Robert D Gaffin & Mariappan Muthuchamy
Cardiovascular Research Institute and Department of Systems Biology and Translational Medicine, Texas A&M Health Science Center, TX, USA.

https://doi.org/10.36866/pn.65.27

Tropomyosin (TM) is a thin filament regulatory protein that is intricately involved in cardiac muscle contraction. Working in conjunction with the troponin complex, TM’s position on actin either blocks or exposes myosin­binding sites, and thus prevents or allows crossbridge formation as outlined in the three state model of muscle contraction (McKillop & Geeves, 1993).

The principal focus of our lab has been to address the role of TM in cardiac muscle function. Initial findings using β-TM transgenic (TG) mouse model demonstrated that β-TM TG hearts displayed diastolic dysfunction including increased time to one-half relaxation and a decreased maximum rate of relaxation in working heart preparations, increased calcium sensitivity in skinned fibres.

Two important charge modifications in β-TM, Ser229Glu and His276Asn, give it a charge that is more negative than α-TM. Since the remaining residue differences between the two isoforms involve rather conservative polar or non-polar changes, we hypothesized that generating β-TM’s two charge modifications on an α-TM background would produce results identical to those seen in β-TM TG mice. Thus, we generated ‘double mutation’ (Ser229Glu and His276Gln) TG mouse lines. These TG mice exhibited decreased Ca2+-sensitivity in skinned fibres as well as a decrease in both the rate of contraction and relaxation in working heart preparations (Gaffin et al. 2004a).

We then wished to see the effects of these two charge changes on an individual basis. α-TMSer229Glu TG mice exhibited a decrease in myofilament calcium sensitivity and a decrease in both +dP/dt and -dP/dt in the working heart while α-TMHis276Asn TG mice did not alter calcium sensitivity but decreased -dP/dt (Gaffin et al. 2004b). Furthermore, TG mouse lines that express a mutant form of α-TM in which the first 275 residues are from α-TM and the last nine amino acids are from β-TM (α-TM9aa∆β) exhibit decreased rates of contraction and relaxation in working heart studies (Gaffin et al. 2006). The myofilaments containing α-TM9aa∆β protein demonstrate a normal pCa-force relationship (Gaffin & Muthuchamy, unpublished data). These data indicate that the function of TM is compartmentalized along its length (Fig. 1).

We propose that residues located near the site where TM interacts with the C-terminus of TnT (which also interacts with the C-terminus of troponin C (TnC), the myofilament’s calcium sensor) primarily affect calcium sensitivity since α-TMSer229Glu caused a decrease while α-TMHis276Asn and α-TM9aa∆β had no effect. On the other hand, α-TMHis276Asn and α-TM9aa∆β mutations located in the carboxy terminus of TM, have altered the rates of contractile dynamics without affecting calcium sensitivity.

To explain this, we point to the fact that the carboxy terminus affects TM-actin affinity in the ‘open’ state of muscle contraction as proposed by Hitchcock­Degregori’s group (Moraczewska et al. 1999). An enhancement of TM-actin affinity during this phase would delay the myofilament’s transition from the ‘open’ to the ‘blocked’ state thus affect contraction and relaxation kinetics in α-TM His276Asn and α-TM9aa∆β TG mice.

Compartmentalization of TM function is further evidenced by the recent work of Dr Wieczorek’s group. TG mice expressing chimeric TM protein (exons 1–8 [residues 1– 257] from α-TM plus exon 9 [residues 258–284] from β-TM) exhibit decreased rates of contraction and relaxation in whole heart studies, and a decrease in calcium sensitivity in skinned fibres (Jagatheesan et al. 2003). In addition, exchanging the TnT binding domains of α-TM amino acids [175–190 and 258–284] with the β-TM regions decreases the rates of contractile dynamics; however, the calcium sensitivity is not altered (Jagatheesan et al. 2004).

These results, along with our data, point out the complexity of the relations between structure and function of TM molecule. Each difference exists between the α- and β-TM molecules such as TnT binding domains, charge residue changes or the changes in the carboxy terminal ends contribute to the contractile dynamic changes and/or calcium sensitization of the myofilaments. However, none of these changes, either individually or in combination within the α-TM molecule, mimic the function that is seen in the β-TM TG mice. In addition, these TG mice functional studies reveal there is a lack of correlation between the force-calcium relationships and the haemodynamic measurements at the myofilaments and the whole heart levels, respectively, indicating a cautious extrapolation/ correlation of the physiological and biochemical parameters of cardiac function. On the other hand, both calcium activation of the thin filament and positive feedback by strong crossbridges mechanisms make significant contributions to each phase of the contractile cycle. Further analytical tools to quantify the contributions of the calcium or crossbridge cooperative effects to each phase of the contractile cycle would be useful to address the mechanisms of TM function in the myofilament activation processes.

In summary, we conclude that the function of α-TM is compartmentalised along its length. Sequences nearer the inner core of TM have a greater effect on calcium sensitivity due to their proximity to the TnT-TnC interface while residues in the carboxy terminus affect either TM-actin affinity or crossbridge kinetics. Furthermore, these studies support the idea that the localised flexibility present in the coiled coil structure of various TM isoforms are different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and differentially modulating the myofilament activation processes.

Acknowledgements

This work was supported by an NIH grant HL-60758 to M M.

References

Gaffin RD, Gokulan K, Sacchettini JC, Hewett T, Klevitsky R, Robbins J & Muthuchamy M (2004a). Charged residue changes in the carboxy-terminus of alpha-tropomyosin alter mouse cardiac muscle contractility. J Physiol 556, 531-543.

Gaffin RD, Gokulan K, Sacchettini JC, Hewett TE, Klevitsky R, Robbins J, Sarin V, Zawieja DC, Meininger GA & Muthuchamy M (2006). Changes in end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics. Am J Physiol Heart Circ Physiol 291, H552-563.

Gaffin RD, Tong CW, Zawieja DC, Hewett TE, Klevitsky R, Robbins J & Muthuchamy M (2004b). Charged residue alterations in the inner­core domain and carboxy-terminus of alpha-tropomyosin differentially affect mouse cardiac muscle contractility. J Physiol 561, 777-791.

Jagatheesan G, Rajan S, Petrashevskaya N, Schwartz A, Boivin G, Arteaga G, de Tombe PP, Solaro RJ & Wieczorek DF (2004). Physiological significance of troponin T binding domains in striated muscle tropomyosin. Am J Physiol Heart Circ Physiol 287, H1484­1494.

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Moraczewska J, Nicholson-Flynn K & Hitchcock-DeGregori SE (1999). The ends of tropomyosin are major determinants of actin affinity and myosin subfragment 1-induced binding to F-actin in the open state. Biochemistry 38, 15885-15892.