Frank U. Müller

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.

Ryanodine Receptor-Mediated Calcium Leak Drives Progressive Development of an Atrial Fibrillation Substrate in a Transgenic Mouse Model

Background— The progression of atrial fibrillation (AF) from paroxysmal to persistent forms remains a major clinical challenge. Abnormal sarcoplasmic reticulum (SR) Ca2+ leak via the ryanodine receptor type 2 (RyR2) has been observed as a source of ectopic activity in various AF models. However, its potential role in progression to long-lasting spontaneous AF (sAF) has never been tested. This study was designed to test the hypothesis that enhanced RyR2-mediated Ca2+ release underlies the development of a substrate for sAF and to elucidate the underlying mechanisms. Methods and Results— CREM-Ib&Dgr;C-X transgenic (CREM) mice developed age-dependent progression from spontaneous atrial ectopy to paroxysmal and eventually long-lasting AF. The development of sAF in CREM mice was preceded by enhanced diastolic Ca2+ release, atrial enlargement, and marked conduction abnormalities. Genetic inhibition of Ca2+/calmodulin-dependent protein kinase II–mediated RyR2-S2814 phosphorylation in CREM mice normalized open probability of RyR2 channels and SR Ca2+ release, delayed the development of spontaneous atrial ectopy, fully prevented sAF, suppressed atrial dilation, and forestalled atrial conduction abnormalities. Hyperactive RyR2 channels directly stimulated the Ca2+-dependent hypertrophic pathway nuclear factor of activated T cell/Rcan1-4, suggesting a role for the nuclear factor of activated T cell/Rcan1-4 system in the development of a substrate for long-lasting AF in CREM mice. Conclusions— RyR2-mediated SR Ca2+ leak directly underlies the development of a substrate for sAF in CREM mice, the first demonstration of a molecular mechanism underlying AF progression and sAF substrate development in an experimental model. Our work demonstrates that the role of abnormal diastolic Ca2+ release in AF may not be restricted to the generation of atrial ectopy but extends to the development of atrial remodeling underlying the AF substrate.

Profiling of histone H3 lysine 9 trimethylation levels predicts transcription factor activity and survival in acute myeloid leukemia.

Acute myeloid leukemia (AML) is commonly associated with alterations in transcription factors because of altered expression or gene mutations. These changes might induce leukemia-specific patterns of histone modifications. We used chromatin-immunoprecipitation on microarray to analyze histone 3 lysine 9 trimethylation (H3K9me3) patterns in primary AML (n = 108), acute lymphoid leukemia (n = 28), CD34(+) cells (n = 21) and white blood cells (n = 15) specimens. Hundreds of promoter regions in AML showed significant alterations in H3K9me3 levels. H3K9me3 deregulation in AML occurred preferentially as a decrease in H3K9me3 levels at core promoter regions. The altered genomic regions showed an overrepresentation of cis-binding sites for ETS and cyclic adenosine monophosphate response elements (CREs) for transcription factors of the CREB/CREM/ATF1 family. The decrease in H3K9me3 levels at CREs was associated with increased CRE-driven promoter activity in AML blasts in vivo. AML-specific H3K9me3 patterns were not associated with known cytogenetic abnormalities. But a signature derived from H3K9me3 patterns predicted event-free survival in AML patients. When the H3K9me3 signature was combined with established clinical prognostic markers, it outperformed prognosis prediction based on clinical parameters alone. These findings demonstrate widespread changes of H3K9me3 levels at gene promoters in AML. Signatures of histone modification patterns are associated with patient prognosis in AML.

Heart-directed Expression of a Human Cardiac Isoform of cAMP-Response Element Modulator in Transgenic Mice

The transcriptional activation mediated by cAMP-response element (CRE) and transcription factors of the CRE-binding protein (CREB)/CRE modulator (CREM) family represents an important mechanism of cAMP-dependent gene regulation possibly implicated in detrimental effects of chronic β-adrenergic stimulation in end-stage heart failure. We studied the cardiac role of CREM in transgenic mice with heart-directed expression of CREM-IbΔC-X, a human cardiac CREM isoform. Transgenic mice displayed atrial enlargement with atrial and ventricular hypertrophy, developed atrial fibrillation, and died prematurely. In vivo hemodynamic assessment revealed increased contractility of transgenic left ventricles probably due to a selective up-regulation of SERCA2, the cardiac Ca2+-ATPase of the sarcoplasmic reticulum. In transgenic ventricles, reduced phosphorylation of phospholamban and of the CREB was associated with increased activity of serine-threonine protein phosphatase 1. The density of β1-adrenoreceptor was increased, and messenger RNAs encoding transcription factor dHAND and small G-protein RhoB were decreased in transgenic hearts as compared with wild-type controls. Our results indicate that heart-directed expression of CREM-IbΔC-X leads to complex cardiac alterations, suggesting CREM as a central regulator of cardiac morphology, function, and gene expression.

Isoprenaline stimulates gene transcription of the inhibitory G protein alpha-subunit Gi alpha-2 in rat heart.

In vitro transcription reactions were performed with isolated ventricular nuclei of adult rats to investigate whether increased mRNA levels of the inhibitory G protein alpha-subunit Gi alpha-2 after prolonged in vivo stimulation with the beta-adrenoceptor agonist isoprenaline are caused by increased transcription. Rats were treated by a 4-day subcutaneous infusion of isoprenaline (2.4 mg/kg per day) or 0.9% NaCl as control. To avoid the influence of developmental expression patterns, adult rats were chosen for all experiments. Signals for Gi alpha-2 and the stimulatory G protein alpha-subunit Gs alpha were specific and due to hybridization of nascent mRNA transcripts. In the isoprenaline group the transcriptional activity of Gi alpha-2 gene increased to 140% of the control value, whereas gene specific hybridization for Gs alpha remained unchanged. These results show that increased Gi alpha-2 mRNA levels after stimulation with isoprenaline are at least partially caused by enhanced transcription of Gi alpha-2 mRNA.

Effects of endotoxin on human myocardial contractility involvement of nitric oxide and peroxynitrite

OBJECTIVES This study examined the effects of endotoxin on cardiac contractility in human myocardium. BACKGROUND In animal myocardium, endotoxin and cytokine treatment led to enhanced inducible nitric oxide synthase (iNOS) expression and contractile dysfunction. Effects in human myocardium are unknown. METHODS Left ventricular myocardial preparations from failing (n = 18) and nonfailing (n = 5) human hearts were incubated for 6 and 12 h in tyrode solution or in tyrode plus lipopolysaccharides (LPS), with LPS plus N(G)-mono-methyl-L-arginine (L-NMMA), with LPS plus hemoglobin or with LPS plus the superoxide scavenger 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron). Force of contraction in response to isoprenaline (0.001 to 3 micromol/liter) was determined in electrically stimulated muscle preparations. The iNOS mRNA expression was examined by in situ hybridization and by polymerase chain reaction. The cyclic guanosine monophosphate (cGMP) levels were determined by radioimmunoassay. RESULTS Isoprenaline concentration dependently increased force of contraction. Six and 12 hours of LPS treatment of failing myocardium decreased maximum inotropic response to isoprenaline by 54% (p = 0.009) and by 69% (p = 0.0023), respectively. In nonfailing myocardium, 12 h of LPS treatment decreased maximum inotropic effect of isoprenaline by 66% (p < 0.001). The LPS effects were attenuated by L-NMMA, hemoglobin and also Tiron. The iNOS mRNA was expressed in all LPS-treated preparations but also in most control myocardial preparations. In situ hybridization revealed iNOS expression within cardiac myocytes. There was no increase in myocardial cGMP content in response to endotoxin. CONCLUSIONS Endotoxin exposure of human myocardium leads to a depression of cardiac contractility, which is mediated by enhanced iNOS activity and release of nitric oxide (NO). Consecutive reaction of NO with superoxide and formation of peroxynitrite may contribute to the decrease in force of contraction.

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.

Molecular aspects of adrenergic signal transduction in cardiac failure

Abstract Abnormal β-adrenergic signal transduction and intracellular Ca2+ handling appear to be a major cause of systolic and diastolic dysfunction in humans with heart failure. The precise mechanisms which cause an alteration in Ca2+ handling have been a subject of investigation in recent years. Several lines of evidence suggest that activation of neurohormonal systems plays a central role. Altered Ca2+-handling (increased diastolic concentrations, reduced systolic Ca2+ release) have a strong impact on diastolic and systolic performance of failing hearts. Sarcoplasmic reticulum Ca2+ ATPase is reduced in activity and in steady-state mRNA concentration. The Na+-Ca2+ exchanger is upregulated at the mRNA and protein levels. Phospholamban depends strongly on cAMP-dependent phosphorylation. A strong sympathetic activation has been shown to desensitize the cAMP system. At the receptor level, there is downregulation of β1-adrenergic receptors. An uncoupling of β2-adrenoceptors has been attributed to an increased activity and gene expression of β-adrenergic receptor kinase in failing myocardium, leading to phosphorylation and uncoupling of receptors. Finally, recent evidence suggests that cAMP-dependent transcription mechanisms may play a role during β-adrenergic stimulation and cardiomyopathy with heart failure – by means of altered actions of cAMP response element binding protein, the cAMP response element modulator, or the activating transcription factor 1. The exact characterization of signal transduction defects could offer novel approaches to the pharmacological treatment of heart failure.

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.

Receptor mechanisms involved in the 5‐HT‐induced inotropic action in the rat isolated atrium

1 The effects of 5‐hydroxytryptamine (5‐HT) in rat cardiac preparations were studied. 5‐HT up to 10 μM failed to affect contractility in papillary muscles. However, in electrically driven (1 Hz) left atria 5‐HT exerted a positive inotropic effect that started at 1 μM and attained its maximum at 10 μM (312±50% of predrug value, n=8). 2 5‐HT 10 μM stimulated the content of inositol‐1,4,5‐trisphosphate but not of cyclic AMP in rat left atria. 3 Plasma and serum levels of 5‐HT amounted to about 0.3 μM and 15 μM, respectively. 4 The selective 5‐HT4 receptor antagonists GR 125487 (10 nM and 1 μM) and SB 203186 (1 μM) did not attenuate the positive inotropic effect of 5‐HT in rat left atria. In contrast, the 5‐HT2 receptor antagonist ketanserin (5 nM, 50 nM, 1 μM) resulted in a concentration‐dependent diminution of the positive inotropic effect of 5‐HT in rat left atria. 5 Reverse transcriptase polymerase chain reaction with specific primers detected mRNA of the 5‐HT2A receptor in rat atria and ventricles, while expression of the 5‐HT4 receptor was confined to atria. 6 It is suggested that the positive inotropic effect of 5‐HT in electrically driven rat left atria is mediated by ketanserin‐sensitive 5‐HT2A receptors and not through 5‐HT4 receptors.