Tuning the Calcium Sensitivity of Cardiac Muscle

It is generally known that muscle contraction is produced by cyclic interaction of myosin heads (crossbridges) of thick filaments with actin thin filaments driven by ATP hydrolysis; a conformational change in an attached crossbridge produces a pulling force on the thin filaments toward the center of a sarcomere (1). The activation of this contractile process in both cardiac and skeletal muscles is regulated by the intracellular calcium ion concentration, [Ca²⁺], and it operates via the thin (actin) filament; when [Ca²⁺] rises, a muscle contracts, and when [Ca²⁺] falls, a muscle relaxes. Troponin (Tn) is a constituent protein of thin filament and the protein to which Ca²⁺ binds to accomplish this regulation. Troponin has three subunits, TnC, TnI, and TnT. When calcium is bound to specific sites on TnC, tropomyosin (Tm, another protein in the thin filament) moves out of the way of the active actin sites in thin filament, so that crossbridges (molecular motors organized in muscle thick filaments) can attach to the thin filament and generate force and/or movement. In the absence of calcium, tropomyosin prevents this crossbridge operation, and therefore muscles remain relaxed.

One of the long running mysteries of the thin filament regulation is why there are so many variants in the N-terminus of TnT. There are muscle-specific isoforms and alternate slice isoforms (four splice variants in fast muscle). Furthermore, why is the N-terminal half of cardiac TnT such a hot spot for cardiomyopathy mutations? At the simple level, the view of TnT’s role is to hold the troponin complex to tropomyosin. More specifically, the N-terminal part of TnT binds along tropomyosin and stabilizes the Tm-Tm-contacts between consecutive Tms along the actin filament. This explains the structural role of TnT, but not why different TnTs are needed; tropomyosin isoforms, after all, do not show the same tissue specificity. αTm is the same in all striated muscle with some variation in the amount of β-Tm expressed. If the structural role of TnT was this simple, how do different Tn isoforms tune muscle function and why do mutations in the region have such a dramatic effect on function?

The implication is that there must be more to TnT and the standard view is that it modulates the cooperativity of the Tm on-off switch of muscle contraction. The work of Gollapudi et al. (2), reported in this issue, gives the first evidence that the N-terminus of cTnT does modulate Tm behavior.

The N-terminus of cardiac troponin T (cTnT) has a distinct N-terminus extension (NTE) of ∼32 amino acids, rich in negative charges and highly conserved in hearts of different mammalian species. This entire 32-amino-acid segment is absent in both fast and slow skeletal muscle TnT. The specific functional role of the NTE has remained unclear despite previous functional studies in which the cTnT was replaced by skeletal TnT. The article by Gollapudi et al. (2) addresses the role of the specific NTE of cardiac TnT using a transgenic mouse model expressing a recombinant chimeric cTnT where the NTE was replaced with the corresponding NTE of fast skeletal TnT. The results show that few mechanical or biochemical parameters are altered in skinned cardiac muscle fiber, except the calcium sensitivity of isometric force.

In detail, chemically skinned papillary muscle fiber bundles isolated from hearts of these transgenic mice were used experimentally to determine a range of dynamic and steady-state mechanical and biochemical parameters at different Ca²⁺ concentrations and at two different sarcomere lengths; a comparison is made between the transgenic (cTnT with altered NTE) and the nontransgenic (control) mouse cardiac muscles with respect to a wide range of physiological and biochemical characteristics such as maximum isometric force, stiffness, maximum shortening velocity, Ca-sensitivity of force, ATPase, etc. Thus, Gollapudi et al., in essence, tested the hypothesis that the NTE of cTnT has a specific regulatory role in cardiac muscle activation.

Their main (positive) finding is shown in Fig. 5 in the article, and it is that the Ca-sensitivity of steady force development is higher in transgenic muscle; in other words, the force versus pCa relation was shifted to the left (low [Ca²⁺]) in transgenic muscle without the cardiac-specific NTE. Equally, or even more important results reported in the study are the negative findings (no significant difference between transgenic and control muscle) from control experiments; thus, chimeric cTnT has no significant effect on maximal Ca-activated force and ATPase rate, stiffness, crossbridge turnover rate, crossbridge detachment rate, and maximum velocity of shortening (see article for details). Thus, most of the other mechanistic and biochemical characteristics that relate to acto-myosin crossbridge cycle were unaltered. It appears that when NTE is altered it affects thin filament activation system directly rather than indirectly via myosin (crossbridge) operation.

Findings are discussed in relation to the mechanism of thin filament activation via three states (i.e., blocked, closed, and open state transition) as previously reported in biochemical studies (see McKillop and Geeves (3)). Indeed, the results would support the hypothesis that the NTE of cTnT modulates the blocked-to-closed state transition of the thin filament via its impact on the underlying allosteric/cooperative mechanisms, as proposed from previous biochemical experiments. Correlated with existent structural data, this study therefore provides novel mechanistic insights by which the unique NTE of cTnT produces regulation of cardiac myofilament. As discussed in the article, the findings may have a bearing on age- and disease-related changes cTnT isoforms, perhaps even in human heart, because functionally distinct cTnT isoforms are thought to be expressed during development and during cardiac muscle disease (see references in the article).

Given the thoroughness of this experimental study, the findings reported therein are clearly significant and conclusive and, as mentioned in the article, they may well relate to age- and disease-related issues of the human heart function. Nevertheless, several issues remain, not the least of which is the molecular mechanism whereby the cardiac-specific N-terminal extension of TnT alters the calcium sensitivity of the thin filament. A recent article by Manning et al. (4) suggests that there is a correlation between the change in flexibility of TnT1 (the N-terminal half of TnT) induced by hypertrophic cardiomyopathy mutations and the change in cooperativity of calcium activation. This raises the possibility that the TnT isoforms have specific flexibility that is tuned to the cellular isoforms of the thin filament. Is the cardiac-specific extension part of this story? The link between cardiomyopathy mutations and protein flexibility is also highlighted in a recent article on tropomyosin (5). Given the close contact between TnT1 and tropomyosin, it is possible that flexibility is a property of the Tm-TnT1 complex.

Inevitably, an in vitro study as that reported here requires further confirmation for the in vivo situation.

First, these experiments are not done exactly under physiological conditions; for example, they were done at 20°C, a much lower temperature than the core physiological temperature of ∼37°C in which the mammalian heart functions in situ. Indeed, it is known that the pCa-force relation is shifted to the right (Ca-sensitivity of thin filament decreased) with an increase of temperature, probably in all types of striated vertebrate muscle (see Stephenson and Williams (6)). Although this probably reflects a TnC property, given the complexity and lack of proper understanding at present of temperature effects on muscle function, it might be one other control (negative) finding worth knowing.

Second, data presented here are from steady-state measurements and from skinned fibers. Although both muscles are striated, vertebrate heart (cardiac muscle) operates as a constant volume pump whereas skeletal muscles operate as force and work producers. It is also not clear whether the function of the in situ heart as a muscular pump is altered in the transgenic mouse, and hence, the functional significance of cTnT NTE requires further elucidation. These are issues for the future.