Cells were transfected with 40 nM of siRNA using INTERFERin for 24h. Transfection media was then replaced with complete Claycomb media and cells were harvested for analysis 48 h following transfection.

Cardiac metabolic inflexibility is driven by robust up-regulation of pyruvate dehydrogenase kinase 4 (PDK4) and phosphorylation-dependent inhibition of pyruvate dehydrogenase (PDH) within a single day of feeding mice a high fat diet. In the current study, we have discovered that PDK4 is a short-lived protein (t1/2 ~1 h) and is specifically degraded by the mitochondrial protease, Lon. Lon does not rapidly degrade PDK1 and 2 indicating specificity toward the PDK isoform that is a potent modulator of metabolic flexibility. Moreover, PDK4 degradation appears regulated by dissociation from the PDH complex dependent on the respiratory state and energetic substrate availability of mouse heart mitochondria. Finally, we demonstrate that pharmacologic inhibition of PDK4 promotes PDK4 degradation in vitro and in vivo. These findings reveal a novel strategy to manipulate PDH activity by selectively targeting PDK4 content through dissociation and proteolysis.