This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 94/4/496    most recent 01.RES.0000117307.57798.F5v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Takimoto, E. Articles by Murphy, A. M. Search for Related Content PubMed PubMed Citation Articles by Takimoto, E. Articles by Murphy, A. M. Pubmed/NCBI databases Gene GEO Profiles HomoloGene Nucleotide Protein UniGene Compound via MeSH Substance via MeSH Hazardous Substances DB CALCIUM COMPOUNDS CALCIUM, ELEMENTAL Related Collections Calcium cycling/excitation-contraction coupling Genetically altered mice Heart failure - basic studies

(Circulation Research. 2004;94:496.)
© 2004 American Heart Association, Inc.

Cellular Biology

Frequency- and Afterload-Dependent Cardiac Modulation In Vivo by Troponin I With Constitutively Active Protein Kinase A Phosphorylation Sites

Eiki Takimoto^*, David G. Soergel^*, Paul M.L. Janssen, Linda B. Stull, David A. Kass, Anne M. Murphy

From the Departments of Pediatrics (D.G.S., L.B.S., A.M.M.) and Medicine (E.T., P.M.L.J., D.A.K.), Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Anne M. Murphy, Ross 1144, Johns Hopkins University School of Medicine, 720 Rutland Ave, Baltimore, MD 21205. E-mail murphy{at}jhmi.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Acute ß-adrenergic stimulation enhances cardiac contractility, accelerates muscle relaxation, and amplifies the inotropic and lusitropic response to increased stimulation frequency. These effects are modulated by phosphorylation of calcium handling and myofilament proteins such as troponin I (TnI) by protein kinase A (PKA). To more directly delineate the role of TnI PKA phosphorylation, transgenic mice were generated that overexpress cardiac TnI in which the serine residues normally targeted by PKA are mutated to aspartic acid to mimic constitutive phosphorylation (TnIDD_22,23). Native cardiac TnI was near completely replaced in one transgenic line as assessed by in vitro phosphorylation, and this led to reduced calcium sensitivity of myofibrillar MgATPase, as expected. TnIDD_22,23 mice had mildly enhanced basal systolic and diastolic function, and displayed marked augmentation of frequency-dependent inotropy and relaxation, with a peak frequency response 2-fold greater in mutants than controls (P<0.005). Increasing afterload prolonged relaxation more in nontransgenic than TnIDD_22,23 (P<0.02), whereas contractile responses to afterload were similar between these strains. Isoproterenol treatment eliminated the differential force-frequency and afterload response between TnIDD_22,23 and controls. In contrast to in vivo studies, isolated isometric trabeculae from nontransgenic and TnIDD_22,23 mice had similar basal, isoproterenol-, and frequency-stimulated function, suggesting that muscle shortening may be important to TnI PKA effects. These results support a novel role for cardiac TnI PKA phosphorylation in the rate-dependent enhancement of systolic and diastolic function in vivo and afterload sensitivity of relaxation. These results have implications for cardiac failure in which force-frequency modulation is blunted and afterload relaxation sensitivity increased in association with diminished PKA TnI phosphorylation.

Key Words: troponin I • protein kinase A • cardiac function • myofilament

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Stimulation of cardiac ß-adrenergic receptors augments myocardial contractility, accelerates the rate of force development and relaxation, and amplifies the positive inotropic and lusitropic effects of higher stimulation frequency.¹ In addition, the load dependence of cardiac relaxation is influenced by cardiac contractility and level of ß-stimulation.^2,3 These effects are thought to be mediated by the phosphorylation of target proteins by protein kinase A (PKA), including the myofilament proteins cardiac troponin I (cTnI) and myosin binding protein-C (MyBP-C), sarcoplasmic reticulum (SR) regulatory proteins phospholamban (PLB) and ryanodine receptor channel, and the sarcolemmal calcium channel. The net result is greater availability of intracellular calcium during systole, more rapid calcium removal from the cytosol into the SR, and enhanced calcium dissociation from myofilaments during diastole (reviewed by Bers⁴). Of these PKA target proteins, strong evidence exists for potent regulation by PLB and calcium channel phosphorylation; however, the role of TnI remains less clear.

TnI is the inhibitory element of the troponin complex that regulates contraction.^5–7 Unlike skeletal TnI, the cardiac isoform contains a unique 31 amino acid domain at the amino terminus with two PKA-targeted phosphorylation sites (positions 22 and 23 in mouse, excluding the initiating methionine). PKA phosphorylation of cTnI desensitizes myofilament MgATPase activity to calcium,⁸ shifting the force-pCa relationship rightward and enhancing relaxation by increasing the dissociation rate of Ca²⁺ from TnC.^9,10 The relative importance of this effect, however, remains somewhat controversial. Li et al¹¹ studied PLB-null mice and found positive lusitropic effects with isoproterenol only at maximal stimulation and higher afterload, suggesting that PLB phosphorylation played the dominant role. In contrast, Kentish et al,¹² studied transgenic mice with slow skeletal TnI replacing cTnI and used flash photolysis of diazo-2 to rapidly remove Ca²⁺ from skinned cardiac muscle fibers. Muscle expressing skeletal TnI (which lacks PKA sites) had no acceleration of relaxation in ß-adrenergic stimulation, suggesting that cTnI phosphorylation played an important role in intrinsic relaxation. Recent studies further suggest that muscle afterload may be important to adrenergic-mediated effects on relaxation.¹³ This may underlie observations by some investigators that PKA or isoproterenol stimulation has minimal to no effect on unloaded shortening velocity in cardiac muscle and myocyte preparations.^14–16

The present study was designed to better elucidate the functional role of PKA-mediated phosphorylation of cTnI both in vivo and in vitro. Based on evidence that aspartic acid (D) substitutions for the serines targeted by PKA are good mimics for changes induced by native phosphorylation¹⁷ and that cTnI specifically altered by such mutations (TnIDD_22,23) exhibits decreased force-calcium sensitivity in vitro,¹⁸ transgenic mice were generated with cardiac overexpression of TnIDD_22,23 using an -myosin heavy chain promoter. Functional analysis was performed in vitro and in vivo to determine the physiological and biochemical influence of this altered protein, permitting a detailed examination of the role of TnI phosphorylation in the intact heart.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Transgenic Model
The expression vector contained the 5.5-kb murine -myosin heavy chain promoter was provided by Dr Jeffrey Robbins (Children’s Hospital Medical Center, Cincinnati, Ohio). A PCR-amplified cDNA encoding rat cardiac TnI with Ser22 and Ser23 changed to aspartic acid residues was cloned into the SalI site (Figure 1A), confirmed by sequencing, and the NotI digested insert was injected into mouse pronuclear embryos (C57BL/6xSJL) as described.¹⁹ Two founders were bred with nontransgenic C57BL/6 mice. Offspring were genotyped as described.¹⁹ Both lines of transgenic mice appeared healthy with no symptoms of heart failure and had normal life spans and breeding potential. For experimental studies, the transgenic mice were compared with sib or age-matched C57BL/6 mice. Studies of the mice were in accordance with institutional guidelines.

View larger version (63K): [in this window] [in a new window]   Figure 1. A, Peptide sequence of the cardiac-specific region of TnI demonstrating the location of the two adjacent serine residues that have been mutated in the transgenic construct to encode aspartic acid residues (D). B, PKA-dependent phosphorylation of myofibrillar proteins. Myofibrils were isolated from hearts of nontransgenic (NTG) control littermates and high-expressing TnIDD22,23 transgenic mice in the presence of protease inhibitors and were preincubated with either alkaline phosphatase (AP) to remove residual endogenous phosphates or sodium fluoride (NaF) to inhibit dephosphorylation. Myofibrils were then washed in buffer, and 100-µg aliquots were exposed to 50 U purified protein kinase A catalytic subunit, 20 µmol/L cAMP, and 32P--ATP for 30 minutes at 30°C. Ten micrograms of protein was separated by gel electrophoresis and exposed to film. Arrows indicate location of myosin binding protein-C (MyBP-C) and TnI on the gels. Only the NTG myofibrils incorporated any significant isotope. Densitometry scans indicated the transgenic line had at least 95% replacement of native TnI with TnIDD22,23 when normalized to MyBP-C incorporation.

Myofibrillar MgATPase Activity Measurement
Myofibrils were prepared from cardiac ventricle as described²⁰ with careful use of protease inhibitors. Assays were performed as described in the expanded Materials and Methods in the online data supplement available at http://circres.ahajournals.org.

Phosphorylation Studies
Myofibrils were isolated as previously described, then resuspended in 50 mmol/L Tris-HCl (pH 7.5) buffer containing protease inhibitors, 20 µmol/L cAMP, and 50 U purified PKA catalytic subunit (Sigma). ³²P--ATP (1 µCi) was added and the samples were incubated at 30°C for 30 minutes. Parallel samples were examined that either had been pretreated with alkaline phosphatase for 20 minutes at 35°C or 10 mmol/L NaF in order to determine the maximal amount of potential phosphorylation. The reaction was stopped by adding SDS-PAGE loading buffer and heating at 65°C for 10 minutes. Proteins were separated by gel electrophoresis and the gel exposed to film. Myofibril preparations were also performed using phosphatase inhibitors 2 nmol/L calyculin A and 0.1 µmol/L orthovanadate in order to maintain endogenous phosphorylation during the myofibril isolation process.

In Vivo Ventricular Function Studies
Left ventricular pressure-volume (PV) studies were performed as previously described.^19,21–23 Assessment of force-frequency and afterload responses, treatment with isoproterenol and statistical methods are described in the expanded Materials and Methods.

Isolated Muscle Studies
Isolated muscle studies were performed as described^24–27 with details in the expanded Materials and Methods.

Phospholamban (PLB) and PLB Phosphorylation
Myocardial protein homogenates were prepared in the presence of protease inhibitors as described above as well as phosphatase inhibitors (Cell Signaling Technologies, Inc). Samples consisting of 5 to 15 µg of total protein were by gel electrophoresis, transferred to nitrocellulose membrane, and stained with mouse anti-phospholamban (Affinity Bioreagents) to determine total PLB followed by rabbit anti–phospho-Ser16-phospholamban (Upstate Cell Signaling Solutions) to determine the degree of phosphorylation at this site. After incubation with secondary antibodies, ECL was performed per manufacturer protocol (Amersham Biosciences).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In Vitro Phosphorylation
Myofilaments isolated from TnIDD_22,23 (high-expressing line) demonstrated markedly lower ³²P incorporation than in nontransgenic mice (NTG) at the molecular weight corresponding to cTnI, whereas the signal corresponding to MyBP-C was similar (Figure 1B). Quantitative analysis indicated TG mice had approximately 95% replacement of native cTnI with mutant TnIDD_22,23. The total amount of TnI protein is similar in TnIDD_22,23 and NTG as determined by Western blot (data not shown). Pretreatment of purified myofibrils with alkaline phosphatase did not significantly increase ³²P incorporation by PKA, indicating the standard myofibril preparation removed most endogenous phosphorylation. However, isolation of myofilaments in the presence of the phosphatase inhibitor NaF (10 mmol/L) did not alter ³²P incorporation in TnIDD_22,23 but reduced ³²P incorporation on average by 17% to 44% (mean=26%, n=3) in NTG hearts when compared with maximal phosphorylation obtained after alkaline phosphatase treatment. This "back phosphorylation" experiment suggested the combined TnI PKA phosphorylation sites were approximately 74% unoccupied at baseline. Similar results were obtained when myofibrils were isolated using the phosphatase inhibitors 2 nmol/L calyculin A and 0.1 µmol/L orthovanadate. Myofibrils isolated from diaphragm (containing ssTnI) and skeletal muscle (containing fsTnI) tested the specificity of the cardiac isoform reaction, and neither demonstrated ³²P incorporation in response to PKA. Taken together these results indicate that the myofilaments from the high-expressing TnIDD_22,23 line had virtually complete replacement of native TnI with the transgenic construct mimicking constitutive phosphorylation.

A second line of mice had approximately 50% replacement of TnI with TnIDD_22,23 as determined by phosphorylation experiments, and this line was utilized only for MgATPase experiments.

Myofibrillar MgATPase Activity
Maximal MgATPase activity was not significantly different in TnIDD_22,23 myofibrils from high-expressing lines (147±11 nmol inorganic phosphate/minute per milligram protein) compared with NTG (135±9 nmol inorganic phosphate/minute per milligram protein). The minimal activities also did not differ between groups (23±2 for the TnIDD_22,23 myofibrils versus 23±3 for the NTG). For the TnIDD_22,23 myofibrils, the mean MgATPase activity had a EC₅₀ for calcium of 5.75±0.32 µmol/L for TG, whereas NTG had an EC₅₀ of 4.75±0.31 µmol/L (P<0.05), demonstrating desensitization of the TG myofilaments to calcium (Figure 2). This EC₅₀ shift is similar to that produced by native PKA phosphorylation in murine skinned fibers^28,29 and by introduction of recombinant TnI PKA site DD mutant in skinned fibers.¹⁸ The Hill coefficient was not significantly different between TnIDD_22,23 lines and NTG. Myofibrils from the lower expressing TnIDD_22,23 line had no significant shift in EC₅₀ compared with NTG myofibrils (data not shown), and physiological experiments were performed on the high-expressing TnIDD_22,23 line.

View larger version (16K): [in this window] [in a new window]   Figure 2. Relationship of MgATPase activity to Ca2+ in myofibrils from NTG and TnIDD22,23 mice. Experiments were performed as described in text. A, Composite curves derived using mean data for EC50 and Hill coefficients from individual experiments. Data are from 4 TG (solid line) and 4 NTG (dashed line) mice after fit of individual experiment to the Hill equation. B, Mean EC50 was 4.64±0.31 µmol/L for NTG and 5.76±0.32 µmol/L for TnIDD22,23 (P<0.05); Hill coefficient NTG 1.52±0.11 and TnIDD22,23 1.62±0.11 (P=NS). Maximal and minimal calcium-activated activity is reported in the text and was not different between groups.

In Vivo Physiology
At baseline, TnIDD_22,23 hearts generated mildly, but significant, enhancement in systolic function (dP/dt_max and dP/dt_max/IP), higher LV end-systolic pressures, and more negative dP/dt_min (Table). The latter suggested enhanced relaxation, which was supported by a significantly reduced relaxation time constant () based on a logistic model. The LV end-systolic pressure was mildly elevated in the TnIDD_22,23.

View this table: [in this window] [in a new window]   Table 1. Hemodynamic Measures on TnIDD22,23 and Nontransgenic (NTG) Mice

In comparison to subtle changes in basal function, marked differences of both systolic and diastolic function were observed at faster pacing rates (Figure 3). Representative steady-state PV loops revealed little difference between TnIDD_22,23 and nontransgenic at lower heart rates, but more enhanced function (leftward shift of the PV loop and end-systolic pressure-volume point) at faster rates in TnIDD_22,23 versus nontransgenic hearts (Figure 3A). Summary data for systolic and diastolic function-heart rate responses are shown in Figure 3B. Both rate-dependent enhancement of systolic load-independent parameters of maximal dP/dt normalized for IP, and maximal power normalized for end diastolic volume, as well as the acceleration of relaxation were enhanced at faster rates in the TnIDD_22,23 animals.

View larger version (20K): [in this window] [in a new window]   Figure 3. In vivo force-frequency response is enhanced in TnIDD22,23 (TG) mice. Hearts were studied using a miniaturized pressure-volume catheter. A, Representative pressure-volume loops in a TnIDD22,23 transgenic (TG) and nontransgenic (NTG) control at atrial pacing rate of 400 and 800 bpm. There is a greater leftward shift (inotropy) with faster rate in the TG animal. B, Mean results for rate response of systolic and diastolic function. dP/dtmax/IP indicates maximal rate of pressure rise normalized to instantaneous developed pressure; PWRmx/EDV, maximal power normalized to end-diastolic volume; dP/dtmin (peak negative dP/dt), and Tau (), relaxation time constant (logistic model). P values are for group interaction term (analysis of covariance). TnIDD22,23 mice showed greater rate-dependent augmentation of systolic and diastolic function versus controls.

We next tested whether nontransgenic and TnIDD_22,23 hearts displayed different systolic and diastolic responses to ß-adrenergic stimulation with isoproterenol. As shown in Figure 4, both types of heart displayed similar systolic augmentation with increasing isoproterenol dose (50% over baseline at 80 ng/kg per minute). Relaxation rates were less affected in both groups, perhaps in part as basal rates were already quite rapid. At maximal isoproterenol stimulation, we varied heart rate over a broad range (using an I_f blocker to inhibit spontaneous sinus rate). Isoproterenol stimulation resulted in a similar flattened force-frequency relationship (FFR) for TnIDD_22,23 and nontransgenic mice, supporting a role for TnI phosphorylation to the normal rate response.

View larger version (26K): [in this window] [in a new window]   Figure 4. Response to isoproterenol in vivo. A, Hearts were studied at a fixed heart rate of 550 bpm and exposed to increasing infusion rates of isoproterenol. Infusion of isoproterenol results in augmentation of dP/dtmax/IP at this fixed heart rate, without significant difference between TG and NTG. Tau response is relatively flat and not different between TG and NTG. B, Rate response in the presence of isoproterenol (at infusion rate of 80 ng/kg per minute). In the presence of isoproterenol, the response to increasing heart rate is relatively flat and there is no significant difference in dP/dtmax/IP nor Tau response to heart rate between TG and NTG.

Afterload Relaxation Dependence
Results of altering ventricular afterload induced by graded aortic constriction on contraction and relaxation are displayed in Figure 5. Raising afterload reduced stroke volume at higher pressures (Figure 5A) as expected and was quantified from the loop data using effective arterial elastance (Ea). At constant heart (used in this protocol), Ea incorporates mean resistive as well as pulsatile loading properties of the vasculature.³⁰ In nontransgenic mice, raising afterload delayed LV relaxation demonstrated by the slower decline in pressure (Figure 5B, data are displayed normalized to peak pressure and cycle length); however, this was far less so in TnIDD_22,23 hearts. Figure 5C shows summary data relating the percent change in afterload to relaxation time constant-normalized to baseline. TnIDD_22,23 mice had significantly shallower relations (covariance analysis) using both the monoexponential (₁) and logistic (₂) relaxation models. In contrast, systolic function responded minimally and similarly to afterload in both mouse strains (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 5. Relaxation dependence on afterload is reduced in TnIDD22,23 (TG) mice in vivo. A, Representative pressure-volume loops demonstrating afterload increase in the murine heart and quantitation of the loading change by effective arterial elastance (Ea). B, Example LV pressure decay time plots for TG versus NTG hearts, with each curve normalized so that peak pressure and cycle length are 1.0. Relaxation became significantly more delayed with increased afterload in NTG compared with TG. C, Regression analyses of relaxation-afterload dependence. Data are percent change in relaxation time constant versus afterload (Ea); Tau(1) and Tau(2) are relaxation time constants based on a monoexponential or logistic decay model, respectively. Relaxation prolonged with higher afterload significantly more in NTG than TG hearts. P values: Load reflects overall relation between Tau and afterload, and Genotype the interaction term reflecting significant differences between NTG and TG. D, Example LV pressure decay time plots for TnIDD22,23 transgenic mice (TG) versus controls (NTG) in the presence of isoproterenol. Isoproterenol abolishes the delay in relaxation with increase afterload in NTG, thus eliminating the differential response between TG and NTG. E, Regression analyses in the presence of isoproterenol. Isoproterenol abolishes the prolonged relaxation in response to afterload (P>0.7 to 0.8), and eliminates the differential response between the TG and NTG (P>0.7 to 0.8).

The lusitropic response to afterload during isoproterenol stimulation is displayed in Figures 5D and 5E. Isoproterenol essentially eliminated relaxation delay induced by afterload in both nontransgenic and TnIDD_22,23 (Figure 5D). This is confirmed by the group covariance analysis (Figure 5E) showing that both TnIDD_22,23 and nontransgenic mice had no significant change in ₁ or ₂ despite increasing afterload. These data support an important role for TnI phosphorylation in the interaction between afterload and chamber relaxation.

Isolated Muscle Studies
In contrast to in vivo studies in which the muscle shortens during systole, studies performed in isometric (high afterload) isolated muscle did not reveal any differences between TnIDD_22,23 and nontransgenic trabeculae. The FFR was positive in both TnIDD_22,23 and nontransgenic trabeculae. Maximum force was 32.5±4.7 mN/mm² at 8 Hz in TnIDD_22,23 versus 34.9±6.7 mN/mm² in nontransgenic. The time to 50% relaxation (RT₅₀) declined similarly in both strains at each of the varied stimulation frequencies (Figures 6A and 6B). Stimulation with isoproterenol (from 1 nmol/L to 1 µmol/L) produced a dose-dependent increase in tension from 20 to 50 mN/mm² that was also similar in TnIDD_22,23 and nontransgenic (Figures 6C and 6D), with an EC₅₀ near 10 nmol/L in both.

View larger version (12K): [in this window] [in a new window]   Figure 6. Effect of increased stimulation frequency and isoproterenol on developed tension and relaxation kinetics in vitro. A and B, Isolated intact trabeculae were stimulated at increasing frequencies. A, Developed force at varying frequency of stimulation. B, RT50 at varying frequency of stimulation. Experiments were performed at 37.5°C and 1.5 mmol/L Ca2+ for 7 trabeculae in each group. ANOVA analysis demonstrated no significant difference between NTG and TnIDD22,23 muscles. C and D, Isoproterenol effect on tension development and relaxation kinetics. Isolated trabeculae were exposed to increasing concentrations of isoproterenol. Developed force (C) and RT50 (D). Experiments were performed at 37.5°C and 1.5 mmol/L Ca2+ for 8 trabeculae in each group. ANOVA analysis demonstrated no significant difference between NTG and TnIDD22,23 muscles.

Assessment of PLB and PLB-Ser 16 Phosphorylation
One potential factor that could alter myocardial behavior in TnIDD_22,23 was secondary changes in the level of PLB content and/or phosphorylation at Ser16, the residue phosphorylated by PKA. Figure 7 displays results of immunoblot studies for PLB and for phosphorylation at PLB-Ser16 (n=3), and reveals no differences in either total or phosphorylated protein.

View larger version (27K): [in this window] [in a new window]   Figure 7. Immunoblot of phospholamban (PLB) content in homogenates from TnIDD22,23 transgenic (TG) and NTG mice. Myofibril preparations from NTG and TG hearts were performed as described. Western blots of samples containing 5, 10, and 15 µg of total protein were performed using primary rabbit anti-phospho-Ser16 phospholamban antibody. ECL was performed after incubation with secondary antibody per manufacturer protocol (Amersham). The membranes were then stripped and reprobed using mouse primary anti-phospholamban antibody, and ECL was repeated after secondary antibody incubation. Bands were scanned and quantified; n=3/group. Representative blots are pictured. There is no significant difference between NTG and TG in amount of total PLB or phospho-Ser16 phospholamban.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study provides novel evidence supporting a prominent role for cardiac TnI PKA phosphorylation in the enhancement in vivo systolic and diastolic function. Whereas basal differences were modest, the TnIDD_22,23 mice had an enhanced response to beat frequency and a modulation of myocardial relaxation in response to afterload. Notably, treatment with isoproterenol in vivo eliminated the differential between TnIDD_22,23 and nontransgenic mice in the response to increased heart rate and imposition of afterload, supporting the role of PKA phosphorylation of TnI in these responses. These findings are of particular significance because of the association of heart failure with decreased phosphorylation of TnI at PKA sites.^31–33

Studies regarding the role of PKA-site TnI phosphorylation have principally focused on cardiac muscle relaxation effects, and the results have been the subject of some debate. Desensitization of myofibrils to Ca²⁺ appears to oppose the inotropic effect produced by PLB and sarcolemmal L-type calcium channel phosphorylation. However, TnI phosphorylation could contribute to augmented function from ß-stimulation by enhancing relaxation¹⁰ and crossbridge cycling,^34,35 and accelerating unloaded shortening velocity.³⁶ Studies in genetically modified animals have suggested that TnI phosphorylation by PKA does have a role in augmenting relaxation³⁷ and the frequency of minimum dynamic stiffness (a surrogate for augmenting crossbridge kinetics¹²). Other studies performed in isolated trabeculae have found that PKA phosphorylation does not alter unloaded shortening,^14,15 and the precise experimental conditions may be important in this regard.

Although most published work has focused on effects of TnI phosphorylation on relaxation, there are data suggesting myofilament protein phosphorylation by PKA can increase skinned myocyte power-output by a mechanism involving the kinetics of loaded shortening.³⁸ Layland and Kentish³⁹ reported positive inotropic effects of ß-adrenergic stimulation in rat trabeculae with pharmacologically inhibited SR. Although, in this particular study, increased sarcolemmal Ca influx from L-type Ca channel phosphorylation may have contributed to the inotropic effect. In contrast, the present model selectively targeted TnI and supports a role for TnI phosphorylation in modulating both systolic and diastolic function in vivo, particularly as a function of heart rate and fterload.

Role of TnI in the Force-Frequency Relationship
Augmentation of systolic and diastolic function in response to higher stimulation frequency is a well-described property of cardiac muscle. In humans, there is nearly a 100% increase in intact heart contractility over the physiological frequency range, and similar responses have been reported in other large mammalian models.^40,41 In mice, the response is diminished, particularly when it is assessed by parameters that minimize the preload-sensitivity with higher frequencies.²² However, ß-adrenergic stimulation has been shown to enhance the FFR, independent of the chronotropic effects of ß-adrenergic stimulation, whereas the relation is profoundly blunted by cardiac failure^1,41,42 in association with altered calcium cycling. Although it has been widely held that phosphorylation of PLB modulating SR-ATPase activity and sarcolemmal calcium channels to enhance Ca²⁺ entry per excitation is central to this synergy,^43,44 the current data supports a key involvement of TnI phosphorylation as well.

It is intriguing that whereas TnI phosphorylation had a prominent effect on the in vivo FFR, there was no impact on this relation in isolated muscle under isometric conditions. One major difference is the lack of muscle shortening in the latter preparation, yet shortening and load-interactions may be central to the mechanisms of TnI modulation. In the study of Layland and Kentish,³⁹ comparisons were made between isometric versus work-loop contracting trabeculae, a methodology that mimics in vivo muscle shortening. Elevation of stimulation frequency had a more notable impact on relaxation in shortening muscles versus those stimulated at fixed length. The increase in crossbridge cycling by TnI phosphorylation was felt to reduce the work required to restretch during diastole at high frequency. The data of Layland and Kentish³⁹ provide a potential explanation for the apparent discrepancy between the enhanced FFR in vivo in our studies with a lack of an enhanced FFR in vitro in nonshortening muscle preparations. However, it is important to note that the muscle studies performed in the present study are limited in that analyses in muscle preparations that permitted shortening and used varying afterload were not performed. Although accurate replication of in vivo loading in isolated muscle is nontrivial, such studies would be needed to directly compare in vitro to in vivo findings. Thus, further experimental testing is needed to understand this apparent discrepancy.

TnI Phosphorylation and the Load Dependence of Relaxation
Elevation of cardiac afterload prolongs cardiac relaxation,^45–47 and this effect is attenuated by ß-adrenergic stimulation.^2,3 Such load dependence of relaxation has been extensively studied for many decades, although underlying mechanisms for the behavior have remained elusive. The phenomenon is important, because delay in relaxation elevates early filling pressures and net filling, and thus may impede diastolic function. Failing hearts display increased relaxation-afterload dependence,^46,47 and such hearts are often exposed to high afterload as a mechanism to sustain arterial pressure, augmenting its impact. The present findings suggest that TnI phosphorylation has a role in the response to afterload, and can help explain reduction of afterload relaxation dependence by ß-adrenergic signaling and its exacerbation in failure hearts. The results are supported by the work of Li et al,¹¹ who found TnI phosphorylation could contribute to ß-adrenergic–stimulated acceleration of relaxation but only in loaded conditions comparing WT to PLB-null myocytes.

Expression of the TnIDD_22,23 did not significantly alter twitch tension or dynamics at physiological temperature. In addition, there was no difference between transgenic and controls in the twitch response to isoproterenol. Although others have suggested that TnI phosphorylation by PKA mediated by isoproterenol may mediate augmented twitch relaxation kinetics in PLB knockout mice, this remains somewhat controversial.^11,48,49 One potentially important difference is that the present experiments were conducted at physiological temperature, whereas prior studies were at lower temperatures that may allow detection of more subtle differences in twitch kinetics. Based on the present findings, the dominant effect of ß-adrenergic stimulation on twitch kinetics in intact isometric muscle would seem more likely due to accelerated removal of Ca²⁺ from the cytosol by PLB phosphorylation. Although the approach used to study intact muscle in these studies is useful in reproducing many aspects of in vivo physiology, ultimately detailed experiments in membrane-depleted muscle preparations will be necessary to determine the intrinsic properties of the TnIDD_22,23 myofilaments separate from the effect of PKA phosphorylation of calcium handling proteins.

In summary, mice with TnI that mimics constitutive phosphorylation at the PKA sites have modest augmentation in baseline ventricular systolic and diastolic function. Most notably these mice have augmented force-frequency response and attenuated afterload-induced prolongation of relaxation, suggesting a role for TnI phosphorylation by PKA in these phenomena in vivo. The results have implications for heart failure in which force-frequency modulation is blunted and afterload relaxation sensitivity increased in association with diminished PKA TnI phosphorylation.

	Acknowledgments

 
This study was funded by the following grants: NIH RO1HL 63038 (A.M.M.), NIH HL F32 HL10401 (D.G.S.), and NIH P50-52307 (DAK). The authors thank John Robinson for expert technical assistance.

	Footnotes

 
*Both authors contributed equally to this study.

This manuscript was sent to Richard A. Walsh, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received May 9, 2003; resubmission received December 15, 2003; accepted January 7, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Ross J Jr, Miura T, Kambayashi M, Eising GP, Ryu KH. Adrenergic control of the force-frequency relation. Circulation. 1995; 92: 2327–2332.[Abstract/Free Full Text]
2. Gillebert TC, Leite-Moreira AF, De Hert SG. Load dependent diastolic dysfunction in heart failure. Heart Fail Rev. 2000; 5: 345–355.[CrossRef][Medline] [Order article via Infotrieve]
3. Su JB, Hittinger L, Laplace M, Crozatier B. Loading determinants of isovolumic pressure fall in closed-chest dogs. Am J Physiol. 1991; 260: H690–H697.[Medline] [Order article via Infotrieve]
4. Bers DM. Cardiac excitation-contraction coupling. Nature. 2002; 415: 198–205.[CrossRef][Medline] [Order article via Infotrieve]
5. Solaro RJ, Rarick HM. Troponin and tropomyosin: proteins that switch on and tune in the activity of cardiac myofilaments. Circ Res. 1998; 83: 471–480.[Abstract/Free Full Text]
6. Solaro RJ, Van Eyk J. Altered interactions among thin filament proteins modulate cardiac function. J Mol Cell Cardiol. 1996; 28: 217–230.[CrossRef][Medline] [Order article via Infotrieve]
7. Van Eyk JE, Thomas LT, Tripet B, Wiesner RJ, Pearlstone JR, Farah CS, Reinach FC, Hodges RS. Distinct regions of troponin I regulate Ca²⁺-dependent activation and Ca²⁺ sensitivity of the acto-S1-TM ATPase activity of the thin filament. J Biol Chem. 1997; 272: 10529–10537.[Abstract/Free Full Text]
8. Holroyde MJ, Howe E, Solaro RJ. Modification of calcium requirements for activation of cardiac myofibrillar ATPase by cyclic AMP dependent phosphorylation. Biochim Biophys Acta. 1979; 586: 63–69.
9. Robertson SP, Johnson JD, Holroyde MJ, Kranias EG, Potter JD, Solaro RJ. The effect of troponin I phosphorylation on the Ca²⁺-binding properties of the Ca²⁺-regulatory site of bovine cardiac troponin. J Biol Chem. 1982; 257: 260–263.[Free Full Text]
10. Zhang R, Zhao J, Mandveno A, Potter JD. Cardiac troponin I phosphorylation increases the rate of cardiac muscle relaxation. Circ Res. 1995; 76: 1028–1035.[Abstract/Free Full Text]
11. Li L, Desantiago J, Chu G, Kranias EG, Bers DM. Phosphorylation of phospholamban and troponin I in ß-adrenergic-induced acceleration of cardiac relaxation. Am J Physiol Heart Circ Physiol. 2000; 278: H769–H779.[Abstract/Free Full Text]
12. Kentish JC, McCloskey DT, Layland J, Palmer S, Leiden JM, Martin AF, Solaro RJ. Phosphorylation of troponin I by protein kinase A accelerates relaxation and crossbridge cycle kinetics in mouse ventricular muscle. Circ Res. 2001; 88: 1059–1065.[Abstract/Free Full Text]
13. Leite-Moreira AF, Correia-Pinto J, Henriques-Coelho T. [Interaction between load and ß-adrenergic stimulation in the modulation of diastolic function]. Rev Port Cardiol. 2001; 20: 57–62.[Medline] [Order article via Infotrieve]
14. Janssen PM, de Tombe PP. Protein kinase A does not alter unloaded velocity of sarcomere shortening in skinned rat cardiac trabeculae. Am J Physiol. 1997; 273: H2415–H2422.[Medline] [Order article via Infotrieve]
15. de Tombe PP, ter Keurs HE. Lack of effect of isoproterenol on unloaded velocity of sarcomere shortening in rat cardiac trabeculae. Circ Res. 1991; 68: 382–391.[Abstract/Free Full Text]
16. Hofmann PA, Lange JH III. Effects of phosphorylation of troponin I and C protein on isometric tension and velocity of unloaded shortening in skinned single cardiac myocytes from rats. Circ Res. 1994; 74: 718–726.[Abstract/Free Full Text]
17. Finley N, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Gasmi-Seabrook G, Howarth JW, Rance M, Solaro RJ, Cheung HC, Rosevear PR. NMR analysis of cardiac troponin C-troponin I complexes: effects of phosphorylation. FEBS Lett. 1999; 453: 107–112.[CrossRef][Medline] [Order article via Infotrieve]
18. Dohet C, al-Hillawi E, Trayer IP, Ruegg JC. Reconstitution of skinned cardiac fibres with human recombinant cardiac troponin-I mutants and troponin-C. FEBS Lett. 1995; 377: 131–134.[CrossRef][Medline] [Order article via Infotrieve]
19. Murphy AM, Kogler H, Georgakopoulos D, McDonough JL, Kass DA, Van Eyk JE, Marban E. Transgenic mouse model of stunned myocardium. Science. 2000; 287: 488–491.[Abstract/Free Full Text]
20. Murphy AM, Solaro RJ. Developmental difference in the stimulation of cardiac myofibrillar Mg²⁺-ATPase activity by calmidazolium. Pediatr Res. 1990; 28: 46–49.[Medline] [Order article via Infotrieve]
21. Georgakopoulos D, Mitzner WA, Chen CH, Byrne BJ, Millar HD, Hare JM, Kass DA. In vivo murine left ventricular pressure-volume relations by miniaturized conductance micromanometry. Am J Physiol. 1998; 274: H1416–H1422.[Medline] [Order article via Infotrieve]
22. Georgakopoulos D, Kass D. Minimal force-frequency modulation of inotropy and relaxation of in situ murine heart. J Physiol. 2001; 534: 535–545.[Abstract/Free Full Text]
23. Georgakopoulos D, Kass DA. Estimation of parallel conductance by dual-frequency conductance catheter in mice. Am J Physiol Heart Circ Physiol. 2000; 279: H443–H450.[Abstract/Free Full Text]
24. Stull LB, Leppo MK, Marban E, Janssen PM. Physiological determinants of contractile force generation and calcium handling in mouse myocardium. J Mol Cell Cardiol. 2002; 34: 1367–1376.[CrossRef][Medline] [Order article via Infotrieve]
25. Janssen PM, Stull LB, Marban E. Myofilament properties comprise the rate-limiting step for cardiac relaxation at body temperature in the rat. Am J Physiol Heart Circ Physiol. 2002; 282: H499–H507.[Abstract/Free Full Text]
26. Backx PH, Gao WD, Azan-Backx MD, Marban E. Mechanism of force inhibition by 2,3-butanedione monoxime in rat cardiac muscle: roles of [Ca²⁺]_i and cross-bridge kinetics. J Physiol (Lond). 1994; 476: 487–500.[Abstract/Free Full Text]
27. Rodriguez EK, Hunter WC, Royce MJ, Leppo MK, Douglas AS, Weisman HF. A method to reconstruct myocardial sarcomere lengths and orientations at transmural sites in beating canine hearts. Am J Physiol. 1992; 263: H293–H306.[Medline] [Order article via Infotrieve]
28. Montgomery DE, Wolska BM, Pyle WG, Roman BB, Dowell JC, Buttrick PM, Koretsky AP, Del Nido P, Solaro RJ. -Adrenergic response and myofilament activity in mouse hearts lacking PKC phosphorylation sites on cardiac TnI. Am J Physiol Heart Circ Physiol. 2002; 282: H2397–H2405.[Abstract/Free Full Text]
29. Zhang R, Zhao J, Potter JD. Phosphorylation of both serine residues in cardiac troponin I is required to decrease the Ca²⁺ affinity of cardiac troponin C. J Biol Chem. 1995; 270: 30773–30780.[Abstract/Free Full Text]
30. Kelly RP, Ting CT, Yang TM, Liu CP, Maughan WL, Chang MS, Kass DA. Effective arterial elastance as index of arterial vascular load in humans. Circulation. 1992; 86: 513–521.[Abstract/Free Full Text]
31. Bodor GS, Oakeley AE, Allen PD, Crimmins DL, Ladenson JH, Anderson PA. Troponin I phosphorylation in the normal and failing adult human heart. Circulation. 1997; 96: 1495–1500.[Abstract/Free Full Text]
32. Wolff MR, Buck SH, Stoker SW, Greaser ML, Mentzer RM. Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies: role of altered ß-adrenergically mediated protein phosphorylation. J Clin Invest. 1996; 98: 167–176.[Medline] [Order article via Infotrieve]
33. Zakhary DR, Moravec CS, Stewart RW, Bond M. Protein kinase A (PKA)-dependent troponin-I phosphorylation and PKA regulatory subunits are decreased in human dilated cardiomyopathy. Circulation. 1999; 99: 505–510.[Abstract/Free Full Text]
34. Hoh JF, Rossmanith GH, Kwan LJ, Hamilton AM. Adrenaline increases the rate of cycling of crossbridges in rat cardiac muscle as measured by pseudo-random binary noise-modulated perturbation analysis. Circ Res. 1988; 62: 452–461.[Abstract/Free Full Text]
35. Turnbull L, Hoh JF, Ludowyke RI, Rossmanith GH. Troponin I phosphorylation enhances crossbridge kinetics during ß-adrenergic stimulation in rat cardiac tissue. J Physiol. 2002; 542: 911–920.[Abstract/Free Full Text]
36. Strang KT, Sweitzer NK, Greaser ML, Moss RL. ß-Adrenergic receptor stimulation increases unloaded shortening velocity of skinned single ventricular myocytes from rats. Circ Res. 1994; 74: 542–549.[Abstract/Free Full Text]
37. Fentzke RC, Buck SH, Patel JR, Lin H, Wolska BM, Stojanovic MO, Martin AF, Solaro RJ, Moss RL, Leiden JM. Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart. J Physiol (Lond). 1999; 517: 143–157.[Abstract/Free Full Text]
38. Herron TJ, Korte FS, McDonald KS. Power output is increased after phosphorylation of myofibrillar proteins in rat skinned cardiac myocytes. Circ Res. 2001; 89: 1184–1190.[Abstract/Free Full Text]
39. Layland J, Kentish JC. Myofilament-based relaxant effect of isoprenaline revealed during work-loop contractions in rat cardiac trabeculae. J Physiol. 2002; 544: 171–182.[Abstract/Free Full Text]
40. Liu CP, Ting CT, Lawrence W, Maughan WL, Chang MS, Kass DA. Diminished contractile response to increased heart rate in intact human left ventricular hypertrophy: systolic versus diastolic determinants. Circulation. 1993; 88: 1893–1906.[Abstract/Free Full Text]
41. Senzaki H, Isoda T, Paolocci N, Ekelund U, Hare JM, Kass DA. Improved mechanoenergetics and cardiac rest and reserve function of in vivo failing heart by calcium sensitizer EMD-57033. Circulation. 2000; 101: 1040–1048.[Abstract/Free Full Text]
42. Hasenfuss G, Pieske B. Calcium cycling in congestive heart failure. J Mol Cell Cardiol. 2002; 34: 951–969.[CrossRef][Medline] [Order article via Infotrieve]
43. Bluhm WF, Kranias EG, Dillmann WH, Meyer M. Phospholamban: a major determinant of the cardiac force-frequency relationship. Am J Physiol Heart Circ Physiol. 2000; 278: H249–H255.[Abstract/Free Full Text]
44. Munch G, Bolck B, Brixius K, Reuter H, Mehlhorn U, Bloch W, Schwinger RH. SERCA2a activity correlates with the force-frequency relationship in human myocardium. Am J Physiol Heart Circ Physiol. 2000; 278: H1924–H1932.[Abstract/Free Full Text]
45. Leite-Moreira AF, Correia-Pinto J, Gillebert TC. Afterload induced changes in myocardial relaxation: a mechanism for diastolic dysfunction. Cardiovasc Res. 1999; 43: 344–353.[Abstract/Free Full Text]
46. Eichhorn EJ, Willard JE, Alvarez L, Kim AS, Glamann DB, Risser RC, Grayburn PA. Are contraction and relaxation coupled in patients with and without congestive heart failure? Circulation. 1992; 85: 2132–2139.[Abstract/Free Full Text]
47. Gillebert TC, Leite-Moreira AF, De Hert SG. Relaxation-systolic pressure relation: a load-independent assessment of left ventricular contractility. Circulation. 1997; 95: 745–752.[Abstract/Free Full Text]
48. Wolska BM, Stojanovic MO, Luo W, Kranias EG, Solaro RJ. Effect of ablation of phospholamban on dynamics of cardiac myocyte contraction and intracellular Ca²⁺. Am J Physiol. 1996; 271: C391–C397.[Medline] [Order article via Infotrieve]
49. Wolska BM, Arteaga GM, Pena JR, Nowak G, Phillips RM, Sahai S, de Tombe PP, Martin AF, Kranias EG, Solaro RJ. Expression of slow skeletal troponin I in hearts of phospholamban knockout mice alters the relaxant effect of ß-adrenergic stimulation. Circ Res. 2002; 90: 882–888.[Abstract/Free Full Text]

This article has been cited by other articles:

				N. C. Lai, T. Tang, M. H. Gao, M. Saito, T. Takahashi, D. M. Roth, and H. K. Hammond Activation of Cardiac Adenylyl Cyclase Expression Increases Function of the Failing Ischemic Heart in Mice J. Am. Coll. Cardiol., April 15, 2008; 51(15): 1490 - 1497. [Abstract] [Full Text] [PDF]

				S. Sadayappan, N. Finley, J. W. Howarth, H. Osinska, R. Klevitsky, J. N. Lorenz, P. R. Rosevear, and J. Robbins Role of the acidic N' region of cardiac troponin I in regulating myocardial function FASEB J, April 1, 2008; 22(4): 1246 - 1257. [Abstract] [Full Text] [PDF]

				M. Avkiran, A. J. Rowland, F. Cuello, and R. S. Haworth Protein Kinase D in the Cardiovascular System: Emerging Roles in Health and Disease Circ. Res., February 1, 2008; 102(2): 157 - 163. [Abstract] [Full Text] [PDF]

				T. Nagayama, E. Takimoto, S. Sadayappan, J. O. Mudd, J.G. Seidman, J. Robbins, and D. A. Kass Control of In Vivo Contraction/Relaxation Kinetics by Myosin Binding Protein C: Protein Kinase A Phosphorylation Dependent and Independent Regulation Circulation, November 20, 2007; 116(21): 2399 - 2408. [Abstract] [Full Text] [PDF]

				K. A. Sheehan, Y. Ke, and R. J. Solaro p21-Activated kinase-1 and its role in integrated regulation of cardiac contractility Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R963 - R973. [Abstract] [Full Text] [PDF]

				J. E. Stelzer, J. R. Patel, J. W. Walker, and R. L. Moss Differential Roles of Cardiac Myosin-Binding Protein C and Cardiac Troponin I in the Myofibrillar Force Responses to Protein Kinase A Phosphorylation Circ. Res., August 31, 2007; 101(5): 503 - 511. [Abstract] [Full Text] [PDF]

				G. A. Ramirez-Correa and A. M. Murphy Is Phospholamban or Troponin I the "Prima Donna" in -Adrenergic Induced Lusitropy? Circ. Res., August 17, 2007; 101(4): 326 - 327. [Full Text] [PDF]

				S.-i. Yasuda, P. Coutu, S. Sadayappan, J. Robbins, and J. M. Metzger Cardiac Transgenic and Gene Transfer Strategies Converge to Support an Important Role for Troponin I in Regulating Relaxation in Cardiac Myocytes Circ. Res., August 17, 2007; 101(4): 377 - 386. [Abstract] [Full Text] [PDF]

				R. R. Lamberts, N. Hamdani, T. W. Soekhoe, N. M. Boontje, R. Zaremba, L. A. Walker, P. P. de Tombe, J. van der Velden, and G. J. M. Stienen Frequency-dependent myofilament Ca2+ desensitization in failing rat myocardium J. Physiol., July 15, 2007; 582(2): 695 - 709. [Abstract] [Full Text] [PDF]

B. J. Biesiadecki, T. Kobayashi, J. S. Walker, R. John Solaro, and P. P. de Tombe The Troponin C G159D Mutation Blunts Myofilament Desensitization Induced by Troponin I Ser23/24 Phosphorylation Circ. Res., May 25, 2007; 100(10): 1486 - 1493.