Critical role of phosphodiesterase 2A in mouse congenital heart defects.

Aims: Phosphodiesterase 2 A (Pde2A), a cAMP-hydrolysing enzyme, is essential for mouse development; however, the cause of Pde2A knockout embryonic lethality is unknown. To understand whether Pde2A plays a role in cardiac development, hearts of Pde2A deficient embryos were analysed at different stage of development. Methods and results: At the stage of four chambers, Pde2A deficient hearts were enlarged compared to the hearts of Pde2A heterozygous and wild-type. Pde2A knockout embryos revealed cardiac defects such as absence of atrial trabeculation, interventricular septum (IVS) defects, hypertrabeculation and thinning of the myocardial wall and in rare cases they had overriding aorta and valves defects. E14.5 Pde2A knockouts showed reduced cardiomyocyte proliferation and increased apoptosis in the IVS and increased proliferation in the ventricular trabeculae. Analyses of E9.5 Pde2A knockout embryos revealed defects in cardiac progenitor and neural crest markers, increase of Islet1 positive and AP2 positive apoptotic cells. The expression of early cTnI and late Mef2c cardiomyocyte differentiation markers was strongly reduced in Pde2A knockout hearts. The master transcription factors of cardiac development, Tbx, were down-regulated in E14.5 Pde2A knockout hearts. Absence of Pde2A caused an increase of intracellular cAMP level, followed by an up-regulation of the inducible cAMP early repressor, Icer in fetal hearts. In vitro experiments on wild-type fetal cardiomyocytes showed that Tbx gene expression is down-regulated by cAMP inducers. Furthermore, Pde2A inhibition in vivo recapitulated the heart defects observed in Pde2A knockout embryos, affecting cardiac progenitor cells. Interestingly, the expression of Pde2A itself was dramatically affected by Pde2A inhibition, suggesting a potential autoregulatory loop. Conclusions: We demonstrated for the first time a direct relationship between Pde2A impairment and the onset of mouse congenital heart defects, highlighting a novel role for cAMP in cardiac development regulation.

ß1-syntrophin modulation by miR-222 in mdx mice.

BACKGROUND: In mdx mice, the absence of dystrophin leads to the deficiency of other components of the dystrophin-glycoprotein complex (DAPC), making skeletal muscle fibers more susceptible to necrosis. The mechanisms involved in the disappearance of the DAPC are not completely understood. The muscles of mdx mice express normal amounts of mRNA for the DAPC components, thus suggesting post-transcriptional regulation. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the hypothesis that DAPC reduction could be associated with the microRNA system. Among the possible microRNAs (miRs) found to be upregulated in the skeletal muscle tissue of mdx compared to wt mice, we demonstrated that miR-222 specifically binds to the 3'-UTR of ß1-syntrophin and participates in the downregulation of ß1-syntrophin. In addition, we documented an altered regulation of the 3'-UTR of ß1-syntrophin in muscle tissue from dystrophic mice. CONCLUSION/SIGNIFICANCE: These results show the importance of the microRNA system in the regulation of DAPC components in dystrophic muscle, and suggest a potential role of miRs in the pathophysiology of dystrophy.

An approachable human adult stem cell source for hard-tissue engineering.

Stem cells were obtained from deciduous dental pulp of healthy subjects, aged 6-10 years. This stem cell population was cultured, expanded, and specifically selected, detecting using a FACsorter, c-kit, CD34, and STRO-1 antigen expression. Then, c-kit+/CD34+/STRO-1+ cells were replaced in the culture medium added of 20% FBS, leading to osteoblast differentiation. In fact, these cells, after a week, showed a large positivity for CD44, osteocalcin, and RUNX-2 markers. To achieve an adipocytic differentiation, cells, after sorting, were challenged with dexamethason 10(-8) mM in the same culture medium. To obtain myotube fusion, sorted cells were co-cultured in ATCC medium with mouse myogenic C2C12 cells and, after a week, human stem cell nuclei were found to be able to fuse, forming myotubes. Differentiated osteoblasts, as assessed by a large positivity to several specific antibodies, after 30 days of culture and already in vitro, started to secrete an extracellular mineralized matrix, which, 2 weeks later, built a considerable number of 3D woven bone samples, which showed a strong positivity to alkaline phosphatase (ALP), alizarin red, calcein, other than to specific antibodies. These bone samples, after in vivo transplantation into immunosuppressed rats, were remodeled in a lamellar bone containing entrapped osteocytes. Therefore, this study provides strong evidence that human deciduous dental pulp is an approachable "niche" of stromal stem cells, and that it is an ideal source of osteoblasts, as well as of mineralized tissue, ready for bone regeneration, transplantation, and tissue-based clinical therapies.

c-Flip expression and function in fetal mouse gonocytes.

Apoptosis is a key mechanism in spermatogenesis, and in testis, most gonocytes degenerate at fetal and postnatal ages to select a cell subset committed to become germ stem cells. The aim of the present study is to investigate mechanisms controlling the massive apoptosis of fetal gonocytes. We evaluated the expression and function of c-Flip, an apoptosis inhibitor known to interfere with the proapoptotic Fas-signaling pathway in a variety of cell types, but never investigated in fetal testis. Expression of c-Flip long isoform (c-FlipL) within fetal testis was localized in gonocytes at 16.5 and 18.5 days post coitum (dpc), both at the mRNA and protein level, while it was weakly expressed or undetectable at earlier stages. Moreover, Fas protein was found in fetal testes at 13.5, 16.5, and 18.5 dpc. Testes at 18.5 dpc, expressing high levels of c-FlipL, were resistant to Fas-induced apoptosis while they became highly sensitive when c-FlipL was inhibited by antisense c-Flip oligos. In addition, there was an inverse relation between gonocyte spontaneous apoptosis sensitivity and c-FlipL levels. Furthermore, caspase-10 activity was inversely related with c-FlipL expression, suggesting that caspase-10 might be a target of c-FlipL. These data represent the first evidence demonstrating c-Flip expression in fetal testes and its role in protecting gonocytes from Fas-dependent apoptosis.

Isolation and expansion of adult cardiac stem cells from human and murine heart.

Cardiac myocytes have been traditionally regarded as terminally differentiated cells that adapt to increased work and compensate for disease exclusively through hypertrophy. However, in the past few years, compelling evidence has accumulated suggesting that the heart has regenerative potential. Recent studies have even surmised the existence of resident cardiac stem cells, endothelial cells generating cardiomyocytes by cell contact or extracardiac progenitors for cardiomyocytes, but these findings are still controversial. We describe the isolation of undifferentiated cells that grow as self-adherent clusters (that we have termed "cardiospheres") from subcultures of postnatal atrial or ventricular human biopsy specimens and from murine hearts. These cells are clonogenic, express stem and endothelial progenitor cell antigens/markers, and appear to have the properties of adult cardiac stem cells. They are capable of long-term self-renewal and can differentiate in vitro and after ectopic (dorsal subcutaneous connective tissue) or orthotopic (myocardial infarction) transplantation in SCID beige mouse to yield the major specialized cell types of the heart: myocytes (ie, cells demonstrating contractile activity and/or showing cardiomyocyte markers) and vascular cells (ie, cells with endothelial or smooth muscle markers).

The block of ryanodine receptors selectively inhibits fetal myoblast differentiation.

Differentiation and morphogenesis of skeletal muscle are complex and asynchronous events that involve various myogenic cell populations and extracellular signals. Embryonic and fetal skeletal myoblasts are responsible for the formation of primary and secondary fibers, respectively, although the mechanism that diversifies their fate is not fully understood. Calcium transients appear to be a signaling mechanism that is widely utilized in differentiation and embryogenesis. In mature skeletal muscle, calcium transients are generated mainly by ryanodine receptors (type 1 and type 3), which are involved in excitation-contraction coupling. However, it is not clear whether the activity of these receptors is important for contractile activity alone or whether it may also play a role in regulating the differentiation/developmental processes. To clarify this point, we first examined the expression of the receptors during development. The results show that the expression of both receptors appears as early as E13 during limb muscle development and parallels the expression of skeletal myosin. The expression and the activity of both receptors is maintained in vitro by all myogenic cell populations isolated from different stages of development, including somitic, embryonic and fetal myoblasts and satellite cells. Blocking ryanodine receptor activity by using ryanodine inhibits in vitro differentiation of fetal myoblasts (judged by the expression of sarcomeric myosin and formation of multinucleated myotubes) but not of somitic or embryonic and satellite muscle cells. This block is caused by the transcriptional inhibition of markers characteristic of terminal differentiation, rather than commitment, as the expression of muscle regulatory factors is not impaired by ryanodine treatment. Taken together, the data reported in this paper demonstrate that, although calcium transients represent a general mechanism for the control of differentiation and development, multiple calcium-dependent pathways may be relevant in different myogenic populations during development. Moreover, since fetal myoblasts are responsible for the formation of secondary fibers during development, and therefore for the building of the bulk of muscular mass, these results suggest that calcium release from ryanodine receptors plays a role in the histogenesis of mammalian skeletal muscle.