Arrhythmogenic cardiomyopathy (ACM) is a genetic disorder mainly due to mutations in desmosomal genes, characterized by progressive fibro-adipose replacement of the myocardium, arrhythmias, and sudden death. It is still unclear which cell type is responsible for fibro-adipose substitution and which molecular mechanisms lead to this structural change. Cardiac mesenchymal stromal cells (C-MSC) are the most abundant cells in the heart, with propensity to differentiate into several cell types, including adipocytes, and their role in ACM is unknown. The aim of the present study was to investigate whether C-MSC contributed to excess adipocytes in patients with ACM. We found that, in ACM patients' explanted heart sections, cells actively differentiating into adipocytes are of mesenchymal origin. Therefore, we isolated C-MSC from endomyocardial biopsies of ACM and from not affected by arrhythmogenic cardiomyopathy (NON-ACM) (control) patients. We found that both ACM and control C-MSC express desmosomal genes, with ACM C-MSC showing lower expression of plakophilin (PKP2) protein vs. controls. Arrhythmogenic cardiomyopathy C-MSC cultured in adipogenic medium accumulated more lipid droplets than controls. Accordingly, the expression of adipogenic genes was higher in ACM vs. NON-ACM C-MSC, while expression of cell cycle and anti-adipogenic genes was lower. Both lipid accumulation and transcription reprogramming were dependent on PKP2 deficiency. Cardiac mesenchymal stromal cells contribute to the adipogenic substitution observed in ACM patients' hearts. Moreover, C-MSC from ACM patients recapitulate the features of ACM adipogenesis, representing a novel, scalable, patient-specific in vitro tool for future mechanistic studies.

The heart contains c-kit⁺ progenitors that maintain cardiac homeostasis. Cardiac c-kit⁺ cells are multipotent and give rise to myocardial, endothelial and smooth muscle cells, both in vitro and in vivo. C-kit⁺ cells have been thoroughly investigated for their stem cell activity, susceptibility to stress conditions and ageing, as well as for their ability to repair the infarcted heart. Recently, expression of mesenchymal stem cell (MSC) markers and MSC differentiation potency have been reported in cardiac progenitor cells. Based on this evidence, we hypothesized that c-kit⁺ cells may have phenotypic and functional features in common with cardiac MSCs. Culture of cells obtained from enzymatic dissociation of heart auricle fragments produced a fast-growing fibroblast-like population expressing mesenchymal markers. C-kit⁺ cells co-expressing MSC markers were identified in this population, sorted by flow cytometry and cultured in the presence or the absence of unselected cardiac cells from the same patients. Subsets of c-kit⁺ cells also co-expressed MSCs markers in vivo, as detected by immunofluorescence analysis of auricle tissue. Ex vivo expanded c-kit⁺ cells produced osteoblasts and adipocytes, although less preferentially than bone marrow-derived MSCs, possessed vascular smooth muscle cell features and were induced to differentiate into endothelium-like and cardiac-like cells. In line with previous findings, our results indicate that c-kit⁺ cardiac progenitors are primitive stem cells endowed with multilineage differentiation ability. They further suggest a possible relationship between these cells and a heart-specific MSC population with cardiovascular commitment potential.

The anthracycline doxorubicin (Dox) is widely used in oncology, but it may cause a cardiomyopathy with bleak prognosis that cannot be effectively prevented. The secretome of human amniotic fluid-derived stem cells (hAFS) has previously been demonstrated to significantly reduce ischemic cardiac damage. Here it is shown that, following hypoxic preconditioning, hAFS conditioned medium (hAFS-CM) antagonizes senescence and apoptosis of cardiomyocytes and cardiac progenitor cells, two major features of Dox cardiotoxicity. Mechanistic studies with mouse neonatal ventricular cardiomyocytes (mNVCM) reveal that hAFS-CM inhibition of Dox-elicited senescence and apoptosis is associated with decreased DNA damage, nuclear translocation of NF-kB and upregulation of the NF-kB controlled genes,Il6andCxcl1, promoting mNVCM survival. Furthermore, hAFS-CM induces expression of the efflux transporter,Abcb1band Dox extrusion from mNVCM. The PI3K/Akt signaling cascade, upstream of NF-kB, is potently activated by hAFS-CM and pre-treatment with a PI3K inhibitor abrogates NF-kB accumulation into the nucleus, modulation ofIl6,Cxcl1andAbcb1band prevention of Dox-initiated senescence and apoptosis in response to hAFS-CM. These results support the concept that hAFS are a valuable source of cardioprotective factors and lay the foundations for the development of a stem cell-based paracrine treatment of chemotherapy-related cardiotoxicity.

Doxorubicin (DOXO) treatment is limited by its cardiotoxicity, since it causes cardiac-progenitor-cell depletion. Although the cardioprotective role of the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF1/CXCR4) axis is well established, its involvement during DOXO-induced cardiotoxicity has never been investigated. We showed that in a mouse model of DOXO-induced cardiomyopathy, CXCR4⁺ cells were increased in response to DOXO, mainly in human cardiac mesenchymal progenitor cells (CmPC), a subpopulation with regenerative potential. Our in vitro results showed a CXCR4 induction after 24 h of DOXO exposure in CmPC. SDF1 administration protected from DOXO-induced cell death and promoted CmPC migration. CXCR4 promoter analysis revealed zinc finger E-box binding homeobox 1 (ZEB1) binding sites. Upon DOXO treatment, ZEB1 binding decreased and RNA-polymerase-II increased, suggesting a DOXO-mediated transcriptional increase in CXCR4. Indeed, DOXO induced the upregulation of miR-200c, that directly targets ZEB1. SDF1 administration in DOXO-treated mice partially reverted the adverse remodeling, decreasing left ventricular (LV) end diastolic volume, LV ejection fraction and LV anterior wall thickness in diastole, recovering LV end systolic pressure and reducing±dP/dt. Moreover, in vivo administration of SDF1 partially reverted DOXO-induced miR-200c and p53 protein upregulation in mouse hearts. In addition, downmodulation of ZEB1 mRNA and protein by DOXO was significantly increased by SDF1. In keeping, p21 mRNA, that is induced by p53 and inhibited by ZEB1, is induced by DOXO treatment and is decreased by SDF1 administration. This study showed new players of the DOXO-induced cardiotoxicity, that can be exploited to ameliorate DOXO-associated cardiomyopathy.

The Pall Celeris system is a filtration-based point-of-care device designed to obtain a high concentrate of peripheral blood total nucleated cells (PB-TNCs). We have characterized the Pall Celeris-derived TNCs for their in vitro and in vivo angiogenic potency. PB-TNCs isolated from healthy donors were characterized through the use of flow cytometry and functional assays, aiming to assess migratory capacity, ability to form capillary-like structures, endothelial trans-differentiation and paracrine factor secretion. In a hind limb ischemia mouse model, we evaluated perfusion immediately and 7 days after surgery, along with capillary, arteriole and regenerative fiber density and local bio-distribution. Human PB-TNCs isolated by use of the Pall Celeris filtration system were shown to secrete a panel of angiogenic factors and migrate in response to vascular endothelial growth factor and stromal-derived factor-1 stimuli. Moreover, after injection in a mouse model of hind limb ischemia, PB-TNCs induced neovascularization by increasing capillary, arteriole and regenerative fiber numbers, with human cells detected in murine tissue up to 7 days after ischemia. The Pall Celeris system may represent a novel, effective and reliable point-of-care device to obtain a PB-derived cell product with adequate potency for therapeutic angiogenesis.

Use of peripheral blood- or bone marrow-derived progenitors for ischemic heart repair is a feasible option to induce neo-vascularization in ischemic tissues. These cells, named Endothelial Progenitors Cells (EPCs), have been extensively characterized phenotypically and functionally. The clinical efficacy of cardiac repair by EPCs cells remains, however, limited, due to cell autonomous defects as a consequence of risk factors. The devise of “enhancement” strategies has been therefore sought to improve repair ability of these cells and increase the clinical benefit. Pharmacologic inhibition of histone deacetylases (HDACs) is known to enhance hematopoietic stem cells engraftment by improvement of self renewal and inhibition of differentiation in the presence of mitogenic stimuli in vitro. In the present study cord blood-derived CD34⁺ were pre-conditioned with the HDAC inhibitor Valproic Acid. This treatment affected stem cell growth and gene expression, and improved ischemic myocardium protection in an immunodeficient mouse model of myocardial infarction. Our results show that HDAC blockade leads to phenotype changes in CD34⁺ cells with enhanced self renewal and cardioprotection.