Role of Cardiac A2A Receptors Under Normal and Pathophysiological Conditions.

This review presents an overview of cardiac A2A-adenosine receptors The localization of A2A-AR in the various cell types that encompass the heart and the role they play in force regulation in various mammalian species are depicted. The putative signal transduction systems of A2A-AR in cells in the living heart, as well as the known interactions of A2A-AR with membrane-bound receptors, will be addressed. The possible role that the receptors play in some relevant cardiac pathologies, such as persistent or transient ischemia, hypoxia, sepsis, hypertension, cardiac hypertrophy, and arrhythmias, will be reviewed. Moreover, the cardiac utility of A2A-AR as therapeutic targets for agonistic and antagonistic drugs will be discussed. Gaps in our knowledge about the cardiac function of A2A-AR and future research needs will be identified and formulated.

Genetic disruption of G proteins, G(i2)alpha or G(o)alpha, does not abolish inotropic and chronotropic effects of stimulating muscarinic cholinoceptors in atrium.

BACKGROUND AND PURPOSE: Classically, stimulation of muscarinic cholinoceptors exerts negative inotropic and chronotropic effects in the atrium of mammalian hearts. These effects are crucial to the vagal regulation of the heart beat. This effect is assumed to be mediated via GTP binding (G) proteins, because they can be abolished by Pertussis toxin. However, it is unknown which G proteins are involved. EXPERIMENTAL APPROACH: We studied contractility in isolated left or right atrium from genetically manipulated mice with deletion of one of two G proteins, either of the alpha subunit of G(i2) protein (G(i2)alpha) or of the alpha subunit of G(o) protein (G(o)alpha). Preparations were stimulated with carbachol alone or after pretreatment with the beta-adrenoceptor agonist isoprenaline. For comparison, the effects of carbachol on L-type Ca(2+)-channels in isolated ventricular cardiomyocytes were studied. KEY RESULTS: The negative inotropic and chronotropic effects of carbachol alone or in the presence of isoprenaline were identical in atria from knockout or wild-type mice. However, the effect of carbachol on isoprenaline-activated L-type Ca(2+)-channel in isolated ventricular cardiomyocytes was greatly attenuated in both types of knockout mice studied. CONCLUSIONS AND IMPLICATIONS: These data imply that there is either redundancy of G proteins for signal transduction or that Pertussis toxin-sensitive proteins other than G(i2)alpha and G(o)alpha mediate the vagal stimulation in the atrium. Moreover, different G proteins mediate the effect of carbachol in ventricle compared with atrium.

Reduced serine-16 and threonine-17 phospholamban phosphorylation in stunning of conscious dogs: no evidence for any involvement of protein kinase A or protein phosphatases.

OBJECTIVE: Cardiac stunning is the consequence of a brief cardiac ischemia. The underlying mechanism is not completely understood. METHODS: Here we induced cardiac transient ischemia in conscious instrumented dogs by means of an occluder in the left anterior descending coronary artery (LAD). Contractile performance, monitored by ultrasound crystals, was reduced during and after ischemia in the LAD area. For control in the same animals cardiac performance was measured in the area of left circumflex coronary artery (Ramus circumflexus, RCx). In the RCx area, no decline in contractility was noted. Tissue was obtained from stunned LAD area and from control areas (RCx). RESULTS: Phospholamban phosphorylation on both serine-16 and threonine-17 was reduced in LAD areas compared to RCx areas. Reduced phosphorylation of PLB is known to inhibit cardiac contractility. While phosphorylation of PLB was reduced, the activity of the appropriate protein phosphatases and protein kinases was not different between tissue obtained from LAD or RCx areas. CONCLUSION: Reduced formation of cAMP might underlie the contractile dysfunction in myocardial stunning.

L-type Ca2+ current downregulation in chronic human atrial fibrillation is associated with increased activity of protein phosphatases.

BACKGROUND: Although downregulation of L-type Ca2+ current (I(Ca,L)) in chronic atrial fibrillation (AF) is an important determinant of electrical remodeling, the molecular mechanisms are not fully understood. Here, we tested whether reduced I(Ca,L) in AF is associated with alterations in phosphorylation-dependent channel regulation. METHODS AND RESULTS: We used whole-cell voltage-clamp technique and biochemical assays to study regulation and expression of I(Ca,L) in myocytes and atrial tissue from 148 patients with sinus rhythm (SR) and chronic AF. Basal I(Ca,L) at +10 mV was smaller in AF than in SR (-3.8+/-0.3 pA/pF, n=138/37 [myocytes/patients] and -7.6+/-0.4 pA/pF, n=276/86, respectively; P<0.001), though protein levels of the pore-forming alpha1c and regulatory beta2a channel subunits were not different. In both groups, norepinephrine (0.01 to 10 micromol/L) increased I(Ca,L) with a similar maximum effect and comparable potency. Selective blockers of kinases revealed that basal I(Ca,L) was enhanced by Ca2+/calmodulin-dependent protein kinase II in SR but not in AF. Norepinephrine-activated I(Ca,L) was larger with protein kinase C block in SR only, suggesting decreased channel phosphorylation in AF. The type 1 and type 2A phosphatase inhibitor okadaic acid increased basal I(Ca,L) more effectively in AF than in SR, which was compatible with increased type 2A phosphatase but not type 1 phosphatase protein expression and higher phosphatase activity in AF. CONCLUSIONS: In AF, increased protein phosphatase activity contributes to impaired basal I(Ca,L). We propose that protein phosphatases may be potential therapeutic targets for AF treatment.

Functional properties of the rat phosphatase 1alpha promoter.

The aim of this study was to investigate the functional properties of the promoter of the protein phosphatase 1alpha catalytic subunit. Luciferase plasmids with different fragments of the rat catalytic subunit of the protein phosphatase 1alpha promoter ranging from -3.7 kbp to -59 bp were transiently transfected into cells by the calcium-phosphate precipitation method. The promoter activity was determined in the absence and presence of inotropic agents which influencing the cAMP-depending pathway. The basal transcriptional activity decreased at fragment -124 bp and shorter fragments. To identify regions of regulatory importance we investigated the cAMP-dependent influence on the transcriptional activity. Stimulation of the complete promoter region with forskolin (1-100 microM) for 6 h led to a concentration-dependent decrease of transcriptional activity. Moreover, regions shorter than 3.7 kbp were inhibited by forskolin (10 microM). Short time stimulation (10 min) with forskolin (10 microM) increased the transcriptional activity of only the 3.7 kbp fragment. The effects were antagonized by Rp-cAMPS, a specific antagonist of protein kinase A, indicating cAMP-dependent effects. The results provide evidence for cAMP-dependent regulation of the protein phosphatase 1alpha promotor.

Early impairment of calcium handling and altered expression of junctin in hearts of mice overexpressing the beta1-adrenergic receptor.

Chronic stimulation of cardiac beta1-adrenergic receptors contributes to disease progression and mortality in patients and animal models of heart failure. To search for the mechanism of adrenergic impairment of cardiac function in vivo, we studied transgenic mice with cardiac-specific overexpression of beta1-adrenergic receptors. Transgenic mice with cardiac overexpression of beta1-adrenergic receptors showed progressive left ventricular fibrosis starting at 4 months of age. Left ventricular catheterization revealed a modest enhancement of contractility and relaxation at 2 months of age, followed by progressive dysfunction in both parameters and ultimately cardiac failure. When the effects of endogenous catecholamines were blocked by the b-receptor antagonist propranolol, maximal rate of contractility (dp/dtmax) and maximal rate of relaxation (dp/dtmin) were significantly blunted in 2-month-old beta1-receptor transgenic mice. Isolated cardiomyocytes from these animals displayed markedly altered calcium transients with significant prolongation of the intracellular calcium transient compared with nontransgenic littermates. We determined the expression of sarcoplasmic reticulum proteins involved in calcium handling by RNase protection assay and by immunoblotting. Although the expression of calsequestrin, triadin, and phospholamban was not altered, we observed a progressive decrease in junctin abundance in beta1-receptor transgenic mice (Pbeta1-adrenergic receptors.

Enhanced protein phosphorylation in hypertensive hypertrophy.

OBJECTIVE: Chronic pressure overload in spontaneously hypertensive rats (SHR) is accompanied by heart hypertrophy and signs of heart failure. Since there is growing evidence for a possible pathophysiological role of altered protein phosphorylation in heart hypertrophy and failure, we studied here cardiac regulatory phosphoproteins and the kinases and phosphatases which regulate their phosphorylation state. METHODS: The experiments were performed in ventricles of SHR (12-13 weeks old) and age-matched normotensive Wistar-Kyoto rats (WKY). RESULTS: Basal as well as isoproterenol (Iso)-stimulated force of contraction (FOC) was markedly decreased in isolated electrically driven papillary muscles of SHR. Iso (3 micromol/l, 10 min) increased FOC by 0.91+/-0.20 mN in SHR and by 3.88+/-0.52 mN in WKY, respectively. Ca(2+)-uptake by sarcoplasmic reticulum (SR) at low ionized Ca(2+)-concentration was increased in homogenates from SHR. This was not due to altered expression of phospholamban (PLB), SR-Ca(2+)-ATPase and calsequestrin. However, PLB-phosphorylation at threonine-17 (PLB-PT-17) and the activity of Ca(2+)/calmodulin dependent protein kinase (Ca(2+)/Cam-PK) was increased in SHR. In addition, we found an enhanced protein kinase A (PKA)-dependent phosphorylation of the inhibitory subunit of troponin (TnI). In contrast, there was no difference in the activity or expression (protein- and mRNA-level) of protein phosphatases type 1 or type 2A between SHR and WKY. CONCLUSIONS: It is suggested that increased Ca(2+)/Cam-PK-activity with resulting increase of PLB-PT-17 enhanced SR-Ca(2+)-uptake in SHR and might contribute to the pathophysiological changes in cardiac hypertrophy of SHR.

Activation and inactivation of cAMP-response element-mediated gene transcription in cardiac myocytes.

OBJECTIVE: Chronic beta-adrenergic stimulation of the cAMP-dependent signalling pathway is implicated in functionally relevant expressional changes in congestive heart failure. We studied activation and inactivation of the cardiac gene transcription mediated by the cAMP-response element (CRE) and the CRE-binding protein (CREB) as an important mechanism of a cAMP-dependent gene regulation. METHODS: We investigated the transcriptional activation by forskolin, an activator of the adenylyl cyclase, in chick embryonic cardiomyocytes transfected with a CRE-controlled luciferase construct in comparison to the phosphorylation and expression of CREB determined on immunoblots. RESULTS: Forskolin (10 micromol/l; 8 h) increased CRE-mediated transcription and phosphorylation of CREB 13- and 1.5-fold, respectively. The phosphorylation was further elevated in combination with cantharidin, an inhibitor of type 1+2A protein phosphatases. The transcriptional response to forskolin was desensitized by pretreatment with forskolin (1 micromol/l; 24 h) while CREB phosphorylation was increased. In forskolin-pretreated cells, total CREB protein levels were decreased. Cantharidin did not restore the attenuated transcriptional response. CONCLUSIONS: In cardiomyocytes, there is an activation of the CRE-mediated gene transcription by forskolin that is attenuated after prolonged stimulation, and this attenuation is not dependent from a dephosphorylation of CREB. We suggest that attenuation of the CRE-mediated transcription through chronic stimulation of the cAMP-pathway, e.g. by elevated catecholamines, contributes to the altered expressional regulation in congestive heart failure.

Hemin, inducer of heme-oxygenase 1, improves functional recovery from myocardial stunning in conscious dogs.

OBJECTIVE: To examine the effects of pretreatment with hemin, an inducer of the potential antioxidative enzyme heme-oxygenase 1 (HO-1) or heat-shock protein 32, on myocardial stunning. DESIGN: Randomized animal study. SETTING: Animal laboratory of a university hospital. PARTICIPANTS: Chronically instrumented mongrel dogs (n = 44). INTERVENTIONS: Dogs underwent chronic instrumentation for measurement of hemodynamics and myocardial wall thickening fraction (WTF). Experiments with 12 dogs were performed on separate days in a crossover fashion: (1) 10 minutes of left anterior descending (LAD) coronary artery occlusion after application of hemin (9 mg/kg/d) for 1 week and (2) 10 minutes of LAD coronary artery occlusion without hemin pretreatment. In control experiments (n = 32), the reversible induction of HO-1, using gel electrophoresis and Western blotting, was determined. MEASUREMENTS AND MAIN RESULTS: WTF was measured as a baseline value before hemin administration and at predetermined time points until complete recovery from stunning. LAD artery occlusion caused a significant reduction in the WTF in the LAD-perfused area with and without hemin, without significant hemodynamic changes. At all time points, after 1 minute of reperfusion, the WTF as percentage of baseline values was significantly higher after hemin pretreatment (p < 0.05). Baseline WTF values were reached after 24 hours with and after >48 hours without hemin pretreatment (p < 0.05). CONCLUSION: Hemin pretreatment attenuates myocardial stunning in conscious dogs.

Effect of Helicobacter pylori eradication on cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) expression during gastric adaptation to aspirin (ASA) in humans.

Gastric adaptation to aspirin is impaired in Helicobacter pylori infection, but the mechanisms underlying this phenomenon are unclear. In this study, we compared gastric mucosal expression of iNOS and COX-2 during 14 days of aspirin ingestion in the same subjects before and 3 months after eradication of H. pylori. Compared to non-infected controls, mucosal expression of COX-2 and iNOS was enhanced before and 3 months after eradication of H. pylori. During aspirin ingestion, mucosal expression of COX-2 remained unchanged before eradication of H. pylori, but increased gradually after successful antimicrobial treatment. Independent of H. pylori status, expression of iNOS increased at the beginning of aspirin intake, but then returned to initial values. We conclude that COX-2 but not iNOS might be involved in gastric adaptation to aspirin in humans and that this mechanism appears to be impaired in H. pylori infection.

Heat shock protein 70 (HSP70) in gastric adaptation to aspirin in Helicobacter pylori infection.

We have recently shown that adaptation of gastric mucosa to aspirin (ASA) is disturbed in Helicobacter pylori (H. pylori)-infected human stomach, but can be restored by eradication of the bacterium. The aim of this study was 1) to evaluate the influence of H. pylori on expression of heat shock protein 70 (HSP70) during ASA ingestion in these subjects and in mice model and 2) to evaluate, whether altered HSP70 expression might be associated with different adaptation to ASA in H. pylori-positive and eradicated subjects. The gastric mucosal HSP 70 gene expression was determined by quantitative RT-PCR and Western blot and immunohistochemistry during 14 days of ASA ingestion (1 g bid) in the same 8 subjects before and 3 months after successful eradication of H. pylori. In addition, HSP70 mRNA and protein expression were examined in 30 mice without and with H. pylori infection and eradication. During 14 days of ASA treatment, human H. pylori-infected mucosa revealed a decrease of HSP70 expression, while after eradication a higher expression and further increase of HSP70 expression during ASA ingestion were observed. Mice inoculated with H. pylori also exhibited decreased gastric mucosal HSP70 mRNA expression that was restored after eradication therapy. Decreased basal and ASA-induced expression of HSP70 may partly be responsible for impaired gastric adaptation to ASA in H. pylori-positive subjects. We conclude that 1. The HSP70 gene and protein expression is reduced during infection with H. pylori in men and mice and that gastric adaptation to ASA in H. pylori eradicated subjects is accompanied by increased HSP70 expression; 2. It is reasonable to assume that decreased HSP70 expression might contribute to disturbed gastric adaptation in H. pylori infection in humans and 3. The expression of HSP70 plays an important role in the mechanism of gastric adaptation to ASA and that H. pylori infection interferes with this adaptation due to decrease of HSP70 expression in gastric mucosal cells.

Role of protein phosphatases in regulation of cardiac inotropy and relaxation.

We studied the effects of the protein phosphatase (PP) inhibitor cantharidin (Cant) on time parameters and force of contraction (FOC) in isometrically contracting electrically driven guinea pig papillary muscles. We correlated the mechanical parameters of contractility with phosphorylation of the inhibitory subunit of troponin (TnI-P) and with the site-specific phosphorylation of phospholamban (PLB) at serine-16 (PLB-Ser-16) and threonine-17 (PLB-Thr-17). Cant (after 30 min) started to increase FOC (112 +/- 4% of control, n = 10) and TnI-P and PLB-Thr-17 (120 +/- 5 and 128 +/- 7% of control) without any alteration of relaxation time. Cant (10 microM) started to increase PLB-Ser-16, but the relaxation was shortened at only 100 microM (from 140 +/- 9 to 116 +/- 12 ms, n = 9). Moreover, 100 microM Cant, 3 min after application, started to increase PLB-Thr-17, TnI-P, and FOC. Cant (100 microM) began to increase PLB-Ser-16 after 20 min. This was accompanied by shortening of relaxation time. Differences in protein kinase activation or different substrate specificities of PP may explain the difference in Cant-induced site-specific phosphorylation of PLB in isometrically contracting papillary muscles. Moreover, PLB-Thr-17 may be important for inotropy, whereas PLB-Ser-16 could be a major determinant of relaxation time.

Regional expression of protein phosphatase type 1 and 2A catalytic subunit isoforms in the human heart.

In mammalian species, including man, the duration of myocardial contraction is shorter in atria than ventricles. Total contraction time depends at least in part on phosphorylation and dephosphorylation of cardiac regulatory proteins. Dephosphorylation reactions are mediated by protein phosphatases. In the mammalian heart more than 90% of the protein phosphatase (PP) activity consists of PP1 and PP2A. Therefore, the aim of this study was to investigate which isoforms of PP1 and PP2A are present in human myocardium and whether their expression is regionally different. RT-PCR and Northern blotting revealed that all isoforms of PP1 and PP2A presently known are expressed in the human heart. Expression levels of PP1 alpha, delta, and gamma as well as 2A alpha were higher in right ventricles than in right atria. However, there was no such difference for PP2A beta. At the protein level PP1 alpha was unchanged, whereas PP2A was by 56% higher in right ventricles compared to atria. The phosphorylation state of TnI was lower in right ventricle than in right atrium. Thus, lower protein expression of PP2A in atrium could contribute to the faster relaxation by increasing the phosphorylation state of TnI. We conclude that expression of PP1 and PP2A isoforms is regionally regulated in the human heart.

Protein phosphatase activity is increased in a rat model of long-term beta-adrenergic stimulation.

We tested the hypothesis that altered phosphorylation of Ca2+ regulatory proteins contributes to contractile anomalies in cardiac hypertrophy. Cardiac hypertrophy was induced in rats by chronic s.c. administration of isoproterenol (Iso, 2.4 mg/kg/day) via osmotic minipumps. On day 2 of Iso treatment the expression of atrial natriuretic factor was increased, time of relaxation in isolated papillary muscles shortened and protein expression of phospholamban (PLB) and sarcoplasmic reticulum Ca2+-ATPase reduced. In addition, the phosphorylation state of PLB at serine-16 and threonine-17 was decreased from (arbitrary units) 2.3+/-0.3 to 1.1+/-0.2 and from 4.1+/-0.6 to 2.1+/-0.2, respectively. This was not accompanied by altered activity of PLB-phosphorylating protein kinases (protein kinase A or Ca2+/calmodulin-dependent protein kinase II), whereas the activity of types 1 and 2A protein phosphatases (PP1 and -2A respectively) was enhanced from 1.1+/-0.08 to 1.71+/-0.13 nmol/mg/min. Iso treatment did not alter the PP1/PP2A activity ratio and 1 nmol/l okadaic acid, a concentration which completely blocks the catalytic subunit of PP2A, inhibited about 40% of total PP activity in all groups studied. These data indicate that the activity of both PP1 and PP2A were increased. All effects of Iso treatment were abolished by co-administration of propranolol (29.7 mg/kg/day). It is concluded that dephosphorylation of PLB is due to enhanced activity of PP1 and PP2A. We suggest that chronic beta-adrenergic stimulation, which occurs in human cardiac hypertrophy and failure, can lead to increased activity of PPs. This may contribute to altered contractile responses in the hypertrophied heart.

Cantharidin enhances norepinephrine-induced vasoconstriction in an endothelium-dependent fashion.

In this study we characterized the effects of the protein phosphatase (PP) type 1 and type 2A inhibitor cantharidin (Cant) and its structural analogs cantharidic acid and endothall on PP activity, force of contraction, and myosin light chain phosphorylation in rat aorta. All compounds inhibited PP activity in homogenates of rat aorta with a rank order of potency of Cant = cantharidic acid > endothall. However, only Cant increased force of contraction and myosin light chain phosphorylation in intact isolated rat aortic rings. Based on these findings, we investigated the effects of Cant on alpha-adrenoceptor-mediated vasoconstriction. Cant (1 and 3 microM) enhanced norepinephrine-induced contraction in endothelium-intact rat aorta. In contrast, Cant did not affect norepinephrine-induced contraction in endothelium-denuded rat aorta. We suggest that inhibition of PP1 and/or PP2A activities by Cant enhances vascular contractility in endothelium-intact rat aorta by increasing the phosphorylation state of endothelial regulatory proteins.

Functional properties of transgenic mouse hearts overexpressing both calsequestrin and the Na(+)-Ca(2+) exchanger.

Overexpression of calsequestrin (CSQ) induces severe cardiac hypertrophy, whereas overexpression of Na(+)-Ca(2+) exchanger (NCX) does not affect cardiac weight. To investigate a possible beneficial effect of NCX in hypertrophy, we produced transgenic mice overexpressing both NCX and CSQ (NCX/CSQ). Surprisingly, these mice developed severe heart failure. The heart/body weight ratio was enhanced and the mRNA expression of ANF, as a marker of hypertrophy, was highest in double transgenic mice. In isolated muscle strips, the basal relaxation time was prolonged in CSQ and NCX/CSQ mice. Moreover, in the presence of caffeine, force of contraction was increased only in CSQ and NCX/CSQ and was accompanied by elevated diastolic tension. In some respects, however, additional overexpression of NCX altered the CSQ phenotype into the wild-type phenotype. The expression of sarcoplasmic reticulum (SR)-Ca(2+)-ATPase and phospholamban, proteins involved in the Ca(2+) uptake of the SR, were only increased in CSQ, indicating a possible influence of NCX in the regulation of SR-Ca(2+) uptake proteins. The Ca(2+) transients and the L-type Ca(2+) currents in the presence of caffeine were very large in CSQ, but smaller increases were noted in double transgenic mice. Therefore, the successful co-overexpression of CSQ and NCX in these mice provides a novel model in which to investigate the interaction of proteins tightly linked to maintain Ca(2+) homeostasis.

On the cardiac contractile, electrophysiological and biochemical effects of endothall, a protein phosphatase inhibitor.

Protein phosphatase inhibitors, e.g. cantharidin, exert positive inotropic effects in mammalian heart preparations. Endothall, a synthetic herbicide which is chemically related to cantharidin, inhibits protein phosphatase activities in mouse liver preparations. However, the cardiac effects of endothall have hitherto not been studied. In guinea pig papillary muscles, endothall (1-100 micromol/l) failed to affect force of contraction, whereas cantharidin (1-100 micromol/l) increased force of contraction maximally to 313.4 +/- 32% of control at 10 micromol/l. In isolated guinea pig ventricular cardiomyocytes, endothall did neither change the free intracellular calcium concentration nor the amplitude of calcium current nor the phosphorylation state of regulatory phosphoproteins like phospholamban. In contrast, cantharidin (30 micromol/l) increased the free intracellular calcium concentration and the L-type calcium current to 149.6 +/- 9% and to 157.6 +/- 12% of control, respectively. Furthermore, cantharidin (1-100 micromol/l) augmented the phosphorylation of phospholamban maximally to 140.8 +/- 7% of control. Nevertheless, in guinea pig ventricular homogenates, both endothall and cantharidin inhibited phosphatase activity with EC(50) values of 1.92 and 0.32 micromol/l, respectively. Thus, in contrast to cantharidin, endothall failed to increase force of contraction, though it inhibited protein phosphatase activity. Clearly, endothall is not an appropriate tool to study the function of protein phosphatases in the mammalian heart.

Biochemical mechanism(s) of stunning in conscious dogs.

The mechanism(s) underlying contractile dysfunction in cardiac stunning is not completely understood. The expression and/or the phosphorylation state of cardiac Ca(2+) homoeostasis-regulating proteins might be altered in stunning. We tested this hypothesis in a well-characterized model of stunning. Conscious dogs were chronically instrumented, and the left anterior descending artery (LAD) was occluded for 10 min. Thereafter, reperfusion of the LAD was initiated. Tissues from reperfused LAD (stunned) and Ramus circumflexus (control) areas were obtained when left ventricular regional wall thickening fraction had recovered by 50%. Northern and Western blotting revealed no differences in the expression of the following genes: phospholamban, calsequestrin, sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a, and the inhibitory subunit of troponin I (TnI). However, the phosphorylation state of TnI and phospholamban were reduced in the LAD area. Fittingly, cAMP levels were reduced by 28% (P < 0.05). It is concluded that the contractile dysfunction in cardiac stunning might be mediated in part by decreased levels of cAMP and subsequently a reduced phosphorylation state of phospholamban and TnI.

The early response genes c-jun and HSP-70 are induced in regional cardiac stunning in conscious mammals.

OBJECTIVES: A reversible contractile dysfunction without necrosis after transient myocardial ischemia has been termed stunning. The molecular mechanisms underlying this phenomenon are only now beginning to be unraveled. It is conceivable that the expression of early-response genes may play a crucial role in stunning. METHODS: The expression of HSP-70, c-jun, and GRP-94 was investigated in a chronically instrumented dog model (n = 9). The left anterior descending coronary artery was occluded temporarily for 10 minutes after the animals had fully recovered from instrumentation. The wall thickening fraction was measured in the left anterior descending coronary artery and the nonischemic ramus circumflex of the left coronary artery-perfused region. When the wall thickening fraction of the left anterior descending coronary artery had recovered to 50% of preocclusion values, tissue samples were obtained from the areas perfused by the left anterior descending coronary artery and the nonischemic ramus circumflex of the left coronary artery. RESULTS: The messenger RNA of HSP-70 was increased to 214% +/- 26% in the area perfused by the left anterior descending artery compared with that perfused by the nonischemic ramus circumflex of the left coronary artery. There was no difference in the messenger RNA of GRP-94. The HSP-70 content was elevated to 130% +/- 14% in the left anterior descending artery compared with the area perfused by the ramus circumflex of the left coronary artery, and the c-jun protein content was 70% +/- 25% higher in the ischemic area compared with the control area. CONCLUSIONS: The induction of early-response genes observed here may indicate that they play an adaptive role in myocardial stunning, even in conscious mammals.

Single-channel activity and expression of atrial L-type Ca(2+) channels in patients with latent hyperthyroidism.

Patients with "latent hyperthyroidism" (suppressed thyroid-stimulating hormone and normal circulating thyroid hormones) are at risk to develop atrial fibrillation. In animal models, hyperthyroidism is associated with increased cardiac L-type Ca(2+) current. Therefore, we assessed L-type channel function and expression in right atria from patients undergoing cardiac surgery. Single L-type channels were studied in the cell-attached condition. Voltage dependence of gating was similar in patients with and without latent hyperthyroidism. With use of a pulse protocol leading to maximum channel availability, single-channel activity was further analyzed. Average peak current was significantly enhanced in latent hyperthyroidism, mainly because of an increased channel availability (P < 0.05). Protein expression was analyzed by Western blot. In latent hyperthyroidism, expression of Ca(2+) channel alpha(1)-subunits was increased more than threefold (P < 0.01). In contrast, sarco(endo)plasmic reticulum Ca(2+)-ATPase and phospholamban levels were not significantly changed. We only observed a trend toward increased sarco(endo)plasmic reticulum Ca(2+)-ATPase expression (P = 0.085). Function and expression of human atrial L-type Ca(2+) channels are increased in latent hyperthyroidism. These endocrine effects on the heart may be clinically relevant.