# Subcellular Proteomic Analyses Reveal REEP5 Knockdown in the Mouse Heart Disrupts Mitochondrial Networks

## TL;DR

This study shows that reducing REEP5 in mouse hearts disrupts mitochondrial networks and increases oxidative stress, highlighting its role in heart cell organelle communication.

## Contribution

The study reveals REEP5's novel role in regulating ER-mitochondria communication and mitochondrial homeostasis in cardiomyocytes.

## Key findings

- Loss of REEP5 leads to fragmented mitochondria and increased reactive oxygen species in cardiomyocytes.
- Subcellular proteomes show altered redox adaptation and depletion of mitochondrial import machinery.
- REEP5 depletion upregulates SR/ER membrane-shaping proteins like RTN4, ATL3, and CKAP4.

## Abstract

Receptor Expression-Enhancing Protein 5 (REEP5) is a cardiac-enriched, membrane-shaping protein localized to the sarco(endo)plasmic reticulum (SR/ER), where it supports membrane network architecture and cardiomyocyte function. While REEP5 has been implicated in calcium handling and contractility, its role in regulating inter-organelle communication and mitochondrial homeostasis remains less well-understood. In this study, we used recombinant adeno-associated virus serotype 9-mediated shRNA knockdown of Reep5 in mouse hearts, combined with subcellular fractionation and data-independent acquisition mass spectrometry, to define proteomic remodeling across microsomal (SR/ER), mitochondrial, and cytosolic compartments. Loss of REEP5 altered the composition of SR/ER membrane-shaping proteins, including upregulation of RTN4, ATL3, and CKAP4, suggesting a partial compensatory response. Microsomal, mitochondrial and cytosolic proteomes exhibited broad reorganization, with enrichment of proteins involved in redox adaptation and proteostasis, alongside depletion of mitochondrial import machinery and antioxidant enzymes. Imaging of isolated cardiomyocytes confirmed fragmented mitochondrial networks and increased reactive oxygen species, consistent with proteomic signatures of disrupted mitochondrial dynamics and oxidative stress. Gene ontology enrichment across all fractions highlighted widespread dysregulation in organelle-specific processes, including translation, protein localization, and metabolic remodeling. Notably, several altered pathways converged on mitochondria-associated membranes, suggesting that REEP5 may support SR/ER-mitochondria tethering and functional crosstalk. These findings position REEP5 as a key regulator of organelle homeostasis in the heart and underscore how its loss disrupts mitochondrial integrity and inter-organelle communication across cellular compartments.

•rAAV9-mediated REEP5 knockdown and subcellular fractionation enabled cardiac organelle-specific proteomic profiling.•SR/ER-shaping proteins are upregulated in response to REEP5 depletion.•Subcellular proteomes reveal altered expression of redox and stress response regulators.•Loss of REEP5 leads to fragmented mitochondria and increased oxidative stress.•REEP5 is a key regulator of mitochondrial homeostasis in cardiomyocytes.

rAAV9-mediated REEP5 knockdown and subcellular fractionation enabled cardiac organelle-specific proteomic profiling.

SR/ER-shaping proteins are upregulated in response to REEP5 depletion.

Subcellular proteomes reveal altered expression of redox and stress response regulators.

Loss of REEP5 leads to fragmented mitochondria and increased oxidative stress.

REEP5 is a key regulator of mitochondrial homeostasis in cardiomyocytes.

Using rAAV9-mediated knockdown and subcellular proteomics, this study uncovers a critical role for REEP5 in maintaining mitochondrial structure and redox balance in the heart. Loss of REEP5 alters organelle-specific proteomes, disrupts mitochondria, and induces oxidative stress. These findings highlight REEP5 as a key regulator of ER–mitochondria communication and cardiac homeostasis.