Browsing by Subject "Skeletal muscle"

Tissue-engineered skeletal muscle presents promising opportunities for developing high-fidelity in vitro models for investigating human muscle biology in the areas of regeneration and disease. In muscle regeneration, satellite cells (SCs) are essential for new muscle fiber formation; however, they lose their native quiescent state upon isolation, making in vitro studies of human SC function challenging. To optimally promote SC quiescence and enable exploration of SC dynamics in vitro, engineered muscle needs to recapitulate the native muscle microenvironment, which is comprised of muscle fibers, extracellular matrix, and other biochemical and mechanical cues. In disease modeling, mechanistic studies and therapeutic development are still extensively evaluated in animal models, which have limited translational relevance to patients. Specifically, Pompe disease is caused by a variety of mutations in the lysosomal enzyme acid alpha-glucosidase (GAA) that varyingly affect residual GAA activity and cannot be captured in the current GAA-/- mouse model. Therefore, human in vitro models are needed to enhance our mechanistic understanding of diseases and stimulate the development of effective therapies. To overcome these limitations, we set the dissertation goals to: 1) generate a pool of quiescent SCs and explore the mechanisms governing their formation and activation using an engineered skeletal muscle microenvironment and 2) develop a high-fidelity tissue-engineered skeletal muscle model for Pompe disease to investigate pathological mechanisms and test candidate therapies. To achieve these goals, we first compared methods for primary human myoblast expansion and found that p38 inhibition significantly increases the formation of Pax7+ cells in engineered 3D skeletal muscle tissues (“myobundles”). Gene expression analysis suggested that within the myobundle environment the Pax7+ cells adopt a quiescent phenotype (3D SCs), characterized by increased Pax7 expression, cell cycle exit, and Notch signaling activation relative to the original 2D expanded myoblasts. We then compared 3D SCs to previously described satellite-like cells that form alongside myotubes in 2D culture, termed reserve cells (RCs). Compared to RCs, 3D SCs showed an advanced quiescent phenotype characterized by a higher Pax7, Spry1, and Notch3 expression, as well as increased functional myogenesis demonstrated by formation of myobundles with higher contractile strength. To examine 3D SC activation, we tested several myobundle injury methods and identified treatment with a bee toxin, melittin, to robustly induce myofiber fragmentation, functional decline, and 3D SC proliferation. To further investigate the transcriptional processes describing how 2D myoblasts acquire 3D SC phenotype (i.e. deactivate) and how 3D SCs respond to injury (i.e. reactivate), we applied single cell RNA-sequencing (scRNA-seq) from which we discovered the existence of two subpools of 3D SCs—“quiescent” (qSC) and “activated” (aSC). The qSC subpool possessed greater expression of quiescence genes Pax7, Spry1, and Hey1, whereas the aSC subpool exhibited increased expression of inflammatory and differentiation markers. Furthermore, we performed trajectory inference along the deactivation process from 2D myoblasts to qSCs and identified deactivation-associated genes, included downregulated genes for proliferation, cytoskeletal reorganization, and myogenic differentiation. In response to tissue injury, we observed a decrease in the proportion of qSCs and an increase in the proportion of aSCs and committed myogenic progenitor cells suggestive of myogenic differentiation. In addition, we observed transcriptional changes within the aSC population reflective of SC activation in vivo, namely increased TNF- signaling, proliferation, and glycolytic and oxidative metabolism. These results strongly suggested that 3D SC heterogeneity and function recapitulate several aspects of native human SCs and could be applied to study human muscle regeneration and disease-associated SC dysfunction. To evaluate the myobundle system in the context of disease modeling, we developed the first 3D tissue-engineered skeletal muscle model of infantile onset Pompe disease (IOPD), the most severe form of Pompe disease. Diseased myobundles demonstrated characteristic GAA enzyme deficiency, accumulation of the GAA target glycogen, and lysosome enlargement. Despite exhibiting these key biochemical and structural hallmarks of disease, IOPD myobundles did not show deficits in contractile force generation or autophagic buildup. We therefore identified metabolic stress conditions that acutely targeted disease-associated abnormalities in the lysosomes and glycogen metabolism, which revealed impairments in contractile function and glycogen mobilization. To further elucidate the biological mechanisms underlying the phenotype of IOPD myobundles, we applied RNA sequencing (RNA-seq) and observed enrichment for terms consistent with Pompe disease phenotype including downregulation of gene sets involved in muscle contraction, increased endoplasmic reticulum stress, and reduced utilization of specific metabolic pathways. We then compared the transcriptomic profiles of GAA-/-¬ and wild-type mice to identify a Pompe disease signature and confirmed the presence of this signature in IOPD myobundles. Finally, treating IOPD myobundles with clinically used recombinant protein (rhGAA) therapy resulted in increased GAA activity, glycogen clearance, and a partial reversal of the disease signature, further confirming the utility of the myobundle system for studies of Pompe disease and therapy. In summary, this dissertation describes novel strategies for the formation and characterization of quiescent human SCs using the myobundle system. We present first-time application of scRNA-seq to engineered skeletal muscle, and uncover transcriptional descriptors of human myoblast deactivation and SC heterogeneity and activation. When utilizing human myobundles as a novel model of Pompe disease, we identified disease hallmarks and responses to therapy consistent with observations in Pompe patients. We anticipate that the findings and methods developed in this work will serve as a useful framework for the future engineering of regenerative human muscle for therapeutic and disease modeling applications.

Continuous nanostructures, such as electrospun nanofibers, embedded with proteins may synergistically present the topographical and biochemical signals to cells for tissue engineering applications. In this dissertation, co-axial electrospinning is introduced as a mean to efficiently encapsulate and release protein and live entities while producing a tissue engineering scaffold with uniaxial topography. In the first specific aim, aligned poly (caprolactone) nanofibers encapsulated with BSA and growth factors were produced to demonstrate controlled release and bioactivity retention properties. Control over release kinetics is achieved by incorporation of poly(ethylene glycol) as a porogen in the shell of the fibers. PEG leaches out in a concentration and molecular weight dependent fashion, leading to BSA release half-lives that range from 1 -20 days. The second specific aim introduces the fabrication of virus and bacterial cell encapsulated electrospun fibers to achieve unique biological functionalization. Adenovirus encoding the gene for green fluorescent protein was efficiently encapsulated into the core of poly(caprolactone) fibers through co-axial electrospinning and subsequently released via the porogen-mediated process. Encapsulated bacterial cells were confined to fibers of varying core sizes, which provided an aqueous core environment for free mobility and allowed the bacterias to proliferate within the fibers.

In the third specific aim, the differentiation of skeletal myoblasts on aligned electrospun polyurethane fibers and in the presence of electromechanical stimulation were systematically studied. Skeletal myoblasts cultured on aligned polyurethane (PU) fibers showed more pronounced elongation, better alignment, upregulation of contractile proteins and higher percentage of striated myotubes compared to those cultured on random PU fibers and film. In the last specific aim, the controlled release aspect of co-axial electrospun fibers were combined with skeletal tissue engineering to serve as a therapeutic implant for the treatment of hemophilia. A non-viral, tissue engineering approach were taken to stimulate local lymphatic or vascular system in order to enhance transport near the FVIII-producing implants to provide effective and sustained treatment for hemophilia A. Stable FVIII-producing clones were engineered from isolated myoblasts and cultured on aligned, protein-releasing electrospun fibers to form skeletal myotubes. The implanted construct rapidly integrated with host tissue and selectively induced angiogenesis or lymphangiogenesis as a result of the encapsulated growth factors. Constructs inducing angiogenesis significantly enhanced the transport of produced FVIII and achieved hemophilia phenotypic correction over two months. The use of co-axial electrospun fibers to serve as controlled delivery and tissue engineering construct furthers the continued pursue of a more sophisticated and medically relevant implant scaffold design.

Electroporation is a biological cell's natural reaction to strong electric fields, where transient pores are created in the cell membrane. While electroporation holds promise of being a safe and effective tool for enhancing molecular delivery in numerous medical applications, it remains largely confined to preclinical research and clinical trials due to an incomplete understanding of the exact mechanisms involved. Muscle fibers are an important delivery target, but traditional theoretical studies of electroporation ignore the individual fiber geometry, making it impossible to study the unique transverse and longitudinal effects from the pulse stimulus. In these long, thin muscle fibers, the total reaction of the fiber to the electric field is due to fundamentally different effects from the constituent longitudinal and transverse components of the electric field generated by the pulse stimulus. While effects from the transverse component have been studied to some degree, the effects from the longitudinal component have not been considered.

This study develops a model of electroporation and delivery of small molecules in muscle tissue that includes effects from both the transverse and longitudinal components of the electric field. First, an asymptotic model of electric potential in an individual muscle fiber is derived that separates the full 3D boundary value problem into transverse and a longitudinal problems. The transverse and longitudinal problems each have their own respective source functions: the new "transverse activating function" and the well known longitudinal activating function (AF). This separation enhances analysis of the different effects from these two AFs and drastically reduces computational intensity. Electroporation is added to the asymptotic fiber model, and simplified two-compartment mass transport equations are derived from the full 3D conservation of mass equations to allow simulation of molecular uptake due to diffusion and the electric field. Special emphasis is placed on choosing model geometry, electrical, and pulsing parameters that are in accordance with experiments that study electroporation-mediated delivery of small molecules in the skeletal muscle of small mammals.

Simulations reveal that for fibers close to the electrodes the transverse AF dominates, but for fibers far from the electrodes the longitudinal AF enhances uptake by as much as 2000%. However, on the macroscopic tissue level, the increase in uptake from the longitudinal AF is no more than 10%, given that fibers far from the electrodes contribute so little to the total uptake in the tissue. The mechanism underlying the smaller effect from the longitudinal AF is found to be unique to the process of electroporation itself. Electroporation occurs on the short time scale of polarization via the transverse AF, drastically increases membrane conductance, and effectively precludes further creation of pores from charging of the membrane via the longitudinal AF. The exact value of enhancement in uptake from the longitudinal AF is shown to depend on pulsing, membrane, and tissue parameters. Finally, simulation results reproduce qualitative, and in some cases quantitative, behavior of uptake observed in experiments.

Overall, percent increase in total tissue uptake from the longitudinal AF is on the order of experimental variability, and this study corroborates previous theoretical models that neglect the effects from the longitudinal AF. However, previous models neglect the longitudinal AF without explanation, while the asymptotic fiber model is able to detail the mechanisms involved. Mechanisms revealed by the model offer insight into interpreting experimental results and increasing efficiency of delivery protocols. The model also rigorously derives a new transverse AF based on individual fiber geometry, which affects the spatial distribution of uptake in tissue differently than predicting uptake based on the magnitude of the electric field, as used in many published models. Results of this study are strictly valid for transport of small molecules through small non-growing pores. For gene therapy applications the model must be extended to transport of large DNA molecules through large pores, which may alter the importance of the longitudinal AF. In broader terms, the asymptotic model also provides a new, computationally efficient tool that may be used in studying the effect of transverse and longitudinal components of the field for other types of membrane dynamics in muscle and nerves.

Rheumatoid Arthritis (RA) is a chronic inflammatory auto-immune disease typically involving the joints, mainly the diarthrodial joint and generally starts between the age of 30 and 60 in women and somewhat later in life in men. It is the most common inflammatory arthritis and about one percent of the population is affected by RA. A complex interaction between various genetic and environmental factors lead to the development of the disease, though the specific cause of RA is not known. The goal of this study is to characterize the maturation of skeletal muscle bundles made with myoblasts isolated from RA patients and compare it with maturation of age-matched controls. Moreover, the engineered myobundles were treated with pro-inflammatory cytokines to assess their effect on the bundle maturation and to replicate the pro-inflammatory phenotype of RA.

Myobundles were prepared with human skeletal muscle (HSkM) samples obtained from young controls, age-matched controls and RA patients through biopsy of vastus lateralis muscle (biopsy of hamstring muscle was taken for young controls). We measured nuclei count, cross-sectional area, Myogenin count, Sarcomeric alpha-actinin (SAA) positive area and the myofiber diameter for each time course studies and cytokine treated bundles.

Contrary to our expectations, the time course study did not indicate significant reduction in fiber formation. This may be due to the effect of medications taken by the RA patient which might be helping the muscle function. Another possible reason might be that the cells could have regained their normal function once they were taken out from the inflammatory environment induced by the pro-inflammatory cytokines. Yet another possible reason may be that the time course considered may not be enough to access changes in the maturation and a longer time period may be required.

We then moved forward to replicate the disease pro-inflammatory phenotype by carrying out cytokine treatments on the engineered myobundles. IFNγ, IFNγ+GMCSF, TNFα+GMCSF and IFNγ+TNFα+GMCSF were chosen for the cytokine treatments. According to our results, the cross-sectional area, nuclei count/CSA, MyoG count/CSA, MyoG/Nuclei count, SAA+ area and the myofiber diameter each decreased with cytokine treatments indicating that the cytokines may indeed affect the regeneration ability of skeletal muscle cells.

The results from cytokines treatment studies indicate that cytokines do play a role in disease development and progression. A longer time course study say for up to 10 days or more post differentiation, more patient data regarding the disease severity and medications might also be helpful in further investigation.

Shear wave elastography imaging (SWEI) of skeletal muscle is of great interest to the medical community, as there is a large need for a non-invasive, quantitative biomarker of muscle health that relates to muscle function. SWEI measures mechanical properties by generating quantitative images of tissue stiffness using an acoustic radiation force (ARF) excitation in the material and measuring the resulting shear waves that propagate outward. Most SWEI tools assume an isotropic, linear, elastic material, however skeletal muscle is commonly modeled as transversely isotropic (TI) due to the alignment of the muscle fibers. This means that shear wave speed (c, SWS) is dependent on the direction of the traveling shear wave relative to the fibers in skeletal muscle.

If muscle is assumed to be incompressible and transversely isotropic (ITI) it can be described with three parameters: the longitudinal shear modulus μ_L, the transverse shear modulus μ_T, and a single parameter combining longitudinal and transverse Young's moduli (E_L and E_T) called tensile anisotropy χ_E. In an elastic ITI material, there are two shear wave modes with different polarizations that can be excited: the shear horizontal (SH) and the shear vertical (SV). Shear moduli μ_L and μ_T can be measured solely based on the observation of the SH mode, however to quantify tensile anisotropy χ_E using SWEI, it is necessary to observe the SV mode. This thesis explores characterizing skeletal muscle as an ITI material and explores factors that affect the measurement of the SV mode wave. In order to evaluate the use of χ_E as a biomarker of muscle health we must understand the factors that affect its measurement using SWEI.

Chapter 3 demonstrates feasibility of measuring both the SH and SV modes using a 3D rotational SWEI system in the vastus lateralis muscle in vivo. We develop and validate methodology to estimate μ_L, μ_T, and χ_E and describe measurements these parameters in vivo.

Chapter 4 explores the factors that affect the SV mode waves, using Green's function simulations to perform a parametric analysis to determine the optimal interrogation parameters to facilitate visualization and quantification of SV waves in muscle. We evaluate the impact of five factors: μ_L, μ_T, and χ_E as well as fiber tilt angle θ_tilt and F-number of the push geometry on SV mode speed, amplitude, and rotational distribution.

Chapter 5 extends the work in Chapter 4 to understand SH and SV wave propagation in 3D by simulating multiple observation tilt angles and all 3 components of displacement. Tilting the observation plane to particular angles allows for maximization of the strength of the SH or SV waves, demonstrating that observation of these tilted planes in in vivo data would increase opportunities for estimation of SH and SV waves.

The work presented in this thesis explores using 3D SWEI to better characterize skeletal muscle as an ITI material, specifically by assessing the SH and SV mode shear wave speed. This work also investigates factors that affect measurement of SV mode waves, and thus the ability to estimate χ_E, towards a better understanding of χ_E for use as a potential biomarker of muscle health.

Muscle fibrosis is caused by muscle injury, dystrophy, sarcopenia, and rheumatoid arthritis. This condition is characterized by hardening and scarring, which impairs contractile muscle function. To understand how fibrotic disease affects muscular function, we created a model of human skeletal muscle fibrosis using three-dimensional engineered skeletal muscle (myobundles). Furthermore, to investigate the effect of skeletal muscle fibrosis on the vascular system, we integrated the fibrotic skeletal muscle with tissue engineered blood vessels. Treating myobundles with Transforming Growth Factor β1 (TGF-β1) reproduced key characteristics of fibrotic skeletal muscle including reduced contractile force, disrupted contractile protein organization, increased stiffness, and expression of profibrotic genes. Treatment with a selective inhibitor (SB525334) of TGF-β1 receptor (ALK5, TGF-βRI) increased contractile function and decreased ECM deposition, consistent with animal studies in the literature. We also observed endogenous secretion of TGF-β1 in our myobundles which is of novel biological significance. siRNA knockdown of TGF-β1 increased contractile force. Testing anti-fibrotic drug Nintedanib in this model, showed an increase in tetanus force production in 2 out of 3 donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Western blot analysis of Nintedanib also providence evidence of its inhibition of TGF-β1 signaling by the reduction of phosphorylated Smad2/3. Repositioned anti-fibrotic drug Suramin treatment of fibrotic myobundles resulted in increase of tetanus force production in all three donors and reduction of pro-fibrotic ECM accumulation of collagen 1 and fibronectin. Suramin’s influence on TGF-β1 signaling in our system was found not to be as targeted as Nintedanib as there was only reduction in Smad3 phosphorylation and not Smad2 phosphorylation. Anti-fibrotic drug testing in our model was also able to wean out donor specific sensitivity to the drugs with donor 3. Skeletal myobundles were integrated with Tissue Engineered Blood Vessels (TEBVs) to identify the effect of skeletal muscle fibrosis on blood vessels or the human vasculature. Integrated TEBVs with 5 ng/ml TGF-β1 dosed myobundles showed reduced function, increased mesenchymal markers such as vimentin and alpha smooth muscle actin, and increased endothelial cell inflammation. Our results suggest a detrimental effect of skeletal muscle fibrosis on blood vessels and show an interaction between the skeletal muscle fibrosis and the human vasculature This model provides a platform to study skeletal muscle fibrosis alone or its effect on the vasculature and allows for testing anti-fibrotic drugs and assessing myobundle function along with disease influence on human vasculature.

Tissue engineering of biomimetic skeletal muscle may lead to development of new therapies for myogenic repair and generation of improved in vitro models for studies of muscle function, regeneration, and disease. For the optimal therapeutic and in vitro results, engineered muscle should recreate the force-generating and regenerative capacities of native muscle, enabled respectively by its two main cellular constituents, the mature myofibers and satellite cells (SCs). Still, after 20 years of research, engineered muscle tissues fall short of mimicking contractile function and self-repair capacity of native skeletal muscle. To overcome this limitation, we set the thesis goals to: 1) generate a highly functional, self-regenerative engineered skeletal muscle and 2) explore mechanisms governing its formation and regeneration in vitro and survival and vascularization in vivo.

By studying myogenic progenitors isolated from neonatal rats, we first discovered advantages of using an adherent cell fraction for engineering of skeletal muscles with robust structure and function and the formation of a SC pool. Specifically, when synergized with dynamic culture conditions, the use of adherent cells yielded muscle constructs capable of replicating the contractile output of native neonatal muscle, generating >40 mN/mm2 of specific force. Moreover, tissue structure and cellular heterogeneity of engineered muscle constructs closely resembled those of native muscle, consisting of aligned, striated myofibers embedded in a matrix of basal lamina proteins and SCs that resided in native-like niches. Importantly, we identified rapid formation of myofibers early during engineered muscle culture as a critical condition leading to SC homing and conversion to a quiescent, non-proliferative state. The SCs retained natural regenerative capacity and activated, proliferated, and differentiated to rebuild damaged myofibers and recover contractile function within 10 days after the muscle was injured by cardiotoxin (CTX). The resulting regenerative response was directly dependent on the abundance of SCs in the engineered muscle that we varied by expanding starting cell population under different levels of basic fibroblast growth factor (bFGF), an inhibitor of myogenic differentiation. Using a dorsal skinfold window chamber model in nude mice, we further demonstrated that within 2 weeks after implantation, initially avascular engineered muscle underwent robust vascularization and perfusion and exhibited improved structure and contractile function beyond what was achievable in vitro.

To enhance translational value of our approach, we transitioned to use of adult rat myogenic cells, but found that despite similar function to that of neonatal constructs, adult-derived muscle lacked regenerative capacity. Using a novel platform for live monitoring of calcium transients during construct culture, we rapidly screened for potential enhancers of regeneration to establish that many known pro-regenerative soluble factors were ineffective in stimulating in vitro engineered muscle recovery from CTX injury. This led us to introduce bone marrow-derived macrophages (BMDMs), an established non-myogenic contributor to muscle repair, to the adult-derived constructs and to demonstrate remarkable recovery of force generation (>80%) and muscle mass (>70%) following CTX injury. Mechanistically, while similar patterns of early SC activation and proliferation upon injury were observed in engineered muscles with and without BMDMs, a significant decrease in injury-induced apoptosis occurred only in the presence of BMDMs. The importance of preventing apoptosis was further demonstrated by showing that application of caspase inhibitor (Q-VD-OPh) yielded myofiber regrowth and functional recovery post-injury. Gene expression analysis suggested muscle-secreted tumor necrosis factor-α (TNFα) as a potential inducer of apoptosis as common for muscle degeneration in diseases and aging in vivo. Finally, we showed that BMDM incorporation in engineered muscle enhanced its growth, angiogenesis, and function following implantation in the dorsal window chambers in nude mice.

In summary, this thesis describes novel strategies to engineer highly contractile and regenerative skeletal muscle tissues starting from neonatal or adult rat myogenic cells. We find that age-dependent differences of myogenic cells distinctly affect the self-repair capacity but not contractile function of engineered muscle. Adult, but not neonatal, myogenic progenitors appear to require co-culture with other cells, such as bone marrow-derived macrophages, to allow robust muscle regeneration in vitro and rapid vascularization in vivo. Regarding the established roles of immune system cells in the repair of various muscle and non-muscle tissues, we expect that our work will stimulate the future applications of immune cells as pro-regenerative or anti-inflammatory constituents of engineered tissue grafts. Furthermore, we expect that rodent studies in this thesis will inspire successful engineering of biomimetic human muscle tissues for use in regenerative therapy and drug discovery applications.

The development of a functional tissue-engineered human skeletal muscle model in vitro would provide an excellent platform on which to study the process of myogenesis, various musculoskeletal disease states, and drugs and therapies for muscle toxicity. We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel under static conditions capable of exerting active contractions. Additionally, we demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of muscle differentiation, elevation of active contractile force amplitudes, and increasing Type II myofiber formation in vitro.

The global hypothesis that motivated this research was that joint inhibition of miR-133a and miR-696 in isolated primary human skeletal myoblasts would lead to accelerated differentiation of tissue-engineered muscle constructs with higher proportion of Type I myofibers and that are capable of significantly increased active contractile forces when subjected to electrical stimulus. The proposed research tested the following specific hypotheses: (1) that HSkM would require different culture conditions than those optimal for C2C12 culture (8% equine serum in differentiation medium on uncoated substrates), as measured by miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in 2D human skeletal muscle cultures with accelerated differentiation and increased Type I muscle fibers compared to control and individual inhibition of each miR, as measured by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696 in this functional 3D human skeletal muscle model would result in active contraction significantly higher than control and individual inhibition by each miR, as measured by isometric force testing, and finally (4) that specific co-culture conditions could support a lamellar co-culture model in 3D of human cord blood-derived endothelial cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force testing and immunofluorescence.

Major results of the dissertation are as follows. Culture conditions of 100 μg/mL growth factor reduced-Matrigel-coated substrates and 2% equine serum in differentiation medium were identified to improve human skeletal myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence of mitochondria and distribution of fiber type did not change with miR transfection in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists a myoblast seeding density threshold for the production of functional muscle. 5 x 106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted in constructs that exerted the highest twitch and tetanus forces. Engineering of human skeletal muscle from transfected cells led to significant increases in force amplitude in joint inhibition compared to negative control (transfection with scrambled miR sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally, co-culture of functional human skeletal muscle with human cord blood-derived endothelial cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases in force amplitudes at 48 and 96 hours of co-culture.

The Muoio lab studies the interplay between lipid whole body energy balance,

mitochondrial function and insulin action in skeletal muscle. Data from our lab suggests that lipid-induced insulin resistance in skeletal muscle may stem from excessive incomplete oxidation of fatty acids, which occurs when high rates of β-­oxidation exceed TCA cycle flux (Koves et al., 2005; Koves et al., 2008). Most notably, we have shown that mice with a genetically engineered decrease in mitochondrial uptake and oxidation of fatty acids are protected against diet-­induced insulin resistance (Koves et al., 2008). This

suggests that an excessive and/or inappropriate metabolic burden on muscle

mitochondria provokes insulin resistance. Our working model predicts that: 1) high rates of incomplete β-oxidation reflect a state of ”mitochondrial stress,” and 2) that energy-overloaded mitochondria generate a yet unidentified signal that mediates insulin

resistance. One possibility is that this putative mitochondrial-derived signal stems from redox imbalance and disruptions in redox sensitive signaling cascades. Therefore, we are interested in identifying molecules that link redox balance, mitochondrial function and insulin action in skeletal muscle. The work described herein identifies thioredoxin-interacting protein (TXNIP) as an attractive candidate that regulates both glucose homeostasis and mitochondrial fuel selection.

TXNIP is a redox sensitive, α-arrestin protein that has been implicated as a negative regulator of glucose control. Mounting evidence suggested that TXNIP might play a key role in regulating mitochondrial function; however, the molecular nature of this relationship was poorly defined. Previous studies in TXNIP knockout mice reported that deficiency of this protein compromises oxidative metabolism, increases glycolytic activity and promotes production of reactive oxygen species (ROS), while also affording protection against insulin resistance. Therefore, we hypothesized that TXNIP might serve as a nutrient sensor that couples cellular redox status to the adjustments in mitochondrial function. We tested this hypothesis by exploiting loss of function models to evaluate the effects of TXNIP deficiency on mitochondrial metabolism and respiratory function.

In chapter 3, we comprehensively evaluated oxidative metabolism, substrate

selection, respiratory kinetics and redox balance in mice with total body and skeletal muscle-­specific TXNIP deficiency. Targeted metabolomics, comprehensive bioenergetics analysis, whole-body respirometry and conventional biochemistry showed that TXNIP deficiency results in reduced exercise tolerance with marked impairments in skeletal muscle oxidative metabolism. The deficits in substrate oxidation were not secondary to decreased mitochondrial mass or increased H₂O₂ emitting potential from the electron transport chain. Instead, the activities of several mitochondrial dehydrogenases involved in branched-chain amino acid and ketone catabolism, the tricarboxylic acid (TCA) cycle and fatty acid β-oxidation were significantly diminished in TXNIP null muscles. These deficits in mitochondrial enzyme activities were accompanied by decreased protein abundance without changes in mRNA expression. Taken together, these results suggest that in skeletal muscle TXNIP plays an essential role in maintaining protein synthesis and/or stability of a subset of mitochondrial dehydrogenase enzymes that permit muscle use of alternate fuels under conditions of glucose deprivation.

Based on these conclusions, we questioned whether additional regulatory

mechanisms could contribute to the reduced oxidative metabolism in the absence ofTXNIP. Several metabolic enzymes of the TCA cycle have been shown to be redox-sensitive protein targets regulated by the thioredoxin (TRX1/TRX2) and glutathione (GSH) redox-mediated circuits. TXNIP has been shown to respond to oxidative stress by shuttling to the mitochondria where it binds to TRX2 and/or other proteins, thus affecting downstream signaling pathways, such as the apoptotic cascade. Therefore, we speculated whether there was a role for redox imbalance in mediating the mitochondrial phenotype of the TXNIP knockout (TKO) mice. In chapter 4, we present preliminary evidence that increased glucose uptake promotes non-mitochondrial ROS production, causing a shift in redox balance, decreased GSH/GSSG, and S-glutathionylation of α-­ketoglutarate dehydrogenase (&alpha-KGD). This post-translational modification protects the protein from permanent oxidative damage, but at the cost of reversible loss of activity and subsequent disruption of TCA cycle flux that contributes, in part, to the diminished oxidative metabolism observed in the TXNIP deficient mice.

In aggregate, this work sheds new light onto the physiological role of TXNIP in

skeletal muscle as it pertains to substrate metabolism and fuel switching in response to nutrient availability. This work has important implications for metabolic diseases such as obesity and type 2 diabetes, which are characterized by marked disruptions in fuel selection.

The research presented here represents a quest to understand and address limitations in the field of skeletal muscle tissue engineering, with hopes to better understand the factors involved in producing viable engineered skeletal muscle tissue. The driving force behind this research was to address two of the many factors important in muscle cell proliferation and differentiation, toward developing mature and functional bioartificial skeletal muscles (BAMs). Our work focused on understanding the individual effects of mechanical stimulation and microRNAs (miRNAs), as well as the synergistic relationship between the two factors. We hypothesized that (1) myoblast proliferation and differentiation are modulated by mechanical stimulation via temporally regulated miRNAs and that (2) modulating these miRNAs can enhance skeletal muscle function in a 3D tissue-engineered system.

We first established a BAM system using C2C12 mouse myoblasts in a collagen gel, showing that these cells were able to produce mature sarcomeres when cultured under steady, passive tension for up to 36 days. Staining muscle-specific proteins and electron microscopy showed distinct striations and myofiber organization as early as 6 days, post-differentiation. At 33 days, cultures contained collagen fibers and showed localization of paxillin at the fiber termini, suggesting that myotendinous junctions were forming.

We then focused on the effects of mechanical stimulation on C2C12 myoblasts in a more simple, 2D system. In particular, we assessed miRNA and muscle-specific gene expression over time and in response to two cyclic stretch regimens using miRNA microarray technology and quantitative real time RT-PCR. Both miRNAs and certain genes, such as SRF and Mef2c, had differential responses to the two regimens. Over-expression and inhibition studies of one muscle-specific miRNA, miR-1, abrogated the stretch response and suggest that a balancing mechanism is in place to avoid large fluctuations in miRNA levels.

Finally, since miRNA modulation quenched the stretch-mediated response in myoblasts, we chose to examine 3D BAM function when miRNA levels were altered to promote differentiation. Using the same collagen gel model established previously, a muscle-specific miRNA, miR-133, known to promote proliferation, was transiently inhibited (anti-miR-133) to encourage differentiation. Forces in the anti-miR-133 BAMs were, on average, 20% higher over the negative control. Further, myofiber diameters were significantly greater and striations were more organized in the anti-miR-133 BAMs, suggesting that transient, exogenous delivery of miRNAs may be a viable approach to create a more fully differentiated muscle.

Transplantation of a functional engineered skeletal muscle substitute is a promising therapeutic option to repair irreversible muscle damage, and, on the other hand, functional muscle tissue constructs can serve as in vitro 3D tissue models that complement the conventional 2D cell cultures and animal models to advance our limited understanding of intrinsic myogenesis and muscle regeneration process. However, the engineering of skeletal muscle constructs with comparable contractile function to the native muscle is hampered by the lack of 1) effective and reproducible methods to form relatively large muscle constructs composed of viable, dense, aligned and matured myofibers, and 2) beneficial microenvironmental cues as well as physiological stimulations that favor the growth, differentiation and maturation of myogenic cells. Thus, in this thesis, I have developed a mesoscopic hydrogel molding approach to fabricate relatively large engineered muscle tissue networks with controllable thickness, pore dimensions, overall myofiber alignment and regional myofiber orientation. I then investigated the effect of variation in pore length on the force generation and passive properties of engineered muscle networks and the potential to improve the contractile function of engineered muscle networks with the treatment of a soluble neurotrophic factor, agrin.

Specifically, high aspect-ratio soft lithography was utilized to precisely fabricate elastomeric molds containing an array of staggered hexagonal posts which created elliptical pores in muscle tissue sheets made from a mixture of primary skeletal myoblasts, fibrin and Matrigel. The improved oxygen and nutrient access through the pores increased the viability of the embedded muscle cells and prevented the formation of an acellular core. The differentiated myofibers were locally aligned in tissue bundles surrounding the elliptical pores. The length and direction of the microfabricate posts arbitrarily determined the length of elliptical pores and the mean orientation of myofibers formed around the pores, which enables the control of pore dimensions and regional myofiber orientation. Contractile force analysis revealed that engineered muscle networks with more elongated pores generated larger contractile force due to the increased myonuclear density and degree of overall myofiber alignment, despite the larger porosity and reduced tissue volume. Furthermore, the introduction of elliptical pores resulted in distinct deformational changes in tissue bundles and node regions that connect the ends of bundles with the applied unaxial macroscopic stretch, but such spatial alteration of local strain field resulted in no significant change in macroscopic length- tension relationships among engineered muscle networks with different pore length.

In addition, supplementing culture medium with soluble recombinant agrin significantly increased contractile force production of engineered muscle networks in the absence of nerve-muscle interaction, primarily or partially due to the agrin-induced upregulation of dystrophin. As expected, alteration in the levels endogenous ACh or ACh-like compound affected the agrin-induced AChR aggregation. Furthermore, increased autocrine AChR stimulation, a novel mechanism underlying survival and maturation of aneural myotubes, attenuated the agrin-induced force increase, while suppressed autocrine AChR stimulation severely comproised the overall force production of engineered muscle networks, of which the underlying mechanisms remains to be elucidated in the future studies.

In summary, a novel tissue engineering methodology that enables the fabrication of relative large muscle tissue constructs with controllable structure and function has been developed in this thesis. Future studies, such as optimizing cell-matrix interaction, incorporating beneficial regulatory proteins in the fibrin-based matrix, and applying specific patterns of electro-mechanical stimulations are expected to further augment the contractile function of engineered muscle networks. The potential application of this versatile tissue fabrication approach to engineer different types of soft tissue might further advance the development of tissue regeneration therapies as well as deepen our understanding of intrinsic tissue morphogenesis and regeneration process.