Catecholamine Desensitization Research Articles

Cyclic nucleotide phosphodiesterases as therapeutic targets in cardiac hypertrophy and heart failure.

Cyclic nucleotide phosphodiesterases (PDEs) modulate the neurohormonal regulation of cardiac function by degrading cAMP and cGMP. In cardiomyocytes, multiple PDE isozymes with different enzymatic properties and subcellular localization regulate local pools of cyclic nucleotides and specific functions. This organization is heavily perturbed during cardiac hypertrophy and heart failure (HF), which can contribute to disease progression. Clinically, PDE inhibition has been considered a promising approach to compensate for the catecholamine desensitization that accompanies HF. Although PDE3 inhibitors, such as milrinone or enoximone, have been used clinically to improve systolic function and alleviate the symptoms of acute HF, their chronic use has proved to be detrimental. Other PDEs, such as PDE1, PDE2, PDE4, PDE5, PDE9 and PDE10, have emerged as new potential targets to treat HF, each having a unique role in local cyclic nucleotide signalling pathways. In this Review, we describe cAMP and cGMP signalling in cardiomyocytes and present the various PDE families expressed in the heart as well as their modifications in pathological cardiac hypertrophy and HF. We also appraise the evidence from preclinical models as well as clinical data pointing to the use of inhibitors or activators of specific PDEs that could have therapeutic potential in HF.

Determinants of contractile reserve in viable, chronically dysfunctional myocardium

There is considerable variability in the sensitivity of inotropic reserve to identify viability in chronically dysfunctional myocardium. This is partially related to the underlying pathophysiology, with more frequent contractile reserve in chronically stunned (with normal resting perfusion) than hibernating myocardium (with reduced flow). This study was undertaken to determine the physiological responses to transient and graded stimulation in chronically stunned and hibernating myocardium to define the relative roles of acute catecholamine desensitization and biphasic responses. Pigs were chronically instrumented with a fixed left anterior descending artery stenosis that resulted in chronically stunned myocardium after 2 mo. One month later, hibernating myocardium was confirmed by regional dysfunction (wall thickening, 3.2 +/- 0.3 vs. 5.5 +/- 5 mm in remote, P=0.01) with reduced resting flow (0.70 +/- 0.07 vs. 0.92 +/- 0.09 ml x min(-1) x g(-1) in remote, P=0.01) without infarction. Wall thickening in dysfunctional regions significantly increased during both graded and transient epinephrine stimulation in chronically stunned (from 3.6 +/- 0.3 to 5.6 +/- 0.5 and 4.9 +/- 0.5 mm, respectively) and hibernating myocardium (from 3.3 +/- 0.3 to 5.4 +/- 0.6 and 5.0 +/- 0.7 mm, respectively) and returned to baseline within 15 min. Although a biphasic response during graded stimulation was common, the subsequent decrement in function was small and similar in both groups (stunned, 0.7 +/- 0.2 mm; hibernating, 1.1 +/- 0.3 mm, P=0.25). We conclude that 1) the extent of contractile reserve during beta-adrenergic stimulation is similar in chronically stunned and hibernating myocardium, 2) there are no significant differences between the responses to transient compared with graded catecholamine stimulation, and 3) submaximal catecholamine stimulation does not induce additional stunning in either chronically stunned or hibernating myocardium.

Influence of β2-Adrenergic Receptor Genotype on Airway Function During Exercise in Healthy Adults

In humans, beta(2)-adrenergic receptors (beta(2)ARs) influence airway tone. There are known functional polymorphisms of the beta(2)AR, such as substitution of glycine for arginine at codon 16. We sought to determine if this variation in genotype differentially influences airway function during exercise. Healthy subjects without asthma who were either homozygous for Arg16 (n = 16; mean age, 29 +/- 2 years [+/- SD]; mean maximum oxygen uptake [Vo(2)], 32 +/- 2 mL/kg/min) or the Gly16 allele (n = 26; mean age, 30 +/- 1 years; mean maximum Vo(2), 33 +/- 1 mL/kg/min) participated in the study. Baseline testing included spirometry and maximal symptom-limited exercise. On a separate day, an arterial cannula was placed to measure catecholamine levels. Subjects then performed exercise at two work levels (40% and 75% of peak work) for 9 min each and performed spirometry at 3-min intervals for assessment of airway function. There were no statistically significant differences between groups in maximum Vo(2) or baseline spirometry (p > 0.05). With both light and heavy exercise, the groups had similar increases in the forced expiratory flow at 50% of vital capacity (FEF(50)). FEF(50) increased by 14 +/- 4% and 15 +/- 3% in arginine and glycine groups, respectively, by end exercise (p > 0.05). During recovery (5 min and 10 min after), the Gly16 homozygotes demonstrated persistent bronchodilation (10 min after FEF(50) = + 7 +/- 2% over pre-exercise) while the Arg16 subjects had a rapid return to baseline (10 min after FEF(50) = - 3 +/- 3%, p = 0.007 between groups). No differences were observed in the catecholamine responses between genotypes, although the increase in epinephrine in the arginine group tended to be higher (p = 0.07). These data suggest that the Arg16Gly polymorphism of the beta(2)AR does not influence airway function during short-duration low- and high-intensity exercise. However, during recovery, the Arg16 genotype is associated with a reduced bronchodilation, possibly due to increased catecholamine desensitization.

Beta-adrenergic receptor signaling: an acute compensatory adjustment-inappropriate for the chronic stress of heart failure? Insights from Gsalpha overexpression and other genetically engineered animal models.

Under normal physiological conditions, the heart must be able to increase its output 5-fold to supply the required blood flow to the coronary circulation and skeletal muscles during severe stress. This is normally met by ≈5-fold increases in myocardial contractility, ≈3-fold increases in heart rate, and additional increases in stroke volume.1 This increased load requires a commensurate increase in myocardial blood flow, because oxygen extraction across the heart is nearly complete, even under normal conditions. Accordingly, the design of the cardiovascular system evolved to conserve myocardial metabolic demand, and consequently coronary blood flow, at rest, but with considerable reserve that can be called on rapidly in times of stress. There is a host of compensatory adjustments, including changes in metabolic substrates and kinetics, as well as oxygen-carrying capacity, that may be recruited in response to stress. However, none is more important than the autonomic nervous system in general, and the sympathetic arm in particular, in terms of providing large, rapid changes in cardiac function. When this compensatory mechanism is unavailable, eg, after treatment with propranolol, the 3-fold increases in heart rate and 5-fold increases in myocardial contractility in response to exercise cannot be achieved.1 In this connection, it is recognized that heart failure is a state characterized by enhanced sympathetic tone, but when the failing myocardium is challenged by β-adrenergic stimulation in vivo or in vitro, the most frequent result is β-adrenergic downregulation or desensitization.2 3 4 5 An impairment of cardiac function leads to autocrine, paracrine, and neurohormonal adjustments, including a strong sympathetic component (Figure 1⇓); under acute conditions, these reflex adjustments are beneficial, as noted above. However, when the sympathetic nervous system is chronically and tonically stimulated, as occurs in the pathogenesis of heart failure, desensitization mechanisms are called into play, such that the …

Voltage-dependent calcium channel promoter restores baroreflex sensitivity in conscious dogs with heart failure.

The aim of this study was to determine the mechanism by which the calcium channel promoter BAY y 5959 affects the control of heart rate and baroreflex sensitivity in conscious dogs with pacing-induced heart failure (HF). We compared responses to BAY y 5959, which increases inotropy and decreases chronotropy, with those to norepinephrine (NE), which coincidentally exerts the same directional effects on inotropy and chronotropy, albeit through different mechanisms, in the presence and absence of ganglionic blockade both in control and in HF. Both BAY y 5959 and NE elicit direct effects on the heart and indirect effects through activation of reflexes, primarily the sinoaortic baroreceptor reflex. BAY y 5959 still reduced heart rate in dogs with arterial baroreceptor denervation, but not after ganglionic blockade. HF induced classic catecholamine desensitization to the inotropic effects of NE and blunted reflex bradycardia. In contrast, inotropic responses to BAY y 5959 were preserved in HF. Surprisingly, the autonomically mediated bradycardia induced by BAY y 5959 was also preserved in HF. Baroreflex sensitivity was assessed in control and in HF by pulse interval-systolic arterial blood pressure (PI/SAP) slopes constructed in response to pharmacological alterations in arterial pressure. HF depressed the PI/SAP slope from 11.5+/-1.3 to 4.8+/-0.9 ms/mm Hg, but during BAY y 5959 infusion in HF, the PI/SAP slope was restored to 24.1+/-5.2 ms/mm Hg. To assess central versus peripheral actions of BAY y 5959, the agent was infused with intra-carotid artery perfusion at a low dose, which acted centrally but did not have an effect peripherally. Under these conditions, it still decreased heart rate and restored baroreflex sensitivity (PI/SAP slope, 12.7+/-2.8 ms/mm Hg). Thus, the calcium promoter restores arterial baroreflex sensitivity in HF. Based on intra-carotid artery experiments, this occurs through a central nervous system and vagal mechanism.

Overexpression of myocardial Gsalpha prevents full expression of catecholamine desensitization despite increased beta-adrenergic receptor kinase.

Inotropic and chronotropic responses to catecholamines in young adult transgenic mice overexpressing myocardial Gsalpha are enhanced. One might predict that over the life of the animal, this chronically enhanced beta-adrenergic receptor stimulation would result in homologous catecholamine desensitization. To test this hypothesis, old transgenic Gsalpha mice and age-matched controls were studied physiologically in terms of responsiveness of left ventricular function (ejection fraction) to isoproterenol in vivo and in vitro in terms of beta-adrenergic receptor signaling. Old transgenic mice still responded to isoproterenol with augmented (P < 0.05) left ventricular ejection fraction (+44+/-3%) compared with age-matched controls (+24+/-1%). Although total beta-adrenergic receptor density was reduced in the old transgenic mice, and G protein receptor kinase 2 (beta-adrenergic receptor kinase) levels were increased, the fraction of receptors binding agonist with high affinity as well as isoproterenol- and G protein-stimulated adenylyl cyclase activities were enhanced. Thus, classical catecholamine desensitization is not effective in attenuation of persistently enhanced responses to sympathetic stimulation in mice overexpressing myocardial Gsalpha. To support this conclusion further, experiments were performed with chronic isoproterenol, which elicited effective desensitization in wild-type controls, but failed to elicit desensitization in overexpressed Gsalpha mice. The results of this study suggest that the lack of protective desensitization mechanisms may be responsible in part for the dilated cardiomyopathy which develops with chronic sympathetic stress over the life of these animals.

Effects of cardiac denervation on development of heart failure and catecholamine desensitization.

Two signatures of heart failure are activation of the sympathetic nervous system and catecholamine desensitization. However, whether or not the elimination of cardiac nerves affects either the progression of heart failure or catecholamine desensitization is not clear. We studied 8 dogs with selective ventricular denervation (VD) (surgical technique) and 10 intact dogs, chronically instrumented for measurement of left ventricular (LV) and arterial pressures, LV dP/dt, LV internal diameter, and wall thickness before and after heart failure was induced by rapid pacing (240 bpm) for 3 to 4 weeks. VD was confirmed by the absence of reflex effects induced by intracardiac veratrine and depletion of tissue norepinephrine and by supersensitive responses to norepinephrine. During the development of heart failure, LV end-systolic and end-diastolic stresses and heart rate increased, while myocardial contractility, as reflected by LV dP/dt and mean velocity of circumferential fiber shortening corrected for heart rate (Vcf(c)), decreased in both intact and VD dogs. However, the increases in LV end-diastolic stress and decreases in LV dP/dt as well as the relationship between LV systolic stress and Vcf(c) in heart failure were less (P<.05) in VD dogs. The responses of LV dP/dt and heart rate to both isoproterenol and norepinephrine in intact dogs were reduced in heart failure. The physiological desensitization to the inotropic effects of isoproterenol and norepinephrine was less in dogs with VD (P<.05), but chronotropic responses were similar because atrial innervation remained intact. Plasma norepinephrine levels were not different in VD dogs (592+/-79 pg/mL) compared with intact dogs (576+/-81 pg/mL) in heart failure. Dogs with selective VD tolerated the development of heart failure better than intact dogs and demonstrated significantly less catecholamine desensitization. The latter indicates that intact ventricular innervation is required for physiological expression of catecholamine desensitization despite comparable elevation of plasma catecholamines during the development of heart failure.

Partial Inhibition of Protein Synthesis byPseudomonasExotoxin A Deranges Catecholamine Sensitivity of Cultured Rat Heart Myocytes

To elucidate cellular mechanisms of myocardial depression inPseudomonassepsis, the effects of sublethal concentrations ofP. aeruginosaexotoxin A—a main virulence factor—were studied in cultured neonatal rat cardiomyocytes. It is known that this toxin exerts its pathogenic effect by inhibition of protein synthesis via ADP-ribosylation and thereby inactivation of elongation factor 2 (EF-2). Within 48–72 h, half maximal inhibition of protein synthesis occurs at 4–10 ng/ml. The toxin prevents theβ-adrenoceptor(AR)-mediated myosin heavy chain isozyme shift (V3/V1), while the T3-induced myosin shift is not suppressed. Whileβ1-AR-downregulation by excess of norepinephrine (NE) is not affected, protein synthesis-dependent receptor upregulation in the recovery period after removal of NE is completely suppressed byP. aeruginosaexotoxin A. Thus, a non-lethal, partial inhibition of global cellular protein synthesis byP. aeruginosaexotoxin A: (1) completely preventsβ1-AR-mediated myosin isozyme shift andβ-AR upregulation; (2) sustains the cardiomyocytes in a catecholamine-refractory contractile state in the recovery period after catecholamine desensitization; (3) suggests cellular mechanisms by whichP. aeruginosaexotoxin A might impair heart function inPseudomonassepsis; and (4) may help reveal the possible influence of endogenous inhibitors of EF-2.

Beta-adrenoceptor desensitization during the development of canine pacing-induced heart failure.

1. The goal of this review is to emphasize four major points regarding the development of catecholamine desensitization in heart failure (HF). 2. Catecholamine desensitization occurs prior to the development of HF (i.e. after 1 day of rapid pacing, physiological responses to beta-adrenoceptor stimulation are depressed by over 50%, yet no evidence of HF is observed for 3-4 weeks of rapid pacing). 3. Multiple mechanisms in the beta-adrenoceptor cascade are involved. In HF there are decreases in beta 1-adrenoceptors, high affinity beta-adrenoceptors, adenylyl cyclase activity and messenger RNA and increases in Gi. 4. Not all mechanisms appear simultaneously (i.e. early decreases occur in high affinity beta-adrenoceptors and adenylyl cyclase; late increases in Gi and decreases in beta-adrenoceptor density evolves). 5. Mechanisms distal to cAMP generation also play a role (i.e. alterations in ryanodine receptor binding and excitation-contraction coupling also occur).

2-Aminopurine inhibits RNA and protein synthesis and reduces catecholamine desensitization in C6-2B rat glioma cells

We previously proposed that intracellular cyclic AMP accumulation induces a putative, rapidly turning over protein inhibitory to further hormone activation of adenylate cyclase. In the present study, 2-aminopurine, which has been reported to selectively block c- fos gene expression, was used to test the hypothesis that c-fos protein might be involved in the desensitization process. Indeed, a reduction in heterologous desensitization to catecholamines was observed in 2-aminopurine-treated C6-2B rat glioma cells. However, we found 2-aminopurine to inhibit, in a concentration-dependent manner, total cellular RNA and protein synthesis in C6-2B. HeLa, Swiss 3T3 and BALB/e cells. mRNA synthesis was also markedly reduced in 2-aminopurine-treated cells. These unexpected findings, while supporting our hypothesis of a protein synthesis-sensitive step in the development of refractoriness, raise concern about the specificity of action of 2-aminopurine to inhibit c- fos induction and thus any cellular process, including desensitization, which might be regulated by c- fos gene expression.

Role of intact cardiac nerves and reflex mechanisms in desensitization to catecholamines in conscious dogs.

To study chronic catecholamine desensitization, mini-osmotic pumps were implanted subcutaneously to deliver NE, (0.5 micrograms/kg/min) or saline over 3-4 wk in dogs instrumented with left ventricular (LV) pressure gauges and arterial and left atrial pressure catheters. An acute challenge to NE (0.4 micrograms/kg/min) in intact, conscious dogs increased LV dP/dt by 1,531 +/- 208 mmHg/s before NE pumps, and by a similar amount, 1,340 +/- 166 mmHg/s, 3-4 wk after NE pumps. In contrast, an acute challenge to isoproterenol (ISO, 0.4 micrograms/kg/min) increased LV dP/dt by 5,344 +/- 532 mmHg/s before NE pumps, and significantly less (P less than 0.05; 2,425 +/- 175 mmHg/s) after NE pumps. In the presence of ganglionic and alpha 1-adrenergic blockades, NE (0.4 micrograms/kg/min) increased LV dP/dt by 3,656 +/- 468 mmHg/s before NE pumps and significantly less (P less than 0.01; 1,459 +/- 200 mmHg/s) after NE pumps. Confirming this, an acute challenge to NE (0.4 micrograms/kg/min) in dogs with arterial baroreceptor denervation increased LV dP/dt by 3,732 +/- 896 mmHg/s before NE pumps, and significantly less (P less than 0.05, 1,725 +/- 408 mmHg/s) after NE pumps. In addition, in cardiac denervated dogs, NE (0.4 micrograms/kg/min) increased LV dP/dt by 9,901 +/- 1,404 mmHg/s before NE pumps and significantly less (P less than 0.01, 2,690 +/- 306 mmHg/s) after NE pumps. Desensitization of heart rate responses to NE challenge was also more apparent in the absence of reflex mechanisms. Thus, neural reflex mechanisms play a major role in physiological expression of cardiac desensitization to catecholamines in conscious dogs.

Diphtheria toxin prevents catecholamine desensitization of A431 human epidermoid carcinoma cells.

We proposed that a rapidly turning over protein, induced in response to catecholamine stimulation of C6-2B rat astrocytoma cells, inhibits subsequent hormonal activation of adenylate cyclase. Studies upon which our hypothesis is based and confirmatory work in a variety of other cell lines and in vivo have utilized actinomycin D and cycloheximide to inhibit RNA and protein synthesis, respectively. These inhibitors, however, are not specific and have been reported also to interfere with other cellular processes. Diphtheria toxin is a specific protein synthesis inhibitor that acts only by ADP-ribosylating elongation factor 2, thus preventing peptide chain elongation. We thus tested whether diphtheria toxin could prevent catecholamine-induced desensitization in A431 human epidermoid carcinoma cells. The toxin inhibited protein synthesis and altered the time course of isoproterenol-stimulated cAMP accumulation as did the less-specific protein synthesis inhibitor cycloheximide. Cellular cAMP content after a 30-min exposure to isoproterenol was similar in control and in toxin-treated cells. However, after 4 hr of treatment with isoproterenol, toxin-treated cells accumulated up to six times more cAMP than controls. When cells or cell-free adenylate cyclase preparations were rechallenged with agonists, toxin-mediated inhibition of protein synthesis prevented desensitization. These results show that diphtheria toxin, a specific inhibitor of protein synthesis, can interfere with the normal physiological regulation of cAMP metabolism in eukaryotic cells and provide compelling evidence that catecholamine stimulation of adenylate cyclase promotes the synthesis of a protein(s) that, in some way, inhibits hormone-stimulated adenylate cyclase.

Beta-adrenergic agonist-mediated desensitization in senescent rats

The capacity of senescent rats to develop the catecholamine refractory state was investigated in CDF (F-344) rats of 3 and 24 months of age. Beta-adrenergic receptor number, receptor-agonist affinity, and adenylate cyclase activity in heart membranes were assessed, following the chronic in vivo administration of the beta-adrenergic agonist, metaproterenol. Drug treatment leads to marked myocardial hypertrophy, receptor down-regulation, and reduced isoproterenol-stimulated adenylate cyclase activity. The extent of catecholamine-refractoriness was not different in the older rats, indicating the catecholamine desensitization of myocardial beta-adrenergic responsiveness is not impaired in senescence. Receptor agonist affinity and the percent of receptors in the high-affinity state decrease with age. These parameters are further reduced by agonist treatment but to a lesser extent in the older animals. Thus, the effects of age and agonist desensitization are not additive and suggest that aged animals may already be partially desensitized.

Agonist-induced internalization of the β adrenergic receptor in a smooth muscle cell line

The mechanism of agonist-induced desensitization of the β adrenergic receptor coupled adenylate cyclase has been studied in a smooth muscle cell line, BC3H-1, which expresses both α and β adrenergic receptors and nicotinic receptors. β receptors have been investigated in intact cells using as radioligand 3HCGP-12177, an hydrophilic compound which labels only surface receptors. The treatment of BC3H-1 cells with the agonist Isoproterenol, at 37° but not at 4°, induced a dose dependent internalization of the β adrenergic receptor. Agonist-induced internalization was very rapid, in the order of few minutes. β adrenergic receptor internalization was very specific: the α adrenergic agonist Phenylefrine had almost no effect on βreceptor levels, while Isoproterenol treatment had no effect on the number of α adrenergic or nicotinic receptors expressed at the cell surface of these cells. β adrenergic receptor internalization is probably the major mechanism responsible for catecholamine desensitization in smooth muscle cells.

Autoradiographic localisation of β-adrenoceptors in human lung

In humans, β2-adrenergic receptors (β2ARs) influence airway tone. There are known functional polymorphisms of the β2AR, such as substitution of glycine for arginine at codon 16. We sought to determine if this variation in genotype differentially influences airway function during exercise.Healthy subjects without asthma who were either homozygous for Arg16 (n = 16; mean age, 29 ± 2 years [± SD]; mean maximum oxygen uptake [V̇o2], 32 ± 2 mL/kg/min) or the Gly16 allele (n = 26; mean age, 30 ± 1 years; mean maximum V̇o2, 33 ± 1 mL/kg/min) participated in the study. Baseline testing included spirometry and maximal symptom-limited exercise. On a separate day, an arterial cannula was placed to measure catecholamine levels. Subjects then performed exercise at two work levels (40% and 75% of peak work) for 9 min each and performed spirometry at 3-min intervals for assessment of airway function.There were no statistically significant differences between groups in maximum V̇o2 or baseline spirometry (p > 0.05). With both light and heavy exercise, the groups had similar increases in the forced expiratory flow at 50% of vital capacity (FEF50). FEF50 increased by 14 ± 4% and 15 ± 3% in arginine and glycine groups, respectively, by end exercise (p > 0.05). During recovery (5 min and 10 min after), the Gly16 homozygotes demonstrated persistent bronchodilation (10 min after FEF50 = + 7 ± 2% over pre-exercise) while the Arg16 subjects had a rapid return to baseline (10 min after FEF50 = − 3 ± 3%, p = 0.007 between groups). No differences were observed in the catecholamine responses between genotypes, although the increase in epinephrine in the arginine group tended to be higher (p = 0.07).These data suggest that the Arg16Gly polymorphism of the β2AR does not influence airway function during short-duration low- and high-intensity exercise. However, during recovery, the Arg16 genotype is associated with a reduced bronchodilation, possibly due to increased catecholamine desensitization.