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Cardiac BIN1 folds T-tubule membrane, controlling ion flux and limiting arrhythmia.

Hong T, Yang H, Zhang SS, Cho HC, Kalashnikova M, Sun B, Zhang H, Bhargava A, Grabe M, Olgin J, Gorelik J, Marbán E, Jan LY, Shaw RM - Nat. Med. (2014)

Bottom Line: Bridging integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is downregulated in failing hearts.We also found that T-tubule inner folds are rescued by expression of the BIN1 isoform BIN1+13+17, which promotes N-WASP-dependent actin polymerization to stabilize the T-tubule membrane at cardiac Z discs.When the amount of the BIN1+13+17 isoform is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered, and arrhythmia can result.

View Article: PubMed Central - PubMed

Affiliation: 1] Cedars-Sinai Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA. [2].

ABSTRACT
Cardiomyocyte T tubules are important for regulating ion flux. Bridging integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is downregulated in failing hearts. Here we find that cardiac T tubules normally contain dense protective inner membrane folds that are formed by a cardiac isoform of BIN1. In mice with cardiac Bin1 deletion, T-tubule folding is decreased, which does not change overall cardiomyocyte morphology but leads to free diffusion of local extracellular calcium and potassium ions, prolonging action-potential duration and increasing susceptibility to ventricular arrhythmias. We also found that T-tubule inner folds are rescued by expression of the BIN1 isoform BIN1+13+17, which promotes N-WASP-dependent actin polymerization to stabilize the T-tubule membrane at cardiac Z discs. BIN1+13+17 recruits actin to fold the T-tubule membrane, creating a 'fuzzy space' that protectively restricts ion flux. When the amount of the BIN1+13+17 isoform is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered, and arrhythmia can result.

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Adult mouse cardiomyocytes express four Bin1 splice variants. (a) Cartoon of Bin1 exons and the splice variants we found in adult mouse cardiomyocytes. BAR, Bin–Amphiphysin–Rvs domain; PI, phosphoinositide binding domain; CLAP, clathrin / AP2 binding region; MDB, myc-binding domain; SH3, SRC Homology 3 domain. (b) Four Bin1 splice variants with alternative inclusion of exon 13 and 17 are detected in adult mouse cardiomyocytes (A.M.C.) using PCR detection with primer sets flanking exon 10–18 or exon 13–18. (c) The percent of each Bin1 variants in adult mouse cardiomyocytes after subcloning and sequencing using PCR primer sets flanking exon 10–18. (d) Quantitative rtPCR analysis of each Bin1 variants (Bin1/HPRT1) in purified neonatal cardiomyocytes (P3, n = 2 litters with 8–10 pups each) and isolated adult mouse cardiomyocytes (n = 5 mice). (e) Western blot analysis confirms the antibody specificity of anti-exon 17 (clone 99D, Sigma) and anti-exon 13 (A#5299, Anaspec) BIN1 antibodies. All four BIN1 isoforms are detected by panBIN1 antibody (rabbit anti BIN1 SH3 domain). (f) Immunofluorescence of anti-exon 17 and anti-exon 13 labeling (red arrow, Z-line/TT region by α-actinin or Cav1.2 co-labeling) in adult mouse cardiomyocytes. (g) Representative confocal images (left, scale bars: 5 µm) and fluorescent profiles (right) of Di-8-ANNEPS membrane labeling in WT and Bin1 HT cardiomyocytes over-expressing GFP, BIN1, BIN1+13, BIN1+17, or BIN1+13+17 (n = 5 cells). Data are presented as mean +/− SEM. *, P < 0.05; **, P < 0.01, and ***, P < 0.001 by student’s t-test or two-way ANOVA.
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Figure 5: Adult mouse cardiomyocytes express four Bin1 splice variants. (a) Cartoon of Bin1 exons and the splice variants we found in adult mouse cardiomyocytes. BAR, Bin–Amphiphysin–Rvs domain; PI, phosphoinositide binding domain; CLAP, clathrin / AP2 binding region; MDB, myc-binding domain; SH3, SRC Homology 3 domain. (b) Four Bin1 splice variants with alternative inclusion of exon 13 and 17 are detected in adult mouse cardiomyocytes (A.M.C.) using PCR detection with primer sets flanking exon 10–18 or exon 13–18. (c) The percent of each Bin1 variants in adult mouse cardiomyocytes after subcloning and sequencing using PCR primer sets flanking exon 10–18. (d) Quantitative rtPCR analysis of each Bin1 variants (Bin1/HPRT1) in purified neonatal cardiomyocytes (P3, n = 2 litters with 8–10 pups each) and isolated adult mouse cardiomyocytes (n = 5 mice). (e) Western blot analysis confirms the antibody specificity of anti-exon 17 (clone 99D, Sigma) and anti-exon 13 (A#5299, Anaspec) BIN1 antibodies. All four BIN1 isoforms are detected by panBIN1 antibody (rabbit anti BIN1 SH3 domain). (f) Immunofluorescence of anti-exon 17 and anti-exon 13 labeling (red arrow, Z-line/TT region by α-actinin or Cav1.2 co-labeling) in adult mouse cardiomyocytes. (g) Representative confocal images (left, scale bars: 5 µm) and fluorescent profiles (right) of Di-8-ANNEPS membrane labeling in WT and Bin1 HT cardiomyocytes over-expressing GFP, BIN1, BIN1+13, BIN1+17, or BIN1+13+17 (n = 5 cells). Data are presented as mean +/− SEM. *, P < 0.05; **, P < 0.01, and ***, P < 0.001 by student’s t-test or two-way ANOVA.

Mentions: To identify the BIN1 isoform(s) responsible for T-tubule membrane folding, we explored cardiac splicing of Bin1, which is a gene encoded by 20 exons (Fig. 5a). Using PCR detection and sequencing of cloned fragments with primers flanking the alternatively spliced region between exon 10 (or exon 13) and 18 (Fig. 5b,c), we found that Bin1 message in adult mouse cardiomyocytes consists of ubiquitous Bin1 (36% of all clones) and Bin1+17 (8%), as well as the alternatively-spliced cardiac variants Bin1+13 (48%) and Bin1+13+17 (8%). Quantitative rt-PCR confirms a similar expression pattern and further identifies an increase of gene expression of all the Bin1 variants in mature adult mouse cardiomyocytes relative to neonatal cardiomyocytes (Fig. 5d). Next, we used exon 13- or 17-specific BIN1 antibodies (Fig. 5e) to localize the distribution of BIN1 isoforms in adult mouse cardiomyocytes. Both anti-BIN1–13 and anti-BIN1–17 antibodies localized to anti-α-actinin and anti-Cav1.2 identified Z-line/T-tubules regions (Fig. 5f), indicating that BIN1+13+17 resides at T-tubules.


Cardiac BIN1 folds T-tubule membrane, controlling ion flux and limiting arrhythmia.

Hong T, Yang H, Zhang SS, Cho HC, Kalashnikova M, Sun B, Zhang H, Bhargava A, Grabe M, Olgin J, Gorelik J, Marbán E, Jan LY, Shaw RM - Nat. Med. (2014)

Adult mouse cardiomyocytes express four Bin1 splice variants. (a) Cartoon of Bin1 exons and the splice variants we found in adult mouse cardiomyocytes. BAR, Bin–Amphiphysin–Rvs domain; PI, phosphoinositide binding domain; CLAP, clathrin / AP2 binding region; MDB, myc-binding domain; SH3, SRC Homology 3 domain. (b) Four Bin1 splice variants with alternative inclusion of exon 13 and 17 are detected in adult mouse cardiomyocytes (A.M.C.) using PCR detection with primer sets flanking exon 10–18 or exon 13–18. (c) The percent of each Bin1 variants in adult mouse cardiomyocytes after subcloning and sequencing using PCR primer sets flanking exon 10–18. (d) Quantitative rtPCR analysis of each Bin1 variants (Bin1/HPRT1) in purified neonatal cardiomyocytes (P3, n = 2 litters with 8–10 pups each) and isolated adult mouse cardiomyocytes (n = 5 mice). (e) Western blot analysis confirms the antibody specificity of anti-exon 17 (clone 99D, Sigma) and anti-exon 13 (A#5299, Anaspec) BIN1 antibodies. All four BIN1 isoforms are detected by panBIN1 antibody (rabbit anti BIN1 SH3 domain). (f) Immunofluorescence of anti-exon 17 and anti-exon 13 labeling (red arrow, Z-line/TT region by α-actinin or Cav1.2 co-labeling) in adult mouse cardiomyocytes. (g) Representative confocal images (left, scale bars: 5 µm) and fluorescent profiles (right) of Di-8-ANNEPS membrane labeling in WT and Bin1 HT cardiomyocytes over-expressing GFP, BIN1, BIN1+13, BIN1+17, or BIN1+13+17 (n = 5 cells). Data are presented as mean +/− SEM. *, P < 0.05; **, P < 0.01, and ***, P < 0.001 by student’s t-test or two-way ANOVA.
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Figure 5: Adult mouse cardiomyocytes express four Bin1 splice variants. (a) Cartoon of Bin1 exons and the splice variants we found in adult mouse cardiomyocytes. BAR, Bin–Amphiphysin–Rvs domain; PI, phosphoinositide binding domain; CLAP, clathrin / AP2 binding region; MDB, myc-binding domain; SH3, SRC Homology 3 domain. (b) Four Bin1 splice variants with alternative inclusion of exon 13 and 17 are detected in adult mouse cardiomyocytes (A.M.C.) using PCR detection with primer sets flanking exon 10–18 or exon 13–18. (c) The percent of each Bin1 variants in adult mouse cardiomyocytes after subcloning and sequencing using PCR primer sets flanking exon 10–18. (d) Quantitative rtPCR analysis of each Bin1 variants (Bin1/HPRT1) in purified neonatal cardiomyocytes (P3, n = 2 litters with 8–10 pups each) and isolated adult mouse cardiomyocytes (n = 5 mice). (e) Western blot analysis confirms the antibody specificity of anti-exon 17 (clone 99D, Sigma) and anti-exon 13 (A#5299, Anaspec) BIN1 antibodies. All four BIN1 isoforms are detected by panBIN1 antibody (rabbit anti BIN1 SH3 domain). (f) Immunofluorescence of anti-exon 17 and anti-exon 13 labeling (red arrow, Z-line/TT region by α-actinin or Cav1.2 co-labeling) in adult mouse cardiomyocytes. (g) Representative confocal images (left, scale bars: 5 µm) and fluorescent profiles (right) of Di-8-ANNEPS membrane labeling in WT and Bin1 HT cardiomyocytes over-expressing GFP, BIN1, BIN1+13, BIN1+17, or BIN1+13+17 (n = 5 cells). Data are presented as mean +/− SEM. *, P < 0.05; **, P < 0.01, and ***, P < 0.001 by student’s t-test or two-way ANOVA.
Mentions: To identify the BIN1 isoform(s) responsible for T-tubule membrane folding, we explored cardiac splicing of Bin1, which is a gene encoded by 20 exons (Fig. 5a). Using PCR detection and sequencing of cloned fragments with primers flanking the alternatively spliced region between exon 10 (or exon 13) and 18 (Fig. 5b,c), we found that Bin1 message in adult mouse cardiomyocytes consists of ubiquitous Bin1 (36% of all clones) and Bin1+17 (8%), as well as the alternatively-spliced cardiac variants Bin1+13 (48%) and Bin1+13+17 (8%). Quantitative rt-PCR confirms a similar expression pattern and further identifies an increase of gene expression of all the Bin1 variants in mature adult mouse cardiomyocytes relative to neonatal cardiomyocytes (Fig. 5d). Next, we used exon 13- or 17-specific BIN1 antibodies (Fig. 5e) to localize the distribution of BIN1 isoforms in adult mouse cardiomyocytes. Both anti-BIN1–13 and anti-BIN1–17 antibodies localized to anti-α-actinin and anti-Cav1.2 identified Z-line/T-tubules regions (Fig. 5f), indicating that BIN1+13+17 resides at T-tubules.

Bottom Line: Bridging integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is downregulated in failing hearts.We also found that T-tubule inner folds are rescued by expression of the BIN1 isoform BIN1+13+17, which promotes N-WASP-dependent actin polymerization to stabilize the T-tubule membrane at cardiac Z discs.When the amount of the BIN1+13+17 isoform is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered, and arrhythmia can result.

View Article: PubMed Central - PubMed

Affiliation: 1] Cedars-Sinai Heart Institute, Cedars-Sinai Medical Center, Los Angeles, California, USA. [2].

ABSTRACT
Cardiomyocyte T tubules are important for regulating ion flux. Bridging integrator 1 (BIN1) is a T-tubule protein associated with calcium channel trafficking that is downregulated in failing hearts. Here we find that cardiac T tubules normally contain dense protective inner membrane folds that are formed by a cardiac isoform of BIN1. In mice with cardiac Bin1 deletion, T-tubule folding is decreased, which does not change overall cardiomyocyte morphology but leads to free diffusion of local extracellular calcium and potassium ions, prolonging action-potential duration and increasing susceptibility to ventricular arrhythmias. We also found that T-tubule inner folds are rescued by expression of the BIN1 isoform BIN1+13+17, which promotes N-WASP-dependent actin polymerization to stabilize the T-tubule membrane at cardiac Z discs. BIN1+13+17 recruits actin to fold the T-tubule membrane, creating a 'fuzzy space' that protectively restricts ion flux. When the amount of the BIN1+13+17 isoform is decreased, as occurs in acquired cardiomyopathy, T-tubule morphology is altered, and arrhythmia can result.

Show MeSH
Related in: MedlinePlus