Aaron D. Kaplan

Patient-specific induced pluripotent stem-cell-derived models of LEOPARD syndrome

The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS–mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.

VEGF administration in chronic myocardial ischemia in pigs

OBJECTIVE Previous investigations have shown the effectiveness of sustained intra- or extravascular administration of vascular endothelial growth factor (VEGF) in chronic myocardial ischemia in improvement of left ventricular function. The present investigations were undertaken in order to evaluate efficacy of a single bolus or local intracoronary delivery. METHODS Yorkshire pigs underwent placement of a left circumflex artery ameroid occluder. Three weeks later the animals were randomized to treatment with VEGF (20 micrograms) accomplished by local intracoronary delivery system (InfusaSleeve, n = 10), intracoronary bolus infusion (n = 7) or by epicardial implantation of an osmotic delivery system (n = 7). An additional group of animals received intracoronary administration of saline and served as a control (n = 9). Three weeks after initiation of therapy, the animals were evaluated with regard to myocardial perfusion and global as well as regional ventricular function. RESULTS All three VEGF treatment groups but not the control animals demonstrated a significant increase in the left-to-left (but not right-to-left) collateral index, myocardial blood flow (pre-therapy LCX vs. LAD (average of all groups): 0.76 +/- 0.35 vs. 0.96 +/- 0.38 ml*min-1*g-1, p = 0.03; post-therapy: LCX vs. LAD: 1.16 +/- 0.39 vs. 1.15 +/- 0.28 ml*min-1*g-1, p = NS) and coronary vasodilatory reserve 3 weeks after growth factor administration. The observed increase in VEGF-induced perfusion correlated with improvement in regional ventricular function in all VEGF-treated groups (pre-therapy vs. post-therapy: i.c. VEGF 20 +/- 5.1 vs. 33 +/- 4.8; local VEGF 16 +/- 2.8 vs. 33.6; pump VEGF 17 +/- 3.8 vs. 34 +/- 4.9 p < 0.05 for all) but not control animals (21 +/- 3.3 vs. 27 +/- 5.8, p = NS). CONCLUSION Single intracoronary delivery (intravascular bolus or local delivery) of VEGF is effective in stimulating physiologically significant angiogenesis in porcine model of chronic myocardial ischemia.

Study familial hypertrophic cardiomyopathy using patient-specific induced pluripotent stem cells

Aims Familial hypertrophic cardiomyopathy (HCM) is one the most common heart disorders, with gene mutations in the cardiac sarcomere. Studying HCM with patient-specific induced pluripotent stem-cell (iPSC)-derived cardiomyocytes (CMs) would benefit the understanding of HCM mechanism, as well as the development of personalized therapeutic strategies. Methods and results To investigate the molecular mechanism underlying the abnormal CM functions in HCM, we derived iPSCs from an HCM patient with a single missense mutation (Arginine442Glycine) in the MYH7 gene. CMs were next enriched from HCM and healthy iPSCs, followed with whole transcriptome sequencing and pathway enrichment analysis. A widespread increase of genes responsible for ‘Cell Proliferation’ was observed in HCM iPSC-CMs when compared with control iPSC-CMs. Additionally, HCM iPSC-CMs exhibited disorganized sarcomeres and electrophysiological irregularities. Furthermore, disease phenotypes of HCM iPSC-CMs were attenuated with pharmaceutical treatments. Conclusion Overall, this study explored the possible patient-specific and mutation-specific disease mechanism of HCM, and demonstrates the potential of using HCM iPSC-CMs for future development of therapeutic strategies. Additionally, the whole methodology established in this study could be utilized to study mechanisms of other human-inherited heart diseases.

Electronic “expression” of the inward rectifier in cardiocytes derived from human-induced pluripotent stem cells

BACKGROUND Human-induced pluripotent stem cell (h-iPSC)-derived cardiac myocytes are a unique model in which human myocyte function and dysfunction are studied, especially those from patients with genetic disorders. They are also considered a major advance for drug safety testing. However, these cells have considerable unexplored potential limitations when applied to quantitative action potential (AP) analysis. One major factor is spontaneous activity and resulting variability and potentially anomalous behavior of AP parameters. OBJECTIVE To demonstrate the effect of using an in silico interface on electronically expressed I(K1), a major component lacking in h-iPSC-derived cardiac myocytes. METHODS An in silico interface was developed to express synthetic I(K1) in cells under whole-cell voltage clamp. RESULTS Electronic I(K1) expression established a physiological resting potential, eliminated spontaneous activity, reduced spontaneous early and delayed afterdepolarizations, and decreased AP variability. The initiated APs had the classic rapid upstroke and spike and dome morphology consistent with data obtained with freshly isolated human myocytes as well as the readily recognizable repolarization attributes of ventricular and atrial cells. The application of 1 µM of BayK-8644 resulted in anomalous AP shortening in h-iPSC-derived cardiac myocytes. When I(K1) was electronically expressed, BayK-8644 prolonged the AP, which is consistent with the existing results on native cardiac myocytes. CONCLUSIONS The electronic expression of I(K1) is a simple and robust method to significantly improve the physiological behavior of the AP and electrical profile of h-iPSC-derived cardiac myocytes. Increased stability enables the use of this preparation for a controlled quantitative analysis of AP parameters, for example, drug responsiveness, genetic disorders, and dynamic behavior restitution profiles.

The Toxoplasma Dense Granule Proteins GRA17 and GRA23 Mediate the Movement of Small Molecules between the Host and the Parasitophorous Vacuole

Toxoplasma gondii is a protozoan pathogen in the phylum Apicomplexa that resides within an intracellular parasitophorous vacuole (PV) that is selectively permeable to small molecules through unidentified mechanisms. We have identified GRA17 as a Toxoplasma-secreted protein that localizes to the parasitophorous vacuole membrane (PVM) and mediates passive transport of small molecules across the PVM. GRA17 is related to the putative Plasmodium translocon protein EXP2 and conserved across PV-residing Apicomplexa. The PVs of GRA17-deficient parasites have aberrant morphology, reduced permeability to small molecules, and structural instability. GRA17-deficient parasites proliferate slowly and are avirulent in mice. These GRA17-deficient phenotypes are rescued by complementation with Plasmodium EXP2. GRA17 functions synergistically with a related protein, GRA23. Exogenous expression of GRA17 or GRA23 alters the membrane conductance properties of Xenopus oocytes in a manner consistent with a large non-selective pore. Thus, GRA17 and GRA23 provide a molecular basis for PVM permeability and nutrient access.

Relaxin Suppresses Atrial Fibrillation by Reversing Fibrosis and Myocyte Hypertrophy, and Increasing Conduction Velocity and Sodium Current in Spontaneously Hypertensive Rat Hearts

Rationale: Atrial fibrillation (AF) contributes significantly to morbidity and mortality in elderly and hypertensive patients and has been correlated to enhanced atrial fibrosis. Despite a lack of direct evidence that fibrosis causes AF, reversal of fibrosis is considered a plausible therapy. Objective: To evaluate the efficacy of the antifibrotic hormone relaxin (RLX) in suppressing AF in spontaneously hypertensive rats (SHR). Methods and Results: Normotensive Wistar-Kyoto (WKY) and SHR were treated for 2 weeks with vehicle (WKY+V and SHR+V) or RLX (0.4 mg/kg per day, SHR+RLX) using implantable mini-pumps. Hearts were perfused, mapped optically to analyze action potential durations, intracellular Ca2+ transients, and restitution kinetics, and tested for AF vulnerability. SHR hearts had slower conduction velocity (CV; P<0.01 versus WKY), steeper CV restitution kinetics, greater collagen deposition, higher levels of transcripts for transforming growth factor-&bgr;, metalloproteinase-2, metalloproteinase-9, collagen I/III, and reduced connexin 43 phosphorylation (P<0.05 versus WKY). Programmed stimulation triggered sustained AF in SHR (n=5/5) and SHR+V (n=4/4), but not in WKY (n=0/5) and SHR+RLX (n=1/8; P<0.01). RLX treatment reversed the transcripts for fibrosis, flattened CV restitution kinetics, reduced action potential duration at 90% recovery to baseline, increased CV (P<0.01), and reversed atrial hypertrophy (P<0.05). Independent of antifibrotic actions, RLX (0.1 µmol/L) increased Na+ current density, INa (≈2-fold in 48 hours) in human cardiomyocytes derived from inducible pluripotent stem cells (n=18/18; P<0.01). Conclusions: RLX treatment suppressed AF in SHR hearts by increasing CV from a combination of reversal of fibrosis and hypertrophy and by increasing INa. The study provides compelling evidence that RLX may provide a novel therapy to manage AF in humans by reversing fibrosis and hypertrophy and by modulating cardiac ionic currents.

Modeling and study of the mechanism of dilated cardiomyopathy using induced pluripotent stem cells derived from individuals with Duchenne muscular dystrophy

ABSTRACT Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD), and is characterized by progressive weakness in skeletal and cardiac muscles. Currently, dilated cardiomyopathy due to cardiac muscle loss is one of the major causes of lethality in late-stage DMD patients. To study the molecular mechanisms underlying dilated cardiomyopathy in DMD heart, we generated cardiomyocytes (CMs) from DMD and healthy control induced pluripotent stem cells (iPSCs). DMD iPSC-derived CMs (iPSC-CMs) displayed dystrophin deficiency, as well as the elevated levels of resting Ca2+, mitochondrial damage and cell apoptosis. Additionally, we found an activated mitochondria-mediated signaling network underlying the enhanced apoptosis in DMD iPSC-CMs. Furthermore, when we treated DMD iPSC-CMs with the membrane sealant Poloxamer 188, it significantly decreased the resting cytosolic Ca2+ level, repressed caspase-3 (CASP3) activation and consequently suppressed apoptosis in DMD iPSC-CMs. Taken together, using DMD patient-derived iPSC-CMs, we established an in vitro model that manifests the major phenotypes of dilated cardiomyopathy in DMD patients, and uncovered a potential new disease mechanism. Our model could be used for the mechanistic study of human muscular dystrophy, as well as future preclinical testing of novel therapeutic compounds for dilated cardiomyopathy in DMD patients. Highlighted Article: Patient-derived induced pluripotent stem cells are used to establish an in vitro model of DMD-associated cardiomyopathy that could be used for future preclinical testing.

Mechanism of automaticity in cardiomyocytes derived from human induced pluripotent stem cells

BACKGROUND AND OBJECTIVES The creation of cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CMs) has spawned broad excitement borne out of the prospects to diagnose and treat cardiovascular diseases based on personalized medicine. A common feature of hiPS-CMs is their spontaneous contractions but the mechanism(s) remain uncertain. METHODS Intrinsic activity was investigated by the voltage-clamp technique, optical mapping of action potentials (APs) and intracellular Ca(2+) (Cai) transients (CaiT) at subcellular-resolution and pharmacological interventions. RESULTS The frequency of spontaneous CaiT (sCaiT) in monolayers of hiPS-CMs was not altered by ivabradine, an inhibitor of the pacemaker current, If despite high levels of HCN transcripts (1-4). HiPS-CMs had negligible If and IK1 (inwardly-rectifying K(+)-current) and a minimum diastolic potential of -59.1±3.3mV (n=18). APs upstrokes were preceded by a depolarizing-foot coincident with a rise of Cai. Subcellular Cai wavelets varied in amplitude, propagated and died-off; larger Cai-waves triggered cellular sCaTs and APs. SCaiTs increased in frequency with [Ca(2+)]out (0.05-to-1.8mM), isoproterenol (1μM) or caffeine (100μM) (n≥5, p<0.05). HiPS-CMs became quiescent with ryanodine receptor stabilizers (K201=2μM); tetracaine; Na-Ca exchange (NCX) inhibition (SEA0400=2μM); higher [K(+)]out (5→8mM), and thiol-reducing agents but could still be electrically stimulated to elicit CaiTs. Cell-cell coupling of hiPS-CM in monolayers was evident from connexin-43 expression and CaiT propagation. SCaiTs from an ensemble of dispersed hiPS-CMs were out-of-phase but became synchronous through the outgrowth of inter-connecting microtubules. CONCLUSIONS Automaticity in hiPS-CMs originates from a Ca(2+)-clock mechanism involving Ca(2+) cycling across the sarcoplasmic reticulum linked to NCX to trigger APs.

Hemodynamic Analysis of Arterial Blood Flow in the Coiled Umbilical Cord

The most significant anatomical structure of the umbilical cord is its level of coiling. The coiled geometry of the umbilical cord largely affects umbilical blood flow that is vital for fetus’s well-being and normal development. In this study, we developed a computational model of steady blood flow through the coiled structure of an umbilical artery. The results showed that the driving pressure for a given blood flow rate is increasing as the number of coils in cord structure increases. The driving gradient pressures also vary with the pitch that dictates the coils’ spreading. The coiled structure is resulting in interwoven streamlines along the helix and wall shear stresses (WSS) with significant spatial gradients along the cross-sectional perimeter anywhere within the helical coil. These gradients may have an adverse effect on the development of the fetus cardiovascular system in cases with over coiling (OC) or under coiling (UC) characteristics. The number of coils does not affect the distribution and levels of WSS. However, when the coils are more spread (eg, larger pitch number), the maximal WSS is significantly smaller. Cases with twisted and OC cords seem to yield very large values and gradients of WSS, which may place the fetus into high risk of abnormal development.

The cardiomyocyte lineage is critical for optimization of stem cell therapy in a mouse model of myocardial infarction

We recently described a murine embryonic stem cell (ESC) line engineered to express the activated Notch 4 receptor in a tetracycline (doxcycline; Dox) regulated fashion (tet‐notch4 ESCs). Notch 4 induction in Flk1+ hematopoietic and vascular progenitors from this line respecified them to a cardiovascular fate. We reasoned that these cells would be ideal for evaluating the contribution of the cardiomyocyte and vascular lineages to the functional improvement noted following stem cell transplantation in infarcted hearts. Flk‐1+ Tet‐notch4 cells from d 3 embryoid bodies exposed to doxycycline (Dox+) were compared to uninduced (Dox−) Flk‐1+ cells. Mice underwent transplantation of 5 × 105 Dox+ cells, Dox− cells, or an equal volume of serum‐free medium after surgically induced myocardial infarction. The mean ejection fraction was 59 ± 15, 46 ± 17, and 39 ± 13% in the Dox+, Dox−, and serum‐free medium groups, respectively (P<0.05 for the differences among all 3 groups). Immunohistochemistry of hearts injected with Dox+ grafts expressed myocardial and vascular markers, whereas grafts of Dox−cells expressed primarily vascular markers. We conclude that cardiovascular progenitors are more effective than vascular progenitors in improving function after myocardial infarction. The transplantation of appropriate cell types is critical for maximizing the benefit of cardiovascular cell therapy.—Adler, E. D., Chen, V. C., Bystrup, A., Kaplan, A. D., Giovannone, S., Eriley‐Saebo, K., Young, W., Kattman, S., Mani, V., Laflamme, M., Zhu, W.‐Z., Fayad, Z., Keller, G. The cardiomyocyte lineage is critical for optimization of stem cell therapy in a mouse model of myocardial infarction. FASEB J. 24, 1073–1081 (2010). www.fasebj.org