|Home | About | Journals | Submit | Contact Us | Français|
The left ventricles (LV) of both rabbits and humans express predominantly β-myosin heavy chain (MHC). Transgenic (TG) rabbits expressing 40% α-MHC are protected against tachycardia-induced cardiomyopathy (TIC), but the normal amount of α-MHC expressed in humans is only 5-7% and its functional importance is questionable. This study was undertaken to identify a myofilament-based mechanism underlying TIC protection and to extrapolate the impact of MHC isoform variation on myofilament function in human hearts.
Papillary muscle strips from TG rabbits expressing 40% (TG40) and 15% α-MHC (TG15) and from non-TG controls expressing ~100% β-MHC (NTG40 and NTG15) were demembranated and calcium activated. Myofilament tension and calcium sensitivity were similar in TGs and respective NTGs. Force-clamp measurements revealed ~50% higher power production in TG40 vs NTG40 (P<0.001) and ~20% higher power in TG15 vs NTG15 (P<0.05). A characteristic of acto-myosin crossbridge kinetics, the “dip” frequency, was significantly higher in TG40 vs NTG40 (0.70±0.04 vs 0.39±0.09 Hz, P<0.01) but not in TG15 vs NTG15. The calculated crossbridge time-on was also significantly shorter in TG40 (102.3±14.2 ms) vs NTG40 (175.7±19.7 ms), but not in TG15 vs NTG15.
The incorporation of 40% α-MHC leads to greater myofilament power production and more rapid crossbridge cycling, which facilitate ejection and relengthening during short cycle intervals and thus protect against TIC. Our results suggest, however, that, even when compared to the virtual absence of α-MHC in the failing heart, the 5-7% α-MHC content of the normal human heart has little if any functional significance.
Expression of the two cardiac myosin heavy chain (MHC) isoforms, α and β, is regulated developmentally and hormonally and in a species-dependent manner.1-3 Rodents express predominantly α-MHC while larger adult mammals, including rabbits and humans, express predominantly β-MHC. Structurally, the two MHC isoforms are 93% identical,4 yet the biochemical and biomechanical properties are markedly different.5 Using the laser trap, crossbridge attachment time (ton) for α-MHC was ~60% that of β-MHC.6 Using the in vitro motility assay, unloaded actin velocity with α-MHC was 2-3 times greater than with β-MHC5, 7, 8 while generating half the isometric force.5 Finally, the ATPase activity of α-MHC is 2-3 times greater than that of β-MHC.9, 10 Despite these major differences, the influence of the two isoforms on crossbridge kinetics and power generation in the intact myofilament is incompletely understood.
Myosin isoform shifts have been reported in end-stage, human heart failure. Miyata et al.,11 Reiser et al.,12 and Noguchi et al.13 all reported reduced expression of α-MHC in failing hearts, from 5-7% of total MHC to virtually undetectable levels. It has been suggested that these modest shifts are functionally significant.11 Moreover, Herron and McDonald14 manipulated thyroid state in rats in order to produce cardiac myofilaments with either 12% or undetectable levels of α-MHC, simulating differences observed in non-failing versus failing human hearts. Using demembranated cardiomyocyte fragments, they reported higher power output in linear proportion to the fraction content of α-MHC, suggesting that small shifts in isoform content within an intact sarcomere have significant functional consequences.15-17 However, while small, the average amount of α-MHC detected in these cardiomyocyte preparations was about twice that in normal human myocardium.11-13 Moreover, manipulation of thyroid state induces various other changes in the myocardium (e.g., alterations in protein phosphorylation18) besides MHC isoform shifting17 that could independently influence myofilament function Arguing against functional significance of MHC isoform shifts in heart failure, we reported that in vitro actin velocity and force generating capacity were similar for cardiac myosin from end-stage, failing human hearts compared with myosin from non-failing hearts,13 but these studies were performed using isolated proteins. Thus, the question of whether MHC isoform shifting is functionally significant in failing human hearts remains unresolved.
We recently produced a transgenic (TG) rabbit that expresses increased α-MHC content in the β-MHC background, thus obviating the need for pharmacologic or thyroid manipulation to modify MHC isoforms.19, 20 These animals do not have detectable changes in other proteins that modulate cardiac contraction. With fractional α-MHC expression levels of ~40% and ~15%, echocardiograms in the resting state did not reveal differences in cardiac dimensions or shortening compared with non-transgenic (NTG) controls, but the presence of 40% α-MHC partially protected these animals from pacing tachycardia-induced cardiomyopathy (TIC).20
The present study was undertaken in demembranated myocardial strips to demonstrate if an identifiable mechanism at the myofilament level could explain protection from TIC and, more generally, to delineate the effects of MHC isoform variation on myofilament contractile function. By extrapolation, we sought to shed additional light on whether MHC isoform shifting is functionally significant in human heart failure.
All procedures were reviewed and approved by the Institutional Animal Care and Use Committees of the University of Vermont and Cincinnati Children's Hospital. Transgenic rabbits with ~40% α-MHC content (TG40) and ~15% α-MHC content (TG15) and the age-matched and genotype-matched non-transgenic controls containing ~97% β-MHC (NTG40 and NTG15, respectively) were examined. Because of the limited availability of TG rabbits, there was a significant age difference between the TG40/NTG40 pair (22.1±0.5 months) and the TG15/NTG15 pair (39.3±2.0 months), but there were no age differences between the TG and genotype-matched controls. All procedures and experiments were performed in a blinded fashion. Rabbits were anesthetized with isoflurane (3-4% induction, 2-3% maintenance) and maintained on a respirator during echocardiographic measures of left ventricular (LV) end-systolic diameter, end-diastolic diameter, interventricular septum and posterior wall thicknesses and fractional shortening.
Concentrations (mmol/L) were formulated by solving equations describing ionic equilibria.21 All reagents were purchased from Sigma (St. Louis, MO) except when noted. Relaxing solution (pCa 8) consisted of 5 MgATP, 40 phosphocreatine, 240 U/mL creatine kinase, 1 free Mg2+, 0.11 CaCl2, 5 EGTA and 20 BES buffer with pH 7.0 and 190 mEq/L ionic strength. Activating solution was the same as relaxing solution with pCa 4.5. Storage solution was the same as relaxing solution with 10 μg/mL leupeptin and 50% (wt/vol) glycerol added. Skinning solution was the same as storage solution with 1% (vol/vol) Triton X-100.
Muscle strip preparation has been described elsewhere.22 Briefly, papillary muscle strips were demembranated in skinning solution for two hours at room temperature, dissected to 140-200 μm diameter and 600-800 μm length, and stored at -20°C for fewer than four days. Strips were attached between a length motor and force gauge using aluminum T-clips, lowered into a 30 μL droplet of relaxing solution maintained at 17°C, stretched to a sarcomere length of 2.2 μm detected by Fourier analysis of video image (IonOptix Corp., Milton, MA), and calcium activated incrementally between pCa 8.0 and 4.5. Recorded forces were normalized to cross-sectional area to provide isometric tension (T). Recorded T minus relaxed tension (Tmin) was normalized to maximum developed tension (Tmax-Tmin) and fit to the Hill equation:
where [Ca2+]50 = calcium concentration at half activation, pCa50 = -log [Ca2+]50, and n = Hill coefficient using a nonlinear least squares algorithm (Sigma Plot 8.0, SPSS, Chicago, IL).
The force-clamp technique was applied at maximal calcium activation. Various mechanical loads were expressed as a fraction of Tmax. Force was maintained constant by feedback control of muscle length.23 The tension-velocity (T-V) relationship was fit to a hyperbolic Hill equation normalized to Tmax:
where T`= T/Tmax, a`= a/Tmax, and a and b are the parameters of the non-normalized hyperbolic Hill equation using a nonlinear least squares algorithm (Sigma Plot 8.0, SPSS, Chicago, IL). The physiological characteristics maximum unloaded shortening velocity (Vmax), velocity at maximum power (Vopt), tension at maximum power (T`opt) and maximum power production (Pmax) were calculated from a` and b, as follows:24
Length perturbations of 0.125% strip length were applied at discrete frequencies over the range 0.1-250 Hz using a microcomputer and custom made software (Igor-Pro 5.0, Wavemetrics, Lake Oswego, OR).22, 25, 26 Length and force signals were digitized, and the elastic and viscous moduli were calculated as the magnitudes of the in-phase and out-of-phase components of the tension response at each frequency divided by the magnitude of the normalized length perturbation. The complex modulus was defined as the elastic and viscous moduli taken as its real and imaginary parts and fitted to the following empirically-determined equation using custom software (IDL 5.5, ITT, Boulder CO):27, 28
where Y(iω) is a complex modulus, and ω = 2π × frequency of perturbation. The mean crossbridge attachment time, ton, was calculated as 1 /(2πc), as demonstrated by Palmer et al.29
All data are presented as mean ± SEM. Results from at least two muscle strips from each rabbit heart were averaged together to provide a single value for each heart used in statistical comparisons. Because of the difference in age between TG40/NTG40 and TG15/NTG15, only comparisons between TG and NTG groups (i.e., TG40 vs NTG40 and TG15 vs NTG15) were made. The unpaired Student t-test was used to compare continuous variables. For force-clamp and sinusoidal analysis studies, a repeated measures analysis of variance (ANOVA) was conducted: fraction of Tmax and frequency of perturbation, repectively, were used as the repeated trial factor. A significant group main effect was followed by a comparison of the TG vs NTG groupings at each trial factor using a Fisher's least significant difference approach to protect against inflated Type I errors. Statistical tests were performed using SPSS 14.0 (Chicago, IL) and were considered significant at the 0.05, 0.01 and 0.001 levels.
Functional evaluation by echocardiography did not reveal any significant differences in ventricular function, fractional shortening or wall thickness between TG and respective NTG groups (Table 1) as reported previously.19, 20 Age and left and right ventricular masses (Table 2) were similar between the TG and NTG pairs. Previously, it was shown that there are no significant differences in transcript levels of atrial natriuretic factor, phospholamban, and SERCA2a in these transgenic rabbits, and histological investigation did not reveal any differences in cell size, fibrosis, or cardiomyocyte orientation.20 The fractional content of α-MHC in the papillary muscles of the TG40 (40.7±4.9%), TG15 (12.5±0.5%) and NTG (2.5±0.9%) hearts were not different from that in the free wall of the same TG40 (39.8±4.4%), TG15 (12.5±3.5%) and NTG (4.3±1.5%) hearts.19, 20
The normalized tension-pCa relationships for each TG/NTG pair were very similar, as shown in Figure 1. There were no statistically significant differences in Tmin, Tdev, pCa50 or n (Hill coefficient of cooperativity) between the TG and respective NTG groups (Table 3).
Results from the force-clamp experiments are shown in Table 3, and tension-velocity and tension-power relationships are shown in Figure 2. Comparisons of force-clamp measurements between TG40 and NTG40 revealed a significant group main effect (P<0.001) and a significant group × fraction of Tmax interaction (P <0.001), indicating an upward shift (greater velocity and power production) in TG40 compared to NTG40 and a change in the shape of the tension-velocity and tension-power relationships (Figures 2A and and2C).2C). Maximum power production, Pmax, and the fraction of tension at Pmax, T`opt, were significantly higher in the TG40 group compared with the NTG40 group (Table 3). Further post-hoc analyses at each fraction of Tmax delineated significant differences in velocity and power between the TG40 and NTG40 groups at fractions of Tmax between 0.3-0.7 (Figure 2C). However, no significant differences in the Vmax and Vopt were observed between TG40 and NTG40 (Table 3).
Repeated-measures comparisons of force-clamp measurements between TG15 and NTG15 revealed a significant group main effect (P<0.05) for velocity and power indicating an overall increase in velocity and power over the entire tension-velocity and tension-power relationships for TG15 compared to NTG15 (Figure 2B and 2D). This increase in velocity and power, however, was subtle and post-hoc analyses did not reveal significant differences between TG15 and NTG15 at any specific fraction of Tmax. There were also no significant differences in the Vmax, Vopt, T`opt or Pmax between the TG15 and NTG15 groups (Table 3).
Figure 3 illustrates the elastic and viscous moduli recorded at maximum calcium activation pCa 4.5. Using repeated-measures analysis, elastic modulus demonstrated a significant group × frequency interaction for both TG40/NTG40 (P<0.01, Figure 3A) and TG15/NTG15 comparisons (P<0.05, Figure 3C). Post-hoc analysis showed that elastic modulus was lower in TG40 compared with NTG40 over the frequency range 0.9-3.8 Hz (Figure 3A) and lower in TG15 compared with NTG15 over 1.8-2.8 Hz (Figure 3C). The viscous modulus likewise demonstrated group × frequency interaction for the TG40/NTG40 comparison (P<0.01, Figure 3B), particularly over 0.3-1.2 Hz (Figure 3B), but not TG15/NTG15 (Figure 3D). The frequency at which the magnitude of the complex modulus is a minimum, or “dip” frequency, characterizes acto-myosin crossbridge kinetics25, 27, 30 and was significantly higher in TG40 compared with NTG40 (0.70±0.04 vs 0.39±0.09 Hz, P<0.01). In contrast, there was no significant difference between TG15 and NTG15 in dip frequency (0.71±0.09 vs 0.67±0.10 Hz, P=NS). The above results for model-independent indices of myofilament performance demonstrate that α-MHC incorporated in the TG40 myocardium significantly enhances crossbridge kinetics.
Table 4 presents model-dependent parameters obtained from fitting the measured elastic and viscous moduli at pCa 4.5 to Equation 3. Parameters A and k, which describe respectively the magnitude and relative viscosity of the passive viscoelastic response of the muscle,29 were not different between TG and NTG groups. Parameters B and C, which respectively describe the magnitudes of the work-generating and work-absorbing responses of the acto-myosin crossbridges,27, 28 were also not different between TG and NTG groups. Parameters b and c, which respectively represent the characteristic frequencies of mechanical work-generating and work-absorbing responses,27, 28 were significantly higher in TG40 compared with NTG40 (Table 4). The mean time period during which myosin crossbridges remain attached to actin, ton, was significantly shorter in TG40 compared with NTG40. These kinetic parameters b, c, and ton were not significantly different between TG15 and NTG15.
Figure 4 illustrates the elastic and viscous moduli recorded under relaxed conditions (pCa 6.5). The elastic modulus was not different between either TG40/NTG40 or TG15/NTG15 pairs (Figures 4A and and4C).4C). However, the viscous modulus was significantly lower in TG40 compared with NTG40 over the frequency range of 0.3-0.7 Hz (Figure 4B) and lower in the TG15 compared to NTG15 over the frequency range of 0.25-0.4 Hz (Figure 4D). These data suggest that even at very low calcium activation conditions, as would be the case during late diastole, crossbridges form and myosin isoform profiles play a significant role in influencing the mechanical properties of the myofilaments at physiological frequencies.
To illustrate how the mechanical properties of the myofilaments can provide protection from tachycardia-induced cardiomyopathy as found in the TG40,20 we used our data to simulate myofilament stress and power over a cardiac cycle of 158 ms, equivalent to the rate of 380 bpm employed in the tachycardia model. It should be noted that, because our data were collected at a temperature 20°C cooler than that in vivo, the frequency characteristics we measured were multiplied by a factor of six (we'd found previously a factor of 5.88 for this temperature difference29) to provide the corresponding frequencies in vivo. For example, the corresponding in vivo frequency ranges over which elastic and viscous module differed between TG40 and NTG40 would be 5.4-22.8 Hz and 1.8-7.2 Hz, respectively. Because the rabbit heart rate ranges between 130-325 bpm (2.2-5.5 Hz), our reported differences in the elastic and viscous moduli between TG40 and NTG40 bear physiological significance.
Figure 5A illustrates the mechanical stress that would arise given the elastic and viscous moduli we measured at pCa 4.5 and an assumed strain. The rise in stress during “systole” (shaded) represents the myofilament resistance to shortening. During “diastole” (unshaded) the stress is reduced as the myofilaments are relengthened. However, those crossbridges formed at peak shortening and still attached later in diastole would impart a resistance to relengthening as indicated most obviously by the negative stress that arises for the NTG40 at time ~120 ms in Figure 5A.
Figure 5B displays power, i.e., the rate of mechanical energy transfer to the myofilaments, over a cardiac cycle. Power is initially positive during shortening, indicating that mechanical energy is transferred to the myofilaments, and then mostly negative during relengthening, indicating recovery of mechanical energy from the myofilaments and facilitating relaxation. As indicated in both Figures 5A and and5B,5B, myofilaments of the TG40 resist shortening less and recover more mechanical energy during relengthening than those of the NTG40. Furthermore, a positive power arises during diastole only in the NTG40 (e.g., time ~120 ms in Figure 5B), which indicates a loss of energy during this late portion of the cardiac cycle. This loss of energy is due to the viscous drag associated with those crossbridges formed during peak shortening and still attached during diastole. Figure 5C depicts a quantitative prediction of the total mechanical energy lost during a single 158 ms cardiac cycle. The energy loss in the TG40 is about half that of NTG40 at all pCa values.
To the best of our knowledge, this is the first study to investigate the mechanical and kinetic properties of intact myofilaments in a transgenic rabbit. A previous study of transgenic mouse cardiomyocytes found that contraction-relaxation function was not affected by an elevation in β-MHC content;31 however, the effects of MHC isoform profile on myofilament function cannot be easily inferred from the intact cardiomyocyte. Other studies performed in rodents assessed the effects of MHC isoform variation on myofilament function after modification of thyroid status in order to convert the predominantly α-MHC rodent heart to variable amounts of β-MHC.1, 3, 4, 14-16 Extrapolation of these results to large mammalian hearts is problematic because altering thyroid status modifies other aspects of myofilament and nonmyofilament function.3, 17, 18 The transgenic rabbit model has the advantage of allowing examination of a relatively physiological condition including normally expressed β-MHC with less possibility of non-MHC mediated alterations in myofilament function.
The primary aims of this study were to identify the mechanism at the myofilament level that explains the protection against tachycardia-induced cardiomyopathy in the transgenic rabbits expressing ~40% α-MHC compared with NTG rabbits and to delineate the impact of variation in the two cardiac MHC isoforms on myocardial performance.19, 20 In conjunction with the latter aim, we also studied transgenic rabbits with ~12.5% α-MHC in the papillary muscles in order to provide data points between the extremes of 40% α-MHC and ~2.5% α-MHC in NTGs and thus allow inferences in regard to the functional significance of the small MHC isoform shifts that occur in failing human hearts.
Force-clamp measurements were used to assess the myofilament contribution to systolic function and revealed that myofilament power production was significantly enhanced on the order of 50% (Figure 2) in the TG40 versus the NTG40. This amounts to power enchancement of ~1.33× per % α-MHC content. There was no difference in maximal isometric tension between TG40 and NTG40 (Table 3); therefore, the enhanced myofilament power production is solely due to the enhanced velocity of loaded shortening (Figure 2A). This enhanced velocity of loaded shortening in the TG40 would assist in ejection over a shorter period of time, as would be necessary to accommodate tachycardia. We did not, however, detect a higher value for unloaded velocity, Vmax, in the TG40 compared to NTG40. This negative finding is not uncommon when Vmax must be extrapolated from the tension-velocity relationship even when other assays, such as the slack test, demonstrate differences in unloaded shortening velocities.14
There was also a significant enhancement of the tension-power relationship in TG15 compared with NTG15, but this change was considerably smaller, i.e., on the order of 20% at maximum power (Figure 2). This amounts to power enhancement of ~2× per % α-MHC content. The combined results for power production in the TG40 and TG15 are reasonably consistent with previous studies that showed a linear relationship between α-MHC content and power output.15, 16 If we assume a linear relationship between α-MHC and power output, our results would predict a percent power enhancement of ~1.33-2× per % α-MHC content. The incorporation of 5-7% α-MHC in the normal human LV would thus result in 7-14% more power compared to no α-MHC in failing hearts.11-13 It is unlikely that such a small difference in power production is physiologically meaningful.
The C-process of our sinusoidal length perturbation analysis demonstrated that increasing the proportion of α-MHC to 40% resulted in a significantly shorter ton, which reflects a more rapid myosin off-rate, i.e., gapp, of a conventional two state model.29 These results are consistent with the higher actin velocity observed for α-MHC in the myosin motility assay, which is thought to be proportional to the reciprocal of ton, and the higher ATPase activity for α-MHC compared with β-MHC.5-10 A shorter ton with the addition of α-MHC was expected; using the laser trap, ton for rabbit α-MHC was measured to be ~60% that of rabbit β-MHC.6 However, a shorter ton for α-MHC cannot account for the lower elastic and viscous moduli in some frequency ranges in the TG populations shown in Figures 3 and and4.4. The lower values for these moduli in the TG must arise from differences in the respective B-processes.
The characteristic frequency b of the B-process was significantly higher for activated myofilaments of the TG40 compared to NTG40 (Table 4) and this result reflects the higher range of frequencies over which α-MHC lowers the elastic and viscous moduli. The molecular mechanisms underlying the B-process are not well understood, although Kawai and colleagues have attributed the value 2πb to a phosphate-dependent, weighted sum of the forward and reverse rates of the myosin power stroke.25, 27 Regardless of this or any other interpretation of the B-process, the phenomenon underlying the B-process clearly lowers the elastic and viscous moduli over physiologically significant frequencies. The incorporation of a significant proportion of α-MHC furthermore protects the myofilaments from the high stresses and energy losses at higher pacing frequencies, as illustrated in Figures 5A-C, and would be expected to protect against tachycardia-induced cardiomyopathy.
There are limitations to our study. First, there was an age difference between TG40-NTG40 group and TG15-NTG15 group. However, age was comparable within each group (i.e., TG vs NTG) and therefore it is reasonable to compare each TG group with its control NTG group. Indeed, our results suggest that age may significantly reduce myofilament performance and explain some of the apparent differences between the NTG40 and NTG15 groups. For example, we found lower measures of velocity, power production and ton in the older NTG15 compared to the younger NTG40. Second, our TG rabbits have greater α-MHC contents (40% and 15%) than non-failing human myocardium. However, as discussed above we believe it is reasonable to use our findings to infer the effects of variations in the two cardiac MHCs in failing human myocardium. Third, we recognize the relatively low statistical power of this study; nevertheless, we believe these data demonstrate the importance of MHC isoform on myofilament mechanical characteristics affecting diastolic function independent of calcium regulation.
In summary, we showed that increasing α-MHC content to ~40% on a β-MHC background in the rabbit results in greater myofilament power production, more rapid rates of crossbridge cycling and lower elastic and viscous moduli at physiologically significant frequencies. In contrast, increasing α-MHC content to ~12.5% does not result in detectable differences in crossbridge cycling kinetics and causes only modest increases in power production. These effects contribute toward protection against functional consequences of prolonged tachycardia in the TG40 rabbits, but the smaller α-MHC content in failing human myocardium is unlikely to have functional significance.
We thank Takamaru Ashikaga, Ph.D., for help in statistical analyses. This research was funded by NIH grants HL50287 (MML, BMP, ZC), HL59408 (MML, BMP, DWM, YW), HL52318 (JR), HL69779 (JR) and HL074728 (JR).
Disclosures There are no conflicts of interest to disclose.