Journal: Scientific reports

Article Title: Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure.

doi: 10.1038/s41598-019-42592-1

Figure Lengend Snippet: Figure 5. Altered protein abundance of putative regulators of cardiac transverse tubule formation. Representative immunoblots (upper) and summary histograms (lower) for: (A) JPH2; (B) TCAP and (C) MTM1. (D) Changes in AmpII protein abundance showing (upper) representative AmpII immunoblot and (lower) summary data (a); correlation between AmpII protein abundance and TT half distance (b) and TT fractional area (c). Solid lines through data in b. and c. are linear regressions with slopes significantly different from zero (*p < 0.05). For immunoblots (A–D) N = control, 7; 4-weeks, 6; HF, 7; tadalafil, 8 and mean of 3 technical replicates. *p < 0.05 vs. control; #p < 0.05 vs. tadalafil; by linear mixed models analysis. For regression analysis: n cells/N hearts; control, 30/ 6; 4-weeks, 25/4; HF, 29/6; tadalafil, 71/6. All data presented as mean ± SEM.

Article Snippet: NRVMs (1–3 days post isolation) were transiently transfected with either variant 8 of human BIN1 (AmpII) (Origene Inc, USA) cloned into pCMV6-AC-mKate2 entry vector (Origene Inc, USA) or pCMV6-mKate2 as a negative control.

Techniques: Quantitative Proteomics, Western Blot, Control

Journal: Scientific reports

Article Title: Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure.

doi: 10.1038/s41598-019-42592-1

Figure Lengend Snippet: Figure 6. Amphiphysin II drives de novo tubule formation in neonatal rat ventricular myocytes and iPSC derived cardiac myocytes. (A) Assessment of sheep myocardial AmpII isoform expression showing (left to right) representative Qiaxcel run and summary data on sheep cardiac isoform abundance. N = 4 hearts. (B) Representative immunoblot (upper) and summary data (lower) showing up-regulation of AmpII protein abundance following transient transfection of NRVMs. N = 2 isolations. (C) Representative images of NRVMs transfected with mKate2 control (a) vector or AmpII expression vector (b–d). Extracellular Oregon Green 488 imaging showing a transfected and non-transfected (NT) cell (c) and overlay of the mKate2 channel and OG488N channel (from c) showing co-localisation of markers and patency of AmpII driven tubules only in the transfected cell (d). (D) Mean data summarizing percentage of non-transfected (NT), mKate2 control vector and AmpII expression vector transfected cells with tubule structures. N = 6 isolations (non-transfected, 67 cells; mKate2, 23 cells; AmpII, 37 cells) cells in each group. (E) Mean data summarising fractional area of cells occupied by tubules in mKate2 control vector and AmpII expression vector transfected cells. N = 5 isolations (mKate2, 20 cells; AmpII, 29 cells). (F) Representative images showing AmpII driven tubule formation in iCell iPSC derived cardiac myocytes. (G) Mean data summarizing percentage of non-transfected (NT), mKate2 control vector and AmpII expression vector transfected iCell iPSC cardiac myocytes with tubule structures (N = 2 cell batches; non-transfected, 45 cells; mKate2, 17 cells; AmpII, 40 cells). (H) Mean data summarizing fractional area occupied by tubules in iCell cardiac myocytes transfected with mKate2 or AmpII vectors. N = 2 cell batches (makte2, 17 cells; AmpII, 29 cells). **p < 0.01 vs mKate2; ***p < 0.001 vs mKate2; #p < 0.05 vs NT; by linear mixed models analysis. Scale bars, 10 µm. Panels A, C, D, F & G show mean ± SEM.

Article Snippet: NRVMs (1–3 days post isolation) were transiently transfected with either variant 8 of human BIN1 (AmpII) (Origene Inc, USA) cloned into pCMV6-AC-mKate2 entry vector (Origene Inc, USA) or pCMV6-mKate2 as a negative control.

Techniques: Derivative Assay, Expressing, Western Blot, Quantitative Proteomics, Transfection, Control, Plasmid Preparation, Imaging

Journal: Molecular Medicine

Article Title: Adverse transverse-tubule remodeling in a rat model of heart failure is attenuated with low-dose triiodothyronine treatment

doi: 10.1186/s10020-019-0120-3

Figure Lengend Snippet: RNA expression of T-tubule and SR genes RT-qPCR analysis of LV tissue for mRNA content of ( a ) bridging integrator-1, BIN1; junctophilin 2, Jph2; L-type calcium channel, LTCC; ryanodine 2, RyR2 and ( b ) SR calcium ATPase, SERCa2; phospholamban, PLN; alpha- and beta-myosin heavy chain, αMHC and βMHC. The binary log of ΔΔCt calculations of PCR cycle amplifications was used to determine the fold-change relative to the mean values of the Sham group. Bar graph shows mean ± SD for n = 4–6 hearts/group. Statistical analysis used one-way ANOVA with post-hoc Tukey’s multi-group comparisons: * p < 0.05 vs sham; ** p < 0.001 vs sham; # p < 0.01 vs MI + Veh.

Article Snippet: Membranes were blocked with TBST/5% milk and incubated overnight at 4 °C with the following primary antibodies (all at 1:500 dilution): rabbit polyclonal anti-Ca v 1.2 antibody (cat. no. ACC-003; Alomone Labs, Jerusalem, Israel), mouse monoclonal (mAb) anti-RyR antibody (cat. no. MA3–916/C3–33; Invitrogen, ThermoFisher), mouse mAb anti-BIN1 (cat no.200–301-E63; Rockland Inc., Limerick PA), mouse mAb anti-Jph2 (cat no.600–401-CC5; Rockland Inc.).

Techniques: RNA Expression, Quantitative RT-PCR

Journal: Molecular Medicine

Article Title: Adverse transverse-tubule remodeling in a rat model of heart failure is attenuated with low-dose triiodothyronine treatment

doi: 10.1186/s10020-019-0120-3

Figure Lengend Snippet: Immunoblot analysis of T-tubule and SR proteins. a Representative immunoblots of microsomal fractions from LV tissues of each study group that were probed with anti-BIN1, Jph2, Ca v 1.2 and RyR2 antibodies. Observed molecular weights (kDa) of each protein are indicated. Ponceau red staining of the immunoblots are shown with molecular weight markers. b , c Bar graphs show the quantitation of the luminescence intensity of the indicated protein bands, normalized to Ponceau red staining and expressed relative to the mean sham values. Values are mean ± SD for n = 4–6 hearts/group. Statistical analysis using one-way ANOVA with post-hoc Tukey’s multiple group comparisons; p- values are indicated

Article Snippet: Membranes were blocked with TBST/5% milk and incubated overnight at 4 °C with the following primary antibodies (all at 1:500 dilution): rabbit polyclonal anti-Ca v 1.2 antibody (cat. no. ACC-003; Alomone Labs, Jerusalem, Israel), mouse monoclonal (mAb) anti-RyR antibody (cat. no. MA3–916/C3–33; Invitrogen, ThermoFisher), mouse mAb anti-BIN1 (cat no.200–301-E63; Rockland Inc., Limerick PA), mouse mAb anti-Jph2 (cat no.600–401-CC5; Rockland Inc.).

Techniques: Western Blot, Staining, Molecular Weight, Quantitation Assay

Journal: Circulation: Heart Failure

Article Title: Plasticity of Surface Structures and β 2 -Adrenergic Receptor Localization in Failing Ventricular Cardiomyocytes During Recovery From Heart Failure

doi: 10.1161/circheartfailure.111.964692

Figure Lengend Snippet: Figure 4. Retubulation is associated with recovery of myocardial BIN1 and TCAP expression but not JPH2. Immunoblots of myo- cardial SERCA2a, JPH2, BIN1, and TCAP expression in control (C), failing (HF), and failing hearts rescued with SERCA2a gene therapy (HFS), with GAPDH as loading control. Quantitative comparison of SERCA2a, JPH2, BIN 1, and TCAP protein levels, showing reduction of all 4 proteins in the failing heart (black) compared with normal controls (blue), and recovery of SERCA2a, BIN 1, and TCAP after AAV9SERCA2a gene therapy (red) (meanSEM except BIN1, medianinterquartile range). Number of hearts: C, n6; HF, n5; HFS, n5. *P0.05, ***P0.001. ISO indicates isoproterenol.

Article Snippet: After 1 hour blocking in 5% milk TBS-T, primary antibodies for JP2 (1/1000), BIN1 (1/500), TCAP (1/500) and GAPDH (1/5000) (respectively Invitrogen, Sigma-Aldrich, ProSci Incorporated and Millipore) were incubated overnight at 4 degrees.

Techniques: Expressing, Western Blot, Control, Comparison

Journal: Communications biology

Article Title: Podosome formation promotes plasma membrane invagination and integrin-β3 endocytosis on a viscous RGD-membrane.

doi: 10.1038/s42003-020-0843-2

Figure Lengend Snippet: Fig. 5 Protrusive F-actin polymerization results in plasma membrane invagination and BIN1 recruitment to the podosome ring. a Interference reflection microscopy (IRM) image of REF52 cell on RGD-membrane. b Intensity profiles of IRM, F-actin, and YFP-paxillin along the dashed line in a. The region of podosome core (F-actin, labeled by CF594-phalloidin) exhibits the lowest intensity in the IRM channel and represents the close contact and protrusive F-actin polymerization towards the substrate. c Kymograph of plasma membrane (PM, labeled by mCherry-KRas-CT), F-actin (labeled by BFP2-UtrCH), and RGD-NA680 during the podosome formation in MEF cell. Plasma membrane becomes enriched and encircles the protrusive F-actin (arrowheads) (see Supplementary Fig. 7c). d Intensity profiles at t1 and t2 in d. The intensities of plasma membrane gradually increase around the F-actin (arrowheads). e Kymograph of BIN1-mCherry, plasma membrane (PM, labeled by PM-GFP), and F-actin (labeled by BFP2-UtrCH) during the podosome formation in MEF cell. BIN1 localizes at the site of plasma membrane invagination and encircles the protrusive F-actin (see Supplementary Fig. 7d and Supplementary Movie 4). f BIN1-mCherry specifically colocalizes with integrin-β3-GFP at the podosome ring and surrounds dot-like F-actin assembly of the podosome core. F-actin is labeled by BPF2-UtrCH in REF52 cell. Inset: the boxed region (5 × 5 μm2). g, h N-BAR-GFP (aa 1–267), not BIN1ΔN-BAR-GFP (aa 268–476) colocalizes with BIN1-mCherry around BFP2-UtrCH labeled podosome core in REF52 cell. Inset: the boxed region (5 × 5 μm2). Scale bars represent 5 µm.

Article Snippet: Scramble and siRNAs targeted Rat for BIN1 (SR511599), DNM2 (SR501378), Dab2 (SR505444), Clathrin heavy chain (Cltc) (SR507164), CIP4 (SR514456), and pRFP-C-RS vector used in shBIN1 were obtained from Origene (Supplementary Table 1).

Techniques: Clinical Proteomics, Membrane, Microscopy, Labeling

Journal: Communications biology

Article Title: Podosome formation promotes plasma membrane invagination and integrin-β3 endocytosis on a viscous RGD-membrane.

doi: 10.1038/s42003-020-0843-2

Figure Lengend Snippet: Fig. 6 BIN1 recruits DNM2 to the podosome ring and promotes RGD endocytosis. a BIN1-mCherry colocalizes with DNM2-GFP at the podosome ring. F-actin is labeled by BPF2-UtrCH in REF52 cell. Inset: the boxed region (4 × 4 μm2) (see Supplementary Movie 5). b Kymograph of the boxed region of a. BIN1 and DNM2 are enriched at the podosome ring in the early phase of podosome formation. c Intensity analyses of BIN1 and DNM2 at the podosome ring. All intensity traces are synchronized by realigning F-actin peak intensity at 400 s. BIN1 and DNM2 both reach the peak intensity before F-actin. Eighteen podosomes from nine cells in three independent experiments are analyzed. d, e Knockdown of BIN1 suppresses RGD-NA488 endocytosis. Three- dimensional confocal images are shown with the z position from 2 to 20 µm (xz and yz view, 500 nm z-step), while the image shown in xy view is at the z position of 5 µm above the adhesion plane. Statistical information is in Supplementary Fig. 10g. f Knockdown of BIN1 by shRNA suppresses the recruitment of DNM2-GFP to the podosome ring. Inset: the boxed region (5 × 5 μm2). Ratiometric insets of DNM2 indicate the recruitment level (see Supplementary Movie 6). g Recruitment level of DNM2-GFP during podosome formation. The recruitment of DNM2 to the podosome ring is reduced when BIN1 expression is suppressed. All intensity traces are synchronized by realigning F-actin peak intensity at 400 s. shScramble samples are analyzed from nine podosomes from six cells in four independent experiments. shBIN1 samples are analyzed from 12 podosomes from seven cells in five independent experiments. Scale bars represent 5 µm. Error estimates are SEM.

Article Snippet: Scramble and siRNAs targeted Rat for BIN1 (SR511599), DNM2 (SR501378), Dab2 (SR505444), Clathrin heavy chain (Cltc) (SR507164), CIP4 (SR514456), and pRFP-C-RS vector used in shBIN1 were obtained from Origene (Supplementary Table 1).

Techniques: Labeling, Knockdown, shRNA, Expressing

Journal: Communications biology

Article Title: Podosome formation promotes plasma membrane invagination and integrin-β3 endocytosis on a viscous RGD-membrane.

doi: 10.1038/s42003-020-0843-2

Figure Lengend Snippet: Fig. 7 Working model of integrin-β3 endocytosis at the podosome. (i) Integrin-β3 form clusters on RGD-membrane. (ii) Podosome formation. Protrusive F-actin polymerization leads to plasma membrane invagination. BIN1 binds the curved membrane at the podosome ring. (iii) BIN1 recruits DNM2 to the membrane invagination site. (iv) DNM2 triggers membrane scission and RGD–integrin-β3 endocytosis.

Article Snippet: Scramble and siRNAs targeted Rat for BIN1 (SR511599), DNM2 (SR501378), Dab2 (SR505444), Clathrin heavy chain (Cltc) (SR507164), CIP4 (SR514456), and pRFP-C-RS vector used in shBIN1 were obtained from Origene (Supplementary Table 1).

Techniques: Membrane, Clinical Proteomics

Journal: Europace

Article Title: Atrial cardiomyocytes contribute to the inflammatory status associated with atrial fibrillation in right heart disease

doi: 10.1093/europace/euae082

Figure Lengend Snippet: Right atrial expression of BIN1, CAV3, and JPH2 proteins. Protein-expression level evaluated by western blot analysis for BIN1 ( A and B ), CAV3 ( A and C ), and JPH2 ( A , D , and E ) in RA from sham and pulmonary artery banding (PAB) rats. (See uncropped gels and total proteins on blot in , and .) (Statistical analysis: data were normally distributed as assessed by Shapiro–Wilk test and statistical difference between the groups was analysed by Student’s t -test. Each point represents the level of expression from an individual animal. n = 5 rats/group.)

Article Snippet: Primary antibodies for immunoblot experiments included: BIN1 (Novus Biologicals, NBP1-89102), CACNA1C (Alomone labs ACC-003), CAV (Novus Biologicals, NBP3-16503), IL6 (ThermoFisher ARC0962), JPH2 (ThermoFisher, PA5-141187), IL1β (ThermoFisher PA5-46956), KCNQ1 (NeuroMab clone N374/10), NLRP3 (Novus Biologicals NBP1-77080SS), SCN5A (Alomone labs ASC-005), RYR2 (Thermofisher MA3-916), SERCA2a (Thermofisher MA3-910).

Techniques: Expressing, Western Blot