Abstract Body: Background: Aging is a hallmark of mitochondrial dysfunction in the heart. Emerging evidence suggests that aberrant alternative splicing (AS) contributes to impaired mitochondrial function. The objective of this study is to examine functional changes in mitochondrial isoforms during cardiac aging between sexes.
Methods: Frozen mouse heart tissues of young adult (4 months) and early aging (20 months) C57BL/6J mice (males and females, n = 5) were lysed in RIPA buffer supplemented with protease/phosphatase inhibitors using bead mill homogenization, followed by sonication and centrifugation (14,000 × g, 15 min, 4°C). A total of 100 μg protein was digested using a filter-assisted protocol with LysC/trypsin. Peptides were labeled with TMT 10-plex reagents as per the manufacturer’s instructions and fractionated by high-pH reversed-phase chromatography. Mass spectrometry data were acquired on an Orbitrap Q-Exactive HF coupled to an Easy-nLC 1200 nano-UPLC system. Raw data were analyzed using FragPipe and searched against the UniProt Swiss-Prot reviewed isoform database for Mus musculus.
Results: A total of 5,181 proteins were identified, including 231 unique AS isoforms (20 mitochondrial isoforms). We focused on Opa1 isoforms 2 and 3, which differ in exon composition and regulatory motifs. Isoform-2 expression was significantly higher in young male hearts compared to young females across both chambers. Isoform-2 contains two motifs, the LQQQIQ motif and the LIG_SUMO_SIM_anti_2 motif at the S2 cleavage site. In contrast, isoform-3 was detected at higher abundance in old female heart chambers than old males and contains the LIG_SUMO_SIM_par_1 motif at the S3 cleavage site. AlphaFold 3 multimer modeling combined with molecular dynamics simulations predicted a high-confidence of interaction between the isoform-3 motif and the peptidase domain of Yme1l. These findings suggest that isoform-3 may undergo enhanced Yme1l -mediated regulation higher in older female hearts, potentially contributing to sex-specific mitochondrial remodeling during cardiac aging.
Conclusion: These results suggest that AS-mediated isoform switching may contribute to the regulation of mitochondrial function of the heart during aging in a sex-dependent manner.