Hypertrophic cardiomyopathy (HCM) is a cardiac disease with relatively high prevalence (1:200) in the general population, which promotes morphological changes of the heart such as left ventricular thickening (Semsarian et al., 2015). The first case of HCM was described in a patient from the 1950's (Teare, 1958) in which the diagnosis of HCM development was solely based on electrocardiogram analyses associated with angiographic/hemodynamic studies. After 1990, the development of DNA-based sequencing methodologies provided a valuable resource which accelerated a new era of HCM diagnosis with the discovery of the first mutation in the ß-myosin heavy chain gene, MYH7 (Geisterfer-Lowrance et al., 1990). Currently, it is widely accepted that mutations in sarcomeric genes lead to intracellular alterations that manifest as cardiac remodeling and pathologic hypertrophy (Force et al., 2010; Marian, 2010; Harvey and Leinwand, 2011; Seidman and Seidman, 2011; Maron and Maron, 2013).

After two decades of extensive genetic investigation and over 1,400 mutations identified, many genes encoding thin and thick filament, and cytoskeletal proteins have been established as the etiological agents of HCM (Maron et al., 2012). The most common mutations are located in thick-filament encoding genes (MYH7 and MBPC3), representing ~50–70% of all HCM patients that exhibit positive genetic test results. Meanwhile, mutations in genes that encode thin-filament proteins, such as the troponin (Tn) complex, have also been shown to occur with relatively high incidence compared to other canonical sarcomeric HCM genes (Willott et al., 2010; Brouwer et al., 2011; Maron et al., 2012). HCM-associated mutations lead to several alterations in cardiac contraction e.g., changes in myosin ATPase activity, actomyosin crossbridge interaction, cooperativity of muscle activation, Ca²⁺ binding to the thin filament, and a host of other HCM predisposing factors (Willott et al., 2010). In general, mutations in the regulatory proteins (tropomyosin and Tn) that are linked to HCM are associated with an increase in myofilament Ca²⁺ sensitivity; thus promoting changes in Ca²⁺ homeostasis and triggering development of cardiac dysfunction and arrhythmias seen in HCM patients (Willott et al., 2010; Brouwer et al., 2011; Landstrom and Ackerman, 2012).

Limited studies of HCM patients' skeletal muscle have identified abnormalities in electromyographic (EMG) and/or histologic analyses (Hootsmans and Meerschwam, 1971; Smith et al., 1976; Lochner et al., 1981; Przybojewski et al., 1981). In 1989, Caforio et al. (1989) showed that skeletal muscle dysfunction occurred in 37% of HCM patients analyzed and found that this dysfunction arose from myogenic origins. This finding was independently verified in skeletal muscle biopsies. The authors also observed selective atrophy of type I muscle fibers in these patients (Caforio et al., 1989). Moreover, a recent clinical study which examined 46 HCM patients by EMG showed evidence of subclinical skeletal muscle myopathy in 13 of the patients (28%) (Karandreas et al., 2000).

Many genes for cardiac sarcomeric proteins are also expressed in some skeletal muscles; these include ß-myosin heavy chain (MYH7), regulatory myosin light chain-2 (MYL2), desmin (DES), and troponin C (TNNC1) (Li et al., 1989; Matsuoka et al., 1989; Macera et al., 1992; Song et al., 1996). Mutations in the MYH7 gene can produce simultaneous abnormalities in cardiac and slow-twitch skeletal muscle function that can culminate as cardiomyopathy and/or myopathy (Hedera et al., 2003; Tajsharghi et al., 2003, 2007; Meredith et al., 2004; Mastaglia et al., 2005; Lamont et al., 2006, 2014; Darin et al., 2007; Overeem et al., 2007; Homayoun et al., 2011). Similarly, the MYL2 gene is expressed in both skeletal and cardiac muscle; mutations in myosin light chain appear to affect the function and morphology of both tissue types (Weterman et al., 2013). Desmin, a sarcomeric protein and a primary component of most intermediate filaments, is also a target of muscle disease. Desmin is expressed in cardiac, skeletal, and smooth muscle, and many studies have shown that mutations in the human DES gene promote adult-onset skeletal myopathy and, depending on which mutation, one of the three types of familial cardiomyopathies, i.e., dilated, hypertrophic, and restrictive (Goldfarb and Dalakas, 2009).

Troponin C (TnC) is a key regulatory protein in striated muscle contraction where its function is to bind Ca²⁺, which subsequently triggers actomyosin interactions and initiates muscle contraction (Farah and Reinach, 1995). The TNNC1 gene expresses TnC in both cardiac and slow skeletal muscle (Song et al., 1996). Seven mutations in TNNC1 (A8V, L29Q, A31S, C84Y, E134D, D145E, and Q122AfsX30) have been identified to date in HCM and restrictive cardiomyopathy patients (Hoffmann et al., 2001; Landstrom et al., 2008; Chung et al., 2011; Parvatiyar et al., 2012; Jaafar et al., 2015; Ploski et al., 2016). The A8V mutation was genetically knocked in and led to HCM in mice (Martins et al., 2015). Unfortunately, no clinical data regarding skeletal muscle abnormalities is available for these patients. With the exception of the E134D mutant, in vitro studies indicate that the above mentioned cTnC mutants alter contractile parameters known to underlie the development of diastolic dysfunction in these patients (Landstrom et al., 2008; Pinto et al., 2009, 2011a; Albury et al., 2012; Parvatiyar et al., 2012; Zot et al., 2016). Despite their extensive characterization in cardiac muscle, however, there are no reports on the effects of these HCM-associated TNNC1 mutations on slow skeletal muscle regulation and function.

The purpose of these in vitro studies was to identify potential pathogenic alterations in slow skeletal muscle regulation arising from the cTnC mutants. Therefore, we evaluated the effects of cTnC A8V, C84Y, E134D, and D145E mutants associated with HCM on the Ca²⁺ sensitivity and maximal force when reconstituted into skinned fibers from rabbit soleus, and maximal and minimal ATPase activities when reconstituted into bovine masseter muscle myofibrils. These muscle types were chosen for this study because they are type I fibers that only express myosin heavy chain I (MHC I) and show sufficient resilience after isolation that is suitable for each type of assay presented. Human TnC was utilized to accentuate translational value of the study. The primary sequence of cTnC is the same among human, porcine and bovine homologs, and rabbit cTnC differs by only one amino acid. Other Tn subunits including slow skeletal troponin I (ssTnI) homologs are largely similar (close to 99% homologous) among human, bovine, porcine, and rabbit. However, it should be noted that rabbit slow skeletal troponin T (ssTnT) isoforms are less homologous to human, porcine, and bovine homologs. Utilization of proteins from different but overall largely homologous species has been widely accepted, although small functional differences cannot be discounted. Overall, we found that these cTnC mutants induce mutation-dependent effects in slow skeletal muscle that were distinct from their effects in cardiac muscle; at least part of that distinction was found to depend on the TnI isoform. Our results are consistent with findings from other major muscle proteins encoded by the same gene in both cardiac and skeletal muscles. The data presented in this manuscript will be discussed in the context of the patients' clinical presentation.

The effects of HCM-associated cTnC mutants on the regulation of cardiac myofilaments have been extensively characterized. In Figure 1A, the schematic indicates the location of which muscle sections were excised from rabbit soleus muscle in order to assess whether different MHC isoforms are expressed within the sections. Figure 1B shows glycerol SDS-PAGE analysis of MHC isoforms in myofibrils isolated from rabbit soleus muscles, illustrating that only the slow-skeletal myosin isoform (i.e., MHC I) was uniformly detectable throughout these muscles. Glycerol SDS-PAGE analysis in Figure 1C shows, similarly, that only MHC I was detectable in bovine masseter muscle myofibrils.

We previously showed that cTnC A8V, C84Y, and D145E mutants increased Ca²⁺ sensitivity of steady-state, isometric force development when reconstituted into skinned cardiac preparations, while the cTnC E134D mutant did not affect Ca²⁺ sensitivity (Table 1) (Landstrom et al., 2008). Here, we determined the effects of the HCM cTnC mutants on the regulation of slow skeletal fibers. To accomplish this, TnC-depleted skinned fibers from rabbit soleus muscle were reconstituted with one of the exogenous cTnC proteins (WT, A8V, C84Y, E134D, or D145E). Only the C84Y mutant displayed a significant increase in the Ca²⁺ sensitivity of force development and a decrease of the n_Hill (t-test and ANOVA/Tukey HSD, Figure 2A and Table 1). The pCa₅₀ (i.e., -log free [Ca²⁺] which yields a half-maximal response, where [Ca²⁺] is in molar units) obtained for cTnC C84Y reconstituted slow skeletal fibers was 6.33 ± 0.07; whereas, the pCa₅₀ of fibers reconstituted with WT cTnC was 6.00 ± 0.03. Moreover, when comparing the Ca²⁺-sensitization associated with the cTnC C84Y mutant between reconstituted slow skeletal and cardiac fibers, the slow skeletal muscle fibers further accentuated this effect. This difference is reflected in the ΔpCa₅₀ (difference between the pCa₅₀ of fibers reconstituted with cTnC C84Y and WT) of slow skeletal muscle (+0.33 log units) and cardiac muscle (+0.27 log units; Landstrom et al., 2008) (Table 1). The n_Hill for slow skeletal fibers containing cTnC C84Y was significantly lower than WT, 1.26 ± 0.05 vs. 1.61 ± 0.07 (Figure 2A and Table 1).

Although reconstitution of the cTnC A8V or D145E mutants into skinned cardiac fibers led to an increase in the Ca²⁺ sensitivity of contraction (Landstrom et al., 2008), this was not observed in the slow skeletal fibers (compared to WT cTnC reconstituted soleus fibers; Figure 2A and Table 1), suggesting that the presence of slow skeletal proteins could play a crucial role in normalizing the Ca²⁺ sensitivity of contraction. The n_Hill values of soleus muscle fibers reconstituted with cTnC A8V or D145E were not statistically different from the WT control, indicating that in slow skeletal muscle, these cTnC mutants do not alter cooperativity (Table 1).

Reconstitution of the cTnC E134D mutant did not alter the Ca²⁺ sensitivity or n_Hill of either skinned cardiac or slow skeletal preparations (Figure 2A and Table 1). Additionally, no significant differences in the maximal force were observed among slow skeletal fibers that were reconstituted with the WT or any of the four HCM cTnC mutants (Figure 2B and Table 1).

To assess the effects of the cTnC mutants on myofibrillar ATPase activity, we performed assays comparing the ATPase activity of both cardiac and slow skeletal myofibrils reconstituted with recombinant WT cTnC or mutants. Supplementary Figure 1 shows the SDS-PAGE analysis of cardiac and slow skeletal myofibrils, demonstrating TnC extraction from native myofibrils followed by cTnC reconstitution. At inhibiting (sub-diastolic) Ca²⁺ concentration (pCa 8.0), cardiac myofibrils reconstituted with cTnC D145E (t-test and ANOVA/Tukey's HSD), or C84Y and D145E (t-tests) displayed significantly higher activities than cardiac myofibrils reconstituted with the WT control (Figure 3A and Table 2). This suggests that the cTnC D145E mutant, and possibly also the C84Y mutant, modifies inhibitory processes within cardiac muscle at low levels of Ca²⁺. At maximal activating Ca²⁺ (pCa 5.0), cardiac myofibrils reconstituted with cTnC A8V, C84Y, or D145E exhibited higher levels of myofibrillar ATPase activity when individually compared to the WT control (t-tests), although no significant differences were identified by ANOVA (Figure 3A and Table 2). Differences in muscle regulation by the cTnC mutants were especially evident for the C84Y and D145E mutants in slow skeletal myofibrils. In the slow skeletal muscle background, cTnC C84Y decreased the myofibrillar ATPase activity at maximally activating Ca²⁺ (pCa 5.0) when compared to the WT control (t-test and ANOVA/Tukey HSD; Figure 3B and Table 2). The effect of reconstituting the D145E mutant in slow skeletal myofibrils showed similar trends in ATPase activity to those observed in cardiac myofibrils: the D145E mutant increased ATPase activity at both low and high Ca²⁺ concentrations when compared to the WT control (t-test and ANOVA/Tukey HSD; Figure 3B and Table 2).

Although cTnC A8V and D145E are capable of altering the physiological properties of cardiac muscle contraction (e.g., ΔpCa₅₀ of +0.36 and +0.24, respectively), these mutants did not substantially alter the corresponding properties of slow skeletal muscle (Table 1). The presence of slow skeletal thin filament proteins may have counteracted the enhancement of apparent Ca²⁺ affinity at cTnC's N-terminal regulatory Ca²⁺ binding site which was observed in the cardiac preparations reconstituted with cTnC A8V or D145E. To better understand the influence of the different sarcomeric isoforms (i.e., cardiac vs. slow skeletal) on these phenomena, we tested whether these effects are partially or totally attributed to the presence of ssTnI. Therefore, we exchanged the endogenous cTn complex of skinned cardiac fibers with exogenous chimeric Tn complexes comprised of cTnC, ssTnI, and cTnT. To assess the amount of displaced endogenous cTn achieved by the cTnT displacement method, we recorded the average values of the cardiac preparation's Ca²⁺-unregulated force (i.e., force generated by preparations that have displaced cTnC and cTnI by incubation with cTnT), which were: 88.5 ± 3.4, 82.1 ± 4.2, 80.2 ± 7.2, 86.7 ± 8.4, and 91.7 ± 3.8% for preparations that were to be reconstituted with cTnC WT, A8V, C84Y, E134D, or D145E, respectively. After cTn displacement, cardiac preparations were incubated with the exogenous binary complex containing ssTnI and WT or mutant cTnC. Finally, the recovered maximal Ca²⁺ regulated force was recorded. The maximal force values were: 81.1 ± 4.7, 82.7 ± 4.6, 91.8 ± 2.4, 92.1 ± 5.0, and 84.4 ± 4.1% of the P₀ for WT, A8V, C84Y, E134D, and D145E, respectively. No significant differences in the Ca²⁺-unregulated force, as well as, the Ca²⁺-regulated maximal force among the five experimental groups existed (t-test and ANOVA; Table 3).

Figure 4 indicates that incorporation of ssTnI into cardiac preparations partially attenuated the effects of cTnC A8V and D145E on the Ca²⁺ sensitivity of force development. The ΔpCa₅₀ values for cardiac preparations reconstituted with cTnC A8V and D145E together with ssTnI were +0.09 and +0.18, respectively (Figure 4 and Table 3); whereas, in skinned cardiac preparations reconstituted with cTnC A8V and D145E alone (i.e., in the presence of endogenous cTnI), the ΔpCa₅₀ values were +0.36 and +0.24, respectively (Table 1) (Landstrom et al., 2008). In contrast, in skinned cardiac preparations reconstituted with binary complex containing ssTnI and cTnC C84Y, the presence of ssTnI did not attenuate the myofilament Ca²⁺ sensitization that was observed in the presence of endogenous cTnI. These cardiac preparations exhibited an even higher Ca²⁺ sensitivity of force development than what was observed in cardiac preparations containing the cardiac isoform of TnI (ΔpCa₅₀ +0.41 or +0.27, respectively; Tables 1, 3). A comparison of n_Hill regression parameter estimates obtained from skinned cardiac preparations reconstituted with ssTnI and HCM cTnC mutants indicates that the C84Y (t-test and ANOVA/Tukey's HSD) and D145E (t-test) mutants significantly reduced apparent cooperativity in comparison to the WT control (1.28 ± 0.04, 1.57 ± 0.03, and 1.74 ± 0.04 for C84Y, D145E, and WT, respectively; Table 3). The cardiac preparations reconstituted with exogenous ssTnI and cTnC E134D showed no significant changes in relation to the pCa₅₀ and n_Hill (t-test and ANOVA/Tukey's HSD) when compared with WT control (Figure 4 and Table 3).

Patients that exhibit cardiomyopathy and possess a positive family history are routinely screened for mutations in a number of thin and thick filament encoding genes. Obvious clinical symptoms such as dyspnea, syncope, chest pain, and arrhythmia—in addition to characteristic cardiac remodeling revealed through imaging—are frequently documented complaints. Many sarcomeric mutations that are identified on the basis of cardiac dysfunction, however, are also expressed in skeletal muscle. With the clinical focus on the heart, more subtle skeletal muscle abnormalities may go undetected (Towbin, 2014). We are interested in whether these mutations may have pathogenic consequences in skeletal muscle that go unreported and/or undetected. Here, we queried whether mutations in TNNC1 that are linked to HCM and expressed in slow skeletal muscle can manifest as a dysfunction or affect the contractile properties of slow skeletal muscle.

While the cardiac effects of TNNC1 mutants were first examined in the context of HCM pathogenic properties and disease causation, they have not been assessed in slow skeletal muscle. This study is the first to examine the effects of mutations present in the TNNC1 gene on slow skeletal muscle regulation (Song et al., 1996). We found that for most of the HCM cTnC mutants examined in this study, their incorporation into slow skeletal muscle ablated the increased Ca²⁺ sensitivity of force development that is seen in cardiac preparations. Only cTnC C84Y maintained its Ca²⁺ sensitizing effects in the slow skeletal muscle fibers. Surprisingly, the effects of this mutation were amplified when incorporated into the slow skeletal myofilament. This suggests that this mutant exerts distinct effects on a common element that exist within both muscle types. Since skeletal muscle comprises a large percentage of a person's mass, and slow type I muscle fibers are found throughout human musculature, it is reasonable to expect that slow muscle dysfunction could impart a substantial burden on the affected individual.

Differences in the protein structure of slow skeletal and cardiac TnI and TnT underlie the functional properties of these muscle types, especially when coupled with the distinctive properties of each mutant cTnC in this study. The overall structures of cardiac and skeletal muscle troponins are similar, at least in troponin's core domain (Takeda et al., 2003; Vinogradova et al., 2005). However, three major differences between skeletal and cardiac troponin structures may contribute to the differential effects: (1) structure of the TnI inhibitory segment is ordered in the skeletal muscle isoform but flexible and disordered in the cardiac isoform (Takeda et al., 2003; Vinogradova et al., 2005); (2) the position and conformation of the TnT2 N-terminal helix (Takeda et al., 2003; Vinogradova et al., 2005); and (3) cTnI contains an N-terminal extension—not present in the skeletal isoforms—which harbors two serine residues capable of undergoing phosphorylation by protein kinase A (Zhang et al., 1995). Based upon the above, the experiments involving ssTnI would not be influenced by phosphorylation of the cardiac-specific, N-terminal extention of cTnI.

The myofibrillar ATPase assays further explored the mechanism underlying the Ca²⁺ sensitization induced by the C84Y mutant in both types of muscle fibers. At saturating Ca²⁺ (pCa 5.0), slow skeletal ATPase activity was significantly reduced, while cardiac ATPase activity trended higher. In cardiac muscle, the residue C84 contacts the cTnI switch region when the N-terminus of cTnC is in the open conformation and the BC loop of cTnC when it is in the closed conformation. Therefore, the C84Y mutant may stabilize the open conformation when bound to cTnI (Li and Hwang, 2015) and affect the critically important positioning of the cTnC N-domain (Hwang et al., 2014). Upon binding of divalent cations, the global structure of cTnC becomes more compact (Jayasundar et al., 2014; Badr et al., 2016), which could likely be influenced by mutations in the cTnC linker region. These alterations in the cTnC N-domain may increase the affinity between cTnC-C84Y and the cTnI switch region and ultimately impact initiation of cardiac contraction. The switch region of cTnI (148–158) is immediately flanked by the inhibitory (136–147) and regulatory (161–209) regions, which anchors to actin during diastole (Tripet et al., 1997) and, in doing so, is largely responsible for inhibition of contractility at diastolic Ca²⁺ levels (Meyer and Chase, 2016). Increased affinity between cTnC-C84Y and the cTnI switch region would therefore be expected to reduce interactions between the cTnI inhibitory and regulatory regions during diastole, permitting actomyosin ATPase activity at subthreshold Ca²⁺ concentrations, thereby promoting greater ATPase activation at higher Ca²⁺ concentrations.

Based upon the study by Westfall et al. (1997) on ssTnI-overexpressing cardiomyocytes, the reduction in Ca²⁺ activation threshold in slow skeletal fibers containing cTnC-C84Y (Figure 2 and Table 1), may arise from altered interactions within individual regulatory units of the thin filament (7 actin monomers, 1 tropomyosin, and 1 cTn complex). Weaker interactions between ssTnI and cTnC-C84Y may lower the threshold for Ca²⁺-activated development of tension and reduce cooperativity in cardiac muscle reconstituted with ssTnI. In addition, it is also possible that mutations in troponin differentially modulate the ATPase activity of myosin (Schoffstall et al., 2011; Chalovich, 2012) on top of their effects on Ca²⁺ activation of the thin filament. While Ca²⁺-activation of the cardiac thin filament is more dependent on cross-bridges than skeletal muscle (Gillis et al., 2007), cycling crossbridges at submaximally activating Ca²⁺ concentrations could result in reduced cooperativity in the presence of Ca²⁺-sensitizing mutations (Westfall et al., 1997). The less flexible TnI switch region in skeletal muscle appears to differently affect Ca²⁺ regulation of myofibrillar ATPase activity in the presence of the C84Y mutation in cTnC. The differential effect of C84Y on myofibrillar ATPase activity between tissue types suggests that the C84Y mutation in cTnC could increase Ca²⁺ sensitivity of contraction in both muscles by distinct mechanisms. In cardiac muscle, it may increase the overall number of cycling cross-bridges; while in the slow skeletal muscle, it may decrease the overall number of cycling cross-bridges and enhance the population of strongly bound cross-bridges.

The other HCM cTnC mutants examined in this study are located in distinct structural regions of cTnC. A8V is present in the cTnC N-helix, which plays a key role in defining the orientation of the cTnC N-domain relative to the troponin complex. As described above, alterations in cTnC N-domain orientation can alter the affinity between the cTnC N-domain and the cTnI switch peptide; these changes are likely responsible for increasing the cardiac myofibrillar ATPase activity at saturated Ca²⁺ concentration in this (Figure 3 and Table 2) and prior studies (Zot et al., 2016). However, cTnC-A8V does not affect slow skeletal myofibrillar ATPase measurements or alter cooperativity in cardiac preparations reconstituted with cTnC.ssTnI.cTnT. Functionally, the D145E mutant is distinct; it is located in the C-domain of cTnC and has been shown to increase the Ca²⁺ affinity of site II of isolated cTnC, cTn complex and the thin filament (Pinto et al., 2009). The D145E mutant has also been shown to increase cTnC binding affinity to the cardiac thin filament (Marques et al., 2017). The affinity between the TnI switch region and cTnC may be increased regardless of which TnI isoform is present, as indicated by increased activity of myofibrillar ATPase in both the slow skeletal and cardiac myofibrillar ATPase at both low and high Ca²⁺ concentrations (Figure 3 and Table 2). The D145E mutant did not affect the Ca²⁺ sensitivity of skinned slow skeletal fibers (Figure 2 and Table 1). Therefore, it could be speculated that new interactions/altered affinity between the cTnC D145E C-domain and ssTnT could potentially normalize the sensitizing effects of the mutant, which could be confirmed by peptide binding studies. The E134D mutant did not affect myofibrillar ATPase activity (Figure 3 and Table 2) or Ca²⁺-sensitivity of force development or cooperativity of thin filament activation (Figure 2 and Table 1) in either skeletal or cardiac preparations, further confirming the likelihood that it is a non-pathogenic variant (Landstrom et al., 2008).

The incorporation of ssTnI into cardiac muscle attenuated the dysfunction of myofilaments containing the HCM-associated cTnC mutants A8V and D145E. This suggests that ssTnI plays a protective role in regard to its ability to mitigate effects of disease-causing cTnC mutants in the heart. However, the protective role of ssTnI in cardiac muscle contraction is not a novel topic. We have previously shown that ssTnI has the ability to attenuate the functional consequences of two mutations in cTnT that are associated with restrictive cardiomyopathy (Pinto et al., 2008a, 2011b). Others have shown that the expression of ssTnI in the murine heart has a protective effect on skinned cardiac preparations and left ventricular function during stress conditions, such as acidosis (Wolska et al., 2001; Urboniene et al., 2005). As mentioned above, the presence of ssTnI in cardiac muscle preparation was not able to completely abolish the effect of the cTnC mutants A8V and D145E, as seen in Figure 4 in cardiac preparations reconstituted with ssTnI. This suggests that the presence of ssTnT in slow skeletal muscle may also play a role in rescuing the cTnC mutants' functional phenotype. Major differences exist between the structure of ssTnT and cTnT isoforms, where a large portion of the N-terminus present in cTnT is absent in ssTnT (Pinto et al., 2012). This region of cTnT has been shown to have important roles in regulating Ca²⁺ sensitization and activation of the skinned fibers due to effects on the dynamics of strong TnT-Tm interactions (Gomes et al., 2002; Gollapudi et al., 2012). Therefore, we speculate that some of the protective effect observed in slow skeletal muscle could be arising from the N-terminus of ssTnT.

While the crossover of mutation effects in both tissue types may lead to pathological consequences in both slow skeletal and cardiac muscle, manifestations may present themselves over distinctive time courses and with varying severities. When comparing the age of diagnosis and the symptoms of HCM patients in the cohort study that identified four variants in the TNNC1 gene, it is interesting to note that the patient bearing the C84Y mutant was diagnosed at the earliest age (8.4 years) and exhibited syncope on exertion (Landstrom et al., 2008). Although syncope is a common event, it can signal more ominous underlying conditions (including HCM and arrhythmogenic right ventricular dysplasia) and outcomes such as sudden death (O'Connor et al., 1999). Alterations in skeletal muscle function may lead to additional downstream consequences that remain unknown. Function may be altered further by globally affecting signaling pathways that influence sarcomeric protein isoform switching and energy consumption. These changes could have profound consequences if the aberrant function arises in multiple muscle types, e.g., the cTnC mutants in slow skeletal muscle—despite slow muscle's fatigue-resistant properties that are associated with differential sensitivity to metabolite levels (Chase and Kushmerick, 1988, 1995) in normal individuals—could also be synergistic with regard to patient fatigue or muscle weakness. The true impact of skeletal and cardiac muscle dysfunction as a result of HCM mutations in the TNNC1 gene will bear out after examination of more cases within the clinic. Individuals affected by mutations in genes expressed more systemically have the potential to influence the time course and severity of disease; therefore, it is of interest to study the broader effects of mutations that may modify disease outcomes when manifesting in multiple muscle types.

Part of this work was presented in the Biophysical Society 56^th Annual Meeting in San Diego, California, USA (February, 2012).

Performed experiments: TV, ML, DG, KJ, CM, and DD. Designed Study: TV, MP, DD, AL, PC, and JP. Analyzed Data: TV, ML, CM, DD, and JP. Wrote/Edited Manuscript: TV, ML, MP, DD, AL, PC, and JP.