Protective effects of intercalated disk protein afadin on chronic pressure overload-induced myocardial damage

Adhesive intercellular connections at cardiomyocyte intercalated disks (IDs) support contractile force and maintain structural integrity of the heart muscle. Disturbances of the proteins at IDs deteriorate cardiac function and morphology. An adaptor protein afadin, one of the components of adherens junctions, is expressed ubiquitously including IDs. At present, the precise role of afadin in cardiac physiology or disease is unknown. To explore this, we generated conditional knockout (cKO) mice with cardiomyocyte-targeted deletion of afadin. Afadin cKO mice were born according to the expected Mendelian ratio and have no detectable changes in cardiac phenotype. On the other hand, chronic pressure overload induced by transverse aortic constriction (TAC) caused systolic dysfunction, enhanced fibrogenesis and apoptosis in afadin cKO mice. Afadin deletion increased macrophage infiltration and monocyte chemoattractant protein-1 expression, and suppressed transforming growth factor (TGF) β receptor signaling early after TAC procedure. Afadin also associated with TGFβ receptor I at IDs. Pharmacological antagonist of TGFβ receptor I (SB431542) augmented mononuclear infiltration and fibrosis in the hearts of TAC-operated control mice. In conclusion, afadin is a critical molecule for cardiac protection against chronic pressure overload. The beneficial effects are likely to be a result from modulation of TGFβ receptor signaling pathways by afadin.

dimensions and pump function were monitored in vivo by transthoracic ultrasonography on Vevo 2100 system (VisualSonics Inc, Canada). During the procedure, mice were anesthetized in supine position on 37°C heating table with isoflurane/air mixture (induction with 4% and maintenance with ~1.5% isoflurane). Two dimensional (B mode) guided M mode measurement of LV parameters was accomplished from parasternal short-axis position at the level of papillary muscles. LV mass (mg) was calculated based on the formula: 1.055 x [(LVDd + LVPWd + IVSd) 3  relative LV wall thickness is calculated as: 2 x LVPWd (mm) / LVDd (mm). Success of TAC operation was confirmed by evaluation of vascular hemodynamics using pulse-wave Doppler mode to measure peak velocity of blood flow in the left and right carotid arteries 2,3 .

Histological staining of heart sections and immunohistochemistry
Mice hearts were snap-frozen by liquid nitrogen within a block of water-soluble medium (Surgipath FSC 22, Leica Biosystems, USA), or fixed with 4% paraformaldehyde and subsequently embedded in paraffin blocks. Cryosections, 10 µm thick, were fixed by 4% paraformaldehyde on the top of poly-L-lysine-coated slides immediately after cutting in a cryostat (Leica Biosystems, USA), then permeabilized with 0.1% Triton X-100, and blocked with 1% bovine serum albumin (BSA). Primary antibodies (Abs) were applied in the BSA

Transmission electron microscopy
Mice myocardium was prepared as described previously 7 . Briefly, mouse myocardium was initially fixed with 4% paraformaldehyde and 2.5% glutaraldehyde, incubated in OsO 4 , dehydrated in stepwise increasing concentrations of ethanol, and embedded in epoxy resin.
Ultrathin sections mounted on mesh grids were double-stained with 2% uranyl acetate / lead citrate, and observed by Hitachi H7500 transmission electron microscope.

Apoptosis assays
To detect apoptotic nuclei, recombinant terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL) reaction was performed on sections of frozen hearts (DeadEnd TM Fluorometric TUNEL System, Promega, USA) and fluorescence of stained nuclei was visualized by confocal microscopy. For each studied group of animals, the percentage of TUNEL-positive nuclei is estimated after evaluation of 1000 nuclei derived from 3 animals.
Activated apoptotic signaling in the cardiomyocytes was assessed by immunostaining of frozen cardiac sections for cleaved caspase 3 and area of positive staining is compared among all groups (3 animals per group).

Western blotting and immunoprecipitation
Mouse heart tissue was homogenized mechanically in the lysis buffer containing 320 mmol/L sucrose, 20 mmol/L Tris-HCl (pH 7.5), 2 mmol/L EDTA, 0.1% Triton X-100, and supplied with protease inhibitors (20 µg/mL aprotinin, 20 µg/mL leupeptin, 1 mmol/L PMSF). After The ratio between phosphorylated and total protein molecule of interest was estimated on the same PVDF membrane: first, primary Ab against phosphorylated protein was probed, then membrane was stripped (WB Stripping Solution, Wako Chemicals, Japan), blocked again with skim milk, and Ab against the total protein was applied. Protein samples for immunoprecipitation, 300 µg each, were pre-cleared with protein G beads (GE Healthcare Life Sciences, UK), incubated with 1.5 µg anti-transforming growth factor (TGF) β receptor I, 2 µg anti-afadin or 2 µg anti-GFP Ab for immunoprecipitation overnight, and finally protein G beads were separated from Ab/protein complex in SDS-PAGE sample buffer heated at 95°C.
For detection, Western blots using primary Abs against the both proteins of interest were performed.