Skeletal muscle derived Musclin protects the heart during pathological overload

Figures

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  Fig. 1. Musclin expression decreases in wasting skeletal muscles during advanced heart failure. a Scheme depicting the experimental time line. Quantification of heart weight/tibia length (HW/TL) ratio, n = 7/group, ****p < 0.0001 (b), lung weight/tibia length (LW/TL) ratio, n = 7/group, ***p = 0.001 (c), left ventricular ejection fraction, n = 5/sham group and n = 8/TAC group, ****p < 0.0001 (d), quadriceps muscle weight, n = 7/group, ****p < 0.0001 (e), gastrocnemius muscle weight n = 7/group, ***p = 0.0003 (f), triceps brachii muscle weight, n = 7/group, **p = 0.0051 (g), plantaris muscle weight, n = 7/group, *p = 0.0175 (h), soleus muscle weight, n = 7/group, *p = 0.0126 (i), and course of body weight (BW) during the experiment until 12 weeks after TAC or sham surgery, n = 4/sham group and n = 5/TAC group, ***p = 0.0001 (j). Inguinal fat weight, **p = 0.0021 (k) in the group of mice that was analyzed by MRI to determine the amount of abdominal fat, *p = 0.0273 (l), psoas muscle, *p = 0.0499 (m) and autochthonal muscle, *p = 0.0412 (n) 12 week after sham or TAC surgery, n = 4/group. Example MRI cross-sections are shown in (o). The red arrows indicate abdominal fat. Scale bar: 5 mm.…

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  Fig. 2. Muscle OSTN mRNA and Musclin serum levels are reduced in patients with heart failure. a OSTN mRNA levels in vastus lateralis muscle biopsy samples from control individuals (n = 9) and from patients with sarcopenia (n = 6) or cachexia (n = 5), *p = 0.0236. b Concentrations of Musclin protein in the serum of healthy control individuals (n = 56) and of patients with severe aortic stenosis (n = 26), *p = 0.0403 (c) and of the same patients excluding the ones with diabetes mellitus (n = 18), **p = 0.0044. Data are shown as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01 as determined by ANOVA followed by the Holms–Sidak’s multiple comparisons test (a) and the two-tailed Mann–Whitney test (b, c). Source data are provided as a source data file.

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  Fig. 3. AAV6-mediated overexpression of Musclin in skeletal muscle attenuates left ventricular dysfunction and myocardial fibrosis during long-term pressure overload. a Scheme depicting the experimental time line. b mRNA level of Ostn (Musclin) 9 weeks after TAC and intramuscular application of AAV6 control (Co) or AAV6 Musclin (Mu) vector (n = 12/group), *p = 0.036 (c) Immunoblot for Musclin 9 weeks after TAC and intramuscular application of AAV6 Co or AAV6 Mu. GAPDH, Actin and Tubulin are loading controls. The size of the proteins is indicated in kDa. d Musclin plasma levels in AAV6 Co (n = 21) or AAV6 Mu (n = 22) treated mice, **p = 0.0029. e Heart weight/tibia length (HW/TL) ratio in AAV6 Co (sham n = 5, TAC n = 7) or AAV6 Mu (sham n = 5, TAC n = 6) treated mice 9 weeks after sham or TAC surgery, ***p = 0.0004, *p = 0.0316. f–h Echocardiographic ejection fraction 3 weeks (all sham n = 5/group, TAC AAV6 Co n = 17, TAC AAV6 Mu n = 15), ****p < 0.0001, **p = 00015 and *p = 0.0293 (f), and 6 weeks after surgery, ****p < 0.0001 in AAV6 Control and AAV6 Musclin groups for sham vs. TAC surgery, **p = 0.0021 (g), and 9 weeks after surgery, ****p < 0.0001, ***p = 0.0006 and **p = 0.0051 (h).…

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  Fig. 4. Adult induced skeletal muscle-specific Musclin deficient mice show exaggerated LV-dysfunction and myocardial fibrosis after short-term TAC. a Scheme depicting the Musclin (Ostn) knockout (KO) targeting strategy. The exon 3 of the Ostn gene is floxed and b deleted through the crossing with double transgenic mice that express Cre recombinase selectively in skeletal muscle and only following doxycycline treatment. c Scheme depicting the experimental time line. d qPCR analysis of Ostn mRNA in the quadriceps muscle and the heart 2 weeks after TAC or sham surgery in KO vs. littermate control (WT) mice (all sham n = 8/group, all TAC n = 12/group), *p = 0.0359. e Immunoblot for Musclin in the quadriceps muscle and the heart 2 weeks after TAC or sham surgery in WT vs. KO mice. GAPDH and Tubulin are loading controls. The size of the proteins is indicated in kDa. f Immunofluorescence staining for Musclin in the quadriceps muscle of WT and KO mice. Scale bar: 100 μm. g Musclin plasma levels of WT (n = 21) and KO mice (n = 16), **p = 0.0014. h Ostn mRNA levels in the indicated organs of the mice as shown (n = 7/group), **p = 0.0027. Quantification of the heart weight/tibia length ratio…

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  Fig. 5. Musclin augments cardiomyocyte contractility by enhancement of CNP/NPR-B signaling. a Scheme depicting the proposed mechanism of augmented cardiomyocyte contractility by Musclin. Scheme (b) and results (c) of the NPR-C/Musclin/CNP competition assay. Increasing levels of recombinant CNP led to Musclin displacement from NPR-C (n = 2 per CNP concentration, mean values are shown). d Representative fluorescence images from Hek293 cells with and without transfection of the NPR-C-GFP construct. Scale bar 100 µm. e CNP concentrations (determined by ELISA) in the supernatant of transfected and untransfected Hek293 cells (as shown in d) 1 h after treatment as indicated (n = 4/condition, *p = 0.0104). f Representatives traces of sarcomere length and g quantification of cell shortening (from traces as shown in f) in isolated wild-type mouse cardiomyocytes treated with recombinant CNP or Musclin as indicated (n = 13 cardiomyocytes/group, **p = 0.0016 for Vehicle vs. CNP (10 nM), **p = 0.0012 for CNP (10 nM) vs. CNP (100 nM) and **p = 0.0026 for CNP (100 nM) vs. CNP (100 nM) plus Musclin (10 nM)). h Representative Fura-2 Ca2+ traces and quantitative analysis (i) in cardiomyocytes treated as…

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  Fig. 6. Musclin increases cGMP and cAMP levels and cardiomyocyte contractility through NPR-B-dependent inhibition of PDE3. Cardiomyocytes were isolated from either cGES-DE5 transgenic (for cGMP measurements) or from Epac2-camps (for cAMP measurements) transgenic mice. a, b Fluorescence resonance energy transfer (FRET)-based measurements of cGMP in single cultured cardiomyocytes (n = 7 cells). The stimulation with CNP and Musclin was conducted as indicated. Representative traces (a) and a quantitative analysis (b) are shown, *p = 0.0342. c, d FRET-based measurements of cAMP in single cardiomyocytes treated as indicated (n = 13 cells). The cells were stimulated with Isoproterenol (Iso), CNP and subsequently Musclin as indicated. The graph illustrates the FRET-responses to CNP in % of the maximal Iso effect. Representative traces (c) and a quantitative analysis (d) are shown, **p = 0.0019. e–h ELISA-based measurements of cGMP and cAMP in cultured cardiomyocytes under the indicated conditions from wild-typ (WT) (e, g), **p = 0.0012 for vehicle-treated cells vs. stimulated with CNP (100 nM), ****p < 0.0001 for cells stimulated with Musclin vs. with CNP (100 nM) and Musclin, **p = 0.0013 for…

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  Fig. 7. Skeletal muscle Musclin regulates cardiac cGMP and cAMP levels and phospholamban phosphorylation at the PKA phosphorylation site (Serine 16). Cardiomyocyte cGMP (a, *p = 0.0268), (c, *p = 0.048) and cAMP (b, *p = 0.029), (d, *p = 0.025) levels (determined by ELISA) after sham and TAC surgery in control (WT) and Musclin knockout (KO) mice after TAC, as well as in Sham and TAC operated mice treated either with AAV6 Control (Co) or AAV6 Musclin (Mu) (WT sham n = 5, WT TAC n = 9, KO TAC n = 10, sham AAV6 Co n = 4, sham AAV6 Mu n = 3, TAC AAV6 Co n = 6, TAC AAV6 Mu n = 7). Immunoblots for the indicated proteins (GAPDH as loading control) from cardiac protein lysate 3 days (e), cardiomyocyte protein lysate 14 days (f) or cardiac protein lysate 9 weeks (g) after TAC or sham surgery in WT or KO mice or in mice treated with AAV6 Co or AAV6 Mu as shown. The size of the proteins is indicated in kDa. Data in bar graphs are shown as mean ± standard error of the mean (SEM). *p < 0.05, determined by one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test. Source data are provided as a source data file.

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  Fig. 8. Musclin inhibits the activation of cardiac fibroblasts through NPR-B dependent activation of protein kinase G (PKG). a Measurement of cardiac fibroblast proliferation by BrDU incorporation ELISA with addition of CNP and/or Musclin (n = 5/condition, **p = 0.0039 for vehicle vs. CNP, **p = 0.0019 for vehicle vs. musclin and ***p = 0.0002). b Assessment of cardiac fibroblast migration through detection of recovery of a scratch wound after 4 h during stimulation as indicated (n = 6/condition, **p = 0.0023, ***p = 0.0003 for vehicle-treated cells vs. stimulated with Musclin, and ***p = 0.0005 vs. cells stimulated with CNP and Musclin). c–e Measurement of cardiac fibroblast proliferation by BrDU incorporation ELISA under the indicated conditions (c–e) (n = 6/condition, except siNPR2 treatment n = 7); *p = 0.0103 for siControl cells, vehicle treated vs. stimulated with CNP, **p = 0.0016 vs. stimulated with Musclin and ***p = 0.0001 vs. stimulated with CNP and Musclin, *p = 0.0103 for siNPR1 cells, vehicle treated vs. stimulated with CNP, ****p < 0.0001 vs. stimulated with Musclin and vs. stimulated with CNP and Musclin (c); *p = 0.0243, **p = 0.0055 and ***p = 0.0003 (d); **p = 0.0021…