Cardiac Ventricular Muscle Research Articles

Modulation of ATP-sensitive K+ channels in skeletal muscle by intracellular protons

Since their discovery in cardiac muscle, ATP-sensitive K+(KATP) channels have been identified in pancreatic beta-cells, skeletal muscle, smooth muscle and central neurons. The activity of KATP channels is inhibited by the presence of cytosolic ATP. Their wide distribution indicates that they could have important physiological roles that may vary between tissues. In muscle cells the role of K+ channels is to control membrane excitability and the duration of the action potential. In anoxic cardiac ventricular muscle KATP channels are believed to be responsible for shortening the action potential, and it has been proposed that a fall in ATP concentration during metabolic exhaustion increases the activity of KATP channels in skeletal muscle, which may reduce excitability. But the intracellular concentration of ATP in muscle is buffered by creatine phosphate to 5-10 mM, and changes little, even during sustained activity. This concentration is much higher than the intracellular ATP concentration required to half block the KATP-channel current in either cardiac muscle (0.1 mM) or skeletal muscle (0.14 mM), indicating that the open-state probability of KATP channels is normally very low in intact muscle. So it is likely that some additional means of regulating the activity of KATP channels exists, such as the binding of nucleotides other than ATP. Here I present evidence that a decrease in intracellular pH (pHi) markedly reduces the inhibitory effect of ATP on these channels in excised patches from frog skeletal muscle. Because sustained muscular activity can decrease pHi by almost 1 unit in the range at which KATP channels are most sensitive to pHi, it is likely that the activity of these channels in skeletal muscle is regulated by intracellular protons under physiological conditions.

Differential response of cultured adult cardiac muscle cells to a tumor promotor: Analysis of myofibrillar organization

Cultured adult rat ventricular cardiac muscle cells were exposed to varying concentrations of 12-0-tetradecanoyl-phorbol-13 acetate (TPA) for two weeks. A considerable number of cardiac myocytes exposed to a medium with less than 200 ng/ml TPA were rich in myofibrils. The rest of the myocytes lacked organized myofibrils; the terminal parts of these myofibrils were transformed into cord-like structures largely consisting of dense Z-band materials. Some of these aberrant myofibrils contained short normal myofibrillar segments, with sarcomeres. A number of myocytes exposed to 200–250 ng/ml TPA contained myofibrils. which terminated in cord-like structures. The Z-band materials appeared as amorphous dense matrices and some sarcomeres were replaced completely or partially by leptomeres; the myocytes contained autophagosomes. The other myocytes did not contain myofibrils when exposed to the above higher concentrations of TPA. The patches of Z-band materials and structures containing Z-band materials attached to thin filaments on either side were scattered throughout the sarcoplasm of the cells, which were packed with myofilaments and 10 nm microfilaments. Some of these myocytes assumed a spindle shape and contained myofilaments. 10 nm microfilaments and leptomeres. Some of the myocytes, treated with TPA for 1–7 days and then allowed to recover in control medium for 7 days, contained various stages of myofibrillar organization, which did not differ significantly from those of the myofibril-containing cells exposed continuously to TPA as discussed above. The rest of the myocytes during the recovery period in control medium did not contain myofibrils. Rough endoplasmic reticulum and Golgi bodies in TPA-treated myocytes were found to be highly developed as compared to the controls. It is evident from these studies that the responsiveness of cardiac myocytes to TPA not only differs from that of skeletal muscle cells studied by others, but also varies within a population of cardiac myocytes.

Culture and characterization of fetal human atrial and ventricular cardiac muscle cells

Atrial and ventricular cardiac muscle cells isolated from 14- to 18-wk old fetal human hearts were grown in culture and characterized. Once established in culture the flattened cells contracted spontaneously and possessed differentiated ultrastructural characteristics including organized sarcomeres, intercalated discs, and transverse tubules with couplings. Atrial granules were present in the cultured atrial cells. Some cultured ventricular myocytes also contained electron-dense granules associated with Golgi cisternae, which were similar in size and appearance to atrial granules. The cultured ventricular myocytes divided and expressed the genes for thymidine kinase, histone H4, myosin heavy chain, muscle-specific creatine kinase, atrial natriuretic factor, and insulin-like growth factor II. These results establish that differentiated fetal human heart muscle cells can be cultured in sufficient quantities for biochemical, molecular, and morphological analyses.

Ultrastructural morphometric analysis of cultured neonatal and adult rat ventricular cardiac muscle cells.

Neonatal and adult rat ventricular cardiac muscle cells cultured on laminin differed from similar myocytes grown on plastic in the amount and distribution of their mitochondria and transverse tubules. Point-count morphometry was used at the electron microscopic level to quantify these differences. Adult myocytes grown on laminin contained more mitochondria per unit volume than adult myocytes grown on plastic. No significant differences were observed in the volume percent of myofibrils in either adult or neonatal ventricular myocytes when grown on laminin and compared to those grown on plastic. The transverse tubule system in neonatal and adult myocytes was reduced significantly when both groups were cultured on laminin. Furthermore, neonatal and adult myocytes cultured on laminin were flatter than those cultured on plastic. This may indicate a relationship between the surface/volume ratio and transverse tubule development in cultured myocytes. These studies establish that point-count morphometry can be used to quantify changes in the organelle volume densities of cultured cardiac muscle cells.

Substrate dependence of energy metabolism in isolated guinea-pig cardiac muscle: a microcalorimetric study.

1. The effects of glucose, pyruvate and lactate on basal metabolism and on contraction-related energy expenditure of thin trabeculae isolated from guinea-pig heart were studied using a microcalorimetric technique. 2. Resting heat rates of cardiac ventricular muscle measured in the presence of substrate-free solution (56 +/- 20 mW (g dry weight)-1), 10 mM-lactate (54 +/- 12 mW (g dry weight)-1) and 10 mM-glucose (63 +/- 24 mW (g dry weight)-1) did not differ significantly. Increasing the external glucose concentration (up to 100 mM) and/or adding insulin (up to 80 units l-1) had virtually no effect on the measured resting heat rate. 3. With 10 mM-pyruvate as substrate resting heat rate was substantially larger (106 +/- 40 mW (g dry weight)-1) than with glucose, lactate or substrate-free solution. The concentrations of pyruvate producing a half-maximal increase in resting heat rate as compared to substrate-free solution ranged between 0.4 and 1.2 mM. 4. In order to test whether the development of an anoxic core contributed to the substrate dependence of resting heat production the critical PO2 (i.e. the PO2 that produced a just-noticeable decrease in heat rate) was determined in cylindrical preparations of various diameters. It was found that none of the preparations had an anoxic core at rest in a solution equilibrated with 100% oxygen. 5. From the dependence of the critical PO2 on the diameter of the preparation the diffusion coefficient of oxygen through cardiac muscle was calculated using a modification of Hill's equation (Hill, 1928). The O2 diffusion coefficient was found to be 1.09 X 10(-5) cm2 s-1. 6. Contraction-related heat production was also found to be dependent on the substrate used. In the presence of 10 mM-pyruvate it was about 60% larger than in the presence of 10 mM-glucose, 10 mM-lactate or with substrate-free solution. 7. Isometric force of contraction showed the same substrate dependence as contraction-related heat production and increased with a similar time course during repetitive stimulation. 8. The possible mechanisms underlying the substrate dependence of myocardial energy metabolism are discussed. It is suggested that the increased energy expenditure observed in the presence of pyruvate may be related to a decrease in intracellular phosphate and/or to an increase in intracellular pH.

Differentiated membrane specializations and myofibrillar breakdown and recovery in cultured adult cardiac myocytes treated with TPA and diacylglycerol.

Cultured adult rat ventricular cardiac muscle cells were treated with either 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or diacylglycerol (DAG) and observed by in situ transmission electron microscopy. Membrane specializations present in untreated cells (intercalated discs, transverse tubules, plasmalemmal couplings) were also present after TPA and DAG treatment. In the case of the transverse tubular system, there was morphological evidence for active growth. Our studies showed that myofilaments began to become disorganized after 12-24 h of TPA treatment and that after 2 days of exposure to TPA the breakdown of sarcomeres was essentially complete. Myocytes that were treated with TPA for 2 days and then allowed to recover in control medium for 5 days contained sarcomeres in various stages of reassembly. These data indicate that TPA-treated cardiac myocytes retain several membrane specializations, suggesting that there are separate controls for myofilament organization and the maintenance of these differentiated plasmalemmal regions. Furthermore, the ability of the myocytes to recover from TPA treatment may provide investigators with a useful model with which to study myofibrillogenesis.

Preproenkephalin mRNA expression in developing rat heart and in cultured ventricular cardiac muscle cells

Heart muscle tissue has previously been reported to have the highest content of preproenkephalin (ppEnk) mRNA of any tissue in the adult rat. We have determined that it is present in the ventricular cardiac muscle cells of the heart and is developmentally regulated. The expression of ppEnk mRNA was observed to be low throughout the first 2 weeks of postnatal development and decreases substantially during week 3. Expression was again low by week 4, but by adulthood (approx. 3 months), it reached a maximum. ppEnk mRNA was actively expressed in primary cardiac muscle cell cultures prepared from both neonatal and adult rats. Its steady-state content in cell cultures was observed to be increased by cyclic AMP and 3-isobutyl-1-methylxanthine. The phorbol ester phorbol 12-myristate 13-acetate elicited a transient effect (i.e. an increase was observed at 4 h and a return to control values by 24 h). We speculate that enkephalin may play a multi-functional role in the differentiation of neonatal cardiac muscle cells and in the terminally differentiated adult heart cell. We demonstrate that the primary culture systems employed in this study will be useful models with which to explore both transcriptional and translational regulation of ppEnk mRNA in the heart.

Distribution of the α-subunit of the Guanine Nucleotide-binding Protein Gi2and its Comparison to Gαo

Site specific antisera against a synthetic peptide corresponding to the sequence 3-17 of G alpha 12 have been raised and the specificity examined using purified homogeneous Go, Gi2 and Gi containing a 41 kDa alpha-subunit. The distribution of G alpha i2 was investigated in plasma membranes from different tissues and cells and compared to the distribution of G alpha o and other pertussis toxin sensitive G alpha. Considerable amounts of G alpha i2 were found in endocrine tissue especially in membranes from the adrenal and thyroid, in leucocytes and platelets where it constitutes the major, if not only, pertussis toxin-sensitive G alpha, as well as in some cell lines (C6, NG 108-15, S49 cyc-); erythrocytes contained a 41 kDa G alpha i which was different from G alpha i2. G alpha o was present abundantly in nervous tissue, adrenal medulla and cortex but also found in low amounts in other membranes except for lung, liver and blood cells. Subcellular fractionation of cardiac ventricular muscle demonstrated the presence of G alpha o and low amounts of G alpha i2 in sarcolemma, but only 41kDa G alpha i was present in sarcoplasmic reticulum. The importance of the distinct distribution in terms of signal transduction is discussed.

Membrane systems of guinea pig myocardium: Ultrastructure and morphometric studies

The structure and quantitative contribution of membrane systems (transverse-axial tubular system [TATS] and sarcoplasmic reticulum [SR]) have been investigated in the heart of the adult guinea pig. Although previous quantitative studies have been made of guinea pig myocardium, this is the first such study that has utilized tissue in which membrane system elements were clearly identified by selective staining (in this case by the osmium-ferrocyanide [OsFeCN] postfixation method). Both membrane systems are highly developed in ventricular cells, but a TATS is essentially absent from atrial myocytes. The ventricular TATS consists principally of large-bore elements which may be oriented transversely, axially, or obliquely, making numerous anastomoses with one another to form a highly interconnected system of extracellular spaces that penetrate to all myoplasmic depths of the ventricular cell. The cell coat that lines the lumina of these tubules is structured, containing fibrillar structures that run along the length of the tubule. The volume fraction (VV) of the ventricular TATS is low (2.5-3.2%), in consideration of the qualitative prominence of the TATS in these cells. The relative total population of sarcoplasmic reticulum is higher in the atria (VV of 10-11%) than in the ventricles (VV of ca. 8%). In all guinea pig myocytes, several major structural divisions of SR can be discerned, which include network SR, junctional SR, corbular SR, and cisternal SR. Junctional SR (J-SR) in the atrial cells is limited almost exclusively to peripheral saccules of junctional SR (PJSR), whereas both interior J-SR and PJSR are present in the ventricle. Two distinct morphological types of PJSR appear in atrial cells, including both flattened and distended saccules, the latter resembling PJSR of lower vertebrate heart. Spheroidal bodies of SR with opaque contents (corbular SR) are prominent at or near Z-line levels of the sarcomeres of atrial and ventricular cells. Cisternal SR is likely a subset of network SR, but some examples appear related to rough endoplasmic reticulum. An overall impression obtained from this study is that guinea pig atria are composed of structurally primitive cells, whereas the ventricular cardiac muscle cells are more highly developed entities.

Growth factors and TPA stimulate DNA synthesis and alter the morphology of cultured terminally differentiated adult rat cardiac muscle cells

Previous studies have established that the terminally differentiated ventricular cardiac muscle cell of the adult rat reinitiates semiconservative DNA replication when grown in culture (W. C. Claycomb and H. D. Bradshaw, Jr., 1983, Dev. Biol. 90, 331–337). Work reported here shows that several growth factors and chemicals will stimulate this DNA synthetic activity in a concentration-dependent manner. Autoradiographic experiments establish that this stimulated DNA synthesis is due to cells not previously synthesizing DNA being induced to enter the S phase of the cell cycle. By far the greatest stimulation (250%) is observed with the tumor promoter 12- O-tetradecanoyl-phorbol-13-acetate (TPA). Fifty ng/ml is the optimal concentration, and the maximal effect is observed 5 days after adding TPA. TPA also substantially increases the protein content of the cultured myocytes. Diacylglycerols (DAG) induce these same changes, indicating that the effect of TPA is mediated by protein kinase C. The morphology of the cultured cardiac muscle cells is profoundly altered by TPA and DAG. TPA- and DAG-treated myocytes spread more thinly on the surface of the culture flask, acquire multiple nuclei, and undergo nucleolar fragmentation. The myofibrillar ultrastructure of the treated cells becomes almost totally disorganized, and intermediate filaments and rough endoplasmic reticulum accumulate in the cytoplasm. These TPA results suggest a possible relationship between the degree of ultrastructural differentiation of the ventricular cardiac muscle cell and DNA synthetic activity. This easily altered cellular plasticity should be very useful for studies of the regulation of cardiac muscle cell proliferation and cell differentiation.

Effects of ryanodine on relaxation in isolated myocardium from different animal species.

Relaxation in mammalian ventricular cardiac muscle is sensitive to the prevailing load. This "load dependence of relaxation" (LD) can be demonstrated only when an efficient sarcoplasmic reticulum (SR) is present. To define further the role of the SR in LD, we studied contraction and relaxation in cat, rat and frog cardiac muscle after exposure to ryanodine. Ryanodine is a selective inhibitor of calcium release from the SR. This view was confirmed in the present study in single cardiac rat myocytes with functioning SR. Ryanodine did not affect LD in multicellular mammalian myocardium even though it had already significantly depressed contractility, suggesting that calcium release from the SR plays no role in establishing LD. Calcium accumulation in the SR as a consequence of the inhibited release can account for the late depression of LD in the presence of ryanodine.

Effects of damaging the endocardial surface on the mechanical performance of isolated cardiac muscle.

The mechanical properties of mammalian ventricular cardiac muscle have been studied in the presence and in the absence of an intact endocardial surface. Isotonic and isometric twitch contractions were obtained from papillary muscles of the right ventricle of cat at 29 degrees and 37 degrees C, at different extracellular calcium concentrations ([Ca2+]o), and at different initial muscle lengths. The endocardial surface was damaged by gentle abrasion of the muscle surface with a plastic blade or by brief immersion for 1 second with 1% Triton X-100. Although there was no evidence of damage to myocardial cells, damaging the endocardial surface resulted in an immediate and irreversible abbreviation of the twitch contractions with, except at the highest ([Ca2+]o, a decrease in peak isometric twitch tension. These changes induced 1) an asymmetrical shift of the tension-[Ca2+]o relation towards increasing [Ca2+]o but with no effect at the highest [Ca2+]o, and 2) a rightward and downward shift of the length-tension relation. Both shifts were significantly more pronounced at 37 degrees C than at 29 degrees C; they were not accompanied by significant changes in Vmax. The asymmetrical shift of the tension-[Ca2+]o relation suggests that the endocardium-mediated chain of events may be mediated by changes in the sensitivity of the contractile proteins to Ca2+. This hypothesis is also supported by the similar pattern of changes (i.e., modulation of the onset of early tension decline) induced by decreasing length at each [Ca2+]o and by the removal of a functional endocardium. Accordingly, the endocardium may help to control the performance of the heart by modulating peak contractile performance and relaxation of the underlying myocardium.

Actions of atrial natriuretic peptide (ANP) on cyclic nucleotide concentrations and phosphatidylinositol turnover in ventricular myocytes

Atrial natriuretic peptide (ANP) stimulates cGMP production in isolated rabbit ventricular myocytes incubated in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (1mM). Half maximal activation was found at 10(-8)M ANP. Cellular cGMP concentrations of around 0.6 pmol/10(6) cells were elevated 4-6 fold by ANP (10(-6)M), 3-4 fold by carbachol (1mM) and around 10 fold by sodium nitroprusside (1mM). ANP had no effect on basal or isoprenaline-stimulated cAMP concentrations or on basal or noradrenaline-stimulated turnover of phosphatidylinositol. From these results we conclude that ANP receptors, coupled to particulate guanylate cyclase, exist in cardiac ventricular muscle. This indicates that ANP may also have a physiological action on ventricular muscle contractility during volume expansion.

Voltage-dependent calcium release in guinea-pig cardiac ventricular muscle as antagonized by magnesium and calcium.

An increase in extracellular potassium concentration from 4 to 16 mmol/l caused a decrease in membrane potential from -92 to -59 mV and selectively diminished the earlier of two contraction components of guinea-pig papillary muscles at 0.2 Hz stimulation frequency in the presence of noradrenaline. The influence on the early contraction component had a threshold of 8 mmol/l K+, corresponding to a membrane potential of -77 mV. However, test contractions elicited 800 ms after the 5 s stimulation interval exhibited an unimpaired early component. Since the activator calcium responsible for the early contraction component is derived, in mammalian ventricular muscle, from the junctional sarcoplasmic reticulum, it is assumed that the release site of the reticulum was filled with calcium shortly (800 ms) after a regular contraction, and lost its calcium at 16 mmol/l extracellular K+, during the 5 s stimulation interval. The potassium-induced depolarization determined the rate of calcium leakage during rest from the intracellular store. The depolarization-induced decline of the early contraction component was equally well antagonized by Mg2+ or Ca2+ without influencing the measured transmembrane potential. Both divalent cations shifted the relation between potassium concentration or membrane potential and the strength of the early contraction component to less negative membrane potentials. In order to reduce the early contraction component by 25% in the presence of 9.6 instead of 1.2 mmol/l Mg2+, the potassium concentration had to be increased from 9.6 to 22.0 mmol/l, with a respective decrease in resting membrane potential from -72.6 to -51.1 mV. The antagonistic effect of both divalent cations is though to result from the neutralization of negative charges outside the sarcolemma with a respective decrease in the outside surface potential.

Interstitial potentials and their change with depth into cardiac tissue

The electrical source strength for an isolated, active, excitable fiber can be taken to be its transmembrane current as an excellent approximation. The transmembrane current can be determined from intracellular potentials only. But for multicellular preparations, particularly cardiac ventricular muscle, the electrical source strength may be changed significantly by the presence of the interstitial potential field. This report examines the size of the interstitial potential field as a function of depth into a semi-infinite tissue structure of cardiac muscle regarded as syncytial. A uniform propagating plane wave of excitation is assumed and the interstitial potential field is found based on consideration of the medium as a continuum (bidomain model). As a whole, the results are inconsistent with any of the limiting cases normally used to represent the volume conductor, and suggest that in only the thinnest of tissue (less than 200 micron) can the interstitial potentials be ignored.

1,25-Dihydroxyvitamin D3 stimulates 45Ca2+ uptake by cultured adult rat ventricular cardiac muscle cells.

This study tested the hypothesis that 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) and its previously described cardiac receptors play roles in regulating intracellular calcium homeostasis in cardiac muscle cells. This question was addressed by assessing whether 1,25-(OH)2D3 influences 45Ca2+ uptake by homogeneous cultures of adult rat ventricular cardiac muscle cells. Twenty-four h prior to the measurement of 45Ca2+ uptake, the cells were transferred to serum-free medium ([Ca2+], 1.0 mM) containing 1.0 nM 1,25(OH)2D3 or vehicle. The cells were then incubated with 45Ca2+ for periods up to 60 min at room temperature, followed by removal of excess external 45Ca2+ by washing repeatedly with La3+. Pretreating the cells with 1,25-(OH)2D3 caused 3-fold stimulation (p less than 0.005) of 45Ca2+ uptake. Stimulation of 45Ca2+ uptake required a prolonged (8-12 h) exposure to 1,25-(OH)2D3, suggesting a receptor-mediated phenomenon. Concentrations of 0.01-10 nM 1,25-(OH)2D3 yielded a dose-response curve which peaked at 1.0 nM and decreased at higher concentrations. Steroid specificity was established by the failure of 1.0 nM levels of 25-hydroxyvitamin D3, estradiol-17 beta, and progesterone to change 45Ca2+ uptake. Sucrose gradient analysis confirmed the presence of a specific 3-4 S 3H-1,25-(OH)2D3 binding component both in freshly isolated and in cultured ventricular cardiac muscle cells. The stimulatory effect of 1,25-(OH)2D3 on 45Ca2+ uptake was abolished by the concomitant incubation of the cells with cycloheximide or actinomycin D, demonstrating a requirement for protein and nucleic acid synthesis. In conclusion, these data demonstrate that 1,25-(OH)2D3 stimulates 45Ca2+ uptake in adult ventricular cardiac muscle cells by a mechanism resembling a receptor-mediated phenomenon.

Effect of class II-III-IV antiarrhythmic drugs on the premature action potential duration (APD) in cardiac Purkinje and ventricular muscle fibers

Effect of class II-III-IV antiarrhythmic drugs on the premature action potential duration (APD) in cardiac Purkinje and ventricular muscle fibers

The cDNA sequence of the major phospholamban mRNA in canine cardiac ventricular muscle.

The cDNA sequence of the major phospholamban mRNA in canine cardiac ventricular muscle.

3-Methylhistidine content and pH dependency of ATPase activity of adult and embryonic chicken cardiac ventricular myosins.

A distinct difference in the 3-methylhistidine (3-MeHis) content and the pH-dependency curve for calcium-activated adenosine triphosphatase (Ca-ATPase) activity was observed between chicken and mammalian cardiac ventricular myosins. The 3-MeHis content and pH dependency of the Ca-ATPase activity of myosins from adult and embryonic chicken cardiac ventricular muscles and chicken fast white and slow red muscles were almost the same.

Two types of calcium channels in guinea pig ventricular myocytes.

In cardiac muscle, Ca2+ plays a key role in regulation of numerous processes, including generation of the action potential and development of tension. The entry of Ca2+ into the cell is regulated primarily by voltage-gated channels in the membrane. Until recently, it was felt that only one type of Ca2+ channel existed in cardiac ventricular muscle. Experiments reported here suggest that in isolated guinea pig ventricular myocytes, there are two distinct types of Ca2+ channels with markedly different activation thresholds, inactivation kinetics, and sensitivities to inorganic and organic Ca2+ channel blockers. The channels were also distinguished based on their response to increased frequency of clamping such that the current through the low-threshold channel decreased while that through the high-threshold channel increased. In a few cells, the current through both channels was enhanced by isoproterenol, a beta-adrenergic agonist, but only the high-threshold channel was enhanced by the Ca2+-channel agonist Bay K 8644. Thus, isolated guinea pig ventricular myocytes appear to have two types of Ca2+ channels distinguished by various criteria.