Hypoxia evokes increased PDI and PDIA6 expression in the infarcted myocardium of ex-germ-free and conventionally raised mice

Abstract

The prototypic protein disulfide isomerase (PDI), encoded by the P4HB gene, has been described as a survival factor in ischemic cardiomyopathy. However, the role of protein disulfide isomerase associated 6 (PDIA6) under hypoxic conditions in the myocardium remains enigmatic, and it is unknown whether the gut microbiota influences the expression of PDI and PDIA6 under conditions of acute myocardial infarction. Here, we revealed that, in addition to the prototypic PDI, the PDI family member PDIA6, a regulator of the unfolded protein response, is upregulated in the mouse cardiomyocyte cell line HL-1 when cultured under hypoxia. In vivo, in the left anterior descending artery (LAD) ligation mouse model of acute myocardial infarction, similar to PDI, PDIA6 protein expression was enhanced in the infarcted area (LAD+) relative to uninfarcted sham tissue or the neighbouring area at risk (LAD-) of C57BL/6J mice. Interestingly, we found that ex-germ-free (ex-GF) mice subjected to the LAD ligation model for 24 h had a reduced ejection fraction compared with their conventionally raised (CONV-R) SPF controls. Furthermore, the LAD+ area in the infarcted heart of ex-GF mice showed reduced PDIA6 expression relative to CONV-R controls, suggesting that the presence of a gut microbiota enhanced LAD ligation-triggered PDIA6 expression. Collectively, our results demonstrate that PDIA6 is upregulated in cardiomyocytes as a consequence of hypoxia. In the LAD mouse model, PDIA6 was also increased in the infarcted area under in vivo conditions, but this increase was suppressed in ex-GF mice relative to CONV-R controls.This article has an associated First Person interview with the first author of the paper.

Keywords: Germ-free; HL-1 cardiomyocytes; Hypoxia; Left anterior descending artery ligation; PDI; PDIA6.

Fig. 1.

Hypoxia-induced upregulation of HIF1α, PDI and PDIA6 in HL-1 cardiomyocytic cells. (A) Hypoxia-dependent (1% O₂) increase in HIF1α protein expression relative to β-actin (n=3, 24 h incubation). (B,C) Hypoxia-dependent (1% O₂) increase in PDI (n=4, 24 h incubation) (B) and PDIA6 (n=3, 12 h incubation) (C) protein levels relative to α-actinin. Normoxia, black bars; hypoxia, grey bars. All data are expressed as mean±s.e.m. Statistical comparisons were performed using the Student's t-test; *P<0.05, ****P<0.0001.

Fig. 2.

The expression of PDI and PDIA6 is increased in the infarcted area (LAD+) in the 24 h LAD ligation mouse model. (A) Long axis (n=6,9) and short axis (n=6,8) of the left ventricle after LAD ligation; SPF C57BL/6J mouse. (B) mRNA expression of the myocardial hypoxia marker PHD3 is increased relative to L32 in the LAD+ area compared with sham or LAD– tissues (n=4,5,5). (C) PDI and PDIA6 mRNA expression relative to L32 is increased in the LAD+ area compared with sham or LAD– tissues (n=5). (D,E) PDI protein levels are increased relative to α-actinin in the LAD+ (n=6) and LAD– (n=6) tissues compared to sham-operated heart tissue (n=5) (western blot) (D) and in plasma (n=6,8) (ELISA) (E). (F) PDIA6 protein levels are increased in LAD+ heart tissue relative to sham or LAD– tissues (ELISA) (n=5). (G) Immunohistochemistry for PDI (top row) and PDIA6 (bottom row) in sham-operated and infarcted LAD+ heart tissue (representative micrographs). Scale bars: 100 µm. LAD+ represents the ligated areas, LAD– represents the area proximal to ligation and sham represents the sham-operated animals. All data are expressed as mean±s.e.m. Statistical comparisons were performed using the Student's t-test and one-way ANOVA; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 3.

24 h LAD ligation model of acute myocardial infarction in germ-free mice. (A) Long axis (n=6,9) and short axis (n=6,8) of the left ventricle after LAD ligation of ex-germ-free (ex-GF) C57BL/6J mice as determined by ultrasound imaging. (B) Ejection fraction % of the long (n=6,3) and short axis (n=6,3) of sham-operated C57BL/6J mice kept under SPF conventionally raised (CONV-R) or ex-GF conditions. (C) Comparison of the long axis (n=9,9) and the short axis (n=8,8) of CONV-R SPF C57BL/6J mice, with ex-GF C57BL/6J mice normalised to sham-operated controls. (D) PDI (n=6,4,6) and PDIA6 (n=6,6,6) mRNA expression in sham-operated, LAD+ and LAD– heart tissues of ex-GF mice. (E) PDI and PDIA6 mRNA expression in heart tissues of sham-operated ex-GF mice compared to SPF CONV-R mice (n=6,3). (F) PDI (n=6) and PDIA6 (n=5) mRNA expression in LAD+ heart tissues of ex-GF mice compared to SPF CONVR mice. LAD+ represents the ligated area, LAD– represents the area proximal to ligation and sham represents the sham-operated animals. All data are expressed as mean±s.e.m. Statistical comparisons were performed using the Student's t-test or one-way ANOVA; n.s., not significant; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.