Abstract-Desmosomes and gap junctions are distinct structural components of the cardiac intercalated disc. Here, we asked whether the presence of plakophilin (PKP)2, a component of the desmosome, is essential for the proper function and distribution of the gap junction protein connexin (Cx)43. We used RNA silencing technology to decrease the expression of PKP2 in cardiac cells (ventricular myocytes, as well as epicardium-derived cells) obtained from neonatal rat hearts.We evaluated the content, distribution, and function of Cx43 gap junctions. Our results show that loss of PKP2 expression led to a decrease in total Cx43 content, a significant redistribution of Cx43 to the intracellular space, and a decrease in dye coupling between cells. Separate experiments showed that Cx43 and PKP2 can coexist in the same macromolecular complex. Our results support the notion of a molecular crosstalk between desmosomal and gap junction proteins is an inherited disease that presents with sustained monomorphic ventricular tachycardia and sudden cardiac death. The disease is characterized by progressive fibrofatty infiltration of the myocardium, most prominent in the free wall of the right ventricle. 1 Recent studies have linked ARVC with mutations in proteins of the cardiac desmosome, 2 a component of the intercalated disc essential for mechanical coupling between cardiac cells. 3 It is estimated that as many as 70% of the mutations linked to familial ARVC are in the gene coding for plakophilin (PKP)2, 4 a 98-kDa desmosomal protein. PKP2 interacts with plakoglobin, desmoplakin, and the desmosomal cadherins via its amino terminal ("head") domain. [5][6] Loss of PKP2 destabilizes the desmosome, 7 and its genetic deletion in mice leads to rupture of the myocardial wall during the embryonic stage. 7 Loss of desmosomal integrity could lead to disruption of mechanical function in hearts afflicted with ARVC; yet, the latter does not directly explain the highly arrhythmogenic nature of the disease, particularly in cases in which lifethreatening arrhythmias occur in the absence of severe displacement of myocardium with fatty or fibrous tissue. 8 Recently, Saffitz and colleagues proposed that disruption of mechanical coupling may lead to loss of gap junctionmediated electrical communication between cells. 8 -10 This hypothesis awaits confirmation in a cellular model in which protein expression can be manipulated and intercellular communication can be assessed directly.Here, we used small interfering (si)RNA technology to silence PKP2 expression in neonatal cardiac cells, and we explored the effect of loss of PKP2 expression on the distribution and function of gap junctions. Our studies focused primarily on 2 cell populations: cardiac myocytes and epicardium-derived cells (EPDCs). Although the importance of cardiac myocytes in the context of ARVC and arrhythmias seems self-evident, a possible role for EPDCs in ARVC has not been described. Yet, as progenitors of the cardiac fibroblast cell lineage, the function of EPDCs deserves atte...

Mutation(s) responsible for ARVD/C in Boxers lead, directly or indirectly, to severe modifications of mechanical and electrical cell-cell interactions. Furthermore, significant reduction in gap junction formation may promote a substrate for malignant ventricular arrhythmias. This model may help to advance our understanding of the molecular basis, pathophysiology, and potential therapeutic approach to patients with ARVD/C.

Objectives We sought to quantify the number and length of desmosomes, gap junctions, and adherens junctions in arrhythmogenic right ventricular cardiomyopathy (ARVC) and non-ARVC dogs, and to determine if ultrastructural changes existed. Animals Hearts from 8 boxer dogs afflicted with histopathologically confirmed ARVC and 6 dogs without ARVC were studied. Methods Quantitative transmission electron microscopy (TEM) and Western blot semi-quantification of α-actinin were used to study the intercalated disc and sarcomere of the right and left ventricles. Results When ARVC dogs were compared to non-ARVC dogs reductions in the number of desmosomes (P = 0.04), adherens junctions (P = 0.04) and gap junctions (P = 0.02) were found. The number of gap junctions (P = 0.04) and adherens junctions (P = 0.04) also were reduced in the left ventricle, while the number of desmosomes was not (P = 0.88). A decrease in the length of desmosomal complexes within LV samples (P=0.04) was found. These findings suggested disruption of proteins providing attachment of the cytoskeleton to the intercalated disc. Immunoblotting did not demonstrate a quantitative reduction in the amount of α-actinin in ARVC afflicted samples. All boxers with ARVC demonstrated the presence of electron dense material originating from the Z band and extending into the sarcomere, apparently at the expense of the cytoskeletal structure. Conclusions These results emphasize the importance of structural integrity of the intercalated disc in the pathogenesis of ARVC. In addition, observed abnormalities in sarcomeric structure suggest a novel link between ARVC and the actin-myosin contractile apparatus.

BACKGROUND-Arrhythmogenic right ventricular cardiomyopathy (ARVC), has been linked to mutations in desmosomal proteins, including plakophilin2 (PKP2). Little is known about the changes in cellular function and structure that follow expression of ARVC-relevant PKP2 mutations.