Immunohistochemical analysis of the adaptation of adult guinea-pig cardiomyocytes in long-term cultures and in cocultures with cardiac neurons: A novel model for studies of myocardial function

In this study, we used laser confocal scanning microscopy and immunofluorescent markers to describe the establishment of long-term cultures of adult guinea-pig cardiomyocytes and their cocultures with adult intrinsic cardiac neurons. We have also investigated the effect of plating density on the adaptation of the myocytes in culture. Providing that the preparation of freshly isolated cardiomyocytes consists mostly (> 80%) of rod-shaped, Ca-tolerant, and quiescent cells and these are plated under optimal conditions and density (105/cm2), these myocytes have the following characteristics: (1) they remain elongated with regular ultrastructural characteristics and quiescent for several days; (2) within 10-14 days, they reestablish their intercellular contacts and resume contractile activity, which becomes synchronous all through the confluent layers; (3) they retain their regular myofibrilar striation all through the adaptation to culture conditions without any sign of dedifferentiation or redifferentiation; (4) these characteristics are lost when the cells are plated at too low (< 104/cm2) or too high (2 × 105/cm2) a density and they exhibit signs of dedifferentiation; (5) the adult ventricular myocytes appear to retain their ability to express atrial natriuretic peptide (ANP), as indicated by immunoreactivity to anti-ANP antibody; (6) this activity seems to be directly related to the surface area of the myocytes in contact with the substrate (i.e. to the stretch of the myocytes); (7) the intrinsic cardiac neurons grow intricate networks of neurites, which form a free-ending type of contact with the cocultured myocytes.abstract typed in here; if it is more than one paragraph use Long-term cultures of adult guinea-pig ventricular myocytes, alone or in their cocultures with cardiac neurons in which both are fully active functionally, provide a valuable experimental model which opens new possibilities for studying the cellular and molecular regulation of myocardial function under acute or chronic effects of various intrinsic and/or extrinsic factors, including neuroregulation.     

The Expression, Phosphorylation, and Localization of Connexin 43 and Gap-Junctional Intercellular Communication during the Establishment of a Synchronized Contraction of Cultured Neonatal Rat Cardiac Myocytes

We analyzed the expression, phosphorylation, and localization of the major cardiac gap-junction protein connexin 43 (Cx43) during the establishment of a synchronized contraction in confluent monolayers of primary cultured neonatal rat cardiac myocytes, combined with a functional assay of gap junctions by the microinjection-dye transfer method. Monitoring of the beating rate and synchronization by Fotonic Sensor showed that at Day 1 of culture cardiac myocytes contracted spontaneously but irregularly, that the contractile rate increased with culture time, and that a synchronized contraction was gradually formed. At Day 7, the confluent cells exhibited synchronous contraction with a relatively constant rate (125 ± 20 beats/min). Cardiac myocytes expressed a large amount of Cx43 mRNA even at Day 1 and maintained the expression until at least Day 7. Immunofluorescence of Cx43 showed that the localization of Cx43-positive spots was mostly restricted to cell-cell contacts between myocytes and that few Cx43-positive spots were present between myocytes and fibroblasts or between fibroblasts. The amount of Cx43 protein, the proportion of phosphorylated forms to the nonphosphorylated one, and the number and total area of Cx43-positive spots increased with culture time. Gap-junctional intercellular communication measured by dye transfer assay was also increased with culture time and correlated well with the number and total area of Cx43-positive spots. Our systematic study suggests that a concerted action of the expression, phosphorylation, and localization of Cx43 and gap-junctional intercellular communication plays a major role in the reestablishment of synchronous beating of cultured neonatal rat cardiac myocytes.     

Calcium signaling mechanisms in dedifferentiated cardiac myocytes: comparison with neonatal andadult cardiomyocytes

Our studies focused on calcium sparking and calcium transients in cultured adult rat cardiomyocytes and compared these findings to those in cultured neonatal and freshly isolated adult cardiomyocytes. Using deconvolution fluorescence microscopy and spec trophotometric image capture, sequence acquisitions were examined for calcium spark intensities, calcium concentrations and whether sparks gave rise to cell contraction events. Observations showed that the preparation of dedifferentiated cardiomyocytes resulted in stellate, neonatal-like cells that exhibited some aspects of calcium transient origination and proliferation similar to events seen in both neonatal and adult myocytes. Ryanodine treatment in freshly isolated adult myocytes blocked the calcium waves, indicating that calcium release at the level of the sarcoplasmic reticulum and t-tubule complex was the initiating factor, and this effect of ryanodine treatment was also seen in cultured-dedifferentiated adult myocytes. However, experiments revealed that in both neonatal and cultured adult myocytes, the inositol triphosphate pathway (IP3) was a major mechanism in the control of intracellular calcium concentrations. In neonatal myocytes, the nucleus and regions adjacent to the plasma membrane we re major sites of calcium release and flux. We conclude: (1) culturing of adult cardiomyocytes leads them to develop mechanisms of calcium homeostasis similar in some aspects to those seen in neonatal cardiomyocytes; (2) neonatal myocytes rely on both extracellular and nuclear calcium for contractile function; and (3) freshly isolated adult myocytes use sarcoplasmic reticulum calcium stores for the initiation of contractile function.     

Natriuretic peptide receptor-B in adult rat ventricle is predominantly confined to the nonmyocyte population

We assessed the cellular localization and relative concentration of the C-type natriuretic peptide (CNP) guanylate cyclase-B (GC-B) receptor in the adult rat heart ventricle by several techniques. In frozen sections of the ventricle, anti-receptor antibody stained the vasculature and cells interstitial to myocytes, but not the myocytes themselves. The same antibody detected GC-B in immunoblots of protein extracts of nonmyocytes, but not myocytes and recognized an equivalent protein in extracts of cultured cardiac fibroblasts, but not A7r5 rat smooth muscle cells. In functional assays, CNP-induced cGMP accumulation per milligram cell protein was an order of magnitude greater in cultured cardiac fibroblasts than in A7r5 smooth muscle cells and two orders of magnitude greater than in freshly isolated cardiac myocytes. Modulation of cGMP accumulation by phosphodiesterases (PDEs) was cell specific as determined by antagonist pharmacological profiles, PDE1 in fibroblasts, PDE2 in A7r5 cells, and PDE3 in myocytes, suggesting that significant but low-level cGMP response to CNP measured in heart myocytes is not due to nonmyocyte contamination. Fibroblasts of cardiac origin do not show an interactive relationship between receptor responsiveness to CNP, cGMP levels, and proliferation-related mitogen-activated signal transduction pathways. Whereas previous reports suggest CNP exerts significant effects in neonatal rat cardiomyocytes, our results suggest that fibroblasts are likely the most responsive cell type (cGMP production) in the adult rat heart.     

Cocultures of fetal and adult cardiomyocytes yield rhythmically beating rod shaped heart cells from adult rats

Summary Different models of isolated cardiomyocytes are generally used for biochemical, biophysical, and pharmacological studies. Fetal cardiomyocytes can be easily cultured for several weeks regaining their ability for rhythmical and synchronous contractions. For investigations, differentiated myocytes derived from adult hearts are closer to the in situ situation. Unfortunately, these cells at best exhibit irregular and asynchronous contractions at very low frequencies. Already 1 d after seeding calcium-tolerant rod-shaped adult cardiomyocytes on a suitable substrate, the differentiated cells begin to dedifferentiate forming a confluent monolayer. After 7–10 d their beating activities are like those of fetal cells. Therefore, we tried to combine the advantages of both cell types to achieve fully differentiated cardiomyocytes, rod-shaped and rhythmically beating, isolated from adult hearts. Using contractile fetal cells as a substrate for the adult cardiomyocytes, freshly seeded differentiated adult myocytes are paced by the contraction frequency of the fetal monolayer. As a consequence, the rod-shaped adult cardiomyocytes reach frequencies of more than 140 cycles/min without external electrical stimulation. This model enables us to study cardiomyocytes in a state very similar to the in situ situation with respect to morphology, integrity, and contractile behavior. An abstract of this article was previously published in Eur. J. Cell Biol. 57 (Suppl.36): 86; 1992.     

Contractile activity and cell-cell contact regulate myofibrillar organization in cultured cardiac myocytes

Adult feline ventricular myocytes cultured on a laminin-coated substratum reestablish intercellular junctions, yet disassemble their myofibrils. Immunofluorescence microscopy reveals that these non- beating heart cells lack vinculin-positive focal adhesions; moreover, intercellular junctions are also devoid of vinculin. When these quiescent myocytes are stimulated to contract with the beta-adrenergic agonist, isoproterenol, extensive vinculin-positive focal adhesions and intercellular junctions emerge. If solitary myocytes are stimulated to beat, an elaborate series of vinculin-positive focal adhesions develop which appear to parallel the reassembly of myofibrils. In cultures where neighboring myocytes reestablish cell-cell contact, myofibrils appear to reassemble from the fascia adherens rather than focal contacts. Activation of beating is accompanied by a significant reduction in the rate of total and cytoskeletal protein synthesis; in fact, myofibrillar reassembly, redevelopment of focal adhesions and fascia adherens junctions require no protein synthesis for at least 24 h, implying the existence of an assembly competent pool of cytoskeletal proteins. Maturation of the fasciae adherens and the appearance of vinculin within Z-line/costameres, does require de novo synthesis of new cytoskeletal proteins. Changes in cytoskeletal protein turnover appear dependent on beta agonist-induced cAMP production, but myofibrillar reassembly is a cAMP-independent event. Such observations suggest that mechanical forces, in the guise of contractile activity, regulate vinculin distribution and myofibrillar order in cultured adult feline heart cells.     

Possible involvement of ATP-purinoceptor signalling in the intercellular synchronization of intracellular Ca2+ oscillation in cultured cardiac myocytes

Isolated and cultured neonatal cardiac myocytes contract spontaneously and cyclically. The contraction rhythms of two isolated cardiac myocytes, each of which beats at different frequencies at first, become synchronized after the establishment of mutual contacts, suggesting that mutual entrainment occurs due to electrical and/or mechanical interactions between two myocytes. The intracellular concentration of free Ca(2+) also changes rhythmically in association with the rhythmic contraction of myocytes (Ca(2+) oscillation), and such a Ca(2+) oscillation was also synchronized among cultured cardiac myocytes. In this study, we investigated whether intercellular communication other than via gap junctions was involved in the intercellular synchronization of intracellular Ca(2+) oscillation in spontaneously beating cultured cardiac myocytes. Treatment with either blockers of gap junction channels or an un-coupler of E-C coupling did not affect the intercellular synchronization of Ca(2+) oscillation. In contrast, treatment with a blocker of P2 purinoceptors resulted in the asynchronization of Ca(2+) oscillatory rhythms among cardiac myocytes. The present study suggested that the extracellular ATP-purinoceptor system was responsible for the intercellular synchronization of Ca(2+) oscillation among cardiac myocytes.     

Multitrack system for superfusing isolated cardiac myocytes

A new system for studying mechanical activity of freshly isolated cardiac myocytes from up to four experimental groups simultaneously is described. Suspensions of cardiac myocytes isolated from adult rat hearts were drawn into microhematocrit capillary tubes, which were then mounted in parallel fashion between two four-channel tubing manifolds placed on the movable stage of an inverted microscope. Within a few minutes, cells settled and attached to the bottom of the tubes and then could be superfused with various test solutions. The system allowed for electrical field stimulation, rapid changes in bathing solutions, control of temperature, and simulation of ischemia and reperfusion with measurements of the effects of such interventions on both populations of cells (low power survey) and individual myocytes (high power). Myocyte responses to these various interventions are described. The primary advantage of this system is the ability to conduct experiments on cardiac myocytes isolated concurrently from multiple experimental groups at the same time and under identical conditions.     

Overexpression of Na+/Ca2+ exchanger alters contractility and SR Ca2+ content in adult rat myocytes

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.     

Myocardial interstitial Cajal-like cells (ICLC) and their nanostructural relationships with intercalated discs: shed vesicles as intermediates

Intercalated discs (ID) are complex junctional units that connect cardiac myocytes mechanically and electrochemically. However, there is limited information concerning the cardiomyocyte interaction with interstitial non-muscle cells. Our previous studies showed that myocardial interstitial Cajal-like cells (ICLC) are located in between cardiomyocytes, blood capillaries and nerve fibres. Typically, ICLC have several very long, moniliform, cytoplasmic processes which establish closed contacts with nerve fibres, as well as each other. We report here ultrastructural evidence concerning the relationships of ICLC processes with ID. The ICLC cytoplasmic prolongations (tens micrometers length) preferentially pass by or stop nearby the ID. Transmission electron microscopy emphasized three distinct connecting features between the tips of ICLC extensions and myocytes at the 'mouth' of ID: free or budding shed vesicles, exocytotic multi-vesicular bodies and direct contacts. In the last case, electron-dense repetitive nanostructures ('pillars') (35-40 nm high and 100-150 nm wide, similar to adhesion molecules) fasten the ICLC to the myocytes. All these features suggest a juxtacrine and/or paracrine intercellular mutual modulation of ICLC and cardiomyocytes in the microenvironment of ID, possibly monitoring the cardiac functions, particularly the electrical activity.     

Comparison of Na(+)/Ca(2+) exchanger current and of its response to isoproterenol between acutely isolated and short-term cultured adult ventricular myocytes

The Na(+)/Ca(2+) exchanger protein is present in the cell membrane of many tissue types and plays key roles in Ca(2+) homeostasis, excitation-contraction coupling, and generation of electrical activity in the heart. The use of adult ventricular myocyte cell culture is important to molecular biological approaches to study the roles and modulation of the cardiac Na(+)/Ca(2+) exchanger. Therefore, we characterised the functional expression of the exchanger in adult guinea-pig ventricular myocytes maintained in short-term culture (for 4 days) and compared the response of ionic current (I(NaCa)) carried by the exchanger from acutely isolated and Day 4 cells to beta-adrenoceptor activation with isoproterenol (ISO). Functional activity of the exchanger was assessed by measuring I(NaCa) using whole cell patch clamp, under selective recording conditions. I(NaCa) amplitude measured at both +60 and -100mV declined significantly by Day 1 of cell culture, showing a further small decline by Day 4. However, cell surface area (assessed by measuring membrane capacitance) also declined over this time-frame. I(NaCa) normalised to membrane capacitance (I(NaCa) density) did not differ significantly between acutely isolated and cells cultured for 4 days. However, although ISO (1 microM) increased I(NaCa) in acutely isolated myocytes, it exerted no significant effect on I(NaCa) from Day 4 cells. This was not due to an inherent inability of these cells to respond to ISO, as L-type calcium current amplitude from Day 4 cells was increased by ISO to a similar extent as that from acutely isolated cells. Our data suggest that the functional expression of the Na/Ca exchanger is well maintained during short-term culture of adult ventricular myocytes. The lack of response to ISO of I(NaCa) from Day 4 cells suggests: (a) that, despite a well-maintained I(NaCa) density, cultured adult myocytes may not necessarily be suitable for studies of exchanger modulation by some agonists and (b) that there may exist subtle differences between beta-adrenergic regulation of the exchanger protein and of L-type Ca channels.     

A primary culture system for sustained expression of a calcium sensor in preserved adult rat ventricular myocytes

For studying heart pathologies on the cellular level, cultured adult cardiac myocytes represent an important approach. We aimed to explore a novel adult rat ventricular myocyte culture system with minimised dedifferentiation allowing extended experimental manipulation of the cells such as expression of exogenous proteins. Various culture conditions were investigated including medium supplement, substrate coating and electrical pacing for one week. Adult myocytes were probed for (i) viability, (ii) morphology, (iii) frequency dependence of contractions, (iv) Ca(2+) transients, and (v) their tolerance towards adenovirus-mediated expression of the Ca(2+) sensor "inverse pericam". Conventionally, in either serum supplemented or serum-free medium, myocytes dedifferentiated into flat cells within 3 days or cell physiology and morphology were impaired, respectively. In contrast, myocytes cultured in medium supplemented with an insulin-transferrin-selenite mixture on substrates coated with extracellular matrix proteins showed an increased cell attachment and a conserved cross-striation. Moreover, these myocytes displayed optimised preservation of their contractile behaviour and Ca(2+) signalling even under conditions of continuous electrical pacing. Sustained expression of inverse pericam did not alter myocyte function and allowed long lasting high speed Ca(2+) imaging of electrically driven adult myocytes. Our single-cell model thus provides a new advance for high-content screening of these highly specialised cells.     

The role of cell death and myofibrillar damage in contractile dysfunction of long-term cultured adult cardiomyocytes exposed to doxorubicin

In failing hearts cardiomyocytes undergo alterations in cytoskeleton structure, contractility and viability. It is not known presently, how stress-induced changes of myofibrils correlate with markers for cell death and contractile function in cardiomyocytes. Therefore, we have studied the progression of contractile dysfunction, myofibrillar damage and cell death in cultured adult cardiomyocytes exposed to the cancer therapy doxorubicin. We demonstrate, that long-term cultured adult cardiomyocytes, a well-established model for the study of myofibrillar structure and effects of growth factors, can also be used to assess contractility and calcium handling. Adult rat ventricular myocytes (ARVM) were isolated and cultured for a total of 14 days in serum containing medium. The organization of calcium-handling proteins and myofibrillar structure in freshly isolated and in long-term cultured adult cardiomyocytes was studied by immunofluorescence and electron microscopy. Excitation contraction-coupling was analyzed by fura 2 and video edge detection in electrically paced cardiomyocytes forming a monolayer, and cell death and viability was measured by TUNEL assay, LDH release, MTT assay, and Western blot for LC3. Adult cardiomyocytes treated with Doxo showed apoptosis and necrosis only at supraclinical concentrations. Treated cells displayed merely alterations in cytoskeleton organization and integrity concomitant with contractile dysfunction and up-regulation of autophagosome formation, but no change in total sarcomeric protein content. We propose, that myofibrillar damage contributes to contractile dysfunction prior to cell death in adult cardiomyocytes exposed to clinically relevant concentrations of anthracyclines.     

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.     

Hepatocyte growth factor protects cardiac myocytes against oxidative stress-induced apoptosis

Hepatocyte growth factor (HGF) has been proposed as an endogenous cardioprotective agent against oxidative stress. The mechanism of HGF action in the heart, however, has not yet been elucidated. The present study demonstrates that HGF protects adult cardiac myocytes against oxidative stress-induced apoptosis. HGF, at the concentrations which can be detected in the plasma of humans subsequent to myocardial infarction, effectively attenuated death of isolated adult rat cardiac myocytes and cultured HL-1 cardiac muscle cells induced by apoptosis-inducing oxidative stress stimuli such as daunorubicin, serum deprivation, and hydrogen peroxide. We identified expression of c-Met HGF receptor in adult cardiac myocytes, which can be rapidly tyrosine phosphorylated in response to HGF treatment. HGF also activated MEK, p44/42 MAPK, and p90RSK. To determine if MEK-MAPK pathway may be involved in the mechanism of HGF-mediated cardiac myocyte protection, effects of a specific MEK inhibitor, PD98059, were studied. Pretreatment of cells with PD98059 partially blocked HGF signaling for protection against hydrogen peroxide-induced cell death. Thus, HGF protects cardiac myocytes against oxidative stress, in part, via activating MEK-MAPK pathway.     

Physical,contractile and calcium handling properties of neonatal cardiac myocytes cultured on different matrices

Extracellular matrix components play a vital role in the determination of heart cell growth, development of spontaneous contractile activity and morphologic differentiation. In this work we studied the physical and contractile changes in neonatal rat cardiac myocytes over the first four days of growth on three different extracellular matrices. We compared commercial laminin and fibronectin, plus a fibroblast-derived extracellular matrix, which we have termed cardiogel. Myocytes cultured on cardiogel were characterized by greater cellular area and volume when compared to cells cultured on the other single-component matrices. Spontaneous contractile activity appeared first in the cells grown on cardiogel, sometimes as early as the first day post-plating, in contrast to day three in the cells cultured on laminin. Measurements of cardiac myocyte contractility i.e. percent shortening and time to peak contraction, were made on each of the first four days in each culture. Myocytes cultured on cardiogel developed maximum shortening more rapidly than the other cultures, and an earlier response to electrical pacing. Histochemical staining for myocyte mitochondrial content, revealed that the cardiogelsupported cells exhibited the earliest development of this organelle and, after four days, the greatest abundance. This reflects both a greater cell size, as well as response to increasing energy demands.

Due to the increase in volume and contractile activity exhibited by the cardiogel grown myocytes, we employed calcium binding and uptake experiments to determine the comparative cellular capacities for calcium and as an indicator of sarcoplasmic reticulum development. Also whole cell phosphorylation in the presence of low detergent was assayed, to correlate calcium uptake with phosphorylation, in an attempt to examine possible increases in calcium pump number and other phosphorylatable proteins. In agreement with our physical and contractile data, we found that the cells grown on cardiogel showed a greater calcium uptake over the first four days of culture, and increased phosphorylation. However, calcium binding was not dramatically different comparing the three culture matrices.

Based on our data, the fibroblast-derived cardiogel is the matrix of choice supporting earliest maturation of neonatal cardiomyocytes, in terms of spontaneous contractions, calcium handling efficiency, cell size and development of a subcellar organelle, the mitochondrion.     

Culture of atrial and ventricular cardiac muscle cells from the adult squirrel monkey Saimiri sciureus

Atrial and ventricular cardiac muscle cells isolated from the adult squirrel monkey Saimiri sciureus were cultured and characterized by light and transmission electron microscopy (TEM). Freshly isolated cells were striated and cylindrical in shape and, when placed in culture, rounded up and lost their highly organized morphology. With prolonged time in culture, they spread out on the surface of the culture flask and reacquired many of the internal ultrastructural characteristics of their in vivo atrial and ventricular cardiac muscle cell counterparts. Autoradiographic experiments indicated that both atrial and ventricular myocytes synthesized DNA when grown in culture. In some binucleated atrial cells only one nucleus became labelled. These studies show that it is now possible to culture cardiac muscle cells isolated from an adult primate.     

Growth and synapse formation among major classes of adult salamander retinal neurons in vitro

Adult neurons, isolated from the salamander retina, were maintained in low-density cell culture and examined for synapse formation by electrophysiological and electron microscopic techniques. Morphologically identifiable rod, cone, horizontal, bipolar, and amacrine/ganglion cells survived for many months, grew processes, and formed numerous cell contacts. Intracellular recordings showed the presence of a variety of voltage- and time-dependent conductances and both electrical and chemical transmission among these cells. At the ultrastructural level, gap junctions, monad ribbon synapses, and conventional synapses, like those present in the intact retina, were observed in sibling cultures. Thus, all major classes of adult retinal neurons, in addition to ganglion cells, are able to regenerate processes and reform synapses. The regenerated synaptic contacts are functional and structurally diverse.     

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.     

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.