The function of mammalian Mss51 is unknown although a putative homolog in yeast is a mitochondrial translational activator. The objective of this work was to characterize mammalian MSS Quantitative RT-PCR and immunoblot of subcellular fractionation were used to determine expression patterns and localization of Mss Mss51 was predominantly expressed in skeletal muscle and in those muscles dominated by fast-twitch fibers.
In vitro , its expression was upregulated upon differentiation of C2C12 myoblasts into myotubes. In human muscle, MSS51 localized to the mitochondria. Rather, Mss51 appears to be one of the effectors of these growth factors on metabolic processes including fatty acid oxidation, glycolysis and oxidative phosphorylation.
Myostatin inhibition has been shown to increase regeneration and decrease fibrosis of skeletal muscle and is thus of high interest for the treatment of acquired and inherited conditions where muscle is not able to regenerate efficiently [ 2— 7 ]. An important additional feature of myostatin inhibition that may indicate potential therapeutic use in insulin resistance and obesity is its modulation of skeletal muscle metabolism [ 8 ].
How to Find the Best Operating System For a Toughbook 31
Myostatin deletion or postnatal inhibition improves insulin sensitivity, increases glucose uptake into skeletal muscle and decreases total body fat [ 12— 14 ]. Treating mice fed high-fat diets with postnatal inhibitors of myostatin reduces the development of insulin resistance and fat accumulation [ 17, 18 ].
In a previous study, we profiled gene expression in skeletal muscle of mice treated with a soluble activin type IIB receptor ActRIIB-Fc , the putative receptor of myostatin and activin A [ 19 ].
One of the genes whose expression was most significantly altered was Mss51 mitochondrial translational activator, named after its putative yeast homolog Mss51 and previously known as Zmynd Mss51 was downregulated 4.
Similar results have been seen in numerous other published studies. Mss51 was downregulated 2. Additionally, Mss51 expression decreased 2. The function of Mss51 in mammals has not been previously reported.
Chapter 28--The Muscular System
In the yeast Saccharomyces cerevisiae , Mss51 couples the synthesis of cytochrome c oxidase subunit 1 COX1 to the assembly of the cytochrome c oxidase complex of the respiratory chain in the mitochondria [ 24 ].
Yeast Mss51 also senses heme and oxygen availability to regulate cytochrome c oxidase biogenesis [ 25 ].
Iterative orthology prediction using Ortho-Profile demonstrated that the human MSS51 protein is a putative homolog of the yeast Mss51 protein, and that human MSS51 has a mitochondrial localization signal [ 26 ]. Here we investigate the expression and function of Mss51 in skeletal muscle.
Louis, MO. Cells were treated with growth factors or inhibitors after three days of differentiation. All animal experiments were conducted in accordance with the guidelines prescribed by the Institutional Animal Care and Use Committee at the Johns Hopkins University School of Medicine. Mice were euthanized by inhalation overdose of isoflurane followed by cervical dislocation. From three mice, the brain, heart, kidney, liver, small intestine, diaphragm, and quadriceps were harvested.
From three other mice, the soleus, diaphragm, long head of the triceps, extensor digitorum longus EDL , and white vastus lateralis WVL were harvested. Written informed consent was obtained for the collection and use of human tissue samples.
Human samples of deltoid muscle were obtained by open muscle biopsy of a living volunteer and from an autopsy donor and were stored frozen prior to subcellular fractionation.
The target sequence within Mss51 , depicted in Fig. In parallel, control cells were transfected with a plasmid expressing functional GFP and subjected to the same cell sorting process as the Mss51 -disrupted cells. Pooled populations of Mss51 -disrupted and control cells were collected and expanded for comparative analysis. Cells were allowed to differentiate into myotubes for six days before analysis unless otherwise stated. At the cut site, a double-strand break occurred and was re-sealed by non-homologous end joining.
Two populations of GFP-positive cells were collected by FACS sorting and expanded for further analysis — Mss51 -disrupted cells were collected as shown and in parallel, control cells were transfected with a GFP expression plasmid and subjected to the same FACS process.
The myofusion index was calculated as the percentage of nuclei that were found in MFpositive fibers. In order to separate nuclear, cytosolic, and mitochondrial fractions from human tissue, subcellular fractionation was performed as previously described [ 29 ] with minor modification. Human tissue was used because at the time of these experiments, there were no available antibodies that selectively recognized mouse Mss The resulting fractions were then heat-denatured and reduced.
To ensure equal protein loading, sample concentrations were measured using the bicinchoninic acid BCA assay. Primer sequences are available upon request.
To assay relative rates of protein synthesis, the surface sensing of translation SUnSET assay was used as previously described [ 30 ]. Plates were kept on ice for 1 hour before extraction of water soluble products using Folch partition as previously described [ 31 ]. Protein was determined in wells of parallel 6-well plates by the method of Lowry et al. Cells were seeded at equal densities and differentiated for 6 days at which point metabolic flux analysis was performed. In both tests, between the addition of each component, the oxygen consumption rate and extracellular acidification were measured as previouslydescribed [ 33 ].
Ch. 11 Muscles
For more than two groups, differences were compared using one-way analysis of variance ANOVA with Bonferroni post hoc comparisons. Probability p values less than 0. Mss51 tissue expression. To examine modulation of Mss51 expression, we first determined the time course of its expression in vitro. Mss51 was expressed at low levels in proliferating C2C12 myoblasts, and significantly higher levels as cells differentiated into myotubes Fig. IGF-1, which has several similar effects on skeletal muscle as myostatin inhibition including increasing proliferation and differentiation in vitro and muscle growth in vivo [ 34 ], also decreased Mss51 expression.
Actaris cf 51 manual muscle
We examined other time points and saw the greatest effects on Mss51 expression levels when the myotubes were differentiated for three days and no significant change seen when proliferating myoblasts were treated data not shown.
This may be because during differentiation, the cells are actively increasing Mss51 expression and are therefore more responsive to exogenous signaling cues. Mss51 expression in vitro. Expression level is normalized to proliferating Day 0 cells using the reference gene TBP. There were no significant differences between brain, heart, kidney, and liver, while expression was higher in the small intestine and diaphragm, approximately fold above the expression level found in the brain.
In the quadriceps, expression was approximately fold higher compared to brain Fig. We also examined the relative expression of Mss51 in different skeletal muscle groups, selected based on their established fiber type distributions [ 35— 37 ], listed from more oxidative to more glycolytic: soleus, diaphragm, long head of the triceps brachii, extensor digitorum longus EDL , and white vastus lateralis WVL Fig.
If you need to hit an individual muscle group every 3 days, then why do bro splits work?
There were significant differences between muscle groups, with lower expression in the more oxidative soleus and diaphragm muscles, intermediate in the mixed-type long head of the triceps brachii, and higher expression in the glycolytic EDL and WVL. Our search revealed MSS51 to be expressed at much higher levels in skeletal muscle than any other tissue examined Fig. To determine cellular localization of MSS51 , human deltoid muscles from an open muscle biopsy of a living donor and from an autopsy were used to perform subcellular fractionation.
These data showed that MSS51 protein co-fractionated with the mitochondrial fraction Fig. Subcellular localization of MSS Subcellular fractionation was performed on human deltoid samples from a biopsy and an autopsy and resulting fractions were subjected to SDS-PAGE. The Mss51 -disrupted myotube population was a pool of many unique Mss51 mutants including cells that have at least one wild-type allele. Relative Mss51 expression in the pooled cells was determined using primers flanking the predicted double strand break, which demonstrated a significant decrease in total Mss51 transcript in the differentiating Mss51 -disrupted myotubes when compared to control cells Fig.
For this reason, we examined cell proliferation and differentiation in Mss51 -disrupted cells.
Proliferation was unchanged compared to control cells, as measured by EdU incorporation Fig. Differentiation was also equivalent in Mss51 -disrupted and control cell populations by myofusion index, the proportion of nuclei found in myosin heavy chain-positive fibers at day 5 post differentiation media Fig.
Creatine kinase enzyme activity, a marker of differentiation, did not differ significantly between control and Mss51 -disrupted myotubes after six days of differentiation Fig.
Myotubes were differentiated for six days to ensure that stable levels of Mss51 transcript and protein were present in control cells, corresponding to the greatest difference between control and disrupted populations.
We also looked at expression of myogenic regulatory factors by qPCR and did not see meaningful differences between control and Mss51 -disrupted cells data not shown.
Protein synthesis, as measured by puromycin incorporation using the SUnSET assay, was not altered in Mss51 -disrupted myotubes after 6 days of differentiation Fig. Mss51 -disrupted myoblasts proliferate and differentiate normally. C Representative myosin heavy chain MF20, green staining of control and Mss51 -disrupted myotubes 2 days after induction of differentiation with nuclei stained by DAPI blue. E Creatine kinase CK activity in myotubes after 6 days of differentiation.
F Protein synthesis rates as measured by puromycin incorporation on the left and total protein stained by SYPRO Ruby on the right after 6 days of differentiation. Differences between groups were not statistically significant. Myostatin knockout and IGF-1 transgene expression lead to an alteration in fiber-type composition with a decrease in type 1 slow oxidative fibers and an increase in type IIB fast glycolytic fibers [ 9, 10, 19, 39— 42 ].
The changes exhibited in Mss51 -disrupted myotubes indicated a shift towards more fast-twitch MyHC expression. Since expression of MyHC isoforms typically coincide with metabolic properties of muscle, we evaluated whether Mss51 modulates the mRNA expression of several key regulators of metabolism Fig. Expression of the glycolytic gene hexokinase 2 Hk2 , which has been shown to be more active in the glycolytic EDL muscle than the oxidative soleus muscle [ 44 ], was significantly increased in Mss51 -disrupted myotubes Fig.
Other genes that had significant changes in expression with decreased Mss51 expression were Cd36 fatty acid translocase , Fasn fatty acid synthase , and Pdk4 pyruvate dehydrogenase lipoamide kinase isozyme 4 , which were all increased.
The products of these genes are all critical in fatty acid utilization. Other genes involved in fatty acid oxidation FAO including HSL hormone sensitive lipase and Acadl long chain specific acyl-coA dehydrogenase were also increased to a lesser extent while other genes involved in FAO were unchanged.
Since yeast Mss51 is a translational activator, we examined changes in protein expression via immunoblotting. Glycolysis and Oxidative Phosphorylation in Mss51 -disrupted cells. C Glycolysis stress test measuring the extracellular acidification rate ECAR in control and Mss51 -disrupted myotubes treated with glucose, oligomycin, and 2-deoxy-D-glucose.
In the glycolysis stress test, the extracellular acidification rate was measured as an indicator of glycolysis when differentiated myotubes were perturbed.
Baseline measurements were taken in glucose-free media before glucose, oligomycin, and 2-deoxy-D-glucose were injected to begin glycolysis, shut down the electron transport chain, and inhibit glycolysis, respectively. Between each treatment, the extracellular acidification rate was measured and plotted Fig. Comparing Mss51 -disrupted and control myotubes, we found that Mss51 -disrupted myotubes had significantly higher glycolysis, glycolytic capacity, and glycolytic reserve than wild-type myotubes Fig.
This allowed for the calculation of basal respiration, ATP production, maximal respiration, and spare respiratory capacity, all measured by changes in oxygen consumption rate Fig. We found that all metrics of mitochondrial respiration excluding proton leak were significantly increased in Mss51 -disrupted cells Fig.
Mammalian Mss51 initially came to attention as one of the most consistently downregulated genes in gene profiling studies of myostatin inhibition [ 19— 23 ].
Mss51 is expressed almost exclusively in skeletal muscle in mice and humans. In mice, it is expressed most abundantly in muscles rich in glycolytic type II fibers and to fold less in those rich in oxidative type I fibers Fig.