Figure 1.
Figure 1. PKC expression, contractile function, and PKC-targeted cTnI phosphorylation in human myocardium before and after ventricular assist device (VAD) support. (A) Representative Western blots for PKCα, PKCδ, and PKCε expression in F human heart tissue collected pre- versus post-VAD treatment along with a silver-stained (Ag stain) portion of the SDS-PAGE gel to indicate protein loading. “A” indicates pre-VAD while “B” indicates post-VAD samples for the labels shown below the blot. (B) Quantitative analysis of the fold change (Δ) in PKCα, δ, and ε protein expression in F compared with NF myocardial tissue (left panel) and the fold change in the ratio of post-/pre-VAD isoform expression (right panel). Results are expressed as mean± SEM for quantitative data shown in B and D–F. An unpaired Student’s t test was used to compare F versus NF samples and post- versus pre-VAD levels, with *, P < 0.05, considered statistically significant (n = number of hearts in each group for B and F). (C) Representative shortening traces collected from F myocytes before VAD (upper panel), or after VAD (middle panel) implantation compared with NF (lower panel) myocytes. Individual traces are shown at stimulation frequencies of 0.2 (black) and 2.0 (red) Hz to illustrate the negative frequency response in myocytes from F human hearts before VAD (Goldberg et al., 2000) and partial restoration after VAD therapy. (D) Quantitative analysis of contractile function stimulated at 0.2 Hz in myocytes collected from six pre- versus seven post-VAD hearts (n = number of cells and *, P < 0.05 vs. pre-VAD for D and E). (E) Analysis of the percent change (%Δ) in the shortening amplitude, rates of shortening and relengthening, plus TTR50% measured at 2.0 versus 0.2 Hz. (F) Representative Western blots show cTnI phosphorylation at residue p-S44 relative to total cTnI expression in pre- and post-VAD samples (left panel), and the previously established decrease in p-S23/24 levels in F hearts (middle panel; Messer et al., 2007). Quantitative analysis of cTnI p-S44 shows significantly higher levels before VAD therapy compared with NF tissue using a one-way ANOVA and post hoc Tukey statistical comparison (right panel; *, P < 0.05). After VAD support, cTnI p-S44 levels are not significantly different from NF or pre-VAD F levels.

PKC expression, contractile function, and PKC-targeted cTnI phosphorylation in human myocardium before and after ventricular assist device (VAD) support. (A) Representative Western blots for PKCα, PKCδ, and PKCε expression in F human heart tissue collected pre- versus post-VAD treatment along with a silver-stained (Ag stain) portion of the SDS-PAGE gel to indicate protein loading. “A” indicates pre-VAD while “B” indicates post-VAD samples for the labels shown below the blot. (B) Quantitative analysis of the fold change (Δ) in PKCα, δ, and ε protein expression in F compared with NF myocardial tissue (left panel) and the fold change in the ratio of post-/pre-VAD isoform expression (right panel). Results are expressed as mean± SEM for quantitative data shown in B and D–F. An unpaired Student’s t test was used to compare F versus NF samples and post- versus pre-VAD levels, with *, P < 0.05, considered statistically significant (n = number of hearts in each group for B and F). (C) Representative shortening traces collected from F myocytes before VAD (upper panel), or after VAD (middle panel) implantation compared with NF (lower panel) myocytes. Individual traces are shown at stimulation frequencies of 0.2 (black) and 2.0 (red) Hz to illustrate the negative frequency response in myocytes from F human hearts before VAD (Goldberg et al., 2000) and partial restoration after VAD therapy. (D) Quantitative analysis of contractile function stimulated at 0.2 Hz in myocytes collected from six pre- versus seven post-VAD hearts (n = number of cells and *, P < 0.05 vs. pre-VAD for D and E). (E) Analysis of the percent change (%Δ) in the shortening amplitude, rates of shortening and relengthening, plus TTR50% measured at 2.0 versus 0.2 Hz. (F) Representative Western blots show cTnI phosphorylation at residue p-S44 relative to total cTnI expression in pre- and post-VAD samples (left panel), and the previously established decrease in p-S23/24 levels in F hearts (middle panel; Messer et al., 2007). Quantitative analysis of cTnI p-S44 shows significantly higher levels before VAD therapy compared with NF tissue using a one-way ANOVA and post hoc Tukey statistical comparison (right panel; *, P < 0.05). After VAD support, cTnI p-S44 levels are not significantly different from NF or pre-VAD F levels.

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