Figure 3: Inhibition of PKCα affects CARP1 expression and localization and decreases MLP phosphorylation.

(a) Effect of PKC inhibitors (10 μM BI I-HCl; BI or 5 μM calphostin C; Ca) or MAP kinase kinase inhibitor (1 μM U0126; U0) treatment for 24 h on PKCα phosphorylation, PKCα and CARP1 expression levels in NMC compared to untreated controls (−). Dotted line indicates that samples were run on the same gel, but non-consecutive. GAPDH was used as loading control. (b) Immunoblot analysis of total cardiac samples from untreated (−), vehicle treated (DMSO, V) or BI treated adult MLP knockout mice (24 h after vehicle or BI-injection), blotted for phospho-PKCα (Thr638/641), PKCα, CARP1 or CARP2. GAPDH was used as a loading control. Shown are representative blots and quantification of changes for PKCα phosphorylation as well as CARP1 and CARP2 protein levels (c). Sample sizes for untreated controls and BI-treated animals were 4 and 3, respectively. P values are indicated in the figure. (d) BI treatment of NRC for 24 h leads to decreased MLP phosphorylation and protein level (see also Supplementary Fig. 1j,k). SDS samples of protein extracts from vehicle treated (V) or BI-treated NRC were run on conventional SDS PAGE (middle) and on 12% polyacrylamide gels containing 50 μM Phostag reagent (top) and immunoblotted for MLP. Phosphorylated MLP proteins (P1) migrate slower due to their interaction with the Phostag reagent, compared to unphosphorylated protein (P0). Phostag profile plot analysis of band intensities (right panel) revealed presence of three distinct P1 MLP phosphorylation bands (P1-a, P1-b and P1-c), which are putatively caused by several distinct phosphorylation sites in MLP. One of these phosphorylation sites (P1-c) is changed significantly upon BI treatment (red curve) compared to vehicle control (blue curve). Shown are normalized average band intensities and s.e. GAPDH was used as loading control (bottom). (e) Representative immunofluorescence images of adult heart sections from BI and vehicle treated MLP knockout mice stained with antibodies against CARP1 (green), α-actinin (blue) and plakoglobin (red). Scale bar=10 μm. (f) Analysis of fluorescence intensity ratios (I ^ID/_S) of CARP1 (left panel) and CARP2 stainings (right panel) between the ID region and the sarcomere (S) of either BI-treated or vehicle-treated 4-month-old MLP mice (see Supplementary Fig. 1d for methodology). Sample sizes (n in base of the bar; from two hearts per sample) and P values are indicated in the figure; $ denotes low expression of CARP1/CARP2 in BI treated hearts (as shown in Fig. 3b). (g,h) Label free impedance analysis of spontaneously contracting WT and MLP knockout NMC using the RTCA cardio system. Representative images of real-time beating activity of WT and MLP mouse cardiomyocytes (g) and quantification of their beating frequency (h). Effect of BI treatment on MLP cardiomyocyte beating frequencies (h, right panel). Bar graphs depict mean values and s.e. The number of independently measured wells (n in base of bar) and P values are indicated.