No abstract

The 13-lined ground squirrel exhibits remarkable cardiac adaptation during hibernation, periods of torpor being interrupted by repeated inter-bout arousal (IBA). During IBA heart rate rises from 2-4 bpm to as great as 300 bpm. This rise in heart rate occurs over just 5 hours and remains elevated for 12-24 hours before returning to the lower heart rate. With such rapid and marked changes in heart rate, and thus filling time, myocardial stiffness and relaxation must be regulated within a short timeframe. The objective of this work was to establish post-translational modifications (PTMs) in two proteins critical to myocardial stiffness and relaxation; titin and cardiac troponin I (TnI). It was specifically hypothesized that phosphorylation during IBA would be similar to that of summer tissue and distinct from tissue isolated during torpor. Left ventricular tissue (summer, n=9; torpor, n=10; IBA, n=7) was solubilized and separated by either 2-12% gradient SDS-Page (titin) or 12.5% SDS-PAGE (TnI). Total titin phosphorylation was measured via Pro-Q Diamond Phosphoprotein/SYPRO Ruby staining. PKA-specific TnI phosphorylation was quantified via western blotting, using a primary antibody specific to Ser 23,24. While there was a tendency toward decreased total titin phosphorylation in IBA, the difference was not significant. In contrast, there were significant group effects in PKA-specific TnI phosphorylation. Phospho-TnI/Total TnI ratios were lower in torpor when compared to summer (0.22±0.12 vs. 0.68±0.,34, p<0.05). While there was no significant difference between summer and IBA (0.62±0.35), the difference between torpor and IBA failed to reach significance. This data supports the hypothesis that rapid changes in heart rates are associated with changes in cardiac troponin-I phosphorylation, a modification that contributes to rapid rates of relaxation. Further dissection of site specific titin phosphorylation will be required to assess the extent to which PTM modify titin associated stiffness. Results from this work may have significant implications in our understanding of altered compliance in human heart failure.