Birgit Bölck

Sarcoplasmic reticulum Ca2+-ATPase modulates cardiac contraction and relaxation.

The cardiac SR Ca(2+)-ATPase (SERCA2a) regulates intracellular Ca(2+)-handling and thus, plays a crucial role in initiating cardiac contraction and relaxation. SERCA2a may be modulated through its accessory phosphoprotein phospholamban or by direct phosphorylation through Ca(2+)/calmodulin dependent protein kinase II (CaMK II). As an inhibitory component phospholamban, in its dephosphorylated form, inhibits the Ca(2+)-dependent SERCA2a function, while protein kinase A dependent phosphorylation of the phospho-residues serine-16 or Ca(2+)/calmodulin-dependent phosphorylation of threonine-17 relieves this inhibition. Recent evidence suggests that direct phosphorylation at residue serine-38 in SERCA2a activates enzyme function and enhances Ca(2+)-reuptake into the sarcoplasmic reticulum (SR). These effects that are mediated through phosphorylation result in an overall increased SR Ca(2+)-load and enhanced contractility. In human heart failure patients, as well as animal models with induced heart failure, these modulations are altered and may result in an attenuated SR Ca(2+)-storage and modulated contractility. It is also believed that abnormalities in Ca(2+)-cycling are responsible for blunting the frequency potentiation of contractile force in the failing human heart. Advanced gene expression and modulatory approaches have focused on enhancing SERCA2a function via overexpressing SERCA2a under physiological and pathophysiological conditions to restore cardiac function, cardiac energetics and survival rate.

Nebivolol, bucindolol, metoprolol and carvedilol are devoid of intrinsic sympathomimetic activity in human myocardium

The present study investigated whether or not there may be differences in the direct cardiac actions of the novel, highly β1‐selective adrenoceptor antagonist nebivolol (NEB) in comparison to metoprolol (MET), bisoprolol (BIS), carvedilol (CAR) and bucindolol (BUC) in human myocardium (n=9). The rank order of β1‐selectivity as judged by competition experiments to 3H‐CGP 12.1777 in the presence of CGP 207.12 A (300 nmol l−1, Kiβ2) or ICI 118.551 (50 nmol l−1, Kiβ1) were NEB(Kiβ2/Kiβ1: 40.7)>BIS(15.6)>MET(4.23)>CAR(0.73)>BUC(0.49). The rank order of the negative inotropic potency of the β‐adrenoceptor antagonists measured in left ventricular trabeculae (dilated cardiomyopathy, DCM) as judged by the concentration needed to induce a 50% decrease in isoprenaline (1 μmol l−1)‐stimulated force (IC50) was: MET (0.6 μmol l−1)>CAR (4.1 μmol l−1)>NEB (7.0 μmol l−1). NEB, BUC, MET and CAR did not not exert an intrinsic sympathomimetic activity (ISA) as determined by measurements of force development in forskolin (0.3 μmol l−1) pre‐treated left ventricular trabeculae, nor by measuring adenylate cyclase activity in forskolin (0.3 μmol l−1)‐stimulated assays (crude membranes). This also holds true for radioligand binding assays with or without guanine nucleotide guanyl‐5′‐yl imidodiphosphate (Gpp(NH)p). Although all studied β‐adrenoceptor antagonists lack intrinsic sympathomimetic activity (ISA), they differ in the β1‐selectivity as well as in their direct negative inotropic action. These differences as well as the mode of extracardiac action may have an impact on outcome of patients treated with β‐adrenoceptor antagonists.

Platelet Sarcoplasmic Endoplasmic Reticulum Ca2+-ATPase and μ-Calpain Activity Are Altered in Type 2 Diabetes Mellitus and Restored by Rosiglitazone

Background— Platelets from patients with type 2 diabetes mellitus display hyperaggregability and increased thrombogenic potential. Methods and Results— In platelets from patients with type 2 diabetes mellitus, we found enhanced tyrosine nitration and inactivation of the sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA-2), elevated platelet [Ca2+]i, and activation of &mgr;-calpain. The tyrosine nitration of SERCA-2 and the activation of &mgr;-calpain in vitro in platelets from healthy volunteers could be evoked in vitro by peroxynitrite. Platelet endothelial cell adhesion molecule-1 was identified as a &mgr;-calpain substrate; its in vitro degradation was stimulated by peroxynitrite and prevented by calpain inhibitors. Calpain activation also was linked to hyperresponsiveness to thrombin and the loss of platelet sensitivity to nitric oxide synthase inhibitors. Platelets from patients with type 2 diabetes mellitus (hemoglobin A1c >6.6%) contained little or no intact platelet endothelial cell adhesion molecule-1, whereas degradation products were detectable. The peroxisome proliferator–activated receptor-&ggr; agonist rosiglitazone increased SERCA-2 expression in megakaryocytes, and treating patients with type 2 diabetes mellitus with rosiglitazone for 12 weeks increased platelet SERCA-2 expression and Ca2+-ATPase activity, decreased SERCA-2 tyrosine nitration, and normalized platelet [Ca2+]i. Rosiglitazone also reduced &mgr;-calpain activity, normalized platelet endothelial cell adhesion molecule-1 levels, and partially restored platelet sensitivity to nitric oxide synthase inhibition. Conclusion— These data identify megakaryocytes/platelets as additional cellular targets for peroxisome proliferator–activated receptor-&ggr; agonists and highlight potential benefits of rosiglitazone therapy in cardiovascular diseases.

Modulation of SERCA: implications for the failing human heart.

Abstract Human heart failure is characterized by distinct alterations in the intracellular homeostasis and key regulators of the sarcoplasmic reticulum Ca2+ sequestration mechanisms. Systolic peak Ca2+ is reduced, diastolic Ca2+ levels are increased and diastolic Ca2+ decay is prolonged. Recently specific changes in the expression, function and modulation of SR Ca2+-ATPase (SERCA) have been elucidated. As such, in a variety of studies SERCA expression appeared to be decreased in the failing human heart, although these findings have been discussed controversially depending on the studied tissue, especially with respect to the non-failing samples and regional variation in the obtained samples. However, consistent findings of a diminished Ca2+ dependent SERCA activation were found. Increasing evidence has been provided that one of the underlying mechanisms for a decreased activation of SERCA is its altered regulation. With respect to this, the modulations through phospholamban and Ca2+-dependent protein kinase II (CaMK II) play a detrimental role in regulating SERCA function. Phospholamban phosphorylation of SERCA at the serine-16 and threonine-17 site is diminished in human heart failure resulting in decreases in the apparent affinity for Ca2+ of the SR Ca2+ uptake rates. In contrast, activation of CaMK II leads to an increased maximal velocity of SR Ca2+ sequestration that may enhance SR Ca2+-load. Additional regulation has been recently elucidated by changes in the apparent coupling ratio of Ca2+ transported per ATP hydrolysed. This review summarizes recent advances in the understanding how SERCA is modulated under physiological and pathophysiological conditions.

The preferential β3‐adrenoceptor agonist BRL 37344 increases force viaβ1‐/β2‐adrenoceptors and induces endothelial nitric oxide synthase viaβ3‐adrenoceptors in human atrial myocardium

The present study investigated the effects of the preferential β3‐AR agonist BRL 37344 (BRL) on force of contraction (FOC), Ca2+‐transient and eNOS‐activity in human right atrial myocardium. BRL concentration‐dependently caused an increase in FOC that was paralleled by an increase in Ca2+‐transient and a shortening of time to half peak relaxation (T0.5T). These effects were abolished in the presence of propranolol (0.3 μM). BRL acted as a competitive antagonist towards isoprenaline and in binding experiments it was shown to have a distinct affinity towards β1/2‐AR. In immunohistochemical experiments BRL (10 μM) increased detection of activated eNOS. This effect remained constant in the presence of propranolol (0.3 μM). BRL increased directly detected NO in DAF‐staining experiments. This increase was significantly smaller in the presence of the NO‐inhibitor L‐NAME. The inotropic effects of BRL were not changed in the presence of L‐NMA. These results suggest that the inotropic effects of BRL in human atrium are mediated via β1/2‐AR, whereas the increase of atrial eNOS‐activity is due to β3‐ adrenergic stimulation. This increase in eNOS‐activity did not influence atrial myocardial contractility. In conclusion, this study shows that β3‐adrenergic stimulation is present in human atrium, but may not be functionally as significant as in the left ventricle.

β1-adrenoceptor selectivity of nebivolol and bisoprolol. A comparison of [3H]CGP 12.177 and [125I]iodocyanopindolol binding studies

There is an ongoing discussion on whether or not high beta(1)-adrenoceptor selectivity of beta-adrenoceptor antagonists may be favorable in the treatment of patients with heart failure. The present study compared the beta(1)-adrenoceptor selectivity of nebivolol and bisoprolol with that of carvedilol in the human myocardium, using a binding assay in conjunction with either the hydrophilic ligand (+/-)-[3H]4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on HCl ([3H]CGP 12.177) or the lipophilic ligand [125I]iodocyanopindolol as radiolabeled compound. Measurements were made using membrane preparations obtained from identical nonfailing donor hearts. beta-adrenoceptor density was found to be slightly higher when [125I]iodocyanopindolol was used compared to [3H]CGP 12.177 (256+/-15 and 213+/-18 fmol/mg protein, respectively). When the highly beta(1)-adrenoceptor-selective compound 2-hydroxy-5-(2-(hydroxy-3-(4((1-methyl-4-trifluoromethyl)-1-H-imidazol-2-yl)-phenoxy)-propyl)-aminoethoxyl)-benzamide (CGP 20.712A) and the highly beta(2)-adrenoceptor-selective compound erythro-(+/-)-1-(7-methylindan-4-yloyl)-3-isopropylaminobutan-2-ol HCl (ICI 118.551) were used in competition experiments, a similar proportion of beta(1)-adrenoceptors was seen for [3H]CGP 12.177 (69.3+/-1.6%) and for [125I]iodocyanopindolol (67.0+/-2.1%). K(i)(beta(1)) and K(i)(beta(2)) were obtained in the presence of 50 nM ICI 118.551 and 300 nM CGP 20.712A. The rank order of beta(1)-adrenoceptor selectivity (K(i)(beta(2))/K(i)(beta(1)) ratio) was nebivolol (for [3H]CGP 12.177 46.1 and for [125I]iodocyanopindolol 22.5)>bisoprolol (13.1 and 6.4)>carvedilol (0.65 and 0.41). To investigate whether in vivo metabolized nebivolol retains high beta(1)-adrenoceptor selectivity, serum specimens were collected before and 2 h after oral administration of 5 mg nebivolol. The samples were used for [125I]iodocyanopindolol binding studies with the myocardial membrane preparations. In these samples, the binding of [125I]iodocyanopindolol to beta(1)-adrenoceptors was inhibited by 46.4+/-5.3%, whereas the binding to beta(2)-adrenoceptors was inhibited by 20.5+/-1.1% compared to that of control samples. It is concluded that nebivolol is approximately 3.5 times more beta(1)-adrenoceptor-selective than bisoprolol in the human myocardium. Furthermore, in vivo metabolized nebivolol retains beta(1)-adrenoceptor selectivity.

Mechanisms of Ca2+-Dependent Calcineurin Activation in Mechanical Stretch-Induced Hypertrophy

Pressure overload is the major stimulus for cardiac hypertrophy. Accumulating evidence suggests an important role for calcium-induced activation of calcineurin in mediating hypertrophic signaling. Hypertrophy is an important risk factor for cardiovascular morbidity and mortality. We therefore employed an in vitro mechanical stretch model of cultured neonatal cardiomyocytes to evaluate proposed mechanisms of calcium-induced calcineurin activation in terms of inhibition of calcineurin activity and hypertrophy. The protein/DNA ratio and ANP gene expression were used as markers for stretch-induced hypertrophy. Stretch increased the calcineurin activity, MCIP1 gene expression and DNA binding of NFATc as well as the protein/DNA ratio and ANP mRNA in a significant manner. The specific inhibitor of calcineurin, cyclosporin A, inhibited the stretch-induced increase in calcineurin activity, MCIP1 gene expression and hypertrophy. The L-type Ca2+ channel blocker nifedipine and a blocker of the Na+/H+ exchanger (cariporide) both suppressed stretch-dependent enhanced calcineurin activity and hypertrophy. Also application of a blocker of the Na+/Ca2+ exchanger (KB-R7943) was effective in preventing calcineurin activation and increases in the protein/DNA ratio. Inhibition of capacitative Ca2+ entry with SKF 96365 was also sufficient to abrogate calcineurin activation and hypertrophy. The blocker of stretch-activated ion channels, streptomycin, was without effect on stretch-induced hypertrophy and calcineurin activity. The present work suggests that of the proposed mechanisms for the calcium-induced activation of calcineurin (L-type Ca2+ channels, capacitative Ca2+ entry, Na+/H+ exchanger, Na+/Ca2+ exchanger and stretch-activated channels) all but stretch-activated channels are possible targets for the inhibition of hypertrophy.

Frequency dependent force generation correlates with sarcoplasmic calcium ATPase activity in human myocardium

AbstractObjective: In congestive heart failure both a decreased function of the sarcoplasmic Ca2+-ATPase and a negative force-frequency relationship have been shown. This study aimed to investigate a possible relationship between frequency potentiation, sarcoplasmic Ca2+-ATPase activity, and SERCA2 protein expression in human myocardium. Methods: Frequency potentiation was studied in electrically stimulated, isometric, left ventricular papillary muscle strip preparations (37°C, 0.5–3.0 Hz) from terminally failing (NYHA IV; n=5, dilated cardiomyopathy) and nonfailing (donor hearts, n=5) human myocardium. In the identical samples the Ca2+-ATPase activity (NADH coupled assay) and the protein expression of sarcoplasmic Ca2+-ATPase (SERCA2), phospholamban, and calsequestrin (western blot) were determined. The frequency dependent change in the force of contraction and Vmax of the Ca2+-ATPase activity and the protein expression of SERCA2 were correlated with each other. Results: In terminally failing myocardium the force-frequency relationship was negative (2.0 Hz vs. 0.5 Hz: –0.2±0.1 ΔmN) contrasting a positive rate dependent potentiation of force in nonfailing tissue (2.0 Hz vs. 0.5 Hz: +0.8±0.2 ΔmN; p<0.01). In failing myocardium the corresponding maximal sarcoplasmic Ca2+-ATPase activity (Vmax) was reduced significantly compared to nonfailing myocardium (174±24 vs. 296±31 nmol ATP/mg·min, p<0.01). The protein expression of SERCA2, phospholamban, and calsequestrin remained unchanged in failing myocardium. The maximal Ca2+-ATPase activity significantly correlated with the frequency dependent change in force of contraction (2 Hz vs. 0.5 Hz: r=0.88, p=0.001; 3 Hz vs. 0.5 Hz: r=0.84, p=0.004). No correlation between protein expression of SERCA2 and Ca2+-ATPase activity or change in force of contraction was observed. Conclusion: Due to a significant correlation between sarcoplasmic Ca2+-ATPase activity and frequency potentiation, the negative rate dependent force potentiation in human heart failure could be at least in part be attributed to decreased function of the sarcoplasmic Ca2+-ATPase.

Increased Expression of Isoform 1 of the Sarcoplasmic Reticulum Ca2+-Release Channel in Failing Human Heart

Background—The sarcoplasmic reticulum (SR) Ca2+-release channel plays a key role in the excitation-contraction coupling of cardiac myocytes. Because respective alterations have been reported in human heart failure, we investigated isoform expression of the SR Ca2+-release channel in human hearts from patients with terminal heart failure (dilated cardiomyopathy [DCM], n=8) and nonfailing organ donors (NF, n=8). Methods and Results—Expression of mRNA of SR Ca2+-release channel isoforms in isolated human cardiomyocytes and myocardial tissue was analyzed by reverse-transcription polymerase chain reaction. Protein expression was quantified in myocardial tissue with [3H]-ryanodine binding and with Western blots, expressed as densitometric units per microgram of protein (DU), and cellular localization was visualized with immunohistochemistry. We found mRNA expression of isoforms 1, 2, and 3 in cardiomyocytes and myocardial tissue both in NF and DCM. Total SR Ca2+-release channel protein expression in NF (Bmax 2.16±0.43 pmol/mg protein) and in DCM (Bmax 2.33±0.22 pmol/mg protein) myocardium was unchanged. Expression of isoform 1 of the SR Ca2+-release channel was significantly (P =0.0037) increased in DCM myocardium (NF 1.97±0.25 versus DCM 3.37±0.31 DU), whereas protein expression of isoform 2 (NF 14.62±0.87 versus DCM 13.52±0.43 DU) and isoform 3 (NF 1.39±0.13 versus DCM 1.35±0.19 DU) was unchanged. All 3 isoforms of the protein could be localized in human ventricular myocytes with fluorescence immunohistochemistry. Conclusions—All 3 isoforms of the SR Ca2+-release channel were determined in human ventricular cardiomyocytes. Increased expression of isoform 1 of the SR Ca2+-release channel could contribute to impaired excitation-contraction coupling in human heart failure.

Reduced sarcoplasmic reticulum Ca2+-ATPase activity and dephosphorylated phospholamban contribute to contractile dysfunction in human hibernating myocardium

Human hibernating myocardium (HHM) is characterized by reversible contractile dysfunction during chronic ischemia. A disturbed calcium-homeostasis is a decisive factor for reduced functional capacity in heart diseases. We therefore investigated calcium-handling proteins in HHM. In 12 patients suffering from multi-vessel coronary artery disease and contractile dysfunction with indication for bypass surgery, HHM was detected preoperatively by thallium scintigraphy, radionuclide ventriculography and dobutamine echocardiography. Transmural biopsies of these regions were taken and analyzed by immunohistochemistry and electron microscopy. Furthermore, SR-calcium ATPase (SERCA2a), phospholamban (PLN), the phosphorylated forms of PLN (PLN-Ser16, PLN-Thr17) as well as sodium-calcium exchanger (NCX) and ryanodine receptor (RyR2) were investigated by RT-PCR and Western-blotting. Additionally, SERCA2a activity was measured by an enzyme-coupled assay. In all patients complete functional recovery could be documented 3 months after revascularization by repeating all preoperative investigations. In HHM maximal SERCA2a activity was significantly reduced (HHM: 424.5± 33.9, control: 609.0± 48.5 nmol ATP mg protein−1 min−1, p≤ 0.05), whereas SERCA2a protein levels were unchanged. mRNA levels (HHM: 1.36± 0.08 vs. control: 0.78± 0.04, p≤ 0.05) and protein amount (HHM:1.67± 0.14 vs. control: 1.00± 0.04, p≤ 0.05) of PLN (A1) were increased resulting in an increased PLN:SERCA2a-ratio. PLN-Ser16 (HHM: 0.60± 0.08 vs. control: 1.00± 0.11, p≤ 0.05) and PLN-Thr17 (HHM: 0.63± 0.11 vs. control: 1.00± 0.06, p≤ 0.05) phosphorylation was significantly decreased. RyR2 and NCX showed no significant alteration. In HHM a decreased activity of SERCA2a due to an impaired phosphorylation of PLN contributes to contractile dysfunction. The increase in the relative ratio of PLN/SERCA2a leads to a decreased calcium affinity of SERCA2a.