Oxphos complex alterations as a pathomechanism in arrhythmogenic cardiomyopathy in knockout cell models

Abstract Introduction Arrhythmogenic cardiomyopathy (ACM) is an inherited cardiac disorder that predominantly affects the right ventricle and is characterized by progressive cardiomyocyte loss and replacement of the myocardium with fibro-fatty tissue, ultimately predisposing individuals to sudden cardiac death (1). Variants in desmosomal genes (PKP2, DSP, DSG2, DSC2, and JUP) constitute the primary genetic causes of ACM, accounting for up to 50% of affected individuals (2). However, some of the molecular pathophysiologic mechanisms are still partially unknown. Recent work pointed that mitochondrial respiration may have an important role in the pathophysiology. In this sense, our previous work demonstrated a reduction in both protein abundance and functional activity of oxidative phosphorylation (OXPHOS) complexes in PKP2-, DSC2-, and DSG2-knockout models (3). Purpose The objective of the present study was to generate to perform an exhaustive study of the mitochondrial respiration in the five desmosomal genes to determine whether these defects reflect a common pathogenic mechanism associated with loss of any of the five desmosomal genes Methods CRISPR/Cas9 editing was used to generate HL1 edited cell models., which were validated by Sanger sequncing (4). Expression profiling was performed by RNA-seq analysis. Protein expression in the resulting knockout clones was assessed by western blot using total protein lysates and an antibody recognizing all five OXPHOS complexes. To evaluate whether changes in OXPHOS levels could be explained by alterations in mitochondrial content, TOM20 expression was quantified in knockout versus wild-type (WT) cells. Results Sequencing confirmed the successful generation of four independent knockout lines per gene, each carrying premature stop codons in both alleles. RNA-seq analysis revealed numerous downregulated pathways related to aerobic respiration, the electron transport chain, oxidative phosphorylation, and ATP synthesis, with gene-specific differences indicating that each desmosomal mutation impacts these metabolic pathways in distinct ways. Western blot analyses revealed no significant differences in the abundance of OXPHOS complex proteins between DSP or JUP knockout cells and WT controls, in contrast to our previously examined PKP2, DSG2, and DSC2 knockout models, which exhibited marked alterations. TOM20 levels did not significantly differ between any of the five knockout groups and WT cells, indicating comparable mitochondrial content. Conclusions These findings suggest that alterations in oxidative phosphorylation contribute differentially to the pathogenic mechanisms driven by distinct desmosomal gene deficiencies in ACM, which may help explain how gene-specific disruptions of oxidative phosphorylation contribute to the variable clinical expression observed among carriers of different desmosomal mutations.