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(Circulation Research. 2005;96:711.)
© 2005 American Heart Association, Inc.

Editorials

Cardiac Hypertrophy Served With Protein Kinase C

Isoform Substitution Available at Additional Cost

Susan F. Steinberg, Mark A. Sussman

From the Department of Pharmacology (S.F.S.), Columbia University, New York, NY; and the Department of Biology (M.A.S.), San Diego State University, Calif.

Correspondence to Mark A. Sussman PhD, San Diego State University, SDSU Heart Institute, Department of Biology, NLS 426, 5500 Campanile Drive San Diego, CA 92182. E-mail sussman{at}heart.sdsu.edu

See related article, pages 748–755

Key Words: protein kinase C • hypertrophy • fibrosis • isoforms

Pressure overload stimulation of the heart stimulates a medley of signaling leading to hypertrophic remodeling. This compensatory effort to increase cardiac output depends on harmonious blending of kinase activities. Participation of protein kinase C (PKC) isoforms in hypertrophic signaling following agonist stimulation or pressure overload is unequivocal, but teasing out the specific roles for various isoform subtypes has been a frustrating endeavor. Researchers have approached this issue by tinkering with PKC isoform kinase activities via overexpression, activating/interfering peptides, or creation of knockout mice.¹ Information gleaned from these studies has made 1 point abundantly clear: PKC isoforms have a propensity for shared activation stimuli and common substrate specificities. As such, altering the activity of 1 PKC isoform almost invariably has consequences for the expression/activation/localization of other family members that normally lead to coordinated PKC responses when the heart is subjected to stress. In the case of a study by Klein et al² in this issue, losing PKC prompts changes in PKC that spell bad news for cardiac structure and function.

Klein et al identify cross-regulation between the novel PKC and PKC isoforms in the heart. They demonstrate that PKC protein expression is similar in normal and knockout PKC^–/– mice under basal conditions and in normal hearts subjected to pressure overload, but that PKC protein expression and phosphorylation levels are increased in PKC^–/– hearts following pressure overload. Prior studies by other groups show that pressure overload-induced hypertrophy leaves PKC unaffected, whereas PKC phosphorylation as well as protein expression level are significantly increased.^3,4 Isoform-specific activation studies using selective peptides that show induction of either PKC or PKC promote similar prohypertrophic effects.⁵ Klein et al found hypertrophic stimulation in the absence of PKC provokes a compensatory rise in PKC activity, reinforcing the cumulative evidence for parallel functional activities of these 2 isoforms. But the isoform substitution is not completely transparent, and problems in cardiac structure and function develop in the PKC^–/– knockout mice.

Removal of the isoform disturbs the harmonious PKC signaling balance and the ensuing shift to PKC-mediated hypertrophy is associated with increased interstitial fibrosis and diastolic dysfunction. Klein et al attribute these maladaptive changes to enhanced production of collagen driven by PKC- and MAPK-dependent signaling. These findings, reminiscent of similar in vitro studies using fibroblasts,^6,7 implicate enhanced PKC activity in promotion of reactive fibrosis following hypertrophic stimulation. Although PKC^–/– knockout mice do show exacerbation of remodeling-induced fibrosis, this is a nonphysiologic milieu that makes definitive conclusions about the true function of PKC difficult to reach. The loss of synergy between the PKC isoforms is an intractable component of genetically engineered mouse models. Of course, examination of pressure overload in mice created with cardiac-specific PKC-overexpression could possibly be a useful adjunct to support PKC‘s role in fibrosis. Paradoxically, such mice have been challenged with another hypertrophic stimulus (angiotensin II), but the authors contend that PKC acts as an antifibrotic agent through inhibition of connective tissue growth factor expression.⁸ Hearts of PKC transgenic mice are noteworthy only by the absence of any overt phenotype aside from mild hypertrophy, as is the case with so many cardiac-specific PKC-overexpressors under basal conditions. Digging further into the literature obscures a coherent picture, because inhibition of PKC promotes fibroblast proliferation.⁹ Furthermore, the use of rottlerin as a specific PKC inhibitor in the studies by Klein et al deserve some comment. Although rottlerin has been touted to be a selective PKC inhibitor, there is recent concern that rottlerin exerts cellular actions (to uncouple mitochondrial respiration from oxidative phosphorylation) via a mechanism that is entirely PKC-independent; many inhibitory actions of rottlerin have been documented in PKC^–/– cell.¹⁰ Recent studies even challenge the efficacy of rottlerin as an in vitro PKC inhibitor. This caveat is not restricted to rottlerin. Indeed, chelerythrine (another frequently used PKC inhibitor) is reported to exert pronounced cellular actions, including to induce cardiomyocyte apoptosis, through a mitochondrial mechanism that is unrelated to PKC inhibition.^11,12 There also is evidence that experiments with even relatively selective PKC inhibitors such as Ro318220 and GF109203X can be misleading, as these "PKC" inhibitors also inhibit RSK and p70 S6 kinase activity (with similar potencies).¹³ Hence, it is important to recognize that studies with rottlerin (and PKC inhibitors in general) must be interpreted with caution. Until these issues can be resolved by further studies, conclusions regarding the impact of PKC on promotion of cardiac fibrosis remain equivocal.

PKC cross-regulation observed by Klein et al shares similarities to phenomena recently identified by Gray et al who showed that PKC protein is upregulated (and PKC partitions to perinuclear structures) even under resting conditions in cardiomyocytes isolated from PKC^–/– mice.¹⁴ The perinuclear localization suggests chronic PKC activation in PKC^–/– hearts; PKC localizes to the nuclei of resting WT cardiomyocytes and translocates to perinuclear structures only following activation with phorbol 12-myristate 13-acetate. These studies bolster the growing literature describing instances of PKC isoform cross regulation. While there has been only limited effort to expose the molecular underpinnings of these regulatory controls, recent advances in our understanding of the mechanisms that regulate PKC expression, phosphorylation, and trafficking suggest several potential areas for regulatory controls that should be considered.

Biochemical and cellular studies over the past decade identify "priming" phosphorylations at highly conserved Ser/Thr residues in the activation loop and C-terminus of PKC and other AGC (protein kinase A, G, and C) superfamily members.^15,16 These "priming" phosphorylations maintain the mature, fully phosphorylated enzyme in a catalytically-competent, protease-/phosphatase-resistant conformation. The PKC-T⁵⁰⁵ phosphorylation site examined in the study by Klein et al corresponds to PKC’s activation loop phosphorylation site. PKC-T⁵⁰⁵ phosphorylation typically is the first, rate-limited phosphorylation event; this phosphorylation generally is attributed to the actions of PDK-1 and (for cPKC isoforms) is prerequisite for the generation of a catalytically competent enzyme.¹⁷ Activated PKC isoforms then undergo additional phosphorylations at C-terminal "turn" and "hydrophobic" motifs. C-terminal phosphorylations (variably attributed to intramolecular autophosphorylation events or phosphorylations by a heterologous kinase complex) function to increase the in vitro thermostability of the enzyme¹³ and generally stabilize the enzyme and to influence PKC isoform trafficking to intracellular membranes and PKC downregulation kinetics.¹¹ However, there also is evidence that PKC plays a more general role to regulate the processing and downregulation of all PKC isoforms by controlling PKC delivery to a phosphatase-containing vesicular compartment.¹⁸ Indeed, a recent study showed that nPKC inhibitors slow the downregulation kinetics for multiple PKC isoforms (PKC, PKC, and PKC) in cardiomyocytes.¹⁹ These mechanisms may contribute to the accumulation of PKC in PKC^–/– hearts.

Traditional models of PKC activation focus on allosteric activation of the mature, fully phosphorylated enzyme. However, recent studies indicate that PKC retains little T⁵⁰⁵ phosphorylation in resting cells (including cardiomyocytes) and that PKC-T⁵⁰⁵ phosphorylation is a dynamically regulated event that is increased during allosteric activation of the enzyme by agonist-activated receptors and lipid cofactors.¹⁹ In cardiomyocytes, acute (agonist-induced) PKC-T⁵⁰⁵ phosphorylation has been attributed to PKC. This result does not necessarily conflict with the prevailing model of PDK-1 as the PKC-T⁵⁰⁵ kinase. Rather, it suggests that there may be a dual mechanism for activation loop phosphorylation, with PDK-1 acting as the activation loop kinase during de novo enzyme synthesis, and PKC fulfilling a more dynamic role to cycle PKC between a more active (activation loop phosphorylated) and a less active (unphosphorylated) state during receptor signaling. In this regard, phorbol ester-sensitive PKC isoforms typically are effectively catalytically inactive without activation loop phosphorylation. However, PKC is unique in that it retains functional kinase activity even without Thr⁵⁰⁵ phosphorylation (presumably because of the presence of an acidic Glu-500 that assumes the role of the phosphorylated T⁵⁰⁵). Nevertheless, T⁵⁰⁵ phosphorylation increases PKC’s catalytic activity. Hence, it may not be entirely serendipitous that PKC-T⁵⁰⁵ phosphorylation (which is not absolutely required for enzyme activity) has evolved to be a component of the dynamically regulated allosteric activation mechanism.

Recognizing that PKC activation is via an elaborate paradigm involving phosphorylation and allosteric activation, Klein et al examine PKC-T⁵⁰⁵ phosphorylation in their study. PDK-1–dependent PKC-T⁵⁰⁵ phosphorylation (during de novo synthesis of the enzyme) would be predicted to be normal in the PKC^–/– hearts. Indeed, similar levels of PKC-T⁵⁰⁵ phosphorylation were identified in resting WT and PKC^–/– hearts. However, PKC-T⁵⁰⁵ phosphorylation was identified as increased (in the context of upregulated PKC protein) in PKC^–/– hearts subjected to transverse aortic construction. The coordinate increase in total and phosphoprotein immunoreactivity renders the interpretation of this particular result uncertain. Based on the aforementioned model, one might have predicted an increase in PKC-T⁵⁰⁵ phosphorylation (because of the Gq/PKC-driving signaling events that contribute to the pathogenesis of transverse aortic construction hypertrophy) in normal hearts, relative to the PKC^–/– hearts. However, such a comparison would require careful normalization to total PKC protein. The PKC^–/– model should constitute an important model system to resolve the specific role of PKC in the dynamic control of PKC-T⁵⁰⁵ phosphorylation in future studies.

The ultimate goal of PKC isoform manipulation from a clinical perspective is to identify suitable candidate molecules for molecular therapeutic interventional approaches designed to mitigate the progression of cardiomyopathic disease. Cross-regulation mechanisms of novel PKC isoforms impose a serious theoretical obstacle to the design of in vivo selective inhibitors. Sophisticated and elegant gene targeting approaches such as those used by Klein et al operate at a highly selective level by removing a single PKC isoform. Nevertheless, collateral effects are evident by compensatory changes reflected in related PKC family members that show altered expression and activity. Thus, studies by Klein et al and others suggest that compounds with even high levels of in vitro PKC isoform specificity may not retain in vivo selectively for a single isoform without impacting on others. Our current inability to interrupt the activity of 1 isoform without impacting others raises the possibility that PKC isoform-selective inhibition may be an inherently unattainable goal. Altering the synergy between PKC isoforms invariably shifts the balance of cardiac signaling, possibly promoting initiation of unanticipated and undesirable remodeling sequelae. This constitutes an important challenge for development of PKC-targeted pharmaceuticals designed to manipulate PKC activity as well as studies intended to dissect apart the specific functional activities of PKC isoforms in the myocardium.

	Acknowledgments

 
This work is supported by NIH R01 grants HL66035 and HL67245 (to M.A.S.) and HL-64639 (to S.F.S.).

	Footnotes

 
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.

	References

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