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1Laboratory of Physiologic Studies, National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, Maryland 20892; 2Inotek Pharmaceuticals Corporation, Beverly, Massachusetts 01915; 3Department of Pharmacology and Pharmacotherapy, Semmelweis University, Budapest, Hungary H-1082; 4Critical Care Division, Department of Internal Medicine, University Hospital, Lausanne 1005, Switzerland; 5Department of Anesthesia and Critical Care, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114; 6Department of Surgery, University of Medicine and Dentistry- New Jersey Medical School, Newark, New Jersey 07103; 7Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences, Budapest H-1083; and 8Institute of Human Physiology and Clinical Experimental Research, Budapest, Hungary H-1082
Submitted 3 May 2004 ; accepted in final form 25 June 2004
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ABSTRACT |
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systolic dysfunction; diastolic dysfunction
The major limitation of the previously used invasive and noninvasive approaches to study the cardiac function in small animal models is that the hemodynamic parameters measured were largely dependent on loading conditions. Pressure-volume (P-V) analysis is a useful approach for examining intact chamber function independently of loading conditions. This analysis has been widely used in large animal studies and in humans (22–24, 27, 31, 36). Recent advances in the development and validation of miniature P-V catheters has made it possible to use this approach for studies in small animals (16, 18, 19, 29, 32, 41). Although P-V loop analysis is widely used in mice, the combined P-V conductance catheter for rats has been introduced only recently, and very limited normative data are available.
Here, we aimed to characterize the baseline cardiac performance and systolic and diastolic function at different preloads in aging F344 male rats using the Millar P-V conductance catheter system.
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METHODS |
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Animals.
Male F344 rats were obtained from the National Institute on Aging at 3 (n = 12) and 24 mo of age (n = 20) and studied 1–2 mo later. Rats were housed two to three per cage, fed a standard laboratory diet and water ad libitum, and maintained on a 12:12-h light-dark cycle. During the observation period, 
50% of the aging and none of young control rats died, and autopsy revealed in most of the cases large dilated hearts and marked fluid accumulation in the chest cavity.
Hemodynamic measurements.
Rats were anesthetized with thiopental sodium (60–80 mg/kg ip): aging animals with heart failure required 15–20% less anesthetic than young rats. The animals were tracheotomized to facilitate breathing and placed on controlled heating pads. The core body temperature measured via a rectal probe was maintained at 37°C. A microtip P-V catheter (SPR-838, Millar Instruments; Houston, TX) was inserted into the right carotid artery and advanced into the LV under pressure control as described previously (32–34). Polyethylene (PE) catheters (PE-50) were inserted into the right femoral artery for measurement of mean arterial pressure (MAP) and the right femoral vein for fluid administration. After stabilization for 20 min, the signals were continuously recorded at sampling rate of 1,000/s using an ARIA P-V conductance system (Millar Instruments) coupled to a PowerLab/4SP A/D converter (AD Instruments; Mountain View, CA) and a personal computer.
HR, maximal LV systolic pressure (LVSP), LV end-diastolic pressure (LVEDP), MAP, maximal slope of systolic pressure increment (+dP/dt) and diastolic pressure decrement (–dP/dt), time constant of LV pressure decay (
), ejection fraction (EF), stroke volume (SV), end-diastolic volume (EDV), CO, and stroke work (SW) were computed using a cardiac P-V analysis program (PVAN3.2, Millar Instruments) as described previously (13, 32). SV and CO were calculated and corrected according to in vitro and in vivo volume calibrations using PVAN3.2 (32). CO and SW were normalized to body weight [cardiac index (CI) and SW index (SWI)]. The total peripheral resistance index (TPRI) was calculated by the following equation: TPRI = MAP/CI.
LV P-V relations were assessed by transiently compressing the inferior vena cava. Indexes of contractility and LV stiffness {preload recruitable stroke work (PRSW), +dP/dt-end-diastolic volume relation (+dP/dt-EDV), slope of end-systolic and end-diastolic P-V relations [ESPVR (Emax) and EDPVR]} as well as the efficiency of LV work [SW/P-V area (PVA)] were calculated using PVAN3.2.
The volume calibration of the conductance system was performed as described previously (32, 31). Briefly, nine cylindrical holes in a block 1 cm deep and with known diameter ranging from 2 to 11 mm were filled with fresh heparinized whole rat blood. In this calibration, the linear volume-conductance regression of the absolute volume in each cylinder versus the raw signal acquired by the conductance catheter was used as the volume calibration formula (29, 32, 41). At the end of each experiment, 50 µl of 15% saline were injected intravenously, and from the shift of P-V relations parallel conductance volume (Vp) was calculated by PVAN 3.2 and used for correction for the cardiac mass volume as described previously (32, 41).
Cardiac remodeling. After the hemodynamic measurements, the animals were killed by exsanguination (transsection of the inferior vena cava). The heart was removed, and the LV and right ventricle were dissected and weighed (the septum was included in the LV weight). The weight of each ventricle was then normalized to body weight. The lungs were also removed and weighed. Wet lung weights were normalized to body weight, and the ratio was used as an index of pulmonary congestion (28, 33).
Statistical analysis. An unpaired two-sided Student's t-test was used to compare tissue weights and hemodynamic parameters of aging and young rats. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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Cardiac function.
Figure 1 shows typical P-V loops (top) and the pressure signal and dP/dt (bottom) obtained from young and aging animals. The shift of the P-V loops to the right and the decreased amplitude and +dP/dt in aging rats indicates a decrease in cardiac contractility. As shown in Table 1, aging was associated with decreased MAP, LVSP, +dP/dt, –dP/dt, EF, SV, CI, and SWI. In contrast, LVEDP, , and EDV were increased in aging animals, indicating diastolic dysfunction. TPRI was also increased in aging rats. There was a slight (11%) but not significant decrease in the HR of aging animals (Table 1).
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We also determined the relation between +dP/dt and EDV. +dP/dt is a classic parameter that is sensitive to changes in contractility but dependent on changes in preload. Analysis of +dP/dt-EDV allowed us to compare +dP/dt of aging and young rats at a given EDV. Figure 3, bottom, shows that the slope of this relation was steeper in young rats, indicating a decreased contractility in aging.
Efficiency of LV work was also decreased in aging rats compared with young rats (Table 1).
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DISCUSSION |
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We found that aging was associated with decreased +dP/dt and EF (Table 1). Although +dP/dt has been widely used as a cardiac contractile parameter, it is well recognized that it is load dependent, especially on changes in preload (22–24, 27). EF is also known to be influenced by both preload and afterload and, therefore, cannot reliably be used to assess the contractile function in the models where both preload and afterload are altered.
Historically, ESPVR (Emax) was proposed as a fairly load-insensitive index of contractility. ESPVR was decreased in aging rats. However, because this relation can be altered not only by changes in inotropic state but also by changes in chamber geometry and other diastolic factors, we also calculated other parameters (22, 23, 31).
PRSW, the linear relation between SW and EDV, may be sensitive to changes in systolic function. It has advantage of being stable in the hearts of different sizes. Although it integrates data from the entire cardiac cycle, it is influenced most of all by systole (22, 23). PRSW was also decreased in aging rats compared with young controls.
Previous investigations (24, 27) have demonstrated that +dP/dt-EDV, another P-V-derived index, represents a sensitive but less load-dependent parameter of chamber contractility. In previous studies, the slope of this relationship increased and shifted leftward with increased contractility, and it decreased and shifted rightward with depressed contractility. We found that this index was also depressed in aging animals compared with young controls.
Our baseline hemodynamic data obtained with the P-V system are in a good agreement with the results of Capasso et al. (9). In that study, F344 rats were anesthetized with chloral hydrate (300 mg/kg ip) followed by measurements of ventricular pressure-derived parameters and ascending aortic flow. From 20 mo of age, there was an elevation in LVEDP, a significant decrease in +dP/dt and –dP/dt, a reduction of SV and CO, and an increase in total peripheral resistance. At 29 mo of age, EF, +dP/dt, –dP/dt, SV, CO, and HR were reduced by 59, 38, 48, 27, 40, and 17%, respectively, whereas LVEDP and total peripheral resistance increased by 275 and 59%, respectively (9). Similarly, in our study in barbiturate-anesthetized F344 male rats using P-V analysis, at 26 mo of age, EF, +dP/dt, –dP/dt, SV, CI, and HR were reduced by 43, 36, 41, 29, 44, and 11%, respectively, whereas LVEDP and total peripheral resistance increased by 139 and 41%, respectively (Table 1). In addition to the baseline parameters of contractility, we showed that all load-independent indexes of LV contractility (see above and also Table 1 and Figs. 2 and 3) demonstrate decreased contractile pump function in aging rats. The indexes of LV failure were accompanied by alterations in ventricular size and shape consisting of increases in the transverse and longitudinal chamber diameters and an abnormal expansion in diastolic and systolic ventricular chamber volumes. Consistent with the expansion of the diastolic chamber volume in the previous report (9), we also observe an increase in EDV in aging F344 rats using the P-V system (Table 1). A similar age-associated decline in cardiac function was also reported in mice using the P-V technique (41).
In a more recent study, Boluyt et al. (5) confirmed both systolic and diastolic dysfunction in isoflurane-anesthetized 30-mo-old female F344 rats using echocardiographic assessment. However, in the latter study, the reduction of systolic performance was less pronounced (EF and fractional shortening were decreased by 9 and 25%, respectively) than it was demonstated by Capasso et al. (9) and by the present investigation (see above). The reason for the less depressed systolic function found by Boluyt et al. (5) can be the protection against development of the cardiac dysfunction and heart failure in female F344 rats compared with male rats used in the previous (9) and our present studies.
One can also argue that aged rats are more sensitive to the cardiovascular depressant effects of barbiturate anesthesia than younger animals, and this could have a significant impact on the magnitude of changes noted in the aged versus younger animals. Although there is no perfect pharmacological agent available for anesthesia in the elderly, dozens of clinical studies have arrived to the same conclusion that no significant difference in outcome can be attributed solely to use of any specific anesthetic agent (4). Barbiturates have been a standard element of anesthesia practice both in the young and elderly patients for more that 50 years. Individual variability in response to thiopental is well known. In elderly patients, an age-related decrease in the initial distribution volume or differences in clearance to the rapidly equilibrating compartments were found (20). Compared with younger patients, geriatric patients require a 20–30% lesser induction dose (12). On the basis of the latter study, we also used a reduced dose of the drug in our aging rats (see METHODS). The predominant cardiovascular effect of thiopental is venodilation, followed by pooling of blood in the periphery (14). Myocardial contractility may be depressed, but not to the extent seen after volatile anesthetics (17). Importantly, barbiturates do not sensitize the heart to cathecholamines. Systemic vascular resistance usually remains unchanged, and no arrhythmia occurs. We have successfully used barbiturates for anesthesia in various rat models of shock, myocardial infarction, and heart failure and reported comparable, if not better, hemodynamic parameters than using inhalation agents (15, 33, 34). In a previous study (35) using 22-mo-old aging Wistar rats, we reported impaired endothelial nitric oxide-mediated dilation of isolated aortic rings without any depression of systolic cardiac function using the same anesthesia protocol. Thus it is unlikely that the increased sensitivity of aging animals to thiopental could have a significant contribution to the hemodynamic changes observed in the present study.
Impaired ventricular relaxation and increased end-diastolic stiffness were also observed in aging animals, as reflected by the decreased –dP/dt, prolonged , and increased LVEDP and EDPVR. Relaxation, an active process, depends mostly on calcium uptake by the sarcoplasmic reticulum during diastole, and under normal conditions usually takes place during the first third of diastole. End-diastolic stiffness is predominantly affected by alterations in myocardial structural components; however, it may also be influenced by various other factors. We observed both delayed relaxation and increased stiffness in aging rats. Consistent with diastolic dysfunction, LVEDP was also increased in aging animals.
Potential limitations of the study. P-V loop analysis, which is widely used in mice, has become a prerequisite for the assessment of LV function because it is the only technique that allows measuring the LV performance independently from loading conditions. The combined pressure-conductance probe for rats has been introduced only recently, and limited normative data are available. The proper measurement of absolute volumes is the most vulnerable part of the P-V technique, especially in a situation that requires the comparison of groups with markedly different ventricular sizes. In these conditions, problems could arise from the potential limitation in the construction of the probe, which has only two pairs of conductance electrodes at the fixed distance from each other. Nevertheless, as our results demonstrate, the baseline P-V data are very similar to the earlier data obtained by using flow measurements in the same aging-associated heart failure model (9). Importantly, our baseline SV data in control young adult rats are very similar to the data recently obtained by MRI (124.3 ± 8.8 vs. 126.8 ± 8 µl) (30), also suggesting that in most earlier studies the rat SV and CO were markedly overestimated.
Because aging is also associated with vascular dysfunction and remodeling, it is conceivable that the impairment of vascular function and the aging-associated decrease in cardiac contractility and heart failure are interrelated: an impairment of endothelial function may lead to global or regional myocardial ischemia, which may secondarily impair cardiac performance. Therefore, myocardial aging in this model should rather be interpreted as a time-dependent biological process that interacts with ischemic heart disease and other pathological conditions, which together define the phenotype.
In conclusion, our data demonstrate decreased systolic performance accompanied by delayed relaxation and increased diastolic stiffness of the heart in the advanced model of aging-associated heart failure using the Millar P-V conductance catheter system. The P-V methodology could be a very useful approach for the assessment of LV function in various pathophysiological conditions associated with cardiac dysfunction and/or heart failure in rats, and, furthermore, it could be successfully applied to evaluate potential pharmacotherapies against these conditions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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) and an aging rats (
). PRSW, preload recruitable SW. Insets: overall slope values as means ± SE of 8–10 experiments in each group. *P < 0.05 vs. young rats. Note that for both relationships, slope values are higher in young than in aging rats, suggesting that systolic performance is decreased in aging rats.