Molecular and Cellular Biochemistry 268: 169–173, 2005.c�2005 Kluwer Academic Publishers. Printed in the Netherlands.

PKCε inhibits the hyperglycemia-inducedapoptosis signal in adult rat ventricular myocytes

Ashwani Malhotra, Barinder P.S. Kang, Sayed Hashmiand Leonard G. MeggsDepartment of Medicine, Division of Nephrology, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark,New Jersey, U.S.A.

Received 17 May 2004; accepted 15 July 2004

Abstract

Recruitment of the protein kinase C (PKC) family of isozymes is an integral component of the signaling events that direct cardiacphenotype expressed during postnatal development and in response to pathologic stimuli. Hyperglycemia is a potent activatingsignal for cardiac PKC isozymes and induces the apoptosis program in cardiac muscle cells. To determine whether cardiacPKC isozymes modulate transmission of the hyperglycemia apoptosis signal, we have employed isozyme-specific peptidemodulators to selectively inhibit (PKC βI/βII, ζ and ε) or activate (PKCε). PKC peptides were delivered to primary culturesof serum starved adult rat ventricular myocytes (ARVM), by conjugation to the homeodomain of drosophila antennapedia.As expected, hyperglycemia induced a 35% increase in ARVM apoptosis. Peptide inhibitors of PKC βI/βII and ζ blockedtransmission of the hyperglycemia apoptosis signal, whereas the isozyme specific inhibitor of PKCε (εV1-2) did not alterthe magnitude of glucose-induced ARVM apoptosis. Alternatively, the PKCε translocation activator (ψεRACK) abolishedhyperglycemia-induced apoptosis, strongly suggesting a cardioprotective role for PKCε in this system. Therefore, we concludethat cardiac PKC isozymes modulate hyperglycemia-induced apoptosis and activation of cardiac PKCε protects ARVM fromthe hyperglycemia-induced death signal. (Mol Cell Biochem 268: 169–173, 2005)

Key words: myocardium, protein kinase C, hyperglycemia, myocytes, peptides

Introduction

An essential component of the PKC signaling cascade istranslocation or redistribution to subcellular compartments,via the attachment to anchoring proteins or receptors for ac-tivated C kinases (RACKs) [1]. The development of PKCmodulating peptides derived from RACK binding sites or(pseudo) ψ-RACK sites has proved to be an effective meansto selectively modify PKC activity in an isozyme-specificmanner [2, 3]. For example, the peptide inhibitor of PKCε

(εV1-2) is derived from the first variable region of the RACKbinding site for rat PKCε (amino acids 2-144) [4]. Conversely,

Address for offprints: Ashwani Malhotra, Division of Nephrology and Hypertension, MSB I – 524, Department of Medicine, UMDNJ-New Jersey MedicalSchool, 185 South Orange Avenue, Newark, New Jersey – 07103, U.S.A. (E-mail: malhotas@umdnj.edu)

the PKCε translocation activator (ψεRACK) corresponds tothe ψ-RACK sequence of rat PKCε (amino acids 85–92) [2].The mechanism for inhibition of PKCε by εV1-2 is due tocompetition of the peptide with endogenous PKCε for bind-ing to its RACK [3, 4], whereas ψεRACK peptide promotesPKCε translocation by displacing the RACK binding site,thereby facilitating the interaction of PKCε with its RACK[2, 4]. Since activation-induced translocation of PKC is pos-tulated to be the primary determinant of isozyme-specificfunction, isozyme-specific peptide translocation modulatorsprovide a powerful tool to study loss or gain of PKCε activ-ity. The ε-isozyme of the PKC family transmits a pro-survival

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signal to protect cardiac muscle cells from ischemic injury [2,5, 6]. Hyperglycemia has been identified as a potent stimulusfor program cell death or apoptosis in cardiac myocytes [7–9].Accordingly, in the present study PKC modulating peptideswere employed to test the hypothesis that selective activationof cardiac PKCε, will induce resistance to the hyperglycemiaapoptosis signal.

Materials and methods

Isolation of adult rat ventricular myocytes and cell culture

Sprague-Dawley rats (body weights 275–300 grams) wereanesthetized with a cocktail mixture containing ketamine andxylazine (80 and 10 mg/kg body weight; i.p.). Hearts were re-moved from the chest cavity, rinsed with minimum essentialmedia (MEM; Sigma Chemical Co.) and cardiac myocytesisolated by enzymatic dissociation with collagenase [10–13].Routinely, 80–90% of cells obtained were viable myocytes, asdetermined by their characteristic morphological shape, stri-ations, and trypan blue staining. Freshly isolated myocyteswere plated in laminin coated petri dishes at a density of2×104 cells/cm2 and incubated in serum-free medium (SFM)at 37 ◦C in an atmosphere containing 5% CO2. Growth factorwithdrawal or culture of cells in serum free media (SFM) isrecognized as a potent stimulus for apoptosis. To examine theeffect of PKCε on cardiomyocyte apoptosis, independent ofserum-derived growth factors [9, 10, 12, 13], primary culturesof ARVM were established under the following conditions;(1) SFM with 5 mM glucose; (2) SFM with 25 mM glucose,for 12 h, unless indicated otherwise. Isozyme specific peptideantagonists for (PKC βI/βII, (ε and ζ ) and agonist (PKCε)were added separately to freshly plated cultures of cardiacmyocytes maintained in SFM containing 5 mM glucose for1 h. The media was then replaced by SFM containing 25 mMglucose + peptide antagonists or agonist (at 0.5 µM) for 12 h.Myocytes were harvested and processed for immunoblotsand biochemical studies. To control for the effect of os-molarity, separate studies were performed with cardiac my-ocytes maintained in SFM containing 5 mM glucose +20 mMmannitol.

Peptide delivery into myocytes

The use of peptides as modulators of intracellular signalinghas been simplified by a recently developed method [2] tointroduce biologically active peptides into cells. The appliedconcentration of the carrier peptide conjugates ranged from0.5–0.75 µM with an internal control consisting of myocytesin which a carrier–carrier dimer was introduced [2]. Myocyteswere treated for 1 h in the presence and absence of the peptide

antagonists or agonist followed by culture; (1) SFM + 25 mMglucose (2) SFM + 25 mM glucose + selective peptide antag-onists or agonist. The intracellular concentration of peptideswas not >10% of the applied concentration and the percent-age of cells in which the peptide is successfully deliveredapproaches 100% [2]. Myocytes were maintained for 12 hunder these conditions and collected by centrifugation at lowspeed (300 rpm).

Analysis of DNA fragmentation by ELISA

Histone-associated DNA fragments were quantified by theCell Death Detection ELISA (Roche Diagnostic) as previ-ously described [14].

DNA isolation and electrophoresis

Isolation of DNA in cardiac myocytes was performed as de-scribed previously [15]. DNA electrophoresis was carried outin 1.5% agarose gels containing 1 ug/ml ethidium bromide,and DNA bands were visualized under UV light.

PKC translocation and PKC activity

The method for extraction, partial purification of PKC andanalysis of different PKC isozymes was performed as de-scribed in our earlier publications [10, 16]. PKC transloca-tion was documented by Western blot analysis with isoformspecific primary antibodies for PKC δ and PKCε.

Statistical analysis

Comparisons between two values were performed by un-paired Students t-test. Statistical significance among multiplecomparisons of independent groups of data was determinedby the Bonferroni method. Values of p ≤ 0.05 were consid-ered significant.

Results

Hyperglycemia-induced ARVM apoptosis

As shown in Fig. 1A, serum-starved ARVM maintained at25 mM glucose exhibit a 35% increase in apoptotic cell death.The ELISA cell death assay was employed to detect histone-associated DNA fragments within the cytoplasmic fractionof cells as a marker of apoptotic cell death. Activation of thedeath program was specific for glucose, as ARVM maintainedat osmolar equivalent conditions (5 mM glucose + 20 mM

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Fig. 1. Effect of 25 mM glucose on adult rat ventricular myocyte (ARVM)apoptosis. A: ARVM were maintained under one of the following conditionsfor 12 h. C = serum free medium (SFM) + 5 mM glucose, H = SFM +25 mM glucose, or M = SFM + mannitol . Data are presented as means± S.E. and represents 3–4 independent observations. ∗ p ≤ 0.05 versus C;+ p ≤ 0.05 versus H. B: DNA gel electrophoresis of serum starved ARVMmaintained at C or H for 12 h.

mannitol), exhibit levels of apoptosis similar to control (5 mMglucose). To confirm the presence of ARVM apoptosis at25 mM glucose, DNA agarose gel electrophoresis (Fig. 1B)was performed with nuclear extracts from ARVM maintainedat 5 and 25 mM glucose.

PKCε inhibits hyperglycemia-induced ARVM apoptosis

We first established the specificity of the peptide inhibitorεV1-2, for cardiac PKCε. εV1-2 was conjugated to thehomeodomain of drosophila antennapedia, which served asthe carrier protein [17, 18]. The εV1-2 peptide abolishedhyperglycemia-induced translocation of PKCε from cytosolto particulate fraction (Fig. 2A and B). As expected [4], theεV1-2 peptide did not influence the redistribution of PKCδ

to particulate fraction. (Fig. 2A lower panel). We next askedwhether selective inhibition of PKCε, PKC βI/βII and ζ

translocation, promotes or inhibits transmission of the hy-perglycemia apoptosis signal. As shown in Fig. 3, the εV1-2peptide did not affect the magnitude of ARVM apoptosisdetected at 25 mM glucose. Parallel experiments with se-lective peptide inhibitors for PKC βI/βII and ζ , detected astriking reduction in ARVM apoptosis at 25 mM glucose,approaching control values (p ≤ 0.05). The aggregate dataimplicate cardiac PKC isozymes in the modulation of thehyperglycemia apoptosis signal in ARVM. To determinewhether translocation-activation of cardiac PKCε inhibitsthe hyperglycemia-induced apoptosis signal, the ψεRACK

Fig. 2. (A) Upper Panel: Immunoelectrophoretic patterns depicting εV1−2

inhibition of PKCε translocation from cytosol to membrane fraction. Cy-tosolic and particulate fractions of ARVM were exposed to 25 mM glucosefor 12 h. C = 5 mM glucose, H = 25 mM glucose, H+ carrier (carr)peptide = carrier peptide negative control for H. Lower Panel: Immunoblotdemonstrating the absence of εV1-2 inhibition of PKCδ translocation. (B)Bar graph represents densitometric analysis of PKCε in cytosolic and mem-brane fractions. Data are presented as means ± S.E. and represents threeindependent observations. ∗ p ≤ 0.05 versus C; # p ≤ 0.05 versus H.

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Fig. 3. Effect of ψε-RACK (agonist) and PKC εV1−2 (antagonist) peptideson glucose induced adult rat ventricular myocyte (ARVM) apoptosis. ARVMwere maintained under one of the following conditions for 12 h.: C = serum-free medium (SFM) + 5 mM glucose, H = SFM + 25 mM glucose, H +ε antagonist = SFM + 25 mM glucose + PKC εV1 − 2, H + βC2-4 Ant= SFM + 25 mM glucose + βC2–4 antagonist and H + ζ pseudo = SFM± 25 mM glucose + PKC ζ pseudo peptide antagonist. A baseline level ofapoptosis was detected in serum starved ARVM under euglycemic conditions(C) by ELISA cell death assay. The histone- associated DNA fragments arepresented as optical density at 405 nm relative to control values. For eachassay, 20 µl of lysate (2 mg/ml) was used. Data are presented as means± S.E. and represents four independent observations. ∗ p ≤ 0.05 versus C;+ p ≤ 0.05 versus H.

peptide was delivered to ARVM prior to exposure at 25 mMglucose (Fig. 4). ARVM expressing the ψεRACK peptideexhibit resistance to the hyperglycemia-induced apoptosissignal, with levels of apoptosis similar to control values. How-ever, inhibition of PKCε with ε-V1-2 did not affect hyper-glycemia induced apoptosis (Fig. 4). Taken together, the dataare in agreement with previous work documenting a cardio-protective role for PKCε [2, 3, 5, 6] and extend this analysisto serum starved ARVM maintained under hyperglycemicconditions.

Discussion

We have shown that resistance to the hyperglycemia apoptosissignal can be induced in serum-starved ARVM by the deliveryof PKC modulating peptides. Here we have employed translo-cation inhibitor peptides for PKC βI/βII, ζ , ε and the translo-cation activator for PKCε, ψεRACK, to identify whichPKC isozymes regulate hyperglycemia-induced apoptosis.

Fig. 4. Effect of ψε-RACK (agonist) and PKC εV1–2 (antagonist) peptideson glucose induced adult rat ventricular myocyte (ARVM) apoptosis. ARVMwere maintained under one of the following conditions for 12 h.: C (−) =serum-free medium (SFM) + 5 mM glucose, C + carr = SFM + 5 mMglucose + carrier peptide (carr), H (−) = SFM + 25 mM glucose, H + ε

agonist = SFM + 25 mM glucose + ψε-RACK and H + ε antagonist =SFM + 25 mM glucose + PKC εV1–2. A baseline level of apoptosis wasdetected in serum starved ARVM under euglycemic conditions (C) by ELISAcell death assay. The histone- associated DNA fragments are presented asoptical density at 405 nm relative to control values. For each assay, 20 µl oflysate (2 mg/ml) was used. Data are presented as means ± S.E. and representsfour independent observations. ∗p ≤ 0.05 versus C.

The rationale for these investigations was based on theparadigm that isozyme-specific peptide inhibitors competewith target PKC isozymes for binding to RACKs, whereastranslocation activators, in this case ψεRACK, facilitate in-teraction of PKCε with its RACK [2, 4]. Of note, the resultingchanges in PKC translocation by either intervention are rel-atively modest. For example, under basal conditions, εV1-2decreased PKCε translocation by 15%, whereas ψεRACKincreased PKCε translocation by 20% [4]. Nonetheless, theeffect of these translocation-modifying peptides on the hyper-glycemia apoptosis signal was striking. The inhibitory pep-tides for PKC βI/βII and ζ abolished hyperglycemia-inducedARVM apoptosis, suggesting a role for these isozymes in thetransmission of the hyperglycemia apoptosis signal. We havealso shown that treatment with the PKCε selective activa-tor peptide also abolished hyperglycemia-induced apoptosis.Therefore, several PKC isozymes regulate the response toinjury of cardiac myocytes by hyperglycemia. Activation ofPKC βI and/or βII and ζ promote the death program in ARVMmaintained at hyperglycemic conditions, whereas selectiveactivation of PKCε abolishes the hyperglycemia apoptosissignal. Although beyond the scope of the present study, itwill be important to identify downstream effectors of eachPKC isozyme in this process. Finally, the approach and rel-evance of ARVM rescue in the in vivo setting remain to beestablished.

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Acknowledgments

This work was supported by a research grant from “Foun-dation of UMDNJ Annual Grants Program (A.M) and thesupport from Summit Area Public Foundation throughthe generosity of Mrs Elaine B. Burnett Fund (LGM).We would like to express our sincere thanks to Dr DariaMochly-Rosen, Department of Molecular Pharmacology,Stanford University; Stanford, CA 94305 and co-founder ofKAI Pharmaceutical for providing us with the peptides andcritically reading of the manuscript.

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