PKCβII inhibition attenuates myocardial infarction induced heart failure and is associated with a reduction of fibrosis and pro-inflammatory responses

Abstract

Protein kinase C βII (PKCβII) levels increase in the myocardium of patients with end-stage heart failure (HF). Also targeted overexpression of PKCβII in the myocardium of mice leads to dilated cardiomyopathy associated with inflammation, fibrosis and myocardial dysfunction. These reports suggest a deleterious role of PKCβII in HF development. Using a post-myocardial infarction (MI) model of HF in rats, we determined the benefit of chronic inhibition of PKCβII on the progression of HF over a period of 6 weeks after the onset of symptoms and the cellular basis for these effects. Four weeks after MI, rats with HF signs that were treated for 6 weeks with the PKCβII selective inhibitor (βIIV5-3 conjugated to TAT(47-57) carrier peptide) (3 mg/kg/day) showed improved fractional shortening (from 21% to 35%) compared to control (TAT(47-57) carrier peptide alone). Formalin-fixed mid-ventricle tissue sections stained with picrosirius red, haematoxylin and eosin and toluidine blue dyes exhibited a 150% decrease in collagen deposition, a two-fold decrease in inflammation and a 30% reduction in mast cell degranulation, respectively, in rat hearts treated with the selective PKCβII inhibitor. Further, a 90% decrease in active TGFβ1 and a significant reduction in SMAD2/3 phosphorylation indicated that the selective inhibition of PKCβII attenuates cardiac remodelling mediated by the TGF-SMAD signalling pathway. Therefore, sustained selective inhibition of PKCβII in a post-MI HF rat model improves cardiac function and is associated with inhibition of pathological myocardial remodelling.

Fig 1

(A) Heart failure (HF) induction and treatment protocol. Twelve-week-old rats were subjected to myocardial infarction (MI) by left anterior descending coronary artery ligation. Four weeks after MI induction, the rats were treated with the PKCβII-specific inhibitor, βIIV5-3, or with control (TAT-carrier peptide). Peptide treatment was continuous (for 6 weeks) using subcutaneous Alzet pump delivery at 3/mg/kg/day. (B) Representative blots showing the levels of cardiac PKCβI and βII in the total and particulate fraction (triton soluble) from sham-operated normal rats or post-MI HF rats treated with TAT or βIIV5-3. Note an increase in PKCβII but not PKCβI in the particulate fraction of failed hearts and a selective loss of PKCβII increase in this fraction from rats treated with βIIV5-3. Association of PKC with the particulate fraction is a measure of PKC activation [36]. The level of cellular PKC distribution between the total and particulate fractions was normalized using GAPDH and Gαo, respectively. (C) Fractional shortening was measured and plotted as graphs before (at week 16) and after treatment (at week 22), n = 6 per group. (D) Cardiac morphology using haematoxylin and eosin stained cardiac slices. (E) Representative photomicrographs of each group (as labelled) depicting cardiomyocyte hypertrophy (increase in cell size). (F) Quantitative analyses of cardiomyocyte width, n = 6 per group. *P < 0.05 sham; ^§P < 0.05 versus post-MI HF treated with βIIV5-3. Data were analysed by the anova with a post hoc testing by Bonferroni.

Fig 2

(A) Collagen deposition was found in both remote and peri-infarcted areas of post-myocardial infarction (MI) hearts as measured by picrosirius staining on week 22, 10 weeks after the myocardial infarction and 6 weeks after peptide treatment. (B) Quantification of cardiac infarcted area in post-MI heart failure (HF) rats treated with TAT or βIIV5-3. Note that no differences were observed between groups. (C–E) Representative photomicrographs (400×) from sham operated age-matched rats, post-MI HF rats treated with TAT or with βIIV5-3 showing collagen deposition in the cardiac remote area (red staining). (F) Quantitative analyses of collagen deposition in the cardiac remote area, n = 6 per group; *P < 0.05 sham; ^§P < 0.05 versus post-MI HF with βIIV5-3. Student’s t-test (one-tailed distribution/two-sample equal variance) was used to compare the effect of PKCβII on infarcted area. Cardiac collagen deposition was analysed by the anova with a post hoc testing by Bonferroni.

Fig 3

(A–C) Phospho SMAD2/3 immunostaining of hearts from sham-operated rats, or from post-myocardial infarction (MI) heart failure (HF) rats treated with TAT or βIIV5-3. A difference in immunostaining (brown) is noted in the peri-vascular region (indicated by box). n = 3 per group. At least 10 high power fields were counted from each slide. (D) Cardiac active and latent TGFβ1 levels were determined by immunoblot. Data are presented as percentage of control (sham) group, n = 6 per group. *P < 0.05 sham; ^§P < 0.05 versus post-MI HF with βIIV5-3. Data were analysed by the anova with a post hoc testing by Bonferroni.

Fig 4

(A–C) Infiltration of inflammatory cells in the epicardial region is shown by haematoxylin and eosin stained myocardial sections of post-myocardial infarction heart failure rats treated with TAT and βIIV5-3_, as well as sham-operated normal rats. 100×. Respective representative photomicrographs are presented, n = 3–5 per group. (A1–C1) Higher magnification of these photomicrographs is shown in the lower panels. 400×. Infiltrated inflammatory cells in the epicardial region are indicated by asterisks. The haematoxylin and eosin was used to identify and to quantify the infiltration of polymorphonuclear cells, based upon the localization of the cell and morphology of the nucleus of the cell.

Fig 5

(A–C) Toluidine blue-positive mast cells are shown (as indicated by black arrowheads) in the endocardium (upper panels) of sham and post-myocardial infarction (MI) rats with heart failure treated with TAT or βIIV5-3. (D) Quantification of total mast cell (MC) numbers including both degranulating and non-degranulating mast cells, n = 3 per group. (E–G) Mast cell degranulation is greater in the epicardial region (lower panels) of post-MI heart failure (HF) rats with TAT treatment as compared with sham-operated rats and post-MI HF rats treated with βIIV5-3. Non-degranulated mast cells, partially degranulated mast cells and completely degranulated mast cells are indicated by black arrowheads, black and white arrows, respectively. The completely degranulated emptied mast cells have only outer cell membrane staining (white arrows). (H) Quantification of degranulated mast cells is presented as percent of total mast cell number, n = 3 per group. *P < 0.05 and **P < 0.01 versus sham; ^#P < 0.05 versus post-MI HF with TAT. Data were analysed by the anova with a post hoc testing by Bonferroni.