0128 : Sca-1 positive cells, but not c-kit positive cells, differentiate into mature cardiomyocytes after brain natriuretic peptide treatment

The Brain Natriuretic Peptide (BNP) is a cardiac hormone, which promotes the recovery of cardiac function and the preservation of cardiac tissue in animal models of heart diseases. Its cardiac protective role in animals was attributed to fibrosis inhibition, as well as to reduction of cardiomyocyte apoptosis and hypertrophy. Recently, we demonstrated that BNP induces heart regeneration via the stimulation of cardiac precursor cell (CPC) proliferation and differentiation into mature cardiomyocytes.The aim of our study was to identify which CPC’s subset is able to respond to BNP stimulation.Cardiac precursor cells identified as being Sca-1+ Nkx2.5+ or c-kit+ Nkx2.5+ cells, expressed in neonatal and adult hearts BNP’s receptors (NPR-A and NPR-B), showing their ability to be activated by BNP treatment. Cell sorting experiments based on the expression of Sca-1 or c-kit were performed on nonmyocyte cells isolated from neonatal wild-type hearts. Sca-1+ and c-kit+ cells were cultured up to 3 weeks with or without BNP in differentiating medium. Sca-1 positive cells, which contained few c-kit+ cells, responded clearly to BNP stimulation by upregulating mRNA levels of genes coding for Nkx2.5, Mlc-2v, c-kit, Sca-1, beta and alpha MHC. Furthermore, higher number of Troponin I+ cells was detected in BNP treated cells compared to untreated cells, suggesting that Sca-1+ cells differentiated after BNP stimulation into mature cardiomyocytes. BNP treatment of c-kit+ cells didn’t induce the upregulation of mRNA coding for cardiomyocyte specific genes. However, we determined that c-kit positive cells spontaneously differentiated into mature cardiomyocytes during the 3 weeks of cell culture without BNP stimulation.To determine which receptor is involved, Sca-1+ cells, isolated from neonatal hearts of NPR-A or NPR-B deficient mice, were treated with BNP. The effects of BNP on wild type and NPR-A KO cells did not differ substantially. However, Sca-1+ cells isolated from NPR-B deficient hearts couldn’t respond anymore to BNP stimulation.Thus, BNP specifically stimulates via NPR-B Sca-1+ cell differentiation into cardiomyocytes. c-kit+ cells display clearly a cardiogenic potential which is BNP independent

0232: A new role of the brain natriuretic peptide in the heart: Modulation of cardiac precursor cell proliferation and differentiation

The actual role of the brain natriuretic peptide (BNP) in the heart remains elusive despite its reported protective effect in ischemic animal hearts. Because recently BNP was shown to control the proliferation and differentiation of murine embryonic stem cells, we asked in this study whether BNP could influence the proliferation and differentiation of cardiac progenitor cells (CPC) in vitro and in vivo. We first identified a c-kit+ Sca-1+ cell population present in neonatal and adult hearts which expressed the NPR-A and NPR-B receptors. In vitro, these cells proliferated and in presence of BNP differentiated into CPCs (c-kit+ Sca-1+ Nkx2.5+) and into mature cardiomyocytes. In parallel, BNP was injected to newborn and adult healthy mice (n=6 mice per group). In the hearts of both neonatal and adult mice, BNP injection increased the number of newly formed cardiomyocytes (neonatal: + 23%, p= 0.009 and adult: +68%, p= 0.005) and the number of CPCs (neonatal: + 142%, p= 0.002 and adult: +134%, p= 0.04). BrdU injection to neonatal BNP treated mice demonstrated that BNP stimulated CPC proliferation. In anticipation that BNP might be used as a therapeutic agent, we injected BNP into mice undergoing myocardial infarction (n=6-7 mice per group). Higher numbers of Nkx2.5+ cells were detected in both the infarcted (+38%, p=0.03) and non infarcted areas (+69%, p=0.02) of BNP treated hearts one week after surgery. Finally, by isolating neonatal cardiac cells from the hearts of NPR-A or NPR-B deficient mice, we demonstrated that BNP modulates the fate of CPCs via NPRB binding and that long term BNP treatment is correlated in vitro and in vivo with decreased Protein Kinase G activity. Our results highlight a new key role for BNP in the control of CPC proliferation and/or differentiation. This new function of BNP should be evaluated in therapies aimed to induce cardiac cell regeneration and should reopen the debate about the therapeutic use of BNP for patients suffering from heart diseases

Brain natriuretic peptide is able to stimulate cardiac progenitor cell proliferation and differentiation in murine hearts after birth

Brain natriuretic peptide (BNP) contributes to heart formation during embryogenesis. After birth, despite a high number of studies aimed at understanding by which mechanism(s) BNP reduces myocardial ischemic injury in animal models, the actual role of this peptide in the heart remains elusive. In this study, we asked whether BNP treatment could modulate the proliferation of endogenous cardiac progenitor cells (CPCs) and/or their differentiation into cardiomyocytes. CPCs expressed the NPR-A and NPR-B receptors in neonatal and adult hearts, suggesting their ability to respond to BNP stimulation. BNP injection into neonatal and adult unmanipulated mice increased the number of newly formed cardiomyocytes (neonatal: +23%, p=0.009 and adult: +68%, p=0.0005) and the number of proliferating CPCs (neonatal: +142%, p=0.002 and adult: +134%, p=0.04). In vitro, BNP stimulated CPC proliferation via NPR-A and CPC differentiation into cardiomyocytes via NPR-B. Finally, as BNP might be used as a therapeutic agent, we injected BNP into mice undergoing myocardial infarction. In pathological conditions, BNP treatment was cardioprotective by increasing heart contractility and reducing cardiac remodelling. At the cellular level, BNP stimulates CPC proliferation in the non-infarcted area of the infarcted hearts. In the infarcted area, BNP modulates the fate of the endogenous CPCs but also of the infiltrating CD45+ cells. These results support for the first time a key role for BNP in controlling the progenitor cell proliferation and differentiation after birth. The administration of BNP might, therefore, be a useful component of therapeutic approaches aimed at inducing heart regeneration