Considerations for Optimizing Warfighter Psychological Health with a Research-Based Flavonoid Approach: A Review.

Optimal nutrition is imperative for psychological health. Oxidative stress and inflammation are underlying etiologies for alterations in psychological health. Warfighters are at risk of health concerns such as depression due to increased stress in austere environments and family separation while deployed. Over the last decade, research has demonstrated the health benefits of flavonoids found in fruits and berries. Berry flavonoids have potent antioxidant and anti-inflammatory properties by inhibiting oxidative stress and inflammation. In this review, the promising effects of various berries rich in bioactive flavonoids are examined. By inhibiting oxidative stress, berry flavonoids have the potential to modulate brain, cardiovascular, and intestinal health. There is a critical need for targeted interventions to address psychological health concerns within the warfighter population, and a berry flavonoid-rich diet and/or berry flavonoid dietary supplement intervention may prove beneficial as an adjunctive therapy. Structured searches of the literature were performed in the PubMed, CINAHL, and EMBASE databases using predetermined keywords. This review focuses on berry flavonoids' critical and fundamental bioactive properties and their potential effects on psychological health in investigations utilizing cell, animal, and human model systems.

Acss2 Deletion Reveals Functional Versatility via Tissue-Specific Roles in Transcriptional Regulation.

The coordination of cellular biological processes is regulated in part via metabolic enzymes acting to match cellular metabolism to current conditions. The acetate activating enzyme, acyl-coenzyme A synthetase short-chain family member 2 (Acss2), has long been considered to have a predominantly lipogenic function. More recent evidence suggests that this enzyme has regulatory functions in addition to its role in providing acetyl-CoA for lipid synthesis. We used Acss2 knockout mice (Acss2-/-) to further investigate the roles this enzyme plays in three physiologically distinct organ systems that make extensive use of lipid synthesis and storage, including the liver, brain, and adipose tissue. We examined the resulting transcriptomic changes resulting from Acss2 deletion and assessed these changes in relation to fatty acid constitution. We find that loss of Acss2 leads to dysregulation of numerous canonical signaling pathways, upstream transcriptional regulatory molecules, cellular processes, and biological functions, which were distinct in the liver, brain, and mesenteric adipose tissues. The detected organ-specific transcriptional regulatory patterns reflect the complementary functional roles of these organ systems within the context of systemic physiology. While alterations in transcriptional states were evident, the loss of Acss2 resulted in few changes in fatty acid constitution in all three organ systems. Overall, we demonstrate that Acss2 loss institutes organ-specific transcriptional regulatory patterns reflecting the complementary functional roles of these organ systems. Collectively, these findings provide further confirmation that Acss2 regulates key transcription factors and pathways under well-fed, non-stressed conditions and acts as a transcriptional regulatory enzyme.

Methadone Blockade of Cardiac Inward Rectifier K+ Current Augments Membrane Instability and Amplifies U Waves on Surface ECGs: A Translational Study.

Background Methadone is associated with a disproportionate risk of sudden death and ventricular tachyarrhythmia despite only modest inhibition of delayed rectifier K+ current (IKr), the principal mechanism of drug-associated arrhythmia. Congenital defects of inward rectifier K+ current (IK1) have been linked to increased U-wave amplitude on ECG and fatal arrhythmia. We hypothesized that methadone may also be a potent inhibitor of IK1, contributing to delayed repolarization and manifesting on surface ECGs as augmented U-wave integrals. Methods and Results Using a whole-cell voltage clamp, methadone inhibited both recombinant and native IK1 with a half-maximal inhibitory concentration IC50) of 1.5 µmol/L, similar to that observed for IKr block (half-maximal inhibitory concentration of 2.9 µmol/L). Methadone modestly increased the action potential duration at 90% repolarization and slowed terminal repolarization at low concentrations. At higher concentrations, action potential duration at 90% repolarization lengthening was abolished, but its effect on terminal repolarization rose steadily and correlated with increased fluctuations of diastolic membrane potential. In parallel, patient ECGs were analyzed before and after methadone initiation, with 68% of patients having a markedly increased U-wave integral compared with premethadone (lead V3; mean +38%±15%, P=0.016), along with increased QT and TPeak to TEnd intervals, likely reflective of IKr block. Conclusions Methadone is a potent IK1 inhibitor that causes augmentation of U waves on surface ECG. We propose that increased membrane instability resulting from IK1 block may better explain methadone's arrhythmia risk beyond IKr inhibition alone. Drug-induced augmentation of U waves may represent evidence of blockade of multiple repolarizing ion channels, and evaluation of the effect of that agent on IK1 may be warranted.

The Effects of Berry Extracts on Oxidative Stress in Cultured Cardiomyocytes and Microglial Cells: A Potential Cardioprotective and Neuroprotective Mechanism.

Oxidative stress is a key underlying factor in cognitive decline and atherosclerosis. Oxidative stress occurs at the cellular level with an imbalance between reactive oxygen species and reactive nitrogen species and a deficiency in antioxidants. Mounting evidence suggests that berry flavonoids may promote cellular health by exerting antioxidant properties. Black currant and various berry extracts were tested in microglia (BV-2) and cardiomyocyte (HL-1) cell lines to study their biological effects. The principal ingredients in black currant and cranberry extract-delphinidin 3-rutinoside (D3R) and cyanidin 3-glucoside (C3G), were also assessed. A menadione-induced oxidative stressor was used, and its output was quantified to detect oxidative stress (CellROXTM). Black currant extract had similar antioxidant effects as N-acetylcysteine (NAC) in HL-1 cells with regard to cellular protection, whereas cranberry extract was ineffective. In contrast, cranberry extract was comparable in effectiveness to black currant extract in BV-2 cells. D3R and C3G also reduced oxidative stress similarly to whole berry extracts, which indicates that these ingredients may confer the antioxidant effects of berries. Black currant and cranberry extracts inhibit oxidative stress in microglial and cardiomyocyte cell lines. Black currant extract was more effective in reducing oxidative stress in the HL-1 cells, whereas cranberry extract was comparable in reducing oxidative stress in the BV-2 cells. The results suggest that berry flavonoids exert neuro- and cardioprotective effects.

Novel cholesterol-dependent regulation of cardiac KATP subunit expression revealed using histone deacetylase inhibitors.

We recently discovered that the histone deacetylase inhibitor, trichostatin A (TSA), increases expression of the sulfonylurea receptor 2 (SUR2; Abcc9) subunit of the ATP-sensitive K+ (KATP ) channel in HL-1 cardiomyocytes. Interestingly, the increase in SUR2 was abolished with exogenous cholesterol, suggesting that cholesterol may regulate channel expression. In the present study, we tested the hypothesis that TSA increases SUR2 by depleting cholesterol and activating the sterol response element binding protein (SREBP) family of transcription factors. Treatment of HL-1 cardiomyocytes with TSA (30 ng/ml) caused a time-dependent increase in SUR2 mRNA expression that correlates with the time course of cholesterol depletion assessed by filipin staining. Consistent with the cholesterol-dependent regulation of SREBP increasing SUR2 mRNA expression, we observe a significant increase in SREBP cleavage and translocation to the nucleus following TSA treatment that is inhibited by exogenous cholesterol. Further supporting the role of SREBP in mediating the effect of TSA on KATP subunit expression, SREBP1 significantly increased luciferase reporter gene expression driven by the upstream SUR2 promoter. Lastly, HL-1 cardiomyocytes treated with the SREBP inhibitor PF429242 significantly suppresses the effect of TSA on SUR2 gene expression. These results demonstrate that SREBP is an important regulator of KATP channel expression and suggest a novel method by which hypercholesterolemia may exert negative effects on the cardiovascular system, namely, by suppressing expression of the KATP channel.

SMN-deficiency disrupts SERCA2 expression and intracellular Ca2+ signaling in cardiomyocytes from SMA mice and patient-derived iPSCs.

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by loss of alpha motor neurons and skeletal muscle atrophy. The disease is caused by mutations of the SMN1 gene that result in reduced functional expression of survival motor neuron (SMN) protein. SMN is ubiquitously expressed, and there have been reports of cardiovascular dysfunction in the most severe SMA patients and animal models of the disease. In this study, we directly assessed the function of cardiomyocytes isolated from a severe SMA model mouse and cardiomyocytes generated from patient-derived IPSCs. Consistent with impaired cardiovascular function at the very early disease stages in mice, heart failure markers such as brain natriuretic peptide were significantly elevated. Functionally, cardiomyocyte relaxation kinetics were markedly slowed and the T50 for Ca2+ sequestration increased to 146 ± 4 ms in SMN-deficient cardiomyocytes from 126 ± 4 ms in wild type cells. Reducing SMN levels in cardiomyocytes from control patient IPSCs slowed calcium reuptake similar to SMA patent-derived cardiac cells. Importantly, restoring SMN increased calcium reuptake rate. Taken together, these results indicate that SMN deficiency impairs cardiomyocyte function at least partially through intracellular Ca2+ cycling dysregulation.

Hemodynamic Effects of Late Sodium Current Inhibitors in a Swine Model of Heart Failure.

OBJECTIVES: To evaluate possible treatment-related hemodynamic changes, we administered ranolazine or mexiletine to swine with heart failure (HF) and to controls. BACKGROUND: Ranolazine and mexiletine potently inhibit depolarizing late Na+ current (INa,late) and Na+ entry into cardiomyocytes. Blocking Na+ entry may increase forward-mode Na/Ca exchange and reduce cellular Ca+2 load, further compromising systolic contraction during HF. METHODS AND RESULTS: Anesthetized tachypaced HF swine received ranolazine (n = 9) or mexiletine (n = 7) as boluses, then as infusions; the same experiments were performed in 10 nonpaced controls. The swine with HF had characteristic elevated left ventricular end-diastolic pressure (LVEDP) and reduced maximal left ventricular pressure rise (+dP/dtmax) and left ventricular peak systolic pressure (LVSP). No significant change occurred after ranolazine dosing for any parameter: LVEDP, +dP/dtmax, LVSP, heart rate, maximal LV pressure fall rate (-dP/dtmax), or time constant for isovolumic relaxation. Similar results seen in additional swine with HF: 7 were given mexiletine, and 7 others were given ranolazine after a 27% rate decrement to maximize INa,late. Patch-clamped HF cardiomyocytes confirmed drug-induced INa,late blockade. CONCLUSIONS: Ranolazine or mexiletine blocking INa,late neither worsened nor improved hemodynamics during advanced HF. Although results must be clinically confirmed, they suggest inhibition of INa,late by ranolazine or mexiletine may not exacerbate HF in patients.

Role of suppression of the inward rectifier current in terminal action potential repolarization in the failing heart.

BACKGROUND: The failing heart exhibits an increased arrhythmia susceptibility that is often attributed to action potential (AP) prolongation due to significant ion channel remodeling. The inwardly rectifying K+ current (IK1) has been reported to be reduced, but its contribution to shaping the AP waveform and cell excitability in the failing heart remains unclear. OBJECTIVE: The purpose of this study was to define the effect of IK1 suppression on the cardiac AP and excitability in the normal and failing hearts. METHODS: We used electrophysiological and pharmacological approaches to investigate IK1 function in a swine tachy-pacing model of heart failure (HF). RESULTS: Terminal repolarization of the AP (TRAP; the time constant of the exponential fit to terminal repolarization) was markedly prolonged in both myocytes and arterially perfused wedges from animals with HF. TRAP was increased by 54.1% in HF myocytes (P < .001) and 26.2% in HF wedges (P = .014). The increase in TRAP was recapitulated by the potent and specific IK1 inhibitor, PA-6 (pentamidine analog 6), indicating that IK1 is the primary determinant of the final phase of repolarization. Moreover, we find that IK1 suppression reduced the ratio of effective refractory period to AP duration at 90% of repolarization, permitting re-excitation before full repolarization, reduction of AP upstroke velocity, and likely promotion of slow conduction. CONCLUSION: Using an objective measure of terminal repolarization, we conclude that IK1 is the major determinant of the terminal repolarization time course. Moreover, suppression of IK1 prolongs repolarization and reduces postrepolarization refractoriness without marked effects on the overall AP duration. Collectively, these findings demonstrate how IK1 suppression may contribute to arrhythmogenesis in the failing heart.

Potent Inhibition of hERG Channels by the Over-the-Counter Antidiarrheal Agent Loperamide.

OBJECTIVES: The aim of this study was to determine the in vitro electrophysiological properties of loperamide. The authors' hypothesis was that loperamide is a potent blocker of the current carried by the human ether-à-go-go-related gene (hERG) potassium channel. BACKGROUND: Loperamide is a peripherally-acting µ-opioid agonist available worldwide as an over-the-counter treatment for diarrhea. Like most opioids, it is not currently known to be proarrhythmic. Recent cases of torsade de pointes in association with high-dose loperamide raise concern given its structural similarity to methadone, another synthetic opioid with an established arrhythmia risk. METHODS: Effects of loperamide on blockade of the hERG potassium channel ion current were assessed in Chinese Hamster Ovary (CHO) cells stably expressing hERG to elucidate current amplitude and kinetics. The concentration required to produce 50% inhibition of hERG current was assessed from the amplitude of tail currents and the impact on action potential duration was assessed in isolated swine ventricular cardiomyocytes. RESULTS: The 50% inhibitory concentration for loperamide inhibition of hERG ionic tail currents was approximately 40 nmol/l. In current-voltage measurements, loperamide reduced steady and tail currents and shifted the current activation to more negative potentials. Loperamide (10 nmol/l) also increased the action potential duration, assessed at 90% of repolarization, in ventricular myocytes by 16.4 ± 1.7% (n = 6; p < 0.004). The maximum rate of rise of phase 0 of the action potential, however, was not significantly altered at any tested concentration of loperamide. CONCLUSIONS: Loperamide is a potent hERG channel blocker. It significantly prolongs the action potential duration and suggests a causal association between loperamide and recent clinical cases of torsade de pointes.

Histone deacetylase inhibitors modulate KATP subunit transcription in HL-1 cardiomyocytes through effects on cholesterol homeostasis.

Histone deacetylase inhibitors (HDIs) are under investigation for the treatment of a number of human health problems. HDIs have proven therapeutic value in refractory cases of cutaneous T-cell lymphoma. Electrocardiographic ST segment morphological changes associated with HDIs were observed during development. Because ST segment morphology is typically linked to changes in ATP sensitive potassium (KATP) channel activity, we tested the hypothesis that HDIs affect cardiac KATP channel subunit expression. Two different HDIs, romidepsin and trichostatin A, caused ~20-fold increase in SUR2 (Abcc9) subunit mRNA expression in HL-1 cardiomyocytes. The effect was specific for the SUR2 subunit as neither compound causes a marked change in SUR1 (Abcc8) expression. Moreover, the effect was cell specific as neither HDI markedly altered KATP subunit expression in MIN6 pancreatic ß-cells. We observe significant enrichment of the H3K9Ac histone mark specifically at the SUR2 promoter consistent with the conclusion that chromatin remodeling at this locus plays a role in increasing SUR2 gene expression. Unexpectedly, however, we also discovered that HDI-dependent depletion of cellular cholesterol is required for the observed effects on SUR2 expression. Taken together, the data in the present study demonstrate that KATP subunit expression can be epigenetically regulated in cardiomyocytes, defines a role for cholesterol homeostasis in mediating epigenetic regulation and suggests a potential molecular basis for the cardiac effects of the HDIs.

Acetate transiently inhibits myocardial contraction by increasing mitochondrial calcium uptake.

BACKGROUND: There is a close relationship between cardiovascular disease and cardiac energy metabolism, and we have previously demonstrated that palmitate inhibits myocyte contraction by increasing Kv channel activity and decreasing the action potential duration. Glucose and long chain fatty acids are the major fuel sources supporting cardiac function; however, cardiac myocytes can utilize a variety of substrates for energy generation, and previous studies demonstrate the acetate is rapidly taken up and oxidized by the heart. In this study, we tested the effects of acetate on contractile function of isolated mouse ventricular myocytes. RESULTS: Acute exposure of myocytes to 10 mM sodium acetate caused a marked, but transient, decrease in systolic sarcomere shortening (1.49 ± 0.20% vs. 5.58 ± 0.49% in control), accompanied by a significant increase in diastolic sarcomere length (1.81 ± 0.01 µm vs. 1.77 ± 0.01 µm in control), with a near linear dose response in the 1-10 mM range. Unlike palmitate, acetate caused no change in action potential duration; however, acetate markedly increased mitochondrial Ca(2+) uptake. Moreover, pretreatment of cells with the mitochondrial Ca(2+) uptake blocker, Ru-360 (10 µM), markedly suppressed the effect of acetate on contraction. CONCLUSIONS: Lehninger and others have previously demonstrated that the anions of weak aliphatic acids such as acetate stimulate Ca(2+) uptake in isolated mitochondria. Here we show that this effect of acetate appears to extend to isolated cardiac myocytes where it transiently modulates cell contraction.

Electrocardiographic studies of romidepsin demonstrate its safety and identify a potential role for K(ATP) channel.

PURPOSE: Romidepsin is a histone deacetylase inhibitor (HDI) approved for the treatment of both cutaneous and peripheral T-cell lymphoma (CTCL and PTCL). During development, a thorough assessment of cardiac toxicity was conducted. EXPERIMENTAL DESIGN: A phase II single-agent nonrandomized study of romidepsin was conducted in patients with CTCL or PTCL who had progressed after at least 1 prior systemic therapy. RESULTS: Results for the first 42 patients enrolled on the NCI 1312 phase II study of romidepsin in CTCL or PTCL showed no cardiac toxicity based on serial electrocardiograms (ECG), troponins, and MUGA scans/echocardiograms. The cardiac assessments reported herein confirm the safety of romidepsin among 131 enrolled patients, while supporting a role for electrolyte replacement. Heart rate increased an average 11 bpm following romidepsin infusion; there was no evidence of increased arrhythmia. Criteria for potassium/magnesium replacement were met before 55% of 1365 romidepsin doses; an association with hypoalbuminemia was confirmed. We propose a mechanism for ST segment flattening and depression, the most common ECG abnormalities observed: HDI-induced alteration of the activity or expression of KATP channels. In addition, examination of the variants of the active transporter of romidepsin, ABCB1, showed a trend toward smaller heart rate changes in the peri-infusion period among wild-type than variant diplotypes. CONCLUSIONS: We conclude that in the context of appropriate attention to electrolyte levels, the data support the cardiac safety of romidepsin.

Promoter DNA methylation regulates murine SUR1 (Abcc8) and SUR2 (Abcc9) expression in HL-1 cardiomyocytes.

Two mammalian genes encode the SURx (SUR1, Abcc8 and SUR2, Abcc9) subunits that combine with Kir6.2 (Kcnj11) subunits to form the ATP-sensitive potassium (KATP) channel in cardiac myocytes. Different isoform combinations endow the channel with distinct physiological and pharmacological properties, and we have recently reported that the molecular composition of sarcolemmal KATP channels is chamber specific in the mouse heart. KATP channel composition is determined by what subunits are expressed in a cell or tissue. In the present study, we explore the role of CpG methylation in regulating SUR1 and SUR2 expression. In HL-1 cardiomyocytes, as in atrial myocytes, SUR1 expression is markedly greater than SUR2. Consistent with CpG methylation-dependent silencing of SUR2 expression, bisulfite sequencing of genomic DNA isolated from HL-1 cells demonstrates that 57.6% of the CpGs in the promoter region of the SUR2 gene are methylated, compared with 0.14% of the the CpG residues in the SUR1 sequence. Moreover, treatment with 10 µM 5-aza-2'-deoxycytidine (Aza-dC) significantly increased both the unmethylated fraction of the SUR2 CpG island and mRNA expression. However, we cannot rule out additional mechanisms of Aza-dC action, as Aza-dC also causes a decrease in SUR1 expression and lower doses of Aza-dC do not alter the unmethylated DNA fraction but do elicit a small increase in SUR2 expression. The conclusion that DNA methylation alone is not the only regulator of SUR subunit expression is also consistent with observations in native myocytes, where the CpG islands of both SUR genes are essentially unmethylated in both atrial and ventricular myocytes. Collectively, these data demonstrate the potential for CpG methylation to regulate SURx subunit expression and raises the possibility that regulated or aberrant CpG methylation might play a role in controlling channel structure and function under different physiological conditions or different species.

Muscle KATP channels: recent insights to energy sensing and myoprotection.

ATP-sensitive potassium (K(ATP)) channels are present in the surface and internal membranes of cardiac, skeletal, and smooth muscle cells and provide a unique feedback between muscle cell metabolism and electrical activity. In so doing, they can play an important role in the control of contractility, particularly when cellular energetics are compromised, protecting the tissue against calcium overload and fiber damage, but the cost of this protection may be enhanced arrhythmic activity. Generated as complexes of Kir6.1 or Kir6.2 pore-forming subunits with regulatory sulfonylurea receptor subunits, SUR1 or SUR2, the differential assembly of K(ATP) channels in different tissues gives rise to tissue-specific physiological and pharmacological regulation, and hence to the tissue-specific pharmacological control of contractility. The last 10 years have provided insights into the regulation and role of muscle K(ATP) channels, in large part driven by studies of mice in which the protein determinants of channel activity have been deleted or modified. As yet, few human diseases have been correlated with altered muscle K(ATP) activity, but genetically modified animals give important insights to likely pathological roles of aberrant channel activity in different muscle types.

Differential K(ATP) channel pharmacology in intact mouse heart.

Classically, cardiac sarcolemmal K(ATP) channels have been thought to be composed of Kir6.2 (KCNJ11) and SUR2A (ABCC9) subunits. However, the evidence is strong that SUR1 (sulfonylurea receptor type 1, ABCC8) subunits are also expressed in the heart and that they play a significant functional role in the atria. To examine this further, we have assessed the effects of isotype-specific potassium channel-opening drugs, diazoxide (specific to SUR1>SUR2A) and pinacidil (SUR2A>SUR1), in intact hearts from wild-type mice (WT, n=6), SUR1(-/-) (n=6), and Kir6.2(-/-) mice (n=5). Action potential durations (APDs) in both atria and ventricles were estimated by optical mapping of the posterior surface of Langendorff-perfused hearts. To confirm the atrial effect of both openers, isolated atrial preparations were mapped in both WT (n=4) and SUR1(-/-) (n=3) mice. The glass microelectrode technique was also used to validate optical action potentials. In WT hearts, diazoxide (300 microM) decreased APD in atria (from 33.8+/-1.9 ms to 24.2+/-1.1 ms, p<0.001) but was without effect in ventricles (APD 60.0+/-7.6 ms vs. 60.8+/-7.5 ms, respectively, NS), consistent with an atrial-specific role for SUR1. The absence of SUR1 resulted in loss of efficacy of diazoxide in SUR1(-/-) atria (APD 36.8+/-1.9 ms vs. 36.8+/-2.8 ms, respectively, NS). In contrast, pinacidil (300 microM) significantly decreased ventricular APD in both WT and SUR1(-/-) hearts (from 60.0+/-7.6 ms to 29.8+/-3.5 ms in WT, p<0.001, and from 63.5+/-2.1 ms to 24.8+/-3.8 ms in SUR1(-/-), p<0.001), but did not decrease atrial APD in either WT or SUR1(-/-) hearts. Glibenclamide (10 microM) reversed the effect of pinacidil in ventricles and restored APD to control values. The absence of Kir6.2 subunits in Kir6.2(-/-) hearts resulted in loss of efficacy of both openers (APD 47.2+/-2.2 ms vs. 47.6+/-2.1 ms and 50.8+/-2.4 ms, and 90.6+/-5.7 ms vs. 93.2+/-6.5 ms and 117.3+/-6.4 ms, for atria and ventricle in control versus diazoxide and pinacidil, respectively). Collectively, these results indicate that in the same mouse heart, significant differential K(ATP) pharmacology in atria and ventricles, resulting from SUR1 predominance in forming the atrial channel, leads to differential effects of potassium channel openers on APD in the two chambers.