A need for NAD+ in muscle development, homeostasis, and aging

Abstract

Skeletal muscle enables posture, breathing, and locomotion. Skeletal muscle also impacts systemic processes such as metabolism, thermoregulation, and immunity. Skeletal muscle is energetically expensive and is a major consumer of glucose and fatty acids. Metabolism of fatty acids and glucose requires NAD+ function as a hydrogen/electron transfer molecule. Therefore, NAD+ plays a vital role in energy production. In addition, NAD+ also functions as a cosubstrate for post-translational modifications such as deacetylation and ADP-ribosylation. Therefore, NAD+ levels influence a myriad of cellular processes including mitochondrial biogenesis, transcription, and organization of the extracellular matrix. Clearly, NAD+ is a major player in skeletal muscle development, regeneration, aging, and disease. The vast majority of studies indicate that lower NAD+ levels are deleterious for muscle health and higher NAD+ levels augment muscle health. However, the downstream mechanisms of NAD+ function throughout different cellular compartments are not well understood. The purpose of this review is to highlight recent studies investigating NAD+ function in muscle development, homeostasis, disease, and regeneration. Emerging research areas include elucidating roles for NAD+ in muscle lysosome function and calcium mobilization, mechanisms controlling fluctuations in NAD+ levels during muscle development and regeneration, and interactions between targets of NAD+ signaling (especially mitochondria and the extracellular matrix). This knowledge should facilitate identification of more precise pharmacological and activity-based interventions to raise NAD+ levels in skeletal muscle, thereby promoting human health and function in normal and disease states.

Fig. 1

NAD+ biosynthetic pathways. NAD+ can be synthesized via de novo, Preiss-Handler, and multiple salvage pathways. NAD+ precursors and metabolites are represented in gray boxes. The enzymes involved in each conversion are listed above the arrows. The cellular outputs regulated by NAD+ or metabolites are listed in white boxes with black outlines. For explanation of the acronyms, please see the abbreviations list in the main body of the text

Fig. 2

Compartmentalization of NAD+ pools in skeletal muscle. Diagram of a striated skeletal muscle fiber. NAD+ is localized to mitochondrial, nuclear, cytosolic, and membrane proximal pools in muscle cells. Additional NAD+ compartments not diagrammed here include vesicular compartments. The NAD+/NADH ratio is higher in the nuclear and cytosolic compartments compared to the mitochondrial compartment. The ratio is unknown in the membrane proximal compartment in muscle. Enzymes that consume NAD+ and their relative subcellular localizations are found within black or white boxes. Integrin receptors and membrane channels that transport NAD+ and calcium across the sarcolemma can be seen in the diagram

Fig. 3

Hypothesized mechanism of membrane proximal NAD+ action in muscle-ECM adhesion. (A–B) Model of membrane proximal NAD+ regulation of subcellular protein localization, post-translational modification, laminin-binding affinity, and ECM organization in muscle. (A) Damage scenario. NRK enzymes localize to cytoplasmic tails of Integrin receptors for laminin and generate an intracellular membrane proximal pool of NAD+. In response to damage, intracellular membrane proximal NAD+ generated by NRK enzymes is actively transported or leaks across the sarcolemma into the extracellular environment. Extracellular membrane proximal NAD+ is consumed by ART enzymes as ADP-ribose moieties are added to Integrin receptors for laminin in mono-ADP-ribosylation reactions. Intracellular localization of Paxillin to cell-matrix adhesion complexes is disrupted. Extracellular organization of laminin is disrupted. (B) NAD+-mediated damage response scenario. Due to the movement of intracellular membrane proximal NAD+ to the extracellular environment, ADP-ribosylation of Integrin receptors for laminin change to a high-affinity binding conformation. Laminin organization is increased, Paxillin localization to cell-matrix adhesion complexes is restored, and Integrin-laminin binding is augmented. (C–E) Reprinted with permission from [33]. c Model of genetic mosaic experiment to determine if wild-type Nrk2b is sufficient in a cell autonomous manner for subcellular localization of beta-Dystroglycan and/or Paxillin to cell-matrix adhesions. Fluorescent dextran labeled wild-type cells were transplanted into a Nrk2b-deficient background and subcellular localization of beta-Dystroglycan and Paxillin were determined by immunohistochemistry. (D–E) Side mount, anterior left, 26 hpf nrk2b morphant hosts with transplanted wild-type cells (red) and beta-Dystroglycan (blue) or Paxillin antibody staining (green). (D-D1) Beta-Dystroglycan robustly localizes to MTJs in nrk2b morphants in the presence and the absence of transplanted wild-type cells. (E-E1) Robust Paxillin localization to cell-matrix adhesion complexes in nrk2b morphants is rescued in the transplanted wild-type cells, but not in the Nrk2b-deficient cells surrounding them. Therefore, Nrk2b is cell autonomously sufficient for subcellular localization of Paxillin, likely through generation of membrane proximal NAD+

Fig. 4

Signaling pathways by which NAD+ regulates mitochondrial biogenesis and homeostasis in muscle. Arrows denote promotion and “T”s signify inhibition of mitochondrial biogenesis or homeostasis. Interventions in green boxes promote NAD+ levels and interventions in red boxes deplete NAD+ levels, thereby regulating mitochondria number and function. For explanation of the acronyms, please see the abbreviations list in the main body of the text

Fig. 5

Model of the pathological observations in aged muscle and their reversal by augmentation of nuclear and mitochondrial NAD+ levels. a Fewer mitochondria, fewer muscle stem cells, reduced levels of NAD+ in nuclei and mitochondria, and reduced nuclear SIRT-1 activity have been observed in aged muscle. b Repletion of NAD+ levels in nuclei and mitochondria via various interventions increases mitochondria, increases muscle stem cells, and increases nuclear and mitochondrial NAD+ levels thereby increasing SIRT-1 activity. One mechanism involves nuclear SIRT-1-mediated regulation of TFAM and PGC1alpha gene expression and their effect on mitochondrial metabolism

Fig. 6

Cellular and molecular mechanisms of amelioration of muscle disease due to NAD+ repletion. (A, B) Model of pathological changes observed in animal models of diseased muscle (A) and the improvement in these disease phenotypes due to provision of dietary NAD+ precursors or NAD+ (B). (C, D) Anterior left, dorsal top, side-mounted, 3 dpf embryos stained with phalloidin to visualize actin. Fiber detachment is readily observed in dag1 morphants (C, white arrowheads), whereas dag1 morphants supplemented with NAD+ display less fiber detachment (D). (E, F) Transmission electron micrographs showing normal muscle ECM (white arrows), disrupted muscle ECM (red arrows), normal sarcomere structure (white arrowheads), and disrupted sarcomere structure (red arrowheads) in a zebrafish model of muscular dystrophy with and without NAD+ supplementation. (E) Dag1-deficient zebrafish. (F) Dag1-deficient zebrafish supplemented with NAD+