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Biomedical subjects
Melanie Y White
Publications and source records attributed to Melanie Y White.
Ischemia-specific phosphorylation and myofilament translocation of heat shock protein 27 precedes alpha B-crystallin and occurs independently of reactive oxygen species in rabbit myocardium.
Heat shock protein 27 (Hsp27) and alpha B-crystallin (alphaBC) are small heat shock proteins that stabilize the myofilament during stress. We utilized two-dimensional gel electrophoresis (2-DE), phospho-fluorescence staining, titanium dioxide (TiO(2)) phosphopeptide purification and mass spectrometry (MS) to fully characterize isoelectric point (pI) variants of Hsp27 and alphaBC in rabbit myocardium subjected to brief ischemia/reperfusion (I/R) injury. Four variants of Hsp27 were detected, two of which were phosphorylated: HSP1 (at three sites, Ser15, Ser78 and Ser82) and HSP2 (at Ser15 and Ser82, but not Ser78). Three variants of alphaBC were detected: alphaBC1 was phosphorylated (at Ser59 alone) and alphaBC2 was deamidated (at Asn146). No modifications were found in the remaining variants. Both phospho-Hsp27 variants increased in abundance in tissue subjected to brief I/R injury (15 min I/60 min R) and ischemia without subsequent reflow (15I/0R), and these increases were not affected by addition of the potent antioxidant, N-(2-mercaptopropionyl) glycine (MPG; 15I/60R + MPG and 15I/0R + MPG). Abundance of native and phosphorylated (but not deamidated) alphaBC was elevated following 15I/60R; however, these increases were ameliorated by the presence of MPG, and did not occur in tissue subjected to 15I/0R. Both phospho-Hsp27 variants and phospho-alphaBC translocated to the myofilament following 15I/60R. Increased myofilament association of phospho-Hsp27 was not influenced by MPG, and there was a greater proportion of HSP2 than HSP1 in this fraction. MPG inhibited phospho-alphaBC translocation and increased alphaBC association with the myofilament did not occur during 15I/0R. Increased phosphorylation of Hsp27 is ischemia-specific and not influenced by reactive oxygen species (ROS), while increased expression and phosphorylation of alphaBC are ROS-dependant.
Actin Cytoskeleton↗
Proteomics of ischemia and reperfusion injuries in rabbit myocardium with and without intervention by an oxygen-free radical scavenger.
A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilized 2-DE and MS to define 57 protein spot changes during brief ischemia/reperfusion (15 min ischemia, 60 min reperfusion; 15I/60R) injury in a rabbit model (White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M. et al., Proteomics 2005, 5, 1395-1410) and shown that the majority of these occur because of physical and/or chemical PTMs. In this study, we subjected rabbit myocardium to 15I/60R in the presence of the OFR scavenger N-(2-mercaptopropionyl) glycine (MPG). Thirty-seven of 57 protein spots altered during 15I/60R remained at control levels in the presence of MPG (15I/60R + MPG). Changes to contractile proteins, including myosin light chain 2 (MLC-2) and troponin C (TnC), were prevented by the addition of MPG. To further investigate the individual effects of ischemia and reperfusion, we generated 2-DE gels from rabbit myocardium subjected to brief ischemia alone (15I/0R), and observed alterations of 33 protein spots, including 18/20 seen in both 15I/60R-treated and 15I/60R + MPG-treated tissue. The tissue was also subjected to ischemia in the presence of MPG (15I/0R + MPG), and 21 spot changes, representing 14 protein variants, remained altered despite the presence of the OFR scavenger. These ischemia-specific proteins comprised those involved in energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin). We conclude that contractile dysfunction associated with myocardial stunning is predominantly caused by OFR damage at the onset of reperfusion, but that OFR-independent damage also occurs during ischemia. These ischemia-specific protein modifications may be indicative of early myocardial injury.
Animals↗
Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems.
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.
Amino Acid Sequence↗
Modifications of myosin-regulatory light chain correlate with function of stunned myocardium.
The precise molecular basis for myocardial stunning remains unresolved, but protein damage within the myofibril is a likely mechanism. We used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to identify protein modifications in stunned myocardium. In isolated, perfused rabbit hearts, low-flow ischemia (1 ml/min) and reperfusion resulted in impaired left-ventricular function (rate-pressure product (RPP) after 15-min ischemia: 65 +/- 5% pre-ischemia). We have characterised the sequence of ventricular myosin-regulatory light chain (MLC-2, 18 kDa) in rabbit myocardium and identified two non-phosphorylated (P(1) and P(2)) and two phosphorylated (P(3) and P(4) at Ser-14) isoelectric point variants. MS revealed that the acidic isoelectric point post-translational modification of P(1) and P(3), resulting in P(2) and P(4) respectively, was due to deamidation of asparagine to aspartate at residue 13, adjacent to Ser-14 phosphorylation site. After 15-min ischemia and reperfusion, a 15-kDa MLC-2 fragment was detected (MLC-2(14-165)), resulting from N-terminal cleavage between Asn/Asp-13 and Ser-14 of non-phosphorylated MLC-2, which accounted for 9.8% of visible non-phosphorylated MLC-2. Subsequent 2-DE of subcellular fractions showed that the fragment was lost from the myofilament. Treatment with an OH radical scavenger, N-(2-mercaptopropionyl) glycine (MPG, 3 mmol/l), preserved contractile function (RPP: 106 +/- 9% pre-ischemia) and prevented cleavage of MLC-2. Proteolytic damage to MLC-2, related to presence of OH radicals during reperfusion, correlates with myocardial stunning and may contribute to impaired contractility.
Animals↗
Duration of ischaemia determines matrix metalloproteinase-2 activation in the reperfused rabbit heart.
It has been hypothesised that activation of matrix metalloproteinase-2 (MMP-2) contributes to reversible myocardial dysfunction (stunning) following short-term ischaemia and reperfusion. Gelatin zymography was used to measure release of both pro-MMP-2 (72 kDa) and MMP-2 (62 kDa), into the coronary effluent from isolated, perfused rabbit hearts during 90 min aerobic perfusion (control), or low-flow ischaemia (15 or 60 min at 1 mL/min), followed by 60 min reperfusion. In controls, pro-MMP-2 was detected in the coronary effluent throughout the first 30 min of aerobic perfusion, but MMP-2 was not detected. In contrast, MMP-2 was detected in the coronary effluent during reperfusion after both 15 and 60 min ischaemia. However, while left ventricular systolic function was impaired after both 15 min and 60 min ischaemia, a significant increase in the release of MMP-2 was only detected in hearts following 60 min ischaemia. The dissociation between mechanical function and MMP-2 levels suggest that MMP-2 does not contribute to myocardial stunning in this model, but may contribute to myocardial dysfunction following prolonged ischaemia.
Animals↗