Incorporation of the Troponin Regulatory Complex of Post-ischemic Stunned Porcine Myocardium Reduces Myofilament Calcium Sensitivity in Rabbit Psoas Skeletal Muscle Fibers

J Mol Cell Cardiol 30, 285–296 (1998)

Incorporation of the Troponin Regulatory Complex of Post-ischemic Stunned Porcine Myocardium Reduces Myofilament Calcium Sensitivity in Rabbit Psoas Skeletal Muscle Fibers Kerry S. McDonald, Richard L. Moss and William P. Miller1 Department of Physiology and 1Section of Cardiology, University of Wisconsin School of Medicine, Madison, WI 53706, USA (Received 28 May 1997, accepted in revised form 7 October 1997) K. S. MD, R. L. M  W. P. M. Incorporation of the Troponin Regulatory Complex of Postischemic Stunned Porcine Myocardium Reduces Myofilament Calcium Sensitivity in Rabbit Psoas Skeletal Muscle Fibers. Journal of Molecular and Cellular Cardiology (1998) 30, 285–296. Decreased calcium sensitivity of tension in post-ischemic myocardium is thought to be a mechanism of depressed function in stunning. The purpose of this study was to determine if the decrease in calcium sensitivity of tension results from ischemia and/or reperfusion-induced alterations in the thin filament regulatory troponin. The experiments utilized an open-chest porcine model of regional LAD myocardial stunning that has previously been shown to cause a decrease in calcium sensitivity of tension in permeabilized myocytes. Stunning was induced by 45 min of low-flow ischemia to the left anterior descending (LAD) coronary artery perfusion bed, which was followed by 30 min of reperfusion. Regional LAD function after reperfusion was 0.5±2.8%, as assessed by systolic wall thickening (v 23.9±4.1% thickening in control, P<0.001). Core biopsy samples from control circumflex and stunned LAD myocardium were acquired from each heart (n=9) after LAD reperfusion, and were used to obtain purified troponin complexes. Isometric tension–pCa relationships were measured in permeabilized psoas skeletal fibers before and after partial exchange of cardiac troponin from either control circumflex (n=6) or stunned LAD (n=8) myocardium for endogenous skeletal troponin. Calcium sensitivity of tension as assessed by pCa50 (i.e. pCa for half-maximal tension) was unchanged after exchange of troponin from control circumflex myocardium (pCa50=5.98±0.02 v 5.96±0.06), but there was a significant decrease in calcium sensitivity of tension after exchange of troponin from stunned LAD myocardium (pCa50=5.97±0.07 v 5.82±0.05, P<0.05). We conclude that the decrease in calcium sensitivity of tension in postischemic stunned myocardium is, in part, due to ischemia and/or reperfusioninduced alterations in the cardiac troponin regulatory complex.  1998 Academic Press Limited K W: Calcium; Contractile proteins; Myocardial contraction; Myocardial ischemia; Myocardial reperfusion injury.

Introduction Myocardial stunning is a form of reversible mechanical dysfunction that persists during reperfusion

following brief periods of ischemia. Myocardial stunning is a clinically relevant phenomenon that occurs after exercise-induced ischemia (Homans et al., 1986; Kloner et al., 1991), coronary angioplasty

Please address all correspondence to: William P. Miller, Section of Cardiology, H6/342, University of Wisconsin, School of Medicine, 600 Highland Avenue, Madison, WI 53792, USA. This work was presented in part at the 67th Scientific Sessions of the American Heart Association, November 1994, in Dallas, Texas.

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(Wijns et al., 1986), cardioplegic arrest for cardiac surgery, and in patients with unstable angina (Nixon et al., 1982; Renkin et al., 1990). Considerable evidence suggests that the etiology of stunning involves the formation of oxygen-derived free radicals and elevated cytosolic calcium levels during ischemia and early reperfusion (for reviews see Bolli, 1990, 1992; Marban, 1991; Kusuoka and Marban, 1992). One potential mechanism for the depressed function in myocardial stunning is a decrease in myofilament sensitivity to calcium. Both Kusuoka et al. (1987) and Carrozza et al. (1992) have reported that maximal calcium-activated pressure (measured during tetani after exposure to ryanodine) was reduced in isolated stunned ferret hearts. Kusuoka et al. (1987) also found that the relationship between developed pressure and perfusate calcium was shifted to higher calcium concentrations in stunned hearts. Recently, Gao et al. (1995) reported data showing a decrease in both maximal calcium-activated tension and calcium sensitivity of tension in trabeculae from stunned rat hearts. These data show that in isolated ferret and rat heart preparations there is generally a decrease in maximal force-generating capacity with stunning and a decrease in calcium-sensitivity of tension. To address the cellular and molecular mechanisms of stunning, our laboratory has employed a clinically-relevant large animal model of stunning. The approach combines an in vivo porcine model of regional myocardial stunning with in vitro measurements of mechanical function on single cellsized preparations of mechanically-disrupted myocardium obtained from small biopsies of the same hearts. Using this approach we have directly demonstrated that there is a decrease in myofilament calcium sensitivity of tension in post-ischemic stunned porcine myocardium (Hofmann et al., 1993; McDonald et al., 1995; Miller et al., 1996). The decrease in calcium sensitivity of tension results from reperfusion injury (Miller et al., 1996) and depends on the degree of ischemia (McDonald et al., 1995). A potential molecular mechanism to account for the decrease in calcium sensitivity of tension associated with stunning is an alteration in the thin filament regulatory protein, troponin. Troponin consists of three subunits: troponin T (TnT), which binds tropomyosin; troponin I (TnI), the inhibitory subunit which binds actin; and troponin C (TnC), the subunit which binds calcium and initiates the series of events leading to contraction. The purpose of this study was to test the hypothesis that the reduction in calcium sensitivity of tension associated with stunning involves al-

terations in the troponin complex. This idea was tested by assessing the calcium sensitivity of tension before and after substitution of cardiac troponin from stunned myocardium into the thin filaments of skeletal muscle fibers. There was a significant decrease in myofilament calcium sensitivity of tension attributable to the troponin regulatory complex from stunned myocardium; this suggests that a defect in troponin contributes to the mechanical dysfunction associated with stunning.

Materials and Methods Protocol for induction of myocardial stunning A previously described open-chest porcine model of myocardial stunning which consistently results in a decrease in calcium sensitivity of isometric tension was used (Hofmann et al., 1993; Miller et al., 1996). Adolescent pigs (n=9) weighing 44±0.7 kg were anesthetized with pentobarbital (35 mg/kg i.v.) and controlled positive-pressure ventilation with supplemental oxygen was established. Additional pentobarbital was given as needed to ensure adequate anesthesia throughout the experiment. Anesthesia, surgery, and general care of the animals conformed strictly to the “Guide for Care and Use of Laboratory Animals” of the National Institutes of Health, and the protocol was approved by the University of Wisconsin Animal Care Committee. The heart was exposed by bilateral thoracotomy with trans-sternotomy. For left-ventricular (LV) pressure measurements, a high-fidelity micromanometer-tipped catheter (Millar Instruments, Houston, TX, USA) was inserted retrogradely from the right internal carotid artery into the left ventricle. After treatment with heparin (20 000 units i.v.), an extracorporeal perfusion circuit was constructed to control flow to the left anterior descending (LAD) coronary artery perfusion bed. Left-ventricular transmural wall thickness was measured by ultrasonic crystals placed midway between the apex and base in the left anterior descending (LAD) coronary artery bed. The LV pressure, LV dP/dt, ECG, and LAD wall thickness were continuously recorded on an eightchannel recorder (Gould Brush 200, Gould Inc., Valley View, OH, USA), and digitized at 10-min intervals throughout the protocol using a microcomputer (Zenith, Zenith Data Systems, Hill Top, MI, USA). Regional LV function was assessed by percent systolic wall thickening, which was defined as the change in wall thickness between end-systole and end-diastole divided by the end-diastolic thickness. Negative values represent a decrease in wall

Troponin From Stunned Myocardium Decreases Calcium Sensitivity

thickness (i.e. thinning) during systole which occurs during ischemia. End-diastole was defined by the onset of positive dP/dt and end-systole was just prior to peak negative LV dP/dt. The LAD flow protocol used to induce stunning was 15 min of control aerobic flow followed by a 45-min period of low-flow ischemia, where flow was reduced to 40% of control. After ischemia, the LAD bed was reperfused for 30 min at control levels of aerobic perfusion. At the end of reperfusion, a core biopsy (2 cm3) was taken from both the stunned LAD bed and control circumflex beds. The myocardium was rapidly frozen in pre-cooled Wollenberg tongs, placed in liquid nitrogen, and stored at −70°C. These myocardial samples were taken from nine heart preparations and resulted in a total of approximately 60 g of both circumflex and stunned LAD myocardium from which whole troponin was purified.

Protein purification Cardiac whole troponin was isolated from circumflex and stunned LAD myocardium using a procedure similar to that previously described by Beier et al. (1988) with several modifications. Porcine myocardium was minced into small pieces (>2 mm) using a scalpel, and suspended in four volumes of 266 m potassium phosphate, 10 m sodium pyrophosphate, 1 m ATP, 0.2 m phenylmethylsulphonylfluoride (PMSF), 0.2 m benzamidine, 5 m EDTA, 1 m NaN3, 0.1 mg/ml Pefabloc and stirred for 20 min at 4°C. The suspension was centrifuged at 7710×g for 20 min and the pellets were resuspended in the above solution. This washing procedure was performed three times. After the third centrifugation, the pellets were suspended in 3 vol of 25 m KCl (pH 7.5), 5 m EDTA, 0.2 m benzamidine, 1 m NaN3, 0.1 mg/ml Pefabloc and stirred for 10 min and then centrifuged at 7710×g for 20 min. The pellets were resuspended in 3 vol of 100 m KCl (pH 7.5), 1 m EDTA, 0.2 m benzamidine, 1 m NaN3, 0.1 mg/ ml Pefabloc and stirred for 10 min, and then filtered through cheesecloth with the residue being saved. This was repeated three more times. The residue was suspended in 20 ml of 2  LiCl and then cold distilled water was added to yield a final LiCl concentration of 0.4 . This solution was then adjusted to contain 0.2 m benzamidine, 1 m NaN3, 10 lg/ ml pepstatin, and 0.1 mg/ml Pefabloc. The pH of this solution was adjusted to 5.1 by adding 1  acetic acid dropwise. The solution was stirred for 2.5 h at 4°C. During this time, pH was checked

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periodically and readjusted to 5.1. This suspension was centrifuged at 7710×g for 20 min and filtered through cheesecloth. The supernatant was saved and its pH was brought to 7.5 using 1  Tris. The supernatant was mixed with 19 g ammonium sulfate/100 ml solution and 84 mg sodium bicarbonate/100 ml solution and centrifuged at 7710×g for 20 min. The pellets were dissolved in 1  KCl, 10 m potassium phosphate (pH 7.0), 15 m 2-mercaptoethanol, 0.1 mg/ml pefabloc and dialysed overnight against this solution. The equilibrated solution was centrifuged at 145 510×g for 30 min and the supernatant was applied to a hydroxyapatite column (7×2.5 cm). The proteins were eluted using a 200 ml phosphate gradient from 1  KCl, 15 m 2-mercaptoethanol, 10 m potassium phosphate (pH 7.0), 1 mg/ml leupeptin to 1  KCl, 15 m 2-mercaptoethanol, 200 m potassium phosphate (pH 7.0), and 1 mg/ml leupeptin. Pooled troponin fractions were mixed with saturated ammonium sulfate to bring final solution to 45% saturation and centrifuged at 145 510×g for 30 min. The pellets were transferred with a minimum volume of troponin exchange solution containing 150 m KCl, 10 m Imidazole (pH 7.0), 3 m MgCl2, 0.5 m EGTA and dialysed against this solution overnight. The troponin-containing solution was centrifuged at 145 510×g for 30 min using an ultracentrifuge to remove any precipitates and the concentration of protein was determined by measuring absorbance at 280 nm. Troponin was adjusted to a concentration of >0.5 mg/ml, aliquoted to 2 ml samples, and stored at −80°C.

Skeletal muscle preparation Bundles of approximately 50 fibers were dissected from the psoas muscle of adult New Zealand white rabbits. Each bundle was tied with 4-0 suture to a glass capillary tube at approximately in situ length and placed in relaxing solution containing 50% glycerol (v/v) for storage at −20°C for up to 4 weeks. On the day of an experiment, a muscle bundle was placed in cold relaxing solution containing 0.5% Brij-58 (polyoxyethylene 20 cetyl ether; Sigma Chemical Co, St Louis, MO, USA). The bundle was then transferred to a dissecting chamber containing cold relaxing solution and single fibers were isolated. One fiber segment was placed in sodium dodecyl sulfate (SDS) sample buffer for later analysis by SDS-polyacrylamide gel electrophoresis. This control fiber segment was obtained from either the same fiber that was mounted to the experimental apparatus or from a near neighbor fiber.

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For mechanical experiments, the ends of a single fiber segment were mounted in an experimental apparatus similar to one previously described (Moss, 1979). Briefly, the fiber segment ends were placed in stainless steel troughs (25 gauge) that were fixed to styluses extending from the active elements of a force transducer (Model 403, Cambridge Technology, Inc., Watertown, MA, USA) and a DC torque motor (model 300, Cambridge Technology, Inc.). The fibers were secured by first overlaying the ends with a 0.5-mm length of 4-0 monofilament nylon suture, and cinching the suture into the troughs with two loops of 10-0 monofilament nylon suture. Muscle fiber force and length signals were digitized at 1000 Hz using a 12-bit A/D converter (AT-MIO16F-5, National Instruments Corp., Austin, TX, USA) and each was displayed and stored on a personal computer using custom software (LabView for Windows, National Instruments Corp.). Muscle length changes were driven by computer generated voltage commands to the torque motor via a 12 bit D/A converter (AT-MIO-16F-5, National Instruments Corp.) using the custom software. Fiber width and sarcomere length were monitored and recorded at 1000× using a video camera and recorder mounted on an inverted microscope (model IMT-2, Olympus Instrument Co., Tokyo, Japan).

Solutions Compositions of relaxing and activating solutions used in mechanical measurements were as follows: 7 m EGTA, 1 m free Mg2+, 20 m imidazole, 4 m MgATP, 14.5 m creatine phosphate, pH 7.0, free Ca2+ concentrations varying between 10−9  (relaxing solution) and 10–4.5  (activating solution), and sufficient KCl to adjust ionic strength to 180 m. The troponin exchange solution contained 150 m KCl, 10 m Imidazole (pH 7.0), 3 m MgCl2, 0.5 m EGTA, and 0.5 mg/ml troponin from either stunned LAD or control circumflex myocardium. All experiments were done at 15°C.

segment was placed in SDS sample buffer for analysis of protein composition using SDS-polyacrylamide gel electrophoresis. Prior to characterization of each tension–pCa relationship, each fiber was repetitively bathed in relaxing solution containing 0.5 mg/ml sTnC for 15 s in order to assure that the thin filaments were fully TnCreplete. Sequential soaks were repeated until no further increases in maximal activation were observed. Skeletal TnC was prepared using the method of Greaser and Gergely (1971). Tension-pCa relationships were characterized by first maximally activating the fiber and subsequently transferring the fiber into a series of submaximal pCa solutions. At each pCa, a steady tension was allowed to develop and the fiber was rapidly slackened to determine total tension. The amount of active tension generated at each pCa was calculated as the difference between total tension and relaxed tension, which was assessed by slackening the fiber while in the relaxed state. Representative force traces are shown in Figure 1 during maximal activation (pCa 4.5) and pCa 9.0 solution in a fiber before and after exchange of troponin. Tensions in submaximally activating solutions were expressed as fractions of peak tension (P0). The P0 value used to normalize submaximal tensions was obtained by linear interpolation between pCa 4.5 activations taken at the beginning and end of the experiments. For purposes of displaying the tension–pCa relationships, data were fitted by computer using least–squares regression analysis with the following equation: Pr=[Ca2+]n/(Kn+[Ca2+]n) where Pr is tension as a fraction of P0, n is the Hill coefficient, and K is calcium concentration at halfmaximal tension. The mid-point (pCa50) and form of the tension–pCa relationship were determined by Hill plot analysis of the data (Shiner and Solaro, 1984). Two separate straight lines were fitted to tension data above and below 0.5P0 by least-squares analysis using the following equation: log[Pr/(1−Pr)]=n(log[Ca2+]+pCa50)

Experimental protocol Two tension–pCa relationships were characterized from each fiber. Following the characterization of a control tension–pCa relationship, the fiber was bathed in troponin exchange solution for 45 min at room temperature. Following troponin exchange, a second tension–pCa relationship was characterized. At the end of the experiment the fiber

The slopes of the two phases of the Hill plot, above and below pCa50, were n1 and n2, respectively.

SDS-polyacrylamide gel electrophoresis (SDS–PAGE) To determine the amount of troponin exchange during these experiments psoas fibers were analysed by SDS-PAGE and subsequent silver staining of the gel (Giulian et al., 1983). Control fiber segments

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(1–2 mm) were transferred directly to SDS sample buffer (10 ll), while fiber segments that were mounted to the apparatus for experimentation were placed in sample buffer at the conclusion of each experiment. Acrylamide concentrations were 12% (200:1 acrylamide:bis ratio) in the separating gel and 3.5% (20:1 acrylamide:bis ratio) in the stacking gel. Approximately 5 nl of each fiber were loaded onto the gels. This volume resulted in silver staining intensities that were in the linear staining range for sTnI and myosin light chain 1 (MLC1), the proteins used to quantify troponin exchange (Fig. 2). Gels were scanned using Biorad Model GS-670 Imaging Densitometer using Molecular Analyst/ PC image analysis software. Relative intensities of protein bands were determined using a spot volume algorithm following subtraction of background.

Statistical analysis Paired t-tests were used to compare tension–pCa relationships before and after exchange of troponin.

All values are means±..., and P<0.05 was chosen as indicating significance.

Results The flow protocol in the LAD coronary bed is shown in Figure 3. After 15 min of control aerobic flow (101±7 ml/min/100 g), ischemia was induced by reducing LAD flow to 40% of control (41±2 ml/ min/100 g). After 45 min of low-flow ischemia, LAD flow was returned to pre-ischemic levels. LAD perfusion bed function, as assessed by systolic wall thickening, was 23.9±4.1% during control, significantly decreased during ischemia (−1.8±2.1%, P<0.001 v control), and recovered slightly, but remained severely depressed following reperfusion (0.5±2.8%, P<0.001 v control). Segments of skeletal muscle fibers were analysed by SDS-polyacrylamide gels to determine the extent of troponin exchange. Figure 4(a) shows an SDSpolyacrylamide gel which contains troponin isolated from circumflex (lane 1) and stunned LAD

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Figure 3 Schematic of LAD coronary artery flow protocol used to induce stunning. After 15 min of control aerobic flow, ischemia was induced by reducing flow to 40% of control. After 45 min of low-flow ischemia, LAD flow was returned to pre-ischemic levels. Regional LAD myocardial function was assessed by percent systolic wall thickening (%Th). During ischemia, %Th was significantly reduced (−1.8±2.1% v control of 23.9±4.1%, P<0.001). After reperfusion, %Th partially recovered (0.5±2.8%), but remained significantly depressed relative to the control value (P<0.001).

myocardium (lane 2). Both these preparations contain the three subunits of cardiac troponin (i.e. troponin T, troponin I, and troponin C). The gel also shows a control psoas fiber segment (lane 4) which does not contain any exogenous cardiac troponin. Lane 3 of the gel contains a fiber segment in which calcium sensitivity of tension was measured before and after the exchange of troponin from stunned LAD myocardium. After exchange (Figs 4(a) and (b)] the fiber contained cardiac troponin, evident by the presence of all three cardiac troponin subunits (cTnT, cTnI, cTnC). To estimate the amount of endogenous cardiac troponin exchanged for skeletal troponin, the skeletal troponin I:myosin light chain 1 (sTnI:LC1) ratio was measured in each fiber. The sTnI:LC1 ratios of all fibers following exchange with cardiac troponin were 0.69±0.01 (n=14) compared to 0.79±0.01 (n=11) in control psoas segments (P<0.001, Student’s t-test), indicating that approximately 13% of the endogenous skeletal troponin was exchanged for exogenous cardiac troponin under our conditions. To examine whether the incorporation of troponin from stunned myocardium results in a change in the calcium sensitivity of tension, tension–pCa relationships were characterized before and after the exchange of cardiac troponin from

either control circumflex myocardium or stunned LAD myocardium. Table 1 summarizes the mechanical properties of psoas fibers before and after exchange with cardiac troponin. The sarcomere length of all fibers was set at approximately 2.60 lm while the fibers were relaxed. During maximal activation in pCa 4.5 solution, sarcomere length in the middle of the fiber shortened by >5% in all preparations as a result of end compliance at the points of attachment to the apparatus. The maximal force generated by these fibers was significantly reduced following exchange of cardiac troponin from both the circumflex (before=312±30 lN; after=226±21 lN) and stunned LAD (before= 292±21 lN; after=200±56 lN) myocardium. Calcium sensitivity of tension, as assessed by pCa50, was unaffected following exchange using troponin from control myocardium. On the other hand, exchange using troponin from stunned LAD myocardium resulted in a significant decrease in calcium sensitivity of tension. Mean pCa50 decreased from 5.97±0.02 to 5.82±0.05 (P<0.001). Figure 5 shows the mean tension pCa relationships and Hill plots from psoas fibers before and after exchange with troponin from either control circumflex myocardium or stunned LAD myocardium. Figure 6 shows the shift in pCa50 following

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troponin exchange in each individual fiber. Following replacement of skeletal troponin with cardiac troponin from the circumflex bed, there was virtually no difference in pCa50 in four of the six fibers, while one fiber showed an increase and another a decrease in pCa50. On the other hand, exchange with cardiac troponin from the stunned LAD myocardium decreased pCa50 (i.e. increased calcium for half-maximal activation) in all fibers, providing evidence that alterations in troponin following ischemia/reperfusion account, at least in part, for the decrease in calcium sensitivity of tension observed in stunning.

Figure 4 (a) Silver-stained SDS-polyacrylamide gel containing: lane 1, troponin isolated from circumflex myocardium; lane 2, troponin isolated from stunned LAD myocardium; lane 3, a psoas fiber following exchange with troponin from stunned LAD myocardium; lane 4, a control psoas fiber. (b) Densitometric scan of lanes 4 (control) and 3 (LAD Tn exchange) shown in panel (a). LC1, LC2, and LC3 are the myosin light chains. cTnI and sTnI are cardiac and skeletal troponin I and cTnC and sTnC are cardiac and skeletal troponin C, respectively. Following LAD troponin exchange, the psoas fiber contained all three subunits of cardiac troponin (cTnT, cTnI, cTnC).

Discussion Myocardial stunning is characterized by reversible contractile dysfunction following brief periods of ischemia. Several lines of evidence suggests that a reduction in myofilament sensitivity to calcium is involved in the depressed myocardial function observed with stunning (Kusuoka et al., 1987; Carrozza et al., 1992; Hofmann et al., 1993; Gao et al., 1995; McDonald et al., 1995; Miller et al., 1996). A variety of mechanisms could potentially elicit a reduction in calcium sensitivity of isometric tension in stunned myocardium. These include: (1)

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Figure 5 Mean tension–pCa relationships and Hill plots for psoas fibers following exchange of either troponin from circumflex myocardium (left panel) or stunned LAD myocardium (right panel). Only exchange of troponin from stunned myocardium resulted in a significant rightward shift in tension–pCa relationship, indicating decreased myofilament calcium sensitivity. (Β) Before exchange; (Χ) after exchange.

decreased calcium binding to troponin C as a result of defects in one or more of the troponin subunits or changes in the phosphorylation states of troponin I or troponin T (Solaro, 1986); (2) decreased binding of force generating cross-bridges normally induced by strongly-bound cross-bridges themselves, i.e. a decrease in cross-bridge induced co-operative activation of the thin filaments (Bremel and Weber, 1972; Gu¨th and Potter, 1987; Swartz and Moss, 1992); and/or (3) defects or alterations in the phosphorylation states of other regulatory or contractile proteins such as C-protein or myosin light chain 2 (Solaro, 1986; Metzger et al., 1989). This study focused on potential mechanisms involving alterations in the troponin molecules per se, which

were addressed by examining whether incorporation of troponin molecules from stunned myocardium into the thin filaments of skeletal muscle fibers reduces calcium sensitivity of tension. In these experiments, we utilized the single-skinned skeletal fiber preparation for the following reasons: (1) because of the size of these fibers, variations in the troponin content of the thin filaments could be easily quantified; and (2) the fibers provided a protein background whereby the presence of exogenous cardiac troponin could be easily detected by SDS-polyacrylamide electrophoresis. We observed a reduction in the calcium sensitivity of tension following incorporation of troponin from stunned myocardium into the thin filaments of

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Troponin From Stunned Myocardium Decreases Calcium Sensitivity Table 1 Characteristics of psoas fibers before and after troponin exchange

Circx Tn exchange (n=6) Before After LAD Tn exchange (n=8) Before After

Sarcomere length (lm) pCa 4.5

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5.98±0.02 5.96±0.06

2.50±0.03 2.42±0.05

29.2±2.1 20.0±5.6∗

5.97±0.02 5.82±0.05∗

∗P>0.05 Paired t-test.

psoas (fast) skeletal fibers. Similar amounts of substitution of troponin from control circumflex myocardium into psoas fibers did not significantly alter calcium-sensitivity of tension. These results are consistent with the idea that ischemia and/or reperfusion modifies the cardiac regulatory protein troponin, which in turn contributes to the reduction in calcium sensitivity of tension associated with stunning. The observed reduction in calcium-sensitivity of tension in skeletal muscle fibers occurred following an apparently small amount of troponin exchange. SDS-gel analysis shows that an average of 13% of the endogenous skeletal troponin was replaced during the exchange protocol [control sTnI/LC1= 0.79±0.02 (n=11); sTnI/LC1 following troponin exchange=0.69±0.01 (n=14)). Of potential importance in explaining these effects, similarly small amounts of troponin extraction has previously been reported to have profound effects on the calcium regulation of contraction in single skinned fibers from rabbit psoas muscle (Moss et al., 1986a). The extraction of 10–20% of whole troponin resulted in the development of significant tension in the absence of calcium (>35% of total tension), caused a marked leftward shift in the tension-pCa relationships (i.e. to lower calcium concentrations), and reduced the slope of the tension–pCa relationship. These previous results and the present findings indicate that relatively small changes in the amount or type of regulatory protein subunits present in the thin filament can significantly alter calcium regulation of tension, at least in the highly co-operative activation system present in fast-twitch skeletal muscle fibers. An additional finding in this study is that the maximum tension generated by skeletal muscle fibers significantly decreased following incorporation of cardiac troponin from either stunned or control myocardium. The cause of this fall in tension is unknown, but is unlikely due to the loss of skeletal troponin C since this was fully

reconstituted in all fibers prior to each series of mechanical measurements. The mechanism underlying the loss of tension likely involves structural disturbances in the myofilament lattice of skinned fibers during the 45-min exchange period in which the fiber was maintained in a rigor condition, despite the fact that the fiber was slackened during the exchange period to reduce the amount of rigor tension. Other factors were discounted since there were no differences in calcium-sensitivity of tension in the control group before and after exchange of cardiac troponin. The run-down in fiber performance also prevented experiments examining whether the decrease in calcium sensitivity found following exchange with troponin from stunned LAD myocardium was reversible upon exchange with troponin from control circumflex myocardium. More compelling experiments to assess the full magnitude of cardiac myofibrillar function in response to troponin isolated from stunned myocardium would involve exchanging greater amounts of endogenous troponin with stunned troponin into cardiac myocytes instead of skeletal muscle fibers, as well as testing the reversibility of these effects upon reconstitution with control troponin. Such experiments are presently not feasible because of the need for methods to distinguish exchanged cardiac troponin, which migrate identically to native troponin on SDS-polyacrylamide gels. There is also a need for methods to alleviate the deterioration of mechanical performance observed in the present study in order to allow for reconstitution of control troponin. Another factor that could potentially influence alterations in calcium sensitivity of tension is variety in the amounts of sTnC/cTnC ratios at various stages of the experimental protocols. Previous studies have shown that partial extraction of sTnC from skeletal muscle fibers and replacment with cTnC decreased the slope of the tension–pCa relationship and shifted the pCa50 to higher calcium concentrations (Moss et al., 1986b). These changes in

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Figure 6 Shifts in pCa50 of individual fibers after partial exchange of troponin for either circumflex (control) myocardium or LAD (stunned) myocardium. (Β) Before exchange; (Χ) after exchange. The offset symbols with the error bars indicate the mean±... pCa50 values before and after troponin exchange.

tension–pCa relationships may have been in part due to deficiencies in TnC, since more recent work reported that near complete repletion (>95%) of partially sTnC extracted fibers with cTnC had no effect on tension–pCa relationships (Moss et al., 1991). In the present study, discrepancies in sTnC/ cTnC ratios are unlikely to account for differences between control and experimental groups since the sTnC/cTnC ratios were similar between the two groups (control exchange, sTnC/cTnC= 1.36±0.18; LAD troponin exchange, sTnC/cTnC= 1.11±0.15). In addition, the maximum calciumactivated tensions were similar in both groups following troponin exchange. The specific alteration in troponin molecules following ischemia and/or reperfusion that causes a reduced calcium sensitivity of tension remains to be determined. One possibility is a defect in one or more of the troponin subunits. It has been proposed that the calcium overload generated during ischemia and/or reperfusion may activate proteases, which in turn degrade regulatory or contractile proteins (Kusuoka and Marban, 1992). Along these lines, 60 min of complete global ischemia in rat hearts resulted in two degradation products that cross-reacted with antitroponin I antibodies in immunoblots (Westfall and Solaro, 1992). However, Barbato et al. (1996) recently identified these bands as two cytosolic

proteins (GAPDH and aB-crystallin) using amino acid sequence analysis. Using our low-flow ischemia porcine model of stunning, processing samples by SDS-polyacrylamide gel electrophoresis and silver staining has not revealed any discernible differences in myofibrillar composition, and no protein bands were evident to suggest degradation of troponin subunits in stunned myocardium (data not shown). However, SDSgels may not detect subtle changes in protein structure which could result in significant functional changes in the troponin regulatory complex without degradation. Another possibility for the decrease in calcium sensitivity of isometric tension is an alteration in the phosphorylation state of either troponin I or troponin T subunits. For example, alterations in the phosphorylation state of troponin I are known to affect calcium sensitivity of actin-activated myosin ATPase and tension (Solaro, 1986; Strang et al., 1994). If alterations in phosphorylation states are found to occur, such results would imply that ischemia disrupted the function of cellular kinases and/ or phosphatases. The time course for synthesis of new myofilament proteins or enzymes to replace those altered in stunning would be consistent with the period of hours to days required to reverse the dysfunction that characterizes stunning (Martin, 1981).

Troponin From Stunned Myocardium Decreases Calcium Sensitivity

Acknowledgments The authors thank Larry Whitesell, Scott Stoker, and Khristen Carlson for technical assistance in performing the in vivo studies, Nadine Lewis for technical assistance in purification of troponin, Dr James Graham for performing the SDS-PAGE on single skeletal muscle fibers, and Dr Marion L. Greaser for expert advice on cardiac muscle regulatory proteins and design of the experiments. This study was supported by NIH Grants R01 HL49375 (W.P.M.) and HL08755 (K.S.M.) and the Rennebohm Foundation of Wisconsion (W.P.M.).

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