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Cardiovascular Research 2007 74(1):124-132; doi:10.1016/j.cardiores.2007.01.019
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Copyright © 2007, European Society of Cardiology

Mn2+-dependent protein phosphatase 1 enhances protein kinase A-induced Ca2+ desensitisation in skinned murine myocardium

Axel Neulena,*, Natascha Blaudecka, Stefan Zittricha, Doris Metzlera, Gabriele Pfitzera,b,* and Robert Stehlea,b

aInstitute of Vegetative Physiology, University of Cologne, Robert-Koch-Strasse 39, D-50931 Köln, Germany
bCenter of Molecular Medicine, University of Cologne, Robert-Koch-Strasse 39, D-50931 Köln, Germany

* Corresponding authors. Institut für Vegetative Physiologie, Universität zu Köln, Robert-Koch-Strasse 39, D-50931 Köln, Germany. Tel.: +49 221 4786950; fax: +49 221 4786965. Email address: axel.neulen{at}uni-koeln.de gabriele.pfitzer{at}uni-koeln.de

Received 10 January 2006; revised 4 December 2006; accepted 23 January 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Objective: Phosphorylation of proteins in cardiac myofilaments is a major determinant in the regulation of the Ca2+ sensitivity of contraction. Whereas most reports have focused on effects of phosphorylation, little is known about reverse effects of dephosphorylation in skinned myocardium. Here we studied the effect of the Mn2+-dependent catalytic subunit of protein phosphatase 1 (PP1c-{alpha}) on the Ca2+ regulation of contraction. In particular, we tested the hypothesis that phosphorylation persists after the skinning procedure and thereby attenuates protein kinase A (PKA)-induced Ca2+ desensitisation.

Methods: Effects of Mn2+ and Mn2+-PP1c on the Ca2+ sensitivity of contraction (pCa50) were investigated in triton-skinned cardiac fibres from mice and compared with those of PKA treatment. Phosphorylation of proteins was monitored by autoradiography.

Results: PKA treatment significantly decreased the pCa50 by 0.04 pCa units. In contrast, treatment with PP1c or Mn2+-containing PP1c buffer significantly increased the pCa50 by 0.26 units or 0.09 units, respectively. These Ca2+ sensitisations were completely reversed by subsequent PKA treatment. Replacement of the endogenous cardiac troponin I (cTnI) in fibres with the phospho-mimicking mutant human cTnIS22/23D abolished the PP1c-induced Ca2+ sensitisation. PP1c removed 32P which had been incorporated into cTnI and cardiac myosin binding protein C by PKA treatment. PKA incorporated twofold more 32P into cTnI in fibres pre-treated with PP1c.

Conclusions: Mn2+-dependent PP1c increases the Ca2+ sensitivity of contraction of skinned cardiac fibres. This can be ascribed to dephosphorylation of PKA-dependent phosphorylation sites. Hence PKA-dependent phosphorylation of sarcomeric proteins persists to a functionally relevant degree after the skinning procedure.

KEYWORDS Contractile function; Protein phosphatases; Protein phosphorylation; Protein kinase A


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Beta(β)-adrenergic stimulation of the heart contributes significantly to the precise adjustment of cardiac function to different demands. Its positive inotropic and lusitropic effects are believed to be mediated by cAMP and activation of protein kinase A (PKA), leading to phosphorylation of proteins involved in Ca2+ handling and of sarcomeric proteins involved in the regulation of actomyosin interaction. Target proteins of PKA in the sarcomere are troponin I (cTnI), the inhibitory subunit of the troponin complex (cTn) [1,2], myosin binding protein C (cMyBP-C) [3], and titin [4]. In particular, it has been hypothesised that phosphorylation of cTnI at Ser-22 and Ser-23 contributes to the positive inotropic and lusitropic effects of β-adrenergic stimulation (reviewed in [1,2]). However, phosphorylation of cMyBP-C may contribute to the PKA-induced modulation of myofibrillar contraction [5,6].

The effects of PKA-dependent phosphorylation on myofilament contractility have been studied extensively in skinned myocardium [7–16]. Such preparations are devoid of Ca2+ fluxes through cell membranes and of a functional sarcoplasmatic reticulum. PKA treatment consistently reduced the Ca2+ sensitivity of contraction. Less consistently, an increase in shortening velocity and in the rate of relaxation was observed [7–12]. Furthermore the reported shifts in pCa50-values are quite variable for unknown reasons, ranging between 0.03 and 0.45 pCa-units [7,15,16]. A simple explanation would be that phosphorylation persists after skinning to a variable extent. It has been reported that sacrifice of animals can lead to an increase in plasma catecholamine levels [17]. If the ensuing phosphorylation of sarcomeric proteins does not decline rapidly, then it may persist in skinned fibres. This notion is supported by the finding that cTnI phosphorylation declines slowly after removal of β-adrenergic agonists in Langendorff perfused hearts [18] and, in fact, may require activation of a phosphatase [18,19]. Such a residual phosphorylation would not only blunt the effect of PKA in skinned fibres but could also mask the effects of phosphorylation of sarcomeric proteins by other protein kinases.

Recently it was shown that the Ca2+-desensitising effects of protein kinase C [20] and of dephosphorylation of the regulatory light chains of myosin [21] were enhanced in skinned myocytes from human failing hearts after treatment with alkaline phosphatase and protein phosphatase 1 (PP1), respectively. In search for a protocol to dephosphorylate the PKA substrates we tested whether the catalytic subunit of PP1 (PP1c) in its Mn2+-dependent form dephosphorylates PKA sites on cTnI. We note that in vivo cTnI is most likely dephosphorylated by a type IIA phosphatase (PP2A) [2,19]. However, PP1c, which has different substrate specificities in its Mn2+-dependent form [22], is easily commercially available with sufficient activity for the use in skinned fibres. Such a protocol is not only of theoretical interest because it has the potential to resolve the controversial issue as regards the effects of PKA on shortening velocity and relaxation kinetics. It is of particular interest for studying the relation between phosphorylation and effects of mutations of troponin subunits linked with familial hypertrophic cardiomyopathy, which, as suggested by biochemical studies, may be complex [23–25].

We show here that Mn2+-dependent PP1c increases the Ca2+ sensitivity of contraction in triton-skinned murine cardiac fibres, an effect which is reversed by PKA. The effects on force were associated with corresponding changes in cTnI phosphorylation. Using skinned fibres in which the endogenous cTn was exchanged with recombinant cTn containing cTnIS22/23D, we determined whether the effects of PP1c on Ca2+ sensitivity can be ascribed to dephosphorylation of Ser-22/-23, the predominant PKA sites on cTnI [1,2]. We focused on cTnI phosphorylation in this proof of principle study because replacement of endogenous cTnI with slow skeletal TnI (ssTnI), which lacks these residues, results in a loss of the Ca2+-desensitising effect of PKA [1,2,7].

A preliminary report of the results of this study was presented at the Annual Meeting of the German Physiological Society in 2005 [26].


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
2.1 Skinned fibres
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Adult male mice of the strain HIM:OF1 were sacrificed by cervical dislocation as approved by the Institutional Animal Care and Use Committee, and skinned fibres (diameter ~200 µm) were dissected from left ventricular papillary muscles as described [27].

2.2 Isometric tension and force data analysis
After mounting the fibres, a test contraction (pCa 4.6) was elicited. Force–pCa relations were obtained, and pCa50 (pCa required for half-maximal tension) and nH (Hill coefficient) were calculated as described [27]. Unless noted otherwise, the experiments were performed at 10 °C controlled by thermo-electric coolers to preserve the structural integrity of fibres during activation [27].

2.3 Phosphatase treatment
Recombinant catalytic subunit of PP1 (PP1c-{alpha}, SIGMA, Deisenhofen, Germany, Cat# P-7937) was stored at –80 °C in (in mmol/L) 50 imidazole, 250 NaCl, 2 DTT, 1 EDTA, 2 MnCl2, 0.025% Tween20, 100 mg/mL trehalose, 20% (v/v) glycerol, pH 7.4 at 20 °C, 5 kU/mL PP1c (manufacturer specifications).

Following a control force–pCa relation, the fibres were equilibrated for 10 min at 10 °C with PP1c buffer containing (in mmol/L) 20 Na2K2Cl2MnATP, 3 MnCl2, 3 K4Cl2EGTA, 10 imidazole, 5 MgCl2, 10 Na2CrP and 2 DTT, pH 6.85 at 20 °C followed by incubation in the same buffer with 0.5 kU/mL PP1c for 90 min. Using the stability constants for MnATP2– and MnEGTA2– given by [28], the PP1c buffer contained ~2.8 mmol/L free Mn2+. PP1c treatment was performed at 10 °C to preserve the integrity of myofibrillar structures since cardiac fibres, like skeletal fibres [28], developed approximately 80% of their initial maximal force in PP1c buffer. Following PP1c treatment the fibres were incubated in relaxing solution containing 50 mmol/L DTT at 20 °C for 30 min to restore the redox state of proteins [29]. This completely reversed the Mn2+-induced contraction. Thereafter a force–pCa relation was recorded. Control fibres were subjected to the same protocol without PP1c.

2.4 PKA phosphorylation
Either recombinant murine (Calbiochem, Bad Soden, Germany, Cat# 539481) or recombinant human catalytic subunit of PKA (kindly provided by K. Jaquet, Bochum, Germany) was used. The recombinant murine C-subunit was stored at –20 °C in (in mmol/L) 20 Tris–HCl (pH 7.5), 50 NaCl, 1 EDTA, 10 2-mercaptoethanol, 50% (v/v) glycerol, and 2.5 MU/ml PKA (manufacturer specifications). The recombinant human C-subunit was stored at 4 °C in (in mmol/L) 1 EDTA, 2 DTT, 5 2-mercaptoethanol, 150 K2HPO4/KH2PO4, pH 6.5, and 14 MU/ml PKA [30].

Both C-subunits were diluted in relaxing solution to give a final activity of 0.5 MU/ml. The fibres were incubated in this solution at 20 °C for 60 min. Control fibres were subjected to the same protocol with the appropriate control buffer (PKA buffer) without C-subunits. All tension experiments were performed with both C-subunits yielding similar results.

2.5 SDS-PAGE and Western Blot
Proteins were either separated using 10% Tris–tricin gels (Anamed, Darmstadt, Germany) or 12.5% SDS polyacrylamide gels which were stained either with Coomassie, Sypro® Ruby (Invitrogen, Karlsruhe, Germany) or silver (Silver Snap® Stain Kit II, Perbio, Bonn, Germany). Fibre lysates from all experimental protocols were examined by SDS-PAGE revealing no major changes in the protein pattern (see online supplement). In Western Blots cTnI was detected using a polyclonal anti-cTnI antibody at a dilution of 1:2000, which was raised by immunisation of rabbits with recombinant murine cTnI (Pineda Antikörper-Service, Berlin, Germany), and a secondary HRP-conjugated donkey anti rabbit antibody (Dianova, Hamburg, Germany) at a dilution of 1:10000. Immunoreactive protein bands were detected with enhanced chemiluminescence (SuperSignal® West Dura, Perbio, Bonn, Germany) and quantified by densitometric scanning of the chemiluminogramms using Phoretix software (Biostep, Jahnsdorf, Germany).

2.6 Protein phosphorylation
Fibres were incubated with PKA C-subunits (0.5 MU/ml) and 20 MBq/ml [{gamma}32P]-ATP (Hartmann Analytic GmbH, Braunschweig, Germany) in a solution containing (in mmol/L) 10 imidazole, 3 K4Cl2EGTA, 2 Na2K2Cl2MgATP, 3 MgCl2, 3 DTT, 138 K-propionate, 5 Tris–HCl, pH 7.0. Enzymatic reactions were terminated by incubation in 15% trichloroacetic acid for 15 min at 20 °C. Proteins of fibre lysates were separated by SDS-PAGE as above followed by autoradiography or phosphorimaging (Amersham Biosciences, Freiburg, Germany).

The amount of 32P incorporated into the endogenous cTnI of fibres was determined using phosphorylated murine cTnI as a standard. Briefly, recombinant murine cardiac troponin complex (1.5 mg/ml) was incubated at 20 °C for 180 min with 0.1 MU/ml PKA (C-Subunit) in a [{gamma}32P]-ATP containing solution composed as the solution used with fibres. Based on previous results [23,25], cTnI was assumed to be bisphosphorylated under this condition. The enzymatic reaction was terminated by transferring the proteins to sample buffer for SDS-PAGE using a desalting spin column (Perbio, Bonn, Germany) and heating the solution to 95 °C for 3 min. The standard was serially diluted and loaded on the same gels as the fibre lysates. cTnI was quantified with Western Blot using an imaging system (Diana III CCD, Raytest, Straubenhardt, Germany) and Phoretix software. The amount of 32P incorporated into cTnI was quantified by phosphorimaging and densitometric analysis (Phoretix software). A phosphate incorporation into the cTnI standard of 2 mol/mol cTnI was taken for the calculation of the amount of phosphate incorporated into the endogenous cTnI of fibre samples.

2.7 Generation of plasmids and protein purification
The plasmids for murine cardiac troponin subunits were obtained as described [31]. Plasmids for human cardiac troponin subunits, pET11chcTnT, pET11chcTnI, and pET11chcTnC, were a generous gift from C. S. Redwood (Oxford, UK). The plasmid pET11chcTnIS22/23D was obtained by site-directed mutagenesis using the Quick Change site-directed mutagenesis kit (Stratagene, Heidelberg, Germany). The correctness of all plasmids was verified by sequencing (MWG Biotech, Ebersberg, Germany). Human and murine cardiac troponin subunits were expressed separately in E. coli, purified, and troponin complexes (Tn) were reconstituted as described [32]. The reconstituted cTn was frozen in liquid nitrogen and stored at –80 °C in (in mmol/L) 300 KCl, 20 DTT.

2.8 Exchange of endogenous cardiac troponin complex (cTn) with exogenous cTn
Fibres were incubated at 10 °C for 15 min in (in mmol/L) 132 NaCl, 5 KCl, 1 MgCl2, 10 Tris, 5 EGTA, 1 NaN3, pH 7.1 (20 °C) followed by incubation in the same buffer containing in addition 3 mg/ml human cTn for 180 min at 20 °C [27,32]. Fibres were then transferred to relaxing solution at 10 °C, and further protocols were applied.

2.9 Statistics
All data were calculated using SIGMA PLOT 4.0 (Systat Software Inc., Richmond, California, USA) and are given as mean±SEM. "n" in figures and tables refers to the number of animals. To analyse differences Student's paired or unpaired t-tests were used as appropriate. p<0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 References
 
Incubation of triton-skinned cardiac fibres with PP1c in Mn2+-containing buffer for 90 min significantly (p<0.001) increased the Ca2+ sensitivity of tension (pCa50) by 0.26 pCa-units (Fig. 1A, Table 1). A total of 16 fibres was incubated with PP1c or PP1c buffer, respectively, and was then divided into different treatment arms. Extending the incubation period to 180 min induced no further significant increase in Ca2+ sensitivity. Although the PP1c buffer itself also increased the Ca2+ sensitivity by 0.09 pCa-units (p<0.001, Fig. 1B, Table 1), the increase in the presence of PP1c was significantly (p<0.001) larger.


Figure 1
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  		Fig. 1 PP1c treatment increases the Ca2+ sensitivity of force generation. Force–pCa relations before (dashed lines, {circ}) and after (solid lines, {bigtriangleup}) incubation with PP1c (A, n=16) or with PP1c buffer (B, n=16). See Table 1 for contractile parameters.

 

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  		Table 1 Contractile parameters of skinned fibres before and after treatment with PP1c or PP1c buffer

 
PP1c treatment only slightly changed other contractile parameters (Table 1). Ca2+-activated force was reduced by ~10% (90 min PP1c). Force in absence of Ca2+ remained low (≤5% of maximal Ca2+-activated force) and Hill coefficients remained high (nH≥4). This confirms that the Ca2+ regulatory system and the cooperative mechanisms of activation retained their full function. Furthermore SDS-PAGE of fibre lysates showed no major changes in the protein pattern after the incubation sequences (see online supplement).

Incubation with PKA but not with PKA buffer completely reversed the PP1c-induced Ca2+ sensitisation (Fig. 2A, B), thereby decreasing the Ca2+ sensitivity by 0.26 pCa-units (p<0.001). The increase in Ca2+ sensitivity induced by Mn2+-containing PP1c buffer (Fig. 1B) was also completely reversed by PKA, suggesting that Mn2+ activated endogenous phosphatase(s) (Fig. 2C, D). In fibres which were not pre-treated with PP1c or PP1c buffer, the PKA-induced decrease in Ca2+ sensitivity was only 0.04 pCa-units (p<0.001, Fig. 2E, F). The Ca2+ sensitivity of fibres treated with PKA was similar irrespective of the pre-treatment (PP1c or PP1c buffer, see Fig. 2A and C). It was only slightly higher ({approx}0.04 pCa-units) than that in fibres treated with PKA only (Fig. 2E, Table 2). These results suggest that the PP1c- and PP1c buffer-induced increase in Ca2+ sensitivity can be ascribed to dephosphorylation of functionally relevant PKA-dependent phosphorylation sites.


Figure 2
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  		Fig. 2 PKA treatment reverses the Ca2+ sensitisation induced by PP1c. Force–pCa relations before (dashed lines, {circ}) and after (solid lines, {bigtriangleup}) incubation with PKA (left panel) or PKA buffer (right panel). See Table 2 for contractile parameters. A, B: PP1c pre-treated fibres (n=5). C, D: PP1c buffer pre-treated fibres (n=5). E, F: not pre-treated fibres (n=7).

 

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  		Table 2 Contractile parameters of skinned fibres before and after treatment with PKA or PKA buffer

 
The long incubation times with PKA and PP1c were chosen to allow for diffusion and to ascertain complete phosphorylation and dephosphorylation based on the kinetics of PKA-induced phosphorylation of cTnI in solution [23,25]. We note, however, that PKA-dependent phosphorylation of cTnI in intact tissue occurs rapidly, i.e. within 30 s [33].

To test whether the PP1c-induced Ca2+ sensitisation of contraction was associated with dephosphorylation of residual endogenous phosphorylation of cTnI, fibres were first incubated with PP1c and then back-phosphorylated with PKA with [{gamma}32P]-ATP as substrate. In PP1c pre-treated fibres 32P incorporation into cTnI was twofold larger than in fibres not pre-treated with PP1c (1.1±0.27 mol phosphate/mol cTnI compared to 0.6±0.09 mol phosphate/mol cTnI in control fibres; Fig. 3A). Incorporation of 32P into cMyBP was enhanced about 1.3-fold in PP1c pre-treated fibres (p<0.05).


Figure 3
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  		Fig. 3 PP1c treatment induces dephosphorylation of PKA sites on cTnI and cMyBP-C. A: Phosphate incorporation into cTnI. In back-phosphorylation experiments fibres were pre-incubated with or without PP1c and then incubated with PKA and [{gamma}32P]-ATP (see Methods). Bars represent means±SEM, n=3. B: All fibres were pre-incubated with PKA and [{gamma}32P]-ATP. Lane 1: Fibres homogenised immediately after PKA treatment. Lane 2: Fibres homogenised after 90 min PP1c buffer. Lanes 3 and 4: Fibres homogenised after 90 min and 180 min of PP1c treatment. A representative of 3 independent experiments is shown.

 
It was also investigated whether PP1c dephosphorylates myofibrillar proteins after incubation with exogenous PKA. In line with previous studies [7,8,11,14], incubation with PKA and [{gamma}32P]-ATP lead to incorporation of 32P into cTnI and cMyBP-C. Subsequent incubation with PP1c for 90 min and 180 min progressively dephosphorylated both proteins (Fig. 3B). This was associated with a concomitant increase in the Ca2+ sensitivity of tension by 0.24 pCa-units (90 min) and 0.35 pCa-units (180 min) from an initial pCa50-value of 5.39±0.02 in fibres pre-treated with PKA (n=3, data not shown). Interestingly, the PP1c-induced increase in Ca2+ sensitivity was larger in these experiments and appeared to be incomplete after 90 min, which is most likely due to the different experimental approach.

To further investigate whether the PP1c-induced increase in Ca2+ sensitivity was due to dephosphorylation of the predominant PKA phosphorylation sites at Ser-22 and Ser-23 of cTnI (human sequence), the endogenous cTn was exchanged with human cardiac troponin (hcTn) which contained either the phospho-mimicking mutant hcTnIS22/23D or wildtype hcTnI (hcTnIWT). Recombinant human troponin was chosen for these experiments because human and murine cTnI have slightly different mobilities on SDS-PAGE gels. This allowed to verify the troponin replacement after the mechanical experiments. In both groups the endogenous cTnI was consistently reduced by ~80% (Fig. 4B) which is in agreement with [34]. Besides the replacement of troponin subunits, no other obvious changes in the protein pattern were induced by the exchange protocol and subsequent incubations with PKA and PP1c (Fig. 4A).


Figure 4
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  		Fig. 4 Exchange of the endogenous murine cardiac Tn with human cardiac Tn. A: Protein patterns of unexchanged and troponin-exchanged fibres. B: The upper part of a 12.5% SDS-PAGE gel was stained with Sypro® Ruby to visualise cTnT, the lower part was subjected to Western blotting and cTnI detected. Lanes 1, 2: recombinant murine/human cTnI. Lanes 3, 4: unexchanged/troponin-exchanged fibres. C: Fibres exchanged with hcTn were incubated with PKA with [{gamma}32P]-ATP as substrate, and fibre lysates were subjected to Western Blotting. 32P incorporation was detected by phosphorimaging. Lane 1: fibre exchanged with hcTnS22/23D. Lane 2: fibre exchanged with hcTnS22/23D + treatment with PP1c. Lane 3: fibre exchanged with hcTnWT. Lane 4: fibre exchanged with hcTnWT + treatment with PP1c. All gels shown are representative of 3 independent experiments. D: 32P incorporation into cTnI (D), normalised to incorporation in not pre-treated skinned fibres; n=3. **No incorporation detected.

 
In the thus exchanged fibres, exogenous PKA phosphorylated hcTnIWT to a similar extent as cTnI in PP1c-treated, unexchanged fibres (Fig. 4C, D), and PKA-induced phosphorylation was not further enhanced by pre-treatment with PP1c. No 32P-incorporation could be detected into hcTnIS22/23D, indicating that PKA phosphorylates only Ser-22/Ser-23 under our experimental conditions.

For the mechanical experiments, exchanged fibres were first incubated with PKA, then with PP1c buffer followed by PP1c. Force–pCa relations were obtained after each incubation step. The pCa50-value after PKA treatment was 5.41±0.01 in hcTnIS22/23D and 5.45±0.02 in hcTnIWT containing fibres. Interestingly, PP1c buffer increased the Ca2+ sensitivity by 0.08 pCa-units in both groups indicating that the buffer-induced increase in Ca2+ sensitivity was not due to dephosphorylation of cTnI at Ser-22 and Ser-23. A further significant increase in Ca2+ sensitivity by 0.12 pCa-units was induced by PP1c in hcTnIWT but not in hcTnIS22/23D containing fibres (Fig. 5, Table 3). Thus, although the PP1c-induced decrease in Ca2+ sensitivity was less than expected based on the experiments with unexchanged fibres, the overall increase in Ca2+ sensitivity (buffer+PP1c) was similar.


Figure 5
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  		Fig. 5 Exchange of the endogenous murine cardiac Tn with human cardiac Tn (hcTnIS22/23D) abolishes the Ca2+-sensitising effect of PP1c. A, B: The endogenous cardiac Tn was exchanged with recombinant human cardiac Tn reconstituted either with hcTnIS22/23D (A, n=5) or with hcTnIWT (B, n=5). The exchanged fibres were treated with PKA (dotted lines, {circ}), followed by sequential incubation with PP1c buffer (dashed lines, {bigtriangleup}) and PP1c (solid lines, {square}) Force–pCa relations were recorded after each incubation step. See Table 3 for contractile parameters. C: Scheme of the experimental protocol.

 

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  		Table 3 Contractile parameters of skinned fibres exchanged with hcTn

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 References
 
The present study demonstrates that phosphorylation of sarcomeric proteins can persist in triton-skinned murine cardiac muscle in a functionally relevant manner as evidenced by the significant increase in Ca2+ sensitivity of contraction (0.26 pCa-units) following treatment with Mn2+-dependent PP1c. The PP1c-induced increase in Ca2+ sensitivity was much larger than that previously reported for incubation with alkaline phosphatase which only induced a small increase in Ca2+ sensitivity by 0.05 [13] or 0.1 pCa-units [12], or no increase [20]. In PP1c pre-treated fibres the Ca2+-desensitising effect of PKA was significantly larger than in not pre-treated fibres, completely reversing the PP1c-induced Ca2+ sensitisation. As pCa50-values were similar following PKA treatment, irrespective of whether the fibres had been pre-treated with Mn2+-PP1c or not, we hypothesised that the PP1c-induced Ca2+ sensitisation is due to dephosphorylation of PKA phosphorylation sites.

The conclusions drawn from the mechanical experiments were supported by the finding that the shifts in pCa50-values were associated with appropriate changes in cTnI and cMyBP-C phosphorylation. Thus, PKA induced a two-fold larger increase in phosphorylation of cTnI after pre-treatment with PP1c. Similar stoichiometries were observed by Winegrad and co-workers in hyperpermeable rat myocardium [35]. Incubation with PP1c removed PKA-induced 32P incorporation from both proteins (cf. Fig. 3). We do not know at present why the difference in PKA-induced phosphorylation between PP1c pre-treated and not pre-treated fibres was less for cMyBP-C than for cTnI. Possibly basal cMyBP-C phosphorylation is less, and therefore cMyBP-C is significantly phosphorylated by PKA without PP1c pre-treatment, or PP1c dephosphorylates cMyBP-C less effectively. While this study was under review, it was reported that Mn2+-PP1c dephosphorylates cardiac titin, the third PKA substrate in the sarcomere [4]. Taken together these data suggest that Mn2+-PP1c dephosphorylates all sarcomeric substrates of PKA.

This may appear surprising because cTnI is a substrate of type 2A phosphatases [2,19] but Mn2+-dependent catalytic subunit of PP1 has different substrate specificities than PP1 under physiological conditions [22]. We cannot exclude the possibility that it also dephosphorylates the regulatory light chains of myosin as these are dephosphorylated by PP1 [21]. We regard this as unlikely because (a) without the targeting subunit the activity of the catalytic PP1 subunit towards dephosphorylation of RLC is low, (b) in unpublished results we found that RLC was not phosphorylated in our skinned fibre preparations (cf. also [36]), and (c) because of the opposing effects of dephosphorylation of cTnI and RLC we would not expect that PKA treatment exactly reverses the PP1c-induced shift in pCa50 if RLC was dephosphorylated. Of interest to our study is that the PP1c-induced shift in pCa50 observed here is of similar magnitude as that observed after activation of PP2A in intact cardiomyocytes [19].

The experiments with fibres exchanged with the phospho-mimicking mutant of human cTnI (hcTnIS22/23D) suggest that the Ca2+-sensitising effect of PP1c can be ascribed at least in part to dephosphorylation of Ser-22/-23 because (1) besides cMyBP-C only hcTnIWT but not hcTnIS22/23D was phosphorylated by PKA and (2) PP1c reversed the PKA-induced decrease in Ca2+ sensitivity in fibres exchanged with hcTnIWT only. It is not clear at present why the PP1c-induced Ca2+ sensitisation was less in hcTnIWT-exchanged fibres compared to unexchanged fibres. This may reflect a limitation of exchanged fibres which, however, have been a valuable tool to study regulation of contraction by different troponins [15,27,32]. We note that FMAX and Hill coefficients were lower in exchanged fibres, indicating that they were not exactly identical to unexchanged fibres. This may be due to (1) loss of proteins – we have no evidence for this –, (2) the recombinant proteins may not exactly substitute for the native proteins, (3) the endogenous troponin complex may not be completely replaced by exogenous hcTn [34].

Interestingly we found that in both, fibres containing hcTnIWT and hcTnIS22/23D, the Mn2+-containing buffer increased the Ca2+ sensitivity to a similar extent as in unexchanged fibres. Rather than this being an oxidation effect (cf. Methods) we assume that Mn2+ itself activates an endogenous phosphatase which dephosphorylates PKA substrates because, in unexchanged fibres, the buffer effect was completely reversed by PKA. Whether or not the Ca2+ sensitisation induced by the putative endogenous phosphatase and exogenous PP1c are truly additive is not known at present. Since the Mn2+-induced Ca2+ sensitisation was not associated with dephosphorylation of cTnI (cf. Fig. 3), our results give rise to the interesting possibility that dephosphorylation of sarcomeric PKA substrates other than cTnI such as cMyBP-C may contribute to the PP1c-induced Ca2+ sensitisation. Further experiments are required to elucidate this point.

Using cMyBP-C deficient mice it was recently proposed that cMyBP-C together with cTnI phosphorylation is required for PKA effects on myofilament activation [6], however, it is not known whether this requires the protein itself or its phosphorylation. It will be interesting to see whether exchanging fibres with the phospho-mimicking mutant of cTnI and manipulating the phosphorylation status of cMyBP-C using Mn2+-PP1c and PKA allows to clarify this point.

A limitation of our study is that we did not investigate whether the effects of other protein kinases such as PKC are affected by PP1c. Hence we do not know whether PKC sites are dephosphorylated by PP1c. However, we do not believe that dephosphorylation of PKC sites contributes to the observed PP1c-induced Ca2+ sensitisation. If this were the case, then PKA, which does not cross-phosphorylate PKC sites on cTnI [37] or on troponin T [2], should not quantitatively reverse the effect of Mn2+-PP1c.

In summary our results show that cTnI phosphorylation can persist in skinned myocardium to a functionally relevant degree. We further provided evidence that Mn2+-PP1c, which is commercially easily available with sufficient activity for the use in skinned fibres, dephosphorylates PKA sites on cTnI. Furthermore our results support the hypothesis that phosphorylation of substrates other than cTnI, e.g. cMyBP-C, may contribute to the PKA-dependent modulation of contraction.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2007.01.019.


   Acknowledgements
 
This work was supported by the DFG (SFB 612-A2), Koeln Fortune (Faculty of Medicine, Cologne), and the Center for Molecular Medicine, University of Cologne.


   Notes
 
Time for primary review 17 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 

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