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% () 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 (); therefore, the enhanced myofilament power production is solely due to the enhanced velocity of loaded shortening (). 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 (). 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 and . 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 () 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 , 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.