Pharmacological- and gene therapy-based inhibition of protein kinase Calpha/beta enhances cardiac contractility and attenuates heart failure

Abstract

Background: The conventional protein kinase C (PKC) isoform alpha functions as a proximal regulator of Ca2+ handling in cardiac myocytes. Deletion of PKCalpha in the mouse results in augmented sarcoplasmic reticulum Ca2+ loading, enhanced Ca2+ transients, and augmented contractility, whereas overexpression of PKCalpha in the heart blunts contractility. Mechanistically, PKCalpha directly regulates Ca2+ handling by altering the phosphorylation status of inhibitor-1, which in turn suppresses protein phosphatase-1 activity, thus modulating phospholamban activity and secondarily, the sarcoplasmic reticulum Ca2+ ATPase.

Methods and results: In the present study, we show that short-term inhibition of the conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work-performing heart preparation in wild-type mice but not in PKCalpha-deficient mice. Ro-32-0432 also increased cardiac contractility in 2 different models of heart failure in vivo. Short-term or long-term treatment with Ro-31-8220 in a mouse model of heart failure due to deletion of the muscle lim protein gene significantly augmented cardiac contractility and restored pump function. Moreover, adenovirus-mediated gene therapy with a dominant-negative PKCalpha cDNA rescued heart failure in a rat model of postinfarction cardiomyopathy. PKCalpha was also determined to be the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology.

Conclusions: Pharmacological inhibition of PKCalpha, or the conventional isoforms in general, may serve as a novel therapeutic strategy for enhancing cardiac contractility in certain stages of heart failure.

Figure 1. Functional assessment of Ro-32-0432 and Ro-31-8220 in the heart

(A) Chemical structures of Ro-32-0432 and Ro-31-8220 used in this study. (B) Western blot analysis for phosphoMARCKS protein to analyze the inhibitory ability of Ro-32-0432 (50 nM) and Ro-31-8220 (50 nM) compared with vehicle (Veh) in cultured neonatal rat cardiomyocytes infected with AdPKCα and treated with PMA. (C) Isolated working heart preparation of cardiac contractility (+dP/dt) *P=0.0052 or (D) left ventricular pressure developed following vehicle infusion (N=3) or Ro-32-0432 (N=4) at 8 × 10⁻⁸ µg/ml *P=0.0012. Statistical significance was assessed by two-sample t tests.

Figure 2. PKCα is the primary contractility regulating PKC isozyme in the heart

(A) Isolated working heart preparation from wildtype (N=4) and PKCα−/− (N=4) mice. Contractility (+dP/dt) was continuously measured as increasing concentration of PMA was infused at the indicated dosages (µg/ml) for 5 minutes each. These data were evaluated for significance using a repeated-measure analysis of variance. All measurements are +/−SEM. (B) Isolated working heart preparation in wildtype (N=6) and PKCα−/− (N=6) mice. Hearts were infused with vehicle (N=6) or Ro-32-0432 (N=6, 8 × 10⁻⁸ µg/ml). Significance was assessed by ANOVA (P=0.0002) followed by a Newman-Keuls post test. *P<0.05 versus Wt vehicle.

Figure 3. Western blot analysis of PKC isozyme content in the adult human heart

(A) Western blot panels and (B) quantitation of the indicated PKC isozymes in the human heart normalized against recombinant protein standards for each. Significance was assessed by ANOVA (P<0.0001) followed by Dunnett’s post test. *P<0.01 α versus βII, βI, γ, and ε.

Figure 4. Ro-32-0432 increases cardiac contractility in Wt and failing mice

(A) Assessment of contractility by invasive hemodynamics in a closed-chest preparation in wildtype mice following vehicle (N=5), dobutamine (N=5, 32 µg/kg/min), or Ro-32-0432 infusion (N=5, 22.5 µg/kg/min). *P<0.001 versus vehicle. (B) Invasive hemodynamic assessment in MLP−/− mice following vehicle (N=3), dobutamine (N=3, 32 µg/kg/min), or Ro-32-0432 infusion (N=3, 22.5 µg/kg/min). *P<0.001 versus vehicle. (C) Invasive hemodynamic assessment in Gαq transgenic mice following vehicle (N=4), dobutamine (N=4, 32 µg/kg/min), or Ro-32-0432 infusion (N=4, 22.5 µg/kg/min). *P<0.001 versus vehicle infusion. Statistical significance was assessed by ANOVA (P<0.0001 for A, B, and C) followed by Newman-Keuls post test.

Figure 5. Ro-31-8220 reverses loss of ventricular performance in MLP−/− mice

(A) Assessment of fractional shortening (FS) in Wt and MLP−/− mice by echocardiography before vehicle (N=8 MLP−/− , N=8 Wt) or Ro-31-8220 (N=10 MLP−/−, N=9 Wt) treatment or 6 weeks after daily s.q injections of vehicle or Ro-31-8220 at 6 mg/kg/day. *P<0.05 MLP−/− versus Wt mice before treatment of after vehicle treatment. #P=0.008 MLP−/− after Ro-31-8220 versus MLP−/− before Ro-31-8220 treatment, #P=0.0036 versus MLP−/− before vehicle treatment, or #P=0.009 versus MLP−/− after vehicle treatment. Statistical significance was assessed by paired t tests for comparisons within the same groups and by two-sample t tests between Ro versus vehicle. ANOVA was used for any comparisons among groups (P<0.0001) followed by a Newman-Keuls post test. (B) Fractional shortening in a separate 4-week study in old Wt (N=8) and MLP−/− mice (14 months, N=8) after vehicle (N=8) or Ro-31-8220 treatment (N=8). Statistical significance was assessed by ANOVA (P=0.04) followed by Newman-Keuls post test. *P<0.05 MLP−/− versus Wt mice. #P<0.05 MLP−/− after Ro-31-8220 versus MLP−/− after vehicle treatment. (C) Fractional shortening in a separate 3-day study in MLP−/− mice treated with vehicle (N=6) or Ro-31-8220 (N=6). #P=0.01 MLP−/− after Ro-31-8220 versus MLP−/− after vehicle treatment. Statistical significance was assessed by a two-sample t test.

Figure 6. Dominant negative PKCα gene transfer reverses heart failure in a rat cryoinfarct model

(A) Invasive hemodynamic assessment of cardiac contractility (+dP/dt) and (B) left ventricular end diastolic pressure using a closed-chest preparation in sham (N=8), Adβgal treated (N=12), or AdPKCα-dn treated rats (N=7). Statistical significance was assessed by ANOVA (P=0.0014 in A, P<0.0001 in B) followed by a Newman-Keuls post test. *P<0.05 Adβgal versus sham. #P<0.05 AdPKCα-dn versus Adβgal.