© 1996 American Heart Association, Inc.
Articles |
An Improved Permeabilization Protocol for the Introduction of Peptides Into Cardiac Myocytes
Application to Protein Kinase C Research
the Department of Molecular Pharmacology (J.A.J., C.-H.C., D.M.-R.), Stanford (Calif) University School of Medicine; the Cardiology Section (M.O.G., J.S.K.), Veterans Affairs Medical Center, and the Department of Medicine Cardiovascular Research Institute (M.O.G., J.S.K.), University of California, San Francisco (Calif).
Correspondence to John A. Johnson, Department of Molecular Pharmacology, Stanford University School of Medicine, Stanford, CA 94305-5332.
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Abstract |
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We have developed an improved, less disruptive procedure for the transient permeabilization of neonatal cardiac myocytes using saponin. The method allows delivery of peptides to a high percentage of cells in culture without effects on long-term cell viability. Permeation was confirmed microscopically by cellular uptake of a fluorescently labeled peptide and biochemically by uptake of 125I-labeled calmodulin and a 20-kD protein kinase C
fragment into the cells. The intracellular molar concentration of the introduced peptide was 
10% of that applied outside. We found no significant effects of permeabilization on spontaneous, phorbol ester–modulated, or norepinephrine-modulated contraction rates. Similarly, the expression of c-fos mRNA (measured 30 minutes after permeabilization) and the incorporation of [14C]phenylalanine following agonist stimulation (measured 3 days after permeabilization) were not altered by saponin permeabilization. Finally, permeabilization of cells in the presence of a protein kinase C pseudosubstrate peptide, but not a control peptide, inhibited phorbol ester–induced [14C]phenylalanine incorporation into proteins by 80%. Our results demonstrate a methodology for the introduction of peptides into neonatal cardiac myocytes that allows study of their actions without substantial compromises in cell integrity.
Key Words: neonatal cardiac myocytes • permeabilization method • contraction • hypertrophy • protein kinase C
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Introduction |
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The introduction of peptides and other cell membrane–impermeable molecules into mammalian cells has been an invaluable biochemical approach in the study of intracellular processes. Numerous methods have been developed to introduce membrane-impermeable molecules into cells. These methods include treating cells with solvents such as toluene or dimethyl sulfoxide, hypotonic buffers, freeze/thaw techniques, electroporation, and various detergents.1 2 3 In addition, microinjection of peptides and proteins into cells has also been used.4 Two major concerns with these methodologies are that the permeabilization or microinjection itself does not compromise cellular intregrity and that a relatively high percentage of cells are permeabilized.
One method of cell permeabilization (referred to as skinning) involves the permeabilization of mammalian cell membranes by incubation of cells with detergents such as saponin. This technique has been used with smooth muscle cells5 and cells or tissue preparations from liver,6 pancreas,3 skeletal muscle,7 cardiac muscle,8 and others.6 This method has also recently been used to indirectly study the calcium dependence of phospholipase C activity9 in neonatal cardiac myocytes. Despite their utility for other purposes, these methods were limited because they either allowed only a small percentage of cells to incorporate the membrane-impermeable molecules4 or, in our hands, severely compromised myocyte functions. Another limitation of these studies was that detergent permeabilizations were induced at room temperature,3 5 7 8 potentially allowing proteolysis and cell-degradative processes to occur. Further, the goal of many of these studies was to permeabilize cells for the duration of the experiments3 5 9 or to allow release of intracellular proteins6 with no expectation that cells would recover and survive for extended periods. Finally, in most permeabilized cell studies, agonists that act intracellularly, such as cAMP or 1,4,5-inositol tris-phosphate, were used.3 5 8 9 With few exceptions,3 the effects of agonists that act on cell surface receptors were either abolished9 or not evaluated.5 7 8 Therefore, improved cell permeabilization technologies would be of great importance for the study of signal transduction in primary cells in culture in general and in cardiac myocytes in particular.
Neonatal rat cardiac myocytes in primary culture are a well-characterized model system often used to study cardiac muscle physiology and biochemistry at the cellular level.10 11 These cells show many similarities with cardiac muscle cells in vivo. For example, they do not divide but do increase in size and accumulate additional protein (hypertrophy) in response to hormonal and other stimuli.11 12 In addition, the cells contract spontaneously in culture at rates similar to the rate of the intact heart in vivo,13 allowing measurement of their beating rate in response to stimuli or drugs.14
Many cardiac responses, such as mRNA accumulation, hypertrophy, and regulation of the force and rate of contraction, involve responses mediated by a family of enzymes collectively referred to as PKC.14 15 16 17 18 19 20 There are at least 10 isozymes in the PKC family,21 six of which are present in neonatal cardiac myocytes.14 22 23 Activation of membrane-bound phospholipases by various hormones and growth factors generates DAGs, which activate most PKC isozymes.24 Experimentally, the PKC-activating drug PMA is often used as a replacement activator for DAG because it is cell permeable and is not metabolized as rapidly as DAG. Each of the PKC isozymes has an autoinhibitory sequence referred to as the pseudosubstrate region. A peptide corresponding to this sequence inhibits the catalytic activity of the enzyme in vitro.25 In addition, a synthetic PKC pseudosubstrate peptide introduced into permeabilized coronary artery smooth muscle cells has been shown to block phorbol ester–induced potentiation of contraction.5
In the present study, we describe a method for introducing bioactive peptides into neonatal cardiac myocytes. This procedure involves saponin permeabilization of the cells on ice in the presence of ATP and has minimal effects on neonatal cardiac myocyte physiology and survival. Furthermore, we found that the introduction of a PKC inhibitory peptide into the cells inhibited a PKC-mediated function, demonstrating the utility of this modified protocol for the introduction of bioactive membrane-impermeable peptides into living cells.
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Materials and Methods |
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Primary Cardiac Myocytes
Primary cell cultures were prepared as described previously.10 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 NMCs, dissociated cells were preplated for 30 minutes onto 100-mm dishes in medium M-199 with Hanks' salt solution (GIBCO) containing 10% fetal bovine serum (Hyclone Laboratories). Most of the cardiac myocytes do not attach under these plating conditions, whereas the NMCs do. Myocytes were cultured in M-199 media supplemented with vitamin B12 (1.5 mmol/L), penicillin G (50 U/mL), and 10% FBS through day 3 for [14C]phenylalanine studies and day 4 for [125I]calmodulin uptake, contraction, c-fos mRNA, trypan blue, and lactate dehydrogenase studies. Cells were plated on 35-mm Corning tissue culture dishes ([125I]calmodulin uptake, contraction, [14C]phenylalanine, trypan blue, and lactate dehydrogenase studies) or 100-mm dishes (c-fos mRNA studies) at a density of 800 cells/mm2 and incubated at 37°C in humidified air with 1% CO2 to maintain pH at 7.3. All cultures were also supplemented with ascorbic acid (80 µmol/L) from day 2 on, regardless of whether FBS was present. The presence of ascorbic acid correlated with an increase in the number of cardiac myocytes that attained stable basal contraction rates within 15 minutes of permeabilization (data not shown). To inhibit NMC proliferation, 0.1 mmol/L bromodeoxyuridine was added to the serum-containing media for the first 4 days of culture. Bromodeoxyuridine does not affect the viability of the myocytes.10 Cells were then placed in defined media (M-199 media containing 50 U/mL penicillin G, 1.5 mmol/L vitamin B12, and 10 µg/mL each of transferrin and insulin) for 1 day ([14C]phenylalanine studies) or 2 days ([125I]calmodulin uptake, contraction, c-fos, trypan blue, and lactate dehydrogenase studies) before the initiation of experiments.
Permeabilization of Cardiac Myocytes
After culturing myocytes in defined media for 1 to 2 days, media were removed, saved, and placed at 37°C (diagram of permeabilization protocol is shown in Fig 1
). These are referred to as conditioned media. The cells were then slowly brought down in temperature by two sequential 2-minute incubations, each with 2 mL (for 35-mm culture dishes) or 5 mL (for 60- and 100-mm culture dishes) of fresh PBS. The first PBS incubation was carried out at room temperature; the second, with chilled PBS in an ice bath. The PBS was discarded, and the cells were incubated with freshly prepared permeabilization buffer (20 mmol/L HEPES, pH 7.4, 10 mmol/L EGTA, 140 mmol/L KCl, 50 µg/mL saponin [Sigma Chemical Co], 5 mmol/L NaN3, and 5 mmol/L oxalic acid dipotassium salt) containing the desired peptides for 10 minutes in an ice bath. The NaN3 was omitted from the permeabilization buffer for c-fos mRNA accumulation and [14C]phenylalanine incorporation experiments. The final volume of permeabilization buffer for 35-, 60-, and 100-mm culture dishes was 1, 1.2, and 3 mL, respectively. It was convenient to prepare a 2x permeabilization buffer so that the additional volume contributed by the peptides could be accommodated without dilution of the permeabilization buffer. ATP was added just before adding the permeabilization buffer to cells (ie, 30 µL of 200 mmol/L ATP, pH 7.4, per milliliter of permeabilization buffer). The cells were then gently washed four times on ice with 2 mL (35-mm tissue culture dishes) or 5 mL (60- or 100-mm tissue culture dishes) of chilled PBS. The final PBS wash was discarded, and an additional 2 or 5 mL of chilled PBS was added to the cells. After a 20-minute recovery on ice, the chilled PBS was removed, 2 or 5 mL of room temperature PBS was added, and the cells were placed at room temperature for 2 minutes. This step was repeated with PBS at 37°C, after which the original cell media (conditioned media) were added back to the cells at 37°C. The cells were further incubated for 15 minutes at 37°C before use in contraction experiments or 30 minutes before use in other studies.
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Ethidium Red Dye Uptake Into All Cells
We wanted to conduct double-labeling immunofluorescence studies for the purpose of determining the percentage of cells incorporating an FITC-labeled peptide after permeabilization steps (Fig 3, Table 1). The strategy was to use a fluorescence microscope equipped with different optical filters to (1) label all cells with one fluorescent molecule (with an emission spectrum different from that of FITC) and (2) determine the percentage of those cells receiving an FITC-labeled peptide in permeabilization steps. We first developed a method for labeling all of the cells with a photofluor, which could be visualized independent of FITC. For this purpose, we used two fluorescent molecules, ethidium red and calcein-AM (Molecular Probes Inc). Calcein-AM is cell permeant and will stain all live cells green. Ethidium red only enters cells with compromised plasma membranes and stains them red. We used calcein-AM to confirm that all saponin-permeabilized cells had incorporated ethidium red. Because each dye emits a characteristic wavelength of light, intracellular staining due to each agent can be visualized independently using different optical filters.
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Uptake of a Fluorescently Labeled Peptide by Permeabilized Cardiac Myocytes
By permeabilizing cells in the presence of ethidium red and FITC-labeled NH2-P-E-W-N-E-T-COOH peptide, we could use a fluorescence microscope equipped with different optical filters to first determine the total number of cells present by ethidium red staining and then determine how many of these cells took up FITC-labeled NH2-P-E-W-N-E-T-COOH peptide. Cells were cultured at a density of 800 cells/mm2 on Labtek culture slides. Saponin permeabilization or mock permeabilization of cells was conducted on ice or at room temperature for 10 minutes in the presence or absence of 6 mmol/L ATP as described in Figs 2 and 3
and the Table. Cells were then allowed to recover on ice, followed by a 10-minute incubation at 37°C and three 37°C PBS washes to reduce nonspecific background staining. Cells were resuspended in conditioned media and viewed using a Zeiss IM35 microscope with a x40 water immersion objective.
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Measurement of 125I-Labeled Calmodulin Uptake
Cells were permeabilized with permeabilization buffer supplemented with BSA (0.25 mg/mL as a carrier protein), 2 µCi of 125I-labeled calmodulin (107 µCi/µg, Amersham), and 150 µg/mL of nonlabeled calmodulin (Sigma) such that the final specific activity of the radiolabeled calmodulin was 2.2x105 cpm/nmol. As a control for nonspecific association of [125I]calmodulin with extracellular surfaces, nonpermeabilized cardiac myocytes were incubated with a saponin-free mock permeabilization buffer consisting of PBS supplemented with 0.25 mg/mL BSA, 2 µCi 125I-labeled calmodulin, and 150 µg/mL nonlabeled calmodulin. Mock permeabilization groups were treated identically to permeabilization groups, except PBS was used in place of the saponin-based permeabilization buffer. Cell-specific intracellular radiolabeling was determined by subtracting the counts per minute value obtained in mock-permeabilized cells from that obtained with saponin-permeabilized cells. After the permeabilization or mock protocols, cells were incubated at 37°C with conditioned media for 30 minutes, washed three times with 2 mL of chilled PBS, and lysed with 1 mL of 1% SDS. After overnight incubation at 37°C, cell extracts were transferred to scintillation vials and 125I-quantified for 10 minutes in a gamma emission counter. Total protein concentrations were determined by the Bio-Rad DC (detergent compatible) protein assay. Cell permeabilization did not significantly alter total cellular protein concentrations (data not shown). However, all 14C counts per minute values obtained were normalized for total cell protein.
Measurement of the Uptake of a Recombinant 20-kD PKC Fragment Into Neonatal Cardiac Myocytes
Cells were cultured on 60-mm tissue culture dishes and subjected to mock or saponin permeabilizations as described above and in the legend of Fig 4. Mock and saponin permeabilizations were carried out on ice or at room temperature in the presence or absence of ATP (as indicated in the legend to Fig 4) in the presence of 300 µg/mL of the 20-kD PKC fragment immunotagged with FLAG sequence (Kodak). Room temperature permeabilizations were conducted in the same manner as chilled permeabilizations, except all chilled washes and permeabilizations were conducted at room temperature. The expression and purification of the recombinant PKC fragment will be published elsewhere. After permeabilization and recovery, cells were incubated at 37°C in 5 mL of conditioned media for 10 minutes to reduce nonspecific association of the 20-kD PKC fragment to extracellular surfaces (data not shown). Media were discarded, and the cells were washed twice with 5 mL of chilled PBS. Cells were harvested and fractionated into cytosolic (supernatant) and particulate fractions. The supernatant and particulate fractions contain the same volume, but the supernatant fractions usually contain more protein than the particulate fractions. Therefore, we normalize all supernatant fractions to one another and all particulate fractions to one another for the total protein in these respective fractions. We feel this is the most accurate method, since the material loaded on our gels represents the total cellular supernatant and particulate fractions minus a small aliquot for protein determinations. Next, cells were subjected to SDS-PAGE (15% acrylamide gels), and proteins were transferred onto nitrocellulose paper (Schleicher & Schuell) as previously described.14 The 20-kD PKC fragment was detected with anti-M2 antisera (Kodak) at 1:1000 dilutions, followed by incubation of blots with a secondary rabbit anti-mouse antibody (Zymed Laboratories, Inc) and then a final incubation with 125I-protein A. The resulting blots were subjected to autoradiography.
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Measurement of Cardiac Myocyte Contraction Rates
Measurements of spontaneous and PMA- and NE-modulated contraction rates were carried out on cells treated as described in the figure legends. The method has been previously described in detail.14 Briefly, the culture dishes were placed in a Medical Systems Corp temperature regulation apparatus positioned on the stage of an inverted microscope (Carl Zeiss Inc) and maintained at 37°C. For studies of the effects of NE or PMA on the rate of contraction (Figs 5 and 6), individual cells were monitored before and after the addition of NE or PMA. Cells were monitored every 2 minutes for a total of 10 minutes to ensure a stable basal rate of contraction. Cells showing a >10% standard error about the mean when their basal contraction rates were averaged were not used in further studies. After permeabilization on ice, in the presence of ATP, >65% of the tested cells attained such a stable rate of contraction and were thus used in further studies. In measurements made after longer times, recoveries approached 100%. NE was prepared in a solution of 10 mmol/L ascorbate193/ µmol/L HCl and diluted in the assays such that the final concentration of ascorbate was 100 µmol/L. Phorbol esters were prepared in dimethyl sulfoxide. The final dimethyl sulfoxide concentration was 0.05% (vol/vol).
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Northern Blot Analysis of c-fos Expression in Cardiac Myocytes
Three 100-mm tissue culture dishes of cells were used for each treatment group. After permeabilization and equilibration in conditioned media, cells were treated as described in the legend to Fig 7. Control and treated cells were then washed with chilled PBS and lysed with 4 mol/L guanidinium isothiocyanate (Fluka). Total RNA was isolated by pelleting the sheared lysates through cesium chloride gradients and fractionated by electrophoresis in a 1% agarose gel containing 0.6 mol/L formaldehyde (15 µg of total RNA per lane). The RNA was then blotted onto a Nytran membrane (Schleicher & Schuell) and hybridized overnight at 65°C with a c-fos cDNA probe (a gift of Dr Tom Curran, St. Jude Children's Research Hospital, Memphis, Tenn) labeled with [
32P]dATP and [32P]dCTP by random priming (Boehringer Mannheim kit). Autoradiographs were then obtained by exposing the hybridized blot to Kodak X-OMAT film for 48 hours at -80°C.
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Measurement of [14C]Phenylalanine Incorporation Into Cardiac Myocyte Proteins
Cells were permeabilized and treated as described in the legends to Figs 9 and 10. Protein synthesis in the cardiac myocytes was monitored in triplicate for each experiment by quantification of [14C]phenylalanine incorporation into trichloroacetic acid–precipitated cellular proteins as described by Simpson.11
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Cell Viability Measurements
Cells were or were not permeabilized and incubated in conditioned media at 37°C for 3 days in 1% CO2. Trypan blue vital dye (GIBCO) was added, and the number of cells excluding or taking up the dye was determined by direct cell counting. Five random fields of cells were monitored in each experiment for each permeabilization condition. The percentage of (viable) cells excluding the dye was then calculated. Cellular LDH analyses were conducted using the Beckman LDH assay system by the Laboratory Medicine facility at the San Francisco Veterans Affairs Medical Center. Briefly, nonpermeabilized and permeabilized cells were frozen in 1 mL PBS overnight at -70°C. Cells were allowed to thaw, scraped from the 35-mm dishes, and transferred to a chilled microfuge tube. The contents of the tube were centrifuged at an Eppendorf microfuge setting of 14 000 for 10 minutes at 4°C. The supernatants were collected, and intracellular LDH activity was measured.
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Utility of Ethidium Red Vital Dye for Labeling Permeabilized Neonatal Cardiac Myocytes
To determine the percentage of permeabilized neonatal cardiac myocytes taking up a fluorescently labeled peptide, we first needed to visualize all of the cells present. We could then compare the number of cells receiving a fluorescently labeled peptide with the total number of cells present and hence calculate the percentage of the total cells receiving the peptide. We first determined that we could label all cells by permeabilizing them as shown in Fig 1
in the presence of ethidium red. To confirm that all cells incorporated ethidium red dye in the permeabilization step, we used a variation of the "LIVE/DEAD" protocol (Molecular Probes Inc). The protocol uses two fluorescent dyes (calcein-AM and ethidium red) that are nonfluorescent when present extracellularly. When they enter cells, each dye fluoresces at different wavelengths of light. This allows independent viewing of staining due to each dye by using a microscope equipped with different optical filters. Calcein-AM is cell permeant and produces a green color in all live cells when wavelengths of light of 495 nm are supplied to the cells. Ethidium red is cell impermeant but stains cells with compromised membranes red (especially nuclei) when viewed at light wavelengths of 590 nm.
We first permeabilized cells as described in Fig 1 in the presence or absence of 1 µmol/L ethidium red. Cells were then allowed to recover and were subsequently incubated with and without 0.3 µmol/L calcein-AM for 10 minutes at 37°C (Fig 2). Cells were then visualized using a fluorescence microscope equipped with different optical filters that allowed independent viewing of calcein-AM and ethidium red staining. As shown in Fig 2a, calcein-AM stained cell bodies and the nuclei of cells. In contrast, no specific cell staining was observed when the same cells were viewed using an optical filter, which provides the wavelength of light needed for visualization of ethidium red (but not calcein-AM) staining (Fig 2d). Similarly, when cells were permeabilized in the presence of ethidium red and not incubated with calcein-AM and then viewed using an optical filter selective for calcein-AM, we found no cell staining (Fig 2b). However, the same cells did show substantial staining due to ethidium red when visualized with the ethidium red–selective optical filter (Fig 2e). Finally, panels c and f of Fig 2 demonstrate that when cells are first permeabilized in the presence of ethidium red and then incubated with calcein-AM for 10 minutes at 37°C, cellular staining produced by each dye can be viewed independently by using the appropriate optical filter. These results indicate that in these double-labeling experiments with calcein-AM and ethidium red (1) staining of neonatal cardiac myocytes with calcein-AM and ethidium red can be visualized independently and exclusive of one another and (2) when both dyes were present, all cells showing calcein-AM staining also showed ethidium red staining (Fig 2, panel c versus panel f). This confirmed that by including ethidium red in the permeabilization buffer, we could label all of the cells with ethidium red.
Note that we needed to use ethidium red to label all of the cells in our permeabilization experiments, because it is viewed with wavelengths of light of 590 nm. The FITC-labeled peptide (NH2-P-E-W-N-E-T-COOH) we discuss below is viewed at the same wavelength of light as calcein-AM; therefore, calcein-AM could not be used to label all of the cells for our peptide uptake experiments, because we could not visualize calcein-AM staining independent of staining due to the FITC-labeled peptide.
Effects of Temperature and ATP on the Uptake of a Fluorescently Labeled Peptide in Saponin-Permeabilized Neonatal Cardiac Myocytes
To compare our protocol (Fig 1) with other methods of cell permeabilization, the intracellular incorporation of a fluorescently labeled peptide (NH2-P-E-W-N-E-T-COOH) was monitored under the conditions shown in Fig 3. In some nonpermeabilized cells, a small amount of background labeling was observed (Fig 3a and 3e). This appeared to be labeling of cell debris. In contrast, the saponin-permeabilized cells incorporated fluorescent labeling in intracellular loci (Fig 3c, 3d, 3g, and 3h).
To directly assess the percentage of cardiac myocytes receiving peptide after permeabilization by various methods, a double-labeling protocol was used. Cells were incubated for 10 minutes at room temperature or on ice with PBS (mock permeabilizations) or saponin-based permeabilization buffer in the presence or absence of 6 mmol/L ATP. Each incubation contained the fluorescein-labeled peptide NH2-P-E-W-N-E-T-COOH and the low-molecular-weight stain ethidium red. Fluorescein yields a green color when examined microscopically using an optical filter that provides wavelengths of 495 nm; hence, cells incorporating the NH2-P-E-W-N-E-T-COOH peptide show intracellular green staining. In contrast, ethidium red gives cells a red color when viewed using a different optical filter. We found no intracellular ethidium red staining of cells incubated with PBS alone on ice or at room temperature. Similarly, less than one half of the cells permeabilized with PBS containing 6 mmol/L ATP showed uptake of ethidium red. All other permeabilization conditions tested showed uptake of ethidium red into all of the cells. This made it possible first to identify the total number of cells taking up ethidium red and then, using a different optical filter, to determine what percentage of these cells also incorporated the fluorescein-labeled peptide NH2-P-E-W-N-E-T-COOH.
In cells incubated for 10 minutes with PBS alone (mock permeabilization), there was no uptake of the NH2-P-E-W-N-E-T-COOH peptide, but a small amount of nonspecific extracellular labeling was observed (Fig 3a and 3e). Since less than half of the cells incubated with ATP alone were labeled with ethidium red, it was not possible to determine the exact percentage of cells taking up the fluorescein-labeled peptide NH2-P-E-W-N-E-T-COOH using this permeabilization condition. However, when cells were incubated for 10 minutes at room temperature or on ice in PBS containing 6 mmol/L ATP, 31±10% (n=3) and 14±1% (n=3), respectively, of the cells incorporating ethidium red also incorporated the fluorescein-labeled NH2-P-E-W-N-E-T-COOH. Since few cells were permeabilized with ATP alone, it was difficult to find representative fields for micrographs. In addition, since uptake was so low, the signal would often fade before taking micrographs. Panels b and f of Fig 3 show the typical low level of uptake seen with these permeabilization conditions, but in most cases far fewer cells showed uptake of the NH2-P-E-W-N-E-T-COOH peptide.
Incubation of cells on ice with saponin in the absence of ATP caused <40% of the cells to incorporate the NH2-P-E-W-N-E-T-COOH peptide (Table, Fig 3c). In contrast, >80% of the cells incorporated fluorescein-labeled NH2-P-E-W-N-E-T-COOH when permeabilized with saponin at room temperature (Table, Fig 3g and 3h) or on ice in the presence of ATP (Table, Fig 3d). These results indicate that saponin permeabilization of neonatal cardiac myocytes for 10 minutes on ice in the presence of ATP or at room temperature leads to nearly identical percentages of the cells incorporating the NH2-P-E-W-N-E-T-COOH peptide.
Uptake of 125I-Labeled Calmodulin by Saponin-Permeabilized Cardiac Myocytes
To determine the intracellular concentration of the introduced peptide, we permeabilized cardiac myocytes as described previously (Fig 1 and "Materials and Methods") in the presence of 125I-labeled calmodulin (2.2x105 cpm/nmol). Permeabilization conditions were the same as those used for studies of contraction rate, c-fos expression, and [14C]phenylalanine incorporation (in the following), except BSA was added to the permeabilization and mock buffers to reduce nonspecific binding of the radioactive peptide to the cell membrane. The total concentration of calmodulin in the extracellular skinning solution was 9 µmol/L. In seven independent experiments, the mean±SE value associated with permeabilized and nonpermeabilized (mock) cells was 518±96 and 306±50 cpm, respectively. After subtraction of counts bound to nonpermeabilized cells from the counts associated with permeabilized cells, the net cell-incorporated radioactivity corresponded to 0.011±0.004% (mean±SE, n=7) of the total 125I-labeled calmodulin pool. The intracellular volume of a single neonatal cardiac myocyte has previously been estimated by Simpson et al26 to be 2 pL. Therefore, the intracellular concentration of the calmodulin in our experiments was calculated to be 774 nmol/L. This corresponds to 10% of the molar extracellular concentration of calmodulin used in these experiments. These studies demonstrate that this modified saponin permeabilization protocol can be effectively used to introduce peptides of at least 16.7 kD into neonatal cardiac myocytes.
Effects of Temperature and ATP on the Uptake of a 20-kD Recombinant PKC Fragment Into Permeabilized Cells
To further examine the uptake of peptide molecules using our protocol and to compare our results with those of a previously published protocol,27 the experiment shown in Fig 4 was conducted. A 20-kD PKC fragment was expressed and purified from bacteria by methods to be published elsewhere. The PKC fragment has FLAG-tagging sequences (Kodak) at the amino and carboxy termini for detection in Western blots. Cells were permeabilized on ice in the presence of ATP as described in Fig 1 and "Materials and Methods" or at room temperature as previously described.27 As shown in Fig 4, most of the PKC fragment in saponin-permeabilized cells was associated with the cell particulate fraction regardless of the permeabilization method used. We hypothesize that this fragment binds to receptors for PKC present in the particulate cell fraction (Reference 28 and authors' unpublished data, 1996). There was more nonspecific cell-associated PKC fragment in cells permeabilized at room temperature (Fig 4, lane 2) than was observed for cells permeabilized on ice in the presence of ATP. Both methods showed comparable levels of PKC fragment uptake up to 10 minutes (Fig 4, lanes 3 and 4). There was substantially more uptake after 30 and 60 minutes for each method, but permeabilization at room temperature yielded higher intracellular fragment amounts. In addition, there was more PKC fragment found in the cytosol of cells permeabilized for 30 to 60 minutes at room temperature (Fig 4, lanes 5 and 6). Still, substantial levels of PKC fragment were achieved in cells permeabilized on ice in the presence of ATP after 10 minutes (Fig 4, lane 4). Of interest, when cells were permeabilized for 10 minutes on ice in the presence of ATP, uptake was substantially greater than in the absence of ATP (Fig 4, lanes 4 versus 7). In the experiment shown in Fig 4, there was no detectable uptake of 20-kD fragment when cells were permeabilized on ice in the absence of ATP. Densitometric analysis of autoradiographs revealed that on average, however, there was a 50±19% (mean±SE, n=5) reduction in uptake when ATP was excluded from permeabilizations carried out on ice. This effect of ATP was not manifested when cells were permeabilized at room temperature. In fact, cells permeabilized for 10 minutes at room temperature in the presence of ATP had the opposite effect. Densitometric analysis of autoradiographs revealed a reduction in peptide uptake of 36±13% (mean±SE, n=5) when compared with uptake levels at room temperature in the absence of ATP. Our results further indicated that there was a time-dependent uptake of PKC fragment in cells permeabilized at room temperature or as described in Fig 1 that appeared to be maximal after 30 minutes.
Saponin Permeabilization on Ice With ATP Has Minimal Effects on Spontaneous, NE-Induced, and Phorbol Ester–Induced Modulation of Contraction Rate
Neonatal cardiac myocytes contract spontaneously when cultured at high densities.14 29 To determine the effect of saponin permeabilization on the spontaneous contraction rate, permeabilized and nonpermeabilized cardiac myocytes were monitored. Within 15 minutes of the procedure, we found, in 31 permeabilization experiments with a total of 548 cells, that 65% of these cells achieved a stable constant beating rate that was nearly identical to the rates of nonpermeabilized cells. In addition, we found no significant differences in the basal spontaneous contraction rates of permeabilized and nonpermeabilized cells. The mean±SE basal spontaneous rates of contraction for nonpermeabilized and permeabilized cells were 50±4 (35 experiments, 383 cells) and 44±3 (28 experiments, 384 cells) contractions per 15 seconds, respectively. The remaining 35% of the cells did not beat at constant rates within the first 15 minutes after permeabilization; however, their long-term survival was virtually unaffected by the permeabilization protocol (see below).
We have recently demonstrated that phorbol ester treatment suppresses the spontaneous contraction rate of neonatal cardiac myocytes.14 Therefore, we determined whether the permeabilization protocol (Fig 1) altered this response. In nonpermeabilized cells, this negative chronotropic effect is maximal after a 20-minute exposure to 3 to 10 nmol/L PMA.14 As shown in Fig 5A and 5B, nonpermeabilized and permeabilized cells had very similar responses to 10 nmol/L PMA treatment. Furthermore, a comparison of the average responses to 3 to 10 nmol/L PMA treatment in permeabilized cells (15 experiments, 75 cells; data not shown) and those reported previously for nonpermeabilized cells14 revealed no significant differences.
NE increases the rate and force of contraction in the heart30 and in ventricular cardiac myocytes. Therefore, we determined the effect of saponin permeabilization on the NE-stimulated increase in contraction rate (Fig 5C and 5D). We used physiological levels of NE (20 nmol/L).31 The time course of 20 nmol/L NE–stimulated contraction was similar for nonpermeabilized and permeabilized cells (Fig 5C and 5D). NE-stimulated contraction rates peaked after a 6-minute exposure to 20 nmol/L NE and returned to baseline levels after 10 minutes (Fig 5C and 5D). The maximal fold increases in contraction rate after exposure to 20 nmol/L NE for nonpermeabilized cells was slightly greater (1.66±0.03-fold versus 1.45±0.02-fold), but this difference was not significant. Together, these results indicate that our saponin permeabilization protocol has minimal or no effects on spontaneous, phorbol ester–modulated, and NE-modulated contraction rates.
Permeabilization of Cardiac Myocytes on Ice in the Presence of ATP Eliminates Deleterious Effects of Room Temperature Permeabilization on Spontaneous and NE-Mediated Contraction Rate
Because we found that greater amounts of peptides can be introduced into the cells after permeabilization at room temperature (Fig 4), we determined the effect of these permeabilization conditions on cell function. We found that after permeabilization at room temperature, spontaneous and NE-induced contraction rates were adversely affected (Fig 6). In four of five experiments, conducted with different myocyte preparations, we observed no recovery of stable spontaneous contraction rate after permeabilization at room temperature without ATP in the permeabilization buffer. These cells also showed either erratic or no response to 1 µmol/L NE, further indicating impaired contractile properties (Fig 6A and data not shown). Further, there was no difference in NE responsiveness of cells permeabilized at room temperature whether NE was added immediately or up to 3 hours after permeabilization, indicating that prolonged recovery periods did not improve cell recovery. In the experiment shown in Fig 6A, only two of the four cells monitored contracted after room temperature permeabilization. The contraction rates of these cells were slow and variable when compared with cells permeabilized on ice in the presence of ATP (Fig 6, panel A versus panel B). In fact, the cells permeabilized according to our procedure (Fig 1) recovered to 100% of their rate before permeabilization, whereas those permeabilized at room temperature recovered to only 40% of their rate before permeabilization. Collectively, these results indicated that room temperature permeabilizations would not be useful to monitor the inhibitory effects of PMA on spontaneous contraction rate, because few cells recover spontaneous contraction, and in those that recovered, the contraction rates were dramatically slowed and highly variable (Fig 6A). In addition, studies involving NE stimulation could not be carried out with cells permeabilized at room temperature, since (as shown in Fig 6A) these cells were not responsive to 1 µmol/L NE. In contrast, cells permeabilized on ice in the presence of ATP recover stable spontaneous contraction and show a typical response to NE and PMA (Figs 5 and 6B).
Effects of ATP in the Permeabilization Buffer on Spontaneous, NE, and Phorbol Ester Modulation of Contraction Rate
Fig 4 demonstrated beneficial effects of ATP on uptake of the PKC fragment in permeabilizations conducted on ice. Therefore, we determined the effect of ATP in the permeabilization buffer on contraction rates after cell permeabilization. We found that inclusion of ATP in room temperature permeabilizations had no beneficial effects on cell recovery. In fact, none of the cells permeabilized at room temperature in the presence of ATP recovered spontaneous or NE-stimulated contraction (Table). However, inclusion of 6 mmol/L ATP during permeabilization on ice greatly facilitated cell recovery for assays of contraction rate (Table). The mean±SE percentage of cells recovering within 15 minutes for use in assays of contraction rate when cells were permeabilized on ice in the absence of ATP was 27±17% (n=5, 32 cells, four different myocyte preparations). In contrast, in the same experiments, the mean±SE recovery for cells permeabilized on ice in the presence of 6 mmol/L ATP was 85±9% (n=5, 44 cells, four different myocyte preparations). Since the recovery of stable spontaneous contraction rate was dramatically reduced in cells permeabilized without ATP, studies of the effects of PMA or NE in these cells were difficult. In addition, when cells did recover, their responses to PMA and NE were different from those of cells permeabilized on ice in the presence of ATP. For example, in two of four experiments with cells permeabilized on ice without ATP, we observed no inhibition of spontaneous contraction rate after exposure to 10 nmol/L PMA, whereas paired controls (cells permeabilized on ice with ATP present) showed typical responses. The other two experiments showed similar inhibitory responses to phorbol ester whether or not ATP was included in the permeabilization step. However, the phorbol ester response was more rapid in onset and greater in magnitude when cells were permeabilized on ice in the absence of ATP.
In addition, in two experiments we compared the NE-stimulated contraction rates of cells permeabilized on ice in the presence and absence of ATP. All of the cells recovered spontaneous contraction, but the cells permeabilized in the absence of ATP showed at least a 30% decline in basal contraction rate over a 10-minute period, whereas cells permeabilized in the presence of ATP showed no decline. Therefore, by our criteria for stable spontaneous contraction rates ("Materials and Methods"), these cells permeabilized without ATP would not be suitable for our assays. Further, when cells permeabilized on ice in the absence of ATP were treated with 1 µmol/L NE, their contraction rate increased to a level below their original basal rate and remained at that level for 1 hour. In contrast, the cells permeabilized on ice in the presence of ATP showed a typical response to NE like that shown in Fig 6B. These results suggest that inclusion of ATP in the chilled saponin permeabilization buffer not only increases peptide uptake (Table, Figs 3 and 4) but also increases the number of cells recovering a stable spontaneous or PMA- and NE-modulated contraction rate (Table).
Stimulated Levels of c-fos mRNA Are Similar in Cardiac Myocytes Permeabilized on Ice in the Presence of ATP and Nonpermeabilized Cardiac Myocytes
Multiple stimuli have been shown to induce c-fos mRNA expression in cardiac myocytes including NE32 and phorbol esters.33 We determined the levels of expression of c-fos mRNA in response to these agonists by Northern blot analysis as another measure of the functional integrity of cardiac myocytes after permeabilization with saponin. As shown in Fig 7, transient permeabilization alone did not increase steady state levels of c-fos mRNA. Furthermore, in permeabilized and nonpermeabilized cells, PMA and NE treatments led to similar elevations of mRNA levels. Equal quantities of mRNA were loaded in these assays, as evidenced by probing the same blots for ß-actin (Fig 7, bottom). These results demonstrate that there was no abnormal rise in c-fos expression as a result of the permeabilization. Most important, the cells permeabilized with saponin using this modified protocol recovered and were capable of elevating c-fos mRNA levels in response to NE and phorbol esters in a manner similar to that of nonpermeabilized cells.
Long-term Viability of Neonatal Cardiac Myocytes After Saponin Permeabilization on Ice in the Presence of ATP
In five or six experiments with different myocyte preparations, we found that when nonpermeabilized cells and cells permeabilized on ice in the presence of ATP were cultured at 37°C in defined media for 3 days, there were no differences in the percentages of cells that excluded trypan blue vital stain (98.7±0.2% versus 99.2±0.4%, n=6), the micrograms of protein per 35-mm dish (198.8±14.4 versus 173.2±20.3 µg, n=5), or the number of cells counted in five random microscope fields (601.3±39.7 versus 617.3±31.6, n=5). Finally, we found no significant differences in cellular LDH enzymatic activities in nonpermeabilized and permeabilized cells when measured immediately after the protocol. In two experiments, each performed in triplicate, the mean±SE total cellular LDH activities per 35-mm dish for nonpermeabilized and permeabilized cells were 530±53 and 500±78 IU/L. Collectively, these results argue strongly that the transient permeabilization protocol described in Fig 1 does not significantly modify the long-term survival of the neonatal cardiac myocytes.
Comparison of Long-term Cell Viability After Saponin Permeabilization of Cells at Room Temperature or on Ice
We found that room temperature permeabilization (with or without ATP present) caused a substantial decrease in cell number, total protein levels, and cell viability as monitored by trypan blue dye exclusion when compared with cells permeabilized on ice in the presence of ATP (Fig 8). Exclusion of ATP from permeabilization on ice caused only a modest reduction in cell number (Fig 8C). These results indicate that of the methods tested the cells permeabilized on ice in the presence of 6 mmol/L ATP show the greatest number of viable cells 3 days after permeabilization. Therefore, this method would be far better than room temperature permeabilization and slightly better than permeabilization on ice in the absence of ATP for long-term studies.
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Saponin-Induced Permeabilization on Ice in the Presence of ATP Does Not Alter PMA-, NE-, and Serum-Stimulated [14C]Phenylalanine Incorporation Into Neonatal Cardiac Myocyte Proteins
Neonatal cardiac myocytes grown in culture undergo hypertrophy after stimulation with NE, PMA, or serum. This response is characterized by an increase in cell size and protein levels.11 To determine whether cardiac myocytes could recover and carry out complex cellular functions for days after permeabilization, we monitored [14C]phenylalanine incorporation in transiently permeabilized and control (nonpermeabilized) cells (Fig 9). We found no effect of permeabilization on PMA-induced, NE-induced, or 5% bovine calf serum–induced [14C]phenylalanine incorporation into cardiac myocyte proteins. These results clearly demonstrate the functional integrity of the myocytes after permeabilization for at least 72 hours.
Introduction of a PKC Pseudosubstrate Peptide Inhibits PMA-Stimulated [14C]Phenylalanine Incorporation Into Neonatal Cardiac Myocyte Proteins
We next demonstrated that the method could be used to introduce a bioactive peptide into the cells. In the experiments shown in Fig 10, cells were permeabilized as described in Fig 1 in the absence of peptide or in the presence of the PKC inhibitory pseudosubstrate peptide (R-F-A-R-K-G-A-L-R-Q-K-N-V) or the control scrambled version of the pseudosubstrate peptide (R-A-L-Q-R-A-K-N-E-V-H-K-V-F-K). After recovery, cells were incubated for 72 hours in the presence of 0.05 µCi /mL [14C]phenylalanine and either vehicle or 3 nmol/L PMA. PMA treatment resulted in a 40% increase in [14C]phenylalanine incorporation into the proteins of cells permeabilized in the absence of peptides (Fig 10). These levels were reduced by 80% in cells permeabilized in the presence of the PKC pseudosubstrate peptide (Fig 10). In contrast, this inhibition was not observed in cells receiving the scrambled pseudosubstrate peptide. These studies suggest that the PMA-induced increases in [14C]phenylalanine incorporation are mediated by PKC and can be antagonized by introduction of the PKC-inhibitor pseudosubstrate peptide. These results also demonstrate the utility of this protocol for the introduction of bioactive peptides into neonatal cardiac myocytes to study their long-term effects on cell responses.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study from this laboratory by Ron et al,34 a chilled permeabilization buffer was added to cells at room temperature for 10 minutes to introduce PKC inhibitory peptides into neonatal cardiac myocytes. In the study of Ron et al,34 which was carried out while this protocol was being developed, only general evaluations of cell functions were made. For example, contracting cells were observed, but there was no attempt to quantify how many cells contracted stably or for how long. The present study describes a much improved protocol with numerous differences from the study briefly described previously by Ron et al34 and provides extensive data on the effects of the protocol on peptide uptake, cell functions, and viability.
The modified saponin permeabilization protocol described in the present study allows peptide delivery to many cells simultaneously with minimal effects on cellular physiology or viability. Studies of contraction rate, hypertrophy, and mRNA accumulation can be conducted after using this protocol. We have also demonstrated that introduction of a PKC pseudosubstrate peptide by this methodology inhibited phorbol ester–mediated [14C]phenylalanine incorporation into neonatal cardiac myocyte proteins. These data provide additional evidence that neonatal cardiac myocyte hypertrophy is mediated, at least in part, by one or more of the PKC isozymes. This protocol should provide a useful tool for investigating the effects of this and other bioactive peptides when introduced into neonatal cardiac myocytes. Such studies will likely provide new insight into the role of biochemical pathways in the modulation of cardiac myocyte functions.
Rationale for Protocol Design
Previous methods27 of saponin-induced permeabilization (J.A. Johnson, unpublished data, 1996) and microinjection4 of contracting cardiac myocytes can cause cell injury, often resulting in hypercontracture and cell death. Improved methodologies used reversible inhibitors of contraction before microinjection.4 Still, a major limitation of microinjection studies is that only a few cells can be successfully injected, and of those only 30% survive.4 This makes comparisons between cell physiology and biochemistry difficult. The method described here (Fig 1) allows delivery of peptides to many cells (Table) with a higher survival rate than previous protocols4 27 (Fig 8) and hence constitutes an improvement over existing methods. Some key changes from other protocols include a gradual decrease in temperature to stop the myocytes from contracting before permeabilization with saponin. Similarly, after permeabilization, cells were slowly brought back to 37°C and incubated with conditioned media that support cell recovery (J.A. Johnson, unpublished data, 1996). It should be noted, however, that similar success with this protocol has been observed in other cell types, such as human endothelial cells (C.-H. Chen, unpublished data, 1996). Therefore, our method may also contribute to decreased proteolysis and other damaging processes that may occur when cell membrane integrity is transiently compromised.
Another improvement was the inclusion of 6 mmol/L ATP in the permeabilization buffer. It has previously been shown that inclusion of nucleotide triphosphates (and other factors) is required in permeabilized cell systems to study DNA repair processes.1 In assays of contraction rate, recovery of spontaneous, PMA-mediated, and NE-mediated responses after permeabilization on ice were greatly improved in cells permeabilized in the presence of ATP (Table). This is consistent with higher muscle cell demand for ATP than other cell types. In addition, ATP alone has been used previously to permeabilize cells,3 and inclusion of it in our permeabilizations carried out on ice aided in cell permeation (Figs 3 and 4, Table), recovery of contraction rate properties (Table), and cell survival (Fig 8C). Inclusion of ATP in room temperature permeabilizations conveyed no benefit with any of these parameters (Table, Figs 3 and 8). We do not believe residual ATP after permeabilization acts on P2 purinergic receptors, because multiple wash steps are included to reduce its concentration ("Materials and Methods") and because the cells that recover from permeabilization contract at rates nearly identical to those found with nonpermeabilized cells. A final improvement was culturing the cells with ascorbic acid ("Materials and Methods"). In cells cultured without ascorbic acid, lower percentages recovered from saponin-induced permeabilization (data not shown). The mechanism by which ascorbic acid enhances recovery is unknown but may be related to its role as a free radical scavenger.
Evidence for Peptide Uptake in Permeabilized Cells
We demonstrated directly that permeabilization of cells as shown in Fig 1 in the presence of a fluorescently labeled peptide allows fluorescent labeling of 80% of the cells, whereas nonpermeabilized cells show very low cell-associated background levels (Figs 3 and 4, Table). In addition, recent studies in our laboratory that examine regulation of the contraction rate with PKC inhibitory peptides demonstrate effects of the peptides in all tested cells (not shown). Therefore, it is likely that the peptides are introduced into most of the myocytes. To further estimate the uptake of peptides following saponin permeabilization of cells, we determined uptake of a radiolabeled calmodulin by scintillation counting and uptake of a 20-kD fragment derived from PKC by Western blot analysis. We obtained reproducible uptake of the 16.7-kD–protein 125I-labeled calmodulin, but cell-associated background labeling was high. We selected this radiolabeled protein because it is commercially available and its size is similar to other proteins that we are currently introducing into cardiac myocytes to inhibit PKC isozymes35 (studies to be published elsewhere). The intracellular molar concentration of calmodulin obtained after transient permeabilization was 10% of that found outside the cells.
Since our calmodulin studies were associated with elevated background labeling, we sought an additional method to monitor peptide uptake. Our studies with the 20-kD fragment of PKC yielded very low levels of nonspecific background labeling of cells; hence, it was a better method for demonstrating peptide uptake and comparing our method with that of Brik et al.27 We found comparable levels of peptide uptake using either method when permeabilizations were carried out for up to 10 minutes (Fig 4). However, at 30- and 60-minute permeabilization times, the method of Brik et al showed enhanced levels of the 20-kD fragment of PKC (Fig 4). Saponin permeabilization at room temperature has previously been shown to allow proteins of 200 kD to leak out of cells.3 In the present study, we have shown that permeabilization of cells on ice in the presence of ATP allows less uptake than room temperature permeabilization methods (Fig 4). Nevertheless, a substantial amount of a 20-kD peptide can be introduced using our method. Further, room temperature permeabilizations were not useful to us because they impaired the functions (Figs 6 and 8, Table) and decreased the viability (Fig 8) of the cardiac myocytes, making measurements of physiology impossible or severely compromised.
Saponin Permeabilization on Ice in the Presence of ATP Has Minimal Effects on Neonatal Cardiac Myocyte Physiology
After demonstrating uptake of 125I-labeled calmodulin, the 20-kD fragment of PKC (Fig 4) and a fluorescently labeled peptide (Fig 3, Table) into permeabilized cells, we next determined how cardiac myocyte functions were altered by permeabilization using our protocol (Fig 1). We evaluated well-characterized responses, such as spontaneous and modulated contraction rates, early gene expression, and hypertrophy, and found no significant differences between nonpermeabilized cells and cells permeabilized on ice in the presence of ATP (Figs 5 through 7 and 9). In contrast, we found substantial impairments of contraction rate properties when cells were permeabilized at room temperature (Fig 6A, Table). Cells often did not contract at all, showed unstable variable contraction rates, and did not respond to NE. This is of major interest, since at 30- and 60-minute permeabilization times the cells appeared to achieve higher levels of peptide uptake (Fig 4) at room temperature but did not recover sufficiently to be used in our contraction rate assays at permeabilization periods of 
5 minutes (Fig 6A, Table, and data not shown). This limited the utility of room temperature permeabilization protocols for the purpose of introducing peptides into cells and monitoring their effects on cell physiology. Therefore, we believe that our protocol (Fig 1) represents a major advance over previous permeabilization procedures.27
Saponin Permeabilization on Ice in the Presence of ATP Does Not Affect Cell Viability
We found virtually no differences between the long-term viability of cardiac myocytes permeabilized on ice in the presence or absence of ATP and nonpermeabilized cardiac myocytes. Cell functions (Figs 5 through 7 and 9), trypan blue exclusion, protein levels, and cell number were very similar for at least 3 days after permeabilization. In cells permeabilized at room temperature, there was a 30% reduction in the number of cells excluding trypan blue dye when compared with cells permeabilized on ice in the presence of ATP (Fig 8A). There was also a 40% decrease in protein levels (Fig 8B) and cell number (Fig 8C) observed in cells permeabilized at room temperature, which makes the effect on cell viability much greater. Cells permeabilized on ice in the absence of ATP also showed a 20% reduction in cell number, indicating that this condition slightly reduced the number of viable cells (Fig 8C). These results indicate that, of the methods tested, the permeabilization method shown in Fig 1 is best suited for both short-term and long-term studies of cell physiology.
Blockade of Function by a PKC Pseudosubstrate Peptide in Permeabilized Neonatal Cardiac Myocytes
We next used the protocol shown in Fig 1 to determine if a known bioactive peptide, the PKC pseudosubstrate peptide, could antagonize PKC-mediated function in neonatal cardiac myocytes. In the experiments shown in Fig 10, the pseudosubstrate peptide antagonized PMA-induced [14C]phenylalanine incorporation into cardiac myocyte proteins by 80%. The effect was specific, because introduction of a scrambled control peptide had no effect on this response (Fig 10). These results demonstrated that this protocol can be used to introduce peptides into neonatal cardiac myocytes, which contribute bioactivity that can be observed for several days. The potent activity of the pseudosubstrate peptide on this response was surprising, because its effect was measured 3 days after it was introduced. Therefore, the pseudosubstrate peptide may have blocked an early event in a cascade, or possibly, it was stable enough to exert effects throughout the 3 days of the experiment. We do not know the intracellular concentration of pseudosubstrate peptide that is required for this inhibition. If the peptide is required for the duration of the experiment, it seems likely that low concentrations would be required, since it is expected that most of the peptide would be lost because of degradation. Further studies will be required to determine the minimal extracellular concentration of applied peptide that will still result in inhibition. Of interest, we have recently found inhibitory effects of the recombinant 20-kD PKC fragment on short-term PMA-induced translocation and the PMA-mediated slowing of contraction rate35 further demonstrating the utility of this protocol.
Our modified saponin permeabilization protocol allows introduction of peptides into neonatal cardiac myocytes with minimal effects on cell physiology or viability. This methodology should allow the use of bioactive peptides in studies to help dissect the roles of cellular enzymes in complex physiological events such as contraction, gene transcription, cellular hypertrophy, and others. A better understanding of these processes may yield novel drugs or strategies to modify cardiac cell biochemistry in pathological states.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from the National Heart, Lung, and Blood Institute to Dr Mochly-Rosen, a Physician Postdoctoral Fellowship from the Howard Hughes Medical Institute to Dr Gray, and a Stanford University Dean's Fellowship to Dr Johnson. We thank Dr Richard Sportsman from Terrapin Technologies Inc for the gift of the fluorescently labeled peptide.
Received November 13, 1995; accepted August 30, 1996.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 K. Hodges, R. Gill, K. Ramaswamy, P. K. Dudeja, and G. Hecht Rapid activation of Na+/H+ exchange by EPEC is PKC mediated Am J Physiol Gastrointest Liver Physiol, November 1, 2006; 291(5): G959 - G968. [Abstract] [Full Text] [PDF]
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 N. Stahmann, A. Woods, D. Carling, and R. Heller Thrombin Activates AMP-Activated Protein Kinase in Endothelial Cells via a Pathway Involving Ca2+/Calmodulin-Dependent Protein Kinase Kinase {beta}. Mol. Cell. Biol., August 1, 2006; 26(16): 5933 - 5945. [Abstract] [Full Text] [PDF]
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 F. Vincent, N. Duquesnes, C. Christov, T. Damy, J.-L. Samuel, and B. Crozatier Dual level of interactions between calcineurin and PKC-{varepsilon} in cardiomyocyte stretch Cardiovasc Res, July 1, 2006; 71(1): 97 - 107. [Abstract] [Full Text] [PDF]
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S. L. Robia, M. Kang, and J. W. Walker Novel determinant of PKC-{epsilon} anchoring at cardiac Z-lines Am J Physiol Heart Circ Physiol, November 1, 2005; 289(5): H1941 - H1950. [Abstract] [Full Text] [PDF]
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S. Shacham, M. N. Cheifetz, M. Fridkin, A. J. Pawson, R. P. Millar, and Z. Naor Identification of Ser153 in ICL2 of the Gonadotropin-releasing Hormone (GnRH) Receptor as a Phosphorylation-independent Site for Inhibition of Gq Coupling J. Biol. Chem., August 12, 2005; 280(32): 28981 - 28988. [Abstract] [Full Text] [PDF]
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M. C. Souroujon, L. Yao, H. Chen, G. Endemann, H. Khaner, V. Geeraert, D. Schechtman, A. S. Gordon, I. Diamond, and D. Mochly-Rosen State-specific Monoclonal Antibodies Identify an Intermediate State in Epsilon Protein Kinase C Activation J. Biol. Chem., April 23, 2004; 279(17): 17617 - 17624. [Abstract] [Full Text] [PDF]
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 G. Abou-Mohamed, J. A. Johnson, L. Jin, A. B. El-Remessy, K. Do, W. H. Kaesemeyer, R. B. Caldwell, and R. W. Caldwell Roles of Superoxide, Peroxynitrite, and Protein Kinase C in the Development of Tolerance to Nitroglycerin J. Pharmacol. Exp. Ther., January 1, 2004; 308(1): 289 - 299. [Abstract] [Full Text] [PDF]
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 W. Pipkin, J. A. Johnson, T. L. Creazzo, J. Burch, P. Komalavilas, and C. Brophy Localization, Macromolecular Associations, and Function of the Small Heat Shock-Related Protein HSP20 in Rat Heart Circulation, January 28, 2003; 107(3): 469 - 476. [Abstract] [Full Text] [PDF]
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A. De, N. Boyadjieva, and D. K. Sarkar Role of Protein Kinase C in Control of Ethanol-Modulated beta -Endorphin Release from Hypothalamic Neurons in Primary Cultures J. Pharmacol. Exp. Ther., April 1, 2002; 301(1): 119 - 128. [Abstract] [Full Text] [PDF]
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 J. C. Braz, O. F. Bueno, L. J. De Windt, and J. D. Molkentin PKC{alpha} regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2) J. Cell Biol., March 4, 2002; 156(5): 905 - 919. [Abstract] [Full Text] [PDF]
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 H.-D. Je, S. S Gangopadhyay, T. D Ashworth, and K. G Morgan Calponin is required for agonist-induced signal transduction - evidence from an antisense approach in ferret smooth muscle J. Physiol., December 1, 2001; 537(2): 567 - 577. [Abstract] [Full Text] [PDF]
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K. Mackay and D. Mochly-Rosen Arachidonic acid protects neonatal rat cardiac myocytes from ischaemic injury through {epsilon} protein kinase C Cardiovasc Res, April 1, 2001; 50(1): 65 - 74. [Abstract] [Full Text] [PDF]
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L. J. De Windt, H. W. Lim, S. Haq, T. Force, and J. D. Molkentin Calcineurin Promotes Protein Kinase C and c-Jun NH2-terminal Kinase Activation in the Heart. CROSS-TALK BETWEEN CARDIAC HYPERTROPHIC SIGNALING PATHWAYS J. Biol. Chem., April 28, 2000; 275(18): 13571 - 13579. [Abstract] [Full Text] [PDF]
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 G. W. Dorn II, M. C. Souroujon, T. Liron, C.-H. Chen, M. O. Gray, H. Z. Zhou, M. Csukai, G. Wu, J. N. Lorenz, and D. Mochly-Rosen Sustained in vivo cardiac protection by a rationally designed peptide that causes varepsilon protein kinase C translocation PNAS, October 26, 1999; 96(22): 12798 - 12803. [Abstract] [Full Text] [PDF]
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 R. A. Hunt, G. J. Bhat, and K. M. Baker Angiotensin II-Stimulated Induction of sis-Inducing Factor Is Mediated by Pertussis Toxin-Insensitive Gq Proteins in Cardiac Myocytes Hypertension, October 1, 1999; 34(4): 603 - 608. [Abstract] [Full Text] [PDF]
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K. Mackay and D. Mochly-Rosen An Inhibitor of p38 Mitogen-activated Protein Kinase Protects Neonatal Cardiac Myocytes from Ischemia J. Biol. Chem., March 5, 1999; 274(10): 6272 - 6279. [Abstract] [Full Text] [PDF]
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O. Feron, C. Dessy, D. J. Opel, M. A. Arstall, R. A. Kelly, and T. Michel Modulation of the Endothelial Nitric-oxide Synthase-Caveolin Interaction in Cardiac Myocytes. IMPLICATIONS FOR THE AUTONOMIC REGULATION OF HEART RATE J. Biol. Chem., November 13, 1998; 273(46): 30249 - 30254. [Abstract] [Full Text] [PDF]
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B. Z. Simkhovich, K. Przyklenk, and R. A. Kloner Role of protein kinase C as a cellular mediator of ischemic preconditioning: a critical review Cardiovasc Res, October 1, 1998; 40(1): 9 - 22. [Abstract] [Full Text] [PDF]
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J. Zhao, O. Renner, L. Wightman, P. H. Sugden, L. Stewart, A. D. Miller, D. S. Latchman, and M. S. Marber The Expression of Constitutively Active Isotypes of Protein Kinase C to Investigate Preconditioning J. Biol. Chem., September 4, 1998; 273(36): 23072 - 23079. [Abstract] [Full Text] [PDF]
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M. O. Gray, J. S. Karliner, and D. Mochly-Rosen A Selective epsilon -Protein Kinase C Antagonist Inhibits Protection of Cardiac Myocytes from Hypoxia-induced Cell Death J. Biol. Chem., December 5, 1997; 272(49): 30945 - 30951. [Abstract] [Full Text] [PDF]
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V. Litvak, D. Tian, Y. D. Shaul, and S. Lev Targeting of PYK2 to Focal Adhesions as a Cellular Mechanism for Convergence between Integrins and G Protein-coupled Receptor Signaling Cascades J. Biol. Chem., October 13, 2000; 275(42): 32736 - 32746. [Abstract] [Full Text] [PDF]
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E. G. Stebbins and D. Mochly-Rosen Binding Specificity for RACK1 Resides in the V5 Region of beta II Protein Kinase C J. Biol. Chem., August 3, 2001; 276(32): 29644 - 29650. [Abstract] [Full Text] [PDF]
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J. C. Braz, O. F. Bueno, L. J. De Windt, and J. D. Molkentin PKC{alpha} regulates the hypertrophic growth of cardiomyocytes through extracellular signal-regulated kinase1/2 (ERK1/2) J. Cell Biol., March 4, 2002; 156(5): 905 - 919. [Abstract] [Full Text] [PDF]
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 R. Ginnan and H. A. Singer CaM kinase II-dependent activation of tyrosine kinases and ERK1/2 in vascular smooth muscle Am J Physiol Cell Physiol, April 1, 2002; 282(4): C754 - C761. [Abstract] [Full Text] [PDF]
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 K. M. Ridge, L. Dada, E. Lecuona, A. M. Bertorello, A. I. Katz, D. Mochly-Rosen, and J. I. Sznajder Dopamine-induced Exocytosis of Na,K-ATPase Is Dependent on Activation of Protein Kinase C-epsilon and -delta Mol. Biol. Cell, April 1, 2002; 13(4): 1381 - 1389. [Abstract] [Full Text] [PDF]
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