The possibility that adult bone marrow cells (BMCs) retain a remarkable degree of developmental plasticity and acquire the cardiomyocyte lineage after infarction has been challenged, and the notion of BMC transdifferentiation has been questioned. The center of the controversy is the lack of unequivocal evidence in favor of myocardial regeneration by the injection of BMCs in the infarcted heart. Because of the interest in cell-based therapy for heart failure, several approaches including gene reporter assay, genetic tagging, cell genotyping, PCR-based detection of donor genes, and direct immunofluorescence with quantum dots were used to prove or disprove BMC transdifferentiation. Our results indicate that BMCs engraft, survive, and grow within the spared myocardium after infarction by forming junctional complexes with resident myocytes. BMCs and myocytes express at their interface connexin 43 and N-cadherin, and this interaction may be critical for BMCs to adopt the cardiomyogenic fate. With time, a large number of myocytes and coronary vessels are generated. Myocytes show a diploid DNA content and carry, at most, two sex chromosomes. Old and new myocytes show synchronicity in calcium transients, providing strong evidence in favor of the functional coupling of these two cell populations. Thus, BMCs transdifferentiate and acquire the cardiomyogenic and vascular phenotypes restoring the infarcted heart. Together, our studies reveal that locally delivered BMCs generate de novo myocardium composed of integrated cardiomyocytes and coronary vessels. This process occurs independently of cell fusion and ameliorates structurally and functionally the outcome of the heart after infarction. myocardial infarction ͉ myocardial regeneration ͉ stem cells ͉ transdifferentiation T o date, the hematopoietic stem cell appears to be the most versatile stem cell in crossing lineage boundaries and the most prone to break the law of tissue fidelity (1). Early studies on c-kit-positive bone marrow cell (BMC) differentiation into myocardium have generated great enthusiasm (2, 3), but other observations have rejected the initial results (4-6) and promoted a wave of skepticism about the therapeutic potential of BMCs for the injured heart. The major criticisms include: (i) lack of utilization of genetic markers for the recognition of donor BMCs and their progeny; (ii) inaccurate interpretation of the original data due to autofluorescence artifacts; and (iii) the possibility that myocyte regeneration is mediated by fusion of BMCs with resident myocytes rather than BMC transdifferentiation. To address these important questions and demonstrate reproducibility of results, four laboratories with complementary expertise have undertaken a series of joined experiments to acquire information on the plasticity of BMCs and their therapeutic potential for the infarcted heart.In this effort, BMCs for myocardial regeneration were obtained from three transgenic mice. In the first, EGFP was driven by the ubiquitous ␤-actin promoter; in the second, EGFP was ...

Cyclic GMP is rapidly formed a few seconds after binding of chemotactic signailing molecules to specific receptors on the cell surface of Dictyostelium amoebae. This phenomenon could be mimicked by addition of a pulse of Ca2+ to ~~eabili~d amoebae. The con~ntration of Ca 2+ for haIf-maximal response was 60 FM. Other ions (K+, Na+, Mg*+ or Mn2+) had no effect. A pulse of 5 PM IP, produced a cyclic GMP response of similar magnitude but IP, elicited no response. The data provide strong support for the hypothesis that cell surface receptor binding induces cyclic GMP formation by liberating Caz+ from internal stores.(Dictyostelium) cyclic GMP Chemotaxis Ca*+ INTRODUCTlONAmoebae of the cellular slime mould, Dictyostelium discoideum, respond chemotactically to signalling molecules such as folate during the foodseeking phase [l], and CAMP during the subsequent starvation phase [2] when they aggregate to form motile multicellular organisms [3]. These chemoattractants bind to separate cell surface receptors, yet appear to induce a group of internal responses common to both types of stimulus 141.Following a pulse of exogenous CAMP or folate, two of the initial responses identified are the polymerisation of actin associated with the cytoskeleton, peaking 3-5 s after stimulation (5lo], and the transient formation of cyclic GMP, which peaks 9-12 s after CAMP or folate binding , and is rapidly degraded by a specific cGMP phosphodiesterase [ 11,121. This cGMP formation has been implicated in chemotactic movement by studies which indicate its rapid accumulation following pulsing of several species of cellular slime mould (including D. ~is~~ide~rn, D. ~~~~e~rn~ and Po~ys~~o~dyfium violaceum) with their specific chemoattractants Abbreviations: IP,, inositol 1,4,5trisphosphate; IP2, inositol 1,4-bisphosphate [ 13,141. It is aIso strongly implicated in chemotaxis by the finding that streamer F mutants of D. discoideum (which lack the specific cCiMP phosphodiesterase and hence produce a large peak of cCMP in response to CAMP-receptor binding that persists for at least 1 min at elevated levels) show a chemotactic movement period that is greatly lengthened compared to the parental wild type f10, 15,16].Recently, studies in this laboratory have produced indirect evidence for the involvement of Ca'+ in chemotaxis in D. discoideum [17-191 and have revealed that IP3, when added to permeabilised amoebae, can mimic the action of chemoattractants on normal intact amoebae in inducing cGMP formation [ZO]. As IP3 in mammalian systems acts via release of intracellular Ca'+ 1211, it seemed worthwhile to study the direct effect of pulses of Ca2+ on the formation of cGMP in permeabilised amoebae. MATERIALS AND METHODS MaterialsIP3 (potassium salt) and IPz (potassium salt) were obtained from Amersham. Saponin and EGTA were obtained from Sigma.

Previous studies of Europe-Finner & Newell indicated that in amoebae of Dictyostelium discoideum, signal transduction used for chemotaxis to cyclic AMP involved transient formation of inositol tris- and polyphosphates. Evidence was also presented for the involvement of a GTP-binding G-protein. Here we report evidence for the involvement of a ras gene product in the D. discoideum inositol phosphate pathway. Use was made of strains of Dictyostelium transformed with a wild-type D. discoideum ras gene (ras-Gly12) or a mutant form of the gene (ras-Thr12). Experiments using separation of soluble inositol phosphates by Dowex anion-exchange resin chromatography indicated that cells transformed with the wild-type ras-Gly12 gene were unaffected in their basal levels of inositol polyphosphates and in the inositol phosphates formed in response to stimulation with the chemotactic agent cyclic AMP. In contrast, cells transformed with the mutant ras-Thr12 gene showed a basal level of inositol polyphosphate that was several-fold elevated over the controls and stimulation of these cells with cyclic AMP produced only a small further elevation. When the inositol phosphates were analysed by h.p.l.c. it was found that the basal level of inositol 1,4,5-trisphosphate was raised three- to fivefold in the ras-Thr12 strain compared to the strain transformed with ras-Gly12, and that inositol hexakisphosphate (which was found to be present in large amounts relative to other inositol phosphates in D. discoideum cells) was also raised to a similar extent in the ras-Thr12-transformed cells. We propose that the Dictyostelium ras gene product codes for a regulatory protein involved in the inositol phosphate chemotactic signal-transduction pathway.

Amoebae of Dictyostelium discoideum show adaptation towards a chemotactic cyclic AMP signal. Within a few seconds of receipt of the signal they are inhibited for a period of 1–2 min from further chemotactic responses to subsequent cyclic AMP signals of similar or smaller magnitude. The site of this adaptation mechanism in the chemotactic transduction pathway was investigated by addition of components of the transduction chain (GTP analogues, myo-inositol-1,4,5-trisphosphate (InsP3) and Ca2+) to permeabilized cells followed by determination of the amount of cyclic GMP formed as a measure of the chemotactic response. This approach was made possible by finding that permeabilization of amoebae with saponin did not uncouple the cell surface cyclic AMP receptors from stimulation of cyclic GMP formation. It was found that InsP3 and Ca2+ were ‘downstream’ from the adaptation mechanism: they could trigger a cyclic GMP response in cyclic AMP-adapted amoebae but could not themselves induce adaptation. In contrast, GTP gamma S was unable to trigger a cyclic GMP response in cyclic AMP-adapted cells, although it could trigger multiple cyclic GMP responses in non-adapted cells. We deduce that the site of adaptation to cyclic AMP stimulation is at the G-protein involved in this signalling pathway. Moreover, as GTP gamma S was found to be unable to induce adaptation, we conclude that the mechanism of adaptation involves an action of the cyclic AMP receptor on the G-protein that is distinct from its commonly reported action of stimulating G-protein binding of GTP.

Cardiac progenitor cells (CPCs) have been identified in the adult heart. However, the molecular mechanisms involved in the commitment of CPCs to the myocyte lineage remain to be determined. Notch-1 is a transmembrane receptor activated by the DSL family of ligands which include Jagged1 and Delta-4. Upon ligand binding, the activated receptor undergoes cleavage by γ-secretase, and its intracellular portion (Notch intracellular domain, NICD) is released, translocates to the nucleus and exerts its function as a transcriptional regulator. The objective of this study was to determine whether the components of the Notch pathway are present in the CPCs of the adult mammalian heart and whether activation of the Notch-1 receptor promotes the differentiation of CPCs into myocytes. For this purpose, c-kit-positive CPCs were isolated from the mouse heart and analyzed by FACS and immunocytochemistry. Notch-1 receptor was detected in ~50% of c-kit-positive CPCs. CPCs were then plated on culture dishes coated with immobilized Jagged1 or Delta-4 and maintained in low-serum medium. Additional groups of cells were similarly exposed to the ligands but were also treated with γ-secretase inhibitor. After 5 days in culture, the number of CPCs was markedly lower in the presence of the Notch ligands and significantly higher in the presence of the γ-secretase inhibitor. After 8 days in culture, cells became confluent and did not express any longer c-kit. With respect to cells treated with the γ-secretase inhibitor, exposure to Jagged1 and Delta-4 resulted respectively in a 10-fold and 20-fold increase in the fraction of CPCs positive for Nkx2.5. These findings were consistent with a positive effect of Notch on CPC differentiation and Nkx2.5 upregulation. To establish whether Notch influenced cardiomyogenesis in vivo, infarcted mice were treated for 11 days with the γ-secretase inhibitor. The regenerative response of the infarcted heart, defined by the percentage of BrdU-positive myocytes distributed in the border zone, was 50% lower in animals that received the γ-secretase inhibitor. Thus, Notch1 modulates CPC differentiation in vitro and myocardial regeneration in vivo after infarction.