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.     

Modulation of myocardial contractility by lysophosphatidic acid (LPA)

Lysophosphatidic acid (LPA) is a phospholipid messenger, which is released from activated platelets and leukocytes. This study examined the effects of LPA on myocardial contractility and characterized the signal transduction pathway involved in these effects. Functional effects of LPA were determined in isolated, electrically driven human myocardial preparations and rat cardiac myocytes. In human atrial and ventricular myocardial preparations, LPA (100 micromol/l) decreased isoprenaline (0.03 micromol/l) enhanced force of contraction by 17 +/- 2% and 28 +/- 3%, respectively. The effect of LPA was attenuated by suramin (1 mmol/l). In isolated rat cardiomyocytes, LPA (1-100 micromol/l) concentration dependently abolished isoprenaline (0.03 micromol/l) induced increase in cell shortening. This antiadrenergic effect was blunted after pretreatment with pertussis toxin (5 microg/ml, 12 h). Forskolin (10 micromol/l) stimulated adenylyl cyclase activity was inhibited by LPA in human myocardial membranes. PCR analysis of human atrial and ventricular cDNAs revealed the expression of two cognate LPA receptors: EDG-2 and EDG-7. Our results suggest that LPA exerts antiadrenergic effects on force of contraction in human and rodent myocardium via a Galpha(i/o) protein-mediated mechanism, most probably by LPA binding to the mammalian LPA receptors EDG-2 and/or EDG-7. This newly discovered action of LPA might be of pathophysiological importance in conditions like myocardial ischemia or inflammatory disorders when LPA release is enhanced.     

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.     

The effect of xamoterol in failing human myocardium

As the dual pharmacological action of partial beta 1-adrenoceptor agonists should improve left ventricular function while also protecting the myocardium against excessive sympathetic stimulation they may be useful in the treatment of heart failure. Therefore, we studied the pharmacological effects of xamoterol (Corwin, ICI 118, 587), a compound with mixed agonist and antagonistic properties at cardiac beta-adrenoceptors in electrically driven human papillary muscle strips from failing human hearts. Specimens were obtained from patients with different grades of myocardial failure who underwent mitral valve replacement (NYHA II-III) or heart transplantation (NYHA IV). Xamoterol (0.0001-100 mumol l-1) produced only negative inotropic effects, as measured by changes in isometric force of contraction in diseased human papillary muscle strips. However, isoprenaline (0.0001-10 mumol l-1) and ouabain (0.01-0.3 mumol l-1) enhanced force of contraction in the same hearts. Prestimulation with noradrenaline (3 mumol l-1) augmented the negative inotropic effect of xamoterol. It is concluded that xamoterol exerts primarily beta-adrenoceptor antagonistic activity in the failing human myocardium.     

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)     

Force/shortening-frequency relationship in multicellular muscle strips and single cardiomyocytes of human failing and nonfailing hearts.

BACKGROUND: Force of contraction (FOC) frequency-dependently increases in multicellular muscle strip preparations of human nonfailing myocardium, whereas FOC declines in human failing myocardium with increasing stimulation frequency. We investigated whether these characteristics can be observed in single isolated myocytes. METHODS AND RESULTS: Isolated multicellular muscle strip preparations and single isolated cardiomyocytes of failing (heart transplants, dilative cardiomyopathy; n = 11) and nonfailing (donor hearts; n = 11) human hearts were studied. The changes in contraction amplitude (cell shortening in micrometers) at increasing frequency of stimulation (0.5-2 Hz) were continuously recorded with a 1-dimensional high-speed camera that detected the cell edges and measured their distance during contraction. The increase in stimulation frequency was associated with a significant decrease in FOC (2 v 0.5 Hz; 68% basal) and a decrease in cell shortening of human left ventricular cardiomyocytes from failing hearts (2 v 0.5 Hz; 65% basal). In contrast, in human nonfailing myocardium, contraction increased at increasing stimulation frequencies (2 v 0.5 Hz; FOC, 180% basal; cell shortening, 129% basal). CONCLUSIONS: The negative force-frequency relationship measured in multicellular preparations of failing human myocardium results from alterations at the single cell level.     

Disproportionate enhancement of myocardial contractility by the xanthine oxidase inhibitor oxypurinol in failing rat myocardium

OBJECTIVE: Xanthine oxidase (XO) inhibitors enhance myofilament Ca(2+) responsiveness of normal rat myocardium. We examined whether this inotropic action is preserved or magnified in failing rat myocardium and whether the magnitude of this effect correlates with tissue xanthine-oxidoreductase (XOR) activity. METHODS: Hearts of 18-20 month-old SHHF (spontaneous hypertensive/heart failure) rats with end-stage heart failure, as well as of normal control rats, were perfused with the XO inhibitor oxypurinol. Afterwards, [Ca(2+)](i) and tension were measured simultaneously in fura-2-loaded intact isolated right ventricular trabeculae. XOR activity was determined fluorometrically in myocardial homogenates. RESULTS: In failing myocardium, 100 microM oxypurinol significantly increased systolic twitch tension (by 87 and 92% at 1.0 and 1.5 mM extracellular [Ca(2+)], respectively), without altering [Ca(2+)](i) transient amplitude. Oxypurinol did not alter the midpoint or cooperativity of the steady-state tension-[Ca(2+)](i) relationship, but significantly enhanced maximum Ca(2+)-activated tension by 75% in failing myocardium. Oxypurinol also exerted a positive inotropic effect in failing myocardium, which was, however, of significantly smaller relative magnitude. Failing rat myocardium exhibited higher XOR activity than nonfailing myocardium, and this activity was largely suppressed in oxypurinol-treated preparations. CONCLUSIONS: The magnitude of functional improvement with XOR inhibitors depends on the initial level of XOR activity. Specifically, the inotropic actions of oxypurinol are more pronounced in failing rat myocardium, a tissue that exhibits enhanced XOR activity. Our findings rationalize how XO inhibitors boost cardiac contractility and improve mechanoenergetic coupling, and why the effects might be relatively 'selective' for heart failure.     

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

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

Force/shortening[ndash ]frequency relationship in multicellular muscle strips and single cardiomyocytes of human failing and nonfailing hearts

Background: Force of contraction (FOC) frequency-dependently increases in multicellular muscle strip preparations of human nonfailing myocardium, whereas FOC declines in human failing myocardium with increasing stimulation frequency. We investigated whether these characteristics can be observed in single isolated myocytes. Methods and Results: Isolated multicellular muscle strip preparations and single isolated cardiomyocytes of failing (heart transplants, dilative cardiomyopathy; N = 11) and nonfailing (donor hearts; N = 11) human hearts were studied. The changes in contraction amplitude (cell shortening in micrometers) at increasing frequency of stimulation (0.5[ndash ]2 Hz) were continuously recorded with a 1-dimensional high-speed camera that detected the cell edges and measured their distance during contraction. The increase in stimulation frequency was associated with a significant decrease in FOC (2 v 0.5 Hz; 68% basal) and a decrease in cell shortening of human left ventricular cardiomyocytes from failing hearts (2 v 0.5 Hz; 65% basal). In contrast, in human nonfailing myocardium, contraction increased at increasing stimulation frequencies (2 v 0.5 Hz; FOC, 180% basal; cell shortening, 129% basal). Conclusions: The negative force-frequency relationship measured in multicellular preparations of failing human myocardium results from alterations at the single cell level.     

Evidence for a sustained effectiveness of sodium-channel activators in failing human myocardium.

Elevation of cytosolic sodium is thought to be correlated with an increase in force of contraction due to an activation of sodium-calcium exchange. We investigated the inotropic response mediated by the new sodium-channel activator BDF 9148 (0.01-100 mumol/l) on failing human myocardium. Force of contraction was studied using electrically driven human papillary muscle strips from moderately (NYHA II-III, mitral valve replacement) and terminally (NYHA IV, heart transplantation) failing hearts. We also investigated the effects in auricular trabeculae from non-failing hearts (aortocoronary bypass operation). Results were compared with inotropic responses to DPI 201-106 (DPI, 0.1-3 mumol/l), Ca2+ (1.8-15 mmol/l) and isoprenaline (0.001-1 mumol/l). Carbachol (100 mumol/l) and adenosine (1000 mumol/l) were examined in the presence of BDF 9148 and isoprenaline. Both sodium-channel activators, BDF 9148 and DPI 201-106, increased force of contraction in a dose-dependent manner in papillary muscle strips as well as in auricular trabeculae. BDF 9148 and DPI 201-106 were more effective (max. PIE NYHA II-III 1.6 +/- 0.2 mN, NYHA IV 5.9 +/- 0.7 mN, P less than 0.05) and more potent (EC50 (in mumol/l): NYHA IV 0.35, 0.19-0.66; NYHA II-III 1.85, 1.37-2.41) in terminally failing as compared to moderately failing left ventricular myocardium. Moreover, the positive inotropic effects of BDF 9148 were greater than those of DPI 201-106 in NYHA IV (max. PIE 2.7 +/- 0.3 mN, P less than 0.05). In NYHA IV, BDF 9148 was as effective as CA2+ (max. PIE 5.1 +/- 0.4 mN). In the same hearts, the positive inotropic effects of isoprenaline were reduced in NYHA IV (max. PIE 2.1 +/- 0.3 mN) compared to NYHA II-III (max. PIE 3.4 +/- 0.4 mN, P less than 0.05). Adenosine as well as carbachol did not affect the positive inotropic response of BDF 9148 or DPI 201-106 but reduced the effectiveness of isoprenaline (P less than 0.05). In myocardial membranes, BDF 9148 was 1000-fold less effective in competition experiments with 3H-ouabain than ouabain. We conclude that (1) sodium-channel activators may produce a significant cAMP-independent positive inotropic effect in left ventricular myocardium from failing human hearts; (2) the inotropic effect of sodium-channel activators were more potent and more effective in NYHA IV as compared to NYHA II-III. The degree of myocardial failure does not reduce the effectiveness of the sodium-channel activator BDF 9148.     

Beta 1- and beta 2-adrenergic-receptor subpopulations in nonfailing and failing human ventricular myocardium: coupling of both receptor subtypes to muscle contraction and selective beta 1-receptor down-regulation in heart failure

We used radioligand binding techniques and measurement of beta-agonist-mediated positive inotropic responses in isolated cardiac tissue to examine beta-adrenergic-receptor subpopulations in nonfailing and failing human left and right ventricular myocardium. In tissue derived from 48 human hearts the receptor subtypes identified in nonfailing ventricle by radioligand binding were beta 1 (77%) and beta 2 (23%), with no evidence of an "atypical" beta-adrenergic receptor. In failing left ventricle the beta 1:beta 2 ratio was markedly different, i.e., 60:38. This decrease in the beta 1 proportion and increase in the beta 2 proportion in the failing ventricles were due to a 62%, "selective" down-regulation of the beta 1 subpopulation, with little or no change in beta 2 receptors. In muscle bath experiments in isolated trabeculae derived from nonfailing and failing right ventricles, both beta 1- and beta 2-adrenergic receptors were coupled to a positive inotropic response. In nonfailing myocardium, beta 1 responses predominated, as the selective beta 1 agonist denopamine produced a response that was 66% of the total contractile response of isoproterenol. In heart failure the beta 1 component was markedly decreased, while the beta 2 component was not significantly diminished. Moreover, in heart failure the beta 2 component increased in prominence, as the contractile response to the selective beta 2 agonist zinterol increased from a minority (39%) to a majority (60%) of the total response generated by isoproterenol. We conclude that failing human ventricular myocardium contains a relatively high proportion of beta 2 receptors, due to selective down-regulation of beta 1 receptors. As a result, in the failing human heart the beta 2-receptor subpopulation is a relatively important mediator of inotropic support in response to nonselective beta-agonist stimulation and is available for inotropic stimulation by selective beta 2 agonists.     

Inotropic and lusitropic dysfunction in myocardium from patients with dilated cardiomyopathy.

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

Excitation-contraction coupling in myocardium of nonhibernating and hibernating chipmunks: effects of isoprenaline, a high calcium medium, and ryanodine

The electromechanical responses to isoprenaline and a high calcium medium on cardiac muscles from nonhibernating and hibernating chipmunks were studied in the presence and the absence of ryanodine. In nonhibernating animal preparations, isoprenaline (5 X 10(-8) M) caused a marked positive inotropic effect with an increase in the amplitude of the action potential plateau and augmented the slow action potential in muscle depolarized with 26 mM K+. In hibernating animal preparations, isoprenaline failed to cause a positive inotropic effect in spite of an increase in the amplitude of action potential plateau and slow action potentials. Similar inotropic effects were caused in the two preparations when extracellular calcium was raised to 6 mM, but action potential plateau and slow action potentials of the two preparations were less affected by this procedure; only in nonhibernating animal preparations was the amplitude of the slow action potential slightly increased. Ryanodine (2 X 10(-6) M) partially inhibited the contraction but augmented the action potential plateau and slow action potentials in nonhibernating animal preparations, while in hibernating animal preparations, it eliminated the contraction and severely inhibited the action potential plateau and slow action potentials. The electrical effects of isoprenaline on the two preparations and of a high calcium medium on nonhibernating animal preparations were more pronounced in the presence of ryanodine than in the absence of it. However, the electrical activity on hibernating animal preparations was unaffected by a high calcium medium in either the presence or absence of ryanodine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cardiac beta-adrenergic mediated chrono- and inotropic effects of imipramine in vitro.

Clinical and experimental studies show that tricyclic antidepressants in "therapeutic plasma concentrations" can increase heart rate, myocardial contractility and blood pressure. Our study was undertaken to analyze the role of beta-adrenergic stimulation in the chronotropic and inotropic effects of imipramine. Strips of rat right atrium including the sinus node, which were beating spontaneously, were used to study chronotropism. Strips of the left atrium, electrically stimulated to beat at 1 Hz, were used to study inotropism. The preparations were superfused in vitro with Tyrode's solution at 37 degrees C and exposed to imipramine while recording membrane potentials or force of contraction. Imipramine exerted dose-dependent biphasic actions. Imipramine 0.8 microM produced positive chronotropic and inotropic actions which were blocked by propranolol. Imipramine 1.6 microM depressed the sinus node automaticity, but it did not modify the force of contraction. Imipramine 3.2 microM depressed both the sinus node automaticity and the myocardial contractility. In conclusion, imipramine in "therapeutic plasma concentrations" produces beta-adrenergic mediated cardiac positive chronotropic and inotropic actions. The possible mechanisms of the depressant effects of imipramine itself on automaticity and contractility are still not clear. The results presented can explain stimulatory and depressant cardiac effects of therapeutic doses and overdoses of tricyclic antidepressants.     

An inhibitor of nitric oxide synthase does not increase contraction or beta-adrenoceptor sensitivity of ventricular myocytes from failing human heart

OBJECTIVE: Nitric oxide (NO) has been implicated in the depression of cardiac function in human heart failure. Some reports have identified iNOS (inducible nitric oxide synthase) within the myocyte component of the failing human heart, and NO is known to decrease the contraction amplitude of isolated ventricular myocytes. We have treated myocytes from failing human ventricle with a NOS inhibitor, NG-monomethyl-L-arginine (L-NMMA), in an attempt to restore contractile function. METHODS AND RESULTS: Myocytes were isolated from failing and non-failing human ventricles and their contraction amplitude was measured during superfusion (32 degrees C, 1-2 mmol/l Ca2+) and electrical stimulation (0.1-2 Hz). The contraction amplitude of myocytes from failing hearts was depressed in a frequency-dependent manner. At 1 Hz, the contraction amplitude of cells from non-failing heart was 4.70 +/- 0.53% cell shortening (mean +/- SEM, n = 13 subjects), compared with 2.18 +/- 0.27% (P < 0.01, 11 patients) from patients with ischaemic heart disease (IHD) or 2.56 +/- 0.74% (P < 0.02, six patients) with dilated cardiomyopathy (DCM). Superfusion with 0.1 mmol/l L-NMMA did not increase contraction amplitude in myocytes from failing heart at either 0.2 Hz (n = 11) or 1 Hz (n = 7). Responses to beta-adrenoceptor stimulation were reduced in myocytes from failing human heart, with contraction amplitude in maximum isoprenaline 0.47 +/- 0.11 of that in high Ca2+ in the same cell (n = 6), compared to 0.99 +/- 0.07 in non-failing heart (n = 14, P < 0.01). The presence of 0.1 mmol/l L-NMMA did not increase the isoprenaline/Ca2+ ratio in myocytes from failing heart (0.40 +/- 0.09, P = NS). CONCLUSION: These results do not suggest a functional role for tonic NO production in the frequency-dependent depression of contraction or beta-adrenoceptor desensitisation in myocytes from failing human ventricle.     

Influence of adenosine on the stimulatory effect of isoprenaline and insulin on myocardial contractility in vivo.

STUDY OBJECTIVE--In vitro investigations have indicated that adenosine can inhibit beta adrenergic stimulated increases in cardiac contractility. The present study was designed to determine the ability of adenosine to inhibit isoprenaline induced increases in contractility in vivo. Adenosine has been reported to exert its inhibitory effects on contractility by inhibiting adenylate cyclase. Thus, adenosine should have no effect on positive inotropic agents that act independently of adenylate cyclase. We therefore assessed the ability of this nucleoside to inhibit the positive inotropic effect of insulin, a hormone that exerts a positive inotropic effect independently of alterations in cyclic AMP. DESIGN--Saline or adenosine (10 mumol.ml-1) was infused into the circumflex artery at 1 ml.min-1 as a background. Isoprenaline (20 or 200 pmol.min-1) was infused into the artery during saline or adenosine infusion. The response to insulin was determined during hyperinsulinaemic euglycaemic clamp. SUBJECTS--16 adult mongrel dogs were anaesthetised with pentobarbitone. Five dogs were used in isoprenaline studies, and 11 dogs in insulin studies. MEASUREMENTS AND MAIN RESULTS--Dogs were instrumented to obtain measurements of mean arterial blood pressure, heart rate, circumflex artery blood flow (Q), instantaneous left ventricular pressure, and posterior left ventricular wall thickness. We used the slope of the end systolic pressure-dimension relationship (Ees) as an index of myocardial contractility, previously shown to reflect changes in myocardial inotropic state independent of influence from afterload and preload. Left ventricular dP/dtmax was derived from left ventricular pressure with respect to time, and Ees was determined from left ventricular pressure and wall thickness. Neither adenosine, isoprenaline, nor insulin alone caused any significant changes in mean arterial pressure or heart rate. Adenosine caused a significant increase in Q. Both left ventricular dP/dtmax and Ees were significantly increased by either insulin or both doses of isoprenaline. Adenosine inhibited the increases in these indices caused by isoprenaline, but not those caused by insulin. CONCLUSIONS--Adenosine is capable of inhibiting the positive inotropic effect of isoprenaline in vivo. The results suggest that adenosine does not inhibit positive inotropic responses that act independently of the stimulation of adenylate cyclase.     

Regulation of basal myocardial function by NO.

The effects of exogenous and endogenous. NO on myocardial functions such as contraction, relaxation and heart rate have recently gained considerable scientific interest. .NO stimulates myocardial soluble guanylate cyclase to produce cGMP, which activates two major target proteins. A small increase in cGMP levels predominantly inhibits phosphodiesterase III, while high cGMP levels activate cGMP-dependent protein kinase. Accordingly, submicromolar .NO concentrations improve myocardial contraction, while submillimolar .NO concentrations decrease contractility. The latter action includes direct inhibitory .NO effects on ATP synthesis and voltage-gated calcium channels. Overall, the inotropic effects of exogenous .NO are small and probably of minor importance for myocardial contractility. Cardiomyocytes are capable of expressing eNOS and iNOS. Endogenous .NO has effects on myocardial contraction, similar to that of exogenous .NO. Various NOS inhibitors can substantially reduce myocardial contractility in vitro and in vivo, suggesting that basal endogenous .NO production supports myocardial contractility. There is also evidence for a .NO-dependent cardiodepressive effect of cytokines that is mediated by expression of iNOS. This is consistent with the negative inotropic effects of .NO at high concentrations. Cardiodepressive actions of endogenous .NO production may play a role in certain forms of heart failure. Finally, .NO also has an effect on heart rate. Physiologic .NO concentrations can stimulate heart rate by activating the hyperpolarization-activated inward current (If) and this effect decreases at submillimolar .NO concentrations. In summary, physiological concentrations of .NO increase contractility and heart rate under basal conditions, while high .NO concentrations induce the opposite effects.     

Increased functional importance of the Na,Ca-exchanger in contracting failing human myocardium but unchanged activity in isolated vesicles

The present study aimed to investigate the hypothesis that the function of the Na,Ca-exchanger (NCX) is of higher importance for contractility and Ca(2+)-homeostasis in left ventricle from terminally failing than from nonfailing human hearts. The effect of decreasing extracellular [Na](e) (140 to 25 mmol/L) on force of contraction in isolated left ventricular papillary muscle strips was studied as a reflection of NCX function in multicellular preparations (terminally failing, DCM, dilated cardiomyopathy, NYHA IV, n = 13; nonfailing, NF, donor hearts, n = 10). Decreasing [Na](e) has previously been shown to increase contractility in vitro secondary to a decreased Ca(2+)-extrusion by the NCX. In addition, the NCX activity was measured as Na(+)-dependent (45)Ca(2+)-uptake into isolated myocardial vesicles as a function of time and Ca(2+)-concentration (DCM n = 8, NF n = 8). Decreasing [Na](e) enhanced the contractility of papillary muscle strips in both DCM and NF, but the contractility of DCM was increased at smaller reductions of [Na](e) than NF. The NCX activity in isolated myocardial vesicles was unchanged as a function of time (T(1/2): DCM 2.4 +/- 0.3 s versus NF 2.5 +/- 0.3 s) and as a function of Ca(2+) (DCM 0.99 +/- 0.08 versus NF 0.96 +/- 0.07 nmol/mg protein x 3 s, K(1/2): DCM 39.2 microM versus NF 38.3 microM). These results demonstrate a higher sensitivity of the failing human myocardium towards Na,Ca-exchanger mediated positive inotropic effects, suggesting a higher significance of the Na,Ca-exchanger for the extrusion of Ca(2+)-ions in intact failing versus nonfailing human myocardium. Since the activity and the Ca (2+)-affinity of the Na,Ca-exchanger in isolated vesicles was unchanged, we propose that alterations in Ca(2+)-and Na(+)-homeostasis (due to impaired function of the sarcoplasmic reticulum and the Na(+), K(+)-ATPase) or the prolonged action potential are the reason for this observation.     

Beta 1- and beta 2-adrenoceptors in sheep cardiac ventricular muscle.

The presence of beta 2-adrenoceptors in the sheep ventricular myocardium was assessed by the radioligand binding technique and functional studies. In membrane preparations, the competition curve between [3H]-dihydroalprenolol and the selective beta 1-antagonist CGP 20712A (0.1 nM-1 mM) was clearly biphasic, and revealed the presence of two different binding sites showing an affinity (pKD) for CGP 20712A of 9.5 +/- 0.9 and 4.5 +/- 0.4, respectively. The relative proportion of beta 1:beta 2 adrenoceptors was about 70:30 in both the right and left ventricle. In ventricular trabeculae driven at 1Hz, isoprenaline (1-300 nM) caused a dose-dependent increase in the force of contraction, the maximum effect being 298 +/- 26 mg, associated with reduction of time to peak tension (t1, clinotropic effect) and relaxation time (t2, 298 +/- 26 mg, associated with reduction of time to peak tension (t1, clinotropic effect) and relaxation time (t2, lusitropic effect). The inotropic dose-response curve for isoprenaline was significantly shifted to the right by pretreatment of the preparations with 0.1 microM CGP 20712A or with the selective beta 2-antagonist ICI 118551 (50 nM). In the presence of CGP 20712A (0.1 microM), isoprenaline, up to a concentration of 10 microM, did not affect either t1 or t2; on the other hand, pretreatment of the preparations with ICI 118551 (50 nM) fully antagonized the clinotropic but not the lusitropic effect of isoprenaline. In the presence of CGP 20712A procaterol (0.01-10 microM), a beta 2-adrenoceptor agonist, induced a positive intropic effect which was not associated with any significant modifications in t1 or t2. This effect was completely abolished by ICI 118551 (50 nM). The positive inotropic action of isoprenaline (1 microM) was associated with a significant decrease in action potential duration measured at -60 mV (220 +/- 8 and 193 +/- 10 ms in the absence and presence of isoprenaline, respectively; P less than 0.05). In the presence of CGP 20712A (0.1 microM) alone, isoprenaline (1 microM) still induced a significant increase in contractility but the action potential profile was only slightly affected. The effects of isoprenaline were fully antagonized by the simultaneous presence of CGP 20712A and ICI 118551 (10 nM). It is concluded that both beta 1- and beta 2-adrenoceptors appear to coexist in sheep ventricular myocardium where their stimulation mediates a positive inotropic effect. However, their functional role on the relaxation phase of the twitch may be different.