GRK5 is a regulator of fibroblast activation and cardiac fibrosis

Significance Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. G protein-coupled receptor (GPCR) kinase 5 (GRK5) has been shown to cause deleterious effects on the cardiomyocyte during HF; however, its effects in cardiac fibroblasts, the crucial cell type responsible for maintaining the structural integrity of the heart, is not understood. Here, we use in vitro and in vivo methods to demonstrate that inhibition of GRK5 inhibits fibroblast activation and attenuates the fibrotic response in the heart.

fetal bovine serum and 12.5 μM CaCl2). Cardiomyocytes were allowed to settle by centrifugation at 300 rpm for 30 seconds. The supernatant was collected and centrifuged at 500 xg for 5 minutes to collect the first fibroblast (non-myocyte) fraction. Cardiomyocytes were resuspended in 10mL DMEM with 10% FBS and subsequently allowed to settle. Supernatant was collected, centrifuged, and combined with the first fibroblast fraction. Fibroblasts were resuspended and plated in growth media (DMEM with 10% FBS) on a 10cm plate coated with 1% gelatin.
Neonatal rat cardiac fibroblasts (NRCFs) were isolated as a byproduct of neonatal rat cardiac myocyte isolation, performed as previously described. 2 Briefly, hearts were isolated from 1-to 2-day-old neonatal rats. Hearts were pre-washed in ADS buffer (NaCl 116 mM, HEPES 20 mM, Na2HPO4 0.8 mM, glucose 5.6 mM, KCl 7 mM, and MgSO4-7H2O 0.8 mM, pH 7.35) to remove blood and then divided and placed in dishes with 7 ml of ADS. They were minced with sterol razor blades in small pieces and then the whole solutions were transferred in flasks and incubated at 37 °C with 7 ml enzyme solution (ADS containing pancreatin 0.6 mg/L, collagenase II 8820 U/L and CaCl2 50 mM) for 10 min. The supernatant from this pre-digestion step was discarded and the pieces were incubated with 15 ml of digestion solution for 15-min intervals at 37 °C. After each interval, the supernatant was collected in 50 ml conical tubes containing 19 ml of F-10 media and 20% FBS pre-heated at 37 °C. The three to six collected fractions were spun down at 1,400g for 10 min, the supernatant was discarded and cells were washed with 5 ml of FBS for each tube. The cells were then centrifuged at 1,400g for 10 min and the supernatant was discarded. The resulting pellet containing the cells was resuspendend in HAM's F10 complete media containing 10% horse serum (HS), 5% FBS and 1% penicillin-streptomycin (P/S), pH 7.4. The cell suspension was filtered through a 70 μm filter and pre-plated on a Nunc Nunclon 100 mm (Thermo Fisher Scientific, Waltham, MA, USA) cell culture dish for 2 h to separate the fibroblasts from the myocyte fraction. The fibroblasts attached to the Nunclon dishes were cultured with DMEM with 10% FBS.

Real-time PCR
Total RNA was isolated from MACFs with TRIzol (Thermo Fisher) according to the company's instructions. After RNA isolation, cDNA was synthesized by reverse transcription of the RNA (iScript cDNA synthesis kit, Bio-Rad). Real-time PCR was performed in triplicated on a CFX96 real0time PCR detection system (Bio-Rad) using SYBR Green mix (Bio-Rad) and specific primers for mouse a-SMA, collagen I, collagen III, MMP2, MMP9, and TGFb. Expression levels were established by comparing to TPT1 expression, which was similar between groups, for normalization and compared using the DDCt method.

Surface Sensing of Translation (SUnSET) Assay
WT and GRK5KO MACFs were stimulated with 1µM AngII for varying amounts of time, followed by a pulse of 1µM puromycin for 30 minutes prior to harvesting. Immunoblotting was performed using an anti-puromycin antibody. 3

Calmodulin Capture Assay
Calmodulin (CaM)-agarose beads (Sigma) were washed with either 2mM CaCl2 or 2mM EDTA in IP buffer (50mM Tris, 150mM NaCl). 250µg of cell lysates were added to the CaM beads with a final concentration of either 2mM CaCl2 or 2mM EDTA IP buffer and rotated overnight. Beads were washed with respective buffers, resuspended in 2X Laemmli buffer, and analyzed by immunoblotting. 4

Immunoblotting
After SDS-PAGE and transfer to nitrocellulose membranes, primary antibody incubations were performed overnight at 4°C. Fluorescent secondary antibodies were obtained from Li-Cor.
Membranes were scanned with the Odyssey infrared imaging system (LI-COR). 5 GRK5 antibody was from Santa Cruz. a-SMA and Puromycin antibody was from Sigma.

In Vivo AngII Infusion and Model of Myocardial Infarction
AngII (1µg/kg/min) dissolved in PBS was continuously infused subcutaneously into mice via an osmotic minipump (ALZET) for 4 weeks. A control group was infused only with PBS. Mice were anesthetized with isofluorane and pumps were implanted subcutaneously through a sub-scapular incision which was then closed using 4.0 silk suture (Ethicon). Tissue was collected 4 weeks post infusion. For our MI model, mice were subjected to permanent ligation of the left main descending coronary artery or a sham surgery as we have described previously and tissue was collected 4 weeks post MI. 6

Transthoracic Echocardiographic Analysis
Transthoracic 2-dimensional echocardiography was performed blinded as previously described using the Vevo 2100 imaging system. 7

Assessment of Myocardial Fibrosis and Hypertrophy
Collagen levels were measured using the Masson's Trichrome staining kit (Sigma HT15) without modifications as previously described. 5 Briefly, mice were euthanized 4 weeks after MI or AngII infusion. Hearts were removed and fixed in 4% paraformaldehyde, embedded in paraffin, and cut into long axis sections 6µM in thickness. Sections were deparaffinized and rehydrated. Before treatment with the Trichrome staining kit, sections were incubated at room temperature in Bouin's solution (HT101128) overnight. Images of the fibrosis were taken on a Nikon DS-Ri1 in a blinded manner. For each area of the heart, at least 10 random fields were measured. Images were quantified using CellProfiler, a cell image analysis software, capable of determining fibrotic area in an unbiased manner. 8 Cardiomyocyte hypertrophy was measured using wheat germ agglutinin (WGA) staining as previously described. 9 Briefly, heart sections were deparaffinized, rehydrated, and washed with phosphate-buffered saline. Sections were then stained with Alexa Fluor 488-conjugated WGA for 1 hour at room temperature in the dark. Sections were washed 3 times for 5 minutes and mounted using Fluoromount-G mounting media containing DAPI nuclear stain (Southern Biotech).

Total Collagen Detection
Hearts were homogenized in 1mg/mL pepsin in .05M acetic acid and incubated at 4° for 48 hours. Supernatant was collected and collagen concentration was detected with a Sirius Red Collagen Detection Kit (Chondrex) without modifications.
Immunofluorescence 1x10 6 MACFs seeded on glass coverslips coated with 1% gelatin were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked with 5% BSA, and incubated with an anti-aSMA antibody in 5% BSA. Cells were then incubated with respective secondary antibodies.

NFAT Luciferase Reporter and Luciferase Assay
Cardiac fibroblasts were infected with an NFAT reporter adenovirus at an MOI of 10.

Medium was changed after 24 hours, and 48 hours after infection cells were stimulated with
AngII for 24 hours. Cells were lysed and luciferase activity measured. 11

Collagen Gel Contraction Assay
Fibroblasts were harvested from a confluent monolayer by Trypsin-EDTA digestion, pelleted, and resuspended in serum free DMEM. Fibroblasts were then seeded into collagen matrices (0.85mg/mL) such that each gel contained 100,000 fibroblasts and cast in 24 well plates. The collagen gels were released from the edges and floating in serum free DMEM with or without AngII. ImageJ software was used to calculate the surface area, which are reported as values normalized to the initial size of the gel. 11

Statistical Tests
Data are expressed as mean ± standard deviation. Statistical significance was determined by ANOVA and Tukey's multiple comparisons test for multivariate experiments and t-test for experiments with two groups.