Cardiac Muscle - dysferlin

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Unformatted text preview: Related Commentary, page 1749 Research article Dysferlin-mediated membrane repair protects the heart from stress-induced left ventricular injury Renzhi Han,1,2,3,4,5 Dimple Bansal,1,2,3,4,5 Katsuya Miyake,6 Viviane P. Muniz,1,2,3,4,5 Robert M. Weiss,2,5 Paul L. McNeil,7 and Kevin P. Campbell1,2,3,4,5 Hughes Medical Institute, 2Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center, of Molecular Physiology and Biophysics, 4Department of Neurology, and 5Department of Internal Medicine, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, Iowa, USA. 6Institute of Molecular Medicine and Genetics and 7Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia, USA. 3Department 1Howard Dilated cardiomyopathy is a life-threatening syndrome that can arise from a myriad of causes, but predisposition toward this malady is inherited in many cases. A number of inherited forms of dilated cardiomyopathy arise from mutations in genes that encode proteins involved in linking the cytoskeleton to the extracellular matrix, and disruption of this link renders the cell membrane more susceptible to injury. Membrane repair is an important cellular mechanism that animal cells have developed to survive membrane disruption. We have previously shown that dysferlin deficiency leads to defective membrane resealing in skeletal muscle and muscle necrosis; however, the function of dysferlin in the heart remains to be determined. Here, we demonstrate that dysferlin is also involved in cardiomyocyte membrane repair and that dysferlin deficiency leads to cardiomyopathy. In particular, stress exercise disturbs left ventricular function in dysferlin-null mice and increases Evans blue dye uptake in dysferlin-deficient cardiomyocytes. Furthermore, a combined deficiency of dystrophin and dysferlin leads to early onset cardiomyopathy. Our results suggest that dysferlin-mediated membrane repair is important for maintaining membrane integrity of cardiomyocytes, particularly under conditions of mechanical stress. Thus, our study establishes what we believe is a novel mechanism underlying the cardiomyopathy that results from a defective membrane repair in the absence of dysferlin. Introduction Dilated cardiomyopathy is the most common type of cardiomyopathy, a condition that can often progress into heart failure and sudden death. Many cases of dilated cardiomyopathy have a genetic etiology. Indeed, inherited forms of idiopathic dilated cardiomyopathy  account for at least 30% of dilated cardiomyopathy cases. These are a  major cause of severe heart failure and necessitate heart transplantation (1). A number of genes encoding sarcomeric and cytoskeletal  proteins have been identified as being responsible for dilated cardiomyopathy: cardiac actin, cardiac troponin T, β-myosin heavy chain,  α-tropomyosin, α-actinin, titin, metavinculin, and desmin (reviewed  in ref. 2). Mutations in genes such as dystrophin (3, 4), α-sarcoglycan (5), β-sarcoglycan (6), γ-sarcoglycan (7), and δ-sarcoglycan (8, 9),  which encode proteins involved in linking the cytoskeleton, sarcolemma, and extracellular matrix, have also been identified as causes  of some familial dilated cardiomyopathy. Such mutations lead to  the disruption of the cytoskeleton-sarcolemma–extracellular matrix  link and thus render the sarcolemma more susceptible to contraction-induced injury (10–12). The plasma membrane provides a physical barrier between the  extracellular and intracellular environments, and the maintenance  of this barrier is crucial for cell survival; however, plasma-membrane disruption occurs physiologically in certain types of cells,  Nonstandard abbreviations used: DGC, dystrophin-glycoprotein complex; DKO,  dysferlin/dystrophin double-mutant (mice); EBD, Evans blue dye; LGMD2B, limb-girdle  muscular dystrophy type 2B. Conflict of interest: The authors have declared that no conflict of interest exists. Citation for this article: J. Clin. Invest. 117:1805–1813 (2007). doi:10.1172/JCI30848. such as skeletal (13) and cardiac muscle (14), due to their large size  and the mechanical stress of contraction and relaxation. The frequency of membrane disruption in skeletal and cardiac muscle cells  is directly dependent on the level of physical activity (13, 14). In  skeletal muscle, membrane disruption is known to initiate a rapid  Ca2+-dependent membrane-repair process (15) that allows the damaged cells to reseal their membranes, which is essential for their  survival. An active membrane repair is therefore required to eliminate the cellular cost of replacing large and/or frequently injured  skeletal muscle cells. Dysferlin has been shown to play a pivotal role  in the membrane-repair pathway of skeletal muscle, and dysferlin  deficiency leads to defective membrane resealing in skeletal muscle  (15, 16). The failure to reseal membrane disruptions and the consequent Ca2+ entry through the disruption sites leads to activation  of Ca2+-triggered proteases and rapid cell death cascade (which can  occur within seconds). In humans, dysferlin deficiency leads to 3  clinically distinct forms of muscular dystrophy: limb-girdle muscular dystrophy type 2B (LGMD2B) (17), Miyoshi myopathy (18),  and a distal myopathy with anterior tibial onset (19). Similarly, dysferlin-deficient mice develop a pronounced limb-girdle muscular  dystrophy (15, 20, 21). Therefore, the rapid resealing of membrane  disruptions in skeletal muscle is required to maintain the plasma  membrane integrity of the cells, which is in turn essential for the  normal structure and function of skeletal muscle. Notably, a 57-year-old Japanese woman with a 3370G→T missense mutation in dysferlin was recently reported to manifest ventricular enlargement and diffuse hypokinesia (22). This finding  suggests that dysferlin deficiency may induce cardiomyopathy  as well as muscular dystrophy. However, as this remains the only  1805 The Journal of Clinical Investigation      Volume 117      Number 7      July 2007  research article Figure 1 Expression and subcellular localization of dysferlin in cardiac muscle. (A) Dysferlin transcripts were amplified by different primer sets (nos. 1–3) from the total RNA extracted from the WT mouse skeletal muscle (SK) and heart (HE). (B) Dysferlin (Dysf) proteins were detected among the SDS-extracted proteins of WT skeletal muscle and heart but not among those extracted from dysferlin-null skeletal muscle and heart. Caveolin-3 (Cav3) was detected and served as the loading control. (C) Subcellular membrane fractionation of heart homogenates from WT mice (see Methods for details) showed that dysferlin is localized in the plasma membrane (fractions 12 and 13; pellet 2) and in the intracellular vesicle fractions (fractions 2–11). α-DG, anti–α-dystroglycan; M, molecular weight marker. report of cardiac involvement in a dysferlin-deficient patient, it  is not clear whether the dysferlin deficiency is the primary cause  of the cardiomyopathy in this case. With respect to this question,  however, a recent meeting abstract reporting elevated tropin levels  in 9 of 11 (81%) European dysferlin patients ranging in age from  19 to 48 (23) is of interest. Although none of these patients had  developed overt cardiomyopathy at the time of reporting, following them for the development of this condition over time as part of  a systematic investigation involving an extended group of patients  of matching age may clarify whether the dysferlin deficiency can  ultimately lead to cardiac abnormality. It is reasonable to conjecture that dysferlin may play an important  role  in  the  heart.  Dysferlin  has  been  shown  to  be  highly  expressed in the rat heart (24), and because cardiac muscle undergoes intensive mechanical activity similar to that experienced by  skeletal muscle, it may also require a robust membrane resealing  mechanism. We thus hypothesized that dysferlin deficiency leads  to defective repair of cardiomyocytes following membrane disruption and thus to cardiomyopathy. To test these hypotheses, we  studied cardiac function and myocyte membrane-repair capacity  in the hearts of dysferlin-null mice. Our data show that dysferlin is  an important component of the Ca2+-dependent membrane-repair  mechanism in cardiomyocytes, that dysferlin deficiency leads to  cardiomyopathy, and that this condition can be worsened by either  stress exercise or the loss of dystrophin. Results Dysferlin is expressed in cardiac muscle. We first examined dysferlin  expression in the hearts of WT mice by RT-PCR and immunoblotting. The dysferlin transcript can be amplified from both the skeletal muscle and heart using a pair of primers designed to complement the 5′ region of the gene (Figure 1A). An alternatively spliced  variant lacking a 42-bp exon (exon 17) had previously been reported in human muscle cells (25). We found that the isoform with  the 42-bp exon is the predominant one in mouse skeletal muscle  whereas cardiac muscle expresses similar levels of both isoforms  (Figure 1A). We detected another isoform of  dysferlin, which we  believe to be novel, that shows an alternatively spliced, in-frame,  93-bp exon (Figure 1A) immediately after exon 5. The presence  1806 of this isoform is very low in adult skeletal muscle but high in  the heart. Immunoblotting of WT heart SDS-extracted proteins  with the dysferlin antibody Hamlet, which was raised against the  dysferlin C terminal region, detected a band of approximately  230 kDa, and hearts from the dysferlin-null mice were completely  devoid of dysferlin protein (Figure 1B). Anti–caveolin-3 antibody  was used as a loading control. To determine the subcellular distribution of dysferlin in normal  cardiomyocytes, sucrose gradient membrane fractionation was performed after homogenizing WT hearts. Dysferlin was detected in  both the fractions that were enriched for plasma membrane and  those that were enriched with intracellular vesicle markers such as  VAMP2 (Figure 1C) and the transferrin receptor (data not shown). Dysferlin is required for the membrane repair of cardiac muscle. Our  data showing that dysferlin is highly expressed incardiac muscle  and that it is present in vesicles as well as in the plasma membrane  lent support to our hypothesis that this protein may be involved  in the repair of cardiac muscle membranes. However, it was not  known whether normal cardiomyocytes possess a Ca2+-dependent  membrane-repair capacity similar to that of skeletal muscle. To  directly address this issue, we developed an in situ membranerepair assay with which to test the cardiomyocytes of the WT  mice. First, the membranes of cardiomyocytes were wounded by  full-power irradiation with a mode-locked infrared laser in the  presence of the membrane-impermeable dye FM 1-43, a watersoluble molecule that becomes fluorescent when it partitions into  a membrane and does not cross it (the assay was conducted on  surface cardiomyocytes of heart slices to assure easy dye accessibility). Upon laser-induced membrane wounding (Figure 2A), the  plasma membranes of WT cardiomyocytes were efficiently resealed  in the presence of Ca2+. This was indicated by the observation that  in the presence of Ca2+, the FM 1-43 fluorescence intensity initially increased but halted within 1–2 minutes whereas the FM 1-43  fluorescence intensity continuously increased in the absence of  Ca2+ (Figure 2, A and B). Thus, like skeletal muscle, cardiac muscle  appears to possess an active and efficient, Ca2+-dependent membrane-repair mechanism. Testing the membrane-repair efficiency  of the cardiomyocytes from the dysferlin-null mice, we found that  even in the presence of Ca2+, the cardiomyocytes from the dysferlinnull heart slices could not effectively reseal the wounded plasma  membranes (Figure 2, A and B). This result clearly suggests that  dysferlin is an important component of the membrane-repair  machinery in the cardiac muscle. Dysferlin-null mice develop mild cardiomyopathy.  To  determine  whether the defective membrane repair in cardiac muscle due to  dysferlin deficiency leads to any cardiac abnormality, we conduct- The Journal of Clinical Investigation      Volume 117      Number 7      July 2007 research article Figure 2 Membrane damage-repair assay in cardiomyocytes in left ventricle slices from normal and dysferlin-null mice. (A) Left panel: WT cardiomyocytes in the presence of physiological Ca2+ (WT + Ca); middle panel: WT cardiomyocytes in the absence of physiological Ca2+ (WT – Ca); right panel: dysferlin-null cardiomyocytes in the presence of physiological Ca2+ (dysf-null + Ca). Arrows indicate sites of laser damage. Scale bar: 50 μm. (B) Quantitative analysis of the membranerepair assay of normal and dysferlin-null cardiomyocytes. ed a histological analysis of heart sections from dysferlin-null mice  and age-matched controls and also measured the serum levels of  cardiac troponin T in these animals. H&E examination of the heart sections from the  dysferlin-null  mice that were less than 32 weeks old did not reveal a remarkable  pathological change (data not shown). However, heart sections  of the dysferlin-null mice that were 56 weeks old showed sporadic necrotic fibers (Figure 3, D and G), and these necrotic fibers  were observed more frequently in 80-week-old dysferlin-null mice    (Figure 3, E and H). Only rarely were necrotic fibers observed in the hearts of the age-matched controls (Figure 3, A and B). As revealed  by Sirius red staining, cardiac fibrosis was present in aged dysferlinnull mice (Figure 3, F and I) but not in aged WT controls (Figure 3C).    Total collagen deposits in aged dysferlin-null mice were significantly higher (~3-fold) than in WT controls (Figure 3J). Troponin T is a cardiac muscle–specific structural protein that is  impermeable to the plasma membrane, and it is undetectable in the  serum under normal physiological conditions. However, in the case of  several abnormal cardiac conditions in which cardiac muscle undergoes active necrosis (including cardiomyopathy), troponin T is released  into the bloodstream and can be detected at high levels in the serum  (26). We measured the resting serum troponin T levels in the dysferlin-null mice of 2 age groups (30 and 70 to 90 weeks old) (Figure 3K).    Of the 8  dysferlin-null mice (30 weeks old), 5 had mildly elevated  (~7-fold increase) serum troponin T levels (0.074 ± 0.015 ng/ml,    n = 5) whereas none of the age-matched controls did (<0.010 ng/ml,  n = 10). In the 70- to 90-week-old group, 17 of the 26 dysferlin-null  mice had increased serum troponin T levels (0.092 ± 0.013 ng/ml,    n = 17) whereas the serum troponin T levels in all of the age-matched  controls was under the level of detection (<0.010 ng/ml, n = 15). Stress exercise exacerbates the cardiomyopathy in dysferlin-null mice.  To determine whether the dysferlin-null mice are likely to develop  a more pronounced cardiac abnormality when their hearts are subjected to mechanical stress, we first recorded echocardiography to  compare the cardiac function of the dysferlin-null mice before and  after stress exercise. When the mice were 54 weeks of age, stress  exercise significantly reduced the heart rate of the  dysferlin-null  mice (668 ± 15 min–1 before and 615 ± 12 min–1 after exercise, n = 9;    P < 0.01) but did not affect that of the control mice (n = 6; P = 0.33)  (Figure 4A). After stress exercise, the dysferlin-null mice had a significantly increased left ventricular end-systolic volume (9.9 ± 1.1 μl    before and 19.0 ± 2.2  μl after exercise;  P < 0.01), which was not  affected by the stress exercise in the control mice (8.3 ± 2.3 μl before  and 8.8 ± 2.2 μl after; P = 0.85) (Figure 4B). Stress exercise also tended to increase the left ventricular end-diastolic volume of the dysferlin-null mice (49.1 ± 3.6 μl before and 60.9 ± 2.5 μl after) although  the difference between exercised and nonexercised animals was not  statistically significant (P = 0.06). However, relative to the left ventricular end-diastolic volume of the exercised control mice, that in  the exercised dysferlin-null mice was considered to be significantly  different (P = 0.003) (Figure 4C). Finally, the left ventricular ejection fraction of the dysferlin-null mice but not of the controls was  significantly decreased by the stress exercise (P = 0.02) (Figure 4D).  These results suggest that the dysferlin-null mice develop exerciseinduced systolic dysfunction and pathological chamber dilation. The Evans blue dye (EBD) uptake analysis of the heart before and  after the stress exercise was also performed in order to determine  whether subtle damage to myocytes occurs at early stages and in  response to exercise. In 24-week-old dysferlin-null mice, which usually do not show any pronounced abnormalities in heart sections, sporadic EBD-positive myocytes were observed (Figure 4E) after stress  exercise. This was not the case in hearts from either the nonstressed  dysferlin-null mice or the stressed WT mice (data not shown). H&E  staining of the matched sections showed fiber necrosis in dysferlinnull mice after stress exercise (Figure 4E). Collectively, these results  suggest that the stress exercise initiates the left ventricular dysfunction at both the cellular and tissue levels in dysferlin-null mice. 1807 The Journal of Clinical Investigation      Volume 117      Number 7      July 2007  research article dysferlin loss. To test this possibility, we generated a DKO mouse model by crossing dysferlin-null  mice with mdx mice, a mouse model of Duchenne  muscular dystrophy lacking dystrophin (31–33).  The latter model is widely used to study Duchenne muscular dystrophy and carries a mutation  that eliminates expression of the 427-kDa muscle  and brain isoforms of dystrophin. The  loss  of  dystrophin  and  dysferlin  in  the  skeletal muscles of DKO mice was confirmed by  immunoblotting and immunofluorescence analysis (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/ JCI30848DS1). This entailed a comparative histological analysis of the cardiac muscles, with heart  sections taken every 200 μm from the apex to the  base so that even highly regionalized histopathological changes to the heart would be detected. Acute  cardiac muscle necrosis and fibrosis were observed  in DKO mice as young as 11 weeks of age (Figure 5,    D–I). In contrast, no apparent necrosis/fibrosis  could  be  detected  in  littermate  mdx  (Figure  5,    A–C) or  dysferlin-null mice (not shown) at even  16 weeks of age. Although small regions of fibrosis could be seen in older  mdx and  dysferlin-null  mice, those about 1 year old (see Figure 3, D–I),  the pathology was never as severe as that observed  in DKO mice. The serum troponin T levels measured  in  10-  and  18-week-old  DKO  mice  were  nearly 20-fold and over 38-fold higher (Figure 5K),  respectively, suggesting that necrosis was actively  occurring in cardiac muscle. At 18 weeks old, both  dysferlin-null and mdx mice showed the same basal  Figure 3 Mild cardiomyopathy in senescent dysferlin-null mice. H&E staining of heart sections levels of serum troponin T levels as WT animals. revealed necrosis and fibrosis in cardiac muscle of dysferlin-null mice (D, E, G, H) but not The mice were also exposed to stress exercise,  in WT mice (A and B). Sirius red staining (C, F, I) showed increased collagen deposits in and this was found to induce large areas of acute  aged dysferlin-null mice. Scale bars: 2 mm (A–F); 500 μm (G–I). H&E and Sirius red stain- necrosis in 9-week-old DKO mice (Figure 6, A–D)  ing images were representatives of at least 4 mice in each group. (J) Quantitative analysis but little to no acute necrosis in mdx or dysferlinof total cardiac collagen deposits revealed about a 3-fold increase in aged dysferlin-null null mice of the same age (data not shown). The  mice (n = 6 for each group; *P = 0.03, unpaired Student’s t test). (K) Serum troponin T was mildly elevated in the dysferlin-null mice at 30 (n = 5) and 70–90 weeks of age (n = 17) but postexercise EBD uptake analysis revealed very  large areas of EBD uptake in the cardiac muscles  not detected (ND) in age-matched WT mice (n = 10 for each age group). of DKO  mice (Figure 6G).  We  again  analyzed  sections taken at 200-μm intervals throughout  Dysferlin-mediated membrane repair minimizes the pathological changes the heart and detected EBD-positive tissue in all sections whereas  in cardiac muscle. In contrast to dysferlin, dystrophin is an integral  EBD-positive tissue was limited to small localized areas in  mdx  component of the dystrophin-glycoprotein complex (DGC), which  (Figure 6F) and dysferlin-null mice (Figure 6E). No EBD-positive  is present on the plasma membrane and connects the extracellular  tissue was seen in WT mice. matrix to the intracellular cytoskeleton (10). Mutations in any of  Dysferlin does not protect ischemic cardiomyopathy. Despite signifithe DGC components disrupt the stability of the DGC within the  cant advances in our understanding of coronary disease over the  plasma membrane (27, 28), which in turn breaks the transmem- past 2 decades, coronary ischemic artery disease remains the prebrane link between the extracellular matrix and the intracellular  dominant cause of premature death (34). To investigate whether  cytoskeleton. Loss of this link renders the muscle membrane sus- dysferlin-mediated membrane repair plays a role in myocardial  ceptible to contraction-induced injuries (10–12, 29, 30). This sug- ischemia, we performed a coronary artery ligation experiment in  gests that a robust membrane-repair capacity may be extremely  WT and dysferlin-null mice. WT (n = 17) and dysferlin-null (n = 24)  important for minimizing the muscle cell loss in the DGC-linked  mice were subjected to myocardial infarction by permanent ligamuscular dystrophies. We therefore hypothesized that the dysferlin/ tion of the proximal left coronary artery. We did not observe any  dystrophin double-mutant (DKO) mice would develop more severe  statistically significant differences between genotypes with respect  cardiac muscle pathology than mice mutant for either gene alone,  to mortality or echocardiographically determined initial infarct  owing to an increased susceptibility to muscle membrane injuries  size, ejection fraction, or the extent of left ventricular remodeling  due to dystrophin loss and to defective membrane repair due to  2 weeks after infarct (Supplemental Figure 2). 1808 The Journal of Clinical Investigation      Volume 117      Number 7      July 2007 research article cardiomyopathy. The observation that stress  exercise and loss of dystrophin greatly exacerbate the cardiac manifestations of disease in  the dysferlin-null mice suggests that the dysferlin-mediated membrane repair is indispensable for maintaining the sarcolemma integrity  of cardiomyocytes. Dysferlin expression in the mouse heart was  confirmed by RT-PCR and immunoblotting in  the present study. The fact that we have identified several splice variants of dysferlin in the  heart muscle implies that dysferlin regulation  is more complex in the heart than in skeletal  muscle and that this protein may have greater  functional diversity than previously appreciated.  Furthermore,  subcellular  membrane  fractionation of the heart muscle suggests  that dysferlin is localized to both the sarcolemma and some unidentified type of vesicles,  which is consistent with a role in the membrane-repair process. However, the nature of  the dysferlin vesicles and the exact mechanism  underlying the dysferlin-mediated membrane  repair remain to be determined. Dysferlin is predicted to have 7 C2 domains,  which in other proteins are known to mediate  Ca2+ and phospholipid binding as well as protein-protein interaction. The first C2 domain  of dysferlin has been shown to bind phospholipid in a Ca2+-dependent manner (35). Dysferlin has also been shown to bind annexins  A1 and A2 following membrane injury in a  Ca2+-dependent manner, and the distribution  of these annexins after injury is significantly  altered in dysferlin-deficient skeletal muscle  (16), suggesting their involvement in the dysferlin-mediated membrane-repair mechanism.  Figure 4 Effect of stress exercise on echocardiography and cardiac uptake of EBD in dysferlin-null The involvement of annexin A1 in membrane  mice. Stress exercise significantly decreased heart rate ( A) and increased end-systolic repair is further supported by a recent report  volume (ESV) (B) in dysferlin-null mice. End-diastolic volume (EDV) (C) also tended to showing that inhibiting its function — using  increase in response to stress exercise in dysferlin-null mice while ejection fraction (EF) an annexin A1 function–blocking antibody,  (D) decreased in these animals. n = 6 for control and n = 9 for dysferlin-null mice. (E) H&E a small peptide  competitor  of  annexin  A1,  (bottom panel) and EBD uptake analysis (top panel) showed individual cardiac muscle fiber or a dominant-negative annexin A1 mutant  necrosis and EBD uptake (marked by asterisks) in heart sections of 24-week-old dysferlinprotein incapable of Ca2+ binding — prevents  null mice following stress exercise. Scale bar: 100 μm. membrane  resealing  (36).  Synaptotagmin  VII–mediated lysosomal exocytosis has also  been implicated in the plasma membrane repair of skeletal muscle,  Discussion In 1998, mutations in dysferlin were reported as the cause of LGM- since synaptotagmin VII–deficient mice developed inflammatory  D2B (17) and Miyoshi myopathy (18), and later they were identi- myopathy with extensive fibrosis, high serum levels of creatine  fied in a distal myopathy with anterior tibial onset (19). Although  kinase, and progressive muscle weakness (37). However, it remains  dysferlin was shown to be widely expressed (with the highest levels  to be clarified whether synaptotagmin VII and dysferlin play simiin skeletal muscle and heart) (24), the involvement of nonskeletal  lar roles in 2 separate membrane-repair mechanisms or different  muscle tissues, especially the heart, in dysferlinopathy patients, has  roles in the same membrane-repair pathway. not been systematically investigated. In the present study, we proOur studies in dysferlin-null mice suggest that dysferlinopathy  vide what we believe is the first evidence in support of the existence  patients may be predisposed to cardiac disease. In support of this,  of a Ca2+-dependent mechanism to repair the membrane wounds  the 57-year-old Japanese woman with a 3370G→T missense mutain cardiomyocytes. In addition, we have shown that dysferlin, which  tion in  dysferlin (resulting in an amino acid change at position  was previously shown to play an important role in the membrane  999 from Trp to Cys) manifested cardiomyopathy after more than  repair of skeletal muscle (15, 16), is also involved in the repair of  20 years of progressive muscle wasting and weakness (22). Intercardiomyocyte membranes and that dysferlin deficiency leads to  estingly, a 53-year-old Japanese man with the same 3370G→T    The Journal of Clinical Investigation      Volume 117      Number 7      July 2007  1809 research article mutations are capable of similar effects. Nevertheless, these clinical reports, together with our present study, suggest that dysferlin deficiency may  potentially cause abnormalities in a variety of tissues. In addition, our study shows that dysferlin  plays an important role in the membrane-repair  pathway in both skeletal and cardiac muscle cells,  suggesting that defective membrane repair may  be a common mechanism that accounts for the  disease manifestations in different tissue types in  dysferlinopathy patients. In spite of a lack of systematic investigation  regarding cardiac involvement in dysferlin-deficient patients, our study clearly shows that dysferlin-deficient cardiomyocytes are defective in  membrane repair and that senescent dysferlin-null  mice indeed develop cardiomyopathy. Although  we cannot currently rule out the possibility of  compensation by other ferlin family proteins (e.g.,  myoferlin; ref. 41) in cardiac muscle, the most likely reason for the mildness of the cardiomyopathy  phenotype in dysferlin-deficient mice is that the  heart does not experience excessive mechanical  stress  under  normal  laboratory  conditions.  In  support of this notion, we observed that stress  exercise exacerbated the cardiac manifestations  in  dysferlin-null mice and that loss of dysferlin  greatly accelerated the progression of cardiomyopathy in mdx mice. These findings suggest that  dysferlinopathy-derived  overt  cardiomyopathy  may require a second process (e.g., physical stress)  Figure 5 or a second cardiomyopathy-linked gene mutaSevere early-onset cardiomyopathy in DKO mice. Histological examination revealed that tion. However, the fact that our results do not  the DKO mice presented large areas of necrosis and fibrosis at 11 (D–F) and 16 (G–I) support a protective role of dysferlin in ischemic  weeks of age whereas mdx mice (A–C) did not present any apparent fiber necrosis or cardiomyopathy indicates that a functional memfibrosis even by 16 weeks of age. Scale bars: 2 mm (A, D, and G); 500 μm (B, C, E, F, brane-repair system is not sufficient for preventH, and I). (J) Quantitative analysis of total cardiac collagen deposits in dysferlin-null, mdx, and DKO mice at 16 weeks of age (n = 5 for each group; P < 0.01 for DKO versus ing myocardial infarction or helping in recovery.  either dysferlin-null or mdx mice). (K) The serum troponin T was dramatically elevated In humans, dysferlinopathy could be expected to  exacerbate coexisting conditions such as hyperin the 10- (n = 5) and 18-week-old DKO mice (n = 5). tension or valvular volume overload states, each  of which imposes hemodynamic stress upon surmutation presented progressive muscle weakness and atrophy as  viving cardiomyocytes. The contribution of dysferlin deficiency  well as progressive choreic movements with no evidence of other  to those processes might not have been accounted for in previous  causes of chorea (38). Very recently, an immunofluorescence study  sporadic assessments of cardiac status in patients with coexisting  detected dysferlin expression in neurons of different cortical lay- cardiac disease states. Taken together, our results suggest that the dysferlin-mediated  ers of normal brain and dysferlin accumulation in the neurites  throughout the Alzheimer brain and colocalization with amyloid  membrane-repair mechanism in the heart becomes very important  β neuritic plaques (39). These studies suggest that, in addition  when coexisting factors lead to more extensive membrane damage  to playing a role in the membrane repair of skeletal and cardi- (e.g., physical exercise) or compromised membrane integrity (e.g.,  ac muscle, dysferlin is important for maintaining normal brain  dystrophin deficiency). Therefore, dysferlin-deficient patients may  function. Expanding the possible roles for this protein even fur- have to avoid excessive physical stress, which could severely accelerther, a French group has found that a patient with a 3370G→T  ate the manifestation of cardiac disease. mutation developed glomerular proteinuria (40). Although these  reports represent individual clinical cases and their significance  Methods is therefore difficult to ascertain, they do point to the possibility  Antibodies. The mouse monoclonal antibody Hamlet (Novocastra Labothat dysferlinopathy may be complicated by manifestations of the  ratories Ltd.), which was raised against dysferlin, was used for Western  disease in a variety of tissues. It is noteworthy that of the 3 cases  blotting and immunofluorescence analysis. Monoclonal antibody IIH6  linked to dysferlin, all share the 3370G→T mutation. However,  against α-dystroglycan, rabbit polyclonal antibody against β-dystroglyit remains unclear whether only this specific mutation can lead  can, and monoclonal antibody against caveolin-3 (BD Biosciences) were  to diseases in tissues other than skeletal muscle or other dysferlin  used for immunofluorescence and Western blot analysis. The anti-lam1810 The Journal of Clinical Investigation      Volume 117      Number 7      July 2007 research article Treadmill exercise. Animals were exercised using the Omnipacer Treadmill (model LC4/M-MGA/AT; AccuScan Instruments Inc.), which had  an adjustable belt speed (0–100 m/min), shock bars with adjustable  amperage, and an on-and-off shock switch for each lane. Animals were  exercised at 12 m/min for 15 minutes and for 24 m/min for 30 minutes.  If an animal became exhausted, the shock bar of its lane was turned  off and the animal was allowed to rest at the back of the treadmill for  a short period of time. WT C57BL/6 (n = 10) and  dysferlin-null mice  (which had been backcrossed with C57BL/6 mice for 6 generations;    n = 10) included approximately equal numbers of males and females.  All mice were injected with EBD (0.5 mg EBD/0.05 ml PBS) i.p. 8 hours  before the exercise. Animals were injected with 50 μl/10 g body weight.  All surviving animals were kept alive for 36–48 hours, and serial sections of cardiac muscle were studied for EBD uptake and histopathological signs of necrosis by routine H&E staining. Echocardiography. Echocardiography was performed on unsedated  54-week-old mice that had been randomly selected from C57BL/6 and  dysferlin-null (backcrossed with C57BL6 for 6 generations) cohorts,  respectively, by an investigator (R.M. Weiss) who was unaware of genotype. Two-dimensional images were acquired in short- and long-axis  planes using a 15-MHz linear array transducer coupled to a Sonos 5500  imager (Philips) at a rate of approximately 200 frames per second and  stored offline for later analysis. Short- and long-axis left ventricular  silhouettes were visually identified and electronically planimetered  by an investigator who was unaware of genotype in order to calculate  Figure 6 end-diastolic and end-systolic volumes using the biplane area-length  Large areas of acute necrosis and EBD uptake in the hearts of DKO mice following stress exercise. Large areas of acute necrosis were detected method previously validated in our laboratory (43). Where applicable,  in the ventricular walls of 9-week-old DKO mice following stress exercise myocardial infarct size was estimated by electronic tracing of the por(A–D). Scale bars: 2 mm (A); 500 μm (B–D). Hearts from DKO mice (G) tion of the left ventricular endocardial silhouette demonstrating akinehad large areas of EBD uptake whereas those of dysferlin-null mice (E) sis and expressed as a percentage of the total of the entire left ventricle.  showed only sporadic EBD uptake and those of mdx mice (F) only small After the initial echocardiography recording was made, the effect of  focused areas of EBD uptake. stress exercise on cardiac function was examined by placing the age-  and sex-matched WT and dysferlin-null mice on an uphill treadmill set  at a 5° vertical incline at a speed of 6 m/min for 3 minutes, after which  inin  α2-chain antibody was purchased from Axxora (Alexis Biochemi- the speed was increased by 3 m/min every 2 minutes until the mice were  cals). Peroxidase-conjugated secondary antibodies were obtained from  exhausted. The echocardiography was then immediately repeated. Roche Applied Science. Heart slice preparation and damage-repair assay. After each mouse (WT and  RT-PCR .  Total  RNA  was  extracted  from  the  skeletal  muscle  and  dysferlin-null) was anesthetized by i.p. injection with 0.1 cc ketamine, the  hearts  of  WT  C57BL/6  mice,  using  RNAzol  B  (Tel-Test  Inc.)  accord- chest was quickly opened and enough ice-cold cardioplegia solution (1–3 ml)    ing  to  the  manufacturer’s  specifications.  Total  RNA  (2  μ g)  was  ran- was injected into the right atrium to stop the heartbeat. The heart was  domly  reverse  transcribed  to  cDNA  using SuperScript  III  Reverse  then removed and a heart slice from the left ventricle was obtained using  Transcriptase (Invitrogen). PCR products were amplified using the fol- 2 fine blades. The heart slices were incubated in the cold PBS solution and  lowing primers: no. 1, 5′-CTCCCCAATGTGAAGAAGGA-3′ (forward),  were warmed up to 37°C in aerated PBS solution before the damage-repair  5 ′ -ACCTCCTGGAGTGGGATCTT-3 ′   (reverse),  product  size:  242  bp;  assay was initiated. The heart slices were placed in PBS containing 2.5 μM  no. 2, 5′-TCGAAAATCTCTGCTACTGGAGGAG-3′ (forward), 5′-CTC- FM1-43 (Invitrogen) with or without 1 mM Ca2+ in a custom-made glass  CACGCGCAGGATATCATCC-3′ (reverse), product size: 306 bp (with a  chamber. Surface cardiomyocytes from the intact side of the slices were  42-bp in-frame splicing exon) or 264 bp; no. 3, 5′-TCAACGCACCTCT- imaged with a 2-photon confocal laser-scanning microscope (LSM 510;  GCTAGAC-3′ (forward), 5′-GAACGGCTCAGCTTCATCTC-3′ (reverse),  Zeiss) coupled to a 10-W Argon/Ti:sapphire laser (Spectra-Physics Lasers  product size: 218 bp or 311 bp (with a novel 93-bp in-frame splicing exon).  Inc.). To induce membrane damage, a 300 μm × 5 μm rectangular area of  The bands were excised and DNA was extracted with the QIAquick Gel  the sarcolemma on the surface cardiomyocytes was irradiated at full power  Extraction Kit (QIAGEN) and sequenced. for 10 seconds starting at t = 20 seconds. Images were captured beginning  Generation of DKO mice . Mice were maintained at The University of  20 seconds before (t = 0) and continuing for 5 minutes after the irradiation  Iowa Animal Care Unit in accordance with animal usage guidelines.  at 10-second intervals. For every image taken, the fluorescence intensity  All animal studies were authorized by the Animal Care Use and Review  at the site of damage was measured using Zeiss LSM 510 imaging softCommittee of The University of Iowa. To generate DKO mice, male  ware (versions 3.2 and 4.0). Fibers that had no membrane resealing showed    dysferlin-null mice (15) were mated to female mdx mice, and F1 offspring  dye entry through the wound site over the entire course of the experiment  were intercrossed to produce the F2 generation. Identification of the  whereas the dye influx halted within 1–2 minutes for the fibers that had  mutant mice was performed by PCR genotyping of genomic DNA pre- resealed under the experimental conditions. pared from mouse tail snips (42). Littermates from the same colony were  Subcellular membrane fractionation of cardiac muscle.  Cardiac  muscle  used for experiments involving DKO mice. membrane fractionation was performed in a manner similar to that pre The Journal of Clinical Investigation      Volume 117      Number 7      July 2007  1811 research article viously described for preparing skeletal muscle membrane fractions (44).  Hearts from C57BL6 mice were removed and rinsed twice in cold PBS.  The hearts were then minced and homogenized on ice 3 times (10 seconds each) using a Polytron homogenizer (Kinematica Inc.) set between  levels 3 and 4 in a buffer containing 20mM HEPES, 250 mM sucrose,  1 mM EDTA, 5 mM benzamidine, 1  μM aprotinin A, 1  μM pepstatin,  1  μ M  leupeptin,  and  1  mM  phenylmethylsulfonyl  fluoride,  pH  7.4.    The homogenate was centrifuged at 2,000  g for 10 minutes. The pellet was discarded, and the supernatant was centrifuged at 9,000  g for  20 minutes. The resulting pellet (P1) was resuspended in PBS with the  standard cocktail of protease inhibitors listed above and then analyzed  for the presence of marker proteins by Western blotting. The supernatant was centrifuged at 180,000  g for 90 minutes. The resulting pellet  was resuspended in PBS with protease inhibitors, loaded on a 10%–30%  (wt/wt) continuous sucrose gradient (3–4 mg protein/5 ml gradient),  and centrifuged at 285,000 g for 55 minutes in a SW40 rotor (Beckman  Coulter). Gradients were separated into 13 fractions, starting from the  top of the tube. The pellet of the sucrose-gradient centrifugation (P2)  was resuspended in PBS and analyzed together with the gradient fractions. All centrifugations were performed at 4°C. H&E staining, Sirius red staining, and immunofluorescence analysis.  Histopathology studies were performed as described previously (8, 45). Several  H&E-stained sections (7  μm) throughout the entire ventricle and lower  part of the atrium were prepared to characterize cardiac muscle pathology. Sirius red staining was performed in a manner similar to that of H&E  staining. For quantitative analysis, images were split into 3 channels (red,  green, and blue) using ImageJ software (version 1.36; gov/ij/). The areas of the red and green regions were then calculated using  ImageJ, and relative collagen was expressed as the ratio of red to green  areas. Immunofluorescence staining was performed as described previously (8, 45). Anti-dysferlin antibody Hamlet was used at 1:40 dilution. Immunoblotting analysis. Fifty sections (7 μm) were prepared from the frozen hearts of different mouse models. The sections were then subjected to  SDS extraction. SDS-extracted proteins were loaded, and Western blotting  was performed as previously described (15).   1. Graham, R.M., and Owens, W.A. 1999. Pathogenesis of inherited forms of dilated cardiomyopathy.  N. Engl. J. Med. 341:1759–1762.   2. Towbin, J.A., and Bowles, N.E. 2006. Dilated cardiomyopathy: a tale of cytoskeletal proteins and  beyond. J. Cardiovasc. Electrophysiol. 17:919–926.   3. Muntoni, F., et al. 1993. Brief report: deletion of  the dystrophin muscle-promoter region associated  with X-linked dilated cardiomyopathy.  N. Engl. J. Med. 329:921–925.   4. Franz, W.M., et al. 2000. Association of nonsense  mutation of dystrophin gene with disruption of sarcoglycan complex in X-linked dilated cardiomyopathy.   Lancet. 355:1781–1785.   5. Piccolo, F., et al. 1995. Primary adhalinopathy:  a common cause of autosomal recessive mus cular dystrophy of variable severity.  Nat. Genet.  10:243–245.   6. Barresi, R., et al. 2000. Disruption of heart sarcoglycan complex and severe cardiomyopathy caused  by  beta  sarcoglycan  mutations.  J. Med. Genet.  37:102–107.   7. Ben Hamida, M., Ben Hamida, C., Zouari, M., Belal,  S., and Hentati, F. 1996. Limb-girdle muscular dystrophy 2C: clinical aspects.  Neuromuscul. Disord.  6:493–494.   8. Coral-Vazquez, R., et al. 1999. Disruption of the  sarcoglycan-sarcospan complex in vascular smooth  muscle: a novel mechanism for cardiomyopathy  and muscular dystrophy. Cell. 98:465–474.   9. Tsubata, S., et al. 2000. Mutations in the human  1812 Determination of cardiac troponin T.  Serum  samples  from  mice  were  obtained by tail bleeding according to The University of Iowa’s institutional guidelines. Cardiac-specific troponin T (cTnT) was measured by the  Chemical Laboratory at the University of Iowa Medical Center. All assays  were performed by technicians blinded to the source of the serum. The  normal range for troponin T levels in the serum was less than 0.03 ng/ml. Statistics. Data are presented as mean ± SEM. Where appropriate, the  significance of differences between multiple experimental groups was  assessed using 1-way ANOVA with Bonferroni’s post-tests, and the significance of differences between 2 experimental groups was assessed by  Student’s t test (paired or unpaired where appropriate). A value of P < 0.05  was accepted as significant. Acknowledgments We would like to thank Steve Moore for his critical comments and  all the members of K.P. Campbell’s laboratory for insightful comments and scientific contributions. We thank the University of  Iowa Roy J. and Lucille A. Carver College of Medicine, the University of Iowa Central Microscopy Research Facility, and the Medical  College of Georgia Imaging Core. This work was supported in part  by the Senator Paul D. Wellstone Muscular Dystrophy Cooperative Research Center grant 1 U54 NS053672. K.P. Campbell is an  investigator of the Howard Hughes Medical Institute. Received  for  publication  November  3,  2006,  and  accepted  in  revised form April 10, 2007. Address correspondence to: Kevin P. Campbell, Howard Hughes  Medical Institute, The University of Iowa, Roy J. and Lucille A. Carver  College of Medicine, 4283 CBRB, 285 Newton Road, Iowa City, Iowa  52242, USA. Phone: (319) 335-7867; Fax: (319) 335-6957; E-mail:  [email protected] Dimple Bansal’s present address is: Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts, USA. girdle muscular dystrophy. Nat. Genet. 20:31–36.   19. Illa, I., et al. 2001. Distal anterior compartment  myopathy:  a  dysferlin  mutation  causing  a  new  muscular  dystrophy  phenotype.  Ann. Neurol.  49:130–134.   20. Bittner, R.E., et al. 1999. Dysferlin deletion in SJL  mice (SJL-Dysf) defines a natural model for limb girdle muscular dystrophy 2B. Nat. Genet. 23:141–142.   21. Ho, M., et al. 2004. Disruption of muscle membrane  and  phenotype  divergence  in  two  novel  mouse models of dysferlin deficiency.  Hum. Mol. Genet. 13:1999–2010.   22. Kuru, S., et al. 2004. A patient with limb girdle muscular dystrophy type 2B (LGMD2B) manifesting  cardiomyopathy. Rinsho Shinkeigaku. 44:375–378.   23. Yilmazer, S., et al. 2006. Cardiac involvement in a  group of patients with dysferlinopathy [abstract].  Neuromuscul. Disord. 16:S110.   24. Anderson, L.V., et al. 1999. Dysferlin is a plasma  membrane protein and is expressed early in human  development. Hum. Mol. Genet. 8:855–861.   25. Salani, S., et al. 2004. Developmental and tissuespecific regulation of a novel dysferlin isoform.  Muscle Nerve. 30:366–374.   26. Soongswang, J., et al. 2002. Cardiac troponin T: its  role in the diagnosis of clinically suspected acute  myocarditis and chronic dilated cardiomyopathy  in children. Pediatr. Cardiol. 23:531–535.   27. Duclos, F. 1998. Progressive muscular dystrophy  in [alpha]-sarcoglycan-deficient mice.  J. Cell Biol.  142:1461–1471. δ-sarcoglycan gene in familial and sporadic dilated  cardiomyopathy. J. Clin. Invest. 106:655–662.   10. Cohn,  R.D.,  and  Campbell,  K.P.  2000.  Molecular  basis of muscular  dystrophies.  Muscle Nerve.  23:1456–1471.   11. Durbeej, M., and Campbell, K.P. 2002. Muscular  dystrophies involving the dystrophin-glycoprotein  complex: an overview of current mouse models.  Curr. Opin. Genet. Dev. 12:349–361.   12. Allen, D.G. 2001. Eccentric muscle damage: mechanisms of early reduction of force.  Acta. Physiol. Scand. 171:311–319.   13. McNeil, P.L., and Khakee, R. 1992. Disruptions of  muscle fiber plasma membranes. Role in exerciseinduced damage. Am. J. Pathol. 140:1097–1109.   14. Clarke, M.S., Caldwell, R.W., Chiao, H., Miyake, K.,  and McNeil, P.L. 1995. Contraction-induced cell  wounding and release of fibroblast growth factor  in heart. Circ. Res. 76:927–934.   15. Bansal, D., et al. 2003. Defective membrane repair  in dysferlin-deficient muscular dystrophy. Nature.  423:168–172.   16. Lennon, N.J., et al. 2003. Dysferlin interacts with  annexins A1 and A2 and mediates sarcolemmal  wound-healing. J. Biol. Chem. 278:50466–50473.   17. Bashir, R., et al. 1998. A gene related to Caenorhabditis elegans spermatogenesis factor fer-1 is mutated in limb-girdle muscular dystrophy type 2B. Nat. Genet. 20:37–42.   18. Liu, J., et al. 1998. Dysferlin, a novel skeletal muscle  gene, is mutated in Miyoshi myopathy and limb  The Journal of Clinical Investigation      Volume 117      Number 7      July 2007 research article   28. Ervasti, J.M., Ohlendieck, K., Kahl, S.D., Gaver,  M.G., and Campbell, K.P. 1990. Deficiency of a glycoprotein component of the dystrophin complex  in dystrophic muscle. Nature. 345:315–319.   29. Petrof,  B.J.,  Shrager,  J.B.,  Stedman,  H.H.,  Kelly,  A.M., and Sweeney, H.L. 1993. Dystrophin protects  the sarcolemma from stresses developed during  muscle contraction.  Proc. Natl. Acad. Sci. U. S. A.  90:3710–3714.   30. Clarke, M.S., Khakee, R., and McNeil, P.L. 1993.  Loss of cytoplasmic basic fibroblast growth factor  from physiologically wounded myofibers of normal  and dystrophic muscle. J. Cell Sci. 106:121–133.   31. Hoffman, E.P., Brown, R.H., Jr., and Kunkel, L.M.  1987. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 51:919–928.   32. Sicinski, P., et al. 1989. The molecular basis of muscular dystrophy in the mdx mouse: a point mutation.   Science. 244:1578–1580.   33. Cox, G.A., Phelps, S.F., Chapman, V.M., and Chamberlain,  J.S.  1993.  New  mdx  mutation  disrupts  expression of muscle and nonmuscle isoforms of  dystrophin. Nat. Genet. 4:87–93.   34. Fuster, V., Badimon, L., Badimon, J.J., and Chesebro, J.H. 1992. The pathogenesis of coronary artery  disease  and  the  acute  coronary  syndromes  (1).   N. Engl. J. Med. 326:242–250.   35. Davis,  D.B.,  Doherty,  K.R.,  Delmonte,  A.J.,  and  McNally, E.M. 2002. Calcium-sensitive phospholipid binding properties of normal and mutant  ferlin C2 domains. J. Biol. Chem. 277:22883–22888.   36. McNeil, A.K., Rescher, U., Gerke, V., and McNeil,  P.L. 2006. Requirement for annexin A1 in plasma  membrane repair. J. Biol. Chem. 281:35202–35207.   37. Chakrabarti, S., et al. 2003. Impaired membrane  resealing and autoimmune myositis in synaptotagmin VII-deficient mice. J. Cell Biol. 162:543–549.   38. Takahashi, T., et al. 2006. A case of dysferlinopathy presenting choreic movements.  Mov. Disord.  21:1513–1515.   39. Galvin, J.E., Palamand, D., Strider, J., Milone, M.,  and Pestronk, A. 2006. The muscle protein dysferlin accumulates in the Alzheimer brain. Acta. Neuropathol. 112:665–671.   40. Izzedine, H., et al. 2006. Loss of podocyte dysferlin expression is associated with minimal change  nephropathy. Am. J. Kidney Dis. 48:143–150.   41. Davis, D.B., Delmonte, A.J., Ly, C.T., and McNally,  E.M. 2000. Myoferlin, a candidate gene and potential modifier of muscular dystrophy.  Hum. Mol. Genet. 9:217–226.   42. Amalfitano, A., and Chamberlain, J.S. 1996. The  mdx-amplification-resistant  mutation  system  assay, a simple and rapid polymerase chain reaction-based detection of the mdx allele. Muscle Nerve.  19:1549–1553.   43. Hill, J.A., et al. 2000. Cardiac hypertrophy is not  a required compensatory response to short-term  pressure overload. Circulation. 101:2863–2869.   44. Zhou, M., et al. 1998. Insulin-dependent protein  trafficking in skeletal muscle cells.  Am. J. Physiol.  275:E187–E196.  45. Cohn, R.D., et al. 2001. Prevention of cardiomyopathy  in  mouse  models  lacking  the  smooth  muscle sarcoglycan-sarcospan complex.  J. Clin. Invest. 107:R1–R7. The Journal of Clinical Investigation      Volume 117      Number 7      July 2007  1813 ...
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