Genetic manipulation of calcium-handling proteins in cardiac myocytes. I. Experimental studies

Two genetic experimental approaches, de novo expression of parvalbumin (Parv) and overexpression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), have been shown to increase relaxation rates in myocardial tissue. However, the relative effect of Parv and SERCA2a on systolic function and on beta-adrenergic responsiveness at varied pacing rates is unknown. We used gene transfer in isolated rat adult cardiac myocytes to gain a fuller understanding of Parv/SERCA2a function. As demonstrated previously, when Parv is expressed in elevated concentration (>0.1 mM), the transduced myocytes showed a reduction in sarcomere-shortening amplitude: 129 +/- 17, 81 +/- 8, and 149 +/- 14 nm for control, Parv, and SERCA2a, respectively. At physiological temperature, shortening amplitude responses of Parv and SERCA2a myocytes to the beta-adrenergic agonist isoproterenol (Iso) were not statistically different from that of control myocytes. However, in SERCA2a myocytes, in which baseline was slightly elevated and the Iso-stimulated value was slightly lower, the increase in shortening was slightly less than in Parv or control myocytes: 108 +/- 14, 169 +/- 39, and 34 +/- 12% for control, Parv, and SERCA2a, respectively. In another test set, Parv myocytes had the strongest early postrest potentiation among all groups studied (rest time = 2-10 s), and SERCA2a myocytes were the least sensitive to variations in stimulation rhythm. To replicate the deficient Ca2+ removal observed in heart failure, we used 150 nM thapsigargin. Under these conditions, control myocytes exhibited slowed relaxation, whereas Parv myocytes retained their rapid kinetics, showing that Parv is still able to control relaxation, even when SERCA2a function is impaired.     

收缩活动促进新生大鼠培养心室肌细胞的^3H—亮氨酸...

To determine whether contraction could influence cell growth, the rate of protein synthesis (3H-leucine incorporation) and cell diameter and volume were measured in cultured neonatal rat cardiac myocytes beating spontaneously or arrested by high potassium. In medium supplemented with 10% calf serum, the 3H-leucine incorporation for 24 h in contracting myocytes (CMC) was significantly higher by 14.2% than that in quiescent myocytes (QMC), i.e. 1,229 +/- 29 cpm/10(5) cells vs. 1,076 +/- 60 cpm/10(5) cells (P < 0.01, n = 5 for each group). The cell diameter and cell volume in QMC group were respectively 15.14 +/- 0.42 microns and 1,842 +/- 123 microns3, while in the CMC group the corresponding figures reached to 16.82 +/- 0.64 microns3 and 2,495 +/- 210 microns3, increased by 11.1% and 35.5% respectively (P < 0.01, n = 6 for each group). With prolongation of culture time, the differences in these parameters between CMC and QMC became even more significant. In all these experiments, there was no significant difference in cell number between the two groups (P > 0.05). It is concluded that contraction per se can accelerate protein synthesis and cell growth in neonatal rat ventricular myocardium.     

Effects of antisense oligonucleotides to the cardiac Na+/Ca2+ exchanger on calcium dynamics in cultured cardiac myocytes.

The present study was designed to explore the role of the Na+/Ca2+ exchanger on spontaneous beating of cultured cardiac myocytes. Antisense oligonucleotides (AS) based on the sequence of the cardiac Na+/Ca2+ exchanger were used to decrease expression of this Ca2+ transporting protein in cardiac myocytes. An application of AS (10 microM) caused an increase in beating rate of myocytes within 6-24 h. After 24 h of exposure, AS increased the beating rate from an average rate of 77 beats/min in control and sense-treated myocytes to 103 beats/min. Moreover, myocytes treated for 24 h with 10 microM AS exhibited an increase in diastolic [Ca2+]i levels. The antisense treatment also led to a approximately 20% decrease in expression of Na+/Ca2+ exchanger proteins within 6-24 h. Changes in mRNA levels following AS treatment could not be detected within 3- to 24-h periods. The results of these studies suggest that the Na+/Ca2+ exchanger plays a potentiating role in spontaneous the beating process by regulating [Ca2+]i dynamics and that even a small reduction in the levels of the exchanger protein has marked effects on the handling of [Ca2+]i during the cardiac cycle.     

The effects of CGRP on calcium transients of dedifferentiating cultured adult rat cardiomyocytes compared to non-cultured adult cardiomyocytes: possible protective and deleterious results in cardiac function

CGRP has potent cardiovascular effects but its role in heart failure is unclear. Effects of CGRP on calcium concentrations in fresh adult rat cardiomyocytes, cultured adult cardiomyocytes and neonatal cardiomyocytes were determined by real time fluorescence spectrophotometry. Treatment of cultured adult cardiomyocytes with CGRP resulted in a rapid cessation of beating and a reduction in intracellular calcium. Similar results were obtained in cultured neonatal myocytes. However, rod-shaped adult cardiomyocytes revealed a number of responses; (a) non-beating cells began to beat with increased intracellular calcium; (b) spontaneously beating cells exhibited increased intracellular calcium content and a faster beating rate or (c), myocytes increased their beating rate and became arrhythmic, suggesting that CGRP action on cultured dedifferentiated adult and neonatal myocytes depletes intracellular calcium, whereas in the rod-shaped mature myocytes calcium is retained, pointing to a different mode of action for CGRP on developing and dedifferentiating cardiomyocytes, compared to fully developed cardiomyocytes.     

Differential alpha- and beta-adrenergic responsiveness of beating rat heart myocytes after stationary and non-stationary cultivation

Spontaneously beating myocytes from the heart ventricles of 1 to 4-day old rats were maintained for 8 days in stationary or in rocker culture under otherwise identical conditions and exposed to phenylephrine and isoproterenol for comparison of their positive chronotropic responses to alpha and to beta-adrenoceptor stimulation, respectively. The heart myocytes in rocked culture were more sensitive to phenylephrine and less sensitive to isoproterenol than were the myocytes in stationary culture and they also displayed a greater maximal response to the alpha-agonist. Addition of 1 mM L(+)-lactate to the rocker cultures abolished alpha-adrenergic responsiveness in one third of the cases while persistently increasing beta-adrenergic sensitivity.     

Regulation of atrial natriuretic factor-(99-126) secretion from neonatal rat primary atrial cultures by activators of protein kinases A and C

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a prohormone (ANF-(1-126] and is cosecretionally processed to the circulating ANF-related peptides, ANF-(1-98) and ANF-(99-126). Recently, we have shown that the cosecretional processing of ANF can be replicated in primary cultures of neonatal rat atrial myocytes maintained under serum-free conditions and that glucocorticoids are responsible for supporting this processing activity. Activators of protein kinase C (phorbol esters and alpha-adrenergic agonists) and of protein kinase A (cAMP analogs, forskolin, and beta-adrenergic agonists) were tested for their abilities to alter the rate of ANF secretion from the primary cultures. ANF secretion was stimulated approximately 4-fold after a 1-h incubation of the cultures with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA); maximal release occurred at about 100 nM TPA. Reversed-phase high performance liquid chromatography analysis of secreted material indicated that the cells efficiently cosecretionally processed ANF under both basal and TPA-stimulated conditions. However, incubating the cultures for more than 1 h with TPA resulted in a blunted secretory response to further TPA challenge and a 40-50% decrease in the quantity of ANF in the cells. The alpha-adrenergic receptor agonist phenylephrine was also capable of stimulating ANF secretion by about 4-fold at a half-maximal dose of about 1 microM. Phenylephrine-stimulated ANF secretion was inhibited by the alpha 1-adrenergic antagonist prazosin with half-maximal inhibition occurring at approximately 1 nM. Forskolin, 8-bromoadenosine 3':5'-cyclic monophosphate, and N6-2(1)-O-dibutyryladenosine 3':5'-cyclic monophosphate inhibited basal, TPA- and phenylephrine-stimulated ANF secretion. The beta-adrenergic agonist isoproterenol partially inhibited phenylephrine-stimulated ANF secretion with the maximal effect occurring at 1 nM. These results indicate that ANF secretion from the neonatal rat atrial cultures is enhanced by activators of protein kinase C, and decreased by activators of protein kinase A, and that these secretory effects may be mediated through the actions of alpha- and beta-adrenergic receptors, respectively.     

Developmental differences in L-type calcium current of human atrial myocytes

We investigated differences in L-type Ca2+ current (ICa) between infant (INF, 1-12 mo old), young adult (YAD, 14-18 yr old), and older adult (AD) myocytes from biopsies of right atrial appendages. Basal ICa was smaller in INF myocytes (1.2 +/- 0.1 pA/pF, n = 29, 6 +/- 1 mo old, 11 patients) than in YAD (2.5 +/- 0.2 pA/pF, n = 20, 16 +/- 1 yr old, 5 patients) or AD (2.6 +/- 0.3 pA/pF, n = 19, 66 +/- 3 yr old, 9 patients) myocytes (P < 0.05). Maximal ICa produced by isoproterenol (Iso) was similar in INF, YAD, and AD cells: 8.4 +/- 1.1, 9.6 +/- 1.0, and 9.2 +/- 1.3 pA/pF, respectively. Efficacy (Emax) was larger in INF (607 +/- 50%) than for YAD (371 +/- 29%) or AD (455 +/- 12%) myocytes. Potency (EC50) was 8- to 10-fold higher in AD (0.82 +/- 0.09 nM) or YAD (0.41 +/- 0.14 nM) than in INF (7.6 +/- 3.5 nM) myocytes. Protein levels were similar for Gialpha2 but much greater for Gialpha3 in INF than in AD or YAD atrial tissue. When Gialpha3 activity was inhibited by inclusion of a Gialpha3 COOH-terminal decapeptide in the pipette, basal ICa and the response to 10 nM Iso were increased in INF, but not in YAD, cells. We propose that basal ICa and the response to low-dose beta-adrenergic stimulation are inhibited in INF (but not YAD or AD) cells as a result of constitutive inhibitory effects of Gialpha3.     

Beta-adrenergic regulation of contractility and protein phosphorylation in spontaneously beating isolated rat myocardial cells

Spontaneously beating heart myocytes were prepared from adult rat ventricular tissues to study the correlation between beta-adrenergic receptor-stimulated changes in contractile performance and protein phosphorylation in vitro. The plasma membrane of isolated myocardial cells was permeabilized by saponin in the presence of EGTA and Mg-ATP. The permeabilized myocytes, which formed a homogeneous cell population, retained the rod-cell morphology of heart cells in situ and showed spontaneous cyclic contractions. Their contractile activity in response to extracellularly added cAMP mimicked the effects caused by beta-adrenergic stimulation of the whole heart: both the frequency and longitudinal velocity of free contraction and relaxation of the cells increased. Similar increases were observed when beta-agonist, isoproterenol, and GTP were added to suspending medium. In addition, isoproterenol maximally enhanced the adenylate cyclase activity of the cells in the presence of GTP. Both of these effects of isoproterenol were completely blocked by the beta-antagonist propranolol. cAMP-mediated phosphorylation of proteins in the permeabilized myocytes was investigated under conditions in which the beating frequency increased. cAMP elevated the phosphorylation level of five proteins; three of them with apparent molecular masses of 24, 15, and 12 kDa were membrane proteins and the other two with apparent molecular masses of 150 and 28 kDa were myofibrillar proteins. The 24-kDa phosphoprotein dissociated into 12-kDa molecules when boiled in sodium dodecyl sulfate, suggesting that these proteins are oligomeric and monomeric forms of phospholamban. The phosphorylation of these five proteins was stimulated by isoproterenol. The effect of isoproterenol was enhanced by GTP but completely blocked by propranolol. The time course of their phosphorylation correlated well with that of the increase in the beating frequency of the cells; both were measured after the administration of isoproterenol and GTP. When propranolol was added after the start of the stimulation by isoproterenol, only phospholamban and the 15-kDa protein were rapidly dephosphorylated in close correlation with the decrease of the beating frequency. These results demonstrate for the first time that the permeabilized myocytes retain the functional beta-adrenergic receptor and cellular responses to beta-adrenergic stimulation. They also suggest that cAMP-mediated phosphorylation of proteins, possibly phospholamban and/or the 15-kDa protein, is involved in the increased contractile activity of permeabilized heart cells.     

Influence of sympathetic innervation on the membrane electrical properties of neonatal rat cardiomyocytes in culture

Co-cultures of rat ventricular myocytes and sympathetic neurons were established. Superior cervical ganglia and ventricles from newborn rats were enzymatically dissociated and plated in a culture dish. Experiments were done between the 3rd (when evidence of neuron-myocyte proximity arises) and the 5th day in culture (before the myocytes become confluent). Simultaneous intracellular recording from a cardiomyocyte and an attached neuron was done using conventional microelectrode techniques (resistance of 60-100 Mohm). The myocytes in co-culture were either quiescent or spontaneously contracting. The contracting cells were either latent pacemaker or ventricular-like myocytes. The action potential (AP) characteristics of cardiomyocytes in co-cultures were comparable to those recorded in cardiomyocytes in pure cultures. Sympathetic innervation of the cardiomyocytes in co-cultures was evidenced by stimulating the neuron and observing an increase in rate of beating in latent pacemaker myocytes (average increase of 19.4 +/- 4.6%). In quiescent cardiomyocytes, neural stimulation evoked a slow depolarization that can reach threshold and initiate APs in the cell. This response is similar to slow excitatory postsynaptic potentials (EPSPs) observed in other synapses. Slow ESPSs could also be recorded in spontaneous beating cells, made quiescent by nifedipine (1x10(-6)-1x10(-7) M). These results indicate that functional synaptic contacts are developed in co-culture of sympathetic neurons and cardiac myocytes, and slow EPSPs can be evoked in cardiomyocytes as well as in other excitable cells. The sympathetic innervation occurring in culture did not significantly modify the spontaneous AP characteristics of the cardiomyocytes.     

Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process

The efficacy of cardiac repair by stem cell administration relies on a successful functional integration of injected cells into the host myocardium. Safety concerns have been raised about the possibility that stem cells may induce foci of arrhythmia in the ischemic myocardium. In a previous work (36), we showed that human cord blood CD34(+) cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34(+) cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process. Umbilical cord blood CD34(+) cells were isolated by a magnetic cell sorting method, transduced with a lentiviral vector carrying the enhanced green fluorescent protein (EGFP) gene, and seeded onto primary cultures of spontaneously beating rat neonatal cardiomyocytes. Cocultured EGFP(+)/CD34(+)-derived cells were analyzed for their electrophysiological features at different time points. After 1 wk in coculture, EGFP(+) cells, in contact with cardiomyocytes, were spontaneously contracting and had a maximum diastolic potential (MDP) of -53.1 mV, while those that remained isolated from the surrounding myocytes did not contract and had a depolarized resting potential of -11.4 mV. Cells were then resuspended and cultured at low density to identify EGFP(+) progenitor cell derivatives. Under these conditions, we observed single EGFP(+) beating cells that had acquired an hyperpolarization-activated current typical of neonatal cardiomyocytes (EGFP(+) cells, -2.24 ± 0.89 pA/pF; myocytes, -1.99 ± 0.63 pA/pF, at -125 mV). To discriminate between cell autonomous differentiation and fusion, EGFP(+)/CD34(+) cells were cocultured with cardiac myocytes infected with a red fluorescence protein-lentiviral vector; under these conditions we found that 100% of EGFP(+) cells were also red fluorescent protein positive, suggesting cell fusion as the mechanism by which cardiac functional features are acquired.     

Imaging nanometer domains of beta-adrenergic receptor complexes on the surface of cardiac myocytes

The contraction of cardiac myocytes is initiated by ligand binding to adrenergic receptors contained in nanoscale multiprotein complexes called signalosomes. The composition and number of functional signalosomes within cardiac myocytes defines the molecular basis of the response to adrenergic stimuli. For the first time, we demonstrated the ability of near-field scanning optical microscopy to visualize beta-adrenergic receptors at the nanoscale in situ. On H9C2 cells, mouse neonatal and mouse embryonic cardiac myocytes, we showed that functional receptors are organized into multiprotein domains of approximately 140 nm average diameter. Colocalization experiments in primary cells at the nanometer scale showed that 15-20% of receptors were preassociated in caveolae. These nanoscale complexes were sufficient to effect changes in ligand-induced contraction rate without the requirement for substantial changes in receptor distribution in the cellular membrane. Using fluorescence intensities associated with these nanodomains, we estimated the receptor density within the observed nanometer features and established a lower limit for the number of receptors in the signalosome.     

血管钠肽抑制异丙肾上腺素增强的大鼠心肌细胞钙瞬变

采用光谱荧光法研究血管钠肽(vasonatrin peptide,VNP)对心肌细胞内钙瞬变的作用及其机制,观察钠尿肽鸟苷酸环化酶(guanylate cyclase,GC)受体的特异性阻断剂(HS-142-1)、8-溴-环磷酸鸟苷(8-Br-cGMP)和镁蓝(methylene blue,MB)对心肌细胞内钙瞬变的影响。结果显示,异丙肾上腺素(isoproterenol,Iso)(10~(-10)～10~(-6)mol/L)可剂量依赖性地引起心肌细胞内钙瞬变增强,相对于对照组分别增强(13±8)%(P>0.05)、(26±13)%(P<0.05)、(66±10)%(P<0.01)、(150±10)%(P<0.01)和(300±25)%(P<0.01)。此效应可被β肾上腺素受体阻断剂普萘洛尔(10~(-6)mol/L)所阻断。VNP(10~(-10)～10~(-6)mol/L)可剂量依赖性地抑制Iso(10~(-8)mol/L)引起的心肌细胞内钙瞬变幅值的升高,相对于Iso(10~(-8)mol/L)分别减弱(99±3)%(P>0.05)、(96±2)%(P<0.05)、(84±6)%(P<0.01)、(66±3)%(P<0.01)和(62±3)%(P<0.01)。8-Br-cGMP(10~(-7)～10~(-3)mol/L)也可剂量依赖性地抑制Iso(10~(-8)mol/L)引起心肌细胞内钙瞬变的增强。HS-142-1(2×10~(-5)mol/L)使VNP的作用几乎完全消失。MB是GC的抑制剂,10~(-5)mol/L MB不但使VNP的作用完全消失,而且增强Iso对心肌细胞内钙瞬变的效应。VNP和HS-142-1本身对心肌细胞内钙瞬变无显著影响。而MB使心     

α1-And β-adrenergic and muscarinic-cholinergic regulation in the spontaneous beating and Ca2+ oscillations in cultured neonatal rat cardiac myocytes

α₁-and β-adrenergic and muscarinic-cholinergic regulation in spontaneous beating and Ca²⁺ oscillations in neonatal rat cardiac myocytes at day 6 of culture was investigated. The spontaneous beating in myocytes decreased in the presence of 10 μM norepinephrine (NE). This negative chronotropic action was antagonized by prazosin. Carbachol (CCh) also showed negative chronotropic action which was inhibited by atropine. On the other hand, isoproterenol (ISP) increased the beating rate which was antagonized by propranolol. NE increased inositol phosphate formation whereas CCh and ISP did not. NE and CCh suppressed the frequency of the spontaneous Ca²⁺ oscillations but ISP increased. The present results suggest that α₁-adrenergic and muscarinic receptors regulate chronotropism to be negative whereas β-adrenoceptor regulates chronotropism to be positive in cultured neonatal rat cardiac myocytes.     

Accelerated rates of ribosomal RNA synthesis during growth of contracting heart cells in culture

Contractile activity of neonatal cardiac myocytes stimulated hypertrophic growth as compared with nonbeating cells that were depolarized with 50 mM KCl. Growth of contracting myocytes was associated with an increased rRNA content as measured by the total RNA/DNA ratio. The fractional rates of rRNA synthesis (K8) and rRNA degradation were determined in contracting and nonbeating myocytes to assess their relative contributions in increasing rRNA content during growth. The values for K8 were calculated from the specific radioactivity of 3'-[3H]UMP in 18 and 28 S rRNA after purification by hybridization to cloned rDNA. The cellular [3H]UTP pool served as the precursor for rRNA synthesis in myocytes that were labeled with 50 microM [3H]uridine. K8 values for 18 and 28 S rRNA in contracting myocytes were accelerated by 59 and 53%, respectively, after 3 days as compared with nonbeating myocytes. Calculations of the rate of cellular rRNA synthesis, which took into account the increased content of myocyte rRNA, revealed that synthesis of both 18 and 28 S rRNA was accelerated 2-fold after 2 days of contraction. The derived values for degradation of 18 and 28 S rRNA were increased marginally in contracting myocytes, but cellular rRNA degradation rates averaged 57% higher. The difference between cellular rates of rRNA synthesis and degradation in contracting myocytes accounted for the 30% increase in rRNA content. These data demonstrated that increased rRNA content in contracting myocytes resulted from acceleration of the fractional rate of rRNA synthesis.     

Brachial artery flow-mediated dilation during handgrip exercise: evidence for endothelial transduction of the mean shear stimulus

Exercise elevates shear stress in the supplying conduit artery. Although this is the most relevant physiological stimulus for flow-mediated dilation (FMD), the fluctuating pattern of shear that occurs may influence the shear stress-FMD stimulus response relationship. This study tested the hypothesis that the brachial artery FMD response to a step increase in shear is influenced by the fluctuating characteristics of the stimulus, as evoked by forearm exercise. In 16 healthy subjects, we examined FMD responses to step increases in shear rate in three conditions: stable shear upstream of heat-induced forearm vasodilation (FHStable); fluctuating shear upstream of heat-induced forearm vasodilation and rhythmic forearm cuff inflation/deflation (FHFluctuating); and fluctuating shear upstream of exercise-induced forearm vasodilation (FEStep Increase). The mean increase in shear rate (+/-SD) was the same in all trials (FHFluctuating): 51.69 +/- 15.70 s(-1); FHStable: 52.16 +/- 14.10 s(-1); FEStep Increase: 50.14 +/- 13.03 s(-1) P = 0.131). However, the FHFluctuating and FEStep Increase trials resulted in a fluctuating shear stress stimulus with rhythmic high and low shear periods that were 96.18 +/- 24.54 and 11.80 +/- 7.30 s(-1), respectively. The initial phase of FMD (phase I) was followed by a second, delayed-onset FMD and was not different between conditions (phase I: FHFluctuating: 5.63 +/- 2.15%; FHStable: 5.33 +/- 1.85%; FEStep Increase: 5.30 +/- 2.03%; end-trial: FHFluctuating: 7.76 +/- 3.40%; FHStable: 7.00 +/- 3.03%; FEStep Increase: 6.68 +/- 3.04%; P = 0.196). Phase I speed also did not differ (P = 0.685). In conclusion, the endothelium transduced the mean shear when exposed to shear fluctuations created by a typical handgrip protocol. Muscle activation did not alter the FMD response. Forearm exercise may provide a viable technique to investigate brachial artery FMD in humans.     

Role of nitric oxide in antagonistic effects of transforming growth factor-beta and interleukin-1 beta on the beating rate of cultured cardiac myocytes.

We have recently shown that transforming growth factor-beta (TGF beta) acts in an autocrine manner to maintain the beating rate of neonatal rat cardiac myocytes cultured in serum-free medium on cardiac fibroblast matrix. Interleukin-1 beta (IL-1 beta) suppresses the myocyte-beating rate, and TGF beta antagonizes this effect. We now show that TGF beta and IL-1 beta also have antagonistic effects on the secretion of nitric oxide (NO) by these myocytes, and that NO secretion, the activity of NO synthase (NOS), and expression of the inducible form of NOS correlate inversely with the effects of these two agents on the beating rate. Western blot analysis shows that treatment of myocytes with TGF beta antagonizes the induction of NOS after treatment with IL-1 beta. Release of NO, induced by IL-1 beta, is dependent upon the availability of the substrate, L-arginine, and is suppressed by a competitive inhibitor, NG-monomethyl-L-arginine. L-Arginine (> 0.25 mM) also suppresses, and NG-monomethyl-L-arginine (> 0.5 mM) enhances the myocyte-beating rate. Treatment with IL-1 beta, but not TGF beta, increases cellular cGMP, presumably by activation of guanylate cyclase by NO. Methylene blue, an inhibitor of guanylate cyclase, reverses the suppression of beating caused by IL-1 beta. Bacterial lipopolysaccharide, present in the serum-free medium, is a coinducer of NO secretion. The suppressive effects of NO on the beating rate can be overcome by altering either the set of cytokines employed to induce NO or the matrix on which the myocytes are cultured, demonstrating that additional parameters are also involved in regulation of the beating rate.     

蛋白激酶Cδ可能参与肥大心肌细胞转向凋亡

为了探讨肥大心肌细胞对凋亡刺激的易感性及蛋白激酶Cδ(protein kinase Cδ,PKCδ)在其中的作用,以内皮素-1 (endothelin-1,ET-1)处理原代培养的新生大鼠心肌细胞,诱导心肌细胞肥大;再用血管紧张素Ⅱ(angiotensin II,Ang II)作为细胞凋亡诱导因子,采用鬼笔环肽(phalloidin)荧光染色与细胞面积测量两种方法检测心肌肥大,Hoechst 33258荧光染色检测细胞凋亡。结果显示：(1)1与10 nmol/L ET-1作用48h,心肌细胞肌原纤维排列整齐、染色增浓,随ET-1浓度增加而愈加明显,心肌细胞表面积分别增加42．5%和67．3%,以此作为轻度和中度心肌细胞肥大模型。(2)正常、轻度肥大与中度肥大心肌细胞受1nmol/L AngⅡ处理24h后,凋亡率分别为(15．54±1．32)%、(20．65±1．40)%与(29．33±3．52)%,三组之间有显著差异(P＜0．05)。(3)受AngⅡ刺激后,PKCδ特异性抑制剂rottlerin不影响正常心肌细胞的凋亡率,却有效抑制了轻度和中度肥大心肌细胞的凋亡。肥大心肌细胞凋亡易感性明显高于正常心肌细胞,抑制PKCδ可以抑制肥大心肌细胞凋亡,提示PKCδ参与肥大心肌细胞凋亡过程。     

NADH fluorescence of isolated ventricular myocytes: effects of pacing, myoglobin, and oxygen supply.

Endogenous fluorescence was used to measure the extent of reduction of mitochondrial NAD in individual, isolated rat cardiac myocytes. NAD reduction was determined from emitted fluorescence at 415 and 470 nm during brief epi-illumination at 365 nm. NAD reduction of resting myocytes, superfused with medium equilibrated with 95% O2/5% CO2, was 27 +/- 3% (SE) (n = 78), comparable to that in beating whole heart. Increasing intracellular Ca2+ did not significantly change NAD reduction. NAD reduction decreased reversibly to 11 +/- 1% (n = 78) in contracting myocytes electrically paced at 5 Hz for 10 min. Oxygen uptake was stimulated fivefold. There was minimal change in sarcoplasmic pH measured by fluorescence of carboxy-seminaphthorhodafluor-1. However, NAD reduction increased reversibly in response to electrically paced contractions when: (a) myoglobin was inactivated with sodium nitrite (37 +/- 7%; n = 48); or (b) cells were more densely layered and gassed with 20% O2/5% CO2 (48 +/- 3%; n = 30). We conclude that (a) the ratio NADH/NAD is decreased in well-oxygenated cells with increased work; (b) steady-state NAD reduction is increased with increased work when oxygen delivery is limited; and (c) functional myoglobin ensures an oxygen supply to the mitochondria of working cells.     

Alveolar and lung liquid clearance in anesthetized rabbits

Alveolar and lung liquid clearance were studied over 8 h in intact anesthetized ventilated rabbits by instillation of either isosmolar Ringer lactate (2 ml/kg) or autologous plasma (2 or 3 ml/kg) into one lower lobe. The half time for lung liquid clearance of the isosmolar Ringer lactate was 3.3 h and that for plasma clearance was 6 h. In the plasma experiments, the alveolar protein concentration after 1 h was 5.2 +/- 0.8 g/dl, which was significantly greater than the initial instilled protein concentration of 4.3 +/- 0.7 g/dl (P less than 0.05). Thus alveolar protein concentration increased by 21 +/- 12% over 1 h, which matched clearance from the entire lung of 19 +/- 11% of the instilled volume. Overall the rate of alveolar and lung liquid clearance in rabbits was significantly faster than in prior studies in dogs and sheep. The fast alveolar liquid clearance rate in rabbits was not due to higher endogenous catecholamine release, because intravenous and alveolar (5 x 10(-5) M) propranolol did not slow the clearance. Also, beta-adrenergic therapy with alveolar terbutaline (10(-5) or 10(-4) M) did not increase the alveolar or lung liquid clearance rates. Phloridzin (10(-3) M) did not slow alveolar liquid clearance. However, amiloride (10(-4) M) inhibited 75% of the basal alveolar liquid clearance in rabbits, thus providing evidence that alveolar liquid clearance in rabbits depends primarily on sodium-dependent transport. This rabbit study provides further evidence for important species differences in the basal rates of alveolar liquid and solute clearance as well as the response to beta-adrenergic agonists and ion transport inhibitors.