Ventricular Cardiac Muscle Cells Research Articles

Marginal zinc deficiency alters the heart proteome of rats.

Zinc deficiency is closely related to cardiovascular diseases (CVDs), but the effects of marginal zinc deficiency (MZD) after birth on the heart are unknown. In this study, 4-week-old male rats were fed a low zinc diet (10 mg kg-1, 1/3 recommended nutrient intake, RNI) for 8 weeks. Echocardiography and histopathology were performed to assess the functional and morphological alterations of the heart. High-throughput proteomics was used to study the effects of MZD on cardiac protein expression. We found that MZD reduced food intake, body weight, serum zinc, and heart weight; however, the coefficient, zinc concentration, function, and histopathology of the heart were not changed. The heart proteome was altered in the marginal zinc-deficient diet group (MZG), compared with the normal zinc diet group (NZG). A total of 310 differentially expressed proteins (P < 0.05) were significantly changed by MZD, among which 163 proteins were up-regulated and 147 were down-regulated. Of these, 43 proteins are related to CVDs and 18 proteins are zinc-associated proteins. Gene Ontology and Pathway analysis revealed that 74 biological processes (BPs) and 37 pathways were significantly changed by MZD. This included six CVD-related BPs, such as regulation of heart rate, cardiac muscle contraction, regulation of ventricular cardiac muscle cell action potential, and regulation of blood pressure, and eight CVD-related pathways, such as dilated cardiomyopathy, diabetic cardiomyopathy, and hypertrophic cardiomyopathy. Our data show that marginal zinc deficiency after birth significantly alters cardiac protein expression and pathways related to CVDs.

Identification of Key Pathways and Genes in Anaplastic Thyroid Carcinoma via Integrated Bioinformatics Analysis.

BackgroundTo provide a better understanding of anaplastic thyroid carcinoma (ATC) at the molecular level, this study aimed to identify the genes and key pathways associated with ATC by using integrated bioinformatics analysis.Material/MethodsBased on the microarray data GSE9115, GSE65144, and GSE53072 derived from the Gene Expression Omnibus, the differentially expressed genes (DEGs) between ATC samples and normal controls were identified. With DEGs, we performed a series of functional enrichment analyses. Then, a protein–protein interaction (PPI) network was constructed and visualized, with which the hub gene nodes were screened out. Finally, modules analysis for the PPI network was performed to further investigate the potential relationships between DEGs and ATC.ResultsA total of 537 common DEGs were screened out from all 3 datasets, among which 247 genes were upregulated and 275 genes were downregulated. GO analysis indicated that upregulated DEGs were mainly involved in cell division and mitotic nuclear division and the downregulated DEGs were significantly enriched in ventricular cardiac muscle cell action potential. KEGG pathway analysis showed that the upregulated DEGs were mainly enriched in cell cycle and ECM-receptor interaction and the downregulated DEGs were mainly enriched in thyroid hormone synthesis, insulin resistance, and pathways in cancer. The top 10 hub genes in the constructed PPI network were CDK1, CCNB1, TOP2A, AURKB, CCNA2, BUB1, AURKA, CDC20, MAD2L1, and BUB1B. The modules analysis showed that genes in the top 2 significant modules of PPI network were mainly associated with mitotic cell cycle and positive regulation of mitosis, respectively.ConclusionsWe identified a series of key genes along with the pathways that were most closely related with ATC initiation and progression. Our results provide a more detailed molecular mechanism for the development of ATC, shedding light on the potential biomarkers and therapeutic targets.

Infiltration of Inflammatory Cells Plays an Important Role in Matrix Metalloproteinase Expression and Activation in the Heart during Sepsis

Septicemia is an emerging pathological condition involving, among other effects, refractory hypotension and heart dysfunction. Here we have investigated the contribution of resident nonmyocytic cells to heart alterations after lipopolysaccharide administration. These cells contributed to the rapid infiltration of additional inflammatory cells that enhance the onset of heart disease through the release of inflammatory mediators. Early activation of resident monocytic cells played a relevant role on the infiltration process, mainly of major histocompatibility complex class II- and CD11b-positive cells. This infiltration was significantly impaired in animals lacking the nitric-oxide synthase-2 (NOS-2) gene or after pharmacological in-hibition of NOS-2 or cylooxygenase-2, suggesting a significant contribution of nitric oxide and prostanoids to the infiltration process. Under these conditions, the expression of NOS-2 and cylooxygenase-2 in the whole organ was attenuated because cardiomyocytes failed to express these enzymes. However, cardiomyocytes expressed and activated matrix metalloproteinase-9 through mechanisms regulated, at least in part, by nitric oxide and prostaglandins in an additive way. These results directly link the inflammatory response in the heart and extracellular matrix remodeling by the matrix metalloproteinases released by the cardiomyocytes, suggesting that activation and recruitment of inflammatory cells to the heart is a major early event in cardiac dysfunction promoted by septicemia.

Gene expression profiling of the aging mouse cardiac myocytes.

Heart disease remains the most frequent cause of death in the general population with increasing incidence in the elderly population. The pathologic failure of the aging heart may be related to structural and functional alterations in cardiac muscle cells. However, the molecular mechanisms underlying the aging-related decline in cardiac muscle function are largely unknown. To provide the first analysis of cardiac aging at the level of gene expression, we established and compared cDNA libraries from apparently healthy young and aged mouse ventricular cardiac muscle cells. We report the identification of genes that exhibit aging-related changes of mRNA levels. Aging expression profiles in aged hearts indicate decreased cellular adaptation and protection against stress-induced injury together with the development of contractile dysfunction. The data suggest reduced activity of the mitochondrial electron transport system and reduced levels of cardiac-specific transcription regulators. The cardiomyocyte aging profile of gene expression displays similarities with known heart disorders. Genes whose mRNA levels change with aging in cardiomyocytes might profoundly affect pathological changes in the heart.

The 21-day postnatal rat ventricular cardiac muscle cell in culture as an experimental model to study adult cardiomyocyte gene expression.

The purpose of this study was to develop and characterize a cardiomyocyte culture system for use as an experimental model to study the mechanism(s) by which cardiac muscle cells permanently exit the cell cycle during early neonatal life. Ventricular cardiomyocytes, isolated by retrograde perfusion of hearts from 21-day-old and adult rats, were compared through 10 days of culture. Expression patterns of genes encoding developmentally programmed proteins were determined to be similar between cardiomyocytes cultured from 21-day-old and adult rats, using the reverse transcription polymerase chain reaction. A lacZ-expressing reporter gene was used to test the efficiency of gene delivery in cultured cardiomyocytes. Transfections using cationic liposomes yielded 24+/-7, 25+/-7 and 10+/-1% cardiomyocytes positive for beta-galactosidase activity in cultured 1-day, 21-day and adult cardiomyocytes, respectively. Direct needle microinjection resulted in 48+/-7, 35+/-6 and 37+/-5% cardiomyocytes positive for enzymatic activity in 1-day, 21-day and adult cardiomyocytes, respectively. Cell cycle-specific cDNA arrays were used to analyze the expression pattern of cell cycle-related genes in 12-O-tetradecanoyl-phorbol-13-acetate (TPA)- and non-TPA-treated cultured 21-day cardiomyocytes. Based on the similarity of cultured 21-day to adult ventricular cardiomyocytes and their high transfection efficiencies, we propose the use of cultured cardiomyocytes from 21-day-old rat ventricles as an experimental model system for the study of adult cardiomyocyte gene expression and cell cycle machinery.

Transfer of macromolecules into living adult cardiomyocytes by microinjection.

Among techniques commonly used to deliver bioactive molecules into living cells, microinjection is a very efficient method. Microinjection has been used extensively for gene transfer into different cell types. We applied the microinjection technique to the adult rat ventricular cardiac muscle cells (AVC) in primary culture and optimized microinjection parameters and the appropriate cell culture conditions. We also optimized the use of particular agents (i.e. 2,3-butanedione monoxime, verapamil) for the prevention of the cell damage caused by the micropuncture. We obtained the expression of a CMV-beta-galactosidase reporter gene in up to 20% of the injected cells with efficient maintenance of long term cell viability. Under our experimental conditions direct microinjection is a very advantageous technique to transfer macromolecules into living adult cardiac muscle cells and a powerful system to study and manipulate the biochemistry and molecular biology of the cardiac myocyte.

Translation of heart preproenkephalin mRNA and secretion of enkephalin peptides from cultured cardiac myocytes.

Rat ventricular cardiac muscle has previously been shown to contain exceptionally high levels of preproenkephalin mRNA (ppEnk mRNA). We have recently determined that the level of ppEnk mRNA is developmentally and hormonally regulated in rat ventricular cardiac muscle tissue and in cultured myocytes (J. P. Springhorn and W. C. Claycomb. Biochem. J. 258: 73-77, 1989). We demonstrate in the current study that heart ppEnk mRNA is structurally identical at the 5' end to brain ppEnk mRNA using a ribonuclease protection assay and that heart ppEnk mRNA can be translated in vitro using a rabbit reticulocyte lysate system. In vitro synthesized preproenkephalin peptides were immunoprecipitated with a polyclonal antibody directed to the carboxy-terminal seven amino acids of preproenkephalin. We have also established by radioimmunoassay that enkephalin-containing peptides are secreted from cultured neonatal and adult rat ventricular cardiac muscle cells. This secretion is linear with respect to time and can be stimulated by phorbol 12-myristate 13-acetate (PMA) and adenosine 3',5'-cyclic monophosphate (cAMP). It was determined by column chromatography that cAMP induced neonatal rat ventricular cardiac muscle cells to secrete Met5-enkephalin-Arg6-Phe7, whereas PMA plus 3-isobutyl-1-methylxanthine induced adult rat ventricular cardiac muscle cells to secrete Met5-enkephalin. These studies establish that ventricular heart muscle ppEnk mRNA can be translated and that enkephalin peptides are secreted from ventricular cardiac muscle cells.

TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult cardiac muscle cells.

The effect of a tumor promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA), on the expression of myosin heavy chain isoforms in cultured rat cardiac ventricular muscle cells was studied. The previous preliminary report [Claycomb WC (1988): "Biology of Isolated Adult Cardiac Myocytes." In Clark WA, Decker RS, Borg TK (eds): New York: Elsevier, pp 284-287] indicated that TPA turns off the expression of myosin heavy chain genes in cultured adult cardiac myocytes. Electrophoretic and immunocytochemical analyses were carried out in the present studies. The myosin heavy chain isoform profiles of cardiac myocytes exposed to TPA at concentrations of 50-250 ng/ml culture medium for varying periods were similar to those of controls that were grown in the absence of TPA, showing predominant isoform V1. Immunofluorescence microscopy with monoclonal antibodies to cardiac ventricular isomyosin revealed the structural organization of myosin in TPA-treated cells. The organization of myosin was variable among different myocytes and within a single myocyte. Immunofluorescence microscopy was extended to the examination of the organization of alpha-actinin which did not differ from that of myosin in some myocytes. In contrast to the previous report [Claycomb, 1988], this study has demonstrated that TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult ventricular cardiac muscle cells.

A ubiquitous factor (HF-1a) and a distinct muscle factor (HF-1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression.

Recent studies have identified a conserved 28-bp element (HF-1) within the rat cardiac MLC-2 gene which confers cardiac muscle-specific and inducible expression during myocardial cell hypertrophy. Utilizing a combination of independent experimental approaches, this study characterizes two cardiac nuclear factors which bind to HF-1, a ubiquitous factor (HF-1a), and an A + T-rich binding factor (HF-1b) which is preferentially expressed in differentiated cardiac and skeletal muscle cells. The HF-1a binding site is located in a core region of the 28-bp conserved element, immediately upstream from the A + T-rich HF-1b site, which is homologous to the MEF-2 site found in a number of muscle genes. By a number of separate criteria (gel mobility shift, competition, and mutagenesis studies), HF-1b and MEF-2 appear to be indistinguishable and thus are either identical or closely related muscle factors. Transient assays of luciferase reporter genes containing point mutations throughout the 28-bp HF-1 regulatory element document the importance of both the HF-1a and HF-1b sites in transient assays in ventricular muscle cells. In the native 250-bp MLC-2 promoter fragment, mutations in the single E box had little effect on cardiac muscle specificity, while point mutations in either the HF-1a or HF-1b binding site significantly reduced promoter activity, underscoring the importance of both the HF-1a and HF-1b sites in the transcriptional activation of this cardiac muscle gene. Thus, this study provides evidence that a novel, ubiquitous factor (HF-1a) and a muscle factor (HF-1b/MEF-2) can form a novel, E-box-independent pathway for muscle-specific expression in ventricular cardiac muscle cells.

A ubiquitous factor (HF-1a) and a distinct muscle factor (HF-1b/MEF-2) form an E-box-independent pathway for cardiac muscle gene expression.

Recent studies have identified a conserved 28-bp element (HF-1) within the rat cardiac MLC-2 gene which confers cardiac muscle-specific and inducible expression during myocardial cell hypertrophy. Utilizing a combination of independent experimental approaches, this study characterizes two cardiac nuclear factors which bind to HF-1, a ubiquitous factor (HF-1a), and an A + T-rich binding factor (HF-1b) which is preferentially expressed in differentiated cardiac and skeletal muscle cells. The HF-1a binding site is located in a core region of the 28-bp conserved element, immediately upstream from the A + T-rich HF-1b site, which is homologous to the MEF-2 site found in a number of muscle genes. By a number of separate criteria (gel mobility shift, competition, and mutagenesis studies), HF-1b and MEF-2 appear to be indistinguishable and thus are either identical or closely related muscle factors. Transient assays of luciferase reporter genes containing point mutations throughout the 28-bp HF-1 regulatory element document the importance of both the HF-1a and HF-1b sites in transient assays in ventricular muscle cells. In the native 250-bp MLC-2 promoter fragment, mutations in the single E box had little effect on cardiac muscle specificity, while point mutations in either the HF-1a or HF-1b binding site significantly reduced promoter activity, underscoring the importance of both the HF-1a and HF-1b sites in the transcriptional activation of this cardiac muscle gene. Thus, this study provides evidence that a novel, ubiquitous factor (HF-1a) and a muscle factor (HF-1b/MEF-2) can form a novel, E-box-independent pathway for muscle-specific expression in ventricular cardiac muscle cells.

Norepinephrine‐induced cardiac hypertrophy of the cat heart

Norepinephrine administration causes progressive hypertrophy of the mammalian heart as measured by myocardial mass. The purpose of this study was to determine the growth response of the myocardial tissue components as well as the myocardial cell itself to norepinephrine. Young, adult cats were given low doses of norepinephrine in dextrose or dextrose alone twice daily for 15 days. On day 16, there were no changes in the animals body weight, right ventricular systolic pressure, right ventricular end-diastolic pressure, heart rate, cardiac index, or blood pressure. However, the right ventricle/body weight, the left ventricle/body weight and the total heart weight/body weight were increased significantly in the norepinephrine treated animals. The increase was on the order of 40%. The cardiac muscle cell was also significantly increased in size and both the right and left ventricular cardiac muscle cells exhibited a dramatic increase in size as measured by cross sectional area. Upon stereological examination it was found that the amount of hypertrophy as seen in the cardiac muscle cells was paralleled by the hypertrophy seen in the other tissue components of the myocardium. The volume density of the muscle cells, the interstitial components, as well as the blood vessel compartment were identical in the control and in the norepinephrine-treated groups. In conclusion, this study demonstrates that the response of the myocardium to norepinephrine is similar to that seen in response to a volume overload rather than that seen in response to pressure overload.

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.

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.

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.

Proto-oncogene expression in proliferating and differentiating cardiac and skeletal muscle

We have examined the expression of 13 proto-oncogenes in proliferating and terminally differentiated cardiac and skeletal muscle. Total RNA was prepared from intact ventricular cardiac-muscle tissue and from purified ventricular cardiac-muscle cells of neonatal and adult rats and from cultured proliferating and terminally differentiated L6A1 rat skeletal-muscle cells. cDNA probes for histone H4, thymidine kinase, myosin heavy chain and M-creatine kinase were used to assess cellular proliferation and differentiation. Oncogenes c-myc, c-raf, c-erb-A, c-ras-H, c-ski, and c-sis were expressed in both proliferating and differentiated cardiac muscle tissue and cells, whereas c-myb expression was not observed in either. c-src was expressed only in neonatal cardiac muscle tissue and cells. c-fms, c-abl, and c-ras-K were expressed in tissue from both neonatal and adult animals but only in purified cells from neonatal animals. c-fes/fps was expressed only in neonatal cardiac muscles cells. c-fos expression was not observed in cardiac-muscle tissue from either neonatal or adult rats, but surprisingly was abundantly expressed in freshly isolated cardiac-muscle cells from animals of both ages. These results emphasize that biochemical analysis using intact cardiac-muscle tissue may not necessarily reflect muscle-specific cell processes. They also show that the expression of c-fos can be activated by the cell isolation procedure. c-myc, c-ski, c-ras-H, c-ras-K, c-abl, c-raf and c-erb-A were expressed in both proliferating and terminally differentiated skeletal-muscle cells, whereas c-myb, c-fos, c-src and c-fms transcripts were observed only in proliferating cells. c-fes/fps and c-sis were not expressed in dividing or fused skeletal-muscle cells. These results demonstrate unique tissue and cell-specific patterns of proto-oncogene expression and suggest that these genes may be involved with the regulation of cellular proliferation and terminal differentiation in striated muscle.

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.