High-Throughput Sequencing of Human Myxomatous Mitral Valve to Dissect Pathophysiological Mechanisms and Progress Toward Therapeutic Treatments

Myxomatous mitral valve degeneration (MMVD) is one of the most common valvular heart diseases, affecting 2% to 5% of the general population, and is the main etiology observed in patients with mitral valve prolapse.^1,2 Although MMVD and the subsequent development of mitral valve prolapse/regurgitation are associated with significant cardiovascular morbidity and mortality, the pathophysiological mechanisms underlying the progressive valve leaflet remodeling remain only sparsely described, leaving invasive treatments, via surgical or transcatheter approaches, as the only option for patients suffering from severe symptomatic disease. ³

MMVD is characterized by a progressive and heterogeneous thickening of the mitral valve leaflets as a consequence of glycosaminoglycan accumulation, with further collagen and elastin fiber fragmentation.^1,2 These phenotypic traits are the results of complex and interrelated mechanisms mediated by the activation of valvular interstitial cells.^1,2 Over the last 20 years, significant efforts have been made to dissect the genetic architecture of MMVD, providing strong evidence regarding the involvement of specific signaling pathways and opening the way to dissect the underlying pathophysiological mechanisms related to the development and progression of this life-threatening disease.^1,2 The activation of the transforming growth factor beta (TGF-β) and bone morphogenic protein pathways, as well as serotonin signaling, is consistently described in various forms of MMVD, across several species, including Marfan syndrome-related MMVD, human carcinoid or drug-induced mitral valve diseases, and canine models developing spontaneous MMVD, such as Cavalier King Charles.^4–7 More recently, these data were also confirmed in the non-syndromic form of MMVD, via the multiomics analysis of a unique Filamin A-mutated rat model. ⁸ However, the translation of these findings to clinical therapeutic applications currently failed to confirm the usefulness of targeting these pathways to mitigate the progression of the disease. In addition, recent studies underlined the contribution of inflammation and the associated chemotaxis and immune cell activation, especially the monocyte-derived macrophages, in the development and progression of the disease.^4,8 Nevertheless, a better understanding of the molecular mechanisms involved in the development and progression of MMVD is still required to extend our knowledge of the intricate regulatory networks leading to MMVD and identify potential therapeutic targets.

High-Throughput Combined mRNA and miRNA to Decipher the Main Signaling Pathways Involved in Human MMVD

In this issue of the Journal of the Heart Valve Society, ⁹ Hagler and collaborators attend to close this gap using high-throughput transcriptomic analyses of human non-syndromic MMVD samples, accounting for the vast majority of patients suffering from mitral valve prolapse: RNA sequencing and miRNA profiling were then performed in 10 MMVD samples and 10 “control” valves. The mRNA analysis firstly confirmed that signaling pathways related to fibrosis, extracellular matrix (ECM) remodeling, ECM–receptor interaction, integrin signaling cascade, focal adhesion, and cellular proliferation, as well as chemotaxis, are central in the pathophysiological processes leading to MMVD (Figure 1).^4,7,8,10 These findings were further corroborated by the miRNA profiling, reinforcing the central role of these pathways in MMVD.

Figure 1.

High-Throughput Sequencing to Unravel Signaling Pathways and Molecular Drivers Involved in Human Myxomatous Mitral Valve Disease. Legend: mRNA and miRNA sequencing analyses using “classical” differentially expressed genes and random forest with boruta feature selection approach identified signaling pathways and molecular drivers of non-syndromic human MMVD: (i) TGF-β, 5-HT, and immune cell activation and chemotaxis pathways are consistently reported, across multiple species, as central contributors in MMVD, (ii) new signaling pathways, such as the circadian rhythm modulation, have been identified and would deserve further investigations, and (iii) specific mediators related to these enriched pathways were reported as potential promising targets in human MMVD.

In an attend to reinforce these findings, the authors analyzed the interactions between mRNA and miRNA and reported the number of miRNA related to the most significant signaling pathways identified in this study: these data highlighted significant interactions between differentially expressed mRNA and miRNA, and pointed out the need of focused studies looking at the role of miRNA in the activation of tissue-specific signaling pathways and derived molecular drivers involved in MMVD.

Boruta Feature Selection Approach to Identify of Most Relevant Pathways.

Interestingly, in order to identify the most relevant pathways and most promising molecular drivers from these complex and interrelated omics databases coming from a relatively small number of heterogeneous samples, the authors used the Random Forests with Boruta Feature Selection approach, a procedure allowing selection of variables based on feature importance derived from iterations of Random Forests algorithms. ¹¹ The selection process is based on the computation of the likelihood of importance for each differentially expressed gene (DEG), from which only the DEGs with a likelihood of importance >95% will then be analyzed. Following the “dimensional reduction” of both mRNA (459 DEGs were conserved over the 2378 identified DEGs after Boruta selection) and miRNA (34 miRNA over 67) databases, functional annotation of the selected variables confirmed the contribution of signaling pathways identified via the standard analytic approach, but also highlighted circadian rhythm modulation as potential new important pathway associated with MMVD.

Circadian Rhythm Involvement in MMVD

This intriguing and novel finding supporting the role of the circadian rhythm in the development of MMVD was validated by quantitative reverse transcription polymerase chain reaction on selected mRNA and miRNA, in an independent cohort of 43 MMVD samples and 43 “control” valves. Interestingly, an interaction between the downregulation of hsa-miR-133a and the upregulation of period circadian regulator 2 has been highlighted by the authors as the potential underlying mechanism for circadian rhythm modulation in MMVD. Authors discussed the potential mechanistic foundation behind this association: the change in the sympathetic and parasympathetic balance related to circadian rhythm alteration modulates heart rate and blood pressure, two factors leading to hemodynamic cardiac adaptations, which in turn can increase mechanical stresses imposed on the valve tissue. Even if the imbalance of the circadian rhythm could lead to systemic hemodynamic changes and increase valve mechanical stresses, the tissue-specific molecular mechanisms governing the development of MMVD remain hypothetical and would deserve further investigations.

Analysis of Human Mitral Valve Samples: Several Advantages but Remaining Limitations

Some limitations need to be considered in interpreting the findings of this study. The mitral valve samples were collected from patients who underwent surgical mitral valve intervention and presented histological evidence of myxomatous remodeling. However, diverse etiologies could lead to surgical mitral valve intervention, resulting in a potentially important heterogeneity of the valve collected and analyzed in this study.^1,2 Indeed, the spectrum of the valve remodeling associated with mitral valve regurgitation is large: patients can present focal mitral leaflet remodeling or important bi-leaflet alterations at the time of surgery, chordae rupture (with minimal leaflet remodeling) leading to massive mitral regurgitation is also an emergency condition requiring mitral valve surgery. This heterogeneity in terms of the expression of the disease could then mask potential interesting molecular mechanisms associated with the development of these different etiologies/diseases. Secondly, the use of mitral valves collected in patients referred for heart transplantation as control tissue is certainly one of the only options to collect “healthy” non-myxomatous mitral valve in such a large quantity, but recent researches have also underlined the effect of mitral valve tethering, derived from dilated left ventricle and papillary muscle displacement, on mitral valve growing and remodeling.^12–15 These phenotypic left ventricle traits, frequently observed in the context of heart failure and heart transplantation, likely induced activation of signaling pathways in the mitral valve^12–15: the potential overlaps between signaling pathways associated with mitral valve remodeling in the context of MMVD versus dilated/dysfunctional left ventricle could then mask important molecular drivers involved in the pathophysiological processes of MMVD. Also, the clinical characteristics of both MMVD and heart transplant patients, such as age, sex, and cardiovascular risk factors, differ significantly, which could highlight molecular differences not restricted to MMVD but rather from the clinical presentation and underlying pathologies of the patients. To overcome these limitations, animal models are an interesting option, but they also present several limitations. ² Finally, regarding the contribution of circadian rhythm in the processes leading to MMVD and the lack of information regarding the time during the day at which the mitral valve samples from MMVD and “control” patients were collected, we cannot rule out a potential confounding effect of this factor. ¹⁶ In any case, the validation of this finding, as well as other molecular drivers highlighted by the authors in this study, will require mechanistic studies to confirm their causal association with the development of MMVD, to further consider potential therapeutic approaches targeting these specific mediators in patients with MMVD.

Multiomics Analysis: A “Game Changer” for the Understanding of Human MMVD Pathophysiology

Nevertheless, the data reported in this manuscript provide highly relevant findings in the field: by confirming in human sporadic samples of MMVD the involvement of traditionally described signaling pathways, such as TGF-β activation, cell adhesion and proliferation, as well as the most recently identified chemotaxis and immune cell activation, the data from Hagler and collaborators reinforce the need for future studies dedicated to deciphering the molecular drivers of these highly relevant pathways. Indeed, the current study emphasizes potential important molecular mediators of these signaling pathways that would deserve further investigations and open new opportunities to target MMVD and specifically modulate the clinical course of the disease. The characterization of differentially regulated miRNAs in MMVD tissue, such as miR1 and miR133a, known to be involved in fibrosis, ECM remodeling, and cell proliferation, also reinforces the relevance of these findings. The large majority of the identified miRNA in mitral valve tissue did not overlap with the circulating miRNA already associated with the presence of MMVD, suggesting that circulating miRNA could serve as biomarkers, but questions their mechanistic effects at the tissue level.

In conclusion, this study highlighted new signaling pathways and molecular drivers involved in human MMVD, using high-throughput sequencing, that contribute to the enrichment of our understanding of MMVD pathophysiology. Collectively with the data already published in the field, this study pointed out the most promising signaling pathways and molecular mediators that could be targeted to develop potential therapeutic treatment for the large population of patients suffering from MMVD.