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Cardiac myosin-binding protein C: a potential early biomarker of myocardial injury.

Baker JO, Tyther R, Liebetrau C, Clark J, Howarth R, Patterson T, Möllmann H, Nef H, Sicard P, Kailey B, Devaraj R, Redwood SR, Kunst G, Weber E, Marber MS - Basic Res. Cardiol. (2015)

Bottom Line: Using novel antibodies raised against the cardiac-specific N-terminus of cMyC, we used confocal microscopy, immunoblotting and immunoassay to document its location and release.Compared to cTnT measured using a contemporary high-sensitivity commercial assay, cMyC peaks earlier (STEMI, 9.3 ± 3.1 vs 11.8 ± 3.4 h, P < 0.007; TASH, 9.7 ± 1.4 vs 21.6 ± 1.4 h, P < 0.0001), accumulates more rapidly (during first 4 h after TASH, 25.8 ± 1.9 vs 4.0 ± 0.4 ng/L/min, P < 0.0001) and disappears more rapidly (post-CABG, decay half-time 5.5 ± 0.8 vs 22 ± 5 h, P < 0.0001).Our results demonstrate that following defined myocardial injury, the rise and fall in the serum of cMyC is more rapid than that of cTnT.

View Article: PubMed Central - PubMed

Affiliation: King's College London BHF Centre, The Rayne Institute, St Thomas' Hospital, 4th Floor Lambeth Wing, Westminster Bridge Road, London, SE1 7EH, UK.

ABSTRACT
Cardiac troponins are released and cleared slowly after myocardial injury, complicating the diagnosis of early, and recurrent, acute myocardial infarction. Cardiac myosin-binding protein C (cMyC) is a similarly cardiac-restricted protein that may have different release/clearance kinetics. Using novel antibodies raised against the cardiac-specific N-terminus of cMyC, we used confocal microscopy, immunoblotting and immunoassay to document its location and release. In rodents, we demonstrate rapid release of cMyC using in vitro and in vivo models of acute myocardial infarction. In patients, with ST elevation myocardial infarction (STEMI, n = 20), undergoing therapeutic ablation of septal hypertrophy (TASH, n = 20) or having coronary artery bypass surgery (CABG, n = 20), serum was collected prospectively and frequently. cMyC appears in the serum as full-length and fragmented protein. Compared to cTnT measured using a contemporary high-sensitivity commercial assay, cMyC peaks earlier (STEMI, 9.3 ± 3.1 vs 11.8 ± 3.4 h, P < 0.007; TASH, 9.7 ± 1.4 vs 21.6 ± 1.4 h, P < 0.0001), accumulates more rapidly (during first 4 h after TASH, 25.8 ± 1.9 vs 4.0 ± 0.4 ng/L/min, P < 0.0001) and disappears more rapidly (post-CABG, decay half-time 5.5 ± 0.8 vs 22 ± 5 h, P < 0.0001). Our results demonstrate that following defined myocardial injury, the rise and fall in the serum of cMyC is more rapid than that of cTnT. We speculate that these characteristics could enable earlier diagnosis of myocardial infarction and reinfarction in suspected non-STEMI, a population not included in this early translational study.

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Development of a quantitative bioassay for human cMyC in serum. a Sequence alignment of cMyC with skeletal myosin binding protein C isoforms. The sequences recognized by monoclonal anti-cMyC antibodies 1A4 and 3H8 are shown in bold. b SPR kinetic sensorgrams demonstrating the kinetic parameters of clone 3H8 (left) and 1A4 (right). c Epitope competition sensorgram of 1A4 and 3H8 binding to a C0C2-conjugated CM5 chip. d Immunoblot of rat and human tissue demonstrating specificity of 3H8 and 1A4 monoclonal antibodies. GAPDH was used as a loading control. Samples 1–9 are various rat tissue (1 ventricle, 2 atria, 3 rectus abdominus, 4 soleus, 5 spleen, 6 kidney, 7 aorta, 8 liver, 9 brain) and 10 is human ventricle. e Representative C0C2 standard curve from cMyC ElectroChemiLuminescent assay indicating the limit of detection (dashed line). f The performance characteristics of the assay for MyC, with a lower limit of quantification at a 10 % coefficient of variation, of approximately 80 ng/L
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Fig3: Development of a quantitative bioassay for human cMyC in serum. a Sequence alignment of cMyC with skeletal myosin binding protein C isoforms. The sequences recognized by monoclonal anti-cMyC antibodies 1A4 and 3H8 are shown in bold. b SPR kinetic sensorgrams demonstrating the kinetic parameters of clone 3H8 (left) and 1A4 (right). c Epitope competition sensorgram of 1A4 and 3H8 binding to a C0C2-conjugated CM5 chip. d Immunoblot of rat and human tissue demonstrating specificity of 3H8 and 1A4 monoclonal antibodies. GAPDH was used as a loading control. Samples 1–9 are various rat tissue (1 ventricle, 2 atria, 3 rectus abdominus, 4 soleus, 5 spleen, 6 kidney, 7 aorta, 8 liver, 9 brain) and 10 is human ventricle. e Representative C0C2 standard curve from cMyC ElectroChemiLuminescent assay indicating the limit of detection (dashed line). f The performance characteristics of the assay for MyC, with a lower limit of quantification at a 10 % coefficient of variation, of approximately 80 ng/L

Mentions: We chose to develop a classic “sandwich” immunoassay format, comprising a single monoclonal capture and a single monoclonal detection antibody, both directed against unique epitopes on the cardiac-specific N-terminal domain of cMyC (see Fig. 3a). An array of 15mer peptides corresponding to overlapping sections of this C0C2 domain was used to map epitope specificity. From 74 viable hybridomas, clones 1A4 and 3H8 were chosen on biophysical characteristics (see below) and mapped to sequences A125AELGESAPSPK and A149PDDPIGLFVM, respectively (see emboldened sequence in Fig. 3a). The 3H8 (capture), and 1A4 (detection) antibodies exhibited strong binding affinity to C0C2 of 9.6 × 10−10 and 4.8 × 10−10 M by surface plasmon resonance (for sensorgram see Fig. 3b). The epitope competition sensorgram (Fig. 3c) shows that 3H8 and 1A4 can bind to C0C2 independently, despite the proximity of their epitopes demonstrated by peptide array (see Fig. 3a). Monoclonal 3H8 bound at 80 % of its maximum binding value in the presence of saturating amounts of 1A4, whereas 1A4 bound at 68 % of its maximum value after saturating injections of 3H8. These high non-competing affinities were accompanied by specificity for cMyC over other isoforms based on immunoblots of rat and human tissue (Fig. 3d).Fig. 3


Cardiac myosin-binding protein C: a potential early biomarker of myocardial injury.

Baker JO, Tyther R, Liebetrau C, Clark J, Howarth R, Patterson T, Möllmann H, Nef H, Sicard P, Kailey B, Devaraj R, Redwood SR, Kunst G, Weber E, Marber MS - Basic Res. Cardiol. (2015)

Development of a quantitative bioassay for human cMyC in serum. a Sequence alignment of cMyC with skeletal myosin binding protein C isoforms. The sequences recognized by monoclonal anti-cMyC antibodies 1A4 and 3H8 are shown in bold. b SPR kinetic sensorgrams demonstrating the kinetic parameters of clone 3H8 (left) and 1A4 (right). c Epitope competition sensorgram of 1A4 and 3H8 binding to a C0C2-conjugated CM5 chip. d Immunoblot of rat and human tissue demonstrating specificity of 3H8 and 1A4 monoclonal antibodies. GAPDH was used as a loading control. Samples 1–9 are various rat tissue (1 ventricle, 2 atria, 3 rectus abdominus, 4 soleus, 5 spleen, 6 kidney, 7 aorta, 8 liver, 9 brain) and 10 is human ventricle. e Representative C0C2 standard curve from cMyC ElectroChemiLuminescent assay indicating the limit of detection (dashed line). f The performance characteristics of the assay for MyC, with a lower limit of quantification at a 10 % coefficient of variation, of approximately 80 ng/L
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Related In: Results  -  Collection

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Fig3: Development of a quantitative bioassay for human cMyC in serum. a Sequence alignment of cMyC with skeletal myosin binding protein C isoforms. The sequences recognized by monoclonal anti-cMyC antibodies 1A4 and 3H8 are shown in bold. b SPR kinetic sensorgrams demonstrating the kinetic parameters of clone 3H8 (left) and 1A4 (right). c Epitope competition sensorgram of 1A4 and 3H8 binding to a C0C2-conjugated CM5 chip. d Immunoblot of rat and human tissue demonstrating specificity of 3H8 and 1A4 monoclonal antibodies. GAPDH was used as a loading control. Samples 1–9 are various rat tissue (1 ventricle, 2 atria, 3 rectus abdominus, 4 soleus, 5 spleen, 6 kidney, 7 aorta, 8 liver, 9 brain) and 10 is human ventricle. e Representative C0C2 standard curve from cMyC ElectroChemiLuminescent assay indicating the limit of detection (dashed line). f The performance characteristics of the assay for MyC, with a lower limit of quantification at a 10 % coefficient of variation, of approximately 80 ng/L
Mentions: We chose to develop a classic “sandwich” immunoassay format, comprising a single monoclonal capture and a single monoclonal detection antibody, both directed against unique epitopes on the cardiac-specific N-terminal domain of cMyC (see Fig. 3a). An array of 15mer peptides corresponding to overlapping sections of this C0C2 domain was used to map epitope specificity. From 74 viable hybridomas, clones 1A4 and 3H8 were chosen on biophysical characteristics (see below) and mapped to sequences A125AELGESAPSPK and A149PDDPIGLFVM, respectively (see emboldened sequence in Fig. 3a). The 3H8 (capture), and 1A4 (detection) antibodies exhibited strong binding affinity to C0C2 of 9.6 × 10−10 and 4.8 × 10−10 M by surface plasmon resonance (for sensorgram see Fig. 3b). The epitope competition sensorgram (Fig. 3c) shows that 3H8 and 1A4 can bind to C0C2 independently, despite the proximity of their epitopes demonstrated by peptide array (see Fig. 3a). Monoclonal 3H8 bound at 80 % of its maximum binding value in the presence of saturating amounts of 1A4, whereas 1A4 bound at 68 % of its maximum value after saturating injections of 3H8. These high non-competing affinities were accompanied by specificity for cMyC over other isoforms based on immunoblots of rat and human tissue (Fig. 3d).Fig. 3

Bottom Line: Using novel antibodies raised against the cardiac-specific N-terminus of cMyC, we used confocal microscopy, immunoblotting and immunoassay to document its location and release.Compared to cTnT measured using a contemporary high-sensitivity commercial assay, cMyC peaks earlier (STEMI, 9.3 ± 3.1 vs 11.8 ± 3.4 h, P < 0.007; TASH, 9.7 ± 1.4 vs 21.6 ± 1.4 h, P < 0.0001), accumulates more rapidly (during first 4 h after TASH, 25.8 ± 1.9 vs 4.0 ± 0.4 ng/L/min, P < 0.0001) and disappears more rapidly (post-CABG, decay half-time 5.5 ± 0.8 vs 22 ± 5 h, P < 0.0001).Our results demonstrate that following defined myocardial injury, the rise and fall in the serum of cMyC is more rapid than that of cTnT.

View Article: PubMed Central - PubMed

Affiliation: King's College London BHF Centre, The Rayne Institute, St Thomas' Hospital, 4th Floor Lambeth Wing, Westminster Bridge Road, London, SE1 7EH, UK.

ABSTRACT
Cardiac troponins are released and cleared slowly after myocardial injury, complicating the diagnosis of early, and recurrent, acute myocardial infarction. Cardiac myosin-binding protein C (cMyC) is a similarly cardiac-restricted protein that may have different release/clearance kinetics. Using novel antibodies raised against the cardiac-specific N-terminus of cMyC, we used confocal microscopy, immunoblotting and immunoassay to document its location and release. In rodents, we demonstrate rapid release of cMyC using in vitro and in vivo models of acute myocardial infarction. In patients, with ST elevation myocardial infarction (STEMI, n = 20), undergoing therapeutic ablation of septal hypertrophy (TASH, n = 20) or having coronary artery bypass surgery (CABG, n = 20), serum was collected prospectively and frequently. cMyC appears in the serum as full-length and fragmented protein. Compared to cTnT measured using a contemporary high-sensitivity commercial assay, cMyC peaks earlier (STEMI, 9.3 ± 3.1 vs 11.8 ± 3.4 h, P < 0.007; TASH, 9.7 ± 1.4 vs 21.6 ± 1.4 h, P < 0.0001), accumulates more rapidly (during first 4 h after TASH, 25.8 ± 1.9 vs 4.0 ± 0.4 ng/L/min, P < 0.0001) and disappears more rapidly (post-CABG, decay half-time 5.5 ± 0.8 vs 22 ± 5 h, P < 0.0001). Our results demonstrate that following defined myocardial injury, the rise and fall in the serum of cMyC is more rapid than that of cTnT. We speculate that these characteristics could enable earlier diagnosis of myocardial infarction and reinfarction in suspected non-STEMI, a population not included in this early translational study.

Show MeSH
Related in: MedlinePlus