LNA oligonucleotide mediates an anti-inflammatory effect in autoimmune myocarditis via targeting lactate dehydrogenase B

Abstract

Treatment of myocarditis is often limited to symptomatic treatment due to unknown pathomechanisms. In order to identify new therapeutic approaches, the contribution of locked nucleic acid antisense oligonucleotides (LNA ASOs) in autoimmune myocarditis was investigated. Hence, A/J mice were immunized with cardiac troponin I (TnI) to induce experimental autoimmune myocarditis (EAM) and treated with LNA ASOs. The results showed an unexpected anti-inflammatory effect for one administered LNA ASO MB_1114 by reducing cardiac inflammation and fibrosis. The target sequence of MB_1114 was identified as lactate dehydrogenase B (mLDHB). For further analysis, mice received mLdhb-specific GapmeR during induction of EAM. Here, mice receiving the mLdhb-specific GapmeR showed increased protein levels of cardiac mLDHB and a reduced cardiac inflammation and fibrosis. The effect of increased cardiac mLDHB protein level was associated with a downregulation of genes of reactive oxygen species (ROS)-associated proteins, indicating a reduction in ROS. Here, the suppression of murine pro-apoptotic Bcl-2-associated X protein (mBax) was also observed. In our study, an unexpected anti-inflammatory effect of LNA ASO MB_1114 and mLdhb-specific GapmeR during induction of EAM could be demonstrated in vivo. This effect was associated with increased protein levels of cardiac mLDHB, mBax suppression and reduced ROS activation. Thus, LDHB and LNA ASOs may be considered as a promising target for directed therapy of myocarditis. Nevertheless, further investigations are necessary to clarify the mechanism of action of anti-inflammatory LDHB-triggered effects.

Keywords: experimental autoimmune myocarditis; inflammation; lactate dehydrogenase B; troponin I.

FIGURE 1

LNA ASO MB_1114 showed an anti‐inflammatory effect in TnI‐induced EAM. (a) Myocardial inflammation of TnI‐immunized mice treated with LNA ASO mmu‐miR‐21a‐5p and LNA ASO mmu‐miR‐146b‐5p, as well as MB_1114 and solvent Ctrl. The following panels show the consolidated results of the initial and repeated experiment. (b, d) Myocardial inflammation and fibrosis of TnI‐immunized mice treated with MB_1114 compared to LNA Ctrl.‐ or solvent Ctrl.‐treated mice. (c) Representative macroscopic pictures (column 1) and histopathological exterminations of HE‐stained (columns 2, 3) and Afog‐stained (columns 4, 5) heart sections. (e) hsTnT serum level of TnI‐immunized mice treated with LNA ASO MB_1114 compared to LNA Ctrl.‐ and solvent Ctrl.‐treated mice. Data are displayed as mean ±SEM. Statistical analysis was performed using one‐way or two‐way ANOVA with the Bonferroni post hoc test. Values of P < 0·05 were considered statistically significant and marked by *P < 0·05, **P < 0·01, ***P < 0·001 and ****P < 0·0001

FIGURE 2

LNA ASO MB_1114 triggered an increased mLDHB protein level potentially mediating anti‐inflammatory in vivo effects. (a) Heart tissue was analysed by Western blot and fold increase in cardiac murine LDHB (mLDHB) of mice immunized with TnI or Control and treated with LNA ASO MB_1114 (n = 19), LNA Ctrl. (n = 10) or solvent Ctrl. (n = 19) normalized against GAPDH expression of TnI‐immunized mice treated with solvent Ctrl. Representative Western blot images of mLDHB protein expression. (b) Western blot analysis of cardiac murine LDHA (mLDHA) protein of different groups normalized against GAPDH expression of TnI‐immunized mice. Representative Western blot images of mLDHA protein expression. Data are displayed as mean ±SEM. Statistical analysis of comparison between groups was performed using two‐way ANOVA with the Bonferroni post hoc test. Values of P < 0·05 were considered statistically significant and marked by **P < 0·01, ***P < 0·001 and ****P < 0·0001

FIGURE 3

mLdhb‐specific GapmeR showed an anti‐inflammatory effect in EAM mice. (a, b) Inflammation and fibrosis of A/J mice, immunized with TnI or Control and treated with GapmeR (n = 9/group) or Ctrl. B (n = 9/group). (c) Representative macroscopic pictures (column 1) and histopathological exterminations of HE‐stained (columns 2, 3) and Afog‐stained (columns 4, 5) heart sections. (d) hsTnT levels in serum of immunized mice treated with GapmeR and Ctrl. B. (e) Heart weight/body weight ratio of examined groups. Data are displayed as mean ±SEM. Statistical analysis of comparison between groups was performed using two‐way ANOVA with the Bonferroni post hoc test. Values of P < 0·05 were considered statistically significant and marked by *P < 0·05, **P < 0·01, ***P < 0·001 and ****P < 0·0001

FIGURE 4

mLdhb‐specific GapmeR‐triggered increased levels of cardiac mLDHB protein expression. (a) Western blot analysis of cardiac murine LDHB (mLDHB) protein expression of A/J mice immunized with TnI or Control and treated with GapmeR (n = 9/group) or Ctrl. B (n = 9/group). mLDHB protein expression was normalized against GAPDH in TnI‐immunized mice treated with Ctrl. B. Representative Western blot images of mLDHB. (b) Western Blot analysis of cardiac murine LDHA (mLDHA) protein level. Protein expression was normalized against GAPDH of Control‐immunized mice treated with Ctrl. B. Representative Western blot images of mLDHA. Data are displayed as mean ±SEM. Statistical analysis of comparison between groups was performed using two‐way ANOVA with the Bonferroni post hoc test. Values of P < 0·05 were considered statistically significant and marked by ***P < 0·001 and ****P < 0·0001

FIGURE 5

mLdhb‐specific GapmeR administration reduced pro‐inflammatory cytokines and led to reduced cardiac mIL‐1α levels in EAM mice. Cytokine levels from supernatant of TnI‐stimulated spleen cell homogenate. (a) mIL‐1β, (b) mIL‐6, (c) mTNF‐α, (d) mIL‐2 and (e) mIL‐17 were analysed using ELISA. Myocardial mRNA expression of (f) mIl‐1α was analysed by qPCR from mice, immunized with TnI or Control and treated with GapmeR (n = 9/group) or Ctrl. B (n = 9/group). qPCR data were normalized against mL32 in Control‐immunized mice treated with Ctrl. B. Data are displayed as mean ±SEM. Statistical analysis of comparison between groups was performed using two‐way ANOVA with the Bonferroni post hoc test. Values of P < 0·05 were considered statistically significant and marked by *P < 0·05, **P < 0·01, ***P < 0·001 and ****P < 0·0001

FIGURE 6

mLdhb‐specific GapmeR‐treated mice showed reduced cardiac levels of ROS‐associated proteins. qPCR analysis of ROS‐associated proteins. (a) mp47 phox, (b) mp67 phox, (c) mNox2, (d) mUcp2 and (e) mSod2 in hearts of mice immunized with TnI or Control and treated with GapmeR (n = 9/group) or Ctrl. B (n = 9/group). mRNA levels were normalized against mL32 and the corresponding mRNA expression level of Control‐immunized mice treated with Ctrl. B. Data are displayed as mean ±SEM. Statistical analysis of comparison between groups was performed using two‐way ANOVA with the Bonferroni post hoc test. Values of P < 0·05 were considered statistically significant and marked by *P < 0·05, **P < 0·01 and ***P < 0·001

FIGURE 7

mLdhb‐specific GapmeR‐treated mice showed reduced protein levels of mBax. Western blot analysis of cardiac mBax protein normalized against GAPDH and the mBax protein level of Control‐immunized mice treated with Ctrl. B. Representative Western blot images of mBax. Data are displayed as mean ±SEM. Statistical analysis of comparison between groups was performed using two‐way ANOVA with the Bonferroni post hoc test. Values of P < 0·05 were considered statistically significant and marked by *P < 0·05 and **P < 0·01