Boosting NAD+ pathways to counteract Doxorubicin-induced cardiotoxicity

Cancer and cardiovascular diseases are among the main causes of death worldwide. Very powerful drugs have been developed in order to cure cancers, and among them we find anthracyclines, which include Doxorubicin (DOX). However, this chemotherapeutic has important side effects, which limit its usage. DOX’s most prevalent side effect is cardiotoxicity; thus, many efforts have been made to find drugs and therapeutic approaches to counteract it. NAD+ is a fundamental coenzyme for the cell, both as a redox co-substrate and as a substrate for enzymes regulating different cellular processes, like post-translational modifications, epigenetic regulation, and so on. NAD+ levels are often decreased in pathologies and during aging, making their restoration a potential strategy to reverse these phenomena. This could be done either by decreasing its consumption, limiting the activity of the aforementioned enzymes, or by boosting its synthesis, by feeding precursors or analogues. LGD SARL is interested in NAD+ boosting approaches to counteract different pathologies, including DOX-induced cardiotoxicity. They have already published that a physiological NAD+ precursor, Nicotinamide Mononucleotide (NMN), is able to counteract Doxorubicin-induced mortality, body weight loss, and cardiotoxicity in mice. Recently, they have become interested in another NAD+-related molecule, whose name and structure cannot be disclosed (called hereafter undisclosed compound, U.C.). Preliminary data in the DOX-induced cardiotoxicity mouse model show that this molecule exerted beneficial biological effects, comparable to those of NMN (which has consequently been used as a positive control). This project aimed to characterize the mechanism of action of U.C., using in vivo and in vitro models and by finding potential U.C. interactors. The first step was characterizing the mechanism underlying the cardioprotection observed in DOX-induced cardiotoxicity in vivo model. Therefore, we measured the levels of NAD+ and its related metabolites in blood, heart and kidneys collected from U.C.- or vehicle-treated animals exposed to DOX. While NMN co-treatment with DOX was able to increase the levels of NAD+ and of its downstream products, N1-Methyl-2-pyridone-5-carboxamide (2PY) and 1-MethylNicotinamide (1-MeNam), reversing the effects of DOX on these metabolites, U.C. co-treatment did not lead to such effects, suggesting a mechanism of action distinct from that of NMN. However, U.C. caused an increase in the levels of ATP and non-significant tendency toward restoring cyclic ADPR (cADPR) levels, suggesting a possible involvement of calcium signaling, which was not observed in NMN-treated animals. In the heart, kidneys and blood of animals treated with U.C. and DOX, a side product of U.C. (called U.C.+1) was formed, which could support U.C.’s mechanism of action. The second aim was to mimic the effects observed in vivo in a cellular system, where we could study a higher number of parameters. We were able to recapitulate these effects in a primary murine cardiomyocyte cell line (mNVCM) treated with DOX, where we found that U.C. reversed the increase in Malondialdehyde (MDA), an oxidative stress biomarker. This suggests that U.C. is not just involved in NAD+ pathways, but also in protection from oxidative stress. Treatment of these cells with DOX and U.C.+1, instead of U.C. itself, showed similar effects, suggesting that it could indeed mediate U.C.’s observed effects. This was further supported in a primary cardiac fibroblast cell line, that massively produced the downstream product rapidly after U.C.’s administration, rather than raising levels of any of the NAD+-pathway classical metabolites. Lastly, we focused on potential U.C.’s interactors, to determine which pathways could be activated after its administration. U.C. is not a substrate or modulator of several NAD+-related enzymes (NMNAT, CD38, SARM-1), as determined on the recombinant form of these enzymes (for NMNAT and SARM-1) or on a cell line (HUVECs) overexpressing CD38. To deepen our understanding of U.C.’s mechanism of action, we administered it to DOX- and H2O2-treated murine myoblasts (C2C12 cell line), which is a non-cardiac muscle cell model. U.C. was not effective in counteracting DOX effects on these cells but successfully prevented H2O2 induced mortality. Thus, these results confirm the involvement of U.C. in protection against oxidative stress, consistently with the decreased levels of MDA measured in mNVCM upon U.C. treatment. Altogether, U.C. appears to modulate cellular energetic and oxidative stress responses independently of NAD⁺ biosynthesis, potentially involving calcium-related signaling pathways, although the molecular mediator remains unidentified. These findings could provide fundamental insights into the prevention of DOX-induced cardiotoxicity.