Vitamin D Supplementation Partially Rescues Breathing Deficits in Mecp2 Hemizygous Null Mice, a Model of Rett Syndrome

Abstract

Rett syndrome (RTT) is a severe X‐linked progressive neurodevelopmental disorder affecting ~1 in 10,000 females. It is the second most common genetic cause of cognitive disability in girls, after Down syndrome. Girls with this devastating disorder develop relatively normally for 6‐18 months, after which they undergo a period of rapid regression. They lose many motor skills, the growth of their brain slows, and they develop repetitive, autistic behaviors and seizures. Autonomic disruptions are a leading cause of early death in RTT, and rescue of breathing deficits is considered a primary goal in the development of therapeutic interventions. We initiated a proof‐of‐concept study by investigating male Mecp2 hemizygous null mice as they have the strongest phenotype which occurs at an early age; male Mecp2‐null (KO) mice have also been characterized thoroughly in the literature. We tested the hypothesis that Vitamin D supplementation (VitD) would ameliorate, or partially rescue breathing and cardiovascular deficits in Mecp2 KO mice. Mice were tested at 4 weeks of age (PRE), then placed on a control (1 IU Vitamin D/g) or VitD (50 IU Vitamin D/g) diet, and re‐tested at 8 weeks of age (POST). To examine our hypothesis, unrestrained barometric plethysmography was used to quantify breathing frequency (breaths/min), tidal volume (VT; mL/breath), VT/inspiratory time (VT/Ti; mL/sec), minute ventilation (VE; mL/min/g) and the ventilatory equivalent for CO2 (VE/VCO2) in both wildtype (WT; n=6) and Mecp2 KO (n=5) littermates. Data Sciences International Ponemah software was used to analyze flow tracings during exposure to air (20.93% O2, balanced N2). The MouseOx Plus by STARR Life Sciences was utilized to collect heart rate (HR) and oxygen saturation in freely moving conscious mice. Data are expressed as mean ± S.D., WT vs. KO. In the PRE group, Mecp2 KO mice had dysregulated breathing and HR as previously demonstrated in the literature (data not shown). In the POST VitD diet groups, VT (0.24±0.02 vs 0.15±0.06 mL/breath; p=0.01) and VT/Ti remained lower (2.085±0.313 vs. 1.291±0.583 mL/sec; p=0.017) in Mecp2 KO. However, breathing frequency (166±21 vs. 166±19 breaths/min), VE/g (1.07±0.12 vs. 0.99±0.57 mL/min/g), ratio of VE/VCO2 (112±47 vs. 103±46) and HR (582±84 vs. 532±59 beats/min) were similar between WT and KO. The number of oxygen desaturations were not significant between WT and KO, but the values were variable across groups and included a number of desaturations (12±7 vs. 5±7 desaturations/30 minutes), similar to findings we have previously described in young control mice. These data reveal a partial rescue of breathing and HR deficits in Mecp2 KO mice following 4 weeks of VitD. The reduced VT/Ti suggests an attenuated neural drive to breath in Mecp2 KO vs. WT that persists after VitD. Current studies are examining the responses to hypoxia and hypercapnia and will identify more information about oxygen and carbon dioxide sensing in these mice following control and VitD diets.