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CARD IOMYOPATHY

Severe dystrophic cardiomyopathy caused by theenteroviral protease 2A–mediated C-terminaldystrophin cleavage fragmentMatthew S. Barnabei, Frances V. Sjaastad, DeWayne Townsend,Fikru B. Bedada, Joseph M. Metzger*

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Enterovirus infection can cause severe cardiomyopathy in humans. The virus-encoded 2A protease is known tocleave the cytoskeletal protein dystrophin. It is unclear, however, whether cardiomyopathy results from the lossof dystrophin or is due to the emergence of a dominant-negative dystrophin cleavage product. We show forthe first time that the 2A protease–mediated carboxyl-terminal dystrophin cleavage fragment (CtermDys) issufficient to cause marked dystrophic cardiomyopathy. The sarcolemma-localized CtermDys fragment causedmyocardial fibrosis, heightened susceptibility to myocardial ischemic injury, and increased mortality duringcardiac stress testing in vivo. CtermDys cardiomyopathy was more severe than in hearts completely lackingdystrophin. In vivo titration of CtermDys peptide content revealed an inverse relationship between the decayof membrane-bound CtermDys and the restoration of full-length dystrophin at the sarcolemma, in support of aphysiologically relevant loss of dystrophin function in this model. CtermDys gene titration and dystrophin re-placement studies further established a target threshold of 50% membrane-bound intact dystrophin necessaryto prevent mice from CtermDys cardiomyopathy. Conversely, the NtermDys fragment did not compete withdystrophin and had no pathological effect. Thus, CtermDys must be localized to the sarcolemma, with intactdystrophin

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binding domains. Here, it is notable that the two products of dystro-phin cleavage by EV-encoded 2Apro are a 2426–amino acidN-terminalfragment (NtermDys) containing dystrophin’s actin binding domainsand a 1251–amino acid C-terminal fragment (CtermDys) containingdystrophin’s b-dystroglycan binding domain (Fig. 1A) (10).

Because the CtermDys cleavage peptide is structurally similar, butnot identical to, the previously described dominant-negative isoformsof dystrophin in skeletal muscle, we tested here the hypothesis that thepresence of this fragment is sufficient to cause dystrophic cardio-myopathy in the heart. Additionally, because EV infection has been as-sociated with increased risk of death after acute myocardial infarction(3), we hypothesized that the CtermDys fragment resulting from dys-trophin cleavage by 2Apro increases susceptibility to ischemic injury andcardiac stress. Insight into dystrophin cleavage fragments as potentialpoison peptides would help instruct future clinical interventions target-ing their decay.

Here, we report that the 2Apro cleavage product CtermDys is stableat the sarcolemma and nucleates key DGC proteins. In otherwisenormal mice, CtermDys—but not NtermDys—caused cardiac fibrosis,increased pump dysfunction during ischemia/reperfusion, and in-creased mortality during cardiac stress testing in vivo. The magnitudeof CtermDys-dependent cardiomyopathy was more severe than inmice with complete dystrophin deficiency. Forced overexpressionof full-length dystrophin dislodged CtermDys from the membraneand prevented cardiomyopathy. In comparison, the NtermDys frag-ment had no effect on dystrophin content and did not cause cardio-myopathy. These findings support a mechanism whereby CtermDysis retained at the membrane, severing intact dystrophin’s connectionto actin and preventing both intact dystrophin and compensatoryutrophin from binding to the sarcolemma. This two-hit model can ex-plain the more severe cardiomyopathy observed for CtermDys com-pared with the cardiomyopathy of complete dystrophin deficiency.On the basis of these results, membrane-bound CtermDys could serve asa new target for therapeutic remediation of Coxsackievirus-mediatedcardiomyopathy.

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RESULTS

Dystrophin peptide fragment expression in the heartIn the first series of experiments, transgenic mice were generated ex-pressing either the N-terminal or the C-terminal product of primarydystrophin cleavage site by 2Apro specifically in the heart (Fig. 1). TheN-terminal transgene-encoded protein (NtermDys) has a predictedmolecular weight of 283 kD and consists of an N-terminal FLAG epi-tope tag and the first 2426 amino acids of dystrophin. This includesthe N-terminal domain of dystrophin and the N-terminal portion ofthe central rod domain (including spectrin-like repeats 1 to 19 andhinges 1, 2, and part of hinge 3) (Fig. 1A). Thus, this fragment retainsboth the N-terminal and rod domain actin binding regions of dystrophin(19). Previous reports have shown that inclusion of an N-terminal FLAGepitope tag does not impair dystrophin’s actin binding function (20).

Western blot of total protein extracts from several lines of trans-genic mice was performed to assess transgene expression. The NtermDystransgene was detected using an anti-FLAG antibody and an anti-dystrophin antibody (recognizing amino acids 1181 to 1388 of dystro-phin, present in both dystrophin and the NtermDys transgene). Fourtransgenic lines (368, 367, 10, and 13) exhibited a range of transgene

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expression (fig. S1A). Line 368 was chosen as the primary transgenicline for further study and will hereafter be referred to as NtermDys un-less otherwise stated.

The C-terminal transgene (CtermDys) has a predicted molecularweight of 145 kD and consists of an N-terminal MYC tag and the last1251 amino acids of dystrophin. This includes dystrophin’s cysteine-rich domain, C-terminal domain, and C-terminal portion of the roddomain (containing part of hinge 3 and all of hinge 4, and spectrin-like repeats 20 to 24) (Fig. 1A). Thus, this transgene retains dystrophin’sdystroglycan binding andC-terminal scaffolding regions. For this trans-gene, two lines of transgenic mice were obtained that bred successfully.Western blot of total protein extracts was carried out to assess transgeneexpression. The CtermDys protein was detected using an anti-MYCantibody and an anti-dystrophin antibody (recognizing amino acids3661 to 3677 of dystrophin, present in both dystrophin and theCtermDystransgene). Epitope tagging of truncated dystrophins causedno apparenteffects (18). No significant difference was detected in expression of theCtermDys transgene between transgenic lines 1 and 4 (fig. S1B). Line 4was chosen for further study due to ease of breeding.

Expression of dystrophin and DGC proteins intransgenic miceDystrophin and DGC proteins are important to investigate because oftheir roles in membrane stability in both acquired and inherited formsof cardiomyopathy (21, 22). In total heart muscle protein extracts,expression of endogenous membrane–bound dystrophin was nor-mal in NtermDys transgenic mice but reduced in CtermDys trans-genic mice (Fig. 1B and fig. S1). To determine the effects of CtermDyson DGC proteins at the membrane, Western blots were performed onpotassium chloride (KCl)–washedmicrosomes extracted from themyo-cardium.Expression levels of dystrophin,a-dystroglycan,a-sarcoglycan,b-dystroglycan, utrophin, neuronal nitric oxide synthase (nNOS), syn-trophin, caveolin-3, and g-sarcoglycan in NtermDys transgenic micewere similar in nontransgenic (NTg) mice, but a-dystrobrevin-2 wassignificantly reduced (Fig. 1, B to H, and fig. S2).

Similar to findings in total protein extracts, dystrophin expression atthe membrane was significantly reduced in CtermDys transgenic miceto about 30% of normal levels (Fig. 1B). Expression of a-dystroglycan,a-sarcoglycan, b-dystroglycan, a-dystrobrevin-2, and g-sarcoglycanwas significantly increased in CtermDys transgenic mice comparedwith that in NTg animals (Fig. 1, D to G, and fig. S2A), whereas expres-sion of syntrophin and caveolin was slightly, but significantly, reduced(fig. S2, B and C). Utrophin and nNOS were unchanged in CtermDystransgenic hearts compared to those inNTg controls (Fig. 1Hand fig. S2D).

The subcellular localization of the NtermDys transgene was as-sessed by immunofluorescence on heart sections of NtermDys transgenicmice. NtermDys transgenic mice were crossed onto the dystrophin-deficient mdx genetic background [NtermDys(mdx)]. The NtermDystransgene localized to the sarcolemma, similar to dystrophin, but wasalso foundmore diffusely throughout cardiacmyocytes (fig. S3). Expressionof the DGC protein b-dystroglycan was unchanged in NtermDys(mdx)hearts (fig. S3A). SomeNtermDyswas detected in the nucleus of cardiacmyocytes, but not in the nuclei of other cells within the heart, such asfibroblasts [dystrophin (mid-rod) in fig. S3]. Subcellular localization ofthe CtermDys transgene was determined by immunofluorescence onheart sections from CtermDys transgenic mice. The CtermDys trans-gene localized at the sarcolemma in transgenic animals (fig. S4), similarto NtermDys.

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Heart structure and membrane integrity inCtermDys miceMdx mice show progressive histopathology withinthe myocardium as seen by inflammation and fibro-sis, although overallmdx heart size is not significant-ly altered (8, 23, 24). In comparison, CtermDys micehad enlarged hearts and significantmyocardial fibrosis,with evidence of inflammation (Fig. 2, A and B). Fibro-sis was significantly greater in CtermDys than in mdxhearts, suggesting that CtermDys transgenic animalshave more severe pathology than both NTg littermatesand mdx mice. Hearts from NtermDys adult miceshowednodifference in fibrosis compared to those fromNTg animals (Fig. 2B). Evans blue dye (EBD) uptakearea was larger than the fibrotic area in both mdxand CtermDys mice (Fig. 2C). Although increasedmembrane permeability is likely the initiating insultleading to fibrosis, there was not a 1:1 correlation be-tween dye-positive area and fibrotic area.

Previous studies have shown that dystrophin de-ficiency predisposes the sarcolemma to mechanicaldisruption during stress with adrenergic agonists, in-cluding isoproterenol (7, 25). To determine whetherexpression of the NtermDys or CtermDys transgenescausesmechanical instability of the cardiac sarcolemma,EBD uptake was assessed during isoproterenol stressin vivo. NTg littermates showed minimal dye uptakein cardiac myocytes during this stress, similar to

Fig. 1. Generation of transgenic mice and expressionof dystrophin and DGC proteins. (A) (Top) Schematic

of dystrophin structure. Hinge domains are indicatedby blue boxes. Spectrin-like repeats are indicated byred boxes, with yellow boxes constituting the rod domain actin binding region of dystrophin. Arrow at hinge3 indicates 2Apro cleavage site. (Bottom) Schematics othe NtermDys and CtermDys transgenes with N-terminaepitope tags. NtermDys contains an N-terminal FLAG epitope tag, the N-terminal domain of dystrophin, and theN-terminal portion of the central rod domain (includingspectrin-like repeats 1 to 19, and hinges 1, 2, and part ohinge 3). CtermDys contains an N-terminal MYC epitopetag, dystrophin’s cysteine (Cys)–rich domain, C-terminadomain, and C-terminal portion of the rod domain (containing part of hinge 3, hinge 4, and spectrin-like repeats20 to 24). (B) Representative Western blot for dystrophinexpression in membrane proteins of NTg, CtermDys, andNtermDys transgenic mice. Quantification of dystrophin expression shown as expression relative to loading normalizedto NTg. (C) Immunofluorescence detection of CtermDys in aheart section of CtermDys mice. Scale bar, 100 mm. InsetMerged laminin (green) and CtermDys (red), indicating thebasal lamina. (D to H) Quantification of b-dystroglycan (D)g-sarcoglycan (E), a-sarcoglycan (F), a-dystroglycan (G)and utrophin (H) expression in membranes isolated fromNTg and transgenic mice. Quantification of protein expression shown as expression relative to loading normalized to NTg. In (B) and (D) to (H), data are means ± SEM(n = 4 to 7 per group); *P < 0.05 versus NTg, analysis of variance (ANOVA) and Dunnett’s multiple comparison test.

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NtermDys transgenic lines 368, 367, 10,and 13 (Fig. 2 and fig. S5). In contrast,CtermDys transgenicmice showed signif-icantly greater dye uptake compared withNTg mice and was similar to dystrophin-deficientmdxmice (Fig. 2C).

Because cleavage of dystrophin by2Apro results in both N- and C-terminalcleavage products, CtermDys (line 4) micewere crossed with NtermDys (line 368)mice to create double transgenic (DTg)mice. DTg mice showed significantlygreater EBD uptake during isoproterenolstress than did NTg mice, but similar tomdx and CtermDys transgenic mice (Fig.2B). Collectively, these results indicatethat expression of CtermDys, but notNtermDys, is sufficient to cause sarco-lemmal instability.

Increased susceptibility toischemic injury and stress-inducedmortality in CtermDystransgenic miceA previous study showed that EV infec-tion may predispose patients to death af-ter acute myocardial infarction (3). Themechanism by which EV infection in-creases susceptibility to ischemic injuryis not known, but given that CtermDysmice show dystrophic cardiomyopathy,we hypothesized that the presence ofthe CtermDys transgene would increasesusceptibility to ischemic injury. To testthis hypothesis, hearts of CtermDys andNTg littermate controls were isolated, per-fused, and subjected to 20min of ischemiaand 60 min of reperfusion. Before ische-mia, NTg and CtermDys transgenic heartshad similar systolic function as measuredby left ventricular developed pressure(LVDP) (Fig. 3A). However, recovery ofsystolic function during reperfusion wassignificantly impaired inCtermDys heartscompared to that in NTg hearts (Fig. 3, Aand B). Additionally, recovery of diastolicfunction during reperfusion was signif-icantly impaired in CtermDys hearts, asshown by elevated left ventricular end-diastolic pressure (LVEDP), compared tothat in NTg hearts (Fig. 3C).

To further examine the cardiomyo-pathy of CtermDys transgenicmice in anin vivo setting,micewere subjected to car-diac stress by isoproterenol administrationfor five consecutive days. Most NTg micesurvived this cardiac challenge (92%),whereas only 40% of mdx survived (Fig.

Fig. 2. Heart morphology, fibrosis, and membrane instability in hearts of CtermDys mice. (A) Whole-heart images (scale bar, 2 mm) and transverse sections [hematoxylin and eosin (H&E)], and heart-to-tibia

length ratio. Data (expressed as ratio of heart weight to tibia length) are means ± SEM (n = 4). P value wasdetermined by t test. The representative H&E-stained heart transverse section indicates inflammation infibrotic area [scale bars, 1 and 0.5 mm (inset)]. (B) Sirius Red–Fast Green staining of representative NTg,NtermDys, CtermDys, and mdx mouse hearts (age 6 months). Scale bar, 200 mm. Fibrotic area was quanti-fied from the stained images. Data are means ± SEM (n = 8 to 15). P values were determined by ANOVA. (C)Representative mosaic images of EBD uptake (18 hours after dye injection) in hearts of NTg, mdx, NtermDys,CtermDys, NtermDys(mdx), CtermDys(mdx), and DTg mice. Scale bars, 1 mm. The dye uptake percentage outof whole-heart area was quantified from the images. Data are means ± SEM (n = 7 to 11 per group). *P <0.05; **P < 0.01; ***P < 0.001 versus NTg, ANOVA.

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3D). All CtermDysmice (n = 10) succumbed within the first 3 days ofthis stress test (Fig. 3D).

Ectopic full-length dystrophin rescuesCtermDys-mediated cardiomyopathyCtermDys mice showed increased mortality during adrenergic stressand increased cardiac fibrosis compared withmdxmice (Figs. 2 and 3),suggesting novel mechanisms for the cardiomyopathy caused by theCtermDys cleavage fragment. To determine whether these pheno-typic differences were founded in aberrant effects on DGC function,or some other direct or nonspecific effect of transgenesis, CtermDystransgenic mice were crossed with two lines of mice transgenicallyexpressing a human intact dystrophin (hDys) transgene. Relativeto native dystrophin, the hDys 600 transgene is expressed at 50% ofnormal dystrophin and the hDys 649 transgene is expressed at lev-els 10-fold greater than normal dystrophin (Fig. 4A). CtermDys-hDys649(mdx) DTg mice demonstrated a significant reduction inCtermDys protein expression (Fig. 4B) with reduced EBD uptakeand complete survival during adrenergic stress similar to NTg mice(Fig. 4, C to E). CtermDys-hDys600(mdx) mice demonstrated no de-crease in CtermDys expression and no improvement in EBD uptake(Fig. 4, B and C). However, there was a significant improvement insurvival during adrenergic stress testing (Fig. 4D), suggesting thattitrating full-length dystrophin levels can confer myocardial protec-tion in CtermDys mice during stress.

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In vivo titration of theCtermDys peptideTo further validate physiological rele-vance, and to establish biological dose-response for the cardiotoxic effects ofCtermDys, floxed CtermDys mice werecrossed with mice expressing a cardiac-specific tamoxifen-inducible Cre recom-binase transgene [Mer-Cre-Mer (MCM)](Fig. 5A) (12). Tamoxifen was adminis-tered to DTg CtermDys ×MCMmice, re-sulting in efficient excision of theCtermDystransgene and a time-dependent reduc-tion in cardiac CtermDys protein (Fig. 5B).Concurrent with decay of the CtermDyspeptide, there was a reciprocal increase infull-length dystrophin at the membrane(Fig. 5B). This is direct evidence of a stoi-chiometric balance between CtermDysand full-length dystrophin at the mem-brane. By assuming fast tamoxifen-basedCtermDys gene excision and subsequentfast CtermDys mRNA decay (estimated24 hours), the half-life of the CtermDyspeptide was calculated to be ~3 to 4 daysin the mouse heart in vivo. If CtermDyslocus excision and mRNA decay wereslower, then the CtermDys peptide half-life would be

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cause severe cardiomyopathy. This finding has implications for humandisease, as discussed below.

Our results provide direct evidence that the CtermDys fragment isstable at the cardiac sarcolemma and efficiently nucleates the DGCin vivo. We propose a two-hit CtermDys-based disease mechanismwhereby (i) membrane-tethered CtermDys mechanically uncouplesthe key actin-DGC linkage and (ii) CtermDys retards full-length dys-trophin recovery and compensatory utrophin binding at the mem-brane to cause marked dystrophic cardiomyopathy (Fig. 6). This modelexplains the more severe cardiomyopathy resulting from CtermDys—in terms of increased cardiac fibrosis and increased mortality duringcardiac stress—than from lack of dystrophin. CtermDys, by blocking

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compensatory up-regulation of the dys-trophin paralog utrophin at themembrane,differs from fully dystrophin-deficientmuscle where utrophin can partially com-pensate for complete lack of dystrophinto lessen disease (29–31). Our results, how-ever, do not exclude other downstreamdeleterious effects of CtermDys. Evidencehere of CtermDys as a loss of dystrophinfunction cleavage fragment provides newmechanistic insight into the clinical presen-tationofEV infection–based cardiomyopathyand establishes a foundation on which toguide new therapeutic interventions.

The physiological relevance of the ex-perimental approaches used here and,by extension, potential clinical insightswarrant discussion. Mechanistic insightsenabled by direct in vivo titration of theCtermDys fragment provide strong sup-port for the CtermDys loss of dystrophinfunction model (Fig. 6). The direct doc-umentation of the CtermDys fragmentdecay rate in the heart in vivo underscoresthe clinical and physiological relevance ofthis experimental model. Specifically, ourresults show a synchronous inverse relation-ship between the decay of the CtermDyspeptide and simultaneous restitution ofmembrane-bound intact dystrophin. Theinverse relationship observed here is takenas direct evidence that CtermDys turnoverand intact dystrophin are in stoichiometricbalance in these hearts in vivo, substantiat-ing this as a valid experimental model ofhuman cardiomyopathy upon EV infec-tion. We propose this CtermDys experi-mental model as physiologically relevantin terms of dissectingmechanisms of pro-teolytic cleavage of dystrophin byEV inwild-type hearts, and therefore reflects whatwould be seen in patients with Coxsackie-virus infection.

Knowlton and colleagues (14, 27) pro-vided evidence that viral infection–mediateddystrophin cleavage causes cardiac muscle

membrane destabilization, leakiness, and dysfunction. The earlier pro-posedmechanistic basis for this effect, stemming fromprevious in vitrostudies, has been that virus-encoded 2Apro cleaves and then displacesdystrophin and severalDGCpartners from the sarcolemma, suggestingthat it is the loss of intact dystrophin at themembrane that causes disease(32). However, quantitative evidence for displacement of membrane-bounddystrophin by 2Apro in hearts in vivowas lacking. This was furthercomplicated by previous findings showing that non–viral-mediated car-diomyopathy causes loss ofmembrane-bound dystrophin (33). Together,these data raised uncertainties regarding the mechanism of how dystro-phin cleavage causes disease. Our data demonstrate that the 2Apro-cleavedCtermDys peptide readily localizes and efficiently nucleates the DGC at

Fig. 4. Expression of full-length functional dystrophin corrects dystrophic cardiomyopathy inCtermDys transgenic mice. (A) Representative Western blots of heart total protein extracts from

NTg and transgenic animals. Dystrophin expression was detected using an antibody that detects bothmouse and human dystrophin. Desmin served as the loading control. (B) Quantification of CtermDysprotein expression in (A) is relative to desmin and normalized to CtermDys mice, and is expressed asmeans ± SEM (n = 4 to 5). P value was determined by ANOVA. (C) Quantification of EBD uptake in NTg,CtermDys, and DTg(mdx) hearts. Data are means ± SEM (n = 4 to 5 per group). P values were determinedby ANOVA. (D) Survival of NTg, CtermDys, CtermDys-hDys649(mdx), and CtermDys-hDys600(mdx) miceduring prolonged adrenergic stress. n = 11 per group. P value comparing CtermDys to NTg and CtermDys-hDys649(mdx) was determined by Mantel-Cox test. (E) Representative images of EBD uptake in NTg,CtermDys, and DTg(mdx) hearts. Scale bars, 1 mm.

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the sarcolemma in vivo, thus permitting direct quantitative analysis ofdystrophin cleavage fragments in the heart. Our findings gain supportfrom previous studies of expression of CtermDys-like truncated dystro-phin isoforms, Dp116 andDp71, shown to be able to localize to skeletalmuscle membranes (17, 18). As discussed below, CtermDys as a defec-

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tive dystrophin fragment attached to thesarcolemma—as opposed to dystrophindeficiency—has implications for the mech-anism of disease and potential therapeutics.

The CtermDys-based loss-of-functionmechanism is supported by our data show-ing that cardiomyopathy can be blocked bycardiac overexpression of intact dystrophinat levels sufficient to displace membrane-boundCtermDys. Further, cardioprotectionis conferred by direct CtermDys peptidetitration in vivo. Here, we calculate that 30%ofnormal levels of intact dystrophin at themembraneare insufficient to confer cardio-protection and estimate a therapeutic targetof >50% membrane-bound dystrophin re-quired to prevent cardiomyopathy. In prin-ciple, as above, a therapeutic dystrophin/CtermDys ratio could be achieved by lim-iting CtermDys formation by infection/dystrophin cleavage suppression (34) and/or by introduction of full-lengthwild-typeor noncleavable dystrophin (14) by genetherapy strategies (23). In this regard, thesefindings are interesting in comparison tothe therapeutic rangeof~20%fordystrophinto prevent DMD in muscle (35). We there-fore predict that the titration of CtermDysaway from the sarcolemma would havebeneficial effects on heart performance.Other therapeutic avenues would involvesmall molecules or other drugs developedto stabilize full-length dystrophin, by lim-iting or preventing its cleavage duringCoxsackievirus infection. Proof of conceptfor this approach has been established intransgenic animals with a modified dystro-phin resistant to cleavage (14).

Our findings of the deleterious effectsof the CtermDys peptide on cardiac mus-cle, together with the unexpected rapidCtermDys turnover kinetics (~3 days), mayprovide new insight into understandingthe clinical presentation and time courseof EV infection–mediated cardiomyopathy.Clinically, EV infection–based cardio-myopathy is challenging to diagnose wheredisease onset can be sudden and severe,and then rapidly resolve (2). This diseasepresentation could be explained in partby CtermDys as a loss-of-function peptidearising rapidly at the onset of infection/dystrophin cleavage. In turn, fast decay

of the CtermDys peptide may help explain the clinical conundrum ofrapid resolution of disease that can occur within days of viral clearance(loss of 2Apro activity) (2, 27). Rapid CtermDys peptide turnover is anunexpected outcome based on estimates of intact dystrophin turnoverrate thought to be on the order of months (26). The basis for fast

Fig. 5. Survival duringphysiological stress testing andgene-dose analysis by in vivo cardiac titrationof the CtermDys peptide. (A) CtermDys mice were crossed with mice expressing a cardiac-specific

tamoxifen-inducible Cre recombinase (MCM). Tamoxifen was administered to the resulting DTg CtermDys ×MCM mice to study the decay of the CtermDys protein in the heart after gene excision in vivo. (B) Repre-sentative Western blot showing CtermDys expression level decay and full-length endogenous dystrophinincrease at the sarcolemma in CtermDys × MCMmice after tamoxifen treatment. Dystrophin and CtermDyswere both detected with the same C-terminal antibody. Desmin was used as loading control. Quantificationof full-lengthdystrophin andCtermDys expression in CtermDys×MCMmice after tamoxifen treatment. Dataaremeans ± SEM (n= 6 per time point). (C) Survival curve of transgenic andNTgmice (n= 8 to 12 per group)starting 5days after tamoxifen treatment, during isoproterenol stress test. (D) Survival curves for transgenicand NTg mice during isoproterenol stress test (no tamoxifen injected) (n = 6 to 8 per group). P value wasdetermined by Mantel-Cox test.

Fig. 6. Two-hitmodel of CtermDys as a loss of dystrophin function peptide. (A) Normal DGC networkin human cardiac muscle. (B) Proposed two-hit model of EV infection–based CtermDys cardiomyopathy

showing severed link to actin and, because CtermDys is anchored to the DGC, prevention of utrophincompensatory binding. (C) Dystrophin-deficient myocyte with capacity for utrophin to bind and compen-sate, in part, for loss of dystrophin.

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CtermDys fragment turnover is not known. The potent effects ofCtermDys may also help explain how fulminant cardiomyopathy andacute heart failure canoccurwith only apparent regional or focal evidenceof infection. Here, CtermDys as a loss-of-function dystrophin fragmentcould help explain the substantial cardiac deficits, such as cardiogenicshock, associated with this disease. Our findings indicate not only thattheCtermDys protein is stable enough to contribute significantly to cardiacdysfunction over the course of days after infection but also that its effectsare more severe than those attributed to full loss of intact dystrophin.

In conclusion, CtermDys is a dominant-negative peptide sufficientto cause severe dystrophic cardiomyopathy. The significance of thisfinding rests in the identification of a 2Apro-mediated dystrophin cleav-age product as a novel target for therapeutic intervention in EV infec-tion. Data support a model whereby CtermDys must be tethered to thesarcolemmatoexert itsdominant loss-of-functioneffects.Thus,membrane-bound CtermDys content serves as a new target for potential therapeuticinterventions. Here, two general approaches for potential translation tohuman health could derive from this work, including gene-based ex-pression of functional dystrophins to dislodgeCtermDys, or using smallmolecules to facilitate faster decay of CtermDys. If successful, theseapproaches would be expected to improve overall heart function inCoxsackievirus-mediated cardiomyopathy.

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MATERIALS AND METHODS

Study designOur hypothesis was that viral-mediated cleavage of dystrophin wouldlead to dominant-negative peptides deleterious to heart structure andfunction in vivo.Wewere interested in testing the hypothesis that cardiacstress would significantly unmask potential dominant-negative effects ofthe dystrophin cleavage fragments. Statistical analysis and sample size jus-tification was derived from our previous animal studies (7, 21, 24) thatwere sufficiently powered to obtain significant insights into the cardio-myopathy of muscular dystrophy. In general, sample sizes averaged 7to 10 per group for heart function and structure. For histological andmouse studies, randomization and blindingwas carried out by using eartag or sample ID numbers, which did not indicate genotype. The personcarrying out the experiment and subsequent analysiswas not aware of thegenotypes associated with the ear tag or sample ID until after data collec-tion was complete, at which time the experimenter was unblinded. Fur-ther details of sample size/replicates are given in the figure legends.

Generation of transgenic miceThe NtermDys and CtermDys transgenic constructs were polymerasechain reaction (PCR)–cloned from themurine dystrophin complementaryDNA containing an N-terminal FLAG tag (gift from J. Ervasti). For theNtermDys transgene, the forward primer 5′-ttttttttgcggccgctacggcaaggtg-ctgtgcacggatctgccc-3′ and reverse primer 5′-ttttttttgcggccgccctgaccgtgc-ccctggactgagcactact-3′ were used to generate a 7.3-kbp (kilo–base pair)productwith flankingNot I restriction sites. For theCtermDys transgene,the forward primer 5′-ttttttttgcggccgcgagcagaacgtgatctcggaggaggacctggga-gcctctgccagtcagactgttactcta-3′ and reverse primer 5′-ttttttttgcggccgcactgaaa-ctaaggactccatcgctctgccc-3′ were used to generate a 3.9-kbp product withflanking Not I restriction sites. PCR mutagenesis was also used to add anN-terminal MYC tag to the CtermDys transgene. These transgenic con-structs were then inserted between loxP sites in a transgenic vector withexpressiondictatedby the cardiac-specifica-myosinheavy chainpromoter.

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These constructs were then injected into (C57BL/6 × SJL)F2 mouse eggs,and potential founders were screened for the transgene by PCR.

AnimalsTransgenic mice were backcrossed onto the C57BL/10SnJ geneticbackground for two to three generations (Jackson Laboratory, #000666).We tracked the hypomorphic dysferlin allele inherited from SJL mice,and this was bred out ofmice used in this study.Male and femalemiceof 2 to 3months of age were analyzed, with the exception ofmice usedfor histological analyses, which were 6 months old. To generate trans-genic (mdx) mice, male transgenic mice were bred with mdx females(Jackson Laboratory, #001801). Transgenicmale pups from these crossesare on themdx background and were then used for experiments to char-acterize the phenotype of CtermDys(mdx) mice.

Total protein extractionTotal cellular proteinwas extracted using amethodwhereby heartswerefrozen in liquid nitrogen and then pulverized and resuspended in 1%SDS, 5mMEGTA, andprotease inhibitors. Sampleswere boiled for 2minand then centrifuged at 14,000g for 2 min, and the supernatant was col-lected. Protein concentration was determined using a BCA protein as-say kit (Thermo Scientific).

Membrane protein isolationHearts were prepared as described previously (23). Briefly, hearts weresimilarly frozen in liquid nitrogen, pulverized and resuspended in abuffer solution lacking detergent, and centrifuged at 14,000g for 25 min.The supernatant was collected and centrifuged at 100,000g for 40 min.The pellet resulting from this spin was then resuspended in buffercontaining 0.6MKCl and washed for 1 hour at 4°C. Samples were thencentrifuged at 150,000g for 40 min, and the resultant pellet was resus-pended in a tris-sucrose buffer. Protein concentration was determinedusing a BCA protein assay kit (Thermo Scientific).

Western blottingFifty micrograms of protein was loaded per sample in 4 to 20% tris-HCl gels for SDS–polyacrylamide gel electrophoresis (Bio-Rad). Proteinwas then transferred to nitrocellulose or polyvinylidene difluoridemem-branes.Membranes were blocked in 5%nonfat drymilk in tris-bufferedsaline, and primary antibodies were applied for 1 hour at room tempera-ture. The following primary antibodieswere used: rabbit anti-dystrophin(C-terminal; Abcam, ab15277, 1:1000),mouse anti-dystrophin (mid-rod;Millipore,mab1692, 1:200),mouse anti–a-dystroglycan (Millipore, 05-593,1:1000), rabbit anti-desmin (Novus Biologicals, NB120-15200, 1:1000),mouse anti-MYC (Cell Signaling Technology, 2276, 1:1000), mouseanti-FLAG (Sigma, F1804, 1:500),mouse anti–a-sarcoglycan (Vector Lab-oratories, VP-A105, 1:100), mouse anti–g-sarcoglycan (Vector Labora-tories, VP-G803, 1:100), mouse anti–b-dystroglycan (Vector Laboratories,VP-B205, 1:100), dystrobrevin (BD Transduction, 610766, 1:500), syn-trophin (Abcam, ab11425, 1:1000), caveolin-3 (BD Transduction, 610420,1:1000), nNOS(Invitrogen/Zymed, 61-7000, 1:250), and utrophin (SantaCruz Biotechnology, 8A4, 1:50). Secondary antibodies were then ap-plied for 1 hour at room temperature, and blots were imaged using anOdyssey infrared scanner (LI-COR).

ImmunofluorescenceHearts of transgenicmicewere embedded in optimal cutting temperature(OCT) and sectioned. Heart sections were fixed in 3% formaldehyde for

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15 min at room temperature, then washed in phosphate-buffered saline(PBS), and blocked in 5%normal goat serum+0.3%TritonX-100 in PBSfor 1 hour at room temperature. Sections were then incubated with pri-mary antibodies in1%bovine serumalbumin (BSA)+ 0.3%TritonX-100in PBS overnight at 4°C. The following primary antibodies were used:rabbit anti-laminin (Sigma, L9393. 1:500),mouse anti-MYC(Cell Signal-ingTechnology, 2276, 1:500),mouse anti-dystrophin (Millipore, mab1692,1:50), and mouse anti–b-dystroglycan (Vector Laboratories, VP-B205,1:50). Sectionswere thenwashed and incubatedwith secondary antibodiesin 1% BSA + 0.3% Triton X-100 in PBS for 1 hour at room temperature.Sections were washed in PBS and mounted using Vectashield mountingmediumwith4′,6-diamidino-2-phenylindole (VectorLaboratories). Slideswere visualized usingZeiss LSM510METAconfocalmicroscope (Carl Zeiss).

EBD uptakeMembrane permeability was assessed using intraperitoneal injec-tions of EBD in PBS at a dose of 200 mg/g. Eighteen hours later, micewere given three injections of isoproterenol at a dose of 500 ng/g at18, 20, and 22 hours after EBD injection. Mice were then euthanized at24 hours after EBD injection. Hearts were then cut into three sectionsacross their short axis and frozen in OCT. Percent Evans blue–positivearea from thin sections of these samples was quantified using ImageJ(National Institutes ofHealth). For each heart, the Evans blue–positivearea reported is an average of three cross sections. Sections were im-aged on a Zeiss AxioObserver Z1 invertedmicroscope.Mosaic imageswere created with AxioVision 4.7 (Carl Zeiss).

HistologyHearts of 6-month-old mice were embedded in OCT or fixed in 10%formalin overnight and embedded in paraffin. Hearts were sectionedand stained with Sirius Red and Fast Green to detect collagen depo-sition within the myocardium and imaged on a Zeiss Axio ObserverZ1 inverted microscope (Carl Zeiss).

Isolated heart preparationMice were injected with 300 U of sodium heparin and anesthetizedwith sodium pentobarbital. The heart and lungs were removed afterthoracotomy and placed immediately in ice-cold Krebs-Henseleitbuffer (118mMNaCl, 4.7 mMKCl, 1.2 mMMgSO4, 1.2 mMKH2PO4,10mMglucose, 25mMNaHCO3, 2.5mMCaCl2, 0.5mMEDTA). Thelungs and thymus were trimmed away to expose the aorta, which wasthen cannulated. Hearts were then perfused at a constant pressure of80 mmHg with Krebs-Henseleit buffer warmed to 37°C and broughtto pH7.4 by bubblingwith 95%O2, 5%CO2.Hearts were paced at 7Hz,and changes in left ventricular pressure were monitored by insertionof a water-filled balloon with an in-line pressure transducer into theleft ventricle. Within the left ventricle, the balloon was inflated to anend-diastolic pressure of 3 to 8mmHg. After 10 to 15min of stabiliza-tion time, hearts were subjected to global no-flow ischemia for 20min.Hearts were not paced during ischemia. Hearts were then reperfusedfor 60min, and pacing was reinitiated at 8min after the end of ischemia(36). Data were collected at a sampling rate of 400 Hz and analyzedusing Chart 6 software (ADInstruments).

Cre-mediated transgene excisionMice were treated with a single dose of tamoxifen (40mg/kg) (Sigma,T5648) by intraperitoneal injection. Tamoxifen was dissolved in peanutoil at a concentration of 10 mg/ml.

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Isoproterenol survival studyMice were administered three doses of isoproterenol or isoprenalineat a dose of 10mg/kg per day for five consecutive days (30mg/kg per day)by intraperitoneal injection. Survival was assessed at 8-hour intervals.

Five days after tamoxifen treatment, the mice received isoproterenol(Sigma, I6504) at a dose of 10mg/kg at 9 a.m., 1 p.m., and 5p.m. for 5 days(30 mg/kg per day) by intraperitoneal injection (Fig. 5). Isoproterenolwas dissolved in normal saline at a concentration of 3 mg/ml. Survivalwas assessed at each injection time.

StatisticsComparisons between two groups were made using Student’s two-tailed t test. Whenmore than two groups were being compared, one-way ANOVA was used with a Newman-Keuls multiple comparisonposttest. Whenmore than one independent variable was tested, two-way ANOVA with a Bonferroni posttest was used to compare groups.Log-rank (Mantel-Cox) test was used to assess significance in survivalstudies. Data are shown as means ± SEM. All statistical analyses werecarried out using Prism (GraphPad Software).

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/294/294ra106/DC1Fig. S1. Transgene expression in hearts of NtermDys and CtermDys transgenic mice.Fig. S2. DGC protein expression in the hearts of transgenic mice.Fig. S3. Subcellular localization of NtermDys protein in the heart.Fig. S4. Subcellular localization of CtermDys protein in the heart.Fig. S5. Evidence of normal membrane integrity in the hearts of NtermDys transgenic lines.

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Acknowledgements: We thank J. Martindale, B. Thompson, J. Ervasti, and K. Prins for additionalresearch support and/or comments on the manuscript. Funding: This work was supported bygrants from NIH and the Muscular Dystrophy Association (J.M.M.). We acknowledge the supportof the Greg Marzolf Jr. Foundation (to M.S.B., Marzolf Fellowship Award) and the Lillehei HeartInstitute in aiding these studies. Author contributions: M.S.B. and J.M.M. wrote the manuscript,designed the experiments, and led the manuscript revisions. M.S.B. conducted most of theexperiments and analyzed the data, including the statistics. F.V.S. performed the in vivo CtermDystitration analysis. F.B.B. performed the heart morphology analysis. D.T. led development of thehDys mice. Competing interests: The authors declare that they have no competing interests.Data and materials availability: All data and materials are available.

Submitted 20 December 2014Accepted 10 June 2015Published 1 July 201510.1126/scitranslmed.aaa4804

Citation: M. S. Barnabei, F. V. Sjaastad, D. Townsend, F. B. Bedada, J. M. Metzger, Severedystrophic cardiomyopathy caused by the enteroviral protease 2A–mediated C-terminaldystrophin cleavage fragment. Sci. Transl. Med. 7, 294ra106 (2015).

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One-sentence summary: Enterovirus-derived C-terminal dystrophin fragment is a dominant-negative peptiderepresenting a new therapeutic target for viral cardiomyopathy remediation.

Editor’s Summary:Two hits in viral cardiomyopathy

Enteroviruses infect millions each year, affecting many organ systems, including the heart. Heart-specific infectionscan cause dystrophic cardiomyopathy and even increase risk of death after heart attack, but the mechanisms behindthese effects are unclear. Barnabei et al. found that the cleavage product of the Coxsackievirus-encoded 2A proteasewas sufficient to cause severe cardiomyopathy inmice, characterized by ischemic injury, poor response to stress, andincreased mortality. The C-terminal—but not N-terminal—fragment of the cleavage product, called CtermDys,worked in a dominant-negative manner in a “two-hit”model: first localizing to the membrane of heart muscle cells,then displacing full-length dystrophin and utrophin. Giving full-length dystrophin rescued the mice from the del-eterious effects of membrane-bound CtermDys, suggesting that both functional dystrophin and disruption ofCtermDys are viable therapeutic approaches for enterovirus-mediated cardiomyopathy.

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C-terminal dystrophin cleavage fragmentmediated−Severe dystrophic cardiomyopathy caused by the enteroviral protease 2A

Matthew S. Barnabei, Frances V. Sjaastad, DeWayne Townsend, Fikru B. Bedada and Joseph M. Metzger

DOI: 10.1126/scitranslmed.aaa4804, 294ra106294ra106.7Sci Transl Med

enterovirus-mediated cardiomyopathy.that both functional dystrophin and disruption of CtermDys are viable therapeutic approaches forfull-length dystrophin rescued the mice from the deleterious effects of membrane-bound CtermDys, suggesting localizing to the membrane of heart muscle cells, then displacing full-length dystrophin and utrophin. Givingof the cleavage product, called CtermDys, worked in a dominant-negative manner in a ''two-hit'' model: first

fragment−−but not N-terminal−−ischemic injury, poor response to stress, and increased mortality. The C-terminal Coxsackievirus-encoded 2A protease was sufficient to cause severe cardiomyopathy in mice, characterized by

found that the cleavage product of the et al.mechanisms behind these effects are unclear. Barnabei infections can cause dystrophic cardiomyopathy and even increase risk of death after heart attack, but the

Enteroviruses infect millions each year, affecting many organ systems, including the heart. Heart-specificTwo hits in viral cardiomyopathy

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