Non-Classical Secretion: A Possible Mechanism to Explain Cardiac Troponin Elevations in the Absence of Acute Myocardial InfarctionTroponin; non-classical secretion; high-sensitivity cardiac troponin assays; exosomes; microvesicles
“It is important to realize that if certain areas of science
appear to be quite mature, others are in the process of
development, and yet others remain to be born.”
Santiago Ramón y Cajal,
Advice for a young investigator
Introduction
Nowadays, high-sensitivity cardiac troponin (hs-cTn) assays are available for clinical use and are part of the definition of acute myocardial infarction (AMI).1 , 2 However, troponin elevation is not limited to AMI, since other conditions related to oxygen demand mismatch, direct myocardial damage, increased myocardial strain, systemic processes (i.e. sepsis), neurological disease and renal failure can also result in its increase.2Troponin can now also be detected in atypical scenarios, such as in strenuous endurance exercise, rapid atrial pacing, and dobutamine stress echocardiography, as well as in 50% to 100% of healthy subjects.2 , 3
From a physiological point of view, troponin is a protein complex that regulates myofibrillar function, consisting of three subunits: I, T and C. In the case of troponin I and troponin T, there are three different tissue-specific isoforms: fast-twitch skeletal, slow-twitch skeletal and cardiac specific (fsTn, ssTn and cTn).4Conversely, TnC has two isoforms, one present in fast-twitch skeletal muscle (fsTnC) and one that expresses in both slow-twitch skeletal and cardiac muscle (ssTnC/cTnC).4
Apart from cell necrosis, several alternative release mechanisms have been postulated for the cardiac isoforms of troponin.3 , 4 However, none of them concisely explains why troponin is released in cases unrelated to myocardial infarction or whether this is indicative of reversible or permanent damage to the cardiac cell. In light of the current lack of knowledge, our main objective was to explore the feasibility of a novel mechanism for cardiac troponin release using a bioinformatical approach.
Materials and Methods
Review of the existing evidence
Before starting the analysis, a comprehensive review of the literature was conducted in search of articles related to troponin secretion. The searched databases were PubMed, bioRxiv and OpenGrey. Articles were first assessed based on their title. If it mentioned troponin and a secretory process, the abstract was read. Articles were selected if the abstract mentioned troponin and a secretory process or pathway. Purely clinical articles that did not investigate a mechanism of troponin release were excluded. The screening and review processes were performed by all authors. Disagreements were solved by consensus during regular meetings.
Sequence analysis
To assess for non-classical secretion of troponin, the SecretomeP 2.0 server was used (http://www.cbs.dtu.dk/services/SecretomeP/).5This tool is applied to predict whether a certain protein undergoes secretion without a signal peptide. It is a sequence-based method that, with the help of neural networks, detects specific features common to extracellular/secreted proteins.5
The canonical sequences from the three subunits of troponin from fast-skeletal, slow-skeletal and cardiac muscle were retrieved from the UniProtKB database (Supplementary File 1). The proteins were first assessed with SignalP 5.0 , a commonly used method for the recognition of signal peptides based on neural networks (http://www.cbs.dtu.dk/services/SignalP/).6This initial step was performed to rule out the classical secretory pathway. Afterwards, the sequences were analyzed with SecretomeP 2.0 . Finally, to assess the possibility of type IV non-classical secretion (used by transmembrane proteins that bypass the Golgi apparatus), the sequences were evaluated using TMHMM 2.0 , a program designed to detect transmembrane helices (http://www.cbs.dtu.dk/services/TMHMM/).7A graphical representation of the followed bioinformatic pipeline can be found in Supplementary Figure 1 .
– Illustration of the cytosol of a cardiomyocyte. The classical pathway of secretion is depicted by the vesicles traveling from the endoplasmic reticulum to the Golgi apparatus; the vesicles arrive at the plasma membrane, where they release their load. The proposed non-classical pathway of secretion is also shown. It starts with the formation of membranous blebs as a result of inflammation and/or cell stress, affecting the cardiomyocyte. Subsequently, unbound cytosolic cardiac troponin (cTn) (representing around 2-4% and 6-8% of the total cTnI and cTnT, respectively)1 enters these blebs and is released as microvesicles. These microvesicles enter the bloodstream, where they can be detected by high-sensitivity cardiac troponin assays. The box on the lower right corner shows the physiopathology of two different conditions where troponin increases. In the first case, a type 1 myocardial infarction case, an acute ischemic event leads to irreversible myocyte necrosis and troponin release. In the second case, rapid atrial pacing induces troponin release. Whether non-classical secretion participates in this process remains an open question. Created with BioRender.com. The 3D structure of the troponin complex was created with data from Protein Data Bank ID 1J1E.
Results
Review of the existing evidence
Nearly 19,900 articles were reviewed, of which 31 were read in full. After abstract and full text assessment, no articles were found to be related to troponin secretion. Hence, to the best of our knowledge, this is the first paper reporting evidence of troponin non-classical secretion.
Sequence analysis
After analyzing the troponin isoforms, SecretomeP 2.0 predicted cTnT, fsTnI, ssTnI, fsTnT and fsTnC to be non-classically secreted. In the 5 cases, the proteins achieved a neural network score (NN-score) greater than 0.6, which is the minimum threshold for mammalian sequences. Additionally, none of the eight troponin isoforms were found to contain a signal peptide or a transmembrane helix, according to SignalP 5.0 and TMHMM 2.0 , respectively. The summarized results are shown in Table 1 . The complete results can be found in Supplementary Table 1 .
Discussion
Non-classical secretion
Non-classical or unconventional secretion is a pathway of protein release. Contrary to classical secretion, it is independent of the Endoplasmic Reticulum (ER)/Golgi apparatus.8Consequently, it does not require a signal peptide, which is a short sequence of amino acids that leads the protein through the classical (ER/Golgi-mediated) secretory process.9Instead, non-classically secreted proteins are released through a myriad of mechanisms that can be classified into 4 groups: type I (pore-dependent transport), type II (ABC transporter-mediated release), type III (released from endosomes/autophagosomes) and type IV (Golgi bypass by transmembrane proteins).8Moreover, other mechanisms, such as exosomes and blebs, have also been recognized to take part in non-classical secretion.9Interestingly, most of the scenarios of unconventional secretion are triggered by cell stress, such as inflammation. Some examples of proteins that use the non-classical pathway are IL-1β/IL-1α, FGF-1, FGF-2, and galectins.8 , 9
Clinical implications of troponin non-classical secretion
Clinically, cTnT non-classical secretion could help to solve the debate around hs-cTn assays. An established secretion pathway for cTn potentially explains why troponins are detected in healthy subjects. In addition, cTn non-classical release might contribute to better define the pathological basis of “myocardial injury”. This term was included in the fourth universal definition of myocardial infarction, with a troponin value above the upper reference limit being the required condition for its diagnosis.1It is not unreasonable to think that an activity condition that generates stress in the cardiac cell results in the release of troponin through a non-classical secretory process ( Figure 1 ). In this regard, troponin elevations might be the result of systemic conditions reflecting on the heart through an inflammatory or cell stress mechanism. This could be the case of patients with sepsis, anemia, cancer, stroke, seizures, or after strenuous exercise.1 , 4 Interestingly, cTnT, but not I, has shown a circadian pattern of release.2cTnT release by tumor cells, possibly by extracellular vesicles, has also been recently reported.10
Proposal limitations
The main limitation of our proposal is that both cardiac troponin I and troponin T are elevated in myocardial injury.1A possible explanation could be that cardiac troponin T, the structural subunit of the troponin complex, carries the other subunits in the form of a dimer or trimer along the non-classical secretory process; however, this is a hypothetical assumption and remains an open but intriguing question.
SecretomeP 2.0 limitations
SecretomeP 2.0 was created in 2004, and after more than 15 years it remains a popular method for the assessment of non-classical secretion.5However, and as it happens with all computational methods, the results obtained are predictions. Thus, they should be interpreted together with the existing experimental body of evidence.
Evidence supporting cTn non-classical secretion
It is worth mentioning that there is some literature supporting our findings. Bleb formation by cardiomyocytes during ischemia, as well as troponin release through microvesicles in multiple cell lines (in both protein and mRNA form) have been previously demonstrated. Available evidence is summarized in Table 2 .
Future perspectives
Further research should aim at demonstrating circulating troponin within microvesicles. This could be tested in patients with acute myocardial infarction (where necrosis should be the leading release mechanism) versus atypical scenarios showing troponin elevation such as strenuous exercise, sepsis and stroke (where non-classical secretion should drive troponin release). After the isolation of plasma microvesicles, a possible approach to validate the non-classical secretion pathway could be the use of mass spectrometry followed by confirmation through specific antibodies.
Conclusion
High-sensitivity assays have brought considerable uncertainty about cTn elevations. Troponin liberation due to non-AMI related causes remains elusive and its exact prognostic significance continues to be investigated. Since our proposal is based on in silico evidence, it should be experimentally confirmed before its exact clinical significance can be ascertained. Whether other troponin variants also enter the non-classical secretory pathway is also an open question. Nonetheless, it is fair to say that troponin non-classical secretion is a promising research field in cardiology that awaits to be explored.
References
- 1 Thygesen K, Alpert JS, Jaffe AS, Chaitman BR, Bax JJ, Morrow DA, et al. Fourth universal definition of myocardial infarction. Eur Heart J.2018;40(3):237-69. Doi: 10.1093/eurheartj/ehy462.
- 2 Chaulin AM, Duplyakov DV. On the potential effect of circadian rhythms of cardiac troponins on the diagnosis of acute myocardial infarction. Signa Vitae.2021;17(3):79-84. Doi: 10.22514/sv.2021.050.
- 3 Hammarsten O, Mair J, Möckel M, Lindahl B, Jaffe AS. Possible mechanisms behind cardiac troponin elevations. Biomarkers.2018;23(8):725-34. Doi: 10.1080/1354750X.2018.1490969.
- 4 Park KC, Gaze DC, Collinson PO, Marber MS. Cardiac troponins: from myocardial infarction to chronic disease. Cardiovasc Res.2017;113(14):1708-18. Doi: 10.1093/cvr/cvx183.
- 5 Bendtsen JD, Jensen LJ, Blom N, von Heijne G, Brunak S. Feature-based prediction of non-classical and leaderless protein secretion. Protein Eng Des Selec. 2004;17(4):349–56. Doi: 10.1093/protein/gzh037.
- 6 Almagro Armenteros JJ, Tsirigos KD, Sønderby CK, Petersen TN, Winther O, Brunak S, et al. SignalP 5.0 improves signal peptide predictions using deep neural networks. Nat Biotechnol .2019;37(4):420–3. Doi: 10.1038/s41587-019-0036-z.
- 7 Sonnhammer ELL, von Heijne G, Krogh A. A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol. 1998;6:175–82.
- 8 Rabouille C. Pathways of Unconventional Protein Secretion. Trends Cell Biol 2017;27(3):230–40. Doi: 10.1016/j.tcb.2016.11.007.
- 9 Doroudgar S, Glembotski CC. The cardiokine story unfolds: ischemic stress-induced protein secretion in the heart. Trends Mol Med.2011;17(4):207-14. Doi: 10.1016/j.molmed.2010.12.003.
- 10 Tsuruda T, Sato Y, Kajihara K, Kawabata T, Kubuki Y, Komaki S, et al. Non-canonical Expression of Cardiac Troponin-T in Neuroendocrine Ethmoid Sinus Carcinoma Following Immune Checkpoint Blockade. Front Cardiovasc Med. 2019;6:124. Doi: 10.3389/fcvm.2019.00124.
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Study AssociationThis study is not associated with any thesis or dissertation work.Ethics approval and consent to participateThis article does not contain any studies with human participants or animals performed by any of the authors.
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Sources of Funding: There were no external funding sources for this study.
Publication Dates
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Publication in this collection
29 Apr 2022 -
Date of issue
Apr 2022
History
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Received
15 June 2021 -
Reviewed
15 Sept 2021 -
Accepted
10 Nov 2021
