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Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We previously reported proof-of-concept data suggesting that both pharmacological inhibition and genetic deletion of KCa3.1 reduces infarction and improves neurologic recovery in rodents by attenuating neuroinflammation. Here we evaluated the potential of repurposing senicapoc for ischemic stroke. In cultured microglia, senicapoc inhibited KCa3.1 currents with an IC50 of 7 nM, reduced Ca2+ signaling induced by the purinergic agonist ATP, suppressed expression of pro-inflammatory cytokines and enzymes (iNOS and COX-2), and prevented induction of the inflammasome component NLRP3. When transient middle cerebral artery occlusion (tMCAO, 60 min) was induced in male C57BL/6 J mice, twice daily administration of senicapoc at 10 and 40 mg/kg starting 12 h after reperfusion dose-dependently reduced infarct area determined by T2-weighted magnetic resonance imaging (MRI) and improved neurological deficit on day 8. Ultra-high-performance liquid chromatography/mass spectrometry analysis of total and free brain concentrations demonstrated sufficient KCa3.1 target engagement. Senicapoc treatment significantly reduced microglia/macrophage and T cell infiltration and activation and attenuated neuronal death. A different treatment paradigm with senicapoc started at 3 h and MRI on day 3 and day 8 revealed that senicapoc reduces secondary infarct growth and suppresses expression of inflammation markers, including T cell cytokines in the brain. Lastly, we demonstrated that senicapoc does not impair the proteolytic activity of tissue plasminogen activator (tPA) in vitro. We suggest that senicapoc could be repurposed as an adjunctive immunocytoprotective agent for combination with reperfusion therapy for ischemic stroke.

Thermosensation, the ability to detect and estimate temperature, is an evolutionarily conserved process that is essential for survival. Thermosensing is impaired in various pain syndromes, resulting in thermal allodynia, the perception of an innocuous temperature as painful, or thermal hyperalgesia, an exacerbated perception of a painful thermal stimulus. Several behavioral assays exist to study thermosensation and thermal pain in rodents, however, most rely on reflexive withdrawal responses or the subjective quantification of spontaneous nocifensive behaviors. Here, we created a new apparatus, the thermal escape box, which can be attached to temperature-controlled plates and used to assess temperature-dependent effort-based decision-making. The apparatus consists of a light chamber with an opening that fits around temperature-controlled plates, and a small entryway into a dark chamber. A mouse must choose to stay in a brightly lit aversive area or traverse the plates to escape to the enclosed dark chamber. We quantified escape latencies of adult C57Bl/6 mice at different plate temperatures from video recordings and found they were significantly longer at 5 °C, 18 °C, and 52 °C, compared to 30 °C, a mouse's preferred ambient temperature. Differences in escape latencies were abolished in male Trpm8-/- mice and in male Trpv1-/- animals. Finally, we show that chronic constriction injury procedures or oxaliplatin treatement significantly increased escape latencies at cold temperatures compared to controls, the later of which was prevented by the analgesic meloxicam. This demonstrates the utility of this assay in detecting cold pain. Collectively, our study has identified a new and effective tool that uses cost-benefit valuations to study thermosensation and thermal pain.

The neurosteroid allopregnanolone (ALLO) is under investigation as a treatment for benzodiazepine-refractory status epilepticus (SE). Here, we assess the cardiopulmonary safety of intravenous ALLO by itself and after a clinically recommended dose of midazolam (MDZ) in two healthy adult beagles. Each dog received ALLO (1 mg/kg, IV), and after a washout period of 2 weeks, each dog was dosed with MDZ (0.2 mg/kg, IV) followed 10 minutes later by ALLO. Behavioral state, vital signs, arterial blood gases, blood chemistries, and plasma ALLO concentrations were monitored for up to 6 hours after dosing. The dogs appeared sleepy but were fully responsive after both treatments. No depression of mean arterial pressure or respiratory rate was noted. Blood gas measurements failed to show evidence of drug-induced acute respiratory acidosis. Estimated maximum plasma ALLO concentrations were in the range of 1500 to 3000 ng/ml. The results indicate that intravenous ALLO can be used safely to treat benzodiazepine-refractory SE, even when administered shortly after a benzodiazepine.

Epileptogenic seizures, or status epilepticus (SE), leads to excitotoxic injury in hippocampal and limbic neurons in the kainic acid (KA) animal model of temporal lobe epilepsy (TLE). Here, we have further characterized neural activity regulated methylaminoisobutryic acid (MeAIB)/glutamine transport activity in mature rat hippocampal neurons in vitro that is inhibited by riluzole (IC50 = 1 μM), an anti-convulsant benzothiazole agent. We screened a library of riluzole derivatives and identified SKA-41 followed by a second screen and synthesized several novel chlorinated aminothiazoles (SKA-377, SKA-378, SKA-379) that are also potent MeAIB transport inhibitors in vitro, and brain penetrant following systemic administration. When administered before KA, SKA-378 did not prevent seizures but still protected the hippocampus and several other limbic areas against SE-induced neurodegeneration at 3d. When SKA-377 - 379, (30 mg/kg) were administered after KA-induced SE, acute neural injury in the CA3, CA1 and CA4/hilus was also largely attenuated. Riluzole (10 mg/kg) blocks acute neural injury. Kinetic analysis of SKA-378 and riluzoles' blockade of Ca2+-regulated MeAIB transport in neurons in vitro indicates that inhibition occurs via a non-competitive, indirect mechanism. Sodium channel NaV1.6 antagonism blocks neural activity regulated MeAIB/Gln transport in vitro (IC50 = 60 nM) and SKA-378 is the most potent inhibitor of NaV1.6 (IC50 = 28 μM) compared to NaV1.2 (IC50 = 118 μM) in heterologous cells. However, pharmacokinetic analysis suggests that sodium channel blockade may not be the predominant mechanism of neuroprotection here. Riluzole and our novel aminothiazoles are agents that attenuate acute neural hippocampal injury following KA-induced SE and may help to understand mechanisms involved in the progression of epileptic disease.

Introduction: Mechanical thrombectomy has improved treatment options and outcomes for acute ischemic stroke with large artery occlusion. However, as the time window of endovascular thrombectomy is extended there is an increasing need to develop immunocytoprotective therapies that can reduce inflammation in the penumbra and prevent reperfusion injury. We previously demonstrated, that by reducing neuroinflammation, KV1.3 inhibitors can improve outcomes not only in young male rodents but also in female and aged animals. To further explore the therapeutic potential of KV1.3 inhibitors for stroke therapy, we here directly compared a peptidic and a small molecule KV1.3 blocker and asked whether KV1.3 inhibition would still be beneficial when started at 72 hours after reperfusion. Methods: Transient middle cerebral artery occlusion (tMCAO, 90-min) was induced in male Wistar rats and neurological deficit assessed daily. On day-8 infarction was determined by T2-weighted MRI and inflammatory marker expression in the brain by quantitative PCR. Potential interactions with tissue plasminogen activator (tPA) were evaluated in-vitro with a chromogenic assay. Results: In a direct comparison with administration started at 2 hours after reperfusion, the small molecule PAP-1 significantly improved outcomes on day-8, while the peptide ShK-223 failed to reduce infarction and neurological deficits despite reducing inflammatory marker expression. PAP-1 still provided benefits when started 72 hours after reperfusion. PAP-1 does not reduce the proteolytic activity of tPA. Discussion: Our studies suggest that KV1.3 inhibition for immunocytoprotection after ischemic stroke has a wide therapeutic window for salvaging the inflammatory penumbra and requires brain-penetrant small molecules.

Background: Secondhand smoke (SHS) is a significant risk factor for cardiovascular morbidity and mortality with an estimated 80% of SHS-related deaths attributed to cardiovascular causes. Public health measures and smoking bans have been successful both in reducing SHS exposure and improving cardiovascular outcomes in non-smokers. Soluble epoxide hydrolase (sEH) inhibitors have been shown to attenuate tobacco exposure-induced lung inflammatory responses, making them a promising target for mitigating SHS exposure-induced cardiovascular outcomes. Objectives: The objectives of this study were to determine 1) effects of environmentally relevant SHS exposure on cardiac autonomic function and blood pressure (BP) regulation and 2) whether prophylactic administration of an sEH inhibitor (TPPU) can reduce the adverse cardiovascular effects of SHS exposure. Methods: Male C57BL/6J mice (11 weeks old) implanted with BP/electrocardiogram (ECG) telemetry devices were exposed to filtered air or 3 mg/m3 of SHS (6 hr/d, 5 d/wk) for 12 weeks, followed by 4 weeks of recovery in filtered air. Some mice received TPPU in drinking water (15 mg/L) throughout SHS exposure. BP, heart rate (HR), HR variability (HRV), baroreflex sensitivity (BRS), and BP variability were determined monthly. Results: SHS exposure significantly decreased 1) short-term HRV by ∼20% (p < 0.05) within 4 weeks; 2) overall HRV with maximum effect at 12 weeks (-15%, p < 0.05); 3) pulse pressure (-8%, p < 0.05) as early as week 4; and 4) BRS with maximum effect at 12 weeks (-11%, p < 0.05). Four weeks of recovery following 12 weeks of SHS ameliorated all SHS-induced cardiovascular detriments. Importantly, mice exposed to TPPU in drinking water during SHS-related exposure were protected from SHS cardiovascular consequences. Discussion: The data suggest that 1) environmental relevant SHS exposure significantly alters cardiac autonomic function and BP regulation; 2) cardiovascular consequences from SHS can be reversed by discontinuing SHS exposure; and 3) inhibiting sEH can prevent SHS-induced cardiovascular consequences.

The voltage-gated sodium NaV1.7 channel plays a key role as a mediator of action potential propagation in C-fiber nociceptors and is an established molecular target for pain therapy. ProTx-II is a potent and moderately selective peptide toxin from tarantula venom that inhibits human NaV1.7 activation. Here we used available structural and experimental data to guide Rosetta design of potent and selective ProTx-II-based peptide inhibitors of human NaV1.7 channels. Functional testing of designed peptides using electrophysiology identified the PTx2-3127 and PTx2-3258 peptides with IC50s of 7 nM and 4 nM for hNaV1.7 and more than 1000-fold selectivity over human NaV1.1, NaV1.3, NaV1.4, NaV1.5, NaV1.8, and NaV1.9 channels. PTx2-3127 inhibits NaV1.7 currents in mouse and human sensory neurons and shows efficacy in rat models of chronic and thermal pain when administered intrathecally. Rationally designed peptide inhibitors of human NaV1.7 channels have transformative potential to define a new class of biologics to treat pain.

ObjectiveThe voltage-gated potassium channel Kv1.3, which is expressed on activated, disease-associated microglia and memory T cells, constitutes an attractive target for immunocytoprotection after endovascular thrombectomy (EVT). Using young male mice and rats we previously demonstrated that the Kv1.3 blocker PAP-1 when started 12 h after reperfusion dose-dependently reduces infarction and improves neurological deficit on day 8. However, these proof-of-concept findings are of limited translational value because the majority of strokes occur in patients over 65 and, when considering overall lifetime risk, in females. Here, we therefore tested whether Kv1.3 deletion or delayed pharmacological therapy would be beneficial in females and aged animals.MethodsTransient middle cerebral artery occlusion (tMCAO, 60 min) was induced in 16-week-old and 80-week-old male and female wild-type C57BL/6J and Kv1.3-/- mice. Stroke outcomes were assessed daily with the 14-score tactile and proprioceptive limp placing test and on day 8 before sacrifice by T2-weighted MRI. Young and old female mice were treated twice daily with 40 mg/kg PAP-1 starting 12 h after reperfusion. Microglia/macrophage activation and T-cell infiltration were evaluated in whole slide scans.ResultsKv1.3 deletion provided no significant benefit in young females but improved outcomes in young males, old males, and old females compared with wild-type controls of the same sex. Delayed PAP-1 treatment improved outcomes in both young and old females. In old females, Kv1.3 deletion and PAP-1 treatment significantly reduced Iba-1 and CD3 staining intensity in the ipsilateral hemisphere.InterpretationOur preclinical studies using aged and female mice further validate Kv1.3 inhibitors as potential adjunctive treatments for reperfusion therapy in stroke by providing both genetic and pharmacological verification.

Myocardial infarction (MI) and osteoporotic fracture are leading causes of morbidity and mortality, and epidemiological evidence linking their incidence suggests possible crosstalk. MI can exacerbate atherosclerosis through the sympathetic nervous system (SNS) activation and β3 adrenoreceptor-mediated release of hematopoietic stem cells, leading to monocytosis. We hypothesized that this same pathway initiates systemic bone loss following MI, since osteoclasts differentiate from monocytes. In this study, MI was created with left anterior descending artery ligation in 12-week-old male mice (n = 24) randomized to β3 -adrenergic receptor (AR) antagonist (SR 59230A) treatment or no treatment for 10 days postoperatively. Additional mice (n = 21, treated and untreated) served as unoperated controls. Bone mineral density (BMD), bone mineral content (BMC), and body composition were quantified at baseline and 10 days post-MI using dual-energy x-ray absorptiometry; circulating monocyte levels were quantified and the L5 vertebral body and femur were analyzed with microcomputed tomography 10 days post-MI. We found that MI led to circulating monocyte levels increases, BMD and BMC decreases at the femur and lumbar spine in MI mice (-6.9% femur BMD, -3.5% lumbar BMD), and trabecular bone volume decreases in MI mice compared with control mice. β3 -AR antagonist treatment appeared to diminish the bone loss response (-5.3% femur BMD, -1.2% lumbar BMD), though these results were somewhat inconsistent. Clinical significance: These results suggest that MI leads to systemic bone loss, but that the SNS may not be a primary modulator of this response; bone loss and increased fracture risk may be important clinical comorbidities following MI or other ischemic injuries.

Background: Secondhand smoke (SHS), a major indoor pollutant, is a significant risk factor for cardiovascular morbidity and mortality including arrhythmias and sudden cardiac death. Exposure to SHS can produce autonomic imbalance, as evidenced by reduced heart rate variability (HRV)-a clinical metric of cardiac vagal regulation. Currently, the mechanisms through which SHS changes the vagal preganglionic neuronal inputs to the heart to produce this remains unknown. Objectives: To characterize the effect of SHS on both the excitability and action potential (AP) characteristics of anatomically identified cardiac vagal neurons (CVNs) in the nucleus ambiguus and examine whether SHS alters small conductance calcium-activated potassium (SK) channel activity of these CVNs. Methods: Adult male mice were exposed to four weeks of filtered air or SHS (3 mg/m3) 6 h/day, 5 day/week. Using patch-clamp recordings on identified CVNs in brainstem slices, we determined neuronal excitability and AP characteristics with depolarizing step- and ramp-current injections. Results: Four weeks of SHS exposure reduced spiking responses to depolarizing current injections and increased AP voltage threshold in CVNs. Perfusion with apamin (20 nM) magnified these SHS-induced effects, suggesting reduced SK channel activity may serve to minimize the SHS-induced decreases in CVNs excitability. Medium afterhyperpolarization (a measurement of SK channel activity) was smaller in the SHS group, further supporting a lower SK channel activity. AP amplitude, rise rate, fast afterhyperpolarization amplitude (a measurement of voltage-gated channel activity), and decay rate were higher in the SHS group at membrane voltages more positive to 0 mV, suggesting altered inactivation properties of voltage-dependent channels underlying APs. Discussion: SHS exposure reduced neuronal excitability of CVNs with compensatory attenuation of SK channel activity and altered AP characteristics. Neuroplasticity of CVNs could blunt regulatory cardiac vagal signaling and contribute to the cardiovascular consequences associated with SHS exposure, including reduced HRV.

Microglia-mediated inflammation exerts adverse effects in ischemic stroke and in neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the voltage-gated potassium channel Kv1.3 is required for microglia activation. Both genetic deletion and pharmacological inhibition of Kv1.3 are effective in reducing microglia activation and the associated inflammatory responses, as well as in improving neurological outcomes in animal models of AD and ischemic stroke. Here we sought to elucidate the molecular mechanisms underlying the therapeutic effects of Kv1.3 inhibition, which remain incompletely understood. Using a combination of whole-cell voltage-clamp electrophysiology and quantitative PCR (qPCR), we first characterized a stimulus-dependent differential expression pattern for Kv1.3 and P2X4, a major ATP-gated cationic channel, both in vitro and in vivo. We then demonstrated by whole-cell current-clamp experiments that Kv1.3 channels contribute not only to setting the resting membrane potential but also play an important role in counteracting excessive membrane potential changes evoked by depolarizing current injections. Similarly, the presence of Kv1.3 channels renders microglia more resistant to depolarization produced by ATP-mediated P2X4 receptor activation. Inhibiting Kv1.3 channels with ShK-223 completely nullified the ability of Kv1.3 to normalize membrane potential changes, resulting in excessive depolarization and reduced calcium transients through P2X4 receptors. Our report thus links Kv1.3 function to P2X4 receptor-mediated signaling as one of the underlying mechanisms by which Kv1.3 blockade reduces microglia-mediated inflammation. While we could confirm previously reported differences between males and females in microglial P2X4 expression, microglial Kv1.3 expression exhibited no gender differences in vitro or in vivo. MAIN POINTS: The voltage-gated K+ channel Kv1.3 regulates microglial membrane potential. Inhibition of Kv1.3 depolarizes microglia and reduces calcium entry mediated by P2X4 receptors by dissipating the electrochemical driving force for calcium.

Acute intoxication with picrotoxin or the rodenticide tetramethylenedisulfotetramine (TETS) can cause seizures that rapidly progress to status epilepticus and death. Both compounds inhibit γ-aminobutyric acid type-A (GABAA) receptors with similar potency. However, TETS is approximately 100 × more lethal than picrotoxin. Here, we directly compared the toxicokinetics of the two compounds following intraperitoneal administration in mice. Using LC/MS analysis we found that picrotoxinin, the active component of picrotoxin, hydrolyses quickly into picrotoxic acid, has a short in vivo half-life, and is moderately brain penetrant (brain/plasma ratio 0.3). TETS, in contrast, is not metabolized by liver microsomes and persists in the body following intoxication. Using both GC/MS and a TETS-selective immunoassay we found that mice administered TETS at the LD50 of 0.2 mg/kg in the presence of rescue medications exhibited serum levels that remained constant around 1.6 μM for 48 h before falling slowly over the next 10 days. TETS showed a similar persistence in tissues. Whole-cell patch-clamp demonstrated that brain and serum extracts prepared from mice at 2 and 14 days after TETS administration significantly blocked heterologously expressed α2β3γ2 GABAA-receptors confirming that TETS remains pharmacodynamically active in vivo. This observed persistence may contribute to the long-lasting and recurrent seizures observed following human exposures. We suggest that countermeasures to neutralize TETS or accelerate its elimination should be explored for this highly dangerous threat agent.

Cerebral edema is exacerbated in diabetic ischemic stroke through poorly understood mechanisms. We showed previously that blood-brain barrier (BBB) Na-K-Cl cotransport (NKCC) and Na/H exchange (NHE) are major contributors to edema formation in normoglycemic ischemic stroke. Here, we investigated whether hyperglycemia-exacerbated edema involves changes in BBB NKCC and NHE expression and/or activity and whether inhibition of NKCC or NHE effectively reduces edema and injury in a type I diabetic model of hyperglycemic stroke. Cerebral microvascular endothelial cell (CMEC) NKCC and NHE abundances and activities were determined by Western blot, radioisotopic flux and microspectrofluorometric methods. Cerebral edema and Na in rats subjected to middle cerebral artery occlusion (MCAO) were assessed by nuclear magnetic resonance methods. Hyperglycemia exposures of 1-7d significantly increased CMEC NKCC and NHE abundance and activity. Subsequent exposure to ischemic factors caused more robust increases in NKCC and NHE activities than in normoglycemic CMEC. MCAO-induced edema and brain Na uptake were greater in hyperglycemic rats. Intravenous bumetanide and HOE-642 significantly attenuated edema, brain Na uptake and ischemic injury. Our findings provide evidence that BBB NKCC and NHE contribute to increased edema in hyperglycemic stroke, suggesting that these Na transporters are promising therapeutic targets for reducing damage in diabetic stroke.

ObjectiveInhibitors of the voltage-gated K+ channel Kv1.3 are currently in development as immunomodulators for the treatment of autoimmune diseases. As Kv1.3 is also expressed on microglia and has been shown to be specifically up-regulated on "M1-like" microglia, we here tested the therapeutic hypothesis that the brain-penetrant small-molecule Kv1.3-inhibitor PAP-1 reduces secondary inflammatory damage after ischemia/reperfusion.MethodsWe studied microglial Kv1.3 expression using electrophysiology and immunohistochemistry, and evaluated PAP-1 in hypoxia-exposed organotypic hippocampal slices and in middle cerebral artery occlusion (MCAO) with 8 days of reperfusion in both adult male C57BL/6J mice (60 min MCAO) and adult male Wistar rats (90 min MCAO). In both models, PAP-1 administration was started 12 h after reperfusion.ResultsWe observed Kv1.3 staining on activated microglia in ischemic infarcts in mice, rats, and humans and found higher Kv1.3 current densities in acutely isolated microglia from the infarcted hemisphere than in microglia isolated from the contralateral hemisphere of MCAO mice. PAP-1 reduced microglia activation and increased neuronal survival in hypoxia-exposed hippocampal slices as effectively as minocycline. In mouse MCAO, PAP-1 dose-dependently reduced infarct area, improved neurological deficit score, and reduced brain levels of IL-1β and IFN-γ without affecting IL-10 and brain-derived nerve growth factor (BDNF) levels or inhibiting ongoing phagocytosis. The beneficial effects on infarct area and neurological deficit score were reproduced in rats providing confirmation in a second species.InterpretationOur findings suggest that Kv1.3 constitutes a promising therapeutic target for preferentially inhibiting "M1-like" inflammatory microglia/macrophage functions in ischemic stroke.

Activated microglia/macrophages significantly contribute to the secondary inflammatory damage in ischemic stroke. Cultured neonatal microglia express the K+ channels Kv1.3 and KCa3.1, both of which have been reported to be involved in microglia-mediated neuronal killing, oxidative burst and cytokine production. However, it is questionable whether neonatal cultures accurately reflect the K+ channel expression of activated microglia in the adult brain. We here subjected mice to middle cerebral artery occlusion with eight days of reperfusion and patch-clamped acutely isolated microglia/macrophages. Microglia from the infarcted area exhibited higher densities of K+ currents with the biophysical and pharmacological properties of Kv1.3, KCa3.1 and Kir2.1 than microglia from non-infarcted control brains. Similarly, immunohistochemistry on human infarcts showed strong Kv1.3 and KCa3.1 immunoreactivity on activated microglia/macrophages. We next investigated the effect of genetic deletion and pharmacological blockade of KCa3.1 in reversible middle cerebral artery occlusion. KCa3.1-/- mice and wild-type mice treated with the KCa3.1 blocker TRAM-34 exhibited significantly smaller infarct areas on day-8 after middle cerebral artery occlusion and improved neurological deficit. Both manipulations reduced microglia/macrophage activation and brain cytokine levels. Our findings suggest KCa3.1 as a pharmacological target for ischemic stroke. Of potential, clinical relevance is that KCa3.1 blockade is still effective when initiated 12 h after the insult.

RationaleCardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes.ObjectiveTo develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes.Methods and resultsFreshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy.ConclusionsMultimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.

Isolation of high quality cardiomyocytes is critically important for achieving successful experiments in many cellular and molecular cardiology studies. Methods for isolating cardiomyocytes from the murine heart generally are time-sensitive and experience-dependent, and often fail to produce high quality cells. Major technical difficulties can be related to the surgical procedures needed to explant the heart and to cannulate the vessel to mount onto the Langendorff system before in vitro reperfusion can begin. During this period, transient hypoxia and ischemia may damage the heart, resulting in low yield and poor quality of cells, especially for heart disease models that have fragile cells. We have developed novel in vivo cannulation methods to minimize hypoxia and ischemia, and fine-tuned the entire protocol to produce high quality ventricular myocytes. The high cell quality has been confirmed using important structural and functional criteria such as morphology, t-tubule structure, action potential morphology, Ca2+ signaling, responsiveness to beta-adrenergic agonist, and ability to have robust contraction under mechanically loaded condition. Together these assessments show the preservation of the cardiac excitation-contraction machinery in cells isolated using this technique. The in vivo cannulation method enables consistent isolation of high-quality cardiomyocytes, even from heart disease models that were notoriously difficult for cell isolation using traditional methods.

Inhibitors of voltage-gated sodium channels (Na(v)) have been used as anticonvulsants since the 1940s, while potassium channel activators have only been investigated more recently. We here describe the discovery of 2-amino-6-trifluoromethylthio-benzothiazole (SKA-19), a thioanalog of riluzole, as a potent, novel anticonvulsant, which combines the two mechanisms. SKA-19 is a use-dependent NaV channel blocker and an activator of small-conductance Ca(2+)-activated K(+) channels. SKA-19 reduces action potential firing and increases medium afterhyperpolarization in CA1 pyramidal neurons in hippocampal slices. SKA-19 is orally bioavailable and shows activity in a broad range of rodent seizure models. SKA-19 protects against maximal electroshock-induced seizures in both rats (ED50 1.6 mg/kg i.p.; 2.3 mg/kg p.o.) and mice (ED50 4.3 mg/kg p.o.), and is also effective in the 6-Hz model in mice (ED50 12.2 mg/kg), Frings audiogenic seizure-susceptible mice (ED50 2.2 mg/kg), and the hippocampal kindled rat model of complex partial seizures (ED50 5.5 mg/kg). Toxicity tests for abnormal neurological status revealed a therapeutic index (TD50/ED50) of 6-9 following intraperitoneal and of 33 following oral administration. SKA-19 further reduced acute pain in the formalin pain model and raised allodynic threshold in a sciatic nerve ligation model. The anticonvulsant profile of SKA-19 is comparable to riluzole, which similarly affects Na(V) and KCa2 channels, except that SKA-19 has a ~4-fold greater duration of action owing to more prolonged brain levels. Based on these findings we propose that compounds combining KCa2 channel-activating and Na(v) channel-blocking activity exert broad-spectrum anticonvulsant and analgesic effects.

Kv1.3 channels regulate the activation/proliferation of effector memory T cells and thus play a critical role in the pathogenesis of autoimmune diseases. Using a combination of immunohistochemistry, confocal microscopy, flow cytometry and electrophysiology methods we observed a significant enrichment of activated Kv1.3(+) memory T cells in psoriasis plaques and synovial fluid from patients with psoriasis/psoriatic arthritis (PsA) compared to non-lesional psoriatic skin, normal skin or peripheral blood lympho-mononuclear cells. In in vitro studies performed with lesional mononuclear cells or T cells derived from skin and joints of psoriatic disease, the small molecule Kv1.3 blocker PAP-1 dose-dependently inhibited proliferation and suppressed IL-2 and IFN-γ production. To further substantiate the pathologic role of Kv1.3 high TEM cells in psoriatic disease we tested whether PAP-1 is able to improve psoriatic disease pathology in the SCID mouse-psoriasis skin xenograft model. Following four weeks of daily treatment with 2% PAP-1 ointment we noticed about 50% reduction in the epidermal thickness (rete peg length) and the number of CD3(+) lymphocytes/mm(2) of dermis decreased by 85%. Vehicle treated and untreated plaques in contrast remained unchanged and showed no reduction in epidermis thickness and infiltrating CD3(+) T cells and HLA-DR(+) T cells. Based on these results we propose the development of Kv1.3 targeted topical immunotherapy for psoriasis and possibly for other inflammatory skin conditions, where effector memory T cells are involved in the pathogenesis.

Cardiomyocytes contract against a mechanical load during each heartbeat, and excessive mechanical stress leads to heart diseases. Using a cell-in-gel system that imposes an afterload during cardiomyocyte contraction, we found that nitric oxide synthase (NOS) was involved in transducing mechanical load to alter Ca(2+) dynamics. In mouse ventricular myocytes, afterload increased the systolic Ca(2+) transient, which enhanced contractility to counter mechanical load but also caused spontaneous Ca(2+) sparks during diastole that could be arrhythmogenic. The increases in the Ca(2+) transient and sparks were attributable to increased ryanodine receptor (RyR) sensitivity because the amount of Ca2(+) in the sarcoplasmic reticulum load was unchanged. Either pharmacological inhibition or genetic deletion of nNOS (or NOS1), but not of eNOS (or NOS3), prevented afterload-induced Ca2(+) sparks. This differential effect may arise from localized NO signaling, arising from the proximity of nNOS to RyR, as determined by super-resolution imaging. Ca(2+)-calmodulin-dependent protein kinase II (CaMKII) and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) also contributed to afterload-induced Ca(2+) sparks. Cardiomyocytes from a mouse model of familial hypertrophic cardiomyopathy exhibited enhanced mechanotransduction and frequent arrhythmogenic Ca(2+) sparks. Inhibiting nNOS and CaMKII, but not NOX2, in cardiomyocytes from this model eliminated the Ca2(+) sparks, suggesting mechanotransduction activated nNOS and CaMKII independently from NOX2. Thus, our data identify nNOS, CaMKII, and NOX2 as key mediators in mechanochemotransduction during cardiac contraction, which provides new therapeutic targets for treating mechanical stress-induced Ca(2+) dysregulation, arrhythmias, and cardiomyopathy.

Senicapoc, a small molecule inhibitor of the calcium-activated potassium channel KCa3.1, was safe and well-tolerated in clinical trials for sickle cell anemia. We previously reported proof-of-concept data suggesting that both pharmacological inhibition and genetic deletion of KCa3.1 reduces infarction and improves neurologic recovery in rodents by attenuating neuroinflammation. Here we evaluated the potential of repurposing senicapoc for ischemic stroke. In cultured microglia, senicapoc inhibited KCa3.1 currents with an IC50 of 7 nM, reduced Ca2+ signaling induced by the purinergic agonist ATP, suppressed expression of pro-inflammatory cytokines and enzymes (iNOS and COX-2), and prevented induction of the inflammasome component NLRP3. When transient middle cerebral artery occlusion (tMCAO, 60 min) was induced in male C57BL/6 J mice, twice daily administration of senicapoc at 10 and 40 mg/kg starting 12 h after reperfusion dose-dependently reduced infarct area determined by T2-weighted magnetic resonance imaging (MRI) and improved neurological deficit on day 8. Ultra-high-performance liquid chromatography/mass spectrometry analysis of total and free brain concentrations demonstrated sufficient KCa3.1 target engagement. Senicapoc treatment significantly reduced microglia/macrophage and T cell infiltration and activation and attenuated neuronal death. A different treatment paradigm with senicapoc started at 3 h and MRI on day 3 and day 8 revealed that senicapoc reduces secondary infarct growth and suppresses expression of inflammation markers, including T cell cytokines in the brain. Lastly, we demonstrated that senicapoc does not impair the proteolytic activity of tissue plasminogen activator (tPA) in vitro. We suggest that senicapoc could be repurposed as an adjunctive immunocytoprotective agent for combination with reperfusion therapy for ischemic stroke. [ABSTRACT FROM AUTHOR]

BackgroundThe calcium-activated potassium channel KCa3.1 is critically involved in T-cell activation as well as in the proliferation of smooth muscle cells and fibroblasts. We sought to investigate whether KCa3.1 contributes to the pathogenesis of obliterative airway disease (OAD) and whether knockout or pharmacologic blockade would prevent the development of OAD.MethodsTracheas from CBA donors were heterotopically transplanted into the omentum of C57Bl/6J wild-type or KCa3.1 mice. C57Bl/6J recipients were either left untreated or received the KCa3.1 blocker TRAM-34 (120 mg/kg/day). Histopathology and immunologic assays were performed on postoperative day 5 or 28.ResultsSubepithelial T-cell and macrophage infiltration on postoperative day 5, as seen in untreated allografts, was significantly reduced in the KCa3.1 and TRAM-34 groups. Also, systemic Th1 activation was significantly and Th2 mildly reduced by KCa3.1 knockout or blockade. After 28 days, luminal obliteration of tracheal allografts was reduced from 89%±21% in untreated recipients to 53%±26% (P=0.010) and 59%±33% (P=0.032) in KCa3.1 and TRAM-34-treated animals, respectively. The airway epithelium was mostly preserved in syngeneic grafts, mostly destroyed in the KCa3.1 and TRAM-34 groups, and absent in untreated allografts. Allografts triggered an antibody response in untreated recipients, which was significantly reduced in KCa3.1 animals. KCa3.1 was detected in T cells, airway epithelial cells, and myofibroblasts. TRAM-34 dose-dependently suppressed proliferation of wild-type C57B/6J splenocytes but did not show any effect on KCa3.1 splenocytes.ConclusionsOur findings suggest that KCa3.1 channels are involved in the pathogenesis of OAD and that KCa3.1 blockade holds promise to reduce OAD development.