Modeling acute myocardial infarction and cardiac fibrosis using human induced pluripotent stem cell-derived multi-cellular heart organoids.

Heart disease involves irreversible myocardial injury that leads to high morbidity and mortality rates. Numerous cell-based cardiac in vitro models have been proposed as complementary approaches to non-clinical animal research. However, most of these approaches struggle to accurately replicate adult human heart conditions, such as myocardial infarction and ventricular remodeling pathology. The intricate interplay between various cell types within the adult heart, including cardiomyocytes, fibroblasts, and endothelial cells, contributes to the complexity of most heart diseases. Consequently, the mechanisms behind heart disease induction cannot be attributed to a single-cell type. Thus, the use of multi-cellular models becomes essential for creating clinically relevant in vitro cell models. This study focuses on generating self-organizing heart organoids (HOs) using human-induced pluripotent stem cells (hiPSCs). These organoids consist of cardiomyocytes, fibroblasts, and endothelial cells, mimicking the cellular composition of the human heart. The multi-cellular composition of HOs was confirmed through various techniques, including immunohistochemistry, flow cytometry, q-PCR, and single-cell RNA sequencing. Subsequently, HOs were subjected to hypoxia-induced ischemia and ischemia-reperfusion (IR) injuries within controlled culture conditions. The resulting phenotypes resembled those of acute myocardial infarction (AMI), characterized by cardiac cell death, biomarker secretion, functional deficits, alterations in calcium ion handling, and changes in beating properties. Additionally, the HOs subjected to IR efficiently exhibited cardiac fibrosis, displaying collagen deposition, disrupted calcium ion handling, and electrophysiological anomalies that emulate heart disease. These findings hold significant implications for the advancement of in vivo-like 3D heart and disease modeling. These disease models present a promising alternative to animal experimentation for studying cardiac diseases, and they also serve as a platform for drug screening to identify potential therapeutic targets.

Novel pharmacotherapeutic avenues for bladder storage dysfunction in men.

INTRODUCTION: Bladder storage dysfunction is associated with low quality of life in men and remains a challenging field in pharmacotherapy because of low persistence followed by patient-perceived lack of efficacy and adverse effects. The persistent desire for the development of novel pharmacotherapy is evident, leading to numerous research efforts based on its pathophysiology. AREAS COVERED: This review describes the pathophysiology, current pharmacotherapeutic strategies, and emerging novel drugs for male bladder storage dysfunction. The section on emerging pharmacotherapy provides an overview of current research, focusing on high-potential target molecules, particularly those being evaluated in ongoing clinical trials. EXPERT OPINION: As pharmacotherapies targeting alpha-adrenergic, beta-adrenergic, and muscarinic receptors - the current primary targets for treating male bladder storage dysfunction - have demonstrated insufficient efficacy and side effects, researchers are exploring various alternative molecular targets. Numerous targets have been identified as central to regulating bladder afferent nerve activity, and their pharmacological effects and potential have been evaluated in animal-based experiments. However, there is a limited number of clinical trials for these new pharmacotherapies, and they have not demonstrated clear superiority over current treatments. Further research is needed to develop new effective pharmacotherapies for bladder storage dysfunction in men.

Improvement of lower urinary tract dysfunction by a monoacylglycerol lipase inhibitor in mice with spinal cord injury.

AIMS: Activation of the endocannabinoid system by monoacylglycerol lipase (MAGL) blockade may affect the lower urinary tract function. We investigated the effect of an MAGL inhibitor, MJN110, on neurogenic lower urinary tract dysfunction (LUTD) in the mouse model of spinal cord injury (SCI). METHODS: Female C57BL/6 mice that underwent spinal cord transection at T8-10 level were divided into three groups consisting of (1) vehicle-treated SCI mice, (2) 5 mg/kg, or (3) 10 mg/kg of MJN110-treated SCI mice. MJN110 and vehicle were administered intraperitoneally for 7 days from 4 weeks after spinal cord transection. We then conducted awake cystometrograms and compared urodynamic parameters between three groups. The expression of cannabinoid (CB) receptors, TRP receptors, and inflammatory cytokines in L6-S1 dorsal root ganglia (DRG) or the bladder mucosa were evaluated and compared among three groups. Changes in the level of serum 2-arachidonoylglycerol (2-AG) and bladder MAGL were also evaluated. RESULTS: In the cystometrogram, detrusor overactivity (DO) parameters, such as the number of nonvoiding contraction (NVC), a ratio of time to the 1st NVC to intercontraction interval (ICI), and NVC integrals were improved by MJN110 treatment, and some effects were dose dependent. Although MJN110 did not improve voiding efficiency, it decreased bladder capacity, ICI, and residual urine volume compared to vehicle injection. MJN110 treatment groups had lower CB2, TRPV1, TRPA1, and inflammatory cytokines mRNA levels in DRG and bladder mucosa. Serum 2-AG was increased, and bladder MAGL was decreased after MAGL inhibitor treatment. CONCLUSIONS: MAGL inhibition improved LUTD including attenuation of DO after SCI. Thus, MAGL can be a therapeutic target for neurogenic LUTD after SCI.

Outcomes of Artificial Urinary Sphincter Implantation in Patients with Detrusor Underactivity and Postprostatectomy Incontinence.

PURPOSE: There is insufficient evidence for postoperative outcomes of artificial urinary sphincter (AUS) implantation for postprostatectomy incontinence (PPI) with detrusor underactivity (DU). Thus, we assessed the impact of preoperative DU on the outcomes of AUS implantation for PPI. MATERIALS AND METHODS: Medical records of men who underwent AUS implantation for PPI were reviewed. Patients who had bladder outlet obstruction surgery before radical prostatectomy or AUS-related complications that required revision of AUS within three months were excluded. Patients were divided into two groups based on the preoperative urodynamic study including pressure flow study, a DU group, and a non-DU group. DU was defined as a bladder contractility index less than 100. The primary outcome was postoperative postvoid residual urine volume (PVR). The secondary outcomes included maximum flow rate (Qmax), postoperative satisfaction, and international prostate symptom score (IPSS). RESULTS: A total of 78 patients with PPI were assessed. The DU group consisted of 55 patients (70.5%) and the non-DU group comprised 23 patients (29.5%). Qmax was lower in the DU group than in the non-DU group and PVR was higher in the DU group as per a urodynamic study before AUS implantation. There was no significant difference in postoperative PVR between the two groups, although the Qmax after AUS implantation was significantly lower in the DU group. While the DU group showed significant improvements in Qmax, PVR, IPSS total score, IPSS storage subscore, and IPSS quality of life (QoL) score after AUS implantation, the non-DU group showed postoperative improvement in IPSS QoL score. CONCLUSION: There was no clinically significant impact of preoperative DU on the outcome of AUS implantation for PPI; thus, surgery can be safely performed in patients with PPI and DU.

Efficacy and Safety of DA-8010, a Novel M3 Antagonist, in Patients With Overactive Bladder: A Randomized, Double-Blind Phase 2 Study.

PURPOSE: DA-8010 is a novel muscarinic M3 receptor antagonist with significant selectivity for bladder over salivary gland in preclinical studies. We evaluated the clinical efficacy and safety of DA-8010 in overactive bladder (OAB) patients. METHODS: This phase 2, randomized, double-blind, parallel-group, active reference- and placebo-controlled trial was conducted at 12 centers in South Korea (NCT03566134). Patients aged ≥19 years with OAB symptoms for ≥3 months were enrolled. Three hundred six patients (30.07% male) were randomized to 12 weeks of treatment among 4 groups; 2 experimental groups (DA-8010 2.5 or 5 mg), an active reference group (solifenacin 5 mg), and a placebo group. The change from the baseline of (=∆) 24-hour frequency at 12 weeks (primary endpoint), episodes of urgency, overall/urgency urinary incontinence, average/ maximum voided volume, nocturia, and patients' subjective responses were analyzed. RESULTS: In the full analysis set, the mean (standard deviation) [median] values for ∆ 24-hour frequency at 12 weeks were -1.01 (2.44) [-1.33] for placebo, -1.22 (2.05) [-1.33] for DA-8010 2.5 mg, and -1.67 (2.25) [-1.67] for DA-8010 5 mg; DA-8010 5 mg showed a significant decrease compared with placebo (P=0.0413). At 4 and 8 weeks, both DA-8010 2.5 mg (P=0.0391 at 4 weeks, P=0.0335 at 8 weeks) and DA-8010 5 mg (P=0.0001 at 4 weeks, P=0.0210 at 8 weeks) showed significant decrease in ∆ 24-hour frequency compared with placebo. DA-8010 5 mg achieved a significant decrease in ∆ number of urgency episodes, compared with placebo at 4 (P=0.0278) and 8 (P=0.0092) weeks. Adverse drug reactions (ADRs) were observed in 3.95% of placebo, 6.67% of DA-8010 2.5 mg, 18.42% of DA-8010 5 mg, and 17.33% of solifenacin 5 mg groups. No serious ADRs were observed in any patient. CONCLUSION: Both DA-8010 2.5 mg and 5 mg showed therapeutic efficacy for OAB without serious ADRs. Therefore, both dosages of DA-8010 can advance to a subsequent large-scale phase 3 trial.

The incidence of testicular torsion and testicular salvage rate in Korea over 10 years: A nationwide population-based study.

PURPOSE: We performed a nationwide epidemiological study of testicular torsion using the National Health Insurance System database for the entire male population of Korea. MATERIALS AND METHODS: Age, sex, socioeconomic status, regional information, and diagnostic codes were retrieved from January 2009 to December 2019. To clearly identify the diagnosis of testicular torsion, patients who had not undergone orchiectomy or orchiopexy were excluded from the study. Multivariable logistic regression models were used to analyze the association between demographic characteristics and testicular loss. RESULTS: The overall incidence of testicular torsion in males was 2.02 cases per 100,000 person-years and 6.99 cases per 100,000 person-years in males under 19 years of age. Testicular torsion most commonly occurred either in infancy or adolescence. The total testicular salvage rate was 75.22% and highest in children at 79.91%. The rate of orchiectomy was high in infancy and in the oldest patients. We determined that age distribution was related to the risk of testicular loss. CONCLUSIONS: This study is the first nationwide epidemiological study of testicular torsion, which contains the entire Korean population. Although the testicular salvage rate in Korea was higher compared to other countries, it is necessary to educate males under 19 years of age on the seriousness of acute testicular pain to minimize the possibility of testicular loss.

Exercise Causes Arrhythmogenic Remodeling of Intracellular Calcium Dynamics in Plakophilin-2-Deficient Hearts.

BACKGROUND: Exercise training, and catecholaminergic stimulation, increase the incidence of arrhythmic events in patients affected with arrhythmogenic right ventricular cardiomyopathy correlated with plakophilin-2 (PKP2) mutations. Separate data show that reduced abundance of PKP2 leads to dysregulation of intracellular Ca2+ (Ca2+i) homeostasis. Here, we study the relation between excercise, catecholaminergic stimulation, Ca2+i homeostasis, and arrhythmogenesis in PKP2-deficient murine hearts. METHODS: Experiments were performed in myocytes from a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout murine line (PKP2cKO). For training, mice underwent 75 minutes of treadmill running once per day, 5 days each week for 6 weeks. We used multiple approaches including imaging, high-resolution mass spectrometry, electrocardiography, and pharmacological challenges to study the functional properties of cells/hearts in vitro and in vivo. RESULTS: In myocytes from PKP2cKO animals, training increased sarcoplasmic reticulum Ca2+ load, increased the frequency and amplitude of spontaneous ryanodine receptor (ryanodine receptor 2)-mediated Ca2+ release events (sparks), and changed the time course of sarcomeric shortening. Phosphoproteomics analysis revealed that training led to hyperphosphorylation of phospholamban in residues 16 and 17, suggesting a catecholaminergic component. Isoproterenol-induced increase in Ca2+i transient amplitude showed a differential response to ß-adrenergic blockade that depended on the purported ability of the blockers to reach intracellular receptors. Additional experiments showed significant reduction of isoproterenol-induced Ca2+i sparks and ventricular arrhythmias in PKP2cKO hearts exposed to an experimental blocker of ryanodine receptor 2 channels. CONCLUSIONS: Exercise disproportionately affects Ca2+i homeostasis in PKP2-deficient hearts in a manner facilitated by stimulation of intracellular ß-adrenergic receptors and hyperphosphorylation of phospholamban. These cellular changes create a proarrhythmogenic state that can be mitigated by ryanodine receptor 2 blockade. Our data unveil an arrhythmogenic mechanism for exercise-induced or catecholaminergic life-threatening arrhythmias in the setting of PKP2 deficit. We suggest that membrane-permeable ß-blockers are potentially more efficient for patients with arrhythmogenic right ventricular cardiomyopathy, highlight the potential for ryanodine receptor 2 channel blockers as treatment for the control of heart rhythm in the population at risk, and propose that PKP2-dependent and phospholamban-dependent arrhythmogenic right ventricular cardiomyopathy-related arrhythmias have a common mechanism.

Changes in transient receptor potential vanilloid 1 and transient receptor potential vanilloid 4 in patients with lower urinary tract dysfunction.

PURPOSE: We investigated the association between transient receptor potential vanilloid (TRPV) expression in human urothelium tissue and lower urinary tract dysfunction (LUTD). MATERIALS AND METHODS: We prospectively enrolled men who planned to undergo surgical treatment for benign prostatic obstruction to analyze TRPV1 and TRPV4 expression in the urothelium using enzyme-linked immunosorbent assay and immunofluorescence staining. Patients were divided into two groups based on urodynamics: the detrusor underactivity (DU) group and the non-DU group. Levels of TRPV1 and TRPV4 were compared between the two groups. We also divided patients into two groups according to degree of subjective urinary urgency symptoms using a 5-point urinary sensation scale and compared the differences in TRPV1 and TRPV4 levels between the two groups. The correlations between urodynamic parameters with TRPV1 or TRPV 4 in all patients were also analyzed. RESULTS: The levels of TRPV1 and TRPV 4 were not significantly different between the DU group (n=10) and the non-DU group (n=11). When we divided the patients according to degree of subjective urgency, the level of TRPV1 was not significantly different between the urgency group (n=10) and the non-urgency group (n =11), but the level of TRPV4 was significantly increased in the urgency group (p=0.029). There was no significant correlation between the level of TRPV1 or TRPV4 and urodynamic parameters in any patients. CONCLUSIONS: TRPV4 could be a useful diagnostic biomarker for patients with LUTD.

Correlation Between Nitric Oxide and Urodynamics in Men With Bladder Outlet Obstruction.

PURPOSE: To investigate the correlation between nitric oxide (NO) and urodynamics in men with bladder outlet obstruction (BOO) by analyzing nitric oxide synthase (NOS) in the urothelium. METHODS: We prospectively enrolled 25 men who planned to undergo surgical treatment for benign prostatic obstruction and identified as BOO in the preoperative urodynamics. Bladder tissue was taken during surgical prostate resection. Expressions of endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS) in the urothelium were analyzed, and their correlation with urodynamic parameters was also assessed in all patients. We also compared the expressions of eNOS, iNOS, and nNOS between BOO with detrusor underactivity (DU) group and BOO without DU group. RESULTS: In all patients, the level of eNOS positively correlated with maximal flow rate and with maximum cystometric capacity (MCC). The level of iNOS positively correlated with MCC. nNOS levels were positively correlated with detrusor pressure at maximal flow and with bladder contractility index in all patients. The level of eNOS, iNOS, and nNOS did not significantly differ between BOO without DU group and BOO with DU group. CONCLUSION: This study suggests that NO was correlated with bladder dysfunction in men with BOO. Particularly, nNOS may reflect the change in detrusor function.

Truncated Milk Fat Globule-EGF-like Factor 8 Ameliorates Liver Fibrosis via Inhibition of Integrin-TGFß Receptor Interaction.

Milk fat globule-EGF factor 8 (MFG-E8) protein is known as an immunomodulator in various diseases, and we previously demonstrated the anti-fibrotic role of MFG-E8 in liver disease. Here, we present a truncated form of MFG-E8 that provides an advanced therapeutic benefit in treating liver fibrosis. The enhanced therapeutic potential of the modified MFG-E8 was demonstrated in various liver fibrosis animal models, and the efficacy was further confirmed in human hepatic stellate cells and a liver spheroid model. In the subsequent analysis, we found that the modified MFG-E8 more efficiently suppressed transforming growth factor ß (TGF-ß) signaling than the original form of MFG-E8, and it deactivated the proliferation of hepatic stellate cells in the liver disease environment through interfering with the interactions between integrins (αvß3 & αvß5) and TGF-ßRI. Furthermore, the protein preferentially delivered in the liver after administration, and the safety profiles of the protein were demonstrated in male and female rat models. Therefore, in conclusion, this modified MFG-E8 provides a promising new therapeutic strategy for treating fibrotic diseases.

Regulation of Survival Motor Neuron Gene Expression by Calcium Signaling.

Spinal muscular atrophy (SMA) is caused by homozygous survival of motor neurons 1 (SMN1) gene deletion, leaving a duplicate gene, SMN2, as the sole source of SMN protein. However, a defect in SMN2 splicing, involving exon 7 skipping, results in a low level of functional SMN protein. Therefore, the upregulation of SMN protein expression from the SMN2 gene is generally considered to be one of the best therapeutic strategies to treat SMA. Most of the SMA drug discovery is based on synthetic compounds, and very few natural compounds have been explored thus far. Here, we performed an unbiased mechanism-independent and image-based screen of a library of microbial metabolites in SMA fibroblasts using an SMN-specific immunoassay. In doing so, we identified brefeldin A (BFA), a well-known inhibitor of ER-Golgi protein trafficking, as a strong inducer of SMN protein. The profound increase in SMN protein was attributed to, in part, the rescue of the SMN2 pre-mRNA splicing defect. Intriguingly, BFA increased the intracellular calcium concentration, and the BFA-induced exon 7 inclusion of SMN2 splicing, was abrogated by the depletion of intracellular calcium and by the pharmacological inhibition of calcium/calmodulin-dependent kinases (CaMKs). Moreover, BFA considerably reduced the expression of Tra2-ß and SRSF9 proteins in SMA fibroblasts and enhanced the binding of PSF and hnRNP M to an exonic splicing enhancer (ESE) of exon 7. Together, our results demonstrate a significant role for calcium and its signaling on the regulation of SMN splicing, probably through modulating the expression/activity of splicing factors.

Modulations of Cardiac Functions and Pathogenesis by Reactive Oxygen Species and Natural Antioxidants.

Homeostasis in the level of reactive oxygen species (ROS) in cardiac myocytes plays a critical role in regulating their physiological functions. Disturbance of balance between generation and removal of ROS is a major cause of cardiac myocyte remodeling, dysfunction, and failure. Cardiac myocytes possess several ROS-producing pathways, such as mitochondrial electron transport chain, NADPH oxidases, and nitric oxide synthases, and have endogenous antioxidation mechanisms. Cardiac Ca2+-signaling toolkit proteins, as well as mitochondrial functions, are largely modulated by ROS under physiological and pathological conditions, thereby producing alterations in contraction, membrane conductivity, cell metabolism and cell growth and death. Mechanical stresses under hypertension, post-myocardial infarction, heart failure, and valve diseases are the main causes for stress-induced cardiac remodeling and functional failure, which are associated with ROS-induced pathogenesis. Experimental evidence demonstrates that many cardioprotective natural antioxidants, enriched in foods or herbs, exert beneficial effects on cardiac functions (Ca2+ signal, contractility and rhythm), myocytes remodeling, inflammation and death in pathological hearts. The review may provide knowledge and insight into the modulation of cardiac pathogenesis by ROS and natural antioxidants.

Structural and Functional Characterization of a Nav1.5-Mitochondrial Couplon.

RATIONALE: The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. OBJECTIVE: To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria. METHODS AND RESULTS: Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A. CONCLUSIONS: We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.

A multicenter prospective study for overactive bladder patient treatment satisfaction with mirabegron after being unsatisfied with antimuscarinic therapy (FAVOR study).

AIM: We investigated the satisfaction and efficacy of mirabegron in patients with overactive bladder (OAB) symptoms who were unsatisfied with previous antimuscarinic treatment. METHODS: This was a 12-week, open-label study of adults with OAB who had been treated with antimuscarinics within 2 years of screening and expressed dissatisfaction over poor efficacy or adverse events of antimuscarinics. All enrolled patients have received mirabegron 50 mg once daily for 12 weeks. The primary outcome was the percentage of patients reporting treatment satisfaction questions (TSQ) at week 12 ("very satisfied" or "somewhat satisfied"). Patients completed voiding diaries, Overactive Bladder Questionnaire short form (OAB-q-SF), Overactive Bladder Symptom Score (OABSS), and the global response assessment (GRA) at baseline, Week 4, and Week 12. At 12-weeks, patients were assessed for willingness to continue treatment. RESULTS: The response rate of treatment satisfaction at 12 weeks was 69.3% (275/397) (95% confidence interval 64.7-73.8). Significant improvements from baseline to weeks 4 and 12 were observed in the frequency, urgency due to urinary incontinence, and urgency episodes per 24 h (all p < .0001). Both OAB-q-SF and OABSS were significantly improved compared to baseline. At 4 and 12 weeks, 27.5% and 41.8% of patients, respectively, responded to the GRA as being moderately or markedly improved. At 12 weeks, 80.8% of patients were willing to continue mirabegron. CONCLUSIONS: Mirabegron improved the rates of treatment satisfaction and symptoms in patients with OAB who were unsatisfied with prior antimuscarinic treatment.

Management of Urinary Incontinence With Underactive Bladder: A Review.

Urinary incontinence is caused by storage function failure, while underactive bladder (UAB) is caused by a decline in detrusor contractility and voiding dysfunction. As the treatment mechanisms for incontinence and UAB are contrary to each other, it is difficult to treat both incontinence and UAB, and the patient's quality of life can be further degraded. Conventional midurethral sling (MUS), such as transobturator tape or retropubic MUS, introduces a risk of postoperative voiding dysfunction in stress urinary incontinence with UAB. However, there have been several reports about the efficacy and safety of conventional MUS. Adjustable sling procedures, such as transobturator adjustable tape or the Remeex system, have better outcomes than conventional MUS because they control tension both during and after surgery. When voiding dysfunction occurs after incontinence treatment with UAB, voiding symptoms can be improved by various therapeutic modalities. Clean intermittent catheterization is recommended for patients with significant increased postvoid residual volumes or urinary retention. Although pharmacotherapy such as with alpha-blockers or parasympathomimetics can be considered for UAB, there is insufficient evidence of their effect on incontinence with UAB. Future therapies, such as stem cell therapy or gene therapy, may be used to treat incontinence with UAB. The possibility of management urgency urinary incontinence that related to detrusor hyperactivity with impaired contractility using sacral neuromodulation has been suggested. Further research is needed to establish evidence for the efficacy and safety of treatments for incontinence with UAB and improve patient quality of life.

Efficacy and Safety of Naftopidil in Patients With Neurogenic Lower Urinary Tract Dysfunction: An 8-Week, Active-Controlled, Stratified-Randomized, Double-Blind, Double-Dummy, Parallel Group, Noninferiority, Multicenter Design.

PURPOSE: The aim of this study was to evaluate the efficacy and safety of naftopidil compared with tamsulosin in patients with neurogenic lower urinary tract dysfunction (LUTD). METHODS: This study was conducted as an 8-week, active-controlled, stratified-randomized, double-blind, double-dummy, parallel group, noninferiority, and multicenter clinical trial. After 2 weeks of screening, eligible subjects were randomly assigned to receive naftopidil (25 mg for 1 week followed by 75 mg for 7 weeks) or tamsulosin (0.2 mg for 8 weeks). Primary endpoint was a change of International Prostatic Symptom Score (IPSS) total score after 8 weeks of treatment. RESULTS: One hundred ninety-four subjects with neurogenic LUTD were included into this trial. There were no differences between the 2 groups in baseline characteristics, including urodynamic study results, subtype of LUTD, pretreatment and concomitant medication, and causes of neurogenic bladder. The medication compliance rate was 94.0% (naftopidil, 93.6%; tamsulosin, 94.4%). There was a statistically significant decrease of IPSS total score at 8 weeks versus baseline in both the naftopidil (-5.64±0.66) and tamsulosin (-6.53±0.65) groups (P<0.0001 each). The mean difference between both groups was 0.89 (upper limit of 95% confidential interval, 2.72), which was lower than the noninferiority limit of 3 points. A subgroup analysis of neurologic lesions and sex found no mean difference of IPSS total score in each group. There was also no difference in safety profiles, including treatment emergent adverse events. CONCLUSION: Naftopidil was not inferior to tamsulosin as a therapeutic drug for patients with neurogenic LUTD and had a similar safety profile.

Changes in uroplakin expression in the urothelium of patients with ulcerative interstitial cystitis/bladder pain syndrome.

Purpose: We evaluated changes in the expression of uroplakin (UP) in the urothelium of patients with ulcerative interstitial cystitis/bladder pain syndrome (IC/BPS). Materials and Methods: Bladder samples were collected from 19 patients with ulcerative IC/BPS who were treated with augmentation ileocystoplasty and from 5 control patients. Frequency-volume charts, the pain visual analogue scale (VAS), and the O'Leary-Sant interstitial cystitis symptom index (ICSI) and problem index (ICPI) were used to evaluate the patients' symptoms preoperatively. The expression levels of UP-Ib and UP-III in the urothelium were compared between the IC/BPS patients and control patients. Results: Sixteen women and three men with IC/BPS were evaluated. Their values for preoperative mean voiding frequency, number of nocturia episodes, and functional bladder capacity as recorded in frequency-volume charts were 21.1±12.8, 5.9±4.2, and 151.1±62.7 mL, respectively. The mean pain VAS, ICSI, and ICPI scores were 8.4±1.3, 17.7±2.2, and 14.7±1.8, respectively. Immunofluorescence staining showed that UP-Ib and UP-III were localized in the urothelium. Upon Western blot analysis, the expression of UP-III was significantly increased in the IC/BPS group compared with the control group. However, expression of UP-Ib did not differ significantly between the IC/BPS and control groups. Conclusions: UP-III was significantly upregulated in patients with ulcerative IC/BPS. UP-III is a potential biomarker for the diagnosis of ulcerative IC/BPS.

Distinct shear-induced Ca2+ signaling in the left and right atrial myocytes: Role of P2 receptor context.

Atrial myocytes are continuously exposed to shear stress during cardiac cycles. Previous reports have shown that shear stress induces two different types of global Ca2+ signaling in atrial myocytes-longitudinal Ca2+ waves (L-waves) and action potential-involved transverse waves (T-waves), and suggested an underlying role of the autocrine activation of P2 receptors. We explored the correlations between ATP release and Ca2+ wave generation in atrial myocytes and investigated why the cells develop two Ca2+-wave types during the same shear force. We examined whether ATP release correlates with different shear-stress (~16 dyn/cm2)-mediated Ca2+ signaling by simultaneous measurement of local Ca2+ and ATP release in individual atrial myocytes using two-dimensional confocal imaging and sniffer patch techniques, respectively. Functional P2X7-receptor-expressing HEK293 cells were established as sniffer cells, which generated currents in real time in response to ATP released from a closely positioned atrial myocyte. Both shear-stress-induced L- and T-waves were preceded by sniffer currents with no difference in the current magnitude. Left atrial (LA) myocytes had two- to three-fold larger sniffer currents than right atrial (RA) cells, as was confirmed by ATP chemiluminescence assay. Shear-stress-induced ATP release was eliminated by connexin (Cx) 43 hemichannel inhibition using La3+, Gap19, or knock-down of Cx43 expression. The level of phosphorylated Cx43 at Ser386 (p-Cx43Ser368), but not total Cx43, was higher in LA versus RA myocytes. Most LA cells (~70%) developed L-waves, whereas most RA myocytes (~80%) presented T-waves. Shear-stress-induced T-waves were completely removed by inhibition of P2X4R, which were most abundant in rat atrial cells. Expression of P2X4R was higher in RA than LA myocytes, whereas expression of P2Y1R, the mediator of L-waves, was higher in LA than RA myocytes. ATP release mainly triggers L-waves in LA myocytes and T-waves in RA myocytes under the same shear force, partly because of the differential expression of P2Y1R and P2X4R between LA and RA myocytes. Higher ATP release in LA myocytes under shear stress may not contribute to determination of the wave pattern.

Clinical guidelines for interstitial cystitis/bladder pain syndrome.

The clinical guidelines for interstitial cystitis and related symptomatic conditions were revised by updating our previous guidelines. The current guidelines define interstitial cystitis/bladder pain syndrome as a condition with chronic pelvic pain, pressure or discomfort perceived to be related to the urinary bladder accompanied by other urinary symptoms, such as persistent urge to void or urinary frequency in the absence of confusable diseases. The characteristic symptom complex is collectively referred as hypersensitive bladder symptoms. Interstitial cystitis/bladder pain syndrome is divided into Hunner-type interstitial cystitis and bladder pain syndrome; Hunner-type interstitial cystitis and bladder pain syndrome represent interstitial cystitis/bladder pain syndrome with Hunner lesions and interstitial cystitis/bladder pain syndrome without Hunner lesions, respectively. So-called non-Hunner-type interstitial cystitis featured by glomerulations or bladder bleeding after distension is included in bladder pain syndrome. The symptoms are virtually indistinguishable between Hunner-type interstitial cystitis and bladder pain syndrome; however, Hunner-type interstitial cystitis and bladder pain syndrome should be considered as a separate entity of disorder. Histopathology totally differs between Hunner-type interstitial cystitis and bladder pain syndrome; Hunner-type interstitial cystitis is associated with severe inflammation of the urinary bladder accompanied by lymphoplasmacytic infiltration and urothelial denudation, whereas bladder pain syndrome shows little pathological changes in the bladder. Pathophysiology would also differ between Hunner-type interstitial cystitis and bladder pain syndrome, involving interaction of multiple factors, such as inflammation, autoimmunity, infection, exogenous substances, urothelial dysfunction, neural hyperactivity and extrabladder disorders. The patients should be treated differently based on the diagnosis of Hunner-type interstitial cystitis or bladder pain syndrome, which requires cystoscopy to determine the presence or absence Hunner lesions. Clinical studies are to be designed to analyze outcomes separately for Hunner-type interstitial cystitis and bladder pain syndrome.

Alterations of Ca2+ signaling and Ca2+ release sites in cultured ventricular myocytes with intact internal Ca2+ storage.

Although cultured adult cardiac myocytes in combination with cell-level genetic modifications have been adopted for the study of protein function, the cellular alterations caused by the culture conditions themselves need to be clarified before we can interpret the effects of genetically altered proteins. We systematically compared the cellular morphology, global Ca2+ signaling, elementary Ca2+ release (sparks), and arrangement of ryanodine receptor (RyR) clusters in short-term (2 days)-cultured adult rat ventricular myocytes with those of freshly isolated myocytes. The transverse (t)-tubules were remarkably decreased (to ∼25%) by culture, and whole-cell capacitance was reduced by ∼35%. The magnitude of depolarization-induced Ca2+ transients decreased to ∼50%, and Ca2+ transient decay was slowed by culture. The culture did not affect sarcoplasmic reticulum (SR) Ca2+ loading. Therefore, fractional Ca2+ release was attenuated by culture. In the cultured cells, the L-type Ca2+ current (ICa) was smaller (∼50% of controls) and its inactivation was slower. In cultured myocytes, there were significantly fewer (∼50% of control) Ca2+ sparks, the local Ca2+ releases through RyR clusters, compared with in freshly isolated cells. Amplitude and kinetics (duration and time-to-peak) of individual sparks were similar, but they showed greater width in cultured cells. Immunolocalization analysis revealed that the cross-striation of RyRs distribution became weaker and less organized, and that the density of RyR clusters decreased in cultured myocytes. Our data suggest that the loss of t-tubules and generation of compromised Ca2+ transients and ICa in short-term adult ventricular cell culture are independent of SR Ca2+ loading status. In addition, the deteriorated arrangement of the RyR-clusters and their decreased density after short-term culture may be partly responsible for fewer Ca2+ sparks and a decrease in global Ca2+ release.