Therapeutic Potential of Emerging NAD+-Increasing Strategies for Cardiovascular Diseases

Cardiovascular diseases are the leading cause of death worldwide. Aging and/or metabolic stress directly impact the cardiovascular system. Over the last few years, the contributions of altered nicotinamide adenine dinucleotide (NAD+) metabolism to aging and other pathological conditions closely related to cardiovascular diseases have been intensively investigated. NAD+ bioavailability decreases with age and cardiometabolic conditions in several mammalian tissues. Compelling data suggest that declining tissue NAD+ is commonly related to mitochondrial dysfunction and might be considered as a therapeutic target. Thus, NAD+ replenishment by either genetic or natural dietary NAD+-increasing strategies has been recently demonstrated to be effective for improving the pathophysiology of cardiac and vascular health in different experimental models, as well as human health, to a lesser extent. Here, we review and discuss recent experimental evidence illustrating that increasing NAD+ bioavailability, particularly by the use of natural NAD+ precursors, may offer hope for new therapeutic strategies to prevent and treat cardiovascular diseases.


Introduction
Cardiovascular diseases are the leading cause of death worldwide according to the World Health Organization. Aging is a primary risk factor for many cardiovascular diseases. With the progressively aging population, the elevated prevalence of cardiovascular diseases undermines human health and longevity [1].

De Novo Synthesis Pathway
The kynurenine pathway drives de novo synthesis of NAD+ from the essential amino acid tryptophan [37] (Figure 1). This metabolic pathway is the dominant route of metabolism for absorbed tryptophan [38], accounting for more than 95% of daily metabolized tryptophan. However, tryptophan is considered an inefficient source of NAD+, as it requires 8 enzymatic reactions and, thus, has lower therapeutic value than other precursors, including NAM, NR, and NMN [39]. Therefore, these last molecules can be considered much more physiologically preferred NAD+ precursors than tryptophan [40]. Although the potential function of the kynurenine pathway of tryptophan metabolism to produce NAD+ has received little attention, emerging evidence suggests potential interplay between this pathway and cardiovascular disease [37,41]. In line with this, the kynurenine pathway has been recently regarded as a potential source of NAD+ in macrophages [42].

Salvage Pathway
NMN is a major intermediate of NAD+ (Figure 1). The salvage synthesis of NMN from NAM is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT), whereas the synthesis of NMN from NR is catalyzed by intracellular NR kinases (NRK1 and NRK2) [43]. Finally, NMN may be subsequently transformed into NAD+ by different subcellular forms of adenylyltransferases (NMNAT1-3) [44].

Other Modulators of Intracellular NAD+ Pools
In addition to these enzymes, others may also influence the tissue abundance of NAD+ ( Figure 1). Specifically, nicotinamide N-methyltransferase (NNMT) catalyzes the Nmethylation reaction of NAM and other analogous molecules using S-adenosyl methionine (SAM) as a donor of methyl groups [45] and produces me-NAM and S-adenosyl homocysteine (SAH) [39,40]. Recent reports suggest that NNMT may also act as a determinant of tissue NAD+ pools in vivo [43]. Interestingly, the subproduct me-NAM has been reported to exert anti-inflammatory, antioxidant, and antidiabetic effects in different experimental models [46][47][48][49][50].

Administration and Bioavailability of NAD+ Precursors
L-tryptophan can be safely supplemented in the diet [51]. This amino acid can be added directly to feedstuff and dissolved into drinking water [38]. As indicated previously, its availability is primarily governed by the kynurenine pathway, which may contribute to NAM production under dietary deficiency of this vitamin form [38].
NAM is highly soluble in water and is considered a safe food supplement in animal nutrition and the predominant circulating form of vitamin B3 in vivo [52]. Its safety margin is at least ten times the dietary requirements and use levels, and it can be administered orally by feeding or drinking (tap water) routes with similar bioequivalence rates. Oral NAM can be absorbed in the stomach and the small intestine either through passive diffusion or Antioxidants 2021, 10, 1939 5 of 41 by Na+-dependent carrier-mediated facilitated transport mechanisms [52], with passive diffusion predominating at pharmacological doses. Similarly, NA absorption into the intestinal mucosa follows the same transport mechanisms as NAM. NA can be further metabolized via NA mononucleotide (NAMN) to form the NA analog of NAD+ through the Preiss-Handler pathway (Figure 1). NAD+ can then be enzymatically cleaved into NAM in the intestinal mucosa and then the liver. The latter tissues contain elevated amounts of NAD+-consuming enzymes, such as NAD+ glycohydrolase, that allow NAM synthesis from NAD+. NAM can be subsequently transported to tissues for the synthesis of NAD+ or its phosphorylated form (NADP+). The principal excretory product is the methylated, water-soluble metabolite me-NAM (Subsection: Other modulators of intracellular NAD+ pools) or some of its oxidation products [53].
NR has a good bioavailability [54]. Unlike NA and NAM, dietary NR cell uptake is mediated by a specific nucleoside transporter [55], where it is transformed into NAD+ through a distinct, two-step biosynthetic pathway.
In contrast to that of NR, the bioavailability of NMN is rather limited [56]. Indeed, extracellular sources of NMN do not efficiently cross the endothelial membrane and require previous dephosphorylation by CD73 (ecto-5 -nucleotidase) to generate NR or must be extracellularly metabolized to NAM by CD38 [57]. Interestingly, a specific NMN transporter (i.e., Slc12a8) exists in murine small intestinal epithelium [58]. This transporter is predominantly expressed in the small intestine and in white adipose tissue and the liver to a lesser extent. However, whether Slc12a8 can also be expressed by cells of the vasculature or cardiomyocytes remains to be determined. NMN can also be obtained from NAM by extracellular NAMPT (eNAMPT, also termed visfatin). In endothelial cells, this conversion process requires the participation of phosphoribosyl-1-pyrophosphate (PRPP) [8]. Finally, extracellular NAD+ can be directly transported into cardiomyocytes through connexin 43 (i.e., Cx43) channels [59]. Taken together, these findings suggest that intracellular transport of NAD+ and NMN may be cell-specific and rely on different transporters.

In Vitro Cardioprotective Effects of NAD+ Sources
Experimental evidence suggests that oxidative stress and inflammation are mechanistically interconnected in driving cardiac and vascular diseases, and both may contribute to disease progression [70]. Increased vascular reactive oxygen species can react with cellular molecules and organelles, which become dysfunctional, leading to inflammation and endothelial dysfunction with devastating consequences on the vasculature and cardiac tissue. Moreover, impaired vascular reactive oxygen species homeostasis and inflammation may be reciprocally exacerbated, thereby resulting in a vicious and chronic cycle.
Current applications for successful control of enhanced oxidative stress and inflammation for the management of such complex chronic diseases are still elusive, partly due to the extensive diversity of oxidative stress pathways and inflammatory cells that can either be cardioprotective or cause cardiac damage. In this context, we next focus on relevant recent research evidence to examine some cardioprotective properties shown by vitamin B3 forms or intermediates in appropriate cell models of disease.

Ex Vivo Properties of NAD+ Precursors Relevant to Cardiovascular Diseases
Reactive oxygen species, including the superoxide anion (O 2 •− ) and the nitric oxide radical (NO • ), are critical bioreactive signaling molecules that may influence both cellular homeostasis and disease. Under physiological conditions, the intracellular pool of such reactive oxygen species is controlled by an intricate array of antioxidant defense systems that include superoxide dismutases, catalase, the glutathione peroxidase/reductase system, and the peroxiredoxin/thioredoxin system [71]. Under pathological conditions, elevated reactive oxygen species can overwhelm cellular antioxidant defenses, impair reactive oxygen species scavenging and, therefore, lead to cardiac dysfunction [72][73][74][75][76].
In addition to the enzymatic antioxidant systems, certain molecules (identified as antioxidants) may serve as direct metabolic scavengers of electrons from reactive radicals and, thus, confer direct protection to biomolecules from oxidative damage by virtue of their chemical ability to accept/donate electrons. In particular, NAM may directly function as an O 2 •− radical scavenger and inhibit the generation of free radicals (e.g., O 2 •− , NO • , and HCLO • ) [77] and prevent protein and lipid oxidation in vitro [78]. Importantly, this scavenging ability may paradoxically enable some vitamins with recognized antioxidant properties to function as pro-oxidants under certain circumstances [79][80][81]. Notably and in contrast to other vitamins with antioxidant abilities, such as vitamins A and C, vitamin B3 (NA) did not induce oxidative damage in vitro at physiologically relevant concentrations [82].
Both the resistance to oxidation of non-HDL and the antioxidant properties of HDL are two of the most important antiatherogenic metrics of lipoproteins ex vivo [83]. We recently reported that isolated plasma non-HDL from mice lacking apolipoprotein (Apo)E (Apoe −/− ) treated with NAM was less susceptible to oxidation ex vivo than that from untreated mice [25]. Notably, incubation of human low-density lipoprotein (LDL) with NAM produced a direct inhibitory effect on lipid peroxidation during an analysis of the oxidation kinetics of exposed lipoproteins [25]. However, the effect of NAM on the HDL antioxidant capacity of Apoe −/− mice could not be evaluated in the latter study due to the limited circulating HDL moieties in this mouse model. Instead, the effect was directly assessed using HDL isolated from wild-type mice on the same genetic background as the Apoe −/− mice. Consistently, our data revealed that HDL isolated from the plasma of NAM-treated mice was more protective against LDL oxidation ex vivo than HDL isolated from untreated mice (Julve et al., 2021, unpublished data, Figure 2), suggesting enhanced atheroprotection by HDL from NAM-treated mice.

Figure 2.
Effect of NAM manipulation on antioxidant ex vivo protection conferred by plasma isolated HDL. (a) Representative diene formation curves of human LDL incubated with mouse HDL isolated from untreated and NAM-treated wildtype C57BL/6J mice in the presence of 2.5 μM CuSO4 at 37 °C. The final oxidation kinetics of the LDL+HDL mixture was shown after subtracting the kinetics of HDL incubated without LDL. (b) HDL antioxidant activity against LDL oxidative modification. Methods used can be found in greater detail in reference [84]. Data were expressed as relative lag phase to LDL oxidized in the presence of HDL isolated from Untreated mice (arbitrary unit = 1). Data were expressed as the median ± interquartile range (n = 4-5 independent pools per group). Differences between the mean values were determined using a nonparametric Mann-Whitney test. * indicates significant differences versus untreated mice (p < 0.05). Abbreviations used were NAM, NAM-treated mice using 1% NAM dissolved in tap water [85]   Methods used can be found in greater detail in reference [84]. Data were expressed as relative lag phase to LDL oxidized in the presence of HDL isolated from Untreated mice (arbitrary unit = 1). Data were expressed as the median ± interquartile range (n = 4-5 independent pools per group). Differences between the mean values were determined using a nonparametric Mann-Whitney test. * indicates significant differences versus untreated mice (p < 0.05). Abbreviations used were NAM, NAM-treated mice using 1% NAM dissolved in tap water [85] (Julve et al., 2021, unpublished data).

Cellular Effects of NAD+-Increasing Strategies Relevant to Cardiovascular Diseases
Compelling experimental evidence suggests a favorable influence of NAD+-restoring strategies on the main cellular mechanisms involved in cardiovascular cell damage.  [78]. This antioxidant property has also been reported for other vitamin B3 derivatives, such as NR [86] and NMN [9], probably suggesting common mechanistic pathways among such different NAD+ precursor intermediates.
The maintenance of intracellular pools of NAD+ is an important vasoprotective mechanism in arterial endothelial cells [86,87]. Consistently, NAD+ depletion is commonly related to age-related vascular dysfunction [88]. Aged endothelial cells exposed to NMN were protected against oxidative stress in a SIRT1-dependent fashion [89]. Similarly, NRmediated restoration of NAD+ contents in the lung and heart protected against oxidative stress and apoptosis in tissues from an animal model of sepsis [86]. NR exposure prevents reactive oxygen species production and apoptosis in endothelial cells induced by conditioned medium collected from LPS-treated macrophages [86], further supporting the beneficial vascular effects of NR. NR is also protective against alcohol-induced oxidative stress and inflammation in cultured macrophages [90]. Importantly, NR effects occurred via NAD+/SIRT1 signaling [86,90].
More recently, NAD+ depletion in lipopolysaccharide (LPS)-mediated activated macrophages was accompanied by reactive oxygen species-mediated DNA damage and by activation of PARP, an NAD+-consuming enzyme [91]. In this study, the salvage pathway was induced upon NAD+ depletion, as revealed by increased NAMPT expression allowing the maintenance of intracellular pools of NAD+ sufficient for fueling glyceraldehyde-3phosphate dehydrogenase activity and Warburg metabolism, which is critical for LPSmediated activation of macrophages [92]. Interestingly, reduced intracellular NAD+ and increased NAMPT expression developed rapidly after inflammatory activation together with DNA damage caused by reactive oxygen species [91].

Inflammation
Insufficient NAD+ leads to vascular inflammation [93]. Accumulating data strongly suggest the contribution of NAD+/SIRT1 signaling to inflammation.
The anti-inflammatory effects of NAM were first described in vitro in multiple immune cell types frequently involved in chronic inflammatory processes, such as atherosclerosis [94][95][96][97][98][99][100][101][102], including activated circulating monocytes and tissue immune cells (i.e., resident macrophages), which are the first inflammatory responders and are ultimately involved in the resolution of inflammation. Notably, NAM can promote the differentiation of monocytes toward macrophages with attenuated inflammatory characteristics, i.e., with strongly reduced proinflammatory features [94]. NA administration may also shift macrophages to M2 polarization in vitro [23]. Similar anti-inflammatory actions in activated macrophages have been reported for NR [90].
Mitochondria play an important role in innate immunity and chronic inflammation [103]. Dysfunctional mitochondria can contribute to the pathophysiology of cardiovascular diseases [104]. Indeed, blockade of de novo synthesis by NAD+ using genetic or pharmacological approaches depleted NAD+ stores, suppressed mitochondrial NAD+-dependent signaling and respiration, and impaired phagocytosis and inflammation resolution [42]. This suggests a critical role for intracellular NAD+ pools in the regulation of macrophage immune function in resting and in aged and immune-challenged macrophages.
Elevated systemic inflammation has been causally linked to mitochondrial dysfunction of peripheral blood mononuclear cells (PBMCs) in subjects with HF, whereas NR-mediated NAD+ elevations improved mitochondrial respiration and attenuated proinflammatory activation of PBMCs isolated from treated volunteers with HF [105].

Apoptosis
Apoptosis may precede cell death and fibrosis and, thus, drive cardiac and vascular dysfunction. Excess reactive oxygen species may damage intracellular proteins, lipids, and DNA and induce apoptotic cell death [106,107]. Under ischemic/hypoxic conditions, NAD+ metabolism is significantly inhibited in myocardial cells and accompanied by reduced cell viability due to enhanced apoptosis [108,109]. Conceivably, strategies to increase NAD+ metabolism may enable cardiomyocytes to evade apoptosis. Supporting this notion, NAM exposure to primary cardiomyocytes and the H9C2 cell line conferred protection against hypoxia-induced apoptosis [108][109][110]. The attenuation of apoptosis alleviated oxidative stress and improved mitochondrial stress in NAM-treated cells [108,109]. Notably, NAMmediated protection from apoptosis was linked to upregulation of autophagy-related proteins and accompanied by concomitant attenuation of the mTOR pathway in another study [110].
Cardiomyocyte mitochondrial dysfunction and mitochondrial DNA instability are increasingly believed to be important features of the progression of various forms of cardiac disease [111]. Under these circumstances, mitochondrial DNA repair mechanisms are commensurately induced, including PARP activity [112]. Elevations in cellular PARP activity may drain cellular NAD+ reserves [113] and lead to mitochondrial dysfunction and, thus, impaired cardiac function. Conversely, NR-mediated restoration of NAD+ pools improved the mitochondrial unfolded protein response and mitochondrial health in cultured cardiomyocytes subjected to mitochondrial stress conditions [114]. The latter finding suggests that NAM not only confers direct cellular protection against oxidation to vascular cells but also prevents cellular apoptosis during free radical exposure and blocks inflammatory vascular cell activation [115] by preventing membrane phosphatidylserine exposure [116], which is an apoptotic inducer [117]. In line with this assertion, NAD+ depletion is linked to macrophage apoptosis [118], whereas its repletion with NAD+ precursors (NA and NAM) protects cells against apoptosis [86,119].

Mitochondrial Stress
Cellular NAD+ depletion may occur upon increased mitochondrial DNA damage due to prolonged PARP action [113], an NAD+-consuming enzyme involved in mitochondrial DNA repair, which may cause inhibition of other NAD+-dependent proteins, especially sirtuins.
NAM pretreatment attenuated mitochondrial stress and protected hypoxic primary rat cardiomyocytes against cell necrosis and apoptosis [108]. In this study, NAM increased the intracellular ATP content in mitochondria, further revealing that NAM protected cardiomyocytes from hypoxia-mediated mitochondrial damage. Importantly, NAM-mediated favorable effects were accompanied by activation of the AMPK pathway [108]. By increasing the intracellular NAD+ content, NAM can also increase the protein abundance of superoxide dismutase and catalase and induce intracellular energy production [109], thus reducing the abundance of mitochondrial reactive oxygen species and preventing the loss of mitochondrial membrane potential.

Autophagy
Autophagy is a cellular process that recycles components of the cytoplasm of cells and plays a key role in tissue remodeling. As previously mentioned, NAM supplementation protected chronic hypoxic myocardial cells against hypoxia-induced apoptosis and increased the protein abundance of autophagy-related proteins by attenuating mTOR pathways [110].
NR exposure promoted autolysosome generation in neonatal and adult mouse cardiomyocytes exposed to doxorubicin (DOX) [120]. In particular, NR can attenuate autophagic flux blockade, autolysosome accumulation, and oxidative stress in DOX-treated cardiomyocytes. Notably, inhibition of lysosomal acidification or SIRT1 abrogated such NRmediated protective effects during DOX-induced cardiotoxicity, thereby suggesting that NR enhances autolysosome clearance via NAD+/SIRT1 signaling to prevent DOX-induced cardiotoxicity.

Fibrosis
Organ fibrosis is characterized by excess deposition of extracellular matrix proteins. Administration of NA, NAM, and NR attenuates experimental liver fibrosis [14,[121][122][123][124], potentially indicating that an insufficient NAD+ supply may underlie organ fibrosis. Similar favorable effects could be conceivably suggested for NAD+-increasing interventions aiming to directly combat myocardial fibrosis. Although such interventions have not yet been explored, this strategy may warrant further investigation.

Atherosclerosis
Immune cell infiltration and activation are a characteristic finding in chronic inflammation processes, such as atherosclerosis [125]. Macrophages and vascular smooth muscle cells are regulated through a coordinated pivotal program during the development of atherosclerotic lesions [126,127]. Regulation of the cholesterol balance in these cell types is critical in atherogenic processes, and the accumulation of LDL-derived cholesterol in foam cells leads to chronic inflammatory responses in the subendothelial arterial intima [128]. The interaction of LDL with proteoglycans initiates LDL retention in the intima, facilitating LDL modifications and increasing their nonregulated uptake by macrophages [129]. In contrast, HDL stimulates cholesterol efflux from macrophages, the first step of the reverse cholesterol transport pathway, which is mechanistically linked to the anti-inflammatory and immunosuppressive functions of HDL [126].
The first evidence that NA administration reduced atherosclerosis was reported in rabbits [130][131][132][133] and minipigs [134] ( Table 1). The antiatherogenic effect of NA was also confirmed in humans [135,136]. Originally, these antiatherogenic effects were mainly attributed to the ability of NA to reduce serum LDL cholesterol (LDL-C) and triglycerides while increasing HDL-C levels. However, these antiatherogenic effects did not always correlate with NA-mediated hypolipidemic effects [130,134]. The development of genetically engineered mice permitted us to evaluate the potential of NA to prevent massive atherosclerosis in different mouse models of hyperlipidemia and investigate the potential atheroprotective mechanisms. NA had a strong lipid-independent antiatherogenic effect on LDL receptor-deficient (Ldlr −/− ) mice, which was mainly mediated by its receptor GPR109A on bone marrow-derived cells [137]. GPR109A activation by NA enhanced ATP binding cassette (ABC) G1-dependent macrophage cholesterol efflux to HDL, inhibited MCP-1-induced macrophage recruitment into the peritoneal cavity, and impaired macrophage homing into atherosclerotic lesions [137]. Furthermore, NA administration in Apoe −/− mice also reduced advanced lesion progression and plaque inflammation independent of plasma lipoprotein levels, rendering advanced atherosclerotic lesions more stable [138]. In line with these findings, an independent study also showed that both Ldlr −/− and Apoe −/− mice treated with NA had significantly lowered aortic cholesterol levels, although this study also reported an NA-mediated hypolipidemic effect [139]. However, these findings were not reproduced in another study conducted in Apoe −/− mice [140], likely because the researchers used a low-fat diet combined with an insufficient NA dose and a short treatment period. On the other hand, Apoe3Leiden transgenic mice overexpressing human cholesteryl ester transfer protein (CETP) treated with NA also showed reduced atherosclerosis development concomitant with enhanced very-low density lipoprotein clearance and low CETP activity resulting in higher HDL-C [141]. In this study, NA also reduced monocyte adhesion and macrophage homing into atherosclerotic lesions but promoted macrophage-to-feces reverse cholesterol transport [141], thereby emphasizing that multiple mechanisms may explain the antiatherogenic effects of NA in vivo.
Other vitamin B3 intermediates, such as NMN and NR, reversed age-related endothelial dysfunction and reduced oxidative stress [15,28]. Although their mechanism of action in atherosclerosis prevention has not been directly evaluated in vivo, the effects of these intermediates can be at least partly explained by their ability to effectively increase the tissue content of NAD+ and, thus, restore SIRT1 activity (reviewed in reference [8]). Supporting this assertion, a host of studies in genetically engineered atherosclerotic mice suggest a protective role of SIRT1, although this effect was mainly related to its expression in vascular cells (reviewed in Reference [142]). However, the potential involvement of a SIRT1-mediated mechanism was reflected only by the upregulation of some aortic targets of liver X receptor (LXR) [143]. Supporting this notion, our group has demonstrated that the gene expression of Abca1 and Abcg1 was upregulated in aortic tissue from Apoe −/− mice and in cultured macrophages exposed to NAM, although only marginally [25]. In the latter study, dietary NAM supplementation prevented aortic atherosclerosis in Apoe −/− mice, which was related to an improvement in the susceptibility of ApoB-containing lipoproteins to oxidation and to upregulation of aortic IL-10 and downregulation of TNFα, suggesting a switch toward anti-inflammatory macrophages [25]. Rather surprisingly, these antiatherogenic effects were accompanied by higher levels of plasma ApoB-containing lipoproteins and impaired macrophage-specific reverse cholesterol transport in vivo [25,144]. Overall, these findings support that NAM-mediated anti-inflammatory effects may prevail in atherogenesis. In line with these findings, me-NAM administration, a major endogenous metabolite of NAM (Section 2), also prevents atherosclerotic plaque progression and inflammation in double Ldlr −/− /Apoe −/− mice [29]. In this study, me-NAM improved endothelial function [29], as was also reported in an independent study conducted in Apoe −/− mice [30].
NAMPT is the rate-limiting enzyme in the NAD+ salvage pathway. However, different studies investigating the effects of NAMPT in atherosclerosis in vivo found conflicting results depending on the localization of NAMPT. Administration of the NAMPT inhibitor FK866 reduced inflammation in atherosclerotic plaques after carotid cast implantation in Apoe −/− mice by reducing CXCL1-mediated activities on neutrophils and improving plaque vulnerability parameters [145]. Additionally, liver-specific deficiency of NAMPT increased plasma HDL-C levels and reduced aortic atherosclerosis, including macrophage homing [146]. These effects were closely related to upregulation of LXR target genes and a consequently enhanced macrophage-to-feces reverse cholesterol transport rate in vivo [146]. Conversely, NAMPT overexpression aggravated atherosclerotic inflammation and enhanced atherosclerosis development in Apoe −/− mice [147]. In contrast, hematopoietic overexpression of human NAMPT attenuated the plaque burden and stabilized lesions in Ldlr −/− mice [148], likely suggesting tissue-specific and possibly species-specific differences to explain such apparently divergent findings. Notably, these effects were found only after modulating intracellular levels of NAMPT and were associated with macrophage resistance to apoptosis, attenuating monocyte intravasation and migration in response to chemotactic signals, thereby skewing monocyte differentiation and macrophage polarization toward an anti-inflammatory M2 phenotype [148].
Finally, NAD+ is also a substrate for poly(ADP-ribose) polymerase, also known as PARP (Section 2). PARP activity leads to NAD+ and ATP depletion after various types of DNA damage [149]. Both PARP pharmacological inhibition and gene deletion reduced plaque sizes in Apoe −/− mice concomitant with reduced macrophage homing in two independent studies [150,151]. In particular, PARP inhibition increased plaque stability without improving the lipoprotein profile. Consistently, PARP inhibition also reduced traits of plaque disruption in a separate study conducted in Apoe −/− mice [152], mainly by decreasing oxidative DNA damage and iNOS-associated protein nitration. In two of these studies, Parp gene deletion protected against oxidant-induced foam cell death [150,152], which may be related to preserved intracellular NAD+ and ATP pools. Moreover, pharmacological inhibition of PARP action also promoted atherosclerotic plaque regression in this mouse model of atherosclerosis by modulating most of these key antiatherogenic factors [153]. Consistent with these findings, a recent report confirmed the in vivo antiatherogenic effects of PARP inhibition in rabbits [154], which were likely due to its ability to improve endothelial function by preserving NAD+ levels.

Ischemic-Infarcted Myocardium or Ischemia-Reperfusion
Myocardial infarction is a common major cardiovascular event that originates from myocardial ischemia in the presence/absence of reperfusion [155]. The term ischemia encompasses a broad range of clinical hypoxia scenarios ranging from angina to permanent occlusion. Although reperfusion intervention is clinically mandatory to treat ischemic injury, the associated cardiac damage caused by this intervention should not be overlooked.
The loss of several dinucleotide species, including NAD+, NADH, NADP+, and NADPH, has been shown in myocardial tissue after ligature of the coronary artery branch [156] in dogs. In the corresponding study, NAD+ decline was accompanied by increased activity of NAD+ glycohydrolase, an NAD+-consuming enzyme (Section 2), which has been reported in response to acute experimental acute infarction [157]. Notably, irreversible postischemic injury occurred when NAD+ depletion was approximately 60-70% [158]. Therefore, NAD+ stabilization appears to be critical for the recovery of mitochondrial and cardiac function. NAD+ mainly acts by reducing oxidative stress and cell death [159]. Importantly, cardiacspecific overexpression of NAMPT increased NAD+ levels in the heart and reduced the size of myocardial infarction in response to prolonged ischemia/reperfusion in mice [160]. In this context, NAD+ supplementation also protected hearts from ischemia/reperfusion injury in mice [161], Bama miniature pigs [162], and rats [159,163] (Table 1). Furthermore, cardiac hypoxia-ischemia injury can be relieved in mice treated with NMN supplementation [164]. NMN also protected against ischemia/reperfusion injury in aged rats [165]. NMN replenished heart NAD+ levels but reduced oxidative stress and mitochondrial ROS levels. Another study also suggested that NRK-2 alleviates ischemia-induced HF through P38 signaling [166]. Finally, NR was also shown to be effective in improving vascular function. NR improved endotheliumdependent relaxation of isolated rat mesenteric arteries in an ischemia-reperfusion model [167]. Overall, these studies strongly suggested that NAD+ and its precursors improve alterations following ischemia/reperfusion injury in vivo.

Cardiomyopathy and Heart Failure (HF)
Cardiomyopathy is one of many causes of HF. Its prevalence continues to rise over time, especially with aging. Dilated cardiomyopathy is characterized by dilatation of the left and right ventricles, leading to systolic dysfunction and a reduced ejection fraction [168]. Susceptibility to dilated cardiomyopathy has been predominantly explained by genetic mechanisms, and, in Western societies, this cardiac condition is the principal indication for heart transplantation.
Various studies have explored HF and NAD+ levels in vivo. Cellular NAD+ is a key regulator of metabolism and bioenergetics, and failure of cardiomyocyte bioenergetics is a critical hallmark of HF. Interestingly, previous studies suggest that mitochondrial dysfunction and metabolic remodeling in HF are associated with an imbalance in the intracellular ratio of NADH to oxidized NAD+ or the NADH/NAD ratio [169,170]. Therefore, controlling intracellular NAD+ levels may improve myocardial bioenergetics and cardiac function. In fact, in a recent paper, normalization of NADH/NAD through supplementation with NAD+ precursors was shown to be associated with cardiac function improvement in an HF mouse model [171]. Specifically, the authors studied the impact of NAD+ precursor supplementation in food (NR) in two different mouse models of dilated cardiomyopathy and found stable myocardial NAD+ levels in the failing heart. Diguet et al. [171] proposed that the benefit observed in their study was due to improved energy metabolism rather than altered protein acetylation. However, previous studies found that NAD+ availability was associated with increased protein acetylation in the failing myocardium, which was attributable to impaired NAD+-dependent protein deacetylation by sirtuins, especially SIRT3 [172][173][174][175]. Another study reported oral supplementation of NR to treat cardiomyopathy caused by laminin A/C gene (Lmna) mutations (LMNA cardiomyopathy) in mice. Mutant mice suffered from dilated cardiomyopathy [176]. The NAD+ salvage pathway has been shown to be altered in the hearts of mice and humans carrying Lmna mutations, leading to alteration of PARP-1, which is an NAD+ cosubstrate enzyme [177]. Oral admin-istration of NR to Lmna H222P/H222P mice resulted in an improvement in the NAD+ cellular content, an increase in PARylation of cardiac proteins, and an improvement in left ventricular structure and function [177]. However, NAM supplementation to Lmna H222P/H222P mice did not change NAD+ contents in the liver or heart or cardiac left ventricular function [177].
In general, approximately half of subjects with HF present with a reduced ejection fraction (HFrEF), and the other half present with a preserved ejection fraction (HEpEF) [178], which is important to emphasize because these presentations reflect different but not entirely distinct pathogenic mechanisms, and their treatments may also be different. For example, mitochondrial dysfunction and metabolic remodeling are known to play important roles in HFrEF, but little is known about the mechanisms of mitochondrial dysfunction in HFpEF. In the myocardium of an HFpEF mouse model, mitochondrial fatty acid oxidation has recently been observed to be impaired and to be associated with hyperacetylation of key enzymes along the pathway. SIRT3 downregulation and NAD+ deficiency secondary to an impaired NAD+ salvage pathway are the main contributors to mitochondrial fatty acid oxidation according to the authors of Reference [179]. Interestingly, supplementation with NAD+ or a direct activator of NAD+ biosynthesis in HFpEF mice with NR ameliorated the HFpEF phenotype by improving mitochondrial function and pathological cardiac remodeling [179].
In the setting of aging, the role of NAD+ has been explored by Tannous et al., who demonstrated that a lack of NRK2 does not influence the NAD+ level in the myocardium in young adults [180]. However, NRK2 expression is required to support the maintenance of NAD+ levels in 24-month-old mouse hearts. At this late stage, the major biosynthetic pathways are repressed, and, in the absence of myocardial NRK2, NAD+ levels fall by 50% [180]. Moreover, Nmrk2-deficient mice, which develop a progressive DCM-like phenotype with aging, are viable but develop progressive cardiac dysfunction and eccentric cardiac remodeling associated with an alteration in laminin deposition in the extracellular matrix starting at 4 months, followed by cardiac fibrosis and structural defects in the adult myocardium [180]. Consistent with these findings, another study suggested that feeding aging mice with NR improved mitochondrial function and preservation of stem cell function, which may contribute to the extended lifespan of NR-treated mice [11]. Additionally, iron overload and iron deficiency have been associated with cardiomyopathy and HF. Inactivation of transferrin receptor (Tfr1) in mice was associated with cardiomegaly, poor cardiac function, failure of mitochondrial respiration and mortality in the second week of life. Interestingly, the lifespan of Tfr1-deficient mice was prolonged by treatment with NR [181]. However, the benefit and safety of NAD+ precursor supplementation require further elucidation. For instance, in another study with a high-dose NR supplementation in a cardiomyocyte mutUNG1 mouse model, which is a mouse model with myocardial mitochondrial DNA damage, sirtuin activity was inhibited rather than increased due to intracellular NAM accumulation, while the addition of NR failed to improve cardiac function [182]. Thus, the authors suggested that high doses of NR should be used with caution, especially when cardiomyopathic symptoms are caused by mitochondrial dysfunction and mitochondrial DNA instability.
As stated above (Section 2), NAMPT, a rate-limiting enzyme in the NAD+ salvage pathway, plays an important role in controlling the level of NAD+. In another mouse model of HF, Byun et al. demonstrated that pressure overload-induced HF is exacerbated in both systemic Nampt-deficient mice and mice with cardiac-specific Nampt overexpression. Unexpectedly, both loss-and gain-of-function models exhibited reduced protein acetylation, suppression of metabolic genes, and mitochondrial energetic dysfunction. Therefore, endogenous NAMPT plays a protective role under pressure overload conditions, but cardiac-specific Nampt overexpression is detrimental rather than therapeutic. The authors suggested that these deleterious effects in NAMPT transgenic mice may be partially due to SIRT1 and suppression of the gene expression of key metabolic targets [183].
Another pathophysiological process promoting HF progression is cardiac fibrosis, which is characterized by increasing ventricular stiffness and a reduced cardiac pumping ca-pacity. In a mouse model of isoproterenol-induced cardiac fibrosis, Wu et al. demonstrated that NMN treatment attenuates cardiac fibrosis in vivo and fibroblast activation in vitro by suppressing oxidative stress and SMAD3 acetylation in an NAD+/SIRT1-dependent fashion [184].
Diabetic cardiomyopathy is another major cause of HF. Subjects with diabetic cardiomyopathy have adverse structural remodeling due to enhanced cardiac hypertrophy and fibrosis, resulting in characteristic cardiac dysfunction. The prevalence of diabetic cardiomyopathy is increasing in parallel with the increase in diabetes mellitus. Accordingly, epidemiological studies show a positive correlation of hyperglycemia with an increase in HF in subjects with either type 1 or type 2 diabetes mellitus. Notably, the manifestation of these conditions is independent of atherosclerotic cardiovascular disease and hypertension [185][186][187]. Additionally, hyperglycemia has been shown to be associated with a reduced NAD+ content, a decreased NAD+/NADH ratio, and promoted protein hyperacetylation and mitochondrial dysfunction in diabetic organs [188][189][190][191][192][193][194]; thus, NAD+ restoration is of therapeutic importance. More recently, NAD+ redox imbalance has been shown to alter protein acetylation and phosphorylation in two different mouse models of diabetic cardiomyopathy [195]. Specifically, an NAD+ redox imbalance in mice with cardiac-specific knockout of complex I subunit Ndufs4 (cKO) hearts causes systolic and diastolic dysfunction due to cardiomyocyte dysfunction. Moreover, diabetic cKO hearts presented increased acetylation of superoxide dismutase 2, protein oxidation, troponin I S150 phosphorylation and impaired energetics. Importantly, all these effects were normalized when cardiac NAD+ levels were elevated using Nampt transgenic mice (diabetic cKO-NAMPT mice), suggesting a beneficial effect of exploiting NAD+ metabolism for diabetic cardiomyopathy. Consistent with this finding, restoration of cardiac NAD+ with NMN supplementation in the myocardium of genetically deficient mice with cardiacspecific expression of Kruppel-like factor 4 (Klf4) (CMK4KO) protected against pressure overload-induced HF induced by transverse aortic constriction [194]. Notably, CMK4KO mice are more prone to pressure overload-induced HF caused by transverse aortic constriction [196,197]. KLF4 belongs to a larger family of zinc-finger transcription factors that regulate critical cellular processes, such as cell survival, apoptosis, and metabolism. Some members of the KLF family influence several cell types belonging to the cardiovascular system [198]. Specifically, KLF4 is a critical regulator of cardiac mitochondrial homeostasis [197]. Conceivably, its cardiac deficiency was associated with cardiac NAD+ deficiency and mitochondrial protein hyperacetylation coupled with reduced SIRT3 in the hearts of mice [198]. Notably, cardiac NAD+ replenishment with NMN supplementation successfully protected these mice from cardiac disease by positively influencing mitochondrial health and oxidative stress and preventing cardiac cell death in treated mice.
Cardiac hypertrophy is also a common feature in many cardiovascular diseases [199]. Although cardiac hypertrophy may develop as a benign, adaptive response to either physiological or pathological situations, chronic cardiac stimulation can cause cardiac hypertrophy, remodeling and dysfunction and eventually lead to HF [200]. Enhanced inflammation via activation of the nucleotide-binding and leucine-rich repeat pyrin domains protein 3 (NLRP3) inflammasome and concomitant secretion of cytokines contributes to the progression of cardiac hypertrophy [201]. Therefore, cardiac hypertrophy inhibition has been considered a possible target for novel therapies. In this context, a recent study demonstrated that NR administration favorably influenced cardiac hypertrophy and dysfunction induced by transverse aortic constriction (TAC) surgery in a mouse model by directly inhibiting myocardial NLRP3 inflammasome activation [202]. Notably, this NR-mediated NAD+ rise in myocardial tissue elevated SIRT3 activity, which resulted in MnSOD deacetylation and TAC-induced myocardial oxidative stress alleviation.

Cardiotoxicity
Cardiotoxicity can occur during cancer treatment and can reduce quality of life and increase the risk of death from cardiac-related causes [203,204]. This condition can appear within days, months, or years after chemotherapy, even after disease remission, which is the case, for example, for doxorubicin (DOX), a drug widely used in chemotherapy for the treatment of several malignancies. Nonetheless, the dose-dependent cardiotoxic effects of DOX, which include increased cardiomyocyte death via apoptosis [205,206], necroptosis [207] and ferroptosis [208], left ventricular dysfunction, dilated cardiomyopathy, and congestive HF in severe cases [209], limit its use in clinical practice; therefore, intensive research to attenuate such adverse effects is currently underway. Importantly, oxidative stress is commonly increased upon DOX treatment and underlies cardiotoxicity. In this context, NAM administration to DOX-treated rats successfully reversed signs of cardiac tissue injury revealed by elevated serum cardiotoxicity molecules and conduction and histopathological abnormalities [210]. Consistently, NR also reduced cardiac injury and myocardial dysfunction in DOX-treated mice by restoring NAD+ levels [120].

Aortic Aneurysm: Vascular Disease
Aortic diseases can be extremely life-threatening and include a variety of clinical entities, the most frequent of which is aortic aneurysm. Aortic aneurysm (AA) refers to progressive localized dilatation and weakening of the aorta, which can occur in the chest, thorax (thoracic aneurysm, TAA) or abdomen (abdominal aortic aneurysm, AAA). Although differences can be found in the pathobiology between TAA and AAA, both are characterized by local chronic inflammation of the aortic wall, apoptosis of smooth muscle cells in the aortic media layer and fragmentation of the extracellular matrix of the aorta at the site of the aneurysm. Increased local expression of proinflammatory cytokines, extracellular matrix degradation, microcalcification, and oxidative stress are biological processes contributing to degeneration of the aorta [211].
The risk factors for developing aortic aneurysms are age, sex, smoking, high blood pressure, peripheral artery disease, genetic disorders (individuals with Marfan, Ehlers-Danlos, Loeys-Dietz, or Turner syndrome can develop TAA), a family history of aneurysmal diseases, inflammatory diseases of the blood vessels (vasculitis), and aortic valve abnormalities. No effective drug therapy is currently available for AA, and 70% of small AAs grow to a size requiring surgical repair before fatal aortic rupture occurs [211]. Accordingly, identifying targets for the development of drug therapies that can effectively limit AA growth and rupture has attracted strong interest.
The sequential pathophysiology of aneurysm formation is unclear, but vascular smooth muscle cells (VSMCs) are known to play a central role in vascular cell vitality and, thus, matrix integrity. NAMPT is expressed in cultured SMCs, and its content and activity decline during advanced smooth muscle cells (SMC) aging [212][213][214]. Knockdown of Nampt in VSMCs has been shown to increase VSMC apoptosis, which has an important influence on AA development [215], whereas overexpression of Nampt can enhance VSMC survival [212], thereby suggesting a potential contribution of the salvage pathway to NAD+ synthesis in this cell type. Although controversial, some reports show that the NAMPT-NAD+ axis control system may favorably influence SMC contractile function [216][217][218].
NAMPT is expressed in periaortic adipose tissue [219], and altering NAD+ metabolism through the diet has been found to suppress age-related aortic dysfunction [220]. Watson and colleagues showed that the aortas of patients with thoracic aortic aneurysms had lower levels of NAMPT than nondilated aortas and correspondingly increased DNA strand breakage in SMCs [217]. They generated mice with homozygous deficiency of NAMPT in SMCs and found that loss of NAMPT rendered the aorta susceptible to aortic dilation and rupture and that their SMCs were more prone to senescence and DNA damage. Finally, they found that the NAMPT promoter was hypermethylated in the aortic media of aortopathy patients, suggesting epigenetically repressed NAMPT production. Together, the results uncovered an endogenous NAD+-fueling system in the aortic media that protects against DNA damage and premature SMC senescence.
The pharmacologic potential of NR in TAAs associated with genetic disorders has been investigated [221]. Using a mouse model with specific deletion of mitochondrial transcription factor A (Tfam) in VSMCs, Tfam-deficient VSMCs were observed to acquire a senescent and proinflammatory phenotype together with impairment in their contractile function [221]. These mice develop aortic aneurysms, medial degeneration, and lethal dissections, and NR supplementation rapidly raised TFAM levels, improved mitochondrial metabolism, and normalized aortic function and diameter in the Marfan mouse model of TAA.
NA inhibits vascular inflammation by decreasing oxidative stress and inhibiting monocyte/macrophage inflammatory responses and inflammatory cytokine production, which are key events in the pathogenesis of AAA [222,223]. Accordingly, NA may of particular interest for the prevention and treatment of aortic aneurysms [224,225].
In this regard, a previous study using two distinct animal models of AAA reported that NA inhibited AAA formation in conjunction with reduced aortic immune cell infiltration, inflammation, and matrix degradation [24]. The beneficial effects of NA are independent of GPR109A signaling, similar to NAM, and likely mediated through NAD+ production leading to SIRT1 activation. These results suggest that boosting NAD+ levels and SIRT1 activity by supplementation with NAM or related biomolecules may represent a promising therapeutic strategy for AAA treatment and prevention [24].
The role of SIRT1 in vascular homeostasis and diseases in mammals has been well documented [226][227][228]. Enhancing NAD+ biosynthesis with NAD+ precursors, such as NMN and NR, increases the activity of SIRT1 [10,12,229,230], improving mitochondrial dysfunction in different pathologies [33,231,232]. Notably, supplementation with NMN restored arterial SIRT1 activity and reversed age-associated arterial dysfunction and oxidative stress in the aortas of aged mice, which was associated with partial normalization of structural proteins in the arterial wall [9]. Additionally, acute NMN incubation in isolated aortas increased NAD+ threefold, supporting the promising potential of NMN supplementation for the treatment of arterial aging [9]. This is consistent with previous reports showing that NMN increases NAD+ in cultured vascular endothelial cells [233] and normalizes the ratio of NAD+/NADH in aortic tissue [234].
Downregulation of SIRT1 might also be mechanistically linked to AAA pathogenesis. Compared with those in adjacent control sections, SIRT1 expression and activity are significantly decreased in human AAA lesions [235]. In Apoe −/− mice infused with Ang II using pumps to induce hypertension (a recognized animal model of aneurysm), the incidence of AAA and the mortality rate were markedly reduced in VSMC-specific, Sirt1 transgenic mice [235]. Even without Apoe deficiency, an increase in both the prevalence and mortality of AAAs was found in VSMC-specific Sirt1 deficient mice compared with wild-type mice. Similar data were obtained in a calcium chloride-induced mouse AAA model in independent studies [235]. The underlying mechanism involves attenuation of vascular senescence by SIRT1 via modulation of the p53/p21 pathway. Moreover, macrophage-specific Sirt1 expression was reported to contribute to AAA protection [236]. Reducing calorie intake protected against AAA formation in Apoe −/− mice by upregulating the expression and activity of SIRT1 in VSMCs [237], and specific knockout of VSMC-derived Sirt1 abolished the prevention of AAA by caloric restriction [235].
In addition to AAAs, SIRT1 is also important in the prevention of TAA. VSMC-specific Sirt1 deficient mice did not influence the incidence of TAA but increased the fatality rate in mice. Moreover, VSMC-specific Sirt1 overexpression markedly blocked TAA development mainly through epigenetic downregulation of MMP-2 [238].
These findings provide evidence that SIRT1 reduction links vascular senescence through vascular inflammation to AAAs and that SIRT1 in VSMCs is a promising therapeutic target for the prevention of life-threatening aortic diseases.
Finally, CD38, another NAD+-consuming enzyme, is a key modulator of NAD+ metabolism and influences cell signaling, aging, and tumor biology, suggesting a role for this enzyme as a target with promising therapeutic potential. CD38 was found to be highly expressed in aortic aneurysm tissue, and circulating CD38 was positively correlated with peak wall stress values, but the mechanisms by which CD38 was involved in AAA remain unclear [239].

Hypertension
Hypertension, also known as high or increased blood pressure, is a condition in which blood vessels are subjected to persistently elevated pressure, eventually causing health problems, such as heart disease [240]. The pathogenesis of essential hypertension is complex and likely multifactorial, involving genetic factors, activation of neurohormonal systems, such as the sympathetic nervous system and renin-angiotensin-aldosterone system, obesity, and increased dietary salt intake. This entity involves the interaction of multiple organs and numerous mechanisms, with the kidney playing a central role, as it is both the contributing and the target organ of the hypertensive processes. In accordance with most major guidelines, a subject with hypertension is defined by a systolic blood pressure (SBP) ≥ 140 mm Hg and/or a diastolic blood pressure (DBP) ≥ 90 mm Hg on repeated examination [241].
Studies exploring NAD+ and hypertension relationships in vivo have shown that endothelial dysfunction by SIRT impairment is involved in vascular homeostasis and cardiovascular disease and is related to hypertension [242]. Several studies in hypertensive mice described that, under the pathological stress of hypertension, endothelial-specific deletion of Sirt6 further exacerbated endothelial dysfunction and cardiorenal injuries, indicating the importance of SIRT6 in maintaining endothelial functions and preventing hypertension and its complications by ensuring endothelial function [242,243]. Inhibition of angiotensin II-activated TGF-β by SIRT1 has been described to contribute to the improvement of vascular remodeling (including reduced vascular inflammation and collagen synthesis) in the aortas of transgenic mice, resulting in a decreased SBP [244]. Other in vivo studies have also demonstrated the vasorelaxing capacity of NAM through inhibition of ADP ribosyl cyclase (a mediator of renal vasoconstriction in vivo through the Ca 2+ signaling pathway) [245][246][247] (Table 1). In a previous study performed in spontaneously hypertensive rats receiving NADH, a lower SPB in treated rats versus nontreated rats was observed after 60 days of treatment [248]. In an in vivo study in rats with renal mass reduction, long-term NA supplementation ameliorated hypertension and partially reversed the upregulation of oxidative, inflammatory, and profibrotic mediators in the remnant kidney [249]. NAM has also been used to treat preeclampsia (high blood pressure and proteinuria in pregnant women) in a reduced uterine perfusion pressure mouse model, which was found to be effective in improving and preventing hypertension, fetal growth restriction and premature birth [250]. In a similar study involving 2 mouse models of preeclampsia, the authors concluded that NAM decreased blood pressure and endotheliosis in the mothers, probably by inhibiting ADP ribosyl cyclase, and also prevented fetal growth restriction, probably by normalizing fetal ATP synthesis via the nucleotide salvage pathway [251]. The same group also reported that a pharmacological dose of NAM normalized blood pressure in mice with impaired eNOS function either pharmacologically or genetically by suppressing inflammation and had partial benefits on kidney damage induced by L-NAME [252]. A previous work carried out in C57BL/6J mice demonstrated that upregulation of monocytederived extracellular NAMPT contributed to preservation of myocardial NAD+ levels and functional compensation against pressure overload, which is crucial to avoid HF [253]. In a recent study regarding HF with a HFpEF, oral supplementation of NAD+ precursors, such as NAM, in Dahl salt-sensitive rats, improved hypertension and diastolic dysfunction, and these effects were mediated partly through alleviated systemic comorbidities, enhanced myocardial bioenergetics and ameliorated cardiomyocyte passive stiffness, and calcium-dependent active relaxation [31].
Nonetheless, most authors conclude that the mechanisms underlying the observed effects explaining how NAD+ precursors reduce blood pressure are still unclear. NAD+consuming enzymes, ADP ribosyl cyclase and SIRTs, and the anti-inflammatory effects of NAD+ have been hypothesized to contribute to blood pressure reduction; however, this assertion requires further research [6]. Only pentaerythritoltetran NA reduced the lipid infiltrated area of the aorta [132] NA and pentaerythritoltetran NA Rabbit Dietary supplementation of NA or pentaerythritoltetran NA (0.5% w/w) in a cholesterol-and coconut oil-containing (1% and 3%, respectively) diet 81 days Both drugs reduced the lipid infiltrated area of the aorta [132] Pentaerythritoltetran NA Rabbit Dietary supplementation of pentaerythritoltetran NA (0.5% w/w) in a coconut oil-containing (8% and 15%) diet 120 and 160 days Reduced lipid infiltration in the aorta [131] Pentaerythritoltetran NA Rabbit Dietary supplementation of pentaerythritoltetran NA (0.75% w/w) in a coconut oil-containing (3%) diet. The concentration of cholesterol in the diet was adjusted for each rabbit andt the animals attained a mean plasma cholesterol level of about 6 mg/mL 160 days Reduced aortic cholesterol [130]

NAD+-Increasing Strategies in Human Heart Health: From Bench to Bed
Clinical interest in NAD+-increasing strategies is growing. The use of these compounds offers exciting new biological insights and therapeutic opportunities. However, the results from clinical trials investigating the impact of vitamin B3 intermediate-based therapies on combatting cardiovascular diseases are still scarce (see ongoing clinical trials in Table 2). Thus, we next discuss current research focusing on the impact of NAD+ precursors on the cardiovascular system.

NA
Historically, NA was one of the first drugs used to treat atherogenic hyperlipidemia by virtue of its contribution to reducing LDL cholesterol levels and increasing HDL-C levels [254] (Section 1). The AIM-HIGH trial did not demonstrate a potential benefit of using NA to reduce cardiovascular events in patients with atherosclerotic cardiovascular disease and optimally controlled LDL cholesterol levels by statin therapy [255]. The potential of NA as an NAD+ source has been elusive in former clinical trials [19], and its clinical use has been progressively reduced mainly due to its adverse side effects, such as flushing [20], despite the generation of new formulations, i.e., a currently used PGD2 receptor subtype 1 (DP1) blocker developed with the purpose of decreasing NA side effects [256].
Pellagra is a systemic disease resulting from severe vitamin B3 deficiency. Although symptomatic cardiac involvement is rather infrequent in subjects with pellagra, a recent case of pellagra in a female child was reported to present signs of dilated cardiomyopathy, as revealed by echocardiography. Notably, NA replacement positively influenced cardiac dysfunction [257].

NAM
The clinical use of NAM may also have benefits in the treatment of clinical myocarditis. This condition refers to heart inflammation that can be caused by infectious (viral)-related mechanisms [258] and displays signs similar to those of other causes of inflamed hearts, including cardiac amyloidosis and hypertrophic cardiomyopathy [259,260]. Previous data in the middle of the 20th century demonstrated that NA was effective in the treatment of myocarditis diagnosed on the basis of abnormal electrocardiograms [261,262].
The latter data might be of particular clinical interest in the context of the COVID-19 pandemic. Indeed, myocarditis is one of the clinical findings observed in subjects with SARS-CoV-2 infection [263], and a positive effect of NAM or any other NAD+ precursor could be hypothesized to prevent this adverse cardiac outcome of COVID-19. Notably, SARS-CoV-2 has been recently reported to directly infect cardiomyocytes of patients with COVID-19 myocarditis but not cardiac macrophages, fibroblasts, or endothelial cells [264]. Similar results have also been found in vitro using iPSC-human derived cardiomyocytes [265]. Interestingly, NAD+ depletion may occur secondarily to the activities induced by NAD+-consuming enzymes, i.e., SIRT1 and PARP, which are directly involved in the activation of intracellular immune defenses upon SARS-CoV-2 infection. These data might suggest that NAD+ depletion is critical in the pathogenesis of viral myocarditis and that administration of NAD+ precursors may serve as a promising therapy. Consistently, vitamin B3 intermediates may be effective in promoting innate and adaptive responses to SARS-CoV-2 infection [266][267][268]. In particular, NAM treatment has been effective for reducing adverse disease outcomes of COVID-19 syndrome in other systems apart from the respiratory system in infected subjects, including the renal system [269]. Therefore, NAM and other NAD+ precursors, i.e., NR, can be hypothesized to contribute to NAD+ replenishment and attenuate myocardial damage. In this context, a recent clinical trial aimed to assess whether NR administration would affect the clinical course of the disease; however, myocardial structure or function was not considered an outcome measure (https://clinicaltrials.gov/ct2/show/NCT04407390, accessed on 20 October 2021). NAD+ elevation using NAM is currently under investigation to assess its influence on myocardial injury in patients undergoing on-pump cardiac surgery (https://clinicaltrials. gov/ct2/show/NCT04750616, accessed on 20 October 2021) ( Table 2).

NR
Because NR may be a more suitable NAD+ precursor than NA and NAM [270], the first clinical trials used NR to assess its pharmacokinetics in humans. Clinical trials involving NR supplementation have shown that orally administered NR is well tolerated with no adverse events [54,271]. Single doses of 100, 300 and 1000 mg of NR produced dose-dependent increases in the blood NAD+ metabolome [54].
Diabetic cardiomyopathy, a risk factor for HF, may occur even in well-controlled asymptomatic diabetic patients [272]. Because exogenous administration of NAD+ improves the mitochondrial NAD+ pool and cardiac function [175], its supplementation either directly or indirectly using NAD+ precursors has been proposed to contribute to increasing the mitochondrial NAD+ pool and mitochondrial homeostasis and in turn benefit myocardial health. However, the potential use of such therapies in obese and diabetic hearts requires further investigation [272].
Oral NR administration attenuated proinflammatory activation of PBMCs in 4 of 19 patients with HF [105]. This observation is consistent with findings following 6-week oral NR administration (1000 mg per day) in healthy middle-aged and older humans, which revealed that such treatment reduced SBP and aortic stiffness, two major independent risk factors for incident cardiovascular events and disease with advancing age [270]. However, these observations have not yet been reproduced in other studies and are being addressed in a currently ongoing clinical trial (https://clinicaltrials.gov/ct2/show/NCT03821623, accessed on 20 October 2021). As mentioned previously, in a long-term human cohort study involving patients with HF with HFpEF, high dietary intake of naturally occurring NAD+ precursors was associated with lower blood pressure and a reduced risk of cardiac mortality, suggesting a potential role of NAD+ precursors in treating diastolic dysfunction and HFpEF in humans [31].
Other therapeutic approaches have already been approved; for instance, a phase II clinical trial to assess the efficiency of NR for HF treatment is expected to start soon (https: //anr.fr/Project-ANR-17-CE17-0015, accessed on 20 October 2021). Interestingly, an interventional clinical trial with 30 participants with systolic HF is underway to determine the safety and tolerability of NR (https://clinicaltrials.gov/ct2/show/NCT03423342, accessed on 20 October 2021) ( Table 2), although no results have been published yet. Another interventional randomized placebo-controlled trial with NR treatment in elective left ventricular assist device surgery has been scheduled to demonstrate whether subjects randomized to receive NR will have higher myocardial NAD+ levels, improved mitochondrial function, restored gene expression, and reduced inflammatory responses compared to participants randomized to the placebo group (https://clinicaltrials.gov/ct2/show/NCT04528004, accessed on 20 October 2021) ( Table 2).

NMN
A recent study performed in 48 young middle-aged recreationally trained runners supplemented with NMN for 6 weeks revealed that exercise training combined with NMN further elevated the ventilatory threshold in amateur runners, most likely due to enhanced O 2 utilization of skeletal muscle rather than cardiac muscle [273]. Although cardiac function was not analyzed in this study, recent data in aged Drosophila demonstrated that exercise training can improve lipotoxic cardiomyopathy induced by a highfat diet [274,275]. Because activation of the cardiac NMNAT/NAD+/SIR2/FOXO and NMNAT/NAD+/SIR2/PGC-1α pathways was identified to mediate the beneficial effects of endurance training in this animal model, NMN administration can be hypothesized to further potentiate exercise resistance to high-fat diet-induced cardiomyopathy. Further investigation is warranted to directly address the potential positive influence of NMN on heart health in future clinical trials.

Concluding Remarks
In addition to the long time since the discovery of NAD+, a huge number of studies have been performed, although more work is required to fully understand its role in human diseases. Compelling evidence suggests that NAD+ homeostasis is required for normal cardiovascular function. Numerous studies suggest that declining NAD+ concomitant with mitochondrial dysfunction is a common trait in myocardial and cardiovascular diseases. Over the last few decades, the contribution of NAD+ precursor supplementation to increasing NAD+ levels and improving cardiovascular function in multiple preclinical animal models of several cardiovascular pathologies has attracted therapeutic interest [276]. Furthermore, except for NA, which causes some adverse clinical outcomes, vitamin B3 intermediates have been demonstrated to be safe and to have promising beneficial effects in combating cardiovascular diseases in vivo. Additional basic and translational research is needed to better understand the underlying mechanisms and consequences for cardiac and vascular structure and function. From a translational perspective and despite considerable current efforts, many questions remain in relation to the use of NAD+ precursors, including administration, optimal and safe doses, and the distribution of NAD+ in different tissues, to achieve the desired therapeutic efficacy. Recent and ongoing clinical trials are predominantly investigating NR, followed by NAM and then NMN, while NA is studied the least. Whether these NAD+-increasing therapies will favorably influence cardiovascular outcomes remains to be determined.