© 1997 American Heart Association, Inc.
Articles |
Involvement of Phosphorylation in Doxorubicin-Mediated Myofibril Degeneration
An Immunofluorescence Microscopy Analysis
the Department of Biochemistry and Molecular Biology and the Institute for Genetic Medicine (M.A.S., S.W., L.K.), University of Southern California School of Medicine, Los Angeles, and the Department of Pharmaceutical Sciences (S.F.H.-A., P.M.V.), University of Southern California School of Pharmacy, Los Angeles.
Correspondence to Dr Larry Kedes, Department of Biochemistry and Molecular Biology and the Institute for Genetic Medicine, University of Southern California School of Medicine, Hoffman Medical Research Bldg #413, 2011 Zonal Ave, Los Angeles, CA 90033. E-mail kedes@zygote.hsc.usc.edu
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Abstract |
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Loss of myofilaments has been observed in both adaptive cardiac responses (ie, hypertrophy) as well as in chemotheraputic use of antineoplastic drugs with cardiotoxic side effects (ie, doxorubicin). An understanding of the degenerative process is a prerequisite for determining approaches to limit the cardiomyopathic changes associated with chronic heart disease or long-term chemotheraputic treatments. However, little is known about the specific events and molecular changes that initiate the degenerative process. To study this process, neonatal rat cardiomyocytes were treated with doxorubicin, which induced rapid and widespread thin-filament degeneration as observed by fluorescence confocal microscopy. which demonstrated deterioration of sarcomeric thin-filament structure. Changes in the spontaneous beating of cardiomyocytes corresponding with myofibrillar degeneration were apparent using differential interference contrast video microscopy. After finding induction of kinase activity by doxorubicin in cultured cardiomyocytes, the protective effects of specific inhibitors of kinase activity were assessed for their ability to inhibit doxorubicin-induced myofibrillar breakdown. Doxorubicin-induced changes appeared similar to the degeneration observed after treatment with a protein kinase activator (phorbol 12-myristate 13-acetate) or a serine-threonine protein phosphatase inhibitor (okadaic acid). Collectively, these results indicate that activation of protein kinase is an important event in the initiation of myofibrillar degeneration by doxorubicin. Further analyses of myofibrillar proteins with respect to biochemical modifications will be necessary to determine if phosphorylation events transmit signal(s) to initiate degeneration.
Key Words: myofibril • cardiomyocyte • doxorubicin • phosphorylation
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Introduction |
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Myofibrillar degeneration is a well-documented characteristic of the cardiomyopathy1 2 and cardiotoxicity associated with various therapeutic drugs.3 4 5 The study of isolated cardiomyocytes in vitro has proved useful for understanding the intracellular changes induced during the degenerative process,6 7 8 but the specific molecular changes in myofibrillar structure that trigger degeneration are unknown.
A variety of hypotheses have been forwarded to account for myofibrillar degeneration induced by the anthracycline drug doxorubicin (reviewed in References 9 through 11). We have recently proposed12 that the effects of doxorubicin are mediated, at least in part, by the induction of a protein kinase pathway that activates the transcription of negative regulators (Id) of tissue-specific transcription factors (E-box binding factors). The transcription repression could be prevented by the overexpression of transcription factor activators (E 2.5). Inhibitors of protein kinase activity blocked the effects of doxorubicin on Id gene induction.13 In the present study, we address the issue of whether protein kinase pathways are also involved in the morphological events that follow exposure of cardiomyocytes to doxorubicin and whether inhibiting these pathways would prevent such events.
Although doxorubicin acts on a variety of cellular processes, only some of these pathways could lead to the myofibrillar degeneration observed within a day of in vitro treatment. The degenerative effect of doxorubicin on myofibrillar structure has been examined by both conventional light and electron microscopy.3 4 8 14 These studies document a variety of effects on both myofibrillar structure and intracellular organelles. With regard to myofibrillar structure, thin-filament complexes are likely to be involved in the early structural changes that accompany degenerative processes. The dynamic nature of actin filament polymerization and breakdown forms the basis for many critical cellular functions,15 and within muscle cells, actin monomers exist in equilibrium with the polymerized thin filaments.16 In addition to rapid incorporation of actin into thin filaments, the structure of thin filaments is particularly sensitive to treatments that lead to myofibril degeneration.17 Since the structural integrity of thin filaments is an excellent marker for myofibrillar integrity, we chose to examine thin-filament organization by fluorescence microscopy to monitor degeneration of both actin filaments and 
-actinin, which cross-links actin together at the Z disk.18 Our findings support the conclusion that thin-filament integrity is highly susceptible to breakdown in the presence of doxorubicin.
Since activation of protein kinase is a common property of both phorbol esters and doxorubicin, the process of myofibrillar degeneration induced by either agent could be mediated via phosphorylation-dependent mechanisms. If this hypothesis is correct, inhibition of kinase activity in cardiomyocytes after treatment with doxorubicin might afford a protective effect from the initiation of myofilament degeneration. Our results indicate that thin-filament organization is preserved by inhibition of kinase activity in doxorubicin-treated cardiomyocytes. Furthermore, experiments performed using a fluorescently tagged inhibitor of kinase activity indicate that doxorubicin-induced activation of kinase occurs to a greater degree in cardiomyocytes than in nonmyocytes within the same heart cell preparation. We conclude that kinase activation in cardiomyocytes is an important step in the intracellular signaling pathway leading to myofilament degeneration.
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Materials and Methods |
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Isolation and Culture of Cardiomyocytes
Rat cardiomyocytes were isolated from 2- to 3-day-old pups as previously described.19 Cells were plated onto glass coverslips coated with laminin (Sigma Chemical Co; catalog No. L 2020, diluted in PBS and used at a concentration of 50 µg/mL) and cultured for 5 to 7 days in MEM medium without L-glutamine containing 2% fetal bovine serum and 1% penicillin/streptomycin (GIBCO/BRL Laboratories) before chemical treatment. Cardiomyocytes used in experiments requiring serum-free medium conditions were changed from medium with serum to DMEM/F12 medium containing GMS-X medium supplement and 1% penicillin/streptomycin (all from GIBCO/BRL)
48 hours before use.
Kinase Activity
Assays were performed on Triton X-100 lysates from identically prepared confluent cardiac cell cultures (10-cm dish per experimental treatment) or cardiac tissue (200 mg of minced hearts per experimental treatment). Treated samples were exposed to 4 µmol/L doxorubicin for 30 minutes before use in assays. Extracts were enriched for PKC activity by Fast Protein Liquid Chromatography (FPLC) ion-exchange using a linear 20 to 300 mmol/L NaCl gradient passed through a Mono Q column. Fractions containing the majority of kinase protein as determined by immunoblot analysis were pooled and concentrated by centrifugation (Microcon 30, Amicon Inc). The concentrated lysate was tested for kinase activity using the PKC assay system (Promega) as directed by the manufacturer, but sheet format streptavidin disks with reduced background labeling (provided by the manufacturer) were used instead of the disks normally included with the kit. Activation of kinase was determined relative to assay background (performed with buffer only) and maximal activity (performed by activating the lysate with 300 µg/mL phosphatidyl serine, 30 µg/mL diacylglycerol, and 0.4 mmol/L CaCl2). Percent kinase activation was calculated as follows: (cpm of lysate in assay buffer-cpm of buffers alone)/(cpm of lysate in the presence of activating solution-cpm of buffers alone).
Drug and Chemical Treatment of Cardiomyocyte Cultures
Cardiomyocyte cultures were treated with PMA, doxorubicin (both from Sigma), or okadaic acid (potassium salt, LC Laboratories). PMA or okadaic acid was used at varying doses as indicated, and doxorubicin was used at a final concentration of 2.0 µmol/L. Unless otherwise noted, cardiomyocytes were treated with PMA or doxorubicin for 24 hours. Okadaic acid treatments were performed as noted in figure legends. Three synthetic isoquinolinesulfonamide derivatives (HA-1004, H-8, and H-7; all from Seikagaku America, Inc), which show selective inhibitory effects upon various kinases by direct binding to the kinase molecule, were chosen.20 These three agents were chosen for their varying specificities: a weak inhibitor useful as a control (HA-1004), a selective but powerful inhibitor of cyclic nucleotide–dependent protein kinase (H-8), and a powerful inhibitor of both cyclic nucleotide–dependent protein kinase and PKC (H-7). Inhibitors were added to cultures immediately before treatment with doxorubicin at a final concentration of 10 µg/mL.
Microscopic Analyses
Cells were prepared for fluorescence microscopy using standard procedures as previously described.21 FITC- or TRITC-conjugated phalloidin (Sigma) was used at a concentration of up to 5 µg/mL, and FIM (Kamiya Biochemical Co) was used at a concentration of 200 nmol/L. This fluorescently tagged bisindolylmaleimide inhibitor retains the selective specificity of the parental compound to inhibit PKC activity by binding to the ATP-catalytic domain.20 The target domain specificity for active catalytic site enables FIM to be used as a valuable tool for observing kinase activation.22 Monoclonal -actinin antibody (Sigma catalog No. A 7811) and anti-PKC
antibody (GIBCO/BRL catalog No. 13198-015) were both used at 1:25 dilution. Secondary fluorescein-conjugated anti-mouse IgG (Boehringer-Mannheim Biochemicals) or Texas red–conjugated anti-rabbit IgG (Jackson Immunoresearch) was used at 1:50 dilution.
Labeled cardiomyocytes were mounted in Vectashield anti-bleaching medium (Vector Laboratories) or ProLong Antifade (Molecular Probes). Photomicrographs of okadaic acid–treated cardiomyocytes were taken using an MC-100 spot camera attached to the port of a Zeiss Axioskop microscope equipped with a x63 and x100/1.3-NA objective and filters for rhodamine and fluorescein fluorescence. Photographs of FIM-labeled and anti-PKC–labeled cells were taken using a Molecular Dynamics CLSM 2010 confocal microscope with image analysis by Imagespace software and printed using a Codonics dye sublimation printer. All other photomicrographs were taken using a Zeiss LSM-1 laser scanning confocal microscope (operated by the Doheny Eye Institute; Dr Janet Blanks, director). Plan Neofluar x40 (NA, 0.75) and x63 (NA, 1.40) oil immersion objectives were used for imaging of fluorescently labeled cells. Image analysis was performed using the standard system operating software provided with the Zeiss LSM microscope (version 2.08). Photomicrographs were taken using a Sony printer connected to the video output of the microscope.
Video-enhanced DIC microscopy was performed as described by Schnapp23 using a Zeiss Axiovert 135 microscope with fiberoptic illumination and a x100/1.3-NA objective attached to a DAGE VE-1000 camera with a Newvicon tube attached to an Argus 10 image processor. Data were recorded on S-VHS cassettes. A temperature-controlled stage maintained the sample at 37°C. Cells were held in a perfusion chamber, which allows for exchange of media and addition of exogenous chemicals and/or drugs. Cardiomyocytes could maintain active beating under observation in this system for over 12 hours (data not shown). The ability to watch myofibrillar degeneration as it occurs is a powerful new approach, since structural deterioration of myofibrils was clearly visible in DIC microscopy images. These observations confirm that treated cardiomyocytes used in the present study were viable and metabolically active.
Spontaneous Beating Analyses
Cardiomyocytes on six-well dishes were monitored until synchronous beating developed, typically within 10 to 12 days after preparation. Doxorubicin from a stock solution in DMEM was diluted into PBS and added to the wells to a final concentration of 2 µmol/L in three of the wells; equivalent volumes of PBS were added to the control wells. After culture for 18 to 24 hours (37°C in 10% CO2), cardiac cell beating was counted per individual fields at 25°C using a Nikon inverted microscope equipped with phase optics at 200-fold magnification. Five to six fields per well were counted, with a total of three control and three doxorubicin-treated wells per experiment. Thus, the total n values per experiment varied from 15 to 18 fields per treatment. In order to control for environmental changes that could affect results within the time taken for the assay (ie, gradual cooling, pH changes), single fields were observed in consecutive wells before cycling through for the five or six fields per well.
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Results |
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Activation of PKC by Doxorubicin
PKC activity was assessed in order to determine whether doxorubicin treatment activated kinase in cardiomyocyte cultures (Table
). Doxorubicin treatment resulted in PKC activation in all of our tests, despite variations in the level of endogenous activity. The fold increase of PKC activity following doxorubicin treatment grew larger with prolonged time in culture (from 2.5- to 12.5-fold). This was presumably due in part to decreasing levels of endogenous PKC activity in untreated controls (from 23.4% to 0.6%), which allowed for detection of doxorubicin-mediated activation. PKC activity increased 3.4-fold after exposure to doxorubicin in 5-day-old cultures, corresponding to the length of culture before use in microscopic analyses. Five-day-old cultures showed comparable levels of PKC activity after treatment with either doxorubicin (30.5%) or PMA (33.1%). Heart tissue treated immediately after harvesting from adult animals gave the highest response (56.5-fold increase), whereas neonatal rat heart lysates assayed directly after harvest without culture showed high endogenous PKC activity (data not shown). High endogenous PKC levels were also present in short-term (2-day) cardiomyocyte cultures, suggesting that elevated PKC levels are present in developing cardiomyocytes.
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Kinase Activation in Mixed Heart Cell Cultures Detected by Fluorescence Analysis After Doxorubicin Treatment
Kinase activation following 30 minutes of exposure to either 2.0 µmol/L doxorubicin or 200 nmol/L PMA was monitored using anti-PKC (the major PKC isoform in the heart) antibody and FIM (a fluorescently tagged kinase inhibitor that selectively binds to activated protein kinase). Untreated control cardiomyocytes show levels of PKC labeling comparable to doxorubicin- or PMA-treated cultures (Fig 1, upper panel, top row). In contrast, the FIM reactivity of untreated cardiomyocytes was low (Fig 1, upper panel, bottom row). Neighboring noncardiomyocytes showed weak labeling compared with the intensity of fluorescence of doxorubicin-treated cardiomyocytes. These results demonstrate that FIM is a useful reagent for detection of activated kinase in cardiomyocytes.
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Recent studies have shown that PKC associates with actin filaments in neonatal rat cardiomyocytes after activation by PMA.24 Untreated control cardiomyocytes show anti-PKC reactivity in peripheral regions of the cell body (Fig 1A
, arrowheads) and discrete clusters of FIM label (Fig 1A, arrows). Kinase activation as evidenced by increased FIM reactivity is apparent in cardiomyocytes after doxorubicin treatment (Fig 1B), when both anti-PKC (Fig 1C) and FIM (Fig 1D) labeling became more intense and widespread throughout the cell. Areas of colocalization were present where intensity of both labels was increased (Fig 1B through 1D, as indicated). The appearance of periodicity in anti-PKC labeling (Fig 1C, as previously observed in Reference 24) prompted us to closely examine FIM staining in relation to Z-disk sarcomeric structure. Sarcomeric actin filament organization concentrated around the Z disk region was revealed by phalloidin label in these cardiomyocytes, and punctate FIM staining with occasional short stretches of periodicity was seen (Fig 1E through 1G). FIM staining was generally not associated with (but rather between) adjacent Z disks, suggesting localization in the region where thin filaments were concentrated (apparent in Fig 1F and 1G, cell on right). Variations in FIM staining patterns included filamentous labeling parallel to myofibrils (Fig 1E, at arrows) and increased perinuclear labeling (Fig 1G, cell on left). Compared with doxorubicin-treated cells, PMA-treated cardiomyocytes showed intense FIM labeling, particularly around (but not restricted to) the perinuclear region (Fig 1H). These experiments indicate that doxorubicin causes a redistribution of kinase and that activated kinase appears coincident with the distribution of PKC.
Effect of Doxorubicin Treatment on Cardiomyocyte Myofibrillar Structure
Neonatal rat cardiomyocytes were treated with 2.0 µmol/L doxorubicin, and the resulting effect on myofibrils was assessed by immunofluorescence microscopy. Fig 2A shows normal cardiomyocytes with well-organized parallel myofibrils that run the length of the cell. In contrast, cardiomyocytes exposed to doxorubicin for 24 hours show profound myofibrillar disorganization and loss of structural integrity (Fig 2B). Higher magnification reveals smearing of -actinin at Z disks and open cytoplasmic regions devoid of myofibrillar structures (Fig 2C). The loss of myofibrils is progressive, and after 96 hours of doxorubicin treatment, virtually all contractile protein organization is gone (Fig 2D). The degenerative effect induced by doxorubicin on myofibrillar structure is not always comparable between individual cells, probably because of cellular variations in the degree of myofibrillar organization in these developing cardiomyocytes. Cardiomyocytes with more developed myofibrillar architecture (Fig 2E and 2G, at large arrows) may take longer to show deterioration than those that were comparatively underdeveloped (Fig 2E and 2G, at arrowheads). Since regions of intact myofibrillar organization could still be found within cardiomyocytes that show extensive structural deterioration (Fig 2E, small arrows), myofibrillar degeneration may proceed along the length of the myofibril rather than occur simultaneously throughout the cell. Cardiomyocytes undergoing degeneration have lost actin filament structure (Fig 2F and 2H), although stress fiber organization in fibroblast cells appears relatively normal (Fig 2G, small arrows). Again, the variation in myofibrillar degeneration between cells appears dependent in part on the extent of cardiomyocyte differentiation (ie, intracellular myofibrillar density) and overall cell density in the culture. These results demonstrate the rapid and dramatic effect of doxorubicin on myofibrillar structure.
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Spontaneous Beating Is Compromised in Doxorubicin-Treated Cardiomyocytes
Myofibrillar degeneration induced by doxorubicin treatment was also observed in living cardiomyocytes in real time using video-enhanced DIC microscopy. The parallel aligned myofibrils that are characteristic of differentiated cardiomyocytes (Fig 3A, at arrows) have well-defined repeating phase-dense regions that correspond to myosin (thick) filament periodicity (Fig 3B, at arrows). After 24 hours of exposure to doxorubicin, myofibrillar structure can still be found (Fig 3C, at arrowheads), but structural changes are clearly evident. Pathological deterioration includes cytoplasmic areas devoid of myofibrils (Fig 3C, at arrows) and regions of myofibrillar disorganization (Fig 3D, at arrow). These degenerative changes are comparable to observations of fixed cells by immunofluorescence microscopy (Fig 2) and indicate that DIC video microscopy can be used to correlate loss of myofibrillar organization and functional changes within living cardiomyocytes.
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To analyze the effects of doxorubicin on cardiomyocyte beating, it was necessary to allow the cells to differentiate in the culture dishes for 10 to 12 days until robust synchronous beating was observed in each field. Multiple fields in each preparation were observed as described (see "Materials and Methods"). Six different cardiomyocyte preparations were analyzed after exposure to 2 µmol/L doxorubicin for 18 to 24 hours. Four of the six doxorubicin-treated cardiomyocyte preparations showed average beating rates that increased from 9.27±1.96 bpm (n=58 fields analyzed from four different preparations) to 42.7±6.44 bpm (n=63 fields analyzed from four different preparations). In conjunction with the enhanced beating, the doxorubicin-treated cultures began to beat asynchronously. Bursts of very rapid beating followed by a brief intermittent cessation of beating were observed in both control and doxorubicin-treated cardiomyocytes. However, although most control cardiomyocytes in each field were beating synchronously, the doxorubicin-treated cultures frequently had individual cells beating at a different rate or out of synchrony with the majority of the population. The burst of faster beating noted 24 hours after treatment was followed by complete cessation of beating of cardiomyocytes treated with doxorubicin for over 48 hours, which paralleled observed loss of myofibril organization. In the remaining two preparations we analyzed, infrequent and sporadic or no observable beating was present after 24 hours of doxorubicin treatment, possibly because of accelerated degradation of myofibrils in these particular cultures.
Partial Prevention of Doxorubicin-Induced Myofibrillar Damage by Addition of Kinase Inhibitors
Neonatal rat cardiomyocytes were treated with 2.0 µmol/L doxorubicin and various inhibitors of protein kinase activity in an attempt to protect myofibrils from the degenerative process. The inhibitors used included three isoquinolinesulfonamide derivatives: HA-1004, H-7, and H-8 (selective inhibitors for PKC-dependent or cyclic nucleotide–dependent protein kinases; all were used at a final concentration of 10 µmol/L). Controls for this experiment include normal cardiomyocytes (Fig 4A) with typical well-organized myofibrillar structure and doxorubicin-treated cells (Fig 4B) showing evidence of myofibrillar degeneration. Myofibrils in cardiomyocytes exposed to a combination of doxorubicin and HA-1004 do not appear significantly better organized than those in the doxorubicin-treated control cardiomyocytes (Fig 4C and 4D). In contrast, the myofibrillar organization of cardiomyocytes treated with doxorubicin in the presence of H-8 (Fig 4E and 4F) or H-7 (Fig 4G and 4H) appears substantially improved over the condition of cells cultured in doxorubicin alone. The myofilament architecture in these H-7– or H-8–treated cells does show subtle signs of myofilament deterioration, suggesting that there may be additional doxorubicin-mediated effects that have an impact on myofilament organization. Control experiments performed to assess the effect of each inhibitory agent alone showed negligible structural differences compared with untreated cells (data not shown). These results indicate that doxorubicin-induced activation of kinase is an important step in myofibrillar degeneration and that inhibition of kinase activity provides partial, but substantial, protection of myofibrillar structure.
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Effect of PMA on Myofibrillar Structure
Neonatal rat cardiomyocytes were treated with 100, 200, or 300 nmol/L PMA, and the resulting effect on myofibrils was assessed by fluorescence confocal microscopy. The normal sarcomeric organization of thin filaments in cardiomyocytes (Fig 5A and 5B) is profoundly disrupted by exposure of the cells to PMA for 24 hours. The smearing of Z disks (Fig 5D) and loss of myofibrillar organization (Fig 5E and 5F) are classic hallmarks of myofilament degeneration similar to observations of doxorubicin-induced changes to myofilament structure.3 4 8 14
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Effect of Okadaic Acid on Myofibrillar Structure
The similarity of the PMA response to the effects elicited by doxorubicin suggested that kinase activation of PKC might be a possible explanation of the effects of doxorubicin. Since protein kinases act in opposition to protein phosphatases, we wanted to see if we could generate a similar response to kinase activation by serine-threonine protein phosphatase inhibition. We used okadaic acid in this context, since it had previously been used in a study that implicated protein phosphatase 2A in regulation of PKC-mediated effects in cardiac tissue.26 Neonatal rat cardiomyocytes were treated with 50 or 500 nmol/L okadaic acid, and the resulting effect on myofibrils was assessed by fluorescence microscopy. The normal sarcomeric organization of thin filaments in cardiomyocytes (Fig 6A) is profoundly disrupted by exposure of the cells to 500 nmol/L okadaic acid for 3 hours (Fig 6B). More pronounced effects are evident after overnight treatment with a 10-fold lower dose (50 nmol/L, Fig 6C). The observed myofibrillar degeneration is similar to that observed in PMA-treated cardiomyocytes (see Fig 5).
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Discussion |
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The present study uses fluorescence and video-enhanced DIC microscopy to reveal structural changes in cardiomyocyte thin-filament organization elicited by exposure to the chemotheraputic agent doxorubicin. Coincubation of cardiomyocytes with the protein kinase inhibitor H-8 partially blocked the degenerative effects on thin-filament structure elicited by doxorubicin, suggesting that the effects of doxorubicin were mediated by protein kinase activity. The present study has compared the effects of doxorubicin treatment with treatment with two other agents that also increase protein phosphorylation, either via increased kinase activity (PMA) or decreased phosphatase activity (okadaic acid). All three agents elicited similar degenerative effects on myofilament structure. Phorbol ester–induced effects on cardiac cells include intracellular kinase activation27 and redistribution of PKC.28 Phorbol ester exposure also caused the disassembly of myofibrils in cultured muscle cells29 30 and the phosphorylation of troponins I and T.31 Phosphorylation of troponin I was also increased after okadaic acid treatment, which exerted a positive inotropic effect on cardiac preparations.32 Membrane and cytosolic preparations of ventricular tissue contain okadaic acid–sensitive phosphatase activity, suggesting that phosphatase 2A may be important in the regulation of phosphorylation of PKC target proteins.26 It is tempting to speculate on the necessity of phosphatase activity for maintenance of myofibril structure, since a requirement for phosphatase has been linked to stability of cytoskeletal structure in nonmitotic cells.33 Thus, the changes in myofibril organization and function observed in the present study are circumstantially correlated with increased protein phosphorylation.
Doxorubicin differs significantly from PMA with respect to inotropic effect: the rate of beating in doxorubicin cultures was increased in four of six separate cardiomyocyte preparations during the course of our assay. The increased frequency of beating after doxorubicin administration is consistent with previous reports of increased heart rate in mice after a series of intraperitoneal doxorubicin injections.34 Since doxorubicin acts as a positive inotropic agent in the present study, it is unlikely that the degenerative changes we observed were due to negative inotropic effects.
Summarizing the effects of treatments performed in the present study (Fig 7), the shared property of increased phosphorylation level suggests a link between kinase induction and myofibrillar degeneration. The complexity of a signal transduction mechanism, such as phosphorylation, complicates a reductionist approach to identifying a specific cause(s) leading to myofibril degeneration in our experiments. Clearly, heart development and function depend on regulated expression levels of kinase in developing heart muscle and differential subcellular localization of isoforms.35 36 37 38 Cardiomyocytes also show PKC isoform shifts in response to in vitro culture conditions.39 40 After exposure to PMA, the expression and localization of PKC in neonatal rat cardiomyocytes is affected, leading to redistribution24 41 as well as downregulation of various PKC isoforms within 1 hour. After treatment, the isoform colocalized with thin filaments, whereas the and ß isoforms were downregulated.35 41 Association of PKC (the predominant PKC isoform in the heart27 ) with thin filaments was proposed as a possible mediator of the negative inotropic effect after PMA exposure.42 This correlation between PKC and thin filaments prompted our comparison, which found that the labeling pattern of PKC was coincident with FIM fluorescence (Fig 2). Interestingly, PKC possesses a cryptic actin-binding motif that may become exposed after kinase activation.43 Sarcomeric proteins that could act as substrates for PKC phosphorylation include troponins I and C, C-protein, and myosin light chain 2.44 45 46 47 Based on these collective observations, it is tempting to speculate on the possible detrimental long-term effects of e isoform localization along myofibrils in the present study. However, PKC-activating stimuli like doxorubicin induce multiple effects (discussed below), which complicate any straightforward cause-and-effect conclusions.
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Myofibril protein phosphorylation mediated by various kinases has been correlated with changes in myocardial or cardiomyocyte function.48 49 Both troponin I and C-protein can be phosphorylated by either PKC or PKA to a similar extent. However, the sites of phosphorylation on troponin I are distinct between PKC and PKA, resulting in different functional consequences for Ca2+ sensitivity and Mg2+-ATPase activity of the myofibril.46 Thus, site-specific kinase activity is an important determinant in the effect of phosphorylation and in target protein specificity.
In the present study, the data indicate that myofibrillar degeneration induced by doxorubicin treatment can be restrained by some classes of kinase inhibitors (Fig 4). Through the use of these agents, we have demonstrated that specific activation of protein kinase is a required step in the induction of myofibrillar degeneration. These alleged "protective" treatments do not appear to alter myofibrillar structure in a 24-hour assay (data not shown). Since the concentrations of inhibitory agents used in the present study are commonly used to study cellular metabolic processes in vitro, it is likely that these doses were appropriate and nontoxic. Induction of kinase activity by doxorubicin was clearly visualized in cardiomyocytes labeled with FIM. Interestingly, our observations of cardiomyocyte cultures labeled with FIM demonstrated that nonmyocytes did not show significant increases in kinase activity after doxorubicin treatment. It is reasonable to speculate that selective cardiotoxic effects of doxorubicin observed in clinical therapy are related, at least in part, to kinase induction in cardiomyocytes. Comparable findings that correlate propensity for kinase activation by doxorubicin with cytotoxic effects on tumor cells have been reported.50
The multiple effects of doxorubicin on cellular processes have confounded the search for a specific pathway(s) that leads to cardiotoxicity. Doxorubicin cardiotoxicity has been attributed, in part, to various effects on gene transcription,8 11 thin-filament proteins,51 52 intracellular Ca2+ release,53 and altered sarcoplasmic reticulum function.54 Doxorubicin also activates PKC in cardiomyocytes (the present study) and other cell types.55 56 Previously, we demonstrated that doxorubicin treatment rapidly leads to the selective inhibition of transcription of muscle-specific genes, including those that encode the thin-filament proteins actin, tropomyosin, and troponin.12 These events are associated with the induced expression of Id, a negative regulator of tissue-specific E-box transcription factors. Thus, myofibril degeneration may be exacerbated by the induction of Id. More recently, doxorubicin has been found to induce a protein kinase pathway that activates transcription of Id.13 Kinase activity involved in regulation of Id gene expression appears distinct from those involved in PKC activation.13 Although the studies described in the present report do not focus on the precise mechanism of doxorubicin action, the similarity of myofibrillar degeneration induced by doxorubicin, PMA, and okadaic acid suggests that kinase activity be targeted for further investigations. The present report supports the idea of a balanced interplay between both kinase and phosphatase activity in the maintenance of myofilament structure.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by Initial Investigator and Grant-in-Aid awards to Drs Sussman and Hamm-Alvarez from the American Heart Association, Greater Los Angeles Affiliate, by a Grant-in-Aid award to Dr Sussman from the American Heart Association, Ohio Affiliate, and by a grant from the National Institutes of Health to Dr Kedes. We are grateful to Masahiko Kurabayashi for helpful discussions, and we thank Silvia da Costa for excellent technical assistance.
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Footnotes |
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Mark A. Sussman, PhD, is currently from The Children's Hospital and Research Foundation, Division of Molecular Cardiovascular Biology, Room 3033, Burnet Avenue, Cincinnati, OH 45229.
Received April 18, 1996; accepted October 15, 1996.
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References |
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