HuR/Cx40 downregulation causes coronary microvascular dysfunction in type 2 diabetes
Rui Si, … , Jason X.-J. Yuan, Ayako Makino
Rui Si, … , Jason X.-J. Yuan, Ayako Makino
Published November 8, 2021
Citation Information: JCI Insight. 2021;6(21):e147982. https://doi.org/10.1172/jci.insight.147982.
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Research Article Vascular biology

HuR/Cx40 downregulation causes coronary microvascular dysfunction in type 2 diabetes

Abstract

Patients with diabetes with coronary microvascular disease (CMD) exhibit higher cardiac mortality than patients without CMD. However, the molecular mechanism by which diabetes promotes CMD is poorly understood. RNA-binding protein human antigen R (HuR) is a key regulator of mRNA stability and translation; therefore, we investigated the role of HuR in the development of CMD in mice with type 2 diabetes. Diabetic mice exhibited decreases in coronary flow velocity reserve (CFVR; a determinant of coronary microvascular function) and capillary density in the left ventricle. HuR levels in cardiac endothelial cells (CECs) were significantly lower in diabetic mice and patients with diabetes than the controls. Endothelial-specific HuR-KO mice also displayed significant reductions in CFVR and capillary density. By examining mRNA levels of 92 genes associated with endothelial function, we found that HuR, Cx40, and Nox4 levels were decreased in CECs from diabetic and HuR-KO mice compared with control mice. Cx40 expression and HuR binding to Cx40 mRNA were downregulated in CECs from diabetic mice. Cx40-KO mice exhibited decreased CFVR and capillary density, whereas endothelium-specific Cx40 overexpression increased capillary density and improved CFVR in diabetic mice. These data suggest that decreased HuR contributes to the development of CMD in diabetes through downregulation of gap junction protein Cx40 in CECs.

Authors

Rui Si, Jody Tori O. Cabrera, Atsumi Tsuji-Hosokawa, Rui Guo, Makiko Watanabe, Lei Gao, Yun Sok Lee, Jae-Su Moon, Brian T. Scott, Jian Wang, Anthony W. Ashton, Jaladanki N. Rao, Jian-Ying Wang, Jason X.-J. Yuan, Ayako Makino

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Figure 4

Identification of HuR-regulated genes and the effect of Cx40 deletion on coronary microvascular function.

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Identification of HuR-regulated genes and the effect of Cx40 deletion on...
(A) mRNA levels were compared between control and diabetic mice (nexperiments = 6 [12 mice per group]) and between WT and Tie2-HuR–/– mice (nexperiments = 6 [12 mice per group]) (Supplemental Tables 4 and 5). Blue, downregulated genes; red, upregulated genes. (B) Cx40 mRNA levels in CECs from WT and Tie2-HuR–/– mice determined by real-time PCR. nmice = 8 per group. (C) Western blot showing Cx40 and GAPDH protein levels in CECs from WT and Tie2-HuR–/– mice (top panel). The bottom dot plot shows Cx40 protein level normalized to GAPDH. nmice = 5 per group. (D) Western blots showing Cx40 and Actin protein levels in CECs from control and diabetic mice (top panel). The bottom dot plot shows Cx40 protein level normalized to Actin. nmice = 5 per group. (E) Binding of Cx40 mRNA to HuR protein determined by ribonucleoprotein immunoprecipitation. mRNA levels were determined by real-time PCR. nmice = 6 per group. (F) Western blots showing Cx40 and Actin protein level in CECs from WT and Cx40–/– mice (top panel). The bottom dot plot shows Cx40 protein level normalized to Actin. nmice = 5 per group. (G) Endothelium-dependent relaxation evaluated by ACh-induced relaxation in CAs. nmice = 7 per group. (H) Endothelium-dependent hyperpolarization–mediated (EDH-mediated) relaxation in CAs determined by ACh administration in the presence of L-NAME (an endothelial NO synthase inhibitor, 1 × 10–4M) and indomethacin (a cyclooxygenase inhibitor, 1 × 10–5M). nmice = 7 per group. (I) Endothelium-independent relaxation evaluated by SNP-induced relaxation in CAs. nmice = 7 per group. (J) CFVR. WT, nmice = 8; Cx40–/–, nmice = 9. (K) Representative photomicrographs (left) and summarized data (right) of capillary density. Scale bar: 50 μm. nmice = 8 per group. Data are presented as mean ± SEM. *P < 0.05 versus Cont or WT. Statistical comparison between dose-response curves was made by 2-way ANOVA with Bonferroni post hoc test (G–I). Unpaired Student’s t test (2-tailed) was used for comparisons of 2 experimental groups (B–F and J–K).