Published online before print November 17, 2005, doi: 10.1161/01.RES.0000196578.15385.bb
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© 2005 American Heart Association, Inc.
Cellular Biology |
Phospholipase C 
Modulates ß-Adrenergic Receptor– Dependent Cardiac Contraction and Inhibits Cardiac Hypertrophy
From the Departments of Pharmacology and Physiology (H.W., E.A.O., R.T.D., B.C.B., A.V.S.) and Biochemistry and Biophysics (H.W., A.V.S.) and the Cardiovascular Research Institute (N.M., T.A.B., B.C.B.), University of Rochester School of Medicine, Rochester, NY; and Departments of Pharmacology (K.L.V., G.G.K.) and Medicine (G.G.K.), State University of New York Upstate Medical University, Syracuse.
Correspondence to Alan V. Smrcka, Department of Pharmacology and Physiology, University of Rochester School of Medicine, 601 Elmwood Ave, Box 711, Rochester, NY 14642. E-mail Alan_Smrcka{at}URMC.rochester.edu
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Abstract |
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Phospholipase C (PLC)
is a recently identified enzyme regulated by a wide range of molecules including Ras family small GTPases, Rho A, G
12/13, and Gß
with primary sites of expression in the heart and lung. In a screen for human signal transduction genes altered during heart failure, we found that PLC mRNA is upregulated. Two murine models of cardiac hypertrophy confirmed upregulation of PLC protein expression or PLC RNA. To identify a role for PLC in cardiac function and pathology, a PLC-deficient mouse strain was created. Echocardiography indicated PLC–/– mice had decreased cardiac function, and direct measurements of left ventricular contraction demonstrated that PLC–/– mice had a decreased contractile response to acute isoproterenol administration. Cardiac myocytes isolated from PLC–/– mice had decreased ß-adrenergic receptor (ßAR)-dependent increases in Ca2+ transient amplitudes, likely accounting for the contractile deficiency in vivo. This defect appears to be independent from the ability of the ßAR system to produce cAMP and regulation of sarcoplasmic reticulum Ca2+ pool size. To address the significance of these functional deficits to cardiac pathology, PLC–/– mice were subjected to a chronic isoproterenol model of hypertrophic stress. PLC–/– mice were more susceptible than wild-type littermates to development of hypertrophy than wild-type littermates. Together, these data suggest a novel PLC-dependent component of ßAR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve and loss of PLC signaling sensitizes the heart to development of hypertrophy in response to chronic cardiac stress.
Key Words: phospholipase C • ß-adrenergic receptor • heart failure • contractility
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Introduction |
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Agonist regulation of intracellular calcium and protein kinase C (PKC) signaling through activation of phospholipase C (PLC) modulates a wide range of physiological responses. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which increase cytosolic calcium concentration and activate PKC, respectively. Five PLC families have been identified as ß,
, 
, 
, and . All contain X and Y domains that form the catalytic core for PIP2-PLC hydrolysis activity, whereas each isoform has unique domains involved in physiological regulation by distinct signaling pathways. PLCß and - isoforms are well-studied enzymes that are regulated by G protein–coupled receptors and receptor tyrosine kinases, respectively.1,2 PLC is a novel PLC isoform that has been shown to be regulated by Ras,3 Rap,3–5 RhoA,5,6 RalA,5 TC21,5 Rac,5 G12/13,5,7 and Gß8 in transfected cells. PLC is also a unique bifunctional enzyme that, in addition to PLC catalytic activity, has an N-terminal domain with homology to the Ras guanine nucleotide exchange factor (GEF) CDC25 and has GEF activity toward Rap.9
A tissue with significant levels of PLC expression is the heart. Roles for specific PLC isoforms in cardiac function have not been well documented. PLC isoforms including PLCß and PLC are expressed in cardiac myocytes, but their respective roles in cardiac biology and disease have not been thoroughly documented. PIP2 hydrolysis pathways have been clearly implicated downstream of Gq-coupled receptors, such as adrenergic receptors and angiotensin II receptors in cardiac myocytes, and presumably involve PLCß isoforms. PLC is not Gq regulated, but it is potentially downstream of G protein–coupled receptors coupled to G12/13 or growth factor tyrosine kinases through Ras. Interestingly, a signaling pathway from ß2-adrenoceptor, through Gs, cAMP, Epac, and Rap2B to PLC and calcium signaling has been proposed based on studies in cultured cells transfected with PLC.10 It remains to be determined whether the in vivo PLC regulatory signaling network in the heart intersects with ß-adrenergic receptor (ßAR) signaling pathways and/or other pathways.
In a screen for signal transduction genes upregulated in human heart failure, we found that PLC is upregulated in human hearts during heart failure and in 2 murine models of hypertrophic stress. We report creation of a genetically modified mouse with targeted disruption of the PLC gene to address the possible role of PLC in cardiac function and disease. These PLC-deficient mice showed decreased responsiveness to ßAR stimulation and have an increased susceptibility to cardiac hypertrophy under chronic stress. We propose that PLC is required for maximum efficacy of the myocardial ßAR system and that its levels may increase in an adaptive response to ßAR downregulation during heart failure.
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Materials and Methods |
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Real-Time PCR
We used 200 ng of total RNA for reverse transcription. The Superscript First-Strand Synthesis System for RT-PCR (Invitrogen) was used according to the instructions of the manufacturer. Human primers sequences for PLC
were designed to have a 530-bp product from either splice variant of PLC and were as follows: forward primer 5'-GGGGCCACGGTCATCCAC-3' and reverse primer 5'-GGGCCTTCATACCGTCCATCCTC-3'. Human GAPDH primer sequences were 5'-GCCAAAAGGGTCATCATCTC-3' and reverse primer 5'-GGCCATCCACAGTCTTCT-3'. Real-time PCR was performed using the ABI Prism 7900 HT Sequence Detection System. For analysis, cycle threshold (Ct) values were calculated for each sample; this value represents the value at which the fluorescent signal rises above background levels. Gene expression was further analyzed by the 2–
Ct method described by Livak and Schmittgen.11
Generation of the PLC Knockout Mouse
A purified PCR product from the 5' end of the GEF domain (amino acids 419 to 752 in the rat PLC 1a protein) was used to generate random primed 32P radioactive probes to identify the appropriate BAC clones on high-density filters spotted with The Children’s Hospital Oakland Research Institute 129S/SvEvTac mouse Bac library. Appropriate clones were identified and obtained from The Children’s Hospital Oakland Research Institute, the appropriate DNA fragments flanking exon 6 were subcloned into the targeting vector as shown in Figure 2B, and the inserted DNA fragments and vector junctions were sequenced and compared with the National Center for Biotechnology Information mouse genomic sequence.
We used a standard homologous recombination replacement strategy (Figure 2B). The targeting vector contained positive (neo) and negative (TK) selection markers and an internal ribosome entry sequence driving expression of LacZ. The linearized targeting construct was electroporated into 129/S6 ES cells and G418- and gancyclovir-resistant colonies were selected. From 200 selected clones, 1 was found to have the correct genomic structure sequence based on Southern blotting of EcoRV digests of genomic DNA, with a probe against the PLC gene, outside the region of recombination (Figure 2B and 2C). These results were confirmed by PCR analysis. These ES cells were injected into blastocysts to create chimeric mice. Six male chimeric mice were bred with C57/B6 females. Heterozygous offspring were identified by brown coat color and subsequent PCR analysis (Figure 2D). These PLC+/– mice were bred to generate homozygous deletion in the F2 generation mice and the line was maintained in a mixed C57/B6, 129/S6 genetic background. PCR analysis was used to identify –/–, –/+, and +/+ animals, and Western blotting for PLC protein was used to confirm loss of PLC protein (Figure 2E). The experiments were approved by the Animal Care and Use Committee of the University of Rochester and followed the guidelines of the National Institutes of Health.
Echocardiography
Transthoracic 2D and M-mode echocardiography analysis was used to assess basal heart function in conscious mice with an Acuson Sequoia C236 echocardiography machine equipped with a 15 MHz frequency probe (Siemens Medical Solutions). Data were collected from 11 animals each of PLC–/– and PLC+/+ animals followed at 2, 4, and 6 months of age and the pooled data analyzed for statistical significance.
Cardiac Hemodynamics
Cardiac hemodynamic parameters were analyzed by inserting a Millar pressure transducer into the left ventricle of anesthetized PLC+/+ and PLC–/– mice as previously described.12
Chronic Isoproterenol Infusion and Analysis of Hypertrophy
Miniosmotic pumps (Alzet Model 1007D) were implanted in mice anesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight). Pumps were filled with isoproterenol or vehicle (0.002% ascorbic acid in PBS) and were set to deliver isoproterenol at 30 mg/kg per day for 7 days each. Mice were subsequently euthanized; their were hearts excised, weighed, and dissected into left atrium, right atrium, right ventricular free wall, and left ventricular (LV) free wall plus septum samples, which were weighed and snap frozen in liquid nitrogen.
Aortic Banding
Eight-week-old male Swiss Webster mice were anesthetized with an IP injection of ketamine (116 mg/kg), xylazine (23 mg/kg), and acepromazine (11 mg/kg) and then intubated. Essentially as described by others,13 a partial transaortic constriction was produced.
Northern Blotting
Tissues were collected three weeks after surgery. Eight micrograms of LV RNA was separated on a formaldehyde-containing agarose gel and electrophoretically transferred to nylon membrane. Membranes were hybridized with random-primed 32P-labeled probes generated using a 376-bp C2 domain fragment of a rat PLC cDNA as a template. This probe recognizes both splice variants of PLC.
Isolation of Cardiac Myocytes
Myocytes were isolated from 4- to 6-month-old wild-type or PLC–/– mice essentially as described,14 except 3 mg/dL Liberase/Blendzyme I (Roche) and 0.01% Trypsin/EDTA replaced collagenase in the perfusion buffer. Cells were plated on laminin coated coverslips at a density of 1x105 cells/35 mm dish.
Analysis of Ca2+ Transients in Cardiac Myocytes
Cells plated on laminin-coated coverslips were loaded with 2 µmol/L Indo-1. Individual cells were excited at 350 nm and the fluorescence emission ratio (405/485 ratio) was collected every 10 ms. The cells were electrically stimulated locally (8V, 50 ms, 1 Hz) by placing an extracellular electrode close to the cell of interest. After collection of data in the absence of ß-adrenergic stimulation, the cells were perfused with 1 µmol/L isoproterenol followed by a train of electrical stimuli. Calcium transient amplitudes were calculated by measuring the change in 405/485 fluorescence with each electrical pulse from 10 to 30 cells for each treatment from each animal. Sarcoplasmic reticulum (SR) Ca2+ content in individual cardiac myocytes was assessed from the magnitude of the myoplasmic Ca2+ transient induced by the application of 10 mmol/L caffeine.
ßAR Binding
Membranes from freshly isolated hearts were removed from 4-month-old PLC–/– and PLC+/+ animals and assayed for 125I-cyanopindolol binding following standard procedures.
Measurement of cAMP
Isolated myocytes (100 000) were suspended in 1 mL of minimum essential medium containing 2.5% FBS, 10 mmol/L 2,3 butanedione monoxime (BDM), and 1 mmol/L isobutyl methyl xanthine (IBMX) and incubated at 37°C for 10 minutes either in the absence or presence of 1 µmol/L isoproterenol. cAMP levels were assayed with a standard radiocompetition assay.
Western Blotting
For immunoprecipitation detection of PLC, rabbit, anti-PLC RA2 (2933) domain antibody, and protein A/G sepharose beads (Santa Cruz Biotechnology) were incubated with soluble NP40 extract containing 1 mg of total protein for 2 hours. Immune complexes were washed 5 times with lysis buffer containing 1% NP40, isolated by centrifugation, and applied to 9% sodium dodecyl sulfate polyacrylamide gel. After resolution, proteins were transferred to polyvinylidene fluoride membranes for 2 hours at 100 V at 4°C. PLC was detected with a rabbit anti-PLC RA1 (2163) domain antibody, followed by horseradish peroxidase–linked anti-rabbit IgG secondary antibody and detection with chemiluminescence reagents (Pico, Pierce).
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Results |
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PLC
Expression Increases During Heart Failure in Humans and Two Murine Models of Cardiac HypertrophyAs part of a screen for human signal transduction genes with altered expression during heart failure, myocardial tissue samples from non–heart failure donors and donors with idiopathic dilated cardiomyopathy before and after support with a LV assist device (LVAD) were assessed for levels of expression of PLC
mRNA by quantitative real-time PCR. LVAD support has been reported to result in salutary reverse remodeling of failing hearts.15 PLC mRNA expression was increased 2-fold in failing human hearts relative to nonfailing hearts (Figure 1A). LVAD mediated reverse remodeling trended toward decreased levels of PLC. These data suggest a potential role for PLC in human cardiac pathology.
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To further investigate whether PLC is upregulated during progression to heart failure and to confirm this result in an animal model, we examined the expression level of PLC protein and mRNA in 2 murine models of hypertrophic stress (Figure 1). Chronic isoproterenol treatment is a hypertrophy model that simulates the elevated sympathetic drive that occurs during progression to heart failure. Mice were implanted with miniosmotic pumps releasing isoproterenol (30 mg/kg per day) or saline control for 1 week. The PLC protein expression level increased in response to chronic isoproterenol treatment compared with the saline-treated controls (Figure 1B and 1C). Expression of PLCß1, PLCß3, and PLC1 did not significantly increase (data not shown). We also examined PLC mRNA levels by Northern blotting after 3 weeks of aortic banding, a pressure overload model of hypertrophic stress. PLC mRNA was upregulated in banded mice compared with sham-operated controls (Figure 1D). Thus, under 2 models of hypertrophic stress in mice, PLC mRNA or protein is upregulated, corroborating the data in human heart failure and indicating that mice are a valid model to investigate a potential role of PLC in human disease.
Generation of a PLC Knockout Mouse
To assess the role of PLC in cardiac function and disease, we created mice with genetic deletion of PLC. Two distinct human PLC cDNA sequences have been identified that are transcribed from a single gene, indicating that PLC has 2 splice variants.4,7 This was further confirmed by RT-PCR and Western blotting.16 The mouse PLC gene covers 300 kb and 35 exons located on chromosome 19. The first 6 exons are represented schematically in Figure 2A. We deleted exon 6 and the surrounding DNA, so that both splice variants and the first functional domain (GEF) would be deleted, replacing the original genomic sequence with the LacZ gene, an SV40 polyadenylyl tail sequence, and neo using a standard homologous recombination/replacement strategy (Figure 2B).
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Western blots were performed to confirm deletion of the PLC protein (Figure 2E). Organs including the heart and lung were collected from PLC+/+ and PLC–/– mice. Because of the apparently low abundance of PLC in native tissues, PLC was immunoprecipitated from organ lysates and then visualized by gel electrophoresis and Western blotting. The antibodies used for immunoprecipitation and Western blotting were directed against the C-terminal RA domains of PLC. Thus, possible splice variants that might bypass splicing of the sixth exon would be detected. The wild-type samples had 2 immunoreactive bands with proper molecular weights (254 kDa and 221 kDa) as well as other lower molecular weight bands probably resulting from proteolysis. Both bands (and apparent proteolytic products) disappeared in knockout samples, indicating successful deletion of both splice variants. Other immunoreactive bands appearing in the knockout lanes resulted from the presence of IgG. To determine whether PLC was present in cardiac myocytes, PLC was immunoprecipitated from lysates from wild-type isolated adult ventricular myocytes or whole heart followed by Western blotting of the immunoprecipitates. Both splice variants of PLC were expressed in the purified ventricular myocytes (Figure 2F).
Basic Characterization of PLC–/– Mice
PLC–/– mice were born at the expected Mendelian ratio and were outwardly normal for at least 1 year. Both the male and female were fertile. No significant differences in body weight were observed at 2, 4, or 6 months. PLC–/– female mice gave birth to up to 6 litters without death during pregnancy. Various organs were collected from the PLC–/– and PLC+/+ mice, including brain, liver, heart, lung, stomach, intestines, testis, and kidneys from wild-type and PLC–/– mice for pathological and morphological examination. There were no significant gross differences between the wild-type and PLC–/– animals. Western blots of lysates from individual organs including heart showed no significant alterations of protein level for PLCß1, PLCß3, PLC1, and PLC1 (data not shown). This indicates that there is no compensatory upregulation of these PLC isoforms in the PLC–/– mice, suggesting that functions of these PLC families are not overlapping. In summary, the PLC–/– mice have no obvious developmental defects.
PLC-Null Mice Have No Evidence of Cardiac Hypertrophy but Have Reduced Cardiac Contractile Reserve
Hearts from PLC–/– animals had normal structure and wall thickness (supplemental Figure I, available online at http://circres.ahajournals.org) and heart weight to body weight ratios (Figure 6A). Trichrome staining showed no traces of interstitial tissue fibrosis (supplemental Figure I). Two molecular markers of hypertrophy, atrial natruretic factor and ß-myosin heavy chain, were measured by real-time PCR and were not significantly increased in PLC–/– mice (data not shown).
Transthoracic echocardiography was used to assess basal heart functions in conscious, unanesthetized mice at 2, 4, and 6 months of age. A representative M-mode trace from a 2-month-old animal is shown in Figure 3A. The 2-month PLC–/– mice had a significantly increased systolic LV dimension, decreased fractional shortening, and a slower mean velocity of circumferential fiber shortening compared with the wild-type littermates (Figure 3B and supplemental Table I). There was no significant progression in cardiac dysfunction at 4 or 6 months compared with the 2-month-old mice (Figure 3B and 3C), suggesting the observed defect is not attributable to persistent stress on the heart and may be a result of loss of PLC in the heart itself. Other cardiovascular parameters such as heart rate, diastolic LV dimension (Figure 3C), ejection time through the aortic valve, and diastolic interventricular wall dimension were not significantly different (supplemental Table I).
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To determine whether the decreased contractile parameters were the result of a loss of responsiveness to sympathetic stimulation (sympathetic drive is likely elevated in the conscious mice undergoing echocardiography) or a more general contractility defect, baseline contraction and acute contractile responses to ß-adrenergic stimulation were directly measured in catheterized mice using a Millar pressure transducer. Increasing doses of isoproterenol were injected directly into the jugular vein of anesthetized mice (eliminating sympathetic inputs) and ventricular pressures were measured within 45 seconds of administration. Baseline LV dP/dt, a measure of the force of contraction, was not significantly lower in PLC–/– mice compared with wild type (Figure 4A); however, the increase in LV dP/dt in response to acute doses of isoproterenol was blunted by nearly 50% in the PLC–/– mice. Maximum LV pressure, minimum LV pressure, and heart rates were not significantly different at any dose of isoproterenol, indicating a very specific effect on ßAR-dependent increases in cardiac force generation (supplemental Figure II). There was no significant difference in ßAR densities in heart membranes (Figure 4B) or in isoproterenol-dependent cAMP production in myocytes isolated from PLC–/– or PLC+/+ animals (Figure 4C).
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Isolated Cardiac Myocytes From PLC–/– Animals Have a Decreased ßAR Stimulation of Electrically Evoked SR Ca2+ Release
Because the acute contractile increase in response to infused isoproterenol was decreased in the PLC–/– animals, it suggested the deficit could be attributable to a direct loss of responsiveness of cardiac myocytes to adrenergic stimulation. To address this hypothesis, cardiac myocytes were isolated from 4- to 6-month-old PLC–/– and PLC+/+ mice and tested for Ca2+ responses to ßAR stimulation. Isoproterenol-stimulated increases in Ca2+ transient amplitudes were significantly lower in the PLC-deleted mice (40% decrease) compared with wild-type littermates (Figure 5A and 5B). There was not a significant difference between PLC–/– and PLC+/+ animals in the baseline amplitude of either electrically-evoked Ca2+ transient amplitudes or caffeine (10 mmol/L) releasable SR Ca2+ (Figure 5C and 5D), suggesting the magnitude of basal SR Ca2+ pools are not different between PLC–/– and PLC+/+ animals. These data are consistent with the data in Figure 4A showing that PLC ablation specifically reduced contractile responses to isoproterenol without significantly altering baseline cardiac contractility.
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ßAR stimulation results in enhanced Ca2+ uptake into the SR through phosphorylation of phospholamban and disinhibition of the SR Ca2+ ATPase. ßAR stimulation of SR pool size, as measured by the increase in caffeine mediated Ca2+ release, was unchanged in the PLC–/– animals (Figure 5E). Isoproterenol stimulation of phosphorylation of phospholamban was also unchanged in the PLC–/– animals (Figure 5F). These data indicate that ßAR regulation of SR Ca2+ ATPase activity is intact in the PLC–/– animals.
Overall, these data strongly suggest that the mechanism underlying the impaired contractile response to isoproterenol in vivo in PLC-deleted mice is the direct result of a defect in the capacity of the ßAR system to increase SR Ca2+ release during membrane excitation. This defect appears to be independent from the ability of the ßAR system to produce cAMP and regulate SR Ca2+ pool size. Together, these data suggest a novel PLC-dependent component of ßAR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve.
Loss of PLC Sensitizes Mice to Development of Stress-Induced Pathological Cardiac Hypertrophy
We hypothesized that PLC might be upregulated to increase cardiac reserve and protect against hypertrophic stress. Alternatively, PLC could be upregulated as secondary consequence of development of hypertrophy. If PLC were protective, then deletion of PLC would increase susceptibility to development of hypertrophy. PLC–/– and PLC+/+ mice were treated in the chronic isoproterenol-induced stress hypertrophic model, and left ventricle weight (LVW) to body weight (BW) ratio was measured after 7 days. There was no significant difference in LVW/BW between PLC+/+ and PLC–/– animals treated with vehicle (Figure 6A). PLC+/+ mice treated with isoproterenol had a higher LVW/BW than vehicle-treated control groups, as expected. However, LVW/BW (Figure 6A) and LV size (Figure 6B) were greatly increased in isoproterenol-treated PLC–/– mice compared with isoproterenol-treated PLC+/+ mice, indicating that loss of PLC sensitizes the animals to hypertrophic stress. PLC+/+ mice treated with isoproterenol had increased fibrosis compared with vehicle treated PLC+/+ mice, whereas isoproterenol-treated PLC–/– mice had the greatest degree of fibrotic lesions (Figure 6C). Thus, PLC deletion results in enhanced susceptibility to cardiac hypertrophy and fibrosis in response to chronic stress, suggesting that PLC protects against development of pathologic hypertrophy under cardiac stress.
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Discussion |
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PLC
gene expression is increased in human heart failure patients, suggesting a possible role of PLC in cardiac disease. PLC protein or mRNA were specifically up regulated after chronic isoproterenol treatment or mechanical stress induced by pressure overload, respectively, in mice. Our data strongly suggest that reduced ßAR-dependent cardiac contraction in vivo in PLC–/– mice is a direct consequence of the decreased ability of ßAR stimulation to increase Ca2+ transients in cardiac myocytes. We propose that, because deletion of PLC reduces cardiac reserve and increases susceptibility to hypertrophy, PLC plays a specific role in enhancing ßAR-dependent contractility. Rather than being a consequence of development of hypertrophy, upregulation of PLC may be a physiological attempt to compensate for ßAR downregulation and desensitization that occurs in heart failure. It is possible that this upregulation could be an initial attempt to protect but is ultimately insufficient to rescue progression to heart failure.
The mechanism for ßAR-dependent increases in Ca2+ transient amplitudes and subsequent contraction has been well studied and has been shown to involve PKA-dependent phosphorylation of phospholamban, the ryanodine receptor, and L-type Ca2+ channels.17,18 This is the first demonstration of a role for a specific PLC isoform, PLC, in ßAR-dependent increases in cardiac contractility. A possible pathway for ßAR regulation of PLC through Gs-AC-cAMP-Epac-Rap2b has been proposed based on experiments in transfected HEK 293 cells.10 A similar pathway could exist in cardiomyocytes. Other mediators might be Gß,8 RhoA, or transactivation of a receptor tyrosine kinase.19
The precise mechanism for how PLC might mediate ßAR-stimulated increases in SR Ca2+ release is unclear. Type 2 IP3 receptors are the only IP3 receptors found in ventricular myocytes, and a recent report describing type 2 IP3 receptor knockout mice indicates that these mice have unaltered Ca2+-handling responses to isoproterenol.20 This does not absolutely rule out a role for IP3 generated by PLC as a mediator of ßAR-dependent increases in contractility, but clearly other mechanisms must be considered. PLC activity also produces diacylglycerol, which can activate PKC, and there are reports that PKC can either positively or negatively affect inotropy21,22 and warrants further investigation in this system.
Reduced cardiac function has not been reported in other PLC knockout mice. PLCß1, PLCß3, and PLC1 are all expressed in the heart; however, none of these isozymes compensated for the loss of PLC. Gq overexpression in myocytes promotes hypertrophy,23 and loss of Gq function inhibits pressure overload–induced hypertrophy.24,25 A primary target of Gq in most cells is PLCß.2 Chronically increased Ca2+ also stimulates hypertrophy through a calcineurin/NFAT-dependent mechanism.18 Thus Gq/PLCß-dependent signaling has been implicated in prohypertrophic signaling. Here we demonstrate that PLC inhibits progression to hypertrophy. Thus, these enzymes appear to play unique and apparently opposite roles in cardiac pathology.
Recently, a different mouse model with apparent loss of PLC activity has been reported.26,27 These mice (called PLCX) were created through deletion of a small portion of the catalytic domain, resulting in production of nearly full-length PLC protein that should lack PLC activity. One of the phenotypes of the PLCX mice is cardiac dysfunction resulting from abnormal development of the aortic and pulmonary valves. These mice did not develop hypertrophy but did have considerably larger hearts than wild-type mice because of dilation of the left ventricle. We did not detect a defect in the aortic valve, indicated by the unchanged ejection time through the aortic valve, and the hearts in the PLC–/– animals are not enlarged in the absence of hypertrophic stress. Thus, it appears that we have characterized a clearly different cardiac phenotype than has been reported. The exact reason for this difference is unknown, but is likely to be attributable, in part, to the difference in the approaches used to suppress PLC function.
These studies have uncovered a novel role for PLC in regulating cardiac function through the ßAR receptor. This pathway is clearly important because impairment of the pathway leads to increased susceptibility to cardiac disease in mice and may play a role in human heart failure. Investigation of the specific mechanistic role of PLC signaling in cardiac myocytes will be the subject of future investigation that could lead to novel therapeutic strategies for increasing cardiac function during heart failure.
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Acknowledgments |
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This work was supported by NIH grants GM053536 (to A.V.S.), DK56294 (to G.G.K.), and AR44657 (to R.T.D.); an American Heart Association Scientist Development Grant (to B.C.B); and Oral Cellular and Molecular Biology Training grant T32 DE07202-15 (to E.O.). We thank Kyle Veenema and Tricia Ludovic for technical assistance.
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Footnotes |
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*Both authors contributed equally to this study.
Original received July 25, 2005; revision received October 12, 2005; accepted November 3, 2005.
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