Intercellular Communication in the Heart: Therapeutic Opportunities for Cardiac Ischemia

The maintenance of tissue, organ, and organism homeostasis relies on an intricate network of players and mechanisms that assist in the different forms of cell–cell communication. Myocardial infarction, following heart ischemia and reperfusion, is associated with profound changes in key processes of intercellular communication, involving gap junctions, extracellular vesicles, and tunneling nanotubes, some of which have been implicated in communication defects associated with cardiac injury, namely arrhythmogenesis and progression into heart failure. Therefore, intercellular communication players have emerged as attractive powerful therapeutic targets aimed at preserving a fine-tuned crosstalk between the different cardiac cells in order to prevent or repair some of harmful consequences of heart ischemia and reperfusion, re-establishing myocardial function

MicroRNAs in the stressed heart: Sorting the signal from the noise

The short noncoding RNAs, known as microRNAs, are of undisputed importance in cellular signaling during differentiation and development, and during adaptive and maladaptive responses of adult tissues, including those that comprise the heart. Cardiac microRNAs are regulated by hemodynamic overload resulting from exercise or hypertension, in the response of surviving myocardium to myocardial infarction, and in response to environmental or systemic disruptions to homeostasis, such as those arising from diabetes. A large body of work has explored microRNA responses in both physiological and pathological contexts but there is still much to learn about their integrated actions on individual mRNAs and signaling pathways. This review will highlight key studies of microRNA regulation in cardiac stress and suggest possible approaches for more precise identification of microRNA targets, with a view to exploiting the resulting data for therapeutic purposes

MicroRNA interactome analysis predicts post-transcriptional regulation of ADRB2 and PPP3R1 in the hypercholesterolemic myocardium

Little is known about the molecular mechanism including microRNAs (miRNA) in hypercholesterolemia-induced cardiac dysfunction. We aimed to explore novel hypercholesterolemia-induced pathway alterations in the heart by an unbiased approach based on miRNA omics, target prediction and validation. With miRNA microarray we identified forty-seven upregulated and ten downregulated miRNAs in hypercholesterolemic rat hearts compared to the normocholesterolemic group. Eleven mRNAs with at least 4 interacting upregulated miRNAs were selected by a network theoretical approach, out of which 3 mRNAs (beta-2 adrenergic receptor [Adrb2], calcineurin B type 1 [Ppp3r1] and calcium/calmodulin-dependent serine protein kinase [Cask]) were validated with qRT-PCR and Western blot. In hypercholesterolemic hearts, the expression of Adrb2 mRNA was significantly decreased. ADRB2 and PPP3R1 protein were significantly downregulated in hypercholesterolemic hearts. The direct interaction of Adrb2 with upregulated miRNAs was demonstrated by luciferase reporter assay. Gene ontology analysis revealed that the majority of the predicted mRNA changes may contribute to the hypercholesterolemia-induced cardiac dysfunction. In summary, the present unbiased target prediction approach based on global cardiac miRNA expression profiling revealed for the first time in the literature that both the mRNA and protein product of Adrb2 and PPP3R1 protein are decreased in the hypercholesterolemic heart

Systems Modeling to Predict Mechano-Chemo Interactions In Cardiac Fibrosis

Cardiac fibrosis poses a central challenge in preventing heart failure for patients who have suffered a cardiac injury such as myocardial infarction or aortic valve stenosis. This chronic condition is characterized by a reduction in contractile function through combined hypertrophy and excessive scar formation, and although currently prescribed therapeutics targeting hypertrophy have shown improvements in patient outcomes, pathological fibrosis remains a leading cause of reduced cardiac function for patients long-term. Cardiac fibroblasts play a key role in regulating scar formation during heart failure progression, and interacting biochemical and biomechanical cues within the myocardium guide the activation of fibroblasts and expression of extracellular matrix proteins. While targeted experimental studies of fibroblast activation have elucidated the roles of individual pathways in fibroblast activation, intracellular crosstalk between mechanotransduction and chemotransduction pathways from multiple biochemical cues has largely confounded efforts to control overall cell behavior within the myocardial environment. Computational networks of intracellular signaling can account for complex interactions between signaling pathways and provide a promising approach for identifying influential mechanisms mediating cell behavior. The overarching goal of this dissertation is to improve our understanding of complex signaling in fibroblasts by investigating the role of mechano-chemo interactions in cardiac fibroblast-mediated fibrosis using a combination of experimental studies and systems-level computational models. Firstly, using an in vitro screen of cardiac fibroblast-secreted proteins in response to combinations of biochemical stimuli and mechanical tension, we found that tension modulated cell sensitivity towards biochemical stimuli, thereby altering cell behavior based on the mechanical context. Secondly, using a curated model of fibroblast intracellular signaling, we expanded model topology to include robust mechanotransduction pathways, improved accuracy of model predictions compared to independent experimental studies, and identified mechanically dependent mechanisms-of- ction and mechano-adaptive drug candidates in a post-infarction scenario. Lastly, using an inferred network of fibroblast transcriptional regulation and model fitting to patient-specific data, we showed the utility of model-based approaches in identifying influential pathways underlying fibrotic protein expression during aortic valve stenosis and predicting patient-specific responses to pharmacological intervention. Our work suggests that computational-based approaches can generate insight into influential mechanisms among complex systems, and such tools may be promising for further therapeutic development and precision medicine

Deep Learning Analyses to Delineate the Molecular Remodeling Process after Myocardial Infarction

Specific proteins and processes have been identified in post-myocardial infarction (MI) pathological remodeling, but a comprehensive understanding of the complete molecular evolution is lacking. We generated microarray data from swine heart biopsies at baseline and 6, 30, and 45 days after infarction to feed machine-learning algorithms. We cross-validated the results using available clinical and experimental information. MI progression was accompanied by the regulation of adipogenesis, fatty acid metabolism, and epithelial-mesenchymal transition. The infarct core region was enriched in processes related to muscle contraction and membrane depolarization. Angiogenesis was among the first morphogenic responses detected as being sustained over time, but other processes suggesting post-ischemic recapitulation of embryogenic processes were also observed. Finally, protein-triggering analysis established the key genes mediating each process at each time point, as well as the complete adverse remodeling response. We modeled the behaviors of these genes, generating a description of the integrative mechanism of action for MI progression. This mechanistic analysis overlapped at different time points; the common pathways between the source proteins and cardiac remodeling involved IGF1R, RAF1, KPCA, JUN, and PTN11 as modulators. Thus, our data delineate a structured and comprehensive picture of the molecular remodeling process, identify new potential biomarkers or therapeutic targets, and establish therapeutic windows during disease progression

Drug-target network in myocardial infarction reveals multiple side effects of unrelated drugs

The systems-level characterization of drug-target associations in myocardial infarction (MI) has not been reported to date. We report a computational approach that combines different sources of drug and protein interaction information to assemble the myocardial infarction drug-target interactome network (My-DTome). My-DTome comprises approved and other drugs interlinked in a single, highly-connected network with modular organization. We show that approved and other drugs may both be highly connected and represent network bottlenecks. This highlights influential roles for such drugs on seemingly unrelated targets and pathways via direct and indirect interactions. My-DTome modules are associated with relevant molecular processes and pathways. We find evidence that these modules may be regulated by microRNAs with potential therapeutic roles in MI. Different drugs can jointly impact a module. We provide systemic insights into cardiovascular effects of non-cardiovascular drugs. My-DTome provides the basis for an alternative approach to investigate new targets and multidrug treatment in MI

Deep Learning Analyses to Delineate the Molecular Remodeling Process after Myocardial Infarction

Specific proteins and processes have been identified in post-myocardial infarction (MI) pathological remodeling, but a comprehensive understanding of the complete molecular evolution is lacking. We generated microarray data from swine heart biopsies at baseline and 6, 30, and 45 days after infarction to feed machine-learning algorithms. We cross-validated the results using available clinical and experimental information. MI progression was accompanied by the regulation of adipogenesis, fatty acid metabolism, and epithelial-mesenchymal transition. The infarct core region was enriched in processes related to muscle contraction and membrane depolarization. Angiogenesis was among the first morphogenic responses detected as being sustained over time, but other processes suggesting post-ischemic recapitulation of embryogenic processes were also observed. Finally, protein-triggering analysis established the key genes mediating each process at each time point, as well as the complete adverse remodeling response. We modeled the behaviors of these genes, generating a description of the integrative mechanism of action for MI progression. This mechanistic analysis overlapped at different time points; the common pathways between the source proteins and cardiac remodeling involved IGF1R, RAF1, KPCA, JUN, and PTN11 as modulators. Thus, our data delineate a structured and comprehensive picture of the molecular remodeling process, identify new potential biomarkers or therapeutic targets, and establish therapeutic windows during disease progression

Immunomodulatory interventions in myocardial infarction and heart failure: a systematic review of clinical trials and meta-analysis of IL-1 inhibition

Following a myocardial infarction (MI), the immune system helps to repair ischaemic damage and restore tissue integrity, but excessive inflammation has been implicated in adverse cardiac remodelling and development towards heart failure (HF). Pre-clinical studies suggest that timely resolution of inflammation may help prevent HF development and progression. Therapeutic attempts to prevent excessive post-MI inflammation in patients have included pharmacological interventions ranging from broad immunosuppression to immunomodulatory approaches targeting specific cell types or factors with the aim to maintain beneficial aspects of the early post-MI immune response. These include the blockade of early initiators of inflammation including reactive oxygen species and complement, inhibition of mast cell degranulation and leucocyte infiltration, blockade of inflammatory cytokines, and inhibition of adaptive B and T-lymphocytes. Herein, we provide a systematic review on post-MI immunomodulation trials and a meta-analysis of studies targeting the inflammatory cytokine Interleukin-1. Despite an enormous effort into a significant number of clinical trials on a variety of targets, a striking heterogeneity in study population, timing and type of treatment, and highly variable endpoints limits the possibility for meaningful meta-analyses. To conclude, we highlight critical considerations for future studies including (i) the therapeutic window of opportunity, (ii) immunological effects of routine post-MI medication, (iii) stratification of the highly diverse post-MI patient population, (iv) the potential benefits of combining immunomodulatory with regenerative therapies, and at last (v) the potential side effects of immunotherapies

Competing Endogenous RNAs, Non-Coding RNAs and Diseases: An Intertwined Story

MicroRNAs (miRNAs), a class of small non-coding RNA molecules, are responsible for RNA silencing and post-transcriptional regulation of gene expression. They can mediate a fine-tuned crosstalk among coding and non-coding RNA molecules sharing miRNA response elements (MREs). In a suitable environment, both coding and non-coding RNA molecules can be targeted by the same miRNAs and can indirectly regulate each other by competing for them. These RNAs, otherwise known as competing endogenous RNAs (ceRNAs), lead to an additional post-transcriptional regulatory layer, where non-coding RNAs can find new significance. The miRNA-mediated interplay among different types of RNA molecules has been observed in many different contexts. The analyses of ceRNA networks in cancer and other pathologies, as well as in other physiological conditions, provide new opportunities for interpreting omics data for the field of personalized medicine. The development of novel computational tools, providing putative predictions of ceRNA interactions, is a rapidly growing field of interest. In this review, I discuss and present the current knowledge of the ceRNA mechanism and its implications in a broad spectrum of different pathologies, such as cardiovascular or autoimmune diseases, cancers and neurodegenerative disorders

Regulation of Cardiomyocyte Proliferation by microRNAs and Small Molecules

Understanding the molecular mechanisms regulating cardiac cell proliferation during the embryonic, fetal and adult life holds a paramount importance in view of developing innovative strategies aimed at inducing myocardial regeneration after cardiac damage. Previous high throughput screening studies in our laboratory identified a series of microRNAs able to trigger cardiomyocyte proliferation and stimulate cardiac regeneration after myocardial infarction. In the first part of this project, we investigated the mechanism of action of the top ten most effective of these miRNAs, revealing an involvement of the Hippo-YAP pathway in their action. We found that all the investigated miRNAs activated YAP-mediated transcription, nuclear localization of active YAP and increased expression of YAP responsive genes. Of notice, miR-199a-3p, one of the most effective miRNAs exerted its direct effect on two mRNA targets impinging on the Hippo pathway, the inhibitory kinase TAOK1 and the E3 ubiquitin ligase, β−TrCP. Most of the miRNAs inducing proliferation (including miR-199a-3p) also modulated the dynamics of the actin cytoskeleton in the treated cardiomyocytes, which displayed a rounded shape and gross bundles of actin fibers at the cytoplasm periphery. Consistent with these observations, we found that the Cofilin2 mRNA was a direct target of four of the investigated miRNAs and that downregulation of Cofilin2 itself was sufficient to promote cardiomyocyte proliferation, activate nuclear translocation of YAP and stimulate transcription of TEAD-responsive genes. The second part of the project was aimed at identifying small molecules exerting a mitogenic effect on neonatal cardiomyocytes through an unbiased high-throughput screening (HTS) of a library of 780 FDA-registered drugs. The neuroactive alkaloid harmine was identified as the most powerful molecule at inducing cardiomyocyte proliferation in vitro and heart regeneration after myocardial infarction in vivo. Harmine exerted its activity through the inhibition of the dual specificity phosphorylation-regulated tyrosine kinase, Dyrk1a and, again, the activation of YAP nuclear translocation. Collectively, these results identify both YAP activation and actin cytoskeleton remodelling as major determinants of cardiomyocyte proliferation and establish the molecular basis for the development of pharmacological therapies to promote heart regeneration through the stimulation of the endogenous capacity of cardiomyocytes to proliferate