不同中医治法对异丙肾上腺素致大鼠心肌肥大的比较研究

目的观察不同中医治法对异丙肾上腺素致大鼠心肌肥大的干预作用。方法以异丙肾上腺素20 mg/kg1、0 mg/kg、5mg/kg递减皮下注射,3 mg/kg维持7 d制备大鼠心肌肥大模型,给予金匮肾气丸、真武汤、补阳还五汤及生脉饮5周,观察大鼠血流动力学指标、心输出量、心脏指数、左心室指数以及血清心房利钠肽(ANP)、脑钠肽(BNP)、血管紧张素Ⅱ(AngⅡ)及去甲肾上腺素(NE)含量的变化。结果异丙肾上腺素造模5周后,模型组大鼠血流动力学处于代偿状态,与正常组比较差异无统计学意义(P>0.05);但心输出量明显降低,血清ANP、BNP含量显著升高,心脏指数及左心室指数增加(P<0.05)。与模型组比较,补阳还五汤组及金匮肾气丸组能够有效提高心输出量,降低血清ANP、BNP水平,降低心脏指数(P<0.05)。生脉饮组和真武汤组能够降低血清ANP含量,对于模型大鼠心脏指数及血清BNP含量均无明显作用(P>0.05),但是有降低心输出量及心肌收缩功能的作用。结论益气活血法和温补肾阳法能够有效改善异丙肾上腺素致大鼠心肌肥大的血流动力学状况,改善过度激活的神经体液水平。     

心复康口服液对心力衰竭大心功能及心肌中链酰基辅酶A脱氢酶基因表达的影响

目的 观察心复康口服液对心肌梗死后心力衰竭大鼠血流动力学、心功能及梗死周围心肌中链酰基辅酶A脱氢酶基因表达的影响.方法 采用结扎SD大鼠左冠状动脉前降支致心肌梗死后心力衰竭大鼠模型,用心复康口服液于术后24 h灌胃给药至6周,以卡托普利作为阳性对照药.给药4周后观察大鼠心脏功能、血流动力学及梗死周围心肌中链酰基辅酶A脱氢酶基因表达的变化.结果 模型组与假手术对照组比较,左心室舒张末压升高,左心室收缩压、心排血量、左心室内压最大上升速率、左心室内压最大下降速率均显著降低,梗死周围心肌中链酰基辅酶A脱氢酶基因表达下调(P＜0.01).与模型组比较,心复康口服液和卡托普利皆可显著降低心梗后心衰大鼠左心室舒张末压(P＜0.05),升高左心室收缩压、心排血量、左心室内压最大上升速率、左心室内压最大下降速率(P＜0.05或P＜0.01);梗死周围心肌中链酰基辅酶A脱氢酶基因表达上调(P＜0.01).结论 心复康口服液可改善心肌梗死后心力衰竭模型大鼠心脏功能,并可上调心肌中链酰基辅酶A脱氢酶基因表达.     

心复康口服液对心力衰竭大鼠心功能及心肌中链酰基辅酶A脱氢酶基因表达的影响

目的观察心复康口服液对心肌梗死后心力衰竭大鼠血流动力学、心功能及梗死周围心肌中链酰基辅酶A脱氢酶基因表达的影响。方法采用结扎SD大鼠左冠状动脉前降支致心肌梗死后心力衰竭大鼠模型,用心复康口服液于术后24h灌胃给药至6周,以卡托普利作为阳性对照药。给药4周后观察大鼠心脏功能、血流动力学及梗死周围心肌中链酰基辅酶A脱氢酶基因表达的变化。结果模型组与假手术对照组比较,左心室舒张末压升高,左心室收缩压、心排血量、左心室内压最大上升速率、左心室内压最大下降速率均显著降低,梗死周围心肌中链酰基辅酶A脱氢酶基因表达下调(P<0.01)。与模型组比较,心复康口服液和卡托普利皆可显著降低心梗后心衰大鼠左心室舒张末压(P<0.05),升高左心室收缩压、心排血量、左心室内压最大上升速率、左心室内压最大下降速率(P<0.05或P<0.01);梗死周围心肌中链酰基辅酶A脱氢酶基因表达上调(P<0.01)。结论心复康口服液可改善心肌梗死后心力衰竭模型大鼠心脏功能,并可上调心肌中链酰基辅酶A脱氢酶基因表达。     

神经胶质细胞生长因子-1β对慢性心衰大鼠心功能及促血管生成素、大麻素受体、蛋白激酶C表达的影响

目的分析神经胶质细胞生长因子-1β(neuregulin-1β)对慢性心衰大鼠心功能及促血管生成素(Ang2)、大麻素受体(CB1)、蛋白激酶C(PKC)表达的影响。方法将30只雄性SD大鼠随机分成正常组、模型组、neuregulin-1β干预组(each n=10)。采用尾静脉注射阿霉素(20 mg/kg)的方法建立慢性心衰大鼠模型,并通过心脏超声确定模型成功。干预组于建模成功后连续1周每天尾静脉给予neuregulin-1β(10μg/kg·d),正常组给予与模型组等体积的生理盐水。三组大鼠均于实验第9周行大鼠心脏超声及血流动力学检测,取心肌组织进行HE染色进行病理学检测,采用Western blotting法检测三组大鼠心肌组织中PKC蛋白量,采用免疫组织化学法检测三组大鼠心肌组织中Ang2、CB1蛋白相对表达量。结果模型组和neuregulin-1β干预组在第8周心脏超声指标均显示慢性心衰大鼠模型建立成功。neuregulin-1β干预1周后,与模型组比较,干预组的心脏超声指标左心室舒张末期内径、左心室收缩末期内径均显著性降低(P0.05),左室缩短率与左室射血分数均显著性升高(P0.05);血流动力学指标左心室收缩压(LVSP)、左心室内压最大上升速率、左心室内压最大下降速率均显著性升高(P0.05),左心室舒张末期压显著性降低(P0.05);PKC相对蛋白量显著减少(P0.05);Ang2和CB1阳性区域评分明显降低(P0.05);同时病理结果显示,neuregulin-1β干预组较模型组心肌病变较轻,除部分心肌水肿外,未见明显的心肌细胞坏死、炎证浸润及明显的血管充血扩张。结论 neuregulin-1β的干预显著改善大鼠心肌细胞的病变情况,其作用机制可能与下调PKC蛋白及Ang2、CB1的表达有关。     

咪达普利对舒张功能不全大鼠心功能的保护与减轻心肌线粒体损伤有关

目的探讨咪达普利对舒张功能不全心力衰竭大鼠心脏功能保护作用的机制。方法30只雄性SD大鼠,随机分为对照组、模型组、用药1组、用药2组和用药3组共5组。采用腹主动脉缩窄建立舒张功能不全心力衰竭模型,对照组只开腹和分离腹主动脉。3个用药组大鼠术后分别灌胃给予咪达普利1.5 mg/(kg.d)、3或6 mg/(kg.d);对照组和模型组大鼠灌胃给予同等量的生理盐水共4周。4周末心脏B超检测心功能,颈动脉插管记录血流动力学变化,取心脏称重,计算与体重的比例,分光光度计检测线粒体丙二醛、超氧化物歧化酶和谷胱甘肽过氧化物酶变化,电镜检测心肌线粒体超微结构改变。结果心脏超声心动图发现舒张功能不全心力衰竭组大鼠室间隔和左心室后壁厚度、E/A比值明显增高,血流动力学检测发现收缩压、舒张压、左心室收缩压、左心室舒张期末压升高,左心室松弛时间常数延长,左心室内压最大下降速率下降,心脏指数和左心室质量指数增加。咪达普利使增加的室间隔和左心室后壁厚度、E/A、收缩压、舒张压、左心室收缩压、左心室舒张期末压、心脏指数和左心室质量指数降低,左心室松弛时间常数缩短,左心室内压最大下降速率升高,随着咪达普利剂量的增加,改变的程度越明显。线粒体功能检测发现,舒张功能不全心力衰竭组大鼠超氧化物歧化酶和谷胱甘肽过氧化物酶下降,丙二醛增加,电镜观察发现心肌细胞肌丝排列不整齐、线粒体肿胀及空泡化等线粒体损伤;咪达普利减轻线粒体结构和功能损伤。结论咪达普利通过减轻心肌细胞和线粒体损伤改善舒张功能不全心力衰竭大鼠的心脏功能。     

血管紧张素Ⅱ-1型受体拮抗剂对心肌梗死大鼠骨桥蛋白表达及胶原沉积的影响

目的:探讨血管紧张素Ⅱ-1型受体拮抗剂对心肌梗死大鼠骨桥蛋白(OPN)的表达及心肌间质胶原沉积的影响。方法:将心肌梗死后24小时存活大鼠随机分为两组:盐水组(16只,5ml/d),厄贝沙坦组[17只,45mg/(kg·d)];另设假手术组(15只)作对照。分别于心肌梗死后4周:导管法测定左心室有创血流动力学及心功能;组织学方法检测非梗死区胶原纤维沉积和心肌细胞横径;Western blot法检测心肌组织骨桥蛋白表达。结果:盐水组与厄贝沙坦组大鼠梗死面积相似,无显著性差异(P>0.05);假手术组大鼠心肌组织Western blot法未检测到骨桥蛋白表达,盐水组大鼠心肌组织有大量骨桥蛋白表达,该上调的蛋白能被厄贝沙坦治疗显著抑制(P<0.01)。与假手术组相比,所有心肌梗死大鼠均出现显著的心肌间质纤维沉积,左心室相对重量增大,非梗死区心肌细胞横径增加,均有显著性差异(P均<0.01);与盐水组相比,厄贝沙坦组心肌间质纤维沉积减轻,左心室相对重量及非梗死区心肌细胞横径降低,均有显著性差异(P均<0.01)。与假手术组相比,所有心肌梗死大鼠在4周后均表现出左心室收缩压和左心室压力最大上升和下降速率显著下降,左心室舒张末压显著上升,均有显著性差异(P均<0.01),提示了显著的左心室收缩和舒张功能不全;与盐水组相比,厄贝沙坦组大鼠心功能显著改善,均有显著性差异(P均<0.01)。结论:心肌梗死后大鼠心肌组织出现大量骨桥蛋白表达,厄贝沙坦治疗显著抑制心肌梗死大鼠骨桥蛋白的表达,并能改善心肌的纤维化,改善心脏功能。     

心肌梗死大鼠左心室收缩与舒张功能的改变及其影响因素

目的 探讨心肌梗死大鼠左心室收缩和舒张功能的改变、以及心室重构对心室舒缩功能的影响.材料和方法结扎Wistar大鼠左冠状动脉、制成心肌梗死模型,6周后测定左室心肌力学指标,心肌胶原含量、血浆及心肌的血管紧张素Ⅱ(Aug Ⅱ)浓度.结果 心肌梗死组与对照组比较,LVPSP、+dp/dt_(max)、dp/dt_(max)绝对值及V_(max)明显降低(P<0.01),LVEDP增加(P<0.01),T值延长(P<0.01),MAP无差异.心肌梗死组与对照组比较、心肌羟脯氨酸和心肌胶原含量明显增高(P均0.01),心肌AngⅡ含量明显升高(P<0.01)、血浆AngⅡ浓度无显著差异.结论 心肌梗死后左室收缩与舒张　功能明显降低,同时出现心肌细胞的肥大和纤维细胞的增生以及间质纤维化、后者可进一步导致和加重心脏泵血功能的异常.     

盐酸埃他卡林对脑卒中易感型自发性高血压大鼠心功能的影响

目的研究盐酸埃他卡林（Ipt）对大鼠无创心功能参数的影响。方法利用清醒无创心功能血流动力学计算机监测系统,对比研究Ipt对正常血压大鼠和脑卒中易感型自发性高血压大鼠（SHRsp）心率、心脏收缩、舒张和泵血功能的影响。结果Ipt0.5mg/kg静注可降低SHRsp心率、室缩波、抑制心肌收缩力指数,延长左室射血期和心肌电机械收缩时间,降低心输出量,延长左室舒张期。Ipt相同剂量对正常血压大鼠心功能参数却无明显影响。结论Ipt可抑制SHRsp大鼠心脏收缩和泵血功能,延长左室舒张期时间,但不影响正常血压大鼠心脏功能,提示Ipt对心功能的影响与血压状态密切相关。     

胸腺肽β4在大鼠心肌梗死模型中介导心脏功能保护机制

目的探讨胸腺肽β4在大鼠心肌梗死模型中介导心脏功能保护机制。方法选择雌性SD大鼠54只,通过左前降支冠状动脉结扎建立心肌梗死模型,随机分为2组:实验组大鼠腹腔内注射胸腺肽β4(5 mg/kg),对照组大鼠腹腔内注射磷酸盐缓冲液,每组27只。2～4周后心脏超声测量大鼠心功能(左心室收缩末内径、左心室舒张末内径、左心室短轴缩短率、左心室射血分数),Western blot检测梗死区生物活性蛋白因子,并测量心肌梗死面积和心肌收缩速率。结果与对照组比较,实验组大鼠注射胸腺肽β4后,心肌缺血组织中血管生长因子相关蛋白表达明显升高,心脏功能与心肌的收缩速率明显升高,而心肌梗死面积明显缩小,差异有统计学意义(P<0.05)。结论胸腺肽β4保护心脏功能防止心肌缺血后器官功能失调。     

环孢素A对儿茶酚胺诱导的大鼠心肌肥大的作用

目的 观察环孢素A（CaA)对儿茶酚胺诱导的大鼠心肌肥大的作用。方法 雌性Wistar大鼠21只,实验分三组,每组7只：（1）单纯肥大组：给大鼠皮下注射异丙肾上腺素（5mg.kg^-1,d^-1),连续10d:(2)CsA治疗组：除注射异丙肾上腺素外,同时腹腔注射CsA(20mg.kg^-1,d^-1),连续10d;（3）对照组：不作特殊处理。三组大鼠计大小,组织形态,心系数以及心肌组织抑制钙调神经磷酸酶CaN,丝裂素活化蛋白激酶（MAPK）及蛋白激酶C（PKC）活性的变化。在培养的大鼠心肌细胞上,观察CsA对去甲肾上腺素（NE）刺激的^3H－亮氨酸掺入的影响。结果 单纯注射异丙肾上腺素组的大鼠心脏明显增大,心肌细胞肥大,排列紊乱,并出现广泛间质纤维化,CaA治疗组大鼠心脏未明显增大,但仍可见部分心肌纤维化,大鼠心重及心系数明显低于单纯肥大组（P＜0．05）。肥大组大鼠心肌组织CaN活性明显高于对照组（P＜0．05）,CaA治疗组大鼠心肌组织CaN活性低于肥大组（P＜005）,三组大鼠心肌组织MAPK活性差异无显著性,但肥大组大鼠心肌组织PKC活性较对照组增高4倍（P＜0．001）,CsA治疗组的大鼠心肌组织PKC活性较肥大组下降50％（P＜0．001）,CsA可明显抑制NE刺激的大鼠心肌细胞^3H-亮氨酸掺入,结论 CaN信号通中可能以儿茶酚胺诱导的心肌肥大中起一定作用,CsA可阻滞儿茶酚胺诱导的心肌肥大,这种作用可能主要通过抑制CaN及PKC活性,阻断CaN和PKC介导的信号传导通路所致。     

Direct activation of mitochondrial K(ATP) channels mimics preconditioning but protein kinase C activation is less effective in middle-aged rat hearts

OBJECTIVES: This study is aimed to determine whether loss of preconditioning (IP) effects in the middle-aged hearts (MA) is due to the failure of protein kinase C (PKC) activation and, if so, whether direct activation of mitochondrial ATP-sensitive potassium channels (m-K(ATP)) or PKC mimics IP. BACKGROUND: PKC is a mediator and m-K(ATP) may be its downstream effector of IP in young adult hearts (YA), but we have demonstrated that IP is not effective in MA. METHODS AND RESULTS: Isolated hearts from YA (12-week) and MA (50-week) Fischer 344 rats were preconditioned by three cycles of ischemia and reperfusion (5 min each), and the translocation of PKC isoforms and the effects on reperfusion injury were assessed. In some hearts activation of m-K(ATP) or PKC by diazoxide or 1, 2-dioctanoyl glycerol (DOG) was performed before 25 min of global ischemia/30 min of reperfusion. IP could improve the recovery of LV function and resulted in higher content of ATP after reperfusion in YA but these beneficial effects of IP was not found in MA. The effects of IP in YA were abolished by 5-hydroxydecanoate. In YA but not in MA, immunohistochemical analysis revealed that IP translocated PKC-alpha and delta from the cytosolic or membrane to the perinuclear region but immunoblotting analysis showed translocation of PKC-alpha, delta and epsilon to the membrane fraction. Pretreatment with diazoxide or DOG mimicked IP and decreased the creatine kinase release in YA. Diazoxide was also effective but effects of DOG were less in MA as compared with in YA. CONCLUSIONS: IP is not effective in MA hearts partly due to failure of translocation of PKC isoforms. Moreover, less efficacy of PKC activation by DOG as compared with activities of m-K(ATP) by diazoxide in MA may suggest that defect(s) of cell signaling downstream to PKC may also be involved in the loss of IP effects in MA.     

Impaired glucose metabolism in the heart of obese Zucker rats after treatment with phorbol ester

OBJECTIVE: To investigate the influence of obesity on the regulation of myocardial glucose metabolism following protein kinase C (PKC) activation in obese (fa/fa) and lean (Fa/?) Zucker rats. DESIGN: Isolated hearts obtained from 17-week-old lean and obese Zucker rats were perfused with 200 nM phorbol 12-myristate 13-acetate (PMA) for different time periods prior to the evaluation of PKC and GLUT-4 translocation. For metabolic studies isolated hearts from 48 h starved Zucker rats were perfused with an erythrocytes-enriched buffer containing increased concentrations (10-100 nM) of PMA. MEASUREMENTS: Immunodetectable PKC isozymes and GLUT-4 were determined by Western blots. Glucose oxidation and glycolysis were evaluated by measuring the myocardial release of 14CO2 and 3H2O from [U-14C]glucose and [5-3H]glucose, respectively. RESULTS: PMA (200 nM) induced maximal translocation of ventricular PKCalpha from the cytosol to the membranes within 10 min. This translocation was 2-fold lower in the heart from obese rats when compared to lean rats. PMA also induced a significant translocation of ventricular GLUT-4 from the microsomal to the sarcolemmal fraction within 60 min in lean but not in obese rats. Rates of basal cardiac glucose oxidation and glycolysis in obese rats were approximately 2-fold lower than those of lean rats. Perfusion with increasing concentrations of PMA (10-100 nM) led to a significant decrease of cardiac glucose oxidation in lean but not in obese rats. CONCLUSION: Our results show that in the heart of the genetically obese Zucker rat, the impairment in PKCalpha activation is in line with a diminished activation of GLUT-4 as well as with the lack of PMA effect on glucose oxidation.     

Preconditioning with heat shock further improved functional recovery in young adult but not in middle-aged rat hearts

Ischemic preconditioning (PC) improves post-ischemic function, and heat shock (HS) mimics delayed PC in young animals. However, PC is not protective and the consequences of HS are not known in the aging hearts. This report examines the efficacy of HS and its synergy with PC in the middle-aged rat hearts. Hearts from 12- or 50-week-old rats were subjected to PC before 25 min ischemia followed by 30 min reperfusion 48 h after HS. HS induced HS proteins (HSP) in both age groups but that PC and HS translocated PKC-alpha and -delta only in young rats. The beneficial effects of HS and PC were additive and enhanced protein kinase C (PKC) translocation in young rats. However, neither HS alone nor in combination with PC conferred any functional advantage or accelerated PKC translocation in old rats. Similarly neither HS alone nor in combination with PC restore PC effects in old rats with impaired PKC activation, despite the induction of HSP, indicating that induction of HSP is insufficient for cytoprotection.     

Expression of activated PKC epsilon (PKC epsilon) protects the ischemic heart, without attenuating ischemic H(+) production

PKC epsilon is a PKC isoform that translocates during preconditioning and may mediate cardioprotection. To investigate whether PKC epsilon activation is cardioprotective, Langendorff-perfused hearts from wild-type (WT) mice and from mice expressing constitutively active mutant PKC epsilon were subjected to 20 min ischemia and 40 min reperfusion while(31)P NMR spectra were acquired. Pre-ischemic glycogen levels were similar in WT and PKC epsilon hearts. During ischemia, ATP fell less in PKC epsilon than in WT hearts. Ischemic intracellular pH, however, was similar in WT and PKC epsilon hearts. During reperfusion, recovery of contractile function and ATP were greater in PKC epsilon than WT hearts. In conclusion, expression of activated PKC epsilon protected hearts from post-ischemic energetic and contractile dysfunction, consistent with the proposed cardioprotective role of PKC epsilon. Protection occurred in the PKC epsilon hearts without attenuation of ischemic H(+) production, implying that, at least in this ischemic model, reduced acidification during ischemia is not necessary for cardioprotection.     

Altered cardiac endothelin receptors and protein kinase C in deoxycorticosterone-salt hypertensive rats

The aim of the present study was to assess the status of ET-1 receptor subtypes (ET(A)and ET(B)) in ventricular myocytes and fibroblasts and to determine the role of PKC-dependent pathways in ET-1-stimulated cardiac cells in deoxycorticosterone acetate (DOCA)-salt hypertensive rats. Systolic blood pressure and relative heart to body weight were significantly increased in DOCA-salt rats. In unilaterally nephrectomized (Uni-Nx) control rats, more than 90% of cardiomyocyte ET receptors were of the ET(A)subtype, whereas in fibroblasts ET(A)and ET(B)receptors were present in a 1:3 ratio. In DOCA-salt rats, the density of the ET(A)receptor subtype was reduced by 31% in cardiomyocytes and in cardiac fibroblasts only ET(B)receptor density was decreased by 29%. Affinity was unchanged. The relative expression of immunoreactive PKC alpha, gamma and epsilon was significantly increased, whereas PKC delta was not altered in cardiac extracts of DOCA-salt rats. In cardiac fibroblasts from DOCA-salt rats PKC delta was significantly increased and PKC epsilon was not translocated after ET-1 stimulation. The hearts of DOCA-salt hypertensive rats are thus characterized by: (1) decreased density of cardiomyocyte ET(A)receptors and fibroblast ET(B)receptors; (2) cell-specific enhanced expression of some PKC isoenzymes (alpha, gamma, delta and epsilon); and (3) unresponsiveness of PKC epsilon to translocate in the presence of ET-1. Together with alterations of ET-1-induced Ca(2+)handling in cardiac myocytes and fibroblasts, which we previously reported, results from the present study indicate a marked modification of the cardiac ET-1 system of DOCA-salt hypertensive rats.     

Hypoxic preconditioning protects rat hearts against ischemia–reperfusion injury via the arachidonate12-lipoxygenase/transient receptor potential vanilloid 1 pathway

Hypoxic preconditioning (HPC) protects rat hearts against ischemia–reperfusion (IR) injury. However, the role of transient receptor potential vanilloid 1 (TRPV1) in HPC-mediated cardioprotection remains unknown. TRPV1 is activated by endovanilloid 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE], which is synthesized by arachidonate 12-lipoxygenase (ALOX12). Therefore, we examined whether HPC protects the myocardium against IR via the ALOX12/TRPV1 pathway. Compared to hearts of rats kept in room air, the hearts of rats kept in air with 10 % oxygen for 4 weeks had better post-ischemic recovery and less tissue damage when subjected to 30-min global ischemia and 4-h reflow in a Langendorff apparatus. Capsazepine, a specific TRPV1 blocker, administered 5 min before reperfusion markedly attenuated the effects of HPC, confirming that TRPV1 is a downstream effector in HPC-mediated cardioprotection. HPC resulted in the upregulation of ALOX12 and myocardial 12(S)-HETE, and prevented IR-induced 12(S)-HETE reduction. In addition, sarcolemmal ALOX12 expression in HPC hearts mainly co-localized with TRPV1 expression. Blockade of ALOX12 by cinnamyl-3,4-dihydroxy-α-cyanocinnamate or baicalein abrogated the effects of HPC, baicalein also decreased 12(S)-HETE expression. Mimicking HPC by given 12(S)-HETE or capsaicin to baicalien-treated hearts enhanced cardiac recovery during reperfusion. The cardiac protein kinase C (PKC) isoforms α, δ, ε, and ζ were preferentially expressed in the sarcolemmal membrane of HPC-treated hearts, indicating their high intrinsic activation state. Capsazepine or co-treatment with baicalein attenuated translocation of PKCα, PKCδ and PKCε, but not that of PKCζ. We conclude that HPC reduces heart susceptibly to IR via ALOX12/TRPV1/PKC pathway, as shown by increased 12(S)-HETE expression in HPC hearts.     

Alterations in protein kinase A and protein kinase C levels in heart failure due to genetic cardiomyopathy.

BACKGROUND: It is becoming evident that both cardiac and skeletal muscles are affected in congestive heart failure. Although protein kinases are known to regulate cardiac function, very little is known about their status in cardiac and skeletal muscles during the development of congestive heart failure. OBJECTIVE: To determine changes in the activities and protein levels of protein kinase A (PKA) and protein kinase C (PKC) in cardiac and skeletal muscles in congestive heart failure due to genetic cardiomyopathy on the basis that PKA and PKC are crucial for protein phosphorylation. ANIMALS AND METHODS: Genetically cardiomyopathic UM-X7.1 hamsters (250 to 300 days old) and age-matched Syrian hamsters were used in this study. PKA and PKC activities were assayed by measuring 32P from [gamma-32P]ATP incorporated into synthetic substrates. Relative protein contents of these protein kinases were obtained by using immunoblot analysis in control and failing hamster hearts and skeletal muscles. RESULTS: PKC activity was significantly increased in the failing hearts compared with control preparations. The relative protein contents of cytosolic PKC-alpha and -epsilon , and of particulate PKC-epsilon isozymes were significantly increased in failing hearts. PKC activity was also markedly increased in cardiomyopathic skeletal muscle. Furthermore, PKA activity and protein level in both cardiac and skeletal muscles were significantly increased in the failing heart group compared with control values. CONCLUSIONS: Increased PKC activity in heart failure may be due to changes in PKC-alpha and -epsilon isozymes in cardiomyopathic hearts. Alterations of PKA and PKC in congestive heart failure were not limited to the heart because similar changes in enzyme activities were evident in skeletal muscle.     

Increased particulate partitioning of PKC epsilon reverses susceptibility of phospholamban knockout hearts to ischemic injury

Cytosolic Ca(2+) overload is a critical mediator of myocardial damage following cardiac ischemia-reperfusion. It has therefore been proposed that normalization of sarcoplasmic reticulum Ca(2+) cycling through inhibition or ablation of the Ca(2+) ATP-ase inhibitor phospholamban (PLN), which shows promise as a treatment for heart failure, could be beneficial in ischemic heart disease. However, a recent study has shown that globally ischemic PLN-deficient hearts exhibit increased ischemic injury, with impaired contractile, ATP, and phosphocreatine recoveries, compared to wild-type hearts. Since protein kinase C (PKC) family members are widely recognized as mediators of both post-ischemic injury and ischemic preconditioning, we assessed PKC levels in PLN-deficient hearts. Compared to genetically normal hearts, PLN-deficient hearts exhibited diminished particulate partitioning of PKC, a known cardioprotective PKC isoform, without alterations in the levels of membrane-associated PKC delta nor PKC alpha. To determine if decreased particulate partitioning of cardioprotective PKC epsilon was a cause of increased ischemic injury in PLN-deficient hearts, PLN-deficient mice were mated with mice expressing a myocardial-specific PKC epsilon translocation activator peptide, pseudo-epsilon receptor for activated kinase C (psi epsilon RACK). In psi epsilon RACK/PLN knockout (KO) hearts, PKC epsilon translocation to membranous cellular structures was augmented and this was associated with a significant acceleration of post-ischemic contraction and relaxation rates, as well as reduction of creatine phosphokinase release, compared to PLN-deficient hearts. Importantly, post-ischemic functional recovery reached pre-ischemic hyperdynamic values in psi epsilon RACK/PLN KO hearts, indicating super-rescue by the combination of PLN ablation and psi epsilon RACK expression. These findings suggest that diminished PKC epsilon particulate partitioning in PLN-deficient hearts is associated with attenuated contractile recovery upon ischemia-reperfusion and that increased translocation of PKC to membranous cellular structures confers full cardioprotection.     

Sarcalumenin alleviates stress-induced cardiac dysfunction by improving Ca2+ handling of the sarcoplasmic reticulum

AIMS: Sarcalumenin (SAR) is a Ca(2+)-binding protein expressed in the longitudinal sarcoplasmic reticulum (SR) of striated muscle cells. Although its Ca(2+)-binding property is similar to that of calsequestrin, its role in the regulation of Ca(2+) cycling remains unclear. METHODS AND RESULTS: To investigate whether SAR plays an important role in maintaining cardiac function under pressure overload stress, SAR-knockout (SAR-KO) mice were subjected to transverse aortic constriction (TAC). To examine the relation of SAR with cardiac type of SR Ca(2+) pump, SERCA2a, we designed cDNA expression using cultured cells. We found that SAR expression was significantly downregulated in hypertrophic hearts from three independent animal models. SAR-KO mice experienced higher mortality than did wild-type (WT) mice after TAC. TAC significantly downregulated SERCA2a protein but not mRNA in the SAR-KO hearts, whereas it minimally did so in hearts from WT mice. Accordingly, SR Ca(2+) uptake and cardiac function were significantly reduced in SAR-KO mice after TAC. Then we found that SAR was co-immunoprecipitated with SERCA2a in cDNA-transfected HEK293T cells and mouse ventricular muscles, and that SERCA2a-mediated Ca(2+) uptake was augmented when SAR was co-expressed in HEK293T cells. Furthermore, SAR significantly prolonged the half-life of SERCA2a protein in HEK293T cells. CONCLUSION: These findings suggest that functional interaction between SAR and SERCA2a enhances protein stability of SERCA2a and facilitates Ca(2+) sequestration into the SR. Thus the SAR-SERCA2a interaction plays an essential role in preserving cardiac function under biomechanical stresses such as pressure overload.