INTRODUCTION

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ACTIVATION OF protein kinase C (PKC) by diacylglycerol has been hypothesized to mediate the positive inotropic response of myocardium to the -adrenergic agonists angiotensin II and endothelin (28, 35). Phorbol esters have been useful in implicating PKC in the regulation of cardiac function (28, 35), but a clear picture of the role of PKC has not emerged, in part, because there are conflicting reports in the literature concerning the effects of phorbol esters on ventricular tissues. Positive (24), negative (5, 15, 21, 42), and both positive and negative inotropy (38) have been described, and these differences cannot be attributed to age or species differences. Moreover, a number of investigators have suggested that the negative inotropic effects of phorbol esters are independent of PKC (38, 39). In an attempt to clarify the role of PKC in ventricular muscle, we developed a method for controlled elevation of diacylglycerols within living cells using light activation of caged diacylglycerol (14). We observed for the first time that the widely used short-chain analog dioctanoylglycerol (diC₈) is capable of initiating a strong positive inotropic effect in ventricular myocytes that is dose dependent, stereospecific, and blocked by PKC antagonists (26).

The magnitude of the response to photoreleased diC₈ rivaled the magnitude of the myocyte response to the -agonist isoproterenol (26). The intracellular mechanisms underlying the -adrenergic response have been well characterized and involve stimulation of Ca²⁺ influx via phosphorylation of the L-type Ca²⁺ channel (10, 13), stimulation of the sarcoplasmic reticulum (SR) Ca²⁺ pump via phosphorylation of phospholamban (17, 40), and desensitization of the myofilaments to Ca²⁺ as a result of phosphorylation of troponin I (19). Thus regulation of excitation-contraction coupling, SR Ca²⁺ content, and myofilament properties all contribute to the cardiomyocyte response to -stimulation. These changes can be mimicked by elevation of cAMP and stimulation of intracellular protein kinase A (PKA) (10, 13, 17, 40).

Diacylglycerol acting through PKC has been hypothesized to regulate some of these same processes such as L-type Ca²⁺ channel activity (8, 21), Ca²⁺ pumping (17, 31), and myofilament Ca²⁺ sensitivity (6, 12, 36). In contrast to the consensus that exists for the mechanisms of the positive inotropic and lusitropic action of PKA, regulation of contractile function by PKC is much less clearly defined. In the present study, we took advantage of the robust positive inotropic response produced by photogeneration of diacylglycerol to characterize PKC-dependent mechanisms with good signal-to-noise ratio in living cardiomyocytes. Reported here are results of measurements of intracellular Ca²⁺ and intracellular pH during this large enhancement of contractility. We found that the vast majority of the positive inotropic response to diacylglycerol is attributable to enhancement of systolic Ca²⁺ as a result of stimulation of the process of excitation-contraction coupling. Some of this work was presented in preliminary form to the Biophysical Society (27).

	MATERIALS AND METHODS

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All reagents were purchased from Sigma Chemical (St. Louis, MO) unless otherwise noted. Endothelin-1, ryanodine, 2,3-butanedione monoxime (BDM), and NH₄Cl were prepared fresh in distilled water. Caffeine was prepared fresh in 1 mM Ca²⁺ Ringer buffer (see below). Caged diC₈ was the -carboxyl-2-nitrobenzyl form synthesized and purified as previously described (14). Fura 2, fluo 3, and 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) were obtained in their acetoxymethyl ester (AM) forms from Molecular Probes (Eugene, OR). Ventricular myocytes were enzymatically dissociated from adult male rats as previously described (26). The yield was ~1-3 × 10⁶ cells per heart, and >80% were rod-shaped and Ca²⁺ tolerant. CaCl₂ was added to Ca²⁺-free Ringer to achieve the indicated Ca²⁺ concentration. Ca²⁺-free Ringer buffer had the following composition (in mM): 125 NaCl, 2 NaH₂PO₄, 5 KCl, 1.2 MgSO₄, 25 HEPES, 5 pyruvate, 11 glucose, and 0.001 insulin, and pH was adjusted to 7.4 with NaOH at room temperature.

Fluorescence measurements. Cells were loaded with fluorescent indicators in their AM forms. Fura 2-AM, fluo 3-AM, or BCECF-AM were separately added to a suspension of 2-3 × 10⁴ cells/ml to a final concentration of 2 µM and then incubated for 1 h at room temperature. Myocytes were then pelleted, washed twice with 0.5 mM Ca²⁺ Ringer solution, and resuspended in the same solution at the same cell density. Cells were then loaded with 700 µM caged diC₈ for 30 min at room temperature as previously described (26) and then transferred to a chamber connected to a perfusion system. All measurements were performed with a DeltaScan D-140 Microscopic Photometer system (Photon Technology, Jersey City, NJ). Fura 2 was excited at 340 and 380 nm, and fluorescence emission was monitored at 510 nm. Fluo 3 was excited at 495 nm with emission at 525 nm. BCECF was excited at 499 and 441 nm with emission at 536 nm. The data sampling rate was 1,200 Hz for fura 2 and fluo 3 and 300 Hz for BCECF. Data were collected, saved, and analyzed with a Pentium personal computer using Felix software (Photon Technology).

Calibration of Ca²⁺ indicators in myocytes. Calibration of signals for conversion of fluorescence to free Ca²⁺ was carried out according to the methods described by Borzak et al. (3). Adult myocytes were individually calibrated in situ by sequential exposure to fura 2 or fluo 3 solutions. Fura 2 solutions were as follows: solution I, 3 mM Ca²⁺ Ringer solution; solution II, solution I with 25 µM ionomycin; and solution III, solution II with 10 mM EGTA replacing CaCl₂. The ratio of 340/380 fluorescence (R) detected at 510 nm was converted to free intracellular Ca²⁺ concentration ([Ca²⁺]_i) using the formula [Ca²⁺]_i = K_d(R  R_min)/(R_max  R), where K_d is the dissociation constant (224 nM), R_min is R in absence of Ca²⁺, and R_max is R at saturating Ca²⁺. Fluo 3 solutions were as follows: solution A, 1 mM Ca²⁺ Ringer solution plus 12.5 µM ionomycin; solution B, solution A with 3 mM ZnCl₂ replacing 1 mM CaCl₂; and solution C, solution B with 10 µM digitonin replacing ZnCl₂. The fluorescence (F) was converted to [Ca²⁺]_i using the formula [Ca²⁺]_i = K_d(F  F_min)/(F_max  F), where K_d = 400 nM and F_min and F_max are minimum and maximum fluorescence, respectively.

Calibration of BCECF in myocytes. BCECF calibration was carried out as described previously (3, 4) using the K⁺/H⁺ exchange activator nigericin to equilibrate intracellular and extracellular pH. At the end of each experiment, 1 mM Ca²⁺ Ringer solution was changed to calibration solution and signals were recorded for three pH standards. Calibration buffers contained 4 mM HEPES-KOH, 120 mM KCl, 0.5 mM EGTA, 5 mM pyruvate, 5.6 mM glucose, 10 mM K-ATP, and the ionophores nigericin (20 µM), ionomycin (4 µM), and carbonyl cyanide m-chlorophenylhydrazone (0.2 µM). pH was adjusted at room temperature with KOH or HCl to standard the pH values 7.42, 6.62, and 7.23.

Twitch shortening. Electrical field stimulation was carried out in a custom-designed 200 µl Plexiglas chamber with a glass floor and two platinum electrodes. The standard stimulation protocol was 0.4 Hz, 1 ms duration, and 40 V with the use of a Grass SD9 stimulator (Quincy, MA) at 20-22°C. The chamber was mounted on a Nikon Diaphot inverted microscope. The myocyte image was created with transmitted light filtered to pass red light. A DM-600 dichroic mirror reflected the red light emerging from the output port up onto a Panasonic charge-coupled device video camera. Individual cell length was monitored with a model VED 104 video edge detector and plotted on an X-Y plotter.

Statistics. Data are expressed as means ± SE. Statistical significance was tested with a Student's paired or unpaired t-test, and P <0.05 was taken as significant.

	RESULTS

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Ca²⁺ transients. Figure 1 summarizes the effects of controlled release of diacylglycerol on electrically paced twitch amplitude and the corresponding intracellular Ca²⁺ transients measured with fluo 3. Release of sufficient diC₈ to increase twitch amplitude by fourfold increased the Ca²⁺ transient amplitude by about twofold (Fig. 1A). The increase of systolic Ca²⁺ developed in parallel with the increase in twitch amplitude (Fig. 1B). Fluo 3 was chosen for these studies because it is excited with visible light, so its use eliminates potential cross talk between fluorophore excitation and photolysis of the caged compound. However, fluo 3 is not a ratiometric indicator, so it is susceptible to artifacts that alter the effective dye concentration, such as dye leakage or cell motion. To control for the effects of cell motion, myocytes were incubated with 20 mM BDM after photorelease of diC₈. This treatment greatly reduced the extent of twitch shortening (by inhibiting actomyosin interactions), but the twofold increase in systolic Ca²⁺ was still observed (Fig. 1C).

View larger version (21K): [in this window] [in a new window]   View larger version (14K): [in this window] [in a new window]   View larger version (20K): [in this window] [in a new window]   Fig. 1.   Effects of dioctanoylglycerol (diC8) on intracellular Ca2+ transients. A: representative traces from a single myocyte. Top traces: 3 control twitches are compared with 3 twitches 5 min after a 20-s pulse of diC8 (inset shows time course of a representative twitch before and after diC8 normalized to same amplitude). Bottom traces: corresponding fluo 3 fluorescence measurements in same cell (inset shows normalized Ca2+ transient time courses before and after diC8). [Ca2+]i, intracellular Ca2+ concentration. B: summary of changes in systolic and diastolic [Ca2+]i caused by diC8 pulses in 15 separate myocytes. Systolic Ca2+ increased on average from 300 to 600 nM (P < 0.01), but diastolic Ca2+ was not significantly elevated in this group of cells. Changes in twitch amplitude for same 15 cells are shown for comparison. Cell shortening averaged 6 µm in control and 19 µm in diC8-treated cells, and average resting cell length was 110 µm. C: effects of cell movement on increase in systolic Ca2+ measured with fluo 3. BDM (20 mM) was used to reduce myocyte twitch amplitude by >80% (top traces), but effect of diC8 to increase systolic Ca2+ was unchanged by BDM (bottom traces).

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effect of diC8 on Ca2+ content of sarcoplasmic reticulum (SR). Measurements were performed on myocytes loaded with fura 2 and caged diC8. Top: 2 basal Ca2+ transients were measured, and then electrical stimulation was halted for 20 s. A rapid brief pulse of 20 mM caffeine was applied onto cell surface to induce SR Ca2+ release. Bottom: same protocol was repeated after a 20-s pulse of diC8 from caged diC8. Top inset: superimposed caffeine transients before and after diC8. Bottom inset: summary of SR Ca2+ content before and after diC8 in 12 myocytes. Mean area under caffeine Ca2+-response curve was similarly unchanged by diC8. Data are means ± SE.

View larger version (28K): [in this window] [in a new window]   View larger version (15K): [in this window] [in a new window]   Fig. 3.   Simultaneous measurements of twitch contractions and fluo 3 fluorescence during postrest potentiation. A: original records of twitch contractions (top) and fluo 3 fluorescence (bottom) showing 2 steady-state twitches followed by a 2-min rest period (break), and then stimulation was resumed until steady state was reestablished. Left: control records. Right: records 5 min after a 20-s diC8 pulse. B: time courses of recovery of twitch amplitude (top) or Ca2+ transient amplitude (bottom) during postrest potentiation before (control) and after diC8. Each data point represents mean ± SE for at least 8 cells. Curves show fits to a monoexponential function as previously described (41), which gave apparent rate constants of 2.2 s1 (control twitch amplitude), 1.8 s1 (diC8 twitch amplitude), 1.9 s1 (control Ca2+ amplitude), and 2.0 s1 (diC8 Ca2+ amplitude). Errors in these values are estimated to be ±15%.

Ca²⁺ sensitivity of myofilaments. Another possible contributor to the positive inotropic action of diC₈ is an increase in the Ca²⁺ responsiveness of the myofilaments. The fact that the twitch amplitude increased by three- to fourfold, whereas the Ca²⁺ transient only increased by twofold, opens the possibility that the Ca²⁺ regulatory system is also more sensitive to Ca²⁺ after diC₈. However, it is also possible that changes in twitch shortening and free Ca²⁺ are not strictly proportional. To investigate Ca²⁺ sensitivity in living myocytes, we first examined the relationship between free Ca²⁺ and twitch shortening during the large swings in these two parameters observed during postrest potentiation. A comparison was then made between control and diC₈ treated cells using this relationship. In most myocytes (13 of 22), there was no detectable difference in the ratio of cell shortening to Ca²⁺ transient amplitude before and after diC₈ (Fig. 4A). In ~40% of myocytes (9 of 22), there was a small leftward shift in this relationship after diC₈ treatment (not shown). The direction of this shift was the same as that observed after treatment with endothelin (Fig. 4B). However, unlike the diC₈ response, the endothelin response was observed in all eight cells tested. The magnitude of this effect was also at least twofold greater in response to endothelin than to diC₈.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of diC8 on myofilament responsiveness to Ca2+. A: relationship between cell twitch amplitude and intracellular Ca2+ amplitude in a control cell derived by plotting values obtained during postrest potentiation experiments as in Fig. 3. Twitch and Ca2+ transient amplitudes were normalized to their respective maxima. The cell was then exposed to a 20-s pulse of diC8, and the new relationship between twitch amplitude and Ca2+ transient amplitude was derived in the same manner. B: relationships between twitch amplitudes and intracellular Ca2+ amplitude before (control) and after 10 nM endothelin (ET). Lines in A and B represent linear regressions. C: plane loops for cell length versus free Ca2+ phase in a myocyte before diC8 (control) and at various times after exposure to diC8. Dotted line is shown to help visualize relengthening trajectory.

Intracellular pH. The positive inotropic actions of a number of agonists in the heart appear to be correlated with an intracellular alkalinization (11, 16, 18, 29). To assess the contribution of pH changes to the mechanism of diC₈ action, we measured intracellular pH with the fluorescent indicator BCECF. Intracellular pH was typically ~7.1-7.2 before photorelease of diC₈, and it did not change significantly under conditions in which diC₈ caused a fourfold increase in twitch amplitude (Fig. 5A). The primary mechanism underlying alkalinization is thought to be an increase in activity of a sarcolemmal Na⁺/H⁺ exchanger (11, 16, 18, 24, 29, 32). We also measured this activity directly by determining the rate of recovery of pH from an acid load (24). First, it was confirmed in these experiments that the pH recovery process was virtually completely inhibited by the amiloride analog N-ethylisopropyl amiloride (EIPA) (not shown), consistent with this intracellular pH change being mediated by the Na⁺/H⁺ exchanger. Figure 6A shows that diC₈ did not accelerate the recovery from an acid load, indicating that diC₈ did not stimulate Na⁺/H⁺ exchange activity. The change in Na⁺/H⁺ exchange activity measured in this way averaged 12 ± 13% (n = 6) after diC₈. Further control experiments showed that regulation of the exchanger was normal in these cells because treatment with endothelin-1 resulted in a measurable alkalinization (Fig. 5B) that was blocked by EIPA (Fig. 5C). Endothelin-1 also stimulated exchange activity by 125 ± 42% (n = 4) (Fig. 6B), consistent with previous reports of stimulation of the Na⁺/H⁺ exchanger by agonists (11, 16, 18, 29).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Measurements of intracellular pH (pHi) in electrically paced myocytes. A: a myocyte was stimulated by photorelease of diC8 (arrow) during a continuous recording of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) fluorescence. N-ethylisopropyl amiloride (EIPA; 5 µM) perfusion had no detectable effect on this response. Similar results were obtained in 7 cells. B: a myocyte was perfused with 10 nM ET (horizontal bar) with continuous recording of BCECF fluorescence. Average pH change was 0.21 ± 0.06 (n = 5). C: experiment in B was repeated but with 5 µM EIPA (lower horizontal bar) applied by perfusion. Upper horizontal bar represents perfusion with ET. Similar results were obtained in 5 cells.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Measurements of Na+/H+ antiport activity. pHi was monitored by BCECF fluorescence. Bars indicate times of exposure of myocytes to 10 mM NH4Cl to induce an acid load (downward pH deflection). Initial rate of recovery (solid line) was taken as a measure of Na+/H+ antiport activity. A: Na+/H+ antiport activity before and after diC8 (arrow) treatment. Mean change in initial rate of recovery for 6 cells was a 12 ± 13% decrease. B: Na+/H+ antiport activity before and after 10 nM ET treatment (bar). Mean change in recovery rate for 5 cells was a 125 ± 42% increase.

View larger version (23K): [in this window] [in a new window]   Fig. 7.   Effects of Na+/H+ exchange inhibitor EIPA on positive inotropic responses to diC8 and ET. A: summary of intracellular Ca2+ transients (systolic and diastolic [Ca2+]) measured with fluo 3 and twitch responses (cell shortening) to diC8 showing effects of EIPA. EIPA (5 µM) was applied by perfusion at 5 min (horizontal bar). B: summary of twitch responses to 10 nM ET (lower horizontal bar) showing effects of EIPA. EIPA (5 µM) was applied by perfusion at 5 min (upper horizontal bar). Error bars represent SE for a minimum of 5 separate cells. * Significant difference (P < 0.05).

	DISCUSSION

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Two well-established mechanisms for increasing the contractility of cardiac muscle are an increase in systolic Ca²⁺ and an increase in responsiveness of the myofilaments to Ca²⁺ (9). Under the conditions of our experiments, the positive inotropic response to diacylglycerol can be accounted for by an increase in the magnitude of the systolic Ca²⁺ transient. Diacylglycerol did not consistently alter the myofilament responsiveness to Ca²⁺. The Ca²⁺ transient can be influenced by the degree of Ca²⁺ loading in the SR (2), but we found no evidence that the SR was "superloaded" with Ca²⁺ as a result of diacylglycerol treatment. Another possibility is that diacylglycerol/PKC stimulated L-type Ca²⁺ channel activity. This channel is a major player in cardiac excitation-contraction coupling, and its activity has been shown to be stimulated by agonists that elevate diacylglycerol (8). Moreover, both the - and ₂-subunits of the cardiac L-type channel have been shown to be good substrates for PKC in vitro (30). The effects of PKC phosphorylation on this channel, however, have been difficult to pin down. Most investigators report inhibitory effects of phorbol esters and diacylglycerol analogs on cardiac L-type channel activity, but these may be PKC independent (7, 33). Other investigators have reported stimulatory or biphasic effects of PKC activators on voltage-gated Ca²⁺ channel function in ventricular tissue (8, 21).

Another candidate protein that could be influenced by diC₈ is the SR Ca²⁺ release channel/ryanodine receptor. This channel has been shown to be regulated by phosphorylation (23), but evidence for regulation by PKC phosphorylation is sparse. Other possibilities include cardiac K channels, which have been shown to be regulated by PKC activators under various conditions (1, 25). A likely candidate is the transient outward K channel, whose inhibition by PKC activators prolongs the action potential (1), which in turn increases the duration of transarcolemmal Ca²⁺ influx. This is typically associated with an increase in duration of the twitch and the Ca²⁺ transient, but we did not observe such increases after photorelease of diC₈. At present we cannot distinguish among these three molecular targets (L-type Ca²⁺ channels, ryanodine receptors, or K channels) with any degree of certainty, so we lump them together as changes in excitation-contraction coupling.

Another major control mechanism shown to occur under both physiological and pathological conditions is alteration of myofilament sensitivity to intracellular Ca²⁺ (9, 11, 12, 18, 29). We employed methods developed by Lakatta and co-workers (34) to assess dynamic interactions between intracellular Ca²⁺ and shortening myofilaments, and we found that diC₈ caused no change in this interaction. A similar lack of an effect of diC₈ on myofilament Ca²⁺ sensitivity was reported in a study using force-pCa measurements in skinned myocytes that had been treated with diC₈ before skinning (22). However, using similar approaches, other groups have detected an increase (36) or a decrease (12) in Ca²⁺ sensitivity after treatment with PKC activators. In none of these experiments was the inotropic response to the activator measured in parallel. In skinned cells or reconstituted myofilaments, treatment with exogenous PKC has consistently failed to enhance the myofilament Ca²⁺ sensitivity (37) unless myosin light chain kinase was also present (6). In experiments analogous to ours in intact myocytes, Capogrossi et al. (5) reported a negative inotropic response and either no change or an increase in myofilament Ca²⁺ responsiveness after diC₈. The cause of the variability was not identified. Therefore, although we cannot completely rule out the possibility that activation of PKC under some conditions increases myofilament Ca²⁺ sensitivity, in many cells we observed a very large positive inotropic response without such a change in Ca²⁺ responsiveness.

To further investigate this important issue, we examined the involvement of another possible target of PKC, namely the Na⁺/H⁺ exchanger. PKC regulation of Na⁺/H⁺ exchange is controversial. It has been demonstrated in many cell types that the growth factor-regulated Na⁺/H⁺ exchanger (NHE-1) is activated by growth factors and phorbol esters in a PKC-dependent manner (32). Sequence analysis of the NHE1 gene however, has revealed no phosphorylation sites for PKC, although sites are present for other kinases. This has led to the proposal that PKC activates the exchanger indirectly for example by activating a kinase cascade. In ventricular tissue, several groups have provided evidence of activation of Na⁺/H⁺ antiport by phorbol esters (24, 28). Growth factor-stimulated alkalinization in the heart has also been blocked by PKC inhibitors. However, these findings have not been confirmed in all cases (29). Our results do not resolve this controversy, but they do show that, under conditions in which diacylglycerol produces a dramatic inotropic response, in some ways mimicking agonist responses (26), the Na⁺/H⁺ exchanger is not activated. It is possible that diC₈ is compartmentalized under our experimental conditions so that certain natural PKC targets are not accessible to the signal. We consider this unlikely because the levels of caged compound in the cell are substantial, the levels of diC₈ produced are at the high end of the physiological range (26), and diC₈ is known to readily diffuse in aqueous and membrane environments (14). Our results suggest that diC₈ alone is not sufficient to stimulate the Na⁺/H⁺ exchanger in ventricular tissue.

Cardiac cross-bridge kinetics is another factor that has been reported to be regulated by PKC phosphorylation. These conclusions were derived from measurements of myosin ATPase activity (37) or myocyte shortening velocity (22), two parameters that were not evaluated here. We did not detect a significant change in the time course of the cardiac twitch, which may be sensitive to changes in cross-bridge kinetics, although this latter point is still under debate. It is also not clear how changes in cross-bridge kinetics might contribute to inotropism in cardiac muscle, although it has been suggested that PKC-mediated inhibition of cross-bridge kinetics may underlie negative inotropic effects (37).

In view of the relatively large number of cardiac proteins suggested to be substrates for, or under the control of, PKC (28, 35), our observation that diacylglycerol predominantly influences one measurable parameter in intact myocytes is surprising. Reasons for the unusual selectivity afforded by this experimental approach are currently unknown. The ability to precisely control diacylglycerol concentration may be one factor. This selectivity may also be due to the species of diacylglycerol used, although there is no indication from in vitro studies that diC₈ is selective for one PKC isoform or directs PKC to a particular substrate or sequence motif. The observed selectivity does indicate that certain PKC substrates are preferred over others; in this case, the preferred substrates appear to be proteins involved in excitation-contraction coupling. The selective action of diacylglycerol on myocyte function contrasts with the pleiotropic effects of agonists that activate phospholipases C and/or D (8, 9, 16, 28, 35, 36). This is an indication that diacylglycerol/PKC signaling in cardiac tissue represents only a part of the complex signaling that occurs downstream of receptor occupation by agonists.

In summary, many cellular processes have been hypothesized to be regulated by PKC in cardiac muscle, including Ca²⁺ channels, K channels, the SR Ca²⁺ pump, the Na⁺/H⁺ exchanger, and the myofilament Ca²⁺ regulatory system. Under conditions in which the diacylglycerol analog diC₈ produced a large positive inotropic effect in isolated rat ventricular myocytes, the only significant and consistent change we detected was an increase in systolic Ca²⁺. Because the SR Ca²⁺ load did not change, these observations suggest that cardiac excitation-contraction coupling is a major locus of regulation by PKC. Details of the molecular mechanism such as the isoform of PKC responsible and the target cardiac proteins involved remain to be determined.