Editor's Note: Commentary based on Roncarati, R., C. Viviani Anselmi, M.A. Losi, L. Papa, et al., Circulating miR-29a, among other up-regulated microRNAs, is the only biomarker for both hypertrophy and fibrosis in patients with hypertrophic cardiomyopathy. J Am Coll Cardiol 2014. 63:920-7.

Background

Hypertrophic cardiomyopathy (HCM) is a primary disorder of the myocardium with a variable natural history, ranging from an asymptomatic and benign course to sudden, premature cardiac death.¹ While clinical signs and risk factors help identify patients at high risk for serious cardiac events, additional imaging or biochemical markers are needed to further discriminate cardiac risk in HCM.2-4 Recently microRNAs (miRNAs) have emerged as highly sensitive and specific circulating biomarkers of cardiomyocyte injury and cardiovascular disease.^5,6 Only one study prior to that of Rancorati et. al. had evaluated circulating miRNA expression in HCM and failed to demonstrate significant plasma miRNA dysregulation in a small number of patients with HCM.^{7, 8}

Study Synopsis

Roncarati et al. aimed to characterize the circulating miRNA profiles in patients with HCM.⁸ In their study of 41 patients with HCM and 41 healthy age and sex matched controls they employed a candidate miRNA approach, profiling 21 miRNAs previously associated with cardiovascular disease, including cardiac or muscle specific miRNAs and miRNAs involved in fibroblast, smooth muscle and endothelial function. miRNAs with significant dysregulation in HCM patients were subsequently correlated with imaging parameters of hypertrophy and fibrosis. Hypertrophy was evaluated by the maximal LV wall thickness and hypertrophy index on transthoracic echocardiogram (TTE), as well as the maximal LV wall thickness and LV mass index (LVMI) on cardiac magnetic resonance (CMR). Cardiac fibrosis was quantified and scored 0-4 based on the degree of late gadolinium enhancement on CMR. Finally, to determine potential differences between HCM and left ventricular hypertrophy (LVH) consequent to pressure overload, miRNA profiling was also performed in 12 high-risk patients with severe symptomatic aortic stenosis (AS).

Of the 21 candidate miRNAs profiled, 12 (miR-27a, -199a-5p, -26a, -145, -133a, -143, -199a-3p, -126-3p, -29a, -155, -30a, and -21) were significantly upregulated in the plasma of patients with HCM compared to controls. Of these, eight miRNAs demonstrated reasonable sensitivity and specificity for patients with HCM (AUCs greater than 0.7). Five miRNAs (miR-29a, -27a, -199a-5p, -21, -155) demonstrated statistically significant correlation with at least one measure of hypertrophy. Of these, miR-29a was the most robust, demonstrating significant correlation with three of four measures of hypertrophy and a trend toward correlation with the fourth measure (LVMI on CMR). Further, miR-29a expression correlated well with degree of fibrosis (r=0.691, p=0.003). Finally, while several miRNAs were similarly upregulated in patients with AS-induced hypertrophy, miR-29a upregulation was specific to patients with HCM. The authors concluded that a circulating miRNA profile can distinguish HCM patients from healthy individuals and that this profile is specific to hypertrophy and fibrosis related to HCM.

Commentary

The utility of circulating miRNAs as biomarkers of cardiovascular disease has attracted considerable attention over recent years. However, the clinical application of miRNAs as disease biomarkers remains limited due to several key obstacles. One of the most significant of these is the lack of consensus regarding circulating miRNA normalization. Although many groups are normalizing serum and plasma miRNA expression to a spiked in synthetic miRNA (as was the case in this study), this does not account for the effects of plasma processing and other pre-analytic variables on circulating miRNA measurement.⁹ Given the high content of many intracellular miRNAs relative to that of plasma, hemolysis and platelet contamination of plasma can introduce significant miRNA variability from one sample to the next.^9,10 While not yet widely used, strategies have been proposed for assessing the degree of RBC-derived miRNA contamination due to hemolysis, as well as minimizing platelet contamination of plasma samples by an additional high speed centrifugation step.^10,11 Though the tissue specific miRNAs reported here (ex. miRs-1, 133a, 195, 214, & 499) are relatively unaffected by these processing variables, miR-29a and others reported here (ex. miRs-16, 21, 30, 126, and 155) are highly expressed in blood cells and may be sensitive to processing conditions inadequately normalized by the synthetic spike-in strategy.12 Despite this, Roncarati et al. present a convincing story for the potential value of miR-29a as a biomarker for cardiac remodeling in HCM. miR-29a not only demonstrated significant upregulation (p<0.0001) with high sensitivity and specificity (AUC = 0.8365), but also correlated with three different measures of hypertrophy assessed by two independent imaging techniques, as well as the degree of fibrosis in patients with HCM.

Note however that the etiology and mechanistic significance of plasma miRNA dysregulation is unknown. While it is clear that many miRNAs (e.g. myo-miRs-208, 133, 1) are released into the plasma following cellular injury, miRNAs are also secreted into the circulation from intact cells bound to stabilizing proteins or packaged within extracellular vesicles. Given evidence of chronic troponin elevations in patients with HCM 2, plasma miRNA expression described herein may in part reflect miRNA release related to chronic myocardial injury.

The miR-29 family members are potent inhibitors of cardiac fibrosis and play a key role in cardiac remodeling.¹³ Plasma miR-29a upregulation has been reported following myocardial injury, where the degree of upregulation correlated with the extent of late remodeling post-MI.¹⁴ While miRNA-29a was not dysregulated in a previous study of hypertrophied cardiac tissue from patients with HCM ¹⁵, this may reflect the regional heterogeneity of miRNA expression that has been reported in cardiovascular disease.¹³ Alternatively, plasma miR-29a expression could reflect secondary effects of HCM on other organs. It is intriguing that miR-29a was not upregulated in patients with concentric hypertrophy secondary to aortic stenosis, although this may reflect insufficient power to detect differential regulation with only 12 patients in the AS group. Though further work is necessary to elucidate the mechanistic significance of the circulating miRNA profile of HCM described herein, Roncarati et al. have provided strong evidence to support the potential role of miRNAs as biomarkers for HCM.

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References

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3. Marian, A.J., On predictors of sudden cardiac death in hypertrophic cardiomyopathy. J Am Coll Cardiol, 2003. 41(6): p. 994-6.
4. Rubinshtein, R., J.F. Glockner, S.R. Ommen, P.A. Araoz, et al., Characteristics and clinical significance of late gadolinium enhancement by contrast-enhanced magnetic resonance imaging in patients with hypertrophic cardiomyopathy. Circ Heart Fail, 2010. 3(1): p. 51-8.
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8. Roncarati, R., C. Viviani Anselmi, M.A. Losi, L. Papa, et al., Circulating miR-29a, among other up-regulated microRNAs, is the only biomarker for both hypertrophy and fibrosis in patients with hypertrophic cardiomyopathy. J Am Coll Cardiol, 2014. 63(9): p. 920-7.
9. McDonald, J.S., D. Milosevic, H.V. Reddi, S.K. Grebe, et al., Analysis of circulating microRNA: preanalytical and analytical challenges. Clin Chem, 2011. 57(6): p. 833-40.
10. Cheng, H.H., H.S. Yi, Y. Kim, E.M. Kroh, et al., Plasma processing conditions substantially influence circulating microRNA biomarker levels. PLoS One, 2013. 8(6): p. e64795.
11. Kirschner, M.B., J.J. Edelman, S.C. Kao, M.P. Vallely, et al., The Impact of Hemolysis on Cell-Free microRNA Biomarkers. Front Genet, 2013. 4: p. 94.
12. Pritchard, C.C., E. Kroh, B. Wood, J.D. Arroyo, et al., Blood cell origin of circulating microRNAs: a cautionary note for cancer biomarker studies.Cancer Prev Res (Phila), 2012. 5(3): p. 492-7.
13. van Rooij, E., L.B. Sutherland, J.E. Thatcher, J.M. DiMaio, et al., Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci U S A, 2008. 105(35): p. 13027-32.
14. Zile, M.R., S.M. Mehurg, J.E. Arroyo, R.E. Stroud, et al., Relationship between the temporal profile of plasma microRNA and left ventricular remodeling in patients after myocardial infarction. Circ Cardiovasc Genet, 2011. 4(6): p. 614-9.
15. Kuster, D.W., J. Mulders, F.J. Ten Cate, M. Michels, et al., MicroRNA transcriptome profiling in cardiac tissue of hypertrophic cardiomyopathy patients with MYBPC3 mutations. J Mol Cell Cardiol, 2013. 65: p. 59-66.

Keywords: Biomarkers, Blood Platelets, Cardiomegaly, Cardiomyopathy, Hypertrophic, Fibrosis, Hypertrophy, Hypertrophy, Left Ventricular, Myocardium, MicroRNAs, Troponin

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