Defining a Unifying Mechanism for Select Cardiomyopathy-Linked Variants of Desmoplakin

Abstract

Arrhythmogenic cardiomyopathy (AC) affects 1 in 2000 Americans every year and segregates with sudden cardiac death (SCD). AC is linked to genetic variants in desmosomal genes, however the molecular mechanisms underlying AC remain unclear. We identified a novel variant (R451G) in the desmoplakin (DSP) gene; heart autopsy revealed pronounced fibrofatty scarring and a significant loss of DSP protein at the intercalated disc (ID). Engineered heart tissues (EHTs) from induced-pluripotent stem cells generated from a symptomatic R451G-positive family member showed a significant loss of DSP protein compared to control EHT. Notably, the R451G-variant sits in a pocket containing several clinical DSP variants and we show that endogenous DSP is cleaved via a calpain-dependent mechanism. From this, we hypothesize that mutant DSP is more susceptible to calpain cleavage, resulting in cellular haploinsufficiency. In previous work, we defined a unifying mechanism for AC-linked variants by assessing the biomolecular properties of DSP variants. While these variants did not significantly perturb the structure of DSP, there were significant changes to stabilizing intramolecular interactions of select variants, which correlated with augmented calpain degradation due to increased exposure of an auto-inhibited calpain-cleavage site. We have now shown that by changing the molecular environment using site-directed mutagenesis, we can ameliorate the calpain sensitivity of clinical variants. Taken together we have identified a unifying mechanism for select AC-linked variants of DSP grounded in increased susceptibility to calpain-cleavage leading to decreased functional DSP. DSP protein expression was also decreased in end-stage failing human hearts compared to control donor hearts. The physiological and biochemical impact of this loss is being further evaluated to define the role DSP haploinsufficiency plays in the fundamental disease mechanism of AC.