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

Schiattarella GG, Cerulo G, De Pasquale V, Cocchiaro P, Paciello O, Avallone L, Belfiore MP, Iacobellis F, Di Napoli D, Magliulo F, Perrino C, Trimarco B, Esposito G, Di Natale P, Pavone LM - PLoS ONE (2015)

Bottom Line: 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.Overall, our findings demonstrate that NAGLU(-/-) mice develop heart disease, valvular abnormalities and cardiac failure associated with an impaired lysosomal autophagic flux.

View Article: PubMed Central - PubMed

Affiliation: Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy.

ABSTRACT
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-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 with an impaired lysosomal autophagic flux.

No MeSH data available.


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Evaluation of lysosomal and autophagy markers in NAGLU-/- hearts.Representative immunoblot and densitometric analysis of LAMP2, BCN1 and LC3-II protein levels in WT and NAGLU-/- hearts at 32 weeks of age (*p<0.05).
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pone.0131662.g006: Evaluation of lysosomal and autophagy markers in NAGLU-/- hearts.Representative immunoblot and densitometric analysis of LAMP2, BCN1 and LC3-II protein levels in WT and NAGLU-/- hearts at 32 weeks of age (*p<0.05).

Mentions: Alterations in the autophagic process have recently been suggested as a pathogenic mechanism common to a variety of lysosomal storage diseases including some MPS IIIA and VI types [12–13]. However, the block of autophagy in MPS has never been studied in connection with cardiac problems. Here, we investigated whether an impairment of autophagy might be associated to cardiac dysfunctions in NAGLU-/- mice together with the lysosomal defects. To this purpose, we first investigated cardiac lysosomal involvement in NAGLU-/- mice by evaluating the protein levels of the lysosomal marker LAMP2 [38]. Cardiac levels of LAMP2 were significantly increased in NAGLU-/- mice compared to WT mice, as shown by Western blotting analysis (Fig 6). We found in LV samples of NAGLU-/- mice an increased expression of Beclin1 (BCN1, homologue of yeast ATG6), a protein of the Class III phosphatidylinositol 3 kinase (PI3K) complex that mediates the early phase of autophagy [39] (Fig 6). BCN1 protein is involved in the formation of autophagosome. The microtubule-associated protein I light chain (LC3-I, homologue of yeast ATG8) is also implicated in this process; LC3-I is cleaved at its carboxy-terminal and further modified to the lipid-conjugated LC3-II which is associated to autophagosome membranes [40]. LC3-II levels are reflective of autophagosome abundance. Western blotting analysis of mouse heart samples showed that LC3-II expression levels were significantly increased in cardiac samples from NAGLU-/- mice compared to WT littermates (Fig 6). These results indicate that the autophagic process is dysfunctional in the heart of NAGLU-/- mice, thus suggesting that dysregulated autophagic capacity can have a detrimental impact in heart tissue, and might represent an important factor contributing to the cardiac disease in MPS IIIB. Indeed, in the heart of NAGLU-/- mice, lysosomal accumulation of HS, due to NAGLU enzyme deficiency, activates autophagy, as demonstrated by increased BCN1 expression levels, but the formed autophagosome cannot be cleared, as shown by the enhanced expression of LC3-II in cardiac samples of NAGLU-/- mice as compared to WT mice. However, further investigations are needed to establish whether the substrate accumulation is the primary mediator of abnormal lysosomal autophagy, and whether the accumulation of autophagosomes rather than the block of the autophagosome-lysosome fusion is responsible for autophagy impairment in the heart of affected mice.


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

Schiattarella GG, Cerulo G, De Pasquale V, Cocchiaro P, Paciello O, Avallone L, Belfiore MP, Iacobellis F, Di Napoli D, Magliulo F, Perrino C, Trimarco B, Esposito G, Di Natale P, Pavone LM - PLoS ONE (2015)

Evaluation of lysosomal and autophagy markers in NAGLU-/- hearts.Representative immunoblot and densitometric analysis of LAMP2, BCN1 and LC3-II protein levels in WT and NAGLU-/- hearts at 32 weeks of age (*p<0.05).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4493027&req=5

pone.0131662.g006: Evaluation of lysosomal and autophagy markers in NAGLU-/- hearts.Representative immunoblot and densitometric analysis of LAMP2, BCN1 and LC3-II protein levels in WT and NAGLU-/- hearts at 32 weeks of age (*p<0.05).
Mentions: Alterations in the autophagic process have recently been suggested as a pathogenic mechanism common to a variety of lysosomal storage diseases including some MPS IIIA and VI types [12–13]. However, the block of autophagy in MPS has never been studied in connection with cardiac problems. Here, we investigated whether an impairment of autophagy might be associated to cardiac dysfunctions in NAGLU-/- mice together with the lysosomal defects. To this purpose, we first investigated cardiac lysosomal involvement in NAGLU-/- mice by evaluating the protein levels of the lysosomal marker LAMP2 [38]. Cardiac levels of LAMP2 were significantly increased in NAGLU-/- mice compared to WT mice, as shown by Western blotting analysis (Fig 6). We found in LV samples of NAGLU-/- mice an increased expression of Beclin1 (BCN1, homologue of yeast ATG6), a protein of the Class III phosphatidylinositol 3 kinase (PI3K) complex that mediates the early phase of autophagy [39] (Fig 6). BCN1 protein is involved in the formation of autophagosome. The microtubule-associated protein I light chain (LC3-I, homologue of yeast ATG8) is also implicated in this process; LC3-I is cleaved at its carboxy-terminal and further modified to the lipid-conjugated LC3-II which is associated to autophagosome membranes [40]. LC3-II levels are reflective of autophagosome abundance. Western blotting analysis of mouse heart samples showed that LC3-II expression levels were significantly increased in cardiac samples from NAGLU-/- mice compared to WT littermates (Fig 6). These results indicate that the autophagic process is dysfunctional in the heart of NAGLU-/- mice, thus suggesting that dysregulated autophagic capacity can have a detrimental impact in heart tissue, and might represent an important factor contributing to the cardiac disease in MPS IIIB. Indeed, in the heart of NAGLU-/- mice, lysosomal accumulation of HS, due to NAGLU enzyme deficiency, activates autophagy, as demonstrated by increased BCN1 expression levels, but the formed autophagosome cannot be cleared, as shown by the enhanced expression of LC3-II in cardiac samples of NAGLU-/- mice as compared to WT mice. However, further investigations are needed to establish whether the substrate accumulation is the primary mediator of abnormal lysosomal autophagy, and whether the accumulation of autophagosomes rather than the block of the autophagosome-lysosome fusion is responsible for autophagy impairment in the heart of affected mice.

Bottom Line: 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.Overall, our findings demonstrate that NAGLU(-/-) mice develop heart disease, valvular abnormalities and cardiac failure associated with an impaired lysosomal autophagic flux.

View Article: PubMed Central - PubMed

Affiliation: Department of Advanced Biomedical Sciences, University of Naples Federico II, Naples, Italy.

ABSTRACT
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-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 with an impaired lysosomal autophagic flux.

No MeSH data available.


Related in: MedlinePlus