Figure 1.
Expression and distribution of TMEM43 in non-transfected HL-1 cells and cells transfected with GFP-tagged wild-type or p.S358L TMEM43.
A. Western blot analysis demonstrating expression of endogenous and exogenous (transfected) TMEM43, with GAPDH as loading control. Rabbit polyclonal TMEM43 antibody specifically detected GFP-TMEM43 and native TMEM43, indicated by the presence of bands at 70 kDa and 43 kDa respectively, in transfected cells and only 43 kDa native TMEM43 in non-transfected HL-1 cells (control). The 70 kDa band indicates the GFP-tagged wild-type (TMEM43-WT) or mutant (TMEM43-S358L) TMEM43 B. Immunofluoresence staining with TMEM43 antibody showed cytoplasmic as well as nuclear envelope distribution of endogenous TMEM43 in non-transfected HL-1 control cells. TMEM43 was detected using anti-TMEM43 antibody and Cy3-labeled anti-rabbit IgG. C. Immunofluorescence staining of TMEM43-WT or TMEM43-S358L cells. Expressed wild-type or mutant TMEM43 were observed by GFP fluorescence (left column); GFP-TMEM43 showed more cytoplasmic distribution. TMEM43 was detected in the cytoplasm of both the stably expressing wild-type (top row) and mutant (bottom row) cell lines. Nuclei are visualized with DAPI (blue, right column). GFP (green, left column) fluorescent, Cy3 (red, middle column) fluorescent and merged images (right column) are shown. Co-localization of signals due to GFP and antibody binding (yellow, right column).
Figure 2.
Transmission Electron Micrograph of transfected HL-1 cells labeled for TMEM43 with immunogold.
A and B. Single immunogold labeling experiments used 15 nm gold particles to label GFP. A. Immunogold-labeled TMEM43-WT cells. The label (large dot) is associated with the endoplasmic reticulum (arrows), nuclear envelope, and the nucleus (N). B. Immunogold-labeled TMEM43-S358L cells. Immunogold particles (large dot) are confined to bundles of cytoplasmic microfilaments (arrows) and what vacuolated endosomal structures (asterisk). The endoplasmic reticulum or nuclear envelope did not show TMEM43 expression. C and D. Double immunogold labeling experiments used 15 nm gold particles to label GFP and 5 nm gold particles to label TMEM43. C. Cytoplasm of a wild-type cell that has been labeled with both TMEM43 (small dot) and GFP (large dot) antibodies. Note the co-localization on the endoplasmic reticulum (arrows). D. Mutant protein labeled with antibody specific for TMEM43 (small dot) or the GFP Tag (large dot) were found in a large vacuolated structure (asterisk) as well as discrete clusters in the cytoplasm (arrows) of stably transfected cells. All bars equal 0.5 µm. E. Quantitative analysis of the distance between large gold particles (GFP-15 nm) and small gold particles (TMEM43-5 nm) in TMEM43-WT (open column) and TMEM43-S358L (filled column) cells. Data are expressed mean ± SD from three independent experiments.
Figure 3.
Expression of IC disc proteins in HL-1 cells transfected with wild-type and mutant TMEM43.
A and B. The stably transfected cells were lysed and Western blot was performed for ZO-1, α-catenin, N-cadherin, β-catenin, JUP, Cx43 and GAPDH. The expression of α-catenin, N-cadherin, β-catenin and JUP were similar in non-transfected HL-1 (control) and stably-transfected cells (TMEM43-WT and TMEM43-S358L). A. The level of ZO-1 was significantly decreased in TMEM43-S358L cells compared to TMEM43-WT and control. B. P2 and P0 represent the phosphorylated and non-phosphorylated forms of Cx43. The level of the non-phosphorylated Cx43 isoform (P0) was increased in mutant cells compared to the control and TMEM43-WT cells. C and D. Densitometry analysis of ZO-1/GAPDH and phosphorylated Cx43 P2/P0. * denotes p<0.05. The densitometry results showed the combined analysis of three separate western blots and the western blot images are representative of three independent cell culture experiments.
Figure 4.
Immunofluorescent staining of TMEM43 and IC disc proteins in HL-1 cells stably expressing wild-type and mutant TMEM43.
A. Immunostaining of Plakoglobin (JUP) was predominantly at the cell-cell border in control and TMEM43-overexpressing (TMEM43-WT) cells, but this significantly decreased at cell border in mutant TMEM43-transfected cells (TMEM43-S358L), with a diffuse expression throughout the cytoplasm instead. TMEM43 (green) was seen in the cytoplasm, as small spots in the TMEM43-WT cells. Greater cytoplasmic distribution was seen in control cells and near the cell edge in the TMEM43-S358L cells. B. α-catenin labeling clearly localized to the border of the cells in control and TMEM43-WT cells but in the TMEM43-S358L cells, α-catenin was clearly located diffusely throughout the cytoplasm, with very little staining specifically at the cell border. C. Immunostaining of ZO-1 labeling showed ZO-1 localization predominantly at the cell-cell junction in control and TMEM43-WT cells, while reduced amounts of ZO-1 were observed in TMEM43-S358L cells. D. Cx43 staining in TMEM43-WT cells demonstrates strong immunoreactive signals at the periphery of the cells and in the cytoplasm which is greater than those levels seen in control cells. In TMEM43-S358L cells, there is diffuse Cx43 staining of the cells throughout the cytoplasm. Images combining the TMEM43 staining/GFP fluorescence with these 4 different proteins (Merge) are shown in the right column including nuclear DAPI staining (blue). All results are representative of three independent cell culture experiments.
Figure 5.
TMEM43 distribution and association with ZO-1 and JUP in the intercalated disc of normal murine myocardium.
A. Electron micrograph of a double immunogold labeled section of the perinuclear region of a cardiac myocyte that has been labeled with TMEM43. Particles were located diffusely throughout the nucleus (N), on the nuclear membrane (arrows) and on components of the sarcotubular network (arrowheads). B. Sarcoplasm of a cardiac myocyte. Note the labeling on the sarcoplasmic reticulum (arrowheads) on vesicular components of the sarcotubular network (asterisk). Figure 4C represents co-localization of TMEM43 (10 nm gold particles) with ZO-1 (15 nm gold particles). Arrows indicate two adherens junctions that contain both ZO-1 and TMEM43. Figure 4D represents co-localization of TMEM43 (10 nm gold particles) with JUP (15 nm gold particles) in desmosomes and adherens junctions (arrows) of the intercalated disc. All bars equal 0.5 µm.
Figure 6.
Effects of TMEM43 on conduction velocity and sodium channel expression.
A. Electrograms recorded from adjacent MEA electrodes in HL-1 cell cultures. All cells beat spontaneously. Control and TMEM43-WT cells beat with a regular, synchronous rhythm. TMEM43-S358L cells beat with an irregular, comparatively slowed rhythm. B. Average velocity illustrating a decrease in conduction velocity of the TMEM43-S358L cells (1.2 cm/s) compared to control cells (2.5 cm/s) and TMEM43-WT cells (2.1 cm/s) (n = 7 control, n = 6 TMEM43-WT and n = 6 TMEM43-S358L, * p<0.01). All results are representative of six independent culture experiments. C. Western blot demonstrating expression of NaV1.5 in control, TMEM43-WT and TMEM43-S358L cells. GAPDH was used as a loading control. Rabbit polyclonal NaV1.5 antibody specifically detected sodium ion channel (NaV1.5) expression in all three cell types. Neither wild-type nor mutant TMEM43 altered the NaV1.5 expression. All results are representative of three independent Western blot experiments.