Cardiotrophic Effects of Protein Kinase C ε
Analysis by In Vivo Modulation of PKCε Translocation
Abstract—Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (εV1) and translocation activator (ψεRACK [receptors for activated C kinase]) specifically targeting PKCε. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCε signaling. As expected for a PKCε RACK binding peptide, confocal microscopy showed that εV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCε by εV1 at lower expression levels upregulated α–skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, εV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCε translocation with ψεRACK resulted in selectively increased β myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCε signaling in normal postnatal maturational myocardial development and suggest the potential for PKCε activators to stimulate “physiological” cardiomyocyte growth.
Protein kinase Cs (PKCs) constitute a large family of phospholipid-dependent serine-threonine kinases with pleiotropic effects. In the heart, clinical and experimental studies have identified a number of conditions such as pathological cardiac hypertrophy, heart failure, and ischemic preconditioning in which PKCs are activated.1 2 3 4 5 Despite these known associations and the importance of PKCs as essential and ubiquitous signaling molecules, their physiological or pathophysiological roles in the heart and other tissues have yet to be established.
In defining the consequences of cardiac PKC activation, one is confronted with the existence of multiple PKC isozymes, each with the potential for distinct physiological and pathological effects. A functional characteristic that distinguishes between different PKC isozymes is the pattern of subcellular redistribution on activation.6 This subcellular redistribution of activated PKC isozymes is a critical determinant of substrate specificity by enforcing proximity of activated isozymes to select substrates. The mechanism for PKC translocation involves recognition and binding of activated PKCs to isozyme-specific anchor proteins collectively termed receptors for activated C kinases, or RACKs.7 Recently, PKC peptides derived from PKC RACK binding or pseudo-RACK sites have been introduced into cardiomyocytes and other cell types, where they act as isozyme-specific translocation inhibitors and activators, respectively.7 8 9 10 In light of these developments, we reasoned that biological roles for individual PKC isozymes could be established by targeted in vivo activation or inhibition of selected endogenous PKCs using peptide translocation modifiers expressed as transgenes. An advantage of this approach is that PKC activity is modified in an isozyme-specific manner, without experimentally altering the stoichiometry of PKC, its upstream activators, or downstream effectors. We recently utilized this approach to create transgenic mice in which endogenous cardiomyocyte PKCε was modestly activated by transgenically expressing the novel PKCε-specific translocation enhancer peptide ψεRACK.10 Ten-week-old mice expressing the ψεRACK octopeptide in cardiac myocytes exhibited increased PKCε partitioning to subcellular particulates (translocation) associated with profound resistance to transient ischemic injury. In the current studies, we have used an opposite approach, that of selectively inhibiting PKCε translocation in vivo with the εV1 peptide, to explore the necessity for PKCε activity in normal physiological postnatal cardiac development. Our results indicate that in vivo inhibition of PKCε translocation blocks an essential cardiomyotrophic function that can result in fatal cardiac insufficiency. In contrast, ψεRACK transgenic mice, in which PKCε is intrinsically activated, undergo hypertrophic cardiac remodeling while retaining normal contractile function.
Materials and Methods
All experiments were performed under a protocol approved by the Institutional Animal Care and Use Committee.
Creation of Transgenic Mice
The PKCε antagonist, corresponding to the first variable region (V1 fragment) of rat PKCε (εV1, amino acids 2 to 144), was previously described.8 An octopeptide corresponding to the pseudo-RACK sequence of rat PKCε (ψεRACK, amino acids 85 to 92) was recently identified as a selective PKCε translocation activator.10 For transgenic expression, the cDNA for each peptide, preceded by an 8–amino acid FLAG epitope,10 was directionally cloned into exon 3 of the full-length mouse α myosin heavy chain (MHC) promoter.11 After separation from vector backbone, transgene constructs were injected into male pronuclei of fertilized FVB/N mouse oocytes. αMHC-FLAG-εV1 (εV1) and αMHC-FLAG-ψεRACK founders were identified by genomic Southern analysis of tail clip DNA.
PKC isozyme expression and translocation were measured by quantitative immunoblot analysis with anti-PKCα (Santa Cruz Biotechnology) and anti-PKCε (Transduction Laboratories) as previously described5 using recombinant human PKCα and PKCε (Calbiochem) as quantitative standards. Western blots were developed using chemifluorescence (Amersham) and quantified on a STORM phosphor imager system.
Detection of Transgenic Peptides
Western blot analysis of εV1 using Sigma anti-FLAG M-2 antibodies was performed using standard techniques. Immunofluorescence studies were carried out with the same monoclonal anti-FLAG antibody and detected with biotinylated anti-mouse antibody (Vector) labeled with avidin D/Texas red (Vector). Phalloidin/Oregon green was from Molecular Probes. Images were analyzed using confocal microscopy.
Assessment of Cardiac Hypertrophy and Function
Morphometric, physiological, and pathological studies utilized standard techniques exactly as previously described.5 12 Cardiac gene expression was assayed by RNA dot–Northern blot analysis of total ventricular RNA (3 μg/dot) using 32P-labeled oligonucleotide probes as described.5 12
Whole-cell currents were recorded by patch-clamp techniques as previously described.13 14 Membrane capacitance was measured using voltage ramps of 0.8 V/s from a holding potential of –50 mV. L-type Ca2+ currents (ICa) were recorded using external and pipette solutions that provided isolation of Ca2+ currents from Na+ and K+ channel currents and Ca2+ flux through the Na+/Ca2+ exchanger.
Transgenic mice and their age-matched nontransgenic (NTG) controls were compared by Student test or ANOVA as appropriate, with P<0.05 considered as significant.
An initial characterization of ψεRACK-overexpressing mice was previously reported.10 At 10 weeks of age, these animals had normal-appearing hearts, with normal contractile function and ≈20% increase in particulate-associated PKCε. Mice expressing the εV1 inhibitory peptide were created using the same full-length αMHC promoter and cloning strategy, and 2 lines were successfully propagated, designated εV1low and εV1med on the basis of transgene copy number and transgenic peptide expression. Expression of the ψεRACK and εV1 peptides was assessed by immunologic techniques using an antibody that recognizes the amino-terminal FLAG epitope. The εV1 peptide migrated at ≈15 kDa, and immunoreactive peptide expression was 83% higher in εV1med (≈40 copies) than in εV1low (≈8 copies), with no detectable immunoreactivity in NTG siblings (Figure 1⇓, top). Three additional εV1 founders and 5 first-generation mice from 2 other εV1 founders had >200 copies of the transgene; all were designated εV1high. Each of these animals died of heart failure, as described below. In 5 individual lines of mice expressing the ψεRACK peptide, transgene copy number ranged from 10 to ≈100 copies, but comparative immunoblotting was not possible because of the small size of the peptide (<2 kDa). Peptide expression was, however, demonstrated by confocal immunostaining using anti-FLAG antibody (Figure 1⇓, bottom). The irregular staining pattern for ψεRACK is presumably due to its affinity for unanchored endogenous PKCε.10
The mechanism for inhibition of PKCε by εV1 is predicted to be competition with endogenous PKCε for binding to its RACK.7 8 9 It was anticipated, therefore, that the εV1 fragment should itself bind to εRACK in the hearts of transgenic mice. Subcellular localization, assayed in εV1low and εV1med cardiac myocytes by anti-FLAG confocal immunomicroscopy, showed that εV1 decorated cell-cell contact areas and intracellular cross-striated structures (Figure 1⇑), recapitulating the pattern of subcellular translocation for activated PKCε reported in cultured neonatal cardiomyocytes6 and adult guinea pig hearts.4
Chronic expression of either PKCε translocation–modifying peptide did not affect the overall amount of PKCε or PKCα in transgenic mouse hearts (Figures 2A⇓ and 2D⇓, respectively). Compared with NTG siblings, however, the amount of PKCε (Figures 2B⇓ and 2C⇓), but not PKCα (Figures 2E⇓ and 2F⇓), associated with the particulate fraction was increased by 20±4% (n=10, P<0.05) in ψεRACK expressors, consistent with the known activity of this peptide as a facilitator of PKCε translocation.10 εV1 had the opposite effect; the amount of PKCε (Figures 2B⇓ and 2C⇓), but not PKCα (Figures 2E⇓ and 2F⇓), in the cardiac particulate fraction decreased by 15±3% compared with NTG siblings (n=10, P<0.05). Neither peptide affected the expression or subcellular partitioning of PKCζ or PKCδ (data not shown).
If PKCε plays a role in normal myocardial development, then chronic reduction in PKCε translocation and activity by εV1 should alter this function. Because transgenes under control of the full-length αMHC promoter are only transiently expressed in the embryonic ventricle,11 it was expected that phenotypic consequences of inhibiting PKCε translocation would evolve during postnatal development. As noted above, of 7 lines of εV1 transgenic mice generated, first-generation mice from 2 lines with an excess of 200 transgene copies had 100% mortality from cardiac insufficiency at an age of 27±2 days (n=5). Before death, these mice became lethargic and developed rapid respirations and cyanosis. Necropsy showed large, thin-walled ventricles (Figure 3A⇓), pulmonary congestion, and ascites. In contrast to typical models of murine dilated cardiomyopathy,15 16 histological examination revealed no evidence of cardiomyocyte dropout or fibrotic replacement but did suggest cardiomyocyte enlargement (Figure 3B⇓). These pathological characteristics and early death were also observed in 3 additional founder mice with >200 copies of the transgene. In contrast, heart size and weight of εV1low and εV1med mice were normal at an age of 15 weeks (or before), as was catheterization-derived peak rate of pressure development (dP/dtmax) and responsiveness to β-adrenergic receptor agonists (Figure 3C⇓). However, echocardiographic left ventricular fractional shortening was slightly, but significantly, depressed in εV1med mice, suggesting mild cardiac dysfunction in this line (Figure 3C⇓, Table⇓).
The above observations suggested a transgene dose effect, wherein a lower copy number of εV1 was well tolerated, an intermediate copy number was associated with subtle cardiac dysfunction, but high copy numbers were lethal at a young age because of inadequate myocardial growth in the perinatal period. It was also possible, however, that 1 or more transgene insertional events were responsible for early lethality, rather than transgene dosage effects of εV1. To distinguish between these 2 possibilities, εV1low and εV1med mice were crossbred to generate dual-transgenic mice with increased levels of εV1 peptide, but without multiplying the effects of transgene insertion. All such dual-transgenic εV1 mouse pups died of heart failure between 25 and 33 days of age with dilated, thin-walled hearts appearing identical to those of εV1high mice (n=6). These studies therefore establish a transgene dose-dependent inhibitory effect of εV1 on normal postnatal myocardial development, the extreme consequence of which can be cardiac failure from myocardial insufficiency.
A common feature of many forms of heart failure is ventricular expression of embryonic cardiac genes.5 17 18 19 Activation of this molecular program is a highly sensitive indicator of myocardial disease and may actually anticipate physiological deterioration. Therefore, levels of these genes were quantified from ventricular RNA of εV1low and εV1med mice. Expression of α–skeletal actin mRNA increased in proportion to the level of expressed εV1 peptide (Figure 3D⇑). No significant change in expression of atrial natriuretic factor (ANF) or a number of other cardiac genes known to be regulated in heart failure was seen in εV1low. βMHC mRNA expression was, however, modestly increased in εV1med hearts, possibly representing a compensatory response to diminished ventricular function (Figure 3C⇑). Taken together, the morphometric, molecular, and functional characteristics of εV1high/med/low and εV1med×low mice are consistent with the notion that a threshold level of PKCε activity is required for normal postnatal myocardial growth.
The antithesis of cardiac PKCε inhibition with εV1 is increased PKCε translocation and activity by ψεRACK.10 Five independent lines of ψεRACK mice displaying essentially identical characteristics were studied. As illustrated in Figure 4B⇓, 8-week-old ψεRACK hearts were normal in size, weight, and function. At 15 weeks, however, ψεRACK hearts were significantly larger than their NTG littermates (Figures 4A⇓ and 4B⇓ and Table⇑). Unlike other forms of cardiac hypertrophy caused by transgenic modification of this signaling pathway,5 20 21 left ventricular systolic and diastolic function measured in ψεRACK mice by echocardiography or in vivo using microminiaturized catheterization techniques were normal (Figure 4B⇓). In a small cohort of ψεRACK mice followed for 6 months, echocardiographic function remained normal (data not shown). Cardiac response to β-adrenergic stimulation was also normal in ψεRACK mice (Figure 4B⇓).
A molecular characteristic of ψεRACK mice that distinguishes it from previously reported forms of murine cardiac hypertrophy caused by activation of endogenous signaling pathways5 17 18 19 20 22 is that ANF gene expression was not increased in ψεRACK hearts. Furthermore, and in contrast to εV1 hearts, α–skeletal actin gene expression was not increased in ψεRACK hearts. Instead, βMHC gene expression was dramatically increased in ψεRACK hearts (Figure 4C⇑).
To examine the effects of PKCε translocation inhibition and activation on individual ventricular cardiac myocytes, independent of chamber geometry and in vivo neurohormonal status, whole-cell patch-clamp studies were performed. Myocyte size, measured as cell capacitance, was significantly smaller than NTG in ψεRACK ventricles, but larger in εV1 ventricles (Figure 5A⇓). In the context of the observed increase in cardiac mass and ventricular wall thickness of ψεRACK mice (Figure 4⇑ and Table⇑), decreased myocyte size suggests an increase in ventricular myocyte number. In contrast, increased cell size of εV1 myocytes is consistent with decreased myocyte number, given that cardiac mass and wall thickness are normal (Figure 3⇑ and Table⇑).
Because previous studies have suggested that PKC activity could regulate Ca2+ channel activity in the heart, we examined L-type Ca2+ current, ICa. Figure 5C⇑ shows representative ICa from NTG, ψεRACK, and εV1med. myocytes. ψεRACK cells had a significantly decreased ICa compared with NTG (Figures 5B⇑ and 5C⇑). In contrast, robust ICa was present in εV1 (Figures 5B⇑ and 5C⇑). There was no change in the current-voltage relationships among the 3 groups (not shown). In all groups, ICa activated around –30 mV and reached its maximum near +10 mV. At the maximum potential, ICa inactivated rapidly during maintained depolarization, but in ψεRACK cells the time to half-decay of the current was prolonged (NTG=18.4±0.6 ms [n=87] versus 21.3±1.1 ms [n=53] ψεRACK; P<0.05); there was no significant change in εV1 myocytes (17.1±1.1 ms, n=29). These differences may, however, reflect the size of Ca2+ influx rather than any change in sarcoplasmic reticulum Ca2+ release, given that the small Ca2+ current amplitude could induce smaller Ca2+-induced Ca2+ inactivation. In either case, these studies demonstrate additional opposing effects of myocardial PKCε translocation activation and inhibition on cardiomyocyte size and function.
The current studies utilized small, catalytically inactive PKCε-derived peptides to selectively modify PKCε subcellular localization, and hence activity in the in vivo mouse heart. The most significant finding is demonstration of a necessary and sufficient role for PKCε during myocardial growth of the normally developing postnatal mouse. Importantly, although transgenic techniques were used to deliver the εV1 and ψεRACK peptides specifically to cardiac myocytes, these are not transgenic studies in the conventional sense, as no active enzyme was overexpressed. Rather, the interaction between PKCε and its membrane anchor proteins, RACKs, was modulated. This had the effect of altering PKCε subcellular trafficking, and hence access to substrates, without affecting its expression level. The resulting changes in basal cardiac PKCε activity were relatively subtle, ie, a 20% increase or 15% decrease in particulate-associated enzyme, and perhaps therefore more physiological than brute force overexpression. This approach of using transgenesis to express an inactive peptide that specifically modifies the activity of a particular signaling pathway without altering the expression of component signaling transducers was previously used by Akhter et al23 to demonstrate that inhibition of receptor-Gαq interactions prevented pressure-overload hypertrophy, thus establishing a necessary role for Gαq signaling in cardiac hypertrophy even though ablation of the Gαq gene has no cardiac phenotype.24 Likewise, the current study establishes a requirement for PKCε activity in cardiomyocyte growth. Consistent with this notion, ψεRACK mice in which enhanced PKCε activity was achieved while the natural stoichiometric relationships between PKC isozymes were maintained, developed increased myocardial mass.
Although it is formally possible that εV1 and ψεRACK modified cardiac growth in our studies through a mechanism(s) other than the predicted alteration of subcellular PKCε trafficking, there is compelling evidence supporting the biochemical activity and isozyme selectivity of these peptides. We have previously shown that εV1 introduced into neonatal cardiac myocytes selectively competes with PKCε for binding to its RACK.8 Under these conditions, εV1 also inhibits phorbol 12-myristate 13-acetate–induced translocation of PKCε, but not PKCδ or PKCβ. The opposite effects were induced by the ψεRACK peptide, which facilitates PKCε translocation to the cardiomyocyte particulate fraction, but does not translocate PKCβ, PKCδ, or PKCη.10 Moreover, the biological effects of ψεRACK were prevented by inhibition of PKC catalytic activity as well as by selectively inhibiting translocation of PKCε translocation, but not by inhibiting the classical PKCs.10
Increased myocardial growth and ventricular remodeling in ψεRACK mice differs in important aspects from the cardiac hypertrophy of transgenic mice overexpressing Gαq, in which PKCε is also activated.5 25 Most striking is normal left ventricular systolic function measured in ψεRACK hearts in vivo or in vitro. This may in part be a consequence of normal responsiveness to β-adrenergic agonists in ψεRACK mice, which contrasts with impaired β-adrenergic receptor signaling in Gαq overexpressors. Indeed, we recently found that modestly increasing cardiac β2-adrenergic receptor expression in Gαq mice improved cardiac function, diminished hypertrophy, and normalized ANF and α–skeletal actin but not βMHC gene expression.22 It is therefore of interest that the “normalized” pattern of gene expression in Gαq/β2AR overexpressors (isolated increase of βMHC without increased ANF or α–skeletal actin) is the same pattern we observed in ψεRACK mice. These data, together with the current studies, suggest that βMHC expression is not the sole determinant of contractile depression in cardiac hypertrophy and support a role for altered Ca2+ signaling26 or βAR responsiveness5 22 in ventricular dysfunction caused by activation of proximal signaling effectors, such as Gαq.
The cellular consequences of PKCε translocation modification confirmed opposing effects of PKCε activation and inhibition on L-type Ca2+ channel function and support the proposition that PKCε signaling can acutely regulate cardiomyocyte function. Perhaps of more relevance to the cardiac phenotypes of these transgenic animals, however, is the indication of smaller ventricular myocyte size in the “hypertrophied” ψεRACK hearts, and larger myocytes in the “hypotrophied” εV1 hearts. A likely explanation is that PKCε signaling contributes to the normal increase in cardiomyocyte number that occurs during early postnatal development, ie, that the ψεRACK “hypertrophy” phenotype is really a consequence of cardiomyocyte hyperplasia. Conversely, ventricular dysfunction and dilated cardiomyopathy in εV1 mice may be a result of inadequate developmental cardiomyocyte hyperplasia. Consistent with this notion is the histological appearance of massively enlarge cardiomyocytes in failing εV1high hearts (Figure 3B⇑). However, further studies are necessary to make a definitive determination.
A fundamental difference in the ψεRACK phenotype and previously described forms of PKC-induced cardiac hypertrophy becomes apparent by comparison with transgenic mice overexpressing PKCβ2, in which pathological hypertrophy is associated with depressed echocardiographic fractional shortening, impaired β-adrenergic receptor function, and myocyte replacement fibrosis.20 Activation of endogenous PKCε by ψεRACK clearly results in a more physiological type of myocardial growth. Differences in experimental design make it impossible to conclude, however, that the distinct phenotypes of PKCβ2 and ψεRACK mice result solely from unique, isozyme-specific PKC functions. PKCβ2 overexpression increased PKC activity 500% to 1000%, compared with a 20% increase in active PKCε in ψεRACK mice. Furthermore, PKCβ2 expression upregulated PKCα, whereas no such collateral effect on this PKC isozyme was seen in ψεRACK mice. Thus, different phenotypes in these 2 models may simply be a consequence of vastly different PKC signaling activities resulting from overexpression versus modulated translocation.
Prior reports of increased PKCε translocation in pressure overload and Gαq-mediated hypertrophy have concluded that PKCε, rather than acting as an agent of “physiological” cardiac growth as reported herein, mediates “pathological” hypertrophy.5 27 We propose that activation of PKCε in these latter cases is indeed a compensatory mechanism that increases muscle mass, but is accompanied by deleterious events (possibly mediated by other PKC isozymes) that ultimately cause cardiac failure. On the basis of current results, it should be feasible to selectively augment the activity of myocardial PKCε in cardiac diseases, such as dilated or ischemic cardiomyopathy, where cardiac insufficiency could be reversed by an increase in healthy myocardial mass. In this regard, a PKCε translocation activator might be used in addition to chronic exercise training or growth hormone to increase cardiac muscle in heart failure.28 29 30 It remains to be determined whether the broad paradigm of altering PKC isozyme function by modulating PKC translocation will also have therapeutic potential in other diseases and organ systems in which specific PKC isozymes are pathological mediators.
This study was supported by National Institutes of Health Grants HL58010 and HL52318 to G.W.D. and HL52141 to D.M.-R.
↵1 Both authors contributed equally to this study.
- Received March 17, 2000.
- Accepted April 17, 2000.
- © 2000 American Heart Association, Inc.
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