Dystrophin deficiency alters the muscle NAD metabolome and energy producing pathways
To assess the biochemical adaptations of muscle to dystrophin deficiency, we analyzed the gastrocnemius muscles of MDX mice before and after eccentric challenge using an untargeted metabolic profiling platform. The platform identified 762 chemical entities, of which 552 were annotated as either polar metabolites or lipids. Principal component analysis of the metabolic profiles demonstrated a clear clustering of samples by time and genotype (Fig. 1A). Furthermore, the MDX samples showed a well-defined chronological progression following damage. The clustering of samples observed 2 hours post-damage showed distinct separation from the other time points, suggesting a period of acute crisis in the muscles. This was the only time at which the majority of significantly altered metabolites appeared depleted when compared to unchallenged controls (Fig. 1B). The pattern of the post-injury sample groups from days 2–7 reflected a transition from the acute response to a repair and recovery stage. By post-injury day 14, the samples had a chemical phenotype closely resembling that of the uninjured MDX muscles (Fig. 1A-B).
Comparing the metabolic profiles of MDX mice to wildtype mice at baseline, we observed a strikingly lower abundance of NAD than almost any other metabolite. A similar pattern emerged in the NAD precursors, nicotinamide (Nam) and nicotinamide riboside (NR), while the methylated derivative of Nam, 1-me-Nam, appeared to be elevated (Fig. 1C). Primary NAD deficiency in mouse muscle and cultured myotubes has been shown to restrict glycolytic flux at the level of GAPDH, resulting in a characteristic buildup of intermediates in the pentose phosphate pathway (8, 18). Consistent with this model, we observed a significant increase in ion counts for dihydroxyacetone phosphate (DHAP), sedoheptulose-7-phosphate (S7P), ribose-5-phosphate, ribulose-5-phosphate, and two isomeric sugar phosphates of the proximal glycolytic pathway, glucose-6-phosphate (G6P) and fructose-6-phosphate (F6P) (Fig. 1C). Also predicted by inhibition of the NAD salvage pathway in myotubes (18) were the depletion of aKG and the appearance of elevated aspartate. In aggregate, this pattern indicates a metabolic shift in MDX muscle at steady state, partially stemming from the loss of the metabolic co-factor, NAD.
The robust response of the global metabolome at 2 hours post-eccentric challenge led us to investigate NAD-related metabolites at this and subsequent time points. As far as the platform could resolve, NAD itself did not appear to respond to the challenge (Fig. 1D). However, Nam was acutely depleted by more than one third after two hours, presumably restricting any residual activity of the NAD salvage pathway, and gradually returned to baseline after four days. Nam homeostasis was further altered by a doubling in the levels of 1-me-Nam in the two days following injury, which only normalized after 14 days. Evidence of a further constriction in glycolysis also emerged post-injury: levels of DHAP, immediately upstream of GAPDH, acutely increased in an opposing pattern to that of lactate, a glycolytic end-product (Fig. 1E). The most striking indicator of the repair phase was the appearance of polyamines, including spermine, spermidine, putrescine, and N-acetylputrescine, which were elevated in the days following injury (Fig. 1C,F). This class of biomolecules serves as a general marker of cellular proliferation and is required for both myocyte differentiation and alternative macrophage activation (19, 20). Consistently, in the case of NAD-related metabolites, glycolytic intermediates, and polyamines, eccentric injury amplified the disparities between MDX and WT muscle.
A novel synthetic CD38 antagonist increases NAD in multiple tissues
Our group previously reported a series of novel chemical entities which potently inhibit CD38, a constitutive NAD-degrading enzyme. Related screening efforts yielded the quinolone dubbed GSK978A, which exhibited 10-fold higher potency against mouse recombinant CD38 than human enzyme (Fig. 2A). This potency is orders of magnitude greater than that of natural products, such as quercetin, and approximates that of “78c”, the best-studied synthetic CD38 inhibitor to-date (11, 14)(Fig. 2A). Yet GSK978A outperformed 78c in a chromosomal stability test of genotoxicity, indicating improved suitability for long-term administration (data not shown). GSK978A was also more soluble and predicted to have low intrinsic clearance in several small animal preclinical species, including mice and rats, but not larger cynomolgus monkeys (Fig. 2B). To assess the suitability of GSK978A for in vivo studies, a pharmacokinetic analysis was performed in blood sampled over 24 hours following doses from 1–30 mg/kg. Doses above 3 mg/kg consistently and durably elevated blood NAD approximately 5-fold within 15 minutes (Fig. 2C). At these doses, the compound was identified within liver, gastrocnemius, adipose, and brain tissues at exposures that well exceeded the IC50 (Fig. 2D). Accordingly, the NAD content of these tissues was found to be elevated by at least 30% after 24 hours (Fig. 2E).
To assess the pharmacodynamics of GSK978A in an eccentric challenge model, an acute study was performed in MDX mice following 5 days of dosing at 3 mg/kg. Dietary nicotinamide riboside, which has been suggested to improve the performance of MDX muscle (10), was included as a comparator. Within 24 hours of eccentric challenge, tetanic strength was lessened by > 50% in all treatment groups, despite preservation of mass in the largest affected gastrocnemius muscles (Fig. 3A-C). Interestingly, the challenged muscles also showed NAD depletion compared to the contralateral side, indicating that the muscle NAD pool does acutely respond to lengthening contractions (Fig. 3D). Mice treated with GSK978A, but not NR, showed a trend toward protection from this effect (Fig. 3E). As a biomarker of muscle repair, total muscle polyamines showed clear elevation in the injured limbs with a trend toward protection by GSK978A, especially when polyamines were normalized to NAD content (Fig. 3F-H). These results indicated that a longer treatment regimen might provide functional improvements to MDX mice via an NAD-sparing mechanism.
Chronic NAD repletion does not provide functional protection from repetitive eccentric challenges
We next designed a long-term study with chronic administration of GSK978A or NR to longitudinally assess the physiology of MDX mice during three stages distinct stages: growth, recovery from eccentric challenge, and recovery from repeated challenge (Fig. 4A). We reasoned that this design would model the efficacy requirements of boys diagnosed with DMD. Beginning at 7–9 weeks of age, during the necrotic bloom phase of muscle pathology, MDX mice were randomized by hindlimb contractility and body weight. Over the course of 20 weeks, hindlimb weakness and increased CK release persisted in the MDX mice compared to WT controls (Fig. 4B-C). The pattern of hindlimb strength and serum creatine kinase (CK) release generally trended downward as MDX animals reached maturity, but remained unchanged in both compound-treated groups, compared to the vehicle-treated controls. MDX mice also accumulated lean mass steadily over the course of the study, reflecting characteristic hypertrophy, in a manner that was treatment-independent (Fig. 4D). At the study conclusion, gastrocnemius muscles from the challenged MDX limbs were found to be ~ 15% less massive than the contralateral side in all treatment groups, reflecting an inability to fully regenerate injured fibers that was not observed in the WT. Surprisingly, contralateral muscles tended to be largest in mice treated with GSK978A (Fig. 4E-F), which may be a consequence of altered gait mechanics to favor the contralateral side, as it was not reflected in total lean mass.
Hindlimb strength was also serially assessed following eccentric challenges beginning at week 10 of treatment, to determine whether treated groups were protected from injury or recovered faster. Compared to WT controls, MDX mice showed approximately double the functional deficit within one day of eccentric challenge, despite similarly shaped tetani, but neither parameter was affected by GSK978A or NR (Fig. 5A-B). Furthermore, MDX hindlimbs showed highly similar recovery kinetics between the first and second challenges, while WT controls rebounded faster after the second bout (Fig. 5C-D). The absence of a protective repeated-bout effect in MDX hindlimbs may reflect that fact that adult dystrophin-deficient muscles are preconditioned to such cycles of damage and repair by activities of normal living.
CD38 inhibition significantly reverts the MDX muscle metabolome to the WT state
Given the lack of physiological protection conferred upon MDX muscle by NAD-modulating compounds, we suspected that the treatments were simply ineffective at correcting the basal metabolic imbalance that formed the basis for our rationale. To address this question, we analyzed the uninjured gastrocnemius muscles from the chronically treated mice using a second untargeted metabolomics platform providing a broader metabolome coverage, which across the treatment groups detected 1,738 ions putatively annotated as up to 3,415 polar metabolites, based on accurate mass. When comparing the metabolites significantly altered between vehicle-treated MDX and WT groups, a reversing pattern emerged in the GSK978A-treated muscles that was not observed in the NR-treated MDX animals (Fig. 6A). This anti-correlation was confirmed with robust significance (R = -0.56, p < 1E^-100) only in the GSK978A treatment group (Fig. 6B-C). Suspecting that such a dramatic reversion effect may have resulted from restoration of a ubiquitous co-factor, like NAD, we interrogated specific NAD-related metabolites and found a robust pattern of restoration in MDX muscle exposed to GSK978A, but few detectable changes in muscles exposed to NR (Fig. 6D-E). The only significantly increased metabolites containing or derived from nicotinamide following NR treatment were the waste products N-methyl-2/4-pyridone-5-carboxamide (2PY or 4PY), which appeared at the same molecular weight and were recently classified as uremic toxins (21). Interestingly, NAADH, the reduced form of a suspected biomarker of NAD repletion (22) or overload, was detected only in GSK978A-treated muscles, while the NAD salvage pathway substrate, phosphoribosyl pyrophosphate (PRPP), showed signs of compensatory depletion. As predicted following NAD restoration, ion counts for components of the proximal glycolytic pathway, including those for glucose-6-phosphate and fructose-1,6,-bisphosphate, were normalized by GSK978A, as were the GAPDH substrate, glyceraldehyde-3-phosphate, and pentose phosphate intermediate, ribose-5-phosphate (Fig. 6F). Collectively, these global metabolomic profiles highlight a restorative effect of CD38 inhibition on multiple metabolic pathways within dystrophin-deficient muscle that is not recapitulated by supplementing an NAD precursor.