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Hypoxia or nutrient restriction during pregnancy in rats leads to progressive cardiac remodeling and impairs postischemic recovery in adult male offspring Yi Xu,*,1 Sarah J. Williams,*,†,1 Darryl O’Brien,* and Sandra T. Davidge*,2 *Departments of Obstetrics/Gynecology and Physiology, Perinatal Research Centre, University of Alberta, Edmonton, Alberta, Canada; and †Physiology, Centre for the Early Origins of Adult Health, School of Molecular and Biomedical Science, University of Adelaide, South Australia, Australia To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4917fje SPECIFIC AIMS A series of epidemiological studies have demonstrated that intrauterine growth restriction (IUGR) is associated with an increased risk of developing cardiovascular disease in adult offspring. The etiology of clinical IUGR is variable; however, in the majority of instances fetal growth is ultimately constrained by a limitation of oxygen and/or nutrient delivery. The mechanisms that underlie the association between prenatal growth and adult cardiovascular health must be defined before therapeutic approaches to reduce the cardiovascular risk associated with IUGR can be developed. Currently, the impact of IUGR on the structure and function of the adult heart is unclear. The aim of this study was to determine the effects of reduced oxygen and/or nutrient supply during late gestation on ultrastructural components of the adult heart, and further, to determine whether such changes are associated with increased cardiac susceptibility to ischemia/reperfusion injury.
PRINCIPAL FINDINGS 1. Left ventricular hypertrophy occurred in male adult offspring where IUGR resulted from maternal hypoxia but not from maternal undernutrition In this study, fetal substrate supply was decreased by reducing maternal inspired oxygen to 12% during the last week of pregnancy (IUGR-H) in rats. Exposure to hypoxia also decreased food intake in pregnant rats. Therefore, in order to assess whether the cardiac effects of IUGR due to prenatal hypoxia combined with reduced food intake (undernutrition) were distinct from the effects of undernutrition alone, we also assessed the cardiac effects of equivalent maternal undernutrition (IUGR-NR) alone. While left ventricular hypertrophy was observed in IUGR-H offspring at 4 and 7 mo, the ratio of left ventricular wt: body wt in IUGR-NR off0892-6638/06/0020-1251 © FASEB
spring was not different from controls at either 4 or 7 mo. Interestingly, these results were consistent with our previous observations in neonatal rat offspring, where maternal hypoxia, but not nutrient restriction, during pregnancy increased relative neonatal heart weight. The present results therefore further suggest that there is a specific impact of prenatal hypoxia, or the combination of hypoxia and undernutrition, on adult heart mass compared to nutrient restriction alone. 2. The cardiac expression of structural proteins implicated in pathological left ventricular remodeling was greater in IUGR offspring When the ultrastructural composition of cardiac tissue was assessed, the left ventricular expression of structural proteins that have been implicated in pathological cardiac remodeling was increased by IUGR in a remarkably consistent pattern. At 4 mo, the ratio of  to ␣ myosin heavy chain (MHC) protein (Fig. 1) and the expression of collagen I and III were all increased in only IUGR-H offspring. At 7 mo, however, the expression of all three proteins was increased in both IUGR-H and IUGR-NR offspring relative to control offspring. When fibrillar collagen structure was assessed by scanning electron microscopy, the collagen weave matrix also demonstrated thickened fibrillar strands, increased density and increased intrafibrillar connections in only IUGR-H offspring at 4 mo, but in both IUGR-H and IUGR-NR offspring at 7 mo. These results suggest that whether fetal growth is restricted by a combined reduction in oxygen and nutrition, or reduced nutrition alone, the adult heart of IUGR offspring undergoes pathological remodeling. 1
These authors contributed equally to this work Correspondence: 232 HMRC, Perinatal Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail:
[email protected] doi: 10.1096/fj.05-4917fje 2
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5. IUGR impaired functional recovery from global ischemia in a manner consistent with the observed remodeling in left ventricular tissue Functional recovery from 20 min global ischemia was impaired in only hearts from IUGR-H offspring at 4 mo relative to control or IUGR-NR at this age (Fig. 2A, B). Similar to the structural and functional differences observed at 7 mo, recovery from ischemia was impaired in hearts from both IUGR-H and IUGR-NR offspring relative to control (Figure 2A).
Figure 1. Effects of maternal hypoxia or nutrient restriction on cardiac protein expression of /␣ MHC in intrauterine growth restriction (IUGR) 4 and 7 mo rats. Different letters (a, b) indicate values that are significantly different from all other groups (P⬍0.05).
3. The cardiac expression of matrix metalloproteinase-2 was reduced in IUGR offspring Consistent with the observed changes in structural protein expression, the expression of matrix metalloproteinase-2 (MMP-2) was lower only in IUGR-H offspring at 4 mo, while at 7 mo, MMP-2 expression was lower in both IUGR-H and IUGR-NR offspring relative to control. A reduction in MMP activity is consistent with the observed accumulation of collagen. 4. IUGR resulted in diastolic dysfunction in hearts from adult offspring consistent with left ventricular stiffening The functional cardiac consequences of impaired prenatal development were assessed using a working heart preparation. At 4 mo, IUGR-H offspring exhibited diastolic dysfunction, indicated by decreased – dp/dt and increased left ventricular end diastolic pressure (LVEDP), which was consistent with left ventricular stiffening. By 7 mo, diastolic dysfunction was observed in both IUGR-H and IUGR-NR offspring relative to control. The impairments in baseline cardiac function in the IUGR groups are consistent and corroborate with the observed pathological structural remodeling in the left ventricular wall.
CONCLUSIONS AND SIGNIFICANCE The significant and novel findings of this study were that the restriction of fetal substrate supply during development resulted in pathological ultrastructural changes in left ventricular cardiac tissue. Furthermore, the changes in cardiac structure observed in all instances were associated functionally with increased left ventricular stiffness consistent with diastolic dysfunction and increased sensitivity to ischemia/reperfusion injury. In this study, comparison of the effects of reduced oxygen combined with reduced nutrition vs. nutrient restriction alone allowed further investigation into how the etiology of fetal growth restriction may influence the adult cardiovascular outcomes. The clinical causes of IUGR are diverse; however, ultimately growth restriction occurs in the majority of cases through a limitation of either fetal oxygen and nutrient delivery, or nutrient delivery alone. While the time course of pathological cardiac remodeling differed in IUGR-H and IUGR-NR offspring, in this study, both protocols led to a similar degree of cardiac impairment by 7 mo of age. These data therefore indicate that whether IUGR results from a reduction in fetal oxygen and nutrient availability, as occurs during placental insufficiency, or from reduced maternal nutrient supply alone, the structure and function of the adult heart is impaired later in life (Fig. 3). Remodeling may therefore represent a central pathway in the pathogenesis of impaired diastolic function and increased cardiac ischemia/ reperfusion injury susceptibility following IUGR. Further studies are required, however, to identify the
Figure 2. Maternal hypoxia or nutrient restriction impaired the recovery of cardiac function following I/R in 4- and 7-mo-old offspring. Percent recovery at 4 mo (A) and 7 mo (B). Perfused isolated hearts were subjected to global ischemia (20 min) followed by reperfusion (40 min). Values are means ⫾ se. Different letters (a, b) indicate values that are significantly different from all other groups (P⬍0.05).
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modeling. This is associated with a change in cardiac functional characteristics, including slower and less efficient contraction. The increased collagen deposition and increased fibrosis observed within left ventricular tissue from IUGR offspring may have further increased ventricular stiffness, and thus impaired diastolic heart function. The ability of the heart to respond to mechanical and chemical stimuli is influenced by the delicate balance between synthesis and degradation of extracellular structural components of extracellular matrix, which therefore has a significant impact on cardiac function. As we observed increased collagen deposition, fibrosis, and reduced MMP-2 expression in both IUGR-H and IUGR-NR offspring, our data suggest that this balance may be perturbed by a reduced oxygen and/or nutrient supply in utero such that inappropriate cardiac remodeling is favored. Heart function and recovery from ischemia/reperfusion injury Figure 3. IUGR resulting from a reduction in fetal oxygen and nutrient availability, as occurs during placental insufficiency, alters the structure and function of the adult heart later in life. Remodeling may, therefore, represent a central pathway in the pathogenesis of impaired diastolic function and increased cardiac ischemia/reperfusion injury susceptibility following IUGR. Although the time course of pathological cardiac remodeling differed in offspring exposed to prenatal hypoxia (IUGR-H) vs. maternal nutrient restriction (IUGR-NR), both conditions led to a similar degree of cardiac impairment by 7 mo of age.
pathways that initiate cardiac remodeling when prenatal growth is impaired, and to define how these pathways may interact with the normal processes of aging. Hypertrophy and remodeling We have previously reported that, at birth, relative heart weight was increased in IUGR-H pups compared to control or IUGR-NR pups. Consistent with this observation, relative left ventricle weight was increased in only IUGR-H offspring, at both ages studied, which may further suggest a specific effect of prenatal hypoxia on heart development. Interestingly, however, pathological left ventricular remodeling occurred in both IUGR-H and IUGR-NR offspring, although hypertrophy was observed only after prenatal hypoxia. Moreover, fetal growth restriction results in changes in the /␣ myosin heavy chain ratio in left ventricular tissue of adult offspring. Although MHC-␣ expression predominates in healthy rat cardiac tissue, a transition to greater MHC- expression occurs during pathological re-
CARDIAC EFFECT OF INTRAUTERINE GROWTH RESTRICTION
The reduced – dp/dt, and higher left ventricular end diastolic pressures observed in IUGR-H offspring at both ages, and IUGR-NR offspring at 7 mo were consistent with increased left ventricular stiffness, and therefore corroborate well with the pattern of ultrastructural changes observed in each group. In addition, in both IUGR-H and IUGR-NR offspring, cardiac remodeling and diastolic dysfunction were associated with impaired functional recovery from ischemia/ reperfusion injury, as well as greater tissue damage. To our knowledge, these data are the first to describe the impact of undernutrition during pregnancy on adult heart function, or sensitivity to ischemia-reperfusion injury. The progressive diastolic dysfunction observed in IUGR-H and IUGR-NR offspring in this study suggests that specific structural remodeling in the left ventricle following impaired prenatal development resulted in the diastolic functional changes observed, and likely contributed to the sensitization toward ischemic tissue injury. Regression of cardiac remodeling improves the prognosis of patients with cardiovascular disease. These results have therefore identified a potentially modifiable mechanism, whereby impaired growth in utero may increase the susceptibility to adverse cardiovascular events in adult life. Further, this mechanism is relevant whether impaired fetal growth is the consequence of reduced oxygen and nutrient supply, as occurs in placental insufficiency, or maternal malnutrition alone, and therefore is relevant to the majority of cases of intrauterine growth restriction. Importantly, the prevention or regression of left ventricular hypertrophy and remodeling may reduce the risk of later cardiovascular disease in individuals whose growth was restricted in utero, and therefore represent one therapeutic approach to reduce the cardiovascular risk associated with IUGR.
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Hypoxia or nutrient restriction during pregnancy in rats leads to progressive cardiac remodeling and impairs postischemic recovery in adult male offspring Yi Xu,*,1 Sarah J. Williams,*,†,1 Darryl O’Brien,* and Sandra T. Davidge*,2 *Departments of Obstetrics/Gynecology and Physiology, Perinatal Research Centre, University of Alberta, Edmonton, Alberta, Canada; and †Physiology, Centre for the Early Origins of Adult Health, School of Molecular and Biomedical Science, University of Adelaide, South Australia, Australia Intrauterine growth restriction (IUGR) increases the risk of developing adult-onset cardiovascular disease. We hypothesized that IUGR resulting from maternal hypoxia or nutrient restriction during late gestation will produce cardiac remodeling and impair cardiac recovery after ischemia/reperfusion (I/R) in adult male offspring aged 4 or 7 mo. SpragueDawley rats were randomized on day 15 of pregnancy to hypoxia (IUGR-H, 12% oxygen), nutrient restriction (IUGR-NR, 40% of control diet) or control (room air) groups. In 4-mo IUGR-H offspring, left ventricular wt/body wt ratio (LVW/BW) and right ventricular wt/BW ratio (RVW/BW) increased, in association with increased collagen I and III expression, beta and alpha myosin heavy chain (/␣MHC) ratio, and decreased matrix metalloproteinase (MMP)-2 activity compared to the other groups. Left ventricular end diastolic pressure was higher in perfused hearts. Functional recovery after I/R was remarkably reduced (10ⴞ3%) compared to both control (39ⴞ5%) and IUGR-NR rats (32ⴞ4%). At 7 mo, both IUGR-H and IUGR-NR offspring had increased LVW/BW, collagen I and III, /␣ MHC ratio, and decreased cardiac recovery and MMP-2 activity compared to control. These findings suggest that hypoxia or undernutrition during development leads to pathological cardiac remodeling, diastolic dysfunction, and increased sensitivity to ischemic injury during adult life.—Xu, Y., Williams, S. J. O’Brien, D., and Davidge, S. T. Hypoxia or nutrient restriction during pregnancy in rats leads to progressive cardiac remodeling and impairs postischemic recovery in adult male offspring. FASEB J. 20, E536 –E545 (2006)
ABSTRACT
Key words: cardiac development 䡠 ischemia 䡠 reperfusion
Substantial epidemiological evidence has demonstrated that intrauterine growth restriction (IUGR) increases the risk of death from cardiovascular disease in adult life (1–3). Experimental investigations have identified a number of mechanisms whereby impaired fetal growth may predispose individuals to the development of hypertension (reviewed in Ref. 4); however, E536
the impact of IUGR on the structure and function of the adult heart is unclear. It has been demonstrated that prenatal hypoxia decreases adult heart tolerance to ischemia/reperfusion (I/R) injury, suggesting that changes in prenatal cardiac development may sensitize the adult heart to injury (5). Clinical studies suggest that human IUGR fetuses undergo left ventricular hypertrophy (6, 7) and remodeling (8), whereas experimental studies have demonstrated that reduced fetal oxygen or nutrient supply influence heart development. We previously observed increased relative heart weight in newborn rats from dams that were exposed to chronic hypoxia, (9) consistent with the reported effects of hypoxia on fetal (10) and neonatal heart size (11). Hypoxia during pregnancy also reduces food intake in pregnant rats; (9, 12–15) however, we observed no change in heart weight in pups from dams nutritionally restricted to a comparative extent (9). In pregnant sheep, undernutrition did increase relative fetal heart weight, and the cardiac expression of several genes that regulate hypertrophy and remodeling by midgestation (16). In contrast, low-protein diet during pregnancy reduced both heart weight and the number of cardiomyocytes in newborn rat pups (17). These data imply that limitation of either oxygen or nutrient supply to the fetus produces short-term structural changes in the neonatal heart. However, less is known regarding the persistence, and long-term consequences of these changes. Left ventricular hypertrophy and remodeling are associated with increased risk of cardiovascular morbidity/mortality, (18 –21), and experimental prevention of these structural changes reduced the risk of cardiovascular disease (18, 21). Phenotypic changes in myocardial tissue where hypertrophy or remodeling has occurred involve modifications in the contractile machinery of cardiomyocytes, including a shift in myosin isoform expression [beta/alpha myosin heavy 1
These authors contributed equally to this work. Correspondence: 232 HMRC, Perinatal Research Centre, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail:
[email protected] doi: 10.1096/fj.05-4917fje 2
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chain, (/␣MHC)], and alterations in the extracellular matrix (ECM) that lead to collagen accumulation (18, 21). These changes promote fibrosis and ventricular stiffness, which may cause electrical and mechanical alterations in heart function, decrease tolerance to episodes of I/R, and predispose the individual to arrhythmia, heart failure, and sudden death (18, 21). The persistence of changes in cardiac structure resulting from hypoxia or undernutrition during fetal development may therefore contribute to the increased risk of later cardiovascular disease. In the present study, we have determined the impact of prenatal hypoxia on markers of cardiac remodeling and further, assessed cardiac function and susceptibility to I/R injury in isolated hearts from adult male offspring. To assess the contribution of decreased appetite in pregnant rats exposed to chronic hypoxia (9, 13–15, 22), we also determined the cardiac effects of equivalent undernutrition in the absence of hypoxia. MATERIALS AND METHODS Animals Female Sprague-Dawley rats were obtained at 3 mo of age (Charles River, Quebec, Canada), acclimatized, and then mated within the animal facility. Throughout pregnancy, rats were housed individually in standard rat cages with 60% humidity, a 12 h light: 12 h darkness light cycle, and ad libitum access to water. All rats received food (standard lab rat chow) ad libitum from day 0 –15 of pregnancy. On day 15, rats were randomized to control (C; n⫽10), maternal hypoxia (IUGR-H; n⫽9) or maternal nutrient restriction (IUGR-NR; n⫽7) protocols and treated as described previously in detail (9). Briefly, the maternal hypoxia group was placed inside a Plexiglass chamber (140 L vol), which was continuously infused with nitrogen and compressed air titrated to maintain an oxygen concentration of 12%. Food intake was determined daily in all rats. Consistent with previous observations (13–15), rats exposed to hypoxia ate less food than control rats (9, 22). To determine the effects of this concentration of undernutrition in the absence of hypoxia, a second group of pregnant rats was nutrient restricted (11.5 g standard rat chow/day) while housed in an identical Plexiglass chamber from day 15–21 of pregnancy. This level of undernutrition was equivalent to the lowest food intake recorded in rats exposed to maternal hypoxia, and represented ⬃40% of control food intake during this time. Litters were reduced to 8 pups/dam at birth to standardize postnatal nutrition. Offspring were weaned at 3 wk, and housed in the animal facilities of the University of Alberta. One to two male offspring from each litter were studied at age 4 mo (C, n⫽15; IUGR-H, n⫽18; IUGR-NR, n⫽18) or 7 mo (C, n⫽15; IUGR-H, n⫽18; IUGR-NR, n⫽18). Male offspring rats were killed by exsanguination while under sodium pentobarbital-induced anesthesia (Somnotol, MTC Pharmaceuticals, Ontario, Canada, ⬃60 mg/kg-body wt). All procedures in this study were approved by the University of Alberta Animal Welfare Committee and were in accordance with the guidelines of the Canadian Council on Animal Care.
Scientific Corporation, Torrington, CT, USA). Rats were trained 2 weeks before blood pressure measurement. Following 5 min acclimatization, blood pressures were recorded approximately every 40 s for 10 min. Data were collected from 1–2 rats/litter for 8 control (5 litters), 10 IUGR-H (5 litters) and 3 IUGR-NR (2 litters) offspring. Measurements were repeated on separate days, with an average of 2 days per animal used to calculate an individual mean blood pressure. Isolated working heart preparation Hearts were excised from anesthetized rats prior to exsanguinations and perfused for 10 min in retrograde Langendorff mode with Krebs-Henseleit solution (37°C) containing (mmol/l) 120 NaCl, 25 NaHCO3, 5 glucose (Glc), 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, and 1.25 CaCl2 (pH 7.4, gassed with 95% O2-5% CO2) against a constant perfusion pressure of 60 mmHg. Hearts were then switched to antegrade working perfusion mode and perfused in a closed recirculating system at 37°C with 100 ml modified Krebs-Henseleit solution containing (mmol/l) 2.5 CaCl2, 5 Glc, 1.2 palmitate prebound to 3% BSA (fraction V), 0.5 lactate, and 100 mU/l insulin. Buffer entered the cannulated left atrium at a pressure equivalent to 11.5 mmHg, and passed to the left ventricle (LV), from which it was spontaneously ejected through the aortic cannula against a pressure equivalent to 80 mmHg (afterload). Pressures were continuously recorded using a 1.4-Fr micromanometer (Millar Instruments, Houston, TX, USA) inserted through the aorta into the left ventricle. After a 10-min equilibration, hearts were paced at 300 beats/min with an electrical stimulator via one silver electrode attached to the right atrium. Heart rate, aortic systolic and diastolic pressures, left ventricular pressure, left ventricular end diastolic pressure, and – dp/dt were recorded using a HSE data acquisition system and IBM computer with HSE isoheart software for windows 2000 (Harvard Apparatus Canada, SaintLaurent, Quebec, Canada). Cardiac output and aortic flow were measured using a TTFM Transit time flowmeter Type 700 (Harvard Apparatus Canada, Saint-Laurent, Quebec, Canada). Cardiac function (systolic pressure⫻cardiac output) and coronary flow were calculated as described previously (23–25). Measurements of cardiac function were carried out every 10 min during baseline and reperfusion. Hearts that showed any cardiac disturbance (ventricle arrhythmia and fibrillation) at any time during the experiment were excluded. All hearts were subjected to a modified working heart protocol: perfusion for a 50-min stabilization period and global ischemia (20 min) followed by reperfusion (40 min). At the end of the experiments, myocardial tissue and perfusate samples were stored at –70°C for subsequent analysis. Measurement of lactate dehydrogenase release in coronary effluent To assess the extent of myocardial injury, effluent perfusate was collected at 10 and 50 min before ischemia and 5, 10, 20, 30, and 40 min during reperfusion. Lactate dehydrogenase (LDH) activity in coronary effluent was determined by spectrophotometry using a commercially available assay (SigmaAldrich, Mississauga, Canada) as described previously (26), and expressed as units per milliliter. Measurement of MMP-2 activity
Blood Pressure Measurement In a subset of rats aged to 6 mo, systolic blood pressure was determined using the tail cuff method (RTBP001-R, Kent CARDIAC EFFECT OF INTRAUTERINE GROWTH RESTRICTION
Zymography was performed as described previously (27). Protein was loaded at 25 g per lane and subjected to 7.5% SDS-PAGE copolymerized with gelatin (2.5 mg/ml). To reE537
veal zones of degradation, gels were stained with Coomassie blue overnight and then placed in a solution containing 20% methanol and 10% acetic acid for 6 h. Pure human MMP-2/ MMP-9 zymography standards (Chemicon) were used as a control. Gels were scanned with Fluor MultiImager (BioRad). Preliminary experiments showed that MMP-2 activity detected in the samples by zymography correlated with MMP-2 protein content detected by Western immunoblot (data not shown). Western blot analyses Samples were resolved by SDS-PAGE, as described previously (23). After electrophoresis, protein was transferred onto nitrocellulose membranes, and membranes were stained with antibodies against collagen I (1:100), collagen III (1:100), (Santa Cruz Biotechnology, Santa Cruz, CA), and ␣,  MHC (1:500, American Type Culture Collection, Beverly, MA). After washing membranes, we applied a peroxidase-conjugated avidin secondary antibody for visualization. To control for differences in protein concentration between samples or loading errors, blots were stripped and reprobed for ␣-actin (1:1000, Santa Cruz Biotechnology) expression. Scanning electron microscope studies Samples of LV tissue were placed in 2.5% glutaraldehyde solution immediately after collection and refrigerated at 4°C until processing. Samples were prepared for scanning electron microscopy (EM) by standard methodology. Briefly, samples were postfixed at room temperature for 1 h in 1% osmium tetroxide in Milonig’s buffer. They were then washed in distilled water and dehydrated (10 min each) in a series of increasing ethanol concentrations (50, 80, and 100%), followed by two additional periods in absolute ethanol. Samples were further dehydrated by critical point drying at 31°C for 5–10 min and then mounted on a specimen holder for drying overnight in a desiccator. Dehydrated samples were sputter coated with gold (Edwards, Model S150B Sputter Coater) and examined with a Hitachi 2500S scanning electron microscope. High-resolution digital images were acquired directly to a computer. Statistics Data are presented as mean ⫾ se. Both one-way and two-way ANOVAs were used to compare groups. Post hoc analysis was performed with Tukey or Student-Newman-Keuls tests, with significance defined as P ⬍ 0.05.
TABLE 1. Characteristics of animal model (4 months)
BW (g) LVW (mg) RVW (mg) LVW/BW (mg/g) RVW/BW (mg/g)
Control
IUGR-H
IUGR-NR
(n⫽16)
(n⫽15)
(n⫽15)
636.9 ⫾ 20.8a 1236 ⫾ 87 334 ⫾ 23
529.6 ⫾ 11.6b 1178.2 ⫾ 34 372 ⫾ 26
632.0 ⫾ 11.9a 1232 ⫾ 45 302 ⫾ 26
1.94 ⫾ 0.01a
2.23 ⫾ 0.08b
1.95 ⫾ 0.06a
0.53 ⫾ 0.03a
0.69 ⫾ 0.03b
0.47 ⫾ 0.04a
Table values are expressed as mean ⫾ se. Different letters indicate values that are significantly different at P ⬍ 0.05. BW, body weight, LVW, left ventricular weight, RVW, right ventricular weight.
IUGR-NR rats, but the RVW/BW ratio was not different (Table 2). These results suggest that LVH had occurred in IUGR-H but not IUGR-NR animals (Tables 1 and 2). Systolic blood pressure was not different in either IUGR-H or IUGR-NR rats compared to control group (C, 129⫾6 mmHg, IUGR-H, 128⫾5 mmHg, IUGR-NR, 134⫾6 mmHg). Expression of /␣ MHC, collagen I and III, and MMP-2 activity Western blots for ␣-actin showed no difference between the samples, suggesting equal loading (data not shown). At 4 mo, the ratio of left ventricular expression of /␣ MHC protein was significantly increased in the IUGR-H group compared to both control and IUGR-NR animals (Fig. 1A, P⬍0.05) whereas at 7 mo the /␣ MHC ratio was markedly increased in both IUGR-H and IUGR-NR rats (Figure 1B). MMP-2 activity was reduced in LV from the IUGR-H group compared to control and IUGR-NR (Figure 1C) at 4 mo. MMP-2 activity was lower in both the IUGR-H and IUGR-NR hearts at 7 mo (Figure 1D). Expression of collagen I and III was increased in left ventricle tissue from IUGR-H offspring at 4 mo compared to control or IUGR-NR offspring (Figures 2A and 2C). In 7-mo offspring, the expression of collagen I and TABLE 2. Characteristics of animal model (7 months)
RESULTS Body weight, blood pressure, and left and right ventricle weight Body weight was lower in the IUGR-H group than control at both 4 and 7 mo (Table 1 and 2). At 4 mo, the left ventricle wt (LVW)/body wt (BW) and right ventricle wt (RVW)/BW ratio were higher in IUGR-H offspring compared with control or IUGR-NR offspring (Table 1). There were no significant changes in absolute LVW and RVW in either 4 or 7 mo offspring (Tables 1 and 2). At 7 mo, the LVW/BW ratio remained higher in IUGR-H offspring compared to control or E538
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BW (g) LVW (mg) RVW (mg) LVW/BW (mg/g) RVW/BW (mg/g)
Control
IUGR-H
IUGR-NR
(n⫽18)
(n⫽16)
(n⫽15)
715.1 ⫾ 14.9a 1299 ⫾ 52 367 ⫾ 21
618.9 ⫾ 21.1b 1381 ⫾ 66 341 ⫾ 11
666.7 ⫾ 20.5a,b 1261 ⫾ 38 323 ⫾ 12
1.82 ⫾ 0.07a
2.26 ⫾ 0.01b
1.91 ⫾ 0.07a
0.52 ⫾ 0.02
0.56 ⫾ 0.02
0.49 ⫾ 0.02
Table values are expressed as mean ⫾ se. Different letters indicate values that are significantly different at P ⬍ 0.05. BW, body weight; LVW, left ventricular weight; RVW, right ventricular weight.
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Figure 1. Representative immunoblots of left ventricular  and ␣ MHC and the /␣ MHC ratio are depicted for offspring from control, maternal hypoxia (IUGR-H), and maternal nutrient restriction (IUGR-NR) groups at age 4 mo (A) and 7 mo (B). Representative zymograms of left ventricular MMP-2 activity and summary graphs are shown for offspring of control, IUGR-H, and IUGR-NR at age 4 mo (C) and 7 mo (D). Values represent means ⫾ se; n ⫽ 7. Different letters (a, b) indicate values that are significantly different from all other groups (P⬍0.05).
III was greater in both IUGR-H and IUGR-NR groups compared to control rats (Fig. 2B and 2D, P⬍0.05). Fibrillar collagen structure and composition were examined by scanning EM in left ventricle from 4- and 7-mo offspring. Left ventricular myocardial sections showed enhanced collagen matrix in IUGR-H rats compared to control and IUGR-NR aged 4 mo (Fig. 3). The collagen weave continued to increase in IUGR-H hearts. In control myocardium, a few fine weave collagen fibers were observed within the interstitial space. After 7 mo, this fine fibrillar collagen weave matrix appeared more dense in both IUGR-H and IUGR-NR rats compared to control. Cardiac function and recovery following I/R injury In 4 mo offspring, – dp/dt was significantly lower in IUGR-H rats compared to both control and IUGR-NR groups (Fig. 4A). LVEDP was also significantly increased in IUGR-H group (Fig. 4B). In 7-mo offspring, both IUGR-H and IUGR-NR rats had decreased – dp/dt (Figure 4C) and increased LVEDP (Figure 4D). These results indicated cardiac diastolic dysfunction in both IUGR-H and IUGR-NR rats at 7 mo. However, in 4- and 7-mo offspring, cardiac systolic function (⫹dp/dt and LVDP) did not differ among groups (data not shown). Baseline cardiac work (Fig. 5A and 5B) and cardiac output (Table 3) values were slightly lower in the CARDIAC EFFECT OF INTRAUTERINE GROWTH RESTRICTION
IUGR-H rats compared to the control and IUGR-NR groups at 4 mo. In addition, baseline function and cardiac output (Table 4) were lower in both IUGR groups at 7 mo (5C and 5D). This baseline function was confirmed in a series of hearts not subjected to ischemia. Following IR, IUGR-NR (32.2⫾4.4%) heart function recovered to the similar level in control offspring hearts (39.9⫾5.7%) at 4 mo; however, hearts from IUGR-H exhibited significantly lower recovery (9.7⫾2.5%, P⬍0.05, Figure 5A and 5B). In 7-mo offspring, recovery of function was significantly depressed in both IUGR-H (8.7⫾1.2%) and IUGR-NR (7.9⫾1.4%) hearts compared to control rats (34.7⫾5.3%, P⬍0.05, Figure 5C and 5D). During reperfusion, heart rate was not significantly different among the groups at either age (Tables 3 and 4). Peak systolic pressure (PSP) was significantly reduced from baseline in all groups (Tables 3 and 4). PSP during reperfusion was decreased in IUGR-H offspring compared to control and IUGR-NR offspring at 4 mo, while at 7 mo PSP was decreased in both IUGR-H and IUGR-NR hearts compared to control. Similarly, cardiac output was significantly decreased in all groups after I/R; however, a significantly greater reduction occurred in only the IUGR-H offspring at 4 mo (Table 3). At 7 mo, both IUGR-H and IUGR-NR offspring had reduced cardiac output compared to control offspring (Table 4). Coronary flow was decreased in the IUGR-H E539
Figure 2. Representative immunoblots of left ventricular collagen I and summary graphs are depicted for offspring from control, maternal hypoxia (IUGR-H), and maternal nutrient restriction (IUGR-NR) groups at age 4 mo (A) and 7 mo (B). Representative immunoblots of left ventricular collagen III and summary graphs are shown for offspring of control, IUGR-H, and IUGR-NR at age 4 mo (C) and 7 mo (D). Values represent means ⫾ se.; n ⫽ 7. Different letters (a, b) indicate values that are significantly different from all other groups (P⬍0.05).
group after I/R at 4 mo but unchanged in control and IUGR-NR groups (Table 3). Coronary flow was significantly depressed in both IUGR-H and IUGR-NR offspring compared to control at 7 mo (Table 4).
LDH release during reperfusion In all groups, following 20 min of ischemia there was a marked elevation in release of LDH into the coronary effluent after 5 min of reperfusion. Furthermore, LDH release in coronary effluent was progressively increased from 20 – 40 min of reperfusion (Fig. 6A and 6B). LDH levels in coronary artery effluent from control hearts were low after I/R, whereas LDH levels from IUGR-H hearts were significantly elevated at 4 mo and were markedly increased in both IUGR-H and IUGR-NR hearts compared to control hearts at 7 mo (Figure 6A and 6B).
DISCUSSION
Figure 3. Fibrillar collagen structure and composition were examined by scanning EM in 4- and 7-mo-old offspring from control, maternal hypoxia (IUGR-H) and maternal nutrient restriction (IUGR-NR) groups. Images of left ventricular myocardial sections showed enhanced collagen matrix in IUGR-H rats compared to control and IUGR-NR aged 4 mo (top). In control myocardium, a few fine-weave collagen fibers were observed within the interstitial space. After 7 mo, this fine fibrillar collagen weave matrix appeared more dense in both IUGR-H and IUGR-NR rats (bottom). E540
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These data demonstrate that the restriction of fetal substrate supply during development resulted in ultrastructural changes in left ventricular cardiac tissue. Furthermore, the changes in cardiac structure observed in all instances correlated functionally with diastolic dysfunction consistent with increased left ventricular stiffness and with increased sensitivity to I/R injury. By assessing both the effects of maternal hypoxia, which also reduced maternal food intake (9), and the effects of equivalent undernutrition alone, this study also addressed the relevance of the nature of substrate restriction in determining later cardiac effects. Maternal hypoxia produced structural and functional changes in cardiac tissue from 4-mo-old offspring that
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Figure 4. Cardiac function is shown for control, maternal hypoxia (IUGR-H), and nutrient restriction (IUGR-NR) from 4-mo and 7-mo-old offspring. The – dp/dt was significantly lower in IUGR-H rats compared to both control and IUGR-NR groups (A). LVEDP was also significantly increased in IUGR-H group (B). At 7 mo, both IUGR-H and IUGR-NR rats had decreased – dp/dt (C) and increased LVEDP (D). Different letters (a, b) indicate values that are significantly different from all other groups (P⬍0.05).
were not observed in offspring from the maternal nutrient restriction group. By 7 mo, however, similar impairment in cardiac structure and function was observed in both IUGR-H and IUGR-NR offspring. These data demonstrate that while the time course of changes may differ, both reduced fetal oxygen and nutrient supply produce a cardiac phenotype, which sensitizes the heart toward ischemic injury. Left ventricular remodeling Consistent with our previous observations in neonatal rat pups (9), relative left ventricle weight was increased
in only IUGR-H offspring, and was observed at both 4 and 7 mo of age, suggesting that left ventricular hypertrophy had occurred in IUGR-H but not IUGR-NR animals. At 4 mo, prenatal hypoxia, but not undernutrition also resulted in significant left ventricular remodeling in adult male offspring, as evidenced by increased /␣ MHC isoform ratio, accumulation of collagen type I and III, and reduced MMP-2 activity in left ventricular tissue from IUGR-H offspring. By 7 mo, these changes were observed in both IUGR-H and IUGR-NR offspring. The earlier occurrence of cardiac changes or left ventricular hypertrophy in IUGR-H offspring may suggest that the interaction of hypoxia
Figure 5. Recovery of cardiac function following ischemic/reperfusion is depicted for control, maternal hypoxia (IUGR-H), and nutrient restriction (IUGR-NR) offspring at 4 and 7 mo of age. Cardiac work (A) and recovery (B, percentage of baseline) at 4 mo, and cardiac work (C) and recovery (D) at 7 mo. Perfused isolated hearts were subjected to global ischemia (20 min) followed by reperfusion (40 min). Values are means ⫾ se. Different letters (a, b) indicate values that are significantly different from all other groups (P⬍0.05).
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TABLE 3. Hemodynamic parameters during baseline and reperfusion periods of ischemia-reperfusion protocol (4 months)
HR (beats/min) Baseline Reperfusion PSP (mmHg) Baseline Reperfusion CO (ml/min/g) Baseline Reperfusion CF (ml/min) Baseline Reperfusion
Control
IUGR-H
IUGR-NR
(n⫽14)
(n⫽10)
(n⫽10)
233 ⫾ 27 207 ⫾ 13
231 ⫾ 12 208 ⫾ 11
235 ⫾ 13 214 ⫾ 11
93 ⫾ 5 65 ⫾ 7a*
103 ⫾ 2 39 ⫾ 8b*
104 ⫾ 3 62 ⫾ 4a*
54 ⫾ 1a 44 ⫾ 1a
39 ⫾ 2b 14 ⫾ 3b*
53 ⫾ 2a 43 ⫾ 3a
20 ⫾ 2 18 ⫾ 0.3a
22 ⫾ 1 11 ⫾ 2b*
20 ⫾ 1 17 ⫾ 1a
Table values are expressed as mean ⫾ se. Two-way ANOVA with a Student-Newman-Keuls post hoc test was used for analysis. Different letters indicate values that are significantly different at P ⬍ 0.05 across the groups. *Significant differences at P ⬍ 0.05 between baseline and reperfusion. HR, heart rate; PSP, peak systolic pressure; CO, cardiac output; CF, coronary flow. Baseline taken after 50-min aerobic perfusion, Reperfusion after 40 min of recovery following ischemia.
and undernutrition constituted a dual insult, thus increasing the severity of the effects on the adult heart. Alternatively, hypoxia may produce specific effects on heart development, either directly (10) or through mechanical force alterations resulting from the hemodynamic adaptations of the fetus. To our knowledge, this is the first report that fetal growth restriction results in changes in /␣ MHC ratio in left ventricular tissue of adult offspring. In the heart, MHC-␣ and MHC- isoforms confer different functional properties (28 –30). In human cardiac tissue, TABLE 4. Hemodynamic parameters during baseline and reperfusion periods of ischemia-reperfusion protocol (7 months)
HR (beats/min) Baseline Reperfusion PSP (mmHg) Baseline Reperfusion CO (ml/min/g) Baseline Reperfusion CF (ml/min) Baseline Reperfusion
Control
IUGR-H
IUGR-NR
(n⫽13)
(n⫽12)
(n⫽13)
222 ⫾ 19 211 ⫾ 19
227 ⫾ 16 218 ⫾ 17
230 ⫾ 14 224 ⫾ 15
100 ⫾ 5 71 ⫾ 5a*
103 ⫾ 2 33 ⫾ 2b*
104 ⫾ 3 38 ⫾ 8b*
56 ⫾ 3a 46 ⫾ 3a
34 ⫾ 2b 14 ⫾ 3b*
33 ⫾ 1b 15 ⫾ 1b*
23 ⫾ 3 17 ⫾ 1a
22 ⫾ 1 9 ⫾ 1b*
23 ⫾ 1 11 ⫾ 1b*
Table values are expressed as mean ⫾ se. Two-way ANOVA with a Student-Newman-Keuls post hoc test was used for analysis. Different letters indicate values that are significantly different at P ⬍ 0.05 across the groups. *Significant differences at P ⬍ 0.05 between baseline and reperfusion. HR, heart rate; PSP, peak systolic pressure; CO, cardiac output; CF, coronary flow. Baseline taken after 50 min aerobic perfusion, Reperfusion after 40 min of recovery following ischemia.
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Figure 6. Lactate dehydrogenase (LDH) release in coronary effluent. A) 4-mo offspring. B) 7-mo offspring. Values are means ⫾ se. n ⫽ 10 to 14. Different letters (a, b) indicate values that are significantly different from all other groups (P⬍0.05).
although the MHC- isoform expression predominates in healthy tissue, there is a further increase in proportional MHC- expression in failing hearts (31). In rats, MHC-␣ isoform expression predominates in healthy cardiac tissue, and a transition to greater MHC- expression occurs during pathological left ventricular remodeling and experimental heart failure, which both slows myocardial contraction and decreases the efficiency of contraction (32, 33). Our data demonstrate that MHC- was increased and MHC-␣ decreased in left ventricular tissue of adult male offspring following either maternal hypoxia or undernutrition in utero. These changes suggest the restriction of fetal substrate supply induced pathological left ventricular remodeling. In the heart, collagen is the primary extracellular protein which supports the myocardium and provides tissue stiffness. A number of studies have indicated that collagen content, in particular collagen type I, increases with age in the normal heart (18, 34, 35). Interestingly, in this study, IUGR-H offspring demonstrated significant collagen accumulation, while relatively young, and in the absence of hypertension. By 7 mo, both IUGR-H and IUGR-NR offspring demon-
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strated significant collagen accumulation, relative to control. Previously, uterine artery ligation in pregnant guinea pigs also increased left ventricular interstitial collagen deposition in young (8 wk old) offspring, further suggesting that impaired prenatal development may result in dysregulated collagen deposition in the heart (36). In contrast to our data, and to the effects of utero-placental insufficiency in the adolescent guinea pig (36), maternal low-sodium diet during pregnancy did not increase staining for collagen in the rat heart at 12 wk of age (37). Ultimately, the nature of substrate deficit before birth may influence later cardiac structure. Along with increased collagen content, MMP-2 activity was reduced in 4-mo-old IUGR-H offspring and in both IUGR-H and -NR offspring at 7 mo. Recent studies have shown that the age-related increase in collagen content was associated with a decrease in MMP-1 and MMP-2 activity, suggesting that suppression of the degradative pathway may be partly responsible for these age-related changes (38). A reduction in MMP-2 activity is consistent with the observed accumulation of collagen in IUGR-H and -NR offspring and with the impaired ventricular relaxation (reduced – dp/dt). The degree of collagen cross-linking increases with age, which may contribute to the recognized age-associated decrease in ventricular function and the progression of LV chamber dilation (18 –20). The ability of the heart to respond to mechanical and chemical stimuli is influenced by the delicate balance between synthesis and degradation of extracellular structural components of ECM, which therefore has a significant impact on cardiac function (18 –20). Our data suggest that this balance may be perturbed by reduced oxygen, or nutrient supply in utero, such that inappropriate cardiac remodeling is favored. Heart function and recovery from ischemia/reperfusion injury Prenatal hypoxia induced diastolic dysfunction in IUGR-H offspring at both 4 and 7 mo, without any observed differences in systolic function. Maternal undernutrition alone did not influence baseline cardiac function in 4-mo-old offspring, but by 7 mo produced diastolic dysfunction in offspring similar to the hypoxia group. The reduced – dp/dt and higher left ventricular end diastolic pressures were consistent with increased left ventricular stiffness and therefore correlate well with the structural changes observed. Interestingly, diastolic dysfunction has previously been described in IUGR fetuses (39) and neonates (40), although the effects in adults are less clear. To our knowledge, these data are the first to describe the effects of undernutrition during pregnancy on adult heart function, or sensitivity to ischemia-reperfusion injury. In rats, the effects of prenatal hypoxia on heart function in adult offspring were assessed using the Langendorff isolated heart function system (5). This method involves perfusion of the left ventricle in a retrograde manner, with CARDIAC EFFECT OF INTRAUTERINE GROWTH RESTRICTION
constant perfusion pressure and controlled end diastolic pressure. Although no differences were observed in – dp/dt values in this study, in contrast to the present study, this may be accounted for by differences between Langendorff and working heart methodologies (41). The working heart system better approximates cardiac fluid dynamics than the Langendorff method (41). In the present study, progressive diastolic dysfunction occurred in IUGR-H and IUGR-NR offspring, suggesting that specific structural remodeling in the left ventricle following impaired prenatal development results in diastolic functional changes. At both ages, hearts from IUGR-H offspring also demonstrated impaired recovery from I/R injury, with greater postischemic release of LDH into coronary effluent. No changes were observed in cardiac function at 4 mo in IUGR-NR offspring; however, by 7 mo, IUGR-NR offspring also demonstrated similar impairment in recovery from I/R injury and greater LDH release. Consistent with these results, it has previously been reported that chronic prenatal hypoxia increased I/R injury in 6-mo-old rat offspring (5). As the effects of decreased maternal nutrition were not assessed in this study, it is not clear whether equivalent undernutrition may also have sensitized hearts by 6 mo of age. Candidate mechanisms identified in this study that may contribute to sensitizing the heart to I/R injury include decreased Hsp 70 and eNOS protein expression, and differential beta-adrenergic receptor expression in hearts from prenatal hypoxia offspring (5). Our data demonstrate that left ventricular remodeling is increased following prenatal hypoxia and suggest that this may also contribute to the sensitization of hearts to ischemia/reperfusion injury. Consistent with our study, low maternal sodium during the last week of pregnancy in rats produced cardiac hypertrophy, and structural changes in cardiomyocytes in 12-wk-old female offspring, further implying that perturbations in the in utero environment influence adult cardiac remodeling (37). In summary, the results of this study highlight the impact of deficits in the prenatal environment on the structure and functioning of the adult heart. In developed countries, placental insufficiency is a major clinical cause of IUGR, which reduces both oxygen and nutrient supply to the fetus (3, 42, 43). The cardiac effects observed in IUGR-H offspring may arise through specific effects of hypoxia, or relate to the interaction of hypoxia and undernutrition during prenatal life. In adulthood, the significant cardiac effects of prenatal hypoxia may be due to changes in heart structure that contribute to the incidence of cardiovascular disease in IUGR individuals. Maternal undernutrition during late pregnancy also impaired cardiac structure and function, although the effects were delayed relative to those resulting from maternal hypoxia. In a cohort study of adults born in southern India, a significant inverse relation was found between birth weight and coronary heart disease (44); however, no association was identified between birth weight and blood pressure, arterial E543
compliance, or left ventricular mass (45). As impaired prenatal growth may be more related to maternal undernutrition in developing than in developed countries (43), it is interesting to speculate that in some instances undernutrition may produce subtle changes in cardiac structure, in the absence of overt hypertension or left ventricular hypertrophy, which increase susceptibility to cardiovascular disease. Importantly, regression of left ventricular hypertrophy and remodeling may reduce the risk of later cardiovascular events (18, 21), and therefore represent one therapeutic avenue to reduce cardiovascular risk in adults whose growth was restricted in utero. This work was supported by a grant from the Canadian Institute of Health Research. S.J.W. is supported by the Premier’s Scholarship for Bioscience (South Australia). S.T.D. is the Canadian Chair in Women’s Cardiovascular Health and a Senior Scholar of Alberta Heritage Foundation for Medical Research.
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