PICOT Inhibits Cardiac Hypertrophy and Enhances Ventricular Function and Cardiomyocyte Contractility
http://www.100md.com
Dongtak Jeong, Hyeseon Cha, Eunyoung Kim
参见附件。
the Department of Life Science (D.J., H.C., E.K., M.K., D.K.Y., J.M.K., P.O.Y., J.G.O., D.H.K., W.J.P.), Global Research Laboratory on Cardiovascular Gene Therapy, Gwangju Institute of Science and Technology, Korea
Cardiovascular Research Center (O.Y.B., S.S., R.J.H.), Massachusetts General Hospital and Harvard Medical School, Charlestown
College of Pharmacy (L.T.T., S.-H.W.), Chungnam National University, Daejeon, Korea
Cardiovascular Division (L.C., R.L.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass
Department of Oral Anatomy (Y.-H.L.), School of Dentistry, Chonbuk National University, Jeonju, Korea.
Abstract
Multiple signaling pathways involving protein kinase C (PKC) have been implicated in the development of cardiac hypertrophy. We observed that a putative PKC inhibitor, PICOT (PKC-Interacting Cousin Of Thioredoxin) was upregulated in response to hypertrophic stimuli both in vitro and in vivo. This suggested that PICOT may act as an endogenous negative feedback regulator of cardiac hypertrophy through its ability to inhibit PKC activity, which is elevated during cardiac hypertrophy. Adenovirus-mediated gene transfer of PICOT completely blocked the hypertrophic response of neonatal rat cardiomyocytes to enthothelin-1 and phenylephrine, as demonstrated by cell size, sarcomere rearrangement, atrial natriuretic factor expression, and rates of protein synthesis. Transgenic mice with cardiac-specific overexpression of PICOT showed that PICOT is a potent inhibitor of cardiac hypertrophy induced by pressure overload. In addition, PICOT overexpression dramatically increased the ventricular function and cardiomyocyte contractility as measured by ejection fraction and end-systolic pressure of transgenic hearts and peak shortening of isolated cardiomyocytes, respectively. Intracellular Ca2+ handing analysis revealed that increases in myofilament Ca2+ responsiveness, together with increased rate of sarcoplasmic reticulum Ca2+ reuptake, are associated with the enhanced contractility in PICOT-overexpressing cardiomyocytes. The inhibition of cardiac remodeling by of PICOT with a concomitant increase in ventricular function and cardiomyocyte contractility suggests that PICOT may provide an efficient modality for treatment of cardiac hypertrophy and heart failure.
Key Words: cardiac hypertrophy protein kinase C PICOT contractility
Introduction
The myocardium undergoes adaptive hypertrophic growth to augment cardiac output in response to a variety of pathological insults. Although this response initially appears to be beneficial, sustained cardiac hypertrophy leads to an increased risk of sudden death or progression to heart failure.1 Therefore, therapies directed at inhibiting or reversing cardiac hypertrophy could be of significant clinical value. Intensive investigations in the past decade have revealed that multiple parallel intracellular signaling pathways can induce cardiac hypertrophy (reviewed previously2–4). No single pathway seems to regulate cardiac hypertrophy alone. Rather, it appears more likely that each pathway operates as a component of an orchestrated hypertrophic network.
In recent years, potential antihypertrophic and inhibitory feedback signaling pathways have been discovered. Their integration into the hypertrophic signaling pathway network adds considerable complexity. Atrial natriuretic factor (ANF), long considered to be a typical molecular marker for cardiac hypertrophy, has been shown to be antihypertrophic by activating cGMP-dependent protein kinase (PKG).5,6 In addition, a calcineurin inhibitory protein, myocyte-enriched calcineurin-interacting protein 1 (MCIP1), has been shown to participate in a negative feedback loop that limits the potentially deleterious effect of unrestrained calcineurin activity during cardiac hypertrophy.7,8 Moreover, SOCS3 (a Suppressor Of Cytokine Signaling 3) binds to janus kinase (JAK) and acts a negative feedback regulator of gp130-mediated cardiac hypertrophy.9 Recently, the transcriptional repressor Nab1 has been shown to intervene in cardiac hypertrophy by repressing early-growth response (Egr) transcription factors.10 These findings suggest that hypertrophic stimuli evoke antihypertrophic or negative feedback pathways, as well as positive hypertrophic signaling pathways. Augmenting these negative regulators, rather than inhibiting the positive regulators, may be a viable antihypertrophic strategy.11
Protein kinase C (PKC) is a ubiquitously expressed serine/threonine kinase and has been implicated in the development of cardiac hypertrophy.12 The rat heart contains at least 5 PKC isozymes (PKC, PKCI, PKC, PKC, and PKC) with distinct expression patterns. The relative importance of these PKC isozymes in hypertrophic signaling remains controversial13–15 and crosstalks between the PKC isozymes may complicate data interpretation.16 In this study, we show that a putative PKC inhibitor, PICOT (PKC-Interacting Cousin Of Thioredoxin), functions as a negative regulator of cardiac hypertrophy. Interestingly, PICOT overexpression dramatically increased ventricular function and cardiomyocyte contractility.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Cell Culture and Hypertrophic Stimulation
Primary cultures of cardiomyocytes from 2-day-old Sprague–Dawley rats were prepared as described.17 Briefly, ventricular tissue was enzymatically dissociated, and the resulting cell suspension was enriched for cardiomyocytes using Percoll (Amersham Pharmacia) step gradients. Cells were plated onto either collagen-coated culture dishes or cover slips and cultured in cardiomyocyte culture medium (DMEM supplemented with 10% FBS and 2 mmol/L L-glutamine; GIBCO BRL). To induce hypertrophy, cardiomyocytes were cultured in serum-free medium for at least 24 hours and then treated with 100 nmol/L enthothelin-1 (ET-1) or 100 μmol/L phenylephrine (PE) for 24 hours.
Generation of Transgenic Mice
A full-length mouse PICOT cDNA with human growth hormone 3' untranslated region was subcloned into a 5.5-kb segment of the –myosin heavy chain (-MHC) promoter. The DNA construct was microinjected into FVB fertilized eggs and transgenic integration was confirmed by Southern blotting (Macrogen, Korea). Transgenic mice and wild-type littermates were analyzed at 8 to 10 weeks of age.
Results
PICOT Is Upregulated During Cardiac Hypertrophy
To identify negative regulators of cardiac hypertrophy, a PCR-based differential screening technique was performed, based on the assumption that antihypertrophic or negative regulators were likely to be induced during cardiac hypertrophy, along with positive hypertrophic regulators. Transverse aortic constriction (TAC) was used to induce cardiac hypertrophy in adult rat hearts. Some novel genes, as well as numerous genes previously found to be associated with cardiac hypertrophy, were isolated (see the Table in the online data supplement). Among the genes that were expressed preferentially in the hypertrophied hearts, as compared with the sham-operated normal hearts, PICOT was of particular interest. PICOT was first identified as PKC-interacting protein by a yeast 2-hybrid screen.18 When transiently overexpressed in T cells, PICOT inhibited the activation of c-jun N-terminal kinase (JNK), activator protein-1 (AP-1), and nuclear factor B (NF-B), suggesting that PICOT functioned as an endogenous inhibitor of PKC.18 Considering that PKC isozymes play important roles in the development of cardiac hypertrophy, the elevated expression of PICOT in hypertrophied hearts suggested a novel negative feedback circuit in cardiac hypertrophy.
Northern blot analysis was used to confirm the induction of PICOT by hypertrophic stimuli in vivo and in vitro. The expression level of PICOT was approximately 3 times higher in hypertrophied hearts than in sham-operated hearts (Figure 1A). PICOT also was induced in neonatal rat cardiomyocytes after exposure to the hypertrophic agonists ET-1 or phenylephrine PE (Figure 1B). In both cases, the extent of PICOT induction was comparable to that of ANF. The induction of PICOT during the progression of cardiac hypertrophy also was examined (Figure 1C). The induction of ANF began 2 days after TAC, peaked after 2 weeks, and declined sharply thereafter. This ANF-induction pattern previously had been shown to correlate well with the onset of cardiac hypertrophy.9 In contrast, the induction of PICOT by TAC was rather slow, beginning approximately 1 week after the induction of ANF and continuing to increase when ANF expression was completely diminished at 3 weeks after banding (Figure 1C). Thus it appeared possible that ANF constituted an acute cardiac hypertrophy inhibitory mechanism, whereas PICOT had a later-onset inhibitory function. Fibroblasts and cardiomyocytes were separately isolated from the sham-operated and hypertrophied hearts (3 weeks after TAC), respectively, and the PICOT level in the protein extracts were determined by immunoblotting (Figure 1D). Approximately 2-fold increase in the PICOT level was detected in the hypertrophied cardiomyocytes but not in the fibroblasts. This indicates that the inhibitory activity of PICOT on the development of cardiac hypertrophy is cell autonomous.
PICOT Inhibits the ET-1– or PE-Induced Hypertrophic Response
To evaluate the ability of PICOT to inhibit cardiac hypertrophy, AdPICOT (PICOT-expressing recombinant adenovirus) was generated. Adenoviral infection routinely resulted in infection of more than 90% of the cultured neonatal rat cardiomyocytes (data not shown). Western blot analyses revealed a protein band in the cardiomyocytes transfected with AdPICOT but not in nontransfected cardiomyocytes or in cardiomyocytes transfected with AdLacZ (-galactosidase-expressing adenovirus) (Figure 2A). The apparent molecular weight of the PICOT band was 40 to 42 kDa, which was slightly higher than the calculated molecular mass (38 kDa), suggesting that PICOT might be posttranslationally modified. At 24 hours after infection, the cardiomyocyte cultures were further stimulated with ET-1 or PE for 24 hours. These treatments significantly increased the size of the AdLacZ-transfected or untransfected cardiomyocytes, as assessed by measuring the surface area of the cells under a microscope. In contrast, no significant ET-1– or PE-induced increase in cell size was observed with the AdPICOT-transfected cardiomyocytes (Figure 2B).
Another feature of the hypertrophic response of cardiomyocytes is a pronounced sarcomeric rearrangement that can be detected by immunostaining with -actinin antibody. Unlike the nontransfected or AdLacZ-transfected cardiomyocytes, the cardiomyocytes transfected with AdPICOT lacked prominent sarcomeric structures after ET-1 or PE treatment (Figure 2C). ANF expression also is a typical marker for cardiac hypertrophy. Treatment with ET-1 or PE significantly increased ANF immunoreactivity in nontransfected or AdLacZ-transfected cardiomyocytes. However, PICOT transfection blocked this elevated ANF immunoreactivity (Figure 2D). Lastly, the increased protein synthesis induced by ET-1 or PE treatment, as determined by 3H-leucine incorporation, was abrogated completely by PICOT transfection (Figure 2E). Taken together, these results indicated that the forced expression of PICOT inhibited agonist-induced cardiac hypertrophy in neonatal rat cardiomyocytes.
PICOT Abrogates Pressure-Overload–Induced Cardiac Hypertrophy
To investigate the role of PICOT as a negative regulator of cardiac hypertrophy in vivo, transgenic mice were generated that expressed PICOT in the heart, under the control of the -MHC promoter (Figure 3A). Two independent lines (TG17 and TG60) were obtained with the -MHC-PICOT transgene. Transgenic lines did not exhibit any obvious gross abnormalities. Western blot analysis revealed that cardiac PICOT expression in both lines was approximately 60% to 80% higher than in the wild-type littermates (Figure 3B). We observed that endogenous PICOT was present both in the cardiomyocytes and fibroblasts of hearts, and the transgene drove PICOT expression in the cardiomyocytes (data not shown).
To determine whether PICOT overexpression antagonized the hypertrophic response to physiologically relevant pressure overload, wild-type littermates and TG17 mice were subjected to TAC. The induction of pressure overload stimulated an &33% increase in the heart weight to body weight ratio in wild type over 14 days. In contrast, hypertrophic growth was blunted significantly (P<0.05) in transgenic mice, with approximately a 15% increase in the heart weight to body weight ratio (Figure 3C). Furthermore, microscopic analysis of histological sections revealed that the increase in the ventricular cross-sectional area was reduced significantly (P<0.01) in transgenic mice (a 25% increase in TAC versus sham) compared with wild type (an 82% increase in TAC versus sham) (Figure 3D). The induction of several fetal genes, including ANF and skeletal actin (SKA), is associated with cardiac hypertrophy. Northern blot analysis showed that the induction of these hypertrophic marker genes was reduced significantly in transgenic mice as compared with wild type (Figure 3E). These results indicated that PICOT abrogated the development of left ventricular hypertrophy induced by pressure overload in vivo. Similar results were obtained with TG60 (data not shown).
PICOT Enhances Ventricular Function and Cardiomyocyte Contractility
Previous reports showed that PKC activation was associated with a reduction of cardiac contractility via the dephosphorylation of phospholamban (PLB)19,20 or the phosphorylation of native thin filaments21 and troponin I (TnI).22,23 Because PICOT has been thought to inhibit PKC, its effects on cardiac contractility were examined. Adult cardiomyocytes were isolated from wild-type and transgenic hearts, and their contractility was measured. An increase in contractility of approximately 89% was observed in transgenic cardiomyocytes (11.0%±1.6) as compared with wild type (5.8%±1.1), as measured by peak shortening (Figure 3F). To determine the in vivo functional consequences of PICOT overexpression, hemodynamic parameters were measured under basal conditions. PICOT transgenic mice showed a 27% increase in ejection fraction and an 18% increase in end-systolic pressure accompanied by a 47% increase in the first derivative of left ventricular systolic pressure rise (+dP/dt) and a 25% increase in the decline (–dP/dT) compared with the wild-type littermates, demonstrating enhanced contraction and relaxation (Figure 3G). Importantly, both heart rate (data not shown) and left ventricular diastolic pressure (Figure 3G), parameters that can affect left ventricular contractility, were indistinguishable in transgenic and wild-type mice. Thus, PICOT not only inhibited the development of cardiac hypertrophy both in vitro and in vivo but also increased ventricular function in vivo in transgenic models.
To examine the effects of PICOT in cardiomyocytes, isolated adult rat cardiomyocytes were transfected with either AdLacZ or AdPICOT, and the consequential mechanical properties were determined using a dual-excitation spectrofluorometer equipped with video-edge detection system. AdPICOT-transfected cardiomyocytes showed an 84% increase in cell shortening, a 57% increase in maximal rate of contraction (–dL/dt), and an 83% increase in the maximal rate of relaxation (+dL/dt) in comparison with AdLacZ-transfected cardiomyocytes (Figure 4A).
To further define the mechanism underlying the enhanced contractility associated with PICOT, intracellular Ca2+ handling was analyzed. Ca2+ transient amplitude and time to peak fluorescence were comparable in AdLacZ- and AdPICOT-transfected cardiomyocytes. Time required to reach 90% baseline fluorescence and time constant of Ca2+ transient decay, , were reduced 15% and 18%, respectively, in AdPICOT-transfected cardiomyocytes (Figure 4B). Thus, PICOT overexpression appears to be coupled with more efficient reuptake of Ca+2 by sarcoplasmic reticulum (SR) during relaxation but not with the enhanced Ca2+ transient amplitude. Cell shortening was plotted against cytosolic Ca2+ content during steady-state contraction (Figure 4C). Upward and leftward shift of the loop in AdPICOT-transfected cardiomyocytes indicated increased cell shortening for any given cytosolic Ca2+ concentration, implying an increase in Ca2+ sensitivity of the myofilaments, compared with AdLacZ-transfected cardiomyocytes. These results indicate that increases in myofilament Ca2+ responsiveness, together with increased rate of SR Ca2+ reuptake, promote an enhanced contractile function in AdPICOT-transfected cardiomyocytes. We observed that phosphorylation at Ser16 of pentameric PLB was significantly increased, whereas total pentameric PLB protein level was significantly reduced in AdPICOT-transfected cardiomyocytes. Therefore, a >2-fold increase in the ratio of phosphorylated PLB to total PLB was observed on PICOT overexpression (Figure 4D). Without a change in expression of SERCA-2 and calsequestrin (data not shown), the hyperphosphorylation of PLB is thought to render SERCA-2 more active, resulting in the enhanced SR Ca2+ reuptake in AdPICOT-transfected cardiomyocytes.
PICOT Suppresses the Activation of PKC and Mitogen-Activated Protein Kinases
PKC activation has been associated with the development of cardiac hypertrophy, and PICOT has been thought to function as an intrinsic inhibitor of PKC. Therefore, the activation of PKC in cardiomyocytes stimulated with ET-1 or PE was tested under the forced expression of PICOT. Using a PKC-specific phosphorylation assay, 2.5- and 4.5-fold increases were observed in the total PKC activity in cardiomyocytes treated with ET-1 or PE, respectively. These increases were abrogated completely by transfection with AdPICOT but not with AdLacZ (Figure 5A). PKC, PKC, and PKC are the most highly expressed isoforms in the adult rat heart.24–26 In addition to these isozymes, PKC also is expressed abundantly in neonatal rat cardiomyocytes and weakly in the adult rat heart (data not shown). The near-complete suppression of total PKC activity by PICOT suggested that the activation of all these PKC isozymes might be abrogated by PICOT transfection. Thus the effect of PICOT overexpression on the specific activation of PKC, PKC, and PKC was evaluated using phospho-PKC, isozyme-specific antibodies. PKC, PKC, and PKC were activated significantly by stimulation with ET-1 or PE, and concomitant PICOT transfection prevented this activation (Figure 5B).
Because mitogen-activated protein (MAP) kinase pathways appear to be a merging point for divergent hypertrophic signaling pathways involving PKC,12,27–30 PICOT inhibition of MAP kinase activation was investigated using antibodies against phospho–extracellular signal-regulated kinase 1/2 (phospho–ERK1/2), phospho-JNK, and phospho-p38. ERK1/2, JNK, and p38 were activated significantly by stimulation with ET-1 or PE. PICOT transfection abrogated the activation of ERK1/2 and JNK but not p38 (Figure 5C). These results indicated that PICOT may counteract hypertrophic stimulation by inhibiting PKC and the downstream ERK1/2 and JNK signaling pathways. These results were in agreement with the previous observation that p38 may play a role in cardiomyocyte survival but not in the development of cardiac hypertrophy.31
Discussion
As is common in most biological systems, it is evident that the hypertrophic signaling pathways are counteracted by a number of antihypertrophic or negative-feedback mechanisms.11 Strategies to stimulate the latter, as well as inhibit the former, may be of significant therapeutic value. Relatively well-characterized negative-feedback regulators of cardiac hypertrophy includes ANF,6 MCIP1,7,8 SOCS3,9 and Nab1.10
In this study, we report that PICOT is upregulated in neonatal cardiomyocytes stimulated with hypertrophic agonists, as well as in pressure-overloaded hearts. Because adenovirus-mediated expression of PICOT in cardiomyocytes or transgenic expression of PICOT in hearts abrogates the hypertrophic responses, it appears that PICOT constitutes a novel negative-feedback circuit for cardiac hypertrophy.
PICOT was first identified as a PKC-interacting protein in a yeast 2-hybrid screen.18 It has an amino-terminal thioredoxin homology (TH) domain that has a 29% amino acid identity and an additional 11% similarity with the thioredoxin family of proteins. Thioredoxin plays a critical role in regulating cardiac oxidative stress and recently has been shown to prevent the development of cardiac hypertrophy through a redox-sensitive mechanism.32 However, in a contrasting report, thioredoxin appears to promote cardiac hypertrophy by serving as a transcriptional cofactor, with its activity regulated by an endogenous inhibitor, thioredoxin-interacting protein (Txnip).33 Thus, it remains to be determined whether thioredoxin function in cardiomyocytes is pro- or antihypertrophic or both. PICOT is less likely to be involved in intracellular redox regulation because the TH domain of PICOT lacks the conserved Cys-Gly-Pro-Cys motif that is essential for catalytic activity; instead, it contains an Ala-Pro-Gln-Cys sequence. Rather, the TH motif of PICOT appears to serve as a structural platform for a specific interaction with PKC isozymes. This notion is supported by the fact that thioredoxin directly interacts with PKC, PKC, PKC, and PKC and inhibits their catalytic activities in vitro.34 However, the possibility that the TH domain of PICOT serves as a dominant negative form of thioredoxin cannot be ruled out.
The carboxy-terminal domain of PICOT contains 2 tandem repeats of an evolutionarily conserved domain of unknown function, referred to as the PICOT homology (PH) domain. We observed that this domain interacts with a number of sarcomeric proteins including MLP and crystallin B (CryB) (D.J., R.J.H., and W.J.P., unpublished data, 2006). Interestingly, these 2 molecules have been implicated in the development of dilated cardiomyopathy.35–37 It is possible that these interactions might be responsible for increased Ca2+ responsiveness of myofilaments. Vigorous genetic and biochemical studies are underway to elucidate a functional link associated with the interaction of PICOT and MLP or CryB.
Deterioration in cardiac contractility is presumed to be a prerequisite for the transition from cardiac hypertrophy to heart failure. PKC-mediated phosphorylation of native thin filaments in failing human hearts is associated with reduced contractility,21 and PKC triggers a chain of interactions that leads to decreased cardiac contractility.19,20 Moreover, inhibition of PKC-mediated phosphorylation of TnI improves cardiac function in vivo.22,23 Therefore, it seems reasonable to speculate that augmentation of PICOT activity in hypertrophied or failing hearts may not only inhibit or reverse hypertrophic conditions but may also help restore cardiac contractility. Indeed, we found that transgenic or adenoviral mediated-overexpression of PICOT dramatically increases the ventricular function of hearts and the contractility of isolated cardiomyocytes. Detailed mechanisms underlying this positive inotropic effect of PICOT remain to be elucidated. Increased phosphorylation of PLB in the PICOT overexpressing cardiomyocytes may reflect a part of the mechanisms. Other mechanisms might include changing phosphorylation status of myofilament proteins such as TnI and modulating function of other sarcomeric proteins such as MLP and CryB.
In conclusion, we have elucidated a novel feedback inhibitory mechanism of cardiac hypertrophy signaling. PICOT, the central molecule in this feedback loop, blocks the development of cardiac hypertrophy in both in vitro and in vivo. PICOT has appeal as a potential therapeutic modality for preventing cardiac hypertrophy and heart failure because it not only blocks hypertrophic signaling but also enhances the inotropic property of cardiomyocytes.
Acknowledgments
Sources of Funding
During this work, W.J.P. was supported by the Global Research Laboratory Program of the Korean Ministry of Science and Technology, a grant (A050472) from the Korean Ministry of Health and Welfare, and a scholarship from the SBS Foundation. D.H.K. was supported by a Systems Biology Research grant (M1–0309-00-0006) from the Korean Ministry of Science and Technology. R.J.H. and W.J.P. were supported by NIH grant HL-080498-01.
Disclosures
None.
Footnotes
These authors contributed equally to this study.
Original received December 5, 2005; revision received June 15, 2006; accepted June 19, 2006.
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Geier C, Perrot A, Ozcelik C, Binner P, Counsell D, Hoffmann K, Pilz B, Martiniak Y, Gehmlich K, van der Ven PF, Furst DO, Vornwald A, von Hodenberg E, Nurnberg P, Scheffold T, Dietz R, Osterziel KJ. Mutations in the human muscle LIM protein gene in families with hypertrophic cardiomyopathy. Circulation. 2003; 107: 1390–1395.
the Department of Life Science (D.J., H.C., E.K., M.K., D.K.Y., J.M.K., P.O.Y., J.G.O., D.H.K., W.J.P.), Global Research Laboratory on Cardiovascular Gene Therapy, Gwangju Institute of Science and Technology, Korea
Cardiovascular Research Center (O.Y.B., S.S., R.J.H.), Massachusetts General Hospital and Harvard Medical School, Charlestown
College of Pharmacy (L.T.T., S.-H.W.), Chungnam National University, Daejeon, Korea
Cardiovascular Division (L.C., R.L.), Brigham and Women’s Hospital and Harvard Medical School, Boston, Mass
Department of Oral Anatomy (Y.-H.L.), School of Dentistry, Chonbuk National University, Jeonju, Korea.
Abstract
Multiple signaling pathways involving protein kinase C (PKC) have been implicated in the development of cardiac hypertrophy. We observed that a putative PKC inhibitor, PICOT (PKC-Interacting Cousin Of Thioredoxin) was upregulated in response to hypertrophic stimuli both in vitro and in vivo. This suggested that PICOT may act as an endogenous negative feedback regulator of cardiac hypertrophy through its ability to inhibit PKC activity, which is elevated during cardiac hypertrophy. Adenovirus-mediated gene transfer of PICOT completely blocked the hypertrophic response of neonatal rat cardiomyocytes to enthothelin-1 and phenylephrine, as demonstrated by cell size, sarcomere rearrangement, atrial natriuretic factor expression, and rates of protein synthesis. Transgenic mice with cardiac-specific overexpression of PICOT showed that PICOT is a potent inhibitor of cardiac hypertrophy induced by pressure overload. In addition, PICOT overexpression dramatically increased the ventricular function and cardiomyocyte contractility as measured by ejection fraction and end-systolic pressure of transgenic hearts and peak shortening of isolated cardiomyocytes, respectively. Intracellular Ca2+ handing analysis revealed that increases in myofilament Ca2+ responsiveness, together with increased rate of sarcoplasmic reticulum Ca2+ reuptake, are associated with the enhanced contractility in PICOT-overexpressing cardiomyocytes. The inhibition of cardiac remodeling by of PICOT with a concomitant increase in ventricular function and cardiomyocyte contractility suggests that PICOT may provide an efficient modality for treatment of cardiac hypertrophy and heart failure.
Key Words: cardiac hypertrophy protein kinase C PICOT contractility
Introduction
The myocardium undergoes adaptive hypertrophic growth to augment cardiac output in response to a variety of pathological insults. Although this response initially appears to be beneficial, sustained cardiac hypertrophy leads to an increased risk of sudden death or progression to heart failure.1 Therefore, therapies directed at inhibiting or reversing cardiac hypertrophy could be of significant clinical value. Intensive investigations in the past decade have revealed that multiple parallel intracellular signaling pathways can induce cardiac hypertrophy (reviewed previously2–4). No single pathway seems to regulate cardiac hypertrophy alone. Rather, it appears more likely that each pathway operates as a component of an orchestrated hypertrophic network.
In recent years, potential antihypertrophic and inhibitory feedback signaling pathways have been discovered. Their integration into the hypertrophic signaling pathway network adds considerable complexity. Atrial natriuretic factor (ANF), long considered to be a typical molecular marker for cardiac hypertrophy, has been shown to be antihypertrophic by activating cGMP-dependent protein kinase (PKG).5,6 In addition, a calcineurin inhibitory protein, myocyte-enriched calcineurin-interacting protein 1 (MCIP1), has been shown to participate in a negative feedback loop that limits the potentially deleterious effect of unrestrained calcineurin activity during cardiac hypertrophy.7,8 Moreover, SOCS3 (a Suppressor Of Cytokine Signaling 3) binds to janus kinase (JAK) and acts a negative feedback regulator of gp130-mediated cardiac hypertrophy.9 Recently, the transcriptional repressor Nab1 has been shown to intervene in cardiac hypertrophy by repressing early-growth response (Egr) transcription factors.10 These findings suggest that hypertrophic stimuli evoke antihypertrophic or negative feedback pathways, as well as positive hypertrophic signaling pathways. Augmenting these negative regulators, rather than inhibiting the positive regulators, may be a viable antihypertrophic strategy.11
Protein kinase C (PKC) is a ubiquitously expressed serine/threonine kinase and has been implicated in the development of cardiac hypertrophy.12 The rat heart contains at least 5 PKC isozymes (PKC, PKCI, PKC, PKC, and PKC) with distinct expression patterns. The relative importance of these PKC isozymes in hypertrophic signaling remains controversial13–15 and crosstalks between the PKC isozymes may complicate data interpretation.16 In this study, we show that a putative PKC inhibitor, PICOT (PKC-Interacting Cousin Of Thioredoxin), functions as a negative regulator of cardiac hypertrophy. Interestingly, PICOT overexpression dramatically increased ventricular function and cardiomyocyte contractility.
Materials and Methods
An expanded Materials and Methods section is available in the online data supplement at http://circres.ahajournals.org.
Cell Culture and Hypertrophic Stimulation
Primary cultures of cardiomyocytes from 2-day-old Sprague–Dawley rats were prepared as described.17 Briefly, ventricular tissue was enzymatically dissociated, and the resulting cell suspension was enriched for cardiomyocytes using Percoll (Amersham Pharmacia) step gradients. Cells were plated onto either collagen-coated culture dishes or cover slips and cultured in cardiomyocyte culture medium (DMEM supplemented with 10% FBS and 2 mmol/L L-glutamine; GIBCO BRL). To induce hypertrophy, cardiomyocytes were cultured in serum-free medium for at least 24 hours and then treated with 100 nmol/L enthothelin-1 (ET-1) or 100 μmol/L phenylephrine (PE) for 24 hours.
Generation of Transgenic Mice
A full-length mouse PICOT cDNA with human growth hormone 3' untranslated region was subcloned into a 5.5-kb segment of the –myosin heavy chain (-MHC) promoter. The DNA construct was microinjected into FVB fertilized eggs and transgenic integration was confirmed by Southern blotting (Macrogen, Korea). Transgenic mice and wild-type littermates were analyzed at 8 to 10 weeks of age.
Results
PICOT Is Upregulated During Cardiac Hypertrophy
To identify negative regulators of cardiac hypertrophy, a PCR-based differential screening technique was performed, based on the assumption that antihypertrophic or negative regulators were likely to be induced during cardiac hypertrophy, along with positive hypertrophic regulators. Transverse aortic constriction (TAC) was used to induce cardiac hypertrophy in adult rat hearts. Some novel genes, as well as numerous genes previously found to be associated with cardiac hypertrophy, were isolated (see the Table in the online data supplement). Among the genes that were expressed preferentially in the hypertrophied hearts, as compared with the sham-operated normal hearts, PICOT was of particular interest. PICOT was first identified as PKC-interacting protein by a yeast 2-hybrid screen.18 When transiently overexpressed in T cells, PICOT inhibited the activation of c-jun N-terminal kinase (JNK), activator protein-1 (AP-1), and nuclear factor B (NF-B), suggesting that PICOT functioned as an endogenous inhibitor of PKC.18 Considering that PKC isozymes play important roles in the development of cardiac hypertrophy, the elevated expression of PICOT in hypertrophied hearts suggested a novel negative feedback circuit in cardiac hypertrophy.
Northern blot analysis was used to confirm the induction of PICOT by hypertrophic stimuli in vivo and in vitro. The expression level of PICOT was approximately 3 times higher in hypertrophied hearts than in sham-operated hearts (Figure 1A). PICOT also was induced in neonatal rat cardiomyocytes after exposure to the hypertrophic agonists ET-1 or phenylephrine PE (Figure 1B). In both cases, the extent of PICOT induction was comparable to that of ANF. The induction of PICOT during the progression of cardiac hypertrophy also was examined (Figure 1C). The induction of ANF began 2 days after TAC, peaked after 2 weeks, and declined sharply thereafter. This ANF-induction pattern previously had been shown to correlate well with the onset of cardiac hypertrophy.9 In contrast, the induction of PICOT by TAC was rather slow, beginning approximately 1 week after the induction of ANF and continuing to increase when ANF expression was completely diminished at 3 weeks after banding (Figure 1C). Thus it appeared possible that ANF constituted an acute cardiac hypertrophy inhibitory mechanism, whereas PICOT had a later-onset inhibitory function. Fibroblasts and cardiomyocytes were separately isolated from the sham-operated and hypertrophied hearts (3 weeks after TAC), respectively, and the PICOT level in the protein extracts were determined by immunoblotting (Figure 1D). Approximately 2-fold increase in the PICOT level was detected in the hypertrophied cardiomyocytes but not in the fibroblasts. This indicates that the inhibitory activity of PICOT on the development of cardiac hypertrophy is cell autonomous.
PICOT Inhibits the ET-1– or PE-Induced Hypertrophic Response
To evaluate the ability of PICOT to inhibit cardiac hypertrophy, AdPICOT (PICOT-expressing recombinant adenovirus) was generated. Adenoviral infection routinely resulted in infection of more than 90% of the cultured neonatal rat cardiomyocytes (data not shown). Western blot analyses revealed a protein band in the cardiomyocytes transfected with AdPICOT but not in nontransfected cardiomyocytes or in cardiomyocytes transfected with AdLacZ (-galactosidase-expressing adenovirus) (Figure 2A). The apparent molecular weight of the PICOT band was 40 to 42 kDa, which was slightly higher than the calculated molecular mass (38 kDa), suggesting that PICOT might be posttranslationally modified. At 24 hours after infection, the cardiomyocyte cultures were further stimulated with ET-1 or PE for 24 hours. These treatments significantly increased the size of the AdLacZ-transfected or untransfected cardiomyocytes, as assessed by measuring the surface area of the cells under a microscope. In contrast, no significant ET-1– or PE-induced increase in cell size was observed with the AdPICOT-transfected cardiomyocytes (Figure 2B).
Another feature of the hypertrophic response of cardiomyocytes is a pronounced sarcomeric rearrangement that can be detected by immunostaining with -actinin antibody. Unlike the nontransfected or AdLacZ-transfected cardiomyocytes, the cardiomyocytes transfected with AdPICOT lacked prominent sarcomeric structures after ET-1 or PE treatment (Figure 2C). ANF expression also is a typical marker for cardiac hypertrophy. Treatment with ET-1 or PE significantly increased ANF immunoreactivity in nontransfected or AdLacZ-transfected cardiomyocytes. However, PICOT transfection blocked this elevated ANF immunoreactivity (Figure 2D). Lastly, the increased protein synthesis induced by ET-1 or PE treatment, as determined by 3H-leucine incorporation, was abrogated completely by PICOT transfection (Figure 2E). Taken together, these results indicated that the forced expression of PICOT inhibited agonist-induced cardiac hypertrophy in neonatal rat cardiomyocytes.
PICOT Abrogates Pressure-Overload–Induced Cardiac Hypertrophy
To investigate the role of PICOT as a negative regulator of cardiac hypertrophy in vivo, transgenic mice were generated that expressed PICOT in the heart, under the control of the -MHC promoter (Figure 3A). Two independent lines (TG17 and TG60) were obtained with the -MHC-PICOT transgene. Transgenic lines did not exhibit any obvious gross abnormalities. Western blot analysis revealed that cardiac PICOT expression in both lines was approximately 60% to 80% higher than in the wild-type littermates (Figure 3B). We observed that endogenous PICOT was present both in the cardiomyocytes and fibroblasts of hearts, and the transgene drove PICOT expression in the cardiomyocytes (data not shown).
To determine whether PICOT overexpression antagonized the hypertrophic response to physiologically relevant pressure overload, wild-type littermates and TG17 mice were subjected to TAC. The induction of pressure overload stimulated an &33% increase in the heart weight to body weight ratio in wild type over 14 days. In contrast, hypertrophic growth was blunted significantly (P<0.05) in transgenic mice, with approximately a 15% increase in the heart weight to body weight ratio (Figure 3C). Furthermore, microscopic analysis of histological sections revealed that the increase in the ventricular cross-sectional area was reduced significantly (P<0.01) in transgenic mice (a 25% increase in TAC versus sham) compared with wild type (an 82% increase in TAC versus sham) (Figure 3D). The induction of several fetal genes, including ANF and skeletal actin (SKA), is associated with cardiac hypertrophy. Northern blot analysis showed that the induction of these hypertrophic marker genes was reduced significantly in transgenic mice as compared with wild type (Figure 3E). These results indicated that PICOT abrogated the development of left ventricular hypertrophy induced by pressure overload in vivo. Similar results were obtained with TG60 (data not shown).
PICOT Enhances Ventricular Function and Cardiomyocyte Contractility
Previous reports showed that PKC activation was associated with a reduction of cardiac contractility via the dephosphorylation of phospholamban (PLB)19,20 or the phosphorylation of native thin filaments21 and troponin I (TnI).22,23 Because PICOT has been thought to inhibit PKC, its effects on cardiac contractility were examined. Adult cardiomyocytes were isolated from wild-type and transgenic hearts, and their contractility was measured. An increase in contractility of approximately 89% was observed in transgenic cardiomyocytes (11.0%±1.6) as compared with wild type (5.8%±1.1), as measured by peak shortening (Figure 3F). To determine the in vivo functional consequences of PICOT overexpression, hemodynamic parameters were measured under basal conditions. PICOT transgenic mice showed a 27% increase in ejection fraction and an 18% increase in end-systolic pressure accompanied by a 47% increase in the first derivative of left ventricular systolic pressure rise (+dP/dt) and a 25% increase in the decline (–dP/dT) compared with the wild-type littermates, demonstrating enhanced contraction and relaxation (Figure 3G). Importantly, both heart rate (data not shown) and left ventricular diastolic pressure (Figure 3G), parameters that can affect left ventricular contractility, were indistinguishable in transgenic and wild-type mice. Thus, PICOT not only inhibited the development of cardiac hypertrophy both in vitro and in vivo but also increased ventricular function in vivo in transgenic models.
To examine the effects of PICOT in cardiomyocytes, isolated adult rat cardiomyocytes were transfected with either AdLacZ or AdPICOT, and the consequential mechanical properties were determined using a dual-excitation spectrofluorometer equipped with video-edge detection system. AdPICOT-transfected cardiomyocytes showed an 84% increase in cell shortening, a 57% increase in maximal rate of contraction (–dL/dt), and an 83% increase in the maximal rate of relaxation (+dL/dt) in comparison with AdLacZ-transfected cardiomyocytes (Figure 4A).
To further define the mechanism underlying the enhanced contractility associated with PICOT, intracellular Ca2+ handling was analyzed. Ca2+ transient amplitude and time to peak fluorescence were comparable in AdLacZ- and AdPICOT-transfected cardiomyocytes. Time required to reach 90% baseline fluorescence and time constant of Ca2+ transient decay, , were reduced 15% and 18%, respectively, in AdPICOT-transfected cardiomyocytes (Figure 4B). Thus, PICOT overexpression appears to be coupled with more efficient reuptake of Ca+2 by sarcoplasmic reticulum (SR) during relaxation but not with the enhanced Ca2+ transient amplitude. Cell shortening was plotted against cytosolic Ca2+ content during steady-state contraction (Figure 4C). Upward and leftward shift of the loop in AdPICOT-transfected cardiomyocytes indicated increased cell shortening for any given cytosolic Ca2+ concentration, implying an increase in Ca2+ sensitivity of the myofilaments, compared with AdLacZ-transfected cardiomyocytes. These results indicate that increases in myofilament Ca2+ responsiveness, together with increased rate of SR Ca2+ reuptake, promote an enhanced contractile function in AdPICOT-transfected cardiomyocytes. We observed that phosphorylation at Ser16 of pentameric PLB was significantly increased, whereas total pentameric PLB protein level was significantly reduced in AdPICOT-transfected cardiomyocytes. Therefore, a >2-fold increase in the ratio of phosphorylated PLB to total PLB was observed on PICOT overexpression (Figure 4D). Without a change in expression of SERCA-2 and calsequestrin (data not shown), the hyperphosphorylation of PLB is thought to render SERCA-2 more active, resulting in the enhanced SR Ca2+ reuptake in AdPICOT-transfected cardiomyocytes.
PICOT Suppresses the Activation of PKC and Mitogen-Activated Protein Kinases
PKC activation has been associated with the development of cardiac hypertrophy, and PICOT has been thought to function as an intrinsic inhibitor of PKC. Therefore, the activation of PKC in cardiomyocytes stimulated with ET-1 or PE was tested under the forced expression of PICOT. Using a PKC-specific phosphorylation assay, 2.5- and 4.5-fold increases were observed in the total PKC activity in cardiomyocytes treated with ET-1 or PE, respectively. These increases were abrogated completely by transfection with AdPICOT but not with AdLacZ (Figure 5A). PKC, PKC, and PKC are the most highly expressed isoforms in the adult rat heart.24–26 In addition to these isozymes, PKC also is expressed abundantly in neonatal rat cardiomyocytes and weakly in the adult rat heart (data not shown). The near-complete suppression of total PKC activity by PICOT suggested that the activation of all these PKC isozymes might be abrogated by PICOT transfection. Thus the effect of PICOT overexpression on the specific activation of PKC, PKC, and PKC was evaluated using phospho-PKC, isozyme-specific antibodies. PKC, PKC, and PKC were activated significantly by stimulation with ET-1 or PE, and concomitant PICOT transfection prevented this activation (Figure 5B).
Because mitogen-activated protein (MAP) kinase pathways appear to be a merging point for divergent hypertrophic signaling pathways involving PKC,12,27–30 PICOT inhibition of MAP kinase activation was investigated using antibodies against phospho–extracellular signal-regulated kinase 1/2 (phospho–ERK1/2), phospho-JNK, and phospho-p38. ERK1/2, JNK, and p38 were activated significantly by stimulation with ET-1 or PE. PICOT transfection abrogated the activation of ERK1/2 and JNK but not p38 (Figure 5C). These results indicated that PICOT may counteract hypertrophic stimulation by inhibiting PKC and the downstream ERK1/2 and JNK signaling pathways. These results were in agreement with the previous observation that p38 may play a role in cardiomyocyte survival but not in the development of cardiac hypertrophy.31
Discussion
As is common in most biological systems, it is evident that the hypertrophic signaling pathways are counteracted by a number of antihypertrophic or negative-feedback mechanisms.11 Strategies to stimulate the latter, as well as inhibit the former, may be of significant therapeutic value. Relatively well-characterized negative-feedback regulators of cardiac hypertrophy includes ANF,6 MCIP1,7,8 SOCS3,9 and Nab1.10
In this study, we report that PICOT is upregulated in neonatal cardiomyocytes stimulated with hypertrophic agonists, as well as in pressure-overloaded hearts. Because adenovirus-mediated expression of PICOT in cardiomyocytes or transgenic expression of PICOT in hearts abrogates the hypertrophic responses, it appears that PICOT constitutes a novel negative-feedback circuit for cardiac hypertrophy.
PICOT was first identified as a PKC-interacting protein in a yeast 2-hybrid screen.18 It has an amino-terminal thioredoxin homology (TH) domain that has a 29% amino acid identity and an additional 11% similarity with the thioredoxin family of proteins. Thioredoxin plays a critical role in regulating cardiac oxidative stress and recently has been shown to prevent the development of cardiac hypertrophy through a redox-sensitive mechanism.32 However, in a contrasting report, thioredoxin appears to promote cardiac hypertrophy by serving as a transcriptional cofactor, with its activity regulated by an endogenous inhibitor, thioredoxin-interacting protein (Txnip).33 Thus, it remains to be determined whether thioredoxin function in cardiomyocytes is pro- or antihypertrophic or both. PICOT is less likely to be involved in intracellular redox regulation because the TH domain of PICOT lacks the conserved Cys-Gly-Pro-Cys motif that is essential for catalytic activity; instead, it contains an Ala-Pro-Gln-Cys sequence. Rather, the TH motif of PICOT appears to serve as a structural platform for a specific interaction with PKC isozymes. This notion is supported by the fact that thioredoxin directly interacts with PKC, PKC, PKC, and PKC and inhibits their catalytic activities in vitro.34 However, the possibility that the TH domain of PICOT serves as a dominant negative form of thioredoxin cannot be ruled out.
The carboxy-terminal domain of PICOT contains 2 tandem repeats of an evolutionarily conserved domain of unknown function, referred to as the PICOT homology (PH) domain. We observed that this domain interacts with a number of sarcomeric proteins including MLP and crystallin B (CryB) (D.J., R.J.H., and W.J.P., unpublished data, 2006). Interestingly, these 2 molecules have been implicated in the development of dilated cardiomyopathy.35–37 It is possible that these interactions might be responsible for increased Ca2+ responsiveness of myofilaments. Vigorous genetic and biochemical studies are underway to elucidate a functional link associated with the interaction of PICOT and MLP or CryB.
Deterioration in cardiac contractility is presumed to be a prerequisite for the transition from cardiac hypertrophy to heart failure. PKC-mediated phosphorylation of native thin filaments in failing human hearts is associated with reduced contractility,21 and PKC triggers a chain of interactions that leads to decreased cardiac contractility.19,20 Moreover, inhibition of PKC-mediated phosphorylation of TnI improves cardiac function in vivo.22,23 Therefore, it seems reasonable to speculate that augmentation of PICOT activity in hypertrophied or failing hearts may not only inhibit or reverse hypertrophic conditions but may also help restore cardiac contractility. Indeed, we found that transgenic or adenoviral mediated-overexpression of PICOT dramatically increases the ventricular function of hearts and the contractility of isolated cardiomyocytes. Detailed mechanisms underlying this positive inotropic effect of PICOT remain to be elucidated. Increased phosphorylation of PLB in the PICOT overexpressing cardiomyocytes may reflect a part of the mechanisms. Other mechanisms might include changing phosphorylation status of myofilament proteins such as TnI and modulating function of other sarcomeric proteins such as MLP and CryB.
In conclusion, we have elucidated a novel feedback inhibitory mechanism of cardiac hypertrophy signaling. PICOT, the central molecule in this feedback loop, blocks the development of cardiac hypertrophy in both in vitro and in vivo. PICOT has appeal as a potential therapeutic modality for preventing cardiac hypertrophy and heart failure because it not only blocks hypertrophic signaling but also enhances the inotropic property of cardiomyocytes.
Acknowledgments
Sources of Funding
During this work, W.J.P. was supported by the Global Research Laboratory Program of the Korean Ministry of Science and Technology, a grant (A050472) from the Korean Ministry of Health and Welfare, and a scholarship from the SBS Foundation. D.H.K. was supported by a Systems Biology Research grant (M1–0309-00-0006) from the Korean Ministry of Science and Technology. R.J.H. and W.J.P. were supported by NIH grant HL-080498-01.
Disclosures
None.
Footnotes
These authors contributed equally to this study.
Original received December 5, 2005; revision received June 15, 2006; accepted June 19, 2006.
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