Hypotheses for the question

Mechanism. Insufficient NAD+-dependent SIRT1 activity alters clock-protein deacetylation, shifting the phase of activation competence relative to inflammatory and growth-factor signals. Cells consequently miss a permissive activation window even when their average daily energy supply remains adequate.

Prediction. The regenerative benefit of a short NAD+ precursor pulse should depend on circadian phase under matched total exposure. Aligning the pulse with the predicted activation window should outperform continuous delivery, and this timing advantage should disappear after muscle stem-cell-specific disruption of the core clock.

Mechanism. Low available NAD+ constrains the NAD+-dependent phosphoglycerate dehydrogenase reaction. Reduced serine synthesis limits one-carbon transfer into purines and thymidylate, causing replication stress before a major decline in cellular ATP becomes apparent.

Prediction. Isotope tracing should reveal reduced glucose-derived serine and nucleotide synthesis in NAD+-depleted activated cells. Nucleoside supplementation should rescue replication and regenerative contribution despite persistently low NAD+, whereas an intervention that restores ATP without restoring nucleotide supply should not.

Mechanism. During activation, PARP1-dependent repair increases nuclear NAD+ demand. In aged cells with limited NAD+ availability, this transient demand prevents SIRT6-dependent removal of H3K9 acetylation at inflammatory enhancers, allowing inflammatory transcription to persist after DNA lesions have been repaired.

Prediction. Restoring nuclear NAD+ specifically during the initial repair period should normalize enhancer acetylation and improve lineage-traced regenerative contribution. This benefit should disappear after catalytic inactivation of SIRT6, despite comparable DNA repair, and delayed NAD+ restoration should be less effective.

Mechanism. Transitioning from quiescence to rapid proliferation requires a massive upregulation of RNA Polymerase I transcription across repetitive 47S rDNA arrays, which generates topological stress and cotranscriptional RNA-DNA hybrids (R-loops). In young MuSCs, NMNAT1-derived nucleolar NAD+ fuels SIRT7 to deacetylate and activate DDX21 to unwind rDNA R-loops; in aged MuSCs, nucleolar NAD+ scarcity leaves DDX21 hyperacetylated and inactive, causing catastrophic rDNA R-loop accumulation, nucleolar fragmentation, and release of free RPL11 to inhibit MDM2 and induce p53-mediated cell cycle arrest.

Prediction. DRIP-qPCR and immunofluorescence in aged MuSCs 48 hours post-injury will show dense R-loop accumulation specifically at 47S rDNA loci, hyperacetylated DDX21, and RPL11-MDM2 complex formation; targeting RNase H1 to the nucleolus or expressing a deacetylation-mimetic DDX21 mutant in aged MuSCs will prevent p53 activation and rescue myoblast proliferation.

Mechanism. NAMPT uses PRPP to convert nicotinamide to NMN. During the rapid cell-cycle burst, diversion of PRPP drops IMP, ATP, and GTP below the level needed for DNA replication even if NAD+ partly recovers.

Prediction. Mild NAMPT inhibition or PRPP supplementation increases EdU-positive myoblasts and regenerated fiber size in aged muscle while lowering NAD+ further. Purine nucleoside supplementation rescues clonal expansion with no rise in NAD+.

Mechanism. The proposed response to low NAD+ is increased nicotinamide turnover and NNMT-dependent methylation of nicotinamide. This consumes S-adenosylmethionine, lowers methylation capacity, and prevents re-establishment of repressive chromatin at differentiation genes when surviving cells attempt to return to quiescence.

Prediction. Lineage-traced cells should show increased nicotinamide methylation flux, a reduced S-adenosylmethionine/S-adenosylhomocysteine ratio, and persistent accessibility of differentiation genes. Transient NNMT inhibition during return to quiescence should improve second-injury regeneration even when NAD+ is experimentally held at the same low level; absent NNMT expression or flux would reject the mechanism.

Mechanism. Injury-associated inflammatory signalling upregulates NNMT in aged SCs, which consumes both NAM (reducing salvage flux) and SAM (reducing methyl donor availability for H3K4/H3K27 methyltransferases and for PRMT-dependent myogenic signalling). The dual drain couples NAD+ loss to an aberrant methylation state that precursors cannot correct.

Prediction. Old SCs show elevated Nnmt mRNA and MNAM; SC-specific Nnmt knockout or a selective NNMT inhibitor restores SC NAD+, SAM/SAH ratio, MyoD induction and fibre regeneration in old mice, and outperforms equimolar NR. Forced NNMT overexpression in young SCs reduces regeneration even with NR co-treatment.

Mechanism. During terminal myogenic differentiation, the Golgi-resident mono-ADP-ribosyltransferase PARP12 requires relatively high cytosolic NAD+ concentrations (due to its high Km for NAD+) to mono-ADP-ribosylate the vesicular tether Golgin-97, which sorts Myomaker (TMEM8C) into Rab11a+ exocytic carriers. Age-related NAD+ decline selectively abolishes PARP12 catalytic activity while sparing lower-Km enzymes, trapping Myomaker in the trans-Golgi network and arresting regeneration at the mononucleated myocyte stage.

Prediction. Differentiating aged myoblasts will exhibit normal Myomaker mRNA and total protein levels but perinuclear Golgi retention of Myomaker alongside loss of Golgin-97 mono-ADP-ribosylation; expressing an engineered low-Km PARP12 variant in aged MuSCs will restore surface Myomaker localization and myotube fusion in vivo.

Mechanism. A fall in nuclear NMNAT1 relative to mitochondrial NMNAT3 moves NAD+ synthesis out of the nucleus. Nuclear ACSS2 and LDH then cannot maintain NAD+ or acetyl-CoA at chromatin, so H3K27ac fails on differentiation enhancers even when mitochondrial respiration is intact.

Prediction. Nucleus-targeted NMNAT1 restores H3K27ac at Myog and myosin enhancers and restores new myofiber formation while mitochondrial NAD+ and oxygen consumption stay low. Mitochondria-targeted NMNAT3 normalizes respiration but does not restore enhancer acetylation or regeneration.

Mechanism. In young quiescent MuSCs, injury cues trigger a local burst of SIRT2 activity at the ciliary basal body that requires cytosolic NAD+ to deacetylate alpha-tubulin and the depolymerizing kinesin KIF2A, driving rapid axonemal disassembly before mitosis. In aged MuSCs, reduced cytosolic NAD+ keeps KIF2A hyperacetylated and catalytically inactive, causing persistent primary cilia during G1/S transition, aberrant partitioning of mother-daughter centrioles, and loss of asymmetric Par3/p38-alpha/beta segregation.

Prediction. Live imaging of injured myofibers from aged mice will reveal persistent Arl13b+ cilia and hyperacetylated KIF2A in dividing Pax7+ cells; expressing a deacetylation-mimetic KIF2A mutant in aged or NAMPT-inhibited MuSCs will restore cilium resorption and rescue the generation of Pax7+/MyoD- self-renewing daughter cells without restoring bulk NAD+ levels.

Mechanism. Nuclear NAD+ is a kinetically separate pool maintained by NMNAT1. Its decline leaves PARP1 unable to complete ADP-ribosylation-dependent eviction of repressive complexes and SIRT6 unable to deacetylate H3K9/H3K56 at myogenic enhancers, so the enhancer landscape stays closed even when total cellular NAD+ is partially restored.

Prediction. Compartment-targeted NAD+ biosensors (nuclear vs mitochondrial PercevalHR/NAD-Snifit) show a selective nuclear drop in old SCs; nucleus-restricted NMNAT1 overexpression rescues MyoD induction and regeneration in old mice, while mitochondria-targeted NMNAT3 overexpression raises mitochondrial NAD+ and respiration without restoring regeneration.

Mechanism. Myofiber necrosis releases abundant heme and labile iron into the regenerative niche, creating intense lipid peroxidation pressure. Young activated MuSCs survive this microenvironment because cytosolic NAD+ fuels NAD kinase (NADK) and provides NADH/NADPH directly to plasma-membrane-localized FSP1 to reduce ubiquinone to radical-trapping ubiquinol; in aged MuSCs, NAD+ depletion exhausts the FSP1-CoQ10H2 antioxidant axis at the sarcolemma, triggering ferroptotic death before the first mitotic division.

Prediction. Aged MuSCs isolated 36 hours after cardiotoxin injury will exhibit high C11-BODIPY lipid peroxidation, an elevated oxidized-to-reduced CoQ10 ratio, and early cell death that is selectively prevented in vivo by liproxstatin-1 or MuSC-specific NADK overexpression, but not by caspase or RIPK1 inhibitors.

Mechanism. NADK phosphorylates NAD+ to NADP+, and pentose-phosphate flux then regenerates NADPH for GPX4. When NADPH falls, lipid peroxides accumulate selectively in progeny that have entered the cell cycle.

Prediction. Lipid peroxidation in aged muscle-stem-cell progeny precedes p16 induction and caspase activation. GPX4 overexpression or a lipophilic radical-trapping antioxidant restores regenerated fiber number despite persistently low NAD+ and low mitochondrial respiration, whereas senolytics, p16 deletion, and mitochondria-targeted antioxidants do not.

Mechanism. SIRT1 must deacetylate ACSS2 before ACSS2 can generate nuclear acetyl-CoA from acetate. Persistently acetylated ACSS2 leaves injury-responsive loci without H3K27ac despite abundant acetate.

Prediction. An acetylation-resistant, constitutively active ACSS2 mutant restores myogenic histone acetylation and regeneration in low-NAD+ aged muscle stem cells without raising NAD+. Acetate supplementation rescues young cells but not aged cells, and overexpression of wild-type ACSS2 does not rescue aged cells.

Mechanism. NNMT methylates nicotinamide to N1-methylnicotinamide, competing with NAMPT, and spends SAM required by SETD2. The resulting loss of H3K36me3 increases cryptic transcription through differentiation genes.

Prediction. Muscle-stem-cell deletion of NNMT restores H3K36me3 and regeneration even when NAD+ recovery is incomplete. Nicotinamide riboside restores NAD+ but not SAM, H3K36me3, or regeneration while NNMT remains high, whereas SAM supplementation restores H3K36me3 and regeneration only together with a modest NAD+ rescue.

Mechanism. To exit quiescence, MuSCs require a rapid surge in autolysosomal degradation to remodel organelles and generate bioenergetic substrates. In young MuSCs, cytosolic NAD+ supports SIRT2-mediated deacetylation of ATP6V1E1 at Lys99, promoting reversible docking of the cytosolic V1 sector onto the membrane V0 sector to acidify lysosomes; in aged MuSCs, hyperacetylated ATP6V1E1 fails to assemble into functional V-ATPase complexes, leading to alkalinized, swollen autolysosomes that leak cathepsin B into the cytosol to degrade Pax7.

Prediction. Proximity ligation assays and ratiometric pH imaging in aged MuSCs 12 hours post-injury will reveal dissociated V1-V0 sectors, elevated lysosomal pH, and cytoplasmic cathepsin B activity; expressing a deacetylation-mimetic ATP6V1E1-K99R mutant in aged MuSCs will restore lysosomal acidification and preserve Pax7 protein levels independently of NAD+ repletion.

Mechanism. SARM1 is allosterically activated by NMN when NAD+ is low. Aging reduces the NAD+ buffer, so an injury-induced NMN pulse that young cells tolerate crosses the SARM1 activation threshold and causes feed-forward NAD+ loss.

Prediction. Muscle-stem-cell deletion of SARM1 preserves NAD+ for the first 48 hours after injury and restores regeneration in aged mice, with little effect in young mice. An NMN-binding-deficient SARM1 mutant phenocopies the deletion, and lowering NAD+ in young cells aborts division only when SARM1 can still bind NMN.

Mechanism. In young MuSCs, high nuclear NAD+ serves as a non-canonical initiating nucleotide for RNA Polymerase II at poised myogenic loci such as Myod1 and Ccnd1, marking these leaky transcripts with a 5'-NAD+ cap that targets them for immediate nuclear degradation by the deNADding exonuclease DXO. Age-related nuclear NAD+ depletion shifts transcription initiation exclusively toward canonical m7G capping, stabilizing leaky Myod1 transcripts and driving premature niche exit prior to injury.

Prediction. NAD-cap sequencing (ONE-seq) of freshly isolated quiescent MuSCs will demonstrate a high ratio of 5'-NAD+ to m7G caps on Myod1 transcripts in young mice that is lost in aged mice, and conditional deletion of Dxo in young Pax7+ MuSCs will phenocopy age-associated loss of quiescence and regenerative failure.

Mechanism. Reduced NAD+-dependent SIRT2 activity changes microtubule acetylation and the routing of recycling endosomes. This reduces polarized delivery of integrin alpha7-beta1 to the cell surface, weakening directed movement along the basal lamina and subsequent niche reoccupation.

Prediction. Restoring polarized integrin recycling should improve niche reoccupation and the response to a second injury while NAD+ remains low. Rescue should track integrin trafficking and migration directionality more closely than proliferation or mitochondrial respiration, and should be lost when alpha7-beta1 engagement is blocked.

Mechanism. Where NAD+ loss also lowers the cytosolic NAD+/NADH ratio, increased NADH-dependent recruitment of the transcriptional corepressor CtBP prolongs repression of DLL1. This changes the timing of ligand presentation between adjacent progenitors and disrupts the balance between differentiation and stem-cell replenishment.

Prediction. Restoring the cytosolic NAD+/NADH ratio should recover DLL1 dynamics and coordinated fate decisions, with little additional benefit after disrupting CtBP recruitment at the DLL1 regulatory region. Rescue should depend on cell contact and should be weaker when Notch stimulation is supplied continuously rather than with physiological timing.

Mechanism. Insufficient NAD+ limits SIRT7-dependent repression of ribosomal DNA transcription during cell-cycle re-entry. Excess transcription at replicating ribosomal DNA creates transcription–replication conflicts, generating nucleolar damage that reduces the number of viable regenerative progenitors.

Prediction. A brief, partial reduction of RNA polymerase I activity during the first S phase should reduce ribosomal DNA damage and restore clonal expansion without restoring NAD+. The same intervention after the first division should provide substantially less benefit, distinguishing this mechanism from a continuous energetic deficit.

Mechanism. NAD+ loss destabilises the mitochondrial transcription/replication balance and opens mPTP/VDAC pores, releasing mtDNA to the cytosol. cGAS–STING activation in SCs induces an IL-6/TGF-β-skewed secretome that drives FAP fibrogenic differentiation, converting a cell-intrinsic metabolic deficit into niche-level fibrosis.

Prediction. Cytosolic mtDNA and STING phosphorylation are elevated in old SCs and reversed by NAD+ precursors; co-transplanting NAD+-depleted (Nampt-deleted) young SCs with wild-type young SCs impairs regeneration of the wild-type cells, and this transmissible defect is abolished by SC-specific Sting1 deletion or by TFAM-stabilising/mtDNA-release blockade without restoring NAD+.

Mechanism. Reduced mitochondrial SIRT5 activity increases succinylation of enzymes controlling succinate turnover, causing activated myogenic progenitors to accumulate and export succinate. Extracellular succinate then activates SUCNR1-dependent profibrotic signaling in responsive fibro-adipogenic progenitors.

Prediction. Conditioned medium from NAD+-depleted myogenic progenitors should promote extracellular matrix production through a succinate-dependent effect. Fibro-adipogenic progenitor-specific SUCNR1 deletion should reduce this fibrosis and improve repair despite persistently low NAD+ in muscle stem cells; absence of functional SUCNR1 in the responding population would reject the proposed pathway.

Mechanism. NAMPT is carried on polarized recycling endosomes that follow the older centrosome into the basal daughter. Age-related centrosome and endosome mis-segregation equalizes NAD+ biosynthetic capacity.

Prediction. An NAD+ biosensor reports sister-cell NAD+ asymmetry after mitosis in young but not aged muscle stem cells. Forcing symmetric NAMPT inheritance in young cells reproduces the aged failure of both reserve-cell formation and differentiation, whereas tethering NAMPT to one daughter restores a Pax7-high reserve from aged cells without changing the pre-mitotic NAD+ pool.

Mechanism. Reduced mitochondrial NAD+ availability limits NADK2-dependent NADP+ production during the increase in mitochondrial activity after injury. Insufficient mitochondrial NADPH regeneration then compromises peroxide detoxification, allowing lipid oxidation to eliminate otherwise competent progenitors.

Prediction. Activated aged cells should show reduced NADP(H) synthesis from mitochondrial NAD+ before lipid peroxide accumulation and cell loss. Temporally restricted ferroptosis inhibition should preserve their regenerative contribution without restoring NAD+, whereas NAD+ replenishment should fail to normalize mitochondrial NADP(H) when NADK2 is disabled.

Mechanism. Extracellular NMN is the main precursor SCs take up after injury (via SLC12A8/dephosphorylation to NR). Aged inflammatory myeloid CD38 and endothelial CD38 consume this interstitial pool, creating a local gradient depletion at the sublaminar niche even when systemic precursors are abundant.

Prediction. Myeloid- or endothelium-specific Cd38 deletion in old mice restores SC NAD+ and fibre regeneration after injury, while SC-specific Cd38 deletion does not; interstitial microdialysis shows restored post-injury NMN/NAD+ in the knockouts. Transplanting old SCs into young muscle rescues them better than young SCs into old muscle treated with NR.

Mechanism. Aged SCs retain NAMPT-derived NMN but lose NMNAT1/2 capacity, so the NMN:NAD+ ratio rises. NMN binding to the SARM1 allosteric site activates its TIR NADase, which hydrolyses NAD+ faster than it is resynthesised, collapsing the pool specifically during the energetically demanding quiescence-exit window and generating cADPR-like signalling products.

Prediction. Sarm1-/- or SC-specific Sarm1 deletion in old mice restores SC NAD+, first-division kinetics and regenerated fibre CSA after cardiotoxin injury, whereas NMN supplementation in old wild-type mice worsens or fails to improve SC NAD+ while NR (which bypasses NMN accumulation less) or NMNAT1 overexpression rescues. Isolated old SCs should show NAD+ loss blocked by a SARM1 TIR inhibitor but not by NAMPT inhibition.

Mechanism. Fusion requires a transient mitochondrial-OXPHOS-dependent and SIRT1-gated step controlling Myomaker/Myomerger surface delivery and PS exposure. NAD+-limited SIRT1 leaves these effectors hyperacetylated and mislocalised, reducing fusion index without changing MyoG+ fraction.

Prediction. In old regenerating muscle, MyoG+ cell number is near-normal while myonuclei per fibre and fusion index are reduced; NAD+ precursor treatment restores fusion index and myonuclear number without changing proliferative output, and Myomerger overexpression rescues old fusion independently of NAD+ status.

Mechanism. NAADP opens TPC2 channels on lysosomes, and the calcium transient recruits mTORC1 to the lysosomal surface. Loss of CD38 base-exchange activity leaves growth-factor pathways intact but disconnects them from quiescence exit.

Prediction. A TPC2 agonist or cell-permeable NAADP restores lysosomal mTORC1 and EdU incorporation after injury while NAD+ remains low. A CD38 mutant that keeps base-exchange activity but lacks NADase activity rescues activation, whereas a hydrolysis-only CD38 mutant does not.

Mechanism. NAD+ caps on selected mRNAs protect them from decapping. Below a threshold NAD+ concentration, this reservoir is lost late in repair, after differentiation has already begun, so the next quiescent pool fails specifically at the re-entry step.

Prediction. NAD+-capped Pax7 and Spry1 mRNAs fall in aged muscle stem cells only during the return-to-quiescence window. Decapping-resistant versions of these transcripts restore the Pax7-positive reserve without increasing NAD+. Raising NAD+ only on days 5–10 after injury preserves both the caps and the reserve, whereas the same increase limited to the first two days does not.

Mechanism. Upon injury, mitochondrial NAD+ imported via SLC25A51 activates SIRT5, which desuccinylates and restrains the E1 subunit of OGDH, maintaining a high alpha-ketoglutarate/succinate ratio required by the Jumonji-domain demethylase KDM6A to strip repressive H3K27me3 marks from the Myog and Mef2c promoters. In aged MuSCs, mitochondrial NAD+ scarcity causes OGDH hypersuccinylation and hyperactivation, converting alpha-ketoglutarate into succinate and epigenetically blocking the proliferation-to-differentiation transition.

Prediction. Activated aged MuSCs will display elevated lysine succinylation on OGDH, a reduced alpha-ketoglutarate/succinate ratio, and persistent H3K27me3 enrichment at the Myog locus; treatment with cell-permeable dimethyl-alpha-ketoglutarate or expression of a succinylation-resistant OGDH mutant will rescue Myog transcription and myofiber maturation in NAD+-depleted MuSCs.

Mechanism. Regenerating MuSCs must squeeze through dense, fibrotic interstitial constrictions along ghost fibers to repopulate injury sites, requiring dynamic nuclear deformability. In young MuSCs, nuclear NAD+ sustains SIRT6 deacetylase activity toward the Ig-fold domain of Lamin A/C, maintaining a viscoelastic nuclear lamina; in aged MuSCs, loss of nuclear NAD+ causes Lamin A/C hyperacetylation and rigidification, resulting in mechanical fracture of the nuclear envelope during confined migration, exposure of genomic DNA to cytosolic cGAS, and type I interferon-driven migratory arrest.

Prediction. Intravital imaging and microfluidic constriction assays of aged MuSCs carrying an NLS-GFP reporter will show frequent nuclear blebbing, cytoplasmic GFP leakage, and perinuclear cGAS foci during migration; MuSC-specific cGAS deletion or expression of a deacetylation-mimetic Lamin A/C variant will restore migration velocity and regeneration in aged muscle.

Mechanism. Quiescence exit requires an ecto-NADase-generated cADPR/ADPR burst acting on RyR and TRPM2 to produce a stereotyped Ca2+ transient that drives p38-MK2 and nuclear MyoD. Even though CD38 protein rises with age, the collapsed local NAD+ substrate pool lowers cADPR output, reducing Ca2+ transient amplitude below the activation threshold in a subset of SCs.

Prediction. GCaMP imaging of SCs in situ shows reduced amplitude and delayed onset of the post-injury Ca2+ transient in old muscle, rescued within minutes by local NAD+ (but not NMN) application or by 8-Br-cADPR-insensitive RyR agonism; TRPM2 or RyR1 agonism rescues old SC activation without raising NAD+, and the fraction of SCs ever entering S phase (not their cycle speed) is what changes.

Mechanism. SIRT2 requires NAD+ to deacetylate α-tubulin at lysine 40. Hyperacetylated microtubules lose anchoring to the basal lamina and uncouple the spindle from the Notch-high niche.

Prediction. Aged muscle stem cells show high α-tubulin K40 acetylation and loss of apicobasal divisions. An acetylation-resistant K40R α-tubulin mutant restores Pax7-positive self-renewal in low-NAD+ aged cells without raising NAD+, whereas a K40 acetylation-mimetic abolishes self-renewal in young cells with normal NAD+.

Mechanism. The proposed mechanism is that MYMK transcripts include a functional NAD-capped subpopulation whose abundance depends on nuclear NAD+ availability. Loss of this cap reduces transcript persistence during differentiation, lowering myomaker protein and limiting fusion independently of progenitor production.

Prediction. Orthogonal cap-sensitive assays should first identify an authentic NAD-capped MYMK population and show its selective depletion in aged cells. A stable, conventionally capped MYMK transcript should rescue fusion without restoring NAD+ or proliferation; failure to detect the proposed cap would reject this mechanism.

Mechanism. PHGDH catalyzes the rate-limiting step of de novo serine synthesis and possesses a high Km for its obligatory cofactor NAD+, making it acutely sensitive to age-related NAD+ decline. In young proliferating MuSCs, robust NAD+-driven PHGDH flux supplies serine to synthesize phosphatidylserine-enriched exosomes that signal via MerTK on Ly6C-high macrophages to induce their conversion into pro-myogenic Ly6C-low macrophages; in aged MuSCs, PHGDH stalling diverts glycolytic intermediates into methylglyoxal while producing phosphatidylserine-deficient vesicles that fail to resolve niche inflammation.

Prediction. 13C6-glucose tracing in aged activated MuSCs will demonstrate a metabolic block at PHGDH with depleted 13C3-serine and reduced Annexin-V binding on MuSC-derived exosomes; dietary serine/phosphatidylserine liposome delivery or MuSC-specific expression of a low-Km PHGDH variant will restore the Ly6C-high-to-low macrophage switch and muscle repair in aged mice.

Mechanism. Reduced NAD+ limits LDH-A reoxidation flux balance, increasing intracellular lactate and lactyl-CoA. p300-dependent H3K18 lactylation accumulates at promoters normally governed by NAD+-dependent sirtuin deacetylation, producing an acylation mark that sirtuins cannot efficiently remove, so the epigenome drifts independently of the acute NAD+ level.

Prediction. Old SCs show elevated H3K18la by CUT&RUN enriched at myogenic/quiescence promoters; p300 inhibition or LDH-A knockdown in old SCs restores regeneration and MyoD/Myogenin kinetics, and NAD+ precursor treatment improves regeneration only when lactylation is also lowered. Forcing lactylation in young SCs (exogenous lactate + p300 activation) phenocopies the aged regenerative deficit without lowering NAD+.

Mechanism. SIRT2 deacetylates tubulin and spindle-associated/polarity components required to position NUMB and to apportion mitochondria asymmetrically. Low NAD+ reduces SIRT2 activity, hyperacetylating these substrates, so planar/symmetric committed divisions replace apical-basal asymmetric divisions.

Prediction. Live imaging of single old SCs on myofibres shows a shift toward symmetric Pax7+/MyoD+ pairs and symmetric mitochondrial inheritance (MitoTimer); acute NAD+ restoration or SIRT2 activation normalises the asymmetric fraction within one division, and SC-specific Sirt2 deletion in young mice reproduces the symmetric-commitment phenotype with normal proliferation rate.

Mechanism. Quiescent MuSCs poise for rapid activation by storing translated-repressed Myf5 mRNA within liquid-like cytoplasmic RNP granules scaffolded by DDX6. Injury-induced signaling normally activates cytoplasmic TNKS1, which consumes NAD+ to PARylate the intrinsically disordered region of DDX6, introducing electrostatic repulsion that dissolves the condensate and releases Myf5 mRNA to ribosomes; in aged MuSCs, NAD+ scarcity prevents DDX6 PARylation, causing RNP granules to mature into non-dynamic hydrogels.

Prediction. Fluorescence recovery after photobleaching (FRAP) and single-molecule FISH in freshly injured aged MuSCs will show immobile DDX6 puncta retaining Myf5 mRNA and lacking poly(ADP-ribose) chains, while acute pharmacological inhibition of TNKS1 with XAV939 in young MuSCs will block Myf5 protein translation without altering Myf5 transcript abundance.

Mechanism. Return to quiescence requires a glycolysis→FAO switch enforced by SIRT3-dependent deacetylation of LCAD/CPT1b and SIRT1-dependent repression of MyoD. With low NAD+, aged SC progeny remain glycolytic and differentiate or die instead of reserving, so the stem pool is consumed by each injury.

Prediction. Pulsed NAD+ precursor delivery restricted to days 5–10 post-injury restores old SC self-renewal (Pax7+ pool recovery, second-injury regenerative capacity) at least as well as continuous dosing, whereas dosing restricted to days 0–2 improves early proliferation but not pool restoration. SC-specific Sirt3 deletion in young mice should phenocopy the aged failure-to-reserve without impairing initial activation.