Dysferlin Deficiency and the Development of Cardiomyopathy in a Mouse Model of Limb-Girdle Muscular Dystrophy 2B
2009; Elsevier BV; Volume: 175; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2009.080930
ISSN1525-2191
AutoresThomas H. Chase, Gregory A. Cox, Lisa Burzenski, Oded Foreman, Leonard D. Shultz,
Tópico(s)RNA Research and Splicing
ResumoLimb-girdle muscular dystrophy 2B, Miyoshi myopathy, and distal myopathy of anterior tibialis are severely debilitating muscular dystrophies caused by genetically determined dysferlin deficiency. In these muscular dystrophies, it is the repair, not the structure, of the plasma membrane that is impaired. Though much is known about the effects of dysferlin deficiency in skeletal muscle, little is known about the role of dysferlin in maintenance of cardiomyocytes. Recent evidence suggests that dysferlin deficiency affects cardiac muscle, leading to cardiomyopathy when stressed. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known. In this study, we examined a mouse model of dysferlinopathy using light and electron microscopy as well as echocardiography and conscious electrocardiography. We determined that dysferlin is normally localized to the intercalated disk and sarcoplasm of the cardiomyocytes. In the absence of dysferlin, cardiomyocyte membrane damage occurs and is localized to the intercalated disk and sarcoplasm. This damage results in transient functional deficits at 10 months of age, but, unlike in skeletal muscle, the cell injury is sublethal and causes only mild cardiomyopathy even at advanced ages. Limb-girdle muscular dystrophy 2B, Miyoshi myopathy, and distal myopathy of anterior tibialis are severely debilitating muscular dystrophies caused by genetically determined dysferlin deficiency. In these muscular dystrophies, it is the repair, not the structure, of the plasma membrane that is impaired. Though much is known about the effects of dysferlin deficiency in skeletal muscle, little is known about the role of dysferlin in maintenance of cardiomyocytes. Recent evidence suggests that dysferlin deficiency affects cardiac muscle, leading to cardiomyopathy when stressed. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known. In this study, we examined a mouse model of dysferlinopathy using light and electron microscopy as well as echocardiography and conscious electrocardiography. We determined that dysferlin is normally localized to the intercalated disk and sarcoplasm of the cardiomyocytes. In the absence of dysferlin, cardiomyocyte membrane damage occurs and is localized to the intercalated disk and sarcoplasm. This damage results in transient functional deficits at 10 months of age, but, unlike in skeletal muscle, the cell injury is sublethal and causes only mild cardiomyopathy even at advanced ages. Plasma membrane damage in mechanically active cells such as the myocyte is inevitable even under normal physiological conditions.1Clarke MS Caldwell RW Chiao H Miyake K McNeil PL Contraction-induced cell wounding and release of fibroblast growth factor in heart.Circ Res. 1995; 76: 927-934Crossref PubMed Scopus (179) Google Scholar, 2Miyake K McNeil PL Mechanical injury and repair of cells.Crit Care Med. 2003; 31: S496-S501Crossref PubMed Google Scholar Since membranes are not self-sealing, effective and efficient repair mechanisms are necessary to maintain cell viability. Dysferlin plays a central role in this active repair mechanism in skeletal muscle. In the absence of dysferlin disruptions of the skeletal muscle plasma membrane are not repaired leading to cell death.3Bansal D Miyake K Vogel SS Groh S Chen CC Williamson R McNeil PL Campbell KP Defective membrane repair in dysferlin-deficient muscular dystrophy.Nature. 2003; 423: 168-172Crossref PubMed Scopus (758) Google Scholar Skeletal muscle can regenerate new cells from satellite cells but eventually even this response is exhausted, and lost myocytes are replaced by fat and fibrosis resulting in debilitating muscular dystrophy. Limb-girdle muscular dystrophy type 2 B (LGMD2B), Miyoshi myopathy, and distal myopathy of anterior tibialis are three clinically distinct forms of muscular dystrophy that are caused by mutations within the dysferlin (DYSF) gene resulting in severe to complete deficiency of dysferlin expression.4Liu J Aoki M Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi Myopathy and limb girdle muscular dystrophy 2B.Nat Genet. 1998; 20: 31-36Crossref PubMed Scopus (744) Google Scholar, 5Bashir R Britton S Strachan T Keers S Vafiadaki E Lako M Richard I Marchand S Bourg N Argov Z Sadeh M Mahjneh I Marconi G Passos-Bueno MR Moreira Ede S Zatz M Beckmann JS Bushby K A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.Nat Genet. 1998; 20: 37-42Crossref PubMed Scopus (553) Google Scholar Clinically, these dysferlinopathies start in young adulthood with progressive muscle weakness and atrophy that advances to severe disability in older adulthood. Dysferlin is a 273 kDa membrane-spanning protein with multiple C2 domains that bind calcium, phospholipids, and proteins to then trigger signaling events, vesicle trafficking, and membrane fusion.6Anderson LV Davison K Moss JA Young C Cullen MJ Walsh J Johnson MA Bashir R Britton S Keers S Argov Z Mahjneh I Fougerousse F Beckmann JS Bushby KM Dysferlin is a plasma membrane protein and is expressed early in human development.Hum Mol Genet. 1999; 8: 855-861Crossref PubMed Scopus (241) Google Scholar, 7Matsuda C Aoki M Hayashi YK Ho MF Arahata K Brown Jr, RH Dysferlin is a surface membrane-associated protein that is absent in Miyoshi myopathy.Neurology. 1999; 53: 1119-1122Crossref PubMed Google Scholar The name "dysferlin" reflects the homology with FER-1, the Caenorhabditis elegans spermatogenesis factor involved in the fusion of vesicles with the plasma membrane, as well as the dystrophic phenotype associated with its deficiency.5Bashir R Britton S Strachan T Keers S Vafiadaki E Lako M Richard I Marchand S Bourg N Argov Z Sadeh M Mahjneh I Marconi G Passos-Bueno MR Moreira Ede S Zatz M Beckmann JS Bushby K A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B.Nat Genet. 1998; 20: 37-42Crossref PubMed Scopus (553) Google Scholar Dysferlin is crucial to calcium dependent membrane repair in muscle cells.3Bansal D Miyake K Vogel SS Groh S Chen CC Williamson R McNeil PL Campbell KP Defective membrane repair in dysferlin-deficient muscular dystrophy.Nature. 2003; 423: 168-172Crossref PubMed Scopus (758) Google Scholar, 8Han R Bansal D Miyake K Muniz VP Weiss RM McNeil PL Campbell KP Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury.J Clin Invest. 2007; 117: 1805-1813Crossref PubMed Scopus (137) Google Scholar In normal skeletal muscle, sarcolemma injuries lead to the accumulation of dysferlin-enriched membrane patches and resealing of the membrane in the presence of Ca2+.3Bansal D Miyake K Vogel SS Groh S Chen CC Williamson R McNeil PL Campbell KP Defective membrane repair in dysferlin-deficient muscular dystrophy.Nature. 2003; 423: 168-172Crossref PubMed Scopus (758) Google Scholar, 9Lennon NJ Kho A Bacskai BJ Perlmutter SL Hyman BT Brown Jr, RH Dysferlin interacts with annexins A1 and A2 and mediates sarcolemmal wound-healing.J Biol Chem. 2003; 278: 50466-50473Crossref PubMed Scopus (310) Google Scholar While the profound effect of dysferlin deficiency in skeletal muscle has been the subject of much investigation, the effect of dysferlin deficiency in cardiac muscle has largely been ignored. However, in 2004, Kuru et al10Kuru S Yasuma F Wakayama T Kimura S Konagaya M Aoki M Tanabe M Takahashi T A patient with limb girdle muscular dystrophy type 2B (LGMD2B) manifesting cardiomyopathy.Rinsho Shinkeigaku. 2004; 44: 375-378PubMed Google Scholar reported on a 57-year-old Japanese woman with LGMD2B and dilated cardiomyopathy; more recently, Wenzel et al11Wenzel K Geier C Qadri F Hubner N Schulz H Erdmann B Gross V Bauer D Dechend R Dietz R Osterziel KJ Spuler S Ozcelik C Dysfunction of dysferlin-deficient hearts.J Mol Med. 2007; 85: 1203-1214Crossref PubMed Scopus (65) Google Scholar described dilated cardiomyopathy in two out of seven patients with LGMD2B and other cardiac abnormalities in three of the others. These observations suggest that dysferlin deficiency can lead to cardiomyopathy as well as to muscular dystrophy. However, neither the morphological location of dysferlin in the cardiomyocyte nor the progression of the disease with age are known. Spontaneous mutations in the mouse are valuable resources in understanding human disease processes. Genetically defined mice develop dysferlinopathies closely resembling LGMD2B, Miyoshi myopathy, and distal myopathy of anterior tibialis.12Ho M Post CM Donahue LR Lidov HG Bronson RT Goolsby H Watkins SC Cox GA Brown Jr, RH Disruption of muscle membrane and phenotype divergence in two novel mouse models of dysferlin deficiency.Hum Mol Genet. 2004; 13: 1999-2010Crossref PubMed Scopus (155) Google Scholar In 2004, Ho et al12Ho M Post CM Donahue LR Lidov HG Bronson RT Goolsby H Watkins SC Cox GA Brown Jr, RH Disruption of muscle membrane and phenotype divergence in two novel mouse models of dysferlin deficiency.Hum Mol Genet. 2004; 13: 1999-2010Crossref PubMed Scopus (155) Google Scholar identified A/J mice as dysferlin deficient. A retrotransposon insertion in the dysferlin gene was found to result in a null allele, resulting in skeletal muscle dystrophy that shows histopathological and ultrastructural features that closely resemble the human dysferlinopathies of LGMD2B, Miyoshi myopathy, and distal myopathy of anterior tibialis.12Ho M Post CM Donahue LR Lidov HG Bronson RT Goolsby H Watkins SC Cox GA Brown Jr, RH Disruption of muscle membrane and phenotype divergence in two novel mouse models of dysferlin deficiency.Hum Mol Genet. 2004; 13: 1999-2010Crossref PubMed Scopus (155) Google Scholar The onset of dystrophic features in A/J mice begins in proximal limb muscles at 4 to 5 months of age and progresses to severe debilitating muscular dystrophy over several months. Ho et al12Ho M Post CM Donahue LR Lidov HG Bronson RT Goolsby H Watkins SC Cox GA Brown Jr, RH Disruption of muscle membrane and phenotype divergence in two novel mouse models of dysferlin deficiency.Hum Mol Genet. 2004; 13: 1999-2010Crossref PubMed Scopus (155) Google Scholar also found that human and murine dysferlin share very similar (approximately 90% identity) amino acid sequences. Cardiac muscle was not included in their study. Recently, Han et al,8Han R Bansal D Miyake K Muniz VP Weiss RM McNeil PL Campbell KP Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury.J Clin Invest. 2007; 117: 1805-1813Crossref PubMed Scopus (137) Google Scholar using sucrose gradient membrane fractionation on homogenates of wild-type C57BL/6J mouse heart muscle, showed that dysferlin is present in the cardiomyocyte plasma membrane and intracellular vesicle fractions. It was proposed that dysferlin is localized to the cardiomyocyte sarcolemma and some unidentified type of vesicles.8Han R Bansal D Miyake K Muniz VP Weiss RM McNeil PL Campbell KP Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury.J Clin Invest. 2007; 117: 1805-1813Crossref PubMed Scopus (137) Google Scholar Han et al8Han R Bansal D Miyake K Muniz VP Weiss RM McNeil PL Campbell KP Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury.J Clin Invest. 2007; 117: 1805-1813Crossref PubMed Scopus (137) Google Scholar in one study and Wenzel et al11Wenzel K Geier C Qadri F Hubner N Schulz H Erdmann B Gross V Bauer D Dechend R Dietz R Osterziel KJ Spuler S Ozcelik C Dysfunction of dysferlin-deficient hearts.J Mol Med. 2007; 85: 1203-1214Crossref PubMed Scopus (65) Google Scholar in another study showed that the induction of significant cardiac stress lead to cardiac dysfunction in dysferlin-deficient mice, but to what extent dysferlin deficiency causes cardiomyopathy by aging alone in patients clinically affected with the debilitating effects of LGMD2B, Miyoshi myopathy, or distal myopathy of anterior tibialis is unknown. In this study, we used the A/J mouse model to study the effects of aging in mice affected by genetically determined dysferlin deficiency by using echocardiography and conscious electrocardiography to determine functional changes in vivo, followed postmortem by light and electron microscopy to determine associated morphological changes. We have determined that the normal primary location for dysferlin in the cardiomyocyte of control A/HeJ mice is the intercalated disk (ID), and to a lesser extent, to a distinctive transverse banding pattern within the sarcoplasm of the cardiomyocyte. We have also determined that in the dysferlin-deficient cardiomyocyte there is evidence of membrane damage at these locations. We also present data that show functional cardiac deficits were present in vivo at around 10 months of age then recovered by 12 months. Histopathology showed that under normal laboratory conditions dysferlin deficiency causes only a mild cardiomyopathy even at advanced ages, suggesting the possibility of dysferlin-independent membrane repair mechanisms in cardiac muscle that do not exist in skeletal muscle. Both A/J and the substrain A/HeJ mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All animals were bred, housed, and maintained in a barrier facility at the Jackson Laboratory under standard conditions with a 12-hour light to 12-hour dark cycle and were provided food (Purina LabDiet Richmond, Indiana; 6%, autoclaved) and water ad libitum. The experimental protocols were approved by the Jackson Laboratory Institutional Animal Care and Use Committee, and are in accordance with accepted institutional and governmental policies. All mice in this study were subjected to necropsy and histopathology. For routine histopathology and immunohistochemical studies cardiac and skeletal muscle tissues as well as other tissues were harvested and fixed in Tellyesniczky/Fekete fixative. Most hearts were sectioned in either the four chamber longitudinal cut or transversely at the equator, base, and apex. For animals to be examined by electron microscopy as well as light microscopy, perfusion with 2% gluteraldehyde, 2% paraformaldehyde, and 0.5% tannic acid was followed by fixation in Tellyesniczky/Fekete fixative overnight. Tissues were then dehydrated and paraffin embedded for routine H&E and Masson's Trichrome staining. The morphometric index most frequently assessed is myocardial fibrosis.13Vasiljevic JD Popovic ZB Otasevic P Popovic ZV Vidakovic R Miric M Neskovic AN Myocardial fibrosis assessment by semiquantitative, point-counting and computer-based methods in patients with heart disease: a comparative study.Histopathology. 2001; 38: 338-343Crossref PubMed Scopus (28) Google Scholar Both semiquantitative and quantitative methods were used as described13Vasiljevic JD Popovic ZB Otasevic P Popovic ZV Vidakovic R Miric M Neskovic AN Myocardial fibrosis assessment by semiquantitative, point-counting and computer-based methods in patients with heart disease: a comparative study.Histopathology. 2001; 38: 338-343Crossref PubMed Scopus (28) Google Scholar, 14Gaspard GJ Pasumarthi KB Quantification of cardiac fibrosis by colour-subtractive computer-assisted image analysis.Clin Exp Pharmacol Physiol. 2008; 35: 679-686Crossref PubMed Scopus (23) Google Scholar to report the condition of the myocardium. Briefly, digital photographs of the myocardium were taken at 400× magnification on an Olympus CX41 (Olympus, Center Valley, PA) microscope equipped with a Leica DX320 (Lelca, Solms, Germany) color camera using Leica software. Color-subtractive computer-assisted image analysis in Image J software (NIH freeware, ) was used to quantify the percentage of collagen as described.14Gaspard GJ Pasumarthi KB Quantification of cardiac fibrosis by colour-subtractive computer-assisted image analysis.Clin Exp Pharmacol Physiol. 2008; 35: 679-686Crossref PubMed Scopus (23) Google Scholar Additionally, tissues sections were examined by a board certified veterinary pathologist specializing in murine pathology (O.F.). A semiquantitative method was used as described13Vasiljevic JD Popovic ZB Otasevic P Popovic ZV Vidakovic R Miric M Neskovic AN Myocardial fibrosis assessment by semiquantitative, point-counting and computer-based methods in patients with heart disease: a comparative study.Histopathology. 2001; 38: 338-343Crossref PubMed Scopus (28) Google Scholar in the assessment of the condition of the myocardium and scored as follows: 0 = normal; 1 = mild degenerative changes, including vacuolation; 2 = moderate degenerative changes, including widespread vacuolation with increased fibrosis; and 3 = severe changes including necrosis, large areas of interstitial fibrosis, and significant loss of cardiomyocytes. The incidental occurrence of the epicardial fibrosis/calcification lesion that is common in A/J and other strains of mice was excluded from consideration in this study. Slides of paraffin embedded sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Retrievagen A (BD Biosciences, San Diego, CA) was used per directions for antigen retrieval. Antigens were denatured with 100 mmol/L glycine in PBS then 0.05% SDS in PBS for 30 minutes at 50°C. Primary antibodies were mouse monoclonal anti-dysferlin (Clone Ham1/7B6) (Lab Vision/NeoMarkers; Fremont, CA) and rabbit polyclonal to Connexin 43/GJA1 (Abcam; Cambridge, MA). To control for nonspecific staining mouse on mouse (MOM) (PK-2200; Vector Laboratories; Burlingame, CA) diluent alone was applied to control sections. Nonspecific secondary binding was blocked and sections were developed by using the peroxidase MOM kit (PK-2200; Vector Laboratories) per directions, then counterstained with Meyer's Hematoxalin. Mice necropsied for transmission electron microscopy (TEM) were perfused with 2% gluteraldehyde, 2% paraformaldehyde, and 0.5% tannic acid in 0.1 M cacodylate buffer, pH 7.4. Tissues for TEM were harvested from the left ventricle, right ventricle, and the interventricular septum, then submerged in EM fix, minced, fixed overnight, then postfixed with 1% osmium tetroxide (0.1 M cacodylate buffer, pH 7.4). The samples were then rinsed in the same buffer, dehydrated in a graded series of ethanols, and embedded in Epon Araldite resin (Electron Microscopy Sciences; Hatfield, PA). Ultrathin sections were poststained with uranyl acetate followed by lead citrate. Samples were imaged on a JEOL JEM-1230 electron microscope (Tokyo, Japan) and images were captured with AMT Advantage CCD 6 Mpix (Danvers, MA) and Image Capture Engine Software version 54.4.2.236. In this longitudinal assessment of cardiovascular function, echocardiography and conscious electrocardiograms were done on groups of control (A/HeJ) and dysferlin-deficient (A/J) mice at 3, 5, 8, 9, 10, 12, 14, and 16 to 18 months of age both male and female. No sex differences were seen. Cardiac electrical activity was recorded from conscious mice gently restrained in a Murine ECG Restrainer plexiglas cylinder (QRS Phenotyping, Inc; Calgary, Canada) by using gel-foot pads (EMPI; St Paul, MN) to collect the signal. Signal acquisition was obtained by a PowerLab ARV module version 5.5.4 and ECG analysis module for chart version 2 (AD Instruments; Golden, CO). Mice were allowed to acclimate for 5 to 10 minutes after which the ECG signal was recorded for up to 5 minutes or until a steady baseline lead I signal was recorded for a 20 to 30 second interval. All of the standard P, QRS, and T wave intervals and amplitudes were measured and the signal was averaged as described.15Maddatu TP Garvey SM Schroeder DG Hampton TG Cox GA Transgenic rescue of neurogenic atrophy in the nmd mouse reveals a role for Ighmbp2 in dilated cardiomyopathy.Hum Mol Genet. 2004; 13: 1105-1115Crossref PubMed Scopus (57) Google Scholar Following the conscious electrocardiograms, echocardiography was performed on the same groups of A/J and A/HeJ mice at these ages from 3 to 18 months. The Vevo 770 High-Frequency Ultrasound (Visualsonics; Toronto, Ontario, Canada) was used to obtain real-time images of cardiac function. A 30 MHz real-time microvisualization scan head yielded ultrasonic images with an infiltration depth of 13 mm, and a capture rate of 100 Hz. Animals were induced individually with 5% isoflurane at 0.8 L/min and maintained on 1 to 1.5% isoflurane at 0.8 L/min. Hair was removed from the ventral thorax with a depilatory cream, just before standard imaging, measurements, and calculations were performed as described.15Maddatu TP Garvey SM Schroeder DG Hampton TG Cox GA Transgenic rescue of neurogenic atrophy in the nmd mouse reveals a role for Ighmbp2 in dilated cardiomyopathy.Hum Mol Genet. 2004; 13: 1105-1115Crossref PubMed Scopus (57) Google Scholar All values are presented as means ± SEM. Student's paired t-test was applied to determine statistical significances. P values <0.05 were considered significant. Statistical analyses were performed by using Prism software (Irvine, CA). Tissue sections of skeletal and cardiac muscle from unmanipulated control A/HeJ and dysferlin-deficient A/J mice were stained with anti-dysferlin antibody and horseradish peroxidase (HRP). In agreement with the known localization, dysferlin staining was localized to the sarcolemma in skeletal muscle in control A/HeJ mice (Figure 1A). In control cardiac muscle, longitudinally sectioned cardiomyocytes showed intense staining of the IDs (Figure 1B). Additionally, in the sarcoplasm there was staining in a transverse striation pattern at regular intervals the length of a sarcomere. Cardiomyocytes oriented in cross section did not show dysferlin staining of the lateral sarcolemma (data not shown). Similar staining patterns were present at various ages and in Balb/cByJ, C57BR/cdJ, and C57BL/6J mice (data not shown). In the dysferlin-deficient A/J mice, staining for dysferlin was negative in both skeletal muscle (Figure 1C) and cardiac muscle (Figure 1D). Examination of H&E and Masson's trichrome stained hearts of 6-month-old A/HeJ mice showed normal cardiomyocytes (Figure 2A) and ID (arrow Figure 2C), whereas dysferlin-deficient A/J mice showed widespread sarcoplasmic vacuolar degeneration of the majority of cardiomyocytes (Figure 2B), often with multiple vacuolations at the ID of cardiomyocytes (arrow Figure 2B), as well as vacuolations in the sarcoplasm (Figure 2D). Since vacuolar degeneration is a manifestation of cell injury, usually due to membrane damage, and the ID is a critical structure in the transmission of electrical impulse from one cardiomyocyte to another, membrane damage at this location could disrupt electrical conduction. Therefore, ECGs were done to assess electrical conduction in the myocardium. The following intervals were measured: P duration (intra-atrial conduction); PR interval (intra-atrial and atrio-ventricular conduction); QRS interval (intraventricular conduction); QT interval (ventricular conduction to repolarization of ventricle); and RR interval (length of full cycle). Results of these ECGs indicated that none of these intervals were significantly different in dysferlin-deficient mice compared with controls at all age intervals tested from 5 to 20 months (data not shown). In addition, none of the amplitudes (P, Q, R, S, or T) were significantly different from those of controls (data not shown). The only parameter that was significantly different from the controls was the increased heart rates of 12- to 16-month-old A/J mice. This increased heart rate peaked at 14 months of age before declining thereafter (Figure 3). There are several possible explanations for this increased heart rate (ie, pain and/or declining cardiovascular conditioning due to skeletal muscle disease, or an early sign of compensation for reduced cardiomyocyte contractility) but it is not reflective of a conduction disturbance. High-frequency ultrasound was used to obtain real-time echocardiographic images to evaluate cardiac function. Ejection fraction (EF) and fractional shortening (FS) are echocardiographic indicators of overall cardiac function. EF, a measure of left ventricular function, is the percentage of blood that is pumped out of the ventricle with each beat. EFs above 60% are considered normal.16Sutton P Measurements in Cardiology. 1999; Google Scholar There was no significant difference in EF between control A/HeJ (75 to 77%) and dysferlin-deficient A/J mice (71 to 79%) from 3 months of age to 8 months of age (Figure 4A). After 8 months the EF of A/J mice declined significantly until by 10 months of age it was reduced to 61%, while A/HeJ mice retained a normal EF (82%). By 12 months of age EF for A/J mice rebounded to 75% and remained nearly at that level for the next 8 months. The EF of A/HeJ mice was only slightly higher at 78% during that time. FS is another measure of left ventricular contractility. It is a ratio of the difference in the diameter of the left ventricle between the contracted and the relaxed states. It has the advantage of being a ratio of actual measurements, whereas ejection fraction is a ratio of three-dimensional quantities derived from two-dimensional measurements. Fractional shortening values greater than 30% are considered normal.16Sutton P Measurements in Cardiology. 1999; Google Scholar As with EF, both A/HeJ and A/J mice had normal FS of 42 to 47% until 8 months of age (Figure 4B). From 8 to 10 months of age the FS of A/J but not A/HeJ decreased to 33%, then rebounded by 12 months of age to 42%. These data suggest that in dysferlin-deficient A/J mice there is a significant deterioration in contractility from 8 to 10 months of age, and a recovery from 10 months of age to 12 months of age. Subsequently, EF and FS of A/J mice remained slightly below that of A/HeJ mice but both continue within the range of normal for the remainder of the study (18 months of age). To further assess the nature of the early morphological changes, TEM was done on A/J and A/HeJ mice beginning at 5 months of age. At 5 months of age the ultrastructure of cardiomyocytes, including the ID, was normal in A/HeJ mice (Figure 5A) as well as A/J dysferlin-deficient mice (Figure 5B). In 6-month-old A/HeJ control mice, the IDs were normal (Figure 5C), while in age-matched dysferlin-deficient A/J mice there were occasionally cardiomyocytes that had membrane disruptions and/or delaminations (long arrow Figure 5D) associated with the fascia adherens (FA) portion of the substructure of the ID. Near the ID there were also occaisonal vacuolations (Figure 5E). Some of these vacuolations were dilations of the sarcoplastic reticulum while others were mitochondria that were swollen to 5 to 10 times normal size with destruction of the cristae (high amplitude swelling). TEM also showed that there were similar vacuolations between myofibrils unassociated with the ID. The desmosomes were normal, and most gap junctions (GJ) of dysferlin-deficient A/J mice were normal but occasionally there were GJ that appeared distorted yet intact (GJ in Figure 5D). This is consistent with the normal electrical conduction seen on the ECGs, and further supported by normal connexin staining on IHC (Supplemental Figure S1, see ). TEM at subsequent ages (8, 10, 12, 14, and 16 months) revealed a persistence of these lesions in A/J mice but not A/HeJ mice, and in addition they had widely scattered individual necrotic cardiomyocytes with myelin figures and vacuolations near the ID (Figure 5F). In the oldest ages (12, 14, and 16 months) there were occasional cardiomyocytes with decreased myofibril density, large accumulations of degenerate mitochondria displacing myofibrils, and collagen in the extracellular matrix (Figure 5G) in both A/J mice and A/HeJ mice. Since one limitation of TEM is the very small sample size, routine histopathology was used to more broadly assess the progression of changes in the myocardia of A/J mice and A/HeJ mice from 2 to 18 months of age. Initial examination of H&E and Masson's trichrome stained hearts of young (2 to 5 month old) mice revealed that both A/J mice and A/HeJ mice had normal hearts (Figure 6, A and D) and normal cardiomyocytes (Figure 6, B and C). Middle aged (6 month) and aged (18 month) A/HeJ cardiomyocytes were normal (Figure 6, F and J), whereas A/J mice often had widespread sarcoplasmic vacuolar degeneration of cardiomyocytes, often with multiple vacuolations at the ID (Figure 6, G and K). Subgrossly, both A/HeJ and A/J hearts appeared to remain normal (Figure 6, E, H, I, and L). We then used two methods to more quantitatively assess the development of cardiomyopathy. The development of cardiomyopathy is associated with an increase in interstitial collagen (fibrosis) as the myocardium responds to the loss of cardiomyocytes. Color-subtractive computer-assisted image analysis and Image J software was used to quantify the percentage of collagen in the myocardium (see Methods). This revealed no significant difference in that both A/HeJ and A/J percentages of collagen was about 2% at 2 months of age and gradually increased to about 3% as they aged to 18 months of age (Figure 6M). This suggests little or no cardiomyocyte death in either A/HeJ mice or A/J mice. Cardiomyopathy is also histopathologically evident as remodeling of the ventricular walls, changes in wall and papillary muscle thickness, degenerative changes that range from vacuolations to necrosis of individual or groups of cardiomyocytes, and changes in nuclear morphology, as well as fibrosis. These more subjective changes can be evaluated for overall significance by an experienced pathologist, so on the same group of sections used for color-subtractive computer-assisted image analysis an experienced patholog
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