Myostatin manipulation in vivo.

<p>Neonatal C57 mice (day 4–5) were treated with either Saline (n = 12), AAV9-LacZ (n = 11), AAV9-dnMSTAT (n = 12), or AAV9-MSTAT (n = 8),via subxiphoid injection to target the heart. A saline injection group was added to control for viral effects. (A) X-gal histochemistry demonstrates distribution of transgene expression. Western blot demonstrates expression of (B) dominant negative myostatin (propeptide antibody), (C) myostatin precursor (∼50 kDa) and processed C-monomer (∼13 kDa) (C-terminus antibody), and (D) phospho-Smad2/3. Samples from myostatin knock-out (MSTN KO) mouse heart and skeletal muscle, as well as from normal (C57) mouse skeletal muscle were also run as controls. The precursor protein is depicted with an arrow at 50 kDa and is present at high levels in the MSTN lane and absent in the MSTN KO lanes. Proposed C-dimer and monomer bands are also depicted with an arrow. Predicted molecular weights for the various forms of myostatin are as follows: full length precursor monomer (43–45 kDa); propeptide (30–32 kDa); C-terminus monomer (12–13 kDa); active C-dimer (24–26 kDa). Glycosylation may causes these forms to run approximately 5–10 kDa higher than predicted on PAGE. (E) Quantification of myostatin full-length precursor (∼50 kDa). *p<0.05 vs. Saline and LacZ (ANOVA). #p<0.05 vs. Saline, LacZ, and dnMSTAT (ANOVA). Scale bar represents 1 mm (lo) or 200 µm (hi).</p

PE increases myostatin translation and activation in NCM's.

<p>Cardiomyocytes were isolated from neonatal rats and grown to confluency. Cells were then stressed with PE (100 uM) or media only (control) for up to 18 hours. Each condition was tested n = 4 times. *p<0.05 vs. 0 min (ANOVA). Western blot was performed using the C-terminal antibody to assess levels of (A) myostatin precursor and (B) active myostatin. (C) A representative myostatin blot is depicted. Predicted molecular weights for the various forms of myostatin are as follows: full length precursor monomer (43–45 kDa); propeptide (30–32 kDa); C-terminus monomer (12–13 kDa); active C-dimer (24–26 kDa). Glycosylation may cause these forms to run approximately 5–10 kDa higher than predicted on PAGE. (D) Phospho-specific Western blot was performed to assess activation of Smad (58 kDa).</p

Myostatin negatively regulates NCM growth and proliferation by modulating signaling through Akt and NFAT3.

<p>NCM's were cultured as described above. Cells were treated with Ad-CMV-LacZ (control), Ad-CMV-dnMSTAT, Ad-CMV-MSTAT at MOI = 100 for 18 hours, then stressed with PE (100 uM) for 0 or 15 minutes. Each condition was tested n = 4 times. Phospho-specific Western blot was performed to assess the activation states of (A) Akt and (B) NFAT3. *p<0.05 vs. LacZ Con (ANOVA).</p

Myostatin negatively regulates cardiac size in vivo.

<p>Neonatal C57 mice (day 4–5) were treated with either Saline, AAV9-LacZ, AAV9-dnMSTAT, or AAV9-MSTAT via subxiphoid injection to target the heart. Mice were followed for 6 and 12 weeks, and the n for each condition is reported on the figure. Relative heart weight is reported as the ratio of heart weight to body weight at (A) 6 weeks and (B) 12 weeks. (C) Cardiomyocyte size at 12 weeks is reported as minimal feret's diameter and was measured in 3 fields using Fovea Pro software 3 months following treatment. Approximately 500–800 cells were counted per condition. *p<0.05 vs. Saline and LacZ (ANOVA). Representative images are displayed. Scale bar represents 40 µm. Magnification is 40X.</p

Myostatin modulates signaling through Akt and NFAT3 pathways in vivo.

<p>Neonatal C57 mice (day 4–5) were treated with either Saline (n = 12), AAV9-LacZ (n = 11), AAV9-dnMSTAT (n = 11), or AAV9-MSTAT (n = 8) via subxiphoid injection to target the heart. Phospho-specific Western blot was performed to determine (A) Akt activity (via GSK-3β phosphorylation) and (B) NFAT3 activity. *p<0.05 vs. Saline and LacZ (ANOVA).</p

Myostatin manipulation in NCM culture.

<p>Cardiomyocytes were isolated from neonatal rats and grown to confluency. Cells were treated with Ad-CMV-LacZ (control), Ad-CMV-dnMSTAT, or Ad-CMV-MSTAT at MOI = 100 for 18 hours in the presence or absence of PE stress (100 uM). A media only control was also performed to control for viral effects. Each condition was tested n = 4 times. (A) X-gal histochemistry demonstrates high efficiency of adenovirus transfection. Scale bar represents 200 µm (lo) or 40 µm (hi). Western blot was performed to confirm expression of (B) dnMSTAT (propeptide antibody) and (C) MSTAT (C-terminus antibody). Samples from myostatin knock-out (MSTN KO) mouse heart and skeletal muscle, as well as from normal (C57) mouse skeletal muscle were also run as controls. Predicted molecular weights for the various forms of myostatin are as follows: full length precursor monomer (43–45 kDa); propeptide (30–32 kDa); C-terminus monomer (12–13 kDa); active C-dimer (24–26 kDa). Glycosylation may cause these forms to run approximately 5–10 kDa higher than predicted on PAGE. (D) Western blot was performed on nuclear extracts to quantify the levels of nuclear Smad2/3. *p<0.05 vs. Media and LacZ (ANOVA).</p

Model of myostatin regulation and function in the heart.

<p>In this study, PE was used to model cardiac stress. This led to early (15 minute) activation of the cardiac growth pathways NFAT and Akt, and an increase in cell size was apparent by 18 hours. Erk was also activated early (15 minutes), and this was shown to be necessary for subsequent MEF-2 phosphorylation and increase in myostatin expression. Myostatin was shown to be able to exert negative feedback on growth by blocking activation of NFAT and Akt. In this model, therefore, PE activates growth via NFAT and Akt, while at the same time activating Erk, which drives MEF-2 dependent upregulation of myostatin, which in turns exerts negative feedback on growth by blocking NFAT and Akt activation. Further studies are needed to determine the mechanism of myostatin-mediated blockade of Akt and NFAT activation, including whether each process is Smad independent vs. Smad dependent.</p

Myostatin upregulation following PE stress is Erk dependent via MEF-2.

<p>Cardiomyocytes were isolated from neonatal rats and grown to confluency. Cells were treated with small molecule inhibitors of PI3K (wortmannin, 5 nM), p38 (SB203580, 3 uM), Erk (PD98059, 50 uM), calcineurin (cyclosporine, 1 uM), or control 1 hour prior to 18 hours of PE stress (100 uM) and compared to baseline (no PE, no inhibition). Each condition was tested n = 4 times. (A) Western blot was performed to assess expression of the myostatin precursor protein using the C-terminus antibody. (B) The assay was repeated with the Erk inhibitor once it was identified as being the most potent inhibitor of myostatin expression to confirm results. (C) Western blot was performed on nuclear extracts to assess the levels of nuclear MEF-2. (D) EMSA was performed to assess MEF-2 DNA binding activity in nuclear extracts. #p<0.05 vs. PE (ANOVA); *p<0.05 vs. Con (ANOVA).</p

Schematic of domains included in each myostatin construct.

<p>(A) The cDNA encoding full-length murine myostatin (MSTAT) that was overexpressed from both adenovirus and AAV vectors contained the following domains: signal sequence, propeptide (N-terminus) containing aspartate at amino acid 76 (D76), RSRR cleavage site, and C-terminus. Myostatin activation requires cleavage at RSRR as an initial step to disrupt the covalent bond between the C-terminus and the propeptide. However, the inhibitory propeptide continues to associate with the C-terminus non-covalently, and full activation of myostatin requires a second cleavage at D76 to free the C-terminus from the propeptide and induce propeptide degradation. (B) The cDNA encoding dominant negative murine myostatin (dnMSTAT) that was overexpressed from both adenovirus and AAV vectors contained the following domains: signal sequence, propeptide (N-terminus) containing a D76A mutation, RSRR cleavage site, and a truncated C-terminus resulting from a premature stop. As a result, no full-length or active myostatin is expressed from this construct. In contrast, a protease-resistant (D76A) inhibitory propeptide that acts in a dominant negative manner is expressed. Corresponding amino acid (AA) positions are indicated below each cartoon. Note that cartoon is not to scale.</p

Myostatin negatively regulates NCM growth and proliferation.

<p>Cardiomyocytes were isolated from neonatal rats and grown to confluency. Cells were treated with Ad-CMV-LacZ (control), Ad-CMV-dnMSTAT, or Ad-CMV-MSTAT at MOI = 100 for 18 hours in the presence or absence of PE stress (100 uM). Each condition was tested n = 4 times. (A) Cell number was counted at 40X in 3 fields for each treatment. (B) NCM surface area was quantified in 3 fields at 40X using Fovea Pro software after laminin staining, and representative images are displayed. Approximately 500–800 cells were counted per condition. *p<0.05 vs. LacZ Con (ANOVA).</p