In the present model, sustained hypertension in salt-sensitive Dahl rats fed a high-salt diet leads to cardiac dysfunction and HF by 17 weeks. Relative to 11 weeks old hypertensive rats, we observed significant pathological cardiac remodeling processes including myocardial fibrosis, vascular intimal thickening, reduction of coronary vessel patency, inflammation and increased cardiac MC number and degranulation. We have recently found that sustained treatment with εV1-2 an εPKC-selective inhibitor [15
] from the age of 11 to 17 weeks, improved cardiac function in these rats [14
]. Here we show that treatment with εV1-2 during this period attenuated MC degranulation without affecting MC density. This treatment also inhibited infiltration of inflammatory cells, vasculopathy and fibrosis in the myocardium of hypertensive DS rats. In contrast, treatment with δV1-1, a δPKC-specific inhibitor [16
] for the same period, did not improve any of these pathological events. Our previous work showed that in vitro
collagen secretion is also dependent on εPKC activation [14
]. Although, we cannot rule out that some of the benefit of εPKC inhibition is indirect, our data are consistent with a deleterious role for εPKC in cardiac fibrosis.
Possible steps in cardiac pathology associated with hypertension that are inhibited by εV1-2 treatment are depicted schematically in . Based on our studies here, we suggests that εV1-2 inhibits MC degranulation, which in turn, decreases TGFβ1 levels, infiltration of inflammatory cells, fibroblast stimulation and intestinal and perivascular fibrosis. ARB also inhibited all these processes, but to a lesser extent as compared with εV1-2. Several other studies suggest a role for MCs in HF progression. The number of degranulated MCs in the infarcted and peri-infarcted regions was found to be higher relative to the non-infarcted region in the hearts of rats [26
] and MCs are found in the fibrotic region of the infarcted myocardium in dogs [27
]. Further, the number of degranulated MCs in the area surrounding the coronary arteries with ruptured plaques is higher in human subjects [28
]. MCs appear to be critical also in remodeling associated with aortocaval fistula-induced HF in rats [29
]. Finally, as we stated earlier, MCs have been implicated in postmyocarditis HF, as was found in our earlier studies in rats [7
] and by others in mice [31
]. Therefore, together with this study in HF of Dahl salt-sensitive rats, it appears that MCs are important component in the pathogenesis of HF irrespective of the etiology.
A scheme demonstrating how MCs in the myocardium may contribute to remodeling events and the role of εPKC in this process
MC degranulation products are pro-inflammatory, hypertrophic and pro-fibrogenic in nature. TGFβ1, a cytokine that is released upon MC stimulation, modulates proliferation of fibroblasts and enhances synthesis and deposition of collagen [6
]. Histamine, tryptase, chymase, tumor necrosis factor-a and stem cell factor, the MC derived mediators, are also involved in remodeling and inhibition of chymase attenuates adverse cardiac remodeling in different HF models [7
]. However, it is not practical to inhibit each mediator of MCs considering the plethora of MC mediators that are released during degranulation. Instead, degranulation of MCs is a convergence point of signaling that leads to cardiac hypertrophy, whose inhibition may provide the desired therapeutic effect. We showed here that inhibition of εPKC correlates with attenuated MC degranulation and thus suggest that such an inhibitor may be useful as a treatment option for heart failure.
A role for PKC in MC activity in the myocardium has not been previously described and conflicting reports on the role of PKC isozymes on MC function in vitro
have been reported. For example, εPKC activation was found to induce c-fos and c-jun gene expression in an activated RBL-2H3 MC line [33
]. However, εPKC activation inhibited antigen-induced hydrolysis of inositol phospholipids by reducing tyrosine phosphorylation of phospholipase C in permeabilized RBL-2H3 cells [34
]. Another study reported that εPKC plays a redundant role on MC degranulation as the extent of degranulation was not significantly different between bone marrow-derived MCs from εPKC null mice and wild type mice following antigen-induced activation [35
]. Similarly, there are conflicting reports which demonstrate positive or negative roles of δPKC on MC degranulation [36
]. Heterogeneity of MCs and use of non-selective PKC modulators may explain some of these conflicting reports on the role of PKC in MC degranulation. Further, genetic manipulations of PKC may also contribute to the conflicting reports due to the compensatory effects by other isozymes. For example, δPKC expression increases in hearts of εPKC KO mice by 60% and it is also more activated in these εPKC KO mice relative to wild type mice [38
]. Similarly, Ways et al
showed that over-expression of αPKC in breast cancer cells increased the expression of βPKC and decreased the expression of δ and ηPKC [39
]. Ping and collaborators found that over-expression of εPKC results in its association with the anchoring protein of βPKC, RACK1, instead of its own RACK, RACK2. They also showed that εPKC-induced hypertrophy involved the recruitment of both PKCβII and εPKC by RACK1. Therefore, the combined effect of PKCβII and εPKC (and not the effect of εPKC, alone) may have led to the observed development of cardiac hypertrophy and failure in AE-εPKC-H mice [40
] that express high levels of εPKC by doing A-to-E point mutation at the pseudo substrate domain. We suggest that genetic manipulation of this family of isozymes results in compensatory changes in other PKC isozymes, making the interpretation of data difficult.
Hence, we used isozyme-selective pharmacological inhibitors in this study, and we applied them once the disease occurred, thus better mimicking the scenario associated with patients care. We show there a direct role for εPKC in MC degranulation and our data are consistent with a deleterious role of sustained εPKC activation in the pathology of HF in these hypertensive rats [14
]. The role of MC degranulation in the progression of myocardial fibrosis of HF in DS rats is further suggested by the co-localization of degranulated MCs in the fibrotic regions (data not shown). Similar association between myocardial fibrosis and MCs are demonstrated in the myocardium of human patients with HF [42
]. In the hypertensive rats, administration of εV1-2 resulted in a decrease in interstitial and perivascular fibrosis as evident from Masson’s-Trichrome staining (see also [14
]). Further, εV1-2 treatment also decreased TGFβ1 levels in the myocardium of these rats, which in turn may contribute to reduced fibrosis. However, there may be other sources for TGFβ1, including cardiomyocytes and inflammatory cells, and the effect of εPKC on fibrosis may thus be indirect.
Olmesartan, an angiotensin receptor blocker, was used as a positive control for the treatment of HF progression in this study. We administered 3 mg/Kg/day of olmesartan, a concentration that did not affect hypertension per se.
Olmesartan treatment also attenuated MC degranulation and inhibited the pathological changes associated with excessive fibrosis. However, εV1-2 appeared to be a more potent MC inhibitor than ARB. In a recent study, we found that εPKC activation was not inhibited by olmesartan in these hypertensive Dahl rats [14
]. Therefore, it εV1-2 and ARB may exert their effects via
In addition to a reduction in myocardial fibrosis, εPKC inhibition attenuated vascular remodeling and infiltration of inflammatory cells. These beneficial effects may also be attributed to the decrease in MC degranulation; as we stated earlier, MC mediators are vasoactive and proinflammatory factors. The observed co-localization of degranulating MCs in the inflammatory milieu and proximity to remodeled vasculature in the samples from TAT or δV1-1 treated samples may indicate the involvement of MCs and their mediators in these processes.
In summary, εPKC inhibition in hypertension-induced HF led to the suppression of pathological remodeling such as myocardial fibrosis, vasculopathy and inflammation that correlated with improvement of myocardial function. We suggest that these beneficial effects of εPKC inhibition on HF progression are, at least in part, due to inhibition of cardiac MC degranulation. Further studies that reveal the time course changes in MC degranulation in HF and the effects of an εPKC inhibitor at these various time points will provide detailed information about the role of εPKC in MC degranulation in HF.