Adrenergic Challenge Research Articles

Correction to: 'Cardiac deficiency of P21-activated kinase 1 promotes atrial arrhythmogenesis in mice following adrenergic challenge'.

Correction to: 'Cardiac deficiency of P21-activated kinase 1 promotes atrial arrhythmogenesis in mice following adrenergic challenge'.

Impact of stress on cardiac phenotypes in mice harboring an ankyrin-B disease variant

Encoded by ANK2, ankyrin-B (AnkB) is a multifunctional adapter protein critical for the expression and targeting of key cardiac ion channels, transporters, cytoskeletal-associated proteins, and signaling molecules. Mice deficient for AnkB expression are neonatal lethal, and mice heterozygous for AnkB expression display cardiac structural and electrical phenotypes. Human ANK2 loss-of-function variants are associated with diverse cardiac manifestations; however, human clinical 'AnkB syndrome' displays incomplete penetrance. To date, animal models for human arrhythmias have generally been knock-out or transgenic overexpression models and thus the direct impact of ANK2 variants on cardiac structure and function invivo is not clearly defined. Here, we directly tested the relationship of a single human ANK2 disease-associated variant with cardiac phenotypes utilizing a novel invivo animal model. At baseline, young AnkBp.E1458G+/+ mice lacked significant structural or electrical abnormalities. However, aged AnkBp.E1458G+/+ mice displayed both electrical and structural phenotypes at baseline including bradycardia and aberrant heart rate variability, structural remodeling, and fibrosis. Young and old AnkBp.E1458G+/+ mice displayed ventricular arrhythmias following acute (adrenergic) stress. In addition, young AnkBp.E1458G+/+ mice displayed structural remodeling following chronic (transverse aortic constriction) stress. Finally, AnkBp.E1458G+/+ myocytes harbored alterations in expression and/or localization of key AnkB-associated partners, consistent with the underlying disease mechanism. In summary, our findings illustrate the critical role of AnkB in invivo cardiac function as well as the impact of single AnkB loss-of-function variants invivo. However, our findings illustrate the contribution and in fact necessity of secondary factors (aging, adrenergic challenge, pressure-overload) to phenotype penetrance and severity.

Cardiac deficiency of P21-activated kinase 1 promotes atrial arrhythmogenesis in mice following adrenergic challenge.

P21-activated kinase 1 (Pak1) signalling plays a vital and overall protective role in the heart. However, the phenotypes of Pak1 deficiency in the cardiac atria have not been well explored. In this study, Pak1 cardiac-conditional knock-out (cKO) mice were studied under baseline and adrenergic challenge conditions. Pak1 cKO mice show atrial arrhythmias including atrial fibrillation (AF) in vivo, detected during anaesthetized electrocardiography without evidence of interstitial fibrosis upon Masson's trichrome staining. Optical mapping of left atrial preparations from Pak1 cKO mice revealed a higher incidence of Ca2+ and action potential alternans under isoprenaline challenge and differences in baseline action potential and calcium transient characteristics. Type-2 ryanodine receptor (RyR2) channels from Pak1 cKO hearts had a higher open probability than those from wild-type. Reverse transcription-quantitative polymerase chain reaction and Western blotting indicated that pCamkIIδ and RyR2 are highly phosphorylated at baseline in the atria of Pak1 cKO mice, while the expression of Slc8a2 and Slc8a3 as a Na+-Ca2+ exchanger, controlling the influx of Ca2+ from outside of the cell and efflux of Na+ from the cytoplasm, are augmented. Chromatin immunoprecipitation study showed that pCreb1 interacts with Slc8a2 and Slc8a3. Our study thus demonstrates that deficiency of Pak1 promotes atrial arrhythmogenesis under adrenergic stress, probably through post-translational and transcriptional modifications of key molecules that are critical to Ca2+ homeostasis. This article is part of the theme issue 'The heartbeat: its molecular basis and physiological mechanisms'.

Altered Expression of Zonula occludens-1 Affects Cardiac Na+ Channels and Increases Susceptibility to Ventricular Arrhythmias

Zonula occludens-1 (ZO-1) is an intracellular scaffolding protein that orchestrates the anchoring of membrane proteins to the cytoskeleton in epithelial and specialized tissue including the heart. There is clear evidence to support the central role of intracellular auxiliary proteins in arrhythmogenesis and previous studies have found altered ZO-1 expression associated with atrioventricular conduction abnormalities. Here, using human cardiac tissues, we identified all three isoforms of ZO-1, canonical (Transcript Variant 1, TV1), CRA_e (Transcript Variant 4, TV4), and an additionally expressed (Transcript Variant 3, TV3) in non-failing myocardium. To investigate the role of ZO-1 on ventricular arrhythmogenesis, we generated a haploinsufficient ZO-1 mouse model (ZO-1+/−). ZO-1+/− mice exhibited dysregulated connexin-43 protein expression and localization at the intercalated disc. While ZO-1+/− mice did not display abnormal cardiac function at baseline, adrenergic challenge resulted in rhythm abnormalities, including premature ventricular contractions and bigeminy. At baseline, ventricular myocytes from the ZO-1+/− mice displayed prolonged action potential duration and spontaneous depolarizations, with ZO-1+/− cells displaying frequent unsolicited (non-paced) diastolic depolarizations leading to spontaneous activity with multiple early afterdepolarizations (EADs). Mechanistically, ZO-1 deficient myocytes displayed a reduction in sodium current density (INa) and an increased sensitivity to isoproterenol stimulation. Further, ZO-1 deficient myocytes displayed remodeling in ICa current, likely a compensatory change. Taken together, our data suggest that ZO-1 deficiency results in myocardial substrate susceptible to triggered arrhythmias.

CaMKII as a therapeutic target in catecholaminergic polymorphic ventricular tachycardia type 1

Abstract Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): South-Eastern Norway Regional Health Authority Background In catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) adrenergic activation of the cardiac ryanodine receptor (RyR2) causes spontaneous calcium release and triggers arrhythmias. Calcium/calmodulin-dependent protein kinase II (CaMKII) can contribute to such arrhythmogenic calcium release and has been proposed as a therapeutic target in CPVT1. To predict the efficacy and safety of this strategy, it is necessary to know whether the mechanism for CaMKII activation is important for its arrhythmogenic effects, and if inhibition has proarrhythmic effects. We tested (1) if oxidation of CaMKII contributes to spontaneous calcium release in CPVT1 and (2) if inhibition of CaMKII in this condition can induce calcium alternans. Methods Mice with the CPVT1-causative mutation RyR2-R2474S (RyR2-RS) were crossed with mice with the CaMKII M281/282V (MMVV) mutation that prevents CaMKII M281/282 oxidation, to create double mutants (RyR2-RSxMMVV). Telemetric ECG surveillance was used to study in vivo arrhythmias following an adrenergic challenge by i.p. administration of the beta-adrenoceptor agonist isoprenaline. Confocal line-scan imaging and whole-cell calcium imaging were used to study arrhythmogenic calcium release in isolated left ventricular cardiomyocytes during stimulation with isoprenaline. Results As expected, RyR2-RS mice exhibited more arrhythmic events and spontaneous calcium release (i.e. calcium sparks and calcium waves) compared to wild-type mice. Treatment of RyR2-RS cardiomyocytes with either the CaMKII inhibitor KN-93 or the antioxidant n-acetyl-cysteine reduced spontaneous calcium release (i.e. calcium sparks and calcium waves, for KN-93 and n-acetyl-cysteine, respectively). Interestingly, CaMKII inhibition by KN-93 also increased both incidence and degree of arrhythmogenic calcium alternans in RyR2-RS cardiomyocytes. This adverse effect was a result of prolonged refractoriness of calcium release. Furthermore, to test whether the protective effect of antioxidant treatment in RyR2-RS was mediated via CaMKII oxidation, we compared arrhythmias and spontaneous calcium release (i.e. calcium waves) in RyR2-RSxMMVV with RyR2-RS. However, these two genotypes did not differ in either incidence or severity of arrhythmias, and showed similar degree of spontaneous calcium release. Conclusions Inhibition of CaMKII protects against spontaneous calcium release in CPVT1, and is a promising therapeutic strategy. However, the fact that such inhibition also induces calcium alternans needs further exploration. Antioxidative agents also attenuate arrhythmogenic calcium release in CPVT1 cardiomyocytes, but this effect does not seem to involve the M281/282 CaMKII oxidation site. Future studies should explore other oxidation sites.

Cardiac response to adrenergic stress differs by sex and across the lifespan.

The aging heart is well-characterized by a diminished responsiveness to adrenergic activation. However, the precise mechanisms by which age and sex impact adrenergic-mediated cardiac function remain poorly described. In the current investigation, we compared the cardiac response to adrenergic stress to gain mechanistic understanding of how the response to an adrenergic challenge differs by sex and age. Juvenile (4 weeks), adult (4-6 months), and aged (18-20 months) male and female mice were treated with the β-agonist isoproterenol (ISO) for 1 week. ISO-induced morphometric changes were age- and sex-dependent as juvenile and adult mice of both sexes had higher left ventricle weights while aged mice did not increase cardiac mass. Adults increased myocyte cell size and deposited fibrotic matrix in response to ISO, while juvenile and aged animals did not show evidence of hypertrophy or fibrosis. Juvenile females and adults underwent expected changes in systolic function with higher heart rate, ejection fraction, and fractional shortening. However, cardiac function in aged animals was not altered in response to ISO. Transcriptomic analysis identified significant differences in gene expression by age and sex, with few overlapping genes and pathways between groups. Fibrotic and adrenergic signaling pathways were upregulated in adult hearts. Juvenile hearts upregulated genes in the adrenergic pathway with few changes in fibrosis, while aged mice robustly upregulated fibrotic gene expression without changes in adrenergic genes. We suggest that the response to adrenergic stress significantly differs across the lifespan and by sex. Mechanistic definition of these age-related pathways by sex is critical for future research aimed at treating age-related cardiac adrenergic desensitization.

Myo–inositol and d-chiro–inositol oral supplementation ameliorate cardiac dysfunction and remodeling in a mouse model of diet-induced obesity

Obesity is an independent risk factor to develop cardiac functional and structural impairments. Here, we investigated the effects of supplementation of inositols on the electrical, structural, and functional cardiac alterations in the mouse model of high fat diet (HFD) induced obesity. Three groups of C57BL6 mice (n = 16 each) were studied: j) HFD feeding; jj) HFD feeding + inositols from week 9 to 13; jjj) standard diet feeding. Study observation period was 13 weeks. Inositols were administered as mixture of myo-inositol and d-chiro-inositol in the drinking water. Effects of inositols were evaluated based on electrical, structural, and functional cardiac features, autonomic sympatho–vagal balance and arrhythmogenic susceptibility to adrenergic challenge. Heart samples were collected for histological evaluations and transcriptional analyses of genes involved in defining the shape and propagation of the action potential, fatty acid metabolism and oxidative stress. Inositol supplementation significantly restored control values of heart rate and QTc interval on ECG and of sympatho–vagal balance. Moreover, it blunted the increase in left ventricular mass and cardiomyocyte hypertrophy, reversed diastolic dysfunction, reduced the susceptibility to arrhythmic events and restored the expression level of cardiac genes altered by HFD.The present study shows, for the first time, how a short period of supplementation with inositols is able to ameliorate the HFD-induced electrical, structural and functional heart alterations including ventricular remodeling. Results provide a new insight into the cardioprotective effect of inositols, which could pave the way for a novel therapeutic approach to the treatment of HFD obesity–induced heart dysfunction.

Murine model of left ventricular diastolic dysfunction and electro-mechanical uncoupling following high-fat diet.

It is well established that obesity is an independent risk factor for cardiac death. In particular various cardiac alterations have been described in obese patients such as long QT on ECG, impaired diastolic filling of the left ventricle (LV), and all-type arrhythmias. In the present study, the above alterations were all reproduced in a mouse model of fat diet-induced obesity. In C57BL6 mice fed on a high fat (n = 20, HF-group) or standard diet (n = 20, C-group) for 13 weeks, balanced by sex and age, we examined heart morphology and function by high-frequency ultrasounds and electric activity by surface ECG. Besides, the autonomic sympathovagal balance (heart-rate variability) and the arrhythmogenic susceptibility to adrenergic challenge (i.p. isoproterenol) were compared in the two groups, as well as glucose tolerance (i.p. glucose test) and liver steatosis (ultrasounds). Body weight in HF-group exceeded C-group at the end of the experiment (+28% p < 0.01). An abnormal ventricular repolarization (long QTc on ECG) together with impaired LV filling rate and increased LV mass was found in HF-group as compared to C. Moreover, HF-group showed higher heart rate, unbalanced autonomic control with adrenergic prevalence and a greater susceptibility to develop rhythm disturbances under adrenergic challenge (i.p. isoprenaline). Impaired glucose tolerance and higher liver fat accumulation were also found in HF mice compared to C. The described murine model of 13 weeks on HF diet, well reproduced the cardiovascular and metabolic disorders reported in clinical obesity, suggesting its potential utility as translational mean suitable for testing new pharmaco-therapeutic approaches to the treatment of obesity and its comorbidity.

Atrial Transcriptional Profiles of Molecular Targets Mediating Electrophysiological Function in Aging and Pgc-1β Deficient Murine Hearts.

BackgroundDeficiencies in the transcriptional co-activator, peroxisome proliferative activated receptor, gamma, coactivator-1β are implicated in deficient mitochondrial function. The latter accompanies clinical conditions including aging, physical inactivity, obesity, and diabetes. Recent electrophysiological studies reported that Pgc-1β-/- mice recapitulate clinical age-dependent atrial pro-arrhythmic phenotypes. They implicated impaired chronotropic responses to adrenergic challenge, compromised action potential (AP) generation and conduction despite normal AP recovery timecourses and background resting potentials, altered intracellular Ca2+ homeostasis, and fibrotic change in the observed arrhythmogenicity.ObjectiveWe explored the extent to which these age-dependent physiological changes correlated with alterations in gene transcription in murine Pgc-1β-/- atria.Methods and ResultsRNA isolated from murine atrial tissue samples from young (12–16 weeks) and aged (>52 weeks of age), wild type (WT) and Pgc-1β-/- mice were studied by pre-probed quantitative PCR array cards. We examined genes encoding sixty ion channels and other strategic atrial electrophysiological proteins. Pgc-1β-/- genotype independently reduced gene transcription underlying Na+-K+-ATPase, sarcoplasmic reticular Ca2+-ATPase, background K+ channel and cholinergic receptor function. Age independently decreased Na+-K+-ATPase and fibrotic markers. Both factors interacted to alter Hcn4 channel activity underlying atrial automaticity. However, neither factor, whether independently or interactively, affected transcription of cardiac Na+, voltage-dependent K+ channels, surface or intracellular Ca2+ channels. Nor were gap junction channels, β-adrenergic receptors or transforming growth factor-β affected.ConclusionThese findings limit the possible roles of gene transcriptional changes in previously reported age-dependent pro-arrhythmic electrophysiologial changes observed in Pgc-1β-/- atria to an altered Ca2+-ATPase (Atp2a2) expression. This directly parallels previously reported arrhythmic mechanism associated with p21-activated kinase type 1 deficiency. This could add to contributions from the direct physiological outcomes of mitochondrial dysfunction, whether through reactive oxygen species (ROS) production or altered Ca2+ homeostasis.

Age-dependent electrocardiographic changes in Pgc-1β deficient murine hearts.

SummaryIncreasing evidence implicates chronic energetic dysfunction in human cardiac arrhythmias. Mitochondrial impairment through Pgc‐1β knockout is known to produce a murine arrhythmic phenotype. However, the cumulative effect of this with advancing age and its electrocardiographic basis have not been previously studied. Young (12‐16 weeks) and aged (>52 weeks), wild type (WT) (n = 5 and 8) and Pgc‐1β−/− (n = 9 and 6), mice were anaesthetised and used for electrocardiographic (ECG) recordings. Time intervals separating successive ECG deflections were analysed for differences between groups before and after β1‐adrenergic (intraperitoneal dobutamine 3 mg/kg) challenge. Heart rates before dobutamine challenge were indistinguishable between groups. The Pgc‐1β−/− genotype however displayed compromised nodal function in response to adrenergic challenge. This manifested as an impaired heart rate response suggesting a functional defect at the level of the sino‐atrial node, and a negative dromotropic response suggesting an atrioventricular conduction defect. Incidences of the latter were most pronounced in the aged Pgc‐1β−/− mice. Moreover, Pgc‐1β−/− mice displayed electrocardiographic features consistent with the existence of a pro‐arrhythmic substrate. Firstly, ventricular activation was prolonged in these mice consistent with slowed action potential conduction and is reported here for the first time. Additionally, Pgc‐1β−/− mice had shorter repolarisation intervals. These were likely attributable to altered K+ conductance properties, ultimately resulting in a shortened QTc interval, which is also known to be associated with increased arrhythmic risk. ECG analysis thus yielded electrophysiological findings bearing on potential arrhythmogenicity in intact Pgc‐1β−/− systems in widespread cardiac regions.

The effects of ageing and adrenergic challenge on electrocardiographic phenotypes in a murine model of long QT syndrome type 3

Long QT Syndrome 3 (LQTS3) arises from gain-of-function Nav1.5 mutations, prolonging action potential repolarisation and electrocardiographic (ECG) QT interval, associated with increased age-dependent risk for major arrhythmic events, and paradoxical responses to β-adrenergic agents. We investigated for independent and interacting effects of age and Scn5a+/ΔKPQ genotype in anaesthetised mice modelling LQTS3 on ECG phenotypes before and following β-agonist challenge, and upon fibrotic change. Prolonged ventricular recovery was independently associated with Scn5a+/ΔKPQ and age. Ventricular activation was prolonged in old Scn5a+/ΔKPQ mice (p = 0.03). We associated Scn5a+/ΔKPQ with increased atrial and ventricular fibrosis (both: p < 0.001). Ventricles also showed increased fibrosis with age (p < 0.001). Age and Scn5a+/ΔKPQ interacted in increasing incidences of repolarisation alternans (p = 0.02). Dobutamine increased ventricular rate (p < 0.001) and reduced both atrioventricular conduction (PR segment-p = 0.02; PR interval-p = 0.02) and incidences of repolarisation alternans (p < 0.001) in all mice. However, in Scn5a+/ΔKPQ mice, dobutamine delayed the changes in ventricular repolarisation following corresponding increases in ventricular rate. The present findings implicate interactions between age and Scn5a+/ΔKPQ in prolonging ventricular activation, correlating them with fibrotic change for the first time, adding activation abnormalities to established recovery abnormalities in LQTS3. These findings, together with dynamic electrophysiological responses to β-adrenergic challenge, have therapeutic implications for ageing LQTS patients.

P505The effects of ageing and adrenergic challenge on electrocardiographic phenotypes in a murine model of long QT syndrome type 3

P505The effects of ageing and adrenergic challenge on electrocardiographic phenotypes in a murine model of long QT syndrome type 3

Altered distribution of adrenergic constrictor responses contributes to skeletal muscle perfusion abnormalities in metabolic syndrome.

Although studies suggest elevated adrenergic activity paralleling metabolic syndrome in OZRs, the moderate hypertension and modest impact on organ perfusion question the multi-scale validity of these data. To understand how adrenergic function contributes to vascular reactivity in OZR, we utilized a multi-scale approach to investigate pressure responses, skeletal muscle blood flow, and vascular reactivity following adrenergic challenge. For OZR, adrenergic challenge resulted in increased pressor responses vs LZRs, mediated via α1 receptors, with minimal contribution by either ROS or NO bioavailability. In situ gastrocnemius muscle of OZR exhibited blunted functional hyperemia, partially restored with α1 inhibition, although improved muscle performance and VO2 required combined treatment with TEMPOL. Within OZR in situ cremaster muscle, proximal arterioles exhibited a more heterogeneous constriction to adrenergic challenge, biased toward hyperresponsiveness, vs LZR. This increasingly heterogeneous pattern was mirrored in ex vivo arterioles, mediated via α1 receptors, with roles for ROS and NO bioavailability evident in hyperresponsive vessels only. These results support the central role of the α1 adrenoreceptor for augmented pressor responses and elevations in vascular resistance, but identify an increased heterogeneity of constrictor reactivity in OZR that is presently of unclear purpose.

18 Metabolically Compromised Murine PGC-1B(-/-) Hearts Show Decreased action Potential Duration and Increased Arrhythmia with Adrenergic Challenge

IntroductionThe incidence of ventricular arrhythmia is predicted to rise due to obesity and diabetes. Disorders in the peroxisome proliferator-activated receptor-γ co-activators, PGC-1α and PGC-1β, are associated with both conditions. Homozygous...

Conduction Slowing Contributes to Spontaneous Ventricular Arrhythmias in Intrinsically Active Murine RyR2‐P2328S Hearts

The familial condition catecholaminergic polymorphic ventricular tachycardia (CPVT) is characterized by episodic bidirectional ventricular tachycardia (BVT), polymorphic ventricular tachycardia (PVT), and ventricular fibrillation following adrenergic challenge. It is associated with mutations involving the cardiac ryanodine receptor (RyR2). We explored for a slowing of myocardial conduction that could potentially result in a substrate for the spontaneous arrhythmogenesis that was observed following introduction of isoproterenol and caffeine in intrinsically beating murine RyR2-P2328S hearts. Such pharmacological challenge increased the number of arrhythmic episodes in electrocardiographic recordings from intact anesthetized mice, with the greatest effects in the homozygote RyR2(S/S). Arrhythmias took the form of bigeminy, BVT, monomorphic ventricular tachycardia, and PVT, as found in human CPVT. Ventricular epicardial conduction velocities (CVs) measured using multielectrode array recordings and maximum action potential upstroke rates, (dV/dt)(max), measured using intracellular microelectrodes were indistinguishable in untreated wild-type (WT) and RyR2(S/S). Pharmacological challenge of RyR2(S/S), but not WT hearts, then reduced CV and (dV/dt)(max) and also revealed a strongly arrhythmic phenotype. There was no evidence of gross structural or fibrotic changes in either RyR2(+/S) or RyR2(S/S) hearts on light microscopy. We associate altered ventricular myocardial CV potentially resulting in arrhythmogenic substrate with arrhythmic properties associated with genetic RyR2 alterations for the first time.

In vivo Electrophysiological Characterization of TASK-1 Deficient Mice

Background/Aims: TASK-1 is a potassium channel predominantly expressed in heart and brain. We have previously shown that anesthetized TASK-1^-/-mice have prolonged QT intervals in surface electrocardiograms (ECGs). In addition, heart rate variability quantified by time and frequency domain parameters was significantly altered in TASK-1^-/-mice with a sympathetic preponderance. Aims of the present study were the analysis of QT intervals by telemetric ECGs, to determine potential influences of anesthesia and β-adrenergic stimulation on repolarization in surface ECGs, to investigate in vivo electrophysiological parameters by intracardiac electrical stimulation and to quantify heart rate turbulence after ischemia/reperfusion or ventricular pacing in TASK-1^+/+ and TASK-1^-/-mice. Methods: Rate corrected QT intervals (QTc) were recorded in conscious mice by telemetry and in surface ECGs following administration of various anesthetics (tribromoethanol (Avertin®), pentobarbital and isoflurane). TASK-1^+/+ and TASK-1^-/mice were characterized by programmed electrical stimulation using an intracardiac octapolar catheter. The baroreceptor reflex was analyzed by heart rate turbulence (turbulence onset and slope) after ischemia/reperfusion and by stimulated premature ventricular contractions. Results: Telemetric and surface ECGs in mice sedated with Avertin®and pentobarbital, showed a significantly lengthened rate corrected QT interval in TASK-1^-/-mice (telemetry: TASK-1^+/+ 43±3ms vs. TASK-1^-/-49±5ms, n=6, p<0.05; Avertin®: TASK-1^+/+ 36±8ms vs. TASK-1^-/-48±4ms, n=13/16, p<0.0001). The prolongation of the QT interval was most pronounced at lower heart rates. Isoflurane, known for its stimulatory effects on the TASK channel family, attenuated the rate corrected QT interval prolongation in TASK-1^-/-mice. Intracardiac electrical stimulation revealed normal values for electrical conduction and refractoriness. No significant arrhythmias after atrial and ventricular burst stimulation were induced before and after adrenergic challenge in both genotypes. Turbulence onset after premature ventricular contraction was significantly altered in TASK-1^-/-mice. Conclusion: TASK-1^-/-mice exhibit a phenotype of QT prolongation, which distinct relation to heart rate. TASK-1 deficiency does neither alter key electrophysiological parameters nor increases atrial/ventricular vulnerability after electrical stimulation. The heart rate response after premature ventricular contractions is significantly abolished indicating a diminished baroreceptor reflex in TASK-1^-/-mice.

Ahnak1 is a tuneable modulator of cardiac Ca(v)1.2 calcium channel activity

Ahnak1 has been implicated in the beta-adrenergic regulation of the cardiac L-type Ca(2+) channel current (I (CaL)) by its binding to the regulatory Cavβ(2) subunit. In this study, we addressed the question whether ahnak1/Cavβ(2) interactions are essential or redundant for beta-adrenergic stimulation of I (CaL). Three naturally occurring ahnak1 variants (V5075M, G5242R, and T5796M) identified by genetic screening of cardiomyopathy patients did essentially not influence the in vitro Cavβ(2) interaction as assessed by recombinant proteins. But, we observed a robust increase in Cavβ(2) binding by mutating Ala at position 4984 to Pro which creates a PxxP consensus motif in the ahnak1 protein fragment. Surface plasmon resonance measurements revealed that this mutation introduced an additional Cavβ(2) binding site. The functionality of A4984P was supported by the specific action of the Pro-containing ahnak1-derived peptide (P4984) in beta-adrenergic regulation of I (CaL). Patch clamp recordings on cardiomyocytes showed that intracellular perfusion of P4984 markedly reduced I (CaL) response to the beta-adrenergic agonist, isoprenaline, while the Ala-containing counterpart failed to affect I (CaL). Interestingly, I (CaL) of ahnak1-deficient cardiomyocytes was not affected by peptide application. Moreover, I (CaL) of ahnak1-deficient cardiomyocytes showed intact beta-adrenergic responsiveness. Similarly isolated ahnak1-deficient mouse hearts responded normally to adrenergic challenge. Our results indicate that ahnak1 is not essential for beta-adrenergic up-regulation of I (CaL) and cardiac contractility in mice. But, tuning ahnak1/Cavβ(2) interaction provides a tool for modulating the beta-adrenergic response of I (CaL).

Alpha-2 adrenergic challenge with guanfacine one month after mild traumatic brain injury: Altered working memory and BOLD response

Alterations in working memory (WM) are common after traumatic brain injury (TBI). Frontal catecholaminergic systems, including the alpha-2 adrenergic system, modulate WM function and may be affected in TBI. We hypothesized that administration of an alpha-2 adrenergic agonist might improve WM after mild TBI (MTBI). Thirteen individuals with MTBI 1 month after injury and 14 healthy controls (HC) were challenged with guanfacine and placebo prior to administration of a verbal WM functional MRI task. Guanfacine was associated with improved WM performance in the MTBI but not the HC group. On guanfacine the MTBI group showed increased activation within a WM task-specific region of interest. Findings are consistent with the hypothesis that alterations in WM after MTBI may be improved with the alpha-2 agonist guanfacine.

E0180 The acute proarrhythmic effects of low concentration BPA on female adult rat and the electrophysiologic mechanisms

ObjectiveTo investigate the acute proarrhythmic effects of low concentration BPA on adult rat and to demonstrate the electrophysiologic mechanisms.Methods and resultsAcute exposure to BPA increased the contractility of cardiac myocytes...

Limited functional and metabolic improvements in hypertrophic and healthy rat heart overexpressing the skeletal muscle isoform of SERCA1 by adenoviral gene transfer in vivo

Adenoviral gene transfer of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a to the hypertrophic heart in vivo has been consistently reported to lead to enhanced myocardial contractility. It is unknown if the faster skeletal muscle isoform, SERCA1, expressed in the whole heart in early failure, leads to similar improvements and whether metabolic requirements are maintained during an adrenergic challenge. In this study, Ad.cmv.SERCA1 was delivered in vivo to aortic banded and sham-operated Sprague-Dawley rat hearts. The total SERCA content increased 34%. At 48-72 h posttransfer, echocardiograms were acquired, hearts were excised and retrograded perfused, and hemodynamics were measured parallel to NMR measures of the phosphocreatine (PCr)-to-ATP ratio (PCr/ATP) and energy substrate selection at basal and high workloads (isoproterenol). In the Langendorff mode, the rate-pressure product was enhanced 27% with SERCA1 in hypertrophic hearts and 10% in shams. The adrenergic response to isoproterenol was significantly potentiated in both groups with SERCA1. 31P NMR analysis of PCr/ATP revealed that the ratio remained low in the hypertrophic group with SERCA1 overexpression and was not further compromised with adrenergic challenge. 13C NMR analysis revealed fat and carbohydrate oxidation were unaffected at basal with SERCA1 expression; however, there was a shift from fats to carbohydrates at higher workloads with SERCA1 in both groups. Transport of NADH-reducing equivalents into the mitochondria via the alpha-ketoglutamate-malate transporter was not affected by either SERCA1 overexpression or adrenergic challenge in both groups. Echocardiograms revealed an important distinction between in vivo versus ex vivo data. In contrast to previous SERCA2a studies, the echocardiogram data revealed that SERCA1 expression compromised function (fractional shortening) in the hypertrophic group. Shams were unaffected. While our ex vivo findings support much of the earlier cardiomyocyte and transgenic data, the in vivo data challenge previous reports of improved cardiac function in heart failure models after SERCA intervention.