INTRODUCTION

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Organ dysfunction is a typical feature of human septic shock and its experimental counterpart, endotoxemia. Although several mechanisms have been suggested to alter organ function after sepsis-related inflammation, many investigators appear to favor a hyperproduction of oxygen-derived free radicals (OFR) as a main mechanism (1). OFR that include superoxide anion (O₂), hydrogen peroxide (H₂O₂), and hydroxyl radical (·OH) are generated by inflammatory cells, mitochondria, and other organelles even during normal function. Moreover, septic shock is characterized by an increased production of nitric oxide (NO). Superoxide anion is known to react with NO to form even more toxic species, peroxynitrite (ONOO) (2). In vital organs such as liver, kidney, and lungs, local production of NO or peroxynitrite can alter protein function in the cytosol and within the mitochondria through the nitration of amino acids such as tyrosine.

Cardiac dysfunction frequently occurs within 24 h of onset of sepsis-related inflammation. It is described as an alteration in both systolic and diastolic functions of both right and left ventricles (4). The role of OFR, NO, and ONOO in sepsis- induced heart dysfunction, and particularly on mitochondrial properties and on cardiac efficiency, is still controversial. An enhanced production of both NO and ONOO has been shown in septic heart (5, 6) as well as after in vitro exposure of myocardium to endotoxin lipopolysaccharide (LPS) (7). In the mitochondria, the direct effect of NO is essentially a reversible inhibition of mitochondrial respiration by competition with oxygen at cytochrome oxidase whereas ONOO induces irreversible alterations in several respiratory chain enzymes via oxidizing reactions (for review, see [8]). Diminished activities of complex I and II of the respiratory chain have been recently shown in the heart of endotoxemic animals (9), though changes in cardiac high-energy phosphate were not detected in two different models of experimental sepsis (10, 11).

At much lower concentrations than that required to inhibit mitochondrial respiration, NO and ONOO may induce a marked loss of cardiac efficiency (i.e., a reduced coupling between ATP production and mechanical work) (12). A similar observation has been made in hearts isolated from LPS-treated animals [11]. Several recent reports have also shown that even low concentrations of NO and ONOO as well as OFR are able to inhibit various isoforms of creatine kinase (CK) (13), an enzyme that plays an important role in the energy transfer in cardiomyocytes. Mitochondrial CK (mi-CK), the CK isoenzyme located at the outer surface of the inner mitochondrial membrane, is coupled with the oxidative phosphorylation, whereas myofibrillar CK (MM-type) is bound to myofilaments and functionally coupled to myosin ATPase. It has been proposed that their location in cardiac muscle facilitates the transduction of high-energy phosphates throughout the cell and acts to fine-tune the regulation of energy utilization and production. Thus, alterations in the CK system, possibly related to NO or ONOO, may lead to energetic and mechanical dysfunction during sepsis.

On the other hand, results of several works have suggested that endotoxemia-dependent myocardial dysfunction could be related to an impairment of the contractile apparatus. In a very severe model of endotoxemia in rabbits, we recently observed a reduced myofibrillar Ca²⁺ sensitivity (17). In addition, changes in Ca²⁺ myofibrillar ATPase activity were shown in chronic peritoneal sepsis in rats (18). In cardiac myocytes incubated with LPS in vitro, Yasuda and Lew (19) recently showed that the decrease in the extent of cell shortening was associated with an increase in cyclic guanosine monophosphate (cGMP) synthesis. They hypothesized that LPS-induced endogenous NO overproduction altered the status of contractile protein phosphorylation through activation of cGMP- dependent protein kinase. However, the precise mechanism responsible for alterations in contractile proteins has not been elucidated in vivo.

As the participation of the different factors may depend on the severity of septic shock, the mechanisms primarily responsible for contractile dysfunction in septic heart remain to be established. In the present study, our aims were thus to determine whether mechanical depression of septic heart is associated (1) with an alteration of energy metabolism by studying mitochondrial and CK functions, or (2) with altered myofilament responsiveness by investigating the mechanisms of decreased Ca²⁺ sensitivity. To achieve these goals, we used a nonlethal model of endotoxemia in rabbits.

	METHODS

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Animal Model

All procedures conformed with the French institutional guidelines in the care and use of laboratory animals. Experiments were carried out in conscious male New Zealand rabbits (2.0 to 2.5 kg). Rabbits were housed individually, acclimatized to a 12-h light-dark cycle, with food and water ad libitum for a 5-d period before and for a 36-h period after the treatment. Conscious rabbits received two random treatments intravenously injected (1.2 ml) through a marginal ear vein: either sterile 0.9% sodium chloride solution (saline) or endotoxin suspension (600 µg/kg; a mixture of equal amount of three types of bacterial LPS, Escherichia coli, Salmonella enteridis, and Salmonella minnesota, all from Sigma Chemical Co., St. Louis, MO) (20). Thirty-six hours after treatment, hearts from the two groups were harvested after anesthesia with pentobarbitone sodium (25 to 35 mg/kg, intravenously) and used for ex vivo experiments.

In a separate set of rabbits, the severity of the model was assessed in in vivo conditions. A fluid-filled catheter was implanted in the left carotid artery under pentobarbitone sodium anesthesia (15 mg/kg, intravenously). Rabbits were allowed to recover for 72 h and then, either endotoxin suspension or saline was injected. Arterial pressure, heart rate (both via the arterial catheter connected to a pressure transducer [Abbott Laboratories, Chicago, IL]), body weight, and temperature were recorded immediately before and up to 36 h after treatment. Hemodynamic data were recorded on a Macintosh personal computer using AcqKnowledge 3.0 software (Biopac Systems, Santa Barbara, CA). Each data point was the mean of 10 consecutive beats recorded in stable conditions. At the same time points, blood (1 ml) was withdrawn through the arterial catheter, for blood gas analysis and ionic composition and replaced by 1 ml of saline.

Isolated Papillary Muscle Studies

Left ventricular papillary muscles were quickly excised and mounted vertically in an organ bath filled with Krebs-Ringer solution containing (mM) NaCl 118, KCl 4.7, MgSO₄ · H₂O 1.2, NaHCO₃ 20, KH₂PO₄ 1.1, dextrose 4.5, and CaCl₂ · 2 H₂O 1.8 (pH 7.4 and temperature 29° C) and bubbled with a gas mixture of 95% O₂/5% CO₂. All buffer solutions contained 20 µM atenolol and 0.1 mM glutathione to prevent any -adrenergic and reactive oxygen species mediated effects (21). The lower end of the muscle was held by a phosphor-bronze clip, and the upper tendinous end was attached to an electromagnetic force- length transducer with a Tevdek 7-0 braided thread. Muscles were stimulated electrically at 0.2 Hz and at a voltage approximately 10% above threshold by rectangular pulses of 5-ms duration through two longitudinally arranged platinum electrodes (21). Muscles were stabilized for at least 2 h at the muscle length at which the maximal active tension was developed (l_max). Measurements of peak active isometric twitch tension were normalized for muscle cross-sectional area, calculated by dividing the wet weight by the length at l_max, and assuming a specific gravity of 1.00.

Functional Properties of Mitochondria and Bound CK

Muscle preparation. Respiratory parameters of the total mitochondrial population were studied in situ in saponin-skinned fibers using the method described earlier (22). Briefly, thin fiber bundles (100 to 250 µm in diameter) were excised from the endocardial surface of the left ventricle and incubated for 30 min at 4° C in skinning solution (S) containing 50 µg/ml saponin, which selectively destroys the integrity of the sarcolemma but preserves the whole population of mitochondria. The bundles were transferred into respiration solution (R) for 10 min to wash out adenine nucleotides and phosphocreatine (PCr). Respiratory rates were determined using a Clark electrode (Strathkelvin Instruments, Glasgow, Scotland) in an oxygraphic cell in 3 ml of solution R (at 22° C with continuous stirring). The solubility of oxygen was taken to be 230 nmol of O₂/ml. After measurements, the fiber bundles were carefully removed and dried. Respiration rates were expressed as µmol O₂/min/g dry weight.

Solutions. Solutions S and R contained (mM): ethyleneglycol-bis- (-aminoethyl ether)-N,N'-tetraacetic acid (EGTA)-CaEGTA buffer 10 (free Ca²⁺ concentration, 100 nM), free Mg²⁺ 1, taurine 20, and imidazole 20. To test the role of possible sulfhydryl group oxidation in endotoxemic mitochondria, 0.5 mM dithiothreitol was added in some experiments but this had no effect on the results obtained. Ionic strength was adjusted to 160 mM by addition of potassium methanesulfonate. Solution S (pH 7.1) also contained 5 mM MgATP and 15 mM PCr. Solution R (pH 7.1) contained 5 mM glutamate, 2 mM malate, 3 mM phosphate, and 2 mg/ml fatty acid free bovine serum albumin, instead of high-energy phosphates.

Protocol. To obtain adenosine diphosphate (ADP) kinetic parameters and assess the functional activity of mi-CK, skinned fibers were exposed to increasing ADP in the presence (20 mM) or in the absence of creatine. The ADP-stimulated respiration above basal oxygen consumption (₀) was plotted to determine the apparent Michaelis constant (K_m) for ADP. The apparent K_m values for ADP were calculated using a nonlinear fitting of the Michaelis-Menten equation. The maximal respiratory capacity (max) of the total mitochondrial population was determined at a high (1 mM) ADP concentration in the presence of 20 mM creatine, and the acceptor control ratio (max/₀), which reflects the extent of coupling between oxidation and phosphorylation in mitochondria, was calculated.

Enzyme Analysis

Frozen tissue samples were weighed, homogenized in ice-cold buffer (30 mg/ml) containing (mM): Hepes 5, EGTA 1, MgCl₂ 5, and Triton X-100 (0.1%), pH 8.7, and then incubated for 60 min at 0° C to ensure complete enzyme extraction. The total activities of CK and myokinase (MK) were assayed (30° C, pH 7.5) using coupled enzyme systems as previously described (23). To determine possible influence of endotoxemia on the oxidation of sulfhydryl residues, no SH-group protector was added into the media.

CK isoenzymes were separated using agarose (1%) gel electrophoresis performed at 200 V for 90 min; individual isoenzymes were resolved by incubating the gels with coupled enzyme system (24). Isoenzyme bands were visualized by the fluorescence of nicotinamide adenine dinucleotide, reduced form (NADH) and quantified using an image analysis system (Bio-Rad, Hercules, CA). Internal standards of commercial MM-CK were run in parallel with tissue samples to ensure linearity in isoenzyme quantification. The specific activity of each isoenzyme was quantified by multiplying each percentage by total activity, as determined spectrophotometrically.

Mechanical Experiments in Triton-skinned Fibers

Muscle preparation. Ventricular fiber bundles (approximately 200 µm in diameter) were dissected from papillary muscles of the left ventricle in a zero-Ca²⁺ Krebs solution (pH 7.4) and incubated for 1 h in a relaxing solution (pCa 9, see SOLUTIONS below) containing 1% Triton X-100 to solubilize the membranes. After the skinning procedure, one bundle was mounted between a fixed end and a force transducer (model AE 801; SensoNor Microelectroniks, Horten, Norway) as previously described (23), adjusted to slack length, stretched by 20%, and subjected to an activation/relaxation cycle. Sarcomere length was measured by laser diffraction and was 2.1 to 2.2 µm. The length and diameter of the muscles were measured by use of a graticule in the dissecting microscope. Fibers were immersed in 2.5-ml chambers continuously stirred at high speed (22° C).

Solutions. Solutions were prepared according to Fabiato and Fabiato (25) as previously described (23) and contained (mM): EGTA 10, imidazole 30, Na⁺ 30.6, Mg²⁺ 3.16, pH 7.1; ionic strength was adjusted to 160 mM (potassium acetate). pCa was 9 in relaxing and rigor solutions and 4.5 in activating solution. Relaxing and activating solutions also contained 3.16 mM MgATP and 12 mM PCr. Rigor solutions were obtained by mixing two solutions of pMgATP 2.5 and 6 (without PCr) or pMgATP 4 and 6 (with PCr). In some experiments, cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA) catalytic subunit from bovine heart (500 IU/ml), alkaline phosphatase (200 IU/ml), S-nitroso-N-acetylpenicillamine (SNAP, 100 µM), or isoproterenol (0.5 µM) (all from Sigma Chemical) were added to solutions.

pMgATP/rigor and pCa/tension relationships. pMgATP/tension or pCa/tension relationships were determined under isometric conditions by stepwise changes in MgATP or Ca²⁺ concentrations until maximal tension was reached. Data were fitted using nonlinear fit of the Hill equation. Slope coefficient nH as well as pMgATP and pCa for half-maximal activation (pMgATP₅₀ and pCa₅₀, respectively) were calculated for each bundle.

Stiffness and kinetic measurements. To determine the kinetic mechanical parameters of the fiber bundle, quick length changes (0.3 to 3% of initial muscle length) were applied in the relaxing and activating solutions as previously described (26). Stiffness was the extreme tension reached during stretching (mN · mm²) divided by the sarcomere length change (µm). A first series of length changes was imposed in the relaxing solution to assess passive properties of each fiber. Resting stiffness was calculated by linear regression analysis on the responses to stretches. Then a second series of length changes was initiated in control activating solution. Tension level before the first stretch was taken as maximal tension and used for normalizations. Active stiffness was calculated as the difference between total stiffness minus resting stiffness. The rate constant of tension recovery after quick stretches was calculated by a least square regression analysis, according to a single exponential model, between 50% and 80% of recovery and using stretches of more than 1% (23).

Statistical Analysis

Values were expressed as mean ± SEM. Comparisons between control and endotoxemic groups were made using Student's t test. When more than two groups of values were compared, an analysis of variance (ANOVA) with subsequent Fisher's protected least significant difference (PLSD) test was used to determine differences among groups. Statistical significance was reached at p < 0.05.

	RESULTS

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Hemodynamic Parameters In Vivo and Papillary Performance Ex Vivo

Endotoxin induced an increase in body temperature (from 38.2 ± 0.2 to 39.8 ± 0.2° C; p < 0.02), a decrease in body weight (from 2.23 ± 0.05 to 2.03 ± 0.06 kg; p < 0.01), and a moderate hypotension (mean arterial pressure from 109 ± 2 to 86 ± 3 mm Hg; p < 0.01) 36 h after treatment in seven rabbits whereas no change was observed in control rabbits (n = 5, data not shown). In addition, blood gases and ionic composition were similar between the two groups (data not shown) and no rabbit died within 36 h after LPS treatment. Measurements of cardiac performance ex vivo showed that active tension of papillary muscles from animals exposed to LPS was depressed: 18.8 ± 3.4 mN/mm² 36 h after LPS treatment (n = 8) versus 27.0 ± 5.4 mN/mm² in control rabbits (n = 5; p < 0.05).

Functional Activity of Mitochondria and mi-CK In Situ

Figure 1 represents typical recordings of the respiratory activity of saponin-skinned ventricular fibers taken from control and endotoxemic rabbits. Basal respiratory rate in the absence of adenine nucleotides was increased after addition of 100 µM ADP. The response of respiratory activity to 20 mM creatine addition, which assesses mi-CK activity, was similar in endotoxemic and control preparations. The maximal respiratory activities in the presence of 1 mM ADP and 20 mM creatine were comparable in control (21.3 ± 1.9 µmol O₂/g dry weight; n = 13 fibers from 4 hearts) and endotoxemic (23.1 ± 1.2 µmol O₂/g dry weight; n = 16 fibers from 6 hearts) preparations. Similarly, acceptor control ratio (max/₀) was the same in endotoxemic fibers (10.7 ± 1.3) compared with control ones (10.5 ± 1.3).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Oxygraph traces of respiratory activities of mitochondria in saponin-skinned fibers taken from control and endotoxemic ventricles. The arrows indicate time of addition of fibers, ADP, and creatine. Values given below the traces are oxygen consumption rates expressed as µmol O2/min/g dry weight.

In the absence of creatine, the apparent K_m values for ADP were 439 ± 56 µM in control (n = 7 fibers from 4 hearts) and 502 ± 43 µM in endotoxemic (n = 11 fibers from 6 hearts) preparations. In these fibers, creatine (20 mM) substantially decreased the K_m for ADP to 63 ± 8 µM in control and to 68 ± 9 µM in endotoxemic preparations, thus showing the unaltered function of mi-CK in endotoxemic ventricular fibers.

CK and MK Activities

CK- and MK-specific activities in ventricular muscle are reported in Table 1. As can be seen, endotoxemia did not induce any alteration in the CK and MK total enzyme activity nor in the CK isoenzyme profile.

Effects of Myofibrillar-bound CK on Rigor Tension

The functional activity of myofibrillar CK was studied by measuring the sensitivity of Triton-skinned fibers to high-energy phosphates in the presence or absence of phosphocreatine. A stepwise decrease in MgATP, in the absence of calcium and phosphocreatine, led to the appearance of typical rigor tension; the force/pMgATP relations in these conditions were identical in control and endotoxemic preparations, as can be seen from the values for pMgATP₅₀ (3.73 ± 0.04 [n = 12 fibers from 5 control hearts] versus 3.84 ± 0.06 [n = 9 fibers from 4 endotoxemic hearts], p = not significant [NS]). In both groups of fibers, the addition of phosphocreatine in the medium very sharply shifted the dependency of rigor tension on MgATP concentration toward a much lower [MgATP] (pMgATP₅₀: 5.54 ± 0.03, and 5.57 ± 0.01, in endotoxemic and control fibers, respectively). These results imply that myofibrillar CK was as efficient in endotoxemic as in control fibers.

Mechanical Characteristics of Triton-skinned Fibers

Table 2 shows that resting tension and maximal calcium-activated tension of control and endotoxemic fibers were similar. Table 2 also shows that crossbridge mechanics (active stiffness and rate constant of tension recovery) were not modified in the myocardium of endotoxemic animals.

To assess the effects of endotoxemia on myofibrillar Ca²⁺ sensitivity in myocardium, tension was measured as a function of pCa. When compared with control, the pCa/tension relationship in skinned fibers from endotoxin-treated rabbits was shifted toward higher Ca²⁺ concentrations, as shown in Figure 2. This was attested by a significant decrease in mean pCa₅₀ by 0.06 pCa unit (p < 0.01, Figure 3) with no significant change in nH (2.18 ± 0.10 [n = 10 fibers from 5 control hearts], versus 2.35 ± 0.05 [n = 11 fibers from 4 endotoxemic hearts]).

View larger version (13K): [in this window] [in a new window]   Figure 2.   Mean isometric tension-pCa relationships obtained in Triton-skinned cardiac fibers from control (solid squares) and endotoxemic (LPS, open circles) rabbits. Values are mean ± SEM of 10 fibers from five control hearts and 11 fibers from four endotoxemic hearts. Continuous curves obtained from Hill equation were fitted by least square fitting procedure. A shift toward lower pCa values in fibers from endotoxemic rabbits indicates a decrease in Ca2+ sensitivity of the contractile proteins. *p < 0.05; **p < 0.01; endotoxemic versus control fibers.

View larger version (13K): [in this window] [in a new window]   Figure 3.   Effects of treatment with either the PKA catalytic subunit (500 IU/ml), or alkaline phosphatase (200 IU/ml) on the pCa50 of Triton-skinned cardiac fibers from control (n = 5) and endotoxemic (LPS; n = 4) rabbits. Both treatments lead to the disappearance in the difference in pCa50 observed in basal conditions between groups. Values are mean ± SEM of 8 to 12 fibers in each experimental condition. *p < 0.01; endotoxemic versus control fibers in the same condition.

To investigate the role of protein phosphorylation in the reduced Ca²⁺ sensitivity of myofilaments in endotoxemic ventricular fibers, we studied the effects of alkaline phosphatase and PKA on the pCa/tension relationship of skinned fibers. After Triton X-100 treatment, some fibers from each control (n = 5) and endotoxemic (n = 4) animals were incubated for 60 min in relaxing solution containing either PKA catalytic subunit from bovine heart, or alkaline phosphatase. Both treatments led to the disappearance of the difference between endotoxemic and control pCa₅₀ values (Figure 3), with no change in nH (endotoxemic and control: 2.27 ± 0.09 [n = 11 fibers] and 2.08 ± 0.09 [n = 8 fibers], respectively, for PKA; 2.15 ± 0.03 [n = 12 fibers] and 2.10 ± 0.03 [n = 8 fibers], respectively, for phosphatase), resting tension, or maximal Ca²⁺-activated tension (Table 2). To assess whether this effect may result from the direct effect of NO, intact myocardial fibers from four control animals were incubated for 10 min with the NO donor SNAP, or isoproterenol (a well-established -adrenergic agonist which induces a PKA-dependent decrease in Ca²⁺ sensitivity of contractile proteins, and thus used as a positive control), or buffer alone. Myocardial bundles were then immediately skinned, and studied for pCa/tension relationship. Myofibrillar Ca²⁺ sensitivity was not altered by SNAP pretreatment, whereas, as expected, pCa₅₀ was decreased in fibers incubated with isoproterenol (Table 3).

	DISCUSSION

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In the present study, we have tested whether sepsis-induced myocardial dysfunction is associated with impairment in cellular energy generation or transport, and investigated the mechanisms of the reduced myofilament sensitivity to Ca²⁺ in rabbit hearts in a nonlethal but hypotensive and cardiodepressive model of endotoxemia. The main results were that, 36 h after endotoxin intravenous injection (at a time when cardiac contractility was consistently decreased in papillary muscles): (1) the intrinsic functional properties of myocardial mitochondria and CK isoform activities were normal; (2) myofilament Ca²⁺ sensitivity of left ventricular skinned fibers was reduced, with no change in passive and maximal Ca²⁺-activated tension nor in crossbridge properties; (3) this decreased Ca²⁺ sensitivity was abolished with phosphatase treatment; (4) tension/Ca²⁺ relations in endotoxemic and control preparations were similar after PKA treatment. Moreover, pretreatment of control fibers with isoproterenol, but not with SNAP, decreased the Ca²⁺ sensitivity to the value measured in endotoxemic preparations in basal conditions. These findings suggest that depression of cardiac contractility may occur without any impairment in cellular energy generation or transport during sepsis. On the other hand, sepsis-related myocardial depression could be explained by a phosphorylation-dependent decrease in myofibrillar Ca²⁺ sensitivity. This effect does not seem to result from a direct action of NO.

Impairment in energy production has been suggested to play a major role in the development of muscular contractile dysfunction during sepsis (3, 9, 27) and, according to recent studies, mitochondrial dysfunction could be related to the generation of OFR and ONOO in septic tissues (2, 3), including myocardium (5, 7). Although our model of in vivo endotoxemia was relatively mild as compared with other experimental studies (9, 17, 28), it must be noted that fluid resuscitation was not provided after LPS administration. Because tissue oxygenation has been shown to be influenced by the presence or absence of replacement fluid during endotoxemia, this could have worsened myocardial oxygen supply and energy production in our model. Despite this, we failed to detect any alteration in cardiac mitochondrial or CK isoenzymes function. Because CK is very sensitive to the effects of OFR and ONOO (14), and because these effects are not reversible in vitro (8, 14, 16), our data suggest that neither OFR nor ONOO were involved in myocardial dysfunction. Previous studies of myocardial energy metabolism in animal models of sepsis have yielded conflicting results (9). Interestingly, it was recently shown that activity of cardiac mitochondrial respiratory chain enzymes was decreased in severe, potentially lethal, endotoxemia, but not after infusion of lower amounts of bacteria (9). Although there is no strict parallelism between enzyme activities, mitochondrial function, and other indices such as PCr/ATP ratios, alterations in energy metabolism are thus likely to depend on the severity of sepsis. Our results show that such alterations are not necessary to the development of myocardial contractile depression during experimental sepsis.

Our data are the first to suggest that decreased myofibrillar Ca²⁺ sensitivity of myocardium from endotoxemic animals may result from protein phosphorylation. The fact that Ca²⁺ sensitivity in endotoxemic preparations in basal conditions was similar to the value measured in control preparations after isoproterenol pretreatment would suggest that this effect may be related to increased PKA-dependent phosphorylation of troponin I. Accordingly, PKA activity has been found to be increased in heart during experimental sepsis (29). Other protein kinases could also alter myofilament Ca²⁺ sensitivity of tension in endotoxemia-induced cardiac dysfunction. Activation of cardiac protein kinase C (PKC) has been reported during endotoxemia (30). Phosphorylation of troponin I by PKC primarily reduces maximal activity of MgATPase activity, but the PKC delta isoform appears to phosphorylate troponin I on its PKA site, resulting in a reduced Ca²⁺sensitivity of MgATPase activity (31). Several reports have suggested that NO, released during endotoxemia by endothelial cells or within myocytes, modifies myocardial contraction by raising cGMP. This latter may reduce the myofilament response to Ca²⁺, by means of the phosphorylation of troponin I by cGMP-dependent protein kinase (32). Accordingly, in vitro incubation of isolated myocytes with endotoxin induces a NO-dependent decrease in the myofilament response to Ca²⁺ (19). However, our experiments using the NO donor SNAP suggest, in accordance with several recent studies (11, 28, 33), that the direct implication of NO in the myocardial dysfunction of in vivo endotoxemia remains to be demonstrated.

Our finding that endotoxin reduces myofilament Ca²⁺ sensitivity by a phosphorylation-dependent mechanism in rabbit heart is consistent with the decreased Ca²⁺ sensitivity of cardiac myofibrillar myosin ATPase activity associated with unchanged amounts of actin, troponin I, troponin T, tropomyosin, and myosin found in a model of chronic peritoneal sepsis in rats (18). In the present study, ex vivo experiments were conducted in hearts excised 36 h after LPS injection because maximal contractile depression has been shown to occur 12 to 48 h after intravenous injection of sublethal doses of LPS in conscious animals (34, 35). Because the mechanisms of LPS-induced myocardial dysfunction may be time-dependent, our results may not be relevant to the earlier stages of endotoxemia. However, we have previously shown that Ca²⁺ sensitivity of skinned cardiac fibers was already decreased (although to a lesser extent) 4 h after LPS injection (17). Alterations leading to decreased myoplasmic Ca²⁺ transient have also been demonstrated in hearts removed from endotoxemic animals (36, 37). The relative importance of the reduced myofilament Ca²⁺ responsiveness compared with altered myoplasmic Ca²⁺ availability cannot be assessed from the present study, but the reduced myocardial contractility over a wide range of extracellular Ca²⁺ concentrations found in intact preparations (36) may be the expression of the reduced responsiveness of myofilament to Ca²⁺.

Our results may have clinical implications. First, reduced myofilament Ca²⁺ sensitivity may constitute the cellular basis of the acute ventricular dilation frequently observed in septic patients (4). Indeed, a reduction in myofilament Ca²⁺ responsiveness is associated with increased length in single cardiac myocytes and increased ventricular distensibility (32, 38). Second, the finding that a decrease in myofilament responsiveness to Ca²⁺ is a determinant of intrinsic myocardial depression in septic shock raises the possibility that Ca²⁺-sensitizing agents might be appropriate treatment to improve heart function in sepsis. Further investigations are needed to test these hypotheses as well as to determine the mechanisms involved in the phosphorylation-dependent reduction of myofilament response to Ca²⁺ in septic heart.