Myostatin plays a pivotal role in skeletal muscle growth, as demonstrated by impressive muscle hypertrophy with inhibition1
or cachexia with overexpression.17
Its potential role in cardiac muscle regulation is only partially understood, and it is unclear whether myostatin is associated with changes in ventricular function, cardiomyocyte size, or clinical status in cardiac disease.
The primary findings of our study in this small cohort of patients were the following: (i) levels of the cardiac myostatin propeptide (marker of activation) were increased in HF and increased after LVAD support in patients with DCM, (ii) levels of myostatin latent complex were increased in the serum of DCM HF patients compared with healthy controls but did not correlate with BMI or exercise tolerance, (iii) Smad phosphorylation and Erk phosphorylation were increased in HF patients, suggestive of increased downstream activity of myostatin, (iv) BMP-1 expression was increased in patients with advanced HF, correlating with higher levels of the cleaved propeptide, (v) myostatin activity was associated both with compensatory myocyte hypertrophy in HF and with myocyte size regression after LVAD support in DCM patients. These findings represent the first report of human myostatin regulation in cardiac disease.
Myostatin may exert a direct effect on cardiomyocyte size and hypertrophy in animal models. As shown by Morissette et al
when myostatin was overexpressed in neonatal cardiomyocytes in the presence of the α-agonist phenylephrine, protein synthesis of the cell and cell size were reduced via the reduction of Akt.18
Lower heart weight is associated with myostatin overexpression in mice,19
consistent with its effects in skeletal muscle; this finding was also confirmed by Artaza et al
who observed lower heart weight in a transgenic model of myostatin overexpression in 7-week-old mice.
The increase in myostatin activation in HF (pre-LVAD), represented by elevated levels of cleaved propeptide and supported by increased Smad phosphorylation, is most likely stimulated by myocyte stretch and increased myocardial stress. A state of pathological stress, induced by pressure–volume overload, produces a maladaptive response to overcome organ hypoperfusion: myocytes hypertrophy, generate greater force per unit stretch, and ultimately the ventricle dilates. Specifically, the process of ventricular hypertrophy in response to pressure–volume overload induces expression of insulin-like growth factor-1 (IGF-1).11,20
Insulin-like growth factor-1 appears to counteract many of the actions of myostatin in vitro
Myostatin activation, we postulate, may be highly dependent on a tightly controlled positive feedback pathway between IGF-1 and myostatin: as pressure–volume-induced stress occurs, IGF-1 increases, and myostatin activation increases as a compensatory function. Insulin-like growth factor-1 may serve to increase cardiomyocyte growth and hypertrophy in an effort to maintain haemodynamics or as a pathological response to myocardial stress, whereas myostatin may act in an opposite fashion to limit unrestrained cellular growth, possibly to prevent the untoward effects of overcompensated myocardial growth as a homeostatic function, as suggested by other investigators.22
This hypothesis is supported by several in vitro
and in vivo
studies. Shyu et al
have demonstrated that cyclic stretch increases IGF-1, but also increases myostatin expression. Further evidence for a counter-regulatory mechanism is found in the control of Akt activation, a serine threonine kinase critical to enhanced myocyte growth and size.18
Myostatin decreases Akt activity,12,18
and IGF-1, via binding to IGF1-R, a tyrosine kinase receptor, ultimately leads to Akt activation.22
With LVAD support and resultant loss in pressure–volume overload stimulus, IGF-1-mediated signalling that limits the actions of myostatin may be lost, allowing the inhibition of myocyte growth and regression of hypertrophy. Indeed, profound changes in myocyte size correlate with the levels of the propeptide in DCM patients—myocyte size was reduced by >30% after LVAD support, a finding that may potentially be attributed to the increased activation of myostatin that was observed. Our hypothesis also predicts reduced myostatin activation after longer durations of mechanical support—as systemic haemodynamics and ventricular stress normalize with longer duration of support, myostatin activation and, conversely, IGF-1 activation are expected to decrease.22
We did, in fact, observe lower myostatin propeptide levels in ICM and DCM patients with LVAD support for >150 days, as predicted by our hypothesis, but larger sample sizes are required to confirm this preliminary finding.
An increase in myostatin activation was not found, however, in the ICM patients. This may be due to the extensive scarring typically found in the apical and ventricular free walls after large myocardial infarction which prevents substantial ventricular reverse remodelling during LVAD support; geometric and size changes are less pronounced in these patients and contribute to the low rate of myocardial recovery after LVAD support.23,24
Finally, it must be noted that age, duration of CHF or LVAD support, pre-LVAD ejection fraction, and other clinical demographics did not correlate with myostatin activation, despite differences in the LVAD, CHF, and normal populations. Prior reports have described no variance in myostatin levels based upon age.25
We observed that levels of full-length myostatin were comparable with normal in both the ICM and DCM groups in the HF state (i.e. pre-LVAD). Cleaved propeptide levels, in contrast, were significantly elevated in both ICM and DCM in HF, suggesting that full-length myostatin is maintained at a constant level with a self-renewal mechanism, regardless of the rate of cleavage (activation); both the myostatin propeptide or the active C-terminal dimer may be involved in feedback stimulus for the full-length protein.26,27
Although Smad2/3 continued to be activated compared with normal controls after mechanical unloading, activation did not increase compared with pre-LVAD levels. However, we did observe an increase in Erk1/2 activation following mechanical unloading, and it has been demonstrated that Erk1/2 can mediate cellular effects downstream of myostatin stimulation.28
These effects may be independent of Smad signalling,28,29
which may explain why a further increase in Smad activation was not observed with mechanical unloading; neither Smad nor Erk signalling are direct markers of myostatin activation. It is also possible that myostatin activates different downstream pathways (Smad vs. Erk) depending on the local environment (HF vs. mechanical unloading).
The increased levels of circulating myostatin latent complex may serve as a reservoir for local activation in the heart by BMP-1, which has been previously implicated as an activator of the myostatin latent complex.3
Indeed, we observed that elevated expression of BMP-1 correlates with increased activation of myostatin in HF. Bone morphogenic protein-1 expression did not further increase with myostatin activation after LVAD support in the DCM patients, but this may indicate that the upregulated BMP-1 enzyme was not yet saturated or that another matrix metalloprotease may be involved in myostatin activation with mechanical unloading vs. HF.
Although the actions of myostatin on the remodelling heart may be beneficial, circulating myostatin may have deleterious effects on skeletal muscle of HF patients, which may contribute to cardiac cachexia.30
Increased serum myostatin has also been observed in other disease states, such as HIV-associated cachexia31
and a subset of patients with Duchenne muscular dystrophy.32
In this study, we could not show a relationship between circulating myostatin and either exercise capacity or BMI. Our study cohort of predominantly obese HF patients do not reflect the cachectic state of more advanced disease. However, these findings remain important, as this is the first report of increased circulating myostatin associated with HF. The role of circulating myostatin in regulating the periphery will require further study in more advanced disease patient populations.
The clinical application of a myostatin inhibitor may not be appropriate in the advanced HF state, but its application after LVAD support to promote myocardial recovery is an intriguing and important concept, as only 5% of patients with chronic HF demonstrate sufficient recovery to allow LVAD explantation without transplantation.24
There is evidence to suggest that prolonged support may not be the optimal strategy for patients to recover function. When examining data from the LVAD Working Group Study,23
ejection fraction initially improves after LVAD implantation, but after 120 days, falls significantly. Although it is currently unknown whether myostatin levels return to pre-HF levels after prolonged support, inhibition of myostatin before further functional deterioration takes place may represent a method to improve heart function. Adjunctive pharmacological therapy during LVAD support formed the basis for the use of clenbuterol (a β-adrenergic agonist with potent anabolic properties) to produce physiological myocyte hypertrophy during LVAD unloading.33
Myostatin inhibition may therefore offer an alternative approach to clenbuterol to promote recovery by altering growth signalling pathways and attenuating cellular changes during LVAD support. Close and cautious study will be required, especially in ICM patients or patients with prolonged LVAD support, as inhibition of compensatory mechanisms could worsen the HF state.
In summary, we have shown that cardiac myostatin propeptide is elevated in HF patients of both ICM and DCM aetiology, and that LVAD support increases propeptide levels in DCM patients. Increased myostatin activation by propeptide cleavage was suggested by the presence of increased Smad phosphorylation and associated with an increase in BMP1 expression. These findings correlate with a reduction in cellular size that may be attributable to downstream effects of myostatin.