Published online before print November 6, 2003, doi: 10.1161/01.RES.0000105087.79373.17
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© 2003 American Heart Association, Inc.
Integrative Physiology |
Protein Kinase C
Negatively Regulates Systolic and Diastolic Function in Pathological Hypertrophy
From the Departments of Internal Medicine/Cardiology (H.S.H., Y.M., A.O., A.S., M.G.Y., K.C.H., G.W.D.), Institute of Molecular Pharmacology and Biophysics (I.B., A.S.), and Internal Medicine/Pulmonary (S.B.L.), University of Cincinnati Medical Center, Cincinnati, Ohio.
Correspondence to G.W. Dorn II, Division of Cardiology, University of Cincinnati Medical Center, 231 Albert B. Sabin Way, Cincinnati, OH 45267-0542. E-mail dorngw{at}ucmail.uc.edu
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Abstract |
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The protein kinase C (PKC) family is implicated in cardiac hypertrophy, contractile failure, and ß-adrenergic receptor (ßAR) dysfunction. Herein, we describe the effects of gain- and loss-of-PKC
function using transgenic expression of conventional PKC isoform translocation modifiers. In contrast to previously studied PKC isoforms, activation of PKC failed to induce cardiac hypertrophy, but instead caused ßAR insensitivity and ventricular dysfunction. PKC inhibition had opposite effects. Because PKC is upregulated in human and experimental cardiac hypertrophy and failure, its effects were also assessed in the context of the Gq overexpression model (in which PKC is transcriptionally upregulated). Normalization (inhibition) of PKC activity in Gq hearts improved systolic and diastolic function, whereas further activation of PKC caused a lethal restrictive cardiomyopathy with marked interstitial fibrosis. These results define pathological roles for PKC as a negative regulator of ventricular systolic and diastolic function.
Key Words: systolic and diastolic heart failure • protein kinase C • interstitial fibrosis • restrictive cardiomyopathy
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Introduction |
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Cardiomyocytes challenged with increased hemodynamic load or neurohormonal stress respond by hypertrophying. A better understanding of the mechanisms that transduce hypertrophy has implications for developing strategies that can prevent functional decompensation. One important signal transducer for development and decompensation of pressure overload cardiac hypertrophy has been determined to be G
q, the -subunit of the Gq heterotrimeric G protein.1,2 Coupled to phospholipase C, Gq is the common signal transducer for multiple heptahelical cardiomyocyte receptors that stimulate cardiomyocyte hypertrophy, such as angiotensin II, endothelin, -adrenergic agonists, and prostaglandin F2.3–6 In vivo genetically manipulated mouse models demonstrate that Gq signaling is both necessary and sufficient for pressure overload hypertrophy,2,7,8 and some critical downstream mediators have been defined.9
Activated by Gq through phospholipase C, the protein kinase C (PKC) family of ubiquitous serine-threonine kinases has long been implicated in cardiac hypertrophy and, more recently, human heart failure.10 The superfamily of twelve PKC isoforms is divided into three subfamilies based on functional and structural characteristics11,12: "conventional" PKCs (cPKC), which include PKC, ßI, ßII, and 
, require both calcium and phospholipid for activation, whereas "novel" PKCs (nPKC), which include PKC
and 
, require phospholipid, but not calcium (atypical PKCs are activated by phosphatidyl serine, and may represent a separate class altogether). Global knockouts of individual PKC isoforms have generally failed to define critical cardiac functions (likely due to opportunistic compensation by related isoforms13–16), whereas overexpression or activation of PKC isoforms has demonstrated that PKCß, , and can each stimulate cardiac hypertrophic phenotypes.1,17–21 PKC has yet to be examined as a mediator of cardiac hypertrophy or failure by either in vivo gain- or loss-of-cardiac-specific function approaches, despite being the most abundant isoform in myocardium, in vitro evidence that it can regulate cardiac myocyte growth,22 and its regulation in human and experimental heart failure.7,10,23
We analyzed the effects of myocardial PKC activity utilizing novel transgenic mice expressing positive and negative regulators of cPKC translocation. Although these peptides are not specific for PKC, its abundance relative to other cPKCs in adult mouse heart (>80% of myocardial cPKC) resulted in a biochemical phenotype of PKC gain and loss of function. We demonstrate that activation of PKC had no effect on hypertrophy. Instead, PKC activation diminished myocardial responsiveness to ß-adrenergic receptor agonists, whereas PKC inhibition enhanced ß-adrenergic responsiveness. In the context of Gq-mediated hypertrophy and upregulated PKC,7,23 further PKC translocation/activation caused a restrictive cardiomyopathy and premature lethality from fulminant heart failure. Gq mice carrying a cPKC inhibitory peptide had modestly improved cardiac function and normal longevity. These observations establish pathological effects of PKC in the adult heart.
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Materials and Methods |
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Generation of Transgenic Mice With Altered PKC
TranslocationTransgenic mice were generated using the cardiomyocyte-specific
-myosin heavy chain (MHC) promoter to express single and concatamerized (three copies) peptides corresponding to either the PKCßC2-C4 region (cPKC inhibitor, amino acids 218 through 226 of rat PKCßII; inhibits translocation of all cPKC isoforms) or homologous regions of PKCß and RACK1 (cPKC activator, enhances translocation of all cPKC isoforms)24 (see Figure 1A, bottom). Founders were identified by genomic Southern analysis of tail clip DNA. Animals were treated in accordance with approved University of Cincinnati IACUC protocols.
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Immunoblot Analysis
Quantitative immunoblot analysis of PKC isoform content used human recombinant PKC isoforms (Sigma) for generation of standard curves.23 PKC isoform partitioning in subcellular fractions was assayed in cytosolic (100 000g supernatant) and Triton X-100-extracted membrane (100 000g pellet) ventricular fractions size-separated on 10% SDS-PAGE gels. Proteins were visualized using enhanced chemifluorescence and quantitated using a Storm PhosphorImager. Antibodies for PKC, ßI, ßII, , , and were obtained from Transduction Laboratories.
Functional Assessments
Two-dimensional guided M-mode echocardiography of conscious unsedated mice was used to measure left ventricular (LV) diastolic and systolic dimensions (LVEDD and LVESD) and septal and posterior wall thickness (SWT and PWT), from which fractional shortening (FS) and LV mass were derived. Pulsed wave Doppler was used to measure aortic ejection time (ET) and calculate velocity of circumferential shortening, Vcf (FS/ET). Diastolic transmitral inflow Doppler indices included the following: peak E wave velocity, peak A wave velocity, E wave deceleration time, and isovolumic relaxation time (IVRT). Invasive hemodynamic studies were performed on anesthetized, spontaneously breathing 6- to 10-week-old transgenic mice, and their nontransgenic littermate controls as described.7
ßAR Density and Adenylyl Cyclase Activity
ßAR density (fmol/mg) was assayed by 125-I-cyanopindolol (CYP) binding, with nonspecific binding determined with 1 µmol/L alprenolol. Adenylyl cyclase activities were determined from membrane preparations as described.7 Reactions were performed for 10 minutes at 37°C with various concentrations of isoproterenol, 10 nmol/L NaF, or 100 µmol/L forskolin. Adenylyl cyclase activities are reported as pmol/min per milligram and as a unitless value after normalization to NaF and forskolin stimulation.
Isolation of Ventricular Myocytes and Electrophysiology
Cardiomyocytes were enzymatically dissociated from ventricles of 4-month-old nontransgenic (NTG) and transgenic mice. Current recordings were obtained in the whole-cell, voltage-clamp configuration of the patch-clamp technique by using 1.60 OD borosilicate glass electrodes (Garner Glass Company), essentially as described.20 Cell capacitance was calculated by integrating the area under an uncompensated capacity transient elicited by a 25-mV hyperpolarizing test pulse (25 ms) from a holding potential of 0 mV.
Implantable Telemetry
Mice were anesthetized and underwent sterile implantation of radio telemetry devices (Data Science Incorporated). Continuous nontethered telemetry was recorded as streaming data to a dedicated computer hard drive for off-line analysis.
Histopathology, Immunohistochemistry, and Apoptosis Studies
Histological examination was performed on formalin-fixed Masson’s trichrome-stained sections. Collagen content of the extracellular matrix was assessed using picrosirius red staining and Image-Pro Plus software (Media Cybernetics). Apoptosis was measured using a TUNEL assay (Promega) imaged on a dual laser Nikon PCM2000 confocal system and a Nikon Eclipse E800 microscope using an emission wavelength of 515±30 nm (fluorescein).
Statistical Analysis
Results are presented as mean±SEM. Experimental groups were compared using Student’s t test or one-way ANOVA. A Bonferroni or Newman-Keuls test was applied to all significant ANOVA results using SigmaStat software. A value of P<0.05 was considered significant.
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Results |
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Effects of Cardiac-Specific cPKC Activator and Inhibitor Peptide Expression on In Vivo PKC Isoform Expression and Activation
Based on our prior experience with in vivo myocardial expression of translocation modifiers for PKC
and PKC,20,21,25–27 we initially generated multiple lines of mice expressing FLAG-epitope tagged cPKC activator and inhibitor peptides. In none of these mice was there detectable modulation of any PKC isoform translocation, nor was there any demonstrable molecular or physiological phenotype (data not shown). Confocal analysis of myocardial samples failed to demonstrate expression of the epitope-tagged peptides, despite abundant mRNA expression (data not shown), suggesting that the small peptides might be unstable. Therefore, the transgenic constructs were reengineered to express thrice-concatamerized versions of the peptides (Figure 1A) and reinjected. Multiple founders were identified for cPKC activator and cPKC inhibitor constructs, and three viable lines were established and phenotyped for each. As transgene expression, PKC translocation, and basal phenotype were similar between lines, baseline data (Table 1) were combined. All other data are from one each cPKC activator and inhibitor line.
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Because the PKCß-derived peptides had the potential to target any conventional PKC isoform,24 we performed quantitative analysis of relative PKC isoform expression in adult FVB/N mouse hearts to determine the most abundant target. As shown in Figure 1B, and consistent with prior reports,23,28 PKC was the most abundant ventricular isoform, followed by PKC. Of the cPKCs, besides PKC, only PKCßI was measurable.
The concatamerized cPKC translocation-modifying peptides had the anticipated effects on PKC subcellular partitioning, without measurable effects on PKCßI or other isoforms (likely due to low expression of the other cPKCs in ventricular tissue; Figure 1C). Compared with nontransgenic controls, basal PKC translocation in cPKC activator hearts, measured by the ratio of immunoreactive partitioning to a particulate subcellular fraction, was increased 44±1.5% (n=8, P=0.013 versus NTG), whereas in cPKC inhibitor hearts, it was decreased by 25±2.6% (n=8, P=0.043 versus NTG). This degree of basal translocation modification is somewhat greater than the 15% to 20% values for our previously described PKC and translocation modifier mice.21,27 PKC, PKCß1, and PKC content were not altered in the transgenic mouse hearts (data not shown).
PKC Translocation Enhancement Is Not Sufficient to Cause Cardiac Hypertrophy In Vivo
Multiple lines of transgenic mice expressing either the cPKC activator or inhibitor peptides were outwardly normal and healthy in all respects. Gross chamber geometry and dimensions (Figure 2A and Table 1), and histological appearance (not shown) were normal. Despite the previously noted association between PKC upregulation and cardiac hypertrophy in the Gq mouse and other pathological cardiac states,7,10,23 and a prior report that PKC expression causes hypertrophy in cardiac myocytes,22 chronically enhanced PKC translocation did not increase gravimetric cardiac mass (Figure 2B), echocardiographically estimated left ventricular mass (Table 1), or isolated myocyte capacitance (Table 1) in mice followed up to 1 year. Although there was no cardiac or cardiomyocyte hypertrophy, enhanced translocation of PKC caused a subtle increase in expression of some fetal cardiac genes that are upregulated in myocardial hypertrophy: Compared with normal controls, ßMHC mRNA was increased 
25 fold in cPKC activator hearts, and ANF mRNA was increased 4 fold (Figures 2C and 2D).
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As with PKC activation, PKC inhibition failed to cause measurable cardiac or cardiomyocyte hypertrophy, but was associated with a 6 fold increase in ßMHC mRNA (Figures 2C and 2D). ANF gene expression, thought to be a marker of cardiac failure rather and possibly of hypertrophy,29 was not increased by chronic PKC inhibition.
Functional Consequences of Myocardial PKC Translocation Modulation
Consistent with the absence of any overt signs of heart failure in cPKC activator and inhibitor mice, there was also no increase in lung/body weight ratios (Table 1). There were no differences in basal left ventricular systolic function (measured as fractional shorting and Vcf) between either of the cPKC translocation modifier mice and their NTG littermates at 10 weeks (see Table 1) or 6 months. cPKC activator mice did, however, develop mild echocardiographic left ventricular dysfunction at 1 year of age (Table 1; P<0.001). Together with increased ANF gene expression in these mice, the time-dependent decrease in left ventricular ejection performance suggested that a subthreshold contractile abnormality could be caused by chronic cPKC activation. Notably, cPKC inhibitor mice retained normal ventricular function through 1 year of age (Table 1).
PKC Activation Impairs ßAR Responsiveness
The functional integrity of the cardiac ß-adrenergic receptor (ßAR) system was assessed in closed chest homodynamic studies of 10- to 12-week-old mice at baseline and during dobutamine infusion. (We typically use dobutamine as the ß1AR provides the majority of inotropic drive in the mouse heart.30) Consistent with the echocardiographic findings, basal left ventricular contractile function of PKC activator or inhibitor mice did not differ from nontransgenic controls (Figure 3A). However, responsiveness to ß1AR stimulation was diminished in cPKC activator mice, with a rightward shift in the dobutamine-response curve (Figure 3A). cPKC inhibitor mice had an antithetic phenotype of enhanced chronotropic and inotropic responses to ß1AR agonist infusion, with a leftward shift in the concentration-response relationship (ED50 [ng/g per minute] for NTG=7.7±2.0, inhibitor=1.9±0.1, activator=17.3±2.3; P<0.05 for activator and inhibitor versus NTG). ßAR density, assessed by 125I-CYP binding, did not differ between cPKC activator, inhibitor, and nontransgenic mice (Figure 3B), ruling out a change in receptor expression. To assess receptor-effector coupling, adenylyl cyclase activity was measured in cardiac membranes at baseline and in response to isoproterenol, NaF, and forskolin. Basal and isoproterenol stimulated adenelyl cyclase activities (pmol/min per milligram) were as follows: NTG, 10.6±1.6 and 28±4.0; cPKC activator, 15.2±1.6 and 34.2±4.5; and cPKC inhibitor, 12.2±2.0 and 25.9±4.1. Both NaF- and forskolin-stimulated activities trended toward being higher in the activator and lower in the inhibitor samples, suggesting regulation at the level of Gs or adenylyl cyclase. Therefore, ßAR function was isolated by normalizing isoproterenol-stimulated activates to NaF and forskolin. This revealed (Figure 3C) a moderate decrease in ßAR function in activator membranes and enhanced function in inhibitor (39±7.6 versus 68±6.7; P<0.03). NTG responses were intermediate (54±9.0). The EC50 values for isoproterenol stimulation of adenylyl cyclase did not differ between the three groups. These results are consistent with previously described effects of PKCs to phosphorylate and uncouple ßAR from adenylyl cyclase.31,32 Indeed, it was not possible to overwhelm the ßAR dysfunction by simply increasing the number of membrane receptors through crossbreeding the PKC translocation modifying mice with transgenic ß2AR overexpressors having nearly maximal ßAR signaling at baseline33 (Figure 3D). Even in the context of a 30-fold increase in ßAR receptor number, ßAR coupling was similarly and reciprocally modulated by the cPKC translocation modifying peptides (Figure 3D), consistent with a potent direct effect of PKC on ßAR coupling.
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Lethal Effects of PKC Activation in Gq-Mediated Hypertrophy
Although the phenotype was relatively subtle by comparison, there were intriguing similarities between the cPKC activator mice and the Gq mice, in which PKC is upregulated and activated.7,23 Both mice exhibit a time-dependent decrease in ventricular function associated with lack of responsiveness to ßAR agonists and an increase in ANF and ßMHC gene expression. However, the cPKC activator mouse has no evidence of hypertrophy, which is the hallmark characteristic of Gq activation in the heart. This suggested that the major function of PKC activation could be to regulate cardiac function, rather than myotrophy. To test whether concurrent Gq-mediated events would unmask a more significant effect of PKC modulation, we expressed both cPKC translocation modifier peptides on the Gq overexpressor background, analogous to our prior studies of Gq in combination with PKC translocation modifiers.25 Our expectation was that the relatively subtle deleterious effects observed with enhanced PKC translocation might be augmented in the context of Gq-mediated myocardial hypertrophy, whereas PKC inhibition might prevent some Gq-mediated pathological effects.
The combination of Gq and the cPKC inhibitor peptide in compound transgenic mice resulted in animals with normal viability and molecular characteristics essentially identical to the parent Gq mouse (Figures 4A and 4B). Compared with Gq, Gq/cPKC inhibitor mice had modest, but significantly smaller hearts, measured as heart weight indexed to body weight (Table 2). A contrasting phenotype was observed in the compound Gq/cPKC activator mice, which displayed stunted growth and premature lethality compared with Gq mice (Table 2, Figure 4C), accompanied by increased heart to body weight ratios (Table 2). Additionally, expression of embryonic cardiac genes was exaggerated compared with Gq alone (Figures 4A and 4B). It was not possible to obtain an unbiased assessment of ventricular function in the Gq/cPKC activator compound transgenics because all male compound transgenic mice died by the age of 5 weeks (Figure 4C). Ventricular tachyarrhythmia was ruled out as the proximate cause of death in these mice, because chronic telemetered EKG monitoring showed only progressive bradycardia and high grade AV block that coincided with tachypnea and lethargy, suggesting that the mice succumbed to heart failure (Figure 4D). Indeed, at 4 weeks of age, male Gq/cPKC activator mice exhibited a substantial decline in LV shortening velocity and depressed heart rate, compared with Gq alone (Table 2), which along with stunted growth (Table 2), are common features of heart failure in juvenile mice.34
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Although aggressive heart failure in male compound Gq/cPKC activator mice precluded a careful assessment of physiological cause and mechanism, we were able to investigate events preceding lethality by studying the females, who exhibited a slower progression of heart failure, dying by 13 weeks of age (Figure 4C). (Similar gender separation has been described in other transgenic mouse heart failure models.35) Serial echocardiographic evaluation of female Gq/cPKC activator mice revealed a progressive decline in LV function that became significant by 7 weeks of age (FS% Gq, 26.17±1.03 versus Gq/cPKC activator, 15.07±2.43; P=0.006; Vcf Gq, 4.0±0.2 circ/s versus Gq/cPKC activator, 1.9±0.31; P=0.001) and increasing lung to body weight ratios in the compound transgenic Gq/cPKC activator mice (Gq 7.83±0.39 versus Gq/cPKC activator 10.41±1.38; P=0.027).
The disparity between progressive heart failure and relatively preserved ventricular dimension and systolic function, ie, the absence of characteristic ventricular dilation measured either by echocardiography or by gross morphometric analysis (see Table 2 and Figure 5), suggested that diastolic functional abnormalities could contribute to lethal heart failure. Fortunately, the inherent bradycardia of Gq mice permitted interrogation of transmitral inflow Doppler patterns for characteristic abnormalities associated with diastolic dysfunction. Compared with Gq, Gq/cPKC activator mice had higher peak E wave velocities with significantly shorter E wave deceleration times (Figure 4E; Gq, 35.02±3.01 ms versus Gq/cPKC activator, 18.02±2.0; P=0.022), reflecting increased left atrial pressures and impaired ventricular filling. These animals completely lacked an A wave, indicating atrial mechanical failure that correlated with extensive atrial thrombosis seen in almost every heart (Figures 5C and 5D). Consistent with these noninvasive studies, invasive hemodynamics revealed that Gq/cPKC activator mice had diminished positive and negative dP/dt, and increased left ventricular end-diastolic pressures (LVEDP), compared with Gq (Table 2). (In contrast, Gq/cPKC inhibitor mice had a modest, but significant improvement in both systolic and diastolic function, measured by positive and negative dP/dt, respectively [Table 2].) Because the above diastolic parameters are relatively load-dependent, we performed pressure-dimension analysis to determine the compliance curves of each ventricle. Gq/cPKC activator mice had a significant left and upward shift in compliance curves, compared with Gq (slope constant Gq, 13.04±1.4 versus Gq/cPKC, 30.1±1.9; P=0.017), representing increased ventricular stiffness.
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Although there were no differences in TUNEL positivity (Gq 0.36±0.12% versus Gq/cPKC activator 0.55±0.08%), a cellular mechanism for combined systolic and diastolic failure in compound transgenic Gq/cPKC activator mice was identified: Masson’s Trichrome staining revealed marked interstitial fibrosis (Figures 5A through 5D) and staining with picrosirius red showed 4-fold increased interstitial collagen content in the Gq/cPKC activator mice, compared with normal, and 2-fold increased compared with Gq (Figure 6). Gq/cPKC inhibitor mice had the reciprocal finding of decreased collagen deposition fraction (Figure 6).
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Discussion |
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To our knowledge, these are the first studies to examine forced gain or loss of PKC
function in the in vivo heart. As with our previous studies of PKC and ,20,21,26,27 the approach was to modify PKC isoform activity by positively or negatively modulating translocation. In contrast to these prior studies, the PKCß-derived peptides used herein lack absolute specificity for the targeted enzyme, but had the potential to modulate translocation of PKC, ß, and .19 However, that adult FVB/N mouse hearts contained vastly more PKC than PKCßI, PKCßII, or PKC, and the biochemical phenotype was therefore that of PKC regulation, with no measurable effects on other PKC isoforms. Nevertheless, the conclusions regarding in vivo PKC effects will need to be confirmed by standard transgenic and knockout studies. In this context, our results suggest that isolated alterations of myocardial PKC activity can modulate ßAR coupling, and hence contractile function, in the heart. Additionally, PKC appears to mediate interstitial fibrosis and restrictive ventricular physiology. Indeed, in our studies, inhibiting PKC translocation in Gq-mediated hypertrophy might be viewed as conferring a modest therapeutic benefit.
In vitro studies have previously implicated PKC-mediated phosphorylation in regulating ßAR receptors.31,32 The reciprocal effects observed herein on ßAR response with PKC activating and inhibiting peptides, and the absence of similar ßAR regulation in analogous models targeting PKC and 20,21,26,27 suggest that the isoform of PKC can be an important modulator of ßAR responsiveness in the heart. Whereas inhibition of PKC enhanced ßAR-mediated inotropic responsiveness to infused dobutamine, activation of PKC resulted in ßAR uncoupling in young mice that was associated with overt basal contractile dysfunction at 1 year of age. Importantly, these effects were independent of cardiac hypertrophy and failure, and thus do not represent actions of endogenous catecholamines. Although the association is correlative only, it suggests the possibility that chronic ßAR uncoupling can be deleterious in the normal heart.
In the present study, myocardial fibrosis and restrictive physiology were related to PKC activation only in the context of Gq-mediated hypertrophy and contractile dysfunction. Myocardial fibrosis is a hallmark of heart failure in human and experimental models, and contributes to functional decompensation because accumulation of interstitial collagen stiffens the ventricle, impairing both systolic and diastolic function.36,37 A physiological connection between PKC and fibrosis is found in the angiotensin system. Yazaki has suggested that angiotensin stimulates cardiomyocytes via Gq/PKC/ERK signaling38 and lack of AT-2 receptors prevents both hypertrophy and myocardial fibrosis in angiotensin-induced hypertension.39 We found increased levels of phosphorylated ERK in cPKC activator hearts (data not shown), confirming the integrity of this signaling pathway in the in vivo heart, and suggesting for the first time a causal relationship between myocyte PKC activity and development of myocardial fibrosis. Furthermore, the nature of the fibrosis observed in Gq/activator mouse hearts was "reactive" or interstitial, which is not associated with cell death, as opposed to "reparative" or replacement fibrosis, which is the scarring that typically follows cell death. An unresolved issue that arises from these observations is how a cardiomyocyte-specific signaling perturbation can result in increase interstitial collagen, which is presumably synthesized not by cardiomyocytes, but by fibroblasts. Again the angiotensin system provides a compelling example. Ichikawa generated chimeric mice with both intact and AT-1 receptor null cells,40 in which infusion of angiotensin caused fibroblast proliferation clustered around the AT-1-expressing cardiac myocytes, providing evidence for local myocyte-to-fibroblast communication. Thus, altered myocyte signaling can contribute to ventricular remodeling through communication with fibroblasts and resulting myocardial fibrosis.
It is useful to compare the results of the current investigations with prior attempts to delineate the roles of PKC isoforms using the approach of translocation modification. PKC seemed to primarily have trophic functions, because its activation produced hyperplastic cardiac enlargement,20,27 and its inhibition cause a hypoplastic dilated cardiomyopathy.20 Increasing PKC activity in the Gq mouse reversed some of the pathological features of that model.25 Thus, PKC seems to be a beneficial isoform, which is supported by conventional transgenic overexpression studies.19 In contrast, modulated translocation of PKC had little measurable effect on myocardial growth or function, but was critical to maintaining proper cytoskeletal structure in cardiac myocytes.26 Activation of PKC, like PKC, resulted in modest, normally functioning cardiomegaly, whereas its inhibition produced a cytoskeletal cardiomyopathy. These results contrast with those for PKC, which seems to affect cardiac function more than growth, notwithstanding in vitro studies that suggested a trophic function.22
Whereas PKC is the most abundant conventional PKC isoform in the adult FVB/N mouse heart (vide supra), PKCß is the major cPKC isoform in human myocardium. Indeed, substantial evidence exists that PKCß mediates certain pathological aspects of human cardiac hypertrophy and heart failure. PKCß expression and activity is increased in human heart failure.10 Expression of PKCß in the mouse heart (more an ectopic expression experiment than overexpression) results in hypertrophy, failure, and death, depending on the age of expression and the activity of the expressed enzyme.17,18 It is interesting to speculate that PKC in the mouse heart has many of the same effects as PKCß in the human, which suggests that cPKC isoforms may be generally pathological in the heart.
In summary, these studies demonstrate that PKC can have profound modulatory effects on cardiac contractility by regulating ß-adrenergic receptors. In the context of Gq-mediated cardiac hypertrophy, PKC appears to stimulate reactive fibrosis, thereby impairing both systolic and diastolic function, and leading to early heart failure and premature lethality. These results suggest that conventional PKC isoforms may be useful therapeutic targets in heart failure syndromes.
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Acknowledgments |
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Acknowledgments
This work was supported by NIH grants HL52310 and POHL22619 and NIH Training Grant HL07382.
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Footnotes |
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Original received June 17, 2003; revision received October 20, 2003; accepted October 23, 2003.
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References |
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