Published online before print July 31, 2006, doi: 10.1161/CIRCULATIONAHA.105.592550
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(Circulation. 2006;114:574-582.)
© 2006 American Heart Association, Inc.
Heart Failure |
Pharmacological- and Gene Therapy-Based Inhibition of Protein Kinase C
/ß Enhances Cardiac Contractility and Attenuates Heart Failure
From the Department of Pediatrics, University of Cincinnati, Cincinnati Children’s Hospital Medical Center (M.H., R.K., T.F.K., T.E.H., J.D.M.) and the Departments of Molecular Genetics (M.H.) and Medicine, University of Cincinnati (H.H., G.W.D.), Cincinnati, Ohio; Procter and Gamble Pharmaceuticals (A.N.C), Cincinnati, Ohio; and the Center for Translational Medicine, Jefferson Medical College (S.T.P., M.C.K., W.J.K), Philadelphia, Pa.
Correspondence to Jeffery D. Molkentin, PhD, Division of Molecular Cardiovascular Biology, Department of Pediatrics, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229-3039. E-mail jeff.molkentin{at}cchmc.org
Received September 30, 2005; revision received May 18, 2006; accepted June 12, 2006.
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Abstract |
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Background— The conventional protein kinase C (PKC) isoform
functions as a proximal regulator of Ca2+ handling in cardiac myocytes. Deletion of PKC in the mouse results in augmented sarcoplasmic reticulum Ca2+ loading, enhanced Ca2+ transients, and augmented contractility, whereas overexpression of PKC in the heart blunts contractility. Mechanistically, PKC directly regulates Ca2+ handling by altering the phosphorylation status of inhibitor-1, which in turn suppresses protein phosphatase-1 activity, thus modulating phospholamban activity and secondarily, the sarcoplasmic reticulum Ca2+ ATPase.
Methods and Results— In the present study, we show that short-term inhibition of the conventional PKC isoforms with Ro-32-0432 or Ro-31-8220 significantly augmented cardiac contractility in vivo or in an isolated work-performing heart preparation in wild-type mice but not in PKC-deficient mice. Ro-32-0432 also increased cardiac contractility in 2 different models of heart failure in vivo. Short-term or long-term treatment with Ro-31-8220 in a mouse model of heart failure due to deletion of the muscle lim protein gene significantly augmented cardiac contractility and restored pump function. Moreover, adenovirus-mediated gene therapy with a dominant-negative PKC cDNA rescued heart failure in a rat model of postinfarction cardiomyopathy. PKC was also determined to be the dominant conventional PKC isoform expressed in the adult human heart, providing potential relevance of these findings to human pathophysiology.
Conclusions— Pharmacological inhibition of PKC, or the conventional isoforms in general, may serve as a novel therapeutic strategy for enhancing cardiac contractility in certain stages of heart failure.
Key Words: heart failure • contractility • protein kinase C • cardiomyopathy
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The protein kinase C (PKC) family of Ca2+ and/or lipid-activated serine-threonine kinases functions downstream of many membrane-associated signal transduction pathways.1 Approximately 10 different isozymes make up the PKC family, and they are broadly classified by their activation characteristics. The conventional PKC isozymes (
, ßI, ßII, and 
) are Ca2+ and lipid activated, whereas the novel isozymes (
, 
, 
, and 
) and atypical isozymes (
and 
) are Ca2+ independent but activated by distinct lipids.2 PKC is the predominant Ca2+-dependent PKC isoform expressed in the mouse and rabbit heart, whereas PKCß and PKC are detectable and may have partially overlapping functions.3,4 With respect to the heart, a number of reports have associated PKC activation with hypertrophy, dilated cardiomyopathy, ischemic injury, or mitogen stimulation.1 Some evidence also exists implicating PKC isozymes as potential regulators of Ca2+ handling and cardiomyocyte contractility. For example, transient stimulation of PKC activity with phorbol 12-myristate 13-acetate (PMA) in high Ca2+ buffer caused a decrease in cardiac myocyte contraction and a decrease in the magnitude of the Ca2+ transient, which was reversed with a PKC inhibitory agent.5,6 PMA stimulation also depressed cardiac contractility in isolated rat hearts and isolated cultured cells, an effect that was also abrogated with PKC inhibitors.7–10
Clinical Perspective p 582
Myocyte contraction and relaxation are directly regulated by intracellular Ca2+ cycling. Ca2+ enters from the voltage-dependent L-type Ca2+ channel within the sarcolemma (plasma membrane), which induces a large release of Ca2+ from the sarcoplasmic reticulum (SR) storage compartment through the ryanodine receptor.11 Myocyte relaxation is initiated by sequestration of Ca2+ within the SR through the activity of the SR Ca2+ ATPase pump and by Na+/Ca2+ exchanger activity at the sarcolemma. The magnitude and timing of Ca2+ release, hence the strength of contraction, is dynamically regulated by ß-adrenergic receptor signaling to adenylyl cyclase, which produces cyclic adenosine monophosphate (cAMP), resulting in protein kinase A activation.11 Once activated, protein kinase A directly phosphorylates nodal Ca2+ regulatory proteins such as the L-type Ca2+ channel, the ryanodine receptor, phospholamban, and inhibitor-1 as a means of regulating protein phosphatase-1.11,12 The failing heart is associated with a dysregulation in Ca2+ handling through many of these proteins and a desensitization in ß-adrenergic receptor signaling.
The loss of contractility that accompanies heart failure is also associated with a general increase in PKC protein content and activity.13–19 We have previously shown that PKC functions as a fundamental regulator of cardiac contractility and Ca2+ handling in myocytes.18,20 For example, PKC gene-deleted mice were shown to be hypercontractile, whereas transgenic mice overexpressing PKC were hypocontractile. Enhancement in cardiac contractility associated with PKC gene deletion protected against pressure overload-induced heart failure and dilated cardiomyopathy associated with deletion of the muscle lim protein (MLP) gene in the mouse.18 Here, we performed a preclinical analysis to determine the efficacy of PKC inhibition as a means of treating heart failure in adult mice and rats.
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Methods |
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Animal Models
The PKC
–/– and MLP–/– mice have been described previously.18,21 Equal ratios of males and females were used in all studies for consistency. Animal experiments were approved by the Institutional Animal Care and Use Committee.
Echocardiography and Physiological Preparations
Mice were anesthetized with isoflurane, and echocardiography was performed with a Hewlett-Packard 5500 instrument (Hewlett-Packard Co, Palo Alto, Calif) with a 15-MHz compact linear-array probe. Echocardiographic measurements were taken on M-mode in triplicate for each mouse. The isolated work-performing heart preparation in the mouse has been described in detail previously.22 Short-term infusion of Ro-32-0432 in the isolated working-heart preparation was performed at a final concentration of 8x10–8 µg/mL for 5 minutes with a stock solution made up in dimethyl sulfoxide, which was infused with the Krebs solution, resulting in a working content of dimethyl sulfoxide below 0.05% (infused at 0.2 to 0.4 mL/min). For invasive hemodynamics in the closed-chest mouse, a 1.4F Millar catheter (Millar Instruments, Houston, Tex) was placed into the left ventricle through the right carotid artery to monitor real-time heart rate, arterial and left ventricular pressures, and +dP/dt (dP/dtmax) and –dP/dt (dP/dtmin) using MacLab software and interface (Mountain View, Calif), as described previously.23 In this preparation, dobutamine was given at 32 µg · kg–1 · min–1, whereas Ro-32-0432 gave a maximal response at 22.5 µg · kg–1 · min–1.
Cryoinfarction Model of Heart Failure in the Rat
The rat cryoinfarct model of heart failure has been described in detail previously.24 Briefly, adult male Sprague-Dawley rats (weight 250 to 300 g; Harlan Sprague-Dawley, Indianapolis, Ind) were anesthetized and mechanically ventilated, and the heart was exposed by a median sternotomy. Twelve of these rats were subjected to cryoinfarction with a liquid nitrogen-cooled probe (8 mm diameter) for 3 freeze-thaw cycles on the left ventricular anterior free wall. Eight other animals underwent a sham procedure.
Rat Catheterization, Invasive Hemodynamics, and Intracoronary Adenovirus Delivery
In vivo cardiac adenoviral gene therapy was performed through an intracoronary route of delivery in the rat as described previously.24,25 Adenovirus was given at 4x1010 plaque-forming units for an adenovirus-encoding ß-galactosidase (Adßgal; n=12, cryoinfarct group) and adenovirus encoding dominant negative PKC (AdPKC-dn) (n=7, cryoinfarct group) in 1.6 mL of saline injected rapidly while the aorta was cross-clamped. There was also a virus-free sham control group (n=8). One week afterward, global function was measured in a closed-chest preparation by cardiac catheterization with a 2F pressure transducer (Millar Instruments), as described previously.25
Cardiac Histological Analysis
Hearts were collected at the indicated times, fixed in 10% formalin containing phosphate-buffered saline, and embedded in paraffin. Serial 9-µm heart sections from each group were analyzed. Samples were stained with hematoxylin and eosin or Masson’s trichrome.
Primary Cardiomyocyte Culture
Adenoviral infection conditions and the generation of primary cardiomyocyte cultures from 1- to 2-day-old Sprague-Dawley rat neonatal hearts were described previously.26 Cardiomyocytes were cultured under serum-free conditions in M199 media supplemented with penicillin/streptomycin (100 U/mL) and L-glutamine (2 mmol/L). Cells were subsequently treated with Ro-32-0432 or Ro-31-8220 at a concentration of 50 nmol/L for 1.5 hours. PMA (200 nmol/L) was also given 1 hour before harvest.
Replication-Deficient Adenoviruses
Dominant-negative PKC was described previously as an L368R mutation.18,27 AdPKC-dn or an adenovirus-encoding ß-galactosidase (Adßgal) was plaque purified, expanded, titered in HEK293, and banded in CsCl for gene therapy in the rat cryoinfarct model described above.
Western Blot Analysis
Western blotting was performed as described previously with primary antibodies against phospho-myristoylated alanine-rich C-kinase substrate (MARCKS), PKC, PKCßI, PKCßII, PKC, and PKC (Santa Cruz Biotechnology, Santa Cruz, Calif).26,27 Recombinant PKC isozyme standards were loaded between 0.1 and 15 ng (Upstate Biotechnologies, Lake Placid, NY). Chemifluorescent detection was performed with the Vistra ECF reagent (RPN 5785; Amersham Pharmacia Biotech, Piscataway, NJ) and scanned with a PhosphorImager (Amersham Pharmacia Biotech).
Statistical Analysis
Two-sample Student t tests were used to compare means between 2 independent groups/samples, and ANOVA was used to compare means among 3 or more independent groups. Paired t tests were used to compare values within mice before and after treatment. Repeated-measures ANOVA was used for analysis of dose-response data. A Newman-Keuls post hoc test was applied whenever multiple comparisons were conducted. Note that caution is warranted when parametric tests such as t tests or ANOVA are used, because they are not robust in small samples, as shown in Figure 1C and 1D.
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All authors had full access to the data and take full responsibility for its integrity. All authors have read and agree to the manuscript as written.
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Results |
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Short-Term Infusion of Ro-32-0432 Increases Cardiac Contractility in Mice
Loss or inhibition of PKC
augments cardiac contractile function at baseline and in models of heart failure.18,20 These results suggested that a nontoxic and tissue-available pharmacological inhibitor with selectivity toward PKC, or the classic isoforms of PKC (, ß, and ) might be of significant therapeutic value. Here, we investigated 2 different bisindolylmaleimide compounds, Ro-32-0432 and Ro-31-8220, which were previously shown to have selectivity and efficacy for classic PKC isoforms, especially PKC28–30 (Figure 1A). To address the ability of these compounds to inhibit PKC in cardiomyocytes, phosphorylation of the MARCKS protein was investigated given its description as a PKC target and its ability to specifically bind the PKC isozyme.31,32 Both compounds specifically inhibited the levels of phosphorylated MARCKS protein at serine 152/156 in cardiomyocytes compared with vehicle after PMA stimulation (Figure 1B). We were unable to identify any phosphorylation target that was absolutely specific to PKC, and intracellular translocation of PKC was not altered by the Ro compounds (data not shown). Although other endogenous PKC isoforms could also phosphorylate MARCKS, this assay was made relatively specific by overexpression of a given PKC isozyme (see online Data Supplement, Figure I). Using this assay, Ro-31-8220 and Ro-32-0432 were determined to be mostly specific for PKC and PKCß but not PKC or PKC (Data Supplement, Figure I). To address the ability of this class of compound to augment intrinsic cardiac contractility in the absence of neurohumoral influences, an isolated working-heart preparation was performed. After equilibration, short-term infusion of Ro-32-0432 significantly enhanced contractility and left ventricular developed pressure in hearts from mice in the FVB genetic background (Figure 1C and 1D).
PKC-Null Mice Demonstrate Specificity of the Inhibitory Effect on Contractility
The data presented above suggested that Ro-32-0432 and Ro-31-8220 could enhance cardiac contractile function, although the relative specificity of this effect as being mediated through conventional PKC isozyme inhibition was uncertain. To more directly ascertain specificity, short-term infusion of PMA or Ro-32-0432 was performed in wild-type or PKC-null hearts in an isolated work-performing heart preparation. PMA functions as a potent activator of PKC isozymes in the heart, largely producing a negative effect on contractility.5–10 In theory, if PKC is the primary regulator of the contractility data presented above, mice deficient in PKC should not respond to Ro-32-0432, or possibly PMA. PMA infusion had no effect on contractility in isolated wild-type hearts from a concentration of 8x10–11 µg/mL through 4x10–9 µg/mL (Figure 2A). Increasing PMA concentration above 8x10–9 µg/mL severely reduced or completely arrested cardiac contractility (Figure 2A). However, PMA infusion in isolated hearts from PKC-null mice had a noticeably different effect. PMA concentrations of 8x10–11 through 4x10–9 µg/mL produced a mild but significant positive inotropic effect and even at the highest concentration of PMA did not reduce contractility in PKC-null hearts below untreated wild-type hearts (Figure 2A).
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Consistent with the PMA infusion data discussed above, infusion of Ro-32-0432 (8x10–8 µg/mL) did not significantly enhance cardiac contractility in work-performing isolated hearts from PKC–/– mice, whereas hearts from wild-type mice (C57BL/6 background) showed a significant increase on infusion (P<0.05; Figure 2B). As expected, hearts from PKC–/– mice continued to show enhanced cardiac contractility at baseline or with vehicle infusion (Figure 2A and 2B). Taken together, these data suggest that the overall contractility effects observed for the bisindolylmaleimide compounds are largely mediated through PKC. Although these compounds can also inhibit PKCß and PKC, these isoforms are significantly lower in total content in the heart than PKC, which further suggests PKC as a biologically important target.3,4 PKC levels also dominate in the human heart compared with PKCß, PKC, and PKC when normalized to total protein content with recombinant standards (Figure 3A and 3B). This result suggests that pharmacological inhibition of PKC with compounds of the bisindolylmaleimide class might also be efficacious as a selective inotrope in heart failure patients.
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Ro-32-0432 Increases Cardiac Contractility in Heart Failure Models
In addition to increasing the cardiac contractile response in an isolated working-heart preparation, catheter-based infusion of Ro-32-0432 in an invasive preparation in anesthetized wild-type mice also increased cardiac contractility (Figure 4A). In this preparation, mice were first challenged with a ß-adrenergic agonist, dobutamine, to assess the upper maximal limit of inotropic effect, equilibrated, then challenged with Ro-32-0432. Although the contractile effect associated with Ro-32-0432 was less robust than with dobutamine, it was nonetheless significant (P<0.001; see Discussion). The same infusion protocol was also used in 2 models of heart failure due to deletion of the MLP gene and overexpression of G protein q subunit (Gq) in the heart (Figure 4B and 4C). Importantly, whereas the response to dobutamine was severely blunted in the 2 heart failure models, consistent with previously reported desensitization of the ß-adrenergic receptors, the inotropic response to Ro-32-0432 was more maintained than in wild-type mice (P<0.05). For example, dobutamine challenge in Gq mice was only 16% that of wild-type mice, whereas the remaining inotropic effect was 57% with Ro-32-0432 infusion (Figure 4C).
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Pharmacological Inhibition of PKC Restores Cardiac Function in MLP–/– Mice
Because long-term treatment with traditional inotropes is associated with adverse outcomes in heart failure patients, in the present study, we investigated the affects of long-term Ro-31-8220 administration over 4 to 6 weeks in MLP–/– heart failure mice. All mice were assessed for ventricular performance by echocardiography at the beginning of the study and 6 weeks later. Ro-31-8220 (or vehicle) was injected subcutaneously once per day at a dosage of 6 mg · kg–1 · d–1. The Ro-31-8220 compound was used for all in vivo studies instead of the Ro-32-0432 compound only because of issues related to expense surrounding large-scale synthesis and the quantity that was needed for long-term use in mice. Pharmacokinetic analysis of Ro-31-8220 in the rat showed a half-life of 5.7 hours, and plasma concentrations of drug were 100-fold greater than the inhibitory concentration 50% (IC50) for PKC at 6 hours (see Discussion). This dosage of Ro-31-8220 was well tolerated in the mouse and had no observable detrimental effects over 6 weeks, nor was body weight affected. Wild-type mice injected with vehicle or Ro-31-8220 showed no change in fractional shortening or any other measures of ventricular dimensions over the 6-week period (Figure 5A; Table 1). In contrast, MLP–/– mice showed a dramatic rescue in fractional shortening after 6 weeks of Ro-31-8220 compared with vehicle treatment or baseline values before treatment (Figure 5A; Table 1). The study shown in Figure 5A was performed in 6-month-old wild-type and MLP–/– mice, although a similar improvement in fractional shortening was observed in aged MLP–/– mice (14 months) compared with vehicle-treated mice over 4 weeks of treatment (Figure 5B; Table 1). Wild-type and MLP–/– mice were also subjected to isolated working-heart preparation, demonstrating a significant increase in cardiac contractility in MLP–/– mice treated with Ro-31-8220, comparable to wild-type mice (Table 2). A similar rescue in diastolic function (–dP/dt) and left ventricular pressure developed was also observed (Table 2).
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Although Ro-31-8220 rescued cardiac contractile performance in MLP–/– mice over 4 or 6 weeks of administration, it did not alter heart weight or reverse cardiac chamber dilation, as assessed by echocardiography, or improve histopathology (data not shown). These observations suggest that part of the increase in cardiac performance associated with long-term Ro-31-8220 treatment might involve a short-term influence on contractility itself. Indeed, injection of Ro-31-8220 in MLP–/– mice for only 3 days improved fractional shortening compared with vehicle treatment (Figure 5C).
Cardiac Adenovirus-Mediated Gene Delivery of Dominant-Negative PKC Attenuates Heart Failure in a Rat Cryoinfarction Injury Model
Although relatively selective for the conventional PKC isoforms, the bisindolylmaleimide compounds used herein have the potential to inhibit other kinases. To further investigate PKC as a primary determinant of the effects described above, we used a dominant-negative PKC adenovirus for gene therapy in rats 12 weeks after cryo-mediated infarction injury. Rats were assessed for ventricular performance by invasive hemodynamics 1 week after adenovirus-mediated gene transfer of a control virus (Adßgal) or AdPKC-dn. Cryoinfarcted rats treated with Adßgal showed a marked depression in cardiac contractility (P<0.05), consistent with our previous observations,24 whereas AdPKC-dn-treated rats showed an augmentation in ventricular performance (P<0.05; Figure 6A). Moreover, elevated left ventricular diastolic pressure that typifies heart failure was largely reversed with AdPKC-dn treatment, without an effect on heart rate (Figure 6B and data not shown). These data support the conclusion that inhibition of PKC in the adult heart augments cardiac contractility, partially relieving heart failure.
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Discussion |
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One potential issue associated with the data presented here is the specificity of the bisindolylmaleimide compounds, Ro-32-0432 and Ro-31-8220. Although both compounds have excellent potency for PKC
, each can also inhibit other kinases,28–30 especially the 2 other classic PKC isozymes, PKCß and PKC. However, given the high degree of sequence conservation among the 3 classic PKC genes, PKCß and PKC may function analogous to PKC in regulating cardiac contractility, so partial or complete inhibition of these isozymes may also be desirable. Indeed, we also observed that administration of LY333531, a PKC/ß-selective inhibitory compound of the dimethylamine class, led to a 28% increase in +dP/dt in wild-type rats (data not shown). Even though this compound was claimed to be PKCß-specific,13 we observed relatively equal inhibitory activity toward PKC (data not shown). Although all 3 conventional isozymes may regulate cardiac contractility, other lines of evidence suggest that PKC is potentially the most critical. For example, PKC is the dominant conventional PKC isozyme expressed in the mouse, rabbit, and human heart, although this observation does not take into account potential differences in activity (Figure 3).3,4 Ro-31-0432 did not significantly augment cardiac contractility in mice lacking PKC, which further supports the conclusion that the biological effect of Ro-31-0432 on contractility is largely due to PKC. Moreover, general activation of both classic and novel PKC isozymes in the heart by short-term infusion of PMA produced a dramatic decrease in contractility in wild-type mice but not in PKC–/– mice. This result suggests that PKC is a primary negative regulator of cardiac contractility after a global activation of PKC isozymes in the heart. The dominance of PKC in regulating cardiac contractility is also supported by adenovirus-mediated gene therapy with dominant-negative PKC in the rat heart. These results are also consistent with our previous observations whereby AdPKC (wild-type) reduced contractility in isolated adult myocytes, whereas AdPKC-dn enhanced it.18 Finally, PKC–/– mice continued to show increased cardiac contractility at baseline, as described previously.18 The dosages of Ro-32-0432 and Ro-31-8220 that were selected for use in vivo were in excess of the calculated in vitro IC50 values. However, calculation of the IC50 value for a given compound does not account for adenosine triphosphate concentration in a cell (which competes with the bisindolylmaleimide compounds), the organ availability of the compound, membrane permeability characteristics, its metabolism, and its selectivity/binding for other kinases, all of which can influence its efficacious dosage in vivo. Hence, an empirical examination of biological effectiveness is probably more important than a rigid adherence to an arbitrary IC50 value.
Ro-32-0432 and Ro-31-8220 each increased cardiac contractility in wild-type and MLP–/– mice without toxic effect or lethality. In fact, none of the 27 wild-type and MLP–/– mice given Ro-31-8220 died during 4 or 6 weeks of long-term treatment at 6 mg · kg–1 · d–1, nor was any lethargy or overt symptoms of drug intolerance observed. Also of interest, long-term and short-term administration of Ro-31-8220 in MLP–/– mice each had an identical effect on contractility, which suggests that PKC inhibition is not subject to significant desensitization as is characteristic of ß-agonists. Indeed, augmented contractility associated with dobutamine administration in the MLP–/– and Gq heart failure models was dramatically blunted compared with wild-type mice, yet the same general contractility enhancement was largely preserved between wild-type mice and the 2 heart failure models when the PKC inhibitory compound was used. Thus, selective targeting of PKC may provide a unique therapeutic approach in patients who are desensitized to ß-agonists, or for more long-term indications where desensitization would normally occur.
One unique aspect associated with PKC inhibition, whether due to gene ablation or the bisindolylmaleimide compounds, is that contractility is only moderately increased. By comparison, ß-agonist or phosphodiesterase inhibitors tend to have a more pronounced effect on cardiac contractility, effects that have been associated with increased incidences of sudden death and more rapid decompensation in heart failure patients.33 Although such data warrant caution when inotropic support is prescribed, there may be important differences in the means of providing such support. The significant, yet less robust augmentation in cardiac contractility associated with PKC inhibition may have a safer profile. PKC also regulates one particular subset of effects that are initiated by ß-adrenergic receptors and cAMP. Specifically, PKC functions through direct phosphorylation of inhibitor-1, which results in altered protein phosphatase-1 activity, which in turn regulates phospholamban phosphorylation. Alterations in phospholamban phosphorylation regulate SR Ca2+ ATPase function in the heart, which controls Ca2+ loading and the magnitude of the Ca2+ transient.11 Larger Ca2+ transients directly enhance myocardial contractility by augmenting the number of cross-bridges generated between the myosin heads and actin-containing thin filaments, generating the power stroke. Thus, pharmacological inhibition of PKC activity would function at the level of SR Ca2+ handling, possibly providing a safer inotropic profile. Indeed, deletion of phospholamban in the mouse augments cardiac contractility only through increased loading of the SR without reducing viability or increasing the propensity toward arrhythmia.34 These observations suggest that inotropic support through PKC inhibition could be safer than using agents that directly activate the ß-adrenergic receptor or increase cAMP. Finally, inhibition of PKC may also benefit a failing myocardium independent of contractility, because PKC is involved in reactive signaling within the heart that participates in hypertrophy, negative remodeling, and decompensation. In conclusion, PKC represents a novel target for treating human heart failure by modulating contractility within a more restrained physiological window by only mediating inotropic effects specifically through SR Ca2+ loading and by possibly reducing reactive signaling through neuroendocrine pathways.
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Acknowledgments |
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We thank Dr Jane F. Djung for pharmacological kinetic analyses.
Sources of Funding
This work was supported by the National Institutes of Health (SCCOR grant P01HL077101) and an Established Investigator Grant from the American Heart Association (Dr Molkentin). M. Hambleton was supported by a National Institutes of Health training grant (No. T32 HL007752). Dr Koch was supported by R01 HL56205 from the National Institutes of Health, and Dr Pleger was supported by a fellowship from the Pennsylvania-Delaware Affiliate of the American Heart Association.
Disclosures
Dr Carr was an employee of Procter and Gamble Pharmaceuticals. Dr Molkentin received research grant support from Proctor & Gamble Pharmaceuticals. Dr Molkentin has a patent pending on the use of PKC inhibitors in treating heart disease. The remaining authors report no conflicts.
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The online-only Data Supplement can be found with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.105.592550/DC1.
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