CaM expression in zebrafish does not affect embryo development or heart morphology. (A) Representative images of 72 hpf embryos from eggs injected with CaM variant mRNA. Embryos overexpressing CaM WT, N97S or N53I display normal morphology and development. (B) Z-stack images of 72 hpf embryonic hearts that were microdissected and immunostained for slow muscle fibres (F59). (C) Developmental rates measured by counting somites at 22 hpf. No significant difference between control, CaM WT, N97S and N53I injected embryos was observed (P = 0.85).

Monitoring of embryo heart contractions. Examples of changing pixel intensities (CPIs) and M-modes for β-adrenergic stimulated embryos injected with CaM WT, N53I or N97S mRNA, respectively. CPIs have peaks at the onset of diastole and systole (stars). M-mode shows embryonic ventricle movement. The mean diastolic interval was 0.173 ± 0.028 s for the control and 0.177 ± 0.017 s for CaM WT compared to 0.156 ± 0.023 s and 0.151 ± 0.025 s for CaM N53I and for CaM N97S (both P < 0.05 compared to WT), respectively. The mean systolic interval was 0.264 ± 0.024 s for the control and 0.263 ± 0.027 CaM WT compared to 0.256 ± 0.031 s for CaM N53I and 0.262 ± 0.027 for CaM N97S. No significant difference in systolic interval was observed (P = 0.9).