Phosphorylation of MYBP-C.

<p>(A) Pro-Q Diamond phosphoprotein staining (Top row) and Deep Purple total protein staining (Bottom row) showing MYBP-C in perfused and ischemic homogenates. The graph represents the phosphoprotein signal divided by the total protein signal. (B) Analyses of MYBP-C phosphorylation in perfused and ischemic (C) fibers with and without 8-Br-cAMP treatment. Data presented are average ± S.E.M. * <i>P</i><0.05 compared to the perfused fibers.</p

Force-Ca²⁺ relationships of ischemia – reperfused fibers.

<p>(A) Force per cross-sectional area versus Ca²⁺ concentration for perfused (•), ischemic (▵) and ischemia-reperfused (□) fibers versus control conditions. (B) Absolute force per cross-sectional area versus Ca²⁺concentration for perfused, ischemic and ischemia – reperfused fibers in the presence of 5 mM added Pi. Data plotted are average ± S.E.M., and the averaged data are fit by the Hill equation for illustrative purposes. Accumulated data from all fibers are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009528#pone-0009528-t001" target="_blank">Table 1</a>.</p

Phosphorylation status of MLC-1 and MLC-2 in perfused and ischemic fibers.

<p>Data presented are average ± S.E.M.</p

Troponin T phosphorylation in perfused and ischemic fibers.

<p>(A) Two-dimensional SDS-PAGE with 3–5.6 NL IPG strips was used to resolve TnT into its phosphoforms. Three representative perfused and ischemic samples are shown with the two predominant phosphoforms labeled P1 (monophosphorylated) and P2 (diphosphorylated). (B) Following densitometric analysis, the percentages of monophosphorylated and diphosphorylated TnT, as a percentage of total TnT phosphoform expression (P1 + P2), are plotted for perfused and ischemic fibers. * <i>P</i><0.05 compared to the corresponding perfused phosphoform.</p

Phosphorylation of TnI at Ser23/24.

<p>(A) Multiplex Western blotting to determine the extent of Ser23/24 phosphorylation of TnI in five perfused and five ischemic samples. Resolved samples were simultaneously labeled using a mouse α-TnI monoclonal antibody (Top row; Cy3 labeled secondary antibody detection) and rabbit phospho-Ser23/24 TnI polyclonal antibody (Bottom row; Cy5 labeled secondary antibody detection). Graph represents densitometry analysis of the Ser23/24 phosphorylated TnI signal divided by the total TnI signal. Fibers from a perfused (B) or ischemic (C) rat heart were left untreated or treated with 8-Br-cAMP, followed by multiplex Western blotting for total and Ser23/24 phosphorylated TnI. Data presented are average ± S.E.M. * <i>P</i><0.05 compared to the respective perfused or control condition.</p

Force and rate constant of force development following 8-Br-cAMP treatment.

<p>(A) The rate constant of force redevelopment at various [Pi] for perfused (circles) and ischemic (triangles) fibers, with (open symbols) and without (closed symbols) 8-Br-cAMP treatment. * <i>P</i><0.05 compared to the control perfused condition. (B) The change in the force per cross-sectional area for perfused and ischemic fibers treated with 8-Br-cAMP. Data presented are average ± S.E.M. * <i>P</i><0.05 compared to untreated ischemic fibers.</p

Activation of fiber contraction by flash photolysis of NPE-ATP.

<p>(A) Typical force transients from a representative perfused and ischemic fiber activated from Ca²⁺-rigor by photoliberation of ATP from NPE-ATP. Single exponential fits to the data traces are shown as dotted lines along with the rate constant of force activation. (B) The rate constant of force activation for perfused (n = 13) and ischemic (n = 12) fibers activated by flash photolysis of NPE-ATP. Data presented are average ± S.E.M.</p

Force redevelopment following length release.

<p>(A) Representative traces of force after length release for a perfused fiber activated by pCa4.5 solution including varying concentrations of added Pi. The force recovery transients were analyzed by single exponential fits (dotted lines) to determine the rate constant of force redevelopment. (B) The rate constant of force redevelopment for perfused (•; n = 7), ischemic (▵; n = 10) and ischemia-reperfused (□; n = 7) fibers. Data presented are average ± S.E.M. * <i>P</i><0.016 compared to perfused.</p

The concentrations of free Ca²⁺ required for half-maximal force (EC₅₀) and the maximal Ca²⁺-activated force per cross-sectional area (Fmax) for three surgery groups under varying experimental conditions.

<p>Data are presented as average ± S.E.M.</p><p>*: denotes <i>P</i><0.016 compared to perfused fibers under identical experimental conditions.</p><p>†: denotes <i>P</i><0.016 compared to perfused and reperfused fibers under identical experimental conditions.</p><p>‡: denotes <i>P</i><0.016 compared to perfused and ischemic fibers under identical experimental conditions.</p

Activation of fiber contraction by Ca²⁺.

<p>(A) Typical force transients from representative perfused, ischemic and ischemia-reperfused fibers activated by Ca²⁺ release following flash photolysis of NP-EGTA. Single exponential fits to the data traces are shown as dotted lines, along with the rate constant of force activation. (B) The rate constant of force activation for perfused (n = 11), ischemic (n = 10) and ischemia – reperfused (n = 9) fibers activated by flash photolysis of NP-EGTA. Data are presented as average ± S.E.M. * <i>P</i><0.016 compared to perfused fibers.</p