Background to the Sarcomere and Troponin

In order to place what we know about troponin T and I in context, it is worth reviewing the fundamentals of troponin expression and biological action. High school biology teaches students that the basic structure of muscle contraction is the sarcomere, a repeating micrometer-sized unit that dictates the anatomy of both cardiac and skeletal striated muscle. The thick (myosin) and thin (actin) filaments within the sarcomere interlock, such that each thick filament is surrounded by six thin ones. Aggregates of sarcomeres assemble linearly into myofibers, and calcium flux causes contraction of the structure.

Tropomyosin is a long, coiled protein that spans most of the actin filament and is an important regulator of muscle contraction, preventing myosin adherence (and hence preventing contraction). Within each tropomyosin protein are complexes of heterotrimeric troponin. These comprise troponin subunits troponin T (TnT), troponin I (TnI), and troponin C (TnC) (Figure 1). TnC binds calcium ions during calcium flux, and this process causes a conformational change in TnI, which in turn exposes the actin filament to myosin and allows muscle contraction. TnT essentially anchors the troponin complex to the tropomoysin structure. Smooth muscle does not contain troponin.

Figure 1. Illustration of the Location of the Troponin Heterotrimer in the Sarcomere of Striated Muscle

TnI is expressed in specific isoforms encoded by three distinct genes: slow skeletal, fast skeletal, and cardiac muscle. TnT also exists as specific isoforms encoded by three distinct genes: slow skeletal, fast skeletal, and cardiac.¹ It is the presence of distinct cardiac isoforms that have allowed development of TnI and TnT immunoassays that are specific for the detection of cardiomyocyte injury and death using a blood test (cTnI and cTnT).

Current Clinical Use of Troponin: Diagnosis of AMI

Although skeletal myocytes can die and re-differentiate, the cardiac tissue is largely viewed as post-mitotic; therefore, protein extrusion from cardiomyocytes is interpreted as a sign of injury and/or necrosis. Clinical assessment, 12-lead electrocardiography and cardiac cTnI or cTnT form the diagnostic cornerstones of patients with acute-onset chest pain. Blood-based cardiac troponin assays have now long been used as the preferred marker of cardiac tissue injury, replacing older assays such as total creatine kinase, creatine kinase-myocardial band, lactate dehydrogenase, and aspartate aminotransferase.²

Historically, low-sensitivity troponin assays yielded results that were interpreted as positive or negative. The National Institute for Health and Care Excellence and the European Society of Cardiology have issued recommendations supporting the use of a new generation of high-sensitivity cardiac troponin (hs-cTn) assays.^3,4 Although yet to be formally endorsed by the Food and Drug Administration, these assays have vastly improved the speed of ruling out acute myocardial infarction (AMI).^5,6 The International Federation of Clinical Chemistry recommends determination of the 99th percentile upper reference limit as diagnostic thresholds for AMI. These recommendations are clearly pragmatic and not biologically equivalent cutoffs given the variation between different assays.⁵ Comparing hs-cTnI and hs-cTnT assays can be challenging due to different reference ranges applied in the general population and different assay-specific pre-analytical and analytical variables. False positives and false negatives can be due to non-repeatable "flyers" or native antibody interference that are assay specific.

Interestingly, the decision regarding whether to measure either hs-cTnT or hs-cTnI in the acute setting is not typically a clinical decision per se. Due to patent, hs-cTnT assays are available from only one manufacturer (Roche Diagnostics). In contrast, several manufacturers market hs-cTnI assays (including Abbott, Siemens, and Beckman Coulter). Therefore, the primary supplier to the local biochemistry laboratory often dictates which of hs-cTnI or hs-cTnT is measured for patient care. It is rare for both hs-cTnT and hs-cTnI to be measured in the same study cohort, and clinicians are often experienced in the clinical interpretation of either hs-cTn assay result but not both.

Comparison of cTnI and cTnT in the Diagnosis of AMI

In the acute setting, the available data suggest that hs-cTn assays perform very similarly in clinical care. The Advantageous Predictors of Acute Coronary Syndromes Evaluation (APACE) study group hypothesized that combining the two biochemical signals from hs-cTnT and hs-cTnI might overcome preanalytical, analytical, and pathophysiological differences between the biomarkers and improve clinical decision-making. They recruited 2,256 patients with suspected AMI (after excluding those with ST-segment elevation myocardial infarction), and 18% were diagnosed with AMI by adjudication. Both hs-cTnT (Roche Diagnostics) and hs-cTnI (Abbott) were measured in the whole cohort at admission, and the correlation between these measures was r = 0.89. The area under the curve (diagnostic accuracy) for both assays at presentation was 0.93 (95% confidence interval, 0.92-0.94). Addition of hs-cTnI to hs-cTnT only marginally improved discrimination for diagnosing AMI, with no evidence that addition of hs-cTnT improved the performance of hs-cTnI.⁷ Combining both troponin measurements (with their respective cutoffs) marginally improved negative predictive values. Further study of the Siemens hs-cTnI assay showed similar diagnostic performance.⁸ The main issue for clinical implementation of the assays is therefore where to draw the diagnostic threshold. Overall, the assays perform similarly, suggesting that acute myocardial injury drives circulating concentrations of both cTn in this setting.

Comparison of hs-cTnI and hs-cTnT in the General Population

Despite apparent broad concordance between troponin assays in the acute setting, strong emerging evidence in the general population suggests that cTnT and cTnI differ meaningfully in their association with disease outcomes. The advent of high-sensitivity assays means that, by definition, the assay should detect troponin in the circulation of at least 50% of the general population. This has led to interest in use of the troponin assays not just in the acute setting but also for cardiovascular disease (CVD) risk prediction.

In previous cohort studies, one of hs-cTnI or hs-cTnT (but rarely both) had been measured, presumably led by a presumed inter-changeability of assays in the acute setting. This has yielded an evidence base lacking direct evidence as to which biomarker might actually be preferable. This is reflected in our recent study-level meta-analysis, where 13 studies measured cTnI and 7 studies measured cTnT; none measured both. In the top versus bottom third of the distribution, the hazard ratio for future CVD events was nominally stronger for cTnT than cTnI (hazard ratio =1.60 versus 1.36; p = 0.171), and cTnT was more strongly associated with fatal CVD (p = 0.027).⁹ However, such direct comparisons of cTn assays are difficult to interpret given the differences in the characteristics of the cohorts between studies.

The cross-sectional Maastricht study provides preliminary evidence that troponin assays may not be interchangeable in the general population.¹⁰ In 1,540 middle-aged individuals from the general population without significant baseline disease, the correlation between hs-cTnI (Abbott) and hs-cTnT (Roche) was only r = 0.54. Clearly, in most biological situations this is a notable correlation, but it is a long way from being considered interchangeable for assays supposedly measuring the same biomarker.

We undertook to compare and contrast hs-cTnI (Abbott) and hs-cTnT (Roche) in the Generation Scotland Scottish Family Health Study prospective cohort study of 19,501 adults from the general population. In cross-sectional data, hs-cTnT and hs-cTnI were detectable in 53.3% and 74.8% of participants, respectively, and were only modestly correlated in unadjusted analyses (r = 0.46), broadly in line with the Maastricht study data (Figure 2).

Figure 2. Scatter Graph of the Distributions of cTnT and cTnI in the Generation Scotland Scottish Family Health Study

However, the correlation between the two troponins was even weaker after adjusting for age and sex (r = 0.31).¹¹ The association of each troponin with traditional CVD risk factors was also distinct, with hs-cTnI showing stronger associations with age, male sex, body mass index, and systolic blood pressure and cTnT showing stronger associations with the presence of diabetes.

We then investigated associations with CVD and non-CVD outcomes. Both troponins had strong univariable associations with CVD outcomes, indicating that circulating levels were probably to some degree related to myocardial injury (Figure 3, left panels). However, after adjusting for traditional CVD risk factors, the strength of the association attenuated for both troponins (Figure 3, right panels) such that the hazard ratio for a 1 standard deviation increase in log troponin was 1.24 (95% confidence interval, 1.17-1.32) for hs-cTnI and 1.11 (1.04-1.19) for hs-cTnT. Both troponins had stronger associations with CVD death than the composite CVD outcome. Of interest, hs-cTnT (but not hs-cTnI) showed an association with non-CVD death.¹²

Figure 3. Association of hs-cTnI and hs-cTnT Unadjusted and Adjusted With Composite Fatal/Non-Fatal CVD Outcome Over Median 7.8 Years (n = 1,177 events)

As a final examination of the possible distinct natures of hs-cTnT and hs-cTnI, we can use genetics to identify potential upstream causes of troponin extrusion into the circulation. Inheritance of specific allelic variants is random at a population level (and therefore not confounded by lifestyle risk factors and circumstance) and is not subject to reverse causality.¹³ The Generation Scotland Scottish Family Health Study indicated distinct patterns of single nucleotide polymorphisms related to hsTnT and hsTnI. For instance, cTnI was associated with single nucleotide polymorphisms in the KLKB1 and F12 genes. These genes encode proteins that are both part of the kallikrein-kinin axis and have shown associations with other vasoactive peptides such as B-type natriuretic peptide. However, these genetic signals were not associated with hs-cTnT. This provides at least some evidence that the genetic determinants of the cTn might not overlap, although larger-scale meta-analyses are required to look at this issue in more detail.

Why Are hs-cTnI and hs-cTnT Signals Different?

The simplest explanation as to why the two cTn appear very similar in the acute setting is that any signal not related to acute cardiomyocyte death or injury is diluted by the overpowering signal derived from the ongoing event. Therefore, most of the cTn (I or T) in the circulation can be attributed to the cardiomyocyte injury. Even then, release kinetics are distinct, and both troponins tend to circulate in distinctive complexes, as well as free forms.¹⁴ There are also heterophilic antibodies that might interfere with either assay. Therefore, the assays will not always be entirely concordant.

It has long been debated that skeletal muscle may in some circumstances express cTnT (but not cTnI).¹⁵ Fetal skeletal muscle expresses cTnT.¹⁶ A recent study in 74 patients with hereditary and acquired skeletal myopathies showed elevations beyond the 99th centile in hs-cTnT in 69% of patients but elevation in cTnI in only 4%. There was also evidence from staining studies that the skeletal muscle tissue itself was the source of circulating cTnT.¹⁷ Other studies have reported similar data.^18,19 In line with this, exercise studies have long reported that intensive aerobic exercise interventions can raise both cTnT and cTnI levels,²⁰ although the effect may be greater for cTnT, suggesting there may also be a non-cardiac source.²¹ This narrative might fit with the data from the Generation Scotland Scottish Family Health Study showing that hs-cTnT is more closely associated with non-CVD death; people with low muscle mass may be undergoing catabolic wasting and are more likely to die of CVD and non-CVD causes.²² This hypothesis requires further elaboration.

Conclusions

Elevations in hs-cTnT and hs-cTnI are important diagnostic tools in the acute clinical setting, and most evidence suggests the tests perform similarly in patients for this purpose. However, there is now growing interest in the potential use of these assays in the general population for prediction of future events. The optimal approach in judicious use of the troponin assay to improve care for apparently healthy patients is not yet clear. However, emerging evidence suggests that selection of a particular troponin assay for a particular clinical task may be an important component of future clinical decision-making.

References

1. Sheng JJ, Jin JP. TNNI1, TNNI2 and TNNI3: Evolution, regulation, and protein structure-function relationships. Gene 2016;576:385-94.
2. Lippi G. Biomarkers: Novel troponin immunoassay for early ACS rule-out. Nat Rev Cardiol 2016;13:9-10.
3. Myocardial infarction (acute): Early rule out using high-sensitivity troponin tests (Elecsys Troponin T high-sensitive, ARCHITECT STAT High Sensitive Troponin-I and AccuTnI+3 assays) Diagnostic guidance (DG)15 (National Institute for Health and Care Excellence website). October 2014. Available at https://www.nice.org.uk/guidance/dg15. Accessed September 5, 2019.
4. Roffi M, Patrono C, Collet JP, et al. 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without persistent ST-segment elevation: Task Force for the Management of Acute Coronary Syndromes in Patients Presenting without Persistent ST-Segment Elevation of the European Society of Cardiology (ESC). Eur Heart J 2016;37:267-315.
5. Apple FS, Sandoval Y, Jaffe AS, Ordonez-Llanos J. Cardiac Troponin Assays: Guide to Understanding Analytical Characteristics and Their Impact on Clinical Care. Clin Chem 2017;63:73-81.
6. Shah AS, Anand A, Sandoval Y, et al. High-sensitivity cardiac troponin I at presentation in patients with suspected acute coronary syndrome: a cohort study. Lancet 2015;386:2481-8.
7. van der Linden N, Wildi K, Twerenbold R, et al. Combining High-Sensitivity Cardiac Troponin I and Cardiac Troponin T in the Early Diagnosis of Acute Myocardial Infarction. Circulation 2018;138:989-99.
8. Boeddinghaus J, Twerenbold R, Nestelberger T, et al. Clinical Validation of a Novel High-Sensitivity Cardiac Troponin I Assay for Early Diagnosis of Acute Myocardial Infarction. Clin Chem 2018;64:1347-60.
9. Willeit P, Welsh P, Evans JDW, et al. High-Sensitivity Cardiac Troponin Concentration and Risk of First-Ever Cardiovascular Outcomes in 154,052 Participants. J Am Coll Cardiol 2017;70:558-68.
10. Kimenai DM, Henry RM, van der Kallen CJ, et al. Direct comparison of clinical decision limits for cardiac troponin T and I. Heart 2016;102:610-6.
11. Welsh P, Preiss D, Shah ASV, et al. Comparison between High-Sensitivity Cardiac Troponin T and Cardiac Troponin I in a Large General Population Cohort. Clin Chem 2018;64:1607-16.
12. Welsh P, Preiss D, Hayward C, et al. Cardiac Troponin T and Troponin I in the General Population. Circulation 2019;139:2754-64.
13. Davies NM, Holmes MV, Davey Smith G. Reading Mendelian randomisation studies: a guide, glossary, and checklist for clinicians. BMJ 2018;362:k601.
14. Park KC, Gaze DC, Collinson PO, Marber MS. Cardiac troponins: from myocardial infarction to chronic disease. Cardiovasc Res 2017;113:1708-18.
15. Jaffe AS, Vasile VC, Milone M, Saenger AK, Olson KN, Apple FS. Diseased skeletal muscle: a noncardiac source of increased circulating concentrations of cardiac troponin T. J Am Coll Cardiol 2011;58:1819-24.
16. Anderson PA, Malouf NN, Oakeley AE, Pagani ED, Allen PD. Troponin T isoform expression in humans. A comparison among normal and failing adult heart, fetal heart, and adult and fetal skeletal muscle. Circ Res 1991;69:1226-33.
17. Schmid J, Liesinger L, Birner-Gruenberger R, et al. Elevated Cardiac Troponin T in Patients With Skeletal Myopathies. J Am Coll Cardiol 2018;71:1540-9.
18. Rittoo D, Jones A, Lecky B, Neithercut D. Elevation of cardiac troponin T, but not cardiac troponin I, in patients with neuromuscular diseases: implications for the diagnosis of myocardial infarction. J Am Coll Cardiol 2014;63:2411-20.
19. Wens SC, Schaaf GJ, Michels M, et al. Elevated Plasma Cardiac Troponin T Levels Caused by Skeletal Muscle Damage in Pompe Disease. Circ Cardiovasc Genet 2016;9:6-13.
20. Klinkenberg LJ, Luyten P, van der Linden N, et al. Cardiac Troponin T and I Release After a 30-km Run. Am J Cardiol 2016;118:281-7.
21. Skadberg Ø, Kleiven Ø, Ørn S, et al. The cardiac troponin response following physical exercise in relation to biomarker criteria for acute myocardial infarction; the North Sea Race Endurance Exercise Study (NEEDED) 2013. Clin Chim Acta 2018;479:155-9.
22. Srikanthan P, Horwich TB, Tseng CH. Relation of Muscle Mass and Fat Mass to Cardiovascular Disease Mortality. Am J Cardiol 2016;117:1355-60.

Clinical Topics: Acute Coronary Syndromes, Cardiovascular Care Team, Dyslipidemia, Prevention, ACS and Cardiac Biomarkers, Lipid Metabolism, Novel Agents, Exercise

Keywords: Actin Cytoskeleton, Acute Coronary Syndrome, Alleles, Body Mass Index, Aspartate Aminotransferases, Chemistry, Clinical, Blood Pressure, Calcium, Cardiovascular Diseases, Cohort Studies, Chest Pain, Confidence Intervals, Confidence Intervals, Creatine Kinase, MB Form, Cross-Sectional Studies, Diabetes Mellitus, Exercise Therapy, Family Health, Hematologic Tests, Immunoassay, Electrocardiography, Antibodies, Heterophile, Kinetics, Lactate Dehydrogenases, Kinins, Kallikreins, Life Style, Ions, Muscle, Smooth, Muscle Contraction, Myocardium, Myocardial Infarction, Myocytes, Cardiac, Muscular Diseases, Myosins, Natriuretic Peptide, Brain, Patient Care, Muscle, Skeletal, Prospective Studies, Protein Isoforms, Risk Factors, Reference Values, Polymorphism, Single Nucleotide, Sarcomeres, Myocardial Infarction, Staining and Labeling, Tropomyosin, Troponin C, Troponin I, Troponin T, United States Food and Drug Administration

< Back to Listings