LDL‑induced NLRC3 inflammasome activation in cardiac fibroblasts contributes to cardiomyocytic dysfunction

Abstract

Heart failure (HF) is a progressive myocardial disease that affects pulse rate. Notably, chronic inflammation serves a crucial role in cardiac dysfunction and HF. Appropriate cardiomyocyte‑fibroblast communication is essential for cardiac function. In addition, cardiac fibroblasts (CFs) are the main cellular population in the cardiac microenvironment; therefore, determining the role of CFs in HF progression and the associated molecular basis is important. In the present study, ELISAs were performed to detect inflammatory factors in the sera of patients with HF and their association with CF activation was analyzed using Pearson's correlation coefficient. The mechanism underlying the proinflammatory phenotype of CFs was investigated via western blotting. Notably, the levels of IL10 and TNF‑α were significantly increased in the sera of patients with HF. Further analysis revealed that CFs were extensively activated in the cardiac tissues of patients with HF and released excessive amounts of cytokines, which could impair the viability of cardiomyocytes. Moreover, low‑density lipoprotein (LDL)‑induced NLRC3 inflammasome was activated in CFs, which gave rise to proinflammatory phenotypes. Targeting LDL in CFs significantly improved the functioning of cardiomyocytes and inhibited apoptosis. These findings highlighted the critical role of LDL in inflammasome activation; to the best of our knowledge, the present study is the first to reveal that CF‑induced microenvironmental inflammation may suppress cardiomyocyte viability. The present study established the cellular basis for CF activation during HF progression and provided information on the cellular interactions important for HF treatment.

Keywords: NLRC3 inflammasome; cardiac fibroblasts; heart failure; low‑density lipoprotein.

Figure 1.

CFs are activated in the heart tissues of patients with HF. (A) Levels of IL1β, IL10, IL18 and TNF-α in the sera from healthy controls (n=20), and patients with mild (n=28) and severe (n=34) HF, as determined by ELISA. (B) Protein expression levels of IL1β, IL10, IL18 and TNF-α in myocardial samples from a control individual (Con-26353-LV1) and patient with HF (HF-15331-LV1), as determined by western blotting. GAPDH was used as the internal control. (C) Hematoxylin and eosin staining and immunohistochemical staining of FAP in myocardial samples from a control individual (Con-26351-LV1) and patient with HF (HF-15328-LV1) (scale bar, 10 µm). (D) mRNA expression levels of IL10 and TNFA in cardiac tissues from LPS-treated mice. (E) Levels of IL10 and TNF-α in the supernatant of CFs treated with LPS. (F) MTT assay showing the viability of neonatal cardiomyocytes treated with IL10 and TNF-α. CFs, cardiac fibroblasts; HF, heart failure; FAP, fibroblast-activated protein.

Figure 2.

NLRC3 inflammasome is activated in the CFs of patients with HF. (A) RT-qPCR analysis of the expression of inflammasome-associated genes (NLRPs and NLRCs) in myocardial samples from control individuals (n=3) and patients with HF (n=3). (B) Representative images of CFs and cardiomyocytes (scale bar, 10 µm). (C) RT-qPCR analysis of NLRC3 mRNA expression in CFs from mice in the LPS treatment or control groups. (D) Immunohistochemical staining of NLRC3 in a myocardial samples from a control individual (Con-26354-LV3) and patient with HF (HF-15329-LV2) (scale bar, 10 µm). (E) Pearson analysis of the correlation between NLRC3 mRNA and IL10 or TNFA mRNA. (F) RT-qPCR analysis of NLRC3 mRNA expression in CFS infected with shRNA-NLRC3 after LPS stimulation. (G) RT-qPCR analysis of IL10 and TNFA mRNA expression in CFs infected with shRNA-NLRC3 after LPS stimulation. CFs, cardiac fibroblasts; HF, heart failure; RT-qPCR, reverse transcription-quantitative PCR; LPS, lipopolysaccharide; shRNA, short hairpin RNA.

Figure 3.

LDL is required for NLRC3 inflammasome activation in CFs. (A) Immunohistochemical staining of LDL in myocardial samples from a control individual (Con-26354-LV3) and patient with HF (HF-15329-LV2) (scale bar, 10 µm). (B) Pearson analysis of the correlation between LDL and NLRC3 mRNA. (C) Western blot analysis of the expression of LDL and NLRC3 in myocardial samples from control individuals (Con-26353-LV1 and Con-26351-LV1) and patient with HF (HF-15331-LV1). GAPDH was chosen as the internal control. (D) Western blot analysis of the expression of LDL in CFs infected with shLDL and shScr. (E) Reverse transcription-quantitative PCR analysis of NLRC3, IL10 and TNFA mRNA expression in CFs infected with shLDL after LPS stimulation. CFs, cardiac fibroblasts; HF, heart failure; LDL, low-density lipoprotein; LPS, lipopolysaccharide; sh, short hairpin RNA; shScr, scrambled shRNA.

Figure 4.

Inhibition of low-density lipoprotein improves the viability of cardiomyocytes. (A) Reverse transcription-quantitative PCR analysis of NLRC3, IL10 and TNFA mRNA expression in CFs treated with a PCSK9 inhibitor after LPS stimulation. (B) MTT assay was used to determine the viability of neonatal cardiomyocytes cultured with a PCSK9 inhibitor in the presence of LPS-stimulated CF supernatant. (C) Immunostaining of Ki-67 in neonatal cardiomyocytes co-cultured with CFs and treated with a PCSK9 inhibitor (scale bar, 10 µm). (D) Pulse analysis of the viability of neonatal cardiomyocytes co-cultured with CFs and treated with a PCSK9 inhibitor. (E) FCM analysis of the apoptosis of neonatal cardiomyocytes cultured with a PCSK9 inhibitor in the presence or absence of LPS-stimulated CF supernatant. (F) FCM analysis of the apoptosis of neonatal cardiomyocytes cultured in the supernatant of CFs from a mouse model of HF with or without PCSK9 inhibitor treatment. CFs, cardiac fibroblasts; HF, heart failure; LPS, lipopolysaccharide; FCM, flow cytometry; PCSK9, proprotein convertase subtilisin/kexin type 9.