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Alterations in protein kinase C isoenzyme expression and autophosphorylation during the progression of pressure overload-induced left ventricular hypertrophy
Cardiomyocytes express several isoenzymes of protein kinase C (PKC), which as a group have been implicated in the induction of left ventricular hypertrophy (LVH) and its transition to heart failure. Individual PKC isoenzymes also require transphosphorylation and autophosphorylation for enzymatic activity. To determine whether PKC isoenzyme expression and autophosphorylation are altered during LVH progression in vivo, suprarenal abdominal aortic coarctation was performed Sprague-Dawley rats. Quantitative Western blotting was performed on LV tissue 1, 8 and 24 weeks after aortic banding, using antibodies specific for total PKCĪ±, PKCĪ“ and PKCĪµ, and their C-terminal autophosphorylation sites. Aortic banding produced sustained hypertension and gradually developing LVH that progressed to diastolic heart failure over time. PKCĪµ levels and autophosphorylation were not significantly different from sham-operated controls during any stage of LVH progression. PKCĪ± expression levels were also unaffected during the induction of LVH, but increased 3.2 Ā± 0.8 fold during the transition to heart failure. In addition, there was a high degree of correlation between PKCĪ± levels and the degree of LVH in 24 week banded animals. However, autophosphorylated PKCĪ± was not increased at any time point. In contrast, PKCĪ“ autophosphorylation was increased prior to the development of LVH, and also during the transition to heart failure. The increased PKCĪ“ autophosphorylation in 1 week banded rats was not accompanied by an increase in total PKCĪ“, whereas total PKCĪ“ levels were markedly increased (6.0 Ā± 1.7 fold) in 24 week banded animals. Furthermore, both phosphorylated and total PKCĪ“ levels were highly correlated with the degree of LVH in 24 week banded rats. In summary, we provide indirect evidence to indicate that PKCĪ“ may be involved in the induction of pressure overload LVH, whereas both PKCĪ“ and PKCĪ± may be involved in the transition to heart failure
PYK2 expression and phosphorylation increases in pressure overload-induced left ventricular hypertrophy
Proline-rich tyrosine kinase 2 (PYK2) is a member of the focal adhesion kinase (FAK) family of nonreceptor protein tyrosine kinases. PYK2 has been implicated in linking G protein-coupled receptors to activation of mitogen-activated protein kinase cascades and cellular growth in a variety of cell types. To determine whether PYK2 expression and phosphorylation is altered in left ventricular (LV) myocardium undergoing LV hypertrophy (LVH) and heart failure in vivo, suprarenal abdominal aortic coarctation was performed in 160-g male Sprague-Dawley rats. Immunohistochemistry and Western blotting were performed on LV tissue 1, 8, and 24 wk after aortic banding. Aortic banding produced sustained hypertension and gradually developing LVH. PYK2 levels were increased 1.8 Ā± 0.2-, 2.7 Ā± 0.6-, and 2.0 Ā± 0.2-fold in 1-, 8-, and 24-wk banded animals compared with their respective sham-operated controls. The increase in PYK2 expression was paralleled by an increase in PYK2 phosphorylation, both of which preceded the development of LVH. Immunohistochemistry revealed that enhanced PYK2 expression occurred predominantly in the cardiomyocyte population. Furthermore, there was a high degree of correlation ( R = 0.75; P< 0.001) between the level of PYK2 and the degree of LVH in 24-wk sham and banded animals. In contrast, FAK levels and FAK phosphorylation were not increased before the development of LVH. However, there was a high degree of correlation (R = 0.68; P < 0.001) between the level of FAK and the degree of LVH in 24-wk sham and banded rats. There was also a significant increase in the ratio of phosphospecific anti-FAK to FAK at this time point. These data are consistent with a role for PYK2 in the induction of pressure overload-induced cardiomyocyte hypertrophy, and suggest that PYK2 and FAK have distinctly different roles in LVH progression