TnI back-phosphorylation. a Results of autoradiography (top) and western immunoblotting (bottom) in a representative TnI back-phosphorylation assay. ³²P-labeled ATP was used to determine PKA-dependent phosphate incorporation into TnI at the pacing and opposite LV sites in control and HF animals following SDS gel electrophoresis. Signal intensities in the autoradiograms were considered to be directly proportional to the extents of inorganic phosphate (P_i) incorporation into TnI in the presence of PKA (+ signs) in vitro, and inversely proportional to the initial levels of phosphorylation of TnI prior to PKA application in vivo. Signal intensities in the absence of PKA were negligible (− signs). Western immunoblots developed with a TnI-sensitive antibody confirmed comparable protein loads during all tests. b Statistical analysis on the results of back-phosphorylation assays under identical conditions suggested a lower mean level of TnI phosphorylation for the HF pacing site than for the control pacing site (columns show means ± SEM; n number of animals, AU optical density in arbitrary units, *P < 0.05 HF pacing vs. control pacing site). c Correlations between TnI phosphorylation and additional proteins at 20 and 150 kDa. Signal intensities at 20 and 150 kDa from pacing and opposite sites of individual animals were plotted in an X–Y diagram as functions of P_i incorporation into TnI in the respective samples. Lines represent relationships between phosphorylation at 20 or 150 kDa and TnI phosphorylation from linear regression analyses, suggesting positive correlations between these variables (r = 0.71, P < 0.0001; r = 0.74, P < 0.0001, respectively)

TnI phosphorylation in experimental animals. Mean levels of [log(P-TnI/TnI)] intensities with 95% confidence intervals for each site of each animal. Horizontal lines indicate average [log(P-TnI/TnI)] values of control and HF pacing sites. The [log(P-TnI/TnI)] values for the control animals were scattered in a narrower range than those for the HF animals

Immunohistochemistry. a Myocardial tissue samples were labeled with specific antibodies against TnI, the phosphorylated form of TnI (P-TnI) and nuclei with DAPI. Disparate amounts of P-TnI intensities resulted in variable yellow intensities on the merged picture of the HF pacing group. b Capturing images of P-TnI stained slices under a confocal microscope revealed cell-to-cell differences: clusters of cardiomyocytes with divergent levels of TnI phosphorylation could be distinguished in the HF animals at the pacing site

Flow cytometry. a Microscopic images of a representative permeabilized cardiomyocyte (top panel) dual-labeled for TnI (red) and P-TnI (green) for flow cytometric evaluation. Signal intensities were plotted in X–Y diagrams, where the values on the X and Y axes reflected TnI and P-TnI intensity levels, respectively (middle and lower panels, n ≈ 20,000 cardiomyocytes from 6 hearts). The wider scatter of the data obtained from the HF pacing site (bottom panel) than those from the control pacing site (middle panel) suggested more heterogeneous TnI phosphorylation levels in the cardiomyocytes of the HF animals in the paced area. b Distribution of relative TnI phosphorylation in isolated cardiomyocytes. Distribution profiles of logarithmically transformed relative signal intensities [log(P-TnI/TnI)] were expressed for the four groups. (The IQRs between the 25 and 75 percentile values of the distribution histograms are highlighted; n number of animals; *P = 0.0071 HF pacing vs. control pacing site; ⁺P = 0.0093 HF pacing vs. control pacing site; ^#P = 0.0047 HF pacing vs. HF opposite site)

Mean Ca²⁺–force relations and distributions of pCa₅₀ values in isolated cardiomyocytes. a Ca²⁺–force relations of isolated cardiomyocytes obtained from the pacing and opposite sites of the HF animals before and after in vitro PKA treatments. The leftward position of the Ca²⁺–force relations at the HF pacing site before PKA treatment illustrated an increased Ca²⁺-sensitivity of force production in cardiomyocytes of this region relative to all others. After PKA treatment, a rightward shift in the Ca²⁺–force relation was observed. b Small differences between the Ca²⁺–force relationships of the control pacing and control opposite groups could be detected before and after in vitro PKA treatment. c Means of pCa₅₀ values in the four groups of cardiomyocytes before and after in vitro PKA treatment. Mean pCa₅₀ at the HF pacing site was significantly higher than in all other groups under basal conditions, and this difference could be eliminated by PKA (*P < 0.05). d PKA-evoked changes in pCa₅₀ values in the four experimental groups. The effect of PKA on mean pCa₅₀ was the highest at the pacing site of HF animals (*P < 0.05). The Hill coefficient did not differ significantly among the four experimental groups before and after PKA exposures (n_Hill ~2.6). e–h Distributions of pCa₅₀ values in cardiomyocytes from the four experimental groups before and after in vitro PKA treatment. pCa₅₀ values of the HF pacing group were dispersed on a wider range than in all other groups. After PKA treatment, a narrower distribution for the pCa₅₀ values could be measured in HF cardiomyocytes at the pacing site