A Proteolytic NH2-terminal Truncation of Cardiac Troponin I That Is Up-regulated in Simulated Microgravity

Abstract

In a tail suspension rat model, we investigated changes in myofilament protein during cardiac adaptation in simulated microgravity. Contractile force and velocity of cardiac muscle were decreased in the tail suspension rats as compared with the control. Ca 2؉ -dependent actomyosin ATPase activity was also decreased; however, sensitivity of cardiac muscle to Ca 2؉ activation was unchanged. There was no change in expression of myosin heavy chain, tropomyosin, troponin T, or troponin I isoforms in hearts of tail suspension rats. A novel finding is a fragment of cardiac troponin I (cTnI) that had increased amounts in the heart of tail suspension rats. Binding of this cTnI fragment by a monoclonal antibody that specifically recognizes the COOH terminus indicates an intact COOH terminus.

Removing the regulatory N-terminal domain of cardiac troponin I diminishes incompatibility during bacterial expression

“…Deletion of this segment (Fig. 4) does not disrupt the core structure of TnI [6,23] and does not alter the overall positive charge of the protein (the isoelectric points of intact and N-terminal truncated mouse cardiac TnI are 9.57 and 9.59, respectively). Therefore, the possible mechanisms may include its direct toxicity to the host metabolism and/ or its effects on the stability and accumulation of the exogenous protein synthesized.…”

“…The chemical environment in bacterial cells is apparently different from that of the muscle cells and may not be suitable for high affinity binding between TnC and TnI. On the other hand, native troponin complex is known to resist to high salt buffers [23]. Therefore, the ineffective binding between TnI and TnC in bacterial cells might not be simply due to the solution environment but result from ineffective folding of the bacterially made troponin proteins (most likely cardiac TnI since the TnC protein was in excess and only a small portion needed to be correctly folded to saturate the co-expressed cardiac TnI).…”

“…The N-terminal segment of cardiac TnI polypeptide is a unique structure that is not present in fast or slow skeletal muscle TnI and may be proteolytically removed in the cardiac muscle during physiological adaptations [23]. The significantly improved expression of N-terminal truncated cardiac TnI in E. coli suggests that the N-terminal segment has a negative effect on bacterial expression.…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…Selective TnI proteolysis at the C terminus has been reported after ischemia/reperfusion injury, also known as myocardial stunning, in rodent hearts [31][32][33]. The N-terminal degradation of cardiac TnI was reported in a tail suspension rat model that simulated microgravity [34]. More interestingly, several groups have reported degradation products of TnI in serum of patients after acute myocardial infarction (AMI) [35][36][37].…”

“…Protein degradation often involves significant molecular weight (MW) reduction, which can be readily detected by 1-D-gel separation followed by Western blot analysis [31,32,[34][35][36][37][38][39][40][41]. Comparative 2-DE was also used to study proteomic changes on a broader scale, and PMF can be employed to identify changed proteins [46].…”