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(Circulation Research. 1995;76:654-663.)
© 1995 American Heart Association, Inc.

Articles

Inhibition of the Spontaneous Rate of Contraction of Neonatal Cardiac Myocytes by Protein Kinase C Isozymes

A Putative Role for the Isozyme

John A. Johnson, Daria Mochly-Rosen

From the Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, Calif.

Correspondence to Daria Mochly-Rosen, Department of Molecular Pharmacology, Stanford University, School of Medicine, Stanford, CA 94305-5332.

	Abstract

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Abstract Protein kinase C (PKC) enzymes regulate numerous cardiac functions. In the present study, we determined the effects of the PKC-activating drug 4-ß phorbol 12-myristate 13-acetate (4-ß PMA) on the rate of contraction and correlated these changes with the distribution and levels of -, ß-, -, -, and -PKC isozymes by using neonatal rat cardiac myocytes in culture. Treatment with 0.3 to 100 nmol/L 4-ß PMA caused negative chronotropic effects on contraction. This effect was maximal at a concentration of 3 nmol/L 4-ß PMA and correlated with redistribution of the - and -PKC isozymes from the cytosolic to the particulate cell fraction. After a 1-hour treatment with 100 nmol/L PMA, the - and ß-PKC isozymes and an 80-kD -like PKC isozyme were greatly diminished (downregulated), yet the negative chronotropic effect was sustained. Therefore, our results are most consistent with a role for the -PKC isozyme in suppressing the contraction rate of neonatal rat cardiac myocytes. Understanding the role(s) of individual PKC isozymes in the modulation of cardiac functions may ultimately yield more selective targets for therapies of cardiac disorders.

Key Words: phorbol ester • protein kinase C isozymes • cardiac myocytes • contraction • heart

	Introduction

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Protein kinase C (PKC) consists of a family of at least eleven closely related isozymes^{1 2} that regulate biochemical processes by phosphorylation of cellular proteins on serine or threonine residues. PKC isozymes require phospholipids such as phosphatidylserine³ and vary in their dependence on calcium for their activity.^{4 5 6 7 8 9} Most PKC isozymes are activated by sn-1,2-diacylglycerols (DGs),^{1 10 11} with the exception of -PKC, which may depend on a novel second-messenger, phosphatidylinositol 3,4,5-trisphosphate.¹² DGs are generated from precursor lipids, such as phosphatidylinositol 4,5-bisphosphate and phosphatidylcholine, via the action of hormonally regulated phopholipases.¹³

The tumor-promoting drug 4-ß phorbol 12-myristate-13-acetate (4-ß PMA) replaces DG as an activator of most PKC isozymes^{1 14} and hence has been extensively used as a tool to evaluate the role of PKC isozymes in cell functions. Activation of PKC isozymes by 4-ß PMA in cells triggers a redistribution or translocation of PKC isozymes from the cytosol to the particulate cell fraction,^{15 16 17 18 19 20 21} where they are thought to regulate the activity of various proteins by phosphorylation. Translocation of PKC isozymes also occurs after treatment of cells with hormones or agonists that stimulate the accumulation of DGs.^{22 23} Immunofluorescence studies demonstrate that 4-ß PMA or hormone treatments cause the translocation of PKC isozymes to distinct cellular loci such as nuclei, cytoskeletal elements, and others.^{24 25 26}

PKC has been implicated in the regulation of cardiac muscle functions by numerous studies. Translocation of PKC from the cytosol to distinct cell loci^{24 25} and the cell particulate fraction^{27 28 29} occurs in cardiac cells after exposure to 4-ß PMA or hormones that activate DG accumulation.^{25 29 30} Furthermore, phosphorylation of substrates,^{31 32 33} modulation of calcium^{31 33 34 35 36} and other ion levels,^{37 38} inotropic and chronotropic effects,^{27 33 34 36 39 40} gene expression,^{41 42 43} secretion of cardiac factors,^{44 45 46} and hypertrophy^{29 47 48} have all been demonstrated after exposure of cardiac muscle cells to PKC-activating stimuli.

Cardiac myocytes mimic many of the normal and pathological aspects of the intact heart⁴⁷ and at the cellular level are more amenable to experimental manipulations than can be achieved when studying the intact heart. Cardiac myocytes contract spontaneously in culture, which allows monitoring of their contractile rate. Thus, physiological as well as biochemical studies can be conducted on these cells in the absence of the neuronal and endocrine inputs found in vivo. In the present study, we treated neonatal rat cardiac myocytes in culture with multiple concentrations of 4-ß PMA to determine if the regulation of the contractile rate in neonatal cardiac myocytes involved distinct PKC isozymes. Short-term activation of PKC isozymes by 4-ß PMA resulted in a reduction in the rate of cardiac myocyte contraction, which correlated with the translocation and lack of downregulation of -PKC. Our studies suggest a role for this isozyme in phorbol ester–induced negative chronotropic effects in neonatal cardiac myocytes.

	Materials and Methods

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Materials
Most chemicals and biochemicals were purchased from Sigma Chemical Co. Phorbol esters were from LC Services; acrylamide, from Fisher Scientific; and trypsin, from Difco. Antisera for Western blots were from GIBCO Life Technologies Inc (anti–-PKC, –-PKC, and –-PKC), Seikagaku America (anti–-PKC and –ß-PKC), and Zymed Laboratories (rabbit anti-mouse IgG, IgA, and IgM).

Primary Cardiac Myocytes
Primary cell cultures were prepared as previously described.⁴⁷ Briefly, cells were obtained from the hearts of 1-day-old Sprague-Dawley rats (Simonsen, Calif) by gentle trypsinization at room temperature. To reduce the number of nonmyocyte cells, dissociated cells were preplated for 30 minutes onto 100-mm dishes in medium 199 (M-199) with Hanks' salts solution (GIBCO) containing 5% fetal bovine serum (Hyclone Laboratories). Most of the cardiac myocytes do not attach under these plating conditions, whereas the nonmyocyte cells do. The nonattached cells were plated on 35-mm (contractility measurements) or 100-mm (Western blots) Corning Petri dishes at a density of 800 cells per square millimeter and incubated at 37°C in humidified air with 1% CO₂. Myocytes were cultured in M-199 supplemented with vitamin B₁₂ (1.5 mmol/L), penicillin G (50 U/mL), and 5% fetal bovine serum through day 4. To inhibit the proliferation of any nonmyocyte cells present, 0.1 mmol/L bromodeoxyuridine (BrDu) was added to the serum-containing medium for the first 4 days of culture. BrDu does not affect the viability of the myocytes.⁴⁷ Cells were then placed in defined medium (M-199 containing 50 U/mL penicillin G, 1.5 mmol/L vitamin B₁₂, and 10 µg/mL each of transferrin and insulin). The resulting cell preparation contained 95% cardiac myocytes.⁴⁷ Cells were treated with and without 4-ß PMA on day 6 of culture, as indicated.

Measurement of Spontaneous Rate of Cardiac Myocyte Contraction
Measurement of the spontaneous contraction rate was carried out on cells grown at a density of 800 cells per square millimeter in 35-mm Corning Petri dishes treated as described in the figure legends. The culture dishes were placed in a temperature-regulation apparatus (Medical Systems Corp) positioned on the stage of an inverted microscope (Carl Zeiss Inc). Cells were brought to 37°C in the apparatus and then equilibrated for 10 to 20 minutes before monitoring the contractile rate. Contractile rates of four cells in one microscopic field were determined every 2 minutes for 15 seconds each. For studies of the effects of PMA on the rate of contraction (Figs 1 and 2), individual cells were monitored before and after the addition of PMA.

View larger version (20K): [in this window] [in a new window]   Figure 1. Graph showing that a short-term exposure to phorbol 12-myristate 13-acetate (PMA) suppresses the rate of spontaneous contraction in neonatal cardiac myocytes. Cells were equilibrated at 37°C as described in "Materials and Methods." Basal rates of contraction were monitored for 20 minutes, and then contraction rates were monitored after exposure to 100 nmol/L concentrations of 4-- or 4-ß PMA. The time of PMA additions is indicated by an arrow. The data shown for each treatment represent the contraction rate of one cell from a single experiment, which is typical of a total of 15 to 20 cells taken from four independent experiments with different myocyte preparations.

View larger version (21K): [in this window] [in a new window]   Figure 2. Graphs showing that 4-ß phorbol 12-myristate 13-acetate (4-ß PMA)–induced reduction in contraction rate is dose dependent. Experimental conditions were the same as described in Fig 1, except 4-ß PMA concentrations of 0.1 to 100 nmol/L were used. The basal (spontaneous) contraction rates were monitored for 10 minutes before PMA treatments, and the average basal contraction rates were calculated and used as 100% of basal contraction rates. Subsequent measurements of the contraction rates of cardiac myocytes treated with either 100 nmol/L 4- PMA (A) or 0.1 (B), 0.3 (C), 1 (D), 3 (E), 10 (F), or 100 (G) nmol/L 4-ß PMA for the times indicated in the figure were then made. Data are presented as the percentage of the average basal rates (before PMA addition). The results shown are the cumulative mean±SEM percentage of basal contraction rates for each time point from multiple experiments. The number of experiments (each from a separate myocyte preparation) used for 4- PMA (100 nmol/L) and 4-ß PMA at concentrations of 0.1, 0.3, 1, 3, 10, and 100 nmol/L were 5, 4, 4, 4, 8, 4, and 6, respectively.

Cell Washes to Reverse PMA-Induced Inhibition of Contraction Rate
Cardiac myocytes often stop contracting if washout experiments are attempted with fresh M-199 alone. There appear to be factors in the medium in which the myocytes have been cultured (conditioned medium) that maintain spontaneous contraction. For this reason, all washouts were conducted with conditioned medium. In addition, it was not possible to completely remove all medium from the myocytes at once without altering their contractile properties. Therefore, we lowered the 4-ß PMA concentration by a multistage dilution protocol. In this protocol, we removed one half of the 4-ß PMA–containing medium and then added back an equivalent amount of conditioned medium. We repeated these dilution steps until the concentration of 4-ß PMA was 0.1 nmol/L.

Cell Harvest and Western Blot Analysis
The media from three 100-mm dishes of cardiac myocytes were aspirated and discarded, and 1.5 mL of chilled homogenization buffer (10 mmol/L Tris-HCl [pH 7.4], 1 mmol/L EDTA, 1 mmol/L EGTA, and 20 µg/mL each of phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, leupeptin, and aprotinin) was added to each dish. The cells were scraped from the plates and triturated three times with a 3-mL syringe and 22-gauge needle. The resulting lysates were centrifuged at 4°C for 30 minutes at 70 000g in a Beckman SW55Ti rotor. The supernatants were concentrated by using a Centricon 30 concentration device (Amicon Corp) to a volume of 250 µL. The pellets were resuspended in 250 µL of homogenization buffer with a tuberculin syringe and 22-gauge needle. Protein concentrations of samples to be loaded on sodium dodecyl sulfate (SDS)–polyacrylamide gels were measured by the Bio-Rad micro protein assay (Bio-Rad Laboratories). The supernatant and pellet solutions were mixed with SDS-Laemlli sample buffer, heated at 90°C for 5 minutes, and subjected to SDS–polyacrylamide gel electrophoresis and Western blot analysis, as previously described.²⁴

There is currently some controversy over which PKC isozymes are expressed in adult and neonatal rat cardiac myocytes.^{25 49 50 51} In addition to the antisera previously mentioned in this section, we have used many antisera to demonstrate the reproducibility of our previous finding that neonatal cardiac myocytes express at least six PKC isozymes.²⁵ Specifically, we used 12 different anti–ß-PKC antibodies, all of which demonstrate the presence of this isozyme in cardiac myocytes. These include the following: a polyclonal anti-recombinant catalytic domain of ß-PKC and an anti-recombinant regulatory domain of ß-PKC, both from Dr Koshland's laboratory (University of California, Berkeley); polyclonal anti–ß_I- and –ß_II-PKC isozymes from Research and Diagnostic Antibodies; anti–-PKC and –ß-PKC monoclonal antibodies from Amersham; polyclonal anti–ß_I-PKC and –ß_II-PKC antibodies from Calbiochem; polyclonal anti–ß-PKC from GIBCO; CK 1.3 monoclonal anti-ß_II antibody that we prepared; monoclonal anti–-PKC and –ß-PKC from Seikagaku; and polyclonal anti–ß_I-PKC and –ß_II-PKC antibodies from Dr Y. Hannun (Duke University). We also used four different anti–-PKC antibodies: our monoclonal antibody CK 1.4 and polyclonal anti–-PKC antibodies from Research and Diagnostic Antibodies, GIBCO, and Biotechnologies. Each of these isozyme-specific sera yielded similar results for their respective PKC isozymes.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Short-term Treatment With 4-ß PMA Decreases Rate of Spontaneous Cardiac Myocyte Contraction
We first determined the effect of 100 nmol/L 4-ß PMA treatment on the rate of contraction of cardiac myocytes. The basal rates of contraction (expressed as the number of contractions per 15 seconds, Fig 1) were monitored at 37°C for 20 minutes to ensure a stable contraction rate. Cells not treated with 4-ß PMA contracted at stable rates (Figs 1 and 2 and data not shown). Cells were then treated with 100 nmol/L of either 4- PMA (a biologically inactive analogue of the phorbol ester 4-ß PMA) or with 4-ß PMA for the times indicated, and contraction rates were recorded. In the experiment shown in Fig 1, the rate of contraction was reduced by 50% after a 20-minute treatment with 100 nmol/L PMA. In six independent experiments, each using different myocyte preparations, the mean±SEM inhibition after a 20-minute exposure to 100 nmol/L PMA was 49±10% (Fig 2). In contrast, 4- PMA (100 nmol/L) had no effect on the contraction rate (Figs 1 and 2).

Inhibition of Contraction Rate by 4-ß PMA Is Concentration Dependent
To examine the dose dependency of the 4-ß PMA–induced inhibition of the contraction rate, cardiac myocytes were treated with 100 nmol/L 4 PMA or 0.1 to 100 nmol/L 4-ß PMA and monitored for inhibition of the contraction rate as described in "Materials and Methods" (Fig 2). Fig 2 shows the mean±SEM results for four to eight experiments. The effect was evident at 0.3 nmol/L 4-ß PMA concentrations (Fig 2C) and was maximal after exposure of the cells to 3 nmol/L 4-ß PMA for 20 minutes (Fig 2E). These results demonstrated that the 4-ß PMA–induced inhibition of the contraction rate occurred in a dose-dependent manner and was maximal after exposure to subdownregulating concentrations of 4-ß PMA.

Inhibition of Contraction Rate After 3 nmol/L 4-ß PMA Treatment Is Reversible
To determine if the 4-ß PMA–induced inhibition of the contraction rate was reversible, the experiments summarized in Fig 3 were conducted. The basal contraction rate of the myocytes was first measured as described in "Materials and Methods." Next, cells were treated for 60 minutes with 3 nmol/L 4-ß PMA, and once again the contraction rate was monitored. These treatment conditions caused maximal levels of 4-ß PMA–induced inhibition of the contraction rate (Fig 2). The cells were then washed to lower the 4-ß PMA concentration to 0.1 nmol/L, as described in "Materials and Methods," and the contraction rate was recorded 10, 20, 30, 60, and 90 minutes after washing was completed. As shown in Fig 2, 0.1 nmol/L 4-ß PMA had no significant effects on the contraction rate. Therefore, dilution of the 4-ß PMA concentration to this level may allow us to determine whether recovery from the 4-ß PMA–induced inhibition of the contraction rate can occur. The 0 time point in Fig 3 reflects the contraction rate after the 60-minute 3 nmol/L 4-ß PMA treatment before washing was initiated and was 53±4% (mean±SEM, n=4). Dilution of 4-ß PMA to 0.1 nmol/L caused a time-dependent reversal of the 3 nmol/L 4-ß PMA–induced inhibition of the contraction rate, which was complete after 90 minutes (Fig 3). These data demonstrate that 3 nmol/L 4-ß PMA–induced inhibition of the contraction rate is reversible and requires the continuous presence of 4-ß PMA for the effect to be observed.

View larger version (14K): [in this window] [in a new window]   Figure 3. Graph showing recovery from 3 nmol/L phorbol 12-myristate 13-acetate (PMA)–induced inhibition of contraction rate in neonatal cardiac myocytes. Spontaneous contraction rate was monitored as described in "Materials and Methods" for 10 minutes. The average of these contraction rates was used as 100% of control, with SEM always being <3%. Cells were then treated with 3 nmol/L PMA for 60 minutes, and the contraction rate of the cells was monitored as in Fig 2. The myocytes were then washed with conditioned medium as described in "Materials and Methods," such that the final PMA concentration was 0.1 nmol/L. Contraction rates of the washed cells were then monitored and recorded at 10, 20, 30, 60, and 90 minutes after washing. Data are plotted as mean±SEM percentage of basal (before PMA addition) contraction rates for four experiments taken from four independent myocyte preparations. The x axis indicates the time after 4-ß PMA removal. The 0 time point represents the percentage of the basal contraction rate observed after a 60-minute treatment with 3 nmol/L 4-ß PMA before washing was initiated.

Differential Effects of 100 nmol/L 4-ß PMA Treatment on PKC Isozyme Levels
We next carried out experiments to determine which PKC isozyme(s) mediates the inhibitory chronotropic response. We examined PKC isozymes after a 0- to 60-minute exposure to 100 nmol/L PMA, since the inhibitory effect on contraction was evident after 5 minutes and was sustained for > 1 hour (Fig 1). Western blot analyses of the cytosolic and particulate fractions with antibodies specific for -, ß-, -, -, and -PKC were next carried out.²⁴ After a 2-minute incubation with 100 nmol/L 4-ß PMA, most of the cytosolic -, ß-, -, and -PKC isozymes translocated to the particulate fraction (Figs 4 and 5), which correlated with the 100 nmol/L 4-ß PMA–induced inhibition of the contraction rate (Figs 1 and 2G). In the experiment shown in Fig 4, -PKC was completely downregulated after a 60-minute exposure to 100 nmol/L 4-ß PMA. Cells used in this experiment were taken from the same preparation of cells as used for the experiment shown in Fig 1; hence, the 100 nmol/L 4-ß PMA–induced inhibition of the contraction rate was unaltered in this experiment despite the loss of -PKC. In addition, one other experiment from a different myocyte preparation showed complete downregulation of the -PKC isozyme after a 60-minute exposure of the cells to 100 nmol/L 4-ß PMA, indicating that in two of four experiments -PKC was completely downregulated. On average, however, densitometric analysis of all four experiments revealed that 15±8% (mean±SEM, n=4) of the total -PKC levels remained after a 60-minute exposure to 100 nmol/L 4-ß PMA (Fig 5). It is interesting to note that the -PKC isozyme was the most sensitive to 4-ß PMA–induced downregulation. We observed a very rapid decrease in total -PKC levels after a 2-minute exposure to 100 nmol/L PMA (Fig 5). The experiment shown in Fig 4 demonstrates that ß-PKC is also downregulated after a 60-minute treatment with 100 nmol/L 4-ß PMA, suggesting that it may also not be necessary for the 100 nmol/L PMA–induced inhibition of the contraction rate. On average, 30±5% (n=4) of the total ß-PKC remained after a 60-minute exposure to 100 nmol/L 4-ß PMA (Fig 5).

View larger version (20K): [in this window] [in a new window]   Figure 4. Effects of 100 nmol/L phorbol 12-myristate 13-acetate (PMA) treatment on the level and distribution of protein kinase C (PKC) isozymes. Cells were cultured as described in "Materials and Methods" and treated with 100 nmol/L 4- or 4-ß PMA for the times indicated in the figure. After fractionation into supernatant (S) and particulate (P) fractions, Western blot analyses were conducted on these fractions with PKC isozyme–selective antibodies against -, ß-, -, -, and -PKC isozymes, as indicated on the right side of the figure. The data shown are from the same cell preparation as used in Fig 1.

View larger version (23K): [in this window] [in a new window]   Figure 5. Graphs showing quantification of protein kinase C (PKC) isozyme levels and distribution after 100 nmol/L 4-ß phorbol 12-myristate 13-acetate (4-ß PMA) treatment. Neonatal cardiac myocytes were treated with 100 nmol/L 4-ß PMA, and PKC isozymes were monitored by Western blot analysis and autoradiography as described in the legend to Fig 4. PKC isozyme levels were then quantified by densitometric scanning of autoradiographs as described in "Materials and Methods." Data are plotted as the percentage of 100 nmol/L 4- PMA levels of each individual PKC isozyme in the cytosol, particulate, and total cell fractions after a 2- to 60-minute exposure to 100 nmol/L 4-ß PMA. The dashed line indicates 100% of 100 nmol/L 4- PMA levels. The results shown are mean±SEM values for PKC isozyme levels at each time point from four independent experiments, each taken from a separate cardiac myocyte preparation. 69 indicates the 69-kD form of -PKC.

In contrast to the - and ß-PKC isozymes, - and -PKC remained in the particulate fraction after 100 nmol/L 4-ß PMA treatment for at least 1 hour, without a substantial decline in their levels (Figs 4 and 5). The cytosolic and particulate levels of the 69-kD form of -PKC (₆₉-PKC) also showed little or no change after exposure of the myocytes to 100 nmol/L 4-ß PMA for 2 to 60 minutes (Figs 4 and 5). However, there was a modest increase in the particulate levels of ₆₉-PKC (see bottom band in Fig 4 and bottom graphs in Fig 5) after a 10- to 30-minute treatment with 100 nmol/L 4-ß PMA. The reason for this increase is unknown, since -PKC is thought to be unresponsive to 4-ß PMA.⁵² In summary, the mean±SEM percentages of total levels of the -, -, and ₆₉-PKC isozymes remaining in four experiments after a 60-minute incubation with 100 nmol/L 4-ß PMA were 75±4%, 109±13%, and 87±10%, respectively.

Finally, exposure of the myocytes to 100 nmol/L 4-ß PMA for 2 minutes also caused the translocation of an 80-kD protein that is recognized by antiserum raised from a peptide sequence thought to be unique to -PKC⁵³ (for simplicity, we referred to it as ₈₀-PKC). The identity of the ₈₀ protein is currently unknown and is beyond the scope of this article. Several lines of evidence suggest that it is indeed a PKC isozyme, possibly -PKC⁵³ or an as-yet-uncharacterized PKC isozyme.

Treatment of Neonatal Cardiac Myocytes With 3 nmol/L 4-ß PMA Induces Differential Patterns of Translocation of PKC Isozymes
For the most part, 100 nmol/L 4-ß PMA did not reveal differential translocation of PKC isozymes. Therefore, we examined the translocation of -, ß-, -, -, and -PKC isozymes after a 0- to 60-minute exposure to 3 nmol/L 4-ß PMA (Figs 6 and 7), a concentration that caused maximal inhibition of the contraction rate (Fig 2). We reasoned that lower 4-ß PMA concentrations may reveal differential translocation of individual PKC isozymes. In the experiment shown in Fig 6, we observed a partial redistribution of the -, -, -, and ₈₀-PKC isozymes from the cytosol to the particulate cell fraction after 3 nmol/L 4-ß PMA treatment. In this experiment, the translocation of the -PKC isozyme (apparent after 5 minutes) correlated best with the time course of the 3 nmol/L 4-ß PMA–induced inhibition of the contraction rate (Fig 2). Note that translocation of the -PKC isozyme in this experiment was not detected until 20 minutes after 3 nmol/L 4-ß PMA exposure, indicating that -PKC translocation did not correlate with the development of the inhibitory effect on the contraction rate (Fig 2). The cumulative results from four independent experiments in which 3 nmol/L 4-ß PMA–induced translocation was measured are presented in Fig 7. On average, only the - and -PKC isozymes were found to translocate to the particulate cell fraction after 3 nmol/L 4-ß PMA treatment. It is important to note that the extent of -PKC translocation correlated well with the 4-ß PMA–induced inhibition of the contraction rate (Figs 1, 2E, and 2G) at 4-ß PMA concentrations of both 3 and 100 nmol/L (Figs 4 through 7). Of interest, 3 nmol/L 4-ß PMA treatment did not cause downregulation of any of the PKC isozymes (Fig 7).

View larger version (47K): [in this window] [in a new window]   Figure 6. Western blot showing the effects of 3 nmol/L phorbol 12-myristate 13-acetate (PMA) treatment on the translocation of protein kinase C (PKC) isozymes. Experimental conditions are the same as described for Fig 4, except 3 nmol/L PMA concentrations and slightly different treatment times were used.

View larger version (22K): [in this window] [in a new window]   Figure 7. Graphs showing quantification of protein kinase C (PKC) isozyme levels and distribution after 3 nmol/L 4-ß phorbol 12-myristate 13-acetate (4-ß PMA) treatment. Experimental conditions and data presentation are exactly as described in the legend to Fig 5, except 3 nmol/L 4-ß PMA concentrations and slightly different treatment times were used. 69 indicates the 69-kD form of -PKC.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Which PKC Isozymes Mediate Cardiac Myocyte Functions Is Controversial
Investigation of the roles of individual PKC isozymes in cardiac myocyte functions is complicated by conflicting reports concerning which PKC isozymes exist in these cells.^{24 25 49 50 51 54 55} In Western blot studies, our laboratory has previously reported the presence of - and ß-PKC isozymes and at the time an unknown PKC isozyme,²⁴ which we later determined was -PKC in neonatal cardiac myocytes.²⁵ Since that time, Bogoyevitch et al⁴⁹ reported that only the -PKC isozyme was expressed in adult rat cardiac myocytes. A recent publication by Puceat et al⁵¹ has reported that both neonatal and adult rat cardiac myocytes express -, -, -, and -PKC isozymes. Further, Rybin and Steinberg⁵⁴ found different patterns of PKC isozyme expression in neonatal (-, -, -, and -PKC) and adult rat (- and -PKC) cardiac myocytes. Church et al⁵⁵ determined by Western blot analysis that neonatal cardiac myocytes contain -, ß-, -, and -PKC but not -PKC isozymes. Finally, Disatnik and Mochly-Rosen²⁵ showed by immunofluorescence studies the presence of -, ß_I-, ß_II-, -, -, and -PKC isozymes in neonatal cardiac myocytes, each of which is translocated to distinct subcellular sites after stimulation with 4-ß PMA or norepinephrine.

There have also been several studies of PKC isozyme translocation and downregulation in cardiac myocytes.^{25 49 50 51 54 55} Puceat et al⁵¹ found that ₁-adrenergic or P₂ purinergic receptor activation in neonatal cardiac myocytes elevated membrane-associated immunoreactivity of - and -PKC. In addition, they found that a short treatment with 100-nmol/L concentrations of 4-ß PMA caused redistribution of -, -, and -PKC isozymes in these cells. Longer 4-ß PMA treatments caused substantial downregulation of the - and -PKC but not -PKC isozymes. These authors also found PKC-mediated increases in myristoylated alanine-rich PKC substrate (MARCKS) phosphorylation and c-fos mRNA accumulation; the former but not the latter function correlated with the persistence of -PKC after PKC downregulation protocols and the redistribution of -PKC to the particulate cell fraction. Our results agree with those of Puceat et al⁵¹ in that we find -PKC to be the most sensitive and -PKC to be one of the least sensitive PKC isozymes to 4-ß PMA–induced downregulation. However, in contrast to Puceat et al, we find the -PKC isozyme to be similarly resistant to 4-ß PMA–induced downregulation (Figs 4 and 5). The reason(s) for all of the above-mentioned discrepancies is at present unknown. It is clear that there are numerous differences in the ages and strains of rats used, methods of myocyte isolation, cell culturing conditions, cell harvest, and antisera that may contribute to the observed differences. For example, Puceat et al isolated neonatal cardiac myocytes from 1- to 2-day-old rat hearts by using collagenase and pancreatin digestion and Percoll gradient techniques. Their cells were then cultured for only 24 hours in 4:1 DMEM/M-199 supplemented with 10% horse serum, 5% fetal bovine serum, ampicillin, and gentamicin. Finally, their cells were serum-starved overnight and used for experiments. In contrast, we digested 1-day-old Sprague-Dawley rat hearts with trypsin and DNase and reduced nonmyocyte cells to <5% by preplating techniques and the inclusion of BrDu in serum-containing culture. Our cells were cultured in M-199 supplemented with 5% fetal bovine serum, penicillin, and vitamin B₁₂ for 4 days, followed by 2 additional days in serum-free medium, after which cells were used for experiments (see "Materials and Methods"). In addition, Rybin and Steinberg⁵⁴ used 2-day-old neonatal Wistar rat hearts, trypsin digestion, preplating methods, and cultured cells in minimal essential medium supplemented with 10% fetal bovine serum and hypoxanthine for an unspecified time for their myocyte preparations but used irradiation to rid cultures of nonmyocyte cells. Finally, as discussed in "Materials and Methods," 12 different anti–ß-PKC antisera and four different anti–-PKC antisera showed immunoreactivity with our neonatal myocyte preparations. In contrast, studies failing to detect the ß-PKC isozyme in their Western blot analyses did not use multiple antisera sources. In summary, differences in tools and techniques between the various studies are the likely explanation for the lack of detection of some of the PKC isozymes.

Relevance of Studies With Phorbol Esters to Those With Physiological Agonists
Another key issue involves the relevance of phorbol ester–induced PKC activation to what occurs when PKC isozymes are activated by physiological agonists via the generation of endogenous DGs. It is generally accepted that activation of PKC isozymes by phorbol esters is more potent and prolonged than that caused by endogenous DGs. Hence, at saturating concentrations, the responses observed after phorbol ester treatments can differ substantially from those caused by hormones that activate PKC via DG production. However, it can be argued that if phorbol esters are administered to cells over a range of treatment times and concentrations, as was done in the present study, one could conceivably achieve cellular responses that more closely mimic physiology. Furthermore, numerous studies that purport to use "physiological agonists" often use them in the presence of antagonists to block the activation of other receptors and use these agonists at several hundred times the circulating levels that are found in vivo. Finally, since isozyme-specific inhibitors are currently unavailable, the methods used in the present study are currently the best tool to implicate specific PKC isozymes in physiological responses.

Use of 4-ß PMA As a Tool to Determine Which PKC Isozyme(s) Mediates the Inhibitory Chronotropic Response in Neonatal Cardiac Myocytes
We have characterized the effects of 4-ß PMA treatment on the rate of contraction and correlated these effects with changes in PKC isozymes in neonatal cardiac myocytes. We reasoned that the activation (translocation) or elimination (downregulation) of individual PKC isozymes might enable us to identify the individual PKC isozymes mediating 4-ß PMA–induced modulation of the contraction rate. Selective differential activation or downmodulation of individual PKC isozymes after 4-ß PMA treatment was not completely achieved. However, candidate PKC isozymes that may mediate regulation of contraction have been identified. Since we did not use adult cardiac myocytes in the present study, our conclusions are restricted to the role of individual isozymes in neonatal cells only.

Translocation of PKC Isozymes Correlates With Inhibition of Contraction Rate
A short-term 4-ß PMA (100 nmol/L) treatment caused a marked inhibition of cardiac myocyte contraction rate (Fig 1) and translocation of -, ß-, -, and -PKC and the putative ₈₀-like PKC isozymes from the cytosolic to the particulate fraction (Figs 4 and 5). The acute effects of 4-ß PMA were specific and involved PKC activation; there was no effect of the inactive 4- PMA on the contraction rate (Figs 1 and 2), PKC isozyme levels, or distribution (Figs 4 through 7). It has previously been reported that 4-ß PMA has inhibitory chronotropic effects on intact heart⁴⁰ and adult cardiomyocytes.²⁷ However, the PKC isozyme(s) that mediates this effect was not determined.

Studies With 100 nmol/L 4-ß PMA Suggest That -PKC Is Not Required for 4-ß PMA–Induced Inhibition of Contraction Rate
Our studies suggest that -, ß-, -, and -PKC and the ₈₀-like PKC all become activated (translocate) after acute exposure to 100 nmol/L 4-ß PMA; hence, all could mediate the 4-ß PMA–induced inhibition of contraction seen in Fig 1. However, -, ß-, and ₈₀-PKC underwent a substantial downregulation after a 1-hour incubation with 100 nmol/L 4-ß PMA (Figs 4 and 5), yet the effect on contraction was sustained for >80 minutes (Fig 1). In addition, in two of four experiments -PKC was found by both visual analysis and densitometric scanning of autoradiographs to be undetected (downregulated) after a 60-minute exposure to 100 nmol/L 4-ß PMA (Fig 4), yet inhibition of contraction was observed in all the experiments (eight of eight independent experiments). The -PKC isozyme was clearly the most sensitive to 4-ß PMA–induced downregulation. In fact, on average, there was already a >50% loss of -PKC immunoreactivity after a 2-minute exposure to 100 nmol/L 4-ß PMA (Fig 5). This decrease was not related to our cell harvest or Western blot protocols, since treatment of cells with 3 nmol/L PMA did not induce downregulation of any of the PKC isozymes tested over a 1-hour time course (Fig 7). These data suggest that -PKC is unlikely to mediate the inhibitory effect on contraction.

Studies With 3 nmol/L 4-ß PMA Implicate - and -PKC in 4-ß PMA–Induced Inhibition of Contraction Rate
Our studies with 3 nmol/L 4-ß PMA suggest that either the - or -PKC isozymes may mediate the 4-ß PMA–induced inhibition of the contraction rate, since increases in the particulate levels of these isozymes but not of the other PKC isozymes (Fig 7) tested correlates with the time course of the 3 nmol/L 4-ß PMA–induced inhibition of the contraction rate (Fig 2). However, together with the results using 100 nmol/L 4-ß PMA, -PKC appears to be the most likely candidate for mediating the 4-ß PMA–induced inhibition of the contraction rate. We argue this since only the extent and time course of -PKC translocation correlated well with 4-ß PMA–induced inhibition of the contraction rate at 4-ß PMA concentrations of both 3 and 100 nmol/L. In addition, in two of four experiments -PKC was completely downregulated after a 60-minute incubation with 100 nmol/L 4-ß PMA, and in four of four experiments an abundance of -PKC remained. Furthermore, in the experiment shown in Fig 6, the translocation of -PKC did not correlate with the 3 nmol/L 4-ß PMA–induced inhibition of the contraction rate, whereas the translocation of -PKC did. Finally, the ß-, -, and ₆₉-PKC isozymes did not translocate after 3 nmol/L PMA treatment, which suggests that they are not involved in the 4-ß PMA–induced inhibition of the contraction rate. It also seems unlikely that the chronotropic effects of 4-ß PMA occur because the elimination of -, ß-, and ₈₀-PKC isozymes relieved stimulatory effects on contraction, since the 4-ß PMA–induced inhibition of contraction (Fig 2) occurred at low nanomolar 4-ß PMA concentrations, which do not cause these isozymes to be downregulated (Fig 7). In summary, our results are consistent with a role for -PKC in the 4-ß PMA–induced inhibition of the contraction rate. However, our studies cannot completely rule out the involvement of other PKC isozymes.

Continuous Activation of One or More PKC Isozymes Is Necessary for 4-ß PMA–Induced Inhibition of Contraction Rate
The 4-ß PMA–induced inhibition of the contraction rate does not appear to involve the initiation of a cascade, which only requires PKC isozymes at the start of the cascade. We demonstrated in Fig 3 that the inhibitory effect on the contraction rate is reversible when the 4-ß PMA is removed. Hence, the effect requires the continuous activation of one or more PKC isozymes. The reversibility of the 4-ß PMA–induced effects were not immediate. However, a progressive recovery that was complete after 90 minutes of washing was observed. Interpretation of these results is complicated by the fact that phorbol esters are notoriously difficult to wash out of cells, so it is quite possible that the actual recovery time would be dramatically shortened if the 4-ß PMA could be eliminated from the cells more quickly.

What Is the Mechanism by Which the -PKC Isozyme Could Regulate the Inhibition of Contraction After Acute PMA Treatment?
Phosphorylation of numerous substrates in heart cells that could directly or indirectly regulate contraction, such as troponins I and T,³² the sarcoplasmic reticulum,³¹ and L-type Ca²⁺ channels,^{33 35} has been reported after treatment with PKC-activating stimuli. It is possible that these phosphorylations play a role in the inhibition of contraction reported in the present study and that -PKC mediated one or more of these phosphotransfer events. It is interesting to note that -PKC localizes to cross-striated structures indistinguishable from myofibrils in neonatal cardiac myocytes after incubation with 4-ß PMA or norepinephrine.²⁵ Therefore, -PKC could be considered a prime candidate for inhibiting contraction by phosphorylating contractile proteins localized to the myofibrils. Finally, 4-ß PMA treatment has been shown to enhance the secretion of prostacyclins from isolated hearts⁵⁶ and cardiac myocytes⁵⁷ as well as the secretion of other factors, such as enkephalin peptides⁴⁶ and TGF ß,⁵⁸ from cardiac myocytes. These factors can alter the cardiac myocyte contraction rate. -PKC also translocates to regions of cell-cell contact when stimuli such as 4-ß PMA are applied to neonatal cardiac myocytes.²⁵ Therefore, it is possible that the -PKC that localizes to cell-cell contacts after activation (possibly intercalated disks) also enhances the secretion of an inhibitory factor, decreases the secretion of a stimulatory factor, or regulates the pathways that respond to these factors. Determination of the precise roles of PKC isozymes in the reduction of contractile rate will require further study.

We have used neonatal cardiac myocytes as a model system to study the involvement of PKC isozymes in the regulation of their rate of contraction. There are at least six PKC isozymes activated by 4-ß PMA in cardiac myocytes. Extensive concentration-response, time-course, and downregulation experiments with 4-ß PMA have suggested that the -PKC isozyme may modulate the contraction rate in neonatal cardiac myocytes. Further studies with specific inhibitors of -PKC and other PKC isozymes will be needed to expand on these results and are currently in progress.

	Acknowledgments

 
This work was supported in part by grants from the National Heart, Lung, and Blood Institute and the March of Dimes to Dr Mochly-Rosen and a Stanford University Dean's Fellowship to Dr Johnson. We thank Dr A. Gordon for constructive discussions.

Received June 27, 1994; accepted December 20, 1994.

	References

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				Z.-H. Zhang, J. A. Johnson, L. Chen, N. El-Sherif, D. Mochly-Rosen, and M. Boutjdir C2 Region–Derived Peptides of ß-Protein Kinase C Regulate Cardiac Ca2+ Channels Circ. Res., May 19, 1997; 80(5): 720 - 729. [Abstract] [Full Text]

				M. A. Sussman, S. F. Hamm-Alvarez, P. M. Vilalta, S. Welch, and L. Kedes Involvement of Phosphorylation in Doxorubicin-Mediated Myofibril Degeneration: An Immunofluorescence Microscopy Analysis Circ. Res., January 1, 1997; 80(1): 52 - 61. [Abstract] [Full Text]

				J. A. Johnson, M. O. Gray, J. S. Karliner, C.-H. Chen, and D. Mochly-Rosen An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes: Application to Protein Kinase C Research Circ. Res., December 1, 1996; 79(6): 1086 - 1099. [Abstract] [Full Text]

				J. A. Johnson, M. O. Gray, C.-H. Chen, and D. Mochly-Rosen A Protein Kinase C Translocation Inhibitor as an Isozyme-selective Antagonist of Cardiac Function J. Biol. Chem., October 4, 1996; 271(40): 24962 - 24966. [Abstract] [Full Text] [PDF]

				V. Rybin and S. F. Steinberg Thyroid Hormone Represses Protein Kinase C Isoform Expression and Activity in Rat Cardiac Myocytes Circ. Res., September 1, 1996; 79(3): 388 - 398. [Abstract] [Full Text]

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