2905 - NLRP3 Inflammasome Activation by mtROS in Cardiomyocytes and Radiation Induced Acute Myocardial Injury

Y. Wu¹, J. Wang¹, S. Wang¹, Y. Zhou², and N. Zhang³; ¹Department of Radiation Oncology, the Fourth Hospital of Hebei Medical University, Shijiazhuang, China, ²Hebei General Hospital, Shijiazhuang, China, ³Shijiazhuang People’s Hospital, Shijiazhuang, China

Purpose/Objective(s): Inflammation and subsequent irreversible cardiac fibrosis are crucial mechanisms of radiation-induced heart disease. The NLRP3 inflammasome mediates caspase-1 activation, following interleukin-1ß and interleukin-18 release in macrophages or immune cells, but its role in cardiomyocytes (CMs) remains unknown. Mitochondrial ROS (mtROS), as one of the damage-associated molecular patterns, triggers the activation of the NLRP3 inflammasome. This study assesses the role of mtROS-enhanced NLRP3 inflammasome in cardiomyocytes, directly promoting radiation-induced myocardial injury. Materials/

Methods: In this study, AC16 human cardiomyocytes were irradiated with a single fraction of 0 Gy, 2 Gy, 6 Gy, 10 Gy, and 16 Gy. Seahorse XF96 was used to examine mitochondrial function. Cell mitochondrial membrane potential and ROS levels were quantitatively analyzed by flow cytometry. Cardiac irradiation was delivered to a mouse model with 6-MV X-ray at single fractions of 20 Gy, 30 Gy, and 45 Gy in three weekly fractions. Cardiac damage was detected on the 14th, 28th, 90th, and 150th day after 20 Gy irradiation. Mortality, transthoracic echocardiography, histological analyses with HE & Masson staining, and transmission electron microscopy were performed to characterize mitochondrial ultrastructure in AC16 human cardiomyocytes and myocardial tissue. NLRP3 inflammasome activation was assessed by western blot (WB) and immunofluorescence (IF).
Results: Cell viability was inhibited after irradiation at 6 Gy, 10 Gy, and 16 Gy, decreasing at 24 h, 48 h, and 72 h with 10 Gy (P < 0.001). Proton leak significantly increased following 10 Gy irradiation at 48 h. Mitochondrial membrane potential significantly decreased (P < 0.05 for all), and ROS increased at 24 h, 48 h, and 72 h after 10 Gy irradiation. Acute mortality was 80% within three weeks after 30 Gy irradiation, compared with 0% in the other groups. On the 21st day, a reduced left ventricular ejection fraction (LVEF) was observed after 45 Gy irradiation in three weekly fractions, while no significant change was noted with 20 Gy. Cardiac troponin I, a marker of myocardial injury, increased significantly in a dose- and time-dependent manner. Histological damage and collagen deposition following irradiation were mainly observed in the endocardium. Transmission electron microscopy revealed decreased electron density, crista fusion and rupture, and vacuolization in AC16 cardiomyocytes at 48 h after 10 Gy, and in mouse heart on the 28th and 150th day after 20 Gy. Meanwhile, after irradiation, NLRP3 inflammasome activity increased in AC16 cardiomyocytes and mouse cardiac tissue, as evidenced by WB and IF images. Consistently, the levels of IL-1ß and IL-18 in cell culture supernates and mouse serum were elevated after irradiation.
Conclusion: In vivo and in vitro, our study explores a novel role of mtROS-enhanced NLRP3 inflammasome in cardiomyocytes, with a mechanistic link to the pathogenesis of irradiation induced myocardial injury.