Myofilament Degradation and Dysfunction of Human Cardiomyocytes in Fabry Disease
2008; Elsevier BV; Volume: 172; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2008.070576
ISSN1525-2191
AutoresCristina Chimenti, Nazha Hamdani, Nicky M. Boontje, Francesco De Cobelli, Antonio Esposito, Jean G.F. Bronzwaer, Ger J.M. Stienen, Matteo Antonio Russo, Walter J. Paulus, Andrea Frustaci, Jolanda van der Velden,
Tópico(s)Studies on Chitinases and Chitosanases
ResumoEarly 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. 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. Fabry disease (FD) is an X-linked lysosomal storage disorder caused by the deficiency of the enzyme α-galactosidase A, resulting in progressive intracellular glycosphingolipid deposition in multiple organ systems, including the heart.1Desnick RJ Ioannou YA Eng CM Alpha-Galactosidase A deficiency: Fabry disease.in: Scriver CR Beaudet AL Sly WS Valle D Kinzler KE Vogelstein B The Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill, New York2001: 3733-3774Crossref Google Scholar In patients with FD, cardiac involvement is characterized by progressive left ventricular (LV) wall thickening, mimicking hypertrophic cardiomyopathy,2Sachdev B Takenaka T Teraguchi H Tei C Lee P McKenna WJ Elliott PM Prevalence of Anderson-Fabry disease in male patients with late onset hypertrophic cardiomyopathy.Circulation. 2002; 105: 1407-1411Crossref PubMed Scopus (478) Google Scholar, 3Chimenti C Pieroni M Morgante E Antuzzi D Russo A Russo MA Maseri A Frustaci A Prevalence of Fabry disease in female patients with late-onset hypertrophic cardiomyopathy.Circulation. 2004; 110: 1047-1053Crossref PubMed Scopus (197) Google Scholar with diastolic LV dysfunction and a preserved LV ejection fraction that may decline in the end stage of the disease.4Weidemann F Breunig F Beer M Sandstede J Stork S Voelker W Ertl G Knoll A Wanner C Strotmann JM The variation of morphological and functional cardiac manifestation in Fabry disease: potential implications for the time course of the disease.Eur Heart J. 2005; 26: 1221-1227Crossref PubMed Scopus (197) Google Scholar The diastolic LV dysfunction has usually been ascribed to myocardial fibrosis in addition to cardiomyocyte hypertrophy and engulfment by glycosphingolipids. Recently however, tissue Doppler imaging (TDI) revealed reduced diastolic and systolic velocities even in the prehypertrophic phase of the disease,5Pieroni M Chimenti C Ricci R Sale P Russo MA Frustaci A Early detection of Fabry cardiomyopathy by tissue Doppler imaging.Circulation. 2003; 107: 1978-1984Crossref PubMed Scopus (212) Google Scholar suggesting an early and direct involvement of cardiomyocyte function.The present study therefore investigated in male patients with untreated FD active and resting tension of isolated cardiomyocytes, myofilament protein composition, myocardial collagen deposition, and glycosphingolipid accumulation and correlated them with TDI myocardial long axis shortening and lengthening velocities.Materials and MethodsPatient PopulationFrom January 1996 to July 2005, 12 consecutive male patients with LV hypertrophy were diagnosed to have FD by means of biochemical, genetic, and endomyocardial biopsy studies. Eight patients had not yet begun enzyme replacement therapy, six of them (47.1 ± 8.3 years) had a complete clinical, morphometric, and force measurement evaluation and constituted our patient population (Table 1). The patients belonged to unrelated families. Reduced peripheral blood α-galactosidase A activity was detected in all patients as previously described6Frustaci A Chimenti C Ricci R Natale L Russo MA Pieroni M Eng CM Desnick RJ Improvement in cardiac function in the cardiac variant of Fabry's disease with galactose-infusion therapy.N Engl J Med. 2001; 345: 25-32Crossref PubMed Scopus (293) Google Scholar and causal mutations were identified by direct sequencing of α-galactosidase A gene in all families. The investigation conforms with the principles outlined in the Declaration of Helsinki.Table 1Characteristics of Fabry Disease PatientsPatient 1Patient 2Patient 3Patient 4Patient 5Patient 6Age (years)504653584135Cardiac manifestations*A, arrhythmias.Dyspnea, chest pain, ADyspnea, chest pain, ADyspnea, chest pain, ADyspnea, chest pain, ADyspnea, chest pain, ADyspnea, chest pain, AExtracardiac manifestations†CNS, central nervous system; AP, acroparesthesias; H, hypohidrosis.Skin, CNS, ears, eyes, kidneys, HSkin, CNS, ears, eyes, kidneys, AP, HSkin, eyes, kidneys, HEyes, ears, kidneysSkin, eyes, ears, kidneysSkin, CNS, eyes, ears, kidneysEnzymatic activity‡Values are the mean (±SD) results of three independent determinations on peripheral blood lymphocytes. (nmol/hour/mg of protein)70.3 ± 9.620.9 ± 1.579.2 ± 8.550.2 ± 5.623.1 ± 2.615.2 ± 0.8LVEDP (mmHg§EDP, left ventricular end diastolic pressure.)262421192022Echocardiographic data¶LV, left ventricular; EDD, end diastolic diameter; MWT, maximal wall thickness; EF, ejection fraction; EDP, left ventricular end diastolic pressure. LVEDD (mm)494644434540 MWT, mm202123191818.5 LVEF (%)505167656365 Fractional shortening (%)384348444047 E/A ratio0.820.930.910.890.940.95 Isovolumic relaxation time (ms)115110108111107108 E-wave deceleration time (ms)285270275240205210MRI data∥MRI, magnetic resonance imaging. LV mass index (g/m2)150.3132.0143.2133.499.9114.1 Late enhancement (%)6.76.347.41.67.9* A, arrhythmias.† CNS, central nervous system; AP, acroparesthesias; H, hypohidrosis.‡ Values are the mean (±SD) results of three independent determinations on peripheral blood lymphocytes.§ EDP, left ventricular end diastolic pressure.¶ LV, left ventricular; EDD, end diastolic diameter; MWT, maximal wall thickness; EF, ejection fraction; EDP, left ventricular end diastolic pressure.∥ MRI, magnetic resonance imaging. Open table in a new tab Clinical StudiesExtensive clinical examination, including the assessment of FD systemic manifestations, electrocardiography, two-dimensional echocardiography with Doppler analysis, and cardiac magnetic resonance imaging (MRI) with late gadolinium enhancement, were performed in all patients. TDI analysis was performed in the pulsed Doppler mode to record mitral annulus velocities at septal and lateral corners.5Pieroni M Chimenti C Ricci R Sale P Russo MA Frustaci A Early detection of Fabry cardiomyopathy by tissue Doppler imaging.Circulation. 2003; 107: 1978-1984Crossref PubMed Scopus (212) Google Scholar, 7Pieroni M Chimenti C DeCobelli F Morgante E Gaudio C Russo MA Frustaci A Fabry cardiomyopathy: echocardiographic detection of endomyocardial glycosphingolipids compartmentalization.J Am Coll Cardiol. 2006; 47: 1663-1671Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Systolic (Sa), early diastolic (Ea), and late diastolic (Aa) velocities were measured and the E/Ea ratio was computed. Maximal wall thickness was defined as the greatest thickness of any segment of the LV wall. Ten age-matched men with no evidence of LV hypertrophy or cardiac and systemic disease were used as controls. MRI was performed as previously described.7Pieroni M Chimenti C DeCobelli F Morgante E Gaudio C Russo MA Frustaci A Fabry cardiomyopathy: echocardiographic detection of endomyocardial glycosphingolipids compartmentalization.J Am Coll Cardiol. 2006; 47: 1663-1671Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar Late enhancement assessment was performed 10 to 15 minutes after injection of gadolinium-DTPA (Shering AG, Berlin, Germany) (0.2 mmol/kg of body weight), by using a three- dimensional inversion recovery T1-weighted sequence.Cardiac Catheterization and Endomyocardial BiopsyAll invasive studies were approved by the ethical committees of our institutions and the patients provided written informed consent. All FD patients underwent coronary and biventricular angiography with biventricular or LV endomyocardial biopsy. LV end-diastolic pressure >16 mmHg was considered as indicative of LV diastolic dysfunction. Eight to ten endomyocardial samples, ∼3 mm3 each, were obtained from each patient. Five to six myocardial samples were processed for routine histological and histochemical analyses. Two samples were fixed in 2% glutaraldehyde in 0.1 mol/L phosphate buffer (pH = 7.3) and embedded in Epon resin; semithin sections were processed for Azur II staining and ultra-thin sections were stained with uranyl acetate and lead hydroxide for transmission electron microscopy.7Pieroni M Chimenti C DeCobelli F Morgante E Gaudio C Russo MA Frustaci A Fabry cardiomyopathy: echocardiographic detection of endomyocardial glycosphingolipids compartmentalization.J Am Coll Cardiol. 2006; 47: 1663-1671Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar One to two endomyocardial biopsy samples were snap-frozen in liquid nitrogen and used for cardiomyocyte force measurements and protein analysis.Morphometric StudiesParaffin-embedded histological sections stained with Masson's trichrome were examined at ×400 magnification with a reticule containing 42 sampling points (no. 105844; Wild Heerbrugg Instruments, Gals, Switzerland) to determine the percent area occupied by cardiomyocytes and by interstitial and replacement fibrosis.8Olivetti G Melissari M Capasso JM Anversa P Cardiomyopathy of the aging human heart: myocyte loss and reactive cellular hypertrophy.Circ Res. 1991; 68: 1560-1568Crossref PubMed Scopus (580) Google Scholar Cardiomyocyte cross-sectional area was computed measuring the cardiomyocyte diameter across the nucleus in 50 to 100 cells cut transversely (78 ± 14 cells; range, 58 to 97).9Anversa P Olivetti G Cellular basis of physiological and pathological myocardial growth.in: Page E Fozzard HA Solaro RJ Handbook of Physiology. The Cardiovascular System. The Heart. Oxford University Press, Oxford2002: 75-144Google Scholar At that level, the diameter of the perinuclear vacuoles was also measured and the percent cardiomyocyte area occupied by vacuoles was computed. In addition, endocardial thickness was determined. These measurements were performed in glutaraldehyde-fixed, Epon resin-embedded, semithin sections stained with Azur II to visualize glycolipid droplets. Images of the histological sections were analyzed using Lucia G software (version 4.82; Nikon, Tokyo, Japan). Morphometric analysis of myofibrillolysis area was performed on ultra-thin sections, stained with uranyl acetate and lead hydroxide. Photographic negatives of transmission electron microscopy sections were analyzed using KS-300 software (Carl Zeiss Co., Oberkochen, Germany).10Frustaci A Perrone GA Gentiloni N Russo MA Morphometry and GH/IGF-1 axis deficiency may identify a form of dilated cardiomyopathy which is corrected by recombinant human growth hormone (rHGH). Reversible dilated cardiomyopathy due to growth hormone deficiency.Am J Clin Pathol. 1992; 97: 503-511Crossref PubMed Scopus (97) Google Scholar Ten surgical specimens from age-matched male patients with mitral stenosis and normal LV function were used as normal controls for morphometric measurements.Force Measurements in Isolated CardiomyocytesBiopsies were stored in liquid nitrogen for up to 41 months (20.2 ± 16.7, range, 3 to 41 months). Previous studies have shown that these samples can be used for force measurements in single cardiomyocytes.11van der Velden J Klein LJ Zaremba R Boontje NM Huybregts MA Stooker W Eijsman L de Jong JW Visser CA Visser FC Stienen GJM Effects of calcium, inorganic phosphate, and pH on isometric force in single skinned cardiomyocytes from donor and failing human hearts.Circulation. 2001; 104: 1140-1146Crossref PubMed Scopus (88) Google Scholar Force measurements were performed in mechanically isolated single cardiomyocytes of the six patients at 15°C as described previously.11van der Velden J Klein LJ Zaremba R Boontje NM Huybregts MA Stooker W Eijsman L de Jong JW Visser CA Visser FC Stienen GJM Effects of calcium, inorganic phosphate, and pH on isometric force in single skinned cardiomyocytes from donor and failing human hearts.Circulation. 2001; 104: 1140-1146Crossref PubMed Scopus (88) Google Scholar, 12van der Velden J Klein LJ van der Bijl M Huybregts MAJM Stooker W Witkop J Eijsman L Visser CA Visser FC Stienen GJM Force production in mechanically isolated cardiac myocytes from human ventricular muscle tissue.Cardiovasc Res. 1998; 38: 414-423Crossref PubMed Scopus (70) Google Scholar, 13Borbély A van der Velden J Papp Z Papp Z Edes I Stienen GJM Paulus WJ Cardiomyocyte stiffness in diastolic heart failure.Circulation. 2005; 111: 774-781Crossref PubMed Scopus (412) Google Scholar The control group consisted of surgical biopsies from five age-matched male patients with mitral stenosis and normal LV end-diastolic pressure, chamber dimensions, and contractile function. Briefly, frozen biopsies were defrosted within 10 seconds in cold relaxing solution (in mmol/L: free Mg, 1; KCl, 100; EGTA, 2; MgATP, 4; imidazole, 10; pH7.0). Cells were mechanically isolated and incubated for 5 minutes in relaxing solution supplemented with 0.5% Triton X-100 to remove all membranes. Thereafter, cells were washed twice in relaxing solution and a single cardiomyocyte was attached between a force transducer and a piezoelectric motor using silicone adhesive (Figure 1, A and B). To enable attachment between the force transducer and motor single preparations were selected for measurements on the basis of cell length (∼100 μm long). Resting sarcomere length of isolated cardiomyocytes was ∼1.7 μm and was adjusted to 2.2 μm for measurements of isometric force. The composition of the relaxing [pCa (−10log{Ca2+}), 9.0] and activating (pCa, 4.5) solution was previously described.14Papp Z Szabo A Barends JP Stienen GJM The mechanism of the force enhancement by MgADP under simulated ischaemic conditions in rat cardiac myocytes.J Physiol. 2002; 543: 177-189Crossref PubMed Scopus (38) Google Scholar All force values were normalized for cardiomyocyte cross-sectional area. A typical contraction-relaxation sequence in a cardiomyocyte from a Fabry sample is shown in Figure 1C. After curing of the glue for 50 minutes, the cardiomyocyte was transferred from the isolating solution on the mounting area to a small temperature-controlled well (volume, 80 μl) containing relaxing solution. Isometric force was measured, after the preparation was transferred, by moving the stage of the inverted microscope to a temperature-controlled well containing activating solution.12van der Velden J Klein LJ van der Bijl M Huybregts MAJM Stooker W Witkop J Eijsman L Visser CA Visser FC Stienen GJM Force production in mechanically isolated cardiac myocytes from human ventricular muscle tissue.Cardiovasc Res. 1998; 38: 414-423Crossref PubMed Scopus (70) Google Scholar On transfer of the cardiomyocyte from relaxing to activating solution, isometric force started to develop. Once a steady-state force level was reached, the cell was shortened within 1 ms to 80% of its original length to determine the base line of the force transducer. The distance between the base line and the steady force level is the total force (Ftotal). After 20 ms the cell was restretched and returned to the relaxing solution, in which a second slack-test of 10 seconds duration was performed to determine resting or passive force (Fpassive). The difference between Ftotal and Fpassive is the active force (Factive) developed by the cardiomyocyte. After measurements of Ftotal and Fpassive, the cardiomyocytes were incubated for 40 minutes at 20°C in relaxing solution containing the catalytic subunit of protein kinase A (PKA) (100 U/ml, batch 12K7495; Sigma, Brooklyn, NY) and 6 mmol/L dithiothreitol (MP Biochemicals, Irvine, CA). Subsequently, force measurements were repeated. Control incubations in relaxing solution with 6 mmol/L dithiothreitol, but without PKA, did not alter Fpassive and Factive of cardiomyocytes.Protein AnalysisProtein analysis was performed on cardiomyocytes, which were not used for force measurements. After isolation and Triton treatment the remaining cell pellet was freeze-dried and homogenized in sample buffer. To detect myofilament proteolysis, myofilament proteins were separated by one-dimensional gel electrophoresis containing 15% total acrylamide (acrylamide to bis-acrylamide ratio, 37.5:1) followed by Western immunoblotting.15van der Velden J Merkus D Klarenbeek BR James AT Boontje NM Dekkers DH Stienen GJM Lamers JM Duncker DJ Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction.Circ Res. 2004; 95: e85-e95Crossref PubMed Google Scholar Five μg (dry weight) of the tissue samples were applied to the gels. Western immunoblot analysis was performed using specific monoclonal antibodies against troponin I (TnI) (clone 8I-7, dilution 1:1000; Spectral Diagnostics Inc., Toronto, Ontario, Canada), desmin (clone DE-U-10, dilution 1:1000; Sigma), myosin light chain 1 (clone F109.16A12, dilution 1:200; Alexis Biochemicals), myosin light chain 2 (clone F109.3E1, dilution 1:200; Alexis Biochemicals, San Diego, CA), and α-actinin (clone EA-53, dilution 1:1000; Sigma) and signals were visualized using a secondary horseradish peroxidase-labeled goat-anti-mouse antibody and enhanced chemiluminescence (ECL Plus Western blotting detection; Amersham Biosciences, Piscataway, NJ).Statistical AnalysisNormal distribution of variables was assessed with Kolmogorov-Smirnov and Shapiro-Wilk tests. Variables showing normal distribution are presented as mean ± SD. Variables not showing normal distribution are presented as median (interquartile range). Categorical variables are presented as proportions or percentages. Continuous variables, showing a normal distribution, were compared with Student's t-test for independent samples (cases versus controls). Continuous variables not showing a normal distribution were compared with Mann-Whitney test (cases versus controls). Bivariate correlations were analyzed by Spearman rho coefficient computation. A two-tailed P < 0.05 was considered statistically significant. Statistical analysis was performed with SPSS version 11.0.1 software (SPSS Inc., Chicago, IL).ResultsClinical StudiesPatients' clinical characteristics, echocardiographic and MRI data are reported in Table 1. α-Galactosidase A activity was very low (mean value, 43.4 ± 7.4 nmol/hour/mg of protein; normal range, 3252 to 1623 nmol/hour/mg of protein) and all patients had extra-cardiac clinical manifestations of the disease. All patients were normotensive, satisfied the electrocardiographic and echocardiographic criteria for LV hypertrophy, and showed an increase in LV mass index. Diastolic function was impaired in all patients but no restrictive filling pattern was detected. Conversely, systolic function, as measured by ejection fraction and fractional shortening, was within the normal range in all FD patients.Gadolinium contrast-enhancement MRI study showed late enhancement in all patients typically localized in the basal or basal-medium segment of the lateral and infero-lateral wall (Figure 2A). Two patients (Table 1, patients 2 and 6) showed additional focal late enhancement in the apex. The mean percentage of myocardium involved was 5.7 ± 2.4% (range, 1.6 to 7.9). On TDI examination all patients had significant reduction of long axis lengthening and shortening velocities measured at the septal and lateral corner of the mitral annulus (Table 2). LV angiography revealed normal wall motion in all patients and coronary angiography showed absence of significant coronary stenoses.Figure 2A: 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 2TDI, Morphometric and Force Measurements in Patients with Fabry Disease Compared with ControlsFabry diseaseControls*Controls for TDI were n = 10, for morphometric studies were n = 10, for force measurements were n = 8.P†P values obtained comparing FD versus controls.TDI velocities Septal Sa, cm/second5.9 ± 0.414.6 ± 1.2<0.001 Septal Ea, cm/second5.7 ± 0.515.5 ± 1.3<0.001 Septal Aa, cm/second6.0 ± 0.510.3 ± 0.9<0.001 Septal E/Ea11.2 ± 0.56.0 ± 1.3<0.001 Lateral Sa, cm/second6.0 ± 0.213.7 ± 2<0.001 Lateral Ea, cm/second5.9 ± 0.414.0 ± 0.4<0.001 Lateral Aa, cm/second6.1 ± 0.610.1 ± 1.5<0.001 Lateral E/Ea10.5 ± 0.35.8 ± 1.2<0.001Morphometric data Fibrosis, %8.7 ± 4.44.1 ± 2.50.02 Interstitial6.7 ± 3.13.4 ± 2.00.02 Replacement1.9 ± 1.50.7 ± 0.60.03 Cardiomyocytes, %81.2 ± 7.288.1 ± 6.10.06 Cardiomyocyte area, μm2861.0 ± 208.4215.4 ± 90.1<0.001 Cardiomyocyte area occupied by vacuoles, %57.3 ± 4.10<0.001 Myofibrillolysis area, %15.2 ± 4.90<0.001Force measurements†P values obtained comparing FD versus controls. Ftotal, kN/m213.7 ± 3.9‡P < 0.001 versus after PKA treatment.17.6 ± 5.3NS Factive, kN/m23.9 ± 3.515.9 ± 5.4<0.001 Fpassive, kN/m29.8 ± 2.3‡P < 0.001 versus after PKA treatment.1.6 ± 0.6<0.001 Ftotal after PKA, kN/m211.1 ± 2.618.2 ± 5.40.02 Factive after PKA, kN/m24.3 ± 3.316.8 ± 5.4<0.001 Fpassive after PKA, kN/m26.6 ± 2.61.4 ± 0.6<0.001Data are present as mean value ± SD. P < 0.05 was considered statistically significant.* Controls for TDI were n = 10, for morphometric studies were n = 10, for force measurements were n = 8.† P values obtained comparing FD versus controls.‡ P < 0.001 versus after PKA treatment. Open table in a new tab Histological and Morphometric StudiesHistological examination of FD endomyocardial biopsies revealed regularly arranged and severely hypertrophied cardiomyocytes with large perinuclear vacuoles containing material that, on frozen sections, stained positively with periodic acid-Schiff and Sudan black stains. Fibrosis was predominantly interstitial (Figure 2B) with focal areas of replacement fibrosis. Computer-assisted histomorphometry showed only a mild, although significant, increase in fibrosis in FD patients compared to controls. The percent tissue area occupied by cardiomyocytes was similar (Figure 2C). Cardiomyocyte cross-sectional area was significantly increased in FD patients and more than 50% of it was occupied by glycosphingolipid vacuoles. The endocardium was thickened (mean FD value = 535 ± 201 μm, normal value = 18 ± 5 μm).On ultrastructural electron microscopic examination intracellular vacuoles appeared to be represented by concentric lamellar structures in single-membrane bound vesicles, indicative of lysosomal glycosphingolipid accumulation. The cytoplasmic inclusions frequently displaced cardiac myofibrils to the periphery of the cell (Figure 3A). Focal areas of myofibrillolysis were also detected (Figure 3B) and myofibrillolysis area was calculated as 15 ± 5%. Lamellar inclusions were present also in the endothelial cells, smooth muscle cells and fibroblasts. Cardiomyocyte area and percent area occupied by glycolipid vacuoles closely correlated (correlation coefficient = 0.99, P < 0.0001). This indicates that the increase of cardiomyocyte size is mainly attributable to intracytoplasmic glycosphingolipids vacuoles.Figure 3A: 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).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Force Measurements in Isolated Cardiomyocytes and Protein AnalysisForce measurement data of six FD patients (number of cardiomyocytes = 17) and of five controls (number of cardiomyocytes = 10) are presented in Table 2 as average value and in Table 3 as single patient value, in addition to the patients' gene mutation. When isolated cardiomyocytes were stretched to a sarcomere length of 2.2 μm a significantly higher Fpassive and a lower Factive was observed in FD patients compared to controls. Treatment with PKA significantly decreased Fpassive, but it remained higher compared to controls. Factive was not altered (Figure 4A). PKA treatment did not alter Fpassive and Factive in controls.Table 3α-Gal A Mutations, Force Measurements, and Protein Analysis in Fabry Disease PatientsForce before PKA (kN/m2)Force after PKA (kN/m2)Patientα-Gal A mutationFtotalFactiveFpassiveFtotalFactiveFpassiveTnI degradation (%)Desmin degradation (%)1M42fsX5516Morrone A Cavicchi C Bardelli T Antuzzi D Parini R Di Rocco M Feriozzi S Gabrielli O Barone R Pistone G Spisni C Ricci R Zammarchi E Fabry disease: molecular studies in Italian patients and X inactivation analysis in manifesting carriers.J Med Genet. 2003; 40: e103Crossref PubMed Scopus (51) Google Scholar14.91.213.711.41.59.90.028.62D315fsX31516Morrone A Cavicchi C Bardelli T Antuzzi D Parini R Di Rocco M Feriozzi S Gabrielli O Barone R Pistone G Spisni C Ricci R Zammarchi E Fabry disease: molecular studies in Italian patients and X inactivation analysis in manifesting carriers.J Med Genet. 2003; 40: e103Crossref PubMed Scopus (51) Google Scholar121.710.311.01.39.70.00.03Y216C*New missense mutation.9.22.07.39.13.16.07.92.24N215S17Eng CM Resnick-Silverman LA Niehaus DJ Astrin KH Desnick RJ Nature and frequency of mutations in the alpha-galactosidase A gene that cause Fabry disease.Am J Hum Genet. 1993; 53: 1186-1197PubMed Google Scholar15.65.610.013.26.26.05.327.45R220X18Meaney C Blanch LC Morris CP A nonsense mutation (R220X) in the alpha-galactosidase A gene detected in a female carrier of Fabry disease.Hum Mol Genet. 1994; 3: 1019-1020Crossref PubMed Scopus (22) Google Scholar10.42.97.67.43.73.70.00.06Q279K19Dominissini S Cariati R Nevyjel M Guerci V Ciana G Bembi B Pittis MG Comparative in vitro expression study of four Fabry disease causing mutations at glutamine 279 of the alpha-galactosidase A protein.Hum Hered. 2004; 57: 138-141Crossref PubMed Scopus (4) Google Scholar19.910.39.614.410.04.45.20.3* New missense mutation. Open table in a new tab Figure 4A: Graph showing total (Ft
Referência(s)