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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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Cardiomyocyte T-tubules are densely folded by BIN1. (α–b) Representative confocal images (a, scale bars: 5 µm) and the fluorescent profiles (b) of live WT and Bin1 HT cardiomyocytes labeled with Di-8-ANNEPS. (c) Quantification of T-tubules peak intensity. (n = 40 from 4–5 cells, P < 0.0001). (d) Cell size normalized membrane capacitance in WT (n = 14) and Bin1 HT (n = 12) cardiomyocytes (P = 0.0181). WC indicates reported whole cell capacitance without T-tubules. (e) 2D transmission electron microscope (TEM) images (Left to right: gross morphology, transverse cross section, and axial cross section) and 3D electron tomography images (right) of WT and Bin1 HT heart sections. Scale bars (left to right): 1 µm, 250 nm, 100 nm, and 100 nm. (f) Electron density profiles (middle) across individual T-tubules marked by the lines in the images above, with average T-tubule electron density in the bottom (n = 75, P < 0.0001). (g) T-tubule lumen area of axial cross sections (n = 80, P < 0.0001). (h) Cardiomyocyte T-tubule contour score (1, circular shape and no folds and spatial complexity; 2, non-circular shape and no folds and spatial complexity; or 3, multiple folds with spatial complexity) distribution (n = 196, P < 0.0001). Data are presented as mean ± SEM, cardiomyocytes are from three mice per genotype, and six left ventricular sections from three hearts per genotype were used for TEM analysis. Student’s t-test and one way-ANOVA were used for statistical analysis.
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Figure 1: Cardiomyocyte T-tubules are densely folded by BIN1. (α–b) Representative confocal images (a, scale bars: 5 µm) and the fluorescent profiles (b) of live WT and Bin1 HT cardiomyocytes labeled with Di-8-ANNEPS. (c) Quantification of T-tubules peak intensity. (n = 40 from 4–5 cells, P < 0.0001). (d) Cell size normalized membrane capacitance in WT (n = 14) and Bin1 HT (n = 12) cardiomyocytes (P = 0.0181). WC indicates reported whole cell capacitance without T-tubules. (e) 2D transmission electron microscope (TEM) images (Left to right: gross morphology, transverse cross section, and axial cross section) and 3D electron tomography images (right) of WT and Bin1 HT heart sections. Scale bars (left to right): 1 µm, 250 nm, 100 nm, and 100 nm. (f) Electron density profiles (middle) across individual T-tubules marked by the lines in the images above, with average T-tubule electron density in the bottom (n = 75, P < 0.0001). (g) T-tubule lumen area of axial cross sections (n = 80, P < 0.0001). (h) Cardiomyocyte T-tubule contour score (1, circular shape and no folds and spatial complexity; 2, non-circular shape and no folds and spatial complexity; or 3, multiple folds with spatial complexity) distribution (n = 196, P < 0.0001). Data are presented as mean ± SEM, cardiomyocytes are from three mice per genotype, and six left ventricular sections from three hearts per genotype were used for TEM analysis. Student’s t-test and one way-ANOVA were used for statistical analysis.

Mentions: Homozygous mice with global Bin1 deletion suffer perinatal death due to cardiomyopathy22. To explore the role of BIN1 in cardiac T-tubule organization, we generated a cardiac-specific deletion of Bin1 using α-myosin heavy chain αMHC-Cre+23 and loxP-flanked Bin1 lines to generate heterozygous (Bin1 HT, Bin1flox/+; αMHC-Cre+) and homozygous (Bin1 HO, Bin1flox/flox; αMHC-Cre+) mice. Use of the αMHC-Cre bypasses embryonic lethality24. At 8–12 weeks, the wildtype (WT), Bin1 HT (reduction in BIN1 similar to that in heart failure19) and Bin1 HO animals have similar overall body and heart phenotypes (Supplementary Figs. 1 and 2), consistent with high cardiac reserve typical for young adult hearts24. Adult cardiomyocytes were isolated from the WT and Bin1 HT animals and labeled with a plasma membrane lipid dye Di-8-ANNEPs for live-cell imaging by spinning disk confocal microscopy (Fig. 1a–c and Supplementary Fig. 3). We found that the distribution and regularity of T-tubules are preserved in cardiomyocytes from Bin1 HT hearts. However, T-tubule fluorescence is decreased in the Bin1 HT cells, indicating less membrane along T-tubule invaginations. Similar results were obtained when membrane structures of freshly-fixed cardiomyocytes were labeled with wheat germ agglutinin (WGA, Supplementary Fig. 3b). Electrophysiological measurement of cardiomyocyte T-tubule capacitance, which is 30% of total cardiomyocyte capacitance25, is decreased by 46% in Bin1 HT (Fig. 1d) cells with normal cell size (Supplementary Fig. 3c), confirming decreased T-tubule membrane in the cardiomyocytes that are deficient in BIN1. The decrease in T-tubule membrane occurs despite preserved cellular content of T-tubule proteins (Supplementary Fig. 4a).


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)

Cardiomyocyte T-tubules are densely folded by BIN1. (α–b) Representative confocal images (a, scale bars: 5 µm) and the fluorescent profiles (b) of live WT and Bin1 HT cardiomyocytes labeled with Di-8-ANNEPS. (c) Quantification of T-tubules peak intensity. (n = 40 from 4–5 cells, P < 0.0001). (d) Cell size normalized membrane capacitance in WT (n = 14) and Bin1 HT (n = 12) cardiomyocytes (P = 0.0181). WC indicates reported whole cell capacitance without T-tubules. (e) 2D transmission electron microscope (TEM) images (Left to right: gross morphology, transverse cross section, and axial cross section) and 3D electron tomography images (right) of WT and Bin1 HT heart sections. Scale bars (left to right): 1 µm, 250 nm, 100 nm, and 100 nm. (f) Electron density profiles (middle) across individual T-tubules marked by the lines in the images above, with average T-tubule electron density in the bottom (n = 75, P < 0.0001). (g) T-tubule lumen area of axial cross sections (n = 80, P < 0.0001). (h) Cardiomyocyte T-tubule contour score (1, circular shape and no folds and spatial complexity; 2, non-circular shape and no folds and spatial complexity; or 3, multiple folds with spatial complexity) distribution (n = 196, P < 0.0001). Data are presented as mean ± SEM, cardiomyocytes are from three mice per genotype, and six left ventricular sections from three hearts per genotype were used for TEM analysis. Student’s t-test and one way-ANOVA were used for statistical analysis.
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Related In: Results  -  Collection

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Figure 1: Cardiomyocyte T-tubules are densely folded by BIN1. (α–b) Representative confocal images (a, scale bars: 5 µm) and the fluorescent profiles (b) of live WT and Bin1 HT cardiomyocytes labeled with Di-8-ANNEPS. (c) Quantification of T-tubules peak intensity. (n = 40 from 4–5 cells, P < 0.0001). (d) Cell size normalized membrane capacitance in WT (n = 14) and Bin1 HT (n = 12) cardiomyocytes (P = 0.0181). WC indicates reported whole cell capacitance without T-tubules. (e) 2D transmission electron microscope (TEM) images (Left to right: gross morphology, transverse cross section, and axial cross section) and 3D electron tomography images (right) of WT and Bin1 HT heart sections. Scale bars (left to right): 1 µm, 250 nm, 100 nm, and 100 nm. (f) Electron density profiles (middle) across individual T-tubules marked by the lines in the images above, with average T-tubule electron density in the bottom (n = 75, P < 0.0001). (g) T-tubule lumen area of axial cross sections (n = 80, P < 0.0001). (h) Cardiomyocyte T-tubule contour score (1, circular shape and no folds and spatial complexity; 2, non-circular shape and no folds and spatial complexity; or 3, multiple folds with spatial complexity) distribution (n = 196, P < 0.0001). Data are presented as mean ± SEM, cardiomyocytes are from three mice per genotype, and six left ventricular sections from three hearts per genotype were used for TEM analysis. Student’s t-test and one way-ANOVA were used for statistical analysis.
Mentions: Homozygous mice with global Bin1 deletion suffer perinatal death due to cardiomyopathy22. To explore the role of BIN1 in cardiac T-tubule organization, we generated a cardiac-specific deletion of Bin1 using α-myosin heavy chain αMHC-Cre+23 and loxP-flanked Bin1 lines to generate heterozygous (Bin1 HT, Bin1flox/+; αMHC-Cre+) and homozygous (Bin1 HO, Bin1flox/flox; αMHC-Cre+) mice. Use of the αMHC-Cre bypasses embryonic lethality24. At 8–12 weeks, the wildtype (WT), Bin1 HT (reduction in BIN1 similar to that in heart failure19) and Bin1 HO animals have similar overall body and heart phenotypes (Supplementary Figs. 1 and 2), consistent with high cardiac reserve typical for young adult hearts24. Adult cardiomyocytes were isolated from the WT and Bin1 HT animals and labeled with a plasma membrane lipid dye Di-8-ANNEPs for live-cell imaging by spinning disk confocal microscopy (Fig. 1a–c and Supplementary Fig. 3). We found that the distribution and regularity of T-tubules are preserved in cardiomyocytes from Bin1 HT hearts. However, T-tubule fluorescence is decreased in the Bin1 HT cells, indicating less membrane along T-tubule invaginations. Similar results were obtained when membrane structures of freshly-fixed cardiomyocytes were labeled with wheat germ agglutinin (WGA, Supplementary Fig. 3b). Electrophysiological measurement of cardiomyocyte T-tubule capacitance, which is 30% of total cardiomyocyte capacitance25, is decreased by 46% in Bin1 HT (Fig. 1d) cells with normal cell size (Supplementary Fig. 3c), confirming decreased T-tubule membrane in the cardiomyocytes that are deficient in BIN1. The decrease in T-tubule membrane occurs despite preserved cellular content of T-tubule proteins (Supplementary Fig. 4a).

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