Jack‐of‐many‐trades: discovering new roles for troponin C

In the mid-1960s Ebashi and Kodama (Ebashi & Kodama, 1966) discovered that the Ca2+ regulation of muscle actomyosin ATP activity required the presence of the thin filament regulatory molecules, tropomyosin and troponin. Less than a decade later, Greaser and Gergely (Greaser & Gergely, 1973) determined that troponin consisted of three subunits, troponin T (TnT), troponin I (TnI) and troponin C (TnC), the latter being the only subunit with a high binding affinity for Ca2+. Canonically, TnC acts as a switch molecule, whereby Ca2+ binding triggers a series of allosteric interactions that initiates contraction and the reversal of these processes permits muscle relaxation. Mammalian striated muscle expresses two TnC isoforms, fast skeletal (fs) and slow skeletal/cardiac (ss/c) TnC. The fsTnC isoform has two N-terminal regulatory domains while the ss/cTnC isoform contains a single N-terminal regulatory domain and has a lower Ca2+ binding affinity than fsTnC. In this issue of The Journal of Physiology, Tikunova et al. (2018) investigated the consequences of expressing ss/cTnC carrying a Ca2+ desensitizing missense mutation on mouse soleus muscle properties. The D73N ss/cTnC mutation was previously shown to elicit cardiac muscle abnormalities similar to dilated cardiomyopathy including cardiac hypertrophy, increased chamber size, thinner walls and reduced ejection fraction (McConnell et al. 2015). Tikunova et al. tested the hypothesis that Ca2+ desensitization of ss/cTnC leads to alterations in skeletal muscle contractile properties that independently augment cardiac dysfunction morbidity. Experiments were designed to systematically examine soleus morphology and function at multiple levels of muscle organization. In accordance with the hypothesis, D73N ss/cTnC induced profound changes in skeletal muscle function, including the conversion of both contractile properties and fatigue profiles to ones more like fast-twitch muscle, which, indeed, could exacerbate morbidity associated with cardiac myopathies. However, as often occurs during rigorous assessment of molecule-to-organ-level studies, Tikunova et al. delivered the muscle research community two unforeseen outcomes. First, and perhaps most intriguing, slow-twitch fibres carrying the D73N ss/cTnC mutation displayed marked changes in morphology; the cross-sectional area (CSA) of these fibres was 60% of soleus slow-twitch fibres from wild-type mice. Interestingly, the size of the fast-twitch fibres in the D73N soleus muscle compensated with a ∼25% increase in average fibre CSA. Still the number of slow-twitch fibres was equivalent between groups and, consequently, the cumulative area occupied by slow-twitch fibres decreased in the experimental group. The higher fast-twitch:slow-twitch area ratio could account for the shift toward fast-twitch contractile properties (i.e. shorter times to peak twitch force and 50% relaxation); however, the fundamental question persists as to how a Ca2+ desensitizing ss/cTnC mutation causes such a profound change in skeletal muscle fibre morphology. The possibilities are many but clarification is limited and the question remains mostly unresolved. Some possibilities include (i) lack of normal fibre development, (ii) activation of muscle fibre atrophy pathways, (iii) shift in the set-point of the force–time integral homeostatic feedback system (Davis et al. 2016), (iv) activation of intracellular [Ca2+]-mediated gene expression processes, and, perhaps even, (v) myofilament regulatory proteins working as trans-activating gene expression factors (Zhang et al. 2013). Whichever fibre morphology control systems are involved, future studies in this regard could have therapeutic implications for muscle development deficiencies, disuse atrophy and ageing-related cachexia. Another exciting result was the marked improvement in relaxation during fatigue tests in D73N isolated soleus muscles. While the total force at the end of the stimulation protocol was lower in D73N soleus muscle, the traces clearly show improvement in the baseline force after each stimulus train over the final half of the fatigue test (Fig. 3 in Tikunova et al. 2018). The exact reasons for altered relaxation properties are unknown and while it could, at least in part, involve the increase in fast-twitch fibre:slow-twitch area ratio, this explanation seems insufficient since the soleus muscles still contained ∼34% slow-twitch fibres. It is becoming clear that striated muscle relaxation is a multifaceted, highly regulated process that involves the integration of three control processes: (i) intracellular Ca2+ decline, (ii) thin filament inactivation, and (iii) myosin cross-bridge interaction kinetics (Luo et al. 2002; Biesiadecki et al. 2014). The observed improvement in relaxation seems rather remarkable given that there was only ∼30% expression of Ca2+ desensitized ss/cTnC and this was in only a fraction of the fibres (∼34%) in the muscle. Such a finding offers a strong scientific premise for continued pursuit of targeting myofilament regulatory proteins as a means to treat diastolic dysfunction and depressed cardiac reserve capacity associated with restrictive cardiomyopathies. Overall, Tikunova et al. uncovered novel roles for TnC, ones that extend beyond its canonical Ca2+ switch role to dimensions where TnC regulates skeletal muscle fibre size and can re-orchestrate relaxation signatures of muscle containing mixed fibre types. The study also provides a robust example of how novel mechanistic insight can be gained from a rigorous and well-designed study that utilizes systematic biochemical and physiology assays across multiple spatial levels of organization. These types of mechanistic studies coupled with proof-of-concept studies showing improved ventricular function in diseased hearts by altering Ca2+ sensing molecules and thin filament activation (Shettigar et al. 2016) illuminate the potentially translational value of continued myofilament research. None declared. Sole author. None.