Arya Kamat
Pronouns: She/Her/Hers
Research Mentor(s): Adam Helms
Research Mentor School/College/Department: Internal Medicine / Cardiovascular Medicine / Medicine
Program: UROPF
Session: Session 1 (9:00am – 9:50am)
Authors: Arya Kamat, Eric Smith, Adam Helms
Abstract
Allelic dominant mutations in the DSP (or desmoplakin) gene are often linked to well-known cardiac disorders, such as dilated or arrhythmogenic right ventricular cardiomyopathy. As mechanisms for this effect remain unclear, we lack material to understand how DSP truncating mutations affect genotype-phenotype correlations for patients with novel DSP. We took DSP deficient lines of ELK and GEM-12, which both have a 90% reduction in desmoplakin levels, to stain and image the desmoplakin and N-cadherin fluorescence in those cell lines; GEM-12 has GFP attached to desmoplakin so it fluoresces without any action while ELK does not. Then, we took the cell lines and treated them with lentivirus that is expressing deactivated Cas-9 targeted to desmoplakin to activate it. Treating with virus can infect the cells and inserting Cas-9 can cause the cells to express deactivated Cas-9 with guide RNA in front of desmoplakin, causing desmoplakin to turn on more and expression to increase. We will then supply an antibiotic to kill all surrounding material without virus, since it imparts antibiotic resistance to cells. Finally, we fixed our cells and were left with desmoplakin-deficient cells that are no longer desmoplakin-deficient. With DSP truncating mutations, there was a loss of DSP at the desmosome. Activation of the DSP gene resulted in the return of DSP protein at the desmosome. Additionally, DSP loss at the desmosome caused the loss of other desmosomal proteins, and the levels of these can be returned to normal with activation of DSP. With this, there was increased fluorescence signaling for desmoplakin as other desmosomal proteins were indirectly activated. A reduction in desmoplakin is going to cause a reduction in other desmoplakin proteins in the desmosome as they’ll have nothing to stick to. The cells are missing only desmoplakin, but when you image the desmosome, you don’t see other proteins since they have nothing to bind to.
