Pharmacological Inhibition of ε-Protein Kinase C Attenuates Cardiac Fibrosis and Dysfunction in Hypertension-Induced Heart Failure
Studies on genetically manipulated mice suggest a role for ε-protein kinase C (εPKC) in cardiac hypertrophy and in heart failure. The potential clinical relevance of these findings was tested here using a pharmacological inhibitor of εPKC activity during the progression to heart failure in hypertensive Dahl rats. Dahl rats, fed an 8% high-salt diet from the age of 6 weeks, exhibited compensatory cardiac hypertrophy by 11 weeks, followed by heart failure at ≈17 weeks and death by the age of ≈20 weeks (123±3 days). Sustained treatment between weeks 11 and 17 with the selective εPKC inhibitor εV1-2 or with an angiotensin II receptor blocker olmesartan prolonged animal survival by ≈5 weeks (εV1-2: 154±7 days; olmesartan: 149±5 days). These treatments resulted in improved fractional shortening (εV1-2: 58±2%; olmesartan: 53±2%; saline: 41±6%) and decreased cardiac parenchymal fibrosis when measured at 17 weeks without lowering blood pressure at any time during the treatment. Combined treatment with εV1-2, together with olmesartan, prolonged animal survival by 5 weeks (37 days) relative to olmesartan alone (from 160±5 to 197±14 days, respectively) and by ≈11 weeks (74 days) on average relative to saline-treated animals, suggesting that the pathway inhibited by εPKC inhibition is not identical to the olmesartan-induced effect. These data suggest that an εPKC-selective inhibitor such as εV1-2 may have a potential in augmenting current therapeutic strategies for the treatment of heart failure in humans.
Although two-thirds of cases of heart failure in the United States are because of myocardial infarction, hypertension is a major contributor to this morbidity. Therefore, we studied hypertension-induced heart failure using hypertensive salt-sensitive Dahl rats.1,2
Because many of the signaling events associated with heart failure involve activation of protein kinase C (PKC),1,3 we determined whether PKC regulation affects disease progression. We focused on εPKC, because there are conflicting reports on its role in cardiac hypertrophy and heart failure based on genetic manipulation of mice.1,4–6 Because the enzyme may have different roles during heart development, we used a pharmacological approach to selectively inhibit it at a defined time during disease.
We previously designed isozyme-selective peptide regulators of εPKC, which function by inhibiting or activating PKC translocation. These peptide regulators are linked to membrane-permeable peptides, TAT47–57, to enable their effective intracellular delivery and are, therefore, useful pharmacological tools. Using the Dahl salt-sensitive hypertension-induced heart failure rat model,1,2 we determined here whether sustained pharmacological inhibition of εPKC with the above-mentioned peptide or with an angiotensin receptor blocker, olmesartan, delays progression to heart failure.
Hypertension-Induced Heart Failure Model
Animal protocols were approved by the Stanford University Institutional Animal Care and Use Committee. Male Dahl salt-sensitive rats on an 8% NaCl-containing diet or on a low-salt diet, as described previously,1,4–6 were treated between the ages of 11 and 17 weeks with εV1-2 or δV1-1 (3 mg/kg per day), with equimolar concentrations of TAT (1.6 mg/kg per day; synthesized as described4,7) or with saline, using osmotic pumps implanted subcutaneously (3 mg/kg per day provides a maximal effect on PKC translocation without causing any adverse effects7). A fourth group was treated with olmesartan (3 mg/kg per day in 0.5% carboxymethylcellulose) by daily gavage (Figure S1A, available online at http://hyper.ahajournals.org). The osmotic pumps were replaced every 2 weeks but were discontinued after the age of 17 weeks, because half of the hypertensive control rats died by that age. Additional groups were treated with both εV1-2 and olmesartan or with olmesartan alone from 11 to 19 weeks of age (Figure S1B). Cardiac functions and blood pressure did not differ between these groups before drug treatment (Figure S1C and S1D). Systolic blood pressure was measured by the tail-cuff method (BP-2000, Visitech Systems), and fractional shortening (FS) was measured by transthoracic echocardiography (Vivid 7, GE). Left ventricle specimens from 17-week-old rats were fixed with 10% buffered formalin, embedded in paraffin, and two 5-μm sections from 3 to 4 animals were stained with Masson’s trichrome. Antitransforming growth factor (TGF)-β1 and anticollagen I goat polyclonal antibodies, anti-GAPDH, and β-actin mouse monoclonal antibodies (Santa Cruz Biotechnology), as well as anti-tissue inhibitor of matrix metalloproteinase (TIMP) 2 rabbit polyclonal antibody (Chemicon), followed by horseradish peroxidase–conjugated goat antirabbit, mouse, or goat IgG antibody were used for immunoblotting. Data were normalized to GAPDH or β-actin. Matrix metalloproteinase (MMP)-2 activity was measured by in-gel zymography, as described previously,2 and quantified using ImageJ 1.35s software (http://rsb.info.nih.gov/ij/).
Collagen Secretion From Primary Cardiac Cultured Fibroblasts
Confluent neonatal cultured fibroblasts8 were serum starved for 48 hours before the addition of TGF-β1 (R&D Systems, 10 ng/mL). εV1-2 (1 μmol/L) was administered every 4 hours and at 15 minutes before the TGF-β1 addition. Culture media were collected after 24 hours, and collagen content was determined using a Sircol soluble collagen assay kit (Biocolor).
In hypertensive Dahl rats, εPKC levels increase by ≈2-fold during the adaptive hypertrophy (between 11 and 17 weeks) and decline to the levels of normotensive animals thereafter.1 To determine whether εPKC plays a positive or negative role in heart failure and death, we treated hypertensive rats with εV1-2 (3 mg/kg per day) or with saline (hypertensive control) from the age of 11 weeks to 17 weeks (Figure S1A). A third group was treated with the δPKC-selective inhibitor δV1-1 as a negative control, because we found that δPKC levels and activity do not change in this model.1 A fourth group of hypertensive rats was treated with the angiotensin II receptor blocker olmesartan, commonly used for the prevention and treatment of heart failure in humans. However, we chose a dose of olmesartan (3 mg/kg per day) that ameliorates heart failure without affecting systolic blood pressure, the etiology of the disease in this model (Figure S1C).
Sustained delivery of εV1-2 or olmesartan (delivered by daily gavage) between weeks 11 to 17 improved animal survival and improved or normalized FS and other parameters of cardiac function (Figures 1 and 2⇓ and Table S1). The relative increase in cardiac weight was attenuated by olmesartan but not by εV1-2 (Figure 1D). (The number of animals in the control-treated group decreased over time, because fewer animals survived without treatment with εV1-2.) As expected, because δPKC levels do not change with the disease in this animal model,1 δPKC inhibition had no effect on the disease progression (Figure 1), indicating the selective effect of εPKC inhibition (we confirmed that the peptide inhibitors selectively inhibited the translocation of their corresponding isozymes and not other PKC isozymes in these hearts; Figure S2). Therefore, while on treatment with the εPKC inhibitor or with olmesartan, cardiac dysfunction because of pressure-overload in hypertensive rats diminished.
Heart failure is associated with increased parenchymal fibrosis,9 and sustained treatment with εV1-2 (and, to a lesser extent, treatment with olmesartan) reduced left ventricle fibrosis relative to hypertensive-control hearts (Figure 3A). The perivascular fibrosis increase in hypertensive rats was greatly attenuated by treatment with εV1-2 but not with olmesartan (Figure 3A, right). Furthermore, collagen I levels in hearts of 17-week–old rats were significantly decreased in the εV1-2-treated group when compared with the hypertensive control group (Figure 3B).
We next measured the levels of one of the major profibrotic cytokines, TGF-β1.10 The ratio of active:latent TGF-β1 was 2-fold higher in the hypertensive group relative to the normotensive age-matched rats. Six weeks of treatment with εV1-2, but not with olmesartan, partially inhibited TGF-β1 activation (Figure 3C). We also found that the activity of MMP2 levels, one of the major MMPs regulating fibrosis and heart failure,2 increased in the 17-week–old hypertensive rats (234±19% compared with normotensive control rats). This increase was greatly inhibited after εPKC inhibition but not by olmesartan (Figure 3D). We next determined whether TIMP2, the major endogenous tissue inhibitor of MMP-2,11 was affected by εPKC inhibition (Figure 3D). The level of TIMP2 increased in the εV1-2-treated group relative to the other groups.
We next determined whether εPKC regulation affects collagen secretion using cultured cardiac fibroblasts (Figure 3E). Collagen secretion increased with TGF-β1 treatment, and εV1-2 inhibited it. Because εV1-2 treatment did not affect collagen secretion under basal conditions (data not shown), the data suggest that εPKC may further contribute to heart failure progression, at least in part, by enhancing TGF-β1–induced collagen release.
Olmesartan treatment did not affect the activity or levels of εPKC (Figure S2A and S2C), and εV1-2 treatment normalized perivascular fibrosis but olmesartan did not (Figure 3). Yet, either treatment increased the life span of the hypertensive rats to a similar extent and decreased the rate of cardiac dysfunction development (Figure 1). These results suggest that the mechanism leading to protection by these 2 agents may be different and that treatment of hypertensive rats with both agents should be additive. Indeed, treatment of the hypertensive rats with both εV1-2 and olmesartan up to week 17 increased the life span of the animals by up to an additional 13 weeks as compared with the treatment of olmesartan alone (average survival was 160±5 days after treatment with olmesartan versus 197±14 days after treatment with olmesartan together with εV1-2; Figure 4A). Again, the high blood pressure remained unchanged (Figure S1D), yet εV1-2 plus olmesartan-treated animals maintained FS similar to the normotensive control rats (Figure 1E and Table S1), even at the age of 24 weeks, 5 weeks after the treatment with εV1-2 and olmesartan terminated (Figure 4B and Table S2). Although the second study did not include a group of animals treated with εV1-2 alone, it is likely that the combined treatment of εV1-2, together with olmesartan (a commonly used drug for patients with heart failure), is superior to treating with each of these agents alone. Therefore, without affecting hypertension, the cause of the disease, treatment with both the εPKC inhibitor and an angiotensin II receptor blocker between weeks 11 and 19 prolonged the lives of the rats by ≤17 weeks with the 2 inhibitors together and enhanced the therapeutic effect obtained by each treatment alone. The molecular basis for this prolonged longevity remains to be determined.
Using a rat model of hypertension-induced heart failure, we demonstrated that pharmacological inhibition of εPKC during the transition from compensatory cardiac hypertrophy to heart failure slowed the progression of heart failure. There was a similar benefit to using the angiotensin II receptor blocker olmesartan. Furthermore, combination treatment with the εPKC inhibitor and olmesartan during transition to heart failure appears to provide a sustained effect; animals survived ≤38 weeks, although the treatment was terminated by week 19. Importantly, these therapeutic effects were not associated with a reduction in blood pressure.
We previously found a transient increase in PKC levels at week 11 of the disease with a return to basal levels by 17 weeks.1 We treated the hypertensive rats with the εPKC inhibitor only during the period when εPKC was elevated, which is also the transition period from the compensatory to the decompensated stage of the disease. On termination of εV1-2 treatment at 17 weeks, there was an increase in end-systolic dimension and a decrease in FS, as measured at 19 weeks (Figure 2), indicating that εPKC inhibition delays the development of the disease in this model.
Histological analysis showed reduced cardiac fibrosis and collagen I levels with εPKC inhibition (Figure 3A and 3B). Yet, εPKC null mice showed increased fibrosis and cardiac dysfunction after pressure-overload–induced heart failure using transaortic constriction.6 However, in that model, the change in pressure-overload is very sudden, and an increase in δPKC levels was observed, an effect that did not occur in our model (Figure S2B and S2D). Furthermore, we reported previously that δPKC activation increases TGF-β1–induced proliferation of cardiac fibroblasts8; perhaps the increase in δPKC mediates the observed fibrosis in the εPKC null mice or may reflect compensation for the complete absence of εPKC during development. We also showed that the levels of active TGF-β1, a major fibroblast-regulating factor, were reduced by εPKC inhibition in vivo. εPKC inhibition also abrogated TGF-β1–induced collagen secretion in vitro. These data are consistent with a profibrotic effect of εPKC in failing heart that we observed in vivo. We also found that εPKC may modulate cardiac fibrosis by regulating metalloprotease activity (Figure 3D). However, εPKC inhibition does not affect fibroblast proliferation.8 Therefore, the changes in active TGF-β1 levels, collagen secretion, and metalloprotease activity may lead to the decreased collagen accumulation and fibrosis that we have observed in the hypertensive rats treated with the εPKC inhibitor. Consistent with other studies, systolic dysfunction develops in these hypertensive rats at 17 weeks and is normalized after treatment with the εPKC inhibitor or olmesartan (Table S1 and Figure S2). Because diastolic function was not measured, it remains to be determined whether the decrease in fibrosis by εPKC inhibition impacts this function. Kidney dysfunction occurring in these animals12 may further contribute to heart failure. Because the εPKC inhibitor was delivered systemically, it may benefit other organs, which, in turn, may prevent progression to heart failure.
Although acute activation of εPKC before ischemia and reperfusion injury in ex vivo rat heart1,13 or before heterotopic heart transplantation14 models are cardioprotective, we already found that a 4-week inhibition of εPKC is protective against chronic rejection in the heterotopic cardiac transplantation model.15 Therefore, εPKC activation is beneficial in acute response to ischemia but is detrimental in chronic cardiac diseases, demonstrating the importance of the use of selective pharmacological tools that can be administered at defined time points and for a defined period.
Heart failure is a complex syndrome with multiple etiologies. Whether our findings in these hypertensive rats are translatable to other models of hypertension and whether the same benefits can be seen also in myocardial infarction–induced models of heart failure have yet to be determined. Furthermore, most of the analyses were carried out at a single time point (17 weeks), when some of the treatment groups had only 50% survival. The data from the surviving animals may represent events related to a slower disease progression or better compensatory mechanisms relative to animals that had succumbed to the disease. We also find it unlikely that fibrosis inhibition is the sole mechanism leading to the benefits afforded by εV1-2. The molecular pathways in both cardiac myocytes and nonmyocytes remain to be elucidated. Finally, we did not determine whether sustained treatment will remain beneficial or whether εPKC inhibition and/or an angiotensin receptor blocker provide only a short-term benefit. Nevertheless, because the period of treatment covered the time from compensated to decompensated hypertrophy in this model, our study suggests that sustained inhibition of εPKC early during decompensatory hypertrophy may delay or inhibit the development of heart failure. Together, our data suggest that an εPKC inhibitor, such as εV1-2, may augment current therapeutic strategies for the treatment of heart failure in humans.
We are grateful to Dr Dan Bernstein for helpful discussions and advice.
Sources of Funding
This study was supported by National Institutes of Health grant HL076675 to D.M.-R. K.I. was supported in part by a fellowship grant from Sankyo Co. N.C.B. was a research fellow supported by the Stanley J. Sarnoff Cardiovascular Research Foundation, Inc. L.S. was supported in part by a Stanford Graduate Fellowship.
D.M.-R. is the founder of KAI Pharmaceuticals, Inc, a company that plans to bring protein kinase C regulators to the clinic. However, none of the work described in this study is based on or supported by the company. The other authors have no disclosures.
The first 2 authors contributed equally to this study.
- Received January 7, 2008.
- Revision received January 23, 2008.
- Accepted March 21, 2008.
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