NMHC IIB is not required in cardiomyocytes for coronary vessel formation.

A: Genomic PCR on tissues isolated from mice carrying the αMHC-Cre transgene and Myh10 flox/+ alleles shows a PCR product of 600 bp in cardiac tissue, consistent with deletion of Myh10 exon 2 from the genome (arrow). This product is not seen in tail, brain, or liver tissue. PCR results from a Myh10∆/+ heterozygous animal are shown for the corresponding tissues, with the 600 bp product present in all tissues. The 1 Kb product represents the Myh10 allele lacking exon 2 deletion. Products were sequenced to confirm specificity of PCR reactions. B: NMHC IIB protein (green) and C: actin (red; phalloidin stain) localisation in fibroblasts cultured from αMHC-Cre; flox/flox hearts. D: Merged image shows DAPI labeling of nuclei (blue). E: NMHC IIB protein (green) and F: actin (red; phalloidin stain) localisation in cardiomyocytes cultured from αMHC-Cre; flox/flox hearts. G: Merged image shows DAPI labelling of nuclei (blue). H: Expression patterns of NMHC IIB (white) and cardiac Troponin T (red), and nuclei stained with DAPI (blue) in αMHC-Cre; Myh10+/+ control embryo with presence of coronary vessel (orange arrow). NMHC IIB expression is seen throughout heart including valve tissue (white arrow). Co-expression of NMHC IIB and cTnT appears pink. I: Higher magnification image of cardiac tissue from αMHC-Cre; Myh10+/+ control embryo. J: Expression patterns of NMHC IIB (white) and cardiac Troponin T (red), and nuclei stained with DAPI (blue) in αMHC-Cre; Myh10 flox/flox control embryo with presence of coronary vessel (orange arrow). NMIIB expression is seen in valve tissue (white arrow) but reduced in myocardial region of heart, and is absent from cTnT positive cells, so that the pink staining indicating NMHC IIB and cTnT co-expression is reduced in the myocardium. K: Higher magnification image of cardiac tissue from αMHC-Cre; Myh10 flox/flox control embryo. Epicardial expression of NMHC IIB persists (white arrows). L: Ventral view of cardiac surface stained with DAB to identify blood cells in αMHC-Cre; Myh10+/+ heart (arrow). M: Dorsal view of cardiac surface stained with DAB to identify blood cells in αMHC-Cre; Myh10+/+ heart (arrows). N: Ventral view of cardiac surface stained with DAB to identify blood cells in αMHC-Cre; Myh10 flox/∆ heart (arrows). O: Dorsal view of cardiac surface stained with DAB to detect endogenous peroxidase activity from blood cells in αMHC-Cre; Myh10+ flox/∆ heart (arrows). Blood cells within vessels are present on the cardiac surface of cardiomyocyte-specific Myh10 mutant hearts. Similar results were seen for αMHC-Cre; Myh10 flox/flox hearts. Scale bars: B-G = 50 μm, H and J = 100 μm, I and K = 15 μm, L-O = 400 μm. Abbreviations: αMHC-Cre: Tg(Myh6-cre)2182Mds/J, CM: cardiomyocyte, cTnT: cardiac troponin T, Fibro: fibroblast, NMIIB: NMHC IIB, ∆: Myh10∆.