Modifications of myosin-regulatory light chain correlate with function of stunned myocardium

MY White, Stuart Cordwell, Hugh C.K. McCarron, Adrian S. Tchen, Brett D. Hambly, Richmond W. Jeremy

Research output: Contribution to journalArticlepeer-review

39 Citations (Scopus)

Abstract

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 (P1 and P2) and two phosphorylated (P3 and P4 at Ser-14) isoelectric point variants. MS revealed that the acidic isoelectric point post-translational modification of P1 and P3, resulting in P2 and P4 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-214-165), 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.
Original languageEnglish
Pages (from-to)833-840
Number of pages8
JournalJournal of Molecular and Cellular Cardiology
Volume35
Issue number7
DOIs
Publication statusPublished - 2003
Externally publishedYes

Keywords

  • Ischemia and reperfusion
  • Myocardium
  • Myosin-regulatory light chain
  • Protein degradation
  • Proteomics

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