EM and 3D-Reconstruction of Thin Filaments Reconstituted with Truncated Troponin I Associated with Myocardial Stunning

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Myocardial “stunning”, a condition resulting from a short period of ischemia followed by reperfusion, can cause cardiac dysfunction, despite the absence of tissue necrosis. Stunning can be associated with rapid proteolytic truncation of the C-terminal 17 acids of TnI (to form “TnI1-192”). Expression of TnI1-192 in transgenic mice is sufficient to account for the stunning phenotype (Murphy et al., 2000), where for example myofibrils containing TnI1-192 and otherwise normal troponin-tropomyosin display increased Ca2+-sensitivity (Narolska et al., 2006; also Foster et al., 2003). In the current study, electron microscopy and 3D-image reconstruction of thin filaments containing cTnI1-192 and control TnC, TnT and tropomyosin was performed to determine if the truncation causes an imbalance in the tropomyosin distribution between different regulatory states. Negatively stained “mutant” filaments showed characteristic periodic troponin projections and tropomyosin strands. Both helical reconstruction and single particle analysis indicated that at low-Ca2+ the tropomyosin localized on the inner aspect of the outer domain of actin. As expected, tropomyosin moves to the inner domain of actin in Ca2+ (Foster et al., 2003). However, truncated TnI appears to promote an extra transition of tropomyosin from the Ca2+- induced, closed position on actin toward the myosin-induced, open-state position. Here, tropomyosin in myosin-free thin filaments appears biased towards the open-state in the presence of only Ca2+. Cross-correlation of filament segments to models of the blocked-, closed-, and open-states (as in Pirani et al., 2005) confirms this open-state bias, which correlates well with the increase in Ca2+-sensitivity observed in in vitro and in fiber assays of function.