AJP - Heart Calcium Transients and Cell-Sarcomere
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Am J Physiol Heart Circ Physiol 292: H318-H325, 2007. First published August 25, 2006; doi:10.1152/ajpheart.00283.2006
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Heart failure-associated alterations in troponin I phosphorylation impair ventricular relaxation-afterload and force-frequency responses and systolic function

Kenneth C. Bilchick ,1,* Jennifer G. Duncan,2,* Rajashree Ravi,3 Eiki Takimoto,1 Hunter C. Champion,1 Wei Dong Gao,2 Linda B. Stull,1,3 David A. Kass,1 and Anne M. Murphy3

1Department of Medicine, 2Department of Anesthesiology and Critical Care Medicine, and 3Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland

Submitted 17 March 2006 ; accepted in final form 22 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Recent studies have found that selective stimulation of troponin (Tn)I protein kinase A (PKA) phosphorylation enhances heart rate-dependent inotropy and blunts relaxation delay coupled to increased afterload. However, in failing hearts, TnI phosphorylation by PKA declines while protein kinase C (PKC) activity is enhanced, potentially augmenting TnI PKC phosphorylation. Accordingly, we hypothesized that these site-specific changes deleteriously affect both rate-responsive cardiac function and afterload dependence of relaxation, both prominent phenotypic features of the failing heart. A transgenic (TG) mouse model was generated in which PKA-TnI sites were mutated to mimic partial dephosphorylation (Ser22 to Ala; Ser23 to Asp) and dominant PKC sites were mutated to mimic constitutive phosphorylation (Ser42 and Ser44 to Asp). The two highest-expressing lines were further characterized. TG mice had reduced fractional shortening of 34.7 ± 1.4% vs. 41.3 ± 2.0% (P = 0.018) and slight chamber dilation on echocardiography. In vivo cardiac pressure-volume studies revealed near doubling of isovolumic relaxation prolongation with increasing afterload in TG animals (P < 0.001), and this remained elevated despite isoproterenol infusion (PKA stimulation). Increasing heart rate from 400 to 700 beats/min elevated contractility 13% in TG hearts, nearly half the response observed in nontransgenic animals (P = 0.005). This blunted frequency response was normalized by isoproterenol infusion. Abnormal TnI phosphorylation observed in cardiac failure may explain exacerbated relaxation delay in response to increased afterload and contribute to blunted chronotropic reserve.

contractility

CARDIOMYOCYTES FROM PATIENTS with heart failure display defects in calcium cycling and response of the myofilament contractile apparatus to calcium. Myofilament function is modulated by phosphorylation of several key regulatory proteins, including the thin filament protein troponin (Tn)I. TnI phosphorylation alters cardiac muscle shortening and relaxation kinetics and the steady-state relationship of calcium to myofilament tension. For the intact left ventricle (LV), this impacts on systolic and diastolic function, rate-responsive function, and the relationship of relaxation kinetics to afterload (48).

TnI undergoes altered phosphorylation in heart failure. Studies have found a modest reduction in phosphorylation of TnI at protein kinase A (PKA) sites in both human and experimental heart failure (28, 49, 50, 52). In addition, the expression of PKC{alpha}, -beta1, and -beta2 isoforms increases in human heart failure (3, 35). Although increased TnI protein kinase C (PKC) site phosphorylation has not been directly reported in the human heart, there are data from animal models suggesting that overexpression of PKC isoforms results in cardiac dysfunction and altered myofilament function (14, 47). In human cardiac muscle it has been noted that dephosphorylation of thin filaments corrects defects in myofilament sliding velocity (19, 44) and maximal force (35), again indirectly suggesting that elevated thin filament phosphorylation by PKC could have deleterious effects.

Functional effects of site-specific phosphorylation of TnI at PKA and PKC sites have been explored in vitro (reviewed in Ref. 22) and in some in vivo studies using combined PKA/PKC phosphorylation mimics or nonphosphorylatable mutations (38–40, 42, 48). However, to date, the in vivo impact of combined lowering of PKA site with enhanced PKC site TnI phosphorylation is unknown. A prior study in which PKA sites were constitutively activated suggests potential relevance for the diminished TnI PKA phosphorylation in the failing heart (48). Specifically, we (48) found that in mice with constitutively active PKA-TnI sites, the cardiac functional response to heart rate (force-frequency relation, FFR) was enhanced, whereas the delay in chamber relaxation in response to increased cardiac afterload was diminished. In failing hearts, both properties behave just the opposite—with a blunted FFR (15, 24, 32, 41) and enhanced load dependence of relaxation (6, 12).

Accordingly, the present study was designed to more directly test the role of site-specific altered TnI phosphorylation on both basal function and FFR and load-dependent relaxation by manipulating PKA and PKC sites to mimic changes in failing hearts. We generated transgenic (TG) mice expressing mutated TnI reflecting such changes and examined their impact on the intact heart by means of pressure-volume analysis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transgenic model. The expression vector contained the 5.5-kb murine {alpha}-myosin heavy chain promoter generously provided by Dr. Jeffrey Robbins Children’s Hospital Research Foundation, Cincinnati, OH. A PCR-amplified cDNA encoding rat cardiac TnI with Ser22 changed to Ala and Ser23 changed to Asp residues to mimic partial dephosphorylation and Ser42 and Ser44 changed to Asp to mimic activation was cloned into the SalI site. The construct was confirmed by sequencing, and the NotI-digested insert was injected into mouse pronuclear embryos (C57BL/6 x SJL) as described previously (34). From multiple founders, lines were bred with nontransgenic (NTG) C57BL/6 mice. We identified two lines with highest expression of TnI mutant, lines 354 and 371. Offspring were genotyped as described previously (34). Both lines of TG mice appeared healthy, with no symptoms of heart failure and normal breeding potential. These lines were used for the present studies. For experimental studies the TG mice were compared with NTG littermates or age-matched C57BL/6 mice. Studies were performed in accordance with institutional guidelines and approval of the IACUC.

Histology. Fixation, embedding, sectioning, and staining of mouse hearts were conducted as described previously (34). Multiple hematoxylin and eosin- and Masson's trichrome-stained sections from base to apex of seven TG and four NTG mice were examined, including mice at 4 mo (3 TG and 2 NTG) and 13–14 mo (4 TG and 2 NTG) with samples from both lines.

Echocardiograms. Echocardiograms were performed on isoflurane-anesthetized TG and NTG mice at 5–6 mo of age with an Agilent imaging system as previously described (5). Mice were initially administered 3% isoflurane for 90–120 s and then maintained with 0.6–1% isoflurane delivered by a mouse ventilator via a conical tube placed over the snout. Rectal temperature was monitored, and mice were externally warmed with a heat lamp to a constant temperature of 37.5°C.

In vivo ventricular function studies. LV pressure-volume studies were performed as described previously (9–11, 34) on mice from 6 to 9 mo of age. Mice were anesthetized with an intraperitoneal injection of urethane (800–1,000 mg/kg), etomidate (7–10 mg/kg), and morphine (1–1.5 mg/kg), intubated orally with a blunt 19-gauge needle attached to plastic tubing, and then paralyzed with an intraperitoneal injection of pancuronium bromide (0.4 mg/kg). Supplemental doses of etomidate or morphine were given as needed if increases in heart rate or blood pressure were observed in response to tail pinch. Ventilation was initiated with 100% oxygen, using a custom-designed constant-flow ventilator delivering a tidal volume of 6.7 µl/g at 120 breaths/min. Force-frequency relations were derived after injection of a funny channel (If) blocker (ULFS-49, 15–20 mg/kg ip) to induce bradycardia without altering LV function and institution of atrial pacing via an esophageal lead. Steady-state data were collected at heart rates ranging from 400 to 700 beats/min. To assess the influence of afterload on cardiac function, hearts were atrially paced at 550 beats/min and graded aortic occlusions were performed by pressing on the descending aorta with a custom manipulator. Steady-state data were collected 2 min after achieving 10-, 30-, and ≥50-mmHg increases in systolic LV pressure.

Mice were then administered a graded infusion of isoproterenol (10–80 ng·kg–1·min–1) via a jugular vein at a constant heart rate (550 beats/min) (48). Data were measured at each dose after 3- to 5-min stabilization. At the peak dose, studies were repeated to test the influence of varying heart rate and afterload.

Measures of LV function were derived as previously reported (9). Parameters included heart rate, LV peak systolic, end-systolic, and end-diastolic pressures and volumes, maximum rates of increase and decrease in LV pressure (dP/dtmax and dP/dtmin), and dP/dtmax normalized for instantaneous pressure (IP), time constant of relaxation (26, 27, 43), and effective arterial elastance, a measure of net LV afterload. Baseline function was compared between groups at a heart rate of 500 beats/min. Basal parameter comparisons were performed by unpaired t-test. Heart rate and afterload dependencies were assessed by analysis of covariance (ANCOVA), with genotype serving as the grouping factor.

Isolated muscle studies. Adult mice (6–11 mo of age) of either sex were anesthetized by intra-abdominal injection of pentobarbital sodium (~10–20 mg), and hearts were rapidly excised via midsternal thoracotomy. Hearts were retrograde perfused with modified Krebs-Henseleit buffer gassed with a 95% O2-5% CO2 gas mixture in a dissection dish at room temperature. Trabeculae or small papillary muscles were quickly dissected from the right ventricle and mounted between a force transducer and a micromanipulator in a perfusion bath as described in earlier investigations (8, 45). Force was measured by a custom-made force transducer from a silicon strain gauge (AEM 801, SensoNor) (1, 7) and expressed as milliNewtons per square millimeter of cross-sectional area.

For skinned fiber studies, trabeculae were treated by 5–10 min of exposure to 1% Triton X-100 in relaxing solution containing (mM) 80 KCl, 25 HEPES, 10 K2EGTA, 15 creatine phosphate sodium salt (Na2CrP), 5 Na2ATP, 5.15 MgCl2, and 0.5 leupeptin. The pH of the above solution was adjusted to 7.2 with KOH. Varied Ca2+ concentrations ([Ca2+]) were achieved by mixing the relaxing solution and activating solution (mM: 10 Ca2+-EGTA, 80 KCl, 25 HEPES, 15 Na2CrP, 5 Na2ATP, 4.75 MgCl2, and 0.5 leupeptin, pH 7.2) in various ratios. [Ca2+] was calculated by a computer program based on the stability constants and the enthalpy values for various reactions from Martell and Smith (25), except for values for Mg-ATP and Ca-ATP reactions from Pettit and Siddiqui (37). Skinned steady-state force-[Ca2+] relations were fit to the Hill equation to yield Fmax, or maximal Ca2+-activated force, Ca50, the [Ca2+] required for 50% of maximal activation, and the Hill coefficient (1, 7).

Phosphorylation with PKA was conducted in skinned trabeculae. Trabeculae were isolated as described above in the presence of 1% Triton X-100 (Fisher) and protease/phosphatase inhibitors. The muscles were then incubated 60 min at room temperature in 300 µl of relaxing solution containing 30 U of the PKA catalytic subunit (Sigma).

Isolation of myofibrils. Myofibrils were isolated as previously described (21) by exposure to 1% Triton X-100 in the presence of protease inhibitors, followed by pelleting of the myofibrils and washing.

Two-dimensional gel electrophoresis. To distinguish the transgene expression from native TnI, the myofibrils were extensively dephosphorylated before separation. Complete dephosphorylation of myofibrils was achieved in three steps by treating the myofibrils 1 h each at 37°C with the kits for alkaline phosphatase (Roche), protein phosphatase (PP)1 (New England Biolabs), and PP2A (Upstate Cell Signaling), carefully following manufacturers' instructions. After the treatment with PP2A, the pellet was dissolved in isoelectric focusing buffer containing 7 M urea, 2 M thiourea, 4% CHAPS, 1% dithiothreitol, 1% destreaking agent, 1.5% ampholytes, and 0.01% bromophenol blue. The samples were stored at –80°C until analysis with two-dimensional electrophoresis. Two-dimensional gel separations of myofilament proteins were conducted as described previously (51).

Phosphorylation of myofibrils with exogenous PKA. Myofibrils were suspended in a 50-µl reaction volume of standard relaxing solution (mM: 60 imidazole, 14.4 KCl, 8.2 MgCl2, 5.5 ATP, 10 EGTA, pH 7.0) containing 30 U of the PKA catalytic subunit, and in vitro phosphorylation was carried out at room temperature for 1 h. The contents were centrifuged after 1 h, and the pellets were dissolved in sample buffer and subjected to SDS-PAGE.

Immunoblotting for phospho-TnI. After SDS-PAGE, the proteins were transferred to nitrocellulose membrane, blocked overnight in 5% nonfat dry milk in Tris-buffered saline with Triton (TBS-T), or in the case of anti-phospho antibody in 2% bovine serum albumin in TBS-T, incubated with primary antibody to total TnI (8I-7, a monoclonal antibody to TnI from Spectral Diagnostics) or with antibody to phospho-TnI (Ser23/Ser24) (Cell Signaling, Danvers, MA) for 1 h at room temperature, followed by incubation with anti-mouse secondary horseradish peroxidase (HRP)-antibody for 8I-7 or anti-rabbit secondary HRP-antibody for phospho-TnI (Upstate Biotechnology, Lake Placid, NY) for 1 h and developed by a chemiluminescence method (Pierce, Rockford IL). Radiographs were densitometrically analyzed with NIH Image software, and the ratio of phospho-TnI to total TnI was calculated.

Determination of phospholamban and sarco(endo)plasmic reticulum Ca2+-ATPase expression by immunoblotting. Myocardial protein homogenates were prepared in histidine buffer [containing (mM) 5 histidine HCl, pH 7.4, 10 EDTA, 50 Na4P2O7, 25 NaF, 0.2 dithiothreitol, and 0.1 PMSF] in the presence of protease and phosphatase inhibitors. Samples of 5–15 µg of total protein were separated by gel electrophoresis, transferred to nitrocellulose membrane, and immunoblotted with mouse anti-phospholamban (PLB) (Affinity Bioreagents, Golden, CO) to determine total PLB, followed by rabbit anti-phospho-Ser-16-PLB (Upstate Biotechnology) to determine the degree of phosphorylation at this site. Anti-phospho-PLB data were normalized to total PLB and total PLB to desmin with NIH Image software. Immunoblotting with sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) antibody (Affinity Bioreagents) to assess expression of SERCA was also performed, and its expression was determined, normalized to sarcomeric {alpha}-actin (Sigma).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of mouse model. Histological examinations of mice hearts from both TG lines did not reveal differences in cellular histology or abnormal fibrosis compared with NTG hearts. The mean of heart weight normalized to body weight did not differ significantly between NTG (4.43 ± 0.55 mg/g, n = 10) and TG (4.23 ± 0.20 mg/g, n = 14) mice, nor did this ratio differ significantly between the 371 (mean 4.33 mg/g) and 354 (mean 4.20 mg/g) TG lines. These lines were found to have equivalent partial expression of mutant protein, with an average of 23% of the total TnI from myofibrils being the mutant PKA/PKC TnI as determined by two-dimensional gel electrophoresis after extensive dephosphorylation (Fig. 1A). In the baseline physiological studies there were no apparent differences between the two TG lines; therefore, the results from the two lines were combined and compared with controls. Although Montgomery et al. (31) noted increased TnT phosphorylation in transgenic mice with TnI PKC site alanine mutations, we did not note any differences in the pattern of TnT on two-dimensional gels in these mice (Fig. 1B).


Figure 1
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  		Fig. 1. Expression of troponin (Tn)I and TnT in nontransgenic (NTG) and transgenic (TG) mice. A: representative immunoblots of 2-dimensional gels stained with anti-TnI antibody. The myofibrils were extensively dephosphorylated before separation to demonstrate the difference in charge species (arrow) between NTG and TG mice derived from 2 different lines. The acidic species used to calculate the % expression for TG is illustrated by the small rectangles on the panels. Densitometry scans demonstrated that the mutant TnI accounted for 23 ± 8% of the total TnI in the TG mice (n = 7), whereas in the NTG mice this acidic species was not detected. B: representative immunoblots of 2-dimensional gels stained with anti-TnT antibody (JLT-12 from Sigma). These myofibrils were left in their native phosphorylation state, and no difference in spot patterns could be detected between NTG and TG mice.

 
TnI PKA/PKC mice have decreased submaximal calcium responsiveness. Steady-state tension-Ca2+ relationships were studied in chemically skinned muscle fibers from TnI PKA/PKC TG and NTG control mice. TG mice had markedly reduced submaximal developed tension versus NTG mice (Fig. 2) and a right shift of the force-Ca2+ relationship (desensitization), with a Ca50 of 2.54 ± 0.36 µM (n = 5) vs. 1.27 ± 0.05 µM in controls (P < 0.001). Treatment of NTG mice with PKA desensitized the myofilaments as expected, with an increased Ca50 (2.63 ± 0.91 µM), whereas similar treatment in TG mice had no effect (2.51 ± 0.207 µM). Fmax was not significantly changed (53.8 ± 8.0 mN/mm2 in TG vs. 58.3 ± 5.2 mN/mm2 in control; n = 5 in each group).


Figure 2
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  		Fig. 2. Relationship between normalized force and calcium concentrations. Skinned NTG and TG trabeculae were treated with various concentrations of calcium in the presence or absence of protein kinase A (PKA), and the measured force is plotted against the calcium concentrations. The calcium concentration required for 50% of maximal activation (Ca50) was 2.542 ± 0.207 µM for TG vs. 1.273 ± 0.052 µM for NTG (P < 0.001). There was no significant difference in Ca50 between NTG and TG when exogenous PKA was added (2.628 ± 0.91 µM for TG compared with 2.514 ± 0.207 µM for NTG). The maximal force was not significantly different between groups (53.77 ± 8.30 mN/mm2 for TG compared with 58.29 ± 5.18 mN/mm2 for NTG).

 
The desensitization and lack of PKA response of the force-Ca2+ relations in TG heart could relate to hyperphosphorylation of remaining endogenous TnI or to primary effects of the PKC site change. To test the former, we determined PKA phosphorylation of TnI under basal and PKA-stimulated conditions in myofibrils (Fig. 3). NTG myofibrils (n = 4) showed baseline phosphorylation at these sites that was increased with exposure to PKA. Basal TnI PKA phosphorylation was also observed in TG myofibrils and, importantly, increased similarly to that in NTG myofibrils in the presence of PKA stimulation. As shown in studies in mice with complete replacement of PKA sites by aspartic acid (data not shown), the phospho-specific TnI antibody recognizes the aspartic acid mutant in the PKA site in the TG protein as well as native phosphorylation of endogenous TnI, thus explaining why at maximal phosphorylation there is little apparent difference in the density on the immunoblot between TG and NTG proteins. These data refute the notion of native PKA hyperphosphorylation in the TG mice and suggest that the baseline rightward shift of the force-Ca2+ relationships in the TG mice was more likely due to dominant effects at the PKC site.


Figure 3
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  		Fig. 3. Phosphorylation status at TnI PKA sites as determined by immunoblotting. Myofibril preparations from NTG and TG mouse hearts (n = 4 in each group) were separated on SDS-PAGE gels with or without phosphorylation by PKA. Each preparation served as its own control. Samples were run on gels simultaneously, and proteins were transferred to membrane and immunoblotted sequentially with antibodies as described in the text. Densitometry scans for the PKA-sensitive antibody were normalized to total TnI antibody. No significant difference in baseline intensity was observed between NTG and TG groups, and intensity increased significantly in both NTG and TG preparations treated with PKA as determined by analysis of variance, single factor. *P < 0.05, #P < 0.001 compared with non-PKA treated.

 
LV chamber characteristics of TnI PKA/PKC mice. TG mice had mild LV dilation reflected in both end-diastolic and systolic dimension, and fractional shortening was mildly but significantly decreased (34.7 ± 3.7% vs. 41.3 ± 5.1%; P = 0.018) (Table 1). Wall thickness was similar between NTG and TG hearts; although the LV free wall was borderline thinned in the TG compared with NTG hearts (0.056 ± 0.01 vs. 0.068 ± 0.01 cm; P = 0.04), the thickness of the ventricular septum did not differ significantly between TG hearts and controls. Although these data revealed subtle systolic dysfunction, invasive pressure-volume data assessed in vivo under different anesthesia did not reveal significant dysfunction in TG mice (Table 2). Thus there was at most a subtle change in baseline function.


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  		Table 1. Mouse echocardiography data

 

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  		Table 2. Baseline parameters in nontransgenic and transgenic mice

 
TnI PKA/PKC mice display reduced LV FFR. In contrast to subtle basal differences, TG mice displayed impaired responses to increased heart rate (Fig. 4). Whereas systolic function (normalized dP/dt for IP, or dP/dt/IP) rose 24% in NTG mice as heart rate was increased from 400 to 700 beats/min, it rose only 13% in TG animals (n = 5 TG and 4 NTG; P = 0.006 by ANCOVA). This is consistent and exactly opposite to findings in mice harboring TnI with the PKA sites constitutively activated (48). When isoproterenol was infused to PKA/PKC TG mice, rate-responsive inotropy was fully restored (Fig. 4). This indicates that although basal PKA phosphorylation of TnI plays an important role in the FFR, other regulating proteins (e.g., PLB) can compensate in response to beta-adrenergic stimulation.


Figure 4
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  		Fig. 4. Force-frequency response of the left ventricle from NTG and TG mice. Augmentation in contractility [maximum rate of increase in left ventricular pressure normalized to instantaneous pressure(dP/dt/IP) in s–1] with increasing heart rate is impaired in TG compared with NTG (P = 0.006 by analysis of covariance). After treatment with isoproterenol (Iso), augmentation of contractility in TG with heart rate is similar to that in NTG. bpm, Beats per minute.

 
We further examined whether PKA/PKC TG mice had basal changes in the level of PLB phosphorylation or in SERCA expression in tissue harvested from unpaced mice at baseline, as both are important modulators of the FFR. Figure 5 displays the immunoblots and reveals no change in total PLB at baseline but a significant increase in the relative level of phospho-PLB normalized to PLB (n = 5 in each group; P < 0.05), which could compensate for the reduced TnI PKA phosphorylation. However, there was also a slight fall in total SERCA expression (n = 5 in each group; P < 0.05), which would potentially have the opposite effect (Fig. 5B). To ensure that differential PLB phosphorylation at maximal frequency (700 beats/min) did not account for the difference in physiological response at this heart rate, hearts from control and TG mice were rapidly excised and flash frozen with the pacing protocol described in MATERIALS AND METHODS. Blots demonstrated no significant difference in PLB phosphorylation between TG and NTG mice when paced at 700 beats/min (Fig. 5C).


Figure 5
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  		Fig. 5. A: immunoblots of phosphorylated phospholamban (PPLB) and phospholamban (PLB) from mice at baseline. B: immunoblots of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). C: immunoblots of PPLB and total PLB from mice paced at 700 beats/min. Each blot was sequentially stripped and labeled with the antibodies noted to normalize for loading. In the case of PLB desmin antibody was used to normalize, whereas the SERCA blots were normalized to sarcomeric {alpha}-actin. Graphs above each blot illustrate the pooled quantitative data.

 
TnI PKA/PKC mice have exacerbated relaxation delay in response to afterload. Figure 6 displays the results for the afterload-relaxation dependence in NTG and TG PKA/PKC mice. TG mice displayed an exaggerated rise in relaxation time constant ({tau}) for a given increase in afterload. The mean slope of this relation increased from 0.52 in NTG mice to 1.01 in TG mice (P < 0.001 by ANCOVA). As with the FFR data, this response is exactly opposite to that previously found in mice harboring PKA-activated TnI (48). However, unlike the FFR, the load-relaxation dependence was not altered by treatment with isoproterenol (Fig. 6), suggesting that TnI phosphorylation was indeed the primary mediator for this regulation.


Figure 6
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  		Fig. 6. Relationship of relaxation time constant ({tau}) to load (arterial elastance, Ea) expressed as % change ({Delta}) after imposition of increasing afterload. TG mice have a more marked slowing of {tau} (more impaired isovolumic relaxation) with increasing load (Ea) on the left ventricle compared with NTG mice (P < 0.001 by analysis of covariance). Slowing of {tau} in TG mice is not improved significantly by isoproterenol. Solid black and gray lines represent linear regression of baseline TG and NTG, respectively, and dotted black line represents linear regression of TG with isoproterenol.

 
beta-Adrenergic response. To test whether the altered TnI phosphorylation state influenced beta-adrenergic contractile and lusitropic response, we performed dose-response studies with isoproterenol (Fig. 7). Contractile function and relaxation rates both improved with isoproterenol, and these responses were similar in NTG and TG mice and not significantly different when compared by linear regression.


Figure 7
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  		Fig. 7. Dose responses of contractile function and relaxation to isoproterenol in NTG and TG mice. A: contractile function (dP/dt/IP, s–1) improves similarly in NTG and TG with increasing isoproterenol dose. B: relaxation improves ({tau} decreases) in both NTG and TG mice with increasing isoproterenol dose, with a trend toward decreased improvement in TG mice.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This study reveals that changes in TnI phosphorylation typical of those potential in dilated failing hearts can impair rate-dependent inotropic reserve and exacerbate prolongation of isovolumic relaxation to increased afterload. These effects are prominent despite only modest expression of the transgene and minimal changes in basal function, and they may reflect a dominant effect of altered PKC phosphorylation. Since both blunted rate-dependent and relaxation phenotypic changes are commonly observed in heart failure, the data suggest a novel explanation and/or target for modulating these adverse effects.

TnI is a thin filament protein that is intimately involved in the regulation of contraction (reviewed in Ref. 20). Muscle contraction is initiated with binding of activator calcium to regulatory binding sites on TnC, increasing the affinity of TnC for TnI and thereby decreasing the affinity of TnI for actin-tropomyosin. As a result, either through the removal of a steric block or allosteric mechanisms, cross-bridge cycling becomes more favorable and muscle contraction ensues. This process is fine-tuned by dynamic phosphorylations of the thin filament proteins TnI and TnT (22, 29), although the mechanical consequences of these changes in vivo have remained unclear.

TnI has at least six phosphorylation sites and may be phosphorylated by PKA, several PKC isoforms, protein kinase D (PKD), protein kinase G, and p-21 activated kinase (reviewed in Ref. 33). Two serines in the cardiac-specific amino terminus of TnI are traditionally considered PKA sites, although recent work indicates that they may also be phosphorylated by PKC and PKD, a downstream kinase to PKC (16). Phosphorylation at these serine residues results in desensitization of steady-state myofibrillar Mg-ATPase and tension to calcium (17). Solution biochemical studies have shown that PKA phosphorylation of TnI increases the off-rate of regulatory calcium from TnC, which may underlie enhanced relaxation by beta-adrenergic stimulation (53). Indeed, recent cardiac muscle fiber studies have confirmed that TnI PKA phosphorylation augments relaxation by increasing the kinetics of this process (18), making this an important regulator of cardiac diastolic function.

The effects of TnI phosphorylation by PKC have also been studied in vitro. In general, phosphorylation by PKC has been found to desensitize the myofilaments to calcium and reduce maximal activation (4, 36), although some studies have yielded conflicting results (39). Detailed skinned muscle studies with in vitro phosphorylation and pseudophosphorylation mutants have shown that the two proximal PKC sites mediate the effect on steady-state calcium desensitization and decreased maximal activation; however, in motility assays the proximal sites had more impact on maximal velocity, whereas the 144 site reduced the calcium sensitivity of filament sliding speed (4). The authors interpreted this data as suggesting that phosphorylation at the PKC sites may decrease the rate of cross-bridge detachment. This is consistent with a study suggesting that the phosphorylation state of TnI may result in depressed sliding velocity of thin filaments isolated from the hearts of patients with end-stage heart failure (19). Thus, although phosphorylation of TnI at PKA and PKC sites both desensitize the myofilaments to calcium in steady-state measurements, the site-specific phosphorylations appear to have different and opposing effects on kinetic properties of the cross-bridge cycle.

Given the effects of PKC phosphorylation on cross-bridge kinetics, it is perhaps not surprising that the TG mice reported here have a blunted FFR. Although the phosphorylation of both PLB and TnI contribute to the FFR in vivo, the results here, as well as previous work (48), confirm the independent contribution of TnI phosphorylation to this response. The FFR is a well-known property of cardiac muscle. beta-Adrenergic stimulation augments the FFR independent of rate effects alone (41), whereas heart failure is associated with a reduced FFR (15, 24, 32, 41). In the current model, impaired rate responsiveness in TG mice could not be attributed to lower phospho-PLB since, if anything, this was increased in the TG animals at baseline, and, indeed, when paced at 700 beats/min the phosphorylation of PLB in TG and NTG mice was equivalent. Our finding that isoproterenol fully reversed the FFR defect in TG mice supports the independent role of alternative PKA targets such as PLB and/or the effects of phosphorylation of the remaining endogenous PKA sites of TnI. SERCA was also slightly reduced in these mice and could contribute to a blunted FFR, although it has been suggested that PLB is the stronger determinant of the FFR (2). SERCA downregulation is often observed in heart failure (30), and the current results suggest the possibility of a downregulation of SERCA in response to abnormalities of TnI phosphorylation.

Prolongation of cardiac relaxation with the imposition of afterload has been well described (6, 13, 23). beta-Adrenergic stimulation attenuates this dependence (12, 46), whereas it is exacerbated in heart failure states (6, 12). Previously, the molecular mechanisms for the interdependence of afterload and relaxation were unknown. However, recent work from our laboratory (48) implicated PKA-mediated phosphorylation of TnI as a major regulator of this response. In these mice with TnI mutations that mimic constitutive PKA phosphorylation, relaxation was far less prolonged despite imposition of afterload compared with nontransgenic controls (48). In the present study, we found that despite the availability of some endogenous TnI PKA sites in the TG mice the exacerbated relaxation delay from afterload was unaltered by isoproterenol administration. These data implicate TnI phosphorylations at PKA and PKC sites as major mediators of load-relaxation interaction and suggest that TnI PKC phosphorylation may dominate over the lusitropic effects of TnI PKA phosphorylation.

In conclusion, we modeled the effects of altered phosphorylation at specific sites of TnI in an attempt to mimic changes that occur in heart failure. Modest expression of this mutant in transgenic mice resulted in very mild changes in baseline LV chamber characteristics but significant depression of the FFR and exacerbated relaxation delay with afterload increase. This supports an important role for altered myofilament phosphorylation in the pathophysiology of heart failure.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported in part by National Institutes of Health Grants R01–HL-63038 and NO1-HV-28180 (A. M. Murphy) and PO1-HL-59408 (D. A. Kass). K. C. Bilchick was supported by T32-HL-07227 and J. G. Duncan by T32-HD-043010.


   ACKNOWLEDGMENTS
 
We thank John Robinson for expert technical assistance.

Present addresses: J. G. Duncan, Washington University School of Medicine, One Children's Place, Suite 5S20, St. Louis, MO 63110; L. B. Stull, Excigen, Inc, 11968 Mays Chapel Rd., Lutherville, MD 21093.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Murphy, Dept. of Pediatrics, Johns Hopkins Univ. School of Medicine, 720 Rutland Ave., Ross Bldg. 1144, Baltimore, MD 21205 (e-mail: murphy{at}jhmi.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* K. C. Bilchick and J. G. Duncan contributed equally to this work. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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