Hearts from 8-week TAC studies were harvested for analyses of CSA and fibrosis. Masson’s trichrome staining was performed as previously described(45 (link)). Briefly, mice were euthanized, and hearts were fixed overnight in 4% paraformaldehyde at 4°C. Hearts were dehydrated and paraffinized followed by embedding in paraffin blocks. Blocks were sectioned at 5 μm and stained with Weigert’s iron hematoxylin and Masson trichrome (Sigma-Aldrich) according to the manufacturer’s instructions. Alternatively, tissue sections were deparaffinized and rehydrated according to the trichrome staining protocol, but after the wash with deionized water, heart sections were stained with Alexa Fluor 594–conjugated wheat germ agglutinin (10μg/mL in 1× PBS) (Invitrogen) for 1 hr at room temperature in a humidified chamber in the dark. Sections were washed 3 times for 5 min with PBS followed by mounting with coverslips using Fluoromount-G mounting media containing DAPI nuclear stain (Southern Biotech). CSA was measured at 20x magnification by tracing individual cells in ImageJ. Fibrosis was assessed by a thresholding method in ImageJ as described previously(46 ).