AJP - Heart Calcium Transients and Cell-Sarcomere
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 290: H886-H893, 2006. First published September 19, 2005; doi:10.1152/ajpheart.00493.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/2/H886    most recent
00493.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gao, W. D.
Right arrow Articles by Nyhan, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gao, W. D.
Right arrow Articles by Nyhan, D.

Increased cross-bridge cycling rate in stunned myocardium

Wei Dong Gao, Tieying Dai, and Daniel Nyhan

Department of Anesthesiology and Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 13 May 2005 ; accepted in final form 13 September 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 GRANTS
 REFERENCES
 
Decreased Ca2+ responsiveness of the myofilaments underlies myocardial stunning. Given that cross-bridge cycling is a major determinant of myofilament behavior, we quantified cross-bridge cycling rate in stunned myocardium. After stabilization, rat hearts were subjected to 20 min of no-flow global ischemia and 30 min of reperfusion at 37°C. Control hearts were perfused continuously at 37°C for 60 min. Trabeculae were dissected and chemically skinned with 1% Triton X-100. The muscles were then activated with solutions of varied Ca2+ concentration ([Ca2+]). Force-[Ca2+] relations, rate of force redevelopment after release (ktr), muscle stiffness (km), and myofilament ATP consumption were determined. Maximal Ca2+-activated force (Fmax) was depressed in stunned myocardium (49 ± 5 vs. 82 ± 5 mN/mm2, P < 0.01). Western immunoblotting showed degradation of troponin I in stunned myocardium. The ktr at Fmax was significantly increased in stunned muscles (19.82 ± 2.74 vs. 13.19 ± 0.96 s–1, 22°C, P < 0.01; 7.49 ± 0.52 vs. 5.81 ± 0.54 s–1, 10°C, P < 0.05). The ratio of km measured at 100 Hz over that at 1 Hz, during Fmax, is lower in stunned muscles (8.22 ± 1.56 vs. 12.94 ± 0.71, P < 0.05). In comparison with km at rigor, km at Fmax is significantly lower in the stunned group (78.82 ± 6.11 vs. 93.27 ± 3.03%, P < 0.05). Myofilament ATP consumption at Fmax did not change in stunned muscles (5,901 ± 952 vs. 5,596 ± 972 pmol·µl–1·min–1, P = 0.49). These results show that cross-bridge cycling is increased in stunned myocardium. Such increases are likely the result of increased transition rate from force-generating states to non-force-generating states. Thus stunned myocardium still maintains ATP consumption in spite of lower force development, rationalizing the long-standing paradox of decreased force but unchanged oxygen consumption in the postischemic heart.

cross-bridge cycling; contraction; myocardial stunning

MYOCARDIAL STUNNING IS CAUSED by brief but severe ischemia followed by reperfusion. This reversible myocardial dysfunction can be seen after acute myocardial infarction, cardiac arrhythmias, and open heart surgery (1). We have previously shown that stunned myocardium is characterized by decreased calcium responsiveness at the cellular level and the primary site of lesion lies at the myofilaments (10, 11). Subsequently, in rat stunned myocardium, we discovered that one of the regulatory contractile proteins, troponin I (TnI), was partially degraded and was responsible for the decreased contraction seen in stunned myocardium (12). These findings were amplified by studies from McDonough et al. (23) and Van Eyk and coworkers (34), who demonstrated loss of 17 residues from the COOH terminus of TnI in stunned rat myocardium. Murphy et al. (25) then created transgenic mice that express cardiac TnI1–193, a truncated TnI lacking 17 residues at the COOH terminus. The hearts of these transgenic mice exhibit the phenotype of stunning both at whole heart and isolated muscle levels. Controversies, however, exist on the role of partial degradation of TnI in causing myocardial stunning (20). In fact, in larger animal models of stunning (e.g., regional stunning in pigs), no proteolysis of troponin I was seen (17). The controversies may reflect the fact that different species (small rodent vs. larger animals), different models (global vs. regional), different experimental conditions, and different cellular processes are involved in stunning (16). Nevertheless, a central role of myofilaments in the pathogenesis of myocardial stunning is likely, since evidence for proteolysis of other contractile proteins have also emerged (9, 28). These structural myofilament alterations, coupled with the phenotypic loss of maximal Ca2+-activated force with little change in sensitivity, make it likely that cross-bridge cycling is abnormal in the stunned heart.

Little is known about cross-bridge behavior in stunned myocardium. McDonald et al. (22) found decreased unloaded shortening velocity in a porcine model of regional stunning. In our previous study (10), stunned muscles were found to have accelerated relaxation. Whether this accelerated relaxation is caused by more rapid dissociation of Ca2+ from troponin C or by a faster rate of cross-bridge cycling is not known. Because the intrinsic rate of relaxation of cardiac myofibrils is governed by the kinetics of the cross-bridge cycling (14, 27), the faster relaxation is likely caused by accelerated detachment of cross bridges from actin. This implies that stunned muscle has a faster cross-bridge cycling rate in spite of decreased force development. However, we did not measure the rates of cross-bridge cycling directly in our previous study.

A number of energetic studies of stunned myocardium indirectly suggest alterations in cross-bridge cycling. Myocardial oxygen consumption (MVO2) remains paradoxically high in spite of decreased contraction (18, 26). The increased MVO2 persists in both global and regional stunning models and is independent of coronary flow and cellular oxygen extraction (31). Furthermore, the increase in MVO2 during stunning is not caused by diminished mitochondrial oxidative phosphorylation (39) but by inefficient ATP utilization. A recent study has examined the various components that contribute to increases in MVO2 (33). Stunned myofilaments were found to be the predominant source of oxygen consumption, and a decreased myofibrillar efficiency resulted in oxygen wastage. However, these studies did not assess cross-bridge cycling rate directly.

In the present study, we performed rapid step release during maximal activations in chemically skinned rat trabeculae from both control and stunned hearts and assessed the rate of force redevelopment (ktr) to determine the rate of cross-bridge cycling. We found that ktr increased significantly in stunned muscles. We also performed muscle stiffness (km) analysis by imposing sinusoidal length perturbations at 1 and 100 Hz and found that the ratio of km at 100 Hz over 1 Hz was reduced in stunned muscles compared with controls. Also in stunned muscles, km during maximal activation was lower than the stiffness during rigor, in which all cross bridges are expected to remain attached because of the absence of ATP. Myofilament ATP consumption was not different in stunned muscles compared with controls. These results show that there is an increased cross-bridge cycling rate in stunned myocardium. Stunned myocardium maintains ATP consumption in spite of decreased force development, likely because of an increased rate of cross-bridge turnover from force-generating states to non-force-generating states.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSIONS
 GRANTS
 REFERENCES
 
Rats. Male LBN-F1 (~250–300 g body wt, n = 30) rats were used in these experiments. The care of the animals and the experiment protocol were approved by the Animal Care and Use Committee of The Johns Hopkins University.

Whole hearts. The hearts were exposed via midsternotomy after the animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (100 mg/kg). 0.3–0.5 ml (300–500 U) heparin was injected in the left atrium, and the aorta was clamped. The heart was then rapidly excised, and the aorta was cannulated. The hearts were perfused retrogradely (~15 ml/min) with Krebs-Henseleit (K-H) solution equilibrated with 95% O2-5% CO2. The K-H solution is composed of (in mmol/l): 120 NaCl, 20 NaHCO3, 5 KCl, 1.2 MgCl, 10 glucose, and 1.0 CaCl2, pH 7.35–7.45. The hearts were beating at 270–300 beats/min. Isovolumic left ventricular pressure was measured with an intracavity balloon filled with water and connected to a pressure transducer. The volume of the balloon was adjusted to a diastolic pressure of ~10 mmHg, which was kept constant throughout the experiments. The hearts were placed in a water-jacketed container to maintain a constant temperature of 37°C. All hearts were perfused at constant flow (15 ml/min). An implantable temperature probe was put inside the ventricle, and the temperature was kept at 37°C. After stabilization, the hearts were divided into the following groups. In control group, the hearts were perfused continuously for 60 min. In the stunned group, the hearts were subjected to 20 min no-flow global ischemia. After ischemia, the hearts were reperfused for 30 min. After each protocol, the hearts were removed from the perfusion apparatus and subsequently perfused with high-K+ (20 mmol/l) K-H solution via the aorta in a dissection dish at room temperature (20–22°C).

Skinned rat trabeculae. Trabeculae from control and stunned groups were quickly dissected from the hearts and were rapidly perfused with high K+ K-H solution. Afterward, the trabeculae were immediately skinned by 15–20 min of exposure to 1% Triton X-100 in relaxing solution containing (in mmol/l) 100 KCl, 25 HEPES, 10 K2EGTA, 15 creatine phosphate sodium salt, 5 Na2ATP, 5.15 MgCl2, and 0.5 leupeptin 0.5 (pH 7.2 with KOH). Activating solution contained (in mmol/l) 10 Ca2+-EGTA, 100 KCl, 25 HEPES, 15 Na2CrP, 5 Na2ATP, 4.75 MgCl2, and 0.5 leupeptin, pH 7.2. Varied Ca2+ concentrations ([Ca2+]) were achieved by mixing the activating solution and relaxing solution in various ratios. [Ca2+] was calculated by a computer program that was based on the stability constants and the enthalpy values for the various reactions from Martell and Smith (21), except values for Mg2+-ATP and Ca2+-ATP reaction from Pettit and Siddiqui (29). Skinning was considered to be complete when the preparation lost its pink color and sarcomeres were visualized. Diastolic sarcomere length was determined by direct visualization under x100 magnification and was set at ~2.2 µm. Resting force was usually 5–10% of maximal activated force at this sarcomere length. The steady state force-[Ca2+] relations were fit with a function of the Hill equation

(1)
where F is the steady-state force at varied [Ca2+] values, Fmax is the maximal Ca2+-activated force, Ca50 is the [Ca2+] required for 50% of maximal activation, and n is the Hill coefficient.

Western immunoblotting of troponin I. In separate experiments, frozen (–80°C) tissue samples (100–200 mg) from control and stunned hearts were ground in liquid nitrogen and decanted into preweighed ice-cold Eppendorf tubes with lysis buffer (6.0 mol/l urea, 2.0 mol/l thiourea, 4% CHAPS, and 40 mmol/l Tris, pH 8.8). The mixture (10% wt/vol) was homogenized by sonication and centrifuged at 12,000 rpm for 40 min at 4°C. The supernatant was collected. Protein contents were determined with the Bio-Rad Protein Assay Kit. Myofilament proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane. After being blotted, the membrane was incubated with anti-TnI (3I-35; epitope 175–189 fragment, dilution 1:30,000; Spectral Diagnostic) for 1 h at room temperature with gentle agitation. After being washed, the membrane was probed with anti-mouse IgG (1:30,000) conjugated to horseradish peroxidase (HRP). HRP activity was visualized with Super Signal Chemiluminescent Substrate(Pierce) for 5 min, and the membranes were exposed to X-ray film (KODAK BioMax XAR Film) for 1–120 s to detect the blot.

Mechanical measurement of cross-bridge kinetics. The quick release/restretch protocol was used to measure ktr. The skinned muscle was mounted between a force transducer and a motor arm via aluminum foil T-clips, the arms of which were folded and crimped directly on both ends of the muscle. The muscle length was decreased by a rapid release step (20% of muscle length) to drop force to zero in 1 ms, held for 25 ms, and then stretched back to the original length. The redeveloped force was recorded. These experiments were performed at both 10 and 22°C. The redeveloped force was fitted with the following single exponential equation

(2)
where Fr is the level of force redevelopment at any time (t) after release and k is ktr.

Measurement of km. The km was measured using small (<0.5% of muscle length) sinusoidal oscillations at 1 and 100 Hz. The ratio of force perturbations over muscle length changes will be used to indicate km.

Measurement of myofilament ATP consumption. Isometric ATPase activity was measured using the following coupled enzyme system (15), which was later adopted by Wannenburg et al. (37) in cardiac trabeculae (37):

1) pyruvate kinase converts: ADP + phosphoenolpyruvate -> ATP + pyruvate;

2) lactate dehydrogenase converts: pyruvate + NADH + H+ -> NAD+ + lactate.

In this coupled system, ATP is added to fuel contraction. ADP thus produced is converted back to ATP by the oxidation of NADH to NAD. Also in this system, one mole of NADH is converted to NAD for one mole of ATP to ADP. Because NADH fluoresces at 470 nm (emission) under ultraviolet radiation at 340 nm (excitation), decreases in fluorescence at 340 nm excitation in the solution reflects a reduction in NADH amount in the solution and thus the rate of ATP hydrolysis by the myofilaments (i.e., ATPase activity). Pyruvate kinase and lactate dehydrogenase, and their substrates (NADH and phosphoenolpyruvate), were added to both relaxing and activating solutions. To allow better diffusion into myofibrils, muscles were bathed in relaxing solution containing these substrates for a few minutes before the next activation. The concentrations of these substrates in both relaxing and activating solutions were as follows: 0.6 mmol/l NADH, 10 mmol/l phosphoenolpyruvate, 100 U/ml pyruvate kinase, 12 U/ml lactate dehydrogenase, 0.2 mmol/l A2P5, and 100 µmol/l leupeptin. To ensure adequate mixing of all substrates, the volume of the bath was limited to 100 µl, and the solution was stirred by air bubbles from a microneedle placed at one end of the bath. The small disturbance in the solution allows adequate mixing but does not interfere with fluorescent measurement. Calibration was performed separately, using ADP (10 nmol/l) that was manually injected in the bath containing the coupled enzyme system. The step drop in fluorescence (in mV) after ADP injections was measured and was later used to calculate ATPase conversion (decrease in mV was used as the denominator). In most of our experiments, ATPase measurements were performed only during maximal activations. We therefore injected ADP in the same maximal activating solution after removing the muscle and calculated ATPase activity based on the drop for that muscle. We also injected ADP in the bath containing the coupled enzyme system during rest. We pooled the data of the injections and determined that the step drop of fluorescence after 10 nmol/l ADP was 6.5 ± 0.3 (SD) mV in <20 s (n = 7). Thus, in our system, there were only small variations in the step drops after ADP injection across experiments. The decreases of fluorescence at 340 nm were monitored during activations, and the rate of NADH decrease (and thus the rate of ATP consumption) was obtained. ATP consumption was normalized to muscle volume and expressed as picomoles per microliters per second. We also calibrated NADH fluorescence over an NADH concentration range of 0–2.0 µmol/l and found that the relationship between NADH concentration and fluorescence intensity was nonlinear. However, a linear relationship was determined in the range of NADH concentrations of 0–400 nmol/l, a range over which our experiments were performed.

Statistics. Student's t-test and one-way ANOVA was used for statistical analysis of the data (32, 38). A value of P < 0.05 was considered to indicate significant differences between groups. Unless otherwise indicated, pooled data are expressed as means ± SE.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSIONS
 GRANTS
 REFERENCES
 
Force-[Ca2+] relations in skinned stunned muscles. Figure 1 shows the pooled data from all muscles studied. In Fig. 1A the data are shown in absolute values, and in Fig. 1B the data are normalized to their maximal values. These results are consistent with our previous findings in stunned muscles (11). Fmax decreased significantly in stunned muscles (49 ± 5 vs. 82 ± 5 mN/mm2, P < 0.01). Ca50 increased in stunned muscles (1.68 ± 0.14 vs. 1.22 ± 0.14 µmol/l in control muscles, P = 0.04), with no changes in the Hill coefficient n (2.5 ± 0.41 in stunned muscles vs. 2.1 ± 0.19 in control muscles, P = 0.5). Thus stunned muscles showed decreased Ca2+ responsiveness.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 1. Force-Ca2+ concentration ([Ca2+]) relationships in control and stunned trabeculae. A: pooled data of force-[Ca2+] relations in control and stunned muscles. These muscle were skinned chemically with Triton (1%), and varied levels of forces were achieved with different [Ca2+] values. Steady-state forces were plotted against [Ca2+] values in both groups. Note that maximal Ca2+-activated force is decreased and [Ca2+] required for 50% of maximal activation is increased in stunned muscles (see text for details). B: normalized force-[Ca2+] relations in the two groups of muscles. Data were normalized to the maximal force in each group. A rightward shift of the force-[Ca2+] relations in stunned muscles is demonstrated (see text for details). Data are means ± SE; n = 8–10 experiments in each group.

 
Western immunoblots of TnI. The role of TnI degradation in the stunned myocardium is controversial. To determine if TnI degradation occurred in our experiments, we performed Western immunoblot analysis of troponin I in tissues from hearts that underwent 20 min global ischemia followed by 30 min reperfusion. Figure 2 reveals a degradation band of TnI in stunned myocardium. Brief ischemia (20 min) alone did not cause TnI degradation. Similar results were obtained in three stunned hearts. These results are consistent with our previous studies (12), confirming that TnI is indeed partially and selectively degraded in stunned myocardium.



View larger version (26K):
[in this window]
[in a new window]
  		Fig. 2. Western immunoblot of troponin I (TnI) of tissue sample from control (ctr), stunned (stun), and 20-min ischemic (isch) hearts. Each lane was loaded with 10 µg sample protein. Note that an additional [degradated (degrad)] band of TnI is seen in the stunned sample but not in control and 20-minute ischemic samples. MW, mol wt.

 
Changes in ktr after stunning in skinned muscles. One of the major changes in stunned myocardium is the depression of Fmax (see Fig. 1A). We thus investigated whether altered kinetic behavior of cross bridges affected force development during maximal activation. Brenner (3, 4) showed that the turnover kinetics of the cross bridges is important in regulating force development. When the turnover rate of the attached cross-bridges changes, the force-Ca relationship changes. We performed quick-step releases during Fmax to assess changes in ktr to probe the behavior of the cross bridges in stunned myocardium. Figure 3A shows the typical traces of force redevelopment after a quick-release step of 25 ms duration. After normalization, it is clear that the rate of force recovery is faster in stunned trabecula than control (Fig. 3, A and B, insets). These force redevelopment traces were fitted with a single exponential equation, and ktr values were determined. Figure 3C shows the pooled data of ktr from both control and stunned muscles. The ktr increased significantly in stunned muscles compared with control muscles (19.82 ± 2.74 vs. 13.19 ± 0.96 s–1, P < 0.02, temperature 22°C). We repeated these experiments at 10°C to verify these changes (Fig. 3, B and D). Despite slowing of ktr as temperature drops, it remains accelerated in stunned muscles (7.49 ± 0.52 vs. 5.81 ± 0.54 s–1, P < 0.05). We also performed quick-release experiments at two submaximal [Ca2+] (Table 1). The ktr was not statistically different between control and stunned groups at these submaximal activations.



View larger version (18K):
[in this window]
[in a new window]
  		Fig. 3. Force redevelopment after a step release during maximal Ca2+ activation. A: representative traces of force redevelopment after a step release during maximal activation in a control and a stunned muscle at 22°C. Inset compares the time courses of the two traces after normalization to each maximal value. The stunned muscle has faster recovery time to the prerelease level. The traces were fitted by a single exponential equation, and the rate constants of force redevelopment (ktr) were obtained in each muscle. [Ca2+] = 24 µmol/l. C: summery of ktr from control and stunned muscles at a temperature of 22°C. B: representative traces of force redevelopment after a step release during maximal activation in a control and a stunned muscle at 10°C. D: summery of ktr from control and stunned muscles at a temperature of 10°C. Please note that, although decreased at 10°C, ktr remains accelerated in stunned muscles. Data are means ± SE; n = 8–10 in each group. *P < 0.01 and **P < 0.03 vs. control.

 

View this table:
[in this window]
[in a new window]
  		Table 1. ktr and myofilament ATPase activity in control and stunned muscles at submaximal activations

 
Because ktr is the sum of fapp (the rate constant for cross bridges from a non-force-generating state to a force-generating state) and gapp (the rate constant from a force-generating state to a non-force-generating state), we derived fapp and gapp based on ktr measured at maximal [Ca2+] and km at rigor and during Fmax (see below). Table 2 shows the derived fapp and gapp in control and stunned muscles. Thus the rate for cross bridges from a force-generating state to a non-force-generating state is increased in stunned muscles.


View this table:
[in this window]
[in a new window]
  		Table 2. fapp and gapp in control and stunned muscles during maximal Ca2+ activation

 
km in stunned myocardium. In this study, we also determined km by use of sinusoidal muscle length perturbations at frequencies of 1 and 100 Hz. In an active muscle, km, when measured at 100 Hz of length perturbations, increases as more cross bridges are attached, whereas km at 1 Hz remains nearly the same compared with a nonactivated muscle (19, 36). We have also shown previously that measurement of the km ratio (100 over 1 Hz) is a sensitive method to establish whether active cross-bridge cycling is present. In each individual muscle during Fmax, km was first measured at 1 Hz and then at 100 Hz, and the ratio of km at 100 Hz over 1 Hz was determined (Fig. 4). The ratio of km at 100 over 1 Hz is greater in control muscles compared with stunned muscles, indicating a higher number of cross bridges during Fmax in control muscles.



View larger version (11K):
[in this window]
[in a new window]
  		Fig. 4. Muscle stiffness measured at 1 and 100 Hz of length perturbations in control and stunned muscles. A: raw traces of muscle stiffness measurement at 1 and 100 Hz muscle length perturbations in a control (top) and a stunned (bottom) muscle. These experiments were performed during maximal Ca2+ activations, and the magnitude of muscle length changes during the perturbations was 0.4% of muscle length at rest. Optimal muscle length was set at a sarcomere length of 2.20 µm and temperature 22°C. B: top, muscle stiffness at 1 and 100 Hz of length perturbations in control and stunned muscles; bottom, pooled data of the ratio of muscle stiffness measured at 100 Hz over 1 Hz in control and stunned groups. Note that the ratio (stiff100Hz/stiff1Hz) is lower in stunned muscles, indicating less cross bridges contributing to force development during maximal Ca2+ activation. Data are means ± SE; n = 5 in each group. *P < 0.05 vs. control.

 
Rigor defines a state when cross bridges are unable to detach when ATP is absent. During rigor, all cross bridges are "locked" and km is expected to be maximal. To determine whether a decreased capability of forming cross bridges is responsible for the decreased Fmax, we induced rigor in stunned muscles by exposing the muscles to ATP-free activating solutions containing maximal Ca2+, and km was measured at 100 Hz. There is no change in km at rigor in stunned muscles (4,799.81 ± 544.83 vs. 5,385.58 ± 887.58 mN·mm–2·µm–1, P = 0.49). This indicates that the total number of cross bridges available for attachment is not different in stunned myocardium compared with control. However, the maximal active km during Fmax decreased over 20% when comparing with km at rigor in stunned muscles (Fig. 5). In control muscles, km during Fmax decreased ~7% in comparison with the km at rigor.



View larger version (13K):
[in this window]
[in a new window]
  		Fig. 5. Muscle stiffness at rigor (A) and during maximal Ca2+ activations (B) in control and stunned muscles. The muscle stiffness was measured at 100-Hz length perturbations with magnitudes of 0.4% of resting length. Muscle stiffness at rigor was not different in control and stunned muscles. Muscle stiffness during maximal Ca2+ activation is not different from that at rigor in control muscles. In stunned muscles, there is an ~20% decrease in muscle stiffness during maximal Ca2+ activations compared with the stiffness at rigor (see text for details). Fmax, maximal Ca2+-activated force. Data are means ± SE; n = 6 in each group. *P < 0.05 vs. control values.

 
Myofilament ATP consumption in stunned myocardium. To complement the above findings, we assessed myofilament actomyosin-ATPase activity in separate groups of skinned muscles (Fig. 6). These measurements would add the analysis of cross bridge in stunned myocardium. Because the major change of Ca2+ responsiveness in stunned myocardium is depression of force development during maximal activations, we focused our measurement of myofilament ATP consumption at Fmax. Figure 7 shows the summary of the data. In spite of significant decreases in Fmax, myofilament ATP consumption was not reduced in stunned muscles. We also measured ATP consumption at two different submaximal activations, and there was no difference between control and stunned groups (Table 1). ATPase activity at rest was not different between control and stunned groups (results not shown).



View larger version (15K):
[in this window]
[in a new window]
  		Fig. 6. Measurement of myofilament ATP consumption. Top: maximal force development during exposure to [Ca2+] in control trabecula. [Ca2+] = 25 µmol/l. Bottom: corresponding changes in NADH fluorescence. Inset: calibration of NADH fluorescence. ADP (10 nmol/l) was manually injected in the bath containing activating solution after the muscle was removed. The step change (in mV) was later used to calculate ATP consumption (see text for details).

 


View larger version (8K):
[in this window]
[in a new window]
  		Fig. 7. Maximal Ca2+-activated force (top) and myofilament ATP consumption (bottom) in control and stunned groups. Maximal Ca2+ activation was achieved by exposing the skinned muscles to activating solution containing 25 µmol/l Ca2+. Note that Fmax decreased significantly, whereas myofilament ATP consumption remained unchanged in stunned muscles. Data are means ± SE; n = 5 in each group. *P < 0.01 vs. control values.

 

   DISCUSSIONS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
GRANTS
 REFERENCES
 
This study in stunned myocardium reveals increased ktr, decreased km during maximal activations with no changes in km at rigor, and no changes in myofilament ATP consumption despite reduced maximal force development. These findings suggest that cross-bridge cycling rate is increased in stunned myocardium. The increased cross-bridge cycling results in decreased Fmax and maintained myofilament ATP consumption.

Cross-bridge cycling in stunned myocardium. We have previously modeled the cross-bridge behavior of stunned myocardium using a cross-bridge model with experimentally constrained rate constants (10). Using measured intracellular [Ca2+] transients as inputs, we predicted that decreased force production could be the result of decreased cross-bridge attachment rates and increased cross-bridge detachment rates. In this study, we assessed cross-bridge cycling directly using a quick-step release method to determine ktr.

Based on the current cross-bridge model (2), ktr obtained from the quick-release experiments (under conditions of low Pi and maximal Ca2+ activation) is the sum of fapp and gapp. Also, force development depends on fapp

(3)
where N indicates the number of cross bridges available, Fo indicates the mean force produced by each strongly attached cross bridge, and fapp/(fapp + gapp) represents the occupancy of force-generating states (2, 5). We have shown in this study that ktr increased significantly in stunned muscles (Fig. 3). Either increased fapp or gapp, or both, would cause the increase in ktr. However, an increase in fapp would result in higher force development. On the other hand, increases in gapp would predict a reduced occupancy of force-generating states of cross bridges and thus lower force development (Table 2).

The ktr did not change significantly at the submaximal activations in stunned muscles tested in this study. However, this does not necessarily mean cross-bridge cycling was not affected. Recent investigations have suggested that cooperative activation of the thin filament because of strong-bound cross bridges plays an important role in force redevelopment (6, 7), especially at submaximal activations. Thus ktr at submaximal Ca2+ is not only dependent on Ca2+ (4, 5) but also on cooperativity (6). More detailed experiments at submaximal Ca2+ will be needed to define cross-bridge behavior at submaximal activations in stunned myocardium.

Changes in km found in this study also support that the occupancy of cross bridges in force-generating states is lower at maximal activation in stunned myocardium. The km at rigor reflects all available cross bridges that are formed and locked in the attached states. Thus km at rigor represents maximal stiffness under the same experimental conditions for the muscle. We have found that km during Fmax is not different from the km at rigor in control muscles. This suggests that the total number of cross bridges available for attachment and contraction is not different in stunned and control myocardium. However, km during Fmax is 23% less than the km at rigor in stunned muscles (Fig. 5), suggesting >20% less attached cross bridges during Fmax. This could be caused by either reduced availability of the cross bridges (i.e., N in Eq. 3) or reduced occupancy of the cross bridges in force-generating states (i.e., the total number of cross bridges engaged in cycling is not reduced but, as a result of increased gapp, the number of cross bridges at any given time is reduced). The fact that the km at rigor is not different between control and stunned muscles indicates that stunned myocardium is capable of recruiting the same total number of cross bridges (i.e., the same N) as control, negating that a reduction in the number of cross bridges available for force generation is responsible for lower force development at maximal activation. Therefore, increased gapp results in less Fmax in stunned myocardium. Our results show that km during Fmax is ~93% of rigor stiffness in control muscles. A similar high ratio of km during Fmax at rigor has also been reported from other laboratories (30). One potential explanation for the high ratio is the formation of rigor-like cross bridges during Fmax (36). If this is true, we could have overestimated the km during Fmax in stunned myocardium. This high ratio also limits our ability to conclude how much fapp and gapp have changed in stunned muscles based on Table 2.

This study, however, cannot rule out the possibility that the force generated by each individual cross bridge is reduced in stunned myocardium (i.e., lower Fo in Eq. 3), thus causing decreased Fmax. Some recent studies have focused on the structural alterations within the cross-bridge complex in stunned myocardium and have suggested decreased strains in the cross bridges formed in the presence of truncated troponin I (8). Direct evidence of decreased force production by each individual cross bridge is still lacking. Moreover, ktr would not necessarily be affected when Fo is decreased.

The results from this study contrast those of McDonald et al. (22), who found decreased unloaded shortening velocity in a porcine model of regional stunning. The discrepancy can be the result of a couple of factors: 1) different models of stunning can cause different changes in myofilaments and thus differences in cross-bridge behavior. For example, in the regional stunning in pigs, no proteolysis of troponin I is seen (17); 2) differences in experimental protocol (e.g., loaded vs. unloaded shortening, slack-length vs. rapid-release stretch back, cell segments vs. intact fibers) could account for the different observations.

Myofilament ATP consumption in stunned myocardium. We have measured myofibrillar ATP consumption during maximal activations and found that the myofibrillar ATP consumption remained unchanged compared with controls, whereas maximal force development decreased significantly (Fig. 7). Our findings are consistent with a number of other studies. It has long been recognized that stunned myocardium consumes more oxygen in spite of decreased contraction (18, 26). The increased MVO2 is independent of coronary flow and tissue oxygen extraction (31). A recent study has shown that oxygen consumption by processes involved in excitation-contraction coupling and by myofibrils increased significantly in stunned myocardium (33). Foster et al. (8) have provided more direct evidence that the myofibrillar ATPase activity was increased in stunned myocardium. Using COOH-terminal truncated cardiac troponin I (cTnI1–192) to reconstruct the troponin complex, they found that there is a >50% increase in Ca2+-activated S1 ATPase activity, whereas force development was ~76% compared with myofilaments composed of reconstructed full-length cTnI. The mechanism of maintained ATP utilization in spite of decreased force in stunned myocardium is not entirely clear. Increases in the rate of cross-bridge detachment, gapp, predict increased ATP usage since cross-bridge detachment occurs when ATP is bound to the actomyosin complex. However, at a molecular level, the nature of the increased cross-bridge detachment remains elusive. We did not calibrate ATPase activity during activations, and this is a limitation to our study. Given that accurate ATPase calibration may be altered by solution volume, light intensity, NADH signal, and basal ATPase activity, we could not completely rule out the contribution of these confounding variables contributing to quantitative changes in ATPase activity.

When viewed in the context of the three-states model of thin filament regulation of contraction (13, 24, 35), the increased ATP consumption in spite of decreased force is likely the result of destabilization of the strongly bound actomyosin intermediates of the cross-bridge cycle (i.e., during the open state). In the absence of Ca2+, troponin stabilizes tropomyosin in a position that blocks myosin binding to actin (the blocked state). In the presence of Ca2+, troponin shifts tropomyosin and allows myosin to bind actin (the closed state). In the presence of myosin binding, tropomyosin adopts a state that permits myosin to bind all exposed sites on actin and hydrolyzes ATP (the open state). It is conceivable that the states of tropomyosin are affected by the troponins. In stunned myocardium, the position of tropomyosin in the open state is likely altered such that the transition to other states is accelerated and the strongly bound cross bridges are not fully supported as a result of degradation of troponin I (12, 23, 34). Thus ATP consumption by the cross bridges would be maintained in spite of decreased force generation.

We conclude that cross-bridge cycling rate is increased in stunned myocardium. The increased cycling rate is caused by an increased rate of transition from force-generating states to non-force-generating states (gapp), resulting in lower force development. Myofilament ATP consumption remained unchanged in spite of decreased force development. These findings have important implications for our understanding and management of stunned myocardium clinically.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Grant HL-44065 (to W. D. Gao).


   ACKNOWLEDGMENTS
 
We thank Eduardo Marbán for helpful discussions throughout the work and for critical reading of the manuscript. We also thank Dr. Pieter de Tombe for suggestions about the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. D. Gao, Dept. of Anesthesiology and Critical Care Medicine, Johns Hopkins Univ. School of Medicine, Tower 711, 600 N. Wolfe St., Baltimore, MD 21287 (e-mail: wgao3{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSIONS
 GRANTS
 REFERENCES
 

  1. Bolli R and Marban E. Molecular and cellular mechanisms of myocardial stunning. Physiol Rev 79: 609–634, 1999.[Abstract/Free Full Text]
  2. Brenner B. Changes in calcium sensitivity at the cross-bridge level. In: Modulation of Cardiac Calcium Sensitivity. A New Approach to Increasing the Strength of the Heart, edited by Lee JA and Allen DG. Oxford, UK: Oxford Univ Press, 1993, p. 197–214.
  3. Brenner B. The cross-bridge cycle in muscle. Mechanical, biochemical, and structural studies on single skinned rabbit fibers to characterize cross-bridge kinetics in muscle for correlation with the actomyosin-ATPase in solution. Basic Res Cardiol 81: 1–15, 1984.
  4. Brenner B. Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: implications for regulation of muscle contraction. Proc Natl Acad Sci USA 85: 3265–3269, 1988.[Abstract/Free Full Text]
  5. Brenner B. Rate of force generation in muscle: correlation with actomyosin ATPase activity in solution. Proc Natl Acad Sci USA 83: 3542–3546, 1986.[Abstract/Free Full Text]
  6. Campbell K. Rate constant of muscle force redevelopment reflects cooperative activation as well as cross-bridge kinetics. Biophys J 72: 254–262, 1997.[Web of Science][Medline]
  7. Fitzsimons DP, Patel JR, and Moss RL. Cross-bridge interaction kinetics in rat myocardium are accelerated by strong binding of myosin to the thin filament. J Physiol 530: 263–272, 2001.[Abstract/Free Full Text]
  8. Foster DB, Noguchi T, VanBuren P, Murphy AM, and Van Eyk JE. C-terminal truncation of cardiac troponin I causes divergent effects on ATPase and force: implications for the pathophysiology of myocardial stunning. Circ Res 93: 917–924, 2003.[Abstract/Free Full Text]
  9. Foster DB and Van Eyk JE. In search of the proteins that cause myocardial stunning. Circ Res 85: 470–472, 1999.[Free Full Text]
  10. Gao WD, Atar D, Backx PH, and Marban E. Relationship between intracellular calcium and contractile force in stunned myocardium. Direct evidence for decreased myofilament Ca2+ responsiveness and altered diastolic function in intact ventricular muscle. Circ Res 76: 1036–1048, 1995.[Abstract/Free Full Text]
  11. Gao WD, Liu Y, Mellgren R, and Marban E. Intrinsic myofilament alterations underlying the decreased contractility of stunned myocardium. A consequence of Ca2+- dependent proteolysis? Circ Res 78: 455–465, 1996.[Abstract/Free Full Text]
  12. Gao WG, Atar DA, Liu Y, Perez NG, Murphy AM, and Marban E. Role of troponin I proteolysis in the pathogenesis of stunned myocardium. Circ Res 80: 393–399, 1997.[Web of Science][Medline]
  13. Geeves MA and Lehrer SS. Dynamics of the muscle thin filament regulatory switch: the size of the cooperative unit. Biophys J 67: 273–282, 1994.[Web of Science][Medline]
  14. Gordon AM, Homsher E, and Regnier M. Regulation of contraction in striated muscle. Physiol Rev 80: 853–924, 2000.[Abstract/Free Full Text]
  15. Griffiths PJ, Guth K, Kuhn HJ, and Ruegg JC. ATPase activity in rapidly activated skinned muscle fibres. Pflügers Arch 387: 167–173, 1980.[CrossRef][Web of Science][Medline]
  16. Kim SJ, Depre C, and Vatner SF. Novel mechanisms mediating stunned myocardium. Heart Fail Rev 8: 143–153, 2003.[CrossRef][Web of Science][Medline]
  17. Kim SJ, Kudej RK, Yatani A, Kim YK, Takagi G, Honda R, Colantonio DA, Van Eyk JE, Vatner DE, Rasmusson RL, and Vatner SF. A novel mechanism for myocardial stunning involving impaired Ca2+ handling. Circ Res 89: 831–837, 2001.[Abstract/Free Full Text]
  18. Krukenkamp IB, Silverman NA, Sorlie D, Pridjian A, Feinberg H, and Levitsky S. Characterization of postischemic myocardial oxygen utilization. Circulation 74: 125–129, 1986.
  19. Leijendekker WJ, Gao WD, and ter Keurs HEDJ. Unstimulated force during hypoxia of rat cardiac muscle: stiffness and calcium dependency. Am J Physiol Heart Circ Physiol 258: H861–H869, 1990.[Abstract/Free Full Text]
  20. Marston SB and Redwood CS. Modulation of thin filament activation by breakdown or isoform switching of thin filament proteins: physiological and pathological implications. Circ Res 93: 1170–1178, 2003.[Abstract/Free Full Text]
  21. Martell AE and Smith RM. Critical Stability Constants, I-IV. New York, NY: Plenum, 1974.
  22. McDonald KS, Mammen PP, Strang KT, Moss RL, and Miller WP. Isometric and dynamic contractile properties of porcine skinned cardiac myocytes after stunning. Circ Res 77: 964–972, 1995.[Abstract/Free Full Text]
  23. McDonough JL, Arrell DK, and Van Eyk JE. Troponin I degradation and covalent complex formation accompanies myocardial ischemia/reperfusion injury. Circ Res 84: 9–20, 1999.[Abstract/Free Full Text]
  24. McKillop DF and Geeves MA. Regulation of the interaction between actin and myosin subfragment 1: evidence for three states of the thin filament. Biophys J 65: 693–701, 1993.[Web of Science][Medline]
  25. Murphy AM, Kogler H, Georgakopoulos D, McDonough JL, Kass DA, Van Eyk JE, and Marban E. Transgenic mouse model of stunned myocardium. Science 287: 488–491, 2000.[Abstract/Free Full Text]
  26. Ohgoshi Y, Goto Y, Futaki S, Taku H, Kawaguchi O, and Suga H. Increased oxygen cost of contractility in canine stunned heart. Circ Res 69: 975–988, 1991.[Abstract/Free Full Text]
  27. Palmer S and Kentish JC. Roles of Ca2+ and crossbridge kinetics in determining the maximum rates of Ca2+ activation and relaxation in rat and guinea pig skinned trabeculae. Circ Res 83: 179–186, 1998.[Abstract/Free Full Text]
  28. Papp Z, van der Velden J, and Stienen GJ. Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart. Cardiovasc Res 45: 981–993, 2000.[Abstract/Free Full Text]
  29. Pettit L and Siddiqui KF. The proton and metal complexes of adenyl-5'-yl imidodiophosphate. Biochem J 159: 169–171, 1976.[Web of Science][Medline]
  30. Prakash YS, Cody MJ, Housmans PR, Hannon JD, and Sieck GC. Comparison of cross-bridge cycling kinetics in neonatal vs. adult rat ventricular muscle. J Muscle Res Cell Motil 20: 717–723, 1999.[CrossRef][Web of Science][Medline]
  31. Silverman NA. Myocardial oxygen consumption after reversible ischemia. J Card Surg 9, Suppl 3: 465–468, 1994.[Web of Science][Medline]
  32. Snedecor GW and Cochran WG. Statistical Methods. Ames, Iowa: Iowa Univ Press, 1989.
  33. Trines SA, Slager CJ, Onderwater TA, Lamers JM, Verdouw PD, and Krams R. Oxygen wastage of stunned myocardium in vivo is due to an increased oxygen cost of contractility and a decreased myofibrillar efficiency. Cardiovasc Res 51: 122–130, 2001.[Abstract/Free Full Text]
  34. Van Eyk JE, Powers F, Law W, Larue C, Hodges RS, and Solaro RJ. Breakdown and release of myofilament proteins during ischemia and ischemia/reperfusion in rat hearts: identification of degradation products and effects on the pCa-force relation. Circ Res 82: 261–271, 1998.[Abstract/Free Full Text]
  35. Vibert P, Craig R, and Lehman W. Steric-model for activation of muscle thin filaments. J Mol Biol 266: 8–14, 1997.[CrossRef][Web of Science][Medline]
  36. Wannenburg T, Heijne GH, Geerdink JH, Van Den Dool HW, Janssen PM, and De Tombe PP. Cross-bridge kinetics in rat myocardium: effect of sarcomere length and calcium activation. Am J Physiol Heart Circ Physiol 279: H779–H790, 2000.[Abstract/Free Full Text]
  37. Wannenburg T, Janssen PM, Fan D, and de Tombe PP. The Frank-Starling mechanism is not mediated by changes in rate of cross-bridge detachment. Am J Physiol Heart Circ Physiol 273: H2428–H2435, 1997.[Abstract/Free Full Text]
  38. Winer BJ. Statistical Principles in Experimental Design (2nd ed.). New York: McGraw-Hill, 1982.
  39. Zuurbier CJ and van Beek JH. Undiminished mitochondrial function during stunning in rabbit heart at 28 degrees C. Cardiovasc Res 35: 113–119, 1997.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
290/2/H886    most recent
00493.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via ISI Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Gao, W. D.
Right arrow Articles by Nyhan, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Gao, W. D.
Right arrow Articles by Nyhan, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.