Jörg Knapp

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.

Postnatal changes in contractile time parameters, calcium regulatory proteins, and phosphatases

Compared with isolated electrically driven neonatal ventricular preparations, the total time of contraction, the time to peak tension, and the time of relaxation were decreased to ∼50% in adult ventricular preparations. The expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was increased to 133% at the protein level and to 154% at the mRNA level in adult vs. neonatal ventricular preparations, whereas phospholamban was unchanged at both the protein and mRNA levels. Moreover, Ca2+ uptake was increased to 180% in adult vs. neonatal ventricular preparations. Phospholamban phosphorylation was enhanced in adult vs. neonatal ventricular preparations. In adult ventricular preparations, phosphatase activity was reduced to 53% of neonatal preparations, the protein levels of the immunologically detectable catalytic subunits of protein phosphatase types 1 and 2A were reduced to 28 and 61% of neonatal preparations, respectively, and the mRNA levels of type 1α, 1β, 1γ, 2Aα, and 2Aβ phosphatase isoforms were decreased to 69, 68, 54, 67, and 63%, respectively. We conclude that in the adult rat heart, the shortened time parameters of contraction can be explained by an elevated expression of SERCA. In addition, an increased phosphorylation state of phospholamban due to reduced phosphatase activity may be involved.Compared with isolated electrically driven neonatal ventricular preparations, the total time of contraction, the time to peak tension, and the time of relaxation were decreased to approximately 50% in adult ventricular preparations. The expression of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) was increased to 133% at the protein level and to 154% at the mRNA level in adult vs. neonatal ventricular preparations, whereas phospholamban was unchanged at both the protein and mRNA levels. Moreover, Ca2+ uptake was increased to 180% in adult vs. neonatal ventricular preparations. Phospholamban phosphorylation was enhanced in adult vs. neonatal ventricular preparations. In adult ventricular preparations, phosphatase activity was reduced to 53% of neonatal preparations, the protein levels of the immunologically detectable catalytic subunits of protein phosphatase types 1 and 2A were reduced to 28 and 61% of neonatal preparations, respectively, and the mRNA levels of type 1alpha, 1beta, 1gamma, 2Aalpha, and 2Abeta phosphatase isoforms were decreased to 69, 68, 54, 67, and 63%, respectively. We conclude that in the adult rat heart, the shortened time parameters of contraction can be explained by an elevated expression of SERCA. In addition, an increased phosphorylation state of phospholamban due to reduced phosphatase activity may be involved.

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.

Identification and expression of a novel isoform of cAMP response element modulator in the human heart

In end‐stage human heart failure, excessive β‐adrenergic stimulation of the cAMP‐dependent signaling pathway due to enhanced endogenous catecholamines is hypothesized to contribute to expressional alterations of myocardial regulatory proteins. The cAMP response element modulator (CREM) regulates the transcription of cAMP‐responsive genes and might be involved in the regulation of cardiac gene expression. Using the reverse transcription polymerase chain reaction, we identified a novel CREM mRNA, CREM‐IbδC‐X, in the human heart. Overexpression of CREM‐IbδC‐X decreased cAMP response element (CRE) ‐mediated gene transcription in HIT‐T15 cells, and this activity was assigned to the part of the sequence encoding putative internally translated proteins. Two of three possible internally translated proteins were immunologically identified in cells overexpressing CREM‐IbδC‐X tagged with the hemagglutinin epitope of the influenza virus. Both proteins were expressed in bacteria and showed CRE‐specific DNA binding, formation of heterodimers with the cAMP response element binding protein (CREB), and inhibition of CREBs binding to the CRE. CREM expression was detected on the mRNA and protein levels in the human heart. We conclude that CREM‐IbδC‐X generates internally translated repressors of CRE‐mediated gene transcription, suggesting the first example for the existence and function of human cardiac CREM.—Müller, F. U., BoknÍK, P., Knapp, Jörg, Neumann, J., Vahlensieck, U., Oetjen, E., Scheld, H. H., Schmitz, W. Identification and expression of a novel isoform of cAMP response element modulator in the human heart. FASEB J. 12, 1191–1199 (1998)

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.

The mechanism of action of cantharidin in smooth muscle

1 The aim of this study was to investigate the mechanism(s) of the vasoconstrictor effect of cantharidin in bovine preparations. 2 Catalytic subunits of protein phosphatase type 1 (PP 1) and type 2A (PP 2A) were immunologically identified in coronary arteries, isolated smooth muscle cells and ventricular myocardium. 3 The mRNAs coding for catalytic subunits of PP 1α, PP 1β and PP 2Aα were identified by hybridization with specific cDNA‐probes in total RNA from coronary arteries, isolated smooth muscle cells and ventricles. 4 The activities of catalytic subunits of PP 1 and PP 2A separated by column chromatography from coronary arteries, isolated smooth muscle cells and ventricles were inhibited by cantharidin in a concentration‐dependent manner. 5 Cantharidin increased the phosphorylation state of smooth muscle proteins including the regulatory light chains of myosin in 32P‐labelled intact smooth muscle cells in a concentration‐dependent manner. 6 Cantharidin did not affect cytosolic calcium concentrations in aortic smooth muscle cells. 7 It is suggested that cantharidin contracts smooth muscle preparations by increasing the phosphorylation state of regulatory proteins due to inhibition of phosphatase activities. Thus, cantharidin might be a useful tool to study the function of phosphatases in smooth muscle.