Abstract
The clinical effects of myocardial ischemia/reperfusion injury (I/R) are typically significantly more adverse in patients with Type 2 Diabetes Mellitus (T2DM). We hypothesize that in T2DM hearts there are underlying molecular adaptations that, even in the absence of reduced functional output, can impact recovery if exposed to I/R. Given the likely rapid molecular response to I/R, we examined how T2DM alters basal myocardial protein phosphorylation to identify pathways associated with such adaptation. We employed a rat model that combined peripheral insulin resistance from energetic overload (high fat diet) and pancreatic insufficiency (streptozotocin), producing elevated blood glucose, serum triglycerides and reduced insulin sensitivity, consistent with a T2DM phenotype. Hearts were subjected to 60 mins ex vivo perfusion followed by protein extraction, trypsin digest, phosphopeptide enrichment and quantitative mass spectrometry. We identified abundance changes in >5400 phosphopeptides despite no evidence of functional perturbation in LV pressure or heart rate between control and T2DM hearts. Differentially abundant phosphosites were mapped to signal pathways. This showed Akt contributed to 36% of pathways with reduced phosphorylation in T2DM hearts. Reduced phosphorylation of Akt at S124/S129 was consistent with the global pathway data (log2 ratio: -1.10). Given that S129 is a casein kinase 2 (CK2) target, we next examined CK2 and CK2 signaling. We observed significantly reduced CK2 abundance (CK2α: 0.41±0.08 and CK2β: 0.54±0.09; n -fold±SEM) and reduced phosphorylation of CK2 targets (CK2 substrate pS/pT-D/E: 0.84±0.01; n -fold±SEM) by Western blot. This was further confirmed by the over-representation of CK2 target sequences (pS/pT-D/E) in the negatively regulated phosphopeptide set (D, 10.45-fold; E 6.82-fold). Reduced phosphorylation at CK2 regulated sites on catenin β1, caspase 9 and eukaryotic translation initiation factor 3, suggests T2DM elicits a pro-apoptotic environment. Increased cleavage of caspases 3 and 9 (2.15±0.28 and 1.32±0.08; n fold±SEM, respectively) confirmed these data. In summary, changes in CK2 signaling may ‘prime’ a pro-apoptotic environment in T2DM hearts that subsequently limits recovery in I/R.
Published Version
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