Effects of piRNA CFAPIR in doxorubicin-induced ferroptosis models of rat and human cardiomyocytes

Abstract

BACKGROUND: Ferroptosis plays a critical role in doxorubicin-induced cardiomyopathy; however, its specific regulatory mechanisms require further elucidation. Piwi-interacting RNA 413 (piRNA413) regulates ferroptosis in doxorubicin-induced cardiomyocytes, designated as cardiac ferroptosis-associated piRNA (CFAPIR). However, the specific regulatory mechanism needs to be further elucidated. OBJECTIVE: To investigate the role and regulatory mechanism of piRNA CFAPIR in doxorubicin-induced cardiomyocyte ferroptosis and cardiomyopathy. METHODS: (1) Intraperitoneal injection of doxorubicin was used to induce cardiomyopathy in mice. The myocardium was in situ injected with CFAPIR knocking down lentivirus. The body mass and survival rate of mice were monitored and recorded; cardiac function, heart volume and mass, inflammation, and cardiac fibrosis were assessed. (2) Doxorubicin was used to induce ferroptosis in AC16 cardiomyocytes, and CFAPIR inhibitor was transfected into cells. Cell damage, ferroptosis (expression levels of ferroptosis markers, iron ion, malondialdehyde, and reduced glutathione content), and mitochondrial dysfunction were detected. The effect of CFAPIR on ABCB8 expression was also examined. RESULTS AND CONCLUSION: (1) CFAPIR levels were significantly upregulated in both doxorubicin-induced cardiomyopathy animal models (P < 0.0001) and cardiomyocyte ferroptosis models (P < 0.01). (2) In vivo, knockdown of CFAPIR significantly alleviated doxorubicin-induced cardiotoxicity, including inhibition of body weight loss (P < 0.05), improved survival rate, improved cardiac function (P < 0.01), reduced cardiac atrophy (P < 0.05), inhibited lactate dehydrogenase activity increase (P < 0.05), and reduced cardiac fibrosis (P < 0.0001). (3) In vitro, knockdown of CFAPIR significantly ameliorated doxorubicin-induced cardiomyocyte ferroptosis, manifested by increased cell viability (P < 0.05), decreased lactate dehydrogenase activity (P < 0.01), upregulated expression of ferroptosis markers xCT (P < 0.01) and glutathione peroxidase 4 (P < 0.001), downregulated mRNA level of prostaglandin-endoperoxide synthase 2 (P < 0.05), reduced iron overload (P < 0.05), decreased malondialdehyde content (P < 0.05), increased reduced glutathione content (P < 0.01), reduced reactive oxygen species accumulation (P < 0.01), and increased mitochondrial membrane potential (P < 0.05). (4) Knockdown of CFAPIR significantly attenuated the doxorubicin-induced decrease in iron transporter ABCB8 expression (P < 0.05). (5) These results indicate that CFAPIR levels are significantly upregulated in both animal models of doxorubicin-induced cardiomyopathy and cellular ferroptosis models, and knockdown of CFAPIR significantly improves doxorubicin-induced cardiotoxicity and cardiomyocyte ferroptosis, possibly by targeting mitochondrial iron transporter ABCB8.

1. Introduction

Cancer is a major public health challenge worldwide and the second leading cause of death after cardiovascular disease [1]. Although novel anticancer therapies such as molecular targeted therapy and immunotherapy have made significant clinical progress [2], chemotherapy remains a primary treatment modality. Doxorubicin, an anthracycline antibiotic, is one of the most commonly used broad-spectrum chemotherapeutic agents, effective against lymphomas, leukemias, Ewing's sarcoma, and solid tumors such as breast cancer [3]. However, doxorubicin treatment is associated with significant toxicities, including cardiotoxicity, hepatotoxicity, nephrotoxicity, myelosuppression, and chemotherapy-induced cognitive impairment [1]. Among these, cardiotoxicity is the most severe adverse effect, greatly limiting its clinical utility [4].

Doxorubicin induces both acute and chronic cardiotoxicity. Acute cardiotoxicity typically occurs within two to three days after chemotherapy, with an incidence of approximately 11%, manifesting as tachycardia, transient left ventricular dysfunction, premature beats, supraventricular arrhythmias, electrocardiographic abnormalities, and myopericarditis, and is generally considered reversible. Chronic cardiotoxicity manifests as cardiomyopathy, characterized by irreversible reduction in ejection fraction and heart failure, which may occur months (early-onset) or years (late-onset) after initial treatment. The cumulative dose of doxorubicin influences the incidence of chronic cardiotoxicity [5-8]. Currently, methods for detecting asymptomatic cardiotoxicity, such as serum biomarkers, electrocardiography, echocardiography, and cardiac magnetic resonance imaging, have been gradually applied in clinical practice. However, these conventional methods only yield positive results after myocardial damage has already occurred [1,9]. Therefore, to enable early intervention and improve patient outcomes, there is an urgent need to identify novel biomarkers and therapeutic targets for doxorubicin-induced cardiotoxicity.