Virginia L. Johnsen

Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily of secreted proteins, is a potent negative regulator of myogenesis. Free myostatin induces the phosphorylation of the Smad family of transcription factors, which, in turn, regulates gene expression, via the canonical TGF-β signaling pathway. There is, however, emerging evidence that myostatin can regulate gene expression independent of Smad signaling. As such, we acquired global gene expression data from the gastrocnemius muscle of C57BL/6 mice following a 6-day treatment with recombinant myostatin compared with vehicle-treated animals. Of the many differentially expressed genes, the myostatin-associated decrease (-11.20-fold; P < 0.05) in the noncoding metastasis-associated lung adenocarcinoma transcript 1 (Malat1) was the most significant and the most intriguing because of numerous reports describing its novel role in regulating cell growth. We therefore sought to further characterize the role of Malat1 expression in skeletal muscle myogenesis. RT-PCR-based quantification of C2C12 and primary human skeletal muscle cells revealed a significant and persistent upregulation (4- to 7-fold; P < 0.05) of Malat1 mRNA during the differentiation of myoblasts into myotubes. Conversely, targeted knockdown of Malat1 using siRNA suppressed myoblast proliferation by arresting cell growth in the G(0)/G(1) phase. These results reveal Malat1 as novel downstream target of myostatin with a considerable ability to regulate myogenesis. The identification of new targets of myostatin will have important repercussions for regenerative biology through inhibition and/or reversal of muscle atrophy and wasting diseases.

O-linked β-N-acetyl glucosamine (O-GlcNAc) is a posttranslational modification consisting of a single N-acetylglucosamine moiety attached by an O-β-glycosidic linkage to serine and threonine residues of both nuclear and cytosolic proteins. Analogous to phosphorylation, the modification is reversible and dynamic, changing in response to stress, nutrients, hormones, and exercise. Aims of this study were to examine differences in O-GlcNAc protein modification in the cardiac tissue of rats artificially selected for low (LCR) or high (HCR) running capacity. Hyperinsulinemic-euglycemic clamps in conscious animals assessed insulin sensitivity while 2-[(14)C] deoxyglucose tracked both whole body and tissue-specific glucose disposal. Immunoblots of cardiac muscle examined global O-GlcNAc modification, enzymes that control its regulation (OGT, OGA), and specific proteins involved in mitochondrial oxidative phosphorylation. LCR rats were insulin resistant disposing of 65% less glucose than HCR. Global tissue O-GlcNAc, OGT, OGA, and citrate synthase were similar between groups. Analysis of cardiac proteins revealed enhanced O-GlcNAcylation of mitochondrial Complex I, Complex IV, VDAC, and SERCA in LCR compared with HCR. These results are the first to establish an increase in specific protein O-GlcNAcylation in LCR animals that may contribute to progressive mitochondrial dysfunction and the pathogenesis of insulin resistance observed in the LCR phenotype.

Homozygosity for a premature stop codon (X) in the ACTN3 "sprinter" gene is common in humans despite the fact that it reduces muscle size, strength and power. Because of the close relationship between skeletal muscle function and cardiometabolic health we examined the influence of ACTN3 R577X polymorphism over cardiovascular and metabolic characteristics of young adults (n = 98 males, n = 102 females; 23 ± 4.2 years) from our Assessing Inherent Markers for Metabolic syndrome in the Young (AIMMY) study. Both males and females with the RR vs XX genotype achieved higher mean VO2 peak scores (47.8 ± 1.5 vs 43.2 ±1.8 ml/O2/min, p = 0.002) and exhibited higher resting systolic (115 ± 2 vs 105 ± mmHg, p = 0.027) and diastolic (69 ± 3 vs 59 ± 3 mmHg, p = 0.005) blood pressure suggesting a role for ACTN3 in the maintenance of vascular tone. We subsequently identified the expression of alpha-actinin 3 protein in pulmonary artery smooth muscle, which may explain the genotype-specific differences in cardiovascular adaptation to acute exercise. In addition, we utilized targeted serum metabolomics to distinguish between RR and XX genotypes, suggesting an additional role for the ACTN3 R577X polymorphism in human metabolism. Taken together, these results identify significant cardiometabolic effects associated with possessing one or more functional copies of the ACTN3 gene.

Type 2 diabetes (T2D) is rapidly rising in prevalence. Characterized by either inadequate insulin production or the inability to utilize insulin produced, T2D results in elevated blood glucose levels. The "gold-standard" in assessing insulin sensitivity is a hyperinsulinemic-euglycemic clamp or insulin clamp. In this procedure, insulin is infused at a constant rate resulting in a drop in blood glucose. To maintain blood glucose at a constant level, exogenous glucose (D50) is infused into the venous circulation. The amount of glucose infused to maintain homeostasis is indicative of insulin sensitivity. Here, we show the basic clamp procedure in the chronically catheterized, unrestrained, conscious rat. This model allows blood to be collected with minimal stress to the animal. Following the induction of anesthesia, a midline incision is made and the left common carotid artery and right jugular vein are catheterized. Inserted catheters are flushed with heparinized saline, then exteriorized and secured. Animals are allowed to recover for 4-5 days prior to experiments, with weight gain monitored daily. Only those animals who regain weight to pre-surgery levels are used for experiments. On the day of the experiment, rats are fasted and connected to pumps containing insulin and D50. Baseline glucose is assessed from the arterial line and used a benchmark throughout the experiment (euglycemia). Following this, insulin is infused at a constant rate into the venous circulation. To match the drop in blood glucose, D50 is infused. If the rate of D50 infusion is greater than the rate of uptake, a rise in glucose will occur. Similarly, if the rate is insufficient to match whole body glucose uptake, a drop will occur. Titration of glucose continues until stable glucose readings are achieved. Glucose levels and glucose infusion rates during this stable period are recorded and reported. Results provide an index of whole body insulin sensitivity. The technique can be refined to meet specific experimental requirements. It is further enhanced by the use of radioactive tracers that can determine tissue specific insulin-stimulated glucose uptake as well as whole body glucose turnover.