180. The Role of the Pyruvate Dehydrogenase Kinase 4 (PDK4) Gene Mutation in the Development of Dilated Cardiomyopathy in Dobermanpinschers and Potential Application of Cardiac Gene Therapy
Authors: Luiz Bolfer, Amara H. Estrada, Brandy Winter, Kathryn Taggart, Francisco Lourenço, Naohiro Terada, Barry J. Byrne, Thomas J. Conlon, Christina A. Pacak
Journal: Molecular Therapy (2016)

Dilated cardiomyopathy (DCM) is estimated to be the third most common inherited type of heart disease in humans reported to affect 35.6 and 0.57 in 100,000 adults and children, respectively. The Doberman Pinscher (DP) is a canine breed affected with idiopathic, non-ischemic DCM that closely resembles the human counterpart acting as a clinically relevant human model. Recently, a 16 base pair deletion in the 5' donor splice site of intron 10 of the pyruvate dehydrogenase kinase 4 (PDK4) gene, encoding for a mitochondrial protein involved in energy metabolism, has been identified but the relationship between the PDK4 mutation, mitochondrial function and phenotypic development of DCM is largely unknown. Our research has focused on understanding the relationship between the consequences of the PDK4 mutation on mitochondrial function and phenotypic development of DCM as well as the potential of cardiac gene therapy to correct the phenotype. A total of 64 DP were tested for the PDK4 mutation and screened for DCM. Healthy dogs and dogs diagnosed with DCM were further divided into 3 groups based on the presence of the genetic mutation: Wild-type (PDK4wt/wt), Heterozygous (PDK4wt/del), and Homozygous (PDK4del/del). Preliminary analyses using isolated mitochondria from skin fibroblasts of these dogs demonstrated a lack of detectable PDK4 protein expression in PDK4del/del, and a significant decrease in PDK4wt/del (48% of normal). Phase contrast microscopy and immunofluorescence studies revealed significantly different morpho-phenotypic characteristics and reduced capacity to adapt to unfavorable metabolic conditions as compared to healthy dogs. Assessment of mitochondrial metabolic potential and function (oxygen consumption rate (OCR) and extracellular acidification rate (ECAR)) were performed using an extracellular flux analyzer. Cells were stressed using serum glucose starvation. Compared to healthy dogs, PDK4wt/del and PDK4del/del revealed a much lower basal respiration rate (OCR reduction of 46% and 68% of normal, respectively) and decreased metabolic potential to meet energy demands (85% and 72% of normal, respectively). Maximal mitochondrial respiration and spare respiratory capacity were all significantly lower in the affected dogs (PDK4wt/del 42% and 14%, and PDK4del/del 40% and 1.4% of normal OCR, respectively). Our results indicate that the mitochondria of affected dogs have a lower metabolic capacity and thus exhaust the two major energy-producing pathways, aerobic and glycolytic metabolism, at a much faster rate when under stress compared to healthy control dogs. Although we detected a significant decrease in PDK4 protein levels, PDK4 transcription levels have yet to be determined in these samples. Our immediate future goals include 1) quantitative PCR to compare PDK4 transcript levels, 2) PCR and sequencing to identify abnormalities in PDK4 mRNA splicing in affected dogs, 3) assessments of mitochondria ATP production and PDK4 protein kinase function. Our long-term goal is to perform in vitro experiments to assess if mitochondrial abnormalities can be improved with gene therapy. To this end, we have cloned canine healthy PDK4 into an adeno-associated virus construct. Ultimately, our efforts will lead us to a cardiac gene therapy clinical trial for Doberman PDK4-DCM.