Increased Expression of the Auxiliary β(2)-subunit of Ventricular L-type Ca(2+) Channels Leads to Single-Channel Activity Characteristic of Heart Failure

BACKGROUND: Increased activity of single ventricular L-type Ca(2+)-channels (L-VDCC) is a hallmark in human heart failure. Recent findings suggest differential modulation by several auxiliary β-subunits as a possible explanation. METHODS AND RESULTS: By molecular and functional analyses of human and murine ventricles, we find that enhanced L-VDCC activity is accompanied by altered expression pattern of auxiliary L-VDCC β-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac β-subunits: Unlike β(1) or β(3) isoforms, β(2a) and β(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, β(2)-subunit mRNA and protein are up-regulated in failing human myocardium. In a model of heart failure we find that mice overexpressing the human cardiac Ca(V)1.2 also reveal increased single-channel activity and sarcolemmal β(2) expression when entering into the maladaptive stage of heart failure. Interestingly, these animals, when still young and non-failing (“Adaptive Phase”), reveal the opposite phenotype, viz : reduced single-channel activity accompanied by lowered β(2) expression. Additional evidence for the cause-effect relationship between β(2)-subunit expression and single L-VDCC activity is provided by newly engineered, double-transgenic mice bearing both constitutive Ca(V)1.2 and inducible β(2) cardiac overexpression. Here in non-failing hearts induction of β(2)-subunit overexpression mimicked the increase of single L-VDCC activity observed in murine and human chronic heart failure. CONCLUSIONS: Our study presents evidence of the pathobiochemical relevance of β(2)-subunits for the electrophysiological phenotype of cardiac L-VDCC and thus provides an explanation for the single L-VDCC gating observed in human and murine heart failure

Charged-particle multiplicities in pp interactions at root s=900 GeV measured with the ATLAS detector at the LHC ATLAS Collaboration

The first measurements from proton–proton collisions recorded with the ATLAS detector at the LHC are presented. Data were collected in December 2009 using a minimum-bias trigger during collisions at a centre-of-mass energy of 900 GeV. The charged-particle multiplicity, its dependence on transverse momentum and pseudorapidity, and the relationship between mean transverse momentum and charged-particle multiplicity are measured for events with at least one charged particle in the kinematic range |η|500 MeVpT>500 MeV. The measurements are compared to Monte Carlo models of proton–proton collisions and to results from other experiments at the same centre-of-mass energy. The charged-particle multiplicity per event and unit of pseudorapidity at η=0η=0 is measured to be 1.333±0.003(stat.)±0.040(syst.)1.333±0.003(stat.)±0.040(syst.), which is 5–15% higher than the Monte Carlo models predict

Diagnostic role of coronary CT angiography in paroxysmal or first diagnosed atrial fibrillation

Objectives The presence of coronary artery disease (CAD) in patients hospitalised with paroxysmal or first diagnosed atrial fibrillation (AF) has major implications for antithrombotic therapy and cardiovascular event rate. Coronary CT angiography (CCTA) is a feasible tool to identify patients with concealed CAD. We aimed to evaluate the diagnostic role of early CCTA in patients hospitalised with paroxysmal or first diagnosed AF.Methods In a 5-year single-centre retrospective analysis, 566 patients with paroxysmal or first diagnosed AF who underwent CCTA were enrolled to investigate the presence of CAD.Results In patients with paroxysmal or first diagnosed AF, CCTA revealed CAD (coronary artery stenosis ≥50%) in 39.2%. Cardiac catheterisation was performed in 31.6%, confirming CAD in 13.1% of all patients. In 8.0% percutaneous coronary intervention and in 0.5% coronary artery bypass grafting was performed. In patients with paroxysmal or first diagnosed AF: (1) angina pectoris per se does not predict CAD; (2) multivariable regression analysis revealed age, male sex and diabetes as risk factors for CAD in AF; (3) Framingham Risk Score for coronary heart disease and CHA2DS2-VASc-Score were relevant risk scores of CAD and (4) the classification of Coronary Artery Calcium score reference values according to the Multi-Ethnic Study of Atherosclerosis was a predictor of CAD.Conclusion Patients with paroxysmal or first diagnosed AF are at risk for CAD, while CCTA is a feasible diagnostic tool for CAD. We recommend to integrate CT calcium scoring and CCTA into the diagnostic workup of patients with new-onset or paroxysmal AF

Subunit expression of cardiac L-VDCC subunits in human myocardial specimens

<p><b>(a)</b> Human specimens from non-failing (NF) and failing (F) myocardium (n = 4–5) were analyzed in immunoblots using specific polyclonal antibodies directed against the particular L-VDCC subunits. <b>(b)</b> L-VDCC subunit expression was normalized to cardiac calsequestrin protein expression in the same sample (number of NF/F specimens was always identical for each subunit; n = 5–8). Quantitative analysis of subunit protein expression is depicted as ratio of F vs. NF. * p<0.001; ** p<0.0001. <b>(c)</b> mRNA expression of β-subunit isoforms (NF: n = 5; F: n = 9–13) was measured by real time PCR, and always normalized to cardiac calsequestrin mRNA expression. * p<0.05.</p

Protein expression of cardiac L-VDCC subunits in old wild-type and tg Ca_V1.2 mice

<div><p><b>(a)</b> Specimens from old wild-type mice and tg Ca_V1.2 in heart failure were analyzed in immunoblots using specific polyclonal antibodies directed against the particular L-VDCC subunits.</p><p><b>(b)</b> Protein expression of L-VDCC subunits was always normalized to cardiac calsequestrin protein expression in the same sample. Quantitative analysis of subunit protein expression is depicted as ratio of 10 months old tg Ca_V1.2 vs. age-matched wild-type. β₁ protein bands were faint, and thus not analyzed quantitatively (number of WT/old tg Ca_V1.2 specimens was always identical for each subunit; n = 4). * p<0.05.</p></div

tg mouse model with an inducible cardiac overexpression of the β_2a under control of a hybrid bombyx-ecdysone receptor

<div><p><b>(a)</b> For cardiac-specific expression the hybrid bombyx-ecdysone receptor (VgBmEcR) was placed under the control of αMHC promoter (for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#s4" target="_blank">Materials and Methods</a>). Transgenic mice (tg_ind β_2a) positive for the hybrid bombyx-ecdysone receptor and the construct of the ecdysone response element (EcRE) and the β_2a, respectively, are identified in Southern blots. The radiolabeled probe specific for the coding sequence of VgBmEcR was generated by SacI digestion. It hybridized to a 3.7 kb band in transgene mouse genomic DNA digested by EcoRI digest; the radiolabeled DNA probe specific for the coding sequence of β_2a was generated by HindIII/KpnI digest. It hybridized to a 2.4 kb band of genomic DNA digested with HindIII/BamHI in tg_ind β_2a but not in WT. </p><p><b>(b)</b> 48 h after treatment with the inducing drug tebufenozide (+T) Western-blot analysis with ventricular tissue from 4–5 month old mice reveals increased expression of β₂-protein in tg_ind β_2a compared to treated wild-type or sham-induced transgenics.</p><p><b>(c)</b> Exemplary traces of single-channel recordings from murine ventricular myocytes. Induction of cardiac overexpression of the β_2a (+T) does not alter single-channel behavior compared to either wild-type mice after treatment with the inducing drug or i.p-application of only the vehicle (water/oil-emulsion) to β_2a-transgenic mice (“sham”). Data were obtained by patch-clamp recordings using cell-attached configuration (charge carrier: 70 mM Ba²⁺; holding potential: −100 mV; test potential: +20 mV for 150 ms). Bottom traces show ensemble average currents from the respective experiment.</p></div

Single L-VDCC gating of young and old tg Ca_V1.2 resembles data obtained from human non-failing and failing ventricle

<p>In a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#pone.0000292-Schroder1" target="_blank">[6]</a> we found single-channel activity to be significantly increased in ventricular myocytes from human hearts failing due to idiopathic dilated cardiomyopathy compared to non-failing ventricles. In excellent agreement the present study reveals activity of single L-VDCC from ≥9 months old, i.e. failing murine hearts overexpressing the human Ca_V1.2 to be significantly increased compared to single-channel activity in 4 months old, <b><i>i.e.</i></b> non-failing young transgenics obtained in a previous study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#pone.0000292-Groner1" target="_blank">[16]</a>. Charge carrier: 70 mM Ba²⁺; holding potential: −100 mV; test potential: +20 mV. Note that Schroder et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#pone.0000292-Schroder1" target="_blank">[6]</a> did not use a depolarizing bath solution, thus potentials are approximate values.</p>*<p>p<0.05 and ⁽*⁾p = 0.07 in a Student's t-test; ^†p<0.05 in a Mann-Whitney test (performed when data failed normality test). Numbers of experiments given in parentheses indicate number of experiments with only one channel in the patch.</p

Gating of single L-VDCC in ventricular myocytes from mice showing cardiac overexpression of Ca²⁺-channel subunits

<div><p><b>(a–e)</b> Single-channel gating parameters of ventricular L-VDCC from murine hearts. Compared to 4–5 months old mice showing a cardiac overexpression of the human Ca_V1.2 (tg Ca_V1.2), the inducing compound tebufenozide (T) significantly increased single L-VDCC activity in ventricular myocytes from age-matched double-transgenics (tg Ca_v1.2×tg_ind β_2a, showing an additional inducible β_2a-overexpression) 48 h after drug administration. Overexpression of the β_2a-subunit without overexpression of the human Ca_v1.2 does not alter single-channel gating (cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000292#pone-0000292-g004" target="_blank">Figure 4c</a>). Tebufenozide treatment does not affect single-channel gating in ventricular myocytes from wild-type mice. Data were obtained by patch-clamp recordings using cell-attached configuration (charge carrier: 70 mM Ba²⁺; holding potential: −100 mV; test potential: +20 mV for 150 ms). *p<0.05 and ⁽*⁾p<0.09 compared to tg Ca_v1.2 in Student's t-test. Number of underlying experiments is given in parentheses.</p><p><b>(f)</b> Exemplary traces of single-channel recordings from murine ventricular myocytes. Activity of single L-VDCC is clearly higher in old (≥9 months, failing) tg Ca_V1.2 compared to channels from young (4–5 months, non-failing) tg Ca_V1.2. Induction of β₂-overexpression in hearts of young tg_ind β_2a×tg Ca_V1.2 by tebufenozide mimicks the heart-failure phenotype of L-VDCC gating otherwise not observed until tg Ca_V1.2 enter the “Maladaptive State” at an age ≥9 months. T = tebufenozide. Data were obtained by patch-clamp recordings using the cell-attached configuration (charge carrier: 70 mM Ba²⁺; holding potential: −100 mV; test potential: +20 mV for 150 ms). Bottom traces show average currents from the respective experiment.</p></div