Expression of Slow Skeletal Troponin I in Hearts of Phospholamban Knockout Mice Alters the Relaxant Effect of β-Adrenergic Stimulation
β-Adrenergic stimulation of the heart results in an enhanced relaxation rate in association with phosphorylation of both cardiac troponin I (cTnI) and phospholamban (PLB). We studied new lines of mice generated by crossbreeding mice that express slow skeletal troponin I (ssTnI) with PLB knockout (PLBKO) mice. This crossbreeding resulted in the generation of PLB/cTnI, PLB/ssTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. Perfusion with isoproterenol (ISO) significantly increased the peak amplitude of fura-2 ratio in PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI groups of mice. However, in the presence of ISO, there were no differences in the peak amplitude of fura-2 ratio among cells isolated from hearts of PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. In cells from PLB/cTnI mice, the extent of shortening was increased and the time of relaxation was significantly decreased during β-adrenergic stimulation. In PLBKO/cTnI cells, stimulation with ISO resulted in an increased extent of shortening and no change in time of relaxation. However, stimulation with ISO in cells isolated from PLBKO/ssTnI mice not only significantly increased the extent of cell shortening but also increased the time of relaxation. We also determined the kinetics of relaxation of papillary muscles isolated from all four groups of animals in the presence and absence of ISO. Perfusion with ISO increased the rate of relaxation only in PLB/cTnI, PLB/ssTnI, and PLBKO/cTnI muscles. During ISO stimulation, the time of relaxation was unchanged in PLBKO/ssTnI muscles. Our data directly demonstrate that phosphorylation of both PLB and cTnI contributes to increased rate of relaxation during β-adrenergic stimulation.
The dynamics of cardiac relaxation are controlled by complex processes involving Ca2+ removal from the sarcoplasm, release of Ca2+ from the myofilaments, the relation between bound Ca2+ and myofilament activation, and the rate of turnover of force-generating crossbridges.1,2⇓ The relative role of each of these processes during relaxation in the basal state and during altered autonomic activity remains unclear. Understanding these processes is critical to our understanding of the alterations that occur physiologically as well as in hypertrophy and failure of the heart, which commonly demonstrate impaired relaxation.3
There is ample evidence that phospholamban (PLB) of the sarcoplasmic reticulum (SR) and the myofilament regulatory protein cardiac troponin I (cTnI) are not only critical as proteins determining relaxation dynamics but also as sites of modulation by signaling cascades. For example, β-adrenergic stimulation of the heart is known to result in an enhanced relaxation rate in association with phosphorylation of both cTnI and PLB.4–6⇓⇓ In turn, in vitro studies demonstrated that phosphorylation of PLB releases the SR Ca2+ pump from a prevailing inhibition, and that phosphorylation of cTnI enhances the off rate for Ca2+ exchange with cTnC7 and increases the rate of cardiac muscle relaxation.8 Based on these findings, our laboratories generated mutant mice (PLBKO) lacking PLB and transgenic mice (TG-ssTnI) expressing slow skeletal TnI (ssTnI), which lacks protein kinase A (PKA) phosphorylation sites.9,10⇓ In the case of PLBKO mice, cardiac relaxation was greatly enhanced and only weakly affected by β-adrenergic stimulation.9,11–13⇓⇓⇓ On the other hand, cardiac relaxation was slowed in TG-ssTnI mice and the relaxant effect of β-adrenergic stimulation was significantly attenuated.10
Although these studies indicated that both PLB and cTnI are major determinants of cardiac dynamics, the role of other sites of modulation by protein phosphorylation remained unclear. β-Adrenergic stimulation results in phosphorylation of L-type Ca2+ channel proteins, the ryanodine receptor, and myosin binding protein C. To determine the relative role of these multiple sites of protein phosphorylation, in work reported here we studied a new line of mice generated by crossbreeding ssTnI-TG mice with PLBKO mice. Our data demonstrate that the relaxant effect of β-adrenergic stimulation is completely absent in heart muscle preparations from PLBKO mice in which cTnI has been replaced with ssTnI.
Materials and Methods
Generation of TG Mice
New TG mouse lines were produced in a PLBKO background9 by crossbreeding existing lines of mice: PLBKO mice, TG mice in which cTnI was replaced by ssTnI (TG-ssTnI),10 and control mice, which express normal levels of PLB and cTnI. The original PLBKO mice were generated in C3H and C57BL strain.9 The TG-ssTnI mice, which were generated in CD1 strain, are heterozygous with respect to the ssTnI gene and homozygous PLB+/+ with respect to the PLB gene. The F1 PLBKO and TG-ssTnI crossbred pups were heterozygous PLB+/− with regard to the PLB gene with ≈50% expressing the ssTnI gene. The second crossbreeding was done using two different mating pairs as follows. We crossbred the mice obtained from the first crossbreeding pair (F1 pups), which were heterozygous with respect to PLB gene and expressed ssTnI genes, with mice that expressed only cTnI and either expressed normal levels of PLB or no PLB (PLBKO). These two crossbreedings gave us a second generation of mice from which we selected (1) mice that were deficient in PLB and either expressed native cTnI (PLBKO/cTnI) or that had cTnI replaced with ssTnI (PLBKO/ssTnI) and (2) mice that expressed normal levels of PLB and expressed either native cTnI (PLB/cTnI) or had native cTnI replaced by ssTnI (PLB/ssTnI).
All of our TG mouse lines were fertile and viable. Moreover, we did not observe any increased mortality or gross cardiovascular pathology in these mice up to 18 months of age, which was the longest time we used the mice for breeding. All experiments were performed in 4- to 6-month-old mice in compliance with animal care policies of the Animal Care Committee of the University of Illinois at Chicago.
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. 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 that were homozygous for the PLB gene, only one 500-bp band was amplified; in homozygous PLB knockout mice, only a 450-bp band was produced, whereas in heterozygous mice for the 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.
Determination of Protein Expression
Myofibrils were isolated from PLB/cTnI, PLB/ssTnI, PLBKO/cTnI, and PLBKO/ssTnI mouse hearts as previously described.4,12,14⇓⇓ They were assayed for protein concentration using a modified Lowry protein assay.15 Proteins (10 and 30 μg) were separated on 12.5% sodium dodecyl sulfate (SDS)/polyacrylamide gels.
Measurement of Cai2+ Transients
Tension Measurement in Isolated Papillary Muscles
The mice were anesthetized with methoxyflurane and the heart was rapidly excised and perfused with a modified Krebs-Henseleit solution. Thin, unbranched, and uniform papillary muscles with the tricuspid valve and a small part of ventricle were carefully dissected from right ventricle.17 We estimated that the cross-sectional area at the base of the muscle was between 0.05 to 0.1 mm2. The muscle preparation was mounted in an experimental chamber and perfused with a modified Krebs-Henseleit solution (0.2 mmol/L Ca2+) at a flow rate of about 2.5 mL/min. The muscle preparations were stimulated at 0.2 Hz via platinum electrodes. The modified Krebs-Henseleit solution had the following composition (in mmol/L): NaCl 118.5, KCl 5.0, MgSO4 1.2, NaH2PO4 2.0, NaHCO3 26, d-glucose 10.0, and Ca2+ as indicated. Solutions used during dissection of the papillary muscles contained 0.2 mmol/L CaCl2; K+ was raised to 15 mmol/L to stop spontaneous beating of the heart. The solutions were equilibrated with a 95% O2/5% CO2 (pH 7.33±0.03) gas mixture by continuous bubbling at 25°C. Temperature in the muscle chamber was kept constant at 25.0±0.1°C. After the initial equilibration, the Ca2+ concentration was gradually increased to 1 mmol/L and the muscle was stretched to generate 90% of maximum developed force. Thereafter, the Ca2+ concentration was gradually increased to 4 mmol/L. At this Ca2+ concentration, the muscles developed between 1 to 3 mN systolic force. Diastolic force did not significantly change with increasing Ca2+ concentration and was <15% of systolic force. The 4 mmol/L Ca2+ concentration was selected to ensure almost identical (<5% increase) amplitude in the developed force before and during isoproterenol (ISO) perfusion. Matching the tension minimized effects of crossbridge feedback on thin filament activation.
SR Vesicle Ca2+ Uptake
Ca2+ uptake into SR vesicles was determined using a method described by Evans et al.18 Hearts were dissected from mice anesthetized with 50 mg/kg body weight sodium pentobarbital and immediately placed in ice-cold saline and trimmed free of atria. The hearts were then transferred to homogenizing buffer (HB) (2 mL/100 mg wet heart), chopped into small pieces with scissors, and homogenized. The HB contained (in mmol/L) KH2PO4 50, NaF 10, EDTA 1.0, sucrose 300, PMSF 0.3, and DTT 0.5 (pH 7.0).
Ca2+ uptake was measured over a range of 10−8 to 10−5mol/L Ca2+ in a reaction mixture that contained (in mmol/L) MgATP2− 5, free Mg2+ 0.5, imidazole 40, creatine phosphate 10, EGTA 0.5, potassium oxalate 5, sodium azide 5, procaine 10, and ruthenium red 0.03. The ionic strength was adjusted to 175 mmol/L with KCl and pH was adjusted to 7.1 with KOH. Ruthenium red and procaine were added to inhibit Ca2+ release from the SR, whereas sodium azide was added to inhibit Ca2+ uptake into mitochondria.19,20⇓ After 2 minutes of preincubation, the reaction was started by adding ventricular homogenate to the reaction mixture at a concentration of 0.20 to 0.25 mg protein/mL and proceeded at 37°C for 2 minutes with constant stirring. Protein concentration was determined using the method of Lowry et al.15 The reaction was stopped by filtration through a 0.45-μm Millipore filter that was washed with ice-cold buffer containing 20 mmol/L Tris and 2 mmol/L EGTA (pH 7.0). Total SR vesicle Ca2+ uptake was calculated from the amount of 45Ca bound to filters as determined by liquid scintillation spectroscopy.
Radioligand Binding Study
Radioligand binding studies were performed as previously described.18 Adrenergic receptor binding (β1 and β2) was determined using a highly selective β1 antagonist, CGP20712A (250 μmol/L), β2 antagonist CI-188,551 (900 μmol/L), and the β-receptor antagonist propranolol (20 μmol/L).21
Data Computation and Statistical Analysis
All results were presented as mean±SE. The significance of differences between the means was evaluated by one-way ANOVA or ANOVA for repeated measurements followed by the Student-Newman-Keuls test. A value of P≤0.05 was the criterion for significance.
The generation of PLB/cTnI, PLB/ssTnI, PLBKO/cTnI, and PLBKO/ssTnI mice is described in detail in the Materials and Methods section and schematically in Figure 1A. Figures 1B and 1C show the ethidium bromide–stained agarose gels of PCR products from the F1 generation mice. All five pups from the litter were heterozygous with regard to the PLB gene, which was determined by the presence of two PCR bands (Figure 1B, lanes 3 through 7) with some pups also expressing the ssTnI gene (Figure 1C, lanes 3, 4, and 7). Figures 1D through 1G show the ethidium bromide–stained agarose gels of PCR products from the F3 generation mice, PLBKO (Figures 1D and 1E), and mice expressing PLB (Figures 1F and 1G). There was only one PCR band (≈450 bp) present in litters from PLBKO mice (Figure 1D, lanes 3 through 10) and another single PCR band (≈500 bp) present in homozygote litters for the PLB gene (Figure 1F, lanes 4 through 8). Figures 1E and 1G show the PCR products of mouse-tail DNA using the primers identifying the ssTnI gene. The F6 or later generations of the mice were used for the studies presented later to eliminate any potential background effects among groups. To assess the level of expression of ssTnI, we isolated myofibrils from hearts of all four groups of mice. Figure 2 shows a representative SDS-PAGE analysis of cardiac myofibrillar preparations at two different loadings. There was no change in expression of any of the myofilament proteins except that cTnI was completely replaced by ssTnI in the PLB/ssTnI (lanes 7 and 8) and PLBKO/ssTnI (lanes 3 and 4) groups of mice.
In the first series of experiments, we isolated myocytes from the PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI hearts and characterized their basal contractility and Ca2+ transients (Figure 3). Compared with cells from PLB/cTnI, myocytes isolated from PLBKO/cTnI and PLBKO/ssTnI hearts showed a significantly increased systolic fura-2 ratio with no changes in diastolic fura-2 ratio (Table). Myocytes isolated from PLBKO/ssTnI hearts also showed a significantly increased extent of shortening compared with PLB/cTnI and PLBKO/cTnI cells (Figure 3). There was no change in systolic fura-2 ratio between cells isolated from PLBKO/cTnI and PLBKO/ssTnI mice; however, PLBKO/ssTnI cells showed a significantly increased extent of shortening. In addition, we found no changes in SR Ca2+ uptake between PLBKO/cTnI and PLBKO/ssTnI mice. However, there was a significant increase in pCa50 and Vmax in the PLB knockout groups (PLBKO/cTnI and PLBKO/ssTnI) compared with the PLB/cTnI group (pCa50 was 6.39±0.02 [n=5] in PLB/cTnI, 6.73±0.02 [n=6] in PLBKO/cTnI, and 6.73±0.01 [n=6] in PLBKO/ssTnI mice; Vmax was 483.6±17.7 nmol/mg per min [n=5] in PLB/cTnI, 588.0±67.6 nmol/mg per min [n=6] in PLBKO/cTnI, and 580.2±44.2 nmol/mg per min [n=6] in PLBKO/ssTnI mice).
Next, we compared the effect of ISO on the systolic and diastolic fura-2 ratio (Table) and on the dynamics of contraction and relaxation (Figure 4) in cardiac myocytes isolated from PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI mice. Perfusion with ISO significantly increased the peak amplitude of fura-2 ratio in all three groups of mice (Table). However, in the presence of ISO, there were no differences in the peak amplitudes of fura-2 ratio among cells isolated from PLB/cTnI, PLBKO/cTnI, and PLBKO/ssTnI group of mice. Figure 4 summarizes the effect of ISO on the extent of shortening (Figure 4A) and the relaxation time (Figure 4B) in cells from all three groups of mice. In PLB/cTnI cells, the extent of shortening was increased and the time at 50% of relaxation was significantly decreased during β-adrenergic stimulation. In PLBKO/cTnI cells, stimulation with ISO resulted in an increased extent of shortening and no change in time to 50% of relaxation. The situation was different in cells isolated from PLBKO/ssTnI mice. Both the extent of cell shortening and the time at 50% relaxation were significantly increased during ISO stimulation. Figure 5 shows a representative example of normalized shortening of cardiac cells not expressing PLB (PLBKO) and either expressing cTnI (Figure 5A) or ssTnI (Figure 5B) before (solid lines) and during 0.05 μmol/L ISO stimulation (dashed lines).
We also determined the kinetics of contraction and relaxation of papillary muscles isolated from all four groups of animals in the presence and absence of ISO. The developed force before and during β-adrenergic stimulation was not significantly different. Figure 6A shows a representative example of the normalized force from mouse papillary muscles expressing native levels of PLB and either expressing cTnI (PLB/cTnI) or ssTnI (PLB/ssTnI). Force was recorded before (solid lines) and during 1 μmol/L ISO stimulation (dashed lines). Under control conditions, the presence of ssTnI was associated with a slower relaxation rate. Perfusion with ISO increased the rates of contraction and relaxation in both groups of mice; however, relaxation rate remained slower in muscles expressing ssTnI compared with muscles expressing cTnI. The effect of ISO on relaxation parameters of papillary muscles from PLB/cTnI and PLB/ssTnI mice is summarized in Figures 6B and 6C. The time to 50% and 75% of relaxation was increased indicating a slower relaxation rate in muscles expressing ssTnI compared with muscles expressing cTnI. Perfusion with ISO resulted in a shorter relaxation time in both groups; however, relaxation of muscles expressing ssTnI (PLB/ssTnI) was still slower when compared with mice expressing cTnI (PLB/cTnI).
Figure 7A shows a representative example of the normalized force from mouse papillary muscles not expressing PLB and either expressing cTnI or ssTnI. Perfusion with ISO increased the rate of relaxation only in mice expressing cTnI. Figures 7B and 7C summarize the effects of ISO on relaxation parameters of papillary muscles not expressing PLB and expressing cTnI or ssTnI. Perfusion with ISO significantly reduced the time to 50% and 75% of relaxation only in papillary muscles expressing cTnI. The time to 50% and 75% of relaxation was unchanged in mice expressing ssTnI during ISO stimulation.
To determine whether the attenuation of ISO stimulation on relaxant parameters between mice expressing cTnI and ssTnI was due to the alteration in expression of β receptors, we measured the total density of β receptors and the distribution between β1 and β2 receptors in all four groups of mice. PLBKO mice resulted in the reduction in the expression of β receptors compared with mice expressing a normal level of PLB. However, the expression of ssTnI did not have any additional effect on the level of expression of β receptors (Bmax was 24.7±1.0 fmol/mg [n=5] in PLB/cTnI, 23.0±1.1 fmol/mg [n=5] in PLB/ssTnI, 15.3±0.5 fmol/mg [n=5] in PLBKO/cTnI, and 18.1±0.8 fmol/mg [n=5] in PLBKO/ssTnI mice). There was no change in Kd or the β1/β2 ratio among any of the groups (data not shown).
A significant new finding reported in the present study is the elimination of the relaxant effect of β-adrenergic stimulation in muscles lacking PLB and TnI PKA-dependent phosphorylation sites. Although it has been known for some time that both PLB and cTnI are phosphorylated during β-adrenergic stimulation of the heart, the relative role, especially of cTnI phosphorylation, has remained unclear. Moreover, there was also evidence reported that in amphibian cardiac muscle, phosphorylation of myosin binding protein C is an important determinant of the relaxant effect of β-adrenergic stimulation.22 In addition, in rat cardiac muscle, Weisberg and Winegrad23 have shown that phosphorylation of myosin binding protein C may extend crossbridges from the backbone of the filament and alter their orientation. These investigators suggested that these changes could result in alteration in rates of crossbridges attachment and detachment. Recently, McClellan et al24 demonstrated that changes in maximum Ca2+-activated force depended on the degree of myosin binding protein C phosphorylation, which could potentially have some effects on the cardiac dynamics. Although myosin light chain 2 may also be phosphorylated during β-adrenergic stimulation, we have previously shown that its level of phosphorylation remains constant at submaximal concentrations of ISO.10,12⇓
Early studies (reviewed in Solaro25) indicated that phosphorylation of cTnI may not be important in the relaxant effect of β-adrenergic stimulation because, in contrast to the case with PLB, levels of phosphorylation of cTnI were not closely correlated with enhanced relaxation during a pulse stimulation with ISO. These issues were resolved by the demonstration that both Ser23 and Ser24 of cTnI had to be phosphorylated for induction of a functional effect on the myofilaments26 and by the demonstration that cTnI phosphorylation alone could account for changes in myofilament Ca2+ sensitivity27 and enhanced crossbridge cycling.28 However, results of studies comparing ventricular pressure dynamics in isolated working heart preparations from wild-type and PLBKO mice indicated that PLB phosphorylation alone is sufficient to cause the relaxant effects of β-adrenergic stimulation.9 In contrast to the case with isolated perfused heart, echocardiographic analysis of cardiac function indicated that relaxation was enhanced with β-adrenergic stimulation.11 Moreover, β-adrenergic stimulation induced an increase in relaxation kinetics of isometrically contracting papillary muscles29 from PLBKO hearts. Studies on hearts of TG-ssTnI mice complemented these data from PLBKO hearts. Fentzke et al10 reported that in situ relaxation of left ventricular pressure was significantly slowed in TG-ssTnI hearts compared with controls. However, Kentish et al28 reported that the rate of relaxation was faster in skinned trabeculae from TG-ssTnI hearts compared with nontransgenic (NTG) hearts. As pointed out by Kentish et al,28 these findings were unexpected, and differences may be due to experimental conditions used for the two types of muscles. To achieve the same level of activation, they had to use the different Ca2+ concentrations for muscles expressing cTnI and ssTnI. They also reported that incubation with PKA increased the rate of relaxation only in preparations from NTG hearts and did not alter fmin, a measure of crossbridge cycling rate, in TG-ssTnI muscles. An obvious approach that we undertook was to crossbreed the PLBKO and ssTnI mice. We conclude from the data presented here that phosphorylation of PLB and cTnI are both important determinants of the relaxant effects of β-adrenergic stimulation.
Results of our studies on heart muscle from PLBKO mice in which ssTnI replaced cTnI also provide new insights into the role of thin filament proteins as a determinant of cardiac dynamics. Although it is generally agreed that shifts in the isoform population of myosin heavy chains may alter relaxation kinetics, a role for thin filament isoform shift has been less well appreciated. Our data provide clear evidence that the state and composition of the thin filament can, in fact, be a significant determinant of relaxation kinetics. In basal conditions, the presence of myofilaments containing ssTnI in place of cTnI significantly slowed relaxation. Moreover, during stimulation with ISO, relaxation was significantly blunted in papillary muscles containing ssTnI in place of cTnI whether or not PLB was present. These results with preparations from hearts of PLBKO/ssTnI mice confirm and extend our earlier data that demonstrated a blunting of the relaxant effect of β-adrenergic stimulation on ssTnI mouse hearts beating in situ.10 Our results also extend data reported by us18 and others30–33⇓⇓⇓ that the presence of mutant forms of thin filament proteins linked to familial hypertrophic cardiomyopathy alters myofilament sensitivity to Ca2+, which results in alteration in relaxation kinetics. The present data add a new dimension and significantly extend these findings by demonstrating that myofilament properties become a significant factor when PLB has been ablated. An important issue is whether expression of the ssTnI transgene influences membrane control of Ca2+ fluxes. Myocytes isolated from PLBKO/ssTnI mouse hearts had enhanced contractility and peak amplitude of the Ca2+ transient compared with PLB/cTnI myocytes. Moreover cells isolated from PLBKO/ssTnI hearts demonstrated the increased extent of shortening compared with PLBKO/cTnI cells, despite no change in the peak amplitude of Ca2+ transient. This is most likely due to increased myofilament sensitivity to Ca2+ observed in hearts expressing ssTnI.10,17,28,34⇓⇓⇓ To test whether expression of ssTnI results in any alterations in SR Ca2+ uptake, we measured the Ca2+ uptake by SR vesicles. Knockout of PLB resulted in increased EC50 and Vmax; however, expression of ssTnI did not affect either EC50 or Vmax, which strongly suggests that the slower rate of relaxation in mice expressing ssTnI is due to enhanced myofilament sensitivity to Ca2+ and not to altered function of SR.
Our data are relevant to other mechanisms of thin filament modulation that enhance Ca2+ sensitivity, including modulation by pharmacological agents and by mutations in thin filament proteins. Pharmacological manipulation of the myofilament response to Ca2+ remains a viable approach in the therapy of cardiac dysfunction. Our data indicate that specific modulation of the interface of cTnI with its neighbors on the thin filament may be an important target for rational drug design. Such an approach has been applied in the case of the development of the Ca2+ sensitizer levosimendan.35 However, levosimendan and other Ca2+ sensitizer agents have other effects such as phosphodiesterase inhibition. It is apparent that identification of the molecular basis for enhancement of the crossbridge cycling and myofilament Ca2+ sensitivity by ssTnI may provide clues for approaches to the development of novel agents useful in adult and pediatric patients.
This research was supported by NIH research grants RO1 HL-58591 (B.M.W.), PO1 HL-62426 (Project 1 [R.J.S., A.F.M.] and Project 4 [P.P.d.T]), R37 HL-22231 (R.J.S.), RO1 HL-52322 (P.P.d.T.), P40RR12358 (E.G.K.), and K01 HL-67709 (G.M.A.) and a Schweppe Career Development Award (B.M.W). B.M.W. and P.P.d.T. are Established Investigators of the American Heart Association.
Original received October 26, 2001; revision received March 18, 2002; accepted March 19, 2002.
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