Post-infarction KLHL40-mediated regulation of cardiac sarcomeric integrity and function

Cardiac sarcomeric remodeling after myocardial infarction (MI) plays a pivotal role in post-injury cardiac dysfunction, yet the molecular mechanisms governing this process remain incompletely understood.

We aimed to elucidate the role of Kelch-like protein 40 (KLHL40) in sarcomeric protein remodeling and calcium signaling regulation after MI.

By integrating transcriptomic and proteomic datasets with experimental validation, we characterized dynamic changes in KLHL40 expression and its effects on sarcomeric components and myocyte state following hypoxia. KLHL40 expression was evaluated in human post-MI heart tissues and hypoxia-induced H9C2 cells. Functional assays revealed its regulation of key Z-disc-associated proteins (MYOT, CAPZA, and TCAP). Mechanistic candidates were identified via His-tag affinity capture (OCTET NTA) and MS-based enrichment analysis. Intracellular calcium levels, calpain activity, inflammasome activation, and apoptosis were assessed, and pathways governing KLHL40 degradation were examined using specific inhibitors.

KLHL40 expression was markedly upregulated within 6 hours (95% CI [-72.53 to -0.9783]; P = 0.04) post-MI and selectively reduced the expression of MYOT (95% CI [0.321-1.08]; P = 0.003), CAPZA (95% CI [0.449-0.902]; P < 0.001), and TCAP (95% CI [0.130-0.689]; P = 0.01) without affecting ACTN2 (95% CI [-1.07 to 1.16]; P > 0.99) or FLNC (95% CI [-0.0673-0.280]; P = 0.22). Mechanistically, KLHL40 was associated with altered handling and calpain activation, including regulation of ATP2A2 and intracellular calcium levels. KLHL40 markedly reduced NLRP3 (95% CI [0.6576-1.145]; P < 0.001) and Cleaved Caspase-1 (95% CI [0.6276-0.8550]; P < 0.001) expression. The BCL2/BAX ratio decreased (95% CI [0.1108-0.8928]; P = 0.02) following KLHL40 knockdown and increased (95% CI [-1.641 to -0.3629]; P = 0.007) with overexpression. Additionally, KLHL40 turnover was regulated via the proteasomal, calpain, and autophagic pathways.

KLHL40 maintains sarcomeric integrity and cardiomyocyte viability after myocardial infarction by modulating calpain signaling and limiting inflammasome activation and apoptosis. KLHL40 expression and stability are tightly regulated through proteasomal, autophagic, and calpain-dependent pathways.