Genetic deletion of myostatin from the heart prevents skeletal muscle atrophy in heart failure

Abstract

Background: Cardiac cachexia is characterized by an exaggerated loss of skeletal muscle, weakness, and exercise intolerance, although the cause of these effects remains unknown. Here, we hypothesized that the heart functions as an endocrine organ in promoting systemic cachexia by secreting peptide factors such as myostatin. Myostatin is a cytokine of the transforming growth factor-beta superfamily that is known to control muscle wasting.

Methods and results: We used a Cre/loxP system to ablate myostatin (Mstn gene) expression in a cell type-specific manner. As expected, elimination of Mstn selectively in skeletal muscle with a myosin light chain 1f (MLC1f)-cre allele induced robust hypertrophy in all skeletal muscle. However, heart-specific deletion of Mstn with an Nkx2.5-cre allele did not alter baseline heart size or secondarily affect skeletal muscle size, but the characteristic wasting and atrophy of skeletal muscle that typify heart failure were not observed in these heart-specific null mice, indicating that myocardial myostatin expression controls muscle atrophy in heart failure. Indeed, myostatin levels in the plasma were significantly increased in wild-type mice subjected to pressure overload-induced cardiac hypertrophy but not in Mstn heart-specific deleted mice. Moreover, cardiac-specific overexpression of myostatin, which increased circulating levels of myostatin by 3- to 4-fold, caused a reduction in weight of the quadriceps, gastrocnemius, soleus, and even the heart itself. Finally, to investigate myostatin as a potential therapeutic target for the treatment of muscle wasting in heart failure, we infused a myostatin blocking antibody (JA-16), which promoted greater maintenance of muscle mass in heart failure.

Conclusions: Myostatin released from cardiomyocytes induces skeletal muscle wasting in heart failure. Targeted inhibition of myostatin in cardiac cachexia might be a therapeutic option in the future.

Figure 1

Genetic deletion of Mstn in skeletal muscle produces hypertrophy. (A) Schematic of the crosses performed to delete the Mstn gene in skeletal muscle with the MLC1f-cre line or in heart with the Nkx2.5-cre line. Muscle weights (MW) normalized to tibia length (TL) of the (B) quadriceps (C) gastrocnemius and (D) soleus from the indicated groups of mice at 2 months of age. The number of mice per group is indicated within the bar *p<0.01 vs. control. (E) Western blot for myostatin protein from the plasma of wildtype (WT) or global Mstn^−/− mice. Recombinant myostatin protein is shown as a control. (F) Western blots for myostatin protein in the plasma of the indicated mice after 2 weeks of TAC stimulation or a control sham procedure. Mstn^−/− plasma is shown as a control.

Figure 2

Mstn deletion from heart prevents skeletal muscle atrophy in heart failure. (A) Representative RT-PCR analysis of myostatin and L7 (control) in heart and quadriceps from control and Mstn fl/fl^Nkx2.5-cre mice. (B) RT-PCR analysis of myostatin and L7 (control) in the quadriceps from Mstn fl/fl^Nkx2.5-cre mice 12 weeks after sham or TAC surgery. (C) RT-PCR analysis of myostatin and L7 (control) in the heart from control and Mstn fl/fl^Nkx2.5-cre mice 12 weeks after sham or TAC surgery. (D) Western blot from quadriceps of Mstn fl/fl and Mstn fl/fl^Nkx2.5-cre mice 12 weeks after sham or TAC surgery. As a control the last lane shows loss of pre-promyostatin from quadriceps in Mstn fl/fl^MLC-cre mice. (E) PCR (30 cycles) from quadriceps genomic DNA isolated from Mstn fl/fl or Mstn fl/fl^Nkx2.5-cre mice 12 weeks after sham or TAC surgery to assay for the recombined allele (Δ, top) or the un-recombined allele (Wt, bottom). The last lane shows recombination of the Mstn locus in Mstn fl/fl^MLC-cre mice from quadriceps and negative control without Cre in the adjacent lane. (F) Fractional shortening (FS) determined by echocardiography in control and Mstn fl/fl^Nkx2.5-cre mice 12 weeks after sham (Sh.) or TAC surgery. *p<0.05 vs. sham. (G) Heart weight normalized to body weight (HW/BW) in control and Mstn fl/fl^Nkx2.5-cre mice 12 weeks after sham (Sh.) or TAC surgery. *p<0.05 vs. sham. (H) Lung (Lg) weight normalized to body weight in control and Mstn fl/fl^Nkx2.5-cre mice 12 weeks after sham (Sh.) or TAC surgery. *p<0.05 vs. sham. (I,J,K) Muscle weights (MW) normalized to tibia length (TL) for quadriceps, gastrocnemius and soleus from control and Mstn fl/fl^Nkx2.5-cre mice 12 weeks after sham or TAC surgery. *p<0.05 vs. sham control, #p<0.05 vs. TAC control. The number of mice per group in panels F-K is indicated within the bars of the graphs.

Figure 3

Cardiomyocyte specific overexpression of myostatin reduces cardiac and skeletal muscle mass. (A) Representative RT-PCR analysis of myostatin and L7 (control) expression in cardiac muscle from non-transgenic (NTG) mice and lines 15 and 27 transgenic mice. (B) Western blot for myostatin protein in plasma of wildtype (WT) and myostatin transgenic mice. Recombinant myostatin protein is shown as a migration control. (C,D,E,F) Muscle weights (MW) or heart weight (HW) normalized to tibia length (TL) from 3 month-old non-transgenic (NTG) and transgenic (TG) mice (Line 27). The number of mice per group is indicated within the bar. *p<0.01 vs. NTG.

Figure 4

Systemic antibody inhibition of myostatin enhances muscle mass in heart failure. (A) Schematic representation of the experimental design for TAC surgery and JA-16 (anti-myostatin) or control antibody (Con AB) infusion and echocardiographic assessment of heart function. (B,C) Cardiac fractional shortening (FS) before treatment at 8 weeks and at sacrifice at 14 weeks after surgery in sham or TAC operated mice treated with Con AB or JA-16. The number of mice per group is indicated within the bar. *p<0.001 vs. sham. (D,E) Muscle weights (MW) normalized to tibia length (TL) were analyzed in quadriceps and gastrocnemius muscles from sham or TAC-operated mice at sacrifice at 14 weeks after surgery. Mice were treated with either Con AB or JA-16. *p<0.05 vs. sham Con AB, # p<0.01 vs. TAC Con AB, † p<0.01 vs. sham JA-16.