Downregulation of Mannose-6-Phosphate Receptors in Fabry Disease Cardiomyopathy: A Potential Target for Enzyme Therapy Enhancement

Abstract

Background: The efficacy of enzyme replacement therapy (ERT) in mobilizing globotryaosylceramide (GB-3) from Fabry cardiomyocytes is limited. The mechanism involved is still obscure. Methods: Assessment of M6Pr, M6Pr-mRNA, and Ubiquitin has been obtained by Western blot analysis and real-time PCR of frozen endomyocardial biopsy samples, from 17 pts with FD, various degree of left ventricular hypertrophy, and maximal wall thickening (MWT) from 11.5 and 20 mm. The diagnosis and severity of FDCM followed definitions of GLA mutation, α-galactosidase A enzyme activity, cardiac magnetic resonance, and left ventricular endomyocardial biopsy with the quantification of myocyte hypertrophy and the extent of Gb-3 accumulation. All patients have received alpha or beta agalsidase for ≥3 years without a reduction in LV mass nor an increase in T1 mapping at CMR. Controls were surgical biopsies from 15 patients undergoing mitral valve replacement. Results: Protein analysis showed mean M6Pr in FDCM to be 5.4-fold lower than in a normal heart (4289 ± 6595 vs. 23,581 ± 4074, p = 0.0996) (p < 0.001): specifically, 9-fold lower in males, p = 0.009, (p < 0.001) and 3-fold lower in females, p = 0.5799, (p < 0.001) showing, at histology, a mosaic of normal and diseased cells. M6Pr-mRNA expression was normal, while ubiquitin showed an increase of 4.6 fold vs. controls (13,284 ± 1723 vs. 2870 ± 690, p = 0.001) suggesting that ubiquitin-dependent post-translational degradation is likely responsible for the reduction of M6Pr in FDCM. Conclusion: M6Pr expression is remarkably reduced in FDCM as a likely result of post-translational degradation. This may explain the reduced efficacy of ERT and be a therapeutic target for the enhancement of ERT activity.

Keywords: Fabry disease; GLA; Mannose-6-phosphate receptors; cardiomyocyte; enzyme replacement therapy; globotriaosylceramide.

Figure 1

Histology (A), electronmicroscopy (B) and immunohistochemistry (C) for M6Pr in a 43-year-old man (pt 1) affected by Fabry disease cardiomyopathy (MWT 14 mm). In panel (C), normal immunostaining of endothelial cells, (black arrows) in contrast with reduced staining of cardiomyocytes, is observed. Panel (D) represents immunohistochemistry for M6Pr in normal myocardium. The pictures suggest a severe reduced expression of M6Pr in Fabry cardiomyocytes vs. controls.

Figure 2

Histology (A), electronmicroscopy (B) and immunohistochemistry (C) for M6Pr in a 54-year-old woman (pt 4) and immunohistochemistry for M6Pr in normal myocardium (D). Comparison of the two pictures (C) vs. (D) suggest a severe reduction in M6Pr expression in Fabry cardiomyocytes. Of note in (C), due to patchy cardiomyocyte involvement, unaffected cells (see arrows) are strongly positive as compared to the affected cells.

Figure 3

Panel (A,B): Western blot analysis of all 17 patients of M6Pr, molecular weight 275 Kd. Panel (A): Lane1 = Markers; Lane 2 = Control; Lane 3 = patient 1; Lane 4 = patient 2; Lane 5 = patient 3; Lane 6 = patient 4; Lane 7 = patient 5; Lane 8 = patient 6; Lane 9 = patient 7; Lane 10 = patient 8. Panel (B): Lane 1 = Markers; Lane 2 = Control; Lane 3 = patient 9; Lane 4 = patient 10; Lane 5 = patient 11; Lane 6 = patient 12; Lane 7 = patient 13; Lane 8 = patient 14; Lane 9 = patient 15; Lane 10 = patient 16; Lane 11 = patient 17. Panel (C–E): Graphs document western blot of M6Pr, in all 17 patients with FDCM showing 5.4-fold lower of M6Pr values in patients versus normal heart (4289 ± 6595 vs. 23,581 ± 4074, p = 0.0996) (p < 0.001), (C), 9-fold lower in male, p = 0.009, (p < 0.001), (D) and 3-fold lower in female p = 0.5799, (p < 0.001), (E). Alpha sarcomeric actin (43 kDa) was used as a loading control. *** = p < 0.001.

Figure 4

Panel (A,B): Western blot analysis of Ubiquitin in 16 out of 17 patients with FDCM, molecular weight 75 Kd. Panel (A): Lane1 = Markers; Lane 2 = Control; Lane 3 = patient 1; Lane 4 = patient 2; Lane 5 = patient 3; Lane 6 = patient 4; Lane 7 = patient 5; Lane 8 = patient 6; Lane 9 = patient 7; Lane 10 = patient 8. Panel (B): Lane 1 = Markers; Lane 2= Control; Lane 3 = patient 9; Lane 4 = patient 10; Lane 5 = patient 11; Lane 6 = patient 12; Lane 7 = patient 13; Lane 8 = patient 14; Lane 9 = patient 15; Lane 10 = patient 16; Panel (C): Graphs report western blot of Ubiquitin in 16 out of 17 patients and show 4.6-fold increase in Ubiquitin values vs. controls (13,284 ± 1723 vs. 2870 ± 690, p < 0.001). *** = p < 0.001.