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Background: Co-curricular (CoC) activity tracking and assessment are required by the Accreditation Council for Pharmacy Education (ACPE) 2016 Standards. The purpose of this manuscript is to evaluate the student-perceived impact of the type of CoC activity chosen. Methods: First-year (P1) through third-year (P3) students in a 4-year Doctor of Pharmacy programme were required to complete two Professional Development Unit (PDUs) activities each semester and two Self-Selected Activities (SSAs) longitudinally through each academic year. PDUs include passive attendance at practitioner presentations, whereas SSAs require more active engagement, such as membership in professional organisations. Students completed surveys for each CoC activity. Survey responses for spring 2020, fall 2020, and spring 2021 were collected through ExamSoft and analysed with PowerBI (2021) and IntellectusStatisticsTM (2019). Results: Students reported positive perceptions for both PDUs and SSAs in the areas of being “different from prior experiences”, “complimenting the curriculum”, “increased my understanding of the profession”, and being of “high quality”. Professional organisation activities were the most commonly selected SSA. Those activities with lower perceived benefit were less clearly aligned with the professional role of a pharmacist, such as intramural sports. Conclusion: Overall, most CoC activities that closely aligned with the profession of pharmacy were perceived to complement the curriculum and enhance students’ understanding of the field. Students reported perceived value in both active and passive CoC activities.

The nicotinamide adenine dinucleotide (NAD(+)) is an essential redox cofactor, involved in various physiological and molecular processes, including energy metabolism, epigenetics, aging and metabolic diseases. NAD(+) repletion ameliorates muscular dystrophy and improves the mitochondrial and muscle stem cell function, and thereby increase lifespan in mice. Accordingly, NAD(+) is considered as an antioxidant and anti-aging molecule. NAD(+) plays a central role in energy metabolism and the energy produced is used for movements, thermoregulation, and defense against foreign bodies. The dietary precursors of NAD(+) synthesis is targeted to improve NAD(+) biosynthesis, however, studies have revealed conflicting results regarding skeletal muscle-specific effects. Recent advances in the activation of nicotinamide phosphoribosyltransferase (NAMPT) in the NAD(+) salvage pathway and supplementation of NAD(+) precursors have led to beneficial effects in skeletal muscle pathophysiology and function during aging and associated metabolic diseases. NAD(+) is also involved in the epigenetic regulation and post-translational modifications of proteins that are involved in various cellular processes to maintain tissue homeostasis. This review provides detailed insights into the roles of NAD(+) along with molecular mechanisms during aging and disease conditions such as the impacts of age-related NAD(+) deficiencies on NAD(+)-dependent enzymes, including poly (ADP-ribose) polymerase (PARPs), CD38, and sirtuins within skeletal muscle, and the most recent studies on the potential of nutritional supplementation and distinct modes of exercise to replenish the NAD(+) pool.

Cardiovascular diseases including cardiac arrhythmias lead to fatal events in patients with coronary artery disease; however, clinical associations from echocardiography, electrocardiography (ECG), and biomarkers remain unknown. We sought to identify the factors that may be related to elevated QRS intervals in patients with risk for coronary artery disease. In this study, we performed analysis of clinical data from 503 patients divided into two groups, i.e., patients with either 50% coronary artery stenosis. We further examined patients with elevated ECG parameters such as QRS > 100 ms and QTc > 440 ms. Patients with >50% coronary artery stenosis exhibited significant increases in age, triglycerides, and troponin levels. Further, ECG parameters demonstrated increased QRS and QTc durations, while echocardiographic parameters highlighted a decrease in ejection fraction (EF) and fractional shortening (FS). Patients with QTc > 440 ms exhibited increased brain natriuretic peptide and creatinine levels with a decrease in estimated glomerular filtration rate clearance rates. Patients with QRS > 100 ms had greater left ventricular (LV) mass and LV internal diameter in systole and diastole. Multimodal logistic regression showed significant relation between QTc, age, and creatinine. These findings suggest that patients with significant coronary stenosis may have lower EF and FS with prolonged QRS intervals, demonstrating greater risk for arrhythmic events.

Diabetes is associated with increased cardiac injury and sudden death. Nicotinamide phosphoribosyltransferase (Nampt) is an essential enzyme for the NAD

Voltage-gated potassium (Kv) channels control myocardial repolarization. Pore-forming Kvα proteins associate with intracellular Kvβ subunits, which bind pyridine nucleotides with high affinity and differentially regulate channel trafficking, plasmalemmal localization and gating properties. Nevertheless, it is unclear how Kvβ subunits regulate myocardial K(+) currents and repolarization. Here, we tested the hypothesis that Kvβ2 subunits regulate the expression of myocardial Kv channels and confer redox sensitivity to Kv current and cardiac repolarization. Co-immunoprecipitation and in situ proximity ligation showed that in cardiac myocytes, Kvβ2 interacts with Kv1.4, Kv1.5, Kv4.2, and Kv4.3. Cardiac myocytes from mice lacking Kcnab2 (Kvβ2(−/−)) had smaller cross sectional areas, reduced sarcolemmal abundance of Kvα binding partners, reduced I(to), I(K,slow1), and I(K,slow2) densities, and prolonged action potential duration compared with myocytes from wild type mice. These differences in Kvβ2(−/−) mice were associated with greater P wave duration and QT interval in electrocardiograms, and lower ejection fraction, fractional shortening, and left ventricular mass in echocardiographic and morphological assessments. Direct intracellular dialysis with a high NAD(P)H:NAD(P)(+) accelerated Kv inactivation in wild type, but not Kvβ2(−/−) myocytes. Furthermore, elevated extracellular levels of lactate increased [NADH](i) and prolonged action potential duration in wild type cardiac myocytes and perfused wild type, but not Kvβ2(−/−), hearts. Taken together, these results suggest that Kvβ2 regulates myocardial electrical activity by supporting the functional expression of proteins that generate I(to) and I(K,slow), and imparting redox and metabolic sensitivity to Kv channels, thereby coupling cardiac repolarization to myocyte metabolism.

Glucocorticoids, such as fluticasone propionate (FP), are used for the treatment of inflammation and alleviation of nasal symptoms and allergies, and as an antipruritic. However, both short- and long-term therapeutic use of glucocorticoids can lead to muscle weakness and atrophy. In the present study, we evaluated the feasibility of the nanodelivery of FP with poly(dl-lactide-co-glycolide) (PLGA) and tested in vitro function. FP-loaded PLGA nanoparticles were prepared via nanoprecipitation and morphological characteristics were studied via scanning electron microscopy. FP-loaded nanoparticles demonstrated an encapsulation efficiency of 68.6% ± 0.5% with a drug loading capacity of 4.6% ± 0.04%, were 128.8 ± 0.6 nm in diameter with a polydispersity index of 0.07 ± 0.008, and displayed a zeta potential of –19.4 ± 0.7. A sustained in vitro drug release pattern was observed for up to 7 days. The use of fluticasone nanoparticle decreased lipopolysaccharide (LPS)-induced lactate dehydrogenase release compared with LPS alone in C2C12 treated cells. FP also decreased expression of LPS-induced inflammatory genes in C2C12 treated cells as compared with LPS alone. Taken together, the present study demonstrates in vitro feasibility of PLGA-FP nanoparticle delivery to the skeletal muscle cells, which may be beneficial for treating inflammation.

Kvβ subunits belong to the aldo-keto reductase superfamily, which plays a significant role in ion channel regulation and modulates the physiological responses. However, the role of Kvβ2 in cardiac pathophysiology was not studied, and therefore, in the present study, we hypothesized that Kvβ2 plays a significant role in cardiovascular pathophysiology by modulating the cardiac excitability and gene responses. We utilized an isoproterenol-infused mouse model to investigate the role of Kvβ2 and the cardiac function, biochemical changes, and molecular responses. The deletion of Kvβ2 attenuated the QTc (corrected QT interval) prolongation at the electrocardiographic (ECG) level after a 14-day isoproterenol infusion, whereas the QTc was significantly prolonged in the littermate wildtype group. Monophasic action potentials verified the ECG changes, suggesting that cardiac changes and responses due to isoproterenol infusion are mediated similarly at both the in vivo and ex vivo levels. Moreover, the echocardiographic function showed no further decrease in the ejection fraction in the isoproterenol-stimulated Kvβ2 knockout (KO) group, whereas the wildtype mice showed significantly decreased function. These experiments revealed that Kvβ2 plays a significant role in cardiovascular pathophysiology. Furthermore, the present study revealed SLC41a3, a major solute carrier transporter affected with a significantly decreased expression in KO vs. wildtype hearts. The electrical function showed that the decreased expression of SLC41a3 in Kvβ2 KO hearts led to decreased Mg2+ responses, whereas, in the wildtype hearts, Mg2+ caused action potential duration (APD) shortening. Based on the in vivo, ex vivo, and molecular evaluations, we identified that the deletion of Kvβ2 altered the cardiac pathophysiology mediated by SLC41a3 and altered the NAD (nicotinamide adenine dinucleotide)-dependent gene responses.

Background Recent studies suggest that alterations in lung microbiome are associated with occurrence of chronic lung diseases and transplant rejection. To investigate the host-microbiome interactions, we characterized the airway microbiome and metabolome of the allograft (transplanted lung) and native lung of single lung transplant recipients. Methods BAL was collected from the allograft and native lungs of SLTs and healthy controls. 16S rRNA microbiome analysis was performed on BAL bacterial pellets and supernatant used for metabolome, cytokines and acetylated proline-glycine-proline (Ac-PGP) measurement by liquid chromatography-high-resolution mass spectrometry. Results In our cohort, the allograft airway microbiome was distinct with a significantly higher bacterial burden and relative abundance of genera Acinetobacter & Pseudomonas. Likewise, the expression of the pro-inflammatory cytokine VEGF and the neutrophil chemoattractant matrikine Ac-PGP in the allograft was significantly higher. Airway metabolome distinguished the native lung from the allografts and an increased concentration of sphingosine-like metabolites that negatively correlated with abundance of bacteria from phyla Proteobacteria. Conclusions Allograft lungs have a distinct microbiome signature, a higher bacterial biomass and an increased Ac-PGP compared to the native lungs in SLTs compared to the native lungs in SLTs. Airway metabolome distinguishes the allografts from native lungs and is associated with distinct microbial communities, suggesting a functional relationship between the local microbiome and metabolome.

Glucocorticoids are utilized for their anti-inflammatory properties in the skeletal muscle and arthritis. However, the major drawback of use of glucocorticoids is that it leads to senescence and toxicity. Therefore, based on the idea that decreasing particle size allows for increased surface area and bioavailability of the drug, in the present study, we hypothesized that nanodelivery of dexamethasone will offer increased efficacy and decreased toxicity. The dexamethasone-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared using nanoprecipitation method. The morphological characteristics of the nanoparticles were studied under scanning electron microscope. The particle size of nanoparticles was 217.5 ± 19.99 nm with polydispersity index of 0.14 ± 0.07. The nanoparticles encapsulation efficiency was 34.57% ± 1.99% with in vitro drug release profile exhibiting a sustained release pattern over 10 days. We identified improved skeletal muscle myoblast performance with improved closure of the wound along with increased cell viability at 10 nmol/L nano-dexamethasone-PLGA. However, dexamethasone solution (1 μmol/L) was injurious to cells because the migration efficiency was decreased. In addition, the use of dexamethasone nanoparticles decreased lipopolysaccharide-induced lactate dehydrogenase release compared with dexamethasone solution. Taken together, the present study clearly demonstrates that delivery of PLGA-dexamethasone nanoparticles to the skeletal muscle cells is beneficial for treating inflammation and skeletal muscle function.

Statins are widely prescribed, yet statin muscle pain limits their use, leading to increased cardiovascular risk. No validated therapy for statin muscle pain exists. The goal of the study was to assess whether metformin was associated with reduced muscle pain. A secondary analysis of data from the ACCORD trial was performed. An ACCORD sub-study assessed patients for muscle cramps and leg/calve pain while walking, typical non-severe statin muscle pain symptoms. We compared muscle pain between patients using a statin (n = 445) or both a statin and metformin (n = 869) at baseline. Overall patient characteristics were balanced between groups. Unadjusted analysis showed fewer reports of muscle cramps (35%) and leg/calve pain while walking (40%) with statins and metformin compared to statin only (muscle cramps, 42%; leg/calve pain while walking, 47%). Multivariable regression demonstrated a 22% odds reduction for muscle cramps (P = 0.049) and a 29% odds reduction for leg/calve pain while walking (P = 0.01). Metformin appears to reduce the risk of non-severe statin muscle pain and additional research is needed to confirm the findings and assess metformin's impact on statin adherence and related cardiovascular outcomes.

We previously demonstrated the role of Kvβ1.1 subunit of voltage-activated potassium channel in heart for its sensory roles in detecting changes in NADH/NAD and modulation of ion channel. However, the pharmacological role for the association of Kvβ1 via its binding to ligands such as cortisone and its analogs remains unknown. Therefore, we investigated the significance of Kvβ1.1 binding to cortisone analogs and AR inhibitor epalrestat. In addition, the aldose reductase (AR) inhibitor epalrestat was identified as a pharmacological target and modulator of cardiac activity via binding to the Kvβ1 subunit. Using a combination of ex vivo cardiac electrophysiology and in silico binding, we identified that Kvβ1 subunit binds and interacts with epalrestat. To identify the specificity of the action potential changes, we studied the sensitivity of the action potential prolongation by probing the electrical changes in the presence of 4-aminopyridine and evaluated the specificity of pharmacological effects in the hearts from Kvβ1.1 knock out mouse. Our results show that pharmacological modulation of cardiac electrical activity by cortisone analogs and epalrestat is mediated by Kvβ1.1.

NEW FINDINGS What is the central question of this study? The goal of this study was to evaluate sex differences and the role of the potassium channel β1 (Kvβ1) subunit in the heart. What is the main finding and its importance? Genetic ablation of Kvβ1.1 in females led to cardiac hypertrophy characterized by increased heart size, prolonged monophasic action potentials, elevated blood pressure and increased myosin heavy chain α (MHCα) expression. In contrast, male mice showed only electrical changes. Kvβ1.1 binds the MHCα isoform at the protein level, and small interfering RNA targeted knockdown of Kvβ1.1 upregulated MHCα. Cardiovascular disease is the leading cause of death and debility in women in the USA, and cardiac arrhythmias are a major concern. Voltage-gated potassium (Kv) channels along with the binding partners; Kvβ subunits are major regulators of the action potential (AP) shape and duration (APD). The regulation of Kv channels by the Kvβ1 subunit is unknown in female hearts. In the present study, we hypothesized that the Kvβ1 subunit is an important regulator of female cardiac physiology. To test this hypothesis, we ablated (knocked out; KO) the KCNAB1 isoform 1 (Kvβ1.1) subunit in mice and evaluated cardiac function and electrical activity by using ECG, monophasic action potential recordings and echocardiography. Our results showed that the female Kvβ1.1 KO mice developed cardiac hypertrophy, and the hearts were structurally different, with enlargement and increased area. The electrical derangements caused by Kvβ1.1 KO in female mice included long QTc and QRS intervals along with increased APD (APD20-90% repolarization). The male Kvβ1.1 KO mice did not develop cardiac hypertrophy, but they showed long QTc and prolonged APD. Molecular analysis showed that several genes that support cardiac hypertrophy were significantly altered in Kvβ1.1 KO female hearts. In particular, myosin heavy chain α expression was significantly elevated in Kvβ1.1 KO mouse heart. Using a small interfering RNA strategy, we identified that knockdown of Kvβ1 increases myosin heavy chain α expression in H9C2 cells. Collectively, changes in molecular and cell signalling pathways clearly point towards a distinct electrical and structural remodelling consistent with cardiac hypertrophy in the Kvβ1.1 KO female mice.

Background Necrotizing enterocolitis (NEC) is an inflammatory bowel necrosis of premature infants. Based on our recent findings of increased Smad7 expression in surgically-resected bowel affected by NEC, we hypothesized that NEC macrophages undergo inflammatory activation because increased Smad7 expression renders these cells resistant to normal, gut-specific, transforming growth factor (TGF)-β-mediated suppression of inflammatory pathways. Methods We used surgically-resected human NEC tissue, murine models of NEC-like injury, bone marrow-derived and intestinal macrophages, and RAW264.7 cells. Smad7 and IκB kinase-beta (IKK-β) were measured by quantitative polymerase chain reaction (qPCR), Western blots, and immunohistochemistry. Promoter activation was confirmed in luciferase reporter and chromatin immunoprecipitation assays. Results NEC macrophages showed increased Smad7 expression, particularly in areas with severe tissue damage and high bacterial load. LPS-induced Smad7 expression suppressed TGF-β signaling and augmented NF-κB activation and cytokine production in macrophages. Smad7-mediated NF-κB activated was likely mediated via increased expression of IKK-β, which, further increased Smad7 expression in a feed-forward loop. We show that Smad7 induced IKK-β expression through direct binding to the IKK-β promoter and its transcriptional activation. Conclusions Smad7 expression in NEC macrophages interrupts TGF-β signaling and promotes NF-κB-mediated inflammatory signaling in these cells through increased expression of IKK-β.

Hypotension, cardiac depression, and elevated microvascular permeability are known problems that complicate resuscitation of patients following traumatic injury, particularly those who are also intoxicated from alcohol consumption. A conscious rat model of combined alcohol intoxication and hemorrhagic shock has been used to study the hemodynamic mechanisms involved. Here, we describe using this model to study microvascular leakage and cardiac electrical activity.

Diabetic retinopathy is a complication of diabetes. Majority of diabetic patients with high blood glucose face the challenge of dealing with retinopathy and macular edema as the disease progresses. Although treatment choices and care are available to manage and symptomatically treat Diabetic retinopathy the current understanding is limited and lacks options for treatment and rescue strategies. Pharmacological options include anti-VEGF treatment strategies and surgical procedures. The present review provides insights in to type of diabetic retinopathy, along with different stages of the disease. In addition, the roles for health care providers, importance of pharmacists for treatment and management of diabetic retinopathy patient care are discussed.

Hyperoxia exposure in mice leads to cardiac hypertrophy and voltage-gated potassium (Kv) channel remodeling. Because redox balance of pyridine nucleotides affects Kv function and hyperoxia alters cellular redox potential, we hypothesized that hyperoxia exposure leads to cardiac ion channel disturbances and redox changes resulting in arrhythmias. In the present study, we investigated the electrical changes and redox abnormalities caused by 72h hyperoxia treatment in mice. Cardiac repolarization changes were assessed by acquiring electrocardiogram (ECG) and cardiac action potentials (AP). Biochemical assays were employed to identify the pyridine nucleotide changes, Kv1.5 expression and myocardial injury. Hyperoxia treatment caused marked bradycardia, arrhythmia and significantly prolonged (ms) the, RR (186.2 ± 10.7 vs. 146.4 ± 6.2), PR (46.8 ± 3.1 vs. 39.3 ± 1.6), QRS (10.8 ± 0.6 vs. 8.5 ± 0.2), QTc (57.1 ± 3.5 vs. 40 ± 1.4) and JT (13.4 ± 2.1 vs. 7.0 ± 0.5) intervals, when compared with normoxia group. Hyperoxia treatment also induced significant increase in cardiac action potential duration (APD) (ex-APD90; 73.8 ± 9.5 vs. 50.9 ± 3.1 ms) and elevated levels of serum markers of myocardial injury; cardiac troponin I (TnI) and lactate dehydrogenase (LDH). Hyperoxia exposure altered cardiac levels of mRNA/protein expression of; Kv1.5, Kvβ subunits and SiRT1, and increased ratios of reduced pyridine nucleotides (NADH/NAD & NADPH/NADP). Inhibition of SiRT1 in H9C2 cells using Splitomicin resulted in decreased SiRT1 and Kv1.5 expression, suggesting that SiRT1 may mediate Kv1.5 downregulation. In conclusion, the cardiotoxic effects of hyperoxia exposure involve ion channel disturbances and redox changes resulting in arrhythmias.

The 2013 American Heart Association Blood Cholesterol Guidelines increased the number of patients recommended for statin therapy in the United States to 56million. Two common statin side effects are muscle pain, referred to as "statin-associated muscle symptoms", and increased risk for new onset type-2-diabetes mellitus. Up to 25% of statin users report muscle symptoms resulting in many patients being switched to lower dose or lower potency statins, or refusing statins altogether. The most likely signaling mechanisms for statin-associated muscle symptoms overlaps with the proposed mechanism of statin-induced insulin resistance. Metformin has outstanding utility in reducing insulin resistance and preventing type-2-diabetes mellitus, but has not been studied for statin-associated muscle symptom rescue or prevention. The overlapping mechanisms of statin-associated muscle symptoms, statin-induced insulin resistance, and metformin intervention offers the potential to address two common and detrimental side effects of statins. As statins are the single best medication class for preventing cardiovascular events the potential for clinical benefit is large given metabolic syndrome's growing prevalence in the United States. Herein we hypothesize that metformin will rescue and prevent patients from statin-associated muscle symptoms. This hypothesis can benefit two patient groups: 1) patients at risk for diabetes who are taking a statin and experiencing muscle symptoms; and 2) patients with diabetes taking metformin who are to be started on a statin. Method to test Group 1) Symptom Rescue: randomized control trial of metformin versus placebo in patients with prediabetes who are already taking a statin, and are experiencing mild-to-moderate muscle symptoms. Method to test Group 2) Symptom Prevention: meta-analysis, of statin randomized control trials, with patient level data, comparing patients taking metformin at baseline to patients not taking metformin when a statin is started. An efficient method to simulate both symptom rescue and symptom prevention is a skeletal muscle cell culture model of statin-associated muscle symptom markers. These experiments would identify if metformin reverses (rescues) or prevents markers of statin-associated muscle symptoms. As metformin is recommended by the American Diabetes Association for type-2-diabetes mellitus prevention, yet not frequently used, validating this hypothesis will lead towards research and practice change including: a) decreases in the frequency of statin-associated muscle symptoms; leading to subsequent increases in statin therapy compliance; b) increases in metformin use in prediabetes with subsequent decrease in the incidence of type-2-diabetes mellitus; and c) decreases in complications of both cardiovascular disease and diabetes due to improved statin compliance and type-2-diabetes mellitus prevention.

Myocardial infarction (MI) is a major cardiovascular disease (CVD) and ranks among the leading causes of morbidity and mortality in humans, worldwide. Despite advances in disease prevention and treatment strategies, majority of the developed and developing world's suffer higher disease burden from MI, and incur billions of dollars in healthcare costs (Murray et al., 2015). Global estimates from 2013 show that MI is the major cardiovascular disease (CVD), and that deaths due to MI accounted for nearly half of the 17 million CVD mortalities (GBD, 2013; Mortality and Causes of Death Collaborators, 2015). Within the United States, MI top's the chart of both communicable and non-communicable diseases in terms of health loss that it is estimated to have inflicted in the population (Murray, et al., 2015). It has been estimated that every 2 minutes, three Americans suffer from myocardial infarction (MI), primary cause of MI being coronary blood flow obstruction and myocardial damage. The annual estimates of MI incidence in USA are approximately three quarter million a year while almost two-thirds of these cases represent new attacks (Mozaffarian, et al., 2015). Collectively, MI continues to lead the charts for CVD incidence rates, health loss, mortalities thereby putting enormous strain on healthcare system.

The present study investigates the physiological role of Kvβ1 subunit for sensing pyridine nucleotide (NADH/NAD+) changes in the heart. We used Kvβ1.1 knockout (KO) or wild-type (WT) mice and established that Kvβ1.1 preferentially binds with Kv4.2 and senses the pyridine nucleotide changes in the heart. The cellular action potential duration (APD) obtained from WT cardiomyocytes showed longer APDs with lactate perfusion, which increases intracellular NADH levels, while the APDs remained unaltered in the Kvβ1.1 KO. Ex vivo monophasic action potentials showed a similar response, in which the APDs were prolonged in WT mouse hearts with lactate perfusion; however, the Kvβ1.1 KO mouse hearts did not show APD changes upon lactate perfusion. COS-7 cells coexpressing Kv4.2 and Kvβ1.1 were used for whole cell patch-clamp recordings to evaluate changes caused by NADH (lactate). These data reveal that Kvβ1.1 is required in the mediated inactivation of Kv4.2 currents, when NADH (lactate) levels are increased. In vivo, isoproterenol infusion led to increased NADH in the heart along with QTc prolongation in wild-type mice; regardless of the approach, our data show that Kvβ1.1 recognizes NADH changes and modulates Kv4.2 currents affecting AP and QTc durations. Overall, this study uses multiple levels of investigation, including the heterologous overexpression system, cardiomyocyte, ex vivo, and ECG, and clearly depicts that Kvβ1.1 is an obligatory sensor of NADH/NAD changes in vivo, with a physiological role in the heart. NEW & NOTEWORTHY Cardiac electrical activity is mediated by ion channels, and Kv4.2 plays a significant role, along with its binding partner, the Kvβ1.1 subunit. In the present study, we identify Kvβ1.1 as a sensor of pyridine nucleotide changes and as a modulator of Kv4.2 gating, action potential duration, and ECG in the mouse heart.

Ventricular arrhythmias account for high mortality in cardiopulmonary patients in intensive care units. Cardiovascular alterations and molecular-level changes in response to the commonly used oxygen treatment remains unknown. In the present study we investigated cardiac hypertrophy and cardiac complications in mice subjected to hyperoxia. Results demonstrate that there is a significant increase in average heart weight to tibia length (22%) in mice subjected to hyperoxia treatment vs. normoxia. Functional assessment was performed in mice subjected to hyperoxic treatment, and results demonstrate impaired cardiac function with decreased cardiac output and heart rate. Staining of transverse cardiac sections clearly demonstrates an increase in the cross-sectional area from hyperoxic hearts compared with control hearts. Quantitative real-time RT-PCR and Western blot analysis indicated differential mRNA and protein expression levels between hyperoxia-treated and control left ventricles for ion channels including Kv4.2 (−2 ± 0.08), Kv2.1 (2.54 ± 0.48), and Scn5a (1.4 ± 0.07); chaperone KChIP2 (−1.7 ± 0.06); transcriptional factors such as GATA4 (−1.5 ± 0.05), Irx5 (5.6 ± 1.74), NFκB1 (4.17 ± 0.43); hypertrophy markers including MHC-6 (2.17 ± 0.36) and MHC-7 (4.62 ± 0.76); gap junction protein Gja1 (4.4 ± 0.8); and microRNA processing enzyme Drosha (4.6 ± 0.58). Taken together, the data presented here clearly indicate that hyperoxia induces left ventricular remodeling and hypertrophy and alters the expression of Kv4.2 and MHC6/7 in the heart.

Recent research suggests that in addition to their role as soluble electron carriers, pyridine nucleotides [NAD(P)(H)] also regulate ion transport mechanisms. This mode of regulation seems to have been conserved through evolution. Several bacterial ion–transporting proteins or their auxiliary subunits possess nucleotide-binding domains. In eukaryotes, the Kv1 and Kv4 channels interact with pyridine nucleotide–binding β-subunits that belong to the aldo-keto reductase superfamily. Binding of NADP + to Kvβ removes N-type inactivation of Kv currents, whereas NADPH stabilizes channel inactivation. Pyridine nucleotides also regulate Slo channels by interacting with their cytosolic regulator of potassium conductance domains that show high sequence homology to the bacterial TrkA family of K + transporters. These nucleotides also have been shown to modify the activity of the plasma membrane K ATP channels, the cystic fibrosis transmembrane conductance regulator, the transient receptor potential M2 channel, and the intracellular ryanodine receptor calcium release channels. In addition, pyridine nucleotides also modulate the voltage-gated sodium channel by supporting the activity of its ancillary subunit—the glycerol-3-phosphate dehydrogenase-like protein. Moreover, the NADP + metabolite, NAADP + , regulates intracellular calcium homeostasis via the 2-pore channel, ryanodine receptor, or transient receptor potential M2 channels. Regulation of ion channels by pyridine nucleotides may be required for integrating cell ion transport to energetics and for sensing oxygen levels or metabolite availability. This mechanism also may be an important component of hypoxic pulmonary vasoconstriction, memory, and circadian rhythms, and disruption of this regulatory axis may be linked to dysregulation of calcium homeostasis and cardiac arrhythmias.

Drug transportation is a vital part of the equation when it comes to aiding in patient therapy. The human anatomy is a collection of many environments that contain complex obstacles, boundaries referred to as “compartments,” which complicate or prevent the transportation of drugs throughout the body. Understanding how a drug maneuvers its way through a particular boundary could unlock the door to possible chemical or formulation development processes that could serve as optimization strategies and could be developed to aid in the drug's diffusion to its intended target. This may seem like a straight-forward concept; however, the time and effort needed to test and analyze multiple scenarios that contain multiple variables weighs heavily in the pharmaceutical industry. To curtail the number of test scenarios needed to be conducted to properly model or simulate the mechanism at which the drug passes through the boundary, the use of artificial neural networks (ANNs) can significantly reduce the number of physically conducted tests. By implementing the ANN modeling approaches, and by reducing the population of test scenarios, error is expected. The use of ANNs can also reduce the amount of error that normally accompanies the results determined via other present day computer-aided modeling approaches. ANNs can more closely predict the outcomes of all other scenarios in the population that were previously not considered in the modeling and design process. Together, the field of ANNs is enhancing and aiding the process of drug transport modeling and simulation, ultimately benefiting the goal of molecule to market.

An ANN is a computational model inspired by networks of biological neurons, wherein the neurons compute output values from inputs. It learns from its past experience and errors in a non-linear parallel processing manner. The neuron is the basic calculating entities which computes from a number of inputs and deliver one output comparing with threshold value and turned on (fired). The computational processing is done by internal structural arrangement consists of hidden layers which utilizes the back propagation and feed forward mechanism to deliver output close to accuracy. The learning is based on reinforcement (supervised) and unsupervised (no target) type. The unsupervised mimics the biological neuron pattern of learning. ANN is widely used in medicine as classifier in cancer applications, drug delivery, pharmaceutical research and Blood-Brain Barrier (BBB) permeability.

Background: Binge drinking combined with trauma is a major cause of mortality in US. It is known that acute alcohol intoxication (AAI) causes deleterious electrical changes in the heart, including involving QTc dispersion. We also recently demonstrated that acute alcohol intoxication (AAI) increases microvascular permeability. In the current study we investigated the consequences of combined hemorrhagic shock and resuscitation (HSR) and AAI on both microvascular permeability and cardiac electrical activity. Hypothesis and Methods: We hypothesized that AAI combined with HSR causes microvascular leakage and electrical dysfunction in the heart. We used an established rat model of AAI/HSR to test the microvascular and cardiac effects. Results: We identified that combined AAI/HSR significantly (p

Background: Voltage-gated potassium channel (Kv) β2 subunits (Kvβ2) belong to the aldoketo-reductase superfamily and associate with the Kv channel modulating its function. Also, Kvβ2 binds pyridine nucleotides (NAD[P] + /NAD[P]H) with high affinity altering Kv channel gating. However, the physiological relevance of Kvβ2 in cardiac health and disease is unknown. Hypothesis: We tested the hypothesis that Kvβ2 is essential to both basal and pyridine nucleotide mediated regulation of cardiac repolarization. Methods and Results: Kvβ2 knockout (KO) and wild type (Wt) mice were utilized for this study. ECG analysis showed prolonged QTc interval (74.7± 3.7 vs. 65.6 ± 3, n=3 , p ) in KO mice when compared to Wt. Monophasic action potential (MAP) data also showed prolonged duration (ms) at 50% repolarization level in KO mouse hearts vs. Wt (MAPD50; 13.2 ± 0.3 vs. 9.7 ± 0.6, n=5-6 , p ). To test the obligatory role of Kvβ2 in pyridine nucleotide modulation of cardiac repolarization, we exposed ex vivo perfused hearts or isolated cardiomyocytes to higher lactate levels. These data revealed that acute lactate exposure leads to significant but similar raise in NADH concentration [NADH] i in both KO and Wt heart tissue. However, marked repolarization deficits appeared only in Wt group when tested utilizing ex vivo perfused hearts (MAPD50 - Wt:11.5 ± 0.7 vs. 8.3 ± 0.5, p ; KO:12.6 ± 0.7 vs. 12± 0.5, p=0.5 , ms) or isolated myocytes (ΔAPD50 – Wt: 4.7±1.6 ms, p ; KO:1.5 ± 0.7 ms, p=0.1 ). Perfusion with high pyruvate levels returned both [NADH] i and MAPD50 to basal levels in Wt mouse hearts. Similarly, we observed that a 14 day isoproterenol treatment also leads to significant increase in cardiac [NADH] i , but only the Wt mouse hearts showed significant repolarization deficits; ex-MAPD50 (5.5 ± 1.6, p< 0.01 ), when compared to no changes in KO (1.9 ± 0.9, p< 0.1) . Conclusions: Our results suggest that Kvβ2 regulates cardiac repolarization. Further, the differential repolarization changes despite similar increases in cardiac [NADH] i indicates an obligatory role of Kvβ2 in the pyridine nucleotide modulation of cardiac repolarization.

AIM: Potassium channel accessory subunits (Kvβ) play a key role in cardiac electrical activity through ion channel modulation. In the present study we hypothesize that Kvβ2 regulates skeletal muscle growth and fiber phenotype via protein-protein interactions. METHODS: Kvβ2 knockout mouse model was used for morphometric, immunohistochemical and biochemical analysis to evaluate the role of Kvβ2 in skeletal muscle physiology. RESULTS: Deletion of Kvβ2 gene in mice (Kvβ2 knockout, KO) leads to significant decrease in body weight along with skeletal muscle size. Key hind limb muscles such as biceps, soleus, and gastrocnemius were significantly smaller in size in KO mice compared to that of wild type. Morphometric measurements and histological analysis clearly point that the fiber size is decreased in each of the muscle type in KO compared with wild type mice. In addition, Kvβ2 deletion contributes to fiber type switching from fast to slow fiber as indicated by more abundant MHCI expressing fibers in gastrocnemius and soleus muscles, which may underscore the smaller muscle size alongside increase in U3 ubiquitin ligase;NEDD4 expression. Using targeted siRNA knockdown approach, we identified that Kvβ2 knockdown do not affect the myoblasts proliferation. However, Pax7 expression was significantly decreased in 4 week old gastrocnemius muscle suggesting that cellular reserve for growth may be deficient in KO mice. This is further supported by decreased migratory capacity of C2C12 cells upon siRNA targeted Kvβ2 knockdown. CONCLUSION: Overall, this is the first report identifying that genetic deletion of Kvβ2 leads to decreased skeletal muscle size along with isotype switching.

Objective. To determine professionalism factors of interest to the University of South Florida (USF) College of Pharmacy (COP) and develop a programmatic plan to monitor the professionalization of students, faculty, preceptors and staff. Methods. In 2013, the USF COP began investigating how best to incorporate processes to capture professionalism across all aspects of the program. A Professionalism Task Force was convened to identify key professionalism factors valued by faculty and preceptors to be incorporated in pharmacy practice experiences, didactic courses, faculty, and preceptor performance evaluations. A factor analysis was performed to identify items for inclusion in professional practice experience evaluations, course syllabi, faculty, preceptor and staff evaluations. Results. The analysis identified 11 factors for practice experiences, three for syllabi, and five for performance evaluations. The information from the factor analysis spurred additional discussion that led to the development of a programmatic plan for professionalization. Conclusion. A brief description of the programmatic professionalization plan is provided, including possible assessment processes. The goal of this endeavor was to ensure appropriate support and development of professionalization across the COP community.

The beta-subunits of the voltage-gated potassium (Kv) channels modulate the kinetics and the gating of Kv channels and assists in channel trafficking and membrane localization. These proteins are members of the AKR6 family. They share a common (alpha/beta)(8) barrel structural fold and avidly bind pyridine nucleotides. Low catalytic activity has been reported for these proteins. Kinetic studies with rat Kvbeta2 revealed that the chemical step is largely responsible for the rate-limitation but nucleotide exchange could also contribute to the overall rate. Herein we report our investigations on the kinetics of cofactor exchange using nucleotide-free preparations of Kvbeta2. Kinetic traces measuring quenching of Kvbeta2 fluorescence by NADP(+) were consistent with a two-step binding mechanism which includes rapid formation of a loose enzyme:cofactor complex followed by a slow conformational rearrangement to form a tight final complex. Closing of the nucleotide enfolding loop, which in the crystal structure folds over the bound cofactor, provides the structural basis for this rearrangement. The rate of the loop opening required to release the cofactor is similar for NADPH and NADP(+) (0.9 min(-1)) and is of the same order of magnitude as the rate of the chemical step estimated previously from kinetic studies with 4-nitrobenzaldehyde (0.3-0.8 min(-1), [S.M. Tipparaju, O.A. Barski, S. Srivastava, A. Bhatnagar, Catalytic mechanism and substrate specificity of the beta-subunit of the voltage-gated potassium channel, Biochemistry 47 (2008) 8840-8854]). Binding of NADPH is accompanied by a second conformational change that might be responsible for a 4-fold higher affinity observed with the reduced cofactor and the resulting difficulty in removing bound NADPH from the protein. These data provide evidence that nucleotide exchange occurs on a seconds-to-minutes time scale and set the upper limit for the maximal possible rate of catalysis by Kvbeta2. Slow cofactor exchange is consistent with the role of the beta-subunit as a metabolic sensor implicated in tonic regulation of potassium currents.

The beta-subunits of voltage-gated potassium (Kv) channels are members of the aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvbeta proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvbeta2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06-0.2 min(-1)) than those of most other AKRs but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvbeta2. The protein catalyzed the reduction of a broad range of carbonyls, including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipids, and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0-3.1) were observed under single- and multiple-turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvbeta proteins are catalytically active AKRs that impart redox sensitivity to Kv channels.

Isolated infant human atrial cells have a slower early repolarization than adult human atrial cells. In addition, from room temperature voltage-clamp studies, infant cells have lower basal L-type calcium currents than adult cells. We hypothesized that the slower repolarization increases the calcium transient of infant human atrial cells. Atrial myocytes were enzymatically dissociated from biopsies of human right atrial appendages of infant (3-8 mo) patients who were undergoing open-heart surgery. Intracellular calcium transients were measured with fluorescence microscopy with application of either square waves or action potential waveforms at physiologic temperature. After repetitive application (1 Hz) of 100-ms duration conditioning depolarizations to 10 mV (from -80 mV), a test pulse of varying duration (DeltaT; 2-100 ms) produced smaller transients (expressed as percentage of the last conditioning pulse) at shorter durations (33 +/- 7% for DeltaT = 2 ms, 80 +/- 4% for DeltaT = 25 ms). With repetitive application of either adult or infant prerecorded action potentials to infant cells, the cells had a decreased calcium transient with the adult action potential (F/F(0) 2.2 +/- 0.4 for infant action potential versus 1.6 +/- 0.2 for adult action potential; n = 7; p < 0.05). The delayed early repolarization of infant cells alters the Ca(2+) transient, which may compensate for the lower availability of basal calcium current in infant cells. The steep relationship that we have demonstrated between test-pulse duration and the calcium transient suggests that modulation of the early repolarization phase of the action potential may be of great significance in modulating excitation-contraction coupling.

We investigated differences in L-type Ca2+ current ( ICa) between infant (INF, 1–12 mo old), young adult (YAD, 14–18 yr old), and older adult (AD) myocytes from biopsies of right atrial appendages. Basal ICa was smaller in INF myocytes (1.2 ± 0.1 pA/pF, n = 29, 6 ± 1 mo old, 11 patients) than in YAD (2.5 ± 0.2 pA/pF, n = 20, 16 ± 1 yr old, 5 patients) or AD (2.6 ± 0.3 pA/pF, n = 19, 66 ± 3 yr old, 9 patients) myocytes ( P < 0.05). Maximal ICa produced by isoproterenol (Iso) was similar in INF, YAD, and AD cells: 8.4 ± 1.1, 9.6 ± 1.0, and 9.2 ± 1.3 pA/pF, respectively. Efficacy (Emax) was larger in INF (607 ± 50%) than for YAD (371 ± 29%) or AD (455 ± 12%) myocytes. Potency (EC50) was 8- to 10-fold higher in AD (0.82 ± 0.09 nM) or YAD (0.41 ± 0.14 nM) than in INF (7.6 ± 3.5 nM) myocytes. Protein levels were similar for Giα2 but much greater for Giα3 in INF than in AD or YAD atrial tissue. When Giα3 activity was inhibited by inclusion of a Giα3 COOH-terminal decapeptide in the pipette, basal ICa and the response to 10 nM Iso were increased in INF, but not in YAD, cells. We propose that basal ICa and the response to low-dose β-adrenergic stimulation are inhibited in INF (but not YAD or AD) cells as a result of constitutive inhibitory effects of Giα3.

Knowledge of postnatal modulation of I to in human atrial myocytes is quite limited. The present study investigated the differences in I to properties between neonatal and adult human atrial myocytes. Methods. – Atrial myocytes were dissociated enzymatically from biopsies of human right atrial appendage. I to and action potentials were recorded by whole-cell patch-clamp technique. The expressed protein levels of Kv4.3 and KChIP2 in atrial tissue were detected by western blot technique. Results. – I to was present in all atrial cells ( n = 37) from 10 neonatal patients (2.5–7 months). The mean value of I to density in neonatal atrial cells was significantly larger than in adult atrial cells. The time constants for I to current decay were significantly faster for neonatal cells, compared to adult cells. I to recovery from inactivation at holding potential of – 80 mV was significantly slower for neonatal atrial cells than for adult atrial cells. There was no difference in the voltage dependence of I to activation between neonatal and adult cells. The voltage-dependent inactivation slope factor was smaller for neonatal compared to adult atrial cells. A more significant frequency-dependent suppression of I to peak current and a more significant lengthening of APD 30 were observed in neonatal atrial cells compared to adult atrial cells. Western blots showed both Kv4.3 and KChIP2 are expressed in neonatal atria, but with significantly higher level of Kv4.3 and lower level of KChIP2 protein compared to adult. Conclusion. – There are significant differences in the properties of I to between neonatal and adult human atrial cells, including a larger current density, faster inactivation and slower recovery from inactivation in the neonatal atrial cells. The physiological differences of I to are consistent with the different expression protein levels of Kv4.3 and KChIP2.

Cardiac Kvβ2 subunit alters gating and expression of potassium channels. However, the physiological significance remains unknown. Therefore, we evaluated the role of Kvβ2 in cardiac injury. We used 14 week male wild type (Wt) or mutant Kvβ2:PM (active site mutant; Y90F) mice, infused with saline or isoproterenol (Iso, 30mg/kg; bw/day) for 14 days. Iso treatment resulted in cardiac hypertrophy in both mice as noted by histomorphological analysis. Echocardiographic assessment revealed significant decrease (p