Please find detailed material information in Additional File 1: Materials.
C57BL/6NHsd male mice were purchased from Envigo RMS Inc (IN, USA). Inducible T2D mice were generated by a single injection of streptozotocin (STZ, 75 mg-kg-1, dissolved in citrate buffer, i.p.) at 6 weeks of age, and given a high-fat diet (60% kcal from fat, Envigo RMS Inc.) from the day of STZ injection. Sixteen weeks after diabetic induction, mice were randomly allocated to experimental groups and used before 26 weeks old (20 weeks after T2D induction). Oral glucose tolerance test (OGTT) and measurements of plasma cholesterol, HDL, and triglyceride levels were performed as described [18, 35, 36]. Male TALLYHO/Jng (TH) mice were purchased from The Jackson Laboratory (ME, USA) and bred in our animal facility. The TH mice are a polygenic T2D model, and male TH mice exhibit hyperglycemia, hyperinsulinemia, hyperlipidemia, and obesity . We used male C57BL/6 mice as wild type (Wt) controls according to The Jackson Laboratory guideline. All mice were fed with a normal laboratory diet (13% kcal from fat, Lab Diet, IN, USA). TH and Wt mice were used for experiments at the age of 16-20 weeks.
HuR floxed mice (HuRfl/fl) were kindly provided by Dr. Jian-Ying Wang from the University of Maryland at Baltimore  and crossed with Tie2-Cre mice (The Jackson Laboratory) to generate endothelial cell (EC)-specific HuR knockout (KO) mice (Tie2-HuR-/- mice, Fig. 3A). HuRwt/wt mice were used as wild-type (Wt) control for Tie2-HuR-/- mice. Hetero (Tie2-HuR+/-) parents were used for the breeding due to the infertility of homo (Tie2-HuR-/-) parents. Genotype rates were: homozygous (17.4%), heterozygous (29.9%), and Wt (52.7%) (Additional File 3: Fig. S1). Wt and Tie2-HuR-/- mice were used after 16 weeks old. Systemic Cx40-KO (Cx40-/-) mice were kindly provided by Dr. Janis Burt from the UA , and C57BL/6J (background strain, The Jackson Laboratory) were used as a Wt control. They were used at 16 weeks old. EC-specific Cx40 overexpression (Cx40Tg) mice were provided by Dr. Anthony Ashton from the University of Sydney . This mouse strain carries the Tie2 (endothelium specific promoter)-driven wild type Cx40 gene with EGFP (Cx40-IRES-EGFP); therefore, the mice will constitutively express Cx40 in ECs. Heterozygous parents were identified by copy number measurement and used as a breeder. Mice without Tie2-Cx40 gene were used as a Wt control of Cx40Tg mice. These mice were bred in the animal facility of the UA and UCSD. Male mice were used for experiments between 22-26 weeks old (16-20 weeks after diabetic induction). The primer sequence information for genotyping is listed in Additional File 2: Table S1 and for real-time PCR in Additional File 2: Table S2. Heart dissection was performed under anesthesia with a mixture of ketamine (100 mg/kg, i.p.) and xylazine (5 mg/kg, i.p.), and all efforts were made to minimize pain.
The age was matched between diabetic or transgenic mice and their control mice. Male mice were used in this study due to the difference in the onset of hyperglycemia and diabetic complications between male and female mice.
Isolation of mouse cardiac endothelial cells (CECs)
Mouse CECs were isolated using a method previously described [11, 12, 18, 35]. Briefly, after flushing blood from the heart, the heart was dissected, minced, and incubated with M199 containing 1 mg/ml collagenase II and 0.6 U/ml dispase II for 1 h at 37°C. The digested material was collected and incubated with magnetic beads that were prepared as follows: Dynabeads® Sheep Anti-Rat IgG were incubated with rat anti-mouse CD31 monoclonal antibody (1 μg/ml) at 4°C overnight. The cell suspension was incubated with beads for 1 h at 4°C, and then CECs were captured and isolated by the Dynal magnet (Thermo Fisher Scientific, MA, USA). The purity of the CEC population in cells isolated from hearts was tested by DiI-acLDL (Thermo-Fisher Scientific) uptake and Bandeiraea Simplicifolia lectin-FITC (BS-l, Sigma Aldrich, Inc. MO, USA) or CD144 staining (Additional File 3: Fig. S2). Efficient isolation yields approximately 104 cells from one heart, with over 80% purity. Western blot and real-time PCR were conducted with freshly isolated CECs from mice. For the immunohistochemistry experiment, we cultured CECs after isolation, and experiments were performed within 5 days without passing the cells.
Human CECs from 4 control and 4 T2D patients were purchased from commercial suppliers (Additional File 1: Materials) and cultured in EC media composed of M199 supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 20 μg/ml ECGS, and 16 U/ml heparin. All experiments were conducted before passage 10.
Isolation of mouse cardiac myocytes and aortic smooth muscle cells
Mouse cardiac myocytes (CMs) were collected after removing ECs from the digested materials of the hearts. After removing ECs, the majority of cells in the digested material are cardiac myocytes; however, other types of cells (i.e., smooth muscle cells [SMCs] and fibroblasts) might be present in the samples at a very small percentage. The samples of aortic SMCs were obtained from an aorta after removing ECs by gently scrubbing the inner layer of the aortic lumen using a cotton tip.
Coronary flow velocity reserve measurement
Coronary flow velocity reserve (CFVR) was used to assess coronary microvascular function , instead of coronary flow reserve, because of the difficulty in precisely measuring coronary arterial diameter in mice . Coronary blood flow velocity (CFV) was measured using a Vevo 2100 system (FUJIFILM Visual Sonics, Inc. Toronto, Canada. Additional File 3: Fig. S3). Mice were anesthetized with isoflurane and kept on the heating pad at 37°C. The resting level of CFV was obtained at 1% isoflurane. CFVR was defined as maximal hyperemic CFV (induced by 2.5% isoflurane) divided by resting CFV (1% isoflurane) [18, 40]. Each experiment was completed within 40 min, and the heart rate was kept above 400 bpm. If the procedure took a longer time, or the heart rate was dropped lower than the criteria, the data was eliminated without analysis.
The evaluation of capillary density and EC apoptosis in the left ventricle (LV) were conducted as described previously [11, 12, 18]. Briefly, the heart was dissected, embedded in OCT compound, frozen in 2-methylbutane precooled with liquid nitrogen, and then kept at -80°C until being sectioned. Sections (6 µm in thickness) were fixed in 4% formaldehyde for 5 min, blocked with 5% BSA for 30 min, and incubated with Bandeiraea Simplicifolia lectin-FITC (BS-l, Sigma Aldrich, Inc.) for 30 min. BS-l was used to probe the terminal β-galactosyl saccharides associated with endothelial cells on the surface of arterioles and venules as well as capillaries. Apoptotic cells were detected using a TUNEL assay (an in situ cell death detection kit, Roche).
After isolation of mouse CECs, cells were stained with HuR antibody and followed by anti-mouse Alexa488. The images were captured with a Nikon Eclipse Ti-E 3D Deconvolution microscope (Nikon Corp. Tokyo, Japan) with a 20x objective lens (for EC apoptosis and capillary density) or 60x objective lens (for HuR staining) in a blinded fashion. The fluorescence intensities were calculated using ImagePro-PLUS 7.0 software (Media Cybernetics Inc. MD, USA).
Western blot analysis
Protein levels were analyzed using SDS-PAGE separation and electrophoretic transfer to nitrocellulose membranes. Primary antibodies used in this study are listed in Additional File 1: Materials.
mRNA from mouse CECs was isolated using a miRNeasy Mini Kit (QIAGEN, CA, USA), and cDNA was made by RT2 First Strand Kit (QIAGEN). We chose 92 genes (including Actb and Gapdh) that are highly expressed in ECs and play crucial roles in endothelial functions for analysis by real-time PCR, including a) endothelium-derived relaxing factors and their regulators; b) modifiers of cytosolic Ca2+ concentration ([Ca2+]), mitochondrial [Ca2+], and endoplasmic reticulum [Ca2+]; and c) regulators of EC proliferation/migration/apoptosis (see Additional File 2: Table S3 for the gene list). The custom PCR plates were made by QIAGEN based on the selected genes (SABIO Number CAPA38128-6:CLAM25240). One 384-well plate includes quadruplicate wells for one gene (for the gene of interest and internal control) and replicates genomic DNA controls, reverse-transcription controls, and positive PCR controls. Primer sets used for the PCR plate are authenticated by the company. Real-time PCR was conducted using the CFX384 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, CA, USA). GAPDH was used as an internal control. The transcript levels of the Gene of Interest were quantified according to the cycle threshold (ΔCt) method. Ct values > 35 were not included in the analysis and were considered as negative. Note that the primer set for Elavl1 (HuR) on the plate (Product # PPM30921A, QIAGEN) detects exon 5, not exon 2; therefore, real-time PCR was repeated using an exon 2-specific primer (Additional File 2: Table S2).
Ribonucleoprotein immunoprecipitation (RIP)
To assess the association of endogenous HuR protein with endogenous Cx40 mRNA, immunoprecipitation (IP) of ribonucleoprotein complexes was performed as previously described  (Additional File 3: Fig. S4). Mouse CECs were isolated and lysed with lysate buffer (100 mM KCl, 5 mM MgCl2, 10 mM HEPES [pH7.0], 0.5% Igepal, 1 mM DTT, 1% protease inhibitor cocktail, 1% phosphatase inhibitor cocktail, 100 U/ml in RNase free water). Prior to RIP, the IP matrix was conjugated with HuR antibody or IgG, and cell lysate was incubated with IP matrix overnight. RNA in IP materials was used for reverse transcription, followed by real-time PCR analysis (Fig. 4). The data of Cx40 mRNA bound to HuR protein was normalized by Cx40 mRNA bound to IgG.
Isometric tension measurement in coronary arterial ring
Isometric tension measurement in coronary arteries (CAs) was performed as described previously [11, 35, 36]. Briefly, third-order small CAs were dissected from the hearts and then cut into 1-mm segments. The CA rings were mounted on a myograph (DMT-USA, Inc. MI, USA) using thin stainless wires (20 μm in diameter), and the resting tension was set at 100 mg. CAs were allowed to equilibrate for 45 min with intermittent washes every 15 min. After equilibration, each CA ring was contracted by treatment with PGF2α to generate a similar contraction level in all groups. The concentration of PGF2α used for precontraction was 5.34 ± 0.19 in Wt and 5.63 ± 0.16 in Cx40-/- mice (shown in –log M). The diameter of the vessels was 134.6 ± 7.1 µm in Wt and 136.1 ± 5.4 µm in Cx40-/- mice. There was no significant difference in either PGF2α concentration or vessel diameter between Wt and Cx40-/- mice. Acetylcholine (ACh) or sodium nitroprusside (SNP, an NO donor) was administrated in a dose-dependent manner (1 nmol/l to 100 μmol/l). The degree of relaxation was shown as a percent of PGF2α-induced contraction.
Tube formation assay in human CECs
We used Cx40 adenovirus (Cx40-Adv) to overexpress the Cx40 gene . HuR downregulation was achieved by HuR siRNA transfection (Santa Cruz Biotechnology Inc. Dallas, TX). Human control CECs (105 cells) were seeded on a 3 cm plate, and Control- or Cx40-Adv was added to the cells at the titer of 100 pfu/cell on the following day. Twenty-four hours later, the viruses were washed, and cells were transfected with control siRNA or HuR siRNA at 100 nM using lipofectamine 3000 reagent (Thermo Fisher Scientific). Specific protein knockdown was verified with Western blotting 48 hours after transfection (Additional File 3: Fig. S5). For tube formation assay , cells were detached, and 4×104 cells were seeded on the Matrigel-coated 4-well chamber. Twenty-four hours after plating cells, 4 microscopic fields, selected at random, were photographed using an EVOS FL Auto Cell Imaging System with 4x objective lens (Thermo Fisher Scientific) in a blinded fashion. Meshes number, total meshes area, junction number, segments number, and total segments length were analyzed using Angiogenesis Analyzer in NIH ImageJ 1.51k software.
Cytosolic ROS measurement in human CECs
HuR and Cx40 were downregulated using HuR siRNA or Cx40 siRNA, respectively (Santa Cruz Biotechnology Inc.). Human control CECs (2 x 104 cells) were seeded on a 4-well glass chamber and then transfected with control-, HuR- or Cx40-siRNA at 100 nM using lipofectamine 3000 reagent. Cytosolic ROS was detected using the fluorescent probe dihydroethidium (DHE). Cells were preloaded with 50 μmol/l DHE for 30 min before capturing images. Cytosolic DHE exhibits blue fluorescence; once it is oxidized by ROS, it illuminates red (ethidium bromide [EB]). The images were captured with a Nikon Eclipse Ti-E 3D Deconvolution microscope with a 60x objective lens in a blinded fashion. The fluorescence intensity was calculated using ImagePro-PLUS 7.0 software. The background fluorescence intensity was subtracted from the cell intensity. The index of cytosolic ROS concentration is described as a ratio of EB and DHE.
We conducted data analysis in a blinded fashion wherever possible and set proper controls for every experimental plan. The mouse numbers and independent experiment numbers are described in the figure legends. Statistical analysis was performed using GraphPad Prism 9 (La Jolla, CA, USA). Data are presented as mean ± SEM. After the data passed a normality test (Kolmogorov-Smirnov), the two-tailed Student’s t-test was used for comparisons of two groups, and one-way ANOVA was used for multiple comparisons. If the data did not pass the normality test, a non-parametric test (Mann-Whitney for two groups, Kruskal-Wallis for multiple comparisons) was used. Bonferroni’s multiple comparisons test was used as a post hoc test for one-way ANOVA and Dunn’s test for the Kruskal-Wallis test. Statistical comparison between dose-response curves was made by two-way ANOVA with Bonferroni post hoc test. Differences were considered to be statistically significant when P<0.05.