Fig. 1.

Hypertrophic myocardium and disrupted sarcomeric structure in the medaka nsh mutant heart. (A,B) Morphology of the wild-type (WT) and nsh mutant heart at 3 days post fertilization (dpf). The mutant showed hypertrophic heart, especially in the atrium (double-headed arrow). The nsh heart lost elasticity and showed increased passive stiffness. Due to diastolic inability (see Movie 2), the blood flow was very slow or clogged around 3 dpf. (C,D) Confocal z-stacked images of the WT and nsh mutant hearts at 3 dpf, in which cardiomyocytes were visualized by the Tg(cmlc2: DsRed2-nuc) signal. These reconstructed confocal images show approximately half of the total cardiomyocytes. (E) Quantification of cardiomyocytes using the transgenic line. Cell counting was performed on the optical slices to attain an accurate count. There was no difference in the number of cardiomyocytes between the WT and nsh mutant hearts. NS, statistically not significant (n=5 for WT and nsh). (F-K) In situ RNA hybridization analysis showing cardiac myosin light chain 2 (cmlc2) (F,G), and atrial and ventricular myosin heavy chains (amhc and vmhc, respectively) (H,I) in the WT and nsh mutant at 3 dpf. Normal sarcomeric protein expression was observed in the nsh mutant. (J,K) Strong expression of brain natriuretic peptide (bnp; arrowheads) was detected at 3 dpf in the WT ventricle (J), whereas bnp expression was strong in the atrium of nsh mutants (K). (L,M) Transmission electron microscopic findings of medaka embryonic cardiomyocytes at 5 dpf. Myofibrillar arrays were evident in the WT heart (L), but myofibrils were reduced with loosely arranged contractile filaments in the nsh mutant (nsh −/−) (M). Thick-filament structures and integrity of the M-line region were disrupted, and Z-discs were not parallel. (N,O) Medaka adult cardiac myocytes in the WT and nsh heterozygotes (nsh +/−) were stained for α-actinin. Expression of sarcomeric α-actinin was maintained in the nsh heterozygous mutant, but fewer striations and patchy staining was observed (O). (P,Q) Transmission electron microscopy showed the rupture of sarcomeric units, as well as irregular and widened Z-discs and M-lines in cardiomyocytes of the nsh heterozygotes. The integrity of the thick filaments is disrupted (Q) Z-discs (arrowheads) and M-lines (arrows) are indicated. a, atrium; v, ventricle; Myo, myocardium. Scale bars: 50 µm (A-D),100 µm (F-K), 1 µm (L,M), 5 µm (N,O), 0.5 µm (P,Q).