Stretch activated ion channels in ventricular myocytes

Patch-clamp recordings from ventricular myocytes of neonatal rats identified ionic channels that open in response to membrane stretch caused by negative pressures (1 to 6 cm Hg) in the electrode. The stretch response, consisting of markedly increased channel opening frequency, was maintained, with some variability, during long (>40 seconds) stretch applications. The channels have a conductance averaging 120 pS in isotonic KCl, have a mean reversal potential 31 mV depolarized from resting membrane potential, and do not require external Ca⁺⁺ for activation. The channels appear to be relatively non-selective for cations. Since they are gated by physiological levels of tension, stretch-activated channels may represent, a cellular control system wherein beat-to-beat tension and/or osmotic balance modulate a portion of membrane conductance.Abbreviations SACs stretch-activated channels - HEPES 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid     

Non-coordinate expression of collagen mRNAs during carbon monoxide-induced cardiac hypertrophy

Collagen gene expression during volume overload-induced cardiac hypertrophy was investigated in adult male rats. Hypertrophy of the left ventricle (22%) and right ventricle (37%) occurred following 27 days continuous exposure to 700 ppm carbon monoxide; hematocrit increased nearly 47%. To examine potential cellular and molecular control of restructuring in the heart, we investigated the expression of two specific procollagen mRNAs for collagen types which have different structural-functional roles [Type I (alpha-1) & Type IV]. Type I (interstitial) mRNA levels increased at least 100% relative to controls within 3 days of initial exposure to 700ppm CO, then declined afterwards; type IV(basement membrane) mRNA levels increased more modestly at first, and increased further afterwards. The ratio of type I/type IV RNA's also increased initially, then later declined, with the greatest differences in the relative responses of type I and IV mRNAs seen in the right ventricle. These data suggest that types I and IV collagen mRNA expression is not coordinately expressed during this type of volume overload-induced hypertrophy in rat heart.     

Stimulation of ribosomal RNA synthesis during hypertrophic growth of cultured heart cells by phorbol ester

Primary cultures of neonatal cardiac myocytes were used to determine the effects of tumor-promoting phorbol esters on ribosomal RNA (rRNA) synthesis during myocyte growth. Treatment of myocytes with phorbol-12,13-dibutyrate (PDBu) increased protein accumulation by 25% and RNA content by 20%. Rates of rRNA synthesis were measured to assess the mechanism by which rRNA accumulated during myocyte growth. Rates of rRNA synthesis were determined from the incorporation of [³H]uridine into UMP of purified rRNA and the specific radioactivity of the cellular UTP pool. After 24h of PDBu treatment, cellular rates of 18S and 28S rRNA synthesis were accelerated by 67% and 64%, respectively. The increased rate of rRNA synthesis accounted for the net increase in myocyte rRNA content after PDBu treatment.     

Sodium-pump activity and its inhibition by extracellular calcium in cardiac myocytes of guinea pigs

Myocardial sodium-pump activity was examined from ouabain-sensitive ⁸⁶Rb⁺ uptake using myocytes isolated from guinea-pig heart. Either sodium loading or the sodium ionophore, monensin, increased ⁸⁶Rb⁺ uptake by over 400%, indicating that the amount of Na⁺ available to the pump is the primary determinant of its activity, and that the sodium pump has a substantial reserve capacity in quiescent myocytes. Moreover, the degree of the above stimulation is markedly higher than corresponding values reported with multicellular preparations, suggesting that diffusion barriers make it impossible to observe the capacity of the sodium pump in the latter preparations. Removal of extracellular Ca²⁺ increased ouabain-sensitive ⁸⁶Rb⁺ uptake, probably by enhancing turnover of the sodium pump rather than increasing availability of Na⁺ to the pump.     

In vitro differentiation of rabbit blastocyst cells

Summary Six- and seven-day post-coitus (p.c.) rabbit embryos have been cultured in an attempt to establish a trophectodermal cell line. Results indicate that cells with epithelial characteristics (i.e. positive staining for cytokeratin) will survive in culture until Passage 3. At that time a fibroblastlike cell becomes predominant. In addition, we have found that the presence of the inner cell mass is required for embryo explants often results in the development of cells that spontaneously contract. These cells stain positively for myosin, which indicates that they may be precardiac cells. Maximum diastolic potential was −59±1.2 mV and the threshold potential was −53±2.3 mV. Spontaneously contracting cells did not respond to atropine, acetylcholine, epinephrine, isoproterenol, or propranolol. Action potential seems to be a result of an inward calcium current, because the beating rate is decreased in a dose-related manner with the calcium channel blocker verapamil, whereas the voltage-sensitive sodium channel blocker tetrodotoxin was without effect. This work was supported by grants HD21302, HD07069, DK31091, and HL37320 from the National Institutes of Health, Bethesda, MD, with additional support from a University of Alabama at Birmingham Cardviovascular Research and Training Center Award.     

内皮衍生舒张因子对缺血（缺氧）,再灌注（复氧）心肌的保护作用

血管内皮产生的内皮衍生舒张因子（ｅｎｄｏｔｈｅｌｉｕｍ－ｄｅｒｉｖｅｄ ｒｅｌａｘｉｎｇ ｆａｃｔｏｒ,ＥＤＲＦ）即一氧化氮（ｎｉｔｒｉｃ ｏｘｉｄｅ,ＮＯ）本工作分别在大鼠Ｌａｎｇｅｎｄｏｒｆｆ离体心脏灌流模型和培养的大鼠心肌细胞上观察了ＮＯ、ＮＯ的前体物质Ｌ－精氨酸（Ｌ－Ａｒｇ）、ＮＯ的前体物质Ｌ－精氨酸（Ｌ－Ａｒｇ）、ＮＯ的合成阻断剂Ｌ－硝基精氨酸（Ｌ－ＮＮＡ）对心肌缺血（缺氧）再灌注（复氧     

Effects of subcultivation and culture medium on differentiation of human fetal cardiac myocytes

Summary Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14–15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin andα-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycinc centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells my provide a useful experimental system for the study of human cardiac muscle cell biology.     

Isolated cardiac myocytes A new cellular model for studying insulin modulation of monosaccharide transport

The usefulness of isolated Ca²⁺-tolerant myocytes as a cellular model system for investigating modulation of monosaccharide transport by insulin was investigated. We have found that the isolation technique described by Haworth et al. (Haworth, R.A., Hunter, D.R. and Berkoff, H.A. (1980) J. Mol. Cell. Cardiol. 12, 715–724), with some minor modifications, consistently gave the highest yield of quiescent, rod-shaped myocytes which maintained their integrity in the presence of 2 mM calcium. Using 3-O-methylglucose, a non-metabolized sugar, transport was shown to possess saturability, substrate stereospecificity, competition and countertransport; all of which have been thoroughly established for d-glucose transport in other systems. The apparent K_m of transport ranged from 2.3 to 3.5 mM. Insulin (10 nM) caused a small but significant increase in K_m and a 2–3-fold increase in V_max. These results suggest that this myocyte preparation will provide a useful model for studying the transport-related effects of insulin as well as current hypotheses regarding the mechanism of insulin modulation of transport at the cellular level.     

T-tubes in cultured mammalian myocardial cells

Summary T-tubes are among the last structural elements of the mammalian myocyte to develop in vivo. We were able to identify T-tubes in early cultures of neonatal rat myocytes. Ventricles were excised from 3- to 4-day-old neonatal rats, incubated overnight in cold trypsin, and treated with sequential changes of collagenase-hyaluronidase. Fractions of cells isolated in this manner were pooled and cultured in plastic petri dishes. In cells prepared for transmission electron microscopy, T-tubes were observed at the cell periphery of cultured myocytes, but were more difficult to identify as the cultures aged and became overgrown by fibroblasts. T-tubes were identified by virtue of their continuity with the sarcolemma, their relatively large diameter, and their regular entry at the level of the Z line. Even at optimal culture ages, T-tubes were not present in every myocyte. At the times T-tubes could be located, myocytes were beating and had begun to establish intercalated discs and gap junctions. The de novo formation of T-tubes in cultured myocytes of neonatal rat heart reflects a duplication of in vivo differentiation by the cultured myocyte. The appropriateness of cultured myocytes in the study of the development and physiology of the heart is emphasized by the in vitro formation of T-tubes.Supported by research grants from the Muscular Dystrophy Association, Inc., The Schlieder Foundation, and USPH-Training Grant HL 07098-04. The authors are indebted to Philip Constantin for assistance in dissociating and culturing heart tissue.     

High-Altitude Pulmonary Hypertension and Signal Transduction in the Cardiovascular System

Estrogen plays a cardioprotective role in female rat hearts subjected to ischemia/reperfusion injury. The its effects are, at least partially, associated with decreased cardiomyocyte contraction and increased expression of β₂-adrenoceptor (β₂-AR). We tested whether β₂-AR could be involved in cardioprotection against ischemic damage and whether the roles of β₂-AR were dependent on estrogenic environment. We first determined the effects of hypoxia/reoxygenation (H/R) on cardiomyocyte shortening in female rats. We then determined the roles of β₂-AR in cardiomyocyte shortening, lactate dehydrogenase (LDH) release in culture medium, and cell death during hypoxia in isolated myocytes from female rats. We further determined the effects of estrogen on the roles of β₂-AR during hypoxia. H/R induced short-term hibernation and stunning at the level of ventricular myocytes from normal female rats. Inhibition of β₂-AR with ICI118,551 significantly elevated adrenergic contractile reserve, myocardial injury, and cell death in normal female rats during hypoxia, whereas ovariectomy (OVX) prominently enhanced myocyte contraction, myocardial injury, and cell death, and deprived the alternations in normal female rats. These changes were restored to normal by estrogen replacement (OVX+E₂). These data suggest that β₂-AR may be involved in the cardioprotection against ischemic damage, and the cardioprotection may depend on estrogenic environment.