Breakdown and Release of Myofilament Proteins During Ischemia and Ischemia/Reperfusion in Rat Hearts

Abstract

Abstract-Our objective in experiments reported here was to identify myofilament proteins of rat hearts either lost or degraded by cardiac ischemia (15-or 60-minute duration) with and without 45 minutes of reperfusion. We correlated these changes with alterations in myofilament sensitivity to Ca 2ϩ and maximum force generation. Protein degradation and loss were assessed by high-performance liquid chromatography, SDS-PAGE, Western blotting analysis, and amino acid sequencing. Compared with nonischemic control hearts, bundles of skinned fibers from hearts subjected to ischemia alone demonstrated a decrease in maximum force generation and an increase in sensitivity to Ca 2ϩ . These changes in function were increased with the duration of the ischemia and with reperfusion. With increasing duration of ischemia, there was an increased loss and degradation of myofibrillar ␣-actinin and troponin I (TnI) at its C-terminus. ␣-Actinin and TnI were most susceptible to ischemia, but with 60 minutes of ischemia/reperfusion, there was also degradation of myosin light chain-1 (MLC1) involving a clip of residues 1 to 19. The MLC1 degradation product was detected in the reperfusion effluent (along with troponin T, tropomyosin, and ␣-actinin) but not in the tissue with 60 minutes of ischemia with no reperfusion. Moreover, with ischemia the following proteins became associated with the myofibrils: GAPDH and proteins of the mitochondrial ATP synthase complex. Our results provide new evidence regarding the mechanism by which ischemia/reperfusion causes myocardial injury and support the hypothesis that an important element in the injury is altered activity and structure of the myofilaments. (Circ Res. 1998;82:261-271.)

Cardiac thin filament regulation

“…Furthermore, Hoffman et al suggested that the mobile domain undergoes a disordered-ordered structural transition when it interacts with actin in the absence of Ca 2+ [32], indicative of an association of this region with actin-Tm (termed "fly-casting mechanism"). It has been shown, at least in rodents, that moderate ischemia/reperfusion induces proteolytic cleavage of 17 residues from the C-terminal of TnI that includes the second actin-interacting sites [80]. Human myofibrils containing truncated cTnI display impaired force relaxation kinetics upon rapid solution switching Ca 2+ deactivation [51].…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…The most studied one is the degradation of TnI, the inhibitory subunit of Tn complex. 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].…”

“…In 2000, TnI and TnT have been approved as the biomarkers for AMI diagnosis [42], and several kits have been commercially developed and used in clinical studies [43][44][45]. In addition to TnI and TnT, other myofilament proteins, including TnC [46], LC2 [46], aactinin [32] and MyBP-C [47], were also reported as useful markers in animal models of ischemia/reperfusion injury. Two mechanisms are believed to be responsible for most myofilament protein degradation.…”

“…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].…”

“…The exact site of degradation can be mapped using either direct sequencing from the N terminus [31,32,34] or MS [33]. Alternatively, the degradation site can be mapped using antibody epitope analysis [62], in which an array of mAb recognizing different regions within protein sequences is used [43][44][45].…”