Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle

<div><p>We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from <i>Ehd1</i>-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining <i>Ehd1</i>-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in <i>Ehd1</i>-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, <i>Ehd1</i>-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. <i>Ehd1</i>-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy.</p></div

<i>Ehd1-</i>heterozygous mice have elevated creatine kinase levels.

<p>(A) Serum creatine kinase (CK) levels are highly elevated in <i>Ehd1-</i>heterozygous (<i>Ehd1+/-</i>) mice at birth (n > 10 of each genotype, p<0.0001). (B) CK levels are consistently elevated in <i>Ehd1+/-</i> 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 <i>Ehd1+/-</i> and WT (n>6 animals per genotype, ns, nonsignificant).</p

Misexpression of triad proteins and expansion of the T-tubule compartment in <i>Ehd1-</i>heterozygous muscle.

<p>(A) BIN1 and DHPR levels are increased +2.25 and +5.4 fold in <i>Ehd1</i>+/- 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 <i>Ehd1+/-</i> 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 <i>Ehd1+/-</i> 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 <i>Ehd1-</i>heterozygous muscle (<i>Ehd1+/-</i>) stained with potassium ferricyanide to color the T-tubule structures black. (E) <i>Ehd1-</i>heterozygous muscle contains duplicated triads containing 2 T-tubules (black arrows) and 3 sarcoplasmic reticulum (SR) in 1 triad unit. Scale 0.5μm. (F) <i>Ehd1-</i>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 <i>Ehd1-</i>heterozygous muscle, 12.5%, compared to 1.7% in control muscle (n>400 structures per genotype, p = 0.04).</p

EHD1 modulates BIN1 mediated tubule formation <i>in vivo</i>.

<p>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.</p

Disordered T-tubules in <i>Ehd1-</i>heterozygous muscle.

<p>Myofibers were immunostained with anti-BIN1 (red) and anti-DHPR (green) antibodies. Representative myofibers are shown. <i>Ehd1-</i>heterozygous (<i>Ehd1+/-</i>) 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. <i>Ehd1</i>-heterozygous muscle with extensive BIN1 fluorescence extending beyond DHPR staining (yellow arrowhead). Scale 5μm.</p

Reduction in EDL force production in <i>Ehd1-</i>heterozygous muscle.

<p>(A) Representative traces from 8-week male WT and <i>Ehd1-</i>heterozygous (<i>Ehd1+/-)</i> EDL muscles with stimulation pulses marked below the force traces. <i>Ehd1+/-</i> muscle has reduced force. (B) <i>Ehd1+/-</i> EDL muscle has reduced twitch force (n > 5 per genotype, p<0.05). (C) <i>Ehd1+/-</i> EDL muscle has reduced maximum tetanic force (n > 5 per genotype, p = 0.05).</p