Inhibition of Senescence-Associated Genes Rb1 and Meis2 in Adult Cardiomyocytes Results in Cell Cycle Reentry and Cardiac Repair Post-Myocardial Infarction.

Background Myocardial infarction results in a large-scale cardiomyocyte loss and heart failure due to subsequent pathological remodeling. Whereas zebrafish and neonatal mice have evident cardiomyocyte expansion following injury, adult mammalian cardiomyocytes are principally nonproliferative. Despite historical presumptions of stem cell-mediated cardiac regeneration, numerous recent studies using advanced lineage-tracing methods demonstrated that the only source of cardiomyocyte renewal originates from the extant myocardium; thus, the augmented proliferation of preexisting adult cardiomyocytes remains a leading therapeutic approach toward cardiac regeneration. In the present study we investigate the significance of suppressing cell cycle inhibitors Rb1 and Meis2 to promote adult cardiomyocyte reentry to the cell cycle. Methods and Results In vitro experiments with small interfering RNA-mediated simultaneous knockdown of Rb1 and Meis2 in both adult rat cardiomyocytes, isolated from 12-week-old Fischer rats, and human induced pluripotent stem cell-derived cardiomyocytes showed a significant increase in cell number, a decrease in cell size, and an increase in mononucleated cardiomyocytes. In vivo, a hydrogel-based delivery method for small interfering RNA-mediated silencing of Rb1 and Meis2 is utilized following myocardial infarction. Immunofluorescent imaging analysis revealed a significant increase in proliferation markers 5-ethynyl-2'-deoxyuridine, PH3, KI67, and Aurora B in adult cardiomyocytes as well as improved cell survivability with the additional benefit of enhanced peri-infarct angiogenesis. Together, this intervention resulted in a reduced infarct size and improved cardiac function post-myocardial infarction. Conclusions Silencing of senescence-inducing pathways in adult cardiomyocytes via inhibition of Rb1 and Meis2 results in marked cardiomyocyte proliferation and increased protection of cardiac function in the setting of ischemic injury.

Adenine acts in the kidney as a signaling factor and causes salt- and water-losing nephropathy: early mechanism of adenine-induced renal injury.

Chronic adenine feeding is extensively used to develop animal models of chronic renal failure with metabolic features resembling those observed in humans. However, the mechanism by which adenine induces renal failure is poorly understood. In this study, we examined the early effects of adenine on water metabolism and salt balance in rats placed in metabolic cages and fed control or adenine-containing diets for 7 days. Molecular and functional studies demonstrated that adenine-fed rats exhibited a significant reduction in food intake, polyuria, polydipsia, decreased urine osmolality, and increased salt wasting. These effects are independent of changes in food intake and result from a coordinated downregulation of water channel aquaporin-2 (AQP2) and salt transporter (Na+-K+-Cl- cotransporter 2; NKCC2) in the collecting duct and medullary thick ascending limb, respectively. As a result, adenine-fed rats exhibited massive volume depletion, as indicated by a significant body weight loss, increased blood urea nitrogen, and increased hematocrit and hemoglobin levels, all of which were significantly corrected with NaCl replacement. Adenine-induced urinary concentrating defect was not corrected by exogenous arginine vasopressin (AVP), and it correlated with reduced cAMP production in vivo and in vitro. In conclusion, adenine acts on renal tubules as a signaling molecule and causes nephrogenic diabetes insipidus with salt wasting, at least, by directly interfering with AVP V2 receptor signaling with subsequent downregulation of NKCC2 and AQP2 in the kidney. The combination of renal fluid loss and decreased food intake with subsequent massive volume depletion likely plays an important role in the development of early prerenal failure that progresses to chronic kidney disease in long-term adenine feeding.

Pre-Conditioning Stem Cells in a Biomimetic Environment for Enhanced Cardiac Tissue Repair: In Vitro and In Vivo Analysis.

INTRODUCTION: Stem cell-based therapies represent a valid approach to restore cardiac function due to their beneficial effect in reducing scar area formation and promoting angiogenesis. However, their translation into the clinic is limited by the poor differentiation and inability to secrete sufficient therapeutic factors. To address this issue, several strategies such as genetic modification and biophysical preconditioning have been used to enhance the efficacy of stem cells for cardiac tissue repair. METHODS: In this study, a biomimetic approach was used to mimic the natural mechanical stimulation of the myocardium tissue. Specifically, human adipose-derived stem cells (hASCs) were cultured on a thin gelatin methacrylamide (GelMA) hydrogel disc and placed on top of a beating cardiomyocyte layer. qPCR studies and metatranscriptomic analysis of hASCs gene expression were investigated to confirm the correlation between mechanical stimuli and cardiomyogenic differentiation. In vivo intramyocardial delivery of pre-conditioned hASCs was carried out to evaluate their efficacy to restore cardiac function in mice hearts post-myocardial infarction. RESULTS: The cyclic strain generated by cardiomyocytes significantly upregulated the expression of both mechanotransduction and cardiomyogenic genes in hASCs as compared to the static control group. The inherent angiogenic secretion profile of hASCs was not hindered by the mechanical stimulation provided by the designed biomimetic system. Finally, in vivo analysis confirmed the regenerative potential of the pre-conditioned hASCs by displaying a significant improvement in cardiac function and enhanced angiogenesis in the peri-infarct region. CONCLUSION: Overall, these findings indicate that cyclic strain provided by the designed biomimetic system is an essential stimulant for hASCs cardiomyogenic differentiation, and therefore can be a potential solution to improve stem-cell based efficacy for cardiovascular repair.

Stem cell-inspired secretome-rich injectable hydrogel to repair injured cardiac tissue.

The objective of this study was to develop an injectable and biocompatible hydrogel that can deliver a cocktail of therapeutic biomolecules (secretome) secreted by human adipose-derived stem cells (hASCs) to the peri-infarct myocardium. Gelatin and Laponite® were combined to formulate a shear-thinning, nanocomposite hydrogel (nSi Gel) as an injectable carrier of secretome (nSi Gel+). The growth factor composition and the pro-angiogenic activity of the secretome were tested in vitro by evaluating the proliferation, migration and tube formation of human umbilical endothelial cells. The therapeutic efficacy of the nSi Gel + system was then investigated in vivo in rats by intramyocardial injection into the peri-infarct region. Subsequently, the inflammatory response, angiogenesis, scar formation, and heart function were assessed. Biocompatibility of the developed nSi Gel was confirmed by quantitative PCR and immunohistochemical tests which showed no significant differences in the level of inflammatory genes, microRNAs, and cell marker expression compared to the untreated control group. In addition, the only group that showed a significant increase in capillary density, reduction in scar area and improved cardiac function was treated with the nSi Gel+. Our in vitro and in vivo findings demonstrate the potential of this new secretome-loaded hydrogel as an alternative strategy to treat myocardial infarction. STATEMENT OF SIGNIFICANCE: Stem cell based-therapies represent a possible solution to repair damaged myocardial tissue by promoting cardioprotection, angiogenesis, and reduced fibrosis. However, recent evidence indicates that most of the positive outcomes are likely due to the release of paracrine factors (cytokines, growth factors, and exosomes) from the cells and not because of the local engraftment of stem cells. This cocktail of essential growth factors and paracrine signals is known as secretome can be isolated in vitro, and the biomolecule composition can be controlled by varying stem-cell culture conditions. Here, we propose a straightforward strategy to deliver secretome produced from hASCs by using a nanocomposite injectable hydrogel made of gelatin and Laponite®. The designed secretome-loaded hydrogel represents a promising alternative to traditional stem cell therapy for the treatment of acute myocardial infarction.

MicroRNA-210-mediated proliferation, survival, and angiogenesis promote cardiac repair post myocardial infarction in rodents.

An innovative approach for cardiac regeneration following injury is to induce endogenous cardiomyocyte (CM) cell cycle re-entry. In the present study, CMs from adult rat hearts were isolated and transfected with cel-miR-67 (control) and rno-miR-210. A significant increase in CM proliferation and mono-nucleation were observed in miR-210 group, in addition to a reduction in CM size, multi-nucleation, and cell death. When compared to control, ß-catenin and Bcl-2 were upregulated while APC (adenomatous polyposis coli), p16, and caspase-3 were downregulated in miR-210 group. In silico analysis predicted cell cycle inhibitor, APC, as a direct target of miR-210 in rodents. Moreover, compared to control, a significant increase in CM survival and proliferation were observed with siRNA-mediated inhibition of APC. Furthermore, miR-210 overexpressing C57BL/6 mice (210-TG) were used for short-term ischemia/reperfusion study, revealing smaller cell size, increased mono-nucleation, decreased multi-nucleation, and increased CM proliferation in 210-TG hearts in contrast to wild-type (NTG). Likewise, myocardial infarction (MI) was created in adult mice, echocardiography was performed, and the hearts were harvested for immunohistochemistry and molecular studies. Compared to NTG, 210-TG hearts showed a significant increase in CM proliferation, reduced apoptosis, upregulated angiogenesis, reduced infarct size, and overall improvement in cardiac function following MI. ß-catenin, Bcl-2, and VEGF (vascular endothelial growth factor) were upregulated while APC, p16, and caspase-3 were downregulated in 210-TG hearts. Overall, constitutive overexpression of miR-210 rescues heart function following cardiac injury in adult mice via promoting CM proliferation, cell survival, and angiogenesis. KEY MESSAGES: MiRNA-210 transfected adult rat CMs show proliferation and reduced cell death in vitro. Cell cycle inhibitor APC is a target of miR-210. MiR-210 overexpressing (210-TG) mouse hearts show CMs cell cycle re-entry and survival post myocardial injury. 210-TG mice show significant neovascularization and angiogenic potential post myocardial infarction. 210-TG hearts show reduced infarct size following ischemic injury.

Nanodiamond-based injectable hydrogel for sustained growth factor release: Preparation, characterization and in vitro analysis.

Nanodiamonds (NDs) represent an emerging class of carbon nanomaterials that possess favorable physical and chemical properties to be used as multifunctional carriers for a variety of bioactive molecules. Here we report the synthesis and characterization of a new injectable ND-based nanocomposite hydrogel which facilitates a controlled release of therapeutic molecules for regenerative applications. In particular, we have formulated a thermosensitive hydrogel using gelatin, chitosan and NDs that provides a sustained release of exogenous human vascular endothelial growth factor (VEGF) for wound healing applications. Addition of NDs improved the mechanical properties of the injectable hydrogels without affecting its thermosensitive gelation properties. Biocompatibility of the generated hydrogel was verified by in vitro assessment of apoptotic gene expressions and anti-inflammatory interleukin productions. NDs were complexed with VEGF and the inclusion of this complex in the hydrogel network enabled the sustained release of the angiogenic growth factor. These results suggest for the first time that NDs can be used to formulate a biocompatible, thermosensitive and multifunctional hydrogel platform that can function both as a filling agent to modulate hydrogel properties, as well as a delivery platform for the controlled release of bioactive molecules and growth factors. STATEMENT OF SIGNIFICANCE: One of the major drawbacks associated with the use of conventional hydrogels as carriers of growth factors is their inability to control the release kinetics of the loaded molecules. In fact, in most cases, a burst release is inevitable leading to diminished therapeutic effects and unsuccessful therapies. As a potential solution to this issue, we hereby propose a strategy of incorporating ND complexes within an injectable hydrogel matrix. The functional groups on the surface of the NDs can establish interactions with the model growth factor VEGF and promote a prolonged release from the polymer network, therefore, providing a longer therapeutic effect. Our strategy demonstrates the efficacy of using NDs as an essential component for the design of a novel injectable nanocomposite system with improved release capabilities.

Stem cell secretome-rich nanoclay hydrogel: a dual action therapy for cardiovascular regeneration.

A nanocomposite hydrogel with photocrosslinkable micro-porous networks and a nanoclay component was successfully prepared to control the release of growth factor-rich stem cell secretome. The proven pro-angiogenic and cardioprotective potential of this new bioactive system provides a valuable therapeutic platform for cardiac tissue repair and regeneration.

MicroRNAs regulating meis1 expression and inducing cardiomyocyte proliferation.

Cardiovascular disease has been the biggest killer in the United States for decades, with almost a million new cases each year. Even though mammalian rodent neonatal cardiomyocytes show proliferative potential for up to 5 days, adult cardiomyocytes lose this ability. Insufficient cardiomyocyte proliferation is one of the major reasons for the lack of regeneration of myocardial tissue, post injury. Several studies have looked at the mechanisms responsible for the arrest in proliferation at an adult stage. Following up on a recent study by Eulalio et al's study on functional screening of 875 miRNAs for neonatal cardiomyocyte proliferation, we recently identified several miRNAs that induce proliferation in naturally senescent adult cardiomyocytes. Additional studies by Mahmood et al 2013 have identified Meis1 as the major regulator of cardiomyocyte cell cycle. In our present study we have identified three of the adult cardiomyocyte proliferation inducing miRNAs to have binding sites on the 3'UTR of Meis1 gene by in-silico analysis and luciferase assay. Additionally we found these miRNAs; miR-548c-3p, miR-509-3p, and miR-23b-3p to induce significant proliferation in adult cardiomyocytes through translational inhibition of Meis1. We found a significant increase in the number of ACMs with each miRNA, in combination, and with siRNA mediated inhibition of Meis1 gene. We confirmed that these microRNAs, through inhibition of Meis1, affect its downstream targets and thereby regulate cell-cycle progression. Further investigating of the mechanism of action of these miRNAs can identify other treatment options for abnormalities associated with the lack of cardiac regeneration post myocardial injury.

MicroRNAs Inducing Proliferation of Quiescent Adult Cardiomyocytes.

In the United States, each year over 700,000 people suffer from a heart attack and over 25% of deaths are related to heart disease, making it the leading cause of death. Following ischemic injury a part of the heart muscle is replaced by a scar tissue, reducing its functioning capacity. Recent advancements in surgical intervention and pharmacotherapy only provide symptomatic relief and do not address the root cause of the problem which is the massive loss of cardiomyocytes (CM). Therefore, the development of novel therapeutic intervention for the repair and regeneration of ischemic myocardium remains an area of intense research. While existing CM in zebra fish and neonatal mice are known to proliferate and replenish the infarcted heart, it has been shown that adult mammalian CM lose this ability, thus preventing regeneration of the scar tissue. There have been many attempts to facilitate regeneration of ischemic heart but have met with limited success. Micro-RNAs (miRNAs) are one of the promising candidates towards this goal as they are known to play important regulatory roles during differentiation and tissue regeneration, and regulate genetic information by post-transcriptional modification as well as regulation of other miRNAs. While previous work by Eulalio et al., showed miRNAs inducing proliferation in neonatal CM (NCM), we here identify miRNAs inducing proliferation of rat adult-CM (ACM). This commentary while analyses recent work by Eulalio et al[1] also shows some new data with microRNAs in rat adult-CMs. Further work into the mechanism of these miRNAs can determine their therapeutic potential towards regenerating cardiac tissue post ischemic injury.

Activation of diverse signaling pathways by ex-vivo delivery of multiple cytokines for myocardial repair.

We tested the hypothesis that simultaneous transgenic overexpression of a select quartet of growth factors activates diverse signaling pathways for mobilization and participation of various stem/progenitor cells for cardiogenesis in the infarcted heart. Human insulin growth factor-1 (IGF-1), vascular endothelial growth factor (VEGF), stromal cell-derived factor-1 (SDF-1a), and hepatocyte growth factor (HGF) plasmids were synthesized and transfected into skeletal myoblasts (SM) from young male wild-type or transgenic rats expressing green fluorescent protein (GFP). Overexpression of growth factors in transfected SM ((Trans)SM) was confirmed by reverse transcription polymerase chain reaction, western blotting, and fluorescence immunostaining. Using our custom-made growth factor array and western blotting, multiple angiogenic and prosurvival factors were detected in (Trans)SM, including secreted frizzled related protein-1,2,4,5, matrix metalloproteinases-3 and 9, connexin-43, netrin-1, Nos-2, Wnt-3, Akt, MAPK42/44, Stat3, nuclear factor kappa B (NFκB), hypoxia-inducible factor 1 (HIF-1α), and protein kinase C (PKC). The conditioned medium (CM) from (Trans)SM was cytoprotective for cardiomyocytes following H(2)O(2) treatment [P<0.01 vs. CM from native SM ((Nat)SM)], promoted a higher transwell migration of human umbilical cord vein endothelial cells (223.3±1.8, P<0.01) and in vitro tube formation (47.8±1.9, P<0.01). Intramyocardial transplantation of 1.5×10(6) (Trans)SM (group-3) in a rat model of acute myocardial infarction induced extensive mobilization of cMet(+), ckit(+), ckit(+)/GATA(4+), CXCR4(+), CD44(+), CD31(+), and CD59(+) cells into the infarcted heart on day 7 and improved integration of (Trans)SM in the heart compared to (Nat)SM (group 2) (P<0.05). Extensive neomyogenesis and angiogenesis in group-3 (P<0.01 vs. group-2), with resultant attenuation of infarct size (P<0.01 vs. group-2) and improvement in global heart function (P<0.01 vs. group-2) was observed at 8 weeks. In conclusion, simultaneous activation of diverse signaling pathways by overexpression of multiple growth factors caused massive mobilization and homing of stem/progenitor cells from peripheral circulation, the bone marrow, and the heart for accelerated repair of the infarcted myocardium.

Cardiac progenitors derived from reprogrammed mesenchymal stem cells contribute to angiomyogenic repair of the infarcted heart.

The strategy to reprogram somatic stem cells to pluripotency status has provided an alternative source of surrogate ES cells (ESC). We report efficient reprogramming of multipotent bone marrow (BM) mesenchymal stem cells (MSC) to pluripotent status and the resultant MSC derived iPS cells (MiPS) and their derived progenitors effectively repaired the infarcted heart. MSC from young, male, Oct4-GFP transgenic mice were reprogrammed by retroviral transduction with Oct4, Sox2, Klf4, and c-Myc stemness factors. MiPS thus generated displayed characteristics of mouse ESC including morphology, surface antigens, gene and miR expression profiles. MiPS also formed spontaneously beating cardiac progenitors which expressed cardiac specific transcription factors and protein markers including Gata4, Mef2c, Nkx2.5, myosin heavy chain, troponin-I, and troponin-T, and showed ultra structural characteristics typical of cardiomyocytes. Intramyocardial delivery of MiPS (group-2) and their derivative cardiac-like cells (MiPS-CP; group-3) in a mouse model of acute myocardial infarction showed extensive survival and engraftment at 4 weeks with resultant attenuation of infarct size (p < 0.001 vs. DMEM injected control; n = 4). Engraftment of MiPS-CP was without cardiac tumorigenesis as compared to 21 % in MiPS transplanted animals. Furthermore, angiogenesis was improved in groups-2 and 3 (p < 0.001 vs. control). Transthoracic echocardiography revealed significantly preserved indices of cardiac contractility (ejection fraction p < 0.001 and fractional shortening p < 0.001 vs. control; n = 7). MSC were successfully reprogrammed into MiPS that displayed ESC-like characteristics and differentiated into spontaneously beating cardiomyocytes. Cardiac progenitors derived from MiPS repopulated the infarcted heart without tumorigenesis and improved global cardiac function.

Activation of IL-11/STAT3 pathway in preconditioned human skeletal myoblasts blocks apoptotic cascade under oxidant stress.

AIM: To determine whether our novel approach of diazoxide-induced stem cell preconditioning might be extrapolated to human skeletal myoblasts to support their survival under lethal oxidant stress. METHODS & RESULTS: Using an in vitro model of H(2)O(2) treatment of human skeletal myoblasts, we report the ability of diazoxide-preconditioned human skeletal myoblasts to express cytokines and growth factors, which act in an autocrine and paracrine fashion to promote their own survival. Preconditioning of skeletal myoblasts was cytoprotective and significantly reduced their apoptotic index (p < 0.05). IL-11 gene and protein expression was significantly increased in preconditioned skeletal myoblasts. Transfection of skeletal myoblasts with IL-11-specific siRNA incurred their death under oxidant stress. The cytoprotective effect of diazoxide preconditioning was blocked by Erk1/2 inhibitor PD98059 (20-100 µM), which abrogated STAT-3 phosphorylation, thus confirming a possible involvement of Erk1/2/STAT3 signaling downstream of IL-11 in cell survival. We also investigated the time course of subcellular changes and signaling pathway of skeletal myoblasts apoptosis under oxidant stress before and after preconditioning. Apoptosis was induced in skeletal myoblasts with 100-500 µM H(2)O(2) for time points ranging from 1 to 24 h. Release of lactate dehydrogenase, disruption of the mitochondrial membrane potential and cytochrome-c translocation into cytoplasm were the earliest signs of apoptosis. Total Akt protein remained unchanged whereas marked reduction in pAkt was observed in the native skeletal myoblasts. Terminal dUTP nick end-labeling and annexin-V positivity were significantly increased after 4 h. Ultra-structure studies showed condensed chromatin, shriveled nuclei and swollen mitochondria. CONCLUSION: These data suggest that skeletal myoblasts undergo apoptosis under oxidant stress in a time-dependent manner and preconditioning of skeletal myoblasts significantly prevented their apoptosis via IL-11/STAT3 signaling.

Cytoprotective and proangiogenic activity of ex-vivo netrin-1 transgene overexpression protects the heart against ischemia/reperfusion injury.

In continuation of a previous work that transgene expression of sonic hedgehog promoted neo-vascularization via netrin-1 release, the current study was aimed at assessing the anti-apoptotic and pro-angiogenic role of netrin-1 transgene overexpression in the ischemic myocardium. pLP-Adeno-X ViralTrak vectors containing netrin-1 cDNA amplified from rat mesenchymal stem cells (Ad-netrin) or without a therapeutic gene (Ad-null) were constructed and transfected into HEK-293 cells to produce Ad-netrin and Ad-null vectors. Sca-1(+)-like cells were isolated and propagated in vitro and were successfully transduced with Ad-netrin transduced Sca-1(+) cells ((Net)Sca-1(+)) and Ad-null transduced Sca-1(+) cells ((Null)Sca-1(+)). Overexpression of netrin-1 in (Net)Sca-1(+) was confirmed by reverse transcription-polymerase chain reaction and western blot. Neonatal cardiomyocytes and rat endothelial cells expressed netrin-1 specific receptor Uncoordinated-5b and the conditioned medium from (Net)Sca-1(+) cells was protective for both the cell types against oxidant stress. For in vivo studies, the rat model of myocardial ischemia/reperfusion injury was developed in female Wistar rats by left anterior descending coronary artery occlusion for 45 min followed by reperfusion. The animals were grouped to receive 70 µL of Dulbecco's modified Eagle's medium without cells (group-1), containing 2×10(6) (Null)Sca-1(+) cells (group-2) and (Net)Sca-1(+) cells (group-3). (Net)Sca-1(+) cells significantly reduced ischemia/reperfusion injury in the heart and preserved the global heart function in group-3 (P<0.05 vs. groups-1 and group-2). Ex-vivo netrin-1 overexpression in the heart increased NOS activity in the heart. Blood vessel density was significantly higher in group-3 (P<0.05 vs. controls). We concluded that netrin-1 decreased apoptosis in cardiomyocytes and endothelial cells via activation of Akt. Netrin-1 transgene expression was proangiogenic and effectively reduced ischemia/reperfusion injury to preserve global heart function.

Neuropeptide y and neuropeptide y y5 receptor interaction restores impaired growth potential of aging bone marrow stromal cells.

Abstract improved growth characteristics of the aging bone marrow cells subsequent to neuropeptide Y (NPY)/neuropeptide Y Y5 receptor (NPY Y5R) ligand-receptor interaction. Bone marrow cells were isolated from neonatal (2-3 weeks), young (8-12 weeks), and old (24-28 months) rats on the basis of their preferential adherence to plastic surface. After culturing the cells at initial seeding density of 1×10(4) cells/cm(2), we found that the proliferation potential of bone marrow cells declined with age. Real-time polymerase chain reaction (PCR) and Western blotting showed that bone marrow cells in different age groups constitutively expressed NPY and NPY receptor subtypes (Y1R, Y2R, and Y5R). However, NPY and Y5R expression increased by more than 130-fold and decreased by 28-fold, respectively, in old bone marrow cells as compared to young bone marrow cells. NPY (10 nM) stimulated the proliferation of all bone marrow cells age groups, and their proliferation was blocked by Y5R antagonist. However, the pro-proliferative effect of NPY on old bone marrow cells was weaker than other cell groups due to lower Y5R expression. Y5R gene transfection of old bone marrow cells with subsequent NPY(3-36) (10 nM) treatment significantly increased proliferation of old bone marrow cells (>56%) as compared to green fluorescence protein-transfected control old bone marrow cells. Stimulation of old bone marrow cells by NPY treatment rejuvenated the growth characteristics of aging bone marrow cells as a result of Y5R overexpression.

Reprogramming of skeletal myoblasts for induction of pluripotency for tumor-free cardiomyogenesis in the infarcted heart.

RATIONALE: Skeletal myoblasts (SMs) with inherent myogenic properties are better candidates for reprogramming to pluripotency. OBJECTIVE: To reprogram SMs to pluripotency and show that reprogrammed SMs (SiPS) express embryonic gene and microRNA profiles and that transplantation of predifferentiated cardiac progenitors reduce tumor formation. METHODS AND RESULTS: The pMXs vector containing mouse cDNAs for Yamanaka's quartet of stemness factors were used for transduction of SMs purified from male Oct4-GFP(+) transgenic mouse. Three weeks later, GFP(+) colonies of SiPS were isolated and propagated in vitro. SiPS were positive for alkaline phosphatase, expressed SSEA1, and displayed a panel of embryonic stem (ES) cell-specific pluripotency markers. Embryoid body formation yielded beating cardiomyocyte-like cells, which expressed early and late cardiac-specific markers. SiPS also had an microRNA profile that was altered during their cardiomyogenic differentiation. Noticeable abrogation of let-7 family and significant up-regulation of miR-200a-c was observed in SiPS and SiPS-derived cardiomyocytes, respectively. In vivo studies in an experimental model of acute myocardial infarction showed extensive survival of SiPS and SiPS-derived cardiomyocytes in mouse heart after transplantation. Our results from 4-week studies in DMEM without cells (group 1), SMs (group-2), SiPS (group-3), and SiPS-derived cardiomyocytes (group 4) showed extensive myogenic integration of the transplanted cells in group 4 with attenuated infarct size and improved cardiac function without tumorgenesis. CONCLUSIONS: Successful reprogramming was achieved in SMs with ES cell-like microRNA profile. Given the tumorgenic nature of SiPS, their predifferentiation into cardiomyocytes would be important for tumor-free cardiogenesis in the heart.

Cardiac tumorigenic potential of induced pluripotent stem cells in an immunocompetent host with myocardial infarction.

AIM: Genetic reprogramming of somatic cells with stemness genes to restore their pluripotent status is being studied extensively to generate pluripotent stem cells as an alternative to embryonic stem cells. This study was designed to examine the effectiveness of skeletal myoblast-derived induced pluripotent stem cells (SkiPS) from young male Oct4/GFP transgenic mice for regeneration of the infarcted heart. METHODS & RESULTS: A mouse model of permanent coronary artery ligation was developed in young female immunocompetent C57BL/6J or C57BL/6x129S4 SV/jae Oct4/GFP mice. SkiPS labeled with Q-dots (3 × 10(5) in 10 µl basal Dulbecco's modified Eagle's medium) were transplanted in and around the area of infarct immediately after coronary artery ligation (n = 16) under direct vision. Control mice (n = 12) were injected with the same number of skeletal myoblasts. Histological studies documented successful engraftment of SkiPS in all the surviving animals 4 weeks later. However, six of the 16 SkiPS-transplanted (37.5%) animal hearts showed intramural teratomas, whereas no tumor growth was observed in the control mice. Q-dot-labeled donor cells were also observed at the site of tumors. Histological studies revealed that teratomas were composed of cells from all of the three embryonic germ layers. Ultra-structure studies confirmed the histological findings and showed regions with well-organized myofibrillar structures in the tumors. CONCLUSION: Undifferentiated induced pluripotent stem cells should not be recommended for cardiac transplantation unless screened for specific teratogenic precursors or predifferentiated into cardiac lineage prior to transplantation.

Phosphodiesterase inhibition with tadalafil provides longer and sustained protection of stem cells.

We hypothesized that inhibition of the cGMP-specific enzyme phosphodiesterase 5A (PDE5A) promoted cGMP/protein kinase G (PKG) activity to condition stem cells for enhanced survival and proliferation. One-time tadalafil treatment (1 µM for 30 min) of mesenchymal stem cells ((Tada)MSCs) provided sustained protection of cells for 36 h. Higher cGMP activity with concomitantly increased PKG1 activity was observed in (Tada)MSCs, which peaked within 12 h after tadalafil treatment. Pretreatment with PKG1 blockers (1 µM KT-5823 or 20 nM K-252a) or transduction with adenoviral PKG1-short-hairpin RNA abolished tadalafil-induced cytoprotection of the cells. A higher proliferation rate was observed in (Tada)MSCs compared with nontreated MSCs ((Cont)MSCs). In a rat model of acute myocardial infarction, (Tada)MSCs transplanted 0 and 24 h after tadalafil treatment showed higher survival compared with (Cont)MSCs on day 2 and day 4 after engraftment. (Tada)MSCs transplanted 48 h after tadalafil treatment lost their protection on both day 2 and day 4 after engraftment, and their rate of survival was similar to (Cont)MSCs. Reduced terminal dUTP nick end-labeling positivity (P < 0.01 vs. (Cont)MSCs) and higher proliferation of (Tada)MSCs (P < 0.01 vs. (Cont)MSCs) was observed in the infarcted heart. Fluorescence immunostaining revealed neomyogenesis in both the infarct and peri-infarct areas. Blood vessel density was significantly increased in group 2 compared with group 1. Transthoracic echocardiographic heart function revealed significant preservation of the indexes of left ventricle contractility and attenuation of remodeling in (Tada)MSC-engrafted animal hearts (group 2) compared with (Cont)MSCs (group 1). PDE5A inhibition using long-acting tadalafil is an innovative approach to promote stem cell survival and proliferation in the infarcted heart.

De novo myocardial regeneration: advances and pitfalls.

The capability of adult tissue-derived stem cells for cardiogenesis has been extensively studied in experimental animals and clinical studies for treatment of postischemic cardiomyopathy. The less-than-anticipated improvement in the heart function in most clinical studies with skeletal myoblasts and bone marrow cells has warranted a search for alternative sources of stem cells. Despite their multilineage differentiation potential, ethical issues, teratogenicity, and tissue rejection are main obstacles in developing clinically feasible methods for embryonic stem cell transplantation into patients. A decade-long research on embryonic stem cells has paved the way for discovery of alternative approaches for generating pluripotent stem cells. Genetic manipulation of somatic cells for pluripotency genes reprograms the cells to pluripotent status. Efforts are currently focused to make reprogramming protocols safer for clinical applications of the reprogrammed cells. We summarize the advancements and complicating features of stem cell therapy and discuss the decade-and-a-half-long efforts made by stem cell researchers for moving the field from bench to the bedside as an adjunct therapy or as an alternative to the contemporary therapeutic modalities for routine clinical application. The review also provides a special focus on the advancements made in the field of somatic cell reprogramming.

Striated muscle tropomyosin isoforms differentially regulate cardiac performance and myofilament calcium sensitivity.

Tropomyosin (TM) plays a central role in calcium mediated striated muscle contraction. There are three muscle TM isoforms: alpha-TM, beta-TM, and gamma-TM. alpha-TM is the predominant cardiac and skeletal muscle isoform. beta-TM is expressed in skeletal and embryonic cardiac muscle. gamma-TM is expressed in slow-twitch musculature, but is not found in the heart. Our previous work established that muscle TM isoforms confer different physiological properties to the cardiac sarcomere. To determine whether one of these isoforms is dominant in dictating its functional properties, we generated single and double transgenic mice expressing beta-TM and/or gamma-TM in the heart, in addition to the endogenously expressed alpha-TM. Results show significant TM protein expression in the betagamma-DTG hearts: alpha-TM: 36%, beta-TM: 32%, and gamma-TM: 32%. These betagamma-DTG mice do not develop pathological abnormalities; however, they exhibit a hyper contractile phenotype with decreased myofilament calcium sensitivity, similar to gamma-TM transgenic hearts. Biophysical studies indicate that gamma-TM is more rigid than either alpha-TM or beta-TM. This is the first report showing that with approximately equivalent levels of expression within the same tissue, there is a functional dominance of gamma-TM over alpha-TM or beta-TM in regulating physiological performance of the striated muscle sarcomere. In addition to the effect expression of gamma-TM has on Ca(2+) activation of the cardiac myofilaments, our data demonstrates an effect on cooperative activation of the thin filament by strongly bound rigor cross-bridges. This is significant in relation to current ideas on the control mechanism of the steep relation between Ca(2+) and tension.

MicroRNA-21 is a key determinant in IL-11/Stat3 anti-apoptotic signalling pathway in preconditioning of skeletal myoblasts.

AIMS: We have previously shown that preconditioning of stem and progenitor cells promotes their survival post-engraftment in the infarcted heart. The present study was designed to (i) delineate the role of microRNA-21 (miR-21) in interleukin-11 (IL-11) signalling during preconditioning of skeletal myoblasts (MY) and (ii) study the long-term fate of preconditioned MY ((PC)MY) post-transplantation in the infarcted heart. METHODS AND RESULTS: We report that pharmacological preconditioning of MY with diazoxide showed robust expression of IL-11 and activation of extracellular signal-regulated kinase 1/2 (Erk1/2) and signal transducers and activators of transcription-3 (Stat3) with concomitantly increased miR-21. These molecular events improved cytoprotection of (PC)MY under oxidant stress in vitro which was compromised by pre-treatment of (PC)MY with IL-11-specific siRNA, Erk1/2 blocker, or anti-miR-21. In vivo studies for sry-gene detection in a female rat heart model of acute myocardial infarction showed two-fold higher survival of male donor (PC)MY 4 and 7 days post-engraftment. Long-term fate of the engrafted cells was determined at 4 months after transplantation. Immunohistological studies revealed that in comparison with (non-PC)MY, (PC)MY improved angiogenic response in the heart which was evident from a higher number of blood vessels per surface area (0.155 mm(2)) and myogenic differentiation of (PC)MY in the heart. Indices of myocardial contractility including ejection fraction and fractional shortening showed significant improvement in (PC)MY-treated animals. CONCLUSION: miR-21 is a key regulator of Erk1/2-Stat3 signalling downstream of IL-11 during preconditioning of MY. The therapeutic benefits of (PC)MY were stable and persisted until 4 months of observation.