Regulatory Proteins

TN-C. The cardiac muscle TN-C (cTN-C) differs from fast skeletal muscle TN-C that it contains only one Ca2+-binding site in the N-terminal domain of the protein. The neighboring site, at the N-terminal does not contain aspartic acid, a prerequisite for coordination of Ca2+. This site is called site 1, whereas the Ca2+-binding site is called site 2 of cTN-C.

TN-I: The cardiac TN-I (cTN-I) differs from the fast skeletal muscle TN-I by containing an N-terminal extension of 32 amino acid residues (Fig. H3).

Fig. H3. Comparison of the overall structure of cardiac muscleTN-I with that of skeletal muscle TN-I. The shaded areas correspond to homologous regions in the structures (From Perry 1996)..

This extension has two adjacent serine residues, No. 22 and 23 in the sequence. Solaro et al. (1976) discovered that TN-I is phosphorylated in perfused rabbit heart stimulated by adrenaline. It was shown later (Mittmann et al.,1990; 1992) that both serines at positions 22 and 23 of rabbit cardiac TN-I can be phosphorylated by cyclic AMP-dependent protein kinase (PKA). Since PKA is activated by adrenaline in the beating heart it appears that PKA is responsible for the diphosphorylation of TN-I in the heart.

Early studies have established that TN-I phosphorylation decreases the Ca2+-sensitivity of cardiac myofibril MgATPase (Ray and England, 1976; Solaro et al., 1976), i.e. the Ca2+ concentration for 50% ATPase activity increases. The motif of two adjacent serine residues was found in hearts from various mammals suggesting that modulation of Ca2+-sensitivity by phospho-TN-I is characteristic for heart muscle. The mechanism of this effect is of interest. Cardiac muscle contraction is initiated by the binding of Ca2+ to site 2 of TN-C as site 1 does not function as Ca2+-binding site (see above). NMR, fluorescence resonance energy transfer, and mutation studies indicate that the N-terminal part of TN-I combines with TN-C. Upon phosphorylation of TN-I, the site 1 of TN-C undergoes a conformational exchange consistent with an equilibrium between closed and opened forms of TN-C (Gaponenko et al., 1999). In addition, TN-I phosphorylation changes the binding of Ca2+ to TN-C, the structure of TN-I, and the cooperative binding of Tn-I to actin-TM (Solaro and Van Eyk, 1996). Previously, phosphorylation has been shown to modulate cardiac function by reducing the Ca2+ affinity for the N-terminal regulatory site of cTN-C (Solaro, 1986). Thus, TN-I phosphorylation is a unique property of the myocardium that plays a key role in cardiac function.

Recently, fluorescence resonance energy transfer was used (Dong et al., 2001) to investigate the global conformation of the inhibitory region of a full-length TN-I mutant from cardiac muscle in the unbound state and in reconstituted complexes with the other cardiac TN subunits. The mutant contained a single tryptophan residue at the position 129 which was used as an energy transfer donor, and a single cysteine residue at the position 152 labeled with IAEDANS as an energy acceptor. The distance between the donor and acceptor sites was found to be 19.4 A and it was insensitive to reconstitution of cTN-I with cTN-T, cTN-C, or cTN-C plus cTN-T, in the absence of bound regulatory Ca2+ in cTN-C. A large increase in the Trp129-Cys152 distance was observed upon saturation of the Ca2+ regulatory site of cTN-C in the complexes. This increase suggests an extended conformation of the inhibitory region in the interface between cTN-C and cTN-I in the holo cardiac troponin, which may pull away the inhibitory region of cTN-I from actin upon Ca2+activation in cardiac muscle.

TN-T: As skeletal TN-T, cardiac TN-T (cTN-T) also has several isoforms (McAuliffe et al., 1990). Two cTN-T isoforms have been identified in adult beef heart, which show differences in their sequence and activation of the actinS1 ATPase. Five isoforms were found in rabbit heart and two in rat heart. During development of rabbit heart, there are shifts in the isotype population of cTN-T and these are related to the differences in the Ca2+ regulation between neonate and adult hearts.

Mutations between residues 92 and 110 of cTN-T impair its TM-dependent functions (Palm et al., 2001).

TM: In skeletal muscle two isoforms of TM, a and p (each under different genetic control) are expressed. In contrast, in the heart only the a-form of TM is expressed. However, using transgenic approaches mice could be produced which overexpressed p-TM in the heart. Novel functions of this TM isoform were detected (Palmiter et al.,1996). Thus, the cardiac myofilaments, containing p-TM demonstrated an increase in the activation of the thin filament by strongly bound cross-bridges, an increase in Ca2+ sensitivity of steady state force, and a decrease in the rightward shift of the Ca2+-force relation induced by cAMP-dependent phosphorylation. These data indicate that switching of TM isoform has a major effect on heart myofilament activity.

Movement of the regulatory proteins in systole versus diastole. Fig. H4 depicts the position of TM and the TN components in the thin filaments. In diastole, TM is fixed in the groove of the actin double helix by TN-T and the C -terminus of TN-I. The position of the TM is such that the myosin cross-bridges (MHC, ELC, RLC) can not react with actin. The binding of Ca2+ to TN-C is the signal for systole, the N-terminus of TN-C interacts with the C-terminus of TN-I. Now TM is free to move on the thin filament removing the steric hindrance of the actin-cross-bridge reaction and systole ensues.

Fig. H4. Illustration of the movement of the regulatory proteins in systole versus diastole (Courtesy of Dr. Helen Rarick). For details see the text.

Fig. H4. Illustration of the movement of the regulatory proteins in systole versus diastole (Courtesy of Dr. Helen Rarick). For details see the text.

In summary, cardiac contraction is a series of interactions between Ca2+, the regulatory proteins, and the actomyosin system. In the resting muscle, at low intracellular free Ca2+concentration, the TN-TM complex inhibits the actin-myosin combination and with an increase in the myoplasmic Ca2+ the inhibition is released. The Ca2+ signaling process starts with the binding of Ca2+ to a single regulatory site of TN-C and by a tight binding of TN-C to TN-I. The signal is transmitted by TN and TM to actin in the thin filament. The final step is the combination of actin with myosin.

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