Secretoneurin to the Rescue? Maybe or Maybe Not

Sudden cardiac death caused by ventricular arrhythmias is a major public health concern due to its increasing incidence and the difficulty in identifying patients at high-risk ¹. Although heart failure, coronary artery disease, and certain genetic syndromes (often caused by mutations in ion channels or gap junctions e.g. long QT syndrome, Brugada syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT)) are well known risk-factors for ventricular arrhythmias and sudden cardiac death, additional risk stratification is warranted. Measurement of biomarkers has revolutionized the practice of cardiology: for instance, natriuretic peptides in the diagnosis and prognosis of heart failure and troponin in the diagnosis of acute myocardial infarction. Yet, for arrhythmic risk, the algorithm to predict the risk of developing ventricular arrhythmias is limited.

Secretoneurin, a 33-amino acid neuropeptide produced by the endoproteolytic cleavage of chromogranin/secretogranin proteins that are principally found in neuroendocrine storage vesicles in a variety of cell types in endocrine and neuronal tissues ², has been proposed as a cardiac biomarker, providing incremental prognostic information to established risk indices in patients with myocardial dysfunction ³. In this issue of Circulation: Arrhythmia and Electrophysiology, Ottesen et al present evidence that circulating secretoneurin is elevated in patients and mice with CPVT compared to healthy controls, and in patients admitted with resuscitated cardiac arrest is elevated compared to subsequent values several days later ⁴. Prior work by the same authors showed that circulating secretoneurin levels predicted mortality in patients with systolic heart failure or out-of-hospital cardiac arrest ³. Furthermore, the authors expand on their prior studies, showing that AAV9-induced overexpression of secretoneurin attenuated induction of ventricular arrhythmias in mice with CPVT, perhaps by inhibiting calmodulin-dependent protein kinase (CaMKII) activity and phosphorylation of ryanodine receptor, and that exogenous secretoneurin decreased the incidence of early after-depolarizations. The authors concluded that upregulation of secretoneurin production is a compensatory mechanism, linked to conditions with abnormal Ca²⁺ regulation in cardiomyocytes, and thus serves a biomarker of the underlying Ca²⁺ dysregulation.

Is secretoneurin a biomarker predictive of malignant ventricular arrhythmias?

A biomarker should add independent information about risk or prognosis, should account for a significant proportion of the risk associated with the disease or condition, should be reproducible, and should be sensitive/specific ⁵. As with secretoneurin, most biological markers are not simply present or absent, but have a wide range of values with overlap between individuals with the disease and those without the disease. The prognostic value of secretoneurin has been previously explored: (a) In patients with severe sepsis, secretoneurin levels at admission provided incremental information to established risk indices for the prediction of mortality and shock, with the optimal cutoff of ~175 pmol/L at ICU admission to predict hospital mortality ⁶. (b) After coronary artery bypass grafting, circulating secretoneurin concentrations were directly correlated with increased risk of worse outcomes and death ⁷. (c) In patients hospitalized for heart failure, secretoneurin levels were closely associated with mortality during follow-up ³. (d) In patients with ventricular arrhythmia-induced cardiac arrest, secretoneurin was associated with short-term mortality ³.

In this study, circulating secretoneurin levels were significantly increased in CPVT patients compared to controls. There is, however, substantial overlap between CPVT patients and controls, and the sample size is quite small. The authors speculate that the elevation in secretoneurin is due to Ca²⁺ mishandling in the heart, but have not yet elucidated the pathways by which abnormalities in Ca²⁺ handling lead to changes in circulating secretoneurin levels. Furthermore, the elevation of secretoneurin could be due to the effects of ryanodine receptor mutations in neuro-endocrine tissue or inflammatory cells. Perhaps future studies could determine whether the elevation of circulating secretoneurin can predict those patients at highest risk for future events and/or response to pharmacological therapies such as α-blockers or flecanide.

In the second use of secretoneurin as a biomarker, the authors expand upon their prior analyses ³ of patients with out-of-hospital cardiac arrest due to ventricular arrhythmias, now stratified to 1-year mortality. Secretoneurin levels decreased through the hospitalization in both survivor and non-survivor groups, and the authors speculate that the increased secretoneurin is a compensatory mechanism during a period in which the risk of subsequent ventricular arrhythmia and mortality are high. Why secretoneurin levels decrease during the hospitalization is unknown, but could be due to many confounding variables including medications.

Downstream effects of secretoneurin in cardiomyocytes

Irrespective of secretoneurin’s utility as a biomarker, an important line of investigation is to explore its role in cardiomyocyte biology. In CPVT mice, ventricular arrhythmias can be induced by stress testing and isoproterenol ⁸. A higher number of arrhythmic events was observed in CPVT mice infected with an AAV-9 expressing scrambled peptide compared to those mice infected with an AAV-9 expressing secretoneurin. The authors chose to perform the statistical comparison to wild-type mice infected with scrambled peptide virus, but a more interesting statistical comparison would be to CPVT mice infected with the scrambled peptide virus. These data support the overall concept that secretoneurin has anti-arrhythmic properties in cardiomyocytes, although the level of exogenous to endogenous secretoneurin is not clear, and whether the level of expression is “physiological” is an important unknown.

Activation of CaMKII in the heart can promote heart failure and arrhythmias ⁹. Previously, this group found that secretoneurin, at concentrations ~10,000-fold greater than were measured in plasma could inhibit CaMKII by 20-30%, and at concentrations ~1000-fold greater than plasma could inhibit CaMKII autophosphorylation ³, suggesting relatively low potency as a direct CaMKII inhibitor ¹⁰. In the current study, they similarly found that high concentrations (2.8 micromoles/L) of secretoneurin inhibited isoproterenol-induced Thr²⁸⁷ autophosphorylation of CaMKII and the phosphorylation of the CaMKII phosphorylation site of ryanodine receptors in Langendorff-perfused hearts. Furthermore, the same high concentration of secretoneurin reduced ryanodine receptor-mediated Ca²⁺ sparks, decreased the incidence of basal Ca²⁺ waves, and isoproterenol-induced Ca²⁺ waves, and early and delayed after-depolarizations. Secretoneurin also reduced L-type Ca²⁺ channel current density, which the authors attributed to CaMKII modulation of the channel subunits. The mechanism by which CaMKII regulates Ca²⁺ channel in the heart is termed facilitation- an increased frequency of action potentials activates CaMKII, which phosphorylates the channel ^{11, 12}. Based upon the limited experiments presented in the paper, secretoneurin may inhibit Ca²⁺ current via signaling pathways unrelated to CaMKII.

A lower concentration of secretoneurin (2.8 nmol/L- which is ~10-fold higher than the highest plasma concentration) had “more modest effects”, although it is not apparent that this concentration of secretoneurin had any statistically significant, biologically-relevant effect (Supplemental Figure 1). One caveat in the interpretation of these data is the comparison of spark frequency in Supplemental Figure 1 and Figure 4. In Figure 4, isoproterenol tripled the spark frequency from ~0.25/second to ~0.75/second, and high concentration of secretoneurin normalized the spark frequency. For the lower concentration of secretoneurin, however, the isoproterenol-induced spark frequency was only ~0.3/second. Perhaps with a more robust isoproterenol-mediated activation of ryanodine receptors, the effect of a low concentration of secretoneurin would have been different than what was observed.

Other downstream targets of secretoneurin have been reported in heart and other tissues. In cardiomyocytes, secretoneurin gene therapy protected against the cardiac hypertrophy induced by isoproterenol treatment through the activation of AMPK and ERK/MAPK pathways, and by upregulating anti-oxidants and suppressing oxidative stress ¹³. It is conceivable that these pathways are involved in limiting arrhythmogenesis in CPVT mice, as chronic leak of Ca²⁺ from the sarcoplasmic reticulum via mutant ryanodine receptors in CPVT mice can induce oxidative stress ¹⁴. Perhaps the modest inhibition of the L-type Ca²⁺ channel by secretoneurin was due to anti-oxidant effects ¹⁵. Secretoneurin also can stimulate proliferation and exert anti-apoptotic effects on endothelial cells via activated PI3K/Akt and mitogen-activated protein kinase pathways ¹⁶, and it acts on neurons after hypoxia and ischemic insult to increase their survival by activating the Jak2/Stat3 pathway ¹⁷. In contrast to its beneficial effects, secretoneurin could also be involved in detrimental activities. For instance, secretoneurin triggers the selective migration of monocytes and fibroblasts, implicating its involvement in inflammatory responses, and perhaps in the acceleration of atherosclerosis. ¹⁸ Secretoneurin acts an endogenous stimulator of vascular endothelial growth factor (VEGF) signaling in coronary endothelial cells by enhancing binding of VEGF to low affinity binding sites and neuropilin-1 and stimulates growth factor receptors such as fibroblast growth factor-3 ¹⁹. Thus, based upon the high concentration of secretoneurin needed to substantially inhibit CaMKII, it is conceivable that the functional and direct downstream target of secretoneurin in cardiomyocytes may not be CaMKII.

Is secretoneurin an endogenous anti-arrhythmic?

For all its beneficial cellular effects, higher levels of secretoneurin are associated with worse survival in patients, indicating that the beneficial effects are not adequate to overcome the pathophysiology of heart failure or CPVT, at least at later stages of the disease process ²⁰. So, secretoneurin may have translational potential for some forms of heart disease, but we shouldn’t expect too much, at least in the short term. Perhaps a modified, re-engineered form of the protein would be more potent inhibitor of CaMKII and arrhythmogenesis. Regardless, more investigation of the cellular effects of secretoneurin, at physiologically-relevant concentrations, is needed to better understand the multi-dimensional attributes of this potential endogenous anti-arrhythmic.