Fig 2: 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.

Fig 3: 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. Cross-sectional fiber area type IIb, **p = 0.0028 for 1000–2000 µm2; *p = 0.0254 for 2000–3000 µm2 and *p = 0.0125 for 3000–4000 µm2 of muscle fiber area from sham vs. TAC group (p), and type IId, ****p < 0.0001 of fiber area <1000 µm2 (q) of quadriceps muscles 12 weeks after sham or TAC surgery, n = 4/group, analyzed from microscopic pictures with ATPase staining (pH 4.2) as shown in (r). Scale bar: 300 μm. s Ostn (Musclin) mRNA levels in different organs 12 weeks after sham or TAC surgery, n = 3/group. t Immunoblot (the size of the proteins is indicated in kDa) and immunofluorescence staining (u) showing Musclin protein levels in the quadriceps muscle of sham and TAC mice. Scale bar: 100 μm. v Musclin plasma levels in mice 12 weeks after sham or TAC surgery, n = 10 for sham and n = 9 for TAC, *p = 0.0115. w Time course (2, 6 and 12 weeks after TAC or sham surgery), n = 4/group, showing the decline of Ostn mRNA levels in the gastrocnemius, triceps, ***p = 0.0007 6 weeks and *p = 0.0241 12 weeks, in quadriceps muscles, **p = 0.002 by 12 weeks after TAC compared to sham mice. Data are shown as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by Kruskal–Wallis test with Dunn’s multiple comparisons test (for s) or two-tailed Student’s t test (all other numerical data containing panels) or p value determined by one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test (p, q). Source data are provided as a source data file.

Fig 4: 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 for siControl cells, vehicle treated vs. stimulated with CNP, ***p = 0.0002 vs. stimulated with Musclin, and ****p < 0.0001 vs. treated with both CNP and Musclin, ***p = 0.0002 for siNPR3 cells, vehicle treated vs. stimulated with CNP, ****p < 0.0001 vs. treated with Musclin and vs. cells treated with both CNP and Musclin (e). Assessment of cardiac fibroblast migration by scratch assay in cardiac fibroblasts (n = 6/condition) treated with siRNA, CNP and Musclin as indicated (f–h), *p = 0.036 for siControl cells, vehicle treated vs. treated with CNP, ***p < 0.0001 vs. Musclin and vs. stimulated with CNP and Musclin, *p = 0.046 for siNPR1 cells, vehicle treated vs. stimulated with CNP, **p = 0.0013 vs. Musclin and ****p < 0.0001 vs. cells treated with both CNP and Musclin (f); *p = 0.0148 for siControl cells, vehicle treated vs. treated with CNP, **p = 0.0013, and *p = 0.0148 between siControl and siNPR2 cells treated with both CNP and Musclin (g); *p = 0.0162 for siControl cells, vehicle treated vs. cells stimulated with CNP, **p = 0.0042 vs. cells treated with Musclin and ***p = 0.0004 vs. cells stimulated with both CNP and Musclin, *p = 0.0162 for siNPR3 cells, vehicle treated vs. stimulated with CNP or Musclin and ***p = 0.0002 vs. stimulated with Musclin and CNP, ***p = 0.0005 for siControl cells treated with Musclin und CNP vs. siNPR3 cells stimulated with Musclin and CNP, ***p = 0.0008 for vehicle-treated siControl cells vs. vehicle-treated siNPR3 cells (h). i, j Assessment of cardiac fibroblast proliferation and migration after stimulation with Musclin, CNP or the PKG inhibitor DT3 as indicated (for proliferation n = 7/condition, **p = 0.0018 for vehicle vs. Musclin treated cells and *p = 0.016 for cells stimulated with Musclin vs. cells treated with Musclin and DT3 (i), and for migration n = 4/condition, **p = 0.0043 for vehicle-treated cells vs. stimulated with Musclin, ***p = 0.0005 for cells treated with Musclin vs. treated with DT3 (j)). k Immunoblot from cardiac fibroblasts’ protein lysate for the indicated proteins after stimulation as indicated. GAPDH was used as loading control. 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, **p < 0.01, ***p < 0.001, ****p < 0.0001 determined by one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test. Mu stands for Musclin. Source data are provided as a source data file.

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 described for (f) (n = 12 cells/group, *p = 0.0168 for Vehicle vs. CNP and *p = 0.0169 for cells stimulated with CNP vs. Musclin). j–l Cell shortening in cardiomyocytes treated as indicated from wild-typ (WT) (vehicle cardiomyocytes n = 46, after CNP stimulation n = 24, treated with Musclin n = 21, cardiomyocytes treated with CNP and Musclin n = 16 cells), ****p < 0.0001, *p = 0.0278 and **p = 0.0091 (j), cardiomyocyte-specific Npr1 knockout (vehicle-treated cardiomyocytes n = 25, after CNP stimulation n = 27, cardiomyocytes treated with Musclin n = 25, cardiomyocytes treated with CNP and Musclin n = 22, *p = 0.01 and ***p = 0.0003 (k), and global Npr2 knockout mice (vehicle-treated cardiomyocytes n = 40, after CNP stimulation n = 39, treated with Musclin n = 31, cardiomyocytes treated with CNP and Musclin n = 28 cells) (l). Data in bar graphs are shown as mean ± standard error of the mean (SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 determined by Kruskal–Wallis with Dunn’s multiple comparisons test (e) or by one-way ANOVA followed by the Holms–Sidak’s multiple comparisons test (all other panels). Source data are provided as a source data file.