B-type natriuretic peptide is upregulated by c-Jun N-terminal kinase and contributes to septic hypotension

Figures

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  Figure 1. Genetic deletion of the Nppb gene delays hypotension in septic mice. (A) Experimental outline for CLP surgery in mice with genetic Nppb deletion and WT controls. (B–D) BNP mRNA (B), plasma BNP (C), plasma cGMP (D), and kidney cGMP (E) in BNP-KO mice 12 hours after CLP surgery. n = 4 WT, n = 3 BNP-KO. *P < 0.05, **P < 0.01 versus WT by t test. (F) Representative parasternal long-axis view and tail-cuff blood pressure trace in BNP-KO mice and matched controls following CLP. (G–K) End-diastolic volume (G), global longitudinal strain (H), cardiac output normalized to body weight (I), mean arterial pressure (J), and xiphoidal surface temperature (K) at baseline, 6 hours after CLP, and 12 hours after CLP. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus WT by 2-way ANOVA for repeated measures with Sidak multiple comparisons. (L) Plasma lactate in BNP-KO mice 12 hours after CLP. n = 4, WT; n = 3, BNP-KO. **P < 0.01 versus WT by t test between treatments.

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  Figure 2. BNP neutralization via 19B3 reverses low preload, increases cardiac output, and protects against hypotension. (A) Experimental outline for CLP surgery in mice followed by administration of 19B3 (50 mg/kg) or IgG (50 mg/kg) 6 hours after CLP. (B–E) BNP mRNA (B), plasma BNP (C), plasma cGMP (D), and kidney cGMP (E) in BNP-KO mice 12 hours after CLP. (F and G) Representative parasternal long-axis view (F) and tail-cuff blood pressure trace (G) in mice following sham or CLP surgery, followed by administration of 19B3 (50 mg/kg) or IgG. (H–M) End-diastolic volume (H), global longitudinal strain (I), cardiac output normalized to body weight (J), mean arterial pressure (K), surface temperature (L), and plasma lactate (M) in mice 12 hours after CLP and 6 hours after 19B3 administration. n = 5, Sham + IgG; n = 5, Sham + 19B3; n = 8, CLP + IgG; n = 8, CLP + 19B3. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus Sham + IgG; ##P < 0.01, ###P < 0.001, ####P < 0.0001 versus Sham + 19B3; $P < 0.05, $$P < 0.01, $$$$P < 0.0001 versus CLP + IgG by 2-way ANOVA with Tukey multiple comparisons between treatments.

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  Figure 3. Activation of JNK signaling increases BNP production. (A) BNP mRNA levels in AC16 cardiomyocytes stimulated with 100 ng/mL or 1 μg/mL LPS for 8 hours. n = 4, control (Ctrl) wells; n = 4, LPS (100 ng/mL) wells; n = 4, LPS(1 μg/mL) wells. *P < 0.05 versus Ctrl by 1-way ANOVA with Tukey multiple comparisons. (B) BNP mRNA levels in AC16 cardiomyocytes stimulated with 1 μg/mL LPS and SP600125 (100nM) for 8 hours. n = 5, Ctrl wells; n = 6, LPS wells; n = 5, LPS + SP600125 wells. **P < 0.01 versus Ctrl, ##P < 0.01 versus LPS by 1-way ANOVA with Tukey multiple comparisons. (C) BNP mRNA levels in AC16 cells infected with Ad-JNK2α2 (MOI, 10; 48 hours). n = 4, Ad-GFP wells; n = 5, Ad-JNK2α2 wells. *P < 0.05 versus Ad-GFP by t test. (D and E) BNP mRNA levels in AC16 cardiomyocytes infected with Ad–c-JunASP (D) or Ad–c-JunALA (E). For D, n = 4, Ad-GFP wells; n = 8, Ad–c-JunASP wells. For E, n = 11, Ad-GFP wells; n = 9, Ad–c-JunALA wells. *P < 0.05, ***P < 0.001 versus Ad-GFP by t test. (F) Schematic representation of the mouse Nppb and human NPPB promoters showing a conserved predicted AP1 motif located –400 bp upstream of the transcription start site, highlighted in yellow. (G) Enrichment of…

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  Figure 4. Cardiac JNK signaling regulates BNP in septic mice. (A) Experimental design for mice included in the study. (B–D) Representative immunoblot (B) and densitometric quantification of phospho–c-Jun at Ser63 (C) and phospho–c-Jun at Ser73 (D). Two separate Western blots with 3 replicates per group were run for a total of n = 6 mice/group. ***P < 0.001 versus Sham, ##P < 0.01 versus CLP + DMSO. (E–G) Cardiac BNP mRNA levels (E), plasma BNP concentration (F), and plasma cGMP levels (G) in mice that underwent CLP or sham surgery followed by administration of JNK inhibitor SP600125 (5 mg/kg) or vehicle (DMSO) 6 hours after CLP. (H) Kidney cGMP levels normalized to kidney weight in mice that underwent CLP or sham surgery with administration of DMSO or JNKi 6 hours after surgery. n = 8, Sham + DMSO; n = 4, Sham + JNKi; n = 8, CLP + DMSO; n = 8, CLP + JNKi. **P < 0.01, ****P < 0.0001 versus Sham + DMSO; ##P < 0.01, ####P < 0.0001 versus Sham + JNKi; $P < 0.05, $$P < 0.01, $$$$P < 0.0001 versus CLP + DMSO by one-way ANOVA with Tukey multiple comparisons.

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  Figure 5. Pharmacological JNK inhibition increases preload and cardiac output and reverses septic hypotension. (A and B) Representative parasternal long-axis view (A) and tail-cuff blood pressure trace (B) in mice following sham or CLP surgery followed by administration of SP600125 ( 5mg/kg) or DMSO. (C–G) End-diastolic volume (C), global longitudinal strain (D), cardiac output normalized to body weight (E), mean arterial pressure (F), and xiphoidal surface temperature (G) at baseline, 6 hours after CLP prior to drug/vehicle administration, and 12 hours after CLP and 6 hours after drug/vehicle administration. n = 8, Sham + DMSO; n = 4, Sham + JNKi; n = 8, CLP + DMSO; n = 8, CLP + JNKi. *P < 0.05, ***P < 0.001, ****P < 0.0001 versus Sham + DMSO; ##P < 0.01, ###P < 0.001, ####P < 0.0001 versus Sham + JNKi; $P < 0.05, $$$P < 0.001, $$$$P < 0.0001 versus CLP + DMSO by 1-way ANOVA with Tukey multiple comparisons. (H) Plasma lactate in mice 12 hours after CLP and 6 hours after drug/vehicle administration at the time of euthanasia. ****P < 0.0001 versus Sham + DMSO; ###P < 0.001, ####P < 0.0001 versus Sham + JNKi; $$$$P < 0.0001 versus CLP + JNKi by 2-way ANOVA with Tukey multiple comparisons…

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  Figure 6. Antibody 19B3 and SP600125 administration increases survival and body temperature in male and female mice treated with antibiotics and fluid. Forty male and 40 female mice were subjected to CLP and assessed for mortality every 12 hours. Mice were allocated beginning 6 hours after CLP to receive a single dose of IgG or 19B3 and a single daily dose of DMSO or SP600125 with saline and ertapenem (70 mg/kg). (A–C) Survival plot assessing 72-hour survival in male mice (A), female mice (B), or mixed-sex (C) cohorts. *P < 0.05, **P < 0.01, ****P < 0.0001 versus CLP + Veh. (D–F) Body surface temperature in male (D), female (E), and combined sex (F) cohorts of mice followed for survival and 6, 24, and 48 hours after CLP. n = 15, CLP + Veh male mice; n = 15, CLP + 19B3 male mice; n = 10, CLP + JNKi male mice; n = 15, CLP + Veh female mice; n = 15, CLP + 19B3 female mice; n = 10, CLP + JNKi female mice. ***P < 0.001, ****P < 0.0001 versus CLP + Veh; #P < 0.05, ##P < 0.01 versus CLP + 19B3 by 2-way ANOVA with Tukey multiple comparisons between treatments.

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  Figure 7. Central illustration. Activation of cardiac JNK in sepsis increases Nppb expression and BNP production and causes cardiac systolic dysfunction, both of which contribute to reduced cardiac output and hypotension.