Mutations in striated muscle contractile proteins have been found to be the cause of a number of inherited muscle diseases; in most cases the mechanism proposed for causing the disease is derangement of the thin filament-based Ca²⁺-regulatory system of the muscle. Hypertrophic cardiomyopathy and hypercontractile diseases of skeletal muscle, such as distal arthrogryposis and “stiff child syndrome,” have been linked to a higher myofilament Ca²⁺-sensitivity (Marston, 2011; Donkervoort et al., 2015). In contrast dilated cardiomyopathy mutations are commonly, but not exclusively, linked to decreased Ca²⁺-sensitivity. Mutations in contractile proteins that are linked to nemaline myopathy and related skeletal muscle myopathies have also been found to be associated with reduced Ca²⁺ sensitivity (Marttila et al., 2012, 2014). The causative connection between myofilament Ca²⁺-sensitivity and muscle dysfunction is a field of intensive research that is too complex to consider in this account. However, when considering the results of such experiments reported over the last 15 years, one feature has been frequently noted, but rarely discussed. The magnitude of changes in myofilament Ca²⁺-sensitivity due to myopathy-causing mutations in skeletal or heart muscle seems to be always in the range 1.5–3x EC₅₀. Such consistency suggests it may be related to a fundamental property of muscle regulation; in this article we will investigate whether this observation is true and consider why this should be so.

Most investigations have found increased Ca²⁺-sensitivity in muscle with hypertrophic cardiomyopathy (HCM) and restrictive cardiomyopathy (RCM)-causing mutations. Our literature search found 71 independent measurements of the mutation-induced change of EC₅₀ ranging from 1.15 to 3.8-fold with a mean of 1.87 ± 0.07 (sem) (Table 1). We also found 11 independent measurements of increased Ca²⁺-sensitivity due to mutations in skeletal muscle proteins ranging from 1.19 to 2.7-fold with a mean of 2.00 ± 0.16 (Table 2).

Dilated cardiomyopathy-causing mutations were initially found to decrease Ca²⁺-sensitivity but more recent studies have indicated the situation is more complex. DCM-linked mutations can both increase and decrease Ca²⁺-sensitivity depending on the individual mutations, moreover the direction of change can be different with a single mutation measured in different systems (Marston, 2011; Memo et al., 2013). This is illustrated in Table 3 where 42 independent determinations show a range of EC₅₀ wt/mutant from 0.3 to 2.3. In addition we found 14 measurements of Ca²⁺-sensitivity changes due skeletal muscle myopathy mutations ranging from 0.39 to 0.63 (Table 4).

Thus, our extensive literature search, although not necessarily complete, found that, indeed, the changes in myofilament Ca²⁺-sensitivity due to disease-causing mutations have a bimodal distribution and that the overall changes in Ca²⁺-sensitivity are quite small and do not extend beyond a 3–4-fold increase or decrease in Ca²⁺-sensitivity. Indeed when all the findings are plotted as a histogram one finds that increases in Ca²⁺-sensitivity on a log scale have an approximately normal distribution with mean increase in Ca²⁺-sensitivity (EC₅₀ wt/mutant) of 1.86-fold (corresponding to ΔpCa₅₀ = 0.255 ± 0.015), whilst the decreases in Ca²⁺ sensitivity have a mean EC₅₀ wt/mutant of 0.54-fold (corresponding to ΔpCa₅₀ of –0.286 ± 0.01; Figure 1A). It is also worth noting that this small Ca²⁺-sensitivity shift is observed independent of the measurement method Figure 1B compares the ΔpCa₅₀ distribution measured by unloaded assays (actomyosin ATPase or in vitro motility) and by loaded assays (force measurements in skinned muscles, cell, and isolated myofibrils). The mean magnitude of the Ca²⁺-sensitivity change is about 20% less when measured in loaded assays.

What could be the underlying reason for this consistent and small effect of mutations on EC₅₀? We will consider two possible mechanisms that are not necessarily mutually exclusive. Firstly, it could be that the limit is set by the capacity of the EC coupling system that supplies activating Ca²⁺ and that striated muscle cannot work in a way compatible with life outside these limits; alternatively it may be due to a fundamental property of the troponin system and the permitted conformational transitions compatible with efficient regulation.

Before attempting to discuss these mechanisms it is worthwhile considering some additional evidence on Ca²⁺-sensitivity shifts. Perhaps the most puzzling observation is that there appears to be no correlation between the Ca²⁺-sensitivity shift and disease severity. Skeletal myopathy mutations that cause life-threating muscle weakness from birth and often require mechanical assistance in breathing (Ravenscroft et al., 2015), have the same Ca²⁺-sensitivity shifts as dilated cardiomyopathy mutations which are considerably less lethal (Hershberger et al., 2013). Whilst heart muscle has compensatory strategies not available in skeletal muscle to account for this difference, the small change in Ca²⁺-sensitivity even in the most severe skeletal muscle disease might be indicative of a fundamental structure-based limit on changes in EC₅₀.

Consideration of the Ca²⁺-sensitivity shifts in cardiomyopathies (Tables 1, 3) do not indicate any correlation with disease severity. Any relationship that may exist is masked by the extreme variability of Ca²⁺-sensitivity shift measurements. For instance, the “severe” TNNI3 R145G HCM/RCM-linked mutation features at both extremes of the Ca²⁺-sensitivity range (1.15x and 3.65x); for the 6 assays in the table the mean is 1.84, close to the mean of all 71 HCM measurements (1.87). The same variability can be seen with other mutations where multiple values are available: ACTC E99K, n = 5, 1.24–2.45 mean 1.85; TPM1 E180G, n = 4, 1.30–2.75, mean 1.78. The second relevant observation is that the physiological modulation of cardiac muscle myofilament Ca²⁺-sensitivity due to phosphorylation of troponin I by protein kinase A has been known to be a 2–3-fold shift for many years (Solaro et al., 2008). Table 5 lists a number of recent determinations of this Ca²⁺-sensitivity shift in several species and measured by both loaded and unloaded assays illustrating its small range. Figure 1C shows how the magnitude and distribution of measured changes is similar to the changes induced by disease-causing mutations. It would be logical to conclude that this represents the range of achievable Ca²⁺ sensitivity shifts in cardiac muscle due to the limitations of the EC coupling system.

In principle, it should be possible to go beyond the Ca²⁺-sensitivity limits set by EC coupling in an in vitro system where Ca²⁺ binding affinity can be much greater or much less than the native troponin. Cardiac troponin C presents extreme examples in a single molecule. Only site II binds Ca²⁺ in the physiologically relevant range (2.5 × 10⁵ M⁻¹) and so is solely responsible for Ca²⁺-regulation (Holroyde et al., 1980). A few amino acid changes in the EF-hand motifs results in sites that do not bind Ca²⁺ (Site I) or sites that bind Ca²⁺ 200x tighter (sites III and IV) and are permanently occupied by Ca²⁺ or Mg²⁺ (Li and Hwang, 2015). Thus, it would seem that neither a very high Ca²⁺ sensitivity nor a very low one are able to participate in regulation. How much deviation of Ca²⁺ affinity from the norm is compatible with muscle regulation?

It is known that for mutations, the small Ca²⁺-sensitivity changes correlate with Ca²⁺ binding affinity to thin filaments (Robinson et al., 2007). In a study of mutations induced in skeletal muscle troponin C, Davis et al. achieved a 243-fold range of Ca²⁺ binding affinities for troponin C. However, this did not translate into such a great range when Ca²⁺-binding was measured in the presence of TnI (96-148) and caused a still smaller shift in the Ca²⁺-sensitivity of force production (Davis et al., 2004). Thus, the most extreme Ca²⁺-sensitizing mutation, V45Q increased TnC Ca²⁺ binding affinity 19-fold, but the increase was only 3.1-fold when measured in the presence of the TnI peptide and Ca²⁺-sensitivity in skinned fibers was just 2.3-fold more than wild-type. This is within the same range of many HCM-causing mutations (Table 1). A similar picture emerges from Cardiac troponin C where the single regulatory Ca²⁺-binding site simplifies the argument: V44Q increases Ca²⁺-binding affinity to TnC 6.5-fold but increases myocyte Ca²⁺-sensitivity by just 3.4-fold (Parvatiyar et al., 2010). Thus, it seems that the structure of troponin and its interactions with the rest of the thin filament does limit the consequences of a modification that increases Ca²⁺ binding affinity.

A slightly different situation arises when Ca²⁺ binding affinity is less than wild-type. Davis et al., noted that the mutations that decreased Ca²⁺ binding affinity the most (F26Q, 63-fold, I37Q, 24-fold and I62Q, 10-fold) could not properly regulate force in skinned fibers since they only produced about 13% of the maximal force of wild-type muscle at saturating Ca²⁺ concentrations. On the other hand, two less extreme mutations, M81Q and F78Q decreased Ca²⁺-sensitivity whilst retaining the same maximum force production as wild type. In these cases, again, the increased Ca²⁺ binding affinity for TnC was substantially greater than the increased Ca²⁺-sensitivity of skinned fibers (5.9x vs. 1.8x for M81Q and 8.4x vs. 4.2x for F78Q). Thus, thin filament structure seems to limit the possible effects of changes in Ca²⁺-binding affinity.

It is self-evident that changing myofilament Ca²⁺ sensitivity will affect contractile output in muscle. It is well-established that EC₅₀ for skinned muscle fibers is about 1 μM and that Ca²⁺-activation of contraction is highly cooperative. Most measurements suggest a five-fold range in free Ca²⁺ concentration during a cardiac muscle contraction. Peak Ca²⁺ concentration is about 600 nM at rest and can be substantially higher during adrenergic stimulation, thus normally muscle is only partially activated (Negretti et al., 1995; Dibb et al., 2007).

Figure 2 shows a real life example: in a mouse model of HCM (ACTC E99K) we measured both the Ca²⁺-activation curve for myofibrils and the contractility of intact papillary muscle as well as the Ca²⁺-transient (Song et al., 2013). Under the conditions of this experiment the Ca²⁺ transient was the same in Wild-type and ACTC E99K muscle, Ca²⁺ sensitivity was 0.8 μM for wild-type and 0.34 μM for ACTC E99K with a Hill coefficient of about 4. The increase in Ca²⁺-sensitivity due to the ACTC E99K HCM mutation corresponds to an approximately four-fold increase in twitch force in the absence of a change in the Ca²⁺-transient that was actually observed.

We can use this model to consider what would happen if Ca²⁺-sensitivity changed beyond the normal range. If myofilament Ca²⁺-sensitivity was 4 times normal, maximum force would reach close to 100%, leaving no range for it to be modulated by adrenergic agents. Moreover, it is likely that the muscle would not fully relax, since, based on the five-fold range of the Ca²⁺ transient even at the lowest Ca²⁺ level force would be 5–10%, a substantial fraction of the peak force of wild-type muscle, thus the hypercontractile phenotype would impose a major defect in relaxation, much more severe than the diastolic dysfunction associated with HCM mutations with only a 1.8-fold average Ca²⁺ sensitivity increase.

If myofilament Ca²⁺-sensitivity were decreased to half the normal, contractility would be very low indeed. The fact that mutations that decrease Ca²⁺-sensitivity are not lethal and indeed in transgenic mice, may exhibit little phenotype, is probably due to a compensatory increase in the Ca²⁺-transient (Du et al., 2007). However, this compensation may not be enough to support normal contraction in the long term, leading to DCM, the phenotype commonly associated with reduced Ca²⁺-sensitivity.