Connie Y. Chang

Proarrhythmic Potential of Mesenchymal Stem Cell Transplantation Revealed in an In Vitro Coculture Model

Background— Mesenchymal stem cells (MSCs) are bone marrow stromal cells that are in phase 1 clinical studies of cellular cardiomyoplasty. However, the electrophysiological effects of MSC transplantation have not been studied. Although improvement of ventricular function would represent a positive outcome of MSC transplantation, focal application of stem cells has the potential downside of creating inhomogeneities that may predispose the heart to reentrant arrhythmias. In the present study we use an MSC and neonatal rat ventricular myocyte (NRVM) coculture system to investigate potential proarrhythmic consequences of MSC transplantation into the heart. Methods and Results— Human MSCs were cocultured with NRVMs in ratios of 1:99, 1:9, and 1:4 and optically mapped. We found that conduction velocity was decreased in cocultures compared with controls, but action potential duration (APD80) was not affected. Reentrant arrhythmias were induced in 86% of cocultures containing 10% and 20% MSCs (n=36) but not in controls (n=7) or cocultures containing only 1% MSCs (n=4). Immunostaining, Western blot, and dye transfer revealed the presence of functional gap junctions involving MSCs. Conclusions— Our results suggest that mixtures of MSCs and NRVMs can produce an arrhythmogenic substrate. The mechanism of reentry is probably increased tissue heterogeneity resulting from electric coupling of inexcitable MSCs with myocytes.

Matrix Rigidity Controls Endothelial Differentiation and Morphogenesis of Cardiac Precursors

Culturing cardiac precursors on a surface with the rigidity of heart tissue increases the yield of endothelial cells. The Heart of the Matrix Cardiosphere-derived cells (CDCs) are adult stem cells with the potential to differentiate into endothelial cells and cardiomyocytes, which are the major cell types of the heart. Their potential to induce cardiac regeneration after myocardial infarct is currently being clinically tested. Kshitiz et al. showed that CDCs cultured on a substrate with a rigidity matching that of normal myocardium yielded higher proportions of cells with the adhesion molecule CD31 (a marker of differentiated endothelial cells) than did those cultured on less or more rigid substrata. CDCs cultured on substratum equivalent in rigidity to the myocardium developed into organized cellular networks reminiscent of blood vessels and appeared to integrate more efficiently into the vasculature of ischemic rat myocardium. The process of sensing substratum rigidity occurred throughout the in vitro culture period, and the signaling pathway involved required the guanosine triphosphatase (GTPase)–activating protein p190RhoGAP acting through various downstream effectors, including the GTPase RhoA. These results could potentially increase the efficacy of regenerative therapies that use CDCs to repair hearts after myocardial infarction. Tissue development and regeneration involve tightly coordinated and integrated processes: selective proliferation of resident stem and precursor cells, differentiation into target somatic cell type, and spatial morphological organization. The role of the mechanical environment in the coordination of these processes is poorly understood. We show that multipotent cells derived from native cardiac tissue continually monitored cell substratum rigidity and showed enhanced proliferation, endothelial differentiation, and morphogenesis when the cell substratum rigidity closely matched that of myocardium. Mechanoregulation of these diverse processes required p190RhoGAP, a guanosine triphosphatase–activating protein for RhoA, acting through RhoA-dependent and -independent mechanisms. Natural or induced decreases in the abundance of p190RhoGAP triggered a series of developmental events by coupling cell-cell and cell-substratum interactions to genetic circuits controlling differentiation.

Hyaluronic acid-human blood hydrogels for stem cell transplantation

Tissue engineering-based approaches have the potential to improve stem cell engraftment by increasing cell delivery to the myocardium. Our objective was to develop and characterize a naturally-derived, autologous, biodegradable hydrogel in order to improve acute stem cell retention in the myocardium. HA-blood hydrogels (HA-BL) were synthesized by mixing in a 1:1(v/v) ratio, lysed whole blood and hyaluronic acid (HA), whose carboxyl groups were functionalized with N-hydroxysuccinimide (NHS) to yield HA succinimidyl succinate (HA-NHS). We performed physical characterization and measured survival/proliferation of cardiosphere-derived cells (CDCs) encapsulated in the hydrogels. Hydrogels were injected intra-myocardially or applied epicardially in rats. NHS-activated carboxyl groups in HA react with primary amines present in blood and myocardium to form amide bonds, resulting in a 3D hydrogel bound to tissue. HA-blood hydrogels had a gelation time of 58±12 s, swelling ratio of 10±0.5, compressive and elastic modulus of 14±3 and 1.75±0.6 kPa respectively. These hydrogels were not degraded at 4 wks by hydrolysis alone. CDC encapsulation promoted their survival and proliferation. Intra-myocardial injection of CDCs encapsulated in these hydrogels greatly increased acute myocardial retention (p=0.001). Epicardial application of HA-blood hydrogels improved left ventricular ejection fraction following myocardial infarction (p=0.01). HA-blood hydrogels are highly adhesive, biodegradable, promote CDC survival and increase cardiac function following epicardial application after myocardial infarction.

Gene Transfer of Connexin43 Mutants Attenuates Coupling in Cardiomyocytes. Novel Basis for Modulation of Cardiac Conduction by Gene Therapy

Modification of electrical conduction would be a useful principle to recruit in preventing or treating certain arrhythmias, notably ventricular tachycardia (VT). Here we pursue a novel gene transfer approach to modulate electrical conduction by reducing gap junctional intercellular communication (GJIC) and hence potentially modify the arrhythmia substrate. The ultimate goal is to develop a nondestructive approach to uncouple zones of slow conduction by focal gene transfer. Lentiviral vectors encoding connexin43 (Cx43) internal loop mutants were produced and studied in vitro. Transduction of neonatal rat ventricular myocytes (NRVMs) revealed the expected subcellular localization of the mutant gene product. Fluorescent dye transfer studies showed a significant reduction of GJIC in NRVMs that had been genetically modified. Additionally, adjacent mutant gene-modified NRVMs displayed delayed calcium transients, indicative of electrical uncoupling. Multi-site optical mapping of action potential (AP) propagation in gene-modified NRVM monolayers revealed a 3-fold slowing of conduction velocity (CV) relative to nontransduced NRVMs. In conclusion, lentiviral vector–mediated gene transfer of Cx43 mutants reduced GJIC in NRVMs. Electrical charge transfer was also reduced as evidenced by delayed calcium transients in adjacent NRVMs and reduced CV in NRVM monolayers. These data validate a molecular tool that opens the prospect for gene transfer targeting gap junctions as an approach to modulate cardiac conduction.

Spiral Waves and Reentry Dynamics in an In Vitro Model of the Healed Infarct Border Zone

Rationale: Reentry underlies most ventricular tachycardias (VTs) seen postmyocardial infarction (MI). Mapping studies reveal that the majority of VTs late post-MI arise from the infarct border zone (IBZ). Objective: To investigate reentry dynamics and the role of individual ion channels on reentry in in vitro models of the “healed” IBZ. Methods and Results: We designed in vitro models of the healed IBZ by coculturing skeletal myotubes with neonatal rat ventricular myocytes and performed optical mapping at high temporal and spatial resolution. In culture, neonatal rat ventricular myocytes mature to form striated myocytes and electrically uncoupled skeletal myotubes simulate fibrosis seen in the healed IBZ. High resolution mapping revealed that skeletal myotubes produced localized slowing of conduction velocity (CV), increased dispersion of CV and directional-dependence of activation delay without affecting myocyte excitability. Reentry was easily induced by rapid pacing in cocultures; treatment with lidocaine, a Na+ channel blocker, significantly decreased reentry rate and CV, increased reentry path length and terminated 30% of reentrant arrhythmias (n=18). In contrast, nitrendipine, an L-type Ca2+ channel blocker terminated 100% of reentry episodes while increasing reentry cycle length and path length and decreasing reentry CV (n=16). K+ channel blockers increased reentry action potential duration but infrequently terminated reentry (n=12). Conclusions: Cocultures reproduce several architectural and electrophysiological features of the healed IBZ. Reentry termination by L-type Ca2+ channel, but not Na+ channel, blockers suggests a greater Ca2+-dependence of propagation. These results may help explain the low efficacy of pure Na+ channel blockers in preventing and terminating clinical VTs late after MI.

Dynamics of Early Afterdepolarization-Mediated Triggered Activity in Cardiac Monolayers

Early afterdepolarizations (EADs) are voltage oscillations that occur during the repolarizing phase of the cardiac action potential and cause cardiac arrhythmias in a variety of clinical settings. EADs occur in the setting of reduced repolarization reserve and increased inward-over-outward currents, which intuitively explains the repolarization delay but does not mechanistically explain the time-dependent voltage oscillations that are characteristic of EADs. In a recent theoretical study, we identified a dual Hopf-homoclinic bifurcation as a dynamical mechanism that causes voltage oscillations during EADs, depending on the amplitude and kinetics of the L-type Ca(2+) channel (LTCC) current relative to the repolarizing K(+) currents. Here we demonstrate this mechanism experimentally. We show that cardiac monolayers exposed to the LTCC agonists BayK8644 and isoproterenol produce EAD bursts that are suppressed by the LTCC blocker nitrendipine but not by the Na(+) current blocker tetrodoxin, depletion of intracellular Ca(2+) stores with thapsigargin and caffeine, or buffering of intracellular Ca(2+) with BAPTA-AM. These EAD bursts exhibited a key dynamical signature of the dual Hopf-homoclinic bifurcation mechanism, namely, a gradual slowing in the frequency of oscillations before burst termination. A detailed cardiac action potential model reproduced the experimental observations, and identified intracellular Na(+) accumulation as the likely mechanism for terminating EAD bursts. Our findings in cardiac monolayers provide direct support for the Hopf-homoclinic bifurcation mechanism of EAD-mediated triggered activity, and raise the possibility that this mechanism may also contribute to EAD formation in clinical settings such as long QT syndromes, heart failure, and increased sympathetic output.

MR Imaging of Normal Hip Anatomy

Understanding normal anatomy of the hip is important for diagnosing its pathology. MR arthrography is more sensitive for the detection of intra-articular pathology than noncontrast MR imaging. Important elements of the osseous structures on MR imaging include the alignment and the marrow. Acetabular ossicles may be present. Normal variations involving the cartilage include the supra-acetabular fossa and the stellate lesion. Important muscles of the hip are the sartorius, rectus femoris, iliopsoas, gluteus minimus and medius, adductors, and hamstrings. The iliofemoral, ischiofemoral, and pubofemoral ligaments represent thickenings of the joint capsule that reinforce and stabilize the hip joint. Normal variations in the labrum include labral sulcus and absent labrum. The largest nerves in the hip and thigh are the sciatic nerve, the femoral nerve, and the obturator nerve.

Evaluation of rapid point-of-care creatinine testing in the radiology service of a large academic medical center: Impact on clinical operations and patient disposition

BACKGROUND Measurement of creatinine and calculation of the estimated glomerular filtration rate (eGFR) are widely employed to identify patients with chronic kidney disease who are at risk for contrast induced acute kidney injury and nephrogenic systemic fibrosis. However, patients may present for radiologic studies without a recent creatinine/eGFR necessitating cancelation of the study or performance of the scan without contrast. Both of these approaches are suboptimal. METHODS We implemented a rapid whole blood point-of-care (POCT) creatinine test (iSTAT, Abbott Point-of-Care) in our radiology department and assessed the impact on clinical operations. RESULTS Over a 7-month period a total of 3087 creatinine tests were performed. Overall 5.3% of patients presenting for scans (441/month) did not have a recent eGFR. An audit of 1 month of creatinine/eGFR values showed that 74% were normal permitting the scan to be performed without further consideration. Of the abnormal values 74% were performed with contrast and 26% without. Of note 78% of patients with an abnormal eGFR had a normal creatinine value. The cost of the POC test was

Association between distal ulnar morphology and extensor carpi ulnaris tendon pathology

10.06 compared to a cost of

Is Biopsying the Paravertebral Soft Tissue as Effective as Biopsying the Disk or Vertebral Endplate? 10-Year Retrospective Review of CT-Guided Biopsy of Diskitis-Osteomyelitis

5.32 (including phlebotomy) for a creatinine performed in the central laboratory. The added incremental cost for the POC test was therefore