Cobalamin and folate protect mitochondrial and contractile functions in a murine model of cardiac pressure overload

    Cobalamin and folate protect mitochondrial and contractile functions in a murine model of cardiac pressure overload J´erˆome Piquereau, Maryline Moulin, Giada Zurlo, Philippe Mateo, M´elanie Gressette, Jean-Louis Paul, Christophe Lemaire, Ren´ee Ventura-Clapier, Vladimir Veksler, Anne Garnier PII: DOI: Reference:

S0022-2828(16)30414-X doi: 10.1016/j.yjmcc.2016.11.010 YJMCC 8488

To appear in:

Journal of Molecular and Cellular Cardiology

Received date: Revised date: Accepted date:

2 May 2016 13 November 2016 18 November 2016

Please cite this article as: Piquereau J´erˆ ome, Moulin Maryline, Zurlo Giada, Mateo Philippe, Gressette M´elanie, Paul Jean-Louis, Lemaire Christophe, Ventura-Clapier Ren´ee, Veksler Vladimir, Garnier Anne, Cobalamin and folate protect mitochondrial and contractile functions in a murine model of cardiac pressure overload, Journal of Molecular and Cellular Cardiology (2016), doi: 10.1016/j.yjmcc.2016.11.010

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Cobalamin and folate protect mitochondrial and contractile functions in a murine model

T

of cardiac pressure overload.

IP

Jérôme Piquereau1, Maryline Moulin1, Giada Zurlo1, Philippe Mateo1, Mélanie Gressette1,

SC R

Jean-Louis Paul2, Christophe Lemaire1, Renée Ventura-Clapier1, Vladimir Veksler1 and Anne Garnier1

UMR-S 1180, Inserm, Univ. Paris-Sud, Université Paris-Saclay, Châtenay-Malabry, France

2

Service de Biochimie, Hôpital Européen Georges Pompidou, Paris, France

MA

NU

1

Address for correspondence: Dr Jérôme Piquereau

TE

D

U-1180 INSERM Faculté de Pharmacie, Université Paris-Sud

CE P

5 rue J-B Clément, 92296 Châtenay-Malabry France

AC

Tel.: (33-1) 46.83.54.53 Fax: (33-1) 46.83.54.75

E-mail: [email protected]

Number of words: 6923

1

ACCEPTED MANUSCRIPT Abstract

T

PGC-1α, a key regulator of energy metabolism, seems to be a relevant therapeutic target to

IP

rectify the energy deficit observed in heart failure (HF). Since our previous work has shown

SC R

positive effects of cobalamin (Cb) on PGC-1α cascade, we investigate the protective role of Cb in pressure overload-induced myocardial dysfunction. Mice were fed with normal diet (ND) or with Cb and folate supplemented diet (SD) 3 weeks before and 4 weeks after

NU

transverse aortic constriction (TAC). At the end, left ventricle hypertrophy and drop of

MA

ejection fraction were significantly lower in SD mice than in ND mice. Alterations in mitochondrial oxidative capacity, fatty acid oxidation and mitochondrial biogenesis transcription cascade were markedly improved by SD. In SD-TAC mice, lower expression

TE

D

level of the acetyltransferase GCN5 and upregulation of the methyltransferase PRMT1 were associated with a lower protein acetylation and a higher protein methylation levels. This was

CE P

accompanied by a sustained expression of genes involved in mitochondrial biogenesis transcription cascade (Tfam, Nrf2, Cox1 and Cox4) after TAC in SD mice, suggesting a

AC

preserved activation of PGC-1α; this could be at least partly due to corrected acetylation/methylation status of this co-activator. The beneficial effect of the treatment would not be due to an effect of Cb and folate on oxidative stress or on homocysteinemia, which were unchanged by SD. These results showed that Cb and folate could protect the failing heart by preserving energy status through maintenance of mitochondrial biogenesis. It reinforces the concept of a metabolic therapy of HF. Keywords myocardial dysfunction; metabolic therapy; cellular energetics; mitochondrial function; b vitamins

2

ACCEPTED MANUSCRIPT

T

1. Introduction

IP

Heart failure (HF), defined as the inability of the heart to provide adequate blood flow

SC R

to meet the needs of the organism, is a major cause of death in industrialized countries. Although the mechanisms leading to this pathology are still shrouded in mystery, it appears

NU

that alterations in energy metabolism of the cardiac muscle cell may play an important role in the onset and the development of this syndrome. Indeed, it has been widely demonstrated that

MA

HF is associated with significant disruption of energy metabolism, including inhibition of free fatty acid oxidation and of oxidative phosphorylation (for review see [1]) so that the failing

D

heart represents an "engine out of fuel" [2].

TE

During the last decade, our team demonstrated that the expression of the peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) is clearly reduced in HF [3].

CE P

This co-activator regulates the function of many transcription factors involved in energy metabolism pathways such as fatty acid oxidation and mitochondrial biogenesis and is

AC

consequently considered as a key regulator of cellular energetics and especially of mitochondrial biogenesis [4, 5]. Thus, alteration of PGC-1α signalling pathways could at least partially explain the metabolic disorders observed in HF patients. Consequently, this coactivator is considered as a relevant therapeutic target, since the stimulation of its expression and/or its function could reasonably be proposed to overcome the chronic energy deficit observed in the failing heart. Recently, a cardiac-specific robotized cellular assay was developed to identify inducers of Pgc-1α gene expression on H9c2 cardiomyoblasts, revealing the positive effect of several compounds on Pgc-1α transcription [6]. The validation of the effect of those molecules in isolated adult cardiomyocytes has shown that cobalamin (Cb) (vitamin B12) exhibited 3

ACCEPTED MANUSCRIPT remarkable results on PGC-1α signalling pathways, characterising this molecule as a potential activator of mitochondrial biogenesis which could be used in a metabolic therapy of HF.

T

A potential role of Cb in cellular metabolism can be suggested regarding previous studies

IP

which showed that deficiency in methyl donors (Cb and folate (vitamin B9)) in rat induces an

SC R

imbalanced methylation/acetylation of PGC-1α by the protein arginine methyltransferase 1 (PRMT1) and the deacetylase sirtuin 1 (SIRT1) in the heart and in the liver [7-9]. In eukaryotic cells, Cb is metabolized into adenosylcobalamin and methylcobalamin, the latter

NU

being the cofactor of methionine synthase in the cytosol which catalyses the remethylation of

MA

homocysteine to methionine using folic acid [10-12]. Knowing that methionine is converted to S-adenosylmethionine (SAM), the universal donor in transmethylation of numerous proteins, Cb is clearly required for the methylation of proteins involved in the regulation of

TE

D

many cellular pathways (Figure 6). The activity of PGC-1α and of proteins controlling its activity like SIRT1 are known to be directly or indirectly regulated by such post-translational

CE P

modifications [7, 13, 14]; therefore, Cb could affect cellular metabolism through this pathway which also requires folate as substrate for methionine synthase.

AC

In the present study we thus tested the hypothesis that Cb and folate could exhibit positive effects on myocardial energy metabolism and cardiac function in an animal model of pressure overload-induced myocardial dysfunction. To this end, cardiac function and energy metabolism phenotype of mice that have undergone transverse aortic constriction (TAC) were assessed under normal diet or a diet supplemented with Cb and folate.

4

ACCEPTED MANUSCRIPT 2. Materials and Methods

T

2.1. Animals

IP

Five week old C57B6J mice were fed with normal diet (ND) or a diet supplemented (SD)

SC R

with Cb (1 mg/kg) and folate (15 mg/kg) for seven weeks. Three weeks after the beginning of diet, mice underwent surgery to induce pressure overload by transverse aortic constriction (TAC). Anaesthesia was induced by intraperitoneal injection of ketamine (50 mg/kg) and

NU

xylazine (8 mg/kg) and silk suture were placed around the aorta using a blunted 27 gauge

MA

needle to generate aortic stenosis. Sacrifice occurred four weeks after surgery. Mice were euthanized by cervical dislocation and heart were rapidly excised, rinsed in cold calcium-free Krebs solution and weighed. A part of the left ventricle was immediately used for

TE

D

mitochondrial function assessment and another part was flash frozen in liquid nitrogen for further biochemical determinations. All animal experimental procedures were approved by

CE P

animal ethics committee of Paris-Sud University, authorized by French government (authorization number: 2013_051) and complied with directive 2010/63/EU of the European

AC

Parliament on the protection of animals used for scientific purposes.

2.2. Echocardiography Surgery and sacrifice were preceded by echocardiography using a 12 Mhz transducer (Vivid 7, General Electric Healthcare) under 2.5% isoflurane gas anaesthesia to assess cardiac function. M-mode echocardiography was used to determine left ventricular mass, fractional shortening and left ventricular ejection fraction.

2.3. Mitochondrial functional assays in permeabilized cardiac fibres

5

ACCEPTED MANUSCRIPT Mitochondrial respiration was studied in situ in saponin-permeabilized cardiac muscle fibres using a Clarke electrode as previously described [15]. A protocol was designed to measure

T

oxygen consumption after successive addition of ADP (2 mM), malate (4 mM), palmitoyl-

IP

CoA and carnitine (100 µM and 2 mM), pyruvate (1 mM), glutamate (10 mM), succinate (15

SC R

mM), and amytal (an inhibitor of complex I, 1 mM) to respiration solution (in mM: 2.77 CaK2 ethyleneglycol tetraacetic acid (EGTA), 7.23 K2EGTA [100 nM free Ca2+], 6.56 MgCl2 [1 mM free Mg2+], 20 taurine, 0.5 dithiothreitol (DTT), 50 K-methane sulfonate [160 mM ionic

NU

strength], 20 imidazole, pH 7.1) at 23 °C. Rates of respiration are given in µmoles O2/min/g

MA

dry weight. A second protocol consisted in determining the respiratory acceptor control ratio (RCR) calculated from basal mitochondrial respiration rate (in the presence of pyruvate/malate 1/4 mM, without ADP) and oxygen consumption after addition of 2mM

CE P

2.4. Enzyme activity

TE

D

ADP.

Frozen tissue samples were weighed, homogenized (Bertin Precellys 24) in ice-cold buffer

AC

(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. Activity of citrate synthase (CS) was determined using standard spectrophotometric assays [16, 17]. Lactate dehydrogenase (LDH) isoenzymes were separated using agarose gel electrophoresis performed at 200 V for 90 minutes. Individual isoenzymes were resolved by incubation of the gels with a coupled enzyme system as previously described [18].

2.5. Immunoblotting Frozen tissue samples were homogenized (Bertin Precellys 24) in ice cold buffer containing HEPES 50 mM, KCl 50 mM, ethylenediaminetetraacetic acid (EDTA) 1 mM, β-

6

ACCEPTED MANUSCRIPT glycerophosphate 5 mM, Triton X-100 0.1%, orthovanadate 1 mM, dithithreitol 1 mM, sodium fluoride 50 mM, Na pyrophosphate 5 mM, phenylmethylsulfonyl fluoride 0.2 mM

T

and antiprotease cocktail set (Calbiochem 539134). Protein extracts were separated on SDS-

IP

polyacrylamide gel (10 to 12%) and then transferred to polyvinylidene difluoride membranes

SC R

for Western blot or were directly applied on nitrocellulose membranes using 96-well BioDot® (Biorad) for dot blot. After 1 hour of blocking in PBS containing TWEEN20 (0.1%) and nonfat milk (5%), the membranes were incubated overnight at 4°C with primary antibody

NU

(superoxide dismutase 2 (SOD, Abcam ab16956), Nrf2 (or nuclear factor erythroid 2-related

MA

factor, NFE2L2) (Santa Cruz sc-722), acetylated-lysine (Ac-Lys) (Cell signaling 9681) and dimethyl-arginine asymmetric antibody (Me-Arg) (Active motif 39231), phospho- and total AMPK (Cell signaling 2531 and 2532), phospho- and total ACC (Cell signaling 3661 and

TE

D

3676), β-actin (Santa-Cruz sc-47778), PPARα (Santa Cruz sc-9000). OxyblotTM protein oxidation detection kit (millipore S7150) was used for protein carbonylation assessment.

CE P

After washing, the membranes were incubated with a secondary antibody coupled with horseradish peroxidase for 1 hour at room temperature and visualized using chemiluminescent

AC

substrate (LuminataTM Western Chemiluminescent HRP Substrates, Millipore). Light emission was detected by autoradiography and quantified using an image-analysis system (Bio-Rad).

2.6. Quantitative real-time PCR Total ventricular RNA was extracted using Trizol reagent (Invitrogen). Oligo-dT first strand cDNA was synthesized from 2 μg of total RNA using iScript cDNA synthesis kit (Bio-rad). 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

7

ACCEPTED MANUSCRIPT were normalized to RPL32 and TBP levels using GeNorm software [19]. Primer sequences

T

are available in supplementary material (Table 1S).

IP

2.7. Preparation of samples for homocysteinemia dosage

SC R

Blood samples immediately collected at sacrifice were placed on ice and plasma was isolated by centrifugation at 2500g for 15 min at 4°C. Plasma total homocysteine defined as the total concentration of homocysteine after quantitative reductive cleavage of all disulfide bonds,

NU

was assayed by using the fluorimetric high-performance liquid chromatography method

MA

described by Fortin et al. [20].

2.8. Cardiomyocyte cultures

TE

D

Twelve-week old Wistar rats were anaesthetized by intraperitoneal injection of pentobarbital (200 mg/kg). Hearts were rapidly excised and perfused (retrograde perfusion) with

CE P

collagenase to isolate individual adult rat ventricular myocytes as described previously.[21] Isolated cells were immediately plated on laminin-coated culture dishes in minimal essential

AC

medium (M4780, Sigma) supplemented with 2.5% foetal bovine serum, penicillin (100units/mL), streptomycin (100µg/mL), and 2% HEPES (pH 7.4) for 1 hour, and switched to serum-free medium during 16 hours before 24 hours incubation in serum-free medium which was not modified (Control) or supplemented with Cb (30µM) and folate (750µM).

2.9. Immunoprecipitation Cells were lysed in a denaturing IP buffer (1%SDS, 50mM Tris HCl, 5mM EDTA, 10mM DTT, 1mM PMSF, 15U/ml DNase I [pH 7.4]) supplemented with a cocktail of protease inhibitors (Roche) and a cocktail of deacetylase inhibitors (Santa Cruz). The lysate was heat at 90°C for 5min and then diluted in non-denaturing buffer to trap SDS (1% triton, 50mM Tris

8

ACCEPTED MANUSCRIPT HCl, 5mM EDTA, 300mM NaCl, 0.02% azide de sodium, 1mM PMSF,pH 7.4) supplemented with a cocktail of protease inhibitor (Roche) and a cocktail of deacetylase inhibitors (Santa

T

Cruz). Lysine-acetylated proteins were immunoprecipitated using anti-acetylated lysine

IP

antibody coated on A magnetic beads (Millipore). Anti-IgG (Santa Cruz) were used as

SC R

control. Imunoprecipitated proteins, input and output were run on SDS-PAGE and were

NU

revealed with anti-PGC-1α antibody (Santa Cruz sc-13067) .

MA

2.10. Statistical analysis

Results are expressed as mean ± SEM. Statistical differences were analyzed using two-way ANOVA; Newman-Keuls post-hoc tests were used to identify significant differences between

AC

CE P

using paired t-test.

TE

D

means. The statistical analysis of immunoprecipitation assay on rat cardiomyocytes was done

9

ACCEPTED MANUSCRIPT 3. Results

T

3.1. Cobalamin and folate supplemented diet attenuated cardiac alterations induced by

IP

pressure overload.

SC R

After 4 weeks of TAC, mice fed without or with SD displayed an increase in heart weight and lung weight (Table 1) compared to their respective sham group, as judged by absolute weight and/or organ weight-to-tibia length ratio. However, the heart weight after TAC was

NU

significantly lower when mice were fed with a diet supplemented with Cb and folate. No

MA

impact of pressure overload was observed on body and kidney weight in any group. Assessment of left ventricle mass by echocardiography also showed that a TAC-induced hypertrophy was significantly less pronounced in SD-TAC group (Figure 1A). Importantly,

TE

D

the decrease in left ventricular ejection fraction (LVEF) and fractional shortening (FS) after 4 weeks of TAC was significantly attenuated by SD (Figures 1B, C and D). The increase in

CE P

systolic and diastolic left ventricular diameter induced by TAC was also less marked in SDTAC group as it is shown in Table 2S presenting the evolution of echocardiographic

AC

parameters four weeks after surgery (absolute values of these parameters are available in Table 3S). Moreover, left ventricular levels of the stress-responsive markers, atrial natriuretic peptide (ANP) and myocyte-enriched calcineurin-interacting protein (MCIP1), mRNA were significantly less upregulated in SD-TAC group than in ND-TAC group (Figure 1E), evidencing a better functional status of the myocardium in TAC in the presence of Cb and folate supplementation.

3.2. Cobalamin and folate supplemented diet protected cardiac energy metabolism in pressure overloaded myocardium.

10

ACCEPTED MANUSCRIPT In order to elucidate the mechanism of the SD protective effect on cardiac function, we sought to determine whether Cb and folate could protect cellular energetics and especially

T

mitochondrial function in this in vivo model. After 4 weeks of TAC in mice fed with ND,

IP

respiration rates in permeabilized fibres measured after cumulative addition of pyruvate,

SC R

glutamate, succinate and amytal were significantly lower compared with ND-sham (Figure 2A). Cobalamin and folate did not change mitochondrial function in healthy animals; however, this diet almost completely prevented the decrease in mitochondrial respiration

NU

observed under pressure overload stress (Figure 2A). The drop in respiration rates observed in

MA

ND-TAC myocardium was combined with a trend (p = 0.125) towards a lower respiratory acceptor control ratio in this group which was not observed in mice treated with SD (Figure 2B). In addition, the activity of citrate synthase (CS), which is currently used as a marker of

TE

D

mitochondrial mass, was significantly reduced in ND group after 4 weeks of TAC, whereas the difference was not significant between sham and TAC mice treated with Cb and folate

CE P

(Figure 2C). Thus, the sum of these data suggests that cardiac protection offered by Cb and folate supplemented diet in pressure overload model is associated with an improved

AC

mitochondrial function.

The protective effect of SD on mitochondrial function under cardiac pressure overload could be linked to a better preservation of metabolism upstream of the mitochondrial oxidative phosphorylation, especially mitochondrial biogenesis cascade as well as free fatty acid oxidation. Indeed, a strong trend towards a higher peroxisome proliferator-activated receptor alpha (Pparα) expression in SD-TAC group in comparison with ND-TAC was revealed by quantitative PCR; this beneficial effect of SD on PPARα expression in mice undergoing TAC has been confirmed by increased protein content in TAC-SD versus TAC-ND (Figure 1SA). Medium chain acyl-CoA dehydrogenase (MCAD) expression was significantly higher in TAC mice fed with SD compared to ND-TAC ones (Figure 2D) and hydroacyl-CoA dehydrogenase

11

ACCEPTED MANUSCRIPT (HADHA) activity tended towards a lesser alteration in SD-TAC group in comparison with ND-TAC group (Figure 2E). These results show that SD improved fatty acid utilization under

T

conditions of TAC. These results are in accordance with the sustained mitochondrial

IP

respiration under palmitoyl-CoA and carnitine in SD-TAC mice which was altered when TAC

SC R

mice were fed with ND (Figure 2F); interestingly, this is not associated with significant changes in Cpt1a and Cpt1b gene expression in any group (Figure 1SB). Finally, the large and significant increase in hexokinase 2 (Hk2) and pyruvate dehydrogenase kinase 4 (Pdk4)

NU

expression as well as prevalence of M-isoform of LDH in ND-TAC group in comparison with

MA

the others groups (Figure 2G and 2H) suggests that the metabolism of TAC mice is remodelled towards glycolysis at the expense of oxidation of fatty acids. SD almost completely normalized these metabolic shifts. This suggests that Cb and folate have

TE

D

generalized effect on the energy metabolism involving different pathways coordinated by

CE P

PGC-1.

3.3. Cobalamin and folate supplemented diet sustained mitochondrial biogenesis

AC

transcription cascade in pressure overloaded myocardium. The pressure overload led to a decrease in the expression of Pgc-1α and Pgc-1β genes and SD did not augment these parameters (Figure 3A). However, SD ensured normalization of downstream PGC-1 targets expression including mitochondrial transcription factor A (Tfam), nuclear respiratory factor 2 (Nrf2), cytochrome c oxidase 1 (Cox1) and cytochrome c oxidase 4 (Cox4) genes (Figure 3B). Given the involvement of Cb in methylation and in acetylation of proteins via methylcobalamin, we studied post-translational regulators of PGC-1α. To investigate the underlying mechanisms allowing an activation of the PGC-1-mediated cascades, we first assessed the combined effect of TAC and diet on post-translational activation/inhibition of PGC-1α. As shown in Figure 4A, Prmt1 gene expression level showed

12

ACCEPTED MANUSCRIPT a large increase after TAC only in mice treated with Cb and folate, evoking a higher methylation capacity in this group. Accordingly, the total methylation level, assessed by

T

immunoblotting, was significantly higher in SD-TAC group than in ND-TAC group (Figure

IP

4B). Interestingly, ND-TAC mice displayed a trend (p = 0.142) towards a lesser methylation

SC R

of proteins in comparison with ND-sham mice (Figure 4B). Whereas gene expression of the deacetylase Sirt1 was not impacted neither by TAC nor by diet, the acetyltransferase general control non-repressed protein 5 (Gcn5) gene expression was decreased in SD-TAC compared

NU

to ND-TAC group (Figure 4A). These results suggest a lower acetylation level of proteins,

MA

and potentially PGC-1α, in SD-TAC mice. In line with these data, a general normalization of elevated total acetylation level in TAC group by SD was observed (Figure 4B). As PGC-1α activity can also be modulated by phosphorylation, AMP-activated protein kinase

TE

D

(AMPK) and acetyl-coA carboxylase (ACC) phosphorylation were analyzed to estimated AMPK activity, another important regulator of PGC-1α activity. Phosphorylation of AMPK

CE P

was significantly increased after TAC with ND or SD, but only protein extracts from SD-TAC mice exhibited a significant and strong increase in ACC phosphorylation after TAC and in

this group.

AC

phosphorylated ACC/total ACC ratio (Figure 4C), revealing a higher activation of AMPK in

In order to confirm that B vitamins can modulate the post-translational status of PGC-1α, adult cardiomyocytes were treated with Cb and folate for studying, by immunoprecipitation assay, the acetylation level of PGC-1α after treatment with these vitamins (Figure 5A). This experiment showed a significant decrease of PGC-1α acetylation level in cardiomyocytes treated with Cb and folate in comparison with non-treated cardiomyocytes (63±23 and 87±26 AU respectively, p = 0.02) thus conferring credit to the hypothesis of the post-translational activation of PGC-1α by these vitamins.

13

ACCEPTED MANUSCRIPT Although in view of the modulations of mitochondrial biogenesis transcriptional cascade described above consequences on mitochondrial mass could be expected, no variation of

T

mitochondrial protein level was noted as showed by respiratory chain complex subunit

SC R

IP

content (C-I-20, C-II-30, C-III-Core2, Cox4, C-V-α), CPT1, VDAC and CS level (Figure 2S).

3.4. The protective effect of SD was not related to Cb and folate action on oxidative stress and homocysteinemia.

NU

Knowing that mitochondria are a major producer and a target of ROS in the heart, assessment

MA

of oxidative stress was also performed. Protein carbonylation level was similar in all groups (Figure 6A) and gene expression (Glutathione peroxidase 1, GPX1) or protein level (Superoxide dismutase 2, SOD2) of antioxidant enzymes as well as protein level of Nuclear

TE

D

factor (erythroid-derived 2)-like 2 (NFE2L2) did not show any significant difference between TAC groups (Figure 6B and C), even if NFE2L2 level tended to be lower in ND-TAC group

CE P

in comparison to the other groups (Figure 6C), suggesting that oxidative stress does not seem to be a major actor in the beneficial effects of SD.

AC

It is also well known that Cb is intimately linked to the concentration of homocysteine in the blood through a stimulation of methionine production from homocysteine and folate (Figure 7). A dysregulation of homocysteinemia has been reported to be associated with cardiac dysfunction. To know whether the beneficial effects of the diet could be due to a decrease in circulating homocysteine,

plasma

homocysteine concentration was

measured.

No

hyperhomocysteinemia was observed in TAC animals, and homocysteinemia was not modified by diet (Figure 6D).

14

ACCEPTED MANUSCRIPT

T

4. Discussion

IP

Alterations of energy metabolism and especially mitochondrial dysfunctions have been

SC R

largely reported in heart failure syndrome for many years [22, 23], suggesting that a metabolic therapy could be a valuable option in this disease. Based on the positive action of Cb on PGC1α in isolated cardiomycytes shown in a previous study[6], we tested the potential protective

NU

effect of Cb and folate during cardiac pressure overload. Here, we show that feeding with SD,

MA

from 3 weeks before TAC to sacrifice, induces beneficial effects in the murine model of myocardial dysfunction. The treatment with Cb and folate clearly minimized the alterations of cardiac contractility classically induced by TAC and the protective effect of these B vitamins

TE

D

was associated with sustained mitochondrial function, and biogenesis transcription cascade as well as improved fatty acid oxidation which seemed to lead to the preservation of an efficient

CE P

mitochondrial function.

overload

AC

4.1. Cb and folate support PGC-1α activity in murine model of cardiac pressure

It has been reported that after a prolonged aortic banding, cardiac dysfunction is associated with decreased oxidative capacity and fatty acid oxidation that could be partly explained by a decrease in PGC-1α expression and an inhibition of its downstream transcription cascade [3, 24]. In the present study, following TAC oxidative capacity and fatty acid oxidation were decreased in ND fed mice while this decrease was prevented by feeding Cb and folate. Moreover, a clear decrease in Pgc-1α transcription was shown in the heart of animals with aortic stenosis. This was associated with a lower transcription level of Tfam, Nrf2, Cox1 and Cox4 in ND-TAC group suggesting inhibition of the PGC-1α cascade. Normalization of the

15

ACCEPTED MANUSCRIPT expression of these latter genes in SD-TAC mice together with improved mitochondrial function evidenced an improvement of PGC-1α activity when TAC mice were fed with SD

T

despite unchanged PGC-1α expression. PGC-1α activity is largely dependent on methylation

IP

and acetylation states of the protein [13, 25, 26]. Those states could be modified by Cb and

SC R

folate, since it has been shown by several studies that methyl donor deficiency in rat induces detrimental effects on energy metabolism through an imbalanced methylation/acetylation of PGC-1α by PRMT1 and SIRT1/GCN5 in the heart and in the liver [7-9]. Methyl donor

NU

deficiency during pregnancy and lactation also induces heart and liver affliction in offsprings because of the same underlying mechanisms [9]. Moreover, one of these studies pointed out

MA

the hypomethylation of PGC-1α to explain the cardiac hypertrophy observed in rats fed without Cb and folate suggesting a major role of energetic status in cardiac remodelling [7].

TE

D

In our study, the expression of Sirt1, Gcn5 and Prmt1 and particularly the assessment of total methylation/acetylation level by immunoblotting suggested a lesser acetylation level and a

CE P

higher methylation level of PGC-1α in SD-TAC mice that are in favour of the activation of this transcriptional co-activator [27, 28]. Besides, the important activity of AMPK in SD-TAC

AC

group revealed in this study could also be an activating mechanism of this key regulator of the cellular energetics [29]. In a convincing manner, these observations allow proposing that the post-translational modifications of PGC-1α were associated with the preservation of mitochondrial function and thus seem to be at least in part responsible for the protection of cardiac energy metabolism observed in SD-TAC group. The impact of Cb and folate on PGC-1α activity can be explained by the role of these compounds in methionine production, the precursor of the universal methyl donor SAM [12, 30], which allows methylation of PGC-1α by PRMT1; but it can also be advanced that SAM is required to modulate the activity of numerous proteins which could secondarily regulate PGC-1α, as it has previously been suggested for SIRT1 [14]. Thus, even if no modification of

16

ACCEPTED MANUSCRIPT Sirt1 expression were noted in our study, a post-translational activation of this enzyme could eventually be hypothesized, reinforcing the idea of a lesser acetylation of PGC-1α suggested

T

by the decrease in Gcn5 expression and in total acetylation level in SD-TAC group. This

IP

hypothesis is supported by the assessment of PGC-1α acetylation state in adult rat

SC R

cardiomyocytes treated with Cb and folate.

One of the major points of the study is the fact that even if the activation level of mitochondrial biogenesis transcriptional cascade is only sustained in SD group when mice

NU

underwent TAC, it is not associated with a significant difference in mitochondrial mass

MA

between both TAC groups as it is shown by mitochondrial protein expression, but an improved mitochondrial function. This observation seems to be the result of a general acceleration of mitochondrial life cycle in which mitophagic processes would also be much

TE

D

more activated in response to SD treatment. This is indeed suggested by the activation of AMPK observed in SD-TAC group, a protein known as an initiator of mitophagy [31]. This

CE P

AMPK activation was associated with a higher content in SD-TAC of Parkin protein (Figure 3S), a protein involved in mitophagy in the cardiomyocyte and playing an important role for

AC

the maintenance of normal cardiac mitochondrial function under stress [32]. Thus, it could be hypothesized that the coordination between mitochondrial biogenesis and mitophagy, which has largely been claimed [33], would lead to the same absolute mitochondrial mass in our different groups but the activation of mitochondrial biogenesis cascade and mitophagy in SDTAC group would allow the preservation of a high quality mitochondrial pool by supporting a more efficient mitochondrial turnover than in ND-TAC group. Interestingly, there is no obvious direct effect of Cb on Pgc-1α transcription in vivo contrary to what had been shown in cells [6]. However, the transposition of what is seen in an acute cellular model to a chronic animal model has to be put in perspective, especially because the

17

ACCEPTED MANUSCRIPT stimulation of Pgc-1α transcription in adult rat cardiomyocytes reported in our previous study was transitory and thus might suggest a complex mechanism.

T

Finally, our study shows that SD treatment prevents the metabolic switch described in TAC-

IP

induced HF at least by preserving the ability to use fatty acids as a substrate to produce

SC R

energy. The decreased capacity to oxidize fatty acids is a hallmark of heart failure. Interestingly, adenosylcobalamin (a derivative of Cb) is the cofactor of methylmalonyl coenzyme A mutase in mitochondria involved in odd-chain fatty acid catabolism [10-12] and

NU

ensures the entry of the last degradation product of these fatty acids, methylmalonyl-CoA,

MA

into the Krebs cycle by its conversion to succinyl-CoA. This should be taken into account, especially as it is likely that the effect of cobalamin on the fatty acid utilization is an important point in the benefit provided by SD. In this regard, it would also be interesting to

TE

D

investigate the effect of this vitamin on fatty acid circulating levels and fatty acid uptake. 4.2. Protective effect of Cb and folate in cardiac pressure overload is independent of its

CE P

role on oxidative stress and on homocysteinemia. Theoretically, the beneficial actions of these two B vitamins could also be explained by

AC

improvement of antioxidant mechanisms. Cb and folate are efficient radical traps [34, 35] and they can prevent oxidative damages in vivo [36, 37]. A deficiency in Cb leads to oxidative stress through methylcobalamin or adenosylcobalamin pathways [38, 39]. Nevertheless, no oxidative damages were observed after four weeks of TAC in our model and no difference in antioxidant elements such as GPX1, SOD2 or NFE2L2, were noted between TAC groups, indicating that a potential antioxidant effect of these B vitamins would not be involved in the protective mechanisms observed here. Cobalamin-mediated oxidative protection is often associated with its role in the control of homocysteinemia. Cb is important to maintain a relatively low homocysteinemia and hyperhomocysteinemia (HHCY) is reported to increase ROS production [40] and to decrease

18

ACCEPTED MANUSCRIPT antioxidant mechanisms [41, 42]. In our study, plasma level of homocysteine was similar in all groups; consequently, it can be considered that Cb did not act through this mechanism in

T

the present work.

IP

The fact that oxidative stress and HHCY previously described in pressure overload model

SC R

[43] were not observed in our study shows that, after four weeks of aortic banding, our mice were at the beginning of the disease. This early stage of the disease certainly explains the fact that no alteration of Cpt1s expression were measured, contrary to what has been previously

NU

reported in a more severe model [44], and no alteration of mitochondrial protein expression

MA

were revealed by Western blot in ND-TAC as it could be expected considering mitochondrial biogenesis alterations (Figure 2S). This latter method is probably not sensitive enough to detect small variations of protein amount. Interestingly, even if the syndrome was not fully

TE

D

developed, the metabolic alterations were noted, demonstrating that these latter occur at an early stage of the disease and that metabolic therapy could consequently be relevant.

CE P

The absence of HHCY in our model is also a critical point to understand the mode of action of Cb and folate in this study. Indeed, even if the underlying mechanisms are not fully

AC

elucidated, it has been described that HHCY can directly or indirectly alter cardiac function. Actually, the vascular alterations induced by HHCY are well known [45, 46], but HHCY can also directly affect contractile properties of the heart through different mechanisms involving mitochondria or not [47, 48]. Knowing that homocysteinemia was normal after four weeks of TAC, this potential protective mechanism of Cb and folate cannot be considered to explain our results. The fact that, in our study, the protection of cardiac function by Cb and folate does not rely on an effect on homocysteinemia is certainly a major point to consider. A few years ago, the ability of these two B vitamins to control homocysteinemia presented these compounds as a potential treatment of human hyperhomocysteinemia-associated diseases, leading to several

19

ACCEPTED MANUSCRIPT clinical trials. Thus, the potential beneficial effects of Cb and folate have extensively been studied in patients who presented hyperhomocysteinemia and cardiovascular risks such as

T

stroke, coronary diseases and ischemic diseases [49, 50]. Despite the normalization of

IP

homocysteinemia of these patients, these studies did not show a prevention of cardiovascular

SC R

events by Cb and folate, conferring a negligible therapeutic interest upon Cb and folate. Nevertheless, these clinical trials focused on homocysteine-lowering effect of Cb and folate and on atherothrombotic events in these particular diseases. The beneficial effects observed in

NU

the present study apply exclusively to a mouse model of pressure overload. To our

MA

knowledge, these vitamins have never been tested in human in the particular context of pressure overload-induced HF. Currently, the use of Cb and folate in the treatment of pressure overload-induced HF and the effect of these vitamins on contractile and metabolic functions

TE

D

in human pathophysiology have been poorly addressed. Our study could suggest a reconsideration of the therapeutic use of Cb and folate in pressure overload-induced HF. This

CE P

is supported by a clinical study led by Witte et al. which showed the improvement of left ventricle function of HF patients treated with a combination of vitamins including Cb and

AC

folate among others [51]. Beyond these therapeutic considerations of vitamins, our study shows that a preservation of mitochondrial function in an animal model of HF is associated with a protection of cardiac function, thus adding support to the concept of metabolic therapy of HF. It is noteworthy to indicate that the present study has several limitations. First, the data suggesting higher free fatty acid utilisation by the mitochondria were obtained under optimal conditions in vitro. A special study in vivo needs to be done to estimate the flux through fatty acid oxidation under Cb and folate treatment. Second, our work does not investigate effect of SD on the global energy state in the myocardium also in vivo. Measurements of the main high energy phosphate concentrations in the heart would be rather informative.

20

ACCEPTED MANUSCRIPT

In summary, we demonstrated here for the first time that the use of a treatment with Cb and

T

folate supplemented diet in a murine model of cardiac pressure overload exhibits beneficial

IP

effects on cardiac function and preservation of mitochondrial function and biogenesis

SC R

transcription cascade. This protective action of Cb and folate is associated with a sustained acetylation/methylation balance potentially involving a post-translational activation of PGC1α. Overall, these results support the notion that, in HF, mitochondrial biogenesis, and

NU

especially PGC-1α, is a relevant target to preserve cardiac energy metabolism and function of

MA

the heart, conferring credit to metabolic therapy.

TE

D

Fundings

Our laboratory is a member of the Laboratory of Excellence LERMIT and is supported by

CE P

grants from “Fondation pour la Recherche Médicale” (to AG, #DPM20121125546), université Paris-Sud (ERM) and CORDDIM (to RVC, #cod110153). RVC is emeritus

AC

scientist at CNRS.

Acknowledgments

We thank Valérie Domergue for preparation of the animals and Claudine Deloménie for her expertise in molecular biology (IFR141-IPSIT). We thank Rodolphe Fischmeister and AnaMaria Gomez for continuous support.

Disclosure: none

21

ACCEPTED MANUSCRIPT References

T

[1] Ventura-Clapier R, Garnier A, Veksler V, Joubert F. Bioenergetics of the failing heart.

IP

Biochimica et biophysica acta. 2011;1813:1360-72.

SC R

[2] Neubauer S. The failing heart--an engine out of fuel. N Engl J Med. 2007;356:1140-51. [3] Garnier A, Fortin D, Delomenie C, Momken I, Veksler V, Ventura-Clapier R. Depressed mitochondrial transcription factors and oxidative capacity in rat failing cardiac and skeletal

NU

muscles. The Journal of physiology. 2003;551:491-501.

MA

[4] Lin J, Handschin C, Spiegelman BM. Metabolic control through the PGC-1 family of transcription coactivators. Cell metabolism. 2005;1:361-70. [5] Patten IS, Arany Z. PGC-1 coactivators in the cardiovascular system. Trends in

TE

D

endocrinology and metabolism: TEM. 2012;23:90-7. [6] Ruiz M, Courilleau D, Jullian JC, Fortin D, Ventura-Clapier R, Blondeau JP, et al. A

CE P

cardiac-specific robotized cellular assay identified families of human ligands as inducers of PGC-1alpha expression and mitochondrial biogenesis. PloS one. 2012;7:e46753.

AC

[7] Garcia MM, Gueant-Rodriguez RM, Pooya S, Brachet P, Alberto JM, Jeannesson E, et al. Methyl donor deficiency induces cardiomyopathy through altered methylation/acetylation of PGC-1alpha by PRMT1 and SIRT1. J Pathol. 2011;225:324-35. [8] Pooya S, Blaise S, Moreno Garcia M, Giudicelli J, Alberto JM, Gueant-Rodriguez RM, et al.

Methyl

donor

deficiency impairs

fatty acid

oxidation

through

PGC-1alpha

hypomethylation and decreased ER-alpha, ERR-alpha, and HNF-4alpha in the rat liver. J Hepatol. 2012;57:344-51. [9] Gueant JL, Elakoum R, Ziegler O, Coelho D, Feigerlova E, Daval JL, et al. Nutritional models of foetal programming and nutrigenomic and epigenomic dysregulations of fatty acid metabolism in the liver and heart. Pflugers Archiv : European journal of physiology. 2013.

22

ACCEPTED MANUSCRIPT [10] Banerjee R, Ragsdale SW. The many faces of vitamin B12: catalysis by cobalamindependent enzymes. Annu Rev Biochem. 2003;72:209-47.

T

[11] Quadros EV. Advances in the understanding of cobalamin assimilation and metabolism.

IP

Br J Haematol. 2010;148:195-204.

SC R

[12] Gueant JL, Caillerez-Fofou M, Battaglia-Hsu S, Alberto JM, Freund JN, Dulluc I, et al. Molecular and cellular effects of vitamin B12 in brain, myocardium and liver through its role as co-factor of methionine synthase. Biochimie. 2013;95:1033-40.

NU

[13] Holness MJ, Caton PW, Sugden MC. Acute and long-term nutrient-led modifications of

MA

gene expression: potential role of SIRT1 as a central co-ordinator of short and longer-term programming of tissue function. Nutrition. 2010;26:491-501. [14] Ghemrawi R, Pooya S, Lorentz S, Gauchotte G, Arnold C, Gueant JL, et al. Decreased

TE

D

vitamin B12 availability induces ER stress through impaired SIRT1-deacetylation of HSF1. Cell Death Dis. 2013;4:e553.

CE P

[15] Kuznetsov AV, Veksler V, Gellerich FN, Saks V, Margreiter R, Kunz WS. Analysis of mitochondrial function in situ in permeabilized muscle fibers, tissues and cells. Nat Protoc.

AC

2008;3:965-76.

[16] Wharton DC, Tzagoloff A. Cytochrome oxidase from beef heart mitochondria. Methods in Enzymology. 1967;10:245-50. [17] Veksler VI, Kuznetsov AV, Anflous K, Mateo P, van Deursen J, Wieringa B, et al. Muscle creatine kinase-deficient mice. II. Cardiac and skeletal muscles exhibit tissue-specific adaptation of the mitochondrial function. The Journal of biological chemistry. 1995;270:19921-9. [18] De Sousa E, Veksler V, Minajeva A, Kaasik A, Mateo P, Mayoux E, et al. Subcellular creatine kinase alterations. Implications in heart failure. Circulation research. 1999;85:68-76.

23

ACCEPTED MANUSCRIPT [19] Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of

T

multiple internal control genes. Genome Biol. 2002;3:research0034.1-.II.

IP

[20] Fortin LJ, Genest J, Jr. Measurement of homocyst(e)ine in the prediction of

SC R

arteriosclerosis. Clin Biochem. 1995;28:155-62.

[21] Verde I, Vandecasteele G, Lezoualc'h F, Fischmeister R. Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type Ca2+ current

NU

in rat ventricular myocytes. British Journal of Pharmacology. 1999;127:65-74.

Journal of physiology. 2004;555:1-13.

MA

[22] Ventura-Clapier R, Garnier A, Veksler V. Energy metabolism in heart failure. The

[23] Mettauer B, Zoll J, Garnier A, Ventura-Clapier R. Heart failure: a model of cardiac and

TE

D

skeletal muscle energetic failure. Pflugers Archiv : European journal of physiology. 2006;452:653-66.

CE P

[24] Witt H, Schubert C, Jaekel J, Fliegner D, Penkalla A, Tiemann K, et al. Sex-specific pathways in early cardiac response to pressure overload in mice. J Mol Med (Berl).

AC

2008;86:1013-24.

[25] Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P. Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. Nature. 2005;434:1138. [26] Wenz T. Regulation of mitochondrial biogenesis and PGC-1alpha under cellular stress. Mitochondrion. 2013;13:134-42. [27] Teyssier C, Ma H, Emter R, Kralli A, Stallcup MR. Activation of nuclear receptor coactivator PGC-1alpha by arginine methylation. Genes Dev. 2005;19:1466-73.

24

ACCEPTED MANUSCRIPT [28] Lerin C, Rodgers JT, Kalume DE, Kim SH, Pandey A, Puigserver P. GCN5 acetyltransferase complex controls glucose metabolism through transcriptional repression of

T

PGC-1alpha. Cell metabolism. 2006;3:429-38.

IP

[29] Jager S, Handschin C, St-Pierre J, Spiegelman BM. AMP-activated protein kinase

SC R

(AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha. Proceedings of the National Academy of Sciences of the United States of America. 2007;104:12017-22. [30] Chiang PK, Gordon RK, Tal J, Zeng GC, Doctor BP, Pardhasaradhi K, et al. S-

NU

Adenosylmethionine and methylation. FASEB journal : official publication of the Federation

MA

of American Societies for Experimental Biology. 1996;10:471-80. [31] Egan DF, Shackelford DB, Mihaylova MM, Gelino S, Kohnz RA, Mair W, et al. Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy

TE

D

sensing to mitophagy. Science. 2011;331:456-61. [32] Piquereau J, Godin R, Deschenes S, Bessi VL, Mofarrahi M, Hussain SN, et al.

CE P

Protective role of PARK2/Parkin in sepsis-induced cardiac contractile and mitochondrial dysfunction. Autophagy. 2013;9.

AC

[33] Palikaras K, Lionaki E, Tavernarakis N. Balancing mitochondrial biogenesis and mitophagy to maintain energy metabolism homeostasis. Cell death and differentiation. 2015;22:1399-401.

[34] Joshi R, Adhikari S, Patro BS, Chattopadhyay S, Mukherjee T. Free radical scavenging behavior of folic acid: evidence for possible antioxidant activity. Free Radic Biol Med. 2001;30:1390-9. [35] Krautler B. Vitamin B12: chemistry and biochemistry. Biochem Soc Trans. 2005;33:80610.

25

ACCEPTED MANUSCRIPT [36] Shaw S, Jayatilleke E, Herbert V, Colman N. Cleavage of folates during ethanol metabolism. Role of acetaldehyde/xanthine oxidase-generated superoxide. Biochem J.

T

1989;257:277-80.

IP

[37] Arriaga-Alba M, Ruiz-Perez NJ, Sanchez-Navarrete J, de Angel BL, Flores-Lozada J,

SC R

Blasco JL. Antimutagenic evaluation of vitamins B1, B6 and B12 in vitro and in vivo, with the Ames test. Food Chem Toxicol. 2013;53:228-34.

[38] Richard E, Alvarez-Barrientos A, Perez B, Desviat LR, Ugarte M. Methylmalonic

NU

acidaemia leads to increased production of reactive oxygen species

and induction of

MA

apoptosis through the mitochondrial/caspase pathway. J Pathol. 2007;213:453-61. [39] Richard E, Desviat LR, Ugarte M, Perez B. Oxidative stress and apoptosis in homocystinuria patients with genetic remethylation defects. Journal of cellular biochemistry.

TE

D

2013;114:183-91.

[40] Tyagi N, Ovechkin AV, Lominadze D, Moshal KS, Tyagi SC. Mitochondrial mechanism

CE P

of microvascular endothelial cells apoptosis in hyperhomocysteinemia. Journal of cellular biochemistry. 2006;98:1150-62.

AC

[41] Streck EL, Vieira PS, Wannmacher CM, Dutra-Filho CS, Wajner M, Wyse AT. In vitro effect of homocysteine on some parameters of oxidative stress in rat hippocampus. Metab Brain Dis. 2003;18:147-54. [42] Matte C, Mackedanz V, Stefanello FM, Scherer EB, Andreazza AC, Zanotto C, et al. Chronic hyperhomocysteinemia alters antioxidant defenses and increases DNA damage in brain and blood of rats: protective effect of folic acid. Neurochem Int. 2009;54:7-13. [43] Hughes WM, Jr., Rodriguez WE, Rosenberger D, Chen J, Sen U, Tyagi N, et al. Role of copper and homocysteine in pressure overload heart failure. Cardiovasc Toxicol. 2008;8:13744.

26

ACCEPTED MANUSCRIPT [44] Lahey R, Wang X, Carley AN, Lewandowski ED. Dietary fat supply to failing hearts determines dynamic lipid signaling for nuclear receptor activation and oxidation of stored

T

triglyceride. Circulation. 2014;130:1790-9.

IP

[45] Vizzardi E, Bonadei I, Zanini G, Frattini S, Fiorina C, Raddino R, et al. Homocysteine

SC R

and heart failure: an overview. Recent patents on cardiovascular drug discovery. 2009;4:1521.

[46] Ganguly P, Alam SF. Role of homocysteine in the development of cardiovascular

NU

disease. Nutr J. 2015;14:6.

MA

[47] Joseph J, Washington A, Joseph L, Koehler L, Fink LM, Hauer-Jensen M, et al. Hyperhomocysteinemia leads to adverse cardiac remodeling in hypertensive rats. American journal of physiology Heart and circulatory physiology. 2002;283:H2567-74.

TE

D

[48] Moshal KS, Tipparaju SM, Vacek TP, Kumar M, Singh M, Frank IE, et al. Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in

CE P

hyperhomocysteinemia. American journal of physiology Heart and circulatory physiology. 2008;295:H890-7.

AC

[49] Joseph J, Loscalzo J. Methoxistasis: integrating the roles of homocysteine and folic acid in cardiovascular pathobiology. Nutrients. 2013;5:3235-56. [50] Clarke R, Halsey J, Lewington S, Lonn E, Armitage J, Manson JE, et al. Effects of lowering homocysteine levels with B vitamins on cardiovascular disease, cancer, and causespecific mortality: Meta-analysis of 8 randomized trials involving 37 485 individuals. Arch Intern Med. 2010;170:1622-31. [51] Witte KK, Nikitin NP, Parker AC, von Haehling S, Volk HD, Anker SD, et al. The effect of micronutrient supplementation on quality-of-life and left ventricular function in elderly patients with chronic heart failure. European heart journal. 2005;26:2238-44.

27

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

28

ACCEPTED MANUSCRIPT Figure legends

IP

T

Table 1: Anatomical characteristics of mice 4 weeks after surgery

SC R

Figure 1: Effect of transverse aortic constriction (TAC) on mouse cardiac function under normal or cobalamin/folate-supplemented diet.

A. Left ventricule mass were assessed by echocardiography before surgery (left panel) and at

NU

sacrifice (right panel) B. Representative echocardiographs at sacrifice C-D. Ejection fraction

MA

and shortening fraction were assessed by echocardiography before surgery (upper panels) and at sacrifice (below panels). E. Markers of cardiac damage 4 weeks after surgery: Anp (atrial natriuretic peptide) and Mcip1 (myocyte-enriched calcineurin interacting protein 1)

TE

D

expression. (n=6 to 8 per experimental group), * p<0.05 sham vs TAC, # p<0.05 ND vs SD.

CE P

Figure 2: Effect of TAC on cardiac energy metabolism under normal or cobalamin/folate-supplemented diet.

AC

A. Rate of respiration measured under phosphorylating conditions (2mM ADP) in the presence of substrates for complex I and II. B. Respiratory acceptor control ratio was calculated as the ratio between maximal respiration rate (ADP 2mM) and basal respiration rate (without ADP) with malate-pyruvate (4-1mM). C. Citrate synthase (CS) enzymatic activity. D. mRNA expression level of the peroxysome proliferator-activated receptor α (Pparα) and the medium-chain acyl-CoA dehydrogenase (Mcad) of fatty acid catabolism. E. Hydroxyacyl-CoA dehydrogenase enzymatic activity. F. Rate of respiration measured under phosphorylating conditions (2mM ADP) in the presence of palmitoyl-CoA and carnitine. G. mRNA expression level of carbohydrate catabolism. H. Proportion of H and M isoforms of

29

ACCEPTED MANUSCRIPT the lactate dehydrogenase (LDH) assessed from activity of isoenzymes. (n=6 to 8 per

T

experimental group), * p<0.05 sham vs TAC, # p<0.05 ND vs SD.

SC R

cobalamin/folate-supplemented diet 4 weeks after surgery.

IP

Figure 3: Markers of mitochondrial biogenesis in heart of mice fed with normal and

A. mRNA expression level of PGC-1 transcriptional coactivators of mitochondrial biogenesis. B. mRNA expression level of key transcriptional factors of mitochondrial biogenesis (Tfam

NU

and nuclear respiratory factor 2, Nrf2) and of mitochondrial complex subunits (Cox1 and

MA

Cox4). (n=6 to 8 per experimental group), * p<0.05 sham vs TAC, # p<0.05 ND vs SD.

TE

normal and supplemented diet.

D

Figure 4: Effect of TAC on post-translational modifications in heart of mice under

A. mRNA expression level of the protein arginine methyltransferase 1 (Prmt1), the sirtuin 1

CE P

(Sirt1) and the general control of amino acid synthesis protein 5-like 2 (Gcn5) involved in protein acetylation/methylation balance. B. total methylation (Me-Arg) and acetylation (Ac-

AC

Lys) of proteins assessed by dot blots. C. Immunoblotting of total AMPK (tAMPK), phosphorylated-AMPK (AMPK-P), total ACC (tACC)/phosphorylated-ACC (ACC-P) (n=6 to 8 per experimental group), * p<0.05 sham vs TAC, # p<0.05 ND vs SD.

Figure 5: PGC-1α acetylation level in adult rat cardiomyocytes after cobalamin and folate treatment. Representative blot after immunoprecipitation of acetylated proteins from adult rat cardiomyocyte lysate followed by immunoblotting with PGC-1α antibody.

30

ACCEPTED MANUSCRIPT Figure 6: Cardiac oxidative status and homocysteinemia of mice fed with normal and cobalamin/folate-supplemented diet 4 weeks after surgery.

T

A. Global carbonylation of left ventricle proteins. B. mRNA expression level of the

IP

glutathione peroxidase 1 (Gpx1). C. Protein level of the superoxide dismutase 2 (SOD2) and

SC R

the nuclear factor (erythroid-derived 2)-like 2 (NFE2L2). D. Total plasma concentration of homocysteine after 4 weeks of surgery (n=6 to 8 per experimental group), * p<0.05 sham vs

NU

TAC, # p<0.05 ND vs SD.

MA

Figure 7: Proposed mechanism

Supplemented diet would increase circulating amount of cobalamin and folate. Consequently, the uptake of these two compounds in the cell would be higher in animal treated with SD

TE

D

leading to an important production of methylcobalamin. This cofactor of methionine synthase allows a higher synthesis of methionine from homocysteine and folate the latter being not

CE P

limiting because of its provision by SD. Finally, this increase S-adenosylmethionine (SAM) amount in the cytosol which would be used for PGC-1α methylation by protein arginine

AC

methyltransferase 1 (PRMT1). The high SAM amount also allows the activation of sirtuin 1 (SIRT1) through the methylation of an unknown actor. SIRT1 activates PGC-1α through direct deacetylation and through indirect activation of AMP-activated protein kinase (AMPK) which phosphorylates PGC-1α. The result would be an activation of PGC-1α activity that supports mitochondrial biogenesis and fatty acid oxidation.

31

ACCEPTED MANUSCRIPT Table 1 Supplemented diet

Diet effect

TAC effect

Interaction

0.59

0.83

242 ± 14 ** ##

0.004

<0.001

0.13

17.3 ± 0.3

0.59

0.65

0.44

9.18 ± 0.3

14 ± 0.7 ** ##

<0.001

<0.001

0.08

Body weight (g)

26.6 ± 1

26.4 ± 1

25.6 ± 0.5

Heart weight (mg)

174 ± 5

292 ± 14

157 ± 6

17.1 ± 0.1

17.3 ± 0.1

17.3 ± 0.2

Heart/Tibia (mg/mm)

10.1 ± 0.4

17 ± 0.9

**

NU

Tibia length (mm)

TAC (n=7)

T

Sham (n=8)

25 ± 1

SC R

TAC (n=7)

**

Statistical analysis

0.17

Sham (n=7)

IP

Normal diet

10.8 ± 0.5

17.4 ± 2.2

10.4 ± 0.3

15.8 ± 2.8

0.78

0.009

0.9

Liver/Tibia (mg/mm)

93.2 ± 3.2

72.2 ± 4.5

75.5 ± 3 #

73.8 ± 3.9

<0.001

0.038

<0.001

*

10.2 ± 0.4

9.8 ± 0.4

8.7 ± 0.5

0.094

0.038

0.3

AC

CE P

* TAC vs Sham; # ND vs SD

D

10.5 ± 0.4

TE

Kidney/Tibia (mg/mm)

MA

Lung/Tibia (mg/mm)

32

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

33

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

34

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

35

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

36

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

37

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

38

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

39

ACCEPTED MANUSCRIPT Highligths

CE P

TE

D

MA

NU

SC R

IP

T

Alterations of energy metabolism are described in mouse model of heart failure. Mitochondrial biogenesis transcriptional cascade is reduced in this model. Diet supplemented with vitamin B12 and B9 prevented these degradations. The mechanism relies in part on the activation of mitochondrial biogenesis genes.

AC

   

40