Potential treatment of cardiac hypertrophy and heart failure by inhibiting the sarcolemmal binding of phospholipase Cbeta1b.

Heart failure, the common end-point of many cardiac diseases, is a major contributor to mortality and morbidity and contributes considerably to health care costs. Current treatment regimens include beta-adrenergic antagonists, angiotensin converting enzyme inhibitors, and inotropic agents are used by some patients. Studies in experimental animals have demonstrated that inhibition of signaling pathways downstream of the heterotrimeric G protein Gq reduce ventricular hypertrophy and protects from the development of heart failure. However, targets identified, to date, have been limited by a lack of tissue specificity. In cardiomyocytes, Gq activates only one splice variant of one subtype of phospholipase Cbeta, specifically phospholipase Cbeta1b (PLCbeta1b) and PLCbeta1b is responsible for Gq mediated hypertrophic and apoptotic responses. PLCbeta1b targets to the sarcolemma via its unique C-terminal sequence and its activation can be inhibited by expressing the C-terminal sequence to compete for sarcolemmal binding. Inhibition of PLCbeta1b by the C-terminal peptide reduces hypertrophic responses in cardiomyocytes. We present the evidence that inhibition of the sarcolemmal association of PLCbeta1b provides a cardiac-specific target for the development of drugs to reduce pathological cardiac hypertrophy and thereby to reduce the burden of heart failure.

Reperfusion-induced Ins(1,4,5)P(3) generation and arrhythmogenesis require activation of the Na(+)/Ca(2+) exchanger.

Reperfusion of globally ischemic rat hearts causes rapid generation of inositol(1,4,5) trisphosphate [Ins(1,4,5)P(3)] and the development of arrhythmias, following stimulation of alpha(1)-adrenergic receptors by norepinephrine released from the cardiac sympathetic nerves. The heightened inositol phosphate response in reperfusion depends on the activation of the Na(+)/H(+) exchanger, which might reflect a central role for increased Ca(2+)following reverse mode activation of the Na(+)/Ca(2+) exchanger (NCX). Isolated, perfused rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and the content of Ins(1,4,5)P(3) measured by mass analysis or by anion-exchange high performance liquid chromatography (HPLC) following [(3)H]inositol labeling. Reperfusion caused generation of Ins(1,4,5)P(3) (1266+/-401 to 3387+/-256 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01) and the development of arrhythmias. Inhibition of NCX either by reperfusion at low Ca(2+) (1133+/-173 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01 relative to reperfusion control) or by adding 10 microm KB-R7943, an inhibitor of reverse mode Na(+)/Ca(2+) exchange, prevented the Ins(1,4,5)P(3) response (1151+/-243 cpm/g tissue, mean+/-s.e.m., n=6, P<0.01 relative to reperfusion control) and the development of ventricular fibrillation. Lower concentrations of KB-R7943 were less effective. Reverse mode activation of NCX is therefore required for the enhanced Ins(1,4,5)P(3) response in early reperfusion, and inhibitors of this transporter may be useful in the prevention of arrhythmias under such conditions.

Evidence for selective coupling of alpha 1-adrenergic receptors to phospholipase C-beta 1 in rat neonatal cardiomyocytes.

Activation of phospholipase C (PLC) in neonatal rat cardiomyocytes (NCM) generates primarily inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) in response to rises in intracellular Ca(2+), or inositol 1,4-bisphosphate (Ins(1,4)P(2)) in response to norepinephrine (NE) (Matkovich, S. J. and Woodcock, E. A. (2000) J. Biol. Chem. 275, 10845-10850). To examine the PLC subtype mediating the alpha(1)-adrenergic receptor response, PLC-beta(1) and PLC-beta(3) were overexpressed in NCM using adenoviral infection (Ad-PLC-beta(1) NCM and Ad-PLC-beta(3) NCM, respectively) and PLC responses assessed from [(3)H]inositol phosphate (InsP) generation in the presence of 10 mm LiCl. The [(3)H]InsP response to NE (100 microm) was enhanced in Ad-PLC-beta(1) NCM relative to cells infected with blank virus (Ad-MX NCM), but was reduced in Ad-PLC-beta(3) NCM. In contrast, the [(3)H]InsP response to ATP (100 microm) was not elevated in Ad-PLC-beta(1) NCM, and was enhanced rather than diminished in Ad-PLC-beta(3) NCM, showing that effects of the two PLC-beta isoforms were specific for particular receptor types. PLC-delta(1) overexpression selectively reduced NE-induced [(3)H]InsP responses, without affecting the ATP stimulation. The reduced NE response was associated with a selective loss of PLC-beta(1) expression in Ad-PLC-delta(1) NCM. alpha(1)-Adrenergic receptor activation caused phosphorylation of PLC-beta(1) but not PLC-beta(3), whereas stimulation by ATP induced phosphorylation of PLC-beta(3) but not PLC-beta(1.) Taken together, these studies provide evidence that NE-stimulated InsP generation in NCM is primarily mediated by PLC-beta(1), despite the presence of both PLC-beta(1) and PLC-beta(3) isoforms.

Targeted alpha(1A)-adrenergic receptor overexpression induces enhanced cardiac contractility but not hypertrophy.

Activation of the alpha(1A)-adrenergic receptor (alpha(1A)-AR)/Gq pathway has been implicated as a critical trigger for the development of cardiac hypertrophy. However, direct evidence from in vivo studies is still lacking. To address this issue, transgenic mice with cardiac-targeted overexpression of the alpha(1A)-AR (4- to 170-fold) were generated, using the rodent alpha-myosin heavy chain promoter. Heterozygous animals displayed marked enhancement of cardiac contractility, evident from increases in dP/dt(max) (80%, P<0.0001), dP/dt(max)/LVP(inst) (76%, P<0.001), dP/dt(max):dP/dt(min) (104%, P<0.0001), and fractional shortening (33%, P<0.05). Moreover, changes in the dP/dt(max)-end-diastolic volume relationship provided load-independent evidence of a primary increase in contractility. Blood pressure and heart rate were largely unchanged, and there was a small increase in (-)norepinephrine-stimulated, but not basal, phospholipase C activity. Increased contractility was directly related to the level of receptor overexpression and could be completely reversed by acute alpha(1A)- but not beta-AR blockade. Despite the robust changes in contractility, transgenic animals displayed no morphological, histological, or echocardiographic evidence of left ventricular hypertrophy. In addition, apart from an increase in atrial natriuretic factor mRNA, expression of other hypertrophy-associated genes was unchanged. To our knowledge, these data provide the first in vivo evidence for an inotropic action of the alpha(1A)-AR.

Age-dependent cardiomyopathy and heart failure phenotype in mice overexpressing beta(2)-adrenergic receptors in the heart.

OBJECTIVE: To explore long-term cardiac phenotype in transgenic (TG) mice with 300-fold overexpression of beta(2)-adrenergic receptors (AR). METHODS: Echocardiography was performed serially on a cohort of wild-type and TG mice (n=26 each) between 4 and 15 months of age. Survival was monitored and autopsy and histological examinations were performed. RESULTS: Heart rate was higher in TG than in wild-type mice throughout the study period. The left ventricular dimensions and fractional shortening were similar between TG and wild-type groups during 4-6 months. Starting at 9 months, however, TG mice showed progressive reduction in fractional shortening and systolic wall thickening, and increase in left ventricular dimensions and left ventricular mass, indicating onset of heart failure, left ventricular hypertrophy and remodeling. Abnormal waveforms in the electrocardiogram and episodes of ventricular ectopic beats were also observed in TG mice. Death of TG mice started at 8.5 months, and the cumulative mortality was 81% by 15 months (P<0.0001 vs. 4% in wild-type mice). The majority of deaths were due to severe heart failure, indicated by cardiac dilatation, lung congestion, pleural effusion and atrial thrombus. Left ventricular sections showed widespread interstitial fibrosis, loss of myocytes and myocyte hypertrophy in TG mice. CONCLUSIONS: A high level of beta(2)AR overexpression results in cardiomyopathy and heart failure. The onset was slower and the expression levels of receptors required are much higher than previously described for the beta(1)AR overexpression.

Activation of the Na(+)/H(+) exchanger is required for reperfusion-induced Ins(1,4,5)P(3) generation.

Post-ischemic reperfusion causes a change in inositol phosphate responses to norepinephrine from primary generation of inositol(1,4) bis phosphate (Ins(1,4)P(2)) to generation of inositol(1,4,5) tris phosphate (Ins(1,4,5)P(3)) that is required for the initiation of reperfusion arrhythmias. The current study was undertaken to investigate the role of Na(+)/H(+)exchange in facilitating this transient change in inositol phosphate response. Rat hearts were subjected to 20 min ischemia followed by 2 min reperfusion and Ins(1, 4,5)P(3)content was measured by mass analysis or by anion-exchange HPLC following [(3)H]inositol labeling. Reperfusion caused generation of [(3)H]Ins(1,4,5)P(3)(1732+/-398 to 3103+/-214, cpm/g tissue, mean+/-S.E.M., n=5, P<0.01) and the development of arrhythmias. Inhibition of Na(+)/H(+)exchange, by reperfusing at pH 6.3 or by pretreating with HOE-694 (10 n M-3 microM) or HOE-642 (3 microM) prevented the [(3)H]Ins(1,4,5)P(3)generation, without causing any suppression of norepinephrine release. Increases in Ins(1,4,5)P(3)mass were similarly reduced by inhibition of Na(+)/H(+)exchange. Thus, activation of Na(+)/H(+)exchange is required for the enhanced Ins(1,4,5)P(3)response observed under reperfusion conditions, and prevention of Ins(1,4,5)P(3)generation may be an important contributor to the anti-arrhythmic actions of inhibitors of Na(+)/H(+)exchange.

Preserved ventricular contractility in infarcted mouse heart overexpressing beta(2)-adrenergic receptors.

Effects of cardiac specific overexpression of beta(2)-adrenergic receptors (beta(2)-AR) on the development of heart failure (HF) were studied in wild-type (WT) and transgenic (TG) mice following myocardial infarction (MI) by coronary artery occlusion. Animals were studied by echocardiography at weeks 7 to 8 and by catheterization at week 9 after surgery. Post-infarct mortality, due to HF or cardiac rupture, was not different among WT mice, and there was no difference in infarct size (IS). Compared with the sham-operated group (all P < 0.01), WT mice with moderate (<36%) and large (>36%) IS developed lung congestion, cardiac hypertrophy, left ventricular (LV) dilatation, elevated LV end-diastolic pressure (LVEDP), and suppressed maximal rate of increase of LV pressure (LV dP/dt(max)) and fractional shortening (FS). Whereas changes in organ weights and echo parameters were similar to those in infarcted WT groups, TG mice had significantly higher levels of LV contractility in both moderate (dP/dt(max) 4,862 +/- 133 vs. 3,694 +/- 191 mmHg/s) and large IS groups (dP/dt(max) 4,556 +/- 252 vs. 3,145 +/- 312 mmHg/s, both P < 0.01). Incidence of pleural effusion (36% vs. 85%, P < 0.05) and LVEDP levels (6 +/- 0.3 vs. 9 +/- 0.8 mmHg, P < 0.05) were also lower in TG than in WT mice with large IS. Thus beta(2)-AR overexpression preserved LV contractility following MI without adverse consequence.

Inositol 1,4,5-trisphosphate and reperfusion arrhythmias.

1. The present review focuses on the role of the Ca2+-releasing second messenger inositol 1,4,5-trisphosphate (IP3) in initiating arrhythmias during early reperfusion following a period of myocardial ischaemia. 2. Evidence for an arrhythmogenic action of IP3 was provided by studies showing a correlation between the extent of the increase in IP3 and the incidence of arrhythmias in early reperfusion. In addition, phospholipase C inhibitors selective for thrombin receptor stimulation were anti-arrhythmic only when arrhythmias were thrombin initiated. 3. Mechanisms by which IP3 could initiate arrhythmias are discussed, with particular emphasis on the role of slow and unscheduled Ca2+ release. 4. The reperfusion-induced IP3 and arrhythmogenic responses can be initiated through either alpha1-adrenoceptors or thrombin receptors, but endothelin receptor stimulation was ineffective. Further studies have provided evidence that the noradrenaline-mediated response was mediated by alpha1A-receptors, while the alpha1B-adrenoceptor subtype appeared to be protective. 5. Reperfusion-induced IP3 responses could be inhibited by procedures known to reduce the incidence of arrhythmias under these conditions, including preconditioning, inhibiting Na+/H+ exchange or by dietary supplementation with n-3 polyunsaturated fatty acids. 6. Inositol 1,4,5-trisphosphate generation in cardiomyocytes can be facilitated by raising intracellular Ca2+ and it seems likely that the rise in Ca2+ in ischaemia and reperfusion is responsible for the generation of IP3, which will, in turn, further exacerbate Ca2+ overload.

Adverse effects of constitutively active alpha(1B)-adrenergic receptors after pressure overload in mouse hearts.

Cardiac hypertrophy and function were studied 6 wk after constriction of the thoracic aorta (TAC) in transgenic (TG) mice expressing constitutively active mutant alpha(1B)-adrenergic receptors (ARs) in the heart. Hearts from sham-operated TG animals and nontransgenic littermates (WT) were similar in size, but hearts from TAC/TG mice were larger than those from TAC/WT mice, and atrial natriuretic peptide mRNA expression was also higher. Lung weight was markedly increased in TAC/TG animals, and the incidence of left atrial thrombus formation was significantly higher. Ventricular contractility in anesthetized animals, although it was increased in TAC/WT hearts, was unchanged in TAC/TG hearts, implying cardiac decompensation and progression to failure in TG mice. There was no increase in alpha(1A)-AR mRNA expression in TAC/WT hearts, and expression was significantly reduced in TAC/TG hearts. These findings show that cardiac expression of constitutively actively mutant alpha(1B)-ARs is detrimental in terms of hypertrophy and cardiac function after pressure overload and that increased alpha(1A)-AR mRNA expression is not a feature of the hypertrophic response in this murine model.

Effects of multisensory environmental stimulation on contextual conditioning in the developing rat.

The effect of increased exposure to multisensory stimulation during development on conditioned freezing to contextual cues in preweanling Sprague-Dawley rats was examined. Rats given increased environmental stimulation exhibited long-term contextual conditioning at a younger age than rats that did not receive such stimulation when there was either low or moderate levels of conditioning (Experiments 1 and 2). These differences in contextual conditioning were not a result of the stimulated rats reacting differently to shock (Experiment 4) or merely freezing more than the nonstimulated rats in all situations (Experiment 3). The role of the glucocorticoid system in the enhanced contextual learning of stimulated preweanling rats and the advantages of the contextual conditioning procedure for studying the effects of environmental stimulation are discussed.

Beta(2)-adrenergic receptor overexpression driven by alpha-MHC promoter is downregulated in hypertrophied and failing myocardium.

OBJECTIVE: The alpha-myosin heavy chain (alpha-MHC) promoter is frequently used to direct cardiac specific transgene expression. We studied whether transgene expression controlled by this promoter was altered under conditions of cardiac hypertrophy and failure. METHODS: Transgenic (TG) mice overexpressing human beta(2)-adrenergic receptors (beta(2)AR) and wild type (WT) controls were subjected to thoracic aortic constriction (TAC) or sham operation and studied at 1, 3 and 8 weeks after surgery. RESULTS: Sham operated TG mice had higher heart rates and left ventricular (LV) contractility than WT (all P<0.01), demonstrating enhanced betaAR activation. TAC at 1, 3 and 8 weeks produced progressive LV hypertrophy which was similar between WT and TG mice. Evidence of heart failure was more marked in TG mice with a greater increase in weights of the right ventricle and lungs and a higher prevalence of atrial thrombus (P<0.05 in each case). In hypertrophied TG hearts, endogenous alpha-MHC mRNA transcripts in LV were maintained at 1 and 3 weeks, but were reduced by approximately 40% relative to the sham-operated group at 8 weeks after TAC. Transgene expression, measured as human beta(2)AR mRNA, was reduced by 45% at 1 and 3 weeks and by 70% at 8 weeks after TAC. beta(2)AR binding sites were reduced by 35, 47 and 65%, respectively, at 1, 3 and 8 weeks. CONCLUSION: Cardiac hypertrophy and failure cause downregulation of the endogenous alpha-MHC as well as cardiac specific overexpression of the transgene directed by an alpha-MHC promoter.

Enhanced negative chronotropy by inhibitory receptors in transgenic heart overexpressing beta(2)-adrenoceptors.

Transgenic (TG) mice overexpressing beta(2)-adrenoceptors (AR) in the heart have enhanced beta-adrenergic activity. Since the degree of beta-adrenergic activation influences the negative chronotropic control of heart rate (HR), we studied the inhibitory effect of cholinergic and purinergic stimulation on HR in TG and wild-type (WT) control mice. Bradycardia in response to vagal nerve stimulation and administration of acetylcholine or adenosine was studied in anesthetised animals and perfused hearts. Basal HR was significantly higher in TG than WT mice (P<0.01). Electrical stimulation of vagal nerves (1-32 Hz) induced a Hz-dependent reduction in HR and the response was more pronounced in TG than WT groups (P<0.01). In perfused hearts, HR reduction by acetylcholine (ACh) was more pronounced with EC(50) 110-fold lower in TG than WT hearts. Adenosine-induced bradycardia, which was abolished by a P(1) antagonist, was more pronounced in TG hearts. After pre-treatment with pertussis toxin (PT, 100 microg/kg), bradycardia by vagal nerve stimulation or ACh remained unchanged in WT, but markedly inhibited in TG hearts (both P<0.01). Conversely, inhibiting guanylyl cyclase with LY83583 (30 microM) or nitric oxide synthase with L-NMMA (100 microM) attenuated HR reduction by vagal nerve stimulation in WT but not in TG hearts. Immunobloting assay showed similar G(ialpha2) abundance in TG and WT hearts. Thus, cardiac overexpression of beta(2)AR with high beta-adrenergic activity leads to hypersensitivity of inhibitory receptors controlling HR due to increase in activity of PT-sensitive G-proteins.

Effects of environmental enrichment on rate of contextual processing and discriminative ability in adult rats.

The effect of environmental enrichment on conditioned freezing to contextual cues in adult Sprague-Dawley rats was examined. The freezing of both enriched-and standard-reared rats increased with the time spent in the chamber prior to shock. Both groups of rats showed equally low levels of contextual conditioning following a preshock period of 4 s and equally high levels following a 120-s preshock period. However, following a preshock period of 16 s enriched rats displayed more contextual conditioning than standard rats. That is, enriched rats appeared to process contextual information faster than their standard-reared counterparts. Enriched- reared rats also showed a greater ability to discriminate between the conditioning context and a similar but distinctive context. Hence, in addition to forming a representation of the context in memory more rapidly than standard-reared rats, enriched-reared rats also appear to form a more complex representation.

beta(2)-adrenergic receptor overexpression exacerbates development of heart failure after aortic stenosis.

BACKGROUND: Beta-adrenergic signaling is downregulated in the failing heart, and the significance of such change remains unclear. METHODS AND RESULTS: To address the role of beta-adrenergic dysfunction in heart failure (HF), aortic stenosis (AS) was induced in wild-type (WT) and transgenic (TG) mice with cardiac targeted overexpression of beta(2)-adrenergic receptors (ARs), and animals were studied 9 weeks later. The extents of increase in systolic arterial pressure (P<0.01 versus controls), left ventricular (LV) hypertrophy (TG, 94+/-6 to 175+/-7 mg; WT, 110+/-6 to 168+/-10 mg; both P<0.01), and expression of ANP mRNA were similar between TG and WT mice with AS. TG mice had higher incidences of premature death and critical illness due to heart failure (75% versus 23%), pleural effusion (81% versus 45%), and left atrial thrombosis (81% versus 36%, all P<0.05). A more extensive focal fibrosis was found in the hypertrophied LV of TG mice (P<0.05). These findings indicate a more severe LV dysfunction in TG mice. In sham-operated mice, LV dP/dt(max) and heart rate were markedly higher in TG than WT mice (both P<0.01). dP/dt(max) was lower in both AS groups than in sham-operated controls, and this tended to be more pronounced in TG than WT mice (-32+/-5% versus -16+/-6%, P=0.059), although dP/dt(max) remained higher in TG than WT groups (P<0.05). CONCLUSIONS: Elevated cardiac beta-adrenergic activity by beta(2)-AR overexpression leads to functional deterioration after pressure overload.

Ca(2+)-activated but not G protein-mediated inositol phosphate responses in rat neonatal cardiomyocytes involve inositol 1,4, 5-trisphosphate generation.

Inositol phosphate (InsP) responses to receptor activation are assumed to involve phospholipase C cleavage of phosphatidylinositol 4,5-bisphosphate to generate Ins(1,4,5)P(3). However, in [(3)H]inositol-labeled rat neonatal cardiomyocytes (NCM) both initial and sustained [(3)H]InsP responses to alpha(1)-adrenergic receptor stimulation with norepinephrine (100 microM) were insensitive to the phosphatidylinositol 4,5-bisphosphate-binding agent neomycin (5 mM). Introduction of 300 microM unlabeled Ins(1,4, 5)P(3) into guanosine 5'-3-O-(thio)triphosphate (GTPgammaS)-stimulated, permeabilized [(3)H]inositol-labeled NCM increased [(3)H]Ins(1,4,5)P(3) slightly but did not significantly reduce levels of its metabolites [(3)H]Ins(1,4)P(2) and [(3)H]Ins(4)P, suggesting that these [(3)H]InsPs are not formed principally from [(3)H]Ins(1,4,5)P(3). In contrast, the calcium ionophore A23187 (10 microM) provoked [(3)H]InsP responses in intact NCM which were sensitive to neomycin, and elevation of free calcium in permeabilized NCM led to [(3)H]InsP responses characterized by marked increases in [(3)H]Ins(1,4,5)P(3) (2.9 +/- 0.2% of total [(3)H]InsPs after 20 min of high Ca(2+) treatment in comparison to 0. 21 +/- 0.05% of total [(3)H]InsPs accumulated after 20 min of GTPgammaS stimulation). These data provide evidence that Ins(1,4, 5)P(3) generation is not a major contributor to G protein-coupled InsP responses in NCM, but that substantial Ins(1,4,5)P(3) generation occurs under conditions of Ca(2+) overload. Thus in NCM, Ca(2+)-induced Ins(1,4,5)P(3) generation has the potential to worsen Ca(2+) overload and thereby aggravate Ca(2+)-induced electrophysiological perturbations.

Inhibition of inositol(1,4,5)Trisphosphate generation by endothelin-1 during postischemic reperfusion: A novel antiarrhythmic mechanism.

BACKGROUND: Reperfusion of ischemic rat hearts in the presence of thrombin or norepinephrine but not endothelin-1 causes the generation of inositol 1,4,5-trisphosphate (Ins 1,4,5P3) and arrhythmias. The present study investigates the effect of endothelin-1 on these responses. METHODS AND RESULTS: Ins 1,4,5P3 generation was quantified by use of [3H] labeling and high-performance liquid chromatography as well as by mass analysis. Twenty minutes of global ischemia followed by 2 minutes of reperfusion increased [3H]Ins 1,4,5P3 from 2828+/-265 to 5033+/-650 cpm/g tissue in the presence of thrombin 2.5 IU/mL and to 4561+/-286 cpm/g tissue in response to release of norepinephrine (n=4, P<0.01) in both cases. Reperfusion in the presence of endothelin-1 alone caused no change in Ins 1,4,5P3 (2762+/-240 cpm/g tissue), but when added together with thrombin or norepinephrine, endothelin-1 reduced the Ins 1,4,5P3 responses to 2313+/-197 and 1764+/-168 cpm/g tissue, respectively (n=4, P<0.01 in both cases). Similar inhibitory interactions between endothelin-1 10 nmol/L and thrombin 2.5 IU/mL were observed under normoxic conditions in nonperfused ventricle, eliminating the possibility that excessive vasoconstriction was responsible. In parallel studies, endothelin-1 suppressed the development of reperfusion arrhythmias initiated by either thrombin (ventricular fibrillation, 75% to 39%, n=16 to 18) or norepinephrine (83% to 8%, n=12 to 22) (P<0.01 in both cases). CONCLUSIONS: Inhibition of Ins 1,4,5P3 generation during myocardial reperfusion by endothelin-1 represents a novel antiarrhythmic mechanism.

Reduced reperfusion-induced Ins(1,4,5)P3 generation and arrhythmias in hearts expressing constitutively active alpha1B-adrenergic receptors.

Reperfusion of globally ischemic rat hearts causes the generation of inositol(1,4,5)trisphosphate [Ins(1,4,5)P3] and the initiation of arrhythmias. These responses are mediated by alpha1-adrenergic receptors (ARs), but the subtype of receptor involved has not been identified. Under normoxic conditions, hearts from transgenic animals expressing constitutively active alpha1B-ARs in heart (alpha1B-constitutively active mutant [CAM]) showed higher [3H] inositol phosphate responses to norepinephrine (2.3-fold) than hearts from nontransgenic animals (alpha1B-WT) (1.6-fold). alpha1B-WT hearts responded to 2 minutes of reperfusion after 20 minutes of global ischemia by generation of Ins(1,4,5)P3 (5301+/-1310 to 11 413+/-1597 CPM/g tissue; mean+/-SEM; n=6; P<0.01 in [3H] labeling studies and 3.8+/-0.2 to 6.3+/-0.6 nmol/g by mass analysis, n=6; P<0.05). In contrast to findings in normoxia, hearts from alpha1B-CAM animals showed no Ins(1,4,5)P3 response in early reperfusion. In parallel studies, alpha1B-WT hearts developed ventricular tachycardia and ventricular premature beats (VPB) during 5 minutes of reperfusion after 20 minutes of ischemia. The incidence of these arrhythmias was reduced in the alpha1B-CAM hearts (95% to 62% for VPB and 47% to 12% for ventricular tachycardia; both P<0.05). The resistance of the alpha1B-CAM hearts was not due to alpha1B-AR-mediated preconditioning, as the Ins(1,4,5)P3 response to thrombin receptor activation during reperfusion was not different between the 2 groups. To investigate the possibility of reduced alpha1A-receptor activity in the alpha1B-CAM hearts, expression of the mRNA for alpha1A- and alpha1B-receptors was measured. alpha1B-WT hearts contained mRNA for both receptor subtypes, but the levels of alpha1B-receptor mRNA were 5-fold higher than alpha1A-receptor mRNA. alpha1B-CAM hearts contained very high levels of alpha1B-receptor mRNA (26-fold increase), but the expression of mRNA for the alpha1A-receptors (0.141+/-0.035 amol/ microg RNA; mean+/-SEM; n=6) was reduced by 50% relative to alpha1B-WT controls (0.276+/-0.046 amol/ microg RNA; n=6; P<0.01). The reduction in arrhythmogenic and Ins(1,4,5)P3 responses in alpha1B-CAM hearts provides evidence that these response are not mediated by alpha1B-receptors.

Protection of neuronal uptake-1 inhibitors in ischemic and anoxic hearts by norepinephrine-dependent and -independent mechanisms.

Cardiac ischemia and anoxia induce massive norepinephrine (NE) release, which is mediated by a reverse operation of uptake-1 and can be suppressed by uptake-1 inhibitors. We studied effects of uptake-1 inhibitors on incidence of ventricular fibrillation (VF%) and myocardial contracture in perfused rat hearts under ischemic or anoxic conditions. NE release occurred in hearts during ischemia or anoxia and was largely inhibited by desipramine, imipramine, and cocaine. The generation of inositol 1,4,5-trisphosphate (InsP3) during reperfusion also was abolished by desipramine. During anoxia/reoxygenation, VF (93 and 71%, respectively) and myocardial contracture occurred and were significantly inhibited by desipramine and by NE depletion. Regional ischemia and reperfusion induced high VF% (86 and 100%, respectively), which was reduced or abolished by desipramine and imipramine at 0.03 and 0.3 microM. During the ischemic phase, cocaine was similarly antiarrhythmic, as was a combination of timolol and prazosin, but NE depletion was not. In NE-depleted hearts, cocaine or the combination of timolol and prazosin showed limited effect on VF%, whereas both desipramine and imipramine abolished VF. In anesthetized rats in vivo, ischemic VF% was reduced by desipramine (30 vs. 92%; p < 0.01). In conclusion, uptake-1 inhibitors protect hearts against ischemia/reperfusion- and anoxia/reoxygenation-induced arrhythmias, partly because of the inhibition of locally mediated NE release. Other actions of desipramine and imipramine may contribute to the overall efficacy.

Selective activation of alpha1A-adrenergic receptors in neonatal cardiac myocytes is sufficient to cause hypertrophy and differential regulation of alpha1-adrenergic receptor subtype mRNAs.

Prolonged stimulation of cardiac alpha1-adrenergic receptors causes myocyte hypertrophy, although the receptor subtypes involved remain controversial. We have used a potent and selective alpha1A agonist, A-61603, to test whether activation of the alpha1A-adrenergic receptor subtype is sufficient to mediate the morphological, biochemical and molecular alterations associated with cardiomyocyte hypertrophy. In neonatal rat cardiomyocyte cultures, 48 h incubation with 50 nm A-61603 led to a marked increase in myocardial cell size that was associated with a significant elevation in the rate of protein synthesis. The increased rate of incorporation of radiolabelled amino acids into protein stimulated by A-61603 was totally abolished by the selective alpha1A antagonist KMD-3213. A-61603 increased ANF secretion three-fold, and ANF mRNA 12-fold above control levels in cardiomyocyte cultures. RNase protection analysis demonstrated a A-61603-mediated two to three-fold increase in alpha1A-adrenergic receptor mRNA with a concomitant 50% decrease in alpha1B mRNA levels by 48 h. Identical responses of differential regulation of alpha1A- and alpha1B-adrenergic receptor mRNA were observed with phenylephrine. Both the stimulation of alpha1A- and repression alpha1B-adrenergic receptor mRNA caused by A-61603 could be abolished by 10-20 nm KMD-3213. The present data provide evidence that selective activation of alpha 1A-adrenergic receptors on cardiomyocytes is sufficient to mediate the phenotypic changes associated with cardiac hypertrophy. In addition, the differential regulation of alpha1A and alpha1B mRNA in response to selective alpha1A-adrenergic receptor stimulation suggests that cross-talk between receptor subtypes may be involved in regulating receptor populations during chronic agonist exposure.