Determination of rate constants for turnover of myosin isoforms in rat myocardium: implications for in vivo contractile kinetics

Abstract

The ventricles of small mammals express mostly α-myosin heavy chain (α-MHC), a fast isoform, whereas the ventricles of large mammals, including humans, express ∼10% α-MHC on a predominately β-MHC (slow isoform) background. In failing human ventricles, the amount of α-MHC is dramatically reduced, leading to the hypothesis that even small amounts of α-MHC on a predominately β-MHC background confer significantly higher rates of force development in healthy ventricles. To test this hypothesis, it is necessary to determine the fundamental rate constants of cross-bridge attachment (f_app) and detachment (g_app) for myosins composed of 100% α-MHC or β-MHC, which can then be used to calculate twitch time courses for muscles expressing variable ratios of MHC isoforms. In the present study, rat skinned trabeculae expressing either 100% α-MHC or 100% β-MHC were used to measure ATPase activity, isometric force, and the rate constant of force redevelopment (k_tr) in solutions of varying Ca²⁺ concentrations. The rate of ATP utilization was ∼2.5-fold higher in preparations expressing 100% α-MHC compared with those expressing only β-MHC, whereas k_tr was 2-fold faster in the α-MHC myocardium. From these variables, we calculated f_app to be approximately threefold higher for α-MHC than β-MHC and g_app to be twofold higher in α-MHC. Mathematical modeling of isometric twitches predicted that small increases in α-MHC significantly increased the rate of force development. These results suggest that low-level expression of α-MHC has significant effects on contraction kinetics.

the myosin molecule, a hexamer composed of two heavy chains (∼220 kDa) and four light chains (16–25 kDa), is the principal component of the thick filament and is responsible for the generation of force in mammalian striated muscle. The rod-shaped COOH-terminal domain of the myosin molecule (light meromyosin) forms the backbone of the thick filament, whereas the globular NH₂-terminus (subfragment 1) contains the actin-binding and catalytic domains (13). Thus, S1 is an important determinant of contractile speed and power developed in the mammalian myocardium.

Two isoforms of myosin heavy chain (MHC), α and β, have been identified in cardiac muscle (23). These MHC isoforms share 93% amino acid sequence homology (32), and yet they confer distinct functional properties to the myocardium: α-MHC has been shown to have markedly higher ATPase activity, faster shortening velocity (V_max), and faster rates of force development (42), whereas β-MHC exhibits a lower tension cost and is thus more efficient (1, 24, 40).

Expression levels of the ventricular MHC isoforms are species dependent and are both developmentally and hormonally regulated (8, 30, 42). The predominant fetal isoform in all mammalian ventricular myocardium is β-MHC, wheras in adults there is a considerable variation in α-MHC content that scales inversely with size: in rodents, the predominant adult isoform is α-MHC (30), whereas the myocardium of larger adult mammals, including humans, expresses predominately β-MHC (29). Several other factors are known to affect the relative proportions of α-MHC and β-MHC in the myocardium. Elevation of thyroid hormone increases the expression of α-MHC, whereas hypothyroidism, aging, and pressure overload all increase the proportion of β-MHC (46). In addition, hypothyroidism completely abolishes α-MHC expression in the rodent ventricular myocardium, whereas hyperthyroidism increases α-MHC expression to virtually 100% of the total MHC content (46). Moreover, MHC content in mammalian myocardium varies regionally, as characterized by the increased expression of α-MHC in the epicardium compared with the endocardium (7).

Animal models of heart failure have often focused on myosin isoform composition as a possible cause of depressed myocardial performance due to the reduced expression of α-MHC. The higher levels of β-MHC in heart failure confer greater contractile efficiency due to reduced tension cost, but this advantage comes at the expense of a slower rate of rise of force and reduced power. It was long believed that the myosin expressed in adult human ventricles was 100% β-MHC (18), and thus it seemed unlikely that alterations in MHC isoform content had any bearing on depressed contractile kinetics in heart failure. However, recent work has shown that normal adult human ventricles express nearly 10% (average of 7% and up to 13% of total myosin content) α-MHC on a background of ∼90% β-MHC, and this distribution switches to 100% β-MHC in heart failure (34). In addition, α-MHC mRNA decreases from 25–35% of total MHC mRNA in normal human hearts to 2% during end-stage ventricular failure (31, 35).

Because of the faster turnover kinetics of α-MHC, we propose that the small amounts of α-MHC on a predominately β-MHC background normally accelerate the rate of myocardial force development and that reduced α-MHC expression (as in heart failure) depresses the kinetics of force development. Consistent with this idea, increased β-MHC expression due to thyroid deficiency significantly slows contractile kinetics. Euthyroid rats expressing ∼80% α-MHC had markedly greater unloaded shortening velocities and rates of force development compared with myocardium from thyroid-deficient rats expressing 100% β-MHC (12), and shortening velocities under load were also faster in euthyroid compared with hypothyroid rats (20). While single molecule experiments have indicated that there is no difference in force per cross-bridge for α-MHC and β-MHC isoforms, the duty cycle (fraction of the cross-bridge cycle spent in force-generating states) of β-MHC is approximately twice that for α-MHC (37). Another study (21) varied MHC isoform expression by thyroid manipulation and showed that the expression of ∼10% α-MHC on a predominately β-MHC background increased shortening velocities at intermediate loads and thus increased power compared with preparations expressing 0% α-MHC.

We tested the idea that low-level expression of α-MHC on a predominately β-MHC background significantly accelerates force development in myocardium. We first measured isometric force, ATP utilization, and the rate constant of force redevelopment (k_tr) in rats expressing either 100% α-MHC or 100% β-MHC and then used those values to derive the rate constants of cross-bridge attachment (f_app) and detachment (g_app) for both MHC isoforms. These rate constants were used to mathematically model an isometric twitch to predict the functional consequences of small changes in the level of expression of α-MHC.

MATERIALS AND METHODS

Experimental Animals

Control and thyroidectomized female Sprague-Dawley rats (Harlan Laboratories, Madison, WI) were housed in groups of 2–3 rats/cage in a light- and temperature-controlled (22–23°C) room. Rats (6–9 mo of age) were divided into two separate groups, and each group was fed a specific diet for a period of 5 wk (27). All thyroidectomized rats (n = 17) were fed a diet of 0.15% propylthiouracil (PTU; Sigma, 0.15 g PTU/100 g rat meal), which has been shown to eliminate any residual plasma triiodothyronine and thyroxine after thyroidectomy (19). Control rats (n = 17) were fed a diet of 0.15% thyroid extract (Sigma, 0.15 g thyroid extract/100 g rat meal). An additional group of 2.5- to 3-mo-old female Sprague-Dawley rats (n = 8) was thyroidectomized but not treated with PTU to generate myocardium with low-level expression of α-MHC, wheras a final group of young (2.5–3 mo old) euthyroid Sprague-Dawley rats (n = 7) was used to obtain myocardium expressing ∼80% α-MHC. All rats had access to food and water ad libitum. No animal showed any signs of distress throughout the feeding protocol, and therefore none of the rats were excluded from the study. Animals were anesthetized by the deep inhalation of isoflurane (15% isoflurane in mineral oil). All animal use was conducted under the strict guidelines and approval of the University of Wisconsin Animal Care and Use Committee.

Experimental Preparations

Skinned trabeculae were used to measure isometric force and ATPase activity. Animals were handled with care to minimize stress levels and limit alterations in contractile protein phosphorylation status. The heart was rapidly removed from the anesthetized animal and placed in a dissecting dish filled with Ringer solution containing (in mM) 120 NaCl, 5 KCl, 1.2 MgSO₄, 1 CaCl₂, 19 NaHCO₃, 1.2 Na₂HPO₄, and 10 glucose. The solution was bubbled with 95% O₂-5% CO₂. Long unbranched trabeculae (1–3 mm × 75–250 μm) were located in the right ventricle and then equilibrated for 10 min in fresh Ringer solution containing 15 mM 2,3-butanedione monoxime before dissection to minimize mechanical damage. Trabeculae were then cut free, tied to wooden dowels with 10-0 suture, and bathed in relaxing solution (4°C) containing 1% Triton X-100. After being skinned for ∼24 h to remove membranes, trabeculae were washed for 60 min in cold relaxing solution and stored at −20°C in 50% glycerol and relaxing solution for up to 5 days before use.

Measurements of k_tr were performed in separate preparations in a setup having a high-speed force transducer. This was necessary because of the low-frequency response of the transducer in the ATPase setup, which necessarily has a stylus of large mass. To prepare skinned myocardial preparations for measurements of k_tr, frozen ventricles that had been rapidly frozen in liquid nitrogen were homogenized in relaxing solution for ∼2 s using a Polytron, which yielded multicellular preparations of 100–250 × 600–900 μm. The homogenate was centrifuged at 120 g for 1 min, and the resulting pellet was washed with fresh relaxing solution and resuspended in relaxing solution containing 1% Triton X-100. After a 30-min permeabilization period, skinned preparations were washed with fresh relaxing solution and then dispersed in relaxing solution in a glass petri dish. The dish was kept on ice at all times except during the selection of preparations for mechanical experiments.

Solutions

The compositions of solutions used for mechanical experiments were calculated using the computer program of Fabiato (9) and the stability constants (corrected to pH 7 and 20°C) listed by Godt and Lindley (17). The relaxing solution (pH 7.0) used for bathing skinned trabeculae contained (in mM) 100 KCl, 20 imidazole, 7 MgCl₂, 2 EGTA, and 4 ATP. In addition, the pCa 9.0 solution contained 5.86 ATP, 6.53 MgCl₂, 20 EGTA, 48.23μM CaCl₂, 100 N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), the pCa 4.5 solution contained 5.89 ATP, 6.01 MgCl₂, 20 EGTA, 19.85 CaCl₂, and 100 BES, and the preactivating solution contained 0.5 EGTA. In additon, pCa 9.0, pCa 4.5, and preactivating solutions contained 5 NaN₃, 0.01 leupeptin, 0.01 P¹-P⁵-di(adenosine-5′)pentaphosphate, and 10 phospho(enol)pyruvic acid. The ionic strength of all solutions was adjusted to 200 mM with potassium propionate. Solutions containing a range of free Ca²⁺ concentrations ([Ca²⁺]_free; i.e., pCa 5.8–5.4) were prepared by mixing the appropriate volumes of pCa 9.0 and pCa 4.5 solutions. Pyruvate kinase (0.91 mg/ml) and lactate dehydrogenase (0.31 mg/ml) were added to all pCa and preactivating solutions before mechanical experiments. Solutions used for the measurements of k_tr were as previously described (43).

Experimental Protocols

ATPase experiments.

While immersed in relaxing solution, a trabecula was attached to the arms of a high-speed length controller (model 312B, Aurora Scientific) and a force transducer (model 403A, Aurora Scientific) by fixing each end to a stainless steel trough with a 0.5-mm length of 4-0 monofilament secured in place with loops of 10-0 monofilament. This procedure was optimized to virtually eliminate compliance at the ends and leave 750–1,000 μm of trabecula exposed to the solution, which allowed accurate measurement of sarcomere length via the diffraction pattern obtained with a He-Ne laser, as described below.

Three variables were measured to calculate f_app and g_app (3): ATPase activity per preparation volume [proportional to (f × g)/(f + g)], tension cost (proportional to g/unified force), which is defined as the rate of ATP turnover divided by isometric force, and k_tr (k_tr = f + g) in response to a brief period of unloaded isotonic shortening followed by rapid restretch. Measurements of preparation dimensions were done during the experiment to allow calculations of sample volume, and cross-sectional area was determined assuming an elliptical geometry.

Isometric force and ATPase activity were recorded in solutions of varying Ca²⁺ concentrations (pCa 5.8–4.5) at 20°C at a mean sarcomere length of 2.1 μm. The active force at each pCa was measured as the difference between total steady-state force and passive force determined by applying a 20% slack step to the preparation in a solution of pCa 9.0. Active force was normalized to the cross-sectional area of the preparation. A He-Ne laser (wavelength 632.8 nm) was directed toward the mounted preparation, and the resulting diffraction pattern was projected onto a screen. Measurements of the distance between the zero- and first-order lines were then used to calculate sarcomere length, a method that is accurate to within 0.1 μm. In addition, careful monitoring of sarcomere length throughout each experiment quickly revealed compliance in the attachments, and, in such cases, the preparation was discarded.

ATP consumption was measured using a NADH-coupled enzyme system (16, 44). ADP production during ATP hydrolysis was measured as the change in NADH concentration:

NADH absorbs UV light at a wavelength of 339 nm, whereas NAD⁺ does not; thus, ATP consumption can be measured as the change in absorbance at 339 nm (16, 44). The experimental chamber consisted of a 15-μl well enclosed on two sides by quartz windows. Near-UV light was directed to the skinned myocardial preparation in the experimental chamber, and the transmitted beam was split and directed to two photodiodes: one to record the transmitted light at 339 nm (for the determination of NADH concentration) and the other to correct the signal for photobleaching and to determine nonspecific absorbance at a reference wavelength other than 339 nm. NADH (70 mM) was injected into the measuring chamber before the incubation of the trabecula while calibration of the ATPase signal was performed with 25-nl injections of 10 mM ADP into the measuring chamber. During experiments, the solution in the measuring chamber was continually stirred via motor-driven displacement of a latex membrane on the bottom of the chamber. Recording of the two signals was necessary to minimize noise that may arise from irregularities due to continuous mixing. The rate of NADH consumption was taken as the ATPase activity and determined by linear regression analysis of the measured absorption trace.

Ca²⁺ sensitivity and kinetics of force redevelopment.

The active force at each pCa was measured as the difference between total steady-state force and passive force determined by applying a 20% slack step to the preparation in a solution of pCa 9.0. Force-pCa relationships were derived by expressing submaximal force (P) at each pCa (range: 6.1–5.5) as a fraction of maximum force (P_o) determined at pCa 4.5 (P/P_o). The force required for half-maximal activation (pCa₅₀) was determined by fitting the force-pCa data with the following Hill equation: P/P_o = [Ca²⁺]^nH/(k^nH + [Ca²⁺]^nH), where n_H is the Hill coefficient and k is the Ca²⁺ concentration required for pCa₅₀.

k_tr was estimated in rat skinned multicellular preparations expressing 100% α-MHC or 100% β-MHC using a modified experimental protocol originally described by Brenner and Eisenberg (5). After a 1-min incubation in preactivating solution, each preparation was transferred to solutions ranging from maximal [Ca²⁺]_free (pCa 4.5) to submaximal [Ca²⁺]_free (pCa 6.1–5.5). Once steady-state force had developed, the preparation was rapidly slackened (<2 ms) by 20% of its original length, resulting in a reduction in force to near zero. After a brief period (10 ms) of unloaded isotonic shortening, the preparation was rapidly restretched to its original length and force redeveloped to the original steady-state level. The apparent k_tr was estimated by linear transformation of the half-time of force redevelopment (t_1/2), i.e., k_tr = 0.693/t_1/2, as previously described (43).

Isolation of single myocytes with enzymatic digestion.

In addition to measurements in trabeculae, single ventricular myocytes were used to measure isometric force, as previously described (45). The thyroid status of female Sprague-Dawley rats was manipulated to generate hypothyroid and hyperthyroid rats expressing 100% β-MHC or 100% α-MHC, respectively. Rats were anesthetized as described above. The heart was rapidly excised, and the aorta was cannulated for retrograde perfusion on a modified Langendorff apparatus. The heart was first perfused for 2–5 min with Ringer solution (pH 7.0) containing (in mM) 118 NaCl, 4.8 KCl, 4.2 NaH₂PO₄, 25 HEPES, 11 glucose, 1.2 MgCl₂, and 1 CaCl₂ followed by perfusion with Ca²⁺-free Ringer solution. After ∼2 min, the heart was perfused with Ca²⁺-free Ringer solution containing 2.5 mg/ml collagenase (type II, Worthington) and 3.0 mg/ml hyaluronidase (Sigma) for 10 min followed by the addition of 250 μl of 0.1 M CaCl₂ and perfusion for an additional 15 min. The heart was then minced into small fragments and incubated in the same solution for 20 min followed by filtration through Teflon mesh. Cells were washed three times in Ringer solution containing 0.2 mM Ca²⁺ and resuspended in 0.3% Triton X-100 relaxing solution for 10 min. After being skinned, cells were washed with relaxing solution, centrifuged for 1 min at low rpm, resuspended in fresh relaxing solution, and then stored on ice until use. All solutions were maintained at 37°C and bubbled with 100% O₂ during the perfusion process.

Single myocyte experiments.

Single rod-shaped myocytes were fixed between thin metal wires with silicone adhesive (Dow Corning) to the stage of a Zeiss inverted microscope. One wire was attached to a force transducer (model 403A, Cambridge Technologies), and the other wire was attached to a length controller (model 6800 HP, Cambridge Technologies). Each element could be maneuvered independently via micromanipulators (Narishige). Isometric force was measured at maximal Ca²⁺ concentration at 20°C and at a mean sarcomere length of 2.1 μm. The active force was measured as the difference between total steady-state force and passive force determined by applying a 20% slack step to the preparation in a solution of pCa 9.0. Active force was normalized to the cross-sectional area of the preparation. Force was sampled (16-bit resolution, DAP5216a, Microstar Laboratories, Bellevue, WA) at rates of 2 kHz using SLControl software developed in our laboratory (6) and saved to data files for analysis.

Quantification of MHC expression.

After each mechanical experiment, the trabecula was cut free at the points of attachment and placed in 10 μl SDS sample buffer (62.5 mM Tris, 75 mM DTT, 25% glycerol, 25 mM SDS, and 0.01% bromophenol blue) and stored at −80°C until the subsequent analysis of MHC isoform content by SDS-PAGE. α- and β-MHC isoforms were separated using 6% acrylamide gels, which were stained using a modified silver stain technique as previously described (15, 47). This method reproducibly detects 2 ng protein/band and has a linearity of stain between 2 and 70 ng protein/band. The proportions of ventricular MHC isoforms were then determined by densitometric analysis of silver-stained gels using an imaging densitometer and LaserPix software (Bio-Rad Laboratories). The proportions of α-MHC and β-MHC were estimated by expressing the area under the peak for each isoform as a fraction of the total area for the two isoforms. To assure the linearity of staining and accuracy of measurements, all samples were serially diluted and run in parallel lanes next to MHC standards from myocardium expressing 100% α-MHC or 100% β-MHC.

Determination of the level of phosphorylation of myofibrillar proteins.

Myofibrillar proteins from control, hyperthyroid, and hypothyroid rat myocardium were separated by SDS-PAGE using 12.5% Tris·HCl Criterion gels (Bio-Rad Laboratories). To determine the level of protein phosphorylation, different volumes (3, 4, 6, and 8 μl) from control, hyperthyroid, and hypothyroid skinned myocardium were loaded on the same gel. Phosphoproteins were detected using a Pro-Q Diamond staining protocol (Molecular Probes) followed by staining with SYPRO ruby (Molecular Probes) for the determination of total protein content. Briefly, gels were fixed in 50% methanol-10% glacial acetic acid for 1.5 h with solution changes every 30 min, washed with double-distilled H₂O six times for 5 min each, stained with Pro-Q Diamond stain for 1.5 h, and then destained with Pro-Q Diamond destaining solution (Molecular Probes) overnight. For the determination of myofibrillar protein content, gels were stained with SYPRO ruby (Molecular Probes) for 3 h and destained with SYPRO ruby destain solution (10% methanol-7% glacial acetic acid) for 2 h with solution changes every 30 min. Immediately after each staining protocol, gels were imaged using a UVP BioImaging System (UVP, Upland, CA) and analyzed using LabWorks 4.5 (UVP). The area of protein and phosphoprotein bands was multiplied by mean optical density and plotted versus volume loaded, and a first-order linear regression was fitted to the data points to determine the slope of the relationship between optical density and volume loaded.

Data analysis.

All data are reported as means ± SE. To test for the significance of effects of altered MHC content on ATP utilization, steady-state force, and k_tr at different levels of Ca²⁺ activation, one-way ANOVA with a Tukey post hoc test for significance (P < 0.05) was used.

RESULTS

Alterations in MHC Expression Due to Manipulation of Thyroid Status

SDS-PAGE analysis of all myocardial preparations used for mechanical measurements revealed that the thyroidectomy of rats and dietary supplementation with PTU completely abolished the expression of α-MHC (Fig. 1) and thus converted the rat myocardium to 100% β-MHC (99.8 ± 0.3%, n = 10). In contrast, euthyroid rats that received dietary supplementation with thyroid extract expressed nearly 100% α-MHC (97.8 ± 0.5%, n = 12). Previous studies (12, 42) using manipulation of rat thyroid status have shown large changes in the expression of ventricular MHC isoforms without altering the expression of myosin light chains (MLC_1V or MLC_2V) or thin filament regulatory proteins. In addition, analysis of phosphoprotein gels using Pro-Q Diamond and SYPRO ruby staining (Fig. 2) indicated that the basal levels of phosphorylation of cardiac myosin-binding protein C, cardiac troponin T, cardiac troponin I, and myosin regulatory light chain were similar in the control, hyperthyroid, and hypothyroid rat myocardium. Thus, the observed differences in kinetics in hyperthyroidism and hypothyroidism can be attributed solely to the distribution of MHC isoforms.

Fig. 1.

Myosin heavy chain (MHC) composition of the rat skinned myocardium. MHC isoform expression was determined with 6% SDS-PAGE. The relative proportions of α-MHC and β-MHC were determined by densitometric analysis (LabWorks software) of silver-stained gels. This representative gel shows that hypothyroid rats typically expressed 100% (99.8 ± 0.2, n = 10) β-MHC (lane 2) and hyperthyroid rats expressed nearly 100% (97.8 ± 0.5, n = 12) α-MHC (lane 3). Lane 1 shows the MHC content of control euthyroid rat myocardium (65% α-MHC and 35% β-MHC).

Fig. 2.

A: determination of protein phosphorylation in the rat myocardium. The level of myofibrillar protein phosphorylation was determined in control, hyperthyroid, and hypothyroid rat myocardium (n = 4 from each group) using SYPRO ruby staining for total protein content (left) and Pro-Q Diamond staining for the determination of protein phosphorylation, as shown in this representative SDS-PAGE gel at a single loading concentration (6 μl; right). Volumes of 3, 4, 6, and 8 μl were loaded in successive lanes for each sample preparation. cMyBP-C, cardiac myosin-binding protein-C; TnT, troponin T; TnI, troponin I; RLC, myosin regulatory light chain. B–E: regression analysis of myocardial protein phosphorylation in rat myocardium. Levels of protein phosphorylation in euthyroid, hyperthyroid, and hypothyroid rat myocardium were compared in Pro-Q Diamond-stained SDS-PAGE gels for the following proteins: cMyBP-C (B), TnT (C), TnI (D), and RLC [myosin light chain 2 (MLC2); E]. Regression lines for optical band intensity versus protein load were determined for both Pro-Q Diamond- and SYPRO ruby (not shown)-stained gels, and the ratio of regression line slopes was calculated. The ratio was normalized to euthyroid rat myocardium for each protein (see insets). AU, arbitrary units.

Steady-State Force Development in the Rat Myocardium With Altered MHC Composition

Table 1 shows the steady-state mechanical properties of rat skinned trabeculae expressing either 100% α-MHC or 100% β-MHC. Altered MHC composition had no effect on the Ca²⁺ sensitivity of force, and the steepness of the force-pCa relationship (n_H) did not vary between the preparations, indicating that MHC composition does not influence the apparent cooperativity of tension development. Our findings did reveal that maximum Ca²⁺-activated force (pCa 4.5) was reduced in preparations expressing 100% β-MHC (Table 1), which differs from previous reports (12, 20, 41, 43) showing that maximum force did not change with the expression of MHC isoforms. However, measurements of maximal force per cross-sectional area in enzymatically digested skinned single myocytes from hypothyroid and hyperthyroid rat myocardium revealed no differences (6.5 ± 1.0 and 7.4 ± 0.7 kN/m², respectively). These results suggest that the depressed force in the trabeculae of hypothyroid rats may be due to changes in the expression of extracellular elements such as collagen, which has been shown to be increased in rats treated with PTU (49). Alterations of force due to increased collagen would not have been evident in homogenized preparations obtained via mechanical disruption, as in previous reports (12, 20, 41, 43).

Table 1.

Effect of MHC composition on steady-state mechanical properties of rat myocardium

	n	Ca2+-Activated Force in Trabeculae, mN/mm2	nH	pCa50
100% α-MHC	12	41.4±2.3	4.06±0.32	5.78±0.01
60% α-MHC	10	41.8±1.6	4.31±0.35	5.78±0.02
0% α-MHC	10	31.3±1.6*	4.13±0.36	5.79±0.01

Data are means ± SE; n, number of cardiac preparations. MHC, myosin heavy chain; Ca²⁺-activated force, difference between maximal force generated at pCa 4.5 and rest force generated at pCa 9.0; n_H, Hill coefficient for the total force-pCa relationship; pCa₅₀, pCa required for half-maximal force generation.

^*Significantly different from hyperthyroid (100% α-MHC) myocardium (P < 0.05).

It should be noted that there were no differences in passive tension between euthyroid (1.46 ± 0.11 mN/mm²), hyperthyroid (1.65 ± 0.16 mN/mm²), and hypothyroid (1.34 ± 0.1 mN/mm²) rat myocardium. Passive tension would most likely be influenced by the distribution and expression of titin isoforms (N2B and N2BA), and while it is unlikely that alterations in titin exist in the hyperthyroid and hypothyroid myocardium used in the present study, titin would not play a role in passive tension until sarcomere lengths greater than those used here (>2.2 μm).

Effect of MHC Content on the Kinetics of Force Redevelopment

A modified release-restretch protocol (5) was used to determine k_tr in steadily Ca²⁺-activated skinned myocardium expressing 100% α-MHC or 100% β-MHC. k_tr, which is equal to f_app + g_app, provides information about the kinetics of cross-bridge transitions between non-force-generating and force-generating states. Consistent with other results (12, 41), expression of α-MHC significantly increased maximal k_tr compared with preparations expressing 100% β-MHC (Table 1). In addition, we measured k_tr in myocardium expressing variable levels of α-MHC (15%, 59%, and 81%) and found that k_tr increased linearly with the expression of α-MHC (Fig. 3). This finding is in agreement with other studies (11, 41) investigating the rate of tension development as a function of MHC isoform expression and suggests that the presence of slower cycling β-MHC cross-bridges is unlikely to impose significant drag on k_tr. k_tr values in the groups expressing intermediate levels of α-MHC (15%, 59%, and 81%) were 12.93 ± 0.21, 17.94 ± 1.4, and 20.78 ± 0.7 s⁻¹, respectively, and the relationship between k_tr and the expression of α-MHC shown in Fig. 3 was well fit by linear regression (r² = 0.994).

Fig. 3.

Effect of the percent α-MHC expression on the maximal rate constant of force redevelopment (k_tr) after a rapid slack-restretch maneuver. k_tr was measured in four groups of rat myocardium with variable expression of α-MHC: 0% α-MHC, hypothyroid [thyroidectomized with propylthiouracil (PTU) treatment; ○]; 15.4% α-MHC, hypothyroid (thyroidectomized without PTU treatment; ▴); 59% α-MHC, euthyroid (□); 81.4% α-MHC (▪); and 97.8% α-MHC, hyperthyroid (thyroid hormone treatment; •). The groups expressing 15.4% and 81.4% α-MHC were obtained from young (2.5–3 mo old) Spraque-Dawley rats. Data are means ± SE.

Effects of MHC content on the Rate of ATP Utilization

The effect of MHC content on ATPase activity was measured using an ATP-enzyme-coupled assay as described above and is shown in Table 2. Linear regression of the absorbance signal of NADH indicated that the preparations expressing 100% α-MHC exhibited rates of ATP utilization that were ∼2.5-fold higher than preparations expressing 100% β-MHC (Fig. 4). This finding is consistent with the k_tr data from preparations of α-MHC or β-MHC, a correlation that presumably manifests in greater rates of cross-bridge cycling in myocardium expressing 100% α-MHC.

Table 2.

Effect of MHC composition on parameters of cross-bridge cycling

	n	ktr, s−1	ATPase, pmol·s−1·mm−3	Tension Cost, pmol·s−1·mm−1·mN−1	fapp	gapp
100% α-MHC	12	23.9±1.2	624.29±23.7	15.09±0.75	6.84	17.06
60% α-MHC	10	17.9±1.4*	531.91±44.2	13.25±1.15	NA	NA
0% α-MHC	10	11.4±0.7*	247.92±13.9*	7.92±0.95*	2.47	8.93

Data are means ± SE; n, number of cardiac preparations. k_tr, rate constant of force redevelopment in response to a rapid slack-restretch maneuver; ATPase, rate of ATP hydrolysis; tension cost, ratio of ATPase to isometric force; f_app, derived rate constant of cross-bridge attachment; g_app, derived rate constant of cross-bridge detachment. Derivations of rate constants were performed only in myocardium expressing either 100% or 0% α-MHC. NA, not applicable.

^*Significantly different from hyperthyroid (100% α-MHC) myocardium (P < 0.05).

Fig. 4.

Effect of MHC composition on the rate of ATP utilization in the rat skinned myocardium. The rate of ATP utilization was measured in the skinned myocardium from euthyroid (∼60% α-MHC, n = 10; □), hyperthyroid (100% α-MHC, n = 12; •), and hypothyroid (0% β-MHC, n = 10; ○) rats and plotted against relative force (P/P_o). Forces recorded at submaximal free Ca²⁺ (P) were expressed relative to the maximal force measured at pCa 4.5 (P_o). Data are means ± SE.

By measuring both isometric force and ATPase activity in each preparation, we were able to calculate tension cost, which is defined as theATPase rate divided by isometric force and is directly proportional to g_app (3). Tension cost was approximately twofold higher in preparations expressing 100% α-MHC compared with those expressing 100% β-MHC (Fig. 5). This finding is consistent with a previous study (41) and indicates that β-MHC is a more economical isoform, i.e., less ATP is hydrolyzed to maintain a given isometric force.

Fig. 5.

Effect of MHC composition on tension cost in rat skinned myocardium. The rate of ATP utilization was measured in the skinned myocardium from euthyroid (∼60% α-MHC, n = 10; □), hyperthyroid (100% α-MHC, n = 12; •), and hypothyroid (0% α-MHC, n = 10; ○) rats and plotted against Ca²⁺-activated isometric force. The slope of the line is equal to tension cost. Force and ATPase were measured simultaneously in solutions of varying free Ca²⁺ concentrations (pCa 4.5 and 5.4–5.8). Data are means ± SE.

Calculation of f_app and g_app and Modeling of an Isometric Twitch

To model the effects of MHC content on twitch kinetics, f_app and g_app were calculated for each isoform assuming the two-state cross-bridge model originally described by Brenner (4). This model assumes that myosin cross-bridges are either attached to the thin filament and generating force or detached and not generating force. We calculated the rate constants of cross-bridge cycling using data obtained from measurements of isometric force [proportional to f/(f + g)], k_tr (k_tr = f + g), and ATPase activity [proportional to (f × g)/(f + g)]. Mechanical measurements yielded ATPase activity per volume of 624 ± 24 pmol·s⁻¹·mm⁻³ for α-MHC and 248 ± 14 pmol·s⁻¹·mm⁻³ for β-MHC, a tension cost of 15.1 ± 0.8 pmol·s⁻¹·mm⁻¹·mN⁻¹ for α-MHC and 7.9 ± 1.0 pmol·s⁻¹·mm⁻¹·mN⁻¹ for β-MHC, and k_tr values of 23.9 ± 1.2 s⁻¹ for α-MHC and 11.4 ± 0.7 s⁻¹ for β-MHC. Together, these values can be used to solve a system of four equations with four unknowns:

(1)

(2)

(3)

(4)

The following rate constants were obtained: f = 6.84 s⁻¹ and g = 17.06 s⁻¹ α-MHC and f = 2.47 s⁻¹ and g = 8.93 s⁻¹ for β-MHC.

These rate constants were then used to model the myocardial twitch in response to a standardized Ca²⁺ transient, calculated as the difference between two exponential functions:

where k₁ and k₂ represent variables that can be altered to vary the duration of the Ca²⁺ transient. Simulations included force responses in the presence of 0%, 10%, 20%, or 30% α-MHC, all of which were calculated using the same Ca²⁺ transient. The mathematical model is in the form of one ordinary differential equation and one algebraic equation, which are solved by the standard fourth-order Runge-Kutta method.

Calculations based on these rate constants predict that the kinetics of the isometric twitch are significantly accelerated as low-level expression of α-MHC is progressively increased on a mostly β-MHC background (Fig. 6). Our model predicts that the maximum rate of rise of force (dF/dt_max) for 10% α-MHC/90% β-MHC is ∼18% faster than for 100% β-MHC (Fig. 7), whereas increases in α-MHC from 0% to 20% and 0% to 30% result in increases of 37% and 54%, respectively, in dF/dt_max. Moreover, the greatest reductions in the time required to reach peak force were observed for α-MHC expression in the range of 0–10% (Table 3).

Fig. 6.

Model predictions of twitch time courses in myocardium with variable ratios of α-MHC and β-MHC. The purple line indicates the Ca²⁺ pulse (in AU) that was used in each of the simulations. The vertical axis indicates the fraction of cross-bridges bound; the horizontal axis is time (in s). Traces represent 0%, 10%, 20%, and 30% α-MHC of the total MHC content.

Fig. 7.

Model predictions for changes in the rate of force development (dF/dt) and relaxation in myocardium with variable expression of α-MHC. dF/dt is shown on the vertical axis; the horizontal axis is time (in s). Traces represent 0%, 10%, 20%, and 30% α-MHC of the total MHC content.

Table 3.

Effect of low-level expression of α-MHC on the time required to reach peak force

	Time Required to Reach Peak Force, ms	Percent Decrease in Time to Peak Force From 0% α-MHC	Fold Decrease in Time to Peak Force From 0% α-MHC
0% α-MHC	76.8		1
10% α-MHC	72.8	5.2	0.95
20% α-MHC	70.0	8.9	0.91
30% α-MHC	67.6	12	0.88

Data were obtained from model predictions of twitch time courses in myocardium with variable ratios of α-MHC and β-MHC. Peak force was determined as the maximal force elicited by a Ca²⁺ transient. The fold decrease in time to peak force from 0% α-MHC was computed as the ratio of the time to peak force from 10%, 20%, and 30% α-MHC to 0% α-MHC. The time to peak force decreased with each incremental addition of α-MHC, with a concomitant increase in the rate of rise of force. The greatest increase in rate was observed when 10% α-MHC was compared with 0% α-MHC. When each incremental addition of α-MHC was compared with the iteration before it (e.g., 20% α-MHC compared with 10% α-MHC), the increase in the rate of rise of force became progressively smaller.

DISCUSSION

In the present study, we modeled an isometric twitch in the rat myocardium as a function of variable, low-level expression of α-MHC on a predominately β-MHC background using derived values for f_app and g_app for each of these isoforms. To accurately predict the rate constants of cross-bridge cycling, we generated myocardium expressing purely (100%) α-MHC or β-MHC by manipulating thyroid status. This method reliably alters the distribution of MHC isoforms without changing the expression levels of other myofibrillar proteins, thereby ensuring that the contractile differences simulated by the model are based solely on alterations in the distribution of MHC isoforms. Our model predicts significant increases in the ability of the myocardium to generate twitch force (in response to a standardized Ca²⁺ transient) as the level of α-MHC increases and agrees conceptually with previous work (22) showing that β-MHC behaves as a negative inotrope in rat cardiac myocytes, possibly due to a decreased number of activated cross-bridges in response to a Ca²⁺ transient. Here, small amounts of α-MHC (10% α-MHC/90% β-MHC) accelerated dF/dt_max by ∼18% compared with myocardium expressing 100% β-MHC. This increase is due to accelerated rates of both cross-bridge attachment (f_app) and detachment (g_app) and, from a functional standpoint, would be expected to increase ejection during systole. In addition, the rate of relaxation, which is based in part on g_app, increased to an even greater extent in preparations expressing α-MHC, which would aid ventricular filling. On the other hand, in conditions such as heart failure, reduced expression of α-MHC and correspondingly slower rates of cross-bridge cycling would increase the economy of contraction by increasing the cross-bridge force-time integral (38) and prolonging the time a cross-bridge remains in the force-generating state, i.e., a longer duty cycle (37). While there is thus an improvement in the energetic efficiency of the pump, the loss of α-MHC would be detrimental to myocardial performance since the rates of force development and relaxation would be slowed, twitch force would be reduced, and work capacity would be diminished (20, 21).

Changes in the Rate of ATP Utilization and Rate Constants of Cross-Bridge Cycling With MHC Expression

As mentioned above, animal models of heart failure and cardiac hypertrophy show a redistribution of myosin isoforms characterized by increased expression of β-MHC and a corresponding reduction in α-MHC, a phenomenon that is especially evident in small mammals such as rodents, which normally express α-MHC as the predominant isoform in adult ventricles (46). In addition, adult human ventricles express ∼10% α-MHC; yet, in conditions of heart failure, there is a significant reduction in α-MHC at both the transcriptional and protein levels (31, 34, 35). Thus, it is possible that the slower kinetics and decreased contractile force characteristic of heart failure involve the redistribution of myosin isoforms to nearly 100% expression of β-MHC and virtually no α-MHC. Our measurements of ATPase activity are consistent with this idea and indicate that increased content of β-MHC in failing hearts would at least account for the reduced twitch kinetics because it is a slower motor protein.

To predict the functional effects of alterations in MHC in the present study, it was necessary to determine f_app and g_app in myocardial preparations expressing 100% α-MHC or 100% β-MHC. Our estimates of these rate constants indicate that myosin composed of α-MHC exhibits a f_app value that was approximately threefold faster than for β-MHC myocardium, whereas the the g_app value was nearly twofold greater in α-MHC myocardium. In a functional context, rapid cross-bridge turnover due to higher levels of α-MHC would increase dF/dt_max in response to a Ca²⁺ transient and enhance power generation during systole, particularly when the heart is operating at or near maximal conditions. This idea is consistent with previous work (28) showing that power output varies linearly with α-MHC in both single myocytes and isolated working heart preparations from the rat. Moreover, an accelerated rate of cross-bridge detachment at the end of systole would enhance force relaxation and increase ventricular filling, thus increasing the volume of blood ejected with each beat. The influence of the myosin isoform on g_app values calculated in the present study is in agreement with other studies. Using the photolabile caged Ca²⁺ chelator diazo-2, Fitzsimons and colleagues (12) showed that tension relaxation, which is determined in part by g_app, was slower in hypothyroid rat myocardium expressing 100% β-MHC than in euthyroid rats expressing predominately α-MHC. It should be noted that a reduction in α-MHC during heart failure is not the sole cause of impaired relaxation. A previous study (2) has shown that Ca²⁺ transients are blunted and prolonged in human heart failure. In conjunction with the loss of α-MHC in heart failure, a reduction in the amplitude of the Ca²⁺ transient would further suppress the rate of force development during systole, whereas an increase in the duration of the Ca²⁺ transient would prolong the twitch, thereby slowing the rate of ventricular relaxation and impairing diastolic filling.

Effect of MHC Isoform Expression on the Ca²⁺ Sensitivity of Force and k_tr in Rat Myocardium

Consistent with previous studies (12, 41, 43), we found no alterations in the slope of the force-pCa relationship or in the Ca²⁺ sensitivity of force in the rat skinned myocardium, indicating that the changes in rate constants did not alter the steady-state numbers of cross-bridges bound to actin at each submaximal Ca²⁺ concentration. Other studies, however, have reported a decrease (33) or an increase (14) in the Ca²⁺ sensitivity of force in hypothyroid rats, and, in both cases, the difference in pCa₅₀ was much larger than that observed here. While the mechanisms underlying these shifts are not known, similar levels of myofibrillar protein content and phosphorylation combined with the lack of change in Ca²⁺ sensitivity of force observed in the present study indicate that hypothyroidism did not alter the isoform expression or phosphorylation states of thin filament regulatory proteins (i.e., troponin T and troponin I).

Although we found no differences in passive tension among euthyroid, hyperthyroid, and hypothyroid rat myocardium, our data did show a significant decrease in the maximum Ca²⁺-activated isometric force in hypothyroid rats (β-MHC), which differs from the findings of others (12, 20, 41, 43). Recent work by Wu et al. (49) has demonstrated an upregulation of type I and type III collagen expression in PTU-treated rats. While we do not completely understand the basis for the reduced force in the present study, it is possible that the preparations selected for this study (trabeculae) exhibit different structural characteristics than the mechanically disrupted preparations used in other studies, a possibility that is reinforced by the fact that our measurements of maximal Ca²⁺-activated force in enzymatically digested skinned single myocytes indicated no change in force per cross-sectional area. If the extracellular collagen matrix remained intact in trabeculae, this would reduce the fraction of cross-sectional area occupied by contractile protein and account for a decrease in force normalized to cross-sectional area. Moreover, while it is possible that the observed changes in force could be attributed to changes in the cytoskeleton, this is unlikely since a previous study (25) has shown that rats treated with PTU do not display alterations in the density of microtubules and yet still exhibit contractile dysfunction.

It is also unlikely that alterations in the distribution of titin isoforms existed in the hyperthyroid and hypothyroid myocardium used here. The study by Wu et al. (49) showed that the expression of the more compliant cardiac titin isoform (N2BA) increased with PTU treatment in rats but did not appear to change significantly for the length of treatment used here (5 wk); the N2BA-to-N2B ratio was ∼0.3 compared with 0.2 in untreated rats. Moreover, alterations in titin isoforms would most likely affect passive tension; we observed no such changes here.

In addition to measurements of ATP utilization and isometric force, a critical component of our estimation of cross-bridge rate constants was the determination of k_tr. k_tr is taken to be the sum of f_app and g_app as myosin cross-bridges transition between force-generating and non-force-generating states (5). Therefore, any change in the magnitude of k_tr is due to a change in one or both of the rate constants. Consistent with recent studies (11, 12, 41, 43), we found that k_tr was significantly slower in preparations expressing 100% β-MHC across all levels of activation compared with preparations expressing 100% α-MHC. In addition, Fig. 3 shows a strong linear correlation between k_tr and the expression of α-MHC, indicating that 1) low-level expression of α-MHC significantly accelerates contractile kinetics over myocardium with no detectable α-MHC; and 2) under steady-state conditions where the number of cycling cross-bridges is constant, there appears to be no undue drag imposed on the faster cycling α-MHC cross-bridges by the presence of β-MHC, either in the form of V2 or V3, or in cases where α-MHC is incorporated in discrete regions of the thick filament (48).

Interestingly, low-level expression of α-MHC increased the rate of force development to a similar extent in both our mathematical simulations and measurements of k_tr. As mentioned above, the derived dF/dt_max was ∼18% greater in myocardium expressing 10% α-MHC compared with 0% α-MHC, whereas we found that k_tr was ∼13% greater in the group of hypothyroid rats expressing ∼15% α-MHC compared with those expressing 0% α-MHC. It is important to note that the measurements of k_tr were performed during conditions of constant Ca²⁺ concentration rather than the limited-duration Ca²⁺ transient used for mathematical modeling. While k_tr would thus underestimate the accelerating effect of a small population of α-MHC cross-bridges due to the presence of a larger number of slower cycling β-MHC cross-bridges than would bind during a Ca²⁺ transient, qualitatively the effect would be similar in that dF/dt_max would be accelerated.

Implications of Altered MHC Expression on the Rate of Rise of Pressure

Our model predictions of the kinetics of an isometric twitch reveal that myocardium with greater expression of α-MHC has a greater peak twitch force in response to a Ca²⁺ transient, as a direct consequence of a larger number of attached cross-bridges. In addition, the time to reach peak force progressively decreases (with a concomitant increase in dF/dt_max) as the levels of α-MHC increase (Table 3). Moreover, the maximum decrease in the time to reach peak force, and thus the greatest increase in rate, is observed when 10% α-MHC is compared with 0% α-MHC. This observation indicates that a small population of fast cycling cross-bridges disproportionately accelerates dF/dt_max in response to a limited-duration Ca²⁺ transient. It is important to note that an increase in α-MHC would have only proportionate effects on steady-state measurements of cross-bridge cycling (i.e., ATPase activity) due to the fact that both α-MHC and β-MHC reach steady-state distributions between attached and detached states.

At the whole organ level, our modeling data indicate that low-level expression of α-MHC on a predominately β-MHC background would accelerate dP/dt_max, thus facilitating both systolic ejection and diastolic filling. Conversely, a reduction in α-MHC content would lead to a decrease in dP/dt_max and a functional deficit, which, to some degree, would be compensated by an increase in sympathetic tone and reexpression of atrial essential MLC-1 in the ventricles of the human failing myocardium (10, 26, 39). While these observations are suggestive of an important role of α-MHC expression in the human myocardium, such an extension of the present results to humans is premature since the ATP turnover rates for both α-MHC and β-MHC are slower in humans and not known for certain. However, recent evidence has suggested that the rate of ATP utilization by β-MHC is nearly fivefold slower in the human myocardium than in the rodent (36). In light of this observation, our model would predict that expression of α-MHC on the order of 10% would dramatically increase the rate of force development and thus enhance dP/dt_max.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grant HL-61635 (to R. L. Moss).