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Serotonin (5-HT) exerts pleiotropic effects in the human cardiovascular system. Some of the effects are thought to be mediated via 5-[HT.sub.4] receptors, which are expressed in the human atrium and in ventricular tissue. However, a true animal model to study these receptors in more detail has been hitherto lacking. Therefore, we generated, for the first time, a transgenic (TG) mouse with cardiac myocyte-specific expression of the human 5-[HT.sub.4] receptor. RT-PCR and immunohistochemistry revealed expression of the receptor at the mRNA and protein levels. Stimulation of isolated cardiac preparations by isoproterenol increased phospholamban phosphorylation at [Ser.sup.16] and [Thr.sup.17] sites. 5-HT increased phosphorylation only in TG mice but not in wild-type (WT) mice. Furthermore, 5-HT increased contractility in isolated perfused hearts from TG mice but not WT mice. These effects of 5-HT could be blocked by the 5-[HT.sub.4] receptor-selective antagonist GR-125487. An intravenous infusion of 5-HT increased left ventricular contractility in TG mice but not in WT mice. Similarly, the increase in contractility by 5-HT in isolated cardiomyocytes from TG mice was accompanied by and probably mediated through an increase in L-type [Ca.sup.2+] channel current and in [Ca.sup.2+] transients. In intact animals, echocardiography revealed an inotropic and chronotropic effect of subcutaneously injected 5-HT in TG mice but not in WT mice. In isolated hearts from TG mice, spontaneous polymorphic atrial arrhythmias were noted. These findings demonstrate the functional expression of 5-[HT.sub.4] receptors in the heart of TG mice, and a potential proarrhythmic effect in the atrium. Therefore, 5-[HT.sub.4] receptorexpressing mice might be a useful model to mimic the human heart, where 5-[HT.sub.4] receptors are present and functional in the atrium and ventricle of the healthy and failing heart, and to investigate the influence of 5-HT in the development of cardiac arrhythmias and heart failure. serotonin; arrhythmia; transgenic mice; signal transduction doi: 10.1152/ajpheart.00691.2009.

Atrial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular [Ca.sup.2+] handling. Diastolic [Ca.sup.2+] release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed [Ryr2.sup.R176Q/+] mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased [Ca.sup.2+]/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in [Ryr2.sup.R176Q/+] mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in [Ryr2.sup.S2814A] knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent [Ca.sup.2+] leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction., Introduction Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia and is a significant contributor to cardiovascular morbidity and possibly mortality. Emerging evidence suggests an important role for abnormal [...]

Cardiac-specific overexpression of the catalytic subunit of protein phosphatase type 1 (PP1) in mice results in hypertrophy, depressed contractility, propensity to heart failure, and premature death. To further address the role of PP1 in heart function, PP1 mice were crossed with mice that overexpress a functional COOH-terminally truncated form of PP1 inhibitor-2 ([I-2.sup.140]). Protein phosphatase activity was increased in PPI mice but was normalized in double transgenic (DT) mice. The maximal rates of contraction (+dP/dt) and of relaxation (-dP/dt) were reduced in catheterized PP1 mice but normalized in DT mice. Similar contractile abnormalities were observed in isolated, perfused work-performing hearts and in whole animals by means of echocardiography. The increased absolute and relative heart weights observed in PP1 mice were normalized in DT mice. Histological analyses indicated that PP1 mice had significant cardiac fibrosis, which was absent in DT mice. Furthermore, PP1 mice exhibited an age-dependent increase in mortality, which was abrogated in DT mice. These results indicate that I-2 overexpression prevents the detrimental effects of PP1 overexpression in the heart and further underscore the fundamental role of PP1 in cardiac function. Therefore, PP1 inhibitors such as I-2 could offer new therapeutic options to ameliorate the deleterious effects of heart failure. transgenic mice; contractility; heart failure; drug target

We studied contractile effects in isolated electrically driven (1 Hz) atrial preparations from patients undergoing cardiac bypass surgery. ATP concentration dependently (10, 30, and 100 [micro]M) and rapidly decreased force of contraction (negative inotropic effect, NIE) and thereafter more slowly increased force of contraction. The maximum positive inotropic effect (PIE) at 100 [micro]M ATP amounted to 152% of the predrug value (n = 9) and was stable and could be washed out fast and completely. The PIE did not affect time parameters of contraction (time to peak tension and time of relaxation). Moreover, a similar NIE and PIE were noted with adenosine 5'-O-(2-thiotriphosphate) (100 [micro]M). In contrast 2-methyl-thio-ATP did not exert a NIE but only a PIE. In a second set of experiments, preparations were first incubated for 30 rain with purinoreceptor antagonists and, in their continuous presence, 100 [micro]M ATP was applied. However, the PIE and NIE of ATP could neither be blocked with suramin (100 and 500 [micro]M), pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (50 [micro]M), nor reactive blue 2 (30, 100, and 500 [micro]M), which are known blockers for subtypes of [P.sub.2] receptors, or 1,3-dipropyl-cyclopentvl-xanthine (1 and 10 [micro]M), a subtype ([A.sub.1] adenosine) [P.sub.1] receptor blocker. Likewise, the inhibitor of phospholipase C (PLC) activity (U-73122) and the inhibitor of adenylate cyclase activity (SQ-022563) (10 [micro]M each) failed to affect the NIE and the PIE of ATP. We tentatively suggest that the PIE of ATP might be mediated via [P.sub.2x4]-1ike receptors. In summary, we describe a novel biphasic effect of ATP on force contraction in the isolated human atrium. It is conceivable that ATP plays a physiological role in the human heart, for instance, after cardiac injury to sustain contractility. human heart; right atrium; adenosine 5'-triphosphate, purinoceptor; inotropy

Triadin is involved in the regulation of cardiac excitation-contraction coupling. However, the extent of its contribution to the regulation of sarcoplasmic reticulum (SR) Ca release remains unclear, because overexpression of triadin in single-transgenic mice was associated with the downregulation of its homologous protein, junctin. In the present study, this problem was circumvented by cross-breeding of mice with heart-directed overexpression of triadin and junctin (JxT). This resulted in a stable approximately threefold expression of total triadin but unchanged junctin protein. Transgenic mice exhibited cardiac hypertrophy and structural abnormalities of myofibrils. Measurement of cardiac function by echocardiography and edge detection in myocytes revealed an impaired relaxation in JxT mice. The stimulation of [beta]-adrenergic receptors resulted in a depressed contractility and an impaired relaxation in catheterized hearts and myocytes of JxT mice. The use of a maximum stimulation frequency (5 Hz) was associated with both a lower shortening and relengthening in isolated myocytes of JxT mice. The contractile effects in JxT myocytes were paralleled by similar changes of the intracellular Ca concentration ([[Ca].sub.i]) peak amplitude and Ca transient decay kinetics at basal conditions, under administration of isoproterenol, and with high-frequency stimulation. Finally, we found a higher caffeine-induced [[Ca].sub.i] peak amplitude in JxT myocytes. Our data show that the stable expression of triadin, independent of junctin expression, resulted in cardiac hypertrophy, prolonged basal relaxation, a depressed response to [beta]-adrenergic agonists, and altered Ca transients. Thus the maintenance of triadin expression is essential for normal SR Ca cycling and contractile function. transgenic mice; sarcoplasmic reticulum; hypertrophy; Ca signaling; cardiac function; force-frequency relationship; [beta]-adrenergic receptor stimulation

Junctin is a transmembrane protein located at the cardiac junctional sarcoplasmic reticulum (SR) and forms a quaternary complex with the [Ca.sup.2+] release channel, triadin and calsequestrin. Impaired protein interactions within this complex may alter the [Ca.sup.2+] sensitivity of the [Ca.sup.2+] release channel and may lead to cardiac dysfunction, including hypertrophy, depressed contractility, and abnormal [Ca.sup.2+] transients. To study the expression of junctin and, for comparison, triadin, in heart failure, we measured the levels of these proteins in SR from normal and failing human hearts. Junctin was below our level of detection in SR membranes from failing human hearts, and triadin was downregulated by 22%. To better understand the role of junctin in the regulation of [Ca.sup.2+] homeostasis and contraction of cardiac myocytes, we used an adenoviral approach to overexpress junctin in isolated rat cardiac myocytes. A recombinant adenovirus encoding the green fluorescent protein served as a control. Infection of myocytes with the junctin-expressing virus resulted in an increased RNA and protein expression of junctin. [Ca.sup.2+] transients showed a decreased maximum [Ca.sup.2+] amplitude, and contractility of myocytes was depressed. Our results demonstrate that an increased expression of junctin is associated with an impaired [Ca.sup.2+] homeostasis. Downregulation of junctin in human heart failure may thus be a compensatory mechanism. junctin; adenovirus; cardiac myocytes; calcium transient; sarcoplasmic reticulum doi:10.1152/ajpheart.01187.2006

Age-dependent biochemical and contractile properties in atrium of transgenic mice overexpressing junctin. Am J Physiol Heart Circ Physiol 287: H2216-H2225, 2004. First published June 17, 2004; doi: 10.1152/ajpheart.00137.2004.--Junctin is a transmembrane protein of the cardiac junctional sarcoplasmic reticulum (SR) that binds to the ryanodine receptor, calsequestrin, and triadin 1. This quaternary protein complex is thought to facilitate SR [Ca.sup.2+] release. To improve our understanding of the contribution of junctin to the regulation of SR function, we examined the age-dependent effects of junctin overexpression in the atrium of 3-, 6-, and 18-wk-old transgenic mice. The ratio of atrial weight and body weight was unchanged between junctin-overexpressing (JCN) and wild-type (WT) mice at all ages investigated (n = 6-8). The protein expression of triadin 1 was decreased starting in 3-wk-old JCN atria (by 69%), whereas the expression of the ryanodine receptor was diminished in 6- (by 48%) and 18-wk-old (by 57%) JCN atria compared with age-matched WT atria. Force of contraction was decreased by 35% in 18-wk-old JCN compared with age-matched WT left atrial muscle strips, which was accompanied by a prolonged time of relaxation (48.1 [+ or -] 0.9 vs. 44.2 [+ or -] 0.8 ms, respectively, n = 6-8, P < 0.05). The spontaneous beating rate of isolated right atria was higher in 18-wk-old JCN mice compared with age-matched WT mice (389 [+ or -] 10 vs. 357 [+ or -] 6 beats/min, respectively, n = 6-8, P < 0.05). Heart rate was lower by 9% in telemetric ECG recordings in 18-wk-old JCN mice during stress tests. Three-week-old JCN atria exhibited a higher potentiation of force of contraction at rest pauses of 30 s (by 13%) and of 300 s (by 35%), suggesting increased SR [Ca.sup.2+] content. This was consistent with the higher force of contraction in 3-wk-old JCN atria (by 29%) compared with age-matched WT atria (by 10%) under the administration of caffeine. We conclude that in 3-wk-old atria, junctin overexpression was associated with a reduced expression of triadin 1 resulting in a higher SR [Ca.sup.2+] load without changes in contractility or heart rate. In 6-wk-old JCN atria, the compensatory downregulation of the ryanodine receptor may offset the effects of junctin overexpression. Finally, the progressive decrease in ryanodine receptor density may contribute to the decreased atrial contractility and lower heart rate during stress in 18-wk-old JCN mice. sarcoplasmic reticulum; calcium handling; force of contraction; protein expression; heart rate

To investigate whether altered function of adenosine receptors could contribute to sinus node or atrioventricular (AV) nodal dysfunction in conscious mammals, we studied transgenic (TG) mice with cardiac-specific overexpression of the [A.sub.1] adenosine receptor ([A.sub.1]AR). A Holter ECG was recorded in seven freely moving littermate pairs of mice during normal activity, exercise (5 min of swimming), and 1 h after exercise. TG mice had lower maximal heart rates (HR) than wild-type (WT) mice (normal activity: 437 [+ or -] 18 vs. 522 [+ or -] 24 beats/min, P < 0.05; exercise: 650 [+ or -] 13 vs. 765 [+ or -] 28 beats/min, P < 0.05; 1 h after exercise: 588 [+ or -] 18 vs. 720 [+ or -] 12 beats/min, P < 0.05; all values are means [+ or -] SE). Mean HR was lower during exercise (589 [+ or -] 16 vs. 698 [+ or -] 34 beats/min, P < 0.05) and after exercise (495 [+ or -] 16 vs. 592 [+ or -] 27 beats/min, P < 0.05). Minimal HR was not different between genotypes. HR variability (SD of RR intervals) was reduced by 30% (P < 0.05) in TG compared with WT mice. Pertussis toxin (n = 4 pairs, 150 [micro]g/kg ip) reversed bradycardia after 48 h. TG mice showed first-degree AV nodal block (PQ interval: 42 [+ or -] 2 vs. 37 [+ or -] 2 ms, P < 0.05), which was diminished but not abolished by pertussis toxin. Isolated Langendorff-perfused TG hearts developed spontaneous atrial arrhythmias (3 of 6 TG mice vs. 0 of 9 WT mice, P < 0.05). In conclusion, [A.sub.1]AR regulate sinus nodal and AV nodal function in the mammalian heart in vivo. Enhanced expression of [A.sub.1]AR causes sinus nodal and AV nodal dysfunction and supraventricular arrhythmias. heart rate regulation; autonomous nervous system; heart rate variability; sinus node dysfunction; atrioventricular block; atrial fibrillation

Triadin 1 is a protein in the cardiac junctional sarcoplasmic reticulum (SR) that interacts with the ryanodine receptor, junctin, and calsequestrin, proteins that are important for [Ca.sup.2+] release. To better understand the role of triadin 1 in SR-[Ca.sup.2+] release, we studied the time-dependent expression of SR proteins and contractility in atria of 3-, 6-, and 18-wk-old transgenic mice overexpressing canine cardiac triadin 1 under control of the [alpha]-myosin heavy chain (MHC) promoter. Three-week-old transgenic atria exhibited mild hypertrophy. Finally, atrial weight was increased by 110% in 18-wk-old transgenic mice. Triadin 1 overexpression was accompanied by time-dependent changes in the protein expression of the ryanodine receptor, junctin, and cardiac/slow-twitch muscle SR [Ca.sup.2+]-ATPase isoform. Force of contraction was already decreased in 3-wk-old transgenic atria. The application of caffeine led to a positive inotropic effect in transgenic atria of 3-wk-old mice. Rest pauses resulted in an increased potentiation of force of contraction after restimulation in 3- and 6-wk-old mice and a reduced potentiation of force of contraction in 18-wk-old transgenic mice. Hence, triadin 1 overexpression triggered time-dependent alterations in SR protein expression, [Ca.sup.2+] homeostasis, and contractility, indicating for the first time an inhibitory function of triadin 1 on SR-[Ca.sup.2+] release in vivo. protein expression; force of contraction; sarcoplasmic reticulum-calcium release

Byline: Roger J. Hajjar (1), Frank U. Muller (2), Wilhelm Schmitz (2), Petra Schnabel (3), M. Bohm (3) Keywords: Key words Heart failure; Signal transduction; G proteins; SERCA; Adrenergic system Abstract: Abnormal [beta]-adrenergic signal transduction and intracellular Ca.sup.2+ handling appear to be a major cause of systolic and diastolic dysfunction in humans with heart failure. The precise mechanisms which cause an alteration in Ca.sup.2+ handling have been a subject of investigation in recent years. Several lines of evidence suggest that activation of neurohormonal systems plays a central role. Altered Ca.sup.2+-handling (increased diastolic concentrations, reduced systolic Ca.sup.2+ release) have a strong impact on diastolic and systolic performance of failing hearts. Sarcoplasmic reticulum Ca.sup.2+ ATPase is reduced in activity and in steady-state mRNA concentration. The Na.sup.+-Ca.sup.2+ exchanger is upregulated at the mRNA and protein levels. Phospholamban depends strongly on cAMP-dependent phosphorylation. A strong sympathetic activation has been shown to desensitize the cAMP system. At the receptor level, there is downregulation of [beta].sub.1-adrenergic receptors. An uncoupling of [beta].sub.2-adrenoceptors has been attributed to an increased activity and gene expression of [beta]-adrenergic receptor kinase in failing myocardium, leading to phosphorylation and uncoupling of receptors. Finally, recent evidence suggests that cAMP-dependent transcription mechanisms may play a role during [beta]-adrenergic stimulation and cardiomyopathy with heart failure -- by means of altered actions of cAMP response element binding protein, the cAMP response element modulator, or the activating transcription factor 1. The exact characterization of signal transduction defects could offer novel approaches to the pharmacological treatment of heart failure. Author Affiliation: (1) Harvard Medical School, Massachusetts General Hospital, Cardiovascular Center, 149 13th Street, Charlestown, MA 02129-2060, USA, US (2) Westfalische Wilhelms-Universitat Munster, Institut fur Pharmakologie und Toxikologie, Domagkstrasse 12, D-48149 Munster, Germany, DE (3) Universitat Koln, Klinik III fur Innere Medizin, Joseph-Stelzmann-Strasse 9, D-50924 Cologne, German, XX Article note: Received: 16 February 1998 / Accepted: 21 August 1998

The possibility that postnatal changes in cardiac phosphatases might occur and modify the role of sarcoplasmic reticulum (SR) proteins was investigated. Cardiac time parameters, SR protein expression and phosphatase expression in neonatal vs adult mammalian cardiac ventricular preparations were measured. Findings indicated that SR Ca(super 2+)-ATPase but not phospholamban expression was altered postnatally and that this increase was accompanied by improved CA(super 2+) uptake.

Chronic beta-adrenergic stimulation in rats causes a downregulation of the phospholamban (PLB) and sarcoplasmic reticulum Ca2+-ATPase (SERCA2) protein expression by 49 and 40% respectively. However, troponin and C protein levels are unaffected. In addition, it reduces cAMP formation and thus PLB phosphorylation in hypertrophied hearts. Chronic beta-adrenergic stimulation decreases the isoproterenol-stimulated positive lusitropic and inotropic effects in papillary muscles.

The chronic iso infusion causes changes in the cardiac gene expression which corresponds to the growth of myocyte hypertrophy and interstital fibrosis. The differences in the hemodynamic parameters do not influence the activation of common mechanisms in pressure overload and beta-adrenergic receptor-mediated cardiac hypertrophy. The mechanisms controlling the gene induce different status of sensitivity to beta-adrenergic receptor stimulation.

A study was conducted to explore the probability that phosphatase inhibition causes the positive inotropic influence of okadaic acid, and to find if the phosphorylation of cardiac proteins influences this inhibition. 30 mu M okadaic acid enhanced the force of contraction to 175% of predrug value in papillary muscles from guinea pigs. By improving channel availability, okadaic acid augmented single Ca2+ channels currents in isolated guinea pig ventricular cardiomyocytes. The enhancement of the phosphorylation state of many proteins including phospholamban (PLB), a 23-LDa protein, and troponin inhibitor (TnI) could mediate the influence of okadaic acid.

Role of protein phosphatases in regulation of cardiac inotropy and relaxation. Am J Physiol Heart Circ Physiol 280: H786-H794, 2001.--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 [+ or -] 4% of control, n = 10) and TnI-P and PLB-Thr-17 (120 [+ or -] 5 and 128 [+ or -] 7% of control) without any alteration of relaxation time. Cant (10 [micro]M) started to increase PLB-Ser-16, but the relaxation was shortened at only 100 [micro]M (from 140 [+ or -] 9 to 116 [+ or -] 12 ms, n = 9). Moreover, 100 [micro]M Cant, 3 min after application, started to increase PLB-Thr-17, TnI-P, and FOC. Cant (100 [micro]M) 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. cantharidin; protein phosphatase inhibitors; protein phosphorylation; phospholamban; inhibitory subunit of troponin

Biochemical mechanism(s) of stunning in conscious dogs. Am J Physiol Heart Circ Physiol 279: H176-H184, 2000.--The mechanism(s) underlying contractile dysfunction in cardiac stunning is not completely understood. The expression and/or the phosphorylation state of cardiac [Ca.sup.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.sup.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 [is less than] 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. myocardial stunning; regulatory proteins; second messengers; phosphorylation