Abstract
A splice site mutation in the canine pyruvate dehydrogenase kinase 4 (PDK4) gene has been shown to be associated with the development of dilated cardiomyopathy (DCM) in Doberman Pinchers (DPs). Subsequent studies have successfully demonstrated the use of dermal fibroblasts isolated from DPs as models for PDK4 deficiency and have shown activation of the intrinsic (mitochondrial mediated) apoptosis pathway in these cells under starvation conditions. For this study, we sought to further explore the functional consequences of PDK4 deficiency in DP fibroblasts representing PDK4wt/wt, PDK4wt/del, and PDK4del/del genotypes. Our results show that starvation conditions cause increased perinuclear localization of mitochondria and decreased cell proliferation, altered expression levels of pyruvate dehydrogenase phosphatase (PDP) and pyruvate dehydrogenase (PDH), dramatically increased PDH activity, and an impaired response to mitochondrial stress in affected cells. In sum, these results show the broad impact of PDK4 deficiency and reveal mechanistic pathways used by these cells in an attempt to compensate for the condition. Our data help to elucidate the mechanisms at play in this extremely prevalent DP disorder and provide further support demonstrating the general importance of metabolic flexibility in cell health.
Highlights
Dilated Cardiomyopathy (DCM) is one of the most prevalent causes of heart disease in humans and canines
To evaluate pyruvate dehydrogenase kinase 4 (PDK4) deficiency in Doberman Pinchers (DPs) fibroblasts, cells were evaluated for cell morphology and mitochondrial localization within cells, PDK expression levels, pyruvate dehydrogenase (PDH) activity and abundance, and response to mitochondrial stress based upon oxygen consumption analyses
Immunofluorescence staining of f-actin in fixed cells revealed that PDK4wt/wt fibroblasts exhibit no significant changes in morphology after 24 hours of starvation (Fig. 1A,D,G)
Summary
Dilated Cardiomyopathy (DCM) is one of the most prevalent causes of heart disease in humans and canines. Previous studies have shown that PDK4 can be upregulated by lipids, for example, those derived from decanoic acid and that PDK4 expression can be regulated by both the PPARβ/δ and TGFβ signaling pathways[26,27,28]. These studies demonstrate that fibroblasts are excellent models for the assessment of PDK4 function. Through phosphorylation of PDH, PDK4 serves as an important regulator of mitochondrial fuel usage by switching away from glucose oxidation under conditions of low glucose availability To better understand this disorder at the molecular level, the present study evaluated healthy and deficient cells in their response to glucose starvation. We have helped to identify the broader mechanistic consequences and compensatory pathways at play in PDK4 deficiency
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