Single Mutation (A162H) in Human Cardiac Troponin I Corrects Acid pH Sensitivity of Ca2+-regulated Actomyosin S1 ATPase

Abstract

In contrast to skeletal muscle, the efficiency of the contractile apparatus of cardiac tissue has long been known to be severely compromised by acid pH as in the ischemia of myocardial infarction and other cardiac my- 2؉ sensitivity of cardiac contractility at low pH (<pH 6.5) is attributable to structural difference(s) in the cardiac and skeletal inhibitory components (TnIs) of their troponins. Here, using a reconstituted Ca 2؉ -regulated human cardiac troponin-tropomyosin actomyosin S1 ATPase assay, we report that a single TnI mutation, A162H, restores Ca 2؉ sensitivity at pH 6.5 to that at pH 7.0. Levels of inhibition (pCa 7.0), activation (pCa 4.0), and cooperativity of ATPase activity were minimally affected. Two other mutations (Q155R and E164V) also previously suggested by us (Pearlstone, J. R., Sykes, B. D., and Smillie, L. B. (1997) Biochemistry 36, 7601-7606) and involving charged residues showed no such effects. With fast skeletal muscle troponin, a single TnI H130A mutation reduced Ca 2؉ sensitivity at pH 6.5 to levels approaching the cardiac system at pH 6.5. These observations provide structural insight into long-standing physiological and clinical phenomena and are of potential relevance to therapeutic treatments of heart disease by gene transfer, stem cell, and cell transplantation approaches.In both cardiac and skeletal muscles, the generation of force through the interaction of thick and thin filaments of the sarcomere is under the control of Ca 2ϩ concentration in the sarcoplasm. The heterotrimeric troponin (Tn) 1 complex through its interaction with the thin filament proteins F-actin (A) and tropomyosin (TM) is the dominant player in this control. Conformational changes associated with Ca 2ϩ binding to the regulatory N domain of troponin C (TnC) alter its interactions with both of the other Tn subunits, troponin I (TnI, inhibitory subunit) and troponin T (TnT, TM binding subunit), and of TnI and TnT with TM and A. In consequence, TM moves from its steric blocking position, facilitating interaction of thick filament myosin heads with the thin filament and generation of ATPase activity and contraction. Sequestration of Ca 2ϩ by the sarcoplasma reticulum reverses the process with ensuing relaxation (for reviews, see Refs. 1 and 2).As key components of this system, the functional and structural relationships of TnC and TnI and their interaction in the presence and absence of Ca 2ϩ have been the subject of intensive investigation over several decades (for reviews, see Refs. 2-5). While high resolution structures of the binary or ternary (with TnT) complexes by x-ray crystallographic or NMR approaches have remained elusive, considerable progress has been made in the mapping of interaction sites of the TnC and TnI components. Two such TnI regions, first described by S. V. Perry and colleagues (for review, see Ref. 5) (see Fig. 1), involve the NH 2 -terminal segment of fast skeletal TnI (fsTnI) (residues ϳ1-30) and the so-called inhibitory peptide or Ip region (residues 96 -116). While the high affin...

Calcium-dependent Changes in the Flexibility of the Regulatory Domain of Troponin C in the Troponin Complex

“…Sample Preparation-Uniformly deuterated cysteineless chicken fast skeletal muscle TnI was expressed and purified as described (24), using a construct provided by Dr. Smillie (26). Substitution of cysteineless TnI for wild-type TnI in the Tn complex does not alter the Ca 2ϩ -dependent regulation of myosin ATPase activity (26).…”

“…Substitution of cysteineless TnI for wild-type TnI in the Tn complex does not alter the Ca 2ϩ -dependent regulation of myosin ATPase activity (26). A uniformly deuterated deletion mutant of chicken skeletal muscle TnT (isoform TnT-3), corresponding to the T2 domain, was expressed and purified as described (25).…”

The unique functions of cardiac troponin I in the control of cardiac muscle contraction and relaxation

“…An interesting aspect of the specialization of the cTnI isoform is an alanine at position 164 near the mobile domain, which is replaced by His at the homologous position in ssTnI. Dargis et al [39] reported that this substitution is of significance in studies in which reconstitution with the mutant, cTnI (A164H), rendered cardiac myofilament preparations relatively more sensitive to Ca 2+ and relatively less sensitive to deactivation by acidic pH. Incorporation of cTnI (A164H) into hearts of transgenic mice recapitulated many of the effects of ssTnI described above, while retaining phosphorylation sites [40].…”

Mechanisms and Use of Calcium-Sensitizing Agents in the Failing Heart

“…23 There is evidence that a difference in a single amino acid (His/Ala) in ssTnI versus cTnI at position 162 is responsible for these functional differences. 24 …”