Cardiovascular Research

Cardiovascular Research 2001 50(3):486-494; doi:10.1016/S0008-6363(01)00225-5
© 2001 by European Society of Cardiology

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Copyright © 2001, European Society of Cardiology

Increased basal contractility of cardiomyocytes overexpressing protein kinase C and blunted positive inotropic response to endothelin-1

Stéphane Baudet^1,a, Jutta Weisser^b, Anita P Janssen^b, Kathrin Beulich^a, Ursula Bieligk^a, Burkert Pieske^b, Jacques Noireaud^c, Paul M.L Janssen^b, Gerd Hasenfuss^b and Juergen Prestle^b,*

^aDepartment of Cardiology and Angiology, University of Freiburg, D-79106 Freiburg, Germany
^bGeorg-August-Universitaet Goettingen, Zentrum Innere Medizin, Department of Cardiology and Pneumology, Robert-Koch-Strasse 40, D-37075 Goettingen, Germany
^cINSERM U533, F-44035 Nantes Cedex 01, France

^* Corresponding author. Tel.: +49-551-396-380; fax: +49-551-392-953 stephane.baudet{at}intervet.com prestle{at}med.uni-goettingen.de

Received 31 March 2000; accepted 11 January 2001

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
Objective: Protein kinase C (PKC) is thought to be involved in the regulation of the mammalian cardiac excitation–contraction coupling process by vasoactive peptides like endothelin-1 (ET-1). However, the demonstration of a causal link between activation of specific PKC isoforms and the increase in contractility mediated by ET-1 is still inferential. Methods: By means of adenovirus-mediated gene transfer, we specifically overexpressed PKC in cultured adult rabbit ventricular myocytes (Ad-PKC). Myocyte shortening and [Ca²⁺]_i transients under basal and ET-1-stimulated conditions were measured in Ad-PKC and Ad-LacZ control transfected cells. Results: Infection with Ad-PKC resulted in a strong, virus dose-dependent increase in PKC protein levels, whereas protein expression of other PKC isoforms remained unchanged. Using a multiplicity of infection of 100 plaque-forming units/myocyte, basal and cofactor-dependent PKC kinase activity was increased 28- and 90-fold, respectively, when compared to control. Myocyte basal fractional shortening and [Ca²⁺]_i transient amplitude were both increased by 21% (P<0.05 each) in Ad-PKC transfected myocytes when compared to Ad-LacZ transfected control myocytes. The positive inotropic effect of ET-1 in control myocytes was markedly blunted in PKC-overexpressing myocytes. Conclusion: Specific overexpression of PKC in rabbit ventricular myocytes increases basal myocyte contractility and [Ca²⁺]_i transients, and modifies their responsiveness to ET-1.

KEYWORDS Contractile function; e–c Coupling; Endothelins; Gene therapy; Myocytes; Protein kinases; Signal transduction

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In contrast with the well-described regulation of mammalian contractility by neurotransmitters like noradrenaline, regulation of this process by the vasoactive peptide endothelin-1 (ET-1) is less understood. Binding of ET-1 to ET_A receptor subtypes generally exerts a positive inotropic effect (PIE), i.e. an increase in contractility of multicellular muscle preparations and isolated cardiomyocytes, with the rabbit exhibiting the highest sensitivity [1–4]. It is now accepted that these acute inotropic effects of ET-1 are due to agonist-receptor-induced activation of the phospholipase C–protein Gq–phosphatidylinositol–biphosphate cascade, leading to the generation of inositol triphosphate and diacylglycerol which subsequently activates protein kinase C (PKC) [4–7]. Activated PKC then phosphorylates numerous target proteins such as ion channels, myofibrillar or Ca²⁺-storage proteins critically involved in regulation of contractility, thereby accounting for the PIE [8–10].

One major difficulty associated with the study of PKC transduction pathways relies in the diversity of the PKC isoforms. In fact, the superfamily of mammalian PKC isotypes is currently comprised of twelve distinct genes [11]. With minor species-differences, almost all PKC isoforms are present in the mammalian myocardium, with PKC and PKC being the most abundant Ca²⁺-sensitive and Ca²⁺-insensitive PKC isoforms, respectively [8,9,12–14]. Despite descriptions of PKC expression pattern in mammalian myocardium of different species, the demonstration of a causal link between the activation of specific PKC isoforms and the PIE of ET-1 is still inferential. PKC activation is usually demonstrated by invasive methods such as an increase in in vitro kinase activity or translocation to membrane compartments and there is only indirect evidence for PKC being one isoform possibly involved in the inotropic effects of ET-1 in mammalian cardiac muscle [15,16]. Demonstration of a causal link would require selective modulation of PKC activity, but the pharmacology of specific PKC isoforms is still poor [17]. Recent strategies have aimed to modulate PKC activity by transgenesis, with the risk that long-term over/underexpression and/or activity of PKC might have affected other important signalling pathways and/or expression/activities of molecules involved in cardiac excitation–contraction coupling [18,19]. Short-term modification of PKC activity has been achieved either by transfection of a peptide inhibiting PKC translocation or by adenovirus-mediated overexpression of PKC [20,21]. The latter approach was used in the present study to overexpress PKC in primary cultures of adult rabbit cardiomyocytes. We demonstrate that specific overexpression of PKC increases basal myocyte contractility and intracellular calcium ([Ca²⁺]_i) transients, and severely blunts the PIE of ET-1.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
2.1 Recombinant adenovirus vector construction
A 2.3-kb EcoRI/EcoRI cDNA fragment of the mouse PKC gene was cloned into the EcoRI site of the plasmid pACCMV·pLpASR^– [22], a E1 region shuttle vector containing a cytomegalovirus (CMV) immediate early promoter and a SV40 polyadenylation signal. This plasmid was co-transfected with pJM17 [22], a plasmid containing the full Ad5 genome plus a 4-Kb fragment of pBR322, into human embryonic kidney (HEK) 293 cells using Superfect^TM (Qiagen) transfection reagent. Viral plaques (Ad-PKC) were picked through agar overlay and propagated in HEK293 cells. Viral stocks were prepared by CsCl ultracentrifugation. The titer of viral stocks was determined by plaque assay using HEK293 cells [22].

An adenovirus carrying the CMV-driven E. coli β-galactosidase gene (Ad-LacZ) was used as a negative control in all experiments.

2.2 Primary culture of rabbit ventricular myocytes and adenovirus infection in vitro
New Zealand white rabbits (2.5–3 kg) were heparinised and anaesthetised with sodium thiopental (50 mg/kg i.v.). The excised heart was mounted on a Langendorff perfusion set-up and perfused with Tyrode I solution (137 mM NaCl, 5.4 mM KCl, 1.2 mM Na₂HPO₄, 1.2 mM MgSO₄, 20 mM HEPES, 15 mM glucose, 1 mM CaCl₂) aerated with 100% O₂ for 5–8 min. Perfusion was then switched to nominally Ca²⁺-free Tyrode solution for 7–10 min (20 ml/min) and digestion was performed by perfusion for 12–15 min (10 ml/min) with Tyrode–enzyme solution containing 1 mg/ml collagenase type II (Worthington), 0.04 mg/ml protease type XIV (Sigma) and 1.25 µM Ca²⁺, 60 mM taurine, 8 mM D,L-glutamic acid and 2 mM D,L-carnitine. Digestion was stopped by perfusion with 100 ml Tyrode solution containing 50 µM Ca²⁺, 2% fatty acid-free type V albumin (Sigma) and 20 mM 2,3-butanedione monoxime (BDM). Atria were cut off and the ventricles were immersed in Tyrode solution containing 20 mM BDM and 50 µM Ca²⁺. The heart was cut into chunks and myocytes were freed by four rounds of mincing and gentle manual agitation. The myocytes were filtered through a sterile nylon gauze (200-µm mesh) and progressively exposed to increasing Ca²⁺ concentrations in Tyrode solution. The final suspension was laid on top of a 6% albumin–M199 medium supplemented with 5 mM D,L-carnitine, 5 mM taurine, 5 mM creatine and antibiotics. After sedimentation, myocytes were plated onto laminin (20 µg/ml)-coated tissue culture plates at a density of 0.5x10⁵ rod-shaped cells/cm². After 4 h, unattached cells were removed by a single wash step with supplemented M199 medium.

Adenoviral infection of myocytes was carried out 16 h after plating by infecting the cells with either Ad-LacZ or Ad-PKC for 1 h at 37°C in a minimal volume of culture medium. Cells were washed twice with supplemented M199 medium and cultured for additional 48 h prior to analysis.

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.3 Myocyte shortening measurements
Cardiomyocytes plated on laminin-coated 35-mm culture dishes were placed in a custom-made, heat-thermostated chamber on a Nikon microscope stage. Myocytes were superfused at a flow-rate of 2.5–3 ml/min with 100% O₂-aerated Tyrode II solution (10 mM HEPES, pH 7.40, at 37°C, 140 mM NaCl, 6 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, 3 mM pyruvate) and electrically stimulated at 1 Hz at about 120% above threshold (typically 4–9 V) by means of platinum electrodes. The image of the myocyte was recorded on a Phillips CCD camera and displayed on a TV monitor. Myocyte shortening was measured by an edge-detection system (Crescent Electronics) at a sampling rate of 240 Hz. On- and off-line analysis was performed with custom-designed LABVIEW software (National Instruments).

Cells selected for data analysis had clear striation, rod-shaped form, stable diastolic length at baseline and a 1:1 pacing capture. The inotropic effects of ET-1 were studied in a cumulative fashion, after a stable baseline twitch amplitude was obtained in a single myocyte. At the end of the dose–response curve the inotropic capacity of the myocyte was tested by exposing the cell to 10^–7 M isoproterenol: all the myocytes exhibited a strong positive inotropic response, i.e. at least a 100% increase in fractional shortening (FS).

2.4 Measurement of Ca²⁺ transients
Myocytes were loaded with fluo-3 acetoxymethylester (AM) (Molecular Probes) at a final concentration of 15 µg/ml for 10 min at room temperature in the culture dish. After loading, the culture dish was mounted on the heated stage of an inverted microscope (Zeiss) and cells were washed for 5 min. Then they were field-stimulated at 0.5 Hz and intermittently illuminated with a 75 W xenon lamp at a wavelength of 480 nm. The emission signal was split in a 505 nm dichroic mirror. Intracellular Ca²⁺ transients were detected at an emission wavelength of 530 nm with a photomultiplier (PMT Hamamatsu R-1527) in photon counts/s and sent to a photometer (Pacific Instruments). The system background was measured by moving the myocyte out of the field and subtracted on an analogue divider. The systolic Ca²⁺ transient was represented as pseudo ratio and calculated as F/F₀ (F=F_total–F_background/F₀).

2.5 Western-immunoblot analysis
For Western-immunoblot analysis, Ad-LacZ and Ad-PKC infected myocytes were homogenised by sonication in lysis buffer (150 mM Tris–Cl, pH 7.4, 2 mM EGTA, 1 mM phenylmethylsulfonylfluoride, 0.05 mM leupeptin, 1 mM iodacetamide, 1 µg/ml aprotinine and 1% Triton X-100). After centrifugation to remove cell debris, protein concentration in the supernatant was determined using the bicinchoninic acid assay (Pierce). A 20-µg amount of total protein was subjected to SDS–polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes by semidry electroblotting. Western-immunoblot analysis of Ad-LacZ and Ad-PKC infected myocytes was performed with a polyclonal anti-PKC (C-15) antibody (Santa Cruz Biotechnology) and monoclonal anti-PKC subtype specific antibodies (Transduction Labs.) and an enhanced chemoluminescence detection system (Amersham) according to the manufacturer's instructions.

2.6 Immunoprecipitation and in vitro kinase assay of PKC
For immunoprecipitation of PKC, 5x10⁵ myocytes infected with either Ad-LacZ at a multiplicity of infection (MOI) of 100 or Ad-PKC at a MOI of 1, 10 and 100 plaque-forming units (pfu)/myocyte were washed three times in ice-cold Dulbecco's phosphate buffer saline (PBS) at 48 h after transfection and were lysed in RIPA buffer (PBS containing 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM phenylmethylsulfonylfluoride, 0.05 mM leupeptin, 1 µg/ml aprotinine, 100 mM sodium orthovanadate and 1 mM sodium fluoride). PKC was immunoprecipitated at 4°C for 4 h with 1 µg/ml of a polyclonal anti-PKC antibody (C-15, Santa Cruz Biotechnology). Immunocomplexes were isolated using protein A–Sepharose beads and washed twice with RIPA buffer and once with kinase buffer (20 mM Tris–Cl, pH 7.5, 5 mM MgSO₄, 2 mM EGTA). Immunocomplexes were resuspended in 30 µl kinase buffer were mixed with 10 µl kinase buffer containing 100 µM final concentration of ATP, 125 µg/ml phosphatidylserine (PS, Sigma), 2.5 µM phorbol-12,13-dibutyrate (PDBu, Sigma), 500 µg/ml PKC peptide as substrate (Biomol), 0.37 MBq [-³²P]ATP (185 TBq/mM, Amersham) and incubated at 30°C for 20 min. The reaction was terminated by adding 100 µl of 75 mM phosphoric acid. The mixture was applied to P81-phosphocellulose units (Pierce) and radioactivity incorporated into PKC peptide was measured by liquid scintillation counting.

2.7 β-Galactosidase assay
Ad-LacZ infected myocytes were washed twice with PBS and fixed with 1% glutaraldehyde in PBS for 10 min at room temperature. The fixative was removed by two wash steps with PBS and cells were stained in PBS (adjusted to pH 8.5) containing 5 mM K₄Fe(CN)₆, 5 mM K₃Fe(CN)₆, 1 mM MgCl₂ and 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) for 2 h at 37°C.

2.8 Statistical analysis
All data are presented as means±S.E.M. Normality of data distribution was analysed by the Kolmogorov–Smirnov procedure. In case of non-normal distribution, data were either log- or square root-transformed and statistical tests were performed on the transformed data. Dose-dependent effects of the compounds on cell shortening parameters were tested by two-way repeated measure analysis of variance (RM-ANOVA), with the concentration as the repeated factor and group (Ad-LacZ vs. Ad-PKC myocytes) as the second factor. A differential effect of the pharmacological compound on a given parameter was indicated by a significant interaction term. When the two-way RM-ANOVA indicated a significant overall concentration effect, a one-way RM-ANOVA on dose effects was independently performed for each group. When the F-test was not significant, the parameter value at all concentrations was averaged; when the F-test was significant, multiple pairwise comparisons were performed between each concentration and the control situation, using the Dunn and Dunnett corrections par non-parametric and parametric ANOVA, respectively. The probability level was set at 5% and all statistical tests were performed with SIGMASTAT software (v2.03, SPSS Science).

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
3.1 Overexpression of PKC in rabbit ventricular myocytes
The transfection efficacy of the adenoviral vector on isolated rabbit ventricular myocytes was initially verified using the Ad-LacZ construct carrying the β-galactosidase gene. As evidenced through β-galactosidase staining, virtually 100% of the myocytes were successfully transfected at a MOI of 100 pfu/myocyte (Fig. 1). The Ad-LacZ virus was further used as a negative control to exclude possible intrinsic effects of the vector itself. Gross morphology of the myocytes was unchanged by either Ad-LacZ or Ad-PKC infection when compared with non-infected myocytes.

View larger version (41K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Transfection efficiency of adenoviral gene transfer in isolated rabbit ventricular myocytes. Myocytes were infected with Ad-LacZ at a MOI of 1, 10 and 100 and were stained for β-galactosidase activity after 48 h culture time as described in Methods. At a MOI of 100 virtually all myocytes were successfully transfected as evidenced through blue staining of the cells (magnification 100x).

 
PKC protein expression was analysed in Ad-PKC_infected myocytes by Western-immunoblotting. Increasing amounts of virus per myocyte resulted in a strong, virus dose-dependent increase in PKC protein expression (Fig. 2A). Equal protein load in each lane was demonstrated by immunological detection of the myocyte specific protein calsequestrin. The specificity of PKC overexpression to the isoform was important to investigate because of previous reports on PKC-mediated control of activity and expression of other PKC isoforms [23–26]. Fig. 2B demonstrates that the expression pattern of other PKCs, such as PKC, , , and PKD, the mouse homologue of human PKCµ [27], was not modified by overexpression of PKC. Therefore, adenovirus-mediated gene transfer of PKC in primary cultures of adult ventricular myocytes was PKC isoform-specific and highly efficient.

View larger version (48K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 PKC protein levels in transfected cardiomyocytes. (A) Western-immunoblot analysis showing PKC protein levels in cardiomyocytes infected with control adenovirus (Ad-LacZ, MOI 100) or Ad-PKC for 48 h with a MOI of 1, 10 and 100. Equal protein load in each lane is indicated by detection of the myocyte specific protein calsequestrin (CS). (B) Western-immunoblot analysis showing PKC isoform protein levels in Ad-PKC (+) or Ad-LacZ (–) transfected cardiomyocytes at a MOI of 100 each.

 
Functional assessment of PKC overexpression was provided by in vitro kinase assay of immunoprecipitated material from Ad-PKC myocytes using PKC specific peptide as substrate. Fig. 3 shows that, compared to Ad-LacZ myocytes, overexpression of PKC was accompanied by a marked, MOI-dependent increase in basal, cofactor-independent as well as in maximal kinase activity (Fig. 3). Since PKC displays low-level kinase activity in the non-activated state, overall PKC enzyme activity is markedly increased in the transfected myocytes even in the absence of an extracellular stimulus.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 In vitro kinase activity of PKC immunoprecipitated from Ad-LacZ- (MOI 100) and Ad-PKC- (MOI 1, 10 and 100) transfected rabbit cardiomyocytes. PKC was immunoprecipitated from cardiomyocytes infected with control adenovirus (Ad-LacZ) or Ad-PKC for 48 h and equal amounts of immunocomplexes were subjected to in vitro kinase assay using PKC peptide as substrate. Basal, cofactor-independent kinase activity was determined in the absence of PS and PDBu (empty bars) and maximum kinase activity was assessed in the presence of 125 µg/ml PS and 2.5 µM PDBu (filled bars). Data represent mean values of three independent experiments and are expressed as means±S.E.M.

 
3.2 Contractile consequences of PKC overexpression in rabbit ventricular myocytes
Basal mechanical parameters of Ad-LacZ- and Ad-PKC-infected myocytes are presented in Table 1.

View this table: [in this window] [in a new window]   Table 1 Basal contractile parameters of Ad-LacZ and Ad-PKC infected adult rabbit ventricular myocytesa

 
The most noticeable result was the significant increase in myocyte FS (expressed in percentage of diastolic cell length) by 21% (P<0.05) in Ad-PKC myocytes compared to Ad-LacZ myocytes. PKC overexpression slightly but significantly prolonged twitch duration: time to peak shortening (TTPS) was increased by 12% (P<0.05), but the relengthening phase, assessed by time to 50% relengthening (RT₅₀), was not statistically significantly different between Ad-LacZ and Ad-PKC myocytes, averaging 163 ms.

ET-1 exerted a pronounced PIE only in Ad-LacZ-infected myocytes. FS maximally increased by 63% at 10^–9 M ET-1 (P<0.001) (Fig. 4). By contrast, the flat dose–response curve in Ad-PKC myocytes indicated that PKC overexpression markedly blunted ET-1-mediated PIE (P for interaction=0.021). The shortening phase of the twitch was not affected in duration, either by ET-1 or by PKC overexpression. Interestingly, a slight but statistically significant interaction (P=0.039) was detected for RT₅₀, which was due to a trend for dose-dependent acceleration of relengthening in Ad-LacZ myocytes and slowing in Ad-PKC myocytes; yet, no concentration effect could be isolated for any group.

View larger version (15K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Dose-dependent effects of ET-1 on twitch amplitude and time course of Ad-LacZ- (basal, n=29; ET-1, n=8 each) or Ad-PKC- (basal, n=36; ET-1, n=13 each) infected cultured adult rabbit ventricular myocytes (MOI 100 each). (Upper panel) positive inotropic effect of ET-1 recorded in Ad-LacZ-infected was significantly blunted in PKC-overexpressing myocytes (p for interaction=0.021). (Middle panel) TTPS was not dose-dependently affected by ET-1, nor was this pattern affected by PKC overexpression. (Lower panel) ET-1 exhibited differential dose-concentration-dependent effects on the relengthening phase, assessed by RT50%, in Ad-LacZ and Ad-PKC infected myocytes (P for interaction=0.039): relaxation became faster in the former while ET-1 tended to slow relaxation in PKC-overexpressing myocytes. #, and ##, P<0.01 and P<0.001, respectively (one-way RM-ANOVA on ET-1 concentration-dependent effects). *, P<0.05 compared to basal (multiple pairwise comparisons; Dunnett correction). Data represent the means±S.E.M.

 
3.3 [Ca²⁺]_i transients in PKC overexpression rabbit ventricular myocytes
In a separate set of experiments, we investigated whether the positive inotropic effect of PKC overexpression was paralleled by a change in intracellular Ca²⁺-handling. Myocyte shortening and [Ca²⁺]_i transients were simultaneously recorded in flou-3 AM loaded myocytes at 48 h after adenovirus infection. Representative recordings in Fig. 5 show that, compared to Ad-LacZ-infected cells, the increase in FS of Ad-PKC-infected myocytes was accompanied by an increased systolic Ca²⁺ transient amplitude. Average results presented in Table 2 demonstrate that the amplitude of [Ca²⁺]_i transients (quantified by F/F₀) was increased by 21%, from 2.04±0.34 in Ad-LacZ-infected myocytes to 2.48±0.16 in Ad-PKC-infected cells (P=0.026). Table 2 also shows that 10^–9 M ET-1 slightly increased, although non-significantly compared to basal, [Ca²⁺]_i transient amplitude in Ad-LacZ-infected myocytes, whereas 10^–9 M ET-1 did not further increase [Ca²⁺]_i transient amplitude in PKC-overexpressing myocytes.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Representative [Ca2+]i transients (assessed by fluo-3 fluorescence records; top traces) and corresponding shortening (bottom traces) in Ad-LacZ- (left traces) and Ad-PKC- (right traces) infected rabbit ventricular myocytes 48 h after transfection (both types of myocytes were infected with a MOI 100 of the respective adenovirus). In the basal situation, [Ca2+]i transient and shortening amplitudes were lower in the LacZ group compared to PKC-overexpressing myocytes. ET-1 at a concentration of 10–9 M increased both cell shortening and [Ca2+]i transient amplitudes only in the LacZ myocytes; F=Ftotal–Fbackground/F0.

 

View this table: [in this window] [in a new window]   Table 2 [Ca2+]i transient amplitudes (F/F0) in Ad-LacZ and Ad-PKC infected adult rabbit ventricular myocytesa

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 
In the present study, we used adenovirus-mediated gene transfer to overexpress PKC in primary cultures of adult rabbit ventricular myocytes. The specificity of PKC overexpression was demonstrated by the unchanged expression pattern of other PKC isoforms, i.e. PKC, , and µ, which are significantly expressed in the rabbit heart [12,13]. Overexpression of PKC in cultured adult rabbit ventricular myocytes was accompanied by: (1) a marked increase in both basal and cofactor-stimulated PKC kinase activity (2) an increased baseline contractility and twitch duration (3) an increase in [Ca²⁺]_i transient amplitude and (4) a marked attenuation of the positive inotropic effects of ET-1.

Mean values in the amplitude of cell shortening were comparably low in both Ad-LacZ- and PKCe-infected myocytes which can be most likely attributed to the long culture time of the myocytes (60 h prior to analysis); in fact, freshly isolated myocytes classically shorten by about 6–12% of their diastolic length ([28–30]; our own observations).

The increased [Ca²⁺]_i transient amplitude most likely accounts for the positive inotropic effect of basal PKC overexpression. Our results are the opposite of those from RA. Takeishi et al. demonstrated decreased amplitude of contraction and [Ca²⁺]_i transients in ventricular myocytes from PKC-overexpressing transgenic mice [19]. Species-differences as well as different duration of transgene expression may explain these apparent contradictory results. Nevertheless, both studies clearly demonstrate that PKC can directly modulate cardiac E–C coupling. A positive inotropic effect upon activation of PKC has also been demonstrated by Pi and Walker. They found that diacylglycerol, the key lipid cofactor for activation of cPKC and nPKC and diC₈, a diacylglycerol analogue, together with cis-unsaturated fatty acids, act synergistically to increase cell shortening and [Ca²⁺]_i transient amplitude in adult rat ventricular myocytes [31,32]. These effects could be blocked by PKC inhibitors.

A limitation of this study is that we have no information on which proteins are being phosphorylated by PKC in our experimental system. The identity of target proteins of PKC is a matter of intense research, because of the increasingly clear role of this PKC isozyme in ischemic preconditioning [33]. In transgenic mice overexpressing a PKC-specific translocation activator peptide (RACK), a decrease in L-type Ca²⁺ current (I_Ca) was observed [18]. Thus, it is unlikely that the increase in [Ca²⁺]_i transient amplitude is due to an increased Ca²⁺-influx via the L-type Ca²⁺ channel.

Independent from regulation of Ca²⁺ homeostasis, phosphorylation of target proteins leading to an increase in myofibrillar Ca²⁺ responsiveness may also contribute to the positive inotropic effect of PKC overexpression. Increased myofibrillar Ca²⁺ responsiveness can be achieved indirectly by intracellular alkalinisation following activation of the sarcolemmal sodium/proton exchanger by PKC-mediated phosphorylation [34]. However, the identity of the respective PKC isoform is still unknown. Additionally, PKC phosphorylates troponin I in vitro and most likely in vivo [35,36]. Yet, increased troponin I phosphorylation in PKC-overexpressing myocytes would be expected to exert a negative rather than a positive inotropic effect [37]. The exact mechanisms by which PKC modulates cardiac excitation–contraction coupling will require in-depth characterisation of its target proteins. Nevertheless, the net inotropic effect of PKC overexpression will be determined by the balance between inhibitory and stimulating influences.

ET-1 exerts PIE in ventricular preparations from several species, the rabbit being the most sensitive [1–4,38]. The underlying cellular mechanisms may be ET-1-mediated intracellular alkalinisation, increased myofibrillar calcium responsiveness and/or potentiation of the calcium current I_Ca,L [4,38]. The observed moderate increase in [Ca²⁺]_i transient amplitude after ET-1 stimulation in the LacZ control cells in the present study is in accordance with previous work showing that the ET-1 mediated increase in [Ca²⁺]_i transient amplitude is much smaller than that produced by elevation of [Ca²⁺]_o or isoproterenol for a given extent of PIE [38]. Our observations of blunted inotropic and [Ca²⁺]_i amplitude responses to ET-1 in PKC-overexpressing myocytes were contrary to our expectation of a potentiation of ET-1 effects on contractility. The mechanisms underlying these blunted effects remain speculative but it seems logical to assume that the marked increase in basal PKC kinase activity would lead to saturation of phosphorylation sites of PKC target proteins, thereby preventing further phosphorylation in the presence of PKC-mobilising compounds such as ET-1. The identity of PKC targets is still elusive but in the context of the inotropic effects of ET-1, recent data demonstrated PKC-mediated ET_A receptor desensitisation in the rat right atria, although the PKC isoform responsible for this effect was not specified [39]. In our experimental set-up, we have no indication that ET_A receptors are downregulated upon overexpression of PKC (data not shown).

In summary, we demonstrated that PKC is directly involved in ET-1 signalling and we provided a first mechanistic insight into the positive inotropic effect exerted by PKC overexpression.

Time for primary review 35 days.

	Acknowledgements

 
We thank H. Mischak for providing us with the cDNA for mouse PKC. We also want to thank W.H. Dillmann for providing us with Ad-LacZ and the plasmids pACCMV·pLpASR^– and pJM17. The authors gratefully acknowledge precious advises in myocyte isolation by S. Lehnart. S. Baudet wants to personally thank all of his colleagues for their constant support and the good time he had in Freiburg.

	Notes

 
¹ Present address: Stéphane Baudet, Intervet Pharma R&D, Internal Medicine II, BP 67131, F-49071 Beaucouzé Cedex, France.

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Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion References

 

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