Cyclic AMP levels in ventricular myocytes from noradrenaline-treated guinea-pigs.

Chronic activation of the sympathetic nervous system in human heart failure is believed to cause cardiac beta-adrenoceptor desensitisation. We have investigated the relationship between beta-adrenoceptor desensitisation and cyclic AMP levels in cardiac myocytes isolated from the ventricle of guinea-pigs chronically infused with noradrenaline hydrochloride for 7 days. Functional beta-adrenoceptor desensitisation was confirmed by a significant decrease in the maximum isoprenaline-stimulated contraction amplitude and an increased EC50 for isoprenaline. In the absence of beta-adrenoceptor stimulation, basal cyclic AMP levels were significantly depressed in populations of myocytes from noradrenaline-treated animals compared to sham-operated controls, and this was not accounted for by myocyte hypertrophy or necrosis. Similarly, there was a significant decrease in cyclic AMP levels at maximally inotropic isoprenaline concentrations. Threshold and maximum inotropic concentrations of the phosphodiesterase inhibitor, 3-isobutyl-l-methylxanthine (IBMX), restored isoprenaline-stimulated cyclic AMP levels in noradrenaline-treated guinea-pig cardiac myocytes, although we have previously reported no increase in maximum inotropic effect of isoprenaline with these compounds.

Contractile dysfunction of isolated ventricular myocytes in experimental uraemia.

In order to clarify the mechanism underlying impaired cardiac performance in uraemia, the contractile function of isolated cardiac myocytes from chronically uraemic and control rats has been compared. Rats were made uraemic by sub-total nephrectomy in a two-stage surgical procedure, and left for 4 weeks. Sham-operated controls were prepared at the same time. Animals were pairfed, and final body weights were not significantly different between the groups. Ventricular myocytes were isolated and their contraction amplitude and velocity were measured using a video-based edge-detection system. Contraction was depressed in myocytes from uraemic animals, with contraction amplitude in maximum Ca2+ reduced from 16.3 +/- 0.6% shortening, to 13.0 +/- 0.8% (p < 0.01, n = 10 animals for each group). There was a concomitant decrease in the velocity of shortening (5.6 +/- 0.4 vs. 3.9 +/- 0.5 micron s-1 change in sarcomere length, p < 0.02) and of relaxation (4.6 +/- 0.4 vs. 3.2 +/- 0.4 micron s-1 p < 0.02). Similar depression was seen at lower perfusate Ca2+ concentrations (1-2 mM) and the EC50 for Ca2+ was unchanged. The response to beta-adrenoceptor stimulation was decreased by the same magnitude as that to Ca2+, with no change in the EC50 for isoproterenol or the ratio of maximum response to isoproterenol or to Ca2+ in the same cell (isoproterenol/Ca2+ ratio). There was no shift in the myosin isozyme composition in uraemic cells, with both groups showing a heterogeneous V1/V2/V3 pattern. We conclude that chronic uraemia is associated with a depression of contractile function in the isolated myocyte but no shift in myosin isoforms or specific beta-adrenoceptor desensitisation.

Regulation of expression of contractile proteins with cardiac hypertrophy and failure.

Transitions in sarcomeric alpha-actin and cardiac myosin heavy chain (MHC) gene expression have been useful as molecular markers for the development of cardiac hypertrophy and failure. In simpler model systems, alpha-actin expression has been useful in delineating some of the molecular pathways responsible for its induction following growth stimulation in vitro. In this study, we report that the effects of adrenergic agonists on alpha-actin expression in neonatal cardiocytes is dependent upon the culture conditions. In cardiocytes plated at 5 x 10(4) cells/cm2, skeletal alpha-actin mRNA levels represent 47%, 37% or 42% of total sarcomeric alpha-actin accumulations following administrations of 4 microM norepinephrine (NE), isoproterenol (Iso), or phenylephrine (PE), respectively. Cultured cardiocytes treated with vehicle (ascorbate) only accumulated 19% skeletal alpha-actin. Under these tissue culture conditions, in contrast to data reported previously, skeletal alpha-actin expression is regulated by both alpha- and beta-adrenergic agonist stimulation. Furthermore, we present data showing that an endogenous anti-beta-MHC transcript is regulated by both pressure-overload- or thyroxine-induced cardiac hypertrophy. Although anti-beta-MHC transcripts do not play a major role in regulating beta-MHC gene expression, the presence of this antisense transcript is associated with a novel set of beta-MHC degradation products. In vitro studies, where oligonucleotides complementary to beta-MHC have been introduced into cardiomyocytes, show that the mRNA levels of beta-MHC are decreased by 14-21% within 72 h after addition of the oligonucleotides. This result together with the presence of beta-MHC degradation products suggest that endogenous anti-beta-MHC transcripts may be involved in a post-transcriptional regulatory mechanism affecting the steady-state levels of beta-MHC expression.

Acceleration of contraction by beta-adrenoceptor stimulation is greater in ventricular myocytes from failing than non-failing human hearts.

Myocytes from failing human ventricle contract and relax more slowly than those from non-failing. This has been suggested to result from the lowering of basal cyclic AMP level in failing myocardium, and the consequent withdrawal of a tonic lusitropic effect. We present data to support this hypothesis by demonstrating that the acceleration of contraction and relaxation by beta-adrenoceptor stimulation is greater in myocytes from failing than non-failing heart. This is despite the desensitisation of the inotropic effect of isoprenaline in the same failing cells. Following beta-adrenoceptor stimulation, speeds of contraction and relaxation are normalised in myocytes from failing heart, with final values not significantly different from non-failing.

Clenbuterol induces hypertrophy of the latissimus dorsi muscle and heart in the rat with molecular and phenotypic changes.

BACKGROUND: Skeletal muscle assistance of the circulation for patients in end-stage heart failure requires electrical training of the latissimus dorsi flap to produce fatigue resistance. This process of electrical transformation and the development of postmobilization atrophy results in a profound loss in peak power generated. The beta 2-adrenoceptor agonist clenbuterol was used to investigate its potential to selectively induce skeletal muscle hypertrophy, particularly the latissimus dorsi muscle (LDM), independent of adverse effects on cardiac muscle. METHODS AND RESULTS: Forty-one male Sprague-Dawley rats were divided into four groups and used in this study. Clenbuterol 2 micrograms.g body wt-1.d-1 was administered subcutaneously for a period of either 5 weeks (group A) or 2 weeks (group A1). Groups B and B1 (controls) were injected with 0.5 mL normal saline once daily. At the end of the experimental period, all rats were weighed and terminally anesthetized for removal of the left LDM, left gastrocnemius-plantaris-soleus (GPS) muscles, and heart. The results showed that the increase in body weight did not differ significantly between the clenbuterol-treated and control groups (P > .5). The ratio of LDM to tibial length (hypertrophic index) for groups A and A1 was significantly greater than controls (P < .01), which represented a 20% to 29% increase. The hypertrophy was more pronounced for hindlimb skeletal muscle (21% to 35% for GPS), and the effects of this relatively high dose of clenbuterol on the heart were less marked (18% to 20% hypertrophy). RNA analyses indicate that ventricles of clenbuterol-treated rats express elevated levels of mRNA to atrial natriuretic factor without a concomitant increase in skeletal alpha-actin and beta-myosin heavy chain, consistent with a "physiological" form of cardiac hypertrophy. CONCLUSIONS: Clenbuterol induces significant hypertrophy of the LDM associated with specific changes in cardiac gene expression.

Abnormalities of the myocytes in ischaemic cardiomyopathy.

Acute myocardial ischemia and subsequent reperfusion result in biochemical and ionic changes in cardiac myocytes which cause contracture of the muscle and a reduced contractile force. Whether changes observed in single myocytes isolated from ischaemic ventricles are a direct consequence of the acute insult, or develop more slowly due to subsequent alterations in load and neurohumoural environment, is controversial. Myocytes from ischemic hearts have a similar contraction amplitude to those from non-failing hearts at physiological or maximally activating levels of ca2+. This could be partly due to the method of cell selection, or could represent the detection of a population of myocytes that have recovered from the original insult. However, there are significant decreases in the velocities of contraction and, particularly, relaxation in myocytes from the ischaemic heart. These resemble alterations caused by anoxia/reperfusion, but similar changes have also been observed in non-ischaemic causes of heart failure. Responses of beta-adrenoceptor stimulation are reduced in single cells from the failing heart, and a post-receptor defect has also been detected. Treatment with pertussis toxin, which reduces the activity of the inhibitory guanine-nucleotide binding protein (Gi) was able to restore beta-adrenoceptor responses to normal. The hypothesis that alterations in the beta-adrenoceptor/Gi/cAMP pathway represent the response of the myocyte to continued exposure to noradrenaline, because of the high sympathetic drive in these patients, is supported by the strong parallels observed with catecholamine-treated animals, and by the fact that non-ischemic aetiologies exhibit similar desensitization. It is concluded that the surviving myocytes in an ischaemic heart are damaged by the neurohumoral alterations that represent the body's attempt to restore cardiac output.

Decreased contractile responses to isoproterenol in isolated cardiac myocytes from aging guinea-pigs.

We have characterized the age-related changes of contractility and beta-adrenoceptor function in isolated cardiac myocytes from guinea-pigs. We used either adult animals from 2 to 14 weeks of age, where body weight increases linearly with age, or senescent ones aged between 53-65 weeks. There was some indication of a decrease in contractility in maximum Ca2+ with age, with significant differences between a young (< or = 4 weeks, weight < 400 g) and aged (> or = 8 weeks, weight > 600 g) group in contraction amplitude expressed as percentage shortening (but not when expressed as micron change in length) or contraction and relaxation velocities. This decline was continued into senescence, and ANOVA showed a significant difference between the three groups for contraction amplitude (percentage shortening, 12.2 +/- 0.9%, young, n = 31; 9.5 +/- 0.6%, n = 28 aged; 6.7 +/- 0.8%, n = 6, senescent; P = 0.005), and contraction or relaxation velocities (P < 0.001). There was a more pronounced decline in maximum response to isoproterenol with age. ANOVA for the maximum isoproterenol response for the three divisions showed significant differences for percentage shortening (11.8 +/- 0.7%, n = 30, young; 7.9 +/- 0.5%, n-28, aged and 5.5 +/- 1.1%, n = 6, senescent; P < 0.001), velocities of contraction (P < 0.001) and relaxation (P < 0.001), and normalized velocities of contraction (P < 0.001) and relaxation (P < 0.01) at maximum isoproterenol, as well as in ISO EC50 (P < 0.001) and isoproterenol/Ca2+ ratio (P < 0.02). A general decrease in contractility of the myocyte occurs as the animal ages, with maximum contraction amplitude being reduced and velocity of contraction and relaxation slowed. The effect was more pronounced for beta-adrenoceptor stimulation than for high Ca2+, suggesting a specific lesion in the adenylate cyclase related pathway. Much of the change occurred between the young adult (< or = 4 weeks) and the aged adult (> or = 8 weeks), although the trend was continued in senescent animals (> 52 weeks).

Contractile function and response to agonists in myocytes from failing human heart.

Ventricular myocytes from failing human hearts have a similar maximum contraction amplitude in high Ca2+ to those from non-failing heart at low stimulation rates (0.2 Hz, 32 degrees C), but do not exhibit the same positive frequency-interval relationship. At higher stimulation rates (1 Hz) therefore, the amplitude is depressed in cells from failing hearts compared to controls. Slow relaxation is seen in myocytes from failing ventricle at all stimulation rates, and contraction velocity is also slightly reduced. beta-adrenoceptor desensitization is evident, and increases with severity of disease. There is also a post-receptor defect in myocytes from failing heart since responses to forskolin and cyclic AMP analogues are reduced, and this is accompanied by decreased cyclic AMP levels in myocardium from patients in end-stage disease. Pertussis toxin treatment, which inactivates Gi, reverses most of the alterations in the beta-adrenoceptor pathway. The role of the sympathetic system is indicated by the parallels between myocytes from failing human heart and those from the noradrenaline-treated guinea-pig, which show beta-adrenoceptor desensitization, a post-receptor defect and reduced basal cyclic AMP levels. However, relaxation velocities are not slowed in these guinea-pig myocytes, indicating that basal cyclic AMP does not have a tonic role in speeding relaxation.

Coexistence of functioning beta 1- and beta 2-adrenoceptors in single myocytes from human ventricle.

BACKGROUND: Both beta 1- and beta 2-adrenoceptors (beta 1 AR and beta 2 AR) are present in human ventricle. This study was designed to determine whether the two subtypes contribute to contraction in single myocytes from human heart. METHODS AND RESULTS: (-)-Epinephrine increased the contraction amplitude and velocity of single myocytes isolated from the ventricles of failing and nonfailing human hearts. Concentration-response curves to (-)-epinephrine were constructed in the presence and absence of selective antagonists for beta 1 AR (CGP 20712A) and beta 2 AR (ICI 118,551). Responses to (-)-epinephrine were antagonized to a variable degree by the blockers, suggesting heterogeneous contribution of beta 1AR and beta 2AR among cells. The most common response in single myocytes was that ICI 118,551 (50 nmol/L) shifted the concentration-response curve less than 10-fold: this was lower than the 100-fold shift expected for a pure beta 2AR effect. Inclusion of CGP 20712A (300 nmol/L) with ICI 118,551 shifted the (-)-epinephrine curve still further. These observations suggest that both beta 1AR and beta 2AR contribute to the increase in contraction amplitude with (-)-epinephrine in this group of myocytes. When 300 nmol/L CGP 20712A was present as the sole antagonist, only a marginal shift of the concentration-response curve for (-)-epinephrine was usually observed, indicating that beta 1AR were not mediating the effect of these low concentrations of (-)-epinephrine. Both beta 1AR and beta 2AR mediated a considerable abbreviation of the time to peak contraction and time to 50% relaxation in the single cells. CONCLUSIONS: beta 1AR and beta 2AR coexist and function on human ventricular myocytes. At low (-)-epinephrine concentrations, contractile responses are predominantly mediated by beta 2AR rather than beta 1AR in myocytes from failing hearts.

Incomplete reversal of beta-adrenoceptor desensitization in human and guinea-pig cardiomyocytes by cyclic nucleotide phosphodiesterase inhibitors.

1. The decreased response to beta-adrenoceptor stimulation seen in heart failure may be related to a defect in cyclic AMP production. The inotropic effects of the selective phosphodiesterase (PDE) III inhibitors, SK&F 94120 and SK&F94836, and the non-selective PDE inhibitor, 3-isobutyl-l-methylxanthine (IBMX), alone and when combined synergistically with isoprenaline, were studied in control and beta-adrenoceptor-desensitized ventricular myocytes. 2. Myocytes isolated from noradrenaline-treated guinea-pigs had a reduced maximum response to isoprenaline compared with control animals (60.0 +/- 2.5%, n = 42 vs 79.5 +/- 1.7% maximum calcium: n = 46, P < 0.001). Together with an approximately 20 fold increase in the isoprenaline EC50, this is indicative of beta-adrenoceptor desensitization as a result with chronic infusion with noradrenaline. 3. The maximum inotropic response of IBMX was depressed following noradrenaline treatment, from 74.9 +/- 4.6% (n = 7) in control, to 61.7 +/- 2.70% (n = 6), as a percentage of maximum calcium in noradrenaline-treated guinea-pig ventricular myocytes (P < 0.02). The pD2 value for IBMX was also reduced (P < 0.02). No significant differences in the inotropic effects of SK&F94120 and SK&F94836 were seen between control and beta-adrenoceptor desensitized myocytes. 4. Threshold inotropic concentrations of SK&F94120 and SK&F94836 caused a five fold decrease in the EC50 of control myocytes for isoprenaline, and an 11 fold decrease in the noradrenaline-treated guinea-pig ventricular myocytes. 5. The maximum response to isoprenaline in myocytes isolated from normal guinea-pigs was unaffected by PDE inhibition; either at threshold or maximum inotropic concentrations, or by CPT cyclic AMP, an analogue of cyclic AMP.6. A significant potentiation of the maximum isoprenaline response by threshold inotropic concentrations was observed with SK&F 94120 (P<0.05), but not with IBMX or SK&F 94836, in myocytes isolated from noradrenaline-treated guinea-pig hearts. This potentiation, however, did not completely restore the response to levels seen in control myocytes.7. The extent of potentiation of the maximum isoprenaline response by maximum inotropic concentrations of either IBMX or CPT cyclic AMP, was no greater than that by threshold concentrations of IBMX, in myocytes isolated from noradrenaline-treated guinea-pig hearts.8. In cardiac myocytes isolated from the explanted hearts of 16 patients with heart failure, threshold concentrations of IBMX and SK&F 94120 decreased the isoprenaline EC50 by a factor of four and six,respectively, but potentiation of the maximum isoprenaline response occurred only with SK&F 94120.The attenuated isoprenaline response was increased from 60.3 +/- 4.5% to 74.3 +/- 4.2% as a % maximum calcium (P<0.05, n = 6), but remained substantially lower than the 116 +/- 7% (P<0.001, n = 6) seen in myocytes isolated from non-failing hearts.9. We conclude that the reduced maximum contraction amplitude with isoprenaline in cardiac myocytes from either patients in end-stage failure, or noradrenaline-treated guinea-pigs, is partly but not solely due to insufficient cyclic AMP levels, since inhibition of cyclic AMP degradation does not result incomplete reversal of the beta-adrenoceptor desensitization.