Agonist activated PKCβII translocation and modulation of cardiac myocyte contractile function

Abstract

Elevated protein kinase C βII (PKCβII) expression develops during heart failure and yet the role of this isoform in modulating contractile function remains controversial. The present study examines the impact of agonist-induced PKCβII activation on contractile function in adult cardiac myocytes. Diminished contractile function develops in response to low dose phenylephrine (PHE, 100 nM) in controls, while function is preserved in response to PHE in PKCβII-expressing myocytes. PHE also caused PKCβII translocation and a punctate distribution pattern in myocytes expressing this isoform. The preserved contractile function and translocation responses to PHE are blocked by the inhibitor, LY379196 (30 nM) in PKCβII-expressing myocytes. Further analysis showed downstream protein kinase D (PKD) phosphorylation and phosphatase activation are associated with the LY379196-sensitive contractile response. PHE also triggered a complex pattern of end-target phosphorylation in PKCβII-expressing myocytes. These patterns are consistent with bifurcated activation of downstream signaling activity by PKCβII.

Figure 1. Adult cardiac myocyte PKCβ_II and PKCβDN expression and contractile function in response to phenylephrine (PHE; 100 nM) or PHE plus the PKCβ inhibitor, LY379196 (LY, 30 nM).

(A). Representative Western blot of PKCβ and PKCβDN expression 2 days after gene transfer compared to non-treated controls. Protein expression is shown under basal conditions (left), in the presence of 100 nM PHE (10 min, middle) and PHE plus LY (10 min, right). (B). Composite shortening traces collected under basal conditions and then 5 and 15 min after the addition of PHE in the absence (left panels) and presence (right panels) of the PKCβ inhibitor, LY. The PHE-induced decrease in shortening amplitude observed in controls (upper left panel; n = 19) and PKCβDN-expressing myocytes (lower left panel; n = 13) is absent in PKCβ_II-expressing (middle left panel, n = 16) myocytes. In PKCβ_II-expressing myocytes (middle right panel; n = 24), the addition of LY379196 with PHE returns the response to the control pattern observed with PHE. LY does not change the PHE-induced shortening response in control (upper right panel; n = 28) or PKCβDN-expressing myocytes (lower right panel; n = 13). Quantitative analysis of contractile function measured before and after PHE or PHE+LY treatment is summarized in Figure 2.

Figure 2. Quantitative analysis of contractile function under basal conditions and in response to PHE or PHE+LY in control, PKCβ_II-, and PKCβDN-expressing myocytes.

(A). Analysis of basal function in the control (n = 46), PKCβ_II-(n = 40), and PKCβDN- (n = 26) expressing myocytes used for the subsequent analysis of PHE and PHE+LY responses (panels B–D). Basal values and the PKCβ_II-induced decreases in shortening and re-lengthening are comparable to values reported in earlier work (16). The response to PHE and PHE+LY is expressed as a percent change (% Δ) from basal values in the remaining panels (B–E). PHE-induced changes in myocyte shortening and re-lengthening were analyzed 1 (B) and 15 (C) min (Control n = 18; PKCβ_II n = 16; PKCβDN n = 13) after PHE, and 15 min after addition of PHE plus LY (D; Control n = 28; PKCβ_II n = 24; PKCβDN n = 13) 2 days after gene transfer. The response to PHE 3 days after gene transfer is shown in (E) (Control n = 15; PKCβ_II n = 16) to demonstrate the consistency of this response in myocytes expressing PKCβ_II relative to controls. Differences in function are identified using a one-way ANOVA and Newman-Keuls post-hoc tests, with p < 0.05 (*) considered significantly different from control values in the present figure and in Figure 3.

Figure 3. Quantitative analysis of basal contractile function before 1 μM PHE or 50 nM phorbol 12,13 myristic acid (PMA) (A; Control n = 39, PKCβ_II n = 35), and in response to 1 (B, D) and 20 (C, E) min of 1 μM PHE (B, C; Control n = 12; PKCβ_II n = 10) or 50 nM PMA (D, E; Control n = 27; PKCβ_II n = 25) in control and PKCβ_II-expressing myocytes.

Agonist-induced decreases in shortening amplitude, and shortening and re-lengthening rates were not different in control and PKCβ_II-expressing myocytes in response to 1 μM PHE (B, C). Comparable decreases in the rates and amplitude of shortening and re-lengthening also were observed in control and PKCβ_II-expressing myocytes after 1 (D) and 20 (E) min of 50 nM PMA.

Figure 4. Western and immunohistochemical analysis of PKCβ phosphorylation, localization and translocation in response to PHE.

(A). Representative classical PKC isoform phosphorylation in response to 0.1–10 μM PHE in control and PKCβ_II-expressing myocytes. The PHE-induced increases in phospho-PKC in control myocytes (left side), which do not express detectable PKCβ_II protein are observed because the phospho-antibody also detects PKCα phosphorylation. Raw blots for this panel are available in supplemental Figure 2. (B). Confocal projection image of PKCβ localization in response to 10 min of PHE (100 nM). PKCβ_II-expressing (upper panel) and control (lower panel) myocytes were immunostained to detect PKCβ with FITC (left panels) and α-actinin with Texas Red (middle panels). Merged images in the far right panels show a similar striated distribution of PKCβ and α-actinin in myocytes expressing PKCβ_II (bars = 10 μm). The punctate distribution of PKCβ_II in response to PHE also overlapped with NCX (results not shown), which is expressed in the t-tubules. (C). Fluorescence image showing PKCβ_II localization in response to 100 nM PHE plus 30 nM LY maintained the perinuclear distribution observed under basal conditions (see; scale bar = 5 μm). (D). Representative fractionation (upper panel) and quantitative analysis (lower panel) of PKCβ_II distribution measured under basal conditions and in response to PHE or PHE+LY after fractionation. In these experiments, PKCβ_II is re-distributed from the cytosol to the myofilament fraction in response to low dose PHE, and this shift is blocked by LY. Results in the lower panel are expressed as mean±SEM (n = 7) and analyzed by one-way ANOVA and post-hoc Newman-Keuls comparisons, with significance set at p < 0.05 (*).

Figure 5. CaMKIIδ phosphorylation (A) and PKD expression and phosphorylation (B, C) in adult rat myocytes treated with PHE.

(A). Representative Western blot showing CaMKIIδ phosphorylation (pCaMKIIδ) under basal conditions and in response to 100 nM PHE (10 min) in the presence and absence of the phosphatase inhibitor, calyculin A (10 nM, calA). Quantitative analysis of basal and PHE-related CaMKIIδ phosphorylation in the absence of calA is shown in the right panel (Basal n = 8/group; PHE n = 3/group). Statistical comparisons were carried out using a 1-way ANOVA and post-hoc Newman-Keuls tests, with (*) p < 0.05 considered significantly different from control (right panel). This blot also shows the same lanes after membranes were re-probed for actin and the gels were silver (Ag)-stained to demonstrate protein loading in each lane. (B). Representative Western blot of phosphorylated PKD (pPKD), PKD and a silver- (Ag) stained portion of the same gel under basal conditions and in response to PHE (10 min, 100 nM PHE). A 2-way ANOVA and Newman-Keuls post-hoc tests are used to analyze the quantitative results shown in the right panel. A p < 0.05 is considered significantly different from control for comparisons among PKC groups (*) and for comparison to basal for groups treated with PHE (**; n = 5–6/group). There are no significant interaction effects. (C). Representative Western blot showing pPKD, PKD, and an Ag-stained portion of the gel in response to LY (30 nM) and PHE plus LY (10 min). The elevated pPKD observed under basal conditions continues to be observed during the PHE+LY response based on one-way ANOVA and Newman-Keuls post-hoc tests, with p < 0.05 (*) considered significantly different (n = 4/group). (D). Representative Western blot showing detection of pPKD, PKD and silver stained portion of the gel in response to PHE plus PKI, LY, and PHE + LY in the presence of calA. Quantitative analysis of the PHE + LY response in the presence of calA is shown in the right panel (n = 6/group) and compared using a 1-way ANOVA (p > 0.05). Raw blots and gel images for panels B and D are available in Supplemental Figure 2.

Figure 6. Phosphorimage analysis (A) and Western blot analysis of cTnI Ser23/24 phosphorylation (pSer23/24) relative to total cTnI expression in response to PHE and PHE+LY (B–E).

(A). Representative phosphorimage showing ³²P protein incorporation under basal conditions, and in response to LY (30 nM), PHE (100 nM) or PHE+LY for control and PKC-β_II expressing myocytes 2 days after gene transfer. Known phosphorylatable proteins are shown on the left side and molecular weight markers on the right. Proteins are separated with 12% SDS-PAGE prior to determining phosphate incorporation with a BioRad Phosphorimager. (B). Representative Western blot of cTnI pSer23/24 phosphorylation relative to cTnI expression in control, PKCβ- and PKCβDN-expressing myocytes under basal conditions and in response to 10 min of PHE (100 nM; 37°C). Experiments were carried out in the absence of calA. These results demonstrate PHE resulted in little change in cTnI pSer23/24 phosphorylation in the absence of phosphatase inhibitor. (C). Representative Western blot of cTnI pSer23/24 phosphorylation and cTnI in control, PKCβ- and PKCβDN-expressing myocytes under basal conditions and in response to 10 min of PHE (100 nM; 37°C) or PHE+LY in the presence of calA. Raw blots and gel images for this panel are available in Supplemental Figure 2. (D). Representative Western blot showing cTnI pSer23/24 and cTnI in controls and myocytes expressing PKCβ_II and PKCβDN treated with PHE and LY for 10 min in the absence (left) and presence (right) of calA. (E). Quantitative analysis of cTnI pSer23/24 phosphorylation relative to cTnI expression in response to PHE or PHE plus LY in the presence of calA. A 1-way ANOVA and post-hoc Newman-Keuls tests showed significant differences (* p < 0.05) when comparing phosphorylation in myocytes expressing PKCβ_II- compared to PKCβDN-expressing or control myocytes during the PHE (left panel; n = 3/group) and PHE+LY (right panel; n = 7/group) responses. The relative increase in cTnI pSer23/24 with PHE treatment detected in myocytes expressing PKCβ_II versus controls is comparable to the enhanced phosphorylation detected in the presence of calA under basal conditions. (F). Representative Western analysis of cTnI pSer23/24 relative to a silver-stained portion of the gel in response to PMA (5–500 nM) in the absence and presence of LY. Phosphorylation of cTnI Ser23/24 is comparable in control and PKCβ_II-expressing myocytes.

Figure 7. Western blot (A) and quantitative (B) analyses of cardiac myosin binding protein C (cMyBP-C) phosphorylation in response to PHE (100 nM) in the presence and absence of LY.

(A). Representative Western blots showing cMyBP-C phosphorylation at residues Ser302 (pSer302; left panel) and Ser282 (pSer282; right panel) relative to total cMyBP-C expression and a Ag-stained portion of each gel under basal, PHE-activated (100 nM; 10 min) and PHE+LY (10 min) treatments in controls, and myocytes expressing PKCβ_II and PKCβDN. Solid lines within each blot indicate a separation of samples on the same blot. Raw blot and gel images for basal and PHE treatments in this panel are available in Supplemental Figure 3. Phosphorylated Ser273 is not detected in these experiments (results not shown) and calA is not present in these experiments and in the quantitative analysis shown in panel B. (B). Quantitative analysis of pSer282 and pSer302 levels relative to total cMyBP-C detected with the pan Ab in response to PHE (left panel; n = 4/group) and PHE+LY (right panel; n = 4/group). A 1-way ANOVA and post-hoc Newman-Keuls tests (* p < 0.05) showed pSer302 phosphorylation is significantly elevated in PKCβ_II-expressing myocytes compared to values in control and PKCβDN-expressing myocytes.

Figure 8. Western blot and quantitative analyses of phospholamban (PLB) Ser16 and Thr17 phosphorylation in response to PHE in the presence and absence of LY.

(A). Representative Western detection of pSer16-PLB (left panels) and pThr17-PLB (right panels) relative to total PLB under basal conditions and in response to PHE (100 nM, 10 min) or PHE plus calA in controls and myocytes expressing PKCβ_II or PKCβDN. Raw blot and gel images for PHE and PHE+CalA are available in Supplemental Figure 3. (B). Quantitative analyses of pSer16-PLB levels relative to total PLB in response to PHE and PHE+LY) in the absence (PHE n = 4–5/group; PHE+LY n = 3/group) and presence of calA (PHE n = 5/group; PHE+LY n = 4/group). Myocytes expressing PKCβ_II are compared to PKCβDN and controls using a 2-way ANOVA and post-hoc Newman-Keuls tests, with p < 0.05 (*, **) considered significant. Statistical comparisons were performed among the PKC group (*; control, PKCβ_II and PKCβDN) and among treatment groups (PHE vs PHE+LY; **) in the absence (left panel) and presence (right panel) of calA. (C). Representative pSer16-PLB and pThr17-PLB levels in response to PHE plus LY relative to actin. Experiments were performed with and without calA in controls and myocytes expressing PKCβ_II and PKCβDN. Quantitative results and the statistical comparisons are shown in panel B.