Cerulo, Giuliana (2016) Difetti cardiovascolari nel modello murino di mucopolisaccaridosi IIIB. [Tesi di dottorato]

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Mucopolysaccharidoses (MPS) comprise a group of 11 distinct lysosomal storage diseases due to inherited deficiencies of the enzymes involved in glycosaminoglycan (GAG) catabolism. The progressive deposition of GAGs in the tissues results in multi-organ system dysfunctions that varies with the specific enzyme mutation present and the particular GAG deposited. The typical symptoms of the diseases include: organomegaly, dysostosis multiplex, mental retardation and developmental delay. There are currently four main therapeutic options for MPS which include hematopoietic stem cells transplantation (HSCT), the replacement of the missing enzyme with its normal recombinant counterpart (enzyme replacement therapy, ERT), substrate reduction therapy, and gene therapy. However, all of these therapeutic approaches, some of which are still under investigation, show a limited efficacy, and MPS patients usually do not survive beyond the second decade of their life. Among the different causes of the early death of MPS patients, cardiac disorders appear to play a key role. Indeed, cardiac dysfunctions have been reported in patients affected by all types of MPS, although early and evident symptoms have been mainly reported for those with MPS I, II, and VI. In particular, cardiac valve thickening, dysfunction (more severe for left-sided than for right-sided valves), and hypertrophy are commonly present, but conduction abnormalities, coronary artery and other vascular involvement have been also observed in these patients. Reported cardiac causes of death include heart failure, sudden death from arrhythmias (including complete atrioventricular block) and coronary occlusion. While cardiac involvement has been well established for most of MPS types, there is a lack of information about cardiac disorders in MPS IIIB. Mucopolysaccharidosis (MPS) IIIB is an autosomal recessive disorder caused by mutations in the gene encoding α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. Patients with MPS IIIB are characterized by profound mental retardation, behavioral problems and death usually in the second decade, along with somatic manifestations that are highly variable among the different phenotypes. In order to get more insights into the cardiac involvement in MPS IIIB disease, in this study we used the murine model of the disease obtained by NAGLU gene disruption (NAGLU knockout mice, NAGLU-/-). These mice exhibit a massive increase of HS deposition in liver and kidney, and at a lesser extent in lung, spleen, thymus, and heart. The only GAG accumulated in NAGLU-/- mouse tissues is represented by HS due to the specific enzymatic defect that characterizes MPS IIIB disease from other MPS syndromes in which accumulation of GAGs is mixed. Here, we investigated cardiac morphology and function in NAGLU-/- mice compared to wild-type (WT) littermates over time using cardiac ultrasound, and histological and biochemical methodologies. As defects in lysosomal autophagy pathway were found in the murine models of MPS IIIA and MPS VI, we also investigated the autophagic process in the heart tissues of NAGLU-/- mice, in order to explore the hypothesis that an abnormal lysosomal autophagic activity might be responsible for cardiac dysfunctions in MPS IIIB. Echocardiographic analysis showed a marked increase in left ventricular (LV) mass, reduced cardiac function and valvular defects in NAGLU-/- mice as compared to wild-type littermates. The NAGLU-/- mice exhibited a significant increase in aortic and mitral annulus dimension with a progressive elongation and thickening of anterior mitral valve leaflet. A severe mitral regurgitation with reduction in mitral inflow E-wave-to-A-wave ratio was observed in 32 week old NAGLU-/- mice. Compared to WT mice, NAGLU-/- mice exhibited a significantly lower survival with increased mortality observed in particular after 25 weeks of age. Histopathological analysis revealed a significant increase of myocardial fiber vacuolization, accumulation of HS in the myocardial vacuoles, recruitment of inflammatory cells and collagen deposition within the myocardium, and an increase of LV fibrosis in NAGLU-/- mice compared to WT mice. Biochemical analysis of heart samples from affected mice showed increased expression levels of cardiac failure hallmarks such as calcium/calmodulin dependent protein kinase II, connexin43, α-smooth muscle actin, α-actinin, atrial and brain natriuretic peptides, and myosin heavy polypeptide 7. Furthermore, heart samples from NAGLU-/- mice showed enhanced expression of the lysosome-associated membrane protein-2 (LAMP2), and the autophagic markers Beclin1 and LC3 isoform II (LC3-II). Overall, our findings demonstrate that NAGLU-/- mice develop heart disease, valvular abnormalities and cardiac failure associated to an impaired lysosomal autophagic activity. Recently, it was reported that diastolic LV function and contraction synchrony were normalized after laronidase treatment in a patient with MPS I. Therapeutic efforts are under way for MPS IIIA patients, including intratechal ERT and gene therapy. On the light of our results on cardiac impairment in MPS IIIB mice, we suggest that it would be interesting to investigate the effect of current therapies on the cardiac function also in MPS IIIB patients.

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