Exploring the mechanisms responsible for energetic dysfunction within the type 2 diabetic heart

Cardiovascular disease accounts for 70% of mortality within type 2 diabetic (T2D) patients. Energetic dysfunction is apparent within the T2D heart, presenting as a decreased phosphocreatine/adenosine triphosphate (PCr/ATP) ratio. This pathophysiology is an independent predictor for mortality risk within the setting of heart failure. While most clinical measurements of cardiac PCr/ATP ratios within T2D patients display a reduced PCr/ATP ratio some disagree, showing no defect. Emerging work indicates that this may be due to simultaneous reductions in PCr and ATP masking a decrease in their ratio. Energetic dysfunction can be primarily attributed to impaired mitochondrial respiration but there is no comprehensive explanation for why T2D cardiac mitochondria produce less ATP despite the excess of fatty acid substrates within the T2D heart.
This work began through the characterisation of energetics within the T2D rodent heart via 31P-MRS in perfused hearts. The addition of a phosphorous standard allowed calculation of absolute ATP and PCr concentrations showing that T2D hearts had an 18% reduction in PCr and a 12% reduction in ATP, with no overall decrease in the PCr/ATP ratio. Reduced ATP turnover was displayed through a 61% reduction in the rate of ATP degradation in T2D hearts, which was associated with a 12-24% reduction in respiratory rates in T2D cardiac mitochondrial populations. Two avenues were investigated to explain this reduction in mitochondrial respiratory rates; acute inhibition of the ATP/ADP carrier (AAC) by long chain acyl-CoAs (LCAC), and post-translational modifications (PTMs) of mitochondrial proteins. Targeted mass spectrometry and ex vivo inhibition assays demonstrated, for the first time, that LCAC-mediated inhibition of the AAC, although present, was not of a sufficient magnitude to explain the inhibition of mitochondrial respiration observed under physiological conditions.
There are many PTMs which are dysregulated in T2D, but perhaps the most relevant to mitochondrial function is that of protein acetylation, which was increased by 50% in T2D mitochondria. The effect of this PTM was elucidated through in vitro acetylation of control mitochondria, which demonstrated that protein acetylation depressed respiratory rates. Honokiol, an activator of the mitochondrial deacetylase Sirt3, was employed in vivo to correct mitochondrial hyperacetylation and displayed complete removal of hyperacetylation following a ten-day dosing regimen. This normalisation of the hyperacetylation within T2D restored respiratory function to that of control mitochondria through a 17-28% increase in respiration. This increased the concentration of ATP and PCr in the T2D heart back to control levels, through a 31% and 29% increase, respectively. In addition, honokiol increased the rate of ATP turnover by 88% in T2D hearts, reverting it to the level of control hearts.
This work demonstrated energetic dysfunction in the absence of a decrease in the PCr/ATP ratio within T2D hearts, providing an explanation for a disparity in the field of cardiac energetics. It also provides the most comprehensive analysis of LCAC inhibition of the AAC within T2D to date, presenting strong evidence that this is not physiologically relevant. Instead, mitochondrial protein hyperacetylation was proposed as the prominent cause of energetic dysfunction within the T2D heart and a safe, bioavailable therapeutic agent was characterised which corrected this hyperacetylation. The pathogenicity of hyperacetylation was demonstrated through its amelioration, which completely corrected; mitochondrial respiration, absolute ATP and PCr concentrations, and ATP turnover within the T2D heart. Overall, evidence is presented which demonstrates that mitochondrial hyperacetylation is a novel, but substantial, aspect of T2D cardiac dysfunction therefore suggesting that honokiol may be a powerful therapeutic agent in preventing cardiovascular mortality.

Advisor: heather, L
Tyler, D