Neonatal Cardiac Muscle Cells Research Articles

Increased expression of UBF is a critical determinant for rRNA synthesis and hypertrophic growth of cardiac myocytes

SPECIFIC AIMSWe tested the hypothesis that increased ribosome biogenesis is a prerequisite for the progression of cardiomyocyte hypertrophic growth and that this process is regulated at the level of ribosomal gene (rDNA) transcription by the nucleolar transcription factor UBF.PRINCIPAL FINDINGS1. Adenovirus-directed expression of UBF antisense RNA inhibits α1-adrenergic- and contraction-induced UBF expression in cardiomyocytesNoncontracting primary cultures of neonatal cardiac muscle cells (cardiomyocytes) were infected at a titer of 1 × 102 PFU/cell with either a control Ad-5-derived virus expressing the nonmammalian, control gene chloramphenicol transferase (Ad-CAT) or an Ad-5 virus expressing UBF cDNA (−267 to+635) in the antisense orientation under control of the CMV promoter (Ad-UBFas). At this titer, 95% of the myocytes were infected as determined by GFP fluorescence. After infection, the cardiomyocytes were stimulated for 3 days with either the α1-adrenergic agonist phenylephrine (PE; 25 μM) or con...

Localization of proliferating cell nuclear antigen in the developing and mature rat heart cell.

The cardiac muscle cell ceases to divide shortly after birth; this cessation is followed by a limited period when DNA synthesis and karyokinesis occur without cytokinesis. The regulation of this process is not known. The purpose of this study is to explore the possible events that could lead to the cessation of cardiac muscle cell division. One protein requisite for DNA synthesis is proliferating cell nuclear antigen (PCNA). This protein is the auxiliary protein of DNA polymerase delta. Rats of fetal age day 18 or days 0, 4, 8, 12, and 16 after birth were obtained. In addition, adult hearts were used for this study. Hearts from the fetal day-18 rats and the day-0 neonatal rats were digested. Cardiac myocytes were isolated and placed in culture for an analysis of DNA synthesis by using tridiated thymidine. Ventricular muscle tissue was isolated from hearts of all ages and frozen in liquid nitrogen for Northern and Western blot analyses. Tridiated thymidine analysis revealed that, although serum stimulation significantly increased the number of labeled fetal cardiac muscle cells, it did not have that effect on neonatal cardiac muscle cells in culture. Northern blot analysis revealed that the steady state levels of mRNA for PCNA remained constant from fetal day 18 through day 4 after birth. Steady state levels declined during the second postnatal week and then reached basal levels by day 16. PCNA message was still present in adult heart tissue. By using indirect immunofluorescence and Western blotting, PCNA protein could be located in the nucleus of cardiac muscle cells during the first 2 weeks after birth. At 16 days after birth, the protein was found in the cytoplasm in very low amounts but was not found in the nucleus. The protein was barely detectable by Western blotting in the cytoplasmic fraction from the adult myocardium. The results of this study suggest that the PCNA message and protein product declined after birth, but both were present at low levels in the adult myocardium. However, the PCNA protein was not translocated to the nucleus in adult myocardial cells. The events involving PCNA correlated closely with the time period when cell division and then DNA synthesis ceased in these cells.

T-type Ca2+ current is expressed in hypertrophied adult feline left ventricular myocytes.

Macroscopic T-type Ca2+ currents, which are often observed in fetal and neonatal cardiac muscle cells, were not found in normal (0 of 17) adult feline ventricular myocytes. However, they were present in most (15 of 21) myocytes isolated from adult feline left ventricles with long-standing pressure-overload-induced hypertrophy. This is the first study to provide evidence in a large mammal, such as the cat, that T-type Ca2+ channels may be reexpressed in adults in association with hypertrophy resulting from slow progressive pressure overload. Importantly, this expression was stable for the duration of the hypertrophy process and was not associated with abrupt pressure overload. T-type Ca2+ currents were separated from L-type Ca2+ currents by exploiting the differences in their voltage dependence of steady-state inactivation. Depolarizations from -80 mV revealed a rapidly activating inward current that peaked in magnitude at -30 mV (-1.8 +/- 0.9 [mean +/- SD] pA/pF) and fully inactivated within 100 milliseconds in 15 of 21 hypertrophied myocytes studied. Further depolarizations activated progressively less T-type Ca2+ current, so that at +10 mV the L-type Ca2+ current predominated. In the hypertrophied myocytes that demonstrated both T-type and L-type Ca2+ currents, two distinct peaks occurred in their current-voltage relations. T-type Ca2+ currents were not evident in any of the 17 normal adult feline left ventricular myocytes studied. The purpose of T-type Ca2+ currents in hypertrophy is unclear. However, their presence may make hypertrophied myocardium more prone to spontaneous action potentials and increase the likelihood for arrhythmias in partially depolarized hypertrophied myocardium.

Microinjection of antibodies and expression vectors into living myocardial cells. Development of a novel approach to identify candidate genes that regulate cardiac growth and hypertrophy.

Microinjection approaches in the cardiac cell context have allowed delivery of various calcium dyes and monitoring of short-term physiological responses. However, unlike other cell types, it has proved difficult to microinject myocardial cells without the concomitant loss of long-term cell viability. An analysis of experimental variables was conducted to adapt microinjection techniques to the neonatal rat ventricular cell context. Among the variables optimized were the selection of culture dishes, plating substrate, microinjection parameters, and a variety of maneuvers to inhibit myocyte hypercontracture, injury, and consequent death after micropuncture. With the modified technique, the percentage of injected cells that maintained long-term viability (48 hours) increased from less than 1% to 30%. Similarly, an increased efficiency of gene transfer and expression (measured as the percentage of injected cells that express the delivered gene) was obtained after either cytoplasmic or nuclear injection of a beta-galactosidase expression vector into cardiac myocytes. Microinjection of marker immunoglobulin G does not interfere with the induction of the hypertrophic response or the expression of a coinjected atrial natriuretic factor promoter-luciferase reporter fusion gene construct. To the best of our knowledge, this study provides the first description of the efficient microinjection of neonatal cardiac muscle cells with maintenance of long-term cell viability. The microinjection technique is now a viable approach to examine cause-and-effect relations between specific gene products and any defined feature or response of cardiac myocytes that can be assayed at a single-cell level.

Effects of caffeine on the DNA and protein synthesis of the protein-energy malnourished neonatal cardiac muscle cells in culture

1. 1. The growth of cardiac cells derived from newborn rats whose dams were either malnourished or malnourished with caffeine during pregnancy was inhibited in culture over the period of 5 days as compared to that of the normally nourished cells. 2. 2. Cells derived from malnourished rats with caffeine added to their diets showed a greater inhibition than those from the malnourished rats not given caffeine. 3. 3. Both DNA and protein synthesis showed an inhibition due to caffeine in a dose-dependent manner using normally nourished cells. 4. 4. In the presence of exogenous 2 mM caffeine, the degree of percent inhibition of DNA and protein synthesis of cells derived from rats malnourished with caffeine was less than that from the rats with malnutrition alone. 5. 5. The present data indicated that malnutrition combined with caffeine during pregnancy exerted a greater negative effect on the nature of cell growth than malnutrition alone and these cells became less sensitive to exogenous caffeine.

Growth factors, growth factor response elements, and the cardiac phenotype.

Fibroblast growth factors (FGF) and type beta-1 transforming growth factor (TGF beta 1) are pleiotropic regulatory peptides which are expressed in myocardium in a precise developmental and spatial program and are up-regulated, in the adult heart, by ischemia or a hemodynamic burden. The accumulation of trophic factors after aortic banding supports the hypothesis that autocrine or paracrine pathways might function to mediate, in part, the consequences of mechanical load. Our laboratory has demonstrated that cardiac muscle cells are targets for the action of peptide growth factors and, more specifically, that modulation of the cardiac phenotype by basic FGF (bFGF) and TGF beta 1 strongly resembles the induction of fetal cardiac genes--including skeletal alpha-actin (SkA), beta-myosin heavy chain, and atrial natriuretic factor--which are characteristic of pressure-overload hypertrophy. Unexpectedly, and despite effects like those of bFGF on five other cardiac genes, acidic FGF (aFGF) was found to repress, rather than stimulate, SkA transcription in neonatal cardiac muscle cells. The proximal 200 nucleotides of a heterologous SkA promoter were sufficient for basal tissue-specific transcription, for induction by bFGF, and for inhibition by aFGF. Thus, both positive and negative regulation by peptide growth factors can be localized to the proximal SkA promoter. Full promoter activity required each of three CC[A/T]6GG motifs similar to the serum response element (SRE) for activation of the c-fos proto-oncogene, as previously shown for SkA transcription in a skeletal muscle background. The most proximal SRE, SRE1, was sufficient in the absence of other SkA promoter sequences for efficient tissue-specific expression in cardiac myocytes (versus cardiac fibroblasts), and was stimulated by bFGF to the same extent as the full-length promoter and endogenous gene. Despite its ability to repress the SkA promoter, aFGF had no significant effect on SRE1. Both FGFs up-regulated the canonical fos SRE, to a comparable degree. Thus, SRE1 can discriminate between signals generated in cardiac myocytes by bFGF and aFGF. In cardiac myocyte extracts, two predominant proteins contact SRE1: serum response factor (SRF) and a second protein, F-ACT-1. Thus, serum response factor and F-ACT-1 are candidate trans-acting factors for basal transcription of the SkA gene in cardiac muscle cells and for induction of SkA by bFGF and, potentially, other trophic signals.

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.

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.

Sarcoplasmic reticulum and intermediate filament organization in cultured neonatal cardiac muscle cells. Studies with reduced osmium ferrocyanide.

Cardiac muscle cells from 3-day-old rat neonates were cultured for periods of 2 to 56 days. In order to facilitate ultrastructural studies on the organization of the sarcoplasmic reticulum, the cells were prepared for transmission electron microscopy according to a regimen including postfixation in reduced osmium ferrocyanide. The nonjunctional sarcoplasmic reticulum (NJSR) was organized as a loose, fenestrated sleeve around the exterior of bundles of myofilaments and was particularly prominent at the level of the Z line. The only recognizable junctional elements of the sarcoplasmic reticulum were in a peripheral location. Reduced osmium ferrocyanide was also useful in distinguishing intermediate (10 nm) filaments, since it understained Z substance, which often obscured these structures. Intermediate filaments were arranged both at the Z line and the intercalated disc, in parallel strands, approximately at right angles to the myofilaments.

Adult mammalian cardiac muscle cells in culture

Adult rat cardiac muscle cells were isolated from the ventricle by a retrograde perfusion technique through the aorta (Nag and Zak, 1979). These single, isolated cardiac muscle cells were cultured for 4 weeks. Throughout the culture period, a small number of muscle cells retained their cylindrical shape, while the rest exhibited alterations in shape and size assuming a flattened body of irregular shape with pseudopodia-like processes and thereby resembling embryonic/neonatal cardiac muscle cells in culture. Transmission electron microscopy revealed that the cylindrical muscle cells contained compactly arranged myofibrils and cellular organelles, similar to those of freshly isolated and in vivo cells. A few irregularly shaped cardiac muscle cells were similar to the cylindrical cells in their internal structural organization. Most of the irregular cells exhibited less myofibrillar content than that of the freshly dissociated and in vivo cells. Myofibrils in the irregular cells were widely spaced and myofilament of some of the myofibrils were loosely bunched. In addition, scattered patches of myofibrils and free myofilaments were observed in many of these cells. The internal structural organization of these irregularly shaped cardiac muscle cells closely resembled the embryonic and neonatal cardiac muscle cells in vitro and in vivo. Most of the muscle cells in culture continued to contract spontaneously, and electron microscope studies clearly indicated that they underwent dedifferentiation. Autoradiography studies demonstrated that the cylindrical and irregularly shaped cardiac muscle cells underwent DNA synthesis and cell division in culture.

Culture of cardiac muscle cells in serum-free media

Cardiac muscle cells from neonatal rats have been cultured in completely defined serum-free media. The most successful system consists of precoating culture flasks with fibronectin at a concentration of 5 μg/cm 2 of surface area and adding fetuin and either dibutyryl cyclic AMP (db-cAMP), cholera toxin, epidermal growth factor or insulin plus dexamethasone to the medium. In order to define a serum- and a hormone or growth factor-free medium, cardiac muscle cells were grown in the presence of fibronectin, fetuin and db-cAMP for 4 days, after which time db-cAMP was omitted from the medium. Under these conditions the cells continue to maintain their differentiated morphology for at least 4 days thereafter. These morphological studies demonstrate that dissociated neonatal cardiac muscle cells are able to grow and differentiate in a chemically defined medium in the absence of animal serum.