Defective BVES-mediated feedback control of cAMP in muscular dystrophy

Fig. 1. BVES disruption compromises the body weight gain and muscle function in mice. All animal experiments were performed in WT and BVES-KO male mice with the C57BL/6N genetic background. a Immunofluorescence images of GA muscles in WT and BVES-KO mice (4 months of age) stained with the antibody against BVES and DAPI. Scale bar: 100 µm. (n = 4 per genotype). b Representative image of WT and BVES-KO male littermates at 4 months of age. c, Body weight gain of male BVES-KO and age/sex-matched WT mice from two to five months of age. Two-tailed paired Student’s t test. d Kaplan–Meier survival curve of WT and BVES-KO male mice. e Voluntary wheel running of BVES-KO and age-matched WT male mice (4 months of age). f, g Endurance capacity test performed by treadmill running showing running distance (f) and time to exhaustion (g) in BVES-KO (n = 5) and WT (n = 5) male mice (4 months of age). Two-tailed unpaired Student’s t test. h The number of dropouts to test the capacity of recovery from muscle injury on the treadmill in BVES-KO and WT male mice (6 months of age). Two-tailed paired Student’s t test. i Tetanic torque measurements of the posterior compartment muscles of BVES-KO and WT male mice…

Fig. 2. BVES deficiency causes muscle atrophy and dystrophy in mice. All animal experiments were performed in WT and BVES-KO male mice with the C57BL/6N genetic background. a Photographs of mice after the removal of skin and muscle tissues dissected from BVES-KO and WT male mice (4 months of age). b, c Net mass of QU (b) and GA (c) muscles in BVES-KO and WT male mice at 4 (QU or GA from WT: n = 10; KO: n = 8) and 8 (QU or GA from WT: n = 12; KO: n = 12) months of age. d, e The relative mass of QU (d) and GA (e) muscles normalized to the body weight at 4 (QU or GA from WT: n = 10; KO: n = 8) and 8 (QU from WT: n = 12; KO: n = 11; GA from WT: n = 12; KO: n = 12) months of age. f H&E-stained images of GA muscle cross-sections from 8-month-old BVES-KO and WT male mice. Scale bar: 100 μm. g The CSA distribution of GA muscle fibers in 8-month-old BVES-KO and WT male mice. CSA was quantified from the entire cross-sections of GA muscles. h Mean CSA of GA muscle fibers at 4 (n = 5 for WT and KO) and 8 (n = 6 for WT and KO) months of age. i Quantification of CNFs in the male GA muscles at 4 (n = 5 for WT and KO) and 8 (n = 6 for WT and KO) months of age. j CK measurements in 8-month-old BVES-KO (n…

Fig. 3. Muscle-specific expression of BVES ameliorates skeletal muscle pathology in BVES-KO mice. All animal experiments were performed in BVES-KO male mice with the C57BL/6N genetic background. a Schematic map of AAV9-BVES constructs. b Representative immunofluorescence images of AAV9-BVES treated and contralateral control GA muscles in BVES-KO male mice (5 months of age) stained with the antibody against HA-tag and DAPI. Scale bar: 100 µm. (n = 6 per treatment). c Western blot of BVES expression detected with anti-BVES and anti-HA antibodies in AAV9-BVES treated and contralateral control GA muscles (5 months of age). d Tetanic torque measurements of the posterior compartment muscles of AAV9-BVES treated and contralateral control GA muscles at 1 (n = 5 for Con and AAV9-BVES) and 3 (n = 4 for Con and AAV9-BVES) months after injection. e, f The relative mass of AAV9-BVES treated and contralateral control GA muscles (e) or untreated QU muscle (f) normalized by the body weight at 1 (n = 5 for Con and AAV9-BVES) and 3 (n = 4 for Con and AAV9-BVES) months after injection. ns, not significant. Two-way ANONA with Tukey’s multiple comparisons test (d–f). g H&E staining of AAV9-BVES treated or…

Fig. 4. BVES interacts with ADCY9 and regulates cAMP/PKA signaling. All animal experiments were performed in WT and BVES-KO male mice with the C57BL/6N genetic background. a Diagram showing the IP-mass spectrometry approach to identify BVES-interacting proteins in mouse skeletal muscle (created with BioRender.com). b, c Gene ontology analysis of the cellular compartment (b) and biological process (c) categorization of co-immunoprecipitated proteins with BVES-HA. d ADCY9 was co-immunoprecipitated with BVES-HA using the Flag antibody from the lysates of Cos-1 cells co-transfected with Flag-ADCY9 and BVES-HA (experiments were repeated twice). e IBMX (500 µM)-induced cAMPr fluorescence changes in ADCY3/6 double mutant HEK293 cells with stable expression of cAMPr, transfected with the indicated plasmids. Cell number: 31, 15, 50, 30 for mCherry, BVES, ADCY9 and ADCY9 + BVES, respectively, from three independent trials per condition. f cAMP measurements in skeletal muscle lysates from BVES-KO (n = 5) and WT (n = 5) male mice (8 months of age). Two-tailed unpaired Student’s t test. g, h Western blot (g) and quantification (h) of p-PKA substrates in skeletal muscle from BVES-KO (n = 4) and WT (n…

Fig. 5. BVES disruption enhances the UPS via FoxO. All animal experiments were performed in WT and BVES-KO male mice with the C57BL/6N genetic background. a–e Western blot of FoxO signaling in total GA muscle extracts from 8-month-old WT (n = 5) and BVES-KO (n = 6) male mice (a) and quantification of FoxO1 (b), FoxO3a (c), MuRF1 (d) and Atrogin-1 (e) normalized to GAPDH. f, g Western blot (f) and quantification (g) of ubiquitination in total GA muscle extracts from 8-month-old WT (n = 5) and BVES-KO (n = 6) male mice. h Immunostaining of FoxO3a and Cav3 (labeling muscle fibers) of GA muscles from WT and BVES-KO male (4 months of age; n = 5 each). Scale bar: 50 µm. i Immunostaining of FoxO3a and Cav3 of GA muscles from AAV9-BVES treated and contralateral control GA muscles at 3 months after injection (n = 5 per treatment). Scale bar: 50 µm. j, k Quantification of FoxO3a positive nuclei per field in WT and BVES-KO (n = 5 each; 4 months of age) GA muscle (j), and AAV9-BVES treated and contralateral control GA muscles in BVES-KO male mice at 3 months after injection (n = 5 each) (k). Three random areas were quantified for each stained section per mouse. l Body weight of BVES-KO male mice at…

Fig. 6. BVES Disruption suppresses the autophagic execution. All animal experiments were performed in WT and BVES-KO male mice with the C57BL/6N genetic background. a–d Western blot (a) of autophagic markers and quantification of LC3A (b), LC3B(c) and p62 (d) normalized to GAPDH in total GA muscle extracts from 8-month-old WT (n = 5) and BVES-KO (n = 6) male mice. e The p62 and Cav3 immunostaining of GA muscles from 4-month-old WT and BVES-KO male mice. (n = 5 biologically independent experiments). Scale bar: 100 µm. f, g, Western blot (f) and quantification (g) of VPS34 in total GA muscle extracts from 8-month-old WT (n = 5) and BVES-KO (n = 5) male mice. h, i Live cell imaging (h) of RFP-LC3B and quantification (i) of LC3B puncta in FDB muscle fibers from 8-month-old WT (n = 22 fibers from 3 mice) and BVES-KO (n = 22 fibers from 3 mice) male mice at 5 days after electroporation with pCMV-RFP-LC3B. j, k Western blot (j) and quantification (k) of LC3B/A in GA muscles from BVES-KO (n = 4 (without colchicine); n = 3 (with colchicine)) and WT (n = 5 each without/with colchicine) male mice without or with colchicine treatment as indicated (6 weeks of age). Blots are representative of two…