Type 5 adenylyl cyclase plays a major role in stabilizing heart rate in response to microgravity induced by parabolic flight

Abstract

It is well known that autonomic nervous activity is altered under microgravity, leading to disturbed regulation of cardiac function, such as heart rate. Autonomic regulation of the heart is mostly determined by β-adrenergic receptors/cAMP signal, which is produced by adenylyl cyclase, in cardiac myocytes. To examine a hypothesis that a major cardiac isoform, type 5 adenylyl cyclase (AC5), plays an important role in regulating heart rate during parabolic flights, we used transgenic mouse models with either disrupted (AC5KO) or overexpressed AC5 in the heart (AC5TG) and analyzed heart rate variability. Heart rate had a tendency to decrease gradually in later phases within one parabola in each genotype group, but the magnitude of decrease was smaller in AC5KO than that in the other groups. The inverse of heart rate, i.e., the R-R interval, was much more variable in AC5KO and less variable in AC5TG than that in wild-type controls. The standard deviation of normal R-R intervals, a marker of total autonomic variability, was significantly greater in microgravity phase in each genotype group, but the magnitude of increase was much greater in AC5KO than that in the other groups, suggesting that heart rate regulation became unstable in the absence of AC5. In all, AC5 plays a major role in stabilizing heat rate under microgravity.

the autonomic nervous system is a major mechanism to regulate cardiac function (8). Norepinephrine released from the synaptic terminal binds to β-adrenergic receptors, leading to the activation of G proteins and thus adenylyl cyclases (AC) to produce cAMP. cAMP, a major second messenger, via activating protein kinase A, initiates a cascade of phosphorylation reactions within cardiac myocytes, leading to increased cardiac function (12). The release of norepinephrine is regulated by the central nervous system while the intracellular cAMP signal is regulated by the activity of AC in the heart. AC is a membrane-bound enzyme and is made of nine isoforms that have distinct biochemical properties and tissue distribution (9–11). It is known that the heart expresses seven AC isoforms (types 2, 3, 4, 5, 6, 7, and 9) while the type 5 isoform (AC5) is a major cardiac isoform in adults and is responsible for approximately one-third of the total enzymatic activity at baseline (22). The expression of this isoform is abundant in the heart while very low in the other peripheral organs (22). Multiple studies have demonstrated that this isoform provides an important mechanism of regulation with the autonomic nervous system under physiological and pathophysiological conditions, such as heart failure (9, 22–24, 28).

The molecular mechanisms to regulate cardiac function through the autonomic nervous system have been extensively studied in the past. However, those under unusual conditions, such as microgravity, have been poorly understood. This is partially because microgravity is a rare condition for living things on earth and the exposure to such an environment is not commonly achieved unless they are subject to space and/or jet flight (35). Currently, functional deterioration of the autonomic nervous system is known to develop under microgravity (3), as exemplified by orthostatic intolerance after space flight (5, 26). The molecular mechanisms for such disturbance remains poorly understood, mostly because the experimental opportunity is limited under microgravity and appropriate models are not readily available for such experiments. As yet, several experimental trials have been conducted under microgravity. The outcome, however, appears to vary according to experimental methods or models exposed to microgravity, partially because phenotypic changes were not always great and thus difficult to make conclusions (1, 4, 20, 26, 32, 34).

To address the above issues, for the first time, we employed the transgenic mouse models in microgravity experiments. We used transgenic mice, in which a single gene product that might play an important role in regulating cardiac function through the autonomic nervous system was either genetically disrupted or overexpressed. We overexpressed AC5, a major cardiac AC isoform, by the use of an α-myosin heavy chain promoter and thus AC activity was increased only in the heart (AC5TG; Ref. 6, 29) and AC5 knock-out (AC5KO) in which AC5 was disrupted. We hypothesized that if this molecule plays an important role in regulating cardiac function through the autonomic nervous system in response to microgravity, distinct phenotypic changes would appear between disruption (AC5KO) and overexpression (AC5TG) of this molecule when they are similarly exposed to microgravity stress (22–24). We exposed these mouse models to microgravity induced by parabolic flights and examined heart rate variability (HRV) as an index of regulating cardiac function by the autonomic nervous system. HRV has been used as such an index in many parabolic flight experiments with healthy human subjects and wild-type rats, although such experiments may suffer from a relatively small number of experimental subjects and the short duration of microgravity (1, 34). We will demonstrate that microgravity-induced changes in HRV were opposite between AC5KO and AC5TG, suggesting the important role of this enzyme isoform in such regulation under microgravity.

METHODS

Experimental animals and animal care.

Generation of AC5KO mice was described previously; mice were backcrossed to C57BL/6 strain for six generations (22). Transgenic mice with cardiac-specific overexpression of AC5 (AC5TG) was made using canine AC5 and the mouse α-myosin heavy chain promoter (14). All experiments were performed in 3-mo-old AC5KO (n = 5), AC5TG (n = 8), and wild-type (WT) controls (n = 9). The care and treatment of the animals were carried out according to the Japanese Government Animal Protection and Management Law (no. 105) and the Guidelines for Animal Experiments of Yokohama City University School of Medicine.

AC assays.

AC activity was measured in the heart of AC5TG (n = 5) and WT (n = 5) controls in advance of parabolic flight experiments. Mice were killed by cervical dislocation, followed by tissue harvesting. Membrane preparations were made as previously described (23, 24), and AC activity was measured by a modification of the method of Salmon et al. (25).

Western blotting.

Western blotting was conducted with commercially available antibodies (24).

Animal preparation and surgery.

ECG recordings were obtained with an implantable telemetric unit (PhysioTel, Data Sciences International, St. Paul, MN; Ref. 31). Mice were anesthetized with 2.5% tribrimoetahnol (0.010–0.015 ml/g) and then an abdominal midline incision was made on the ventral surface for implantation. Two other smaller incisions, one on each side, were also made in the pectoral region for suturing of the leads. Subcutaneously, these leads were directed cranially from the abdominal incision with a trochar sleeve and sutured into the pectoral muscles after the sleeve was removed. The 3.5-gauge wireless radiofrequency transmitter was then inserted into the peritoneal cavity. To anchor the implant in place, it was sutured to the abdominal muscles. Skin incisions were sutured, and a warming lamp maintained body temperature for recovery.

Parabolic flight experiment.

Parabolic flight experiments were performed using a jet airplane operated by the Diamond Air Service in Nagoya, Japan. In one parabolic flight, three or four conscious mice were placed separately in double-walled plastic cages in an airplane and they were freely floating during parabolic flight. Six parabolic flights were performed in this study. In one parabolic flight, which lasted for ∼1 h, 8–12 parabolas were performed in total with a 4- to 6-min interval between consecutive parabolas. At the end of each parabolic flight, mice were killed by cervical dislocation and replaced with the next mice.

Parabolic flight was divided into five phases. The first phase (I) was in normogravity [1 gravity (G)], the second phase (II) was in hypergravity (1.3 G), the third phase (III) was in microgravity (0.03 G), the fourth phase (IV) was in hypergravity (1.8 G), and the fifth phase (V) was in normogravity (1 G; Fig. 1). Each parabola provided 15–20 s of microgravity (phase III) and we evaluated data from the first parabola, except in Figs. 5B and 6B, in which the G force in the hypergravity phase (phase II) before the microgravity phase was kept below 1.3 G to avoid an excessive gravity stress on mice and the effect from the previous parabolas. We then compared changes in heart rate (HR) among phases and genotypes. During the experiment, the room temperature in the plane was kept at 22 ± 2°C and air pressure was 0.9 ± 0.1 atm.

Fig. 1.

Schematic representation of parabolic flight. Each parabola provided ∼20 s of 0.03 gravity (G). Parabolic flight was divided into 5 phases. The first phase (I) was normogravity (1 G), the second phase (II) was hypergravity (1.3 G), the third phase (III) was microgravity (0.03 G), the fourth phase (IV) was hypergravity (1.8 G), and the fifth phase (V) was normogravity (1 G). y-Axis indicates changes in gravity.

Fig. 5.

Comparison of R-R interval under microgravity. A: R-R interval under microgravity (phase III) from the first parabola is shown by the plot of time (s) vs. R-R interval (ms) in WT (left), AC5KO (middle), and AC5TG (right) (n = 4). B: R-R interval under microgravity (phase III) from the eighth parabola is shown by the plot of time (s) vs. R-R interval (ms) in WT (left), AC5KO (middle), and AC5TG (right) (n = 4).

Fig. 6.

Comparison of R-R interval under hypergravity. A: R-R interval under hypergravity (phase IV) from the first parabola is shown by the plot of time (s) vs. R-R interval (ms) in WT (left), AC5KO (middle), and AC5TG (right) (n = 4). B: R-R interval under hypergravity (phase IV) from the eighth parabola is shown by the plot of time (s) vs. R-R interval (ms) in WT (left), AC5KO (middle), and AC5TG (right) (n = 4).

Data acquisition and analysis.

Conscious mice were separately placed in double-walled plastic cages, which were placed on a receiver in a special rack within the aircraft, and data from the freely moving mice were recorded by the data acquisition system during flights. ECG signals were recorded from the telemetric unit with the use of an under-cage receiver (Data Sciences International, St. Paul, MN), digitized at a sampling rate of 2 kHz, and fed into a microcomputer-based data acquisition system (Power Lab System, AD Instruments, Milford, MA). ECG signal processing was performed with the software program Chart v5.0 and HRV analysis with the HRV plug-in for Chart v5.0 (AD Instruments). This software detects R waves from all ECG recorded leads after they are passed through a filter that eliminates noise and an algorithm that detects ECG fiducial points. All portions of R-R interval data were screened on the computer to confirm the sinus origin of the rhythm. After this, all questionable portions containing ectopic beats were excluded manually and only segments containing solely qualified beats were included in the final analysis. HRV measurements and analyses were conducted following published guidelines (19), and standard time-domain indexes were derived.

HRV measurements.

Indexes studied in the HRV measurements included mean HR, R-R interval (mean, max, minimum), the standard deviation of normal R-R intervals (SDNN), and the square root of the mean squared differences of successive R-R intervals (RMSSD). The SDNN index is a measure of total autonomic variability (16, 19). Note that the standard terminology in HRV analysis for the R-R interval is NN, indicating that the measurement is derived from two normal or sinus beats. The RMSSD index correlate with the high-frequency components from power spectral density and as such is a measure of parasympathetic modulation (18). The different G phases causes important changes in the R-R interval. Therefore, we also used the coefficient of variation (CV; in %) as a normalized index of SDNN (CV-SDNN) and RMSSD (CV-RMSSD) (1, 2): SDNN (ms) = SD of all normal R-R intervals; CV-SDNN (%) = SDNN/mean R-R × 100; RMSSD (ms) = square root of the mean squared differences of successive R-R intervals; CV-RMSSD (%) = RMSSD/mean R-R × 100.

Statistical analysis.

All data are reported as mean ± SD. Comparison of data was performed by two-tailed Student's t-test (Fig. 2A) or Kruskal-Wallis nonparametric test followed by Dunn test (Figs. 3, 4A, and 7). Differences were considered significant at P < 0.05.

Fig. 2.

Adenylyl cyclase (AC) activity and Western blotting in transgenic mice with AC5 overexpression (AC5TG). A: AC measured in wild-type (WT; n = 5) and AC5TG (n = 5). Stimulation was performed at the level of AC catalytic unit with forskolin (100 μM; *P < 0.01; 2-tailed Student's t-test). B: example of a Western blot analysis for protein expression of Gsα, Giα, Gβ, Gγ, and GRK2 obtained in WT (n = 4) and AC5TG (n = 4).

Fig. 3.

Comparison of heart rate (HR) during parabolic flights. Comparison of HR among phases I, II, III, IV, and V in each genotype group. HR was compared between phase I and phase II, III, IV, or V in each genotype group (*P < 0.01; nonparametric) and also between WT (A) and AC5 knockout (AC5KO; B) or AC5TG (C; +P < 0.05; nonparametric). bpm, Beats/min.

Fig. 4.

Comparison of R-R interval during parabolic flights. A: comparison of mean (mean), maximum (Max), and minimum (min) R-R intervals among phases I, II, III, IV, and V in each genotype group. Maximum R-R interval was compared with minimum R-R interval in each genotype group (*P < 0.05; nonparametric). B: representative ECG recordings in phase I and phase III in each genotype group.

Fig. 7.

Comparison of SDNN, CV-RMSSD, RMSSD, CV-RMSSD during parabolic flights. The standard deviation of normal R-R intervals (SDNN) (A), coefficient of variation (CV) in % as a normalized index of SDNN (CV-SDNN) (B), square root of the mean squared differences of successive R-R intervals (RMSSD) (C), and coefficient of variation (CV) in % as a normalized index of RMSSD (CV-RMSSD) (D) in phases I, II, III, IV, and V. These indexes were compared between phase I and other phases in WT (+), AC5KO (*), and AC5TG (#) (P < 0.05 vs. phase I; nonparametric).

RESULTS

AC assays and Western blotting.

The steady-state AC activity was determined in AC5TG as the maximal cAMP production over 15 min in the presence of 100 μM forskolin (Fsk), which represents the total amount of AC activity with minimal involvement of the receptor and G proteins (13; Fig. 2A). Fsk-stimulated AC activity showed an eightfold increase in AC5TG compared with that in WT (WT, 534 ± 20; AC5TG, 4,266 ± 812 mol·15 min⁻¹·mg⁻¹, P < 0.01). For comparison, AC activity was decreased in AC5KO relative to that in WT by 30–40%, as shown by us and others (22, 28). We also examined the expression of Gsα, Giα, Gβ, Gγ, and GRK2 by Western blotting, which was not different in AC5TG (Fig. 2B), as well as in AC5KO from that in WT, as also recently shown by us (22).

The effect of parabolic flight on HR.

Mean HR in each genotype group was evaluated in each phase of parabolic flight. The HR in phase I in AC5KO and AC5TG was significantly higher than that in WT (539 ± 65 vs. AC5KO; 599 ± 77 vs. AC5TG; 643 ± 74 beats/min, P < 0.05; Fig. 3), suggesting that basal HR was increased in these groups as previously shown by others and us [AC5KO (22, 28) and AC5TG (30)]. HR showed a trend of decrease in later phases (phases II-VI) in WT and AC5TG (Fig. 3, A and C) while not in AC5KO (Fig. 3B). Thus HR changes in response to gravity change were attenuated in AC5KO.

The effect of parabolic flight on R-R interval of HRV.

Maximum, mean, and minimum R-R interval in each genotype group were next examined during parabolic flight (Fig. 4). Maximum, mean, and minimum R-R intervals, in general, increased gradually in later phases (I through IV) in WT and AC5TG (Fig. 4A, left and right). When maximum, mean, and minimum R-R intervals were compared in each genotype group, we found that the degree of variance of the R-R interval was smaller in AC5TG and much greater in AC5KO. In particular, we found that the difference between maximum and minimum R-R interval was extremely large in phase III of AC5KO (Fig. 4A, middle). For further analysis, the R-R intervals under microgravity (phase III) (Fig. 5) and hypergravity (phase IV; Fig. 6) were plotted by time (s) vs. R-R interval (ms) in each genotype group from first (Figs. 5A and 6A) and the following, i.e., eighth parabola (Figs. 5B and 6B), showing that R-R interval during microgravity of the first parabola was much more variable in AC5KO (Fig. 5A, middle) while more stable in AC5TG than that in WT not only under microgravity (Fig. 5) but also under hypergravity (Fig. 6) in both the first and the following parabola. It is thus tempting to speculate that the lack of AC5 unstabilized R-R interval under microgravity and the overexpression of AC5 stabilized R-R intervals under hypergravity as well as under microgravity during parabolic flight.

The effect of parabolic flight on SDNN and RMSSD of HRV.

The above findings implicated the presence of difference in HR regulation under microgravity when the amount of AC5 expression was altered. Because HR under microgravity is most likely regulated by the autonomic nervous system, we compared SDNN as a measure of total autonomic instability (16). SDNN (Fig. 7A) and also another parameter CV-SDNN (Fig. 7B) were significantly greater under microgravity (phase III) and hypergravity (phase II and IV) than that in phase I (normogravity) in each genotype group. Notably, when these changes were expressed as fold-increase from that in phase I, the magnitude of increase in SDNN was strikingly greater in AC5KO under micro (∼40-fold)- and hypergravity (∼9-fold) while the degree of such increases was much smaller in both WT and AC5TG (∼3-fold), suggesting that HR was more variable under micro- and hypergravity in AC5KO. It was thus tempting to speculate that the lack of AC5 unstabilized HR regulation not only under microgravity but also under hypergravity. We also examined RMSSD (Fig. 7C) and also another parameter CV-RMSSD (Fig. 7D), which correlates with the high-frequency components from power spectral density and as such is a measure of parasympathetic modulation, showing that these indexes demonstrated very strong positive correlation to SDNN or CV-SDNN as previously demonstrated (17).

DISCUSSION

Results from the current study suggested that AC5 may stabilize HR control because R-R interval was extremely variable under microgravity in AC5KO, compared with that in WT and AC5TG. More important, total autonomic variability index (SDNN) was increased to a greater degree in AC5KO than in WT and AC5TG under microgravity and its increase was smaller in AC5TG than in WT and AC5KO. Accordingly, our findings suggest that the amount of AC5 expression is strongly related to the degree of the stability of HR regulation under microgravity; when AC5 expression is abundant, HR stability was increased, and when scarce, the stability was decreased.

When AC5 was disrupted in AC5KO, basal cardiac function was not decreased paradoxically, despite AC5 being responsible for at least one-third of cardiac AC catalytic activity, as shown recently by us and others (22, 28). In contrast, overexpression of AC5 enhanced basal HR as shown by a previous report (30) and also by this study. Accordingly, AC5 may not contribute to the maintenance of basal HR, but may play a major role in regulating HR when autonomic nerve activity is changed under pathological conditions such as heart failure (23, 24) or under extraordinary conditions such as microgravity. We do not completely understand the mechanisms that contribute to increased basal HR in AC5KO (22, 28). However, our previous studies indicated the lack of muscarinic inhibition in AC5KO that normally occurs in WT, which might lead to increased basal HR. In support of this concept, abnormal baroreflex response in HR was also shown in AC5KO (22). The current report agrees with this concept, at least in part, in that the lack of AC5 attenuated the stability of HR control in response to microgravity. Previous studies suggest that baroreceptors were activated under microgravity to suppress sympathetic activity in standing positions of healthy human subjects and anesthetized rats to eliminate emotional stress (1, 7, 34).

Our study also demonstrated the usefulness of genetically engineered mouse models in examining functional changes under such extraordinary conditions. Indeed, so far as we are concerned, this is the first study to use genetically engineered models for such a study despite multiple studies conducted in the past half century (33). These studies tried to examine changes in autonomic nerve activity under microgravity during parabolic flight. Studies using rats demonstrated that HR was decreased (27) or increased (34), and arterial pressures were also decreased (4) or increased (34) despite similar experimental protocols. These inconsistent results were because, at least partially, responses to microgravity were never great in their magnitude and only limited numbers of animals can be examined per flight. Use of genetically engineered mouse models might overcome this problem, at least partially, because genetic manipulation may be able to amplify the magnitude of such responses if a single gene product that may play an important role in such regulation is selectively overexpressed or disrupted. If this is the case, such findings would confirm the importance of the gene product in such regulation, as implicated in our study.

We do not know whether a short exposure to microgravity during parabolic flight can really mimic changes during space flight, which may last much longer, even if findings in our study are similar to those obtained in human study during parabolic flight (2, 15, 21). We also are aware that parabolic flight study has major limitations such as the small number of subjects, transient and short duration of microgravity (∼20 s), and influence of hypergravity phase, even if such a study can provide an opportunity to study human and animal adaptation to prolonged exposure to microgravity (1, 2, 34).

Nevertheless, our study demonstrated that genetic manipulation of AC5, which can be achieved in mice, amplified cardiac responses under microgravity, and strongly suggested that AC5 plays an important role in stabilizing HR under microgravity.

GRANTS

This study was supported in part by the grants from the Japan Space Forum (Y. Ishikawa); the Japanese Ministry of Education, Culture, Sports, Science, and Technology (Y. Ishikawa and S. Okumura); the Kitsuen Kagaku Research Foundation (Y. Ishikawa); the National Institutes of Health (GM-067773 and HL-059139; Y. Ishikawa); Fukuda Foundation for Medical Technology (S. Okumura); Takeda Science Foundation (S. Okumura); Grant for Research on Autonomic Nervous System and Hypertension from Kimura Memorial Foundation/Pfizer Pharmaceuticals (S. Okumura); Yokohama Foundation for Advancement of Medical Science (S. Okumura); Grant for 2006–2007 Strategic Research Project (No. K19027) of Yokohama City University, Japan (S. Okumura); and Mitsubishi Pharma Research Foundation (S. Okumura).