Structure of the NH2-terminal variable region of cardiac troponin T determines its sensitivity to restrictive cleavage in pathophysiological adaptation

The troponin complex in sarcomeric thin filaments plays a central role in the Ca2+-activation of cardiac and skeletal muscle contraction [1]. Troponin T (TnT) is the tropomyosin-binding subunit of the troponin complex [2]. Three muscle type-specific (fast skeletal, slow skeletal and cardiac) TnT genes have evolved in vertebrates and alternative RNA splicing further produces multiple protein isoforms [3, 4]. The TnT isoforms and alternatively spliced variants differ mainly in their NH2-terminal region of which the alternative splicing is regulated during development [5, 6] and skeletal muscle adaptations to unloading [7] or abnormal neurological stimuli [8]. Aberrant splicing of cardiac TnT (cTnT) in the NH2-terminal region has been found in hypertrophic and failing human hearts [9] and animals with dilated cardiomyopathy [10, 11]. The alternatively spliced NH2-terminal variable region of TnT does not contain binding sites for any known proteins in the myofilaments [12–14], and its removal did not abolish the core function of troponin [15–18]. However, structural variations in the NH2-terminal region of TnT affected the Ca2+-sensitivity in myosin ATPase activation and force development [19, 20]. Protein conformation and binding studies showed that NH2-terminal alterations in TnT altered the molecular conformation of the conserved middle and COOH-terminal regions [21, 22], and the binding affinities for troponin I (TnI), troponin C (TnC) and tropomyosin [21, 23]. Therefore, the NH2-terminal variable region of TnT is a regulatory structure that modulates muscle contractility and cardiac function. Whereas TnT isoform expression and alternative splicing provide chronic regulations, the NH2-terminal variable region of cTnT can also be restrictively cleaved by myofibril-associated μ-calpain in acute responses to stress conditions such as myocardial ischemia-reperfusion [24] and ventricular pressure overload [25]. The NH2-terminal truncated cTnT (cTnT-ND) remains in the myofilaments and is physiologically functional. Studies of cardiac function demonstrated that transgenic mouse hearts over-expressing cTnT-ND exhibited a unique change of ~5–10% slower systolic velocity, which did not decrease but increased cardiac output by elongating the time of ventricular rapid ejection phase [25]. This novel function suggested a plausible adaptive value of the production of cTnT-ND in compensating for cardiac function under energetic crisis due to ischemia and/or pressure overload. The mechanisms that activate the restrictive NH2-terminal proteolysis of cTnT remain to be investigated. Intracellular calcium overload is known to be a cause of myocardial injury in stress conditions such as ischemia-reperfusion [26]. On the other hand, the rhythm contraction of cardiac muscle is based on Ca2+-induced allosteric changes in the myofilament [1], therefore, cytosolic free Ca2+ in cardiomyocytes normally rises to near micromolar concentration during contraction-relaxation cycles. In the present study, we investigated the induction of cTnT-ND in the heart and in isolated cardiomyocytes for underlying mechanisms. Pathophysiological calcium overloading conditions induced the production of cTnT-ND independent of the phosphorylation states of cTnT. In contrast, the structure of the NH2-terminal variable region exhibited a strong correlation to the production of cTnT-ND in vivo. The results suggest that calcium overload in cardiomyocytes may convey an increased sensitivity of cTnT to the restrictive μ-calpain cleavage, in which the NH2-terminal structure may play a determining role.