In this study, we analyzed myocyte hypertrophy, myocardial fibrosis, cardiac inflammation and mast cell degranulation in a region remote from the infarcted area. We hypothesized that remodeling of the remote area as induced by MI contributed to cardiac dysfunction. Indeed, with the use of the PKCβII -selective inhibitor, βIIV5-3, remodeling in the remote area was blunted and cardiac function was improved in a rat model of post-MI HF.
In this model, rats were subjected to MI and developed symptoms of end-stage HF. A six-week treatment with βIIV5-3, initiated four weeks after MI induction, attenuated cardiac dysfunction, myocyte hypertrophy, collagen deposition, myocardial inflammation and mast cell density and degranulation. In addition, PKCβII inhibition normalized both right and left ventricle wall thickness ( and ).
In hearts of patients with HF, both PKCβI and II levels are higher relative to age-matched normal hearts [8
]. A more recent study also reported increased expression of PKCβII in end-stage human HF (dilated cardiomyopathy) [10
]. Similarly, we also found a 50% increase in PKCβII activity in the myocardium of rats with end-stage HF (10 weeks after MI) as compared with sham-operated rats; treatment with βIIV5-3 for 6 weeks selectively decreased PKCβII levels close to the levels in the sham rats () . As expected, after six-week treatment, with the PKCβII -specific inhibitor βIIV5-3 did not affect the levels or activity of PKCβI, whereas it completely blunted the activation of PKCβII ().
The role of PKCβI in HF in mice is presently controversial. Since PKCβI and βII s are alternatively spliced forms of the same gene product, some of the studies did not distinguish between the two isozymes. Though it was found that adult mice express little PKCβ in the myocardium [9
], several reports using a variety of tools demonstrate that PKCβ is an important isozyme in cardiac disease. In fact, targeted over-expression of PKCβII in mice resulted in cardiac hypertrophy with myocardial dysfunction, fibrosis, inflammation and fetal gene expression [11
]. Moreover, activated over-expression of PKCβII was lethal in neonatal mice, whereas in adult mice it induced hypertrophy and affected cardiac contractility [27
]. These studies show the importance of PKCβII in cardiac hypertrophy in mice. In contrast, in one study, PKCβ KO mice showed no cardiac symptoms [28
]. This may reflect compensatory effects by other PKC isozymes or other signaling enzymes. Further a number of studies suggest that mice are not ideally suitable as models of human diseases. Similar to HF in humas, in a hypertension-induced HF model in rats, PKCβII levels increased in the late HF stage and not during the early hypertrophy stage [5
]. Here we show an increase in fractional shortening following a six week βIIV5-3 treatment in rats that developed end-stage pathological hypertrophy four weeks after MI. We also found that a reduction in cardiomyocyte width and heart/body weight ratio. These data indicate that PKCβII activation contributes to the development of cardiac hypertrophy and dysfunction in rats. Since PKCβII levels and activity are elevated in failed human hearts, we suggest that PKCβII inhibition is a potential target for the treatment of heart failure.
Supporting this suggestion is the study of Boyle et al.,
who reported that a four week treatment with ruboxistaurin (LY333531), an inhibitor of PKCβ, beginning a week after induction of MI, inhibited cardiac fibrosis in rats [29
]. A similar reduction in myocardial fibrosis (collagen deposition) following treatment with a specific PKCβII inhibitor has been observed by us in the MI-induced end-stage HF in rats. Moreover, βIIV5-3 treatment resulted in decreased TGFβ1 levels and SMAD2/3 phosphorylation in the hearts of post-MI HF rats. TGFβ1 ligation to its receptor leads to SMAD 2/3 phosphorylation in the cytoplasm and translocation of phospho SMAD 2/3 into the nucleus to stimulate proliferation through increased transcription and protein synthesis of select genes [23
]. We found that fibroblast stimulation and collagen release was inhibited by chronic treatment with βIIV5-3 (), presumably by limiting the TGF-SMAD signaling pathway.
A six week βIIV5-3 treatment also caused a reduction in infiltration of inflammatory cells into post MI myocardium. Previous studies reported that PKCβII activation is involved in a variety of inflammatory processes. PKCβII activation by interferon gamma leads to translocation of PKCβII into the nucleus and activation of the transcription factors that regulate the expression of critical inflammatory genes such as major histocompatability complex II in microglial cells [30
]. High glucose-induced interleukin (IL)-6 release from monocytes was attenuated by PKCβII inhibition [31
]. We also found a reduction in mast cell number and degranulation after treating rats with βIIV5-3. A role for PKCβII in different stages of the MC degranulation process has been previously suggested by in vitro
studies. Aggregation of the FCεRI on mast cells elicits a PKCβ -dependent fos
binding of DNA and resulting gene transcription in mast cell culture [32
]. PKC-βII regulates Akt activity by directly phosphorylating it on Ser-473 in antigen-IgE-stimulated mast cells [33
] and PKCβII -deficient mast cells exhibit reduced IL-6 production [34
]. Stimulation by antigen or PMA and a calcium ionophore induces Ser (1917) phosphorylation of myosin heavy chain II in mast cells, which coincides with the exocytosis process in these cells, an effect that is mediated by PKCβII [35
]. These reports are consistent with our findings here on the role of PKCβII in mast cell activation in vivo
. However the molecular mechanism leading to the beneficial effects of PKCβII inhibition remains to be elucidated.
In conclusion, we show that sustained selective inhibition of PKCβII by βIIV5-3 attenuated adverse cardiac remodeling and improved myocardial function in an end-stage HF model induced by MI in rats. If proven to be safe, such a specific inhibitor of PKCβII may benefit human patients with MI-induced HF.