Depletion of HuR in murine skeletal muscle enhances exercise endurance and prevents cancer-induced muscle atrophy.

The master posttranscriptional regulator HuR promotes muscle fiber formation in cultured muscle cells. However, its impact on muscle physiology and function in vivo is still unclear. Here, we show that muscle-specific HuR knockout (muHuR-KO) mice have high exercise endurance that is associated with enhanced oxygen consumption and carbon dioxide production. muHuR-KO mice exhibit a significant increase in the proportion of oxidative type I fibers in several skeletal muscles. HuR mediates these effects by collaborating with the mRNA decay factor KSRP to destabilize the PGC-1α mRNA. The type I fiber-enriched phenotype of muHuR-KO mice protects against cancer cachexia-induced muscle loss. Therefore, our study uncovers that under normal conditions HuR modulates muscle fiber type specification by promoting the formation of glycolytic type II fibers. We also provide a proof-of-principle that HuR expression can be targeted therapeutically in skeletal muscles to combat cancer-induced muscle wasting.

Inactivation of the ubiquitin-specific protease 19 deubiquitinating enzyme protects against muscle wasting.

The ubiquitin system plays a critical role in muscle wasting. Previous work has focused on the roles of ubiquitination. However, a role for deubiquitination in this process has not been established. Because ubiquitin-specific protease (USP)19 deubiquitinating enzyme is induced in skeletal muscle in many catabolic conditions, we generated USP19 knockout (KO) mice. These mice lost less muscle mass than wild-type (WT) animals in response to glucocorticoids, a common systemic cause of muscle atrophy as well as in response to denervation, a model of disuse atrophy. KO mice retained more strength and had less myofiber atrophy with both type I and type IIb fibers being protected. Rates of muscle protein synthesis were similar in WT and KO mice, suggesting that the sparing of atrophy was attributed to suppressed protein degradation. Consistent with this, expression of the ubiquitin ligases MuRF1 and MAFbx/atrogin-1 as well as several autophagy genes was decreased in the muscles of catabolic KO mice. Expression of USP19 correlates with that of MuRF1 and MAFbx/atrogin-1 in skeletal muscles from patients with lung cancer or gastrointestinal cancer, suggesting that USP19 is involved in human muscle wasting. Inhibition of USP19 may be a useful approach to the treatment of many muscle-wasting conditions.

Strong muscle-specific regulatory cassettes based on multiple copies of the human slow troponin I gene upstream enhancer.

High-level tissue-specific expression of recombinant proteins in muscle is an important issue for several therapeutic applications. To achieve this goal, we generated several constructs containing one to five copies of the upstream enhancer (USE) of 160-bp of the human slow troponin I gene, linked to that gene's minimal promoter. We also tested constructs made with one to four copies of a 100-bp deletion of USE (DeltaUSE) reported to drive pan-muscle-specific expression in transgenic mice. These constructs were evaluated by measuring the activity of the reporter gene beta-galactosidase (beta-gal). In cell culture, these multimerized enhancers retained tissue specificity and their transcriptional strength increased with the number of enhancer copies. In myotube cultures (which still contain nondifferentiated cells), constructs containing four and five USE copies were stronger than the cytomegalovirus (CMV) early enhancer/promoter and slightly weaker than the hybrid CMV enhancer/beta-actin (CB) promoter. Those containing three USE, or four DeltaUSE copies were similar in strength to CMV. After electrotransfer of plasmid DNA into the mouse tibialis anterior muscle, the strengths of the two constructs (USEx3 and DeltaUSEx3) were tested; as measured by beta-gal activity in the total muscle lysate and by the number of transduced fibers, they were similar to CMV and CB. Muscle fiber typing, after electrotransfer of the soleus muscle, showed that DeltaUSEx3 and USEx3 were active in slow and fast fibers. The tissue specificity of these two constructs was also evaluated by hydrodynamic plasmid injection through the tail vein. Although significant beta-gal expression was measured in the liver when CMV was tested, no expression above background level was detected with USEx3 and DeltaUSEx3. The strength, muscle specificity, and small size of these transcriptional elements render them very attractive for gene therapy applications.

TnIfast IRE enhancer: multistep developmental regulation during skeletal muscle fiber type differentiation.

To identify developmental steps leading to adult skeletal muscle fiber-type-specific gene expression, we carried out transgenic mouse studies of the IRE enhancer of the quail TnIfast gene. Histochemical analysis of IRE/herpesvirus tk promoter/beta-galactosidase reporter transgene expression in adult muscle directly demonstrated IRE-driven fast vs. slow fiber-type specificity, and IIB>IIX>IIA differential expression among the fast fiber types: patterns similar to those of native-promoter TnIfast constructs. These tissue- and cell-type specificities are autonomous to the IRE and do not depend on interactions with a muscle gene promoter. Developmental studies showed that the adult pattern of IRE-driven transgene expression emerges in three steps: (1) activation during the formation of primary embryonic (presumptive slow) muscle fibers; (2) activation, to markedly higher levels, during formation of secondary (presumptive fast) fibers, and (3) differential augmentation of expression during early postnatal maturation of the IIB, IIX, IIA fast fiber types. These results provide insight into the roles of gene activation and gene repression mechanisms in fiber-type specificity and can account for apparently disparate results obtained in previous studies of TnI isoform expression in development. Each of the three IRE-driven developmental steps is spatiotemporally associated with a different major regulatory event at the fast myosin heavy chain gene cluster, suggesting that diverse muscle gene families respond to common, or tightly integrated, regulatory signals during multiple steps of muscle fiber differentiation.

Coregulation of fast contractile protein transgene and glycolytic enzyme expression in mouse skeletal muscle.

Little is known of the gene regulatory mechanisms that coordinate the contractile and metabolic specializations of skeletal muscle fibers. Here we report a novel connection between fast isoform contractile protein transgene and glycolytic enzyme expression. In quantitative histochemical studies of transgenic mouse muscle fibers, we found extensive coregulation of the glycolytic enzyme glycerol-3-phosphate dehydrogenase (GPDH) and transgene constructs based on the fast skeletal muscle troponin I (TnIfast) gene. In addition to a common IIB > IIX > IIA fiber type pattern, TnIfast transgenes and GPDH showed correlated fiber-to-fiber variation within each fast fiber type, concerted emergence of high-level expression during early postnatal muscle maturation, and parallel responses to muscle under- or overloading. Regulatory information for GPDH-coregulated expression is carried by the TnIfast first-intron enhancer (IRE). These results identify an unexpected contractile/metabolic gene regulatory link that is amenable to further molecular characterization. They also raise the possibility that the equal expression in all fast fiber types observed for the endogenous TnIfast gene may be driven by different metabolically coordinated mechanisms in glycolytic (IIB) vs. oxidative (IIA) fast fibers.