Glucocorticoids play a key role in maturation and structural organisation of the developing zebrafish cardiovascular system

Glucocorticoids (GCs) are essential for organ system development during embryogenesis, with complete global knock out of the GC receptor (GR) in rodents resulting in multi-organ defects. Less is known about the subtle, transient effects of GC alterations during early fetal development and the influence on cardiac phenotype. We have explored the effects of mild alterations in GC dynamics in the zebrafish focusing on structure and function of the heart during the first 120 hours post fertilisation. The zebrafish GC system was modulated from the 2-cell stage until 120 hours post fertilisation (hpf) this was achieved by targeting GR pharmacologically, using either the GR-selective agonist dexamethasone (dex) [100μM] or the GR antagonist mifepristone (RU486) [10μM], or molecularly by transiently knocking-down GR mRNA with targeted morpholino (Mo). GCs were measured by ELISA and mass spectrometry. Assessment of cardiac structure and function during early development was achieved using transgenic zebrafish expressing green fluorescent protein under the cardiomyosin light chain 2 promoter. Temporal and spatial gene and protein expression studies were carried out using qRT-PCR, western blot analysis and immunohistochemistry. Reduced GR activity, in both RU486 and Mo groups, resulted in smaller (p<0.05) hearts at 120hpf (67±3.5 and 75±2.4μm, respectively) compared to controls (90.2±0.8μm) with impaired cardiac function (heart rate and ejection fraction). Isolated hearts showed an immature cardiac phenotype, comprising abnormal myofibril organisation with reduced striation pattern and smaller, less dense nuclei. Gene expression analysis of growth factors and cardiac structural proteins suggested these hearts were developmentally less mature than controls (ventricular myosin heavy chain and insulin like growth factor expression were reduced (p<0.001 and p<0.05 respectively) at 120hpf. GR stimulation with dex resulted in larger hearts (107.2±4.5 compared to 90.2±0.8μm p=0.048) with increased ejection fraction. Isolated hearts had enhanced myofibril organisation with clear striation but had fewer cardiomyocytes compared to controls (p=0.034) suggesting cardiac hypertrophy. These results show that GCs, acting through GR, contribute to regulation of cardiac structure and function during maturation of the zebrafish. In demonstrating this relationship we show that subtle alterations in GC dynamics are significant enough to alter cardiac phenotype, which may in turn lead to cardiac disease or disorder in later life.