The influence of trout cardiac troponin I and PKA phosphorylation on the Ca²⁺ affinity of the cardiac troponin complex

When the temperature of the vertebrate heart is reduced, its ability to contract and generate force is impaired. This effect of temperature change is due to a reduction in the activity of Ca²⁺ handling proteins, such as the sarcolemmal Na⁺/Ca²⁺ exchanger (Elias et al., 2001), and to the contractile element losing its ability to respond to changes in intracellular Ca²⁺ (Churcott et al., 1994; Gillis et al., 2000; Harrison and Bers, 1989). Ca²⁺ is the intracellular signal that initiates contraction by binding to the thin filament via cardiac troponin C (cTnC). The Ca²⁺ activation of cTnC initiates a series of conformational changes through the components of the thin filament, resulting in the formation of force-generating cross-bridges between actin and myosin.

For ectothermic species, such as rainbow trout, the desensitizing effect of temperature on contractile function represents a potential challenge. Trout live, and remain active, in waters where temperatures range seasonally between 4 and 20°C (Black, 1950). Such temperatures would cause cardioplegia in the mammalian heart (Harrison and Bers, 1989; Churcott et al., 1994). One characteristic of the trout heart that enables it to function at these low temperatures is that it is 10 times more sensitive to Ca²⁺ than mammalian hearts when measured at the same temperature (Churcott et al., 1994). This helps to compensate for the desensitizing effect of low temperature, as the Ca²⁺ sensitivity of the trout heart and mammalian heart are similar at their respective physiological temperatures (Churcott et al., 1994; Gillis and Tibbits, 2002). The comparatively high Ca²⁺ sensitivity of the trout heart is due in part to trout cTnC having 2.3-fold the Ca²⁺ affinity of human cTnC (Gillis et al., 2000). The residues in trout cTnC responsible for its comparatively high Ca²⁺ affinity are Asn2, Ile28, Gln29 and Asp30 (Gillis et al., 2005). When human cTnC was mutated to contain these residues and the mutant protein was incorporated into rabbit cardiac myocytes, the Ca²⁺ sensitivity of force generation was increased 2-fold (Gillis et al., 2005). This clearly shows that trout cTnC can increase the Ca²⁺ sensitivity of force generation but indicates that other mechanisms are involved in the comparatively high Ca²⁺ sensitivity of the trout heart.

A second candidate for increasing the Ca²⁺ sensitivity of the trout heart is cardiac troponin I (cTnI). This regulatory protein, along with cTnC and cardiac troponin T (cTnT), form the cardiac troponin (cTn) complex. When the heart is relaxed, the inhibitory peptide of cTnI (residues 128–147) interacts with actin and prevents the movement of tropomyosin (Tm) across the surface of the thin filament. In this state Tm is thought to sterically block the formation of force-producing cross-bridges between actin and myosin (Kentish et al., 2001; Li et al., 2004; Rarick et al., 1999; Solaro and Rarick, 1998). Following the release of Ca²⁺ from the sarcoplasmic reticulum, Ca²⁺ binds to the regulatory domain of cTnC (site II). The resulting conformational change triggers the movement of the switch peptide of cTnI (residues 147–163) towards a hydrophobic cleft that has been exposed within the N-terminus of cTnC (Kentish et al., 2001; Li et al., 2004; Rarick et al., 1999; Solaro and Rarick, 1998). This movement of the switch peptide pulls the adjacent inhibitory peptide away from the actin–Tm complex (Kentish et al., 2001; Li et al., 2004; Rarick et al., 1999; Solaro and Rarick, 1998). As a result, Tm is released and it is able to roll across the surface of actin and expose myosin binding sites (Gordon et al., 2000).

In the mammalian heart, cTnI is a critical site for the regulation of thin filament function. It is targeted for phosphorylation by protein kinase A (PKA), protein kinase C (PKC) and protein kinase D (PKD) following adrenergic stimulation (Kobayashi and Solaro, 2005; Cuello et al., 2007; Haworth et al., 2004). One domain of cTnI found in mammalian species that is targeted for phosphorylation is the N-terminal extension (residues 1–32), which contains two phosphorylatable residues (Ser23 and Ser24). This region of cTnI interacts with the N-terminal domain of cTnC and the strength of the interaction increases during Ca²⁺ activation (Robertson et al., 1982). When Ser23 and Ser24 are phosphorylated, this interaction between cTnI and cTnC is thought to weaken, leading to a reduction in the Ca²⁺ affinity of the regulatory domain of cTnC (Chandra et al., 1997; Robertson et al., 1982; Solaro et al., 2008). This translates into a decrease in the Ca²⁺ sensitivity of force generation and increased rates of muscle relaxation (Kentish et al., 2001; Li et al., 2004; Matsuba et al., 2009; Rarick et al., 1999; Solaro and Rarick, 1998). The change in cTnC Ca²⁺ affinity upon cTnI phosphorylation illustrates the fact that changes in the structural properties of cTnI can alter the ability of the cTn complex to be activated by Ca²⁺.

The purpose of this study was to determine how trout cTnI influences the Ca²⁺ affinity of a troponin complex containing mammalian components (cTnC, cTnT, cTnI) and to characterize how PKA phosphorylation modulates this. To do this, we cloned cTnI from a trout heart cDNA library, expressed the protein and then complexed it with rat cTnT and the human cTnC mutant C35S, T53C, C84S human cTnC (T53C human cTnC) labeled with anilinonapthalenesulfote iodoacetamide (IAANS) at Cys53. IAANS is a fluorescent molecule and by titrating this complex with Ca²⁺ and measuring fluorescence we were able to monitor the change in cTnC conformation that occurs during Ca²⁺ activation. Using this technique we compared the Ca²⁺ affinity of the cTn complex containing trout cTnI with that of one containing rat cTnI. In addition, we characterized how the Ca²⁺ affinity of these two protein complexes is altered following PKA treatment.

Nested PCR with degenerate primers was first utilized to clone an interior region of the cTnI cDNA and then RACE-PCR was used to clone the 5′ and 3′ ends of the gene. The degenerate primers were designed to target a region of high conservation identified using known cTnI sequences from the zebrafish (Danio rerio), and two species of puffer fish (Takifugu rubripes and Tetraodon nigroviridis). One forward (F) and two reverse primers (R1 and R2) were chosen. The primer sequences are as follows: cTnI-F: GAC RWR GTG GAT GAR GAG MGR TAY GA, cTnI-R1: CTT CTT CAC CKK CTT GAG GTK GG, cTnI-R2: CSA YRT TCT TRC GCC AGT CKS YCA C; where R, W, M, Y, K and S correspond to standardized coding used to indicate specific, multiple nucleotides in the same position within degenerate primers.

The first PCR reaction was performed using trout heart cDNA as the template with F and R2 as the primers. The second PCR reaction was performed using the product of the first PCR reaction with F and R1 as the primers. Following PCR, the DNA was inserted into the StrataClone™ PCR cloning vector pSC-A (Stratagene, Agilent Technologies Inc., La Jolla, CA, USA) for sequencing.

RACE-PCR was completed using primers cTnI-F and cTnI-R2 and the SMART™ RACE cDNA amplification kit (Clontech, Mountain View, CA, USA). Two different samples of RACE-ready cDNA were created from different hearts and used as separate templates for each reaction. The products of each RACE-PCR reaction were inserted into the StrataClone™ PCR cloning vector and sequenced.

Primers were designed for the 5′ and 3′ ends of each gene based on the sequences obtained via RACE-PCR. PCR was performed with multiple samples of trout heart cDNA and the products were cloned into the sequencing plasmid. Sequencing results demonstrate that each PCR product was the same.

The full-length trout cTnI cDNA was inserted into a pET-24a(+) expression vector (Novagen, San Diego, CA, USA) by engineering NdeI and EcoRI restriction sites into the 5′ and 3′ ends of the gene, respectively. The sequence of the primers used was: cTnI-NdeI-F: C ATA TGC CAG AGC AAG TAC AAG AGA AGC G and cTnI-EcoRI-R: G AAT TCT ACT GAG GGG TGC CTT TGG C. The PCR product was subsequently digested with EcoRI and NdeI, then gel purified. The insert was ligated into linearized pET-24a(+) using T4 DNA ligase (New England Biolabs, Ipswich, MA, USA). The ligated plasmid was transfected into NovaBlue competent cells (Novagen). Liquid cultures were grown overnight and isolated plasmids were sent for sequencing to confirm the presence of the insert.

To confirm that the protein resulting from the cloned gene corresponds to that expressed in the trout heart, western blotting and mass spectrometry were used to characterize the cTnI present in trout cardiac myofilament proteins. Multiple lanes of trout myofilament proteins were separated using SDS-PAGE with recombinant rat cTnI used as the standard. We took care to ensure that equal amounts of protein were loaded per lane. The SDS-PAGE gel was transferred onto Hybond-P PVDF membranes using an ECL semi-dry transfer unit (GE Healthcare, Piscataway, NJ, USA). The primary antibody was a goat polyclonal raised against the C-terminus of human cTnI (sc-8118, Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). This antibody also binds fast skeletal (fsTnI) and slow skeletal TnI (ssTnI) from a number of vertebrate species (T.E.G., unpublished) (Santa Cruz Biotechnology product literature). The secondary antibody was a rabbit anti-goat IgG conjugated to horseradish peroxidase (AP106P; Millipore, Etobicoke, ON, Canada). The western blot identified rat cTnI on the membrane as well as a single protein band in the trout heart sample that was of lower mass (Fig. 1).

A sample of the trout myofilament proteins used in western blotting was separated using SDS-PAGE and then transferred onto Hybond-P PVDF membranes. The membrane was stained with Coomassie R250 and the single band, from multiple lanes, corresponding to that identified using western blotting was excised and prepared for trypsin digestion and mass spectroscopy analysis at the Advanced Analysis Center at the University of Guelph. (Note that there was only a single band on the protein gel in the region of the band that was bound by the western antibody; see Fig. 1.) Briefly, the excised gel bands were destained until colorless, and dried using a SpeedVac. Following reduction with dithiothreitol (DTT) and alkylation with iodoacetamide, the protein was digested with 10 ng of sequencing grade trypsin (Calbiochem, La Jolla, CA, USA) in 25 mmol l^–1 NH₄HCO₃ (pH 7.6) at 37°C overnight. The proteolytic peptides were extracted, and cleaned up by a C18 Ziptip (Millipore). MALDI data were acquired using an Applied Biosystems/MDS Sciex QStar XL quadrupole time-of-flight (QqTOF) mass spectrometer under a nitrogen laser (337 nm), and 2,5-dihydroxybenzoic acid was used as the matrix. The peptide fingerprinting masses (m/z) on the MS spectrum were compared with the theoretical values generated in silico by MS-Digest, a ProteinProspector program developed at the UCSF Mass Spectrometry Facility (http://prospector.ucsf.edu/). Using this method the peptide fingerprint generated from the protein band was compared with those for proteins within the National Center for Biotechnology Information (NCBI) protein database, including trout cTnI.

The cDNA for rat cTnI and rat cTnT were provided by Dr M. Regnier (University of Washington) while the cDNA for T53C human cTnC was provided by Dr J. P. Davis (University of Ohio). All proteins were expressed using BL21 Gold (DE3) competent E. coli cells (Agilent Technologies Inc.) with expression being initiated with IPTG (0.4 mmol l^–1). Each protein was purified using an AKTA FPLC (GE Healthcare) and standard procedures. T53C human cTnC was purified as detailed previously (Gillis et al., 2007), rat cTnT was purified as in Chandra et al. (Chandra et al., 2006), while rat cTnI and trout cTnI were purified as in Guo et al. (Guo et al., 1994). One modification to optimize the purification of trout cTnI was that all buffers used on the CM cation exchange column had a pH of 7.0 instead of 8.0. Following purification, the identity of each protein was confirmed using peptide sequence analysis as described above. All proteins were lyophilized and then stored at –20°C.

Purified T53C human cTnC was labeled as in Davis et al. (Davis et al., 2007). In brief, T53C human cTnC was dialyzed into (in mmol l^–1): 50 Tris, 6000 urea, 90 KCl, 1 EGTA, pH 7.5. Protein concentration was determined using a Bradford assay and then 5-fold molar excess of IAANS was added to the solution. The reaction was allowed to occur for 8 h in the dark, and then stopped with 2 mmol l^–1 DTT. IAANS-labeled T53C human cTnC (human cTnC^IAANS) was then dialyzed against (in mmol l^–1): 10 MOPS (pH 7.0), 4600 urea, 1 DTT, with 0.01% NaN₃. IAANS concentration was calculated from its absorbance at 325 nm using its extinction coefficient of 24,900 l mol^–1 cm^–1. The efficiency of binding was calculated by dividing the concentration of human cTnC^IAANS by the IAANS concentration (binding efficiency was 92%).

Tn complexes were prepared following Davis et al. (Davis et al., 2007). Tn subunits were dialyzed separately into (in mmol l^–1): 10 MOPS (pH 7.0), 4600 urea, 1 DTT, with 0.01% NaN₃. Bradford assays were utilized and the subunits were mixed for 20 min at a molar ratio of 1.5:1:1.5 (TnI:TnC:TnT). The control group contained rat cTnI/human cTnC^IAANS/rat cTnT (mammalian cTn^IAANS) and the experimental group contained trout cTnI/human cTnC^IAANS/rat cTnT (trout cTnI-cTn^IAANS). The sequence of rat cTnC varies from that of human cTnC by 1 residue out of 161. This difference is a methionine instead of an isoleucine at residue 119. This is a conservative substitution that should not alter the function of the protein. Therefore, the proteins in the mammalian cTn complex would be interacting with each other within a ‘rat’ conformation. The complexes were dialyzed against a series of buffers containing (in mmol l^–1): 10 MOPS (pH 7.0), 0.5 DTT, 3 MgCl₂, with 0.01% NaN₃. The first buffer contained 2 mol l^–1 urea and 1 mol l^–1 KCl; the second contained 1 mol l^–1 KCl; and the third contained 0.15 mol l^–1 KCl (repeated three times). The complexes were then dialyzed against a titration buffer containing (in mmol l^–1): 200 MOPS (pH 7.0), 150 KCl, 3 MgCl₂, 1 DTT, 2 EGTA, with 0.02% Tween-20.

The PKA treatment was completed when the proteins were in a complex so that they would be in a ‘physiological’ conformation interacting with cTnC and cTnT. This was done to ensure that only targets that are phosphorylated in vitro would be targeted in this experiment. Samples of trout cTnI-cTn^IAANS and mammalian cTn^IAANS were each diluted to 0.35 mg ml^–1 by the addition of titration buffer. PMSF and Na₂ATP were then added to final concentrations of 0.25 and 10 mmol l^–1, respectively. The catalytic subunit of PKA (20 units) was added to 1 ml volumes of each protein complex. The mixtures were incubated for 1 h at 25°C and then dialyzed against titration buffer. The control groups were treated in the same way but without the addition of PKA or Na₂ATP. Phosphorylated trout cTnI-cTn^IAANS and mammalian cTn^IAANS will be designated as trout cTnI-cTn^IAANS*P and mammalian cTn^IAANS*P.

The protein complexes were separated using SDS-PAGE and the gels were stained using Pro-Q Diamond phosphoprotein stain following the manufacturer's instructions (Molecular Probes, Eugene, OR, USA). Stained gels were imaged using a Typhoon 9410 scanner (GE Healthcare) with an excitation wavelength of 532 nm and an emission filter of 560 nm. Following imagining of Pro-Q-stained gels, gels were stained for total protein using Coomassie R250.

Differences between the K_F1/2 values and Hill coefficients (n_H) generated by fitting each titration curve with the Hill equation were analyzed using two-way ANOVA followed by Bonferonni post hoc tests (P<0.05). Data are represented as means ± s.e.

The cDNA for trout cTnI is 570 nucleotides (GenBank accession number HM012798). The protein encoded by this gene is 189 amino acids, which is 21 residues shorter than human cTnI. Interestingly, trout cTnI lacks the cardiac-specific N-terminal extension that contains the PKA/PKC/PKD phosphorylation targets (serine residues) at positions 23 and 24 (Fig. 2). All other cTnIs cloned from fish species also lack this extension (Shaffer and Gillis, 2010). Trout cTnI does, however, contain Ser residues at positions 14 and 16. The corresponding residues in human cTnI are the PKC targets Ser42 and Ser44 (Fig. 2). In addition, there is also a Ser at residue 115 in trout cTnI that corresponds to the PKC target Thr143 in human cTnI (Fig. 2). At the amino acid level trout cTnI displays 59% identity to the human cTnI paralog while it is 78% identical to human ssTnI.

The single TnI band in the trout cardiac myofilament protein sample was identified as trout cTnI with a display score of 88% with 72% coverage. This was the best score obtained for any protein within the NCBI protein database. The identified peptides obtained from this analysis are mapped onto the protein sequence of trout cTnI in Fig. 2. This analysis clearly indicates that the TnI gene we cloned from trout heart cDNA corresponds to the TnI protein expressed in the heart.

The treatment of trout cTnI-cTn and mammalian cTn with PKA resulted in the phosphorylation of trout cTnI and rat cTnI. Fig. 3 is a composite image establishing the phosphorylation state of rat cTnI and trout cTnI following PKA treatment. The lanes in Fig. 3A show recombinant rat cTnI and trout cTnI stained with Coomassie R250, demonstrating the difference in protein mass. The western blot in Fig. 3B identifies recombinant rat cTnI and trout cTnI using the human cTnI antibody sc-8118. Fig. 3C is a comparison image of control and phosphorylated rat cTnI and trout cTnI stained with Pro-Q Diamond stain and Coomassie R250. The levels of these images have not been modified. From Fig. 3C it is evident that there is no Pro-Q signal from either of the non-phosphorylated bands while there is a clear signal from the phosphorylated bands. From this it is evident that the phosphorylation of mammalian cTn and trout cTnI-cTn increases the strength of each TnI band on the Pro-Q stained gel. The Coomassie-stained bands indicate that there was equal loading of protein between the control and PKA-treated samples. As expected, there was no evidence that either cTnC or cTnT was phosphorylated by PKA treatment (data not shown). To determine whether there were differences in the levels to which trout cTnI and rat cTnI were phosphorylated by PKA, we utilized ImageJ (National Institutes of Health, Bethesda, MD, USA) to analyze the density of the TnI bands on the protein gels stained with Pro-Q and Coomassie R250. To correct for differences in the amount of protein in each band, we standardized the density of each band stained with Pro-Q to the density of the same band stained with Coomassie. This analysis demonstrated that the Pro-Q-stained bands of phosphorylated rat cTnI had a higher density than the Pro-Q-stained bands of phosphorylated trout cTnI. For rat cTnI the standardized density was 1.03±0.02 and that for trout cTnI was 0.85±0.07. This indicates that the level of phosphorylation of the trout cTnI sample was ∼82.5% that of the rat cTnI sample. As the same concentrations of the trout cTnI-cTn and mammalian cTn complexes were treated with the same number of units of PKA, this difference indicates that more phosphate groups were incorporated into rat cTnI than into trout cTnI. This can be interpreted as there being more phosphorylatable targets in rat cTnI than in trout cTnI. This result reflects the fact that trout cTnI does not contain the N-terminal extension but that it does contain other putative PKA targets.

The Ca²⁺ titration of each Tn complex produced sigmoidal curves that reached an asymptote upon saturation (Fig. 4). The data were fitted well by the Hill equation with a goodness of fit for all curves being greater than 0.89 (Table 1). The titration curve for trout cTnI-cTn^IAANS is positioned to the left of that for mammalian cTn^IAANS (Fig. 4). The K_F1/2 for trout cTnI-cTn^IAANS was significantly less than that of mammalian cTn^IAANS (1.46±0.01 vs 2.62±0.35 μmol l^–1) (Table 1). This difference indicates that the Ca²⁺ affinity of trout cTnI-cTn^IAANS is 1.80-fold that of mammalian cTn^IAANS. Phosphorylation of the trout cTnI-cTn^IAANS and mammalian cTn^IAANS caused the titration curves to shift to the right (Fig. 5), indicating that the Ca²⁺ affinity of the complexes was reduced by this treatment. The K_F1/2 of trout cTnI-cTn^IAANS*P shifted 2.06 μmol l^–1 while that for mammalian cTn^IAANS*P shifted 4.51 μmol l^–1. This clearly shows that the presence of trout cTnI reduces the influence of PKA treatment on the Ca²⁺ affinity of the Tn complex. The Hill coefficients calculated for the titration curves of trout cTnI-cTn^IAANS and mammalian cTn^IAANS were less than 1 (Table 1) and there was no effect of PKA phosphorylation on these values.

The use of IAANS-labeled T53C human cTnC to study the Ca²⁺ activation of the cTn complex is a technique that was established by Davis et al. (Davis et al., 2007). These authors demonstrated that the conservative mutations (C35S, T53C, C84S) required to allow the labeling of Cys53 with IAANS and the subsequent attachment of IAANS to Cys53 did not significantly alter the functional characteristics of the Tn complex (Davis et al., 2007). The shift in IAANS fluorescence is caused by the change in the conformation of cTnC that occurs upon Ca²⁺ binding. The residue to which IAANS is attached (Cys53) is located within the BC subdomain of cTnC, which moves away from the NAD subdomain as the regulatory domain (N-terminus) of cTnC binds Ca²⁺ and interacts with cTnI (Davis et al., 2007). In the current model of the Ca²⁺ activation of cTnC, and subsequent interaction with cTnI, the BC domain does not interact with cTnI during Ca²⁺ activation (Takeda et al., 2003). Therefore, the change in fluorescence that was monitored reports on the change in conformation of cTnC and not on the interaction between cTnC and cTnI. In addition, the K_d of the labeled cTn complex (0.65 μmol l^–1) measured by these authors is within the range of intracellular Ca²⁺ concentrations during muscle activation and the rate of Ca²⁺ disassociation from the labeled complex (k_off) is almost identical to that of the native complex (36.0±0.8 vs 36.5±0.4 s^–1) measured using a fluorescent chelator (Quinn 2) (Davis et al., 2007). As a result, this method is an effective technique to monitor the Ca²⁺ activation of the cTn complex.

In this study we have demonstrated that the TnI expressed in the trout heart lacks the N-terminal extension present in cTnI from all other vertebrate groups. By replacing rat cTnI in a mammalian cTn complex with trout cTnI we have demonstrated that this protein increases the Ca²⁺ affinity of the complex and also reduces the influence of PKA phosphorylation on the Ca²⁺ affinity of the cTn complex. These results are significant as they provide insight into the role that trout cTnI plays in regulating thin filament function in a lower vertebrate and how this protein has changed during the evolution of the heart and its function in vertebrate species.

In the current study, the K_F1/2 (equivalent to K_d) of mammalian cTn^IAANS was 2.62±0.35 μmol l^–1. This value is within the range of intracellular Ca²⁺ concentrations during muscle activation. The difference between this value and that from the Davis et al. study (Davis et al., 2007) (0.65 μmol l^–1), where the same techniques were used, likely reflects the fact that the studies were completed in different laboratories using buffers made from different stock solutions. The shift in the K_F1/2 of the complex that occurred when rat cTnI was replaced with trout cTnI indicates that this protein increases the Ca²⁺ affinity of the cTn complex. A similar shift in K_F1/2 was found to occur when human cTnI was replaced in a cTn complex with mammalian ssTnI (Davis et al., 2007), which is consistent with previous experiments demonstrating that ssTnI increases the Ca²⁺ sensitivity of force generation in mammalian cardiac tissue (Fentzke et al., 1999; Metzger et al., 2003; Westfall and Metzger, 2007). The results of the current study therefore suggest that trout cTnI does play a role in the comparatively high Ca²⁺ sensitivity of the trout heart. The similar shift in K_F1/2 caused by the replacement of rat cTnI with either trout cTnI (present study) or rat ssTnI (Fentzke et al., 1999; Metzger et al., 2003; Westfall and Metzger, 2007) is also evidence that these two proteins are more similar to each other than trout cTnI is to rat cTnI. This result is interesting as both rat ssTnI and trout cTnI lack the N-terminal extension, present in rat cTnI, that interacts with the N-terminal domain of cTnC in a Ca²⁺-dependent manner.

To more completely characterize the influence of trout cTnI on the Ca²⁺ affinity of the Tn complex, further work needs to be completed with this protein complexed with trout cTnC and trout cTnT. Previous work has illustrated that each of the troponin components influences the Ca²⁺ binding characteristics of the Tn complex (Abbott et al., 2001; Davis et al., 2007; Liang et al., 2008; McAuliffe et al., 1990; McCall et al., 2006; Robertson et al., 1982). It may be, therefore, that all trout components need to be working together for the full effect of the functional characteristics of the proteins to be realized. In addition, the reconstituted troponin may differ in unpredictable ways from a native trout or rat complex because of potential differences in interactions between heterologous subunits.

Following a phylogenetic study of all known TnI isoforms, we have recently proposed that the three muscle-specific isoforms of TnI (cardiac, ss and fs) were produced via two genome duplications that occurred in an ancestor of all extant vertebrate groups (Shaffer and Gillis, 2010). Our analysis indicated that a fsTnI ‘like’ paralog is the ancestral form of the protein, which was then replicated to produce two isoforms via genome duplication. One of these evolved to become cTnI. The gene for cTnI was subsequently replicated and one of these evolved to become ssTnI. A similar scheme for the evolution of other contractile proteins has also been suggested by Oota and Saitou (Ota and Saitou, 1999). We have also suggested that the similarity between cTnI from fish species and ssTnI indicates that there has been little change in the sequence of fish cTnI since this group diverged from the vertebrate lineage (Shaffer and Gillis, 2010). The results of the current study demonstrating that trout cTnI increases the Ca²⁺ affinity of the cTn complex, as does ssTnI, support this hypothesis.

The result that PKA treatment causes a reduction in the Ca²⁺ affinity of mammalian cTn^IAANS is supported by previous studies characterizing cTn function in solution (Chandra et al., 1997; Robertson et al., 1982). In a study by Robertson and colleagues (Robertson et al., 1982) where native cTnC was labeled with IAANS, the pCa₅₀ (equivalent to the –log of K_F1/2) of the cTn complex decreased from 6.71 to 6.51. This is equal to a 1.14 μmol l^–1 increase in the amount of Ca²⁺ required to half-saturate the protein complex upon PKA phosphorylation. In the current study, this increase in K_F1/2 was equal to 4.51 μmol l^–1. The difference between the two studies is likely due to differences in the position of the IAANS probe within the cTn complex. In the Robertson et al. study (Robertson et al., 1982), native bovine cTnC was used, so IAANS would have been bound to Cys35 and Cys84. In the current study, IAANS was attached to residue 53, which, because of its location within the molecule, does not interact with other regions of cTnC or with cTnI/cTnT during Ca²⁺ activation (Davis et al., 2007). The decrease in the Ca²⁺ affinity of the mammalian cTn complex with PKA treatment is consistent with the decrease in Ca²⁺ sensitivity of force generation that occurs when mammalian cardiac tissue is treated with PKA (Fentzke et al., 1999; Matsuba et al., 2009). This change in cTn function is thought to be responsible for the increased rate of muscle relaxation that occurs following β-adrenergic stimulation in the heart (Kentish et al., 2001; Li et al., 2004; Rarick et al., 1999; Solaro and Rarick, 1998).

The treatment of trout cTnI-cTn with PKA results in the phosphorylation of trout cTnI and causes a decrease in the Ca²⁺ affinity of the cTn complex. Because of the experimental design it was not possible to determine whether this shift in K_F1/2 (2.06 μmol l^–1) was significantly different from that caused by similar treatment of mammalian cTn (4.51 μmol l^–1). As trout cTnI lacks the PKA targets at positions 23 and 24, this indicates that another residue or residues was/were targeted for phosphorylation and this caused the change in Ca²⁺ affinity. The most likely candidates are the serines at residues 14 and 16 of trout cTnI (Fig. 2). These residues correspond to Ser43 and Ser45 in mammalian cTnI isoforms, which are targets for phosphorylation by PKC (Fig. 2). Analysis of trout cTnI protein sequence using NetPhos (Blom et al., 1999) gives each of these residues a high score (>98%), indicating that these are putative phosphorylation sites. In mouse cardiac preparations the phosphorylation, or pseudo-phosphorylation, of these sites causes a decrease in the Ca²⁺ sensitivity of force generation, a decrease in maximum tension and a reduction in thin filament sliding speed (Burkart et al., 2003; Noland et al., 1995). These effects at the cellular level are due to the cTn complex losing its ability to be activated by Ca²⁺. The fact that the Ca²⁺ affinity of the trout cTnI-cTn shifted by a smaller value following PKA treatment suggests that the function of the complex is affected to a lesser degree than that containing rat cTnI.

In the accompanying study (Gillis and Klaiman, 2011), we demonstrated that the treatment of skinned trout cardiac trabeculae with PKA reduces force generation and the rate of cross-bridge cycling at maximum Ca²⁺ activation. There was no influence, however, on the Ca²⁺ sensitivity of force generation. When the trabeculae were analyzed using Pro-Q diamond stain it was found that the only protein to increase in phosphate content was myosin regulatory light chain. There was no change in the level of cTnI phosphorylation. The accompanying study demonstrates that trout cardiac tissue does not respond to PKA treatment in the same way as mammalian tissue and this is likely because trout cTnI is not phosphorylated by PKA when functioning within the trout contractile element. Our result in the current study demonstrating that trout cTnI is phosphorylated by PKA is likely due to the protein being more accessible to PKA when it is functioning in solution than when it is functioning within an intact contractile element.

The results of this study demonstrate that trout cTnI can increase the Ca²⁺ affinity of a mammalian cTn complex and that it also reduces the influence of PKA phosphorylation on cTn function. These results provide evidence that cTnI from fish can be viewed as ancestral or primitive forms of the paralogous protein in mammalian cardiac tissue. This hypothesis is supported by previous phylogenetic studies of vertebrate TnI (Shaffer and Gillis, 2010). The sensitizing effect of trout cTnI on the mammalian cTn complex suggests that this protein is involved in the comparatively high Ca²⁺ sensitivity of the trout heart and therefore helps enable cardiac function at low temperatures. The limited response to PKA phosphorylation of the cTn complex containing trout cTnI is due to the lack of the N-terminal extension in the protein. The N-terminal extension present in cTnI from all other vertebrate groups allows for greater regulation of cTn function and it evolved after fish diverged from the vertebrate lineage (Shaffer and Gillis, 2010). The lack of this protein domain in cTnI therefore limits the ability of the animal to regulate cardiac contractility.