Troglitazone -induced inhibition of L -type calcium currents is augmented in streptozotocin -induced diabetic rat cardiac ventricular myocytes

Troglitazone -induced inhibition of L -type calcium currents is augmented in streptozotocin -induced diabetic rat cardiac ventricular myocytes     

Expression and regulation of connexins in cultured ventricular myocytes isolated from adult rat hearts

Gap junctions were assayed during re-differentiation of adult rat cardiomyocytes in long-term culture to gain insight into the processes of remodeling. Double immunostaining allowed the localization of connexins Cx40, Cx43, and Cx45 between myocytes and demonstrated co-expression and co-localization in individual cells and gap junction plaques, respectively. Immunoblots showed differential time-dependent changes in connexin expression and phosphorylation. The total amount of connexins and the ratio of phosphorylated/non-phosphorylated isoforms gradually increased during the re-establishment of intercellular communication. Dual voltage-clamp studies showed the involvement of several types of gap junction channels. Multichannel currents yielded diverse spectra of g(j,inst)=f( V(j)) and g(j,ss)=f( V(j)) relationships ( g(j,inst): instantaneous gap junction conductance; g(j,ss): conductance at steady state; V(j): transjunctional voltage), indicative of homotypic and heterotypic channels. Single-channel currents revealed two prominent conductances reflecting gamma(j,main) and gamma(j,residual). The histograms of gamma(j,main) showed four discrete peaks (41-44, 59-61, 70-76, and 100-107 pS) attributable to a combination of Cx45-Cx45, Cx40-Cx45 and Cx43-Cx45 channels (1st peak), Cx43-Cx43 and Cx40-Cx43 channels (2nd peak), Cx43-Cx43 channels (3rd peak) and Cx40-Cx40 and Cx40-Cx43 channels (4th peak). However, the presence of heteromeric channels cannot be excluded. The data are consistent with an up-regulation of Cx45 and Cx43 during re-differentiation.     

Physiological concentration of nitric oxide induces positive inotropic effects through cGMP pathway in isolated rat ventricular myocytes

We used authentic NO or NO from NO donors to show that the physiological levels of NO (<1 microM) induce a positive inotropic effect and demonstrated that the effect is evoked through a cGMP-dependent pathway. In isolated rat ventricular myocytes, authentic NO at 588 nM increased both cell shortening and the intracellular Ca(2+) ([Ca(2+)]i) transient (133 and 117%, respectively; p < 0.05 vs. baseline), and 0.16-1.7 microM NO elicited reproducible dose-dependent increases in cell shortening. NOC18 (0.1 mM: actual NO concentration 673 nM) or SNAP (0.1 mM: actual NO concentration 285 nM) showed similar effects (shortening 215% and [Ca(2+)]i transient 160% increases, and shortening 148% and [Ca(2+)]i transient 117% increases, respectively). The NO-induced increases in cell shortening and the [Ca(2+)]i transient were inhibited by an inhibitor of soluble guanylate cyclase (ODQ, 30 microM) or by an inhibitor of cAMP-dependent protein kinase (KT5720, 0.1 microM). In the presence of an inhibitor of cGMP-inhibited cAMP-phosphodiesterase (milrinone, 10 microM), NO failed to increase both cell shortening and the [Ca(2+)]i transient. These results suggest that physiological levels of NO induce positive inotropy through a cGMP-dependent pathway.     

白细胞介素-2对缺氧/复氧心肌细胞[Ca2+]i的作用

目的:观察白细胞介素-2(IL-2)对心肌细胞在缺氧/复氧过程中电刺激诱导的[Ca²⁺]i的作用。方法:采用酶解分离成年大鼠心室肌细胞化学缺氧模型, 以Fura-2/AM为钙探针, 用细胞内双波长钙荧光系统检测心肌[Ca²⁺]i的变化。结果:①缺氧/复氧过程中, 缺氧5min时, 心肌[Ca²⁺]i幅度降低、舒张末期[Ca²⁺]i升高, [Ca²⁺]i达峰时间(TTP)延长, 恢复时间(RT)延长。复氧10min后, 心肌[Ca²⁺]i幅度、舒张末期[Ca²⁺]i、TTP及RT逐渐回复, 但不能完全恢复到对照水平;②在缺氧期间加入IL-2(2×10⁵U/L), 复氧期间[Ca²⁺]i各参数回复减慢;③用κ-阿片受体拮抗剂nor-BNI(10^-8mol/L)预处理后, 缺氧+IL-2对复氧时[Ca²⁺]i作用的影响被减弱, 而δ-阿片受体拮抗剂纳曲吲哚(10^-6mol/L)预处理则无此作用。结论:缺氧时同时存在IL-2, 可加剧复氧时心肌[Ca²⁺]i的变化, 其机制可能是IL-2通过心肌κ-阿片受体而发挥作用。     

VIP and secretin augment cardiac L-type calcium channel currents in isolated adult rat ventricular myocytes

Vasoactive intestinal peptide (VIP) is colocalized in parasympathetic nerve terminals in the heart and coreleased from these nerve terminals with the “classical” neurotransmitter acetylcholine (Ach). VIP also exerts a positive inotropic effect on the intact heart and enhances adenylyl cyclase activity in isolated heart membranes. Using the whole-cell patch-clamp technique, we show here that VIP enhances Ca²⁺ and Ba²⁺ currents (I _Ba) through voltage-dependent L-type Ca²⁺ channels in adult rat ventricular myocytes. Neither the kinetics nor the voltage-dependent properties of the currents are affected. The effect of VIP on I _Ba is dose dependent with a half-maximal concentration of approximately 0.4 μM. The onset of the effect of VIP and the recovery phase are slow, suggesting the involvement of an intracellular second messenger. The effect of VIP on I _Ba is antagonized by a peptide analog of the growth hormone releasing factor ([Ac-Tyr¹, D-Phe²]-GRF) which belongs to the same peptide family as VIP. Although VIP and the β-adrenergic receptor agonist isoproterenol (ISO) enhance I _Ba peak amplitudes to approximately the same extent, the effect of VIP is not seen on all cells. Only approximately 50% of the isolated myocytes respond to 5 μM VIP, whereas 95% of the cells respond to ISO. Similar results were obtained using the amphotericin B perforated-patch whole-cell-recording technique, suggesting that the variable response to VIP does not reflect the loss of a pivotal intracellular regulator. The gastrointestinal hormone secretin, a peptide structurally related to VIP, also potentiates I _Ba in adult rat ventricular myocytes, although secretin is substantially more potent than VIP (half-maximal concentration for secretin is about 0.7 nM). Taken together, these results suggest that the VIP- (and secretin-) induced potentiation of I _Ba in adult rat ventricular myocytes is mediated through a non-VIP-preferring class of VIP receptors. Received: 7 December 1995/Received after revision and accepted: 31 May 1996     

Mechanisms associated with the negative inotropic effect of deuterium oxide in single rat ventricular myocytes

Deuterium oxide (D2O) is known to cause a negative inotropic effect in muscle although the mechanisms associated with this response in cardiac muscle are not well understood. We studied the effects of D2O in single rat ventricular myocytes in order to characterise the mechanisms associated with its negative inotropic effect and to assess its possible use as an acute modulator of microtubules. D2O rapidly reduced the magnitude of contraction in rat ventricular myocytes, and there was some recovery of contraction in the presence of D2O. Colchicine, an agent known to depolymerise microtubules, did not modify the effect of D2O. D2O decreased the L-type Ca2+ current (ICa), measured under whole cell and perforated patch clamp conditions. Slowing of the time to peak and a delay in inactivation of ICa were observed. Intracellular calcium ([Ca2+]i) and sodium ([Na+]i) were measured using the fluorescent indicators fura-2 and SBFI, respectively. The fall in contraction upon exposure to D2O was not associated with a fall in the [Ca2+]i transient; this response is indicative of a reduction in myofilament Ca2+ sensitivity. Both the [Ca2+]i transient and [Na+]i increased during the partial recovery of contraction in the presence of D2O. We conclude that a decrease in the myofilament sensitivity for Ca2+ and a reduction in Ca2+ influx via ICa are principally responsible for the negative inotropic effect of D2O in cardiac muscle. We found no evidence to explain the negative inotropic effect of D2O in terms of microtubule proliferation. In addition we suggest that acute application of D2O is not a useful procedure for the investigation of the role of microtubules in excitation-contraction coupling in cardiac muscle.     

l-Arginine currents in rat cardiac ventricular myocytes

l -Arginine ( l -Arg) is a basic amino acid that plays a central role in the biosynthesis of nitric oxide, creatine, agmantine, polyamines, proline and glutamate. Most tissues, including myocardium, must import l -Arg from the circulation to ensure adequate intracellular levels of this amino acid. This study reports novel l -Arg-activated inward currents in whole-cell voltage-clamped rat ventricular cardiomyocytes. Ion-substitution experiments identified extracellular l -Arg as the charge-carrying cationic species responsible for these currents, which, thus, represent l -Arg import into cardiac myocytes. This result was independently confirmed by an increase in myocyte nitric oxide production upon extracellular application of l -Arg. The inward movement of Arg molecules was found to be passive and independent of Na²⁺, K²⁺, Ca²⁺ and Mg²⁺. The process displayed saturation and membrane potential ( V _m)-dependent kinetics, with a K _0.5 for l -Arg that increased from 5 m m at hyperpolarizing V _m to 20 m m at +40 mV. l -Lysine and l -ornithine but not d -Arg produced currents with characteristics similar to that activated by l -Arg indicating that the transport process is stereospecific for cationic l -amino acids. l -Arg current was fully blocked after brief incubation with 0.2 m m N -ethylmaleimide. These features suggest that the activity of the low-affinity, high-capacity CAT-2A member of the y²⁺ family of transporters is responsible for l -Arg currents in acutely isolated cardiomyocytes. Regardless of the mechanism, we hypothesize that a low-affinity arginine transport process in heart, by ensuring substrate availability for sustained NO production, might play a cardio-protective role during catabolic states known to increase Arg plasma levels severalfold.     

cGMP facilitates calcium current via cGMP-dependent protein kinase in isolated rabbit ventricular myocytes

 The effect of guanosine 3′,5′-cyclic monophosphate (cGMP) on L-type Ca current (I _Ca) was investigated in a study of rabbit ventricular myocytes using the whole-cell patch-clamp technique. Intracellular application of cGMP (100 μM) increased I _Ca in the absence of isoprenaline or forskolin. 8-Bromo-cGMP (100 μM) and 8-(4-chlorophenylthio)-cGMP (8-pCPT-cGMP, 400 μM), relatively specific stimulators of cGMP-dependent protein kinase (cGMP-PK), also increased I _Ca. The stimulatory effect of 8-pCPT-cGMP was suppressed by Rp-8-chlorophenylthio-cGMP (400 μM), a phosphodiesterase-resistant cGMP-PK inhibitor. When I _Ca was increased by bath application of the non-specific phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX, 100 μM), 8-pCPT-cGMP (400 μM) resulted in additional stimulation of I _Ca. In the presence of 8-pCPT-cGMP, additional applications of isoprenaline (1 μM) or forskolin (1 μM) induced a further increase in I _Ca. From these results, it could be concluded that the activation of cGMP-dependent protein kinase is involved in the facilitation of I _Ca by cGMP in rabbit ventricular myocytes. Received: 17 March 1997 / Received after revision: 28 August 1997 / Accepted: 16 September 1997     

低氧后心肌钙瞬变变化与β-肾上腺素受体脱敏的关系

目的:慢性低氧时,心脏对β-肾上腺素受体激动的反应出现脱敏现象,细胞内钙[Ca²⁺]_i瞬变的幅度降低、时程延长,本研究观察上述变化在低氧时发生和发展的时间过程和对应关系。方法:在年龄对等的正常及慢性低氧1d、3d、1周、2周、3周、4周和8周的大鼠,分别分离正常及慢性低氧条件下的心室肌细胞,以Fura-2为[Ca²⁺]_i的指示剂,用光谱荧光法测定心肌细胞的[Ca²⁺]_i瞬变及其对心肌β-受体激动后反应的变化。结果:在低氧1d、3d、1周等时间内,电刺激引起的[Ca²⁺]_i瞬变、咖啡因引起的[Ca²⁺]_i瞬变及电刺激引起的[Ca²⁺]_i瞬变对β-受体激动剂异丙肾上腺素的增加反应无明显变化;在低氧2周以上时,电刺激引起的[Ca²⁺]_i瞬变的幅度开始降低,而时程开始延长,其对异丙肾上腺素的反应也开始降低。咖啡因引起的[Ca²⁺]_i瞬变幅度也开始降低;在低氧3周和4周时,上述变化程度逐渐加重;在低氧8周时各参数的变化有所恢复,但与4周时的变化程度无明显差异。结论:低氧2-4周时,心脏的β-肾上腺素受体发生脱敏现象,脱敏的机制与3种调节[Ca²⁺]_i瞬变的蛋白质:L-型钙通道、ryanodine受体操纵的钙通道和钙泵等活性的降低有关,后者也可能是低氧时心脏功能降低的重要机制。低氧4-8周时,心脏对低氧产生了适应和代偿。     

Distribution of ryanodine receptors in rat ventricular myocytes

Ryanodine receptors (RyRs) are the major ion channels in the sarcoplasmic reticulum responsible for Ca²⁺ release in muscle cells. Localization of RyRs is therefore critical to our understanding of Ca²⁺ cycling and Ca²⁺-dependent processes within ventricular cells. Recently, RyRs were reportedly found in non-classical locations in the middle of the sarcomere, between perinuclear mitochondria and in the inner mitochondrial membrane of cardiac mitochondria. However, for multiple reasons these reports could not be considered conclusive. Therefore, we modified immunogold labeling to visualize the distribution of RyRs in ventricular myocytes. Using antibodies to the voltage-dependent anion channel (i.e. VDAC) or cytochrome c along with our labeling method, we showed that these mitochondrial proteins were appropriately localized to the mitochondrial outer and inner membrane respectively. Immunogold labeling of ultrathin sections of intact and permeabilized ventricular myocytes with antibodies to three types of RyRs confirmed the existence of RyRs between the Z-lines and around the perinuclear mitochondria. However, we did not find any evidence to support localization of RyRs to the mitochondrial inner membrane.     

Influence of genetically predisposed diabetes on ethanol-induced depression of cardiac contraction in adult rat ventricular myocytes

Diabetes mellitus and alcohol (ethanol) intake are two positively correlated major risk factors for cardiovascular abnormalities. However, the interaction of the two on cardiac function is largely unknown. The purpose of the present study was to examine the impact of genetically predisposed diabetes on acute ethanol exposure-induced cardiac contractile depression at the myocyte level. Ventricular myocytes from spontaneously biobreeding diabetes-prone (BBDP) rats and their diabetes-resistant littermates (BBDR) were stimulated to contract at 0.5 Hz. Contractile properties analysed include: peak shortening amplitude (PS), time-to-PS (TPS), time-to-90 % relengthening (TR(90)) and maximal velocities of shortening/relengthening (+/- dL/dt). BBDP rats displayed hyperglycaemia, reduced body weight gain and increased cardiac, hepatic and renal size. Myocytes isolated from BBDP rat hearts exhibited prolonged TPS and TR(90) associated with normal PS and +/- dL/dt, compared with myocytes from the BBDR group. Acute ethanol exposure (80-640 mg dl(-1)) caused a concentration-dependent inhibition of PS in both BBDR and BBDP myocytes. However, the degree of inhibition of PS was significantly reduced in BBDP myocytes compared to that of BBDR myocytes. The maximal inhibition was 52.9 % and 28.4 % in BBDR and BBDP groups, respectively. Ethanol significantly depressed +/- dL/dt in both BBDR and BBDP myocytes. In addition, ethanol did not affect TPS or TR(90) in either the BBDR or BBDP group. Collectively, these results suggest that the ethanol-induced depression in cardiac myocyte contraction may be 'shadowed' by genetically predisposed diabetes.     

Rapid electrical stimulation induces early activation of kinase signal transduction pathways and apoptosis in adult rat ventricular myocytes

Chronic tachycardia in patients and rapid pacing in animal models induce myocardial dysfunction and initiate a cascade of compensatory adaptations that are ultimately unsustainable, leading to ventricular enlargement and failure. The molecular pathogenesis during the early stages of tachycardia-induced cardiomyopathy, however, remains unclear. We utilized our previously reported cell culture pacing system to directly assess phosphatidylinositol-3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signalling of adult rat ventricular myocytes (ARVM) in response to rapid electrical stimulation. Freshly isolated ARVMs were maintained quiescent (0 Hz), or continuously stimulated at 5 (normofrequency) and 8 Hz (rapid frequency). Pacing resulted in an increase in mitochondrial respiration, assessed by mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 48 h. Rapid pacing at 8 Hz significantly increased cell injury and death as assessed by Trypan Blue uptake, creatine phosphokinase release, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Pacing at 5 Hz induced early, but weak, activation of Akt and protein kinase 38 (p38). Rapid pacing further augmented the early activation of Akt and p38, and induced extracellular signal-related kinase (Erk) and c-jun amino terminal kinase (JNK) activation. Incubation of ARVM with PI3K inhibitor LY294002 resulted in a twofold increase of TUNEL-positive cells under all pacing conditions examined. In conclusion, rapid pacing has immediate and detrimental consequences for cardiomyocyte survival, with pro-apoptotic pathways (e.g. JNK, p38) able to overwhelm antiapoptotic signalling (PI3K/Akt, Erk). The rapid pacing methodology described in this report will be particularly useful in determination of cell signalling pathways associated with tachycardia-induced cardiomyopathy.     

The dual effects of ouabain on45Ca2+ transport and contractility in adult rat ventricular myocytes

We used isolated ventricular myocytes to study⁴⁵Ca²⁺ transport in the presence of three concentrations of ouabain (10 nM, 1 M, and 100 M) in Tyrode solution containing 1 mM CaCl₂. The cells were quiescent and during⁴⁵Ca²⁺ uptake and⁴⁵Ca²⁺ efflux experiments 10 nM ouabain decreased Ca²⁺ content, 1 M, didn't change it appreciably, and 100 M increased it significantly. Qualitatively, the same results were obtained at 22°C and 35°C. Ouabain did not significantly affect the electrical activity of isolated, electrically stimulated myocytes, but it increased the amplitude of shortenings of these myocytes in a dose-dependent manner. Thus, the positive inotropic effect of ouabain at therapeutic doses (10 nM) occurs in spite of decreased Ca²⁺ content, while at high toxic doses the positive inotropic effect is accompanied by an increment in Ca²⁺ content. These data support the hypothesis that the mechanisms of positive inotropy of ouabain are different at therapeutic and toxic concentrations of this drug. Finally, our study demonstrates that the effects of low doses of ouabain are independent of the release of endogenous catecholamines.     

K(+) current distribution in rat sub-epicardial ventricular myocytes

In the rat ventricle, the transient outward K(+) current ( I(TO)) is carried by heteromeric channels composed of Kv4.2 and Kv4.3. However its distribution in the cell membrane is unclear: immunohistochemical studies of Kv4.2 distribution in the cardiac ventricular cell membrane have given equivocal results, and there are no corresponding studies of Kv4.3. We therefore used detubulated cardiac cells to investigate the functional distribution of I(TO) between the t-tubules and surface membrane. I(TO), the delayed rectifier ( I(K)), the inward rectifier ( I(K1)) and steady-state ( I(SS)) K(+) currents were monitored using the patch-clamp technique in control and formamide-treated (detubulated) cells from rat left ventricular sub-epicardium. Formamide treatment decreased cell capacitance by 20%, did not significantly change the density of I(TO), I(K) or I(K1) but decreased the density of I(SS) and L-type Ca current ( I(Ca)). These data suggest that I(TO), I(K), and I(K1) are uniformly distributed between the surface and t-tubule membranes, but that I(SS) and I(Ca) are concentrated in the t-tubules.     

Transformation of adult rat cardiac myocytes in primary culture

We characterized the morphological, electrical and mechanical alterations of cardiomyocytes in long-term cell culture. Morphometric parameters, sarcomere length, T-tubule density, cell capacitance, L-type calcium current (I(Ca,L)), inward rectifier potassium current (I(K1)), cytosolic calcium transients, action potential and contractile parameters of adult rat ventricular myocytes were determined on each day of 5 days in culture. We also analysed the health of the myocytes using an apoptotic/necrotic viability assay. The data show that myocytes undergo profound morphological and functional changes during culture. We observed a progressive reduction in the cell area (from 2502 +/- 70 microm(2) on day 0 to 1432 +/- 50 microm(2) on day 5), T-tubule density, systolic shortening (from 0.11 +/- 0.02 to 0.05 +/- 0.01 microm) and amplitude of calcium transients (from 1.54 +/- 0.19 to 0.67 +/- 0.19) over 5 days of culture. The negative force-frequency relationship, characteristic of rat myocardium, was maintained during the first 2 days but diminished thereafter. Cell capacitance (from 156 +/- 8 to 105 +/- 11 pF) and membrane currents were also reduced (I(Ca,L), from 3.98 +/- 0.39 to 2.12 +/- 0.37 pA pF; and I(K1), from 34.34p +/- 2.31 to 18.00 +/- 5.97 pA pF(-1)). We observed progressive depolarization of the resting membrane potential during culture (from 77.3 +/- 2.5 to 34.2 +/- 5.9 mV) and, consequently, action potential morphology was profoundly altered as well. The results of the viability assays indicate that these alterations could not be attributed to either apoptosis or necrosis but are rather an adaptation to the culture conditions over time.     

Measurement of calcium entry and exit in quiescent rat ventricular myocytes

The aim of this work was to obtain the first quantitative measurements of Ca2+ influx and efflux in quiescent cardiac cells. The relationship between free and total Ca2+ was obtained during a caffeine application. This buffering curve was then used to calculate changes of total Ca2+ from measurements of free cytosolic [Ca2+] ([Ca2+]i) made with Indo-1. The rate of Ca2+ removal from the cytoplasm was calculated by differentiating total Ca2+ with respect to time. The dependence of d(total Ca2+)/dt on [Ca2+]i was hyperbolic. Inhibition of either Na+-Ca2+ exchange (by addition of 10 mmol l(-1) NiCl2 or removal of external Na+) or the sarcolemmal Ca2+-activated adenosine triphosphatase (Ca2+-ATPase) (with carboxyeosin) decreased the calculated efflux. In both cases, the main effect was on the apparent maximum rate (Vmax) with little effect on the Michaelis-Menten constant (Km). These results suggest that the Na+-Ca2+ exchange and Ca2+-ATPase have very similar affinities for [Ca2+]i and that their fractional contributions do not change over the systolic range of [Ca2+]i. Ca2+ influx was quantified in two ways. The first method was to extrapolate the curve relating Ca2+ efflux to [Ca2+]i to zero [Ca2+]i. This gave a value of 4.49+/-0.54 micromol l(-1) s(-1) which was reduced to zero by either removal of external Ca2+ or addition of Ni2+. In other experiments external Ca2+ was removed and the maximum rate of fall of total Ca2+ calculated as 2.53+/-0.93 micromol l(-1) s(-1). This approach can be used to provide a quantitative analysis of the control of resting [Ca2+]i.     

负压创面疗法对慢性创面肉芽组织生长及白细胞介素-6的影响

目的研究负压创面疗法对慢性创面血管内皮细胞、增生期细胞及白细胞介素-6的影响。方法将40例慢性创面患者随机分为负压治疗组与常规治疗组。观察两组创面肉芽组织生长情况,并于治疗后1、4、7、14d取创面组织进行HE染色和免疫组化染色,观察血管内皮细胞（标志物采用兔抗人八因子抗原）和增生期细胞（标志物采用鼠抗人Ki-67抗原）数目,负压治疗组于上述时间点收集创面引流液,应用酶联免疫吸附试验法测定自细胞介素-6的含量。结果负压治疗组患者的治疗时间明显缩短,肉芽组织生长迅速,内皮细胞及增生期细胞数较常规治疗组显著增高（P〈0．05）,负压治疗组白细胞介素石迅速下降。结论负压创面疗法能促进创面肉芽组织生长,加速内皮细胞生成,刺激细胞增生,降低创面白细胞介素-6含量。