Protamine can induce adverse hemodynamic effects that could be related to a decrease in systemic vascular resistance and/or a negative inotropic effect (1,2). These hemodynamic effects can be because of direct effects on the cardiovascular system or indirect effects through the release of mediators and even anaphylactic reaction (3,4). Although Park et al. (5) have provided convincing arguments that protamine could induce an alteration in cardiac membrane ionic conductance, leading to intracellular calcium overload, the precise mechanisms by which protamine impairs myocardial function are not completely understood (6). A recent study suggested that the cardiac effects of protamine might be at least partly indirect through the release of mediators such as tumor necrosis factor and that these effects could be inhibited by prostacyclin (4). Moreover, although hypocalcemia may occur and calcium is commonly used in cardiac surgery, the role of extracellular calcium concentration on the myocardial effects of protamine remains unknown. The potential for heparin to reverse the myocardial effects of protamine also remains controversial (6,7).
We therefore examined the direct inotropic and lusitropic effects of protamine at different extracellular Ca2+ concentrations and studied the interaction of protamine with heparin in isolated rat cardiac papillary muscles.
We used adult Wistar rats weighing 250–300 g. Care of the animals conformed to the recommendations of the Helsinki Declaration, and the study was performed in accordance with the regulations of the official edict of the French Ministry of Agriculture.
Left ventricular papillary muscles were studied in a Krebs-Henseleit bicarbonate buffer solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 1.1 mM KH2PO4, 25 mM NaHCO3, 2.5 mM CaCl2, and 4.5 mM glucose) maintained at 29°C, as previously reported (8). Preparations were field stimulated at 12 pulses/min by two platinum electrodes with rectangular wave pulses lasting 5 ms just above threshold. The bathing solution was bubbled with 95% oxygen and 5% carbon dioxide, resulting in a pH of 7.4. After a 60-min stabilization period at the initial muscle length at the apex of the length-active isometric tension curve (Lmax), papillary muscles recovered their optimal mechanical performance. The extracellular calcium concentration ([Ca2+]o) was decreased from 2.5 to 1 or 0.5 mM because rat myocardial contractility is nearly maximum at 2.5 mM (8).
In control groups (1 or 0.5 mM [Ca2+]o, n = 10 in each group), we studied the effects of increasing concentrations of protamine(Sanofi Winthrop, Gentilly, France) in a cumulative manner (final concentrations of 10, 30, 100, and 300 μg · mL−1). The total volume of drugs did not exceed 3% of the bath volume. We verified that the largest concentration of protamine did not significantly modify ionized calcium concentration (98 ± 2% of baseline, not significant [NS]) (BG3 System; Instrumentation Laboratory, Saint-Mandé, France). The inotropic and lusitropic responses were recorded 15 min after each dose was added to the bathing solution. In addition, we studied the postrest potentiation (n = 8) for each concentration of protamine and at 0.5 mM [Ca2+]o because a postrest potentiation study is more sensitive at a small [Ca2+]o.
Since we noted a positive inotropic effect at small concentrations of protamine and at 0.5 mM [Ca2+]o, we investigated the mechanism of this effect in additional experiments. In a first group (n = 6), α- and β-adrenoceptors were blocked with phentolamine (1 μM) and propranolol (1 μM); in a second group (n = 6), Ca2+ channels (ICa) were blocked with nifedipine (10-7 M) in dimethyl sulfoxide; in a third group (n = 6), we tested dimethyl sulfoxide alone. All these drugs were added to the bathing solution at the end of the stabilization period at 0.5 mM [Ca2+]o. In an additional group (n = 6), rats were pretreated with reserpine (4 mg · kg−1 injected intraperitoneally 24 h before killing), which completely depletes intramyocardial catecholamine stores in the rat myocardium (9). Protamine (10 μg · mL−1) was then added to the bathing solution and its effect was compared to the effect of protamine (10 μg · mL−1) alone (n = 8). All the drugs were purchased from Sigma-Aldrich Chimie (L’Isle d’Abeau Chesnes, France).
Lastly, we studied the interaction between heparin (30 IU · mL−1) and protamine (300 μg · mL−1). This concentration of heparin was considered sufficient to neutralize that amount of protamine. In separate groups of muscles (n = 6 in each group), we studied the effects of heparin alone, heparin after protamine, and protamine after heparin at 1 mM [Ca2+]o.
Electromagnetic Lever System and Recording
The electromagnetic lever system has been described previously (8). Briefly, the load applied to the muscle was determined using a servomechanism-controlled current through the coil of an electromagnet. Muscular shortening induced a displacement of the lever, which modulated the light intensity of a photoelectric transducer. All analyses were made from digital records of force and length obtained with a computer as previously described (8).
Conventional mechanical variables at Lmax were calculated from three twitches. The first twitch was isotonic and was loaded with the preload corresponding to Lmax; maximum shortening (maxVc) and lengthening (maxVr) velocities were determined from this twitch. The second twitch was abruptly clamped to zero-load just after the electrical stimulus with a critical damping to slow the first and rapid shortening overshoot resulting from the recoil of series passive elastic components (8); the maximum unloaded shortening velocity (Vmax) was determined from this twitch. The third twitch was fully isometric at Lmax; maximum isometric active force normalized per cross-sectional area (AF), and the peak of the positive (+dF · dt−1) and the negative (−dF · dt−1) force derivatives at Lmax normalized per cross-sectional area were determined from this twitch. Vmax and AF tested the inotropic state under low (isotony) and high (isometry) loads, respectively. In this model, because preload was maintained constant, a contracture could be observed only in isotonic conditions and was expressed as % of Lmax. Because changes in the contraction phase induce coordinated changes in the relaxation phase, maxVr and -dF · dt−1 cannot assess lusitropy, and thus variations in contraction and relaxation must be considered simultaneously to quantify drug-induced changes in lusitropy (10). The coefficient R1 = maxVc/maxVr evaluates the coupling between contraction and relaxation under low load, and thus the lusitropy under low load (10). Under isotonic conditions, the amplitude of sarcomere shortening is more than that observed under isometric conditions (11). Because of the lower sensitivity of myofilaments for calcium when cardiac muscle is markedly shortened under low load, relaxation proceeds more rapidly than contraction, apparently because of the rapid uptake of calcium by the sarcoplasmic reticulum (SR) (8,12). Thus, in rat myocardium, R1 tests SR uptake function. The coefficient R2 = (+dF · dt−1/-dF · dt−1) evaluates the coupling between contraction and relaxation under high load, and thus the lusitropy under high load in a manner that is less dependent on inotropic changes. When the muscle contracts isometrically, sarcomeres shorten very little (11). Because of the increased sensitivity of myofilaments for calcium, the time course of relaxation is determined by calcium unbinding from troponin C rather than by calcium sequestration by the SR (12). Thus R2 indirectly reflects myofilament calcium sensitivity (10). A decrease in R1 or R2 indicates a positive lusitropic effect.
The effects of protamine on postrest potentiation was also studied. During rest in the rat myocardium, SR accumulates calcium above and beyond that accumulated with regular stimulation, and the first beat after the rest interval (B1) is more forceful that the last beat before the rest interval (B0) (8,13). During postrest recovery (B1, B2, B3 …), the SR-dependent part of activator calcium decreases somewhat towards a steady state, which is achieved in a few beats. The study of postrest potentiated contraction may provide insight into SR functions in a biochemically intact preparation. The maximal isometric AF during postrest recovery was studied at a [Ca2+]o of 0.5 mM, after a 1-min rest duration, and the rate constant of the exponential decay of AF was determined (8). τ is the number of beats required for the postrest potentiation to decay to one-tenth of its maximum, and is assumed to represent the time required for the SR to reset itself (13).
At the end of the study, the muscle cross-sectional area was calculated from the length and weight of papillary muscle, assuming a density of 1.
Data are expressed as mean ± sd. Control values between groups were compared by analysis of variance. The Student’s t-test was used to compare two means. Comparison of several means was performed using a repeated-measures analysis of variance and the Newman-Keuls test. The beat-to-beat decay of active isometric force during postrest recovery was plotted against the number of beats and fitted to an exponential curve, and regression was performed using the least squares method (8). All probability values were two-tailed, and P values < 0.05 were required to reject the null hypothesis.
We studied 76 left ventricular papillary muscles. The mean Lmax was 5.9 ± 1.2 mm, the mean cross-sectional area was 0.52 ± 0.22 mm2, the mean ratio of resting force to total force was 0.09 ± 0.03, the mean R1 was 0.74 ± 0.07, at a [Ca2+]o of 2.5 mM. A decrease in contractility was observed as [Ca2+]o was decreased from 2.5 to 1 or 0.5 mM. The decrease in Vmax (86 ± 7% of baseline at a [Ca2+]o of 1 mM and 63 ± 10% of baseline at a [Ca2+]o of 0.5 mM) and AF (88 ± 8% of baseline at a [Ca2+]o of 1 mM and 55 ± 12% of baseline at a [Ca2+]o of 0.5 mM) were consistent with those previously reported (8,14).
At a [Ca2+]o of 1 mM, protamine induced a negative inotropic effect, as shown by the significant decreases in Vmax and AF (Fig. 1). At a [Ca2+]o of 0.5 mM, protamine induced a moderate positive inotropic effect at small concentrations, then a negative inotropic effect at large concentrations (Fig. 1). At a [Ca2+]o of 1 mM, protamine induced a marked contracture of the muscle at concentrations of 100 μg · mL−1 (4 ± 3% of Lmax, P < 0.05) and 300 μg · mL−1 (12 ± 3% of Lmax, P < 0.05) (Fig. 2). In contrast, at a [Ca2+]o of 0.5 mM, protamine induced no detectable contracture.
Protamine induced a decrease in postrest potentiation (Table 1). At each concentration studied, protamine did not significantly modify τ (Table 1).
Under low load (R1), protamine induced a negative lusitropic effect that was much more pronounced at a [Ca2+]o of 0.5 mM compared with that at a [Ca2+]o of 1 mM (Fig. 3). Under high load (R2), a significant positive lusitropic effect was only noted at a [Ca2+]o of 1 mM and for the largest concentration (300 μg · mL−1) of protamine (Fig. 3). Because a decrease in AF per se induces a decrease in R2 (14), we compared the change in R2 observed with 300 μg · mL−1 of protamine and at a [Ca2+]o of 1 mM with that induced by decreasing [Ca2+]o to obtain the same decrease in AF. We found no significant difference in the decrease in R2 with protamine (80 ± 15% of baseline) and decreasing [Ca2+]o (92 ± 10% of baseline).
Mechanism of the Positive Inotropic Effect of Protamine
Because we noted that protamine induced a positive inotropic effect at a [Ca2+]o of 0.5 mM and at small concentrations (10 and 30 μg · mL−1) (Fig. 1), we investigated the mechanism of this effect. We observed that this positive inotropic effect was not significantly modified by α and β-adrenoceptor blockade or pretreatment by reserpine. In contrast, in the presence of nifedipine, protamine induced a negative inotropic effect (Fig. 4). Dimethyl sulfoxide, which was used as a solvent of nifedipine, had no significant inotropic effect and did not significantly modify the positive inotropic effect of protamine (data not shown).
Interaction with Heparin
Heparin (30 IU · mL−1) alone had no effect on contractility under low (Vmax: 101 ± 3% of baseline, NS) or high load (AF: 96 ± 4% of baseline, NS). When heparin was introduced before protamine, the negative inotropic effect of protamine was not observed (Fig. 5). Conversely, when heparin was added after protamine, heparin was able to reverse the negative inotropic effect of protamine (Fig. 6). Nevertheless, heparin did not reverse the negative lusitropic effect of protamine under low load (Fig. 6).
In the present study, we observed the following: 1) at a high [Ca2+]o, protamine induced a negative inotropic effect that was associated with a marked contracture of the muscle; 2) at a low [Ca2+]o and at small concentrations, protamine induced a moderate positive inotropic effect that was blocked by nifedipine; 3) protamine induced an impairment in mechanical variables that reflect SR function (R1, postrest potentiation); 4) heparin was able to block or significantly reverse the negative inotropic effect of protamine.
Several investigators have reported a depressant effect of protamine in isolated ventricular myocardium of various species, the rabbit (7,15), the Guinea-pig (5), the pig (6), and the human (16). No data about the effects of protamine on myocardial relaxation are available. The precise mechanism by which protamine induces a negative inotropic effect is not fully understood. Park et al. (5) have suggested that protamine may alter membrane ionic permeability and impair the Na+/K+ ATPase and Na+/Ca2+ exchange, leading to intracellular calcium overload with a subsequent increase of resting force and an eventual contracture. Moreover, calcium overload could be responsible for a decrease in contractility and impairment in the SR (17). Our data show that protamine induced a negative inotropic effect under low or high load (Fig. 1). The magnitude of this negative inotropic effect depended on [Ca2+]o, being more at 1 mM [Ca2+]o and generally parallel to the magnitude of the contracture. Although intracellular calcium was not directly measured, these results agree with the hypothesis of Park et al. (5) that the negative inotropic effect of protamine is mainly attributable to calcium overload. In contrast, at a [Ca2+] of 0.5 mM, small concentrations of protamine induced a moderate positive inotropic effect that was blocked by nifedipine, a calcium channel blocker. This result suggests that protamine can also enhance Ca2+ inward current (ICa), an effect that may also have contributed to calcium overload at high [Ca2+]o at larger concentrations. The modulation of the inotropic effects of protamine by calcium concentration might have important clinical consequences. Although its use in cardiac surgery remains controversial (18), administration of calcium might enhance the negative inotropic effects of protamine. Protamine could enhance the diastolic dysfunction observed during calcium administration after cardiopulmonary bypass (19).
Protamine induced a significant negative lusitropic effect under low load (R1), that was more pronounced at a [Ca2+]o of 0.5 mM (Fig. 3). However, it is difficult to interpret R1 at a [Ca2+]o of 1 mM because of the marked contracture of the muscle. Indeed, calcium overload probably alters SR uptake, as previously observed in rat myocardium (20). Because we did not observe any contracture at a low [Ca2+]o, the negative lusitropic effect (i.e., increase in R1), strongly suggests that protamine impairs calcium uptake by the SR. This effect on SR might have contributed to the negative inotropic effect observed at a low [Ca2+]o and high concentrations of protamine, or to calcium overload at a high [Ca2+]o.
Protamine also significantly decreased postrest potentiation without alteration in postrest recovery (Table 1). The diminished postrest potentiation suggests that protamine either decreases SR calcium release, and/or decreases the capacity of SR to accumulate calcium during rest. In contrast, postrest recovery was not modified, suggesting that the capacity of SR to reset itself was not modified by protamine. However, our results indicate that most SR functions were impaired by protamine.
At a [Ca2+]o of 0.5 mM, we did not observe any significant effect of protamine on R2. A positive lusitropic effect under high load of protamine was observed at a [Ca2+]o of 1 mM, but only at a large concentration (>300 μg · mL−1) (Fig. 3). We have previously demonstrated that a marked decrease in AF per se can induce a moderate decrease in R2 (14). This phenomenon agrees with the cooperativity concept whereby the myofilament calcium sensitivity is modified by calcium itself. We observed that the protamine-induced decrease in R2 was comparable to that observed by decreasing [Ca2+]o. Thus, our results suggest that protamine did not directly influence the myofilament calcium sensitivity.
Our results provide evidence that heparin or complex heparin-protamine had no significant inotropic effect (Fig. 5), as previously reported (7). In contrast, whether heparin can or cannot reverse the myocardial effects of protamine remains controversial. In the isolated rabbit heart, Wakefield et al. (7) observed that heparin is able to reverse the negative inotropic effect of protamine, whereas in isolated pig cardiomyocyte, Hird et al. (6) did not observe any significant reversal. In rat myocardium, we observed that heparin could relatively rapidly reverse the negative inotropic effect of protamine (Fig. 6). These results are important because these experiments were designed to mimic a clinically relevant scenario, including the concentrations of drugs tested.
What are the precise mechanisms of action of protamine? Because protamine is a polycation, it is thought to not enter into the cell. Most of its effects are thought to occur through screening of negative sarcolemmal surface charges (21); this includes modifications of the function of sarcolemmal channels such as ATP-sensitive potassium channels (22). Our results also suggest that protamine is able to modify the functions of an intracellular compartment, i.e., the SR. If protamine cannot enter the myocyte, our results suggest that the protamine-induced sarcolemmal surface changes are able to interfere with the SR, which is predictable considering the close relationship between SR and sarcolemma. However, when heparin was added to reverse the effects of protamine, we observed a dissociation between inotropic and lusitropic variables. Indeed, heparin reversed the negative inotropic effect but not the negative lusitropic effect of protamine (Fig. 6). These results suggest that the mechanisms of the protamine-induced impairment in SR function differ from those involved in its negative inotropic effects, and thus may not be related to surface charge screening. Further studies are required to confirm this hypothesis.
The following points must be considered in the assessment of the clinical relevance of our results. First, because this study was conducted in vitro, it only evaluated intrinsic myocardial contractility. Observed changes in cardiac function after in vivo protamine administration also depend on modifications in venous return, afterload, and compensatory mechanisms. For example, heparin can block protamine-induced myocardial depression, but not its severe hypotensive effects in dogs (23). Moreover, there is some evidence that protamine can also have indirect cardiac effects through the release of mediators such as tumor necrosis factor (4). Second, this study was conducted at 29°C and at a low-stimulation frequency. Papillary muscles must be studied at this temperature because stability of mechanical variables is not sufficient at 37°C, and at a low frequency because high-stimulation frequency induces core hypoxia (24). Third, it was performed in rat myocardium, which differs from human myocardium. Fourth, after administration in the human, a large amount of protamine can bind to heparin or albumin, and thus the concentrations of 100–300 μg · mL−1 that result in a toxic myocardial effect would rarely be achieved clinically.
In conclusion, in isolated rat myocardium, protamine induced a negative inotropic effect associated with muscle contracture, probably related to calcium overload at a large calcium concentration, whereas protamine induced a moderate positive inotropic effect probably related to calcium channel activation at a small calcium concentration. Protamine also altered SR functions, an effect that may contribute to calcium overload. Extracellular calcium seems to play a key role in the myocardial effects of protamine, and our results suggest that calcium administration might be not indicated in the presence of protamine-induced myocardial dysfunction. Lastly, heparin was able to inhibit or reverse the myocardial effect of protamine.
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