An Inhibitor of the δPKC Interaction with the d Subunit of F1Fo ATP Synthase Reduces Cardiac Troponin I Release from Ischemic Rat Hearts: Utility of a Novel Ammonium Sulfate Precipitation Technique

Abstract Background: There are numerous potential sources of interference in immunoassays. Our aim was to identify the blood component that causes negative interference in cardiac troponin I (cTnI) immunoassays based on antibodies against the central part of cTnI. Methods: We isolated an interfering factor (IF) from a sample with low recovery of added cTnI, using several consecutive purification steps: caprylic acid precipitation, ammonium sulfate precipitation, and purification on Cibacron Blue gel and protein G columns. Purified IF was identified by gel electrophoresis and mass spectrometric analysis of protein bands. For the direct detection of human antibodies to cardiac troponin in serum samples, we developed immunoassays using three different anti-human immunoglobulin antibodies and measured troponin antibodies in samples with low and normal cTnI recovery. Results: Treatment with caprylic acid did not precipitate IF, but IF precipitated at 40% ammonium sulfate saturation. IF bound to a Cibacron Blue gel column, from which it was eluted with a linear salt gradient; it also bound to protein G. Gel electrophoresis of purified IF showed two major bands with molecular masses corresponding to the heavy (∼50 kDa) and light chains (∼25 kDa) of immunoglobulin, and their identities were confirmed by mass spectrometry. The presence of troponin-specific autoantibodies was confirmed in samples with low recoveries of cTnI by three different immunoassays. The median signals were significantly higher in 10 samples with low recovery than in 10 samples with normal recovery of cTnI (P ≤ 0.007). Conclusions: Circulating autoantibodies to cTnI or other proteins of the troponin complex can be a source of negative interference in cTnI immunoassays.

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Degradation of rat cardiac troponin I during ischemia independent of reperfusion

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00149.2004 ◽

2004 ◽

Vol 287 (3) ◽

pp. H1269-H1275 ◽

Cited By ~ 11

Author(s):

Brian S. Palmer ◽

Paul F. Klawitter ◽

Peter J. Reiser ◽

Mark G. Angelos

Keyword(s):

Cardiac Troponin ◽

Troponin I ◽

Progressive Increase ◽

Protective Role ◽

Left Ventricular ◽

Cardiac Troponin I ◽

Global Ischemia ◽

Stunned Myocardium ◽

Rat Hearts ◽

Developed Pressure

Cardiac troponin I (cTnI) degradation has been noted in the stunned myocardium of rodents after ischemia and reperfusion and is one proposed mechanism for the decreased left ventricular (LV) contractility in postischemic hearts. cTnI degradation has been best described after reperfusion of the ischemic myocardium. The effect of ischemia, independent of reperfusion, on cTnI breakdown has not been well characterized. We tested the hypothesis that progressive cTnI degradation occurs with increasing durations of ischemia and that this ischemia-based degradation is, in part, oxidant mediated. Isolated perfused rat hearts underwent global ischemia of 15, 20, or 25 min with and without reperfusion. A second series of hearts was treated with the antioxidants tiron (10 mM) and N-acetylcysteine (4 mM) before 20 min of global ischemia without reperfusion. cTnI degradation was measured using a cTnI-specific antibody and Western blot analyses. A progressive increase in cTnI degradation was seen with increasing duration of ischemia (no reperfusion), which correlated with the return of LV developed pressure during reperfusion. The extent of cTnI degradation was increased in hearts pretreated with antioxidants, although the qualitative degradation pattern was not altered. We conclude that a time-dependent cTnI breakdown occurs during global ischemia that is independent of reperfusion. cTnI breakdown during ischemia is further increased in the presence of antioxidants, suggesting ROS generated during ischemia may play a cTnI protective role.

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Abstract 36: Disrupting the Delta Protein Kinase C Interaction with the “d” Subunit of F1Fo ATP Synthase Protects Against Ischemia-Reperfusion Injury in Perfused Rat Hearts

Circulation Research ◽

10.1161/res.111.suppl_1.a36 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

John A Johnson ◽

Mourad Ogbi ◽

Robert W Caldwell

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Infarct Size ◽

Atp Synthase ◽

Ischemia Reperfusion ◽

Global Ischemia ◽

F1fo Atp Synthase ◽

Kinase C ◽

Rat Hearts ◽

D Subunit

Cardiac ischemia / reperfusion (IR) injury is associated with severe energy deprivation and is the number one cause of death world-wide. Mitochondrial F1Fo ATP synthase produces >90% of cardiac energy in mammals, yet few studies have targeted its role in IR injury. Previously, we identified a hypoxia-induced interaction of delta protein kinase C (dPKC) with the “d” subunit of F1Fo ATP synthase (dF1Fo) in neonatal cardiac myocytes, which inhibits F1Fo function. In the present work we investigated the hypothesis that a novel peptide inhibitor of the dPKC-dF1Fo interaction would preserve ATP and reduce infarct-size in isolated rat hearts subjected to IR injury. This peptide [NH2-YGRKKRRQRRRMLATRALSLIGKRAISTSVC-COOH] contains HIV-Tat protein transduction and mitochondrial targeting domains, the dPKC-dF1Fo inhibitor sequence, and a FLAG epitope. In hearts exposed to global ischemia, or IR, dPKC co-immuno-precipitated with dF1Fo. Pretreatment with the dPKC-dF1Fo inhibitor exacerbated cardiac ATP loss by 1.9-fold (n=5, p<0.03) following 10 min of global ischemia. However; following a pro-longed IR exposure ATP levels were enhanced by 2.1-fold (p<0.02, n=5). These opposing effects of the[[Unable to Display Character: ]]dPKC-dF1Fo inhibitor on ATP levels are likely due to relief of dPKC inhibition of the different modes of the F1Fo complex during ischemia (ATPase) and oxygenated reperfusion (ATP synthase). We next used 2,3,5, tetrazolium chloride staining techniques to determine if the dPKC-dF1Fo inhibitor had infarct-sparing effects following prolonged IR. In hearts exposed to 30 min of global ischemia and 150 min of reperfusion the dPKC-dF1Fo inhibitor reduced infarct size, (expressed as the percentage of total LV area) from 45 + 3 % (n=6) to 22 + 3 % (n=6, p < 0.01). Delivery and stability of the dPKC-dF1Fo inhibitor in hearts was confirmed by FLAG immunoreactivity in western blots conducted on mitochondria isolated the left ventricle. This is the first demonstration that perfusion with the dPKC-dF1Fo inhibitor prior to IR improves ATP recovery and reduces infarction in intact mammalian hearts. Our results support the potential for this peptide as a first-in-class translational agent for combating cardiac IR injury.

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