Direct measurements of luminal Ca 2+ with endo-lysosomal GFP-aequorin reveal functional IP 3 receptors.

Endo-lysosomes are considered acidic Ca 2+ stores but direct measurements of luminal Ca 2+ within them are limited. Here we report that the Ca 2+ -sensitive luminescent protein aequorin does not reconstitute with its cofactor at highly acidic pH but that a significant fraction of the probe is functional within a mildly acidic compartment when targeted to the endo-lysosomal system. We leveraged this probe (ELGA) to report Ca 2+ dynamics in this compartment. We show that Ca 2+ uptake is ATP-dependent and sensitive to blockers of endoplasmic reticulum Ca 2+ pumps. We find that the Ca 2+ mobilizing messenger IP 3 which typically targets the endoplasmic reticulum evokes robust luminal responses in wild type cells, but not in IP 3 receptor knock-out cells. Responses were comparable to those evoked by activation of the endo-lysosomal ion channel TRPML1. Stimulation with IP 3 -forming agonists also mobilized the store in intact cells. Super-resolution microscopy analysis confirmed the presence of IP 3 receptors within the endo-lysosomal system, both in live and fixed cells. Our data reveal a physiologically-relevant, IP 3 -sensitive store of Ca 2+ within the endo-lysosomal system.

A method for determining the dependence of calcium oscillations on inositol trisphosphate oscillations.

In some cell types, oscillations in the concentration of free intracellular calcium ([Ca2+]) are accompanied by oscillations in the concentration of inositol 1,4,5-trisphosphate ([IP3]). However, in most cell types it is still an open question as to whether oscillations in [IP3] are necessary for Ca2+ oscillations in vivo, or whether they merely follow passively. Using a wide range of models, we show that the response to an artificially applied pulse of IP3 can be used to distinguish between these two cases. Hence, we show that muscarinic receptor-mediated, long-period Ca2+ oscillations in pancreatic acinar cells depend on [IP3] oscillations, whereas short-period Ca2+ oscillations in airway smooth muscle do not.

Calcium oscillations in a triplet of pancreatic acinar cells.

We use a mathematical model of calcium dynamics in pancreatic acinar cells to investigate calcium oscillations in a ring of three coupled cells. A connected group of cells is modeled in two different ways: 1), as coupled point oscillators, each oscillator being described by a spatially homogeneous model; and 2), as spatially distributed cells coupled along their common boundaries by gap-junctional diffusion of inositol trisphosphate and/or calcium. We show that, although the point-oscillator model gives a reasonably accurate general picture, the behavior of the spatially distributed cells cannot always be predicted from the simpler analysis; spatially distributed diffusion and cell geometry both play important roles in determining behavior. In particular, oscillations in which two cells are in synchrony, with the third phase-locked but not synchronous, appears to be more dominant in the spatially distributed model than in the point-oscillator model. In both types of model, intercellular coupling leads to a variety of synchronous, phase-locked, or asynchronous behaviors. For some parameter values there are multiple, simultaneous stable types of oscillation. We predict 1), that intercellular calcium diffusion is necessary and sufficient to coordinate the responses in neighboring cells; 2), that the function of intercellular inositol trisphosphate diffusion is to smooth out any concentration differences between the cells, thus making it easier for the diffusion of calcium to synchronize the oscillations; 3), that groups of coupled cells will tend to respond in a clumped manner, with groups of synchronized cells, rather than with regular phase-locked periodic intercellular waves; and 4), that enzyme secretion is maximized by the presence of a pacemaker cell in each cluster which drives the other cells at a frequency greater than their intrinsic frequency.

Control of calcium oscillations by membrane fluxes.

It is known that Ca(2+) influx plays an important role in the modulation of inositol trisphosphate-generated Ca(2+) oscillations, but controversy over the mechanisms underlying these effects exists. In addition, the effects of blocking membrane transport or reducing Ca(2+) entry vary from one cell type to another; in some cell types oscillations persist in the absence of Ca(2+) entry (although their frequency is affected), whereas in other cell types oscillations depend on Ca(2+) entry. We present theoretical and experimental evidence that membrane transport can control oscillations by controlling the total amount of Ca(2+) in the cell (the Ca(2+) load). Our model predicts that the cell can be balanced at a point where small changes in the Ca(2+) load can move the cell into or out of oscillatory regions, resulting in the appearance or disappearance of oscillations. Our theoretical predictions are verified by experimental results from HEK293 cells. We predict that the role of Ca(2+) influx during an oscillation is to replenish the Ca(2+) load of the cell. Despite this prediction, even during the peak of an oscillation the cell or the endoplasmic reticulum may not be measurably depleted of Ca(2+).

Modulation of Ca2+ oscillations by phosphorylation of Ins(1,4,5)P3 receptors.

Activation of InsP(3)Rs (InsP(3) receptors) represents the major mechanism underlying intracellular calcium release in non-excitable cells such as hepatocytes and exocrine cells from the pancreas and salivary glands. Modulation of calcium release through InsP(3)Rs is therefore a major route whereby the temporal and spatial characteristics of calcium waves and oscillations can potentially be 'shaped'. In this study, the functional consequences of phosphoregulation of InsP(3)Rs were investigated. Pancreatic and parotid acinar cells express all three types of InsP(3)R in differing abundance, and all are potential substrates for phosphoregulation. PKA (protein kinase A)-mediated phosphorylation of InsP(3)Rs in pancreatic acinar cells resulted in slowed kinetics of calcium release following photo-release of InsP(3). In contrast, activation of PKA in parotid cells resulted in a marked potentiation of calcium release. In pancreatic acinar cells the predominant InsP(3)R isoform phosphorylated was the type 3 receptor, while the type 2 receptor was markedly phosphorylated in parotid acinar cells. In order to further decipher the effects of phosphorylation on individual InsP(3)R subtypes, DT-40 cell lines expressing homotetramers of a single isoform of InsP(3)R were utilized. These data demonstrate that phosphoregulation of InsP(3)Rs results in subtype-specific effects and may play a role in the specificity of calcium signals by 'shaping' the spatio-temporal profile of the response.

A model of calcium waves in pancreatic and parotid acinar cells.

We construct a mathematical model of Ca(2+) wave propagation in pancreatic and parotid acinar cells. Ca(2+) release is via inositol trisphosphate receptors and ryanodine receptors that are distributed heterogeneously through the cell. The apical and basal regions are separated by a region containing the mitochondria. In response to a whole-cell, homogeneous application of inositol trisphosphate (IP(3)), the model predicts that 1), at lower concentrations of IP(3), the intracellular waves in pancreatic cells begin in the apical region and are actively propagated across the basal region by Ca(2+) release through ryanodine receptors; 2), at higher [IP(3)], the waves in pancreatic and parotid cells are not true waves but rather apparent waves, formed as the result of sequential activation of inositol trisphosphate receptors in the apical and basal regions; 3), the differences in wave propagation in pancreatic and parotid cells can be explained in part by differences in inositol trisphosphate receptor density; 4), in pancreatic cells, increased Ca(2+) uptake by the mitochondria is capable of restricting Ca(2+) responses to the apical region, but that this happens only for a relatively narrow range of [IP(3)]; and 5), at higher [IP(3)], the apical and basal regions of the cell act as coupled Ca(2+) oscillators, with the basal region partially entrained to the apical region.

Identification of a ryanodine receptor in rat heart mitochondria.

Recent studies have shown that, in a wide variety of cells, mitochondria respond dynamically to physiological changes in cytosolic Ca(2+) concentrations ([Ca(2+)](c)). Mitochondrial Ca(2+) uptake occurs via a ruthenium red-sensitive calcium uniporter and a rapid mode of Ca(2+) uptake. Surprisingly, the molecular identity of these Ca(2+) transport proteins is still unknown. Using electron microscopy and Western blotting, we identified a ryanodine receptor in the inner mitochondrial membrane with a molecular mass of approximately 600 kDa in mitochondria isolated from the rat heart. [(3)H]Ryanodine binds to this mitochondrial ryanodine receptor with high affinity. This binding is modulated by Ca(2+) but not caffeine and is inhibited by Mg(2+) and ruthenium red in the assay medium. In the presence of ryanodine, Ca(2+) uptake into isolated heart mitochondria is suppressed. In addition, ryanodine inhibited mitochondrial swelling induced by Ca(2+) overload. This swelling effect was not observed when Ca(2+) was applied to the cytosolic fraction containing sarcoplasmic reticulum. These results are the first to identify a mitochondrial Ca(2+) transport protein that has characteristics similar to the ryanodine receptor. This mitochondrial ryanodine receptor is likely to play an essential role in the dynamic uptake of Ca(2+) into mitochondria during Ca(2+) oscillations.

Calcium wave propagation in pancreatic acinar cells: functional interaction of inositol 1,4,5-trisphosphate receptors, ryanodine receptors, and mitochondria.

In pancreatic acinar cells, inositol 1,4,5-trisphosphate (InsP(3))-dependent cytosolic calcium ([Ca(2+)](i)) increases resulting from agonist stimulation are initiated in an apical "trigger zone," where the vast majority of InsP(3) receptors (InsP(3)R) are localized. At threshold stimulation, [Ca(2+)](i) signals are confined to this region, whereas at concentrations of agonists that optimally evoke secretion, a global Ca(2+) wave results. Simple diffusion of Ca(2+) from the trigger zone is unlikely to account for a global [Ca(2+)](i) elevation. Furthermore, mitochondrial import has been reported to limit Ca(2+) diffusion from the trigger zone. As such, there is no consensus as to how local [Ca(2+)](i) signals become global responses. This study therefore investigated the mechanism responsible for these events. Agonist-evoked [Ca(2+)](i) oscillations were converted to sustained [Ca(2+)](i) increases after inhibition of mitochondrial Ca(2+) import. These [Ca(2+)](i) increases were dependent on Ca(2+) release from the endoplasmic reticulum and were blocked by 100 microM ryanodine. Similarly, "uncaging" of physiological [Ca(2+)](i) levels in whole-cell patch-clamped cells resulted in rapid activation of a Ca(2+)-activated current, the recovery of which was prolonged by inhibition of mitochondrial import. This effect was also abolished by ryanodine receptor (RyR) blockade. Photolysis of d-myo InsP(3) P(4(5))-1-(2-nitrophenyl)-ethyl ester (caged InsP(3)) produced either apically localized or global [Ca(2+)](i) increases in a dose-dependent manner, as visualized by digital imaging. Mitochondrial inhibition permitted apically localized increases to propagate throughout the cell as a wave, but this propagation was inhibited by ryanodine and was not seen for minimal control responses resembling [Ca(2+)](i) puffs. Global [Ca(2+)](i) rises initiated by InsP(3) were also reduced by ryanodine, limiting the increase to a region slightly larger than the trigger zone. These data suggest that, while Ca(2+) release is initially triggered through InsP(3)R, release by RyRs is the dominant mechanism for propagating global waves. In addition, mitochondrial Ca(2+) import controls the spread of Ca(2+) throughout acinar cells by modulating RyR activation.

Targeted phosphorylation of inositol 1,4,5-trisphosphate receptors selectively inhibits localized Ca2+ release and shapes oscillatory Ca2+ signals.

The current study provides biochemical and functional evidence that the targeting of protein kinase A (PKA) to sites of localized Ca(2+) release confers rapid, specific phosphoregulation of Ca(2+) signaling in pancreatic acinar cells. Regulatory control of Ca(2+) release by PKA-dependent phosphorylation of inositol 1,4, 5-trisphosphate (InsP(3)) receptors was investigated by monitoring Ca(2+) dynamics in pancreatic acinar cells evoked by the flash photolysis of caged InsP(3) prior to and following PKA activation. Ca(2+) dynamics were imaged with high temporal resolution by digital imaging and electrophysiological methods. The whole cell patch clamp technique was used to introduce caged compounds and to record the activity of a Ca(2+)-activated Cl(-) current. Photolysis of low concentrations of caged InsP(3) evoked Cl(-) currents that were inhibited by treatment with dibutryl-cAMP or forskolin. In contrast, PKA activators had no significant inhibitory effect on the activation of Cl(-) current evoked by uncaging Ca(2+) or by the photolytic release of higher concentrations of InsP(3). Treatment with Rp-adenosine-3',5'-cyclic monophoshorothioate, a selective inhibitor of PKA, or with Ht31, a peptide known to disrupt the targeting of PKA, largely abolished forskolin-induced inhibition of Ca(2+) release. Further evidence for the targeting of PKA to the sites of Ca(2+) mobilization was revealed using immunocytochemical methods demonstrating that the R(IIbeta) subunit of PKA was localized to the apical regions of acinar cells and co-immunoprecipitated with the type III but not the type I or type II InsP(3) receptors. Finally, we demonstrate that the pattern of signaling evoked by acetylcholine can be converted to one that is more "CCK-like" by raising cAMP levels. Our data provide a simple mechanism by which distinct oscillatory Ca(2+) patterns can be shaped.

Agonist-dependent phosphorylation of the inositol 1,4,5-trisphosphate receptor: A possible mechanism for agonist-specific calcium oscillations in pancreatic acinar cells.

The properties of inositol 1,4,5-trisphosphate (IP3)-dependent intracellular calcium oscillations in pancreatic acinar cells depend crucially on the agonist used to stimulate them. Acetylcholine or carbachol (CCh) cause high-frequency (10-12-s period) calcium oscillations that are superimposed on a raised baseline, while cholecystokinin (CCK) causes long-period (>100-s period) baseline spiking. We show that physiological concentrations of CCK induce rapid phosphorylation of the IP3 receptor, which is not true of physiological concentrations of CCh. Based on this and other experimental data, we construct a mathematical model of agonist-specific intracellular calcium oscillations in pancreatic acinar cells. Model simulations agree with previous experimental work on the rates of activation and inactivation of the IP3 receptor by calcium (DuFour, J.-F., I.M. Arias, and T.J. Turner. 1997. J. Biol. Chem. 272:2675-2681), and reproduce both short-period, raised baseline oscillations, and long-period baseline spiking. The steady state open probability curve of the model IP3 receptor is an increasing function of calcium concentration, as found for type-III IP3 receptors by Hagar et al. (Hagar, R.E., A.D. Burgstahler, M.H. Nathanson, and B.E. Ehrlich. 1998. Nature. 396:81-84). We use the model to predict the effect of the removal of external calcium, and this prediction is confirmed experimentally. We also predict that, for type-III IP3 receptors, the steady state open probability curve will shift to lower calcium concentrations as the background IP3 concentration increases. We conclude that the differences between CCh- and CCK-induced calcium oscillations in pancreatic acinar cells can be explained by two principal mechanisms: (a) CCK causes more phosphorylation of the IP3 receptor than does CCh, and the phosphorylated receptor cannot pass calcium current; and (b) the rate of calcium ATPase pumping and the rate of calcium influx from the outside the cell are greater in the presence of CCh than in the presence of CCK.

Secretagogues cause ubiquitination and down-regulation of inositol 1, 4,5-trisphosphate receptors in rat pancreatic acinar cells.

BACKGROUND & AIMS: The action of several exocrine pancreas secretagogues depends on the second messenger inositol 1,4, 5-trisphosphate (IP3), which, via endoplasmic reticulum-located IP3 receptors, mobilizes intracellular Ca2+ stores. Signaling pathways like this one are regulated at multiple loci. To determine whether IP3 receptors are one of these loci, we measured IP3 receptor concentration, distribution, and modification in secretagogue-stimulated rat pancreatic acinar cells. METHODS: Isolated rat pancreatic acinar cells were exposed to cholecystokinin and other secretagogues, or rats were injected intraperitoneally with cerulein. Then samples of cells or pancreata were probed for IP3 receptor content and distribution as well as for ubiquitin association with IP3 receptors. RESULTS: Secretagogues rapidly down-regulated acinar cell IP3 receptors both in vitro and in vivo. They also elicited receptor redistribution and caused receptors to become ubiquitinated, indicating that the ubiquitin/proteasome proteolytic pathway contributes to the down-regulation. Surprisingly, however, proteasome inhibitors did not block IP3 receptor down-regulation, and phospholipase Cbeta1 and protein kinase C also were down-regulated. Thus, secretagogues simultaneously activate an additional proteolytic pathway. CONCLUSIONS: Secretagogues rapidly down-regulate IP3 receptors and other proteins involved in intracellular signaling by a mechanism that involves, but is not limited to, the ubiquitin/proteasome pathway. Loss of these proteins may account for the disruption of Ca2+ mobilization that occurs in models of acute pancreatitis, and may contribute to cell adaptation under physiological conditions.

Calcium signaling in rat pancreatic acinar cells: a role for Galphaq, Galpha11, and Galpha14.

Stimulus-secretion coupling in the pancreatic acinar cell is initiated by the secretagogues CCK and ACh and results in the secretion by exocytosis of the contents of zymogen granules. A key event in this pathway is the G protein-activated production of second messengers and the subsequent elevation of cytosolic-free Ca2+. The aim of this study was therefore to define the heterotrimeric G protein alpha-subunits present and participating in this pathway in rat pancreatic acinar cells. RT-PCR products were amplified from pancreatic acinar cell mRNA with primers specific for Galphaq, Galpha11, and Galpha14 but were not amplified with primers specific for Galpha15. The sequences of these PCR products confirmed them to be portions of the rat homologues of Galphaq, Galpha11, and Galpha14. The pancreatic-derived cell line AR42J similarly expressed Galphaq, Galpha11, and Galpha14; however, the Chinese hamster ovary (CHO) cell line only expressed Galpha11 and Galphaq. These data indicate that caution should be exercised when comparing signal transduction pathways between different cell types. The expression of these proteins in acinar cells was confirmed by immunoblotting samples of acinar membrane protein using specific antisera to the individual G protein alpha-subunits. The role of these proteins in Ca2+ signaling events was investigated by microinjecting a neutralizing antibody directed against a homologous sequence in Galphaq, Galpha11, and Galpha14 into acinar cells and CHO cells. Ca2+ signaling was inhibited in acinar cells and receptor-bearing CHO cells in response to both physiological and supermaximal concentrations of agonists. The inhibition was >75% in both cell types. These data indicate a role for Galphaq and/or Galpha11 in intracellular Ca2+ concentration signaling in CHO cells, and in addition to Galphaq and Galpha11, Galpha14 may also fulfill this role in rat pancreatic acinar cells.

Optical measurement of stimulus-evoked membrane dynamics in single pancreatic acinar cells.

Stimulation of pancreatic acinar cells induces the release of digestive enzymes via the exocytotic fusion of zymogen granules and activates postfusion granule membrane retrieval and receptor cycling. In the present study, changes in membrane surface area of rat single pancreatic acinar cells were monitored by cell membrane capacitance (Cm) measurements and by the membrane fluorescent dye FM1-43. When measured with the Cm method, agonist treatment evoked a graded, transient increase in acinar cell surface area averaging 3. 5%. In contrast, a 13% increase in surface area was estimated using FM1-43, corresponding to the fusion of 48 zymogen granules at a rate of 0.5 s-1. After removal of FM1-43 from the surface-accessible membrane, a residual fluorescence signal was shown by confocal microscopy to be localized in endosome-like structures and confined to the apical regions of acinar cells. The development of an optical method for monitoring the membrane turnover of single acinar cells, in combination with measurements of Cm changes, reveals coincidence of exocytotic and endocytotic activity in acinar cells after hormonal stimulation.

Heterotrimeric G-protein Gq/11 localized on pancreatic zymogen granules is involved in calcium-regulated amylase secretion.

The heterotrimeric G-protein Gq/11 was identified on pancreatic acinar zymogen granules and its function in calcium-regulated exocytosis was examined. Western blotting showed alphaq/11, but not alphas or alphao, to be localized to the zymogen granule membrane along with G-protein beta-subunit; all three alpha subunits were present in a plasma membrane fraction and the alphaq/11 signal was 30-fold more enriched in the plasma membrane as compared with granule membrane. Neither CCK receptors nor alpha subunits of the sodium pump, both plasma membrane markers were present on granule membranes. Immunohistochemistry of pancreatic lobules showed that alphaq/11 localized to the zymogen granule-rich apical region of acinar cells together with a much stronger signal at the basolateral plasma membrane. When the substance-P-related peptide GPAnt-2a, an antagonist of Gq/11, was introduced into streptolysin-O permeabilized acini to bypass the plasma membrane, the amylase release induced by 10 microM free calcium was potentiated in a concentration-dependent manner. By contrast, another substance-P-related peptide, GPAnt-1, an antagonist of Go and Gi, showed no effect on calcium-induced amylase release from permeabilized acini. GPAnt-2a peptide also exerted an inhibitory effect on the total GTPase activity of the purified zymogen granules and a larger inhibitory effect on the GTPase activity of the Gq/11 protein immunopurified from zymogen granules. GPAnt-1, however, did not inhibit GTPase activity of either zymogen granules or immunopurified Gq/11. These results suggest that GPAnt-2a peptide augmented calcium-induced amylase release from permeabilized acini by inhibiting GTPase activity of the Gq/11 protein on zymogen granules. We conclude that Gq/11 protein on zymogen granules plays a tonic inhibitory role in calcium-regulated amylase secretion from pancreatic acini.

Endothelin-activated calcium signaling in enteric glia derived from neonatal guinea pig.

The ability of guinea pig enteric glia to respond to endothelins was examined using fura 2-based digital microscopy in glial cells derived from guinea pig taenia coli. Each isoform of endothelin (ET-1, ET-2, ET-3) evoked dose-dependent and equipotent increases in intracellular Ca2+ concentration ([Ca2+]i) and in percentage of cells responding, 4alaEt-1, an ETB receptor agonist, elicited similar [Ca2+]i increments. BQ-788, an ETB antagonist, inhibited [Ca2+]i responses to endothelin. Preincubation of glia with U-73122 a phospholipase C inhibitor, abolished the [Ca2+]i response to ET-3 exposure. Thapsigargin also eliminated ET-3-evoked Ca2+ signaling. The inositol 1,4,5-trisphosphate (IP3) receptor antagonist heparin, introduced into glial cells by radio frequency electroporation, blocked [Ca2+]i responses to ET-3 (100 nM) in 63% of glia. Sustained elevation in [Ca2+]i was abolished by removal of Ca2+ from the buffer and inhibited 85. -3% by Ni2+ (1 mM). Preincubation of glia with 100 nM phorbol 12-myristate 13-acetate (24 h) also inhibited sustained increments in [Ca2+]i by 87%. The presence of IP3 receptors in enteric glia was confirmed by immunofluorescent confocal microscopy.

Evidence that zymogen granules are not a physiologically relevant calcium pool. Defining the distribution of inositol 1,4,5-trisphosphate receptors in pancreatic acinar cells.

A key event leading to exocytosis of pancreatic acinar cell zymogen granules is the inositol 1,4,5-trisphosphate (InsP3)-mediated release of Ca2+ from intracellular stores. Studies using digital imaging microscopy and laser-scanning confocal microscopy have indicated that the initial release of Ca2+ is localized to the apical region of the acinar cell, an area of the cell dominated by secretory granules. Moreover, a recent study has shown that InsP3 is capable of releasing Ca2+ from a preparation enriched in secretory granules (Gerasimenko, O., Gerasimenko, J., Belan, P., and Petersen, O. H., (1996) Cell 84, 473-480). In the present study, we have investigated the possibility that zymogen granules express InsP3 receptors and are thus Ca2+ release sites. Immunofluorescence staining, obtained with antisera specific to types I, II, or III InsP3 receptors and analyzed by confocal fluorescence microscopy revealed that all InsP3 receptor types were present in acinar cells. The type II receptor localized exclusively to an area close to or at the luminal plasma membrane. While types I and III InsP3 receptors displayed a similar luminal distribution, these receptors were also present at low levels in nuclei. The localization of InsP3 receptor was in marked contrast to the distribution of amylase, a zymogen granule content protein. In a zymogen granule fraction prepared in an identical manner to the aforementioned report demonstrating InsP3-induced Ca2+ release, immunoblotting demonstrated the presence of types I, II, and III InsP3 receptors. Ca2+ release from this preparation in response to InsP3, but not thapsigargin, could also be demonstrated. In contrast, when the zymogen granules were further purified on a Percoll gradient, InsP3 receptors were undetectable, and InsP3 failed to release Ca2+. Transmission electron microscopy performed on both preparations showed that the Percoll-purified granule preparation consisted of essentially pure zymogen granules, whereas the granules prepared without this step were enriched in granules but also contained significant contamination by mitochondria, endoplasmic reticulum, and nuclei. It is concluded that zymogen granules do not express InsP3 receptors and thus are not a site of Ca2+ release relevant to the secretory process in the pancreatic acinar cell.

Ultrastructure and cell-cell coupling of cardiac myocytes differentiating in embryonic stem cell cultures.

Differentiation cultures of embryonic stem (ES) cells can be a useful in vitro system for understanding cardiac myocyte development. However, cell morphometry, sarcomere development, and functional cell-cell junction formation have not been examined in detail to determine whether ES cell-derived cardiac myocytes exhibit structural and functional characteristics similar to cardiac myocytes within the developing heart. Therefore, we examined cellular dimensions, sarcomere formation, and cell-cell contacts in differentiating cardiac myocytes derived from mouse D3-ES cell cultures. Cells exhibited rod-shaped morphology and had single centrally located nuclei, typical of maturing cardiac myocytes. The cellular dimensions of 59 individual cardiac myocytes within contracting foci of ES cell cultures were analyzed (length = 42.2 +/- 2.1 microns, area = 197 +/- 19 microns2, and diameter = 5.5 +/- 0.3 microns) and found to be similar to myocytes in vivo. Transmission electron micrographs of ES cell-derived cardiac myocytes indicated myofibrillar architecture ranged from sparse and disorganized to densely packed, parallel arrays of myofibrils organized into mature sarcomeres. This pattern of myofibrillar assembly in maturing sarcomeres was similar to that observed during in vivo myocyte differentiation. Another hallmark of cardiac development is the formation of intercalated discs, which functionally couple adjacent cardiac myocytes. Electron micrographs indicated nascent intercalated discs were forming in foci of ES cell-derived cardiac myocytes. In addition, indirect immunostaining with anti-connexin 43 antibody (Ab), a monoclonal Ab to the gap junction component of the intercalated disc, indicated that gap junctions were present in contracting ES cell foci. Furthermore, microinjection of single cardiac myocytes with Lucifer yellow (2.5 microM) resulted in the spread of fluorescence to adjacent cells within a contracting focus, an indication of functional cell-cell coupling across these gap junctions. Together, these results indicate ES cell-derived cardiac myocytes exhibit cell morphology, sarcomere formation, and cell-cell junctions similar to those observed in cardiac myocytes developing in vivo.