Blebbistatin Research Articles

RACK1 contributes to the upregulation of embryonic genes in a model of cardiac hypertrophy

Receptors for activated C kinases (RACKs) have been shown to coordinate PKC-mediated hypertrophic signalling in mice. However, little information is available on its participation in embryonic gene expression. This study investigated the involvement of RACK1 in the expression of embryonic genes in a zebrafish (ZF) ex vivo heart culture model by using phenylephrine (PE) or a growth factors cocktail (GFs) as a prohypertrophic/regeneration stimulus. Blebbistatin (BL) inhibition has also been studied for its ability to block the signal transduction actions of some PEs. qRT‒PCR and immunoblot analyses confirmed the upregulation of RACK1 in the PE- and GFs-treated groups. BL administration counteracted PE-induced hypertrophy and downregulated RACK1 expression. Immunohistochemical analyses of the heart revealed the colocalization of RACK1 and embryonic genes, namely, Gata4, Wt1, and Nfat2, under stimulation, whereas these genes were expressed at lower levels in the BL treatment group. Culturing ZF heart cells activated via GFs treatment increased the expression of RACK1. The overexpression of RACK1 induced by the transfection of recombinant RACK1 cDNA in ZF heart cells increased the expression of embryonic genes, especially after one week of GFs treatment. In summary, these results support the involvement of RACK1 in the induction of embryonic genes during cardiac hypertrophy/GFs stimulation in a fish heart model, which can be used as an alternative study model for mammals.

Mechanotransduction in response to ECM stiffening impairs cGAS immune signaling in tumor cells

The tumor microenvironment (TME) plays decisive roles in disabling Tcell-mediated antitumor immunity, butthe immunoregulatory functions of its biophysical properties remain elusive. Extracellular matrix (ECM) stiffening is a hallmark of solid tumors. Here, we report that the stiffened ECM contributes to the immunosuppression in TME via activating the Rho-associated coiled-coil-containing protein kinase (ROCK)-myosin IIA-filamentous actin (F-actin) mechanosignaling pathway in tumor cells to promote the generation of TRIM14-scavenging nonmuscle myosin heavy chain IIA (NMHC-IIA)-F-actin stress fibers, thus accelerating the autophagic degradation of cyclic guanosine monophosphate (GMP)-AMP synthase (cGAS) to deprive tumor cyclic GMP-AMP (cGAMP) and further attenuating tumor immunogenicity. Pharmacological inhibition of myosin IIA effector molecules with blebbistatin (BLEB) or the RhoA upstream regulator of this pathway with simvastatin (SIM) restored tumor-intrinsic cGAS-mediated cGAMP production and enhanced antitumor immunity. Our work identifies that ECM stiffness is an important biophysical cue to regulate tumor immunogenicity via the ROCK-myosin IIA-F-actin axis and that inhibiting this mechanosignaling pathway could boost immunotherapeutic efficacy for effective solid tumor treatment.

Altered contractility in mutation-specific hypertrophic cardiomyopathy: A mechano-energetic in silico study with pharmacological insights.

Introduction: Mavacamten (MAVA), Blebbistatin (BLEB), and Omecamtiv mecarbil (OM) are promising drugs directly targeting sarcomere dynamics, with demonstrated efficacy against hypertrophic cardiomyopathy (HCM) in (pre)clinical trials. However, the molecular mechanism affecting cardiac contractility regulation, and the diseased cell mechano-energetics are not fully understood yet. Methods: We present a new metabolite-sensitive computational model of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) electromechanics to investigate the pathology of R403Q HCM mutation and the effect of MAVA, BLEB, and OM on the cell mechano-energetics. Results: We offer a mechano-energetic HCM calibration of the model, capturing the prolonged contractile relaxation due to R403Q mutation (∼33%), without assuming any further modifications such as an additional Ca2+ flux to the thin filaments. The HCM model variant correctly predicts the negligible alteration in ATPase activity in R403Q HCM condition compared to normal hiPSC-CMs. The simulated inotropic effects of MAVA, OM, and BLEB, along with the ATPase activities in the control and HCM model variant agree with in vitro results from different labs. The proposed model recapitulates the tension-Ca2+ relationship and action potential duration change due to 1µM OM and 5µM BLEB, consistently with in vitro data. Finally, our model replicates the experimental dose-dependent effect of OM and BLEB on the normalized isometric tension. Conclusion: This work is a step toward deep-phenotyping the mutation-specific HCM pathophysiology, manifesting as altered interfilament kinetics. Accordingly, the modeling efforts lend original insights into the MAVA, BLEB, and OM contributions to a new interfilament balance resulting in a cardioprotective effect.

Can Blebbistatin block the hypertrophy status in the zebrafish ex vivo cardiac model?

Ex-vivo simple models are powered tools to study cardiac hypertrophy. It is possible to control the activation of critical genes and thus test the effects of drug therapies before the in vivo tests. A zebrafish cardiac hypertrophy developed by 500 μM phenylephrine (PE) treatment in ex vivo culture has been demonstrated to activate the essential expression of the embryonal genes. These genes are the same as those described in several previous pieces of research on hypertrophic pathology in humans. The efficacy of the chemical drug Blebbistatin (BL) on hypertrophy induced ex vivo cultured hearts is studied in this research. BL can inhibit the myosins and the calcium wave in counteracting the hypertrophy status caused by PE. Samples treated with PE, BL and PE simultaneously, or pre/post-treatment with BL, have been analysed for the embryonal gene activation concerning the hypertrophy status. The qRTPCR has shown an inhibitory effect of BL treatments on the microRNAs downregulation with the consequent low expression of essential embryonal genes. In particular, BL seems to be effective in blocking the hyperplasia of the epicardium but less effective in myocardium hypertrophy. The model can make it possible to obtain knowledge on the transduction pathways activated by BL and investigate the potential use of this drug in treating cardiac hypertrophy in humans.

Metabolic Stress is Not Sufficient to Increase Skeletal Muscle Tissue Extracellular Vesicle Secretion in an Ex Vivo System

Skeletal muscle (SkM) is a metabolically active tissue and accounts for ~35% of total human body mass. Exercise imposes acute metabolic stress on SkM myofibers, and contractile proteins require energy sustained through both glycolytic and oxidative metabolism. Increased metabolic demand during contraction promotes intercellular communication through secreted factors that include extracellular vesicles (EVs). However, the mechanisms linking metabolic flux and EV secretion are currently unknown. We hypothesized that mimicing metabolic stress by blocking glycolysis with 2-deoxyglucose (2-DG), or oxidative phosphorylation with BAM15, a mitochondrial uncoupler, would increase EV secretion in SkM. Additionally, we aimed to characterize the effects of contractile inhibition with blebbistatin (BLEB) to better understand the role of contraction on EV release. We tested our hypothesis using an ex vivo EV secretion assay. Vastus medialis muscle (VM) from male C57Bl/6J mice (n=6) and soleus (SOL) and plantaris (PLA) muscle from female mT/mG mice (n=6) was cut into ~5 mg pieces and added to 12 well plates (~50 mg/well) filled with 750mL of serum-free DMEM and placed in a cell culture incubator at 37°C for 24 hours. The effect of 2-DG on VM EV secretion was measured during a log dose response (0.1nM – 100mM). SOL and PLA from female mice were treated with either 10mM of BLEB, or 10mM of BAM15. After incubation, tissue mass was measured and conditioned media was centrifuged (3,000 x g for 15 min) to remove cell debris. EVs were isolated using ExoQuick-TC. NTA was performed on isolated EVs using a Horiba Viewsizer 3000. EV secretion was normalized to tissue mass and culture media volume and reported as ([Particle]/mL/mg tissue). Statistical comparisons for 2-DG experiments were made using a repeated measures 1-way ANOVA with Bonferroni post-hoc testing. BLEB (n=6) and BAM15 (n=4) experiments were analyzed using a paired Student's t-test. No effect of 2-DG was observed in regard to EV abundance (p = .07) or diameter (p = 0.37). BAM15 did not affect EV abundance in SOL (p = 0.64) or PLA (p = 0.78). BLEB did not alter EV abundance (p = 0.69; CON: 1.32E+09 ± 4.86E+08 vs BLEB 1.67E+09 ± 5.87E+08), but significantly reduced EV mean diameter (p = 0.007; DMSO: 114.9 ± 5.1 vs. BLEB: 103.9 ± 4.2). Contrary to our hypothesis, inducing metabolic stress did not significantly increase SkM EV secretion. Mitochondrial uncoupling did not increase EV secretion in oxidative SkM such as the soleus, which is predominately comprised of fibers with high mitochondrial density. Inhibition of glycolysis with 2-DG did not increase SkM EV secretion. In the presence of BLEB, EV mean diameter was reduced but not EV abundance, suggesting that inhibiting contractile function may affect specific pathways of EV release. Collectively, our data demonstrate that metabolic stress may not be sufficient to increase EV secretion from SkM in an ex vivo system. Future studies might explore the specific role of contraction or extracellular factors as mediators of SkM EV secretion.

Synergistic effects of EMPs and PMPs on pulmonary vascular leakage and lung injury after ischemia/reperfusion

BackgroundVascular leakage is an important pathophysiological process of critical conditions such as shock and ischemia–reperfusion (I/R)-induced lung injury. Microparticles (MPs), including endothelial cell-derived microparticles (EMPs), platelet-derived microparticles (PMPs) and leukocyte-derived microparticles (LMPs), have been shown to participate in many diseases. Whether and which of these MPs take part in pulmonary vascular leakage and lung injury after I/R and whether these MPs have synergistic effect and the underlying mechanism are not known.MethodsUsing hemorrhage/transfusion (Hemo/Trans) and aorta abdominalis occlusion-induced I/R rat models, the role of EMPs, PMPs and LMPs and the mechanisms in pulmonary vascular leakage and lung injury were observed.ResultsThe concentrations of EMPs, PMPs and LMPs were significantly increased after I/R. Intravenous administration of EMPs and PMPs but not LMPs induced pulmonary vascular leakage and lung injury. Furthermore, EMPs induced pulmonary sequestration of platelets and promoted more PMPs production, and played a synergistic effect on pulmonary vascular leakage. MiR-1, miR-155 and miR-542 in EMPs, and miR-126 and miR-29 in PMPs, were significantly increased after hypoxia/reoxygenation (H/R). Of which, inhibition of miR-155 in EMPs and miR-126 in PMPs alleviated the detrimental effects of EMPs and PMPs on vascular barrier function and lung injury. Overexpression of miR-155 in EMPs down-regulated the expression of tight junction related proteins such as ZO-1 and claudin-5, while overexpression of miR-126 up-regulated the expression of caveolin-1 (Cav-1), the trans-cellular transportation related protein such as caveolin-1 (Cav-1). Inhibiting EMPs and PMPs production with blebbistatin (BLE) and amitriptyline (AMI) alleviated I/R induced pulmonary vascular leakage and lung injury.ConclusionsEMPs and PMPs contribute to the pulmonary vascular leakage and lung injury after I/R. EMPs mediate pulmonary sequestration of platelets, producing more PMPs to play synergistic effect. Mechanically, EMPs carrying miR-155 that down-regulates ZO-1 and claudin-5 and PMPs carrying miR-126 that up-regulates Cav-1, synergistically mediate pulmonary vascular leakage and lung injury after I/R.Graphic abstract8k8MRpqBub_wL9wmZ4ez89Video

Blebbistatin reveals beneficial effects on the cystometric parameters in an animal model of detrusor overactivity

The aims of the study were to determine the effectiveness of blebbistatin (BLEB) on detrusor overactivity (DO) in an animal model induced by retinyl acetate (RA) and, because of potential urothelial permeability, to evaluate the degenerative impact of BLEB on the urothelium. Three days after RA instillation into the urinary bladder, BLEB was administered into the bladder and immediately after cystometric assessment was performed. Furthermore, Evans Blue extravasation into bladder tissue and urothelium thickness were measured. Sixty female Wistar rats were used and randomly assigned to one of four groups (n = 15 in each group): (1) control, (2) RA, (3) BLEB, and (4) RA + BLEB. RA administration induced changes in cystometric parameters reflecting DO, as previously reported. Treatment with BLEB did not significantly alter cystometric parameters in rats which did not receive RA. Administration of BLEB to rats pretreated with RA reversed changes in cystometric parameters induced by RA in basal pressure, threshold pressure, detrusor overactivity index, amplitude of nonvoiding contractions, frequency of nonvoiding contractions, voided volume, volume threshold, intercontraction interval, bladder compliance, and volume threshold to elicit nonvoiding contractions. There were no significant differences in Evans Blue extravasation into bladder tissue or urothelium thickness between the groups. The current research provides new data on the possible utility of blebbistatin in the pharmacotherapy of DO, which is an important feature of overactive bladder (OAB). Further studies in human patients with DO/OAB are warranted to confirm these preclinical results.

Omecamtiv mercabil and blebbistatin modulate cardiac contractility by perturbing the regulatory state of the myosin filament.

Key points Omecamtiv mecarbil and blebbistatin perturb the regulatory state of the thick filament in heart muscle.Omecamtiv mecarbil increases contractility at low levels of activation by stabilizing the ON state of the thick filament.Omecamtiv mecarbil decreases contractility at high levels of activation by disrupting the acto‐myosin ATPase cycle.Blebbistatin reduces contractility by stabilizing the thick filament OFF state and inhibiting acto‐myosin ATPase.Thick filament regulation is a promising target for novel therapeutics in heart disease. Contraction of heart muscle is triggered by a transient rise in intracellular free calcium concentration linked to a change in the structure of the actin‐containing thin filaments that allows the head or motor domains of myosin from the thick filaments to bind to them and induce filament sliding. It is becoming increasingly clear that cardiac contractility is also regulated through structural changes in the thick filaments, although the molecular mechanisms underlying thick filament regulation are still relatively poorly understood. Here we investigated those mechanisms using small molecules – omecamtiv mecarbil (OM) and blebbistatin (BS) – that bind specifically to myosin and respectively activate or inhibit contractility in demembranated cardiac muscle cells. We measured isometric force and ATP utilization at different calcium and small‐molecule concentrations in parallel with in situ structural changes determined using fluorescent probes on the myosin regulatory light chain in the thick filaments and on troponin C in the thin filaments. The results show that BS inhibits contractility and actin‐myosin ATPase by stabilizing the OFF state of the thick filament in which myosin head domains are more parallel to the filament axis. In contrast, OM stabilizes the ON state of the thick filament, but inhibits contractility at high intracellular calcium concentration by disrupting the actin‐myosin ATPase pathway. The effects of BS and OM on the calcium sensitivity of isometric force and filament structural changes suggest that the co‐operativity of calcium activation in physiological conditions is due to positive coupling between the regulatory states of the thin and thick filaments.

Role of both actin–myosin cross bridges and NO-cGMP pathway modulators in the contraction and relaxation of human placental stem villi

IntroductionHuman placental stem villi (PSV) present contractile properties. We studied the role of actin–myosin cross bridges (CBs) and the effects of NO-cGMP pathway modulators in the PSV contraction and relaxation. MethodsIn vitro contractile properties were investigated in 71 PSV from term human placentas studied according to their long axis. Contraction was induced by both KCl and electrical tetanic stimulation. Relaxation was induced by inhibiting the CB cycle with either 2,3-butanedione monoxime (BDM) or blebbistatin (BLE) and by activating the NO-cGMP pathway with isosorbide dinitrate (ISDN), sildenafil (SIL) or ISDN + SIL. ResultsPSV tension slowly increased by 140% of the basal tone after KCl exposure and by 85% after tetanus. The addition of BDM, BLE, ISDN, SIL and ISDN + SIL induced a relaxation of PSV, the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). the overall time course of relaxation (in s) was respectively (means ± SD) 3412 ± 1904, 14,250 ± 3095*, 3813 ± 1383, 2883 ± 1188 and 2440 ± 477; significantly longer in BLE compared with BDM, ISDN, SIL and ISDN + SIL:*p < 0.001). These relaxation kinetics were particularly slow. Other relaxation parametres, i.e., maximum lengthening, -peak dT/dt, and resting tension, did not differ between these 5 subgroups. Discussion and conclusionIsolated human PSV were able to contract after both KCl exposure and tetanus. This increase in contractility was reversed by inhibiting the CB cycle with BDM or BLE and by stimulating the NO-cGMP pathway with ISDN or SIL. The association ISDN + SIL did not potentiate the relaxing processes

The mechanical uncoupler blebbistatin is associated with significant electrophysiological effects in the isolated rabbit heart

Blebbistatin (BS) is a recently discovered inhibitor of the myosin II isoform and has been adopted as the mechanical uncoupler of choice for optical mapping, because previous studies suggest that BS has no significant cardiac electrophysiological effects in a number of species. The aim of this study was to determine whether BS affects cardiac electrophysiology in isolated New Zealand White rabbit hearts. Langendorff-perfused hearts (n= 39) in constant-flow mode had left ventricular monophasic action potential duration (MAPD) measured at apical and basal regions during constant pacing (300 ms cycle length). Standard action potential duration restitution was obtained using the single extrastimulus method with measurement of the maximal restitution slope. Ventricular fibrillation threshold was measured as the minimal current inducing sustained ventricular fibrillation with burst pacing (30 stimuli, at 30 ms intervals). Optical action potentials were recorded using the voltage-sensitive dye di-4-ANEPPS. Measurements were taken at baseline and after 60 min perfusion with BS (5 μm). Blebbistatin significantly prolonged left ventricular apical (mean ± SEM; from 129.9 ± 2.9 to 170.7 ± 4.1 ms, P < 0.001, n= 8) and basal MAPD (from 135.0 ± 2.3 to 163.3 ± 5.6 ms, P < 0.001) and effective refractory period (from 141.3 ± 4.8 to 175.6 ± 3.7 ms, P < 0.001) whilst increasing the maximal slope of restitution (apex, from 0.79 ± 0.09 to 1.57 ± 0.16, P < 0.001; and base, from 0.71 ± 0.06 to 1.44 ± 0.24, P < 0.001) and ventricular fibrillation threshold (from 5.3 ± 1.1 to 17.0 ± 2.9 mA, P < 0.001). In other hearts, blebbistatin significantly prolonged optically recorded action potentials (from 136.5 ± 6.3 to 173.0 ± 7.9 ms, P < 0.05, n= 4). In control experiments, the increase of MAPD with blebbistatin was present whether the hearts were perfused in constant-pressure mode (n= 5) or in unloaded conditions (n= 5). These data show that blebbistatin significantly affects cardiac electrophysiology. Its use in optical mapping studies should be treated with caution.

Effects of polar cortical cytoskeleton and unbalanced cortical surface tension on intercellular bridge thinning during cytokinesis

To probe the contributions of polar cortical cytoskeleton and the surface tension of daughter cells to intercellular bridge thinning dynamics during cytokinesis, we applied cytochalasin D (CD) or colchicine (COLC) in a highly localized manner to polar regions of dividing normal rat kidney (NRK) cells. We observed cellular morphological changes and analyzed the intercellular bridge thinning trajectories of dividing cells with different polar cortical characteristics. Global blebbistatin (BS) application was used to obtain cells losing active contractile force groups. Our results show that locally released CD or colchicine at the polar region caused inhibition of cytokinesis before ingression. Similar treatment at phases after ingression allowed completion of cytokinesis but dramatically influenced the trajectories of intercellular bridge thinning. Disturbing single polar cortical actin induced transformation of the intercellular bridge thinning process, and polar cortical tension controlled deformation time of intercellular bridges. Our study provides a feasible framework to induce and analyze the effects of local changes in mechanical properties of cellular components on single cellular cytokinesis.

Blebbistain, a Myosin II Inhibitor, as a Novel Strategy to Regulate Detrusor Contractility in a Rat Model of Partial Bladder Outlet Obstruction

Partial bladder outlet obstruction (PBOO), a common urologic pathology mostly caused by benign prostatic hyperplasia, can coexist in 40–45% of patients with overactive bladder (OAB) and is associated with detrusor overactivity (DO). PBOO that induces DO results in alteration in bladder myosin II type and isoform composition. Blebbistatin (BLEB) is a myosin II inhibitor we recently demonstrated potently relaxed normal detrusor smooth muscle (SM) and reports suggest varied BLEB efficacy for different SM myosin (SMM) isoforms and/or SMM vs nonmuscle myosin (NMM). We hypothesize BLEB inhibition of myosin II as a novel contraction protein targeted strategy to regulate DO. Using a surgically-induced male rat PBOO model, organ bath contractility, competitive and Real-Time-RT-PCR were performed. It was found that obstructed-bladder weight significantly increased 2.74-fold while in vitro contractility of detrusor to various stimuli was impaired ∼50% along with decreased shortening velocity. Obstruction also altered detrusor spontaneous activities with significantly increased amplitude but depressed frequency. PBOO switched bladder from a phasic-type to a more tonic-type SM. Expression of 5’ myosin heavy chain (MHC) alternatively spliced isoform SM-A (associated with tonic-type SM) increased 3-fold while 3’ MHC SM1 and essential light chain isoform MLC17b also exhibited increased relative expression. Total SMMHC expression was decreased by 25% while the expression of NMM IIB (SMemb) was greatly increased by 4.5-fold. BLEB was found to completely relax detrusor strips from both sham-operated and PBOO rats pre-contracted with KCl, carbachol or electrical field stimulation although sensitivity was slightly decreased (20%) only at lower doses for PBOO. Thus we provide the first thorough characterization of the response of rat bladder myosin to PBOO and demonstrate complete BLEB-induced PBOO bladder SM relaxation. Furthermore, the present study provides valuable evidence that BLEB may be a novel type of potential therapeutic agent for regulation of myogenic and nerve-evoked DO in OAB.

Smooth muscle myosin expression, isoform composition, and functional activities in rat corpus cavernosum altered by the streptozotocin-induced type 1 diabetes

Diabetes mellitus (DM) is a quite common chronic disease, and the prevalence of erectile dysfunction (ED) is three times higher in this large population. Although diabetes-related ED has been studied extensively, the actin-myosin contractile apparatus was not examined. The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and essential light chains (LC(17)) exist as several different alternatively spliced isoforms with distinct contractile properties. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed corpus cavernosum smooth muscle (CCSM). In this study, we examine whether diabetes alters SMM expression, alternative splicing, and/or functional activities, including sensitivity to BLEB. By using streptozotocin (STZ)-induced 2-mo diabetic rats, functional activities were tested in vivo by intracavernous pressure (ICP) recording during cavernous nerve stimulation and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin, and embryonic SMM (SMemb) expression by real-time RT-PCR. Results revealed that the blood glucose level of STZ rats was 407.0 vs. 129.5 mg/dl (control). STZ rats exhibited ED confirmed by significantly increased CCSM contractile response to phenylephrine and decreased ICP response. For STZ rats, SM-B, LC(17a) and SM2 isoforms, total SMM, and myocardin expression increased, whereas SM-A, LC(17b), and SM1 isoforms were decreased, with SMemb unchanged. BLEB was significantly more effective in relaxing STZ CCSM both in vitro and in vivo. Thus we demonstrated a novel diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype was supported further by total SMM expression increase. Moreover, the change in CCSM phenotype was associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED.

Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle.

Assessment of mitochondrial ADP-stimulated respiratory kinetics in PmFBs (permeabilized fibre bundles) is increasingly used in clinical diagnostic and basic research settings. However, estimates of the Km for ADP vary considerably (~20-300 μM) and tend to overestimate respiration at rest. Noting that PmFBs spontaneously contract during respiration experiments, we systematically determined the impact of contraction, temperature and oxygenation on ADP-stimulated respiratory kinetics. BLEB (blebbistatin), a myosin II ATPase inhibitor, blocked contraction under all conditions and yielded high Km values for ADP of >~250 and ~80 μM in red and white rat PmFBs respectively. In the absence of BLEB, PmFBs contracted and the Km for ADP decreased ~2-10-fold in a temperature-dependent manner. PmFBs were sensitive to hyperoxia (increased Km) in the absence of BLEB (contracted) at 30 °C but not 37 °C. In PmFBs from humans, contraction elicited high sensitivity to ADP (Km<100 μM), whereas blocking contraction (+BLEB) and including a phosphocreatine/creatine ratio of 2:1 to mimic the resting energetic state yielded a Km for ADP of ~1560 μM, consistent with estimates of in vivo resting respiratory rates of <1% maximum. These results demonstrate that the sensitivity of muscle to ADP varies over a wide range in relation to contractile state and cellular energy charge, providing evidence that enzymatic coupling of energy transfer within skeletal muscle becomes more efficient in the working state.

1130 STREPTOZOTOCIN-INDUCED TYPE 1 DIABETES IN RAT ALTERS SMOOTH MUSCLE MYOSIN ISOFORM COMPOSITION TO A MORE CONTRACTILE PHENOTYPE AND INCREASES SENSITIVITY TO THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 20111130 STREPTOZOTOCIN-INDUCED TYPE 1 DIABETES IN RAT ALTERS SMOOTH MUSCLE MYOSIN ISOFORM COMPOSITION TO A MORE CONTRACTILE PHENOTYPE AND INCREASES SENSITIVITY TO THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN Xinhua Zhang, Arnold Melman, and Michael DiSanto Xinhua ZhangXinhua Zhang Camden, NJ More articles by this author , Arnold MelmanArnold Melman Bronx, NY More articles by this author , and Michael DiSantoMichael DiSanto Camden, NJ More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.727AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The mRNAs encoding smooth muscle myosin (SMM) heavy chains (MHC) and the 17 kDa essential light chains (LC17) exist as several different isoforms with distinct contractile properties, produced via alternative splicing mechanisms. Recently, we provided novel data that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed both rat and human corpus cavernosum smooth muscle (CCSM)(J Sexual Med, 2009). In this study, we determine if diabetes alters SMM expression, alternative splicing and/or functional activities including sensitivity to BLEB. METHODS Fisher 344 male streptozotocin (STZ)-induced diabetic rats were kept for 2 months. Functional activities were then tested in vivo by intracavernous pressure (ICP) recording and in vitro via organ bath contractility studies. SMM isoform composition was analyzed by competitive RT-PCR and total SMM, myocardin (a transcriptional regulator of SMM) and embryonic SMM (SMemb) expression by Real-time RT-PCR. RESULTS Average blood glucose levels of STZ rats were 407 vs 129.5 mg/dl in rats injected with saline (SHAM). STZ rats showed severe erectile dysfunction (ED) confirmed by decreased ICP response to electrical stimulation of cavernous nerve. For STZ rats, LC17a and SM-B isoforms (both associated with increased contractility and faster shortening velocity) and total SMM and myocardin expression increased while LC17b and SM-A isoforms (both associated with decreased contractility and slower shortening velocity) were decreased with SM1/2 SMM MHC isoforms and SMemb unchanged. Interestingly, BLEB was more effective in relaxing STZ CCSM both in vitro and in vivo. CONCLUSIONS We show for the first time to our knowledge a diabetes-specific effect on alternative splicing of the SMM heavy chain and essential light chain genes to a SMM isoform composition favoring a heightened contractility and ED. A switch to a more contractile phenotype is further supported by total SMM expression increase. Moreover, we provide novel data that the change in CCSM phenotype is associated with an increased sensitivity to BLEB, which may serve as a novel pharmacotherapy for ED with specificity to the faster type myosin isoforms found in the diabetic rat CCSM. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e454 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xinhua Zhang Camden, NJ More articles by this author Arnold Melman Bronx, NY More articles by this author Michael DiSanto Camden, NJ More articles by this author Expand All Advertisement Advertisement PDF DownloadLoading ...

1124 PATHOPHYSIOLOGY OF PRIAPISM WITH VARIED DURATION: A NOVEL RAT MODEL STUDY USING THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN

You have accessJournal of UrologySexual Function/Dysfunction/Andrology: Basic Research1 Apr 20111124 PATHOPHYSIOLOGY OF PRIAPISM WITH VARIED DURATION: A NOVEL RAT MODEL STUDY USING THE MYOSIN II SPECIFIC INHIBITOR BLEBBISTATIN Xinhua Zhang, Arnold Melman, and Michael DiSanto Xinhua ZhangXinhua Zhang Camden, NJ More articles by this author , Arnold MelmanArnold Melman Bronx, NY More articles by this author , and Michael DiSantoMichael DiSanto Camden, NJ More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.721AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Priapism is an important but understudied and underfunded disease that is highly prevalent in the sickle cell population. The exact molecular basis for priapism remains unclear. Dog and rat models involving pharmacologically induced (e.g. papaverine) and vacuum-induced ischemia have been developed but do not fully recapitulate the human condition. Recently, transgenic mice have contributed to understanding this disorder, but weaknesses include low percent priapism, cost and unknown initiation/duration. Last year, we reported that blebbistatin (BLEB), a specific myosin II inhibitor, potently relaxed rat and human corpus cavernosum smooth muscle (CCSM) (J Sexual Med, 2009). Here, we validate a novel, extended duration BLEB-induced rat priapism model in which the penis can maintain erection for up to 4 days. METHODS BLEB was intracavernously injected (ICI) into adult rats. After 2h or 4h under anesthesia or 4d or 1wk in awake rats, intracavernous pressure (ICP) recording and histology (Hematoxylin & Eosin or Masson's Trichrome staining) were performed. Expression of molecules in major priapism-linked or well known CCSM contractile pathways was determined by Real-time RT-PCR. RESULTS ICI of 1.25 μmols BLEB induced priapism validated by visual full erection and ICP/MAP (∼0.75) up to 4d. At 2h & 4h, CC vasocongestion and ICP can almost be returned to baseline by flushing. Endothelial nitric oxide synthase (eNOS) expression decreased while phosphodiesterase V (PDE5), Rho-kinase alpha (ROKα), endothelin receptor A (ETA) and SM myosin heavy chain (SMMHC) increased. In contrast, 4d rat ICP is refractory to flushing exhibiting CC thrombosis and early fibrosis. eNOS, nNOS and endothelin receptor B (ETB) increased while PDE5, RhoA, ROKα, ETA, SMMHC and adrenergic receptors α1c & α1d decreased. At 1wk, there was severe fibroplasia preventing ICP recording and CC atrophy with SMMHC 10-fold decreased, TGFβ1 2.5-fold increased, NOS and ET receptors unchanged, ROKα/β and α1c decreased. Hypoxia inducible factor (HIF-1α) decreased at 4h but increased 2- and 4.5-fold at 4d and 1wk. CONCLUSIONS We established a novel, easily induced, long duration BLEB-induced rat priapism model. Importantly, this model showed CCSM contractile molecules upregulated and relaxation molecules downregulated with ICP reversible in the early compensated stage while these pathways were opposite (contraction decreased and relaxation increased with ICP irreversible) in the later decompensated stage with eventual severe fibrosis and atrophy. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e452 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Xinhua Zhang Camden, NJ More articles by this author Arnold Melman Bronx, NY More articles by this author Michael DiSanto Camden, NJ More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Blebbistatin Protects Rodent Myocytes from Death in Primary Culture via Inhibiting Na/Ca Exchange

Introduction: It has been long recognized that rodent myoyctes die during long-term primary culture, which limits the use of genetically altered myocytes for signaling studies. Blebbistatin (BLB), a myosin II ATPase inhibitor, has been used to protect rodent myocytes. However, the mechanisms underlying the protective effects of this drug are not clear and are the topics of this study. Materials & Methods: Adult rat ventricular myocytes (ARVM) were isolated and cultured with or without BLB (10μM) and BDM(10mM) for 72 hours. Myocyte death was evaluated by trypan blue staining. The effects of these two drugs on myocyte contraction, intracellular Ca transient ([Ca]i, Indo-1,410/480), SR Ca content, L-type calcium and Na/Ca exchanger currents were studied acutely. Results: 1, Both BDM (61.5± 6.4%) and BLB (74.0±3.2%) promoted myocyte survival in culture at 72 hours (control: 7.0±1.8%); 2. ARVM fractional shortening was reduced by BLB (1.7±0.4%) and BDM (0.5±0.1%, control: 6.5±0.7%); 3, Acutely, the amplitude of [Ca]i (Δ[Ca]i) was depressed by both BDM (0.038±0.005) and BLB (0.065±0.008) comparing to control (0.130±0.010). 4, Diastolic Ca was significantly increased by BLB (0.90±0.06) but not by BDM (0.73±0.06) comparing to control(0.70±0.05). 5. BLB and BDM significantly reduced the SR Ca content(Δ[Ca]i in BLB vs. BDM vs. control: 0.16±0.016, 0.09±0.01, 0.24±0.03). 5. The mechanisms of the protective effect of BDM and BLB are different in that BDM mainly reduced Ca influx through the L-type Ca channel (85% reduction) and Na/Ca exchanger (60% reduction) while BLB inhibited Na/Ca exchanger (100% inhibition) without altering the LTCC(<5% reduction). Conclusion: These results suggest both BDM and BLB protects rodent myocytes in culture by preventing cytosolic and SR Ca overload by both common and different mechanisms: both BDM and BLB inhibit NCX while BDM, but not BLB, reduces ICa-L.

In Vitro and In Vivo Relaxation of Corpus Cavernosum Smooth Muscle by the Selective Myosin II Inhibitor, Blebbistatin

Blebbistatin (BLEB) is a small cell permeable molecule originally reported as a selective inhibitor of myosin II isoforms expressed by striated muscle and non-muscle cells (IC(50) = 0.5-5 microM) with poor inhibition of turkey gizzard smooth muscle (SM) myosin II (IC(50) approximately 80 microM). However, recently it was found that BLEB can potently inhibit mammalian arterial SM (IC(50) approximately 5 microM). To investigate the effect of BLEB on corpus cavernosum SM (CCSM) tone and erectile function (EF). CC tissue obtained from penile implant patients along with CC, aorta and bladder from adult male rats were used for BLEB organ bath studies. Intracavernosal BLEB was administered to rats and EF was assessed via intracavernous pressure (ICP). Effects of BLEB on agonist-induced CCSM, aorta and bladder contraction in vitro and ICP in vivo. BLEB completely relaxed human CCSM pre-contracted with phenylephrine (PE) in a dose-dependent manner decreasing tension by 76.5% at 10 microM. BLEB pre-incubation attenuated PE-induced contraction of human CC by approximately 85%. Human CC strips pre-contracted with endothelin-1 or KCl were almost completely relaxed by BLEB. Rat CCSM pre-contracted with PE showed BLEB relaxation comparable to human CCSM. BLEB inhibition was similar for rat aorta but slower for bladder. Both maximal ICP and ICP/mean arterial pressure were dose-dependently increased by BLEB intracavernous injections with full erection at 1 micromole. Our novel data reveals that BLEB nearly completely relaxes rat and human CCSM pre-contracted with a variety of potent agonists and exhibits tissue selectivity. Coupled with our in vivo data in which nanomole doses of BLEB significantly increase ICP, our data substantiates an important role for the SM contractile apparatus in the molecular mechanism for EF and suggests the possibility of BLEB binding at myosin II as a therapeutic treatment for ED by targeting SM contractile pathways.

Effects Of Blebbistatin And BDM (2,3-Butanedione Monoxime) On The Short-range Mechanical Properties Of Murine Diaphragm Muscle Fibers

Blebbistatin (BLEB) and 2,3-butanedione monoxime (BDM) are well-know inhibitors of myofilament force production and useful tools in structural and functional studies of cell motility and muscle contraction. In this study, we investigated the effects of BLEB and BDM on the short-range mechanical properties of single chemically permeabilized murine diaphragm fibers. BLEB and BDM were used in separate sets of experiments to reduce isometric force in saturating Ca2+ solution to approximately 50% of the control value. Muscle fibers were subjected to repeated triangular length changes (paired ramp stretches/releases, 0.04 l0, 0.33 l0 s−1) imposed under fiber length control in solutions with free Ca2+ concentrations ranging from pCa 9.0 to pCa 4.5. Short-range stiffness values were calculated from the slopes of regression lines fitted to the first 15 ms of XY plots of force against muscle length for each stretch response and expressed as Young's Moduli. Analysis of results obtained in control Ca2+ solutions (without BLEB or BDM) showed that short-range stiffness increased proportionately with the level of isometric force. Experiments performed with BLEB showed that short-range stiffness declined in proportion with the reduction in isometric force (no change in the stiffness/force ratio). In contrast, BDM produced a disproportionately large decrease in isometric force (that is, the stiffness/force ratio increased in the presence of BDM, ANACOVA test, p<0.001). These results support the hypothesis that BLEB and BDM reduce isometric force in skeletal muscles by different mechanisms. BLEB seems to prevent myosin heads from attaching to the thin filament while BDM probably reduces force by decreasing the rate at which myosin heads undergo tension-generating biochemical transitions.

Strain-induced orientation response of endothelial cells: effect of substratum adhesiveness and actin-myosin contractile level.

Endothelial cells subjected to cyclic stretching change orientation so as to be aligned perpendicular to the direction of applied strain in a magnitude and time-dependent manner. Although this type of response is not the same as motility, it could be governed by motility-related factors such as substratum adhesiveness and actin-myosin contractile level. To examine this possibility, human aortic endothelial cells (HAEC) were uniaxially, cyclically stretched on silicone rubber membranes coated with various concentrations of fibronectin, collagen type IV and laminin to produce differing amounts of adhesiveness (measured using a radial flow detachment assay). Cells were subjected to 10% pure cyclic uniaxial stretching for three hours at a rate of 10%/sec. Time-lapse images revealed that cells underwent large morphological changes without moving. For each type of protein there was a parabolic dependence on initial adhesiveness with optimal cell orientation occurring at very similar adhesive strengths. The effect of actin-myosin contractile level was examined by stretching cells treated with different doses of 2,3-butanedione monoxime (BDM) and Blebbistatin. Each drug induced a dose-dependent decrease in orientation angles after three hours of cyclic stretching. Furthermore, cell and stress fiber orientations were tightly coupled for untreated and Blebbistatin-treated cells but were uncoupled for BDM-treated cells. Even though orientation response to cyclic stretching is not a spontaneous motile response, it is determined, in large part, by the same factors that affect spontaneous motility--the cell-substratum adhesiveness and actin-myosin contractile level.