Activation of calcium signaling through Trpv1 by nnos and peroxynitrite as a key trigger of skeletal muscle hypertrophy

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1 Activation of calcium signaling through Trpv y nnos and peroxynitrite as a key trigger of skeletal muscle hypertrophy Naoki Ito, Urs T. Ruegg, Akira Kudo, Yuko Miyagoe-Suzuki, and Shin ichi Takeda Supplementary Information a Sham c Sham Nos +/+ Nos / d 6 2 Capsaicin e Hindlim-suspension Capsaicin f Denervation Capsaicin 6 2 Supplementary Figure Quantitative histological analysis related to Figure and Figure. (a c) Average size of the cross-sectional areas (CSA) of muscle fiers from wild-type and nnos-null mice (a), L-NAME administered mice () and 7-nitroindazole administered mice (c) (n > 3 for all groups). (d f) Left: The CSA distriutions of muscle fiers from capsaicin-administered mice (d), from hindlim-suspended mice (e) and from denervated mice (f). At least 2, fiers were counted. Right: Average size of the CSA (n = for all groups). P <.5, P <. y Student s t-test (d f) or y ANOVA with Tukey-Kramer test (a c). Error ars indicate s.e.m.

2 a Total protein content (µg) Total protein content (µg) sham Day 7 Day sham Day 7 Day c Total protein content (µg) Trpv +/+ Trpv / Supplementary Figure 2 -induced increase of protein contents is impaired in nnos-null, FeTPPS-administered or Trpv-null mice. (a c) Total protein contents of plantaris muscle from wild-type and nnos-null mice (a), FeTPPS-administered mice () and Trpv-null mice (c) (n > for all groups). P <.5, P <., P <. y ANOVA with Tukey-Kramer test. Error ars indicate s.e.m. 2

3 a 2A 2X 2B Sham Sham Nos +/+ Nos / Type 2A Type 2B Type Nos +/+ Nos / Nos +/+ Nos / Nos +/+ Nos / c Synergist alation (d) d BrdU pulses Nos +/+ Nos / e BrdU (+) nuclei / fier laminin α2 BrdU f F/ (+) cells / section g Ly-6G (+) cells / section Nos +/+ Nos / Nos +/+ Nos / Nos +/+ Nos / 3

4 Supplementary Figure 3 The transition of myosin heavy chain (MyHC) isoforms, activation of satellite cells and inflammatory response induced y overload is not impaired in nnos-null mice. (a) Representative silver stainings of MyHC isoforms separated y glycerol-containing gels. () The percentage of MyHC type IIA-, IIB- and I-positive fiers in wild-type and nnos-null plantaris muscle as measured y immunostaining with MyHC isoformspecific antiodies (n = 3 for all groups). (c) Schematic diagram for the BrdU pulsechase experiment. (d) Representative muscle sections showing immunostainings with anti-laminin a2 (green) and anti-brdu (red) antiodies. The BrdU-positive nuclei inside the muscle asal lamina (arrows) were interpreted as eing activated satellite cells. Scale ar: µm. (e) The numer of activated satellite cells in muscle sections from sham and overloaded wild-type and nnos-null mice (n > 3). (f and g) The numer of F/ positive macrophages (f) and Ly-6G positive neutrophils (g) in muscle sections from sham and overloaded wild-type and nnos-null mice (n = for all groups). P <. y ANOVA with Tukey-Kramer test. Error ars indicate s.e.m., not significant.

5 a (mg g ) (mg g ) Nos / overload Supplementary Figure Progression of hypertrophy y nitric oxide is independent of the sgc-cgmp pathway. (a) Muscle weights 7 d after synergist alation from the cell-permeale cgmp mimetic, -Br-cGMP-, and the cgmp-specific phosphodiesterase inhiitors, tadalafilor sildenafil-administered nnos-null mice (n > ). () Muscle weights 7 d after synergist alation from the guanylate cyclase inhiitor, ODQ administered wild-type mice (n > ). Statistical analysis was performed using ANOVA with Tukey-Kramer test. Error ars indicate s.e.m., not significant. ODQ 5

6 a colon c (mg g ) NOX NOX2 NOX3 NOX TBP rain (mg g ) RT(-) Nos +/+ plantaris d e Sham f (mg g ) sham overload GKT369 GKT369 g h i 2 Sham: pre-treatment : pre-treatment. : post-treatment.6 (mg g ) DPI Nox2 +/Y cpm (normalized to normal load)..2 Nox2 /Y GKT369 Sham (- Nox +/+ Nox / Normal load 3 min j p-p7s6k(thr39) p7s6k p-(ser73) Nox +/+ Nox / 6

7 Supplementary Figure 5 NOX promotes overload-induced muscle hypertrophy. (a) Muscle weights 7 d after synergist alation from the mitochondrial respiratory chain inhiitor, rotenone-, the xanthine oxidase inhiitor, allopurinol-, or the gloal NOX inhiitor, apocynin-administered wild-type mice (n > ). () RT-PCR analysis of NOX isoform expression in four samples of plantaris muscle. Colon and rain tissue samples were used as positive controls for NOX and NOX3, respectively. TATAinding protein (TBP) was used as a loading control. (c) Muscle weights 7 d after synergist alation in Nox2-null or Nox-null mice (n > 3). (d) Muscle weights 7 d after synergist alation from mice administered with either vehicle or the NOX inhiitor, GKT369 (n > 3). (e) Representative H&E stainings of transverse sections of the plantaris muscle from vehicle- or GKT369-administered mice. Scale ar: µm. (f) The cross-sectional area (CSA) distriutions are shown as frequency histograms. At least 2, fiers were counted. (g) The gloal NOX inhiitor, DPI, was administered intraperitoneally 3 min efore (pre-treatment) or 6 min after (post-treatment) synergist alation. Muscle weights 7 d after synergist alation were measured (n > ). (h) The CSA distriutions of muscle fiers from DPI-administered mice. (i) Superoxide production as measured y a lucigenin-derived chemiluminescence assay using homogenates of muscles exposed to normal load or overload for 3 min with or without DPI, GKT369 and the antioxidant N-acetyl cysteine (NAC) (n > 5). (j) Western lot analysis for phosphorylation of p7s6k (Thr39) and (Ser73) of muscles from normal load or overloaded for 3 min in wild-type or Nox-null mice (n > 3). P <.5, P<., P <. y ANOVA with Tukey-Kramer test. Error ars indicate s.e.m., not significant. 7

8 a p7s6k Rapamycin c (mg g ) Rapamycin p7s6k Nos +/+ Nos / FeTPPS Supplementary Figure 6 The immediate phosphorylation of p7s6k and the susequent increase in muscle weight induced y overload is mtor-dependent. (a) Three minutes after the onset of overload, plantaris muscles were taken from vehicle- or rapamycin-administered C57BL/6 mice. Phosphorylation of (Ser73) and p7s6k (Thr39) was evaluated y Western lots. () Plantaris muscles weights 7 d after synergist alation from vehicle- or rapamycin-administered mice (n > 3). (c) nnos-null or FeTPPS-administered mice were forced to run on a treadmill. Phosphorylation of (Ser73) and p7s6k (Thr39) was evaluated y Western lots. P <.5, P <. y ANOVA with Tukey-Kramer test. Error ars indicate s.e.m.

9 a Sham Day Day 3 Day 7 p7s6k Nos +/+ Nos / IGF- C57BL/6 Nos / Trpv / p- (Ser73) Nos +/+ Nos / p-p7s6k (Thr39) Nos +/+ Nos / Supplementary Figure 7 The response to IGF- is not impaired in nnos-null or Trpv-null mice. (a) Western lot analysis indicating the effect of IGF- on phosphorylation of p7s6k (Thr39) and (Ser 73) in C57BL/6, nnos-null and Trpv-null mice (n = ). () Western lot analysis indicating phosphorylation of p7s6k and in nnos-null at, 3 and 7 d after synergist alation. 9

10 s after s after a Thapsigargin.2 Peak magnitude of the Ca 2+ response (ΔF/F) SIN- c p7s6k p7s6k p7s6k p7s6k Thapsigargin Ionomycin A237 SIN- d Dantrolene Nifedipine e p7s6k p- (Ser73) Dantrolene f p7s6k Nifedipine g (mg g ) Nos +/+

11 Supplementary Figure SIN- induces intracellular calcium release from the sarcoplasmic reticulum y regulating a RyR- or DHPR-independent pathway. (a) Left: Pseudocolor Ca 2+ images of C2C2 myotues s following SIN- treatment in a Ca 2+ uffer in order to diminish extracellular Ca 2+, or pre-treated with thapsigargin to deplete Ca 2+ stores. Scale ar: µm. Right: Quantitative analysis of the effect of Ca 2+ uffer or thapsigargin on the peak magnitude of the SIN-- induced increase of [Ca 2+ ] i (n > 3). () Western lot analysis indicating the effects of thapsigargin, ionomycin or A237 with or without BAPTA-AM on phosphorylation of (Ser73) and p7s6k (Thr39). (c) Western lot analysis indicating the effect of SIN- with or without BAPTA-AM on phosphorylation of and p7s6k. (d) Left: Pseudocolor Ca 2+ images of C2C2 myotues s following SIN- treatment with or without dantrolene or nifedipine to inhiit RyR or DHPR. Scale ar: µm. Right: Quantitative analysis of the effect of dantrolene or nifedipine on the peak magnitude of the SIN--induced increase of [Ca 2+ ] i (n > 3). (e and f) Western lot analysis indicating the effects of dantrolene (e) or nifedipine (f) on the overload-induced phosphorylation of p7s6k and (n > 3 for all groups). (g) Muscle weights 7 d after synergist alation in vehicle-, dantrolene- or nifedipine-administered mice (n = ). P <. y ANOVA with Tukey-Kramer test., not significant.