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CARDIOVASCULAR ANESTHESIA

Isoproterenol Enhances Myofilament Ca²⁺ Sensitivity During Hypothermia in Isolated Guinea Pig Beating Hearts

Yuri Nakae, MD PhD^*, Satoshi Fujita, MD PhD, and Akiyoshi Namiki, MD PhD^*

*Department of Anesthesiology, Sapporo Medical University School of Medicine, Sapporo, Japan; and Department of Anesthesia, Hokkaido Keiaikai Minami 1-jyo Hospital, Sapporo, Japan

Address correspondence and reprint requests to Yuri Nakae, MD, PhD, Department of Anesthesiology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226-0509. Address e-mail to ynakae{at}mcw.edu

	Abstract

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Isoproterenol is often required to treat acute left ventricular dysfunction during separation from cardiopulmonary bypass for cardiac surgery. We hypothesized that heart rate and intracellular Ca²⁺ concentration ([Ca²⁺]_i) homeostasis may be important factors when isoproterenol improves the cardiac function during hypothermia. Accordingly, we investigated the effect of isoproterenol on the cardiac functional variables, [Ca²⁺]_i, and myofilament Ca²⁺ sensitivity under spontaneous beating during hypothermia. Intact guinea pig hearts were perfused with a modified Krebs-Ringer solution (baseline) and Krebs-Ringer solution containing isoproterenol (1 nM) at 37°C, 32°C, and 27°C while all cardiac variables and [Ca²⁺]_i were recorded. Isoproterenol increased developed left ventricular pressure (LVP), maximum rate of increase in LVP, and coronary inflow at 27°C, and it also increased heart rate and maximum rate of decrease in LVP at each temperature (P < 0.05). Isoproterenol produced a leftward shift of the curve of developed LVP as a function of available [Ca²⁺]_i at 32°C and 27°C (P < 0.05), without changing available [Ca²⁺]_i. Isoproterenol improves the cardiac function, especially systolic ventricular function, by enhancement of myofilament Ca²⁺ sensitivity under spontaneous beating during hypothermia in intact guinea pig hearts.

IMPLICATIONS: Enhancement of myofilament Ca²⁺ sensitivity is involved in the improvement of cardiac function by isoproterenol under spontaneous beating during hypothermia.

	Introduction

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Hypothermia as low as 27°C is widely used to protect hearts during cardiac surgery. Acute left ventricular (LV) dysfunction is often encountered on separation from cardiopulmonary bypass (CPB), and the ß-adrenoceptor agonist isoproterenol is useful for treating this condition (1,2). Experimental evidence has demonstrated that hypothermia alters ventricular contractility and intracellular Ca²⁺ concentration ([Ca²⁺]_i) homeostasis (3–11). A previous study has shown that the direct effect of hypothermia on ventricular contractility is particularly dependent on the frequency of contraction in isolated paced rabbit heart (12). Because most of the previous studies apply to heart rate (HR) pacing, we were interested in the effect of hypothermia on cardiac function under spontaneous beating. We hypothesized that the HR and [Ca²⁺]_i homeostasis may be important factors when isoproterenol improves cardiac function under spontaneous beating during hypothermia. Accordingly, we investigated the effect of isoproterenol on the cardiac functional variables, [Ca²⁺]_i, and myofilament Ca²⁺ sensitivity under spontaneous beating during hypothermia. We selected an intact guinea pig spontaneous beating heart model because we could evaluate myofilament Ca²⁺ sensitivity by measuring [Ca²⁺]_i and left ventricular pressure (LVP) simultaneously under conditions that excluded neural and endocrine effects.

	Methods

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Approval from the Institutional Animal Care Committee of Sapporo Medical University was obtained before initiation of this study. English short-haired guinea pigs (n = 27) weighing 250–300 g were injected intraperitoneally with 20 mg of ketamine (Sigma-Aldrich, Tokyo, Japan) and 1000 U of heparin (Sigma-Aldrich) and were decapitated after they became unresponsive to noxious stimulation. Each heart was isolated and perfused in a retrograde manner through the aorta at 55 mm Hg by the method reported previously (13,14). The perfusate, a modified Krebs-Ringer (KR) solution (in mM: 137 Na⁺, 5 K⁺, 1.2 Mg²⁺, 2.5 Ca²⁺, 134 Cl^-, 15.5 HCO₃^-, 1.2 H₂PO₄^-, 11.5 glucose, 2 pyruvate, 16 mannitol, and 0.05 EDTA, with 5 U/L insulin), was equilibrated with a gas mixture of 97% oxygen and 3% carbon dioxide (CO₂) at a flow rate of 1 L/min, resulting in a pH of 7.4. Perfusate and bath temperatures were regulated at 37°C ± 0.1°C by a thermostatically controlled water circulator (Thermo Pump^TM; Taitec Co., Koshiya, Japan). A thermometer probe attached with water circulator was placed in contact with the left atrium in the bath. Isovolemic developed (systolic - diastolic) LVP, systolic LVP, diastolic LVP, maximum rates of increase and decrease in LVP (+dP/dt_max and -dP/dt_max), spontaneous atrial HR, and aortic inflow, which indicates coronary inflow, were measured continuously. LVP was measured by a pressure transducer (DTX Plus Press^TM; Ohmeda, Madison, WI) connected to a thin, saline-filled latex balloon (LB-3^TM; Mars, Inc., Sapporo, Japan) inserted into the LV through the left atrium. The balloon volume was adjusted to maintain a diastolic LVP of 0 mm Hg during the initial control period. Coronary inflow was measured at constant temperature and constant perfusion pressure (55 mm Hg) by using a self-calibrating, in-line, ultrasonic flowmeter (T106; Transonic, Tokyo, Japan) placed directly into the aortic inflow line. To determine maximal coronary inflow, adenosine (0.2 mL of 200 µM stock solution) was injected into the aortic root cannula during the initial control period and after the last control reading. HR pacing was not applied to examine the effects of hypothermia and drug under spontaneous beating. When arrhythmia occurred during decreasing the temperature, these cases were excluded.

Transmural LV phasic [Ca²⁺]_i was measured by the fluorescence ratio of indo-1 emission at 385 nm and 456 nm (F385/F456) by using a fiber bundle (FB-50; Japan Spectroscopic, Tokyo, Japan) connected to a luminescence spectrometer (CAF-110; Japan Spectroscopic). Our methods have been described in detail previously (13,14). Briefly, the excitation light was derived from a 75 W xenon arc lamp through a 350-nm filter, an interference filter (bandwidth, 12 nm), and a visible light-blocking filter, and it penetrated transmurally (5 mm). The light emitted from the heart was collected by the outgoing fiber bundle and filtered at 385 and 456 nm. The end of the fiberoptic cable (tip surface area, 38.5 mm²) was placed against the LV epicardial surface through a hole in the bath. To reduce cardiac motion artifacts, the heart was held to the fiberoptic tip with nylon mesh.

After perfusion for 15 min by using the standard perfusate, the hearts were loaded in an ambient light-free room at 25°C ± 0.5°C for 20–30 min with 166 mL of recirculating standard perfusate containing free Ca²⁺ fluorescent indicator indo-1 AM (6 µM, cell-permeable ester form of indo-1, Sigma-Aldrich). Loading of indo-1 was stopped when F385/F456 intensities had increased by approximately 10-fold. Residual indo-1 was washed out by perfusing the heart with standard perfusate for at least 20 min. To avoid blanching of indo-1, the arc lamp shutter was opened only for 2.5-s recording intervals for a total exposure of <125 s for each indo-1-loaded heart. Fluorescence of the indo-1 dye is independent of the tissue oxygenation state at these two isobestic wavelengths (15,16).

After correcting for autofluorescence and noncytosolic (primarily mitochondria) uptake, F385/F456 was calibrated to [Ca²⁺]_i transients (in nM) at 37°C, 32°C, and 27°C by the standard equation for fluorescent indicators: [Ca²⁺]_i = S456 · K_d[(R - R_min)/(R_max - R)], where K_d is the indo-1 dissociation constant for Ca²⁺, S456 is the ratio between the fluorescence intensities measured at 456 nm in the absence of Ca²⁺ and in the presence of saturating Ca²⁺, R is the ratio of detected indo-1 fluorescence intensities, and R_min and R_max are the fluorescence ratios in the absence of Ca²⁺ and in the presence of saturating Ca²⁺, respectively (15,16). Five additional hearts (1.4 ± 0.2 g) were used for calculation of K_d. K_d of indo-1 for Ca²⁺ was calculated to be 220 ± 37 nM, 236 ± 39 nM, and 249 ± 40 nM at 37°C, 32°C, and 27°C, respectively, and the average value of K_d at each temperature was used in the equation for calculation of [Ca²⁺]_i. Noncytosolic (primarily mitochondria) fluorescence was measured at the end of each experiment by perfusing hearts with 17.5 µM MnCl₂ to quench fluorescence derived from the cytosolic compartment (17). Mn²⁺ quenching obliterates the cytosolic-derived Ca²⁺ single phasic transient, but not the beat-to-beat phasic change in LVP, and results in a Ca²⁺ signal greater than the diastolic but less than the systolic - cytosolic Ca²⁺ signal. For each of 14 hearts measured [Ca²⁺]_i, background autofluorescence was corrected at each wavelength at 37°C after initial perfusion and equilibration and before indo-1 loading. In pilot studies (n = 4), the drug used did not alter basal autofluorescence and Mn²⁺-corrected noncytosolic Ca²⁺, and autofluorescence was unaffected over the 3-h testing period with normal oxygenation. Also, the drug used did not interact with the fluorescence of indo-1 AM. The [Ca²⁺]_i transients during each contraction were defined as available [Ca²⁺]_i (systolic - diastolic, s - d[Ca²⁺]_i). The [Ca²⁺]_i transients can be measured reliably for at least 3 h after washout of extracellular indo-1 AM. The data were digitized by a MacLab^TM system (AD Instruments, NSW, Australia) and stored in a personal computer.

Eighteen hearts were used during these protocols. At first, we studied the effects of hypothermia on cardiac functional variables and [Ca²⁺]_i. After loading and residual washout of indo-1 AM, seven hearts were perfused with KR solution (vehicle) at 37°C, 32°C, and 27°C. At the same time, the concentration of extracellular Ca²⁺ ([CaCl₂]_e) was increased incrementally from 0.3 to 4.5 mM for an 11-min period, during which all cardiac variables and [Ca²⁺]_i were measured at 1-min intervals until, under any protocol, LVP reached its maximal value. The hearts were perfused and stabilized at each temperature for 20 min before the measurements were started, and each temperature was applied in random fashion in the same heart. Before starting the study, we confirmed that the temperature of perfusate reached a steady state within 20 min after changing from one temperature to another by in-line measurement at the coronary outflow. Each heart underwent a change in [CaCl₂]_e three times. Normalized developed LVP was plotted as a function of s - d[Ca²⁺]_i. Normalized developed LVP was expressed as a percentage of maximal developed LVP.

Next, we studied the dose-dependent effect of isoproterenol on cardiac functional variables to determine the administration dose at 2.5 mM of [CaCl₂]_e at 37°C. Initial control measurements were obtained during perfusion of KR solution (CaCl₂ 2.5 mM) after a 30-min stabilization period. Four hearts were perfused with KR solution (vehicle, CaCl₂ 2.5 mM) containing isoproterenol (Sigma-Aldrich; 0.1, 1, and 10 nM) for 4 min at 37°C. All cardiac variables were measured after a 4-min equilibration period. After control measurements, different concentrations of isoproterenol were tested in random fashion in the same heart with a 20-min washout period.

Finally, we examined the effects of isoproterenol on cardiac functional variables and [Ca²⁺]_i during hypothermia. Seven hearts were perfused with KR solution (vehicle) and KR solution containing isoproterenol (1 nM) at 37°C, 32°C, and 27°C. At the same time, [CaCl₂]_e was increased incrementally as described in the first protocol. The hearts were perfused and stabilized at each temperature for 20 min before the measurements started, and each temperature was applied in random fashion in the same heart. Each heart underwent a change in [CaCl₂]_e six times, three times in the absence of drug (baseline), serving as its own control at each temperature. We confirmed that the drug effect was maximal within 3 min and stable during drug perfusion, and that 20 min after discontinuation of the drug, baseline conditions had returned before changing to a different temperature. Postcontrol values (n = 4) were measured at 37°C in the same heart at the end of all interventions. Altering [CaCl₂]_e did not significantly alter noncytosolic compartment Ca²⁺ for a 20-min period, as previously reported (18).

All data are expressed as means ± SD except when noted. Cardiac functional data were compared by using Tukey’s comparison of means test after analysis of variance for repeated measures. Curves of normalized developed LVP as a function of s - d[Ca²⁺]_i were compared by using nonlinear regression analyses with Boltzmann’s sigmoidal equations (Prism^TM; GraphPad Software, Inc., CA), where all correlation coefficients (R) were >0.98. Slopes, 95% confidence intervals (CI₉₅), 50% maximum values of normalized developed LV (ED₅₀), and slope comparisons were determined for each curve. Differences between means were considered significant when P was <0.05.

	Results

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The maximal coronary inflow after administration of adenosine was increased 22 ± 2 mL/min compared with before adenosine (17 ± 1 mL/min) during the initial control period and 18 ± 2 mL/min compared with before adenosine (15 ± 2 mL/min) after the last control reading, respectively, for all experimental protocols (P < 0.05).

Table 1 summarizes the effects of hypothermia on cardiac functional variables and [Ca²⁺]_i at 0.3 mM (minimal), 2.5 mM (normal), and 4.5 mM (maximal) of [CaCl₂]_e. At 2.5 mM of [CaCl₂]_e, HR at 32°C and 27°C were decreased less than that at 37°C (P < 0.05). Systolic LVP and developed LVP at 27°C were increased more than those at 37°C, whereas -dP/dt_max and coronary inflow at 27°C were decreased compared with those at 37°C (P < 0.05). At 0.3 mM of [CaCl₂]_e, diastolic LVP at 32°C and 27°C was increased compared with that at 37°C, whereas HR at 32°C and 27°C was decreased compared with that at 37°C (P < 0.05). Systolic LVP, developed LVP, coronary inflow, +dP/dt_max, and -dP/dt_max did not change. At 4.5 mM of [CaCl₂]_e, systolic LVP and developed LVP at 27°C were increased compared with those at 37°C, whereas coronary inflow and -dP/dt_max at 27°C were decreased compared with those at 37°C (P < 0.05). Also, HR at 32°C and 27°C was decreased compared with that at 37°C (P < 0.05), and diastolic LVP and +dP/dt_max did not change.

View larger version (23K): [in this window] [in a new window]   Figure 1. Changes in normalized developed left ventricular pressure (LVP [%]) with increases in systolic - diastolic [Ca2+]i (s - d[Ca2+]i) (nM) while extracellular Ca2+ was increased incrementally from 0.3 to 4.5 mM at 37°C, 32°C, and 27°C. The relation curves at 32°C and 27°C were shifted to the right compared with those at 37°C (P < 0.05). Data are presented as mean ± SD (n = 7).

Table 2 summarizes the effects of isoproterenol (1 nM) on cardiac functional variables and [Ca²⁺]_i at 2.5 mM of [CaCl₂]_e at each temperature. Isoproterenol (1 nM) increased systolic LVP, developed LVP, and +dP/dt_max compared with baseline values at 27°C and 32°C (P < 0.05). Isoproterenol (1 nM) increased -dp/dt_max and HR compared with baseline values at each temperature, and it also increased coronary inflow compared with baseline values at 27°C (P < 0.05). Postcontrol values at 37°C at 2.5 mM of [CaCl₂]_e showed the following: systolic LVP, 66 ± 7 mm Hg; diastolic LVP, 3 ± 1 mm Hg; developed LVP, 63 ± 6 mm Hg; coronary inflow, 10.3 ± 0.7 mL · g^-1 · min^-1; +dP/dt_max, 53 ± 21 mm Hg/s; -dP/dt_max, 41 ± 15 mm Hg/s; HR, 189 ± 10 bpm; s[Ca²⁺]_i, 680 ± 15 nM; d[Ca²⁺]_i, 278 ± 15 nM; and s - d[Ca²⁺]_i, 402 ± 16 nM. There were no differences between baseline and postcontrol values.

View larger version (18K): [in this window] [in a new window]   Figure 2. Changes in normalized developed left ventricular pressure (LVP [%]) with increases in systolic - diastolic [Ca2+]i (s - d[Ca2+]i) (nM) while extracellular Ca2+ was increased incrementally from 0.3 to 4.5 mM in the perfusate of vehicle and isoproterenol (1 nM) at 37°C (A), 32°C (B), and 27°C (C). Isoproterenol (1 nM) shifted to the left compared with baseline at 32°C (B) and 27°C (C, P < 0.05), but not at 37°C (A). Isoproterenol also caused a gentler slope in the relation curve compared with baseline at 32°C (B, P < 0.05). Data are presented as mean ± SD (n = 7 for each figure). Baseline values present during vehicle administration at each temperature.

	Discussion

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Hypothermia decreased -dP/dt_max, an indication for the velocity of relaxation, and this finding indicates that hypothermia induced the diastolic ventricular dysfunction. The dP/dt_max varies to some extent with preload and HR. The decrease of -dP/dt_max would be explained by the marked decrease of HR because hypothermia did not change diastolic LVP. It is still uncertain whether hypothermia has an inotropic effect. There are differences in animal species, experimental models, and experimental conditions that include the frequency of contraction and the degree of hypothermia among previous studies (3–11). A previous study has shown that the inotropic action of mild hypothermia is directly related to contraction frequency (12). The heart is able to maintain a good LVP at rates slower than those encountered physiologically. However, diastolic ventricular dysfunction occurs together with systolic dysfunction when the HR increases. Our results demonstrated that hypothermia did not change +dP/dt_max, an indication for the velocity of contraction, despite increasing systolic LVP and developed LVP. These findings indicate that hypothermia would not improve the systolic ventricular function in spontaneously beating heart.

Hypothermia increased s[Ca²⁺]_i, d[Ca²⁺]_i, and available [Ca²⁺]_i. The increase of s[Ca²⁺]_i reflects that hypothermia increases transsarcolemmal Ca²⁺ influx via Ca²⁺ channels and other ion channels and Ca²⁺ release from the sarcoplasmic reticulum (24). Also, the increase of d[Ca²⁺]_i reflects that hypothermia inhibits Ca²⁺ uptake rate and Ca²⁺-adenosine triphosphatase activity in the sarcoplasmic reticulum (25). Because hypothermia increased Ca²⁺ transients without improving the systolic ventricular function, this finding may suggest that it induces other effects that counterbalance its effect on Ca²⁺ transients. Hypothermia shifted to the right the curve of developed LVP as a function of available [Ca²⁺]_i. This finding indicates that hypothermia attenuates myofilament Ca²⁺ sensitivity. Mechanisms of inotropic effect are explained by the alterations of available [Ca²⁺]_i, time course of Ca²⁺ transients, myofilament Ca²⁺ sensitivity, and efficacy or rate of individual cross-bridge cycle formation (20–23). Therefore, hypothermia might not improve the systolic ventricular function as a result of attenuation of myofilament Ca²⁺ sensitivity, despite increasing available [Ca²⁺]_i.

In this study, we monitored temperature at the site in contact with the left atrium in the bath, instead of intramyocardial temperature. The thermometer probe attached with a water controller helped to assess a steady myocardial temperature by the feedback regulation. In addition, because the temperature of perfusate at the coronary outflow reached a steady state before each measurement, we confirmed that the temperatures applied in this study would closely reflect the intramyocardial temperature.

We used the perfusate, a modified KR solution, that was equilibrated with a gas mixture of 97% oxygen and 3% CO₂. A high partial pressure of oxygen (95% oxygen) affects coronary blood flow in a blood-perfused isolated rabbit heart (26). In this study, adenosine was injected to determine maximal coronary inflow during the initial control period and after the last control reading. Because the coronary inflow significantly increased compared with the initial value after the administration of adenosine, we confirmed that coronary vasoreactivity was preserved under a high partial pressure of oxygen (97% oxygen) at 37°C. Moreover, because the coronary inflow responded to the change of temperature and exposure to isoproterenol, the coronary vascular tone would be preserved throughout the study.

Potential etiologies of post-CPB cardiac dysfunction include severe preoperative systolic and diastolic ventricular dysfunction, surgically uncorrectable disease, inadequate myocardial protection, continuing myocardial ischemia, myocardial stunning, infarction, and acute myocardial ß-adrenergic receptor desensitization (1,2,27). Our studies demonstrated the effect of hypothermia on cardiac function and [Ca²⁺]_i dynamics under conditions that excluded neural and endocrine effects. However, our results may be applicable only to the clinical situation because the range of temperature and presence of spontaneous beating are similar to conditions during rewarming from CPB.

In conclusion, our results indicate that isoproterenol improves cardiac function, especially systolic ventricular function, by enhancement of myofilament Ca²⁺ sensitivity under spontaneous beating during hypothermia in intact guinea pig hearts.

	Acknowledgments

 
This work was supported in part by a Grant-in-Aid for Scientific Research (10671434) from the Ministry of Education, Japan.

	Footnotes

 
Presented in part at the annual meeting of the American Society of Anesthesiologists, San Francisco, CA, October 14–18, 2000.

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