Myostatin Regulates Energy Homeostasis in the Heart and Prevents Heart Failure ”

Response to Letter Regarding Article, “Myostatin Regulates Energy Homeostasis in the Heart and Prevents Heart Failure” In the article referred to by the letter of Neumann et al, we found that inactivation of myostatin in the adult heart caused a metabolic switch toward glycolysis and glycogen accumulation, cardiac hypertrophy, and heart failure, which was associated with massive activation of AMP-activated kinase (AMPK). Because pharmacological inhibition of AMPK in vitro and in vivo reversed several aspects of this phenotype, we concluded that myostatin-mediated repression of AMPK is required to maintain a mature aerobic energy metabolism in the heart and to prevent cardiac hypertrophy. Neumann et al challenge this conclusion arguing that activation of AMPK might represent the reaction to metabolic alterations rather than the cause. Furthermore, they postulate that increased transphosphorylation of protein kinase D observed in our study after inactivation of myostatin in the adult heart might explain increases in glucose utilization and uptake and cardiac hypertrophy by phosphorylation of HDAC5 (histone deacetylase 5) and subsequent deinhibition of myocyte enhancer factor 2 (MEF2). We share the skepticism expressed by Neumann et al on the use of pharmacological inhibitors such as the AMPK-inhibitor compound C, because many inhibitors exert off-target effects. To cope with this problem, we took advantage of a second inhibitor Ara-A, which is structurally and mechanistically different from compound C. Ara-A had exactly the same effect as compound C in rescuing the metabolic phenotype after myostatin deletion. In addition, we found that the treatment of primary cardiomyocytes with compound C followed by real-time respirometry leads to decreased glycolytic capacity, whereas AICAR (5-aminoimidazole4-carboxamide ribonucleotide), an AMPK activator, increased the glycolytic capacity. We also demonstrated that either inhibitor blocked increased phosphorylation of AMPK at the activating phosphorylation site T172 after inactivation of myostatin. In our view these observations clearly indicate that the inactivation of myostatin directly causes AMPK activation. To further validate activation of AMPK, we monitored phosphorylation of 2 AMPK targets, acetyl-CoA carboxylase, and glycogen synthase (GS). Neumann et al speculate that phosphorylation of GS at S641 might have been caused by GS kinase 3 and not by AMPK. Of course, GS kinase 3 does also phosphorylate GS at Ser641. However, increase of GS phosphorylation at Ser641 closely correlated with increased AMPK activity in our study, whereas inactivation of AMPK results in decreased Ser641 phosphorylation indicating that AMPK targets GS. Moreover, activation of GS kinase 3 suppresses and not induces cardiac hypertrophy, which argues against functionally relevant GS kinase 3 activation in myostatin mutant mice. Based on the general assumption that AMPK is a safeguard and not a culprit when it comes to cardiac hypertrophy, Neumann et al challenge our hypothesis that excessive activation of AMPK contributes to cardiac hypertrophy. Yet, treatment of adult myostatin mutant mice with either compound C or Ara-A prevents cardiac hypertrophy in our experimental setting. We are aware of the fact that AMPK is generally seen as inhibitor of protein synthesis, which seems to contradict a role of AMPK activation in promoting cardiac hypertrophy. However, point mutations in the nucleotide-binding region of the AMPK γ2-subunit, encoded by the Prkag2 gene (eg, the N488I allele) resulting in aberrant increase of kinase activity, cause cardiac hypertrophy probably via mechanistic target of rapamycin (mTOR) and FoxO (forkhead box O) signaling pathways independent of glycogen storage. Moreover, the expression of a kinase-dead version of AMPK-α2 mice in compound-heterozygous mice carrying the Prkag2 allele N488I, which increases AMPK activity, blocks cardiac hypertrophy and decreases enhanced glycogen storage. In further support of the prohypertrophic role of aberrant AMPK activity, we identified several new interaction partners of AMPK involved in hypertrophic signaling by coimmunoprecipitation. Therefore, a link seems to exist between myostatin, AMPK, and cardiac hypertrophy, which would fit to the well-known role of myostatin as inhibitor of Akt and mTOR signaling in heart and skeletal muscle. Neumann et al correctly state that most of the PRKAG2 mutations render AMPK insensitive to AMP, but cause increased AMPK activity, glycogen storage, and cardiac hypertrophy (see also paragraph above). We cannot see that these facts collide with our notion that PRKAG2 “mimics aspects of the myostatin phenotype,” which is characterized by increased AMPK activity, glycogen storage, and cardiac hypertrophy. Instead, the rescue of the PRKAG2 N4881 phenotype associated with increased AMPK activity by kinase-dead AMPK-α2 seems to phenocopy the effects of pharmacological inhibitors of AMPK in hearts of myostatin mutants with increased AMPK activity. Neumann et al reason that activation of AMPK after inactivation of myostatin in the heart might represent the reaction to metabolic alterations rather than the cause, which is a reasonable concern. However, we demonstrated several direct effects of myostatin signaling on AMPK: (1) we showed that cardiacspecific overexpression of myostatin reduces AMPK activity in vivo and (2) we demonstrated that treatment of adult primary cardiomyocytes with recombinant myostatin blocks AMPK phosphorylation after 10 minutes in vitro. Moreover, other groups also observed activation of AMPK in skeletal muscle and fat after inactivation of myostatin. Regulation of AMPK activity is complex and does not solely depend on the cellular energy status as indicated by the activation of AMPK via CamKKβ (calcium/ calmodulin-dependent protein kinase kinase beta). Similarly, we are just at the beginning of understanding the pleiotropic effects of AMPK in the heart in physiological and pathological conditions. Neumann et al point out that the stimulation of AMPK in cardiomyocytes upregulates glucose and fatty acid uptake as well as their utilization but did not mention that AMPK activation in left-ventricular hypertrophy activates glucose uptake and in parallel decreases β-oxidation. In our article, we described decreased interaction of AMPK with all major enzymes involved