Toad heart utilizes exclusively slow skeletal muscle troponin T: an evolutionary adaptation with potential functional benefits

Abstract

The three isoforms of vertebrate troponin T (TnT) are normally expressed in a muscle type-specific manner. Here we report an exception that the cardiac muscle of toad (Bufo) expresses exclusively slow skeletal muscle TnT (ssTnT) together with cardiac forms of troponin I and myosin as determined using immunoblotting, cDNA cloning, and/or LC-MS/MS. Using RT-PCR and 3'- and 5'-rapid amplification of cDNA ends on toad cardiac mRNA, we cloned full-length cDNAs encoding two alternatively spliced variants of ssTnT. Expression of the cloned cDNAs in Escherichia coli confirmed that the toad cardiac muscle expresses solely ssTnT, predominantly the low molecular weight variant with the exon 5-encoded NH(2)-terminal segment spliced out. Functional studies were performed in ex vivo working toad hearts and compared with the frog (Rana) hearts. The results showed that toad hearts had higher contractile and relaxation velocities and were able to work against a significantly higher afterload than that of frog hearts. Therefore, the unique evolutionary adaptation of utilizing exclusively ssTnT in toad cardiac muscle corresponded to a fitness value from improving systolic function of the heart. The data demonstrated a physiological importance of the functional diversity of TnT isoforms. The structure-function relationship of TnT may be explored for the development of new treatment of heart failure.

FIGURE 1.

mAb 4B8 specific to cardiac TnT across vertebrate species. Western blots using 14% SDS-PAGE with acrylamide:bisacrylamide ratio of 180:1 characterized the isoform specificity of anti-TnT mAb 4B8 generated against human cardiac TnT immunogen. mAb 4B8 recognized TnT in adult cardiac but not skeletal muscles from all mammalian, avian, amphibian, and fish species examined except for one variant in fish red muscle. The cardiac TnT bands recognized by mAb 4B8 were confirmed in Western blots using mAb CT3 that recognizes both cardiac TnT and ssTnT and blots using mAb 2C8 that recognizes all three muscled type isoforms of TnT across vertebrate species. Western blot using mAb TnI-1 detected cardiac TnI, slow skeletal muscle TnI, and fast skeletal muscle TnI in the muscle samples, verifying the presence of all three fiber types. The results demonstrate that mAb 4B8 is a highly valuable cardiac TnT-specific antibody and can be used for studies in all vertebrate species.

FIGURE 2.

Toad cardiac muscle expresses solely ssTnT in the absence of cardiac TnT. A single TnT band was detected in toad heart with a significantly lower molecular weight than that of frog cardiac TnT as shown in the Western blot using mAb 2C8 recognizing all three muscle-type isoforms of TnT (Fig. 1). This TnT band in toad heart was recognized in the Western blot using mAb CT3 that reacts to both cardiac TnT and ssTnT with a molecular weight similar to that of ssTnT in toad and frog skeletal muscles. In contrast, it was not recognized in Western blots using the cardiac TnT-specific mAb 4B8. Reblotting of the 4B8 blot with mAb CT3 confirmed its presence on the membrane. The results demonstrated that the sole TnT expressed in toad heart is ssTnT.

FIGURE 3.

Exclusive expression of ssTnT in toad tadpole heart. The SDS-PAGE gels and Western blots showed expression of ssTnT in the heart of toad tadpoles in the absence of cardiac TnT. Frog tadpole heart and adult toad and frog hearts were used as controls. Same as that in adult toad heart, cardiac TnI was expressed in toad and frog tadpole hearts.

FIGURE 4.

Cloning of cDNAs encoding ssTnT from toad cardiac mRNA. A, two degenerated primers derived from amino acid sequences conserved in vertebrate ssTnT were used in RT-PCR to clone ssTnT cDNA from total RNA extracted from adult toad cardiac muscle. Extended using 3′- and 5′-rapid amplification of cDNA ends, two full-length ssTnT cDNA variants were cloned. DNA sequencing revealed their difference in the inclusion or exclusion of the exon 5-encoded segment (DYGEHIEE) in the NH₂-terminal variable region. B, mAb CT3 Western blots showed that ssTnT proteins expressed from the cloned cDNAs had identical apparent molecular weights (MW) to that of the ssTnT bands in toad cardiac and skeletal muscles. The low M_r ssTnT is the only variant detectable with Western blot in the toad cardiac muscle.

FIGURE 5.

Phylogenetic and sequence studies of toad ssTnT. A, the phylogenetic tree was constructed from aligning amino acid sequences of vertebrate cardiac TnT and ssTnT isoforms using DNA Star Clustal W software. The result demonstrated a conserved sequence of toad ssTnT, which is distinct from cardiac TnT (cTnT). B, alignment of amino acid sequences of toad ssTnT isoforms, Xenopus ssTnT, and Xenopus cardiac TnT. The results showed that toad ssTnT is similar to Xenopus ssTnT but significantly different from cardiac TnT in the NH₂-terminal variable region and a lack of six amino acids near the COOH terminus. GenBank accession numbers: Xenopus ssTnT, NM_001092738.1; Xenopus cardiac TnT high molecular weight (MW) isoform, AF467919.1; Xenopus cardiac TnT low M_r isoform, AF467920.1.

FIGURE 6.

Baseline function of ex vivo toad and frog working hearts. A, at identical heart rates and normalized to heart weight, toad hearts exhibited a trend of smaller stroke volume than that of frog hearts. This difference was primarily due to the larger size of the toad heart normalized to body weights (Table 1), whereas the actual stoke volumes of toad and frog hearts were similar if normalized to the body weight. B, toad and frog hearts exhibited similar maximum aortic and ventricular pressures (AP and VP, respectively). C, toad hearts had faster systolic and diastolic contractile velocities (±dP/dt) than that of frog hearts. D, toad hearts had shorter ejection time than that of frog hearts. n = 3 in frog and n = 4 in toad groups. Values are presented as mean ± S.E. *, p < 0.05 versus frog heart in two-tail Student's t test.

FIGURE 7.

Toad hearts were able to produce output against higher afterload than that of frog hearts. A, at 25 cm H₂O afterload, positive stroke volume response to preloads from 2 to 8 cm H₂O was detected in both toad and frog hearts. B, at 4 cmH₂O preload, toad hearts continued to produce aortic output when afterload was increased from the base line of 25 cm H₂O up to 65 cm H₂O, whereas frog hearts failed to produce aortic output at afterload above 45 cm H₂O (p < 0.05 for the maximum afterload at which aortic output was detectable in Student's t test). n = 3 in frog and n = 3 in toad groups. Values are presented in mean ± S.E.

FIGURE 8.

Toad heart expresses normal cardiac TnI. A, Western blots detected only cardiac TnI (cTnI) in toad and frog hearts, whereas slow and fast skeletal muscle TnI were detectable under the same conditions in skeletal muscles. No skeletal muscle TnI was detected in the toad heart. B, amino acid sequence alignment outlined the similarities and differences between toad cardiac TnI and cardiac TnI of Xenopus, human, and mouse. The main difference between amphibian and mammalian cardiac TnI is in the NH₂-terminal extension and a 6-amino acid extension at the COOH terminus. The conserved protein kinase A phosphorylation sites as found in human and mouse cardiac TnI are outlined with a gray box. GenBank accession numbers: Xenopus cardiac TnI, L25721.1; human cardiac TnI, X54163.1; mouse cardiac TnI, NM_009406.

FIGURE 9.

Toad heart expresses cardiac MHC. A, MHC isoforms in the hearts and skeletal muscles of toad, frog, and Xenopus were resolved in glycerol/SDS gel. Mouse diaphragm muscle was analyzed in parallel as control. A single MHC band was detected in toad heart with a slightly different gel mobility from that of frog cardiac MHC or the MHC bands in toad rectum abdominis and sartorius muscles. B, mAb FA2 specific to cardiac α- and β-MHC (cardiac β-MHC is the same as MHC I in slow skeletal muscle) recognized the MHC band in both toad and frog hearts, supporting their nature as cardiac MHC.

FIGURE 10.

Sequence coverage of toad MHC from LC-MS/MS analyses using four different proteases. A database search showed that cardiac-like myosin heavy polypeptide 15 of X. laevis (gi 148222862) was the top match. Therefore, this sequence was used as a template to demonstrate the peptide mapping results for toad MHC. Amino acids marked with black letters represent sequences detected in LC-MS/MS analyses. The boxed lowercase letters represent amino acids that are different from the Xenopus MHC 15 sequence. X represents amino acid residues in the MHC of the Xenopus MHC 15 sequence, which are not covered in the LC-MS/MS results. Together with a match of the toad heart MHC sequence with other cardiac MHC proteins in the NCBInr database as described in the text, the data confirmed that the MHC in toad heart is a cardiac isoform.