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1Department of Biomedical Engineering, 2Department of Medicine, Cardiology Division, and 3Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland
Submitted 25 October 2004 ; accepted in final form 16 March 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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2 wk were determined. Heart rate (HR) and mean arterial pressure (MAP) responses to TBCO were measured. In control mice, TBCO increased HR (15 ± 2 beats/min, P < 0.05) and MAP (17 ± 2 mmHg, P < 0.05). These responses were markedly potentiated in denervated control (DC) mice, in which the aortic depressor nerve and sympathetic trunk were sectioned before measurement. Baroreflex responses to TBCO were eliminated by blockade with hexamethonium bromide (10 µg/kg). In HLU (denervated) mice, HR and MAP responses were reduced 70% compared with DC mice. In part 2, myocardial contractile responses to TBCO were measured with a left ventricular micromanometer-conductance catheter. TBCO in DC mice increased the slope of the end-systolic pressure-volume relation (end-systolic elastance) by 86 ± 13%. This inotropic response was attenuated (14 ± 10%, P < 0.005) after HLU. In part 3, contractile responses to isoproterenol were impaired in myocytes isolated from HLU mice. In conclusion, selective carotid baroreceptor unloading stimulates HR, blood pressure, and myocardial contractility, and HLU attenuates each response. These findings have important implications for the management of OI in astronauts, the elderly, and individuals subjected to prolonged bed rest.
hindlimb unweighting; microgravity; baroreflex; orthostatic hypotension; mouse; cardiovascular deconditioning
There are several unanswered questions regarding autonomic reflex control of blood pressure after exposure to microgravity. Specifically, the contribution of altered myocardial contractility to impaired autonomic control of blood pressure in microgravity remains unknown. In addition, most investigations of autonomic reflex responses in models of microgravity before our study have used closed-loop methodologies to assess baroreflex control of heart rate (HR). Previous methodologies measure the HR response after alteration of peripheral resistance with vasoconstrictor or dilator agents. The differentiation of HR from blood pressure baroreflex responses, possible only via an open-loop method, is critical to our understanding of OI, inasmuch as each parameter may be independently influenced in pathophysiological scenarios (22).
We developed techniques to assess open-loop baroreflexes in the mouse. Isolation of the carotid sinus baroreceptor region and transient bilateral carotid occlusion (TBCO) with resultant selective carotid arterial baroreceptor unloading have enabled us to determine the individual roles played by baroreflex mechanisms in control of HR and mean arterial pressure (MAP) in mice. We have used this model to test the hypothesis that baroreflex control of HR and MAP is attenuated in a mouse model simulating microgravity. Moreover, by measuring left ventricular (LV) pressure-volume relations (integrated cardiovascular function), we tested the prediction that myocardial contractile responses are also attenuated in HLU mice. In addition, we measured contractile reserve in isolated cardiac myocytes from HLU mice. Together, our findings demonstrate that HLU leads to a combined impairment in HR, MAP, and LV contractile responses after selective carotid baroreceptor unloading.
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METHODS |
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45°C. Part 1: Common Protocol
After halothane (3%) induction in an anesthetic chamber, mice were anesthetized using a dedicated vaporizer with 1.5% halothane added to a constant flow of 1:1 O2-N2, which was delivered using an adapted face mask. Anesthesia was maintained with halothane during the entire surgery, and 1:4 albumin-saline was administered via the right jugular vein with a 30-gauge needle. The cervical region was exposed via a midline ventral incision, the right and left thyroid lobes were retracted, and the sternomastoid muscles were retracted bilaterally. The sternoyhoid was looped with 6-0 silk and retracted over the trachea to expose the common carotid sinus and artery region. Carotid arteries parallel to the trachea were carefully isolated. After 5 min of steady-state MAP and HR monitoring and measurement, a baroreflex response was induced by selective carotid baroreceptor unloading through transient occlusion of both carotid arteries (TBCO) with microclips (Accurate Surgical and Scientific Instruments, Westbury, NY). After 10–15 s of TBCO, MAP and HR were measured. Microclips were released, and steady-state MAP and HR readings were maintained before another trial was attempted. This was repeated four to six times in each mouse.
The afferent neural signal was generated by a change in blood pressure sensed by the carotid baroreceptors, distal to the point of carotid occlusion. However, other baroreceptors in the aortic arch and heart that lie proximal to the point of occlusion will not sense a decrease in arterial pressure and, thus, will buffer the cardiovascular effector responses. Hence, there is a need for aortic arch baroreceptor deafferentation. The relative locations of the aortic depressor nerve (ADN) and the sympathetic trunk (ST), the two major buffering afferents, have been previously reported (24).
Groups of mice. One group of mice acted as controls (n = 9), in which the above-described protocol was performed with intact ADN and ST. A second subgroup of mice acted as denervated controls (DC, n = 7). In these mice, afferent signals from the aortic arch and cardiopulmonary baroreceptors were eliminated by bilateral sectioning of the ADN and ST (25), allowing for complete isolation of the carotid baroreceptor reflex.
After denervation of the ADN and ST, the ganglionic blocker hexamethonium bromide (10 µg/kg in 200 µl of saline) was administered to a third subgroup of mice (Hex, n = 4). Hexamethonium was administered via the right jugular venous line, and 5 min were allowed for effect before any procedures were done.
A fourth group of mice (HLU, n = 6) was subjected to HLU for 14–21 days before physiological assessment. HLU mice were briefly anesthetized with halothane to minimize discomfort to the animal during the process. The tail was cleaned, and a light coat of tincture of benzoine was applied. The tail was air-dried until it was tacky. The adhesive strip tapes were looped through a swivel harness and applied to a freely rotating ball-bearing line, allowing free 360° rotation. The hindlimb of the mouse was elevated to create a 35° angle with the ground, so that only the front limbs were in contact with the floor. After the specified duration of HLU, the mice were lowered, weighed, and subjected to cardiovascular parameters measured in response to TBCO with ADN and ST denervation. All animals in the HLU group were denervated before physiological assessment to provide unbuffered comparison with DC mice.
In all subgroups of mice mentioned above, MAP and HR were measured before, during, and after TBCO, as described above. MAP was measured via a femoral arterial line (Tapered R-FAC microrenathane tubing, Brain Tree Scientific, Braintree, MA) linked to a pressure transducer (Statham P23 Db), and HR was obtained from the blood pressure trace via a Biotach/ECG transducer. The data were collected at 1,000 Hz (Biopac Data Acquisition, Biopac Systems, Santa Barbara, CA) and digitally stored.
Part 2: Measurement of In Vivo Myocardial Contractile Responses (Pressure-Volume Loops)
In a study separate from part 1, a different group of mice was subjected to HLU for 14–21 days (n = 6) as described above and were compared with caged control mice that were not subjected to HLU (DC, n = 20). None of the mice in this study were used in part 1 or part 3. Mice were anesthetized with a combination cocktail of morphine (80 µg/kg), urethane (200 µg/kg), and etomidate (120 µg/kg) and were ventilated (inspired O2 fraction = 100%, tidal volume = 200 µl) with a sinusoidal solenoid valve after tracheostomy. Buffering nerves (ADN and ST) were denervated, and carotid arteries were isolated as described above. After a substernal lateral thoracotomy, a 1.4-Fr micromanometer-conductance catheter (Millar Instruments, Houston, TX) was advanced retrogradely into the LV by an apical stab wound made with a 30-gauge needle through the longitudinal cardiac axis, as previously described (4, 38). The catheter was advanced until the distal tip was placed in the aortic root and the proximal electrode just within the endocardial wall of the LV apex. Albumin-saline (1:4) was administered via the right jugular vein with a 30-gauge needle. Offset calibration of the recorded volume signal was obtained by a saline-washin technique, and stroke volume calibration was derived from direct measurement of aortic blood flow, obtained by using a flow probe (model AT01RB, Transonics, Ithaca, NY) (4, 38). Pressure, volume, and flow signals were digitized at 1 kHz, stored on a disk, and analyzed with custom software.
Indexes of myocardial systolic and diastolic performance were derived from pressure-volume data obtained at steady state and during transient unloading via vena caval occlusion (VCO) of the heart. Baseline and VCO readings were acquired before and after TBCO for HLU and DC mice.
VCO data were used to determine the slope of the end-systolic pressure-volume relation (ESPVR) and the first derivative of the pressure (dP/dt)-end-diastolic volume relation. Myocardial contractility was indexed by the peak rate of rise in LV pressure (dP/dt) divided by instantaneous pressure and the load-independent end-systolic elastance (Ees), i.e., the slope of the ESPVR. Although the ESPVR is nonlinear in mice, over the range of data obtained by preload and afterload changes, the ESPVR can be considered linear (21). End systole is measured as the point of peak elastance [peak pressure-to-volume (P-V) ratio] and are plotted for each P-V loop. A linear regression line is fit through these points, and the slope is derived. Over the range of the data obtained by VCO, the ESPVR was found to be linear with minimal error. Baseline cardiac preload was indexed as the LV end-diastolic volume and end-diastolic pressure. Cardiac afterload was evaluated with effective arterial elastance (Ea, i.e., the ratio of LV systolic pressure to stroke volume).
Part 3: In Vitro Myocyte Studies
Mice for this in vitro study were not used for the in vivo studies mentioned above. Myocytes (n = 27 for each group) from HLU (n = 3) and age-matched control mice (control, n = 3) were isolated. Hearts were perfused with Ca2+-free buffer containing (in mmol/l) 120 NaCl, 5.4 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 5.6 glucose, 20 NaHCO3, 10 2,3-butanedione monoxime (Sigma), and 5 taurine (Sigma), gassed with 95% O2-5% CO2, and then subjected to enzymatic digestion with collagenase type 2 (1 mg/ml; Worthington) and protease type XIV (0.1 mg/ml; Sigma). Myocytes were obtained by mechanical disruption of digested hearts, filtration, centrifugation, and resuspension in 0.125 mmol/l Ca2+-Tyrode solution containing (in mmol/l) 144 NaCl, 1 MgCl2, 10 HEPES, 5.6 glucose, and 5 KCl, with pH adjusted to 7.4 with NaOH. Myocytes were resuspended in 0.25 mmol/l Ca2+-Tyrode solution and then in 0.5 mmol/l Ca2+-Tyrode solution and stored in Tyrode solution containing 0.5 mmol/l probenecid and 1.8 mmol/l Ca2+.
Myocytes were incubated with 5 µmol/l fura 2-AM (Molecular Probes, Eugene, OR) and then transferred to a Lucite chamber on the stage of an inverted microscope (model TE 200, Nikon) and continuously superfused with Tyrode solution containing 1.8 mmol/l Ca2+ and 0.5 mmol/l probenecid. Sarcomere length and Ca2+ transients (3) were measured in myocytes stimulated with increasing doses of isoproterenol. These experiments were conducted in room air (PO2 21%).
Sarcomere length was recorded with a charge-coupled device camera (model iCCD, IonOptix). Change in average sarcomere length was determined by fast Fourier transform of the Z-line density trace to the frequency domain. Intracellular Ca2+ concentration ([Ca2+]i) was measured using the Ca2+-sensitive dye fura 2 and a dual-excitation spectrofluorometer (IonOptix, Milton, MA) alternately excited with a xenon lamp at wavelengths of 365 and 380 nm. The emission fluorescence was reflected through a barrier filter (510 ± 15 nm) to a photomultiplier tube. The fura 2 fluorescence ratio, i.e., the ratio of the photon live count detected by excitation at 365 nm to that detected by excitation at 380 nm, represents [Ca2+]i.
Statistics
Statistical analyses were performed using StatView (SAS Institute). Values are means ± SE unless otherwise stated. The differences between each group were determined using ANOVA. Post hoc analyses were performed by using Student-Newman-Keuls test. P < 0.05 was considered significant.
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RESULTS |
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HR and blood pressure responses to TBCO.
Baseline MAP and HR were similar in control and DC mice (Table 1). As expected, TBCO increased MAP and HR in control mice, whereas the TBCO-stimulated increase in MAP and HR was substantially enhanced in DC compared with control mice (
MAP = 40 ± 2 vs. 17 ± 2 mmHg, HR = 33 ± 3 vs. 15 ± 2 beats/min, n = 9, P < 0.01 for both; Fig. 1A, Table 1). Representative HR and MAP traces are shown in Fig. 1B for control and DC mice. The carotid sinus baroreflex-mediated HR and MAP increases are substantially enhanced after ADN and ST deafferentation, consistent with the concept that these nerve trunks were buffering the response to baroreceptor unloading of the TBCO.
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MAP = 8 ± 2 mmHg, HR = 9 ± 1 beats/min, n = 4, P < 0.001 vs. DC; Table 1, Fig. 1A).
Impact of HLU on baroreflex function.
In all HLU mice, buffering nerves were denervated before TBCO. Baseline HR and MAP were similar in HLU and DC mice. However, in HLU mice, HR and MAP responses to TBCO were markedly attenuated: MAP = 14 ± 1 mmHg and HR = 10 ± 2 beats/min (n = 6, P < 0.01 vs. DC; Table 1, Fig. 2).
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Table 2 summarizes heart weight-to-body weight ratios as well as baseline hemodynamic data from DC (all animals undergoing integrated cardiovascular interrogation were denervated) and HLU mice. The mice were instrumented with a micromanometer-conductance catheter. HR and systolic pressure were lower in HLU than in DC mice (Table 2). Similarly, baseline indexes of myocardial contractility were lower in HLU than in DC mice. Furthermore, there was a significant decrease in the heart weight-to-body weight ratio in HLU mice, suggesting a component of myocardial atrophy in this model (Table 2).
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The demonstration that activation of the endogenous sympathetic response resulted in not only a markedly attenuated pressor response but also a depressed contractility response prompted us to study the contractile reserve in isolated cardiac myocytes from HLU and control mice. Myocytes were isolated from a separate group of control and HLU mice. Simultaneous sarcomere shortening and [Ca2+]i were measured in isolated myocytes in response to increasing concentrations of the 
-agonist isoproterenol. Isoproterenol increased sarcomere shortening in a dose-dependent manner in control and HLU mice (Fig. 4). However, the contractile response to isoproterenol was significantly attenuated in the HLU mice (Fig. 4). Although the percent change in sarcomere shortening was significantly depressed in HLU mice, there was no significant difference in Ca2+ transients in response to -adrenergic receptor activation in control and HLU mice. This suggests that the mechanism responsible for depressed contractility in HLU mice involves alterations in Ca2+ sensitivity or decreased myocardial mass, rather than alterations in [Ca2+]i.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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A major new finding of this study is that myocardial contractility is clearly augmented in response to TBCO and that this effect is profoundly impaired by HLU. Data obtained in space and in ground-based human and animal studies have been at odds with regard to cardiac contractile impairment in microgravity-associated OI. Koenig et al. (23) demonstrated reduced cardiac output and MAP during lower body negative pressure associated with lowered peak +dP/dt in rhesus monkeys exposed to head-down tilt for 4 days. On the other hand, Ray et al. (33) demonstrated no change in baseline dP/dt in HLU rats. Our study using isovolumic phase (peak +dP/dt) and end-ejection (Ees) indexes of myocardial contractility and isolated myocyte studies suggest that HLU leads to reduced baseline and baroreflex-stimulated LV contraction. These findings cannot be attributed solely to reduced pressor responses (afterload), inasmuch as we confirmed reduced contractility with the preload- and afterload-independent index Ees. In addition, isolated myocytes demonstrate impaired contractile responses to -adrenergic receptor activation (reduced contractile reserve). The observation that depressed sarcomere shortening is associated with no alteration in [Ca2+]i suggests a mechanism other than that involving dysregulation of Ca2+ cycling but, perhaps, an alteration of contractile sensitivity or mechanical alterations in the contractile apparatus itself. This is consistent with the finding that cardiac mass as a percentage of total body weight is decreased, providing a potential explanation for the depressed contractile reserve without an effect on the Ca2+ transient.
Our study supports the hypothesis that the contractile responses of the heart may be involved in OI. Adaptational changes in cardiac structure may have a significant influence on cardiac function and are supported by previous findings in humans and animals. The loss in ventricular mass found in HLU agreed with the echocardiographic measurements from the Skylab 4 mission. Arbeille et al. (2) showed that the LV wall thickness (measured by echocardiography) was significantly reduced in subjects after 6 wk of bed rest. In addition, Levine et al. (24) found that, after 2 wk of bed rest, normal subjects showed a significant reduction in ventricular mass (measured by MRI). Moreover, studies with rats in real or simulated microgravity also showed significant differences with respect to cardiac structure. Goldstein et al. (20) reported that the average cross-sectional area of LV papillary muscle fibers was significantly decreased by 20% in rats flown on COSMOS 2044 for 14 days. Thus the decreased LV mass found in HLU mice correlates well with the cardiac echocardiographic data found in astronauts and the heart weights of rats after HLU. Our findings demonstrating a reduced heart weight-to-body weight ratio as well as a [Ca2+]i-independent mechanism explaining impaired contractile reserve are congruent with the hypothesis regarding cardiac atrophy.
Our findings with regard to HR attenuation are consistent with observations in humans after exposure to microgravity and prolonged bed rest. Fritsch-Yelle et al. (18) reported decreased slope, range, and operational point of HR responses to baroreceptor activation (by using a neck pressure device) in astronauts, whereas Kamiya et al. (22) demonstrated similar findings in individuals after prolonged (120 days) bed rest. This is further confirmed and highlighted by the Neurolab findings (8). Although the diminished HR response after HLU in mice agrees with human bed rest and microgravity studies, attenuated HR responses are less obvious in rats. Overton et al. (30) and Tipton (37) examined HR responses to various sympathomimetic agents and lower body negative pressure in HLU rats and found that HLU affected baroreflex pressor, but not HR, responses. Nevertheless, it is possible that this discrepancy may be a function of the difference in methods used to elicit baroreflex responses (lower body negative pressure vs. TBCO) or the duration of HLU (2 days vs. 21 days).
Similarly, diminished pressor responses have also been observed in animal models and in bed rest and postflight studies. In a subset of presyncopal astronauts, Fritsch-Yelle et al. (19) demonstrated attenuated norepinephrine release after orthostatic challenge after return to the 1-g environment, which could contribute to diminished orthostatic tolerance. Also consistent with this finding, Shoemaker et al. (35) demonstrated decreased mean sympathetic neural activity in response to an orthostatic challenge after bed rest in a group of OI subjects. On the other hand, Cox et al. (8) demonstrated that sympathetic baroreflex gain was not impaired after microgravity, raising the possibility that end-organ hyporesponsiveness contributes to OI. End-organ hyporesponsiveness is further supported by animal data demonstrating impaired responses in isolated arteries and veins (13) to norepinephrine and other contractile agonists (9, 10, 12, 13, 32).
Astronauts, patients exposed to bed rest, and animal models of microgravity are subject to 15–18% plasma volume loss (14). This can contribute to the OI that astronauts experience on their return. However, hypovolemia alone is unlikely to be responsible for the hemodynamic changes observed in humans and in animal models of microgravity. If loss of circulating volume alone were responsible for the attenuated MAP observed with baroreflex activation observed in our mice, one would expect to see an enhanced HR response. For example, hypovolemia increases baroreflex sensitivity and cardiopulmonary baroreflex gain (7). Therefore, the attenuated HR response after HLU is driven primarily by changes in baroreflex regulation itself, whether it is due to autonomic nervous balance- and/or end-organ hyporesponsiveness.
It is important to note that previous studies examining baroreflex regulation of HR in the mouse have primarily used closed-loop methodologies, whereby MAP and HR changes are directly influenced by vasoconstrictor and dilatory agents (27). Thus HR responses are measured in response to changes in MAP. With a closed-loop method, only the HR baroreflex response is mediated by the carotid sinus baroreceptor. A technically unique feature of our study is that the carotid sinus baroreflex is exclusively isolated to determine the HR, MAP, and integrated cardiovascular parameters, such as contractility, afterload, and preload responses to baroreceptor activation, in the mouse. This is particularly critical when there is a divergence of sympathetic and vagal efferent baroreflexes, as is likely in the exposure to microgravity (8).
Limitations.
Our study was potentially limited by other effects of TBCO. It is possible that TBCO caused cerebral ischemia and low PO2 exposure of chemoreceptor regions in the carotid sinus and brain (causing a chemoreceptor-mediated baroreflex response). This could have potentially activated additional effector responses, which would alter the baroreflex responses to HR and MAP. Yang et al. (40) reported that C57BL/6 mice are more susceptible than other strains to global cerebral ischemic injury. However, 
15 min of TBCO was needed to produce evidence of irreversible neuronal cell damage in this strain of mice. Also, mice in our study that were ventilated with 100% oxygen still showed a significant baroreflex response, which most likely excludes the chemoreceptor response as a major player in the response that we observed after TBCO.
Another possible limitation of the study is the length of HLU. Although, for the majority of experiments examining the effects of simulated microgravity on the cardiovascular and musculoskeletal system in rodents, we used 14–21 days (1, 9, 11–13, 15, 26, 34, 39), it remains unclear whether this amount of time represents an appropriate simulation. Furthermore, it remains to be determined whether this represents long- or short-term exposure, given the life span of rodents (2 yr). Time-response experiments will be necessary to determine the differential effect of duration of HLU on cardiovascular changes.
A further potential limitation is that TBCO represents an extreme form of baroreceptor unloading. However, to our knowledge, there is no technique available to test the blood pressure response to unloading other than this open-loop technique. Our laboratory has previously performed a graded unloading technique by completely isolating the carotid sinus baroreceptor in a rat (36). In this study, the MAP-carotid sinus pressure relation was determined by stepwise changes in the carotid sinus pressure. The average peak gain (Pa/ISP, where Pa is arterial pressure and ISP is intrasinus pressure) was obtained from the maximum slope (point to point) of the arterial pressure-carotid sinus pressure relation. The peak gain in the rat is 2.0 and occurs at a carotid sinus pressure of 110 mmHg. The responses are completely flat at <60 and >160 mmHg. If this can be extrapolated to the mouse, we can surmise that although TBCO may seem to represent an extreme perturbation, the responses suggest that the system is responding over its defined operating range. Thus, even if the carotid sinus pressure is below 60 mmHg during TBCO, we would not predict any further response.
Indeed, it would be ideal to be able to completely isolate the carotid sinus of the mouse and generate MAP-carotid sinus pressure relations. These experiments, in combination with pressure-volume loops, would be extremely challenging technically and are beyond the scope of this study.
The dose of isoproterenol (10–6 M) that caused a divergence in responses between control and HLU myocytes in part 3 is worthy of discussion. Although this dose may be considered a nonphysiological stimulus that is endogenously unattainable in relation to serum levels (16, 17), it is widely accepted that 10–6 M isoproterenol is needed to show maximal adrenergic responses in vitro (31, 41). It is unclear why there are such differences between endogenous levels of catecholamines and exogenous doses to provide maximal responses, but this is beyond the scope of this study.
In conclusion, we have examined the individual contributions of MAP, HR, and contractility baroreflex responses by using a novel baroreflex activation procedure in the mouse (TBCO). This physiological stimulus was employed to activate carotid baroreceptors and was used to demonstrate that baroreceptors in mice significantly influence all three cardiovascular responses: HR, MAP, and myocardial contractility. Importantly, all three responses were markedly impaired in animals subjected to HLU. HLU mice have a lower baseline inotropic state and also demonstrate attenuated HR, blood pressure, Ea, and cardiac contractility responses to TBCO. Therefore, the chronotropic, inotropic, and pressor contributions to the overall baroreflex response can have further crucial implications in providing countermeasures for OI in astronauts and in patients subjected to prolonged bed rest.
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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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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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O2 max and regional blood flow responses to exercise. J Appl Physiol 66: 653–659, 1989.3-Adrenoceptor deficiency blocks nitric oxide-dependent inhibition of myocardial contractility. J Clin Invest 106: 697–703, 2000.[ISI][Medline]
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