Myosin Light Chain–Actin Interaction Regulates Cardiac Contractility
Abstract The amino-terminal domain of the essential myosin light chain (MLC-1) binds to the carboxy terminus of the actin molecule. We studied the functional role of this interaction by two approaches: first, incubation of intact and chemically skinned human heart fibers with synthetic peptide corresponding to the sequences 5 through 14 (P5-14), 5 through 8 (P5-8), and 5 through 10 (P5-10) of the human ventricular MLC-1 (VLC-1) to saturate actin-binding sites, and second, incubation of skinned human heart fibers with a monoclonal antibody (MabVLC-1) raised against the actin-interacting N-terminal domain of human VLC-1 using P5-14 as antigen to deteriorate VLC-1 binding to actin. P5-14 increased isometric tension generation of skinned human heart fibers at both submaximal and maximal Ca2+ activation, the maximal effective peptide dosage being in the nanomolar range. A scrambled peptide of P5-14 with random sequence had no effects up to 10−8 mol/L, ie, where P5-14 was maximally effective. P5-8 and P5-10 increased isometric force to the same extent as P5-14, but micromolar concentrations were required. Amplitude of isometric twitch contraction, rate of tension development, rate of relaxation, and shortening velocity at near-zero load of electrically driven intact human atrial fibers increased significantly on incubation with P5-14. These alterations were not associated with modulation of intracellular Ca2+ transients as monitored by fura 2 fluorescence measurements. Incubation of skinned human heart fibers with MabVLC-1 increased isometric tension at both submaximal and maximal Ca2+ activation levels, having a maximal effective concentration in the femtomolar range. We conclude that VLC-1–actin interaction modulates cardiac contractility and may be a target for new inotropic intervention.
The cardiac myosin molecule is composed of two heavy chain (MHC) isoforms, α-MHC and β-MHC, with molecular weights of ≈200 kD each. The globular portions of the MHC are noncovalently linked with two types of light chain (MLC) isoforms with molecular weights of 18 or 19 kD (MLC-2), so-called regulatory or phosphorylatable MLC, and 25 to 28 kD (MLC-1), so-called essential MLC.1
In striated muscle, different MHC isoforms are expressed, encoded by at least seven different genes.2 It is well established that the expression of a defined MHC isoform is associated with the contractile and energetic behavior of the muscle.3 Phosphorylation of the MLC-2 increased the rate constant for the transition of cross-bridges from non–force-generating into force-generating states, thus increasing the ability of muscle to produce force.4 5 The role of the essential MLC, however, is less well understood. In the human heart, two MLC-1 isoforms are encoded by different genes, leading to the expression of the 28-kD atrial-specific (ALC-1) form and the 27-kD ventricular-specific (VLC-1) form.6 The MLC-1 forms comprise a C-terminal MHC-binding domain and an unusual proline-rich N-terminal domain. It could be demonstrated that synthetic peptides7 8 or proteolytic products9 of the last amino terminus of MLC-1 bind to a specific carboxy-terminal domain of actin. To contribute to the understanding of MLC-1–actin interaction, we deteriorated the actin binding of the N-terminus of the VLC-1 using synthetic peptides corresponding to the N-terminal domain of human VLC-1 and monoclonal antibodies raised against the N-terminal MLC-1 domain (MabVLC-1). We demonstrate in this work that these interventions increased cardiac contractility of both skinned and intact tissue at given Ca2+ concentrations.
Materials and Methods
Peptide Synthesis and Antibody Preparation
Based on the following sequence of the amino terminus of human VLC-16 : 1Met Ala Pro Lys Lys Pro Glu Pro Lys Lys Asp Asp Ala Lys Ala Ala Pro Lys Ala Ala20, peptides comprising the sequences 5 through 14 (P5-14; underlined), 5 through 8 (P5-8), and 5 through 10 (P5-10) were synthesized. Furthermore, a scrambled peptide of P5-14 with random sequence (Pro-Lys-Asp-Lys-Glu-Ala-Lys-Pro-Lys-Asp) was produced. In short, fully protected peptide resin was assembled on a resin support by stepwise Merrifield solid-phase synthesis peptide synthesizer using N-tert-butoxycarbonyl chemistry (Applied Biosystems Inc). Crude free peptide was cleaved from the resin and was then purified by reverse-phase high-performance liquid chromatography (HPLC). The composition of the peptide was determined on a Beckman 6300 amino acid analyzer after acid hydrolysis. Molecular weights of crude and purified peptides were determined by PDMS.
BALB/c mice immunized with P5-14 conjugated with keyhole limpet hemocyanin were chosen for somatic cell fusion with myeloma cells.10 Positive hybridomas selected by ELISA were subcloned two or three times and grown in RPMI 1640 medium supplemented with fetal calf serum (15%). Monoclonal antibodies were purified by chromatography on protein A Sepharose.
All experiments were performed with human heart preparations. Tissue for skinned fibers was obtained during cardiac transplantation. Patients suffered from heart failure clinically classified as New York Heart Association (NYHA) functional class IV as judged by the attending cardiologist just before operation. Tissue for experiments with intact fibers was obtained from the right auricle of patients operated on for mitral valve replacement or combined coronary artery bypass graft. All patients gave written informed consent before surgery.
Chemically Skinned Fibers
Skinned fibers were prepared by incubation of small papillary fiber bundles (1 mm in cross section) in a solution containing (mmol/L) imidazole 20, ATP 5, NaN3 10, MgCl2 5, EGTA 4, dithioerythritol (DTE) 2, and 50% glycerol, pH 7.0, at +4°C for 1 hour and subsequently in the same solution containing, in addition, 1% Triton X-100 at +4°C for 24 hours. The fibers were then stored in the same solution without Triton at −20°C. Skinned fibers (150 to 200 μm in cross section and 4 to 6 mm long) were mounted on a force transducer (AME AE 801, SensoNor), and a glass rod was attached to a micromanipulator with a fast-setting glue. Fiber length was adjusted to an extent that resting tension was just at threshold.
Fibers were first immersed in relaxing solution (pCa 8) and then transferred to activating solution. Relaxing solution contained (mmol/L) imidazole 20, ATP 10, creatine phosphate 10, NaN3 5, EGTA 5, MgCl2 12.5, DTE 1, and leupeptine 0.01, pH 7.0. Contraction solution had the same composition, except that EGTA was substituted by 5 mmol/L CaEGTA. The desired Ca2+ concentration was obtained by mixing relaxation with contraction solution in the appropriate proportions. Experiments were performed at 21°C to 23°C. Free Ca2+ concentrations were calculated by use of the binding constants of Fabiato and Fabiato11 and are given as pCa (−log of free [Ca2+]).
Intact Atrial Fibers
Atrial myocardium was obtained from the right auricle of patients operated on for mitral valve replacement or combined coronary artery bypass graft. The tissue was transported in +4°C oxygenated Krebs-Henseleit solution (mmol/L: NaCl 119, NaHCO3 25, KCl 4.6, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.2, and glucose 11, containing 30 mmol/L 2,3-butanedione monoxime). Atrial trabeculae (about 4 mm long and 0.7 mm in maximal initial diameter) were dissected and mounted between an optoelectronic force transducer and a length-step generator by microsyringes. The trabeculae were electrically stimulated at 1 Hz and 5 V (5-ms duration). Fiber length was adjusted to optimal length, ie, length at which the trabeculae generated maximal tension (maximal fiber diameter, 0.5 mm). Trabeculae were stimulated for 5 minutes until steady-state tension developed. During isometric tension generation, isotonic releases at near-zero load were performed on the preparations. The experimental setup and the computer-controlled feedback circuit were purchased from Scientific Instruments. Length and tension signals were simultaneously recorded with a storage oscilloscope and digitized in an IBM-compatible PC.
Intracellular Ca2+ measurements in human trabeculae were performed as previously described12 with commercially available equipment (K. Güth, Scientific Instruments). In short, trabeculae were prepared as described above but incubated for 6 hours in oxygenated Ringer’s solution (mmol/L concentrations: NaCl 147, KCl 4, CaCl2 2.2) containing 5 μmol/L fura 2, 0.5% Cremophor, and 10 μmol/L P5-14 at 22°C. The same procedure was applied to allow the peptide P5-14 to penetrate the cells. The fibers were subsequently electrically stimulated in Krebs-Henseleit solution without fura 2 or peptide as described above at 37°C. Fiber length was adjusted to optimal length. Intracellular fura 2 was excited (xenon lamp) at 340 and 380 nm by use of a rotating wheel (rotating frequency, 125 Hz). Fluorescence emission (510 nm) was collected and sorted in the respective channels of a photomultiplier. Fluorescence signal and tension signal were simultaneously recorded on a storage oscilloscope and sampled online on an IBM-compatible PC.
Human atrial and ventricular fibers were homogenized in 2% SDS, 20 mmol/L Tris/glycine-buffer, 1% β-mercaptoethanol, pH 8.0, and boiled in a water bath for 1 minute. Fifty micrograms per sample was applied to a gel. Disk SDS-polyacrylamide gel electrophoresis was performed for 4 hours at 120 V (constant) (cf Reference 1313 ). Proteins were transferred from SDS gels to a nitrocellulose membrane (30 minutes, 3 mA/cm2), and the proteins were visualized by incubation with Ponceau red. The membrane was subsequently incubated with MabVLC-1 (primary antibody) for 2 hours and then with the second (biotinylated) antibody (anti-mouse) for 1 hour at room temperature. Proteins were visualized by the streptavidin–horseradish peroxidase complex/chloronaphthol reaction. MabVLC-1 recognized the essential VLC-1 but not the essential ALC-1, which has a higher molecular weight and thus a lower mobility in the gel than the VLC-1.6 The faint signal observed in the atrial skinned fiber extract represents the essential VLC, which is normally expressed in low amounts in the atrium.14
Values are expressed as mean±SEM. Significance analysis was performed with Student’s t test.
Chemically Skinned Human Ventricular Fibers
In a first experimental approach, we investigated MLC-1–actin interaction by incubation of skinned ventricular fibers with the synthetic peptide corresponding to the P5-14 amino acid sequence (Lys-Pro-Glu-Pro-Lys-Lys-Asp-Asp-Ala-Lys) of human ventricular MLC-1 (VLC-1).6
To identify the incubation time required for the peptide to diffuse into the myofibrillar network, we incubated chemically skinned fiber bundles in relaxation solution at 22°C with 1.6×10−9 mol/L P5-14. Purity of the peptide was 99.9%. At given times, the fibers were activated with submaximal (pCa 5.5) and maximal (pCa 4.5) Ca2+ activation solution and subsequently relaxed at pCa 8. Isometric tension elicited at both pCa 5.5 and pCa 4.5 rose with incubation time: at 45 minutes of incubation with the peptide, there was a pronounced force-enhancing effect (Fig 1⇓). In the experiment shown in Fig 1⇓, tension rose by 73% and 32% at submaximal and maximal Ca2+ activation, respectively, compared with the first relaxation-contraction cycle without peptide. Considering these results, all subsequent experiments with peptides and skinned fibers were performed after 45 minutes of incubation at 22°C in relaxing solution.
In a second set of experiments, we investigated dose-effect relations. Skinned human ventricular fibers were incubated with various concentrations of P5-14 for 45 minutes at 22°C in relaxing solution (three fibers per peptide dose). Half-maximal effective dose was in the femtomolar range; threshold concentration of P5-14 was 10−13 mol/L (Fig 2⇓). Maximal effective peptide dose was ≈10−11 mol/L. In this particular experiment, tension rose to 38.5±3% (n=3) and 10.4±4% (n=3) of the tension obtained without peptide at pCa 5.5 and 4.5, respectively.
In additional experiments, we investigated the effects of maximal doses of a random peptide (Pro-Lys-Asp-Lys-Glu-Ala-Lys-Pro-Lys-Asp) and P5-14 on both submaximal and maximal Ca2+-activated isometric tension. Taking force before the incubation period as 100%, incubation with a maximal effective P5-14 dose (6.4×10−9 mol/L) increased isometric tension by 24.55±5% (n=31) at maximal (pCa 4.5) and 58±9% (n=31) at submaximal (pCa 5.5) Ca2+ activation levels (mean±SEM; number of skinned human heart fibers in parentheses). The P5-14 effect was reversible. A normalization of force development could be observed after 90 minutes of washout (incubation in relaxation solution; not shown). The random peptide was without any effect up to a concentration of 10−8 mol/L (not shown), ie, when P5-14 was maximally effective.
P5-8 and P5-10 peptides were without any effect on isometric tension up to a concentration of 4.5×10−7 mol/L peptide. At higher concentrations, however, both P5-8 and P5-10 revealed force-enhancing effects comparable to those of P5-14. Maximal effective concentrations of both P5-8 and P5-10 were 5×10−5 mol/L, when force increased by 43±4% (n=4) and 46±8% (n=4), respectively, at pCa 5.5. Force obtained at pCa 4.5 increased by 12±3% (n=4) and 14±5% (n=4) with P5-8 and P5-10, respectively (not shown).
In an additional approach, we studied MLC-1–actin interaction by incubation of skinned ventricular fibers with MabVLC-1 raised against the N-terminal domain of VLC-1 with P5-14 as antigen. To demonstrate its specificity, we performed a Western blot analysis using skinned human atrial and ventricular fibers. As demonstrated in Fig 3⇓, MabVLC-1 showed virtually no cross-reactivity with the atrial MLC-1 or rabbit psoas LC-1 isoforms. Only the VLC-1 was recognized by the antibody, reflecting the sequence differences in the amino-terminal region of atrial and ventricular MLC-1 in the human heart.6
The dose-effect relation of MabVLC-1 was obtained by the same experimental protocol as mentioned above, except that P5-14 was replaced by MabVLC-1. Threshold concentration was about 5×10−12 mol/L (pCa 6) with a half-maximal effective dose in the femtomolar range. Incubation with a maximal effective antibody concentration (10−9 mol/L) increased tension by 3.5%, 17%, and 27.5% at pCa 4.5, 5.6, and 6.0, respectively (Fig 4⇓). Incubation of human atrial fibers or rabbit psoas fibers with MabVLC-1 had no effect on tension development (not shown).
Intact Human Trabeculae
To demonstrate that P5-14 also is effective in intact human myocardium, we incubated human atrial trabeculae with P5-14 dissolved in oxygenated Krebs-Henseleit buffer. To ensure the penetration of the hydrophilic P5-14 into the cardiac cells, we used a relatively high exogenous peptide concentration (10 μmol/L) and coincubated the atrial trabeculae with the detergent Cremophor (0.5%), ie, the same procedure that allows the calcium indicator fura 2 to penetrate the cells. Using this method, we found pronounced positive inotropic effects of intact trabeculae incubated with P5-14. In fact, incubation of intact human heart fibers with P5-14 alone (without detergent) or with detergent alone (without peptide) revealed no effect (not shown).
There was a time-dependent increase of isometric tension generation, maximal rate of tension development (+dP/dtmax), maximal rate of relaxation (−dP/dtmax), and shortening velocity at near-zero load on incubation of intact human atrial trabeculae with P5-14 (Fig 5⇓). After 2 hours of incubation of human atrial trabeculae with 10 μmol/L peptide, amplitude of isometric tension rose from 16.1 mN/mm2 before incubation to 24.2 mN/mm2. In addition to increased force amplitude, time to peak tension decreased from 106 ms before incubation to 82.3 ms after incubation with the peptide. +dP/dtmax and −dP/dtmax rose from 4.5 and 3.1 g/s before incubation to 8.6 and 4.2 g/s after 2 hours in the presence of 10 μmol/L P5-14, respectively. Furthermore, shortening velocity at near-zero load increased from 4.2 muscle lengths per second (ML/s) to 6.11 ML/s after 2 hours of incubation with 10 μmol/L P5-14 (Fig 6⇓).
These effects became more pronounced on incubation of atrial trabeculae with 10 μmol/L P5-14 for 6 hours: tension, time to peak tension, +dP/dtmax, and −dP/dtmax were 33.8±3.6 mN/mm2, 84±3.9 ms, 12.6±1.2 g/s, and 6.0 g/s, respectively (six different trabeculae). All these values were statistically significant at the P<.01 level if compared with atrial trabeculae incubated for 6 hours without peptide: tension, time to peak tension, +dP/dtmax, and −dP/dtmax were 14.5±3.5 mN/mm2, 101.5±4.8 ms, 5.27±1.0 g/s, and 2.4±0.4 g/s, respectively (eight different trabeculae) (Fig 6⇑).
The initial high tension development of intact atrial trabeculae incubated for 6 hours with P5-14 decreased by about 45% after 45 minutes of stimulation subsequent to the incubation period (Fig 7⇓). In contrast, tension generation of atrial trabeculae not incubated with peptide remained constant during this stimulation period (not shown). Interestingly, although there was a time-dependent decrease of tension, intracellular Ca2+ concentration monitored by registration of the fura 2 fluorescence signal remained constant during the period investigated (Fig 7⇓).
The main finding of the present work is that incubation of both chemically skinned and intact human heart fibers with a peptide corresponding to the amino acid sequence 5 through 14 of the N-terminal domain of the human VLC-1 (P5-14) increased cardiac contractility at given free [Ca2+]. We suggest that VLC-1–actin interaction regulates force production of the heart muscle and that P5-14 deteriorates this interaction by blocking the appropriate VLC-1 binding domain on the C-terminus of actin. This deterioration increases force production. We did not directly show that the peptide used binds to the carboxy-terminal actin domain. However, existing evidence demonstrates that peptides bind to the desired domains both in the soluble system and in structurally intact muscle fibers. Thus, skinned muscle fibers or soluble actin-S1 incubated with synthetic peptides corresponding to the MHC domains known to interact with actin bind to the desired actin sites and inhibit actin-myosin interaction, thus reducing force of contraction15 and ATPase activity,16 17 respectively (for review of peptide competition, see Reference 1818 ). In fact, it could be demonstrated that peptides corresponding to the N-terminal domain of the MLC-1 have a specific binding domain at the last C-terminus of the actin molecule.7 8 Likewise, the N-terminal extension of MLC-1 cleaved from the molecule by controlled proteolysis binds to actin.9 In our hypothesis, binding of the peptide to the carboxy-terminal domain of actin will deteriorate the native MLC-1–actin interaction. With a similar approach, deterioration of the VLC-1–actin interaction in skinned human heart fibers by MabVLC-1 caused a force-enhancing effect similar to that of the peptide. Furthermore, since the peptide P5-14 used in this study was highly purified (purity checked by HPLC) and effective in the nanomolar range, we can exclude the possibility that the positive inotropic effect of P5-14 was due to contaminants or unspecific binding. It seems to be the specific sequence that is required for the inotropic action, since a scrambled peptide with the same amino acids as P5-14 but random sequence was without any effect up to 10−8 mol/L.
It is surprising that P5-14 was effective in those very low concentrations, since in comparable studies using skinned fibers as a model, micromolar peptide doses were required.15 16 18 However, P5-14 contains two proline and four lysine residues and therefore may have, by itself, a tertiary structure and be highly charged. This may facilitate specific binding of P5-14 to the appropriate actin binding site. Peptides having less advantageous primary sequences consequently bind with lower effectiveness and, therefore, higher concentrations are required. In fact, P5-14 fragments (P5-8 and P5-10) still increased isometric force, but in micromolar doses. Even those peptides with less advantageous sequences could be effective, since they may contain sufficient information to form the appropriate structure in the presence of a suitable template, as previously suggested.16
The three-dimensional structure of myosin subfragment 1 (S1) revealed a close association of the MLC-1 at the head-rod junction of the MHC.19 20 From these structure studies, MLC-1–actin interaction seems to be unlikely. However, it is not clear whether the essential light chain shown in the Rayment model represents the MLC-1 (A1) or the MLC-3 (A2). Since the essential light chain in the Rayment model comprised amino acids 5 through 149, we suggest that it is MLC-3, since MLC-1 has about 190 amino acids.21 Thus, more than 40 amino-terminal amino acids, ie, the domain that interacts with actin, are not represented in the Rayment model. Furthermore, since myosin S1 was prepared for crystal x-ray diffraction studies by proteolytic papain digestion, possible proteolytic breaks into the light chains could not be ruled out,19 limiting the resolution of the native essential light chain structure. Thus, in our hands, the interaction of MLC-1 with actin cannot be ruled out by the myosin structure obtained by recent crystal x-ray diffraction studies.19 20 Indeed, crystallographic analysis demonstrated that myosin-S1 is very close to the thin filament, not in the relaxed state but in the rigor state,22 which is assumed to be close to the conformation of active cycling cross-bridges in the force-generating state. The same studies22 suggest that MLC-1 and myosin-S1 binding to the thin filament may occur at different actin monomers.
Even in intact human atrial trabeculae, P5-14 induced a positive inotropic effect. Interestingly, using the same procedure that allows the Ca2+ indicator fura 2 to penetrate the cell membrane (coincubation with a low concentration of detergent) also allowed P5-14 to penetrate the myocyte of the atrial fibers, leading to a pronounced inotropic effect. Incubation with P5-14 alone or with detergent alone was without any effect, indicating that the peptide crossed the cell-membrane barrier. The extent of P5-14–induced inotropic effect increased with the incubation period. Thus, the amplitude of isometric twitch contraction rose to 150% after 2 hours and to about 210% after 6 hours of incubation.
The peptide-induced high initial tension development of intact atrial trabeculae incubated for 6 hours with P5-14 decreased by about 45% after 45 minutes of stimulation. Those atrial trabeculae not loaded with P5-14 remained constant during this period. The decline of the positive inotropic effects with time could be due to intracellular cytoplasmic degradation of P5-14 by endogenous peptidases, hence decreasing the peptide-induced inotropic effect. It is interesting that this decrease of tension was not related to a decrease of the Ca2+ signal. Cessation of the inotropic effect without changing intracellular Ca2+ is in agreement with the effects of P5-14 observed in skinned fibers, namely, increased isometric tension in the presence and decreased isometric tension on 90 minutes of washout of P5-14 at given free Ca2+ concentrations.
The pronounced inotropic effect observed upon deterioration of the MLC-1–actin interaction demonstrates the important role of this process in regulation of cardiac contraction. Further evidence for this assumption came from the recent observation that an increased expression of the ALC-1 in the ventricle of patients with cardiomyopathy correlated with increased Ca2+ sensitivity of the contractile apparatus,23 possibly because the primary sequences of the two MLC-1 isoforms are different in the N-terminus.6 Furthermore, substitution of the skeletal MLC-1 by MLC-3, which lacks the amino-terminal actin binding region,21 increased shortening velocity in the in vitro motility assay.24 25 Thus, differential interaction between MLC-1 and actin, either isoform-dependent or by pharmacological intervention, may modulate cardiac contractile behavior.
The observed effects allow some theoretical considerations regarding the mechanism of the inotropic effect seen on inhibition of MLC-1–actin interaction. Maximal shortening velocity is a function of the detachment rate constant of cross-bridges bearing negative tension (“g2” in Reference 2626 ). Thus, inhibition of MLC-1–actin interaction modulates cross-bridge cycling kinetics by increasing detachment rate. Furthermore, the high detachment rate constant may be involved in the elevated relaxation rate (−dP/dtmax) observed on inhibition of MLC-1–actin interaction. Furthermore, force amplitude rose and the time required to peak force decreased, ie, the rate of isometric force development (ktd) increased on P5-14 incubation. ktd is given by (f+g),27 ie, the sum of attachment and detachment rate constants. We cannot conclude precisely from our study which one of the two rate constants of the cross-bridge cycle changed, but judging from the effects of P5-14 on shortening velocity, we suggest that at least the detachment rate was upregulated. Besides affecting cross-bridge cycling kinetics, deterioration of MLC-1–actin interaction may increase tension output per cross-bridge.
In conclusion, interaction between MLC-1 and actin seems to be a very effective modulator of the contractile behavior of cardiac contraction in vivo. This interaction may be a target for a new positive inotropic intervention omitting the deleterious side effects of enhanced Ca2+ in the cardiac cell.
This work was supported by Sonderforschungsbereich SFB 320 C/5 (Dr Morano) and Deutsche Forschungsgemeinschaft DFG Va-1 (Dr Vahl).
- Received September 26, 1994.
- Accepted February 13, 1995.
- © 1995 American Heart Association, Inc.
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