[Developing a Chinese PI-RADS v2-based nomogram for predicting clinically significant prostate cancer in patients with a prior negative biopsy].

Objective: To develop a nomogram based on prostate imaging reporting and data system version 2 (PI-RADS v2) to predict clinically significant prostate cancer in patients with a prior negative prostate biopsy. Methods: The clinical and pathological data of 231 patients who underwent repeat prostate biopsy and multiparametric MRI (mpMRI) were reviewed. Based on PI-RADS v2, the mpMRI results were assigned as PI-RADS grade from 0 to 2. A Logistic regression nomogram for predicting the probabilities of clinically significant prostate cancer were constructed. The performances of the nomogram were assessed using area under the receiver operating characteristic (ROC) curve, calibrations and decision curve analysis. Results: Of the total 231 repeat prostate biopsy patients, clinically significant prostate cancer was detected in 59 cases(25.5%). In multivariate Logistic regression analysis, age, prostate specific antigen (PSA), prostate volume (PV), digital rectal examination (DRE) and mpMRI results were significant independent predictors of the diagnosis of clinically significant prostate cancer (P<0.05). The nomogram with super predictive accuracy were constructed (AUC=0.927, P<0.001), and exhibited excellent calibration. Decision curve analysis also demonstrated a high net benefit across a wide range of threshold probabilities . Conclusions: PI-RADS v2 combined with age, PSA, PV and DRE can predict the probability of clinically significant prostate cancer in patients with negative initial biopsies. The nomogram generated may help the decision-making process in patients with prior benign histology before the performance of repeat biopsy.

[Development of a Chinese nomogram based on muti-parametric magnetic resonance for predicting the probability of prostate cancer in patients after initial negative biopsy].

Objective: To develop a predictive nomogram based on multi-parametric magnetic resonance imaging (mpMRI) information to identify men more likely to have a cancer diagnosed on repeat prostate biopsy. Methods: The clinical data of 237 patients who received repeat prostate biopsy after initial negative biopsy from Department of Urology of Peking University First Hospital between January 2001 and August 2016 was reviewed. Patient age, body mass index (BMI), serum total prostate-specific antigen (PSA), percent free PSA (f/t), prostate volume (PV), PSA density (PSAD), PSA velocity (PSAV), digital rectal examination (DRE), transrectal ultrasound (TRUS)and mpMRI results were included in the univariate and multivariate analysis. A nomogram was developed using selected variables and the area under the receiver operating characteristic (ROC) curve was calculated as a measure of discrimination. Results: A total of 76 patients (32.07%) had prostate cancer (PCa) detected on repeat biopsy. Based on univariate and multivariate logistic regression analysis, the patient age, PSA, PV, DRE and mpMRI results were independent predictors for the diagnosis of PCa on repeat biopsy. The current nomogram performed well (AUC=0.910) and showed excellent calibration. Conclusions: Multi-parametric magnetic resonance imaging combined with age, PSA, PV and DRE can predict the probability of PCa in patients with initial negative biopsy. The nomogram might help in decision-making for men with prior benign histology before the performance of repeat biopsy.

[Predictive factor analysis of time to progression of castration-resistant prostate cancer after androgen deprivation therapy].

OBJECTIVE: To explore risk factors including prostate-specific antigen (PSA) kinetics for the prediction of castration-resistant prostate cancer (CRPC), and to build a practical model for predicting the progression to CRPC after androgen deprivation therapy (ADT) so as to facilitate clinicians in decision-making for prostate cancer patients receiving ADT. METHODS: A total of 185 patients with prostate cancer who had received ADT as the primary therapy in Department of Urology of Peking University First Hospital from 2003 to 2014 were enrolled retrospectively. All the patients were diagnosed with prostate cancer via prostate biopsy and followed up every four weeks from the initiation of ADT. All the patients received ADT with luteinizing hormone-releasing hormone agonists (LHRH-A) or surgical castration accompanied with an antiandrogen (bicalutamide or flutamide, combined androgen blockade). The clinical information of the patients were collected including age, clinical TNM stage, Gleason score (GS), risk groups of prostate cancer, PSA at the initiation of ADT, PSA nadir after ADT, PSA decline velocity, and the time to PSA nadir. The end point of this study was the diagnosis of CRPC, which was based on the European Association of Urology (EAU) Guideline 2016. Cox proportional hazards regression models were established to analyze and estimate their effects on the time of progression to CRPC. RESULTS: In this study, 185 patients with prostate cancer who had received ADT as the primary therapy were included. The mean age was (71.02±8.67) years. The median time to progression to CRPC in this cohort was 38 months (ranging from 4 to 158 months). On univariate analysis, we found clinical T stage, N stage, the metastasis state before ADT, risk groups of prostate cancer, PSA decline velocity, and PSA nadir were all related to the time to CRPC progression, P<0.01 for all the above variables. And on multivariate analysis, the presence of distant metastasis before ADT (HR=6.030, 95% CI: 3.229-11.263, P=0.001), higher PSA nadir (HR=1.185, 95% CI: 1.080-1.301, P=0.001), higher PSA decline velocity>11 µg/(L×month) (HR=2.124, 95% CI: 1.195-3.750, P=0.001), and time to PSA nadir ≤9 months (HR=3.623, 95% CI: 1.640-4.817, P=0.004) were found to be significantly associated with an increased risk of progression to CRPC. CONCLUSION: Patients with rapid decreasing of PSA in the initial ADT were more likely to progress to CRPC.

[Clinical and pathological characteristics of prostate cancer patients younger than 55 years old].

Objective: Prostate cancer is commonly diagnosed among old men while younger men are rarely diagnosed with prostate cancer. In this study we identify clinical and pathological features of 154 patients with prostate cancer younger than 55 years old and to assist intreatment decisions. Methods: The medical records of 154 prostate cancer patients younger than 55 years old in Peking University First Hospital from Feb 1953 to Jun 2016 were reviewed, retrospectively. Data was collected including symptoms, digital rectal examination (DRE), prostate-specific antigen (PSA), Gleason score, tumor stage, treatment strategies. Results: The mean age was 50.9±4.5, and 25.3% patients were between 40-50 years. Fifty-six (36.4%) patients initially presented with lower urinary tract symptoms. A solid mass could be found by digital rectal examination in 48(31.2%) patients. All patients were diagnosed by pathology of biopsy or surgery. The median Gleason score was 8. Gleason 2-6, 3+ 4, 4+ 3, 8, 9-10 were 15 cases(9.7%), 28 cases(18.2%), 21 cases(13.6%), 15 cases(9.7%), 51 cases(33.1%), respectively. Based on 2009 AJCC TNM Classification criteria the distribution of tumor stage was T1, T2, T3, and T4 in 2(1.3%), 54 (35.1%), 60 (39.0%), and 37 (24.0%) patients. Forty patients (25.9%) were found with bone metastasis and four (2.5%) suffered from visceral metastasis. Fifty-three(34.4%)underwent hormonal therapy and 79(51.3%) underwent radical prostatectomy. Conclusion: Younger prostate caner patients usually presented with LUTS symptoms and were featured for higher tumor stage and aggressiveness. More optimal and personalized risk-based therapy options are required.

[Prostate biopsy results and clinicopathological characteristics of the 55 years or younger patients].

Objective: To explore the clinical features, biopsy results and risk factors of patients underwent prostate biopsy with age≤55, and to help make the proper treatment strategies of this cohort of patients. Methods: The data of patients with age≤55 underwent prostate biopsy between Jan 2011 and Nov 2013 was retrospectively reviewed. Clinical factors including prostate-specific antigen, digital rectal examination, ultrasonography and magnetic resonance imaging were recorded. Biopsy positive results were defined as the presence of prostate cancer and high-grade prostate cancer (Gleason score≥7). Results: There were all together 82 patients in this cohort, with the median age of 51 years old and median prostate-specific antigen of 8.62 µg/L. Among them 71 patients (86.6%) underwent pre-biopsy magnetic resonance imaging test, with 25 positive (35.2%), 18 suspicious (25.4%) and 28 negative results (39.4%). Pathology confirmed prostate cancer in 26 patients (31.7%), including 23 (28.0%) high-grade prostate cancer. Higher prostate-specific antigen, positive ultrasonography and positive magnetic resonance imaging were risk factors for prostate cancer and high-grade prostate cancer. For patients with prostate-specific antigen between 4 and 10 µg/L, 15.0% were diagnosed with prostate cancer, and positive ultrasound and magnetic resonance imaging were predictive for biopsy results. Conclusions: The positive rate of prostate biopsy in men younger than 55 years old is 31.7%, and the risk for prostate cancer of this cohort of patients shouldn't be neglected. Prostate-specific antigen value, ultrasonography and magnetic resonance imaging could help predict biopsy results.

Functional characteristics of ES cell-derived cardiac precursor cells identified by tissue-specific expression of the green fluorescent protein.

In contrast to terminally differentiated cardiomyocytes, relatively little is known about the characteristics of mammalian cardiac cells before the initiation of spontaneous contractions (precursor cells). Functional studies on these cells have so far been impossible because murine embryos of the corresponding stage are very small, and cardiac precursor cells cannot be identified because of the lack of cross striation and spontaneous contractions. In the present study, we have used the murine embryonic stem (ES, D3 cell line) cell system for the in vitro differentiation of cardiomyocytes. To identify the cardiac precursor cells, we have generated stably transfected ES cells with a vector containing the gene of the green fluorescent protein (GFP) under control of the cardiac alpha-actin promoter. First, fluorescent areas in ES cell-derived cell aggregates (embryoid bodies [EBs]) were detected 2 d before the initiation of contractions. Since Ca2+ homeostasis plays a key role in cardiac function, we investigated how Ca2+ channels and Ca2+ release sites were built up in these GFP-labeled cardiac precursor cells and early stage cardiomyocytes. Patch clamp and Ca2+ imaging experiments proved the functional expression of the L-type Ca2+ current (ICa) starting from day 7 of EB development. On day 7, using 10 mM Ca2+ as charge carrier, ICa was expressed at very low densities 4 pA/pF. The biophysical and pharmacological properties of ICa proved similar to terminally differentiated cardiomyocytes. In cardiac precursor cells, ICa was found to be already under control of cAMP-dependent phosphorylation since intracellular infusion of the catalytic subunit of protein kinase A resulted in a 1.7-fold stimulation. The adenylyl cyclase activator forskolin was without effect. IP3-sensitive intracellular Ca2+ stores and Ca2+-ATPases are present during all stages of differentiation in both GFP-positive and GFP-negative cells. Functional ryanodine-sensitive Ca2+ stores, detected by caffeine-induced Ca2+ release, appeared in most GFP-positive cells 1-2 d after ICa. Coexpression of both ICa and ryanodine-sensitive Ca2+ stores at day 10 of development coincided with the beginning of spontaneous contractions in most EBs. Thus, the functional expression of voltage-dependent L-type Ca2+ channel (VDCC) is a hallmark of early cardiomyogenesis, whereas IP3 receptors and sarcoplasmic Ca2+-ATPases are expressed before the initiation of cardiomyogenesis. Interestingly, the functional expression of ryanodine receptors/sensitive stores is delayed as compared with VDCC.

Identifying Corticothalamic Network Epicenters in Patients with Idiopathic Generalized Epilepsy.

BACKGROUND AND PURPOSE: Corticothalamic networks are considered core pathologic substrates for idiopathic generalized epilepsy; however, the predominant epileptogenic epicenters within these networks are still largely unknown. The current study aims to identify these epicenters by resting-state functional connectivity. MATERIALS AND METHODS: To identify epicenters within the corticothalamic networks in idiopathic generalized epilepsy, we retrospectively studied a large cohort of patients with this condition (n = 97) along with healthy controls (n = 123) by resting-state functional MR imaging. The thalamus was functionally divided into subregions corresponding to distinct cortical lobes for 5 parallel corticothalamic networks. The functional connectivity between each voxel in the cortical lobe and the corresponding thalamic subregion was calculated, and functional connectivity strength was used to evaluate the interconnectivity of voxels in the cortex and thalamus. RESULTS: The projection of 5 cortical lobes to the thalamus is consistent with previous histologic findings in humans. Compared with controls, patients with idiopathic generalized epilepsy showed increased functional connectivity strength in 4 corticothalamic networks: 1) the supplementary motor area, pulvinar, and ventral anterior nucleus in the prefrontal-thalamic network; 2) the premotor cortex and ventrolateral nucleus in motor/premotor-thalamic networks; 3) the visual cortex, posterior default mode regions, and pulvinar in parietal/occipital-thalamic networks; and 4) the middle temporal gyrus in the temporal-thalamic network. CONCLUSIONS: Several key nodes were distinguished in 4 corticothalamic networks. The identification of these epicenters refines the corticothalamic network theory and provides insight into the pathophysiology of idiopathic generalized epilepsy.

Block effects of IHC-72 on calcium current in single guinea pig cardiac myocytes.

Block of the calcium current by IHC-72 in single ventricular myocytes isolated from guinea pig was studied by using the whole-cell patch clamp technique. IHC-72 in micromolar concentrations depressed the calcium current in a concentration-dependent manner, with an IC50 of 0.06 +/- 0.02 mcmol/l. After application of IHC-72 the peak calcium current began to decrease within 1 min and reached a steady state within 10 min. The current-voltage relationship for the calcium current peak was not changed by IHC-72, but the amplitude was significantly suppressed. Although IHC-72 did not alter the time course of inactivation of the calcium current, it shifted its steady state.

[Comparison of antiarrhythmic effects of IHC-72 (an iodonium-72), lidocaine and verapamil].

The antiarrhythmic actions of 3,6-dimethylamino-dibenzopyridonium edetate (IHC-72), lidocaine (Lid) and verapamil (Ver) on several models Were compared at equitoxic doses (equal fraction of LD50). The action of IHC-72 against aconitine induced arrhythmia was similar to that of Lid but stronger than that of Ver in anesthetized rats. The effect of IHC-72 on ouabain induced arrhythmia was also similar to that of Lid, but weaker than that of Ver in anesthetized guinea pigs. The activity of IHC-72 to raise electrical ventricular fibrillation thresholds (VFT) was weaker than that of Lid and Ver. The effects of IHC-72 in decreasing the incidence of ventricular premature beat(VP B), ventricular tachycardia (VT), ventricular fibrillation (VF) and shortening the duration of VT yielded by reperfusion were similar to those of Lid anf Ver in vivo.

Effects of 3,6-dimethylamino-dibenzopyriodonium edetate on action potentials in guinea pig papillary muscles.

The effects of 3,6-dimethylamino-dibenzopyriodonium edetate (IHC-72) on action potentials (AP) and slow response action potentials of guinea pig papillary muscles were studied with intracellular microelectrodes. IHC-72 12.7, 25.4, and 50.8 mumol.L-1 decreased the maximal upstroke velocity (Vmax), amplitude of action potential (APA), over shot (OS), and resting potential (RP) while prolonged the action potential duration at 30%, 50%, 90%, and 100% repolarization (APD30, APD50, APD90, and APD100). IHC-72 25.4 and 50.8 mumol.L-1 decreased the APA, Vmax, and prolonged APD50 and APD90 under high K+ superfusion. IHC-72 25.4 and 50.8 mumol.L-1 depressed the automaticity, APA, and maximal diastolic potential (MDP) of the slow response action potentials induced by BaCl2. The results indicated that IHC-72 might nonspecifically inhibit the transmembrane movement of Ca2+, Na+, and K+.

Establishment of beta-adrenergic modulation of L-type Ca2+ current in the early stages of cardiomyocyte development.

beta-Adrenergic modulation of the L-type Ca2+ current (ICaL) was characterized for different developmental stages in murine embryonic stem cell-derived cardiomyocytes using the whole-cell patch-clamp technique at 37 degreesC. Cardiomyocytes first appeared in embryonic stem cell-derived embryoid bodies grown for 7 days (7d). ICaL was insensitive to isoproterenol, forskolin, and 8-bromo-cAMP in very early developmental stage (VEDS) cardiomyocytes (from 7+1d to 7+2d) but highly stimulated by these substances in late developmental stage (LDS) cardiomyocytes (from 7+9d to 7+12d), indicating that all signaling cascade components became functionally coupled during development. In early developmental stage (EDS) cells (from 7+3d to 7+5d), the stimulatory response to forskolin and 8-bromo-cAMP was relatively weak. The forskolin effect was strongly augmented by ATP-gamma-S. At this stage, basal ICaL was stimulated by the nonselective phosphodiesterase (PDE) inhibitor isobutylmethylxanthine, by PDE inhibitors selective for the PDE II, III, and IV isoforms, as well as by the phosphatase inhibitor okadaic acid. Stimulation of ICaL by the catalytic subunit of the cAMP-dependent protein kinase A (PKA) was found to be similar (about 3 times) throughout development and in adult mouse ventricular cardiomyocytes, indicating that no structural changes of the Ca2+ channel related to phosphorylation occurred during development. ICaL was stimulated by isoproterenol in the presence of a PKA inhibitor and GTP-gamma-S in LDS but not VEDS cardiomyocytes, suggesting the development of a membrane-delimited stimulatory pathway mediated through the stimulatory GTP binding protein, Gs. We conclude that uncoupling and/or low expression of Gs protein accounted for the ICaL insensitivity to beta-adrenergic stimulation in VEDS cardiomyocytes. Furthermore, in EDS cells at the 7+4d stage, the reduced beta-adrenergic response is due, at least in part, to high intrinsic PDE and phosphatase activities.

Protective effects of 3,6-dimethylamino-dibenzopyriodonium edetate on global ischemia reperfused isolated rat hearts.

The effects of 3,6-dimethylamino-dibenzopyriodonium edetate (IHC-72) on global ischemia reperfused rat hearts were investigated. In the isolated working rat heart, 40-min global ischemia followed by 30-min reperfusion resulted in increases of ventricular tachycardia (VT) and ventricular fibrillation (VF), increases of creatine kinase (CK) release and malondialdehyde (MDA) contents, but decreased superoxide dismutase (SOD) activity. Following ischemia and reperfusion, the accumulation of myocardial calcium increased. IHC-72 50 mumol.L-1 given 10 min before ischemia and during reperfusion decreased the cardiac CK release, VT, and VF, reduced the MDA contents, prevented the reduction of SOD activity and attenuated the accumulation of myocardial calcium and sodium vs control. These results indicated that IHC-72 protected myocardial reperfused injury.

Improvement in sensitivity of fast atom bombardment mass spectrometry of amino acids by di-isopropylphosphorylation.

Positive ion fast atom bombardment mass spectrometric sensitivities of seven amino acids and the corresponding di-isopropylphosphorylated derivatives were carefully compared. Results showed that an improvement in sensitivity by factors of 4-29, mostly above 10, were achieved after the derivatization. The chemical noise derived from glycerol matrix was also greatly reduced by this derivatization.

Functional expression and regulation of the hyperpolarization activated non-selective cation current in embryonic stem cell-derived cardiomyocytes.

1. The biophysical and pharmacological characteristics of the hyperpolarization activated non- selective cation current (If) were recorded using whole-cell voltage clamp in embryonic stem (ES) cell-derived cardiomyocytes at different stages of development. 2. The cation current was detected in a large percentage (65 %) of early stage (EDS, differentiated for 7 + 3-4 days) cells at a current density of 11.4 +/- 0.6 pA pF-1 (n = 47). In late stage (LDS, differentiated for 7 + 9-12 days) cells the percentage of cells expressing If decreased (45 %), but If densities (15.5 +/- 0.9 pA pF-1, n = 20) were increased. 3. The muscarinic agonist carbachol (CCh, 1 microM) depressed basal If in EDS cells by 45.7 +/- 6.5 %, n = 5) and was without effect in LDS cardiomyocytes (n = 4). The beta-adrenoceptor agonist isoprenaline (ISO, 1 microM) stimulated If in LDS cells by 33 +/- 5.2 % (n = 6) but not in EDS cells (n = 5). 4. Cell infusion with the catalytic subunit of the cAMP-dependent protein kinase (PKA, 7 microM) stimulated If in EDS cells by 37.0 +/- 2.9 %, (n = 4), but subsequent superfusion of 8-bromo-cAMP (200 microM) was without effect. Intracellular perfusion of LDS cardiomyocytes with the highly selective peptide inhibitor of PKA (PKI, 20 microM) completely inhibited the stimulation of the L-type Ca2+ current (ICa,L) as well as of If by ISO (1 microM). 5. Extracellular superfusion with phosphodiesterase (PDE) inhibitors - IBMX, a non-selective antagonist, Erythro-9-(2-hydoxy-3-nonyl)adenine (EHNA), a PDE2 antagonist and rolipram, a PDE4 antagonist - resulted in stimulation of ICa,L and If in EDS cells. By contrast, milrinone and cilostamide, two PDE3 antagonists, stimulated ICa,L, but not If. 6. The present work demonstrates that If is functionally expressed during early cardiomyogenesis. Similar to ICa,L, If is regulated during embryonic development by phosphorylation via PKA. In contrast to ICa,L, If is not regulated by PDE3 suggesting different localization of these ion channels with respect to PDE3.

Intracellular Ca2+ oscillations drive spontaneous contractions in cardiomyocytes during early development.

Activity of cardiac pacemaker cells is caused by a balanced interplay of ion channels. However, it is not known how the rhythmic beating is initiated during early stages of cardiomyogenesis, when the expression of ion channels is still incomplete. Based on the observation that early-stage embryonic stem cell-derived cardiomyocytes continuously contracted in high extracellular K+ solution, here we provide experimental evidence that the spontaneous activity of these cells is not generated by transmembrane ion currents, but by intracellular [Ca2+]i oscillations. This early activity was clearly independent of voltage dependent L-type Ca2+ channels and the interplay between these and ryanodine sensitive Ca2+ stores. We also show that intracellular Ca2+ oscillations evoke small membrane depolarizations and that these can trigger L-type Ca2+ channel driven action potentials.

Regulation of the L-type Ca2+ channel during cardiomyogenesis: switch from NO to adenylyl cyclase-mediated inhibition.

In adult mammalian cardiomyocytes, stimulation of muscarinic receptors counterbalances the beta-adrenoceptor-mediated increase in myocardial contractility and heart rate by decreasing the L-type Ca2+ current (ICa) (1, 2). This effect is mediated via inhibition of adenylyl cyclase and subsequent reduction of cAMP-dependent phosphorylation of voltage-dependent L-type Ca2+ channels (3). Little is known, however, about the nature and origin of this pivotal inhibitory pathway. Using embryonic stem cells as an in vitro model of cardiomyogenesis, we found that muscarinic agonists depress ICa by 58 +/-3% (n=34) in early stage cardiomyocytes lacking functional beta-adrenoceptors. The cholinergic inhibition is mediated by the nitric oxide (NO)/cGMP system since it was abolished by application of NOS inhibitors (L-NMA, L-NAME), an inhibitor of the soluble guanylyl cyclase (ODQ), and a selective phosphodiesterase type II antagonist (EHNA). The NO/cGMP-mediated ICa depression was dependent on a reduction of cAMP/protein kinase A (PKA) levels since application of the catalytic subunit of PKA or of the PKA inhibitor PK) prevented the carbachol effect. In late development stage cells, as reported for ventricular cardiomyocytes (2, 4), muscarinic agonists had no effect on basal ICa but antagonized beta-adrenoceptor-stimulated ICa by 43 +/-4% (n=16). This switch in signaling pathways during development is associated with distinct changes in expression of the two NO-producing isoenzymes, eNOS and iNOS, respectively. These findings indicate a fundamental role for NO as a signaling molecule during early embryonic development and demonstrate a switch in the signaling cascades governing ICa regulation.

X-ray photoelectron spectra study on the derivatives of N-beta-phenethyl amine and N-beta-phenethyl glycine.

The X-ray photoelectron spectra of sixteen derivatives of N-beta-phenethyl amine and N-beta-phenethyl glycine have been studied. The effect of different structures on N1s binding energy and that of their Pauling's atomic charge density on the nitrogen atoms are investigated. The experimental binding energy of N1s is proportional to the calculated Pauling's atomic charge density. It shows that the phosphoryl group is the strongest nitrogen lone pair electron localizing group as compared with the corresponding sulfonyl and acyl derivatives. The XPS results agree with the data that there is no decarbonylation during the synthesis of N-dialkylphosphoryl-tetrahydro-3-benzazepin-1-one. In addition, since the N1s in P-N bond is smaller than that in S-N and C-N bonds, the phosphoryl group can be removed under much milder conditions. These results provide a semi-empirical evidence for the synthesis of the skeleton of the cephalotaxine. It also differentiates the reaction paths for these three different amino protecting groups.

Guanylyl cyclase stimulatory coupling to K(Ca) channels.

We coexpressed the human large-conductance, calcium-activated K (K(Ca)) channel (alpha- and beta-subunits) and rat atrial natriuretic peptide (ANP) receptor genes in Xenopus oocytes to examine the mechanism of guanylyl cyclase stimulatory coupling to the channel. Exposure of oocytes to ANP stimulated whole cell K(Ca) currents by 21 +/- 3% (at 60 mV), without altering current kinetics. Similarly, spermine NONOate, a nitric oxide donor, increased K(Ca) currents (20 +/- 4% at 60 mV) in oocytes expressing the channel subunits alone. Stimulation of K(Ca) currents by ANP was inhibited in a concentration-dependent manner by a peptide inhibitor of cGMP-dependent protein kinase (PKG). Receptor/channel stimulatory coupling was not completely abolished by mutating the cAMP-dependent protein kinase phosphorylation site on the alpha-subunit (S869; Nars M, Dhulipals PD, Wang YX, and Kotlikoff MI. J Biol Chem 273: 14920-14924, 1998) or by mutating a neighboring consensus PKG site (S855), but mutation of both residues virtually abolished coupling. Spermine NONOate also failed to stimulate channels expressed from the double mutant cRNAs. These data indicate that nitric oxide donors stimulate K(Ca) channels through cGMP-dependent phosphorylation and that two serine residues (855 and 869) underlie this stimulatory coupling.

Selection of ventricular-like cardiomyocytes from ES cells in vitro.

Ischemic disorders of the heart can cause an irreversible loss of cardiomyocytes resulting in a substantial decrease of cardiac output. The therapy of choice is heart transplantation, a technique that is hampered by the low number of donor organs. In the present study, we describe the specific labeling, rapid but gentle purification and characterization of cardiomyocytes derived from mouse pluripotent embryonic stem (ES) cells. To isolate the subpopulation of ventricular-like cardiomyocytes, ES cells were stable transfected with the enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh).). First fluorescent cells were detected at day 6 + 8 of differentiation within EBs. Four weeks after initiation of differentiation 25% of the cardiomyocyte population displayed fluorescence. Immunohistochemistry revealed the exclusive cardiomyogenic nature of EGFP-positive cells. This was further corroborated by electrophysiological studies where preferentially ventricular phenotypes, but no pacemaker-like cardiomyocytes, were detected among the EGFP-positive population. The enzymatic digestion of EBs, followed by Percoll gradient centrifugation and fluorescence-activated cell sorting, resulted in a 97% pure population of cardiomyocytes. Based on this study, ventricular-like cardiomyocytes can be generated in vitro from EBs and labeled using CMV(enh)./MLC-2v-driven marker genes facilitating an efficient purification. This method may become an important tool for future cell replacement therapy of ischemic cardiomyopathy especially after the proof of somatic differentiation of human ES cells in vitro.