Pacsin 2 is required for the maintenance of a normal cardiac function in the developing mouse heart.

The Pacsin proteins (Pacsin 1, 2 and 3) play an important role in intracellular trafficking and thereby signal transduction in many cells types. This study was designed to examine the role of Pacsin 2 in cardiac development and function. We investigated the development and electrophysiological properties of Pacsin 2 knockout (P2KO) hearts and single cardiomyocytes isolated from 11.5 and 15.5days old fetal mice. Immunofluorescence experiments confirmed the lack of Pacsin 2 protein expression in P2KO cardiac myocytes in comparison to wildtype (WT). Western blotting demonstrates low expression levels of connexin 43 and T-box 3 proteins in P2KO compared to wildtype (WT). Electrophysiology measurements including online Multi-Electrode Array (MEA) based field potential (FP) recordings on isolated whole heart of P2KO mice showed a prolonged AV-conduction time. Patch clamp measurements of P2KO cardiomyocytes revealed differences in action potential (AP) parameters and decreased pacemaker funny channel (If), as well as L-type Ca2+ channel (ICaL), and sodium channel (INa). These findings demonstrate that Pacsin 2 is necessary for cardiac development and function in mouse embryos, which will enhance our knowledge to better understand the genesis of cardiovascular diseases.

Effects of synthetic neural adhesion molecule mimetic peptides and related proteins on the cardiomyogenic differentiation of mouse embryonic stem cells.

BACKGROUND/AIMS: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. METHODS: In the present study, using a transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP), we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGL(L), hNgf_C2, EnkaminE, Plannexin and C3) on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. RESULTS: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF) peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. CONCLUSION: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to cardiac pathologies where BDNF levels are impaired.

Functional expression and regulation of hyperpolarization-activated cyclic nucleotide-gated channels (HCN) in mouse iPS cell-derived cardiomyocytes after UTF1 -neo selection.

BACKGROUND/AIMS: In vitro reprogramming of somatic cells holds great potential to serve as an autologous source of cells for tissue repair. However, major difficulties in achieving this potential include obtaining homogeneous and stable cells for transplantation. High electrical activity of cells such as cardiomyocytes (CMs) is crucial for both, safety and efficiency of cell replacement therapy. Moreover, the function of the cardiac pacemaker is controlled by the activities of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Here we have examined changes in HCN gene expression and function during cardiomyogenesis. METHODS: We differentiated murine iPS cells selected by an undifferentiated transcription factor 1 (UTF1) -promoter-driven G418 resistance to CMs in vitro and characterized them by RT-PCR, immunocytochemistry, and electrophysiology. RESULTS: As key cardiac markers alpha-actinin and cardiac troponin T could be identified in derived CMs. Immunocytochemical staining of CMs showed the presence of all HCN subunits (HCN1-4). Electrophysiology experiments revealed developmental changes of action potentials and If currents as well as functional hormonal regulation and sensitivity to If channel blockers. CONCLUSION: We conclude that iPS cells derived from UTF-selection give rise to functional CMs in vitro, with established hormonal regulation pathways and functionally expressed If current in a development-dependent manner; and have all phenotypes with the pacemaker as predominant subtype. This might be of great importance for transplantation purposes.

Specific inhibition of epithelial Na+ channels by antisense oligonucleotides for the treatment of Na+ hyperabsorption in cystic fibrosis.

BACKGROUND: Cystic fibrosis (CF) respiratory epithelia are characterized by a defect Cl(-) secretion and an increased Na(+) absorption through epithelial Na(+) channels (ENaC). The present study aimed to find an effective inhibitor of human ENaC with respect to replacing amiloride therapy for CF patients. Therefore, we developed specific antisense oligonucleotides (AON) that efficiently suppress Na(+) hyperabsorption by inhibiting the expression of the alpha-ENaC subunit. METHODS: We heterologously expressed ENaC in oocytes of Xenopus laevis for mass screening of AON. Additionally, primary cultures of human nasal epithelia were transfected with AON and were used for Ussing chamber experiments, as well as biochemical and fluorescence optical analyses. RESULTS: Screening of several AON by co-injection or sequential microinjection of AON and ENaC mRNA in X. laevis oocytes led to a sustained decrease in amiloride-sensitive current and conductance. Using primary cultures of human nasal epithelia, we show that AON effectively suppress amiloride-sensitive Na(+) absorption mediated by ENaC in CF and non-CF tissues. In western blot experiments, it could be shown that the amount of ENaC protein is effectively reduced after AON transfection. CONCLUSIONS: Our data comprise an initial step towards a preclinical test with AON to reduce Na(+) hyperabsorption in CF epithelia.

Altered Functional Expression of ß-Adrenergic Receptors in Rhesus Monkey Embryonic Stem Cell-Derived Cardiomyocytes.

Pluripotent stem cells have demonstrated the potential to generate large numbers of functional cardiomyocytes (CMs) from different cell sources. Besides Wnt signaling, additional pathways are involved in early cardiac development and function. To date however, no study exists showing the effects of perturbing the canonical Wnt pathway using nonhuman primate embryonic stem (ES) cells. In this study, we investigated the effect of canonical Wnt inhibition during differentiation of nonhuman primate ES cell-derived CMs under defined, growth factor conditions. Rhesus monkey ES (rES) cells were differentiated into spontaneously beating CMs in the absence (control) or presence (treated) of Wnt inhibitor Dickkopf1 (DKK1), vascular endothelial growth factor, and basic fibroblast growth factor combined or added in a sequential manner during differentiation. Quantification and functional characterization of CMs were assessed by molecular and electrophysiological techniques. Analysis revealed no difference in average ratio of spontaneously beating clusters in both control and treated groups. However, the percentage of CMs was significantly reduced and the expressions of specific cardiac markers tested were also decreased in the treated group. Interestingly, we found that in CMs obtained from treated group, ß-adrenergic receptors (ß-ARs) were less expressed, their function was altered and electrophysiological studies revealed differences in action potential responsiveness to ß-AR stimulation. We demonstrated that the Wnt/ß-catenin pathway inhibitor, DKK1 associated with other growth factors repressed functional expression of ß-ARs in rES cell-derived CMs. Thus, control of this pathway in each cell line and source is important for proper basic research and further cell therapy applications.

Dynamic Support Culture of Murine Skeletal Muscle-Derived Stem Cells Improves Their Cardiogenic Potential In Vitro.

Ischemic heart disease is the main cause of death in western countries and its burden is increasing worldwide. It typically involves irreversible degeneration and loss of myocardial tissue leading to poor prognosis and fatal outcome. Autologous cells with the potential to regenerate damaged heart tissue would be an ideal source for cell therapeutic approaches. Here, we compared different methods of conditional culture for increasing the yield and cardiogenic potential of murine skeletal muscle-derived stem cells. A subpopulation of nonadherent cells was isolated from skeletal muscle by preplating and applying cell culture conditions differing in support of cluster formation. In contrast to static culture conditions, dynamic culture with or without previous hanging drop preculture led to significantly increased cluster diameters and the expression of cardiac specific markers on the protein and mRNA level. Whole-cell patch-clamp studies revealed similarities to pacemaker action potentials and responsiveness to cardiac specific pharmacological stimuli. This data indicates that skeletal muscle-derived stem cells are capable of adopting enhanced cardiac muscle cell-like properties by applying specific culture conditions. Choosing this route for the establishment of a sustainable, autologous source of cells for cardiac therapies holds the potential of being clinically more acceptable than transgenic manipulation of cells.

Modulation of L-type calcium current by intracellular magnesium in differentiating cardiomyocytes derived from induced pluripotent stem cells.

Intracellular Mg(2+), which is implicated in arrhythmogenesis and transient cardiac ischemia, inhibits L-type Ca(2+) calcium channel current (ICaL) of adult cardiomyocytes (CMs). We take the advantage of an in vitro model of CMs based on induced pluripotent stem cells to investigate the effects of intracellular Mg(2+) on the phosphorylation or dephosphorylation processes of L-type Ca(2+) channels (LTCCs) at early and late stages of cardiac cell differentiation. Using the whole-cell patch-clamp technique, we demonstrate that increasing intracellular Mg(2+) concentration [Mg(2+)]i from 0.2 to 5 mM markedly reduced the peak of ICaL density, showing less effect on both the activation and inactivation properties in the late differentiation stage (LDS) of CMs more so than in the early differentiation stage (EDS). Increasing the [Mg(2+)]i from 0.2 to 2 mM in the presence of cAMP-dependent protein kinase A significantly decreased ICaL in LDS (70%) and in EDS (36%) CMs. In addition, the effect of forskolin was greatly attenuated in the presence of 2 mM [Mg(2+)]i in LDS but not in EDS CMs. The effect of forskolin was enhanced in the presence of ATP-γ-S in LDS CMs compared with EDS CMs. The exposure of both EDS and LDS CMs to 2 mM [Mg(2+)]i considerably reduced the effects of isobutylmethylxanthine (IBMX) and okadaic acid on ICaL. Our results provide evidence for differential regulation of LTCCs activities by cytosolic Mg(2+) concentration in developing cardiac cells and confirm that Mg(2+) acts under conditions that favor opening of the LTCCs caused by channel phosphorylation.