INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

MYOFIBRILLAR PROTEINS COMPRISE three major classes of proteins, namely contractile proteins (myosin and actin), regulatory proteins (tropomyosin and troponin complex), and structural proteins (C protein, -actinin, etc.). Interactions among these major classes of myofibrillar proteins determine the contractile properties of the heart (22, 27, 35). Contractile proteins convert the chemical energy of ATP hydrolysis into mechanical work. Regulatory proteins regulate the actin-myosin cross-bridge attachment. Structural proteins provide some mechanical linkage and stability properties to the contractile and regulatory proteins (22, 35). Protein phosphorylation has been recognized as an important mechanism for regulating cellular function and metabolism. Recent advances in the studies of cardiovascular pathophysiology have indicated that changes in the phosphorylation states of myofibrillar proteins underlie cardiac dysfunction under various disease conditions including ischemia, hypertrophy, and heart failure (3, 15, 16, 18, 19, 22, 30). Myocardial function was significantly altered in patients and in animal models during sepsis (21, 26, 32). Altered myocardial function during sepsis involves complex regulatory mechanisms. Although changes in myofibrillar proteins have been implicated to be an etiologic factor contributing to cardiac dysfunction in endotoxin and septic shock, the observations were confined to the histological examination of the myofilaments (17, 31) and the measurement of myofibrillar Mg²⁺-ATPase activities (4, 8, 21). Because the phosphorylation and the Ca²⁺ sensitivity of myofibrillar proteins play an important role in the regulation of cardiac function, the present study was undertaken to correlate sepsis-induced alterations in the phosphorylation and Ca²⁺ sensitivity of the three major classes of myofibrillar proteins with changes in cardiac contractility during different phases of sepsis.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Animal model. All animal experiments in this study were performed with the approval of the Animal Care Committee of Saint Louis University School of Medicine and in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Male Sprague-Dawley rats weighing from 275 to 300 g were used. All animals were fasted overnight with free access to water. Sepsis was induced by cecal ligation and puncture (CLP) as described by Wichterman et al. (34) with minor modification. While the rats were under anesthesia with halothane, a laparotomy was performed, and the cecum was ligated just distally to the ileocecal valve to avoid any intestinal obstruction and punctured twice with an 18-gauge needle. The cecum was then returned to the peritoneal cavity, and the abdomen was closed in two layers. Control rats were sham operated (a laparotomy was performed, and the cecum was manipulated but neither ligated nor punctured). All animals were resuscitated subcutaneously with 4 ml/100 g body wt of normal saline at the completion of surgery and also at 7 h postsurgery. Early and late sepsis refers to those animals killed at 9 and 18 h, respectively, after CLP. The mortality rates were 0% for control, 3% for early sepsis, and 29% for late sepsis.

Preparation of cardiac myofibrils and phosphorylation of myofibrillar proteins. Hearts removed from control or septic rats under urethane anesthesia were retrogradely perfused by the Langendorff technique under constant pressure (80 cmH₂O) at 37°C with Krebs-Henseleit buffer (in mM: 118 NaCl, 4.7 KCl, 1.2 MgSO, 2.5 CaCl₂, 0.1 KH₂PO, pH 7.4, 25 NaHCO, and 10 glucose) for 10 min (first perfusion). The perfusion circuit was then switched to a recirculating system containing the same buffer in the presence of [³²P]orthophosphoric acid (2.0 mCi/50 ml) and proceeded for 30 min (second perfusion). The second perfusion allowed ³²P incorporation into cellular ATP, which acts as the phosphate donor for protein phosphorylation (23). At the end of the second perfusion, the hearts were perfused for 5 min with nonradioactive buffer to wash out radioactivity (third perfusion). When the effects of isopreterenol or propranolol were studied in some experiments, atropine (0.1 µM) and prazosin (0.1 µM) were present in the perfusion media to block muscarinic and ₁-adrenergic receptors, respectively. All of the perfusion solutions were saturated with 95% O₂-5% CO₂. At the end of the third perfusion, hearts were freeze-clamped with aluminum clamps precooled in liquid nitrogen, and the samples were then used for the isolation of cardiac myofibrils.

Assay of myofibrillar ATPase activity. The activities of myofibrillar ATPase were measured as described by Resink et al. (23). To determine myofibrillar Mg²⁺-ATPase activity, the reaction mixture contained 30 mM KCl, 5 mM NaN₃, 30 mM imidazole, pH 7.0, 3 mM MgCl₂, 1 mM DTT, various concentrations of free Ca²⁺, and cardiac myofibril preparation (150 µg protein) in a final volume of 1 ml. The free Ca²⁺ concentrations were calculated using an EGTA-CaCl₂-buffering system as described by Fabiato (6). To determine myosin Ca²⁺-ATPase activity, the reaction mixture contained 0.65 M KCl, 5 mM NaN₃, 5 mM CaCl₂, 30 mM imidazole, pH 7.5, 1 mM DTT, and cardiac myofibril preparation (150 µg protein) in a final volume of 1 ml. To determine myosin K⁺, EDTA-ATPase activity, the reaction mixture contained 0.65 M KCl, 5 mM NaN₃, 1 mM DTT, 30 mM imidazole, pH 7.5, 2 mM EDTA, and cardiac myofibril preparation (150 µg protein) in a final volume of 1 ml. Phosphoenolpyruvate (3 mM) and pyruvate kinase (3 U) were included in all of the reaction mixtures as an ATP-regenerating system. The ATPase reaction was initiated by the addition of ATP and allowed to proceed for 10 min at 37°C. The reaction was then terminated by the addition of 2.5 ml of stop solution (31 mM sodium bisulfite, 8.3 mM p-methylaminophenol sulfate, 2.9 mM molybdic acid, and 350 mM H₂SO). The amount of inorganic phosphate liberated from ATP hydrolysis was measured with a spectrophotometer.

Measurement of cardiac contractility. Hearts obtained from control and septic rats were perfused with nonradioactive buffer by the same protocol as described above. A saline-filled latex balloon was inserted into the left ventricle through the left atrium and connected to a pressure transducer (Statham P23 DB) for the measurement of left ventricular pressure. The left ventricular end-diastolic pressure was adjusted at 6 mmHg at the beginning of the experiments. The maximal rate of increase (+dP/dt_max) and decrease (dP/dt_max) of left ventricular pressure development was monitored on a multichannel polygraph (Grass Instruments model 79D). At the end of each experiment, the heart was freeze-clamped, stored at 70°C, and then used for the assay of myofibrillar ATPase activity.

Other assays and statistical analysis. For measurement of heart cAMP content, 100 mg of frozen tissue sample from nonradioactive perfused hearts were homogenized at 4°C in a buffer containing 50 mM Tris, pH 7.5, and 4 mM EGTA. The homogenates were heated for 10 min in a boiling water bath. After centrifugation, the cAMP content in the supernatant was determined by radioimmunoassay with a commercial [³H]cAMP assay kit (Amersham). Na⁺-K⁺-ATPase, Ca²⁺-ATPase, and azide-sensitive ATPase activities were measured according to the method of Jones and Besch (12). The protein content was determined by the method of Lowry et al. (14). Results are expressed as means ± SE. Statistical analysis of the data was performed by using one-way ANOVA followed by Student-Newman-Keuls test. P < 0.05 was considered statistically significant.

Materials. ³²P-labeled orthophosphoric acid and [-³²P]ATP were purchased from ICN Biomedicals (Costa Mesa, CA). Na₂ATP, isoproterenol, prazosin, propranolol, atropine, leupeptin, pepstatin A, aprotinin, Triton X-100, and molecular weight standard proteins were purchased from Sigma Chemical (St. Louis, MO). Other chemicals and reagents were of analytic grade.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Table 1 depicts marker enzyme activities in cardiac homogenates and myofibrils isolated from control and septic rat hearts. The yield of myofibrils was similar among control, early septic, and late septic groups. Na⁺-K⁺-ATPase serves as a marker for sarcolemmal membrane, Ca²⁺-ATPase serves as a marker for sarcoplasmic reticulum, and azide-sensitive ATPase serves as a marker for mitochondria membrane (12). In the control group, the activities of Na⁺-K⁺-ATPase, Ca²⁺-ATPase, and azide-sensitive ATPase in myofibril preparations were reduced by 88.7, 67.4, and 96.1%, respectively, compared with the homogenate preparations. Similar results were obtained in the early and late septic groups. There were no differences in the marker enzyme activities among control, early, and late septic groups. These results indicate that the myofibrils isolated from both the control and the septic rat hearts were minimally contaminated by sarcolemma, sarcoplasmic reticulum, and mitochondria membranes.

Figure 1 depicts a representative experiment of the autoradiography from SDS-PAGE of the myofibrillar proteins labeled with ³²P in the control and septic groups. Perfusion of intact beating hearts with ³²P-labeled solution shows basal ³²P incorporation into numerous proteins, including C protein (140 kDa), troponin T (TnT) (39 kDa), TnI (27 kDa), and myosin light chain-2 (MLC-2) (19 kDa). When hearts were perfused in the presence of isoproterenol, phosphorylation of TnI and C protein was enhanced, whereas the phosphorylation of TnT and MLC-2 remained unaffected in all three experimental groups. When hearts were perfused in the presence of propranolol, the phosphorylation of TnI and C protein was diminished. These results are in agreement with previous findings that TnI and C protein were responsive, whereas TnT and MLC-2 were unresponsive to -adrenergic stimulation and inhibition (2, 16, 30).

View larger version (58K): [in this window] [in a new window]   Fig. 1.   Identification of 32P-labeled myofibrillar proteins in rat hearts. Hearts from control and septic rats were perfused with 32P-labeled buffer. Cardiac myofibrils were prepared, subjected to SDS-PAGE in 5-20% gel, and autoradiographied as described in MATERIALS AND METHODS. + Signifies that hearts were perfused in the presence, and  signifies they were perfused in the absence of isoproterenol (0.5 µM) or propranolol (1 µM) during the second and third perfusion periods. Early and late sepsis refers to experiments carried out at 9 and 18 h, respectively, after cecal ligation and puncture. Left: molecular mass standards in kDa. TnT, troponin T; TnI, troponin I; MLC-2, myosin light chain-2.

Figure 2 shows quantitative analysis of ³²P incorporation into TnI and C protein in control, early, and late septic rat hearts. Phosphorylation of TnI was increased by 268% (P < 0.01; Fig. 2A, column 3 vs. 1) during the early hyperdynamic phase, whereas it was decreased by 46% (P < 0.05; Fig. 2A, column 6 vs. 1) during the late hypodynamic phase of sepsis. Phosphorylation of C protein was increased by 76% (P < 0.01; Fig. 2B, column 3 vs. 1) during the early phase, whereas it was decreased by 41% (P < 0.05; Fig. 2B, column 6 vs. 1) during the late phase of sepsis. Phosphorylation of TnI was significantly stimulated by isoproterenol within each respective group (Fig. 2A, column 2 vs. 1, column 4 vs. 3, and column 7 vs. 6). The isoproterenol-stimulated phosphorylation of TnI was increased by 30% (P < 0.01) (Fig. 2A, column 4 vs. 2) during early sepsis, whereas it was decreased by 50% (P < 0.01) during late sepsis (Fig. 2A, column 7 vs. 2). Similar results were obtained for phosphorylation of C protein in regard to isoproterenol stimulation. It is noteworthy that the observed increases in the phosphorylation of TnI and C protein during early sepsis were completely blocked by propranolol (Fig. 2A, comparison among columns 5, 3, and 1; Fig. 2B, comparison among columns 5, 3, and 1). These findings indicate that phosphorylation of TnI and C protein underwent a biphasic change, i.e., an increase during the early phase followed by a decrease during the late phase of sepsis. Furthermore, the sepsis-induced alterations in the phosphorylation of TnI and C protein were mediated through a -adrenergic receptor pathway.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Changes in the phosphorylation of TnI (A) and C protein (B) during the progression of sepsis. The experiments were carried out as described in MATERIALS AND METHODS. The number of experiments was 6 for each group. *P < 0.05 and **P < 0.01 compared with control; P < 0.05 and P < 0.01 compared with early sepsis; P < 0.05 and P < 0.01 compared with late sepsis. + And  as in Fig. 1.

Figure 3 depicts quantitative analysis of ³²P incorporation into TnT and MLC-2 in the rat hearts during the progression of sepsis. As shown in Fig. 3A, phosphorylation of TnT remained relatively unchanged during the early and late phases of sepsis (comparison among columns 1, 3, and 6). Isoproterenol and propranolol had no effect on the phosphorylation of TnT (comparison among columns 1-7). As shown in Fig. 3B, phosphorylation of MLC-2 was relatively unchanged during early sepsis, but it was reduced by 38% (P < 0.05; column 6 vs. 1) during late sepsis. Neither isoproterenol nor propranolol affected phosphorylation of MLC-2 (comparison among columns 1-7). These data indicate that phosphorylation of TnT remained unaffected during the progression of sepsis. Although the phosphorylation of MLC-2 was intact during the early phase, it was significantly decreased during the late phase of sepsis. Furthermore, -adrenergic stimulation or inhibition had no effect on the phosphorylation of TnT and MLC-2.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Alterations in the phosphorylation of TnT (A) and MLC-2 (B) during the early and late phases of sepsis. The experiments were carried out as described in MATERIALS AND METHODS. The number of experiments was 6 for each group. *P < 0.05 compared with control. + And  as in Fig. 1.

Figure 4 shows the effect of different Ca²⁺ concentrations, and Table 2 depicts the effects of -adrenergic agonist and antagonist on the activities of myofibrillar Mg²⁺-ATPase in the control, early sepsis, and late sepsis. A sigmoid relationship between myofibrillar Mg²⁺-ATPase activities and Ca²⁺ concentrations was observed both in the control and septic groups. During the early phase of sepsis, myofibrillar Mg²⁺-ATPase activities were inhibited by 19-36% (P < 0.05) at lower Ca²⁺ concentrations (<10⁶ M) but were relatively unchanged at high Ca²⁺ concentrations (>10⁶ M). During the late phase of sepsis, myofibrillar Mg²⁺-ATPase activities were inhibited at low and high Ca²⁺ concentrations (10⁸ to 2.5 × 10⁵ M). Analysis of data using Eadie-Hofstee plots reveals that the V_max for Ca²⁺ was not significantly affected during the early phase, but it was decreased by 21% (P < 0.05) during the late phase of sepsis. The K_m value for Ca²⁺ was increased by 48% (P < 0.05) and 109% (P < 0.01) during the early and late phases, respectively, of sepsis (Table 2). The V_max for Ca²⁺ for myofibrillar Mg²⁺-ATPase was unaffected by isoproterenol. The K_m values for Ca²⁺ were increased (+39%, P < 0.05) in the control, decreased (54%, P < 0.01) in the late sepsis, but unaffected in the early sepsis by isoproterenol. These data indicate that myofibrillar Mg²⁺-ATPase was impaired during the early and late phases of sepsis, and the impairment was associated with a decrease in the Ca²⁺ sensitivity of the myofilament.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Changes in myofibrillar Mg2+-ATPase activities as a function of different concentrations of Ca2+ during different phases of sepsis. Myofibrillar Mg2+-ATPase activities were assayed as described in MATERIALS AND METHODS. Free Ca2+ concentrations were varied as indicated on the abscissa.

View this table: [in this window] [in a new window]   Table 2.   Effect of sepsis on myofibrillar Mg2+-ATPase activity and influence of -adrenergic agonist and antagonist in rat hearts

Figure 5 depicts the effects of sepsis on the myosin Ca²⁺-ATPase and K⁺-EDTA-ATPase activities. Myosin Ca²⁺-ATPase activity was unchanged during early sepsis but was decreased by 16% (P < 0.05) during late sepsis (Fig. 5A). Myosin K⁺-EDTA-ATPase activity was unaffected during the early phase but was reduced by 24% (P < 0.05) during the late phase of sepsis (Fig. 5B). Isoproterenol and propranolol had no effect on regulating myosin ATPase activities (data not shown). These results indicate that myosin Ca²⁺-ATPase and K⁺-EDTA-ATPase activities were relatively unchanged during the early phase, but they were impaired during the late phase of sepsis. The impairment in myosin Ca²⁺-ATPase and K⁺-EDTA-ATPase was not associated with -adrenergic mediation.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Changes in the activities of myosin Ca2+- ATPase (A) and myosin K+-EDTA-ATPase (B) during the progression of sepsis. Myosin Ca2+-ATPase and myosin K+-EDTA-ATPase activities were measured as described in MATERIALS AND METHODS. The number of experiments was 8 for each group. *P < 0.05 compared with control.

Figure 6 depicts changes of cardiac contractile parameters during the progression of sepsis. Myocardiac +dP/dt_max was increased by 19% (P < 0.05; Fig. 6A, column 3 vs. 1) during the early phase of sepsis, whereas it was reduced by 40% (P < 0.01; Fig. 6A, column 6 vs. 1) during the late phase of sepsis. The dP/dt_max remained relatively unchanged (Fig. 6B, column 3 vs. 1) during early sepsis, but it was decreased by 47% (P < 0.01; Fig. 6B, column 6 vs. 1) during late sepsis. The +dP/dt_max was stimulated significantly by isoproterenol within each respective group (Fig. 6A, column 2 vs. 1, column 4 vs. 3, and column 7 vs. 6). There were no differences in the isoproterenol-stimulated +dP/dt_max among control, early sepsis, and late sepsis groups (Fig. 6A, columns 2, 4, and 7). The dP/dt_max was significantly increased by isoproterenol within each respective group (Fig. 6B, column 2 vs. 1, column 4 vs. 3, and column 7 vs. 6). The isoproterenol-stimulated dP/dt_max was increased by 16% (P < 0.05) (Fig. 2A, column 4 vs. 2) during early sepsis, whereas it was decreased by 24% (P < 0.01) during late sepsis (Fig. 2A, column 7 vs. 2). The observed increases in the +dP/dt_max during early sepsis were completely blocked by propranolol (Fig. 6A, comparison among columns 5, 3, and 1). These data indicated that during the early hyperdynamic phase of sepsis, cardiac contraction was increased, whereas relaxation remained unaffected. During the late hypodynamic phase of sepsis, contraction and relaxation were both impaired. Furthermore, the sepsis-induced alterations in myocardial contraction and relaxation were mediated through a -adrenergic receptor pathway.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Effect of sepsis on cardiac contractile parameters in perfused hearts isolated from control and septic rats. A: changes in the maximal rate of increase of left ventricular pressure development (+dP/dtmax). B: changes in the maximal rate of decrease of left ventricular pressure (dP/dtmax). The number of experiments was 8 for each group. *P < 0.05 and **P < 0.01 compared with control; P < 0.05 and P < 0.01 compared with early sepsis; P < 0.01 compared with late sepsis. + And  as in Fig. 1.

Figure 7 shows alterations in cardiac cAMP levels during the early and late phases of sepsis. Tissue cAMP level was increased by 34% (P < 0.01; column 3 vs. 1) during early sepsis, whereas it was decreased by 24% (P < 0.01; column 6 vs. 1) during late sepsis. Isoproterenol significantly increased cAMP levels within each respective group (column 2 vs. 1, column 4 vs. 3, and column 7 vs. 6). The isoproterenol-stimulated cAMP level was increased by 17% (P < 0.05) (column 4 vs. 2) during early sepsis but was decreased by 19% (P < 0.05) (column 7 vs. 2) during late sepsis. The observed increase in tissue cAMP level during early sepsis was completely blocked by propranolol (comparison among columns 5, 3, and 1). These results indicate that myocardial cAMP content underwent a biphasic change during the progression of sepsis, i.e., an increase during the early phase and a decrease during the late phase of sepsis. Furthermore, the sepsis-induced alterations in myocardial cAMP level were mediated through a -adrenergic receptor pathway.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Effect of sepsis on tissue cAMP levels in perfused hearts isolated from control and septic rats. Cardiac cAMP contents were determined as described in MATERIALS AND METHODS. The number of experiments was 6 for each group. **P < 0.01 compared with control; P < 0.05 and P < 0.01 compared with early sepsis; P < 0.01 compared with late sepsis. + And  as in Fig. 1.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Data presented in the present study demonstrate that phosphorylation of TnI and C protein underwent a biphasic change during the progression of sepsis, i.e., an increase during the early phase followed by a decrease during the late phase of sepsis. The increases in the phosphorylation of TnI and C protein during early sepsis were associated with the decrease in the Ca²⁺ sensitivity of myofilaments and the increases in myocardial +dP/dt_max and cAMP level. The decreases in the phosphorylation of TnI and C protein during late sepsis coincided with the declines in the activities of myofibrillar ATPase, Ca²⁺ sensitivity of myofilaments, myocardial ±dP/dt_max, and cAMP content. The increases and decreases in the phosphorylation of TnI and C protein, ±dP/dt_max, and tissue cAMP level were sensitive to isoproterenol stimulation and propranolol inhibition. These findings suggest that alterations in the phosphorylation of myofibrillar proteins such as TnI, C protein, and MLC-2, and changes in the activities and the Ca²⁺ sensitivity of myofibrillar ATPase may contribute to the altered cardiac function during the progression of sepsis. Furthermore, the altered phosphorylation and Ca²⁺ sensitivity of cardiac myofibrillar proteins during the progression of sepsis were mediated through a -adrenergic receptor pathway.

Cardiac TnI acts as a molecular switch during the contraction and relaxation cycle (22, 28). During the diastole in which intracellular Ca²⁺ concentration is below the threshold for binding to TnC, TnI binds to actin-tropomyosin (actin-Tm) and inhibits interaction between myosin and actin through steric blocking of myosin-binding sites on actin and by inhibiting a kinetic step in the myofibrillar ATP hydrolysis cycle from the actin-myosin complex (11, 30). During the systole, a rise in intracellular Ca²⁺ concentration and subsequent binding of Ca²⁺ to TnC result in an increased affinity of TnC for the ATPase-inhibiting region of TnI and eventually functional detachment of TnI from actin. This allows TnI to move away from actin-Tm and ultimately leads to activation of myofibrillar Mg²⁺-ATPase and muscle contraction (28). Studies on myofibrillar protein phosphorylation have indicated that phosphorylation of TnI exerts an allosteric effect on Ca²⁺ binding to TnC, decreasing the sensitivity of TnC for Ca²⁺, and thus increasing the amount of Ca²⁺ that is required to bind to TnC to produce contraction (22). In so doing, TnI phosphorylation facilitates the rate of myocardial relaxation (30). Because TnI phosphorylation plays an important role in regulating cardiac function, our finding that TnI phosphorylation was increased during early but decreased during the late phases of sepsis thus provides a molecular basis for explaining why myocardium is in hyperdynamic state during the early phase of sepsis, whereas it is in hypodynamic state during the late phase of sepsis.

Phosphorylation of TnI has been defined as the basis for the decrease in myofilament sensitivity to Ca²⁺ that follows -adrenergic receptor stimulation (7, 10, 27). In the present study, the inverse relationship between TnI phosphorylation and Ca²⁺ sensitivity of myofilaments with isoproterenol stimulation was observed only in the control and early septic rat hearts. In late septic rat hearts, a decrease in TnI phosphorylation was accompanied by a decrease in Ca²⁺ sensitivity of myofibrillar Mg²⁺-ATPase. The mechanism responsible for the reduction in the Ca²⁺ sensitivity of myofibrillar Mg²⁺-ATPase during the late phase of sepsis may involve an intrinsic defect of myofilaments. Histological examination of ventricular tissue from septic animals suggested that alterations in myofilament proteins occurred during sepsis (17, 31). Myofibrillar disruption and edema were observed in the histological section obtained from canine left ventricle with chronic peritonitis (31). Interfiber and intrafiber edema was reported to occur within the ventricular myocardium of endotoxic dogs (17). These changes in myofilament structure and spacing may explain why a decrease in TnI phosphorylation was accompanied by an increase in the Ca²⁺ sensitivity of myofibrillar Mg²⁺-ATPase during the late phase of sepsis. Similar situations in regard to the dissociation between TnI phosphorylation and Ca²⁺ sensitivity of myofibrillar Mg²⁺-ATPase were also observed under other pathological conditions such as ischemia (1, 9, 33).

C protein is one of the structural proteins in the heart. The function of C protein is unclear, although both structural and regulating roles have been suggested (2, 22). In the present study, C protein phosphorylation was increased during early but decreased during the late phases of sepsis. Although alterations of C protein phosphorylation were associated with changes in cardiac dynamics during the progression of sepsis, it is less clear whether C protein phosphorylation plays a significant role in regulating cardiac performance during the progression of sepsis.

TnT is the largest component of the troponin complex. TnT has been shown to be phosphorylated by protein kinase C in cardiac myocytes (20, 22), and the phosphorylation of TnT may lead to a reduction of Ca²⁺-stimulated myofibrillar Mg²⁺-ATPase activity (20, 22) and depression of tension development (13). Our results that TnT phosphorylation remained unaffected during the early and late phases of sepsis suggest that TnT does not play a role in regulating the altered cardiac function during the progression of sepsis.

Myosin molecule is an asymmetric hexamer, consisting of a pair of heavy chains and two pairs of nonidentical light chains (24). In cardiac muscle myosin, two types of light chains are found, one with a molecular mass of 27 kDa and termed MLC-1 (essential light chain) and the other with a molecular mass of 19 kDa and referred to as MLC-2 (regulatory light chain) or phosphorylated light chain (22). The myosin molecule is generally divided into motor domain (the globular head) and tail domain (the rod). These two domains function independently in that the head retains the motor or enzymatic activity and the tail retains the ability to self-assemble into filament-like structures (24). The binding sites for actin, ATP, and the two types of light chains are located in the myosin head. Phosphorylation of MLC-2 correlates with increases in the V_max of myofibrillar Mg²⁺-ATPase and myosin Ca²⁺-ATPase (23). In an intact heart, phosphorylation of MLC-2 has been reported to correlate with increased maximum tension or dP/dt values (19, 27). In the present study, we found that MLC-2 phosphorylation was reduced during the late phase of sepsis, and this reduction was associated with decreases in the activities of myofibriller Mg²⁺-ATPase, myosin Ca²⁺-ATPase, K⁺-EDTA-ATPase, and ±dP/dt_max. These data indicate that the reduction in the phosphorylation of MLC-2 contributes to the depressed cardiac function as observed during the late phase of sepsis. Furthermore, it is noteworthy that myosin K⁺-EDTA-ATPase activity has been used as an index for the integrity of S1 and S2 thiol groups of the enzyme (25). Our finding that the observed decrease in the myosin Ca²⁺-ATPase activity coupled with the decrease in K⁺-EDTA-ATPase activity suggests that S1 and S2 thiol groups of the enzyme may be compromised during the late phase of sepsis.

The mechanism responsible for the alterations in the phosphorylation of TnI, C protein, and MLC-2 during the progression of sepsis is not completely understood. Cardiac TnI and C protein are excellent substrates for protein kinase A (10, 27), whereas MLC-2 is phosphorylated by a specific enzyme, myosin light chain kinase (2). Physiologically, TnI and C protein phosphorylation is regulated by -adrenergic receptor stimulation (30). The observed alterations in the phosphorylation of TnI and C protein during the progression of sepsis are most likely due to the altered expression of -adrenergic receptors. This contention is supported by our previous findings that -adrenergic receptors were overexpressed during the early phase but underexpressed during the late phase of sepsis (32). In current studies, phosphorylation of TnI and C protein was responsive to isoproterenol stimulation and propranolol inhibition within each respective experimental group including control, early sepsis, and late sepsis. The isoproterenol-stimulated phosphorylation of TnI and C protein remained significantly increased during the early phase but decreased during the late phase of sepsis. The altered phosphorylation of TnI and C protein with isoproterenol stimulation also correlated with changes in tissue levels of cAMP. These data reinforce the contention that the altered TnI and C protein phosphorylation as observed during the progression of sepsis is mediated by changes in the expression of -adrenergic receptors. The mechanism responsible for the reduced phosphorylation of MLC-2 during the late phase of sepsis may involve structural disruption of myofilaments as reported previously (17, 31) and changes in myosin light chain kinase activity. Further investigation is required to clarify the exact mechanism leading to the altered phosphorylation of MLC-2 during sepsis.

Perspectives