Blebbistatin extends culture life of adult mouse cardiac myocytes and allows efficient and stable transgene expression

The characterization of cellular phenotypes of heart disorders can be achieved by isolating cardiac myocytes from mouse models or genetically modifying wild-type cells in culture. However, adult mouse cardiac myocytes show extremely low tolerance to isolation and primary culture conditions. Previous studies indicate that 2,3-butanedione monoximine (BDM), a nonspecific excitation-contraction coupling inhibitor, can improve the viability of isolated adult mouse cardiac myocytes. The mechanisms of the beneficial and unwanted nonspecific actions of BDM on cardiac myocytes are not understood. To understand what contributes to murine adult cardiac myocyte stability in primary culture and improve this model system for experimental use, the specific myosin II inhibitor blebbistatin was explored as a media supplement to inhibit mouse myocyte contraction. Enzymatically isolated adult mouse cardiac myocytes were cultured with blebbistatin or BDM as a media supplement. Micromolar concentrations of blebbistatin significantly increased the viability, membrane integrity, and morphology of adult cardiac myocytes compared with cells treated with previously described 10 mM BDM. Cells treated with blebbistatin also showed efficient adenovirus gene transfer and stable transgene expression, and unlike BDM, blebbistatin does not appear to interfere with cell adhesion. Higher concentrations of BDM actually worsened myocyte membrane integrity and transgene expression. Therefore, the specific inhibition of myosin II activity by blebbistatin has significant beneficial effects on the long-term viability of adult mouse cardiac myocytes. Furthermore, the unwanted effects of BDM on adult mouse cardiac myocytes, perhaps due to its nonspecific activities or action as a chemical phosphatase, can be avoided by using blebbistatin.     

Electrical, contractile, and ultrastructural properties of adult rat and guinea-pig ventricular myocytes in long-term primary cultures

The electrical, contractile, and morphological characteristics of ventricular myocytes isolated from adult rat and guinea-pig hearts and maintained in cultures for 7-24 days are described. These cultured cells form different networks, depending on the species, when plated at certain density and maintained under specific conditions; the cells within the networks appear to be electrically coupled. Their resting and action potentials, their contractile activity (shortenings), and their pharmacological responses qualitatively resemble those of freshly isolated myocytes. Cultured cells from both species exhibit near-normal ultrastructural organization of sarcomeres, myofilaments, and mitochondria, as well as formation of intercellular contacts, or gap junctions. These data indicate that cultured adult rat and guinea-pig myocardial cells that make intercellular contacts possess electrical, contractile, and ultrastructural properties and responses to pharmacological agents similar to those of the respective adult myocardial tissues and the functionally intact freshly isolated cells from which these cultures are prepared. Thus, this study indicates that long-term cultures (7-24 days) of networked cardiac myocytes could be used as a valuable experimental model in various investigations of excitation-contraction coupling in cardiac muscle.     

Treatment of cardiac myocytes with 8-(4-chlorophenylthio)-adenosine 3'',5''-cyclic monophosphate, forskolin or cholera toxin does not stimulate cellular or heparin-releasable lipoprotein lipase activities.

Incubation of isolated cardiac myocytes with 500 microM-8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) or 100 microM-forskolin for 2 1/2 h did not increase the heparin-induced release of lipoprotein lipase (LPL) into the medium. When LPL activity in cardiac myocytes was depleted by treatment of rats with cycloheximide (2 mg/kg; 2.5 h) and inclusion of the protein-synthesis inhibitor in the isolation solutions, incubation with CPT-cAMP or forskolin did not influence the rate of repletion of LPL activity in cells or the recovery of heparin-releasable LPL activity. Although the administration of cholera toxin (0.5 mg/kg; 16-17 h) to rats increased LPL activity in a low-speed supernatant fraction from heparin-perfused hearts, LPL activity was not increased in cardiac myocytes from cholera-toxin-treated rat hearts, and the heparin-induced release of LPL was unchanged. Incubation of cultured ventricular myocytes with 1 microgram of cholera toxin/ml or 500 microM-CPT-cAMP for 24 h did not increase cellular LPL activity or LPL released into the culture medium after a 40 min incubation with heparin. Therefore interventions that stimulate adenylate cyclase activity (forskolin, cholera toxin) or incubation with CPT-cAMP do not increase cellular LPL activity or promote the translocation of LPL to a heparin-releasable fraction in cardiac myocytes.     

Myocyte enlargement, differentiation, and proliferation kinetics in the fetal sheep heart.

The generation of new myocytes is an essential process of in utero heart growth. Most, or all, cardiac myocytes lose their capacity for proliferation during the perinatal period through the process of terminal differentiation. An increasing number of studies focus on how experimental interventions affect cardiac myocyte growth in the fetal sheep. Nevertheless, fundamental questions about normal growth of the fetal heart remain unanswered. In this study, we determined that during the last third of gestation the hearts of fetal sheep grew primarily by four processes. 1) Myocyte proliferation contributed substantially to daily cardiac mass gain, and the number of cardiac myocytes continued to increase to term. 2) The (hitherto unrecognized) contribution to cardiac growth by the increase in myocyte size associated with the transition from mononucleation to binucleation (terminal differentiation) became considerable from approximately 115 days of gestational age (dGA) until term (145dGA). Because binucleation became the more frequent outcome of myocyte cell cycle activity after approximately 115dGA, the number of binucleated myocytes increased at the expense of the number of mononucleated myocytes. Both the interval between nuclear divisions and the duration of cell cycle activity in myocytes decreased substantially during this same period. Finally, cardiac growth was in part due to enlargement of 3) mononucleated and 4) binucleated myocytes, which grew in cross-sectional diameter but not length during the last third of gestation. These data on normal cardiac growth may enable a more detailed understanding of the consequences of experimental and pathological interventions in prenatal life.     

Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process

The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34(+) cells were cocultured with cardiac myocytes infected with a red fluorescence protein-lentiviral vector; under these conditions we found that 100% of EGFP(+) cells were also red fluorescent protein positive, suggesting cell fusion as the mechanism by which cardiac functional features are acquired.     

Embryonic stem cells form an organized, functional cardiac conduction system in vitro

A functional pacemaking-conduction system is essential for maintaining normal cardiac function. However, no reproducible model system exists for studying the specialized cardiac pacemaking-conduction system in vitro. Although several molecular markers have been shown to delineate components of the cardiac conduction system in vivo, the functional characteristics of the cells expressing these markers remain unknown. The ability to accurately identify cells that function as cardiac pacemaking cells is crucial for being able to study their molecular phenotype. In differentiating murine embryonic stem cells, we demonstrate the development of an organized cardiac pacemaking-conduction system in vitro using the coexpression of the minK-lacZ transgene and the chicken GATA6 (cGATA6) enhancer. These markers identify clusters of pacemaking "nodes" that are functionally coupled with adjacent contracting regions. cGATA6-positive cell clusters spontaneously depolarize, emitting calcium signals to surrounding contracting regions. Physically separating cGATA6-positive cells from nearby contracting regions reduces the rate of spontaneous contraction or abolishes them altogether. cGATA6/minK copositive cells isolated from embryoid cells display characteristics of specialized pacemaking-conducting cardiac myocytes with regard to morphology, action potential waveform, and expression of a hyperpolarization-activated depolarizing current. Using the cGATA6 enhancer, we have isolated cells that exhibit electrophysiological and genetic properties of cardiac pacemaking myocytes. Using molecular markers, we have generated a novel model system that can be used to study the functional properties of an organized pacemaking-conducting contracting system in vitro. Moreover, we have used a molecular marker to isolate a renewable population of cells that exhibit characteristics of cardiac pacemaking myocytes.     

Effect of exercise training on intracellular free Ca2+ transients in ventricular myocytes of rats.

The purpose of this study was to test the hypothesis that exercise training induces enhanced intracellular free Ca2+ (Cai) availability to the contractile elements of cardiac cells. Cai transients were directly measured in single isolated contracting ventricular myocytes from exercise-trained (EX) and sedentary control (SED) rats. Male Sprague-Dawley rats underwent 16 wk of progressive treadmill exercise (32 m/min, 8% grade, 1.5 h/day) (EX) or were cage confined (SED). EX rats had lower resting heart rate and elevated skeletal muscle oxidative capacity. Cai was measured with the fluorescent Cai indicator fura-2. Simultaneous video monitoring indicated that myocytes suspended in physiological salt solution were quiescent until stimulated electrically at a frequency of 0.2 Hz (12-36 V, 2-ms duration). Stimulated Cai transients, measured from changes in fura-2 fluorescence, were similar in cells from EX and SED groups. Peak shortening, time to peak shortening, velocity of shortening, contraction duration, and time to half-relaxation were also similar in cells from EX and SED rats. Ryanodine (10 microM) was applied to eliminate the contribution of Ca2+ release from sarcoplasmic reticulum to the Cai transient. Verapamil was applied to eliminate the contribution of voltage-gated Ca2+ channels to Cai transients. Cai transients were also similar in cells from EX and SED groups after these pharmacological interventions. These results suggest that treadmill training of rats does not alter Cai availability to the contractile elements in isolated ventricular myocytes.     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.     

Abolition of reperfusion-induced arrhythmias in hearts from thiamine-deficient rats

Extensive work has been done regarding the impact of thiamine deprivation on the nervous system. In cardiac tissue, chronic thiamine deficiency is described to cause changes in the myocardium that can be associated with arrhythmias. However, compared with the brain, very little is known about the effects of thiamine deficiency on the heart. Thus this study was undertaken to explore whether thiamine deprivation has a role in cardiac arrhythmogenesis. We examined hearts isolated from thiamine-deprived and control rats. We measured heart rate, diastolic and systolic tension, and contraction and relaxation rates. Whole cell voltage clamp was performed in rat isolated cardiac myocytes to measure L-type Ca(2+) current. In addition, we investigated the global intracellular calcium transients by using confocal microscopy in the line-scan mode. The hearts from thiamine-deficient rats did not degenerate into ventricular fibrillation during 30 min of reperfusion after 15 min of coronary occlusion. The antiarrhythmogenic effects were characterized by the arrhythmia severity index. Our results suggest that hearts from thiamine-deficient rats did not experience irreversible arrhythmias. There was no change in L-type Ca(2+) current density. Inactivation kinetics of this current in Ca(2+)-buffered cells was retarded in thiamine-deficient cardiac myocytes. The global Ca(2+) release was significantly reduced in thiamine-deficient cardiac myocytes. The amplitude of caffeine-releasable Ca(2+) was lower in thiamine-deficient myocytes. In summary, we have found that thiamine deprivation attenuates the incidence and severity of postischemic arrhythmias, possibly through a mechanism involving a decrease in global Ca(2+) release.     

Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types including spontaneously contracting cardiac myocytes. We have utilized the ES cell differentiation culture system to study the development of the cardiac contractile apparatus in vitro. Difficulties associated with the cellular and developmental heterogeneity of this system have been overcome by establishing attached cultures of differentiating ES cells, and by the micro-dissection of the contracting cardiac myocytes from culture. The time of onset and duration of continuous contractile activity of the individual contracting myocytes was determined by daily visual inspection of the cultures. A functional assay was used to directly measure force production in ES cell-derived cardiac myocyte preparations. The forces produced during spontaneous contractions in the membrane intact preparation, and during activation by Ca2+ subsequent to chemical permeabilization of the surface membranes were determined in the same preparation. Results showed a transition in contractile sensitivity to Ca2+ in ES cell-derived cardiac myocytes during development in vitro. Cardiac preparations isolated from culture following the initiation of spontaneous contractile activity showed marked sensitivity of the contractile apparatus to activation by Ca2+. However, the Ca2+ sensitivity of tension development was significantly decreased in preparations isolated from culture following prolonged continuous contractile activity in vitro. The alteration in Ca2+ sensitivity obtained in vitro paralleled that observed during murine cardiac myocyte development in vivo. This provides functional evidence that ES cell-derived cardiac myocytes recapitulate cardiogenesis in vitro. Alterations in Ca2+ sensitivity could be important in optimizing the cardiac contractile response to variations in the myoplasmic Ca2+ transient during embryogenesis. The potential to stably transfect ES cells with cardiac regulatory genes, together with the availability of a functional assay using control and genetically modified ES cell- derived cardiac myocytes, will permit determination of the functional significance of altered cardiac gene expression during cardiogenesis in vitro.     

Metabolic communication from cardiac myocytes to vascular endothelial cells

The endothelium releases substances that affect both vascular and cardiac myocytes. However, under conditions of augmented metabolic demands and cardiac work, signals from the cardiac myocytes may be critical for the endothelium to fulfill its secretory and regulatory function in the vascular bed. Therefore, we hypothesized that cardiac myocytes produce substances that alter the resting membrane potential of endothelial cells and thus vascular tone. Isolated rat cardiac myocytes were electrically stimulated at the rate of 0 and 400 beats/min (Po2 = 150 mmHg), and supernatants were collected from each group (Sup-0; control) and (Sup-400) and used within 6 mo. These supernatants were applied to human coronary endothelial cells that were subsequently analyzed by using the whole cell and cell-attached patch-clamp modes. Sup-0 had no effect on the whole cell current and the zero-current potential. The Sup-0 from myocytes treated with aprotinin, an inhibitor of kallikrein and serine protease, reduced whole cell current between -120 and -60 mV. Sup-400 depolarized endothelial cells from the resting membrane potential of -45 to -5 mV (P < 0.05), increased the magnitude of an inward current, and activated an outward current. Moreover, Sup-400 cells assayed in cell-attached patches increased single channel amplitude and the probability of a channel being in the open state. These effects were reversed by the Sup-400 from aprotinin-treated cells. We conclude that under certain metabolic conditions, isolated cardiac myocytes produce and release vasoactive substances that alter the function of K+ channels in vascular endothelial cells. Thus cardiac myocytes seem to communicate metabolic information to the endothelium, which could potentially influence vascular tone.     

Identification of cardiac stem cells within mature cardiac myocytes

Cardiac stem cells are described in a number of mammalian species including humans. Cardiac stem cell clusters consisting of both lineage-negative and partially committed cells are generally identified between contracting cardiac myocytes. In the present study, c-kit⁺, Sca⁺, and Isl1⁺ stem cells were revealed to be located inside the sarcoplasm of cardiac myocytes in myocardial cell cultures derived from newborn, 20-, and 40-day-old rats. Intracellularly localized cardiac stem cells had a coating or capsule with a few pores that opened into the host cell sarcoplasm. The similar structures were also identified in the suspension of freshly isolated myocardial cells (ex vivo) of 20- and 40-day-old rats. The results from this study provide direct evidence for the replicative division of encapsulated stem cells, followed by their partial cardiomyogenic differentiation. The latter is substantiated by the release of multiple transient amplifying cells following the capsule rupture. In conclusion, functional cardiac stem cells can reside not only exterior to but also within cardiomyocytes.     

Beta-myosin heavy chain myocytes are more resistant to changes in power output induced by ischemic conditions

During ischemia intracellular concentrations of P(i) and H+ increase. Also, changes in myosin heavy chain (MHC) isoform toward beta-MHC have been reported after ischemia and infarction associated with coronary artery disease. The purpose of this study was to investigate the effects of myoplasmic changes of P(i) and H+ on the loaded shortening velocity and power output of cardiac myocytes expressing either alpha- or beta-MHC. Skinned cardiac myocyte preparations were obtained from adult male Sprague-Dawley rats (control or treated with 5-n-propyl-2-thiouracil to induce beta-MHC) and mounted between a force transducer and servomotor system. Myocyte preparations were subjected to a series of isotonic force clamps to determine shortening velocity and power output during Ca2+ activations in each of the following solutions: 1) pCa 4.5 and pH 7.0; 2) pCa 4.5, pH 7.0, and 5 mM P(i); 3) pCa 4.5 and pH 6.6; and 4) pCa 4.5, pH 6.6, and 5 mM P(i). Added P(i) and lowered pH each caused isometric force to decline to the same extent in alpha-MHC and beta-MHC myocytes; however, beta-MHC myocytes were more resistant to changes in absolute power output. For example, peak absolute power output fell 53% in alpha-MHC myocytes, whereas power fell only 38% in beta-MHC myocytes in response to elevated P(i) and lowered pH (i.e., solution 4). The reduced effect on power output was the result of a greater increase in loaded shortening velocity induced by P(i) in beta-MHC myocytes and an increase in loaded shortening velocity at pH 6.6 that occurred only in beta-MHC myocytes. We conclude that the functional response to elevated P(i) and lowered pH during ischemia is MHC isoform-dependent with beta-MHC myocytes being more resistant to declines in power output.     

Altered single cell force-velocity and power properties in exercise-trained rat myocardium.

Myocardial function is enhanced by endurance exercise training, but the cellular mechanisms underlying this improved function remain unclear. The ability of the myocardium to perform external work is a critical aspect of ventricular function, but previous studies of myocardial adaptation to exercise training have been limited to measurements of isometric tension or unloaded shortening velocity, conditions in which work output is zero. We measured force-velocity properties in single permeabilized myocyte preparations to determine the effect of exercise training on loaded shortening and power output. Female Sprague-Dawley rats were divided into sedentary control (C) and exercise trained (T) groups. T rats underwent 11 wk of progressive treadmill exercise. Myocytes were isolated from T and C hearts, chemically skinned, and attached to a force transducer. Shortening velocity was determined during loaded contractions at 15 degrees C by using a force-clamp technique. Power output was calculated by multiplying force times velocity values. We found that unloaded shortening velocity was not significantly different in T vs. C myocytes (T = 1.43 muscle lengths/s, n = 46 myocytes; C = 1.12 muscle lengths/s, n = 43 myocytes). Training increased the velocity of loaded shortening and increased peak power output (peak power = 0.16 P/P(o) x muscle length/s for T myocytes; peak power = 0.10 P/P(o) x muscle length/s for C myocytes, where P/P(o) is relative tension). We found no effect of training on myosin heavy chain isoform content. These results suggest that training alters power output properties of single cardiac myocytes and that this adaptation may improve the work capacity of the myocardium.     

Intact individual heart cells isolated from human ventricular tissue.

The intricate architecture of heart muscle, comprising irregularly shaped cells which interdigitate in a complex three-dimensional array, has often compromised clear interpretation of experimental data obtained from the whole organ. One approach to minimise some of the difficulties is to use individual muscle cells in suspension, and data have already been reported using myocytes isolated from mammalian ventricles. It is difficult, however, to extrapolate results obtained from animal tissues to situations of medical relevance in man. Intact isolated muscle cells were obtained from human ventricular tissue by modifications of methods used for isolating smooth muscle, atrial, and ventricular tissue from animals. Electrical studies showed that these myocytes had functional characteristics similar to those observed in the whole heart. Such cells will prove a useful preparation for studies on both the mechanisms underlying myocardial performance in normal and diseased states and the response of heart tissue at the cellular level to conditions found during cardiac surgery.     

Comparisons of different stages of chick embryonic development by the physiological regulatory response to hyposmotic challenge

Cardiac myocytes isolated and cultured from 11 day chick embryos present a Ca(2+)-dependent regulatory volume decrease (RVD) when exposed to hyposmotic stimulus. The RVD of myocytes from different embryonic stages were analyzed to evaluate their physiological performance through development. Among the several embryonic stages analyzed (6, 11, 16 and 19 days) only 19 day cardiac myocytes present a greater RVD when compared with 11 day (considered as control), the other ages showed no difference in the regulatory response. As it is known that RVD is Ca(2+) dependent, we decided to investigate the transient free Ca(2+) response during the hyposmotic swelling of the 11 and 19 day stages. The 11 day cardiac myocyte showed a transient 40% increase in intracellular free Ca(2+) when submitted to hyposmotic solutions, and the free Ca(2+) returned to baseline levels while the cells remained in hyposmotic buffer. However, the intracellular free Ca(2+) transient in the 19 day cells during hyposmotic challenge increases 100% and instead of returning to baseline levels, declines to 55% above control, well after the 11 day transient has returned to baseline. Also, quantitative fluorescence microscopy revealed that 19 day cardiac myocytes have more sarcoplasmic reticulum (SR) Ca(2+) ATPase sites per cell as compared to the 11 day cells. Our findings suggest that 19 day cells have more developed intracellular Ca(2+) stores (SR). By evoking the mechanism of Ca(2+) induced Ca(2+) release, the cells have more free Ca(2+) available for signaling the RVD during hyposmotic swelling.     

Chronic stretch of engineered heart tissue induces hypertrophy and functional improvement.

To examine the influence of chronic mechanical stretch on functional behavior of cardiac myocytes, we reconstituted embryonic chick or neonatal rat cardiac myocytes to a 3-dimensional engineered heart tissue (EHT) by mixing freshly isolated cells with neutralized collagen I and culturing them between two Velcro-coated silicone tubes, held at a fixed distance with a metal spacer. After 4 days, EHTs were subjected to a phasic unidirectional stretch for 6 days in serum-containing medium. Compared to unstretched controls, RNA/DNA and protein/cell ratios increased by 100% and 50%, respectively. ANF mRNA and alpha-sarcomeric actin increased by 98% and 40%, respectively. Morphologically, stretched EHTs exhibited improved organization of cardiac myocytes into parallel arrays of rod-shaped cells, increased cell length and width, longer myofilaments, and increased mitochondrial density. Thus, stretch induced phenotypic changes, generally referred to as hypertrophy. Concomitantly, force of contraction was two- to fourfold higher both under basal conditions and after stimulation with calcium or the beta-adrenergic agonist isoprenaline. Contraction kinetics were accelerated with a 14-44% decrease in twitch duration under all those conditions. In summary, we have developed a new in vitro model that allows morphological, molecular, and functional consequences of stretch to be studied under defined conditions. The main finding was that stretch of EHTs induced cardiac myocyte hypertrophy, which was accompanied by marked improvement of contractile function.     

A method to collect isolated myocytes and whole tissue from the same heart

A technique for isolation of cardiac myocytes and collection of whole heart tissue from individual hearts of adult rats is described in this study. After excision of the apical half of the left ventricle (LV) and cauterization of the cut edge, aortas were cannulated and high-quality isolated cardiac myocytes were collected after collagenase perfusion of the basal portion. Myocyte dimensions from the basal portion of cauterized and noncauterized hearts from matching rats were identical. Additionally, myocyte dimensions from the basal and apical halves of the LV were compared with the use of whole heart-isolated myocyte preps. No regional differences between basal and apical LV myocyte size were found. Therefore, this cauterization method can be used to collect isolated myocytes from the basal half and whole heart tissue from the apical half, with each half being representative of the other with respect to myocyte dimensions.     

大鼠甲状腺毒症心肌肥大的细胞内钙离子分布的研究

采用人工大鼠腹腔注射Ｌ－甲状腺素３周、４周不等,发现实验组大鼠心肌细胞线粒体内有大量Ｃａ２＋沉淀物积聚,多呈弥漫性散在分布,亦可沿线粒体嵴呈线样排列。结果提示：细胞内Ｃａ２＋积聚和异常分布与细胞损害程度密切相关。     

Heart ventricular cells of neonatal rats in a culture: DNA synthesis, ultrastructure and localization of atrial natriuretic peptide

We determined the optimal conditions suitable for expanding cardiac cells in vitro for their future use in experimental transplantation into injured myocardium of adult animals. Ventricular cardiac cells were isolated enzymatically from 2-3 day-old rats and cultured at different cell densities within 5-7 days to 4 weeks. Mixed cultures of muscle and non-muscle cells were examined by light autoradiography, electron microscopy, and immunogold method. The best results were obtained at a density of 3 x 10(5) cells/ml in the medium, consisting of 90% DMEM and 10% fetal calf serum, during 5-7 days of cultivation. In such cultures myocytes made 62.5 +/- 7.9%. After a 24 h incubation with 3H-thymidine, 22.0 +/- 2.2% of myocytes were labeled. Muscle cells contact with each other and with non-muscle cells, contain myofibrils, contract and display atrial natriuretic peptide (ANP)-like immunoreactivity.