Fig. 3

3-OST-7 regulates tpm4-driven myofibrillogenesis, sarcomere assembly and ventricular contraction.

Whole mount IHC was performed on fixed 48(injected with control 3-OST-3Z MO) or 3-OST-7 morphant embryos to detect cardiac sarcomere proteins (n = 30 for each group). The heart was then dissected out of the embryo, mounted on cover slips, and imaged using a confocal microscope (thus, the dorso-ventral orientation of the mounted hearts was random). IHC using anti-Tnnt2 antibody and anti-Tpm antibody revealed levels of these proteins were greatly reduced in 3-OST-7 morphants (B and F, dashed lines outline the hearts) compared to control (A and E) embryos. TEM of control (C) and 3-OST-7 morphant (G) hearts show the presence of organized myofibrils (red arrowheads) in control and absence in morphants. ISH for tpm4 showed tpm4 transcript levels were decreased in 3-OST-7 morphants (H) compared to control embryos (D) at 48 hpf. (D and H) are ventral views with anterior on top; n = 40 for each group. Overexpression of tpm4 rescues the expression of Tnnt2 (I) and Tpm (J) proteins, assembly of myofibrils (K), and the noncontracting ventricle phenotype in 3-OST-7 morphant embryos as assessed by ejection area measurements (L, p<0.05, *). The ejection area, a measure of contractility, was obtained by computing the difference between systolic and diastolic area for either atrium or ventricle. At, atrium; V, ventricle; error bars, SEM.