Ischemia/reperfusion-induced myosin light chain 1 phosphorylation increases its degradation by matrix metalloproteinase-2

Summary

Degradation of myosin light chain 1 (MLC1) by matrix metalloproteinase-2 (MMP-2) during myocardial ischemia/reperfusion (I/R) injury has been established. However, the exact mechanisms controlling this process remain unknown. I/R increases the phosphorylation of MLC1, but the consequences of this modification are not known. We hypothesized that phosphorylation of MLC1 plays an important role in its degradation by MMP-2. To examine this, isolated perfused rat hearts were subjected to 20 min global ischemia followed by 30 min of aerobic reperfusion. I/R increased phosphorylation of MLC1 (as measured by mass spectrometry). If hearts were subjected to I/R in the presence of ML-7 (a myosin light chain kinase (MLCK) inhibitor) or doxycycline (a MMP inhibitor) an improved recovery of contractile function was seen compared to aerobic hearts and MLC1 was protected from degradation. Enzyme kinetic studies revealed an increased affinity of MMP-2 for the phosphorylated form of MLC1 compared to non-phosphorylated MLC1. We conclude that MLC1 phosphorylation is important mechanism controlling the intracellular action of MMP-2 and promoting the degradation of MLC1. These results further support previous findings implicating posttranslational modifications of contractile proteins as a key factor in the pathology of cardiac dysfunction during and following ischemia.

Introduction

Myosin light chain 1 (MLC1), also known as myosin essential light chain is an integral structural component of the actomyosin cross-bridge, but it also plays a role in force development and muscle contraction (for review see [1–3]). The functional importance of MLC1 is further implicated by the recent identification of several missense mutations in the human ventricular MLC1 isoform, which are associated with hypertrophic cardiomyopathy [1]. However, the mechanism underlying the role of MLC1 in heart disease is not known.

Matrix metalloproteinase-2 (MMP-2) is a metalloproteinase initially described to be involved in both physiological and pathological remodeling/degradation of extracellular matrix components [4]. Over a decade ago studies also revealed a novel action of MMP-2 at the cellular level, with a time frame of action of minutes rather than days [5, 6].

The novel actions of MMP-2 include the intracellular degradation of heart contractile proteins, such as troponin I [7], titin [8] and MLC1 [9–11], during ischemia reperfusion (I/R) injury. This intracellular substrate cleavage action of MMPs, and their role in biology and pathology, have been recently reviewed by Cauwe and Opdenakker [12].

Although cardiac MLC1 phosphorylation has been previously described [13], the functional implications of this modification remain unknown. Our previous studies showed that post-translational modification of MLC1 during hypoxia-reoxygenation (I/R) resulted in a decrease of MLC1 level and was also associated with an increase in MMP-2 activity [10]. Based on these observations we hypothesize that phosphorylation of cardiac MLC1 triggered by I/R may lead to an increase in its degradation by MMP-2, and that this degradation may underlie contractile dysfunction of I/R hearts.

In this study we show that increased phosphorylation of the MLC1 during I/R in isolated perfused rat hearts is associated with a decrease in cardiac function. Furthermore, inhibition of this phosphorylation, secondary to inhibiting myosin light chain kinase (MLCK), results in an improved mechanical function of the myocardium during reperfusion. Moreover, we show that the in vitro phosphorylation of MLC1 increases the affinity of MMP-2 for MLC1. As a result, establishing the molecular mechanisms responsible for the degradation of contractile proteins in the development of heart injury, triggered by I/R or other conditions that cause oxidative stress, may lead to the development of new therapeutic strategies aimed at preserving contractile function of the heart.

Results

MLC2 in I/R hearts

In the heart, myosin regulatory light chain (MLC2) is the major target of MLCK. Therefore, it was important to assess the level of MLC2 as well as its phosphorylation in the hearts subjected to I/R. MLC2 levels, as well as its phosphorylation status, were measured in control hearts, in the hearts subjected to I/R, and in I/R hearts perfused with MLCK inhibitor, ML-7. As shown in Fig. 2a and 2b, no changes in MLC2 protein levels measured either by 2-dimensional electrophoresis (2-DE) or immunobloting were observed in any of the analyzed heart groups. Also, as analyzed by mass spectrometry (data not shown), there were no visible changes in the MLC2 phosphorylation in any of the experimental groups.

Figure 2.

Densitometric analysis of the levels of cardiac MLC2 determined by 2-DE (a) and by immunoblotting (b) in heart homogenates (n=4/group for 2-DE and n=3/group for immunoblotting).

Although ML-7 is considered a selective inhibitor of MLCK, there are reports showing that ML-7 inhibits other kinases [14]. According to the supplier information the K_i of ML-7 is approximately 70 times higher for protein kinase A (21 µM) and 140 times higher for protein kinase C (42 µM) than for MLCK (0.3 µM). To rule out potential effects of ML-7 on PKC, we measured PKC activity in the 3 experimental groups. PKC activity was significantly increased in response to I/R. Five micromolar of ML-7 did not affect PKC activity in hearts subjected to I/R (Fig. 3a). We then confirmed the inhibitory effect of ML-7 on MLCK activity (Fig. 3b). While I/R did not change MLCK activity, administration of 5 µM of ML-7 to I/R hearts significantly reduced MLCK activity by more than 60 % (Fig. 3b).

Figure 3.

Effect of 5 µM of ML-7 on protein kinases in isolated rat hearts. (a) Protein kinase C activity in isolated perfused rat hearts under aerobic conditions or subjected to I/R in the presence or absence of the myosin light chain kinase inhibitor ML-7. (n=4). (b) Myosin light chain kinase activity in isolated perfused rat hearts under aerobic conditions or subjected to I/R in the presence or absence of the myosin light chain kinase inhibitor ML-7 (n=4–6).

*p < 0.05 vs. aerobic control; ^#p < 0.05 vs. I/R.

Because we did not observe any changes in MLC2 protein levels or its phosphorylation status in any of the experimental groups, and because of ML-7 concentration used in the assay (5 µM in ex vivo conditions, that is below the K_i for other kinases), we assume that in our experimental model MLCK was acting on MLC1 as a main target.

Inhibition of MLCK-dependent phosphorylation of myocardial MLC1 during I/R

Heart function, assessed by rate pressure product (RPP), was significantly decreased in hearts reperfused following ischemia, in comparison to hearts perfused aerobically for 75 minutes (10.6±2.4 vs. 27.4±1.3 bpm*mmHg*10³; respectively; p<0.05) (Fig. 4a). Pharmacological inhibition of MLCK by ML-7 resulted in an increased functional recovery of hearts during reperfusion. The protection seen by ML-7 occurred in a concentration-dependent manner. Using 5 µM ML-7 resulted in an increased recovery of contractile function (Fig. 4a) and, in addition, it also prevented the degradation of MLC1 protein during I/R (Fig. 4b). A protective effect of ML-7 against MLC1 degradation was confirmed by 2-DE (Fig. 4c). This was accompanied by the decreased formation of the truncated form of MLC1 in hearts treated with ML-7 in comparison to MLC1 from I/R hearts (Fig. 4c).

Figure 4.

Protection of cardiac mechanical function and MLC1 levels by ML-7. (a) Mechanical function (determined as RPP) of hearts submitted to I/R perfused in the presence or absence of ML-7 (MLC kinase inhibitor) (n=6–9/group). (b) Densitometric analysis of the levels of MLC1 in heart homogenates analyzed by immunoblotting (n=3/group). (c) Densitometric analysis of the levels of truncated MLC1 as determined by 2-DE (n=4/group).

RPP – rate pressure product; MLC1 – Myosin light chain 1; I/R – ischemia/reperfusion.

*p < 0.05 vs. aerobic control; ^#p < 0.05 vs. I/R.

Analysis of in vitro and ex vivo MLC1 phosphorylation

Since rat MLC1 protein was not commercially available we used a recombinant human cardiac MLC1 (hMLC1) in our in vitro studies. Phosphorylation status of hMLC1 phosphorylated in vitro with MLCK was compared with the MLC1 from ex vivo I/R heart (Fig. 5). In vitro incubation of hMLC1 with MLCK resulted in phosphorylation of either threonine 127 (T127) or T129 or tyrosine 130 (Y130) (T127/T129/Y130). Because these amino acids are localized in the same tryptic peptide it is difficult to determine, by analysis of the tryptic peptides, which residue is phosphorylated. In addition, phosphorylation of serine 179 (S179) and Y185 was detected. Inhibition of MLCK by ML-7 abolished phosphorylation of S179 and Y186, but did not inhibit phosphorylation of T127/T129/Y130 site (Table 1).

Figure 5.

Comparative analysis of MLC1 sequences from rat I/R hearts and human recombinant MLC1 phosphorylated in vitro with myosin light chain kinase and respective phosphorylation sites as determined by mass spectrometry (phosphorylated amino acid residues are shown in red). Homology between rat and human MLC1 is 93.5% identity between rat (200 aminoacids) and human (195 aminoacids).

Insert in box shows P and P’ positions of MMP-2 cleavage site within the C-terminal of rat and human MLC1.

MMP-2 – matrix metallopreoteinase-2; MLC1 – myosin light chain 1.

* - phosphorylated residues.

: – represents full homology between amino acids

. – represents partial homology between amino acids

Table 1.

In vitro phosphorylation of human recombinant MLC1 with MLC kinase (MLCK) and its inhibition by ML-7, an inhibitor of MLCK.

Identified phosphorylation sites in MLC1
Control	+ MLCK*
		+ ML-7** (5µM)
	n/d	T 127 or T 129 or Y 130	T 127 or T 129 or Y 130
n/d	S 179	n/d
n/d	Y 186	n/d

T – Threonine; Y – Tyrosine; S – Serine

n/d – not detected

^*Human recombinant MLC1 (7.5 µg) was incubated with MLCK (25 ng) and ATP (1.4 µg) for 20 min, as described in the Methods section.

^**ML-7 (5 µM) was added to the reaction mixture 5 min before ATP.

MLC1 from rat hearts subjected to I/R was phosphorylated at either T69 and T77, or Y78 (T77/Y78). As mentioned above, and similarly to human MLC1, the exact identification of which amino acid is phosphorylated is difficult. Also, T132/T134/Y135, T164, S184, and Y190 were identified as phosphorylation sites (Fig. 5 and Table 2).

Table 2.

Phosphorylation status of MLC1 from rat I/R hearts perfused with ML-7 an inhibitor of MLC kinase (MLCK)

Identified phosphorylation sites in myocardial MLC1
Aerobic heart	I/R heart (20/30)
		+ ML-7 (5 µM)
T 69	T 69	n/d
n/d	T 77 or Y 78	T 77 or Y 78
T 132 or T 134 or Y 135	T 132 or T 134 or Y 135	T 132 or T 134 or Y 135
n/d	T 164	n/d
n/d	S 184	n/d
n/d	Y 190	n/d

T – Threonine; Y – Tyrosine; S – Serine

n/d – not detected

The two phosphorylated amino acids located at the C-terminus [S184 (rat MLC1) and S179 (hMLC1) together with Y190 (rat MLC1) and Y185 (hMLC1)] were found to be located very close (S in P6 position) or next to (Y in P’1 position), the known MMP-2 cleavage site. The cleavage site for MMP-2 is localized between asparagine N189 (P1) and tyrosine Y190 (P’1) in rat MLC1, and N184 (P1) and Y185 (P’1) in hMLC1 (see inset in Fig. 5).

Effect of phosphorylated MLC1 on MMP-2 kinetic

We also determined what effect MLC1 phosphorylation had on the affinity of MLC1 for MMP-2, and the consequent degradation by MMP-2. Kinetic analysis of the MMP-2 reaction with OmniMMP substrate, in presence of MLCK-phosphorylated hMLC1 revealed a higher affinity of phospho-MLC1 to MMP-2 (decreased V_max for OmniMMP substrate) compared to non-phosphorylated MLC1 (V_max not changed).

Co-localization of MLC1 with MLCK in I/R hearts

Potential co-localization of MLC1 with MLCK was evaluated by separating protein complexes from heart homogenates under non–reducing and non-denaturing conditions (using native gels), followed by dual-immunoblotting (Fig. 7). Under non-reducing conditions, two distinct bands of MLC1 (red) were seen in aerobic control, I/R and I/R+ML-7 heart homogenates (Fig. 7a, middle panel). Blotting for MLCK revealed only one band (green) (Fig.7a, top panel). Overlap of both blots revealed co-localization of MLCK with MLC1 (yellow) (Fig. 7a, bottom panel). Quantitative analysis of the blots showed a slightly increased co-localization of MLC1 with MLCK in I/R hearts compared to aerobic controls, but the difference was not statistically significant (p=0.0661, Fig. 7b). Administration of ML-7 significantly reduced the co-localization of MLC1 with MLCK in comparison to non-treated hearts subjected to I/R (Fig. 7b).

Figure 7.

Co-localization of MLC1 with MLCK and MLCP. (a) Analysis of co-localization of MLC1 with MLCK and MLCP by dual immunoblotting. (b) Quantitative analysis of MLC1 levels co-localized with MLCK – myosin light chain 1; MLCK – myosin light chain kinase; I/R – ischemia/reperfusion. *p < 0.05 vs. aerobic control; n=3/group.

Protection of contractile function and MLC1 protein levels by doxycycline

Apart from its antibacterial function, doxycylcine has also been shown to be an inhibitor of MMPs [15]. It was also shown that doxycylcine mediated inhibition of MMP-2 improves cardiac functional recovery following I/R [7, 9, 16, 17]. We therefore used doxycyclin in our studies to inhibit the activity of MMP-2. We found that mechanical function of I/R hearts was protected by doxycycline in a concentration-dependent manner (Fig. 8a). Administration of 30 µM doxycycline resulted in full recovery of contractile function during reperfusion (Fig. 8a) and also prevented the degradation of MLC1 protein during I/R (Fig. 8b).

Figure 8.

Protection of cardiac mechanical function and MLC1 levels by doxycycline (Doxy), an inhibitor of MMP-2. (a) Mechanical function (determined as RPP) of hearts subjected to I/R and perfused in the presence or absence of Doxy (n=6–9/group). (b) Densitometric analysis of the levels of MLC1 in heart homogenates determined by immunoblotting (n=3/group).

RPP – rate pressure product; MLC1 – Myosin light chain 1; I/R – ischemia/reperfusion. *p < 0.05 vs. aerobic control; ^#p < 0.05 vs. I/R.

Discussion

Cardiac contraction has been well studied and many of the molecular elements involved in the physiology of contraction identified. Myosin light chain 1 is an important contractile protein involved in the structural and functional stability of the contractile apparatus. Here we show that MLC1 phosphorylation plays an important role in the pathology of cardiac contractile dysfunction following ischemia-reperfusion. MLC1 is phosphorylated during I/R, possibly by MLCK, and this phosphorylation results in increased degradation of MLC1 by MMP-2 that is associated with the development of contractile dysfunction. Our data show that phosphorylation of MLC1 increases the affinity of MMP-2 for MLC1. This was evident by a decrease in V_max in a competition assay with the phosphorylated MLC1 compared to the non-phosphorylated counterpart. In addition we show that either inhibition of MLC1 phosphorylation or inhibition of MMP-2 activity in I/R hearts results in the protection of contractile function during reperfusion, as well as the preservation of MLC1 protein levels. Moreover, our results from the in vitro study confirm what has been previously suggested [13, 18] that in addition to MLC2, MLC1 can also be a suitable substrate for phosphorylation by MLCK.

Mass spectrometry analysis of human recombinant MLC1 (hMLC1) phosphorylated in vitro revealed three phosphorylation sites located on the C-terminus of hMLC1. Since these phosphorylation sites are located in the P6 and P’1 positions of the known MMP-2 cleavage site [9], it is likely that phosphorylation of these residues results in a conformational change of MLC1 exposing the cleavage site of MLC1 for MMP-2. This is supported by our data showing an increased affinity of phosphorylated MLC1 to MMP-2.

Since we observed no changes in the phosphorylation status of MLC2 with I/R in the presence or absence of ML-7, and we used concentrations of ML-7 below the k_i for other kinases, we can infer that the beneficial effects of MLCK inhibition are due to the inhibition of MLC1 phosphorylation and consequent preservation of its protein levels. Inhibition of MLCK with ML-7 prevented the phosphorylation of amino acid residues from P6 and P’1 positions but failed to inhibit phosphorylation of T127/T129/Y130. Indeed, mass spectrometry analysis of MLC1 from perfused rat hearts revealed four phosphorylation sites that can be attributed to MLCK, two of which are in the vicinity of the MMP-2 cleavage site and are I/R-dependent. Interestingly, we also found two phosphorylation sites on MLC1 in aerobically perfused hearts and of these two only one (T69) appears to be MLCK-dependent. This suggests that other kinases apart of MLCK may be involved in the process of MLC1 phosphorylation. The analysis of the T127/T129/Y130 phosphorylation site of human MLC1 and T132/T134/Y135 phosphorylation site of rat MLC1 for kinase consensuses sequence indicate AMPK (AMP-activated protein kinase) and PKC as two other potential mediators. Indeed, activation of these kinases has been reported in I/R [19, 20]. Phosphorylation of T69 and T132/T134/Y135 is observed in MLC1 from aerobically perfused hearts (controls). This suggests that phosphorylation of these sites may be important in physiological conditions.

Recently, a cardiac specific MLCK has been identified in human heart failure and described as the protein kinase responsible for phosphorylating MLC2 and regulating cardiac contractility [21, 22]. Here we report a slightly increased co-localization of MLCK with MLC1, triggered by I/R, with no changes in MLCK activity. To date, changes in cardiac MLCK activity have not been described in response to I/R. Our results confirm that MLCK activity does not change in response to I/R but that 5 µM ML-7 administration significantly reduce MLCK activity.

Although ML-7 has been suggested to be a specific MLCK inhibitor, it has been reported to affect the activity of other kinases, similar to what occurs with the vast majority of protein kinase inhibitors (for review see [14]). Inhibition of PKC by ML-7 has been shown in vitro at high concentrations [14]. We have measured the activity of PKC in all our groups. PKC activity was significantly increased with I/R but unaffected by 5 µM ML-7, which inhibits MLCK activity. PKC has been described to potentially phosphorylate MLC1 (and MLC-2) [23] and increase its activity in response to I/R [24]. Our data show a potential phosphorylation site on MLC1 under PKC regulation (T127/T129/Y130 site of human MLC1 and T132/T134/Y135 site of rat MLC1). This site is phosphorylated under both physiological (aerobic) and pathological (I/R) conditions and is unaffected by ML-7 administration. This indicates that phosphorylation of MLC1 by PKC is independent of I/R and may regulate MLC1 structure and/or function.

MLCK has been described as being the enzyme responsible for phosphorylating MLC2 at S15 of the N-terminus, and this phosphorylation is considered responsible for the regulatory character of MLC2 in skeletal and cardiac muscle contraction [25–28]. However, results from our in vitro studies demonstrate that MLC1 is also a substrate for MLCK. Studies of protein-protein interactions, where samples were run under non-reducing conditions to determine localization of free proteins or co-localized with protein complexes, showed that in isolated perfused heart homogenates MLC1 is present in two protein bands. However, only one of these bands corresponds to a complex of MLC1 with MLCK. These observations suggest the existence of two pools of intracellular MLC1: one associated with, and under regulation of, MLCK and another independent of MLCK. Also, the second MLC1 band might correspond to free MLC1. Another possible explanation is that the second band of MLC1, detected by immunoblotting under non-reducing and non-denaturing conditions, corresponds to degraded MLC1. Interestingly we observed a slightly increased co-localization of MLC1 and MLCK triggered by I/R, with no changes in MLCK activity, with this effect being inhibited by ML-7. This suggests that part of the mechanism of cardioprotection by ML-7 involves the inhibition of complex formation of MLCK with MLC1, in addition to inhibition of MLCK activity.

Isolated perfused rat hearts, subjected to I/R in the presence of doxycycline show a significant increase in contractile function recovery during reperfusion, in comparison to I/R alone. Moreover, this protection in cardiac contractile function is associated with a preservation of MLC1 levels, similar to what was observed with ML-7 administration.

In summary these results together with our previous studies show an important role of post-translational modifications triggered by I/R, such as phosphorylation, on contractile protein MLC1, and its degradation by MMP-2 and association of this process with contractile dysfunction.

Materials and Methods

This investigation conforms to the Guide to the Care and Use of Experimental Animals published by the Canadian Council on Animal Care.

Heart preparations

Male Sprague-Dawley rats (300g to 350g) were anesthetized with sodium pentobarbital (40 mg/kg, i.p.). The hearts were rapidly excised and briefly rinsed by immersion in ice-cold Krebs-Henseleit solution. Spontaneously beating hearts were placed in a water-jacketed chamber (EMKA Technologies, Paris, France) and maintained at 37°C. Hearts were perfused in the Langendorff mode at a constant pressure of 60 mmHg with modified Krebs-Henseleit buffer at 37°C containing (in mM): NaCl (118), KCl (4.7), KH₂PO₄ (1.2), MgSO₄ (1.2), CaCl₂ (3.0), NaHCO₃ (25), glucose (11), and EDTA (0.5), and gassed continuously with 95% O₂/5% CO₂ (pH 7.4).

A water-filled latex balloon connected to a pressure transducer was inserted through an incision in the left atrium into the left ventricle through the mitral valve. The volume was adjusted to achieve an end diastolic pressure of 10 mmHg. Coronary flow, heart rate, and left ventricular pressure were monitored using an EMKA recording system with IOX2 software (EMKA Technologies, Paris, France). Left ventricular developed pressure (LVDP) was calculated as the difference between systolic and diastolic pressures of the left ventricular pressure trace. The rate pressure product (RPP) was calculated as the product of heart rate and LVDP. Stock solutions (140×) of various reagents were infused into the heart via a side-port proximal to the aortic cannula at the rate 140 times lower than coronary flow (CF), usually 0.1 mL min⁻¹ with a Gilson mini pump Minipuls 3 (Gilson, Middleton, WI, USA).

I/R protocol

Control hearts (aerobic control, n=12) were perfused aerobically for 75 min. Ischemic hearts (I/R, n=9), after 25 min of aerobic perfusion, were subjected to 20 min global no-flow ischemia (by closing of the aortic inflow line), followed by 30 min of aerobic reperfusion. In two separate groups of I/R hearts (n=6 each) either ML-7 (1–5 µM [Merck, Mississauga, Canada]), myosin light chain kinase (MLCK) inhibitor or doxycycline (Doxycycline, 1–30 µM [Sigma, Taufkirchen, Germany]), an inhibitor of MMP-2, were infused 10 min before of onset of ischemia and for the first 10 min of reperfusion. Water was used as vehicle for doxycycline while ethanol (50% (v/v) was the vehicle for ML-7. The maximal concentration of ethanol infused during the heart was less than or equal to 0.025% (v/v). The scheme of the experimental protocol is shown in Figure 1. At the end of perfusion the hearts were freeze clamped in liquid nitrogen and used for biochemical studies.

Figure 1.

Schematic representation of the perfusion protocol for the isolated Langendorff perfused rat hearts. When present, ML-7 (an inhibitor of MLC kinase) or doxycycline, an inhibitor of MMP were administered 10 min before the onset of ischemia, during ischemia and for the first 10 min of reperfusion.

Preparation of heart protein extracts

Protein samples for 2-dimensional gel electrophoresis (2-DE) were prepared at room temperature by mixing frozen (−80°C), powdered heart tissue (40 to 60 mg wet weight) with 200 µL rehydration buffer (8 mol/L urea, 4% CHAPS, 10 mmol/L DTT, 0.2% Bio-Lytes 3/10 [BioRad, Hercules, CA, USA]) at room temperature. Samples were sonicated twice for 5 sec and centrifuged for 10 min at 10,000×g to remove any insoluble particles. Protein content of the heart extract in rehydration buffer was measured with the BioRad Bradford protein assay.

For other biochemical studies, frozen heart tissue powder was homogenized on ice in 150 mM NaCl, 50 mmol/L Tris-HCl (pH 7.4) containing protease inhibitor cocktail (Sigma, St Louis, MO, USA) and 0.1% Triton X-100. Homogenates were centrifuged at 10 000×g at 4°C for 10 min and the supernatant was collected and stored at −80°C until use.

Two-Dimensional gel electrophoresis (2-DE)

Protein samples (0.4 mg) were applied to each of 11 cm immobilized linear pH gradient (5–8) strips (IPG, BioRad, Hercules, CA, USA), with rehydration for 16–18 h at 20°C. For isoelectrofocusing, the BioRad Protean IEF cell was used with the following conditions at 20°C with fast voltage ramping: step 1: 15 min with end voltage at 250 V; step 2: 150 min with end voltage at 8000 V; step 3: 35 000 V-hours (approximately 260 min). After isoelectrofocusing, the strips were equilibrated according to the manufacturer’s instructions. The second dimension of 2-DE was then carried out with Criterion pre-cast gels (8 – 16%) (BioRad). After separation, proteins were detected with Coomassie Briliant Blue R250 (BioRad). To minimize variations in resolving proteins during the 2-DE run, 12 gels were run simultaneously using a Criterion Dodeca Cell (BioRad, Hercules, CA, USA). Because of this limitation for 2-DE analysis we used 4 hearts from each group. All the gels were stained in the same bath and next scanned with a calibrated densitometer GS-800 (BioRad, Hercules, CA, USA). Quantitative analysis of MLC1 and MLC2 spot intensities from 2-DE were measured with PDQuest 7.1 measurement software (BioRad, Hercules, CA, USA).

Mass Spectrometry

MLC1 and MLC2 protein spots were manually excised from the 2-DE gel. These spots were then processed using a MassPrep Station (Waters, Milford, MA, USA) using the methods supplied by the manufacturer. The excised gel fragment containing the protein spot was first destained in 200 µl of 50% acetonitrile with 50 mM ammonium bicarbonate at 37°C for 30 min. Next, the gel was washed twice with water. The protein extraction was performed overnight at room temperature with 50 µL of a mixture of formic acid, water, and isopropanol (1:3:2, vol:vol). The resulting solution was then subjected to trypsin digestion and mass spectrometry analysis. For electrospray, quadruple time-of-flight (Q-TOF) analysis, 1 µl of the solution was used. Liquid chromatography/mass spectrometry (LC/MS) was performed on a CapLC high-performance liquid chromatography unit (Waters, Milford, MA, USA) coupled with Q-TOF-2 mass spectrometer (Waters, Milford, MA, USA). A mass deviation of 0.2 was tolerated and one missed cleavage site was allowed. Resulting values from mass spectrometry (MS/MS) analysis were used to search against the NCBInr and SwissProt databases with Mammalia specified. We used the Mascot (www.matrixscience.com) search engine to search the protein database.

MLCK activity assay

Frozen ventricular tissue was homogenized in a solution containing 20 mM Tris-HCl (pH 7.4 at 4°C), 50 mM NaCl, 50 mM NaF, 5 mM Na pyrophosphate, 0.25 mM sucrose, protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and 1 mM dithiothrietol. Protein contents of the homogenates were determined using the Bradford protein assay. MLCK activity was assessed using 0.3 mg of total ventricular protein, incubated in a solution containing 40 mM HEPES, 80 mM NaCl, 8% (w/v) glycerol, 0.2 mM EDTA, 500 mM CaCl₂ 0.01 % triton X-100, 0.8 mM DTT, 10 mM MgCl₂, 200 mM [γ³²P]ATP, and 200 mM Ziptide (KKLNRTLSFAEPG), in the absence or presence of 20 mM EGTA. Reactions were incubated for 10 min at 30°C, and terminated with addition of H₃PO₄ (3% v/v final concentration). An aliquot of each reaction was spotted on P81-phosphocellulose, which was washed in 1% H₃PO₄ (5–6×, 10 min each wash), and subsequently acetone (2×, 5 min each wash). The incorporation of γ³²P into the Ziptide substrate was determined by scintillation counting.

PKC activity assay

Protein kinase C (PKC) activity was determined using a commercially available PKC assay kit from Promega (Madison, WI, USA) according to the supplier’s instructions. Briefly, crude heart homogenates from frozen heart samples were homogenized in PKC extraction buffer using a sonicator. PKC was semi-purified using a 1 ml DEAE cellulose column. The PKC enriched fraction (9µg protein/reaction) was incubated with PepTag® C1 peptide and incubated for 90 min at 30°C. The reaction was stopped by placing tubes in a boiling water bath. After cooling, the phosphorylated and non-phosphorylated peptides were separated in a 0.8% agarose gel at 100V for 30 min. Detection of PepTag® C1 peptide was performed using the VersaDoc 5000 and Quantity One software (BioRad, Hercules, CA, USA). The decrease in non-phosphorylated PepTag® C1 peptide was used as an indicator of increased PKC activity.

Cloning, expression and purification of human cardiac MLC1

The cDNA clone for the human ventricular myosin essential light chains (MLC1), a product of the MYL3 gene, was isolated as previously described [11, 29]. Briefly, total adult human heart RNA was purchased from Stratagene (Agilent, Santa Clara, CA, USA), Oligo (dT)15 (Promega, Madison, WI, USA) and specific primers were designed based on the published nucleotide sequence from NCBI: NG_007555. Restriction sites for NcoI and BamHI were inserted to facilitate ligation into the pET 3D plasmid (Merck KGaA, Darmstadt, Germany). PCR was performed according to the Omniscript manual and a resulting PCR product was gel purified using a Qiaex II kit (Qiagen, Valencia, CA, USA). The eluted DNA was then digested with Nco I and BamHI and ligated into similarly digested pET 3d plasmid using T4 DNA Ligase (NEBiolabs, Ipswich, MA, USA). Subcloning efficiency DH5 α competent cells (Invitrogen, Eugene, OR, USA) were transformed and plated onto LB-CB plates. Resultant colonies were screened for the MLC1 insert and the positive colonies were selected. The isolated plasmid DNA was sent for sequencing (Cardiovascular Facility, University of Miami, Miami, Florida USA) and the clones were confirmed to have the correct MLC1 sequence (Swiss-Prot: P08590). Confirmed DNA was used to transform BL21 (DE3) Codon Plus competent cells (Agilent, Santa Clara, CA, USA) for MLC1 wild-type protein expression. MLC1 protein was expressed in 8l of enriched media consisting of 30g of peptone/l, 20g of select yeast extract/l, and M9 minimal salts 10g/l with 20 µg/ml ampicillin and purified using column chromatography S-Sepharose followed by DEAE-Sephacel.

In vitro phosporylation of MLC1

MLC1 (7.5 µg) was incubated with 25 ng of the active form of myosin light chain kinase (MLCK) (SignalChem, Richmond, ON, Canada) and 1.4 µg of ATP (Sigma, St Louis, MO, USA) for 20 min at 37° C in 40 mM MOPS-NaOH reaction buffer with pH 7.0, containing 0.5 mM CaCl₂ and 1 µM calmodulin (Upstate, Temecula, CA, USA). The total volume of reaction mixture was 30 µl. In vitro phosphorylation of MLC1 was verified by mass spectrometry. In some experiments MLCK was incubated for 5 min with 10 µM of ML-7 (inhibitor of MLCK) before ATP was added to the reaction mixture.

Degradation of phosphorylated MLC1 by MMP-2

Phosphorylated or non-phosphorylated MLC1 (7.5 µg) was incubated at 37^oC for 60 minutes with 0.2 µg of 64 kDa MMP-2 (Calbiochem, Merck KGaA, Darmstadt, Germany) in 50 mM Tris-HCl buffer, pH 7.6 and containing 5 mM CaCl₂ and 150 mM NaCl, total volume 60 µL) [9]. The reaction mixture was analyzed by 15% SDS-PAGE under reducing conditions and visualized by immunoblot analysis with anti-MLC1 IgG (Abcam, Cambridge, MA, USA).

Kinetic analysis of MMP-2 activity in presence of phosphorylated and non-phosphorylated MLC1 (competition assay)

The hydrolysis of OmniMMP fluorogenic substrate (Enzo Life Sciences, PA, USA, 0–25 µM) by MMP-2 was measured at 37°C in a continuous plate reader-based protocol [30]. Briefly, the sample containing MMP-2 (0.2 nM), substrate and either phosphorylated or non-phosphorylated MLC1 (0–25 µM,) was measured every 30 sec for 1 h (λ_ex 328 nm, λ_em 393 nm). The rate of product formation was determined through linear regression of the fluorescence-time data by the plate reader software. Linear reaction rates for control data was fitted to the Michaelis-Menten equation in order to obtain K_M and V_max values. Linear reaction rates from assays containing MLC1 were fitted to an equation for linear competitive inhibition in order to determine K_M, V_max and K_i values for the MLC1 samples.

Immunoblot analysis

Recombinant MLC1, its degradation products, MLC1, MLC2 and phospho-MLC2 content in the heart extracts were determined by immunoblotting. Equal volumes of MLC samples from the in vitro experiments or 20µg of protein from each heart extract were analyzed by SDS-PAGE using 15% gels. After electrophoresis protein was transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, USA). MLC1, MLC2 and phospho-MLC2 were identified using mouse monoclonal anti-MLC1, rabbit polyclonal anti-MLC2 and rabbit polyclonal anti-phospo-MLC2 antibodies (Abcam, Cambridge, MA, USA).

Co-localization of myocardial MLC1 with myocardial MLCK or MLCP was analyzed by separating protein complexes in non-reducing and non-denaturing conditions on native mini-PROTEAN TGX pre-cast gels (BioRad, Hercules, CA, USA), followed by dual-immunoblot detection. MLC1 was detected with mouse monoclonal anti-MLC1 antibody (Abcam, Cambridge, MA, USA) using goat anti-mouse IgG tagged with Alexa fluor 488 (Invitrogen, Eugene, OR, USA) as secondary antibody. MLCK was detected with rabbit monoclonal antibody (Abcam, Cambridge, MA, USA) with goat anti rabbit IgG tagged with Alexa fluor 647 (Invitrogen, Eugene, OR, USA) as a secondary antibody.

Band densities were measured with VersaDoc 5000 and Quantity One software (BioRad, Hercules, CA, USA).

Statistical Analysis

Protein spot intensity was measured using PDQuest software (BioRad, Hercules, CA, USA) and evaluated by the Kruskal Walis and Mann-Whitney U tests. ANOVA or Kruskal-Wallis tests were used in functional studies (followed by Tukey’s post-hoc test) and an unpaired t-test was used in immunoblot analysis. Data are expressed as mean±SEM. A p < 0.05 was considered statistically significant.

Figure 6.

Effect of MLC1 phosphorylation on the maximal velocity of MMP-2 activity (V_max) in a competition assay (n=3–4).