Increased neuronal nitric oxide synthase-derived NO production in the failing human heart

Experimental data suggest that nitric oxide (NO) generated from neuronal NO synthase (nNOS) modulates the myocardial inotropic state. To assess the contribution of NO, derived from endothelial and neuronal isoforms, to the pathophysiology of congestive heart failure in human beings, we compared expression, localisation, and specific activity of NOS isoforms in myocardium from patients with dilated cardiomyopathy with those in controls who had died from head trauma or intracranial bleeds. Diseased hearts had a significant increase in nNOS mRNA and protein expression, and activity associated with the translocation of nNOS to the sarcolemma through interactions with caveolin 3. Enhanced nNOS activity counteracted a decrease in eNOS expression and activity. Our results provide evidence of increased nNOS-derived NO in the failing human heart. Such altered regulation may be important in the pathophysiology of cardiac dysfunction in human congestive heart failure.     

An inhibitor of nitric oxide synthase does not increase contraction or beta-adrenoceptor sensitivity of ventricular myocytes from failing human heart

OBJECTIVE: Nitric oxide (NO) has been implicated in the depression of cardiac function in human heart failure. Some reports have identified iNOS (inducible nitric oxide synthase) within the myocyte component of the failing human heart, and NO is known to decrease the contraction amplitude of isolated ventricular myocytes. We have treated myocytes from failing human ventricle with a NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), in an attempt to restore contractile function. METHODS AND RESULTS: Myocytes were isolated from failing and non-failing human ventricles and their contraction amplitude was measured during superfusion (32 degrees C, 1-2 mmol/l Ca2+) and electrical stimulation (0.1-2 Hz). The contraction amplitude of myocytes from failing hearts was depressed in a frequency-dependent manner. At 1 Hz, the contraction amplitude of cells from non-failing heart was 4.70 +/- 0.53% cell shortening (mean +/- SEM, n = 13 subjects), compared with 2.18 +/- 0.27% (P < 0.01, 11 patients) from patients with ischaemic heart disease (IHD) or 2.56 +/- 0.74% (P < 0.02, six patients) with dilated cardiomyopathy (DCM). Superfusion with 0.1 mmol/l L-NMMA did not increase contraction amplitude in myocytes from failing heart at either 0.2 Hz (n = 11) or 1 Hz (n = 7). Responses to beta-adrenoceptor stimulation were reduced in myocytes from failing human heart, with contraction amplitude in maximum isoprenaline 0.47 +/- 0.11 of that in high Ca2+ in the same cell (n = 6), compared to 0.99 +/- 0.07 in non-failing heart (n = 14, P < 0.01). The presence of 0.1 mmol/l L-NMMA did not increase the isoprenaline/Ca2+ ratio in myocytes from failing heart (0.40 +/- 0.09, P = NS). CONCLUSION: These results do not suggest a functional role for tonic NO production in the frequency-dependent depression of contraction or beta-adrenoceptor desensitisation in myocytes from failing human ventricle.     

Evidence for calcineurin-mediated regulation of SERCA 2a activity in human myocardium

Compromised SERCA 2a activity is a key malfunction leading to the Ca(2+) cycling alterations in failing human myocardium. SERCA 2a activity is regulated by the Ca(2+)/calmodulin-dependent protein kinase (CaM-kinase) but alterations of the CaM-kinase pathway regarding SERCA 2a in heart failure are unresolved. Therefore we investigated the CaM-kinase and phosphatase calcineurin mediated regulation of SERCA 2a in failing and non-failing human myocardium. We studied human myocardial preparations from explanted hearts from non-failing organ donors (NF, n=8) and from patients with terminal heart failure undergoing cardiac transplantation (dilated cardiomyopathy, DCM, n=8). SERCA 2a activity was determined using a NADH-coupled enzyme assay [expressed in nmol ATP/(mg protein x min)] and by(45)Ca(2+) uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/microg protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [gamma -(32)P] (expressed as pmol(32)P/mg). Maximal(45)Ca(2+) uptake (in pmol/mg/min) (NF: 3402+/-174; DCM: 2488+/-189) and maximal SERCA 2a activity were reduced in DCM compared to NF (V(max): NF: 125+/-9; DCM: 98+/-5). The V(max) reduction could be mimicked by calcineurin in vitro in NF (NF(control): 72.1+/-3.7; NF(+calcineurin): 49.8+/-2.9) and restored in DCM by CaM-kinase in vitro (DCM(control): 98+/-5; DCM(+CaM-kinase): 120+/-6). Protein expression of SERCA 2a, phospholamban and calsequestrin remained similar, but calcineurin expression was significantly increased in failing human hearts (NF: 11.6+/-1.5 v DCM: 17.1+/-1.6). Although the capacity of endogenous CaM-kinase to phosphorylate PT17 was significantly higher in DCM (DCM(control): 128+/-36; DCM(+endogenous CaM-kinase): 205+/-20) compared to NF myocardium (NF(control): 273+/-37; NF(+endogenous CaM-kinase): 254+/-31), net phosphorylation at threonine-17 phospholamban was significantly lower in DCM (DCM 130+/-11 v NF 170+/-11). A calcineurin-dependent dephosphorylation of phospholamban could be mimicked in vitro by incubation of NF preparations with calcineurin (NF(control) 80.7+/-4.4 v NF(+calcineurin) 30.7+/-4.1, P<0.05). In human myocardium, the V(max) of SERCA 2a and the phosphorylation of phospholamban is modulated by CaM-kinase and calcineurin, at least in vitro. In failing human myocardium, despite increased CaM-kinase activity, calcineurin dephosphorylation leads to decreased net phosphorylation of threonine-17 phospholamban in vivo. Increased calcineurin activity contributes to the impaired V(max) of SERCA 2a in failing human myocardium and the disorder in Ca(2+)-handling in heart failure.     

Myocardial contractile effects of nitric oxide

Recent experimental and clinical research solved some of the controversies surrounding the myocardial contractile effects of NO. These controversies were: (1) does NO exert a contractile effect at baseline? (2) is NO a positive or a negative inotrope? (3) Are the contractile effects of NO similar when NO is derived from NO-donors or from the different isoforms of NO synthases (NOS)? (4) Does NO exert the same effects in hypertrophied, failing or ischemic myocardium? Transgenic mice with cardioselective overexpression of NOS revealed NO to produce a small reduction in basal developed LV pressure and a LV relaxation-hastening effect mainly through myofilamentary desensitization. Similar findings had previously been reported during intracoronary infusions of NO-donors in isolated rodent hearts and in humans. The LV relaxation hastening effect was accompanied by increased diastolic LV distensibility, which augmented LV preload reserve especially in heart failure patients. This beneficial effect on diastolic LV function always overrode the small NO-induced attenuation in LV developed pressure in terms of overall LV performance. In most experimental and clinical conditions, contractile effects of NO were similar when NO was derived from NO-donors or produced by the different isoforms of NOS. Because expression of inducible NOS (NOS2) is frequently accompanied by elevated oxidative stress, NO produced by NOS2 can lead to peroxynitrite-induced contractile impairment as observed in ischemic or septic myocardium. Finally, shifts in isoforms or in concentrations of myofilaments can affect NO-mediated myofilamentary desensitization and alter the myocardial contractile effects of NO in hypertrophied or failing myocardium.     

Increased messenger RNA level of the inhibitory G protein alpha subunit Gi alpha-2 in human end-stage heart failure.

In human heart failure the positive inotropic and cAMP-elevating effects of both beta-adrenoceptor agonists and phosphodiesterase inhibitors are diminished. This has been explained at least in part by an increase in the inhibitory signal-transducing G protein (Gi) and unchanged stimulatory G protein (Gs). In the present study we determined the mRNA expression pattern of the alpha subunits of Gi-1, Gi-2, Gi-3, and Gs in myocardial tissue samples of patients undergoing heart transplantation. Northern blot analysis of total RNA extracted from left ventricles with 32P-labeled cDNAs demonstrated expression of Gi alpha-2, Gi alpha-3, and Gs alpha mRNA. In contrast, Gi alpha-1 mRNA was not detectable. To investigate whether the increased ratio of Gi/Gs might be due to altered gene expression, we compared mRNA levels of Gi alpha-2, Gi alpha-3, and Gs alpha in left ventricular myocardium from failing hearts with idiopathic dilated cardiomyopathy (n = 8) and ischemic cardiomyopathy (n = 6) and from nonfailing hearts from transplant donors (n = 8). Compared with nonfailing control hearts, the Gi alpha-2 mRNA was increased by 75 +/- 26% (p less than 0.05) in idiopathic dilated cardiomyopathy hearts and 90 +/- 26% (p less than 0.05) in ischemic cardiomyopathy hearts. Gi alpha-3 and Gs alpha mRNA levels were similar in the three groups. The results suggest that as in other mammalian species, Gi alpha-2 and Gi alpha-3 mRNA are the predominant Gi alpha mRNA subtypes in human ventricular myocardium.(ABSTRACT TRUNCATED AT 250 WORDS)     

钙调神经磷酸酶在人类心肌中的表达与分布

目的 了解钙调神经磷酸酶(CaN)在人类健康心肌和衰竭心肌中的表达与分布.方法 以12例行移植手术的终末期衰竭心脏和5例因车祸丧生的"健康供体"心脏为研究对象,分别利用免疫组化和Western blot技术对左、右心室中CaN的表达与分布进行研究.结果 CaN在心脏的心肌细胞、纤维母细胞、心外膜间皮细胞中染色阳性,且Western blot实验在后两种细胞中均检测到单一的58 000带,心肌内血管内皮细胞和平滑肌细胞中CaN染色阴性.与"供体"心肌比较,无论左心室还是右心室,衰竭心肌的CaN蛋白水平差异均无统计学意义(衰竭右心室和供体右心室电泳带强度分别为130.20±8.66和139.87±6.21,P=0.33.衰竭左心室和供体左心室电泳带强度分别为106.45和126.34±12.09),且左右心室肌CaN蛋白水平差异也无统计学意义(衰竭心肌左、右心室电泳带强度分别为96.99±10.67和104.58±13.18,P=0.63.供体心肌左、右心室电泳带强度分别为132.12和120.74).结论 CaN存在于人类心脏多数类型细胞中,包括心肌细胞、纤维母细胞和心外膜间皮细胞.心力衰竭时,CaN蛋白水平没有改变.     

Regional expression and functional characterization of the L-type Ca2+-channel in myocardium from patients with end-stage heart failure and in non-failing human hearts

The purpose of the present study was to investigate the expression and functional relevance of sarcolemmal L-type Ca2+-channels in failing and non-failing human myocardium. The protein expression of sarcolemmal L-type Ca2+-channels was determined with 3H-(+)-PN 200-110-binding experiments and Western blot analysis using a specific antibody against the alpha1-subunit in membrane preparations of ventricular and atrial myocardium from both failing (n = 15) and non-failing hearts (n = 8). The gene expression of the ion conducting pore of the L-type Ca2+-channel was examined with Northern blot technique in human failing and non-failing RNA. For normalization the RNA expression of calsequestrin was used. In electrically driven ventricular papillary muscle strips and auricular trabeculae, the responses to nifedipine and Ca2+ as parameters of myocardial function were studied. The protein expression as measured by 3H-(+)-PN 200-110-binding (Bmax) and Western Blot analysis with calsequestrin as reference was similar in left ventricular failing and non-failing myocardium. However, both were reduced in atrial compared to ventricular tissue in failing and non-failing hearts. The KD remained unchanged. Calsequestrin levels were unaltered in failing and non-failing hearts. The gene expression of the alpha1-subunit was similar in human failing and non-failing hearts. The L-type Ca2+-channel antagonist nifedipine reduced force of contraction with the same potency and efficiency in ventricular failing and non-failing myocardium. In contrast, the potency of nifedipine was higher in atrial than in ventricular tissue. Consistently, atrial myocardium from patients with dilated cardiomyopathy was more sensitive towards Ca2+ than those of the control group. In conclusion, the altered Ca2+-homeostasis in failing human myocardium may be less due to changes in sarcolemmal L-type Ca2+-channel expression or function than due to an altered intracellular Ca2+-handling.     

Angiotensin II receptors in normal and failing human hearts

To demonstrate the existence and help clarify the function of angiotensin II (Ang II) receptors in the human heart, we characterized the cardiac Ang II receptor and examined the levels and distribution of ventricular Ang II receptors in normal (n = 6) and failing (n = 14) hearts. Ang II receptors were characterized using the Ang II receptor agonist [125I]Ang II. Cardiac [125I]Ang II-binding sites were of high affinity (Kd, approximately 1 nmol/L) and low capacity (Bmax, approximately 3 fmol/mg membrane protein) and were pharmacologically specific [IC50 values for Ang II, [Sar1,Ile8]Ang II, and Ang III were 1.2, 3.0, and 400 nmol/L, respectively; the inactive Ang II metabolite Ang-(1-5), at a concentration of 1 mumol/L, inhibited [125I]Ang II binding by less than 10%]. These characteristics of cardiac [125I]Ang II-binding sites are similar to those of previously characterized mammalian heart Ang II receptors. In normal adult donor hearts (n = 5), Ang II receptor density in the left ventricle [LV, 2.90 +/- 1.40 (+/- SE) fmol/mg] was similar to that in the right ventricle (RV, 3.82 +/- 1.10 fmol/mg). The ventricular Ang II receptor density in adult patients with idiopathic (LV, 1.77 +/- 0.35 fmol/mg; RV, 1.58 +/- 0.29 fmol/mg; n = 8) or dilated cardiomyopathy (LV, 2.00 +/- 0.58 fmol/mg; RV, 2.56 +/- 0.52 fmol/mg n = 5) was similar to that in the normal heart. Ventricular Ang II receptors, localized by autoradiography using the Ang II receptor antagonist [125I]-[Sar1,Ile8]Ang II, were consistently found in the myocardium, cardiac adrenergic nerves, and coronary vessels of normal and failing ventricles. In human ventricles Ang II receptor levels were not correlated with age. Because ventricular Ang II receptor density in a normal neonatal human heart and that in a heart from an adolescent patient with idiopathic cardiomyopathy were more than 10-fold and more than 5-fold higher, respectively, than in normal adult ventricles, we investigated whether postnatal changes occur in ventricular Ang II receptors in rats. In male and female rats ventricular Ang II receptor density was about 2-fold higher in 1-day-old rats compared to that in 10-day-old or peripubertal rats. These data suggest developmental regulation of ventricular Ang II receptors. Our findings suggest that direct and neural angiotensinergic inputs to the myocardium play a role in the regulation of cardiac function in man and that these inputs are preserved in the failing heart.     

Angiotensin II-forming pathways in normal and failing human hearts

Reduced preload and afterload to the heart are important effects of angiotensin converting enzyme (ACE) inhibitors in the treatment of congestive heart failure. However, since angiotensin II (Ang II) directly increases the strength of myocardial contraction, suppression of Ang II formation by ACE inhibitors could potentially reduce the beneficial effects of Ang II on the failing heart. To study how ACE inhibition suppresses cardiac Ang II formation in man, we characterized ACE-dependent and ACE-independent Ang II-forming pathways in eight normal and 24 failing human hearts obtained at cardiac transplantation. Ang II-forming activity in left ventricular (LV) membrane preparations was assessed by measuring the conversion of [125I]angiotensin I (Ang I) to [125I]Ang II. LV [125I]Ang II-forming activity in normal hearts (35.5 +/- 2.7 fmol/min/mg, n = 8) was not different from that in hearts from patients with ischemic cardiomyopathy (25.5 +/- 2.9 fmol/min/mg, n = 9) and was 48% lower (p less than 0.001) in hearts from patients with idiopathic cardiomyopathy (18.5 +/- 1.9 fmol/min/mg, n = 15).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

GH-independent cardiotropic activities of hexarelin in patients with severe left ventricular dysfunction due to dilated and ischemic cardiomyopathy

AIM: To investigate acute cardiotropic activities of hexarelin in patients with severe left ventricular dysfunction due to ischemic (iCMP) and dilated cardiomyopathy (dCMP). METHODS AND RESULTS: We studied the effect of intravenous hexarelin administration on growth hormone (GH) levels and left ventricular ejection fraction (LVEF) evaluated by radionuclide angiography in eight patients with dCMP (age 53.0+/-2.8, LVEF 16.7+/-2.1%) and five patients with iCMP (age 52.0+/-2.8 years, LVEF 22.6+/-2.1). Results were compared with a group of seven normal subjects (age 37.4+/-3.4 years, LVEF 64.0+/-1.5%) and seven patients with severe growth-hormone deficiency (GHD; age 42.0+/-4.4 years, LVEF 50.0+/-1.9%) previously studied with the same methodology. In dCMP and iCMP patients hexarelin induced a similar significant (P<0.05) increase in GH levels. In iCMP patients hexarelin induced a LVEF increase (peak LVEF 26.2+/-2.5%, P<0.05) as observed in normals and GHD, while in dCMP LVEF was unchanged (peak LVEF 17.7+/-1.7, P=NS). In all groups other hemodynamic parameters were unchanged. CONCLUSIONS: Acute hexarelin administration increases LVEF in iCMP patients (as in normals and GHD) but not in dCMP patients in spite of a similar GH releasing effect and basal LVEF. A possible explanation of the positive inotropic effect of hexarelin in iCMP could be a direct stimulation on viable myocardium or myocardial contractile reserve.     

Decreased protein and phosphorylation level of the protein phosphatase inhibitor-1 in failing human hearts

OBJECTIVE: The protein phosphatase inhibitor-1 (I-1) is a highly specific and potent inhibitor of type 1 phosphatases (PP1) that is active only in its protein kinase A (PKA)-phosphorylated form. I-1 ablation decreases, I-1 overexpression sensitizes beta-adrenergic signaling in the heart. It is controversial whether I-1 expression is altered in human heart failure (HF), likely because its detection in heart is difficult due to its low abundance. METHODS AND RESULTS: I-1 was >500-fold enriched from left ventricular myocardium (LVM) from patients with terminal HF (n=16) and non-failing controls (NF, n=5) and quantified with an affinity-purified I-1 and a I-1 phosphospecific antiserum. In non-failing I-1 protein levels amounted to 126 fmol/mg protein. In failing hearts, I-1 protein levels were reduced by 58% and I-1 phosphorylation by 77% (P<0.001 vs. NF). I-1 phosphorylation correlated well with serine-16 phosphorylation of phospholamban (PLB) in the same hearts (P<0.001). In contrast, PLB, troponin I (TnI) and PP1 protein and TnI phosphorylation levels did not differ between HF and NF. CONCLUSIONS: The results suggest that the reduction in I-1 protein and phosphorylation in failing human hearts leads to increased phosphatase activity which in turn may result in reduced phosphorylation of cardiac proteins such as PLB.     

Myocardial catecholamine and neuropeptide Y depletion in failing ventricles of patients with idiopathic dilated cardiomyopathy. Correlation with beta-adrenergic receptor downregulation.

BACKGROUND. Myocardial adrenergic neurotransmitters and beta-adrenergic receptor levels were measured in left and right ventricular myocardial specimens obtained from 30 patients with biventricular failure resulting from idiopathic dilated cardiomyopathy. METHODS AND RESULTS. Nonfailing myocardium obtained from 12 organ donors provided control data. Norepinephrine, dopamine, and neuropeptide Y concentrations were significantly decreased in failing compared with nonfailing control hearts. The mean ratio of dopamine to norepinephrine and of dopamine to neuropeptide Y in failing hearts was also significantly decreased compared with nonfailing control hearts. Compared with nonfailing control hearts, Bmax and beta 1-receptor density were significantly decreased in failing hearts and there were weak but significantly positive correlations of Bmax and beta 1-adrenergic receptors with norepinephrine, dopamine, and neuropeptide Y. CONCLUSIONS. Norepinephrine and its cotransmitter neuropeptide Y are depleted in failing human ventricular myocardium. Decreased norepinephrine stores correlate weakly with beta 1-adrenergic receptor downregulation consistent with the hypothesis that norepinephrine depletion occurs in response to increased adrenergic drive. Decreased dopamine relative to norepinephrine implies that an abnormality of dopamine conversion to norepinephrine is not present in failing human heart.     

The gene expression fingerprint of human heart failure

Multiple pathways are responsible for transducing mechanical and hormonal stimuli into changes in gene expression during heart failure. In this study our goals were (i) to develop a sound statistical method to establish a comprehensive cutoff point for identification of differentially expressed genes, (ii) to identify a gene expression fingerprint for heart failure, (iii) to attempt to distinguish different etiologies of heart failure by their gene expression fingerprint, and (iv) to identify gene clusters that show coordinated up- or down-regulation in human heart failure. We used oligonucleotide microarrays to profile seven nonfailing (NF) and eight failing (F) human hearts with a diagnosis of end-stage dilated cardiomyopathy. Biological and experimental variability of the hybridization data were analyzed, and then a statistical analysis procedure was developed, including Student's t test after log-transformation and Wilcoxon Mann-Whitney test. A comprehensive cutoff point composed of fold change, average difference, and absolute call was then established and validated by TaqMan PCR. Of 6,606 genes on the GeneChip, 103 genes in 10 functional groups were differentially expressed between F and NF hearts. A dendrogram identified a gene expression fingerprint of F and NF hearts and also distinguished two F hearts with distinct etiologies (familial and alcoholic cardiomyopathy, respectively) with different expression patterns. K means clustering also revealed two potentially novel pathways associated with up-regulation of atrial natriuretic factor and brain natriuretic peptide and with increased expression of extracellular matrix proteins. Gene expression fingerprints may be useful indicators of heart failure etiologies.