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(Circulation Research. 1995;76:1028-1035.)
© 1995 American Heart Association, Inc.

Articles

Cardiac Troponin I Phosphorylation Increases the Rate of Cardiac Muscle Relaxation

Ren Zhang, Jiaju Zhao, Alan Mandveno, James D. Potter

From the Department of Molecular and Cellular Pharmacology, University of Miami (Fla) School of Medicine.

Correspondence to Dr James D. Potter, Department of Molecular and Cellular Pharmacology, University of Miami School of Medicine, PO Box 016189, Miami, FL 33101.

	Abstract

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Abstract Cardiac troponin (Tn) I (CTnI), compared with skeletal TnI, contains extra amino acids (32 to 33) at its amino terminus, including two adjacent serine residues. These two serine residues are believed to be phosphorylated by protein kinase A (PKA) upon stimulation of the heart by ß-agonists. In this study, we found that phosphorylation of a cardiac skinned muscle preparation by PKA, mainly at CTnI, results in a decrease in the Ca²⁺ sensitivity of muscle contraction. The pCa₅₀ decreased by 0.27±0.06 pCa units upon phosphorylation. To study cardiac muscle relaxation, we used diazo-2, a photolabile Ca²⁺ chelator with a low Ca²⁺ affinity in its intact form that is converted to a high-affinity form after photolysis. We found that the rate of cardiac muscle relaxation increased from a time of half-relaxation (t_1/2)=110±10 milliseconds to t_1/2=70±8 milliseconds after CTnI phosphorylation. This result demonstrates that CTnI phosphorylation can be linked with the increased rate of muscle relaxation in a relatively intact muscle preparation. Since CTnI phosphorylation has been shown previously to affect the Ca²⁺ affinity and Ca²⁺ off-rate of CTnC in vitro, it is likely that the faster relaxation seen here reflects faster dissociation of Ca²⁺ from cardiac TnC (CTnC). Model calculations show that increased dissociation of Ca²⁺ from CTnC, coupled with the faster uptake of Ca²⁺ by the sarcoplasmic reticulum stimulated by PKA phosphorylation of phospholamban, can account for the faster relaxation seen in the inotropic response of the heart to catecholamines.

Key Words: cardiac troponin I • cardiac skinned muscle

	Introduction

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Troponin (Tn) I is a subunit of the Tn complex that regulates muscle contraction. Functionally, TnI inhibits actomyosin ATPase when muscle is in the relaxed state. When Ca²⁺ binds to the low-affinity site(s) of TnC, the inhibition by TnI of the actomyosin ATPase is removed by protein-protein interactions among the Tn subunits, tropomyosin, and actin, allowing muscle to contract.¹ TnI has three isoforms: fast and slow skeletal, and cardiac TnI (CTnI).² The major difference between cardiac and skeletal TnI is that CTnI contains 32 to 33 extra amino acids at its amino terminus, including two adjacent serine residues at positions 22 and 23 or 23 and 24, depending on the animal species.^{4 5 6} The importance of this additional amino acid stretch was realized after reports that these serine residues are able to be phosphorylated by protein kinase A (PKA).^{7 8} The extent of CTnI phosphorylation seemed to correlate well with the degree of positive inotropy of rabbit heart stimulated by catecholamines.⁹ Several lines of experimental evidence suggest that phosphorylation of CTnI may have a unique and important regulatory role in controlling cardiac function.¹⁰ Studies with beating hearts showed that the covalent phosphate content increased by 1 to 2 mol phosphate per mole protein after perfusion with maximally activating concentrations of ß-adrenergic agonists.^{8 9 11} Physiologically, the phosphorylation of TnI appears to influence the Ca²⁺ regulation of cardiac myofibrils. Measurements of the Ca²⁺-activated ATPase of isolated myofibrils and the Ca²⁺-activated force developed by hyperpermeable trabecular preparations suggest that phosphorylation of TnI results in a decrease in the pCa at which half-maximal ATPase or force is achieved.^{12 13} Reconstitution experiments in which CTnI and cardiac TnT (CTnT) were complexed with cardiac TnC (CTnC) labeled with IAANS showed that the Ca²⁺ affinity to the single regulatory Ca²⁺-specific site of CTnC was decreased when CTnI was phosphorylated by PKA.¹⁴ By use of stopped-flow techniques and computer modeling, it was also demonstrated that phosphorylation of CTnI had little effect on the time to peak or the time to half-peak Ca²⁺ saturation of CTnC. However, the time to half-relaxation (t_1/2) decreased dramatically, from 135.8 to 97.6 milliseconds.¹⁴ This suggested that phosphorylation of CTnI, which occurs in response to ß-adrenergic stimulation in beating hearts, may be partly responsible for the increased rate of relaxation seen after this inotropic intervention. The exact mechanism as to how CTnI phosphorylation affects the dissociation of Ca²⁺ is still not clear, but it may involve a change in the interaction between CTnI and CTnC.¹⁵ PKA can phosphorylate phospholamban (PL) in the sarcoplasmic reticulum (SR) and CTnI and C protein in the myofilaments in the perfused-heart experiments.^{16 17 18} Phosphorylation of C protein does not seem to have any effect on myofibrillar Ca²⁺ sensitivity.¹² However, it is very difficult to separate the effects of CTnI phosphorylation from the effects of PL phosphorylation, which greatly affects the Ca²⁺ transient. So far, there is no direct evidence showing how CTnI phosphorylation may be involved in the inotropic change brought about by ß-agonist stimulation. To sort this out, we have taken the following approach.

Diazo-2, a photolabile derivative of BAPTA, is able to rapidly convert from a chelator with a low affinity for Ca²⁺ (K_d=2.2 µmol/L) to one with high affinity (0.073 µmol/L) upon photolysis.^{19 20} We used this technique to study the change in the rate of muscle relaxation brought about by phosphorylation with PKA. We found that when a cardiac skinned muscle (CSM) preparation was exposed to PKA, only CTnI was heavily phosphorylated, whereas C protein was only lightly phosphorylated. The Ca²⁺ sensitivity of muscle contraction decreased, and more important, the relaxation time shortened. This finding is consistent with what has been proposed for the possible physiological function of CTnI phosphorylation.²¹ This is the first report in which CTnI phosphorylation has been directly linked with the rate of muscle relaxation in a relatively intact muscle preparation and may account in part for the faster relaxation seen in the inotropic response of the heart to catecholamines.

	Materials and Methods

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CSM Preparation
Freshly isolated porcine hearts were quickly immersed into an O₂-saturated solution containing (mmol/L) NaCl 140, KCl 4, CaCl₂ 1.8, MgCl₂ 1.0, NaHPO₄ 1.8, glucose 5.5, and HEPES 5, pH 7.4. Cardiac muscle bundles were removed by dissection from the left ventricle of these hearts (1 to 2 cm in length and 2 to 3 mm in diameter) and chemically skinned by incubating with 50% glycerol and 1% Triton X-100 in the relaxing solution (pCa 8.0) containing 10^-8 mol/L Ca²⁺, 5 mmol/L Mg²⁺, 7 mmol/L EGTA, 20 mmol/L imidazole, 5 mmol/L MgATP²⁺, 20 mmol/L creatine phosphate, and 15 U/mL creatine phosphokinase, pH 7.0, at an ionic strength of 150 mmol/L at 4°C for 24 hours. This functionally skinned (CSM) preparation was then stored at -20°C in the same solution without Triton X-100 and was dissected into small bundles (100 to 200 µmol/L in diameter, 2 to 4 mmol/L in length) before use. The maximal storage time before use for these preparations was 3 weeks.

Phosphorylation of the CSM
The CSM was phosphorylated for 1 hour at room temperature in a solution containing 50 mmol/L phosphate, pH 6.8, 10 mmol/L MgCl₂, 50 mmol/L NaCl, 10 mmol/L Mg-ATP, and 0.5 U/µL of the catalytic subunit of PKA (Sigma Chemical Co). After phosphorylation, the enzyme was removed by incubation of CSM in the pCa 8 relaxing solution. In the PKA inhibition experiment, the CSM was incubated in the PKA solution described above with the addition of 1 U/µL of type III protein kinase inhibitor (Sigma). The phosphorylation of CSM was monitored by adding [³²P]-ATP to the reaction. The radiolabeled CSM was solubilized in SDS sample buffer (2% SDS, 5 mmol/L Tris-glycine, pH 6.8, 2% mercaptoethanol, 20% sucrose, and 0.05% bromophenol blue) and analyzed for the level of phosphorylation by SDS-PAGE (15%) and autoradiography.

Ca²⁺ Dependence of Force Development
The CSM preparation (100 to 200 µmol/L in diameter) was mounted with stainless steel clips to a force transducer²² and immersed in the contracting solution to measure the initial force. The composition of the contraction solution (pCa 4) was the same as the relaxation buffer, except the Ca²⁺ concentration was 10^-4 mol/L. To determine the Ca²⁺ dependence of force development, the contraction of CSM was tested in solutions with intermediate concentrations of Ca²⁺. The Ca²⁺ dependence was determined before and after CSM phosphorylation. The results represent the average of 10 independent experiments. The Ca²⁺ dependence data were fit to the Hill equation with SIGMAPLOT (Jandel Scientific): relative force (%)=[Ca²⁺]ⁿ/([Ca²⁺]ⁿ+pKⁿ), where pK is the midpoint (pCa₅₀) and n is the Hill coefficient.

Measurement of Cardiac Muscle Relaxation Using Diazo-2
The CSM preparation (100 to 200 µmol/L in diameter), attached to a force transducer system previously described,²² was incubated with a solution containing (mmol/L) diazo-2 2 (Molecular Probes), CaCl₂ 0.5, TES 60, pH 7.0, MgATP 5.0, Mg²⁺ 1.0, and creatine phosphate 10, along with 15 U/mL creatine phosphokinase in the cuvette surrounding the fiber.²⁰ The ionic strength of the solution was adjusted to 200 mmol/L by adding potassium propionate. At the ratio of total added Ca²⁺ to diazo-2 given above, the resulting average initial force was 80% of the initial force seen in the pCa 4 contraction solution. This ratio provided the greatest extent of relaxation after photolysis of the diazo-2. When force reached equilibrium, the cuvette surrounding the CSM preparation was slid laterally out of the path of the flash lamp, suspending the CSM in air, and exposed to a UV flash produced by a xenon flash lamp²³ passed through a UG11 cutoff filter (Newport Corp). The cuvette was then moved back over the CSM preparation and washed with pCa 8 solution. The total time the preparation was suspended in air was 5 seconds, as indicated in Fig 4. The length of the pulse of each flash was 1 millisecond, with the total energy for the flash equal to 30 mJ. The photolysis-induced relaxation of the CSM could be measured many times without significantly altering the relaxation parameters. The force transients were recorded on an IBM-compatible computer using ASYSTANT+ (Macmillan Software). Two protocols were used to measure the rate of muscle relaxation before and after phosphorylation: For protocol 1, the rate of muscle relaxation was determined without measuring the Ca²⁺ dependence of force development before or after phosphorylation. For protocol 2, the Ca²⁺ dependence of force development and the rate of muscle relaxation were determined first, followed by muscle phosphorylation with PKA for 1 hour. Then the rate of relaxation and Ca²⁺ dependence of force development were measured again.

View larger version (17K): [in this window] [in a new window]   Figure 4. Computer modeling of the time course of Ca2+ binding to the Ca2+-specific site of cardiac troponin (CTn). The Ca2+ transient was generated by the following equation: pCa=8-a(e-FT-e-Rt), where R and F are the rising and falling constants, respectively, and a is an amplitude factor. The resting level pCa was arbitrarily set at 8. The change in Ca2+ binding to the single Ca2+-specific site was calculated from the following equation: dx/dt=koff [10(pCa50-pCa)(100-x)-x], where time (t) is in seconds, koff is the rate of Ca2+ dissociation from CTn (calculated from the following equation: koff=0.693/t1/2), x is the percentage Ca2+ saturation of the single Ca2+-specific site of CTn, and t1/2 is the actual time to half-relaxation derived from the experiment described in Fig 3. Top, The change of Ca2+ transient before and after phosphorylation of phospholamban. The values for dephosphorylation (solid line) are as follows: a=4.00, F=10, and R=100. The values for phosphorylation (dashed line) are as follows: a=4.73, F=15, and R=200. Bottom, Ca2+ saturation of troponin before and after phosphorylation of phospholamban and/or CTnI. Solid line indicates Ca2+ saturation of troponin before phosphorylation of phospholamban and CTnI, koff value of 6.3 s-1, and pCa50 of 5.56; dashed line, Ca2+ saturation of troponin after phosphorylation of phospholamban but before phosphorylation of CTnI; and dotted line, Ca2+ saturation after phosphorylation of both phospholamban and CTnI, koff value of 9.9 s-1, and pCa50 of 5.29.

	Results

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Phosphorylation of CSM by PKA
To determine the extent of CTnI phosphorylation in CSM from adult porcine heart, the CSM was phosphorylated as described in "Materials and Methods," solubilized in SDS sample buffer, and analyzed by SDS-PAGE (Fig 1, lane b). Myosin heavy chain (205 kD), C protein (150 kD), actin (45 kD), and CTnI (30 kD) are as indicated. Bovine CTnI³ was used as a standard, as shown in Fig 1, lane a. The autoradiogram shown in Fig 1, lane c, indicates that the primary site of phosphorylation of CSM, by PKA phosphorylation, is CTnI. There is no additional phosphorylation after 1 hour of incubation of the CSM in the phosphorylation solution, suggesting that CTnI is fully phosphorylated under our experimental conditions. There is also a very faint band at the top of the gel that appears to correspond to C protein. Consistent with reported findings,¹² we have shown that phosphorylation of CSM by PKA occurs primarily on CTnI and to a lesser extent on C protein. In contrast to other reported results,^{12 24} CTnI was phosphorylated to a significantly higher extent than C protein under the conditions used in our experiments and is consistent with a report by Venema and Kuo.²⁵ It is worth noting that the amount of C-protein phosphorylation varied from lightly to barely phosphorylated, whereas the CTnI phosphorylation remained high.

View larger version (93K): [in this window] [in a new window]   Figure 1. SDS-PAGE and autoradiogram of skinned cardiac muscle phosphorylated by protein kinase A (PKA) in vitro. The photographs represent a Coomassie blue–stained gel and autoradiogram of the same gel of solubilized cardiac skinned muscle (CSM) phosphorylated by PKA. Phosphorylation of CSM was performed in a solution containing 50 mmol/L NaCl, 10 mmol/L MgCl2, 50 mmol/L sodium phosphate, pH 6.8, 100 µmol/L ATP, 50 µCi/mL [32P]-ATP, and 0.5 U/mL of the catalytic subunit of PKA, at 30°C for 60 minutes. Lanes are as follows: a, purified bovine cardiac troponin I (CTnI) as standard; b, phosphorylated cardiac myofibrils; and c, autoradiograph of the CSM sample shown in lane b.

Effect of CTnI Phosphorylation on the Ca²⁺ Dependence of Muscle Contraction
In Fig 2, top and bottom left, the effect of CTnI phosphorylation on the Ca²⁺ dependence of force development in the CSM is illustrated. Phosphorylation by PKA did not change the maximal force at pCa 4 (Fig 2, top); however, the pCa₅₀ decreased (Fig 2, bottom left) by 0.27 pCa unit (Table 1), suggesting a decreased Ca²⁺ sensitivity of the CSM after phosphorylation. The effect of PKA on the Ca²⁺ dependence of muscle contraction of CSM could be inhibited by the type III protein kinase inhibitor. The Ca²⁺ sensitivity of force development was not changed when muscle fibers were simultaneously incubated with PKA and type III protein kinase inhibitor, as shown in Fig 2, bottom right. Thus, the change in Ca²⁺ sensitivity of force development brought about by phosphorylation of cardiac fibers is specific for PKA. This also excludes the possibility that the shift in Ca²⁺ sensitivity results from some nonspecific effect of components in the phosphorylation reaction solution. These results showing the decreased Ca²⁺ dependence of force development brought about by PKA phosphorylation are consistent with another study reported by Hofmann and Lange.²⁴ The absence of the catalytic subunit of PKA in the phosphorylation solution did not change the Ca²⁺ dependence of force development (data not shown), again showing the specificity of CSM phosphorylation for PKA. The latter also excludes the argument that the Ca²⁺ sensitivity of the CSM would gradually decrease with time independent of phosphorylation. Therefore, the change in Ca²⁺ sensitivity seen is clearly dependent on phosphorylation of the CSM. In another study (unpublished data), we have shown that replacing CTnI with a bacterially expressed mutant CTnI whose two serine residues (22 and 23) have been converted to alanine prevented the change in Ca²⁺ sensitivity brought about by PKA phosphorylation. Thus, CTnI appears to be the major phosphorylation site linked with the effects seen on the Ca²⁺ dependence of cardiac muscle contraction.

View larger version (19K): [in this window] [in a new window]   Figure 2. Recording (top) and graphs (bottom) showing the effect of protein kinase A (PKA) phosphorylation of cardiac troponin I (CTnI) on the Ca2+ dependence of force development in cardiac skinned muscle (CSM). Top, CSM was tested for its initial force in the pCa 4.0 solution. The CSM was then tested in solutions containing intermediate Ca2+ concentrations as indicated. After the initial Ca2+ dependence of force development was determined, the CSM was incubated in the phosphorylation solution described in "Materials and Methods." After the phosphorylation step, the Ca2+ dependence of force development was determined again on the same CSM preparation. *Baseline was changed. **The CSM was phosphorylated for 1 hour, and the phosphorylation solution was washed out with pCa 8.0 solution. A fresh set of Ca2+ solutions starting with pCa 8.0 was used to measure the Ca2+ dependence after phosphorylation. Bottom left, The results from 10 of these experiments were averaged (mean±SEM), fitted with the Hill equation (see Table 1), and summarized here. indicates measurement of Ca2+ dependence of force development before phosphorylation; , measurement of Ca2+ dependence of force development after phosphorylation. Bottom right, The CSM was tested for its Ca2+ dependence of force development before the PKA treatment, as described for the bottom left panel. The CSM was then incubated in the phosphorylation solution containing 0.5 U/µL catalytic subunit of PKA and 1 U/µL protein kinase inhibitor type III for 60 minutes. The same CSM was again tested for its Ca2+ dependence of force development. The data were fitted with the Hill equation and represent an average of four experiments (mean±SEM). indicates measurement of Ca2+ dependence of force development before the treatment; , measurement of Ca2+ dependence of force development after the treatment.

View this table: [in this window] [in a new window]   Table 1. Comparison of the Ca2+ Dependence of Force Development and the Rate of Muscle Relaxation Before and After Phosphorylation

Effect of Phosphorylation on the Rate of Muscle Relaxation
The effect of the photolysis of 2 mmol/L diazo-2 on the rate of relaxation of the CSM is illustrated in Fig 3. When exposed to UV light, diazo-2, a photolabile Ca²⁺ chelator, rapidly changes its affinity for Ca²⁺ from K_a=4.5x10⁵ (mol/L)^-1 to K_a=1.3x10⁷ (mol/L)^-1. The CSM was first incubated in a solution containing 0.5 mmol/L Ca²⁺ and 2 mmol/L diazo-2 (Fig 3, left). After tension developed to the steady state, the muscle was placed in air, followed by rapid exposure to a UV pulse produced by a xenon flash lamp (see "Materials and Methods"). The tension declined rapidly (Fig 3) and reached a new steady state that was due to the rapid Ca²⁺ chelation by the activated diazo-2. In these experiments, the extent of relaxation varied from 20% to 80% of the initial force tested in pCa 4. Even though the extent of relaxation varied in these experiments, the rate of relaxation did not (Table 2). These data are consistent with previous studies showing that the extent of relaxation has no effect on the rate of relaxation.²⁶ In addition, the initial force in the diazo-2 solution used in these experiments before photolysis varied somewhat. However, this also had no effect on the rate of relaxation in these experiments (Table 2). The same was also true when we intentionally altered the ratio of Ca²⁺ total to diazo-2 (data included in Table 2). Taking all of the above into consideration, we have shown that the average rate of relaxation (Table 1) increased from t_1/2 of 110 milliseconds to t_1/2 of 70 milliseconds upon CTnI phosphorylation. The Ca²⁺ dependence of force development before and after phosphorylation was also determined for the CSM used to measure the rate of relaxation (protocol 2, "Materials and Methods"). The change in pCa₅₀ due to the CSM phosphorylation was not significantly different from the data described in Fig 2, bottom left and Table 1. The changes in the rate of muscle relaxation were the same whether protocol 1 or 2 was applied (see "Materials and Methods").

View larger version (17K): [in this window] [in a new window]   Figure 3. Recordings showing force relaxation after photolysis of diazo-2. Left, The cardiac skinned muscle (CSM) preparation was tested for its initial contraction and relaxation by using the pCa 4.0 and pCa 8.0 solutions, respectively. The CSM was then incubated in a solution containing 0.5 mmol/L CaCl2 and 2.0 mmol/L diazo-2. (*The pen recorder was stopped when the diazo-2 solution was applied as indicated in the figure.) After the force reached a steady state, the CSM preparation was placed in air, followed by a change in the chart speed of the recorder and the initiation of the UV flash (see "Materials and Methods"). The muscle relaxed as Ca2+ was chelated by diazo-2 that was converted into its high–Ca2+ affinity form by the photolysis. The data were collected and analyzed as described in "Materials and Methods" and shown in right panel. Right, The rates of muscle relaxation before and after phosphorylation were normalized and plotted as described in "Materials and Methods." The time to half-relaxation (t1/2) values are indicated before and after phosphorylation for the same CSM preparation.

View this table: [in this window] [in a new window]   Table 2. Effect of the Extent of Relaxation and the Initial Force on the Time to Half-Relaxation

	Discussion

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The results of the present study show that when CSM is incubated with the catalytic subunit of PKA, essentially only two proteins are phosphorylated. They are CTnI and C protein, with CTnI being the most heavily phosphorylated. The amount of C-protein phosphorylation varied from lightly to barely phosphorylated in other independent experiments (Fig 1). The Ca²⁺ dependence of force development was decreased as a result of this phosphorylation. This result is consistent with previous reports showing that PKA phosphorylates predominantly CTnI and C protein in the myofilaments.¹² Previous in vitro experiments have indicated that C protein is also a substrate for Ca²⁺/calmodulin-dependent protein kinase II, phosphorylase kinase, and protein kinase C.¹⁰ Phosphorylation of C protein does not appear to have any effect on myofibrillar Ca²⁺ sensitivity in a reconstituted system,¹² although there is evidence suggesting that C protein can stimulate or inhibit myosin ATPase activity, depending on the ratio of C protein to myosin in the preparation.²⁷ In our experiments, C protein was only lightly phosphorylated compared with CTnI phosphorylation. We have also shown that substituting bacterially expressed CTnI, whose two NH₂-terminal serines (22 and 23) have been changed to alanine, into CTnI-depleted cardiac fibers abolishes changes in the Ca²⁺ dependence of muscle contraction of the substituted fibers in response to PKA phosphorylation,²⁸ even though C-protein phosphorylation by PKA occurs in these preparations. These results indicate that C-protein phosphorylation has no effect on the Ca²⁺ dependence of muscle contraction. Taken together, these findings show that phosphorylation of CTnI by PKA and not C protein is primarily responsible for affecting the Ca²⁺ sensitivity of cardiac muscle contraction.

Our studies have shown that in the CSM, CTnI phosphorylation by PKA caused a decrease in the Ca²⁺ sensitivity of force development, suggesting that the affinity of Ca²⁺ binding to the single Ca²⁺-specific site of CTnC is decreased. Previous experiments on the effect of CTnI phosphorylation on the Ca²⁺ affinity of this site were conducted in reconstituted systems, such as estimating Ca²⁺ binding to CTnC using CTnC labeled with IAANS by fluorescence¹⁴ or measuring actomyosin ATPase activity at different Ca²⁺ concentrations.^{11 29} In the present study, we measured directly the change of Ca²⁺ sensitivity of force development upon CTnI phosphorylation by PKA by using a CSM preparation. Our data are consistent with the previous hypothesis that CTnI phosphorylation by PKA is responsible for the decreased Ca²⁺ affinity of CTnC and therefore the Ca²⁺ sensitivity of force development.

Our kinetic studies on the rate of muscle relaxation provide further evidence of how CSM phosphorylation by PKA affects cardiac muscle function. There has been a lot of interest in how ß-adrenergic stimulation regulates cardiac muscle contraction by the activation of PKA. It is generally believed that phosphorylation of the following proteins are involved in this regulation¹⁸ : phosphorylation of PL on the SR, phosphorylation of CTnI on the thin filament, and phosphorylation of C protein on the thick filament.³⁰ Phosphorylation of PL has been linked to faster Ca²⁺ pumping by the SR at submaximal Ca²⁺ concentrations, leading to an increase in the amplitude and a change in the time course of the Ca²⁺ transient, which results in higher peak force and faster contraction and relaxation. Phosphorylation of C protein has been related to faster muscle relaxation in frog atrial muscle. Phosphorylation of CTnI decreased the Ca²⁺ affinity to CTnC in in vitro experiments and has been thought to contribute to the characteristic faster relaxation.¹⁴ However, there are some reports that tend to contradict the latter idea. Previous in vivo studies have shown that CTnI remained half phosphorylated even after ß-agonists were washed out and force returned to the basal level. On the other hand, dephosphorylation of serine 16 on PL correlated well with the decrease in the level of cAMP.³¹ Since CTnI dephosphorylation does not correlate well with the removal of ß-agonists, it could suggest that CTnI phosphorylation is not involved in increasing the rate of relaxation and casts doubt on the possible physiological significance of CTnI phosphorylation by PKA. Therefore, it has been suggested that cAMP-dependent phosphorylation of PL might be the dominant player responsible for the inotropic and chronotropic effects seen during ß-adrenergic stimulation of intact hearts.³¹

Since PL, C protein, and CTnI are phosphorylated in the perfused-heart experiments upon stimulation by ß-agonists, it is very difficult to determine the exact physiological role of CTnI phosphorylation by use of this technique. Although many experiments have shown that phosphorylation of CTnI decreases the Ca²⁺ affinity of CTnC in vitro,²¹ these data alone are still not enough to justify the physiological role of CTnI phosphorylation in cardiac muscle regulation. Only an intact contractile system, in which muscle contraction and relaxation can be measured and in which the effects of CTnI phosphorylation can be separated from the effects of phosphorylation of the other proteins, will be able to provide plausible evidence for the functional role of CTnI phosphorylation in cardiac muscle regulation. The advantages of our CSM system are that contraction and relaxation can be easily monitored and PL involvement in the PKA pathway is excluded, since in the CSM preparation we used, the membrane systems were destroyed by detergent treatment. Therefore, we could easily and reliably estimate the role of PKA phosphorylation of CTnI and C protein in this system.

In the present study, we have shown that phosphorylation of CTnI and C protein increased the rate of cardiac muscle relaxation, which was unrelated to the phosphorylation of PL on the SR. Ideally, if we could substitute the endogenous CTnI with phosphorylated CTnI in our preparation and then observe the change in the rate of muscle relaxation brought about by the phosphorylated CTnI, we could definitively rule out a contribution from C-protein phosphorylation. We have tried to use the vanadate extraction and reconstitution method³² to perform this experiment. However, the kinetics of muscle contraction and relaxation were not the same as those before extraction of CTnI. We have not explored the possible mechanism for this phenomenon. However, it is possible that myosin light chain 2 is also extracted along with CTnC, as observed when the EDTA extraction method is used, and affects the kinetics of force development.^{33 34} In spite of this limitation, our results, along with other reports, appear sufficient to demonstrate the functional role of CTnI phosphorylation. Under our experimental conditions, the amount of C-protein phosphorylation varied from lightly to barely phosphorylated. Since cardiac C protein can be phosphorylated with up to 5 to 6 mol phosphate per mole protein,³⁵ it appears that in our preparation there is only marginal phosphorylation of C protein. Also, as pointed out above, the level of C-protein phosphorylation that we have observed in this preparation has no effect on the Ca²⁺ dependence of contraction, whereas CTnI phosphorylation does. In addition, if the rate of cardiac muscle relaxation is proportional to the amount of C-protein phosphorylation, proposed as a major mechanism for regulation of frog atrial cardiac muscle relaxation by Hartzell,³⁰ C-protein phosphorylation still would not be a factor in our experiments, since it is essentially unphosphorylated. Another similar study has shown that besides C-protein phosphorylation, another protein with M_r of 36 000 was also phosphorylated by PKA in toad cardiac muscle and that this protein is most likely to be TnI.³⁶ This is in contrast to Hartzell's observation that only C protein is phosphorylated in amphibian atrial muscle³⁰ in response to ß-agonists. Moreover, the regulation of contraction and relaxation in mammalian cardiac muscle is quite different from amphibian cardiac muscle, where phosphorylation of PL is not involved in muscle relaxation.³⁶ Comparing the aged with the normal adult heart, it was found that the faster relaxation induced by ß-agonist stimulation was diminished along with the decreased phosphorylation of CTnI and PL, whereas the level of C-protein phosphorylation remained the same.³⁷ These data suggested that C-protein phosphorylation played a minor role in the inotropic effect brought about by ß-agonist stimulation in mammalian cardiac muscle. Previous reports have shown that newborn rabbit and dog myocardium is less sensitive to isoproterenol than is adult myocardium^{38 39} and that the relaxation time does not shorten in the newborn heart when stimulated by isoproterenol. Since PL and C protein are expressed throughout cardiac development^{40 41} and CTnI is only expressed in the late stage of cardiac development,⁴² these data suggest the importance of the phosphorylation of CTnI in the cardiac response to the stimulation by catecholamines. On the other hand, the change of muscle relaxation by PKA phosphorylation in the present study is basically the same as the change of the rate of Ca²⁺ removal from TnC due to cardiac TnI phosphorylation.¹⁴ If it is assumed that the rate of cardiac muscle relaxation is directly correlated with the dissociation of Ca²⁺ from the single Ca²⁺-specific site of CTnC (in CTn) and that the Ca²⁺ on-rate to the Ca²⁺-specific site of CTnC does not change, then the predicted change in pCa₅₀ (apparent pK_Ca) for this site would decrease by 1.6 fold or by 0.20 pCa units. Since the measured decrease in pCa₅₀, 0.27- or 1.8-fold, is close to this, it is likely that the observed change in relaxation rate is primarily due to changes in the Ca²⁺ off-rate of CTnC (Table 1). Therefore, we would suggest that the observed increase in the rate of cardiac muscle relaxation is mainly caused by CTnI phosphorylation. This is the first evidence showing that PKA phosphorylation of CTnI can be shown to cause faster relaxation of cardiac muscle in a relatively intact contractile system.

To estimate the physiological significance of these parameters, we have used the measured rate constants from this study to model^{14 43} the time course of Ca²⁺ binding to the single Ca²⁺-specific regulatory site of CTn in response to different simulated Ca²⁺ transients. The results of these calculations are shown in Fig 4. Two Ca²⁺ transients are illustrated in Fig 4, top. One represents the expected Ca²⁺ transient in the absence of PKA phosphorylation of PL. The other, with phosphorylation of PL, results in a faster release of Ca²⁺, a higher peak Ca²⁺ amplitude, and faster Ca²⁺ uptake. These two Ca²⁺ transients were used to derive the calculation of the amount of Ca²⁺ bound to the single Ca²⁺-specific regulatory site of CTnC in CTn in Fig 4, bottom, as a function of time. If there is a direct relation between the binding of Ca²⁺ to CTnC and force, then force would track Ca²⁺ binding. To estimate the contribution of CTnI phosphorylation to the rate of relaxation, we have compared the CTnC Ca²⁺ binding transients of nonphosphorylated and phosphorylated CTnI (Fig 4, bottom). This shows that the change of t_1/2 in response to just PL phosphorylation is only from 141.9 to 126.6 milliseconds, or a change of 15.3 milliseconds. However, when CTnI is, in addition, phosphorylated, t_1/2 drops to 85.6 milliseconds. It is clear from these calculations that the calculated rate of relaxation, in response to PKA phosphorylation, results in only a minor change, unless the effects of CTnI phosphorylation are included in the simulation. Thus, these calculations show that even if the SR is able to reduce the Ca²⁺ transient faster, it will not speed up relaxation very much unless Ca²⁺ also dissociates faster from CTnC, keeping pace with the faster pumping of the SR.

Combining our results with previous studies, we believe that (1) both CTnI and PL phosphorylation by PKA are involved in modulating cardiac muscle regulation in response to ß-adrenergic stimulation, (2) phosphorylation of PL in the SR is responsible for the increase of SR Ca²⁺ transport and the greater release of Ca²⁺, thus changing the time course of the Ca²⁺ transient and increasing the speed of contraction/relaxation, and (3) CTnI phosphorylation directly increases the turnover of Ca²⁺ binding to the Ca²⁺-specific regulatory site on CTnC and is in part responsible for the faster cardiac muscle relaxation, hence facilitating the increase of heart rate. Thus, phosphorylation of PL and CTnI by PKA works synergistically in producing the observed inotropic effects. Obviously, as proposed previously¹⁴ and supported by recent experiments with a PL "knockout" mouse model,⁴⁴ PL phosphorylation plays a predominant role in the inotropic response.

Since CTnI phosphorylation appears to be physiologically significant, the question arises as to why dephosphorylation has not been shown to follow the same time course as the dephosphorylation of PL. CTnI contains two adjacent serine residues next to three arginine residues, R-R-R-S-S. This sequence arrangement makes both serines meet the minimal sequence requirement (which is R-R-X-S) for PKA phosphorylation.⁴⁵ CTnI isolated from heart tissue often contains 0.4 to 1.5 mol phosphate per mole protein,^{46 47} suggesting that one of the serine residues is phosphorylated in the resting state or that both serine residues are half-phosphorylated. Peptide studies have indicated that the kinetics of phosphorylation for each serine are different. The rate constant for the first serine phosphorylation is 13-fold slower than the second serine in these peptides,⁴⁸ suggesting that the physiological properties of those two serine phosphorylations may be different. It is also possible to isolate intact CTnI in which only one of the two serines is phosphorylated.⁴⁹ If the peptide studies reflect how CTnI is phosphorylated in vivo, the first serine residue phosphorylation could be a rate-limiting step, or one of them may be constitutively phosphorylated. This also suggests that phosphorylation of both serine residues is probably required for the effects seen with CTnI phosphorylation. In addition, this may also explain why CTnI dephosphorylation does not correlate well with the removal of ß-agonists, since the dephosphorylation of the two serines may also have different time courses. Further study will be needed to determine the functional role of the phosphorylation of these two serines.

Summary
This is the first report in which CTnI phosphorylation has been directly linked with the rate of muscle relaxation independent of the effects of PL phosphorylation and, through the faster dissociation of Ca²⁺ from CTnC, may account in part for the faster relaxation seen in the inotropic response of the heart to catecholamines.

	Acknowledgments

 
This study was supported by National Institutes of Health grants AR-40727, AR-37701, and HL-42325.

Received August 24, 1994; accepted February 14, 1995.

	References

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