Arrythmogenic Cardiomyopathy Prevention: MicroRNA as Diagnostic Biomarkers
Prof. Chiara Turchi opened her presentation by thanking Professor Moroncini for organizing the event, emphasizing the importance of such occasions: “I am a biologist, and I very often stay within the laboratory walls, so having opportunities like these is absolutely very, very important.”
The project stems from a collaboration that the speaker herself described as “perhaps somewhat unusual” — between forensic medicine and cardiology. This synergy between two seemingly distant disciplines has proven particularly fruitful in tackling a complex condition like arrhythmogenic cardiomyopathy, which demands expertise in both molecular biology and cardiac electrophysiology.
Arrhythmogenic Cardiomyopathy: A Rare but Devastating Disease
Arrhythmogenic cardiomyopathy is a rare, genetically heterogeneous disease characterized by two fundamental features. From a morphological standpoint, there is progressive replacement of normal cardiac tissue with fibroadipose tissue. Clinically, the disease is marked by the onset of significant and potentially lethal arrhythmias.
As Prof. Turchi emphasized, this condition is one of the leading causes of cardiac death in young people, and particularly in young athletes. Although rare, it carries an extremely significant clinical and social impact, affecting young, apparently healthy individuals during physical activity.
Genetic Heterogeneity and the Diagnostic Challenge
Arrhythmogenic cardiomyopathy is defined as genetically heterogeneous for several reasons. The genes responsible — or partially responsible — for the disease are numerous and varied, with mutations distributed across many different genes. A particularly complicating factor is that the penetrance of these mutations is incomplete, generating considerable phenotypic variability.
One of the greatest challenges in managing this disease is that only 50% of patients with arrhythmogenic cardiomyopathy carry identifiable genetic mutations. This finding suggests the involvement of additional factors and biomarkers that may play a role both in disease onset and in its clinical course.
MicroRNAs: New Candidate Biomarkers
The scientific literature suggests that, among all potential biomarkers, microRNAs may be the most promising candidates. These are well-characterized molecules with a crucial role in the post-transcriptional regulation of gene expression.
MicroRNAs function by binding to target messenger RNA molecules and can act in two ways: by blocking the translation of messenger RNA into protein, or by causing its degradation. This fine-tuned mechanism of gene expression regulation makes them particularly interesting biomarkers for complex diseases such as arrhythmogenic cardiomyopathy.
Project Objectives
The aim of the project was to investigate differential microRNA expression patterns in disease manifestation and, as a final goal, to evaluate their potential use as biomarkers in clinical settings. A diagnostic test based on microRNAs could represent a fundamental tool for early diagnosis and monitoring of disease progression.
Study Design: From Discovery Phase to Validation
The project included a critical preliminary phase of sample collection, followed by a first stage referred to as the Discovery Phase. In this phase, researchers sequenced the entire miRNome — the complete set of microRNAs — from the enrolled samples. The results of this first phase were then subjected to validation in a second phase, which was still ongoing at the time of the presentation.
Sample Collection: Innovation Through Electroanatomical Mapping
The sample collection phase was particularly important and innovative. To date, approximately 20 subjects with suspected arrhythmogenic cardiomyopathy have been enrolled in the study, from whom both blood samples and cardiac tissue via biopsy were collected.
A distinctive feature of this study is that biopsies were performed in a targeted manner, guided by electroanatomical mapping — a technique developed and refined specifically within this project by the Cardiology and Arrhythmology Clinic directed by Professor Dello Russo.
Electroanatomical Mapping: Identifying Diseased Tissue
Electroanatomical mapping is performed using catheters that record the heart’s electrical potentials. Two parameters are essentially measured: the variation in electrical signal amplitude and the timing with which the impulse is recorded. It has been observed that low-amplitude and late potentials — those conducted more slowly — are characteristic of diseased tissue.
As illustrated by Prof. Turchi, the system generates color-coded maps. In the voltage map, red indicates areas of low-amplitude potentials, representing potentially diseased zones. In the late potential map, purple marks the problematic areas. ECG recordings highlight in yellow the potentials corresponding to the affected myocardial regions.
This mapping enabled targeted biopsies, sampling tissue from areas most likely to be diseased. This is crucial because fibroadipose replacement does not occur uniformly throughout the myocardium, but only at specific sites. Without this guidance, there would be a real risk of inadvertently sampling healthy tissue, rendering the analysis meaningless.
Study Controls
Regarding controls, volunteer subjects were recruited for blood samples, while control cardiac tissue was obtained from cadavers, enabling a direct comparison with diseased tissue.
Analytical Phases: RNA Extraction and Quantification
The first analytical phase of the study involved total RNA extraction from both plasma and cardiac tissue, using two different kits specifically designed for each sample type. RNA quantification was also performed using distinct methods for each.
Standard techniques such as TapeStation and Qubit were not sufficiently sensitive to detect the RNA extracted from plasma, which is particularly scarce in this type of sample. The group therefore adopted a recently commercialized PCR technique, offering greater sensitivity and better suited to the purpose.
Sequencing the Entire miRNome
The pivotal phase of the study was sequencing of the entire miRNome using Illumina technology. A total of 30 samples were sequenced, comprising both cases and controls. The initial sequencing results were highly encouraging: data quality was good and the number of reads obtained was optimal for the study’s objectives.
Challenges Encountered: An Essential Aspect of Research
Before presenting the results, Prof. Turchi wished to share the difficulties encountered during the study, offering a meaningful reflection: “Challenges are part of every laboratory journey, and I always say: if we don’t find problems, perhaps we haven’t looked carefully enough, and we haven’t done our laboratory work with sufficient attention.”
Among the main challenges faced:
Plasma separation: must be performed immediately, within one hour of sample collection. Beyond this timeframe, researchers found it impossible to extract RNA effectively.
Final extract volume: proved insufficient for all planned analyses, requiring modifications to standard protocols.
MicroRNA quantification in plasma: represented a significant technical challenge, resolved through the adoption of more sensitive kits.
Library quality for sequencing: particularly in libraries derived from cadaveric tissue, quality was not always optimal, forcing researchers to exclude two samples from subsequent analysis.
Discovery Phase Results: Differentially Expressed MicroRNAs
The results of the first phase, visualized through volcano plots, showed in red the overexpressed microRNAs and in blue the underexpressed ones that reached statistical significance in cases compared to controls.
Analysis of cardiac tissue revealed particularly interesting findings. Expression heatmaps showed that case samples displayed a homogeneous internal expression pattern, clearly distinct from that of controls. Cases, clustered together in the analysis, showed a clear separation from controls, indicating a distinctive molecular signature of the disease.
Analysis of Plasma Samples
The same analysis was performed on plasma samples. Here too, microRNAs potentially useful for diagnostic purposes were identified, although fewer in number compared to those found in cardiac tissue. Furthermore, plasma expression patterns were less homogeneous than those observed in cardiac tissue, suggesting greater biological variability in the circulating compartment.
Selection of Candidate MicroRNAs
The subsequent phase involved a cross-comparison of results obtained from tissue and plasma, enabling the selection of approximately 10 microRNAs for the second validation phase. Among these, one microRNA stood out as particularly noteworthy: it was underexpressed by approximately threefold compared to healthy controls in both plasma and cardiac tissue.
This consistency across the two biological compartments is highly significant, as it suggests that this microRNA could genuinely serve as a plasma-based diagnostic biomarker, making a non-invasive test feasible.
Toward the Goal: The Validation Phase
The phase currently underway involves validation of the microRNAs selected in the Discovery Phase using Digital PCR. As Prof. Turchi emphasized, the ultimate objective is to develop and validate an assay applicable in clinical diagnostics.
A fundamental consideration is that a diagnostic test cannot rely on cardiac biopsy, because by that stage of clinical progression it would already be “too late.” The goal is therefore to develop a plasma-based diagnostic test, studying circulating microRNAs, that would enable non-invasive and potentially early diagnosis.
Digital PCR for Validation
Digital PCR technology was selected for the validation phase — a highly sensitive and quantitative technique that enables absolute measurement of microRNA levels even when present at very low concentrations, as is the case in plasma. This technology is particularly well-suited for biomarker validation intended for diagnostic use, given its high precision and reproducibility.
Clinical Perspectives and Expected Impact
The development of a diagnostic test based on circulating microRNAs could represent a breakthrough in the management of arrhythmogenic cardiomyopathy. Current diagnosis relies on clinical, electrocardiographic, and imaging criteria that often allow identification of the disease only when it is already at an advanced stage.
A plasma-based test enabling early diagnosis could make it possible to: identify at-risk individuals before the occurrence of major arrhythmic events; non-invasively monitor disease progression; stratify risk in relatives of affected patients; guide therapeutic decisions, such as implantation of an implantable cardioverter defibrillator (ICD).
This is particularly relevant given that arrhythmogenic cardiomyopathy predominantly affects young people and athletes — populations in which early diagnosis could literally save lives through preventive identification and appropriate arrhythmic risk management.
A Model for Cardiovascular Precision Medicine
This project represents a paradigmatic example of how precision medicine can be applied to rare cardiovascular diseases. The integrated approach combines: advanced electroanatomical mapping techniques for targeted biopsies; next-generation sequencing for analysis of the entire miRNome; validation with ultrasensitive technologies such as Digital PCR; and a focus on developing non-invasive diagnostic tests applicable in clinical practice.
This model demonstrates how translational research must begin from a real clinical problem — the difficulty of early diagnosis in arrhythmogenic cardiomyopathy — leverage the most advanced technologies available (sequencing, Digital PCR), while always keeping sight of the ultimate goal: returning to clinical practice with concrete, applicable tools (a plasma-based diagnostic test).
The Collaboration Between Forensic Medicine and Cardiology
A distinctive feature of this project is the interdisciplinary collaboration between forensic medicine and cardiology. Forensic medicine contributed expertise in molecular biology, sample management, and laboratory analysis, while cardiology provided clinical and electrophysiological knowledge along with innovative electroanatomical mapping techniques.
This synergy enabled the development of an approach that neither discipline could have achieved alone, once again demonstrating that precision medicine inherently requires the convergence of diverse and complementary expertise.
Conclusions and Future Impact
The work presented by Prof. Turchi represents a significant contribution to the understanding and diagnosis of arrhythmogenic cardiomyopathy. The identification of microRNAs specifically altered in the disease opens the way for the development of diagnostic biomarkers that could transform the clinical management of this devastating condition.
The ongoing validation phase using Digital PCR is a crucial step toward clinical translation. If the results confirm the Discovery Phase data, it will be possible to move toward larger clinical studies and, eventually, toward the development of a commercially available diagnostic test.
This would have a significant impact not only for patients with arrhythmogenic cardiomyopathy, but also for their family members, who could benefit from genetic and molecular screening to identify predisposition to the disease at an early stage — enabling preventive interventions before the onset of potentially lethal arrhythmic events.
The project fits perfectly within the objectives of Spoke 7 of HEAL Italia, demonstrating how precision medicine research can yield concrete results in the prevention of cardiovascular diseases — one of the leading causes of mortality in developed countries, and an area where early diagnosis can truly make the difference between life and death, especially in young individuals.
