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

Area of Science:

• Cardiovascular Biology
• Cellular Biology
• Proteomics

Background:

• Receptor Expression-Enhancing Protein 5 (REEP5) is a cardiac protein crucial for sarco(endo)plasmic reticulum (SR/ER) structure and cardiomyocyte function.
• Its role in inter-organelle communication and mitochondrial homeostasis is not fully understood.

Purpose of the Study:

• To investigate the function of REEP5 in regulating cardiac organelle homeostasis and inter-organelle communication.
• To define the proteomic changes in SR/ER, mitochondria, and cytosol upon REEP5 loss.

Main Methods:

• Recombinant adeno-associated virus serotype 9 (rAAV9)-mediated shRNA knockdown of Reep5 in mouse hearts.
• Subcellular fractionation coupled with data-independent acquisition mass spectrometry (DIA-MS).
• Cardiomyocyte imaging to assess mitochondrial morphology and reactive oxygen species (ROS).

Main Results:

• REEP5 loss altered SR/ER membrane protein composition, with compensatory upregulation of RTN4, ATL3, and CKAP4.
• Proteomic analysis revealed broad reorganization in microsomal, mitochondrial, and cytosolic compartments, including altered redox adaptation and proteostasis.
• Mitochondrial fragmentation, increased ROS, and dysregulation of pathways converging on mitochondria-associated membranes (MAMs) were observed.

Conclusions:

• REEP5 is a key regulator of cardiac organelle homeostasis.
• Loss of REEP5 disrupts mitochondrial integrity, promotes oxidative stress, and impairs inter-organelle communication, particularly between the SR/ER and mitochondria.
• These findings highlight REEP5's importance in maintaining cardiomyocyte function and SR/ER-mitochondrial crosstalk.