**1. Introduction**

The adenine nucleotide translocase (ANT), one of the most abundant proteins of the inner mitochondrial membrane, exchanges the matrix ATP for ADP in the intermembrane space and thus, links mitochondrial ATP production with cellular energetics. Several studies have demonstrated a crucial role of ANT in the pathogenesis of cardiac diseases. Downregulation of ANT1, the main ANT isoform in the heart and skeletal muscle [1,2], has been found in patients with hypertrophic cardiomyopathy, and lactic acidosis [3]. Mice lacking *ANT1* developed cardiac hypertrophy and lactic acidosis [2], and a substantial decline in cardiac function compared to wildtype (WT) animals [4]. Heart- and muscle-specific *ANT1* knockout (KO) mice exhibit deficiency in mitochondrial bioenergetics associated with mitochondrial myopathy and hypertrophic cardiomyopathy [5]. Additionally, *ANT1* KO

mice display an increase in reactive oxygen species (ROS) production and inhibition of oxidative phosphorylation (OXPHOS) in cardiac mitochondria [6]. Moreover, cardiac ischemia-reperfusion (IR) reduced *ANT1* expression whereas cardiac-specific overexpression of *ANT1* attenuated IR injury and reduced infarct size in rats [7]. In rat neonatal cardiomyocytes, overexpression of ANT1 protected against hypoxia-induced cell death, loss of mitochondrial membrane potential (ΔΨ m), and increased ROS production [7]. Therefore, understanding the role of ANT in the regulation of mitochondrial bioenergetics can provide a novel insight into mitochondrial-based cardiac therapies.

ANT has been shown to interact with various subunits of the electron transport chain (ETC) complexes in HEK293 cells [8] and in yeas<sup>t</sup> [9]. Several studies, the earliest one in 2000, demonstrated that ETC individual complexes can be assembled in large supramolecular structures known as respiratory chain supercomplexes (RCS) [10]. The main RCS is the respirasome, which is composed of complexes I, III, and IV in various stoichiometries. It has been proposed that the respirasome facilitates electron transfer, reduces electron leakage and mitochondrial ROS (mtROS) production, maintains structural organization of ETC complexes, and provides an e fficient ATP production [11].

The assembly mechanisms and the structural identity of RCS remain to be elucidated. The role of ANT in RCS formation was recently proposed after it was observed that ANT interacts with RCS and that this interaction is conserved from yeas<sup>t</sup> to higher eukaryotes [8], potentially implicating a crucial role of ANT in mitochondrial bioenergetics. However, these studies were mostly done in yeas<sup>t</sup> and HEK293 cells; the RCS and ANT interactome has not been reported in mammalian tissues, particularly, in the heart. We have shown that pharmacological inhibition of ANT by atractyloside provoked RCS disintegration in cardiac mitochondria in vitro [12]. These studies sugges<sup>t</sup> that ANT may have a structural interaction with RCS and/or play a regulatory role in RCS. Furthermore, post-translational modifications on ANT may a ffect its regulatory and structural capability in RCS assembly. Indeed, acetylation has been demonstrated to regulate the activity of ETC complexes [13,14] and thus, might a ffect the RCS stability.

Here, we investigated the role of ANT1 in RCS assembly in H9c2 cardiomyoblasts. *ANT1* KD cells demonstrated increased total cellular ATP levels, with a reduction in ΔΨ m and no changes in mitochondrial ATP production. However, *ANT1* KD did not a ffect the enzymatic activity of individual ETC complexes nor mitochondrial oxygen consumption. Deficiency in *ANT1* expression induced disassembly of RCS, particularly the respirasome, suggesting a potential role of ANT in RCS formation. Also, we found that ANT1 is not hyperacetylated in *SIRT3* KO mice although RCS levels in these animals were lower than in WT counterparts.

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