Generation and characterization of a Myh6-driven Cre knockin mouse line

Fig. 1. Strategy for generating Myh6-Cre mice. a The strategy for generating Myh6-Cre mice. The IRES-Cre-wpre-polyA cassette was inserted between the translational stop codon and the 3'UTR. b PCR results show that homologous recombination was detected in the F0 generation mice. c Locations of the genotyping primers and an example of genotyping result showing the PCR products

Fig. 2. Myh6-Cre labels cardiomyocytes in embryonic hearts. (a, c) Whole-mount view of the Myh6-Cre/ + ;R26-tdTomato/ + embryos at E8.5 and E9.5. b Immunostaining for tdTomato and TNNI3 on embryonic sections at E9.5. The arrows in (a–c) indicate hearts with enriched tdTomato signals. d Whole-mount view of the organs from Myh6-Cre/ + ;R26-tdTomato/ + mice at E13.5. (e–g) Immunostaining for tdTomato and TNNI3, WT1 or CDH5 on heart sections from Myh6-Cre/ + ;R26-tdTomato/ + mice at E13.5. Boxed regions are magnified on the right. 4 embryos were examined for each embryonic stage. Red scale bars, 1 mm; White scale bars, 100 µm

Fig. 3. Myh6-Cre labels cardiomyocytes in infant mice. (a, b) Whole-mount view of the hearts from Myh6-Cre/ + ;R26-tdTomato/ + mice at P2W. Scale bars, 5 mm. (c, d) Immunostaining for tdTomato and TNNI3 or PDGFRa on heart sections from Myh6-Cre/ + ;R26-tdTomato/ + mice at P2W. (e, f) Immunostaining for tdTomato, CDH5 and PDGFRb or aSMA on heart sections from Myh6-Cre/ + ;R26-tdTomato/ + mice at P2W. Boxed regions in (c–f) are magnified on the right. Scale bars in (c–f), 100 µm. Each picture is representative of 3 individual mouse samples

Fig. 4. Myh6-Cre labels cardiomyocytes and a small portion of smooth muscle cells in adult hearts. (a, b) Whole-mount view of the hearts from adult Myh6-Cre/ + ;R26-tdTomato/ + mice. Scale bars, 2 mm. (c, d) Immunostaining for tdTomato and TNNI3 or PDGFRa on heart sections from adult Myh6-Cre/ + ;R26-tdTomato/ + mice. (e, f) Immunostaining for tdTomato, CDH5 and PDGFRb or aSMA on heart sections from adult Myh6-Cre/ + ;R26-tdTomato/ + mice. The arrows in (f) indicate tdTomato-labelled smooth muscle cell. g Immunostaining on heart sections from adult Myh6-MerCreMer + ;R26-tdTomato/ + mice, which were administered with tamoxifen 48 h before harvest. The arrows in (g) indicate cardiomyocytes with weak tdTomato signal in the cytoplasm. Boxed regions are magnified as indicated. Scale bars in (c–g), 100 µm. Each picture is representative of 3 individual mouse samples

Fig. 5. Myh6-Cre targets some other organs outside the heart and affects the expression of endogenous Myh6 in heart. a tdTomato was detected in some other organs from the adult Myh6-Cre/ + ;R26-tdTomato/ + mice at P8W. 3 mouse samples were examined. b ECG analyses of the P8W hearts with indicated genotypes. n = 6 mice per group. LVESD, left ventricular end-systolic dimension; LVEDD, left ventricular end-diastolic dimension; ESV, end-systolic volume; EDV, end-diastolic volume; EF, ejection fraction; FS, fractional shortening. c Whole bodies of the mice with indicated genotypes at P8W. d Whole-mount view of the hearts with indicated genotypes at P8W. e The relative heart weight was increased in the Myh6-Cre/Cre mice at P8W. HW, heart weight. BW, body weight. f HE staining shows the thinner left ventricular wall and ventricular septum in the hearts of Myh6-Cre/Cre mice at P8W. g WGA staining on heart sections. h The expression level of Myh6 in the adult hearts at P8W. n = 3 mice per group in (c–h). White scale bars, 5 mm; Black scale bars, 2 mm; Red scale bars, 200 µm; Yellow scale bars, 100 µm. NS, non-significant; *p < 0.05; ***p < 0.001

Fig. 6. Inactivation of the Dsp gene in cardiomyocytes using Myh6-Cre. a The strategy for inactivating the Dsp gene in cardiomyocytes using Myh6-Cre. Primers P5 and P6 were used to detect exon 2. b The relative expression level of Dsp mRNA in hearts or non-cardiac tissues at E10.5. *** p < 0.001; NS, non-significant. n = 3 hearts per group. (c, d) Whole-mount view of embryos and hearts at E11.5 and E13.5. n = 4 embryos per group. Scale bars, 2 mm