Phospholipase C ε Modulates β-Adrenergic Receptor– Dependent Cardiac Contraction and Inhibits Cardiac Hypertrophy
Phospholipase C (PLC) ε is a recently identified enzyme regulated by a wide range of molecules including Ras family small GTPases, Rho A, Gα12/13, and Gβγ 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 PLCε mRNA is upregulated. Two murine models of cardiac hypertrophy confirmed upregulation of PLCε protein expression or PLCε RNA. To identify a role for PLCε in cardiac function and pathology, a PLCε-deficient mouse strain was created. Echocardiography indicated PLCε−/− mice had decreased cardiac function, and direct measurements of left ventricular contraction demonstrated that PLCε−/− mice had a decreased contractile response to acute isoproterenol administration. Cardiac myocytes isolated from PLCε−/− mice had decreased β-adrenergic receptor (βAR)-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 βAR system to produce cAMP and regulation of sarcoplasmic reticulum Ca2+ pool size. To address the significance of these functional deficits to cardiac pathology, PLCε−/− mice were subjected to a chronic isoproterenol model of hypertrophic stress. PLCε−/− 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 βAR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve and loss of PLCε signaling sensitizes the heart to development of hypertrophy in response to chronic cardiac stress.
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 β, γ, δ, ζ, and ε. 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. PLCβ and -γ isoforms are well-studied enzymes that are regulated by G protein–coupled receptors and receptor tyrosine kinases, respectively.1,2 PLCε 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 Gβγ8 in transfected cells. PLCε 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 PLCε expression is the heart. Roles for specific PLC isoforms in cardiac function have not been well documented. PLC isoforms including PLCβ and PLCγ 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 α adrenergic receptors and angiotensin II receptors in cardiac myocytes, and presumably involve PLCβ isoforms. PLCε 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 β2-adrenoceptor, through Gs, cAMP, Epac, and Rap2B to PLCε and calcium signaling has been proposed based on studies in cultured cells transfected with PLCε.10 It remains to be determined whether the in vivo PLCε regulatory signaling network in the heart intersects with β-adrenergic receptor (βAR) signaling pathways and/or other pathways.
In a screen for signal transduction genes upregulated in human heart failure, we found that PLCε 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 PLCε gene to address the possible role of PLCε in cardiac function and disease. These PLCε-deficient mice showed decreased responsiveness to βAR stimulation and have an increased susceptibility to cardiac hypertrophy under chronic stress. We propose that PLCε is required for maximum efficacy of the myocardial βAR system and that its levels may increase in an adaptive response to βAR downregulation during heart failure.
Materials and Methods
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 PLCε were designed to have a 530-bp product from either splice variant of PLCε 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−ΔΔCt method described by Livak and Schmittgen.11
Generation of the PLCε Knockout Mouse
A purified PCR product from the 5′ end of the GEF domain (amino acids 419 to 752 in the rat PLCε 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 PLCε 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 PLCε+/− 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 PLCε protein was used to confirm loss of PLCε 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.
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 PLCε−/− and PLCε+/+ animals followed at 2, 4, and 6 months of age and the pooled data analyzed for statistical significance.
Cardiac hemodynamic parameters were analyzed by inserting a Millar pressure transducer into the left ventricle of anesthetized PLCε+/+ and PLCε−/− 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.
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.
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 PLCε cDNA as a template. This probe recognizes both splice variants of PLCε.
Isolation of Cardiac Myocytes
Myocytes were isolated from 4- to 6-month-old wild-type or PLCε−/− 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 1×105 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 β-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.
Membranes from freshly isolated hearts were removed from 4-month-old PLCε−/− and PLCε+/+ 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.
For immunoprecipitation detection of PLCε, rabbit, anti-PLCε 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. PLCε was detected with a rabbit anti-PLCε RA1 (2163) domain antibody, followed by horseradish peroxidase–linked anti-rabbit IgG secondary antibody and detection with chemiluminescence reagents (Pico, Pierce).
PLCε Expression Increases During Heart Failure in Humans and Two Murine Models of Cardiac Hypertrophy
As 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 PLCε mRNA by quantitative real-time PCR. LVAD support has been reported to result in salutary reverse remodeling of failing hearts.15 PLCε 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 PLCε. These data suggest a potential role for PLCε in human cardiac pathology.
To further investigate whether PLCε is upregulated during progression to heart failure and to confirm this result in an animal model, we examined the expression level of PLCε 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 PLCε protein expression level increased in response to chronic isoproterenol treatment compared with the saline-treated controls (Figure 1B and 1C). Expression of PLCβ1, PLCβ3, and PLCγ1 did not significantly increase (data not shown). We also examined PLCε mRNA levels by Northern blotting after 3 weeks of aortic banding, a pressure overload model of hypertrophic stress. PLCε mRNA was upregulated in banded mice compared with sham-operated controls (Figure 1D). Thus, under 2 models of hypertrophic stress in mice, PLCε 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 PLCε in human disease.
Generation of a PLCε Knockout Mouse
To assess the role of PLCε in cardiac function and disease, we created mice with genetic deletion of PLCε. Two distinct human PLCε cDNA sequences have been identified that are transcribed from a single gene, indicating that PLCε has 2 splice variants.4,7 This was further confirmed by RT-PCR and Western blotting.16 The mouse PLCε 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).
Western blots were performed to confirm deletion of the PLCε protein (Figure 2E). Organs including the heart and lung were collected from PLCε+/+ and PLCε−/− mice. Because of the apparently low abundance of PLCε in native tissues, PLCε 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 PLCε. 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 PLCε was present in cardiac myocytes, PLCε 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 PLCε were expressed in the purified ventricular myocytes (Figure 2F).
Basic Characterization of PLCε−/− Mice
PLCε−/− 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. PLCε−/− female mice gave birth to up to 6 litters without death during pregnancy. Various organs were collected from the PLCε−/− and PLCε+/+ mice, including brain, liver, heart, lung, stomach, intestines, testis, and kidneys from wild-type and PLCε−/− mice for pathological and morphological examination. There were no significant gross differences between the wild-type and PLCε−/− animals. Western blots of lysates from individual organs including heart showed no significant alterations of protein level for PLCβ1, PLCβ3, PLCγ1, and PLCδ1 (data not shown). This indicates that there is no compensatory upregulation of these PLC isoforms in the PLCε−/− mice, suggesting that functions of these PLC families are not overlapping. In summary, the PLCε−/− mice have no obvious developmental defects.
PLCε-Null Mice Have No Evidence of Cardiac Hypertrophy but Have Reduced Cardiac Contractile Reserve
Hearts from PLCε−/− 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 β-myosin heavy chain, were measured by real-time PCR and were not significantly increased in PLCε−/− 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 PLCε−/− 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 PLCε 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).
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 β-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 PLCε−/− 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 PLCε−/− 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 βAR-dependent increases in cardiac force generation (supplemental Figure II). There was no significant difference in βAR densities in heart membranes (Figure 4B) or in isoproterenol-dependent cAMP production in myocytes isolated from PLCε−/− or PLCε+/+ animals (Figure 4C).
Isolated Cardiac Myocytes From PLCε−/− Animals Have a Decreased βAR Stimulation of Electrically Evoked SR Ca2+ Release
Because the acute contractile increase in response to infused isoproterenol was decreased in the PLCε−/− 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 PLCε−/− and PLCε+/+ mice and tested for Ca2+ responses to βAR stimulation. Isoproterenol-stimulated increases in Ca2+ transient amplitudes were significantly lower in the PLCε-deleted mice (40% decrease) compared with wild-type littermates (Figure 5A and 5B). There was not a significant difference between PLCε−/− and PLCε+/+ 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 PLCε−/− and PLCε+/+ animals. These data are consistent with the data in Figure 4A showing that PLCε ablation specifically reduced contractile responses to isoproterenol without significantly altering baseline cardiac contractility.
βAR stimulation results in enhanced Ca2+ uptake into the SR through phosphorylation of phospholamban and disinhibition of the SR Ca2+ ATPase. βAR stimulation of SR pool size, as measured by the increase in caffeine mediated Ca2+ release, was unchanged in the PLCε−/− animals (Figure 5E). Isoproterenol stimulation of phosphorylation of phospholamban was also unchanged in the PLCε−/− animals (Figure 5F). These data indicate that βAR regulation of SR Ca2+ ATPase activity is intact in the PLCε−/− animals.
Overall, these data strongly suggest that the mechanism underlying the impaired contractile response to isoproterenol in vivo in PLCε-deleted mice is the direct result of a defect in the capacity of the βAR system to increase SR Ca2+ release during membrane excitation. This defect appears to be independent from the ability of the βAR system to produce cAMP and regulate SR Ca2+ pool size. Together, these data suggest a novel PLC-dependent component of βAR signaling in cardiac myocytes responsible for maintenance of maximal contractile reserve.
Loss of PLCε Sensitizes Mice to Development of Stress-Induced Pathological Cardiac Hypertrophy
We hypothesized that PLCε might be upregulated to increase cardiac reserve and protect against hypertrophic stress. Alternatively, PLCε could be upregulated as secondary consequence of development of hypertrophy. If PLCε were protective, then deletion of PLCε would increase susceptibility to development of hypertrophy. PLCε−/− and PLCε+/+ 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 PLCε+/+ and PLCε−/− animals treated with vehicle (Figure 6A). PLCε+/+ 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 PLCε−/− mice compared with isoproterenol-treated PLCε+/+ mice, indicating that loss of PLCε sensitizes the animals to hypertrophic stress. PLCε+/+ mice treated with isoproterenol had increased fibrosis compared with vehicle treated PLCε+/+ mice, whereas isoproterenol-treated PLCε−/− mice had the greatest degree of fibrotic lesions (Figure 6C). Thus, PLCε deletion results in enhanced susceptibility to cardiac hypertrophy and fibrosis in response to chronic stress, suggesting that PLCε protects against development of pathologic hypertrophy under cardiac stress.
PLCε gene expression is increased in human heart failure patients, suggesting a possible role of PLCε in cardiac disease. PLCε 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 βAR-dependent cardiac contraction in vivo in PLCε−/− mice is a direct consequence of the decreased ability of βAR stimulation to increase Ca2+ transients in cardiac myocytes. We propose that, because deletion of PLCε reduces cardiac reserve and increases susceptibility to hypertrophy, PLCε plays a specific role in enhancing βAR-dependent contractility. Rather than being a consequence of development of hypertrophy, upregulation of PLCε may be a physiological attempt to compensate for βAR 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 βAR-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, PLCε, in βAR-dependent increases in cardiac contractility. A possible pathway for βAR regulation of PLCε 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 Gβγ,8 RhoA, or transactivation of a receptor tyrosine kinase.19
The precise mechanism for how PLCε might mediate βAR-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 PLCε as a mediator of βAR-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. PLCβ1, PLCβ3, and PLCγ1 are all expressed in the heart; however, none of these isozymes compensated for the loss of PLCε. Gαq overexpression in myocytes promotes hypertrophy,23 and loss of Gαq function inhibits pressure overload–induced hypertrophy.24,25 A primary target of Gαq in most cells is PLCβ.2 Chronically increased Ca2+ also stimulates hypertrophy through a calcineurin/NFAT-dependent mechanism.18 Thus Gq/PLCβ-dependent signaling has been implicated in prohypertrophic signaling. Here we demonstrate that PLCε 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 PLCε activity has been reported.26,27 These mice (called PLCεΔX) were created through deletion of a small portion of the catalytic domain, resulting in production of nearly full-length PLCε protein that should lack PLCε activity. One of the phenotypes of the PLCεΔX 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 PLCε−/− 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 PLCε function.
These studies have uncovered a novel role for PLCε in regulating cardiac function through the βAR 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 PLCε 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.
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.
↵*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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