Protein Kinase C Isoform Expression in Normal and Failing Rabbit Hearts

Abstract

Protein kinase C (PKC) is activated by alpha-adrenergic stimulation. Molecular analysis showed that PKC consists of a family of at least 12 isozymes. Studies of their distribution in the heart showed conflicting results. The first goal of our study was thus to characterize cardiac PKC in normal rabbits. PKC plays an important role in gene expression, cell growth, and differentiation and is involved in the hypertrophy phase of cardiac overload, but since its expression has never been evaluated in heart failure, the second goal of our study was to evaluate PKC activity and isoform expression in rabbits with heart failure induced by a double hemodynamic overload (aortic insufficiency followed by an aortic stenosis). In the first part of the study, PKC isoform expression analyzed in normal rabbits by immunoblotting showed that isoforms alpha, beta, epsilon, and zeta were expressed along with PKC gamma, which had never been detected in the heart. PKC gamma expression was also identified by polymerase chain reaction, and immunofluorescence techniques showed a localization on intercalated disks associated with the membrane localization observed with the other isoforms. In the second part of the study, PKC activity, content, and isoform expression showed a decrease of 37% in the failing group. PKC immunodetection with a monoclonal antibody (Mab 1.9) recognizing the catalytic domain of all PKC isoforms revealed a 20% decrease in the failing ventricles compared with normal left ventricles. Expressed PKC isoforms quantified by Western blot showed, in the failing heart group compared with the control group, a decrease of 27%, 32%, 16%, and 9% of PKC alpha, PKC beta 1, PKC gamma, and PKC epsilon, respectively, whereas PKC zeta was not significantly modified. These results show that, in heart failure, PKC activity and expression of Ca(2+)-dependent PKC isoforms are decreased. This may lead to alterations of PKC-induced phosphorylations.

Differential Induction of Protein Kinase C Isoforms at the Cardiac Hypertrophy Stage and Congestive Heart Failure Stage in Dahl Salt-Sensitive Rats

“…PKCε increased significantly in spontaneously hypertensive rats (SHR), which exhibit genetic hypertension similar to that of Dahl salt-sensitive rats (37). In congestive heart failure, PKCα, β, γ, and ε decreased and PKCζ was not significantly modified when rabbits underwent heart failure induced by a double hemodynamic overload (aortic insufficiency followed by an aortic stenosis) (38). PKCα and β increased and PKCε was not significantly changed in the failing human heart due to dilated and ischemic cardiomyopathy (15).…”

Role of Protein Kinase C in the Signal Pathways That Link Na+/K+-ATPase to ERK1/2

“…PKC activities were determined by slight modifications of previously described procedures (13)(14)(15)(16). Briefly, the cytosolic and the particulate fractions (above) were partially purified by passage through DE-52 cellulose columns (15,16) and assayed either for Ca 2ϩ -stimulated PKC activity using histone H 1 as substrate (histone kinase) or for Ca 2ϩ -independent PKC activity using the synthetic substrate of PKC ⑀ , Epep (Epep kinase).…”

“…Briefly, the cytosolic and the particulate fractions (above) were partially purified by passage through DE-52 cellulose columns (15,16) and assayed either for Ca 2ϩ -stimulated PKC activity using histone H 1 as substrate (histone kinase) or for Ca 2ϩ -independent PKC activity using the synthetic substrate of PKC ⑀ , Epep (Epep kinase). The reaction mixtures contained 20 mM Tris-HCl (pH 7.5), 10 M [␥-32 P]ATP, 10 mM magnesium acetate, 0.75 mM CaCl 2 , 50 g/ml leupeptin, either 100 g/ml histone or 50 g/ml Epep, with or without 30 M phosphatidylserine, and 0.5 M dioleyl-sn-glycerol.…”

“…To assay the translocation of the various PKC isoforms, the cytosolic and the particulate fractions were subjected to immunoblot analysis, using the indicated isoform-specific antibodies and appropriate secondary antibodies, as described before (15,16). Quantitation of the relative intensities of the immunoblots were done using chemiluminescence and exposure to x-ray film.…”

The role of protein kinase C-mediated phosphorylation of sarcomeric proteins in the heart—detrimental or beneficial?

“…In contrast, PKCε expression in the cytosol and membrane was not significantly different between failing and non-failing hearts (Bowling et al 1999). PKCα, β, ε, ζ, and γ were found to be expressed in healthy rabbit hearts, but when heart failure was induced, western blotting showed a decreased PKC activity and expression of the PKC isoforms α, β 1 , γ, and ε (Rouet- Benzineb et al 1996). Belin et al (2007) studied PKCα quantity, activity, and signaling to myofilaments in rat with early and end-stage congestive heart failure and found that PKCα expression and activation was unaltered in early heart failure, but it did increase in endstage heart failure.…”