The Murine Model of Mucopolysaccharidosis IIIB Develops Cardiopathies over Time Leading to Heart Failure

RESEARCH ARTICLE The Murine Model of Mucopolysaccharidosis IIIB Develops Cardiopathies over Time Leading to Heart Failure Gabriele Giacomo Schiattarella2☯, Giuliana Cerulo1,3☯, Valeria De Pasquale1, Pasquale Cocchiaro1,3, Orlando Paciello3, Luigi Avallone3, Maria Paola Belfiore4, Francesca Iacobellis4, Daniele Di Napoli5, Fabio Magliulo2, Cinzia Perrino2, Bruno Trimarco2, Giovanni Esposito2, Paola Di Natale1, Luigi Michele Pavone1* a11111 1 Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy, 2 Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy, 3 Department of Veterinary Medicine and Animal Productions, University of Naples Federico II, Naples, Italy, 4 Radiology Department, Second University of Naples, Naples, Italy, 5 Biotechnology Centre, AORN Cardarelli, Naples, Italy ☯ These authors contributed equally to this work. * Abstract OPEN ACCESS Citation: Schiattarella GG, Cerulo G, De Pasquale V, Cocchiaro P, Paciello O, Avallone L, et al. (2015) The Murine Model of Mucopolysaccharidosis IIIB Develops Cardiopathies over Time Leading to Heart Failure. PLoS ONE 10(7): e0131662. doi:10.1371/ journal.pone.0131662 Editor: Lachlan J. Smith, University of Pennsylvania, UNITED STATES Received: March 4, 2015 Accepted: June 4, 2015 Published: July 6, 2015 Copyright: © 2015 Schiattarella et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported in part by grants from the Italian Ministry of Health–Young Researcher Grant (2009) and from the Italian Ministry of Health– FIRB 2012 Grant (Futuro in Ricerca) to C.P. Competing Interests: The authors have declared that no competing interests exist. Mucopolysaccharidosis (MPS) IIIB is a lysosomal disease due to the deficiency of the enzyme α-N-acetylglucosaminidase (NAGLU) required for heparan sulfate (HS) degradation. The disease is characterized by mild somatic features and severe neurological disorders. Very little is known on the cardiac dysfunctions in MPS IIIB. In this study, we used the murine model of MPS IIIB (NAGLU knockout mice, NAGLU-/-) in order to investigate the cardiac involvement in the disease. 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 (WT) 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-toA-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 (LC3II). Overall, our findings demonstrate that NAGLU-/- mice develop heart disease, valvular abnormalities and cardiac failure associated with an impaired lysosomal autophagic flux. PLOS ONE | DOI:10.1371/journal.pone.0131662 July 6, 2015 1 / 15 Cardiac Disease in the Murine Model of Mucopolysaccharidosis IIIB Introduction Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases characterized by the accumulation of glycosaminoglycans (GAGs) in various organs of affected patients [1]. The storage defect is due to the absence of lysosomal enzymes involved in GAG catabolism. Deposition of GAGs in the heart of individuals with MPS causes cardiac dysfunctions [2]. Although advances in MPS treatment, including enzyme replacement therapy [3], hematopoietic stem cell transplantation [4] and gene therapy [5], have significantly improved the outcome of these disorders, death from cardiac causes continues to be common among these patients. The cardiac valves, usually mitral and aortic, the heart muscle itself and coronary arteries are characteristically affected in MPS patients [6–9]. However, the onset and extent of cardiac involvement varies depending upon the type of MPS. Children with MPS I show the earliest and most severe cardiac disorders, whereas cardiac involvement in individuals with MPS VII has been reported in adulthood [6] as well as in young individuals [10]. While cardiac involvement in patients with MPS I, II and VI has been well described [9], less is known about heart dysfunctions in MPS III. MPS III includes four distinct diseases (A, B, C, D) due to the deficiency of enzymes involved in heparan sulfate (HS) degradation; in particular, MPS IIIB (Sanfilippo type B syndrome) is due to the deficiency of the lysosomal enzyme α-N-acetylglucosaminidase (NAGLU). 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. Few studies have been reported on cardiac disorders in MPS IIIB patients [2, 8]. In order to get more insight into 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-/-) [11]. These mice exhibit a massive increase in HS deposition in the liver and kidney, and, at a lesser extent, in the lung, spleen, thymus and heart. 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 analyses. Furthermore, as an impairment of autophagy was found in embryonic fibroblasts and brain tissues from MPS IIIA mice [12], in human skin fibroblasts from MPS VI patients and in the liver, spleen, and kidney tissues from MPS VI rats [13], we also investigated the autophagic marker levels in the heart tissues of NAGLU-/- mice in order to verify whether abnormal autophagy might be involved in heart dysfunction in MPS IIIB. Materials and Methods (...truncated)