代谢性抑制预处理中Na+/Ca2+交换体反向转运的激活触发预处理后的心肌保护作用（英文）

目的研究大鼠心室肌细胞在代谢性抑制预处理中钠/钙交换体（NCX）反向转运的活性,以及NCX反向转运抑制剂是否可以阻止代谢性抑制预处理后的心肌保护作用。方法酶解法分离制备钙耐受心肌细胞,用Fura2/AM负载,采用双激发荧光光电倍增系统（IonOptixPhotometrySystem）检测钙信号,用单心肌细胞动缘探测技术观察心肌细胞收缩/舒张功能,台盼蓝染色法检测细胞存活率。结果在代谢性抑制预处理30min时,NCX反向转运被激活。NCX反向转运抑制剂KBR7943（0.5μmol/L）可以抑制代谢性抑制预处理对心肌细胞收缩功能和细胞存活率的作用。NCX激动剂E4031（1μmol/L）可以模拟代谢性抑制预处理后对心肌细胞收缩功能的保护作用,这一作用也可被KBR7943阻断。结论代谢性抑制预处理中,NCX反向转运的激活触发了代谢性抑制预处理后的心肌保护作用;NCX的抑制剂可以阻止代谢性抑制预处理后的心肌保护作用。     

代谢性抑制预处理中Na^＋／Ca^2＋交换体反向转运的激活触发预处理后的心肌保护作用

目的 研究大鼠心室肌细胞在代谢性抑制预处理中钠／钙交换体（NCX）反向转运的活性，以及NCX反向转运抑制剂是否可以阻止代谢性抑制预处理后的心肌保护作用。方法酶解法分离制备钙耐受心肌细胞，用Fura-2／AM负载，采用双激发荧光光电倍增系统（IonOptix Photometry System）检测钙信号，用单心肌细胞动缘探测技术观察心肌细胞收缩／舒张功能，台盼蓝染色法检测细胞存活率。结果 在代谢性抑制预处理30min时，NCX反向转运被激活。NCX反向转运抑制剂KB-R7943（0．5μmol／L）可以抑制代谢性抑制预处理对心肌细胞收缩功能和细胞存活率的作用。NCX激动剂E4031（μmol／L）可以模拟代谢性抑制预处理后对心肌细胞收缩功能的保护作用，这一作用也可被KB-R7943阻断。结论 代谢性抑制预处理中，NCX反向转运的激活触发了代谢性抑制预处理后的心肌保护作用；NCX的抑制剂可以阻止代谢性抑制预处理后的心肌保护作用。     

代谢抑制处理后大鼠心室肌细胞反向Na+/Ca2+交换体转运功能的变化

目的:利用荧光标记法观察代谢抑制处理后,大鼠心肌细胞反向Na+/Ca2+交换体（NCX）转运功能的变化。方法:酶解法分离制备钙耐受心肌细胞用Fura-2/AM负载,采用双激发荧光光电倍增系统（IonOptix Photom etry Sys-tem）检测钙信号。结果:细胞置于无Na+液后,可见[Ca2+]i逐渐升高,L-型Ca2+通道阻断剂n ifed ip ine在浓度为1μmol/L时,不影响此现象;而NCX的抑制剂N i2+,在浓度为1 mmol/L时,则完全阻断[Ca2+]i的升高。采用20mmol/L乳酸加10 mmol/L脱氧葡萄糖作为代谢抑制物处理心肌细胞不同时间,正常Tyrode液灌流10 m in,之后检测无Na+液引发[Ca2+]i升高效应的变化,发现5 m in处理与对照组无显著性差异,10和30 m in处理后此效应逐渐减弱。结论:首次发现,代谢抑制处理后心肌NCX的反向转运功能被抑制,阐明其调节机制,将为心肌缺血/再灌注损伤的治疗提供新思路。     

依达拉奉保护H9c2心肌细胞对抗化学性低氧引起的损伤

目的探讨新型的自由基清除剂依达拉奉(EDA)能否保护H9c2心肌细胞对抗化学性低氧引起的损伤。方法应用化学性低氧模拟剂氯化钴(CoCl2)处理H9c2心肌细胞以建立化学性低氧损伤模型。应用CCK-8比色法检测细胞存活率;Hoechst 33258核染色法检测凋亡细胞的形态学及数量变化;双氯荧光素(DCFH-DA)染色荧光显微镜照像测定细胞内活性氧(ROS)水平;JC-1染色荧光显微镜照像检测线粒体膜电位(MMP)。结果应用100～1000μmol/L CoCl2处理H9c2心肌细胞24 h,呈浓度依赖性地降低细胞存活率;在12～36 h范围内,800μmol/L CoCl2呈时间依赖性地抑制细胞存活率;10～40μmol/L EDA或500～2000μmol/L N-乙酰半胱氨酸(NAC,为ROS的清除剂)预处理H9c2心肌细胞1 h呈浓度依赖性地对抗CoCl2对细胞存活率的抑制作用;在800μmol/LCoCl2处理H9c2心肌细胞前1 h,应用40μmol/L EDA预处理细胞不仅可明显的抑制CoCl2诱导的细胞内ROS生成增多,还能抑制CoCl2的致细胞凋亡作用及MMP的损伤作用。结论 EDA能保护心肌细胞对抗CoCl2诱导的损伤作用,此心肌细胞保护作用可能与其抗氧化作用及保护MMP有关。     

钠钙交换蛋白与心肌保护

钠钙交换蛋白在心肌细胞内Ca2 浓度的调节中起着重要作用。在心肌缺血再灌注损伤中,钠钙交换蛋白被认为是钙超载的主要路径,从而导致心肌细胞的机械和电活动功能障碍。钠钙交换蛋白抑制剂benzyloxyphenyl衍生物(KB-R7943、SEA0400和SN-6)能特异地、有效地抑制钠钙交换蛋白的Ca2 内流模式,提供良好的心肌保护作用。     

κ阿片受体激动通过钙调神经磷酸酶和胞外信号调节激酶信号抑制异丙肾上腺素诱导的乳鼠心肌肥大

目的探讨κ阿片受体(κ-OR)激动抑制异丙肾上腺素(Iso)诱导的乳鼠心肌细胞肥大的信号转导机制。方法利用体外培养模型,以Iso 10μmol/L诱导心肌细胞肥大,观察κ-OR激动剂U50488 H1μmol/L的作用,并进一步探索在钙调神经磷酸酶(CaN)特异性抑制剂环孢菌素A(CsA)、细胞外信号调节激酶(ERK)抑制剂U0126、L型钙通道阻滞剂维拉帕米及β肾上腺素受体阻断剂普萘洛尔存在的情况下,κ-OR的激活对心肌肥大的影响。Lowry法测心肌细胞蛋白含量;消化分离法及计算机图像分析系统测细胞体积;采用Till阳离子测定系统,以Fura-2/AM为荧光探针,观察胞内[Ca2+]i瞬间变化;Western blot法测CaN、ERK表达。结果U50488 H1μmol/L可以明显抑制Iso诱导的心肌蛋白含量增加、体积增大和胞内[Ca2+]i瞬间变化的增高,其抑制程度与CsA、U0126、维拉帕米及普萘洛尔相似;U50488 H可以抑制Iso诱导的CaN表达增加和ERK磷酸化增加;CaN抑制剂CsA可以抑制Iso诱导的ERK磷酸化增加,ERK抑制剂U0126可以抑制Iso诱导的CaN表达增加。结论κ-OR...     

新型硫化氢供体8L通过上调NF-E2相关因子2的表达拮抗H2O2损伤H9c2心肌细胞

目的探讨新型硫化氢供体8L对氧化应激诱导心肌细胞损伤的保护作用及NF-E2相关因子2(Nrf2)有关的分子机制。方法用活性氧供体过氧化氢(H2O2)处理培养的大鼠H9c2心肌细胞,建立心肌细胞损伤的体外模型以模拟急性缺血再灌注诱导的心肌损伤;在H2O2处理前给予8L预处理观察其对心肌细胞的保护作用。为了明确Nrf2的作用,在H2O2处理或8L预处理前给予其选择性抑制剂鸦胆苦醇预处理。细胞计数试剂盒8比色法检测细胞存活率,试剂盒法检测乳酸脱氢酶(LDH)的释放,罗丹明123染色结合荧光照相术检测线粒体膜电位(MMP),Western Blot法检测核内Nrf2的表达。结果 H9c2心肌细胞经0~600μmol/L H2O2处理6 h可浓度依赖性地降低细胞存活率,且半数有效浓度约为400μmol/L。400μmol/L H2O2处理H9c2心肌细胞6 h可使LDH释放增加,MMP降低,并增加细胞核内Nrf2的表达(P均0.01)。在用400μmol/L H2O2处理前,先用50、100和200μmol/L 8L预处理1 h,可将细胞存活率从(52.6±4.3)%分别提高至(72.5±6.3)%、(83.1±5.2)%和(85.7±4.9)%。200μmol/L 8L预处理1 h还可明显抑制H2O2诱导的LDH释放(P0.01)及MMP受损(P0.05),但可易化H2O2诱导的Nrf2表达上调(P0.01)。另外,10μmol/L鸦胆苦醇预处理1 h不但可加重H2O2诱导的心肌细胞损伤(P0.01),还可拮抗8L的心肌细胞保护作用(P0.01)。结论硫化氢供体8L可减轻氧化应激诱导的H9c2心肌细胞损伤,其机制可能与上调Nrf2有关。     

蛋白激酶C和Ca2+在心肌细胞预处理中的作用

目的：探讨心肌细胞缺氧预处理、蛋白激酶C（PKC）和细胞内钙离子在心肌细胞预处理中的作用。方法：在培养乳鼠心肌细胞缺氧预处理的模型上，观察缺氧预处理以及PKC抑制剂Chelerythrine和钙离子螯合剂BAPTA／AM对缺氧预处理的影响。结果：缺氧预处理可以减少缺氧／复氧对心肌细胞的损伤。PKC抑制剂Chelerythrine和钙离子螯合剂BAPTA／AM可以抑制缺氧预处理的心肌保护作用。结论：PKC和钙离子介导心肌细胞的缺氧预处理。     

阿霉素中毒心肌细胞钙离子调节功能的变化

本文采用Fura-2/AM荧光标记同位素~(45)Ca~(2+)负载示踪法测定了心肌细胞内钙浓度及肌浆网摄钙功能,探讨了阿霉素对心肌细胞内钙调节功能的影响,同时还观察了维拉帕米预处理10min后,阿霉素对心肌细胞内钙浓度及钙调节功能的影响.结果显示,阿霉素作用早期(10min)能够增加心肌细胞外钙的快相内流,使心肌细胞内游离钙浓度增高,肌浆网摄钙量也有轻度增加,阿霉素作用60min后,与对照组相比细胞内~(45)Ca~(2+)总量增加,而肌浆网对~(45)Ca~(2+)的摄取明显减低,细胞浆内游离钙浓度明显升高.维拉帕米预处理组,维拉帕米部分阻断了阿霉素对细胞内钙的调节作用.结果提示,阿霉素通过增加心肌细胞钙内流速度和抑制肌浆网摄钙量,导致心肌细胞钙过负荷,进而影响心肌细胞的收缩舒张功能.     

丝裂素活化蛋白激酶参与心肌缺氧预处理的延迟保护作用

目的　探讨丝裂素活化蛋白激酶 (mitogen- activated protein kinase,MAPK)在心肌缺氧预处理延迟保护中的作用。方法　在培养乳鼠心肌细胞缺氧预处理的模型上 ,检测预处理后即刻、1h、6 h和 12 h的 MAPK活性变化 ,观察细胞缺氧 /复氧损伤后、延迟预处理后及蛋白激酶 C(PKC)抑制剂 chelerythrine(Ch)干预后的细胞存活率、L DH的释放、MDA含量和 SOD活性。结果　 MAPK活性在预处理后即刻明显增加 (P<0 .0 1) ,在 6 h后降至或接近对照水平。与未预处理组心肌细胞缺氧 /复氧损伤相比较 ,预处理后 2 4 h心肌细胞存活率增高 [(5 8.6 4± 5 .5 3) %vs (44 .2 9± 4 .2 7) % ,P<0 .0 1],L DH[(5 9.5 0± 11.0 8) U/ L vs(83.17± 13.6 9) U/ L,P<0 .0 1]和 MDA含量[(2 .33± 0 .4 9) nmol/ L vs(3.2 9± 0 .2 6 ) nmol/ L,P<0 .0 1]均降低 ,SOD活性增加 [(2 1.5 3± 3.6 3) n U/ m l vs(12 .86± 2 .6 8) n U/ ml,P<0 .0 1]。PKC抑制剂 chelerythrine可消除预处理的延迟保护作用。结论　预处理 2 4 h心肌细胞对再次缺氧 /复氧有保护作用 ,PKC、MAPK均参与心肌细胞预处理后的延迟保护作用     

Cardioprotection of preconditioning by metabolic inhibition in the rat ventricular myocyte. Involvement of kappa-opioid receptor.

To determine whether opioid receptors (ORs) are involved in the delayed cardioprotection of ischemic preconditioning (IP), the effect of severe metabolic inhibition (MI) with a glucose-free buffer that contained sodium cyanide and 2-deoxy-D-glucose on the viability of isolated rat ventricular myocytes was first determined 20 hours after preconditioning with a sublethal metabolic inhibition (MIP) with a glucose-free buffer that contained 2-deoxy-D-glucose and lactate for 30 minutes in the presence of OR antagonists. With the use of trypan blue exclusion as an index of cell viability, severe MI killed >60% of the cells and the value increased significantly after MIP. In the presence of 5x10(-6) mol/L nor-binaltorphimine (nor-BNI), a selective kappa-OR antagonist, but not 5x10(-6) mol/L CTOP, a selective mu-OR antagonist, or 5x10(-6) mol/L naltrindole, a selective delta-OR antagonist, the cardioprotection of MIP was significantly attenuated. To verify the role of kappa-OR, we studied the effects of severe MI after pretreatment with the kappa-OR agonist U50,488H (UP) for 30 minutes. U50,488H at 3x10(-6) to 1x10(-4) mol/L increased cell viability concentration-dependently with an EC50 of 3.311x10(-6) mol/L. In the presence of 5x10(-6) nor-BNI, the cardioprotection of UP (3x10(-5) mol/L) was blocked. A time course study showed that UP-induced cardioprotection occurred in 2 windows: the first occurred approximately 1 hour later and the other occurred 16 to 20 hours later. Additional studies on cell contraction and intracellular Ca2+ ([Ca2+]i) revealed that both UP and MIP attenuated the inhibitory effects of severe MI on contractility and electrically induced [Ca2+]i transient in single ventricular myocytes. On blockade of protein kinase C, the delayed cardioprotections of UP and MIP were significantly attenuated. In conclusion, the results of the present study have provided evidence that kappa-OR mediates the cardioprotection of MIP, which may involve protein kinase C and [Ca2+]i.     

KB-R7943对豚鼠心室肌细胞振荡式Na~+/Ca~(2+)交换电流的影响

目的观察Na+/Ca2+交换体阻滞剂KB-R7943(KBR)对豚鼠心室肌细胞振荡式Na+/Ca2+交换电流(INCX)的影响。方法采用全细胞膜片钳技术,利用去极化电压脉冲刺激诱发细胞膜产生振荡式电流,在不同的离子环境下,记录KBR对这一电流的影响。结果该振荡式电流的产生与细胞内钙超载引起的肌质网钙释放有关,它的主要成分是Na+/Ca2+交换电流,与细胞膜的阴离子通道无关。KBR对振荡式INCX的抑制作用具有剂量依赖性,对外向和内向振荡电流的抑制率无差异,两者的半数抑制浓度均约为4μmol/L。结论KBR可抑制振荡式INCX,其抑制作用与实验条件下的离子环境有关,与Na+/Ca2+交换体的运转模式无关。     

Cardiac sodium-calcium exchanger is regulated by allosteric calcium and exchanger inhibitory peptide at distinct sites

The sarcolemmal Na+-Ca2+ exchanger (NCX) is the main Ca2+ extrusion mechanism in cardiac myocytes and is thus essential for the regulation of Ca2+ homeostasis and contractile function. A cytosolic region (f-loop) of the protein mediates regulation of NCX function by intracellular factors including inhibition by exchanger inhibitory peptide (XIP), a 20 amino acid peptide matching the sequence of an autoinhibitory region involved in allosteric regulation of NCX by intracellular Na+, Ca2+, and phosphatidylinositol-4,5-biphosphate (PIP2). Previous evidence indicates that the XIP interaction domain can be eliminated by large deletions of the f-loop that also remove activation of NCX by intracellular Ca2+. By whole-cell voltage clamping experiments, we demonstrate that deletion of residues 562-679, but not 440- 456, 498-510, or 680-685 of the f-loop selectively eliminates XIP-mediated inhibition of NCX expressed either heterologously (HEK293 and A549 cells) or in guinea pig cardiac myocytes. In contrast, by plotting I(NCX) against reverse-mode NCX-mediated Ca2+ transients in myocytes, we demonstrate that Ca2+-dependent regulation of NCX is preserved in Delta562-679, but significantly reduced in the other three deletion mutants. The findings indicate that f-loop residues 562-679 may contain the regulatory site for endogenous XIP, but this site is distinct from the Ca2+-regulatory domains of the NCX. Because regulation of the NCX by Na+ and PIP2 involves the endogenous XIP region, the Delta562-679 mutant NCX may be a useful tool to investigate this regulation in the context of the whole cardiac myocyte.     

Cardiac-specific ablation of the Na+-Ca2+ exchanger confers protection against ischemia/reperfusion injury

During ischemia and reperfusion, with an increase in intracellular Na+ and a depolarized membrane potential, Ca2+ may enter the myocyte in exchange for intracellular Na+ via reverse-mode Na+-Ca2+ exchange (NCX). To test the role of Ca2+ entry via NCX during ischemia and reperfusion, we studied mice with cardiac-specific ablation of NCX (NCX-KO) and demonstrated that reverse-mode Ca2+ influx is absent in the NCX-KO myocytes. Langendorff perfused hearts were subjected to 20 minutes of global ischemia followed by 2 hours of reperfusion, during which time we monitored high-energy phosphates using 31P-NMR and left-ventricular developed pressure. In another group of hearts, we monitored intracellular Na+ using 23Na-NMR. Consistent with Ca2+ entry via NCX during ischemia, we found that hearts lacking NCX exhibited less of a decline in ATP during ischemia, delayed ischemic contracture, and reduced maximum contracture. Furthermore, on reperfusion following ischemia, NCX-KO hearts had much less necrosis, better recovery of left-ventricular developed pressure, improved phosphocreatine recovery, and reduced Na+ overload. The improved recovery of function following ischemia in NCX-KO hearts was not attributable to the reduced preischemic contractility in NCX-KO hearts, because when the preischemic workload was matched by treatment with isoproterenol, NCX-KO hearts still exhibited improved postischemic function compared with wild-type hearts. Thus, NCX-KO hearts were significantly protected against ischemia-reperfusion injury, suggesting that Ca2+ entry via reverse-mode NCX is a major cause of ischemia/reperfusion injury.     

Role of sodium-calcium exchanger in modulating the action potential of ventricular myocytes from normal and failing hearts

Increased Na+-Ca2+ exchange (NCX) activity in heart failure and hypertrophy may compensate for depressed sarcoplasmic reticular Ca2+ uptake, provide inotropic support through reverse-mode Ca2+ entry, and/or deplete intracellular Ca2+ stores. NCX is electrogenic and depends on Na+ and Ca2+ transmembrane gradients, making it difficult to predict its effect on the action potential (AP). Here, we examine the effect of [Na+]i on the AP in myocytes from normal and pacing-induced failing canine hearts and estimate the direction of the NCX driving force using simultaneously recorded APs and Ca2+ transients. AP duration shortened with increasing [Na+]i and was correlated with a shift in the reversal point of the NCX driving force. At [Na+]i > or =10 mmol/L, outward NCX current during the plateau facilitated repolarization, whereas at 5 mmol/L [Na+]i, NCX had a depolarizing effect, confirmed by partially inhibiting NCX with exchange inhibitory peptide. Exchange inhibitory peptide shortened the AP duration at 5 mmol/L [Na+]i and prolonged it at [Na+]i > or =10 mmol/L. With K+ currents blocked, total membrane current was outward during the late plateau of an AP clamp at 10 mmol/L [Na+]i and became inward close to the predicted reversal point for the NCX driving force. The results were reproduced using a computer model. These results indicate that NCX plays an important role in shaping the AP of the canine myocyte, helping it to repolarize at high [Na+]i, especially in the failing heart, but contributing a depolarizing, potentially arrhythmogenic, influence at low [Na+]i.     

Stretch-elicited Na+/H+ exchanger activation: the autocrine/paracrine loop and its mechanical counterpart

The stretch of the cardiac muscle is immediately followed by an increase in the contraction strength after which occurs a slow force increase (SFR) that takes several minutes to fully develop. The SFR was detected in a wide variety of experimental preparations including isolated myocytes, papillary muscles and/or trabeculae, left ventricle strips of failing human myocardium, in vitro isovolumic and in vivo volume-loaded hearts. It was established that the initial increase in force is due to an increase in myofilament Ca2+ responsiveness, whereas the SFR results from an increase in the Ca2+ transient. However, the mechanism(s) for this increase in the Ca2+ transient has remained undefined until the proposal of Na+/H+ exchanger (NHE) activation by stretch. Studies in multicellular cardiac muscle preparations from cat, rabbit, rat and failing human heart have shown evidence that the stretch induces a rise in intracellular Na+ ([Na+]i) through NHE activation, which subsequently leads to an increase in Ca2+ transient via reverse-mode Na+/Ca2+ (NCX) exchange. These experimental data agree with a theoretical ionic model of cardiomyocytes that predicted an increased Na+ influx and a concurrent increase in Ca2+ entry through NCX as the cause of the SFR to muscle stretch. However, there are aspects that await definitive demonstration, and perhaps subjected to species-related differences like the possibility of an autocrine/paracrine loop involving angiotensin II and endothelin as the underlying mechanism for stretch-induced NHE activation leading to the rise in [Na+]i and reverse-mode NCX.     

Calcium entry via Na/Ca exchange during the action potential directly contributes to contraction of failing human ventricular myocytes

Prolongation of the Ca2+ transient and action potential (AP) durations are two characteristic changes in myocyte physiology in the failing human heart. The hypothesis of this study is that Ca2+ influx via reverse mode Na+/Ca2+ exchanger (NCX) or via L-type Ca2+ channels directly activates contraction in failing human myocytes while in normal myocytes this Ca2+ is transported into the sarcoplasmic reticulum (SR) to regulate SR Ca2+ stores. METHODS: Myocytes were isolated from failing human (n=6), nonfailing human (n=3) and normal feline hearts (n=9) and whole cell current and voltage clamp techniques were used to evoke and increase the duration of APs (0.5 Hz, 37 degrees C). Cyclopiazonic acid (CPA 10(-6) M), nifedipine (NIF;10(-6) M) and KB-R 7943 (KB-R; 3x10(-6) M) were used to reduce SR Ca2+ uptake, Ca2+ influx via the L-type Ca2+ current and reverse mode NCX, respectively. [Na+)i was changed by dialyzing myocytes with 0, 10 and 20 mM Na(+) pipette solutions. RESULTS: Prolongation of the AP duration caused an immediate prolongation of contraction and Ca2+ transient durations in failing myocytes. The first beat after the prolonged AP was potentiated by 21+/-5 and 27+/-5% in nonfailing human and normal feline myocytes, respectively (P<0.05), but there was no significant effect in failing human myocytes (+5+/-4% vs. steady state). CPA blunted the potentiation of the first beat after AP prolongation in normal feline and nonfailing human myocytes, mimicking the failing phenotype. NIF reduced steady state contraction in feline myocytes but the potentiation of the first beat after AP prolongation was unaltered (21+/-3% vs. base, P<0.05). KB-R reduced basal contractility and abolished the potentiation of the first beat after AP prolongation (2+/-1% vs. steady state). Increasing [Na+]i shortened AP, Ca2+ transient and contraction durations and increased steady state and post AP prolongation contractions. Dialysis with 0 Na+ eliminated these effects. CONCLUSIONS: Ca2+ enters both normal and failing cardiac myocytes during the late portion of the AP plateau via reverse mode NCX. In (normal) myocytes with good SR function, this Ca(2+) influx helps maintain and regulate SR Ca2+ load. In (failing) human myocytes with poor SR function this Ca2+ influx directly contributes to contraction. These studies suggest that the Ca2+ transient of the failing human ventricular myocytes has a higher than normal reliance on Ca2+ influx via the reverse mode of the NCX during the terminal phases of the AP.     

Inhibition by KB-r7943 of the reverse mode of the Na+/Ca2+ exchanger reduces Ca2+ overload in ischemic-reperfused rat hearts.

Ca2+ influx via the Na+/Ca2+ exchanger (NCX) may lead to Ca2+ overload and myocardial injury in ischemia-reperfusion. Direct evidence that increased cytoplasmic Ca2+ concentration ([Ca2+]i) is mediated by the reverse mode of the NCX is limited, so in the present study the [Ca2+]i dynamics and left ventricular pressure were monitored in perfused beating hearts. The effects of KB-R7943 (KBR), a selective inhibitor of the NCX in the reverse mode, were analyzed during low-Na+ exposure and ischemia-reperfusion. Hearts from Sprague-Dawley rats were retrogradely perfused and loaded with 4 micromol/L fura-2 to measure the fluorescence ratio as an index of [Ca2+]i. To evaluate KBR effects on the reverse mode exchanger, the increase in [Ca2+]i induced by low-Na+ exposure (Na+: 30 mmol/L, 10 mmol/L caffeine pre-treatment) was measured with and without 10 micromol/L KBR (n=5). In another series, the hearts were subjected to 10 min of low-flow ischemia with pacing, followed by reperfusion in the absence (n=6) or in the presence of 10 micromol/L KBR (n=6). Background autofluorescence was subtracted to estimate the ratio in the ischemia-reperfusion protocol. KBR significantly suppressed the increase in [Ca2+]i induced by low-Na+ (40.2 +/- 11.2% of control condition, p=0.014), as well as on increase in diastolic [Ca2+]i during ischemia (% increase from pre-ischemia in [Ca2+]i at 10 min: KBR, 17.9 +/- 6.4%; no KBR, 44.4 +/- 7.7%; p=0.024). After reperfusion, diastolic [Ca2+]i normalized more rapidly in KBR-treated hearts (% increase at 1 min: KBR, 4.5 +/- 7.0%; no KBR, 39.8 +/- 12.2%; p=0.03). Treatment with KBR also accelerated recovery of the rate-pressure product on reperfusion (1 min: KBR, 8,944 +/- 1,554 min(-1) mmHg; no KBR, 4,970 +/- 1,325; p<0.05). Thus, inhibition of the reverse mode exchanger by KBR reduced ischemic Ca2+ overload and possibly improved functional myocardial recovery during reperfusion in a whole heart model.     

Endogenous hydrogen sulfide contributes to the cardioprotection by metabolic inhibition preconditioning in the rat ventricular myocytes

The possible role of hydrogen sulfide (H2S) in cardioprotection was investigated in isolated rat ventricular myocytes exposed to severe metabolic inhibition (MI) in glucose-free buffer containing 2-deoxy-D-glucose (2-DOG), an inhibitor of glycolysis. Pretreatment (30 min) with NaHS (a H2S donor) at concentrations of 10(-5) to 10(-4) mol/L caused a concentration related increase in cell viability and the ratio of rod-shaped cells. A time course study showed that NaHS-induced cardioprotection occurred in 2 time windows (approximately 1 h and 16-28 h). To observe whether endogenous H2S may be involved in the delayed cardioprotection response of IP, DL-propargylglycine (PAG) and beta-cyano-L-alanine (BCA; two inhibitors of H2S biosynthesis) were used. Both drugs significantly attenuated the cardioprotection produced by MI using cell viability, cellular injury index, and electrically-induced [Ca2+]i transients as end-points. These data suggest that endogenous H2S plays an important role in the cardioprotection following MI preconditioning. In an attempt to determine the mechanism of the cardioprotective effect of H2S, we examined the effect of blocking KATP channels with glibenclamide (a non-selective KATP channel blocker), 5-hydroxydecanoic acid (5-HD, a mitochondrial KATP blocker), and HMR-1098 (a sarcolemmal KATP blocker). The cardioprotective effects of NaHS were significantly attenuated by glibenclamide and HMR-1098 treatment but not by 5-HD. Inhibition of NO production with L-NG nitroarginine methyl ester (L-NAME) also attenuated the cardioprotection of NaHS. In conclusion, our findings provide the first evidence that H2S may protect the heart most probably by activating sarcolemmal KATP channels and/or provoking NO release and the cardioprotective effects of metabolic ischemic preconditioning is, at least partially, mediated by endogenous H2S.     

The sarcoplasmic reticulum and the Na+/Ca2+ exchanger both contribute to the Ca2+ transient of failing human ventricular myocytes

Our objective was to determine the respective roles of the sarcoplasmic reticulum (SR) and the Na+/Ca2+ exchanger in the small, slowly decaying Ca2+ transients of failing human ventricular myocytes. Left ventricular myocytes were isolated from explanted hearts of patients with severe heart failure (n=18). Cytosolic Ca2+, contraction, and action potentials were measured by using indo-1, edge detection, and patch pipettes, respectively. Selective inhibitors of SR Ca2+ transport (thapsigargin) and reverse-mode Na+/Ca2+ exchange activity (No. 7943, Kanebo Ltd) were used to define the respective contribution of these processes to the Ca2+ transient. Ca2+ transients and contractions induced by action potentials (AP transients) at 0.5 Hz exhibited phasic and tonic components. The duration of the tonic component was determined by the action potential duration. Ca2+ transients induced by caffeine (Caf transients) exhibited only a phasic component with a rapid rate of decay that was dependent on extracellular Na+. The SR Ca2+-ATPase inhibitor thapsigargin abolished the phasic component of the AP Ca2+ transient and of the Caf transient but had no significant effect on the tonic component of the AP transient. The Na+/Ca2+ exchange inhibitor No. 7943 eliminated the tonic component of the AP transient and reduced the magnitude of the phasic component. In failing human myocytes, Ca2+ transients and contractions exhibit an SR-related, phasic component and a slow, reverse-mode Na+/Ca2+ exchange-related tonic component. These findings suggest that Ca2+ influx via reverse-mode Na+/Ca2+ exchange during the action potential may contribute to the slow decay of the Ca2+ transient in failing human myocytes.