**1. Introduction**

Equine atypical myopathy (AM) is a severe environmental intoxication linked to the ingestion of certain maple (*Acer*) seeds and seedlings. In Europe, the incriminated tree is the sycamore maple (*Acer pseudoplatanus*) [1], whereas in the United States, the box elder (*Acer negundo*) has been linked to the poisoning [2]. Two toxins, hypoglycin A (HGA) [3–5] and methylenecyclopropylglycine (MCPrG) [6] are involved in the poisoning [7]. These molecules are not toxic per se but once in the body are transformed into their active metabolites, methylenecyclopropylacetyl-CoA (MCPA-CoA) and methylenecyclopropylformyl-CoA (MCPF-CoA), respectively [8–10]. Both toxins are known inhibitors of fatty-acid ß-oxidation, which results in an impaired capacity of energy production using oxidative metabolism [10–13]. The MCPA-CoA inhibits also dehydrogenases involved in the degradation of branched-chain amino acids [10]. The ingestion of maple toxins led to the detection of toxins, conjugated toxic metabolites, and fatty esters in blood [1,2,5,7,14–17].

**Citation:** Kruse, C.-J.; Stern, D.; Mouithys-Mickalad, A.; Niesten, A.; Art, T.; Lemieux, H.; Votion, D.-M. In Vitro Assays for the Assessment of Impaired Mitochondrial Bioenergetics in Equine Atypical Myopathy. *Life* **2021**, *11*, 719. https:// doi.org/10.3390/life11070719

Academic Editors: Giorgio Lenaz, Salvatore Nesci and Gopal J. Babu

Received: 4 May 2021 Accepted: 15 July 2021 Published: 20 July 2021

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Clinical signs in intoxicated horses include muscle weakness and stiffness, eventual recumbency, and, in 74% of cases, death [18]. Macroscopic, histologic, and histochemical analyses confirm multifocal degeneration and necrosis with variable severity between cases and muscles [19]. Indeed, muscular lesions seem to be more constant and severe in the myocardium and respiratory and postural muscles [19,20], therefore, oxidative muscle fibers appear to be particularly affected by the toxin [19]. Transmission electron microscopy revealed several ultrastructural changes affecting especially mitochondria, as matrix loss and cristae fragmentation [19].

Previous studies performed on skeletal muscle show that structural alterations are associated with mitochondrial functional consequences [13]. Using high-resolution respirometry (HRR), a severe depression of mitochondrial oxidative phosphorylation (OXPHOS) and electron transfer system capacities (ET capacity) in AM affected horses was found [13] using standardized substrate-uncoupler-inhibitor titration (SUIT) protocols validated for respirometric assessments of equine muscle cells [21].

Since AM outbreaks are seasonal (i.e., autumnal and spring outbreaks resulting from the consumption of seeds and seedlings, respectively) and do not occur to the same extend every year [22], in vivo sampling is naturally limited. Also, because of the acute nature of AM and the rapid progression of the condition with a mean survival time of 38 h [23], complementary examinations and sampling might be difficult to perform. Additionally, the use of surrogate animals to study AM may not be valid, since both rodents and rabbits display damage of different organs than horses after HGA intoxication and do not show signs of rhabdomyolysis [24,25]. Because of these obstacles, an in vitro model was designed attempting to reproduce mitochondrial dysfunction by adding methylenecyclopropylacetyl (MCPA) (i.e., the sole toxin commercially available) during HRR experiments. Ultimately, our final goal will be to define a treatment for AM based on the ability of the drug to restore an adequate mitochondrial function. The aim of the present study was: (1) to reproduce specific changes in OXPHOS capacity and respiratory control patterns observed in skeletal muscle of AM affected horses using conventional SUIT protocols [13]; (2) to measure the effect of MCPA on fatty acids utilization, and (3) to determine the cytotoxicity and viability of equine primary muscle cells subjected to HGA and MCPA.

#### **2. Materials and Methods**

#### *2.1. Cell Culture of Equine Primary Myoblasts*

Equine primary skeletal myoblast cultures were purchased from RevaTis® (RevaTis, Aye, Belgium). A vial containing 2.5 million cells was thawed at 37 ◦C in a water bath. The cells were then cultivated in an 80 cm<sup>2</sup> culture Tflask and multiplied from passage 5 to passage 8. All manipulations were performed under streamline flow hood.

Cells were cultured in the maintenance media Dulbecco's modified Eagles's lowglucose DMEM 1 g/L (Lonza, Verviers, Belgium) supplemented with 20% fetal bovine serum, 1% L-glutamine, 1% penicillin-streptomycin, and 0.5% amphotericin B (Thermo Fisher Scientific, Karlsruhe, Germany). When 80% confluence was reached, culture medium was removed, and the flasks were washed using Dulbecco's phosphate-buffered saline (DPBS) without Ca2+ and Mg2+. Cells were subsequently trypsinized (TrypLETM Express, Thermo Fisher Scientific, Karlsruhe, Germany), centrifuged, and counted according to Ceusters et al. [26]. The equivalent of one 175 cm<sup>2</sup> culture T-flask was used to continue cell culture to further passages.

At each passage, part of the cells was immediately processed for HRR. Ten thousand cells per well were seeded in a 96 clear bottom white plate (Cellstar® Greiner Bio-One, Vilvoorde, Belgium) and supplemented with DMEM medium until further analysis. Plates and flasks were incubated at 37 ◦C and 5% CO<sup>2</sup> in a humidified incubator.

#### *2.2. Toxicity Assays*

The cell toxicity of MCPA was assessed using the CellToxTM Green assay (Promega Benelux, Leiden, The Netherlands). This assay was developed to determine toxic effects by binding DNA of cells with impaired membrane integrity.

Plated cells were kept in a humidified incubator at 37 ◦C and 5% CO<sup>2</sup> until 80% of confluence was reached. Confluence was assessed by light microscopy after 24 to 36 h. Once confluent, the cells were washed with DPBS without Ca2+ and Mg2+. The cells were then exposed to different concentrations of MCPA (Merck, Darmstadt, Germany) with DMSO as solvent and HGA (Toronto Research Chemicals, Ontario M3J2K8, Canada) suspended in DMEM medium without phenol red. CellToxTM Green dye was added to each well. The bottom of the plate was covered with a BrightMaxTM seal (Greiner Bio-One, Vilvoorde, Belgium) and subsequently placed in the EnSpire® Multimode Plate Reader (PerkinElmer, Waltham, MA, USA) for reading. Data was recorded for 24 h after toxin exposure in order to determine the evolution of cytotoxicity.

Additionally, a real-time viability assay through reducing potential measurement was performed. The nonlytic dye contained in the RealTime-GloTM MT Cell Viability Assay (Promega Benelux, Leiden, The Netherlands) allows for continuous reading and is based on the continuous reduction of the viability substrate by the viable cells contained in each well. The DMSO concentrations in each well were of 0.5% in order to exclude DMSO induced toxicity to the cells.

#### *2.3. High-Resolution Respirometry*

After centrifugation, the cells were counted and then placed in MiR05 mitochondrial respiration medium (0.5 mM EGTA, 3 mM MgCl2. 6 H2O, 20 mM taurine, 10 mM KH2PO4, 20 mM HEPES, 1 g/L BSA, 60 mM potassium-lactobionate, 110 mM sucrose, pH 7.1) at 37 ◦C. In total, 2.5 to 3 million cells were added to each 2 mL Oxygraph-2k chamber (Oroboros Instruments, Innsbruck, Austria). Oxygen concentration (µM) and oxygen flux per million cells (pmol O2·s −1 ·10<sup>6</sup> cells−<sup>1</sup> ) were recorded online using DatLab software (Oroboros Instruments, Austria). Experiments were conducted using specific SUIT protocols and oxygen levels were maintained between 200 and 500 µM O<sup>2</sup> to avoid any oxygen-related limitations of respiration and to align with previous studies [13,21,27]. Oxygen flux was expressed as respiration per million cells (pmol O2·s −1 ·10<sup>6</sup> cells−<sup>1</sup> ), or as control ratios, namely flux control ratios (*FCR*). These ratios have the advantage of being independent of cell count, mitochondrial content, and density, indicating qualitative changes of mitochondrial respiratory control [28].

Several SUIT protocols were applied, using both fatty acids and reduced nicotinamide adenine dinucleotide (NADH)-linked substrates in order to assess mitochondrial pathways at different levels of integration. In order to have a global overview, the electron transfer (ET) pathway was fed by the NADH junction (N-junction) substrates pyruvate, glutamate in the presence of malate. Fatty acids are catabolized via ß-oxidation and support ET pathways through both reduced flavin adenine dinucleotide (FADH2) junction (F-junction) and N-junction (see level 5; Figure 1). Therefore, ß-oxidation relies on the combination of F-junction and N-junction pathways.

Muscle cells suspended in MiR05 were added to the Oxygraph-2k chambers containing a final volume of 2 mL per chamber maintained at 37.0 ◦C. In SUIT1, electron flow was sustained by the NADH-linked substrate glutamate and co-substrate malate (GM; 10 and 2 mM, respectively) followed by a saturating concentration of ADP (D; 2.5 mM). In SUIT2, the initial substrate was pyruvate with malate as co-substrate (PM; 5 and 2 mM, respectively) with subsequent addition of digitonin (Dig; 10 µg·10<sup>6</sup> cells−<sup>1</sup> ) and ADP (D; 2.5 mM) followed by addition of glutamate (G; 10 mM). Three fatty acids' protocols were performed and started with the addition of acetylcarnitine (Act; 5 mM; SUIT3), octanoylcarnitine (Oct; 0.5 mM; SUIT4), and palmitoylcarnitine (Pal; 0.04 mM; SUIT5), and malate (2 mM; SUIT3, SUIT4, SUIT5) as co-substrate. In SUIT3 to 5, electrons from both the electron-transferring flavoprotein complex (CETF) and the Complex I (CI) entered the Q-junction. In all proto-

cols, digitonin (Dig; 0.01 mM) was added before ADP. Optimal digitonin concentration was determined by careful titration experiments as previously described [29]. In all SUIT protocols, ADP-stimulated respiration represents OXPHOS capacity, *P* whereas ET capacity, *E* was obtained by addition of the uncoupler FCCP, rendering this state as not limited by the capacity of the phosphorylation system [28].

**Figure 1.** ET-substrate types are linked to ET-pathway types in substrate-uncoupler-inhibitor titration (SUIT) protocols. Electrons from multiple upstream origins feed into the Q junction. These origins include (5) fatty acid ß-oxidation (FAO) providing electrons from FADH<sup>2</sup> to the electron-transferring flavoprotein complex (CETF; F pathway), (4) dehydrogenases and enzymes converging at the N-junction and providing electrons from NADH to complex I (N pathway), (3) succinate (S) providing electrons from FADH<sup>2</sup> to Complex II (CII; S pathway). From the Q junction, electrons are then transferred to Complex III, cytochrome c and complex IV, before their transfer to O<sup>2</sup> to form H2O. Figure from Gnaiger (2020) with permission [28]. Abbreviations: FADH<sup>2</sup> = flavin adenine dinucleotide; NADH = Nicotinamide adenine dinucleotide.

μ ∙ − In every protocol, succinate (S; 10 mM) was subsequently added for electron flow from Complex II (CII) into the Q-junction to give the flux through the NS-pathway (N and S pathway combined) or the F- and S-pathway combined. By stepwise addition of the non-physiological uncoupler FCCP (U; 0.05 µM, followed by 0.025 µM steps until maximal oxygen flux was reached), ET-capacity, *E*, was obtained. Electron input into the Q-junction through CII only was subsequently measured by inhibition of CI with rotenone (Rot; 0.5 µM). Finally, residual oxygen consumption state was obtained by addition of antimycin A (Ama; 2.5 µM) to inhibit Complex III (CIII). For each protocol, MCPA [9 mM] was added to a parallel chamber after ADP addition. The concentration was defined beforehand by toxicity assays. All protocols are summarized in Table 1.

For all protocols, a second oxygraphic run was performed to establish if cytochrome *c* (Cyt *c*; 10 µM) addition induced an increase in O<sup>2</sup> flux. Cytochrome *c* release is considered as an essential quality control because of the possible limitation of active respiration when the outer mitochondrial membrane has been damaged by the laboratory procedures, allowing the loss of cytochrome *c* located in the intermembrane space [28].

μ μ


**Table 1.** Substrate-uncoupler-inhibitor titration (SUIT) protocols performed.

<sup>1</sup> NS-pathway. <sup>2</sup> F-pathway and S-pathway. Abbreviations: GM = Glutamate & malate; PM = Pyruvate & malate; ActM = Acetylcarnitine & malate; OctM = Octanoylcarnitine & malate; PalM = Palmitoylcarnitine & malate; D = ADP; G = Glutamate; S = Succinate; U = Uncoupler (FCCP); Rot = Rotenone; Ama = Antimycine A.
