Axolotls are useful models for the study of cardiac development because of a unique, naturally occurring, recessive mutation in what is called gene c, which results in cardiac non-function. The gene c mutation causes failure of the heart to beat and causes death of embryos by stage 42. Mutant hearts lack expression of tropomyosin, as well as having significantly decreased expression of the tropomyosin binding subunit of the troponin complex, Troponin T (TnT).
The troponin complex is made up of three subunits: the inhibitory complex (Troponin I), the calcium binding complex (Troponin C) and the tropomyosin binding subunit (Troponin T). Troponin T is necessary for myofibril formation, and isoforms specific to each type of muscle have been characterized. Cardiac TnT (cTnT), slow skeletal TnT (ssTnT) and fast skeletal TnT (fsTnT), the three main TnT isoforms, also undergo alternative splicing, resulting in formation of additional isoforms, especially different stages of development (Zhang et al. 2007).
Humans with mutations in cardiac Troponin T mutations present with a variety of syndromes, including dilated cardiomyopathy and hypertrophy(Zhang et al. 2007).
cTnT Expression in Developing Axolotl Embryos
Immunohistochemical analyses of mutant embryo hearts using a TnT antibody that recognized skeletal and cardiac TnT showed expression in the heart at all stages of development. Further investigations by Zhang et al. 2007, however, used an antibody specific only to cardiac TnT, in order to eliminate the possibility of reacting with skeletal TnT, at various stages of development in normal and mutant Axolotl embryos.
The heart in a normal axolotl embryo begins beating at stage 34, and it is at this stage that c/c mutants are first distinguishable. After the initial heartbeat, cTnt expression is significantly increased in normal embryos and distinct staining patterns from cTnT antibody are visible on fully developed myofibrils. In the mutant embryos, the heart fails to beat at this stage and pools of cTnT are visible, concentrated in the conus region.
Expression of cTnT increases and an organized, striated staining patten becomes apparent in normal embryos as cTnT in incorporated into the myofibrils of the developing heart structure. Mutant embryos show diffuse and random staining patterns localized to peripheral membranes.
Multiple layers of well-developed myocardial cells show an organized staining pattern and a strong regular heartbeat in normal embryos at this stage. Mutant hearts have only one layer of myocardial cells and obtain oxygen only through diffusion. Yolk platelets are still present in the heart sue to lack of metabolism and the heart is distended. Mutant embryos do not survive past this stage.
These studies concluded that while reduced, substantial expression of cTnT exists in the developing heart in the mutant embryo, but in dramatically altered expression patterns from wild-type embryos. The majority of the cTnT expression in mutant hearts was restricted to the conus area, with significantly decreased expression in the ventricular area. The analyses support the existence of two isoforms ( cardiac and skeletal) of TnT in the developing heart and suggest that the cardiac isoform has critical role in myofibrillar organization of the axolotl heart. The paper raises the question of whether the decreased expression of cTnT RNA in mutant hearts is due to fewer myocardial cells present or due to a decrease of cTnT RNA expression in each cell.
Sferrazza et al. 2007 present a possible explanation for the decrease in cTnT expression, proposing that the decrease is due to abnormal splicing of cTnT in mutant embryos.
This hypothesis was tested by cloning four different cTnT isoforms (TNNT2-1,2,3,4) from a developing axolotl heart. These were sequenced and it was determined that each of the three shorter isoforms was created by the splicing out of a single segment of the longer molecule (TNNT2-1) (Sferrazza et al. 2007).
All four isoforms can be amplified from a normal axolotl heart, while TNNT2-2 is absent from c/c mutant hearts, as shown in Figure 2 above. Conversely, TNNT2-3 is expressed at near wild type levels in c/c mutant axolotl hearts. It was determined through these experiments that mutant hearts show both significantly decreased expression of cTNT and differential isoform expression patterns compared to normal axolotls (Sferrazza et al. 2007).
The cause of decreased cTnT expression, and ultimately failure of normal heart development, in the axolotl cardiac mutant has not been completely characterized, however abnormal splicing events and decreased mRNA provide good starting points for further investigation.
Sferrazza GF, Zhang C, Jia P, Athauda G, Dube S, Lemanski SL, Dube DK, Lemanski LF. Role of Myofibril-Inducing RNA in cardiac TnT expression in developing Mexican axolotl. Biochem Biophys Res Com. 2007;357:32–37. doi: 10.1016/j.bbrc.2007.03.064
Zhang, C., Pietras, K., Sferrazza, G., Jia, P., Athauda, G., Rueda-de-Leon, E., Maier, J., Dube, D., Lemanski, S. and Lemanski, L. (2007), Molecular and immunohistochemical analyses of cardiac troponin T during cardiac development in the Mexican axolotl, Ambystoma mexicanum. J. Cell. Biochem., 100: 1–15. doi: 10.1002/jcb.20918