Tissue procurement strategies affect the protein biochemistry of human heart samples.

Author(s): Walker LA, Medway AM, Walker JS, Cleveland JC Jr, Buttrick PM

Publication: J Muscle Res Cell Motil, 2011, Vol. 31, Page 309-14

PubMed ID: 21184256 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The aim of this study was to assess the potential impact of cold ischemia time, temperature, and storage buffer on select phosphorlyated proteins in heart specimens. Five failing heart specimens procured from cardiac transplant recipients were subjected to a cold ischemia time of 60 min in MOPS or cardioplegia buffer on wet ice or at room temperature, or 30 min in the absence of buffer on wet ice or at room temperature. All specimens were snap frozen by immersion in liquid nitrogen and stored at -80 degrees C until analysis. The authors note that the time from excision to dissection was no more 20 minutes and was not included in their reported cold ischemia time. Phosphoprotein gel stain signals were normalized to the total protein signal for the essential myosin light chain.

   Summary of Findings:

   Cold ischemia time, temperature, and storage buffer did not alter levels of phosphorylated MyBP-C or phosphorylated desmin, as determined by protein mass and a phosphoprotein gel stain, although significant differences in the levels of phosphorylated MLC-2, phosporylated TnT, and phosphorylated TnI were reported (p<0.05). Levels of phosphorylated MLC-2 were 15% lower in specimens subjected to an ischemia time of 60 min in cardioplegia buffer on wet ice when compared to case-matched controls that were snap frozen immediately (p<0.05), and a significant decline was also observed in specimens stored on ice in the absence of buffer for 30 min (p<0.05). Phosphorylated TnT levels were also significantly lower in specimens subjected to an ischemia time of 60 min in cardioplegia buffer regardless of temperature when compared to case-matched specimens snap frozen immediately (p<0.05). However, levels of phosphorylated TnI were significantly greater in specimens subjected to an ischemia time of 60 min at room temperature when stored in either cardioplegia or MOPS buffer in comparison to case-matched specimens snap frozen immediately (p<0.05). Western blot analysis of beta-actin, MyBP-C, total TnI and serine 22/23 phosphorylation of TnI failed to reveal any ischemia-associated changes.

   Biospecimens

   • Tissue - Heart

   Preservative Types

   • Frozen

   Diagnoses:

   • Cardiovascular Disease

   Platform:

   Analyte	Technology Platform
   Protein	1D/2D gels
   Protein	Western blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Western blot Specific	Targeted peptide/protein	Phosphotroponin I (S22/23) Total troponin I Myosin binding protein C Beta-actin
   Biospecimen Acquisition	Cold ischemia time	0 min 30 min 60 min
   Biospecimen Acquisition	Pre-preservation condition	No buffer MOPS buffer Cardioplegia buffer
   Storage	Storage temperature	Room temperature Wet ice

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