Maximal Actomyosin ATPase Activity and In Vitro Myosin Motility Are Unaltered in Human Mitral Regurgitation Heart Failure
Myofibrillar but not actomyosin ATPase is depressed in failing myocardium from patients with dilated cardiomyopathy. Since there is a similar depression of myofibrillar ATPase in mitral regurgitation myocardium, we investigated whether or not the hydrolytic and mechanical performances of myosin are altered by comparing the maximal actomyosin ATPase activity and the in vitro myosin motility of myocardial myosin from patients with mitral regurgitation heart failure with that of patients with normal ventricular function. The results show that there is no significant difference (P>.05) between nonfailing and failing values for either the maximal actomyosin ATPase activity (0.3 s−1·head−1) or the myosin motility (1 μm/s). These observations suggest that changes, other than in the myosin heavy chain, contribute to the altered myocardial performance in mitral regurgitation myocardium.
Heart failure is a deadly syndrome with a high rate of morbidity and mortality. Patients with severe MR have abnormal LV function, with in vivo maximum velocity of circumferential fiber shortening being decreased by ≈40% in the presence of normal wall stress.1 2 Although depressed excitation-contraction coupling may contribute to this defect,3 4 crossbridge function is also altered with a 50% reduction in myofibrillar Ca2+-activated ATPase activity.5 6 In addition, we have previously shown that the crossbridge force-time integral during the isometric twitch is increased by 90% in MR myocardium compared with nonfailing myocardium.3 Similar myocardial and crossbridge abnormalities are seen in end-stage failure in DCM.7 These findings of altered crossbridge function may relate to changes in the myosin heavy chain,8 the myosin light chains,9 10 11 or the actin filament regulatory proteins.12 13 Although Ca2+-activated myofibrillar ATPase is depressed to 70% of control values in DCM and heart failure secondary to hypertensive heart disease (pressure overload),5 6 12 14 15 16 17 Ca2+–myosin ATPase is not different from normal.6 16 This finding suggests that unlike small animals, the human myocardium responds to stress by altering the interaction between myosin and the thin filament without recourse to changes in the intrinsic enzymatic activity of myosin. The actomyosin ATPase activity of purified myosin from human MR failing myocardium has not been measured. Hence, the objective of the present study was to fill this gap and to investigate further whether, in the absence of altered myosin ATPase activity, there may still be an alteration in motion-producing ability in MR myosin.
Information about LV free wall myocardium in MR heart failure is limited because of the inability to obtain large tissue samples from humans for detailed biochemical and mechanical studies. In the present study, we report a simple isolation procedure for myosin that provides protein from minute human cardiac biopsy samples (<2 mg) in sufficient quantity to quantify the enzymatic and motion-generating capacity of myosin through actin-activated myosin ATPase and in vitro myosin motility measurements.
Materials and Methods
Collection of Human Ventricular Samples
Subepicardial tissue was obtained from seven MR patients (New York Heart Association class III failure; mean LV ejection fraction, 0.66±0.06; mean LV end-diastolic pressure, 16±2 mm Hg; four MR patients had severe regurgitation and three had moderately severe regurgitation) and from eight coronary artery bypass patients who had normal LV wall motion and normal ventricular function (ejection fraction, 0.68±0.02). Because of insufficient biopsy size, tissue from three of the eight nonfailing hearts and from two of the seven MR hearts was pooled to give the sixth sample in each category. All tissue was obtained by surgical biopsy from the anterior segment of the LV subepicardial wall shortly after cardioplegic arrest.18 Patients gave informed written consent before participating in the study, which was approved by the Committee on Human Research of the University of Vermont. There were no complications resulting from the biopsy procedure in any patient. The excised tissue was immediately submerged at room temperature, preoxygenated with BDM protective solution, and after a 60-minute recovery from surgical trauma, it was dissected into thin connective tissue–free strips ≈0.2 to 0.5 mm in diameter.18 19 The strips were then frozen by immersion in liquid nitrogen and stored at −71°C for subsequent myosin ATPase and motility measurements.
Preparation of Contractile Proteins
Myosin was isolated from minute samples dissected from the human ventricular biopsy tissue as follows: Each sample (≈2 mg wet weight) was homogenized using a glass micro–tissue grinder in 0.25 mL of 0.3 mol/L KCl, 0.01 mol/L Na4P2O7·10H2O, 1 mmol/L MgCl2, 1 mmol/L DTT, and 0.15 mol/L imidazole (pH 6.8). The homogenate was then stirred on ice for 90 minutes and subsequently centrifuged at 140 000g for 60 minutes in a Beckman TLX-100 ultracentrifuge to remove muscle residue and actin. The supernatant was diluted with 4 mL of 1 mmol/L DTT and allowed to stand on ice for 60 minutes to precipitate myosin. Myosin was collected by centrifugation at 20 000g for 20 minutes in a Beckman SW-50.1 rotor, and the pellet was dissolved in myosin buffer (0.3 mol/L KCl, 1 mmol/L H4EGTA, 4 mmol/L MgCl2, 1 mmol/L DTT, and 25 mmol/L imidazole, pH 7.4). Protein concentrations were determined with a Bio-Rad assay kit using BSA as a standard. The purification took ≈4 hours and yielded 43±16 μg (mean±SD, n=9) of myosin per 2 mg of tissue sample. The myosin was used immediately, with ≈15 μg of myosin used in the motility assay and the rest used for the actin-activated myosin ATPase assay.
Actin was extracted from chicken pectoralis acetone powder, stored in filamentous form, and fluorescently labeled overnight with tetramethylrhodamine B isothiocyanate–phalloidin (Sigma Chemical Co) at 4°C as previously described.20
Actomyosin ATPase Activity
The actomyosin ATPase activity at 30°C was determined from the rate of Pi release in a mixture containing 0.05 mg/mL myosin, 10 to 50 μmol/L actin, 25 mmol/L KCl, 0.4 mmol/L H4EGTA, 4 mmol/L MgCl2, 1 mmol/L DTT, 2 mmol/L Na2ATP, and 10 mmol/L imidazole (pH 7.0). Pi was detected with malachite green reagent.21 The maximal actomyosin ATPase activity and Km were extrapolated from a double-reciprocal plot of the ATPase rate versus actin concentration (ie, Lineweaver-Burk plot; see Fig 2⇓).
In Vitro Motility Assay
The in vitro motility assay was performed as described previously.20 In brief, actin movement by myosin was observed at 30°C in assay buffer consisting of 25 mmol/L KCl, 25 mmol/L imidazole, 1 mmol/L H4EGTA, 4 mmol/L MgCl2, 10 mmol/L DTT, 0.1 mg/mL glucose oxidase, 0.018 mg/mL catalase, and 2.3 mg/mL glucose (pH 7.4). The latter three reagents were used to remove oxygen and retard photobleaching. A 10-μL flow cell was constructed from a nitrocellulose-coated coverslip, two glass spacers, and a glass microscope slide. First, 10 μL of myosin (100 μg/mL in myosin buffer) was infused into the flow cell and allowed to bind to nitrocellulose surface. After 60 seconds, 20 μL of BSA (0.5 mg/mL in myosin buffer) was perfused through to wash out unbound myosin and to block any exposed nitrocellulose surface. Then 10 μL of fluorescently labeled actin (5 μg/mL in assay buffer) was introduced and allowed to bind to myosin for 60 seconds. Unbound actin was washed with 20 μL of assay buffer. Finally, to initiate actin movement by myosin, 30 μL of assay buffer containing 1 mmol/L MgATP and 0.5% (wt/vol) methyl cellulose was perfused through the flow cell. Actin filament movement was observed through an inverted microscope equipped for rhodamine epifluorescence.20 22 Actin images were obtained with an image-intensified video camera (Hamamatsu C2400-97), corrected for uneven background illumination with a digital image processor (Hamamatsu, Argus 10), and finally recorded on a VHS videotape (Panasonic AG-7300).22 Videotaped images of actin filament motion were digitized by a video grabber card (Oculus 300, Coreco Inc) in a personal computer. For each actin filament, 10 images were digitized at an interval of 0.5 to 2 seconds. Actin filament velocity was then calculated from these images using a computer program developed in our laboratory.22
A novel aspect of the present study is the ability to isolate myosin from <2 mg of human myocardium with sufficient purified myosin recovered to run SDS-polyacrylamide gels, actomyosin ATPase activity measurements, and in vitro motility assays. SDS-PAGE (Fig 1⇓) shows that myosin preparations isolated from ventricular tissues of patients with NF and MR were similar in their composition of heavy chains and light chains. No evidence existed for loss of either the essential (LC1) or the regulatory (LC2) light chains from MR or NF preparations. The purity of the preparation was excellent, with only slight amounts of actin and tropomyosin remaining.
The effect of actin on the myosin ATPase activity for human ventricular myosin isolated from NF and MR myocardium is shown in Fig 2⇓ in the form of ATPase rates versus actin concentration. These data are also presented as Lineweaver-Burk plots in the insets. The inverse of the y-axis intercept of the Lineweaver-Burk plots is an estimate of the maximal actomyosin ATPase activity rate at saturating actin concentration, whereas the inverse of the x-axis intercept provides an estimate for the association constant of myosin for actin (Km). The maximal actomyosin ATPase activities and Km values for NF and MR did not differ significantly from each other (P>.05, Table). Maximal ATPase rates for individual samples from both myosins ranged from 0.20 to 0.41·s−1·head−1, whereas Km ranged from 2 to 7 μmol/L actin (Table⇓).
Actin filament movement over NF and MR myosins was observed with the quality of movement judged to be excellent, with >85% of all filaments moving continuously within a visual field. Composite histograms of actin filament velocities over NF and MR myosins from all hearts (NF, 619 filaments; MR, 446 filaments) are shown in Fig 3⇓. The NF (solid bars) and MR (striped bars) histograms are not statistically different (P>.05), with velocity means and SDs of 1.3±0.4 and 1.0±0.3 μm/s for NF and MR, respectively. Actin filament velocities for NF and MR myosins were also compared by determining the mean velocity for all filaments from each heart and then averaging these mean values (n=6 for both NF and MR) to get a mean for each group. Using this approach, once again actin filament velocities were not different for NF and MR myosin (see Table).
The present study shows that both the hydrolytic and the mechanical performances of human ventricular myosin are unaltered in heart failure due to MR. This conclusion is supported by both the maximal actomyosin ATPase activities and the in vitro myosin motility being the same for control and failing human myocardium (Figs 2 and 3⇑⇑ and the Table⇑). This conclusion is consistent with previous reports of unchanged myosin ATPase activity in hypertrophied23 24 25 and end-stage failing human myocardium.6 16 Our study extends this finding to MR failing myosin and includes the new finding that its motion-producing capacity is also unaltered.
The present results do not rule out the possibility that ventricular myosin is structurally modified in MR heart failure. For example, an atrial-like LC1 has been detected in the ventricles of hypertrophied human hearts.10 11 Rupp and Jacob8 have reported that in an improved pyrophosphate gel system, human ventricular myosin migrates down the gel as two distinct bands (VA and VB). VA migrated faster than rat cardiac V1, and VB comigrated with rat cardiac V3. Both bands were observed in normal and diseased hearts (MR and mitral stenosis), but the VA-to-VB ratio was influenced by the preload on the heart.
In addition to heavy and light chain isoform differences, alterations in the level of LC2 phosphorylation may play a role in heart failure. If LC2 phosphorylation is required for normal mechanical activity, then the observation that LC2s were completely dephosphorylated in end-stage failing myocardium (DCM)9 might explain the altered mechanics of the heart. The effect of phosphorylation could not be addressed in the present study because of the small tissue sample size. In addition, the human biopsy samples were preincubated and stored (>2 hours) in a solution containing 30 mmol/L BDM, which is likely to have completely dephosphorylated the LC2,18 given the apparent inhibition by BDM of myosin light chain kinase activity.26
The similarities in actin filament velocities over myosin from nonfailing and failing MR human hearts are not consistent with the data of Hajjar and Gwathmey27 from end-stage DCM hearts. These investigators found that both the rate of crossbridge cycling, determined from dynamic stiffness measurement, and the maximum unloaded shortening velocity (Vo) were reduced by 40% in human DCM myocardium. Although technical differences may be present (eg, the DCM fibers may not have been fully activated, reducing Vo because of the existence of a viscoelastic internal load),28 this depression may also reflect the more severe heart failure (class IV) than in the present study (class III) or the difference in etiology or ventricular stress in DCM compared with MR.
Myofibrillar ATPase activity is significantly reduced in congestive heart failure due to coronary artery disease, mitral valve insufficiency, or idiopathic cardiomyopathy.6 12 16 These data in conjunction with our observation that the actin-activated myosin ATPase activity is unaltered in myosin from class III MR failing human hearts suggest that a change in the regulatory proteins of the thin filament may underlie depression of myofibrillar ATPase activity. Indeed, Anderson and colleagues12 13 have shown that the relative expression of the troponin T isoform 4 (formerly troponin T-2) is increased in end-stage DCM failing human myocardium and that the increase correlates well with reduced myofibrillar ATPase activity.
It is interesting to note that the reduced myofibrillar ATPase activity observed by Pagani et al6 for MR myocardium (21.6±4.8 versus 41.8±2.2 nmol Pi·mg protein−1·min−1 at 30°C, pCa 5, for MR and NF, respectively) is comparable (ie, 0.25 s−1·head−1, calculated for MR using their values of percentage of myofibrillar protein per milligram tissue weight) to the actomyosin ATPase rates per myosin head we report for both MR and NF myocardium (see the Table⇑). Since the similar ATPase rates in the present study were obtained using unregulated actin filaments, the 1.9-fold higher myofibrillar ATPase rates found in NF myocardium by Pagani et al suggest that actin-linked regulatory proteins that are expressed in normal myocardium are capable of enhancing the hydrolytic activity of myosin. Therefore, it is possible that the reduced myofibrillar ATPase activity in MR is the result of a shift to actin-linked regulatory protein isoforms that do not have the ability to enhance the hydrolytic activity of myosin. In fact, in MR, the loss of enhanced enzymatic activity results in a myofibril that behaves effectively as an unregulated system, in which kinetics of the crossbridge cycle are significantly different than in NF myocardium.
Our previous observations showing3 29 30 that the average crossbridge isometric force-time integral is increased in MR class II-III failing human myocardium as well as in end-stage DCM failing myocardium may also be explained to some degree on the basis of altered crossbridge kinetics. The crossbridge force-time integral is the product of the crossbridge isometric unitary force and the time the crossbridge remains attached during its power stroke. The higher crossbridge force-time integral in failing hearts can result from each myosin crossbridge either generating a higher unitary force, or by being attached for a longer time, or by a combination of the two. It is conceivable that altered actin-linked regulatory protein isoforms in MR myocardium decreases the crossbridge detachment rate from actin. This decrease could contribute to both increased crossbridge force-time integral and to decreased myofibrillar ATPase activity in MR myocardium.
It is difficult to infer from our ATPase rate and actin filament velocity data, obtained under unloaded conditions, whether the isometric force producing capacity is altered in myosin from failing human hearts under isometric conditions. However, in vitro force measurements at the level of a single myosin molecule31 may answer this question.
Selected Abbreviations and Acronyms
|LC1, LC2||=||myosin light chain-1 and -2|
|NF||=||normal LV function|
This study was supported by funds from the National Institutes of Health to Dr Warshaw (HL-45161) and Dr Alpert (HL-28001) and by grants from the Alberta Heart and Stroke Foundation to Dr ter Keurs. Dr ter Keurs is a Medical Scientist of the Alberta Heritage Foundation for Medical Research (AHFMR). The-Tin Nguyen holds a studentship of the AHFMR.
Reprint requests to Dr David M. Warshaw, Department of Molecular Physiology and Biophysics, College of Medicine, University of Vermont, Burlington, VT 05405. E-mail firstname.lastname@example.org.
- Received January 26, 1996.
- Accepted May 2, 1996.
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