Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems

Abstract

Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.

Increased Cross-bridge Cycling Kinetics after Exchange of C-terminal Truncated Troponin I in Skinned Rat Cardiac Muscle

“…creatine kinase), and proteins regulating redox homeostasis (e.g. NADH-Uq) have also been reported to be altered in myocardial stunning (26). Whether these pathways affect myofilament chemo-mechanical transduction in myocardial stunning cannot be determined from the current study.…”

Mitochondria: A mirror into cellular dysfunction in heart disease

“…As a result, the OxPhos pathway of the mitochondria has been the subject of numerous investigations. Complex 1 of the OxPhos pathway has been shown to be affected by I/R [20,[90][91][92][93], whereby the activity of the complex is significantly reduced, with concurrent changes in the abundance of several complex 1 subunits, as identified by proteomic techniques including 2-DE [76,94]. To investigate the discrete compositional alterations with I/R, isolated mitochondria treated with elevated levels of ROS and/or NO resulted in the inactivation of complex 1 [95][96][97][98][99][100][101], with in vitro preparations showing a similar inactivation of complex 1 by increased concentration of both ROS and Ca 21 [102].…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…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.…”

“…Comparative 2-DE was also used to study proteomic changes on a broader scale, and PMF can be employed to identify changed proteins [46]. Specific 2-DE methods have been developed for analyzing large [22] and basic heart proteins [59].…”

“…Interestingly, phosphorylation of TnI by PKA and PKC changed its susceptibility to calpain-induced degradation conversely [124]. As another example of communications between different types of modifications, degradation of MLC-2 and TnC after ischemia/reperfusion could be prevented by the addition of an oxygen-free radical scavenger [46]. Different modifications occur at different time points after an extrinsic insult.…”