Calcium desensitization in cardiac troponin C: a novel role in mouse skeletal muscle

Voluntary movement, postural stability, heat production and metabolism are all essential homeostatic processes in which the role of skeletal muscle is essential. Thus, it is important to consider the potential widespread implications of skeletal muscle pathology. In a paper recently published in The Journal of Physiology, Tikunova et al. (2018) provide convincing evidence of functional changes within skeletal muscle fibres upon alteration of cardiac troponin C (TnC) (Tikunova et al. 2018). Ultimately, this study highlights the multifactorial nature of diseases such as dilatated cardiomyopathy (DCM), and emphasizes the need for critical consideration of multiple organ systems when determining pathogenesis and symptom management. The function of skeletal muscle is mediated by a combination of fibres, broadly categorized into slow or fast twitch. Each plays an important role in mediating a wide range of movements, with slow-twitch fibres undergoing slower contraction and relaxation and exhibiting increased fatigue resistance compared to fast-twitch fibres (Berchtold et al. 2000). These differences result from unique kinetic and biochemical properties, including the composition of their respective myosin heavy chain (MHC) isoforms. Specifically, the expression of different troponin isoforms in these subtypes, with differing Ca2+ binding and exchange capacities, regulates the contractility of striated muscle (Berchtold et al. 2000). During skeletal muscle contraction, Ca2+ binding to TnC induces a conformational change within the troponin complex, revealing actin-binding sites on myosin and allowing cross-bridge formation to occur. The slow skeletal and cardiac muscle-specific isoform of TnC (ss/cTnC) is predominantly found in cardiac and skeletal slow-twitch fibres, regulating muscle contraction by binding Ca2+ through a regulatory EF-hand motif. While ss/cTnC contains four EF-hand motifs (two N-terminal and two C-terminal), the N-terminal EF-hand II (i.e. residues 65–76) plays a critical role in the regulation of muscle contraction (Takeda et al. 2003). While phenotypic skeletal muscle abnormalities have previously been observed in patients with DCM, it is currently unknown whether these abnormalities occur secondary to impaired cardiac function. Consequently, the current study examined the physiological consequences of desensitization of the regulatory N-terminal domain of ss/cTnC to Ca2+ in mice. Specifically, Tikunova et al. (2018) investigated mice soleus skeletal muscle, where ss/cTnC-containing slow-twitch fibres are prevalent, to provide insight into how potential skeletal muscle aberrations are associated with morbidities resulting from impaired cardiac function. This study utilized heterozygous D73N knock-in mice to examine the effects of desensitizing the regulatory domain of ss/cTnC to Ca2+ on the contractile properties of slow skeletal muscle. Heterozygous D73N knock-in mice were backcrossed with C57BL/6J mice, as a homozygous genotype was developmentally lethal within 1 day of life. RT-PCR analysis was used to confirm that the amount of ss/cTnC transcript in isolated D73N soleus muscle was similar to estimated values in left ventricular muscle. The researchers examined various contractile properties of the soleus muscle, including the force–stimulation frequency relationship, fatigue resistance, and times to peak force and relaxation during isometric twitches and tetani. Utilizing isolated single fibres, the force versus pCa relationship in slow- and fast-twitch fibres was also studied. Finally, MHC and troponin-T isoform compositions were determined using gel electrophoresis and western blots, respectively, and slow- and fast-type fibres were identified using immunohistochemistry. To determine if the D73N mutation could elicit pathologies associated with DCM, heart biomass from wild-type and knock-in mice heterozygous for the ss/cTnC D73N mutation was measured. Consistent with a previous study, D73N mice had significantly increased heart mass compared to WT mice, suggesting impaired cardiac function. Next, a muscle-apparatus lever system was used to determine the force output from wild-type and D73N soleus muscle to assess muscle contractile properties. All measured kinetic properties (i.e. time to peak force, time to 50% relaxation, time to 90% relaxation) during both twitch and tetanus were significantly shorter in soleus harbouring the D73N mutation. It is interesting that following normalization with muscle cross-sectional area, peak twitch and tetanus forces did not differ between wild-type and D73N mice. Wild-type and D73N soleus were also stimulated (i.e. from 10 to 100 Hz) to elucidate the force–frequency relationship. At lower stimulation frequencies (i.e. from 10 to 50 Hz), D73N soleus generated less force than wild-type soleus. Finally, the muscle was stimulated with trains of stimuli as a fatigue test. It was observed that D73N soleus had a greater reduction in peak force generation compared to wild-type soleus, indicating lower fatigue resistance. To determine the fibre type composition of the soleus muscle, digested protein lysates from single fibre and whole soleus muscle from wild-type and D73N mice were immunoblotted, and MHC composition was visualized. In D73N soleus, slow-type MHC1 was significantly lower relative to total MHC, concomitant with a smaller, but significant, increase in fast-type MHC2 relative to total muscle MHC. Moreover, cryosections from isolated wild-type and D73N soleus were used to identify slow- and fast-twitch fibres. While fibre composition did not differ, mean cross-sectional area of slow-twitch fibres was significantly reduced (∼40%) in D73N soleus. Lastly, using isolated single fibres from wild-type and D73N soleus, the concentration of Ca2+ required to reach 50% of max contraction (pCa50) was measured to investigate the relationship between force and Ca2+ concentration. As expected, the pCa50 was significantly lower for D73N slow-twitch fibres compared to wild-type, with no differences in pCa50 for fast-twitch fibres between D73N and wild-type mice. This work assesses the physiological consequences of Ca2+-desensitized ss/cTnC in skeletal muscle, to evaluate its potential contributory role in cardiac disease and morbidity. By engineering a D73N mutation within the Ca2+ binding loop of the ss/cTnC regulatory EF-hand domain (Fig. 1), Tikunova et al. (2018) were able to investigate the various effects of Ca2+-desensitized ss/cTnC on skeletal muscle fibre composition, cross-sectional area and contractile force. Backbone cartoon representation. The N-terminal lobe is highlighted in blue, while the C-terminal lobe is highlighted in green. A short linker region is highlighted in black. The residues comprising the regulatory EF-hand are highlighted in red, with the exception of the Asp73 residue, which is highlighted in orange. All images were rendered using the PDB ID 2JT3 in PyMol. This study further suggests that mutations to ss/cTnC contribute to cardiac muscle abnormalities similar to those in human patients with DCM-like symptoms. It has long been established that skeletal muscle aberrations, including decreased oxidative capacity and slower rates of contraction and relaxation, are likely secondary to chronic heart failure (Drexler et al. 1992). However, this study provides novel evidence that skeletal muscle from mice with impaired cardiac function contracts faster, suggesting a primary role for this effect. Another common consequence of heart failure is skeletal muscle atrophy (Mancini et al. 1992), but this was not observed in D73N mice. As a result, the link between chronic heart failure and skeletal myopathies may be more complex than originally thought. While this study is the first to describe the effects of desensitizing ss/cTnC to Ca2+ on the physiological properties of skeletal muscle, there is one notable limitation. The use of only adult male mice excludes the potential to highlight sex differences. With that said, the effects of female hormones on cardiovascular and skeletal physiology make these studies difficult. Potential sexual dimorphisms may remain elusive, complicating the translation of this research. Future studies should seek to determine if the phenotypic consequences observed in D73N mice skeletal muscle are a direct consequence of changes in the contractile parameters of slow-twitch fibres. Specifically, engineering multiple point mutations within the N-terminal regulatory EF-hand of ss/cTnC has the potential to exacerbate this phenomenon (Fig. 1). More direct Ca2+ measurements such as Ca2+ binding assays would also aid in clarifying the magnitude of Ca2+ desensitization in D73N ss/cTnC. Cardiomyopathies remain a significant source of morbidity and a tremendous burden on healthcare systems worldwide. The prevalence of this condition continues to increase as medical advances positively affect life expectancy. Detrimental musculoskeletal abnormalities are thought to arise as a secondary consequence of cardiomyopathies, including DCM, but this may not be entirely accurate. Tikunova et al. (2018) show that the D73N mutation of ss/cTnC in mice soleus muscle results in decreased fatigue resistance and Ca2+ sensitivity, subsequently leading to faster skeletal muscle contraction and relaxation. These functional changes are associated with an increase in fast-twitch fibre cross-sectional area, but may be due, at least in part, to altered slow fibre contraction kinetics due to the reduced affinity of D73N ss/cTnC for Ca2+. The results of this study suggest that skeletal abnormalities in mice with impaired cardiac function may not only be a secondary consequence of cardiomyopathies, but have the potential to arise primarily via mechanisms that warrant further investigation. None. M.J.N., M.Z., H.E.S. and Q.L. drafted the work and revised it critically for important intellectual content. All authors have read and approved the final version of this manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. None.

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