell masses were observed in vitro culture and delayed human EG cells, which beat spontaneously from 20-120 times per minute and maintained beating for 2-15 days, periodic acid's staining (PAS), Myoglobin and a-actin immunological histology positive were all positive and reacted with K+, Ca2+ and adrenalin. Relatively unorganized myofibrillar bundles or more organized sarcomeres, z-bands or a gap junction, the presence of desmosomes in a few cells of the cell masses was observed with transmision electron microscope, which initially demonstrated that these cells were cardiomyocytes. We could not get rhythmly beating cardiomyocytes with 0.75%-1% DMSO, 10-7 mM RA and 20% cardiomyocytes conditioned medium,but in which the percentage of cardiac alpha-actin immunostaining positive cells were increased. The results first demonstrated that human EG cells can differentiate into rhythmic beating cardiomyocytes in vitro and suggests that human EG cells may represent a new potent resource for cardiomyocytes transplantation therapy for myocardium infarction.

Cell Culture Techniques ; methods ; Cell Differentiation ; Embryo, Mammalian ; cytology ; Germ Cells ; cytology ; Humans ; Myocytes, Cardiac ; cytology ; Pluripotent Stem Cells ; 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