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. 2016 Apr 15;129(8):1661-70.
doi: 10.1242/jcs.179887. Epub 2016 Feb 29.

VSports app下载 - HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles

Liang-Yi Juo  1 , Wern-Chir Liao  1 , Yen-Ling Shih  2 , Bih-Ying Yang  2 , An-Bang Liu  3 , Yu-Ting Yan  4
Affiliations

HSPB7 interacts with dimerized FLNC and its absence results in progressive myopathy in skeletal muscles

Liang-Yi Juo et al. J Cell Sci. .

Abstract

HSPB7 belongs to the small heat-shock protein (sHSP) family, and its expression is restricted to cardiac and skeletal muscles from embryonic stages to adulthood. Here, we found that skeletal-muscle-specific ablation of the HspB7 does not affect myogenesis during embryonic stages to postnatal day 1 (P1), but causes subsequent postnatal death owing to a respiration defect, with progressive myopathy phenotypes in the diaphragm. Deficiency of HSPB7 in the diaphragm muscle resulted in muscle fibrosis, sarcomere disarray and sarcolemma integrity loss. We identified dimerized filamin C (FLNC) as an interacting partner of HSPB7. Immunofluorescence studies demonstrated that the aggregation and mislocalization of FLNC occurred in the muscle of HspB7 mutant adult mice VSports手机版. Furthermore, the components of dystrophin glycoprotein complex, γ- and δ-sarcoglycan, but not dystrophin, were abnormally upregulated and mislocalized in HSPB7 mutant muscle. Collectively, our findings suggest that HSPB7 is essential for maintaining muscle integrity, which is achieved through its interaction with FLNC, in order to prevent the occurrence and progression of myopathy. .

Keywords: FLNC; HSPB7; Myopathy V体育安卓版. .

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Expression and localization of HSPB7 in skeletal muscle. (A) Western blot analysis of calf muscle showing HSPB7 expression from embryonic stages to adulthood (E14.5 to P56). (B) Immunohistochemical analysis of HSPB7 and MHC expression at E13.5 (arrows, muscle tissue; arrowheads, heart). (C) Western blot analysis of HSPB7 expression in the heart and various skeletal muscles. GAPDH was used as loading control. EDL, extensor digitorum longorum; TA, tibialis anterior. (D) Subcellular localization of HSPB7 in the soleus muscle of adult mice. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and desmin (green), sarcomeric α-actinin (green) or γ-sarcoglycan (green). The nucleus was visualized by Hoechst 33342 staining. The arrows in A and C represent the expression of HSPB7. Scale bars: 500 μm (B, upper and middle panel); 200 μm (B, lower panel); 10 μm (D).
Fig. 2.
Fig. 2.
Characterization of HspB7 CKO mice. (A) Survival curve of HspB7 CKO and control (Flox/Flox) mice. (B) Abdominal view of dissected diaphragms from 12-week-old HspB7 CKO and control mice. A costal-sided diaphragmatic defect (arrow) was observed in HspB7 CKO mice. (C) The growth curve of CKO and control mice. Mice of each genotype were weighed every 4 weeks. A significant difference in body weight (BW) was observed between CKO and control mice after the age of 36 weeks (n=5; mean±s.e.m.). (D) Significant decreases in diaphragm weight (DW) of 12-week-old CKO mice (n=6; mean±s.e.m.). (E) A decrease in forelimb grip strength was observed in CKO mice (n=4; mean±s.d.). (F) At 36 weeks of age, a significant increase in serum creatine kinase levels was observed in CKO mice (n=8 for CKO and n=6 for Flox/Flox; mean±s.d.). Scale bars: 4 mm (B). *P<0.05 (Student's t-test).
Fig. 3.
Fig. 3.
Diaphragm muscle defects in HspB7 CKO mice. (A) Diaphragm muscle sections from control and CKO mice were stained with Masson trichrome. The diaphragm muscle cross-section on P1 revealed no defects in CKO mice. At 12–24 weeks (W) of age, Masson trichrome staining revealed interstitial fibrosis (aniline blue stain). (B) H&E-stained diaphragm histological sections from Flox/Flox and CKO mice at 12 weeks. (C) Transmission electron micrographs (TEMs) of longitudinally sectioned diaphragm muscles from control (Flox/Flox) and CKO mice. (i) Control (Flox/Flox). Z-line streaming (arrows in ii), causing filament disruption, (arrows in iii) and Z-line disruption (vi) were observed. Focal rearrangements (arrows in iv), small rod bodies (arrowheads in iv) and central muscle nuclei (arrow in v) were observed in the diaphragm of CKO mice. (D) Quantification of central nucleation in the diaphragm of control (Flox/Flox) and CKO mice: the number of muscle fibers with central nuclei is represented as a percentage of the total number of muscle fibers analyzed (mean±s.d.; n=10 fields, 3 mice per group). (E) Myofiber size (CSA) distribution in the diaphragm muscle of 12-week-old control (Flox/Flox) and CKO mice (n=8 fields, 3 mice per group). ***P<0.001 (Student's t-test). Scale bars: 100 μm (A); 20 μm (B); 1 μm (C).
Fig. 4.
Fig. 4.
HSPB7 interacts and colocalizes with FLNC. (A) Diaphragm muscle from adult Hspb7Flox/Flox and wild-type (WT) mice was lysed and incubated with anti-FLAG M2 affinity gel for co-immunoprecipitation (IP) of proteins binding to HSPB7, and analyzed by western blotting. (B) The colocalization of HSPB7 and FLNC was assessed by confocal microscopy. The muscle sections were co-immunostained with antibodies against HSPB7 (red) and FLNC (green). The nucleus was visualized by Hoechst 33342 staining. (C) A construct of FLNC lacking the dimerization region (HA–FLNC-CΔ24) was overexpressed in HEK293T cells. The domains mediating interaction between HSPB7 and FLNC were assessed through co-immunoprecipitation in vitro. (D) The dimerization region deletion and mutations of FLNC-C (HA–W2711X and HA–M2670D) were overexpressed in HEK293T cells. The domains mediating interaction between HSPB7 and FLNC were assessed through co-immunoprecipitation in vitro. Scale bars: 10 μm.
Fig. 5.
Fig. 5.
Loss of HSPB7 results in FLNC and γ-sarcoglycan protein aggregation. Confocal micrographs of longitudinal sections (A) and cross-sections (B,C) of the diaphragm of 12-week-old mice. Specific antibodies were used to identify the distributions of the sarcomeric components α-actinin (Z-line), FLNC and γ-sarcoglycan as indicated. FLNC (arrows in A,C) and γ-sarcoglycan (arrows in B) aggregation were observed in the diaphragm muscle. The arrangement of α-actinin was normal (arrowheads in A). However, Z-line disarray was also observed in the diaphragm of CKO mice (asterisks in A). FLNC and γ-sarcoglycan were discontinuous or missing in some regions of the sarcolemma (arrowheads in B,C). Extracellular matrix accumulation was labeled with wheat germ agglutinin (WGA). FLNC mislocalizes on the sarcolemma (arrowheads in C). The nucleus was visualized by Hoechst 33342 staining. (D) Western blot analysis of expression levels of FLNC and α-actinin in the diaphragm muscle. The muscle homogenate supernatant (S) and pellet (P) fractions from control (Flox/Flox) and CKO mice at 12 and 36 weeks of age. GAPDH was used to verify the loading amount in the supernatant. Coomassie Blue staining (45–75 kDa) was used to verify the loading amount in the pellet. The graph shows the mean±s.d. densitometry results (n=3). *P<0.05; **P<0.01 (Student's t-test). Scale bars: 10 μm (A); 50 μm (B); 20 µm (C)
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