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J Appl Physiol 101: 40-46, 2006. First published March 24, 2006; doi:10.1152/japplphysiol.00798.2005
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Porcine cardiac myocyte power output is increased after chronic exercise training

Aaron C. Hinken, F. Steven Korte, and Kerry S. McDonald

Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri

Submitted 5 July 2005 ; accepted in final form 15 March 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chronic exercise training increases the functional capacity of the heart, perhaps by increased myocyte contractile function, as has been observed in rodent exercise models. We examined whether cardiac myocyte function is enhanced after chronic exercise training in Yucatan miniature swine, whose heart characteristics are similar to humans. Animals were designated as either sedentary (Sed), i.e., cage confined, or exercise trained (Ex), i.e., underwent 16–20 wk of progressive treadmill training. Exercise training efficacy was shown with significantly increased heart weight-to-body weight ratios, skeletal muscle citrate synthase activity, and exercise tolerance. Force-velocity properties were measured by attaching skinned cardiac myocytes between a force transducer and position motor, and shortening velocities were measured over a range of loads during maximal Ca2+ activation. Myocytes (n = 9) from nine Ex pigs had comparable force production but a ~30% increase in peak power output compared with myocytes (n = 8) from eight Sed. Interestingly, Ex myofibrillar samples also had higher baseline PKA-induced phosphorylation levels of cardiac troponin I, which may contribute to the increase in power. Overall, these results suggest that enhanced power-generating capacity of porcine cardiac myofibrils contributes to improved cardiac function after chronic exercise training.

Yucatan miniature swine; progressive treadmill training; cardiac troponin I; cardiac myocytes

CHRONIC EXERCISE IS KNOWN to improve cardiac function as indexed by increases in maximal levels of cardiac output (CO) and stroke volume (SV) (28). Whereas increased CO is an ultimate goal of exercise training, enhanced SV is a primary adaptation given the fact lower heart rates (HR) occur at a given workload after chronic exercise training (7). The increase in SV is thought to be due, in part, to training-induced alterations of the intrinsic contractile function of the myocardium (9, 16, 25). Alterations of contractile function after exercise are accompanied by increases in force production due potentially to myocyte hypertrophy (27), increased Ca2+ sensitivity of force (6), and possibly increased shortening velocities (32). There also have been a few studies that focused on the effects of exercise training on the entire force-velocity relationship because it is velocity of shortening under a load that is most applicable to in vivo ventricular function during ejection. Two studies utilizing a rat model of chronic exercise training reported increased loaded shortening velocities in myocardial preparations from trained animals (3, 25). However, alterations in loaded shortening have not been investigated in other animal models, specifically larger animal models such as swine that may more closely mimic human cardiovascular physiology.

The purpose of this study was to evaluate cardiac myocytes from a pig model of exercise training for mechanical changes intrinsic to the myofilaments. The use of a porcine model of exercise training may be applicable to humans because pigs and humans have similar heart morphology and adaptive responses to chronic exercise with respect to maximal oxygen consumption, SV, HR, and skeletal muscle oxidation capacity (for review see Ref. 23). This model previously has been shown to produce increased exercise tolerance, moderate cardiac hypertrophy, increased coronary blood flow capacity, increased oxidative capacity of skeletal muscle, and lower heart rates during exercise at submaximal intensities (20). These changes occurred without any apparent alterations in myofibrillar ATPase, Ca2+ regulatory systems, or the metabolic system (20). Importantly, as CO was sustained with lower HR at submaximal workloads an increase in SV occurred. Increased SV could be the result of a number of things, including less aortic resistance, greater ventricular volume, and/or increased myocardial contractility. The main objective of this study was to determine whether cardiac myofibrillar contractility differs between sedentary (Sed) and exercise-trained (Ex) pig myocytes to account, at least in part, for the increased SV. Contractile function was assessed in permeabilized cardiac myocyte preparations by measuring isometric force, loaded shortening velocity, power output, and rate of force development during maximal Ca2+ activation. Another objective was to determine whether alterations in cardiac myofibrillar proteins may account for any observed functional differences. Specifically, we examined relative myosin heavy chain (MHC) isoform contents, relative cardiac troponin T (cTnT) isoform contents, and PKA-induced baseline phosphorylation of the myofibrillar proteins cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI).


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal model.   Adult male miniature swine weighing 25–40 kg were obtained from the breeder (Charles River) and familiarized with treadmill exercise over a 1- to 2-wk period of time. Treadmill performance tests were administered to each animal to evaluate exercise tolerance as previously described (20). Pigs were randomly assigned to Sed (n = 8) or Ex (n = 9) groups, with the Ex group completing the 16- to 20-wk endurance training program described previously (20, 21). Pigs assigned to the Sed group were restricted to their enclosures (2 x 4 m) and did not exercise. Treadmill performance tests were again completed at the end of 18–20 wk to determine the effectiveness of the training protocol on exercise duration. The overall efficacy of the training program was determined by comparing the exercise tolerance (as reflected in the treadmill performance test), heart weight (HW)-to-body weight (BW) ratios (HW:BW), and skeletal muscle oxidative capacity of trained and control groups. At the time of death, samples were taken from the middle of deltoid muscles that were stored frozen at –70°C until citrate synthase analysis. Citrate synthase activity was measured from whole muscle homogenate by the spectrophotometric method of Srere (30). Animal care and use procedures complied with the Guide for the Care and Use of Laboratory Animals issued by the National Institutes of Health and were approved by the University of Missouri Animal Care and Use Committee.

Isolation of cardiac myocytes.   The heart was excised from the experimental animal after administration of a preanesthetic mixture of ketamine (35 mg/kg) and xylazine (2.25 mg/kg) and the general anesthesia thiopental (25 mg/kg), and myocytes were isolated as previously described (19). Briefly, a section of left ventricular free wall (~10 cm3) near the left anterior descending coronary artery was removed, and half was rapidly frozen in liquid nitrogen for biochemical analyses and the other half was placed in ice-cold relaxing solution for myocyte experiments. The piece in relaxing solution was cut into smaller pieces (2–3 mm) and homogenized with a hand blender. The resultant slurry was centrifuged 75 s at 165 g, and the pellet was suspended for 3 min in 0.5% ultrapure Triton X-100 (Pierce Chemical) in relaxing solution. The permeabilized myocytes were washed and centrifuged twice with cold relaxing solution with the final suspension kept on ice during the day of the experiment. One myocyte per pig was used, and myocytes were used within 12 h of isolation.

Solutions.   Relaxing solution in which the ventricles were disrupted, skinned, and suspended contained (in mmol/l) 2 EGTA, 5 MgCl2, 4 ATP, 10 imidazole, and 100 KCl at pH 7.0. Compositions of relaxing and activating solutions used in mechanical measurements were as follows (in mmol/l): 7 EGTA, 5 MgCl2, 20 imidazole, 4 ATP, 14.5 creatine phosphate, pH 7.0, Ca2+ concentrations of 10–9 M (relaxing solution) and 10–4.5 M (maximal activating solution), and sufficient KCl to adjust ionic strength to 180 mM. The final concentrations of each metal, ligand, and metal-ligand complex at 12°C were determined with the computer program of Fabiato (8). Immediately preceding activations, muscle preparations were immersed for 60 s in a solution of reduced Ca2+-EGTA buffering capacity, identical to normal relaxing solution except that EGTA is reduced to 0.5 mM. This protocol resulted in more rapid steady-state force development and helped preserve the striation pattern during activation.

Experimental apparatus and mechanical measurements.   All mechanical measurements were made at 12–14°C with an apparatus described previously (22). In short, myocyte preparations were attached between a force transducer and torque motor by gently placing the ends of the myocyte into stainless steel troughs. The ends of the myocyte were secured by overlaying a piece of 3-0 monofilament nylon suture onto each end of the myocyte and tying the suture into the troughs with two loops of 10-0 monofilament suture. The apparatus was then mounted on the stage of an inverted microscope resting on a pneumatic vibration table. Force transducer and motor were driven by voltage commands from a personal computer with force and length output signals digitized, displayed, and stored on a personal computer using custom software based on LabView for Windows (National Instruments). Images of the myocyte preparations were recorded digitally while relaxed and during activation by use of an imaging system (IonOptix) and video snapshot software (Play); myocyte length and width measurements for cross-sectional area calculations were taken from these images. Videomicroscopy was completed by using a x40 objective (Olympus UWD 40) and x25 intermediate lenses. Sarcomere length of the myocyte was set initially to yield passive forces near zero and monitored during experiments by a videomicroscopy system utilizing fast Fourier transform (IonOptix, Milton, MA).

Power output of single skinned myocyte preparations was determined at varied loads as described earlier (22). Briefly, myocytes were placed in activating solution, and once steady-state force developed a series of force clamps (less than steady-state force) was performed to determine isotonic shortening velocities. By using a servo-system, force was maintained constant for a designated period of time (150–250 ms) while the length change was continuously monitored. After the force clamp, the myocyte preparation was slackened to reduce force to near zero to allow estimation of the relative load sustained during isotonic shortening; the myocyte was subsequently reextended to its initial length.

The kinetics of force redevelopment were obtained by using a procedure previously described for skinned cardiac myocyte preparations (14). While in Ca2+ activating solution, the myocyte preparation was rapidly shortened by ~15% of initial length (Lo), reducing force to zero. The myocyte preparation was then allowed to shorten for ~20 ms, after which the preparation was rapidly restretched to a value slightly greater than Lo for 2 ms and then returned to Lo. The slack-restretch maneuver caused dissociation of cross bridges, and subsequent force redevelopment was due to reattachment of cross bridges and transition to force-generating states (1). Force redevelopment measurements were carried out before the series of loaded contractions.

Data analysis.   Myocyte preparation length traces during loaded shortening were fit to a single decaying exponential equation:

Formula 1(1)
where L is cell length at time t, A and C are constants with dimensions of length, and k is the rate constant of shortening (kshortening). Velocity of shortening at any given time t was determined as the slope of the tangent to the fitted curve at that time point. In this study, velocities of shortening were calculated by extrapolation of the fitted curve to the onset of the force clamp (i.e., t = 0).

Hyperbolic force-velocity curves were fit to the relative force-velocity data by using the Hill equation (13):

Formula 2(2)
where P is force during shortening at velocity V; Po is the peak isometric force; and a and b are constants with dimensions of force and velocity, respectively. Power-load curves were obtained by multiplying force x velocity at each load on the force-velocity curve. The optimum force for mechanical power output (Fopt) was calculated by using the equation (33)

Formula 3(3)
Curve fitting was performed by using a customized program written in Qbasic, as well as commercial software (Sigmaplot).

Force redevelopment traces were fit by a single exponential function:

Formula 4(4)
where F is tension at time t, Fmax is maximal tension, and ktr is the rate constant of tension redevelopment. Fres represents any residual tension present immediately after the slack-restretch maneuver.

SDS-PAGE, Western blot, and autoradiography.   After mechanical measurements, MHC isoform expression was determined for each myocyte preparation. The single myocyte was removed from the experimental apparatus, suspended in 8 µl of SDS sample buffer, and stored at –80°C for subsequent SDS-PAGE analysis. The gel electrophoresis procedure was similar to one previously described (17, 24). The gels for SDS-PAGE were prepared with a 3.5% acrylamide stacking gel and a 12% acrylamide resolving gel. Samples were separated by SDS-PAGE at constant voltage (250 V) for 8 h. Gels were initially fixed in a 10% acetic acid-50% ethanol solution, followed by 2% glutaraldehyde. MHC isoforms were visualized by ultrasensitive silver staining, and gels were subsequently dried between Mylar sheets.

Western blot analysis was completed as previously described (19). Briefly, serial dilutions (2, 1, 0.5 µg) of left ventricular homogenates (n = 8 for Sed, n = 9 for Ex) were prepared in SDS sample buffer and separated by SDS-PAGE for 3 h. Protein was then transferred from gel to nitrocellulose membrane by using a semidry blot apparatus for 45–60 min at constant current (100 mA). The nitrocellulose blots were then placed in a blocking buffer [3% BSA in Tris-buffered saline plus Tween 20 (TTBS)] and rocked overnight at 4°C. The blocking buffer was removed and blots washed in TTBS. The primary cTnT antibody (Advanced Immunochemical) (cTnT 1:2,000 in 0.6% BSA in TTBS) was allowed to react with blots for 1 h followed by washing in TTBS. Secondary antibody (S-adenosyl-L-methionine-IgG 1:2,500 in 0.3% BSA in TTBS) reacted for 1 h followed by TTBS washes. On completion of the final wash, blots were coated for 1 min with enhanced chemiluminescent substrate (Amersham). Blots were then placed between two pieces of clear acetate and exposed to Kodak X-OMAT photography film, followed by film development. The relative amounts of cTnT protein isoforms were determined by scanning the film with an Epson scanner and measuring the areas under the peaks with QuantiScan (Biosoft) software.

PKA-induced phosphate incorporation into myofibrillar substrates was determined as described previously (18). Briefly, skinned cardiac myocytes (10 µg) were incubated with the catalytic subunit of PKA (3 to 5 µg/ml) and 50 µCi [{gamma}-32P]ATP at room temperature (21–23°C) for 30 min. The reaction was stopped by the addition of electrophoresis sample buffer and heating at 95°C for 3 min. The samples (n = 8 for Sed, n = 9 for Ex) were then separated by SDS-PAGE for 2.5 h at 15 mA, silver stained, dried, and subsequently exposed to X-ray film for visualization. Individual radiolabeled protein bands were then excised from the gel and quantified using a scintillation counter (Packard 1900 TR). Stoichiometric cTnI phosphate incorporation was calculated using the following equation: cpm cTnI/[cTnI]·[Pi]/cpm total = [Pi]/[cTnI], where all concentrations (denoted by brackets) are in moles and cpm is counts per minute.

Statistics.   Comparisons between sedentary and exercised samples for force, normalized and absolute power outputs, ktr, and all biochemical analysis were made by using a Student's t-test. P < 0.05 was chosen as indicating significance. All values are expressed as means ± SD unless otherwise noted.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Animal model.   Exercise-trained miniature Yucatan swine (n = 9) had lower BW than sedentary counterparts (n = 8) (~20%; P = 0.003) with no difference in HW (Table 1). However, because of the reduction in BW there was a ~25% increase in HW:BW (P = 0.005). In addition to increased HW:BW, the efficacy of the training protocol was characterized by an increase in exercise tolerance, as indexed by an increase in time to exhaustion during treadmill performance tests, and increased deltoid muscle citrate synthase activity (Table 1). Taken together these central and peripheral markers of training provide confirmation that the treadmill training protocol was effective in producing a trained state.


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  		Table 1. Animal morphologies with indexes of training and myocyte measurements

 
Myocyte studies.   Ventricular hypertrophy often occurs in animals undergoing stringent training paradigms and is commonly due to the enlargement and elongation of individual myocytes (for review see Ref. 26). The myocyte preparations in this study were, however, not different in length and width between Sed (n = 8) and Ex (n = 9) (Table 1). However, the possibility of enlargement or elongation exists as a quantitative assessment of enzymatically isolated single myocytes was not performed in our study. Nevertheless, mechanical measurements are expressed relative to myocyte size when applicable to correct for any size-induced variability. Myocyte preparation mechanical measurements are summarized in Table 2. Previous studies in cardiac myocytes have reported either no change (3) or increased maximal force (32) after exercise training. In this study, we found no difference in absolute maximal Ca2+-activated force production between myocytes from Sed (28.6 ± 10 kN·m2) and Ex (28.4 ± 13 kN·m2) animals.


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  		Table 2. Myocyte mechanical properties from sedentary and exercise-trained miniature Yucatan swine

 
The primary aim of the study was to assess differences in power output between myocytes from sedentary and exercise-trained Yucatan miniature swine. We found peak absolute power output in myocytes from exercise-trained animals to be ~57% higher than myocytes from sedentary animals (Sed = 0.42 ± 0.2 µW/mg vs. Ex = 0.67 ± 0.2 µW/mg, P = 0.02). Figure 1A shows a representative force-velocity and power-load relationship of a permeabilized cardiac myocyte preparation taken from either exercise-trained or sedentary myocardium. The force-velocity relationship (Fig. 1, top) displays similar maximal isometric force production between myocytes, but the myocyte from exercise-trained myocardium has less curvature of the force-velocity relationship resulting from faster rates of shortening at intermediate loads. Figure 1, bottom, shows the power-load relationship for these two preparations. The Ex myocyte had higher power output at most loads with the greatest difference occurring at peak power output. Figure 1, right, shows a plot of peak power output values from each Sed and Ex myocyte preparation; this illustrates that peak power output was consistently greater in Ex myocytes.


Figure 1
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  		Fig. 1. Representative absolute force-velocity and power-load relationships in sedentary (Sed) and exercise-trained (Ex) myocyte preparations. Left: absolute force-velocity and power-load relationships of myocytes from Sed ({circ}) and Ex (bullet) pigs. Isometric force was similar between preparations but loaded shortening velocity was faster in the Ex myocyte, resulting in greater peak power output. Right: plot of peak power data points for all cells. Sed myocytes displayed lower power values than Ex myocytes. Bar indicates mean ± SD for data points.

 
Another way to observe changes in loaded shortening and power output is to normalize the data to maximal isometric force of each preparation; this allows assessment of shortening velocity at a given load. Sed and Ex myocytes when normalized to isometric force show similar loaded shortening velocities at most high and low loads. However, shortening velocity between 10 and 25% relative force was significantly higher in myocytes from exercise-trained pigs (Fig. 2). These loads correspond to peak power output and are most physiologically relevant as they are thought to be the loads encountered by the heart in vivo. Normalized peak power output also was significantly higher in myocytes from Ex pigs (Sed 0.016 ± 0.004 P/Po·ML·s–1, Ex 0.022 ± 0.004 P/Po·ML·s–1, where ML is muscle length). A similar increase in normalized peak power output, as well as a similar decrease in curvature (a/Po) of the force-velocity relationship, has been reported in rat cardiac myocytes after exercise training (3).


Figure 2
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  		Fig. 2. Normalized force-velocity and power-load relationships of myocytes from Sed (open symbols) and Ex (solid symbols) pigs. Interestingly, loaded shortening and thus power output was similar between groups over most loads with the exception of loads producing maximal power output (where the ventricles are thought to work in vivo). Values are means ± SE.

 
We also addressed the effects of exercise training on the rate of myocyte force development after a mechanical perturbation. The rate constant of force redevelopment (ktr) was determined in the presence of maximal Ca2+ and is thought to be limited by the rate that cross bridges activate the thin filament (31). An increase in ktr would allow more rapid ventricular pressure development and thus more time to be spent in the ejection phase of the cardiac cycle. We found no difference in ktr between myocytes from sedentary and exercise-trained animals (Sed 1.6 ± 0.3·ML·s–1; Ex 1.3 ± 0.5·ML·s–1), suggesting that the rate of force development independent of Ca2+ handling plays a minimal role in augmenting ventricular performance after exercise training.

Biochemical analysis.   MHC expression was identical between Sed and Ex myocyte preparations used in mechanical measurements with the beta-MHC isoform accounting for all MHC content (Fig. 3A). In addition, because changes in cTnT isoform content have been implicated in functional changes that occur in a diabetic dyslipidemic exercise pig model, we examined whether relative cTnT isoform contents were different between pig populations. Three distinct isoforms of cTnT were expressed (so named cTnT1, cTnT2, and cTnT3 in accordance with each isoform's SDS-PAGE migration pattern). Relative cTnT isoform content was similar between Sed (58 ± 7% cTnT1, 29 ± 5% cTnT2, and 13 ± 4% cTnT3) and Ex myocardium (57 ± 7% cTnT1, 30 ± 5% cTnT2, and 13 ± 5% cTnT3) (Fig. 3B).


Figure 3
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  		Fig. 3. Representative silver-stained SDS-PAGE of myosin heavy chain (MHC) and Western blot of cTnT from Sed and Ex myocytes. A: MHC separated by SDS-PAGE and subsequently silver stained from atrial and ventricular pig myocytes and Sed and Ex myocyte preparations. Atrial myocytes contained small amounts of {alpha}-MHC. All Sed and Ex myocytes that were tested mechanically contained only beta-MHC. B: representative Western blot of cardiac troponin T (cTnT) from Sed and Ex myocytes showed similar cTnT isoforms. Bar plot summarizes relative isoform distribution.

 
Basal PKA-induced phosphorylation of myofibrillar proteins was assessed using a back-phosphorylation assay (18, 19). Autoradiograms after SDS-PAGE separation displayed phosphorylation of cMyBP-C and cTnI (Fig. 4). There was also phosphate incorporation into other myofibrillar proteins, which displayed a similar pattern to that previously observed in porcine myofibrils (see Fig. 3 of Ref. 19). These other proteins were not identified and routinely exhibited highly variable phosphate incorporation. Stoichiometric analysis of phosphate incorporation into cTnI yielded measurements of 1.2 ± 0.2 and 0.7 ± 0.2 µmol of Pi/µmol protein (P < 0.05) in Sed and Ex samples, respectively. Decreased Pi incorporation indicates a higher baseline phosphorylation of this protein. Pi incorporation into cMyBP-C followed the same trend as cTnI with Ex animals having less incorporation than the Sed control (Sed 2.2 ± 0.8 µmol of Pi/µmol protein, Ex 1.5 ± 0.5 µmol of Pi/µmol protein); however, this difference in phosphorylation was not statistically significant (P = 0.10). The finding of higher baseline cTnI phosphorylation may contribute to increased loaded shortening and power output in Ex myocytes as PKA phosphorylation has previously been shown to increase power output in rat myocytes (12).


Figure 4
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  		Fig. 4. Effect of PKA-induced phosphorylation on Sed and Ex myocyte preparations. A: silver-stained SDS-PAGE (left) and corresponding autoradiogram (right) of myocytes from Sed and Ex pigs showing representative 32P incorporation into myofibrillar proteins after PKA treatment. Lane 1 contains cardiac myocytes (~10 µg) obtained from an Ex pig, whereas lane 2 contains myocytes (~10 µg) from a Sed pig. B: stoichiometric analysis of cardiac myosin binding protein-C (cMyBP-C) and cardiac troponin I (cTnI) bands yielded lower PKA-induced phosphorylation of cTnI from Ex samples. These lower levels of phosphate incorporation indicate higher levels of PKA-induced baseline phosphorylation of cTnI in Ex compared with Sed preparations.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Effects of exercise on force-velocity properties and power output.   The purpose of this study was to evaluate mechanical changes of porcine cardiac myofilaments in response to exercise training. We found that permeabilized myocytes obtained from exercise-trained myocardium generated greater peak power output than sedentary counterparts despite producing similar isometric force (after normalization for differences in myocyte preparation size). This is important because it defines that the difference in power output was a consequence of altered cross-bridge interaction kinetics of the myofilaments as opposed to greater force simply due to myocyte hypertrophy.

The exact reason(s) are unknown for the faster cross-bridge interaction kinetics and subsequent increase in loaded shortening velocity after exercise training. There are several possibilities to explain these differences, including protein isoform changes and phosphorylation levels of myofibrillar proteins. Regarding protein isoforms shifts, small increases in {alpha}-MHC protein expression in rat skinned cardiac myocytes have been shown to significantly increase power output capacity (11); however, we did not detect any {alpha}-MHC in our functionally tested myocytes; this is in agreement with a recent study that found an increase in rat myocyte power output after treadmill training with no change in MHC expression (3). A second possibility is cardiac TnT isoform composition. We recently found a correlation between myocardial cTnT isoform expression and ventricular fractional shortening in a pig model of exercise in combination with diabetes dyslipidemia (19). With diabetes, cTnT content shifted toward greater expression of lower molecular weight cTnT isoforms (cTnT2 and cTnT3) compared with control pigs, and moderate endurance exercise prevented the cTnT isoform shift and improved fractional shortening of the ventricle. In the present study, however, Sed and Ex myocardium had similar cTnT isoform contents, indicating that cTnT isoform expression was not likely a contributor to the greater power output reported in Ex myocytes.

Another possibility is changes in the phosphorylation levels of myofibrillar proteins. Because multiple studies have shown increased left ventricular responsiveness to beta-adrenergic stimulation after exercise training (10, 29), we examined whether PKA-induced phosphorylation of myofibrillar proteins is altered in response to endurance exercise training. We did find greater exercise-induced basal PKA-mediated phosphorylation of the myofibrillar apparatus at cTnI and a trend for higher phosphorylation in cMyBP-C. We have previously shown that phosphorylation of the myofibrillar proteins cTnI and cMyBP-C by PKA produced an increase in absolute power in rat skinned cardiac myocytes (12), but these increases arose from both greater isometric force and faster loaded shortening, whereas there was no increase in force in this study. In addition, increases in loaded shortening velocity after PKA stimulation were seen over a greater range of loads in rat myocytes than those observed from exercised pig myocytes in this study. Although the exact reasons for these differences are not known, they may arise from differences in phosphorylation levels of myofibrillar proteins (including cTnI, MyBP-C, titin, and myosin light chain 2) induced by other kinases (e.g., PKC and myosin light chain kinase) or variations in combinatorial myofibrillar protein isoform content including MHC (e.g., {alpha}-MHC in rat vs. beta-MHC in pig), cardiac troponin T (15, 19), and titin (2).

Although our results suggest a role for PKA-induced phosphorylation, there certainly are other possibilities that may contribute to the increase in power output in Ex myocytes that were not examined in this study. Such factors include altered phosphorylation status induced by other kinases and/or altered myofibrillar isoform content of proteins such as titin and myosin light chain 1 (MLC1). In this regard, Diffee and colleagues (4, 5) showed an increased expression of atrial MLC1 in exercised rat hearts, and this increase was more pronounced in the endocardial region compared with the subepicardial region. Such regional distributions of varied myofibrillar proteins may also exist in the pig heart, and, because myocytes in this study were not screened for regional origin, this may be a source of the difference between Sed and Ex myocytes. However, this seems unlikely because the variability in function within Sed or Ex myocyte groups was low, and there is a low probability that randomly selected myocytes originated exclusively from either the endocardial or subepicardial region.

In conclusion, we found peak power-generating capacity to be greater in skinned myocytes from chronically exercise-trained pigs compared with sedentary counterparts. The mechanisms creating this difference are not known, but a potential contributor may be greater PKA-induced phosphorylation of myofilament proteins cTnI and cMyBP-C in Ex myocytes. Overall, the results suggest that changes in contractile properties intrinsic to the myofibrils may contribute, at least in part, to the enhanced ventricular function associated with exercise training.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-57852 and HL-52490 to M. H. Laughlin and R01 HL-57852 and K02 HL-71550 (to K. S. McDonald).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. S. McDonald, One Hospital Dr., MA415 MSB, Columbia, MO 65212 (e-mail: mcdonaldks{at}missouri.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
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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