Increased Ca2+ Affinity of Cardiac Thin Filaments Reconstituted with Cardiomyopathy-related Mutant Cardiac Troponin I

Abstract

To understand the molecular mechanisms whereby cardiomyopathy-related cardiac troponin I (cTnI) mutations affect myofilament activity, we have investigated the Ca 2؉ binding properties of various assemblies of the regulatory components that contain one of the cardiomyopahty-related mutant cTnI. Acto-S1 ATPase activities in reconstituted systems were also determined. We investigated R145G and R145W mutations from the inhibitory region and D190H and R192H mutations from the second actin-tropomyosinbinding site. Each of the four mutations sensitized the acto-S1 ATPase to Ca 2؉ . Whereas the mutations from the inhibitory region increased the basal level of ATPase activity, those from the second actin-tropomyosin-binding site did not. The effects on the Ca 2؉ binding properties of the troponin ternary complex and the troponin-tropomyosin complex with one of four mutations were either desensitization or no effect compared with those with wild-type cTnI. All of the mutations, however, affected the Ca 2؉ sensitivities of the reconstituted thin filaments in the same direction as the acto-S1 ATPase activity. Also the thin filaments with one of the mutant cTnIs bound Ca 2؉ with less cooperativity compared with those with wild-type cTnI. These data indicate that the mutations found in the inhibitory region and those from the second actintropomyosin site shift the equilibrium of the states of the thin filaments differently. Moreover, the increased Ca 2؉ bound to myofilaments containing the mutant cTnIs may be an important factor in triggered arrhythmias associated with the cardiomyopathy.In experiments reported here, we have investigated the functional significance of mutations of cardiac troponin I (cTnI) 2 linked to cardiomyopathy. The Ca 2ϩ -dependent interaction of TnI with actin and TnC is one of the most important events in the regulation of striated muscle contraction. Ca 2ϩ binding to troponin C (TnC) triggers a series of conformational transitions among thin filament proteins that activate the thin filament (for review, see Refs. 1-4). A current model for the regulatory mechanism of muscle contraction, originally derived from biochemical analysis, involves three states of the thin filament:blocked (B-state), closed (C-state), and open (M-state) (5-7). The three states have been defined to reflect different interactions between actin and the myosin head. The B-state, which the majority of the thin filaments occupy when the cytoplasmic [Ca 2ϩ ] is low and Ca 2ϩ is not bound to the regulatory site(s) on Tn (5,8,9), is stabilized by the interaction between TnI and actin. With Ca 2ϩ binding to the regulatory site(s) of TnC, a hydrophobic patch is exposed in the N-terminal regulatory site of TnC (10, 11). In the case of cardiac TnC (cTnC), this structural change requires cTnI (12-14). The newly exposed hydrophobic patch interacts with the regulatory region of TnI (12, 15-17) and the C-terminal half of the TnI molecule moves away from actin (18 -22). In the C-state, non-force-generating cross-bridges bind weakly to actin...

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

“…These observations indicated that the binding of the inhibitory region, but not the second actin binding site, induces a structural change in actin and prevents actin-myosin strong interaction. On the basis of results of experiments with cardiomyopathy-linked mutations of cTnI, we proposed that the functional role of the second actin-binding site may be to increase the local concentration of the cTnI molecule for actin to facilitate the interaction of the inhibitory region and actin [34].…”

Cardiac thin filament regulation

“…As might be expected, these amino acid replacement(s) in the inhibitory region result in impaired inhibitory function at diastole. Furthermore, the weakened interaction between mutated TnI and actin-Tm causes sensitization of the thin filaments to Ca 2+ [38] and associated slowed relaxation of force kinetics [44]. Consistent with this notion, thin filaments that contain cTnI with a HCM/RCMlinked mutation in the inhibitory region bind Ca 2+ stronger than their wild-type counterpart [38,61].…”

“…It is of note that Ca 2+ binding to isolated cardiac troponin complex in solution is not cooperative, as would be expected based on the presence of a single-regulatory low-affinity Ca 2+ -binding site on cTnC [13,38]. Furthermore, although the head-to-tail interactions between Tm molecules is considered to be important in cooperative S1 binding, this does not extend to Ca 2+ binding to the Tn-Tm complex.…”

“…Furthermore, although the head-to-tail interactions between Tm molecules is considered to be important in cooperative S1 binding, this does not extend to Ca 2+ binding to the Tn-Tm complex. Only when the Tn complex is incorporated into the thin filament does the single Ca 2+ -binding site on TnC bind Ca 2+ in a cooperative manner, indicating the presence of a positive cooperative feedback mechanism that is transmitted along the thin filament to neighboring regulatory units, a process that likely involves Tm-actin-Tn interaction [13,38]. Finally, we have found that the relationship between activator [Ca 2+ ] and myofilament force is highly cooperative in both cardiac and fast skeletal muscle but not in slow skeletal muscle despite the fact that the slow skeletal sarcomere contains cTnC [41].…”

ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease

“…In addition, it could represent a pathomechanism underlying the diastolic dysfunction seen in both disease states. Solution studies with mutant troponin proteins, which are known to cause HCM, showed a reduction in the B state at low-Ca 2+ conditions compared with wild-type troponin proteins (18,19). Mutation-induced irregularities in troponin-tropomyosin interactions disrupt the B state and shift the thin filament to the C state, increasing the available myosin-binding sites on actin.…”