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(Circulation. 1999;99:505-510.)
© 1999 American Heart Association, Inc.
Clinical Investigation and Reports |
Protein Kinase A (PKA)-Dependent Troponin-I Phosphorylation and PKA Regulatory Subunits Are Decreased in Human Dilated Cardiomyopathy
From the Department of Physiology and Biophysics (D.R.Z., C.S.M., M.B.), Case Western Reserve University School of Medicine, Cleveland, Ohio; Department of Molecular Cardiology (D.R.Z., M.B.), Lerner Research Institute and Department of Thoracic and Cardiovascular Surgery (R.W.S.) and Center for Anesthesiology Research (C.S.M.), Cleveland Clinic Foundation, Cleveland, Ohio. The current affiliation for Dr Stewart is the University Hospitals of Cleveland, Ohio.
Correspondence to Meredith Bond, PhD, Department of Molecular Cardiology FF10, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195. E-mail bondm{at}cesmtp.ccf.org
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Abstract |
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Background—Most studies indicate that failing human hearts have greater baseline myofibrillar Ca2+ sensitivity of tension development than nonfailing hearts. Phosphorylation of cardiac troponin I (TnI) by cAMP-dependent protein kinase (PKA) decreases the affinity of the troponin complex for Ca2+, thus altering the Ca2+ sensitivity of force production. We tested the hypothesis that PKA-dependent TnI phosphorylation is altered in the failing human heart and investigated changes in PKA regulatory subunits as a potential mechanism.
Methods and Results—Using in vitro
back-phosphorylation with [
-32P]ATP,
we demonstrated a significant (P<0.05) 
25%
reduction in baseline PKA-dependent TnI phosphorylation
in human hearts with dilated cardiomyopathy (DCM)
compared with nonfailing (NF) human hearts. There was no significant
difference in cAMP content or maximal PKA activity between DCM and NF
hearts, but expression of the regulatory subunits of PKA-I (RI) and
PKA-II (RII) was significantly decreased in DCM versus NF hearts (RI by
40%, P<0.05; RII by 30%,
P<0.01).
Conclusions—PKA activity is regulated at the substrate level through interactions of PKA regulatory subunits with A-kinase anchoring proteins. The reduced baseline PKA-dependent phosphorylation of TnI in DCM may be due to decreased expression of RI and RII and consequently reduced anchoring of PKA holoenzyme. These findings provide new evidence of deficiencies in downstream regulation of the ß-adrenergic pathway in the failing human heart and may account for increased baseline myofibrillar Ca2+ sensitivity.
Key Words: cardiomyopathy • troponin • enzymes • proteins • receptors, adrenergic, beta
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Introduction |
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It has been estimated that the prevalence of congestive heart failure in the United States is as high as 3 million individuals, with 700 000 new cases diagnosed annually.1 Approximately half of these congestive heart failure patients are diagnosed with nonischemic dilated cardiomyopathy (DCM), yet the underlying causes are unknown. Several reports indicate that tension development by cardiac myofibrils is more sensitive to calcium in failing relative to nonfailing (NF) human hearts.2 3 In addition, because protein kinase A (PKA)-dependent phosphorylation of cardiac troponin I (TnI) controls myofibrillar sensitivity to calcium,4 TnI phosphorylation may be altered in failing hearts.3 5 6
Although it is generally accepted that ß-adrenoceptor density is reduced in the failing human myocardium,7 evidence for the existence of defects distal to the receptor/adenylyl cyclase complex is rapidly accumulating. For example, even after stimulation by positive inotropic agents that act beyond the receptor, such as dibutyryl-cAMP and cAMP-phosphodiesterase inhibitors, the decreased positive inotropic response in failing hearts is still observed.8 PKA activity is regulated at the substrate level by PKA binding to A-kinase anchoring proteins (AKAPs) through interactions with the regulatory subunit (R).9 Two classes of the R subunit exist, RI and RII, which form type I and type II PKA, respectively. PKA-I is primarily cytosolic, whereas up to 75% of PKA-II is compartmentalized by binding to AKAPs.9 RI may also bind to AKAPs.10 Recently, this laboratory has demonstrated that AKAP100 and RII colocalize in adult cardiac myocytes.11 The potential for local regulation of PKA activity implies that changes in PKA substrate phosphorylation are not necessarily due to changes in receptor-effector coupling. The purpose of the present study was to investigate whether PKA-dependent TnI phosphorylation is decreased in human DCM and to test the hypothesis that alterations in RI or RII may contribute to the differences observed.
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Methods |
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Human Myocardial Tissue
Failing human cardiac muscle was obtained from explanted hearts of cardiac transplant recipients at the Cleveland Clinic Foundation. NF human heart tissue was obtained from unmatched organ donors through Life Banc of Northeast Ohio. All failing hearts had left ventricular ejection fractions <25% (15±2%), and all NF hearts had ejection fractions >60% (65±3%) (Table 1
). Mean age was 48±4 years for
patients with DCM and 42±8 years for NF patients. Effects of age or
sex (DCM, 4 men and 4 women; NF, 5 men and 2 women) were therefore
excluded. Seven of 8 patients in heart failure had been treated with
digoxin and 6 of 8 with amiodarone. All NF patients were on
inotropic support (dopamine or norepinephrine). All hearts
were arrested and transported in ice-cold, oxygenated
cardioplegic solution (in mmol/L): NaCl 147.16,
MgCl2 16.00, KCl 20.0,
NaHCO3 10.0, CaCl2 2.25, pH
7.4. Once in the laboratory, tissue was flash-frozen in liquid
N2 and stored at -80°C. This protocol was
approved by the Cleveland Clinic Institutional Review Board.
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Preparation of Cardiac Myofibrils
Samples of cardiac left ventricular free wall
(5 g) from patients with end-stage (New York Heart Association class
IV) idiopathic DCM and from NF donor hearts were
homogenized in 5 vol of MOPS buffer (10 mmol/L, pH
7.4, 4°C) containing sucrose (290 mmol/L),
NaN3 (3 mmol/L), DTT (1 mmol/L), NaF
(20 mmol/L), pepstatin A (1 µmol/L), leupeptin (1
µmol/L), and PMSF (0.8 mmol/L) by use of three 20-second
bursts and 40-second cooling intervals, position 6, with a
Polytron homogenizer. This yielded total heart
homogenate (THH). An equivalent volume of extraction
buffer, 50 mmol/L Tris (pH 7.5) containing Triton X-100 (0.1%),
NaF (20 mmol/L), DTT (0.05 mmol/L),
MgCl2 (0.5 mmol/L), EDTA (0.125
mmol/L), antipain (5 µg/mL), leupeptin (10 µg/mL), pepstatin A (5
µg/mL), and PMSF (43 µg/mL), was added to the THH, and the mixture
was centrifuged at 10 000g for 5 minutes at 4°C.
The detergent-solubilized supernatant was set aside, and the pellet was
resuspended in an equivalent volume of extraction buffer and washed
twice again. The resultant myofibrillar fraction was resuspended in
Ca2+-free extraction buffer and stored at
-20°C.
Determination of TnI Concentration by ELISA
Equal concentrations of myofibrillar proteins were denatured in
8 mol/L urea, then coated onto 96-well microplates (50 µL/well in
quadruplicate) in coating buffer (45.3 mmol/L
NaHCO3, 18.2 mmol/L
Na2CO3, pH 9.6) overnight
at 4°C. The plates were blocked with PBS (pH 7.2) containing 3% BSA
and 0.05% Tween 20 for 1 hour at 37°C. Bound TnI was detected with
an anti-cardiac TnI monoclonal antibody (1:1000) (gift of S.
Schiaffino, University of Padua, Padua, Italy) for 2 hours at 37°C,
followed by incubation with goat anti-mouse IgG alkaline phosphatase
(AP)-linked secondary antibody (1:5000) for 1 hour at 37°C.
Absorbance was monitored with p-nitrophenyl phosphate (1
mg/mL in 11.8% diethanolamine, pH 9.6) at 405 nm. A standard curve of
OD405 versus concentration of purified
recombinant human cardiac TnI (gift of I. Trayer, University of
Birmingham, Birmingham, UK) was generated to allow determination
of absolute TnI concentrations. TnI protein was not different between
DCM and NF hearts (Table 2).
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Back-Phosphorylation
Fractions enriched in myofibrils from failing and NF hearts were
back-phosphorylated according to the method of
Karczewski et al.12 Briefly, proteins were incubated in
50 mmol/L MOPS (pH 7.0) containing 20 mmol/L NaF, 1
mmol/L DTT, 10 mmol/L MgCl2,
[-32P]ATP (200 µmol/L, 5
µCi/reaction, reaction volume 40 µL), and the catalytic subunit of
cAMP-dependent protein kinase (PKAcat, 250 U/mL)
for 45 minutes at 30°C. To ensure that the reaction reached
completion and that kinase activity was not depleted, the myofibrillar
fraction was incubated for 45 minutes with excess
PKAcat plus NaF to block endogenous
phosphatases. Phosphorylation was terminated by boiling
for 5 minutes in gel-loading buffer (50 mmol/L Tris-HCl, pH 6.8,
1% SDS, 1.5% DTT, 5% glycerol, 0.05% bromophenol blue). Control
samples were incubated without PKAcat or with
PKAcat plus protein kinase inhibitor
PKI(5–24). Proteins were then separated by
12.5% SDS-PAGE. Gels were stained with Coomassie blue, destained,
dried at 80°C, exposed to storage phosphor screens, and developed
overnight. Protein loading was normalized to actin, as quantified by
scanning densitometry. The radioactive signal corresponding to the
bands was quantified by volume analysis of background-corrected
pixel intensities by use of a StormImager in phosphor mode and
ImageQuant software. The measured phosphorylation
increased linearly with increasing protein concentration (0.5 to 5.0
mg/mL). To calculate phosphorylation stoichiometry,
[-32P]ATP standards of known specific
activity (from 4.2x10-5 to
4.2x10-3 µCi/µL) prepared from the same
aliquot as the phosphorylation assay were spotted onto
nitrocellulose and developed with the gels. This generated a standard
curve from which specific activity and moles of
32P were calculated.
Maximal PKA-dependent phosphorylation was determined by
pretreatment with AP to remove bound phosphate, then treatment with
PKAcat and [-32P]ATP
to rephosphorylate only PKA sites. Briefly, samples were
preincubated in 25 mmol/L Tris (pH 7.8), 25 mmol/L KCl,
10 mmol/L MgCl2, 0.5 mmol/L DTT for 10
minutes at 30°C. Dephosphorylation was initiated by
addition of AP (1:100 enzyme:protein) and allowed to proceed for 20
minutes at 35°C. In these experiments, NaF was omitted, then added
after dephosphorylation to terminate the reaction.
Maximal phosphate incorporation was achieved after 20 minutes of AP
pretreatment. These conditions resulted in a statistically significant
increase in 32P incorporation after
rephosphorylation in the AP-treated compared with
untreated myofibrils from every heart studied (P=0.001).
32P incorporation into proteins, as measured by
back-phosphorylation, is inversely related to the
extent of phosphorylation of the proteins in vivo.
Thus, bands on the autoradiogram that were very intense
in the AP-treated lanes but of low intensity in the untreated lanes
represent proteins highly phosphorylated in
vivo.
cAMP Assay
Supernatant fractions were prepared from total
homogenates of DCM and NF hearts, as described above.
Proteins were precipitated with 6% TCA and centrifuged at
2500g for 30 minutes. The supernatants were extracted 4
times with 5 vol of water-saturated ether, then dried under vacuum.
cAMP content was determined by use of a 125I-cAMP
double-antibody radioimmunoassay kit. Acid precipitates from the
samples were dissolved in NaOH, and protein content was determined by a
bicinchoninic acid protein assay.
PKA Activity
Phosphorylation was monitored with a Gibco PKA
assay system with kemptide as substrate. Supernatant fractions were
incubated in 50 mmol/L Tris (pH 7.5) containing 10 mmol/L
MgCl2, 0.25 mg/mL BSA,
[-32P]ATP (100 µmol/L, 1 to
2x105 dpm/nmol; 4 nmol/assay tube) with 50
µmol/L kemptide at 30°C for 10 minutes. The reaction was quenched
by spotting the sample mixture onto phosphocellulose discs followed by
a 1% (vol/vol) phosphoric acid wash. 32P
incorporation was measured by liquid scintillation counting. Total PKA
activity (PKA-I and PKA-II) was measured with a maximally effective
concentration of cAMP (10 µmol/L) in the reaction buffer.
Samples were run in duplicate, counts were averaged, and protein
concentration was determined. Activities were corrected for nonspecific
32P incorporation by subtracting the cpm of
paired reactions containing PKI(6–22) amide
(1 µmol/L). Background counts (without sample)
represented <0.1% of total counts.
Immunoblot Analysis
Samples from DCM and NF human hearts were first adjusted for
total protein. Protein loading was then normalized to actin and
quantified by scanning densitometry of the Coomassie-stained gel.
Proteins were denatured for 5 minutes at 95°C in 50 mmol/L
Tris-HCl, pH 6.8, 1% SDS, 1.5% DTT, 5% glycerol, 0.05% bromophenol
blue; separated by 10% SDS-PAGE at 125 V, 90 mA for 1 hour; then
transferred to nitrocellulose membranes at 100 V, 320 mA, for 1 hour 45
minutes. The membranes were blocked in 3% fish gelatin (wt/vol) in
Tris-buffered saline containing 0.1% Tween-20 (TBST). After two
10-minute washes in TBST, the membranes were incubated overnight with a
monoclonal anti-RI antibody (Transduction Laboratories) or polyclonal
anti-RII antibody (Santa Cruz Biotechnology). An AP-linked anti-mouse
or anti-rabbit IgG was the secondary antibody, and the membranes were
developed with Vistra ECF substrate. Fluorescence corresponding
to the bands was quantified by StormImager analysis.
Statistical Analysis
Paired and unpaired t tests were used to evaluate the
effect of treatments on individual samples and to compare differences
between DCM and NF, respectively. Differences at P<0.05
were considered significant. Results are expressed as mean±SEM.
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Results |
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TnI in each fraction from the myofibrillar extraction was identified as the phosphorylated protein running at the same position (28 kDa) as purified recombinant TnI (Figure 1
). Phospholamban (PLB) was identified as
the phosphorylated protein running at 8 kDa. Identity
of both proteins was confirmed by Western blot analysis.
Although TnI was present in the supernatant after detergent
extraction, the suspension of the pellet after the third extraction
(myofibrillar fraction) was enriched in TnI relative to other
fractions. This fraction also contained PLB. The purpose of this
extraction was not to obtain a pure myofibrillar preparation but rather
to obtain a fraction enriched for TnI. This fraction was free of other
phosphorylated proteins in the regions of TnI and PLB
and thus provided the best signal-to-noise ratio for measurement.
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PKA-Dependent Phosphorylation
To determine whether altered phosphorylation of
TnI may contribute to the increased Ca2+
sensitivity observed in the failing human heart, we measured
stoichiometry of PKA-mediated TnI phosphorylation by
back-phosphorylation.12 Other techniques,
such as estimating protein phosphorylation levels based
on band-shifting in SDS gels, are less quantitative and measure only
total nonspecific phosphate incorporation.5
Back-phosphorylation has the significant advantage of
allowing measurement of phosphorylation catalyzed
specifically by PKA. In the presence of
PKI(5–24), no phosphate incorporation could be
detected, further substantiating the specificity of this method.
We first determined whether differences in maximal
phosphorylation of TnI and PLB existed in DCM versus NF
hearts. Figure 2 shows a
representative autoradiogram of
phosphorylated myofibrillar proteins from DCM and NF
hearts before and after AP treatment. Pretreatment with AP resulted in
a significant increase (P<0.001) in
PKAcat-catalyzed 32P
incorporation into TnI and PLB in all preparations of myofibrils from
failing and NF hearts. Maximal PKA-dependent
phosphorylation (total sites
phosphorylated) of either protein was not significantly
different between DCM and NF hearts (Table 2
). Baseline
phosphorylation of PLB was not significantly different
between DCM and NF hearts (Table 2). However, baseline TnI
phosphorylation was significantly decreased
(P<0.05) in DCM compared with NF hearts (Figure 3).
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cAMP Levels and PKA Activity
To investigate whether decreased TnI
phosphorylation in DCM could result from differences in
cAMP content, we measured cAMP in the same group of DCM and NF hearts
by radioimmunoassay. There was no significant difference in cAMP
content between DCM and NF hearts (Table 2). Total PKA activity,
ie, activity in the presence of a maximally effective concentration of
cAMP, was not significantly different between DCM and NF hearts (Table 2).
Quantification of PKA Regulatory Subunits
The catalytic subunit (C) is ultimately responsible for
phosphorylation of target proteins; however, because of
the fixed stoichiometry of
R2C2, the amount of R in a
particular location will determine the local concentration of
C.13 To examine the role of PKA anchoring in DCM, we
quantified protein levels of RI and RII in DCM and NF hearts by
immunoblot analysis. Because it is not known which
PKA isoform is primarily responsible for TnI
phosphorylation in vivo, we measured protein levels of
both RI and RII. RI showed a single major band at 48 kDa (Figure 4A), and RII showed a single major band
at 55 kDa (Figure 5A). RI was
significantly decreased (by 40%; P<0.001) in DCM versus
NF hearts (Figure 4B). RII was also significantly decreased (by
30%; P<0.05) in DCM versus NF hearts (Figure 5B).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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The main findings in this study are that baseline TnI phosphorylation is diminished in myocardial samples from patients with end-stage DCM and that PKA-RI and RII protein levels are decreased in these same hearts. Decreased TnI phosphorylation would lead to an increase in the Ca2+ affinity of troponin C (TnC) and subsequent decreased off-rate of Ca2+ from TnC.4 This results in an increased Ca2+ sensitivity of force development. Decreased baseline PKA phosphorylation of TnI in the failing human heart is consistent with our previous observations in spontaneously hypertensive rat (SHR). In the 76-week-old SHR, a model of decompensated cardiac hypertrophy and precursor to heart failure, baseline TnI phosphorylation was significantly reduced versus Wistar-Kyoto (WKY) controls.6 This was associated with increased Ca2+ sensitivity of actomyosin ATPase activity. Although PKA-dependent TnI phosphorylation under baseline conditions in the 76-week old SHR was decreased, the ß-adrenergic pathway was not attenuated because maximal TnI phosphorylation similar to WKY controls was attained.6 Similarly, in the present study, we observed that maximal PKA activity was not different between failing and NF human hearts. Thus, despite decreased ß-receptor density in SHR and human hearts, our results indicate that there is sufficient reserve in these pathways that downstream components can still be activated to levels comparable to controls. Interestingly, the 26-week old SHR, which demonstrate compensated hypertrophy, did not show differences in basal TnI phosphorylation.14 Therefore, these findings, together with the current results, suggest that altered PKA-dependent TnI phosphorylation may be involved in development or progression of heart failure.
There is considerable opportunity for regulation of PKA activity at sites distal to cAMP production or breakdown. Early studies established that several pools of cAMP exist in the cardiomyocyte, implying that only a small proportion of total cAMP is directly involved in activation of specific PKA-dependent pathways.15 16 There is now increasing evidence for local regulation of PKA activity by binding of PKA to AKAPs through interactions with its regulatory subunits.9 10 11 PKA anchoring can effectively increase local concentrations of the enzyme, thus permitting selective substrate phosphorylation. For example, we found that PKA-mediated phosphorylation of PLB was not decreased in the same failing human hearts in which TnI phosphorylation was decreased. This is consistent with our SHR results, in which changes in TnI phosphorylation were not coupled to changes in PLB phosphorylation.14 Furthermore, Bohm et al17 found no difference in PLB phosphorylation in failing human hearts, although total cAMP was decreased. This suggests compartmentalization of PKA in human hearts, which may be disrupted in heart failure.
In the human heart, an RII binding protein (AKAP100) has been detected by Northern blot analysis.18 Recent work from this laboratory showed AKAP100 localization to the T-tubule/junctional sarcoplasmic reticulum region in rat cardiomyocytes.11 Confocal images showed that RII colocalizes with AKAP100. It is estimated that the distance from the T-tubule/junctional sarcoplasmic reticulum region to TnI in the A-band overlap zone of the myofibrils (500 to 700 nm) is probably sufficient for effective diffusion of C from the sites of AKAP-tethered PKA. However, this hypothesis remains to be tested.
We report, for the first time, decreased RI and RII protein in DCM hearts compared with NF control hearts. This could result from regulated proteolysis of R19 or down-regulation of R transcription. Future experiments will address this question. Decreased R implies decreased PKA holoenzyme (R2C2) targeting and therefore decreased local pools of C. This may contribute to the decreased baseline TnI phosphorylation observed in the dilated hearts.
There are potential limitations of this study concerning use of failing and NF human hearts. Because brain death may be associated with massive catecholamine release,20 NF hearts would presumably have been "treated" differently from DCM and so may not represent the ideal control. In addition, donor hearts were given inotropic support (dopamine or norepinephrine). Elevated circulating catecholamines are believed to trigger downregulation of ß-receptors7 ; however, myocardial ß-receptor downregulation has been shown to take several weeks or months to develop.21 For example, in dogs, several weeks of high-dose norepinephrine causes a decreased mechanical response to isoproterenol but does not involve decreased ß-receptor density.22 Nevertheless, ß-receptor levels are not our principal focus in the present study, given the importance of downstream regulation of the ß-pathway.
These limitations are inherent in any study of failing human myocardium and are difficult to address experimentally. However, animal models of experimental heart failure and hypertrophy do provide qualitatively similar results. In the dog model of cardiomyopathy, Ca2+ sensitivity of isometric tension was increased,23 as was also found in failing human hearts.3 In the 76-week-old SHR, we demonstrated that decreased TnI phosphorylation was associated with increased Ca2+ sensitivity.6 This suggests that the functional differences and the altered PKA-dependent TnI phosphorylation between DCM and NF hearts reflect changes that occur during heart failure instead of alterations in the NF human myocardium. Together, these results suggest that the differences observed are a consequence of heart failure per se and not the result of life support or trauma.
In summary, our findings suggest that alterations in the ß-adrenergic pathway, distal to cAMP and unrelated to downregulation of the ß-receptor/adenylate cyclase complex, may contribute to the decreased ß-adrenergic response observed in failing human hearts. In other words, rather than changes at the level of the receptor, altered RI or RII levels may ultimately explain the decreased phosphorylation of TnI, although not necessarily other PKA targets, in the failing heart.
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Acknowledgments |
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This work was supported by NHLBI grants HL-56256 (to Dr Bond) and HL-49929 (to Dr Moravec) and by an AHA Established Investigator Award and Grant-in-Aid to Dr Moravec. We thank Dr Norman Ratliff of the Department of Pathology, Cleveland Clinic Foundation, and Life Banc of Northeast Ohio for providing human tissue. We also thank Drs Frank Brozovich, Tom Egelhoff, and J.P. Jin for helpful discussions, and Steve Schomisch and Mike Trentanelli for expert technical assistance.
Received August 7, 1998; revision received September 24, 1998; accepted October 13, 1998.
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 B. K. McConnell, Z. Popovic, N. Mal, K. Lee, J. Bautista, F. Forudi, R. Schwartzman, J.-P. Jin, M. Penn, and M. Bond Disruption of Protein Kinase A Interaction with A-kinase-anchoring Proteins in the Heart in Vivo: EFFECTS ON CARDIAC CONTRACTILITY, PROTEIN KINASE A PHOSPHORYLATION, AND TROPONIN I PROTEOLYSIS J. Biol. Chem., January 16, 2009; 284(3): 1583 - 1592. [Abstract] [Full Text] [PDF]
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H.-Z. Feng, M. Chen, L. S. Weinstein, and J.-P. Jin Removal of the N-terminal Extension of Cardiac Troponin I as a Functional Compensation for Impaired Myocardial {beta}-Adrenergic Signaling J. Biol. Chem., November 28, 2008; 283(48): 33384 - 33393. [Abstract] [Full Text] [PDF]
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 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]
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 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]
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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. [Abstract] [Full Text] [PDF]
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E. E. Jweied, R. D. McKinney, L. A. Walker, I. Brodsky, A. S. Geha, M. G. Massad, P. M. Buttrick, and P. P. de Tombe Depressed cardiac myofilament function in human diabetes mellitus Am J Physiol Heart Circ Physiol, December 1, 2005; 289(6): H2478 - H2483. [Abstract] [Full Text] [PDF]
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 J. Layland, R. J. Solaro, and A. M. Shah Regulation of cardiac contractile function by troponin I phosphorylation Cardiovasc Res, April 1, 2005; 66(1): 12 - 21. [Abstract] [Full Text] [PDF]
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 F. S. Korte, E. A. Mokelke, M. Sturek, and K. S. McDonald Exercise improves impaired ventricular function and alterations of cardiac myofibrillar proteins in diabetic dyslipidemic pigs J Appl Physiol, February 1, 2005; 98(2): 461 - 467. [Abstract] [Full Text] [PDF]
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A. M. Murphy Another New Kinase Targets Troponin I Circ. Res., November 26, 2004; 95(11): 1043 - 1045. [Full Text] [PDF]
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U. Gergs, P. Boknik, I. Buchwalow, L. Fabritz, M. Matus, I. Justus, G. Hanske, W. Schmitz, and J. Neumann Overexpression of the Catalytic Subunit of Protein Phosphatase 2A Impairs Cardiac Function J. Biol. Chem., September 24, 2004; 279(39): 40827 - 40834. [Abstract] [Full Text] [PDF]
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E. Takimoto, D. G. Soergel, P. M.L. Janssen, L. B. Stull, D. A. Kass, and A. M. Murphy Frequency- and Afterload-Dependent Cardiac Modulation In Vivo by Troponin I With Constitutively Active Protein Kinase A Phosphorylation Sites Circ. Res., March 5, 2004; 94(4): 496 - 504. [Abstract] [Full Text] [PDF]
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A. El-Armouche, T. Pamminger, D. Ditz, O. Zolk, and T. Eschenhagen Decreased protein and phosphorylation level of the protein phosphatase inhibitor-1 in failing human hearts Cardiovasc Res, January 1, 2004; 61(1): 87 - 93. [Abstract] [Full Text] [PDF]
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S. Mishra, H. N. Sabbah, J. C. Jain, and R. C. Gupta Reduced Ca2+-calmodulin-dependent protein kinase activity and expression in LV myocardium of dogs with heart failure Am J Physiol Heart Circ Physiol, March 1, 2003; 284(3): H876 - H883. [Abstract] [Full Text] [PDF]
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A. M. Murphy Troponin I: In Sickness and In Health--and Normal Development Circ. Res., September 20, 2002; 91(6): 449 - 450. [Full Text] [PDF]
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A. V. Gomes, G. Guzman, J. Zhao, and J. D. Potter Cardiac Troponin T Isoforms Affect the Ca2+ Sensitivity and Inhibition of Force Development. INSIGHTS INTO THE ROLE OF TROPONIN T ISOFORMS IN THE HEART J. Biol. Chem., September 13, 2002; 277(38): 35341 - 35349. [Abstract] [Full Text] [PDF]
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W. G. Pyle, M. P. Sumandea, R. J. Solaro, and P. P. De Tombe Troponin I serines 43/45 and regulation of cardiac myofilament function Am J Physiol Heart Circ Physiol, September 1, 2002; 283(3): H1215 - H1224. [Abstract] [Full Text] [PDF]
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 F.-L. Tan, C. S. Moravec, J. Li, C. Apperson-Hansen, P. M. McCarthy, J. B. Young, and M. Bond The gene expression fingerprint of human heart failure PNAS, August 20, 2002; 99(17): 11387 - 11392. [Abstract] [Full Text] [PDF]
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X. Wang, M. X. Li, and B. D. Sykes Structure of the Regulatory N-domain of Human Cardiac Troponin C in Complex with Human Cardiac Troponin I147-163 and Bepridil J. Biol. Chem., August 16, 2002; 277(34): 31124 - 31133. [Abstract] [Full Text] [PDF]
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A. A. Armoundas, G. F. Tomaselli, and H. D. Esperer Pathophysiological basis and clinical application of T-wave alternans J. Am. Coll. Cardiol., July 17, 2002; 40(2): 207 - 217. [Abstract] [Full Text] [PDF]
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 A. N. Carr, A. G. Schmidt, Y. Suzuki, F. del Monte, Y. Sato, C. Lanner, K. Breeden, S.-L. Jing, P. B. Allen, P. Greengard, et al. Type 1 Phosphatase, a Negative Regulator of Cardiac Function Mol. Cell. Biol., June 15, 2002; 22(12): 4124 - 4135. [Abstract] [Full Text] [PDF]
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J. B Sande, I. Sjaastad, I. B Hoen, J. Bokenes, T. Tonnessen, E. Holt, P. K Lunde, and G. Christensen Reduced level of serine16 phosphorylated phospholamban in the failing rat myocardium: a major contributor to reduced SERCA2 activity Cardiovasc Res, February 1, 2002; 53(2): 382 - 391. [Abstract] [Full Text] [PDF]
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 M. V. Westfall and J. M. Metzger Troponin I Isoforms and Chimeras: Tuning the Molecular Switch of Cardiac Contraction Physiology, December 1, 2001; 16(6): 278 - 281. [Abstract] [Full Text] [PDF]
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P. Boknik, I. Heinroth-Hoffmann, U. Kirchhefer, J. Knapp, B. Linck, H. Luss, T. Muller, W. Schmitz, O.-E. Brodde, and J. Neumann Enhanced protein phosphorylation in hypertensive hypertrophy Cardiovasc Res, September 1, 2001; 51(4): 717 - 728. [Abstract] [Full Text] [PDF]
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 K.-Y. Hwang, B. S. Schwartz, B.-K. Lee, P. T. Strickland, A. C. Todd, and J. P. Bressler Associations of Lead Exposure and Dose Measures with Erythrocyte Protein Kinase C Activity in 212 Current Korean Lead Workers Toxicol. Sci., August 1, 2001; 62(2): 280 - 288. [Abstract] [Full Text] [PDF]
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Q. Yang, T. E. Hewett, R. Klevitsky, A. Sanbe, X. Wang, and J. Robbins PKA-dependent phosphorylation of cardiac myosin binding protein C in transgenic mice Cardiovasc Res, July 1, 2001; 51(1): 80 - 88. [Abstract] [Full Text] [PDF]
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J. Yang, C. S. Moravec, M. A. Sussman, N. R. DiPaola, D. Fu, L. Hawthorn, C. A. Mitchell, J. B. Young, G. S. Francis, P. M. McCarthy, et al. Decreased SLIM1 Expression and Increased Gelsolin Expression in Failing Human Hearts Measured by High-Density Oligonucleotide Arrays Circulation, December 19, 2000; 102(25): 3046 - 3052. [Abstract] [Full Text] [PDF]
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 P. Dorigo, M. Floreani, G. Santostasi, I. Maragno, D. Danieli-Betto, E. Germinario, S. M. Magno, G. Primofiore, A. M. Marini, and F. Da Settimo Pharmacological Characterization of a New Ca2+ Sensitizer J. Pharmacol. Exp. Ther., December 1, 2000; 295(3): 994 - 1004. [Abstract] [Full Text]
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A. Sanbe, J. Gulick, E. Hayes, D. Warshaw, H. Osinska, C.-B. Chan, R. Klevitsky, and J. Robbins Myosin light chain replacement in the heart Am J Physiol Heart Circ Physiol, September 1, 2000; 279(3): H1355 - H1364. [Abstract] [Full Text] [PDF]
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D. R. Zakhary, C. S. Moravec, and M. Bond Regulation of PKA Binding to AKAPs in the Heart : Alterations in Human Heart Failure Circulation, March 28, 2000; 101(12): 1459 - 1464. [Abstract] [Full Text] [PDF]
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M. L. Ruehr, D. R. Zakhary, D. S. Damron, and M. Bond Cyclic AMP-dependent Protein Kinase Binding to A-kinase Anchoring Proteins in Living Cells by Fluorescence Resonance Energy Transfer of Green Fluorescent Protein Fusion Proteins J. Biol. Chem., November 12, 1999; 274(46): 33092 - 33096. [Abstract] [Full Text] [PDF]
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D. R. Zakhary, M. A. Fink, M. L. Ruehr, and M. Bond Selectivity and Regulation of A-kinase Anchoring Proteins in the Heart. THE ROLE OF AUTOPHOSPHORYLATION OF THE TYPE II REGULATORY SUBUNIT OF cAMP-DEPENDENT PROTEIN KINASE J. Biol. Chem., December 22, 2000; 275(52): 41389 - 41395. [Abstract] [Full Text] [PDF]
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Z.-B. Yu, L.-F. Zhang, and J.-P. Jin A Proteolytic NH2-terminal Truncation of Cardiac Troponin I That Is Up-regulated in Simulated Microgravity J. Biol. Chem., May 4, 2001; 276(19): 15753 - 15760. [Abstract] [Full Text] [PDF]
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