Hematopoietic Id Deletion Triggers Endomyocardial Fibrotic and Vascular Defects in the Adult Heart

Inhibitor of DNA binding (Id) proteins play important roles in regulating cardiac development via paracrine signaling. Id1/Id3 knockout mice die at mid-gestation with multiple cardiac defects. Single Id knockout studies have not reported cardiomyopathies. To bypass embryonic lethality we used Tie2CRE-mediated recombination to conditionally delete Id1 against global Id3 ablation (Id cDKOs), which develops adult-onset dilated cardiomyopathy. We confirm upregulation of thrombospondin-1 (TSP1) in Id cDKO hearts. Colocalization studies reveal increased TSP1 expression in the vicinity of endothelial cells and near regions of endocardial fibrosis/disruption. Downstream fibrotic molecules were upregulated. Endocardial capillary density was reduced with evidence of vascular distention. Treatment of Id cDKO cardiac explants with LSKL, a peptide antagonist of TSP1 activation of TGFβ, reversed the increased expression of fibrotic molecules. We conducted bone marrow transplant experiments in which we transferred bone marrow cells from Id cDKO mice into lethally irradiated WT mice. The majority of WT recipients of Id cDKO bone marrow cells phenocopied Id cDKO cardiac fibrosis 4 months post-transplantation. Injection of LSKL into adult Id cDKO mice led to downregulation of fibrotic molecules. The results prompt caution when bone marrow transfers from individuals potentially carrying mutations in the Id axis are applied in clinical settings.


Serum Isolation/Preparation
Whole blood was collected from the retroorbital isnus into untreated 1.5 mL eppendorf tubes and allowed to sit at room temperature for 1 hr. Samples were then centrifuged at 3000 rpm (1500 x g) for 15 mins in 4°C. The serum was carefully removed from the top leaving behind the interphase buggy coat and red blood cells. Serum was aliquoted and stored in -80°C.

Western Blot Analysis
Snap frozen left ventricular tissue was immediately suspended in RIPA buffer (containing 150 nM sodium chloride, 1.0% NP-40, 0.5% sodium deoxycholate, 0.5% SDS, 50 nM Tris pH 8.0) and supplemented with PhosStop phosphatase inhibitor (Roche) and cOmplete Ultra protease inhibitor cocktail (Roche). Samples were homogenized over ice and spun down at 12000 rpm in 4°C to remove insoluble debris. Samples were loaded with 4X Laemmli buffer (BioRad) and protease inhibitor (Sigma) and heated at 99°C for 10 mins before loading onto 4-20% SDS gradient gels. Gels were run at 120 mV and transferred onto nitrocellulose at 75 mV in 4°C for 2 hrs. Nitrocellulose membranes were stained with Ponceau S stain to check for even loading prior to antibody probing.

Real Time qPCR
Snap frozen tissue samples were immediately suspended in Trizol. Total RNA was extracted according to manufacturer's protocol and 1 μg of total RNA was reverse transcribed using Superscript III reverse transcriptase (Invitrogen).
Primers were designed from mouse sequences corresponding to the genes of interest provided below: as-5'-GTCAGTGGCAAAAGCTCCTC-3' Excel and the number of regions containing endomyocardial fibrosis was normalized by the number of regions containing muscle as follows: (blue)/(red) x 100, which represents (number of regions containing fibrosis)/(number of regions containing cardiac muscle) x 100. WTBMT: n = 2 and RBMT: n = 4. n = number of mice. All coronal sections were obtained at mid-ventricular level.
To quantify collagen content, hydroxyproline content was determined with 10 mg of homogenized WTBMT and RBMT cardiac tissue using the hydroxyproline Assay Kit (Sigma-Aldrich), according to the manufacturer's recommendation.

Myocyte Cross Sectional Area Analysis
Following deparaffinization and hydration to ddH 2 O and 1X PBS, sections were incubated for 30 mins with wheat germ agglutinin conjugated with Alexa Fluor 546 (red) diluted at 1:1000 in Hank's Buffered Saline Solution (HBSS) and washed in two changes of 1X PBS prior to mounting with Vectashield Mounting Medium with DAPI counterstain (Vector). Images were acquired using a Nikon Eclipse 80i microscope with NIS Elements AR software. Images were analyzed for myocyte cross sectional area using ImageJ software. Measurements were obtained from five randomly selected fields within the endocardial region of the heart.

TSP1 Immunohistochemistry
Following deparaffinization and hydration to ddH 2 O, sections were immersed in citrate buffer pH 6.0 (Thermo Scientific) and microwaved for 1.5 mins and subsequently placed in the pressure cooker for 20 mins followed by cooling at 20 mins at room temperature. Elements of the LP system kit were used for this stain (Thermo Scientific

CD31 Quantification
Following deparaffinization and hydration to ddH 2 O, sections were immersed in citrate buffer pH 6.0 (Thermo Scientific) and microwaved for 1.5 mins and subsequently placed in the pressure cooker for 20 mins followed by cooling at 20 mins at room temperature. Sections were then treated with 3% hydrogen peroxide for 10 mins at room temperature to quench endogenous peroxidase activity followed by permeabilization with 0.3% Triton X-100 in 0.05%

TSP1/CD31 Dual Immunostain and Confocal Microscopy
Following deparaffinization and hydration to ddH 2 O and 1X PBS, sections were immersed in citrate buffer pH 6.0 (Thermo Scientific) and placed in the pressure cooker for 20 mins followed by cooling at room temperature for 20 mins.
Sections were then treated with 3% hydrogen peroxide (Fisher Scientific) for 10 mins to quench endogenous peroxidase activity. Permeabilization was achieved by incubating sections in 0.3% Triton X-100 in 1X PBST for 10 mins.

GFP/CD31 Dual Immunostain
Following deparaffinization and hydration to ddH 2 O and 1X PBS, sections were immersed in citrate buffer pH 6.0 (Thermo Scientific) and placed in the pressure cooker for 20 mins followed by cooling at room temperature for 20 mins.