Difference in the cardioprotective mechanisms between ischemic preconditioning and pharmacological preconditioning by diazoxide in rat hearts.

BACKGROUND: Recent studies have implicated the opening of mitochondrial K(ATP) (mitoK(ATP)) channels and the production of reactive oxygen species (ROS) in the cardioprotective mechanism of ischemic preconditioning (IPC). METHODS AND RESULTS: The involvement of mitoK(ATP) channels and ROS in the cardioprotective effects of both IPC and the mitoK(ATP) channel opener diazoxide (DZ) was investigated in ischemic/reperfused rat hearts. The effects of IPC and DZ on myocardial high-energy phosphate concentrations and intracellular pH (pH(i)) were also examined using (31)P nuclear magnetic resonance spectroscopy. Although both the mitoK(ATP) channel inhibitor 5-hydroxydecanoate and the antioxidant N-acetylcysteine abolished the postischemic recovery of contractile function by DZ, neither of them inhibited that by IPC. IPC attenuated the decline in pHi during ischemia, but DZ did not (6.28+/-0.04 in IPC, p<0.05, and 6.02+/-0.05 in DZ vs 6.02 +/-0.06 in control hearts). DZ, but not IPC, reduced the decrease in ATP levels during ischemia (ATP levels at 20-min ischemia: 26.3+/-3.4% of initial value in DZ, p<0.05, and 8.1+/-3.0% in IPC vs 15.1+/-1.3% in control hearts). CONCLUSIONS: These results suggest that DZ-induced cardioprotection is related to ROS production and reduced ATP degradation during ischemia, whereas attenuated acidification during ischemia is involved in IPC-induced cardioprotection, which is not mediated through mitoK(ATP) channel opening or ROS production.     

Age-related loss of cardiac preconditioning: impact of protein kinase A

Helium induces preconditioning (He-PC) by mitochondrial calcium-sensitive potassium (mK_Ca) channel-activation, but this effect is lost in the aged myocardium. Both, the upstream signalling pathway of He-PC and the underlying mechanisms for an age-related loss of preconditioning are unknown. A possible candidate as upstream regulator of mK_Ca channels is protein kinase A (PKA).We investigated whether 1) regulation of PKA is involved in He-PC and 2) regulation of PKA is age-dependent.Young (2-3 months) and aged (22-24 months) Wistar rats were randomised to eight groups (each n = 8). All animals underwent 25 min regional myocardial ischemia and 120 min reperfusion. Control (Con, Age Con) animals were not further treated. Young rats inhaled 70% helium for 3 × 5min (He-PC). The PKA-blocker H-89 (10 μg/kg) was administered with and without helium (He-PC + H-89, H-89). Furthermore, we tested the effect of direct activation of mK_Ca channels with NS1619. The adenylyl cyclase activator forskolin (For) was administered in young (300 μg/kg) and aged animals (300 and 1000 μg/kg).He-PC reduced infarct size from 60 ± 4% (Con) to 37 ± 10% (p < 0.05). Infarct size reduction was completely abolished by H-89 (58 ± 5%; p < 0.05), but H-89 alone had no effect (57 ± 2%). NS1619 reduced infarct size in the same concentration in both, young and aged rats (35 ± 6%; p < 0.05 vs. Con and 34 ± 8%; p < 0.05 vs. Age Con). Forskolin in a concentration of 300 μg/kg reduced infarct size in young (37 ± 6%; p < 0.05) but not in aged rats (48 ± 13%; n.s.). In contrast, 1000 μg/kg Forskolin reduced infarct size also in aged rats (28 ± 3%; p < 0.05).He-PC is mediated by activation of PKA. Alterations in PKA regulation might be an underlying mechanism for the age-dependent loss of preconditioning.     

Distinct effect of diazoxide on insulin secretion stimulated by protein kinase A and protein kinase C in rat pancreatic islets

Protein kinase activation is known to stimulate glucose-induced insulin secretion in the presence of diazoxide. Diazoxide opens the ATP-sensitive K(+) channel and inhibits FAD-linked glycerophosphate dehydrogenase activity in a concentration-dependent manner. In the present study, we examined the effect of lower (100 microM) and higher (250 microM) concentrations of diazoxide on insulin release by protein kinase A (PKA) and protein kinase C (PKC) activation. Forced depolarization by a high potassium concentration, augmented the intracellular Ca(2+) concentration ([Ca(2+)](i)) similarly in the presence of both concentrations of diazoxide. Under this condition, 250 microM diazoxide inhibited insulin release enhanced by PKA activation but not that by PKC. Under a basal concentration of [Ca(2+)](i), PKC activation elicited glucose-induced insulin secretion at 100 and 250 microM diazoxide, while PKA activation did so only at 100 microM. These augmentations were completely inhibited by mannoheptulose, a glucokinase inhibitor. Glyceraldehyde, in place of glucose, enhanced insulin secretion by PKC activation under both concentrations of diazoxide. On the other hand, it did not affect PKA-stimulated insulin release under either conditions, but in the case of 100 microM, glucose augmented the insulin secretion in the presence of glyceraldehyde and db-cAMP concentration-dependently. These data suggest that insulin release stimulated by PKA and PKC activation under diazoxide is dependent on glucose metabolism, and that a signal derived from proximal steps in glycolysis may be necessary for the secretion by PKA activation.     

Regulation of cardiac L-type calcium channels by protein kinase A and protein kinase C

Voltage-dependent L-type Ca(2+) channels are multisubunit transmembrane proteins, which allow the influx of Ca(2+) (I:(Ca)) essential for normal excitability and excitation-contraction coupling in cardiac myocytes. A variety of different receptors and signaling pathways provide dynamic regulation of I:(Ca) in the intact heart. The present review focuses on recent evidence describing the molecular details of regulation of L-type Ca(2+) channels by protein kinase A (PKA) and protein kinase C (PKC) pathways. Multiple G protein-coupled receptors act through cAMP/PKA pathways to regulate L-type channels. ss-Adrenergic receptor stimulation results in a marked increase in I:(Ca), which is mediated by a cAMP/PKA pathway. Growing evidence points to an important role of localized signaling complexes involved in the PKA-mediated regulation of I:(Ca), including A-kinase anchor proteins and binding of phosphatase PP2a to the carboxyl terminus of the alpha(1C) (Ca(v)1.2) subunit. Both alpha(1C) and ss(2a) subunits of the channel are substrates for PKA in vivo. The regulation of L-type Ca(2+) channels by Gq-linked receptors and associated PKC activation is complex, with both stimulation and inhibition of I:(Ca) being observed. The amino terminus of the alpha(1C) subunit is critically involved in PKC regulation. Crosstalk between PKA and PKC pathways occurs in the modulation of I:(Ca). Ultimately, precise regulation of I:(Ca) is needed for normal cardiac function, and alterations in these regulatory pathways may prove important in heart disease.     

Connexin 43 in cardiomyocyte mitochondria and its increase by ischemic preconditioning

OBJECTIVE: Connexin 43 (Cx43) is involved in infarct size reduction by ischemic preconditioning (IP); the underlying mechanism of protection, however, is unknown. Since mitochondria have been proposed to be involved in IP's protection, the present study analyzed whether Cx43 is localized at mitochondria of cardiomyocytes and whether such localization is affected by IP. METHODS AND RESULTS: Western blot analysis on mitochondrial preparations isolated from rat, mouse, pig, and human hearts showed the presence of Cx43. The preparations were not contaminated with markers for other cell compartments. The localization of Cx43 to mitochondria was also confirmed by FACS sorting (double staining with MitoTracker Red and Cx43) and immuno-electron and confocal microscopy. To study the role of Cx43 in IP, mitochondria were isolated from the ischemic anterior wall (AW) and the control posterior wall (PW) of pig myocardium at the end of 90 min low-flow ischemia without (n=13) or with (n=13) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion. With IP, the mitochondrial Cx43/adenine nucleotide transporter ratio was 3.4+/-0.7 fold greater in AW than in PW, whereas the ratio remained unchanged in non-preconditioned myocardium (1.1+/-0.2, p<0.05). The enhancement of the mitochondrial Cx43 protein level occurred rapidly, since an increase of mitochondrial Cx43 was already detected with two cycles of 5 min ischemia/reperfusion in isolated rat hearts to 262+/-63% of baseline. CONCLUSION: These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.     

缺氧预适应通过蛋白激酶C途径上调心肌细胞促血管生成因子的表达

目的 :研究缺氧预适应 （HPC）对心肌细胞血管内皮生长因子 （VEGF）和碱性成纤维细胞生长因子表达的影响。方法 :建立体外培养的新生大鼠心肌细胞 HPC模型 ,免疫组织化学法结合计算机图像分析 ,研究 HPC对心肌细胞血管内皮生长因子 （VEGF）和碱性成纤维细胞生长因子 （b FGF）表达的影响以及蛋白激酶 C（PKC）在此过程中的作用。结果 :心肌细胞 HPC后 ,VEGF和 b FGF表达显著增多 ,阳性染色吸光度值明显升高。 PKC抑制剂 chel-erythrine chloride（Che）可拮抗 HPC促进心肌细胞表达 VEGF和 b FGF的作用。结论 :HPC可促进心肌细胞VEGF和 b FGF的表达 ,此作用是通过激活 PKC途径介导的     

No evidence for mediation of ischemic preconditioning by alpha1-adrenergic signal transduction pathway or protein kinase C in the isolated rat heart

Summary The purpose of this study was to elucidate the role of activation of the alpha₁-adrenergic signal transduction pathway and of protein kinase C (PKC) in the mechanism of protection of functional recovery by ischemic preconditioning in the isolated perfused rat heart. After a stabilization period, nonpreconditioned and preconditioned isolated perfused rat hearts were subjected to sustained ischemia for 25 and 30 minutes of reperfusion. Preconditioning consisted of three episodes of 5 minutes of ischemia, interspersed with 5 minutes of reperfusion. The endpoint was postischemic functional recovery. The effectiveness of preconditioning in the presence of the alpha₁-adrenergic blocker prazosin, the selective PKC blockers chelerythrine and bisindolylmaleimide (BIM), and the ability of repetitive alpha₁-adrenergic activation to mimic preconditioning were compared with the appropriate nonpreconditioned and preconditioned control groups. Alpha₁-adrenergic blockade with prazosin (3×10^-7 M) during the preconditioning phase did not abolish the protective effect of preconditioning on functional recovery, and repeated intermittent alpha₁-adrenergic activation with phenylephrine in different concentrations (1×10^-8 to 3× 10^-5 M) did not mimic the protective effect of preconditioning. PKC blockade with the selective PKC inhibitors, chelerythrine (10 M) and BIM (4 M), did not abolish the protective effect of preconditioning on functional recovery is isolated perfused rat hearts when given either during the preconditioning phase or shortly before the onset of sustained ischemia. The characteristic metabolic changes of preconditioning during sustained ischemia, namely, energy sparing as manifested in reduced accumulation of lactate, were also not abolished by preconditioning in the presence of selective PKC blockers. We conclude that no evidence could be found for alpha₁-adrenergic or PKC activation in the mechanism of ischemic preconditioning in the isolated rat heart.     

Redox regulation of cardiac protein kinase C

The two extremes of redox stress imposed on cardiac tissue under ischemia and reperfusion change the redox potential of the cells and affect numerous redox-sensitive molecules, including the ones involved in intracellular communication. Protein kinase C (PKC), a key signalling kinase, is one of those subject to redox control. Activation of PKC by oxidation represents a new paradigm of the alternate signalling principle. Reactive oxygen species act directly on PKC, releasing chelated Zn²⁺ ions from the zinc finger of the regulatory domain. Zn²⁺ release from PKC by oxidative stress has been shown at the level of isolated protein fragments, PKC immune complexes and single cells. Zn²⁺ movements have been further characterized in cryosections prepared from adult rat hearts subjected to in vivo stress by global ischemia followed by reperfusion. The morphology of labile zinc in cardiac tissue and zinc release following PKC stimulation with lipid activator are described. The studies lead to an unexpected and intriguing result, suggesting that in addition to serving a structural function, Zn²⁺ ions are likely to play a dynamic regulatory role in PKC. The cysteine-rich domains of the serine/threonine kinases are identified as redox sensors. Thus, being an integrated composite of redox signalling systems, free Zn²⁺ reflects the protein redox status and serves as a valid biomarker of stressed tissue and its capacity to respond to stimuli.     

蛋白激酶C激动剂致心律失常作用机制的研究

目的:观察蛋白激酶C激动剂佛波酯(PMA)对兔室性心律失常的影响并探讨其作用机制。方法:制备左室楔形心肌块灌注模型,随机分为正常对照组(灌流台氏液),PMA组(灌流台氏液+PMA500nmol/L)。采用浮置玻璃微电极法同步记录心内膜、心外膜心肌细胞跨膜动作电位及跨室壁心电图,给予程序性期前刺激诱发室性心动过速的发生。通过免疫印迹法(Western blot)检测心肌细胞缝隙连接蛋白43(Cx43)总量及其S368位点去磷酸化(NP-Cx43S368)蛋白水平的变化。结果:与正常对照组相比,PMA组QT间期显著缩短[(260±25)ms:(302±21)ms,P0.01],T波顶点至终点的间期、T波顶点至终点的间期与QT间期的比值显著增大[(61±13)ms:(51±7)ms、0.24±0.05:0.17±0.02,均P0.01],Cx43的总量、NP-Cx43S368蛋白水平显著减少(分别为P0.05、P0.01);与正常对照组相比,PMA组明显增加了室性心动过速诱发率(70.0%:0%,P0.05)。结论:PMA通过下调心肌缝隙连接的总量及功能从而导致心律失常的发生。     

Remote ischemic preconditioning preserves mitochondrial function and activates pro-survival protein kinase Akt in the left ventricle during cardiac surgery: A randomized trial

Background

Understanding the intracellular mechanisms induced by remote ischemic preconditioning (RIPC) in the human left ventricle opens new possibilities for development of pharmacological cardioprotection against ischemia and reperfusion injury. In this study we investigated the effects of RIPC on mitochondrial function, activation of pro-survival protein kinase Akt and microRNA expression in left ventricular biopsies from patients undergoing coronary artery bypass surgery (CABG).

Methods

Sixty patients were randomized to control (n = 30) or RIPC (n = 30). A blood pressure cuff was applied to the arm of all patients preoperatively. The cuff remained deflated in control group, whereas RIPC was performed by 3 cycles of cuff inflation to 200 mm Hg for 5 min, separated by 5 min deflation intervals. Left ventricular biopsies were obtained before and 15 min after aortic declamping. The primary outcome was mitochondrial respiration measured in situ. Secondary outcomes were activation of protein kinase Akt, assessed by western immunoblotting, and expression of microRNAs assessed by array and real-time polymerase chain reaction.

Results

Mitochondrial respiration was preserved during surgery in patients receiving RIPC (+ 0.2 μmol O₂/min/g, p = 0.69), and reduced by 15% in controls (− 1.5 μmol O₂/min/g, p = 0.02). Furthermore, RIPC activated protein kinase Akt before aortic clamping (difference from control + 43.3%, p = 0.04), followed by increased phosphorylation of Akt substrates at reperfusion (+ 26.8%, p < 0.01). No differences were observed in microRNA expression.

Conclusions

RIPC preserves mitochondrial function and activates pro-survival protein kinase Akt in left ventricle of patients undergoing CABG. Modulation of mitochondrial function and Akt activation should be further explored as cardioprotective drug targets.Clinical Trial Registration: http://www.clinicaltrials.gov, unique identifier: NCT01308138.     

Ischemic preconditioning activates phosphatidylinositol-3-kinase upstream of protein kinase C

The present study is designed to test whether phosphatidylinositol 3-kinase (PI3-kinase) has a role in the signaling pathway in ischemic preconditioning (PC) and whether it is proximal or distal to protein kinase C (PKC). Before 20 minutes of global ischemia, Langendorff-perfused rat hearts were perfused for 20 minutes (control); preconditioned with 4 cycles of 5-minute ischemia and 5-minute reflow (PC); treated with either wortmannin (WM) or LY 294002 (LY), each of which is a PI3-kinase inhibitor, for 5 minutes before and throughout PC; treated with 1,2-dioctanoyl-sn-glycerol (DOG), an activator of PKC for 10 minutes (DOG); treated identically to the DOG group except with WM added 10 minutes before and during perfusion with DOG; or treated with either WM or LY for 25 minutes. Recovery of left ventricular developed pressure (LVDP; percentage of initial preischemic LVDP), measured after 30 minutes of reflow, was improved by PC (72+/-2% versus 36+/-4% in control; P<0.001), and this was blocked by WM and LY (41+/-4% and 43+/-5%, respectively; P<0.05 compared with PC). DOG addition improved postischemic LVDP (67+/-6%; P<0.001 compared with control), but in contrast to its effect on PC, WM did not completely eliminate the protective effect of DOG (52+/-4%; P>0.05 compared with DOG; P<0.05 compared with control). PC induced phosphorylation of protein kinase B and translocation of PKC epsilon, and it increased NO production, and these effects were blocked by WM, which suggests a role for PI3-kinase in PC upstream of PKC and NO.     

Acceleration of crossbridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function

Normal cardiac function requires dynamic modulation of contraction. beta1-adrenergic-induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non-PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, k(rel) (s-1), and delayed force development, k(df) (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline k(rel) and k(df) similar to WT myocardium, but, unlike WT, k(rel) and k(df) were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction.     

Carvedilol protects ischemic cardiac mitochondria by preventing oxidative stress.

Ischemia negatively affects mitochondrial function by inducing the mitochondrial permeability transition (MPT). The MPT is triggered by oxidative stress, which occurs in mitochondria during ischemia as a result of diminished antioxidant defenses and increased reactive oxygen species production. It causes mitochondrial dysfunction and can ultimately lead to cell death. Therefore, drugs able to minimize mitochondrial damage induced by ischemia may prove to be clinically effective. We analyzed the effect of carvedilol, a beta-blocker with antioxidant properties, on mitochondrial dysfunction. Carvedilol decreased levels of TBARS (thiobarbituric acid reactive substances), an indicator of oxidative stress, which is consistent with its antioxidant properties. Regarding cell death by apoptosis, although ischemia did increase caspase-8-like activity, there were no changes in caspase-3-like activity, which is activated downstream of caspase-8; this may indicate that the apoptotic cascade is not activated by 60 minutes of ischemia. We conclude that carvedilol protects ischemic mitochondria by preventing oxidative mitochondrial damage, and, by so doing, it may also inhibit the formation of the MPT pore.     

缺血预适应对兔心脏电生理参数的影响

目的　缺血预适应是指心肌短暂缺血再灌注后 ,使心肌产生适应性反应 ,对随后持续缺血的耐受能力明显增强 ,使持续缺血造成的心肌梗塞范围缩小 ,减轻缺血再灌注心律失常和心功能异常。我们用在体兔心脏缺血预适应模型 ,初步观察了预适应心脏保护的电生理基础 ,探讨其在减少恶性心律失常中的可能作用。方法　将 30只健康家兔随机分成对照组、缺血组、预适应组 ,运用Franz心外膜MAP探针记录技术 ,观察约 40分钟稳定的心电图和MAP图形 ,此后行程序期前刺激测定心室肌有效不应期。结果　缺血组缺血 1分钟内 ,即出现心肌细胞MAPA和dv dtmax明显下降 (P <0 0 1)。此后 ,随缺血时间延长 ,MAP形态逐渐变小。分别于再灌注 5、10和 2 0分钟 ,MAPD5 0 、MAPA和dv dtmax才逐渐恢复接近正常 (P >0 0 5 )。缺血组持续缺血前 5分钟和整个再灌注期 ,MAPD90 和基础值比较变化不大 (P >0 0 5 ) ,而持续缺血 5～ 2 0分钟 ,才发现MAPD90 明显缩短。持续缺血早期 ,ST段逐渐升高 ,但随缺血时间的进一步延长 ,至缺血 15分钟后 ,ST段反而下降。在上述参数动态变化过程中 ,预适应可明显减轻缺血造成的MAPA、dv dtmax下降程度 ,相对延长MAPD5 0 和MAPD90 ,减少ST段升高幅度。各实验组ERP MAPD90 分别为 :C组 1 0 5± 0 0 3;IS组     

多黏菌素B阻断蛋白激酶C活化后的缺血心肌保护作用

目的 通过硫酸多黏菌素B抑制蛋白激酶C(PKC)活化,探讨PKC活化在缺血心肌保护中的作用.方法 将健康家兔30只随机分为缺血组(IS组,10只)、缺血预适应组(IPC组,10只)和硫酸多黏菌素B组(PMB组,10只),按照文献方法建立缺血和预适应模型.用多导电生理记录仪同步记录左室压力数据、曲线及单向动作电位(MAP)图形.结果 各实验组不改变缺血兔缺血和再灌注期心率,IPC组心率和收缩压乘积明显高于其他组.PKC活化对左室收缩功能有明显保护作用,但对左室舒张功能保护作用不明显.IS组、PMB组MAP振幅(MAPA)、除极最大速率(dv/dt max)在缺血期5～20 min明显缩短;IPC组变化不明显,而PMB可以抵消IPC保护作用,导致心肌细胞传导速度延缓和复极离散度增加.PMB并不能增加电交替、室性早搏和心室颤动的发生率.结论 PKC活化可以降低心肌坏死、改善左室收缩功能,但并不能降低电交替、室性早搏和心室颤动的发生,表明IPC抗心肌缺血坏死和抗心律失常分属不同的机制,PKC活化并不参与抗心律失常作用.