Desmoplakin (DP) is a component of desmosomes, critical adhesive complexes found in cardiac intercalated discs. DP is often mutated in arrhythmogenic cardiomyopathy (AC), an inherited disorder that is a frequent cause of sudden cardiac death. The disease progression of AC is characterized by cardiomyocyte cell death and replacement of the healthy myocardium by fibro-fatty deposits. Furthermore, AC patients display contractility and conduction defects that can eventually result in sudden cardiac death. Gap junctions, comprised of connexin (Cx) molecules are specialized intercellular channels that are indispensable in the myocardium where they coordinate the propagation of electrical signals between cardiomyocytes. Here, we elucidate a mechanism by which DP regulates Cx43 expression levels. DP loss was found to cause robust activation of ERK1/2 MAPK. Analysis of Cx43 phospho-sites in DP-deficient cardiomyocytes revealed an increase in MAPK sites S279/282, previously shown to signal internalization and degradation of Cx43. RNA-sequencing revealed Ras-GTPases as candidates for DP-dependent activation of the ERK1/2 MAPK pathway. Using a novel reagent (RRSP) that cleaves Ras, we showed a restoration of Cx43 loss in DP-deficient cardiomyocytes. Our results reveal a novel mechanism for the regulation of Cx43 in cardiac disease caused by DP loss. Loss of DP was also found to perturb the junctional localization and activation of RhoA GTPase signaling in cardiomyocytes. The small GTPase RhoA plays a fundamental role in regulating actomyosin contractility via its various downstream effectors. We identify that the Rho GEF Ect2, a functional regulator of RhoA activity, is a novel interacting partner of DP at cardiac cell junctions. We demonstrate that DP is required for the localization of Ect2 to cardiac cell junctions and that Ect2’s activation is dependent upon DP’s ability to scaffold the kinase PKCα at the cell membrane to specifically phosphorylate Ect2. Depletion of either DP or Ect2 leads to defects in actomyosin driven contractility of cardiomyocytes, providing a plausible mechanism for contractility defects observed in AC patients. Finally, we investigated Plakophilin-2 (PKP2), a desmosome component that is critical for DP expression and localization in cardiac cells, and its role in promoting fibrotic signaling pathways in cardiac cells. Loss of PKP2 led to the activation of TGF-β (a known activator of fibrosis) as well as increases in transcript levels of various downstream pro fibrotic signaling components. This activation of TGF-β signaling by PKP2 was found to be a byproduct of DP loss as PKP2 depletion resulted in the destabilization and degradation of DP. Together our collective results indicate that DP is a crucial regulator of cardiomyocyte homeostasis and that its loss can lead to severe cardiac outcomes such as AC.

Last modified

• 11/19/2019

Creator

• Chen Yuan Kam

DOI

• https://doi.org/10.21985/n2-mcq7-5e73

Keyword

• Desmosome
• Desmoplakin
• Rho
• Arrhythmogenic
• Connexin
• Cardiocutaneous

Date created

• 2018-01-01

Resource type

• Dissertation

Rights statement

• In Copyright