Left Ventricular Stretch Stimulates Angiotensin II– Mediated Phosphatidylinositol Hydrolysis and Protein Kinase C ε Isoform Translocation in Adult Guinea Pig Hearts
Abstract Stretch of neonatal cardiomyocytes activates phospholipase C with production of inositol trisphosphate and diacylglycerol in part by formation of angiotensin II (Ang II). However, the response of this pathway to physical stimuli in the adult heart is poorly understood. Thus, in isovolumic perfused guinea pig hearts, we characterized stretch-mediated phosphatidylinositol (PI) hydrolysis and protein kinase C (PKC) isoform translocation using elevated diastolic pressure. Balloon dilatation (minimum diastolic pressure, 25 mm Hg) of the left ventricle (LV) stimulated PI hydrolysis. Pretreatment of stretched hearts with the specific angiotensin (AT1) receptor antagonist losartan abolished stretch-mediated accumulation of inositol phosphates. To examine PKC isoform expression and activation under these conditions, whole-heart extracts were examined by immunoblot analysis. Ang II translocated PKCε to the particulate fraction. 4β-Phorbol 12-myristate 13-acetate but not an inactive congener translocated PKCε to the particulate fraction and produced a decrease in myocardial contractile function. Mechanical stretch also translocated PKCε to the particulate fraction; however, this was attenuated but not abolished by losartan. We conclude that in the adult heart, LV dilatation produced stretch-mediated activation of phospholipase C, which resulted in PI hydrolysis and PKCε activation in part by stimulation of the local renin angiotensin system. In contrast to stretch-mediated inositol phosphate accumulation, PKCε translocation is not prevented by AT1 receptor blockade, indicating that this PKC isoform can be activated in response to mechanical deformation by an Ang II–independent mechanism in the adult myocardium.
Cardiac hypertrophy is a process wherein an increase in mass occurs in response to augmented global or regional chamber work largely as a consequence of an increase in the size of terminally differentiated cardiomyocytes. It has been known for some time that stretch can lead to an increase in protein synthesis in cardiac muscle.1 The mechanism(s) by which this physical stimulus is converted to biochemical events that lead to a change in the cardiac phenotype is poorly understood.2 3
It has been shown that in neonatal cardiac myocytes mechanical deformation can activate a number of intracellular second messengers and in particular the inositol phosphate–PKC (phospholipase C) pathway.4 5 6 Cardiac myocytes and fibroblasts possess G protein–coupled AT1 receptors for Ang II that activate multiple intracellular signaling pathways. After binding of Ang II to its specific receptor, the signal is transduced by a specific heterotrimeric G protein (Gq), which undergoes dissociation to the GTP-bound Gαq subunit. Gαq subsequently activates the effector enzyme phosphoinositide-specific phospholipase Cβ, which is bound to the cytoplasmic face of the plasma membrane and hydrolyzes the plasma membrane lipid, phosphatidyl inositol 4,5-bisphosphate. This process generates two biologically active intracellular messengers, DAG and IP3. DAG activates phospholipid-dependent PKC, a potent mediator of transcriptional regulation and hypertrophy in a variety of cell types, and IP3, which in part maintains cellular Ca2+ homeostasis by releasing Ca2+ from endoplasmic reticulum stores. Although the cardiomyocyte sarcoplasmic reticulum contains IP3 receptors, the role of this polyphosphate in cardiac excitation-contraction coupling is uncertain.
PKC isoenzyme expression has been characterized in cardiomyocytes,7 8 9 vascular smooth muscle,10 11 endothelium,12 and platelets.13 Presently, 11 isoenzymes of PKC have been identified, and their cDNAs have been cloned from different tissues and cell lines. The isoenzymes are classified into three subfamilies: conventional PKCs (α, β1/β2, and γ, Ca2+ dependent), novel PKCs (δ, ε, η, and θ, Ca2+ independent), and atypical PKCs (ζ, λ/ι, and μ, which do not bind or respond to phorbol esters). Recent observations suggest that PKC may participate in neonatal myocyte growth in response to mechanical stretch, which stimulates cells to hypertrophy.4
The tumor-promoting drug PMA can replace DAG as an activator of conventional and novel PKC isozymes and has been used to evaluate the role of PKC isozymes in cell functions.14 Activation of PKC isozymes by PMA or hormones that activate DAG causes translocation of PKC from the cytosol to a particulate cell fraction, where the kinase regulates the activity of a number of proteins by phosphorylation. It has been shown that phosphorylation of substrates by PKC modulates Ca2+ and other ion levels, produces inotropic and chronotropic effects, alters gene expression, and induces secretion of cardiac growth factors and hypertrophy.9
Whether and to what extent mechanical deformation activates the phospholipase C pathway in the adult LV is unknown. We designed the present study to test the hypothesis that pathophysiological levels of mechanical stretch of the adult guinea pig LV can activate this ubiquitous bifurcated cell signaling pathway by an Ang II–dependent mechanism.
Materials and Methods
Myo-[1,2-3H]inositol (45.6 Ci/nmol) was obtained from Dupont/NEN. Ang II, PMA, and PDD were purchased from Sigma Chemical Co. Enalaprilat and losartan were obtained from Merck Pharmaceutical Co.
Polyclonal anti-PKC antibodies, which recognize C-terminal amino acids 313 to 326 of PKCα, 726 to 737 of PKCε, and 577 to 592 of PKCζ, and respective inhibitory peptides were purchased from Life Technologies. Alkaline phosphatase immunoblot developing and chemiluminescence immunoblot developing systems were from Bio-Rad. All other chemicals and reagents were purchased from Fisher Scientific Corp.
Isolated Perfused Heart Preparation
Adult male Hartley strain guinea pigs (Charles River Laboratories, Wilmington, Mass) were anesthetized with intraperitoneal ketamine (54 mg/kg), acepromazine maleate (1.8 mg/kg), and xylazine (10.9 mg/kg) and heparinized by injecting 200 U heparin sodium (1000 U/mL) into the abdominal aorta. Beating hearts were quickly excised, weighed, and then perfused using a modified Langendorff preparation with the ascending aorta terminally cannulated as previously described.15 16 Briefly, hearts were perfused at a constant flow rate of 10 mL/g per minute with oxygenated Krebs-Henseleit buffer containing (mmol/L) NaCl 113.8, KCl 4.7, MgSO4 · 7H2O 1.10, KH2PO4 0.12, NaHCO3 23.6, CaCl2 2.5, mannitol 6.0, and glucose 11.0, pH 7.4 to 7.5 at 37°C. A water-filled latex balloon attached to the end of a 3F Millar high-fidelity micromanometer catheter (Millar Instruments) was inserted into the LV through the mitral valve orifice for pressure measurements and to induce LV stretch. All hearts were paced at a constant heart rate of 250 to 300 bpm. The right ventricle was vented, and the LV balloon was inflated sufficiently to obtain an initial end-diastolic pressure of 3 to 5 mm Hg and was isovolumic during initial perfusion.
LV pressure and heart rate were continuously monitored on a multichannel recorder (MK 200A, Gould) interfaced to an IBM computer. Analog signals were digitized at a sampling frequency of 1000 Hz, and hemodynamic parameters were derived from software developed in our laboratory.15 Fifteen to 20 cardiac cycles were averaged from each condition, and premature contractions were excluded from analysis. LV developed pressure (LV maximum−LV minimum) was measured, and the maximal rate of isovolumic pressure development (maximum dP/dt) was derived and used as an index of LV contractility; minimum dP/dt was chosen as an indicator of the rate of LV isovolumic relaxation.
Phosphatidylinositol Hydrolysis in Response to Ang II and Mechanical Stretch
After acquisition of baseline cardiac mechanics, a fixed volume of recirculated LV perfusate (150 mL) was supplemented with 0.75 μCi/mL [3H]myoinositol for 2 hours to label phospholipids, and the LV was perfused. Lithium chloride (5 mmol/L) was added 10 minutes before the addition of Ang II or LV stretch to inhibit dephosphorylation of inositol phosphates. In studies in which the effects of Ang II on phosphoinositide hydrolysis were assessed, Ang II was added in the indicated concentrations (0.1 to 10 μmol/L) to the perfusate and circulated for 30 minutes. In experiments examining the effect of LV stretch, the LV balloon was inflated to achieve a minimum diastolic pressure of 25 mm Hg for 30 minutes. In some experiments, the AT1 receptor antagonist losartan (1 μmol/L) or the ACE inhibitor enalaprilat (1 μmol/L) was added to the perfusate 10 minutes before mechanical stretch. After completion of the studies, whole hearts were quickly frozen with liquid nitrogen–cooled Wollenberger clamps and stored at −70°C until analysis of inositol phosphates or PKC. For measurement of inositol phosphates, frozen hearts were pulverized and homogenized in 4 mL of 10% trichloroacetic acid using a Tekmar tissue homogenizer. Water-soluble 3H-labeled products were extracted with water-saturated ether (vol/vol) twice, with chloroform (vol/vol) once, and then again with water-saturated ether (vol/vol). The labeled inositol phosphates were resolved by ion-exchange HPLC using a Whatman Partisil SAX column and a linear gradient of 0 to 1 mol/L ammonium formate as previously described.17 Inositol phosphates were identified by elution profiles compared with authentic standards. AMP, ADP, and ATP served as internal markers. Inositol phosphates were quantified by integrating the area under their respective peaks (Fig 1⇓). Since isotopic equilibrium may not have been reached by the 2- to 3-hour perfusion period, the data are corrected for heart weight, and incorporation of myoinositol was assessed by counting radioactivity in the whole-heart homogenates before extraction. The results represent total inositol phosphate accumulation (IP, IP2, and IP3).
Immunoblot Analysis of PKC Isoform Translocation
Membrane and cytosolic fractions of detergent-extracted PKC were prepared according to methods previously described.18 19 Briefly, each whole guinea pig heart was pulverized, and the powdered tissue from each heart was homogenized in 7 mL ice-cold lysis buffer composed of (mmol/L) Tris-HCl 25, EGTA 5, EDTA 2, and NaF 100 (pH 7.4) containing (μmol/L) leupeptin 20, E64 10, pepstatin 120, and phenylmethylsulfonyl fluoride 200, plus 5 mmol/L dithiothreitol. An 800g crude particulate fraction was discarded, and the supernatant was centrifuged at 100 000g for 60 minutes. The pellet constituted the membrane-particulate fraction, and the particulate-free supernatant constituted the cytosolic fraction. Particulate fractions were resuspended in homogenizing buffer containing 0.5% Triton X-100 and centrifuged at 100 000g for 60 minutes, and the resulting detergent-treated supernatant was the membrane fraction. Protein content was estimated by a modification of the method of Lowry (see Kiss et al15 ).
Cytosolic and membrane proteins were analyzed for PKC isoform content by one-dimensional electrophoresis on 10% SDS-polyacrylamide gels by a modification of the method of Laemmli (see Kiss et al15 ). Gels were electrophoretically transferred to Immunlite blotting membranes, blocked with 5% nonfat milk in Tris-buffered saline at room temperature, and incubated overnight with primary antibodies against specific PKC isoforms. Bound antibody was detected by enhanced chemiluminescence. Quantification of PKCα, PKCε, and PKCζ translocation was performed by laser densitometric analysis of autoradiographs.
The amount of loading of tritiated myoinositol in each heart was quantified by determining the total amount of 3H (cpm) and dividing by the heart weight (g) and radioactivity in the whole-heart homogenate. PKC translocation was calculated by comparing the relative densities of PKC isoform immunoblots in particulate and cytosolic fractions in arbitrary units.
All data are presented as mean±SEM. Inositol phosphate accumulation, cardiac mechanics, and PKC translocation were directly compared between control and stretched LV groups by Student’s unpaired t test. One- or two-way ANOVAs were used as appropriate for multiple comparisons, followed by Dunnett’s test for post hoc analyses. Significance was accepted at P<.05.
Morphometric and Cardiac Mechanics
As shown in Table 1⇓, There were no significant differences in LV weight–to–body weight or lung weight–to–body weight ratios among the treatment groups. Likewise, isovolumic LV mechanical parameters at a physiological LV minimum pressure (5 mm Hg) were similar among the groups.
Effects of Ang II on Inositol Phosphate Accumulation in Isolated Perfused Guinea Pig Hearts
In an attempt to determine the effects of angiotensin on the adult guinea pig myocardium, we perfused hearts with 0, 0.1, 1, and 10 μmol/L Ang II. Control hearts showed little accumulation of inositol phosphates over the 30-minute study period. In contrast, Ang II increased inositol phosphate accumulation in a dose-related fashion (Fig 2⇓).
Effects of LV Stretch on Inositol Phosphate Accumulation
Fig 1⇑ demonstrates representative chromatographs from an LV homogenate in the absence of stretch or drug intervention compared with a heart in which the LV was stretched at 25 mm Hg for 30 minutes. Hearts that underwent stretch (Fig 3⇓) manifested a 256% increase in inositol phosphate accumulation relative to nonstretched hearts (3510±1007 versus 8991±2332 cpm/g heart wt for nonstretched versus stretched hearts, respectively; P<.05). Ang II (10 μmol/L) stimulation similarly augmented inositol phosphate accumulation (6176±1068 versus 8991±2332 cpm/g heart wt for nonstretched versus stretched hearts, respectively).
Effects of AT1 Receptor Blockade and ACE Inhibition on Stretch-Mediated Inositol Phosphate Accumulation
Ang II has been implicated as a critical mediator of the stretch-induced hypertrophic responses in neonatal cardiac myocytes.20 In an attempt to elucidate the mechanism of stretch-induced inositol phosphate accumulation, the effects of the specific AT1 Ang II receptor antagonist losartan were explored. Losartan (1 μmol/L) effectively inhibited the stretch-mediated inositol phosphate accumulation (Fig 3⇑). Inositol phosphate accumulation was significantly lower in the losartan plus stretch group than in the stretched group (losartan plus stretch, 703±164 cpm/g heart wt; stretch, 8891±2332 cpm/g heart wt; P<.05). Inositol phosphate accumulation also was lower with AT1 blockade (losartan plus stretch, 703±164; no stretch, 3510±1007 cpm/g heart wt; P<.05) (Fig 3⇑). However, addition of losartan to nonstretched buffer-perfused hearts (n=3) failed to lower inositol phosphate levels (3411±227 cpm/g heart wt) below those of unstretched hearts (3510±1007 cpm/g heart wt, n=7 hearts) in the absence of AT1 blockade. Therefore, it does not appear that Ang II generation occurred in the unstretched heart under these experimental conditions.
We have previously demonstrated ACE-independent generation of Ang II in the primate myocardium.21 In order to determine whether and to what extent ACE-independent generation of Ang II contributes to stretch-mediated phosphatidylinositol hydrolysis, we measured inositol phosphate accumulation in the presence of the ACE inhibitor enalaprilat. Enalaprilat (1 μmol/L) also completely inhibited stretch-mediated inositol phosphate accumulation (Fig 3⇑).
PKC Isoforms in Adult Guinea Pig Heart: Effects of Phorbol Ester and Hormonal Stimulation
Using Western blot analyses, we detected α, ε, and ζ isoforms of PKC in lysates prepared from adult hearts. The bands detected by immunoblotting had molecular masses of ≈80, 96, and 78 kD, corresponding to those of PKC isoforms α, ε, and ζ, respectively. Antibodies directed against the β, δ, and γ isoforms of PKC did not show immunoreactivity (data not shown). In subsequent experiments, we assessed the effects of Ang II and PMA by their ability to increase membrane-associated immunoreactivity for each of these PKC isoforms.4 PMA treatment increased the amount of immunoreactivity in PKCα and PKCε, but not in PKCζ, in membranes of adult hearts.
PMA (100 nmol/L) stimulation depressed isovolumic LV mechanics compared with baseline values (Fig 4⇓). There was a 67% decrease in developed pressure during PMA stimulation (baseline, 118±4 mm Hg; PMA, 39±5 mm Hg; P<.05). Similarly, there were 62% and 63% decreases in the rates of contractility and relaxation (+dP/dt: baseline, 2336±51 mm Hg/s; PMA, 893±51 mm Hg/s; −dP/dt: baseline, −1924±70 mm Hg/s; PMA, −720±50 mm Hg/s; each, P<.05). Stimulation with inactive phorbol ester PDD had no effect on baseline isovolumic LV mechanics.
In order to determine the effects of Ang II stimulation on PKC activation, we measured PKC translocation from the cytosol to the membrane. Adult hearts were perfused with 10 μmol/L Ang II for 30 minutes after equilibration on a modified Langendorff preparation. PKCα and PKCζ showed no translocation to the membrane with Ang II stimulation. In contrast, PKCε translocated with Ang II stimulation (Fig 5⇓).
Effects of Stretch on Protein Kinase Translocation
Additional hearts were studied to examine the effects of LV stretch on PKC translocation. The hearts were excised and placed on a modified Langendorff preparation. The LV was preloaded with 25 mm Hg minimum diastolic pressure after a 30-minute equilibration period. A time course of 0, 2.5, 5, 7.5, 10, and 20 minutes showed maximal translocation at 7.5 minutes, which was still detectable at 20 minutes (Fig 6⇓). Whole hearts that were nonstretched (n=5) and stretched (n=5) for 7.5 minutes at 25 mm Hg were perfused, extracted, and probed with PKCα, PKCε, and PKCζ antibodies. PKCε translocation in the stretched hearts increased 5.5-fold from cytosol to membrane over baseline values (from 0.304±0.123 to 1.681±0.475 arbitrary units, P<.05). Neither PKCζ (from 0.265±0.031 to 0.548±0.213 arbitrary units) nor PKCα underwent significant translocation (Table 2⇓).
Effects of AT1 Receptor Blockade on PKC Translocation
In order to elucidate the potential role of Ang II on stretch-mediated PKC translocation, 1 μmol/L losartan was added to the perfusate, and the LV balloon was inflated to 25 mm Hg minimum diastolic pressure for 7.5 minutes. In contrast to the effect on phosphatidylinositol hydrolyses, the same degree of AT1 receptor blockade with losartan attenuated but did not abolish stretch-induced PKC translocation. There was a 3.5-fold increase in PKCε translocation compared with baseline (1.05±0.201 versus 0.304±0.123 arbitrary units, P<.05) (Fig 7⇓) (Table 2⇑).
The principal result of the present study is that pathophysiological distension of the adult LV activates the phospholipase C signal transduction pathway. The key observations include the following: (1) Ang II, phorbol ester stimulation, and mechanical deformation stimulate phosphatidylinositol hydrolysis and translocate PKCε in LV myocardium. (2) AT1 receptor inhibition (losartan) and ACE inhibition (enalaprilat) completely blocked inositol phosphate hydrolysis but not PKC translocation; to our knowledge, these are the first data in the adult heart that demonstrate ex vivo localized production of Ang II and resultant coupling to downstream signal transduction. (3) PKC activation by phorbol ester stimulation produced a negative inotropic effect in the isovolumically contracting guinea pig LV.
A variety of cell-signaling pathways have been demonstrated to be activated by cardiovascular mechanical deformation.22 These include stretch-activated ion channels, an extracellular matrix protein/integrin–linked pathway, the Na+-H+ antiporter, phospholipases C, D, and A2, protein kinase C, tyrosine kinases, P21ras, mitogen-activated protein kinases, and 90-kD S6 kinase. This variety of signal transduction pathways and the potential cross talk among them has largely been studied in cultured neonatal rat cardiomyocytes and avian myocytes.23 The determinants of mechanotransduction in the adult heart have been more difficult to study because of the inability to passage terminally differentiated myocytes in culture. As a corollary, there are few data regarding the existence and importance of various stretch-activated signal transduction pathways in the adult LV, despite the possibility that these pathways may be importantly affected by developmental regulation and the multicellular environment of the whole organ.
In particular, Sadoshima et al5 have demonstrated the importance of Ang II in the stretch-induced production of the hypertrophy phenotype in the cultured neonatal cardiomyocyte. They demonstrated that the addition of angiotensin under these conditions increased protein synthesis and induced immediate-early genes and growth factors and that AT1, but not AT2, receptor blockade prevented these actions.20 Static stretch of neonatal cardiocytes recapitulated these events, whereas stretch-induced hypertrophy was prevented by AT1 receptor blockade. Immunoelectron microscopy suggested that this was mediated in part by an autocrine action of Ang II.20 The present study extends these observations to the adult LV. We have demonstrated that pathophysiological distension (25 mm Hg) of the isolated isovolumically contracting LV activates phospholipase C–mediated accumulation of inositol phosphates and activation of PKC. AT1 receptor blockade completely inhibited IP3 accumulation but not PKC translocation. In this regard, it is interesting that chelation of intracellular Ca2+, but not downregulation of PKC, suppressed Ang II–mediated activation of mitogen-activated protein kinase and 90-kD S6 kinase.24 These important downstream components of tyrosine kinase signaling pathways may be critically affected by stretch, Ang II–stimulated accumulation of IP3, and the resultant modulation of intramyocyte Ca2+ stores. Taken together, the results of the present study demonstrate autocrine and/or paracrine production of Ang II by the adult heart and the importance of the local renin-angiotensin system in cardiac mechanotransduction.
Komuro et al4 have also used cultured neonatal cardiomyocytes to examine the role of stretch-induced Ang II–mediated activation of the phospholipase C signal transduction pathway. Static stretch of these myocytes to 20% of resting length (corresponding to a 1.73 increase in chamber volume [ie, 1.23]) increased inositol phosphate levels, whereas the accumulation of c-fos was attenuated by PKC inhibition. In the present study, we demonstrate that LV distension in the adult heart to levels seen in pathological states directly induces PKC translocation. The incomplete inhibition of PKC activation by AT1 receptor blockade suggests the presence of an Ang II–independent process in mature myocardium. It is possible that other Gq-coupled receptors, such as the endothelin or the α1-adrenergic receptor, are activated by stretch in the adult heart. Alternatively, stretch may stimulate phospholipase D–mediated hydrolysis of phosphatidylcholine. The resultant formation of phosphatidic acid and its metabolism via a phosphohydrolase to DAG may activate PKC.25 The cellular origin of putative signaling peptides cannot be deduced from the present study. However, the isolated isovolumic heart preparation permits the study of these processes without the confounding effects of neurally modulated or circulating hormonal factors and establishes their presence and importance at the whole-organ level.
We immunologically identified three major isoforms of PKC present in the adult guinea pig whole-heart homogenates. Whole-heart preparations expressed α, ε, and ζ isoforms, with the Ca2+-independent isoform ε being the most immunoreactive. Others have shown that multiple PKC isoforms are expressed in the rat heart in an age-dependent fashion.26 PKCα, PKCδ, PKCε, and PKCζ were detected in whole extracts from neonatal ventricle and cultured neonatal ventricular myocytes. However, only two PKC isoforms (PKCδ and PKCε) were detected in total protein extracts from isolated adult ventricular myocytes.9 We detected small amounts of PKCα and large amounts of PKCζ in total protein extracts from whole adult guinea pig hearts. This finding most likely represents PKCα and PKCζ derived from nonmuscle cells in the heart or from the atria, since whole-heart lysates were used in the present study.
It has recently been demonstrated that AT-1 cells (a transplantable tumor lineage cell line derived from transgenic mouse atrial cardiomyocytes) express PKCα, PKCδ, and PKCε isoforms27 ). Stimulation with endothelin induces selective membrane association of only the Ca2+-independent PKCε; translocation of PKCα was not detected.27 Our findings are consistent with the hypothesis that in cardiac myocytes, receptors coupled to phospholipase C–mediated phosphoinositide hydrolysis selectively induce the translocation of PKCε (but not PKCα) to the particulate fraction, whereas in noncardiac myocytes receptors coupled to phosphatidylinositide hydrolysis increase the membrane association of both PKCα and PKCε isoforms. It should be emphasized that PKCα, PKCε, and PKCζ comigrated at their expected molecular weights and that affinity-purified isoform-specific antibody binding was either blocked or attenuated by inhibitory peptides. We were unable to precisely quantify the reciprocal decrease of PKCε in the soluble fraction and increase in the particulate fraction because of variations in the background of the gels among experiments.
The effect of phorbol esters to translocate PKC from the soluble to the particulate fraction has been used as an indicator of PKC activation.28 Thirty-minute stimulation of the heart with PMA translocated PKCε to the particulate fraction. A number of laboratories have reported that phorbol esters modulate contractile function of neonatal and adult rat cardiomyocytes and perfused hearts.18 29 30 In the present study, we have shown that PMA produces significant negative inotropy in the isolated isovolumic buffer-perfused guinea pig LV. There was a 67% decrease in developed pressure and equivalent decreases in the rates of contraction and relaxation (62% and 63% reduction, respectively). There are several mechanisms by which phorbol ester stimulation of PKC may contribute to myocyte contractile depression. There is some evidence that PMA stimulation causes a decrease in the Ca2+ transient in adult murine cardiomyocytes.30 In embryonic chicken dorsal root ganglion neurons, both a short-acting DAG analogue and a phorbol ester decrease the Ca2+ current.23 Since phorbol esters induce translocation of certain PKC isoforms to the myofilaments,31 some investigators have focused on direct phosphorylation of proteins within the contractile machinery as a mechanism for PKC-dependent modulation of contractile function. Cardiac troponins I and C have been identified as endogenous substrates for phosphorylation by PKC in the heart.32 Phosphorylization of troponin I leads to a decrease in myofilament sensitivity to Ca2+ and to a reduction in myofibrillar actin myosin ATPase activity and could contribute to a negative inotropic response. Troponin T has been shown to be a target for phorbol ester–induced phosphorylation in neonatal, but not adult, ventricular myocytes.33 The functional role of C-protein phosphorylation is not known. Finally, there is recent evidence that PKC phosphorylates myosin light chain 2.32 34 Whether PKC-mediated phosphorylation of myosin light chain 2 alters myofibrillar Ca2+ sensitivity is unclear. It is possible that enhanced constitutive19 and/or stretch-activated phospholipase C hydrolysis may contribute to altered function of the hypertrophied and failing adult heart. Experiments are under way in our laboratories to directly address this issue by cardiac-specific overexpression of Gαq in genetically engineered mice.
In summary, we have demonstrated that mechanical distension of the adult LV resulted in inositol phosphate accumulation and isoform-specific translocation of PKCε from the soluble to the particulate fraction in guinea pig whole-heart lysates. Inositol phosphate accumulation but not translocation of PKC appears to be completely mediated by Ang II–coupled Gαq-linked phospholipase C hydrolysis. Taken together, the results of the present study suggest that stretch-induced activation of the phospholipase C signal transduction pathway during pathological states may play a critical role in modulating growth and contractile function in the intact LV.
Selected Abbreviations and Acronyms
|Ang II||=||angiotensin II|
|IP, IP2, IP3||=||inositol monophosphate, 4,5-bisphosphate, and 1,4,5-trisphosphate|
|LV||=||left ventricle (ventricular)|
|PKC||=||protein kinase C|
|PMA||=||4β-phorbol 12-myristate 13-acetate|
This study was supported in part by National Institutes of Health grants SCOR in Heart Failure (P50 HL-52318, Dr Walsh), HL-33579 (Dr Walsh), and HL-49267 (Dr Dorn) and by the Veterans Administration. Dr Dorn is an Established Investigator of the American Heart Association.
- Received October 25, 1996.
- Accepted July 5, 1997.
- © 1997 American Heart Association, Inc.
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