Introduction
Significant progress has been made in identifying sin- gle gene mutations responsible for causing human car- diomyopathies. Although many of these monogenic cardiomyopathies are rare, insights into pathogenesis by identification of the responsible mutations can provide clues about mechanisms in more common forms of heart disease. We have studied a group of hu- man cardiomyopathies caused by mutations in genes encoding proteins that function as linkers in cell-cell adhesion junctions. These heart diseases, which we have termed cell-cell junction cardiomyopathies, are caused by mutations in intracellular proteins that link adhesion molecules at adherens junctions and desmo- somes to the myocyte cytoskeleton. Among the genes implicated in these diseases are those encoding desmo- plakin, plakoglobin, and plakophilin-2. These muta- tions have both dominant and recessive patterns of in- heritance and are associated with clinical phenotypes of arrhythmogenic right ventricular cardiomyopathy/
dysplasia (ARVC/D) or dilat cardiomyopathy (DCM), with or without hair and skin abnormalities [1]. Com- mon features of the cell-cell junction cardiomy- opathies are a high incidence of syncope, ventricular arrhythmias, and sudden cardiac death. This observa- tion suggests that alterations in intercellular adhesion caused by defects in cell-cell mechanical junctions may create anatomic substrates that are particularly con- ducive to the development of lethal ventricular ar- rhythmias. Our work in this area has focused on the hypothesis that defective mechanical linkage in the cell-cell junction cardiomyopathies causes remodeling of gap junctions, which, in turn, can give rise to con- duction abnormalities that may contribute to the high incidence of sudden death in these patients.
Electrical Coupling at Gap Junctions in the Heart
Cardiac muscle is not a true electrical syncytium.
Rather, it is composed of individual cells, each in-
vested with a continuous lipid bilayer which provides a considerable degree of electrical insulation. Elec- trical activation of the heart requires intercellular transfer of current, a process that can only occur at gap junctions [2]. Thus, the number, size, and distri- bution of gap junctions are important determinants of impulse propagation in cardiac muscle. Further- more, alterations in the structure or function of gap junctions can give rise to conduction disturbances that may contribute to arrhythmogenesis.
There is an intimate structural, functional, and spatial relationship between gap junctions and me- chanical junctions in cardiac myocytes. Cardiac my- ocytes are connected by extremely large intercellular junctions, which, presumably, have evolved to sub- serve the specialized electrical and contractile func- tion of these cells. For example, because cardiac my- ocytes contract, they require more extensive and ro- bust adhesion junctions than noncontractile cells in other solid organs. It is no surprise, therefore, that ad- herens junctions and desmosomes, organelles re- sponsible for physically connecting one cardiac my- ocyte to another, are highly concentrated at the ends of individual cells where they form elaborate com- plexes that can be readily identified at the light mi- croscopic level of resolution and which have been giv- en a specific name – the intercalated disk. Intercalat- ed disks are composed mainly of arrays of adherens junctions, each located at the end of a row of sar- comeres. These junctions act as bridges which link actin filaments within sarcomeres of neighboring cells. Interspersed among the adherens junctions are desmosomes, which also provide mechanical coupling and link cell-cell adhesion junctions to desmin fila- ments of the cytoskeleton. Because electrical activa- tion of the heart requires intercellular transfer of cur- rent, cardiac myocytes also have a special requirement for extensive electrical coupling. Indeed, gap junctions interconnecting ventricular myocytes are among the largest in living systems, presumably reflecting a need for many low-resistance electrical communicating channels between cells to ensure safe conduction.
CELL ADHESION PATHOLOGY
Jeffrey E. Saffitz
CHAPTER
5
tions, we have proposed that the extent to which car- diac myocytes can become electrically coupled de- pends on their degree of mechanical coupling [3]. As shown in Fig. 5.1, this hypothesis provides a mecha- nistic link between contractile dysfunction and elec- trical dysfunction in the cell-cell junction cardiomy- opathies. A genetic defect in a protein in cell-cell ad- hesion junctions may lead to unstable mechanical linkage between cells and/or discontinuities between
calated disk proteins in cardiac tissues from patients and have shown that, as a general rule, the cell-cell junction cardiomyopathies are associated with re- modeling of gap junctions. Second, we have identified electrophysiological phenotypes in genetically engi- neered mouse models of the human cardiomy- opathies. Third, we have elucidated signaling path- ways that coordinately regulate expression of me- chanical and electrical junction proteins in cardiac myocytes in response to mechanical stress.
Altered Expression and Distribution of Cell-Cell Junction Proteins in Human Cardiomyopathies
To test the hypothesis that defects in the adhesion junction-cytoskeleton network disrupt gap junctions, we analyzed ventricular tissues from patients with Naxos disease, a cardiocutaneous syndrome consist- ing of the clinical triad of woolly hair, palmoplantar keratoderma, and ARVC/D [4]. Patients with Naxos disease are readily identified early in life because of the distinctive cutaneous features (the disease is 100% penetrant) [5, 6]. Naxos disease is associated with a particularly high incidence of sudden cardiac death. Approximately 60% of affected children pre- sent with syncope and/or aborted sudden death, and the annual risk of arrhythmic death is 2.3% [6]. Nax- os disease is caused by a recessive mutation in the gene encoding plakoglobin [7]. The mutation is a deletion of nucleotides 2157 and 2158 which causes a frame-shift resulting in premature termination and expression of a truncated protein lacking 56 residues at the C-terminus [7]. We characterized the distrib- ution of cell-cell junction proteins in fixed ventricu- lar tissues from autopsy of Naxos disease patients us- ing confocal immunofluorescence microscopy, and also performed immunoblotting analysis on frozen, unfixed cardiac tissue from an affected child who died of acute leukemia before overt clinical or patho- logical evidence of ARVC/D had developed. Im- Linking Electrical and Contractile Dysfunction in the
Cell-Cell Junction Cardiomyopathies
Genetic Defect in a Protein in Cell-Cell Adhesion Junctions
Diminished Force Transmission
Myocyte Injury Myocardial Remodeling
Contractile Dysfunction Cardiomyopathy
Gap Junction Remodeling Destabilization of Sarcolemmas of Adjacent Cells
Electrical Dysfunction Slow Conduction
Arrhythmias Sudden Death Abnormal Mechanical Linkage
at Cell-Cell Junctions and/or Discontinuities Between Junctions
and the Cytoskeleton
Fig. 5.1 • A flow chart illustrating a proposed mechanism linking electrical and contractile dysfunction in the cell-cell junction cardiomyopathies
munoreactive signal for plakoglobin, the mutant protein in Naxos disease, was dramatically reduced at intercalated disks in both ventricles from all Naxos disease patients whereas signals for N-cadherin, desmoplakin and desmocollin-2 appeared to be nor- mal (Fig. 5.2) [4]. There was also a striking reduction in the amount of junctional signal for connexin43 (C×43), the major ventricular gap junction protein, in both the right and left ventricles in Naxos disease, including the individual who died before ARVC/D had become manifest clinically or pathologically (Fig. 5.2) [4]. Electron microscopy revealed smaller and fewer gap junctions interconnecting ventricular myocytes, providing independent evidence of gap junction remodeling. Immunoblotting revealed that truncated plakoglobin was expressed abundantly in the myocardium even though it failed to localize nor- mally at intercellular junctions. Similarly, although C×43 signal at gap junctions was dramatically re- duced, total C×43 protein content assessed by im- munoblotting showed little or no reduction. How- ever, the highly phosphorylated P2-isoform of C×43, which is selectively located in the junctional pool, was missing [4]. These observations suggest that remod- eling of gap junctions in Naxos disease is not related to changes in C×43 expression per se, but rather to an inability to assemble and/or maintain large gap junc- tion channel arrays. The degree of gap junction re- modeling observed in Naxos disease patients is suf- ficient to cause conduction slowing, which could contribute to the characteristic widening of the QRS complex in the right precordial leads. Although un- coupling at gap junctions may not, by itself, cause ar- rhythmias, it could produce a substrate that pro- motes arrhythmias when combined with a “second
insult” such as the pathological changes in the right ventricle in ARVC/D.
We have also characterized the distribution of cell-cell junction proteins in Carvajal syndrome, a car- diocutaneous syndrome characterized by woolly hair, palmoplantar keratoderma, and a diffuse cardiomy- opathy that is distinct from ARVC/D [8]. Complex ventricular arrhythmias and conduction disturbances are prominent in Carvajal syndrome. Affected chil- dren usually die before the age of 20, apparently due to both pump dysfunction and lethal arrhythmias [8].
Carvajal syndrome is caused by a recessive single nu- cleotide deletion mutation in desmoplakin leading to a premature stop-codon and truncation of the C-ter- minal desmin-binding domain [9]. We described the pathology of Carvajal syndrome and analyzed the dis- tribution of cell-cell junction proteins in the heart of an 11-year-old girl from Ecuador [10]. The heart was markedly enlarged. The left ventricle was widely di- lated and showed shallow posterior and antero-sep- tal aneurysms with mural thrombosis (Fig. 5.3). The right ventricle also showed discrete aneurysms in- volving inferior, atypical, and infundibular regions (Fig. 5.3). Interestingly, these same right ventricular areas also show the greatest abnormalities in ARVC/D (the so-called triangle of dysplasia), but there was no gross or microscopic evidence of fatty replacement of right or left ventricular muscle in Carvajal Syndrome.
Confocal microscopy revealed that immunoreactive signals for both desmoplakin (the mutant protein) and plakoglobin were markedly diminished at inter- calated disks, presumably reflecting altered interac- tions between these two binding partners [10]. The intermediate filament protein desmin was distributed in a normal sarcomeric pattern but it failed to local- Fig. 5.2 • Representative confocal microscopy images of left ventricle from control and Naxos disease stained with specific antibodies against selected intercellular junction proteins
ize properly at intercalated disks. This indicates that interactions between desmin and desmoplakin are disrupted in Carvajal Syndrome, which, according to our hypothesis, would cause remodeling of gap junc- tions. As predicted, C×43 signal at junctions was markedly diminished. These results provide further evidence that abnormal protein-protein interactions at intercellular junctions cause both contractile and electrical dysfunction in Carvajal Syndrome.
Taken together, our studies of human cell-cell junction cardiomyopathies have led to two major conclusions: (1) remodeling of gap junctions is a con- sistent and prominent feature of the cell-cell junction cardiomyopathies, and (2) specific patterns of ab- normal localization of mechanical junction proteins at intercalated disks correlate with cardiomyopathy disease phenotypes.
Mouse Models of Human Cardiomyopathies
Delineation of structural and molecular pathology in human tissues is essential to understanding the cell- cell junction cardiomyopathies; yet to elucidate mech- anisms of disease, we have turned to analysis of mouse models.
The first mouse line we characterized was a mod- el of human desmin-related cardiomyopathy created by X.J. Wang in the laboratory of Jeffrey Robbins [11]. We were attracted to this model because we an- ticipated that it might exhibit altered desmin-desmo- some interactions and, therefore, recapitulate Carva- jal syndrome. Human desmin-related skeletal and cardiomyopathies have been attributed to a 7-amino acid deletion mutation (R173-E179) and several mis- sense mutations in desmin. To determine whether the R173-E179 deletion was sufficient to cause desmin- related cardiomyopathy, Wang et al. [11] produced transgenic mice with cardiac-specific expression of the 7-amino acid deletion mutation in desmin (D7- des) implicated in the human disease. In their initial description of this model, they showed that D7-des mice exhibit features of human desmin-related car- diomyopathy including intracellular accumulation of desmin, disruption of the desmin filament network, misalignment of myofibrils, and diminished respon- siveness to α-adrenergic agonist stimulation [11].
To test the hypothesis that expression of D7-des disrupts the linkage between desmosomes and the cy- toskeleton and leads to remodeling of gap junctions, we characterized the expression and localization of in- tercellular junction proteins and searched for an elec- Fig. 5.3 • Gross photographs of the right (a, b) and left (c-e) sides of the heart in Carvajal syndrome. Both ventricles con- tained discrete regions of aneurysmal wall thinning. Affected areas included the subtricuspid posterior right ventricle (tran- silluminated area in b), the posterior basal portion of the left ventricle (area marked by asterisk in d), and the antero-sep- tal portion of the left ventricle which was lined by a mural thrombus (e)
d e
trophysiological phenotype [12]. As predicted by stud- ies of the human cell-cell junction cardiomyopathies, C×43 signal at intercalated disks was decreased by ap- proximately threefold in D7-des hearts due to signifi- cant reductions in both the number and mean size of individual gap junctions (Fig. 5.4). The amount of im- munoreactive signal at intercalated disks was also re- duced significantly for selected adhesion molecules and linker proteins of both desmosomes and adherens junctions, and desmin-desmosomal interactions were completely disrupted [12]. Quantitative electron mi- croscopy showed decreased gap junction density in D7-des mice, providing independent evidence of gap junction remodeling, but immunoblotting showed no reduction in the total tissue content of C×43 and me- chanical junction proteins. These observations are consistent with findings in Naxos disease, suggesting that diminished localization of cell-cell junction pro- teins at intercalated disks is not due to insufficient pro- tein expression but, rather, to failure of these proteins to assemble properly within electrical and mechanical junctions. We also showed, using optical mapping, that remodeling of gap junctions in D7-des mice slows ven- tricular conduction [12]. These results indicate, there- fore, that a defect in a protein conventionally thought to fulfill a strictly mechanical function in the heart can also lead to electrophysiological alterations that may contribute to arrhythmogenesis.
Mechanisms Regulating Expression of Cell-Cell Junction Proteins in Response to Mechanical Load
To elucidate mechanisms regulating expression of in- tercellular junction proteins, we developed an in vit- ro system in which monolayers of neonatal rat ven-
tricular myocytes are grown on silicone membranes and subjected to uniaxial pulsatile stretch [13]. Im- position of this mechanical load rapidly induces a hy- pertrophic response which can be rigorously quanti- fied and characterized. An important feature of this response is a marked increase in C×43 expression and enhanced intercellular coupling. After only 1h of stretch (110% of resting length at 3 Hz), expression of C×43 is increased by approximately twofold, re- sulting in a significant increase in both the number of gap junctions and the velocity of impulse propa- gation [13]. We have shown previously that upregu- lation of C×43 expression is mediated by stretch-in- duced secretion of vascular endothelial growth fac- tor (VEGF) which acts in an autocrine fashion [14].
Incubation of cells with exogenous VEGF for 1h in- creases C×43 expression by an amount roughly equal to that seen after 1h of pulsatile stretch. Moreover, stretch-induced upregulation of C×43 expression can be blocked by stretching cells in the presence of anti-VEGF or anti-VEGF receptor antibodies.
To determine whether stretch-induced formation of new gap junctions requires concomitant assembly of new mechanical junctions, we measured changes in mechanical junction protein expression in cells sub- jected to stretch [15]. The amounts of plakoglobin, desmoplakin, and N-cadherin at cell-cell junctions all increased by at least twofold in myocytes subjected to 1h of pulsatile stretch (Fig. 5.5). However, VEGF se- cretion plays no role in this process. For example, ad- dition of exogenous VEGF does not affect plakoglo- bin, desmoplakin, or N-cadherin expression, nor is stretch-induced upregulation of these proteins blocked by anti-VEGF antibodies. To further define the responsible mechanisms, we studied the role of fo- cal adhesion kinase (FAK), which is phosphorylated in response to integrin engagement and activates Fig. 5.4 • Representative confocal immunofluorescence images showing the amount of C×43 immunoreactive signal at cell-cell junctions in left ventricular myocardium from a nontransgenic control mouse (Con), a transgenic mouse expressing wild-ype desmin (WT-des), and a transgenic mouse expressing D7-des
multiple intracellular signaling molecules including src kinase. We infected cardiac myocytes with an ade- novirus containing GFP-FRNK, a GFP-tagged domi- nant-negative inhibitor of FAK-dependant signaling, and then subjected cells to pulsatile stretch [15].
FRNK blocked stretch-induced upregulation of both electrical (C×43) and mechanical (N-cadherin, desmoplakin, and plakoglobin) junction proteins. In- fection of cells with virus expressing GFP alone had no effect. Addition of exogenous VEGF to FRNK-in- fected cells upregulated expression of C×43 but not mechanical junction proteins. Conditioned medium removed from uninfected cells after 1h of stretch in- creased C×43 expression when added to nonstretched cells, and this effect was blocked by anti-VEGF anti- bodies, but stretch-conditioned medium from FRNK- infected cells had no effect on C×43 expression. Thus, secretion of VEGF in response to stretch requires ac- tivation of FAK. Finally, the src kinase inhibitor PP2 blocked stretch-induced upregulation of mechanical junction proteins but not C×43 [15]. These results in- dicate that mechanical load regulates expression of both electrical and mechanical junctions proteins, but
by disparate mechanisms. C×43 expression is regu- lated by autocrine actions of chemical mediators se- creted during stretch, whereas adhesion junction pro- teins are regulated by intracellular mechanotrans- duction pathways initiated via FAK and dependent on downstream activation of src kinase.
Conclusions
We have proposed a unified hypothesis that links con- tractile and electrical dysfunction in the cell-cell junc- tion cardiomyopathies. It is based on the premise that the extent to which cardiac myocytes are coupled me- chanically at cell-cell adhesion junctions is a key de- terminant of the extent to which they can be coupled electrically at gap junctions. Our observations in sev- eral human cardiomyopathies indicate that genetic de- fects in linker proteins such as desmoplakin and plako- globin can create anatomic substrates of sudden death by remodeling gap junctions. Molecular mechanisms responsible for gap junction remodeling in the cell-cell junction cardiomyopathies are unknown. One possi-
0 2 4 6 8 10 12
C x 4 3 P l a k o g l o b i n D e s m o p l a k i n N - c a d h e r i n Co n tr o l
S tre tc h
*
*
*
*
b
bility is that rates of C×43 synthesis and degradation are unaffected but connexin molecules are unable to assemble properly in gap junctions. It must also be considered, however, that C×43 gene expression may be altered in the cell-cell junction cardiomyopathies.
Plakoglobin and other members of the catenin fami- ly fulfill both structural and nuclear signaling roles [16]. Disease-related mutations may shift the relative proportions of these proteins within junctional and cytosolic pools, which, in turn, could affect nuclear sig- naling mediated by plakoglobin, or β-catenin or oth- er related proteins. If, for example,β-catenin substi- tutes for mutant plakoglobin within cell-cell junctions, then the resultant decrease in the cytosolic pool of β-catenin could lead to diminished expression of C×43 and other proteins under the control ofβ-catenin sig- naling. Thus, it is possible and perhaps even likely that both altered mechanical integrity and altered nuclear signaling underlie the pathogenesis of contractile and electrical dysfunction in heart muscle diseases caused by mutations in cell-cell junction proteins.
Acknowledgements
Work in the author’s laboratory was supported by grants from the National Institutes of Health, March of Dimes, American Heart Association, and the Sarnoff Endowment.
References
1. Protonotarios N, Tsatsopoulou A (2004) Naxos disease and Carvajal syndrome: cardiocutaneous disorders that highlight the pathogenesis and broaden the spec- trum of arrhythmogenic right ventricular cardiomy- opathy. Cardiovasc Pathol 13:185-194
2. Saffitz JE, Lerner DL, Yamada KA (2004) Gap junction distribution and regulation in the heart. In: Zipes DP, Jalife J, (eds) Cardiac electrophysiology: From cell to bedside, 4th edn. Saunders, Philadelphia pp 181-191 3. Saffitz JE (2005) Dependence of electrical coupling on
mechanical coupling in cardiac myocytes: insights gained from cardiomyopathies caused by defects in cell-cell connections. Ann N Y Acad Sci 1047:336-344
4. Kaplan SR, Gard JJ, Protonotarios N et al (2004) Re- modeling of myocyte gap junctions in arrhythmogenic right ventricular cardiomyopathy due to a deletion in plakoglobin (Naxos disease). Heart Rhythm 1:3-11 5. Protonotarios N, Tsatsopoulou AA, Gatzoulis KA
(2002) Arrhythmogenic right ventricular cardiomy- opathy caused by a deletion in plakoglobin (Naxos dis- ease). Card Electrophysiol Rev 6:72-80
6. Protonotarios N, Tsatsopoulou A, Anastasakis A et al (2001) Genotype-phenotype assessment in autosomal recessive arrhythmogenic right ventricular cardiomy- opathy (Naxos disease) caused by a deletion in plako- globin. J Am Coll Cardiol 38:1477-1484
7. McKoy G, Protonotarios N, Crosby A et al (2000) Identification of a deletion in plakoglobin in arrhyth- mogenic right ventricular cardiomyopathy with pal- moplantar keratoderma and woolly hair (Naxos dis- ease). Lancet 355:2119-2124
8. Carvajal-Huerta L (1998) Epidermolytic palmoplantar keratoderma with woolly hair and dilated cardiomy- opathy. J Am Acad Dermatol 39:418-421
9. Norgett EE, Hatsell SJ, Carvajal-Huerta L et al (2000) Recessive mutation in desmoplakin disrupts desmo- plakin-intermediate filament interactions and causes dilated cardiomyopathy, woolly hair and keratoderma.
Hum Mol Genet 9:2671-2766
10. Kaplan SR, Gard JJ, Carvajal-Huerta L et al (2004) Structural and molecular pathology of the heart in Carvajal syndrome. Cardiovasc Pathol 13:26-32 11. Wang X, Osinska H, Dorn GW 2nd et al (2001) Mouse
model of desmin-related cardiomyopathy. Circulation 103:2402-2407
12. Gard JJ, Yamada K, Green KG et al (2005) Remodeling of gap junctions and slow conduction in a mouse model of desmin-related cardiomyopathy. Cardiovasc Res 67:539-547
13. Zhuang J, Yamada KA, Saffitz JE et al (2000) Pulsatile stretch remodels cell-to-cell communication in cul- tured myocytes. Circ Res 87:316-322
14. Pimentel RC, Yamada KA, Kléber AG et al (2002) Au- tocrine regulation of C×43 expression by VEGF. Circ Res 90:671-677
15. Yamada K, Green KG, Samarel AM et al (2005) Distinct pathways regulate expression of cardiac electrical and mechanical junctions proteins in response to stretch.
Circ Res 97:346-353
16. Conacci-Sorrell M, Zhurinsky J, Ben-Ze’ev A (2002) The cadherin-catenin adhesion system in signaling and cancer. J Clin Invest 109:987-991
