Isoflurane (ISO) is known to depress cardiac contraction. Here, we hypothesized that decreasing myofilament Ca2+ responsiveness is central to ISO-induced reduction in cardiac force development. Moreover, we also tested whether the nitroxyl (HNO) donor 1-nitrosocyclohexyl acetate (NCA), acting as a myofilament Ca2+ sensitizer, restores force in the presence of ISO. Trabeculae from the right ventricles of LBN/F1 rats were superfused with Krebs-Henseleit solution at room temperature, and force and intracellular Ca2+ ([Ca2+]i) were measured. Steady-state activations were achieved by stimulating the muscles at 10 Hz in the presence of ryanodine. The same muscles were chemically skinned with 1% Triton X-100, and the force-Ca2+ relation measurements were repeated. ISO depressed force in a dose-dependent manner without significantly altering [Ca2+]i. At 1.5%, force was reduced over 50%, whereas [Ca2+]i remained unaffected. At 3%, contraction was decreased by ∼75% with [Ca2+]i reduced by only 15%. During steady-state activation, 1.5% ISO depressed maximal Ca2+-activated force (Fmax) and increased the [Ca2+]i required for 50% activation (Ca50) without affecting the Hill coefficient. After skinning, the same muscles showed similar decreases in Fmax and increases in Ca50 in the presence of ISO. NCA restored force in the presence of ISO without affecting [Ca2+]i. These results show that 1) ISO depresses cardiac force development by decreasing myofilament Ca2+ responsiveness, and 2) myofilament Ca2+ sensitization by NCA can effectively restore force development without further increases in [Ca2+]i. The present findings have potential translational value because of the efficiency and efficacy of HNO on ISO-induced myocardial contractile dysfunction.
The effects of inhalational anesthetic agents on myocardial contraction have been the focus of extensive investigations for many years, and the depression of force development by different inhalational agents has been repeatedly shown. At the cellular level, altered cardiac excitation-contraction coupling has been identified as a major vehicle of impaired cardiac performance. In fact, inhalational anesthetics have been shown to decrease sarcolemmal L-type Ca2+ channel function (Terrar and Victory, 1988), decrease sarcoplasmic reticular Ca2+ storage (Davies et al., 2000), Ca2+ release (Jiang and Julian, 1998), and decrease myofilament Ca2+ sensitivity (Bosnjak et al., 1992; Davies et al., 2000). More importantly, it appears that the impact of inhalational agents on myocardium is agent- and dose-dependent, with decreasing Ca2+ availability being viewed as the major mode of action based on previous studies (Rusy and Komai, 1987; Housmans and Murat, 1988; Housmans et al., 2000).
However, other recent studies have shown that, at clinical dosages (i.e., 0.5–1.5 minimum alveolar concentration), isoflurane (ISO) or sevoflurane differentially decrease contractile force development while producing little inhibition of Ca2+ release. This point is proven by the lack of change in the amplitudes of intracellular Ca2+ transients (Hanley and Loiselle, 1998; Bartunek and Housmans, 2000; Davies et al., 2000; Graham et al., 2005). This clearly hints at the possibility that ISO directly decreases myofilament Ca2+ responsiveness. Nonetheless, early studies that used skinned cardiac muscle preparations yielded variable results; some failed to show any effect of ISO at low concentrations (i.e., clinically relevant doses: 1–3%) (Su and Kerrick, 1980; Su and Bell, 1986), and others showed either increased or decreased myofilament Ca2+ sensitivity (Murat et al., 1988; Herland et al., 1993). The contention was that the presence of a cellular membrane system could influence the modulation of cardiac inotropy by inhalational agents (Herland et al., 1993). Thus, it remains unclear whether ISO affects myofilament function per se, i.e., independently from changes induced at the sarcolemmal level.
Nitroxyl (HNO), the protonated one-electron reduction product of nitric oxide (Tocchetti et al., 2011), augments cardiac inotropy and improves relaxation in dog hearts, both under normal and heart failure conditions (Paolocci et al., 2001, 2003). The positive inotropic and lusitropic effects of HNO are independent of β-adrenergic stimulation but are sensitive to the redox states of the cardiac cell (Tocchetti et al., 2007; Froehlich et al., 2008). Recently, we found that HNO augments cardiac contractility by increasing Ca2+ responsiveness of the myofilaments (Dai et al., 2007). This enhancement of force is not associated with an increase in diastolic tension or myofibrillar Mg-ATPase activities. Subsequently, we have confirmed that HNO, by interacting with specific, highly reactive thiols in myofilament proteins, modifies the redox states of key contractile proteins, thus promoting an increase in force production after Ca2+ binds to troponins (Tn) (Murray et al., 2009).
In this study, we tested two main hypotheses. First, we aimed to verify whether ISO is able to exert a direct effect on myofilament sensitization to Ca2+. To do so, we determined the steady-state force-Ca2+ relations of individual muscles, before and after skinning. Our second aim was to test whether the HNO donor, 1-nitrosocyclohexyl acetate (NCA) (Sha et al., 2006; Shoman et al., 2011), can restore cardiac force development in the presence of ISO by virtue of its Ca2+-sensitizing effect (Dai et al., 2007). We hope to demonstrate that HNO, donated by NCA, can restore myocardial contractility during ISO-induced anesthesia and that HNO donors are probably able to provide an inotropic counterbalancing effect in patients displaying compromised cardiac function due to inhalation of volatile anesthetics such as ISO, thus justifying the use of HNO donors for patients with heart failure undergoing inhalational anesthesia.
Materials and Methods
LBN/F1 rats (250–300 g; Harlan, Indianapolis, IN) were used in these experiments. Animal care and experimental protocols were approved by the Animal Care and Use Committee of The Johns Hopkins University School of Medicine.
Trabecular Muscle Preparation.
The rats were anesthetized via intra-abdominal injection with pentobarbital (100 mg/kg); the heart was exposed by mid-sternotomy, rapidly excised, and placed in a dissection dish. The aorta was cannulated, and the heart was perfused in a retrograde fashion (∼15 ml/min) with dissecting Krebs-Henseleit (K-H) solution equilibrated with 95% O2, 5% CO2. The dissecting K-H solution was composed of 120 mM NaCl, 20 mM NaHCO3, 5 mM KCl, 1.2 mM MgCl2, 10 mM glucose, 0.5 mM CaCl2, and 20 mM 2,3-butanedione monoxime, pH 7.35 to 7.45 at room temperature (21–22°C). Trabecular muscle from the right ventricle of the heart was dissected and mounted between a force transducer and a motor arm, superfused with K-H solution without 2,3-butanedione monoxime at a rate of ∼10 ml/min, and stimulated at 0.5 Hz.
Force was measured by a force transducer system (KG7; Scientific Instruments GmbH, Heidelberg, Germany) and expressed in millinewtons per square millimeter of cross-sectional area. The muscles underwent isometric contractions with the resting muscle length set, such that resting force was 15% of total force development (i.e., optimal muscle length). This resting muscle length, corresponding to resting sarcomere length of 2.20 to 2.30 μm as determined by laser diffraction (Gao et al., 1996), was maintained throughout the experiments. ISO was delivered via an ISO-specific vaporizer (calibrated by a vapor analyzer) to the K-H solution along with the 95% O2, 5% CO2 gas mixture at a constant flow rate (1.0 l/min). The K-H solution was bubbled through a fine-porosity gas distribution tube with desired doses (vol %) of ISO for at least 15 min before use. Because of the volatile nature of ISO, the K-H solution was constantly bubbled with the gas mixture saturated with ISO, and the reservoir was covered to maintain the desired percentage of ISO throughout the experiments. In this design, the muscles were superfused with K-H buffer saturated with desired percentage of ISO (i.e., not via inhalation). NCA was dissolved in dimethyl sulfoxide to make a stock solution (0.06 M) and stored in −20°C until use. All experiments were performed at room temperature (20°C–22°C).
Measurement of [Ca2+]i.
Intracellular Ca2+ ([Ca2+]i) was measured by using the free acid form of fura-2 as described previously (Gao et al., 1994; Dai et al., 2007). Fura-2 potassium salt was microinjected iontophoretically into one cell and allowed to spread throughout the whole muscle (via gap junctions). The tip of the electrode (∼0.2 μm in diameter) was filled with fura-2 salt (1 mM), and the remainder of the electrode was filled with 150 mM KCl. After a successful impalement into a superficial cell in nonstimulated muscle, a hyperpolarizing current of 5 to 10 nA was passed continuously for approximately 15 min. In some muscles, multiple injections (up to 3–4) were applied at different sites, with duration of the injection limited to <10 min at each site to achieve an optimal signal-to-noise ratio. As previously established, this loading did not affect force development. Fura-2 epifluorescence was measured by excitation at 380 and 340 nm. Fluorescent light was collected at 510 nm by a photomultiplier tube (R1527; Hamamatsu Photonics, Hamamatsu City, Japan). The output of the photomultiplier was collected and digitized. [Ca2+]i was given by the following equation (after subtraction of the autofluorescence) (eq. 1): where R is the observed ratio of fluorescence (340 nm/380 nm), K′d is the apparent dissociation constant, Rmax is the ratio of 340 nm/380 nm at saturating Ca2+, and Rmin is the ratio of 340 nm/380 nm at 0 Ca2+. The values of K′d, Rmax, and Rmin were determined by in vivo calibrations as described previously (Gao et al., 1994, 1998).
Steady-State Activation of Trabeculae.
Ryanodine (1.0 μM) was used to enable steady-state activation. After the muscles were exposed to ryanodine for 15 min, different levels of tetanizations were induced briefly (∼4–8 s) by stimulating the muscles at 10 Hz at various concentrations of extracellular Ca2+ ([Ca2+]o; 0.5–20 mM). The steady-state force-[Ca2+] relations were fit with a function of the Hill equation (eq. 2): where F is the steady-state force at various Ca2+ concentrations, Fmax is the maximal Ca2+-activated force, Ca50 is the Ca2+ concentration required to achieve 50% Fmax, and n is the Hill coefficient.
After the steady-state experiments, the same trabeculae were immediately skinned by 15 to 20 min of exposure to 1% Triton X-100 in relaxing solution containing 100 mM KCl, 25 mM HEPES, 10 mM K2EGTA, 15 mM creatine phosphate sodium salt (Na2CrP), 5 mM Na2ATP, 5.15 mM MgCl2, and 0.5 mM leupeptin, pH 7.2 with KOH. Activating solution contained 10 mM Ca2+-EGTA, 100 mM KCl, 25 mM HEPES, 15 mM Na2CrP, 5 mM Na2ATP, 4.75 mM MgCl2, and 0.5 mM leupeptin, pH 7.2. Different Ca2+ concentrations were achieved by mixing the activating solution and relaxing solution at different ratios. Skinning was considered to be complete when the preparation lost its pink color and sarcomeres were visible. Diastolic sarcomere length was determined by direct visualization under 100× magnification and was set at ∼2.2 μm. Resting force was usually 10 to 15% of maximal activated force at this sarcomere length. The force-Ca2+ relationship was obtained by exposing the skinned muscles to activating solutions with various Ca2+ concentrations and were fit with the Hill equation as described above.
Paired Student's t test and multivariate ANOVA were used for statistical analysis of the data (Systat 10.2.01; Systat Software Inc., San Jose, CA). A value of P < 0.05 was considered to indicate significant differences between groups. Unless otherwise indicated, pooled data are expressed as means ± S.E.M.
Isoflurane Depresses Twitch Force without Affecting [Ca2+]i Transients.
We initially studied the effect of ISO on force development and [Ca2+]i transients in isolated intact trabecular muscles. Figure 1A depicts a representative trace of the typical raw recordings of force and [Ca2+]i transients at different ISO concentrations, whereas Fig. 1B shows the pooled data of all of the muscles tested. ISO depressed force development in a dose-dependent manner without significantly altering the amplitude of [Ca2+]i transient. Moreover, at doses between 0.5 and 1.5%, developed force was reduced by more than 50% (from 18.0 ± 1.2 to 8.6 ± 0.5 mN/mm2, P < 0.001), whereas [Ca2+]i transient amplitudes remained unaffected ([Ca2+]i = 0.58 ± 0.06 μM at 0.5% and 0.55 ± 0.06 μM at 1.5% ISO, P > 0.05). At 3% ISO, contraction was decreased by approximately 75% (to 4.9 mN/mm2), whereas [Ca2+]i was reduced by only 15% (to 0.5 ± 0.05 μm, P > 0.05). Thus, after treatment with ISO, the reduction of force seen seems to stem from changes in the properties of the myofilament (i.e., decreased myofilament Ca2+ responsiveness), rather than from decreased Ca2+ cycling/availability. In addition, relaxation of twitch force, as measured by time to half-relaxation from peak force, was faster in the presence of ISO (Table 1).
Isoflurane Decreases Myofilament Ca2+ Responsiveness.
The data presented above only indirectly supports the contention that myofilament properties change under the influence of ISO given that, during activation and contraction, peak force development and peak amplitude of the [Ca2+]i transient are not at equilibrium. Thus, to unequivocally prove that the anesthetic has a primary effect on myofilaments, we measured the steady-state force-[Ca2+]i relation before and during 1.5% ISO exposure. In the presence of ISO, Fmax decreased, whereas the [Ca2+]i, required for 50% activation (Ca50), was increased (Fig. 2A; Table 2). The Hill coefficient was not affected in these muscles. Overall, there is a significant difference between the two relationships (P < 0.001) when multivariate ANOVA was performed among the parameters of Fmax, Ca50, and Hill coefficient. Hence, the action of ISO in the intact muscle was to decrease Fmax and increase Ca50.
In the intact muscle, the myofilaments are bathed in a cellular milieu that contains many factors that modulate the steady-state force-[Ca2+]i relationship (Gao et al., 1997). ISO could potentially modify these factors, thereby decreasing myofilament Ca2+ responsiveness. Therefore, to rule out all potential confounding factors, we skinned the same trabecular muscle in which the steady-state force-[Ca2+]i relations had been obtained. This enabled us to obtain force-[Ca2+] relations devoid of potential soluble confounding factors in these muscles. Likewise, in these skinned muscles, the Fmax and Ca50 were significantly depressed (Fig. 2B; Table 2) in the presence of ISO. Multivariate ANOVA also showed significant difference between the two relationships (P < 0.001). Thus, the depressing effect of ISO on the steady-state force-Ca2+ relation persisted in skinned muscles. We also performed experiments in which trabeculae were chemically skinned immediately after stabilization and the force-Ca2+ relationship was obtained in the absence or presence of 1.5% ISO. These experiments served as internal checkpoints for the tetanization protocol performed to verify that the protocol itself did not affect the force-Ca2+ in the (same) skinned muscles afterward. Indeed, these experiments showed identical force-Ca2+ relations as in Fig. 2B, indicating that tetanization protocol had no effect on subsequent force-Ca2+ relations in skinned muscles (Fig. 3). Thus, all together these data strongly support our initial hypothesis that the ISO-induced negative effect on myocardial contraction largely stems from a direct adverse impact on the myofilament.
Reversal of the Negative Inotropic Effect of Isoflurane.
If ISO inhibits force generation by directly decreasing myofilament Ca2+ responsiveness, agents that increase myofilament Ca2+ responsiveness (so-called Ca2+ sensitizers) should be able to restore force in the presence of this anesthetic agent. We tested this hypothesis by exposing the muscles to the new HNO donor, NCA (Sha et al., 2006; DuMond and King, 2011; Shoman et al., 2011). As we learned earlier, NCA increases force development by increasing myofilament sensitivity to Ca2+ (Murray et al., 2009). At 5 μM (slightly higher than the EC50 of NCA as determined in cardiac muscle (Murray et al., 2009), NCA increased force development significantly without affecting twitch dynamics (Table 1). It is noteworthy that NCA completely restored force development in the presence of 1.5% ISO, with little increase in the amplitude of the [Ca2+]i transient (Fig. 4). To show that NCA was indeed effective, we compared force recovery and [Ca2+]i transient amplitude when [Ca2+]o was raised (Fig. 5). Although ISO-dependent force depression could be fully restored by increasing the [Ca2+]o, the [Ca2+]i more than doubled. In contrast to Ca2+, NCA produced a full recovery in force without increasing [Ca2+]i. It is noteworthy that levosimendan (Levo), another Ca2+-sensitizing agent, which binds to cardiac TnC (Haikala et al., 1995), hardly restored force in the presence of ISO (Fig. 6), although it was able to increase force development in the absence of it as expected (Sato et al., 1998). This suggests that ISO affects steps downstream of Tn activation in myofilament contraction.
In this study, we provide two novel findings. First, we demonstrated that ISO is able to reduce force development without affecting the intracellular Ca2+ transient. For the first time, we show that ISO decreased Fmax and increased Ca50 of the steady-state force-Ca2+ relation in individual muscles, both before and after they were chemically skinned. Second, we demonstrated the ability of HNO donors such as NCA to rescue force development in ISO-treated muscles by virtue of their sensitizing action to Ca2+ at the myofilament level. This study extended the results from previous investigations on the effect of ISO on myocardial contractility while deepening our understanding of how ISO may dampen myocardial function.
The myofilament desensitizing effect of ISO (and/or sevoflurane) has been shown by a number of prior studies in intact cardiac muscles and myocytes (Hanley and Loiselle, 1998; Jiang and Julian, 1998; Graham et al., 2005). ISO may decrease myofilament Ca2+ responsiveness by 1) directly interacting with the contractile proteins to affect activation/contraction processes and 2) modifying soluble cytoplasmic factors that may influence the behavior of the myofilaments. These pathways can be removed by skinning the muscle preparation; thus, the environment bathing the myofilaments proteins can be controlled. By equalizing the composition of solutions bathing the myofibrils, any of the effects of ISO observed is the result of a direct interaction between the agents and the contractile proteins. On the other hand, the results of previous studies using skinned muscle preparations have been variable; some have shown that myofilament Ca2+ responsiveness to inhalational anesthetics decreased (Herland et al., 1993), whereas others have shown no change or increased myofilament Ca2+ responsiveness (Su and Kerrick, 1980; Su and Bell, 1986; Murat et al., 1988). Taken together, these studies suggest that, at least in part, the myofilament desensitization may reflect changes in the soluble cytoplasmic factors. Therefore, we chose to try another approach to evaluate the effect of ISO on myofilament Ca2+ responsiveness using the same muscle before and after skinning. This is a technically challenging experimental design that allows us to neatly distinguish the relative roles of soluble cytoplasmic factors and myofilament proteins in ISO-induced force depression, directly with increased reliability. The decreased myofilament Ca2+ responsiveness induced by ISO in intact muscles was reproducible in the same skinned muscles, confirming a direct myofilament effect of ISO.
The mechanism by which ISO interacts with myofilaments to decrease force development is not known at the moment. In cardiac muscle, force development depends on this sequence of events: Ca2+ binding to TnC (Krudy et al., 1994), interactions between Tn subunits (Kleerekoper et al., 1995; Dong et al., 2003), Tn-tropomyosin-actin interactions (McKillop and Geeves, 1993; Gordon et al., 2000; Lehman et al., 2001), and cross-bridge formation to generate force (Geeves and Holmes, 2005; Fujita-Becker et al., 2006). ISO has been shown to increase TnC affinity for Ca2+ and to promote conformational changes within Tn (Breukelmann and Housmans, 2007): however, such changes would not be expected to decrease force. In in vitro motility assays, ISO does not interfere with cross-bridge formation between isolated myosin and actin (Vivien et al., 2005). The inability of Levo, another known myofilament Ca2+ sensitizer that binds TnC and stabilizes the conformation of Ca2+-Tn complex (Haikala et al., 1995), to restore force in the presence of ISO suggests that ISO probably targets process(es) after TnC activation. Thus, given the above considerations, actin-tropomyosin interactions are most probably targeted by ISO. The fact that HNO provides complete (and competitive) reversal of ISO-induced force depression without additional increases in intracellular Ca2+ provides strong support to our view that HNO increases force development by increasing myofilament Ca2+ responsiveness (Dai et al., 2007; El-Armouche et al., 2010). Recently, we have shown that HNO promotes formation of disulfide bridges between actin and tropomyosin. These disulfide bridges prime tropomyosin for exposing more force-generating cross-bridge binding sites on actin so that more force could be produced (Murray et al., 2009). However, the molecular nature of isoflurane's action on myofilament proteins is unknown. Ongoing studies in our laboratory are focusing on identifying the myofilament protein(s) that are targeted by ISO.
Our current findings have some important pragmatic implications. The negative inotropic effect of ISO is probably to depress contractility further in patients with heart failure under anesthesia. Routinely, vasoconstrictors (i.e., phenylephrine) and crystalloid are administered to restore blood pressure and maintain cardiac output. In spite of these therapies, the perioperative morbidity and mortality for patient with heart failure is surprisingly high (i.e., 11.7%) (Hernandez et al., 2004). It is conceivable that vasoconstrictors and fluid therapy may impair the capability of the heart to contract due to an increase in pre- and postload. On the other hand, positive inotropic agents are expected to be more effective because they can augment cardiac contractility but they tend to produce Ca2+ overload. Ca2+ sensitizers, which represent a new class of inotropic agents, may be an effective alternative (Kass and Solaro, 2006). Their potential is especially relevant for patients with heart failure who require anesthesia. Decreased myofilament Ca2+ responsiveness as a result of alterations in the function of myofilament regulatory proteins is a significant contributor to the severely decreased contractility of failing myocardium (Noguchi et al., 2004; VanBuren and Okada, 2005; Murphy, 2006). Thus, inotropic agents with Ca2+-sensitizing properties are more desirable and probably more efficient for restoring contractility than conventional inotropic agents that increase contractility simply by increasing the availability of Ca2+ (Brixius et al., 2005; Hasenfuss and Teerlink, 2011). In addition, because of the exaggerated Ca2+ overload, the use of conventional inotropic agents is associated with worsening morbidity and mortality in heart failure patients (Cohn et al., 1998; Cuffe et al., 2002). Another aspect that singles NCA/HNO out among other inotropes is that it does not alter ISO-induced impact on lusitropy (Table 1).
In summary, we demonstrated that, at clinically relevant doses, the inhalational anesthetic ISO directly depresses myofilament contractility by decreasing Ca2+ responsiveness. The force depression can be effectively and fully restored by the novel myofilament Ca2+ sensitizer, NCA. Ca2+ can also restore the loss in force depression, but only with concomitant doubling of [Ca2+]i. Although the molecular nature of ISO's myofilament action is still under investigation, our findings indicate that cardiac depression during anesthesia, especially in patients with heart failure, is more effectively and efficiently treated with positive inotropic agents that possess Ca2+-sensitizing properties.
Participated in research design: Ding, Li, Shen, Martin, Sivakumaran, Paolocci, and Gao.
Conducted experiments: Ding, Li, Shen, and Gao.
Contributed new reagents or analytic tools: King and Martin.
Performed data analysis: Ding, Li, Shen, and Paolocci.
Wrote or contributed to the writing of the manuscript: Ding, Li, Shen, Martin, Sivakumaran, Paolocci, and Gao.
This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants R01-HL091923, R01-HL075265]; and American Heart Association Mid-Atlantic [Grant 0855439E]. N.P. is a founder and stock owner at Cardioxyl Pharmaceutical, Inc.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
- 1-nitrosocyclohexyl acetate
- analysis of variance
- maximal Ca2+-activated force
- Ca2+ required to achieve 50% of Fmax
- intracellular Ca2+
- extracellular Ca2+
- troponin C.
- Received June 21, 2011.
- Accepted August 23, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics