Open Access

Elisa Martin

• The maintenance of protein homeostasis in muscle by degradation systems, e.g. the autophagy lysosomal pathway (ALP) and the ubiquitin-proteasome system (UPS), is of great importance. It prevents the accumulation of nonfunctioning and not properly folded proteins, which can lead to protein aggregate myopathies (PAMs) and several other protein storage diseases. Degradation by the UPS depends on the transfer of ubiquitin to a target protein. This happens in a cascade of E1-E2-E3 proteins. This process is also involved in protein location and regulation of protein activity. E3 ligases are often tissue specific. Muscle RING-finger proteins (MuRFs) are a family of really interesting new gene (RING)-Finger E3 ubiquitin ligases, that are almost exclusively expressed in the striated muscle. They play a role in muscle wasting, but are also important for the maintenance of the structure of striated muscle. MuRF proteins are also involved in the regulation of the striated muscle energy metabolism. Previous work has demonstrated that MuRF1/MuRF3 DKO mice show a protein surplus myopathy characterized by an accumulation of myosin heavy chain proteins in striated muscles and a reduction in function of both heart and skeletal muscle. The aim of this study was to test the hypothesis that the myopathic phenotype of MuRF1/MuRF3 DKO mice is mediated by a disturbed energy homeostasis in the heart and skeletal muscle, with focus on mitochondrial function. Because sex-specific differences have not been investigated in these mice so far, a further aim was to investigate any differences between male and female mice. To test these hypotheses, we measured the weight of the heart and the hindlimb muscles tibialis anterior and soleus to detect a possible hypertrophy in the DKO mice. Hematoxylin and eosin staining of histological cross sections of the tibialis anterior were performed to investigate protein accumulations. Muscle function was quantitated via grip strength and specific force measurements. Possible changes in protein amounts were detected via mass spectrometry analyses and western blot analyses. Changes in gene expression were investigated by qRT-PCR. Coimmunoprecipitation was used to determine direct interactions between proteins. Protein stability and ubiquitination were investigated by cycloheximide (CHX) and ubiquitination assays, respectively. DKO mice showed an increase in heart and skeletal muscle weights. Grip strength assays revealed limb weakness of DKO mice. H&E staining of histological cross sections of the tibialis anterior muscle (TA) showed protein aggregates within myofibers. Mass spectrometry analyses of proteins isolated from TA and heart muscle revealed an increase of muscle stress markers and structural proteins in DKO mice, while proteins involved in the energy metabolism were reduced. Especially interesting here were the proteins of the mitochondrial electron transport chain (ETC), which play a major role in the energy production of the mitochondria by catalyzing the phosphorylation of ADP to ATP, the universal energy carrier in all living organisms. These changes were more pronounced in TA compared to heart. Western blot and qRT-PCR results of ETC subunits supported our proteome data. They also revealed a sex-specific difference, in which the reduction ETC subunits was more pronounced in females than males. In female TA NDUFB8, SDHB, UQCRC2, MTCO1 and ATP5 were significantly reduced compared to controls, while only UQCRC2 and ATP5 were decreased in male TA compared to controls. A significant reduction in gene expression of Ndufb8, Sdhb, Mtco1 and Atp5 was detected in TA of female mice compared to controls, while only Ndufb8, Sdhb and Atp5 were decreased in male TA compared to controls. We observed the same pattern in Heart of male (protein: NDUFB8; mRNA: Mtco1) and female (protein: UQCRC2, MTCO1, ATP5; mRNA: Sdhb, Mtco1) DKO mice compared to their controls. The reduction in ETC subunits was paralleled by a reduction in complex I and complex III activity in the TA of DKO mice, but not in heart. However, this was only significant in the TA of female but not male mice. Mechanistical analyses using coimmunoprecipitation, cycloheximide chase and ubiquitination assays showed that MuRF1 physically interacted with the transcriptional repressor histone deacetylase 5 (HDAC5), mediated its ubiquitination as well as its UPS-dependent degradation. The absence of MuRF1 and MuRF3 in DKO mice let to an increase in the amounts of HDAC5 in TA. Because HDAC5 binds to PGC-1α, the master regulator of mitochondrial biogenesis (encoded by Ppargc1a), we investigated its gene expression in DKO muscle and found it to be reduced. These data connect MuRF1 and MuRF3 directly to the striated muscle energy metabolism, by regulating mitochondrial function. The results provide insights into the development of PAMs and possibly other protein storage diseases, where a decrease of mitochondrial function has already been described.