Comparison of PKC expression and localization, contractile function, and cTnI phosphorylation in hearts from PO- versus sham-treated rats. (A) Representative Western blots for PKCα (upper left panel) and phosphorylated PKCα T638 (PKCα p-T638; lower left panel) in PO compared with sham rat hearts. A silver-stained (Ag-stain) portion of the gel also is shown as a loading control. The quantitative analyses of PKCα and PKCα p-T638 are shown in the upper and lower right panels, respectively. There were increases in the ratio of PKCα p-S657/total PKCα in two samples from PO (1.42, 2.64) when normalized to the sham control ratio (1.00, 1.00). Quantitative data presented in A and D–H are expressed as mean ± SEM. The n values in A and F–H equal the number of rat samples. Statistical analysis in A, D, F, and G used an unpaired Student’s t test (*, P < 0.05). (B) Fluorescence images collected with comparable exposure times after immunostaining for PKCα show the redistribution of PKCα in two sham (panels 1, 2) compared with two PO (panels 3, 4) rat hearts. Scale bars, 10 µm. (C) Representative Westerns show PKCδ and PKCε expression in PO compared with sham-treated rat hearts. Actin immunostaining is shown as a loading control for each lane. (D) Composite shortening traces collected from sham (black) and PO (red) myocytes at 0.2 Hz (upper panel) and the quantitative analysis of traces showed PO produced a significant reduction in shortening amplitude versus sham myocytes (lower panel; *, P < 0.05; n = number of myocytes in D and E). No significant changes were detected in resting sarcomere length (SL) or the rates of shortening and relengthening (data not shown). (E) The percent change (%Δ) in resting SL, peak shortening amplitude, and the rates of shortening and relengthening at 0.5, 1, and 2 Hz were compared with the response at 0.2 Hz in sham and PO myocytes. Results are analyzed with a two-way ANOVA and Tukey’s post hoc tests, with significance set to P < 0.05 (*, versus 0.2 Hz response; ♦, versus sham at the same frequency)_. PO and stimulation frequency each caused significant effects in the peak amplitude, shortening, and relengthening rates, and there were interaction effects for peak amplitude and relengthening rate. Overall, shortening amplitude and relengthening rate are significantly more impaired at higher frequencies in PO compared with sham myocytes. (F) Representative cTnI p-S45 and cTnI Western blots (left panels) and the quantitative analysis of expression (middle, right panels) show that PO increases cTnI p-S45 without a change in total cTnI compared with sham rat myocytes. The cTnI expression is normalized to a silver-stained band in each gel. *, P < 0.05 versus sham.(G) Representative Western blot for cTnI p-T144 and an silver-stained portion of the gel, which is used as a loading control (left panels) and quantitative analysis of cTnI p-T144/silver (Ag) ratio (right panel) show there are no significant differences in cTnI p-T144 levels for PO compared with sham rat myocytes. (H) Representative Western blots for cTnI p-S23/24 and a silver-stained portion of the gel (left panels) and quantitative analysis of cTnI p-S23/24 levels (right panel) in sham and PO rat myocytes under basal conditions and in response to 1, 10, and 100 nM of the β-adrenergic receptor agonist ISO, and/or the β-antagonist, propranolol (PRO; 10 µM). Results are analyzed with a two-way ANOVA and post hoc Tukey’s test. The quantitative analysis shows that 10 nM ISO produced a significant increase in p-S23/24 above basal levels in sham myocytes (*, P < 0.05 versus basal sham), but not PO myocytes (P > 0.05 versus basal PO), and this ISO response is reduced in PO compared with sham myocytes (♦, P < 0.05 versus sham+ISO).