Original article
Inhibition of protein kinase C reduces left ventricular fibrosis and dysfunction following myocardial infarction

https://doi.org/10.1016/j.yjmcc.2005.03.008Get rights and content

Abstract

Despite current therapies, chronic heart failure (CHF) remains a major complication of myocardial infarction (MI). The pathological changes that follow MI extend to regions remote from the site of infarction (non-infarct zone, NIZ) where fibrosis is a prominent finding. Although the mechanisms underling this adverse remodeling are incompletely understood, activation of protein kinase C has recently been implicated in its pathogenesis. MI was induced in Sprague–Dawley rats by ligation of the left anterior descending coronary artery. One week post-MI, animals were randomized to receive the PKC-inhibitor, ruboxistaurin (LY333531) for 4 weeks, or no treatment. When compared with sham-operated animals, post-MI rats showed a 33 ± 7% reduction in fractional shortening over a 4 weeks period, that was attenuated by treatment with ruboxistaurin (6 ± 11%, P < 0.05). Increased matrix deposition was noted in the NIZ, particularly in the subendocardial region of post-MI rats, in association with elevated expression of the profibrotic growth factor, transforming growth factor-beta. These findings were also significantly reduced by ruboxistaurin. PKC-inhibition with ruboxistaurin led to attenuation in both the pathological fibrosis and impaired cardiac function that follow experimental MI, suggesting a possible role for this agent in preventing post-infarction heart failure.

Introduction

Chronic heart failure (CHF) is a major complication of myocardial infarction (MI) that substantially worsens prognosis. Although there have been major therapeutic advances in the management of MI, post-infarction CHF remains a common cause of morbidity, hospitalization and premature death [1].

The ischemic necrosis of MI is followed by a complex sequence of structural changes involving the left ventricle, referred to as post-infarction remodeling [1]. These changes include progressive chamber dilatation, eccentric hypertrophy and fibrosis [2]. Although, there has been substantial investigation into the roles of hypertrophy and dilatation, more recent studies have highlighted the importance of fibrosis, remote from the site of infarction, in the pathogenesis of post-infarction cardiac dysfunction [3]. Indeed, the predilection for fibrosis in the subendocardium of the non-infract zone (NIZ) is viewed as a major contributor to both mechanical dysfunction [4] and the propensity to dysrhythmia [5] following MI.

A range of neurohumoral, as well as mechanical factors have been associated with the maladaptive remodeling that occurs in response to cardiac injury. While seemingly diverse, factors such as angiotensin II, endothelin, pro-inflammatory cytokines and mechanical stretch all activate common intracellular signaling pathways that include the activation of protein kinase C (PKC) [6]. Indeed, recent studies have shown that, following MI, a number of P KC isoenzymes are activated, including alpha, beta, epsilon and delta [7], [8].

Ruboxistaurin (LY 333531) is an inhibitor of PKC that is currently in Phase II clinical trials, where its investigation to date has been confined to the complications of diabetes [9]. Ruboxistaurin, inhibits PKC activity with an IC50 for the beta isoforms of 4.7 nM for beta I and 5.9 nM for beta II, compared with 200 nM for PKC-alpha and more than 1 μM for other PKC isoforms and non-PKC kinases [10]. However, despite its relative specificity for PKC-beta, there is approximately 50-fold more PKC-alpha in the left ventricle of the rat than PKC-beta [7], such that in the cardiac setting, ruboxistaurin may be viewed as inhibiting both PKC beta and alpha isoenzymes.

As in post-MI remodeling and heart failure, the pathological accumulation of excess matrix is also a feature of diabetic nephropathy where ruboxistaurin has been shown to reduce the expression of the profibrotic growth factor, transforming growth factor-beta (TGF-beta) with a concomitant decrease in kidney fibrosis [11]. Accordingly, the aims of the present study were twofold. We firstly sought to firstly determine the effects of PKC-inhibition on the cardiac dysfunction that develops post-MI and secondly to examine the pathological fibrosis and TGF-beta overexpression that occurs in this setting.

Section snippets

Animal model

MI was induced in 15 male Sprague–Dawley rats, aged 10 weeks, by ligation of the left anterior descending (LAD) coronary artery, as previously described [12]. Animals were anesthetized with xylazine 1 mg/100 g, ketamine 7.5 mg/100 g and atropine 0.006 mg/100 g intraperitoneally (IP) and were given subcutaneous carprofen 5 mg/kg for analgesia. Sham animals underwent thoracotomy and incision of pericardial sac, but not LAD ligation. One week post-operatively, animals underwent echocardiography

Clinical parameters

Left ventricular weight, heart weight, lung weight and heart:body weight ratio were all increased following MI. The increases in heart: body weight ratio and lung weight were significantly attenuated with ruboxistaurin treatment (Table 1).

Infarct size and mortality

Myocardial infarct size was the same in the treated and untreated MI groups (35.70 ± 5.07% and 38.97 ± 2.25%, respectively; P = 0.57). There was no mortality in either the treated or untreated MI groups after randomization at 1 week post MI and no deaths at any

Discussion

Following MI, fibrosis is an integral component of the reparative process, maintaining structural integrity of the necrotic area by the elaboration of a connective tissue scar. However, the accumulation of this collagenous material remote from the site of infarction is maladaptive and is associated with both reduced myocardial elasticity and impaired contractility [27], [28]. In the present study, we demonstrate that following MI, inhibition of PKC with ruboxistaurin, leads not only to a

Acknowledgements

This project was supported by grants from the National Health and Medical Research Council of Australia (NHMRC) and the Juvenile Diabetes Research Foundation (JDRF). Ruboxistaurin was provided by Eli Lilly and Company. Andrew Boyle is the recipient of a medical postgraduate scholarship from the NHMRC and Darren Kelly is a recipient of a Career Development Award from JDRF. The authors would like to thank Mariana Pacheco and Sylwia Glowacka for expert technical assistance, and David Prior, for

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