Proteomic and transcriptomic analyses in cardiomyocyte-specific OGT knockout hearts reveal reduced mitochondrial protein networks and proteostatic adaptation

Clara C. Cho¹, Wenxi Zhang¹, Aidan Dunphy¹, Rohit Satish¹, Sogol Sedighi¹, Brian O’Rourke¹, D. Brian Foster¹, Natasha E. Zachara^2,3, & Kyriakos N. Papanicolaou ^1,*

¹Division of Cardiology, Department of Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

²Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

³Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA

Abstract

Background: O-Linked N-acetylglucosamine transferase (OGT) adds O-GlcNAc to serine/threonine residues on thousands of nuclear, mitochondrial, and cytosolic proteins, influencing protein homeostasis and metabolic set points. To define the roles of OGT in the heart, we generated an inducible, cardiomyocyte-specific OGT knockout (icko) mouse model and profiled myocardial protein and RNA changes after gene deletion.

Methods: Adult mice received tamoxifen to induce cardiomyocyte-restricted OGT deletion; efficacy was confirmed by immunoblotting for OGT and global O-GlcNAc. Myocardial proteins were quantified by label-free proteomics in MaxQuant (MaxLFQ) at 6, 9, and 16, weeks post-tamoxifen and analyzed in R (filtering/normalization; stats/visualization with tidyverse/limma). Pathway enrichment analysis was performed with clusterProfiler. RNA-seq was performed in hearts at 9 weeks post-tamoxifen and data were analyzed using DESeq for differential expression followed by clusterProfiler enrichment analysis.

Results: Proteomics at 6, 9, and 16 weeks showed coherent down-regulation of mitochondrial pathways in icko versus controls, including oxidative phosphorylation complexes, tricarboxylic-acid cycle enzymes, and fatty-acid β-oxidation components. The 9-week RNA-seq showed concordant directionality for key metabolic modules, supporting a coordinated remodeling toward reduced oxidative capacity. In parallel, enrichment analyses and confirmatory western blot analyses indicated upregulation of protein homeostasis/folding programs and increased abundance of select heat-shock proteins (HSPs) in icko hearts. We also observed enhanced extracellular matrix (ECM) organization/remodeling signatures in icko hearts across datasets.

Conclusions: Loss of OGT is associated with a concerted shift toward reduced abundance of mitochondrial oxidative-metabolism proteins, reinforced proteostasis, and enhanced ECM organization. Taken together, the data support a model in which OGT/O-GlcNAc help maintain mitochondrial protein homeostasis and sustain protein-quality control in the adult myocardium.

Keywords: OGT, cardiomyocyte, proteomics, RNA-seq, DESeq, clusterProfiler, heat-shock proteins, mitochondria, oxidative phosphorylation, proteostasis, extracellular matrix.