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Reducing brain TACE activity improves neuroinflammation and cardiac function in heart failure rats



doi: 10.3389/fphys.2022.1052304.


eCollection 2022.

Affiliations

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Yang Yu et al.


Front Physiol.


.

Abstract

Tumor necrosis factor (TNF)-α converting enzyme (TACE) is a key metalloprotease mediating ectodomain shedding of a variety of inflammatory mediators, substrates, and growth factors. We previously reported that TACE-mediated production of TNF-α in the hypothalamic paraventricular nucleus (PVN) contributes to sympathetic excitation in heart failure (HF). Here, we sought to determine whether central interventions in TACE activity attenuate neuroinflammation and improve cardiac function in heart failure. Myocardial infarction-induced HF or sham-operated (SHAM) rats were treated with bilateral paraventricular nucleus microinjection of a TACE siRNA or a 4-week intracerebroventricular (ICV) infusion of the TACE inhibitor TAPI-0. Compared with SHAM rats, scrambled siRNA-treated HF rats had higher TACE levels in the PVN along with increased mRNA levels of TNF-α, TNF-α receptor 1 and cyclooxygenase-2. The protein levels of TNF-α in cerebrospinal fluid and phosphorylated (p-) NF-κB p65 and extracellular signal-regulated protein kinase (ERK)1/2 in the PVN were also elevated in HF rats treated with scrambled siRNA. The expression of these inflammatory mediators and signaling molecules in the PVN of HF rats were significantly attenuated by TACE siRNA. Interestingly, the mRNA level of TNF-α receptor 2 in the PVN was increased in HF treated with TACE siRNA. Moreover, sympathetic excitation, left ventricular end-diastolic pressure, pulmonary congestion, and cardiac hypertrophy and fibrosis were reduced by PVN microinjection of TACE siRNA. A 4-week treatment with intracerebroventricular TAPI-0 had similar effects to ameliorate these variables in HF rats. These data indicate that interventions suppressing TACE activity in the brain mitigate neuroinflammation, sympathetic activation and cardiac dysfunction in HF rats.


Keywords:

ADAM17; hypothalamic paraventricular nucleus; inflammation; myocardial infarction; sympathetic nervous system; tumor necrosis factor-α.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures



FIGURE 1

Schematic showing the experimental procedures in protocol I (A) and Protocol II (B).


FIGURE 2


FIGURE 2

Validation of PVN transfection and TACE knockdown. (A): Expression of green fluorescent protein (GFP) in the PVN in a normal rat 1 week after bilateral PVN microinjections of an adeno-associated virus (AAV) vector encoding TACE siRNA and GFP and a control rat without treatment with AAV vector. GFP was distributed diffusely throughout the PVN. (B and C): mRNA and protein expression of TACE in the PVN of rats 1 week after PVN microinjections of TACE siRNA or scrambled (Scr) siRNA. Non-treated normal rats served as control (CON). Values are mean ± SEM (n = 6 for each group). One-way ANOVA followed by Tukey’s post hoc tests was used for data analysis. mRNA data are expressed as a fold change compared to CON. *p < 0.05.


FIGURE 3


FIGURE 3

mRNA and protein levels of TACE in the PVN (A and B), soluble tumor necrosis factor (sTNF)-α levels in the cerebrospinal fluid (CSF, (C), and mRNA expression of inflammatory mediators TNF-α (D), TNF-α receptor 1 (TNFR1, (E), TNFR2 (F), cyclooxygenase (COX)-2 (G) and COX-1 (H) in the PVN in HF or SHAM rats pretreated with PVN microinjection of TACE siRNA or Scr siRNA. Values are mean ± SEM (n = 6-8 for each group). Two-way ANOVA followed by Tukey’s post hoc tests was used for data analysis. mRNA data are expressed as a fold change compared to SHAM + Scr siRNA. *p < 0.05.


FIGURE 4


FIGURE 4

Protein levels of phosphorylated (p-) NF-κB p65 (A) and its inhibitor IκB-α (B), p-ERK1/2 (C), and mRNA expression of c-Fos (a marker of neuronal activity, (D) in the PVN, and levels of plasma norepinephrine (NE, a marker of sympathetic nerve activity, (E), in HF or SHAM rats pretreated with PVN microinjection of TACE siRNA or Scr siRNA. Values are mean ± SEM (n = 6-8 for each group). Two-way ANOVA followed by Tukey’s post hoc tests was used for data analysis. mRNA data are expressed as a fold change compared to SHAM + Scr siRNA. *p < 0.05.


FIGURE 5


FIGURE 5

(A) Representative M-mode echocardiographic images from HF or SHAM rats pretreated with PVN microinjection of TACE siRNA or Scr siRNA (4 weeks after coronary artery ligation). (B–E): Quantitative comparison of echocardiographic parameters including ischemic zone as a percent of left ventricular (LV) circumference (% IZ), LV ejection fraction (LVEF), LV end-systolic volume (LVESV) and LV end-diastolic volume (LVEDV) 24 h and 4 weeks after coronary artery ligation. (F–I): Quantitative comparison of LV hemodynamic parameters including heart rate, systolic blood pressure (SBP), maximum rate of rise of LV pressure (LV dP/dtmax) and LV end-diastolic pressure (LVEDP) from four treatment groups 4 weeks after coronary artery ligation. Values are mean ± SEM (n = 7-8 for each group). Two-way ANOVA followed by Tukey’s post hoc tests was used for data analysis. #p < 0.05 vs SHAM + Scr siRNA; §p < 0.05 vs same group at 24 h; †p < 0.05, HF+TACE siRNA vs HF + Scr siRNA; *p < 0.05.


FIGURE 6


FIGURE 6

(A) Representative heart and lung images from HF or SHAM rats pretreated with PVN microinjection of TACE siRNA or Scr siRNA (4 weeks after coronary artery ligation). (B–D): Anatomic measurements including body weight (BW), ratios of heart weight or lung weight to BW. (E) Representative wheat-germ-agglutinin staining (WGA) showing cardiomyocyte area from four treatment groups. (F–I): Quantitative comparison of the cross-sectional area of myocytes and mRNA expression of pro-hypertrophic markers atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and β-myosin heavy chain (β-MHC), in the peri-infarct areas of the left ventricular in treatment groups. Two-way ANOVA followed by Tukey’s post hoc tests was used for data analysis. Values are mean ± SEM (n = 6-8 for each group). *p < 0.05.


FIGURE 7


FIGURE 7

(A) Representative Masson’s Trichrome staining showing cardiac fibrosis from HF or SHAM rats pretreated with PVN microinjection of TACE siRNA or Scr siRNA (4 weeks after coronary artery ligation). (B–G): Quantitative comparison of the collagen deposition and mRNA expression of pro-fibrotic markers α-smooth muscle actin (α-SMA), transforming growth factor (TGF)-β1, fibronectin, collagen-I and collagen-III, in the peri-infarct areas of the left ventricular in four treatment groups. Values are mean ± SEM (n = 6-8 for each group). Two-way ANOVA followed by Tukey’s post hoc tests was used for data analysis. *p < 0.05.


FIGURE 8


FIGURE 8

Quantitative comparison of the levels of inflammatory mediators including TNF-α in CSF (A), mRNA of TNF-α (B), TNFR1 (C), TNFR2 (D) and COX-2 (E), protein of p-NF-κB p65 (F) and its inhibitor IκB-α (G), and p-ERK1/2 (H), and mRNA of neuronal excitatory marker c-Fos (I) in the PVN, and levels of sympathetic excitatory marker norepinephrine (NE, (J) in plasma, in HF rats treated with a 4-week ICV TACE inhibitor TAPI-0 or vehicle (VEH). Values are mean ± SEM (n = 6-8 for each group). Student’s t-test was used for data analysis. mRNA data are expressed as a fold change compared to HF+VEH. *p < 0.05.


FIGURE 9


FIGURE 9

Quantitative comparison of echocardiographic parameters including ischemic zone as a percent of left ventricular (LV) circumference (% IZ, (A)), LV ejection fraction (LVEF, (B)), LV end-systolic volume (LVESV, (C)) and LV end-diastolic volume (LVEDV, (D)), and LV hemodynamic parameters including heart rate (E), systolic blood pressure (SBP, (F)), maximum rate of rise of LV pressure (LV dP/dtmax, (G)) and LV end-diastolic pressure (LVEDP, (H)), from HF rats treated with TACE inhibitor TAPI-0 or vehicle (VEH). Values are mean ± SEM (n = 7-8 for each group). Two-way ANOVA followed by Tukey’s post hoc tests was used for echocardiography data analysis. Student’s t-test was used for hemodynamic data analysis. §p < 0.05 vs same group at 24 h; †p < 0.05, HF+TAPI0 vs HF+VEH; *p < 0.05.


FIGURE 10


FIGURE 10

Representative heart and lung images (A) and anatomic measurements (B,C and D) including body weight (BW), ratios of heart weight or lung weight to BW from HF rats treated with TACE inhibitor TAPI-0 or vehicle (VEH). (E and F) Representative wheat-germ-agglutinin staining (WGA) showing cardiomyocyte area and quantitative comparison of the cross-sectional area of myocytes in the peri-infarct areas of the left ventricular in the two treatment groups. (G and H) Representative Masson’s Trichrome staining showing cardiac fibrosis and quantitative comparison of the collagen deposition in the peri-infarct areas of the left ventricular in the two treatment groups. Student’s t-test was used for data analysis. Values are mean ± SEM (n = 6 for each group). *p < 0.05.

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