# Sex-specific benefits of a combined supplementation of B vitamins, nicotinamide riboside, folate and cobalamin, in a murine model of heart failure

Solène E. Boitard¹, Morgane Delouche¹, Ahmed Karoui¹†, Mélanie Gressette¹†, Iman Momken¹,², Bertrand Bouchard³, Françoise Mercier-Nomé⁵, Apolline Imbard⁶, Christophe Lemaire¹,⁷, Anne Garnier¹, Matthieu Ruiz³,<fnref n="4" />, Mathias Mericskay¹*† and Jérôme Piquereau¹,⁸*†

## Abstract

Despite a substantial therapeutic arsenal to treat patients affected by heart failure (HF), no treatment specifically targets alterations of cardiac energy metabolism described in HF. Based on the results of previous studies demonstrating the cardiac preventive effects of B vitamins when introduced before inducing cardiac pressure overload in mice, we investigated the efficacy of a diet supplemented with a B vitamin cocktail (B3, B9 and B12 (3VitB)) to restore energy metabolism and improve cardiac function in an animal model of established HF. Four weeks after transverse aortic constriction (TAC) induction, male and female mice were treated with 3VitB. 3VitB increased life expectancy and reduced the TAC-induced alterations of cardiac parameters in males. Although these effects on survival and cardiac function were less clear in females due to their higher resistance to TAC, the 3VitB cocktail was beneficial in females as 8 weeks of treatment improved physical capacities and led to milder cardiomyocyte stress-induced hypertrophy in similar ways to those observed in males. In both sexes, 3VitB protected cardiac mitochondrial oxidative capacities, at least by supporting the process of mitochondrial biogenesis. Interestingly, our results revealed sex-specificities not only in response to cardiac pressure overload but also in response to 3VitB treatment. Overall, this study demonstrated the efficacy of 3VitB to preserved cardiac function and energy metabolism in an established HF model, especially in males that are more sensitive to cardiac pressure overload. This confers credit to vitamin supplementations and to metabolic therapy as new strategies to treat HF.

Full list of author information is available at the end of the article

<figure><img src="image_1.png" /></figure>

BMC

© The Author(s) 2025. **Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

<footnote>†Ahmed Karoui and Mélanie Gressette contributed equally to this work.</footnote>

<footnote n="4">⁴Mathias Mericskay and Jérôme Piquereau contributed equally to this work.</footnote>

<footnote>*Correspondence:
Mathias Mericskay
mathias.mericskay@inserm.fr
Jérôme Piquereau
jerome.piquereau@universite-paris-saclay.fr</footnote>
**Plain english summary**

Heart failure (HF) is a severe condition in which the heart struggles to pump blood effectively. While many treatments exist, none specifically target the changes in cardiac energy production processes (energy metabolism) that occur in this disease. In the present study, it has been explored whether a diet enriched with a cocktail of B vitamins (nicotinamide riboside (B3), folate (B9) and cobalamin (B12)), called 3VitB, could improve cardiac energy metabolism and function in a mouse model of established HF induced by cardiac pressure overload. Four weeks after cardiac stress induction, mice were treated with 3VitB. In male mice, this vitamin cocktail increased survival rates, improved heart function and physical capacities. Female mice, which naturally resist cardiac stress better, showed less marked improvements in survival and cardiac function, but after several weeks of treatment, they exhibited better physical performance and less harmful heart cell hypertrophy. In both sexes, 3VitB helped protect the heart’s mitochondria, the main energy producers of the cardiac cell, by supporting their renewal and function. The study also found differences between males and females in how their hearts responded to both stress and vitamin treatment. Overall, the findings of this study suggest that 3VitB supplementation helps preserve heart energy metabolism and function in HF, especially in males which are more vulnerable to cardiac stress. This supports the idea that vitamin supplements and metabolic therapies could be promising new approaches to treat HF.

**Highlights**

* Alterations of energy metabolism and physical capacities are described in mouse model of established heart failure in males and females. Diet supplemented with nicotinamide riboside, vitamin B12 and B9 improved mitochondrial oxidative capacities and physical performance when introduced at an advance stage of heart failure. Males are more sensitive than females to the B vitamin cocktail treatment. Beneficial effects of these three B vitamins are associated with maintenance of mitochondrial biogenesis in both sexes.

**Introduction**

Despite constant advances in medical science, prevalence of heart failure (HF) is still high and is expected to rise in the world with ageing of the population and the spread of western diet and associated comorbidities. This syndrome, which is often the terminal stage of diverse chronic cardiovascular diseases (CVD), is a major cause of death worldwide and cannot be fully cured in spite of important efforts to improve therapeutics these last decades.

It is now well-established that profound modulations of energy metabolism are involved in the development of HF. The failing heart is in an energy-depleted state [1], which is associated with a shift from predominant fatty-acid oxidation to an increase in ketone bodies and glucose uptake, although for the latter its use remains confined to glycolysis and ancillary pathways and disconnected of further oxidation due to the severe mitochondrial dysfunction occurring in HF [1, 2]. While the underlying reasons for such alterations remain only partially understood, the knowledge acquired from HF animal models and HF patients on the dysregulations of mitochondrial life cycle [3] and the disruption of nico-

2 inhibitors inhibitor, specifically addressed the issue of altered cardiac energy metabolism. More recently, the use of sodium-glucose-cotransporter 2 inhibitors or glucagon-like peptide-1 receptor agonists that were originally designed as first-line therapies for diabetes but also emerged as novel treatment options for HF therapy, revived the idea that modulators of energy metabolism could be of the greatest medical significance for the treatment of this syndrome. These new clinical findings prompted the research community to pursue the metabolic strategy and design strategies to stimulate cardiac energy metabolism.

Micronutrient deficiency has long been known as one of the feature characterizing patients with HF, particularly those with advanced disease who are prone to cachexia and malnutrition due to multiple causes [6, 7]. Among micronutrient B vitamins are essential precursors of the cofactors required for mitochondrial enzymes involved in energy metabolism [8]. Vitamin supplementation has so far mostly been assessed as a prophylactic measure to reduce the risk of developing cardiovascular disease. These studies, many of them underpowered, showed modest benefits at best for cardiovascular diseases and even adverse effects for some like vitamin E
slow down the progression of established HF and modify
the outcome by restoring mitochondrial function.

In this context, our team has previously demonstrated
the therapeutic capacity of B vitamins in mouse mod-
els of HF [10, 11]. Our first study [10] showed that the
protective effects of cobalamin (B12) and folate (B9) that
impact on the homocysteine/methionine cycle preserves
left ventricular function and mitochondrial oxidative
capacity in a mouse model of pressure overload triggered
by transverse aorta constriction (TAC). We also showed
that nicotinamide riboside (NR), a nucleoside form of the
vitamin B3 that functions as a precursor for the nicotin-
amide adenine dinucleotide (NAD) cofactor, is also ben-
eficial to reduce cardiac dilatation and improves outcome
in a model of non-ischemic dilated cardiomyopathy as
well as in the TAC model [11] and as also shown by oth-
ers [12–14].

Beside their role in mitochondrial metabolism, vita-
mins B3, B9 and B12 also have signaling function as pre-
cursors for NAD⁺-mediated deacetylation by sirtuins
family members and for S-adenosine methionine (SAM),
the methyl donor used by methyl transferases [8]. They
can thus converge on the activation of the peroxisome
proliferator-activated receptor gamma co-activator 1 α
(PGC-1α), a master regulator of energy metabolism and
mitochondrial life cycle, by promoting its methylation
by protein arginine methyltransferase 1 (PRMT1) and its
deacetylation by sirtuin-1 (SIRT1), two activating post-
translational modulations of this co-activator [15, 16].

SIRT1 is a deacetylase that regulates many facets of
energy metabolism and its central role makes it a prime
target for the development of metabolic therapies of HF.
However, its activity requires NAD⁺ [17] and the loss of
myocardial NAD⁺ in HF [4] could be a hindrance to the
development of such a therapy. Indeed, this lack of NAD⁺
could limit the effect of SIRT1 activation strategies on the
one hand and on the other hand fully efficient catabolism
requires NAD⁺ at many stages, especially for mitochon-
drial functions [18]; this implies that suitable NAD⁺ level
would be a prerequisite for any effective metabolic ther-
apy. In an interesting way, in our second aforementioned
study [11] our team showed that treatment with NR
restores cardiac NAD⁺ level and protects cardiac func-
tion in mouse models of dilated cardiomyopathy or TAC
induced-pressure overload. According to our analysis,
in these models NR would take advantage of an increase
in the expression of the NMRK2 enzyme that catalyzes
the synthesis of nicotinamide mononucleotide (NMN)
(the immediate upstream precursor of NAD⁺) from NR,
thereby compensating for the decrease in nicotinamide
phosphoribosyltransferase (NAMPT) activity which
ensures NAD⁺ production from nicotinamide (NAM) in
the healthy heart [19].

Based on these encouraging results, we chose to assess
in the present study the effect of a vitamin cocktail con-
taining NR, folate and cobalamin in a murine model of
pressure overload-induced HF. While the effects of these
three B vitamins were assessed by preventive administra-
tion at early stage before the onset of the first HF symp-
toms in our previous studies, here we decided to assess
the efficacy of the vitamin B3-9-12 cocktail (3VitB) in a
model of established HF to reveal its potential effects at a
more advanced stage of the disease, as would be the case
with HF patients. In addition, considering the key impor-
tance of studying the potential differences in response
to treatments between males and females in preclinical
research to improve translational potential of our find-
ing, we decided to include males and females separately
in our study, especially because until now females had
not been assessed in our preclinical studies with these
vitamins.

**Materials and methods**

Animals

All animal experimental procedures were approved by animal ethics committee of Paris-Sud University, authorized by French government (authorization number: APAFIS#24778-2020032416337862) and complied with directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. Heart failure was induced in male and female C57BL/6NCrl mice (Charles River Laboratories, breeding site Italy) subjected to pressure overload by surgical transverse aortic constriction (TAC) performed according to surgery procedure described in supplementary information.

Treatment administration on non-synthetic diet

Four weeks after surgical transverse aortic constric-
tion (TAC), mice were randomized based on different
inclusion criteria assessed by trans-thoracic echocar-
diography: pressure gradient above 60mmHg (Figure
S2B), decrease of 10% of left ventricular ejection frac-
tion (LVEF) and increase of 30% of left ventricular mass
(Figure S2C). Mice that did not match two or more of
these criteria were excluded from the study. After ran-
domization, male and female mice were divided into
three groups: (1) sham-operated mice receiving standard
SAFE® A04 non-synthetic diet (SAFE, Augy, France), (2)
TAC mice receiving standard SAFE® A04 non-synthetic
diet called TAC-ND, (3) TAC mice receiving customized
SAFE® A04 supplemented with NR (4 g/kg), cobalamin
(1 mg/kg) and folate (20 mg/kg) called TAC-3VitB in this
manuscript. Animals were housed under temperature-
controlled conditions (21 °C) and had free access to water
and to their respective food. Supplementation with vita-
mins started four weeks after TAC surgery and two dif-
ferent treatment times were evaluated in two batches
(Figure S1A and B): (1) 20 weeks of vitamins treatment for survival and cardiac function study in males (Sham *n* = 11; TAC-ND *n* = 20; TAC-Vit *n* = 19) and females (Sham *n* = 11; TAC-ND *n* = 16; TAC-Vit *n* = 20); (2) 8 weeks of vitamins treatment for studying physical capacities, metabolomic, histologic, and molecular analysis in males (Sham *n* = 9; TAC-ND *n* = 11; TAC-Vit *n* = 11) and females (Sham *n* = 6; TAC-ND; *n* = 10; TAC-Vit *n* = 10).

## Survival

A survival study was carried out during 20 weeks of treatment to evaluate the impact of treatment on lifetime in males and females. A regular monitoring of body weight (each week) and cardiac function by trans-thoracic echocardiography (every four weeks) were done and correlated to end points determined on ethical project. When mice lost more than 20% of total body weight, had a left ventricular ejection fraction under 20% or reached end points, mice were euthanized and marked on survival curve as an event.

## Effort test on treadmill

Before introduction and after 8 weeks of treatment, the ability of the heart to adapt to physical effort was evaluated by an exertion test on treadmill (Ugo Basile, France) in male and female mice. Three days before the effort test, an adaptation phase was carried out with the following program: three and two days before the test, the mice were subjected to the following training session during which the treadmill started at a 3 m.min⁻¹ speed before being increased by 1 m.min⁻¹ every 2 min for 10 min without slope. After a day off, the exhaustion test was performed with the following program: after an acceleration of 30 min, the treadmill started at a 3 m.min⁻¹ speed that was increased by 3 m.min⁻¹ every 3 min without slope to determine the maximal running time under increasing speed.

## Echocardiography

Every four weeks, cardiac function was evaluated by trans-thoracic echocardiography with a Vevo 3100 device (FUJIFILM Visualsonics Inc., Toronto, Canada) equipped with a linear array 22–55 MHz MicroScan mouse cardiovascular transducer (MS550). Echocardiographic measures were performed on anesthetized mice using isoflurane (Centravet, Plancoet, France), 3% for induction and 1 to 2% for the maintenance of anesthesia (see supplementary information for further details). Images were analyzed using VevoLab Software (FUJIFILM Visualsonics Inc., Toronto, Canada).

## Histological analysis

Hearts were fixed in 4% paraformaldehyde, paraffin embedded and serially sectioned (3 µm).

Sections were stained with Sirius red (Ab150681; Abcam) for studying fibrosis or with FITC-labeled wheat germ agglutinin (WGA) to label cell membranes. The slides were scanned using NanoZoomer2.0-RS* (Hammatsu Photonics) digital scanner. Using ImageJ software, fibrosis and cross-sectional area of the fibers were quantified from 12 to 15 fields of a transversal section of the left ventricle for each animal.

## Mitochondrial oxidative capacities assays

Mitochondrial respiration was studied in situ in saponin-permeabilized cardiac muscle fibers using a Clarke electrode (O2k-Fluorespirometer, Oroboros Instruments) as previously described [20]. Preparation of permeabilized fibers is detailed in supplementary information. Oxygen consumption of the fibers were measured after successive addition of pyruvate 1 mM, malate (4 mM), ADP (2 mM), succinate (15 mM), amytal (an inhibitor of complex I, 1 mM) and N, N, N',N'-tetramethyl-p-phenylenediamine dihydrochloride (TMPD)-ascorbate (0.9:9mM) to respiration solution. Rates of respiration are given in nmoles O2/min/mg dry weight. The respiratory acceptor control ratio (ACR) was calculated from basal mitochondrial respiration rate (in the presence of pyruvate/malate 1/4 mM, without ADP) and oxygen consumption after addition of 2mM ADP.

## Enzyme activity

Frozen tissue samples were weighed, homogenized (Bertin Precellys 24) in ice-cold buffer (50 mg/ml) containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 5 mM (pH 8.7), EGTA 1 mM, DTT 1mM and 0.1% Triton X-100. Enzymatic activities were measured spectrophotometrically using standard coupled enzymes assays at 30°C. Citrate synthase (CS) activity was measured in a Tris-HCl buffer (pH 8) containing acetyl-Coa (10 mM), oxaloacetic acid (10 mM) and 5,5'-ditiobis-(2-Nitrobenzenoic acid) (DTNB) that allows, through coupled reactions involving CS, the production of the C₆O₄S₂⁻ ion the apparition of which was followed at 412 nm. Complex I (NADH-CoQ reductase) activity was determined by monitoring the disappearance of NADH at 340 nm in a 50 mM phosphate buffer (KH₂PO₄ (0.411 mM), K₂HPO₄ (0.088 mM), pH 7.4) containing 3.75 mg/ml BSA, 100 µM NADH and 100 µM of decyl-ubiquinone that played the role of electron acceptor. Results are given in IU/g protein.

## Quantitative Real-time PCR

Frozen tissue samples were weighed and homogenized (Bertin Precellys 24) in Trizol reagent (Invitrogen) allowing total ventricular RNA extraction. After addition of chloroform, samples were centrifugated at 10.000 g at 4 °C to obtain three phases: phenol-chloroform fraction,
interphase and aqueous phase containing proteins, DNA and RNA respectively. Total RNAs were then precipitated by the addition of isopropanol to the aqueous phase, centrifugated at 10.000 g and resuspended in DNase/RNase free water. Oligo-dT first strand cDNA was synthesized from 1 µg of total RNAs using iScript cDNA synthesis kit (Bio-rad). Negative controls without reverse transcriptase enzyme were performed to verify the absence of genomic DNA in the samples. Real-time PCR was performed using the SYBR® green method on CFX96 TouchTM Real Time PCR Detection system (Bio-Rad) from 2.5 ng cDNA. mRNA levels for all target genes were normalized to Tyrosine 3-Monooxygenase/Tryptophan 5-Monooxygenase Activation Protein Zeta (YWHAZ) expression level. Primer sequences are available in Table S1.

## Vitamin B12 dosage

Frozen myocardium tissue samples were weighed and homogenized in HCl (1 N). Two-step immunoassay for the quantitative determination of vitamin B12 using chemiluminescence microparticle immunoassay (CMIA) on Alinity i\* multiparametric instrument (Abbott, IL, USA) was performed.

## Liquid Chromatography-Mass spectrometry

A liquid-chromatography coupled to mass spectrometry analysis was developed and carried out to quantified Methyl-NAM. This procedure is detailed in supplementary information.

## Statistical analysis

The results are presented as the means ± standard error of the mean (SEM), with individual values for each condition. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software, RITME, France). The significance of the differences between groups was evaluated first after verifying the normality of the distribution by a Shapiro-Wilk normality test. If the distribution was normal, an ordinary one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test was done. If it was not normal, a nonparametric Kruskal-Wallis test followed by an uncorrected Dunn's test was chosen. To compare significance on two parameters such as treatment and sex, two-way analysis of variance (ANOVA) followed by uncorrected Fisher's LSD test was done. P values ≤ 0.05 were considered statistically significant. For the survival curve, to compare two groups, a Logrank (Mantel-Cox test) analysis was done.

## Results

### 1. The 3VitB cocktail improves survival and physical capacities of mice subjected to cardiac pressure overload

Cardiac pressure overload induced mortality over time in mice fed with non-supplemented diet (ND) or diet supplemented with 3VitB cocktail when compared with sham groups in both sexes (Fig. 1A). Survival was significantly improved in males fed with 3VitB as shown by Kaplan-Meier curve and the significant increase in median survival (Fig. 1B). These effects were combined with a beneficial impact on the normal body weight gain in males with ageing that was impaired in male TAC-ND group (Fig. 1C). Females also showed a trend towards a lower mortality rate and a higher median survival in TAC-3VitB group in comparison with TAC-ND group, although it remained below study's defined significance level (Fig. 1A and B). This difference might be explained by the higher median survival in TAC-ND group in females than in males (Fig. 1B and supplementary table S3, *p* = 0.03). Also of note, TAC did not affect weight gain in females suggesting they resist better to the cardiac injury caused by TAC (Fig. 1C).

Exercise stress testing is an important noninvasive test to diagnose and risk stratify HF in patients. Although analysis of exercise capacities before treatment at 4 weeks after TAC showed an equal reduction in the animals randomly assigned to ND and 3VitB treatment, in females as well as in males, exercise capacities were significantly improved by 8 weeks of 3VitB treatment in both sexes (Fig. 1D) and females showed higher capacities than males (Table S3, *p* = 0.003).

### 2. Cardiac function of mice affected by HF is protected by the 3VitB cocktail, especially in males

Since physical capacities are used by New York Heart Association to stratify the stage of HF, the improved exercise capacities on treadmill in TAC-3VitB groups could suggest a better preservation of cardiac function after 3VitB treatment introduction in this model. Importantly, we verified and confirmed that the TAC procedure triggered the same level of constriction and pressure gradient across the aortic cross in males and females before treatment as assessed by pulse wave Doppler analysis (Supplementary Figure S2A and B and Table S3, *p* > 0.99), resulting in similar increase in LV mass and reduction in LVEF in both sexes at 4 weeks before treatment (Supplementary Figure S2C), ruling out any differences in early TAC response as a cause of subsequent evolution in cardiac function.

In male TAC-3VitB group, the degradation of left ventricular ejection fraction (LVEF), stroke volume (SV) and the increase of LV end-systolic (LVDs) and -diastolic diameters (LVDd) over the time course of the survival study, were slower than in TAC-ND group (Fig. 2A-D),
<figure><img src="image_2.png" /><figcaption>**Fig. 1** The 3VitB cocktail improves survival and physical capacities of mice subjected to cardiac pressure overload. (A) Survival curve analysis of males and females treated with ND or 3VitB compared using log-rank test. (B) Median survival after diet introduction. (C) Body weight gain from surgery to death or to sacrifice 24 weeks after surgery. (D) Maximal of running time on increasing speed on treadmill before and 8 weeks after treatment introduction. (n = 11 to 20), ** p < 0.01, *** p < 0.001</figcaption></figure>

thereby confirming a benefit of these vitamins on the
preservation of LV function and remodeling. In females,
LVEF, SV and LVDs were similarly affected in TAC-ND
group as compared to males (Fig. 2A-C and Table S3,
p > 0.05 between sexes in TAC-ND groups) although

LVDd was less increased (Fig. 2D, Table S3, *p* = 0.01
between males TAC-ND and females TAC-ND). The
3VitB treatment did not improved these parameters in
females. Interestingly, the evaluation of the late diastolic
transmitral flow velocity (E/A ratio) showed a similar
<figure><img src="image_3.png" /><figcaption>**Fig. 2** Cardiac protective effects of the 3VitB cocktail. A. Ejection fraction (EF) progression from surgery to death or to sacrifice 24 weeks after surgery. B. Stroke volume (SV) progression from surgery to death or to sacrifice 24 weeks after surgery. C. End-systolic left ventricle diameter (LVDs) progression from surgery to death or to sacrifice 24 weeks after surgery. D. End-diastolic left ventricle diameter (LVDd) progression from surgery to death or to sacrifice 24 weeks after surgery (E) Representative echocardiographs and E wave/A wave ratio (E/A ratio) 8 weeks after treatment introduction (F) mRNA expression level of β-myosin heavy chain (β-Mhc), α-myosin heavy chain (α-Mhc) and brain natriuretic peptide (Bnp). (n = 11 to 20), \* *p* < 0.05, \*\* \*p* < 0.01, \*\*\* \*p* < 0.001</figcaption></figure>

increase 8 weeks after TAC in males and females (Fig. 2E and Table S3, *p* = 0.17 between sexes in TAC-ND groups) indicative of LV diastolic dysfunction and 3VitB treatment reduced this ratio to normal levels in both sexes.

The quantification in LV myocardium of transcripts of molecular markers of HF in mice showed the characteristic shift from cardiac fast α-myosin heavy chain (Mhc) to slow βMhc and the increase in brain natriuretic
peptide (Bnp) gene expression associated with LV dilation in males TAC-ND group; and the 3VitB treatment robustly reduced these markers (Fig. 2F). In females, the βMhc/αMhc ratio increased less after TAC than in males (Fig. 2F and Table S3, *p* = 0.02 between sexes in TAC-ND groups) and it was rescued by the 3VitB treatment. Bnp expression was much less induced by TAC in females in agreement with the milder dilation of the LV chamber.

**3. The 3VitB cocktail prevents the development of cardiac hypertrophy and fibrosis induced by cardiac pressure overload**

In order to study the potential effects of the 3VitB cocktail on cardiac structure, a series of animals was especially designed to be sacrificed after 8 weeks of B vitamins supplementation, i.e. 12 weeks after TAC (Figure S1B). In males, heart weight-to-tibia length ratio (HWTL) at sacrifice was significantly increased in TAC-ND group and reduced in the TAC-3VitB group (Fig. 3A). In females, HWTL was significantly increased in TAC-ND group but much less than in males and the 3VitB treatment did not further reduced this parameter (Fig. 3A, and Table S3, *p* < 0.001 *p* = 0.02 between sexes in TAC-ND groups). In contrast, TAC increased lung weight-to-tibia length ratio in both sexes suggesting similar levels of pulmonary oedema and the 3VitB had same beneficial impact in both sexes (Fig. 3B). In spite of the lower increase of HWTL ratio triggered by TAC in females compared to males, the mean cross-sectional area of cardiac fibers and the proportion of very thick cardiomyocytes showed similar and significant increase in both sexes in TAC-ND groups (Fig. 3C and D). This confirmed the activation of the hypertrophy process in these groups that was counter-balanced by 3VitB treatment as judged by the “treatment effect” revealed by the 2-way ANOVA statistical analysis including males and females together (Table S3, treatment factor *p* < 0.001). This histological analysis was completed with Sirius red staining that showed a significant rise in cardiac fibrosis in TAC-ND group in males that was prevented by the 3VitB supplementation (Fig. 4A and B). This observation is reinforced by the measurement of the expression of collagen I that showed a significant increase in the TAC group fed with ND (Fig. 4C) while no increase was reported when mice were under 3VitB treatment. No expression change in collagen III was measured in TAC groups. Remarkably, no such increase in cardiac fibrosis and collagen I expression was observed in the female TAC-ND group (Fig. 4A, B and C).

**4. Energy metabolism of mice affected by HF is preserved by the 3VitB cocktail, especially in males**

Considering the major role of B vitamins in energy
metabolism and mitochondrial metabolism in particular,
key elements of energy metabolism were characterized in

the myocardium of the animals sacrificed 8 weeks after
the beginning of the treatment. In males, mitochondrial
oxidative capacities measured in the presence of sub-
strates feeding complex I and II and direct activation of
complex IV were altered in the TAC-ND group and fully
rescued by the 3VitB treatment (Fig. 5A). In females,
mitochondrial respiration rates were only mildly affected
in the TAC-ND group, although they were not signifi-
cantly different than in TAC-ND males (Table S3). Yet
they were significantly increased by the 3VitB treatment.

Interestingly, the beneficial impact of the 3VitB treat-
ment on mitochondrial oxidative capacities was also
revealed by the higher respiratory acceptor control
ratio (ACR) as measured in NAD-dependent complex
I driven respiration in both males and females in com-
parison with Sham and TAC-ND groups (Fig. 5A). The
decrease in mitochondrial oxidative capacities in TAC-
ND males was associated with significant reduction of
complex I biochemical activity (Fig. 5B) and citrate syn-
thase (CS) activity and protein level (Fig. 5C and D), CS
being usually used as a marker of mitochondrial mass;
these alterations were prevented by 3VitB supplementa-
tion. In females, despite their lesser sensitivity to TAC,
the activities of these enzymes and CS protein level were
affected in the TAC-ND group (Fig. 5B, C and D) and not
restored by 3VitB cocktail. The measurement of the mito-
chondrial superoxide dismutase 2 (SOD2) protein level
revealed a rise in protein level in both TAC-3VitB groups
that suggests that the 3VitB treatment could promote an
antioxidant system (Fig. 5E). Higher oxidative capacities
in TAC-3VitB males were associated with prevention of
NAD level drop induced by TAC (Fig. 5F). Incidentally,
the assessment of NAD and cobalamin level in myocard-
dium showed significant increases in TAC-3VitB groups
that demonstrated the assimilation of NR and vitamin
B12 by the 3VitB treated mice (Fig. 5F and G). Methyl-
NAM metabolite that represents the end-pathway prod-
uct of both vitamin B3 elimination (nicotinamide) and
vitamins B12/9 action on methylation, was induced by
the 3VitB diet in both sexes (Fig. 5H).

**5. The 3VitB cocktail sustained mitochondrial biogenesis transcription cascade in pressure overloaded myocardium in males and females**

Although alterations and preservation of oxidative capac-
ities were observed in TAC-ND and TAC-3VitB males
respectively, no significant modulation of *Pgc-1α*, *Pgc-1β* and *Nrf1* transcripts level was noticed (Fig. 6A). The
expression of these three genes involved in mitochondrial
biogenesis was not altered in TAC-ND females neither
when compared to sham group and only a slight increase
in *Nrf1* expression was noticed in TAC-3VitB female
group in comparison with Sham and TAC-ND groups.
The 3VitB cocktail globally normalized the expression of
<figure><img src="image_4.png" /><figcaption>**Fig. 3** The 3VitB cocktail prevents the development of cardiac hypertrophy. (A) Heart weight to tibia length ratio (HWTL) at sacrifice 8 weeks after 3VitB treatment introduction. (B) Lung weight to tibia length ratio (LXTL) at sacrifice 8 weeks after 3VitB treatment introduction. (C) Representative pictures of cardiomyocyte hypertrophy analysis by fluorescein isothiocyanate (FITC)-labeled wheat germ agglutinin (WGA) after 8 weeks of 3VitB treatment. (D) proportion of cardiomyocytes by cross sectional size category, mean cross sectional area. (n=6 to 8 animals per group), \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001</figcaption></figure>

mitochondrial transcription factor A (*Tfam*), cytochrome c oxidase 4 (*Cox4*) and medium-chain acyl-CoA dehydrogenase (*Mcad*) in both sexes, with some minor differences (Fig. 6A). At the protein level, PGC-1α protein level was not altered by surgery or treatment but a significant

increase in NRF1 protein level was observed in TAC-3VitB groups in both males and females (Fig. 6B).

While the increase in the expression of PGC-1α downstream cascade genes in male and female TAC-3VitB groups (Fig. 6A) could explain the preservation of
<figure><img src="image_5.png" /><figcaption>**Fig. 4** Myocardial fibrosis after 8 weeks of treatment. (A) Representative pictures of myocardial fibrosis analysis by Sirius red after 8 weeks of 3VitB treatment. (B) Proportion of total fibrosis in the different experimental groups at sacrifice 8 weeks after 3VitB treatment introduction. (C) Left ventricular mRNA expression level of collagen I (Col1a) and collagen III (Col3a). (n=6 to 8 animals per group), \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001</figcaption></figure>

mitochondrial oxidative capacities (Fig. 5A), the absence
of any variation in PGC-1α gene expression and protein
level between the different experimental groups sug-
gested a post-translational activation of this transcrip-
tional coactivator. Amongst the strongest activators of
PGC-1α, SIRT1 requires NAD⁺ for its deacetylase activ-
ity which is thus largely dependent on NAD metabolism.
3VitB cocktail prevented the significant drop of NAD
level induced by TAC in males while NAD level was not
altered by TAC in females (Fig. 5F). Whereas the vitamin
treatment could help maintain NAD level, a prerequi-
site for optimal SIRT1 activity, this deacetylase did not
show any significant changes in protein level in the differ-
ent groups in both sexes, even though a trend towards a
higher SIRT1 content was noticed in females after treat-
ment (Fig. 7A). The acetylation level of P53, a target of
SIRT1, tended to increase in both TAC-ND groups and
was significantly reduced after 8 weeks of vitamins treat-
ment in females only, revealing the activation of SIRT1
by the 3VitB cocktail in females (Fig. 7A). The absence of
p53 deacetylation in males suggested that another energy
signaling pathway is preferentially activated. As we pre-
viously showed that mitochondrial functions in males
are especially sensitive to AMP-activated protein kinase

(AMPK) while this pathway is dispensable in females
[21], activation of this kinase was assessed by measuring
phosphorylated-AMPK (P-AMPK) and phosphorylated-
acetyl-CoA carboxylase (P-ACC), a major AMPK target
used to reveal AMPK activation. P-AMPK/total-AMPK
ratio was unchanged in all male groups, but TAC-3VitB
group exhibited significantly higher P-ACC/total-ACC
ratio after 8 weeks of 3VitB treatment, unveiling the
activation of AMPK in this group (Fig. 7B). In contrast,
AMPK phosphorylation level was repressed by TAC
in females and no activation by the 3VitB cocktail was
noticed as measured by ACC phosphorylation level (Fig.
7B).

Discussion

These last years, intensive research led in the field of
HF has allowed a better understanding of the physiopa-
thology of this syndrome. Although grey areas remain,
the research community agrees that energy metabo-
lism alterations in the myocardium plays important
roles in cardiac decompensation and should be a target
of future therapies [3, 22]. Despite this, so far, no meta-
bolic therapy of HF is routinely used in clinics to spe-
cifically improve failing myocardium energy metabolism,
<figure><img src="image_6.png" /><figcaption>**Fig. 5** Cardiac mitochondrial oxidative capacities of mice affected by HF is preserved by 8 weeks of 3VitB cocktail treatment. (A) Rate of respiration measured under phosphorylating conditions (2mM ADP) in the presence of substrates for complex I (P/M), complexes I+II (succinate), complex II (Amytal) and complex IV (TMPD). (B) Complex I enzymatic activity. (C) Citrate synthase (CS) enzymatic activity. (D) Citrate synthase (CS) protein level. (E) SOD2 protein level. (F) Myocardial NAD level. (G) Myocardial cobalamin level. (H) Myocardial Methyl-NAM (MeNAM) level. (n=6 to 8 animals per group), \* *p* < 0.05, \*\* *p* < 0.001</figcaption></figure>

especially mitochondrial functions [23]. Based on the protective action of three B vitamins (NR, folate and cobalamin) on energy metabolism of the heart in animals facing cardiac pressure overload when administrated

separately and preventively in our previous studies [10, 11], we tested the potential positive effects of a cocktail of these vitamins (3VitB) in a murine model of established HF to investigate its curative use. Here, we showed that
<figure><img src="image_7.png" /><figcaption>**Fig. 6** Cardiac mitochondrial oxidative capacities of mice affected by HF is preserved by 8 weeks of 3VitB cocktail treatment. (A) mRNA expression level peroxisome proliferator-activated receptor gamma co-activator 1 α (*Pgc-1α*), peroxisome proliferator-activated receptor gamma co-activator 1 β (*Pgc-1β*), nuclear respiratory factor 1 (*Nrf1*), mitochondrial transcription factor A (*Tfam*), cytochrome oxidase subunit 4 (*Cox4*) and the medium-chain acyl-CoA dehydrogenase (*Mcad*). (B) *Pgc-1α* and *Nrf1* protein level</figcaption></figure>

the introduction of a diet supplemented with B3, B9 and B12 in mice with established cardiac dysfunction exhibits beneficial effects with stronger impacts in males although numerous beneficial effects were also seen in females. Precisely, this study shows that this combination of three vitamins slows down the degradation of cardiac function induced by pressure overload, improves the physical capacities of the animals and protects myocardium energy metabolism, especially mitochondrial oxidative capacities; these effects are more pronounced in males with significant improvement of survival in these mice that are more severely affected by TAC in comparison with females.

In recent years, the studies aiming at understanding the metabolic alterations encountered in HF demonstrated that structural and functional alterations in mitochondria are associated with major deregulations of NAD homeostasis [4, 24]. Beyond the fact that NAD as a coenzyme is required to support fuel substrate oxidation, TCA cycle and oxidative phosphorylation in mitochondria to generate ATP, loss of NAD homeostasis has also been reported to impact enzymes with NAD⁺-dependent activity, such as Sirtuins, that could be involved in cardiac disease progression [25–27]. It was therefore with the idea of proposing a treatment that could both stimulate the fundamental processes of energy metabolism and support cellular levels of NAD that the combination of B vitamins
<figure><img src="image_8.png" /><figcaption>**Fig. 7** Cardiac mitochondrial oxidative capacities of mice affected by HF is preserved by 8 weeks of 3VitB cocktail treatment. (A) Immunoblotting of PGC-1α, total-P53 (P53) and acetylated P53 (P53-acetyl). (B) Immunoblotting of total AMPK (tAMPK), phosphorylated-AMPK (AMPK-P), total ACC (tACC)/ phosphorylated-ACC (ACC-P)</figcaption></figure>

tested in this study was developed, hypothesizing that the addition of the respective effects of these vitamins on mitochondrial biogenesis and NAD metabolism [10, 11] could constitute a new tool in the therapeutic arsenal for treating HF. While the significant reduction in mortality in males is a strong argument in favor of the efficacy of this B vitamin cocktail in slowing the progression of the disease, the improvement in physical capacities observed in both sexes is also highly significant, since it demonstrates better adaptation of the treated animals when the body's energy demand increases. By extrapolation, this would be akin to a better exercise test score in HF patients, a predominant indicator in determining the stage of HF [28]. Moreover, as exercise training and cardiac rehabilitation are considered as major non-pharmaceutical beneficial approaches in the treatment of HF [29], the increased physical capacities conferred by the 3VitB cocktail could enhance the benefit of these approaches. Thus, the subtler effects of this treatment on mortality and cardiac systolic function in the females, resulting from a milder phenotype that was expected due to widely-reported cardioprotective mechanisms in this sex [30], should not obscure its benefits in female mice.

Incidentally, the improvement in diastolic function, the reduction in pathological β-MHC isoform expression, and the milder cardiomyocytes hypertrophy in TAC females after treatment, comparable to what was observed in males, demonstrates the sensitivity of the females to these vitamins. Interestingly, 3VitB treatment improved physical capacities in exercise stress test in females despite a lack of clear improvement of systolic cardiac function although the latter was less affected than males. Moreover, we note that diastolic dysfunction as assessed by E/A was improved in females as in males. Obviously, physical exercise capacities integrate a number of regulatory physiological processes and we measured the cardiac function parameters at rest since limitation of the murine model precludes to perform echocardiography while exercising. Therefore, we cannot know if the 3VitB treatment improves cardiac adaptation to exercise or other parameters as well including vascular resistance as reported for NR [31], skeletal muscle bioenergetics or respiratory function, as suggested by the reduced level of pulmonary oedema obtained with the 3VitB treatment.
In the cardiac left ventricle, the study of the mito-
chondrial respiratory chain on permeabilized ventricu-
lar fibers clearly highlighted an improvement in the
oxidative capacity of mitochondria in treated males and
females. In the latter, these improved oxidative capaci-
ties are not associated with any significant effects of the
vitamins on citrate synthase enzyme activity, a marker of
functional mitochondrial mass, that remains degraded in
3VitB treated females compared to sham females. This
was not the case in males, where the 3VitB cocktail nor-
malized the activity of this enzyme, suggesting again that
the treatment acts through different pathways according
to sex. However, the activation of the expression of genes
that are under the control of PGC-1α such as Tfam, Cox4
and Mcad [32], shows that the support of mitochondrial
oxidative capacities, involves mechanisms that stimulate
genes involved in energy metabolism in both sexes. It is
now well known that PGC-1α plays a central role in the
regulation of energy metabolism and that its expression
and activity can be modulated through many ways [33].
The various mechanisms leading to the activation of this
major regulator of energy metabolism and mitochon-
drial life cycle in particular could therefore be differently
stimulated by the B vitamins in males and females in the
present model (Fig. 8). These mechanisms could involve
SIRT1, which appears to be activated only in treated
females. SIRT1 is a deacetylase whose actions on PGC-1α
activity and on metabolism in general has been high-
lighted in several studies [17, 34, 35], and it could there-
fore be at the heart of mechanisms protecting energy
metabolism. Deregulations of pathways involving SIRT1
have been described in the pathophysiology of HF [36],
and the effects of a combination of cobalamin and folate
[10, 37] and NR [38] on the activity of this enzyme could
play a crucial role in the benefits observed in females. In
males, SIRT1 does not seem to be activated, suggesting
other pathways turn on to mediate the protective effect
of the 3VitB therapy. Among these pathways, the stimu-
lation of AMPK revealed in males in the present study
could play a role in improving the survival of the animals
included in this study. AMPK, a key metabolic sensor, is
known for its cardioprotective effects [39–41] and it has
already been shown that AMPK phosphorylation or/and
activity is increased under NR or Cobalamin-folate treat-
ment in several tissues/cell types, although the underly-
ing mechanisms have not been identified [10, 42, 43]. We
showed that AMPK controls the regulatory mechanisms
of energy metabolism in a sex-specific manner, being
essential in males to reduce cardiac fibrosis and maintain
mitochondrial function and structure but dispensable in
females [21]. Its activation only in males in the present
study could thus partly explain the differences observed
between both sexes, its involvement in the sex-specific
effects of the 3VitB cocktail will thus be the subject of a

future work. Interestingly, the differences in the response
between males and females could also involve complex
interactions with other components of the diet. Indeed,
in a recently published collaborative study by our teams
[44], this same 3VitB cocktail was mixed with a synthetic
diet whose composition (amino acids, fat, carbohydrates,
minerals and vitamins) matching the composition of the
natural diet that we used in the present study. The 3VitB
synthetic diet was provided to mice of the same genetic
substrain C57BL6N, starting 4 weeks after TAC as the
present study. Interestingly, mixing the 3VitB cocktail
with the synthetic diet blunted the benefit of the treat-
ment in males but not in females, which, on the contrary,
responded positively to the synthetic diet. The 3VitB syn-
thetic diet notably normalized the circulating lipid profile
compared to untreated TAC females suggesting improved
energy metabolism although mitochondrial function was
not measured in this study [44]. Altogether, the results of
David et al. [44], and the present study, show that some
components in the natural diet that we used in the pres-
ent study synergize with the 3VitB cocktail to unlock its
beneficial action for males and those components are not
necessary for the females. We therefore hypothesize that
such components could be the phytoestrogens derived
from soya that are present in the natural diet and possess
estrogen-like activity, and those phytoestrogens would
not be necessary in females that have a robust estrogen
signaling. An alternative, but not mutually exclusive
hypothesis is that differences in the intestinal microbiota
between the Canadian [44] and French (present study)
mice cohorts contribute to a different response in males
and females. Future research will be required to decipher
between those complex mechanisms to better under-
stand the interaction between B vitamins and environ-
mental factors. Nevertheless, it's important to underline
that in two independent studies, the supplementation
with 3VitB cocktail provides a benefit compared to non-
supplemented diet to at least one sex, further supporting
the high translational potential of this approach.

A few years ago, the potential beneficial effects of B
vitamins, especially cobalamin and folate, in the context
of human cardiovascular diseases associated with hyper-
homocysteinemia (HHCY) were questioned and led to
several clinical trials [45, 46]. HHCY is often due to vita-
min B9, and/or B12 deficiency and although the ability
of cobalamin and folate to reduce homocysteinemia in
patients was confirmed by these studies, the prevention
of cardiovascular events by these vitamins was not con-
clusive enough to recommend treating patients at car-
diovascular risk with these compounds. However, these
studies only assessed the consequences of the homocys-
teine-reducing effect of cobalamin and folate on athero-
thrombotic events in specific cardiovascular diseases;
they did not assess vitamin B9 and B12 supplementation
<figure><img src="image_9.png" /><figcaption>**Fig. 8** Potential molecular mechanisms activated by 3VitB treatment to support cardiac energy metabolism. In males, the effect of the B vitamins on cardiac metabolism and function would involve AMPK that could be activated by the 3VitB cocktail through pathways that remain to be explored. This activation of AMPK would induce a stimulation of PGC-1α through its phosphorylation. This would stimulate mitochondrial biogenesis and diverse facets of cardiac energy metabolism, thereby helping the heart cope with pressure overload. In females, 3VitB treatment activates SIRT1 that could be a key element in the beneficial effects of these vitamins. The support of SIRT1 activity in this model could be due to a synergistic action of the three vitamins, leading to the stabilization of SIRT1 which would deacetylate and activate PGC-1α with subsequent effects on mitochondrial biogenesis and cardiac energy metabolism. (Ac, acetyl moiety; ADPR, adenosine diphosphate ribose; Me, methyl moiety; Nnmt, nicotinamide N-methyl-transferase; THF, tetrahydrofolate)</figcaption></figure>

in HF patients and they did not take into account the roles of these vitamins in energy metabolism [8] that could be of great value in the context of HF. HHCY has been reported in HF regardless of etiology and is associated with clinical variables [47], and although folate and cobalamin deficiencies seem rare in HF, data suggest that subclinical B12 deficiency may be underestimated in this pathology [48]. Regardless of cause, HHCY may

contribute to HF progression since it exerts harmful vascular effects and may directly impair cardiac function (for review see [8]). Therefore, the use of these two vitamins in the treatment of HF to lower hyperhomocysteinemia and stimulate energy metabolism seems to be a sensible strategy, especially since cardiovascular beneficial effects of cobalamin and folate have been revealed in patients older than 69 in Towgifhi's study after stratifying treated
population [49]. Moreover, two meta-analyses of folate supplementation found reductions in cardiovascular events in patients with cardiovascular disease [50, 51]. While these studies give credit to cobalamin and folate as therapeutic agents, the beneficial effects of NR on the cardiovascular system reported by a significant number of preclinical studies have raised hopes for its clinical applications (for review see [8]). Based on evidence of alterations in NAD homeostasis in HF in animal models and HF patients [4, 11], the NAD replenishment strategy has recently led to a clinical trial involving patients with HF with reduced ejection fraction (NCT03423342). This trial demonstrated the safety of NR, which increased total blood NAD levels, improved mitochondrial respiration capacities in peripheral blood mononuclear cells that also expressed less pro-inflammatory cytokines in comparison to placebo [52]. Thus, the use of B vitamins as therapeutic agents to treat cardiovascular diseases seems credible. To date the combination of folate, cobalamin and NR has never been tested in humans in the specific context of HF with reduced ejection fraction, the results produced by the present work demonstrating the support of energy metabolism by the 3VitB cocktail in a model of heart failure induced by pressure overload, are strongly in favor of considering the therapeutic use of these B vitamins in heart failure.

In its advanced stages, HF is a multi-organ syndrome in which systemic inflammation and dysfunction in the kidneys, liver, lungs, endothelium, or skeletal muscle compound the primary cardiac impairment [53]. While our study demonstrated clear effects of 3VitB treatment on cardiac energy metabolism, we cannot exclude the possibility that these vitamins also improved survival and/or physical capacities through actions on other organs or systems. For instance, systemic inflammation, which plays a key role in HF progression, particularly in cardiac fibrosis and coronary microvascular endothelial dysfunction [54], has been shown to be reduced by NR treatment [55, 56]. In a model of sepsis-induced cardiac dysfunction, NAD repletion via NAD precursors lowered circulating proinflammatory cytokines and was associated with reduced damage to the kidneys, liver, and vascular endothelium [57]. Protective effects of NR on the liver and kidneys have also been observed in animal models of hepatic stress and renal stress [58, 59], demonstrating that NR maintains favorable circulating lipid profile and protects renal function from damage commonly seen after myocardial infarction. Obviously, all these actions could be beneficial in the context of our murine model of established HF, could be involved in the positive effects of the 3VitB treatment and could be complementary to the benefits on cardiac energy metabolism reported in the present study.

## Conclusion

In summary, the present work shows, in line with our previous studies, that the oral supplementation with a cocktail mixing folate, cobalamin and NR improves heart function, mitochondrial function and survival in mice facing cardiac pressure overload with a higher sensitivity in males. These vitamins could be an initial tool for targeting metabolic alterations in HF and may be a new option to treat this disease. Despite the considerable work carried out during this study, the precise mechanisms involved in the benefits conferred by this treatment have not been clearly identified, and this remains a major limitation of our study. However, AMPK in males and SIRT1 in females could play key roles that will be investigated in future works. Of course, therapeutic use of this compounds will also require further studies to determine pharmacokinetics of these vitamins and to assess safety and efficacy in human before translation to clinics.

## Abbreviations

<table><tr><td>3VitB</td><td>Three B vitamins</td></tr><tr><td>ACC</td><td>Acetyl-CoA carboxylase</td></tr><tr><td>ACR</td><td>Acceptor control ratio</td></tr><tr><td>AMPK</td><td>AMP-activated protein kinase</td></tr><tr><td>BNP</td><td>Brain natriuretic peptide</td></tr><tr><td>COX4</td><td>Cytochrome c oxidase 4</td></tr><tr><td>CS</td><td>Citrate synthase</td></tr><tr><td>CVD</td><td>Cardiovascular disease</td></tr><tr><td>HF</td><td>Heart failure</td></tr><tr><td>HWTL</td><td>Heart weight-to-tibia length ratio</td></tr><tr><td>LV</td><td>Left ventricle</td></tr><tr><td>LVDd</td><td>Diastolic left ventricular diameter</td></tr><tr><td>LVDs</td><td>Systolic left ventricular diameter</td></tr><tr><td>LVEF</td><td>Left Ventricular Ejection fraction</td></tr><tr><td>MHC</td><td>Myosin heavy chain</td></tr><tr><td>NAD</td><td>Nicotinamide adenine dinucleotide</td></tr><tr><td>NAM</td><td>Nicotinamide</td></tr><tr><td>NAMPT</td><td>Nicotinamide phosphoribosyltransferase</td></tr><tr><td>MCAD</td><td>Medium-chain acyl-CoA dehydrogenase</td></tr><tr><td>NMN</td><td>Nicotinamide mononucleotide</td></tr><tr><td>NMRK2</td><td>Nicotinamide riboside kinase 2</td></tr><tr><td>ND</td><td>Normal diet</td></tr><tr><td>NR</td><td>Nicotinamide riboside</td></tr><tr><td>NRF1</td><td>Nuclear respiratory factor 1</td></tr><tr><td>P-ACC</td><td>Phosphorylated-acetyl-CoA carboxylase</td></tr><tr><td>P-AMPK</td><td>Phosphorylated-AMPK-activated protein kinase</td></tr><tr><td>PGC-1</td><td>Peroxisome proliferator-activated receptor gamma co-activator 1</td></tr><tr><td>PRMT1</td><td>Protein arginine methyltransferase 1</td></tr><tr><td>SAM</td><td>S-adenosine methionine</td></tr><tr><td>SIRT1</td><td>Sirtuin-1</td></tr><tr><td>SV</td><td>Stroke volume</td></tr><tr><td>SOD2</td><td>Superoxide dismutase 2</td></tr><tr><td>TAC</td><td>Transverse aortic constriction</td></tr><tr><td>TFAM</td><td>Mitochondrial transcription factor A</td></tr></table>

## Supplementary Information

The online version contains supplementary material available at https://doi.org/10.1186/s13293-025-00764-x.

Supplementary Material 1

## Acknowledgements

We thank Valérie Domergue and the Animex team (animal experimentation), facility of the "Ingénierie et Plateforme au Sercice de l'Innovation
Thérapeutique" UMS IPSIT of Université Paris Saclay. We also acknowledge Dr Renée Ventura-Clapier and Pr. Vladimir Veksler for continuous support.

**Author contributions**

J.P. and M.M. designed the study. S.E.B., M.D., A.K., M.G., I.M., B.B., F.M-N., A.J and J.P. carried out the experimental design and execution of experiments. S.E.B., M.D., A.K., M.G., I.M., B.B., F.M-N., A.J, M.R. and J.P. analyzed the data. S.E.B., J.P., M.M., A.G., C.L. and M.R. contributed to data interpretation. J.P., M.M. and S.E.B. wrote the manuscript. J.P., M.M. and M.R. critically revised the manuscript.

**Funding**

This work was funded by Université Paris Saclay and Institut National de la Santé et de la Recherche Médicale (INSERM). Our laboratory is a member of the Laboratory of Excellence LERMIT and is supported by European Research Area Network on Cardiovascular Diseases (to Dr Matthieu Ruiz and to Dr. Jérôme Piquereau (#ANR-19-ECVD-0007-01)). Solène E. Boitard was supported by a postdoctoral fellowship from Lefoulon-Delalande Foundation.

**Data availability**

The datasets used and/or analysed during the current study are available from the corresponding authors on reasonable request.

**Declarations**

**Ethics approval and consent to participate**

Not applicable.

**Consent for publication**

Not applicable.

**Competing interests**

The authors declare no competing interests.

**Author details**

¹Signalling and Cardiovascular Pathophysiology, Inserm, UMR-S 1180, Université Paris-Saclay, Orsay, France

²Université d'Evry, Université Paris Saclay, Evry-Courcouronnes, France

³Montreal Heart Institute Research Centre, Montreal, Canada

⁴Department of Nutrition, Faculty of medicine, Université de Montréal, Montreal, Canada

⁵US31-UMS3679-Plateforme PHIC, Ingénierie et Plateformes au Service de l'Innovation Thérapeutique (IPSIT), INSERM, CNRS, Université Paris- Saclay, Orsay, France

⁶Department of Biochemistry, Assistance Publique-Hôpitaux de Paris, Paris, France

⁷Université Versailles St-Quentin, Université Paris-Saclay, Orsay, France

⁸Laboratoire PRÉTI UR 24184, Université de Poitiers, Poitiers, France

Received: 20 May 2025 / Accepted: 19 September 2025

Published online: 21 October 2025

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