High fat diet-induced obesity increases myocardial injury and alters cardiac STAT3 signaling in mice after polymicrobial sepsis

Abstract

Little is known about how obesity affects the heart during sepsis and we sought to investigate the obesity-induced cardiac effects that occur during polymicrobial sepsis. Six-week old C57BL/6 mice were randomized to a high fat (HFD) (60% kcal fat) or normal diet (ND) (16% kcal fat). After 6weeks of feeding, mice were anesthetized with isoflurane and polymicrobial sepsis was induced by cecal ligation and puncture (CLP). Plasma and cardiac tissue were obtained for analysis. Echocardiography was performed on a separate cohort of mice at 0 and 18h after CLP. Following 6-weeks of dietary intervention, plasma cardiac troponin I was elevated in obese mice at baseline compared to non-obese mice but troponin increased only in non-obese septic mice. IL-17a expression was 27-fold higher in obese septic mice versus non-obese septic mice. Cardiac phosphorylation of STAT3 at Ser727 was increased at baseline in obese mice and increased further only in obese septic mice. Phosphorylation of STAT3 at Tyr705 was similar in both groups at baseline and increased after sepsis. SOCS3, a downstream protein and negative regulator of STAT3, was elevated in obese mice at baseline compared to non-obese mice. After sepsis non-obese mice had an increase in SOCS3 expression that was not observed in obese mice. Taken together, we show that obesity affects cardiac function and leads to cardiac injury. Furthermore, myocardial injury in obese mice during sepsis may occur through alteration of the STAT3 pathway.

Keywords: Cardiac inflammation; Obesity; STAT3; Sepsis.

Figure 1. EchoMRI to determine body composition of mice fed normal and high-fat diets for 6 weeks

Changes in body fat, lean mass, free water, total water and total weight prior to and after dietary intervention. Values are means ± SEM. White bars=non-obese mice fed normal diet, Black bars=obese mice fed high-fat diet. n=12–13/group. *p<0.05 compared to normal diet.

Figure 2. Plasma levels of troponin I and myeloperoxidase activity in obese and non-obese mice subjected to polymicrobial sepsis

(A) Plasma cardiac troponin I and (B) cardiac neutrophil infiltration as determined by myeloperoxidase assay (MPO), white bars/boxes=non-obese mice, grey bars/boxes=obese mice. *p<0.05 vs. time 0, #p<0.05 vs. non-obese mice by 2-way ANOVA. n=4/group for MPO and n=6–8/group for troponin.

Figure 3. Cardiac gene expression in obese and non-obese mice after sepsis

Gene expression profile of 90 genes related to IL-6/STAT3 signaling were evaluated at 6h after CLP in obese and non-obese mice. (A) Center dots represent unchanged gene expression. Boundary lines indicate 4-fold gene regulation cut-off between septic obese and septic non-obese samples (red line, 4-fold upregulation; green line 4-fold downregulation). IL-17a, Csf2 and IL-10 demonstrate differences that represent more than a doubling (log 2 changes of >1) and that were statistically significant at P≤0.05. (B) Relative expression of IL-17a, Csf2 and IL-10 in obese septic mice normalized to non-obese septic mice. Grey bar = obese septic mice, White bar = non-obese septic mice. Dashed line is placed at 1 for reference. n=4 mice/group.

Figure 4. Phosphorylation of cardiac STAT3

(A) changes in phosphorylation of STAT3 at serine 727 and (B) tyrosine 705, total STAT3 and actin expression in heart nuclear extracts by Western blot at 0 and 6h after CLP. Densitometric analysis of (C) pSTAT3 (Ser727) to STAT3 to Actin ratio log transformed and (D) pSTAT3 (Tyr705) to STAT3 to Actin ratio. *p<0.05 vs. time 0, #p<0.05 vs. non-obese mice by 2-way ANOVA. White bars=non-obese, Black bars=obese. n=3-4mice/group for densitometric analysis.

Figure 5. Cardiac expression of SOCS3

(A) Expression of SOCS3 and actin in heart nuclear extracts by Western blot analysis at 0 and 6h after CLP. (B) Densitometric analysis of SOCS to actin ratio. *p<0.05 vs. time 0, #p<0.05 vs non-obese group by 2-way ANOVA. White bars=non-obese, Black bars=obese. n=3–4mice/group for densitometric analysis.

Figure 6. Cardiac expression of gp130

(A) Expression of gp130 and actin in heart cytosol extracts by Western blot analysis at 0 and 6h after CLP. (B) Densitometric analysis of gp130 to actin ratio. #p<0.05 vs non-obese group by 2-way ANOVA. White bars=non-obese, Black bars=obese. n=3–4mice/group for densitometric analysis.