We are pleased to submit our manuscript, which highlights a perspective direction in hypertrophic cardiomyopathy research. Our study focuses on non-coding RNAs, specifically microRNAs, as promising cardiac biomarkers, and advanced cardiac magnetic resonance (CMR) imaging as a research tool that could facilitate the discovery of novel circulating miR biomarkers. The current biomarkers are not specific for HCM, and their use is limited by risk stratification for heart failure death. By reviewing recent literature, we discuss the potential to identify specific circulating microRNA signatures linked to adverse microanatomical features of HCM observed using advanced CMR. We aim to engage the HCM scientific community in future interdisciplinary collaborations. The brief review of evolving modalities already applied in some areas of clinical practice may be of interest to a broad audience of practitioners, including cardiologists, radiologists, laboratory specialists, and genetics.

Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disorder, characterized by left ventricular (LV) hypertrophy in the absence of abnormal loading conditions [1]. In addition to cardiomyocyte hypertrophy, histological hallmarks include myocardial fibrosis (focal and diffuse), extensive disarray, and microvascular disease (MVD), all linked to arrhythmias, sudden cardiac death (SCD), and heart failure [2–6]. HCM exhibits significant heterogeneity in LV morphology [7], clinical course [8], and genetic etiology, involving both monogenic and polygenic components. Over recent decades, genetic studies have focused on protein-coding genes, identifying sarcomeric gene variants as the primary cause in approximately half of HCM patients [9–11]. This has established sarcomere dysfunction as a crucial mechanism in HCM [12], prompting the development of new treatments, such as cardiac myosin inhibitors [13]. Despite notable advancements, challenges remain in altering disease progression [14]. In clinical practice, there is a need for disease-specific plasma biomarkers to differentiate HCM from secondary LV hypertrophy, enhance risk stratification, and monitor phenotype evolution in preclinical mutation carriers. A fundamental question is how to identify the signaling pathways and regulatory networks that mediate the phenotypic expression of HCM’s complex genetics.

This paper reviews micro non-coding ribonucleic acids (microRNAs or miRs), known as negative controllers of gene expression, as promising biomarkers for HCM. A special focus is placed on a) myocardial microanatomical features of HCM as a research field for disease-specific biomarkers; b) whether cardiac magnetic resonance (CMR) tissue characterization techniques hold potential to advance the discovery of circulating biomarkers, such as miRs, in HCM (Figure 1).

Since myocardial biopsy is not typically used in managing HCM, biomarkers are sought from cardiac imaging, clinical features, and circulating molecules. Similar to other cardiovascular diseases, a multi-parametric approach is employed for the diagnosis and risk stratification in HCM.

The only validated diagnostic marker for HCM is LV myocardial thickness, defined as ≥15 mm in adult probands and routinely assessed using echocardiography or CMR [1]. However, this marker has limitations: it cannot always reliably distinguish HCM from secondary LV hypertrophy or metabolic disorders and is insufficient to detect preclinical and early-stage disease. Furthermore, the quality of echocardiographic image may be compromised by patient-related factors, while CMR, although more precise, is not universally accessible and has procedural restrictions. Supporting diagnostic tools include electrocardiogram, family screening, genetic testing, and clinical assessment to exclude phenocopies. Currently, there are no specific blood biomarkers for HCM. While several molecules reflecting myocardial wall stretch, fibrosis, inflammation, apoptosis, necrosis, and endothelial dysfunction have demonstrated correlations with imaging findings [15], they have not been integrated into clinical practice due to insufficient evidence of specificity, thereby limiting their diagnostic utility. Moreover, none of these biomarkers exhibit adequate sensitivity to detect subclinical HCM [16].

Few imaging markers and clinical features - such as ventricular tachycardia, unexplained syncope, and a family history of SCD - are incorporated into SCD risk stratification algorithms [17, 18]. However, these models do not include blood biomarkers and show modest predictive value. Current European guidelines recommend the use of N-terminal pro-B-type natriuretic peptide (NT-proBNP) and troponin T for assessing mortality risk in overt HCM, particularly for death due to heart failure [1]. Additionally, NT-proBNP can serve as a surrogate marker for evaluating the therapeutic efficacy of cardiac myosin inhibitors [19, 20]. However, no risk models are currently designed to predict heart failure or other non-SCD-related mortality in HCM.

Challenges in identifying blood biomarkers for HCM may be attributed to historically small sample sizes in cardiomyopathy studies, inaccurate phenotype characterization, and truly weak biomarker correlations. Furthermore, simplistic approach based on a limited set of pre-selected biomarkers may not capture the complex underlying pathways.

Recent advancements in omics technologies, enabling the quantification of thousands of low-abundance circulating molecules (e.g., RNA, proteins, metabolites), have led to the discovery of novel biomarkers. Studies have demonstrated the ability of compiled protein and miR panels to differentiate HCM from hypertensive heart disease [21, 22] and subclinical HCM from controls [23, 24].

Advances in cardiovascular genetics have shifted scientific interest toward the non-protein-coding genome and its non-coding RNA (ncRNA) transcripts [25]. The foremost function of ncRNAs is to regulate protein-coding gene expression, thereby affecting fundamental processes such as growth, differentiation, metabolism, apoptosis, and autophagy [26–29]. With growing evidence on the involvement of ncRNAs in heart diseases [30] and the advent of advanced computational methods, researchers can now explore the diagnostic, prognostic, and therapeutic potential of ncRNAs as biomarkers and novel targets for intervention [31].

Among various types of ncRNAs [32], miRs are the most abundant [33] and of particular interest in human diseases, including HCM [34]. MiRs are small molecules (∼20–22 nucleotides) that form a coordinated regulatory system and control gene expression post-transcriptionally by binding to their messenger RNAs, resulting in cleavage or translational repression [35]. A single miR can have numerous high- and low-affinity gene targets, and a single gene can be regulated by multiple miRs.

MiRs are detected not only in tissues but also in the bloodstream, making them attractive biomarkers for cardiac diseases, where biopsy is uncommon. They enter circulation from living and dead cells via active secretion or passive release. Despite the RNase-rich environment of blood, circulating miRs remain stable [36], protected within extracellular vesicles or bound to RNA-binding proteins [37, 38]. As conserved regulators of gene expression, miRs serve as dynamic biomarkers that reflect disease stages [39]. In heart failure studies, circulating miRs significantly improved the diagnostic power of NT-proBNP, which may be particularly beneficial for identifying heart failure with preserved ejection fraction, where standard clinical assessment, imaging, and NT-proBNP alone may not be definitive [40, 41].

Microanatomical changes in the myocardium, such as fibrosis, disarray, and MVD, are closely associated with HCM and its major clinical outcomes, as demonstrated in early histological studies [2–6], making them attractive substrates for the discovery of non-invasive biomarkers. Importantly, these changes are not merely secondary to LV hypertrophy but are also present in non-hypertrophied LV segments [55] and at the preclinical stage [56–58]. In mouse models, early administration of mavacamten, a first-in-class cardiac myosin inhibitor, suppressed the development of myocardial disarray and fibrosis by attenuating hypertrophic and profibrotic gene expression [59]. This highlights the role of specific signaling pathways in these adverse microanatomical changes in HCM. The identification of miR signatures associated with these features appears to be a promising avenue for HCM research.

CMR, with its ability to accurately track pathological processes in the myocardium, is increasingly used in clinical trials of new therapeutics for non-ischemic cardiomyopathies [76]. CMR imaging series serve as surrogate markers of treatment efficacy and, as a merit, provide mechanistic insights into the molecular pathways of natural (placebo group) and treatment response over shorter time periods. In HCM, cardiac myosin inhibitors have been evaluated in clinical trials using surrogate clinical and imaging endpoints. The EXPLORER-HCM CMR substudy showed significant reductions in LV mass, wall thickness, and left atrial volume index, suggesting that mavacamten alters HCM pathophysiology [20]. Meanwhile, LGE and ECV were not significantly changed, supporting the irreversible nature of myocardial fibrosis. Recent research by Joy et al. showed associations of CMR-derived disarray and MVD with stages of phenotype evolution [67], suggesting potential future applications of these techniques in HCM research before and after therapeutic interventions. Notably, in overt disease, the presence versus absence of sarcomeric mutation has different effects on microstructure and microvasculature [67]. Stress perfusion CMR has recently been used as a validation tool for another potentially more cost-effective and clinically practical marker of MVD – impaired myocardial work on echocardiogram [77].

MiRs are promising biomarkers, as their altering profiles can reflect distinct molecular processes in the heart. Given the heterogeneity of HCM, likely due to a cascade of molecular and structural changes, it is essential to investigate biomarkers across all stages of the disease. Before the onset of overt LV hypertrophy, HCM is characterized by abnormalities in myocardial microstructure, which emphasize their relevance in early pathogenesis. We presume that circulating molecules associated with these specific changes could be the strong candidates for further validation in larger studies as both disease-specific and prognostic biomarkers.

Given the limitations and biases of myocardial biopsy, CMR serves as a valuable tool for cardiac research. Advanced CMR technologies, currently being validated in preclinical models and human histology, enable the non-invasive visualization and quantification of myocardial microanatomical changes at all stages of the disease, including the preclinical phase. Preclinical HCM appears to be of particular interest for biomarker discovery, offering a unique opportunity to explore ncRNA signatures and potentially uncover disease-specific pathways without the confounding influence of secondary changes related to hemodynamic abnormalities. The expanding availability of genetic testing, including cascade family screening, is facilitating the identification of mutation carriers, making such studies feasible. When conducting discovery studies in overt HCM, the genotype of patients should be considered, as both miRs and myocardial microstructure are sensitive to the genotype status.

Novel HCM therapeutics, such as myosin inhibitors and gene editing, hold the potential to reverse the disease phenotype. Incorporating candidate miR panels into such self-controlled trials could enhance biomarker discovery by identifying those that reflect reversible adverse myocardial changes. A complementary imaging and genome-based biomarker approach could deeper insights into the complex underlying processes and identify novel targets for emerging therapeutic technologies.

HCM is a slowly progressive disease with a low event rate, making prospective studies is costly and time-consuming. Nevertheless, longitudinal studies with adequate sample size are essential to evaluate the prognostic power of candidate miR panels. Although conducting such studies in HCM is challenging, growing awareness and diagnostic expertise among professionals, emerging international cardiomyopathy collaborations, and advances in potentially curative therapies foster optimism.

Besides miRs, there are two other types of ncRNAs in the scope of interest in HCM: long non-coding RNAs (lncRNAs), which account for 80%–90% of all ncRNAs, and circular RNAs (circRNAs), a newer class of ncRNAs known for their stability due to a closed ring structure [89, 90]. To the best of our knowledge, only one study has investigated myocardial fibrosis in HCM by integrating CMR with relevant circulating lncRNAs [88]. This field remains largely unexplored, and significant efforts are required to advance our understanding.

Enhanced myocardial characterization and staging of HCM using advanced CMR techniques holds promise for identifying circulating miRs as biomarkers. MiR signatures associated with adverse microanatomical changes detected by CMR could be the strong candidates for longitudinal validation studies. To ensure comprehensive and reliable results, future research should consider patients’ genetic status.