Verapamil Treatment Research Articles

Target population for a selective cardiac myosin inhibitor in hypertrophic obstructive cardiomyopathy: Real-life estimation from the French register of hypertrophic cardiomyopathy (REMY)

BackgroundThe efficacy of current pharmacological therapies in hypertrophic cardiomyopathy is limited. A cardiac myosin inhibitor, mavacamten, has recently been approved as a first-in-class treatment for symptomatic hypertrophic obstructive cardiomyopathy. AimsTo assess the profile and burden of cardiac myosin inhibitor candidates in the hypertrophic cardiomyopathy prospective Register of hypertrophic cardiomyopathy (REMY) held by the French Society of Cardiology. MethodsData were collected at baseline and during follow-up from patients with hypertrophic cardiomyopathy enrolled in REMY by the three largest participating centres. ResultsAmong 1059 adults with hypertrophic cardiomyopathy, 461 (43.5%) had obstruction; 325 (30.7%) of these were also symptomatic, forming the “cardiac myosin inhibitor candidates” group. Baseline features of this group were: age 58±15years; male sex (n=196; 60.3%); diagnosis-to-inclusion delay 5 (1–12)years; maximum wall thickness 20±6mm; left ventricular ejection fraction 69±6%; family history of hypertrophic cardiomyopathy or sudden cardiac death (n=133; 40.9%); presence of a pathogenic sarcomere gene mutation (n=101; 31.1%); beta-blocker or verapamil treatment (n=304; 93.8%), combined with disopyramide (n=28; 8.7%); and eligibility for septal reduction therapy (n=96; 29%). At the end of a median follow-up of 66 (34–106) months, 319 (98.2%) were treated for obstruction (n=43 [13.2%] received disopyramide), 46 (14.2%) underwent septal reduction therapy and the all-cause mortality rate was 1.9/100 person-years (95% confidence interval 1.4–2.6) (46 deaths). Moreover, 41 (8.9%) patients from the initial hypertrophic obstructive cardiomyopathy group became eligible for a cardiac myosin inhibitor. ConclusionsIn this cohort of patients with hypertrophic cardiomyopathy selected from the REMY registry, one third were eligible for a cardiac myosin inhibitor.

The importance of heteroresistance and efflux pumps in bedaquiline-resistant Mycobacterium tuberculosis isolates from Iran

BackgroundTuberculosis (TB) continues to pose a threat to communities worldwide and remains a significant public health issue in several countries. We assessed the role of heteroresistance and efflux pumps in bedaquiline (BDQ)-resistant Mycobacterium tuberculosis isolates.MethodsNineteen clinical isolates were included in the study, of which fifteen isolates were classified as MDR or XDR, while four isolates were fully susceptible. To evaluate BDQ heteroresistance, the Microplate Alamar Blue Assay (MABA) method was employed. For screening mixed infections, MIRU-VNTR was performed on clinical isolates. Mutations in the atpE and Rv0678 genes were determined based on next-generation sequencing data. Additionally, real-time PCR was applied to assess the expression of efflux pump genes in the absence and presence of verapamil (VP).ResultsAll 15 drug-resistant isolates displayed resistance to BDQ. Among the 19 total isolates, 21.05% (4/19) exhibited a heteroresistance pattern to BDQ. None of the isolates carried a mutation of the atpE and Rv0678 genes associated with BDQ resistance. Regarding the MIRU-VNTR analysis, most isolates (94.73%) showed the Beijing genotype. Fifteen (78.9%) isolates showed a significant reduction in BDQ MIC after VP treatment. The efflux pump genes of Rv0676c, Rv1258c, Rv1410c, Rv1634, Rv1819, Rv2459, Rv2846, and Rv3065 were overexpressed in the presence of BDQ.ConclusionsOur results clearly demonstrated the crucial role of heteroresistance and efflux pumps in BDQ resistance. Additionally, we established a direct link between the Rv0676c gene and BDQ resistance. The inclusion of VP significantly reduced the MIC of BDQ in both drug-susceptible and drug-resistant clinical isolates.

391 Intraarterial Verapamil for Neuroprotection in Ischemic Stroke

INTRODUCTION: Verapamil is an FDA-approved calcium channel blocker administered for the standard-of-care treatment of vasospasm. Preclinical studies have shown that verapamil reduces inflammation and protects penumbra neurons from further damage and eventual death. METHODS: Patients were recruited based on inclusion and exclusion criteria in a clinical protocol reviewed and approved by two institutional review boards. The final cohort comprised 42 emergency care patients with an acute onset focal neurological deficit consistent with ischemic stroke and/or a computed tomographic scan consistent with acute cerebral ischemia. All patients were 18 years of age or older that presented within 24 hours of symptom onset and had a significant but not severe neurological deficit, measured by a National Institutes of Health Stroke Scale (NIHSS) score greater than four but less than 22. All patients received a mechanical thrombectomy intervention after identifying a large vessel occlusion (LVO). RESULTS: The Verapamil-treated group (N = 23) included 22 patients with middle cerebral artery (MCA) occlusions and 1 with a basilar posterior cerebral artery (PCA) occlusion. In these patients, investigators administered 10 mg of Verapamil intraarterially following mechanical thrombectomy. Patients exposed to control conditions (N = 19) were matched for age, biological sex, admission NIHSS score, occlusion location, administration of tPA, and achieved TICI score. Intraarterial Verapamil treatment did not significantly increase sICH, hemorrhagic transformation, or death compared to standard of care treatment, demonstrating safety in an acute ischemic stroke patient population. Verapamil treatment was associated with significantly greater changes in mRS score, improved recovery time, and shortened length of hospital stay compared to standard-of-care treatment. CONCLUSIONS: This study suggests that verapamil is a safe and effective neuroprotective drug in ischemic stroke clinical populations that require mechanical thrombectomy for treatment.

Vitexin along with verapamil downregulates efflux pump P-glycoprotein in macrophages and potentiate M1 to M2 switching via TLR4-NF-κB-TNFR2 pathway in lipopolysaccharide treated mice

The lipopolysaccharide, a microbial toxin, is one of the major causative agents of sepsis. P-gp expression and its functions are altered during inflammation. LPS has been known to impair the functions of P-gp, an efflux transporter. But the effect of LPS on P-gp expression in murine peritoneal macrophages is poorly understood. Molecular docking studies reveal that vitexin is a potent substrate and verapamil a potent inhibitor of P-gp. In the present experimental study, the curative potential of vitexin as a fruit component and verapamil treated as a control inhibitor of P-gp was examined in a murine LPS sepsis model. The effects of vitexin and verapamil on P-gp expression in macrophages correlating with changes in macrophage polarization and associated functional responses during LPS induced sepsis were studied. Peritoneal macrophages of LPS (10 mg/kg body weight) challenged mice exhibited elevated levels of H2O2, superoxide, and NO in parallel with lower antioxidant activity. LPS treatment increased P-gp expression through increased TLR4/expression. However, LPS challenged mice treated with vitexin (5 mg/kg body weight) + verapamil (5 mg/kg body weight) showed higher anti-oxidant enzyme activity (SOD, CAT and GRx) resulting in reduced oxidative stress. This combination treatment also elevated TNFR2, concomitant with down-regulation of TLR4, NF-κB and P-gp expression in murine peritoneal macrophages, resulting in a switch from M1 to M2 polarisation of macrophages and reduced inflammatory responses. In conclusion, combined vitexin and verapamil treatment could be used as a promising therapy to regulate P-gp expression and protection against LPS mediated sepsis and inflammatory damages.

Pharmacological Modulation of the Ca2+/cAMP/Adenosine Signaling in Cardiac Cells as a New Cardioprotective Strategy to Reduce Severe Arrhythmias in Myocardial Infarction.

Acute myocardial infarction (AMI) is the main cause of morbidity and mortality worldwide and is characterized by severe and fatal arrhythmias induced by cardiac ischemia/reperfusion (CIR). However, the molecular mechanisms involved in these arrhythmias are still little understood. To investigate the cardioprotective role of the cardiac Ca2+/cAMP/adenosine signaling pathway in AMI, L-type Ca2+ channels (LTCC) were blocked with either nifedipine (NIF) or verapamil (VER), with or without A1-adenosine (ADO), receptors (A1R), antagonist (DPCPX), or cAMP efflux blocker probenecid (PROB), and the incidence of ventricular arrhythmias (VA), atrioventricular block (AVB), and lethality (LET) induced by CIR in rats was evaluated. VA, AVB and LET incidences were evaluated by ECG analysis and compared between control (CIR group) and intravenously treated 5 min before CIR with NIF 1, 10, and 30 mg/kg and VER 1 mg/kg in the presence or absence of PROB 100 mg/kg or DPCPX 100 µg/kg. The serum levels of cardiac injury biomarkers total creatine kinase (CK) and CK-MB were quantified. Both NIF and VER treatment were able to attenuate cardiac arrhythmias caused by CIR; however, these antiarrhythmic effects were abolished by pretreatment with PROB and DPCPX. The total serum CK and CK-MB were similar in all groups. These results indicate that the pharmacological modulation of Ca2+/cAMP/ADO in cardiac cells by means of attenuation of Ca2+ influx via LTCC and the activation of A1R by endogenous ADO could be a promising therapeutic strategy to reduce the incidence of severe and fatal arrhythmias caused by AMI in humans.

Chronic Administration of Ion Channel Blockers Impact Microglia Morphology and Function in a Murine Model of Alzheimer's Disease.

As the population ages, a high prevalence of multimorbidity will affect the way physicians need to think about drug interactions. With microglia's important involvement in the pathology and progression of Alzheimer's disease (AD), understanding whether systemically administered drugs intended for other affections could impact microglia function, already impacted by the presence of beta-amyloid, is important. The aim of this study was to evaluate morphological changes of microglia, using in vivo 2-photon laser scanning microscopy, in a murine model of AD under systemic administration of sodium or calcium ion channel blockers in order to establish potential effects that these drugs might have on microglia under neuro-inflammatory conditions. A total of 30 mice (age 14-16 weeks, weight 20-25 g) were used, with 25 APP randomly divided into three groups. The remaining animals were CX3CR1GFP/GFP male mice (n = 5) used as WT controls. After baseline behavior testing, all animals received daily intraperitoneal injections for 30 days according to the assigned group [WT (n = 5), Control (n = 5), Carbamazepine (n = 10), and Verapamil (n = 10)]. The results showed that the Verapamil treatment improved short-term memory and enhanced exploratory behavior in APP mice. The Carbamazepine treatment also improved short-term memory but did not elicit significant changes in anxiety-related behavior. Both Verapamil and Carbamazepine reduced the surveillance speed of microglia processes and changed microglia morphology in the cortex compared to the Control group. Due to their complex molecular machinery, microglia are potentially affected by drugs that do not target them specifically, and, as such, investigating these interactions could prove beneficial in our management of neurodegenerative pathologies.

1761-P: Verapamil Increases ß-Cells Proliferation in MIN6 Cells and ß-Cell Mass in Zebrafish Model

Recent clinical studies highlighted a significant clinical utility for verapamil in both type 1 (T1D) and type 2 (T2D) diabetes. Verapamil treatment in T1D lowered dependency on exogenous insulin. In T2D, addition of verapamil to metformin therapy improved glycemic control. These findings led us to further study the molecular and cellular mechanisms of verapamil in the treatment of T1D and T2D using an in vitro (MIN6 mouse β cell-line) and in vivo zebrafish (Danio rerio) model. In this study, MIN6 cells were cultured in growth media containing 5.6 mM glucose (mimicking normoglycemia) and supplemented with 2% BSA to eliminate any proliferative effect of high glucose in the media, and growth factors in FBS. The cells were treated with different concentrations of verapamil. The levels of cell proliferation (MTT assay), cell growth (cell counts), Ki67 expression (IF), and cell cycle (flow cytometry) were studied in MIN6 cells treated with the optimal dose detected (50 μM) of verapamil. Larvae (24hpf) of the Ins:NfsB-mCherry transgenic zebrafish (expressing mCherry fluorescence reporter under the control of insulin proximal promoter) were treated with 10 uM of verapamil for 3 days. Full larvae images were acquired using a stereo microscope. Our results show that verapamil induced significantly increased proliferation of MIN6 cells. Cell cycle analysis shows initiation of the proliferative effect during an early time point (1-4 hours) after verapamil treatment. Higher Ki67 expression indicated enhanced cellular proliferation at 24 hours and growth curve data confirmed that the proliferative effect of verapamil lasted for 7 days. As evident from increased mCherry fluorescence intensity in zebrafish larvae, verapamil treatment increased the β cell mass during the exposure period of three days. Taken together, our findings support the positive impact of verapamil on mouse β cell proliferation in vitro as well as on increased β cell mass in Zebrafish model. Disclosure H.Arefanian: None. S.E.John: None. S.Jacob: None. M.Alhusayan: None. S.J.Kurian: None. S.P.Kochumon: None. S.T.K.Sindhu: None. F.Alzaid: None. M.Abu-farha: None. J.Abubaker: None. A.T.Thangavel: None. A.Al madhoun: None. R.Ahmad: None. F.Almulla: None. F.Alrashed: None. F.Bahman: None. H.Alsaeed: None. N.A.Abukhalaf: None. S.Shenouda: None. M.R.Williams: None. R.Nizam: None. Funding Kuwait Foundation for the Advancement of Sciences (RACB-2019-001)

1763-P: Pretreatment with Verapamil Increases MIN6 Mouse ß-Cell Survival after Exposure to Cytotoxic Stressors

Verapamil was found to lower the risk of developing new-onset diabetes and to lower fasting glucose levels in persons with diabetes. While previous studies have focused on treatment with verapamil, our objective in this study was to determine the effect of verapamil pre-treatment on MIN6 mouse β cell-line viability in response to different stressors mimicking conditions of in vitro models of type 1 (T1D) and type 2 diabetes (T2D). MIN6 cells were treated with either streptozotocin (STZ), T1D-cytomix containing IL-1β, TNF-α, and INF-γ, or a T2D-cytomix containing IL-1β, TNF-α, and palmitic acid. MTT assay was performed to determine viability of the cells pre-treated with verapamil (50 µM) for 24 hours, followed by each stressor treatment for another 24 hours, under normal (5.6 mM) glucose conditions. For all stressors, pre-treatment with verapamil significantly increased the cell viability, compared to verapamil-untreated controls. To study the synergistic effect of verapamil pre-treatment on the combined treatment of verapamil plus stressors, MIN6 cells were pre-treated with verapamil, then exposed to co-treatment conditions. We found that co-treatment of verapamil with each stressor significantly improved the cell survival, compared to cells treated with the stressor only. In case of STZ and T2D-cytomix, pre-treatment of verapamil significantly increased the cell viability, compared to cells that only received co-treatment alone. No significant synergistic effect of verapamil was observed as pre-treatment on co-treatment of verapamil and T1D-cytomix. In all cases showing protective effect of verapamil pre-treatment, verapamil improved cell viability in a dose-dependent manner. These results suggest that verapamil may have cytoprotective effect in MIN6 β cells under conditions mimicking T1D/T2D insults, and pre-treatment with verapamil, in particular, offers higher protection. Disclosure H.Arefanian: None. M.Alhusayan: None. N.A.Abukhalaf: None. S.Shenouda: None. H.Alsaeed: None. S.J.Kurian: None. M.R.Williams: None. F.Alzaid: None. M.Abu-farha: None. J.Abubaker: None. A.T.Thangavel: None. F.Bahman: None. R.Ahmad: None. F.Almulla: None. A.Al madhoun: None. S.T.Sindhu: None. F.Alrashed: None. S.P.Kochumon: None. R.Nizam: None. S.E.John: None. S.Jacob: None. Funding Kuwait Foundation for the Advancement of Sciences (RACB-2019-001)

1774-P: Verapamil Prevents Decline of IGF-1 in Subjects with T1D

We have previously found that the calcium channel blocker and anti-hypertensive, verapamil, promotes functional beta cell mass and improves glucose homeostasis in diabetic mice and in humans with recent-onset type 1 diabetes (T1D). Surprisingly, we also discovered that verapamil reversed the T1D-associated elevation in T-follicular-helper cell markers in peripheral blood monocytes, suggesting that it also has immunomodulatory effects. Now, a global proteomics analysis using liquid chromatography-tandem mass spectrometry (LC-MS/MS) of serum samples from T1D subjects at baseline and after 1 year of receiving verapamil or placebo identified insulin-like growth factor 1 (IGF-1) as one of the top proteins with significantly changed abundance over time. Specifically, IGF-1 abundance decreased during year 1 after diagnosis in subjects with T1D receiving placebo, which is consistent with the lower IGF-1 levels reported previously in individuals with T1D as compared to age- and sex-matched healthy subjects. Interestingly, this decline was blunted in subjects receiving verapamil. To further confirm the LC-MS/MS results, we also measured patient serum levels of IGF-1 by ELISA. The results were very much in alignment and again revealed significantly decreased IGF-1 levels in the placebo group a year after diagnosis, but no such effect in the verapamil treatment group, suggesting that verapamil prevents the decline of IGF-1 in subjects with T1D. Of note, IGF-1, which is primarily produced in liver, acts on many tissues, including pancreatic islets promoting pathways involved in cell growth, differentiation and glucose metabolism. In beta cells, IGF-1 has also been shown to control apoptosis. Thus, our results reveal yet another previously unappreciated pathway affected by verapamil that may contribute to the beneficial effects observed with this treatment in the context of T1D. Disclosure G.Xu: None. J.Chen: None. B.Lu: None. W.Qian: None. A.Shalev: Other Relationship; TIXiMED. Funding National Institutes of Health (R01DK078752, U01DK120379)

Intra-arterial verapamil improves functional outcomes of thrombectomy in a preclinical model of extended hyperglycemic stroke.

The abrupt hyperglycemic reperfusion following thrombectomy has been shown to harm the efficacy of the intervention in stroke patients with large vessel occlusion. Studies of ours and others have shown thioredoxin-interacting protein (TXNIP) is critically involved in hyperglycemic stroke injury. We recently found verapamil ameliorates cerebrovascular toxicity of tissue plasminogen activators in hyperglycemic stroke. The present study aims to answer if verapamil exerts direct neuroprotective effects and alleviates glucose toxicity following thrombectomy in a preclinical model of hyperglycemic stroke. Primary cortical neural (PCN) cultures were exposed to hyperglycemic reperfusion following oxygen-glucose deprivation (OGD), with or without verapamil treatment. In a mouse model of intraluminal stroke, animals were subjected to 4h middle cerebral artery occlusion (MCAO) and intravenous glucose infusion. Glucose infusion lasted one more hour at reperfusion, along with intra-arterial (i.a.) verapamil infusion. Animals were subjected to sensorimotor function tests and histological analysis of microglial phenotype at 72h post-stroke. According to our findings, glucose concentrations (2.5-20mM) directly correlated with TXNIP expression in OGD-exposed PCN cultures. Verapamil (100nM) effectively improved PCN cell neurite growth and reduced TXNIP expression as well as interaction with NOD-like receptor pyrin domain-containing-3 (NLRP3) inflammasome, as determined by immunoblotting and immunoprecipitation. In our mouse model of extended hyperglycemic MCAO, i.a. verapamil (0.5mg/kg) could attenuate neurological deficits induced by hyperglycemic stroke. This was associated with reduced microglial pro-inflammatory transition. This finding encourages pertinent studies in hyperglycemic patients undergoing thrombectomy where the robust reperfusion may exacerbate glucose toxicity.

Inhibitory Effect of Verapamil on the Growth of Human Airway Granulation Fibroblasts.

To explore the inhibitory effect of verapamil, a calcium channel blocker, on the growth of human airway granulation fibroblasts to provide an experimental basis for the clinical use of calcium channel blockers in preventing and treating benign airway stenosis. Primary human airway normal fibroblasts and human airway granulation fibroblasts were cultured by tissue block attachment culture method, and the experimental studies were carried out using 3-8 generation cells. Cell Counting Kit-8 (CCK-8) was used to test the proliferation of human normal airway fibroblasts and human airway granulation fibroblasts and the semi-inhibitory concentration of verapamil on normal airway fibroblasts and airway granulation fibroblasts. A scratch test detected the migration effect of verapamil on human airway granulation fibroblasts. The mRNA relative expression levels of related factors were detected by PCR to compare the differences between normal airway fibroblasts and airway granulation fibroblasts. Western blot was used to detect the relative amount of related proteins and compare the differences between normal airway fibroblasts and granulation airway fibroblasts. After 48 hours of treatment with half of the inhibitory concentration of Vera Pammy for granulation airway fibroblasts, the relative expression levels of related factors on mRNA and protein were observed. Human normal airway fibroblasts and human airway granulation fibroblasts with a purity of more than 95% could be obtained from primary culture by tissue block adherence method. CCK8 results showed that the proliferation rate of human airway granulation fibroblasts was faster than that of the normal human airway fibroblasts. The semi-inhibitory concentration of verapamil on human normal airway fibroblasts was 92.81 ug/ml, while the semi-inhibitory concentration on human airway granulation fibroblasts was 69.57 ug/ml. The scratch test indicated that the cell migration rate of human airway granulation fibroblasts treated with verapamil decreased significantly (P < 0.05). PCR results showed that the mRNA relative expression levels of TGFβ1, COL1A1, Smad2/3, VEGFA, IL6, and IL8 in human airway granulation fibroblasts were significantly higher than those in normal human airway fibroblasts (P < 0.05). The mRNA relative expressions of TGFβ1, smad2/3, and COL1A1 in human airway granulation fibroblasts treated with semi-inhibited verapamil for 48h were down-regulated (P < 0.05), while the mRNA relative expressions of VEGFA, IL6 and IL8 had no significant changes (P > 0.05). WB test showed that the relative protein expressions of TGFβ1, Smad2, and VEGFC in human airway granulation fibroblasts were upregulated (P < 0.05) but downregulated after verapamil treatment compared with before treatment (P < 0.05). Calcium channel blockers can inhibit the proliferation of human airway granulation fibroblasts through TGFβ1/ Smad pathway, which may be a method to prevent and treat benign airway stenosis.

Verapamil inhibits TXNIP-NLRP3 inflammasome activation and preserves functional recovery after intracerebral hemorrhage in mice

Intracerebral hemorrhage (ICH) is the second most common type of stroke with no satisfactory treatment. Recent studies from our group and others indicated a potential positive effect of verapamil, a commonly prescribed calcium channel blocker, with thioredoxin-interacting protein (TXNIP) inhibitor properties, in ischemic stroke and cognitive disorders. It is unclear whether there would be a beneficial effect of verapamil administration in ICH. Therefore, this study was designed to determine the neuroprotective effects of verapamil in a murine ICH model. ICH was induced by stereotactic injection of collagenase type VII (0.075 U) into the right striatum of adult male C57BL/6 mice. Verapamil (0.15 mg/kg) or saline was administered intravenously at 1 h post-ICH followed by oral (1 mg/kg/d) administration in drinking water for 28 days. Motor and cognitive function were assessed using established tests for motor coordination, spatial learning, short- and long-term memory. A subset of animals was sacrificed at 72 h after ICH for molecular analysis. Verapamil treatment reduced expression of TXNIP and NOD-like receptor pyrin domain-containing-3 inflammasome activation in the perihematomal area. These protective effects of verapamil were associated with decreased proinflammatory mediators, microglial activation, and blood-brain barrier permeability markers and paralleled less phosphorylated nuclear factor kappa B level. Our findings also demonstrate that long-term low-dose verapamil effectively attenuated motor and cognitive impairments. Taken together, these data indicate that verapamil has therapeutic potential in improving acute motor function after ICH. Further investigations are needed to confirm whether verapamil treatment could be a promising candidate for clinical trials.

Sex steroid hormones and insulin sensitivity: the role of KATP sensitive channels

The Association of sex steroid hormones and insulin secretion has been reported in a number of studies, however, the mechanism behind the effects of sex steroid hormones on insulin secretion is still unclear. The present study investigated the effects of KATP sensitive channels blocker (verapamil) or opener (diazoxide) on insulin sensitivity in male and female rats. In this experimental-laboratory study, rats were divided to control (no treatment) and gonadectomized male and female rats, and male rats treated with testosterone enanthate, diazoxide and veramapil, and female rats treated with estradiol valerate, progesterone, diazoxide and veramapil. Serum glucose and insulin level were measured 4 weeks after surgery or treatment. Data were analyzed by one-way analysis of variance (ANOVA). Ttestosterone, verapamil, and co-administration of testosterone and verapamil resulted in a significant decrease in insulin sensitivity in male rats. Treatment of female rats with progesterone, diazoxide, and co-administration of progesterone and diazoxide or verapamil, led to a significant increase in insulin sensitivity, however, estradiol and verapamil treatment resulted in a significant decrease in insulin sensitivity. Our findings indicated that testosterone and estradiol were insulin sensitivity reducers and progesterone was an insulin sensitivity enhancer in rats. Despite progesterone, testosterone did not significantly affect KATP channels blocker (verapamil) or opener (diazoxide) action on insulin sensitivity.

In Vitro Drug Screening Using iPSC-Derived Cardiomyocytes of a Long QT-Syndrome Patient Carrying KCNQ1 & TRPM4 Dual Mutation: An Experimental Personalized Treatment.

Congenital long QT syndrome is a type of inherited cardiovascular disorder characterized by prolonged QT interval. Patient often suffer from syncopal episodes, electrocardiographic abnormalities and life-threatening arrhythmia. Given the complexity of the root cause of the disease, a combination of clinical diagnosis and drug screening using patient-derived cardiomyocytes represents a more effective way to identify potential cures. We identified a long QT syndrome patient carrying a heterozygous KCNQ1 c.656G>A mutation and a heterozygous TRPM4 c.479C>T mutation. Implantation of implantable cardioverter defibrillator in combination with conventional medication demonstrated limited success in ameliorating long-QT-syndrome-related symptoms. Frequent defibrillator discharge also caused deterioration of patient quality of life. Aiming to identify better therapeutic agents and treatment strategy, we established a patient-specific iPSC line carrying the dual mutations and differentiated these patient-specific iPSCs into cardiomyocytes. We discovered that both verapamil and lidocaine substantially shortened the QT interval of the long QT syndrome patient-specific cardiomyocytes. Verapamil treatment was successful in reducing defibrillator discharge frequency of the KCNQ1/TRPM4 dual mutation patient. These results suggested that verapamil and lidocaine could be alternative therapeutic agents for long QT syndrome patients that do not respond well to conventional treatments. In conclusion, our approach indicated the usefulness of the in vitro disease model based on patient-specific iPSCs in identifying pharmacological mechanisms and drug screening. The long QT patient-specific iPSC line carrying KCNQ1/TRPM4 dual mutations also represents a tool for further understanding long QT syndrome pathogenesis.

Verapamil inhibited the development of ureteral stricture by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.

Ureteral stricture (US) is a fibrotic process that leads to urinary tract obstruction and even kidney damage, with the characteristic of reduced extracellular matrix (ECM) degradation and increased collagen synthesis. Verapamil, as a calcium channel blocker, was reported to prevent scar formation. Our work aimed to investigate the biological effects and mechanism of verapamil in US. Fibroblasts were subjected to transforming growth factor-beta 1 (TGF-β1) to stimulate collagen synthesis, and the messenger ribonucleic acid (mRNA) and protein expressions in fibroblasts were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. The location of phosphorylation-signal transducer and activator of transcription 3 (p-STAT3) and Jund proto-oncogene subunit (JunD) in fibroblasts were determined by immunofluorescence (IF). The binding relationship between signal transducer and activator of transcription 3 (STAT3) and collagen type I alpha1 (COL1A1)/collagen type III alpha 1 chain (COL3A1) and the binding relationship between JunD and tissue inhibitor of metalloproteinases-1 (TIMP-1) were verified by dual luciferase reporter gene and chromatin Immunoprecipitation (ChIP) assays. Herein, we found that verapamil could inhibit TGF-β1/Ca2 + ⁄calmodulin-dependent protein kinase II (CaMK II)-mediated STAT3 activation in fibroblasts, and STAT3 inhibition repressed collagen production. In addition, verapamil could inhibit TGF-β1/CaMK II-mediated Mothers against DPP homolog 3 (Smad3)/JunD pathway activation in fibroblasts, and JunD silencing inhibited TIMP1 (a matrix metalloproteinase inhibitor) expression. Our subsequent experiments revealed that STAT3 bound with COL1A1 promoter and COL3A1 promoter and activated their transcription, and JunD bound with TIMP1 promoter and activated its transcription. Moreover, as expected, STAT3 activation could eliminate the inhibitory effect of verapamil treatment on TGF-β1-induced collagen production in fibroblasts, and JunD overexpression reversed the inhibitory effect of verapamil treatment on TGF-β1-induced TIMP1 expression in fibroblasts. Verapamil inhibited collagen production and TIMP-1 expression in US by blocking CaMK II-mediated STAT3 and Smad3/JunD pathways.

Improved fatigue properties, bone microstructure and blood glucose in type 2 diabetic rats with verapamil treatment

BackgroundType 2 diabetes mellitus is a global epidemic disease, which leads to a severe complication named increased bone fracture risk. This study aimed to explore if verapamil treatment could improve bone quality of type 2 diabetes mellitus. MethodsRat models of control, diabetes and verapamil treatment with 4/12/24/48 mg/kg/d were established, respectively. Blood glucose was monitored during 12-week treatment, and bilateral tibiae were collected. Microstructural images of bilateral metaphyseal cancellous bone and high-resolution images of cortical bone of left tibial shafts were obtained by micro-computed tomography. Fatigue properties of bone were evaluated via cyclic compressive tests of right tibial shafts. FindingsVerapamil treatment had no significant effect on blood glucose, but blood glucose tended to decline with the increase of verapamil-treated time and dose. Compared with controls, osteocyte lacunar and canal porosities in diabetes and verapamil-treated groups were significantly decreased (P < 0.05), trabecular separation and degree of anisotropy were significantly increased (P < 0.05), while trabecular tissue mineral density, trabecular bone volume fraction and trabecular number in verapamil-treated (48 mg/kg/d) group were significantly higher than those in diabetes (P < 0.05). Compared with diabetes, initial compressive elastic moduli in verapamil-treated (12/24/48 mg/kg/d) groups were significantly increased (P < 0.05), while secant modulus degradations in verapamil-treated (24/48 mg/kg/d) groups were significantly decreased (P < 0.05). InterpretationVerapamil could improve bone microstructure and fatigue properties in type 2 diabetic rats; and high-dose verapamil presented a significant effect on improving bone quality. These findings provided a new possibility for preventing the high bone fracture risk of type 2 diabetes mellitus in clinics.

Wnt Signaling Interactor WTIP (Wilms Tumor Interacting Protein) Underlies Novel Mechanism for Cardiac Hypertrophy.

The study of hypertrophic cardiomyopathy (HCM) can yield insight into the mechanisms underlying the complex trait of cardiac hypertrophy. To date, most genetic variants associated with HCM have been found in sarcomeric genes. Here, we describe a novel HCM-associated variant in the noncanonical Wnt signaling interactor WTIP (Wilms tumor interacting protein) and provide evidence of a role for WTIP in complex disease. In a family affected by HCM, we used exome sequencing and identity-by-descent analysis to identify a novel variant in WTIP (p.Y233F). We knocked down WTIP in isolated neonatal rat ventricular myocytes with lentivirally delivered short hairpin ribonucleic acids and in Danio rerio via morpholino injection. We performed weighted gene coexpression network analysis for WTIP in human cardiac tissue, as well as association analysis for WTIP variation and left ventricular hypertrophy. Finally, we generated induced pluripotent stem cell-derived cardiomyocytes from patient tissue, characterized size and calcium cycling, and determined the effect of verapamil treatment on calcium dynamics. WTIP knockdown caused hypertrophy in neonatal rat ventricular myocytes and increased cardiac hypertrophy, peak calcium, and resting calcium in D rerio. Network analysis of human cardiac tissue indicated WTIP as a central coordinator of prohypertrophic networks, while common variation at the WTIP locus was associated with human left ventricular hypertrophy. Patient-derived WTIP p.Y233F-induced pluripotent stem cell-derived cardiomyocytes recapitulated cellular hypertrophy and increased resting calcium, which was ameliorated by verapamil. We demonstrate that a novel genetic variant found in a family with HCM disrupts binding to a known Wnt signaling protein, misregulating cardiomyocyte calcium dynamics. Further, in orthogonal model systems, we show that expression of the gene WTIP is important in complex cardiac hypertrophy phenotypes. These findings, derived from the observation of a rare Mendelian disease variant, uncover a novel disease mechanism with implications across diverse forms of cardiac hypertrophy.

Increased Glycolysis and ATP Production Are Not Prerequisites for Cardiomyocytes Cell Cycle Induction

BackgroundIt has been reported that metabolic switch from fatty acid oxidation to glycolysis is associated with cardiomyocyte cell cycle induction. However, it is unknown if increased glycolysis and ATP production are indispensable for cardiomyocyte cell cycle induction. Here, we aim to address this question.MethodsCell cycle was induced by overexpression of CCND2 gene or treatment of FGF1 (100 ng/mL) and CHIR99021 (5 µM) on human induced pluripotent stem cells‐derived cardiomyocytes (hiPSC‐CMs). Contraction inhibition was induced by treatment of verapamil (0.5 μM). Cell cycle was evaluated by immunostaining using antibodies against cell cycle markers. Real‐time ATP production and mitochondrial oxidative phosphorylation were determined by Seahorse assay. Metabolic profile was assessed by liquid chromatography–mass spectrometry.ResultsTreatment of FGF1 and CHIR99021 induces cell cycle of hiPSC‐CMs, reduced carbohydrate metabolism and real‐time ATP production from both mitochondria and cytoplasm. Data from RNA Sequencing showed this treatment increased expression of CCND2 and CDK4 and decreased expression of sarcomeric contractile related genes. Overexpression of CCND2 in hiPSC‐CMs also induces cell cycle, increased aerobic glycolysis (Warburg effect) and no change of real‐time ATP production. Inhibition of contraction via treatment of verapamil resulted in reduced overall metabolism and real‐time ATP production.ConclusionOur data suggest that cardiomyocyte cell cycle may be induced without increased glycolysis or real‐time ATP production.

Exploratory study reveals far reaching systemic and cellular effects of verapamil treatment in subjects with type 1 diabetes

Currently, no oral medications are available for type 1 diabetes (T1D). While our recent randomized placebo-controlled T1D trial revealed that oral verapamil had short-term beneficial effects, their duration and underlying mechanisms remained elusive. Now, our global T1D serum proteomics analysis identified chromogranin A (CHGA), a T1D-autoantigen, as the top protein altered by verapamil and as a potential therapeutic marker and revealed that verapamil normalizes serum CHGA levels and reverses T1D-induced elevations in circulating proinflammatory T-follicular-helper cell markers. RNA-sequencing further confirmed that verapamil regulates the thioredoxin system and promotes an anti-oxidative, anti-apoptotic and immunomodulatory gene expression profile in human islets. Moreover, continuous use of oral verapamil delayed T1D progression, promoted endogenous beta-cell function and lowered insulin requirements and serum CHGA levels for at least 2 years and these benefits were lost upon discontinuation. Thus, the current studies provide crucial mechanistic and clinical insight into the beneficial effects of verapamil in T1D.

Biliary organoids uncover delayed epithelial development and barrier function in biliary atresia.

Biliary atresia is a severe inflammatory and fibrosing cholangiopathy of neonates of unknown etiology. The onset of cholestasis at birth implies a prenatal onset of liver dysfunction. Our aim was to investigate the mechanisms linked to abnormal cholangiocyte development. We generated biliary organoids from liver biopsies of infants with biliary atresia and normal and diseased controls. Organoids emerged from biliary atresia livers and controls and grew as lumen-containing spheres with an epithelial lining of cytokeratin-19pos albuminneg SOX17neg cholangiocyte-like cells. Spheres had similar gross morphology in all three groups and expressed cholangiocyte-enriched genes. In biliary atresia, cholangiocyte-like cells lacked a basal positioning of the nucleus, expressed fewer developmental and functional markers, and displayed misorientation of cilia. They aberrantly expressed F-actin, β-catenin, and Ezrin, had low signals for the tight junction protein zonula occludens-1 (ZO-1), and displayed increased permeability as evidenced by a higher Rhodamine-123 (R123) signal inside organoids after verapamil treatment. Biliary atresia organoids had decreased expression of genes related to EGF signaling and FGF2 signaling. When treated with EGF+FGF2, biliary atresia organoids expressed differentiation (cytokeratin 7 and hepatocyte nuclear factor 1 homeobox B) and functional (somatostatin receptor 2, cystic fibrosis transmembrane conductance regulator [CFTR], aquaporin 1) markers, restored polarity with improved localization of F-actin, β-catenin and ZO-1, increased CFTR function, and decreased uptake of R123. Organoids from biliary atresia are viable and have evidence of halted epithelial development. The induction of developmental markers, improved cell-cell junction, and decreased epithelial permeability by EGF and FGF2 identifies potential strategies to promote epithelial maturation and function.