FOXO1-NMNAT3 Axis Drives Chemo Heart Damage — NAD+ May Be the Fix

Doxorubicin (DOX) remains one of oncology's most effective chemotherapeutic agents, but its dose-dependent cardiotoxicity is a major clinical barrier. The heart is uniquely vulnerable because it cannot synthesize NAD+ from scratch and relies almost entirely on salvage pathways. This study investigated whether NAD+ metabolic dysregulation is a central mechanism in DOX-induced cardiotoxicity (DIC) and whether restoring NAD+ could be a viable cardioprotective strategy.

Using human cardiomyocytes (AC16), mouse atrial myocytes (HL-1), and C57BL/6 mice, the researchers established robust DIC models. In vivo, mice received a single 20 mg/kg DOX injection, a dose calibrated to approximate a clinically relevant 60 mg/m² human dose. Key cardiac outcomes measured included ejection fraction, fractional shortening, histopathology (H&E and WGA staining), serum biomarkers (cTnI, BNP, CK-MB), ROS levels, mitochondrial membrane potential, and NAD+/NADH ratios. Mechanistic studies employed siRNA knockdown, pharmacological inhibition, overexpression constructs, dual-luciferase reporter assays, and ChIP-qPCR.

DOX exposure caused significant NAD+ depletion alongside elevated oxidative stress markers in both cell lines and mouse hearts. Exogenous NAD+ supplementation (200 mg/kg i.p.) and NMN administration (500 mg/kg i.p.) given before DOX injection substantially preserved cardiac function, reduced cardiomyocyte death, and attenuated redox imbalance. Echocardiography confirmed improved ejection fraction and fractional shortening in treated mice. In contrast, pharmacological inhibition or genetic silencing of CD38—an NAD+-consuming ectoenzyme often implicated in NAD+ depletion—failed to restore NAD+ levels in this context, suggesting CD38 is not the primary driver here.

The mechanistic core of the study centers on NMNAT3, the mitochondrial isoform of the nicotinamide mononucleotide adenylyltransferase family, which catalyzes the final step of mitochondrial NAD+ synthesis. NMNAT3 expression was significantly downregulated by DOX. Overexpression of NMNAT3 rescued mitochondrial NAD+ and reduced oxidative damage. Using computational transcription factor binding analysis, dual-luciferase reporter assays, and ChIP-qPCR, the team identified FOXO1 as a direct transcriptional repressor of NMNAT3. DOX activates FOXO1, which then binds the NMNAT3 promoter to suppress its expression—creating a cascade from oxidative stress to NAD+ depletion to further oxidative injury. Inhibiting FOXO1 with AS1842856 de-repressed NMNAT3 and partially rescued NAD+ levels.

This work establishes the FOXO1→NMNAT3 suppression→NAD+ depletion→oxidative stress axis as a previously unrecognized driver of DIC. Importantly, NMN, which is orally bioavailable and already commercially available as a supplement, demonstrated cardioprotective efficacy in this model, suggesting a potentially translatable intervention. The findings reframe NAD+ replenishment not merely as a general metabolic support strategy but as a mechanism-targeted redox therapy for chemotherapy-related cardiac injury.