Craniofacial asymmetry is a dentofacial deformity with genetic influences. The genes PITX2, ENPP1 and ESR1 have multiple genetic associations with functional properties in muscle and bone. The objectives of this study are to investigate how PITX2, ENPP1 and ESR1 gene expression associates with four subclassifications of craniofacial asymmetry, temporomandibular disorders and fiber type differences compared between right and left masseter muscles. We developed an asymmetry classification that diagnosed four types of asymmetry with distinctive growth patterns: Group 1 – menton deviation without ramal difference (“mandibular body asymmetry”); Group 2 –menton deviation with shorter ramal height on the deviated side (“typical asymmetry”); Group 3 – shorter ramal height on the opposite side of menton deviation (“atypical asymmetry”); Group 4 – menton deviation with shorter ramal height and maxillary canting on the deviated side (“C-shaped asymmetry”). Some of these patients are at high risk for TMD; therefore, temporomandibular joint functioning is assessed as a routine part of the pre-surgical evaluation. TMD was diagnosed using the Diagnostic Criteria for TMD (DC/TMD). The clinical examination includes mandibular range of motion, palpation for pain, joint noise and bruxism. In addition, the Jaw Pain and Function (JPF) questionnaire was used to assess patient reported symptoms as an indication of perceived severity before and one year after orthognathic surgery. Masseter muscle samples were collected from 174 subjects undergoing surgical treatment for correction of malocclusion. Muscle serial cross-sections were mounted for immunostaining with five antibodies specific for myosin heavy chain (MyHC) isoform. We classified masseter fibers into 4 fiber type groups: type I, type I/II hybrid, type IIA and/or IIX, neonatal and atrial. With the remaining muscle samples, total RNA was isolated and PITX2, ENPP1, and ESR1 expression was quantified using TaqMan qRT-PCR. Average relative quantity gene expression values and percent differences between left and right masseter samples were calculated. In this population, there is a high prevalence of facial asymmetry (48%). Pre-surgical mean JPF scores are significantly different between symmetric (JPF=1.97) and asymmetric (JPF=6.9; p<0.001) patients; with scores ≥ 6 diagnostic for presence of TMD. ENPP1 and ESR1 expression is differentially expressed between right and left masseter muscle in patients with asymmetry. ENPP1 is differentially expressed in asymmetry group 4 (p=0.01) and ESR1 is differentially expressed in asymmetry group 1 (p=0.048), group 2 (p=0.004) and group 4 (p=0.02). Masseter fiber type properties of type I, type I/II hybrid and type II fibers associate with facial asymmetry and specific subclassifications, suggesting functional differences between type I, type I/II and type II fibers may be important factors in the development of symmetry between facial sides. There are significant differences in the left-right percent differences of fiber area of type I fibers in asymmetry group 3 (p=0.05), type I/II hybrid fibers in group 3 (p=0.02), and type II fibers in asymmetric patients (p=0.03), asymmetry group 2 (p=0.05) and group 4 (p=0.005). Additionally, there are significant differences in the left-right percent differences of percent occupancy of type I fibers in asymmetric patients (p=0.04), asymmetry group 2 (p=0.01) and group 3 (p=0.05) and type II fibers in asymmetry group 2 (p=0.04). By comparing gene expression with masseter muscle fiber type properties, we found significant results for PITX2 and ENPP1 suggesting their roles as genetic factors influencing jaw bone length and masticatory muscle strength in malocclusion. There are significant positive correlations between left-right percent differences of PITX2 and type I fiber area (r=0.86; p=0.03), type I/II hybrid fiber area (r=0.94; p=0.006), and type I/II hybrid fiber percent occupancy (r=0.90; p=0.01). Also, there are positive correlations approaching significance between left-right percent differences of ENPP1 and type I fiber area (r=0.80; p=0.06) and type I/II hybrid fiber area (r=0.75; p=0.09). Given the high prevalence of TMD in a population of patients with facial asymmetry, we compared differences in gene expression in masseter muscle of patients with specific TMD diagnostic conditions. Average PITX2 expression is significantly increased (p=0.0375) and average ENPP1 is increased, but not significantly, in all TMD patients diagnosed by the clinician. Average ESR1 is slightly increased compared to JPF scores and may be an essential factor for patient reported TMD symptoms. With these results, PITX2, ENPP1, and ESR1 should be considered biomarkers for asymmetry and TMD; however, further studies are needed to provide a more thorough understanding of the genetic influences on the craniofacial complex. / Oral Biology

The SH3 and cysteine rich domain 3 (Stac3) gene is expressed specifically in skeletal muscle and essential for skeletal muscle contraction and postnatal life in mice. In this dissertation project, I conducted two studies to further understand the role of STAC3 in skeletal muscle development, growth, and contraction. In the first study, I compared the contractile responses of hindlimb muscles of Stac3 knockout and control mice to electrical stimulation, high [K+]-induced membrane depolarization, and caffeine and 4-chloro-m-cresol (4-CMC) activation of ryanodine receptor (RyR). Frequent electrostimulation-, high [K+]-, 4-CMC- and caffeine-induced maximal tensions in Stac3-deleted muscles were approximately 20%, 29%, 58% and 55% of those in control muscles, respectively. 4-CMC- and caffeine-induced increases in intracellular calcium were not different between Stac3-deleted and control myotubes. Myosin-ATPase and NADH-tetrazolium reductase staining as well as gene expression analyses revealed that Stac3-deleted hindlimb muscles contained more slow type-like fibers than control muscles. These data together confirm a role of STAC3 in EC coupling but also suggest that defective EC coupling is only partially responsible for the significantly reduced contractility in Stac3-deleted hindlimb muscles. In the second study, I determined the potential role of STAC3 in postnatal skeletal muscle growth, fiber composition, and contraction by disrupting Stac3 gene expression in postnatal mice through the Flp-FRT and tamoxifen-inducible Cre-loxP systems. Postnatal Stac3 deletion inhibited body and limb muscle mass gains. Histological staining and gene expression analyses revealed that postnatal Stac3 deletion decreased the size of myofibers and increased the percentage of myofibers containing centralized nuclei without affecting the total myofiber number. Postnatal Stac3 deletion decreased limb muscle strength. Postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced maximal force output in limb muscles. Similarly, postnatal Stac3 deletion reduced electrostimulation- but not caffeine-induced calcium release from the sarcoplasmic reticulum. These results demonstrate that STAC3 is important to myofiber hypertrophy, myofiber type composition, contraction, and EC coupling in postnatal skeletal muscle. / Ph. D.

Postnatal skeletal muscle growth occurs through myonuclear accretion and high protein turnover rate. While fiber type composition of the muscle could affect protein turnover rate, less is known about how fiber type influences the regulation of protein synthesis and degradation signaling pathways. Thus, the hypothesis of this work was that variation in fiber type composition will differentially affect the regulation of signaling pathways related to protein turnover in skeletal muscle hypertrophy in growing pigs. Downregulated protein synthesis signaling and reduced expression of type II MyHC isoforms have been reported in skeletal muscles of low birth weight (LBWT) neonatal pigs. Therefore, we sought to determine whether these changes are sustained until weaning and would explain the reduction in LBWT pig growth compared to their normal birth weight (NBWT) sibling at weaning. Another objective was to determine whether the regulation of protein turnover signaling pathways are correlated to fiber type differences in skeletal muscles. Our data suggest that the longissimus dorsi (LD, glycolytic) muscle of LBWT pigs experienced compensatory growth while the soleus (oxidative) remained proportionally smaller. Growth of the LD was accompanied by upregulation of translation initiation. Additionally, there was no difference in expression of MyHC isoforms between NBWT and LBWT pigs. These data suggest the rapid growth of the LD of LBWT pigs may be attributed to an upregulation of protein synthesis signaling and occurred only in glycolytic muscles. A caveat in LBWT pig model is that the reduction in type II MyHC at birth is not the only factor that could influence muscle growth, and that other factors may have confounded our results. This is why we aimed to use β-adrenergic agonist as a means to induce a shift fiber type in muscles to a more glycolytic phenotype. Our objective was to determine the influence of the β-adrenergic agonist Ractopamine (RAC) induced slow-to-fast fiber type transformation on the regulation of protein synthesis and degradation pathways. Although supplementation improved translational capacity, enhanced S6K1 phosphorylation, and reduced the abundance of calcium-dependent proteases, RAC feeding had no effect on body or muscle weights. These results suggest that a fiber type transformation without other physiological influences does not alter protein turnover signaling in favor of hypertrophy in growing pigs. / Master of Science / Skeletal muscles grow by increasing the amount of protein contained within them. The amount of protein deposited is determined by the net balance between the rates at which proteins are synthesized and degraded. However, not all skeletal muscles grow at the same rate. One factor that is thought to influence protein synthesis and degradation rates is the types of muscle fibers that are present within a muscle. These fibers can display a range of contractile and metabolic characteristics, from slow-twitch oxidative fibers to fast-twitch glycolytic fibers. In the presented studies, we sought to determine whether changes in fiber type composition result in difference to the signaling pathways the regulate protein synthesis and degradation, ultimately leading to differences in the muscle growth of young pigs. We have previously shown reduced activation of the protein synthesis pathway in the skeletal muscle of low birth weight (LBWT) newborn pigs. These pigs also had lower expression of glycolytic fibers. In experiment 1, we aimed to compare the signaling pathways regulating protein synthesis and degradation in LBWT and normal birth weight (NBWT) pigs at weaning. We also sought to determine if the regulation of these signaling pathways changed between muscles with differing fiber type compositions. The glycolytic longissimus dorsi (LD) muscle of LBWT pigs grew rapidly between birth and weaning whereas the highly oxidative soleus did not. In addition, the LD of LBWT pigs had greater protein synthesis signaling and similar expression of muscle fibers compared with NBWT pigs, suggesting the improvement in protein synthesis signaling of LBWT pigs between birth and weaning may be related to a shift in fiber type. In experiment 2, we used a compound called ractopamine hydrochloride (RAC) to promote a slow-to-fast fiber type switch in the muscle of young pigs. With this study, we sought to determine the effect of this fiber type transformation, without the influence of birth weight, on the regulation of protein synthesis and degradation pathways. Although RAC-fed pigs showed some minor changes that could improve protein synthesis and decrease protein degradation, RAC feeding had no observable effect on body weight or muscle growth. These results suggest that a fiber type transformation alone is not enough to promote muscle growth in growing pigs.

The purpose of this investigation was to explore the phenotypic and performance outcomes associated with ACTN3 and ACE polymorphisms. Ten trained men (age = 25.8 ± 3.0 years, height = 183.3 ± 4.1 cm, body mass = 92.3 ± 9.3 kg, and back squat to body mass ratio = 1.8 ± 0.3) participated. Blood samples were analyzed to determine ACTN3 and ACE polymorphisms. Standing ultrasonography images of the vastus lateralis (VL) were collected to determine whole muscle cross-sectional area (CSA-M), and a percutaneous muscle biopsy of the VL was collected to determine type I–specific CSA (CSA-T1), type II–specific CSA (CSA-T2), and type II to type I CSA ratio (CSA-R). Isometric squats were performed on force platforms with data used to determine peak force (IPF), allometrically scaled peak force (IPFa), and rate of force development (RFD) at various timepoints. One repetition maximum back squats were performed, whereby allometrically scaled dynamic strength (DSa) was determined. Cohen's d effect sizes revealed ACTN3 RR and ACE DD tended to result in greater CSA-M but differ in how they contribute to performance. ACTN3 RR's influence seems to be in the type II fibers, altering maximal strength, and ACE DD may influence RFD capabilities through a favorable CSA-R. Although the findings of the current investigation are limited by the sample size, the findings demonstrate the potential influence of ACTN3 and ACE polymorphisms on isometric and dynamic strength testing. This study may serve as a framework to generate hypotheses regarding the effect of genetics on performance.

This thesis determined whether changes in adenosine monophosphate-activated protein kinase (AMPK) activity would influence sarcoplasmic reticulum Ca2+-ATPase (SERCA) content and function in left ventricle (LV) and skeletal muscle isolated from sedentary or exercise trained mice. The data indicate that AMPKα2 kinase dead transgenic (KD) mice, as compared to wild-type (WT) mice, were characterized by reduced SERCA1a, SERCA2a and higher phospholamban (PLN) protein levels in both cardiac and skeletal muscle. Notably, exercise-training up-regulated myocardial SERCA2a protein content by 43%, as compared to sedentary WT mice. In contrast, exercise-training did not alter myocardial SERCA2a protein content in KD mice. Even so, exercise-training up-regulated SERCA1a protein content in skeletal muscle in both WT and KD mice. Based on these data, it appears that an AMPKα2-mediated mechanism influences SERCA2a content and function in the heart and skeletal muscle, which may contribute to the pathophysiology of models characterized by impaired AMPK activity and impaired calcium-cycling.

This thesis determined whether changes in adenosine monophosphate-activated protein kinase (AMPK) activity would influence sarcoplasmic reticulum Ca2+-ATPase (SERCA) content and function in left ventricle (LV) and skeletal muscle isolated from sedentary or exercise trained mice. The data indicate that AMPKα2 kinase dead transgenic (KD) mice, as compared to wild-type (WT) mice, were characterized by reduced SERCA1a, SERCA2a and higher phospholamban (PLN) protein levels in both cardiac and skeletal muscle. Notably, exercise-training up-regulated myocardial SERCA2a protein content by 43%, as compared to sedentary WT mice. In contrast, exercise-training did not alter myocardial SERCA2a protein content in KD mice. Even so, exercise-training up-regulated SERCA1a protein content in skeletal muscle in both WT and KD mice. Based on these data, it appears that an AMPKα2-mediated mechanism influences SERCA2a content and function in the heart and skeletal muscle, which may contribute to the pathophysiology of models characterized by impaired AMPK activity and impaired calcium-cycling.

Muscle architecture, innervation pattern and fiber type distribution of lumbar multifidus (LMT) throughout its volume was quantified. Musculotendinous (n=10) and neural components (n=3) were dissected and digitized from thirteen embalmed cadaveric specimens. The data were imported into Autodesk® Maya® 2008 to generate 3D neuromuscular models of each specimen. Architectural parameters (fiber bundle length, FBL; fiber bundle angle, FBA; tendon length) were quantified from the models using customized software. The medial branch of the posterior rami (L1-L5) was traced through LMT to determine its distribution. Using immunohistochemistry, Type I/II muscle fibers were identified in 29 muscle biopsies from one fresh frozen specimen. The total area and number of each cell type was calculated using Visiopharm® (image analysis software). Architectural and fiber type data were analyzed using ANOVA with Tukey’s post-hoc test (p ≤ 0.05). From L1-L4, LMT had three architecturally distinct regions: superficial, intermediate and deep. At L5, intermediate LMT was absent. Mean FBL decreased significantly from superficial (5.8 ± 1.6cm) to deep regions (2.9 ± 1.1cm) as did volume (superficial, 5.6 ± 2.3ml; deep, 0.7 ± 0.3ml). In contrast, mean FBA increased from superficial to deep. The medial branch of the posterior ramus (L1-L5) supplied the five bands of LMT. Each medial branch in turn divided to supply the deep, intermediate and superficial regions separately. The area occupied by Type I fibers was significantly less (p< 0.01) in the deep (56%) compared with the superficial regions (75%). Based on architecture and morphology, superficial LMT with the longest FBL and relatively small FBA is well designed for torque production and controlling the lumbar lordosis. Intermediate LMT with significantly longer FBL compared with the deep region and with its caudal to cranial line of action may help to control intersegmental stability. Furthermore, the absence of intermediate LMT at L5 and may contribute to the higher incidence of instability observed at the lumbosacral junction. Deep LMT with its short FBL, large FBA and proximity to the axis of spinal rotation may function to provide proprioceptive input to the CNS rather than a primary stabilizer of the lumbar spine.

Muscle architecture, innervation pattern and fiber type distribution of lumbar multifidus (LMT) throughout its volume was quantified. Musculotendinous (n=10) and neural components (n=3) were dissected and digitized from thirteen embalmed cadaveric specimens. The data were imported into Autodesk® Maya® 2008 to generate 3D neuromuscular models of each specimen. Architectural parameters (fiber bundle length, FBL; fiber bundle angle, FBA; tendon length) were quantified from the models using customized software. The medial branch of the posterior rami (L1-L5) was traced through LMT to determine its distribution. Using immunohistochemistry, Type I/II muscle fibers were identified in 29 muscle biopsies from one fresh frozen specimen. The total area and number of each cell type was calculated using Visiopharm® (image analysis software). Architectural and fiber type data were analyzed using ANOVA with Tukey’s post-hoc test (p ≤ 0.05). From L1-L4, LMT had three architecturally distinct regions: superficial, intermediate and deep. At L5, intermediate LMT was absent. Mean FBL decreased significantly from superficial (5.8 ± 1.6cm) to deep regions (2.9 ± 1.1cm) as did volume (superficial, 5.6 ± 2.3ml; deep, 0.7 ± 0.3ml). In contrast, mean FBA increased from superficial to deep. The medial branch of the posterior ramus (L1-L5) supplied the five bands of LMT. Each medial branch in turn divided to supply the deep, intermediate and superficial regions separately. The area occupied by Type I fibers was significantly less (p< 0.01) in the deep (56%) compared with the superficial regions (75%). Based on architecture and morphology, superficial LMT with the longest FBL and relatively small FBA is well designed for torque production and controlling the lumbar lordosis. Intermediate LMT with significantly longer FBL compared with the deep region and with its caudal to cranial line of action may help to control intersegmental stability. Furthermore, the absence of intermediate LMT at L5 and may contribute to the higher incidence of instability observed at the lumbosacral junction. Deep LMT with its short FBL, large FBA and proximity to the axis of spinal rotation may function to provide proprioceptive input to the CNS rather than a primary stabilizer of the lumbar spine.

Factors that stimulate mitochondrial biogenesis in skeletal muscle include AMPK, calcium, and circulating FFAs. Chronic treatment with either AICAR, a chemical activator of AMPK, or increasing circulating FFAs with a high fat diet increases mitochondria in rat skeletal muscle. The purpose of this study was to determine whether the combination of chronic chemical activation of AMPK and high fat feeding would have an additive effect on skeletal muscle mitochondria levels. We treated Wistar male rats with a high fat diet (HF), AICAR injections (AICAR), or a high fat diet and AICAR injections (HF+AICAR) for six weeks. At the end of the treatment period, markers of mitochondrial content were examined in white quadriceps, red quadriceps, and soleus muscles, predominantly composed of unique muscle-fiber types. In white quadriceps, there was a cumulative effect of treatments on LCAD, cytochrome c, and PGC-α protein, as well as on citrate synthase and β-HAD activity. In contrast, no additive effect was noted in the soleus and in the red quadriceps only β-HAD activity increased additively. The additive increase of mitochondrial markers observed in the white quadriceps may be explained by a combined effect of two separate mechanisms: high fat diet-induced post transcriptional increase in PGC-α protein and AMPK mediated increase in PGC-α protein via a transcriptional mechanism. These data show that chronic chemical activation of AMPK and a high fat diet have a muscle type specific additive effect on markers of fatty acid oxidation, the citric acid cycle, the electron transport chain, and transcriptional regulation.

No description available.