Figure 7. SM22α expression is increased in cells present in the subepicardium and shallow myocardium of Pod1^−/− mouse hearts.

(A–D) Immunohistochemistry (IHC) was performed on E14.5 and E17.5 mouse heart sections using anti-SM22α antibody (brown). (A,C) SM22α is primarily expressed in coronary vascular SM (asterisks) of Pod1^+/− controls at E14.5 (A) and E17.5 (C). SM22α is rarely expressed in the subepicardium (arrows, insets) of Pod1^+/− hearts. (B,D) In Pod1^−/− embryos, SM22α is expressed in EPDCs (arrows, insets) and in interstitial cells (arrowheads, insets) at E14.5 (B) and E17.5 (D). (E,F) Differentiation of Pod1-deficient EPDCs into SM was analyzed by double IF using anti-βGal (green) and anti-SM22α (red) antibodies. (E) In E17.5 Pod1^+/− mice, heterozygous for the Pod1/LacZ locus, βGal⁺ subepicardial cells (green) are distinct from SM22α⁺ cells (red) in the subepicardium (arrow, inset) and in coronary vascular SM (asterisk). (F) In Pod1^−/− hearts, Pod1-deficient βGal⁺ cells co-express SM22α (yellow) in the subepicardium (arrowheads, inset). (G) Total SM22α⁺ subepicardial and shallow intramyocardial cells per section at E17.5, labeled by IHC as in C–D, were quantified. Statistical significance of differences between Pod1^+/− controls and Pod1^−/− was determined by Student’s t-test (n=3). *P•0.01, #P•0.05.