NAD⁺ Replenishment Mitigates Cardiomyocyte Senescence and Corrects Heart Failure with Preserved Ejection Fraction in Aged Mice

NAD⁺ Replenishment Mitigates Cardiomyocyte Senescence and Corrects Heart Failure with Preserved Ejection Fraction in Aged Mice | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results NAD⁺ Replenishment Mitigates Cardiomyocyte Senescence and Corrects Heart Failure with Preserved Ejection Fraction in Aged Mice Qingxia Huang , Yongjie Wang , Karina Farias , Xinliu Zeng , Qiuying Chen , Huiyong Cheng , Steven S. Gross , Jacob Geri , Anthony A. Sauve , Yue Yang doi: https://doi.org/10.1101/2025.07.15.664959 Qingxia Huang 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 2 Research Center of Traditional Chinese Medicine, College of Traditional Chinese Medicine, Northeast Asia Research Institute of Traditional Chinese Medicine, Changchun University of Chinese Medicine , Changchun, Jilin, 130021, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yongjie Wang 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 3 Department of Animal Sciences, North Carolina A&T State University , Greensboro, NC 27411 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Karina Farias 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Xinliu Zeng 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 4 Department of Obstetrics and Gynecology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology , Wuhan 430022, China Find this author on Google Scholar Find this author on PubMed Search for this author on this site Qiuying Chen 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Huiyong Cheng 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Steven S. Gross 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jacob Geri 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Anthony A. Sauve 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yue Yang 1 Department of Pharmacology, Weill Cornell Medicine , New York, NY 10021 Find this author on Google Scholar Find this author on PubMed Search for this author on this site For correspondence: yuy2010{at}med.cornell.edu Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Cardiomyocyte senescence, characterized by elevated cell cycle inhibitor expression, persistent DNA damage response, and mitochondrial dysfunction, contributes to myocardial stiffness and the progression of heart failure with preserved ejection fraction (HFpEF), the most common form of heart failure affecting individuals over 65. In this study, we investigated the role of NAD⁺ metabolism in cardiomyocyte senescence and cardiac function. Aged mice exhibited reduced cardiac NAD⁺ levels, impaired NAD⁺ biosynthesis and mobilization, and increased consumption, leading to suppressed SIRT1/6 activity and accumulation of senescent cardiomyocytes. This was accompanied by diastolic dysfunction consistent with HFpEF. In senescent AC16 cardiomyocytes, NAD⁺ depletion promoted senescence, which was reversed by the NAD⁺ precursors nicotinamide riboside (NR) and dihydronicotinamide riboside (NRH). In aged mice, two months of NR or NRH treatment improved diastolic function and reduced cardiomyocyte senescence. While NR primarily activated SIRT1 to suppress cell cycle arrest markers, NRH more robustly activated both SIRT1 and SIRT6, enhancing DNA damage repair. Acetylated H2AX, a SIRT6 substrate elevated in aged hearts and senescent cells, was selectively deacetylated by NRH. These findings identify NAD⁺ availability as a critical regulator of cardiac senescence and support NAD⁺ precursors, particularly NRH, as promising senescence-reducing therapies for treating aging-associated HFpEF. Introduction Cardiomyocytes make up approximately 80% of the cellular volume and 30%-40% of the total cell population in the heart, serving as the primary cell type responsible for normal cardiac function 1 . Although cardiomyocytes are postmitotic and terminally differentiated cells, prolonged stress, such as mitochondrial dysfunction, reactive oxygen species (ROS), and DNA damage response (DDR), can induce senescence-like phenotypes 1 , 2 . Senescent cardiomyocytes exhibit typical senescence markers, including elevated levels of DNA damage marker phosphorylated H2AX at S-139 (γH2AX), cell cycle inhibitors p16 INK4A and p21 WAF1/CIP1 , and senescence-associated beta-galactosidase (SA-β gal) activities from lysosome 3 – 5 . Increased percentage of senescent cardiomyocytes has been found in both aged human and mouse hearts 3 . They exhibit functional decline, including reduced contractility, mitochondrial dysfunction, and hypertrophy, all of which contribute to diastolic dysfunction 6 and the development of heart failure with preserved ejection fraction (HFpEF) 1 , 7 . HFpEF, the most common type of HF in population over 65 years old, is a leading cause of mortality, morbidity, and hospitalization 8 – 10 . Nonetheless, HFpEF still lacks an effective treatment 11 – 14 , and research targeting cardiomyocyte senescence in age-associated HFpEF remains limited. Treatment with senolytic compounds or genetic clearance of senescent cells has been shown to reduce cardiomyocyte hypertrophy and cardiac fibrosis in aged mice 3 , 15 , suggesting that targeting cardiomyocyte senescence is a promising strategy for treating HFpEF. NAD + is one of the most abundant energy-sensing metabolites that functions as a redox cofactor in hundreds metabolic reactions 16 . While the extent of age-related decline in cardiac NAD + levels varies between studies 17 , NAD + depletion has been reported in myocardial tissues from both human and mice with HFpEF 18 , 19 . Low NAD + levels can potentiate mitochondrial dysfunction and cellular senescence 20 , partially through reducing the activity of sirtuins (SIRTs) 21 . SIRTs are a family of NAD + -dependent deacetylases, with nuclear SIRTs playing key roles on inflammation, DNA repairment, and senescence 22 . SIRT1 regulates the transcription of cell cycle inhibitors, through regulating p53-p21 and Rb-p16 23 , 24 pathways. It also controls the release of senescence-associated secretory phenotype (SASP), a wide array of cytokines and chemokines that drives chronic inflammation and tissue remodling, by regulating p53 activity 22 , 25 . Another nuclear isoform, SIRT6, more closely regulate chromatin remodeling, telomere preservation, DNA damage response (DDR) and genomic stability 26 . SIRT6 is more abundantly expressed in the cardiac tissue than SIRT1, and it has higher affinity to NAD + when using histone as substrates, suggesting that SIRT6’s key function in regulating cardiac aging. Knocking out SIRT6 leads to cardiomyocyte senescence and premature death in mice 27 , 28 . Additionally, mitochondrial SIRT3 helps maintain mitochondrial homeostasis and energy metabolism, both of which are known to decline in HFpEF 29 . Recent studies indicate that supplementation with NAD + precursors, such as nicotinamide (NAM), can improve diastolic dysfunction associated with aging, hypertension, or cardiometabolic syndrome 19 . Similarly, treatment with nicotinamide riboside (NR), another NAD + precursor, has been shown to reverse the hyperacetylation of key fatty acid oxidation enzymes, improve mitochondrial function, and ameliorate the HFpEF phenotype 30 – 32 . However, these studies did not examined the cardiac senescence status related to NAD + change. Additionally, these HF models were induced with cardiac pressure overload or other co-morbidities, and they may not faithfully represent the pathogenesis of aging-induced HFpEF. Therefore, there is still a significant knowledge gap in how NAD + levels regulate senescence as a treatment for aging-related HFpEF. To address this gap, we aimed to characterize the impacts of NAD + changes on cardiac senescence and function in aged mice. Additionally, we compared the treatment effect of a novel NAD + precursor, dihydronicotinamide riboside (NRH), one of the most potent NAD + precursors discovered in our lab 33 , to the effect of commonly used NAD + precursor NR. Comparison of NRH and NR treatments provided insights into how different levels of NAD⁺ induction influence their treatment effectiveness and ability to activate distinct SIRT isoforms. Material and Methods Materials and methods are described in the Supplemental Material. Results NAD + balance was largely disturbed in the heart of male aged mice Maintaining NAD + balance is key to preserve normal metabolic activities as well as regulating SIRTs, but the reports on the cardiac NAD⁺ with aging varied significantly. Also, it remains unclear what metabolic shifts are responsible for the disruption of NAD + balance in aged hearts. To characterize the specific alteration in NAD + metabolism in aged hearts, we performed untargeted metabolomic analysis from the cardiac extracts of young (2-month-old) and aged (24-month-old) male C57BL/6JN mice that express both wildtype and mutated Nnt. Nicotinate and nicotinamide metabolism was identified as one of the most significantly impacted pathways in aged heart ( Fig. 1A , S. Table 1 for KEGG pathway analysis). We further confirmed that NAD + concentration has been reduced by 1/3 in aged heart compared to young heart in these mice ( Fig. 1B ). Besides NAD + levels, other NAD⁺-related metabolites, including NADH and NADP⁺, were also significantly reduced and the NAD + /NADH redox ratio was not significantly disturbed ( Fig. 1A ). Additionally, upstream precursors such as NAM, NMN, and NaMN declined, while the NAD⁺ degradation product MeNAM increased, indicating both impaired synthesis and accelerated degradation of NAD⁺. Download figure Open in new tab Figure 1. NAD + balance was largely disturbed in the heart of aged mice. (A) Untargeted metabolomic analysis revealed the top differentially impacted pathways between young and aged hearts (left). Heatmap on the right shows the relative abundance of metabolites as fold over control (FOC) in the Nicotinate and nicotinamide metabolism pathway, N = 3. (B) Cardiac NAD + levels in young (2-4 month) and aged (22-24-month) male C57BL/6 mice, N = 7 per group. (C) Western blot and densitometry quantification of NAD + biosynthesis and mitochondrial storage enzymes in young and aged hearts. (D) Protein expression of NAD + consumers, along with the formation of poly-ADPR proteins (PAR) in the heart. (E) Western blots of SIRT1, 3 and 6, PGC1α and pan acetyl-lysine (Ac-K) modification. (F) Immunoblots of SIRT1/6 targets Ac-p53 (K373/382) and Histone H3 (K9, H3K9), and the quantifications of (D) to (F) blots. N = 6 per group. (G) Illustration of mechanism leading to NAD + depletion in the aged heart. Downregulated metabolites and proteins are shown in red, and upregulated metabolites and proteins are shown in green. All data are shown in mean ± SD, * indicates P < 0.05, **, P < 0.01, ***, P < 0.001 and ****, P < 0.0001. To delineate the sources of NAD⁺ imbalance, we examined the protein levels of key biosynthesis and degradation enzymes. In aged hearts, NAMPT, the rate-limiting enzyme converting NAM to NMN, and NMNAT, which catalyze the conversion of NMN to NAD⁺ in the salvage pathway and of NaMN to NaAD in the Preiss-Handler and the de novo pathways, were all significantly reduced ( Fig. 1C ). ADK, responsible for NRH phosphorylation in NAD⁺ biosynthesis 34 , was also downregulated. Recent discovery of the mitochondrial NAD transporter SLC25A51 also suggests that the mitochondrial NAD⁺ storage can help to replenish depleted subcellular pools and supports overall NAD⁺ homeostasis 35 , 36 . Here we found SLC25A51, along with NMNAT3, which facilitate the interchange between NAD and the storage form NMN in mitochondria, were both decreased ( Fig. 1C ) , suggesting impaired storage and mobilization of mitochondrial NAD⁺ reservoir under deficiency. Collectively, these findings indicate that multiple pathways involved in NAD⁺ synthesis and storage were compromised in the aged heart. NAD⁺ serves as a substrate for SIRTs, poly (ADP-ribose) polymerases (PARPs), and hydrolases/cyclases such as CD38 and SARM1 20 . In aged heart, CD38 and SARM1 proteins were both upregulated ( Fig. 1D ) , consistent with previous studies identifying them as key contributors to age-associated NAD⁺ decline 37 . While PARP1 protein levels remained unchanged, its activation, shown by the formation of poly-ADP-ribose (PAR) on target proteins, was significantly elevated ( Fig. 1D ) , implying increased DDR and NAD⁺ consumption. SIRT activity depends on cellular NAD⁺ availability, but their affinities to NAD⁺ are lower than those of CD38 or PARP1. Activation of other NAD⁺ consumers often depletes NAD⁺ availability leading to inactivation of SIRTs. We then assessed the expression and activities of SIRTs. While the protein levels of SIRT1, SIRT3, and SIRT6 remained unchanged, aged hearts exhibited decreased overall deacetylase activity, as indicated by increased global lysine acetylation (Ac-K). PGC1α, a co-activator of SIRT1 and regulator of mitochondrial biogenesis, was reduced ( Fig. 1E ). Furthermore, increased acetylation of p53 at K382 and histone H3 at K9 (H3K9), known substrates of SIRT1 and SIRT6 38 , 39 , were increased ( Fig. 1F ), further confirmed diminished SIRT activation. Thus, severe NAD⁺ depletion in the aged heart was a combined result from reduced biosynthesis, impaired mitochondrial NAD⁺ mobilization, and increased consumption from CD38, SARM1 and PARP1. As a result, SIRT activities, rather than expression, were compromised due to NAD⁺ deficiency ( Fig. 1G ). NAD + deficient hearts exhibited upregulation of senescence in cardiomyocytes Since SIRT1 and SIRT6 plays key regulating roles on senescence, we next examined the exhibition of senescent markers in aged hearts with NAD + deficiency and inactivated nuclear SIRTs. Whole-heart lysates from aged mice exhibited elevated protein expression of cell cycle arrest markers p16, p21, and the DNA damage marker γH2AX ( Fig. 2A ), alongside increased lysosomal SA-β gal activityies ( Fig. 2B ), indicating an increased presence of senescent cells. Since cardiomyocytes represent the majority mass of the heart, we propose that the elevation in senescent signals is mainly driven by senescence in cardiomyocyte 3 . To test this, we co-staining of γH2AX with WGA which defines cell boundaries, as well as p16 with the cardiomyocyte marker cTnT. In both cases, the positive signal of γH2AX and p16 were confined to the nuclei of WGA-surrounding cardiomyocytes or cTnT positive cells ( Fig. 2C ), confirming increased senescence in cardiomyocytes. Additionally, immunohistochemical staining of p16 confirmed these increased cell cycle arrest markers are expressed in large and elongated nuclei of cardiomyocytes ( Fig. 2D ). Whole heart mRNA further demonstrated that cell cycle arrest markers, including Cdkn1a (p16), Cdkn2a (p21), Cdkn2b (p15), and Gadd45a , were significantly upregulated in the aged heart ( Fig. 2E ). Secretion of SASP is a key feature of senescence and the composition of SASP significantly differ in cell types 3 . Aged mice exhibited elevated expression of Gdf15 , a cardiomyocyte specific SASP gene, and other SASP genes Tnfa , Il10 and Cxcl10 ( Fig. 2E ). These findings suggest that the accumulation of senescent cardiomyocyte is potentially contributing to reducing contractility, local inflammation and increased stiffness in aged ventricular walls 4 , 40 . Download figure Open in new tab Figure 2. Senescence in cardiomyocytes was upregulated in aged hearts. (A) Expression of senescence markers in whole heart lysates. N = 6/group. (B) Whole heart lysate SA-β gal activity measured by ONPG hydrolysis. (C) Immunofluorescent staining and quantification of γH2AX/WGA/DAPI and p16/cTnT/DAPI to identify CM as the source of senescence upregulation. (D) Immunohistochemical DAB staining with Hematoxylin counterstaining and quantification of p16 in the ventricle sections. (E) mRNA expression of common cell cycle inhibitors and SASP component between young and aged heart. N = 5 for (B) - (E) . All data are shown in mean ± SD, * indicates P < 0.05, **, P < 0.01, ***, P < 0.001 and ****, P < 0.0001. Mice with cardiomyocyte senescence exhibited HFpEF phenotypes with mitochondrial dysfunction We next examined cardiac function between young and aged mice. Echocardiography indicated that aged male mice had similar LVEF and fractional shortening (FS) percentages compared to young mice, suggesting preserved systolic function. However, their diastolic functions were impaired, as evidenced by elevated E/e’ ratios ( Fig. 3A , S. Table 2 for detailed parameters). These aged mice exhibited increased heart and lung weights compared to young mice, implying cardiomyocyte hypertrophy and pulmonary congestion. Serum N-terminal prohormone of brain natriuretic peptide (pro-BNP) levels, a clinical marker of heart failure, was also increased in old mice ( Fig. 2B ). Wheat germ agglutinin (WGA) staining revealed increased cardiomyocyte sizes ( Fig. 2C ), while Masson’s trichrome staining indicated more fibrotic area ( Fig. 2D ) in aged ventricles, further confirming that these mice exhibit clinical features of HFpEF. Download figure Open in new tab Figure 3. Mice with cardiomyocyte senescence exhibited HFpEF phenotypes with mitochondrial dysfunction. (A) Representative echocardiography-derived M-mode (top), pulsed-wave Doppler (middle) and tissue Doppler (bottom) tracings in young and aged male mice. Ejection fraction (EF) and fractional shortening (FS) percentages assess left ventricular systolic function, while the E/e’ ratios evaluate diastolic function. (B) The ratios of heart weight to tibia length (HW/TL), lung weight to tibia length (LW/TL) and the serum pro-BNP concentrations between young and aged mice. N = 7-8 for (A) & (B). (C) WGA staining of the left ventricle slide (scale bars = 20 μm) and quantification of cardiomyocyte sizes. (D) Masson’s trichrome staining of left ventricular cross-sections (scale bars = 50 μm) and quantification of interstitial and perivascular fibrosis. (E) Mitochondrial quality examined by the protein expression of 5 OxPhos complexes and VDAC1, and the mitochondrial DNA (mtDNA) over nuclear DNA (HK2) ratios. (F) Presence of oxidative stress in heart lysate examined by immunoblot of lipid peroxidation product 4-HNE, and the quantification of the MDA activity assay between young and aged hearts. N = 5-6 per group. All data are shown in mean ± SD, * indicates P < 0.05, **, P < 0.01, ***, P < 0.001 and ****, P < 0.0001. Mitochondrial dysfunction and excessive ROS production are also key features of HFpEF and cardiomyocyte senescence 1 . To assess mitochondrial integrity, we quantified oxidative phosphorylation (OxPhos) complex levels and found that all five complexes were downregulated in aged hearts ( Fig. 2E ). Additionally, VDAC1, a mitochondrial membrane transporter, was reduced, implying a decrease in mitochondrial quantity, which was further confirmed by marked reduction in mtDNA copy numbers ( Fig. 2E ). Disrupted OxPhos can cause ROS production and oxidative stress 41 . Levels of 4-hydroxynonenal (4-HNE), a lipid peroxidation byproduct, were elevated in aged hearts, indicating oxidative damage. Similarly, malondialdehyde (MDA), another oxidative stress marker, was also increased ( Fig. 2F ). Therefore, HFpEF mice with cardiomyocyte senescence exhibited phenotypes with mitochondrial dysfunction and oxidative stress in aged heart. NAD + levels were driving factors for in vitro cardiomyocyte senescence A low NAD + /NADH ratio is associated with the cell cycle arrest and mitochondrial dysfunction which can promote senescence in fibroblasts 42 . However, whether deficiency in NAD + alone is sufficient to induce senescence in cardiomyocytes has not been evaluated. To address this, we incubated AC16, a human ventricular cardiomyocyte cell line, with FK866, an inhibitor of the NAD + synthesis enzyme NAMPT, to reduce cellular NAD + content ( Fig. 4A ). FK866 treatment led to a 50% reduction in NAD⁺ levels and a significant increase in SA-β gal staining compared to control cells ( Fig. 4B ). The cell cycle arrest marker p16 was also increased in NAD⁺-deficient cells ( Fig. 4C ). Furthermore, doxorubicin (DOX), a known cardiotoxin that induces senescence in AC16 cells 43 – 45 , suppressed cellular NAD⁺ levels ( Fig. 4A ). The combination of FK866 and DOX further reduced NAD⁺ levels and increased p16 expression ( Fig. 4B&C ). These findings confirm that the extent of NAD⁺ depletion is associated with the exacerbation of senescence in AC16 cells. Download figure Open in new tab Figure 4. NAD levels were driving factors for in vitro cardiomyocyte senescence. (A) NAD + levels in AC16 cardiomyocytes treated with 500 nM FK866 (NAMPT inhibitor) or/and 125 nM DOX for 48 hr. N = 4. (B) Representative image of SA-β gal staining in cells treated with FK866 or/and DOX, with blue color developed with X-Gal (scale bars = 200 μm) or fluorescent probe (scale bars = 50 μm). (C) The protein expression of p16 in FK866 or/and DOX treated cell lysates. (D) NAD + levels in AC16 cells treated with 250 μM NRH or NR together with 500 nM FK866 for 48 hr. N = 8. (E) SA-β gal ONPG hydrolysis activities from cells treated with FK866 together with either NRH or NR for 48 hr. (F) Representative IF staining of γH2AX with quantification. Scale bars = 50 μm, N = 3. (G) NAD + levels in AC16 cells treated with 250 μM NRH or NR together with 125 nM DOX for 48 hr. N = 8. (H) Quantified area of SA-β gal staining with X-Gal in AC16 cells treated with DOX together with either NRH or NR. (I) Representative images of SA-β gal staining shown in (H) (scale bars = 200 μm), and IF staining of γH2AX (scale bars = 50 μm). The immunoblots of p16 and p21, and the quantification of γH2AX, p16 and p21, N=3. (J) The gene expression of cell cycle arrest markers. (K) Western blot and quantification of OxPhos Complexes. All data are shown in mean ± SD. Different letter indicates P < 0.05 between groups. If NAD⁺ deficiency drives senescence, then restoring NAD⁺ levels should reverse its progression. To test this hypothesis, we co-treated AC16 cells with NAD⁺ precursors, NRH or NR, alongside FK866. While NRH and NR can both bypass NAMPT to enhance NAD⁺ synthesis, NRH is the more potent NAD⁺ booster, increasing NAD⁺ levels by an average 5-fold, whereas NR increases them by approximately 1.5-fold ( Fig. 4D ). The FK866-induced increase in SA-β gal activity was completely blocked by both NRH and NR treatments ( Fig. 4E ), and both precursors significantly reduced the accumulation of γH2AX in FK866-treated cells ( Fig. 4F ), confirming that FK866-induced senescence originated from NAD⁺ deficiency. Furthermore, NRH more effectively restored NAD⁺ levels compared to NR in DOX-treated cells ( Fig. 4G ). Both NRH and NR prevented DOX-induced SA-β gal staining and γH2AX accumulation ( Fig. 4H&I ). Cell cycle arrest markers were similarly suppressed by NRH and NR at protein and mRNA levels ( Fig. 4I&J ). Additionally, DOX-induced senescence was accompanied by mitochondrial dysfunction, as demonstrated by downregulation of OxPhos complexes I, III, and IV, which were rescued by either NAD⁺ precursor treatments ( Fig. 4K ). Together, these findings indicate that changes in cardiomyocyte NAD⁺ levels are directly associated with senescence progression, and that replenishing NAD⁺ can reduce cardiomyocyte senescence. NAD + precursor treatments improved diastolic function and reduced cardiomyocyte senescence in aged mice To examine if the treatment of NAD + precursors can effectively reduce cardiomyocyte senescence and rescue diastolic functions in the in vivo models of aging-related HFpEF, we administered treatments in aged mice with pre-existing diastolic dysfunction. 22-24-old male C57BL/6 mice were randomized to different groups with matching average E/e’ ratios. The treatment groups received 250 mg/kg NRH or NR through IP injection, 3 times a week for 8 weeks ( Fig. 5A ). Both treatments were well tolerated, as the food intake and body weight change were similar between groups, so were blood glucose and serum alanine aminotransferase (ALT) levels ( S. Fig 1A&B ). Since NRH is a newly discovered compound that does not have a GRAS status like NR, we further examined serum panel for liver and kidney function and found no changes in NRH treated mice ( S. Table 3 ). Additionally, 3/10, 1/10 and 2/8 mice from Ctrl, NRH or NR group were euthanized due to treatment-independent causes during the treatment period, showing the treatments did not significant impact on the survival curve. Download figure Open in new tab Figure 5. NAD + precursor treatments lead to improved diastolic function and reduced cardiac senescence in aged mice. (A) Aged mice were randomly divided into 3 groups based on E/e’ ratio and subsequently administered 250 mg/kg NRH, NR, or PBS through IP injection 3 times a week for 8 weeks. (B-C) Representative echocardiography at week 8 and EF, FS and E/e’ ratios at week 0,4 and 8, * indicate p<0.05 between Ctrl and NRH group. (D) Heart weight and lung weight ratios, and serum pro-BNP levels by the end of 8 weeks. N = 6-10 for (C) & (D). (E) Representative images and quantification of WGA-defined cardiomyocytes size (upper, scale bars = 20 μm) and Masson’s Trichrome stained cardiac fibrotic areas (lower, scale bars = 50 μm). (F ) Representative IF co-staining of γH2AX and WGA, and IHC of p16, and their quantifications. Scale bar = 50 μm. (G) SA-β gal ONPG hydrolysis activities, as well as protein expressions and quantifications of p16, p21 and γH2AX in whole heart lysates. (H) Relative mRNA expression of cell cycle arrest and SASP-related genes in the heart. (I) Quantification of 4-HNE and MDA activities in aged hearts. (J) Protein levels of OxPhos Complexes and mtDNA copy numbers in aged control and treated hearts. N = 5 for (E) - (J) . Data are shown in mean ± SD. Different letter shows P < 0.05 between groups. Echocardiography revealed significant reduction in E/e’ ratios in NRH-treated mice after 8 weeks, and a strong trend (p=0.08) in NR-treated mice, indicating improved diastolic function by both precursors. Systolic function, represented by LVEF and FS, remained unchanged throughout the treatment period ( Fig 5B&C ). Heart and lung weights were reduced, and serum pro-BNP levels were lowered in both treatment groups ( Fig 5D ). Cardiomyocyte sizes and fibrotic areas ( Fig 5E ) were also diminished following treatment of either NAD + precursors, confirming that NRH and NR were comparably effective in reducing hypertrophy and fibrosis. We then evaluated the presence of senescent cardiomyocytes. Immunohistochemistry (IHC) staining revealed both NRH and NR reduced p16 labeling in cardiomyocytes ( Fig 5F ). Similarly, in whole-heart lysates, SA-β gal activity and protein levels of p16 and p21 were decreased by both treatments ( Fig 5G ). However, NRH was more effectively than NR in suppressing γH2AX expression ( Fig 5F&G ). mRNA analysis supports this by showing that while both compounds downregulated Cdkn1a , Cdkn2a , Gadd45a and Cxcl10 , only NRH significantly reduced the cardiomyocyte-specific SASP Gdf15 and another SASP, Il10 ( Fig 5H ). These findings indicate that both NRH and NR can attenuate cell cycle arrest, with NRH showing better efficacy in alleviating DNA damage in cardiomyocytes. Mitochondrial function and antioxidant defense were also enhanced by both NAD + replenishing treatments. NRH markedly increased the protein levels of OxPhos complexes I, IV and V, whereas NR elevated only complex V ( Fig 5J ). This pattern was consistent with mtDNA copy numbers analysis, where NRH induced greater increases in all three mitochondrial markers Rnr2 , Nd1 and Cytb , compared to NR, which elevated mtDNA copy numbers to a lesser extent ( Fig 5J ). Additionally, both NRH and NR significantly reduced oxidative stress markers 4-HNE and MDA ( Fig 5I ), suggesting decreased ROS production and/or enhanced ROS scavenging in the heart. Taken together, while both NRH and NR effectively suppressed cardiomyocyte senescence and improved diastolic function in aged mice, NRH demonstrated greater potency in reducing DNA damage marker and enhancing mitochondrial function. NAD + precursors reprogrammed NAD + metabolism and boosted up cardiac SIRT activities Following IP injection of either NRH or NR, ventricular NAD + levels were significantly elevated, with NRH inducing more increase than NR ( Fig 6A ). To understand their uptake pathways into the heart, we performed metabolomic analysis 30 min after IP injection. NRH treatment led to a robust upregulation of nearly all NAD + metabolites, including an 8-fold increase in NRH itself, supporting our previous finding that NRH can directly enter cardiac cells 46 . NR levels were also significantly elevated by 5-fold, suggesting NRH may be oxidized to NR to join NAD + synthesis following cellular uptake. Similarly NMN was increased by 2.5-fold by NRH but with minimal changes by NR ( Fig 6B&C ). In should be noted that NMN not only serves as an intermediate in NAD biosynthesis, but also as a storage form for mitochondrial NAD, so the increase in NMN could potentially represent elevation in both processes. In contrast, NR treatment did not significantly increase NR or NMN levels in the heart but resulted in a marked elevation of NaMN and a more substantial increase in NaAD, suggesting that NA, derived from NR degradation into NAM followed by microbiome-mediated deamidation 47 , serves as the primary precursor for NAD⁺ synthesis in cardiac cells ( Fig 6B&C ). Download figure Open in new tab Figure 6. NAD + precursor treatments reprogramed NAD + metabolism and enhanced SIRT activities in the aged hearts. (A) Ventricle NAD + levels 30-min after IP injection of 250 mg/kg NRH or NR in young C57BL/6 mice. N = 6-7 per group. (B-C) Relative abundances of metabolites expressed as FOC in the Nicotinate and nicotinamide metabolism pathway extracted from the left ventricles 30-min post IP injection of NRH or NR. N = 3. (D) Western blots and quantification of NAD + biosynthesis, storage and consumption-related enzymes. (E) SIRT-related proteins in the whole heart lysates. (F) Cardiac expressions of p53 and H3K9 acetylation, and AMPK phosphorylation, normalized to their total proteins. N = 5 for (D) to (F) . All data are shown in mean ± SD. Different letter shows P < 0.05 between groups. We next investigated how two months of treatment impact on NAD⁺ metabolizing enzymes involved in both biosynthesis/storage and consumption. While NR did not alter the expression of key participants in these pathways, NRH specifically upregulated the mitochondrial NAD⁺ transporter SLC25A51 and NMNAT3 ( Fig 6D ), which catalyzes the exchange between NAD⁺ and NMN for mitochondrial storage 36 . These increases imply enhanced ability to store and mobilize NAD + in and from the mitochondrial pool. NRH also increased levels of ADK, the kinase required for its phosphorylation, possibly through a feedback mechanism. In term of NAD⁺ consumption, NRH significantly reduced the protein levels of PARP1 ( Fig 6D ), potentially further reducing NAD⁺ depletion rate. We then evaluated SIRT expressions and activities. Protein levels of SIRT1, SIRT3, and SIRT6 remained unchanged in both NRH- and NR-treated hearts, yet global lysine acetylation was significantly reduced, and PGC1α expression was upregulated by NRH treatment, indicating augmented SIRT activities ( Fig 6E ). Both NRH and NR treatment lead to deacetylation of p53, with NRH showing stronger effect ( Fig 6F ). Acetylation of p53 is known to enhances its stability and transcriptional activity, therefore promoting the expression of downstream cell cycle arrest markers p16 and p21 and SASP genes 42 , 48 – 50 . Therefore, nuclear SIRT activation mediated p53 deacetylation can potentially block the senescence, especially during early onset. On the contrary, acetylation of H3K9 was significantly repressed by NRH but not NR treatment. H3K9 is primarily a SIRT6 target and localizes at promoter regions of multiple genes involved in cell cycle arrest and SASP, where its acetylation correlates with transcriptional activation 51 . Thus, the deacetylation of H3K9 induced by NRH likely contributes to further reduction in senescent markers. Additionally, given the prior report that NAD + enhancement can promote SASP secretion in existing senescent fibroblasts through AMPK inhibition 49 , we examined AMPK status in the cardiac tissues. Both NAD + precursors increased AMPK phosphorylation ( Fig 6F ), indicating activation, rather than inhibition, in response to NAD + enhancement, potentially downstream of the SIRT1-LKB1 pathway or increased AMP/ATP ratio 52 . Collectively, both NRH and NR appear to activate SIRT1, while only NRH activated SIRT6, leading to different levels of suppression of senescence pathways. Both NRH and NR induced deacetylation in cardiac proteome SIRTs have extensive substrates in the whole proteome in all different cellular compartments. To further elucidate the mechanism behind NAD + precursors’ protective effect against diastolic dysfunction and senescence, we performed proteomic analysis using acetyl-lysine enriched lysates from the whole heart. Principal Component Analysis of the proteomic data revealed distinctive changes between all three groups ( Fig 7A ). Among the differentially expressed proteins, NRH group has 167 proteins and NR group has 247 proteins that are downregulated (FOC<0.75, P<0.05) compared to aged controls. Among the 115 commonly suppressed proteins, KEGG pathway analysis revealed the top altered pathway being Cytoskeleton in muscle cells, which highly superimpose with other top hits in Focal adhesion, Motor proteins and Muscle contraction, strongly indicating a major modification on the structural components of cardiomyocytes ( Fig 7B ). Download figure Open in new tab Figure 7. NAD + precursors induced deacetylation in cardiac proteome. (A) Principal component analysis (PCA) of acetyl-lysine enriched proteome from Aged, NRH and NR treated hearts. (B) Venn diagram showing commonly downregulated proteins (FOC < 0.75, P < 0.05) in acetyl-lysine–enriched lysates from aged hearts treated with NRH or NR. KEGG pathways show the most significantly downregulated pathways by both NAD + precursor treatments. (C) Heatmap showing the relative expression levels (Log2FC) of participants in the Cytoskeleton in muscle cells pathway. (D) Titin and MYL4 protein levels in acetyl-lysine immunoprecipitated lysates. (E) Volcano plot showing proteins differentially regulated by NRH and NR treatment-induced deacetylation on lysines. (F) The top 10 biological processes more significantly suppressed by NRH than NR (FOC < 0.75, P < 0.05). (G) Heatmap showing the expression levels of proteins participating in the Nucleosome assembly process. *, # , ✝ indicates P < 0.05 between indicated groups. (H) H2AX expressions in acetyl-lysine immunoprecipitated lysates (upper) and immunoblot and quantification of H2AX acetylation at K-5 in whole heart lysates (lower). Different letter shows P<0.05 between groups. N = 6. (I) IF images of Ac-H2AX in left ventricle, scale bar = 20 µm. (J) IF staining of Ac-H2AX in AC16 cells following treatment with NRH or NR, together with 125 nM DOX for 48 hr, scale bar = 20 µm. All data are shown in mean ± SD, different letter shows P < 0.05 between groups From the list of downregulated proteins participating in the cytoskeleton pathway ( Fig 7C ), we identified the giant sarcomeric protein Titin (TTN), which has been previously recognized as a key regulator of diastolic heart function and its performance can be improved through NAD + -SIRT1 mediated deacetylation 19 . We validated decreased TTN levels in acetyl-lysine immunoprecipitated lysates in both NAD + precursor treated groups ( Fig 7D ). Additionally, in the pathway targets we found several members of myosin filaments: MYH4, MYH9, MYL2, MYL4, MYL7, MYL9. Among these, MYL4 has been reported to be related to myocardial hypertrophy 53 , 54 and has predicted acetylation sites at K-10 and K-140 55 . We also observed that MYL4 levels were reduced in acetyl-lysine immunoprecipitated lysates ( Fig 7D ), implying that NAD + precursors mediated SIRT activation can deacetylate these filament proteins and reduce hypertrophy in cardiomyocytes. Despite major increases in mitochondrial DNA and proteins, neither NRH nor NR induced significant mitochondrial protein deacetylation. Among all mitochondrial proteins identified in the acetyl-lysine immunoprecipitated samples, only 14 were commonly decreased by both treatments ( S. Fig 2A ). In contrast, 57 proteins were commonly increased, with the TCA cycle as the most significantly enriched pathway among these targets ( S. Fig 2B ). We suspect this reflects an overall increase in the abundance of mitochondrial proteins, rather than decreased SIRT3-mediated deacetylation. Supporting this, most of the upregulated proteins are key enzymes involved in the TCA cycle, oxidative phosphorylation, fatty acid oxidation, and mitochondrial translation ( S. Fig 2C ), processes aligned with improved mitochondrial function and increased mitochondrial content. The mitochondrial transcription factor TFAM, and the mitochondrial SIRT3 and SIRT5, were also enriched by both treatments in the immunoprecipitated pools ( S. Fig 2D ). To further determine if the changes observed in the acetyl-lysine enriched proteome reflect true differences in acetylation status, we conducted secondary analysis using MSFragger to identify acetyl-modification on the peptides 56 . We detected the acetylation of IDH2, a SIRT3 target, at K-272, and ATP5PB, a subunit of Complex V, at K-225. Both IDH2 and ATP5PB levels were unchanged in the original dataset, but their acetylated peptides were lowered by both NRH and NR ( S. Fig 2E ). These findings suggest that although NAD⁺ replenishment may activate SIRT3-mediated deacetylation, this effect could be partially masked by the concurrent increase in total mitochondrial protein abundance. NRH specifically deacetylated H2AX Although NRH and NR showed many common targets, they also have distinct regulation on 102 proteins ( Fig 7E ). NRH significantly downregulated 53 targets more than NR, with most affected genes located in nucleus and cytosol. GO analysis revealed that Nucleosome assembly was the most differentially impacted biological process by NRH and NR ( Fig 7F ). We then traced back to proteins participating in nucleosome assembly and found 16 proteins in acetyl-enriched dataset ( Fig 7G ), which covered all four core histones, indicating the nucleosome is likely to be immunoprecipitated as a complex. H3-3, the most abundant form of histone H3, was among the differentially regulated targets, supporting our earlier observation that NRH exhibits stronger deacetylation activity on H3K9 compared to NR. Additionally, in NRH-treated heart, less histone H2A, including H2AC20, H2AX and H2AZ, were pulled down by acetyl-lysine antibody, while NR-treated heart displayed slight increase in these proteins, indicating a major differential regulation. Among these H2A subunits, H2AX has well-characterized acetylation sites at K-5 and K-37, which are associated with cell cycle arrest and DDR 57 , 58 . To validate this change, we examined H2AX levels in acetyl-lysine immunoprecipitated lysate and found H2AX level reduced by NRH but not by NR ( Fig 7H ). Similar pattern was observed in AcH2AX(K-5) levels in the whole lysates ( Fig 7H ) and in cardiac sections ( Fig 7I ). In AC16 cells, DOX significantly increased H2AX acetylation, and this modification was reversed by co-treatment with NRH, but not NR ( Fig 7J ). Together, we found that NRH and NR displayed distinct ability to deacetylate histone proteins, and NRH is more effective in deacetylating core histone proteins in cardiomyocytes. SIRT6 mediates the differential impact of NRH and NR Given that nuclear substrates were most differentially affected by NRH and NR, we hypothesized that SIRT1 and SIRT6 were key mediators of the observed changes in senescence pathways. To test this, we used inhibitors and siRNA knockdown to assess their roles in DOX-induced senescent AC16 cells. The NRH-induced reduction in SA-β gal staining was completely abolished by either pharmacological inhibition (EX527 for SIRT1 and OSS_128167 for SIRT6) or knockdown of SIRT1 or SIRT6 ( Fig 8A and S. Fig 3 for validation), indicating NRH-induced strong NAD + elevation activated both SIRT1 and 6 to deliver anti-senescent effects. In contrast, the effect of NR was blocked solely by the absence of SIRT1, not SIRT6, implying that NR-induced moderate NAD + enhancement only activated SIRT1 to reduce senescence ( Fig. 8A ). Download figure Open in new tab Figure 8. NRH specifically deacetylated H2AX through SIRT6. (A) SA-β gal staining with X-gal in AC16 cells treated with DOX and NRH/NR, together with SIRT1 inhibitor (EX527) or SIRT6 inhibitor (OSS_128167), after 48 hr (upper panel, scale bars = 200 μm), and their quantification (bottom left). Lower panels show the SA-β gal staining with fluorescent probe in AC16 cells treated with DOX and NRH/NR, together with siRNA against SIRT1 or SIRT6 for 48 hr (scale bars = 50 μm), and their quantifications (bottom right). (B) Immunoblots of p53 and H3K9 acetylation (left), and their quantifications (right) in DOX-treated AC16 cells incubated with NAD + precursors together with siRNA against SIRT1 or SIRT6. (C) IF staining (scale bar = 20 µm) and quantification of γH2AX and Ac-H2AX expression in AC16 cells treated with DOX and NAD + precursors, with or without siRNA against SIRT1 or SIRT6. (D) H2AX or SIRT1 expression in SIRT6-immunoprecipitated lysates in AC16 cells treated with DOX and NRH or NR. All data are shown in mean ± SD, N = 3. Different letter shows P < 0.05 between groups. We further examined the effects of NRH and NR treatments on the acetylation status of nuclear SIRT targets p53 and H3K9 59 , 60 . We found p53 and H3K9 acetylation reduced by both NRH and NR, and NRH-induced deacetylation was counteracted by the knockdown of either SIRT1 or SIRT6, suggesting the SIRT6 activity is also dependent upon SIRT1 activation. In contrast, the deacetylation effect of NR was only lost upon SIRT1 knockdown ( Fig. 8B ). We then focused on how H2AX acetylation and phosphorylation were influenced by SIRT1 and SIRT6 ( Fig. 8C ). AC6 cells treated with DOX exhibited concurrent increases in acetylation and phosphorylation on H2AX. NRH treatment led to suppression of ac-H2AX and γH2AX at the same time, which were blocked with both SIRT1 and SIRT6 siRNA treatments. In contrast, NR had no effect on H2AX acetylation but moderately reduced H2AX phosphorylation, which was reversed by SIRT1 knockdown but was unaffected by SIRT6, suggesting again that while NR can activate SIRT1 to reduce DNA damage, it cannot sufficiently activate SIRT6 and deacetylate H2AX ( Fig. 8C ), implying that ac-H2AX is a direct target of SIRT6 but not SIRT1. To validate this, we immunoprecipitated SIRT6 in AC16 cells. In control cells, H2AX was immunoprecipitated together with SIRT6. This interaction was diminished following DOX treatment but restored by NRH, not NR ( Fig. 8D ). Additionally, SIRT1 was also pulled down with SIRT6 in control and NRH treated cells, but this interaction was diminished by DOX treatment, with or without NR ( Fig 8D ) . This observation agrees with previous report that SIRT1-mediated SIRT6 deacetylation is a pre-requisite for SIRT6-mediated DDR 60 , and the depletion of either isoform can abolish the deacetylation effect on H2AX induced by NRH treatment. Discussion HFpEF is the most common form of HF among the elderlies, yet it remains without effective treatment 61 . Although many studies use interventions to induce HFpEF by exacerbating comorbidities such as hypertension, obesity, and diabetes, these models do not fully recapitulate the aging-related mechanisms underlying the disease 62 . In this study, we demonstrated that aged male C57BL/6 mice develop HFpEF phenotypes independent of metabolic disturbances but driven by NAD⁺ deficiency-induced cardiomyocyte senescence, which was reversed through NAD⁺ restoration. In aged hearts, impaired NAD⁺ homeostasis, driven by reduced synthesis and increased consumption, leads to SIRT inactivation. The repressed activities, especially on nuclear SIRT1 and SIRT6, promote cell cycle arrest, persistent DDR, mitochondrial dysfunction, and oxidative stress, collectively inducing cardiomyocyte senescence. The accumulation of senescent cells contributes to hypertrophy and fibrosis, increasing ventricular stiffness and leading to diastolic dysfunction and HF. Importantly, restoring cardiac NAD⁺ levels reactivated SIRTs, resulting in deacetylation of p53 and histone proteins, which in turn suppressed cardiomyocyte senescence. Reduction in senescence alleviated hypertrophy, attenuated SASP secretion, lessened fibrosis, leading to improved myocardial relaxation and cardiac function. To our knowledge, this is the first study identifying NAD⁺ deficiency as a central driver of age-related cardiomyocyte senescence and demonstrating that NAD⁺ restoration can mitigate senescence and improve diastolic dysfunction in aged mice. Notably, NRH, a more potent NAD⁺ precursor capable of directly entering cardiomyocytes, exhibited superior activation of SIRT6 and histone deacetylation, offering enhanced anti-senescence benefits. In recent years, accumulating evidence has supported the beneficial effects of NAD⁺ supplementation in various HF models. Supplementation with NAM reduced cardiomyocyte passive stiffness in aged C57BL/6J mice and rats with cardiometabolic syndrome, through SIRT1-mediated TTN deacetylation 19 . Consistent with these findings, we found that both NRH and NR significantly reduced TTN and MYL4 acetylation, confirming that NAD⁺ enhancing treatments can reduce cardiomyocyte hypertrophy and alleviate myocardial stiffness. Additionally, the benefits of NAD⁺ precursors such as NR in HFpEF models, induced by high-fat diet plus L-NAME, have been linked to enhanced SIRT3 activity and improved mitochondrial function 30 . However, another study also suggest that NR-induced mitochondrial improvement can be achieved independent of SIRT3 in chronic pressure overload-induced HF 31 . Consistent with the latter report, our study observed improved mitochondrial quality without significant mitochondrial proteins deacetylation following NRH and NR treatment. Multiple proteins participating in mitochondrial biogenesis or mitochondrial functions were increased in acetyl-lysine enriched lysates, which may reflect overall increase in mitochondrial protein amount by both treatments. Notably, NRH treatment, in particular, enhanced protein levels of complexes I, IV, and V, and upregulated the mitochondrial NAD⁺ transporter SLC25A51 and its partner NMNAT3, suggesting a robust impact on mitochondrial metabolism. While the transcriptional regulation of SLC25A51 and NMNAT3 remains to be understood, it is possible that their upregulation reflects broader activation of PGC1α-driven mitochondrial biogenesis 63 . Increase in PGC1α expression, particularly by NRH treatment, corroborate with this possibility. In general, our findings align with and extend previous work by showing that NAD⁺ precursors improve diastolic function and enhanced mitochondrial biogenesis in the aged heart. The link between NAD⁺ levels and cardiomyocyte senescence were less characterized by previous works. Senescence, a hallmark of aging, is defined by permanent cell cycle arrest in proliferative cells 5 . In terminally differentiated cardiomyocytes, a senescence-like phenotype has been reported in aged human and mouse hearts 3 , as well as in mouse hearts injured by ischemia/reperfusion 15 . These cardiomyocytes exhibit elevated SA-β gal activity, increased expression of cell cycle arrest markers, and elevated secretion of SASP factors such as Gdf15 , Tgfb2 , and Edn3 3 . γH2AX expression was enriched in telomere-associated foci within senescent cardiomyocytes 3 . Consistent with these findings, our analysis of aged mouse hearts showed that NAD⁺ deficiency was associated with increased SA-β gal activity, elevated protein levels of p16 and p21, and accumulation of γH2AX. Although these senescence markers could also arise from other cardiac cell types, the localization of γH2AX and p16 predominantly within cardiomyocyte nuclei suggests that the observed changes primarily reflect cardiomyocyte senescence. Supporting a causal relationship, NAD⁺ depletion in AC16 cells, induced either by inhibiting NAD⁺ synthesis or by DOX, both promoted senescence. Together, these findings indicate that NAD⁺ depletion is sufficient to trigger cardiomyocyte senescence, likely through deacetylation of key transcription factors such as p53 and histone proteins H3 51 , thereby promoting the transcription of cell cycle arrest genes and prolonging DDR. Reducing cardiomyocyte senescence has emerged as a promising strategy to improve cardiac function and survival in aged mice 64 . Among potential interventions, SIRT activators are particularly attractive as they can mitigate senescence without causing substantial structural changes 40 . SIRTs regulate multiple hallmarks of cellular senescence, including genome integrity, mitochondrial dysfunction, inflammation, oxidative stress, ER stress, and metabolic reprogramming 22 , thereby protecting against HF. Inhibition of the NADase CD38 protected cardiomyocytes from senescence through SIRT1 and SIRT6 activation, reducing cardiac hypertrophy and fibrosis in aging models 65 . On the other hand, while NAD⁺ precursor compounds, such as NAM, NR and NRH, are well-established SIRT activators, their specific metabolism, potency, and differential activation of SIRT isoforms in cardiac tissue have not been fully characterized. Our findings show that NAD⁺ precursors with distinct NAD⁺-enhancing capacities exert differential impacts on SIRT1 and SIRT6. Partial NAD⁺ restoration by NR was sufficient to activate SIRT1, deacetylate TTN and inhibit p53-dependent cell cycle arrest markers. In contrast, more potent NAD⁺ replenishment by NRH additionally activated SIRT6, which preferentially deacetylates histones and facilitates more effective DNA damage reduction. SIRT6 plays a crucial role in aging and senescence by binding to H3K9 and γH2AX to recruit DNA repair machinery, including PARP1 60 , 65 . In this study, we identified H2AX is a direct target of SIRT6. Acetylation of H2AX at K5 is essential for the recruitment of DNA repair factors to damaged chromatin, including ATM, which phosphorylates H2AX at S139 to form γH2AX 57 . However, in aged hearts and NAD + deficient cardiomyocytes, both acetylation and phosphorylation levels of H2AX were elevated, suggesting that SIRT6 inactivation-induced H2AX hyperacetylation may prolong DNA damage signaling and promote cellular senescence. Additionally, hyperacetylated H2AX can facilitate the formation of senescence-associated heterochromatin foci (SAHF) 66 . Notably, NRH more effectively reduced the expression of both macroH2A subunits which are markers of SAHF, in acetyl-lysine enriched lysates compared to NR, indicating that NRH may better suppress senescence stabilization. Furthermore, although NRH caused H2AX deacetylation through SIRT6 direct binding, SIRT6 cannot be activated without the presence of SIRT1, which aligned with previous findings that SIRT1-mediated deacetylation of SIRT6 at K33 enhances its recruitment to DNA damage sites 60 . These findings demonstrate a synergistic relationship between SIRT1 and SIRT6 and highlight the importance of potent NAD⁺ replenishment to activate both SIRT1 and SIRT6 for effective mitigation of cellular senescence. In conclusion, our study reveals a central role for NAD⁺ balance in regulating cardiomyocyte senescence and diastolic dysfunction, establishing NAD⁺ as a key metabolic determinant of cardiac aging. We demonstrated that supplementation with NAD⁺ precursors, particularly NRH, a novel compound capable of directly entering cardiomyocytes, could strongly activate SIRT1 and SIRT6. This synergistic activation suppressed the senescence program and mitigated sustained DNA damage responses. Building on our previous findings that NRH confers metabolic benefits by alleviating obesity, hyperglycemia, and hyperlipidemia 46 , this work positions NRH as a promising therapeutic candidate for HFpEF, with the potential to not only reverse aging-related cardiac decline but also reduce cardiovascular risk in older patients. Sources of Funding This work was funded by the NIH/NIA grant R01AG066192 to Y.Y. Disclosures Y.Y. and A.A.S. had intellectual property related to methods of production of NRH. Author Contribution Q.H. collected the data, performed the analysis, wrote the paper; Y.W., K.F., X. Z., and H.C. collected the data; Q.C., S.S.G., J.G. provided consultant and resources for the analysis; A.A.S. obtained initial funding; Y.Y. conceived and designed the experiment, collected the data, wrote the paper and supervised the project. All authors discussed the results and contributed to the final manuscript. Supplemental Materials Supplemental methods Supplemental Table 1-7 Supplemental Figure 1-3 Funder Information Declared National Institutes of Health, https://ror.org/01cwqze88 , R01AG066192 Footnotes ↵ 5 Deceased Nonstandard Abbreviations and Acronyms HFpEF heart failure with preserved ejection fraction SASP senescence-associated secretory phenotype NRH dihydronicotinamide riboside NR nicotinamide riboside SIRT sirtuin HF heart failure LV left ventricle EF ejection fraction FS fractional shortening ROS reactive oxygen species SA-β gal senescence-associated beta-galactosidase NAM nicotinamide NA nicotinic acid H3K9 histone H3 at K9 pro-BNP N-terminal prohormone of brain natriuretic peptide WGA wheat germ agglutinin DOX doxorubicin ALT alanine aminotransferase DDR DNA damage repair SAHF senescence-associated heterochromatin foci MDA malondialdehyde 4-HNE 4-hydroxynonenal OxPhos oxidative phosphorylation Ac-K lysine acetylation Reference ↵ Luan , Y. et al. Cardiac cell senescence: molecular mechanisms, key proteins and therapeutic targets . Cell Death Discov 10 , 78 , doi: 10.1038/s41420-023-01792-5 ( 2024 ). OpenUrl CrossRef PubMed ↵ Grootaert , M. O. J . Cell senescence in cardiometabolic diseases . NPJ Aging 10 , 46 , doi: 10.1038/s41514-024-00170-4 ( 2024 ). OpenUrl CrossRef ↵ Anderson , R. et al. Length-independent telomere damage drives post-mitotic cardiomyocyte senescence . EMBO J 38 , doi: 10.15252/embj.2018100492 ( 2019 ). OpenUrl Abstract / FREE Full Text ↵ Tang , X. , Li , P. H. & Chen , H. Z . Cardiomyocyte Senescence and Cellular Communications Within Myocardial Microenvironments . Front Endocrinol (Lausanne) 11 , 280 , doi: 10.3389/fendo.2020.00280 ( 2020 ). OpenUrl CrossRef PubMed ↵ Suryadevara , V. et al. SenNet recommendations for detecting senescent cells in different tissues . Nat Rev Mol Cell Biol 25 , 1001 – 1023 , doi: 10.1038/s41580-024-00738-8 ( 2024 ). OpenUrl CrossRef PubMed ↵ Zhang , T. Y. , Zhao , B. J. , Wang , T. & Wang , J . Effect of aging and sex on cardiovascular structure and function in wildtype mice assessed with echocardiography . Sci Rep 11 , 22800 , doi: 10.1038/s41598-021-02196-0 ( 2021 ). OpenUrl CrossRef PubMed ↵ Kumar , M. , Yan , P. , Kuchel , G. A. & Xu , M . Cellular Senescence as a Targetable Risk Factor for Cardiovascular Diseases: Therapeutic Implications: JACC Family Series . JACC Basic Transl Sci 9 , 522 – 534 , doi: 10.1016/j.jacbts.2023.12.003 ( 2024 ). OpenUrl CrossRef PubMed ↵ Butrous , H. & Hummel , S. L . Heart Failure in Older Adults . Can J Cardiol 32 , 1140 – 1147 , doi: 10.1016/j.cjca.2016.05.005 ( 2016 ). OpenUrl CrossRef PubMed Stewart , M. W. , Traylor , A. C. & Bratzke , L. C . Nutrition and Cognition in Older Adults With Heart Failure: A Systematic Review . J Gerontol Nurs 41 , 50 – 59 , doi: 10.3928/00989134-20151015-06 ( 2015 ). OpenUrl CrossRef ↵ Savarese , G. & Lund , L. H . Global Public Health Burden of Heart Failure . Card Fail Rev 3 , 7 – 11 , doi: 10.15420/cfr.2016:25:2 ( 2017 ). OpenUrl CrossRef PubMed ↵ Roh , J. , Hill , J. A. , Singh , A. , Valero-Munoz , M. & Sam , F . Heart Failure With Preserved Ejection Fraction: Heterogeneous Syndrome, Diverse Preclinical Models . Circ Res 130 , 1906 – 1925 , doi: 10.1161/CIRCRESAHA.122.320257 ( 2022 ). OpenUrl CrossRef PubMed Shah , S. J. et al. Research Priorities for Heart Failure With Preserved Ejection Fraction: National Heart, Lung, and Blood Institute Working Group Summary . Circulation 141 , 1001 – 1026 , doi: 10.1161/CIRCULATIONAHA.119.041886 ( 2020 ). OpenUrl CrossRef Jasinska-Piadlo , A. & Campbell , P . Management of patients with heart failure and preserved ejection fraction . Heart 109 , 874 – 883 , doi: 10.1136/heartjnl-2022-321097 ( 2023 ). OpenUrl FREE Full Text ↵ Yang , M. et al. Impact of Multimorbidity on Mortality in Heart Failure With Mildly Reduced and Preserved Ejection Fraction . Circ Heart Fail , e011598 , doi: 10.1161/CIRCHEARTFAILURE.124.011598 ( 2025 ). OpenUrl CrossRef ↵ Walaszczyk , A. et al. Pharmacological clearance of senescent cells improves survival and recovery in aged mice following acute myocardial infarction . Aging Cell 18 , e12945 , doi: 10.1111/acel.12945 ( 2019 ). OpenUrl CrossRef PubMed ↵ Yang , Y. & Sauve , A. A . NAD(+) metabolism: Bioenergetics, signaling and manipulation for therapy . Biochim Biophys Acta 1864 , 1787 – 1800 , doi: 10.1016/j.bbapap.2016.06.014 ( 2016 ). OpenUrl CrossRef PubMed ↵ Abdellatif , M. , Sedej , S. & Kroemer , G . NAD(+) Metabolism in Cardiac Health, Aging, and Disease . Circulation 144 , 1795 – 1817 , doi: 10.1161/CIRCULATIONAHA.121.056589 ( 2021 ). OpenUrl CrossRef PubMed ↵ Koay , Y. C. et al. The Efficacy of Risk Factor Modification Compared to NAD(+) Repletion in Diastolic Heart Failure . JACC Basic Transl Sci 9 , 733 – 750 , doi: 10.1016/j.jacbts.2024.01.011 ( 2024 ). OpenUrl CrossRef PubMed ↵ Abdellatif , M. et al. Nicotinamide for the treatment of heart failure with preserved ejection fraction . Sci Transl Med 13 , doi: 10.1126/scitranslmed.abd7064 ( 2021 ). OpenUrl Abstract / FREE Full Text ↵ Chini , C. C. S. , Cordeiro , H. S. , Tran , N. L. K. & Chini , E. N . NAD metabolism: Role in senescence regulation and aging . Aging Cell 23 , e13920 , doi: 10.1111/acel.13920 ( 2024 ). OpenUrl CrossRef ↵ Akar , F. G. & Young , L. H . NAD Repletion Therapy: A Silver Bullet for HFpEF? Circ Res 128 , 1642 – 1645 , doi: 10.1161/CIRCRESAHA.121.319308 ( 2021 ). OpenUrl CrossRef PubMed ↵ Ding , Y. N. , Wang , H. Y. , Chen , X. F. , Tang , X. & Chen , H. Z . Roles of Sirtuins in Cardiovascular Diseases: Mechanisms and Therapeutics . Circ Res 136 , 524 – 550 , doi: 10.1161/CIRCRESAHA.124.325440 ( 2025 ). OpenUrl CrossRef PubMed ↵ Li , Y. & Tollefsbol , T. O . p16(INK4a) suppression by glucose restriction contributes to human cellular lifespan extension through SIRT1-mediated epigenetic and genetic mechanisms . PLoS One 6 , e17421 , doi: 10.1371/journal.pone.0017421 ( 2011 ). OpenUrl CrossRef PubMed ↵ Huang , J. et al. SIRT1 overexpression antagonizes cellular senescence with activated ERK/S6k1 signaling in human diploid fibroblasts . PLoS One 3 , e1710 , doi: 10.1371/journal.pone.0001710 ( 2008 ). OpenUrl CrossRef PubMed ↵ Ungurianu , A. , Zanfirescu , A. & Margina , D . Sirtuins, resveratrol and the intertwining cellular pathways connecting them . Ageing Res Rev 88 , 101936 , doi: 10.1016/j.arr.2023.101936 ( 2023 ). OpenUrl CrossRef PubMed ↵ Ren , S. C. et al. SIRT6 in Vascular Diseases, from Bench to Bedside . Aging Dis 13 , 1015 – 1029 , doi: 10.14336/AD.2021.1204 ( 2022 ). OpenUrl CrossRef PubMed ↵ Pillai , V. B. , Samant , S. , Hund , S. , Gupta , M. & Gupta , M. P . The nuclear sirtuin SIRT6 protects the heart from developing aging-associated myocyte senescence and cardiac hypertrophy . Aging (Albany NY) 13 , 12334 – 12358 , doi: 10.18632/aging.203027 ( 2021 ). OpenUrl CrossRef PubMed ↵ Li , X. et al. The transcription factor GATA6 accelerates vascular smooth muscle cell senescence-related arterial calcification by counteracting the role of anti-aging factor SIRT6 and impeding DNA damage repair . Kidney Int 105 , 115 – 131 , doi: 10.1016/j.kint.2023.09.028 ( 2024 ). OpenUrl CrossRef PubMed ↵ Diao , Z. et al. SIRT3 consolidates heterochromatin and counteracts senescence . Nucleic Acids Res 49 , 4203 – 4219 , doi: 10.1093/nar/gkab161 ( 2021 ). OpenUrl CrossRef PubMed ↵ Tong , D. et al. NAD(+) Repletion Reverses Heart Failure With Preserved Ejection Fraction . Circ Res 128 , 1629 – 1641 , doi: 10.1161/CIRCRESAHA.120.317046 ( 2021 ). OpenUrl CrossRef PubMed ↵ Walker , M. A. et al. Raising NAD(+) Level Stimulates Short-Chain Dehydrogenase/Reductase Proteins to Alleviate Heart Failure Independent of Mitochondrial Protein Deacetylation . Circulation 148 , 2038 – 2057 , doi: 10.1161/CIRCULATIONAHA.123.066039 ( 2023 ). OpenUrl CrossRef PubMed ↵ Diguet , N. et al. Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy . Circulation 137 , 2256 – 2273 , doi: 10.1161/CIRCULATIONAHA.116.026099 ( 2018 ). OpenUrl Abstract / FREE Full Text ↵ Yang , Y. , Mohammed , F. S. , Zhang , N. & Sauve , A. A . Dihydronicotinamide riboside is a potent NAD(+) concentration enhancer in vitro and in vivo . J Biol Chem 294 , 9295 – 9307 , doi: 10.1074/jbc.RA118.005772 ( 2019 ). OpenUrl Abstract / FREE Full Text ↵ Yang , Y. , Zhang , N. , Zhang , G. & Sauve , A. A . NRH salvage and conversion to NAD(+) requires NRH kinase activity by adenosine kinase . Nat Metab 2 , 364 – 379 , doi: 10.1038/s42255-020-0194-9 ( 2020 ). OpenUrl CrossRef ↵ Luongo , T. S. et al. SLC25A51 is a mammalian mitochondrial NAD(+) transporter . Nature 588 , 174 – 179 , doi: 10.1038/s41586-020-2741-7 ( 2020 ). OpenUrl CrossRef PubMed ↵ Hoyland , L. E. et al. Subcellular NAD(+) pools are interconnected and buffered by mitochondrial NAD() . Nat Metab 6 , 2319 – 2337 , doi: 10.1038/s42255-024-01174-w ( 2024 ). OpenUrl CrossRef PubMed ↵ Chini , C. C. S. et al. CD38 ecto-enzyme in immune cells is induced during aging and regulates NAD(+) and NMN levels . Nat Metab 2 , 1284 – 1304 , doi: 10.1038/s42255-020-00298-z ( 2020 ). OpenUrl CrossRef PubMed ↵ Zhang , C. et al. Resveratrol attenuates doxorubicin-induced cardiomyocyte apoptosis in mice through SIRT1-mediated deacetylation of p53 . Cardiovasc Res 90 , 538 – 545 , doi: 10.1093/cvr/cvr022 ( 2011 ). OpenUrl CrossRef PubMed ↵ Wood , M. , Rymarchyk , S. , Zheng , S. & Cen , Y . Trichostatin A inhibits deacetylation of histone H3 and p53 by SIRT6 . Arch Biochem Biophys 638 , 8 – 17 , doi: 10.1016/j.abb.2017.12.009 ( 2018 ). OpenUrl CrossRef PubMed ↵ Zhai , P. & Sadoshima , J . Cardiomyocyte senescence and the potential therapeutic role of senolytics in the heart . J Cardiovasc Aging 4 , doi: 10.20517/jca.2024.06 ( 2024 ). OpenUrl CrossRef ↵ Murphy , M. P . How mitochondria produce reactive oxygen species . Biochem J 417 , 1 – 13 , doi: 10.1042/BJ20081386 ( 2009 ). OpenUrl Abstract / FREE Full Text ↵ Wiley , C. D. et al. Mitochondrial Dysfunction Induces Senescence with a Distinct Secretory Phenotype . Cell Metab 23 , 303 – 314 , doi: 10.1016/j.cmet.2015.11.011 ( 2016 ). OpenUrl CrossRef PubMed ↵ Gewirtz , D. A . A critical evaluation of the mechanisms of action proposed for the antitumor effects of the anthracycline antibiotics adriamycin and daunorubicin . Biochem Pharmacol 57 , 727 – 741 , doi: 10.1016/s0006-2952(98)00307-4 ( 1999 ). OpenUrl CrossRef PubMed Web of Science Octavia , Y. et al. Doxorubicin-induced cardiomyopathy: from molecular mechanisms to therapeutic strategies . J Mol Cell Cardiol 52 , 1213 – 1225 , doi: 10.1016/j.yjmcc.2012.03.006 ( 2012 ). OpenUrl CrossRef PubMed ↵ Dhingra , R. et al. Bnip3 mediates doxorubicin-induced cardiac myocyte necrosis and mortality through changes in mitochondrial signaling . Proc Natl Acad Sci U S A 111 , E5537 – 5544 , doi: 10.1073/pnas.1414665111 ( 2014 ). OpenUrl Abstract / FREE Full Text ↵ Zeng , X. et al. NRH, a potent NAD(+) enhancer, improves glucose homeostasis and lipid metabolism in diet-induced obese mice through an active adenosine kinase pathway . Metabolism 164 , 156110 , doi: 10.1016/j.metabol.2024.156110 ( 2025 ). OpenUrl CrossRef PubMed ↵ Liu , L. et al. Quantitative Analysis of NAD Synthesis-Breakdown Fluxes . Cell Metab 27 , 1067 – 1080 e1065 , doi: 10.1016/j.cmet.2018.03.018 ( 2018 ). OpenUrl CrossRef PubMed ↵ Zhao , Y. et al. Acetylation of p53 at lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces expression of p21(Waf1/Cip1) . Mol Cell Biol 26 , 2782 – 2790 , doi: 10.1128/MCB.26.7.2782-2790.2006 ( 2006 ). OpenUrl Abstract / FREE Full Text ↵ Nacarelli , T. et al. NAD(+) metabolism governs the proinflammatory senescence-associated secretome . Nat Cell Biol 21 , 397 – 407 , doi: 10.1038/s41556-019-0287-4 ( 2019 ). OpenUrl CrossRef PubMed ↵ Brooks , C. L. & Gu , W . The impact of acetylation and deacetylation on the p53 pathway . Protein Cell 2 , 456 – 462 , doi: 10.1007/s13238-011-1063-9 ( 2011 ). OpenUrl CrossRef PubMed ↵ Michishita , E. et al. SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin . Nature 452 , 492 – 496 , doi: 10.1038/nature06736 ( 2008 ). OpenUrl CrossRef PubMed Web of Science ↵ Hou , X. et al. SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase . J Biol Chem 283 , 20015 – 20026 , doi: 10.1074/jbc.M802187200 ( 2008 ). OpenUrl Abstract / FREE Full Text ↵ Bajpai , A. K. et al. Exploring the Regulation and Function of Rpl3l in the Development of Early-Onset Dilated Cardiomyopathy and Congestive Heart Failure Using Systems Genetics Approach . Genes (Basel) 15 , doi: 10.3390/genes15010053 ( 2023 ). OpenUrl CrossRef PubMed ↵ Zhong , Y. et al. Myosin light-chain 4 gene-transfer attenuates atrial fibrosis while correcting autophagic flux dysregulation . Redox Biol 60 , 102606 , doi: 10.1016/j.redox.2023.102606 ( 2023 ). OpenUrl CrossRef PubMed ↵ Yang , L. et al. The fasted/fed mouse metabolic acetylome: N6-acetylation differences suggest acetylation coordinates organ-specific fuel switching . J Proteome Res 10 , 4134 – 4149 , doi: 10.1021/pr200313x ( 2011 ). OpenUrl CrossRef PubMed Web of Science ↵ Kong , A. T. , Leprevost , F. V. , Avtonomov , D. M. , Mellacheruvu , D. & Nesvizhskii , A. I . MSFragger: ultrafast and comprehensive peptide identification in mass spectrometry-based proteomics . Nat Methods 14 , 513 – 520 , doi: 10.1038/nmeth.4256 ( 2017 ). OpenUrl CrossRef PubMed ↵ Ikura , M. et al. Acetylation of Histone H2AX at Lys 5 by the TIP60 Histone Acetyltransferase Complex Is Essential for the Dynamic Binding of NBS1 to Damaged Chromatin . Mol Cell Biol 35 , 4147 – 4157 , doi: 10.1128/MCB.00757-15 ( 2015 ). OpenUrl Abstract / FREE Full Text ↵ Kuno , A. et al. SIRT1 in the cardiomyocyte counteracts doxorubicin-induced cardiotoxicity via regulating histone H2AX . Cardiovasc Res 118 , 3360 – 3373 , doi: 10.1093/cvr/cvac026 ( 2023 ). OpenUrl CrossRef PubMed ↵ Zhang , P. et al. Tumor suppressor p53 cooperates with SIRT6 to regulate gluconeogenesis by promoting FoxO1 nuclear exclusion . Proc Natl Acad Sci U S A 111 , 10684 – 10689 , doi: 10.1073/pnas.1411026111 ( 2014 ). OpenUrl Abstract / FREE Full Text ↵ Meng , F. et al. Synergy between SIRT1 and SIRT6 helps recognize DNA breaks and potentiates the DNA damage response and repair in humans and mice . Elife 9 , doi: 10.7554/eLife.55828 ( 2020 ). OpenUrl CrossRef PubMed ↵ He , S. et al. The role of cardiomyocyte senescence in cardiovascular diseases: A molecular biology update . Eur J Pharmacol 983 , 176961 , doi: 10.1016/j.ejphar.2024.176961 ( 2024 ). OpenUrl CrossRef PubMed ↵ van Ham , W. B. et al. Clinical Phenotypes of Heart Failure With Preserved Ejection Fraction to Select Preclinical Animal Models . JACC Basic Transl Sci 7 , 844 – 857 , doi: 10.1016/j.jacbts.2021.12.009 ( 2022 ). OpenUrl CrossRef PubMed ↵ Le Couteur , D. G. et al. Nutritional reprogramming of mouse liver proteome is dampened by metformin, resveratrol, and rapamycin . Cell Metab 33 , 2367 – 2379 e2364 , doi: 10.1016/j.cmet.2021.10.016 ( 2021 ). OpenUrl CrossRef PubMed ↵ Xu , M. et al. Senolytics improve physical function and increase lifespan in old age . Nat Med 24 , 1246 – 1256 , doi: 10.1038/s41591-018-0092-9 ( 2018 ). OpenUrl CrossRef PubMed ↵ Zhou , H. et al. Downregulation of Sirt6 by CD38 promotes cell senescence and aging . Aging (Albany NY) 14 , 9730 – 9757 , doi: 10.18632/aging.204425 ( 2022 ). OpenUrl CrossRef PubMed ↵ Corpet , A. & Stucki , M . Chromatin maintenance and dynamics in senescence: a spotlight on SAHF formation and the epigenome of senescent cells . Chromosoma 123 , 423 – 436 , doi: 10.1007/s00412-014-0469-6 ( 2014 ). OpenUrl CrossRef PubMed View the discussion thread. Back to top Previous Next Posted July 18, 2025. Download PDF Supplementary Material Email Thank you for your interest in spreading the word about bioRxiv. NOTE: Your email address is requested solely to identify you as the sender of this article. Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. 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Sauve , Yue Yang bioRxiv 2025.07.15.664959; doi: https://doi.org/10.1101/2025.07.15.664959 Share This Article: Copy Citation Tools NAD⁺ Replenishment Mitigates Cardiomyocyte Senescence and Corrects Heart Failure with Preserved Ejection Fraction in Aged Mice Qingxia Huang , Yongjie Wang , Karina Farias , Xinliu Zeng , Qiuying Chen , Huiyong Cheng , Steven S. Gross , Jacob Geri , Anthony A. 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