Ruoya Ho

The cAMP-responsive Rap1 Guanine Nucleotide Exchange Factor, Epac, Induces Smooth Muscle Relaxation by Down-regulation of RhoA Activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC20) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2′-O-Me-cAMP (“007”), significantly reduced agonist-induced contractile force, RLC20, and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI2 analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca2+ desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

Smooth and Cardiac Muscle-selective Knock-out of Krüppel-like Factor 4 Causes Postnatal Death and Growth Retardation

Krüppel-like factor 4 (Klf4) is a transcription factor involved in differentiation and proliferation in multiple tissues. We demonstrated previously that tamoxifen-induced deletion of the Klf4 gene in mice accelerated neointimal formation but delayed down-regulation of smooth muscle cell differentiation markers in carotid arteries following injury. To further determine the role of Klf4 in the cardiovascular system, we herein derived mice deficient for the Klf4 gene in smooth and cardiac muscle using the SM22α promoter (SM22α-CreKI+/Klf4loxP/loxP mice). SM22α-CreKI+/Klf4loxP/loxP mice were born at the expected Mendelian ratio, but they gradually died after birth. Although ∼40% of SM22α-CreKI+/Klf4loxP/loxP mice survived beyond postnatal day 28, they exhibited marked growth retardation. In wild-type mice, Klf4 was expressed in the heart from late embryonic development through adulthood, whereas it was not expressed in smooth muscle. No changes were observed in morphology or expression of smooth muscle cell differentiation markers in vessels of SM22α-CreKI+/Klf4loxP/loxP mice. Of interest, cardiac output was significantly decreased in SM22α-CreKI+/Klf4loxP/loxP mice, as determined by magnetic resonance imaging. Moreover, a lack of Klf4 in the heart resulted in the reduction in expression of multiple cardiac genes, including Gata4. In vivo chromatin immunoprecipitation assays on the heart revealed that Klf4 bound to the promoter region of the Gata4 gene. Results provide novel evidence that Klf4 plays a key role in late fetal and/or postnatal cardiac development.

Intracellular Chloride Accumulation and Subcellular Elemental Distribution During Atrial Fibrillation

Background—Ion channel remodeling occurs during atrial fibrillation (AF); however, the extent of alteration in the subcellular distribution of elements (Na, K, Cl, Ca, Mg, P) is unknown. Electron probe microanalysis was used to determine the total (free+bound) in vivo subcellular concentration of these elements during AF. Methods and Results—The left atrial appendage (LAA) was snap-frozen in situ after pacing (640 bpm) for 3 minutes (n=5 dogs), 30 minutes (n=3), or 48 hours (n=5). Dogs in sinus rhythm (n=3) served as controls. Whole-cell, cytosolic, and mitochondrial elemental concentrations were measured in cryosections. LAA effective refractory period (ERP) was measured before and after pacing. LAA ERP decreased significantly after 48 hours (116±3 to 88±10 ms, P =0.02). Whole-cell Cl increased by 9.0 mmol/L and 17 mmol/L after 3 and 30 minutes of pacing, respectively (P <0.0001), without a concomitant increase in Na. However, at 48 hours, whole-cell Na was reduced by 51% (P <0.01). Cytosolic Ca increased by 1.1 mmol/kg dry wt after 3 minutes (P <0.005), but mitochondrial Ca remained low and unchanged. Cell size measured in transverse cryosections increased after 3 minutes of pacing (75±5 to 109±11 &mgr;m2, P =0.007) but returned to baseline by 30 minutes (66±5 &mgr;m2). Conclusions—Intracellular Cl accumulation induced by rapid pacing is a novel finding and may play a role in AF pathogenesis by causing resting membrane depolarization and ERP reduction. There was no evidence of cellular or mitochondrial Ca overload despite the development of electrical remodeling and transient increase in cytoplasmic Ca.

The Actin-associated Protein Palladin Is Required for Development of Normal Contractile Properties of Smooth Muscle Cells Derived from Embryoid Bodies

Palladin is a widely expressed actin-associated protein localized at stress fibers, focal adhesions, and other actin-based structures, playing a significant role in cell adhesion and cell motility. Knockout of Palladin in mice is embryonic lethal, demonstrating the importance of Palladin in development yet its role in the vasculature is not known. In the present study, smooth muscle cell (SMC) markers, such as myosin, actin, caldesmon, calponin, and LPP, were down-regulated in embryoid bodies (EBs) derived from embryonic stem cells lacking Palladin. Transgenic embryonic stem cell lines were generated that stably expressed a puromycin-resistance gene under the control of a SM α-actin (SMA) promoter. Negative selection was then used to purify SMCs from EBs. Purified SMCs expressing multiple SMC markers were designated APSCs (SMA-puromycin-selected cells). Palladin null APSCs express significantly less myosin, actin, calponin, and h-caldesmon. The filamentous (F) to globular (G) actin ratio, known to regulate myocardin family transcription factors, was also decreased. Palladin null APSCs showed increased cell adhesion and decreased cell motility. Importantly, Palladin null APSCs within collagen gels generated less maximum contractile force when stimulated with endothelin-1, sphingosine 1-phosphate (S1P), and thrombin. Myosin light chains (MLC20) were phosphorylated by lysophosphatidic acid to the same extent in Palladin null and wild type APSCs but myosin content/total protein was reduced by >50%, consistent with the observed decreases in contractility. All together, these results suggest that Palladin is essential for expression of the full complement of contractile proteins necessary for optimal force development of SMCs derived from EBs.

Signaling Pathways That Control Rho Kinase Activity Maintain the Embryonic Epicardial Progenitor State

Background: Epicardial cells are a potential source of progenitor cells for revascularization of the injured heart. Results: Decreased p63RhoGEF and GEF-H1 and increased Epac, p190RhoGAP, and Rnds activities suppress RhoA signaling in epicardial progenitors. Conclusion: The embryonic epicardial progenitor state is maintained by signaling pathways that control RhoA activity. Significance: Manipulation of these signaling molecules might promote cardiac revascularization. This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury.

Parallel electron energy-loss spectroscopy free from gain variation

A new method is presented for removing the effect of the gain variation of parallel detectors used for the quantitation of trace elements with electron energy loss spectroscopy (EELS). Use of the ratio of two first-difference spectra eliminates the effect of gain variation of the detector, therefore eliminating the need for gain normalization and dark current subtraction. This method is particularly suitable for revealing small signals superimposed on a large background, a typical scenario for trace element quantitation of both biological and inorganic materials. This method has been tested on a system with a cooled CCD camera as the parallel detector and illustrated by the analysis of low concentration Ca in an organic matrix. The method is expected to be generally applicable to spectral analysis affected by gain variations of parallel detectors.

Calcium content of peripheral and central mitochondria in the guinea pig myocardium: electron probe analysis

We quantitated subcellular elemental concentrations in stimulated and resting guinea pig myocardium to determine whether species-specific properties of guinea pigs or the subcellular localization of mitochondria accounted for reports of higher mitochondrial Ca in guinea pigs than in other species. Small papillary muscles or trabeculae isolated from guinea pig ventricles were stimulated to raise cytosolic [Ca(2+)](i) by two methods: (1). tetanizing by rapid pacing preparations in which Ca(2+) uptake by the sarcoplasmic reticulum was inhibited with cyclopiazonic acid or (2). freeze trapping paced muscles near-peak systole. Electron probe X-ray microanalysis showed no significant difference between the (low, approximately 0.4 mmol/kg dry weight) mitochondrial Ca content of stimulated guinea pig hearts, compared to mitochondria of other species, such as rat and hamsters, and the Ca contents of peripheral and central mitochondria were also not significantly different.

Thickness determination of biological thin sections by multiple-least-squares fitting of the carbon K-edge in the electron energy-loss spectrum.

We show that the local thickness of biological thin sections can be measured by fitting the electron energy-loss spectrum of the plurally scattered carbon K-edge, and that such measurements are consistent with the results obtained by a conventional method based on the relationship t/lambda alpha ln(It/I0). The new method allows the simultaneous measurement and/or mapping of both local thickness and core-level excitations (e.g. Ca L-edge) in this energy range (280-400 eV) without requiring the measurement of the zero-loss and the valence/plasmon excitations.

A high-sensitivity CCD system for parallel electron energy-loss spectroscopy (CCD for EELS)

A cooled frame transfer CCD camera system was developed and tested as a parallel detector in an electron energy‐loss spectrometer mounted on a transmission electron microscope. The use of a shutterless camera with a frame transfer CCD collected virtually 100% of the photon signal with a reasonably fast acquisition time. The system detective quantum efficiency was over 90% under normal experimental conditions. Because of the low channel to channel gain variations in the CCD, the signal‐to‐noise ratio and the detection limit were substantially better than that obtained with a silicon intensified target (SIT) camera, and direct fitting to the standard data was feasible. Quantitation at the phosphorus L edge generated from a phosphoprotein, Phosvitin, showed that, under identical experimental conditions, direct fitting of spectra obtained with this CCD system gave better sensitivity than that given by the SIT camera system. Because of its larger pixel charge well, the CCD system can also operate at a much higher beam current, resulting in a significant reduction in the time required for elemental mapping at a given sensitivity.

Calcium Quantitation with a Parallel Electron Energy Loss Spectroscopy/Cooled Charge-Coupled Device/200 keV System

: We evaluated factors affected the accuracy and precision of quantitating trace concentration of Ca with electron energy loss spectroscopy (EELS). These factors include internal reflection in the spectrometer, precision of correlation between standards and experimental spectra, and radiation damage-induced spectral changes. We present methods of correcting for these effects and improving the reliability of trace Ca quantitation. A two-step fitting procedure is described that improves the retrieval of small Ca signals from the large background common to biological specimens. After optimizing the experimental conditions and data processing procedures, our current system can detect about 2.2 mmol/kg Ca in a 730-Å thick specimen at a total dose of about 410 nA. sec at 95% confidence level by fitting the first difference spectra. Because of the 0.1% residual gain variation after gain normalization, the first difference spectrum fitting is still the preferred method for trace Ca quantitation. Our study also demonstrates the clear advantage of using a 200 keV system, instead of 120 keV or lower accelerating voltages, for EELS analysis of relatively thick biological cryosections.