Assessment of Risk Factors for Chronic Heart Failure: Toward Cardiovascular Precision Medicine

Heart failure — and cardiac decompensation in particular — represents a complex clinical condition characterized by multiple and often poorly understood risk factors, which affect men and women differently. The condition carries a high mortality rate and is associated with frequent hospitalizations, generating a considerable impact on both healthcare systems and economic resources.

Patients with heart failure present extremely heterogeneous clinical profiles, differing in ejection fraction, the underlying disease that led to its development, circulating catecholamine levels, and response to beta-blocker therapy. Despite this variability, all these patients share one common feature: a reduced response to beta-adrenergic stimulation, particularly at the beta-1 receptor. Yet the significance of this receptor downregulation and desensitization remains an active area of investigation.

From an epidemiological standpoint, data show that heart failure with preserved ejection fraction is more prevalent in elderly individuals and in women. This raises critical questions about the existence of gender- or sex-related factors that might explain this differential incidence — including estrogenic signaling, differences in chromosomal composition (XX versus XY), and genetic factors such as microRNAs linked to both estrogenic stimulation and the X chromosome.

The Beta-Adrenergic Receptor Paradox

One of the most intriguing aspects of this research concerns the interpretation of alterations in the cardiac beta-adrenergic system. Competing theories exist regarding the pathophysiological significance of these changes. On one hand, attenuation of the beta-adrenergic signal through desensitization and downregulation could exert a beneficial effect, protecting the heart from excessive adrenergic stimulation in an adaptive and protective mechanism. On the other hand, this attenuation could play a detrimental pathogenetic role, contributing to the deterioration of cardiac function through the loss of catecholamine-driven contractility and myocardial relaxation.

In cardiomyocytes, the predominant beta-adrenergic receptor is the beta-1 subtype, accounting for approximately 70% of the total, with the remaining 30% represented by the beta-2 receptor. The beta-3 receptor also appears to be present in human cardiomyocytes, albeit in very small quantities and with a role that remains uncertain, particularly in cardiac disease.

Numerous studies have documented a significant reduction in the beta-1 subtype, at both the protein and messenger RNA level, reaching up to 50%, with a severity that closely correlates with disease progression. By contrast, beta-2 receptors remain substantially unchanged in most studies. Many investigations also report significant alterations in the proteins that constitute the signaling system located beneath the cell membrane of the beta-adrenergic receptors.

These findings give rise to a clinical paradox: how can the efficacy of certain beta-blockers in heart failure be explained, given that the beta-1 receptor is already significantly downregulated? This question largely remains unanswered.

The Limitations of Current Experimental Models

A critical issue highlighted by this research concerns the limitations of currently available experimental models. The desensitization and downregulation of beta-adrenergic receptors have been studied predominantly in heterologous systems — that is, human tumor cell lines in which these receptors are artificially overexpressed.

Studies conducted in these models demonstrate that, while the beta-2 adrenergic receptor is readily desensitized and downregulated following catecholamine stimulation, the beta-1 receptor is not. This finding appears to contradict what is observed in the failing human heart, where it is precisely the beta-1 receptor that undergoes downregulation, while the beta-2 remains substantially unchanged.

This discrepancy between results obtained in heterologous systems and clinical observations underscores the urgent need to develop validated experimental models — both in vitro and in vivo, and ideally suited to sex-specific studies — in order to adequately investigate the phenomena of beta-adrenergic receptor sensitization and desensitization.

Research Objectives and Key Milestones

The research project presented by Dr. Matarrese was structured around well-defined milestones, with the primary objective of identifying a quantitative or semi-quantitative methodology capable of unequivocally discriminating between the two receptor types and distinguishing membrane-bound from internalized receptors.

A second crucial objective was to validate an alternative experimental model, identifying peripheral blood monocytes as a potentially suitable system for studying these phenomena. In parallel, the research team worked on the development and comparison of sex-specific in vitro models to investigate multiple aspects of cardiomyocyte physiology, and on the creation of in vivo models using mice carrying genetically induced alterations at the level of beta-adrenergic receptors or sub-membrane signaling proteins.

Specific Scientific Objectives

The scientific objectives of the project aimed to answer fundamental questions in the understanding of heart failure pathophysiology. First and foremost: whether the reduction in beta-1 receptor density observed in patients is simply an epiphenomenon, or whether it plays an active pathogenetic role in disease development.

A second objective concerned the validation of monocytes as a potential clinical biomarker for monitoring disease progression and therapeutic response, with the future prospect of developing a diagnostic kit for clinical use. Finally, the research sought to determine whether estrogenic signaling or other sex-related factors might play a role in the downregulation of the human beta-1 adrenergic receptor.

Monocytes as an Innovative Experimental Model

The choice of peripheral blood monocytes as an experimental model represents one of the most innovative aspects of this research. This decision was based on scientific evidence suggesting that these cells may mirror, for certain biochemical and molecular alterations, what occurs in cardiomyocytes — both in patients and in animal models.

To validate this approach, the researchers first developed a quantitative — specifically semi-quantitative — methodology capable of unequivocally distinguishing the two receptor types. Using wild-type mice and those specifically knocked out for the beta-1, beta-2, or both receptors, the team validated antibodies capable of clearly differentiating the two receptor subtypes, employing monocytes from these animals as positive and negative controls.

The validated antibodies were then tested on receptor expression in peripheral blood monocytes from both male and female human subjects. The results revealed an important initial difference compared to cardiomyocytes: while the latter express approximately 70% beta-1 and 30% beta-2 receptors, monocytes — as in most other non-cardiac cells — display exactly the opposite ratio. Nevertheless, expression levels proved sufficient to enable the study of receptor downregulation.

The Role of Oxidative Stress and TSPO

A fundamental finding to emerge from the research concerns the role of oxidative stress in the regulation of beta-adrenergic receptors. An increase in oxidative stress and reactive oxygen species (ROS) has been extensively documented in both animal models and patients with cardiac dysfunction. It is also well established that beta-blocker treatment is capable of reducing oxidative stress.

Given that approximately 90% of ROS produced — both under physiological and pathological conditions — originate at the mitochondrial level, the researchers focused their attention on specific mitochondrial proteins. In particular, the study concentrated on the translocator protein (TSPO), a protein directly involved in the regulation of mitochondrial ROS production.

The rationale for investigating this protein was supported by several factors: its abundant expression in the cardiovascular system and the numerous studies indicating that its downregulation is associated with cardiac dysfunction. The researchers therefore investigated whether beta-1 receptor density might be regulated through TSPO signaling.

Using diazepam — a reference benzodiazepine — as a stimulus, it was observed that this compound produced a reduction in beta-1 adrenergic receptor expression on the monocyte surface, but not in beta-2 expression. To confirm that this reduction was specifically attributable to the interaction between TSPO and its ligand, rather than to a non-specific effect, the researchers employed a specific TSPO antagonist, which proved capable of restoring receptor density on the cell surface.

Experimental Results: Selective Receptor Desensitization

The experiments conducted revealed differential regulation patterns for the two beta-adrenergic receptor subtypes. It emerged that catecholamines are unable to induce desensitization of the beta-1 receptor, while they are effective in regulating the beta-2. Conversely, oxidative stress — as previously observed in mouse cardiomyocytes — was found to selectively downregulate the beta-1 receptor on the monocyte surface, but not the beta-2.

These data strongly suggest that monocytes may indeed serve as a mirror of what occurs in cardiomyocytes, and that this capacity to reflect cardiac alterations is selective and specific. Direct stimulation with catecholamines or isoproterenol, which activates both beta-1 and beta-2 receptors, demonstrated specificity for the beta-2 but not for the beta-1.

This finding carries potentially significant clinical implications. Peripheral blood monocytes could be proposed as biomarkers both for disease progression monitoring and for predicting response to beta-blocker therapy. This is particularly meaningful given that, currently, beta-blocker therapy is managed in an entirely empirical manner in clinical practice, without objective tools to predict individual therapeutic response.

Development of Sex-Specific Cellular Models

Through funding from the HEAL ITALIA project, the research team succeeded in developing and validating both XX (female) and XY (male) cardiomyocyte models that preserve the typical cytoskeletal characteristics of cardiomyocytes and meet the requirements for studying beta-adrenergic system downregulation. These models will enable sex-specific studies aimed at understanding what the downregulation of the beta-1 receptor may represent at the pathogenetic level.

In parallel, a miRNomic analysis was initiated on these cellular systems. As highlighted in the preceding presentation at the same conference, many microRNAs — including those linked to the X chromosome — are altered in cardiovascular diseases. These microRNAs could explain the different clinical and epidemiological characteristics observed between the sexes in cardiovascular conditions.

The objective of this analysis is to isolate microRNAs that may be of interest both as biomarkers — potentially even more rapid and accessible to identify than monocytes, which require isolation from peripheral blood — and as innovative, sex-specific therapeutic targets.

Preclinical Animal Models: Investigating the Pathogenetic Role

To determine whether beta-adrenergic receptors play a pathogenetic role in heart failure or simply represent a disease marker, the use of preclinical animal models is essential. The research team adopted two complementary experimental strategies.

The first involved modifying beta-adrenergic signal transduction components by introducing non-desensitizable receptors exclusively into the myocardium. The second strategy completely eliminated beta-adrenergic receptors through the generation of specific knockout mice.

In both models, pressure overload was induced through thoracic aortic coarctation — a procedure that induces arterial hypertension and progressively leads to cardiac hypertrophy, cardiac dysfunction, and ultimately overt heart failure.

Results from Non-Desensitizable Receptor Models

In the first experimental approach, mice carrying non-desensitizable beta-adrenergic receptors developed heart failure at the same time points and in the same manner as wild-type mice. This result suggests that the pathophysiological problem does not appear to be directly linked to the phenomenon of beta-adrenergic receptor desensitization per se.

However, these data do not fully answer the fundamental question: are the very presence of the beta-adrenergic receptor and its capacity for desensitization indispensable to the development of pathological cardiac hypertrophy? Could the beta-adrenergic receptor — and the beta-1 subtype in particular — play an active pathogenetic role, rather than being simply a marker of cardiac disease?

Results from Knockout Models

To address this critical question, the team used mice completely lacking beta-adrenergic receptors. In this case too, the development of cardiac hypertrophy proved virtually identical in knockout mice compared to wild-type animals. This surprising finding indicates that beta-adrenergic receptors do not appear to be obligatory for the development of pathological cardiac hypertrophy.

This conclusion, however unexpected, leaves the enigma of beta-blocker efficacy in heart failure unresolved. If beta-adrenergic receptors are not essential to the development of the disease, through what mechanism do beta-blockers exert their beneficial effect in some patients?

Clinical Implications and Future Perspectives

The research presented by Dr. Matarrese carries profound implications for precision medicine in cardiovascular disease. As emphasized by Prof. Moroncini in his concluding remarks, the study fits perfectly within the direction of precision medicine, challenging well-established paradigms in clinical practice.

Current guidelines for the treatment of heart failure recommend beta-blockers with a Class 1A level of evidence — the highest available — suggesting their administration to virtually all patients with heart failure. However, as the research highlights, this universal therapeutic strategy may not be appropriate for all patients.

In some cases, beta-blockers may exert no preventive effect whatsoever, while in others they could actually worsen the clinical condition. It therefore becomes essential to identify specific subgroups within the broad and heterogeneous category of heart failure patients, in order to tailor therapeutic strategies to individual characteristics.

Toward Personalized Clinical Biomarkers

Peripheral blood monocytes emerge from this research as highly promising potential clinical biomarkers. Their capacity to reflect the status of cardiac beta-adrenergic receptors could enable:

• Non-invasive monitoring of disease progression
• Prediction of individual response to beta-blockers
• Personalization of pharmacological therapy
• Future development of diagnostic kits for clinical use

This approach would represent a significant step forward compared to the currently empirical management of beta-blocker therapy, providing clinicians with objective tools to guide therapeutic decisions.

The Importance of Sex Differences

A particularly innovative aspect of this research is the attention devoted to sex differences in the pathophysiology of heart failure. The development of sex-specific cellular models and the analysis of X-chromosome-linked microRNAs represent promising approaches for understanding why heart failure with preserved ejection fraction is more prevalent in women and in older individuals.

These studies could identify innovative, sex-specific therapeutic targets, paving the way for preventive and therapeutic strategies personalized not only on the basis of individual patient characteristics, but also taking into account biological differences related to sex.

Conclusions

The research presented by Dr. Paola Matarrese within the HEAL ITALIA project represents a significant contribution to the understanding of the pathophysiological mechanisms of heart failure and to the identification of new approaches for cardiovascular precision medicine.

The results obtained — while leaving fundamental questions about the pathogenetic role of beta-adrenergic receptors open — have enabled the validation of new experimental models, the identification of potential clinical biomarkers, and the development of tools for sex-specific research. The paradox of beta-blocker efficacy in heart failure remains an enigma yet to be solved, but this very contradiction underscores the complexity of the condition and the necessity of moving away from universal therapeutic approaches in favor of personalized strategies.

As highlighted throughout the conference, precision medicine is not simply an abstract concept, but a concrete clinical necessity for improving patient outcomes and optimizing the use of healthcare resources. The HEAL ITALIA project, with its eight thematic areas (spokes) dedicated to different aspects of precision medicine, represents an exemplary model of how translational research can help transform clinical practice.

As Dr. Matarrese emphasized, the hope is that this significant investment in research may have a future — completing the studies already underway and delivering tangible benefits to patients with heart failure, while contributing to the development of a national precision medicine platform accessible to all Italian citizens, from the north to the south of the country.