Fig 1.
Myocilin transcript is found in skeletal muscle and heart in humans and mice.
(A) GTEx database shows that MYOC gene is expressed in human skeletal muscle and heart; and (B) Real-time PCR (RT-PCR) confirms that Myoc transcript is in wild-type mouse skeletal muscle and heart (+/- SD). Note that the myocilin data used for Fig 1A were obtained from the GTEx Portal (https://www.gtexportal.org/home/) in May 2017.
Fig 2.
Transgenic mice had human mutant MYOC expressed in skeletal muscle and human MYOC is detected by Western blot as a doublet due to partial N-glycosylation.
(A) Western blots for human MYOC protein in adult transgenic mouse gastrocnemius muscle lysates. Human MYOC protein was detected in the transgenic mouse skeletal muscle and in the heart using R&D Systems anti-MYOC antibody (1:1000). Loading was 40μg tissue lysate per well of a 10% SDS-PAGE gel. Total mouse number for this representative western blot is N = 6 different animals. (B) NTM5 cells were transiently-transfected with a plasmid with cDNA for human MYOC-FLAG or a plasmid with cDNA for mouse Myoc-FLAG. 20μg cell lysates were treated with PNGase to remove N-glycosylation followed by Western blot analysis using anti-FLAG (1:1000). (C) Weights of gastrocnemius muscles and hearts harvested from equal numbers of male and female wt and CMV-MYOC-Y437H transgenic mice did not differ between groups. N per group was ≥ 6. +/- SD is indicated and t-test were p>0.1. Abbreviations–wild-type, wt; transgenic, Tg.
Fig 3.
Western blots for ER protein expression in wt and CMV-MYOC-Y437H transgenic muscle lysates (40μg per lane).
(A) Over-expression of CMV-MYOC-Y437H mutant protein in the transgenic animal has little impact on expression of ER proteins in skeletal muscle or heart. (B) Western blots examining CMV-Y437H mouse skeletal muscle lysates for pro-apoptotic proteins associated with ER stress showed no differences between the wt and transgenic mice. Control cell extracts for CASP-3 (10μL per well) were from Cell Signaling Technology (9663). Controls for CASP-12 were cell lysates (40μg lysates/well) obtained from HeLa cells +/- 1μM staurosporine (Abcam, ab146588) treatment for 18 hours. Note that all representative Western blots have wells loaded with lysates obtained from different animals (N = 2 wt and 2 transgenic per blot).
Fig 4.
Electron micrographs of gastrocnemius muscle from 4 to 6 month old female wt and CMV-MYOC-Y437H transgenic mouse skeletal muscle.
(A) No ER/SR expansion was observed in transgenic mouse skeletal muscle. Transgenic mouse skeletal muscle was observed to have multiple M-line bands (highlighted by dashed line box) within the H zone of the sarcomeres. Direct magnification was 4800X. (B) Additional electron micrographs from CMV-MYOC-Y437H transgenics have been enlarged to show the diffuse M-line in sarcomeres. Direct magnification was 18500X. (C) The number of mitochondria per three sarcomere was quantified from four micrographs and there was no significant difference between wt and CMV-MYOC-Y437H transgenics. +/- SD; t-test p>0.1.
Fig 5.
Western blots for expression of M-band proteins in wt and CMV-MYOC-Y437H mice.
(A) Skeletal muscle lysates (40μg per lane) from MYOC Y437H mutant transgenic mice indicated lower expression of both MYOM1 and CKM in comparison to wild-type animals. Note that representative Western blot wells are loaded with tissue lysates from different animals (N = 2 wt and 2 transgenic per blot). (B) Densitometry was completed to measure amount of protein in MYOM1 and CKM Western blot sample lysates. Error bars are +/- SEM and * represents t-test p<0.05. Abbreviations–Ubiquitin, Ub.
Fig 6.
Western blot following immunoprecipitation (IP) indicates that CKM binds MYOC.
The Western blot was stripped and treated with anti-CKM to show CKM input.
Fig 7.
NTM5 cells transiently-transfected for 48 hours with plasmids which had MYOC cDNAs were analyzed by Western blot.
(A) Wild-type (wt) MYOC is predominantly secreted while mutant CMV-MYOC-Y437H is not secreted and has a larger non-soluble fraction when compared to wt MYOC. Anti-MYOC goat antibody from R&D Systems was utilized. (B) Percentage of MYOC in each cell fraction was estimated based on Western blot band intensity. Error bars are +/-SD.
Fig 8.
Model of mutant MYOC pathology in mouse skeletal muscle sarcomere.
(A) Wt sarcomere has CKM binding myomesins (MYOMs) to form a dense M-line. (B) Mutant MYOC can bind CKM causing the M-line to become disperse. It is possible that there may be accumulation of CKM bound to MYOC at a location beyond the M-band region.