1 Supplemental Methods in Vivo Mouse Studies
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Supplemental Methods In vivo mouse studies – A subset of WT and qv4J animals were administered the STAT3 inhibitor S3I-201 in-vivo at 20mg/kg daily intraperitoneal for 14 days (1). Transaortic constriction (TAC) was performed on a subset of WT mice for 6 weeks to induce chronic pressure-overload and heart failure, as described (1, 2). Cardiac performance was assessed prior to surgery and at 6 weeks post TAC using Vevo 2100 (Visualsonics). Specifically, cardiac function was determined using the MS-400 transducer in the short axis M-mode. For heart extractions, mice were anesthetized with 2.5% gaseous isoflurane and oxygen and sacrificed with confirmation by the absence of response to probing of the lower extremities. Immunoblot analysis – Primary ventricular cardiac fibroblast lysate was analyzed using SDS-PAGE and immunoblotting, as described (1, 3, 4). Briefly, equal protein loading was achieved using standard BCA protein assay protocols and verified by Ponceau staining of immunoblots. The following antibodies were used for immunoblotting: Total STAT3 (1:1000, Cell Signaling, Catalog #: 4904), vimentin (1:1000, Abcam, Catalog # ab92547:) and GAPDH (1:5000, Fitzgerald, Catalog #: 10R-G109A). Immunofluorescence – Cardiac fibroblasts were seeded in 35 mm Matex glass bottom dishes until ~50-70% confluency. Cells were then fixed in 4% paraformaldehyde and permeabilized with TritonX-100 for 10 mins. Non-specific binding was blocked using blocking buffer comprised of 1.5% bovine serum albumin (BSA), 3% goat serum in 1xPBS ° (Invitrogen) overnight at 4 C. Primary antibodies [βIV-spectrin (1:100, Millipore, Clone# N393/76), total STAT3 (1:100, Cell Signaling, Catalog#: 4904), plakoglobin/anti-gamma catenin (1:400, Abcam, Catalog #: ab15153)] were prepared in blocking buffer and added for overnight incubation at 4°C. After washing, secondary Alexa Flour 568 anti-mouse (1:200, Invitrogen, Catalog #: A11004), 466 anti-rabbit (1:200, Invitrogen, Catalog #: A11008), and 633 phalloidin (1:200, Invitrogen, Catalog #: A22284) antibodies were prepared in blocking buffer and incubated for 2 hrs at 4°C. After washing, DAPI (Vecta Laboratories) was applied and dishes were stored at 4°C in the dark until they were imaged using confocal microscopy. Multiple random cells were selected from each field/preparation for analysis. STAT3 localization analysis was conducted by extracting color bands (blue and green) from images and removing background noise with a 3x3 median filter in MATLAB. Threshold values were set, and binary images were generated, as described (3). Histology – Whole hearts were fixed in 10% formalin, trimmed to reveal ventricles, processed into paraffin, and serially sectioned using microtome into 5 microns. Longitudinal heart sections were then stained using Masson’s Trichrome to evaluate amounts of interstitial and perivascular fibrosis. Percent fibrosis was quantified across the entire longitudinal section (not including atria) using custom software in MATLAB. Quantitative real-time PCR (qPCR) – Total RNA from mouse or human primary cardiac fibroblasts was extracted with TRIzol Reagent following manufacturer’s instructions. RNA 1 concentration was measured using Agilent 2100 Bioanalyzer (Aligent Technologies). SuperScript III reverse transcriptase VILO cDNA Synthesis kit (Invitrogen) was used for the first-strand complementary DNA synthesis (20ng/μL) (primers for mouse and human targets provided in Tables S4 and S5). qPCR reactions were performed in triplicate on cDNA samples in 96-well optical plates with SYBR Green Gene Expression Assays and SYBR Green Universal PCR Master Mix (Invitrogen). qPCR was performed at 95°C for 3 mins., 40 cycles of 95°C for 15 s. and 60°C for 1 min. on Applied Biosystems 7900HT Fast Real-Time PCR System or StepOnePlus Real-Time PCR System (Life Technologies). qPCR data were analyzed using relative standard curve method and 2 delta Ct was used to calculate fold changes in relative gene expression. qPCR products were confirmed by melt-curve analysis, amplicon length, and DNA sequencing. Rpl-7 levels were used as a normalization control. Experiments were conducted in technical triplicates. RNA sequencing - RNA was isolated from male and female wildtype (WT) and qv4J mouse primary cardiac fibroblasts and sequenced with Ocean Ridge BioSciences (Deerfield Beach, FL). Mice were sacrificed using isoflurane overdose, hearts were immediately excised, and cardiac fibroblasts were isolated and cultured at passage 1 conditions as previously described. Total RNA was isolated from the cardiac fibroblasts using the TRI Reagent® (Molecular Research Center). Total RNA was quantified by O.D. measurement and assessed for quality on a 1% agarose – 2% formaldehyde gel. The RNA was then treated with RNase free DNase I (Epicentre) and re-purified on RNeasy MinElute columns (Qiagen). The newly digested RNA samples were then assessed for quality by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer RNA 6000 Pico assays (Agilent Technologies). Amplified cDNA libraries suitable for sequencing were prepared from 200 ng of DNA-free total RNA using the Universal Plus mRNA-Seq Library Prep Kit (NuGEN Technologies, Inc.). The quality and size distribution of the amplified libraries were determined by chip-based capillary electrophoresis on Agilent 2100 Bioanalyzer High Sensitivity DNA assays (Agilent Technologies). Libraries were quantified using the Takara Library Quantification Kit. The libraries were pooled at equimolar concentrations and diluted prior to loading onto a flow-cell on an Illumina cBot. The libraries were extended and bridge amplified to create sequence clusters. The flow cell was transferred to the Illumina HiSeq 4000 instrument and sequenced using 150 nt paired-end reads plus a single index read. Quality-filtered and base-trimmed reads were used for alignment. Sequence alignment was performed using HISAT2 version 2.0.5. The read summarization program featureCounts2 version 1.5.1 was used for exon- and gene- level counting. Normalized RPKM values were calculated from the raw featureCounts read, and were then filtered to retain a list of genes with a minimum of approximately 50 mapped reads in 25% or more samples. The threshold of 50 mapped reads is considered the Reliable Quantification Threshold. The resulting gene list with calculated fold changes was analyzed using Ingenuity Pathway Analysis [QIAGEN (5)]. The software was used to identify highly indicated upstream regulators and disease and function networks. 2 Supplemental References 1. Unudurthi SD, et al. betaIV-Spectrin regulates STAT3 targeting to tune cardiac response to pressure overload. J Clin Invest. 2018;128(12):5561-72. 2. Glynn P, et al. Voltage-Gated Sodium Channel Phosphorylation at Ser571 Regulates Late Current, Arrhythmia, and Cardiac Function In Vivo. Circulation. 2015;132(7):567-77. 3. Hund TJ, et al. A betaIV spectrin/CaMKII signaling complex is essential for membrane excitability in mice. J Clin Invest. 2010;120(10):3508-19. 4. Hund TJ, et al. beta(IV)-Spectrin regulates TREK-1 membrane targeting in the heart. Cardiovasc Res. 2014;102(1):166-75. 5. Kramer A, Green J, Pollard J, Jr., and Tugendreich S. Causal analysis approaches in Ingenuity Pathway Analysis. Bioinformatics. 2014;30(4):523-30. 3 Supplemental Table 1: Echocardiographic features in wildtype (WT) and qv4J mice at baseline and WT mice subjected to 6 weeks of transaortic constriction (TAC) WT qv4J WT TAC (N=5) (N=5) (N=5) HR (bpm) 446.71±11.83 392.14±23.24 448.46±8.78 EF (%) 63.24±0.60 49.49±0.85 34.89±2.20 LVID,d (mm) 2.94±0.03 3.34± 0.08* 4.28±0.12*# LVID,s (mm) 2.02±0.07 2.53±0.07* 3.58±0.13*# FS (%) 33.24±0.37 24.34±0.47* 16.55±1.19*# LVAW,d (mm) 0.75±0.02 0.62±0.03 0.95±0.07*# LVAW,s (mm) 1.19±0.04 0.83±0.05* 1.16±0.09# LVPW,d (mm) 0.78±0.17 0.632±0.06 0.80±0.10 LVPW,s (mm) 0.96±0.07 0.71±0.03 0.91±0.09 HR = heart rate; EF = ejection fraction; LVID,d = left ventricular inner chamber diameter in diastole; LVID,s = left ventricular inner chamber diameter in systole; FS = fractional shortening; LVAW,d = LV anterior wall thickness in diastole; LVAW,s = LV anterior wall thickness in systole; LVPW,d = LV posterior wall thickness in diastole; LVPW,s = LV posterior wall thickness in systole; *P<0.05 vs. WT #P<0.05 vs. qv4J Supplemental Table 2: Differentially regulated genes in qv4J fibroblasts with more than twofold difference from wildtype (WT) Gene Entrez fold change name Description gene ID from WT Serpina3n serine (or cysteine) peptidase inhibitor, clade A, member 3N 20716 14.74341 Ang2 angiogenin, ribonuclease A family, member 2 11731 14.61122** Lrrn4 leucine rich repeat neuronal 4 320974 12.29352 Iglj1 immunoglobulin lambda joining 1 404737 11.31642 Angptl7 angiopoietin-like 7 654812 10.29877 Upk3b uroplakin 3B 100647 8.24076 4 Rnase2a ribonuclease, RNase A family, 2A (liver, eosinophil-derived 93726 7.21412* neurotoxin) Atp10d ATPase, class V, type 10D 231287 5.85188* Traj32 T cell receptor alpha joining 32 100124357 5.80643* Erdr1 erythroid differentiation regulator 1 170942 5.74261** Col28a1 collagen, type XXVIII, alpha 1 213945 5.49104* Tagap T cell activation Rho GTPase activating protein 72536 5.37396* Rtn4rl2 reticulon 4 receptor-like 2 269295 5.06487* Ighd1-1 immunoglobulin heavy diversity 1-1 777657 5.00956 Rbp4 retinol binding protein 4, plasma 19662 4.927054 C3 complement component 3 12266 4.688854 Serpina3g serine (or cysteine) peptidase inhibitor, clade A, member 3G 20715 4.572000 Scn7a sodium channel, voltage-gated, type VII, alpha 20272 4.526735*