Carbon Monoxide Levels Experienced by Heavy Smokers Impair Aerobic Capacity and Cardiac Contractility and Induce Pathological Hypertrophy

Cigarette smoke contains hundreds of potentially toxic compounds and is an important risk factor for cardiovascular disease. However, the key components responsible for endothelial and myocardial dysfunction have not been fully identified. The objective of the present study was to determine the cardiovascular effects of long-term inhalation of carbon monoxide (CO) administrated to give concentrations in the blood similar to those observed in heavy smokers. Female rats were exposed to either CO or air (control group) (n = 12). The CO group was exposed to 200 ppm CO (100 h/wk) for 18 mo. Rats exposed to CO had 24% lower maximal oxygen uptake, longer (145 vs. 123 μ m) and wider (47 vs. 25 μ m) cardiomyocytes, reduced cardiomyocyte fractional shortening (12 vs. 7%), and 26% longer time to 50% re-lengthening than controls. In addition, cardiomyocytes from CO-exposed rats had 48% lower intracellular calcium (Ca^{2 +}) amplitude, 22% longer time to Ca^{2 +} decay, 34% lower capacity of sarcoplasmic reticulum Ca^{2 +}-ATPase (SERCA2a), and 37% less t-tubule area compared to controls. Phosphorylation levels of phospholamban at Ser16 and Thr17 were significantly reduced in the CO group, whereas total concentration of phospholamban and SERCA2a were unchanged. Cardiac atrial natriuretic peptide, vascular endothelial growth factor, cyclic guanosine monophosphate, calcineurin, calmodulin, pERK, and pS6 increased, whereas pAkt and pCaMKII δ remained unchanged by CO. Endothelial function and systemic blood pressure were not affected by CO exposure. Long-term CO exposure reduces aerobe capacity and contractile function and leads to pathological hypertrophy. Impaired Ca^{2 +} handling and increased growth factor signaling seem to be responsible for these pathological changes.     

Effect of endothelin antagonism on contractility, intracellular calcium regulation and calcium regulatory protein expression in right ventricular hypertrophy of the rat

We have documented the effects of long-term endothelin receptor antagonism on intracellular Ca2+ regulation and Ca2+ regulatory protein expression in rat hearts with right ventricular hypertrophy without signs of heart failure. Rats were given either a single injection of monocrotaline (50 mg/kg, n=9) resulting in pulmonary hypertension-induced myocardial hypertrophy, or monocrotaline followed by daily administration of the endothelin subtype-A receptor antagonist 2-benzo(1,3)dioxol-5-yl-3-benzyl-4-(4-methoxy-phenyl-)-4-oxobut-2-enoate-Na (PD 155080, 50 mg/kg) over 9 weeks (n=8). Hearts from saline-injected rats served as controls (n=9). Monocrotaline-treated animals developed marked right-sided hypertrophy without fibrosis as evident from hydroxyproline measurements, systolic contractility was increased, fully compensating for the increased afterload, but diastolic function was impaired as evident from protracted relaxation and slowed diastolic intracellular Ca2+ handling (measured by aequorin bioluminescence). In hypertrophic hearts, quantitative immunoblotting analyses showed increased levels both of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and phosphorylated phospholamban, along with decreased levels of total phospholamban, which is in line with strengthened right ventricular systolic function. PD 155080 reversed abnormalities in Ca2+ handling, although SERCA and phospholamban protein levels were not altered (P=not significant versus monocrotaline group). Thus, endothelin-A receptor antagonism attenuates right ventricular remodeling and improves myocardial Ca2+ handling, but has no discernable effect on elevated expression of SERCA and phospholamban observed in hypertrophic hearts. These data indicate that the hypotensive action of PD 155080 is independent of its effects, if any, on SERCA and its regulation.     

Astragaloside IV Alleviates Hypoxia/Reoxygenation-Induced Neonatal Rat Cardiomyocyte Injury via the Protein Kinase A Pathway

Background: Astragaloside IV (As-IV) exerts beneficial effects on hypoxia/reoxygenation (H/R)-induced cardiomyocyte injury, possibly through normalization of sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) function. The exact mechanisms remain unknown. This study was designed to investigate the role of protein kinase A (PKA) in the protective effect of As-IV on SERCA2a function. Methods: Cultured cardiomyocytes from neonatal rats were exposed to 6 h of hypoxia followed by 3 h of reoxygenation (H/R) with or without As-IV treatment. Myocyte injury was determined by the creatine kinase (CK)-MB fraction in supernatant. Myocardial SERCA2a activity and PKA kinase activity were assessed. PKA subunit mRNA expression and Ser(16) phosphorylated phospholamban (Ser(16)-PLN) protein expression were detected by real-time PCR and Western blot, respectively. Results: The administration of As-IV significantly decreased CK-MB release and restored SERCA2a activity in H/R cardiomyocytes. The mRNAs of PKA subunits, PKA-RIα, PKA-RIIα, PKA-RIIβ, PKA-Cα and PKA-Cβ, were downregulated in H/R cardiomyocytes. However, PKA-Cα mRNA expression was significantly increased after As-IV treatment. Meanwhile, there was a tendency to recovery of the H/R-induced PKA kinase activity decrease after As-IV treatment. The expression of Ser(16)-PLN protein, which is specifically phosphorylated by PKA, was upregulated in As-IV-treated H/R cardiomyocytes. Conclusions: These results suggest that the cardioprotection of As-IV may be through the upregulation of PKA and Ser(16)-PLN, thereby restoring SERCA2a function in H/R injury.     

Modification of alterations in cardiac function and sarcoplasmic reticulum by astragaloside IV in myocardial injury in vivo

Astragaloside IV, the primary pure saponin isolated from Astragalus membranaceus has been found to have potent cardioprotective effects. In this study, we aim to investigate if the beneficial effects of astragaloside IV on cardiac function are associated with improvement in sarcoplasmic reticulum Ca(2+)-pump function in myocardial injury in vivo. Myocardial injury in rats was induced by subcutaneous injection of a high dose of isoproterenol, and the therapeutic effect of astragaloside IV was observed. Isoproterenol-treated rats showed widespread subendocardial necrosis, a rise in serum lactate dehydrogenase and creatine kinase, formation of lipid oxide product malondialdehyde and inhibition of left ventricular diastolic and systolic function, which suggested severe myocardial injury and acute heart failure. Moreover, sarcoplasmic reticulum Ca(2+)-uptake ability and Ca(2+)-ATPase (SERCA2a) activity were significantly reduced. And the level of SERCA2a mRNA and protein expression was also markedly decreased, associated with a decrease in Ser(16)-phosphorylated phospholamban protein expression, while total phospholamban level was unchanged in the isoproterenol-treated group compared with controls. However, these biochemical and hemodynamic changes in the acute failing hearts were prevented by treatment of isoproterenol-induced rats with astragaloside IV. Likewise, the observed reductions in sarcoplasmic reticulum Ca(2+)-pump function as well as in SERCA2a mRNA and protein levels and the phosphorylation level of phospholamban in the injured hearts were attenuated by astragaloside IV treatment. These results suggest that the beneficial effect of astragaloside IV on isoproterenol-induced myocardial injury may be due to its ability to prevent changes of SERCA2a and Ser(16)-phosphorylated phospholamban protein expression and, thus, may prevent the depression in sarcoplasmic reticulum Ca(2+) transport and improve cardiac function.     

Insulin improves cardiomyocyte contractile function through enhancement of SERCA2a activity in simulated ischemia/reperfusion

AIM: Insulin exerts anti-apoptotic effects in both cardiomyocytes and coronary endothelial cells following ischemia/reperfusion (I/R) via the Akt-endothelial nitric oxide synthase survival signal pathway. This important insulin signaling might further contribute to the improvement of cardiac function after reperfusion. In this study, we tested the hypothesis that sarcoplasmic reticulum calcium-ATPase (SERCA2a) is involved in the insulin-induced improvement of cardiac contractile function following I/R. METHODS: Ventricular myocytes were enzymatically isolated from adult SD rats. Simulated I/R was induced by perfusing cells with chemical anoxic solution for 15 min followed by reperfusion with Tyrode's solution with or without insulin for 30 min. Myocyte shortening and intracellular calcium transients were assessed and underlying mechanisms were investigated. RESULTS: Reperfusion with insulin (10(-7) mol/L) significantly improved the recovery of contractile function (n=15-20 myocytes from 6-8 hearts, P<0.05), and increased calcium transients, as evidenced by the increased calcium [Ca2+] fluorescence ratio, shortened time to peak Ca2+ and time to 50% diastolic Ca2+, compared with those in cells reperfused with vehicle (P<0.05). In addition, Akt phosphorylation and SERCA2a activity were both increased in insulin-treated I/R cardiomyocytes, which were markedly inhibited by pretreatment of cells with a specific Akt inhibitor. Moreover, inhibition of Akt activity abolished insulin-induced positive contractile and calcium transients responses in I/R cardiomyocytes. CONCLUSION: These data demonstrated for the first time that insulin improves the recovery of contractile function in simulated I/R cardiomyocytes in an Akt-dependent and SERCA2a-mediated fashion.     

Regulation of phospholamban and sarcoplasmic reticulum ca<Superscript>2+</Superscript>-atpase by atorvastatin: implication for cardiac hypertrophy

Abnormal intracellular Ca2+ homeostasis in the myocardium has been suggested as the cause of cardiac hypertrophy, and this process can be prevented by the HMG-CoA reductase inhibitors, statins. In the present study, the effect of atorvastatin on left ventricular hypertrophy was investigated, and then whether the underlying mechanism was related to a defect in intracellular Ca2+ homeostasis explored. Twelve spontaneously hypertensive rats (SHR), at 8 weeks old, were used in this study, and received either distilled water or atorvastatin for ten weeks, with age-matched normotensive Wistar-Kyoto rats (WKY) used as controls. RT-PCR and western blotting were used to detect the mRNA and protein expressions of phospholamban (PLB) and sarcoplasmic reticulum Ca2+-ATPase (SERCA2a), respectively, and a colorimetric method used to examine the SERCA2a activity. Additionally, cardiac hypertrophic indices, such as the cardiosomatic ratio, left ventricular weight to body weight (LVW/BW) ratio and cardiomyocytes transverse diameter (TDM), together with the systolic blood pressure (SBP) and serum lipids levels were also examined. After ten weeks, significant decreases were observed in both the mRNA and protein expression levels of SERCA2a, as well as its activity, in the hypertrophied hearts of the SHR. The administration of atorvastatin to the same strains of rats effectively inhibited these decreases, and the above cardiac hypertrophic indices, as well as the SBP and serum lipids levels were significantly decreased. However, no significant changes in the expressions of PLB were observed in WKY, SHR and atorvastatin-treated SHR. These findings demonstrated that through regulation of the PLB and SERCA2a levels in the hearts of SHR, atorvastatin can prevent the cardiac hypertrophy caused due to pressure overload, which provides a relatively new insight into the mechanism of atorvastatin in the prevention of cardiac hypertrophy.     

Acute methamphetamine exposure inhibits cardiac contractile function

Methamphetamine, a commonly seen substance of abuse, has been reported to exert detrimental effect on bodily function including the cardiovascular system although its mechanism of action is poorly understood. This study was designed to examine the direct impact of methamphetamine on isolated whole heart and single cardiomyocyte contractile function. Murine hearts and isolated cardiomyocytes from adult FVB mice were exposed to various concentrations of methamphetamine for 30 min prior to the assessment of mechanical function using a Langendroff apparatus and an IonOptix Myocam^® system, respectively. Cardiac contractile properties analyzed included maximal velocity of left ventricular pressure development and decline (±dP/dt), peak shortening amplitude (PS), maximal velocity of shortening/relengthening (±dLdt), time-to-PS (TPS), time-to-90% relengthening (TR₉₀), resting and electrically stimulated increase of intracellular Ca²⁺ as well as intracellular Ca²⁺ decay. Our results revealed that acute methamphetamine exposure depressed ±dP/dt, PS and rise of intracellular Ca²⁺ without affecting ±dLdt, TPS, TR₉₀, resting intracellular Ca²⁺ and intracellular Ca²⁺ decay. Furthermore, methamphetamine nullified the adrenergic agonist norepinephrine-elicited positive cardiomyocyte contractile response, including elevated PS, ±dLdt and shortened TR₉₀ without affecting TPS. Western blot analysis showed unchanged expression of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA2a) and phospholamban, associated with upregulated Na⁺–Ca²⁺ exchanger levels following acute methamphetamine exposure. In addition, methamphetamine promoted overt cardiomyocyte protein damage evaluated by carbonyl formation. Taken together, these results demonstrate direct cardiac depressant effect of methamphetamine in myocardium and isolated cardiomyocytes, possibly associated with protein damage and dampened adrenergic response.     

Imbalance between CaM kinase II and calcineurin activities impairs caffeine-induced calcium release in hypertrophic cardiomyocytes

Cardiac hypertrophy impairs Ca(2+) handling in the sarcoplasmic reticulum, thereby impairing cardiac contraction. To identify the mechanisms underlying impaired Ca(2+) release from the sarcoplasmic reticulum in hypertrophic cardiomyocytes, we assessed Ca(2+)-dependent signaling and the phosphorylation of phospholamban, which regulates Ca(2+) uptake during myocardial relaxation and is in turn regulated by Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and calcineurin. In cultured rat cardiomyocytes, treatment with endothelin-1, angiotensin II, and phenylephrine-induced hypertrophy and increased CaMKII autophosphorylation and calcineurin expression. The calcineurin level reached its maximum at 72h and remained elevated for at least 96h after endothelin-1 or angiotensin II treatment. By contrast, CaMKII autophosphorylation, phospholamban phosphorylation, and caffeine-induced Ca(2+) mobilization all peaked 48h after these treatments. By 96h after treatment, CaMKII autophosphorylation and phospholamban phosphorylation had returned to baseline, and caffeine-induced Ca(2+) mobilization was impaired relative to baseline. A similar biphasic change was observed in dystrophin levels in endothelin-1-induced hypertrophic cardiomyocytes, and treatment with the novel CaM antagonists DY-9760e and DY-9836 significantly inhibited the hypertrophy-induced dystrophin breakdown. Taken together, the abnormal Ca(2+) regulation in cardiomyocytes following hypertrophy is in part mediated by an imbalance in calcineurin and CaMKII activities, which leads to abnormal phospholamban activity.     

左旋氨氯地平对缺氧心肌细胞钙信号系统的影响

目的:观察缺氧对心肌细胞内游离钙离子浓度和细胞膜及肌浆网上钙离子泵,即Ca2+ AT Pase表达的影响及左旋氨氯地平的干预作用。方法:制备大鼠心肌细胞缺氧模型,随机分为正常对照组、缺氧损伤组和左旋氨氯地平组。测定心肌细胞内游离钙离子的浓度(FURA2 /AM荧光探针法)、细胞膜上Ca2+ ATPase表达水平(RT PCR法)以及肌浆网上钙泵(SERCA2 )含量(WesternBlot法)。结果:缺氧损伤组心肌细胞内游离钙离子浓度[ (716±s164)nmol·L-1 ]较正常对照组[ (208±32)nmol·L-1 ]显著上升(P<0. 01 ),细胞膜及肌浆网上钙泵的表达(0. 54±0. 12和158±15)较正常对照组( 0. 86±0. 07和210±12 )均明显降低(均P<0. 01),经左旋氨氯地平干预后细胞内游离钙浓度降低至(359±75 )nmol·L-1,P<0. 01,细胞膜上及肌浆网上钙泵的表达(0. 81±0. 11和184±11)均明显增强(均P<0. 01)。结论:左旋氨氯地平可能通过增加细胞膜及肌浆网上钙泵的表达有效减轻心肌细胞内的钙超载,对缺氧心肌具有保护作用。     

Comparison of low and high doses of carvedilol on restoration of cardiac function and calcium-handling proteins in rat failing heart

1. The beta-adrenoceptor antagonist carvedilol reverses cardiac dysfunction in the failing heart. A recent study showed that beta-adrenoceptor antagonists indirectly normalize Ca(2+)-regulatory proteins. The relationship between these two phenomena and the suitable dosage of carvedilol remains unclear. 2. We investigated the change in left ventricular (LV) remodelling and function in a rat model of heart failure due to myocardial infarction (MI) with or without carvedilol (30 or 2 mg/kg per day) treatment for 6 weeks. The expression of mRNA and proteins of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLB) in cardiomyocytes was also measured. 3. There was significant LV remodelling and cardiac contractile dysfunction in MI rats. The expression of SERCA mRNA and protein were downregulated (P < 0.01), but the expression of PLB mRNA and protein were upregulated (P < 0.01) in MI rats compared with sham-operated rats. After treatment with carvedilol, LV remodelling and cardiac contractile dysfunction were clearly improved. Low-dose carvedilol was better at improving some parameters of LV remodelling and function than the high dose. Carvedilol partially restored the low expression of SERCA (P < 0.05), but had no effect on PLB expression (P > 0.05). Moreover, low-dose carvedilol induced a more significant improvement in SERCA expression than did the high dose (P < 0.05). 4. The results of the present study suggest that carvedilol is effective in improving LV remodelling and cardiac contractile dysfunction after MI. This may be related to the normalization of SERCA expression.     

黄芪苷Ⅳ对血管紧张素Ⅱ诱导新生大鼠心肌细胞肥大的保护作用

目的研究黄芪苷Ⅳ(astragalosideⅣ,AST)对血管紧张素Ⅱ(angiotensinⅡ,AngⅡ)诱导新生大鼠心肌细胞肥大的保护作用并探讨其作用机制。方法AngⅡ刺激新生大鼠心肌细胞,制备心肌细胞肥大模型,分别用AST10mg·mL-1和20mg·mL-1进行预防性治疗。检测心肌细胞直径及细胞总蛋白含量,测定心肌细胞[Ca2+]i、肌浆网钙泵(SERCA)活力和钙调神经磷酸酶(CaN)活性。结果AngⅡ组细胞直径增大,总蛋白含量增加,与对照组相比差异有显著性(P<0.01);AST10mg·mL-1和20mg·mL-1能够降低心肌细胞肥大程度(P<0.05或P<0.01)、[Ca2+]i(P<0.01)及CaN活性(P<0.05或P<0.01)、提高SERCA活力(P<0.01)。结论AST对AngⅡ诱导心肌细胞肥大有明显的保护作用,可能与其降低[Ca2+]i、提高心肌SERCA活力及降低CaN活性有关。     

A novel endothelin receptor antagonist CPU0213 improves diabetic cardiac insufficiency attributed to up-regulation of the expression of FKBP12.6, SERCA2a, and PLB in rats

The depressed sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a) and Ca2+-release channels (ryanodine receptor RyR2) are involved in the diabetic cardiomyopathy. However, an implication of a down-regulation of FK506-binding protein or calstabin-2 (FKBP12.6) is undefined. It was hypothesized that the down-regulation of FKBP12.6 and SERCA2a of the intracellular calcium handling system is closely related to an up-regulated endothelin (ET) system. An ET receptor antagonist CPU0213 is newly discovered and expected to ameliorate cardiac insufficiency which is mediated by the depressed FKBP12.6 and SERCA2a in diabetic rat heart. Diabetes was developed in male Sprague-Dawley rats 8 weeks after an injection of streptozotocin (60 mg/kg IP), and CPU0213 was instituted 30 mg/kg, SC in the last 4 weeks. The assessment of the cardiac function, cardiac calcium handling proteins, endothelin system, and redox enzyme system were conducted. The compromised cardiac function in diabetic rats was accompanied by a significant down-regulation of expression of FKBP12.6 as well as SERCA2a and phospholamban. These were closely linked with an increased ET-1 and up-regulation of endothelin converting enzyme, PropreET1, and inducible nitric oxide synthase mRNA in diabetic cardiomyopathy. After 4-week treatment, CPU0213 was capable to attenuate completely the down-regulated FKBP12.6 and SERCA2a, and up-regulated ET system in association with a recovery of the cardiac insufficiency of diabetic cardiomyopathy.     

正性肌力药物作用靶点的研究进展

正性肌力药物能影响心肌收缩力,是治疗心力衰竭的主要药物之一,用于治疗心力衰竭的药物疗效及用药患者终期生存率取决于作用靶点。与心肌收缩力有关的靶点包括:β肾上腺素受体、磷酸二酯酶、L-型钙通道、蛋白激酶A、蛋白激酶C、蛋白磷酸酶、Na+-Ca2+交换体、Na+-K+-ATP酶、肌浆网钙泵、受磷蛋白、兰尼碱受体、三磷酸肌醇受体、与钙增敏剂相关的心肌收缩蛋白等。文中对心肌收缩力相关的作用靶点进行综述,分析临床现今使用的和未来的正性肌力药物应具备的靶点特征。     

Sevoflurane and bradykinin-induced calcium mobilization in pulmonary arterial valvular endothelial cells in situ: sevoflurane stimulates plasmalemmal calcium influx into endothelial cells

Kinins locally synthesized in the cardiovascular tissue are believed to contribute to the regulation of cardiovascular homeostasis by stimulating the endothelial cells to release nitric oxide, prostacyclin, or a hyperpolarizing factor via autocrine-paracrine mechanisms. This study was designed to investigate the action of sevoflurane on bradykinin-induced Ca2+ mobilization in endothelial cells in situ. Utilizing fura-2-loaded rat pulmonary arterial valve leaflets, the effects of sevoflurane were examined on bradykinin-induced increases in intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ. In the presence of extracellular Ca2+ (1.5 mM), bradykinin (3-30 microM) produced an initial phasic and a subsequent tonic increase in [Ca2+]i in a concentration-dependent manner. However, it produced only the phasic increase in [Ca2+]i in the absence of extracellular Ca2+. Sevoflurane (5%, 0.67 mM) inhibited both the phasic and tonic responses to bradykinin. In these experiments, sevoflurane (3-5%) generated sustained increases (approximately 20-40% of the bradykinin-induced maximal increase in [Ca2+]i) in the resting [Ca2+]i level. Sevoflurane still increased [Ca2+]i after depletion of the intracellular Ca stores with ionomycin (0.1 microM ). However, the sevoflurane-induced increase in [Ca2+]i was eliminated by removal of the extracellular Ca and attenuated by NiCl (1-3 mM). In conclusion, in the pulmonary arterial valvular endothelial cells, sevoflurane inhibits both bradykinin-induced Ca2+ release from the intracellular stores and bradykinin-induced plasmalemmal Ca2+ influx. In addition, sevoflurane appears to stimulate the plasmalemmal Ca2+ influx and thereby increase the endothelial [Ca2+]i level. Sevoflurane might influence the pulmonary vascular tone through its direct action on the pulmonary arterial valvular endothelial cells.     

Allopurinol modulates reactive oxygen species generation and Ca2+ overload in ischemia-reperfused heart and hypoxia-reoxygenated cardiomyocytes

Myocardial oxidative stress and Ca2+ overload induced by ischemia-reperfusion may be involved in the development and progression of myocardial dysfunction in heart failure. Xanthine oxidase, which is capable of producing reactive oxygen species, is considered as a culprit regarding ischemia-reperfusion injury of cardiomyocytes. Even though inhibition of xanthine oxidase by allopurinol in failing hearts improves cardiac performance, the regulatory mechanisms are not known in detail. We therefore hypothesized that allopurinol may prevent the xanthine oxidase-induced reactive oxygen species production and Ca2+ overload, leading to decreased calcium-responsive signaling in myocardial dysfunction. Allopurinol reversed the increased xanthine oxidase activity in ischemia-reperfusion injury of neonatal rat hearts. Hypoxia-reoxygenation injury, which simulates ischemia-reperfusion injury, of neonatal rat cardiomyocytes resulted in activation of xanthine oxidase relative to that of the control, indicating that intracellular xanthine oxidase exists in neonatal rat cardiomyocytes and that hypoxia-reoxygenation induces xanthine oxidase activity. Allopurinol (10 microM) treatment suppressed xanthine oxidase activity induced by hypoxia-reoxygenation injury and the production of reactive oxygen species. Allopurinol also decreased the concentration of intracellular Ca2+ increased by enhanced xanthine oxidase activity. Enhanced xanthine oxidase activity resulted in decreased expression of protein kinase C and sarcoendoplasmic reticulum calcium ATPase and increased the phosphorylation of extracellular signal-regulated protein kinase and p38 kinase. Xanthine oxidase activity was increased in both ischemia-reperfusion-injured rat hearts and hypoxia-reoxygenation-injured cardiomyocytes, leading to reactive oxygen species production and intracellular Ca2+ overload through mechanisms involving p38 kinase and extracellular signal-regulated protein kinase (ERK) via sarcoendoplasmic reticulum calcium ATPase (SERCA) and protein kinase C (PKC). Xanthine oxidase inhibition with allopurinol modulates reactive oxygen species production and intracellular Ca2+ overload in hypoxia-reoxygenation-injured neonatal rat cardiomyocytes.     

Method-related effects of adenovirus-mediated LacZ and SERCA1 gene transfer on contractile behavior of cultured failing human cardiomyocytes

INTRODUCTION: Adenovirus-mediated gene transfer into cardiomyocytes has emerged as an interesting tool to study functional effects of single proteins. However, the functional consequences of cell isolation, cell culture per se and adenovirus-mediated transfer of the LacZ or SERCA1 gene in failing human cardiomyocytes warrant further investigation. METHODS: Primary cell culture was performed without or after adenovirus-mediated gene transfer of LacZ or SERCA1. Functional behavior of myocytes was assessed under basal conditions (field stimulation, 0.5 Hz, 37 degrees C), and during inotropic stimulation with isoproterenol (ISO; 10(-9)-10(-5) M), [Ca(2+)](o) (1.5-15 mM) or increasing stimulation rates (0.25-2.5 Hz). Results were compared to trabeculae from the same hearts. RESULTS: Freshly isolated myocytes showed full inotropic competence as compared to multicellular preparations. The response to stimulation with ISO and [Ca(2+)](o), as well as changes in stimulation rate resulted in a maximal increase in fractional cell shortening (FS) to 215+/-24% and 291+/-34%, and a frequency-dependent decline in FS to 46+/-5% of the basal value, respectively. After 48 h of cell culture, basal FS did not change significantly compared to fresh cells but both time to peak shortening and time to 50% relengthening were prolonged. After culture, the concentration-response curve for ISO was significantly shifted to the left (EC(50) 5.16 x 10(-8) vs. 1.12 x 10(-8) M, p<0.05). LacZ gene transfer caused efficient beta-Gal expression without affecting the inotropic responses to ISO or stimulation rate but impaired the contractile amplitude. SERCA1 gene transfer increased FS by 68% vs. LacZ and accelerated relengthening kinetics (+dL/dt 93+/-13 vs. 61+/-8 mum/s, p<0.05 vs. LacZ). DISCUSSION: Contractile responses of isolated human myocytes are comparable to multicellular preparations. The use of primary cell culture and adenovirus infection with CMV-promoter-mediated LacZ expression per se modulates contractile behavior in failing human myocytes. SERCA1 expression markedly improves contractile function. The method-related changes in contractile behavior observed here need to be taken into account in further studies.     

SERCA2 activity is involved in the CNP-mediated functional responses in failing rat myocardium

BACKGROUND AND PURPOSES

Myocardial C-type natriuretic peptide (CNP) levels are increased in heart failure. CNP can induce negative inotropic (NIR) and positive lusitropic responses (LR) in normal hearts, but its effects in failing hearts are not known. We studied the mechanism of CNP-induced NIR and LR in failing hearts and determined whether sarcoplasmatic reticulum Ca²⁺ ATPase2 (SERCA2) activity is essential for these responses.

EXPERIMENTAL APPROACH

Contractility, cGMP levels, Ca²⁺ transient amplitudes and protein phosphorylation were measured in left ventricular muscle strips or ventricular cardiomyocytes from failing hearts of Wistar rats 6 weeks after myocardial infarction.

KEY RESULTS

CNP increased cGMP levels, evoked a NIR and LR in muscle strips, and caused phospholamban (PLB) Ser¹⁶ and troponin I (TnI) Ser^23/24 phosphorylation in cardiomyocytes. Both the NIR and LR induced by CNP were reduced in the presence of a PKG blocker/cGMP analogue (Rp-8-Br-Pet-cGMPS) and the SERCA inhibitor thapsigargin. CNP increased the amplitude of the Ca²⁺ transient and increased SERCA2 activity in cardiomyocytes. The CNP-elicited NIR and LR were not affected by the L-type Ca²⁺ channel activator BAY-K8644, but were abolished in the presence of isoprenaline (induces maximal activation of cAMP pathway). This suggests that phosphorylation of PLB and TnI by CNP causes both a NIR and LR. The NIR to CNP in mouse heart was abolished 8 weeks after cardiomyocyte-specific inactivation of the SERCA2 gene.

CONCLUSIONS AND IMPLICATIONS

We conclude that CNP-induced PLB and TnI phosphorylation by PKG in concert mediate both a predictable LR as well as the less expected NIR in failing hearts.     

Paradoxical effects of ginkgolide B on cardiomyocyte contractile function in normal and high-glucose environments

AIM: Ginkgo biloba extract is a natural product used widely for cerebral and cardiovascular diseases. It is mainly composed of terpene lactones (ginkgolide A and B) and flavone glycosides (eg quercetin and kaempferol). To better understand the cardiac electromechanical action of Ginkgo biloba extract in normal and diabetic states, this study was designed to examine the effect of ginkgolide B on cardiomyocyte contractile function under normal and high-glucose environments. METHODS: Isolated adult rat ventricular myocytes were cultured for 6 h in a serum-free medium containing either normal (NG; 5.5 mmol/L) or high (HG; 25.5 mmol/L) glucose with or without ginkgolide B (0.5-2.0 microg/mL). Mechanical properties were evaluated using the IonOptix MyoCam system. Contractile properties analyzed included peak shortening (PS), maximal velocity of shortening/relengthening (+/-dl/dt), time-to-PS (TPS) and time-to-90% relengthening (TR90). Levels of essential Ca(2+) regulatory proteins sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), phospholamban (PLB) and Na(+)-Ca(2+) exchanger (NCX) were assessed by Western blotting. RESULTS: Ginkgolide B nullified HG-induced prolongation in TR90. However, ginkgolide B depressed PS, +/-dl/dt and shortened TPS in NG and HG cells. Ginkgolide B also prolonged TR90 in NG cells. Western blot analysis revealed that HG upregulated SERCA2a and downregulated PLB expression without affecting that of NCX. Ginkgolide B disrupted the NG-HG response pattern in SERCA2a and NCX without affecting that of PLB. CONCLUSION: Ginkgolide B affects cardiomyocyte contractile function under NG or HG environments in a paradoxical manner, which may be attributed to uneven action on Ca(2+) regulatory proteins under NG and HG conditions.