Fig 1.
Disordered T-tubules in Ehd1-heterozygous muscle.
Myofibers were immunostained with anti-BIN1 (red) and anti-DHPR (green) antibodies. Representative myofibers are shown. Ehd1-heterozygous (Ehd1+/-) fibers displayed disorganized (white arrowhead) and aggregated (white arrow) T-tubule structures in 27% of myofibers, marked by DHPR, also evidenced in DIC images compared to 0% in control fibers. Ehd1-heterozygous muscle with extensive BIN1 fluorescence extending beyond DHPR staining (yellow arrowhead). Scale 5μm.
Fig 2.
Misexpression of triad proteins and expansion of the T-tubule compartment in Ehd1-heterozygous muscle.
(A) BIN1 and DHPR levels are increased +2.25 and +5.4 fold in Ehd1+/- quadriceps muscle compared to WT correlating with the increase in T-tubule structures. Gel code bands are shown as a loading control (LC). (B) Junctophilin 2 (JP2) protein levels were decreased 13-fold in Ehd1+/- quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (C) Junctophilin 1 (JP1) protein levels were similar in Ehd1+/- quadriceps muscle compared to wildtype controls. Gel code stained bands are shown as a loading control (LC). (D) Ultrastructural analysis reveals ectopic (dotted arrow) and elongated (arrow) T-tubules in 8-week-old Ehd1-heterozygous muscle (Ehd1+/-) stained with potassium ferricyanide to color the T-tubule structures black. (E) Ehd1-heterozygous muscle contains duplicated triads containing 2 T-tubules (black arrows) and 3 sarcoplasmic reticulum (SR) in 1 triad unit. Scale 0.5μm. (F) Ehd1-heterozygous muscle stained with potassium ferricyanide, outlines duplicated T-tubule structures (two black arrows). Scale 0.5μm. (G) Ultrastructural analysis of 2-D images reveals increased tubule abnormalities in Ehd1-heterozygous muscle, 12.5%, compared to 1.7% in control muscle (n>400 structures per genotype, p = 0.04).
Fig 3.
Reduction in EDL force production in Ehd1-heterozygous muscle.
(A) Representative traces from 8-week male WT and Ehd1-heterozygous (Ehd1+/-) EDL muscles with stimulation pulses marked below the force traces. Ehd1+/- muscle has reduced force. (B) Ehd1+/- EDL muscle has reduced twitch force (n > 5 per genotype, p<0.05). (C) Ehd1+/- EDL muscle has reduced maximum tetanic force (n > 5 per genotype, p = 0.05).
Fig 4.
Ehd1-heterozygous mice have elevated creatine kinase levels.
(A) Serum creatine kinase (CK) levels are highly elevated in Ehd1-heterozygous (Ehd1+/-) mice at birth (n > 10 of each genotype, p<0.0001). (B) CK levels are consistently elevated in Ehd1+/- mice at 8wk, 6m, and 14m compared to WT controls (n ≥ 6 of each genotype, p<0.0002). (C) Evans blue dye uptake was measured from excised tissues (absorbance per mg tissue). Graph expressed as an average of all tissues. Evans blue dye uptake (measured as absorbance) is similar in Ehd1+/- and WT (n>6 animals per genotype, ns, nonsignificant).
Fig 5.
EHD1 modulates BIN1 mediated tubule formation in vivo.
Myofibers were electroporated with BIN1-GFP and wildtype EHD1-mCherry or EHD1T72A-mCherry. Imaging occurred one week post-electroporation. (A & B) EHD1 and BIN1 normally align in ordered T-tubules in live skeletal muscle. Expression of EHD1T72A results in mislocalization of EHD1T72A and ectopic tubule formation (white arrow), marked with BIN1 staining. Low magnification images are shown below. Scale 5μm. BIN1 mislocalization occurred in 11/11 EHD1T72A myofibers, while 0/11 EHD1 myofibers expressed BIN1 mislocalization.
Fig 6.
EHD1T72A is a negative regulator of BIN1 mediated tubule formation in vivo.
Representative images of myofibers coelectroporated with BIN1-GFP and EHD1-mCherry or EHD1T72A-mCherry in wildtype myofibers. Images were processed identically in Fiji and are shown with T-tubules running horizontally. When coelectroporated with EHD1, BIN1 localizes to ordered T-tubules. Graphically this corresponds to the horizontal axis clustering around 0 degrees. Coexpression of EHD1T72A results in mislocalization of BIN1 tubules, causing lateral extensions between longitudinal tubules. Quantification shows a cluster of tubules both at 0 degrees, T-tubules, and at 90 degrees, L-tubules (arrow). Scale 5μm.