Cardioprotection of Ischemia/Reperfusion Injury by Cholesterol-Dependent MG53-Mediated Membrane Repair
Rationale: Unrepaired cardiomyocyte membrane injury causes irreplaceable cell loss, leading to myocardial fibrosis and eventually heart failure. However, the cellular and molecular mechanisms of cardiac membrane repair are largely unknown. MG53, a newly identified striated muscle-specific protein, is involved in skeletal muscle membrane repair. But the role of MG53 in the heart has not been determined.
Objective: We sought to investigate whether MG53 mediates membrane repair in cardiomyocytes and, if so, the cellular and molecular mechanism underlying MG53-mediated membrane repair in cardiomyocytes. Moreover, we determined possible cardioprotective effect of MG53-mediated membrane repair.
Methods and Results: We demonstrated that MG53 is crucial to the emergency membrane repair response in cardiomyocytes and protects the heart from stress-induced loss of cardiomyocytes. Disruption of the sarcolemmal membrane by mechanical, electric, chemical, or metabolic insults caused rapid and robust translocation of MG53 toward the injury sites. Ablation of MG53 prevented sarcolemmal resealing after infrared laser–induced membrane damage in intact heart, and exacerbated mitochondrial dysfunction and loss of cardiomyocytes during ischemia/reperfusion injury. Unexpectedly, the MG53-mediated cardiac membrane repair was mediated by a cholesterol-dependent mechanism: depletion of membrane cholesterol abolished, and its recovery restored injury-induced membrane translocation of MG53. The redox status of MG53 did not affect initiation of MG53 translocation, whereas MG53 oxidation conferred stability to the membrane repair patch.
Conclusions: Thus, cholesterol-dependent MG53-mediated membrane repair is a vital, heretofore unappreciated cardioprotective mechanism against a multitude of insults and may bear important therapeutic implications.
In eukaryotic cells, the plasma membrane partitions a ≈10 000-fold Ca2+ gradient and prevents loss of vital intracellular constituents, thus representing the last line of defense for cell integrity, homeostasis, and function. Physical, chemical or metabolic disruption of the plasma membrane leads to a repair-or-die emergency of the cell. Although the natural tendency to reseal the lipid biomembrane acts constitutively, recent studies indicate that plasma membrane disruption requires active emergency response mechanisms to mend the broken membrane.1 In the heart, plasma membrane repair is of particular importance because cardiomyocytes are terminally differentiated cells, displaying only very limited self-renewal capability.2 Cardiomyocytes undergo transient membrane injuries that occur as accidents under physiological conditions and can be exacerbated by various pathophysiological stresses.3 Progressive necrotic and apoptotic cell death causes onset of myocardial fibrosis and undermines cardiac contractile and electrophysiological performance, ultimately leading to heart failure.4,5
Ironically, little was known about cardiac membrane repair until recently when several molecular components in striated muscles were identified.6–10 In particular, 2 muscle-specific proteins, dysferlin7 and MG53,9 have been implicated in the repair of sarcolemmal membrane in skeletal muscles. Similar to the skeletal muscle, dysferlin-deficient mice display defective cardiomyocyte membrane repair ability that is linked to increased susceptibility to cardiomyopathy,6 whereas the molecular function of dysferlin in membrane repair has not been fully elucidated. For example, it is unknown whether the formation of a membrane repair patch is associated with translocation of dysferlin toward the injury site in cardiac muscle. Our previous studies have shown that MG53 is an essential component of membrane repair machinery in skeletal muscle, as MG53 ablation results in defective membrane repair and progressive skeletal myopathy.9 In addition, we have found that MG53 can interact with dysferlin and is required for dysferlin movement to the acute membrane injury sites.11 However, the role of MG53 in the heart has not been determined and cardiac membrane repair remains largely elusive.
Here, we investigate the cellular and molecular mechanism of MG53-mediated membrane repair in the heart and demonstrate its cardioprotective role against various insults. We show that genetic ablation of mg53 prevents membrane resealing and increases susceptibility to ischemia/reperfusion (I/R)-induced myocardial damage. Rapid and robust MG53 translocation toward the injury sites occurs in cardiomyocytes subjected to either local or global plasma membrane disruption, whereas physiological process of excitation-contraction coupling or membrane deformation is unable to trigger such MG53 translocation. Furthermore, we show that unfurled membrane cholesterol is indispensable in initiating MG53-mediated membrane repair, uncovering a novel signaling role of membrane cholesterol. As an important cardioprotective response to stress, the cholesterol-dependent MG53-mediated membrane repair may provide a valuable therapeutic target for the treatment of heart disease.
Animals were treated in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996), and protocols were approved by the Institutional Animal Care and Use Committee of Peking University accredited by AAALAC International. The mg53−/− mice were generated as described9 and wild-type (WT) littermate mice were used as controls. Single ventricular myocytes were enzymatically isolated from the hearts as described previously.12 Adenovirus containing mg53 or mg53(C242A) mutant gene was used to infect cardiomyocytes. Plasma membrane injury was elicited mechanically, electrically, or chemically in cardiomyocytes. The whole-heart membrane repair assay using two-photon excitation microscopy was as described by Han et al,6 with minor modifications, and the I/R protocol used was as described previously.13
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
MG53 Mediates Membrane Repair in Intact Heart
MG53 protein expression in mouse heart was detectable during embryonic development and was progressively increased during the first month after birth (Online Figure I, A). At the age of 12 to 14 weeks, abundant expression of MG53 was found in muscular walls of all 4 chambers (right and left atria and ventricles) (Online Figure I, B). To test whether MG53 participates in membrane repair in cardiac muscle, we used a membrane repair assay modified from Han et al6 (Figure 1) and a mouse model with genetic ablation of mg53.9 Langendorff-perfused beating hearts from mg53−/− and WT littermate mice were subjected to infrared laser irradiation applied to a band of 500 μm2 (910 nm, 650 mW, 57 seconds). The process of membrane resealing was visualized by local accumulation of FM1-43, a membrane-impermeable dye that becomes fluorescent when it partitions into a membrane. In WT heart, time-lapse two-photon imaging revealed that local FM1-43 fluorescence leveled off after an initial rise following laser irradiation, indicative of a halt in entry of FM1-43 after membrane resealing. In contrast, mg53−/− mouse hearts exhibited a continuous, high-rate elevation of FM1-43 fluorescence over much broader bands at the injury site (Figure 1), indicating an impairment of membrane resealing. These results establish an important role of MG53 in membrane repair in intact heart.
Deficiency of MG53 Exacerbates I/R-Induced Loss of Cardiomyocytes
We next sought to determine the possible cardioprotective function of MG53-mediated membrane repair in the heart. In this regard, mg53−/− mice at young age (12 to 14 weeks old) did not exhibit detectable changes in cardiac morphometrics and contractile functions when compared to WT littermate controls (Online Figure II). Histological analysis failed to reveal any significant myocardial pathology, either (Online Figure III). The absence of overt cardiac phenotypes in young mg53−/− mice raised under normal laboratory conditions is consistent with the finding in dysferlin-deficiency mice6 and supports the hypothesis that membrane repair serves as an emergency response against detrimental stimuli to the heart.
To assess potential MG53-mediated cardioprotection under stress conditions, we subjected the Langendorff-perfused mouse hearts to an I/R protocol (30 minutes/30 minutes) and assessed cardiac damage in the presence and absence of MG53-mediated membrane repair. Because loss of mitochondrial membrane potential (ΔΨm) is an early event of cell death,13,14 we used ΔΨm as a sensitive readout of cardiomyocyte damage and performed simultaneous confocal measurement of tetramethyl rhodamine methyl ester (TMRM) fluorescence (index for ΔΨm) and Evans blue dye (EBD) uptake (index for loss of membrane integrity) (Figure 2A). Before the I/R treatment, there was a trend of increased number of cardiomyocytes showing a loss of ΔΨm in mg53−/− compared to WT mouse hearts (10.15±0.95% in mg53−/− versus 5.54±2.00% in WT, P>0.05, n=6 hearts with 220∼250 image frames for each group), but the difference was not statistically significant (Figure 2B). Confocal microscopy revealed that, after the I/R injury, local multicellular areas showing complete loss of ΔΨm were interspersed with areas of intact ΔΨm, giving rise to a sharply delimited mosaic pattern (Figure 2A). Importantly, whereas the I/R injury caused a significant increase in the number of ΔΨm-lost cardiomyocytes in both mg53−/− and WT mouse hearts, the number of ΔΨm-lost cardiomyocytes in mg53−/− hearts was greater than that in WT controls by 140% (Figure 2B), indicating a cardioprotective role of MG53. Detailed analysis further revealed that a higher percentage of cardiomyocytes that had lost ΔΨm was EBD-positive in the absence of MG53 (69.62±5.94% in mg53−/− versus 44.23±8.18% in WT, P<0.05, n=6 hearts with 220∼250 image frames for each group) (Figure 2B), suggesting that MG53 retards the transition from reversible to irreversible cardiomyocyte damage.
Furthermore, we used conventional assessments to evaluate the cardioprotective effect of MG53 in response to I/R injury. As shown in Online Figure IV, MG53 ablation increased infarct size (42.32±3.06% in mg53−/− versus 31.73±2.11% in WT, assessed in Langendorff-perfused heart subjected to global I/R injury, P<0.05, n=6 hearts for each group), TUNEL-positive nuclei (15.64±1.60% in mg53−/− versus 9.14±1.17% in WT, P<0.01, n=6 hearts for each group) and also lactate dehydrogenase release by 2-fold. Importantly, plasma membrane translocation of endogenous MG53 did occur when the intact heart was subjected to I/R insult (Figure 2C), suggesting that MG53-mediated repair mechanism is activated by this stress.
MG53 Translocation During Acute Membrane Injury
As shown in Figure 2C, MG53 translocated and formed extensive patches on the sarcolemma in clusters of cardiomyocytes in WT heart after the I/R injury, supporting that MG53 plays a crucial role in mending broken membrane. To investigate the underlying mechanism, we used adenoviral expression of a MG53 fusion protein with an amino-terminal GFP (GFP-MG53) in cultured rat cardiomyocytes (Online Figure V). Under the present experimental conditions, GFP-MG53 was expressed at a level similar to that of endogenous MG53 (Online Figure V, A), and high-resolution confocal microscopy allowed for real-time visualization of intracellular GFP-MG53 trafficking. Figure 3A shows that focal electroporation of the sarcolemmal membrane caused rapid enrichment of GFP-MG53 at the wound site. Local GFP-MG53 fluorescence rose biexponentially with time constants of 12.0 and 166 s, respectively (Figure 3B and Online Video I). Mechanical injury by microelectrode penetration of the plasma membrane also triggered GFP-MG53 translocation to the injury site, and repetitive injuries at different locations resulted in multiple patches of enriched GFP-MG53 (Online Figure VI), indicating that MG53 translocation ensues wherever and whenever the plasma membrane is disrupted.
Global membrane permeabilization by a chemical detergent, saponin (50 μg/mL), induced robust GFP-MG53 translocation over the entire sarcolemmal membrane (Figure 3C and Online Video II). The gain of GFP-MG53 at the membrane mirrored a near-complete loss of GFP-MG53 in the cytoplasm, both displaying a time constant of ≈4.4 seconds (Figure 3D). This result indicates that most of the GFP-MG53 molecules are available and responsive when damage occurs at the sarcolemmal membrane of the cardiomyocytes. In GFP-expressing cells, however, complete loss of GFP following chemical permeabilization occurred without any appreciable staining of the sarcolemmal membrane (Figure 3C and Online Video III). Similar results were obtained with another membrane disruptive reagent, Triton X-100 (0.1% vol/vol) (Online Figure VII). Notably, immunofluorescence of native MG53 was concentrated at the damaged plasma membrane in a manner similar to that of GFP-MG53 (Figure 3E). Furthermore, in GFP-MG53-expressing cells, the intrinsic fluorescence from GFP-MG53 overlapped with the anti-MG53 immunofluorescence with a colocalization coefficient of 0.91±0.02 (n=9 cells). Similarly, immunofluorescence of endogenous MG53 was also found to concentrate at membrane injury sites wounded by electroporation (Online Figure VIII, A) and microelectrode penetration (Online Figure VIII, B). These results corroborate that GFP-MG53 and endogenous MG53 behave similarly during the process of membrane translocation.
Furthermore, consistent with Figure 2C, hypoxia-reoxygenation treatment on cardiomyocytes induced membrane translocation of GFP-MG53 (Online Figure IX), substantiating that MG53-mediated membrane repair protects heart from I/R induced injury.
MG53 Translocation Requires Membrane Disruption
What is the exact signal that is sensed by MG53 and promotes its plasma membrane translocation? To this end, the above experiments in Figure 3 were performed in a Ca2+-free external solution (with the benefit of preventing cell contracture after membrane damage), suggesting that Ca2+ entry is not a prerequisite for MG53 translocation in cardiomyocytes, as is the case in skeletal muscle.9 To further test whether intracellular Ca2+ release participates in regulating MG53 translocation, we used caffeine (20 mmol/L) to deplete the intracellular sarcoplasmic reticulum Ca2+ store before cell wounding. Figure 4A shows that GFP-MG53 translocation induced by saponin treatment remained intact even in the absence of intracellular Ca2+ release. Conversely, we also found no accumulation of GFP-MG53 at the sarcolemmal membrane despite physiological Ca2+ transients and cell contraction elicited by electric pacing at 1 or 5 Hz (Figure 4B). Taken together, we conclude that neither Ca2+ entry nor intracellular Ca2+ transients are necessary or sufficient for GFP-MG53 translocation. This finding reinforces the idea that MG53 translocation is an early event before Ca2+-dependent vesicle trafficking and membrane fusion steps in the membrane repair cascade.15–18
Further studies showed that membrane stretching or altered membrane curvature does not result in MG53 translocation to the cell membrane. Here we used a patch pipette to create the “Ω” shaped membrane deformation by negative pressure, as is the practice during the formation of a gigaseal for electrophysiological recording. No GFP-MG53 localization was evident in spite of the sharp membrane deformation and local stretch stress (Figure 4C). Only after the membrane was ruptured by suction did GFP-MG53 translocate to membrane at the patch pipette tip (Figure 4C). Thus, these data show that plasma membrane disruption is a prerequisite for membrane translocation of MG53.
Oxidation Confers Stability to the MG53 Repair Patch in Cardiomyocytes
Our previous study in skeletal muscle has shown that entry of extracellular oxidants is necessary for MG53 translocation, and the cysteine residue of 242 amino acid of MG53 serves a redox-sensor for MG53 oligomerization or crosslinking.9 In this study, we extended this finding by showing that redox regulation exerts differential effects at different phases of MG53 translocation in cardiomyocytes. Specifically, application of the membrane permeable oxidant H2O2 with different concentrations (0.1, 0.5, 1 mmol/L) failed to recruit GFP-MG53 to the plasma membrane (Online Figure X), even at a high dose (5 mmol/L) that caused cell damage and contracture (Figure 5A). Inclusion of the reducing reagent dithiothreitol (DTT) did not affect the initial GFP-MG53 translocation, either; nevertheless, the nascent GFP-MG53 matrix was quickly dissociated from the membrane in the presence of DTT (Figure 5B), suggesting that oxidation is required to stabilize the MG53 repair patch. To further investigate this phenomenon, we expressed GFP-MG53(C242A) mutant (the cysteine of 242 amino acid was mutated to alanine), which lacks the redox-sensing ability,9 to a MG53-null background of cardiomyocytes from mg53−/− mouse heart (to avoid possible interference of endogenous MG53). In response to saponin-induced membrane disruption, GFP-MG53(C242A) robustly translocated to the membrane but was unable to form a stable repair patch (Figure 5C), as was the case with DTT treatment. Hence our data suggest that initial movement of MG53 on membrane disruption is redox-independent, whereas oxidative regulation of MG53 at cysteine of 242 amino acid plays a critical role in stabilizing the MG53 repair patch.
Membrane Cholesterol Is Necessary to Signal MG53 Translocation
Given that MG53 discriminates between intact and injured membrane, we hypothesized that MG53 interacts with the hydrophobic core of the lipid bilayer membrane, which is exposed only in the event of membrane disruption. In this regard, cholesterol came into the picture because it is embedded in the hydrophobic core and accounts for ≈40% of the lipid molecules of the plasma membrane in animal cells.19 Besides, the majority (≈90%) of it is enriched in plasma versus organelle membranes.19,20 We therefore tested the hypothesis that membrane cholesterol, when unfurled, signals MG53 to translocate to the membrane.
Staining cholesterol with filipin uncovered a dense colocalization of GFP-MG53 and cholesterol to the membrane site injured by electrode penetration, but an absence of GFP-MG53 in intact cholesterol-rich plasma membrane (Figure 6A). Strikingly, when membrane cholesterol was depleted with methyl-β-cyclodextrin (M-β-CD), the GFP-MG53 translocation after focal electroporation was completely abolished (Figure 6B and 6C). The effects of Triton X-100 as a membrane-disrupting reagent were similar to that of electroporation (Figure 6B). Restoration of cholesterol by incubation the cells with M-β-CD complexed with cholesterol largely revived the GFP-MG53 translocation to damaged membranes (Figure 6B and 6C).
Because cholesterol is particularly enriched in the membrane microdomains called lipid rafts and caveolae,21,22 we investigated whether such substructures are required for cholesterol regulation of MG53 translocation. By filipin disruption of the lipid rafts and caveolae without depleting membrane cholesterol, we found that GFP-MG53 translocation to membrane damage sites remained unaffected (Figure 6B and 6C), suggesting that MG53 recognizes unfurled membrane cholesterol regardless of the integrity of lipid rafts and caveolae. Thus, membrane cholesterol is obligatory for MG53 translocation, and exposure of membrane cholesterol likely acts as an initial step of the MG53–mediated membrane repair.
Cardioprotection by MG53-Mediated Membrane Repair
As a muscular pump undergoing constant contractile activity, the heart is an organ under intense mechanical stress with high metabolic demands. As a result, cardiomyocytes are under constant threat of mechanical injury, oxidative stress, and metabolic insult. Because cardiomyocytes cannot be replaced in large numbers, lost cardiomyocytes are replaced by fibroblasts, resulting in myocardial fibrosis in response to a number of etiologies, including myocardial infarction caused by coronary heart disease. Thus, the heart must develop intrinsic defensive mechanism to survive such injuries by preventing cardiomyocytes death. In this regard, the present study has identified MG53 as a potent cardiac protector: MG53 ablation exacerbated the I/R-induced myocardial damage, as manifested by markedly worsened mitochondrial dysfunction and loss of cardiomyocytes.
Several lines of evidence have demonstrated that cardioprotection by MG53 is mediated, at least in part, by its membrane repair function. First, we have shown that local or global membrane disruption evokes MG53 to translocate exclusively to the injured membrane in cardiomyocytes. Second, a membrane repair assay using FM1-43 fluorescence revealed that MG53 deficiency impaired membrane resealing after two-photon irradiation damage in intact heart. That MG53 is inert to physiological Ca2+ transients and cardiac contraction is also consistent with the idea that MG53 is normally a bystander but plays a key role in the emergency response to rescue the cell from disastrous membrane disruption.
One other muscle specific protein thought to contribute to this membrane repair response in striated muscle is dysferlin.6–8 Dysferlin has been shown to be essential for membrane repair in skeletal muscle7; however it requires the function of MG53 to translocate to sites of membrane injury.11 Thus, MG53 can nucleate the assembly of repair machinery at sites of membrane damage, whereas dysferlin likely operates at a downstream stage of the membrane resealing process. Our findings that MG53 is important in protecting the heart from I/R injury is in contrast to previous report that dysferlin is unable to protect the heart from cardiomyopathy induced by coronary ligation in mice.6 The contrasting phenotypes of MG53 and dysferlin deficiencies suggest that MG53 and dysferlin play different cardioprotective roles, or that the dysferlin-mediated Ca2+-dependent membrane repair mechanism is partly redundant of MG53-mediated repair mechanism in the heart. Future investigation is required for better understanding the relationship and relative contributions of these distinctive coexisting repair mechanisms.
It is also noteworthy that MG53 ablation did not induce any abnormality for heart structure and function in young adult mice raised under normal laboratory conditions. This finding is in agreement with cardiac phenotypes of dysferlin deficiency: myocardial fibrosis does not begin in young adult mice (less than 32 weeks) and is mild in senescent animals (≥1 year old).6 These previous and present findings underscore the idea that membrane repair mechanisms may not be essential in young, healthy cardiomyocytes. In fact, membrane deformation that does not rupture the membrane, intracellular Ca2+ release or contraction induced by electric pacing, and even membrane deformation and stretching were not sufficient to trigger the MG53 translocation process. These results are in general agreement with the present thought in this field that membrane repair process consists of molecular components that act as an “emergency repair” response system.1 As such, MG53 safeguards the heart from both cumulative loss of cardiomyocytes under normal wear-and-tear conditions and from massive acute cardiomyocyte death in the event of myocardial I/R.
Role of Membrane Cholesterol in MG53-Mediated Membrane Repair
In searching for the exact signal that triggers MG53 translocation, we have demonstrated, for the first time, that membrane cholesterol is an indispensable molecular player for the initiation of MG53 translocation in cardiac membrane repair. As highly hydrophobic, compactly ring-rigid structure, cholesterol is an important component of the plasma membrane. It is embedded in the hydrophobic core of the lipid bilayer, and thus acts to rigidify the membrane, reduce the passive permeability, and increase the mechanical durability.23 Recent investigation of lipid rafts has unraveled an important role of cholesterol in assembling these membrane microdomains critical for the regulation of signal transduction and membrane trafficking.21,22 Here we have identified yet another fundamental role of membrane cholesterol in signaling membrane repair. Depletion of membrane cholesterol abolished, and its recovery restored MG53 translocation on membrane electroporation or chemical permeabilization. Remarkably, cholesterol regulation of MG53 translocation remained intact even after filipin disruption of membrane lipid rafts or caveolae, suggesting that cholesterol-mediated hydrophobic interaction between MG53 and the disrupted membrane is not confined to specialized membrane microdomains. Our previous study in skeletal muscle showed that MG53 mediates membrane repair through direct interaction with membrane phosphatidylserine,9 here we further found that cholesterol, as another membrane lipid, is obligatory for MG53-mediated membrane repair. Because MG53 can interact with phosphatidylserine directly but not cholesterol in the lipid profiling assay,9 cholesterol-dependent secondary membrane structure or other yet-to-be identified partner may contribute to this role of cholesterol.
In determining whether the cholesterol-dependent MG53 translocation is sensitive to redox regulation as promoted by studies in skeletal muscle,9 we demonstrated 2 steps for formation of the MG53 repair patch in cardiomyocytes: trigger of MG53 translocation and stabilization of the translocated MG53. The initial MG53 translocation to membrane injury sites is redox-independent, but MG53 oxidation at cysteine of 242 amino acid appears to be necessary for stabilizing the nascent MG53 repair patch. In skeletal muscle, the initial MG53 translocation phase appears to be very brief or the MG53 repair patch is very unstable, such that MG53 translocation was seen as only a small blip in the time course plot under reducing conditions (see figure 4f and 4g in the article by Cai et al9). Also, the Ca2+ independence of MG53 translocation in cardiomyocytes is consistent to that in skeletal muscle which showed that extracellular Ca2+ entry is not essential for MG53 translocation to membrane injury sites (see figure 6e and 6f in the article by Cai et al9).
Intuitively, this cholesterol dependence of MG53 translocation serving to initiate cardiac membrane repair would be robust and fool-proof, because membrane cholesterol is exposed if and only if the membrane is disrupted. Given the abundance of membrane cholesterol, it confers one additional advantage of efficiency to membrane repair. Both robustness and effectiveness are important features in a situation where speed and fidelity of the response is a matter of survival of the cell.
In summary, we have shown that MG53 membrane translocation constitutes the initial step of repairing disrupted cardiomyocyte plasma membrane in the heart. Mechanistically, membrane cholesterol exposed in the disrupted membrane signals MG53 translocation in a redox-independent manner, whereas MG53 oxidation confers stability to the repair patch. As a result, cholesterol-dependent MG53-mediated membrane repair protects heart from loss of mitochondrial function and subsequent irreplaceable loss of cardiomyocytes under stress conditions such as myocardial I/R injury. These findings uncover an important heretofore unappreciated cardioprotective mechanism that involves cardiac membrane repair, and might help to develop therapeutic strategies to ameliorate cardiomyopathy and to retard, or potentially reverse, the progression of heart failure.
We thank I. C. Bruce, A. W. Cheng, and C. L. Wei for critical discussion and C. M. Cao, Y. Zhang, H. Q. Fang, H. L. Zhang, and N. Hou for technical assistance.
Sources of Funding
This work was supported by the Major State Basic Research Development Program of China (2007CB512100) and the National Natural Science Foundation of China (30630021, 30628009, and 30900264).
Pasumarthi KB, Field LJ. Cardiomyocyte cell cycle regulation. Circ Res. 2002; 90: 1044–1054.
Clarke 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: 927–934.
Kostin S, Pool L, Elsasser A, Hein S, Drexler HC, Arnon E, Hayakawa Y, Zimmermann R, Bauer E, Klovekorn WP, Schaper J. Myocytes die by multiple mechanisms in failing human hearts. Circ Res. 2003; 92: 715–724.
Cai C, Weisleder N, Ko JK, Komazaki S, Sunada Y, Nishi M, Takeshima H, Ma J. Membrane repair defects in muscular dystrophy are linked to altered interaction between MG53, caveolin-3, and dysferlin. J Biol Chem. 2009; 284: 15894–15902.
Cheng H, Lederer WJ, Cannell MB. Calcium sparks: elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993; 262: 740–744.
Matsumoto-Ida M, Akao M, Takeda T, Kato M, Kita T. Real-time 2-photon imaging of mitochondrial function in perfused rat hearts subjected to ischemia/reperfusion. Circulation. 2006; 114: 1497–1503.
Crompton M. The mitochondrial permeability transition pore and its role in cell death. Biochem J. 1999; 341 (Pt 2): 233–249.
Steinhardt RA, Bi G, Alderton JM. Cell membrane resealing by a vesicular mechanism similar to neurotransmitter release. Science. 1994; 263: 390–393.
Bi GQ, Alderton JM, Steinhardt RA. Calcium-regulated exocytosis is required for cell membrane resealing. J Cell Biol. 1995; 131: 1747–1758.
Miyake K, McNeil PL. Vesicle accumulation and exocytosis at sites of plasma membrane disruption. J Cell Biol. 1995; 131: 1737–1745.
McNeil PL, Steinhardt RA. Loss, restoration, and maintenance of plasma membrane integrity. J Cell Biol. 1997; 137: 1–4.
Simons K, Ikonen E. How cells handle cholesterol. Science. 2000; 290: 1721–1726.
Novelty and Significance
What Is Known?
Plasma membrane repair is an active emergency response to mend broken membrane.
MG53 is an essential component of membrane repair machinery in skeletal muscle.
Deficiency of dysferlin-mediated membrane repair leads to cardiomyopathy.
What New Information Does This Article Contribute?
MG53 plays crucial roles in membrane repair response of heart.
MG53-mediated membrane repair protects the heart from ischemia/reperfusion injury.
Cholesterol is necessary for signaling MG53-mediated membrane repair response.
Cardiomyocytes undergo membrane injury, which happens under physiological conditions and this injury could be exacerbated by pathophysiological stress. Unrepaired cardiomyocyte membrane injury causes irreplaceable cell loss, leading to myocardial fibrosis and eventually heart failure. However, the cellular and molecular mechanisms of cardiac membrane repair are largely unclear. In this study, we demonstrate that MG53, a newly identified striated muscle-specific protein, constitutes an essential component of the membrane repair machinery in heart. MG53-mediated membrane repair serves as a powerful cardioprotection mechanism in preventing ischemia-reperfusion injury. Furthermore, we find that the redox status of MG53 does not affect initiation of MG53 translocation, whereas MG53 oxidation confers stability to the membrane repair patch. Unexpectedly, we reveal a crucial role of membrane cholesterol in signaling MG53 to mend the broken membrane. We uncover a new role of plasma cholesterol and, for the first time, link cholesterol, MG53, and membrane repair with endogenous mechanisms defending against the life-threatening cardiac accident. As an important cardioprotective response to stress, the cholesterol-dependent MG53-mediated membrane repair may provide a valuable therapeutic target for the treatment of heart disease.
↵*These authors contributed equally to this work.
Original received December 30, 2009; revision received April 23, 2010; accepted April 30, 2010. In March 2010, the average time from submission to first decision for all original research papers submitted to Circulation Research was 13.3 days.