Nongenetic Optical Modulation of Pluripotent Stem Cells Derived Cardiomyocytes Function in the Red Spectral Range.

Optical stimulation in the red/near infrared range recently gained increasing interest, as a not-invasive tool to control cardiac cell activity and repair in disease conditions. Translation of this approach to therapy is hampered by scarce efficacy and selectivity. The use of smart biocompatible materials, capable to act as local, NIR-sensitive interfaces with cardiac cells, may represent a valuable solution, capable to overcome these limitations. In this work, a far red-responsive conjugated polymer, namely poly[2,1,3-benzothiadiazole-4,7-diyl[4,4-bis(2-ethylhexyl)-4H-cyclopenta[2,1-b:3,4-b']dithiophene-2,6-diyl]] (PCPDTBT) is proposed for the realization of photoactive interfaces with cardiomyocytes derived from pluripotent stem cells (hPSC-CMs). Optical excitation of the polymer turns into effective ionic and electrical modulation of hPSC-CMs, in particular by fastening Ca2+ dynamics, inducing action potential shortening, accelerating the spontaneous beating frequency. The involvement in the phototransduction pathway of Sarco-Endoplasmic Reticulum Calcium ATPase (SERCA) and Na+ /Ca2+ exchanger (NCX) is proven by pharmacological assays and is correlated with physical/chemical processes occurring at the polymer surface upon photoexcitation. Very interestingly, an antiarrhythmogenic effect, unequivocally triggered by polymer photoexcitation, is also observed. Overall, red-light excitation of conjugated polymers may represent an unprecedented opportunity for fine control of hPSC-CMs functionality and can be considered as a perspective, noninvasive approach to treat arrhythmias.

Myocardial hypoxic stress mediates functional cardiac extracellular vesicle release.

AIMS: Increased shedding of extracellular vesicles (EVs)-small, lipid bilayer-delimited particles with a role in paracrine signalling-has been associated with human pathologies, e.g. atherosclerosis, but whether this is true for cardiac diseases is unknown. METHODS AND RESULTS: Here, we used the surface antigen CD172a as a specific marker of cardiomyocyte (CM)-derived EVs; the CM origin of CD172a+ EVs was supported by their content of cardiac-specific proteins and heart-enriched microRNAs. We found that patients with aortic stenosis, ischaemic heart disease, or cardiomyopathy had higher circulating CD172a+ cardiac EV counts than did healthy subjects. Cellular stress was a major determinant of EV release from CMs, with hypoxia increasing shedding in in vitro and in vivo experiments. At the functional level, EVs isolated from the supernatant of CMs derived from human-induced pluripotent stem cells and cultured in a hypoxic atmosphere elicited a positive inotropic response in unstressed CMs, an effect we found to be dependent on an increase in the number of EVs expressing ceramide on their surface. Of potential clinical relevance, aortic stenosis patients with the highest counts of circulating cardiac CD172a+ EVs had a more favourable prognosis for transcatheter aortic valve replacement than those with lower counts. CONCLUSION: We identified circulating CD172a+ EVs as cardiac derived, showing their release and function and providing evidence for their prognostic potential in aortic stenosis patients.

Toward Cardiac Regeneration: Combination of Pluripotent Stem Cell-Based Therapies and Bioengineering Strategies.

Cardiovascular diseases represent the major cause of morbidity and mortality worldwide. Multiple studies have been conducted so far in order to develop treatments able to prevent the progression of these pathologies. Despite progress made in the last decade, current therapies are still hampered by poor translation into actual clinical applications. The major drawback of such strategies is represented by the limited regenerative capacity of the cardiac tissue. Indeed, after an ischaemic insult, the formation of fibrotic scar takes place, interfering with mechanical and electrical functions of the heart. Hence, the ability of the heart to recover after ischaemic injury depends on several molecular and cellular pathways, and the imbalance between them results into adverse remodeling, culminating in heart failure. In this complex scenario, a new chapter of regenerative medicine has been opened over the past 20 years with the discovery of induced pluripotent stem cells (iPSCs). These cells share the same characteristic of embryonic stem cells (ESCs), but are generated from patient-specific somatic cells, overcoming the ethical limitations related to ESC use and providing an autologous source of human cells. Similarly to ESCs, iPSCs are able to efficiently differentiate into cardiomyocytes (CMs), and thus hold a real regenerative potential for future clinical applications. However, cell-based therapies are subjected to poor grafting and may cause adverse effects in the failing heart. Thus, over the last years, bioengineering technologies focused their attention on the improvement of both survival and functionality of iPSC-derived CMs. The combination of these two fields of study has burst the development of cell-based three-dimensional (3D) structures and organoids which mimic, more realistically, the in vivo cell behavior. Toward the same path, the possibility to directly induce conversion of fibroblasts into CMs has recently emerged as a promising area for in situ cardiac regeneration. In this review we provide an up-to-date overview of the latest advancements in the application of pluripotent stem cells and tissue-engineering for therapeutically relevant cardiac regenerative approaches, aiming to highlight outcomes, limitations and future perspectives for their clinical translation.

Subchronic exposure to titanium dioxide nanoparticles modifies cardiac structure and performance in spontaneously hypertensive rats.

BACKGROUND: Non-communicable diseases, intended as the results of a combination of inherited, environmental and biological factors, kill 40 million people each year, equivalent to roughly 70% of all premature deaths globally. The possibility that manufactured nanoparticles (NPs) may affect cardiac performance, has led to recognize NPs-exposure not only as a major Public Health concern, but also as an occupational hazard. In volunteers, NPs-exposure is problematic to quantify. We recently found that inhaled titanium dioxide NPs, one of the most produced engineered nanomaterials, acutely increased cardiac excitability and promoted arrhythmogenesis in normotensive rats by a direct interaction with cardiac cells. We hypothesized that such scenario can be exacerbated by latent cardiovascular disorders such as hypertension. RESULTS: We monitored cardiac electromechanical performance in spontaneously hypertensive rats (SHRs) exposed to titanium dioxide NPs for 6 weeks using a combination of cardiac functional measurements associated with toxicological, immunological, physical and genetic assays. Longitudinal radio-telemetry ECG recordings and multiple-lead epicardial potential mapping revealed that atrial activation times significantly increased as well as proneness to arrhythmia. At the third week of nanoparticles administration, the lung and cardiac tissue encountered a maladaptive irreversible structural remodelling starting with increased pro-inflammatory cytokines levels and lipid peroxidation, resulting in upregulation of the main pro-fibrotic cardiac genes. At the end of the exposure, the majority of spontaneous arrhythmic events terminated, while cardiac hemodynamic deteriorated and a significant accumulation of fibrotic tissue occurred as compared to control untreated SHRs. Titanium dioxide nanoparticles were quantified in the heart tissue although without definite accumulation as revealed by particle-induced X-ray emission and ultrastructural analysis. CONCLUSIONS: The co-morbidity of hypertension and inhaled nanoparticles induces irreversible hemodynamic impairment associated with cardiac structural damage potentially leading to heart failure. The time-dependence of exposure indicates a non-return point that needs to be taken into account in hypertensive subjects daily exposed to nanoparticles.

An autofluorescence-based method for the isolation of highly purified ventricular cardiomyocytes.

AIMS: The aim of our study was to set up a simple and reliable isolation method of living ventricular cardiomyocytes (vCMs) for molecular and biological studies. METHODS AND RESULTS: A standard technique for the retrograde perfusion of an enzymatic solution was used to isolate cardiac cells from adult mouse heart. Fluorescence-activated cell sorting (FACS) on adult murine cardiac ventricle cells was performed, comparing the intrinsic autofluorescence in the FITC channel and the forward scatter (FSC) parameter in order to isolate highly fluorescent cells. The expression of cell-specific mRNAs was assessed with real-time PCR in cells sorted on the basis of their FITC and FSC characteristics. We identified two distinct subpopulations of cells harvested after retrograde perfusion of wild-type heart: FITChigh/FSCdim and FITCdim/FSChigh. Immunophenotyping and mRNA analysis (qPCR and RNA sequencing) revealed that only FITChigh/FSCdim cells were highly enriched in CM markers. Genes with high expression in endothelial cells and fibroblasts were enriched in the FITCdim/FSChigh subpopulation. With the use of tdTomatofl/fl-α-myosin heavy chain MerCreMer+/-mouse heart, we found that tdTomato-positive vCMs were present in the FITChigh/FSCdim region but were only rare in the FITCdim/FSChigh fraction. CONCLUSION: We have developed a simple and reliable method for the isolation of highly purified vCMs from the adult murine myocardium, avoiding fixation and permeabilization steps. These isolated vCMs can be used in particular for detailed molecular studies, avoiding contamination with other myocardial cell types.

Growth hormone-releasing hormone attenuates cardiac hypertrophy and improves heart function in pressure overload-induced heart failure.

It has been shown that growth hormone-releasing hormone (GHRH) reduces cardiomyocyte (CM) apoptosis, prevents ischemia/reperfusion injury, and improves cardiac function in ischemic rat hearts. However, it is still not known whether GHRH would be beneficial for life-threatening pathological conditions, like cardiac hypertrophy and heart failure (HF). Thus, we tested the myocardial therapeutic potential of GHRH stimulation in vitro and in vivo, using GHRH or its agonistic analog MR-409. We show that in vitro, GHRH(1-44)NH2 attenuates phenylephrine-induced hypertrophy in H9c2 cardiac cells, adult rat ventricular myocytes, and human induced pluripotent stem cell-derived CMs, decreasing expression of hypertrophic genes and regulating hypertrophic pathways. Underlying mechanisms included blockade of Gq signaling and its downstream components phospholipase Cß, protein kinase Cε, calcineurin, and phospholamban. The receptor-dependent effects of GHRH also involved activation of Gαs and cAMP/PKA, and inhibition of increase in exchange protein directly activated by cAMP1 (Epac1). In vivo, MR-409 mitigated cardiac hypertrophy in mice subjected to transverse aortic constriction and improved cardiac function. Moreover, CMs isolated from transverse aortic constriction mice treated with MR-409 showed improved contractility and reversal of sarcolemmal structure. Overall, these results identify GHRH as an antihypertrophic regulator, underlying its therapeutic potential for HF, and suggest possible beneficial use of its analogs for treatment of pathological cardiac hypertrophy.

Histone Methyltransferase G9a Is Required for Cardiomyocyte Homeostasis and Hypertrophy.

BACKGROUND: Correct gene expression programming of the cardiomyocyte underlies the normal functioning of the heart. Alterations to this can lead to the loss of cardiac homeostasis, triggering heart dysfunction. Although the role of some histone methyltransferases in establishing the transcriptional program of postnatal cardiomyocytes during heart development has been shown, the function of this class of epigenetic enzymes is largely unexplored in the adult heart. In this study, we investigated the role of G9a/Ehmt2, a histone methyltransferase that defines a repressive epigenetic signature, in defining the transcriptional program for cardiomyocyte homeostasis and cardiac hypertrophy. METHODS: We investigated the function of G9a in normal and stressed cardiomyocytes with the use of a conditional, cardiac-specific G9a knockout mouse, a specific G9a inhibitor, and high-throughput approaches for the study of the epigenome (chromatin immunoprecipitation sequencing) and transcriptome (RNA sequencing); traditional methods were used to assess cardiac function and cardiovascular disease. RESULTS: We found that G9a is required for cardiomyocyte homeostasis in the adult heart by mediating the repression of key genes regulating cardiomyocyte function via dimethylation of H3 lysine 9 and interaction with enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2, and MEF2C-dependent gene expression by forming a complex with this transcription factor. The G9a-MEF2C complex was found to be required also for the maintenance of heterochromatin needed for the silencing of developmental genes in the adult heart. Moreover, G9a promoted cardiac hypertrophy by repressing antihypertrophic genes. CONCLUSIONS: Taken together, our findings demonstrate that G9a orchestrates critical epigenetic changes in cardiomyocytes in physiological and pathological conditions, thereby providing novel therapeutic avenues for cardiac pathologies associated with dysregulation of these mechanisms.