The Role of Adenine Nucleotide Translocase in the Assembly of Respiratory Supercomplexes in Cardiac Cells

The physiological significance of the RCS is still under debate [35]. The mitochondrial RCS have been suggested to increase the effectiveness of electron transport through the ETC complexes, optimize ATP production, and reduce mtROS production by reducing electron leakage [11]. Disassembly of the RCSs, particularly the respirasome, was observed in cardiovascular diseases such as IR [16] and heart failure [36]. However, the mechanisms underlying the assembly of the RCSs, as well as their physiological role in the heart, are not fully understood. Our previous studies demonstrated that high Ca2+ and pharmacological/genetic inhibition of complex I (Figure 4C,D) stimulate disruption of the RCS in H9c2 cells and isolated mitochondria [12,37]. These studies suggested crosstalk between RCS assembly and permeability transition pore (PTP) opening as Ca2+ is the strong inducer of pore opening and complex I is the PTP regulator. This point is further supported by the current study that demonstrates that genetic downregulation of ANT, a PTP regulator, induces disorganization of the RCS. However, the cause–effect relationship between RCS and PTP seems to be more complex. Despite RCS disassembly, inhibition of complex I by rotenone prevented Ca2+-induced PTP opening in cardiac mitochondria [12], and ANT1 KD did not increase mtROS, a PTP inducer in H9c2 cells (Figure 2D). Finally, we demonstrate that acetylation of mitochondrial proteins due to SIRT3 deficiency induces RCS disassembly in an ANT-independent manner because there was no difference in ANT acetylation between WT and SIRT3−/− mitochondria (Figure 5).

Disruption of the RCS could be a result of direct mechanisms involving disruption of protein–protein interactions due to changes in lysine residue charges, or indirect mechanisms through inactivation of RCS regulatory proteins (e.g., RCS assembly factors) due to their hyperacetylation.

In conclusion, this study suggests that ANT is involved in RCS assembly, although RCS may not be solely dependent on ANT. ANT may physically interact with ETC complexes I, III, and IV [8] and thus, be involved in the respirasome structure or play a regulatory role in the formation/maintenance of the RCS assembly. Further studies are required to elucidate the role of ANT in the structural integrity and regulation of RCS and other mitochondrial supercomplexes (e.g., ATP synthasome) in cardiac cells.

We elucidated the contribution of only ANT1 downregulation to mitochondrial bioenergetics and RCS assembly. ANT family proteins contain four isoforms (ANT1-4) that play a differential role and perform distinctly opposite functions in cell life and death. We were not able to verify protein expression of other ANT isoforms in ANT1 KD H9c2 cells due to lack of reliable ANT2, ANT3, and ANT4 antibodies. Functional redundancy of other ANT isoforms could compensate for the effects induced by ANT1 deficiency.