Abstract-The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS Ϫ/Ϫ mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca 2ϩ -channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (nϭ12; PϽ0.01). Measuring of total NOS activity by conversion of [3 H]-L-arginine to [ 3 H]-L-citrulline showed a 30% increase in nNOS overexpressing mice (nϭ18; PϽ0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17Ϯ3% decrease of ϩdp/dt max compared with noninduced mice (PϽ0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; nϭ15; PϽ0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca 2ϩ ATPase and additionally with L-type Ca 2ϩ -channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I Ca,L density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca 2ϩ -transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca 2ϩ -channels and in turn reduces Ca 2ϩ -transients which accounts for the negative inotropic effect. (Circ Res. 2007;100:e32-e44.) Key Words: nNOS Ⅲ contractility Ⅲ excitation Ⅲ contraction coupling Ⅲ conditional overexpression S everal studies have demonstrated neuronal NO synthase (nNOS) protein expression within cardiac myocytes. 1 Specifically, nNOS has been localized to the sarcolemma 2,3 and the sarcoplasmatic reticulum (SR), 4 where it has been shown to be in close proximity to the SR Ca 2ϩ -release channel (RyR2) 5 and the SR Ca 2ϩ ATPase. However, the impact of nNOS on myocardial contractility remains largely controversial. Results from nNOS Ϫ/Ϫ mice and from pharmacological inhibition of nNOS provided insights into the role of nNOS in the cardiovascular system. But these approaches suffer from complete nNOS blockade and did not take into account a possible translocation of nNOS to specific subcellular sites. Some authors have shown, that inhibition of nNOS activity, via gene disruption or by pharmacological inhibition, enhanced basal contractility. 7,8 In the latter study, the positive inotropic effects of nNOS inhibition or gene disruption were related to ...

AimsClinical observations in patients with long QT syndrome carrying sodium channel mutations (LQT3) suggest that bradycardia caused by parasympathetic stimulation may provoke torsades de pointes (TdP). β-Adrenoceptor blockers appear less effective in LQT3 than in other forms of the disease.Methods and resultsWe studied effects of autonomic modulation on arrhythmias in vivo and in vitro and quantified sympathetic innervation by autoradiography in heterozygous mice with a knock-in deletion (ΔKPQ) in the Scn5a gene coding for the cardiac sodium channel and increased late sodium current (LQT3 mice). Cholinergic stimulation by carbachol provoked bigemini and TdP in freely roaming LQT3 mice. No arrhythmias were provoked by physical stress, mental stress, isoproterenol, or atropine. In isolated, beating hearts, carbachol did not prolong action potentials per se, but caused bradycardia and rate-dependent action potential prolongation. The muscarinic inhibitor AFDX116 prevented effects of carbachol on heart rate and arrhythmias. β-Adrenoceptor stimulation suppressed arrhythmias, shortened rate-corrected action potential duration, increased rate, and minimized difference in late sodium current between genotypes. β-Adrenoceptor density was reduced in LQT3 hearts. Acute β-adrenoceptor blockade by esmolol, propranolol or chronic propranolol in vivo did not suppress arrhythmias. Chronic flecainide pre-treatment prevented arrhythmias (all P < 0.05).ConclusionCholinergic stimulation provokes arrhythmias in this model of LQT3 by triggering bradycardia. β-Adrenoceptor density is reduced, and β-adrenoceptor blockade does not prevent arrhythmias. Sodium channel blockade and β-adrenoceptor stimulation suppress arrhythmias by shortening repolarization and minimizing difference in late sodium current.

Background/Aims: The aim of the study was to characterize the whole cell current of the two-pore domain potassium channel TASK-1 (K2P3) in mouse ventricular cardiomyocytes (I_TASK-1) and to analyze the cardiac phenotype of the TASK-1^-/- mice. Methods and Results: We have quantified the ventricular I_TASK-1 current using the blocker A293 and TASK-1^-/- mice. Surface electrocardiogram recordings of TASK-1^-/- mice showed a prolonged QTc interval and a broadened QRS complex. The differences in electrocardiograms between wild type and TASK-1^-/- mice disappeared during sympathetic stimulation of the animals. Quantitative RT-PCR, patch clamp recordings and measurements of hemodynamic performance of TASK-1^-/- mice revealed no major compensatory changes in ion channel transcription. Action potential recordings of TASK-1^-/- mouse cardiomyocytes indicated that I_TASK-1 modulates action potential duration. Our in vivo electrophysiological studies showed that isoflurane, which activates TASK-1, slowed heart rate and atrioventricular conduction of wild-type but not of TASK-1^-/- mice. Conclusion: The results of an invasive electrophysiological catheter protocol in combination with the observed QRS time prolongation in the surface electrocardiogram point towards a regulatory role of TASK-1 in the cardiac conduction system.

Voltage-gated sodium channels composed of a pore-forming α subunit and auxiliary β subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used immunohistochemical methods to define the level of expression and the subcellular localization of sodium channel α and β subunits in human atrial myocytes. Nav1.2 channels are located in highest density at intercalated disks where β1 and β3 subunits are also expressed. Nav1.4 and the predominant Nav1.5 channels are located in a striated pattern on the cell surface at the z-lines together with β2 subunits. Nav1.1, Nav1.3, and Nav1.6 channels are located in scattered puncta on the cell surface in a pattern similar to β3 and β4 subunits. Nav1.5 comprised approximately 88% of the total sodium channel staining, as assessed by quantitative immunohistochemistry. Functional studies using whole cell patch-clamp recording and measurements of contractility in human atrial cells and tissue showed that TTX-sensitive (non-Nav1.5) α subunit isoforms account for up to 27% of total sodium current in human atrium and are required for maximal contractility. Overall, our results show that multiple sodium channel α and β subunits are differentially localized in subcellular compartments in human atrial myocytes, suggesting that they play distinct roles in initiation and conduction of the action potential and in excitation–contraction coupling. TTX-sensitive sodium channel isoforms, even though expressed at low levels relative to TTX-sensitive Nav1.5, contribute substantially to total cardiac sodium current and are required for normal contractility. This article is part of a Special Issue entitled “Na+ Regulation in Cardiac Myocytes”.

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Changes of Z show a strong inverse correlation with changes of directly measured EVLWI. This allows the application of Z as a measure of intrathoracic fluid status and has the potential to optimize patient care, especially in the context of evolving telemedicine concepts.

Introduction: We previously demonstrated that conditional overexpression of the neuronal nitric oxide synthase (nNOS, NOS1) inhibited L-type Ca 2+ -channels. We now hypothesize that nNOS overexpression has an impact on myocardial contractility and acts cardioprotective after ischemia-reperfusion. Methods and results: We assessed cardiac function in the newly established transgenic mouse model with conditional, myocardial nNOS overexpression. NOS-activity (22 ± 1.5 vs. 29 ± 1μM /sec, n=18, p<0.05) was significantly enhanced after nNOS overexpression. Co-immunoprecipitation experiments indicated interaction of nNOS with SR Ca 2+ ATPase and additionally with L-type Ca 2+ -channels in nNOS overexpressing animals. I ca,L (reduction of 40±6 rel. %, n=12, p<0.05) as well as intracellular Ca 2+ -transients and fractional shortening in cardiomyocytes were clearly impaired in nNOS overexpressing mice (3.0 ± 0.4F/F 0 vs. 2.2 ± 0.2F/F 0 , n=13, p<0.005 and 7.7 ± 1.3% vs. 3.8 ± 0.5%, n=13, p<0.05). In vivo examinations of the nNOS overexpressing mice showed a decrease of +dp/dt max (reduction for 52 ± 17%, n=12, p<0.05) as well as a reduced ejection fraction (43±5% vs. 63±9%, n=12, p<0.05). Ischemia-reperfusion experiments showed a cardio-protective effect of nNOS overexpression (30 min post-ischemia, LVDP 20±6 in non-induced animals vs. 60±11 mmHg in nNOS overexpressing animals, n=6, p<0.05). Discussion: In summary, we demonstrated that under basline conditions, conditional transgenic overexpression of nNOS resulted in a mild reduction of myocardial contractility, mainly due to inhibition of the L-type Ca 2+ -channel. In contrast, under pathophysiological conditions (i.e. ischemia-reperfusion) nNOS overexpression acts cardioprotective. These effects might be caused by a reduction of myocardial Ca 2+ -overload after reperfusion.

Abstract-The role of the neuronal NO synthase (nNOS or NOS1) enzyme in the control of cardiac function still remains unclear. Results from nNOS Ϫ/Ϫ mice or from pharmacological inhibition of nNOS are contradictory and do not pay tribute to the fact that probably spatial confinement of the nNOS enzyme is of major importance. We hypothesize that the close proximity of nNOS and certain effector molecules like L-type Ca 2ϩ -channels has an impact on myocardial contractility. To test this, we generated a new transgenic mouse model allowing conditional, myocardial specific nNOS overexpression. Western blot analysis of transgenic nNOS overexpression showed a 6-fold increase in nNOS protein expression compared with noninduced littermates (nϭ12; PϽ0.01). Measuring of total NOS activity by conversion of [3 H]-L-arginine to [ 3 H]-L-citrulline showed a 30% increase in nNOS overexpressing mice (nϭ18; PϽ0.05). After a 2 week induction, nNOS overexpression mice showed reduced myocardial contractility. In vivo examinations of the nNOS overexpressing mice revealed a 17Ϯ3% decrease of ϩdp/dt max compared with noninduced mice (PϽ0.05). Likewise, ejection fraction was reduced significantly (42% versus 65%; nϭ15; PϽ0.05). Interestingly, coimmunoprecipitation experiments indicated interaction of nNOS with SR Ca 2ϩ ATPase and additionally with L-type Ca 2ϩ -channels in nNOS overexpressing animals. Accordingly, in adult isolated cardiac myocytes, I Ca,L density was significantly decreased in the nNOS overexpressing cells. Intracellular Ca 2ϩ -transients and fractional shortening in cardiomyocytes were also clearly impaired in nNOS overexpressing mice versus noninduced littermates. In conclusion, conditional myocardial specific overexpression of nNOS in a transgenic animal model reduced myocardial contractility. We suggest that nNOS might suppress the function of L-type Ca 2ϩ -channels and in turn reduces Ca 2ϩ -transients which accounts for the negative inotropic effect. (Circ Res. 2007;100:e32-e44.) Key Words: nNOS Ⅲ contractility Ⅲ excitation Ⅲ contraction coupling Ⅲ conditional overexpression S everal studies have demonstrated neuronal NO synthase (nNOS) protein expression within cardiac myocytes. 1 Specifically, nNOS has been localized to the sarcolemma 2,3 and the sarcoplasmatic reticulum (SR), 4 where it has been shown to be in close proximity to the SR Ca 2ϩ -release channel (RyR2) 5 and the SR Ca 2ϩ ATPase. However, the impact of nNOS on myocardial contractility remains largely controversial. Results from nNOS Ϫ/Ϫ mice and from pharmacological inhibition of nNOS provided insights into the role of nNOS in the cardiovascular system. But these approaches suffer from complete nNOS blockade and did not take into account a possible translocation of nNOS to specific subcellular sites. Some authors have shown, that inhibition of nNOS activity, via gene disruption or by pharmacological inhibition, enhanced basal contractility. 7,8 In the latter study, the positive inotropic effects of nNOS inhibition or gene disruption were related to ...