Michael Böhm

Increase of Gi alpha in human hearts with dilated but not ischemic cardiomyopathy.

In myocardial membranes from hearts with dilated cardiomyopathy (DCM), there was a 37% increase of the Gi alpha-protein as measured by 32P-ADP-ribosylation of a approximately 40 kDa pertussis toxin substrate. Immunoblotting techniques also showed increased amounts of Gi alpha in DCM. In hearts with ischemic cardiomyopathy (ICM), Gi alpha was not altered compared with nonfailing myocardium (NF). Basal and Gpp(NH)p-stimulated adenylate cyclase activity was reduced in DCM but not in ICM. The number of beta-adrenoceptors was similarly reduced both in DCM and ICM compared with NF. Alterations of m-cholinoceptors or A1-adenosine receptors did not occur. Consistently, indirect negative inotropic effects of the m-cholinoceptor agonist carbachol and the A1-adenosine receptor agonist R-PIA were not different in ICM, DCM, and nonfailing myocardium. In ICM and DCM, there was a marked reduction of the positive inotropic responses to isoprenaline and milrinone. However, there was a further reduction in DCM compared with ICM. It is concluded that the increase of Gi alpha is accompanied by a reduction of basal and guanine-nucleotide-stimulated adenylate cyclase activity. Alterations of m-cholinoceptors and A1-adenosine receptors do not appear to be involved. The further decrease of the positive inotropic effects of isoprenaline and milrinone in DCM provides evidence that the increase of Gi alpha is functionally relevant in DCM but not ICM and hence might contribute to the reduced effects of endogenous catecholamines and exogenous cAMP-dependent positive inotropic agents in the former but not the latter condition.

Cardiomyocytes of Noncardiac Origin in Myocardial Biopsies of Human Transplanted Hearts

Background—Cell replacement therapy with stem cells able to differentiate into cardiomyocytes has been discussed as a method for remodeling damaged myocardium. A physiological or pathophysiological situation in which this phenomenon might be relevant is not known. We studied the origin of cardiomyocytes in myocardial biopsies of male patients that had undergone sex-mismatched cardiac transplantation to determine whether cells containing a Y chromosome (and therefore being of recipient origin) are able to differentiate into cardiomyocytes. Methods and Results—Myocardial biopsies (n=21) were obtained from the right ventricles of male patients (n=13) who had undergone sex-mismatched heart transplantation. Tissue from 1 nontransplanted male and myocardial biopsies from sex-matched heart–transplanted patients served as controls. Cells from donor and recipient origins were identified by fluorescence in situ hybridization with the use of specific probes for X and Y chromosomes on paraffin sections of the biopsies. Cell types were identified by using immunostaining procedures on the same tissue sections. Cardiomyocytes of recipient origin were detected in 8 of 13 male recipients of female hearts. They were connected by gap junctions with adjacent myocytes. Of the cardiomyocyte nuclei, 0.16±0.04% (mean±SEM, median 0.09%) contained the Y-chromosomal marker. There was no detectable correlation with the extent or number of rejection episodes, time of transplantation, or medical treatment regimen. Conclusions—These results show that regeneration by cells of noncardiac origin (differentiated into cardiomyocytes and physiologically linked to neighboring myocytes) can be detected even in small myocardial biopsies. This may lead to new diagnostic and therapeutic strategies in the treatment of myocardial infarction, inflammatory heart disease, and/or heart failure.

Contribution of cAMP-phosphodiesterase inhibition and sensitization of the contractile proteins for calcium to the inotropic effect of pimobendan in the failing human myocardium.

Previous studies have shown reduced effects of cAMP-dependent positive inotropic agents in the failing human myocardium; thus other cAMP-independent mechanisms of action may be useful to increase force of contraction in this condition. The purpose of this investigation was to determine whether a positive inotropic effect of the cAMP-phosphodiesterase (PDE) inhibitor pimobendan is observed in the failing human myocardium and to study whether other factors, such as an increase in the Ca2+ sensitivity of myofilaments, play a functional role in the increase in force of contraction. Pimobendan produced a positive inotropic effect in isolated preparations from nonfailing donor hearts; however, in moderately (New York Heart Association class II-III, NYHA II-III) and severely (NYHA IV) failing myocardium, this effect was reduced. In addition, in NYHA IV specimens pimobendan inhibited the crude cAMP-PDE (crude PDE) and the isoenzymes I-III (PDE I-III) in a concentration-dependent way. As judged from the IC50 values found in this tissue for the inhibition of PDE III and of crude PDE, the potency of the compound was 18.1 times greater on PDE III. Consistent with a cAMP-PDE-dependent mechanism of action, the positive inotropic effect was potentiated by isoproterenol and inhibited by adenosine in failing myocardium. In failing myocardium, pimobendan also increased the sensitivity of skinned cardiac fibers to Ca2+ and shifted the Ca(2+)-tension relation to the left. This sensitizing effect began at 0.01 mumol/l in NYHA II-III and NYHA IV and rose to about 200% at 300 mumol/l in both groups. In contrast, the demethylated metabolite UD-CG 212 Cl failed to produce positive inotropic effects in failing myocardium alone, but in the presence of isoproterenol, it exerted an increase in force of contraction. The potency of UD-CG 212 Cl for PDE III inhibition in NYHA IV was greater than that of pimobendan. The metabolite pronouncedly decreased the sensitivity of skinned cardiac fibers to Ca2+ at 30-300 mumol/l in NYHA II-III and NYHA IV. It is concluded that in the failing human heart pimobendan inhibited PDE III and sensitized contractile proteins for Ca2+. Both effects appear to be involved in the positive inotropic effect of the compound, because its metabolite, UD-CG 212 Cl, had no effect on force of contraction and on the Ca2+ sensitivity of skinned cardiac fibers but inhibited PDE III even more potently than pimobendan.(ABSTRACT TRUNCATED AT 400 WORDS)

Desensitization of adenylate cyclase and increase of Gi alpha in cardiac hypertrophy due to acquired hypertension.

The present study investigated whether reduced adenylate cyclase activity and an increase in inhibitory guanine nucleotide binding proteins (Gi alpha), which have been observed in the failing human heart, already occur in myocardial hypertrophy before the stage of heart failure. In membranes of hypertrophic hearts from rats with different forms of experimentally induced hypertension without heart failure (one-kidney, one clip rats, deoxycorticosterone-treated rats, and rats with reduced renal mass), basal as well as isoprenaline-, 5-guanylylimidodiphosphate-, and forskolin-stimulated adenylate cyclase activity was reduced. The activity of the catalyst was depressed in deoxycorticosterone but unchanged in one-kidney, one clip and reduced renal mass compared with controls. The number of beta-adrenergic receptors was similar in all groups. Radioimmunological quantification of Gi alpha proteins revealed an increase by 73% in one-kidney, one clip, 67% in reduced renal mass, but only 20% in deoxycorticosterone compared with sham-operated, age-matched control rats. The increase of Gi alpha was accompanied by smaller changes of pertussis toxin-induced [32P]ADP-ribosylation of a 40-kd membrane protein. It is concluded that Gi alpha contributes to the reduced adenylate cyclase activity in cardiac hypertrophy in one-kidney, one clip and reduced renal mass and to a smaller extent in deoxycorticosterone. It is suggested that an enhanced expression of Gi alpha could occur not only in severe heart failure but also in cardiac hypertrophy and could, therefore, contribute to myocardial depression and progression of disease in heart failure. In addition, Gi alpha might represent an important regulatory mechanism for cardiac adenylate cyclase activity and thus, might play an important role in various cardiac diseases.

Characterization of A1 adenosine receptors in atrial and ventricular myocardium from diseased human hearts.

The purpose of the present study was to characterize adenosine receptors in human atrial and ventricular myocardium. In isolated electrically driven preparations, adenosine produced „direct” negative inotropic effects in atrial myocardium (AT). In ventricular myocardium (VE), it only had negative Inotropic properties when force of contraction had been stimulated with isoprenaline (“indirect” effect), but it has no inotropic effect alone. The adenosine receptor antagonist 8-phenyltheophylline antagonized the „direct” and „indirect” effects; these findings indicated that both effects were mediated by adenosine receptors. In cardiac membranes from human AT and VE, adenosine receptors were characterized with [3H]-8-cyclopentyl-l,3-dipropylxanthine (DPCPX) binding. The effects of agonists. R-(-)-N6-phenylisopropyladenosinc (R-PIA), S-(+)-N6-phenylisopropyladenosuie (S-PIA), and 5′-(N-ethylcarboxamido)adenosine (NECA) and the effects of guanine nucleotides [Gpp(NH)p] were studied also. The antagonist affinities as judged from the apparent affinity, Kd, of [3H] DPCPX were similar in AT (2.2 nmol/1; 95% confidence limits, 1.4–3.7) and VE (1.8 nmol/1; 95% confidence limits, 1.0–3.0). The number of adenosine receptors was 1.7 times greater in AT (26.9 ± 2.33 fmol/mg protein; n =5) than in VE (16.2 ± 23 fmol/mg protein; n =5). High and low affinity states of adenosine receptors evaluated with the influence of Gpp(NH)p on agonist competition with R-PIA were similar in AT or VE. The rank orders of potency for agonists (R-PIA>S-PIA>NECA) and antagonists (DPCPX>8-phenyltheophylline>theophylline) were characteristic for the A, receptor subtype. It is concluded that A, adenosine receptors exist in the human myocardium. Since binding properties were similar in AT and VE, the same A1 adenosine receptor probably couples to different effectors in a similar guanine nucleotide-dependent way. [3H] DPCPX is the first radiolabeled antagonist ligand that allows detection of A, adenosine receptors and their coupling in the human myocardium.

Inotropic and lusitropic dysfunction in myocardium from patients with dilated cardiomyopathy

Isometric force of contraction (DT), peak rate of tension increase (+T), peak rate of tension decrease (-T), time to peak tension (TPT), and time to half-relaxation (T 1/2 T) were measured in electrically driven human papillary muscle strips (New York Heart Association [NYHA] class IV heart transplants, dilated cardiomyopathy; nonfailing (NF) donor hearts, brain dead) (1 Hz, 37 degrees C) under basal conditions (1.8 mmol/L Ca2+) and after stimulation with isoprenaline, ouabain, and Ca2+. There was no difference in the isometric contraction (+T, -T, TPT, and T 1/2 T) between NYHA IV hearts and NF hearts under basal conditions. Inotropic stimulation above 300% of basal DT increased -T significantly more in NF hearts (p less than 0.05) compared with NYHA IV hearts. The effectiveness of ouabain and Ca2+ to increase DT was not significantly changed in NYHA IV hearts compared with NF hearts. The isoprenaline-mediated increase in DT was reduced (p less than 0.05) in NYHA IV hearts to a similar extent (70%) as beta-adrenoceptors were downregulated. When the rate of stimulation was increased to 3 Hz (force-frequency relationship), force of contraction increased only in NF preparations, whereas it decreased in NYHA IV myocardium (p less than 0.05). It was concluded that the contractile apparatus in terminally failing human myocardium is sufficient to maximally increase DT. During inotropic stimulation, abnormalities in diastolic rather than systolic contraction become evident. This may indicate abnormal intracellular Ca2+ handling.

Cardiac adenylyl cyclase, beta-adrenergic receptors, and G proteins in salt-sensitive hypertension.

The present study investigated whether high salt intake (8%) in Dahl salt-sensitive and salt-resistant rats with and without hypertension produces a heterologous desensitization of cardiac adenylyl cyclase as observed in various types of hypertension and human heart failure. In membranes from Dahl salt-sensitive rats on a high-salt diet (8%) basal, isoproterenol-, 5-guanylylimidodiphosphate-, and forskolin-stimulated adenylyl cyclase was reduced compared with the low-salt (0.4%) group and Dahl salt-resistant rats on either 0.4% or 8% sodium chloride. The activity of the catalyst was depressed, and the expression of the immunodetectable inhibitory G proteins Gi alpha was increased in Dahl salt-sensitive rats on 8% sodium chloride, whereas the density of beta-adrenergic receptors and the activity of the stimulatory G protein Gs alpha reconstituted into Gs alpha-deficient S49 cyc- mouse lymphoma cell membranes were unchanged in any condition studied. We conclude that high salt intake in salt-sensitive hypertensive Dahl rats produces hypertension, cardiac hypertrophy, and heterologous desensitization of cardiac adenylyl cyclase. The latter alteration is due to an increase of Gi alpha proteins and a depressed catalyst activity of adenylyl cyclase. The results demonstrate that heterologous adenylyl cyclase desensitization can precede the development of contractile dysfunction in later stages and can occur independently of changes in beta-adrenergic receptors.

Improvement of cholera toxin-catalyzed ADP-ribosylation by endogenous ADP-ribosylation factor from bovine brain provides evidence for an unchanged amount of Gsα in failing human myocardium

The aim of the present study was to investigate whether or not alterations of Gs alpha can be detected with cholera toxin-induced ADP-ribosylation in myocardial membranes from patients with heart failure. Therefore, Gs alpha was radiolabeled by cholera toxin-catalzyed (32P)ADP-ribosylation with (32P)NAD as substrate. In membranes from left ventricular myocardium of six patients with dilated cardiomyopathy classified as NYHA IV and three samples from two non-failing donor hearts, labeling was too weak to allow detection of possible changes in the amount of Gs alpha. Therefore, the cytosolic small molecular weight G protein ARF (ADP-ribosylation factor), a cofactor for cholera toxin-induced ADP-ribosylation of Gs alpha, was partially purified from bovine cerebral cortex. ARF activity was quantified by its ability to enhance auto-ADP-ribosylation of cholera toxin A1-subunit. Gs alpha was identified by comparing the ADP-ribosylation patterns of myocardial membranes, membranes prepared from human leukemia (HL 60) and S 49 mouse lymphoma wild type cells (45 kDa-band present) with membranes of the Gs alpha-deficient S 49 variant cyc- (45 kDa-band missing). In the presence of ARF, specific radiolabeling of the Mr 45,000 subtype of Gs alpha was markedly enhanced. The amounts of Gs alpha as measured by cholera toxin-dependent (32P)-ADP-ribosylation in the presence of ARR were similar in failing and nonfailing human hearts. It is concluded that factors other than Gs alpha are responsible for the altered regulation of the adenylate cyclase complex in heart failure. Moreover, by enhancing cholera toxin-catalyzed ADP-ribosylation, endogenous ADP-ribosylation factor from bovine brain appears to be a useful tool to study Gs alpha even in tissues in which the labeling of Gs alpha is rather weak.

Calcium sensitivity and myosin light chain pattern of atrial and ventricular skinned cardiac fibers from patients with various kinds of cardiac disease

In the present study, the Ca2(+)-sensitivity and myosin light chain patterns of skinned fibers of right atrium and left papillary muscles of 27 patients suffering from mitral valve disease (MVD, moderate heart failure), ischemic cardiomyopathy (ICM, severe heart failure), dilated cardiomyopathy (DCM, severe heart failure), and coronary heart disease (CHD, no heart failure, no atrial hypertrophy) were investigated. Myosin light chains of both chemically skinned and intact samples were studied by two-dimensional gel electrophoresis (2D-PAGE). Ca2(+)-sensitivity of ventricular fibers was about 0.14 pCa-units higher than that of atrial fibers in all groups except dilated cardiomyopathy where this difference was markedly diminished (only 0.06 pCa-units). Generally, Ca2(+)-sensitivity of skinned ventricular fibers was the same among the different heart diseases. Skinned atrial fibers from patients with dilated cardiomyopathy, however, were significantly (about 0.08 pCa-units) more sensitive for Ca2+ than those of the other groups (coronary heart disease, mitral valve disease or ischemic cardiomyopathy) which showed similar Ca2(+)-tension relationships. Ventricle-specific P-light chain forms could be observed in atrial samples from patients of all groups, whereas no atrium-specific light chain forms were detectable in any ventricular sample. It is concluded that there is no difference in Ca2(+)-sensitivity of the ventricular contractile elements of the human heart in different heart diseases. In atrial myocardium, there is an increased Ca2(+)-sensitivity of skinned fibers from hearts with dilated cardiomyopathy which is probably related to an elevation of right atrial pressure.

Different cardiodepressant potency of various calcium antagonists in human myocardium

Abstract Calcium antagonists play an important role in the therapy of systemic hypertension 1 and coronary artery disease. 2 In heart failure, it has been reported that calcium antagonists produce beneficial hemodynamic effects by reducing afterload. 3 These compounds also produce negative inotropism, which can be of potential benefit in coronary artery disease by reducing energy expenditure of the heart or in hypercontractile states, such as some forms of hypertrophic cardiomyopathy. The therapeutic potential is often limited by their negative inotropic effects, which, in addition to their vasodilatory properties, can lead to severe hypotension. Direct negative inotropic effects of calcium antagonists have been studied in various laboratory animals and species differences were observed. 4 Hitherto, data on the human heart have not been available. This study was performed to compare the negative inotropic effects of 1,4 dihydropyridines (nifedipine, nitrendipine), diltiazem and verapamil on diseased human myocardium. Isradipine, a new 1,4 dihydropyridine with supposedly minor negative inotropic actions, was also studied.