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Title: Investigation of phenotypic rescue of Mybpc3 deficient mouse
Author: Abou Al Saud, Sara
ISNI:       0000 0004 6346 8882
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2015
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Mutations in the myosin binding protein C gene (MYBPC3) are a frequent cause of hypertrophic cardiomyopathy (HCM) and calcineurin plays a major role in hypertrophic remodelling. However, a functional link between MYBPC3 mutations and calcineurin has not been investigated. Mybpc3 knock out (KO) mice were generated and an increase in the regulator of calcineurin 1 (Rcan1) mRNA expression was detected, indicating an increase in calcineurin activity (Knöll et al. unpublished data). Accordingly, it was hypothesised that calcineurin, particularly its major β-isoform (CnAβ), plays a role in the pathogenesis of these mice. Therefore we investigated Mybpc3/CnAβ double KO (dKO) mice. Our results confirmed that the severe heart failure (HF) phenotype observed in Mybpc3 KO mice is completely rescued by additional ablation of CnAβ as judged by echocardiography, gravimetric analysis, histology and electron microscopy studies. We also have measured muscle contractility in skinned cardiac trabeculae from dKO mice and demonstrated that the rescue was present at the level of the contractile apparatus. Moreover, this rescue was specific to Mybpc3 KO mice as the phenotype of a mouse model expressing the apical hypertrophic cardiomyopathy-causing mutation ACTC E99K actin was more severe in ACTC E99K/ CnAβ double transgenic (dTG) mice. Crucially, it was found that ventricular myosin light chain (MLC2v) was hyperphosphorylated in the Mybpc3/CnAβ dKO mice. Furthermore, calcineurin was shown to dephosphorylate MLC2v in vitro. We therefore investigated whether MLC2v hyperphosphorylation per se could rescue the Mybpc3 KO phenotype. Mybpc3 KO mice were injected with AAV9 overexpressing pseudophosphorylated MLC2v (S14/15D; AAV9-pMLC2v), cardiomyocytes were transfected with adv-pMLC2v, and a construct to create a TG mice overexpressing pMLC2v was made. 3 | P a g e The AAV9-pMLC2v injected mice were studied in detail. A maximal improvement in cardiac function was observed 6 weeks after injection. Echocardiography demonstrated ~20%EF and ~25%FS increase in the Mybpc3 KO hearts whilst there was a trend to diminished performance in WT. However, two different concentrations of AAV9-pMLC2v resulted in gravimetric parameters, left ventricle (LV) dimensions, and typical HCM abnormalities indistinguishable from untreated Mybpc3 KO mice. The beneficial effects of MLC2v hyperphosphorylation on the regulation and enhancement of myocardial contractile function point to a possible molecular mechanism in attenuating the hypertrophy and enhancing myocardial function in the dKO mice. Genetic testing combined with inhibition of calcineurin and/or modification of MLC2v phosphorylation in individuals affected by MYBPC3 mutations, might provide a novel strategy to prevent and/or treat specifically this otherwise serious condition.
Supervisor: Marston, Steven ; Knoell, Ralph ; Ferenczi, Mike Sponsor: Jāmiʻat al-Malik Saʻūd
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available