Subthreshold Thermal Stress Aggravates Methamphetamine-Induced Cardiomyocyte Pyroptosis via the Mitochondrial ROS/BAX/mtDNA/NLRP3 Pathway

Figures

• 

  Figure 1. Subthreshold thermal stress exacerbates methamphetamine-induced cardiac injury. (A) Experimental timeline of drug administration and cardiac functional assessments. Panels show core temperature measurements ((B); n = 10), myocardial cTnI level ((C); n = 6), representative image of echocardiography and cardiac function indicators ((D,E); n = 6, the green and red dotted lines respectively represent the end-diastolic and end-systolic inner diameters of the left ventricle), and representative HE staining ((F); scale bar = 50 μm) in the NC-Sal, STS-Sal groups, NC-METH and STS-METH groups. (G,H) CCK8 assays under varying concentrations of METH (0.05–1.5 mM), normal (37 °C) and high (39 °C) ambient temperatures, and different treatment time (n = 3, the dotted lines represent that the cell survival rate is 75%). Representative images of bubble-like protrusions under light microscopy ((I); scale bar = 20 μm) in H9c2 cells exposed to METH. Data presented as mean ± SEM. */# p < 0.05; **/## p < 0.01; ***/### p < 0.001; ns, not significant.

• 

  Figure 2. METH/STS induces cardiomyocyte pyroptosis and inflammatory injury in vivo. (A,B) GO and KEGG analyses between the STS-Sal and STS-METH groups. The heatmap ((C); n = 3) for visualizing the mRNA levels of the pyroptosis-related genes, Western blot analysis ((D,E); n = 4–6) of pyroptosis effector proteins expression under different conditions. Western blot analysis of pyroptosis effector proteins expression ((F,G); n = 5–6), representative graphs of echocardiography and cardiac function indicators ((H,I); n = 6, the green and red dotted lines respectively represent the end-diastolic and end-systolic inner diameters of the left ventricle), myocardial cTnI levels ((J); n = 6) after METH/STS exposure with or without pyroptosis inhibitor pretreatment (VX765 or DSF). Data presented as mean ± SEM. */# p < 0.05; **/## p < 0.01; ***/### p < 0.001; ns, not significant.

• 

  Figure 3. METH/STS triggers cardiomyocyte pyroptosis via mtDNA escape. Representative TEM images ((A), scale bar = 1 or 2 μm), quantitative analysis of cytosol Cyt C (B) and mtDNA content (C) exposed to METH and/or high ambient temperature (n = 6). Quantitative analysis of mtDNA content ((D); n = 6), myocardial cTnI level ((E); n = 6), representative graphs of echocardiography and cardiac function indicators ((F,G); n = 6, the green and red dotted lines respectively represent the end-diastolic and end-systolic inner diameters of the left ventricle), myocardial IL-1β level ((H); n = 6), pyroptosis effector proteins expression ((I,J); n = 5), and representative HE staining ((K); scale bar = 50 μm) under METH/STS co-exposure after ddc pretreatment (25 or 50 mg/kg). Data presented as mean ± SEM. */# p < 0.05; **/## p < 0.01; ***/### p < 0.001; ns, not significant.

• 

  Figure 4. METH/STS activates cardiomyocyte pyroptosis via BAX-mediated mtDNA escape. Heatmaps for visualizing the mRNA levels of the pyroptosis-related genes exposed to METH and/or high ambient temperature ((A); n = 3). Immunoblotting analysis of BCL-2-related protein expression exposed to METH and/or high ambient temperature ((B,C); n = 5). Immunoblotting analysis of BCL-2-related protein expression ((D,E); n = 5), myocardial cTnI level ((F); n = 6), representative graphs of echocardiography and cardiac function indicators ((G,H); n = 6, the green and red dotted lines respectively represent the end-diastolic and end-systolic inner diameters of the left ventricle), myocardial IL-1β level ((I); n = 6), pyroptosis effector protein expression ((J,K); n = 5–6), and representative HE staining ((L); scale bar = 50 μm) after METH/STS exposure with or without Bip V5 pretreatment. Data presented as mean ± SEM. */# p < 0.05; **/## p < 0.01; ***/### p < 0.001; ns, not significant.

• 

  Figure 5. METH/STS inhibits mitochondrial respiratory chain complex I activity and induces oxidative stress. The heatmap for visualizing ((A); n = 3) and the relative mRNA expression levels ((B); n = 6) of the mitochondrial complex I-related genes (Ndufv2/Ndufs1/Ndufs4), and quantitative analysis of complex I activity, MDA, SOD and ATP levels exposed to METH and/or STS ((C–E); n = 6 or 8). Quantitative analysis of complex I activity ((F); n = 9), representative graphs and fluorescence intensity of cellular ROS ((G,H); n = 9) and mtROS ((I,J); n = 9), and ATP levels ((K); n = 12) in METH/STS-challenge H9c2 cells. Data presented as mean ± SEM. */# p < 0.05; **/## p < 0.01; ***/### p < 0.001; ns, not significant.

• 

  Figure 6. MitoQ ameliorates METH/STS-induced cardiomyocyte pyroptosis by inhibiting ROS/BAX/mtDNA escape. Representative graphs and fluorescence intensity of mtROS ((A,B); n = 9), cell viability ((C); n = 3), BAX/BCL-2 expression ((D,E); n = 4), cytosol mtDNA content ((F); n = 6) and pyroptosis effector proteins expression ((D,G); n = 3) in METH/STS-challenge H9c2 cells with/without MitoQ intervention. Representative graphs of echocardiography and cardiac function indicators ((H,I); n = 6, the green and red dotted lines respectively represent the end-diastolic and end-systolic inner diameters of the left ventricle), myocardial cTnI level ((J); n = 6), BAX/BCL-2 expression ((K,L); n = 3), cytosol mtDNA content ((M); n = 6), pyroptosis effector proteins expression ((K,N); n = 3), and IL-1β ((O); n = 6) after METH/STS exposure with or without mitoQ intervention. Data presented as mean ± SEM. * p < 0.05; **/## p < 0.01; *** p < 0.001; ns, not significant.

• 

  Figure 7. Subthreshold Thermal Stress Aggravates Methamphetamine-Induced Cardiomyocyte Pyroptosis via the Mitochondrial ROS/BAX/mtDNA/NLRP3 Pathway. The graphical abstract provides an overview of the core molecular pathways involved in METH/STS causing cardiac damage. METH/STS exposure induces a burst of mitochondrial ROS by inhibiting mitochondrial respiratory chain complex I, activates BAX and causes mtDNA leakage into the cytoplasm, and therefore activates the NLRP3 inflammasome and caspase-1/GSDMD-dependent pyroptosis, while MitoQ reversed METH/STS-induced myocardial injury by alleviating oxidative stress. The graphical abstract was created using BioRender.com.