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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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cMyC and cTnT accumulation during ST elevation myocardial infarction. a cMyC immunoblot of serum collected from an individual patient with the corresponding laboratory-determined cTnT concentrations appearing as an overlay. The controls are C1, recombinant C0C2 domain of cMyC; C2, fresh mouse heart homogenate and C, control human serum. Soon after chest pain onset cMyC appears in the serum predominantly as the full-length protein (~140 kDa) which then diminishes as a shorter fragment accumulates (~40 kDa). These fragments seem to reach maximum abundance in advance of cTnT. b Relative timings of cMyC, measured by densitometry of full-length protein (closed circles) and dominant fragment (open circles), compared to cTnT (filled squares) measured in our clinical laboratory. The immunoblot detection technique is relatively crude and insensitive (see text for details), so band densities are expressed as % of the maximum density achieved at a single time point on the individual patient’s immunoblot (see a for example). Complete values for cTnT were obtained after immunoblot analyses and normalized similarly. The peak concentrations of full-length (7.9 ± 4.4 h) and fragment (9.3 ± 3.1 h) forms of cMyC occurred earlier than those of Troponin (11.8 ± 3.4 h, P < 0.007 and P < 0.001, respectively). The figure comprises analyses from 18 patients for cMyC fragment and cTnT (n = 18). In two patients, there was a serum sample missing at the peak of troponin concentration and in two patients no clear cMyC immunoreactivity could be discerned. Full-length cMyC could only be detected in the serum of 9 of the 18 patients in whom a fragment was visible
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Fig2: cMyC and cTnT accumulation during ST elevation myocardial infarction. a cMyC immunoblot of serum collected from an individual patient with the corresponding laboratory-determined cTnT concentrations appearing as an overlay. The controls are C1, recombinant C0C2 domain of cMyC; C2, fresh mouse heart homogenate and C, control human serum. Soon after chest pain onset cMyC appears in the serum predominantly as the full-length protein (~140 kDa) which then diminishes as a shorter fragment accumulates (~40 kDa). These fragments seem to reach maximum abundance in advance of cTnT. b Relative timings of cMyC, measured by densitometry of full-length protein (closed circles) and dominant fragment (open circles), compared to cTnT (filled squares) measured in our clinical laboratory. The immunoblot detection technique is relatively crude and insensitive (see text for details), so band densities are expressed as % of the maximum density achieved at a single time point on the individual patient’s immunoblot (see a for example). Complete values for cTnT were obtained after immunoblot analyses and normalized similarly. The peak concentrations of full-length (7.9 ± 4.4 h) and fragment (9.3 ± 3.1 h) forms of cMyC occurred earlier than those of Troponin (11.8 ± 3.4 h, P < 0.007 and P < 0.001, respectively). The figure comprises analyses from 18 patients for cMyC fragment and cTnT (n = 18). In two patients, there was a serum sample missing at the peak of troponin concentration and in two patients no clear cMyC immunoreactivity could be discerned. Full-length cMyC could only be detected in the serum of 9 of the 18 patients in whom a fragment was visible

Mentions: We compared release of cMyC to cTnT by examining the time to peak concentration in serum samples from 20 STEMI patients presenting early after chest pain onset (patient characteristics are shown in Table 1 of the online supplement) with a cTnT concentration 12 h after admission >2000 ng/L. Venous blood was collected on admission and then every 2 h until 18 h after symptom onset and then once again at 24 h. The main purpose was to visualize any cMyC-circulating fragments. Since we were unable to reliably detect cMyC immunoreactivity if cTnT peak concentration was less than 2000 ng/L, we estimate the difference between the lower limit of detection of cMyC by immunoblot and cTnT by commercial electrochemiluminescent high-sensitivity immunoassay (Roche Elecsys E170) to be at least two orders of magnitude. This difference precluded a comparison based on time at which the biomarker was first detected. For this reason, our a priori intent was to compare time to peak concentration of cMyC and cTnT. Figure 2a shows the pattern of cMyC immunoreactivity in a patient presenting 180 min after chest pain onset. In this particular patient, the peak concentration of cMyC occurred much earlier than that of cTnT, an advantage particularly apparent for the full-length protein (approx. 140 kDa, see upper arrow). We performed an identical analysis in patients presenting early with suspected STEMI. Of the 20 STEMI patients’ serum we analyzed, there were two in which there were no clear cMyC bands despite a substantial cTnT rise. Thus, 18 patients subsequently proved to have sufficient myocardial infarction to allow detection of at least the 40 kDa fragment of cMyC (see lower band on Fig. 2a). Figure 2b shows the relative concentration profiles from time of presentation after chest pain onset (2–14 h, mean 3.3 h) to 24 h. This information was collected in advance of the laboratory determination of cTnT in the research samples. There is fairly rapid accumulation of the full-length cMyC protein (filled circles) with a mean time to peak of 7.9 ± 4.4 h that precedes the more easily detected 40 kDa fragment (open circles) with a mean time to peak of 9.3 ± 3.1 h. Both compare favorably with cTnT (closed squares) (time to peak of 11.8 ± 3.4 h, P > 0.007 and P > 0.001, respectively). In every patient’s profile, the time of peak concentration of full-length cMyC preceded that of cTnT (9/9) (P > 0.007) whilst for the 40 kDa fragment it coincided or preceded it in 16/16 (P > 0.001). Although our immunoblotting technique for cMyC detection was relatively crude and insensitive, it suggested that the cMyC release kinetics compare favorably with cTnT, prompting investment in a more sensitive and quantitative assay.Fig. 2


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)

cMyC and cTnT accumulation during ST elevation myocardial infarction. a cMyC immunoblot of serum collected from an individual patient with the corresponding laboratory-determined cTnT concentrations appearing as an overlay. The controls are C1, recombinant C0C2 domain of cMyC; C2, fresh mouse heart homogenate and C, control human serum. Soon after chest pain onset cMyC appears in the serum predominantly as the full-length protein (~140 kDa) which then diminishes as a shorter fragment accumulates (~40 kDa). These fragments seem to reach maximum abundance in advance of cTnT. b Relative timings of cMyC, measured by densitometry of full-length protein (closed circles) and dominant fragment (open circles), compared to cTnT (filled squares) measured in our clinical laboratory. The immunoblot detection technique is relatively crude and insensitive (see text for details), so band densities are expressed as % of the maximum density achieved at a single time point on the individual patient’s immunoblot (see a for example). Complete values for cTnT were obtained after immunoblot analyses and normalized similarly. The peak concentrations of full-length (7.9 ± 4.4 h) and fragment (9.3 ± 3.1 h) forms of cMyC occurred earlier than those of Troponin (11.8 ± 3.4 h, P < 0.007 and P < 0.001, respectively). The figure comprises analyses from 18 patients for cMyC fragment and cTnT (n = 18). In two patients, there was a serum sample missing at the peak of troponin concentration and in two patients no clear cMyC immunoreactivity could be discerned. Full-length cMyC could only be detected in the serum of 9 of the 18 patients in whom a fragment was visible
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4383815&req=5

Fig2: cMyC and cTnT accumulation during ST elevation myocardial infarction. a cMyC immunoblot of serum collected from an individual patient with the corresponding laboratory-determined cTnT concentrations appearing as an overlay. The controls are C1, recombinant C0C2 domain of cMyC; C2, fresh mouse heart homogenate and C, control human serum. Soon after chest pain onset cMyC appears in the serum predominantly as the full-length protein (~140 kDa) which then diminishes as a shorter fragment accumulates (~40 kDa). These fragments seem to reach maximum abundance in advance of cTnT. b Relative timings of cMyC, measured by densitometry of full-length protein (closed circles) and dominant fragment (open circles), compared to cTnT (filled squares) measured in our clinical laboratory. The immunoblot detection technique is relatively crude and insensitive (see text for details), so band densities are expressed as % of the maximum density achieved at a single time point on the individual patient’s immunoblot (see a for example). Complete values for cTnT were obtained after immunoblot analyses and normalized similarly. The peak concentrations of full-length (7.9 ± 4.4 h) and fragment (9.3 ± 3.1 h) forms of cMyC occurred earlier than those of Troponin (11.8 ± 3.4 h, P < 0.007 and P < 0.001, respectively). The figure comprises analyses from 18 patients for cMyC fragment and cTnT (n = 18). In two patients, there was a serum sample missing at the peak of troponin concentration and in two patients no clear cMyC immunoreactivity could be discerned. Full-length cMyC could only be detected in the serum of 9 of the 18 patients in whom a fragment was visible
Mentions: We compared release of cMyC to cTnT by examining the time to peak concentration in serum samples from 20 STEMI patients presenting early after chest pain onset (patient characteristics are shown in Table 1 of the online supplement) with a cTnT concentration 12 h after admission >2000 ng/L. Venous blood was collected on admission and then every 2 h until 18 h after symptom onset and then once again at 24 h. The main purpose was to visualize any cMyC-circulating fragments. Since we were unable to reliably detect cMyC immunoreactivity if cTnT peak concentration was less than 2000 ng/L, we estimate the difference between the lower limit of detection of cMyC by immunoblot and cTnT by commercial electrochemiluminescent high-sensitivity immunoassay (Roche Elecsys E170) to be at least two orders of magnitude. This difference precluded a comparison based on time at which the biomarker was first detected. For this reason, our a priori intent was to compare time to peak concentration of cMyC and cTnT. Figure 2a shows the pattern of cMyC immunoreactivity in a patient presenting 180 min after chest pain onset. In this particular patient, the peak concentration of cMyC occurred much earlier than that of cTnT, an advantage particularly apparent for the full-length protein (approx. 140 kDa, see upper arrow). We performed an identical analysis in patients presenting early with suspected STEMI. Of the 20 STEMI patients’ serum we analyzed, there were two in which there were no clear cMyC bands despite a substantial cTnT rise. Thus, 18 patients subsequently proved to have sufficient myocardial infarction to allow detection of at least the 40 kDa fragment of cMyC (see lower band on Fig. 2a). Figure 2b shows the relative concentration profiles from time of presentation after chest pain onset (2–14 h, mean 3.3 h) to 24 h. This information was collected in advance of the laboratory determination of cTnT in the research samples. There is fairly rapid accumulation of the full-length cMyC protein (filled circles) with a mean time to peak of 7.9 ± 4.4 h that precedes the more easily detected 40 kDa fragment (open circles) with a mean time to peak of 9.3 ± 3.1 h. Both compare favorably with cTnT (closed squares) (time to peak of 11.8 ± 3.4 h, P > 0.007 and P > 0.001, respectively). In every patient’s profile, the time of peak concentration of full-length cMyC preceded that of cTnT (9/9) (P > 0.007) whilst for the 40 kDa fragment it coincided or preceded it in 16/16 (P > 0.001). Although our immunoblotting technique for cMyC detection was relatively crude and insensitive, it suggested that the cMyC release kinetics compare favorably with cTnT, prompting investment in a more sensitive and quantitative assay.Fig. 2

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