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(Circulation Research. 2007;100:408.)
© 2007 American Heart Association, Inc.

Cellular Biology

p38-MAPK Induced Dephosphorylation of -Tropomyosin Is Associated With Depression of Myocardial Sarcomeric Tension and ATPase Activity

Susan Vahebi^*, Asuka Ota^*, Manxiang Li, Chad M. Warren, Pieter P. de Tombe, Yibin Wang, R. John Solaro

From the Department of Physiology and Biophysics (S.V., C.M.W., P.P.d.T., R.J.S.), Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago; Departments of Anesthesiology and Medicine (A.O., M.L., Y.W.), David Geffen School of Medicine, University of California at Los Angeles.

Correspondence to R. John Solaro, PhD, Department of Physiology and Biophysics, (M/C 901), College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-7342. E-mail solarorj{at}uic.edu

	Abstract

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Our objective in work presented here was to understand the mechanisms by which activated p38 MAPK depresses myocardial contractility. To test the hypothesis that activation of p38 MAPK directly influences sarcomeric function, we used transgenic mouse models with hearts in which p38 MAPK was constitutively turned on by an upstream activator (MKK6bE). These hearts demonstrated a significant depression in ejection fraction after induction of the transgene. We also studied hearts of mice expressing a dominant negative p38 MAPK. Simultaneous determination of tension and ATPase activity of detergent-skinned fiber bundles from left ventricular papillary muscle demonstrated a significant inhibition of both maximum tension and ATPase activity in the transgenic-MKK6bE hearts. Fibers from hearts expressing dominant negative p38 MAPK demonstrated no significant change in tension or ATPase activity. There were no significant changes in phosphorylation level of troponin-T3 and troponin-T4, or myosin light chain 2. However, compared with controls, there was a significant depression in levels of phosphorylation of -tropomyosin and troponin I in fiber bundles from transgenic-MKK6bE hearts, but not from dominant negative p38 MAPK hearts. Our experiments also showed that p38 MAPK colocalizes with -actinin at the Z-disc and complexes with protein phosphatases (PP2, PP2ß). These data are the first to indicate that chronic activation of p38 MAPK directly depresses sarcomeric function in association with decreased phosphorylation of -tropomyosin.

Key Words: heart failure • myofilaments • protein phosphatase • tropomyosin kinase

	Introduction

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The response of the myocardium to stressors that signal hypertrophy, dilation, and failure represents integrated effects of pathways regulating myocyte growth, death, and contractility.¹ A potentially important signaling cascade involves the mitogen activated protein kinase, p38 MAPK. The p38 MAPKs are activated by G-protein coupled receptors, as well as chemical, physical, osmotic, and radiation stresses.^2,3 In vivo studies demonstrated that p38 MAPK activity is increased during hypertrophy induced by pressure overload, and is potentially important in transduction of mechanical signals.³ Further evidence for p38 MAPK as a stress mediating factor has come from the observation of induction of apoptosis, hypertrophy, and the hypertrophic marker ANF in neonatal cardiac myocytes over-expressing MKK6bE and MKK3bE, the upstream activators of p38 MAPKs.^4,5,6

Activation of p38 MAPK activity in intact hearts also resulted in early lethal cardiomyopathy characterized by impaired contractility.⁷ Acute activation of p38 MAPK with arsenite has also been demonstrated to depress tension generated in detergent extracted cardiac myocytes.⁸ Moreover, prolonged activation of p38 MAPK activity is also associated with a negative inotropic effect that occurs without alterations in cellular Ca²⁺ fluxes.⁹ Although these studies suggest a general linkage between p38 MAPK activation and depressed contractility at the level of the sarcomere, the underlying cellular mechanism remains unclear.

In the present experiments we tested the hypothesis that activation of p38 MAPK alters contractility by a mechanism involving an altered phosphorylation and mechano-energetics of cardiac myofilaments. Our approach involved the use of transgenic (TG) mouse models with either conditional expression of MKK6bE, an upstream activator of p38^9,10 or a dominant negative mutant of p38.¹¹ Our data show that compared with controls, activation of p38 MAPK in TG-MKK6bE hearts resulted in depression of myofilament maximum tension and ATPase activity with no effect on the ratio of tension to ATPase activity (tension cost). The depressed tension generation was associated with dephosphorylation of -tropomyosin (-Tm) and troponin I (TnI) in TG-MKK6bE hearts. Our data also demonstrated p38 MAPK localization to the cardiac sarcomere structure in complex with protein phosphatases. To our knowledge this is the first evidence that dephosphorylation of -Tm may be important as a determinant of cardiac function and may play a role in p38 mediated contractile dysfunction in stressed myocardium.

	Materials and Methods

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Animal Model and Mechano-Energetic Measurements on Fiber Bundles
We studied preparations from 3 month nontransgenic (NTG) control mouse hearts, and from age matched TG hearts expressing constitutively-activated MKK6bE, which was generated by replacing Ser 207 and Thr 211 with Glu as described previously.⁹ The transgene was induced by tamoxifen for 2 to 4 weeks as described.¹⁰ We also studied TG hearts expressing a dominant negative p38 MAPK (DN-p38 [] MAPK) in which p38 was mutated at Thr180 with Ala and Tyr182 with Phe, as described previously.¹¹ Fiber bundles from left ventricular papillary muscle were dissected and prepared as described previously.^12,13,14 We measured isometric tension and actomyosin Mg-ATPase activity simultaneously in skinned fiber bundles at sarcomere length 2.2 µm as described previously.^12.13,14 The isometric tension and ATPase activity were determined simultaneously at 20°C in the presence of variable Ca²⁺ concentrations as described.¹⁴ Only fiber bundles retaining 95% of their initial maximum tension after the series of measurements were retained for analysis. Detailed electrophoresis and staining methods are in the online data supplment available at http://circres.ahajournals.org.

Analysis of Proteins by Gel Electrophoresis, Coimmuno-Precipitation, and Mass Spectrometry
We used several techniques including 1D and 2D, gel electrophoresis^15–17 and NEIEF¹⁸ to analyze protein content and phosphorylation¹⁹ in left ventricular tissue and skinned fiber bundles. In coimmuno-precipitation experiments aimed at determination of protein complexed with p38 MAPK, we used FLAG tagged DN-p38[]MAPK TG mice and the FLAG antibody. Proteins eluted in the co-IP were analyzed by MS/MS.²⁰ These methods are presented in detail in the online data supplement available at http://circres.ahajournals.org.

Data Analysis and Statistical Evaluation
Individual tension-Ca²⁺ and ATPase-Ca²⁺ relations were fit to a modified Hill equation using Graphpad Prism Software. Tension cost was derived by linear regression of the tension-ATPase relationship. Statistical evaluation was by one way ANOVA and unpaired t-test as appropriate; P<0.05 was considered significant. Data obtained from 1-D and 2-D electrophoresis were analyzed by two tailed t-test with significance set at P<0.05.

	Results

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Depression of Cardiac and Myofilament Function With Activation of p38 MAPK
Table 1 summarizes the significant depression in ejection fraction after induction of the transgene. Data in the online supplement summarize the progressive changes that occurred in NTG and TG-MKK6bE mice in heart weight/body weight and atrial weight/body weight. The transgene expression and specific activation of p38 MAPK activity was confirmed by RT-PCR and Western blotting using phospho-specific antibodies as previously described.²⁰ To determine whether this depression in function involves direct modifications at the level of the sarcomere, we compared Ca²⁺ activation of tension and ATPase rate in detergent extracted fiber bundles from hearts of NTG controls and TG- MKK6bE mice (Figure 1 A–C). Data plotted in Figure 1A indicate that activation of p38 MAPK in this TG mouse model resulted in a significant depression in myofilament maximum tension. Activation of p38 MAPK also induced a significant depression in maximum ATPase activity (Figure 1B). Figure 1C shows a plot of tension as a function of the simultaneously determined ATPase activity. The slope of this relation, which provides a measure of the energetic cost of tension (economy), was the same for controls and TG- MKK6bE fibers. Table 2 summarizes the parameters obtained from the experiments. Data in Table 2 also show that we found no significant differences in either the maximum tension or ATPase rate or response to Ca²⁺ between skinned fibers from hearts of NTG mice and mice expressing dominant negative p38 MAPK mice. Although we did these experiments with the idea of demonstrating an increased myofilament force and ATPase rate, these results indicate that basal activity of p38 MAPK is likely to be low in these mice under our experimental conditions.

View this table: [in this window] [in a new window]   Table 1. Progressive Loss of Contractile Function in TG-MKK6bE Hearts

View larger version (20K): [in this window] [in a new window]   Figure 1. Comparison of response to Ca2+ and mechano-energetic characteristics of skinned fiber bundles from hearts of NTG () and TG-MKK6bE () mice. A, Steady-state isometric tension as a function of pCa. B, Actomyosin Mg-ATPase activity determined simultaneously. C, Tension cost as determined from the slope of the relation between ATPase activity and tension. Data are given as means±SEM (*P<0.05) for n=10 fiber bundles from left ventricular papillary muscles of 4 separate hearts. See Table 2 for summary of parameters.

View this table: [in this window] [in a new window]   Table 2. Parameters Describing Mechanical and Energetic Characteristics of Skinned Fiber Bundles of Left Ventricular Papillary Muscles From NTG Controls, TG-MKK6bE, and TG-DN[]p38 MAPK

Activation of p38 MAPK Decreases Phosphorylation of -Tm and TnI
One possible mechanism by which the signaling process engaging p38 MAPK might affect myofilament activity is by altered levels of protein phosphorylation. Figure 2 (A–D) shows results of 12%–2D IEF/SDS analysis of left ventricular samples from TG-MKK6bE (A and C) and from NTG controls (B and D). In the gels shown in Figure 2, total protein from TG-MKK6bE (Figure 2A) and NTG (Figure 2B) heart samples was visualized with SYPRO Ruby stain, which revealed no differences between TG and NTG hearts in total protein for Tm (spots 1 and 2), myosin light chain 2 (MLC2) (spots 3 and 4), MLC1 (spot 5), troponin-T3 (TnT3) (spots 6 and 7) and TnT4 (spots 8 and 9). Comparison of the density of phospho-protein staining of spot 1 in Figure 2C and 2D, visualized with PRO-Q Diamond Stain, demonstrated that the phosphorylated form of Tm was reduced in TG hearts compared with NTG controls. The density of staining of phosphorylated forms of MLC2 (spot 3), TnT3 (spot 6) and TnT4 (spot 8) was not significantly different between the TG (Figure 2C) and NTG hearts (Figure 2D). Data in Figure 3A summarize our quantification of phosphorylated myofilament proteins and indicate that elevated p38 MAPK activity is associated with a significant de-phosphorylation of -Tm. Although earlier studies⁹ indicated no change in levels of TnI phosphorylation with activation of p38 MAPK. We also measured these levels using a recently developed NEIEF method.¹⁸ As summarized in Figure 3B, there was a small but significant decrease in the level of TnI phosphorylation in TG-MKK6bE hearts compared with controls. This relatively small depression in TnI phosphorylation was not sufficient to alter the pCa₅₀ for activation of force or ATPase rate (Table 2). Moreover, a decrease in TnI phosphorylation would be expected to either increase tension or have no effect.²¹ Thus our data indicate that the depression in Tm phosphorylation may be the dominant factor in the depression of tension induced by activation of p38 MAPK.

View larger version (54K): [in this window] [in a new window]   Figure 2. Two dimensional 12% gel electrophoresis analysis of proteins from NTG and TG-MKK6bE hearts. SYPRO Ruby stained gels for total protein from left ventricular of TG-MKK6bE hearts (A), and NTG control hearts (B). Pro Q Diamond stained gels specific for phospho-proteins of TG-MKK6bE hearts (C), and NTG hearts (D). Phosphorylated form of -Tm (spot 1), nonphosphorylated form of -Tm (spot 2), phosphorylated myosin light chain-2 (spot 3), nonphosphorylated myosin light chain-2 (spot 4), myosin light chain-1 (spot 5), phosphorylated TnT3 (spot 6), nonphosphorylated TnT3 (spot 7); 8, phosphorylated TnT4 (spot 8), nonphosphorylated troponin T4 (spot 9).

View larger version (19K): [in this window] [in a new window]   Figure 3. Comparison of relative levels of phosphorylation of myofilament proteins from NTG and TG-MKK6bE hearts. A, Values are given as means±SEM (*P<0.05) n=3 for determinations from 2-D gel electrophoresis and demonstrate a significant reduction in phosphorylation of Tm. B, Values are given as means±SEM (*P<0.05) n=4 for determinations from NEIEF Western blot and demonstrate a significant reduction in phosphorylation of TnI.

Activation of p38 MAPK activity has been shown to activate transcriptional factors with functional changes during onset or progression of heart failure.²² To determine whether p38 MAPK induced Tm isoform switching, we probed the samples from TG -MKK6bE and NTG hearts for ß-Tm. Our analysis indicated exclusive expression of the -Tm isoform in these hearts (Data Supplement). In view of evidence that p38 MAPK activity has been identified as a key activator of proteases during the early stages of apoptosis,²³ we tested whether the depression in tension in skinned fiber bundles from the TG-MKK6bE hearts could be attributed to cleavage of cTnT. We have reported that activation of caspase-3 resulted in a truncation of TnT and a depression of maximum tension and ATPase activity in skinned fibers.²⁴ However, our Western blot analysis did not show truncation of cTnT.

Activation of p38 MAPK Does Not Induce Translocation of HSP 25/27
Acute activation of p38 MAPK by arsenite in cardiac myocytes has been reported to induce a decrease in maximum tension associated with translocation of HSP 27.⁸ To test whether depression of maximum force in vivo in our TG-MKK6bE mouse model is associated with translocation of HSP25/27, we probed the heart samples from the TG and control mouse for the presence of HSP25/27 in Triton X-100 skinned fiber bundles and nonskinned fibers. We found no difference in the level of HSP25/27 between these preparations from either TG MKK6bE or NTG hearts (data supplement).

M-Protein and Protein Phosphatases 2C- and 2C-ß Complex With p38 MAPK in Heart Muscle Cells
To provide more mechanistic insights into p38 mediated regulation of contractile function, we analyzed proteins that interact with p38 MAPK in hearts by coimmuno-precipitation followed by molecular analysis by mass spectrometry (MS). To take advantage of the property of enhanced binding affinity by a kinase dead mutant, we expressed a FLAG tagged p38 kinase dead mutant in mouse heart. As described in the data supplement, the p38 MAPK interacting proteins were isolated from an anti-FLAG affinity column and the protein complexes were separated on SDS gels. The same procedure was applied to NTG heart samples. Compared with these controls, we identified several specific protein species in the p38 MAPK complex by LC/MS/MS (data supplement). Among them are M-protein and two protein phosphatases (PP2C- and PP2C-ß); each was matched with multiple peptide sequences and highly specific MOWSE scores (Figure 4). M-protein was identified from one of the high molecular weight bands with a MOWSE score of 1,350. M-protein is a 165 kDa protein that belongs to immunoglobulin super-group along with myomesin and titin.²⁵ M-protein, which is expressed highly in fast skeletal and heart muscle cells, is localized in the center of the A- band and thought to be important in sarcomeric organization by linking myosin thick filaments to elastic molecules, such as titin. Among other proteins that were coimmuno-precipitated were magnesium-dependent protein phosphatases, PP2C- and PP2C-ß (Figure 4). Both PP2C- and PP2C-ß were shown by others to negatively regulate the MAPK pathway, including p38 and JNK.²⁶ Proteins^26–30 identified in our analysis are listed in Table 3. Although more extensive studies are required, this finding along with changes in Tm phosphorylation status in TG-MKK6bE hearts raises the possibility that PP2C isoforms are part of p38 MAPK signaling complex, which is targeted to sarcomeres and contributes to the reduction of Tm phosphorylation level.

View larger version (122K): [in this window] [in a new window]   Figure 4. MS identification of M-protein, PP2C-, and PP2C-ß. A-C, Peptide sequences of M-protein, PP2C-, and PP2C-ß are shown; peptides that correspond to the identified peptides from MS/MS are highlighted. Search parameters were set to account for oxidation, fragment mass tolerance was set set to ±0.8 Da, and peptide mass tolerance was set to ±2 Da. See Table 3 for a summary of identified proteins from p38 complex in heart with number of matched peptides and total MOWSE score. MOWSE score of over 41 has probability value <0.05.

View this table: [in this window] [in a new window]   Table 3. Cardiac Proteins in Complex With 38 MAPK Identified by MS/MS

Localization of p38 MAPK on Sarcomeres of Heart Muscle Cells
The impact of p38 MAPK activation on force generation may involve direct interactions with sarcomere proteins as supported by mass-spectrometry studies. By immuno-fluorescent labeling of heart tissue from either control or TG mice with targeted expression of Flag-tagged p38[] mutant protein (Figure 5), we identified p38[] MAPK protein to be distributed in a striated pattern with significant colocalization with -actinin located at Z-disc.

View larger version (77K): [in this window] [in a new window]   Figure 5. Intracellular localization of p38 in adult heart. Confocal images of immuno-fluorescent labeling of p38 (red) and -actinin (green) and merged images (right panels) from ventricular sections of NTG and TG-p38[]DN hearts are shown as indicated.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Our data are the first to provide evidence that specific activation of p38 MAPK directly induces a depression of myofilament maximum force and ATPase rate. Data presented here are also the first to demonstrate altered cardiac and myofilament function occurs together with a change in Tm phosphorylation in association with a sarcomeric localization of p38 MAPK possibly in complex with protein phosphatases. These results significantly extend earlier data indicating a depression in myofilament activity associated with activation of p38 MAPK using a transgenic approach⁹ or chemical stimulation using arsenite.⁸ Results from both of these approaches indirectly indicated a reduction in tension generation by myofilaments, but there were no direct measurements of myofilament function. Inasmuch as our experiments were performed under identical conditions for controls and TG myofilaments, our data demonstrate that the depression in cardiac function occurs at the level of the sarcomeres.

There are several lines of evidence that support our hypothesis that dephosphorylation of Tm might affect tension. Tm is critical in transmitting the signal initiated by Ca²⁺ binding to cTnC to actin. Tm binds to cTnT and cTnI as well as actin. Moreover Tm binds to itself forming a head to tail polymer extending along the actin helix. At least one phosphorylation site for Tm at Ser 283 has been identified.³¹ This site of phosphorylation is within the C-terminal region, which participates in Tm polymerization and that interacts strongly with the N-terminal region of cTnT.²¹ Ser 283 phosphorylation has been reported to strengthen the binding of Tm to TnT and to the Tn complex.³² Compared with de-phosphorylated controls, thin filaments reconstituted with phosphorylated Tm demonstrate a significant increase in actin activated myosin S-1 ATPase activity.³² Thus, although effects of phosphorylation of Tm on force generation in the sarcomere lattice have not been defined, existing in vitro data indicate that phosphorylation may enhance tension. Moreover results of studies of myofilaments from transgenic mice expressing either ß-Tm or mutant forms of -Tm strongly indicate that charge changes introduced into the C-terminus of -Tm, as occurs with phosphorylation, affect myofilament force and ATPase rate and cardiac function.^33,34

A depression in Tm phosphorylation could result from either an inhibition of kinase or a stimulation of phosphatase activity. Tm kinase expression is high early in cardiac development but wanes with maturity.³⁵ Our results showed that Tm is 40% phosphorylated in the NTG adult mouse heart, which agrees with earlier data.³⁵ Although a downregulation of expression or activity of Tm kinase might occur, our finding that p38 MAPK exists in complex with PP2C- and PP2C-ß strongly indicates that activation of a phosphatase is responsible for our finding of a depression in Tm phosphorylation. Activation of phosphatases has previously been associated with the p38 MAPK pathway. PP2C has been demonstrated to interact with phosphorylated p38 MAPK in mammalian cells and to inhibit activation of p38 MAPK following an environmental stress.^26,27 It has also been reported³⁶ that activation of cardiac p38 MAPK through Gi-guanylyl cyclase resulted in activation of protein phosphatase 2A (PP2A), and that PP2A may directly interact with p38 MAPK in human platelets.³⁷

An important question is: what is the physiologic and patho-physiological significance of p38 MAPK signaling mediated depression of tension? Pathways upstream of p38 MAPK are well recognized to be significant in the long-term regulation of cardiac function and cardiac remodeling in stress responses.³⁸ Our data indicate that together with remodeling and apoptosis stimulated by p38 MAPK activation, there is also a depression of cardiac reserve as a result of inhibition of maximum tension generation and ATPase activity. Whether this change is adaptive or maladaptive is not certain. A depression in ATPase rate would spare energy consumption during stress, but also, in the long term, potentially lead to a vicious cycle of growth, remodeling, and depressed contractility. Inhibition of p38-MAPK with SB-203580 has been reported to worsen function in hearts subjected to ischemia and reperfusion.^38,39 Yet the role of p38-MAPK in ischemia/reperfusion injury is controversial.^40,41,42 Sumida et al⁴³ reported a blunting of sarcolemma damage and a cardio-protective effect that was enhanced over that of perfusion with SB-203580 alone, when they transiently inhibited force generation with butane dione monoxime during treatment with the inhibitor. They hypothesized that the sudden reversal of the depression in cardiac function associated with p38 MAPK inhibition may produce a strain on cellular structures and induce mechanical damage.

These results from studies of pathological models and inhibitors emphasize the importance of understanding the mechanism of the negative inotropic effects of p38 MAPK activation. Our data show that there is a direct effect of activation of p38 MAPK on myofilament tension and ATPase rate that is independent of translocation of heat shock protein and proteolysis, and likely to be dependent on activation of a phosphatase that induces a dephosphorylation of Tm. Although a detailed mechanism for de-phosphorylation of -Tm in hearts with activation of the p38 MAPK signaling pathway has yet to be demonstrated, on the basis of data presented here, it will be important to re-examine some of the models described above for alterations in -Tm phosphorylation and to more thoroughly understand relations between Tm phosphorylation and sarcomere mechanics. Our finding of a sarcomere localization of p38 MAPK also indicates the importance of a precise definition of binding partners for p38 MAPK in the sarcomere. Experience with other sarcomeric proteins has shown that other kinases have points of contact with Z-disc proteins such as -actinin that sense strain on the sarcomere and may be mobilized by ventricular stress associated with elevated end diastolic volume.⁴⁴ An exiting new direction in research on p38 MAPK is investigation of its role in stabilizing sarcomeric structure by manipulating and transmitting mechanical stress via other signaling molecules.

	Acknowledgments

 
Sources of Funding

This research was supported in part by NIH grants PO1 HL62426 (R.J.S.,P.P.d.T.), RO1 HL64035 (R.J.S), HL 062311 (Y.W.), and an AHA Scientist Development Grant (T.K.). S.V. was supported by an AHA pre-doctoral fellowship and NIH training grant T32 HL07692–14-15. Y.W. is a current Established Investigator of AHA. These studies were submitted in partial fulfillment for the degree of doctor of philosophy (S.V.).

Disclosures

None.

	Footnotes

 
*Both authors contributed equally to this work.

Original received July 15, 2005; resubmission received October 25, 2006; revised resubmission received December 18, 2006; January 5, 2007.

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