Our results suggest that endogenous MAFbx/atrogin-1 plays a key role in mediating the development of pathological hypertrophy. Expression of MAFbx is increased in response to PO, and hypertrophy induced by either PO or β-adrenergic stimulation is significantly inhibited in MAFbx KO mice. Furthermore, downregulation of MAFbx inhibits LV dysfunction in response to PO and β-adrenergic stimulation. These findings are in contrast with the role of MAFbx in skeletal muscle, where MAFbx mediates denervation-induced atrophy 9
, as well as with the inhibitory role of MAFbx in exercise-induced cardiac hypertrophy in the heart 11
. We propose that MAFbx mediates pathological hypertrophy in part through proteasomal degradation of IκB-α and stabilization of NF-κB.
MAFbx is strongly upregulated by PO in the heart at both the mRNA and protein levels. MAFbx was originally identified as a gene upregulated when skeletal muscle undergoes denervation-induced atrophy 11
. However, mRNA expression of fetal type genes and genes encoding the UPS are generally upregulated in the heart during both hypertrophy and atrophy 16
. In cardiomyocytes, both FoxO3a 17
and TNF-α 18
upregulate expression of MAFbx, whereas Akt 19
and exercise 19
reduce it. FoxO3a induces atrophy of the heart 17
. Since FoxO3a is phosphorylated and presumably excluded from the nucleus by PO, it is unlikely that FoxO3a mediates upregulation of MAFbx in our TAC model. The level of TNF-α is increased by PO and in the failing heart, and TNF-α could stimulate hypertrophy 20
. Thus, it will be interesting to elucidate the role of TNF-α in mediating upregulation of MAFbx in response to PO.
Our results suggest that upregulation of MAFbx during PO positively mediates hypertrophy and cardiac dysfunction. To our knowledge, the role of MAFbx in mediating pathological hypertrophy has not been investigated with a loss-of-function mouse model. The fact that the lack of MAFbx reverses the changes in the gene expression profile that accompany PO-induced hypertrophy, as evaluated by unbiased DNA microarray analyses, confirms the reversal of the hypertrophy phenotype in the MAFbx KO mice, supporting our hypothesis. Since selective knock-down of MAFbx also inhibited PE-induced cardiac hypertrophy in cardiomyocytes in vitro, the role of MAFbx in mediating cardiac hypertrophy must be cell autonomous.
It should be noted that Li et al
reported that overexpression of MAFbx in the heart inhibits TAC-induced cardiac hypertrophy. Overexpressed MAFbx directly binds to calcineurin and promotes its degradation 12
. Since calcineurin plays an important role in mediating cardiac hypertrophy, the authors proposed that overexpression of MAFbx may inhibit cardiac hypertrophy through suppression of calcineurin 21
. These results are in marked contrast with our finding that MAFbx mediates cardiac hypertrophy in response to PO. Furthermore, upregulation of calcineurin in response to PO was significantly inhibited in MAFbx KO mice in our hands (Online Fig. VI
). This difference may be due to the fact that Li et al
used a gain-of-function model 12
, while we used a loss-of-function model of MAFbx. In fact, we found that overexpression of MAFbx partially inhibited PE-induced hypertrophy in cultured cardiomyocytes despite marked accumulation of p65-NF-κB. This raises a possibility that exogenously overexpressedMAFbx regulates additional targets, thereby inhibiting cardiac hypertrophy. Although expression of MAFbx is low at baseline, it is markedly increased by PO. The loss-of-function model would be more useful for evaluating the role of endogenous MAFbx upregulation in regulating hypertrophy during PO. Li et al
also demonstrated that MAFbx negatively regulates physiological hypertrophy using MAFbx KO mice 11
. Previous studies showed that several molecules, such as Akt 22
and ASK 23
, not only promote physiological hypertrophy but also suppress pathological hypertrophy. Thus, it is possible that the role of MAFbx in suppression of physiological hypertrophy and mediation of PO-induced hypertrophy may involve a common mechanism. At present, however, this hypothesis remains to be tested.
In this report, we showed that MAFbx regulates pathological hypertrophy in part through activation of the NF-κB pathway. Transcription factor binding site analyses showed that genes with DNA-binding sites for the NF-κB family were significantly downregulated compared to genes without NF-κB binding sites. Subsequent analyses showed that ubiquitination of IκB-α, a negative regulator of NF-κB, in the PO heart was significantly attenuated in MAFbx KO mice. Furthermore, MAFbx and IκB-α physically interact with one another and MAFbx induces proteasomal degradation of IκB-α. MAFbx directly uniquitinates IkB in vitro. Although further investigation is needed to prove that IκB-α is a direct and physiological substrate of MAFbx in the heart in vivo, these results suggest that endogenous MAFbx regulates UPS-induced degradation of IκB-α in cardiomyocytes.
Pharmacologic blockade of NF-κB activation with pyrrolidine dithiocarbamate (PDTC) inhibits cardiac hypertrophy and cardiac remodeling 24
. Additionally, cardiac-specific NF-κB inhibition by expression of a stabilized IκB-α mutant attenuates angiotensin II-induced hypertrophy 25
. Furthermore, the targeted disruption of the p50 subunit of NF-κB has been shown to attenuate myocardial hypertrophy 26
. These findings suggest that NF-κB plays an important role in the development of cardiac hypertrophy, as well as cardiac dysfunction. It should be noted that the heart weight and cardiomyocyte size in MAFbx KO mice are not significantly different from those in WT mice at baseline, despite significant accumulation of IκB-α. We speculate that other mechanisms override the MAFbx - NF-κB pathway to prevent baseline hypertrophy. NF-κB is one of the most important signaling pathways linked to the loss of skeletal muscle mass during aging, disuse, space travel, AIDS, sepsis, cancer, and chronic heart failure 27
. This dichotomic function of NF-κB in cardiac and skeletal muscle may partially explain why MAFbx KO attenuates pathological hypertrophy in cardiac muscles but attenuates atrophy in skeletal muscle.
Although the complete suppression of PE-induced cardiac hypertrophy in the presence of MAFbx knock-down was reversed when NF-κB was overexpressed, the reversal was partial. We speculate that MAFbx may regulate additional molecules to mediate cardiac hypertrophy. Recent evidence suggests that MAFbx regulates expression of MPK-1, a phosphatase, thereby inducing persistent activation of JNKs and apoptosis of cardiomyocytes 28
. We have shown previously, however, that activation of the MEKK1-JNK pathway during PO negatively regulates cardiac hypertrophy and cardiac dysfunction 29
. Thus, the role of the MPK1-JNK pathway in mediating the effect of MAFbx during PO remains to be elucidated.
Our findings have significant clinical implications. First, endogenous MAFbx not only mediates hypertrophy, but also induces cardiac dysfunction during PO. Second, expression of MAFbx is increased in skeletal muscle in chronic heart failure 30
. MAFbx is implicated in the development of muscle atrophy during cardiac cachexia 30
. We therefore speculate that intervention to attenuate the function of MAFbx during cardiac hypertrophy and heart failure may not only ameliorate cardiac remodeling and dysfunction, but also prevent skeletal muscle atrophy, a significant complication in heart failure patients.
In conclusion, endogenous MAFbx plays an essential role in mediating pathological cardiac hypertrophy in response to PO, in part through nuclear accumulation of NF-κB (Online Fig. VII
). We propose that MAFbx and its downstream signaling molecules, including NF-κB, could be important targets for future treatment of heart failure patients.