rapid communication Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection?

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1 Am J Physiol Heart Circ Physiol 278: H1395 H1400, rapid communication Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection? JOHN E. BAKER, 1,2 EUGENE A. KONOREV, 3 GARRETT J. GROSS, 2 WILLIAM M. CHILIAN, 4 AND HOWARD J. JACOB 4 1 Division of Pediatric Surgery, 3 Biophysics Research Institute, and Departments of 2 Pharmacology and Toxicology and 4 Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin Baker, John E., Eugene A. Konorev, Garrett J. Gross, William M. Chilian, and Howard J. Jacob. Resistance to myocardial ischemia in five rat strains: is there a genetic component of cardioprotection? Am J Physiol Heart Circ Physiol 278: H1395 H1400, There is a need to develop new and more consistent animal models of cardioprotection. Traditionally, outbred dogs, rabbits, and rats have been studied. We determined resistance to ischemia in isolated hearts from inbred strains of rats. Hearts from inbred rats: SS/Mcw (Dahl S, Dahl salt-sensitive), DA/Hsd (Dark Agouti), LEW/Hsd (Lewis), and BN/SsN/Mcw (Brown Norway); and from an outbred rat: Hsd:WIST (Wistar) were subjected to 27 min of global, no-flow ischemia, followed by 3hofreperfusion. Infarct size in the Brown Norway rat was 2.5 times less than that observed in the Dahl S rat, with the Dark Agouti, Lewis, and Wistar rats intermediate in response. Hearts from Brown Norway rats were also most resistant to ischemia in terms of postischemic enzyme leakage and contractile and vascular function compared with other strains. The average polymorphism rate between strains revealed that such strains were genetically diverse. This study demonstrates strain differences in resistance to myocardial ischemia, suggesting these rats could be used to study a genetic and/or environmental basis for these differences and to provide new animal models for the physiological study of cardioprotection. cardiovascular diseases; genetics STUDIES OF ISCHEMIA and reperfusion in the heart frequently involve the use of animal models, traditionally outbred dogs, rabbits, and rats. To improve our understanding of cardioprotection, there is a need to develop better animal models to study the physiology and biochemistry of the heart during the cycle of ischemia and reperfusion. The isolated heart model has been used extensively in the development of cardioprotective strategies to protect the heart against the deleterious effects of ischemia and reperfusion. Differences exist between species in resistance of the isolated heart to ischemia (8). However, it is unknown to what degree differences in resistance of the isolated heart to ischemia exist within a single species, such as the rat. If strain differences exist, they could be used to facilitate the development of better models to study resistance of the heart to ischemia and would facilitate the use of genetic strategies to differentiate between genetic and environmental factors involved in this process. Evidence that different strains may differ with respect to resistance to myocardial ischemia was derived from studies which showed that renal ischemia and reperfusion induced less infiltration and major histocompatibility complex (MHC) class II expression in PVG and Wistar Furth rats in comparison with Lewis and Dark Agouti rats (11). These investigations (11) suggested an underlying genetic component may be responsible for resistance to renal ischemia. Systolic blood pressure is higher in Dahl S rats compared with Brown Norway rats, and genetic segregation studies (10) revealed an underlying genetic component responsible for hypertension. To facilitate the study of molecular genetics in disease states, the NIH and international funding agencies have supported the rat genome project, which has completed the initial genetic infrastructure of the rat with over 6,000 genetic markers identified and over 100,000 rat cdna clones sequenced from 12 normalized libraries (17). The objective of our study was to determine resistance to myocardial ischemia in four strains of inbred rats: SS/Mcw (Dahl S, Dahl salt-sensitive), DA/Hsd (Dark Agouti), LEW/Hsd (Lewis), and BN/SsN/Mcw (Brown Norway); and one strain of outbred rat: Hsd: WIST (Wistar), using well-accepted, multiple, independent end points of tissue injury representative of the phenotypic response of the myocardium to ischemia. METHODS The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Animals. Animals used in this study received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, by the National Research Council. All rats were housed in identical cages and were fed an identical /00 $5.00 Copyright 2000 the American Physiological Society H1395

2 H1396 GENETICS AND MYOCARDIAL ISCHEMIA standard diet. Male rats (8 wk, g) were used for the study. We selected the SS/Mcw and BN/SsN/Mcw rats because of our initial work with these strains. Furthermore, we (HJ Jacob) have conducted a genome scan for an F 2 intercross between these two strains for hypertension. Therefore, we have the ability to distinguish between susceptibility and direct action of hypertension on resistance to ischemia in the BN/SsN/Mcw rat. The other three strains, Dark Agouti, Lewis, and Wistar, were selected because of their previous use to study renal ischemia (11). Isolated heart perfusion. Isolated rat hearts perfused in a retrograde manner, with a balloon inflated in the left ventricle to control intracavity pressure, were instrumented as previously described (2). The standard perfusate was modified Krebs-Henseleit bicarbonate buffer (2) in which the calcium content was reduced to 1.8 mmol/l. Glucose (11.1 mmol/l) was added to the perfusate. Before use, all perfusion fluids were filtered through cellulose acetate membranes with pore size 5.0 µm to remove particulate matter. The hearts were kept in temperature-controlled chambers to maintain myocardial temperature at 37 C. Assessment of ventricular function. Left ventricular function was monitored continuously throughout each experiment as previously described (2). End-diastolic pressure was set to 5 mmhg and developed pressure was recorded during steady-state conditions. Coronary flow rate was measured throughout the experiment by timed collections of the coronary effluent from the right side of the heart into a graduated cylinder. Coronary flow rate was expressed as milliliters per minute per gram wet weight. Contracture development during ischemia was defined as an increase in intracavity pressure of 4 mmhg above end-diastolic values. Perfusion sequence. The following experiments were performed initially in a random order using five hearts from five strains to test the null hypothesis that resistance to myocardial ischemia is independent of the strain of rat investigated. Following these initial studies, we increased the number of hearts studied in the two groups that exhibited the greatest difference in resistance to myocardial ischemia to 10. Immediately after aortic cannulation, hearts from SS/Mcw, DA/Hsd, LEW/Hsd, BN/SsN/Mcw, and Hsd:WIST rats were perfused in the Langendorff mode (2) for 30 min at a constant perfusion pressure of 80 mmhg. Ventricular function and coronary flow rate were recorded under steady-state conditions. Hearts were subjected to 27-min global, no-flow ischemia and 3-h reperfusion. During the initial 40-min reperfusion period, indices of cardiac function were measured under steady-state conditions, and the entire coronary effluent was collected for the determination of lactate dehydrogenase activity (14). At the end of the 3-h reperfusion period, hearts were processed and stained with triphenyltetrazolium chloride dye for infarct size determination. The 27-min period of ischemia was selected from preliminary studies which determined the duration of ischemia that resulted in 50 60% infarct size upon reperfusion in the SS/Mcw rats. By determining the time period resulting in 50 60% infarct size in the SS/Mcw rats, we were able to detect whether hearts from DA/Hsd, LEW/Hsd, BN/SsN/ Mcw, and Hsd:WIST rats were more or less resistant to ischemia relative to SS/Mcw rats. Infarct size determination. After 3-h reperfusion, hearts were rapidly removed from the perfusion apparatus and sliced across the long axis of the left ventricle, from apex to base, into 2-mm-thick transverse sections and then incubated in 1% triphenyltetrazolium chloride (Sigma Chemical, St. Louis, MO) in phosphate buffer (ph 7.4) at 38 C for 20 min. Infarct areas were visually enhanced by storage in 10% formaldehyde solution for 24 h before final measurement. In the globally ischemic heart, the entire ventricle is at risk of infarction and therefore measurement of collateral flow and the area at risk is not required. Global ischemia resulted in multiple small areas of triphenyltetrazolium chloride staining. We were careful to separate the areas of viable and necrotic tissue using a surgical blade. The tissues were then weighed by an independent observer (GJ Gross) who did not know the origin of the hearts. The volumes of the infarcted zone and the area at risk were then calculated by multiplying the weight of the planimetered areas by the slice thickness. Infarct volume was expressed as a percentage of left ventricular volume for each heart (13). All hearts that were entered into the study were included in the analysis. Recovery of developed pressure and coronary flow rate were expressed as a percentage of their preischemic values. Results were expressed as means SE. Statistical analysis was performed by use of repeated-measures ANOVA with the Greenhouse-Geisser adjustment used to correct for the inflated risk of a type I error (16) and, where this proved significant, Mann-Whitney s rank sum test was used as a second step to identify which groups were significantly different. After ANOVA, the data were reanalyzed for differences related to multiple comparisons using the Bonferroni procedure (1). Significance was accepted at a level of P Genetic relationship between strains. We assessed the genetic distance among the five strains investigated in this study by two methods. 1) We used the average rate of polymorphism between the different strains, because a polymorphism is a sequence variant and therefore provides an estimate of genetic distance. For these calculations, we used the average polymorphism rate for 4,328 genetic markers (12) greater than 3 base pairs. The average polymorphism rate provides a whole genome estimate of genetic distance. 2) To determine how closely related the strains are, we used polymorphism data as alleles for constructing haplotypes for chromosome 1 ( 10% of the rat genome). The allele data were then used to generate haplotypes (conserved regions of the genome) to compare how similar the two strains are to each other. For this purpose, we declared a haplotype when three or more markers in a row or within the same bin ( 1.3 cm) had the same allele size. Evaluation of a random set of haplotypes revealed that long ( 10 markers in order) haplotypes were more prevalent in closely related strains known to be derived from the same progenitor. As would be expected from these observations, the greater the rate of polymorphism between two strains, the fewer the number of haplotypes ( 3 markers with the same allele size) in common. These data suggest that linkage disequilibrium could be used to study these different inbred strains, as well as to determine the evolutionary relationships between strains. RESULTS Preischemic function. Table 1 gives baseline functional data for the five strains of rats studied. Preischemic values for heart rate in the SS/Mcw rat of beats/s were no different from the values determined in the four other strains studied. Heart rate is an index of electrical function of the heart. Coronary flow rate in the SS/Mcw rat was ml min 1 g wet wt 1. This value was no different from that observed in the BN/SsN/Mcw, DA/Hsd, LEW/Hsd, and Hsd:WIST rat. Coronary flow rate is an index of vascular function of the heart. Coronary flow rate can also be affected by mechanical forces such as contracture.

3 GENETICS AND MYOCARDIAL ISCHEMIA H1397 Table 1. Hemodynamic values for each group of rats Groups n Heart rate, beats/s Coronary flow rate, ml min 1 wet wt g 1 Preischemic Ischemic Postischemic Reperfusion (40 min) Left ventricular developed pressure, mmhg Heart wet weight, g Onset of contracture, min Time to peak contracture, min Peak contracture, mmhg Heart rate, beats/s Coronary flow rate, ml min 1 wet wt 1 Left ventricular developed pressure, mmhg 1. SS/Mcw * * 63 4* 2. DA/Hsd * 88 5* 3. LEW/Hsd * 79 5* 4. Hsd: WIST * 77 8* 5. BN/SsN/Mcw * Values are means SE. SS/Mcw, Dahl salt-sensitive; DA/Hsd, Dark Agouti; LEW/Hsd, Lewis; Hsd:WIST, Wistar; BN/SsN/Mcw, Brown Norway. *P 0.05, preischemic vs. postischemic; P 0.05, vs. SS/Mcw. Preischemic values for left ventricular developed pressure in the SS/Mcw rat of mmhg were no different from the values determined in the four other strains. Developed pressure is an index of contractile function of the heart. Heart weight (wet) in the SS/Mcw rat was g. This value was not different from the BN/SsN/Mcw, DA/Hsd, and LEW/Hsd rats but was 18% lower than that observed in the Hsd:WIST rat (P 0.05). These data suggest that cardiac weight, as an index of hypertrophy, is equal among four of the five strains studied and that these hearts were not hypertrophied. The absolute values for heart rate, coronary flow rate, and left ventricular developed pressure in the five strains of rat studied were all similar to values reported for other rat strains (6, 8, 14). Contracture development during ischemia. The onset of contracture development in the SS/Mcw rat heart occurred after min of global ischemia. This value was not different from the four other strains of rat. Contracture of the heart is an index of ischemiainduced high-energy phosphate depletion and intracellular calcium overload. The time to peak contracture in the SS/Mcw rat was min following the onset of global ischemia. This value was no different from that observed in the DA/Hsd, LEW/Hsd, and Hsd:WIST rats but was shorter than that found in the BN/SsN/ Mcw rat by 11% (P 0.05). Peak contracture development in the SS/Mcw heart was 57 1 mmhg. This value was no different from that observed in the Hsd:WIST rat but was greater than that observed in the DA/Hsd, LEW/Hsd, and BN/SsN/Mcw rats by 12%, 16%, and 19%, respectively (P 0.05). Postischemic function. Resistance to myocardial ischemia was measured using multiple, independent end points of tissue injury. Figure 1 shows that infarct size as a percentage of the left ventricle in the SS/Mcw rat was 53 2%. This value was no different from that observed in the DA/Hsd rat, but was 2.5 times greater than that observed in the BN/SsN/Mcw rat (P 0.02), with the LEW/Hsd and Hsd:WIST rats intermediate in response. Infarct size is an index of irreversible myocardial injury resulting in cell death. Figure 2 shows that cumulative leakage of lactate dehydrogenase from the SS/Mcw heart during the first 40 min of reperfusion was 24 1 IU/g wet wt. This value was no different from that observed in the DA/Hsd rat, but was 2.2 times greater than that observed in the BN/SsN/Mcw rat (P 0.02), with the LEW/Hsd and Hsd:WIST rats intermediate in responses (P 0.05). Enzyme leakage is a measure of severe myocardial injury that has yet to progress to irreversible cell injury. Figure 3 and Table 1 show that recovery of developed pressure in the left ventricle of hearts from SS/Mcw rats was 51 3% of its preischemic value. Recovery of developed pressure was significantly less than values found in the DA/Hsd, LEW/Hsd, BN/SsN/Mcw, and Hsd:WIST rats by 43%, 31%, 29%, and 59%, respectively (P 0.05). Table 1 shows that recovery of coronary flow rate in the SS/ Mcw rat was 62 3% of its preischemic value. This value was no different from that observed in the LEW/Hsd and Hsd:WIST rats but was significantly less than values observed in the DA/Hsd and BN/SsN/Mcw rats by 23% and 52%, respectively (P 0.05). Hearts from all strains returned spontaneously to sinus rhythm upon reperfusion. There was no incidence of ventricular fibrillation. Genetic relationship among strains. Table 2 illustrates the average polymorphism rate, which provides a simplistic estimate of genetic diversity among the five strains studied. These data also reveal that the BN/SsN/ Mcw rat is as genetically diverse from SS/Mcw as from WIST/Nhg (inbred Wistar) rats (18). Moreover, these data demonstrate the rate of polymorphism is sufficiently high that we could use a genetic cross between the BN/SsN/Mcw and SS/Mcw rats in conjunction with a total genome scan (i.e., test a set of 180 Fig. 1. Infarct size following 27-min global, no-flow ischemia and 3-h reperfusion in five strains of rat [SS/Mcw, Dahl S (Dahl saltsensitive); DA/Hsd, Dark Agouti; LEW/Hsd, Lewis; WIST, Wistar (Hsd:WIST); and BN/SsN/Mcw, Brown Norway]. Data are means SE. IS, infarct size; LV, left ventricle. *P 0.05 vs. SS/Mcw rats.

4 H1398 GENETICS AND MYOCARDIAL ISCHEMIA Table 2. Average polymorphism rate among strains BN/SsN/Mcw DA/Hsd LEW/Hsd SS/Mcw WIST/Nhg BN/SsN/Mcw DA/Hsd LEW/Hsd SS/Mcw 41 Data for this table are taken from data reported by Steen et al. (18) for 4,328 genetic markers used to determine allele sizes. The WIST/ Nhg (Wistar) rat used by Steen et al. was inbred and, therefore, captured only a subset of the alleles segregating in an outbred Hsd:WIST colony. Fig. 2. Postischemic leakage of lactate dehydrogenase following 27-min global, no-flow ischemia and 40-min reperfusion in five strains of rat. Data are means SE. *P 0.05 vs. SS/Mcw rats. genetic markers distributed across the rat genome) to identify the region(s) of the rat genome responsible for increased resistance to myocardial ischemia. Similar data were found for the haplotype analysis for chromosome 1 (data not shown). DISCUSSION Inbred rat strains may be useful in the development of better physiological and biochemical models to study resistance to myocardial ischemia, because these strains should yield less interanimal variation. A secondary advantage would be to identify strains with different responses to ischemia. We have demonstrated striking differences in resistance to myocardial ischemia in five strains of rat using an experimental design in which potentially confounding environmental effects are minimized before hearts are subjected to global ischemia and reperfusion. Based upon our assessment of strain relatedness, we believe there are likely to be genetic components responsible for resistance to myocardial ischemia. Fig. 3. Recovery of developed pressure in LV following 27-min global, no-flow ischemia and 40-min reperfusion in five strains of rat. Data are means SE. LVDP, left ventricular developed pressure. *P 0.05 vs. SS/Mcw rats. There were no differences in preischemic function among the five strains studied that could be used to reliably predict postischemic recovery of function. However, increased time to peak contracture in the BN/SsN/ Mcw rat compared with other strains was an index of increased resistance to ischemia. For all end points used to determine resistance to myocardial ischemia, there were significant strain-dependent differences with respect to infarct size, postischemic enzyme leakage, and postischemic contractile and vascular function. Hearts from BN/SsN/Mcw rats were consistently more resistant to myocardial ischemia compared with all other strains studied, regardless of the end point used to measure tissue injury. We observed a 2.5-fold reduction in infarct size and a 2.2-fold reduction in postischemic enzyme leakage between hearts from BN/SsN/ Mcw rats compared with SS/Mcw rats, with 10 hearts studied in each group. The extent of the difference observed between these two strains, together with the relatively small variation within each strain, suggests that these phenotypic traits are amenable to genetic analysis. Evidence for a genetic component of ischemiareperfusion injury. Transient renal ischemia followed by reperfusion in the rat results in acute leukocyte infiltration and upregulation of MHC class II expression (11). Genetic differences in MHC class II expression have been reported in other tissues. Interstitial cell expression of MHC class II expression in rat hearts is under genetic control (5). In addition, the extent to which MHC class II expression is upregulated by brain endothelial cells in response to interferon- (IFN- ) is genetically controlled in rats (15). The spontaneously hypertensive rat and the stroke-prone substrain of the spontaneously hypertensive rat have much larger and less variable infarcts after middle cerebral artery occlusion than all other rat strains (3, 4, 7). This increased sensitivity to cerebral ischemia in the absence of reperfusion in the stroke-prone spontaneously hypertensive rat is genetically determined with susceptibility to cerebral infarction inherited as a dominant trait (9). Species differences in resistance of isolated hearts to ischemia and their responsiveness to myocardial protection have been described (8). However, to the best of our knowledge, this is the first study within a single species, the rat, to suggest a genetic basis responsible for resistance to myocardial ischemia. We considered that the reduced resistance to myocardial ischemia in the SS/Mcw rat was related to the

5 GENETICS AND MYOCARDIAL ISCHEMIA H1399 genetic predisposition of this inbred strain to systemic hypertension. However, myocardial injury in the normotensive DA/Hsd rat as assessed by infarct size and postischemic enzyme leakage was comparable with that seen in the SS/Mcw rat. Increased resistance to myocardial ischemia would be expected to delay the evolution from reversible to irreversible injury and necrosis. Resistance to ischemia would be manifest phenotypically as a reduction in infarct size, arrhythmias, enzyme leakage, and loss of contractile and vascular function. These well-accepted end points of tissue injury reflect the state of myocardium during the evolution from reversible to irreversible injury. The changes observed in the magnitude of increased resistance to myocardial ischemia in the hearts from the BN/SsN/Mcw rat relative to the SS/ Mcw rat are comparable to those seen in hearts following ischemic preconditioning (2) and adaptation of hearts to chronic hypoxia before sustained ischemia (1). We have suggested that adaptation to chronic hypoxia represents a unique form of preconditioning (1). We propose that hearts from BN/SsN/Mcw rats may already be preconditioned compared with SS/Mcw rats as a consequence of a genetic component resulting in increased resistance to ischemia. Our findings suggest that Brown Norway and Dahl S rats may be suitable as models to study resistance and susceptibility to myocardial ischemia, respectively. Use of the isolated heart to study resistance to ischemia. The isolated heart preparation enables the effective, reproducible, and economic study of mechanical function, metabolism, and ultrastructure in small mammalian hearts such as the rat over a wide range of experimental conditions, providing detailed physiological characterization that can be studied further using molecular genetic tools. For the study of resistance to ischemia, the isolated heart can be readily subjected to conditions of global, no-flow ischemia. This situation avoids the potential contribution of collateral flow supplied from nonischemic beds in conditions of regional ischemia that may influence resistance to ischemia. Thus all hearts from the five strains investigated were subjected to a virtually identical ischemic insult with the imposition of global rather than regional ischemia, which avoids problems with variable risk areas present in models of regional ischemia. Molecular genetics and resistance to myocardial ischemia. Within the last several years, there has been a virtual explosion of information about interventions that can produce cardioprotection and mimic the protective effects of ischemic preconditioning. This research has yielded valuable information, but there are still critical issues that require resolution. For example, pharmacological cardioprotection can be produced by a myriad of seemingly unrelated substances, including opioids, adrenergic agonists, nitric oxide donors, muscarinic agonists, ATP-sensitive potassium, channel openers, growth factors, and protein kinase C activators. Furthermore, the salutary effects of ischemic preconditioning can be blocked by as many apparently unrelated substances. This information has generated intense interest, but our assessment is that these results are incomplete. Common pathways that likely link the response of the many effective antagonists and agonists have eluded discovery. The use of molecular genetics to localize the gene(s) responsible for resistance to myocardial ischemia may permit us to combine the standard pharmacological approach in genetically defined and derived models that display considerable differences in their response to ischemia-reperfusion injury. In this study we report dramatic strain differences. Interestingly, transient renal ischemia followed by reperfusion in several of the rat strains used in this study results in acute leukocyte infiltration and also appears to be under genetic control (11). Consequently, it is likely that the strain differences we observed in our studies have a genetic component; therefore, these genetic components can be defined and used to help identify environmental components that are also likely to play a role in this response. In conclusion, molecular genetic strategies can be used to further our understanding of ischemia-reperfusion injury and hopefully lead to better clinical outcomes in the future. We are grateful to Drs. Allen W. Cowley, Jr., and Anne Kwitek- Black for stimulating discussion and to Mary Lynne Koenig for secretarial assistance. This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-54075, HL-08311, HL-54508, and HL Address for reprint requests and other correspondence: J. E. Baker, Division of Pediatric Surgery, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI ( jbaker@mcw.edu). Received 24 August 1999; accepted in final form 28 December REFERENCES 1. Baker JE, Curry BD, Olinger GN, and Gross GJ. Increased tolerance of the chronically hypoxic immature heart to ischemia: Contribution of the K ATP channel. Circulation 95: , Baker JE, Holman P, and Gross GJ. Preconditioning in immature rabbit hearts: role of K ATP channels. Circulation 99: , Coyle P. Outcomes to middle cerebral artery occlusion in hypertensive and normotensive rats. Hypertension 6, Suppl I: 69 74, Coyle P. 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