Fig. 6

Loss of MBNL1 leads to changes in matrix composition and TGFβ signaling in the heart valves. a High magnification view of the septal leaflet of the tricuspid valve on a coronal section of an adult Mbnl1 ^∆E3/∆E3 heart stained with Masson’s trichrome reveals swellings of collagen-poor tissue in the valve (black arrowhead). The inset shows the corresponding valve leaflet in a wild type heart. Histological analysis was performed on sections from both male and female wild type (n = 7) and knockout (n = 12) mice ranging in age from 4 to 15 months. Representative images shown are from 5 month-old mice. b High magnification view of the mural leaflet of the tricuspid valve on a coronal section of an adult Mbnl1 ^∆E3/∆E3 heart stained with Movat’s pentachrome reveals normal stratification but reduced mucin staining indicative of glycosaminoglycans in the regions of swelling. The inset shows a comparable wild type leaflet. Movat’s pentachrome staining was performed on heart sections from three mice for each group ranging from 10 to 15 months old; representative images shown are from 15 month-old mice. c Western blot analysis confirms loss of MBNL1 protein, and shows a decrease in chondroitin sulfate proteoglycans (CSPG) and increases in fibronectin, phosphorylated SMAD2 (pSMAD2), and MBNL2 in Mbnl1 ^∆E3/∆E3 heart valves compared to wild type. Protein integrity and loading were also confirmed by Ponceau S staining (data not shown). Representatives of three independent sets using samples from 5–6 month-old male mice are shown. d Transcript levels in Mbnl1 ^∆E3/∆E3 heart valves were compared to those of wild type heart valves by real-time RT-PCR (n = 5 to 6 each, ~10 month-old males). e Accumulations of fibronectin (red arrowheads) were seen in the Mbnl1 ^∆E3/∆E3 tricuspid valve by immunohistochemistry. The inset shows a tricuspid valve leaflet from a wild type heart. Immunohistochemistry was performed on heart sections from three mice for each group ranging from 4 to 10 months old; representative images shown are from 4 month-old mice