Published online before print August 2, 2004, doi: 10.1161/01.CIR.0000138110.84758.BB
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(Circulation. 2004;110:700-704.)
© 2004 American Heart Association, Inc.
Original Articles |
Targeted Inactivation of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channel Gene Prevents Ischemic Preconditioning in Isolated Mouse Heart
From the Department of Pharmacology and Center of Biomedical Research Excellence, University of Nevada School of Medicine, Reno. Dr. Hong Chen is now at the Department of Pharmacology and Shanghai Institute of Hypertension, Shanghai Second Medical University, Shanghai, China.
Correspondence to Dayue Duan, MD, PhD, FAHA, Associate Professor, Center of Biomedical Research Excellence, Department of Pharmacology, University of Nevada School of Medicine, Manville Medical Building Room 9/MS 318, Reno, NV 89557-0270. E-mail dduan{at}med.unr.edu
Received November 20, 2003; de novo received February 6, 2004; revision received March 10, 2004; accepted March 17, 2004.
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Abstract |
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Background— Recent evidence suggests that chloride channels may be involved in ischemic preconditioning (IPC). In this study, we tested whether the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels, which are expressed in the heart and activated by protein kinase A and protein kinase C, are important for IPC in isolated heart preparations from wild-type (WT) and CFTR knockout (CFTR–/–) mice.
Methods and Results— Hearts were isolated from age-matched WT or CFTR–/– (B6.129P2-Cftrtm1Unc and STOCKCftrtm1Unc-TgN 1Jaw) mice and perfused in the Langendorff or working-heart mode. All hearts were allowed to stabilize for 10 minutes before they were subjected to 30 or 45 minutes of global ischemia followed by 40 minutes of reperfusion (control group) or 3 cycles of 5 minutes of ischemia and reperfusion (IPC group) before 30 or 45 minutes of global ischemia and 40 minutes of reperfusion. Hemodynamic indices were recorded to evaluate cardiac functions. Release of creatine phosphate kinase (CPK) in the samples of coronary effluent and infarct size of the ventricles were used to estimate myocardial tissue injury. In WT adult hearts, IPC protected cardiac function during reperfusion and significantly decreased ischemia-induced CPK release and infarct size. A selective CFTR channel blocker, gemfibrozil, abrogated the protective effect of IPC. Furthermore, targeted inactivation of the CFTR gene in 2 different strains of CFTR–/– mice also prevented IPC’s protection of cardiac function and myocardial injury against sustained ischemia.
Conclusions— CFTR Cl– channels may serve as novel and crucial mediators in mouse heart IPC.
Key Words: ischemia • myocardial infarction • hemodynamics • ion channels • genes
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Introduction |
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Numerous studies have examined the signaling pathways involved in ischemic preconditioning (IPC), a phenomenon in which brief ischemic episodes elicit protection of the heart against sustained ischemia. It has been suggested that both sarcolemmal and mitochondrial ATP-sensitive potassium channels (sarc-KATP and mito-KATP, respectively) may serve as triggers or end-effectors.1 Protein kinase C (PKC) may link cellular signal events during ischemia to the activation of end-effectors.2,3 The precise mechanism of IPC, however, remains to be elucidated.
Recent studies have pointed to a potential role of anion channels in IPC. A cell swelling–induced Cl– current (ICl,swell) has been suggested to contribute to IPC in rabbit heart,4 although the causal role for ICl,swell in IPC has yet to be confirmed.5 Previous studies have demonstrated that a different class of anion channels, cystic fibrosis transmembrane conductance regulator (CFTR), are expressed in mammalian heart.6,7 Several lines of evidence suggest that CFTR channels may be involved in IPC, including the possibilities that (1) sarc-KATP blockers, such as glibenclamide, which suppress IPC protection, also block CFTR channels in noncardiac8 and cardiac cells;6,9 (2) PKC and PKA, 2 essential second messengers in IPC,2,3 can activate CFTR channels;6,7 and (3) triggers of IPC (nitric oxide, opioids, adenosine, etc) can all regulate CFTR channel function.10 Because much of the evidence favoring a role of CFTR in IPC is circumstantial, we tested this hypothesis directly by studying hemodynamics and tissue injury of hearts isolated from wild-type (WT) and 2 strains of CFTR-knockout (CFTR–/–) mice subjected to ischemia and reperfusion. The results from these experiments are consistent with the conclusion that CFTR Cl– channels may serve as novel and important mediators of IPC.
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Methods |
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This investigation conforms to the Guide for the Care and Use of Laboratory Animals (US National Institute of Health publication No. 85-23, revised 1996) and was in accordance with the institutional guidelines for animal care and use approved by the University of Nevada Institutional Animal Care and Use Committee.
CFTR–/– Mice
Two strains of CFTR–/– mice were used. Homozygous B6.129P2-Cftrtm1Unc (B6-CFTR–/–) mice11 were generated by mating heterozygous (CFTR+/–) breeders (JAX Laboratory) to CFTR+/– siblings. B6-CFTR–/– offspring 3 to 8 weeks old and their age-matched CFTR+/– and WT C57BL/6J littermates were used in this study. The STOCKCftrtm1Unc-TgN (FABPCFTR) 1Jaw12 breeders were obtained from Dr Jeffrey Whitsett (Children’s Hospital Medical Center, Cincinnati, Ohio) and bred in our transgenic animal facility. Eight-week-old FABPCFTR–/– offspring and their age-matched CFTR+/– and WT FVB/NJ littermates were used. Genotyping in both knockout strains was performed by polymerase chain reaction on tail DNA using a 3-primer assay resulting in a 357-bp band for CFTR–/–, a 5260-bp band for CFTR+/+, and both bands for CFTR+/–.
Isolated Langendorff and Working Heart Preparations
Hearts were removed rapidly from male mice anesthetized with pentobarbital sodium (50 mg/kg IP injection). The aorta was cannulated with a cannula connected to the HSE isolated heart perfusion system (model IH-1, Harvard Apparatus, Inc). Retrograde (Langendorff) perfusion was started instantly with modified Krebs-Henseleit buffer, which contained (in mmol/L) NaCl 118.0, KCl 4.7, CaCl2 2.5, MgSO4 1.2, KH2PO4 1.2, NaHCO3 25.0, pyruvate 2.0, glucose 11.0, and EDTA-Na2 0.5. Buffer was continuously bubbled with 95% O2 and 5% CO2 (pH 7.4, 37°C) throughout the perfusion period. The left atrium was then cannulated, and the hearts were perfused antegradely through the atrial cannula (working-heart mode; afterload, 60 mm Hg; preload, 10 mm Hg). Left ventricular pressure (LVP), LV developed pressure (LVDP), and LV end-diastolic pressure (LVEDP) were measured by a Millar tip catheter (1.4/0.8F pressure transducer, Millar) inserted into the LV cavity through the aorta or an open-ended PE-50 polyethylene cannula (OD, 0.965 mm) inserted into the LV cavity through the apex. Measurements of functional performance (aortic pressure, LVP, LVEDP, LVDP, first derivative of LVDP, ±dP/dt, and heart rate) were recorded continuously during the experiment and analyzed offline by an HSE data acquisition system (HSE Haemodyn, Harvard Apparatus). Cardiac output was calculated as the sum of coronary flow and aortic outflow collected during perfusion.
Assessment of Tissue Injury and Viability
Total global ischemia produced by clamping both atrial inflow and aortic outflow was used for induction of sustained ischemia as well as for IPC. Myocardial tissue injury was determined by measuring CPK release and infarct size. CPK activity in the coronary effluent collected during 40 minutes of reperfusion was measured with a spectrophotometer using a Sigma CPK kit according to the manufacturer’s instructions (Sigma Diagnostics). At the end of the experimental protocols, the hearts were perfused in the Langendorff mode for an additional 80 minutes and then frozen at –80°C, cut transversely into slices 
600 µm thick, and stained with 1% triphenyl tetrazolium chloride in phosphate buffer (pH 7.4) at 37°C for 15 minutes. After fixation in 10% neutral buffered formaldehyde overnight, the slices were scanned and saved as JPEG files. The viable myocardium stained deep red, and necrotic tissues stained pale. The entire risk area (the sum of total ventricular area minus cavities) was calculated in pixels using computer software (SimplePCI, Compix, Inc), and infarct areas were compared with the entire area at risk in a gray-scale manner.
Statistical Analysis
All group data are presented as mean±SEM. ANOVA and Student’s t test were used to determine statistical significance. A 2-tailed probability value of P
0.05 was considered statistically significant.
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Results |
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To test whether CFTR Cl– channels are involved in IPC, we first applied gemfibrozil (Gem), a selective blocker of CFTR channels in guinea pig13 and mouse heart (D. Duan, unpublished data, 2000), to isolated WT mouse hearts perfused in the working-heart mode. Hearts from age- and size-matched male C57BL/6J mice were divided randomly into control, IPC, and IPC+Gem (200 µmol/L) groups and subjected to the experimental protocols shown in Figure 1A. Under control conditions, 30 minutes of ischemia caused a significant decrease in cardiac function as estimated by LVP, LVDP, +dP/dt, –dP/dt, and cardiac output measured at the end of 40 minutes of reperfusion. Postischemic heart function recovered to the same level as that before sustained ischemia in IPC hearts. Administration of Gem during the preconditioning reperfusion abolished IPC-induced heart function recovery with a significant decrease in LVDP, +dP/dt, and –dP/dt (Figure 1, B through D). Table 1 summarizes the hemodynamic changes in the control, IPC, and IPC+Gem groups. Consistent with these functional changes, CPK release after sustained ischemia was significantly reduced in the IPC hearts. CPK release in the IPC+Gem group, however, remained as high as in the control group (Figure 1E). These results suggest that blockade of CFTR channels may prevent IPC in mouse heart.
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To confirm that the effects of Gem on IPC are a result of the blockade of CFTR channels and are not of other effects, such activation of peroxisome proliferator–activated nuclear receptors,14 we first used B6-CFTR–/– mice11 to examine the effect of IPC on ischemia-induced myocardial CPK release and infarct size. B6-CFTR–/– mice on normal diet are significantly smaller in both body and heart than WT and CFTR+/– mice (Table 2) and can live for only up to 5 weeks because of severe intestinal disease.11 Therefore, experiments were performed using 3- to 4-week-old WT (C57BL/6J), CFTR+/–, and CFTR–/– littermates. These hearts, especially the hearts of CFTR–/– mice, are too small to be perfused in working-heart mode. Therefore, these hearts were perfused only in the Langendorff mode, and no hemodynamic data were collected. For the same reason, it is impossible to accurately measure the infarct size of these small hearts, so CPK release (units per heart tissue weight per minute) was used as the index for myocardial injury. As shown in Table 2, IPC effectively reduced myocardial CPK release in WT and CFTR+/– mice but not in the age-matched CFTR–/– mice. To test whether the failure to precondition the CFTR–/– mouse heart is because of severe intestinal disease and lower body and heart weights, a special liquid diet food, peptamin, was used to feed the B6-CFTR–/– mice so that they were able to survive up to 8 weeks with body and heart weights (20.3±1.0 g and 103.4±3.5 mg, respectively, n=6) similar to those of WT mice (21.9±0.5 g and 104.3±2.9 mg, respectively, n=6, P>0.05). Hearts from these mice were also perfused only in the Langendorff mode, and infarct sizes were measured (Figure 1F). Consistent with the results in the 3- to 4-week-old mice, IPC failed to reduce the infarct size induced by 30 minutes of sustained global ischemia in the 8-week-old CFTR–/– mice, whereas it significantly limited the infarct size in both WT and CFTR+/– mouse hearts, suggesting that targeted inactivation of the CFTR gene prevented IPC in isolated mouse heart.
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To confirm these observations and ensure that the results in the peptamin-fed CFTR–/– mice are not because of the special diet, we used another strain of CFTR–/– mice. Homozygous FABPCFTR–/– mice are produced from FVB/NJ background and can live on normal mouse chow to a normal age and body weight, because the lethal intestinal defect seen in the B6-CFTR–/– mice is corrected.12 Hearts from these mice therefore offered an alternative approach to assess the effects of CFTR gene disruption on IPC-induced reduction in infarct size and protection of cardiac function. Hearts from age- and size-matched male WT (FVB/NJ), FABPCFTR+/–, and FABPCFTR–/– mice were perfused in the working-heart mode, and the infarct sizes of the ventricles were measured. In the FVB/NJ WT mouse hearts, 30-minute sustained ischemia caused a significantly smaller infarct size (19.1±2.3%, n=5) than that in the C57BL/6J WT mouse (31.4±2.5%, n=6, P=0.006). Furthermore, IPC in FVB/NJ WT mice caused only a mild reduction in the infarction (17.5±2.3%, P>0.5) and no significant recovery in cardiac function compared with the ischemia group (83.1±1.4% versus 84.4±1.6% in LVDP, 64.4±0.3% versus 62.6±3.7% in +dP/dt, and 80.6±2.4% versus 70.3±4.6% in –dP/dt, P=NS). Apparently, the response to ischemia and IPC varies in different genetic backgrounds of mice. Similar observations of different response to ischemia/reperfusion injury and preconditioning in different mouse strains have been reported by Bao et al.15 In fact, variations in cardiovascular traits occur naturally among inbred mouse strains.16 Therefore, we used a 45-minute sustained global ischemia protocol (Figure 2A). Postischemic cardiac function (LVDP and ±dP/dt) recovered significantly to a level close to that before 45 minutes of sustained ischemia in ischemic preconditioned hearts of WT FVB/NJ and CFTR+/– mice but not of CFTR–/– mice (Figure 2, B through D). Consistent with these functional changes, IPC significantly decreased myocardial infarct size in both WT and CFTR+/– mice but not in the CFTR–/– mice (Figure 2, E and F).
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Discussion |
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In this report, we present compelling evidence for an important functional role of CFTR Cl– channels in IPC of isolated mouse heart.
The CFTR Cl– channel protein is a member of the ATP-binding cassette (ABC) superfamily of transporters. Members in the ABC superfamily, including the sulfonylurea receptor, which combines with inward rectifier K+ (Kir6.1, Kir6.2) channel subunits to form functional sarc-KATP channels, share similar structure of transmembrane domains and nucleotide binding domains. However, CFTR seems to be unique in forming a PKA- and PKC-regulated anion channel.
It is highly unlikely that the ex vivo IPC response of CFTR–/– hearts are the result of chronic hemodynamic effects caused by hypoxia or pulmonary hypertension, because these CFTR–/– mice do not develop any lung disease or pulmonary hypertension, although lung disease is the leading cause of mortality and morbidity in humans with cystic fibrosis.11,12 Several mechanisms may be responsible for a functional role of CFTR channels in mouse heart IPC. (1) It has been demonstrated that cardiac CFTR plays a role in early action potential shortening during hypoxia and ischemia.17 Activation of CFTR will also decrease resting membrane potential and action potential duration, thereby limiting intracellular Ca2+ overload and cell damage.7 (2) The CFTR channel is an important transporter of sphingosine 1-phosphate (S-1-P),18 which has recently emerged as an important lipid messenger involved in IPC.19 (3) CFTR is permeable not only to Cl– but also to larger organic ions, as well as reduced and oxidized forms of glutathione.20 Therefore, CFTR may contribute to the control of oxygen stress–induced apoptosis and the regulation of inflammation and the immune responses. (4) CFTR may decrease intracellular pH and modulate apoptosis.21 (5) CFTR functions as a regulator of volume-dependent homeostatic cell mechanisms in cell proliferation and apoptosis.22 We are currently in the process of investigating these potential mechanisms and the relative role of CFTR in early and late preconditioning.
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Acknowledgments |
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This study was supported by National Center for Research Resources grant P-20-RR-15581 and National Heart, Lung, and Blood Institute grant HL-63914. We thank Dr Joseph R. Hume for excellent suggestions and critical reading of the manuscript.
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Footnotes |
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*The first 2 authors contributed equally to this work.
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References |
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