NLRP3 Inflammasome Inhibition Reduces Ischemia/Reperfusion Injury in Female but not Male Rats

Background: Myocardial infarction (MI), or “heart attack”, remains a leading cause of death worldwide. MI is caused by prolonged coronary artery ischemia (I; blockage of blood flow) leading to cell death. While reperfusion (R) is therapeutic, continued activation of cell death pathways through distinct and overlapping mechanisms exacerbates damage beyond that due to I. The NLR family pyrin domain containing 3 (NLRP3) inflammasome is activated during I/R, leading to pyroptosis. However, NLRP3 inhibition beyond 2 hr R fails to reduce MI size. Therefore, we sought to determine whether limiting NLRP3 priming prior to I is sufficient to reduce I/R injury and MI size. Purpose: To investigate the protective efficacy of NLRP3 gene knockout (KO) and loss of NLRP3 priming on MI size in male vs female rats. Methods: NLRP3 KO DAHL/SS- Nlrp3 em2Mcwi rats were generated via CRISPR-Cas9 (14 base pair deletion in exon 1 of NLRP3 gene) in Dahl salt sensitive rats. Wildtype (WT) and NLRP3 KO rats received Teklad 7034 low salt diet (0.12% NaCl) ad libitum to delay the development of hypertension. In-vivo coronary artery ligation (CAL; 31 min I) was used to induce MI in male (n=7) and female (n=13) WT (n=8) and NLRP3 KO (n=12) rats aged 90-135 days, with sham surgery as a non-ischemic control. Cardiac puncture was used to collect blood following 3 hr R to assess IL-18 levels and complete blood count (CBC). The left ventricle was harvested to determine area at risk (AAR) and infarct size using Evans Blue dye perfusion and 2,3,5-Triphenyltetrazolium chloride (TTC) staining, respectively. AAR and infarct size quantification was conducted using ImageJ, with data analyzed via 2-way ANOVA (Minitab). Data were presented with mean ± SEM. Results: Left ventricle weight (LVW)/right tibia length (RTL) was increased in NLRP3KO (male: 246.41 ± 5.51mg/cm; female: 201.98 ± 4.49 mg/cm) vs. WT (male: 233.26 ± 4.34 mg/cm; female: 191.11 ± 3.69 mg/cm; p < 0.05). Surprisingly, baseline plasma IL-18 levels were elevated in NLRP3 KO (male: 21.54 ± 2.00 pg/mL; female: 19.36 ± 1.80 pg/mL) vs. WT (male: 9.06 ± 2.01 pg/mL; female: 5.13 ± 1.55 pg/mL; p < 0.05). Following MI, NLRP3 KO rats had lower IL-18 levels (male: 148.26 ± 16.81 pg/mL; female: 170.18 ± 59.04 pg/mL) vs. WT (male: 252.01 ± 30.92 pg/mL; female: 330.39 ± 51.18 pg/mL; p < 0.05). Infarct size was reduced in NLRP3 KO vs WT females (KO vs. WT: 26.27% vs. 19.08%), but not males (KO vs. WT: 27.72% vs. 27.41%). Conclusion: IL-18 levels were reduced in NLRP3 KO groups with I/R as expected. The absence of NLPR3 may be cardioprotective in female but not male rats. Elevated IL-18 levels at baseline in NLRP3 KO require further investigation, but may suggest dysregulated Interferon-γ expression and/or alternative inflammasome activation, such as AIM2. Understanding divergent roles of NLRP3 inflammasome activation may inform sex-specific therapeutic interventions for the treatment of cardiac inflammation and MI. Our data also suggest a possible direct role for NLRP3 in the regulation of cardiac growth. This work was supported by NIH R21 AG070723-01 (DHK) and NIH training program T32GM108563 and T32GM154124. Animal protocols were approved by the Penn State IACUC #43534. This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.