In most vertebrates studied until now, i.e. mammals, birds and teleosts, two MyHC isoforms, MYH6 and MYH7, are expressed in the adult heart. Orthologs of MYH7 are predominantly expressed in the ventricle, whereas orthologs of MYH6 are predominantly expressed in the atrium and, in fish species, also in the sinus venosus. This data was obtained by means of in situ hybridization [4, 6, 49, 59, 60] and electrophoretic separation [34, 39]. One exception is Xenopus, in which, as Garriock et al.  described after sequence comparison and in situ hybridization, an ortholog of MYH15 is the main ventricular MyHC isoform. MYH15 has become a pseudogene in mammals . However, the isomyosin distribution in the heart of chondrichthyans, the earliest phylogenetical group of living gnatosthomes, remains unknown. Knowledge of MYH isoform expression in the chondrichthyan heart would inform about (1) what was the primitive condition of the trait in gnatosthomes, and (2) when the cardiac MYH distribution shared by most gnatosthomes did appear.
In an ongoing immunohistochemical study of the chondrichthyan heart using the dogfish as an animal model, we detected a striking pattern of immunohistochemical signals with the two anti-pan-MyHC antibodies MF20 and A4.1025. While MF20 signals were homogeneously distributed in the four myocardial segments of the chondrichthyan heart, namely, the sinus venosus, atrium, ventricle, and conus arteriosus, A4.1025 signals were strong in the sinus venosus and atrium, but faint in the conus arteriosus and the ventricle (Fig. 1). This segment-specific immunohistochemical pattern differs from the homogeneous pattern detected with A4.1025 in all the vertebrate species examined, i.e. hamsters (present results), mice [29, 52], Xenopus [1, 44, 48] and zebrafish [22, 24, 44]. This data already suggests that the cardiac segments of the dogfish heart express distinct MyHC isoforms that show differential affinity for MF20 and A4.1025 antibodies.
In order to corroborate our immunohistochemical results, we performed western blot analyses using MF20 and A4.1025 antibodies. Single 220 kDa bands, the approximate molecular weight of MyHC , were obtained after applying a myosin-specific extraction method in dogfish and hamster hearts. This confirms the specificity of both antibodies for MyHC in both species. In addition, the different intensity between the A4.1025 bands corresponding to the inflow and outflow myocardial segments of the dogfish heart strengthens the immunohistochemical findings. Western blot results were corroborated by colorimetric quantification of bands obtained in slot blot analyses. In the dogfish, the intensity of A4.1025 signals in the outflow segments was almost three–fold significantly lower than that in the inflow segments, whereas the intensity of MF20 signals did not significantly differ between blots.
The wall of the bulbus arteriosus does not contain myocardium. It is mainly composed of smooth muscle cells, elastin and collagen [13, 30]. However, our western blot analyses of bulbus extracts showed a narrow band using MF20 or A4.1025 antibodies (Fig. 3). In this regard, we cannot exclude that myocardial tissue of the distal portion of the conus arteriosus was removed together with the bulbus during dissection for protein extraction.
MF20 and A4.1025 antibodies recognize different epitopes of the MyHC protein. MF20 is specific for the tail region , whereas the epitope recognized by A4.1025 is in the head region [7, 10]. Thus, our immunohistochemical, western blot and slot blot results strongly suggest that in contrast to the mammalian heart, in the dogfish heart there is at least one MyHC isoform with a different affinity for MF20 and A4.1025 antibodies. In addition, it can be deduced that this or these isoforms are differentially expressed in the inflow versus the outflow myocardial segments in this species.
To test our hypothesis and identify the MyHC isoform/s differentially expressed in the dogfish cardiac segments, we performed HPLC-ESI-MS/MS analyses in the samples of the inflow and outflow segments. The genomic DNA of the dogfish has not been fully sequenced, and there is no record of the peptide sequence of the MyHC isoforms in this species. Although the whole genome of three additional elasmobranch species (Chiloscyllium punctatum, Scyliorhinus torazame and Rhincodon typus) has been recently sequenced , these sequences are unassembled, and hence they are not accessible to the MS search engine. Therefore, we compared the trypsinised peptides of the inflow and the outflow tract myocardium of the dogfish with protein sequences obtained from the genomic database of the elephant shark (Callorhinchus milii) provided by Venkatesh et al. . C. milii, which belongs to the holocephalans, the sister group of the elasmobranchs, is the only chondrichthyan with the MYH protein sequences included in the UniProtKB/Swiss-Prot database. The MS search engine used in this study (MASCOT and SEQUEST) acquires its maximal efficiency when the highly reliable UniProtKB/Swiss-Prot database is used for comparisons. Samples from both the inflow and outflow segments of the dogfish contained peptides corresponding to MYH10, MYH2, MYH6 and MYH7, whereas peptides corresponding to MYH7B were only detected in the outflow segments (Table 1). In order to confirm these results, we used a chordate peptide database. As it might be expected taking into account the high number of entries in the chordate database and the high level of conservation of MyHC isoforms, several additional sequences matched with peptides in our samples (MYH1B, MYH4, MYH8, MYH9, MYH13, MYHemb and MYSC). Despite this, MYH7B was again found to match with peptides from the outflow segments but not with those from the inflow segments (Table 1).
In order to confirm the distribution of MyHC isoforms revealed by HPLC-ESI-MS/MS and to quantify the relative abundance of these isoforms, we performed ESI-Quadrupole-Orbitrap assays in additional dogfish samples. The results confirmed the data obtained by the previous experiments. The dogfish cardiac outflow segments contain four main sarcomeric isomyosins: MYH2, MYH6, MYH7 and MYH7B, whereas the inflow segments contain only MYH2, MYH6 and MYH7. In addition, the relative abundance of these isoforms is segment-specific. The fast-twitch MYH2 and MYH6 account for more than 75% of the total MyHC isoforms in the inflow segments, where MYH7 (22%) is the only slow-twitch isoform. By contrast, the two slow-twitch isoforms MYH7 and MYH7B are predominant in the outflow segments (70%), whereas MYH2 and MYH6 account for less than 30% of total isomyosins in these segments.
According to the contraction speed, there are two types of skeletal isomyosins: slow and fast-twitch isoforms. In all the vertebrates studied until now, the fast-twitch MyHC that characterizes the inflow segments is MYH6, whereas the slow-twitch MYH7 predominates in the ventricle . Our results show that the myocardium of chondrichthyans contains two additional MyHC isoforms, which are not expressed in the heart of other vertebrates, MYH2 (fast-twitch), abundant in the inflow segments, and MYH7B (slow-twitch), concentrated in the outflow segments. Nevertheless, the myocardium of chondrichthyans shows the same segment-specific composition of isomyosins as other vertebrates with respect to their contraction speed: the inflow segments contains predominantly fast-twitch isomyosins (MYH2 and MYH6; 77%), while the outflow segments contains predominantly slow-twitch isomyosins (MYH7 and MYH7B; 70%). Thus, this segment-specific distribution of fast vs. slow MyHC isoforms can be considered a shared trait of Gnathostomes. New studies in cyclostomes may help to elucidate whether this trait is a synapomorphy or a symplesiomorphy for Gnathostomes. However, the composition of specific cardiac isomyosins has changed during the evolution of the group, reducing its variability in derived taxa.
MYH2 is a fast-twitch skeletal muscle MyHC (), which has not been detected in the myocardium of any adult vertebrate studied until now. Our results point to two possible evolutionary scenarios. In early gnathostomes, MYH2 may have been a cardiac myosin isoform that lost its function in cardiac contraction throughout evolution. Given that MYH2 is absent in the heart of both mammals and teleosts, loss of cardiac expression of this isomyosin might have taken place before the origin of Osteichthyes. Alternatively, MYH2 cardiac expression may have been absent in early gnathostomes, being an independent acquisition in chondrichthyans. MYH7B (firstly known as MYH14) is a poorly understood slow tonic MyHC isoform which, together with MYH15 and MYH16 is considered an ancient MyHC isoform that could have given rise to the current main cardiac isomyosins, MYH6 and MYH7, after a duplication process [12, 53]. Although MYH7B transcripts have been detected in the heart of multiple vertebrate species, including human, mouse , rat , chick, Xenopus  and zebrafish , the cardiac expression of the protein has been the subject of debate. Warkman et al.  self-dismissed a previous positive result in the adult human heart due to a failure in their early experimental design. In addition, it was not clear if an ortholog of MYH7B (ssMHC) is really expressed in the cardiac conduction system of the chick , because later experiments were not able to reproduce this result . In mammals, MYH7B protein has only been found in the extraocular musculature of humans and rats , and in the soleus muscle of mice . Absence of MYH7B expression in the cardiac muscle relies on the presence of a premature termination codon in exon 7 of the gene. The activation of a splicing machinery in skeletal muscle that excludes this exon allows MYH7B translation in this tissue . Therefore, our results clearly show for the first time the expression of MYH2 and MYH7B protein in the adult myocardium of an extant vertebrate, namely, the lesser spotted dogfish.
Similar to myh6 and myh7, myh7b DNA sequence includes an intronic miRNA (MyomiR) named miR-499 , which is present in the myh7b gene of most vertebrate species . The importance of miR-499 relies on its function as a post-transcriptional regulation element of myh7b . It has been also proposed that miR-499 is able to interact with Sox6 to regulate the pathway responsible for the differentiation of the slow and the fast musculature . We propose that in teleosts, birds and mammals MYH6 and MYH7 became mainly restricted to the inflow and outflow segments respectively, whereas MYH2 and MYH7B disappeared from the heart. The mRNA of MYH7B probably acquired a regulatory role in cardiac and extracardiac myogenesis through its intronic miRNA, miR-499 [5, 28, 35, 55].
Using a combination of Orbitrap-Quadrupole and A4.1025 immunoprecipitation methodologies, we were able to calculate the relative affinity of the MyHC isoforms of the dogfish heart for the A4.1025 antibody. The slow-twitch MyHC isoforms showed a reduced affinity compared to the fast-twitch isoforms, being MYH7B particularly unable to bind the antibody (Table 3). These data allow interpretation of the A4.1025 immunohistochemical results in terms of MyHC isoform content. The strikingly low A4.1025 signals in the cardiac outflow segments of the dogfish (Fig. 1) is most probably due to three factors: 1) the high proportion of slow-twitch MyHC isoforms in these segments; 2) the low affinity of these isoforms (particularly MYH7B) for A4.1025; 3) the low concentration of total MyHC in the outflow compared with the inflow segments (see ).
If we assume that the A4.1025 antibody has similar affinities for MyHC isoforms of different gnathostomes, given the high level of conservation of the ATP-binding site in the MyHC protein sequences among vertebrates, our initial immunohistochemical study of different vertebrate species (Fig.
) can be reinterpreted in terms of specific isomyosin abundance. The sand ray and the gulper shark showed the same A4.1025 immunohistochemical pattern as the dogfish, suggesting a distinctly shared MyHC distribution in the heart of chondrichthyans (MYH2 and MYH6 in the inflow and MYH7 and MYH7B in the outflow segments). The silver arowana, the sturgeon, the zebrafish and the hamster showed the expected A4.1025 homogeneous immunohistochemical pattern in all the myocardial segments, suggesting that the conventional MyHC distribution (MYH6 in the inflow and MYH7 in the outflow segments) appeared at the base of Osteichthyes. By contrast, the gray bichir, a representative of polypteriforms, the earliest diverged group of extant actinopterygians [
], showed a different A4.1025 immunohistochemical pattern of staining compared to all the other species studied. A4.1025 signals were intense in all the myocardial segments except for the ventricle. This can be interpreted as a relatively high abundance of slow-twitch isomyosins, particularly MYH7B, only in the ventricle but not in the conus arteriosus. From the evolutionary viewpoint, this finding implies a reacquisition of MYH7B expression in the heart of polypteriforms (Fig.
). In addition, it will be interesting to investigate possible differences in the type of myocardial contraction between bichirs and other actinopterygians. Further studies are required to test the hypothesis depicted in fig.
, by analyzing the MyHC isoform distribution in the heart of additional vertebrate species, particularly in sarcopterygians and other early diverged actinopterygians (as lepisosteiforms) would be of high interest.
In gnathostomes there are two basic cardiac anatomical designs, primitive and modern with respect to the type of cardiac contraction [18, 30]. In mammals, birds and most teleosts, which show the modern design, the main contractile segments are the atrium and the ventricle, whereas in chondrichthyans and early actinopterygians, which show the primitive design, the conus arteriosus is interposed between the ventricle and the bulbus arteriosus. While atrium and ventricle work as synchronous, fast-contracting segments, the conus arteriosus is a slow-contracting segment with peristaltoid contraction. It has been hypothesized that the conus arteriosus serves as an accessory pumping chamber , as an elastic reservoir to minimize pressure fluctuations [9, 26], and to allow a correct function of the conal valvular apparatus  in elasmobranchs. It has been also proposed that the myocardium of the conus arteriosus of teleosts plays an active function in the performance of the outflow tract valves . Birds and mammals also show the primitive cardiac anatomical design at early embryonic stages [11, 16, 37]. In these embryos, MYH isoform coexpression in the ventricle and the conus arteriosus, including MYH7B at least in mammals , allows a slow conduction velocity and a peristaltoid contraction . After cardiac septation, a shift to segment-specific MYH single isoform expression allows development of the fast conduction velocity and synchronous contraction of atrium and ventricle, necessary for the adult type of one-way valves that start to develop at the end of septation (Moorman & Lammers, 1994). According to our results, MYH7B is expressed in the ventricle and/or the conus arteriosus of Chondrichthyes and Polypteriforms, which are fish with a primitive cardiac anatomical design, i.e. with a well-developed conus arteriosus. It can then be hypothesized that the slow tonic MYH7B is required for the peristaltic contraction of the conus arteriosus in fish with a primitive cardiac anatomical design. MYH7B peptides would have disappeared from the adult heart once the modern design, including a synchronous and fast contraction, has developed in tetrapods and teleosts. The MyHC isoform expression pattern in polypteriforms and acipenseriforms, both with a long conus but a different MYH7B cardiac content compared to chondrichthyans, may represent transitional conditions between the primitive and the modern cardiac anatomical design. In this context, it would be interesting to further investigate the anatomy, type of contraction and precise MYH isoform composition in these two actinopterygian groups.