A novel purification method of murine embryonic stem cell- and human-induced pluripotent stem cell-derived cardiomyocytes by simple manual dissociation

Cardiomyocytes derived from embryonic stem cells (ES-CMs) and induced pluripotent stem cells (iPS-CMs) are useful for toxicity and pharmacology screening. In the present study, we found that cardiomyocyte-rich beating cell clusters (CCs) emerged from murine embryonic stem cell (mESC)-derived beating EBs and from human-induced pluripotent stem cell (hiPSC)-derived beating EBs dissociated by gentle pipetting with a thin glass pipette. The percentage of cardiac troponin T (cTnT)-positive cells in the beating CCs obtained from mESC-derived and hiPSC-derived beating EBs was higher (81.5% and 91.6%, respectively) than in beating-undissociated EBs (13.7% and 67.1%, respectively). For mESCs, the yield of cTnT-positive cells from beating CCs was estimated to be 1.6 times higher than that of beating EBs. The bromodeoxyuridine labeling index of mouse ES-CMs and human iPS-CMs in beating CCs was 1.5- and 3.2-fold, respectively, greater than those in beating EBs. To investigate the utility of the cells in toxicity assessment, we showed that doxorubicin, a cardiotoxic drug, induced myofilament disruption in cardiomyocytes isolated by this method. This simple method enables preparation of mouse ES-CMs and human iPS-CMs with better proliferative activity than beating EBs not dissociated by pipetting, and the cardiomyocytes are useful for drug-induced myocardial toxicity testing.     

Early-age-dependent selective decrease of differentiation potential of hair-follicle-associated pluripotent (HAP) stem cells to beating cardiac-muscle cells

We have previously discovered nestin-expressing hair-follicle-associated pluripotent (HAP) stem cells and have shown that they can differentiate to neurons, glia, and many other cell types. HAP stem cells can be used for nerve and spinal cord repair. We have recently shown the HAP stem cells can differentiate to beating heart-muscle cells and tissue sheets of beating heart-muscle cells. In the present study, we determined the efficiency of HAP stem cells from mouse vibrissa hair follicles of various ages to differentiate to beating heart-muscle cells. We observed that the whiskers located near the ear were more efficient to differentiate to cardiac-muscle cells compared to whiskers located near the nose. Differentiation to cardiac-muscle cells from HAP stem cells in cultured whiskers in 4-week-old mice was significantly greater than in 10-, 20-, and 40-week-old mice. There was a strong decrease in differentiation potential of HAP stem cells to cardiac-muscle cells by 10 weeks of age. In contrast, the differentiation potential of HAP stem cells to other cell types did not decrease with age. The possibility of rejuvenation of HAP stem cells to differentiate at high efficiency to cardiac-muscle cells is discussed.     

Further studies on preservation of the beating rhythm of myocardial cells in culture

1. 1. Single myocardial cells from fetal mouse heart beat spontaneously in monolayer culture. In standard medium they maintained a constant beating rate for at least 5 h. After the beating rate of individual cells had been accelerated for a short time by electrical stimulation, the original beating rate could be immediately restored by interrupting the stimulation. Quiescent myocardial cells from neonatal mouse atrium could be induced to beat by electrical stimulation and most of them ceased to beat again immediately by interrupting the stimulation.

2. 2. After the spontaneous beating of individual myocardial cells had been stopped or slowed down for a short time by incubation in medium of low temperature or high potassium or low calcium concentration, the original beating rate could be restored by replacing the cells in the original, normal medium.

3. 3. After the spontaneous beating of individual myocardial cells had been stopped by adding a metabolic inhibitor, such as 2,4-dinitrophenol or 2-deoxyglucose, the original beating rate could be restored by replacing the cells in the original, normal medium.

4. 4. Both single myocardial cells and cell clusters showed arrhythmia, including flutter and fibrillation, in medium of low potassium or high calcium concentration. After a short period of arrhythmia, the original beating rate could be restored by replacing the cells in the original, normal medium. The arrhythmia of cell clusters produced in either low potassium or high calcium medium was also corrected immediately by addition of quinidine sulfate.

     

Exploring the implicit interlayer regulatory mechanism between cells and tissue: Stochastic mathematical analyses of the spontaneous ordering in beating synchronization

The present study focused on beating synchronization, and tried to elucidate the interlayer regulatory mechanisms between the cells and clump in beating synchronization with using the stochastic simulations which realize the beating synchronizations in beating cells with low cell–cell conductance. Firstly, the fluctuation in interbeat intervals (IBIs) of beating cells encouraged the process of beating synchronization, which was identified as the stochastic resonance. Secondly, fluctuation in the synchronized IBIs of a clump decreased as the number of beating cells increased. The decrease in IBI fluctuation due to clump formation implied both a decline of the electrophysiological plasticity of each beating cell and an enhancement of the electrophysiological stability of the clump. These findings were identified as the community effects. Because IBI fluctuation and the community effect facilitated the beating stability of the cell and clump, these factors contributed to the spontaneous ordering in beating synchronization. Thirdly, the cellular layouts in clump affected the synchronized beating rhythms. The synchronized beating rhythm in clump was implicitly regulated by a complicated synergistic effect among IBI fluctuation of each beating cell, the community effect and the cellular layout. This finding was indispensable for leading an elucidation of mechanism of emergence. The stochastic simulations showed the necessity of considering the synergistic effect, to elucidate the interlayer regulatory mechanisms in biological system.     

EFFECTS OF CORTISOL ON CULTURED RAT HEART CELLS : Lipase Activity, Fatty Acid Oxidation, Glycogen Metabolism, and ATP Levels as Related to the Beating Phenomenon

This paper reports the determination of the ability of rat heart cells in culture to release [¹⁴C]palmitate from its triglyceride and to oxidize this fatty acid and free [¹⁴C]palmitate to ¹⁴CO₂ when the cells are actively beating and when they stop beating after aging in culture. In addition, the levels of glucose, glycogen, and ATP were determined to relate the concentration of these metabolites with beating and with cessation of beating. When young rat heart cells in culture are actively beating, they oxidize free fatty acids at a rate parallel with cellular ATP production. Both fatty acid oxidation and ATP production remain constant while the cells continue to beat. Furthermore, glucose is removed from the growth medium by the cells and stored as glycogen. When cultured cells stop beating, a decrease is seen in their ability to oxidize free fatty acids and to release them from their corresponding triglycerides. Concomitant with decreased fatty acid oxidation is a decrease in cellular levels of ATP until beating ceases. Midway between initiation of cultures and cessation of beating the cells begin to mobilize the stored glycogen. When the growth medium is supplemented with cortisol acetate and given to cultures which have ceased to beat, reinitiation of beating occurs. Furthermore, all decreases previously observed in ATP levels, fatty acid oxidation, and esterase activity are restored.     

Effects of cardioactive drugs on the beating activity of myocardial cell cultures isolated from offspring of trained and untrained pregant rats

Summary Primary cultures of beating myocardial cells were obtained from 5-d-old offspring of trained (T) and untrained (UT), pregnant, Sprague-Dawley rats. The myocardial cells from the T and UT groups were evaluated for their beating responses to three cardioactive drugs: verapamil (V), isoproterenol (ISO), and propranolol (PRO). The myocardial cell cultures from the UT group showed complete loss of beating when the calcium (Ca⁺⁺) antagonist, V, was added to the cultures for 1 h or more; the T group was able to show some beating at comparable concentrations and durations of exposure with V. The beta agonist, ISO, markedly stimulated the beating rate of both the T and UT groups, but the beating rates were higher in the UT group at comparable concentrations and durations of exposure than with the T group. When the cultures were pretreated with the beta blocker, PRO, before treatment with ISO, a concentration inhibitory effect on the beating rate was observed in both groups. However, the T cultures were more sensitive to the inhibitory effects of PRO. These results demonstrate that primary cultures of rat myocardial cells isolated from the offspring of trained and untrained pregnant rats show differential beating responses to three well-known cardioactive drugs. This study was supported by grants from the American Heart Association, Texas Affiliate, and the University of Texas Research Institute.     

A backward swimming mutant of Chlamydomonas reinhardii

A backward swimming mutant (RL-10) was isolated from Chlamydomonas reinhardii. In contrast to the wild-type flagellum which usually displays a ciliary type beating pattern, the flagella in the RL-10 cells always propagated such undulating waves as found in sperm flagella. This abnormal beating pattern was maintained after the cell was demembranated by a non-ionic detergent (Nonidet P40) and reactivated with ATP. Reactivated axonemes (demembranated flagella) of the wild-type cells changed the beating pattern from the ciliary type to the flagellar type when the Ca²⁺ concentration was increased from 10⁻⁷ to 10⁻⁶ M. However, the RL-10 axonemes did not show such a Ca-dependent change in the beating pattern. Hence the RL-10 flagella might carry defects in the controlling mechanisms of flagellar beating pattern, at sites other than the membrane.     

A study on the preservation of the beating rhythm of single myocardial cells in vitro

Single myocardial cells from fetal mouse heart beat spontaneously in monolayer culture. In standard medium they maintained constant beating rates for at least 5 h at 37 °C. When the beats of single myocardial cells were stopped for a short time by treatment with EGTA, or slowed down by incubating the cells in medium of low pH, the original beating rates could be restored by replacing the cells in the original medium. The same procedure also restored the rates after they had been disturbed by incubating the cells in medium of low sodium and high potassium ion content. Moreover, the original beating rates could be restored after keeping the cells at 10 °C for 22–24 h, but not after keeping them at 37 °C for 22–24 h.     

The role of mononuclear phagocytes in cardiac allograft rejection in the rat: III. The effect of cells extracted from rat cardiac allografts upon beating heart cell cultures

Cells extracted from rat cardiac allografts were able to bring about cessation of beating of heart cell culture monolayers nonspecifically. Nonadherent populations, depleted of macrophages, were consistently less potent than unseparated cells in this assay. Cells extracted from isografts were totally ineffective. Allogeneically stimulated peritoneal cells were also nonspecifically active. Again, nonadherent cells were less efficient than unseparated cells at stopping heart cell monolayers from beating, while adherent cells, enriched for macrophages, were more efficient. Activated bone marrow culture macrophages syngeneic or allogeneic to the heart cultures were also highly potent in beating heart cell assays. Thus in all cases the predominant effector cell type was adherent and nonspecific in its action and therefore presumably a macrophage. Supernatants from wells in which no beating cells remained following incubation with each type of effector population tested were transferred undiluted to fresh wells. In all cases there was no effect at all upon the beating of heart cell monolayers. Antirat heart antiserum plus complement was able to bring about the cessation of beating of heart culture monolayers at a dilution of 1:64. Alloantibody plus complement did not bring about cessation of beating at any dilution, although nonmyocardial cells were killed. The possibility that macrophages are the chief effector cell type in a DTH-like mechanism for cardiac allograft rejection is discussed.     

Prostaglandins have limited actions on abnormalities of beating induced in cultured heart cells

Prostaglandins are antiarrhythmic in a variety of situations including ischaemic arrhythmias, but the mechanisms involved are not known. In view of this, the protective actions of prostaglandins A₂, E₂, F_1α, F_2β, and I₂ against abnormalities of beating induced in cultured heart cells were investigated. Abnormalities of beating were induced in single cells by a variety of agents including ouabain, Ca⁺⁺, K⁺, dinitrophenol (DNP), and toxic material from the jellyfish . Abnormalities were assessed in terms of rate, rate range, subjective arrhythmic behaviour and percent cells beating. The prostaglandins (at 10⁻⁷-10⁻⁵ M) were added with the arrhythmogenic agent to test for their ability to modify agent-induced beating abnormalities and were compared with lidocaine and quinidine. Prostaglandins alone had minimal direct effects on the cells and only minimally reduced responses to arrhythmogenic agents. The most protective prostaglandins, PGE₂ and PGF_1α, tended to normalise beating behaviour most noticeably in DNP-treated cells, unlike lidocaine and quinidine which were effective against Ca⁺⁺-induced changes while worsening those of K⁺. Thus, a general ability to protect disturbed cardiac cells is not seen with high concentrations of prostaglandins.     

The effect of 5-bromodeoxyuridine (BrdU) on cardiac muscle differentiation

Cultured cardiac muscle cells undergo cell division and form beating progeny. Incorporation of BrdU into the nuclei of daughter cells does not suppress their ability to beat and form cross-striated myofibrils. Fluorescence microscopy of clones derived from single beating cells fed with BrdU-treated medium for over 2 weeks reveal cytoplasmic fibrils stainable with fluorescein-labeled antimyosin. The effect of BrdU on the emergence of cardiac muscle phenotype was also investigated by utilizing cardiac myogenic precursor cells from precardiac mesoderm in early embryos (stage 4–stage 9). These studies show that the cardiac myogenic cells fall into the following categories with respect to their ability to express the differentiated phenotype in the presence of BrdU: (1) precardiac mesodermal cells that are inhibited; (2) precardiac mesodermal cells that are not inhibited; and (3) beating cardiac muscle cells that are not inhibited. The entry of precardiac cells from the first category to the second and to the third appears to be unsynchronized.     

Role of the community effect of cardiomyocyte in the entrainment and reestablishment of stable beating rhythms

To investigate the roles that the community effect and entrainment function of cultured cardiomyocyte play in decreasing beating fluctuation and reestablishing synchronized beating, we developed a single-cell-based two-dimensional network culture assay to measure and compare the dynamics of beating rhythm synchronization of individual cells before and after they form networks. Studying the formation of two-cell networks, we found that their synchronized beating tended to be determined by the cardiomyocyte whose beat rate fluctuated less than that of the other cardiomyocyte. We further found that the strength of this tendency increased with the number of cells in the network. These results indicate that (1) beating fluctuation is one of the important factors influencing the reestablishment of a stable synchronous beating rhythm, (2) the larger networks reduce fluctuation, and (3) the formation of a spatial network can itself stabilize cardiomyocyte beat rates.     

The effects of CGRP on calcium transients of dedifferentiating cultured adult rat cardiomyocytes compared to non-cultured adult cardiomyocytes: possible protective and deleterious results in cardiac function

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.     

Beating of multinucleated giant myocardial cells in culture

Cultured mouse myocardial cells grown as cell sheets in Petri dishes fused together and formed multinucleated giant cells on treatment with HVJ (Sendai virus). The giant cells had well organized myofibrils and beat spontaneously and rhythmically. The spontaneous beating activity of the giant cells changed in response to changes of the external potassium and calcium concentrations and on addition of ouabain in the same way as the beating of cultured myocardial cells not treated with HVJ. When a microelectrode was inserted into giant cells that exhibited spontaneous beating, action potentials were easily recorded.     

Age-related loss of DNA repair synthesis in isolated rat myocardial cells

Singly isolated beating heart cells from newborn rats performed unscheduled DNA synthesis (‘repair’ synthesis) when irradiated with ultraviolet light in vitro. Repair synthesis could not be induced in beating cardiac cells isolated from adult animals (3–12 months old).     

Beating activity of heterokaryons between myocardial and non-myocardial cells in culture

Cultured mouse myocardial cells grown as monolayers fused upon treatment with HVJ (Sendai virus). The myocardial cells also fused with quail myocardial cells, neuroblastoma cells and non-excitable cells, such as KB cells. The beating activity of these heterokaryons was studied in the present work. Heterokaryons composed of myocardial cells from different species maintained spontaneous beating activity for 2 days or more. Those of one myocardial and one neuroblastoma cell maintained the activity for 22-26 h, while those of one myocardial and one non-excitable cell, such as KB cell, lost the activity within 2-4 h after addition of HVJ. Heterokaryons that had stopped spontaneous beating did not contract on application of electrical-field stimulation. The ration of non-myocardial cells in the heterokaryons increased in inverse proportion to the decrease in beating activity of the heterokaryons. Study of the rapid disappearance of beating activity in heterokaryons composed of one myocardial and one KB cell showed that both excitability of the cell membrane and myofibril organization were rapidly lost.     

Studies of in vitro cultivated cells from the smooth muscle organs. 3. Effectiveness of some drugs on pulsation frequency of isolated smooth muscle cells of the chicken amnion]

The effects of some drugs on the beating frequency of isolated cells of the chick amnion cultivated on cover slips were investigated. Cholinergic and adrenergic agonists and antagonists, serotonine, antispasmodics, coronary dilatants and local anesthetics influenced the beating frequency significantly. The isolated chick amnion cells equal in their pharmacological behaviour the intact chick amnion and smooth muscle cells of mammals but differ from isolated beating heart cells.     

Prostaglandins have limited actions on abnormalities of beating induced in cultured heart cells.

Prostaglandins are antiarrhythmic in a variety of situations including ischaemic arrhythmias, but the mechanisms involved are not known. In view of this, the protective actions of prostaglandins A2, E2, F1 alpha, F2 beta, and I2 against abnormalities of beating induced in cultured heart cells were investigated. Abnormalities of beating were induced in single cells by variety of agents including ouabain Ca++, K+, dinitrophenol (DNP), and toxic material from the jellyfish Cyanea. Abnormalities were assessed in terms of rate, rate range, subjective arrhythmic behaviour and percent cells beating. The prostaglandins (at 10(-7)-10(-5) M) were added with the arrhythmogenic agent to test for their ability to modify agent-induced beating abnormalities and were compared with lidocaine and quinidine. Prostaglandins alone had minimal direct effects on the cells and only minimally reduced responses to arrhythmogenic agents. The most protective prostaglandins, PGE2 and PGF1 alpha, tended to normalise beating behaviour most noticeably in DNP-treated cells, unlike lidocaine and quinidine which were effective against Ca++-induced changes while worsening those of K+. Thus, a general ability to protect disturbed cardiac cells is not seen with high concentrations of prostaglandins.