# WASHC3 knockout disrupts mitochondrial protein homeostasis and energy metabolism in cardiomyocytes

## TL;DR

This study shows that WASHC3 is important for heart cell energy and mitochondrial health, linking its absence to both neurological and cardiac issues.

## Contribution

The novel role of WASHC3 in cardiac mitochondrial protein homeostasis and bioenergetics is identified.

## Key findings

- WASHC3 knockout leads to downregulation of mitochondrial proteins and impaired respiration in cardiomyocytes.
- Loss of WASHC3 causes progressive heart degeneration in aged zebrafish.
- Findings connect WASHC3 to both neuromuscular and cardiac mitochondrial dysfunction.

## Abstract

The WASH complex regulates endosomal actin dynamics and vesicular trafficking and is essential for neuronal integrity and motor function. Although variants in WASHC4, WASHC5, and WASHC3 are linked to neurodevelopmental abnormalities, the role of WASHC3 beyond the nervous system, particularly in cardiac mitochondrial regulation, remains unclear.

We modeled WASHC3 loss of function in zebrafish and human cardiomyocytes. Washc3 was suppressed in zebrafish embryos by antisense oligonucleotide-mediated knockdown, and a stable Washc3 knockout line was generated using CRISPR/Cas9. Washc3-deficient zebrafish hearts were analyzed by quantitative LC-MS/MS proteomics with GO/KEGG enrichment and transcript-level assays. Mitochondrial bioenergetics was assessed by Seahorse XF assays in primary zebrafish cardiomyocytes and in human AC16 cardiomyocytes following AAV-shRNA-mediated WASHC3 knockdown.

Washc3 knockdown embryos exhibited neuromuscular degeneration, impaired locomotion, and early cardiac dysfunction. In contrast, Washc3 knockout zebrafish showed normal early development but developed progressive pericardial degeneration and epicardial remodeling in aged animals. Cardiac proteomics revealed downregulation of mitochondrial proteins, particularly oxidative phosphorylation components, supported by pathway enrichment and concordant transcript-level findings. Mitochondrial respiration was significantly impaired in both Washc3-deficient zebrafish cardiomyocytes and WASHC3-depleted human AC16 cardiomyocytes.

These findings identify a previously unrecognized role for WASHC3 in maintaining cardiac mitochondrial protein homeostasis and bioenergetic function and provide a framework linking neuromuscular and cardiac phenotypes to impaired mitochondrial bioenergetics in energy-demanding tissues.