Fabry cardiomyopathy: myocardial fibrosis, inflammation and reduced mannose6phosphate receptors cause low accessibility to enzyme therapy

Abstract Background Clinical impact of enzyme replacement therapy (ERT) on advanced Fabry disease cardiomyopathy (FDCM) appears limited. Purpose Clarify the pathologic mechanisms responsible of ERT inefficacy in the advanced FDCM. Methods Ten male patients with advanced FDCM (echocardiographic maximal wall thickness 19.3 ± 2.1 mm) underwent left ventricular endomyocardial biopsy before and 4 hours after beta-agalsidase infusion. Comparative studies between pre and post infusion samples included: histology and electron microscopy; assessment of myocardial alpha-galactosidase A activity; immunohistochemistry for alpha-galactosidase A and semiquantitative evaluation (from 0 to 3) of its cardiomyocyte content; Ultrastructural immunogold analysis with anti-alpha-galactosidase A ab. Western Blot (WB) quantification of mannose-6-phosphate receptors (M6Pr). Controls were surgical left ventricular biopsies from patients with mitral stenosis. Results Histologic and Ultrastructural evaluation showed no removal of storage material while myocardial fibrosis was 9.8 ± 6.8 vs 3.8 ± 2.0 of controls and virus-negative lymphocytic inflammation was observed in 7 out of 10 patients. At Ultrastructural immunogold analysis, Myocardial alpha-galactosidase A activity increased in post infusion samples by overall 1.89-fold. Alpha-galactosidase A immunostaining in cardiomyocytes was absent at baseline in all patients and did not significantly improve in post-infusion samples. Immunogold particles increased by 1.33-fold remaining far from normal controls (86.9 ± 6.6). Protein analysis showed M6Pr in advanced FDCM to be 81% lower than in normal heart. Conclusions Our study shows a low accessibility to ERT of cardiomyocytes affected by advanced FDCM. It is sustained by myocardial fibrosis, inflammation and severe down-regulation of M6Pr. Figure Legend Histologic and ultrastructural and mannose6phosphate receptors characteristics of patients with severe Fabry Disease Cardiomyopathy. Panel A shows severe cellular glycolipid infiltration associated with diffuse interstitial fibrosis (Masson trichrome, 100x). Panel B reveals an overlapping lymphocytic CD45Ro+ myocardial inflammation with focal necrosis of adjacent cardiomyocytes. Insert(i), Ultrastructural detail showing vacuoles (black arrows) to consist of myelin bodies,( scale bar=3μm). Panel C (upper) Western blot analysis of all 10 patients of Mannose-6-phosphate Receptor, molecular weight 275 Kd. Alpha sarcomeric actin (43 kDa) was used as a loading control. (Panel C -lower) Panel D: Graphs document western blot of Mannose-6-phosphate Receptor, in all 10 patients with severe FDCM showing 3-fold lower of Mannose-6-phosphate Receptor values in patients versus normal heart (4.830 ± 1116,724 vs 14.737± 1145,386; p < 0.001).