This study reveals several key findings with regard to muscle wasting in cancer cachexia. The first, and most important, is that pro-cachectic factors do appear to have a high degree of selectivity as to which skeletal muscle gene product is targeted for downregulation. We found that of the myofibrillar proteins examined, MyHC was pronouncedly downregulated, while no significant changes occurred in the expression levels of tropomyosin, troponin, sarcomeric actin, actinin, and myosin light chain. MyHC selectivity was shown in cultured myotubes and mouse muscles treated with TNF/IFN, as well as in mice bearing C-26 tumors. In addition to these cachectic factors, we have extended our analysis by investigating the effects of other known mediators of muscle wasting. Results showed that myotubes treated with TNF and either high concentrations of IL-6 or medium conditioned with G361 melanoma cells also caused the selective downregulation of MyHC (Supplemental Figure 2). Furthermore, selective targeting of MyHC was also observed in mice bearing G361 melanomas, tumors previously shown to secrete the muscle proteolysis factor proteolysis-inducing factor (PIF) (53
). Although other myofibrillar proteins remain to be investigated, these data strongly support that MyHC is a preferred target of multiple pro-cachectic factors. Given that MyHC accounts for approximately 40% of myofibrillar protein content in mature muscle (37
), and mice lacking this gene display growth and muscle defects (54
), it is reasonable to propose that loss of MyHC would contribute significantly to the reduction in lean mass associated with a cachectic state.
A second finding revealed by this study is that inflammatory cytokines are capable of repressing MyHC at the RNA level. The data showed that TNF alone was not sufficient to regulate MyHC mRNA expression (Figure ), which is consistent with the notion that in vivo TNF functions in concert with other inflammatory cytokines and/or tumor factors to promote muscle wasting (26
). Although our findings demonstrate the dual requirement of TNF and IFN for suppression of MyHC, it should be noted that some reports do maintain the individual role of TNF in muscle wasting (56
). In addition, administration of TNF in animals has been shown to lead to the reduction of MyHC mRNA (58
), although under these conditions the effect may be secondary to the direct activity of the cytokine. Whether circulating levels of TNF or IFN are elevated in cancer patients is controversial, but studies do support their relevance in wasting (11
). There is also evidence that cytokines may rise as a consequence of the cancer therapy itself. Cachexia-like symptoms have been reported as common side effects of IFN therapy in cancer patients (59
), and elevated IFN levels were also reported in patients receiving IL-2 and IFN-β therapy (60
). We envision that regulation of myosin at the RNA level may be a contributing mechanism to muscle breakdown in conditions that favor persistent signaling from both TNF and IFN.
Which signaling molecules mediate the cooperative activity of TNF and IFN is not yet known, but there is good reason to suspect the involvement of the NF-κB pathway. NF-κB is strongly activated by TNF, and recently its activity was shown to be required for cytokine-mediated repression of MyHC protein (25
). NF-κB was also shown to be required for TNF and IFN to downregulate the expression of MyoD protein in C2C12 myotubes (25
). Consistent with these findings, the data presented in this study showed that TNF/IFN could dramatically reduce MyoD mRNA expression, and that rescued expression of MyoD in C2C12 myotubes was sufficient to block TNF/IFN–mediated repression of the MyHC IIb promoter. Given that MyoD binding to the MyHC IIb promoter is required for myosin expression in fast-twitch muscles (41
), it is tempting to speculate that cytokine-induced suppression of MyHC IIb transcription is regulated by NF-κB through the inhibited synthesis of MyoD. However, it is possible that in addition to MyoD other myogenic regulators can be targeted by cytokines, leading to the further downregulation of myosin.
Lastly, findings of this study demonstrate that selective targeting of key muscle gene products, such as MyHC, can occur through different regulatory mechanisms. In contrast to TNF/IFN–treated myotubes, selective targeting of MyHC in mice bearing C-26 tumors occurred at the protein level. Induction of ubiquitin and E3 ligase genes, in conjunction with the presence of ubiquitin-MyHC conjugates, supports the involvement of the ubiquitin-dependent proteasome system in the regulation of this myofibrillar protein. Thus, these data imply that MyHC is a preferred substrate of ubiquitin ligases. In vitro, core myofibrillar proteins have been shown to serve as efficient substrates for ubiquitin conjugation, resulting in their degradation by the ATP-dependent 26S proteasome (62
). Importantly, the rate of this proteolysis was significantly reduced when core myofibrillar proteins were preassembled into a myofibril complex. This implied that the dissociation of myofibrillar proteins in the contractile apparatus is the rate-limiting step for ubiquitination and subsequent ATP-dependent proteolysis. Consistent with this thinking, sepsis-induced muscle cachexia was reported to be associated with the disruption of sarcomere architecture and the subsequent release of myofilaments, although in this model these effects were largely found to be regulated by a calcium-dependent proteolysis pathway (63
). In line with these findings, we predict that C-26 tumor–induced muscle wasting is associated with the disorganization of the myofibrillar network, and that the specificity of MyHC downregulation in C-26 tumor–bearing mice derives from the partial dissociation of MyHC from actin and the remaining components of the sarcomeric complex. Upon dissociation of the myofibril, MyHC would thus be susceptible to ubiquitin ligase activity and primed for proteasome-dependent degradation. The absence of actin, troponin, or tropomyosin degradation may signify that these proteins remain bound to each other, thereby minimizing their polyubiquitination. Although our current data demonstrate that myosin and actin association is lost in cachectic muscle (Figure ), it will be interesting to test whether interactions are compromised between myosin and the other core myofibrillar proteins, as well as the interactions of the core proteins not found to be susceptible to proteolysis.
Based on these current results, we propose that muscle wasting is not a process regulated by the downregulation of a general number of myofibrillar proteins but rather is highly selective as to the targeting of key muscle gene products. Data presented here from different models of cytokine- and tumor-induced cachexia indicate that MyHC is a selective target irrespective of the pro-cachectic factor. The identification of MyHC as one of these selective targets may be useful in the design of future cancer-cachexia therapies.