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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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Subunit expression of cardiac L-VDCC subunits in human myocardial specimens(a) 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) 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. (c) 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.
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pone-0000292-g001: Subunit expression of cardiac L-VDCC subunits in human myocardial specimens(a) 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) 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. (c) 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.

Mentions: Protein expression of CaV1.2, α2δ, low molecular weight β1, and β3 was similar in non-failing and failing human myocardium, but we found a significant up-regulation of β2 (Figure 1a,b). There was no difference in gene expression of the CaV1.2, and the α2δ at the protein level (mRNA data not shown). At least two β1-subunit isoforms (β1a,c), four β2-subunit isoforms (β2a–d), and two β3-subunit isoforms (β3a,trunc) are expressed at relevant levels in human myocardium [12]. β1a (GenBank No NM_199247) and β1c (GenBank_199248) are sequence-identical except for replacement of exon 7a by exon 7b in β1c, consistent with previous work [23]. β2a–d isoforms differ only with respect to the N-terminal region (D1 domain). Quantitation by real-time PCR revealed an increased expression of β1c and all β2 isoforms in heart failure, in line with the protein data (Figure 1c).


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)

Subunit expression of cardiac L-VDCC subunits in human myocardial specimens(a) 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) 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. (c) 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC1808423&req=5

pone-0000292-g001: Subunit expression of cardiac L-VDCC subunits in human myocardial specimens(a) 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) 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. (c) 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.
Mentions: Protein expression of CaV1.2, α2δ, low molecular weight β1, and β3 was similar in non-failing and failing human myocardium, but we found a significant up-regulation of β2 (Figure 1a,b). There was no difference in gene expression of the CaV1.2, and the α2δ at the protein level (mRNA data not shown). At least two β1-subunit isoforms (β1a,c), four β2-subunit isoforms (β2a–d), and two β3-subunit isoforms (β3a,trunc) are expressed at relevant levels in human myocardium [12]. β1a (GenBank No NM_199247) and β1c (GenBank_199248) are sequence-identical except for replacement of exon 7a by exon 7b in β1c, consistent with previous work [23]. β2a–d isoforms differ only with respect to the N-terminal region (D1 domain). Quantitation by real-time PCR revealed an increased expression of β1c and all β2 isoforms in heart failure, in line with the protein data (Figure 1c).

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