Shama R. Iyer

NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation

NAD+ treatment can reverse the functional decline in degenerating muscles. Making muscle work better Degenerating muscle—whether from muscular dystrophies, myopathies, or other diseases—loses its mitochondria (the energy supply) and an essential cofactor nicotinamide adenine dinucleotide (NAD+), while gaining an extra load of enzymes that use up NAD+, as reported by Ryu and colleagues. The resulting loss of NAD+ is exacerbated by a drop in NAD+ biosynthetic enzymes, such as NAMPT. Restoration of NAD+ levels in either mice or worms with disease-like degenerating muscles improved muscle function, a consequence of more mitochondria, more muscle structural proteins, and a decrease in inflammation. The authors suggest that NAD+ repletion may be a successful therapeutic approach for a number of muscle-wasting diseases. Neuromuscular diseases are often caused by inherited mutations that lead to progressive skeletal muscle weakness and degeneration. In diverse populations of normal healthy mice, we observed correlations between the abundance of mRNA transcripts related to mitochondrial biogenesis, the dystrophin-sarcoglycan complex, and nicotinamide adenine dinucleotide (NAD+) synthesis, consistent with a potential role for the essential cofactor NAD+ in protecting muscle from metabolic and structural degeneration. Furthermore, the skeletal muscle transcriptomes of patients with Duchene’s muscular dystrophy (DMD) and other muscle diseases were enriched for various poly[adenosine 5′-diphosphate (ADP)–ribose] polymerases (PARPs) and for nicotinamide N-methyltransferase (NNMT), enzymes that are major consumers of NAD+ and are involved in pleiotropic events, including inflammation. In the mdx mouse model of DMD, we observed significant reductions in muscle NAD+ levels, concurrent increases in PARP activity, and reduced expression of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme for NAD+ biosynthesis. Replenishing NAD+ stores with dietary nicotinamide riboside supplementation improved muscle function and heart pathology in mdx and mdx/Utr−/− mice and reversed pathology in Caenorhabditis elegans models of DMD. The effects of NAD+ repletion in mdx mice relied on the improvement in mitochondrial function and structural protein expression (α-dystrobrevin and δ-sarcoglycan) and on the reductions in general poly(ADP)-ribosylation, inflammation, and fibrosis. In combination, these studies suggest that the replenishment of NAD+ may benefit patients with muscular dystrophies or other neuromuscular degenerative conditions characterized by the PARP/NNMT gene expression signatures.

Defective signaling, osteoblastogenesis and bone remodeling in a mouse model of connexin 43 C-terminal truncation

ABSTRACT In skeletal tissue, loss or mutation of the gap junction protein connexin 43 (Cx43, also known as GJA1) in cells of the osteoblast lineage leads to a profound cortical bone phenotype and defective tissue remodeling. There is mounting evidence in bone cells that the C-terminus (CT) of Cx43 is a docking platform for signaling effectors and is required for efficient downstream signaling. Here, we examined this function, using a mouse model of Cx43 CT-truncation (Gja1 K258Stop). Relative to Gja1+/− controls, male Gja1−/K258Stop mice have a cortical bone phenotype that is remarkably similar to those reported for deletion of the entire Cx43 gene in osteoblasts. Furthermore, we show that the Cx43 CT binds several signaling proteins that are required for optimal osteoblast function, including PKCδ, ERK1 and ERK2 (ERK1/2, also known as MAPK3 and MAPK1, respectively) and β-catenin. Deletion of the Cx43 CT domain affects these signaling cascades, impacting osteoblast proliferation, differentiation, and collagen processing and organization. These data imply that, at least in bone, Cx43 gap junctions not only exchange signals, but also recruit the appropriate effector molecules to the Cx43 CT in order to efficiently activate signaling cascades that affect cell function and bone acquisition. Highlighted Article: The C-terminal domain of Cx43 in skeletal tissue affects signaling cascades and osteoblast function. Truncation of this domain recapitulates key aspects of Cx43 conditional deletion in bone.

Altered nuclear dynamics in MDX myofibers

Duchenne muscular dystrophy (DMD) is a genetic disorder in which the absence of dystrophin leads to progressive muscle degeneration and weakness. Although the genetic basis is known, the pathophysiology of dystrophic skeletal muscle remains unclear. We examined nuclear movement in wild-type (WT) and muscular dystrophy mouse model for DMD (MDX) (dystrophin-null) mouse myofibers. We also examined expression of proteins in the linkers of nucleoskeleton and cytoskeleton (LINC) complex, as well as nuclear transcriptional activity via histone H3 acetylation and polyadenylate-binding nuclear protein-1. Because movement of nuclei is not only LINC dependent but also microtubule dependent, we analyzed microtubule density and organization in WT and MDX myofibers, including the application of a unique 3D tool to assess microtubule core structure. Nuclei in MDX myofibers were more mobile than in WT myofibers for both distance traveled and velocity. MDX muscle shows reduced expression and labeling intensity of nesprin-1, a LINC protein that attaches the nucleus to the microtubule and actin cytoskeleton. MDX nuclei also showed altered transcriptional activity. Previous studies established that microtubule structure at the cortex is disrupted in MDX myofibers; our analyses extend these findings by showing that microtubule structure in the core is also disrupted. In addition, we studied malformed MDX myofibers to better understand the role of altered myofiber morphology vs. microtubule architecture in the underlying susceptibility to injury seen in dystrophic muscles. We incorporated morphological and microtubule architectural concepts into a simplified finite element mathematical model of myofiber mechanics, which suggests a greater contribution of myofiber morphology than microtubule structure to muscle biomechanical performance.NEW & NOTEWORTHY Microtubules provide the means for nuclear movement but show altered organization in the muscular dystrophy mouse model (MDX) (dystrophin-null) muscle. Here, MDX myofibers show increased nuclear movement, altered transcriptional activity, and altered linkers of nucleoskeleton and cytoskeleton complex expression compared with healthy myofibers. Microtubule architecture was incorporated in finite element modeling of passive stretch, revealing a role of fiber malformation, commonly found in MDX muscle. The results suggest that alterations in microtubule architecture in MDX muscle affect nuclear movement, which is essential for muscle function.

Novel multi-functional fluid flow device for studying cellular mechanotransduction

Cells respond to their mechanical environment by initiating multiple mechanotransduction signaling pathways. Defects in mechanotransduction have been implicated in a number of pathologies; thus, there is need for convenient and efficient methods for studying the mechanisms underlying these processes. A widely used and accepted technique for mechanically stimulating cells in culture is the introduction of fluid flow on cell monolayers. Here, we describe a novel, multifunctional fluid flow device for exposing cells to fluid flow in culture. This device integrates with common lab equipment including routine cell culture plates and peristaltic pumps. Further, it allows the fluid flow treated cells to be examined with outcomes at the cell and molecular level. We validated the device using the biologic response of cultured UMR-106 osteoblast-like cells in comparison to a commercially available system of laminar sheer stress to track live cell calcium influx in response to fluid flow. In addition, we demonstrate the fluid flow-dependent activation of phospho-ERK in these cells, consistent with the findings in other fluid flow devices. This device provides a low cost, multi-functional alternative to currently available systems, while still providing the ability to generate physiologically relevant conditions for studying processes involved in mechanotransduction in vitro.

Alternating bipolar field stimulation identifies muscle fibers with defective excitability but maintained local Ca 2+ signals and contraction

BackgroundMost cultured enzymatically dissociated adult myofibers exhibit spatially uniform (UNI) contractile responses and Ca2+ transients over the entire myofiber in response to electric field stimuli of either polarity applied via bipolar electrodes. However, some myofibers only exhibit contraction and Ca2+ transients at alternating (ALT) ends in response to alternating polarity field stimulation. Here, we present for the first time the methodology for identification of ALT myofibers in primary cultures and isolated muscles, as well as a study of their electrophysiological properties.ResultsWe used high-speed confocal microscopic Ca2+ imaging, electric field stimulation, microelectrode recordings, immunostaining, and confocal microscopy to characterize the properties of action potential-induced Ca2+ transients, contractility, resting membrane potential, and staining of T-tubule voltage-gated Na+ channel distribution applied to cultured adult myofibers. Here, we show for the first time, with high temporal and spatial resolution, that normal control myofibers with UNI responses can be converted to ALT response myofibers by TTX addition or by removal of Na+ from the bathing medium, with reappearance of the UNI response on return of Na+. Our results suggest disrupted excitability as the cause of ALT behavior and indicate that the ALT response is due to local depolarization-induced Ca2+ release, whereas the UNI response is triggered by action potential propagation over the entire myofiber. Consistent with this interpretation, local depolarizing monopolar stimuli give uniform (propagated) responses in UNI myofibers, but only local responses at the electrode in ALT myofibers. The ALT responses in electrically inexcitable myofibers are consistent with expectations of current spread between bipolar stimulating electrodes, entering (hyperpolarizing) one end of a myofiber and leaving (depolarizing) the other end of the myofiber. ALT responses were also detected in some myofibers within intact isolated whole muscles from wild-type and MDX mice, demonstrating that ALT responses can be present before enzymatic dissociation.ConclusionsWe suggest that checking for ALT myofiber responsiveness by looking at the end of a myofiber during alternating polarity stimuli provides a test for compromised excitability of myofibers, and could be used to identify inexcitable, damaged or diseased myofibers by ALT behavior in healthy and diseased muscle.

Superparamagnetic Iron Oxide Nanoparticles in Musculoskeletal Biology.

The use of platelet-rich plasma and mesenchymal stem cells has garnered much attention in orthopedic medicine, focusing on the biological aspects of cell function. However, shortly after systemic delivery, or even a local injection, few of the transplanted stem cells or platelets remain at the target site. Improvement in delivery, and the ability to track and monitor injected cells, would greatly improve clinical translation. Nanoparticles can effectively and quickly label most cells in vitro, and evidence to date suggests such labeling does not compromise the proliferation or differentiation of cells. A specific type of nanoparticle, the superparamagnetic iron oxide nanoparticle (SPION), is already employed as a magnetic resonance imaging (MRI) contrast agent. SPIONs can be coupled with cells or bioactive molecules (antibodies, proteins, drugs, etc.) to form an injectable complex for in vivo use. The biocompatibility, magnetic properties, small size, and custom-made surface coatings also enable SPIONs to be used for delivering and monitoring of small molecules, drugs, and cells, specifically to muscle, bone, or cartilage. Because SPIONs consist of cores made of iron oxides, targeting of SPIONs to a specific muscle, bone, or joint in the body can be enhanced with the help of applied gradient magnetic fields. Moreover, MRI has a high sensitivity to SPIONs and can be used for noninvasive determination of successful delivery and monitoring distribution in vivo. Gaps remain in understanding how the physical and chemical properties of nanomaterials affect biological systems. Nonetheless, SPIONs hold great promise for regenerative medicine, and progress is being made rapidly toward clinical applications in orthopedic medicine.

Engineering functional and histological regeneration of vascularized skeletal muscle.

Tissue engineering strategies to treat patients with volumetric muscle loss (VML) aim to recover the structure and contractile function of lost muscle tissue. Here, we assessed the capacity of novel electrospun fibrin hydrogel scaffolds seeded with murine myoblasts to regenerate the structure and function of damaged muscle within VML defects to the mouse tibialis anterior muscle. The electrospun fibrin scaffolds provide pro-myogenic alignment and stiffness cues, myomimetic hierarchical structure, suturability, and scale-up capabilities. Myoblast-seeded scaffolds enabled remarkable muscle regeneration with high myofiber and vascular densities after 2 and 4 weeks, mimicking that of native skeletal muscle, while acellular scaffolds lacked muscle regeneration. Both myoblast-seeded and acellular scaffolds fully recovered muscle contractile function to uninjured values after 2 and 4 weeks. Electrospun scaffolds pre-vascularized with co-cultured human endothelial cells and human adipose-derived stem cells implanted into VML defects for 2 weeks anastomosed with host vasculature and were perfused with host red blood cells. These data demonstrate the significant potential of electrospun fibrin scaffolds seeded with myoblasts to fully regenerate the structure and function of volumetric muscle defects and these scaffolds offer a promising treatment option for patients with VML.

Non-invasive assessment of muscle injury in healthy and dystrophic animals with electrical impedance myography

Dystrophic muscle is particularly susceptible to eccentric contraction–induced injury. We tested the hypothesis that electrical impedance myography (EIM) can detect injury induced by maximal‐force lengthening contractions.

Optical recording of action potential initiation and propagation in mouse skeletal muscle fibers

Individual skeletal muscle fibers have been used to examine a wide variety of cellular functions and pathologies. Among other parameters, skeletal muscle action potential propagation has been measured to assess the integrity and function of skeletal muscle. In this paper, we utilize Di-8-ANEPPS, a potentiometric dye and mag-fluo-4, a low-affinity intracellular calcium indicator to non-invasively and reliably measure action potential conduction velocity in skeletal muscle. We used an extracellular bipolar electrode to generate an electric field that will initiate an action potential at one end of the fiber or the other. Using enzymatically dissociated flexor digitorum brevis (FDB) fibers, we demonstrate the strength and applicability of this technique. Using high-speed line scans, we estimate the conduction velocity to be approximately 0.4 m/s. In addition to measuring the conduction velocity, we can also measure the passive electrotonic potentials elicited by pulses by either applying tetrodotoxin (TTX) or reducing the bath sodium levels. We applied these methodologies to FDB fibers under elevated extracellular potassium conditions, and found that the conduction velocity is significantly reduced compared to our control concentration. Lastly, we have constructed a circuit model of a skeletal muscle in order to predict passive polarization of the fiber by the field stimuli. Our predictions from the model fiber closely resemble the recordings acquired from in vitro assays. With these techniques, we can examine how many different pathologies and mutations affect skeletal muscle action potential propagation. Our work demonstrates the utility of using Di-8-ANEPPS or mag-fluo-4 to non-invasively measure action potential conduction velocity.

Imaging Analysis of the Neuromuscular Junction in Dystrophic Muscle

Duchenne muscular dystrophy (DMD), caused by the absence of the protein dystrophin, is characterized as a neuromuscular disease in which muscle weakness, increased susceptibility to muscle injury, and inadequate repair appear to underlie the pathology. Considerable attention has been dedicated to studying muscle fiber damage, but there is little information to determine if damage from contraction-induced injury also occurs at or near the nerve terminal axon. Interestingly, both human patients and the mouse model for DMD (the mdx mouse) present fragmented neuromuscular junction (NMJ) morphology. Studies of mdx mice have revealed presynaptic and postsynaptic abnormalities, nerve terminal discontinuity, as well as increased susceptibility of the NMJ to contraction-induced injury with corresponding functional changes in neuromuscular transmission and nerve-evoked electromyography. Focusing on the NMJ as a contributor to functional deficits in the muscle represents a paradigm shift from the more prevalent myocentric perspectives. Further studies are needed to determine the extent to which the nerve-muscle interaction is disrupted in DMD and the role of the NMJ in the dystrophic progression. This chapter lists the tools needed for nerve terminal and NMJ structural analysis using fluorescence imaging, and provides a step-by-step outline for how to stain, image, and analyze the NMJ in skeletal muscle, with specific attention to mdx muscle.