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(Circulation Research. 1997;81:92-100.)
© 1997 American Heart Association, Inc.

Articles

Positive Inotropy Mediated by Diacylglycerol in Rat Ventricular Myocytes

YeQing Pi, R. Sreekumar, XuPei Huang, , Jeffery W. Walker

From the Department of Physiology, University of Wisconsin, Madison.

Correspondence to Jeffery W. Walker, PhD, Department of Physiology, University of Wisconsin School of Medicine, 1300 University Ave, Madison, WI 53706. E-mail jwwalker{at}facstaff.wisc.edu

	Abstract

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Abstract Many neurohormones stimulate phospholipid hydrolysis and elevate diacylglycerol in the mammalian heart, but the physiological consequences of these intracellular events are unclear. Regulation of myocardial contraction by diacylglycerol was investigated in the present study by releasing the diacylglycerol analogue dioctanoylglycerol (diC₈) within adult rat ventricular myocytes by using a light-sensitive caged compound. This approach permitted us to avoid exposure of myocytes to extracellular diC₈ and yet to control the amount of diC₈ released into the cells. Photorelease of diC₈ produced a slowly developing (half-time, 1.9±0.1 minute; n=26) but robust (406±42%) enhancement of twitch amplitude in electrically paced myocytes (0.5 Hz, 1 mmol/L Ca²⁺ Ringer's solution [pH 7.4], 22°C). This positive inotropic effect was dose dependent, stereospecific for the S-enantiomer of diC₈, synergistically enhanced by arachidonic acid, and blocked by the protein kinase C inhibitor chelerythrine. The data provide evidence that diacylglycerol can induce a strong positive inotropic effect in mammalian ventricular muscle, possibly by activating protein kinase C. By contrast, perfusion of diC₈ extracellularly onto myocytes caused a 42±2% decline in twitch amplitude, in accordance with previous reports. To account for this dependence on how diC₈ is applied, we postulate that diC₈ has distinct physiological actions at intracellular and extracellular sites. The peptide neurohormone endothelin-1, which elevates diacylglycerol in cardiac tissues, produced a positive inotropic effect that was similar to the response to photoreleased diC₈. The diacylglycerol/protein kinase C pathway has now become a good candidate for mediator of at least a component of the positive inotropy associated with agents that stimulate phospholipid turnover in adult mammalian myocardium.

Key Words: positive inotropy • endothelin • myocyte • diacylglycerol • protein kinase C

	Introduction

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Mammalian myocardial tissues express many different cell surface receptors that, when activated, cause PI turnover and elevate DAG (reviewed in Reference 1¹ ). DAG, in turn, activates PKC,² which can phosphorylate a spectrum of cardiac proteins that control myocardial excitability and contraction (reviewed in Reference 3³ ). The precise roles of PI hydrolysis and DAG signaling in cardiac regulation remain open to question, partly because agonists that stimulate PI turnover do not influence contractility in a consistent manner. For instance, -adrenergic agonists^{4 5} and endothelin^{6 7 8 9 10} enhance contractility, whereas opiates¹¹ and platelet-activating factor¹² depress contractility, even though all stimulate PI turnover. Other agonists, including adenosine,^{13 14} acetylcholine (muscarinic),^{13 15} and angiotensin II (in some species)^{16 17} cause PI hydrolysis without obvious changes in contractility, suggesting an uncoupling of DAG formation and contractile regulation in some cases. It is therefore possible that PI hydrolysis and the resulting elevation of DAG are not significant short-term signaling events in cardiac tissue but that they primarily regulate gene expression and growth.^{18 19}

To establish a direct link between intracellular DAG and changes in myocardial contraction, a number of investigators have characterized the physiological effects of the perfusion of DAG analogues and phorbol esters in whole hearts,^{20 21 22 23} papillary muscles,²⁴ and isolated neonatal^{25 26} and adult ventricular myocytes.^{27 28} The general conclusion from these studies is that these agents promote negative inotropy (for a review, see Reference 29²⁹ ), which is paradoxical because many neurohormones associated with DAG signaling initiate large positive inotropic responses (eg, -adrenergic agonists,^{4 5} endothelin,^{6 7 8 9 10} ATP,³⁰ insulin,³¹ and angiotensin II¹⁸ ).

Considerable complexity exists in these signaling pathways, including the likelihood that agonists stimulate a variety of signaling pathways in parallel and mobilize several second messengers.^{18 19} It is therefore necessary to systematically examine cellular responses to each putative messenger, alone and in combination with others, to obtain a complete understanding of agonist actions. In the case of DAG, additional complexity arises because of multiple routes for its production: phospholipase C acting on PI or phospholipase D acting on phosphatidylcholine followed by hydrolysis of phosphatidic acid.³² Each route may produce distinct species of DAG and operate in distinct time domains. Finally, DAGs (either directly or via PKC) may regulate many cardiac proteins, including troponin I,³³ the sarcoplasmic reticulum Ca²⁺ pump,²⁶ the Na⁺-H⁺ exchanger,^{23 28} and the L-type Ca²⁺ channel.^{25 34 35 36 37 38 39 40} Alterations in the function of these proteins have been hypothesized to underlie negative inotropy,^{26 33 39 40} positive inotropy,^{4 10 34} or biphasic inotropic responses.^{36 38} Overall, a detailed understanding of DAG action in myocardium will depend on being able to gain control over the dosage, species, timing, and location of DAG produced within myocardial cells.

The goal of the present study was to evaluate the effects of intracellular DAG on living adult ventricular myocytes containing intact signaling and regulatory pathways. By using intact myocytes, we could assess the integrated regulation of many interdependent cellular processes by monitoring a single physiological output, ie, twitch amplitude. Our approach used a light-sensitive caged DAG compound that permitted intracellular DAG to be systematically varied using near-UV light.⁴¹ We identified conditions under which diC₈ can initiate a substantial positive inotropic response in adult myocytes. The involvement of PKC was tested by determining the stereospecificity of diC₈ action, by examining the effects of other lipid PKC activators that interact with DAG, and by use of the relatively specific PKC antagonist chelerythrine.⁴² A comparison was also made between myocyte responses to diC₈ and to endothelin-1. Some of the present study has been presented in preliminary form to the Biophysical Society.⁴³

	Materials and Methods

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Reagents were purchased from Sigma Chemical Co unless otherwise stated. Chelerythrine chloride and endothelin-1 stock solutions were prepared in distilled water. Arachidonic acid (lot 95H 49501) was stored at -20°C under nitrogen, and stock solutions were prepared fresh in ethanol for each experiment (final ethanol concentration, 0.025%). Caged diC₈ was synthesized and purified as described previously.⁴⁴

Myocyte Isolation
Ventricular myocytes were isolated from adult male Sprague- Dawley rats (200 to 250 g) euthanized with metofane as previously described.⁴¹ Briefly, hearts were quickly excised, cannulated, and subjected to retrograde perfusion on a Langendorff apparatus at 37°C with oxygenated Ringer's buffer of the following composition (mmol/L): NaCl 125, NaH₂PO₄ 2, KCl 5, MgSO₄ 1.2, HEPES 25 (pH 7.4), pyruvate 5, glucose 11, insulin 0.001, and CaCl₂ 1. A brief perfusion with Ca²⁺-free Ringer's buffer was followed by 0.6 mg/mL collagenase and 0.36 mg/mL hyaluronidase in Ca²⁺-free Ringer's buffer. The left ventricle was cut away from the rest of the tissue and further incubated in the enzyme solution. Isolated myocytes were washed, pelleted in a tabletop centrifuge, and resuspended in 0.5 mmol/L Ca²⁺ Ringer's buffer at room temperature at a density of 10⁵ cells/mL. The yield of the cells was 1 to 1.5x10⁶ cells per heart, with a viability of typically 85% rod-shaped cells. Myocytes displayed normal responsiveness to electrical field stimulation for up to 6 to 8 hours after isolation.

Measurement of Twitch Shortening
Electrical field stimulation was carried out in a custom-designed 200-µL Plexiglas chamber with a glass floor and two 10x2-mm platinum electrodes along opposite walls. The stimulation protocol was 0.5 Hz, 1-millisecond duration, and 40 V at 20°C to 22°C; a Grass SD9 stimulator was used. The chamber was mounted on a Nikon Diaphot inverted microscope, and individual cells were monitored with a model VED 104 video-based edge detector (Crescent Electronics) and plotted on an X-Y plotter (Plotomatic).

Caged diC₈ in Myocytes
Myocytes at a density of 3 to 6x10⁴ cells/mL were incubated with 400 to 800 µmol/L -carboxyl caged diC₈⁴¹ dissolved in DMSO (final DMSO concentration, 0.05%) in a siliconized Eppendorf tube for 15 minutes. Myocytes were then allowed to settle onto the glass floor of the stimulation chamber and perfused at 0.5 mL/min with a Labconco multistatic pump for at least 10 minutes to remove DMSO and unincorporated caged diC₈. The beam of a Nikon 75-W xenon arc lamp attached to the epifluorescence arm was passed through a UG11 band-pass filter (300 to 380 nm, Chroma Technologies) and onto the cell via a DM400 dichroic mirror and Nikon 0.55 NA x40 objective. After the myocyte twitch became stable for at least 2 minutes, illumination was initiated, and exposure time was controlled by a Uniblitz electronic shutter in the light path. Perfusion at 0.5 mL/min was continued throughout the experiment. Each myocyte response was carried out on a single cell from a fresh aliquot of cells, and the chamber was thoroughly cleaned between measurements. Myocytes from one heart were used each experimental day, with data collected from three to five myocytes per day. All data sets were obtained with cells isolated from a minimum of three separate hearts.

Effects of UV Light
Myoyctes were exposed for up to 9 minutes to the UV light protocol with no discernible effects on twitch amplitude or duration (eg, see Figs 2A and 3D). Cell damage due to UV light is most pronounced with chronic exposure (hours) to UV-B (280 to 320 nm). In the present study, we used a 300- to 400-nm cutoff filter (UG-11, Chroma Technologies) and glass microscope optics to select for UV-A (also called near-UV, 340 to 400 nm), and we limited exposures to seconds or minutes. UV light intensities were similar to those widely used for fura 2 measurements in myocytes. Even so, caution must be exercised with this experimental approach, since we found that a 5-fold increase in light intensity caused some rundown of twitches.

View larger version (24K): [in this window] [in a new window]   Figure 2. Control experiments illustrating the effects of UV light itself and the caged diC8 compound itself on myocyte twitches. A, A representative original record of a myocyte exposed continuously for 9 minutes to the standard UV light intensity used throughout. B, Original record of a myocyte loaded with 750 µmol/L caged diC8 and then perfused extracellularly with 7.5 µmol/L diC8. No UV light was applied during this experiment. C, Original record of a myocyte loaded with 750 µmol/L caged diC8 and then perfused with 10 nmol/L endothelin. Again, no UV light was applied. Similar results were obtained in five cells for each condition.

View larger version (22K): [in this window] [in a new window]   Figure 3. Stereospecificity of diC8 action in myocytes. Myocytes were loaded with 375 µmol/L S-caged diC8, electrically paced, and illuminated for 10 seconds (arrow in panel A) or 3 minutes (solid bar in panel B) while changes in myocyte cell length were monitored. After the abrupt inhibition of contractility (* in panel B), the light was terminated, and cell twitches resumed (** in panel B). A separate population of myocytes was loaded with 375 µmol/L R-caged diC8 and illuminated for 10 seconds (arrow in panel C) or 3 minutes (solid bar in panel D). Similar results were observed in six cells for each condition.

Statistical Analysis
Data are presented as mean±SEM, with n values representing the number of myocytes in the data set. Statistical significance between two points was determined by a paired Student's t test.

	Results

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DiC₈ pulses were created in cardiac myocytes by controlled photolysis of caged diC₈ incorporated into the cells. Fig 1 shows representative responses in three cells to different diC₈ concentrations produced by systematically varying the illumination time. Exposure to near-UV light (340 to 400 nm) for 5 seconds produced little change in twitch amplitude, indicating the existence of a threshold below which diC₈ was ineffective. Exposure for 10 seconds, however, gave rise to a significant enhancement of contractility (Fig 1B), and exposure for 20 seconds gave a larger response (Fig 1C). Individual cardiac myocytes responded to these brief intracellular diC₈ pulses in a reproducible and characteristic manner for at least 2 hours after caged diC₈ loading, which is consistent with the caged compound being stable to metabolism over this time range.⁴¹ The large positive inotropic effects observed here contrast rather dramatically with the effects of diC₈ applied in the extracellular solution by perfusion, which consistently show a decrease in contractility (Fig 1D). The negative inotropic response to perfusion of 7.5 µmol/L diC₈ was a 42±2% (n=7) reduction in twitch amplitude.

View larger version (33K): [in this window] [in a new window]   Figure 1. Original records showing effects of photoreleasing different amounts of diC8 on myocyte contractility. Traces show time courses of changes in myocyte cell length monitored by a video edge detector. A through C, A population of myocytes was loaded with 750 µmol/L caged diC8 as described in "Materials and Methods," and then single myocytes were illuminated on the microscope stage for the indicated times: 5 seconds (A), 10 seconds (B), and 20 seconds (C). D, Twitch response is shown of a myocyte perfused extracellularly with 7.5 µmol/L diC8 (solid bar) under the same conditions (0.5 Hz, 1 mmol/L Ca2+ Ringer's solution [pH 7.4], 20°C to 22°C).

Control experiments were carried out to establish that exposure to UV light itself was without effect on myocyte twitches (Fig 2A). Other control experiments showed that the presence of caged diC₈ or the loading procedure using DMSO did not alter the myocytes in any way. Myocytes loaded with caged diC₈ gave the expected 41±5% (n=5) reduction in twitch amplitude in response to perfusion of diC₈ (Fig 2B). Furthermore, myocytes loaded with caged diC₈ responded as expected to the test agonists endothelin-1 (Fig 2C) and isoproterenol (not shown).

We next addressed the question of specificity. Since the principal known target of diC₈ is PKC² and since activation of this kinase is stereospecific for S-diglycerides,⁴⁴ we tested whether the positive inotropy observed with caged diC₈ was stereospecific. R- and S-caged diC₈ were prepared as described previously⁴⁴ and then loaded into separate myocyte populations under identical conditions. With use of diC₈, the 10-second pulse protocol gave a result very similar to that described above, a slow-onset 2-fold enhancement of twitch amplitude (Fig 3A), but there was no change when the R-caged diC₈ isomer was used (Fig 3C). Also shown are responses to a 3-minute photolysis protocol in the presence of S and R isomers. This prolonged photolysis of S-caged diC₈ was used to establish the maximum response to diC₈ (Fig 3B) and also shows the presence of an abrupt inhibition of contractility identified previously.⁴¹ The R-caged diC₈ gave no change in myocyte contractility, even for exposure times up to 3 minutes (Fig 3D). The positive inotropic response to photoreleased diC₈ is therefore stereospecific, and release of by-products into the myocytes did not interfere in a detectable way.

Use of caged diC₈ provides a straightforward way to establish a dose-response relationship for diC₈. Uncertainties associated with incorporation efficiency at different diC₈ concentrations are eliminated because caged diC₈ loading is constant, whereas the amount of diC₈ produced is proportional to illumination time.⁴¹ Composite responses derived from averaging traces from at least 10 cells for each exposure time show general characteristics, including amplitudes, onset times, and durations (Fig 4A). The dose-response relationship shown in Fig 4B was obtained by identifying the peak of positive inotropy, which varied somewhat from cell to cell. The relationship reveals a rather steep dependence of the positive inotropic effect on the amount of photoreleased diC₈. The diC₈ levels that gave rise to a minimum versus a maximum enhancement of contractility differed by only a factor of 4 (based on illumination time). Twenty seconds of irradiation was taken as a maximum because longer times did not give larger responses (see Fig 3B), and the 20-second response was similar in magnitude to the response to 5 nmol/L isoproterenol (Fig 5E).

View larger version (28K): [in this window] [in a new window]   Figure 4. Summary of myocyte responses to different doses of diC8. A, Composite responses averaged at 2-minute intervals show mean time courses, amplitudes, and durations for the 5-second exposure (, n=10), 10-second exposure (, n=13), and 20-second exposure (, n=24). B, Relative diC8 dose-response relationship for the positive inotropic effect. Bars show mean±SEM (n) of maximum twitch amplitudes for each exposure time. Data sets are larger in panel B than in panel A because they include cells whose entire time course was not recorded. *P<.001 vs all others.

View larger version (22K): [in this window] [in a new window]   Figure 5. Effects of PKC inhibition on responses to endothelin, diC8, and isoproterenol. The top two traces show myocyte twitch responses to 10 nmol/L endothelin-1 (solid bars) perfused onto the cells in the absence (A) and presence (B) of 4 µmol/L chelerythrine. The middle two traces show myocyte twitch responses to a 10-second pulse of photoreleased diC8 in the absence (C) and presence (D) of 4 µmol/L chelerythrine. The bottom two traces show myocyte responses to 5 nmol/L isoproterenol (solid bars) in the absence (E) and presence (F) of 4 µmol/L chelerythrine. Magnitudes of twitch responses for isoproterenol-treated cells were 526±49% (n=9) increase (no inhibitor) and 510±67% (n=5) increase (with chelerythrine).

An intriguing feature of these positive inotropic responses was their slow onset, displaying half-times of 2 to 3 minutes (Table) and reaching peak amplitudes in 5 to 6 minutes. There was no significant difference in the half-time for a 10-second pulse versus a 20-second pulse of diC₈ (Table). Responses were also sustained for many minutes, as indicated by a noticeable plateau once the response peaked. Responses did eventually fully reverse after a mean duration of 18 minutes for a 10-second diC₈ exposure and 34 minutes for a 20-second exposure (Table). That such brief exposure to diC₈ gave rise to slowly developing and sustained responses suggests that diC₈ was not rapidly washed out or metabolized after photorelease. Since most experiments were carried out with constant perfusion at 0.5 mL/min, the time course and duration of the response are consistent with the effects taking place within the cells rather than at extracellular sites or in the outer leaflet of the plasma membrane, where the diC₈ would be subject to rapid washout.

View this table: [in this window] [in a new window]   Table 1. Summary of Amplitude and Time Courses for Positive Inotropic Effects

One of the goals of these experiments was to determine whether photorelease of diC₈ can mimic myocyte responses to specific agonists. Fig 5A shows the effects on contractility of perfusion of these cells with 10 nmol/L endothelin. Endothelin caused a slowly developing sustained positive inotropy (130±12% increase), which was similar in amplitude to that seen after a 10-second pulse of diC₈ (Fig 5). Evidence for the involvement of PKC in these positive inotropic responses was provided by preincubation of myocytes with the PKC inhibitor chelerythrine chloride. Chelerythrine was chosen because it displays a good selectivity for inhibition of PKC versus other protein kinases⁴² and because it is insensitive to near-UV light. In control experiments, we found that 4 µmol/L chelerythrine had no effect on basal twitch amplitude or on the large positive inotropic response of myocytes to 5 nmol/L isoproterenol (Fig 5E and 5F). In contrast, chelerythrine pretreatment largely eliminated the response to perfusion of 10 nmol/L endothelin (Fig 5B) and to photoreleased diC₈ (Fig 5D). A quantitative comparison between myocyte responses to photoreleased diC₈ and to 10 nmol/L endothelin is summarized in Fig 6 and the Table. Although amplitudes of positive inotropic responses were similar for these two treatments (140% versus 130% increase), the endothelin response differed in two important ways. First, the time course of onset was slower by 2- to 3-fold (half-time, 2.1 versus 5.7 minutes). Second, the inotropic response was more sustained for endothelin (duration, >35 versus 18 minutes) (Fig 5 and the Table). It is apparent that endothelin does more that generate a short burst of intracellular DAG.

View larger version (27K): [in this window] [in a new window]   Figure 6. Summary of myocyte responses to endothelin and diC8. A, Composite traces taken at 2-minute intervals from cells exposed to 10 nmol/L endothelin (, n=11) or a 10-second diC8 pulse (, n=13 unless otherwise indicated). B, Magnitude of positive inotropic effects of endothelin±4 µmol/L chelerythrine measured at its peak (11 to 15 minutes) and photoreleased diC8±4 µmol/L chelerythrine measured at its peak (5 to 9 minutes). Bars indicate mean±SEM (n) maximal increases in twitch amplitude over basal twitch amplitude. Basal twitches measured before drug application or illumination were 5% to 10% of resting cell length. *P<.01; **P<.001.

Long-chain unsaturated fatty acids have been shown to be good activators of PKC, especially in combination with DAG.⁴⁵ Therefore, we tested whether DAG and cis-unsaturated fatty acids would enhance myocyte contractility in a synergistic manner. First, we established conditions whereby perfusion with the cis-unsaturated fatty acid arachidonic acid by itself gave no inotropic response (Fig 7A). We then confirmed that a 5-second pulse of diC₈ gave no response on its own (Fig 7B). In combination, arachidonic acid and diC₈ gave a strong positive inotropic response that was similar to the response to a higher concentration of photoreleased diC₈ (Fig 7C). The response was slow in onset (half-time, 2.9±0.4 minutes) and sustained (duration, >15 minutes) (Table). In addition, this synergistic response to diC₈ and arachidonic acid was blocked in the presence of the PKC inhibitor chelerythrine chloride (Fig 7D). These data are summarized in Fig 8 and the Table.

View larger version (44K): [in this window] [in a new window]   Figure 7. Effects of arachidonic acid and diC8 on myocyte contractility when applied separately or in combination. A, A typical myocyte response to 25 µmol/L arachidonic acid applied by extracellular perfusion (solid bar). B, A typical response (in a separate cell) to a 5-second pulse of photoreleased diC8 (arrow). C, Effects of the combination of 25 µmol/L arachidonic acid (solid bar) and a 5-second pulse of diC8 (arrow). D, The response to arachidonic acid and photoreleased diC8 in the presence of 4 µmol/L chelerythrine. Similar results were obtained in a minimum of six cells.

View larger version (27K): [in this window] [in a new window]   Figure 8. Summary of synergistic enhancement of myocyte contractility by arachidonic acid and photoreleased diC8. Bars represent mean±SEM (n). *P<.001 vs all others.

The effects of arachidonic acid on myocyte contractility were not dependent on its metabolism to oxygenated species, because preincubation with indomethacin did not abolish the observed effects (not shown). Moreover, the synergistic action of arachidonic acid was reproduced by other cis-unsaturated fatty acids, including oleic acid, linoleic acid, and linolenic acid (not shown). Stearic acid (a saturated fatty acid), at concentrations up to 200 µmol/L, was without effect on myocyte contractility either alone or in combination with diC₈ (not shown).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have used a caged DAG compound to release the DAG analogue diC₈ within cardiac myocytes in a controlled way using near-UV light.⁴¹ Photogenerated diC₈ produced a positive inotropic effect, which was stereospecific and dose dependent, with an 4-fold enhancement of contractility under optimal conditions. This contractile response was modulated by the presence of cis-unsaturated fatty acids and blocked by the PKC inhibitor chelerythrine. This is the first direct demonstration, to our knowledge, of positive inotropy being initiated by a DAG analogue in mammalian adult ventricular muscle.

A few investigators have resolved smaller increases (12%²² and 116%²⁸ ) in response to phorbol esters, but most investigators report dose-dependent negative ino-tropy after phorbol ester or DAG treatment.^{20 21 22 23 39 40} A strong case has been made that DAG mediates a physiologically important negative inotropy in ventricular tissues under some conditions.^{11 18 29} Our method of releasing diC₈ from a caged compound appears to favor mechanisms that lead to positive rather than negative inotropy. We tested the possibility that this difference was simply a reflection of different effective doses of DAG by photoreleasing higher concentrations of diC₈. Interestingly, high concentrations of photoreleased diC₈ produced another effect on myocyte twitches (see Fig 3B), which we refer to as a loss of excitability. In our original report,⁴¹ we called this sudden inhibitory effect a negative inotropic response, but given the abrupt onset of inhibition and the fact that higher stimulation intensity often overcomes this inhibition, it is likely that this effect is more related to a sudden loss of excitability than to a true negative inotropy. The underlying mechanism of this loss of myocyte excitability at high intracellular diC₈ levels remains to be elucidated.

Why does the photorelease protocol we have used reveal a positive inotropic effect, when perfusion of DAG analogues, including diC₈, consistently produces negative inotropic effects in the same cells?^{27 39 40} We confirmed that perfusion of diC₈ produces negative inotropy under our experimental conditions, ruling out the possibility that our myocyte preparation or buffer conditions are fundamentally different from those used by other investigators. One unique aspect of the photorelease approach is that diC₈ is not produced in the solution surrounding the cells because myocytes are extensively washed before and during illumination. Therefore, although we have no independent proof that the diC₈ effects that we observed are intracellular, we infer an intracellular site of action on the basis of the loading and washing protocol used. We propose that extracellular or surface membrane actions of diC₈ are responsible for the negative inotropic effects observed during diC₈ perfusion, whereas controlled release of diC₈ from a caged compound reveals a distinct intracellular effect of diC₈. Interestingly, two recent reports reveal inhibitory effects of diC₈ on L-type Ca²⁺ channel function that appear to be extracellular and PKC independent.^{39 40} It should be noted that no such extracellular inhibitory effects have been reported previously or observed in the present study for arachidonic acid, even though it was also applied by extracellular perfusion. Arachidonic acid and diC₈ appear to be different in this respect. Further experiments are required to determine precisely where DAGs and cis-unsaturated fatty acids exert their inotropic effects in cardiac tissues.

It is a concern when applying amphipathic lipids to cells that nonspecific detergent-like effects on cell membranes are responsible for observed changes in cell physiology. The positive inotropic effects reported in the present study were observed only with the S-enantiomer of diC₈; the R-enantiomer was inactive, supporting the idea that diC₈ functions by binding to a target protein rather than by altering the physical state of myocyte membranes. It is also important to consider how much diC₈ is required within the myocytes to effect this physiological response. Quantification of caged diC₈ content of myocytes and the extent of uncaging by photolysis indicates that a 10-second light pulse produces 0.5 fmol of intracellular diC₈ (based on 50% incorporation of 400 µmol/L caged diC₈ in 10⁵ cells/mL and a measured value of 0.2% photolysis of the compound per minute). This 0.5 fmol compares favorably with 0.2 fmol of DAG per cell measured in a population of neonatal cardiac myoyctes after angiotensin II stimulation (60 pmol/3x10⁵ cells).¹⁹ For reference, if 0.5 fmol was distributed uniformly within a cylindrical myocyte with a length of 100 µm and radius of 10 µm, then the average intracellular concentration would be reasonable at 15 µmol/L: 5x10^-13 mmol/(10^-3 cm)²(10^-2 cm). If, on the other hand, diC₈ was completely dissolved in phospholipid bilayers, then the ratio of diC₈ molecules to phospholipids would be a modest 1 in 2000 (assuming a value of 1 pmol of phospholipids per myocyte⁴¹ ). The amount of diC₈ that accumulates in myocyte membranes during perfusion is unknown. There is also no information concerning the DAG levels produced after endothelin stimulation, but it is likely that this value is similar to the 0.2 fmol per cell mobilized by angiotensin II.¹⁹

Comparison of contractile responses to photoreleased diC₈ and to an optimal dose of endothelin revealed some interesting differences. Although both gave positive inotropic effects, diC₈ produced a larger response than did the neurohormone. This observation implies that endothelin does not fully activate the DAG/PKC pathway or that inhibitory (eg, G_i-mediated) influences are also switched on by this agonist. Interestingly, the maximum enhancement of contractility observed with photoreleased diC₈ was 4-fold, or nearly 80%, of that seen with an optimal dose of isoproterenol in these cells (Fig 5E), indicating that the DAG/PKC pathway could potentially be as important a modulator of contractility as the classic cAMP-dependent protein kinase (protein kinase A) pathway.

The half-time for development of positive inotropy following the photorelease of diC₈ was slow and not dependent on the dose of diC₈, suggesting that the onset rate is limited by intrinsically slow processes downstream from diC₈ elevation. The time course of the inotropic effect of endothelin was even slower, which can be explained by the additional slow steps associated with the formation of DAG by phospholipase C (and possibly phospholipase D). The positive inotropic response to photoreleased diC₈ appeared to be fully reversible, regardless of the amount of diC₈ produced. The somewhat sustained nature of the response to photoreleased diC₈ is consistent with the rate of metabolism of diC₈ measured to be in the time domain of tens of minutes (half-time, 10 minutes at 37°C).⁴⁶ By contrast, the response to endothelin was maintained over the 35-minute observation period and was even sustained for at least 5 minutes after washout, with no hint of recovery. Elucidating the cellular mechanisms underlying this prolonged response to endothelin may be a key to understanding its long-term effects on myocardium.

Our observation that DAG and arachidonic acid synergistically enhanced myocyte twitch amplitude is consistent with the response being mediated by PKC, because PKC is synergistically activated by these lipid messengers in vitro.^{2 45 47} DAG and arachidonic acid have been shown to be elevated concomitantly as a result of agonist stimulation in cardiac myocytes.¹⁸ Their synergistic effects on contractility mean that when both DAG and cis-unsaturated fatty acids are elevated, the likelihood of switching on intracellular PKC will be increased; thus, PKC will appear to be more tightly coupled to PI turnover under these conditions. Therefore, cis-unsaturated fatty acids may play an important role as a modulator of the responsiveness of cardiac tissues to intracellular DAG. It should be emphasized, however, that it is currently unknown whether cis-unsaturated fatty acids contribute to the inotropic response to endothelin. It is also important to qualify our observations with regard to the role of PKC in myocyte responses to diC₈. DiC₈ may exert its action through its known ability to activate PKC, but we cannot rule out the possibility that diC₈ acts stereospecifically and synergistically with arachidonic acid to activate other regulatory proteins in myocytes and that the effects of the PKC inhibitor chelerythrine are somewhat nonspecific.

Regardless of its precise cellular and molecular mechanisms, it is clear that the effects of DAG on cardiac excitability and contractility are complex. Three distinct myocyte responses to diC₈ have now been identified: a negative inotropy when perfused extracellularly, a positive inotropy when released intracellularly, and a loss of excitability at high doses. Caged DAG provides one way to control the dose, timing, and location of DAG within living cells, in order to begin to identify its many mechanisms of action.

In summary, we have tested the hypothesis that the DAG/PKC signaling pathway in cardiac myocytes mediates responses to agonists that stimulate phospholipid hydrolysis. Many of these agonists, such as endothelin, angiotensin II, phenylephrine, and insulin, lead to slowly developing sustained positive inotropic responses, but attempts to mimic such responses by applying DAG analogues or phorbol esters has led to the rather puzzling paradox that these compounds consistently promote negative rather than positive inotropic responses in various cardiac preparations. Using a caged DAG compound to control intracellular DAG levels, we now show that DAG signaling can lead to a strong positive inotropic response in isolated adult rat ventricular myocytes. The results provide evidence to directly link phospholipid turnover, DAG formation, and an increase in contractility in ventricular myocytes. It is likely that DAG and its known target protein PKC play a major role in short-term regulation of contractility and may, at least in part, mediate the response to many positive inotropic agents that stimulate phospholipid turnover in the mammalian heart.

	Selected Abbreviations and Acronyms

 

DAG = diacylglycerol diC8 = dioctanoylglycerol DMSO = dimethyl sulfoxide PI = phosphoinositide PKC = protein kinase C

	Acknowledgments

 
This study was funded by National Institutes of Health grants P01 HL-47053 (Dr Walker) and NSF MCB-922082 (Dr Walker). Dr Huang was supported by a postdoctoral fellowship from the American Heart Association, Wisconsin Affiliate, Inc. Dr Walker was supported by a National Institutes of Health Research Career Development Award (K04 HL-03119). Dr Pi is a Visiting Scholar from Hunan Medical University, PRC. We thank Dr J.R. Patel for helpful discussions and assistance with instrumentation.

	Footnotes

 
Previously presented as preliminary results in abstract form (Biophys J. 1997;72:A298).

Received November 20, 1996; accepted April 18, 1997.

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