Induced Pluripotent Stem Cell-Derived Cardiomyocytes Provide In Vivo Biological Pacemaker Function.

BACKGROUND: Although multiple approaches have been used to create biological pacemakers in animal models, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have not been investigated for this purpose. We now report pacemaker function of iPSC-CMs in a canine model. METHODS AND RESULTS: Embryoid bodies were derived from human keratinocytes, their action potential characteristics determined, and their gene expression profiles and markers of differentiation identified. Atrioventricular blocked dogs were immunosuppressed, instrumented with VVI pacemakers, and injected subepicardially into the anterobasal left ventricle with 40 to 75 rhythmically contracting embryoid bodies (totaling 1.3-2×106 cells). ECG and 24-hour Holter monitoring were performed biweekly. After 4 to 13 weeks, epinephrine (1 µg kg-1 min-1) was infused, and the heart removed for histological or electrophysiological study. iPSC-CMs largely lost the markers of pluripotency, became positive for cardiac-specific markers. and manifested If-dependent automaticity. Epicardial pacing of the injection site identified matching beats arising from that site by week 1 after implantation. By week 4, 20% of beats were electronically paced, 60% to 80% of beats were matching, and mean and maximal biological pacemaker rates were 45 and 75 beats per minute. Maximum night and day rates of matching beats were 53±6.9 and 69±10.4 beats per minute, respectively, at 4 weeks. Epinephrine increased rate of matching beats from 35±4.3 to 65±4.0 beats per minute. Incubation of embryoid bodies with the vital dye, Dil, revealed the persistence of injected cells at the site of administration. CONCLUSIONS: iPSC-CMs can integrate into host myocardium and create a biological pacemaker. Although this is a promising development, rate and rhythm of the iPSC-CMs pacemakers remain to be optimized.

Autocrine A2 in the T-system of ventricular myocytes creates transmural gradients in ion transport: a mechanism to match contraction with load?

Transmural heterogeneities in Na/K pump current (IP), transient outward K(+)-current (Ito), and Ca(2+)-current (ICaL) play an important role in regulating electrical and contractile activities in the ventricular myocardium. Prior studies indicated angiotensin II (A2) may determine the transmural gradient in Ito, but the effects of A2 on IP and ICaL were unknown. In this study, myocytes were isolated from five muscle layers between epicardium and endocardium. We found a monotonic gradient in both Ip and Ito, with the lowest currents in ENDO. When AT1Rs were inhibited, EPI currents were unaffected, but ENDO currents increased, suggesting endogenous extracellular A2 inhibits both currents in ENDO. IP- and Ito-inhibition by A2 yielded essentially the same K0.5 values, so they may both be regulated by the same mechanism. A2/AT1R-mediated inhibition of IP or Ito or stimulation of ICaL persisted for hours in isolated myocytes, suggesting continuous autocrine secretion of A2 into a restricted diffusion compartment, like the T-system. Detubulation brought EPI IP to its low ENDO value and eliminated A2 sensitivity, so the T-system lumen may indeed be the restricted diffusion compartment. These studies showed that 33-50% of IP, 57-65% of Ito, and a significant fraction of ICaL reside in T-tubule membranes where they are transmurally regulated by autocrine secretion of A2 into the T-system lumen and activation of AT1Rs. Increased AT1R activation regulates each of these currents in a direction expected to increase contractility. Endogenous A2 activation of AT1Rs increases monotonically from EPI to ENDO in a manner similar to reported increases in passive tension when the ventricular chamber fills with blood. We therefore hypothesize load is the signal that regulates A2-activation of AT1Rs, which create a contractile gradient that matches the gradient in load.

Caspases and p38 MAPK regulate endothelial cell adhesiveness for mesenchymal stem cells.

Mesenchymal stem cells natively circulating or delivered into the blood stream home to sites of injury. The mechanism of mesenchymal stem cell homing to sites of injury is poorly understood. We have shown that the development of apoptosis in endothelial cells stimulates endothelial cell adhesiveness for mesenchymal stem cells. Adhesion of mesenchymal stem cells to apoptotic endothelial cells depends on the activation of endothelial caspases and p38 MAPK. Activation of p38 MAPK in endothelial cells has a primary effect while the activation of caspases potentiates the mesenchymal stem cell adhesion. Overall, our study of the mesenchymal stem cell interaction with endothelial cells indicates that mesenchymal stem cells recognize and specifically adhere to distressed/apoptotic endothelial cells.

Von willebrand factor increases endothelial cell adhesiveness for human mesenchymal stem cells by activating p38 mitogen-activated protein kinase.

INTRODUCTION: Delivered systemically or natively circulating mesenchymal stem cells accumulate in injured tissues. During homing mesenchymal stem cells adhere to endothelial cells and infiltrate underlying tissue. Previously we have shown that adhesiveness of endothelial cells for mesenchymal stem cells correlates with the inhibition of mitochondrial function of endothelial cells and secretion of von Willebrand factor. We hypothesized that von Willebrand factor is an auto/paracrine regulator of endothelial cell adhesiveness and studied the effect of von Willebrand factor on adhesion of mesenchymal stem cells to endothelial cells. METHODS: We used Affymetrix DNA microarrays, human protein phospho-MAPK array, Western blot, cell-based ELISA and flow cytometry analysis to study the activation of endothelial cells by von Willebrand factor. Cell adhesion assay and protein kinase inhibitors were used to evaluate the role of mitogen-activated protein kinases in the regulation of endothelial cell adhesiveness for mesenchymal stem cell. RESULTS: Treatment of endothelial cells with von Willebrand factor stimulated the mesenchymal stem cell adhesion in a time- and concentration-dependent manner. Mesenchymal stem cells did not adhere to immobilized von Willebrand factor and did not express receptors for von Willebrand factor suggesting that the stimulation of the mesenchymal stem cell adhesion is a result of endothelial cell activation with von Willebrand factor. Treatment of endothelial cells with von Willebrand factor activated ERK-1,2 and p38 MAPK without an effect on gene or cell surface expression of E-selectin, P-selectin, VCAM1 and ICAM1. Inhibition of p38 MAPK, but not ERK-1,2, in endothelial cells completely abrogated the stimulation of the mesenchymal stem cell adhesion by von Willebrand factor. CONCLUSIONS: Von Willebrand factor is an auto/paracrine regulator of endothelial cells. Activation of p38 MAPK in endothelial cells by von Willebrand factor is responsible for the regulation of endothelial cell adhesiveness for mesenchymal stem cells.

Increased myocyte content and mechanical function within a tissue-engineered myocardial patch following implantation.

During the past few years, studies involving the implantation of stem cells, chemical factors, and scaffolds have demonstrated the ability to augment the mammalian heart's native regenerative capacity. Scaffolds comprised of extracellular matrix (ECM) have been used to repair myocardial defects. These scaffolds become populated with myocytes and provide regional contractile function, but quantification of the myocyte population has not yet been conducted. The purpose of this study was to quantitate the myocyte content within the ECM bioscaffold and to correlate this cell population with the regional mechanical function over time. Xenogenic ECM scaffolds derived from porcine urinary bladder were implanted into a full-thickness, surgically induced, right ventricular-free wall defect in a dog model. Zero, 2, and 8 weeks following implantation, regional function and myocyte content were determined in each patch region. Regional function did not significantly increase from 0 to 2 weeks. At 8 weeks, however, regional stroke work increased to 3.7 +/- 0.7% and systolic contraction increased to 4.4 +/- 1.2%. The myocyte content also significantly increased during that period generating a linear relationship between regional function and myocyte content. In conclusion, ECM used as a myocardial patch increases both the regional function and the myocyte content over time. The mechanical function generated in the patch region is correlated with the quantity of local tissue myocytes.

Apoptotic endothelial cells demonstrate increased adhesiveness for human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) participate in the wound healing process in mammalians. Adhesion of MSCs to endothelium is a key step in the homing of MSCs circulating in the bloodstream to the sites of injury and inflammation. Because endothelial cells (ECs) may become apoptotic under certain pro-inflammatory conditions, we investigated the effects of pro-inflammatory, TNF-alpha and IL-1 beta, and pro-apoptotic agents, actinomycin D, cycloheximide, okadaic acid, wortmannin, and staurosporine, on human MSCs (hMSCs) adhesion to ECs. Treatment of ECs with pro-apoptotic agents markedly increased adhesion of hMSCs to ECs. This adhesion correlated with reduction of mitochondrial membrane potential, inhibition of NADH dehydrogenases, and release of von Willebrand factor (vWF) by ECs. Treatment of ECs with exogenous vWF also stimulated hMSC adhesion. These data provide evidence that apoptosis of ECs may regulate homing of hMSCs to the sites of tissue injury. These results are consistent with the hypothesis that activation of apoptotic signaling pathways in ECs releases vWF which regulates hMSC adhesion to ECs.

Enhanced recovery of mechanical function in the canine heart by seeding an extracellular matrix patch with mesenchymal stem cells committed to a cardiac lineage.

The need to regenerate tissue is paramount, especially for the heart that lacks the ability to regenerate after injury. The urinary bladder extracellular matrix (ECM), when used to repair a right ventricular defect, successfully regenerated some mechanical function. The objective of the current study was to determine whether the regenerative effect of ECM could be improved by seeding the patch with human mesenchymal stem cells (hMSCs) enhanced to differentiate down a cardiac linage. hMSCs were used to form three-dimensional spheroids. The expression of cardiac proteins was determined in cells exposed to the spheroid formation and compared with nonmanipulated hMSCs. To determine whether functional calcium channels were present, the cells were patch clamped. To evaluate the ability of these cells to regenerate mechanical function, the spheroids were seeded on ECM and then implanted into the canine heart to repair a full-thickness right ventricular defect. As a result, many of the cells spreading from the spheroids expressed cardiac-specific proteins, including sarcomeric alpha-actinin, cardiotin, and atrial natriuretic peptide, as well as the cell cycle markers cyclin D1 and proliferating cell nuclear antigen. A calcium current similar in amplitude to that of ventricular myocytes was present in 16% of the cells. The cardiogenic cell-seeded scaffolds increased the regional mechanical function in the canine heart compared with the unmanipulated hMSC-seeded scaffolds. In addition, the cells prelabeled with fluorescent markers demonstrated myocyte-specific actinin staining with sarcomere spacing similar to that of normal myocytes. In conclusion, the spheroid-derived cells express cardiac-specific proteins and demonstrate a calcium current similar to adult ventricular myocytes. When these cells are implanted into the canine heart, some of these cells appear striated and mechanical function is improved compared with the unmanipulated hMSCs. Further investigation will be required to determine whether the increased mechanical function is due to a differentiation of the cardiogenic cells to myocytes or to other effects.

Culturing of human mesenchymal stem cells as three-dimensional aggregates induces functional expression of CXCR4 that regulates adhesion to endothelial cells.

Culture-expanded human mesenchymal stem cells (hMSCs) are increasingly used in a variety of preclinical and clinical studies. However, these cells have a low rate of engraftment to bone marrow or damaged tissues. Several laboratories have shown that during isolation and subculturing mesenchymal stem cells quickly lose the expression of CXCR4, the key receptor responsible for lymphocytes and hematopoietic stem cell homing. Here we show that culturing of hMSCs as three-dimensional aggregates (hMSC spheroids) restores CXCR4 functional expression. Expression of CXCR4 inversely correlates with the secretion of SDF-1 by hMSCs. Cells from hMSC spheroids up-regulate expression of CD49b, the alpha2 integrin subunit, and suppress the expression of CD49d, the alpha4 integrin subunit. Transfer of cells from the spheroids back to a monolayer suppresses the expression of CXCR4 and CD49b and restores the expression of CD49d. Treatment of cells from the spheroids with SDF-1 leads to CXCR4 internalization and activation of ERK-1,2. Adhesion of hMSCs to human umbilical vein endothelial cells (HUVECs) was investigated. SDF-1, AMD-3100, or exposure of HUVECs to hypoxia did not affect adhesion of hMSCs from a monolayer to HUVECs. Adhesion of cells from hMSC spheroids to HUVECs was stimulated by SDF-1, AMD-3100, or by exposure of HUVECs to hypoxia. Stimulatory effects of hypoxia and addition of SDF-1 or AMD-3100 were not additive. Overall, our data indicate that the expression of CXCR4 by hMSCs regulates hMSC adhesion to endothelial cells.

Replacing damaged myocardium.

Heart failure survival after diagnosis has barely changed for more than half a century. Recently, investigation has focused on differentiation of stem cells in vitro and their delivery for use in vivo as replacement cardiac contractile elements. Here we report preliminary results using mesenchymal stem cells partially differentiated to a cardiac lineage in vitro. When delivered to the canine heart on an extracellular matrix patch to replace a full-thickness ventricular defect in vivo, they improve regional mechanical function. The delivered cells were also tracked, and some became myocytes with mature sarcomeres.

Voltage-gated ion channel Kv4.3 is associated with Rap guanine nucleotide exchange factors and regulates angiotensin receptor type 1 signaling to small G-protein Rap.

The voltage-gated potassium channel Kv4.3 was coexpressed with its beta-subunit Kv channel-interacting protein 2 and the angiotensin type 1 receptor in HEK-293 cells. Proteomic analysis of proteins coimmunoprecipitated with Kv4.3 revealed that Kv4.3 is associated with Rap guanine nucleotide exchange factors MR-GEF and EPAC-1. Previously, we demonstrated that Kv4.3 interacts with the angiotensin type 1 receptor in HE293 cells and cardiac myocytes. On the basis of this, we investigated the angiotensin type 1 receptor signaling to small G-proteins Ras and Rap-1 in the presence and absence of the Kv4.3-Kv channel-interacting protein 2 macromolecular complex. Ras activation was not significantly affected by coexpression of Kv4.3 and Kv channel-interacting protein 2. Ras exhibited a rapid activation-inactivation pattern with maximum activity at 2.5 min after addition of angiotensin II. In contrast, activation of Rap-1 was affected dramatically by coexpression of Kv4.3 and Kv channel-interacting protein 2 with the angiotensin type 1 receptor. In the absence of Kv4.3 and Kv channel-interacting protein 2, stimulation of the angiotensin type 1 receptor resulted in steady activation of Rap-1 that reached a plateau 25 min after addition of angiotensin II. In the presence of Kv4.3 and Kv channel-interacting protein 2, Rap-1 reaches a maximum activity 2.5 min after addition of angiotensin II and then deactivates rapidly, demonstrating a pattern of activation similar to that of Ras. Our findings show that Kv4.3 regulates angiotensin type 1 receptor signaling to the small G-protein Rap-1.

Xenografted adult human mesenchymal stem cells provide a platform for sustained biological pacemaker function in canine heart.

BACKGROUND: Biological pacemaking has been performed with viral vectors, human embryonic stem cells, and adult human mesenchymal stem cells (hMSCs) as delivery systems. Only with human embryonic stem cells are data available regarding stability for >2 to 3 weeks, and here, immunosuppression has been used to facilitate survival of xenografts. The purpose of the present study was to determine whether hMSCs provide stable impulse initiation over 6 weeks without the use of immunosuppression, the "dose" of hMSCs that ensures function over this period, and the catecholamine responsiveness of hMSC-packaged pacemakers. METHODS AND RESULTS: A full-length mHCN2 cDNA subcloned in a pIRES2-EGFP vector was electroporated into hMSCs. Transfection efficiency was estimated by GFP expression. I(HCN2) was measured with patch clamp, and cells were administered into the left ventricular anterior wall of adult dogs in complete heart block and with backup electronic pacemakers. Studies encompassed 6 weeks. I(HCN2) for all cells was 32.1+/-1.3 pA/pF (mean+/-SE) at -150 mV. Pacemaker function in intact dogs required 10 to 12 days to fully stabilize and persisted consistently through day 42 in dogs receiving > or =700,000 hMSCs (approximately 40% of which carried current). Rhythms were catecholamine responsive. Tissues from animals killed at 42 days manifested neither apoptosis nor humoral or cellular rejection. CONCLUSIONS: hMSCs provide a means for administering catecholamine-responsive biological pacemakers that function stably for 6 weeks and manifest no cellular or humoral rejection at that time. Cell doses >700,000 are sufficient for pacemaking when administered to left ventricular myocardium.

Finding fluorescent needles in the cardiac haystack: tracking human mesenchymal stem cells labeled with quantum dots for quantitative in vivo three-dimensional fluorescence analysis.

Stem cells show promise for repair of damaged cardiac tissue. Little is known with certainty, however, about the distribution of these cells once introduced in vivo. Previous attempts at tracking delivered stem cells have been hampered by the autofluorescence of host tissue and limitations of existing labeling techniques. We have developed a novel loading approach to stably label human mesenchymal stem cells with quantum dot (QD) nanoparticles. We report the optimization and validation of this long-term tracking technique and highlight several important biological applications by delivering labeled cells to the mammalian heart. The bright QD crystals illuminate exogenous stem cells in histologic sections for at least 8 weeks following delivery and permit, for the first time, the complete three-dimensional reconstruction of the locations of all stem cells following injection into the heart. Disclosure of potential conflicts of interest is found at the end of this article.

Mesenchymal stem cells support migration, extracellular matrix invasion, proliferation, and survival of endothelial cells in vitro.

We investigated effects of the paracrine factors secreted by human mesenchymal stem cells (hMSCs) on endothelial cell migration, extracellular matrix invasion, proliferation, and survival in vitro. Human mesenchymal stem cells were cultured as a monolayer or as three-dimensional aggregates in hanging drops (hMSC spheroids). We performed analysis of paracrine factors in medium conditioned by a monolayer of hMSCs and hMSC spheroids. Concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, angiogenin, procathepsin B, interleukin (IL)-11, and bone morphogenic protein 2 were increased 5-20 times in medium conditioned by hMSC spheroids, whereas concentrations of IL-6, IL-8, and monocyte hemoattractant protein-1 were not increased. Concentrations of VEGF and angiogenin in medium conditioned by hMSC spheroids showed a weak dependence on the presence of serum, which allows serum-free conditioned medium with elevated concentrations of angiogenic cytokines to be obtained. Medium conditioned by hMSC spheroids was more effective in stimulation of umbilical vein endothelial cell proliferation, migration, and basement membrane invasion than medium conditioned by a monolayer of hMSCs. This medium also promotes endothelial cell survival in vitro. We suggest that culturing of hMSCs as three-dimensional cellular aggregates provides a method to concentrate proangiogenic factors secreted by hMSCs and allows for reduction of serum concentration in conditioned medium. Our data support the hypothesis that hMSCs serve as trophic mediators for endothelial cells. Disclosure of potential conflicts of interest is found at the end of this article.

Angiotensin receptor type 1 forms a complex with the transient outward potassium channel Kv4.3 and regulates its gating properties and intracellular localization.

We report a novel signal transduction complex of the angiotensin receptor type 1. In this complex the angiotensin receptor type 1 associates with the potassium channel alpha-subunit Kv4.3 and regulates its intracellular distribution and gating properties. Co-localization of Kv4.3 with angiotensin receptor type 1 and fluorescent resonance energy transfer between those two proteins labeled with cyan and yellow-green variants of green fluorescent protein revealed that Kv4.3 and angiotensin receptor type I are located in close proximity to each other in the cell. The angiotensin receptor type 1 also co-immunoprecipitates with Kv4.3 from canine ventricle or when co-expressed with Kv4.3 and its beta-subunit KChIP2 in human embryonic kidney 293 cells. Treatment of the cells with angiotensin II results in the internalization of Kv4.3 in a complex with the angiotensin receptor type 1. When stimulated with angiotensin II, angiotensin receptors type 1 modulate gating properties of the remaining Kv4.3 channels on the cell surface by shifting their activation voltage threshold to more positive values. We hypothesize that the angiotensin receptor type 1 provides its internalization molecular scaffold to Kv4.3 and in this way regulates the cell surface representation of the ion channel.

MiRP1 modulates HCN2 channel expression and gating in cardiac myocytes.

MinK-related protein (MiRP1 or KCNE2) interacts with the hyperpolarization-activated, cyclic nucleotide-gated (HCN) family of pacemaker channels to alter channel gating in heterologous expression systems. Given the high expression levels of MiRP1 and HCN subunits in the cardiac sinoatrial node and the contribution of pacemaker channel function to impulse initiation in that tissue, such an interaction could be of considerable physiological significance. However, the functional evidence for MiRP1/HCN interactions in heterologous expression studies has been accompanied by inconsistencies between studies in terms of the specific effects on channel function. To evaluate the effect of MiRP1 on HCN expression and function in a physiological context, we used an adenovirus approach to overexpress a hemagglutinin (HA)-tagged MiRP1 (HAMiRP1) and HCN2 in neonatal rat ventricular myocytes, a cell type that expresses both MiRP1 and HCN2 message at low levels. HA-MiRP1 co-expression with HCN2 resulted in a 4-fold increase in maximal conductance of pacemaker currents compared with HCN2 expression alone. HCN2 activation and deactivation kinetics also changed, being significantly more rapid for voltages between -60 and -95 mV when HA-MiRP1 was co-expressed with HCN2. However, the voltage dependence of activation was not affected. Co-immunoprecipitation experiments demonstrated that expressed HA-MiRP1 and HCN2, as well as endogenous MiRP1 and HCN2, co-assemble in ventricular myocytes. The results indicate that MiRP1 acts as a beta subunit for HCN2 pacemaker channel subunits and alters channel gating at physiologically relevant voltages in cardiac cells.