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 consisting of the clinical triad of woolly hair, palmoplantar keratoderma, and ARVC/D . 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 present with syncope and/or aborted sudden death, and the annual risk of arrhythmic death is 2.3% . Nax-os disease is caused by a recessive mutation in the gene encoding plakoglobin . 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 . We characterized the distribution of cell-cell junction proteins in fixed ventricular tissues from autopsy of Naxos disease patients using 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 pathological evidence of ARVC/D had developed. Im-
Cx43 Plakoglobin N-Cadherin Desmoplakin Desmocollin
Cx43 Plakoglobin N-Cadherin Desmoplakin Desmocollin
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 normal (Fig. 5.2) . There was also a striking reduction in the amount of junctional signal for connexin43 (Cx43), 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) . 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 normally at intercellular junctions. Similarly, although Cx43 signal at gap junctions was dramatically reduced, total Cx43 protein content assessed by immunoblotting showed little or no reduction. However, the highly phosphorylated P2-isoform of Cx43, which is selectively located in the junctional pool, was missing . These observations suggest that remodeling of gap junctions in Naxos disease is not related to changes in Cx43 expression per se,but rather to an inability to assemble and/or maintain large gap junction channel arrays. The degree of gap junction remodeling observed in Naxos disease patients is sufficient to cause conduction slowing, which could contribute to the characteristic widening of the QRS complex in the right precordial leads. Although uncoupling at gap junctions may not, by itself, cause arrhythmias, it could produce a substrate that promotes 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 . Complex ventricular arrhythmias and conduction disturbances are prominent in Carvajal syndrome. Affected children usually die before the age of 20, apparently due to both pump dysfunction and lethal arrhythmias . 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 . We described the pathology of Carvajal syndrome and analyzed the distribution of cell-cell junction proteins in the heart of an 11-year-old girl from Ecuador . The heart was markedly enlarged. The left ventricle was widely dilated and showed shallow posterior and antero-sep-tal aneurysms with mural thrombosis (Fig. 5.3). The right ventricle also showed discrete aneurysms involving 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 intercalated disks, presumably reflecting altered interactions between these two binding partners . The intermediate filament protein desmin was distributed in a normal sarcomeric pattern but it failed to local
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 junctions. As predicted, Cx43 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 consistent and prominent feature of the cell-cell junction cardiomyopathies, and (2) specific patterns of abnormal localization of mechanical junction proteins at intercalated disks correlate with cardiomyopathy disease phenotypes.
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