A1-adenosine receptor inhibition of adenylate cyclase in failing and nonfailing human ventricular myocardium

BACKGROUND. Receptors that couple via the stimulatory G protein, Gs, to adenylate cyclase and to a positive inotropic response have been extensively investigated in falling human heart. In contrast, much less is known about receptors, such as the A1-adenosine receptor, that couple to adenylate cyclase via the inhibitory G protein, Gi, to give a negative inotropic response. Activation of such Gi-coupled receptors might worsen heart failure. Furthermore, alpha Gi is increased in failing human ventricular myocardium, which may enhance inhibitory receptor coupling to adenylate cyclase. METHODS AND RESULTS. A1-Adenosine receptor inhibition of adenylate cyclase was examined in crude particulate preparations derived from 12 nonfailing and 12 failing human left ventricles. Experimental conditions were designed for maximal inhibitory responses. Dose-response curves were performed with the selective A1-adenosine receptor agonist R-phenylisopropyl-adenosine (R-PIA). No differences in nonfailing versus failing heart were observed for basal adenylate cyclase activity (49.0 +/- 4.1 versus 45.7 +/- 2.6 pmol cyclic AMP/min/mg), maximal R-PIA-mediated inhibition (31.1 +/- 2.6 versus 30.2 +/- 1.6 pmol cyclic AMP/min/mg), ED50 (R-PIA x 10(-7) 1.28 +/- 0.10 versus 1.36 +/- 0.08), or slope (1.06 +/- 0.06 versus 1.03 +/- 0.10), respectively. Furthermore, fluoride, forskolin, and manganese adenylate cyclase activation were not different in failing heart, which is consistent with no change in the catalytic unit of adenylate cyclase. The inhibitory G protein alpha Gi, as quantitated by pertussis toxin-catalyzed ADP-ribosylation, was increased in failing heart (105.7 +/- 5.8 versus 132.7 +/- 3.4 optical density units, p less than 0.003). Basal adenylate cyclase activity was reduced in failing heart (7.8 +/- 0.8 versus 4.5 +/- 0.4 pmol cyclic AMP/min/mg, p less than 0.005) with assay conditions designed to assess G protein effects. CONCLUSIONS. The A1-adenosine receptor pathway exerts a major inhibitory effect on human myocardial adenylate cyclase activity. Although alpha Gi was increased in failing heart, A1-adenosine receptor inhibition of adenylate cyclase was not altered in preparations of failing versus nonfailing human ventricular myocardium.     

Beta 2 receptors on myocardial cells in human ventricular myocardium

Beta 2-adrenergic receptors comprise 40% of the total beta-receptor population in failing human right ventricles, as deduced from computer modeling of iodine-125 iodocyanopindolol-ICI 118,551 competition curves. A myocardial cell origin of at least some of the beta 2 subpopulation was demonstrated by documenting a beta 2-receptor mediated positive inotropic response to the selective beta 2 agonist zinterol. It is concluded that beta 2-adrenergic receptors are present on human ventricular myocardial cells.     

Neurotransmitter depletion compromises the ability of indirect-acting amines to provide inotropic support in the failing human heart

To test the hypothesis that cardiac norepinephrine depletion related to heart failure alters contractile responses to beta-adrenergic agonists with a component of "indirect" action (acting by release of neuronal norepinephrine), we examined the inotropic potential of several pharmacologically distinct beta-agonists. Contractile responses to the nonselective beta-agonist isoproterenol, the beta 2-selective agonist zinterol, and the direct- and indirect-acting agonists dopamine and dopexamine were compared in isolated right ventricular trabeculae removed from failing, nonfailing innervated, and previously transplanted and, therefore, denervated nonfailing human hearts. In failing hearts, the contractile response to isoproterenol was significantly lower (41%) than that in nonfailing innervated hearts. The responses to the mixed agonists dopamine and dopexamine were even more attenuated in failing hearts, to a level 76-90% lower than those of nonfailing innervated hearts. In denervated, previously transplanted, nonfailing hearts, the contractile responses to the mixed agonists dopamine and dopexamine were 66-72% lower than those in the nonfailing innervated group, but the response to isoproterenol was not significantly different. The response to zinterol was not significantly different among the three groups. In subjects with severe heart failure, in vivo hemodynamic responses to dopexamine were compared with those of the direct-acting beta-agonist dobutamine. Responses to dopexamine and dobutamine were measured before and after prolonged continuous infusions of each drug. The response to dopexamine, but not to dobutamine, diminished over time. We conclude that a large component of the inotropic response to dopamine and dopexamine in human hearts is due to the ability of these agonists to promote the release of neuronal norepinephrine; when neuronal norepinephrine is depleted, indirect-acting agonists are less able to produce an inotropic response.     

The functional importance of beta 1 and beta 2 adrenoceptors in the human heart

Radioligand binding studies have demonstrated convincingly the coexistence of beta 1 and beta 2 adrenoceptors in the human heart. Both subtypes are involved in the increase in tissue levels of cyclic adenosine monophosphate in isolated, electrically driven, human right atria and in the activation of adenylate cyclase in human cardiac membrane preparations. In isolated, electrically driven strips of human right atria, isoproterenol increased contractile force through stimulation of both beta 1 and beta 2 adrenoceptors, while the selective beta 2-adrenoceptor agonist, procaterol, caused its positive inotropic effect predominantly through beta 2-adrenoceptor stimulation. Norepinephrine, however, increased contractile force solely via beta 1-adrenoceptor stimulation. In this preparation, dobutamine also acted as a full agonist, producing a positive inotropic effect through stimulation of both beta-adrenoceptor subtypes. Dopexamine hydrochloride, on the other hand, having an approximately 10-fold greater affinity for right atrial beta 2 than for beta 1 adrenoceptors, acted as a partial agonist (maximal positive inotropic effect: about 30% that of isoproterenol). Similar effects have been obtained in human right and left ventricular strips; thus, there can be no doubt that cardiac beta 2 adrenoceptors can contribute to the positive inotropic effects of beta-adrenoceptor agonists in the human heart. Besides mediating positive inotropic effects, right atrial beta 2 adrenoceptors may be involved in the regulation of heart rate since, in healthy volunteers, the selective beta 2-adrenoceptor antagonist, ICI 118,551, was more potent than the selective beta 1-adrenoceptor antagonist, bisoprolol, in antagonizing isoproterenol-induced tachycardia, when both antagonists were administered in doses that selectively occupied more than 90% of beta 2 and beta 1 adrenoceptors, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasoactive intestinal peptide receptor in failing human ventricular myocardium exhibits increased affinity and decreased density

We investigated vasoactive intestinal peptide (VIP)-receptor pharmacology in failing and nonfailing human ventricular myocardium by examining [125I]VIP binding in membrane fractions of left ventricle and inotropic effects of VIP in isolated right ventricular trabeculae mounted in tissue baths. [125I]VIP binding demonstrated upwardly concave, curvilinear Scatchard plots consistent with two classes of binding sites. Only the high-affinity (dissociation constant [Kd] 400-800 pM) site could be regulated by guanine nucleotides. Compared with nonfailing heart, membranes derived from failing heart exhibited a twofold reduction in the Kd of the high-affinity VIP binding site, whereas the receptor density (Bmax) was decreased by 62%. In concordance with this decreased receptor density and increased affinity, the maximal contractile response of right ventricular trabeculae from failing right ventricles was decreased by 61%, and the dose-response curve to VIP was left-shifted approximately threefold. We conclude that the VIP receptor in failing human ventricular myocardium exhibits novel regulatory behavior consisting of increased receptor affinity and decreased receptor density.     

Myocardial catecholamine and neuropeptide Y depletion in failing ventricles of patients with idiopathic dilated cardiomyopathy. Correlation with beta-adrenergic receptor downregulation.

BACKGROUND. Myocardial adrenergic neurotransmitters and beta-adrenergic receptor levels were measured in left and right ventricular myocardial specimens obtained from 30 patients with biventricular failure resulting from idiopathic dilated cardiomyopathy. METHODS AND RESULTS. Nonfailing myocardium obtained from 12 organ donors provided control data. Norepinephrine, dopamine, and neuropeptide Y concentrations were significantly decreased in failing compared with nonfailing control hearts. The mean ratio of dopamine to norepinephrine and of dopamine to neuropeptide Y in failing hearts was also significantly decreased compared with nonfailing control hearts. Compared with nonfailing control hearts, Bmax and beta 1-receptor density were significantly decreased in failing hearts and there were weak but significantly positive correlations of Bmax and beta 1-adrenergic receptors with norepinephrine, dopamine, and neuropeptide Y. CONCLUSIONS. Norepinephrine and its cotransmitter neuropeptide Y are depleted in failing human ventricular myocardium. Decreased norepinephrine stores correlate weakly with beta 1-adrenergic receptor downregulation consistent with the hypothesis that norepinephrine depletion occurs in response to increased adrenergic drive. Decreased dopamine relative to norepinephrine implies that an abnormality of dopamine conversion to norepinephrine is not present in failing human heart.     

Myocardial expression of CC- and CXC-chemokines and their receptors in human end-stage heart failure

OBJECTIVES: Chemokines regulate several biological processes, such as chemotaxis, collagen turnover, angiogenesis and apoptosis. Based on the persistent immune activation with elevated circulating levels of chemokines in patients with congestive heart failure (CHF), we have hypothesised a pathogenic role for chemokines in the development of CHF. The objective of this study was to examine mRNA levels and cellular localisation of chemokines and chemokine receptors in human CHF. METHODS: We examined explanted hearts from ten patients with end-stage heart failure (all chambers) and in ten organ donors using an RNase protection assays and immunohistochemical techniques. RESULTS: Our main findings were: (i) expression of eight chemokine and nine chemokine receptor genes in both failing and nonfailing myocardium, (ii) particularly high mRNA levels of monocyte chemoattractant protein (MCP)-1 and CXC-chemokine receptor 4 (CXCR4), in both chronic failing and nonfailing myocardium, (iii) decreased mRNA levels of MCP-1 and interleukin (IL)-8 in the failing left ventricles compared to failing left atria, (iv) decreased chemokine (e.g., MCP-1 and IL-8) and increased chemokine receptor (e.g., CCR2, CXCR1) mRNA levels in failing left ventricles and failing left atria compared to corresponding chambers in the nonfailing hearts and (v) immunolocalisation of MCP-1, IL-8 and CXCR4 to cardiomyocytes. CONCLUSION: The present study demonstrates for the first time chemokine and chemokine receptor gene expression and protein localisation in the human myocardium, introducing a new family of mediators with potentially important effects on the myocardium. The observation of chemokine dysregulation in human end-stage heart failure may represent a previously unknown mechanism involved in progression of chronic heart failure.     

Angiotensin II Receptors in the Normal and Failing Heart

Mammalian myocardium expresses two major subtypes of angiotensin II receptors, namely, AT1 and AT2. These two subtypes mediate a diverse set of Ang II actions on the myocardium in multiple types of cells. AT1 receptors mediate most of the conventionally known angiotensin II actions such as vasoconstrictive, inotropic, chronotropic, and trophic–mitogenic effects. The function(s) of AT2 receptors remains obscure in the myocardium. Expression of the two receptor subtypes appears to be developmentally regulated, with high expression in the fetus followed by decreased expression in the early postnatal period. In end-stage failing human heart, expression of the two subtypes is differentially regulated, with selective downregulation of the AT1 subtype and unchanged or relative upregulation of the AT2 subtype. The selective AT1 receptor downregulation in failing human ventricular myocardium is similar to the behavior of 1-adrenergic receptors, and is chamber-specific or only present in failing chambers in the failing human heart. AT1 receptor downregulation in failing ventricular myocardium is likely caused directly or indirectly by activation of the cardiac renin–angiotensin system. Cardiac AT1 receptor downregulation is present in subjects treated with angiotensin-converting enzyme inhibitors, which suggests that blockade of AT1 receptors may be necessary to optimally inhibit adverse effects of angiotensin II. The overall effectiveness of selective AT1 receptor antagonist therapy will likely depend on the probable enhancement of unblocked AT2 receptor-mediated effects of Ang II in the failing human heart, the consequences of which are yet to be elucidated.     

A polymorphism within a conserved beta(1)-adrenergic receptor motif alters cardiac function and beta-blocker response in human heart failure

Heterogeneity of heart failure (HF) phenotypes indicates contributions from underlying common polymorphisms. We considered polymorphisms in the beta(1)-adrenergic receptor (beta(1)AR), a beta-blocker target, as candidate pharmacogenomic loci. Transfected cells, genotyped human nonfailing and failing ventricles, and a clinical trial were used to ascertain phenotype and mechanism. In nonfailing and failing isolated ventricles, beta(1)-Arg-389 had respective 2.8 +/- 0.3- and 4.3 +/- 2.1-fold greater agonist-promoted contractility vs. beta(1)-Gly-389, defining enhanced physiologic coupling under relevant conditions of endogenous expression and HF. The beta-blocker bucindolol was an inverse agonist in failing Arg, but not Gly, ventricles, without partial agonist activity at either receptor; carvedilol was a genotype-independent neutral antagonist. In transfected cells, bucindolol antagonized agonist-stimulated cAMP, with a greater absolute decrease observed for Arg-389 (435 +/- 80 vs. 115 +/- 23 fmol per well). Potential pathophysiologic correlates were assessed in a placebo-controlled trial of bucindolol in 1,040 HF patients. No outcome was associated with genotype in the placebo group, indicating little impact on the natural course of HF. However, the Arg-389 homozygotes treated with bucindolol had an age-, sex-, and race-adjusted 38% reduction in mortality (P = 0.03) and 34% reduction in mortality or hospitalization (P = 0.004) vs. placebo. In contrast, Gly-389 carriers had no clinical response to bucindolol compared with placebo. Those with Arg-389 and high baseline norepinephrine levels trended toward improved survival, but no advantage with this allele and exaggerated sympatholysis was identified. We conclude that beta(1)AR-389 variation alters signaling in multiple models and affects the beta-blocker therapeutic response in HF and, thus, might be used to individualize treatment of the syndrome.     

Myosin heavy chain isoform expression in the failing and nonfailing human heart

In the heart, the relative proportions of the 2 forms of the motor protein myosin heavy chain (MyHC) have been shown to be affected by a wide variety of pathological and physiological stimuli. Hearts that express the faster MyHC motor protein, alpha, produce more power than those expressing the slower MyHC motor protein, beta, leading to the hypothesis that MyHC isoforms play a major role in the determination of cardiac contractility. We showed previously that a significant amount of alphaMyHC mRNA is expressed in nonfailing human ventricular myocardium and that alphaMyHC mRNA expression is decreased 15-fold in end-stage failing left ventricles. In the present study, we determined the MyHC protein isoform content of human heart samples of known MyHC mRNA composition. We demonstrate that alphaMyHC protein was easily detectable in 12 nonfailing hearts. alphaMyHC protein represented 7.2+/-3.2% of total MyHC protein (compared with approximately 35% of the MyHC mRNA), suggesting that translational regulation may be operative; in contrast, there was effectively no detectable alphaMyHC protein in the left ventricles of 10 end-stage failing human hearts.     

Differences in beta-adrenergic neuroeffector mechanisms in ischemic versus idiopathic dilated cardiomyopathy.

BACKGROUND. We measured the content and activities of components of the beta-adrenergic receptor-G protein-adenylate cyclase complex and adrenergic neurotransmitter levels in left and right ventricular myocardial preparations derived from 77 end-stage failing human hearts from patients with idiopathic dilated cardiomyopathy (IDC) or ischemic dilated cardiomyopathy (ISCDC). METHODS AND RESULTS. The results were compared with data obtained in 21 nonfailing hearts removed from organ donors. Compared with ISCDC ventricles, IDC left and right ventricles exhibited a greater degree of total beta- or beta 1-receptor downregulation. In contrast, compared with IDC right ventricles, isolated tissue preparations of ISCDC right ventricles exhibited a greater degree of subsensitivity to the inotropic effect of isoproterenol, indicating a relatively greater degree of functional uncoupling of right ventricular ISCDC beta-receptors from mechanical response. In addition, relative to IDC left ventricles, preparations of ISCDC left ventricle exhibited greater subsensitivity to beta-agonist-mediated adenylate cyclase stimulation, indicating functional uncoupling of left ventricular ISCDC beta-receptors from cyclic AMP generation. The uncoupling of beta-receptors in ISCDC left and right ventricles may have been a result of abnormalities in G protein activation of adenylate cyclase; compared with age- and cardiac function-matched respective left or right IDC ventricles, ISCDC left ventricles exhibited less stimulation of adenylate cyclase by NaF or forskolin but no change in Mn2+ stimulation, whereas ISCDC right ventricles exhibited less stimulation by the nonhydrolyzable guanine nucleotide Gpp (NH)p. Also, IDC right ventricles exhibited a "selective" (not present in IDC left ventricles or ISCDC ventricles) decrease in stimulation of adenylate cyclase by Mn2+. Tissue neurotransmitter levels and pertussis toxin-catalyzed ADP ribosylation were altered to similar extents in IDC and ISCDC. CONCLUSIONS. These data indicate that potentially important differences exist in the regulatory behavior of components of the beta-adrenergic receptor-G protein-adenylate cyclase complex in IDC versus ISCDC, differences that presumably relate to the distinct pathophysiologies of these two types of heart muscle disease.     

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°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 EC₅₀values 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 μmol/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 ∼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.     

Evidence against spare or uncoupled beta-adrenoceptors in the human heart

It is well established that increasing degrees of heart failure are accompanied by a reduced density of myocardial beta-adrenoceptors. It is unclear, however, whether all beta-adrenoceptors in the cardiac cell membrane are coupled to the effector system or whether "spare receptors" or "uncoupled" beta-adrenoceptors also exist. To investigate this, we measured the density of beta-adrenoceptors and the positive inotropic response to isoprenaline in preparations from the same human hearts. The myocardium from nonfailing hearts had significantly (p less than 0.01) higher numbers of beta-adrenoceptors (104 +/- 7 fmol/mg protein) compared with tissue from moderately (mitral valve disease, New York Heart Association [NYHA] class II to III, 60 +/- 2.8 fmol/mg protein) and terminally (dilated cardiomyopathy, NYHA class IV, 35 +/- 2.7 fmol/mg protein) failing human hearts. The KD values of the drug-receptor complexes did not differ within the different patient groups. There was a linear relationship (r = 0.97) between the beta-adrenoceptor density measured and the maximally obtainable positive inotropic effect elicited by isoprenaline in the three groups tested. Thus there seem to be no spare beta-adrenoceptors, that is, receptors not required for the production of the maximal inotropic response in the left ventricular human myocardium, and there are no uncoupled beta-adrenoceptors. The beta-adrenoceptors associated with the plasma membrane (marker: 3H-ouabain binding sites) remained functionally active. In addition, these results indicate that either there is no amplifier system behind the receptor level or it remains unchanged in the failing left ventricular human myocardium under the conditions tested.     

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)     

The role of SR Ca(2+)-content in blunted inotropic responsiveness of failing human myocardium

The effects of inotropic agents are blunted in end-stage failing human myocardium. This has been related to a number of subcellular alterations including desensitization of the beta -adrenergic system. However, it is unknown whether alterations in SR Ca(2+)-handling contribute to blunted inotropic responsiveness of failing myocardium. We tested the hypothesis that the reduced effectiveness of Ca(2+)-dependent inotropic interventions results from the inability of the SR to sufficiently increase its Ca(2+)-content in failing human myocardium. Experiments were performed in ventricular muscle preparations from a total of four non-failing and 18 end-stage failing hearts. Isometric twitch force and SR Ca(2+)-content (using rapid cooling contractures; RCCs) were assessed under basal experimental conditions (1 Hz, 37 degrees C, [Ca(2+)](o) 2.5 mmol/l), and at increasing [Ca(2+)](o) (1.25-15 mmol/l), increasing concentrations of the beta -adrenergic agonist isoproterenol (ISO; 0.01-10 micromol/l), or the glycolytic substrate pyruvate (5-15 mmol/l). In addition, paired RCCs were evoked in a subset of experiments to investigate the relative contribution of SR Ca(2+)-uptake v Na(+)/Ca(2+)-exchange to cytosolic Ca(2+)-elimination. In non-failing human myocardium, Ca(2+), ISO, and pyruvate exerted significant positive inotropic effects (increase in twitch force by maximally 396%, 437%, and 82%, respectively). The inotropic effects were associated with increasing RCCs (by 147%, 193%, and 51%, respectively). In failing myocardium, the inotropic effects of Ca(2+) and ISO were significantly less pronounced (with maximal increases in twitch force by 226% and 138%, respectively), associated with blunted effects on RCCs (increase by 33% and 79%, respectively). In contrast, the inotropic effect of pyruvate was unchanged in failing myocardium (increase by 66%), while the corresponding RCCs increased only by 30%. We conclude that the inotropic effects of Ca(2+), ISO, and pyruvate are associated with a significant increase in SR Ca(2+)-content in non-failing human myocardium. In end-stage failing myocardium, the reduced inotropic response to Ca(2+) and ISO is associated with the inability of the SR to appropriately increase its Ca(2+)-content, possibly related to decreased SR Ca(2+)-ATPase and increased Na(+)/Ca(2+)-exchanger expression. In contrast, the maintained inotropic response to pyruvate despite reduced SR Ca(2+)-loading points to additional subcellular effects such as enhanced myofilament Ca(2+)-responsiveness.     

Deficient production of cyclic AMP: pharmacologic evidence of an important cause of contractile dysfunction in patients with end-stage heart failure

We studied the effects of different classes of inotropic drugs on human working myocardium in vitro that was isolated from the hearts of patients with end-stage heart failure, and compared the responses to these drugs with those noted in muscles from nonfailing control hearts. Although peak isometric force generated in response to increased extracellular calcium reached control levels in the muscles from patients with heart failure, the time course of contraction and rate of relaxation were greatly prolonged. The inotropic effectiveness of the beta-adrenergic agonist isoproterenol and the phosphodiesterase inhibitors milrinone, caffeine, and isobutylmethylxanthine was markedly reduced in muscles from the patients with heart failure. In contrast, the effectiveness of inotropic stimulation with acetylstrophanthidin and the adenylate cyclase activator forskolin was preserved. After a minimally effective dose of forskolin was given to elevate intracellular cyclic AMP levels, the inotropic responses of muscles from the failing hearts to phosphodiesterase inhibitors were markedly potentiated. These data indicate that an abnormality in cyclic AMP production may be a fundamental defect present in patients with end-stage heart failure that can markedly diminish the effectiveness of agents that depend on generation of this nucleotide for production of a positive inotropic effect.     

Cardiac overexpression of the norepinephrine transporter uptake-1 results in marked improvement of heart failure

A hyperadrenergic state is one of the key features of human and experimental heart failure. Decreased densities and activities of the presynaptic neuronal norepinephrine (NE) transporter uptake-1 occur both in patients and animal models. It is currently unclear to what extent the reduction of uptake-1 contributes to the deterioration of heart failure. Therefore, we investigated the effects of myocardial overexpression of uptake-1 in both nonfailing rabbit hearts and in an animal model of heart failure. Heart failure was induced in rabbits by rapid ventricular pacing. Adenoviral gene transfer was used to overexpress uptake-1 in the myocardium. Uptake-1 overexpression led to increased NE uptake capacity into the myocardium. In contrast, systemic plasma NE levels in uptake-1-overexpressing failing rabbits (uptake-1-CHF) did not differ from controls. Downregulation of SERCA-2 and beta-adrenergic receptors in the failing myocardium was significantly reversed after uptake-1 overexpression. Uptake-1 overexpression significantly improved left ventricular (LV) diameters (LV end-diastolic diameter: in GCP-overexpressing failing rabbits (GFP-CHF), 17.4+/-0.4 mm; in uptake-1-CHF rabbits, 15.6+/-0.6 mm) and systolic contractility (fractional shortening: GFP-CHF, 20.7+/-0.6%; uptake-1-CHF, 27.3+/-0.7%), as assessed by echocardiography at the end of the heart failure protocol. Intraventricular tip catheter measurements revealed enhanced contractile reserve (dP/dt max with isoproterenol 1.0 microg/kg: GFP-CHF, 6964+/-230 mm Hg/sec; uptake-1-CHF, 7660+/-315 mm Hg/sec) and LV relaxation (dP/dt min with isoproterenol 1.0 microg/kg: GFP-CHF: -3960+/-260 mm Hg/sec; uptake-1-CHF, -4910+/-490 mm Hg/sec). End-diastolic filling pressures (GFP-CHF, 8.5+/-1.2 mm Hg; uptake-1-CHF, 5.6+/-0.7 mm Hg) tended to be lower in uptake-1 overexpressing animals. In summary, local overexpression of uptake-1 in the myocardium results in marked structural and functional improvement of heart failure, thus underlining the importance of uptake-1 as a key protein in heart failure.     

Activation of cardiac beta 2 adrenoceptors in the human heart

Comparative pharmacologic studies have indicated that the cardiac beta 2 adrenoceptors of vertebrate species are "adrenaline" receptors; i.e., the distribution of beta 2 receptors in the heart seems to be related to the amounts of adrenaline in the sympathetic nerves and in the circulation, and the beta 2 receptors seem to be stimulated mainly by adrenaline. In the human right atrium the order of potency for the agonists and the blocking agents indicate a relatively high proportion of active beta 2 receptors. These findings are in agreement with radioligand binding studies demonstrating up to 50% beta 2 receptors in myocardial membrane preparations. The pharmacologic studies thus add support to the assumption that these beta 2 receptors are functionally active and not merely experimental oddities. It is hypothesized that in normal situations the beta 2-receptor effects are additive to the beta 1 effects. However, during acute stress situations the large amounts of released adrenaline are assumed to increase markedly both inotropy and chronotropy in the heart via beta 2 receptors. It is postulated that only unselective beta blockers can abolish all beta-receptor effects in the heart during stress reactions with profound catecholamine stimulation.