Phillyrin protects against myocardial ischemia/reperfusion injury by promoting KNL1 K605 acetylation to inhibit the p53/p21 pathway

Abstract

Reperfusion therapy is critical for acute myocardial infarction but is often accompanied by myocardial ischemia/reperfusion injury (MIRI). Phillyrin, a natural lignan from Forsythia suspensa, exerts anti-inflammatory and antioxidant effects; however, its role and mechanism in MIRI remain unclear. In this study, HL-1 cardiomyocytes are subjected to oxygen-glucose deprivation/reperfusion (OGD/R). Cell viability, apoptosis, oxidative stress, and inflammation are measured after phillyrin treatment. Multiomics (mRNA-seq, proteomics, and acetylproteomics) is used to identify key targets and pathways. Molecular docking, co-immunoprecipitation, site-directed mutagenesis, and western blot analysis are used to validate posttranslational regulation. A mouse MIRI model is established to confirm the in vivo cardioprotective effects of phillyrin. Phillyrin preserves cell viability and reduces apoptosis, oxidative stress, and inflammation in OGD/R-injured HL-1 cells. Multiomics integration reveals that phillyrin acts primarily through posttranslational regulation and highlights kinetochore scaffold 1 (KNL1) as the only protein that is both upregulated and hyperacetylated at lysine 605 (K605). Mechanistically, phillyrin may bind to the KNL1 C-terminus and enhance the interaction between KNL1 and acetyltransferase p300/CBP. KNL1 K605R mutation and Knl1 knockdown reduce KNL1 protein expression and reverse the inhibitory effects of phillyrin on p53 pathway-mediated apoptosis, oxidative stress, and inflammation. In mouse MIRI models, phillyrin reduces infarct size, myocardial damage, and cardiomyocyte apoptosis; these effects are abolished by knockdown of Knl1. Therefore, phillyrin promotes KNL1 acetylation at K605 to increase KNL1 protein expression, thereby inhibiting p53 signaling and alleviating apoptosis, oxidative stress, and inflammation in MIRI. This study identifies KNL1 acetylation at K605 as a novel posttranslational modification target for cardioprotection.

1. Introduction

Acute myocardial infarction (AMI) is a severe cardiovascular disease that significantly threatens human health. Reperfusion therapy, which rapidly restores coronary blood flow, facilitates the metabolic and functional recovery of cardiomyocytes, and ameliorates the pathophysiological alterations induced by myocardial ischemia, represents the most effective strategy for reducing ischemic damage and limiting infarct size [1]. However, myocardial ischemia/reperfusion may lead to a range of adverse effects, including myocardial injury, arrhythmias, and inflammatory responses [2–4], which can further exacerbate ischemic damage [5]. To date, although several therapeutic strategies (e.g., beta-blockers and remote ischemic conditioning) have been applied to alleviate myocardial ischemia/reperfusion injury (MIRI) in clinical practice, their efficacy remains limited by factors such as narrow application scope, potential side effects, or incomplete inhibition of MIRI-related pathological processes [6,7]. Therefore, more effective and safe therapeutic approaches to improve MIRI outcomes are urgently needed.

The pathophysiology and mechanisms underlying MIRI are complex and multifactorial and involve reactive oxygen species (ROS) generation, apoptosis, autophagy, inflammatory responses, mitochondrial dysfunction, and immune reactions [2,8,9]. Although these pathological processes have long been recognized as MIRI drivers, recent advances have highlighted that epigenetic regulation acts as a “master switch” to orchestrate their progression: it not only modulates the development of cardiovascular diseases such as cardiac hypertrophy, hypertension, and heart failure but also plays an indispensable role in MIRI by fine-tuning the expression and function of key regulatory proteins [4,10–12]. This has made epigenetic targeting a highly promising therapeutic strategy for MIRI.

Among diverse epigenetic and posttranslational modifications (PTMs), protein acetylation stands out as a central, dynamically regulated mechanism in cardioprotection against MIRI. Acetylation is a rapid, energy-efficient PTM that governs protein stability, enzymatic activity, and protein–protein interactions and plays critical roles in cell cycle progression, apoptosis, oxidative stress, and inflammation. In MIRI, ischemia/reperfusion disrupts acetyl-CoA homeostasis and acetyltransferase/deacetylase activity, leading to the aberrant acetylation of mitochondrial, cytoskeletal, and signaling proteins. This dysregulation directly promotes cardiomyocyte apoptosis, cell cycle arrest, oxidative stress, and inflammation. For instance, the acetylation of mitochondrial enzymes enhances their antioxidant capacity and [text truncated]