Repetitive use of levosimendan for treatment of chronic advanced heart failure: Clinical evidence, practical considerations, and perspectives: An expert panel consensus

Background The intravenous inodilator levosimendan was developed for the treatment of patients with acutely decompensated heart failure. In the last decade scientific and clinical interest has arisen for its repetitive or intermittent use in patients with advanced chronic, but not necessarily acutely decompensated, heart failure. Recent studies have suggested long-lasting favourable effects of levosimendan when administered repetitively, in terms of haemodynamic parameters, neurohormonal and inflammatory markers, and clinical outcomes. The existing data, however, requires further exploration to allow for definitive conclusions on the safety and clinical efficacy of repetitive use of levosimendan. Methods and results A panel of 30 experts from 15 countries convened to review and discuss the existing data, and agreed on the patient groups that can be considered to potentially benefit from intermittent treatment with levosimendan. The panel gave recommendations regarding patient dosing and monitoring, derived from the available evidence and from clinical experience. Conclusions The current data suggest that in selected patients and support out-of-hospital care, intermittent/repetitive levosimendan can be used in advanced heart failure to maintain patient stability. Further studies are needed to focus on morbidity and mortality outcomes, dosing intervals, and patient monitoring. Recommendations for the design of further clinical studies are made

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 Ca2+/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×min)] and by45Ca2+ uptake. Protein expression of SERCA 2a, phospholamban, calsequestrin and calcineurin was assessed by Western blotting (expressed as densitometric units/μ g protein); phosphorylation of cardiac proteins was detected with specific phospho-antibodies for phospholamban at threonine-17 (PT17) or by incorporation of [ γ -32P] (expressed as pmol32P/mg). Maximal45Ca2+ uptake (in pmol/mg/min) (NF: 3402±174; DCM: 2488±189) and maximal SERCA 2a activity were reduced in DCM compared to NF (Vmax: NF: 125±9; DCM: 98±5). The Vmax reduction could be mimicked by calcineurin in vitro in NF (NFcontrol: 72.1±3.7; NF+calcineurin: 49.8±2.9) and restored in DCM by CaM-kinase in vitro (DCMcontrol: 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 (DCMcontrol: 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 (NFcontrol 80.7±4.4 v NF+calcineurin 30.7±4.1, P<0.05). In human myocardium, the Vmax 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 Vmax of SERCA 2a in failing human myocardium and the disorder in Ca2+-handling in heart failure

Reduced Ca2+-sensitivity of SERCA 2a in failing human myocardium due to reduced serin-16 phospholamban phosphorylation

It is still a matter of debate, whether decreased protein expression of SERCA 2a and phospholamban (PLB), or alterations in the phosphorylation state of PLB are responsible for the reduced SERCA 2a function in failing human myocardium. Thus, in membrane preparations from patients with terminal heart failure due to idiopathic dilated cardiomyopathy (NYHA IV. heart transplants) and control hearts (NF), SERCA 2a activity was measured with an NADH coupled assay with as well as without stimulation with protein kinase A (PKA). The protein expression of SERCA 2a, PLB and calsequestrin as well as the phosphorylation status of PLB (Back-phosphorylation technique: Serine-16-PLB specific antibody) were analysed using Western blotting technique and specific antibodies. In NF, the maximal activity (Vmax) and the Ca(2+)-sensitivity of SERCA 2a activity were significantly higher compared to NYHA IV. Protein expression of SERCA 2a, PLB and calsequestrin were unchanged, whereas both, the phosphorylation status of PLB as well as serine-16-PLB-phosphorylation, were significantly reduced in NYHA IV. After stimulation with PKA only the Ca(2+)-sensitivity, but not Vmax increased concentration-dependently. Therefore, in human myocardium, the Ca(2+)-sensitivity but not the Vmax of SERCA 2a is regulated by cAMP-dependent phosphorylation of phospholamban at position serine-16. Threonine-17-PLB-phosphorylation or direct phosphorylation of SERCA 2a may be candidates for regulation of maximal SERCA 2a activity in human myocardium

Unchanged protein levels of SERCA II and phospholamban but reduced Ca2+uptake and Ca2+-ATPase activity of cardiac sarcoplasmic reticulum from dilated cardiomyopathy patients compared with patients with nonfailing hearts

BACKGROUND: The aim of the present study was to investigate whether Ca2+ uptake into the sarcoplasmic reticulum (SR) is altered in failing human myocardium resulting from dilated cardiomyopathy. METHODS AND RESULTS: Ca(2+)-ATPase (SERCA II) activity and Ca(2+)-dependent 45Ca2+ uptake (oxalate supported, steady state) in isolated vesicles from the SR (VSR) and in crude membrane preparations (CSR) (free Ca2+, 0.01 to 100 mumol/L) from nonfailing (donor hearts, n = 13) and terminally failing (heart transplants, dilated cardiomyopathy, n = 17) human myocardium were studied. In the same hearts, protein levels (Western blot analysis) and mRNA levels (Northern blot analysis) of SERCA II and phospholamban were measured. Increasing concentrations of Ca2+ were followed by an increased Ca(2+)-ATPase activity and Ca2+ uptake. Ca2+ uptake activity and Ca(2+)-ATPase activity in CSR preparations from failing myocardium were significantly reduced compared with nonfailing hearts (Ca(2+)-ATPase, 163 +/- 8 and 125 +/- 7 nmol ATP/mg protein per minute for nonfailing tissue and failing tissue in New York Heart Association [NYHA] class IV, respectively; Ca2+ uptake, 7.1 +/- 0.8 and 3.5 +/- 0.3 nmol/mg protein per minute in CSR from nonfailing and NYHA class IV hearts, respectively P < .05). In contrast, no significant difference was measured in VSR. In the same preparations (CSR and VSR), both SERCA II and phospholamban levels (Western blot technique with monoclonal antibodies) were unchanged in failing compared with nonfailing tissue. mRNA expression relative to GAPDH mRNA for SERCA IIa and for phospholamban was significantly reduced in failing human myocardium (P < .05). CONCLUSIONS: These findings provide evidence that in failing human myocardium caused by dilated cardiomyopathy, protein levels of SERCA II and phospholamban are unchanged even though mRNA levels for SERCA II and phospholamban and the SERCA II function are reduced compared with nonfailing myocardium