Clinical and Experimental Pharmacology and Physiology (2010) 37, 460–465
doi: 10.1111/j.1440-1681.2009.05323.x
Cisplatin compromises myocardial contractile function and mitochondrial ultrastructure: Role of endoplasmic reticulum stress Heng Ma,*† Kyla R Jones,* Rui Guo,* Peisheng Xu,* Youqing Shen‡ and Jun Ren*† *Division of Pharmaceutical Science, Center for Cardiovascular Research and Alternative Medicine, ‡Department of Chemical and Petroleum Engineering, University of Wyoming, Laramie, WY, USA and †Department of Physiology, Fourth Military Medical University, Xi’an, China
SUMMARY
INTRODUCTION
1. Cisplatin is a potent chemotherapeutic agent with broadspectrum antineoplastic activity against various types of tumours. However, a major factor limiting treatment with cisplatin is its acute and cumulative cardiotoxicity. The aim of the present study was to explore the effect of cisplatin on myocardial contractile function and the possible underlying cellular mechanisms. 2. C57 mice were treated with cisplatin (10 mg ⁄ kg per day, i.v.) or vehicle (0.9% NaCl) for 1 week and myocardial function was assessed using the Langendorff and cardiomyocyte edgedetection systems. Transmission electron microscopy, mitochondrial membrane potential, indices of endoplasmic reticulum (ER) stress and caspase 3 activity were evaluated. 3. Cisplatin-treated mice developed myocardial contractile dysfunction, as evidenced by a reduction in left ventricular developed pressure (LVDP) and the first derivative of LVDP (+ ⁄ )dP ⁄ dt). Cisplatin treatment significantly prolonged time to 90% relengthening, depressed peak shortening, maximal velocity of shortening ⁄ relengthening (+ ⁄ )dL ⁄ dt) and augmented the frequency-elicited depression in peak shortening. The JC-1 fluorescent assay demonstrated that cispatin-induced cardiac dysfunction was associated with mitochondrial membrane depolarization. Transmission electron microscopy revealed that cisplatin induces ultrastructural abnormalities of the mitochondria. Following cisplatin treatment, cardiomyocytes show activation of the ER stress response, increased caspase 3 activity and increased terminal deoxyribonucleotidyl transferase-mediated dUTP– digoxigenin nick end-labelling (TUNEL) staining. 4. The data indicate that cisplatin is cardiotoxic and may lead to left ventricular dysfunction and depressed cardiomyocyte contraction associated with mitochondrial abnormalities, enhanced ER stress and apoptosis. This work should shed some light on the management of cisplatin-induced cardiac injury. Key words: cardiotoxicity, cisplatin, endoplasmic reticulum stress, mitochondrial abnormalities.
Cisplatin is a potent chemotherapeutic agent with broad-spectrum antineoplastic activity against various types of tumours.1 Cisplatin treatment results in remissions of both adult and paediatric malignancies, including of the lung, head and neck,2 and especially those of the genito-urinary system.3,4 Several clinical trials, as well as in vitro models, have demonstrated a steep dose–response relationship for cisplatin in a variety of tumour types, but numerous toxic side-effects have been reported, limiting its use. Some of the reported side-effects include bilateral high-frequency hearing loss5 and nephrotoxicity.6 A major factor limiting cisplatin treatment is concern regarding its acute and cumulative cardiotoxicity.7,8 Clinical reports regarding acute cisplatin treatment have identified myocardial infarction and severe ventricular arrhythmias in young patients.9,10 Those studies suggested that the use of cisplatin can be a risk factor for coronary heart disease. Cardiotoxicity remains a major problem because of a high correlation between the degree of heart injury and the dose of cisplatin used.7 The mechanisms underlying the antitumour effects of cisplatin are relatively well understood; however, the cellular and molecular mechanisms involved in the toxic side-effects of cisplatin in the heart remain unknown. As one of the most energy demanding tissues in the body, the heart is almost completely dependent upon oxidative phosphorylation to supply the large amount of ATP required for contraction and relaxation.11 The mitochondria are important for these metabolic processes. These vital organelles generate over 90% of the cellular ATP via oxidative phosphorylation, using energy derived from oxidation in the respiratory chain.12 This process is driven by the consumption of molecular oxygen. Thus, because of their critical role in cell survival, mitochondria serve as targets for cellular toxins and chemotherapeutic agents.13 Previous studies indicated that the mitochondria are also likely to be a major target for cisplatin in cancer cells.14 Cisplatin binding results in a significant decrease in the mitochondrial function of melanoma cells15 and changes in mitochondrial function have been implicated in cancer cell resistance to chemotherapeutic agents.16 Determining whether the mitochondria are the primary targets of cisplatin chemotherapy is important for an understanding of the mechanism underlying the cardiotoxic action of cisplatin. However, the effect of cisplatin on myocardial mitochondrial function remains unclear and warrants further investigation. The endoplasmic reticulum (ER) stress response (also known as the unfolded protein response) is known to be closely associated with mitochondrial function and contributes to cardiac contractile dysfunction,17 yet there is little if any evidence that ER stress mediates the toxic effects of
Correspondence: Dr Heng Ma, Division of Pharmaceutical Science, School of Pharmacy, Department 3375, University of Wyoming, 1000 E University Ave, Laramie, WY 82071, USA. Email:
[email protected] Received 23 September 2009; revision 25 October 2009; accepted 26 October 2009. 2010 The Authors Journal compilation 2010 Blackwell Publishing Asia Pty Ltd
Cisplatin and cardiac dysfunction cisplatin in cardiomyocytes. Based on these observations, we hypothesized that cisplatin may be directly toxic or that ER stress may play an important role in the cardiotoxic effects of cisplatin. The aim of the present study was to explore the effect sof cisplatin on myocardial contractile function, mitochondrial function and the possible mechanisms involved. Crucial protein markers of ER stress, such as eukaryotic translation inhibition factor (eIF) 2a, were also monitored in the myocardium following cisplatin treatment.
METHODS Experimental animals and cisplatin treatment All animal procedures were approved by the University of Wyoming Institutional Animal Care and Use Committee in accordance with National Institutes of Health standards. All animals were housed in a temperature-controlled room under a 12 h light–dark cycle with ad libitum access to tap water. Male C57BL ⁄ 6 mice, 4 months of age (NIA, Baltimore, MD, USA) were used in the study. Age-matched C57 mice were treated with either cisplatin injected into the tail vein (10 mg ⁄ kg per day18,19) or vehicle (0.9% NaCl) for 1 week. The cisplatin solution (1 mg ⁄ mL) was prepared in phosphate-buffered saline (PBS) and each mouse received an injection of 0.2 mL. Mice were killed 1 week after cisplatin injection for collection of cardiac tissues.
Mouse heart perfusion Isolated hearts were perfused retrogradely with a Krebs’–Henseleit buffer (composition (in mmol ⁄ L): NaCl 118; KCl 4.7; MgSO4 1.2; KH2PO4 1.2; NaHCO3 25; CaCl2 1.4; glucose 7) containing 0.4 oleate, 1% bovine servum albumin (BSA) and a low fasting concentration of insulin (10 lU ⁄ mL). Hearts were perfused at a constant flow of 4 mL ⁄ min (equal to an aortic pressure of 80 cmH2O) for 60 min. A fluid-filled latex balloon connected to a solid-state pressure transducer was inserted into the left ventricle through a left atriotomy to measure pressure. Left ventricular developed pressure (LVDP), the first derivative of LVDP (+ ⁄ )dP ⁄ dt) and heart rate were recorded using a digital acquisition system at a balloon volume that resulted in a baseline left ventricular (LV) end-diastolic pressure of 5 mmHg.20
Cardiomyocyte isolation Individual ventricular cardiomyocytes were isolated from adult C57 mice and cisplatin-treated mice as described previously.21 Brifely, after mice had been anaesthetized with a solution of ketamine plus xylazine, hearts were removed and perfused with Krebs’–Henseleit bicarbonate (KHB) buffer (composition (in mmol ⁄ L): NaCl 118; KCl 4.7; MgSO4 1.2; KH2PO4 1.2; NaHCO3 25; HEPES 10; glucose 11.1). Hearts were digested with 10 mg ⁄ mL Liberase (Roche Diagnostics, Indianapolis, IN, USA) for 20 min before the left ventricles were removed and minced before being filtered. The myocyte yield was approximately 75% and was not affected by cisplatin treatment. Only rod-shaped myocytes with clear edges were selected for mechanical studies.
Cell shortening and relengthening Mechanical properties of the cardiomyocytes were evaluated using a SoftEdge MyoCam system (IonOptix, Milton, MA, USA).22 Briefly, cardiomyocytes were visualized under an inverted microscope (IX-70; Olympus Optical, Tokyo, Japan) and stimulated with a voltage frequency of 0.5 Hz. The myocyte being observed was shown on a computer monitor using an IonOptix MyoCam camera. IonOptix SoftEdge software was used to capture changes in cell shortening and relengthening. The indices considered were peak shortening amplitude (PS), time to peak shortening (TPS), time to 90% relengthening (TR90), maximal velocity of shortening and relengthening (+ ⁄ )dL ⁄ dt).21 When the stimulus was changed from 0.1 to 5.0 Hz, a steady state contraction
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of myocytes was achieved (usually after the first five to six beats) before PS was recorded.
Transmission electron microscopy Samples of heart tissue were fixed in 2.5% glutaraldehyde in 0.1 mol ⁄ L PBS (pH 7.4) for 1 h. After fixation, samples were rinsed with 0.1 mol ⁄ L PBS (pH 7.4), post-fixed in 1% osmium tetroxide in the same buffer for 1 h at 4C, dehydrated in graded ethanol and embedded in araldite. For histomorphometric analysis, semithin sections (0.5 lm) were generated and stained with 0.1% Toluidine blue. For ultrastructural analysis, ultrathin sections (50 nm) were prepared after the tissue blocks had been trimmed. Sections, mounted on copper grids stained with saturated uranyl acetate and 1% lead citrate, were examined under a transmission electron microscope.
Measurement of mitochondrial membrane potential Cardiomyocytes were suspended in HEPES–saline buffer and mitochondrial membrane potential (Wm) was detected as described previously.23 Briefly, following 10 min pre-incubation with 5 lmol ⁄ L JC-1 at 37C, cells were rinsed twice using HEPES–saline (HS) buffer (composition (in mmol ⁄ L): NaCl 154; KCl 5; MgSO4 1.2; NaH2PO4 1.2; CaCl2 1.0; Na-HEPES 10, pH 7.4) free of JC-1. The fluorescence of each sample was read at an excitation wavelength of 490 nm and emission wavelengths of 530 and 590 nm using a spectrofluorometer (Spectra MaxGeminiXS, Atlanta, GA, USA) at 10 s intervals. Fluorescence intensity is expressed as the ratio of emission at 590 to 530 nm. The mitochondrial uncoupler sodium azide (NaN3; 10 mmol ⁄ L) was used as a positive control for measurements of mitochondrial Wm.
Western blot analysis Total protein was prepared as described previously.24 Briefly, ventricular tissues were homogenzied and sonicated in lysis buffer containing 20 mmol ⁄ L Tris (pH 7.4), 150 mmol ⁄ L NaCl, 1 mmol ⁄ L EDTA, 1 mmol ⁄ L EGTA, 1% Triton, 0.1% sodium dodecyl sulphate (SDS) and 1% protease inhibitor cocktail. Protein assay reagent (Bio-Rad Laboratories, Hercules, CA, USA) was used to assess the heart protein concentration in the supernatant. Equal amounts (30 lg protein ⁄ lane) of proteins or prestained molecular weight markers (SeeBlue Plus2; Invitrogen, Carlsbad, CA, USA) were separated on 10 or 15% SDS–polyacrylamide gels in a minigel apparatus (Mini-PROTEAN II; Bio-Rad) and were then transferred electrophoretically to nitrocellulose membranes (0.2 lm pore size; Bio-Rad). Membranes were incubated for 1 h in a blocking solution containing 5% milk in Tris-buffered saline (TBS). Membranes were then washed in TBS and incubated overnight at 4C with anti-eIF2a, anti-glucose regulated 78 kDa protein (GRP78) and anti-GAPDH antibodies (all dilutions 1 : 1000). After blots had been washed with Tris-buffered saline Tween-20 (TBST) to remove excess primary antibody, they were incubated for 1 h with horseradish peroxidase (HRP)conjugated anti-rabbit secondary antibody (1 : 5000). Antibody binding was detected using enhanced chemiluminescence (Amersham Pharmacia, Piscataway, NJ, USA), the film scanned and the intensity of immunoblot bands detected with a Bio-Rad Calibrated Densitometer (model GS-800). All immunoblotting was repeated at least twice to ensure reproducibility of the results. Manufacturer-recommended positive controls were used to ensure the accuracy of the bands.
Caspase 3 activity Caspase 3 is an enzyme activated during the induction of apoptosis. In the present study, caspase 3 activity was determined according to methods described elsewhere.25 Briefly, myocytes were lysed in 100 lL ice-cold cell lysis buffer (50 mmol ⁄ L HEPES, 0.1% CHAPS, 1 mmol ⁄ L dithiothreitol, 0.1 mmol ⁄ L EDTA, 0.1% Nonidet P-40). Following cell lysis, 70 lL reaction buffer and 20 lL caspase 3 colourimetric substrate (Ac-DEVD-p¢1NA) were added to the cell lysate and incubated for 1 h at 37C; during this time, the
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caspase enzyme in the sample was allowed to cleave the chromophore pNA from its substrate molecule. Absorbence was detected at 405 nm with caspase 3 activity being proportional to colour reaction. Protein content was determined using the Bradford method.26 Caspase 3 activity is expressed as pmol p-nitroanilide released per lg protein per min.
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Terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling
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Terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL) staining using fluorescein-labelled dUTP (Roche Diagnostics, Indianapolis, IN, USA) and counterstaining with 0.5 lg ⁄ mL propidium iodide was performed on 5 lm sections from hearts fixed by paraformaldehyde perfusion and processed as described previously.20 Slides were examined in a blinded fashion for apoptotic nuclei using a Zeiss LSM410 confocal microscope (Carl Zeiss, Thornwood, NY, USA).
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Statistical analysis Data are given as the mean ± SEM. The statistical significance (P < 0.05) was estimated for each variable by Student’s t-test. Statistical analysis for frequency and mitochondrial Wm was performed using multifactorial analysis of variance (ANOVA) followed by Dunnett’s post hoc analysis.
RESULTS Effects of cisplatin on C57 mice As indicated in Table 1, cisplatin treatment for 1 week had no effect on bodyweight and organ weights in C57 mice, although heart weight was less than that in control mice. This resulted in a decreased heart weight ⁄ bodyweight ratio (P < 0.05 vs control), suggesting harmful effects of cisplatin on the heart. Furthermore, cisplatin treatment of C57 mice decreased the function of isolated hearts, including LVDP and + ⁄ )dP ⁄ dt.
Fig. 1 Effect of cisplatin on cell shortening. (a) Resting cell length, (b) peak shortening (PS) normolized against cell length, (c) maximal velocity of shortening (+dL ⁄ dt), (d) maximal velocity of relengthening ()dL ⁄ dt), (e) time to peak shortening (TPS) and (f) time to 90% relengthening (TR90) in control and cisplatin-treated mice. Data are the mean ± SEM (n = 150–200 cells from three mice per group). *P < 0.05 compared with the control group.
Effects of cisplatin on cardiomyocyte mechanics Assessment of cardiomyocyte mechanics revealed that cisplatin significantly depressed PS amplitude and + ⁄ )dL ⁄ dt, but prolonged TR90 without affecting TPS (Fig. 1). To evaluate the potential contribution of the sarcoplasmic reticulum27 in the cardiac contractile
Table 1 General features of the control and cisplatin-treated mice Control Bodyweight (g) Heart weight (mg) Heart weight ⁄ bodyweight (mg ⁄ g) Liver weight (g) Liver weight ⁄ bodyweight (mg ⁄ g) Kidney weight (mg) Kidney weight ⁄ bodyweight (mg ⁄ g) Heart rate (ex vivo; b.p.m.) LVDP (mmHg) +dP ⁄ dt (mmHg ⁄ s) )dP ⁄ dt (mmHg ⁄ s)
25.2 164 6.49 1.48 58.7 407 16.1 308 86.1 4062 2639
± ± ± ± ± ± ± ± ± ± ±
0.5 10 0.30 0.10 1.3 17 0.4 13 2.9 178 87
Cisplatin treated 24.1 134 5.56 1.58 61.4 424 16.5 298 76.2 3764 2080
± ± ± ± ± ± ± ± ± ± ±
1.0 5* 0.16* 0.10 1.6 18 0.3 15 3.3* 160* 131*
Data are the mean ± SEM (n = 10 mice per group). *P < 0.05 compared with control. LVDP, left ventricular developed pressure.
Fig. 2 Effect of cisplatin exposure on frequency (0.1–5.0 Hz)-associated responses in peak shortening (PS) amplitude in control (h) and cisplatintreated ( ) cardiomyocytes. Peak shortening is shown as percentage change from the value obtained at 0.1 Hz in the same cell. Data are the mean ± SEM (n = 25 cells). *P < 0.05 compared with the control group.
respons to cisplatin, untreated or cisplatin-treated cardiomyocytes were paced at higher stimulating frequencies to examine the SR Ca2+ handling capacity. Cells were initially stimulated to contract at 0.5 Hz for 5 min to ensure steady state prior to increasing the stimulating frequency to 5.0 Hz. Figure 2 shows a comparable negative staircase (frequency-associated response) in PS with increased stimulus frequency between the cisplatin-treated and control groups. Cisplatin exposure significantly exaggerated stimulus frequency elicited depression of PS. Thus, administration of cisplatin can induce cardiomyocyte contractile dysfunction, culminating in cardiotoxicity.
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Cisplatin and cardiac dysfunction
Effects of cisplatin on ER stress markers, caspase 3 activity and apoptosis
Fig. 3 Isolated cardiomyocyte mitochondrial membrane potential from control (h) and cisplatin-treated ( ) groups. The mitochondrial uncoupler 10 mmol ⁄ L sodium azide (NaN3) was used as a positive control (s). Data are the mean ± SEM (n = 3 independent experiments per group). *P < 0.05 compared with the control group.
Effects of cisplatin on cardiomyocyte mitochondrial Wm Given that mitochondrial function is essential to cardiomyocyte viability and function,28,29 the cationic lipophilic probe JC-1 was used to monitor changes in mitochondrial Wm in response to cisplatin treatment (Fig. 3). Dynamic changes in Wm were evidenced by changes in the ratio between red (aggregated JC-1) and green (monomeric form of JC-1) fluorescence. We found that, compared with the control group, cisplatin treatment produced a significant collapse of the mitochondrial Wm, indicating functional abnormalities of mitochondria after cisplatin treatment. Effects of cisplatin on the ultrastructure of cardiomyocyte mitochondria Transmission electron microscopy analysis of the ultrastructural characteristics of cardiomyocytes in both the control and cisplatin-treated groups demonstrated that cisplatin treatment for 1 week had no effect on sarcomeres, which remained intact, although the mitochondrial matrix, area of cristae and matrix volume were significantly increased (Fig. 4). Other abnormalities included dilated ER with membrane debris. Thus, cisplatin induces ultrastructural abnormalities of the mitochondria in the heart and these abnormalities are associated with functional mitochondrial abnormalities.
Fig. 4 Transmission electron microscopy analysis of the ultrastructural characteristics of cardiomyocytes in the control (a) and cisplatin-treated (b,c) groups. The arrow indicates dilated endoplasmic reticulum. Bars, 0.5 lm.
We next examined the mechanisms regulating cisplatin-induced cardiotoxicity. Because of the accumulation of membranous debris within and dilation of the ER in hearts of cisplatin-treated mice, we tested whether the ER stress response pathway was recruited in the response. We evaluated protein expression of the ER stress markers eIF2a and GRP78. As shown in Fig. 5a,b, phosphorylation of eIF2a and expression of GRP78 was significantly upregulated in the myocardium of cisplatin-treated mice. To determine whether the increase in ER stress induced by cisplatin was triggering cardiomyocyte death, we evaluated caspase 3 activity. As shown in Fig. 5c,d, caspase 3 activity and TUNEL-positive nuclei were significantly increased in cardiomyocytes following cisplatin treatment. Altogether, the data from the present study indicate a potential role for ER stress in the cardiac damage seen following cisplatin treatment.
DISCUSSION The present study provides evidence that cisplatin treatment is associated with depressed myocardial contractile function, as demonstrated by depressed LVDP of the whole heart and PS of isolated ventricular myocytes. Furthermore, the cisplatin-induced depression of PS is exacerbated with increasing frequency of stimulation. The cardiotoxicity elicited by cisplatin may be associated with direct damage to cardiomyocyte mitochondrial function in addition to disturbances to the mitochondrial ultrastructure. Notably, further studies revealed that the severity of ER stress following cisplatin treatment may contribute to cisplatin-induced deficits in myocardial function and apoptosis. These data strongly support the notion that the mitochondria may be the primary target of cisplatin in the pathogenesis of deleterious effect in the heart and indicate a potential role for ER stress in cisplatininduced cardiac damage. Clinical studies have reported that cisplatin therapy is usually associated with cardiac toxicity. Cardiac events following cisplatin intake may include arrhythmias, myocarditis, cardiomyopathy and congestive heart failure.8,30 One clinical study reported the case of a young patient with head–neck cancer in whom a continuous electrocardiogram (ECG) recording was performed. The ECG documented serious ventricular arrhythmias in the presence of myocardial ischaemia during infusion of 5-fluorouracil and cisplatin.9 However, more than a decade after its clinical introduction, the exact mechanism underlying the effects of cisplatin on the heart is not fully known. The present study revealed that cisplatin treatment decreased the heart weight ⁄ bodyweight ratio and triggered deterioration of heart contractile function, as evidenced by reduced + ⁄ )dP ⁄ dt and LVDP, prolonged
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Fig. 5 Effect sof cisplatin exposure on (a) the endoplasmic reticulum (ER) stress marker eukaryotic initiation factor (eIF) 2a, (b) glucose-regulated protein (GRP) 78, (c) caspase 3 activity and (d) apoptosis. (a,b) Representative gels are shown using specific antibodies. Data are the mean ± SEM (n = 5 mice per group). *P < 0.05 compared with the control group.
TR90 and depressed PS and + ⁄ )dL ⁄ dt associated with normal TPS. The results of the present study consolidate the notion that cisplatin directly interrupts cardiac excitation–contraction coupling. Most research into the cytotoxicity of cisplatin over the past three decades has focused on the effects of cisplatin on inter- and intrastrand DNA cross-links, which may lead to cell death in sensitive cells. However, recent studies have shown that cisplatin may have significant direct effects on mitochondria14 that can induce apoptosis6 and may account for the clinical effects of the drug, such as cardiotoxicity. The interactions between cisplatin and mitochondria should provide a better understanding of this class of drugs and the development of new therapeutic approaches. The present study demonstrated that cisplatin significantly decreases mitochondrial Wm in cardiac tissues. Consistently, electron micrographs showed mitochondrial abnormalities, including of the mitochondrial matrix and cristae and significantly increased matrix volume. In addition, membranous debris and dilation of the ER were noted in cardiomyocytes. Thus, cisplatin induces ultrastructural abnormalities of the mitochondria and these are associated with the functional abnormalities of mitochondria in cardiomyocytes. Although changes in mitochondrial function seem to be the ultimate cause of cisplatin-induced toxicity, our data suggest activation of the ER stress response in the myocardium following cisplatin intake. The ER is a widespread intracellular membranous network involved in important steps in the folding and modification of proteins, as well as protein glycosylation and trafficking of membrane and secretary proteins. Recent evidence indicates that many factors, such as infectious agents, environmental toxins and adverse metabolic conditions, can interfere with protein folding, which can lead to ER stress.31 The ER stress contributes to several diseases, such as neurodegenerative disorders, diabetes and ischaemia–reperfusioninduced heart damage.32 Three different classes of ER stress proximal sensors have been identified, namely inositol-requiring protein-1 (IRE1), the protein kinase RNA (PKR)-like ER kinase (PERK)-translation initiation factor eIF-2a pathway and activating transcription
factor-6 (ATF6). Each of the three ER stress transducers governs a distinct arm of the ER stress-induced unfolded protein response (UPR).33 Activation of the UPR causes upregulation of genes that express ER chaperone proteins, such as GRP78, which increases protein folding activity and prevents protein aggregation.34 Data from the present study indicate that cisplatin initiates the ER stress response because phosphorylation of eIF2a, a marker of PERK activation, was significantly increased in hearts from cisplatin-treated mice. The data demonstrate that cisplatin induction of the UPR is highly dependent on the PERK ⁄ eIF2a pathway. In addition, we have shown that cisplatin significantly upregulates the ER chaperone GRP78 (also known as immunoglobulin-binding protein (BiP)) in cardiac tissues. It is believed that upregulation of GRP78 is pivotal for cell survival to facilitate folding and assembly of ER proteins and to prevent them from aggregation during ER stress.34 The ER can initiate apoptosis following the accumulation of unfolded proteins or inhibition of ER–Golgi transport resulting from the so-called ER stress response.35 Our data suggest that activation of caspase 3 is largely responsible for cisplatin-induced cardiomyocyte toxicity. Although the present study may not directly explain the interaction between mitochondrial abnormalities and ER stress following cisplatin intake, ER stress has been confirmed to trigger apoptosis in several diseases.33 Moreover, preliminary evidence indicates that ER stress may directly lead to cardiomyocyte contractile dysfunction via an Akt-dependent pathway.36 This is consistent with the dampened heart function observed in the present study. Therefore, one can assume that ER stress plays an important role in cisplatin-induced cardiotoxicity. In summary, the present study provides convincing evidence that cisplatin directly impairs cardiac excitation–contraction coupling, possibly related to mitochondrial damage and ER stress. It can lead to LV dysfunction and depressed cardiomyocyte contraction associated with mitochondrial abnormalities, enhanced ER stress and apoptosis. Intervention that restores mitochondrial function or inhibits ER stress in the heart may be a novel way in which to attenuate cisplatininduced cardiomyocyte injury. It would be beneficial to study the effects of cardiotoxicity in cisplatin-treated cancer patients in the hope of reducing the side-effects associated with the use of cisplatin.
ACKNOWLEDGEMENTS This work was supported, in part, by the National Institutes of Health, University of Wyoming Northern Rockies Regional INBRE (5P20RR016474 to JR), AHA Postdoctoral Fellowship (09POST2250477 to HM) and the National Science Foundation of China (30700263 to HM; 30728023 to JR).
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