miR-322 regulates insulin signaling pathway and protects against metabolic syndrome-induced cardiac dysfunction in mice

    miR-322 regulates insulin signaling pathway and protects against metabolic syndrome-induced cardiac dysfunction in mice Alexandre Marchand, Fabrice Atassi, Nathalie Mougenot, Michel Clergue, Veronica Codoni, Jeremy Berthuin, Carole Proust, David-Alexandre Tr´egou¨et, Jean-S´ebastien Hulot, Anne-Marie Lompr´e PII: DOI: Reference:

S0925-4439(16)00011-9 doi: 10.1016/j.bbadis.2016.01.010 BBADIS 64407

To appear in:

BBA - Molecular Basis of Disease

Received date: Revised date: Accepted date:

28 August 2015 21 November 2015 6 January 2016

Please cite this article as: Alexandre Marchand, Fabrice Atassi, Nathalie Mougenot, Michel Clergue, Veronica Codoni, Jeremy Berthuin, Carole Proust, David-Alexandre Tr´egou¨et, Jean-S´ebastien Hulot, Anne-Marie Lompr´e, miR-322 regulates insulin signaling pathway and protects against metabolic syndrome-induced cardiac dysfunction in mice, BBA - Molecular Basis of Disease (2016), doi: 10.1016/j.bbadis.2016.01.010

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ACCEPTED MANUSCRIPT miR-322 regulates insulin signaling pathway and protects against

Proust BSc. 1,2,3

1,2,3

1,2,3

, Veronica Codoni Msc.

1,2,3

1,2,3

, Nathalie Mougenot PhD. 4,

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Michel Clergue Bsc.

PhD.1, Fabrice Atassi BSc.

, Jeremy Berthuin Bsc.

1,2,3

, Carole

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Alexandre Marchand

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metabolic syndrome-induced cardiac dysfunction in mice.

, David-Alexandre Trégouët PhD.

, and Anne-Marie Lompré PhD. 1,2,3.

1,2,3

, Jean-Sébastien Hulot MD. PhD.

Institute for Cardiometabolism and Nutrition (ICAN), Paris, F-75013, France.

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INSERM UMR-S 1166, Paris, F-75013, France.

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Sorbonne Universités, Université Pierre et Marie Curie -UPMC Univ Paris 06, UMR-S

- PECMV Platform, Sorbonne Universités, UPMC Univ Paris 06, Paris, F-75013, France

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1166, Paris, F-75013, France.

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Corresponding author: Anne-Marie LOMPRE INSERM UMRS 1166, Faculté de Médecine Pierre et Marie Curie, 91 boulevard de l’Hôpital, 75634 Paris Cedex 13, France Fax: +33 1 40 77 96 45

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Phone: +33 1 40 77 96 91

e-mail: [email protected]

Running title: miR-322 protects from HFD cardiac dysfunction

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ACCEPTED MANUSCRIPT ABSTRACT We identified murine miR-322, orthologous to human miR-424, as a new regulator of

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insulin receptor, IGF-1 receptor and sirtuin 4 mRNA in vitro and in vivo in the heart and

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found that miR-322/424 is highly expressed in the heart of mice. C57Bl/6N mice fed 10

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weeks of high fat diet (HFD) presented signs of cardiomyopathy and a stable miR-322 cardiac level while cardiac function was slightly affected in 11 weeks-old ob/ob which overexpressed miR-322. We thus hypothesized that mmu-miR-322 could be protective against cardiac

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consequences of hyperinsulinemia and hyperlipidemia. We overexpressed or knocked-down

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mmu-miR-322 using AAV9 and monitored cardiac function in wild-type C57Bl/6N mice fed a control diet (CD) or a HFD and in ob/ob mice. The fractional shortening progressively declined while the left ventricle systolic diameter increased in HFD mice infected with an

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AAVcontrol or with an AAVsponge (decreasing miR-322 bioavailability) but also in ob/ob mice infected with AAVsponge. Similar observations were also found in CD-fed mice

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infected with AAVsponge. On the contrary over-expressing miR-322 with AAVmiR-322 was efficient in protecting the heart from HFD effects in C57Bl/6N mice. This cardioprotection

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could be associated with the regulation of identified targets IGF1R, INSR and CD1, a decrease in insulin signaling pathway and an enrichment of genes involved in mitochondrial function and fatty acid oxidation as demonstrated by transcriptome analysis. Altogether, these results emphasize miR-322 as a new potential therapeutic target against cardiac consequences of metabolic syndrome, which represents an increasing burden in the western countries.

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ACCEPTED MANUSCRIPT Highlights  miR-322 targets key receptors of insulin signaling pathway and sirtuin 4.  miR-322 over-expression protects the heart from high fat diet consequences.

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 miR-322 inhibition induces cardiac dysfunction in control diet fed and ob/ob mice

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 miR-322 promotes mitochondrial respiratory chain and fatty acid metabolism genes.

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Keywords: microRNA, cardiomyopathy, high fat diet, insulin pathway, metabolic syndrome.

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Abbreviations: miR: microRNA; HFD: high fat diet; CD: control diet; IGF-1: insulin growth factor-1; IGF1R: insulin growth factor-1 receptor; INSR: Insulin receptor; SIRT4: sirtuin 4,

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CD1: cyclin D1; VSMC: vascular smooth muscle cell; FS: fractional shortening; LVESD: left

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ventricle end systolic diameter; LVEDD: left ventricle end diastolic diameter.

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ACCEPTED MANUSCRIPT 1. INTRODUCTION Metabolic syndrome (obesity, inflammation, insulin-resistance, high cholesterol),

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which increases the risk of heart disease, stroke and diabetes, is a leading cause of mortality

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and morbidity worldwide [1]. Despite progress in the characterization and comprehension of

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the cardiovascular consequences of metabolic syndrome and diabetes, the mechanisms implicated are still not clearly understood. Cardiac insulin resistance is an early adaptive event in response to obesity, which develops before the onset of insulin resistance in

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peripheral organs and as early as 10 days of high fat feeding in C57BL/6 mice [2]. It is

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associated with blunted Akt-mediated insulin signaling and GLUT4 levels, involves defects in glucose transport and glycogen storage in cardiomyocytes as well as cardiac remodeling and systolic dysfunction [2]. The major causes of cardiac insulin resistance are: 1) dyslipidemia

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and lipotoxicity, 2) reduction in mitochondrial oxidative capacity and increased mitochondrial

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uncoupling, 3) inflammation and cytokines, 4) activation of the stress kinase signaling [3].

An intriguing field of investigation is epigenetics, particularly the post-transcriptional

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regulation by microRNAs (miRNAs). Several miRNAs are involved in the control of metabolism and energy homeostasis [4] with a tissue-specific impact on insulin sensitivity and insulin signaling pathway. For example, inhibition of the let-7 family members and miR143 provides protection from insulin resistance-associated obesity by targeting either insulin receptor (INSR), insulin receptor substrate 2 (IRS2) [5] or oxysterol-binding-protein-related protein (ORP) 8 [6]. MiR-103/107 stabilizes insulin receptors at the membrane in the liver and adipose tissue by targeting caveolin-1 [7], whereas miR-802 impairs glucose metabolism through the silencing of Hnf1b in the liver [8]. MiR-223, which is upregulated in insulinresistant human hearts, induces GLUT4 protein expression and increases glucose uptake when overexpressed in cardiomyocytes [9]. More surprisingly, cardiac-specific miR-208a inhibition

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ACCEPTED MANUSCRIPT in mice confers resistance to obesity, improves glucose homeostasis and lowers plasma lipids levels, which is an effect linked to the regulation of systemic energy homeostasis via its target

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MED13 [10].

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MiR-322 (and its human ortholog miR-424) is expressed in a broad range of tissues. It shows anti-proliferative and pro-differentiation effects in many different cell types [11-13]. We recently showed that miR-424/322 inhibits vascular smooth muscle cells (VSMC)

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pathological proliferation by targeting Ca2+ regulating proteins and cyclin D1 [14]. Here we

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present evidences that miR-322 also regulates insulin pathway in the heart and has a cardioprotective effect in a model of high fat diet-induced metabolic syndrome and in leptindeficient obese mice (ob/ob). This microRNA could represent a new therapeutic strategy to

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protect the heart from consequences of metabolic syndrome.

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ACCEPTED MANUSCRIPT 2. MATERIALS AND METHODS 2.1. Generation of adenovirus associated virus (AAV)

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A detailed description of AAV-miR322 (AAV322), AAV-control (AAVCtl) and

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AAV-sponge anti-miR-322 (AAVsponge) constructs is provided in the supplemental

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experimental procedures.

2.2. In vivo experimental protocol

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C57Bl6/N, leptin deficient mice (ob/ob) and littermates (ob/+) mice as well as Wistar

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rats were obtained from Janvier-labs (Le Genest St isle, France). The Ethics Committee in Animal Experiment Charles Darwin approved this study (Ce5/2012/124). Five-week old C57Bl/6N mice were subjected to a control diet (CD: 10% calories from fat, no sucrose

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3.85kcal/g, D12450K) or a high-fat diet (HFD: 60% calories from fat, 7% sucrose, 5.24Kcal/g, D12492) from Research Diets (Broogarden, Lynge, Denmark) for 16 weeks.

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Mice were housed in a climate-controlled environment with a 12h light/dark cycle and free access to food and water ad libitum. Body weight and food intake were monitored weekly.

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After six weeks of diet, animals were anesthetized using intraperitoneal (IP) injection of ketamine (Parke Davis, France) and xylazine (Bayer, France) (75 and 10mg/kg respectively). They were injected with 1.1011 AAV particles (either AAV322, AAVsponge or AAVCtl) through the tail vein. AAV treatment did not modify food intake (3g/mice/week approximately). Six weeks-old ob/ob mice were also injected with AAVsponge or AAVctl (1.1011).

2.3. Blood glucose, insulin measurements and glucose or insulin tolerance tests See supplemental experimental procedures

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2.4. Echocardiographic analysis

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Echocardiographic measurements were performed on isoflurane anesthetized animals,

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using echocardiography-Doppler (General Electric Medical systems Co, Vivid 7

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Dimension/Vivid 7 PRO) with a probe emitting ultrasounds with 9-14 MHz frequency. Mmode LV end-systolic and end-diastolic dimensions as well as LV percent fractional shortening were averaged from at least 4–5 sets of measures obtained from different cardiac

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cycles. All measures were made on mice with similar heart rates (500-600 bpm).

2.5. Rat VSMC culture and treatment

Rat VSMC were prepared using aortas from 6 week-old male wistar rats as previously

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described [14] and detailed in supplemental experimental procedures.

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2.6. RNA isolation and RT-PCR

Total RNA including miRNA was isolated with the mirVana miRNA isolation kit (Life technologies, Villebon sur Yvette, France) or using the RNeasy Mini kit from Qiagen

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(Courtaboeuf, France) according to the manufacturer’s instructions. Total RNA Reverse Transcriptase-PCR analysis was performed using the Absolute QPCR SYBR green mix (ABgene, Courtaboeuf, France) on an MX3005P QPCR system (Stratagene, Agilent Technologies, Massy, France). The list of primers is included in the supplemental methods.

2.7. Protein preparation and western blot. Protein extracts were prepared using the Promokine Mammalian Whole Cell Extraction kit (PromoCell GMBH, Heidelberg, Germany) and phosphatase inhibitors (SigmaAldrich). The list of antibodies used is detailed in the supplemental methods. Densitometric analysis was performed with the NIH Image/ImageJ software. 7

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2.8. Luciferase reporter constructs and miRNA target validation by luciferase assay. The psiCHECK-2 vector (Promega, Charbonnieres, France) containing both firefly

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and renilla luciferase genes was used to introduce 3’UTR sequence immediately downstream

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of the stop codon of the renilla luciferase gene. See details in the supplemental methods.

2.9. Statistical Analysis

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Data are expressed as means ± SEM. Normality was tested for each group using the Shapiro-Wilk test. Experiments with two groups were analyzed with a paired t-test or with the

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non parametric Mann-Whitney test when the number of observations was too small to pass the normality test. When the normality test failed, multiple comparisons were analyzed by

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Kruskal–Wallis one-way analysis of variance (ANOVA) test followed by the Dunn’s multiple

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comparison test. Repeated measures two-way ANOVA followed by the Bonferroni’s multiple

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comparison test was used for paired samples with normal distribution. Statistical analysis was performed using the Prism software. When p values were below 0.05, differences were

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considered significant.

2.10. Transcriptome analysis Transcriptome analysis of total RNA samples was performed using the Illumina MouseWG-6 v2.0 Expression BeadChip Kit (www.illumina.com). Differential gene expression analyses were performed using the LIMMA R package [15]. To identify gene sets differentially associated with the tested conditions, a gene set enrichment analysis was conducted using the GeneSetDB tool [16] as described in the supplemental methods.

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ACCEPTED MANUSCRIPT 3. RESULTS

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3.1. miR-322 targets several members of the insulin signaling pathway.

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We used prediction algorithms (miRanda: http://www.microrna.org and Targetscan

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6.2: http://www.targetscan.org) to identify new miR-424/322 target mRNAs conserved in human and rodents and found that Insulin receptor (INSR), IGF-1 receptor (IGF1R) and the downstream effector of the insulin signaling pathway MAP2K1 (MEK1) were predicted

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targets. Sirtuin 4 (SIRT4), a protein implicated in fatty acid oxidation and insulin secretion

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[17, 18] was also a potential target (supplemental Figure 1). These predicted targets suggested involvement of miR-322 in the control of metabolic pathways. To confirm the potency of miR-322 on these targets, miR-322 levels were modulated

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in a primary culture of rat vascular smooth muscle cells (VSMC). Over-expression of miR322 (Figure 1A) was associated with a significant decrease in INSR, IGF1R, cyclin D1 (CD1)

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and SIRT4 protein levels whereas the level of MEK1 was not significantly changed (Figure 1B). Conversely, anti-miR-322 transfection significantly decreased endogenous miR-322

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level (Figure 1C) and increased IGF1R, CD1, and SIRT4 protein although the increase was not significant for INSR (Figure 1D). A miR-322 sponge (allowing the capture of miR-322 by an artificial mRNA) inhibited the effect of miR-322 as shown by a reporter luciferase assay (supplemental Fig 2A) and thus decreased miR-322 availability for its biological targets. It had the same effect than the anti-miR on gene expression (supplemental Figure 2B). The miR322 effect was not correlated to mRNA regulation except for CD1 and to a smaller extent SIRT4 (supplemental Figure 3A). To further check the direct interaction between miR-322 and its targets, various 3’UTR constructs encompassing miR-322 complementary binding sites, wild-type or mutated in the miR-322 seed sequence recognition site, were tested in a reporter luciferase assay. IGF1R, INSR and SIRT4 3’UTR induced a significant decrease in

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ACCEPTED MANUSCRIPT Renilla luciferase expression and activity when miR-322 was over-expressed. This inhibitory effect was lost when the 7bp seed sequence recognition site in the 3’UTR was mutated, thus

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indicating a direct interaction between miR-322 and these mRNAs (Figure 2E). As expected,

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MEK1 3’UTR did not affect Renilla luciferase activity (not shown).

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We then checked the effect of miR-322 over-expression on insulin signaling by measuring insulin-induced Akt phosphorylation in VSMC. In agreement with the decrease in IGF1R and INSR expression, miR-322 over-expression was associated with a strong

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inhibition of Akt phosphorylation (supplemental Figure 3B). Moreover insulin-induced

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glucose uptake (supplemental Figure 3C) and insulin-induced VSMC proliferation (supplemental Figure 3D) were prevented.

Altogether, the results pointed towards a potential role of miR-322 as a regulator of

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insulin signaling and lipid metabolism.

type-2 diabetes.

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3.2. miR-322 is highly expressed in the heart and is up-regulated in animal models of

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We assessed expression level of miR-322 in tissues collected from two different rodent species (Wistar rats and C57Bl/6N mice). In both species, expression was lower in pancreas than in liver but highly expressed in insulin responsive-tissues: muscles, adipose tissue and heart (Figure 2A-B). Since miR-322 was highly expressed in the heart, and was able to regulate in vitro insulin signaling pathway and SIRT4 we hypothesized that its modulation in the heart could play a role in heart function changes associated with metabolic syndrome.

3.3. Cardiac function in mice fed a high fat diet and in ob/ob mice.

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ACCEPTED MANUSCRIPT As a model of metabolic syndrome we choose to feed mice with a high fat diet (HFD) as this model seemed closer to human pathologies and to compare it with another model:

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leptin-deficient obese mice (ob/ob).

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C57Bl6/N mice were fed a control diet (CD) or a HFD for 16 weeks. The HFD (60%

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fat) was chosen to induce progressive obesity and hyperinsulinemia as a model of metabolic syndrome and pre-diabetic state [19]. As expected, HFD induced a progressive increase in body weight and in blood insulin level, whereas glycaemia did not increase significantly at

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least until 14 weeks of HFD (supplemental Figure 4A). Meanwhile fractional shortening (FS)

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markedly declined whereas both left ventricular end systolic diameter (LVESD) and left ventricular end diastolic diameter (LVEDD) were increased indicating cardiac dysfunction

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(supplemental Figure 4B).

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We also used a genetic model of obesity: the ob/ob mice. Eleven weeks-old ob/ob

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mice were compared to aged-matched ob+ mice. Their body weight was 41.7 ± 0.9g and 23.6 ± 0.2g respectively. As expected the ob/ob mice were highly hyperglycemic and hyperinsulinemic and a GTT test revealed their glucose intolerance (supplemental Figure 5A).

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However, cardiac parameters (fractional shortening as well as LVESD and LVEDD) were not altered at this stage (supplemental Figure 5B). Interestingly cardiac miR-322 expression was significantly increased in ob/ob mice (Figure 2C), but not in HFD fed-mice (Figure 2D) when compared to their control counterparts. These data showed that, at least at this stage, the ob/ob mice did not display cardiac dysfunction unlike mice fed a HFD of the same age. Because cardiac miR-322 level was not different in CD and HFD groups while it was higher in ob/ob mice than in their control littermates, we decided to assess the influence of long-term modulation of miR-322 expression (overexpression and inhibition) in the heart of these two models.

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ACCEPTED MANUSCRIPT 3.4. miR-322 overexpression using cardiotropic AAV regulates cardiac insulin signaling pathway in control mice.

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To better understand the role of miR-322/424 in the heart in vivo, we generated a

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recombinant serotype 9 Adeno Associated Viruses (AAV) encoding rodent miR-322 gene

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(AAV322), the same AAV without transgene (AAVCtl) and an AAVsponge inhibiting miR322 effects. This AAV serotype was chosen because it is known to potently infect the heart and specifically targets cardiomyocytes [20].

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The efficiency of miR-322 over-expression after AAV322 injection was firstly

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assessed in C57Bl6/N mice on normal diet 4 weeks after administration. AAV322 injection allowed a 10-fold increase in cardiac miR-322 expression compared to AAVCtl (Figure 3A). Expression of the predicted target proteins IGF1R, INSR, CD1 were significantly decreased

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while the decrease was not significant for SIRT4 (Figure 3B). Half of the mice were additionally challenged with insulin 10 min before sacrifice. Interestingly, AAV322-injected

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mice did not respond to the burst of insulin whereas AAVCtl-injected animals did, as indicated by Akt-phosphorylation (Figure 3C). These results suggested that insulin signaling

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was blunted in the heart after miR-322 over-expression. It suggested that miR-322 overexpression could reduce insulin signaling in the heart in a pathological context of hyperinsulinemia like the one occurring during HFD.

3.5. miR-322 modulation using cardiotropic AAV has no impact on general metabolism in control diet or high fat diet. After six weeks of diet, AAV322, AAVCtl or AAVsponge were injected via the tail vein. Mice were followed-up for 10 more weeks and metabolic parameters as well as cardiac function were monitored. There were no significant differences in body weight (Figure 4A), fasting glycaemia (Figure 4B) and fasting insulinemia (Figure 4C) between mice treated with

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ACCEPTED MANUSCRIPT AAVCtl, AAV322 and AAVsponge in CD. HFD fed mice treated with AAVsponge showed an early increase in fasting insulin level (Figure 4C) but the same level was reached for all

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AAV-treated mice at the end of the protocol. A glucose tolerance test (GTT) showed a

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delayed reabsorption of glucose (Figure 4D) and an insulin tolerance test (ITT) a reduced

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effect of insulin in HFD fed animals (Figure 4E). Similar observations were made for all three AAV treated mouse groups.

At the end of the experiment, after 16 weeks of diet and 10 weeks after AAV

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administration, despite significant differences between CD and HFD, neither AAV322 nor

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AAVsponge–injected mice had altered body weight (supplemental Figure 6A), fasting glycaemia (supplemental Figure 6B) or fasting insulinemia (supplemental Figure 6D) when compared to AAVCtl-injected animals.

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Altogether these results indicate that independently of the AAV injected, all mice responded similarly to the HFD: developing progressively hyperinsulinemia and alterations in

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glucose reabsorption. However differences between AAV groups were observed on cardiac

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function monitored at the same time.

3.6. miR-322 over-expression protects cardiac function in HFD fed mice whereas its inhibition in CD fed or ob/ob mice is deleterious. In CD fed mice FS, LVESD and LVEDD from AAV322-injected animals were not different from the values observed in AAVCtl-injected animals, showing stable and normal cardiac function during the course of the experiment. However mice injected with AAVsponge showed a rapid decrease in FS as well as an increase in LVESD and LVEDD reaching levels that were similar to those in HFD fed mice (Figure 5 A-C-E). A typical echocardiogram from a mice representative of each group taken 10 weeks after AAV injection is presented in supplemental Figure 7. These results suggested that the absence of miR-322

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ACCEPTED MANUSCRIPT was deleterious for the normal heart, although its over-expression did not modify heart function under physiological conditions. Insulin signaling was analyzed by evaluating Akt

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phosphorylation in the heart in response to an ip injection of insulin (supplemental Figure

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8A). There was no significant difference in the basal level of Akt phosphorylation between

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groups. However, while insulin induced Akt phosphorylation in the AAVCtl group and AAVsponge treated groups, Akt phosphorylation was blunted in the AAV322 treated group in agreement with our previous experiment (Figure 3A).

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Under HFD a decline in FS and an increase in LVESD and to a lesser extent in

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LVEDD were observed compared to CD fed mice before AAV injection confirming previous observations in the experiment without AAV (see supplemental Figure 4). Mice were then randomly divided in three groups and injected with AAVCtl, AAV322 or AAVsponge. A

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progressive decrease in FS as well as an increase in LVESD were observed in AAVCtl- and AAVsponge-injected mice (Figure 5 B-D-F and supplemental Figure 7) in HFD. On the

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contrary during the experiment HFD-fed mice injected with AAV322 showed improved FS as well as a smaller increase in LVESD as compared to AAVCtl- or AAVsponge-treated mice.

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Ten weeks after AAV injection, FS in HFD fed animals treated with AAV322 was similar to FS values before AAV injection (Figure 5B). This suggested that miR-322 overexpression inhibited heart function degradation induced by HFD. Akt phosphorylation without insulin stimulation was not different between the three groups despite a tendency to a lower level of phospho-Akt in AAV322 treated mice. Insulin effect was more variable than in CD mice and insulin injection did not induce significant AKT hyperphosphorylation (Supplemental Figure 8B) in agreement with the hyperinsulinemia of HFD fed animals.

After sacrifice, at the end of the experiment, all groups of HFD fed mice showed a significant and similar increase in heart weight/tibia length ratio (supplemental Figure 6C).

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ACCEPTED MANUSCRIPT However hearts overexpressing miR-322 interestingly showed less fibrosis than hearts in which miR-322 action was decreased (supplemental Figure 6E). This was in good agreement

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with our observations on cardiac function and emphasizes the potential benefit of having a

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high miR-322 cardiac expression in a HFD context.

We then performed a similar experiment in ob/ob mice which had shown elevated miR-322 cardiac expression and protected cardiac function to evaluate the effect of miR-322

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inhibition on this model. Six weeks-old ob/ob mice were injected with saline, AAVCtl and

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AAVsponge and followed-up for 5 more weeks. No differences in weight were observed between the three groups along the experiment (data not shown). As expected at the end of the experiments ob/ob mice with saline were highly hyperglycaemic, hyperinsulinemic and highly

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intolerant to glucose but did not present cardiac function alterations. Similar results were observed with AAVCtl mice. However ob/ob mice injected with AAVsponge presented a

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severe decline in FS and an increase in LVESD and LVEDD when compared to animals treated with saline or AAVCtl (supplemental Figure 5C). This indicated that decreasing miR-

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322 bioavailability by AAVsponge deteriorates cardiac function in ob/ob mice as well as in CD- and HFD-fed mice. It suggested that a minimal level of miR-322 in the heart is necessary to maintain optimal cardiac function. To better understand the mechanism involved in the effects of miR-322 on cardiac function in a pathological context, we analyzed miR-322 targets expression at the end of the HFD experimental protocol.

3.7. miR-322 modulates signaling pathways in the heart to preserve cardiac function. First we verified the efficiency of the AAV to modulate cardiac miR-322 expression. Ten weeks after AAV injection, we found that HFD mice injected with AAV322 still

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ACCEPTED MANUSCRIPT displayed a 1.6-fold increase, and HFD mice injected with AAVsponge a 1.6-fold decrease in cardiac miR-322 expression (Figure 6A). As expected from in vitro and in vivo results (Figure

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1 and figure 3) the levels of miR-322 targets INSR, IGF1R and CD1 were significantly

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decreased in AAV322-injected hearts (Figure 6B) (not significantly for SIRT4) while IGF1R,

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CD1 and SIRT4 increased in AAVsponge-injected ones (not significantly for INSR) (Figure 6C). This strengthens the idea that these targets are important for cardiac function homeostasis. However many other direct and indirect targets of miR-322 could also be

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involved.

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To go further and obtain a comprehensive insight into the gene network influenced by miR-322, a transcriptome analysis was performed comparing hearts of mice having the highest and lowest expression in miR-322 in the experiment. Sixteen weeks HFD fed mice

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injected either with AAV322 (which displayed preserved cardiac function, n=3), or with AAVsponge (in which the cardiac function was altered the most, n=3) were compared. After

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pre-processing the gene expression data, a total of 23,897 probes, corresponding to 17,196 distinct genes, remained for statistical comparisons between AAV322 and AAVsponge

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groups (see supplemental data). No single gene expression satisfied the Bonferroni threshold of 2.09 10-6 (= 0.05/23,897) for declaring statistical difference between the two groups. Nevertheless, a gene-set enrichment analysis identified two pathways significantly enriched, at the 0.05 False Discovery Rate (FDR): the "respiratory electron transport" and the "mitochondrial fatty acid beta-oxidation" pathways with FDR of 2.3 10-4 and 9.3 10-4, respectively (Figure 7). The former included 17 genes (out of 65 genes that define this pathway) and the second 7 genes (among 13) with p-values < 0,05 for association with miR322 over-expression (see legend of Figure 7).

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ACCEPTED MANUSCRIPT Interestingly these results suggested that miR-322 plays a role either directly or indirectly in improving mitochondrial respiratory capacities and fatty acid metabolism, which

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could help the heart to face the increase in fatty acids induced by the HFD.

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4. DISCUSSION

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In this study we present for the first time to our knowledge a role for miR-322/424 in

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regulating heart function in a model of metabolic syndrome and validated new targets INSR,

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IGF1R and SIRT4 conserved between human and rodents. Few studies have explored a possible link between miR-322/424 and the metabolic status. MiR-424 is overexpressed in the human cornea of type 2 diabetic patients [21]. Human corneal epithelial cells over-expressing

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miR-424 have decreased migration capacities and decreased phospho-p38, phospho-EGFR and phospho-Akt levels [21] in good agreement with our results. Concerning the heart, miR-

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424 is overexpressed in myocardial tissue from children with non syndromic Tetralogy of Fallot [22]. Surprisingly, in this study miR-424 was shown to promote cell proliferation and

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inhibit migration in primary embryonic mouse cardiomyocytes. These findings suggest that miR-424 affects differently embryonic and adult heart probably due to different contexts with

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regulatory elements,

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(interaction

modifications). For this reason, and since adult cardiomyocytes are not transfectable, we used

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VSMC from adult rats instead of neonatal cardiomyocytes (mostly used) for our in vitro experiments. Indeed VSMC are also contractile cells and share many pathways with adult cardiomyocytes.

We provide evidence that miR-322 has a cardio-protective effect. We show that in a HFD context associated with hyperinsulinemia but not hyperglycaemia, miR-322 downregulates IGF1R and INSR thus decreasing Akt phosphorylation and insulin action [23, 24]. Such a direct regulation of insulin signaling pathway by a microRNA has already been described with the let-7 family, which mediated repression of IGF1R, INSR and IRS2 in liver,

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ACCEPTED MANUSCRIPT fat, skin and muscle [25]. Let-7g induction resulted in peripheral insulin resistance and glucose intolerance but the cardiac effects of this miRNA family have not been investigated.

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Another important function regulated by miR-322 that could participate to its

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protective effects on heart function is mitochondrial capacity to deal with fatty acids. We

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identified SIRT4, a mitochondrial protein that uses NAD to ribosylate protein substrates, as a new miR-322 target. It has previously been shown that SIRT4 inhibition increases expression

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of mitochondrial and fatty acid metabolism enzymes [17]. While the effect of miR-322 on SIRT4 expression was clear in vitro, in vivo regulation in the heart was more difficult to

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assess as it was variable between animals, possibly due to other interfering regulations. However increasing the capacities to deal with fatty acids seems an important consequence of

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miR-322 in HFD mice as revealed by our results from hearts transcriptome. MiR-322 directly

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or indirectly upregulates many genes implicated in pathways allowing a better fatty acid

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metabolism and improving mitochondrial respiratory capacities. This could contribute to attenuate fatty acid toxicity and oxidative stress, which have been shown to play an important role in the pathophysiology of cardiac remodeling. Interestingly, an association between

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impaired mitochondrial function and myocardial contractile dysfunction was recently described in type 2 diabetic patients [26].

In this study we knocked down miR-322 using a miR-322 sponge. This sponge induced cardiac dysfunction in CD-fed and ob/ob mice, thus underlining the potential importance of miR-322 for maintaining cardiac function homeostasis. While we proved that it was efficient to trap and decrease miR-322 bioavailability (supplemental Figure 2), due to its structure (an artificial mRNA with 5 successive miR-322 target sites in its 3’UTR) we cannot exclude that the sponge construct also traps other microRNAs with close sequences (such as members of the miR-16 family which are related to miR-322) and that the strong effects

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ACCEPTED MANUSCRIPT observed on the normal heart reflect not only the loss of miR-322 but also of some other miRNAs. However, clear opposite effects on the targets of miR-322 and on cardiac function

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were observed between specific miR-322 overexpression and the sponge treatment arguing

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for a predominant impact of the sponge on miR-322. Future research using an inducible and

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conditional knock out model of miR-322 in the heart are needed to clarify its function in the normal heart.

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An interesting point is that the HFD model we used was more a model for metabolic syndrome or “pre-diabetic” state than an acute model for diabetes. Indeed, the progressive

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increase in body weight and insulinemia were not accompanied by a significant rise in glycaemia until the end of the experiment (16 weeks of diet). However, our results showed a

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rapid decrease in cardiac function as early as after 6 weeks of diet, which could be related to

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hyperlipidemia and progressive development of hyperinsulinemia.

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While been highly intolerant to glucose and obese, ob/ob mice of the same age did not display cardiac dysfunction. This is in agreement with previous results showing that cardiac

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contractile and hemodynamic parameters were preserved up to 15 weeks in ob/ob mice [27]. This could be explained by the numerous adaptations exhibited by hearts from ob/ob mice to maintain high oxygen consumption and fatty acid oxidation rates in a context of increased fatty acid supply and hyperinsulinemia [28].

5. CONCLUSIONS In conclusion, miR-322 seems essential for heart function homeostasis. Our study also highlights its role as a cardiac protector in a HFD context of hyperinsulinemia and high fatty acids content. It protects heart function possibly by regulating insulin signaling (via INSR and 20

ACCEPTED MANUSCRIPT IGF1R inhibition) and by improving fatty acid oxidation and energy production (via SIRT4 inhibition and/or global gene pathways regulations).

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Altogether these data suggest that miR-424/322 over-expression represents a

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promising new therapeutic strategy for stabilizing heart function in patients with metabolic

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syndrome and diabetes.

ACKNOWLEDGMENTS

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This work was supported by Institute of Cardiometabolism and Nutrition (ICAN)

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(ANR-10-IAHU-05), by Fondation de France (2012-00029516) and by AFM-Téléthon (camIR). VC is supported by a grant from the Fondation Leducq (grant 12CVD02). A. M. and A-M. L. are the guarantors of this work and, as such, had full access to all

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the data in the study and take responsibility for the integrity of the data and the accuracy of

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the data analysis.

Conflict of interest: AM and AML have a patent pending Inserm EP13306704.1:

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methods for the prevention and treatment of diabetic cardiomyopathy.

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ACCEPTED MANUSCRIPT 6. FIGURES LEGENDS: Figure 1: IGF1R, INSR, and SIRT4 are new targets of miR-322. Rat VSMC were

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transfected either with 30 nM PreNeg (negative control) or PremiR-322 to overexpress miR-

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322. They were transfected with 100nM AntiNeg (negative control) or AntimiR-322 to inhibit

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its expression. A, C: level of miR-322 normalized to U87 in VSMC after transfection with PremiR-322 and AntimiR-322 respectively. B, D Representative Western-Blots and quantification showing protein expression of predicted targets of miR-322 normalized to

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GAPDH, B: after over-expression of PremiR-322 (P322) compared to similar transfection of

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a negative control (PNeg). D: after inhibition of miR-322 with Anti-miR (A322) compared to similar transfection of a negative control (ANeg). For each culture (n=4 to 6) gene expression values were expressed as % of the value obtained for the same gene in the control (PNeg or

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ANeg arbitrarily set as 100). E. Various 3’UTR constructs (see supplemental materials) were subcloned dowstream of the renilla luciferase gene in the psiCHECK2 vector. Renilla

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luciferase was normalized to Firefly luciferase and the ratio obtained in P322 transfected/PNeg transfected cells (%) is presented for each construct (n=5 or 6 for each target

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3’UTR). Data are expressed as mean+/- SEM. Comparison was performed with one sample ttest to determine values significantly different from 100: *: p<0.05, **: p<0.01, ***: p<0.001.

Figure 2: miR-322 is highly expressed in the heart and overexpressed in ob/ob mice but not HFD-fed mice. A. Detection of miR-322 expression normalized to small nucleolar RNA U87 in various tissues from 8-months old non diabetic Wistar rats (n=5). B. Detection of miR-322 level normalized to small nucleolar RNA 202 (snoR202) in various tissues of 22 weeks old C57Bl/6N mice (n=8). C. Detection of miR-322 level normalized to snoR202 in the heart of 12-weeks old ob/ob mice or wild type littermates (ob/+) (n=4/group), D. Expression of miR-322 normalized to snoR202 in the hearts of C57Bl/6N mice fed during 16

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ACCEPTED MANUSCRIPT weeks with a high-fat diet (HFD) or a control diet (CD) (n=6/group). All data are expressed as mean+/- SEM. Comparison between two groups was done using non parametric Mann-

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Whitney test *: p<0.05.

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Figure 3: Strong miR-322 over-expression in the heart after 1 month AAV inhibits protein targets and Akt phosphorylation in control diet. Mice were infected with AAVmiR-322 (n=8) or AAVCtl (n=12) by tail-vein injection. A. After 1 month, mice were

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sacrificed and miR-322 level was detected in the hearts. MiR-322 level was normalized to

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snoR202. B. Representative Western-Blot of mir-322 protein targets in the hearts of AAV infected mice. C. For half of the mice, insulin (1UI/kg) was injected 10 min before sacrifice and hearts were flash frozen after removal. After protein extraction, 50µg were loaded on

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SDS-PAGE. A representative Western-Blot shows Akt phosphorylation on serine 473. Quantification was determined by normalizing to total Akt in control or insulin treated mice.

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All data are expressed as mean+/- SEM. Non parametric Mann-Whitney test were used for

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statistical analysis. Statistical significance is indicated as: *: p<0.05.

Figure 4: Modulation of miR-322 by AAV9 has no impact on weight gain, or glucose metabolism in response to high fat diet. Three groups of mice (n=11-12/ group) were fed a HFD and 3 groups (n=9-12/ group) a CD for 16 weeks. After 6 weeks of diet, 1x1011 AAV9 particles (either AAVCtl, AAV322 or AAVsponge) were injected through the tail vein. A. Body weight was measured once a week for all groups and compared to CD-AAVCtl. .B. A sample of blood was taken after a night of fasting at 8, 11 and 16 weeks of diet (corresponding to 2, 5, 10 weeks post AAV injection) and fasting glycaemia was measured. All groups were compared to CD-AAVCtl and HFD-AAVCtl (HFD-AAVCtl vs CD-AAVCtl at 11 weeks diet: ** p<0.01; HFD-AAV322 vs Ctl-AAVCtl at 16 weeks and HFD-

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ACCEPTED MANUSCRIPT AAVsponge vs CD-AAVCtl at 16 weeks: * p<0.05). C. Plasma was used to measure fasting insulin at 8-11 and 16 weeks of diet (2-5-10 weeks + AAV) (HFD-AAVsponge vs CD-

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AAVCtl at 5 and 10 weeks+AAV: ***p<0.001; HFD-AAVCtl and HFD-AAV322 vs CD-

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AAVCtl at 10 weeks + AAV: **p<0.01: HFD-AAVsponge vs HFD-AAV322 at 5

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weeks+AAV: $$p<0.01). D. After 11 weeks of diet (5 weeks post AAV injection) a GTT was performed. After a night of fasting, the mice received 2g/kg of D-glucose and glycaemia was monitored at 0, 15, 30, 60, 90 and 120 minutes. For statistical significance, all groups were

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compared to CD-AAVCtl and HFD-AAVCtl (HFD-AAV322 vs CD-AAVCtl at 30, 60 and 90

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min: * p<0.05; HFD-AAVsponge vs CD-AAVCtl at 90 min:* p<0.05). E. After 14 weeks of diet (8 weeks post AAV injection), an ITT was performed. After a night of fasting, the mice received 0.75 UI/kg insulin by IP injection and glycaemia was monitored at 0, 15, 30, 60, 90

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and 120 minutes. For statistical significance, all groups were compared to CD-AAVCtl and HFD-AAVCtl (HFD-AAV322 vs Ctl-AAVCtl at 15, 90 and 120 min: * p<0.05). All data are

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expressed as mean+/- SEM. Two-way ANOVA followed by the Bonferroni post hoc test

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were used for all statistical analysis: *, p<0.05, ** and $$ p<0.01, ***p<0.001

Figure 5: miR-322 is important for cardiac function and protects the heart from HFD consequences. Three groups of mice were fed a HFD (n=11-12/ group) and 3 groups a CD (n=9-12/ group) for 16 weeks. After 6 weeks of diet, AAVCtl, AAV322 or AAVsponge were injected through the tail vein. Echocardiograms were recorded after 6, 7, 10, 13 and 16 weeks of diet (corresponding to just before AAV injection=time 0 and 1, 4, 7, 10 weeks post AAV). A and B. Percentage of fractional shortening (FS) in CD and HFD groups respectively. C and D. Left ventricular end systolic diameter (LVESD) in CD and HFD groups respectively. E and F. Left ventricular end diastolic diameter (LVEDD) in CD and HFD groups respectively. All data are expressed as mean+/- SEM. Two-way repeated measures ANOVA

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ACCEPTED MANUSCRIPT followed by the Bonferroni post hoc test were used for statistical analysis after verification of the Gaussian distribution of the groups. AAVsponge vs AAVctl *, AAV322 vs AAVctl $,

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AAV322 vs AAVsponge £. *, $ and £ : p<0.05; **, $$ and ££: p<0.01; *** , $$$ and £££ :

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p<0.001. The evolution of each parameter during the experiment was analyzed by a paired

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student t- test. For each animal the parameters at 10 weeks post-AAV were compared to their values before AAV injection; &&: p<0.01, &&&: p<0.001.

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Figure 6: MiR-322 modulation in the heart during HFD affects its targets IGF1R, INSR,

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SIRT4 and CD1. After 16 weeks of diet and 10 weeks post-AAV, mice were sacrificed and the hearts were withdrawn for further analysis. A. Cardiac miR-322 expression was detected in each group of HFD-fed mice and normalized to snoR202 (n=6 or 7/group). All groups were

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compared to HFD-AAVCtl. B. Representative Western Blot showing miR-322 targets detected in hearts from HFD-AAVCtl and HFD-AAV322 mice. Protein levels were quantified

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and normalized to tubulin expression (n=3 for AAVctl, n= 4 for AAV322) (SIRT4 antibody revealed 2 close bands which were quantified together). HFD-AAV322 group was compared

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to HFD-AAVCtl. C. Representative Western-Blots showing miR-322 targets detected in hearts from HFD-AAVCtl and HFD-AAVsponge mice. Protein levels were quantified and normalized to tubulin expression (n=4 for AAVctl and n=5 for AAVsponge). HFDAAVsponge group was compared to HFD-AAVCtl. All data are expressed as mean+/- SEM. Comparison of protein expression in hearts infected with AAV322 or AAVsponge and hearts infected with AAVctl was performed using the non parametric Mann-Whitney test. One-way ANOVA followed by the Bonferroni multiple comparison test was used for miR-322 cardiac expression. *: p<0.05, **: p<0.01.

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ACCEPTED MANUSCRIPT Figure 7: Hierarchical clustering of cardiac gene expression modulated by miR-322 over- expression.

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Heatmap representation of normalized expression levels observed in 3 HFD-AAV32 and 3

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HFD-AAV sponge mice. For ease of presentation, data shown were normalized to the mean

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sample observed in the AAVsponge mice. Top block represents expression data from genes belonging to the "mitochondrial fatty acid beta-oxydation" pathway. Middle block includes expression levels for genes belonging to the "respiratory electron transport" pathway while

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bottom block shows expression data for a random subset of 100 genes. The variations of

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expression between AAV322 and AAVsponge treated mice are represented by green and red colours. The higher expressed genes in AAV32 compared to AAVsponge mice are represented in red. Squares point to genes with expression levels that differ at p <0.05

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between the two conditions. These are Acadl, Acads, Acadvl, Echs1, Mcee, Pcca and Pccb for the "mitochondrial fatty acid beta-oxydation" pathway and Atp5a1, Cox7b, Etfa, Etfb,

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Ndufa3, Ndufa5, Ndufa7, Ndufb10, Ndufb2, Ndufb7, Ndufb9, Ndufs2, Ndufs4, Ndufs8,

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Ndufv3, Sdhb and Ucp3 for the "respiratory electron transport" gene set.

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Graphical abstract

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