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Cardiovascular Research 2004 61(4):756-763; doi:10.1016/j.cardiores.2003.12.019
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Troponin I phosphorylation plays an important role in the relaxant effect of β-adrenergic stimulation in mouse hearts

James R Peñaa and Beata M Wolska*,a,b

aProgram in Cardiovascular Sciences, Department of Medicine, Section of Cardiology, University of Illinois at Chicago, Chicago, IL 60612, USA
bDepartment of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL 60612, USA

* Corresponding author. Department of Medicine, Section of Cardiology (M/C 715), University of Illinois, 840 S. Wood Street, Chicago, IL 60612, USA. Tel.: +1-312-4130240; fax: +1-312-9965062. bwolska{at}uic.edu

Received 9 May 2003; revised 13 December 2003; accepted 18 December 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The present study was designed to address the question of the contribution of cardiac troponin I (cTnI) phosphorylation to the enhanced rate of relaxation during β-adrenergic stimulation in hearts in situ. Methods: In situ hemodynamic measurements were performed in mouse hearts that (1) express normal level of phospholamban (PLB) and either express cTnI (PLB/cTnI) or the slow skeletal isoform of TnI (PLB/ssTnI) that cannot be phosphorylated by protein kinase A (PKA) or (2) do not express PLB and either express cTnI (PLBKO/cTnI) or ssTnI (PLBKO/ssTnI). Results: In the basal state, there was no difference in heart rate (HR), developed pressure (DP), left ventricular end-diastolic pressure (LVEDP) or rate of contraction (+dP/dt) between PLB/cTnI and PLB/ssTnI groups. However, hearts expressing ssTnI (PLB/ssTnI) showed a significantly decreased rate of relaxation (–dP/dt) when compared with hearts expressing cTnI (PLB/cTnI). In response to β-adrenergic agonist, isoproterenol (ISO), HR increased similarly in both groups. At the two highest doses of ISO, the rate of relaxation (–dP/dt) was significantly smaller in PLB/ssTnI than in PLB/cTnI hearts. In the basal state, there was no difference in HR, DP, LVEDP,+dP/dt and –dP/dt between PLBKO/cTnI and PLBKO/ssTnI hearts. In response to ISO, HR increased similarly in both groups and was only slightly smaller in PLBKO/ssTnI group at the lowest dose of ISO. However, during ISO perfusion, when cTnI was phosphorylated, the rate of relaxation was significantly slower in PLBKO/ssTnI compared to PLBKO/cTnI hearts. Conclusion: Our data support the hypothesis that phosphorylation of cTnI significantly contributes to the enhanced rate of relaxation during β-adrenergic stimulation.

KEYWORDS Troponin I; Phospholamban; β-Adrenergic stimulation; Relaxation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
β-Adrenergic stimulation plays a major role in the regulation of rates of cardiac contraction and relaxation. Stimulation of β-adrenergic receptors activates adenylyl cyclase, increases production of cAMP and activates protein kinase A (PKA). It is known that during β-adrenergic stimulation, multiple proteins in the sarcolemma, sarcoplasmic reticulum (SR) and myofilament are phosphorylated at multiple sites. Phospholamban (PLB) appears to be the most prominent among the proteins responsible for the enhanced rate of relaxation [1–4]. However, the role of other proteins cannot be excluded. Recently, it has been shown that titin also contain sites for PKA-dependent phosphorylation [5].

Early studies showed that phosphorylation of the thin filament protein, troponin I (TnI), reduces the affinity of TnI to troponin C (TnC) [6] and decreases myofilament sensitivity to Ca2+ [7–9]. Some data also suggest that phosphorylation of TnI by PKA can directly increase cross-bridge kinetics [10–14] and thus contributes to the enhanced rate of relaxation during β-adrenergic stimulation. It has also been suggested that phosphorylation of myosin-binding protein C (MyBP-C) affects relaxation by extending cross-bridges from the backbone of the thick filament, which changes their orientation and increases the probability of cross-bridges to form weak attachments to thin filaments in the absence of activation [15–17]. In addition, McClellan et al. [18] have shown that changes in maximum Ca2+-activated force of cardiac cells correlated well with the degree of phosphorylation of MyBP-C.

The present study was designed to determine the contribution of cTnI phosphorylation to the enhanced rate of relaxation during β-adrenergic stimulation in hearts in situ. The differences in the response to isoproterenol (ISO) between studies using PLBKO mice in isolated cardiac myocytes and papillary muscle [2,3,19] emphasize the importance of using the intact, in situ heart for studies addressing this question. Additionally, it has been shown recently that the contribution of myofilament properties to the relaxant effect of β-adrenergic stimulation may be greater when force and length are changing simultaneously (as occurs in the whole heart in situ) than during isometric conditions [20]. Having the advantage of a new mouse model [19], we were able to investigate how the β-adrenergic pathway modulates cardiac dynamics in situ in mouse hearts that (1) express normal level of PLB and either express cTnI or the slow skeletal isoform of TnI (ssTnI) that cannot be phosphorylated by PKA or (2) do not express PLB and express either cTnI or ssTnI.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
This investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of health (NIH Publication No. 85-23, revised 1996).

2.1 Transgenic mice
New TG mouse lines were produced in a PLBKO background by cross-breeding existing lines of mice: PLBKO mice, TG mice in which cTnI was replaced by ssTnI (TG-ssTnI) and control mice, which express normal levels of PLB and cTnI as previously described [19]. Four groups of mice were used: (1) mice that were deficient in PLB and either express native cTnI (PLBKO/cTnI) or that had cTnI replaced with ssTnI (PLBKO/ssTnI) and (2) mice that express normal levels of PLB and either express native cTnI (PLB/cTnI) or have native cTnI replaced by ssTnI (PLB/ssTnI). All experiments were performed in at least 15-generation mice at 4–6 months of age.

2.2 Genotype analysis and offspring selection
The genotype of the litters was determined by polymerase chain reaction (PCR) analysis on genomic DNA isolated from tail biopsy samples [19]. We used two sets of PCR primers to identify expression of the PLB gene—one set for identifying the wild-type allele that gives a 500-bp PCR product and the other set for identifying the targeted allele that gives a 450-bp product. In mice which were homozygous for the PLB gene, only one 500-bp band was amplified; in PLBKO mice, only a 450-bp band was produced, whereas in heterozygous mice for PLB gene, both bands were generated. To identify expression of ssTnI gene, we used one set of primers, which gives a 500-bp PCR product.

2.3 In situ measurements
In situ measurements were performed as previously described by Evans et al. [21] and Lorenz [22]. Mice were anesthetized by intraperitoneal (i.p.) injection of 50 µg/g body weight (b.w.) of ketamine and 100 µg/g b.w. of sodium thiobutabarbital (Inactin, Research Biochemicals International, Natik, MA). The level of anesthesia was assessed by toe pinch. When mice required additional anesthesia, they were given one-fifth of the original dose of both anesthetics intraperitoneally. Mice were placed supine on a thermally controlled warming plate and their body temperature maintained at 38 °C. A tracheotomy was performed and a short segment of PE-120 tubing was inserted into the airway and secured with suture. The right carotid artery was isolated and the distal end tied off. The artery was cannulated with a 1.4-F Millar MIKRO-TIP pressure transducer (model SPR-671, Millar Instruments, Houston, TX). Using the continuous pressure display as a guide, the transducer was advanced retrogradely down the right carotid artery, into the aorta, through the aortic valve and into the left ventricle (LV). When a stable LV pressure wave form was noted, the transducer cable was secured in place with suture. Prior to each catheterization, the transducer was calibrated in warm saline in a sealed chamber at 20 and 200 mm Hg, as recommended by the manufacturer. At the end of each experiment, the catheter was immediately re-zeroed in warm saline to check for drift. To gain venous access, the right femoral vein was isolated, the distal end tied off and the proximal end catheterized with stretched PE 10 tubing. After a short length of tubing was advanced into the femoral vein and secured in place, the free end was connected to a 100 µl Hamilton glass syringe mounted on a PHD2000 micro-infusion/withdrawal pump (Harvard Apparatus, Holliston, MA). All surgical incisions were covered with saline-soaked gauze to minimize evaporation, and mice were allowed to stabilize after completion of surgery for 30 min prior to beginning the experimental protocol.

In order to analyze the myocardial response to β-adrenergic stimulation, we infused increasing concentrations of ISO (0.08, 0.16 and 0.32 ng ISO/g b.w./min), given over 3 min at 0.1 µl/g b.w. via the femoral venous catheter. The infusion vehicle consisted of 0.9% saline with 10 U/ml heparin added to prevent clotting of the venous line. Mice were allowed to recover to baseline for 10–15 min between doses. The raw data signal was amplified on the internal amplifier in a Gould WindowGraf Chart Recorder (Gould Instrument Systems, Valley View, OH), recorded at 2000 Hz and analyzed using the Left Ventricular Pressure Module of the Pone-mah Digital Acquisition Analysis and Archive System software (Gould Instrument Systems) on a personal computer. This program performs calculations of heart rate (HR), left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), developed pressure (DP), positive dP/dt (+dP/dt; maximum rate of pressure development) and negative dP/dt (–dP/dt; maximum rate of pressure decay) every 5 s. The data taken at baseline (control) was the average at the time points during the 5 min prior to ISO infusion. The data presented for ISO infusion were the average of all the data points taken during the final 2 min of infusion, when the LV parameters had reached a steady state. In addition, to examine the impact of infusing a volume of 0.1 µl/g b.w. over 3 min on cardiac function, we infused vehicle alone and found no effect.

2.4 Myofibrillar protein phosphorylation
Following an in situ experiment, the ventricles were removed, rinsed in ice-cold saline, flash frozen in liquid nitrogen and stored at –80 °C until needed. Myofibrils were isolated from PLB/cTnI, PLB/ssTnI, PLBKO/cTnI and PLBKO/ssTnI mouse hearts using a modified protocol as previously described [21]. The ventricles were cut into small cubes in fresh relax buffer (75 mM KCl, 10 mM imidazole pH 7.2, 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3, 4 mM creatine phosphate, 1 mM ATP, 50 mM BDM, 1 mM DTT, 1 mM benzamidine–HCl, 0.1 mM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin, 1% Triton X-100) on ice. The cut ventricular tissue was suspended in 2 ml of cold relax buffer with 10 mM EDTA and homogenized (Ultra-Turrax T8, IKA). The homogenate was centrifuged at 4 °C, 5000 rpm for 8 min, resuspended in 7 ml of rigor buffer (75 mM KCl, 10 mM imidazole pH 7.2, 2 mM MgCl2, 2 mM EGTA, 1 mM NaN3, 1% Triton X-100) and further processed in a glass dounce tissue grinder. The homogenate was centrifuged at 4 °C, 5000 rpm for 8 min, resuspended with 7 ml of ice-cold rigor buffer, placed on ice for 5 min and repeated. The pellet was then washed with 7 ml of rigor buffer minus Triton X-100, centrifuged 5000 rpm at 4 °C for 5 min and repeated. The pellet was then resuspended with 7 ml of ice-cold K60 buffer (2 M KCl, 0.5 M MOPS, 100 mM MgCl2, pH 7.0), centrifuged 3000 rpm for 8 min at 4 °C. The final pellet was resuspended with 0.2 ml of K60 buffer and myofibril protein concentration determined (BioRad Protein Assay). Endogenous levels of PKA phosphorylation were determined using a backphosphorylation technique modified from Karczewski et al. [23]. Briefly, 40 µg (5 µl) of myofibril protein was used for each phosphorylation reaction. The protein was incubated at 30 °C for 2 min with 12.5 µl of 2 x buffer (80 mM histidine–HCl, 20 mM MgCl2, 30 mM NaF, 2 mM EGTA) followed by the addition of 5 µl (12.5 U) of PKA (Sigma P-2645). The phosphorylation reaction was initiated by the addition of 1 µl of 32P-ATP (NEN, Dupont) and carried out for 40 min at 30 °C. The reaction was stopped by addition of 8 µl of 4 X gel-loading buffer. The samples were immediately boiled in a water bath for 10 min and separated on a 12% SDS–polyacrylamide gel. The gel was stained with Coomassie brilliant blue for 20 min and destained for 2 h. Destained gels were then exposed to a phosphor screen overnight for quantification of 32P incorporation (Molecular Dynamics, STORM). Equal gel loading was verified from the Coomassie-stained gel.

2.5 Data computation and statistical analysis
All results were presented as mean±S.E.M. The significance of differences between the means was evaluated by one-way ANOVA followed by the Student–Newman–Keul's test. A value of P≤0.05 was the criterion for significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
To distinguish the role of PLB phosphorylation from cTnI phosphorylation on cardiac function, we measured LV pressure in situ in PLB/cTnI, PLB/ssTnI, PLBKO/cTnI and PLBKO/ssTnI hearts during control conditions and during β-adrenergic stimulation. We compared PLBKO and PLB groups separately because expression of both β-receptors and ryanodine receptors (RyRs) are downregulated in PLBKO hearts [19,24]. Masaki et al. [25] also reported that the Ca-dependent inactivation of L-type Ca2+ current is increased in PLBKO cells. In the first series of experiments, we compared the hemodynamic parameters in PLB/cTnI and PLB/ssTnI mouse hearts. There were no differences in HR, DP or positive dP/dt between PLB/cTnI and PLB/ssTnI groups in the basal state (Figs. 1 and 2)Go. In addition, LVEDP was not different between the PLB/cTnI (3.6±0.9 mm Hg; n = 14) and PLB/ssTnI (4.1±1.5 mm Hg; n = 16) groups. However, hearts expressing ssTnI showed a significantly decreased rate of relaxation (–dP/dt) when compared with hearts expressing cTnI (Fig. 2B). Next, we compared the responses of these two groups of animals to increasing doses of ISO. In response to β-adrenergic stimulation, HR increased significantly in both groups of animals in a dose-dependent manner (Fig. 1A). At each dose of ISO, there was no difference in HR between the PLB/cTnI and PLB/ssTnI group of animals. The DP increased significantly in PLB/ssTnI hearts, but only at the two highest doses of ISO (Fig. 1B). In response to β-adrenergic stimulation, positive dP/dt increased significantly in both groups of animals in a dose-dependent manner (Fig. 2A). At the lowest dose of ISO (0.08 ng/g b.w./min) the increase in positive dP/dt was significantly greater in hearts expressing ssTnI than in hearts expressing cTnI (Fig. 2A). At the two highest doses of ISO (0.16 and 0.32 ng/g b.w./min), the rate of relaxation (–dP/dt) and its percent change were significantly smaller in hearts expressing ssTnI than in hearts expressing cTnI (Figs. 2B and 3)Go. At the highest dose of ISO, LVEDP was 1.5±1.1 mm Hg (n = 5) in PLB/cTnI group and 2.0±1.2 mm Hg (n = 8) in PLB/ssTnI group.


Figure 1
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  		Fig. 1 Effects of increasing doses of ISO on heart rate (HR) (panel A) and developed pressure (DP) (panel B) in hearts from PLB/cTnI (zero ISO n = 14; ISO n = 5) and PLB/ssTnI (zero ISO n = 16; ISO n = 8) mice. Values are presented as means±S.E.M. *Significant difference from PLB/ssTnI zero ISO; # significant difference from PLB/cTnI zero ISO.

 

Figure 2
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  		Fig. 2 Effects of increasing doses of ISO on rates of contraction (dP/dt) (panel A) and relaxation (–dP/dt) (panel B) in hearts from PLB/cTnI (zero ISO n = 14; ISO n = 5) and PLB/ssTnI (zero ISO n = 16; ISO n = 8) mice. Values are presented as means±S.E.M. *Significant difference from PLB/ssTnI zero ISO; # significant difference from PLB/cTnI zero ISO; {diamondsuit} significant difference from PLB/cTnI at the same dose of ISO.

 

Figure 3
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  		Fig. 3 Effects of increasing doses of ISO on rates of relaxation (–dP/dt) in hearts from PLB/cTnI (n = 5) and PLB/ssTnI (n = 8) mice. Values are presented as means±S.E.M. {diamondsuit} Significant difference from PLB/cTnI at the same dose of ISO.

 
We also compared the basal contractility and the effect of ISO stimulation in the hearts that do not express PLB and either express cTnI or ssTnI. In the basal state, there was no difference in DP, HR, positive dP/dt and negative dP/dt between the PLBKO/cTnI and PLBKO/ssTnI hearts (Figs. 4 and 5)Go. In addition, LVEDP was not different between PLBKO/cTnI (4.4±1.7 mm Hg; n = 10) and PLBKO/ssTnI (5.2±1.9 mm Hg; n = 12) groups. In response to β-adrenergic stimulation, HR increased similarly in both groups and was only slightly smaller in PLBKO/ssTnI group at 0.08 ng/g b.w./min of ISO (Fig. 4A). The positive dP/dt was not significantly different at any dose of ISO between these two groups (Fig. 5A). At the highest dose of ISO, LVEDP was 2.3±1.8 mm Hg (n = 6) in PLBKO/cTnI group and 3.4±2.0 mm Hg (n = 8) in PLBKO/ssTnI group. However, we observed that at any dose of ISO, the rate of relaxation was significantly slower in PLBKO/ssTnI compared to PLBKO/cTnI mice (Fig. 5B). Fig. 6A shows the representative raw data of dP/dt in PLBKO/cTnI and PLBKO/ssTnI hearts before ISO and during ISO stimulation (0.16 ng/g b.w./min). Fig. 6B summarizes the percentage change in the negative dP/dt at different ISO concentrations in PLBKO/cTnI and PLBKO/ssTnI mice. The response to ISO was significantly different between these two groups of mice. We also compared another index of relaxation (–dP/dt/DP), which is less load dependent in PLBKO/cTnI and PLBKO/ssTnI mice. At zero ISO, this index was not different between groups (95.1±3.6 s–1 in PLBKO/cTnI mice and 83.3±4.7 s–1 in PLBKO/ssTnI mice), but it became significantly different during ISO stimulation (88.6±3.6 s–1 in PLBKO/cTnI mice and 73.2±2.2 s–1 in PLBKO/ssTnI mice), further confirming the role of cTnI phosphorylation in the relaxant effect of β-adrenergic stimulation.


Figure 4
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  		Fig. 4 Effects of increasing doses of ISO on heart rate (HR) (panel A) and developed pressure (DP) (panel B) in hearts from PLBKO/cTnI (zero ISO n = 10; ISO n = 6) and PLBKO/ssTnI (zero ISO n = 12; ISO n = 8) mice. Values are presented as means±S.E.M. *Significant difference from PLB/ssTnI zero ISO; # significant difference from PLB/cTnI zero ISO; {diamondsuit} significant difference from PLB/cTnI at the same dose of ISO.

 

Figure 5
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  		Fig. 5 Effects of increasing doses of ISO on rates of contraction (dP/dt) (panel A) and relaxation (–dP/dt) (panel B) in hearts from PLBKO/cTnI (zero ISO n = 10; ISO n = 6) and PLBKO/ssTnI (zero ISO n = 12; ISO n = 8) mice. Values are presented as means±S.E.M. {diamondsuit} Significant difference from PLB/cTnI at the same dose of ISO.

 

Figure 6
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  		Fig. 6 (A) Representative example of effects of ISO (0.16 ng/g b.w./min) on the ±dP/dt in PLBKO/cTnI and PLBKO/ssTnI mice. (B) Effects of increasing doses of ISO on relaxation rates (–dP/dt) in hearts from PLBKO/cTnI (n = 6) and PLBKO/ssTnI (n = 8) mice. Values are presented as means±S.E.M. {diamondsuit} Significant difference from PLBKO/cTnI at the same dose of ISO.

 
The back-phosphorylation technique was used to confirm that cTnI is phosphorylated in PLB/cTnI and PLBKO/cTnI hearts during ISO (0.32 ng/g b.w./min) stimulation in mice subjected to in situ experiments (Fig. 7). These experiments demonstrated that in the basal state, cTnI is only slightly phosphorylated (Fig. 7A, lane 3, and B, lane 5), in PLB/cTnI and PLBKO/cTnI hearts, but perfusion with ISO resulted in almost complete phosphorylation of cTnI (Fig. 7A, lane 4, and B, lane 6). Moreover, we were not able to detect any phosphorylated form of cTnI in PLB/ssTnI (Fig. 7B, lane 3 and 4) and PLBKO/ssTnI (Fig. 7B, lanes 7 and 8) mice, confirming our previous finding that cTnI is completely replaced by ssTnI in those two groups of mice [19].


Figure 7
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  		Fig. 7 Phosphoimage showing the effects of back-phosphorylation by PKA on representative myofibrillar protein samples prepared from PLB/cTnI, PLB/ssTnI, PLBKO/cTnI and PLBKO/ssTnI mouse hearts frozen after in situ measurements. During the in situ measurements, mice were infused with either vehicle alone or ISO. (A) Myofibrillar protein samples from PLB/cTnI (lanes 3 and 4) in the presence of PKA. Lanes 1 and 2 show a recombinant cTnI (r) in the presence or absence of PKA. (B) Myofibrillar protein samples from PLB/ssTnI (lanes 3 and 4), PLBKO/cTnI (lanes 5 and 6) and PLBKO/ssTnI (lanes 7 and 8) hearts in the presence of PKA. Lanes 1 and 2 show a recombinant cTnI (r) in the presence or absence of PKA.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Utilizing mice that express ssTnI in a PLBKO background, we directly demonstrate that residual effects of β-adrenergic stimulation in PLBKO mice are due to the presence of cTnI and that cTnI is essential for the full effect of β-adrenergic stimulation on cardiac relaxation. Our study thus demonstrates that both altered myofilament properties and Ca2+ homeostasis have significant effects on heart function in situ.

Although there is general agreement for the role of PLB phosphorylation in the enhanced rate of relaxation during β-adrenergic stimulation, there is no clear consensus for the role of cTnI phosphorylation. Moreover, the first report from PLBKO mice showed that catecholamines are without effect on contraction and relaxation dynamics in work-performing hearts [1]. The lack of effect of ISO on relaxation in work-performing PLBKO preparations indicated that PLB is the exclusive protein responsible for the enhanced rate of relaxation during β-adrenergic stimulation. However, in vivo echocardiographic data showed that ISO increased fractional shortening in both wild-type and PLBKO mice [26]. Studies in closed-chest mice also demonstrated that the effects of ISO on positive and negative dP/dt were present although blunted in PLBKO mouse hearts compared to wild type [27]. Experiments with isolated myocytes from PLBKO mouse hearts did not completely resolve the role of TnI phosphorylation in the rate of relaxation during β-adrenergic stimulation. In the first report from PLBKO cells, we have shown that ISO increased the extent of myocyte shortening and maximal shortening and relengthening velocity [2]. In contrast, Li et al. [3] demonstrated that in the absence of an external load, there is a lack of effect of β-adrenergic activation on relaxation in PLBKO cells. The discrepancy between data presented by Wolska et al. [2] and Li et al. [3] may be due to the differences in experimental conditions. On the other hand, both Li et al. [3] and Wolska et al. [19] showed that during isometric contractions of papillary muscles cTnI phosphorylation contributes to the enhanced relaxation.

A significant new step in determining the role of the cTnI phosphorylation in the lusitropic effect of β-stimulation came with the development of new mouse models that were produced by crossbreeding PLBKO mice with mice either expressing ssTnI [19] or mice expressing mutated cTnI lacking phosphorylation sites for PKC and PKA[28]. We [19] have shown that during β-adrenergic stimulation, the time of relaxation was significantly faster in papillary muscles isolated from PLB/cTnI compared to PLB/ssTnI mice, which correlates well with the observed difference in the rate of relaxation (–dP/dt) between PLB/cTnI and PLB/ssTnI hearts in situ (Fig. 2B). In addition, earlier studies using control and transgenic mice showed that cells expressing ssTnI had a slower rate of relaxation than control cells both at baseline and during β-adrenergic stimulation [9]. Using our new mouse model, we [19] also demonstrated that in the basal state the rate of relaxation was not different between PLBKO/cTnI and PLBKO/ssTnI cells or papillary muscles, which correlates well with our current in situ data (Fig. 5B). During ISO perfusion, the time of relaxation was unchanged in cells and decreased in papillary muscles isolated from PLBKO/cTnI mice. Perfusion with ISO prolonged the time of relaxation in cells but not in papillary muscles from PLBKO/ssTnI mice. Moreover, during ISO stimulation, the time of relaxation was significantly longer in papillary muscles isolated from PLBKO/ssTnI compared to PLBKO/cTnI hearts [19]. This observation matches our finding for negative dP/dt in the in situ experiments (Fig. 5B). Although our previous data [9,19] and data by Pi et al. [28] strongly suggested that TnI phosphorylation plays an important role in enhancing the rate of relaxation at room temperature in unloaded cells and isometrically contracting papillary muscle preparations, its contribution in the intact heart remained unclear.

In the present study, we therefore took advantage of the PLBKO mice that either express cTnI or ssTnI to examine the impact of TnI phosphorylation on the in situ hearts. In the intact preparation, both Ca2+ extrusion from the cytosol and intrinsic properties of the myofilaments are important components of myocardial relaxation. Both Ca2+ regulation and myofilament properties are altered by temperature and loading conditions. In isolated cardiac myocyte shortening under unloaded conditions, Puglisi et al. [29]estimated that the relative contributions of all Ca2+ transporting systems involved in cardiac myocyte relaxation, such as the sarcoplasmic reticulum Ca2+ pump, Na+/Ca2+ exchanger, sarcolemmal Ca2+ pump and the mitochondrial Ca2+ uniporter, remain similar at 25 and 35 °C. However, recently, Janssen et al. [30] reported that temperature independently affects contraction and relaxation in rat right ventricle trabeculae. They suggested that at temperature 37.5 °C, cross-bridge cycling kinetics become rate limiting for cardiac relaxation. The in situ method allowed us not only to perform experiments at physiological temperature but also under conditions where the hearts were blood perfused and were under neuronal and hormonal input. We found that expression of ssTnI in PLB and PLBKO hearts resulted in alterations of some basal hemodynamic parameters and responses to β-adrenergic stimulation. The slower rate of relaxation in basal conditions observed in PLB/ssTnI compared to PLB/cTnI hearts can be attributed to the fact that myofilaments expressing ssTnI have an increased sensitivity to Ca2+ [31]. Moreover, during ISO stimulation, only cTnI is phosphorylated (Fig. 7), and therefore, myofilament sensitivity decreases, further enhancing the difference in Ca2+ sensitivity between PLB/cTnI and PLB/ssTnI myofilaments. Interestingly, in PLBKO hearts, we found no significant difference in basal hemodynamic parameters in hearts expressing cTnI and ssTnI (Figs. 4 and 5)Go. During ISO perfusion, the HR increased in a similar way in both groups of animals showing a slight but significant difference only at the lowest dose of ISO. However, our major finding is that at the whole heart level, similar basal relaxation rate becomes significantly faster in PLBKO/cTnI compared to PLBKO/ssTnI hearts during ISO stimulation (Fig. 5B and 6)Go when cTnI is phosphorylated (Fig. 7B).

In summary, our data support the hypothesis that phosphorylation of cTnI significantly contributes to the enhanced rate of relaxation during β-adrenergic stimulation in the closed-chest preparation. Moreover, our current data together with previous studies show that the phosphorylation of TnI acts in concert with PLB phosphorylation to regulate cardiac relaxation both in unloaded and loaded preparations.


   Acknowledgements
 
This research was supported by NIH research grants RO1 HL-58591 and HL-64209 (BMW). BMW is an Established Investigator of the American Heart Association.


   Notes
 
Time for primary review 22 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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