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

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

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

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