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Am J Physiol Heart Circ Physiol 278: H2076-H2083, 2000;
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Vol. 278, Issue 6, H2076-H2083, June 2000

Changes of beta -adrenergic signaling in compensated human cardiac hypertrophy depend on the underlying disease

Ulrich Schotten1, Karsten Filzmaier1, Britta Borghardt1, Simone Kulka1, Friedrich Schoendube2, Carlos Schumacher1, and Peter Hanrath1

1 Department of Cardiology and 2 Department of Thoracic and Cardiovascular Surgery, University Hospital Aachen, D-52057 Aachen, Germany


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In human heart failure, desensitization of the beta -adrenergic signal transduction has been reported to be one of the main pathophysiological alterations. However, data on the beta -adrenergic system in human compensated cardiac hypertrophy are very limited. Therefore, we studied the myocardial beta -adrenergic signaling in patients suffering from hypertrophic obstructive cardiomyopathy (HOCM, n = 9) or from aortic valve stenosis (AoSt, n = 8). beta -Adrenoceptor density determined by [125I]iodocyanopindolol binding was reduced in HOCM and AoSt compared with nonhypertrophied, nonfailing myocardium (NF) of seven organ donors. In HOCM the protein expression of stimulatory G protein alpha -subunit (Gsalpha ) measured by immunoblotting was unchanged, whereas the inhibitory G protein alpha -subunit (Galpha i-2) was increased. In contrast, in AoSt, Galpha i-2 protein was unchanged, but Gsalpha protein was increased. Adenylyl cyclase stimulation by isoproterenol was reduced in HOCM but not in AoSt. Plasma catecholamine levels were normal in all patients. In conclusion, both forms of hypertrophy are associated with beta -adrenoceptor downregulation but with different changes at the G protein level that occur before symptomatic heart failure due to progressive dilatation of the left ventricle develops and are not due to elevated plasma catecholamine levels.

aortic valve stenosis; G proteins; hypertrophic obstructive cardiomyopathy; beta -adrenoceptors; signal transduction


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

FORCE OF CONTRACTION IN HUMAN ventricular myocardium is mainly regulated by beta -adrenergic stimulation (10). We recently showed that in hypertrophic obstructive cardiomyopathy (HOCM) the positive inotropic effect of beta -adrenergic stimulation is reduced (38). A comparable reduction in the beta -adrenergic positive inotropism was previously reported in congestive heart failure, e.g., due to dilated cardiomyopathy (9). In dilated cardiomyopathy, this is known to be due to changes in the transmembrane beta -adrenergic signal transduction pathway. beta -Adrenoceptor density is reduced, and most probably there is an increase in the inhibitory G protein (Gi) alpha -subunit (Gialpha ) (19, 31). These changes are assumed to be due to a negative-feedback regulation activated by chronic catecholaminergic overstimulation (18). The aim of the present study was to determine whether comparable changes underlie the reduced positive inotropic effect of beta -adrenergic stimulation in HOCM. To test whether these changes are a common feature of hypertrophied human myocardium or occur specifically in primary myocardial hypertrophy due to HOCM, we investigated secondary hypertrophied myocardium of patients with acquired, severe aortic valve stenosis (AoSt) for comparison. In the literature there are very few data regarding the beta -adrenergic system in hypertrophied human myocardium, probably because of the limited access to appropriate tissue samples.

In primary cardiac hypertrophy due to HOCM, myocardial beta -adrenoceptor density has been found to be reduced in radioligand binding experiments (38) and with the use of positron emission tomography with 11C-labeled 4-(3-tertiarybutylamino-2-hydroxypropoxyl)-benzimidazole-2-one (CGP-12177) as tracer (28). However, comparative studies of myocardial G protein expression and adenylyl cyclase (AC) activity between nonfailing control myocardium and hypertrophied myocardium due to HOCM have not yet been performed.

In secondary cardiac hypertrophy due to AoSt, right atrial beta -adrenoceptor density has been reported to be reduced depending on the severity of clinical symptoms (11, 30). In patients with symptoms of severe heart failure (NY Heart Association III-IV), Steinfath et al. (40) reported a reduced beta -adrenoceptor density in septal biopsies obtained during aortic valve replacement. A study of Galinier et al. (21) confirms the reduced left ventricular beta -adrenoceptor density in AoSt and demonstrates an impaired expression of Gi proteins, but the control group consisted of patients with coronary artery disease with a reduced myocardial beta -adrenoceptor density and patients with mitral valve regurgitation suffering from symptomatic heart failure.

Therefore, it was the aim of the present study to investigate myocardial beta -adrenoceptor density and subtype distribution, G protein levels, and AC activity in primary and secondary left ventricular hypertrophy. The results were compared with those obtained from nonhypertrophied, nonfailing (NF) control myocardium of organ donors with no cardiovascular history but whose hearts could not be transplanted for technical reasons. Because the study focuses on compensated cardiac hypertrophy, patients with reduced left ventricular pump function were excluded.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Patients. Hypertrophied septal myocardium was obtained from nine patients (6 women and 3 men, mean age 45 ± 5 yr) suffering from HOCM. All patients had asymmetric hypertrophy of the basal interventricular septum (thickness as determined by M-mode echocardiography = 23 ± 2 mm). In these patients, hypertrophy was not caused by additional arterial hypertension or valvular stenosis. The patients underwent septal myectomy (29) for symptomatic left ventricular outflow tract obstruction (chest pain and syncope) with a mean pressure gradient of 71 ± 8 mmHg. From the hearts of eight symptomatic patients (5 women and 3 men, mean age 67 ± 1 yr) suffering from acquired, severe AoSt (mean pressure gradient = 84 ± 1 mmHg), hypertrophied septal myocardium was obtained during valve replacement. All the patients (HOCM as well as AoSt) had normal left ventricular systolic pump function at rest, as proven by echocardiography and ventriculography. Cardiac indexes (3.0 ± 0.2 and 3.2 ± 0.3 l · min-1 · m-2 for HOCM and AoSt, respectively; not significant) and mean pulmonary capillary wedge pressures (13 ± 2 and 12 ± 3 mmHg for HOCM and AoSt, respectively, not significant) were within the normal range. None of the patients had received beta -adrenoceptor blocking agents or angiotensin-converting enzyme inhibitors for >= 1 yr before operation. Patients with angiographically proven coronary artery disease, moderate or severe mitral regurgitation, or hypertension were excluded from the study. Nonfailing, nonhypertrophied left ventricular septal myocardium was obtained from the hearts of seven multiorgan donors who died from cerebral trauma (n = 5) or cerebral hemorrhage (n = 2). Their hearts could not be transplanted for technical reasons. There was no history of cardiac disease in these patients, and they did not receive catecholamines before heart explantation. Informed written consent was obtained from all patients before operation; in the case of heart explantation, consent was obtained from the relatives. The investigation conforms with the principles outlined in the Declaration of Helsinki.

beta -Adrenoceptors. Immediately after surgical resection, tissue samples were frozen in liquid nitrogen and stored at -80°C until use. beta -Adrenoceptor density and subtype distribution were determined by radioligand binding experiments according to Brodde et al. (12) with minor modifications. From each heart, 110 mg of myocardium (all steps at 4°C) were homogenized with a Turrax homogenizer (IKA) in 1 mmol/l KHCO3. After low-speed centrifugation (60 g for 20 min; Kontron), the supernatant was filtered through four layers of gauze and recentrifuged at 50,000 g for 20 min. The resulting pellet was resuspended in binding buffer [10 mmol/l Tris · HCl, 154 mmol/l NaCl, 0.01% (wt/vol) ascorbic acid, pH 7.4], giving a final protein concentration of 160 µg/ml in 250 µl of assay volume. Incubation with [125I]iodocyanopindolol (ICYP; 7 concentrations in the range 4-350 pmol/l) reached equilibrium within 90 min at 37°C. The reaction was stopped by the addition of 6 ml of 4°C buffer and vacuum filtration through GF/C filters (Whatman, Maidstone, UK) with a Brandel cell harvester (Dunn). Filters were washed twice with 6 ml of the buffer, and retained radioactivity was counted in a gamma counter (Berthold). Nonspecific binding was determined in the presence of 1 µmol/l CGP-12177 and was ~15% of total binding at 10 pmol/l ICYP. Radioligand binding to beta 1-adrenoceptors was discriminated from binding to beta 2-adrenoceptors by displacement of ICYP with different concentrations of the 10,000-fold selective beta 1-adrenoceptor blocker 1-(2-(3-carbamoyl-4-hydroxy)phenoxyethylamino)-3-(4-(-1-methyl-4-trifluoro-methyl-2-imidazolyl)phenoxy)-2-propanolol-methansulfonate (CGP-20712A, 10-11-10-4 mol/l) (17).

Maximal beta -adrenoceptor number and ICYP affinity were determined by Scatchard analysis (35) of saturation binding experiments. Saturation curves, Scatchard analysis, and displacement experiments were calculated with computer software designed by GraphPad (San Diego, CA). Protein content was measured by the method of Bradford (8), with gamma -globulin as standard. Noncollagenic protein, defined as not precipitating in 0.05 mol/l NaOH, was additionally determined by the same method.

G proteins. From each heart, 120 mg of myocardium were homogenized in 10 mmol/l Trizma, 1 mmol/l EDTA, 255 mmol/l sucrose, and 1 mmol/l phenylmethylsulfonyl fluoride (pH 7.4). Electrophoresis of homogenate aliquots containing 80 µg protein/lane was performed according to Laemmli (27). Electrophoretic transfer on nitrocellulose (0.45 µm) was performed by tank blotting [Bio-Rad; transfer buffer: 25 mmol/l Trizma, 192 mmol/l glycine, and 20% (vol/vol) methanol] (41) and was complete after 66 min at 1 A. Nitrocellulose was blocked with 5% nonfat milk for 2 h at room temperature and subsequently exposed to commercially available polyclonal rabbit antisera (NEN DuPont) directed against the alpha -subunits of the stimulatory G (Gs) protein (antiserum RM/1) and the Gi protein (antiserum AS/7). Radioactive labeling was performed with 125I-labeled protein A. Then autoradiography membranes were cut, and single band signals were quantified in a gamma counter. Specificity of both antisera used in this study has been demonstrated elsewhere (22, 23, 39).

In both proteins, there was a linear correlation between the amount of protein subjected to gel electrophoresis and the radioactive signals. The G protein signals were linear up to 120 µg loaded in each lane, indicating that quantification with 80 µg/lane was performed within the linear range.

AC. AC activity was determined according to the method described by Salomon et al. (34). Sixty milligrams of myocardium were homogenized in 3 ml of buffer (5 mmol/l Tris, 1 mmol/l EGTA, 250 mmol/l sucrose, pH 7.4) with a Turrax homogenizer. After the addition of 10 ml of buffer, homogenates were centrifuged twice at 2,200 g for 20 min. Pellets were resuspended in 100 mmol/l Tris and 100 mmol/l sucrose (pH 7.4), giving a protein concentration of 1.5 mg/ml. Enzyme activity was determined in assays (100 µl) containing 75 mmol/l Tris, 50 mmol/l sucrose, 5 mmol/l MgCl2, 5 mmol/l creatine phosphate, 2.5 U of creatine kinase, 1 mmol/l IBMX, 0.1 mmol/l cAMP ([3H]cAMP, 60,000 cpm), and 1 mmol/l Na2-ATP ([32P]ATP, 1 µCi). To assess maximal catalyst activity, experiments were performed in the presence of 5 mmol/l MnCl2, which is known to uncouple AC from G proteins (15). In these cases, MgCl2 was replaced by 5 mmol/l MnCl2. When the effect of isoproterenol was studied, 5'-guanylyl imidodiphosphate [Gpp(NH)p, 10 µmol/l] was added to the assay solution. The reaction was started by the addition of the homogenates (75 µg of protein) to the reaction mixture. After 20 min (5 min in the case of assays containing MnCl2), incubation was stopped by the addition of 100 µl of 1 mol/l HCl and subsequent heating to 95°C for 5 min. After the addition of 800 µl of 125 mmol/l KOH and centrifugation for 2 min at 12,000 g, the supernatants were applied to Dowex columns (AG 50W 4X, Bio-Rad) and subsequently to aluminum columns. After elution with 6 ml of 100 mmol/l imidazole (pH 7.5) and addition of 11 ml of scintillation cocktail (Ultima Gold XR, Canberra Packard), radioactivity was counted.

Under the experimental conditions reported, AC activity was linear with respect to incubation time (up to 30 min) and protein concentration (up to 150 µg/assay), indicating that determination of AC activity with 75 µg of protein and 20 min of incubation time was within the linear range.

Myosin content. Myosin content was measured in the same homogenates that were used for Western blot analysis and AC assays, as described previously (36). Homogenates were diluted in 10 mmol/l Trizma, 1 mmol/l EDTA, 255 mmol/l sucrose, and 1 mmol/l phenylmethylsulfonyl fluoride (pH 7.4) to a final concentration of 2 µg/100 µl. Electrophoretic separation of the proteins was performed as described above but with 2 µg protein/lane. The gels were stained in Coomassie brilliant blue G (Bio-Rad, Hamburg, Germany). After the gels were destained for 24 h, the remaining protein bands were scanned with a laser densitometer (Ultro Scan XL Laser Densitometer, Pharmacia, Freiburg, Germany). Myosin bands were identified by the same mobility as a commercially available myosin marker (Bio-Rad) at 205 kDa. Linearity between amounts of protein and densitometric signals was proven by plotting different amounts of protein against corresponding densitometric units.

Catecholamines. Catecholamines were quantified by reverse-phase high-pressure liquid chromatography with electrochemical detection (Pharmacia, Freiburg, Germany) (16, 26). Plasma catecholamine blood samples were collected between 6 and 7 AM, after a resting phase of >= 12 h. Two patients with HOCM did not fulfill these conditions and, therefore, were excluded.

Statistics. Values are means ± SE. Statistical significance of mean differences was determined by one-way ANOVA and Newman-Keuls multiple comparison tests. P < 0.05 was considered significant.

Materials. CGP-12177 and CGP-20712A were gifts from Ciba-Geigy (Basel, Switzerland), IBMX from Aldrich Chemie (Steinheim, Germany), and isoproterenol from Boehringer Ingelheim.

Trizma, Tween 20, and ATP · Tris were obtained from Sigma Chemical (Deisenhofen, Germany), nitrocellulose from Schleicher and Schuell, catecholamine kit from Chromsystems (München, Germany), [125I]ICYP and antibodies RM/1 and AS/7 from NEN, DuPont de Nemours (Bad Homburg, Germany), and 125I-protein A from Amersham Buchler (Braunschweig, Germany).

All chemicals were of analytic or best commercial grade available. Deionized and twice-distilled water was used throughout the experiments.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

beta -Adrenoceptors. Representative saturation binding experiments are depicted in Fig. 1A. As shown in Fig. 1B, Scatchard plots were linear, indicating that ICYP bound to a single binding site. The slopes of the Scatchard plots were similar in all three groups, reflecting similar affinities of the radioligand to the receptor binding sites (range 5-17 pmol/l). ICYP displacement with the highly selective beta 1-adrenoceptor antagonist CGP-20712A revealed a reduced percentage of beta 1-adrenoceptor subtype in HOCM and AoSt compared with NF (Fig. 1C).


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  		Fig. 1.   Representative binding experiments. Representative saturation binding curves (A) and Scatchard plot (B) of [125I]iodocyanopindolol (ICYP) in nonfailing (NF) and hypertrophied myocardium due to hypertrophic obstructive cardiomyopathy (HOCM) and aortic valve stenosis (AoSt) are shown. Specific ICYP binding showed saturation characteristics, and Scatchard plots were linear, indicating that ICYP bound to 1 distinct receptor population. Discrimination of beta 1- and beta 2-adrenoceptor subtype was shown by ICYP displacement with CGP-20712A (C).

In NF the beta -adrenoceptor density was 74 fmol/mg protein (Fig. 2). This receptor population consisted of 77% beta 1- and 23% beta 2-adrenoceptors. In HOCM and AoSt, beta -adrenoceptor density was reduced to 59 and 52%, respectively (Fig. 2A; P < 0.05). The downregulation was also observable when the beta -adrenoceptor density was related to noncollagenic protein instead of total protein content. In both diseases the reduced beta -adrenoceptor density was due to a selective loss of beta 1-adrenoceptors, whereas beta 2-adrenoceptor density was unchanged (Fig. 2B).


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  		Fig. 2.   beta -Adrenoceptor density of hypertrophied myocardium. A: beta -adrenoceptor density (Bmax) was reduced in HOCM and AoSt compared with NF controls. B: in HOCM, as well as in AoSt, beta 1-adrenoceptors were selectively downregulated, whereas beta 2-adrenoceptor densities were unchanged in comparison with NF myocardium. Numbers in parentheses represent number of patients. Values are means ± SE.

G proteins. Figure 3 shows the results of representative immunoblot experiments. The polyclonal antiserum RM/1 specifically bound to two proteins (52 and 45 kDa) representing the alpha -subunits of the Gs protein, Gsalpha -long (52 kDa) and Gsalpha -short (45 kDa) (39). The antiserum AS/7 is directed against the alpha -subunit of the Gi proteins, Galpha i-1 and Galpha i-2. In human myocardium it bound specifically to a 40-kDa protein, i.e., to Galpha i-2 (22, 23).


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  		Fig. 3.   Representative autoradiographs. Autoradiographs of Western blots (80 µg protein/lane) with different antibodies directed against alpha -subunits of G proteins are shown. Bound antibodies were detected with 125I-protein A. A standard sample of an NF control (Std) was applied to each gel to promote comparability of determination from different blots. Antibody RM/1 directed against 2 splicing forms of stimulatory G protein alpha -subunit (Gsalpha ) specifically marked 2 bands at 52 and 45 kDa. Antibody AS/7 directed against Galpha i-1 and Galpha i-2 showed a band at 40 kDa. Gsalpha signals were more pronounced in AoSt than in HOCM and NF; Gi signals were increased in HOCM but not in AoSt.

The myocardial expression of Gsalpha (52 kDa) protein was increased in AoSt compared with NF and HOCM (Fig. 4; P < 0.05), whereas there was no difference between HOCM and NF. With respect to Gsalpha (45 kDa) protein, there was no difference in the myocardial level in all three groups. In contrast, there was an increase in the myocardial level of Galpha i-2 protein in HOCM compared with NF and AoSt (Fig. 5; P < 0.05). Gsalpha protein upregulation in AoSt and Gialpha protein upregulation in HOCM were also observable when the protein expression was related to the myosin content of the homogenates (Table 1).


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  		Fig. 4.   Gs protein level in hypertrophied myocardium. Radioactive signal intensities of Gsalpha are shown (means ± SE). A: Gsalpha (52 kDa) protein was increased in AoSt compared with NF controls and HOCM. B: Gsalpha (45 kDa) protein did not differ in all 3 diagnoses. Numbers in parentheses represent number of patients.



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  		Fig. 5.   Gi protein level in hypertrophied myocardium. Radioactive signal intensities of Galpha i-2 protein are shown (means ± SE). There was an increase in Galpha i-2 protein in HOCM in comparison with NF myocardium; Galpha i-2 concentration in AoSt was the same as in NF. Numbers in parentheses represent number of patients.


                              
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  		Table 1.   G protein expression related to myosin content of homogenates

AC activity. Table 2 gives the results of the AC activity assays. Absolute baseline activities as well as maximal AC activity revealed by stimulation with MnCl2 were the same in NF, HOCM, and AoSt. Isoproterenol exerted the lowest AC stimulation in HOCM. Additionally, related to maximal enzyme stimulation by MnCl2, isoproterenol stimulation was significantly reduced in HOCM (increased MnCl2-to-isoproterenol ratio) but unchanged in AoSt compared with NF. In contrast, AC stimulation by Gpp(NH)p, a nonhydrolyzable GTP analog, was higher in AoSt than in HOCM. When related to maximal AC activity in the presence of MnCl2, stimulation by Gpp(NH)p was significantly increased in AoSt compared with NF [decreased MnCl2-to-Gpp(NH)p ratio] but decreased in HOCM [increased MnCl2-to-Gpp(NH)p ratio]. Stimulation of Gs protein activity by NaF resulted in a higher AC activity in AoSt than in HOCM and NF in absolute values or when related to maximal AC activity in the presence of MnCl2. All differences in AC activity between patient groups that were present when AC activity was related to total protein were also observable when AC activity was related to the myosin content of the homogenates.

                              
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  		Table 2.   Myocardial adenylyl cyclase activity

Catecholamine levels. Plasma levels of norepinephrine and epinephrine were normal in HOCM as well as in AoSt (Table 3).

                              
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  		Table 3.   Plasma catecholamine levels


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Unlike studies on beta -adrenergic signal transduction in failing human myocardium due to dilated or ischemic cardiomyopathy that can be performed on explanted hearts, studies on signal transduction in hypertrophied myocardium have mainly been performed in animal models (2, 4, 20). The results of the present study confirm previous reports of beta -adrenoceptor downregulation in AoSt (21, 40) and HOCM (24, 28, 38). Furthermore, the study demonstrates that, in compensated cardiac hypertrophy in the absence of symptomatic heart failure due to depressed systolic left ventricular pump function and elevated plasma catecholamine levels, additional alterations of the beta -adrenergic signal transduction pathway occur at the level of G proteins. These alterations of the beta -adrenergic system are of functional relevance with respect to AC activity and differ in primary hypertrophied myocardium from patients with HOCM and in secondary hypertrophied myocardium from patients with acquired severe AoSt.

HOCM. In hypertrophied septal left ventricular myocardium of patients suffering from HOCM, the beta -adrenoceptor density is reduced as a result of selective loss of beta 1-adrenoceptors. The beta -adrenoceptor downregulation cannot be explained by an increased protein content of the extracellular matrix due to fibrosis, because the beta 1-adrenoceptor density was also diminished when related to noncollagenic protein and because only beta 1- but not beta 2-adrenoceptor density was reduced. Therefore, the selective reduction of beta 1-adrenoceptor density is due to a selective lack of increase in beta 1-adrenoceptor gene expression during the development of cardiac hypertrophy (1) or to downregulation as a consequence of catecholaminergic overstimulation.

The myocardial level of the Gsalpha (52 and 45 kDa) protein is unchanged in HOCM patients, whereas there is an increase in protein expression of Gialpha protein. The changes of beta -adrenoceptor density as well as of the Gi protein level are directed toward a desensitization of the beta -adrenergic signal transduction. This assumption is supported by the present data on AC. In HOCM, absolute isoproterenol-stimulated AC activity was reduced and was only half as much as in NF, when related to maximal achievable AC activity. Most probably these changes contribute to the reduced inotropic effect of isoproterenol in HOCM (38).

AoSt. Similar to our observation in HOCM, in compensated cardiac hypertrophy due to acquired severe AoSt, beta 1-adrenoceptors were selectively downregulated, whereas beta 2-adrenoceptor density was unchanged. Interestingly, in the same myocardial specimens, Gsalpha (52 kDa) protein was increased, whereas Gialpha protein was similar to that in NF. From a functional point of view, myocardial beta -adrenoceptor density and G protein levels are therefore changed counteractively in AoSt. The functional relevance of increased Gsalpha protein in hypertrophied myocardium of AoSt patients is supported by the observation that specific stimulation of Gs protein by NaF exerts a more pronounced AC stimulation in AoSt than in HOCM and NF. Furthermore, in relation to maximal AC activity, stimulation of Gs and Gi proteins by Gpp(NH)p results in a higher AC activity in AoSt than in HOCM and NF.

HOCM vs. AoSt. In congestive heart failure, beta -adrenoceptor downregulation is most probably due to a systemic catecholaminergic overstimulation determined by increased plasma catecholamine levels. This mechanism can be excluded as the cause of beta -adrenoceptor downregulation in HOCM and AoSt, since plasma catecholamine levels reported here and by others (21, 25, 38) were within the normal range. However, even in the absence of elevated plasma catecholamine levels, beta -adrenoceptor downregulation might occur as a consequence of locally enhanced sympathetic activation. In an animal model of hypertensive cardiac hypertrophy (3, 7), it has been demonstrated that myocardial sympathetic nerve terminals become depleted of epinephrine and neuropeptide Y, which is coreleased with norepinephrine (33) before plasma catecholamine levels increase. Furthermore, in HOCM, Brush et al. (13) showed a reduced myocardial neuronal catecholamine reuptake, which might result in a local beta 1-adrenoceptor overstimulation. Therefore, local sympathetic stimulation might explain beta -adrenoceptor downregulation in hypertrophied human myocardium, although plasma catecholamine levels are not elevated. Alternatively, beta -adrenoceptor downregulation might be due to a selective lack of increase in beta 1-adrenoceptor expression during development of hypertrophy, as was hypothesized previously (1).

The most interesting differences between HOCM and AoSt were the different changes in myocardial G protein levels with respective alterations in AC activity. It is noteworthy that all differences in G protein levels and AC activity were observable in relation to total protein content as well as in relation to the myosin content of the homogenates, indicating that altered protein content of the extracellular matrix does not account for the biochemical alterations observed. In HOCM, Gialpha protein expression is higher than in AoSt and in NF, whereas in AoSt, Gsalpha protein is increased. This difference is of functional importance. Activation of G proteins by Gpp(NH)p results in a decreased AC activity in HOCM but an increased AC activity in AoSt in relation to maximal AC activity. NaF-induced activation of Gs protein led to a more pronounced AC activation in AoSt than in HOCM and NF, indicating that Gs protein upregulation might lead to a higher probability of Gsalpha -AC complex formation. Furthermore, whereas absolute isoproterenol-stimulated AC activity is reduced in HOCM, there is no significant difference between AoSt and NF. This implies that increased Gsalpha protein in AoSt may functionally antagonize beta -adrenoceptor downregulation. Although this hypothesis is challenged by a recent report about a large stoichiometric excess of Gs protein molecules relative to beta -adrenoceptors and AC in rat ventricular myocytes (32), this report does not necessarily indicate that Gs protein upregulation could not be of functional relevance. First, although Gs protein might also be in great excess in humans, it may not be in functional excess if affinity of Gs protein for AC is low. Furthermore, high levels of Gs protein might compete with other G proteins for binding sites at AC, which would mean that AC activity is in part a function of the relative quantities of different G proteins.

The hypothesis that Gs protein upregulation might antagonize beta -adrenoceptor downregulation is further supported by results of contraction experiments. We previously showed that the positive inotropic potency of isoproterenol is reduced in HOCM (EC50 = 0.21 µmol/l) compared with NF (EC50 = 0.019 µmol/l) (38). Recently, in isometric contraction experiments with myocardial preparations obtained from patients with AoSt, we observed a similar positive inotropic potency of isoproterenol [EC50 = 0.036 µmol/l, (37)], as in NF myocardium. This interesting observation supports the hypothesis that increment of myocardial Gi protein level is one of the key alterations underlying catecholamine refractoriness in several pathophysiological conditions (5). Similar to the situation in heart failure, myocardial Gi protein content has been found to be increased in catecholamine-refractory cardiogenic and septic shock (5, 6) as well as in hypertensive cardiomyopathy (14). In our study, increased Gialpha protein expression has only been found in HOCM in which the positive inotropic potency of catecholamines and the stimulatory effect of isoproterenol on AC were reduced. In contrast, in AoSt, no increase of Gialpha protein was observable, and AC was not desensitized toward isoproterenol.

In summary, the study shows that, in compensated human cardiac hypertrophy, changes of the beta -adrenergic signal transduction pathway occur; these changes are associated with a selective beta 1-adrenoceptor downregulation and differ in primary and secondary hypertrophy with respect to G protein expression and AC activity. These alterations are of functional relevance and occurred in the absence of elevated plasma catecholamine levels and before symptomatic heart failure due to progressive dilation of the left ventricle develops.


    ACKNOWLEDGEMENTS

We thank Mireille van Helden for excellent technical assistance.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: U. Schotten, Medical Clinic I, University Hospital Aachen, Pauwelsstrasse 30, D-52057 Aachen, Germany (E-mail: usch{at}pcserver.mk1.rwth-aachen.de).

Received 30 August 1999; accepted in final form 22 December 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Boheler, KR, and Schwartz K. Gene expression in cardiac hypertrophy. Trends Cardiovasc Med 2: 176-182, 1992[ISI].

2.   Böhm, M, Castellano M, Agabiti RE, Flesch M, Paul M, and Erdmann E. Dose-dependent dissociation of ACE inhibitor effects on blood pressure, cardiac hypertrophy, and beta -adrenergic signal transduction. Circulation 92: 3006-3013, 1995[Abstract/Free Full Text].

3.   Böhm, M, Ettelbrueck S, Flesch M, van Gilst W, Knorr A, Maack C, Pinto Y, Paul M, Teisman A, and Zolk O. beta -Adrenergic signal transduction following carvedilol treatment in hypertensive cardiac hypertrophy. Cardiovasc Res 40: 146-155, 1998[ISI][Medline].

4.   Böhm, M, Gierschik P, Knorr A, Larisch K, Weismann K, and Erdmann E. Role of altered G-protein expression in the regulation of myocardial adenylate cyclase activity and force of contraction in spontaneous hypertensive cardiomyopathy in rats. J Hypertens 10: 1115-1128, 1992[ISI][Medline].

5.   Böhm, M, Kirchmayr R, and Erdmann E. Myocardial Gialpha protein levels in patients with hypertensive cardiac hypertrophy, ischemic heart disease and cardiogenic shock. Cardiovasc Res 30: 611-618, 1995[ISI][Medline].

6.   Böhm, M, Kirchmayr R, Gierschik P, and Erdmann E. Increase of myocardial inhibitory G-proteins in catecholamine-refractory septic shock or in septic multiorgan failure. Am J Med 98: 183-186, 1995[ISI][Medline].

7.   Böhm, M, Moll M, Schmid B, Paul M, Ganten D, Castellano M, and Erdmann E. beta -Adrenergic neuroeffector mechanisms in cardiac hypertrophy of renin transgenic rats. Hypertension 24: 653-662, 1994[Abstract/Free Full Text].

8.   Bradford, MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976[ISI][Medline].

9.   Bristow, MR, Ginsburg R, Minobe W, Cubicciotti RS, Sageman WS, Lurie K, Billingham ME, Harrison DC, and Stinson EB. Decreased catecholamine sensitivity and beta -adrenergic-receptor density in failing human hearts. N Engl J Med 307: 205-211, 1982[Abstract].

10.   Bristow, MR, Kantrowitz NE, Ginsburg R, and Fowler MB. beta -Adrenergic function in heart muscle disease and heart failure. J Mol Cell Cardiol 17 Suppl2: 41-52, 1985.

11.   Brodde, OE. Pathophysiology of the beta -adrenoceptor system in chronic heart failure: consequences for treatment with agonists, partial agonists or antagonists? Eur Heart J 12 Suppl F: 54-62, 1991.

12.   Brodde, OE, Karad K, Zerkowski HR, Rohm N, and Reidemeister JC. Coexistence of beta 1- and beta 2-adrenoceptors in human right atrium. Direct identification by (+/-)-[125I]iodocyanopindolol binding. Circ Res 53: 752-758, 1983[Abstract/Free Full Text].

13.   Brush, JEJ, Eisenhofer G, Garty M, Stull R, Maron BJ, Cannon RO, Panza JA, Epstein SE, and Goldstein DS. Cardiac norepinephrine kinetics in hypertrophic cardiomyopathy. Circulation 79: 836-844, 1989[Abstract/Free Full Text].

14.   Castellano, M, and Böhm M. The cardiac beta -adrenoceptor-mediated signaling pathway and its alterations in hypertensive heart disease. Hypertension 29: 715-722, 1997[Abstract/Free Full Text].

15.   Cech, S, Broaddus W, and Maguire M. Adenylate cyclase: the role of magnesium and other divalent cations. Mol Cell Biochem 33: 67-92, 1980[ISI][Medline].

16.   Davis, GC, Kissinger PT, and Shoup RE. Strategies for determination of serum or plasma norepinephrine by reverse-phase liquid chromatography. Anal Chem 53: 156-159, 1981[Medline].

17.   Dooley, DJ, Bittiger H, and Reymann NC. CGP 20712A: a useful tool for quantitating beta 1- and beta 2-adrenoceptors. Eur J Pharmacol 130: 137-139, 1986[ISI][Medline].

18.   Feldman, AM. Modulation of adrenergic receptors and G-transduction proteins in failing human ventricular myocardium. Circulation 87: IV27-IV34, 1993.

19.   Feldman, AM, Cates AE, Veazey WB, Hershberger RE, Bristow MR, Baughman KL, Baumgartner WA, and Van Dop C. Increase of the 40,000-mol wt pertussis toxin substrate (G protein) in the failing human heart. J Clin Invest 82: 189-197, 1988.

20.   Flesch, M, Erdmann E, and Böhm M. Changes in beta -adrenoceptors and G-proteins during the transition from cardiac hypertrophy to heart failure. J Card Fail 2: S35-S43, 1996[Medline].

21.   Galinier, M, Senard JM, Valet P, Arias A, Daviaud D, Glock Y, Bounhoure JP, and Montastruc JL. Cardiac beta -adrenoceptors and adenylyl cyclase activity in human left ventricular hypertrophy due to pressure overload. Fundam Clin Pharmacol 8: 90-99, 1994[ISI][Medline].

22.   Goldsmith, P, Gierschik P, Milligan G, Unson CG, Vinitsky R, Malech HL, and Spiegel AM. Antibodies directed against synthetic peptides distinguish between GTP-binding proteins in neutrophil and brain. J Biol Chem 262: 14683-14688, 1987[Abstract/Free Full Text].

23.   Goldsmith, P, Rossiter K, Carter A, Simonds W, Unson CG, Vinitsky R, and Spiegel AM. Identification of the GTP-binding protein encoded by Gi3 complementary DNA. J Biol Chem 263: 6476-6479, 1988[Abstract/Free Full Text].

24.   Golf, S, Myhre E, Abdelnoor M, Andersen D, and Hansson V. Hypertrophic cardiomyopathy characterised by beta -adrenoceptor density, relative amount of beta -adrenoceptor subtypes and adenylate cyclase activity. Cardiovasc Res 19: 693-699, 1985[ISI][Medline].

25.   Kawai, C, Yui Y, Hoshino T, Sasayama S, and Matsumori A. Myocardial catecholamines in hypertrophic and dilated (congestive) cardiomyopathy: a biopsy study. J Am Coll Cardiol 2: 834-840, 1983[Abstract].

26.   Keller, R, Oke A, Mefford I, and Adams RN. Liquid chromatographic analysis of catecholamines: routine assay for regional brain mapping. Life Sci 19: 995-1003, 1976[ISI][Medline].

27.   Laemmli, UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970[Medline].

28.   Lefroy, DC, De SR, Choudhury L, Uren NG, Crake T, Rhodes CG, Lammertsma AA, Boyd H, Patsalos PN, and Nihoyannopoulos P. Diffuse reduction of myocardial beta -adrenoceptors in hypertrophic cardiomyopathy: a study with positron emission tomography. J Am Coll Cardiol 22: 1653-1660, 1993[Abstract].

29.   Messmer, BJ. Extended myectomy for hypertrophic obstructive cardiomyopathy. Ann Thorac Surg 58: 575-577, 1994[Abstract].

30.   Michel, MC, Maisel AS, and Brodde OE. Mitigation of beta 1- and/or beta 2-adrenoceptor function in human heart failure. Br J Clin Pharmacol 30 Suppl1: 37S-42S, 1990.

31.   Neumann, J, Schmitz W, Scholz H, von Meyerinck L, Doring V, and Kalmar P. Increase in myocardial Gi proteins in heart failure. Lancet 2: 936-937, 1988[ISI][Medline].

32.   Post, S, Hilal DR, Urasawa K, Brunton L, and Insel PA. Quantification of signaling components and amplification in the beta -adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes. Biochem J 311: 75-80, 1995.

33.   Purjeranta, M, Rechardt L, Pelto HM, and Kyösola K. Light and electron microscopic demonstration of neuropeptide Y-like immunoreactive nerves in human cardiac muscle. Virchows Arch 410: 147-151, 1986.

34.   Salomon, Y, Londos C, and Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541-548, 1974[ISI][Medline].

35.   Scatchard, G. The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51: 660-672, 1949[ISI].

36.   Schotten, U, Koenigs B, Schoendube F, Boknik P, Schmitz W, and Hanrath P. Reduced sarcoplasmic reticulum Ca2+-ATPase protein expression in compensated primary and secondary cardiac hypertrophy. J Mol Cell Cardiol 31: 1483-1494, 1999[ISI][Medline].

37.   Schotten, U, Schumacher C, Koenigs B, Hense M, Schoendube FA, and Hanrath P. Increased inhibitory G protein level rather than beta -adrenoceptor downregulation determines catecholamine refractoriness in human hypertrophied myocardium (Abstract). J Mol Med 76: B29, 1998.

38.   Schumacher, C, Becker H, Conrads R, Schotten U, Pott S, Kellinghaus M, Sigmund M, Schondube F, Preusse C, Schulte HD, and Hanrath P. Hypertrophic cardiomyopathy: a desensitized cardiac beta -adrenergic system in the presence of normal plasma catecholamine concentrations. Naunyn Schmiedebergs Arch Pharmacol 351: 398-407, 1995[ISI][Medline].

39.   Simonds, WF, Goldsmith PK, Woodard CJ, Unson CG, and Spiegel AM. Receptor and effector interactions of Gs. Functional studies with antibodies to the alpha s carboxyl-terminal decapeptide. FEBS Lett 249: 189-194, 1989[ISI][Medline].

40.   Steinfath, M, Geertz B, Schmitz W, Scholz H, Haverich A, Breil I, Hanrath P, Reupcke C, Sigmund M, and Lo HB. Distinct down-regulation of cardiac beta 1- and beta 2-adrenoceptors in different human heart diseases. Naunyn Schmiedebergs Arch Pharmacol 343: 217-220, 1991[ISI][Medline].

41.   Towbin, H, Staehelin T, and Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350-4354, 1979[Abstract/Free Full Text].

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