MiR-133a and miR-1 are known as muscle-specific microRNAs that are involved in cardiac development and pathophysiology. We have shown that both miR-1 and miR-133a are early and progressively upregulated during in vitro cardiac differentiation of adult cardiac progenitor cells (CPCs), but only miR-133a expression was enhanced under in vitro oxidative stress. miR-1 was demonstrated to favor differentiation of CPCs, whereas miR-133a overexpression protected CPCs against cell death, targeting, among others, the proapoptotic genes Bim and Bmf. miR-133a-CPCs clearly improved cardiac function in a rat myocardial infarction model by reducing fibrosis and hypertrophy and increasing vascularization and cardiomyocyte proliferation. The beneficial effects of miR-133a-CPCs seem to correlate with the upregulated expression of several relevant paracrine factors and the plausible cooperative secretion of miR-133a via exosomal transport. Finally, an in vitro heart muscle model confirmed the antiapoptotic effects of miR-133a-CPCs, favoring the structuration and contractile functionality of the artificial tissue.

Background: The inability of the adult mammalian heart to replace cells lost after severe cardiac injury compromises organ function. Although the heart is one of the least regenerative organs in the body, evidence accumulated in recent decades indicates a certain degree of renewal after injury. We have evaluated the role of cardiac Bmi1 ⁺ progenitor cells (Bmi1-CPC) following acute myocardial infarction (AMI). Methods: Bmi1^Cre/+;Rosa26^YFP/+ (Bmi1-YFP) mice were used for lineage tracing strategy. After tamoxifen (TM) induction, yellow fluorescent protein (YFP) is expressed under the control of Rosa26 regulatory sequences in Bmi1 ⁺ cells. YFP⁺ cells were tracked following myocardial infarction. Additionally, whole transcriptome analysis of isolated YFP⁺ cells was performed in unchallenged hearts and after myocardial infarction. Results: Deep-sequencing analysis of Bmi1-CPC from unchallenged hearts suggests that this population expresses high levels of pluripotency markers. Conversely, transcriptome evaluation of Bmi1-CPC following AMI shows a rich representation of genes related to cell proliferation, movement, and cell cycle. Lineage-tracing studies after cardiac infarction show that the progeny of Bmi1-expressing cells contribute to de novo cardiomyocytes (CM) (13.8 ± 5 % new YFP⁺ CM compared to 4.7 ± 0.9 % in age-paired non-infarcted hearts). However, apical resection of TM-induced day 1 Bmi1-YFP pups indicated a very minor contribution of Bmi1-derived cells to de novo CM. Conclusions: Cardiac Bmi1 progenitor cells respond to cardiac injury, contributing to the generation of de novo CM in the adult mouse heart.

Background: Insulin-like growth factor 1 (IGF-1) and hepatocyte growth factor (HGF) are among the most promising growth factors for promoting cardiorepair. Here, we evaluated the combination of cell- and gene-based therapy using mesenchymal stem cells (MSC) genetically modified to overexpress IGF-1 or HGF to treat acute myocardial infarction (AMI) in a porcine model. Methods: Pig MSC from adipose tissue (paMSC) were genetically modified for evaluation of different therapeutic strategies to improve AMI treatment. Three groups of infarcted Large White pigs were compared (I, control, non-transplanted; II, transplanted with paMSC-GFP (green fluorescent protein); III, transplanted with paMSC-IGF-1/HGF). Cardiac function was evaluated non-invasively using magnetic resonance imaging (MRI) for 1 month. After euthanasia and sampling of the animal, infarcted areas were studied by histology and immunohistochemistry. Results: Intramyocardial transplant in a porcine infarct model demonstrated the safety of paMSC in short-term treatments. Treatment with paMSC-IGF-1/HGF (1:1) compared with the other groups showed a clear reduction in inflammation in some sections analyzed and promoted angiogenic processes in ischemic tissue. Although cardiac function parameters were not significantly improved, cell retention and IGF-1 overexpression was confirmed within the myocardium. Conclusions: The simultaneous administration of IGF-1- and HGF-overexpressing paMSC appears not to promote a synergistic effect or effective repair. The combined enhancement of neovascularization and fibrosis in paMSC-IGF-1/HGF-treated animals nonetheless suggests that sustained exposure to high IGF-1 + HGF levels promotes beneficial as well as deleterious effects that do not improve overall cardiac regeneration.

Studies in recent years have established that the principal effects in cardiac cell therapy are associated with paracrine/autocrine factors. We combined several complementary techniques to define human cardiac progenitor cell (CPC) secretome constituted by 914 proteins/genes; 51% of these are associated with the exosomal compartment. To define the set of proteins specifically or highly differentially secreted by CPC, we compared human mesenchymal stem cells and dermal fibroblasts; the study defined a group of growth factors, cytokines and chemokines expressed at high to medium levels by CPC. Among them, IL-1, GROa (CXCL1), CXCL6 (GCP2) and IL-8 are examples whose expression was confirmed by most techniques used. ELISA showed that CXCL6 is significantly overexpressed in CPC conditioned medium (CM) (18- to 26-fold) and western blot confirmed expression of its receptors CXCR1 and CXCR2. Addition of anti-CXCL6 completely abolished migration in CPC-CM compared with anti-CXCR2, which promoted partial inhibition, and anti-CXCR1, which was inefficient. Anti-CXCL6 also significantly inhibited CPC CM angiogenic activity. In vivo evaluation also supported a relevant role for angiogenesis. Altogether, these results suggest a notable angiogenic potential in CPC-CM and identify CXCL6 as an important paracrine factor for CPC that signals mainly through CXCR2.

Accumulation of reactive oxygen species (ROS) is associated with several cardiovascular pathologies and with cell cycle exit by neonanatal cardiomyocytes, a key limiting factor in the regenerative capacity of the adult mammalian heart. The polycomb complex component BMI1 is linked to adult progenitors and is an important partner in DNA repair and redox regulation. We found that high BMI1 expression is associated with an adult Sca1⁺ cardiac progenitor sub-population with low ROS levels. In homeostasis, BMI1 repressed cell fate genes, including a cardiogenic differentiation program. Oxidative damage nonetheless modified BMI1 activity in vivo by derepressing canonical target genes in favor of their antioxidant and anticlastogenic functions. This redox-mediated mechanism is not restricted to damage situations, however, and we report ROS-associated differentiation of cardiac progenitors in steady state. These findings demonstrate how redox status influences the cardiac progenitor response, and identify redox-mediated BMI1 regulation with implications in maintenance of cellular identity in vivo.