miR-99a-5p: A Potential New Therapy for Atherosclerosis by Targeting mTOR and Then Inhibiting NLRP3 Inflammasome Activation and Promoting Macrophage Autophagy.

Objective: MicroRNAs have been revealed to be involved in the development of atherosclerosis. The present study is aimed at exploring the potential of miR-99a-5p as a therapy for atherosclerosis. We suspected that miR-99a-5p might inhibit NLRP3 inflammasome activation and promote macrophage autophagy via constraining mTOR, therefore, alleviating atherosclerosis. Methods: The cell viability in ox-LDL-induced THP-1 macrophages was assessed by CCK-8 assay. Bioinformatic analysis was used to predict the target genes of miR-99a-5p. The binding between miR-99a-5p and mTOR was confirmed by luciferase reporter assay. In vivo, a high-fat-diet-induced atherosclerosis model was established in apolipoprotein E knockout mice. Hematoxylin-eosin, oil red O, and Sirius red staining were performed for the determination of atherosclerotic lesions. MTOR and associated protein levels were detected by Western blot analysis. Results: miR-99a-5p inhibited NLRP3 inflammasome activation and promoted macrophage autophagy by targeting mTOR. Enforced miR-99a-5p significantly reduced the levels of inflammasome complex and inflammatory cytokines. Furthermore, miR-99a-5p overexpression inhibited the expression of mTOR, whereas mTOR overexpression reversed the trend of the above behaviors. In vivo, the specific overexpression of miR-99a-5p significantly reduced atherosclerotic lesions, accompanied by a significant downregulation of autophagy marker CD68 protein expression. Conclusion: We demonstrated for the first time that miR-99a-5p may be considered a therapy for atherosclerosis. The present study has revealed that miR-99a-5p might inhibit NLRP3 inflammasome activation and promote macrophage autophagy by targeting mTOR, therefore, alleviating atherosclerosis.

Uric acid is associated with cardiac death in patients with hypertrophic obstructive cardiomyopathy.

BACKGROUND: The role of uric acid (UA) in survival of patients with hypertrophic obstructive cardiomyopathy (HOCM) has not been fully evaluated. This study aimed to determine whether UA could be an independent risk factor of cardiac death in patients with HOCM. METHODS: A total of 317 patients with HOCM, who were receiving conservative treatment in Fuwai Hospital from October 2009 to December 2014, all of them completed UA evaluations, were analyzed. Patients were divided into three groups according to the UA levels: Tertile 1 (≤ 318 µmol/L, n = 106), Tertile 2 (319 to 397 µmol/L, n = 105), and Tertile 3 (≥ 398 µmol/L, n = 106). RESULTS: During a median follow-up of 45 months, 29 cardiac deaths (9.1%) occurred, including 6 sudden cardiac deaths and 23 heart failure-related deaths. Cardiac death in Tertile 3 (n = 16, 55.2%) was significantly higher than in Tertile 1 (n = 6, 20.7%) and Tertile 2 (n = 7, 24.1%). In univariate model, UA level (continuous value) showed predictive value of cardiac death [hazard ratio (HR) = 1.006, 95% CI: 1.003-1.009,P = 0.009]. Univariate Cox survival analysis had shown a significant higher property of cardiac death in patients of Tertile 3 when compared with those of Tertile 1, but cardiac death in patients of Tertile 2 did not show significant prognositic value compared with those of Tertile 1 (HR = 3.927, 95% CI: 0.666-23.162,P = 0.131). UA was found to be an independent risk factor (HR = 1.005, 95% CI: 1.001-1.009,P = 0.009) of cardiac death in the multivariate regression analysis after the adjustment for age, body mass index, atrial fibrillation, hemoglobin, creatinine, high-sensitivity C-reactive protein, interventricular septum/left ventricular posterior wall ratio, left ventricular outflow tract and left ventricular ejection fraction. CONCLUSIONS: UA concentration was found to be independently associated with cardiac death in HOCM patients receiving conservative treatment. Randomized trials of UA-lowering agents for HOCM patients are warranted.

Real world effectiveness of PCSK-9 inhibitors combined with statins versus statins-based therapy among patients with very high risk of atherosclerotic cardiovascular disease in China (RWE-PCSK study).

BACKGROUND: The efficacy and safety of proprotein convertase subtilisin/kexin type 9 (PCSK-9) inhibitors were confirmed by several clinical trials, but its effectiveness in routine clinical practice in China has not been evaluated. This study aims to describe the real world effectiveness of PCSK-9 inhibitors combined with statins compared with statins-based therapy among patients with very high risk of atherosclerotic cardiovascular disease (ASCVD). METHODS: This is a multi-center observational study, enrolled patients from 32 hospitals who underwent percutaneous coronary intervention (PCI) from January to June in 2019. There are 453 patients treated with PCSK-9 inhibitors combined with statins in PCSK-9 inhibitor group and 2,610 patients treated with statins-based lipid lowering therapies in statins-based group. The lipid control rate and incidence of major adverse cardiovascular events (MACE) over six months were compared between two groups. A propensity score-matched (PSM) analysis was used to balance two groups on confounding factors. Survival analysis using Kaplan-Meier methods was applied for MACE. RESULTS: In a total of 3,063 patients, 89.91% of patients had received moderate or high-intensity statins-based therapy before PCI, but only 9.47% of patients had low-density lipoprotein cholesterol (LDL-C) levels below 1.4 mmol/L at baseline. In the PSM selected patients, LDL-C level was reduced by 42.57% in PCSK-9 inhibitor group and 30.81% (P < 0.001) in statins-based group after six months. The proportion of LDL-C ≤ 1.0 mmol/L increased from 5.29% to 29.26% in PCSK-9 inhibitor group and 0.23% to 6.11% in statins-based group, and the proportion of LDL-C ≤ 1.4 mmol/L increased from 10.36% to 47.69% in PCSK-9 inhibitor group and 2.99% to 18.43% in statins-based group ( P < 0.001 for both). There was no significant difference between PCSK-9 inhibitor and statins-based treatment in reducing the risk of MACE (hazard ratio = 2.52, 95% CI: 0.49-12.97, P = 0.250). CONCLUSIONS: In the real world, PCSK-9 inhibitors combined with statins could significantly reduce LDL-C levels among patients with very high risk of ASCVD in China. The long-term clinical benefits for patients received PCSK-9 inhibitor to reduce the risk of MACE is still unclear and requires further study.

The impact of subclinical hypothyroidism on long-term outcomes in older patients undergoing percutaneous coronary intervention.

BACKGROUND: Subclinical hypothyroidism (SCH) is reportedly associated with an increased risk of adverse events in patients undergoing percutaneous coronary intervention (PCI). The prognostic significance of SCH in the elderly was poorly defined. The purpose of this study was to evaluate the association between SCH and long-term outcomes in older patients undergoing PCI. METHODS: Three thousand one hundred sixty-eight patients aged 65 years or older who underwent PCI from January 2012 to October 2014 were included. Patients were divided into SCH group (n = 320) and euthyroidism (ET) group (n = 2848) based on thyroid function test. Cox proportional hazard regression analyses were used to estimate the relative risks (RRs) of all-cause death and cardiac death for patients with SCH during a 4-year follow-up period. RESULTS: There were 227 deaths during the follow-up period including 124 deaths caused by cardiac events. There was no significant difference in mortality rate between the SCH group and the ET group (p > 0.05). After adjustment for covariates, compared with patients with ET, the RRs of death from all-cause and cardiac in patients with SCH were 1.261 (95%CI: 0.802-1.982, p = 0.315) and 1.231 (95%CI: 0.650-2.334, p = 0.524), respectively. When SCH was stratified by age, gender, and degree of thyroid-stimulating hormone elevation, no significant associations were also found in any stratum. CONCLUSION: Our investigation revealed that SCH was negatively associated with the outcome of PCI in older patients.

Cardiac specific transcription factor Csx/Nkx2.5 regulates transient-outward K+ channel expression in pluripotent P19 cell-derived cardiomyocytes.

The homeobox-containing gene Csx/Nkx2.5 codes several cardiac transcription factors and plays a critical role in early cardiogenesis. We investigated the effect of Csx/Nkx2.5 on the expression of cardiac ion channels using P19-derived cardiomyocytes. P19CL6 cells and P19CL6 cells with Csx/Nkx2.5 overexpression (P19CL6-Csx cells) were induced to differentiate into cardiomyocytes by treatment with dimethyl sulfoxide. Action potentials and membrane currents were measured by whole cell patch clamp at different differentiation stage: the early stage (1-5 days after beating had begun) and the late stage (10-15 days after beating). Expression of Csx/Nkx2.5 mRNA was increased as the differentiation stages advanced in both P19CL6 and P19CL6-Csx cells. In action potential configuration, maximal diastolic potentials in P19CL6-Csx cells exhibited more hyperpolarized potential (‒ 64.2 mV) than those in P19CL6 cells (‒ 54.8 mV, p < 0.01) in the early stage. In P19CL6 cells, among 6 different voltage-gated and ligand-operated K+ channels expressed during the early stage, the transient-outward K+ channel was most predominant. By overexpression of Csx/Nkx2.5, developmental decrease in the transient-outward K+ channel was suppressed. Homeobox-containing gene Csx/Nkx2.5 modifies the amount of distinct ionic channels, during differentiation periods, predominantly changing the expression of the transient-outward K+ channel.

Homocysteine as a potential predictive factor for high major adverse cardiovascular events risk in female patients with premature acute coronary syndrome.

We aimed to investigate the correlation of homocysteine (Hcy) level with clinical characteristics, and explore its predictive value for major adverse cardiovascular events (MACE) risk in female patients with premature acute coronary syndrome (ACS).The serum Hcy level was detected from 1299 female patients with premature ACS. According to the tertile of Hcy level, patients were divided into 3 groups: lowest tertile group (≤9.1 µmol/L), middle tertile group (9.2-11.6 µmol/L) and highest tertile group (>11.6 µmol/L). MACE incidence was recorded and MACE-free survival was caculated with the median follow-up duration of 28.3 months.Increased Hcy correlated with older age (P < .001), higher creatinine level (P < .001), and enhanced uric acid level (P = .001), while reduced fasting glucose concentration (P < .001). MACE incidence was 10.7% and it was highest in highest tertile group (22.1%), followed by middle tertile group (7.7%) and lowest tertile group (2.4%) (P < .001). Receiver operating characteristic curve showed that Hcy distinguished MACE patients from non-MACE patients with the area under the curve of 0.789 (95% CI: 0.742-0.835). Kaplan-Meier curves revealed that MACE-free survival was shortest in Hcy highest tertile group, followed by middle tertile group and lowest tertile group (P < .001). Multivariate Cox analyses further showed that higher Hcy level was an independent predictive factor for poor MACE-free survival (middle tertile vs lowest tertile (P = .001, HR: 3.615, 95% CI: 1.661-7.864); highest tertile vs lowest tertile (P < .001, HR: 11.023, 95% CI: 5.356-22.684)).Hcy serves as a potential predictive factor for increased MACE risk in female patients with premature ACS.

Tribenuron-methyl resistance and mutation diversity of Pro197 in flixweed (Descurainia Sophia L.) accessions from China.

Flixweed (Descurainia Sophia L.) is a problematic weed in winter wheat fields in China, which causes great loss of wheat yield. A total of 46 flixweed accessions from winter wheat-planting areas were collected and used for the survey of resistance to tribenuron-methyl and Pro197 mutation diversity. According to the "R" resistance rating system, 16 flixweed accessions have evolved resistance to tribenuron-methyl, 13 accessions have high risk of developing resistance to this herbicide and 17 accessions are susceptible. The mutation of Pro197 codon (CCT) changed proline (Pro) into leucine (Leu) (homozygous, RR), serine (Ser, RR), histidine (His, RR), threonine (Thr, RR), Pro/Leu (heterozygous, RS), Pro/Ser (RS), Pro/His, Pro/Thr (RS) and Pro/Tyr (RS). Among these amino acid changes, a Pro197-Pro/Tyr (heterozygous, RS) substitution caused by the mutation of two successive nucleotides was identified for the first time in resistant weed species. In addition, the Pro197-His and Pro197-Pro/His mutations have not been reported previously in flixweed. Finally, a CPAS marker was developed to identify flixweed plants with or without Pro197 mutation.

Different cross-resistance patterns to AHAS herbicides of two tribenuron-methyl resistant flixweed (Descurainiasophia L.) biotypes in China.

Flixweed (Descurainiasophia L.) is a troublesome weed in winter wheat fields in China. Two flixweed accessions, HB08 and HB16 with a Pro-197-Leu and Pro-197-Ser AHAS-mutation respectively, have evolved very high levels resistance to sulfonylurea (SU) herbicide, tribenuron-methyl. Cross resistance of HB08 and HB16 to AHAS herbicides of SU, imidazolinone (IMI), triazolopyrimidine (TP) and pyrimidinyl-thiobenozoate (PTB) families was investigated by dose-response experiments. In addition, the effects of AHAS herbicides on the activity of AHAS extracted from HB08 and HB16 plants were evaluated. HB16 exhibited cross resistance to SU herbicides halosulfuron-methyl and triasulfuron, TP herbicides flumetsulam and penoxsulam, but displayed more sensitivity to IMI herbicide imazethapyr. By contrast, HB08 only showed cross resistance to SU herbicides halosulfuron-methyl and triasulfuron. The in vitro sensitivity of AHAS to AHAS herbicides is consistent with the results of dose-response experiments and the estimated Pearson's r values for HB08 and HB16 are 0.996 and 0.912 respectively. These indicated that altered AHAS sensitivity was responsible mainly for cross resistance patterns observed in the two resistant biotypes.

Pyruvate restores ß-adrenergic sensitivity of L-type Ca(2+) channels in failing rat heart: role of protein phosphatase.

Oxidative stress plays a major role in the pathogenesis of heart failure, where the contractile response to ß-adrenergic stimulation is profoundly depressed. This condition involves L-type Ca(2+) channels, but the mechanisms underlying their impaired adrenergic regulation are unclear. Thus the present study explored the basis for impaired adrenergic control of Ca(2+) channels in a rat infarction model of heart failure. Patch-clamp recordings of L-type Ca(2+) current (I(Ca,L)) from ventricular myocytes isolated from infarcted hearts showed a blunted response to intracellular cAMP that was reversed by treatment with exogenous pyruvate. Biochemical studies showed that basal and cAMP-stimulated protein kinase A activities were similar in infarcted and sham-operated hearts, whereas molecular analysis also found that binding of protein kinase A to the α(1C) subunit of voltage-gated Ca(2+) channel isoform 1.2 was not different between groups. By contrast, protein phosphatase 2A (PP2A) activity and binding to α(1C) were significantly less in infarcted hearts. The PP2A inhibitor okadaic acid markedly increased I(Ca,L) in sham-operated myocytes, but this response was significantly less in myocytes from infarcted hearts. However, pyruvate normalized I(Ca,L) stimulation by okadaic acid, and this effect was blocked by inhibitors of thioredoxin reductase, implicating a functional role for the redox-active thioredoxin system. Our data suggest that blunted ß-adrenergic stimulation of I(CaL) in failing hearts results from hyperphosphorylation of Ca(2+) channels secondary to oxidation-induced impairment of PP2A function. We propose that the redox state of Ca(2+) channels or PP2A is controlled by the thioredoxin system which plays a key role in Ca(2+) channel remodeling of the failing heart.

[Regulation of T-type Ca(2+) channel in lysophosphatidylcholine-stimulated cardiomyocytes].

OBJECTIVE: To study the effect of lysophosphatidylcholine (LPC) on T-type calcium channel currents (I(Ca.T)) in cardiomyocytes, and identify the mechanism by which LPC accumulation in intracellular and/or interstitial space may uptake tachycardia and various arrhythmias during cardiac ischemia. METHODS: Neonatal rat cardiomyocytes from 1 to 3-day-old Wistar rats and hypertrophied ventricular myocytes from Wistar rats were prepared. Human cardiac T-type calcium channel α1 subunits, Ca(V3.1) and Ca(V3.2), were stably expressed in HEK293 cells. In this study, cardiomyocytes and heterologous expression of human Ca(V3.1) and Ca(V3.2) components were measureed by whole-cell patch clamp to study the up-regulation of I(Ca.T) by LPC. RESULTS: LPC markedly accelerated the spontaneous beating rates of neonatal rat cardiomyocytes from (42±8) beats/min in control to (64 ±8) beats/min after LPC application for 5 min at the physiological [Ca(2+)](i) concentration (pCa=7.2). In neonatal cardiomyocytes, I(Ca.T) was significantly increased by 10 µmol/L LPC by 21.5% when [Ca(2+)](i) was high (pCa=7). Intracellular Ca(2+)-dependent augmentation of I(Ca.T) by LPC was confirmed not only in neonatal cardiomyocytes but also in adult ventricular myocytes from the hypertrophied hearts. In this experiment, I(Ca.T) was significantly increased by 10 µmol/L LPC by 23.5% when [Ca(2+)](i) was high (pCa=7), although it was unchanged when [Ca(2+)](i) was low (pCa=11), control: (3.8±0.2) pA/pF, n=16; LPC: (3.7±0.4) pA/pF, n=10. LPC exerted no effect on the Ca(V3.1) T-type Ca(2+) channel current (I(Ca(V)3.1)) regardless of the [Ca(2+)](i) concentration at a pCa of 7 or at a pCa of 11. In contrast, LPC up-regulated the Ca(V3.2) T-type Ca(2+) channel current (I(Ca(V)3.2)), which was much larger at a pCa of 7 [LPC=10 µmol/L: (68.8±2.1) pA/pF, n=10; LPC=50 µmol/L: (78.4±4.8) pA/pF, n=9)] than that at a pCa of 11 [(38.5±2.1) pA/pF, n=11]. CONCLUSION: The present study indicates that LPC up-regulates the cardiac I(Ca.T) in physiological or higher [Ca(2+)](i) concentration, which may accelerate the pathophysiological cardiac automaticity and trigger tachyarrhythmias as novel ischemia-related mechanism.

Role of apoptosis signal-regulating kinase-1-c-Jun NH2-terminal kinase-p38 signaling in voltage-gated K+ channel remodeling of the failing heart: regulation by thioredoxin.

c-Jun NH(2)-terminal kinase (JNK) and p38 kinase are key regulators of cardiac hypertrophy and apoptosis during pathological stress, but their role in regulating ion channels in the diseased heart is unclear. Thus, we compared the kinase profile and electrophysiological phenotype of the rat ventricle 6-8 weeks after myocardial infarction (MI). Molecular analyses showed that JNK and p38 activities were markedly increased in post-MI hearts, while parallel voltage-clamp studies in ventricular myocytes revealed a characteristic downregulation of transient outward K(+) current (I(to)) density. When post-MI myocytes were treated with JNK or p38 inhibitors, I(to) density increased to control levels. Upregulation of I(to) was also elicited by insulin-like growth factor-1, which decreased JNK/p38 activity in post-MI hearts, and these changes were blocked by the thioredoxin (Trx) reductase inhibitor auranofin. Consistent with activation of JNK-p38 signaling, binding of apoptosis signal-regulating kinase-1 with Trx1 was also markedly decreased post-MI, and was reversed by insulin-like growth factor-1 in an auranofin-sensitive manner. We conclude that expression of ventricular K(+) channels is redox regulated and that chronic impairment of the Trx system in the post-MI heart contributes to I(to) remodeling through sustained activation of apoptosis signal-regulating kinase-1-JNK-p38 signaling. The cardiac Trx system may thus be a novel therapeutic target to reverse or prevent ventricular arrhythmias in the failing heart.

Role of gamma-glutamyl transpeptidase in redox regulation of K+ channel remodeling in postmyocardial infarction rat hearts.

gamma-Glutamyl transpeptidase (gamma-GT) is a key enzyme in GSH metabolism that regulates intracellular GSH levels in response to extracellular GSH (GSH(o)). The objective of this study was to identify the role of gamma-GT in reversing pathogenic K(+) channel remodeling in the diseased heart. Chronic ventricular dysfunction was induced in rats by myocardial infarction (MI), and studies were done after 6-8 wk. Biochemical assays of tissue extracts from post-MI hearts revealed significant increases in gamma-GT activity in left ventricle (47%) and septum (28%) compared with sham hearts, which paralleled increases in protein abundance and mRNA. Voltage-clamp studies of isolated left ventricular myocytes from post-MI hearts showed that downregulation of transient outward K(+) current (I(to)) was reversed after 4-5 h by 10 mmol/l GSH(o) or N-acetylcysteine (NAC(o)), and that the effect of GSH(o) but not NAC(o) was blocked by the gamma-GT inhibitors, acivicin or S-hexyl-GSH. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine did not prevent upregulation of I(to) by GSH(o), suggesting that intracellular synthesis of GSH was not directly involved. However, pretreatment of post-MI myocytes with an SOD mimetic [manganese (III) tetrapyridylporphyrin] and catalase completely blocked recovery of I(to) by GSH(o). Confocal microscopy using the fluorogenic dye 2',7'-dichlorodihydrofluorescein diacetate confirmed that GSH(o) increased reactive oxygen species (ROS) generation by post-MI myocytes and to a lesser extent in myocytes from sham hearts. Furthermore, GSH(o)-mediated upregulation of I(to) was blocked by inhibitors of tyrosine kinase (genistein, lavendustin A, and AG1024) and thioredoxin reductase (auranofin and 13-cis-retinoic acid). These data suggest that GSH(o) elicits gamma-GT- and ROS-dependent transactivation of tyrosine kinase signaling that upregulates K(+) channel activity or expression via redox-mediated mechanisms. The signaling events stimulated by gamma-GT catalysis of GSH(o) may be a therapeutic target to reverse pathogenic electrical remodeling of the failing heart.

Glutathione homeostasis in ventricular myocytes from rat hearts with chronic myocardial infarction.

The ubiquitous tripeptide glutathione (GSH) is an essential factor in many biological processes, thus its depletion has a major impact on cell function and survival. In this study, we examined regulation of GSH in cardiomyocytes under chronic oxidative stress elicited by myocardial infarction (MI). Cardiac dysfunction was induced in rats by coronary artery ligation, and experiments were conducted in myocytes isolated from non-infarcted left ventricle and septum after 6-8 weeks. Fluorescence microscopy studies using the probe monochlorobimane showed that [GSH] in myocytes from post-MI hearts was 42% less than in sham control hearts (P < 0.05). However, depleted GSH levels were normalized after 5-6 h by an insulin mimetic (bis-peroxovanadium-1,10-phenanthroline, bpV(phen); 10 micromol l(-1)) or by exogenous pyruvate (5 mmol l(-1)). The increase in [GSH] by bpV(phen) was partly inhibited by buthionine sulphoximine (BSO; 50 micromol l(-1)), a blocker of GSH synthesis, and by 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU; 100 micromol l(-1)), an inhibitor of glutathione disulphide reductase. By comparison, the effect of pyruvate was not altered by BSO but was completely blocked by BCNU. Studies using inhibitors of signalling cascades indicated that upregulation of [GSH] by bpV(phen) in myocytes from post-MI hearts was mediated by mitogen activated protein kinase/extracellular signal-regulated kinase kinase 1/2 and p38 mitogen-activated protein kinase but not by phosphatidylinositol 3-kinase. The effect of pyruvate was not altered by any kinase inhibitor tested. In cells loaded with the probe TEMPO-9-AC to monitor superoxide anion, baseline fluorescence was 2.3-fold greater in post-MI myocytes than in sham control myocytes (P < 0.05) and was markedly decreased by diphenyleneiodonium (30 micromol l(-1)), an inhibitor of NADPH oxidase, exogenous GSH (10 mmol l(-1)) or bpV(phen). In parallel studies, [GSH] in post-MI myocytes was also normalized by diphenyleneiodonium or exogenous GSH. These data show that GSH is differentially regulated by receptor tyrosine kinase-dependent and -independent agonists that maintain functional GSH levels necessary to neutralize excess generation of reactive oxygen species in the failing heart.

Oxidoreductase regulation of Kv currents in rat ventricle.

Oxidative stress contributes to the arrhythmogenic substrate created by myocardial ischemia-reperfusion partly through a shift in cell redox state, a key modulator of protein function. The activity of many oxidation-sensitive proteins is controlled by oxidoreductase systems that regulate the redox state of cysteine thiol groups, but the impact of these systems on ion channel function is not well defined. Thus, we examined the roles of the thioredoxin and glutaredoxin systems in controlling K(+) channels in the ventricle. An oxidative shift in redox state was elicited in isolated rat ventricular myocytes by brief exposure to diamide, a thiol-specific, membrane-permeable oxidant. Voltage-clamp studies showed that diamide decreased peak outward K(+) current (I(peak)) evoked by depolarizing test pulses by 41% (+60 mV; p<0.05) while steady-state outward current (I(ss)) measured at the end of the test pulse was decreased by 45% (p<0.05). These electrophysiological effects were not prevented by protein kinase C blockers, but the tyrosine kinase inhibitors genistein or lavendustin A blocked the suppression of both K(+) currents by diamide. Moreover, inhibition of I(peak) and I(ss) by diamide was reversed by dichloroacetate and an insulin-mimetic. The effect of dichloroacetate to normalize I(peak) after diamide was blocked by the thioredoxin system inhibitors auranofin or 13-cis-retinoic acid, but I(ss) was not affected by either compound. A pan-specific inhibitor of glutaredoxin and thioredoxin systems, 1,3-bis-(2-chloroethyl)-1-nitrosourea, also blocked the dichloroacetate effect on I(peak) but only partially inhibited the recovery of I(ss). These data suggest that acute regulation of cardiac K(+) channels by oxidoreductase systems is mediated by redox-sensitive tyrosine kinase/phosphatase pathways. The pathways controlling I(peak) channels are targets of the thioredoxin system whereas those regulating I(ss) channels are likely controlled by the glutaredoxin system. Thus, cardiac oxidoreductase systems may be important regulators of ion channels affected by pathogenic oxidative stress.

Diversity of acetyl-coenzyme A carboxylase mutations in resistant Lolium populations: evaluation using clethodim.

The acetyl-coenzyme A carboxylase (ACCase)-inhibiting cyclohexanedione herbicide clethodim is used to control grass weeds infesting dicot crops. In Australia clethodim is widely used to control the weed Lolium rigidum. However, clethodim-resistant Lolium populations have appeared over the last 5 years and now are present in many populations across the western Australian wheat (Triticum aestivum) belt. An aspartate-2078-glycine (Gly) mutation in the plastidic ACCase enzyme has been identified as the only known mutation endowing clethodim resistance. Here, with 14 clethodim-resistant Lolium populations we revealed diversity and complexity in the molecular basis of resistance to ACCase-inhibiting herbicides (clethodim in particular). Several known ACCase mutations (isoleucine-1781-leucine [Leu], tryptophan-2027-cysteine [Cys], isoleucine-2041-asparagine, and aspartate-2078-Gly) and in particular, a new mutation of Cys to arginine at position 2088, were identified in plants surviving the Australian clethodim field rate (60 g ha(-1)). Twelve combination patterns of mutant alleles were revealed in relation to clethodim resistance. Through a molecular, biochemical, and biological approach, we established that the mutation 2078-Gly or 2088-arginine endows sufficient level of resistance to clethodim at the field rate, and in addition, combinations of two mutant 1781-Leu alleles, or two different mutant alleles (i.e. 1781-Leu/2027-Cys, 1781-Leu/2041-asparagine), also confer clethodim resistance. Plants homozygous for the mutant 1781, 2078, or 2088 alleles were found to be clethodim resistant and cross resistant to a number of other ACCase inhibitor herbicides including clodinafop, diclofop, fluazifop, haloxyfop, butroxydim, sethoxydim, tralkoxydim, and pinoxaden. We established that the specific mutation, the homo/heterozygous status of a plant for a specific mutation, and combinations of different resistant alleles plus herbicide rates all are important in contributing to the overall level of herbicide resistance in genetically diverse, cross-pollinated Lolium species.