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. 2006 Jul 7;99(1):15-24.
doi: 10.1161/01.RES.0000231290.45676.d4. Epub 2006 Jun 8.

Myostatin regulates cardiomyocyte growth through modulation of Akt signaling

Michael R Morissette  1 , Stuart A Cook, ShiYin Foo, Godfrina McKoy, Noboru Ashida, Mikhail Novikov, Marielle Scherrer-Crosbie, Ling Li, Takashi Matsui, Gavin Brooks, Anthony Rosenzweig
Affiliations

Myostatin regulates cardiomyocyte growth through modulation of Akt signaling

Michael R Morissette et al. Circ Res. .

Abstract

Myostatin is a highly conserved, potent negative regulator of skeletal muscle hypertrophy in many species, from rodents to humans, although its mechanisms of action are incompletely understood. Transcript profiling of hearts from a genetic model of cardiac hypertrophy revealed dramatic upregulation of myostatin, not previously recognized to play a role in the heart. Here we show that myostatin abrogates the cardiomyocyte growth response to phenylephrine in vitro through inhibition of p38 and the serine-threonine kinase Akt, a critical determinant of cell size in many species from drosophila to mammals. Evaluation of male myostatin-null mice revealed that their cardiomyocytes and hearts overall were slightly smaller at baseline than littermate controls but exhibited more exuberant growth in response to chronic phenylephrine infusion. The increased cardiac growth in myostatin-null mice corresponded with increased p38 phosphorylation and Akt activation in vivo after phenylephrine treatment. Together, these data demonstrate that myostatin is dynamically regulated in the heart and acts more broadly than previously appreciated to regulate growth of multiple types of striated muscle.

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Figures

Figure 1
Figure 1
MSTN inhibits PE-mediated cardiomyocyte hypertrophy. A, Confocal microscopy. Cardiomyocytes transduced (multiplicities of infection [MOI]=100) with Ad.GFP, Ad.MSTN, or Ad.MSTN+Ad.dnMSTN were PE stimulated (100 μmol/L, 24 hours) where indicated before fixation, staining with rhodamine-phalloidin, and examination by ×40 laser-scanning confocal microscopy in at least 6 independent experiments for each condition. B, Confocal microscopy. Laser-scanning confocal microscopy (×80) of cardiomyocytes transduced with Ad.GFP or Ad.dnMSTN reveals enhanced sarcomere organization in dnMSTN-expressing cardiomyocytes. C, Cell size. Cell size in cardiomyocytes transduced and stimulated as above was quantified from digital micrographs analyzed with NIH Image (***P<0.001 vs unstimulated, Ad.GFP transduced; ###P<0.001 vs PE stimulated, Ad.GFP transduced). Cumulative data shown from 7 independent experiments with 250 to 750 cells quantitated for each condition from N independent observations: GFP (N=7); GFP+PE (N=3); MSTN (N=3); MSTN+PE (N=4); dnMSTN (N=5). D, Protein synthesis. Protein synthesis was measured by [3H]-leucine incorporation in cells treated as above and normalized to unstimulated controls (***P<0.001, **P<0.01 vs Ad.GFP infected, unstimulated; ###P<0.001 vs Ad.GFP transduced, PE stimulated). Cumulative data from 7 independent experiments shown with each condition tested N times: GFP (N=11); GFP+PE (N=11); MSTN (N=9); MSTN+PE (N=9); dnMSTN (N=4). E, Cell proliferation. Cell number was quantitated in 4 independent experiments for each of the treatment conditions at the same time point used for cell size and protein synthesis.
Figure 2
Figure 2
MSTN regulates Akt signaling. A, Akt and GSK3β phosphorylation. Immunoblots of protein from cardiomyocytes transduced with Ad.GFP or Ad.MSTN and stimulated with PE (100 μmol/L, 1 hour) where indicated, for phospho- and total Akt(473) and phospho-GSK3β(9/21). Representative data from 1 of 7 independent experiments are shown. B, dnMSTN increases Akt activation. Cardiomyocyte protein lysates after infection with Ad.GFP or Ad.MSTN were immunoblotted for phospho- or total Akt or used in an immunocomplex kinase assay (bottom). Representative data are shown from 1 of 5 to 7 independent experiments. C, Quantification of Akt phosphorylation. Densitometry of phospho-Akt(473) immunoblots normalized to total Akt was performed for each of the following conditions tested N times and normalized to GFP within each experiment: GFP (N=13); GFP+PE (N=5); MSTN (N=13); MSTN+PE (N=5); dnMSTN (N=4). ***P<0.001, *P<0.01 vs Ad.GFP infected, unstimulated; ##P<0.001 vs Ad.GFP transduced, PE stimulated.
Figure 3
Figure 3
Role of Akt signaling in PE and MSTN effects. A, PE-induced protein synthesis. Protein synthesis was measured by [3H]-leucine incorporation as above and normalized to maximal (100%) PE-stimulated incorporation (***P<0.001 vs Ad.GFP; ###P<0.001 vs Ad.GFP PE-stimulated). Cumulative data from 11 (GFP±PE) or 4 (dnAkt±PE) independent experiments are shown. B, PE-induced cell size. Cell size was measured as above (***P<0.001 vs unstimulated Ad.GFP, ###P<0.001 vs Ad.GFP PE stimulated). Cumulative data from 2 independent experiments with 163 to 221 cells measured for each condition shown. C, MSTN-inhibited cell size. Cell size was quantitated in cardiomyocytes treated as above (***P<0.001, +++P<0.001, ###P<0.001). Cumulative results from 7 independent experiments are shown.
Figure 4
Figure 4
LIF- and IGF-I–induced protein synthesis and Akt phosphorylation. A and B, Protein synthesis. Protein synthesis was measured by [3H]-leucine incorporation in cells treated as above and normalized to maximal (100%) LIF- or IGF-I–stimulated protein synthesis (**P<0.01 vs unstimulated, Ad.GFP transduced). Cumulative data from 7 (LIF) and 9 (IGF-I) independent experiments are shown. C and D, Akt phosphorylation. The ratio of immunoreactive phospho- to total Akt was quantified by densitometry and normalized to Ad.GFP control (***P<0.001 or *P<0.05 vs Ad.GFP). Each condition was tested N times: GFP (N=13); GFP+LIF (N=5); GFP+IGF-I (N=4); MSTN (N=13); MSTN+LIF (N=7); MSTN+IGF-I (N=4).
Figure 5
Figure 5
MAP kinase signaling and ANF expression. A, Erk (p44/42) phosphorylation. Cells infected as above were stimulated with PE, LIF, or IGF-I (30 minutes) before immunoblotting. Representative immunoblot and cumulative densitometry data from 7 to 9 independent experiments are shown (**P<0.01, *P<0.05 vs GFP). B, Jnk (p46/54) phosphorylation. Representative immunoblot and cumulative densitometry data from 4 independent experiments are shown. C, p38 phosphorylation. Representative immunoblot and cumulative densitometry data from 4 independent experiments are shown (*P<0.05 vs GFP). D, ANF expression. ANF mRNA was quantitated by quantitative RT-PCR in Ad.GFP- or Ad.MSTN-infected cardiomyocytes±PE stimulation (100 μmol/L, 24 hours). Results from 3 independent experiments normalized to GFP-infected control are shown (**P<0.01 vs GFP; ##P<0.01 vs GFP+PE).
Figure 6
Figure 6
Role of p38. A, Akt phosphorylation. Cardiomyocytes infected as above were pretreated (1 hour) with SB239063 (10 μmol/L) before stimulation (PE, 100μmol/L, 1 hour). Representative immunoblot and cumulative densitometry data, normalized to total Akt and GFP control, from 5 independent experiments are shown (***P<0.001 vs GFP; ##P<0.01 or ###P<0.001 vs GFP+PE). B, Akt and p38 phosphorylation. Cardiomyocytes were infected and stimulated as above. Representative immunoblot and cumulative densitometry, normalized to GAPDH and GFP controls, for 5 independent experiments are shown (*P<0.05, **P<0.01, or ***P<0.001 vs GFP).
Figure 7
Figure 7
MSTN−/− mice. A, Heart weight. PE (75 mg/kg per day) was infused for 14 days in MSTN+/+ and MSTN−/− mice before the animals were euthanized and heart weight was determined. Percentage increase in heart weight after PE infusion is shown compared with age- and genotype-matched controls receiving vehicle only. Number of mice studied: MSTN+/+ (N=27); MSTN−/− (N=21); male MSTN+/+ (N=18); male MSTN−/− (N=12); female MSTN+/+ (N=9); female MSTN−/− (N=9) (*P<0.04 vs unstimulated MSTN−/− ; #P<0.05 vs unstimulated, male MSTN−/− ). B, Akt activation. Three minutes after PE injection, hearts from male MSTN+/+ and MSTN−/− littermates were harvested. Representative immunoblots and cumulative quantification from Akt kinase assays (whole-heart lysates) are shown (N=8 mice; *P<0.04 vs MSTN+/+ ). C, p38 phosphorylation. Whole-heart lysates from male mice treated as above immunoblotted with anti–phospho-p38 antibody. Representative immunoblot and cumulative quantitative data from 6 mice shown (*P<0.05 vs MSTN+/+ ).
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