Bettina Linck

Messenger RNA expression and immunological quantification of phospholamban and SR-Ca2+-ATPase in failing and nonfailing human hearts

OBJECTIVES Human heart failure is associated with prolonged relaxation and prolonged Ca2+ transients which indicates an impaired function of the sarcoplasmic reticulum (SR) and may be detrimental for cardiac function. Controversy exists whether the altered SR function is accompanied by changes in the expression of phospholamban (PLB) and cardiac SR-Ca(2+)-ATPase (SERCA2) on mRNA and/or protein levels. METHODS We determined mRNA and/or protein levels for PLB and SERCA2 in the same left ventricular tissue of patients undergoing heart transplantation due to idiopathic dilated cardiomyopathy (IDC) or ischemic cardiomyopathy (ICM) in comparison to left ventricular tissue from nonfailing human hearts (NF). Total protein extracts were prepared and subjected to SDS gel electrophoresis. PLB and SERCA2 were identified with specific antibodies. Total RNA was isolated and hybridized with 32P-labeled cDNAs for human PLB and rat SERCA2. RESULTS Hybridization revealed the three expected mRNAs with the PLB probe (3.3 kb, 1.9 kb and 0.6 kb) and a single band with the SERCA2 probe (4.5 kb). Determination of respective proteins by immunoblotting revealed unchanged protein levels for PLB and SERCA2, whereas the mRNA levels for PLB and SERCA2 were reduced by about 30% and 50%, respectively. CONCLUSIONS These data show the level of SERCA2 and PLB protein and mRNA in the same hearts. The reduced mRNA level of SERCA2 and PLB is in accordance with previous data. However, the unchanged protein levels may indicate that the diminished RNA expression is not accompanied by a corresponding decrease for these proteins in human heart failure. These data also show that the altered SR function in human heart failure cannot be explained by altered protein levels of PLB and SERCA2. Furthermore, it is suggested that extrapolations from cardiac mRNA levels to protein expression may be misleading.

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.

Enhanced protein phosphorylation in hypertensive hypertrophy.

OBJECTIVE Chronic pressure overload in spontaneously hypertensive rats (SHR) is accompanied by heart hypertrophy and signs of heart failure. Since there is growing evidence for a possible pathophysiological role of altered protein phosphorylation in heart hypertrophy and failure, we studied here cardiac regulatory phosphoproteins and the kinases and phosphatases which regulate their phosphorylation state. METHODS The experiments were performed in ventricles of SHR (12-13 weeks old) and age-matched normotensive Wistar-Kyoto rats (WKY). RESULTS Basal as well as isoproterenol (Iso)-stimulated force of contraction (FOC) was markedly decreased in isolated electrically driven papillary muscles of SHR. Iso (3 micromol/l, 10 min) increased FOC by 0.91+/-0.20 mN in SHR and by 3.88+/-0.52 mN in WKY, respectively. Ca(2+)-uptake by sarcoplasmic reticulum (SR) at low ionized Ca(2+)-concentration was increased in homogenates from SHR. This was not due to altered expression of phospholamban (PLB), SR-Ca(2+)-ATPase and calsequestrin. However, PLB-phosphorylation at threonine-17 (PLB-PT-17) and the activity of Ca(2+)/calmodulin dependent protein kinase (Ca(2+)/Cam-PK) was increased in SHR. In addition, we found an enhanced protein kinase A (PKA)-dependent phosphorylation of the inhibitory subunit of troponin (TnI). In contrast, there was no difference in the activity or expression (protein- and mRNA-level) of protein phosphatases type 1 or type 2A between SHR and WKY. CONCLUSIONS It is suggested that increased Ca(2+)/Cam-PK-activity with resulting increase of PLB-PT-17 enhanced SR-Ca(2+)-uptake in SHR and might contribute to the pathophysiological changes in cardiac hypertrophy of SHR.

cAMP Response Element Binding Protein Is Expressed and Phosphorylated in the Human Heart

BACKGROUND In end-stage failing human hearts and in rat hearts after prolonged in vivo beta-adrenergic treatment, several proteins involved in the cAMP-dependent signal transduction are altered on the protein, mRNA, or transcriptional level, eg, beta-adrenoceptors, G-proteins, or proteins of Ca2+ homeostasis. In many tissues, cAMP-dependent transcriptional regulation occurs through the cAMP response element binding protein (CREB) and related transcription factors binding as dimers to cAMP response elements (CREs) in the promoter regions of regulated genes. METHODS AND RESULTS To investigate a possible role of CREB in the human heart, nuclear protein of explanted failing and nonfailing human hearts was used to test for CRE specific binding properties in gel mobility shift assays. CRE specific binding was found in competition studies, and CREB and its phosphorylated form were immunologically identified in supershift experiments. The alternatively spliced CREB isoforms CREB327 and CREB341 were found to be expressed on the mRNA level by the reverse transcriptase-polymerase chain reaction. CONCLUSIONS We conclude that in the failing and nonfailing human heart, CREB is expressed on the protein and mRNA levels and that CREB is phosphorylated and able to bind to CREs, indicating a functional role of CREB in the human heart.

Negative chronotropic and inotropic effects exerted by diadenosine hexaphosphate (AP6A) via A1‐adenosine receptors

1 Diadenosine hexaphosphate (AP6A) exerts vasoconstrictive effects. The purpose of this study was to investigate whether AP6A has any effect on cardiac function. 2 The effects of AP6A (0.1 −100 μm) on cardiac contractility and frequency were studied in guinea‐pig and human isolated cardiac preparations. Furthermore, the effects of AP6A on the amplitude of the L‐type calcium current, on the adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) content and on the phosphorylation of regulatory phosphoproteins, i.e. phospholamban and troponin inhibitor, were investigated in guinea‐pig isolated ventricular myocytes. 3 In isolated spontaneously beating right atria of the guinea‐pig AP6A exerted a negative chronotropic effect and reduced the rate of contraction maximally by 35% (IC20 = 35 μm). 4 In isolated electrically driven left atria of the guinea‐pig AP6A exerted a negative inotropic effect and reduced force of contraction maximally by 23% (IC20 = 70 μm). 5 In isolated electrically driven papillary muscles of the guinea‐pig AP6A alone was ineffective, but attenuated isoprenaline‐stimulated force of contraction maximally by 23% (IC20 = 60 μm). Furthermore, AP6A attenuated the relaxant effect of isoprenaline. 6 In human isolated electrically driven ventricular preparations AP6A alone was ineffective, but attenuated isoprenaline‐stimulated force of contraction by maximally 42% (IC20 = 18 μm). Moreover, AP6A attenuated the relaxant effect of isoprenaline. 7 All these effects of AP6A were abolished by the selective A‐‐adenosine receptor antagonist 1,3‐dipropyl‐cyclopentyl‐xanthine (DPCPX, 0.3 μm), whereas the M‐cholinoceptor antagonist atropine (10 μm) and the P2‐purinoceptor antagonist suramin (300 μm) failed to abolish the effects of AP6A. 8 AP6A 100 μm had no effect on the amplitude of the L‐type calcium current, but attenuated isoprenaline‐stimulated L‐type calcium current. The maximum of the current‐voltage relationship (I‐V curve) was shifted to the left by isoprenaline and additional application of AP6A shifted the I‐V curve back to the right to the control value. The phosphorylation state of phospholamban and the troponin inhibitor was unchanged by AP6A alone, but was markedly attenuated by AP6A in the presence of isoprenaline. Cyclic AMP levels remained unchanged by AP6A, even after stimulation with isoprenaline. 9 In summary, AP6A exerts negative chronotropic and inotropic effects in guinea‐pig and human cardiac preparations. These effects are mediated via A1‐adenosine receptors as all effects were sensitive to the selective A‐‐adenosine receptor antagonist DPCPX. Furthermore, the effects of AP6A on cyclic AMP levels, protein phosphorylation and the L‐type calcium current are in accordance with stimulation of A1‐adenosine receptors.

Activation and inactivation of cAMP-response element-mediated gene transcription in cardiac myocytes

OBJECTIVE Chronic beta-adrenergic stimulation of the cAMP-dependent signalling pathway is implicated in functionally relevant expressional changes in congestive heart failure. We studied activation and inactivation of the cardiac gene transcription mediated by the cAMP-response element (CRE) and the CRE-binding protein (CREB) as an important mechanism of a cAMP-dependent gene regulation. METHODS We investigated the transcriptional activation by forskolin, an activator of the adenylyl cyclase, in chick embryonic cardiomyocytes transfected with a CRE-controlled luciferase construct in comparison to the phosphorylation and expression of CREB determined on immunoblots. RESULTS Forskolin (10 micromol/l; 8 h) increased CRE-mediated transcription and phosphorylation of CREB 13- and 1.5-fold, respectively. The phosphorylation was further elevated in combination with cantharidin, an inhibitor of type 1+2A protein phosphatases. The transcriptional response to forskolin was desensitized by pretreatment with forskolin (1 micromol/l; 24 h) while CREB phosphorylation was increased. In forskolin-pretreated cells, total CREB protein levels were decreased. Cantharidin did not restore the attenuated transcriptional response. CONCLUSIONS In cardiomyocytes, there is an activation of the CRE-mediated gene transcription by forskolin that is attenuated after prolonged stimulation, and this attenuation is not dependent from a dephosphorylation of CREB. We suggest that attenuation of the CRE-mediated transcription through chronic stimulation of the cAMP-pathway, e.g. by elevated catecholamines, contributes to the altered expressional regulation in congestive heart failure.

Effects of cantharidin on force of contraction and phosphatase activity in nonfailing and failing human hearts

1 The effect of the phosphatase inhibitor, cantharidin (3–300 μm) on force of contraction was studied in isolated electrically driven right ventricular trabeculae carneae from human myocardium. 2 The positive inotropic effect of cantharidin started at a concentration of 100 μm with a positive inotropic effect to 199% and to 276% of the predrug value in nonfailing and failing human hearts, respectively. 3 Under basal conditions the contraction time parameters were prolonged in human heart failure vs. nonfailing preparations. However, the positive inotropic effect of cantharidin did not affect contraction time parameters. Thus, time to peak tension, time of relaxation and total contraction time were not shortened by cantharidin in nonfailing and failing preparations. 4 The phosphatase activity was unchanged in preparations from failing hearts compared to nonfailing hearts. 5 Cantharidin inhibited phosphatase activity in a concentration‐dependent manner. The IC50 value of cantharidin was about 3 μm in both nonfailing and failing human myocardium. 6 The positive inotropic effect of cantharidin was similar in nonfailing and failing human hearts, accompanied by a similar inhibitory effect of cantharidin on the phosphatase activity. The positive inotropic effect of cantharidin in failing hearts was as strong as the effect of isoprenaline in nonfailing hearts. 7 It is concluded that the treatment with a phosphatase inhibitor may offer a new positive inotropic modality for the treatment of human heart failure.

The Inhibitory Effect of Bupivacaine on Prostaglandin E 2 (EP 1 ) Receptor Functioning: Mechanism of Action

Prostaglandin E2 receptors, subtype EP1 (PGE2EP1) have been linked to several physiologic responses, such as fever, inflammation, and mechanical hyperalgesia. Local anesthetics modulate these responses, which may be due to direct interaction of local anesthetics with PGE2EP1 receptor signaling. We sought to characterize the local anesthetic effects on PGE2EP1 signaling and elucidate mechanisms of anesthetic action. In Xenopus laevis oocytes, recombinant expressed PGE2EP1 receptors were functional (half maximal effect concentration, 2.09 ± 0.98 × 10−6 M). Bupivacaine, after incubation for 10 min, inhibited concentration-dependent PGE2EP1 receptor functioning (half-maximal inhibitory effect concentration, 3.06 ± 1.26 × 10−6 M). Prolonged incubation in bupivacaine (24 h) inhibited PGE2-induced calcium-dependent chloride currrents (ICl(Ca)) even more. Intracellular pathways were not significantly inhibited after 10 min of incubation in bupivacaine. But ICl(Ca) activated by intracellular injection of GTP&ggr;S (a nonhydrolyzable guanosine triphosphate [GTP] analog that activates G proteins, irreversible because it cannot be dephosphorylated by the intrinsic GTPase activity of the &agr; subunit of the G protein) was reduced after 24 h of incubation in bupivacaine, indicating a G protein-dependent effect. However, inositol 1,4,5-trisphosphate- and CaCl2- induced ICl(Ca) were unaffected by bupivacaine at any time points tested. Therefore, bupivacaine’s effect is at phospholipase C or at the G protein or the PGE2EP1 receptor. All inhibitory effects were reversible. We conclude that bupivacaine inhibited PGE2EP1 receptor signaling at clinically relevant concentrations. These effects could, at least in part, explain how local anesthetics affect physiologic responses such as fever, inflammation, and hyperalgesia during the perioperative period.

Contractility and inhibition of protein phosphatases by cantharidin

1. Cantharidin is a natural defensive toxicant produced by blister beetles. 2. Cantharidin shares structural similarity with highly toxic commercial herbicides (e.g., endothall, endothall anhydride and endothall thioanhydride). 3. Cantharidin inhibits the activity of purified catalytic subunits of serine/threonine protein phosphatases (PP) type 1 and type 2A. 4. Cantharidin increases force of contraction in isolated myocardial and vascular preparations. 5. Cantharidin enhances the phosphorylation state of myocardial and vascular regulatory proteins. 6. Cantharidin is a valuable tool for studying the function of PP in regulatory phosphorylation-dephosphorylation events.

Functional studies in atrium overexpressing A1-adenosine receptors

Adenosine and the A1‐adenosine receptor agonist R‐PIA, exerted a negative inotropic effect in isolated, electrically driven left atria of wild‐type mice. In left atria of mice overexpressing the A1‐adenosine receptor, adenosine and R‐PIA exerted a positive inotropic effect. The positive inotropic effect of adenosine and R‐PIA in transgenic atria could be blocked by the A1‐adenosine receptor antagonist DPCPX. In the presence of isoprenaline, adenosine exerted a negative inotropic effect in wild‐type atria but a positive inotropic effect in atria from A1‐adenosine receptor overexpressing mice. The rate of beating in right atria was lower in mice overexpressing A1‐adenosine receptors compared with wild‐type. Adenosine exerted comparable negative chronotropic effects in right atria from both A1‐adenosine receptor overexpressing and wild‐type mice. A1‐adenosine receptor overexpression in the mouse heart can reverse the inotropic but not the chronotropic effects of adenosine, implying different receptor‐effector coupling mechanisms.