Myofilament degradation and dysfunction of human cardiomyocytes in Fabry disease

Abstract

Early detection of myocardial dysfunction in Fabry disease (FD) cardiomyopathy suggests the contribution of myofilament structural alterations. Six males with untreated FD cardiomyopathy submitted to cardiac studies, including tissue Doppler imaging and left ventricular endomyocardial biopsy. Active and resting tensions before and after treatment with protein kinase A (PKA) were determined in isolated Triton-permeabilized cardiomyocytes. Cardiomyocyte cross-sectional area, glycosphingolipid vacuole area, myofibrillolysis, and extent of fibrosis were also determined. Biopsies of mitral stenosis in patients with normal left ventricles served as controls. Active tension was four times lower in FD cardiomyocytes and correlated with extent of myofibrillolysis. Resting tension was six times higher in FD cardiomyocytes than in controls. PKA treatment decreased resting tension but did not affect active force. Protein analysis revealed troponin I and desmin degradation products. FD cardiomyocytes were significantly larger and filled with glycosphingolipids. Fibrosis was mildly increased compared with controls. Tissue Doppler imaging lengthening and shortening velocities were reduced in FD cardiomyocytes compared with controls, correlating with resting and active tensions, respectively, but not with cardiomyocyte area, percentage of glycosphingolipids, or extent of fibrosis. In conclusion, myofilament degradation and dysfunction contribute to FD cardiomyopathy. Partial reversal of high resting tension after pharmacological PKA treatment of cardiomyocytes suggests potential benefits from enzyme replacement therapy and/or energy-releasing agents.

Figure 1

Single FD (A) and control (B) cardiomyocyte attached between a force transducer and a piezoelectric motor. C: Contraction-relaxation sequence in FD cardiomyocyte before (left) and after (right) treatment with PKA. The abrupt changes in force mark the transitions of the preparation through the interface between solution and air (ie, transfers between wells containing relaxing and activating solutions).

Figure 2

A: Cardiac MRI short (left) and long (right) axis view showing basal infero-lateral hyperenhancement in FD patient 3 of Table 2. B: LV endomyocardial biopsy from the same patient showing mild interstitial and focal replacement fibrosis (F) and severe glycolipid engulfment of cardiomyocytes (Masson staining). C: Volume composition of the myocardium in endomyocardial biopsies of FD compared with controls revealing no significant difference in percentage of cardiomyocytes (M) and other interstitial components (O), whereas interstitial (IF) and replacement (RF) fibrosis showed a mild significant increase. Results are mean ± SD. *P < 0.05 compared with controls. Original magnification, ×40 (B).

Figure 3

A: Electron microscopy showing comparison of the normal arrangement of myofilaments in a normal cardiomyocyte (A) and myofilament dislodgment and disarray attributable to glycosphingolipid accumulation in FD cardiomyopathy (B). B: Electron microscopy of FD cardiomyopathy showing myofibrillolysis areas (M) around glycolipid deposits (G).

Figure 4

A: Graph showing total (F_total), active (F_active), and passive (F_passive) force before and after PKA in FD cardiomyocytes (P = 0.02 total force before versus after, P = 0.4 F_active before versus after P = 0.004, F_passive before versus after. B: Protein analysis using one-dimensional gel electrophoresis. Ponceau-stained blot of molecular weight marker (M) and myofilament proteins (P) (1). Degradation products of TnI (2) and desmin (3) in a control sample (nonfailing donor heart), kept at 20°C for 1 hour, and in FD samples, in which similar degradation products are evident.

Figure 5

Scatter plot of the correlations between active force and septal Sa (A: correlation coefficient = 0.99, P < 0.001), active force and lateral Sa (B: correlation coefficient 0.90, P < 0.05), passive force and septal E/Ea (C: correlation coefficient = 0.99, P < 0.001), passive force and lateral E/Ea (D: correlation coefficient = 0.94, P < 0.05), active force and myofibrillolysis area (E: correlation coefficient = 0.94, P < 0.05), passive force after PKA treatment and area of glycosphingolipid vacuoles (F: correlation coefficient = 0.99, P > 0.001).