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Increased expression of the auxiliary beta(2)-subunit of ventricular L-type Ca(2)+ channels leads to single-channel activity characteristic of heart failure.

Hullin R, Matthes J, von Vietinghoff S, Bodi I, Rubio M, D'Souza K, Friedrich Khan I, Rottländer D, Hoppe UC, Mohacsi P, Schmitteckert E, Gilsbach R, Bünemann M, Hein L, Schwartz A, Herzig S - PLoS ONE (2007)

Bottom Line: In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes.In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium.Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Swiss Heart Center Bern, University Hospital, Bern, Switzerland. roger.hullin@insel.ch

ABSTRACT

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 beta-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 beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(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 beta(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 beta(2) expression. Additional evidence for the cause-effect relationship between beta(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 beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(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 beta(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.

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Related in: MedlinePlus

Gating of single L-VDCC in ventricular myocytes from mice showing cardiac overexpression of Ca2+-channel subunits(a–e) 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 CaV1.2 (tg CaV1.2), the inducing compound tebufenozide (T) significantly increased single L-VDCC activity in ventricular myocytes from age-matched double-transgenics (tg Cav1.2×tgind β2a, showing an additional inducible β2a-overexpression) 48 h after drug administration. Overexpression of the β2a-subunit without overexpression of the human Cav1.2 does not alter single-channel gating (cp. Figure 4c). 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 Cav1.2 in Student's t-test. Number of underlying experiments is given in parentheses.(f) Exemplary traces of single-channel recordings from murine ventricular myocytes. Activity of single L-VDCC is clearly higher in old (≥9 months, failing) tg CaV1.2 compared to channels from young (4–5 months, non-failing) tg CaV1.2. Induction of β2-overexpression in hearts of young tgind β2a×tg CaV1.2 by tebufenozide mimicks the heart-failure phenotype of L-VDCC gating otherwise not observed until tg CaV1.2 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.
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pone-0000292-g005: Gating of single L-VDCC in ventricular myocytes from mice showing cardiac overexpression of Ca2+-channel subunits(a–e) 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 CaV1.2 (tg CaV1.2), the inducing compound tebufenozide (T) significantly increased single L-VDCC activity in ventricular myocytes from age-matched double-transgenics (tg Cav1.2×tgind β2a, showing an additional inducible β2a-overexpression) 48 h after drug administration. Overexpression of the β2a-subunit without overexpression of the human Cav1.2 does not alter single-channel gating (cp. Figure 4c). 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 Cav1.2 in Student's t-test. Number of underlying experiments is given in parentheses.(f) Exemplary traces of single-channel recordings from murine ventricular myocytes. Activity of single L-VDCC is clearly higher in old (≥9 months, failing) tg CaV1.2 compared to channels from young (4–5 months, non-failing) tg CaV1.2. Induction of β2-overexpression in hearts of young tgind β2a×tg CaV1.2 by tebufenozide mimicks the heart-failure phenotype of L-VDCC gating otherwise not observed until tg CaV1.2 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.

Mentions: Our functional analyses support the idea of pathophysiological relevance of β2-subunit up-regulation, but the parallel biophysical and biochemical changes in cardiomyocytes may still be coincidental. Rather than following the natural course of gene expression changes, transgene-controlled β2-subunit overexpression should prove its causative role in native tissue. A hybrid drosophila-bombyx ecdysone receptor (VgBmEcR) when coupled to an αMHC promoter should combine strictly drug-controlled, transgene-specific, and cardiac tissue-specific gene induction. We generated this double transgenic model of αMHC VgBmEcR and the (rat) β2a gene, hence referred to as tgind β2a, under control of the ecdysone response element (Figure 4a). Mice carrying both transgenic constructs developed normally and did not show any signs of developmental or cardiac dysfunction. In vivo induction with tebufenozide clearly increased cardiac β2-subunit expression in tgind β2a mice at the protein level (Figure 4b) proofing functionality of drug-controlled gene expression. However, the single L-VDCC phenotype after induction was not altered in this mouse when compared with either tebufenozide treated wild-type mice or sham-induced tgind β2a (Figure 4c, Figure 5a–e).


Increased expression of the auxiliary beta(2)-subunit of ventricular L-type Ca(2)+ channels leads to single-channel activity characteristic of heart failure.

Hullin R, Matthes J, von Vietinghoff S, Bodi I, Rubio M, D'Souza K, Friedrich Khan I, Rottländer D, Hoppe UC, Mohacsi P, Schmitteckert E, Gilsbach R, Bünemann M, Hein L, Schwartz A, Herzig S - PLoS ONE (2007)

Gating of single L-VDCC in ventricular myocytes from mice showing cardiac overexpression of Ca2+-channel subunits(a–e) 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 CaV1.2 (tg CaV1.2), the inducing compound tebufenozide (T) significantly increased single L-VDCC activity in ventricular myocytes from age-matched double-transgenics (tg Cav1.2×tgind β2a, showing an additional inducible β2a-overexpression) 48 h after drug administration. Overexpression of the β2a-subunit without overexpression of the human Cav1.2 does not alter single-channel gating (cp. Figure 4c). 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 Cav1.2 in Student's t-test. Number of underlying experiments is given in parentheses.(f) Exemplary traces of single-channel recordings from murine ventricular myocytes. Activity of single L-VDCC is clearly higher in old (≥9 months, failing) tg CaV1.2 compared to channels from young (4–5 months, non-failing) tg CaV1.2. Induction of β2-overexpression in hearts of young tgind β2a×tg CaV1.2 by tebufenozide mimicks the heart-failure phenotype of L-VDCC gating otherwise not observed until tg CaV1.2 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.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1808423&req=5

pone-0000292-g005: Gating of single L-VDCC in ventricular myocytes from mice showing cardiac overexpression of Ca2+-channel subunits(a–e) 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 CaV1.2 (tg CaV1.2), the inducing compound tebufenozide (T) significantly increased single L-VDCC activity in ventricular myocytes from age-matched double-transgenics (tg Cav1.2×tgind β2a, showing an additional inducible β2a-overexpression) 48 h after drug administration. Overexpression of the β2a-subunit without overexpression of the human Cav1.2 does not alter single-channel gating (cp. Figure 4c). 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 Cav1.2 in Student's t-test. Number of underlying experiments is given in parentheses.(f) Exemplary traces of single-channel recordings from murine ventricular myocytes. Activity of single L-VDCC is clearly higher in old (≥9 months, failing) tg CaV1.2 compared to channels from young (4–5 months, non-failing) tg CaV1.2. Induction of β2-overexpression in hearts of young tgind β2a×tg CaV1.2 by tebufenozide mimicks the heart-failure phenotype of L-VDCC gating otherwise not observed until tg CaV1.2 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.
Mentions: Our functional analyses support the idea of pathophysiological relevance of β2-subunit up-regulation, but the parallel biophysical and biochemical changes in cardiomyocytes may still be coincidental. Rather than following the natural course of gene expression changes, transgene-controlled β2-subunit overexpression should prove its causative role in native tissue. A hybrid drosophila-bombyx ecdysone receptor (VgBmEcR) when coupled to an αMHC promoter should combine strictly drug-controlled, transgene-specific, and cardiac tissue-specific gene induction. We generated this double transgenic model of αMHC VgBmEcR and the (rat) β2a gene, hence referred to as tgind β2a, under control of the ecdysone response element (Figure 4a). Mice carrying both transgenic constructs developed normally and did not show any signs of developmental or cardiac dysfunction. In vivo induction with tebufenozide clearly increased cardiac β2-subunit expression in tgind β2a mice at the protein level (Figure 4b) proofing functionality of drug-controlled gene expression. However, the single L-VDCC phenotype after induction was not altered in this mouse when compared with either tebufenozide treated wild-type mice or sham-induced tgind β2a (Figure 4c, Figure 5a–e).

Bottom Line: In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes.In accordance, beta(2)-subunit mRNA and protein are up-regulated in failing human myocardium.Interestingly, these animals, when still young and non-failing ("Adaptive Phase"), reveal the opposite phenotype, viz: reduced single-channel activity accompanied by lowered beta(2) expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, Swiss Heart Center Bern, University Hospital, Bern, Switzerland. roger.hullin@insel.ch

ABSTRACT

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 beta-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 beta-subunit gene products. In HEK293-cells we show differential modulation of single L-VDCC activity by coexpression of several human cardiac beta-subunits: Unlike beta(1) or beta(3) isoforms, beta(2a) and beta(2b) induce a high-activity channel behavior typical of failing myocytes. In accordance, beta(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 beta(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 beta(2) expression. Additional evidence for the cause-effect relationship between beta(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 beta(2) cardiac overexpression. Here in non-failing hearts induction of beta(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 beta(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.

Show MeSH
Related in: MedlinePlus