Nicotinamide for the treatment of heart failure with preserved ejection fraction

Abstract

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent and intractable form of cardiac decompensation commonly associated with diastolic dysfunction. Here, we show that diastolic dysfunction in patients with HFpEF is associated with a cardiac deficit in nicotinamide adenine dinucleotide (NAD⁺). Elevating NAD⁺ by oral supplementation of its precursor, nicotinamide, improved diastolic dysfunction induced by aging (in 2-year-old C57BL/6J mice), hypertension (in Dahl salt-sensitive rats), or cardiometabolic syndrome (in ZSF1 obese rats). This effect was mediated partly through alleviated systemic comorbidities and enhanced myocardial bioenergetics. Simultaneously, nicotinamide directly improved cardiomyocyte passive stiffness and calcium-dependent active relaxation through increased deacetylation of titin and the sarcoplasmic reticulum calcium adenosine triphosphatase 2a, respectively. In a long-term human cohort study, high dietary intake of naturally occurring NAD⁺ precursors was associated with lower blood pressure and reduced risk of cardiac mortality. Collectively, these results suggest NAD⁺ precursors, and especially nicotinamide, as potential therapeutic agents to treat diastolic dysfunction and HFpEF in humans.

Fig. 1. Oral supplementation of NAM improves diastolic dysfunction in ZSF1 obese rats.

(A) Schematic overview of nicotinamide (NAM) feeding protocol to male and female ZSF1 obese rats with cardiometabolic syndrome and HFpEF. NAM (40 mM) was added to the drinking water starting at the age of 8 weeks and after 3 months cardiac parameters were assessed. (B) Representative echocardiography-derived M-mode tracings, (C) left ventricular mass indexed to body surface area (LVmassi), (D) ejection fraction (EF), (E) representative pulsed-wave Doppler (top) and tissue Doppler (bottom) tracings, and (F) ratio of peak early Doppler transmitral flow velocity (E) to myocardial tissue Doppler velocity (e’), (n=8/9/10 and 8/9/11 rats for lean/obese/obese+NAM in males and females, respectively). (G) Invasive hemodynamic assessment of left ventricular end-diastolic pressure (EDP), (H) myocardial stiffness constant (indexed end-diastolic pressure volume exponential relationship coefficient β. EDPVR β_i), (I and J) end-systolic and end-diastolic pressure volume relationships generated by inferior vena cava occlusions with corresponding pressure-volume loops in (I) male and (J) female ZSF1 rats, (n=5/9/8 and 5/6/7 rats for lean/obese/obese+NAM in males and females, respectively). (K) Lung weight normalized to tibia length (LW/TL), (n=8/9/11 rats for lean/obese/obese+NAM in either sex). (L) Maximal oxygen consumption (VO²max), (M) running distance, and (N) workload in ZSF1 rats during exercise exhaustion testing, (n=6/8/10 and 8/9/9 rats for lean/obese/obese+NAM in males and females, respectively). All results were generated from 2 independent cohorts. Indicated P values on top of panels (C and D, F to H, and K to N) represent factor comparisons by a linear mixed model including sex (s) and group (g) as fixed factors and the cohort as a random factor; following comparisons between different groups within the respective sex are indicated by Bonferroni post-hoc test. EDPVR α_i was included in the model as a covariate in (F). Bars and error bars show means and SEM, respectively, with individual data points superimposed.

Fig. 2. NAM ameliorates blood pressure control and adiposity in ZSF1 obese rats.

(A and B) Systolic arterial blood pressure, non-invasively measured by the tail-cuff method, in (A) male and (B) female ZSF1 obese rats treated or not with nicotinamide (NAM) starting from 8 weeks. (n=10/11 and 9/11 rats in obese/obese+NAM in males and females, respectively). (C and D) Acetylcholine-induced vasodilation measured ex vivo in the aortic rings of (C) male and (D) female rats; Inset graphs denote corresponding EC₅₀ of single rings, (n=20/24 and 22/23 rings obtained from 4 obese/obese+NAM in males and females, respectively). (E and F) Body weight gain of (E) male and (F) female ZSF1 obese rats and their lean controls, (n=8/9/11 and 10/9/11 rats in lean/obese/obese+NAM for males and females, respectively). (G) Visceral white adipose tissue (WAT) normalized to tibia length (TL), (n=8/9/11 rats for lean/obese/obese+NAM in either gender). (H) Feeding efficiency (body weight gain per consumed kcal energy) measured during the first 2-3 weeks of treatment before the obvious body weight difference thereafter, (n=8/14/11 and 8/13/8 rats in lean/obese/obese+NAM for males and females, respectively). (I) Energy expenditure (shown as a function of body weight) evaluated by indirect calorimetry, and (J) estimated energy expenditure (during dark and light cycles of the day) at a hypothetical equal body weight (490.6 g) in all groups as derived from corresponding analysis of covariance (ANCOVA), (n=4 male rats per group). L, light; D, dark. All results except in I and J were generated from 2 independent cohorts. (A to F) P values were calculated by two-way repeated measures ANOVA, followed by Bonferroni-corrected pairwise comparisons; (C and D) EC₅₀ were compared by Mann-Whitney test. (G and H) Indicated P values on top of panels represent factor comparisons by a linear mixed model including sex (s) and group (g) as fixed factors and the cohort as a random factor, and following comparisons between different groups within respective sex are indicated by Bonferroni post-hoc tests. (J) Factorial ANCOVA was used including group (g) and time of the day (t; light vs. dark 12h cycles) as fixed factors and body weight as a covariate, followed by Bonferroni-corrected pairwise comparisons. Circles/Bars and error bars show means and SEM, respectively, with individual data points superimposed, except for (J) where the means and SEM were derived from ANCOVA and corresponding individual data points are shown in (I).

Fig. 3. NAM-mediated metabolic reprogramming improves energy homeostasis in HFpEF.

(A) Heatmap depicting relative abundance of significantly differentiated metabolites by nicotinamide (NAM) in the plasma of 12-week-old ZSF1 obese rats (4 weeks of NAM treatment) that were fasted for 6 hours, (n=3/7/7 rats in lean/obese/obese+NAM). (B) Correlation of NAM-induced metabolomic changes vs. the difference between ZSF1 lean and obese controls (Log₂[fold-change, FC]). (C) Volcano plot showing up- and down-regulated metabolites of NAM-treated vs. control ZSF1 obese rats. (D and E) Relative difference in selected circulating metabolites related to (D) branched-chain amino acids metabolism (left) and glycolysis (right), as well as (E) lipolysis and ketogenesis (shown are polyunsaturated fatty acids and ketone bodies, please see also fig. S7 for other related metabolites), (n=3/7/7 rats in lean/obese/obese+NAM). (F) Relative abundance of tricarboxylic acid cycle (TCA) metabolites in left ventricular tissue of 20-week-old NAM-treated ZSF1 obese rats compared to age-matched obese and lean controls, (n=3/6/8 rats in lean/obese/obese+NAM). (G) Heatmaps of cardiac transcripts showing the expression (red=high, blue=low) of differentially regulated genes involved in TCA cycle in ZSF1 lean, obese and NAM-treated obese rats, (n=4 rats per group). (H and I) High-energy phosphate compounds in (H) the heart and (I) skeletal muscle (i.e., gastrocnemius) of ZSF1 rats, (n=4/7/4 and 11/12/21 rats in lean/obese/obese+NAM for heart and skeletal muscle, respectively). PCr, phosphocreatine; ATP, adenosine triphosphate; ADP, adenosine diphosphate. (J) Schematic representation of affected metabolic pathways by NAM supplementation. Abbreviations: BCAA, branched-chain amino acids; BCKA, branched-chain keto acids; G6P, glucose-6-phosphate. P values were calculated by (B) Pearson correlation, (C) Welch t-test or (D to F and H to I) ANOVA with Dunnett’s post-hoc test. Bars and error bars show means and SEM, respectively, with individual data points superimposed.

Fig. 4. SERCA2a deacetylation contributes to improved diastolic function by NAM.

(A) Venn diagram (left) showing the overlap of detected acetylated proteins in the hearts of control and NAM-treated 20-week-old male ZSF1 obese rats, along with their subcellular localization in a pie chart (right). (B) Distribution of NAM-induced changes in acetylation of cardiac peptides (mitochondrial and non-mitochondrial); the dashed line denotes acetylation in control ZSF1 obese rats. The inset figure denotes the sum of peptides with up- or down-regulated acetylation. (C) Relative signal intensity difference of significantly regulated acetylation sites in titin and SERCA2a (Atp2a3). Note that negative values indicate deacetylation and positive ones indicate acetylation, (n=4 obese+NAM compared to the average of 3 obese). (D) Diastolic (Dia) calcium and (E) changes in calcium transient amplitude as indicated by Fura-2/AM ratio (340:380 nm), (F) time to 90% decay (DT₉₀), along with (G) simultaneously measured sarcomere shortening, (H) time to 90% relaxation (RT₉₀) and (I) maximal relaxation rate of electrically-paced adult ZSF1 obese and lean cardiomyocytes that were pre-incubated or not with NAM (100 μM) for one hour, (n=18/20/18/23 cardiomyocytes in lean/lean+NAM/obese/obese+NAM isolated from 4 lean and 4 obese ZSF1 rats at the age of 20 weeks). P values were calculated by (C) one-sample t-test or (D to I) two-way repeated measures ANOVA. Bars/circles and error bars show means and SEM, respectively, with individual data points superimposed.

Fig. 5. Titin deacetylation is sufficient to improve cardiomyocyte passive stiffness.

(A) The relationship between passive force (F_passive) and sarcomere length, indicative of passive stiffness, in skinned cardiomyocytes isolated from 20-week-old ZSF1 lean and obese rats treated or not with nicotinamide (NAM). Exponential curves are fitted to the group average (n=18/16/21 cardiomyocytes in lean/obese/obese+NAM isolated from 4/3/4 rats, respectively). (B and C) Representative Western blot (top) and quantification (bottom) of (B) cardiac titin acetylation (normalized to GAPDH) in 20-week-old ZSF1 rats, (n=7/18/17 rats in lean/obese/obese+NAM), (C) In vitro deacetylation of titin N2B isoform in skinned cardiomyocytes from healthy 20-week-old Wistar-Kyoto rats. Cells were incubated with recombinant SIRT1 for 2 hours at 30°C. Representative Western blot (top panels) probed with acetylated-lysine-specific antibodies for detection of total N2B acetylation; GAPDH antibody was used as a loading control. Lower panel shows quantification of titin-N2B acetylation normalized to GAPDH (N=4 rats per group). (D) Relative change in passive force of isolated skinned cardiomyocytes upon deacetylation with recombinant SIRT1 compared to control cells force that is measured in the same buffer but without SIRT1, (n=3-8 cardiomyocytes per condition and per time point). Data points are fitted using a one-phase decay curve fit. (E) Representative micrographs and (F) quantification of left ventricular (LV) fibrotic remodelling due to collagen accumulation, as evaluated by picrosirius red staining in 20-week-old male and female ZSF1 rats, (n=4/4/6 and 4/4/5 for lean/obese/obese+NAM in males and females, respectively). P values were calculated by (A) two-way repeated measures ANOVA with Dunnett’s post-hoc test, (B) ANOVA with Dunnett’s post-hoc test, (C and D) Welch’s t-test, or two-way independent ANOVA including group (g) and sex (s) as fixed factors with Bonferroni-corrected pairwise comparisons. Bars and error bars show means and SEM, respectively, with individual data points superimposed.

Fig. 6. NAM improves diastolic dysfunction in hypertensive Dahl rats.

(A) Schematic representation of nicotinamide (NAM) supplementation to hypertensive Dahl salt-sensitive rats. Seven-week-old male rats were fed a high-salt diet (8% NaCl) for 5 weeks followed by treatment in the form of a single intraperitoneal injection of furosemide (10 mg/kg body weight) and a shift to low-salt diet (0.3% NaCl) combined with 40 mM NAM in the drinking water (high-salt+NAM) or not (high-salt). Healthy controls were fed the low-salt diet throughout the experiment (low-salt ctrl). (B) Systolic, (C) diastolic and (D) mean arterial blood pressure, non-invasively measured by the tail-cuff method. (E) Representative echocardiography-derived M-mode tracings, (F) left ventricular ejection fraction (EF), (G) representative pulsed-wave Doppler (top) and tissue Doppler (bottom) tracings, and (H) ratio of peak early Doppler transmitral flow velocity (E) to myocardial tissue Doppler velocity (e’). (I) End-diastolic pressure (EDP) invasively measured by intracardiac catheterization. (J) Heart weight (HW; left) and lung weight (LW; right) normalized to tibia length (TL). (K) Maximal oxygen consumption (VO2max), (L) running distance, and (M) workload during exercise exhaustion testing. (N) Representative Western blot (top) and quantification (bottom) of cardiac SERCA2a acetylation (normalized to total SERCA2a expression), and (O) titin acetylation (normalized to GAPDH). (P) Relative abundance of cardiac unsaturated fatty acids differentially regulated by NAM in 18-week-old Dahl salt-sensitive rats, (n=4-6 rats per group). P values were calculated by (B to D) factorial repeated measures ANOVA (including age and groups as fixed factors) followed by Games-Howell post-hoc test or (F and H to P) ANOVA with Dunnett’s post-hoc test. Bars/line and error bars show means and SEM, respectively, with individual data points superimposed.

Fig. 7. NAM attenuates age-related diastolic function decline in C57BL/6J mice.

(A) Schematic overview of nicotinamide (NAM) feeding protocol to 24-month-old C57BL/6J male mice. NAM (24 mM) was added to the drinking water starting at the age of 20 months, and after 4 months cardiac parameters were assessed as shown. (B) Representative echocardiography-derived M-mode tracings, and (C) left ventricular ejection fraction (EF). (D) Heart weight normalized to tibia length (HW/TL). (E) Representative echocardiographic pulsed-wave Doppler (top) and tissue Doppler (bottom) tracings, and measures of diastolic dysfunction, including (F) the ratio of peak early Doppler transmitral flow velocity (E) to myocardial tissue Doppler velocity (e’) and (G) isovolumic relaxation time (IVRT), (n=6/11/11 mice in young/aged/aged+NAM). (H and I) Representative Western blot (top) and quantification (bottom) of (H) cardiac SERCA2a acetylation (normalized to total SERCA2a expression), and (I) titin acetylation (normalized to GAPDH), (n=7-8 mice per group). P values were calculated by ANOVA with Dunnett’s post-hoc test. Bars/line and error bars show means and SEM, respectively, with individual data points superimposed.

Fig. 8. Higher NAD⁺ is associated with lower risk of HFpEF and cardiac death in humans.

(A and B) Association of baseline (year 1995) dietary intake of niacin and niacin equivalents (NE) with (A) all-cause (n=335) and cardiac (n=77) mortality during a 20-year follow-up (1995-2015) or (B) systolic and diastolic blood pressure in the Bruneck Study (n=815). (A) Hazard ratios and (B) effect sizes were calculated for a 1-SD unit increase of calorie-adjusted loge-transformed niacin or NE intake. Model 1 (M1) was adjusted for total caloric intake; Model 2 (M2) was additionally adjusted for age, sex, current smoking, diabetes, alcohol intake, hypertension, body-mass index and total cholesterol. (C and D) Relative abundance of (C) cardiac NAD⁺ and (D) plasma NAM in human HFpEF and non-failing hearts (n=12/10 and 10/9 samples, respectively, in non-failing and HFpEF). Bars and error bars show means and SEM, respectively, with individual data points superimposed. P values were calculated by (A) Cox regression analysis, (B) linear regression analysis or (C and D) Welch t-test.