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.
White
Melanie Y
MY
Department of Medicine, Blackburn Building D06, The University of Sydney, 2006, NSW, Sydney, Australia.
Cordwell
Stuart J
SJ
McCarron
Hugh C K
HC
Tchen
Adrian S
AS
Hambly
Brett D
BD
Jeremy
Richmond W
RW
D000818:Animals; D024722:Cardiac Myosins / Q000235:genetics* / Q000378:metabolism; D006321:Heart / Q000502:physiology; D008297:Male; D017682:Myocardial Stunning / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism; D018994:Myosin Light Chains / Q000235:genetics* / Q000378:metabolism; D020033:Protein Isoforms; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D018994:Myosin Light Chains; D020033:Protein Isoforms; C107184:myosin light chain 2; D024722:Cardiac Myosins