心力衰竭家兔心肌细胞钙调控蛋白表达的异常

目的探讨心力衰竭(心衰)心肌细胞钙调控蛋白表达异常的临床意义。方法 16只家兔随机分为两组,假手术组和心衰组各8只。通过超容量负荷联合压力负荷建立家兔心衰模型,利用心导管术和心脏多普勒观察手术前后家兔血流动力学及心脏结构和功能的变化。采用蛋白免疫印迹(Western blot)法测定心肌组织 L 型钙通道(LTCC)、肌浆网钙释放通道(RyR2)、肌浆网钙泵(SERCA2a)以及钠钙交换体(NCX)表达水平。结果家兔心衰组与假手术组相比,左室/体重比值、心率、左室舒张末压明显增加(P<0.01);左室短轴缩短率[(21.3±4.00)%与(36.5±1.36)%]和左室射血分数(0.45±0.07与0.70±0.02)降低(P<0.01);心肌组织 LTCC、RyR2表达下降(R_(LTCC/actin):0.287±0.029与0.624±0.009,R_(RyR2/actin):0.106±0.001与0.203±0.011,P<0.01);SERCA2a、NCX 表达增加(R_(NCX/actin:0.497±0.015与0.221±0.014,R_(SERCA2a/actin:0.611±0.036与0.433±0.008,P<0.01)。结论 LTCC 和 RyR2表达下调是心衰心肌收缩力降低的因素之一,而心衰早期 SERCA2a、NCX 表达增加可能有利于心肌舒缩功能的保持。     

Alteration of myocardial sarcoplasmic reticulum Ca2+-ATPase and Na+-Ca2+ exchanger expression in human left ventricular volume overload

BACKGROUND: Reduced myocardial contractility is often attributed to altered Ca(2+) transients and expression of Ca(2+)-ATPase of the SR (SERCA) and Na+/Ca(2+)exchanger (NCX) genes. AIMS: To assess myocardial expression of SERCA and NCX protein levels in left ventricular (LV) remodelling due to chronic severe mitral regurgitation (MR). METHODS: Myocardial expression of SERCA/NCX in biopsy specimens obtained during mitral surgery was assessed in 36 MR patients with LV remodelling and plasma neurohumoral/cytokine activation and in four non-failing hearts (NFH). RESULTS: Myocardial protein levels of SERCA were significantly (20%) lower in the MR group than in NFH group (p=0.016). No significant changes in NCX were observed. However, a lack of homogeneity with regard to SERCA/NCX proteins was observed. Moreover, SERCA was negatively correlated with BNP (r=-0.49, p=0.02), TNFalpha (r=-0.68, p=0.0005) and IL-6 (r=-0.52, p=0.02), whereas NCX was only negatively correlated with TNFalpha (r=-0.62, p=0.002). CONCLUSIONS: MR patients showed wide variations in SERCA/NCX protein expression. Myocardial protein levels of SERCA were significantly lower in the MR population. Moreover, a correlation between BNP, cytokines (IL-6, TNFalpha) and the expression of SERCA/NCX proteins was observed.     

培哚普利对心力衰竭大鼠心肌细胞钙瞬变及其调控蛋白的影响

目的研究培哚普利在心力衰竭(心衰)治疗中对心肌细胞的收缩特性、钙瞬变及其调控蛋白的影响。方法通过缩窄雄性Wistar大鼠的腹主动脉,制成压力负荷增高性心衰模型。随机分成培哚普利治疗组(培哚普利组,3mg·kg-1·d-1)、心衰对照组和假手术对照组(假手术组)。12周后分离左室心肌细胞,测定单个心肌细胞在电刺激时的缩短分数、钙瞬变以及钠-钙交换蛋白(NCX1)、内质网Ca2+-ATPase(SERCA2)、受磷蛋白等钙调控蛋白的转录与翻译水平。结果心肌细胞的缩短分数(FS)和[Ca2+]imax在心衰对照组均明显低于假手术组[FS(%):7.51±1.15与13.21±1.49,[Ca2+]imax(nmol/L):330.85±50.05与498.16±14.07;均为P<0.01]。给予培哚普利治疗后,培哚普利组的FS和[Ca2+]imax分别为(10.89±1.18)%和(488.72±22.27)nmol/L,明显高于心衰对照组(均为P<0.01)。在心衰对照组,NCX1和受磷蛋白的mRNA水平显著高于假手术组,而SERCA2的mRNA却明显低于假手术组。在培哚普利组,其NCX1和SERCA2的mRNA水平则介于心衰对照组和假手术组之间,差异有统计学意义(均为P<0.05)。在心衰对照组及培哚普利组,NCX1的蛋白表达量分别是假手术组的(1.141±0.047)倍(P<0.01)和(1.074±0.081)倍(P=0.018),SERCA2的表达量则是假手术组的(0.803±0.100)倍(P<0.01)和(0.893±0.084)倍(P=0.003)。结论培哚普利抗心衰治疗能够减轻心肌细胞的钙瞬变及其调控蛋白的异常变化,使心衰中单个心肌细胞的收缩特性得到保护。     

Left ventricular assist device support reverses altered cardiac expression and function of natriuretic peptides and receptors in end-stage heart failure

OBJECTIVE: Atrial (ANP) and B-type natriuretics peptides (BNP) via their guanylyl cyclase-A (GC-A) receptor not only regulate arterial blood pressure and volume but also exert local antihypertrophic, antifibrotic and lusitropic effects in the heart. To elucidate whether cardiac hypertrophy/insufficiency and reversal is associated with changes in the local responsiveness to NPs, we compared the mRNA expression of ANP, BNP and receptors and the responsiveness of GC-A to ANP in left ventricular tissue obtained from 10 patients with congestive heart failure (CHF) before and after hemodynamic unloading by left ventricular assist device (LVAD) support. METHODS AND RESULTS: Quantitative "real time" RT-PCR demonstrated that the mRNA expression levels of ANP, BNP and the NP-metabolizing NPR-C receptor were both markedly increased in human failing hearts. GC-A mRNA expression levels were not different from nonfailing hearts, but cGMP production by GC-A in response to ANP was nearly abolished. Reversal of cardiomyocyte hypertrophy during LVAD support was accompanied by normalization of ANP, BNP and NPR-C mRNA levels and a significant recovery of GC-A responsiveness to ANP. CONCLUSION: In CHF patients, increased local clearance by NPR-C receptors and diminished responsiveness of cardiac GC-A might impair the local antihypertrophic effects of natriuretic peptides and contribute to the progression of cardiac hypertrophy and insufficiency. Reverse remodeling during LVAD support reverses these changes and can thereby recuperate the local protective effects of ANP and BNP.     

Increased functional importance of the Na,Ca-exchanger in contracting failing human myocardium but unchanged activity in isolated vesicles

The present study aimed to investigate the hypothesis that the function of the Na,Ca-exchanger (NCX) is of higher importance for contractility and Ca(2+)-homeostasis in left ventricle from terminally failing than from nonfailing human hearts. The effect of decreasing extracellular [Na](e) (140 to 25 mmol/L) on force of contraction in isolated left ventricular papillary muscle strips was studied as a reflection of NCX function in multicellular preparations (terminally failing, DCM, dilated cardiomyopathy, NYHA IV, n = 13; nonfailing, NF, donor hearts, n = 10). Decreasing [Na](e) has previously been shown to increase contractility in vitro secondary to a decreased Ca(2+)-extrusion by the NCX. In addition, the NCX activity was measured as Na(+)-dependent (45)Ca(2+)-uptake into isolated myocardial vesicles as a function of time and Ca(2+)-concentration (DCM n = 8, NF n = 8). Decreasing [Na](e) enhanced the contractility of papillary muscle strips in both DCM and NF, but the contractility of DCM was increased at smaller reductions of [Na](e) than NF. The NCX activity in isolated myocardial vesicles was unchanged as a function of time (T(1/2): DCM 2.4 +/- 0.3 s versus NF 2.5 +/- 0.3 s) and as a function of Ca(2+) (DCM 0.99 +/- 0.08 versus NF 0.96 +/- 0.07 nmol/mg protein x 3 s, K(1/2): DCM 39.2 microM versus NF 38.3 microM). These results demonstrate a higher sensitivity of the failing human myocardium towards Na,Ca-exchanger mediated positive inotropic effects, suggesting a higher significance of the Na,Ca-exchanger for the extrusion of Ca(2+)-ions in intact failing versus nonfailing human myocardium. Since the activity and the Ca (2+)-affinity of the Na,Ca-exchanger in isolated vesicles was unchanged, we propose that alterations in Ca(2+)-and Na(+)-homeostasis (due to impaired function of the sarcoplasmic reticulum and the Na(+), K(+)-ATPase) or the prolonged action potential are the reason for this observation.     

Adenoviral gene transfer of SERCA2a improves left-ventricular function in aortic-banded rats in transition to heart failure

In human and experimental models of heart failure, sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) activity is decreased, resulting in abnormal calcium handling. The disturbances in calcium metabolism have been shown to contribute significantly to the contractile dysfunction observed in heart failure. We investigated whether increasing SERCA2a expression can improve ventricular function in an animal model of heart failure obtained by creating ascending aortic constriction in rats. After 19-23 wk of banding during the transition from compensated hypertrophy to heart failure (documented by >25% decrease in fractional shortening), rats were randomized to receive either an adenovirus carrying the SERCA2a gene (Ad.SERCA2a, n = 13) or beta-galactosidase (Ad.betagal, n = 14) by using a catheter-based technique. The failing hearts infected with Ad. betagal were characterized by a significant decrease in SERCA2a expression and a decrease in SERCA2a activity compared with nonfailing sham-operated rats (n = 11). In addition, these failing hearts had reduced left-ventricular systolic pressure, maximal rate of left-ventricular pressure rise and decline (+dP/dt, -dP/dt), and rate of isovolumic relaxation (tau). Overexpression of SERCA2a restored both SERCA2a expression and ATPase activity to nonfailing levels. Furthermore, rats infected with Ad.SERCA2a had significant improvement in left-ventricular systolic pressure, +dP/dt, -dP/dt, and rate of isovolumic relaxation (tau) normalizing them back to levels comparable to sham-operated rats. In this study, we show that in an animal model of heart failure where SERCA2a protein levels and activity are decreased and severe contractile dysfunction is present, overexpression of SERCA2a in vivo restores both systolic and diastolic function to normal levels.     

Early expression of natriuretic peptides and SERCA in mild heart failure: association with severity of the disease

BACKGROUND: We investigated changes in genetic expression of atrial and brain natriuretic peptides (ANP and BNP) and sarcoplasmic reticulum Ca(2+)-ATPase (SERCA) in patients with stable mild to moderate chronic heart failure (CHF), since data on this topic were primarily obtained in end-stage CHF. METHODS: We studied tissue from 25 patients with idiopathic dilated cardiomyopathy (IDC) in New York Heart Association (NYHA) class II (n=12) and III-IV (n=13). Myocardial tissue from normal hearts (n=10) served as controls. Messenger RNA (mRNA) expression of ANP, BNP, and SERCA was isolated, and correlated with severity of CHF, left ventricular function (LVEF), peak oxygen uptake (peak VO(2)), and wedge pressure. RESULTS: A significant trend for gradual changes in mRNA expression according to increasing NYHA class was found for ANP, BNP (P<0.0001) and SERCA (P=0.04), with a marked increase in patients with more advanced CHF (ANP and BNP: P<0.01 vs. controls; SERCA: NS) and less pronounced changes in patients with mild CHF. mRNA of ANP and BNP correlated strongly with LVEF (-0.621 and -0.621, respectively, both P<0.01) and peak VO(2) (-0.625 and -0.555, respectively, both P<0.01) and, to a lesser extent, with wedge pressure (0.440 and 0.488, respectively, both P<0.05). SERCA correlated most strongly with wedge pressure (-0.623, P<0.01), and weak, non-significant correlations with LVEF and peak VO(2) were found. CONCLUSIONS: Genetic expression of ANP, BNP, and SERCA is progressively altered in proportion to the severity of CHF, although this is more marked for ANP and to a lesser extent BNP, than for SERCA. These changes support the concept that already early in CHF, genetic expression is affected, which has implications for the understanding of the pathophysiology of CHF.     

Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2a) activity and impairs cardiac function in mice

Sarcolipin (SLN) inhibits the cardiac sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA2a) by direct binding and is superinhibitory if it binds through phospholamban (PLN). To determine whether overexpression of SLN in the heart might impair cardiac function, transgenic (TG) mice were generated with cardiac-specific overexpression of NF-SLN (SLN tagged at its N terminus with the FLAG epitope). The level of NF-SLN expression (the NF-SLN/PLN expression ratio) was equivalent to that which induces profound superinhibition when coexpressed with PLN and SERCA2a in HEK-293 cells. In TG hearts, the apparent affinity of SERCA2a for Ca(2+) was decreased compared with non-TG littermate control hearts. Invasive hemodynamic and echocardiographic analyses revealed impaired cardiac contractility and ventricular hypertrophy in TG mice. Basal PLN phosphorylation was reduced. In isolated papillary muscle subjected to isometric tension, peak amplitudes of Ca(2+) transients and peak tensions were reduced, whereas decay times of Ca(2+) transients and relaxation times of tension were increased in TG mice. Isoproterenol largely restored contractility in papillary muscle and stimulated PLN phosphorylation to wild-type levels in intact hearts. No compensatory changes in expression of SERCA2a, PLN, ryanodine receptor, and calsequestrin were observed in TG hearts. Coimmunoprecipitation indicated that overexpressed NF-SLN was bound to both SERCA2a and PLN, forming a ternary complex. These data suggest that NF-SLN overexpression inhibits SERCA2a through stabilization of SERCA2a-PLN interaction in the absence of PLN phosphorylation and through the inhibition of PLN phosphorylation. Inhibition of SERCA2a impairs contractility and calcium cycling, but responsiveness to beta-adrenergic agonists may prevent progression to heart failure.     

Expression of A-, B-, and C-type natriuretic peptide genes in failing and developing human ventricles. Correlation with expression of the Ca(2+)-ATPase gene.

Brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are novel natriuretic peptides, originally isolated from porcine brain. Similar to atrial natriuretic peptide (ANP), BNP is also synthesized in and secreted from cardiocytes, but CNP is not expressed at significant levels in normal adult myocardium. Previous studies have indicated that the serum level and ventricular expression of the ANP gene were augmented in patients with heart failure. Recently, the serum level of BNP was also reported to increase in human heart failure. To examine whether or not the expression of these natriuretic peptides is regulated in ventricular myocardium in a concordant manner, we performed Northern blot analysis using total cellular RNA isolated from the diseased left ventricles of 30 cardiac transplant recipients with end-stage heart failure, seven ventricles from organ donors (control group), and two ventricles of artificially aborted 17- and 19-week-old fetuses. The levels of mRNAs encoding both BNP and ANP increased significantly (p less than 0.01) in the left ventricular myocardium from the patients with end-stage heart failure as compared with the control group. The levels of BNP mRNA correlated positively with those of ANP mRNA (r = 0.73, p less than 0.01) and negatively with those of sarcoplasmic reticulum Ca(2+)-ATPase mRNA (r = -0.66, p less than 0.01) in the left ventricular myocardium from the patients with heart failure. There was also a negative correlation between the levels of ANP and the sarcoplasmic reticulum Ca(2+)-ATPase mRNAs (r = -0.65, p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Altered myocardial Ca2+ cycling after left ventricular assist device support in the failing human heart

OBJECTIVES: The objective of the present study was to determine whether improved contractility after left ventricular assist device (LVAD) support reflects altered myocyte calcium cycling and changes in calcium-handling proteins. BACKGROUND: Previous reports demonstrate that LVAD support induces sustained unloading of the heart with regression of pathologic hypertrophy and improvements in contractile performance. METHODS: In the human myocardium of subjects with heart failure (HF), with non-failing hearts (NF), and with LVAD-supported failing hearts (HF-LVAD), intracellular calcium ([Ca(2+)](i)) transients were measured in isolated myocytes at 0.5 Hz, and frequency-dependent force generation was measured in multicellular preparations (trabeculae). Abundance of sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA), Na(+)/Ca(2+) exchanger (NCX), and phospholamban was assessed by Western analysis. RESULTS: Compared with NF myocytes, HF myocytes exhibited a slowed terminal decay of the Ca(2+) transient (DT(terminal), 376 +/- 18 ms vs. 270 +/- 21 ms, HF vs. NF, p < 0.0008), and HF-LVAD myocytes exhibited a DT(terminal) that was much shorter than that observed in HF myocytes (278 +/- 10 ms, HF vs. HF-LVAD, p < 0.0001). Trabeculae from HF showed a negative force-frequency relationship, compared with a positive relationship in NF, whereas a neutral relationship was observed in HF-LVAD. Although decreased SERCA abundance in HF was not altered by LVAD support, improvements in [Ca(2+)](i) transients and frequency-dependent contractile function were associated with a significant decrease in NCX abundance and activity from HF to HF-LVAD. CONCLUSIONS: Improvement in rate-dependent contractility in LVAD-supported failing human hearts is associated with a faster decay of the myocyte calcium transient. These improvements reflect decreases in NCX abundance and transport capacity without significant changes in SERCA after LVAD support. Our results suggest that reverse remodeling may involve selective, rather than global, normalization of the pathologic patterns associated with the failing heart.     

Gene expression of antioxidative enzymes in the human heart: increased expression of catalase in the end-stage failing heart

BACKGROUND: An increase in oxidative stress is suggested to be intimately involved in the pathogenesis of heart failure. However, gene expression of enzymes that metabolize reactive oxygen metabolites has not been investigated in the human heart. METHODS AND RESULTS: Myocardial tissue homogenates of the left ventricular wall from hearts in end-stage failure due to dilated (DCM) or ischemic (ICM) cardiomyopathy (n=12 each), as well as from nonfailing donor hearts (n=12), were analyzed for mRNA levels of manganese superoxide dismutase (MnSOD), copper-zinc superoxide dismutase (CuZnSOD), glutathione peroxidase (GPX), and catalase by Northern blot analyses. Protein levels of MnSOD, CuZnSOD, and catalase were determined by Western blot or ELISA. MnSOD, CuZnSOD, and GPX mRNA levels were similar in all 3 groups. In contrast, catalase mRNA levels were found to be increased by 123+/-23% in DCM hearts and by 93+/-10% in ICM hearts (P<0.01 each) compared with control hearts. Likewise, catalase protein levels were found to be increased in failing hearts (DCM by 90+/-10%, ICM by 90+/-13%; P<0. 05 each) compared with control hearts. In addition, the observed upregulation of catalase mRNA and protein in failing hearts was attended by an increased catalase enzyme activity (DCM by 124+/-16%, ICM by 117+/-15%; P<0.01 each), whereas MnSOD, CuZnSOD, and GPX enzyme activity levels were unchanged in failing compared with nonfailing myocardium. CONCLUSIONS: Increased oxidative stress in human end-stage heart failure may result in a specific upregulation of catalase gene expression as a compensatory mechanism, whereas SOD and GPX gene expression remain unaffected.     

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.     

Multiple alterations in Ca2+ handling determine the negative staircase in a cellular heart failure model

BackgroundThe flat or negative force frequency relationship (FFR) is a hallmark of the failing heart. Either decreases in SERCA2a expression, increases in Na⁺/Ca²⁺ exchanger (NCX) expression or elevated Na⁺_i have been independently proposed as mediators of the negative FFR.Methods and ResultsTo determine whether each one of these mechanisms is sufficient to account for the negative FFR of the failing heart or on the contrary, various mechanisms, acting in concert are required. SERCA2a was pharmacologically inhibited with thapsigargin (TG) or cyclopiazonic acid (CPA) or by using siRNA technology; Na⁺_i was increased with either ouabain (Oua) or monensin and NCX protein was overexpressed by gene transfer (Ad.NCX), to mimic in nonfailing cat myocytes the phenotype of the failing heart and examine their effect on the FFR. The positive FFR of healthy myocytes remained unaffected after either SERCA2a inhibition, Na⁺_i elevation, or NCX overexpression. However, the combination of TG + Oua, Oua + Ad.NCX, or TG + Ad.NCX, converted the positive FFR to negative. Moreover, the FFR became negative at lower frequencies, when the 3 interventions were combined.ConclusionsCa²⁺ handling has to be altered at several levels to explain the negative FFR of the failing heart. These anomalies in Ca²⁺ homeostasis acting in synergy have additive effects.     

Regional expression of phospholamban in the human heart.

BACKGROUND: Several independent lines of evidence indicate that phospholamban (PLB) expression correlates positively with depression of force of contraction and duration of contraction in isolated cardiac preparations of several animal species. Here, we studied whether PLB levels correlate with attenuation of contractility and enhancement of contractile time parameters in different parts of the human heart. METHODS: Force of contraction was measured in isolated electrically driven atrial and ventricular preparations from human hearts. Ca(2+)-uptake by human atrial and ventricular homogenates was assayed at different ionized Ca(2+)-concentrations. Protein expression of PLB and the sarcoplasmic Ca(2+)-ATPase (SERCA) was measured in homogenates by quantitative immunoblotting using specific antibodies. PLB mRNA expression was quantified in human cardiac preparations by Northern blot analysis. RESULTS: The duration of contraction in isolated preparations of human right ventricle (RV) was double that found in right atrial preparations (RA) (620 +/- 25 ms versus 308 +/- 15 ms). In RA, PLB expression was reduced by 44% at the protein level and by 34% at the mRNA level compared to RV. In contrast, the SERCA protein content was increased by 104% in RA compared to RV. Ca(2+)-uptake at low ionized Ca(2+)-concentration, where the inhibiting effect of PLB is maximal, amounted to 1.39 +/- 0.28 nmol Ca2+/mg protein in RA and to 0.62 +/- 0.09 nmol Ca2+/mg protein in RV (n = 6 both). CONCLUSIONS: It is suggested that duration of contraction is shorter in human atrium versus ventricle due to the combined effect of decreased PLB levels (which inhibits SERCA function) and increased SERCA levels. The lower relative ratio of PLB to SERCA leads to less inhibition of SERCA and increased Ca(2+)-uptake which enhances relaxation and contraction in human atrium.     

Restoration of contractile function in isolated cardiomyocytes from failing human hearts by gene transfer of SERCA2a

BACKGROUND: Failing human myocardium is characterized by abnormal relaxation, a deficient sarcoplasmic reticulum (SR) Ca(2+) uptake, and a negative frequency response, which have all been related to a deficiency in the SR Ca(2+) ATPase (SERCA2a) pump. METHODS AND RESULTS: To test the hypothesis that an increase in SERCA2a could improve contractile function in cardiomyocytes, we overexpressed SERCA2a in human ventricular myocytes from 10 patients with end-stage heart failure and examined intracellular Ca(2+) handling and contractile function. Overexpression of SERCA2a resulted in an increase in both protein expression and pump activity and induced a faster contraction velocity (26.7+/-6.7% versus 16.6+/-2.7% shortening per second, P<0.005) and enhanced relaxation velocity (32. 0+/-10.1% versus 15.1+/-2.4%, P<0.005). Diastolic Ca(2+) was decreased in failing cardiomyocytes overexpressing SERCA2a (270+/-26 versus 347+/-30 nmol/L, P<0.005), whereas systolic Ca(2+) was increased (601+/-38 versus 508+/-25 nmol/L, P<0.05). In addition, the frequency response was normalized in cardiomyocytes overexpressing SERCA2a. CONCLUSIONS: These results support the premise that gene-based therapies and targeting of specific pathways in human heart failure may offer a new modality for the treatment of this disease.     

腺相关病毒介导心肌肌浆网Ca2+-ATPase 2a基因转导治疗大鼠慢性心力衰竭

目的 研究腺相关病毒为载体的心肌肌浆网Ca^2＋ -ATPase 2a（sarcoplasmic reticulum Ca^2＋ -ATPase,SERCA2a）基因转导对慢性心力衰竭（HF）大鼠的治疗作用,并探讨其多种可能的机理.方法 采用腹主动脉缩窄术建立HF大鼠模型,应用经腹心包腔内注射术分别将生理盐水、携带eGFP基因和携带SERCA2a基因的重组腺相关病毒导入HF、HF＋EGFP和HF＋SERCA2a组大鼠心脏.于导入30天,检测各组大鼠的心脏功能、SERCA2a蛋白表达和活性;比较HF组和HF＋SERCA2a组大鼠心肌蛋白质组表达的差异;检测各组大鼠心肌肌球蛋白重链（MHC）亚型的表达.结果 HF大鼠心脏内转导入SERCA2a基因30天,心脏收缩和舒张功能达到对照组大鼠水平,并且HF＋SERCA2a组左室重/体重比值显著降低;SERCA2a蛋白表达和活性明显升高至对照组大鼠水平;多种能量代谢酶表达明显增加;α-MHC、β-MHC的表达以及α-MHC/β-MHC恢复至对照组大鼠水平.结论 以重组腺相关病毒2作为载体,SERCA2a基因转导可以增强衰竭心脏的SERCA2a功能,增加心脏能量代谢,纠正MHC亚型的异常表达;在临床方面表现为显著改善心脏收缩和舒张功能,可能能够减轻心脏肥厚等病理性结构改变.     

抑制NADPH氧化酶对心力衰竭兔心肌钙调节蛋白的影响

目的 探讨抑制NADPH氧化酶对心力衰竭(心衰)兔心功能的影响及其机制.方法 通过超容最负荷联合压力负荷建立家兔心衰模型,以NADPH氧化酶特异性抑制剂apocynin给予干预.8周后,观察兔心脏结构和功能的变化,并检测心肌NADPH氧化酶活性以及心肌肌浆网钙泵(sarcoplasmic reticulum Ca~(2+) ATPase,SERCA2a)、雷诺定受体2(ryanodine receptor 2,RyR2)、受磷蛋白(phospholamban,PLB)和钠钙交换体(sodium-calcium exchanger,NCX)基因表达的变化.结果 心衰兔出现明显的心脏扩大和心功能异常,这些改变与心肌NADPH氧化酶活性增加、SERCA2a、RyR2和PLB mRNA表达降低以及NCX mRNA表达增加相关.apocynin显著抑制心衰兔心肌NADPH氧化酶活性(P<0.05),上调SERCA2a、RyR2和PLB mRNA表达(SERCA2a/GAPDH比值:0.63±0.11与0.34±0.08,RyR2/GAPDH比值:0.23±0.04与0.17±0.06,PLB/GAPDH比值:1.28±0.13与0.95±0.09,P<0.05),下调NCX mRNA表达(NCX/GAPDH比值:0.67±0.10与0.95±0.12,P<0.05),并改善心功能[左室射血分数(60.06±10.07)%与(38.87±3.31)%,左室短轴缩短率(30.12±6.56)%与(17.40±2.45)%,P<0.05].结论 抑制NADPH氧化酶能够调节心衰时心肌钙调节蛋白异常表达,这可能是其改善心衰时心功能的分子机制.     

Impaired SERCA function contributes to cardiomyocyte dysfunction in insulin resistant rats

Ventricular dysfunction in type 2 diabetic patients is becoming apparent early after diagnosis of diabetes, but the cellular mechanisms contributing to this dysfunction are not well established. Our group has recently identified cardiomyocyte dysfunction in diet-induced insulin resistant rats that have not developed type 2 diabetes. The present investigation was designed to determine cellular mechanisms contributing to slowed cardiomyocyte relaxation in sucrose (SU)-fed rats. SU-feeding was used to induce whole-body insulin resistance. After 9-12 weeks on diet, isolated ventricular myocyte shortening/relengthening were slower in SU-fed adult male Wistar rats (42-63%) compared to starch (ST)-fed controls. Cytosolic Ca2+ removal attributable to Na+/Ca2+ exchange (NCX) and to sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was evaluated with fluo-3/AM. Caffeine-releasable Ca2+ and cytosolic Ca2+ clearing through NCX were normal, whereas Ca2+ uptake by SERCA was significantly slower in SU myocytes (330+/-29 ms) compared to ST cells (253+/-16 ms). Protein levels for SERCA, NCX and phospholamban were not affected by SU-feeding. Manipulating intracellular Ca2+ with various positive inotropic interventions (e.g. post-rest potentiation, isoproterenol) and changes in stimulus frequency demonstrated that mechanical properties can be improved in subsets of myocytes. Thus, we conclude that impaired SERCA activity (with normal protein content) contributes to cardiomyocyte dysfunction in insulin resistant animals, whereas NCX function and expression are normal. These results suggest that subtle changes in Ca2+ regulation which occur prior to overt ventricular dysfunction/failure, may be common to early stages of a number of disorders involving insulin resistance (e.g. diabetes, obesity, syndrome X and hypertension).     

Modulation of cardiac contractility by the phospholamban/SERCA2a regulatome

Heart disease remains the leading cause of death and disability in the Western world. Current therapies aim at treating the symptoms rather than the subcellular mechanisms, underlying the etiology and pathological remodeling in heart failure. A universal characteristic, contributing to the decreased contractile performance in human and experimental failing hearts, is impaired calcium sequestration into the sarcoplasmic reticulum (SR). SR calcium uptake is mediated by a Ca(2+)-ATPase (SERCA2), whose activity is reversibly regulated by phospholamban (PLN). Dephosphorylated PLN is an inhibitor of SERCA and phosphorylation of PLN relieves this inhibition. However, the initial simple view of a PLN/SERCA regulatory complex has been modified by our recent identification of SUMO, S100 and the histidine-rich Ca-binding protein as regulators of SERCA activity. In addition, PLN activity is regulated by 2 phosphoproteins, the inhibitor-1 of protein phosphatase 1 and the small heat shock protein 20, which affect the overall SERCA-mediated Ca-transport. This review will highlight the regulatory mechanisms of cardiac contractility by the multimeric SERCA/PLN-ensemble and the potential for new therapeutic avenues targeting this complex by using small molecules and gene transfer methods.