Levosimendan increases the phosphorylation state of phospholamban in the isolated human atrium

Fig. 1. Scheme: Potential mechanism(s) of action of levosimendan in the human and mouse cardiomyocytes. Stimulation of the activity of β-adrenoceptors (β-AR) by endogenous noradrenaline or exogenous isoprenaline leads via stimulatory GTP-binding proteins (Gs) to an increase of adenylyl cyclase (AC) activity. Adenylyl cyclase increases the formation of 3′,5′-cyclic adenosine monophosphate (cAMP) that stimulates cAMP-protein kinase (PKA). PKA phosphorylates and thus activates phospholamban (PLB) at the amino acid serine 16, the inhibitory subunit of troponin (TnI), the ryanodine receptor (RYR) and the L-type calcium channel (LTCC). The formed cAMP can be degraded to inactive 5′-AMP and pyrophosphate by isoenzymes of the phosphodiesterase family of proteins (PDE). Cilostamide and rolipram (bottom: structural formulae) inactivate phosphodiesterases 3 and 4, respectively. The β-adrenoceptor is blocked by β-adrenoceptor blockers like timolol or propranolol. Calcium cations (Ca2+) are stored on calsequestrin (CSQ) in the sarcoplasmic reticulum and are released via RYR from the sarcoplasmic reticulum (SR). These released calcium cations bind to troponin C on thin myofilaments, and as a result,…

Fig. 2. Positive inotropic effects of levosimendan in mouse atrium only in the presence of rolipram. (A) Original recordings: Levosimendan ((-)-(R)-[[4-(1,4,5,6-tetrahydro-4-methyl-6-oxo-3-pyridazinyl)phenyl]hydrazono]propane-dinitrile) (10 nM to 10 µM) was cumulative applied alone (A, top) or the presence 0.1 µM rolipram ((RS)-4-(3-cyclopentyloxy-4-methoxy-phenyl)pyrrolidin-2-on) (A, bottom). Rolipram was used to inhibit the activity of phosphodiesterase IV. The positive inotropic effect of levosimendan in presence of rolipram was abrogated by subsequently applied 10 µM propranolol. Vertical bars indicate force in millinewton (mN). Horizontal bars indicate time in minutes (min). (B) Effects of isoprenaline (Iso: 1 µM), levosimendan (Levo: 10 µM), or both (Roli + Levo) in single high-speed contractions compared to control conditions (no drug addition: Ctr). Please note that we normalized in the ordinate the maximum developed force of contraction under all conditions as 100% in order to facilitate detection of changes in contraction and relaxation times (abscissa in milliseconds (ms)). Concentration-dependent effects of increasing concentrations of levosimendan on force of contraction, or…

Fig. 3. Positive chronotropic effect of levosimendan in the mouse atrium in the presence of rolipram. Original recording and summarized data. Levosimendan has a positive chronotropic effect in the mouse heart that is potentiated by the phosphodiesterase IV inhibitor rolipram. Ordinates in (A) indicate the effect of levosimendan on force of contraction or in beats per minute (B: bpm) in isolated spontaneously beating right atrial preparations from mouse hearts. Horizontal bars in (A) indicate time in minutes (min). Vertical bars in (A) indicate force of contraction in millinewton (mN). *p < 0.05 versus Ctr (t-test). Numbers in brackets indicate the number of experiments

Fig. 4. Levosimendan increases phospholamban phosphorylation in the mouse atrium only in the presence of rolipram. (A) The panel depicts a typical Western blot of electrically stimulated left atrial preparations (LA) or spontaneously beating right atrial preparations (RA) from mouse heart. Horizontal arrows indicate the apparent molecular weight in kilodalton (kDa) of phospholamban phosphorylated at serine 16 (PS16-PLB) or calsequestrin (CSQ), used as a loading control or sarcoplasmic calcium ATPase (SERCA: compare scheme in Fig. 1 for subcellular localization). Samples were treated with 1 µM levosimendan alone, 0.1 µM rolipram alone, or 1 µM levosimendan and 0.1 µM rolipram (Ro) combined (as indicated by + in the respective lanes). Apparent molecular weight standards are depicted in the left-hand first lane. “Boiled” means that a mouse LA sample was treated with 1 µM isoprenaline as a positive control (+ Ctr) for phosphorylation of phospholamban, and then loaded to the gel either after boiling (second lane from the left) or unboiled (third lane from the left). The typical mobility change for phospholamban is seen when comparing the high (p, pentameric) or low (m, monomeric) molecular…

Fig. 5. Inotropic effects of levosimendan. (A) Original recording of the inotropic effect of levosimendan (Levo, 1 µM) and its inhibition by propranolol (Prop, 10 µM) (upper recording). The lower recording shows a propranolol control, and on the right side, the quantification of force of contraction of samples from five patients is shown. Data are normalized to control conditions (no drug addition, Ctr). *p < 0.05 versus Ctr; #p < 0.05 versus Levo (1-way ANOVA, n = 5). (B) The positive inotropic effect of levosimendan does not necessitate the concomitant inhibition of phosphodiesterases and is abrogated in the concomitant inhibition of phosphodiesterase III by 1 µM cilostamide in electrically stimulated muscle strips from human right atrium. The positive inotropic effects were abrogated by 10 µM propranolol. Subsequent application of a very high concentration of isoprenaline (10 µM) surmounted the effect of propranolol, indicating that the sample was properly contracting and that the efficacy of 10 µM isoprenaline was superior to that of 1 µM levosimendan. Vertical bars indicate force in millinewton (mN). Horizontal bars indicate time in minutes (min). (C) Typical sweeps of muscle…

Fig. 6. Levosimendan alone increases phosphorylation of phospholamban in the human heart. (A) Western blot of phosphorylated phospholamban at serine 16 (PS16-PLB) and the phosphorylated inhibitory subunit of troponin (P-TnI) in contracting human atrium. Horizontal arrows indicate the apparent molecular weight of phospholamban phosphorylated at serine 16 or phosphorylated inhibitory subunit of troponin (P-TnI), calsequestrin (CSQ), and SERCA were used as a loading control. Levosimendan 0.1 µM, 1 µM, or 10 µM or additional 10 µM propranolol was added to contracting human right atrial muscle strips, as indicated and freeze-clamped at the plateau of the inotropic effect. Controls for PLB phosphorylation were omission of drugs or application of propranolol alone. Apparent molecular weight standards are depicted in the left-hand first lane. Samples were not boiled (compare Fig. 4). Therefore, always a high (p) and low (m) molecular weight form of phospholamban is detectable. (B) Quantification of Western blots for PS16-PLB normalized to the CSQ signal as loading control. *p < 0.05 versus levosimendan (t-test, n = 3); Levo, levosimendan; Prop, propranolol