Total beta-adrenoceptor deficiency results in cardiac hypotrophy and negative inotropy.

The present study investigated cardiac function in hearts of mice with total deficiency of the beta1-, beta2- and beta3-adrenoceptors (TKO) in comparison to wildtype mice (WT). We investigated cardiac morphology and echocardiographic function, measured protein expression of Ca2+-regulatory proteins, SERCA 2a activity, myofibrillar function, and performed running wheel tests. Heart weight and heart-to-body weight ratio were significantly smaller in TKO as compared to WT. This was accompanied by a decrease in the size of the cardiomyocytes in TKO. Heart rate and ejection fraction were significantly diminished in TKO as compared to WT. Protein expressions of SERCA 2a, ryanodine receptor and Na+/Ca2)-exchanger were similar in TKO and WT mice, but phospholamban protein expression was increased. PKA-dependent phosphorylation of phospholamban at serine 16 was absent and CaMKII-dependent phosphorylation at threonine 17 was decreased in TKO. All alterations were paralleled by a decrease in SERCA 2a-activity. A similar maximal calcium-dependent tension but an increased myofibrillar calcium-sensitivity was measured in TKO as compared to WT. We did not observe relevant functional impairments of TKO in running wheel tests. In the absence of beta-agonistic stimulation, SERCA 2a activity is mainly regulated by alterations of phospholamban expression and phosphorylation. The decreased SERCA 2a activity following beta-adrenoceptor deficiency may be partly compensated by an increased myofibrillar calcium-sensitivity.

Regional expression of sodium pump subunits isoforms and Na+-Ca++ exchanger in the human heart.

Cardiac glycosides exert a positive inotropic effect by inhibiting sodium pump (Na,K-ATPase) activity, decreasing the driving force for Na+-Ca++ exchange, and increasing cellular content and release of Ca++ during depolarization. Since the inotropic response will be a function of the level of expression of sodium pumps, which are alpha(beta) heterodimers, and of Na+-Ca++ exchangers, this study aimed to determine the regional pattern of expression of these transporters in the heart. Immunoblot assays of homogenate from atria, ventricles, and septa of 14 nonfailing human hearts established expression of Na,K-ATPase alpha1, alpha2, alpha3, beta1, and Na+-Ca++ exchangers in all regions. Na,K-ATPase beta2 expression is negligible, indicating that the human cardiac glycoside receptors are alpha1beta1, alpha2beta1, and alpha3beta1. alpha3, beta1, sodium pump activity, and Na+-Ca++ exchanger levels were 30-50% lower in atria compared to ventricles and/or septum; differences between ventricles and septum were insignificant. Functionally, the EC50 of the sodium channel activator BDF 9148 to increase force of contraction was lower in atria than ventricle muscle strips (0.36 vs. 1.54 microM). These results define the distribution of the cardiac glycoside receptor isoforms in the human heart and they demonstrate that atria have fewer sodium pumps, fewer Na+-Ca++ exchangers, and enhanced sensitivity to inotropic stimulation compared to ventricles.

Loss of annexin A7 leads to alterations in frequency-induced shortening of isolated murine cardiomyocytes.

Annexin A7 has been proposed to function in the fusion of vesicles, acting as a Ca(2+) channel and as Ca(2+)-activated GTPase, thus inducing Ca(2+)/GTP-dependent secretory events. To understand the function of annexin A7, we have performed targeted disruption of the Anxa7 gene in mice. Matings between heterozygous mice produced offspring showing a normal Mendelian pattern of inheritance, indicating that the loss of annexin A7 did not interfere with viability in utero. Mice lacking annexin A7 showed no obvious phenotype and were fertile. To assay for exocytosis, insulin secretion from isolated islets of Langerhans was examined. Ca(2+)-induced and cyclic AMP-mediated potentiation of insulin secretion was unchanged in the absence of annexin A7, suggesting that it is not directly implicated in vesicle fusion. Ca(2+) regulation studied in isolated cardiomyocytes, showed that while cells from early embryos displayed intact Ca(2+) homeostasis and expressed all of the components required for excitation-contraction coupling, cardiomyocytes from adult Anxa7(-/-) mice exhibited an altered cell shortening-frequency relationship when stimulated with high frequencies. This suggests a function for annexin A7 in electromechanical coupling, probably through Ca(2+) homoeostasis.

Nebivolol induces eNOS activation and NO-liberation in murine corpus cavernosum.

Erectile function is critically dependent upon the activation of the endothelial nitric oxide synthase (eNOS) in the smooth muscle cells of penile corpus cavernosum tissue. Nebivolol is a beta(1)-selective beta-adrenoceptor blocker (beta-ARB) with additional vasodilating properties, which have been attributed to eNOS-activation. Our study investigated whether nebivolol is able to increase eNOS activity in erectile tissue. Murine penile tissue was incubated in an organ bath under control conditions and in the presence of nebivolol or metoprolol. Immunofluorescence staining was performed using specific antibodies against eNOS-activation or eNOS-serine 1177 phosphorylation. Corpus cavernosum smooth muscle tissue was identified using a smooth muscle actin antibody. In addition, slices of murine erectile tissue were incubated with diaminofluorescein (DAF), a specific fluorescence marker for NO-liberation. Under control conditions and after application of metoprolol, we observed a small eNOS-activation and serine 1177-phosphorylation in murine corpus cavernosum tissue. A significant increase in eNOS-activation and serine 1177-phosphorylation of eNOS was observed only in the presence of nebivolol (10 muM). These alterations of the eNOS protein induced after application of nebivolol were associated with a time-dependent increase in DAF fluorescence in murine erectile tissue. We conclude that beta-adrenoceptor blockers differentially influence erectile tissue. Since cardiovascular diseases are often associated with the development of erectile dysfunction, the nebivolol-induced eNOS-activation in corpus cavernosum may be beneficial when treating patients suffering from cardiovascular disease.

Beta3-adrenergic stimulation in the human heart: signal transduction, functional implications and therapeutic perspectives.

Next to beta1- and beta2-adrenoceptors, a third beta-adrenoceptor population is expressed in the human heart, the beta3-adrenoceptor. In mammalian ventricular myocytes, beta3-adrenergic stimulation leads to a decrease in contractility via a release of nitric oxide (NO). Recently, different molecular mechanisms of beta3-adrenergic activation of endothelial nitric oxide synthase (eNOS) have been uncovered in cardiac myocytes. In the non-failing and especially the failing heart, beta3-adrenergic stimulation may offer protection against excessive catecholaminergic beta1-adrenoceptor stimulation. In this context, the beta3-adrenoceptor is discussed as a novel target for the pharmacological therapy of heart failure.

Increased expression of isoform 1 of the sarcoplasmic reticulum Ca(2+)-release channel in failing human heart.

BACKGROUND: The sarcoplasmic reticulum (SR) Ca(2+)-release channel plays a key role in the excitation-contraction coupling of cardiac myocytes. Because respective alterations have been reported in human heart failure, we investigated isoform expression of the SR Ca(2+)-release channel in human hearts from patients with terminal heart failure (dilated cardiomyopathy [DCM], n=8) and nonfailing organ donors (NF, n=8). METHODS AND RESULTS: Expression of mRNA of SR Ca(2+)-release channel isoforms in isolated human cardiomyocytes and myocardial tissue was analyzed by reverse-transcription polymerase chain reaction. Protein expression was quantified in myocardial tissue with [(3)H]-ryanodine binding and with Western blots, expressed as densitometric units per microgram of protein (DU), and cellular localization was visualized with immunohistochemistry. We found mRNA expression of isoforms 1, 2, and 3 in cardiomyocytes and myocardial tissue both in NF and DCM. Total SR Ca(2+)-release channel protein expression in NF (B(max) 2.16+/-0.43 pmol/mg protein) and in DCM (B(max) 2.33+/-0.22 pmol/mg protein) myocardium was unchanged. Expression of isoform 1 of the SR Ca(2+)-release channel was significantly (P=0.0037) increased in DCM myocardium (NF 1.97+/-0.25 versus DCM 3.37+/-0.31 DU), whereas protein expression of isoform 2 (NF 14.62+/-0.87 versus DCM 13.52+/-0.43 DU) and isoform 3 (NF 1.39+/-0.13 versus DCM 1.35+/-0.19 DU) was unchanged. All 3 isoforms of the protein could be localized in human ventricular myocytes with fluorescence immunohistochemistry. CONCLUSIONS: All 3 isoforms of the SR Ca(2+)-release channel were determined in human ventricular cardiomyocytes. Increased expression of isoform 1 of the SR Ca(2+)-release channel could contribute to impaired excitation-contraction coupling in human heart failure.

Reduced sodium pump alpha1, alpha3, and beta1-isoform protein levels and Na+,K+-ATPase activity but unchanged Na+-Ca2+ exchanger protein levels in human heart failure.

BACKGROUND: Cardiac glycosides initiate an increase in force of contraction by inhibiting the sarcolemmal sodium pump (Na+, K+-ATPase), thereby decreasing Ca2+ extrusion by the Na+-Ca2+ exchanger, which increases the cellular content of Ca2+. In patients with heart failure the sensitivity toward cardiac glycosides is enhanced. METHODS AND RESULTS: Because the inotropic effect of cardiac glycosides may be a function of the sodium pump and Na+-Ca2+ exchanger (NCE) expression levels, the present study aimed to investigate protein expression of both transporters (immunoblot with specific antibodies against the sodium pump catalytic alpha1-, alpha2-, alpha3-, and glycoprotein beta1-isoforms and against NCE) in left ventricle from failing (heart transplantations, New York Heart Association class IV, n=21) compared with nonfailing (donor hearts, NF, n=22) human myocardium. The density of 3H-ouabain-binding sites (Bmax) and the Na+,K+-ATPase activity were also measured. In NYHA class IV, protein levels of Na+,K+-ATPase alpha1- (0.62+/-0.06 of control), alpha3- (0.70+/-0.09), and beta1- (0.61+/-0.04) but not alpha2-isoforms were significantly reduced (P<0.01), whereas levels of NCE (0.92+/-0.13 of control) and calsequestrin (0.98+/-0.06) remained unchanged. Both Na+,K+-ATPase activity (NF: 1.9+/-0.29; NYHA class IV: 1.1+/-0.17 micromol ATP/min per milligram of protein) and the 3H-ouabain binding sites (Bmax NF: 15.9+/-1.9 pmol/mg protein; NYHA class IV: 9.7+/-1.5) were reduced in NYHA class IV and correlated significantly to each other (r2=0. 73; P<0.0001), as did beta1-subunit expression. In left ventricular papillary muscle strips from NYHA class IV compared with nonfailing tissue the Na+-channel modulator BDF 9198 exerted an increase in force of contraction with unchanged effectiveness but enhanced potency. CONCLUSIONS: The enhanced sensitivity of failing human myocardium toward cardiac glycosides may be, at least in part, attributed to a reduced protein expression and activity of the sarcolemmal Na+,K+-ATPase without a change in Na+-Ca2+ exchanger protein expression.

Myofilament calcium regulation in human myocardium.

BACKGROUND: We investigated whether decreased myofilament calcium contractile activation may, in part, contribute to heart failure. METHODS AND RESULTS: Calcium concentration required for 50% activation and Hill coefficient for fibers from nonfailing and failing human hearts at pH 7.1 were not different. Maximum calcium-activated force (F(max)) was also not different. However, at pH 6.8 and 6.9, differences were seen in myofilament calcium activation between nonfailing and failing hearts. At lower pH, failing myocardium was shifted left on the calcium axis compared with nonfailing myocardium, which suggested an increase in myofilament calcium responsiveness. Increased inorganic phosphate concentration decreased maximal force development by 56% in nonfailing and 36% in failing myocardium and shifted the calcium-force relationship by 2.01+/-0.22 versus 0.86+/-0.13 micromol/L, respectively (P<0.05). Addition of cAMP resulted in a 0. 56 micromol/L shift toward higher intracellular calcium concentrations in nonfailing myocardium and a 1.04 micromol/L shift in failing myocardium. Protein kinase A in the presence of cAMP resulted in a further rightward shift in nonfailing human myocardium but did not further shift the calcium-force relationship in fibers from failing hearts. cGMP also resulted in a greater decrease in myofilament calcium sensitivity in fibers from failing hearts. CONCLUSIONS: We propose that changes at the level of the thin myofilaments result in differential responses to changes in the intracellular milieu in nonfailing versus failing myocardium.

Evidence for reduction of norepinephrine uptake sites in the failing human heart.

OBJECTIVES: This study investigated the role of neuronal uptake of norepinephrine (uptake-1) in human heart failure as a local factor for altering concentrations of norepinephrine at the cardiac myocyte membranes. BACKGROUND: Several beta-adrenergic neuroeffector defects occur in heart failure. Whether an alteration in norepinephrine uptake-1 occurs is still unresolved. METHODS: The role of norepinephrine uptake-1 was studied in electrically stimulated (1 Hz, 37 degrees C) human ventricular cardiac preparations and isolated myocardial membranes. RESULTS: The effectiveness of norepinephrine in increasing the force of contraction was decreased in relation to the degree of heart failure. In contrast, the potency of norepinephrine was increased in failing hearts (New York Heart Association functional class IV) in relation to the concentrations producing 50% of the maximal effect (EC50). The EC50 values for isoproterenol, which is not a substrate for norepinephrine uptake-1, were reduced in myocardium in functional classes II to III and IV compared with those in nonfailing myocardium. The uptake inhibitors cocaine and desipramine (3 mumol/liter) potentiated the positive inotropic effects of norepinephrine in nonfailing myocardium (p < 0.05) but not in functional class IV myocardium. Radioligand binding experiments using the uptake inhibitor hydrogen-3 mazindol revealed a significant decrease by approximately 30% in norepinephrine uptake-1 carrier density in functional classes II to III and IV myocardium versus nonfailing myocardium (p < 0.05). CONCLUSIONS: In human heart failure, there is a presynaptic defect in the sympathetic nervous system, leading to reduced uptake-1 activity. This defect in the failing heart can be mimicked by the effects of uptake blocking agents, such as cocaine and desipramine, in the nonfailing heart only. Compromised norepinephrine uptake-1 in functional class IV cannot be further increased by cocaine and desipramine. The pathophysiologic consequences could be an increased synaptic concentration of norepinephrine predisposing to adenylyl cyclase desensitization.

Isoform expression of the sarcoplasmic reticulum Ca2+ release channel (ryanodine channel) in human myocardium.

The Ca2+ release channel of the sarcoplasmic reticulum (SR) is essential for the release of Ca2+ from intracellular stores and is expressed widely in various excitable cells. It plays a key role particularly in excitation contraction coupling in myocytes in skeletal and cardiac muscle. Three isoforms of the SR Ca2+ release channel have been cloned. Recently coexpression of different isoforms was reported in different animal species and various tissues. In human cardiac tissue, however, isoform expression is not yet established. Therefore the aim of this study was to characterize isoform expression of the SR Ca2+ release channel in the human heart. We examined specific isoform expression of mRNA and proteins of the SR Ca2+ release channel in the four different chambers of the heart and the interventricular septum from explanted human hearts from nonfailing organ donors (n=8). Reverse transcriptase PCR from total cardiac RNA with isoform specific primers and western blots from myocardial homogenates with isoform specific antibodies were performed. Quantification of protein expression was achieved by densitometric scanning and computer analysis and is expressed as densitometric units per microgram of protein. A single band DNA signal was detected by reverse transcriptase PCR for the skeletal isoform 1 and the cardiac isoform 2 and isoform 3 in all regions of the human heart investigated. Specific protein expression was detected in all five myocardial regions of the human heart in western blots for the skeletal isoform I and cardiac isoform 2, and a weaker specific band was also detectable for isoform 3 of the SR Ca2+ release channel. Quantification of protein expression showed significant (P=0.008) lower expression of isoform 1 in the right ventricle (42+/-4 densitometric units/g tissue) and similar expression in all other regions (right atrium 58+/-3; septum 51+/-5, left atrium 54+/-5; left ventricle 51+/-6). Isoform 2 of the SR Ca2+ release channel was also significantly lower (P=0.001) in the right ventricle (33+/-4 densitometric/g tissue) and similar in the other heart chambers (right atrium 42+/-5: septum 41+/-3, left atrium 52+/-6, left ventricle 42+/-3). Differences in isoform 3 of the SR Ca2+ release channel for the various myocardial regions did not reach significant levels (right atrium 45+/-6, right ventricle 38+/-5, septum 49+/-8, left atrium 46+/-7, and in left ventricle 45+/-3 densitometric units/g tissue). In conclusion, all three isoforms of the SR Ca2+ release channel were determined in the human heart at both mRNA and protein levels with different quantitative expression in the different heart chambers. Coexpression of the three different isoforms with different functional properties might increase the complexity of regulation of excitation contraction coupling in the human heart in a chamber specific mode.

Unchanged sarcoplasmic reticulum Ca2+-ATPase activity, reduced Ca2+ sensitivity, and negative force-frequency relationship in transgenic rats overexpressing the mouse renin gene.

Transgenic rats overexpressing the mouse Ren-2 gene [TG(mREN2)27 rats, TGR] were used to characterize alterations in force generation and relaxation following cardiac hypertrophy. Age-matched Sprague-Dawley rats were used as the control group. The beta-adrenoceptor dependent increase in force of contraction was reduced in the transgenic animals but not the Ca2+-dependent increase in force generation. Additionally, force of contraction decreased after increasing stimulation frequencies (up to 7 Hz), but the frequency-dependent decrease in force of contraction was significantly more pronounced in the transgenic group. The Ca2+ sensitivity in chemically skinned fiber preparations of TGR was reduced than that in Sprague-Dawley rats while maximum effectiveness was the same. Unexpectedly, the sarcoplasmic reticulum Ca2+-ATPase activity measured in crude membrane preparations from TGR did not differ from that in Sprague-Dawley rats; however, the activity of the Na+/K+-ATPase was less while the Na+/Ca2+-exchanger activity was significantly greater. In the same preparations the protein expression of SERCA2 was reduced in TGR while expression of phospholamban and calsequestrin remained the same. Thus in the model of cardiac hypertrophy harboring the mouse Ren-2 gene the hypothesized correlation between SERCA2 function and force-frequency relationship was not observed. Possible reasons for the more negative force-frequency relationship in TGR included changes at the level of the myofilaments and altered intracellular Na+ homeostasis which may result from the reciprocal changes in the Na+/K+-ATPase and the Na+/Ca2+-exchanger activity.

Unchanged protein expression of sarcoplasmic reticulum Ca2+-ATPase, phospholamban, and calsequestrin in terminally failing human myocardium.

The enhanced diastolic Ca2+ levels observed in cardiac myocytes from patients with idiopathic dilated cardiomyopathy (DCM) may be either a consequence of functional impairment of sarcoplasmic reticulum calcium-ATPase (SERCA 2) and its regulator protein phospholamban or due to a reduction in the number of SERCA 2 proteins. As different myocardial membrane preparations may lead to different accumulation of proteins, the present study evaluated two different membrane preparations, in human failing and nonfailing myocardium for comparison of SERCA 2 activity and the protein expression of SERCA 2 and phospholamban. Crude membranes and tissue homo-genates without any centrifugation steps were prepared from human nonfailing hearts (donor hearts, NF, n=18) and terminally failing hearts (heart transplant, DCM, n=18). Calsequestrin protein expression was used as an internal control for overall protein expression. In both crude membranes and homogenates maximal SERCA 2 activity (Vmax) was significantly reduced in failing heart preparations (NF crude membranes, 130+/-8; DCM crude membranes, 102+/-5 nmol ATP/mg protein per minute). In contrast, the protein expression of SERCA 2 (NF crude membranes, 488+/-35; DCM crude membranes, 494+/-42; P=0.92), phospholamban (NF crude membranes, 497+/-51; DCM crude membranes, 496+/-45; P=0.98) and calsequestrin (NF crude membranes, 109+/-06; DCM crude membranes, 107+/-08; P=0.84) was unchanged in NF and DCM hearts in both preparation methods. This was also the case when the protein expression was normalized to calsequestrin protein levels. Preparation of sarcoplasmic reticulum in crude membranes led to enhanced purification and consequently higher SERCA 2, phospholamban, and calsequestrin protein levels in crude membranes than in the homogenates, which was paralleled by an increase in SERCA 2 enzyme activity. In conclusion, the altered Ca2+ handling in DCM may be a consequence of reduced SERCA 2 enzyme activity and not the result of differences in protein expression of the Ca2+ regulating proteins SERCA 2, phospholamban, and calsequestrin in human myocardium. The present study emphasizes the importance of different myocardial membrane preparations with respect to quantitative investigations of protein expression and function.

Calcineurin independent development of myocardial hypertrophy in transgenic rats overexpressing the mouse renin gene, TGR(mREN2)27.

Myocardial hypertrophy is an independent risk factor for development of heart failure. The intracellular calcium homeostasis is altered in myocardial hypertrophy, and recent studies in animal models have confirmed an interaction between the Ca2+/calmodulin-dependent calcineurin signaling cascade and development of cardiac hypertrophy. There is evidence for the involvement of various pathways in development of hypertrophy. A transgenic rat model overexpressing the mouse renin gene, TGR(mREN2)27 has been shown to progress profound cardiac hypertrophy, possibly due to a monogenetic disorder. However, the exact mode of action is not known. To study a possible involvement of calcineurin and its downstream pathway in development of cardiac hypertrophy in this transgenic rat model we measured the protein expression of marker proteins of the calcineurin cascade (calcineurin, NFAT-3, GATA-4) and calcineurin phosphatase activity and GATA-4 DNA binding in TGR ( n=10) compared to age-matched Sprague-Dawley rats ( n=10). In our study there was no significant difference in calcineurin activity between the transgenic hearts and the hearts of Sprague-Dawley rats. Furthermore, we found neither an increase in protein expression of calcineurin B nor a rise in nuclear translocated NFAT-3 DU. Interestingly, the protein expression of GATA-4 and its DNA binding activity were significantly higher in hypertrophied myocardium than in control hearts. In transgenic rats overexpressing the mouse renin gene and thereby developing pronounced cardiac hypertrophy [TGR(mREN2)27] we thus found no activation of calcineurin or its downstream pathway. However, the expression of the transcriptional factor GATA-4 and its DNA binding activity were significantly increased in hearts of transgenic rats. Thus GATA-4 seems to be a marker of hypertrophy independently of calcineurin activation, possibly activated by various pathways.

Sarcoplasmic reticulum Ca2+ATPase and phospholamban mRNA and protein levels in end-stage heart failure due to ischemic or dilated cardiomyopathy.

Abnormalities in intracellular Ca2+ handling play a crucial role in the pathogenesis of heart failure. The reduced capacity of failing human myocardium to restore low resting Ca2+ levels during diastole has been explained by the impairment of Ca2+ uptake into the sarcoplasmic reticulum (SR) via the SR Ca2+ATPase. It is unclear whether Ca2+ATPase function, protein levels, and mRNA steady-state levels correspond to one other, and whether the cause of heart failure, namely idiopathic dilated or ischemic cardiomyopathy, produces different changes. The present study examined SR Ca2+ATPase activity and both mRNA and protein levels of SR Ca2+ATPase, phospholamban, and Gi alpha 2 in left ventricular myocardium from eight nonfailing hearts, from eight hearts of patients with idiopathic dilated cardiomyopathy (DCM), and from six hearts from patients with ischemic cardiomyopathy (ICM). Compared to nonfailing myocardium, the activity of the SR Ca2+ATPase was significantly reduced in failing myocardium from patients with DCM (36%, P < 0.01) and from patients with ICM (37%, P < 0.001). Significantly lower levels of SR Ca2+ATPase mRNA levels (55% and -56%, P < 0.001 for DCM and ICM, respectively) and phospholamban mRNA (45%, P < 0.001 for DCM; 31%, P < 0.05 for ICM) were observed in failing than in nonfailing myocardium. In contrast, no significant changes were observed at the level of proteins, Gi alpha 2 mRNA and protein levels were both significantly increased in failing myocardium. There were no differences between idiopathic dilated and ischemic cardiomyopathy concerning the examined parameter. It is concluded that reduced SR Ca2+ATPase activity contributes to an altered intracellular Ca2+ handling by the SR in both dilated and ischemic cardiomyopathic hearts. However, changes in SR Ca2+ATPase and phospholamban steady-state protein levels do not contribute to these alterations. The dissociation between protein and mRNA levels provides evidence for a posttranscriptional or post-translational regulation of these proteins. The observed alterations are not dependent on the underlying cause of end-stage heart failure.

cAMP concentrations, cAMP dependent protein kinase activity, and phospholamban in non-failing and failing myocardium.

OBJECTIVE: Several signal transduction defects such as a reduction of myocardial cAMP formation and an altered intracellular Ca2+ handling have been observed in the failing human myocardium. The aim of the study was to obtain data on changes beyond cAMP formation involving cAMP dependent protein kinase and its substrates. METHODS: cAMP dependent protein kinase activity and cAMP concentrations were measured in the particulate and soluble fraction of failing human hearts (ischaemic, and dilated cardiomyopathy) and non-failing donor hearts. Phospholamban was quantified by cAMP dependent phosphorylation using 32P-ATP as substrate and on western blots using a monoclonal antibody. RESULTS: cAMP concentrations were reduced in the particulate fraction in both ischaemic and dilated cardiomyopathy and in the soluble fraction in dilated cardiomyopathy, but there was no difference in cAMP dependent protein kinase activity. Both phospholamban levels and cAMP dependent phosphorylation of phospholamban were similar in non-failing myocardium and in both ischaemic and dilated cardiomyopathy. CONCLUSIONS: These findings show that the reduction of cAMP formation is the predominant alteration in heart failure, but cAMP dependent protein kinase and phospholamban are evidently unchanged.

Nebivolol, bucindolol, metoprolol and carvedilol are devoid of intrinsic sympathomimetic activity in human myocardium.

1. The present study investigated whether or not there may be differences in the direct cardiac actions of the novel, highly beta(1)-selective adrenoceptor antagonist nebivolol (NEB) in comparison to metoprolol (MET), bisoprolol (BIS), carvedilol (CAR) and bucindolol (BUC) in human myocardium (n=9). 2. The rank order of beta(1)-selectivity as judged by competition experiments to (3)H-CGP 12.1777 in the presence of CGP 207.12 A (300 nmol l(-1), K(i)beta(2)) or ICI 118.551 (50 nmol l(-1), K(i)beta(1)) were NEB(K(i)beta(2)/K(i)beta(1): 40.7) > BIS(15.6) > MET(4.23) > CAR(0.73) > BUC(0.49). 3. The rank order of the negative inotropic potency of the beta-adrenoceptor antagonists measured in left ventricular trabeculae (dilated cardiomyopathy, DCM) as judged by the concentration needed to induce a 50% decrease in isoprenaline (1 micromol l(-1))-stimulated force (IC(50)) was: MET (0.6 micromol l(-1)) > CAR (4.1 micromol l(-1)) > NEB (7.0 micromol l(-1)). 4. NEB, BUC, MET and CAR did not not exert an intrinsic sympathomimetic activity (ISA) as determined by measurements of force development in forskolin (0.3 micromol l(-1)) pre-treated left ventricular trabeculae, nor by measuring adenylate cyclase activity in forskolin (0.3 micromol l(-1))-stimulated assays (crude membranes). This also holds true for radioligand binding assays with or without guanine nucleotide guanyl-5'-yl imidodiphosphate (Gpp(NH)p). 5. Although all studied beta-adrenoceptor antagonists lack intrinsic sympathomimetic activity (ISA), they differ in the beta(1)-selectivity as well as in their direct negative inotropic action. These differences as well as the mode of extracardiac action may have an impact on outcome of patients treated with beta-adrenoceptor antagonists.

Potent vasodilatory with minor cardiodepressant actions of mibefradil in human cardiac tissue.

1. The present study compared the cardiovascular effects of mibefradil (MIB), a novel Ca2+-channel antagonist with high selectivity for T-type Ca2+-channels to the effect of the L-type Ca2+-channel-antagonists nifedipine (NIF) and diltiazem (DIL) in left ventricular myocardium and coronary arteries of hearts obtained from patients suffering from dilated cardiomyopathy (NYHA IV). Right atrial myocardium from patients undergoing aortocoronary bypass surgery without signs of cardiac failure was studied as well. 2. NIF and DIL (100 micromol l(-1)) completely depressed force of contraction (FOC) in electrically driven left ventricular myocardium (NIF 6.5+/-1.4% and DIL 7.1+/-1.2% of control), whereas a similar concentration of MIB only reduced force of contraction to 55.1+/-4.0% of the basal FOC. The negative inotropic potency as measured by the concentration needed to reduce basal FOC for 25% was NIF (0.0095 micromol l(-1))>DIL (0.041 micromol l(-1))>MIB (9.47 micromol l(-1)). 3. All three Ca2+-channel antagonists were more potent in human atrial compared to human left ventricular myocardium to reduce FOC. 4. The rank order of Ca+-antagonistic moiety as measured by the decrease of the intracellular Ca2+-transient (fura-2 ratio method) was NIF>DIL>MIB. 5. All Ca2+-channel antagonists completely relaxed human coronary arteries (% of papaverine effect: MIB 81.7+/-5.5%, DIL 91.3+/-0.9%, NIF 96.4+/-3.7%) precontracted with PGF2alpha (0.3 micromol l(-1)). The rank order of vasodilatory potency was NIF (EC50; 0.02 micromol l(-1))>DIL (0.13 micromol l(-1))>MIB (2.05 micromol l(-1)). 6. The vasoselectivity measured by the ratio of the concentration needed to achieve a 25% decrease in force and the concentration needed for 25% vasodilatation was 316 for MIB, 1.5 for NIF and 1.0 for DIL. 7. The present study provides evidence that blockade of T-type Ca2+-channels (e.g. mibefradil) results in potent vasodilatory properties with only minor cardiodepressant effects.

Studies of the nucleoside transporter inhibitor, draflazine, in the human myocardium.

1. The aim of the present study was to determine the effect of the nucleoside transporter inhibitor, draflazine, on the force of contraction in human myocardium and the affinity of the compound for the nucleoside transporter. Nucleoside transport inhibitors, like draflazine, are of potential importance for cardiopreservation of donor hearts for heart transplantation. 2. Functional experiments were performed in isolated electrically driven (1 Hz, 1.8 mmol l-1 Ca2+) human atrial trabeculae and ventricular papillary muscle strips. The affinity of draflazine for the myocardial nucleoside transporter was studied in isolated membranes from human ventricular myocardium and human erythrocytes in radioligand binding experiments using [3H]-nitrobenzylthioinosine ([3H]-NBTI). Dipyridamole was studied for comparison. 3. In membranes from human myocardium and erythrocytes, [3H]-NTBI labelled 1.18 pmol mg-1 protein and 23.0 pmol mg-1 protein, respectively, nucleoside transporter molecules with a KD value of 0.8 nmol l-1. Draflazine concentration-dependently inhibited binding of [3H]-NBTI to myocardial and erythrocyte membranes with a K(i)-value of 4.5 nmol l-1. The potency as judged from the K(i) values was ten times greater than that of dipyridamole in both myocardial and erythrocyte membranes. 4. Draflazine, at concentrations up to 100 mumol l-1, did not produce negative inotropic effects in atrial and ventricular myocardium. (-)-N6-phenylisopropyladenosine (R-PIA) and carbachol did not reduce force of contraction in ventricular myocardium, but exerted concentration-dependent direct negative inotropic effects in atrial myocardium. 5. The data provide evidence that draflazine specifically binds to the nucleoside transporter of the human heart and erythrocytes with high affinity. The compound does not produce negative inotropic effects at concentrations as high as 100 micromol 1-1.6. Draflazine could be a useful agent for cardio preservation because it does not produce cardio depressant effects. Thus, it may be possible to perfuse explanted hearts directly with this agent without the hazard of cardiodepression.

The preferential beta3-adrenoceptor agonist BRL 37344 increases force via beta1-/beta2-adrenoceptors and induces endothelial nitric oxide synthase via beta3-adrenoceptors in human atrial myocardium.

1 The present study investigated the effects of the preferential beta(3)-AR agonist BRL 37344 (BRL) on force of contraction (FOC), Ca(2+)-transient and eNOS-activity in human right atrial myocardium. 2 BRL concentration-dependently caused an increase in FOC that was paralleled by an increase in Ca(2+)-transient and a shortening of time to half peak relaxation (T0.5T). These effects were abolished in the presence of propranolol (0.3 micro M). 3 BRL acted as a competitive antagonist towards isoprenaline and in binding experiments it was shown to have a distinct affinity towards beta(1/2)-AR. 4 In immunohistochemical experiments BRL (10 micro M) increased detection of activated eNOS. This effect remained constant in the presence of propranolol (0.3 micro M). 5 BRL increased directly detected NO in DAF-staining experiments. This increase was significantly smaller in the presence of the NO-inhibitor L-NAME. 6 The inotropic effects of BRL were not changed in the presence of L-NMA. 7 These results suggest that the inotropic effects of BRL in human atrium are mediated via beta(1/2)-AR, whereas the increase of atrial eNOS-activity is due to beta(3)- adrenergic stimulation. This increase in eNOS-activity did not influence atrial myocardial contractility. In conclusion, this study shows that beta(3)-adrenergic stimulation is present in human atrium, but may not be functionally as significant as in the left ventricle.

Negative inotropic activity of the calcium antagonists isradipine, nifedipine, diltiazem, and verapamil in diseased human myocardium.

The use of calcium antagonists in the treatment of cardiac failure is limited by the negative inotropic effects they exert. The comparative inotropic activity of four calcium antagonists was therefore investigated, using electrically driven human papillary muscle strips and human atrial trabeculae. These inotropic effects were studied with cumulative concentration-response curves. The four calcium antagonists included in this study were all found to significantly (P less than or equal to .05) diminish the force of contraction above 0.01 mumol/L, but with different potencies. On the basis of the relationship between therapeutic vasoactive plasma concentrations and the negative inotropic effective concentrations in our system in vitro, the ranking order of potential negative inotropism in the treatment of cardiac failure with calcium antagonists was verapamil greater than nifedipine greater than diltiazem greater than isradipine. Thus, to minimize cardiodepressant risk, the use of calcium antagonists with weak negative inotropic effects at relevant concentrations should be preferred, especially in the case of patients with compromised cardiac function.