In the current report, we describe our investigation on myosin motor function and the cross-bridge kinetics in skinned papillary muscle strips from transgenic mice expressing ELC mutant Δ43. The Δ43 truncation mutant was generated to investigate the functional significance of the long ELC isoform (A1) and its unique N-terminal extension (Kazmierczak et al. 2009
). This is the first study which comprehensively characterizes all the elementary steps of the cross-bridge cycle consisting of six states by applying sinusoidal length perturbations and by measuring the tension transients at varying concentrations of ATP, ADP and Pi. In this way, the significance of the N-terminal extension of ELC could be characterized. Our study is an extension of the earlier study (Muthu et al. 2011
), in which the same mouse models were used. In this report, the ADP study has been added, the rate constants of the elementary steps have been resolved, the number of data pool has been increased, and the cross-bridge distribution has been added. Therefore, a complete picture of the cross-bridge cycle is presented in this report.
We studied the effect of Ca2+
on tension and the rate constant 2πb
(), and found that there were no significant differences between Δ43 and WT (). However, the rate constant 2πc
was significantly faster in Δ43 than WT at the high level of activation (pCa ≤ 5.7) (). These results indicate that the N-terminal extension of ELC does not affect the regulatory system, but affects the cross-bridge detachment step. The observation that 2πb
increases and saturates by ~70 % tension in mouse papillary fibers () is similar to that of rabbit psoas fibres (Kawai et al. 1981
) and the shortening velocity in frog semitendinosus fibres (Julian and Moss 1981
). These results indicate that each cross-bridge is not independent, many cross-bridges work together cooperatively, and their cooperativity breaks down at a low level activation.
Compared with a previous report (Kazmierczak et al.2009
), the tension () produced in skinned muscle strips in the current study is less. This is because 200 mM ionic strength (IS) and 8 mM Pi were used in the standard activating solution, whereas 150 mM IS and no added Pi were used previously. The addition of 8 mM Pi reduces isometric tension to ~0.59× by decreasing the strongly attached cross-bridge number (Kawai and Halvorson 1991
; Dantzig et al. 1992
; Fortune et al. 1991
) (), and a 50 mM increase in IS is known to reduce isometric tension by ~0.29× by the modification of the rapid equilibrium between the detached state and the weakly attached state (Kawai et al. 1990
). In addition, the sensitivity of tension and stiffness to IS may be increased by Pi (Iwamoto 2000
). The higher IS and Pi are also factors causing increased cooperativity (nH
) (Gordon et al. 2000
), which may explain significantly larger values of nH
in our study compared to previously reported, which used different solutions (lower [IS] without Pi) (Kazmierczak et al. 2009
). The composition of the solution used in the current study reflects more physiological conditions because of the physiological [Pi
] present in active cardiomyocytes is 4–9 mM (Opie et al.1971
), 4–6 mM, in active human calf (skeletal) muscles (Roth et al. 1989
), and the IS is reported to be ~215 mM in frog skeletal muscle (Godt and Maughan 1988
). Our results demonstrate that the ELC’s N-terminal extension does not affect the Ca2+
sensitivity in the physiological solution (, ). However, the cooperativity decreased significantly with the N-terminal truncation ().
Under the standard activation, the Δ43 mutant muscle strips exhibited altered cross-bridge kinetics: in frequency plots, complex modulus plots were shifted right compared to those for WT (). This observation demonstrates that the cross-bridge kinetics became faster in mutant strips than in WT (see also at [MgATP] ≥ 1 mM; at pCa ≤ 5.6). This shift is significant, as shown by the significant increase in 2πc
in Δ43 compared to WT (). Our finding with Δ43 is consistent with the conclusion from previous studies demonstrating that skeletal muscle fibres reconstituted with only the long ELC form (A1) had a slower shortening velocity than those reconstituted with the short ELC form (A2) (Bottinelli et al. 1994
; Sweeney 1995
). Similar results were obtained in an in vitro motility assay (Lowey et al. 1993
), suggesting that the absence of the N-terminal extension in Δ43 mice may account for increased cross-bridge kinetics relative to those determined in the Tg-WT model. Our measurements demonstrate an increase in the rate constant k2
of the cross-bridge detachment step (), accounts for this acceleration.
Isometric tension was 13 % less in the Δ43 model than in the WT model, and this result is consistent with the earlier observation (Kazmierczak et al. 2009
). Possible causes of reduced tension are based on (a) a decrease in the number of force generating cross-bridges owing to the changes in the kinetic constants (b) a decrease in force/cross-bridge, and (c) a decrease in the myosin content (Kazmierczak et al. 2009
). Our result demonstrating that T5
was not different between Δ43 and WT models excludes (b). T5
is force supported by the AM*DP state, which is equal to force supported by the AM*D state, and both of these are major force generating states (). (a) is consistent with our finding that the number of strongly attached states is less in Δ43 than WT models by ~9 % (= 1–50/55 %) (). If reason (c) is the solitary cause, one group of parameters (tension, stiffness) should decrease proportionately to the myosin content, whereas other group of parameters (kinetic constants) should remain the same. From our results () we can conclude that reason (c) is not the solitary cause, but it may somewhat contribute to the results, hence (a) must be the major cause. Interestingly, stiffness measured during the standard activation was less by 18 % and after rigor induction by 7 % in Δ43 than in WT (). The reduction in stiffness during activation must be partially due to the reduction of the number of strongly attached cross-bridges (). The reduction in rigor stiffness suggests that the stiffness of a cross-bridge is also modified by the N-terminal truncation.
It has been hypothesized that the N-terminal domain of a long ELC may function as a “tether” between the myosin head and actin, restricting binding of myosin to actin (Kazmierczak et al. 2009
; Lowey et al. 2007
; Morano 1999a
; Sweeney 1995
). The data in this report support this hypothesis, because a removal of this sequence leads to faster cross-bridge kinetics, and reduced stiffness during activation and after rigor induction. The rigor is a static state in which the number of attached cross-bridges is maximized and no cross-bridge cycling takes place. Numerous studies indicate that, during striated muscle contraction, the N-terminus of ELC and actin may interact directly (Winstanley et al. 1977
; Sutoh 1982
; Henry et al.1985
; Timson et al. 1999
; Milligan et al. 1990
; Morano et al. 1995
; Miyanishi et al. 2002
). It has been hypothesized that the positively charged N-terminus of the long ELC isoform (A1) makes a contact with the negatively charged C-terminus of actin (Sutoh 1982
; Timson et al. 1999
) and lead to alterations in force development during contraction (Morano et al. 1995
; Miller et al. 2005
; Ritter et al. 1999
). Three-dimensional maps of vertebrate muscle thin filaments obtained by cryo EM revealed that the N-terminal extension of the ELC is in a position to make a molecular contact with the C-terminus of the actin monomer (Milligan et al. 1990
). The recent structural modeling study from the Morano group (Aydt et al. 2007
) depicted the N-terminal domain of ELC-A1 as a rod-like 9.1 nm-long extension that can function as a bridge between the ELC core of the myosin head and the binding site of the ELC on the actin filament. Using cryo EM in conjunction with light-scattering and fluorescence analysis, Lowey et al. (Lowey et al. 2007
) demonstrated bindings of the N-terminal extension of the ELC to the SH3 domain of MHC, and subsequently to actin. Consistent with our previous investigation (Kazmierczak et al. 2009
), we found decreased tension and stiffness in the Δ43 mouse model compared with WT mice. Consequently, we conclude that the stiffness of rigor cross-bridges is reduced in Δ43 by 7 % because of the lack of the N-terminal extension of ELC, and stiffness increases in WT because of the N-terminal extension that tethers actin. As a result, the reduction of the active stiffness (18 %) must be the sum of the tether (7 %) and the number of strongly attached cross-bridges (9 %). The near exact match of the sum of these numbers could be fortuitous, however, because each value is associated with measurement errors.
are less in Δ43 than in WT (), because these are scaled with tension and represent the number of cycling cross-bridges, much like stiffness. It is conceivable that the reduced tension measured in muscle strips of Δ43 mouse model is responsible for inducing the compensatory hypertrophy observed in the hearts bearing the Δ43 truncation mutation of ELC, especially in aged mice (Muthu et al. 2011
; Kazmierczak et al. 2009
Our data on stiffness and tension are in accord with those by Micheal et al. (Michael et al. 2012
) published during the revision process of this manuscript . The authors used muscle strips from 7 month old female Tg-WT and Tg-Δ43 mice, the same mouse models used in the current report. In agreement with the original study (Kazmierczak et al. 2009
) and the current investigation on Tg-Δ43 mice, Micheal et al. (Michael et al. 2012
) showed that the deletion of 1-43 amino acids in cardiac ELC leads to impaired tension development and decreased instantaneous stiffness parameter (ED
) that is a measure of the muscle fibre stiffness due to the strain of strongly-bound cross-bridges. They concluded that a decrease in ED
in Tg-Δ43 fibres is due to a decrease in the number of strongly-bound cross-bridges in Tg-Δ43 mice, the same conclusion that was reached in the current report. However, the increase in Δ43 cross-bridge kinetics observed in our study stays in apparent disagreement with their result of a mutation induced decrease in the cross-bridge detachment kinetics (Michael et al. 2012
). But, their estimated rate constant of muscle length mediated cross-bridge distortion (in s−1
), used as a measure of the rate of cross-bridge detachment, was only smaller in Tg-Δ43 versus Tg-WT fibres for the sarcomere length SL = 1.9 μm. This was not true for SL = 2.13 μm, the sarcomere length observed in the fibres for which our cross-bridge kinetics were determined. Despite different approaches used in both investigations, it is possible that the sarcomere length is critical to the N-terminus induced alterations in the myosin cross-bridge kinetics.
The phosphorylation status of myofilament proteins influence muscle contraction in many different ways as reviewed in Gordon et al. (2000)
. In this report, we found that the phosphorylation status of myofilament proteins were not significantly different between WT and Δ43, demonstrating that the changes observed in Δ43 mice cannot be attributed to the changes in the phosphorylation levels.
In summary, the current study found that the Δ43 mutation in ELC produces faster myosin cross-bridge kinetics. The Δ43 model exhibited decreased force and stiffness, but the force/cross-bridge was not changed. The number of strongly attached cross-bridges is decreased, causing a decrease in tension and stiffness. In addition, a decrease in active and rigor stiffness in Δ43 model supports the notion that the N-terminus of ELC works as a molecular tether between actin and the myosin head. We conclude that the ELC’s N-terminal domain plays a role in stabilizing myosin motor function to optimize cardiac performance, and that any structural perturbations in this region of myosin results in changes in both cross-bridge kinetics and the amount of force generated.