# Spatiotemporal AMPKα2 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only

Lucile Grimbert, Maria-Nieves Sanz, Mélanie Gressette, Catherine

Rucker-Martin, Marta Novotova, Audrey Solgadi, Ahmed Karoui, Susana

Gomez, Kaveen Bedouet, Eric Jacquet, et al.

► To cite this version:

Lucile Grimbert, Maria-Nieves Sanz, Mélanie Gressette, Catherine Rucker-Martin, Marta Novotova, et al.. Spatiotemporal AMPKα2 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only. Biology of Sex Differences, 2021, 12 (1), 10.1186/s13293-021-00394-z. http://hal-03426886

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# Spatiotemporal AMPKα2 deletion in mice induces cardiac dysfunction, fibrosis and cardiolipin remodeling associated with mitochondrial dysfunction in males only

Lucile Grimbert<fnref n="1" />, Maria-Nieves Sanz<fnref n="1" />, Mélanie Gressette<fnref n="1" />, Catherine Rucker-Martin², Marta Novotova³, Audrey Solgadi⁴, Ahmed Karoui<fnref n="1" />, Susana Gomez<fnref n="1" />, Kaveen Bedouet<fnref n="1" />, Eric Jacquet⁵, Christophe Lemaire<fnref n="1" />,⁶, Vladimir Veksler<fnref n="1" />, Mathias Mericskay<fnref n="1" />, Renée Ventura-Clapier<fnref n="1" />, Jérôme Piquereau<fnref n="1" />*† and Anne Garnier<fnref n="1" />†

**Abstract**

**Background:** The AMP-activated protein kinase (AMPK) is a major regulator of cellular energetics which plays key role in acute metabolic response and in long-term adaptation to stress. Recent works have also suggested non-metabolic effects.

**Methods:** To decipher AMPK roles in the heart, we generated a cardio-specific inducible model of gene deletion of the main cardiac catalytic subunit of AMPK (Ampka2) in mice. This allowed us to avoid the eventual impact of AMPK-KO in peripheral organs.

**Results:** Cardio-specific Ampka2 deficiency led to a progressive left ventricular systolic dysfunction and the development of cardiac fibrosis in males. We observed a reduction in complex I-driven respiration without change in mitochondrial mass or in vitro complex I activity, associated with a rearrangement of the cardiolipins and reduced integration of complex I into the electron transport chain supercomplexes. Strikingly, none of these defects were present in females. Interestingly, suppression of estradiol signaling by ovariectomy partially mimicked the male sensitivity to AMPK loss, notably the cardiac fibrosis and the rearrangement of cardiolipins, but not the cardiac function that remained protected.

**Conclusion:** Our results confirm the close link between AMPK and cardiac mitochondrial function, but also highlight links with cardiac fibrosis. Importantly, we show that AMPK is differently involved in these processes in males and females, which may have clinical implications for the use of AMPK activators in the treatment of heart failure.

**Highlights**

* AMPK is a metabolic sensor of cellular energy which regulates energy homeostasis.

<figure><img src="image_3.png" /></figure>

BMC

© The Author(s) 2021. **Open Access** This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

<footnote>*Correspondence: jerome.piquereau@universite-paris-saclay.fr</footnote>

<footnote>†Jérôme Piquereau and Anne Garnier contributed equally to this work</footnote>

<footnote n="1">¹ Faculté de Pharmacie, UMR-S1180, INSERM, Université Paris-Saclay, 5 rue J-B Clément, 92296 Châtenay-Malabry, France</footnote>

<footnote>Full list of author information is available at the end of the article</footnote>
* We generated a cardiac-specific inducible deletion of Ampkα2 and demonstrated that this deletion induces mild cardiac dysfunction in male only.

* Cardiac dysfunction observed in males was associated with cardiac fibrosis and cardiac cardiolipin remodeling that are not seen in females.

* Although no significant cardiac function alteration was noticed in ovariectomized female Ampkα2ciKO mice, these latter exhibited cardiac fibrosis and mild cardiolipins remodeling.

* Our results show a higher dependence on AMPK signaling fibrosis and cardiolipin biosynthesis/maturation in males, either due to the absence of female hormones protection or/and to the action of male hormones. This may contribute to the known difference in cardiovascular risk and outcome between sexes.

**Keywords:** Heart, AMP-activated protein kinase, Fibrosis, Cardiolipins, Energy metabolism

**Introduction**

The AMP-activated protein kinase (AMPK) is a ubiquitous serine/threonine kinase which acts as a cellular “fuel gauge” regulating energy homeostasis [1]. This kinase is composed of a catalytic subunit (α) and two regulatory subunits (β and γ), each including several isoforms exhibiting differential tissue expression. In murine heart, the α-subunit type 2 (α2) is the major catalytic subunit isoform (70–80% of the total AMPK activity) and is mostly expressed in the cardiomyocytes, the α-subunit type 1 (α1) isoform being predominant in the non-myocyte cells [2, 3]. AMPK is activated under conditions of metabolic stress that deplete ATP [1] and modulate a number of physiological processes aiming at restoring energy balance [4]. Its activation requires allosteric stimulation by AMP and the α-subunit Thr172 phosphorylation by upstream kinases [5] such as Liver Kinase B1 (LKB1) [6] or Ca²⁺/calmodulin-dependent kinase kinase (CamKK) [7]. AMPK regulates energy metabolism by directly modulating the activity of key enzymes of cellular energetics and impacts expression of many genes involved in the energy production/consumption processes [8]. For instance, AMPK phosphorylates the peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α), a master regulator of energy metabolism which, amongst others, stimulates mitochondrial biogenesis. It also enhances glucose and fatty acid uptake through the translocation of GLUT4 and CD36 transporters to the plasma membrane [9], boosts glycolysis by directly activating 6-phosphofructokinase 2 (PFK2) [10], and increases fatty acid oxidation by phosphorylating and inhibiting acetyl-CoA carboxylase (ACC) [11]. In a nutshell, AMPK stimulates catabolic processes producing ATP and inhibits anabolic processes consuming ATP [12].

Although the metabolic regulations of AMPK have been the main subject of a number of studies these last years, the role of AMPK extends beyond this energetic aspect. In the heart, it is known that this enzyme also

exhibits non-metabolic effects like regulation of myocardial fibrosis and inhibition of cytosolic reactive oxygen species (ROS) production by NADPH oxidase [13–16]; this has to be considered when deciphering the part played by AMPK in cardiac physiology and pathophysiology. Our team has shown in a global and constitutive male *Ampkα2* knockout mouse model [17] that *Ampkα2* suppression led to significant decrease in mitochondrial oxidative capacities associated with a decrease in cardiolipin (CL) content suggesting a link between AMPK and the mitochondrial membrane components. Knowing the importance of mitochondrial phospholipid composition for suitable mitochondrial functions [18–20], a potential impact of AMPK activity on mitochondrial respiratory function through a regulation of CL biosynthesis/maturation could be proposed. Intriguingly, we also highlighted a concomitant decrease in AMPK activity and in CL content in cardiac left ventricle of male rats treated with doxorubicin [21]. In this study, doxorubicin treated female rats exhibited lesser alterations in cardiac function and mitochondrial oxidative capacities than males and showed preserved AMPK activity and CL content. Beyond the possible relationship between AMPK, CL and mitochondrial function, these interesting results could also evoke a possible role for AMPK in the sexual dimorphism extensively reported in cardiovascular diseases [22].

This last decade, AMPK has often been presented as a potential therapeutic target in many cardiovascular diseases since its pharmacological stimulation has been shown to be beneficial for the pathological heart under many circumstances such as ischemia/reperfusion, diabetic cardiomyopathy, pathological hypertrophy or heart failure (HF) [23–26]. ATP production deficiency has been extensively demonstrated in these pathologies [27] and, given the role of AMPK in energy metabolism, it is quite rational to consider the activation of AMPK as a plausible strategy to improve myocardial energetics in patients affected by these diseases. Yet, before going further in the
development of such a therapy, a better understanding of AMPK role in cardiomyocyte homeostasis is required. The clarification of the connection between AMPK and CLs is of particular interest since our knowledge of this phenomenon is almost nonexistent in the one hand and, on the other hand the AMPK/CL axis could be part of the mechanisms responsible for the higher resistance of the female heart to cardiac diseases, at least before menopause.

Currently, inducible and tissue-specific deletion of a given protein is one of the most powerful tools to understand its cell autonomous roles in mature organs. Thus, we generated an original model of cardiac-specific *Ampka2* knockout mouse inducible in adult by tamoxifen injection (1) to study the consequences of cardiomyocyte specific *Ampka2* deletion on cardiac function, structure and energy metabolism; (2) to investigate the interplay between AMPK and cardiac CL and (3) to identify sex-specific AMPK-dependent regulations.

# Materials and methods

## Animals

*Ampka2*<sup>floxΔE6/floxΔE6</sup> (AMPKα2<sup>f/f</sup>) homozygous mice (kind gift of Dr. Viollet [28]) and α-MHC-MerCreMer (α-MHC-Cre) mice were crossed to create cardiac-specific and inducible knock-out (ciKO) mice (α-MHC-MerCreMer x *Ampka2*<sup>floxΔE6/floxΔE6</sup> (α-MHC-Cre/*Ampka2*<sup>f/f</sup>)). Male and female α-MHC-Cre/*Ampka2*<sup>f/f</sup> mice were injected with tamoxifen (40 mg/kg i.p daily × 2 days) at the age of 8 weeks to induce *Ampka2* deletion, thereby generating *Ampka2* cardiac-specific inducible mice called *Ampka2ciKO* or KO in this study. Littermate *Ampka2*<sup>floxΔE6/floxΔE6</sup> mice not carrying α-MHC-MerCreMer transgene were subjected to the same tamoxifen treatment and were used as control mice (*Ampka2*<sup>f/f</sup> or CT). Mice were euthanized 16 weeks after tamoxifen injection and hearts were rapidly excised, rinsed in cold calcium-free Krebs solution and weighed. A part of the left ventricle (LV) was immediately used for mitochondrial function assessment and another part was flash frozen in liquid nitrogen for further biochemical determinations. For ovariectomy, females had surgery at the age of 7 weeks (a week before tamoxifen injection). Anesthesia was induced by intraperitoneal injection of ketamine (50 mg/kg) and xylazine (8 mg/kg) and temperature during surgery was maintained at 37 °C using a heating pad. After incision in the pelvic region, ligation was performed between each uterine horn and ovary before excising both ovaries. Mice were sutured using absorbable suture silk (6-0) then placed under a heat lamp until they woke up. At awaking, mice were treated with buprenorphine (0.05 mg/kg, subcutaneous) to avoid post-surgery pain. All animal

experimental procedures were approved by animal ethics committee of Paris-Saclay University, authorized by French government (Authorization Number: B9201901) and complied with directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

## Echocardiography

Echocardiography was done using a 12-MHz transducer (Vivid 7, General Electric Healthcare) under 2.5% isoflurane gas anesthesia to assess cardiac function. The body temperature was maintained with a heating pad. M-mode echocardiography was used to determine left ventricular mass, fractional shortening and left ventricular ejection fraction.

## Histological analysis

Hearts were fixed in 4% paraformaldehyde, paraffin embedded and serially sectioned (5 µm).

Sections were stained with Sirius red. Fibrosis quantification was performed on 3–4 Sections (5–10 fields/section) per animal using Image J software.

## Mitochondrial functional assays in permeabilized cardiac fibers

Fibers prepared from the left ventricle were permeabilized with saponin as previously described [29] and kept on ice until use in a buffer containing in mM: 2.77 CaK₂ ethyleneglycol tetraacetic acid (EGTA), 7.23 K₂EGTA [100 nM free Ca²⁺], 6.56 MgCl₂ [1 mM free Mg²⁺], 5.7 Na₂ATP, 15 phosphocreatine, 20 taurine, 0.5 dithiothreitol (DTT), 50 K-methane sulfonate [160 mM ionic strength], 20 imidazole, pH 7.1. Measurements aiming at determining mitochondrial parameters were expressed per gram of dry fiber weight.

## Mitochondrial respiration

Mitochondrial respiratory function was studied in situ in saponin-permeabilized cardiac muscle fibers using a Clarke electrode as previously described [29]. A protocol was designed to measure oxygen consumption after successive addition of ADP (2 mM), malate (4 mM), L-glycerol-3-phosphate (4 mM), palmitoyl-CoA and carnitine (100 µM and 2 mM), pyruvate (1 mM), glutamate (10 mM), succinate (15 mM), amytal (an inhibitor of complex I, 1 mM) and the complex IV substrates N,N',N'-tetramethyl-phenylenediamine dihydrochloride (TMPD)-ascorbate (0.5 mM–2 mM) (activator of complex IV) to a solution containing in mM: 2.77 CaK₂ ethyleneglycol tetraacetic acid (EGTA), 7.23 K₂EGTA [100 nM free Ca²⁺], 1.38 MgCl₂, 3 K₂HPO₄, 20 taurine, 0.5 dithiothreitol (DTT), 90 K-methane sulfonate and
10 Na-methane sulfonate, 20 imidazole, pH 7.1 at 23 °C. Rates of respiration are given in µmoles O₂/min/g dry weight.

## Enzyme activity

Frozen tissue samples were weighed, homogenized (Bertin Precellys 24) in ice-cold buffer (50 mg/ml) containing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 5 mM (pH 8.7), EGTA 1 mM, DTT 1 mM and 0.1% Triton X-100. Activities of citrate synthase (CS), cytochrome c oxidase (COX) and complex I were determined using standard spectrophotometric assays [30, 31].

## 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-glycerophosphate 5 mM, Triton X-100 0.1%, orthovanadate 1 mM, dithiothreitol 1 mM, sodium fluoride 50 mM, Na pyrophosphate 5 mM, phenylmethylsulfonyl fluoride 0.2 mM and antiprotease cocktail set (Calbiochem 539,134). Protein extracts were separated on SDS-polyacrylamide gel (8 to 12%) and then transferred to polyvinylidene difluoride membranes for Western blot. After an hour of blocking in PBS containing Tween 20 (0.1%) and non-fat milk (5%), the membranes were incubated overnight at 4 °C with primary antibody (Table 1). After washing, the membranes were incubated with a secondary antibody coupled with horseradish peroxidase for 1 h at room temperature and visualized using chemiluminescent substrate (Luminata™ Western Chemiluminescent HRP Substrates, Millipore). Light emission was detected by autoradiography and quantified using an image-analysis system (iBright FL1000, Invitrogen).

<table><caption>**Table 1** Antibodies</caption><thead><tr><th>Antibody</th><th>Supplier</th><th>Reference</th><th>Dilution</th></tr></thead><tbody><tr><td>Actin</td><td>Santa Cruz</td><td>sc47778</td><td>1/10000</td></tr><tr><td>ACC</td><td>Cell Signaling</td><td>3676</td><td>1/1000</td></tr><tr><td>Phospho ACC</td><td>Cell Signaling</td><td>3661</td><td>1/1000</td></tr><tr><td>AMPKa1</td><td>Santa Cruz</td><td>sc19126</td><td>1/1000</td></tr><tr><td>AMPKa2</td><td>Santa Cruz</td><td>Sc19129</td><td>1/1000</td></tr><tr><td>Phospho AMPK</td><td>Cell signaling</td><td>2531</td><td>1/500</td></tr><tr><td>AMPK total</td><td>Cell signaling</td><td>2532</td><td>1/500</td></tr><tr><td>CS</td><td>Abcam</td><td>Ab96600</td><td>1/1000</td></tr><tr><td>mi-CK</td><td></td><td></td><td></td></tr><tr><td>Grim19 (NDUFA13)</td><td>Abcam</td><td>ab110240</td><td>1/1000</td></tr><tr><td>Oxphos</td><td>Mitoscience</td><td>Ms604</td><td>1/500</td></tr><tr><td>Tubulin</td><td>Abcam</td><td>Ab7291</td><td>1/10000</td></tr><tr><td>Vinculin</td><td>Sigma</td><td>V9131</td><td>1/900</td></tr></tbody></table>

## Blue-native page

Mitochondria and mitochondrial electron transfer chain complexes were isolated from heart tissue using digitonin 5% (from Native Page Sample Prep Kit, Invitrogen) [32]. Fifty micrograms of proteins were loaded on a 3–16% acrylamide native gel (Invitrogen). After electrophoresis, proteins were transferred to a PVDF membrane. Immunoblotting was performed with Grim-19 antibody (Table 1) raised against NDUFA13 complex I subunit.

## Real-time quantitative RT-PCR analysis

Frozen tissue samples were weighed and homogenized (Bertin Precellys 24) in ice-cold buffer. Total ventricular RNA was extracted using standard procedure with Trizol reagent (Invitrogen). cDNAs were synthesized from 2 µg total RNA according to the protocol provided with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, France). Real-time PCR was performed using TaqMan Low Density Array (TLDA) technology as previously described [33]. Pre-designed TaqMan probe and primer sets for target genes were chosen from an on-line catalog (Applied Biosystems, France). TLDA were designed to amplify 26 cDNA for each sample as follows: 21 target genes involved in energy metabolism, mitochondrial function and cardiac remodeling and 5 housekeeping genes (Table 2). Each PCR reaction was performed on 4 ng of cDNA in a volume of 1 µl. The thermal cycling conditions were 2 min at 50 °C and 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Quantification was achieved using the ΔΔC<sub>t</sub> method. The average C<sub>t</sub> obtained in CT group was used as a calibrator and the geometric mean of the 5 housekeeping genes was used as the reference for normalization. The amplification efficiency of each probe is estimated maximal as described by the manufacturer.

Quantification of Col1a1 (forward 5'-CTCAAGATG TGCCACTCTGACT-3', reverse 5'-CTCCATGTTGCA GTAGACCTTG-3'), and Col3a1 (5'-GAT GGAAACCCT GGATCAGA -3', 5'-GCACCAGGAGAACCATTTTC-3') mRNA were assessed using the SYBR® Green method on a LightCycler rapid thermal cycler (Roche Diagnostics) as previously described [34]. For each target gene, a standard curve was constructed from the analysis of a fivefold cDNA serial dilution and used for samples concentration calculation. Ywhaz (5'-AGACGGAAGGTGCTG AGAAA-3', 5'-GAAGCATTGGGGATCAAGAA-3') was used as housekeeping gene as its expression did not differ between groups.

## Electron microscopy

Left ventricular papillary muscles were isolated from three control and three KO mouse hearts, fixed with
<table><caption>**Table 2** Primers</caption><thead><tr><th>Gene name</th><th>Gene symbol</th><th>Function</th><th>Identification number (applied biosystems)</th></tr></thead><tbody><tr><td>Natriuretic peptide type B</td><td>Nppb</td><td>Hypertrophy</td><td>Mm01255770_g1</td></tr><tr><td>Peroxisome proliferator-activated receptor, gamma, coactivator 1 alpha</td><td>Ppargc1a</td><td>Mitochondrial biogenesis</td><td>Mm00447181_m1</td></tr><tr><td>Peroxisome proliferator-activated receptor, gamma, coactivator 1 beta</td><td>Ppargc1b</td><td>Mitochondrial biogenesis</td><td>Mm00504730_m1</td></tr><tr><td>GA repeat binding protein, alpha</td><td>Gabpa</td><td>Mitochondrial biogenesis</td><td>Mm00484598_m1</td></tr><tr><td>Estrogen related receptor, alpha</td><td>Esrra</td><td>Mitochondrial biogenesis</td><td>Mm00433143_m1</td></tr><tr><td>Peroxisome proliferator-activated receptor alpha</td><td>Ppara</td><td>Mitochondrial biogenesis</td><td>Mm00440939_m1</td></tr><tr><td>Transcription factor A, mitochondrial</td><td>Tfam</td><td>Mitochondrial biogenesis</td><td>Mm00447485_m1</td></tr><tr><td>Mitochondrially encoded cytochrome c oxidase I</td><td>COX1</td><td>Mitochondrial biogenesis</td><td>Mm04225243_g1</td></tr><tr><td>Cytochrome c oxidase subunit IV isoform 1</td><td>Cox4i1</td><td>Mitochondrial biogenesis</td><td>Mm01250094_m1</td></tr><tr><td>Optic atrophy 1</td><td>Opa1</td><td>Mitochondrial dynamics</td><td>Mm00453879_m1</td></tr><tr><td>Mitofusin 1</td><td>Mfn1</td><td>Mitochondrial dynamics</td><td>Mm00612599_m1</td></tr><tr><td>Mitofusin 2</td><td>Mfn2</td><td>Mitochondrial dynamics</td><td>Mm00500120_m1</td></tr><tr><td>Dynamin 1-like</td><td>Dnm1l</td><td>Mitochondrial dynamics</td><td>Mm01342903_m1</td></tr><tr><td>Superoxide dismutase 2, mitochondrial</td><td>Sod2</td><td>Oxidative stress</td><td>Mm01313000_m1</td></tr><tr><td>Catalase</td><td>Cat</td><td>Oxidative stress</td><td>Mm00437992_m1</td></tr><tr><td>Glutathione peroxidase 1</td><td>Gpx1</td><td>Oxidative stress</td><td>Mm00656767_g1</td></tr><tr><td>Acyl-CoA synthetase long-chain family member 1</td><td>Acsl1</td><td>Cardiolipins biosynthesis</td><td>Mm00484217_m1</td></tr><tr><td>CDP-diacylglycerol synthase 1</td><td>Cds1</td><td>Cardiolipins biosynthesis</td><td>Mm01208328_m1</td></tr><tr><td>Cardiolipin synthase 1</td><td>Crls1</td><td>Cardiolipins biosynthesis</td><td>Mm00503002_m1</td></tr><tr><td>Tafazzin</td><td>Taz</td><td>Cardiolipins biosynthesis</td><td>Mm00504978_m1</td></tr><tr><td>Elongation of very long chain fatty acids-like 2</td><td>Elovl2</td><td>Cardiolipins biosynthesis</td><td>Mm00517086_m1</td></tr><tr><td>ELOVL family member 5, elongation of long chain fatty acids</td><td>Elovl5</td><td>Cardiolipins biosynthesis</td><td>Mm00506717_m1</td></tr><tr><td>Fatty acid desaturase 1 (delta 5 desaturase)</td><td>Fads1</td><td>Cardiolipins biosynthesis</td><td>Mm00507605_m1</td></tr><tr><td>Fatty acid desaturase 2 (delta 6 desaturase)</td><td>Fads2</td><td>Cardiolipins biosynthesis</td><td>Mm00517221_m1</td></tr><tr><td>Beta-2 microglobulin</td><td>B2m</td><td>Reference genes</td><td>Mm00437764_m1</td></tr><tr><td>Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide</td><td>Ywhaz</td><td>Reference genes</td><td>Mm03950126_s1</td></tr><tr><td>Ribosomal protein, large P2</td><td>Rplp2</td><td>Reference genes</td><td>Mm00782638_s1</td></tr><tr><td>Polymerase (RNA) II (DNA directed) polypeptide A</td><td>Polr2a</td><td>Reference genes</td><td>Mm00839493_m1</td></tr><tr><td></td><td>18S</td><td>Reference genes</td><td>Hs99999901_s1</td></tr></tbody></table>

2% glutaraldehyde in cacodylate buffer (in mM: 150 Na-cacodylate, 2CaCl2, pH 7.3) for 1 h, post-fixated by 1% osmium tetroxide in cacodylate buffer for 30 min and stained with 1% aqueous solution of uranyl acetate. After dehydration in graded ethanol series and acetone, the tissue was embedded in Durcupan (ACM Fluka). Ultrathin (58–60 nm) longitudinal sections were cut using an ultramicro-tome (Power-Tome MT-XL, RMC/Sorvall, Tucson, AZ, USA). The sections were mounted on formvar-coated copper grids, contrasted with lead citrate and examined with a JEM 1200 electron microscope (Jeol, Tokyo, Japan) at 80 kV. Random images of cardiomyocytes were recorded with a CCD camera (Gatan DualVision 300 W) at a magnification of 15,000 and analyzed using Graphic Cell Analyzer14.

**Cardiolipin content**

Cardiolipin analysis was performed by liquid chromatography using corona-CAD detector as previously described [21]. As cardiolipins are almost exclusively located in the inner mitochondrial membrane, lipids were extracted from 10 to 20 mg of heart homogenized in PBS using the Folch method [35]. Total lipids were extracted by adding 1.5 mL of methanol and 3 mL of chloroform to the tissue suspension. After centrifugation at 1000 g for 10 min, the lower phase containing total lipids was collected and evaporated to dryness at room temperature under nitrogen gas. The samples were resuspended in 100 µl of chloroform per 10 mg of heart and subsequently analyzed. Quantification of cardiolipins was performed on a Dionex U-3000 RSLC system (ThermoFisher
Scientific) equipped with a Corona-CAD Ultra (ThermoFisher Scientific). Separation of lipids was performed with a PVA-Sil column (150 × 2.1 mm I.D., 120 Å) (YMC Europe GmbH) at 35 °C. Chromatographic method was inspired from the method developed by Imbert et al. [36]. The flow rate was set at 0.400 mL/min and 5 µl of sample were injected. The corona-CAD nebulizer was set at 30 °C and the nitrogen pressure was set at 5 bars. Standard curve from 0.5 to 0.025 mg/mL of cardiolipin sodium salt from bovine heart (98% purity from Sigma-Aldrich) was used.

## Statistical analysis

All results are expressed as mean ± SEM. To assess significance, we performed Student's t test when the experimental design compared only 2 groups or two-way ANOVA for independent factors when appropriate for the experimental design; Tukey post hoc tests were used to identify significant differences between means. Results were considered statistically different when p-value was strictly lower than 0.05. Symbols referring to statistics are defined in each figure legend.

## Results

### Cardiac-specific tamoxifen-induced loss of Ampkα2 in male and female mice

At 8 weeks of age, α-MHC-Cre/$Ampkα2^{fl/f}$ mice were given tamoxifen to induce exon 6 excision from the floxed *Ampkα2* alleles. Sixteen weeks after *Ampkα2* deletion, AMPKα2 protein level in left ventricle (LV) homogenates exhibited reductions of 95±1% in *Ampkα2ciKO* males and 90±3% in females when compared with the respective control *Ampkα2fl/f* mice (Fig. 1A). This drastic decrease in AMPKα2 content was not compensated by any change in AMPKα1 expression (Fig. 1A) and led to an important decrease in total AMPKα content in the LV of both sexes (Fig. 1B). The antibody recognizing both isoforms showed up to at least 75% reduction of signal in the *Ampkα2ciKO* heart (Fig. 1B, total AMPK), further demonstrating the predominant expression of AMPKα2 over AMPKα1 when considering the whole myocardium. As AMPKα2 is depleted over 95%, we calculated that AMPKα1 accounts for 80% of the remnant total AMPK signal in the *Ampkα2ciKO* heart. Whether in males or females, the important decrease in AMPKα2 content was associated with a lower amount of phosphorylated AMPKα (Thr 172), the active form of AMPK. The antibody recognizing both phosphorylated AMPKα1 (Thr 183), and AMPKα2 (Thr 172) showed a 30% reduction of signal. Thus, the remnant 70% phospho-AMPK signal comes most probably mostly from phosphorylated AMPKα1. Even though this set of data could suggest a decrease in AMPK activity, phosphorylation of

acetyl-CoA carboxylase (ACC), a target of AMPKα1/AMPKα2, was not significantly different between all groups while the total amount of ACC was unchanged in *Ampkα2ciKO* mice (Fig. 1B). The cardiac specificity of *Ampkα2* deletion in the present animal model was confirmed by the similar protein level of AMPKα2 and AMPKα1 in skeletal muscles of *Ampkα2ciKO* and *Ampkα2fl/f* male mice (Fig. 1C).

### Ampkα2ciKO male mutants develop a mild left ventricular systolic dysfunction

Sixteen weeks after *Ampkα2* deletion, mutant mice did not display any change in heart weight as judged by absolute organ weight and heart weight-to-body weight ratio (Fig. 2A and B). The expression of the gene encoding brain natriuretic peptide (Bnp) was significantly increased after *Ampkα2* deletion and was significantly lower in females than in males (Fig. 2C). Cardiac function of *Ampkα2ciKO* mice and their control littermates was assessed by serial echocardiography 3, 7, 10, 13 and 16 weeks after the first tamoxifen injection in males and by a single echocardiography at 16 weeks in females. In males, while echocardiography parameters did not show any difference between control and mutant mice 3 weeks after induction of *Ampkα2* deletion, significant decreases in LV ejection fraction (EF) and fractional shortening (FS) as well as a significant increase in end-systolic left ventricular internal diameter (LVIDs) were observed starting 7 weeks after tamoxifen treatment (Fig. 2D–G and Table 3). However, these alterations of cardiac systolic function were still slight after 16 weeks and no significant impact on cardiac output was noticed at this time point (Table 3). In these animals, diastolic function seemed to be more preserved since most of the diastolic echocardiography parameters were similar in CT and *Ampkα2ciKO* mice at 16 weeks. Nevertheless, left ventricular end-diastolic volume was significantly increased in *Ampkα2* deleted male mice at this time point (Table 3) and LVIDd tended to be higher in this group, even showing a significant difference with *Ampkα2fl/f* at 7 weeks (Fig. 2G). In females, echocardiography did not reveal any significant modifications between CT and KO in LV ejection fraction (Fig. 2H), fractional shortening (Fig. 2I) and ventricular internal diameters (Fig. 2J–K and Table 4). This is in contrast with the mild left ventricular systolic dysfunction observed in KO male mice and suggests that female heart could be more resistant to *Ampkα2* deletion.

### Cardiac-specific Ampkα2 deletion is associated with ventricular fibrosis and mitochondrial alterations in males

Inasmuch as ventricular dysfunction has been reported to be often associated with fibrosis which can change
A **Left ventricle - 16 weeks**

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B **Left ventricle - 16 weeks**

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C **Soleus- 16 weeks**

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**Fig. 1** Cardiac-specific *Ampka2* inactivation 16 weeks after tamoxifen injection in adult male and female mice. **A** Protein content of AMPKα2 and AMPKα1 in left ventricle (LV) homogenates. Tubulin is used as a loading control. **B** Immunoblotting of total AMPK (tAMPK), total ACC (tACC), phosphorylated-AMPK (pAMPK) and phosphorylated-ACC (pACC) in LV. Tubulin is used as a loading control. **C** Protein content of AMPKα2 and AMPKα1 in skeletal muscle. Actin is used as a loading control. (n = 3 to 4 per experimental group). ANOVA: $\phi p \le 0.05$, $\phi\phi p \le 0.01$, $\phi\phi\phi p \le 0.001$ for the genotype effect. Post hoc Tukey test: \*p < 0.05, \*\*p < 0.001 Ampka2<sup>f/f</sup> vs Ampka2ciKO
<figure><img src="image_7.png" /></figure>

tissue properties and negatively affect contraction and/or relaxation, the consequences of *Ampka2* deletion on fibrosis have been assessed using Sirius red staining. In *Ampka2ciKO* males, after 16 weeks of deletion, total fibrosis of LV was markedly increased and collagen

deposition was observed in the vicinity of the vessels (perivascular fibrosis) as well as around the cardiomyocytes (interstitial fibrosis) (Fig. 3A). No cardiac fibrosis was observed in female KO mice compared to CT mice. In line with these observations, the expression level of
<table><caption>**Table 3** Echocardiographic parameters in males 16 weeks after induction of *Ampka2* deletion by tamoxifen injection</caption><thead><tr><th>Echocardiographic parameters</th><th>Ampka2<sup>ff</sup> n = 7</th><th>Ampka2ciKO n = 7</th></tr></thead><tbody><tr><td>HR (beats/min)</td><td>519 ± 42</td><td>533 ± 47</td></tr><tr><td>IVSd (mm)</td><td>0.781 ± 0.158</td><td>0.831 ± 0.148</td></tr><tr><td>IVSs (mm)</td><td>1.276 ± 0.191</td><td>1.330 ± 0.191</td></tr><tr><td>LVIDd (mm)</td><td>3.84 ± 0.30</td><td>4.13 ± 0.38</td></tr><tr><td>LVIDs (mm)</td><td>2.26 ± 0.28</td><td><strong>2.67 ± 0.49*</strong></td></tr><tr><td>LVPWd (mm)</td><td>0.738 ± 0.219</td><td>0.741 ± 0.192</td></tr><tr><td>LVPWs (mm)</td><td>1.286 ± 0.153</td><td>1.196 ± 0.181</td></tr><tr><td>EDV (ml)</td><td>0.14 ± 0.03</td><td><strong>0.18 ± 0.05*</strong></td></tr><tr><td>ESV (ml)</td><td>0.03 ± 0.01</td><td><strong>0.06 ± 0.04*</strong></td></tr><tr><td>EF (%)</td><td>78.3 ± 4.53</td><td><strong>71.1 ± 8.61*</strong></td></tr><tr><td>SV (ml)</td><td>0.11 ± 0.02</td><td>0.12 ± 0.02</td></tr><tr><td>FS (%)</td><td>41.32 ± 4.10</td><td><strong>35.62 ± 6.48*</strong></td></tr><tr><td>CO (ml/min)</td><td>58.3 ± 12.5</td><td>65.9 ± 11.7</td></tr></tbody></table>

Statistical differences are highlighted in bold

HR, heart rate; IVSd, interventricular septal thickness at end-diastole; IVSs, interventricular septal thickness at end-systole; LVIDd, left ventricular internal dimension at end-diastole; LVIDs, left ventricular internal dimension at end-systole; LVWPD, left ventricular posterior wall thickness at end-diastole; LVWPs, left ventricular posterior wall thickness at end-systole; EDV, end-diastolic volume; ESV, end-systolic volume; EF, left ventricular ejection fraction; SV, stroke volume; FS, left ventricular fractional shortening; CO, cardiac output; *p < 0.05 Ampka2<sup>ff</sup> vs Ampka2ciKO

<table><caption>**Table 4** Echocardiographic parameters in females 16 weeks after induction of *Ampka2* deletion by tamoxifen injection</caption><thead><tr><th>Echocardiographic parameters</th><th>Ampka2<sup>ff</sup> n = 7</th><th>Ampka2ciKO n = 6</th></tr></thead><tbody><tr><td>HR (beats/min)</td><td>384 ± 30</td><td>447 ± 35</td></tr><tr><td>IVSd (mm)</td><td>0.97 ± 0.12</td><td>0.99 ± 0.09</td></tr><tr><td>IVSs (mm)</td><td>1.49 ± 0.12</td><td>1.56 ± 0.15</td></tr><tr><td>LVIDd (mm)</td><td>2.76 ± 0.21</td><td>2.55 ± 0.16</td></tr><tr><td>LVIDs (mm)</td><td>1.50 ± 0.16</td><td>1.30 ± 0.12</td></tr><tr><td>LVPWd (mm)</td><td>1.10 ± 0.07</td><td>1.03 ± 0.03</td></tr><tr><td>LVPWs (mm)</td><td>1.48 ± 0.08</td><td>1.46 ± 0.10</td></tr><tr><td>EDV (ml)</td><td>0.07 ± 0.01</td><td>0.05 ± 0.01</td></tr><tr><td>ESV (ml)</td><td>0.12 ± 0.02</td><td>0.09 ± 0.01</td></tr><tr><td>EF (%)</td><td>81.1 ± 4.4</td><td>82.4 ± 0.4</td></tr><tr><td>SV (ml)</td><td>0.05 ± 0.01</td><td>0.05 ± 0.01</td></tr><tr><td>FS (%)</td><td>45.4 ± 4.7</td><td>47.1 ± 1.0</td></tr><tr><td>CO (ml/min)</td><td>18.4 ± 4.3</td><td>21.2 ± 3.6</td></tr></tbody></table>

HR, heart rate; IVSd, interventricular septal thickness at end-diastole; IVSs, interventricular septal thickness at end-systole; LVIDd, left ventricular internal dimension at end-diastole; LVIDs, left ventricular internal dimension at end-systole; LVWPD, left ventricular posterior wall thickness at end-diastole; LVWPs, left ventricular posterior wall thickness at end-systole; EDV, end-diastolic volume; ESV, end-systolic volume; EF, left ventricular ejection fraction; SV, stroke volume; FS, left ventricular fractional shortening; CO, cardiac output

*Col3a1* was significantly increased and *Col1a1* expression tended to be higher in mutant males in comparison with CT males while CT and KO female mice exhibited similar expression level of these genes (Fig. 3B). Interestingly, a significant correlation between EF and percentage of total fibrosis has been highlighted in males (Fig. 3C).

The loss of AMPKα2 negatively impacted mitochondrial function only in males since respiration rates measured in permeabilized fibers in presence of malate, pyruvate and glutamate were largely decreased in *Ampka2ciKO* male mice (Fig. 4A), whereas these parameters were similar to controls in female *Ampka2ciKO* mice (Fig. 4A). In males, the subsequent addition of succinate with or without amytal (inhibitor of complex I) led to the normalization of mitochondrial O₂ consumption to control values in mutant mice suggesting that complex II activity can compensate for the deficit in complex I-respiration in the context of saturating succinate concentration (Fig. 4A). This indicates that maximal mitochondrial oxidative capacities were comparable in both male groups, what was confirmed by the equivalent TMPD-stimulated cytochrome C-dependent respiration rate (Fig. 4A) and the similar activity of cytochrome oxidase (COX) and citrate synthase (CS) (Fig. 4B), the latter being traditionally used as a marker of mitochondrial mass. In line with enzyme activity, no significant difference in CS protein level was noticed in male mutant mice compared to *Ampka2<sup>ff</sup>* mice either (Fig. 4C). Altogether, these results suggest that the decrease in mitochondrial respiration observed in males when malate, pyruvate and glutamate were added in the respiration chamber would be due to alterations of complex I of electron transfer chain (ETC). Yet, activity of complex I measured on protein homogenate of LV and the protein content of C-I-20 (a complex I subunit) were not changed by the loss of AMPKα2 (Fig. 4B and D). Incidentally, none of the subunits of the five complexes of ETC showed alterations 16 weeks after *Ampka2* deletion in males (Fig. 4D). Regarding females, although COX and complex I activities were significantly lower than in males (regardless genotype), no enzyme activity (CS, COX, complex I) or mitochondrial protein expression (CS and subunits of the ETC complexes) measured in this study were altered by 16 weeks of *Ampka2* deletion (Fig. 4B–D).

Despite the fact that mitochondrial mass and maximal oxidative capacities seemed to be similar in both male groups, the significant decrease in *Nrf2* and *Cox1* expression and the trend towards lower expression of *Erra*, *Tfam*, *Pparα* and *Cox4* displayed by male mutant mice in comparison with controls could suggest a slight alteration of mitochondrial biogenesis while *Pgc-1α* and *Pgc-1β* expression seemed not to be affected by the loss of AMPKα2 (Fig. 4E). Expression of all these genes was
<figure><img src="image_8.png" /></figure>

similar to control after 16 weeks of *Ampka2* deletion in females (Fig. 4E). Note that the expression of *Pgc-1β*, *Nrf2*, *Ppara* and *Cox1* showed clear sexual dimorphism (Fig. 4E).

As cardiac function and energy metabolism are strongly related to cardiomyocytes ultrastructure, cyto-architecture analyses of cardiomyocytes have been

thoroughly done using TEM. This revealed that, beyond mitochondrial biogenesis, mitochondrial life cycle could be more largely disturbed by *Ampka2* deletion. Indeed, in LV of male *Ampka2ciKO* mice, mitochondria were not as well organized along the myofilaments as in controls (Fig. 5Aa and Ab), exhibited a marked heterogeneity in size (Fig. 5Ab and Ac) and were frequently organized in
(See figure on next page.)

**Fig. 4** Left ventricle mitochondrial phenotype 16 weeks after cardiac-specific *Ampka2* deletion in male and female mice. **A** Rate of respiration after successive addition of pyruvate (1 mM), glutamate (10 mM), succinate (15 mM), amytal (1 mM) and TMPD-ascorbate (0.5:2 mM). **B** Citrate synthase (CS), cytochrome c oxidase (COX) and complex I enzymatic activities. **C** Immunoblotting of citrate synthase (CS) and mitochondrial creatine kinase (mCK). Vinculin is used as a loading control. **D** Total protein content of 5 subunits of oxidative phosphorylation complexes: C-I-20 (complex I), C-II-30 (complex II), C-III-Core 2 (complex III), C-IV-COXI (complex IV) and C-V-α (complex V). **E** mRNA expression level of genes related to mitochondrial biogenesis: *Pgc-1α*, *Pgc-1β*, *Nrf2* (*gabpa*), *Erra*, *Ppara*, *Tfam*, *Cox1* and *Cox4*. (n = 5 to 7 per experimental group). ANOVA: $\$p < 0.05$ for the genotype effect; $\mu p < 0.05$, $\muμ p < 0.01$, $\muμμ p < 0.001$ for the sex effect; $\$p < 0.05$ for interaction; Post hoc Tukey test: \*\*p < 0.05 Ampka2f/f vs Ampka2ciKO (same sex); $\$αp < 0.05$, $\$ααp < 0.01$ males vs females (same genotype)

clusters (Fig. 5Ac). Important disorganization of cristae (Fig. 5Ad and Ae) and “giant” mitochondria (Fig. 5Af) were also frequently noticed in male mutant mice. The latter point could be partly explained by a potential alteration of mitochondrial dynamics process since the expression of the pro-fission gene *Drp1* was lower when AMPKα2 was not expressed while the expressions of *Mfn1*, *Mfn2* and *Opa1*, involved in mitochondrial fusion, were not significantly changed by the deletion (Fig. 5B). Of note, no alteration in the expression of all these genes was noticed in females (Fig. 5B).

## Inner mitochondrial membrane is altered in male *Ampka2ciKO* mice

Whereas the alteration of complex I function 16 weeks after *Ampka2* deletion was one of the most noticeable result, the apparent discrepancy between the respiration data indicating a complex I deficiency and the biochemical data showing normal complex I activity and protein content (subunit C-I-20) raised questions. We thus sought to determine whether the microenvironment surrounding complex I within the inner mitochondrial membrane could be different in mutant mice and could explain its lack of efficiency within the ETC. Sixteen weeks after tamoxifen injection, *Ampka2ciKO* male mice clearly showed a marked rearrangement in the mitochondrial membrane cardiolipin (CL) profile (Fig. 6). Although the total CL content in KO was similar to the *Ampka2f/f* mice in both sexes (Fig. 6A and B), the amount of several CL species was affected by the loss of AMPKα2 in males only (Fig. 6C and D). The proportion of CL containing 20-carbon acyl chains was significantly changed in *Ampka2ciKO* since a large reduction of CL with eicosadienoic acid (20:2) chains and a slight increase in CL containing eicosatrienoic acid (20:3) or eicosatetraenoic acid (20:4) were observed (Fig. 6C). Male KO mice also exhibited a much larger fraction of CL containing docosahexaenoic (22:6) or docosapentaenoic (22:5) acyl chains. Interestingly, these alterations in mitochondrial membrane composition were associated with significant decreases in expression of *Aclsl1* and *Cds1* as well as a trend towards a lower expression of *Crls1* in males only (Fig. 6E), these three genes encoding important enzymes

involved in CL synthesis. None of the enzymes involved in the production of CL was significantly impacted in the female *Ampka2ciKO* mice 16 weeks after deletion when compared to female CT mice. Of note, the expression of many genes encoding enzymes involved in CL biosynthesis/maturation showed sexual dimorphism. As complexes of the respiratory chain are usually arranged in supercomplexes, we thought to investigate the assembly of complex I in these structures. In males, the CL remodeling was associated with a lower propensity of complex I to interact physically with the other complexes of ETC. Indeed, a lower proportion of complex I took part in the organization of mitochondrial supercomplexes and a higher part of complex I was found isolated; no such changes were revealed in female mice (Fig. 7A and B).

## Ovariectomy makes female mice more sensitive to the loss of AMPKα2

Obviously, one major difference between males and females lies in hormonal status which is incidentally known to be largely involved in the sexual dimorphism reported in cardiovascular diseases [22]. To investigate the potential interactions between AMPK and female hormones that might explain the differences observed between males and females in the present mouse model, ovariectomy (OVX) was performed in CT and *Ampka2ciKO* mice. Seventeen weeks after ovariectomy, mice were sacrificed and the success of the surgery was confirmed by the large reduction of uterus weight in both OVX groups (Fig. 8A). Interestingly, although ablation of the ovaries did not induce clear modulations of heart weight, cardiac *Bnp* expression and heart function in *Ampka2ciKO* mice (Fig. 8B–G), this surgery led to a significant increase in total fibrosis in *Ampka2ciKO* mice only (Fig. 8H–I). In this group, total fibrosis reached 5.9±0.3% of the tissue (Fig. 8I), so a level like the one observed in the male *Ampka2ciKO* mice in which total fibrosis reached 4.6±0.4% of the tissue (Fig. 3A).

Since the loss of AMPKα2 led to drastic changes in CL profile in males, mitochondrial membrane CL content/profile has been assessed after ovary excision. Whereas the total CL content and the major CL species were not affected by the genotype or the hormone
<figure><img src="image_9.png" /><figcaption>**Fig. 4** (See legend on previous page.)</figcaption></figure>
A

a

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b

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c

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d

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e

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f

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B

<figure><img src="image_16.png" /><figcaption>**Fig. 5** Cardiomyocyte cytoarchitecture and mitochondrial morphology 16 weeks after cardiac-specific *Ampka2* deletion in male mice. **A** Transmission electron micrograph of left ventricle of *Ampka2fl/fl* (Aa) and *Ampka2ciKO* (Ab-Af) mice. **B** mRNA expression level of genes encoding mitochondrial dynamics proteins: optic atrophy protein 1 (Opa1), Mitofusin 1 and 2 (Mfn1 and Mfn2) and dynamin-related protein 1 (Drp1). ANOVA: μμ *p* < 0.01 for the sex effect; $ *p* < 0.05 for interaction; Post hoc Tukey test: \* *p* < 0.05 *Ampka2fl/fl* vs *AmpkciKO* (same sex); \*\* *p* < 0.01 males vs females (same genotype)</figcaption></figure>
(See figure on next page.)

**Fig. 6** Left ventricular cardiolipin profile in male and female mice 16 weeks after cardiac-specific *Ampka2* deletion. **A** Total cardiolipin content in male mice. **B** Total cardiolipin content in female mice. **C** Relative content of each identified cardiolipin species in male mice. **D** Relative content of each identified cardiolipin species in female mice. **E** Expression of genes related to cardiolipin biosynthesis and maturation: acyl-CoA synthetase long-chain family member 1 (Acs1l), CDP-diacylglycerol synthase 1 (Cds1), cardiolipin synthase 1 (Crls1), tafazzin (Taz), elongation of very long chain fatty acids-like 2 (Elovl2), elongation of long chain fatty acids (Elovl5), fatty acid desaturase 1 (Fads1) and fatty acid desaturase 2 (Fads2). ANOVA: μ *p* < 0.05, μμ *p* ≤ 0.01 for the sex effect; $p < 0.05$, $$p < 0.01$ for interaction; Post hoc Tukey test: \* *p* < 0.05; \*\* *p* < 0.01, \*\*\* *p* < 0.001 *Ampka2l/f* vs *Ampka2ciKO* (same sex); $\alpha p < 0.05$, $\alpha\alpha p < 0.01$ males vs females (same genotype)

deficiency (Fig. 9A and B), many minor CL species were impacted by ovariectomy in a genotype-dependent manner. Indeed, even if no significant changes were noticed when each OVX group was compared to its respective sham group (Fig. 9C), the proportion of CL with eicosadienoic acid (20:2) and CL containing eicosatrienoic acid (20:3) or eicosatetraenoic acid (20:4) slightly changed following ovariectomy. Notably, the mild modulations noticed in KO-OVX mice compared to KO-sham group were systematically the opposite of the slight alterations displayed by CT-OVX group when compared to CT-sham one. This led to significant lower proportion of CL with eicosadienoic acid (20:2) and a trend towards a higher proportion of CL containing eicosatrienoic acid (20:3) or eicosatetraenoic acid (20:4) in KO-OVX mice than in CT-OVX ones (Fig. 9C), mimicking the pattern observed in KO males versus CT males (Fig. 6C). These OVX induced-CL profile modulations could thus be under the control of AMPK and this was confirmed by the two-way ANOVA statistical analysis which indicated an interaction between surgery and genotype for the variations of the proportion of CL with eicosadienoic acid (20:2) (Fig. 9C). These differences between OVX groups was hardly explained by the expression of the enzymes involved in the synthesis/maturation of the CL since only the expression of *Crls1* was significantly lower in KO-OVX group while the expression of the other enzymes of this biosynthesis pathway was neither affected by surgery nor by genotype (Fig. 9D). Although the alterations observed in mitochondrial membrane composition in KO-OVX mice is reminiscent of what was described in male *Ampka2ciKO* mice, they were less marked than in males and seemed not to be sufficient to clearly affect mitochondrial function since mitochondrial respiratory assay did not show significant alterations in KO-OVX group in comparison with the other groups (Fig. 9E).

Discussion

Although the role of AMPK has largely been studied for many years, its role in cardiac physiology and pathophysiology is still not completely understood. With the aim of clarifying the place of this kinase in cardiomyocyte homeostasis, we generated an original inducible cardiac-specific *Ampka2* knockout mouse model

using Cre recombinase. Of note, none of the deleterious effects on cardiac function observed in males were due to a potential toxicity of Cre recombinase since our team already demonstrated that the tamoxifen conditions used for inducing gene deletion do not impact cardiac function in αMHC-MerCreMer mice [37]. Using this inducible cardiac-specific *Ampka2* knockout mouse model, we show that specific cardiac deficiency of AMPKα2 at adult age in males (1) did not induce cardiac hypertrophy; (2) led to a mild left ventricular dysfunction; (3) resulted in the development of cardiac fibrosis; (4) reduced complex I-driven respiration without changes in mitochondrial mass or in *in vitro* complex I activity; (5) was associated with a rearrangement of the CL species and a reduced integration of complex I into the ETC supercomplexes. Importantly, the effects of the cardiac-specific *Ampka2* deletion observed in males were not found in females at basal state, suggesting that alternative pathways to AMPK are active in female mice. Ovariectomy of female KO mice suggest that their lower sensitivity to cardiac *Ampka2* deletion could partly be explained by the blunting effects of female hormones on the development of cardiac fibrosis and by a role in the maturation of cardiolipins; however alterations in these females remained quite mild since they displayed normal mitochondrial function and were asymptomatic as regard to systolic function.

Owing to the AMPK structure based on the assembly of several subunits, suppression of AMPK activity in genetically modified animal models can result from different strategies. Many *Ampk* null mice have already been generated using KO or dominant negative technologies targeting one or the other subunit of AMPK and gave variable results on AMPK activity and compensatory mechanisms between the diverse isoforms [24, 38–40]. In the present murine model of cardiac specific and inducible *Ampka2* deletion, the AMPKα2 protein content in LV was drastically decreased and was associated with a reduction in phosphorylated AMPK level. Yet, phosphorylation of ACC, a direct target of AMPK, was not modified. This result which can be surprising at first sight, can actually be explained by the fact that the experiments have been done in non-stressing conditions which are not expected to induce strong AMPK activity
<figure><img src="image_17.png" /><figcaption>**Fig. 6** (See legend on previous page.)</figcaption></figure>
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B

<figure><img src="image_19.png" /><figcaption>**Fig. 7** Left ventricular integration of electron transfer chain complex I within the mitochondrial supercomplexes 16 weeks after cardiac-specific *Ampka2* deletion induction in male and female mice. **A** Mitochondrial supercomplexes were first separated by BN-PAGE (left panels), then transferred to PVDF membrane and incubated with complex I antibody (right panels) in CT and KO males (upper panels) and females (lower panels). **B** Quantification of isolated complex I and complex I including supercomplexes in males (left panel) and females (right panel). (n = 4 per experimental group). \*p < 0.05 *Ampka2II* vs *Ampka2ciKO* (same sex)</figcaption></figure>

and consequently a high ACC phosphorylation level. Furthermore, this indicates that the tiny residual amount of AMPKα2 together with AMPKα1, the expression of which was not changed in the present model, could maintain the amount of phosphorylated ACC at basal level. Indeed, ACC is a common target of both AMPKα

isoforms and a small amount of AMPKα1/2 could be sufficient to ensure phosphorylation status of this enzyme when the animal is not subjected to stress [41]. Nevertheless, as AMPK phosphorylates many other proteins and as the affinity of AMPKα1 and AMPKα2 for their targets differs and gives them a relative specificity [6], the

(See figure on next page.)

**Fig. 8** Cardiac function and myocardial fibrosis in control (Sham) and ovariectomized (OVX) female mice 16 weeks after cardiac-specific *Ampka2* deletion. **A** Uterus weight. **B** Heart weight. **C** brain natriuretic peptide (Bnp) gene expression in left ventricle. **D** Left ventricular ejection fraction. **E** Left ventricular fractional shortening. **F** Left ventricular internal dimension at end-systolic (LVIDs). **G** Left ventricular internal dimension at end-diastolic (LVIDd). **H** Representative pictures of fibrosis analysis by Sirius red staining of subequatorial heart section. **I** Percentage of total fibrosis. ANOVA: \# *p* < 0.05, ### *p* < 0.001 for the OVX effect; §§*p* < 0.01 for the genotype effect, §§*p* < 0.01 interaction. Post hoc Tukey test: \* *p* < 0.05, \*\* *p* < 0.01 Sham vs OVX
<figure><img src="image_20.png" /><figcaption>**Fig. 8** (See legend on previous page.)</figcaption></figure>
(See figure on next page.)

**Fig. 9** Cardiolipin profile and oxidative capacities 16 weeks after cardiac-specific *Ampka2* deletion in control (Sham) and ovariectomized (OVX) female mice. **A** Total cardiolipin content. **B** Relative content of major cardiolipin species. **C** Relative content of minor cardiolipin species. **D** Expression of genes related to cardiolipin biosynthesis and maturation: acyl-CoA synthetase long-chain family member 1 (Acs1l), CDP-diacylglycerol synthase 1 (Cds1), cardiolipin synthase 1 (Crls1), tafazzin (Taz), elongation of very long chain fatty acids-like 2 (Elov12), elongation of long chain fatty acids (Elov15), fatty acid desaturase 1 (Fads1) and fatty acid desaturase 2 (Fads2). **E** Rate of respiration after successive addition of pyruvate (1 mM), glutamate (10 mM), succinate (15 mM), amytal (1 mM). (n = 4 to 7 per experimental group). ANOVA: \*p ≤ 0.05 for the OVX effect; \*\*p < 0.05 for the genotype effect, \*\*\*p < 0.01 CT-OVX vs KO-OVX

drastic decrease in AMPKα2 content in *Ampka2ciKO* males may impact the signaling pathways downstream of AMPK because the residual AMPKα2 could not ensure phosphorylation of all the targets, some of which would not be phosphorylated by AMPKα1 as efficiently as by AMPKα2.

In male *Ampka2ciKO* mice, the modest alterations of several cardiac contractile parameters (EF, FS, LVIDd, LVIDs) suggest a mild left ventricular systolic dysfunction which is in accordance with the cardiac impairment described in global and constitutive *Ampka2* deletion [17, 25, 39]. The degradation of cardiac function was clearly evidenced 7 weeks after deletion, got worst as the mice deleted for *Ampka2* got older, but was not associated with important anatomical changes when compared to control. Several studies showed narrow links between AMPK and protein synthesis regulation [42, 43] and suggested an anti-hypertrophic action of AMPK under various stresses [39, 44, 45]. However, the present work did not show any modifications of the cardiac mass neither in *Ampka2ciKO* male nor in *Ampka2ciKO* female mice when the animals were not subjected to any stress. This result, which incidentally recalls what was observed in other dominant negative or global *Ampka2* KO mice [17, 39], suggests that this AMPK isoform does not play critical role in cardiac hypertrophy/growth at basal state. For the understanding of this study, it seems important to note that the absence of cardiac dysfunction after 16 weeks of *Ampka2* deletion in females probably does not mean that females are totally insensible to AMPKα2 loss. Indeed, although not statistically significant the slight increase in *Bnp* expression in *Ampka2ciKO* females after 16 weeks of deletion could suggest that females were not fully protected from this deletion but rather more resistant. It cannot be excluded that these females would develop significant cardiac dysfunction at a later age and this point will require further investigations.

Interestingly, the ventricular dysfunction observed in males was associated with a significant increase of myocardial fibrosis that was correlated with EF. Although this could imply that fibrosis affects ventricular mechanical properties and function in this model, the fact that KO-OVX mice exhibited a comparable cardiac fibrosis level without any changes in cardiac function compels

us to consider that fibrosis is not the only factor explaining cardiac dysfunction in males. Be that as it may, the development of fibrosis in *Ampka2ciKO* male and in KO-OVX female mice could suggest that AMPKα2 is linked to fibrosis. It is generally thought that the cardiac fibrotic response is essentially mediated by cardiac fibroblasts; yet in this cardiac-specific *Ampka2* KO model, *Ampka2* was only deleted in cardiomyocytes and it could be suggested that this deletion would stimulate cardiac fibrotic response by cardiac fibroblasts through communication between both cell types. Even though the link between AMPK and cardiac fibrosis has already been suggested since the activation of AMPK can suppress fibrosis induced by various stresses (for review see [16]), regulation of the fibrotic response by AMPKα2 is still poorly understood, on the contrary to the role of AMPKα1, the major AMPK isoform in cardiac fibroblasts, which has clearly been demonstrated to control the proliferation of cardiac fibroblasts and the development of fibrosis especially through the TGF-β1/p38 axis [15]. From another side, it cannot be excluded that fibrosis observed in males was linked to mitochondrial dysfunction since it has recently been shown that oxidative stress plays a key role in myocardial fibrosis development [46, 47] and excessive ROS production is a well-known feature of altered mitochondria. Nevertheless, cardiac fibrosis in OVX group was not associated with clear alteration of electron transfer chain, rather suggesting that cardiac fibrosis was an earlier event than important mitochondrial perturbations in these mice and giving weight to the hypothesis of a link between AMPK and fibrosis. It could also reasonably be proposed that mitochondrial dysfunction could at least secondarily participates in fibrosis establishment in this model, all the more so mitochondrial dysfunction could be at the origin of cell death within the myocardium which is known to trigger reparative fibrosis to prevent rupture of the ventricular wall.

In this study, cardiac *Ampka2* deletion provoked mitochondrial complex I-driven respiration dysfunction only in male LV. This was not associated with any significant changes in maximal mitochondrial oxidative capacity and mitochondrial mass, even though gene expression of several markers of mitochondrial biogenesis were significantly reduced (*Nrf-2* and *Cox1*) or tended to be lower
<figure><img src="image_21.png" /><figcaption>**Fig. 9** (See legend on previous page.)</figcaption></figure>
(ERRα, Tfam, Ppara and Cox4) in KO. This is in accordance with the observation made in the mouse model of global and constitutive deletion of Ampka2 [17] and suggests that this kinase would only play a minor role in mitochondrial biogenesis regulation in the non-stressed heart. Similarly, cardio-specific or constitutive deletion of Ampka2 led to complex I-driven respiration impairment without any decrease in complex I enzymatic activity measured in total heart extract, thereby suggesting an alteration of in situ regulation of this complex.

It is known that the in situ activity of ETC complexes can be affected by the phospholipid content of inner mitochondrial membrane and that complex I is particularly sensitive to its microenvironment [48]. Cardiolipin, a phospholipid which represents 15 to 20% of total mitochondrial phospholipid is essentially found in the inner membrane of the mitochondria [17]. It is formed of two phosphate moieties connected with a glycerol backbone. It thus contains four acyl groups and carries negative charges. Cardiolipin mainly contain (18:2)4 acyl chains, but the length and composition of acyl chains can vary enormously. In the global constitutive Ampka2 KO mice, the alteration of complex I-driven respiration was associated with a clear reduction in LV content of CL [17]. Given that CLs play a key role in many processes of mitochondrial metabolism [49], this suggested that the decrease in CL could partly explain the ETC defects observed in this model. In the present work, cardiac-specific Ampka2 deletion in males induced a decrease in the expression of several key enzymes of CL biosynthesis (Cds1, Acsl1 and Crls1) and substantial CL rearrangement even though the total CL content was similar to control. The difference between global constitutive Ampka2 KO mice and the present model could come from the different timing and duration of the deletion, from the beginning of in utero life in the constitutive KO versus a loss of AMPK starting in the adult life in Ampka2ciKO mice, from an established pool of CL in mitochondria. Nevertheless, our present study and a previous one by our team on doxorubicin-induced cardiomyopathy further bring evidence that AMPK loss has an impact on mitochondrial CL profile [17, 21]. Recently, it has been shown that CL remodeling alters lipid membrane properties and assembly of complex subunits [50]. The increase in CL containing docosahexaenoic acyl chain (22:6) in KO males is particularly interesting as it has been reported that a higher proportion of tetradocosahexaenoyl-CL in the heart disrupts the formation of microdomains and phospholipid-protein contacts regulating the mitochondrial enzymes, especially complex I and IV [51], thereby demonstrating a strong link between CL and ETC. Rather than existing as individual complexes, respiratory chain complexes physically interact

in highly organized structures allowing functional link between ETC components resulting in a more efficient electron transfer [52]. Given that CL create the environment required for the assembly of complex subunits and the formation of supercomplexes allowing an efficient complex-I-linked and complex II-linked mitochondrial respiration [53], the CL profile modulations observed in KO males is probably part of the mechanism leading to ETC alterations in our model.

The reduction in Cds1 expression in Ampka2ciKO mice suggests a role for AMPK signaling in the regulation of genes involved in CL biosynthesis. Interestingly, the expression of this enzyme has been shown to be under the control of PGC-1α/ERRα axis [54]. As a major regulator of PGC-1α, AMPK could thus modulate CL metabolism through this pathway. The trend towards a reduction in Erra expression in Ampka2ciKO males in comparison with Ampka2fl/fl males is in favor of this hypothesis. The present study shows that this alteration of mitochondrial membrane CL composition was not observed in female KO mice in which complex I-driven respiration was similar to control. This once again highlights the connection between complex I activity and mitochondrial membrane CL composition [55, 56] and also suggests a sexual dimorphism in the regulation of mitochondrial membrane composition. Such a sexual dimorphism in CL composition has already been observed in rats [57]. Surprisingly, ablation of the ovaries in female Ampka2ciKO mice did not affect the mitochondrial membrane CL composition as strongly as in males. Nevertheless, cardiac mitochondrial CL profile in ovariectomized females was modified according to a pattern reminding the rearrangements observed in Ampka2ciKO males, strengthening the idea that female hormones normally participate in the maintenance of inner mitochondrial CL composition and compensate for the lack of Ampka2 in KO-sham females. Even so, the CL alterations in KO-OVX mice are quite mild and appear insufficient to impact mitochondrial function as in the males, suggesting that the duration of female hormone deficiency was not sufficient to totally suppress the higher resistance of the Ampka2ciKO females. This could also be explained by the fact that OVX mice had surgery at the age of 7 weeks and a first impregnation with female hormones cannot be excluded as 6-week-old mice may be able to reproduce. These mice could have been exposed to female hormones during one or two weeks and it could interfere with the development of the phenotype of Ampka2ciKO mice and explain the milder effect of Ampka2 deletion in OVX mice than in males. In addition, even after ovariectomy, females continue to display a different global morphology than males and are not exposed to high level of male hormones. The fact that Ampka2ciKO OVX females did
not perfectly reproduced the CL rearrangement observed in males may not be that surprising since male hormones could also differently regulate CL biosynthesis [58]. In the heart, the profound disparities in CL content/species between male and female rats subjected to doxorubicin treatment pleads in favor of a sexual dimorphism in CL metabolism [57]. Together these studies suggest that the role of male and female hormones in CL biosynthesis/ maturation requires further investigations. So far, the role of AMPK in the sexual dimorphism of CL metabolism is still elusive. Although it might have been interesting to study the reversibility of the *Ampka2* deletion-induced alterations in OVX females by 17-β estradiol treatment to better understand the underlying phenomena, the present study clearly confirms that AMPK is involved in CL biosynthesis and remodeling in a sex-dependent manner.

CLs play many roles in mitochondrial membrane organization and are crucial for mitochondrial super-complex formation [59]. In male *Ampka2* KO mice, the proportion of complex I not enclosed in supercomplexes was increased and these mice displayed less cardiac complex-I-containing supercomplexes. When considering the fact that neither mitochondrial respiration nor CLs profile nor the amount of complex-I-containing supercomplexes were significantly modulated in female KO mice, one can suggest that the concomitant alterations of ETC functioning, mitochondrial membrane CL composition and supercomplex disorganization in males, together with modest systolic dysfunction are in no way coincidental. In X-linked Barth syndrome induced by a mutation in the gene encoding tafazzin, an enzyme responsible for CL maturation, mature CLs are lost and this leads to an instability of respiratory chain supercomplexes which affects complex I activity [60], thereby highlighting the importance of CLs for complex I-containing supercomplexes formation and function. In male *Ampka2* KO mice, the alterations of CL profile and complex I-containing supercomplexes formation undeniably affects the ETC functioning and this at least explains in part the alteration of complex-I-driven respiration observed in this model.

## Limitations of the study

The fact that *Ampka2* deletion is induced by injections of tamoxifen which interacts with estrogen receptor could be a limitation of this study that was focused on sex differences. However, the tamoxifen dose used was chosen after a long period of development and study of the literature to determine the lowest dose of tamoxifen to achieve an optimal level of deletion of *Ampka2* and limit side-effects. Thus, each mouse was injected with only two 40 mg/kg doses which correspond to 2 doses of 1 mg tamoxifen for a 25 g mouse. A recent study testing

the clearance of tamoxifen in mice [61] showed that after high dose tamoxifen (5-days treatment with 3 mg or 6 mg tamoxifen daily (intraperitoneal)) the concentration of tamoxifen in serum is very low 10 days after the last injection. The doses used in this study were 7.5 or 15 times higher than in the present study, it can be assumed that our mice were not exposed to tamoxifen more than a few days. As most of the measurements realized in our study have been done 16 weeks after the last tamoxifen injection, the potential impacts of tamoxifen through estrogen signaling interaction on the produced results were probably limited.

Naturally, AMPK is involved in many signaling pathways and the phenotype of *Ampka2ciKO* mice was the result of a set of perturbations. We did not extensively study AMPK roles which are particularly intricate and there were undoubtedly many other disturbances that had not been uncovered in this work. Nevertheless, this study has the merit of highlighting a poorly understood role for AMPK, which partly explains the disturbances in energy metabolism observed in this model and which certainly played its part in the phenotype resulting from the deletion of *Ampka2* in the heart.

## Perspectives and significance

Cardiac-specific inducible *Ampka2* deletion model proved helpful in deciphering the role of AMPK in basal conditions. In this model, *Ampka2* deletion induced a progressive cardiac dysfunction associated with cardiac fibrosis and a mitochondrial dysfunction associated with cardiolipin remodeling in males only, thereby suggesting a role for AMPKα2 in these processes. Although the involvement of AMPKα2 in the regulation of myocardial fibrosis and cardiolipin biosynthesis and maturation requires further studies, our results highlight a higher dependence on AMPK signaling for these processes in males, either due to the absence of female hormones protection or to counteract the action of male hormones, and that may contribute to the known difference in cardiovascular risk and outcome between sexes.

## Abbreviations

ACC: Acetyl-CoA carboxylase; AMPK: AMP-activated protein kinase; *Ampka2ciKO*: *Ampka2* Cardiac-specific inducible mice; CL: Cardiolipin; COX: Cytochrome c oxidase; CS: Citrate synthase; CT: Control mice; EF: Ejection fraction; ETC: Electron transfer chain; FS: Fractional shortening; HF: Heart failure; LV: Left ventricle; OVX: Ovariectomy; ROS: Reactive oxygen species.

## Acknowledgements

We thank Valérie Domergue and Pauline Robert for preparation of the animals as well as Claudine Deloménie for her expertise in molecular biology (IFR141-IPSIT). We thank Rodolphe Fischmeister and Ana-Maria Gomez for continuous support and Maryline Moulin for her valuable help which has been of great value to perform and interpret cardiolipin profiling. We thank Ladislav Novota for his help during TEM experiments and Anne Lombès for her help to perform blue native page experiments. Maria-Nieves Sanz's current address:
Department of Cardiovascular Surgery, Inselspital, Bern University Hospital, Bern, Switzerland and Department for BioMedical Research, University of Bern, Bern, Switzerland.

**Authors' contributions**

Study conception and design: LG, VV, MM, RV-C, JP and AG. Acquisition of data: LG, M-NS, MG, CR-M, MN, AK, AS, SG, KB, EJ and JP. Analysis and interpretation of data: LG, M-NS, MG, CR-M, MN, AS, KB, EJ, CL, VV, MM, RV-C, JP and AG. Drafting of manuscript and critical revision: LG, M-NS, MG, CR-M, MN, AS, KB, EJ, CL, VV, MMe, RV-C, JP and AG. All authors read and approved the final manuscript.

**Funding**

Our laboratory is a member of the Laboratory of Excellence LERMIT and is supported by grants from "Fondation pour la Recherche Médicale" (to A.G., #DPM20121125546), European Research Area Network on Cardiovascular Diseases (to J.P, #ANR-19-ECVD-0007-01), Fondation de France (to A.G., 00086500), Université Paris-Sud (ERM) and CORDDIM (to R.V.-C, #cod110153), STEFANIK (to C. L. and M. N., 35814NK). This study was supported by the Slovak Research and Development Agency grant (M.N., APVV-15-0302), and by the Slovak Scientific Grant Agency (M.N., VEGA 2/0091/19). R.V.-C is emeritus scientist at CNRS.

**Availability of data and materials**

The data that support the findings of this study are available from the corresponding author upon reasonable request.

**Declarations**

**Ethics approval and consent to participate**

Not applicable.

**Consent for publication**

Not applicable.

**Competing interests**

No potential conflicts of interest were disclosed.

**Author details**

¹Faculté de Pharmacie, UMR-S1180, INSERM, Université Paris-Saclay, 5 rue J-B Clément, 92296 Châtenay-Malabry, France. ²Université Paris-Saclay, Inserm, Hypertension Artérielle Pulmonaire: Physiopathologie et Innovation Thérapeutique, 92350 Le Plessis Robinson, France. ³Department of Cellular Cardiology, Institute of Experimental Endocrinology, Biomedical Research Center, University Science Park for Biomedicine, Slovak Academy of Sciences, Bratislava, Slovakia. ⁴Service d'Analyse des Médicaments et Métabolites, Université Paris-Saclay, Inserm, CNRS, Institut Paris Saclay d'Innovation Thérapeutique, 92296 Châtenay-Malabry, France. ⁵Université Paris-Saclay, CNRS, Institut de Chimie Des Substances Naturelles, UPR 2301, 91198 Gif-sur-Yvette, France. ⁶Université Versailles St-Quentin, Université Paris-Saclay, Inserm, UMR-S 1180, 92296 Châtenay-Malabry, France.

Received: 26 April 2021 Accepted: 29 August 2021

Published online: 17 September 2021

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