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Skeletal muscle energetics and function in sickle cell disease murine model: a synergetic effect of hydroxyurea and endurance training?1Aix-Marseille Univ, CNRS, CRMBM, Marseille, France, 2Laboratoire Interuniversitaire de Biologie de la Motricité, Chambéry, France
Synopsis
In sickle cell disease (SCD), hydroxyurea can increase foetal haemoglobin production and restore oxygen-carrying capacity of patients. As endurance training is also known to improve muscle aerobic capacity, one can wonder whether hydroxyurea and endurance training can have a synergetic effect. Townes mice (model of SCD) have been trained with and without hydroxyurea for 8 weeks. Muscle energetics and function were assessed in response to a rest-stimulation-recovery protocol before and after interventions. While training increased force production, mitochondrial function was improved when training was combined to hydroxyurea supplementation. In SCD, this combination would have a synergetic effect in muscle energetics.
Introduction
Sickle cell disease (SCD) is an inherited hemoglobinopathy resulting in a production of sickle Hb (HbS) which can polymerize in deoxygenated conditions, eventually leading to the sickling of red blood cells (RBC)1. SCD patients suffer from severe chronic haemolytic anaemia2 and arterial desaturation3,4, resulting in tissue hypoxia5,6. All organs ache from these alterations, including skeletal muscle7–9. The decreased oxygen-carrying capacity can alter energetics and function in response to exercise in patients and mice model. Any strategy which could reverse metabolic and functional defects could be of interest.Hydroxyurea (HU) is commonly used is SCD in order to produce foetal haemoglobin (HbF)10,11, reduce sickle haemoglobin level and ultimately alleviate SCD-related symptoms12. HU treatment has also been related to an increase in anaerobic and aerobic capacities in patients13.
Regular physical activity can also be considered as an interesting therapeutic strategy. Endurance training has already been proven as efficient for improving oxygen supply14,15. In SCD patients, moderate-intensity endurance-exercise has been reported safe in adults and linked to an improved functional capacity16. These results have been further confirmed in an SCD animal model17.
Overall, one can wonder whether endurance training and hydroxyurea supplementation can have a synergetic effect on muscle function and more specifically on muscle energetics considering the tight link between energetics and oxygenation18.
Methods
Townes mice (the SCD murine model) are expressing the human haemoglobin and display the typical clinical phenotype of SCD patients such as anaemia, sickling and organs dysfunction19. Skeletal muscle energetics and function of twenty-one mice (2 ± 0.5 months-olds) were assessed in response to a standardized Rest-Stimulation(1Hz)-Recovery protocol before and after interventions. Investigations were conducted using an homebuilt experimental setup that has been designed to be operational inside the PharmaScan® gradient insert (Bruker BioSpin MRI Gmbh, Ettlinge, Germany)20. The setup allows (i) magnetic resonance imaging (MRI), (ii) muscle force measurements and (iii) 31P-magnetic resonance spectroscopy (31P-MRS). MRI was used to get anatomical information about the hindlimb. The posterior hindlimb muscle mechanical performance was assessed with a dedicated homemade ergometer consisting of a foot pedal coupled to a force transducer. 31P-MRS was used to dynamically monitor the levels of high-energy phosphorylated compounds and acidosis.Nine mice (END) were subjected to an 8-weeks endurance training on a treadmill, with 55 minutes sessions five days a week, including one-week acclimatation17. Training started at 15m/min and increased progressively during the weeks up to 17m/min. Twelve mice (END-HU) were submitted to an 8-weeks hydroxyurea supplementation in drinking water at 50 mg/kg/day21, associated with endurance training. Assessments conducted before intervention are indicated through number 1 (END1) and post-intervention session with number 2 (END2).
Results
As illustrated in figure 1, total specific peak force was significantly higher in the post-intervention groups (END2 and END-HU2, +46% and +39% respectively, p < 0.05) as compared to the corresponding pre-intervention conditions (END1 and END-HU1), with no other significant difference between groups.The rate of PCr recovery (VPCrrec) was accelerated in END-HU2 as compared to END-HU1 (+83%, figure 2, p < 0.05). No other significant difference was found after endurance intervention only.
Discussion
Both type of interventions allowed to increase total specific peak force as compared to their pre-intervention condition. It has been previously reported that HU enhanced anaerobic and aerobic capacity in patients13 and that endurance training also increased skeletal muscle function16. The present result further supports the beneficial effects of each and both interventions. However, a synergetic effect was not identified.Of interest, the rate of PCr recovery, a well-known index of mitochondrial function22, was accelerated as a result of the combination between HU and endurance training whereas the corresponding kinetics was not modified by the endurance training modality. One must keep in mind that PCr recovery kinetics is tightly linked to oxygenation. Indeed, in hyperoxic conditions, PCr recovery kinetics can be accelerated18, whereas hypoxia led to a reduced rate of PCr resynthesis22. Considering the anaemic status of Townes mice, one could have expected a reduced VPCrrec19. Recent studies reported an unchanged recovery kinetics in women with SCD23 and Townes mice24 compared to their control counterparts thereby illustrating that the mitochondrial function might not be compromised in SCD.
The synergetic effect of both interventions can be compared to the effects of hyperoxia in a trained population. It has been reported that trained subject can benefit from hyperoxia given their larger mitochondrial content. More specifically, an hyperoxia-mediated accelerated PCr recovery has been measured in trained subjects whereas it was not in sedentary controls22. Endurance training would lead to an increased mitochondrial content25,26, so that the beneficial effect in hyperoxia could be observed. Such an effect would not be observed when mitochondrial content is unchanged. In our case, HU might create the hyperoxic condition given the increased oxygen delivery to skeletal muscle related to the increased production of HbF27 and NO10. Training only would not be effective in Townes mice likely because of the anaemic status.
Overall, the combination of endurance training and hydroxyurea supplementation did improve the PCr recovery kinetics of Townes mice.
Conclusion
In conclusion, a potential synergetic effect of endurance training and hydroxyurea treatment has been demonstrated for skeletal muscle energetic and function in a sickle cell disease mice model.Acknowledgements
This work was partly funded by France Life Imaging (grant ANR-11-INBS-0006), AFM and Region Sud.
Hydroxyurea has been kindly provided by the ADDMEDICA company.
Constance MICHEL is a PhD student supported by Aix-Marseille University.
CRMBM is a research lab funded by CNRS.
Laboratoire Inter Universitaire de Biologie de la Motricité is a research group funded by the Université Savoie Mont-Blanc.
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Figures
Specific total force production from the 6 minutes moderate exercise of each condition before and after intervention. Results are presented in boxplot. Statistical results are assessed on the boxplot.
VPCrrec, the rate of PCr recovery, after the moderate exercise of each condition, before and after intervention. Results are presented in boxplot. Statistical results are displayed on the boxplot.
Time-dependant changes in specific peak force throughout the moderate exercise in each condition. Results are presented as mean ± SEM.
