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Am J Physiol Heart Circ Physiol 280: H2732-H2739, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 6, H2732-H2739, June 2001

PKA accelerates rate of force development in murine skinned myocardium expressing alpha - or beta -tropomyosin

Jitandrakumar R. Patel1, Daniel P. Fitzsimons2, Scott H. Buck3, Mariappan Muthuchamy3, David F. Wieczorek1, and Richard L. Moss1

1 Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706; 2 Department of Pediatric Cardiology, Children's Memorial Hospital, Chicago, Illinios 60614; and 3 Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

In myocardium, protein kinase A (PKA) is known to phosphorylate troponin I (TnI) and myosin-binding protein-C (MyBP-C). Here, we used skinned myocardial preparations from nontransgenic (NTG) mouse hearts expressing 100% alpha -tropomyosin (alpha -Tm) to examine the effects of phosphorylated TnI and MyBP-C on Ca2+ sensitivity of force and the rate constant of force redevelopment (ktr). Experiments were also done using transgenic (TG) myocardium expressing ~60% beta -Tm to test the idea that the alpha -Tm isoform is required to observe the mechanical effects of PKA phosphorylation. Compared with NTG myocardium, TG myocardium exhibited greater Ca2+ sensitivity of force and developed submaximal forces at faster rates. Treatment with PKA reduced Ca2+ sensitivity of force in NTG and TG myocardium, had no effect on maximum ktr in either NTG or TG myocardium, and increased the rates of submaximal force development in both kinds of myocardium. These results show that PKA-mediated phosphorylation of myofibrillar proteins significantly alters the static and dynamic mechanical properties of myocardium, and these effects occur regardless of the type of Tm expressed.

protein phosphorylation; Ca2+ sensitivity of force; skinned myocardium


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

AGONIST BINDING TO beta -ADRENERGIC RECEPTORS in the mammalian heart activates cAMP-dependent protein kinase (protein kinase A; PKA), which in turn mediates the phosphorylation of several proteins, including L-type Ca2+ channels (14, 33), intracellular Ca2+ release channels (29, 32, 37), phospholamban (12, 18), and the myofibrillar proteins troponin I (TnI) and myosin-binding protein C (MyBP-C) (12, 27). Phosphorylation-induced changes in the function of L-type and ryanodine-sensitive Ca2+ channels and phospholamban appear to explain much of the increase in amplitude and the more rapid decline of Ca2+ transient in beta -agonist-treated myocardium; however, it is not clear whether phosphorylation of TnI or C protein or both contributes to altered twitch kinetics under these conditions.

Phosphorylation-induced changes in the function of TnI and MyBP-C have previously been examined using purified proteins in solution and skinned myocardial preparations. For example, Robertson et al. (26) found that phosphorylation of TnI decreases the binding affinity of troponin C (TnC) for Ca2+, whereas Weisberg and Winegrad (34) demonstrated that phosphorylation of MyBP-C induces movement of myosin heads away from the thick-filament backbone. From such effects, one would anticipate 1) a decrease in Ca2+ sensitivity of force and faster relaxation [most likely due to the faster off rate of Ca2+ from TnC (koff), because the association constant = (kon/koff), where kon is the on rate of Ca2+ to TnC] due to TnI phosphorylation; and 2) a faster rate of force development due to MyBP-C phosphorylation. With the exception of myocardium expressing beta -tropomyosin (beta -Tm), in which TnI and C protein phosphorylation was found to have no significant effect on Ca2+ sensitivity of force (24), PKA has consistently been found to reduce the Ca2+ sensitivity of force in skinned myocardium (8, 15, 30). However, studies of the possible effects on activation or relaxation kinetics in skinned myocardium due to TnI and/or MyBP-C phosphorylation have yielded variable results. For example, Zhang et al. (38) reported an increase, whereas Johns et al. (17) reported no change, in the rate of relaxation, and Araujo and Walker (1) found an increase, whereas Fitzsimons and Wolff (11) found no change, in the rate of force development.

The primary purpose of the present study was to assess the effects of PKA-mediated phosphorylations of TnI and MyBP-C on the activation dependence of cross-bridge turnover kinetics in skinned myocardium from mouse hearts. Experiments were done using tissue from nontransgenic (NTG) hearts expressing predominantly alpha -Tm and from transgenic (TG) hearts expressing both alpha - and beta -Tm to test the idea that the effects of PKA on myofibrillar function require the alpha  (cardiac)-isoform of Tm.


    METHODS AND MATERIALS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Transgenic mice expressing beta -Tm. TG mice (FVBN strain) expressing beta -Tm in the heart were generated as described previously (21). Expression of the transgene was driven by the murine alpha -myosin heavy chain promoter, which restricts expression of beta -Tm to cardiac muscle. Compared with littermate NTG controls, the TG mice used in the present study exhibited no gross phenotypic abnormalities, no change in mortality, and no visible evidence of abnormalities or hypertrophy of the heart.

Skinned myocardial preparations. NTG and TG mice (4-7 mo old) of either sex were injected intraperitoneally with 5,000 U heparin/kg body wt. After 15 min, the mice were anesthetized with inhaled isoflurane (15% isoflurane in mineral oil) in accordance with institutional animal care guidelines. Hearts were excised, and right and left ventricles were dissected free in Ringer solution [containing (in mmol/l) 118 NaCl, 4.8 KCl, 2 NaH2PO4, 1.2 MgCl2, 25 HEPES, and 11 glucose; pH 7.4 at 22°C]. Both ventricles were rapidly frozen in liquid nitrogen, a step that was essential for good quality preparations. To obtain multicellular preparations (600-900 × 100-250 µm), the frozen pieces of ventricle were thawed and homogenized for ~4 s in ice-cold relaxing solution [containing (in mmol/l) 100 KCl, 10 imidazole, 5 MgCl2, 2 EGTA, and 4 ATP; pH 7.0] using a Polytron homogenizer. The cellular homogenate was centrifuged at 120 g for 1 min, and the resulting pellet was washed with fresh relaxing solution and then resuspended in relaxing solution containing 250 µg/ml saponin and 1% Triton X-100. After 30 min, the skinned preparations were washed three times with fresh relaxing solution and dispersed in ~50 ml of relaxing solution in a glass petri dish. The dish was kept on ice at all times except during the selection of preparations for experiments.

Experimental solutions, apparatus, and protocol. All chemicals were purchased from Sigma except for CaCl2 (Orion Research), propionic acid (Fluka), creatine phosphate (ICN), and ATP (Boehringer). Solution compositions were calculated using the computer program of Fabiato (9) and the stability constants (corrected to pH 7.0 and 15°C) listed by Godt and Lindley (13). All solutions contained (in mmol/l) 100 N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid, 14.5 creatine phosphate, and 5 dithiothreitol. In addition, the following pCa solutions were used: 1) pCa 9.0 solution, containing (in mmol/l) 7 EGTA, 0.02 CaCl2, 5.42 MgCl2, and 4.74 ATP; 2) pCa 4.5 solution, containing (in mmol/l) 7 EGTA, 7.01 CaCl2, 5.26 MgCl2, and 4.81 ATP; and 3) preactivating solution, containing (in mmol/l) 0.07 EGTA, 5.42 MgCl2, and 4.74 ATP. Ionic strength of all solutions was adjusted to 180 mmol/l using potassium propionate. A range of solutions containing different free [Ca2+] (i.e., pCa 6.5-5.5) for determining Ca2+ sensitivity of force and the rate constant of force redevelopment (ktr) were prepared by mixing solutions of pCa 9.0 and 4.5.

Skinned preparations with well-defined edges and no free ends of cells evident in the middle region were transferred from the petri dish to a stainless steel experimental chamber (19) containing relaxing solution. The ends of each of the preparations were attached to the arms of a motor (model 350, Cambridge Technology; Cambridge, MA) and a force transducer (model 403, Cambridge Technology) as described earlier (19). The chamber assembly was then placed on the stage of an inverted microscope (Olympus) fitted with a ×40 objective and a CCTV camera (model WV-BL600, Panasonic). Light from a halogen lamp was passed through a cut-off filter (transmission >620 nm) and was used to illuminate the skinned preparations. Video images of the preparations were recorded and then used to assess mean sarcomere length during the course of each experiment. Changes in force were recorded on a chart recorder (Allen Datagraph) using a slow time base and on an oscilloscope (Nicolet 310) using a faster time base.

At the start of each experiment, the skinned myocardial preparations were stretched to a mean sarcomere length of ~2.35 µm. The protocol for simultaneous determination of Ca2+ activated force and ktr was a modification of the multistep protocol developed by Brenner and Eisenberg (6). After force had reached a steady level in activating solution, the preparation was rapidly slackened by ~20%, held for ~26 ms, and then restretched back to its original length. Typical changes in force recorded during this protocol are shown in Fig. 1 for pCa 4.5 and 9.0. After a rapid decrease in muscle length, steady-state force abruptly fell to zero and remained at zero until the preparation was restretched back to its original length. The drop in force recorded in the 9.0 pCa solution was taken as resting force and was therefore subtracted from the drop in total force at 4.5 pCa to yield maximum Ca2+-activated force. As a consequence of restretch, there was an initial transient increase, followed by a decrease in force (seen as a spike in the force trace) and subsequent slower recovery of force to near the initial steady-state level. The ktr reported in the present study is the rate constant of force redevelopment after the spike. After force had recovered to its steady-state level, the preparation was transferred back to relaxing solution. With the use of the same procedure, Ca2+-activated force and ktr were determined for a range of Ca2+ concentrations. At the end of each experiment, steady-state force at pCa 4.5 was remeasured to determine whether significant rundown had occurred. The preparations were then cut free at the points of attachment, placed in SDS sample buffer, and stored at -80°C until subsequent analysis of contractile protein content analysis by 12% SDS-PAGE and ultrasensitive silver staining (31). Gels were then scanned using a densitometer (Molecular Analyst, Bio-Rad) and commercially available software.


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  		Fig. 1.   Experimental protocol for simultaneous determination of Ca2+-activated force and rate constant of force redevelopment (ktr) in skinned myocardial preparations. Bottom: changes in force recorded before, during, and after a step change in length (top) of skinned myocardium. Once active force reached steady state in the 4.5 pCa solution, muscle length was rapidly decreased by 20%. The Ca2+-activated force was determined by subtracting the resting force measured at pCa 9.0 from the total force generated at pCa 4.5. After ~26 ms of unloaded shortening, the preparation was restretched to its original length. ktr is the rate constant of force redevelopment after the spike.

In experiments assessing the effects of PKA on Ca2+ sensitivity of force and ktr, the skinned preparations were first incubated for 1 h (22-25°C) in the 9.0 pCa solution with 1 unit of catalytic subunit of bovine cardiac PKA per microliter. Force and ktr were then measured as described above.

To determine which proteins in NTG and TG myocardium were phosphorylated by PKA, skinned preparations were suspended in relaxing solution containing 1 mmol/l ATP and 25 µCi [gamma -32P]ATP/µl. Phosphorylation was initiated by adding 1 unit of catalytic subunit of bovine cardiac PKA per microliter. After 30 min at 22-25°C, the reaction was first quenched by the addition of electrophoresis sample buffer (8 mol/l urea, 2 mol/l thiourea, 75 mmol/l dithiothreitol, 3% SDS, 1% bromophenol blue, and 50 mmol/l Tris; pH 6.8), and the buffer was then heated for 3 min at 100°C. Sample buffer (10 µl) was loaded onto gels for SDS-PAGE. Gels were subsequently stained with Coomassie brilliant blue, dried, and exposed to X-ray film (X-OMAT AR, Eastman Kodak) for 8 h.

Data analysis. Cross-sectional areas of skinned preparations were calculated by assuming that the preparations were cylindrical and by equating the width, measured from video images of the mounted preparations, to diameter. Each submaximal Ca2+-activated force (P) was expressed as a fraction of the maximum Ca2+-activated force (Po) generated by the preparations at pCa 4.5, i.e., P/Po. To determine the Ca2+ sensitivity of isometric force (pCa50), the force-pCa data were fitted with a Hill equation: P/Po = [Ca2+]N/(kN + [Ca2+]N), where N is slope (Hill coefficient) and k is the Ca2+ concentration required for half-maximal activation (pCa50). ktr values were determined by linear transformation of the half-time (t1/2) of force recovery [ktr = -ln 0.5 × (t1/2)-1] as previously described (7, 25).

All data are presented as means ± SE. Statistical analysis of the data was done using an unpaired t-test. P values <0.05 were taken as indicating significant differences.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

Maximum force, Ca2+ sensitivity of force, and rate of force redevelopment in untreated and PKA-treated NTG myocardium. The effects of PKA treatment on mechanical properties were initially assessed in NTG myocardium (Table 1). Maximum Ca2+-activated force (at pCa 4.5) was unaffected by PKA: untreated NTG myocardium generated a maximum force of 18.3 ± 1.5 mN/mm2 (n = 11), whereas PKA-treated NTG myocardium generated 15.4 ± 1.6 mN/mm2 (n = 10). The force-pCa relationships from both untreated and PKA-treated NTG myocardium were sigmoidal and were fit using the Hill equation (data not shown). The pCa50 and Hill coefficient were 5.86 ± 0.02 and 4.0 ± 0.2, respectively, in untreated NTG myocardium and 5.74 ± 0.02 and 3.7 ± 0.1 in PKA-treated NTG myocardium, respectively. Statistical analysis indicated that pCa50 was significantly reduced by PKA treatment (P < 0.001) but the Hill coefficient was unchanged (P = 0.193). Thus PKA reduced the Ca2+ sensitivity of force but had no affect on apparent cooperativity in the activation of force.

                              
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  		Table 1.   Summary of mechanical properties of untreated and PKA-treated NTG and TG myocardium

Measurements of the rate of force redevelopment revealed free [Ca2+] (and force)-dependent changes in ktr in both untreated and PKA-treated skinned preparations from NTG mouse hearts. To illustrate the effects of activation on ktr, records of force redevelopment at various levels of free [Ca2+] are shown in Fig. 2 for both untreated (A) and PKA-treated NTG myocardium (B), where steady forces at each pCa were normalized to 1.0 to provide better visualization of the variations in kinetics of force redevelopment. In all preparations studied, the maximum value of ktr (at pCa 4.5) was similar before and after treatment with PKA (Table 1). At submaximal activation, PKA reduced ktr at each pCa (Fig. 3A); however, when activation was expressed in terms of isometric force as a fraction of maximum (Fig. 3B), it became evident that PKA treatment accelerated ktr at submaximal levels of activation. The apparent differences in the effects of PKA in Fig. 3, A and B, are due to the inactivating effects of PKA treatment on submaximal force, which are evident by the greater pCa50 after PKA treatment (Table 1). Once this is taken into account, it is evident that PKA treatment accelerated ktr at submaximal forces.


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  		Fig. 2.   Tension records obtained during measurements of ktr in nontransgenic (NTG) myocardium before and after treatment with PKA. ktr was determined at submaximal (pCa 6.0-5.5) and maximal (pCa 4.5) free [Ca2+] in untreated (A; -) and protein kinase A (PKA; +)-treated NTG myocardium (B). The force transients are replotted here relative to the peak steady-state force attained after the step change in muscle length to facilitate direct visualization of differences in rates of force recovery. Maximum Ca2+-activated force (Po) and ktr values at each pCa are shown in parentheses.



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  		Fig. 3.   Effects of PKA on the activation dependence of ktr in NTG and transgenic (TG) myocardium before (-) and after treatment with PKA (+). ktr was measured at varying levels of activation in untreated (n = 11) and PKA-treated NTG (n = 10) myocardium and in untreated (n = 10) and PKA-treated (n = 8) TG myocardium. In each case, ktr values determined during submaximal activations were expressed relative to the maximum ktr measured in the same preparation at pCa 4.5 and then plotted against pCa (A) or against the steady isometric force (B) before the release/restretch protocol. Data are plotted as means ± SE. *Significant difference between the value of ktr measured in either NTG or TG myocardium after PKA treatment compared with control.

Protein content of skinned preparations from NTG and TG hearts. In the experiments assessing the possible roles of Tm isoforms in the regulation of static and dynamic mechanical properties, the expression of Tm was assessed in NTG and TG ventricular preparations used for mechanical measurements. SDS-PAGE of these samples (Fig. 4) showed that NTG preparations expressed virtually 100% alpha -Tm, whereas TG preparations expressed a mixture of alpha - and beta -Tm, i.e., in TG myocardium, beta -Tm accounted for 58 ± 3% of total Tm. Importantly, expression of the beta -Tm transgene did not alter the expression of other myofibrillar proteins in TG myocardium.


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  		Fig. 4.   Myofibrillar protein content of NTG and TG myocardium determined by SDS-PAGE. Myofibrillar protein content was determined in NTG and TG preparations using 12% SDS-PAGE. The following myofibrillar proteins were identified in order of increasing mobility: myosin heavy chain (MHC), tropomyosin (Tm), cardiac troponin I (cTnI), ventricular myosin light chain 1 (MLC1), and ventricular myosin light chain 2 (MLC2).

Maximum force and Ca2+ sensitivity of force in myocardium expressing beta -Tm. The maximum Ca2+-activated force generated by TG myocardium did not differ significantly from the force generated by NTG myocardium (Table 1). In each case, the force-pCa relationship was sigmoidal and was well fit using the Hill equation (data not shown). This fit yielded a pCa50 of 5.95 ± 0.02 and a Hill coefficient of 3.7 ± 0.1 in TG myocardium compared with a pCa50 of 5.86 ± 0.02 and a Hill coefficient of 4.0 ± 0.2 in NTG myocardium. Statistical analysis indicates that the expression of beta -Tm significantly increased the pCa50 (P < 0.001), i.e., increased the Ca2+ sensitivity of force, but had no effect on the Hill coefficient (P = 0.186). These results are consistent with earlier findings using a related mouse line (36), although the change in pCa50 (0.15 pCa unit) was greater in that study perhaps as a result of somewhat greater expression of beta -Tm (~65%).

ktr in myocardium expressing beta -TM. Expression of beta -Tm had no effect on maximum ktr measured at pCa 4.5 compared with TG myocardium expressing predominantly alpha -Tm (Table 1). However, the activation dependence of ktr differed in TG and NTG myocardium in that ktr was greater in TG myocardium at each submaximal [Ca2+] between pCa 6.0 and 5.8 (Fig. 3A) and at each submaximal force (Fig. 3B).

Maximum force, Ca2+ sensitivity of force, and rate of force redevelopment in PKA-treated TG myocardium. PKA treatment had no effect on the maximum Ca2+-activated force in TG myocardium in that force was 16.5 ± 1.8 mN/mm2 (n = 8) in PKA-treated myocardium versus 17.8 ± 1.9 mN/mm2 (n = 8) in untreated myocardium. Analysis of force-pCa relationships before and after PKA treatment yielded a pCa50 of 5.95 ± 0.02 and a Hill coefficient of 3.7 ± 0.1 for untreated TG myocardium and a pCa50 of 5.85 ± 0.01 and a Hill coefficient of 3.5 ± 0.1 for PKA-treated TG myocardium. Thus PKA significantly reduced the pCa50 (P < 0.001) in TG myocardium, i.e., reduced the Ca2+ sensitivity of force, but had no effect on the Hill coefficient (P = 0.33).

PKA treatment had no effect on maximum ktr in TG myocardium (Table 1). PKA treatment was found to have no effect on ktr when plotted against pCa (Fig. 3A), but when activation was expressed in terms of isometric tension as a percentage of the maximum (Fig. 3B), PKA increased ktr at submaximal levels of activation. Thus, when the inactivating effect of PKA treatment on submaximal tension (Table 1) is taken into account, it is clear that phosphorylation of myofibrillar proteins accelerates cross-bridge interaction kinetics.

Phosphorylation of myofibrillar proteins due to treatment of NTG and TG myocardium with PKA. Autoradiography was used to assess PKA-mediated incorporation of 32P into myofibrillar proteins in both TG and NTG myocardium. Figure 5 shows SDS-PAGE (lanes 1 and 2) and the corresponding autoradiograph of the same skinned preparations (lanes 3 and 4) treated with PKA in the presence of [gamma -32P]ATP. Under the conditions used here, PKA treatment resulted in the phosphorylation of predominantly MyBP-C and TnI. The ratios of band intensities on the autoradiographs relative to those on SDS gels were 5.5 ± 0.6 for MyBP-C and 5.4 ± 0.5 for TnI in NTG myocardium (n = 3 different hearts) and 5.0 ± 0.3 and 4.6 ± 0.7 for TG myocardium (n = 3 different hearts). Therefore, the degree of phosphorylation of MyBP-C and TnI was similar in TG and NTG myocardium.


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  		Fig. 5.   Phosphorylation of myofibrillar proteins in TG and NTG skinned myocardium due to treatment with catalytic subunit of PKA. A Coomassie blue-stained SDS-PAGE gel (lanes 1 and 2) and autoradiographs (lanes 3 and 4) of PKA-treated NTG myocardium (lanes 1 and 3) and TG myocardium (lanes 2 and 4) in the presence of [gamma -32P]ATP are shown. PKA induced significant increases in the radiolabeling of both cardiac troponin I (TnI) and myosin-binding protein (MyBP)-C in both NTG and TG myocardium.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

The primary results of the present study show that PKA-mediated phosphorylation of TnI and MyBP-C, as well as expression of beta -Tm, have profound effects on the contractile properties of mouse skinned myocardium. The rate of force development by myocardium during submaximal activation was increased by PKA-mediated phosphorylation of TnI and MyBP-C or by expression of beta -Tm. We also found that PKA reduced the Ca2+ sensitivity of force, confirming earlier results from both mouse (24) and rat myocardium (8, 15, 16, 30), whereas expression of beta -Tm increased the Ca2+ sensitivity of force, similar to earlier results (24, 36). The effects of PKA on mechanical properties occurred regardless of the Tm isoform present, i.e., 100% alpha -Tm or 58% beta -Tm/42% alpha -Tm. These results suggest that the activation of the thin filament in terms of numbers of cross-bridges and kinetics of cross-bridge binding varies with regulatory protein phosphorylation state and isoform expression.

Effects of PKA on mechanical properties of NTG myocardium. PKA treatment was found to increase the rate of force redevelopment at submaximal levels of Ca2+ activation but had no effect on cross-bridge turnover kinetics in maximally activated preparations. Importantly, this effect of protein phosphorylation on kinetics would not have been apparent from plots of ktr versus pCa (Fig. 3A), because such plots do not take into account the inactivation of submaximal tensions due to TnI phosphorylation. When ktr is plotted against isometric force expressed as a fraction of the maximum in the same preparations, the stimulatory effects of PKA on kinetics become apparent (Fig. 3B). From these results, it is plausible to think that PKA-induced phosphorylation of myofibrillar proteins contributes to alterations in twitch kinetics in the living myocardium due to application of beta -agonists. While the mechanism of effects of myofibrillar protein phosphorylation on cross-bridge kinetics is not known, it is likely that this involves a phosphorylation-induced alteration in regulatory protein interactions within the thin filament (28).

Effects of beta -Tm expression on myocardial contraction in absence of treatment with PKA. Our interest in the possible effects of Tm isoforms on contractile properties arose from earlier studies (24, 36) in which expression of ~65% beta -Tm caused an increase in Ca2+ sensitivity of force, a ~30% decrease in maximum force, and a ~35% decrease in the Hill coefficient. In the present study, expression of ~58% beta -Tm in mouse myocardium also increased the Ca2+ sensitivity of force (0.08 pCa units vs. ~0.15 pCa units in the earlier studies) but had no effect on maximum force or the steepness of the tension-pCa relationship (Table 1). A new finding here is that expression of beta -Tm was also associated with an increase in ktr at submaximal levels of activation but not during maximal activation (Fig. 3). The differences in static mechanical effects of beta -Tm between this and earlier studies might be due to expression of ~58% beta -Tm in our study versus ~65% beta -Tm in the earlier work. Whereas this difference in beta -Tm content seems small, other studies have observed significant differences in twitch kinetics in myocardium expressing 55% Tm (21) or 75% Tm (20). Furthermore, greater expression of beta -Tm has been associated with pathological abnormalities in the heart, which were not seen in hearts expressing lower levels of beta -Tm expression. In another study (22), expression of >40% mutated alpha -Tm (Asp175Asn) was required before functional changes in both the work-performing heart and skinned myocardial preparations became apparent. Other possibilities, such as differences in temperature (20 vs. 15°C), ionic strength (200 vs. 180 mmol/l), pH (7.1 vs. 7.0), or method of isolation of skinned preparations, seem unlikely but are not ruled out by our results.

Whatever the reason for these discrepancies, our results support earlier conclusions by Palmiter et al. (24) and Wolska et al. (36): that the Tm isoform contributes to the sensitivity of the thin filament to Ca2+ and presumably to other activators such as strong-binding cross-bridges. Furthermore, the idea that the observed increase in sensitivity is mediated by a change in cross-bridge kinetics at submaximal activations (36) is supported by our finding that the rate of force redevelopment is increased in submaximally activated preparations expressing beta -Tm. According to a two-state model of cross-bridge interaction (5), steady isometric force is proportional to [fapp/(fapp + gapp)], where fapp and gapp are the forward and reverse rate constants for the transition from force-generating to nonforce-generating states. The greater ktr values observed in beta -Tm TG myocardium at submaximal levels of activation support the idea that fapp is increased. ktr is equal to the sum of fapp and gapp, and earlier studies (36) have reported no change in gapp in beta -Tm TG myocardium; therefore, those investigators concluded that beta -Tm increased fapp.

Effects of PKA on contraction in TG myocardium expressing beta -Tm. We also investigated the possibility that the PKA effects on contractility in mouse skinned myocardium depend on the presence of alpha -Tm, which is the only Tm isoform in the adult mouse ventricle. Palmiter et al. (24) reported that TG myocardium expressing 65% beta -Tm did not exhibit the characteristic rightward shift in the tension-pCa relationship due to treatment with PKA. In contrast, we found that PKA treatment of skinned TG myocardium expressing 58% beta -Tm resulted in an ~0.10-pCa unit rightward shift in the tension-pCa relationship (Table 1), which was similar to the shift seen in NTG control myocardium. Furthermore, PKA increased ktr in TG myocardium at submaximal (but not maximal) levels of activation (Fig. 3B), and the amount of increase was similar to that seen in NTG controls. These results suggest that PKA effects occur with either alpha -Tm or beta -Tm, i.e., there does not seem to be a requirement for the cardiac-specific isoform.

At present, we do not know the reason(s) for the discrepancy between our results from PKA-treated TG beta -Tm myocardium (reduced Ca2+ sensitivity of force) and those obtained by Palmiter et al. (24) (no change in Ca2+ sensitivity of force), although there are several possible explanations. For example, because the decrease in Ca2+ sensitivity of force is likely to be due to phosphorylation of TnI, it is possible that the effect was masked in their study due to higher baseline levels of TnI phosphorylation before PKA treatment. Alternatively, the PKA-induced decrease in Ca2+ sensitivity of force may be masked by the significantly greater increase in Ca2+ sensitivity of force (0.15 vs. 0.09 pCa units in the present study) in their study as a result of the higher level of beta -Tm expressed in their preparations. While it is difficult to reach firm conclusions about such possibilities, it is plausible that the use of different phosphorylation protocols resulted in differing degrees of phosphorylation of myofibrillar proteins in the two studies, which in turn would affect the amount of right shift in the tension-pCa relationship.

Irrespective of these differences regarding Ca2+ sensitivity of force, we also found that ktr at submaximal levels of activation increased in both NTG and TG preparations after treatment with PKA. Both before and after application of PKA, we observed an activation-dependent increase in ktr that was ~10-fold over the whole range of activation in NTG preparations and 5-fold in the TG preparations. Previous studies have also reported activation-dependent variations in cross-bridge kinetics in rat and guinea pig skinned myocardium using a photolabile Ca2+ chelator (2, 10, 23) and in intact rat myocardium (3) and in rat (35), guinea pig (23), and frog (4) skinned myocardium using a rapid release and restretch maneuver. In the present study, the difference in activation-dependent potentiation of ktr between NTG and TG myocardium is a manifestation of stimulation of submaximal values of ktr by expression of beta -Tm in the TG myocardium.

Possible mechanism of PKA effects on mechanical properties. Our finding that PKA accelerated ktr at submaximal (but not at maximal) levels of activation suggests that phosphorylation of TnI and/or MyBP-C increases the cross-bridge cycling rate in partially activated preparations, an idea that is consistent with earlier conclusions (1) that PKA-mediated phosphorylation accelerates cross-bridge attachment rate. The mechanism by which phosphorylation speeds kinetics is likely to involve enhanced activation of the thin filament either by altering the activation of the thin-filament regulatory strand (in the case of TnI) or by increasing the probability of cross-bridge binding and thus the number of cross-bridges bound to actin (in the case of MyBP-C). With regard to the latter possibility, Winegrad and colleagues (34) have shown in isolated thick filaments that phosphorylation of MyBP-C causes cross-bridges to extend away from the thick-filament backbone. In an intact filament lattice, such a change might be expected to increase the likelihood of cross-bridge binding to the thin filament.

Whereas it is possible to separately explain each of the mechanical effects of PKA treatment on the basis of alterations in cross-bridge rate constants, a simple two-state model cannot simultaneously explain all the effects. For example, an increase in cross-bridge attachment rate (fapp) explains the increase in ktr at low levels of activation but would be expected to increase, not decrease, force at low [Ca2+]. However, the decrease in force due to TnI phosphorylation is likely to be due to the decrease in Ca2+-binding affinity to TnC, reported previously (26), which reduces force at each [Ca2+] regardless of effects on cross-bridge rate constants. In contrast, the previously reported effect of PKA treatment to increase unloaded shortening velocity (30) suggests that cross-bridge detachment rate (gapp) is also increased by phosphorylation of myofibrillar proteins. Increases in gapp would be expected to contribute to the PKA-dependent increase in ktr.


    ACKNOWLEDGEMENTS

We thank Dr. James Graham and Cinder Krema for SDS-PAGE analysis of the myocardial preparations. We also thank Karen Marquardt and Scott Stoker for technical assistance.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants P01 HL-47053 (to R. L. Moss), K08 HL-03134 (to S. H. Buck), and HL-54912/PO1 HL-22619 (to D. F. Wieczorek).

Address for reprint requests and other correspondence: J. R. Patel, Dept. of Physiology, University of Wisconsin Medical School, 1300 University Ave., Madison, WI 53706 (E-mail: jrpatel{at}physiology.wisc.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.

Received 6 November 2000; accepted in final form 9 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Heart Circ Physiol 280(6):H2732-H2739
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