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Am J Physiol Regul Integr Comp Physiol 302: R68–R74, 2012. First published October 19, 2011; doi:10.1152/ajpregu.00340.2011.

Innovative Methodology

Ventricular function during exercise in mice and rats Heidi L. Lujan, Hussein Janbaih, Han-Zhong Feng, Jian-Ping Jin, and Stephen E. DiCarlo Department of Physiology, Wayne State University School of Medicine, Detroit, Michigan Submitted 29 June 2011; accepted in final form 13 October 2011

Lujan HL, Janbaih H, Feng HZ, Jin JP, DiCarlo SE. Ventricular function during exercise in mice and rats. Am J Physiol Regul Integr Comp Physiol 302: R68 –R74, 2012. First published October 19, 2011; doi:10.1152/ajpregu.00340.2011.—The mouse has many advantages over other experimental models for the molecular investigation of left ventricular (LV) function. Accordingly, there is a keen interest in, as well as an intense need for, a conscious, chronically instrumented, freely moving mouse model for the determination of cardiac function. To address this need, we used a telemetry device for repeated measurements of LV function in conscious mice at rest and during exercise. For reference, we compared the responses in mice to the responses in identically instrumented conscious rats. The transmitter body of the telemetry device (rat PA-C40; mouse PA-C10; Data Sciences International, St. Paul, MN) was placed in the intraperitoneal space through a ventral abdominal approach (rat) or subcutaneously on the left flank (mouse). The pressure sensor, located within the tip of a catheter, was inserted into the left ventricle through an apical stab wound (18 gauge for rat; 21 gauge for mouse) for continuous, nontethered, recordings of pulsatile LV pressure. A minimum of 1 wk was allowed for recovery and for the animals to regain their presurgical weight. During the recovery period, the animals were handled, weighed, and acclimatized to the laboratory, treadmill, and investigators. Subsequently, LV parameters were recorded at rest and during a graded exercise test. The results document, for the first time, serial assessment of ventricular function during exercise in conscious mice and rats. This methodology may be adopted for advancing the concepts and ideas that drive cardiovascular research.

However, the original, pioneering work recording ventricular function in conscious mice required that the mouse be restrained and/or tethered to the recording system (14, 17, 38). These procedures prevented the study of left ventricular (LV) function during activity or exercise. It is especially important to evaluate the cardiovascular system during exercise, since more can be learned about how a system operates when it is forced to perform than when it is idle (33). Specifically, exertion may reveal cardiovascular phenotypes that are not apparent under resting conditions (11). Furthermore, restraint is a powerful stressor (34). Accordingly, the previous studies would be significantly advanced with a model that includes a permanently implanted pressure sensor and the use of telemetry for continuous measurements of left ventricle pressure (LVP) during exercise or in the freely moving, unrestrained, mouse. This model would be of major importance for advancing the concepts and methods that drive cardiovascular research. Therefore, to extend the earlier pioneering work, we used a permanently implanted pressure sensor and the use of telemetry for repeated measurements of LV hemodynamics in conscious mice at rest and during exercise. For reference, we compared the responses in mice to the responses in identically chronically instrumented conscious rats.

gene-manipulated; mouse models; embryonic stem cell technology

MATERIALS AND METHODS

Surgical Procedures THE MOUSE HAS SIGNIFICANT advantages over other experimental models for the molecular investigation of cardiac function. For example, with the exception of man, significantly more is known about the genetics of mice than any other mammal (5–9, 25, 29, 32). Furthermore, investigators have identified spontaneous mouse mutants and used embryonic stem cell technology to design mice with mutations in many nonlethal genes to understand cardiac function. Specifically, several gene-manipulated mouse models of cardiac hypertrophy and cardiac failure are now available (10, 13, 22, 28, 37, 40). Accordingly, gene-manipulated mice are an important model in cardiovascular research (1, 4, 18 –20, 27, 30, 37). These ground-breaking efforts for gene-manipulated mouse models have generated an interest in, as well as a need for, a conscious, chronically instrumented, freely moving mouse model for the determination of cardiac function (36, 38). Original work has made it possible to record ventricular pressure (14), as well as ventricular pressure and volume (17) in conscious, restrained mice (the mice were restrained in a soft plastic cone). These pioneering studies overcame the confounding influences of anesthesia and surgical trauma.

Address for reprint requests and other correspondence: S. E. DiCarlo, Wayne State Univ. School of Medicine, 540 E. Canfield Ave., Detroit, MI 48201 (e-mail: [email protected]). R68

Experimental procedures and protocols were reviewed and approved by the Animal Care and Use Committee of Wayne State University and complied with The American Physiological Society’s “Guiding Principles in the Care and Use of Animals”. Six adult, male C57BL/6 mice and seven adult, male Sprague-Dawley rats were studied to determine LV function at rest and during exercise. Six additional adult, male C57BL/6 mice were studied to determine the time to recover day/night variations in heart rate and LVP following surgical trauma and anesthesia, as well as the effects of acute anesthesia on heart rate and LV parameters. All surgical procedures were performed using aseptic surgical techniques. Mice and rats were anesthetized with pentobarbital sodium (50 mg/kg ip), atropinized (0.05 mg/kg ip), intubated, and prepared for aseptic surgery. Supplemental doses of pentobarbital sodium (10 –20 mg/kg ip) were administered when the subjects regained the blink reflex or responded during the surgical procedures. Radiotelemetry Implantation After anesthesia was induced, all animals were intubated and supported on a ventilator. The heart was approached via a left thoracotomy through the 3rd (mouse) or 4th (rat) intercostal space. The heart was positioned to visualize the apex. A loose purse-string suture (8 – 0 silk mouse and 6 – 0 silk rat) was placed around the apex. We used a 21-gauge or 18-gauge needle (for mouse and rat, respectively) to puncture the myocardium in the center of the purse-string suture. Subsequently, the catheter of a telemetry device (mouse PA-C10 and rat PA-C40; Data Sciences International) was routed

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Innovative Methodology LVP IN CONSCIOUS RATS AND MICE

through the 6th intercostal space and chronically inserted into the left ventricle through the apical stab wound for continuous, nontethered, recording of pulsatile LV pressure. The LV pressure signal was monitored for proper placement. Once verified, the purse string suture was drawn closed, and the heart was returned to its normal position. The ribs were approximated, the chest was closed in layers, and the skin was closed. The transmitter body was placed in the intraperitoneal space through a ventral abdominal approach (rat) or subcutaneously on the left flank (mouse). A minimum of 1 wk was allowed for recovery and for the animals to regain their presurgical weight. During the recovery period, the subjects were handled, weighed, and acclimatized to the laboratory, treadmill, and investigators. A postmortem analysis of the catheter placement and positioning was conducted in all animals. Catheter placement was within the mid-left ventricle in every animal. Experimental Procedures Heart rate and LV hemodynamic parameters at rest and during exercise. Following recovery, the subjects were brought to the laboratory and allowed to adapt to the environment for ⬃1 h to ensure stable hemodynamic conditions. After the stabilization period, beat-

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by-beat, steady-state hemodynamic variables were recorded over 10 –15 s. Subsequently, each subject ran continuously, without aversive stimuli on a motorized treadmill at 5, 10, 15, and 20 m/min on a 10% grade for ⬃3 min at each workload (23). The steady-state LV parameters and heart rate responses were recorded during the 3rd min at workloads 5–20 m/min (Fig. 1). Although no aversive stimuli were used, all subjects required gentle coaxing, (i.e., tapping on the hindquarters) during the highest workload. By using these relatively low workloads without aversive stimuli and providing training sessions, we feel we are truly studying a response to exercise rather than a response to stress. Time to recover day/night variations in heart rate and LV hemodynamic parameters following surgical trauma and anesthesia in mice. Once fully awake from anesthesia following the procedures to implant the telemetry device, the mice were transferred to a dedicated animal room with limited access. The mice were housed individually in standard cages. The room was controlled with a 12:12-h day (6 AM– 6 PM)/ night (6 PM– 6 AM) light cycle, and room temperature ranged between 27 and 29°C. In this setting, LV pressure and heart rate were recorded for 10 s every 5 min for 11 consecutive days starting the night of the surgery. This procedure was implemented to

Fig. 1. Second analog recordings of LV pressure and dP/dt at rest and during the 3rd min of a graded exercise protocol (5, 10, 15, and 20 m/min) in a mouse (A) and in a rat (B) are shown. AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00340.2011 • www.ajpregu.org

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determine the time required to obtain the normal day/night variations in heart rate and LV pressure following surgical trauma and anesthesia. Effect of anesthesia on resting heart rate and LV hemodynamic parameters in mice. Following recovery (2 wk) from the surgical procedures for implanting the radio telemetry transmitter, heart rate and LV parameters were recorded before and following anesthesia with pentobarbital sodium (50 mg/kg ip). Briefly, the mice were brought to the laboratory and allowed to adapt to the environment for ⬃1 h to ensure stable hemodynamic conditions. After the stabilization period, beat-by-beat, steady-state hemodynamic variables were recorded over 10 –15 s. Subsequently, each mouse received an injection of pentobarbital sodium (50 mg/kg ip). Once anesthetized, the mice were placed on a feedback-based temperature control system (model no. 40 –90-8; FHC, Bowdoin, ME) for monitoring and maintaining body temperature within the physiological range. Thirty minutes following the anesthesia, steady-state LV parameters and heart rate responses were recorded. This procedure was implemented to determine the effect of acute anesthesia on heart rate and LV function parameters in mice. Data analysis. All recordings were sampled at 2 kHz, and the data were expressed as means ⫾ SE. All exercise and anesthesia data were the average of every beat during the last 10 –15 s of the period. To determine the time to recover day/night variations in heart rate and LV hemodynamic parameters, data were recorded for 10 s every 5 min for 11 consecutive days and 12-h day and night averages were obtained. A two-factor ANOVA with repeated measures on one factor with post hoc Holm-Sidak method was used to compare LV function parameters and heart rate at rest and during exercise for the conscious mice and rats (Figs. 2 and 3). The force-frequency relationship for mice and rats was determined by plotting the relationship between heart rate and the percent change from rest for LV systolic pressure, dP/dt⫹, and dP/dt⫺. Plots with linear regressions and slopes were obtained for each animal. Subse-

quently, group (mice and rats) averages were calculated for slopes and the maximum percent change for each variable. Group averages were compared with an unpaired Student’s t-test. RESULTS

Heart Rate and LV Hemodynamic Parameters at Rest and During Exercise Figure 2A, presents heart rate, while Fig. 2B presents the percent change in heart rate from rest during graded treadmill exercise (5, 10, 15, and 20 m/min) for mice and rats. Heart rate was significantly higher in mice compared with rats at rest and at each workload (significant group effect). Furthermore, heart rate was significantly higher during exercise compared with rest in both mice and rats (significant exercise effect and group ⫻ exercise interaction). However, there was no difference in the percent change in heart rate from rest between mice and rats (Fig. 2B). Figure 3A presents LV systolic and diastolic pressures, while the percent change from rest in LV systolic pressure is presented in Fig. 3B and the percent change from rest in LV diastolic pressure (Fig. 3C) during graded treadmill exercise (5, 10, 15 and 20 m/min) is presented for mice and rats. LV systolic pressure was significantly higher in mice compared with rats at each workload (significant group effect). Furthermore, LV systolic pressure was significantly higher during exercise compared with rest (significant exercise effect). In contrast, there was no difference in LV diastolic pressure between mice and rats. Furthermore, exercise increased LV diastolic pressure only at the 3 highest workloads. Finally, mice had a significantly greater percent change from rest in LV systolic pressure (Fig. 3B) and diastolic pressure (Fig. 3C) at each workload (significant group effect). Figure 4A presents LV dP/dt⫹ and dP/dt⫺ and the percent change from rest for dP/dt⫹ (Fig. 4B) and dP/dt⫺ (Fig. 4C) during graded treadmill exercise (5, 10, 15, and 20 m/min) for mice and rats. There was a significant group effect for dP/dt⫹ and dP/dt⫺ (Fig. 4A). Specifically, both dP/dt⫹ and dP/dt⫺ were greater in mice compared with rats. Similarly, both dP/dt⫹ and dP/dt⫺ were greater during exercise compared with rest (significant exercise effect). Finally, mice had a significantly greater percent change from rest for dP/dt⫺ (Fig. 4C) at each workload (significant group effect) but not dP/dt⫹ (Fig. 4B). Figure 5 presents the force-frequency relationship, i.e., the relationship between the percent change from rest for LV pressure (Fig.5A), dP/dt⫹ (Fig. 5B), and dP/dt⫺ (Fig. 5C), and heart rate for mice and rats. The range of acceleration for heart rate was significantly greater for mice [233 ⫾ 19 beats per minute (bpm)] compared with rats (151 ⫾ 8 bpm). In contrast, the slope or gain (percent/bpm) of the force-frequency relation for LV systolic pressure (0.127 ⫾ 0.025 vs. 0.125 ⫾ 0.018) and dP/dt⫺ (0.226 ⫾ 0.019 vs. 0.198 ⫾ 0.030) was similar for rats and mice, respectively. In contrast, the slope or gain (percent/bpm) of the forcefrequency relation for dP/dt⫹ (0.396 ⫾ 0.047 vs. 0.267 ⫾ 0.027 P ⫽ 0.04) was higher for mice. Time for Mice to Recover Day/Night Variations in Heart Rate and LV Pressure

Fig. 2. Heart rate (A) and the percent change in heart rate from rest (B) during graded treadmill exercise (5, 10, 15, and 20 m/min) for mice and rats are shown. *P ⬍ 0.05, mice vs. rats. #P ⬍ 0.05, exercise vs. rest.

Figure 6 presents the time for mice to recover night/day variations in heart rate and LV pressure. Specifically, 12:12-h

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Fig. 3. LV systolic and diastolic pressures (A) and the percent change from rest in LV systolic (B) and diastolic (C) pressures during graded treadmill exercise (5, 10, 15, and 20 m/min) for mice and rats. *P ⬍ 0.05, mice vs. rats. #P ⬍ 0.05, exercise vs. rest.

night and day averages for heart rate (Fig. 6A) and mean LV pressure (Fig. 6B) in mice, beginning the night following the procedures for implanting the telemetry device and continuing for 11 days are presented. The normal night/day variation in heart rate began the 8th night following the anesthesia and surgical trauma (Fig. 6A). This timing is similar for results recently reported for mice and rats (2, 15, 21). In contrast, normal night/day variation in mean LV pressure began the 4th night following the anesthesia and surgical trauma (Fig. 6B).

Effect of Anesthesia on Resting Heart Rate and LV Hemodynamic Parameters in Mice Figure 7 presents the effect of pentobarbital sodium anesthesia on resting heart rate and LV hemodynamic parameters in mice. Specifically, baseline values and the percent change from baseline following 30 min of anesthesia (pentobarbital sodium, 50 mg/kg ip) for heart rate, LV systolic pressure, dP/dt⫹, and dP/dt⫺, in mice, are presented. As expected, pentobarbital sodium anesthesia markedly reduced heart rate and LV hemodynamic parameters.

Fig. 4. LV dP/dt⫹ and dP/dt⫺ (A) and the percent change from rest for dP/dt⫹ (B) and dP/dt⫺ (C) during graded treadmill exercise (5, 10, 15, and 20 m/min) for mice and rats are presented. *P ⬍ 0.05, mice vs. rats. #P ⬍ 0.05, exercise vs. rest.

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more, significant efforts were required to maintain body temperature within the physiological range during the anesthesia. These results confirm the work of Fitzgerald et al. (11). These investigators pointed out that although the physiological heart rate (HR) range for the conscious mouse is 500 –700 bpm, the induction of anesthesia reduces HR by more than 50% depending on the agent used (11). Similarly, resting cardiac output in the conscious mouse (20 ml/min) is substantially reduced by the induction of anesthesia (7–11 ml/min) (3, 11). It is important to note that mice are very responsive to variations in ambient temperature and are often housed and studied in rooms maintained between 21 and 23°C, which is markedly below their thermoneutral zone of 30°C (35). Studies conducted in rooms with temperatures below thermoneutrality serve as a mild cold stress, and the mice respond with tachycardia due to a withdrawal of cardiac vagal tone and increased sympathetic tone (35). Accordingly, as emphasized by Swoap et al. (35), these findings support the need for carefully controlling ambient temperature during the assessment of cardiovascular function in mice. In this context, core body temperature of the anesthetized mouse also drops by ⬃0.5°C/min at room temperature (11). Anesthesia also significantly alters the autonomic nervous system. This is a concern because disturbances in cardiac autonomic balance play a critical role in cardiovascular diseases. Specifically, reductions in parasympathetic activity or

Fig. 5. Force-frequency relationship, i.e., the relationship between the percent change from rest for LV pressure (A), dP/dt⫹ (B), and dP/dt⫺ (C) and heart rate, for mice and rats is presented.

DISCUSSION

Worldwide efforts are currently under way to knock out specific genes in the offspring of mice, to knock in genes with specific mutations and/or to selectively silence or express gene products in a variety of tissues in a quest to understand, prevent, and treat cardiovascular diseases (24). Often, the phenotype of genetically engineered mice is not fully appreciated, due, in part, to the difficulty of measuring physiological responses in the intact, conscious freely moving state. To address this concern, as well as provide a resource for investigators using available spontaneous or engineered mouse mutants, we assessed LV function at rest and during exercise in mice and rats. Whenever possible, studies of cardiovascular physiology and pathophysiology should be conducted in conscious animals to avoid the complications associated with the use of anesthesia. Specifically, it is well known that anesthesia dramatically depresses the cardiovascular system and compromises the ability to regulate body temperature. For example, Fig. 7 documents markedly depressed heart rate and LV function parameters during pentobarbital sodium anesthesia. Further-

Fig. 6. Time for mice to recover night/day variations in heart rate and LV pressure is presented. Specifically, 12-h day and night averages for heart rate (A) and mean LV pressure (B), in mice, beginning the night following the procedures for implanting the telemetry device and continuing for 11 days are presented.

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resting and exercise heart rate. In contrast, the increase is only about 50% for the rat and only 10% for the mouse (16). Similarly, Georgakopoulos and Kass (12) reported only modestly enhanced contraction and relaxation (13–15%) at heart rates between 400 and 500 bpm with a flat force-frequency relation with heart rate above 600 bpm in anesthetized, openchest mice. Although, our findings are in general agreement with results from rat trabeculae, the results from the conscious, exercising mouse were surprisingly robust. Limitations

Fig. 7. Effect of anesthesia on resting heart rate and LV hemodynamic parameters in mice is presented. Specifically, baseline values and the percent change from baseline following 30 min of anesthesia (pentobarbital sodium, 50 mg/kg ip) for heart rate, LV systolic pressure, dP/dt⫹ and dP/dt⫺, in mice, are presented.

increases in sympathetic activity increase the morbidity and mortality associated with cardiovascular diseases. Accordingly, avoiding these complications associated with the use of anesthesia has the potential to improve the validity and clinical significance of the results. Furthermore, the effects of the surgical trauma and anesthesia can markedly alter cardiovascular parameters during the recovery period, and it can take from 5 to 7 days before the return of normal circadian patterns (2, 15, 21). This fact is documented in Fig. 6, showing the effect of surgical trauma and anesthesia on the recovery of day/night variations in heart rate and LV function. The normal day/night variations in heart rate and LV required 4 to 8 days for recovery. This timing is similar to results recently reported for mice and rats (15, 21). An exercise protocol should also be used whenever possible in the study of cardiovascular function since exertion may reveal cardiovascular phenotypes that are not apparent under resting conditions (11). That is, more can be learned about how a system operates when it is forced to perform than when it is idle (33). For example, Yang et al. (39) documented that although young adult mice carrying a mutated cardiac myosin binding protein C (MyBP-C) were apparently healthy, while resting with no signs of early mortality and older mice only displayed mild cardiac hypertrophy, when these mutated MyBP-C mice performed submaximal treadmill exercise, they were significantly compromised in their ability to exercise and, several mice died suddenly during the testing procedure (11). Accordingly, an exercise paradigm has the potential to reveal unexpected cardiovascular phenotypes that are not apparent under resting conditions possibly due, in part, to the increased functional demands made on the system during physical stress. Mice/Rat Comparisons Mice, compared with rats, had a greater percent change in LV systolic pressure, LV diastolic pressure and dP/dt⫺, as well as a larger range in heart rate during a graded exercise test. However, the percent change in dP/dt⫹ was not different between mice and rats during exercise. In a recent review, Janssen and Periasamy (16) reported that the relative increase in isometric force, of small and thin ventricular trabeculae, within the in vivo frequency range for man, dog, and rabbit are similar, nearly doubling between

LV pressure and its derivative, dP/dt, are complex functions that are markedly dependent on ventricular preload and afterload. Accordingly, alterations in ventricular end-diastolic pressure and arterial diastolic pressure can alter LVP and dP/dt independent of the inotropic state. During exercise, changes in preload and afterload accompany alterations in the inotropic state. In this situation, the dP/dt per se may be of limited value as an independent measure of myocardial contractility. To overcome this limitation, investigators have proposed the use of the LV dP/dt at 40 mmHg developed pressure (LVdP/dt40) as an index of myocardial contractility, since this index is minimally affected by alterations in preload or afterload (26). Unexpectedly, the mice did not demonstrate a linear relationship between heart rate and exercise intensity. This may be the result of the mice being excited due to our failure to provide a longer period of adaptation. Accordingly, the “novel stress” of exercise, rather than the “physical stress” of exercise may explain the lack of graded heart rate response. Thus, there is an essential need for assuring adequate exposure to the experimental conditions during the assessment of cardiovascular function in mice. Perspectives and Significance This paper describes the application of existing technology, surgical techniques, and experimental protocols for the study of LV function during exercise in the mouse. The methodology allows for the accurate and reliable interpretation of LV parameters never before recorded in the freely moving mouse. The techniques are of particular value for repeated measurements of LV hemodynamics in conscious mice at rest and during exercise. This is especially important if anesthesia or ex vivo preparations confound the results. Finally, this procedure avoids the acute effects of surgical trauma because the animals were allowed 7 or more days for recovery. In this context, blood loss of only 300 ␮l can result in hypovolemic shock (11, 31). Investigators may be encouraged to adopt these existing procedures to their investigations of cardiac function since highly reliable data can be obtained in mice under physiological conditions. GRANTS This study was supported by National Heart, Lung, and Blood Institute Grants HL-88615 (to S. E. DiCarlo) and HL-98945 (to J.-P. Jin). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS Author contributions: H.L.L., H.-Z.F., J.-P.J., and S.E.D. conception and design of research; H.L.L. and H.J. analyzed data; H.L.L. and S.E.D. inter-

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