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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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BIN1+13+17 uses F-actin to connect to Z-disc α-actinin. (a–b) HeLa cells expressing GFP tagged BIN1, BIN1+13, BIN1+17, and BIN1+13+17 (scale bars: 10 µm) (a), with the length of folds like structure (linear streaks) quantified in (b). (Mean ± SEM; n = 20 folds from 5 cells; *** indicates P < 0.001 by one-way ANOVA). (c) TEM confirms that BIN1+13+17 but not BIN1+17 induces elongated membrane folds in HeLa cells. Scale bars: 1 µm (left) and 0.5 µm (right two panels). (d) HeLa cells expressing isoforms of GFP-BIN1 (green) and LifeAct-mCherry (red) (scale bars: 10 µm). (e) GST pulldown of GST-BIN1 isoforms and N-WASP-V5 in HeLa cells. (f) In vitro pyrene-actin polymerization assay using purified Arp2/3, N-WASP and BIN1 isoforms. Left, representative tracing of actin polymerization kinetics. Right, the Vmax data of polymerization kinetics. Data are presented as mean ± SEM (n = 5, * indicates P < 0.05 by one-way ANOVA). The negative control contains pyrene-actin alone with a GST control protein (GST-GFP, bottom black line indicated by the bottom arrow), the positive control contains pyrene-actin supplemented with Arp2/3 and VCA (active domain of N-WASP, top black line indicate by the top arrow), and the rest samples contain pyrene-actin supplemented with Arp2/3, N-WASP with GST-GFP or 1 µM GST-BIN1 isoforms. (g) Purified GST-BIN1 fusion protein pre-coated glutathione beads were added to adult heart lysates for pulldowns of α-actinin (right) or F-actin (left). (h) Schematic illustration of BIN1+13+17 forming an extracellular ionic diffusion barrier inside T-tubules.
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Figure 6: BIN1+13+17 uses F-actin to connect to Z-disc α-actinin. (a–b) HeLa cells expressing GFP tagged BIN1, BIN1+13, BIN1+17, and BIN1+13+17 (scale bars: 10 µm) (a), with the length of folds like structure (linear streaks) quantified in (b). (Mean ± SEM; n = 20 folds from 5 cells; *** indicates P < 0.001 by one-way ANOVA). (c) TEM confirms that BIN1+13+17 but not BIN1+17 induces elongated membrane folds in HeLa cells. Scale bars: 1 µm (left) and 0.5 µm (right two panels). (d) HeLa cells expressing isoforms of GFP-BIN1 (green) and LifeAct-mCherry (red) (scale bars: 10 µm). (e) GST pulldown of GST-BIN1 isoforms and N-WASP-V5 in HeLa cells. (f) In vitro pyrene-actin polymerization assay using purified Arp2/3, N-WASP and BIN1 isoforms. Left, representative tracing of actin polymerization kinetics. Right, the Vmax data of polymerization kinetics. Data are presented as mean ± SEM (n = 5, * indicates P < 0.05 by one-way ANOVA). The negative control contains pyrene-actin alone with a GST control protein (GST-GFP, bottom black line indicated by the bottom arrow), the positive control contains pyrene-actin supplemented with Arp2/3 and VCA (active domain of N-WASP, top black line indicate by the top arrow), and the rest samples contain pyrene-actin supplemented with Arp2/3, N-WASP with GST-GFP or 1 µM GST-BIN1 isoforms. (g) Purified GST-BIN1 fusion protein pre-coated glutathione beads were added to adult heart lysates for pulldowns of α-actinin (right) or F-actin (left). (h) Schematic illustration of BIN1+13+17 forming an extracellular ionic diffusion barrier inside T-tubules.

Mentions: To understand how BIN1+13+17 organizes T-tubule membrane, we expressed GFP-tagged BIN1 isoforms in HeLa cells. Both confocal and TEM (Fig. 6a–c) imaging identified that only BIN1+13+17 induces formation of elongated, F-actin associated, membrane folds (Fig. 6d), indicating a role of actin in BIN1+13+17-mediated fold formation. Cytochalasin D induced stabilization of the barbed ends of F-actin at Z-discs is known to help preserve T-tubule structure of cultured cardiomyocytes34. We found that actin stabilization by cytochalasin D increases, whereas actin disruption by latrunculin A decreases, cardiac T-tubule membrane intensity (Supplementary Fig. 6). To further explore the differential roles of BIN1 isoforms in organizing actin, we studied their interaction with the actin polymerizing protein N-WASP, a known binding partner of the BAR domain protein Amphiphysin 135. Interestingly, although BIN1+13 does not bind to N-WASP (Fig. 6e), BIN1+13+17 does and can activate N-WASP to promote Arp2/3 nucleated actin polymerization (Fig. 6f). Biochemical GST pull-down confirms that BIN1+13+17 is associated with F-actin and α-actinin in adult mouse hearts (Fig. 6g). These data indicate that BIN1+13+17 binds to and activates N-WASP function to form elongated F-actin polymers for the development of membrane folds, and binds to Z-disc α-actinin to maintain these membrane folds, creating an ionic diffusion barrier within cardiac T-tubules (cartoon in Fig. 6h).


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)

BIN1+13+17 uses F-actin to connect to Z-disc α-actinin. (a–b) HeLa cells expressing GFP tagged BIN1, BIN1+13, BIN1+17, and BIN1+13+17 (scale bars: 10 µm) (a), with the length of folds like structure (linear streaks) quantified in (b). (Mean ± SEM; n = 20 folds from 5 cells; *** indicates P < 0.001 by one-way ANOVA). (c) TEM confirms that BIN1+13+17 but not BIN1+17 induces elongated membrane folds in HeLa cells. Scale bars: 1 µm (left) and 0.5 µm (right two panels). (d) HeLa cells expressing isoforms of GFP-BIN1 (green) and LifeAct-mCherry (red) (scale bars: 10 µm). (e) GST pulldown of GST-BIN1 isoforms and N-WASP-V5 in HeLa cells. (f) In vitro pyrene-actin polymerization assay using purified Arp2/3, N-WASP and BIN1 isoforms. Left, representative tracing of actin polymerization kinetics. Right, the Vmax data of polymerization kinetics. Data are presented as mean ± SEM (n = 5, * indicates P < 0.05 by one-way ANOVA). The negative control contains pyrene-actin alone with a GST control protein (GST-GFP, bottom black line indicated by the bottom arrow), the positive control contains pyrene-actin supplemented with Arp2/3 and VCA (active domain of N-WASP, top black line indicate by the top arrow), and the rest samples contain pyrene-actin supplemented with Arp2/3, N-WASP with GST-GFP or 1 µM GST-BIN1 isoforms. (g) Purified GST-BIN1 fusion protein pre-coated glutathione beads were added to adult heart lysates for pulldowns of α-actinin (right) or F-actin (left). (h) Schematic illustration of BIN1+13+17 forming an extracellular ionic diffusion barrier inside T-tubules.
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Figure 6: BIN1+13+17 uses F-actin to connect to Z-disc α-actinin. (a–b) HeLa cells expressing GFP tagged BIN1, BIN1+13, BIN1+17, and BIN1+13+17 (scale bars: 10 µm) (a), with the length of folds like structure (linear streaks) quantified in (b). (Mean ± SEM; n = 20 folds from 5 cells; *** indicates P < 0.001 by one-way ANOVA). (c) TEM confirms that BIN1+13+17 but not BIN1+17 induces elongated membrane folds in HeLa cells. Scale bars: 1 µm (left) and 0.5 µm (right two panels). (d) HeLa cells expressing isoforms of GFP-BIN1 (green) and LifeAct-mCherry (red) (scale bars: 10 µm). (e) GST pulldown of GST-BIN1 isoforms and N-WASP-V5 in HeLa cells. (f) In vitro pyrene-actin polymerization assay using purified Arp2/3, N-WASP and BIN1 isoforms. Left, representative tracing of actin polymerization kinetics. Right, the Vmax data of polymerization kinetics. Data are presented as mean ± SEM (n = 5, * indicates P < 0.05 by one-way ANOVA). The negative control contains pyrene-actin alone with a GST control protein (GST-GFP, bottom black line indicated by the bottom arrow), the positive control contains pyrene-actin supplemented with Arp2/3 and VCA (active domain of N-WASP, top black line indicate by the top arrow), and the rest samples contain pyrene-actin supplemented with Arp2/3, N-WASP with GST-GFP or 1 µM GST-BIN1 isoforms. (g) Purified GST-BIN1 fusion protein pre-coated glutathione beads were added to adult heart lysates for pulldowns of α-actinin (right) or F-actin (left). (h) Schematic illustration of BIN1+13+17 forming an extracellular ionic diffusion barrier inside T-tubules.
Mentions: To understand how BIN1+13+17 organizes T-tubule membrane, we expressed GFP-tagged BIN1 isoforms in HeLa cells. Both confocal and TEM (Fig. 6a–c) imaging identified that only BIN1+13+17 induces formation of elongated, F-actin associated, membrane folds (Fig. 6d), indicating a role of actin in BIN1+13+17-mediated fold formation. Cytochalasin D induced stabilization of the barbed ends of F-actin at Z-discs is known to help preserve T-tubule structure of cultured cardiomyocytes34. We found that actin stabilization by cytochalasin D increases, whereas actin disruption by latrunculin A decreases, cardiac T-tubule membrane intensity (Supplementary Fig. 6). To further explore the differential roles of BIN1 isoforms in organizing actin, we studied their interaction with the actin polymerizing protein N-WASP, a known binding partner of the BAR domain protein Amphiphysin 135. Interestingly, although BIN1+13 does not bind to N-WASP (Fig. 6e), BIN1+13+17 does and can activate N-WASP to promote Arp2/3 nucleated actin polymerization (Fig. 6f). Biochemical GST pull-down confirms that BIN1+13+17 is associated with F-actin and α-actinin in adult mouse hearts (Fig. 6g). These data indicate that BIN1+13+17 binds to and activates N-WASP function to form elongated F-actin polymers for the development of membrane folds, and binds to Z-disc α-actinin to maintain these membrane folds, creating an ionic diffusion barrier within cardiac T-tubules (cartoon in Fig. 6h).

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