ABSTRACT
BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease associated with ventricular arrhythmias and an increased risk of sudden cardiac death. Currently, there are no approved treatments that address the underlying genetic cause of this disease, representing a significant unmet need. Mutations in Plakophilin-2 (PKP2), encoding a desmosomal protein, account for approximately 40% of ARVC cases and result in reduced gene expression. METHODS: Our goal is to examine the feasibility and the efficacy of adeno-associated virus 9 (AAV9)-mediated restoration of PKP2 expression in a cardiac specific knock-out mouse model of Pkp2. RESULTS: We show that a single dose of AAV9:PKP2 gene delivery prevents disease development before the onset of cardiomyopathy and attenuates disease progression after overt cardiomyopathy. Restoration of PKP2 expression leads to a significant extension of lifespan by restoring cellular structures of desmosomes and gap junctions, preventing or halting decline in left ventricular ejection fraction, preventing or reversing dilation of the right ventricle, ameliorating ventricular arrhythmia event frequency and severity, and preventing adverse fibrotic remodeling. RNA sequencing analyses show that restoration of PKP2 expression leads to highly coordinated and durable correction of PKP2-associated transcriptional networks beyond desmosomes, revealing a broad spectrum of biological perturbances behind ARVC disease etiology. CONCLUSIONS: We identify fundamental mechanisms of PKP2-associated ARVC beyond disruption of desmosome function. The observed PKP2 dose-function relationship indicates that cardiac-selective AAV9:PKP2 gene therapy may be a promising therapeutic approach to treat ARVC patients with PKP2 mutations.
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heart disease that leads to abnormal heartbeats and a higher risk of sudden cardiac death. ARVC is often caused by changes in a gene called PKP2, that then makes less PKP2 protein. PKP2 protein is important for the normal structure and function of the heart. Human ARVC characteristics can be mimicked in a mouse model missing this gene. Given no therapeutic option, our goal was to test if adding a working copy of PKP2 gene in the heart of this mouse model, using a technique called gene therapy that can deliver genes to cells, could improve heart function. Here, we show that a single dose of PKP2 gene therapy can improve heart function and heartbeats as well as extend lifespan in mice. PKP2 gene therapy may be a promising approach to treat ARVC patients with PKP2 mutations.
ABSTRACT
Heat-induced cell death appears to be a cell-specific event. Chronic heat stress was lethal to human colon cancer cells (Caco-2, HT29, and HCT116), but not to normal diploid fibroblasts and other cancer cells (BJ-T, WI38, HeLa, ovarian 2008, WI38VA). Acute heat stress (45-51 degrees C, 30 min) caused cell death of colon cancer cells during recovery at physiological temperature. Thermal killing of Caco-2 cells was not mediated via oxidative stress since Caco-2 cells were much more resistant than HeLa and other cancer cells to H(2)O(2)-induced cell death. Acute heat stress caused a striking loss of eukaryotic initiation factor 5A (eIF5A) in colon cancer cells, but not in HeLa and other normal or transformed human fibroblasts. The heat-induced loss of eIF5A is likely to be due to changes in the protein stability. The half-life of eIF5A was changed from >20 h to less than 30 min during the acute heat stress. Sequence analysis of the eIF5A gene from Caco-2 and HeLa cells did not reveal any difference, suggesting that the change in stability in Caco-2 cells was not due to any eIF5A mutation. Pretreatment of cells with protease inhibitors such as phenylmethyl sulfonyl fluoride (PMSF) partially blocked the heat-induced loss of eIF5A and prevented heat-induced cell death. In light of the essential role of eIF5A in cell survival and proliferation, our results suggest that the stability of eIF5A may have an important role in determining the fate of the particular cell type after severe heat stress.
