Myocardial glycophagy flux dysregulation and glycogen accumulation characterize diabetic cardiomyopathy.

Diabetic heart disease morbidity and mortality is escalating. No specific therapeutics exist and mechanistic understanding of diabetic cardiomyopathy etiology is lacking. While lipid accumulation is a recognized cardiomyocyte phenotype of diabetes, less is known about glycolytic fuel handling and storage. Based on in vitro studies, we postulated the operation of an autophagy pathway in the myocardium specific for glycogen homeostasis - glycophagy. Here we visualize occurrence of cardiac glycophagy and show that the diabetic myocardium is characterized by marked glycogen elevation and altered cardiomyocyte glycogen localization. We establish that cardiac glycophagy flux is disturbed in diabetes. Glycophagy may represent a potential therapeutic target for alleviating the myocardial impacts of metabolic disruption in diabetic heart disease.

The cardiomyocyte origins of diastolic dysfunction: cellular components of myocardial "stiffness".

The impaired ability of the heart to relax and stretch to accommodate venous return is generally understood to represent a state of "diastolic dysfunction" and often described using the all-purpose noun "stiffness." Despite the now common qualitative usage of this term in fields of cardiac patho/physiology, the specific quantitative concept of stiffness as a molecular and biophysical entity with real practical interpretation in healthy and diseased hearts is sometimes obscure. The focus of this review is to characterize the concept of cardiomyocyte stiffness and to develop interpretation of "stiffness" attributes at the cellular and molecular levels. Here, we consider "stiffness"-related terminology interpretation and make links between cardiomyocyte stiffness and aspects of functional and structural cardiac performance. We discuss cross bridge-derived stiffness sources, considering the contributions of diastolic myofilament activation and impaired relaxation. This includes commentary relating to the role of cardiomyocyte Ca2+ flux and Ca2+ levels in diastole, the troponin-tropomyosin complex role as a Ca2+ effector in diastole, the myosin ADP dissociation rate as a modulator of cross bridge attachment and regulation of cross-bridge attachment by myosin binding protein C. We also discuss non-cross bridge-derived stiffness sources, including the titin sarcomeric spring protein, microtubule and intermediate filaments, and cytoskeletal extracellular matrix interactions. As the prevalence of conditions involving diastolic heart failure has escalated, a more sophisticated understanding of the molecular, cellular, and tissue determinants of cardiomyocyte stiffness offers potential to develop imaging and molecular intervention tools.

Genome assembly and transcriptomic analysis to elucidate the ability of Nasonovia ribisnigri to break host plant resistance.

Aphid genomic resources enable the study of complex life history traits and provide information on vector biology, host adaption and speciation. The currant-lettuce aphid (Nasonovia ribisnigri (Hemiptera: Aphididae) (Mosley)) is a cosmopolitan pest of outdoor lettuce (Lactuca sativa (Asterales: Asteraceae) (Linnaeus)). Until recently, the use of resistant cultivars was an effective method for managing N. ribisnigri. A resistant cultivar containing a single gene (Nr-locus), introduced in the 1980s, conferred complete resistance to feeding. Overreliance of this Nr-locus in lettuce resulted in N. ribisnigri's ability to break resistance mechanism, with first reports during 2003. Our work attempts to understand which candidate gene(s) are associated with this resistance-breaking mechanism. We present two de novo draft assembles for N. ribisnigri genomes, corresponding to both avirulent (Nr-locus susceptible) and virulent (Nr-locus resistant) biotypes. Changes in gene expression of the two N. ribisnigri biotypes were investigated using transcriptomic analyses of RNA-sequencing (RNA-seq) data to understand the potential mechanisms of resistance to the Nr-locus in lettuce. The draft genome assemblies were 94.2% and 91.4% complete for the avirulent and virulent biotypes, respectively. Out of the 18,872 differentially expressed genes, a single gene/locus was identified in N. ribisnigri that was shared between two resistant-breaking biotypes. This locus was further explored and validated in Real-Time Quantitative Reverse Transcription PCR (qRT-PCR) experiments and has predicted localisations in both the cytoplasm and nucleus. This is the first study to provide evidence that a single gene/locus is likely responsible for the ability of N. ribisnigri to overcome the Nr-locus resistance in the lettuce host.

Phenological sensitivity to climate across taxa and trophic levels.

Differences in phenological responses to climate change among species can desynchronise ecological interactions and thereby threaten ecosystem function. To assess these threats, we must quantify the relative impact of climate change on species at different trophic levels. Here, we apply a Climate Sensitivity Profile approach to 10,003 terrestrial and aquatic phenological data sets, spatially matched to temperature and precipitation data, to quantify variation in climate sensitivity. The direction, magnitude and timing of climate sensitivity varied markedly among organisms within taxonomic and trophic groups. Despite this variability, we detected systematic variation in the direction and magnitude of phenological climate sensitivity. Secondary consumers showed consistently lower climate sensitivity than other groups. We used mid-century climate change projections to estimate that the timing of phenological events could change more for primary consumers than for species in other trophic levels (6.2 versus 2.5-2.9 days earlier on average), with substantial taxonomic variation (1.1-14.8 days earlier on average).

Cardiomyocyte functional screening: interrogating comparative electrophysiology of high-throughput model cell systems.

Cardiac arrhythmias of both atrial and ventricular origin are an important feature of cardiovascular disease. Novel antiarrhythmic therapies are required to overcome current drug limitations related to effectiveness and pro-arrhythmia risk in some contexts. Cardiomyocyte culture models provide a high-throughput platform for screening antiarrhythmic compounds, but comparative information about electrophysiological properties of commonly used types of cardiomyocyte preparations is lacking. Standardization of cultured cardiomyocyte microelectrode array (MEA) experimentation is required for its application as a high-throughput platform for antiarrhythmic drug development. The aim of this study was to directly compare the electrophysiological properties and responses to isoproterenol of three commonly used cardiac cultures. Neonatal rat ventricular myocytes (NRVMs), immortalized atrial HL-1 cells, and custom-generated human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were cultured on microelectrode arrays for 48-120 h. Extracellular field potentials were recorded, and conduction velocity was mapped in the presence/absence of the ß-adrenoceptor agonist isoproterenol (1 µM). Field potential amplitude and conduction velocity were greatest in NRVMs and did not differ in cardiomyocytes isolated from male/female hearts. Both NRVMs and hiPSC-CMs exhibited longer field potential durations with rate dependence and were responsive to isoproterenol. In contrast, HL-1 cells exhibited slower conduction and shorter field potential durations and did not respond to 1 µM isoproterenol. This is the first study to compare the intrinsic electrophysiologic properties of cultured cardiomyocyte preparations commonly used for in vitro electrophysiology assessment. These findings offer important comparative data to inform methodological approaches in the use of MEA and other techniques relating to cardiomyocyte functional screening investigations of particular relevance to arrhythmogenesis.

Spatial and habitat variation in aphid, butterfly, moth and bird phenologies over the last half century.

Global warming has advanced the timing of biological events, potentially leading to disruption across trophic levels. The potential importance of phenological change as a driver of population trends has been suggested. To fully understand the possible impacts, there is a need to quantify the scale of these changes spatially and according to habitat type. We studied the relationship between phenological trends, space and habitat type between 1965 and 2012 using an extensive UK dataset comprising 269 aphid, bird, butterfly and moth species. We modelled phenologies using generalized additive mixed models that included covariates for geographical (latitude, longitude, altitude), temporal (year, season) and habitat terms (woodland, scrub, grassland). Model selection showed that a baseline model with geographical and temporal components explained the variation in phenologies better than either a model in which space and time interacted or a habitat model without spatial terms. This baseline model showed strongly that phenologies shifted progressively earlier over time, that increasing altitude produced later phenologies and that a strong spatial component determined phenological timings, particularly latitude. The seasonal timing of a phenological event, in terms of whether it fell in the first or second half of the year, did not result in substantially different trends for butterflies. For moths, early season phenologies advanced more rapidly than those recorded later. Whilst temporal trends across all habitats resulted in earlier phenologies over time, agricultural habitats produced significantly later phenologies than most other habitats studied, probably because of nonclimatic drivers. A model with a significant habitat-time interaction was the best-fitting model for birds, moths and butterflies, emphasizing that the rates of phenological advance also differ among habitats for these groups. Our results suggest the presence of strong spatial gradients in mean seasonal timing and nonlinear trends towards earlier seasonal timing that varies in form and rate among habitat types.

The sensitivity of breeding songbirds to changes in seasonal timing is linked to population change but cannot be directly attributed to the effects of trophic asynchrony on productivity.

A consequence of climate change has been an advance in the timing of seasonal events. Differences in the rate of advance between trophic levels may result in predators becoming mismatched with prey availability, reducing fitness and potentially driving population declines. Such "trophic asynchrony" is hypothesized to have contributed to recent population declines of long-distance migratory birds in particular. Using spatially extensive survey data from 1983 to 2010 to estimate variation in spring phenology from 280 plant and insect species and the egg-laying phenology of 21 British songbird species, we explored the effects of trophic asynchrony on avian population trends and potential underlying demographic mechanisms. Species which advanced their laying dates least over the last three decades, and were therefore at greatest risk of asynchrony, exhibited the most negative population trends. We expressed asynchrony as the annual variation in bird phenology relative to spring phenology, and related asynchrony to annual avian productivity. In warmer springs, birds were more asynchronous, but productivity was only marginally reduced; long-distance migrants, short-distance migrants and resident bird species all exhibited effects of similar magnitude. Long-term population, but not productivity, declines were greatest among those species whose annual productivity was most greatly reduced by asynchrony. This suggests that population change is not mechanistically driven by the negative effects of asynchrony on productivity. The apparent effects of asynchrony on population trends are therefore either more likely to be strongly expressed via other demographic pathways, or alternatively, are a surrogate for species' sensitivity to other environmental pressures which are the ultimate cause of decline.

Pericardial adipose and aromatase: A new translational target for aging, obesity and arrhythmogenesis?

A correlation exists between the extent of pericardial adipose and atrial fibrillation (AF) risk, though the underlying mechanisms remain unclear. Selected adipose depots express high levels of aromatase, capable of converting androgens to estrogens - no studies have investigated aromatase occurrence/expression regulation in pericardial adipose. The Women's Health Initiative reported that estrogen-only therapy in women elevated AF incidence, indicating augmented estrogenic influence may exacerbate cardiac vulnerability. The aim of this study was to identify the occurrence of pericardial adipose aromatase, evaluate the age- and sex-dependency of local cardiac steroid synthesis capacity and seek preliminary experimental evidence of a link between pericardial adipose aromatase capacity and arrhythmogenic vulnerability. Both human atrial appendage and epicardial adipose exhibited immunoblot aromatase expression. In rodents, myocardium and pericardial adipose aromatase expression increased >20-fold relative to young controls. Comparing young, aged and aged-high fat diet animals, a significant positive correlation was determined between the total aromatase content of pericardial adipose and the occurrence/duration of triggered atrial arrhythmias. Incidence and duration of arrhythmias were increased in hearts perfused with 17ß-estradiol. This study provides novel report of pericardial adipose aromatase expression. We show that aromatase expression is remarkably upregulated with aging, and aromatase estrogen conversion capacity significantly elevated with obesity-related cardiac adiposity. Our studies suggest an association between adiposity, aromatase estrogenic capacity and atrial arrhythmogenicity - additional investigation is required to establish causality. The potential impact of these findings may be considerable, and suggests that focus on local cardiac steroid conversion (rather than systemic levels) may yield translational outcomes.

Insights into the role of maladaptive hexosamine biosynthesis and O-GlcNAcylation in development of diabetic cardiac complications.

Diabetes mellitus significantly increases the risk of heart failure, independent of coronary artery disease. The mechanisms implicated in the development of diabetic heart disease, commonly termed diabetic cardiomyopathy, are complex, but much of the impact of diabetes on the heart can be attributed to impaired glucose handling. It has been shown that the maladaptive nutrient-sensing hexosamine biosynthesis pathway (HBP) contributes to diabetic complications in many non-cardiac tissues. Glucose metabolism by the HBP leads to enzymatically-regulated, O-linked attachment of a sugar moiety molecule, ß-N-acetylglucosamine (O-GlcNAc), to proteins, affecting their biological activity (similar to phosphorylation). In normal physiology, transient activation of HBP/O-GlcNAc mechanisms is an adaptive, protective means to enhance cell survival; interventions that acutely suppress this pathway decrease tolerance to stress. Conversely, chronic dysregulation of HBP/O-GlcNAc mechanisms has been shown to be detrimental in certain pathological settings, including diabetes and cancer. Most of our understanding of the impact of sustained maladaptive HBP and O-GlcNAc protein modifications has been derived from adipose tissue, skeletal muscle and other non-cardiac tissues, as a contributing mechanism to insulin resistance and progression of diabetic complications. However, the long-term consequences of persistent activation of cardiac HBP and O-GlcNAc are not well-understood; therefore, the goal of this timely review is to highlight current understanding of the role of the HBP pathway in development of diabetic cardiomyopathy.

Dietary omega-6 fatty acid replacement selectively impairs cardiac functional recovery after ischemia in female (but not male) rats.

A definitive understanding of the role of dietary lipids in determining cardioprotection (or cardiodetriment) has been elusive. Randomized trial findings have been variable and sex specificity of dietary interventions has not been determined. In this investigation the sex-selective cardiac functional effects of three diets enriched by omega-3 or omega-6 polyunsaturated fatty acids (PUFA) or enriched to an equivalent extent in saturated fatty acid components were examined in rats after an 8-wk treatment period. In females the myocardial membrane omega-6:omega-3 PUFA ratio was twofold higher than males in the omega-6 diet replacement group. In diets specified to be high in omega-3 PUFA or in saturated fat, this sex difference was not apparent. Isolated cardiomyocyte and heart Langendorff perfusion experiments were performed, and molecular measures of cell viability were assessed. Under basal conditions the contractile performance of omega-6 fed female cardiomyocytes and hearts was reduced compared with males. Omega-6 fed females exhibited impaired systolic resilience after ischemic insult. This response was associated with increased postischemia necrotic cell damage evaluated by coronary lactate dehydrogenase during reperfusion in omega-6 fed females. Cardiac and myocyte functional parameters were not different between omega-3 and saturated fat dietary groups and within these groups there were no discernible sex differences. Our data provide evidence at both the cardiac and cardiomyocyte levels that dietary saturated fatty acid intake replacement with an omega-6 (but not omega-3) enriched diet has selective adverse cardiac effect in females. This finding has potential relevance in relation to women, cardiac risk, and dietary management.

The Effect of Farmers' Decisions on Pest Control with Bt Crops: A Billion Dollar Game of Strategy.

A farmer's decision on whether to control a pest is usually based on the perceived threat of the pest locally and the guidance of commercial advisors. Therefore, farmers in a region are often influenced by similar circumstances, and this can create a coordinated response for pest control that is effective at a landscape scale. This coordinated response is not intentional, but is an emergent property of the system. We propose a framework for understanding the intrinsic feedback mechanisms between the actions of humans and the dynamics of pest populations and demonstrate this framework using the European corn borer, a serious pest in maize crops. We link a model of the European corn borer and a parasite in a landscape with a model that simulates the decisions of individual farmers on what type of maize to grow. Farmers chose whether to grow Bt-maize, which is toxic to the corn borer, or conventional maize for which the seed is cheaper. The problem is akin to the snow-drift problem in game theory; that is to say, if enough farmers choose to grow Bt maize then because the pest is suppressed an individual may benefit from growing conventional maize. We show that the communication network between farmers' and their perceptions of profit and loss affects landscape scale patterns in pest dynamics. We found that although adoption of Bt maize often brings increased financial returns, these rewards oscillate in response to the prevalence of pests.

The role of CaMKII in diabetic heart dysfunction.

Diabetes mellitus (DM) is an increasing epidemic that places a significant burden on health services worldwide. The incidence of heart failure (HF) is significantly higher in diabetic patients compared to non-diabetic patients. One underlying mechanism proposed for the link between DM and HF is activation of calmodulin-dependent protein kinase (CaMKIIδ). CaMKIIδ mediates ion channel function and Ca(2+) handling during excitation-contraction and excitation-transcription coupling in the myocardium. CaMKIIδ activity is up-regulated in the myocardium of diabetic patients and mouse models of diabetes, where it promotes pathological signaling that includes hypertrophy, fibrosis and apoptosis. Pharmacological inhibition and knockout models of CaMKIIδ have shown some promise of a potential therapeutic benefit of CaMKIIδ inhibition, with protection against cardiac hypertrophy and apoptosis reported. This review will highlight the pathological role of CaMKIIδ in diabetes and discuss CaMKIIδ as a therapeutic target in DM, and also the effects of exercise on CaMKIIδ.

Long-term phenological trends, species accumulation rates, aphid traits and climate: five decades of change in migrating aphids.

Aphids represent a significant challenge to food production. The Rothamsted Insect Survey (RIS) runs a network of 12·2-m suction-traps throughout the year to collect migrating aphids. In 2014, the RIS celebrated its 50th anniversary. This paper marks that achievement with an extensive spatiotemporal analysis and the provision of the first British annotated checklist of aphids since 1964. Our main aim was to elucidate mechanisms that advance aphid phenology under climate change and explain these using life-history traits. We then highlight emerging pests using accumulation patterns. Linear and nonlinear mixed-effect models estimated the average rate of change per annum and effects of climate on annual counts, first and last flights and length of flight season since 1965. Two climate drivers were used: the accumulated day degrees above 16 °C (ADD16) indicated the potential for migration during the aphid season; the North Atlantic Oscillation (NAO) signalled the severity of the winter before migration took place. All 55 species studied had earlier first flight trends at rate of ß = -0·611 ± SE 0·015 days year(-1). Of these species, 49% had earlier last flights, but the average species effect appeared relatively stationary (ß = -0·010 ± SE 0·022 days year(-1)). Most species (85%) showed increasing duration of their flight season (ß = 0·336 ± SE 0·026 days year(-1)), even though only 54% increased their log annual count (ß = 0·002 ± SE <0·001 year(-1)). The ADD16 and NAO were shown to drive patterns in aphid phenology in a spatiotemporal context. Early in the year when the first aphids were migrating, the effect of the winter NAO was highly significant. Further into the year, ADD16 was a strong predictor. Latitude had a near linear effect on first flights, whereas longitude produced a generally less-clear effect on all responses. Aphids that are anholocyclic (permanently parthenogenetic) or are monoecious (non-host-alternating) were advancing their phenology faster than those that were not. Climate drives phenology and traits help explain how this takes place biologically. Phenology and trait ecology are critical to understanding the threat posed by emerging pests such as Myzus persicae nicotianae and Aphis fabae cirsiiacanthoidis, as revealed by the species accumulation analysis.

CaMKIIδ and cardiomyocyte Ca(2+) signalling new perspectives on splice variant targeting.

Control of cardiomyocyte cytosolic Ca(2+) levels is crucial in determining inotropic status and ischemia/reperfusion stress response. Responsive to fluctuations in cellular Ca(2+), Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is a serine/threonine kinase integral to the processes regulating cardiomyocyte Ca(2+) channels/transporters. CaMKII is primarily expressed either in the δB or δC splice variant forms, which may mediate differential influences on cardiomyocyte function and pathological response mechanisms. Increases in myocyte Ca(2+) levels promote the binding of a Ca(2+)/calmodulin complex to CaMKII, to activate the kinase. Activity is also maintained through a series of post-translational modifications within a critical region of the regulatory domain of the protein. Recent data indicate that the post-translational modification status of CaMKIIδB/δC variants may have an important influence on reperfusion outcomes. This study provided the first evidence that the specific type of CaMKII post-translational modification has a role in determining target selectivity of downstream Ca(2+) transporters. The study was also able to demonstrate that the phosphorylated form of CaMKII closely co-localizes with CaMKIIδB in the nuclear/myofilament fraction, contrasting with a co-enrichment of oxidized CaMKII in the membrane fraction with CaMKIIδC . It has also been possible to conclude that a hyper-phosphorylation of CaMKII (Thr287) in reperfused hearts represents a hyper-activation of the CaMKIIδB , which exerts anti-arrhythmic actions through an enhanced capacity to selectively increase sarcoplasmic reticulum Ca(2+) uptake and maintain cytosolic Ca(2+) levels. This suggests that suppression of global CaMKIIδ may not be an efficacious approach to developing optimal pharmacological interventions for the vulnerable heart.

Seasonal migration to high latitudes results in major reproductive benefits in an insect.

Little is known of the population dynamics of long-range insect migrants, and it has been suggested that the annual journeys of billions of nonhardy insects to exploit temperate zones during summer represent a sink from which future generations seldom return (the "Pied Piper" effect). We combine data from entomological radars and ground-based light traps to show that annual migrations are highly adaptive in the noctuid moth Autographa gamma (silver Y), a major agricultural pest. We estimate that 10-240 million immigrants reach the United Kingdom each spring, but that summer breeding results in a fourfold increase in the abundance of the subsequent generation of adults, all of which emigrate southward in the fall. Trajectory simulations show that 80% of emigrants will reach regions suitable for winter breeding in the Mediterranean Basin, for which our population dynamics model predicts a winter carrying capacity only 20% of that of northern Europe during the summer. We conclude not only that poleward insect migrations in spring result in major population increases, but also that the persistence of such species is dependent on summer breeding in high-latitude regions, which requires a fundamental change in our understanding of insect migration.

Aromatase transgenic upregulation modulates basal cardiac performance and the response to ischemic stress in male mice.

Estrogen in females is conventionally considered a cardioprotective influence, but a role for estrogen in male cardioprotection has yet to be defined. Estrogen biosynthesis from testosterone is regulated by aromatase. Aromatase has recently been shown to be expressed in the adult heart, although little is known about its involvement in the regulation of myocardial function and stress responses. The goal of this study was to determine whether upregulation of tissue aromatase expression could improve ischemic resilience in male hearts. Isolated hearts from male transgenic aromatase-overexpressing (AROM(+); high estrogen, low testosterone) mice and wild-type (WT) mice (12 wk) were Langendorff perfused and subjected to ischemia-reperfusion (25 min ischemia and 60 min of reperfusion). Basal systolic function was lower in AROM(+) hearts (dP/dtmax: 4,121 ± 255 vs. 4,992 ± 283 mmHg/s, P < 0.05) and associated with augmented Akt phosphorylation, consistent with a suppressor action of estrogen on contractility. Ischemic contracture was attenuated in AROM(+) hearts (43 ± 3 vs. 55 ± 4 mmHg, P < 0.05), yet AROM(+) hearts were more arrhythmic in early reperfusion. At the end of 60 min of reperfusion, AROM(+) systolic functional recovery was lower (left ventricular developed pressure: 39 ± 6 vs. 56 ± 5 %basal, P < 0.05) and diastolic dysfunction was accentuated (36 ± 4 vs. 24 ± 2 mmHg, P < 0.05). This is the first study to show that in vivo aromatase upregulation modulates basal cardiac performance and the response to ischemic stress. These data suggest that while chronic exposure to enhanced estrogenic influence may have benefits in limiting ischemic contracture severity, acute functional recovery in reperfusion is compromised. A temporally targeted, tissue-specific intervention combining aromatase treatment with inotropic support may offer therapeutic potential for men and women.

Ca(2+) /calmodulin dependent kinase II: a critical mediator in determining reperfusion outcomes in the heart?

Ischaemic heart disease is a major cause of death and disability in the Western world, and a substantial health burden. Cardiomyocyte Ca(2+) overload is known to significantly contribute to contractile dysfunction and myocyte death in ischaemia and reperfusion, and significant advancements have been made in identifying the downstream mediators and cellular origins of this Ca(2+) mismanagement. Ca(2+) /calmodulin-dependent kinase II (CaMKII) is recognized as an important mediator linking pathological changes in subcellular environments to modifications in cardiomyocyte Ca(2+) handling. Activated in response to fluctuations in cellular Ca(2+) and to various post-translational modifications, CaMKII targets numerous Ca(2+) channels/transporters involved in Ca(2+) handling and contractile function regulation. CaMKII is activated early in reperfusion, where it exacerbates Ca(2+) leak from the sarcoplasmic reticulum and promotes the onset of ventricular arrhythmias. Inhibiting CaMKII can increase functional recovery in reperfusion and reduce apoptotic/necrotic death, at least partly through indirect and direct influences on mitochondrial Ca(2+) levels and function. Yet, CaMKII can also have beneficial actions in ischaemia and reperfusion, in part by providing inotropic support for the stunned myocardium and contributing as an intermediate to cardioprotective preconditioning signalling cascades. There is considerable potential in targeting CaMKII as a part of a surgical reperfusion strategy, though further mechanistic understanding of the relationship between CaMKII activation status and the extent of ischaemia/reperfusion injury are required to fully establish an optimal pharmacological approach.

Cardioprotective 3',4'-dihydroxyflavonol attenuation of JNK and p38(MAPK) signalling involves CaMKII inhibition.

DiOHF (3',4'-dihydroxyflavonol) is cardioprotective against I/R (ischaemia/reperfusion) injury. The biological activities of flavonols are associated with kinase modulation to alter cell signalling. We thus investigated the effects of DiOHF on the activation of MAPKs (mitogen-activated protein kinases) that regulate the cardiac stress response. In an ovine model of I/R, JNK (c-Jun N-terminal kinase), p38(MAPK), ERK (extracellular-signal-regulated kinase) and Akt were activated, and NP202, a pro-drug of DiOHF, reduced infarct size and inhibited JNK and p38(MAPK) activation, whereas ERK and Akt phosphorylation were unaltered. Similarly, in cultured myoblasts, DiOHF pre-treatment preserved viability and inhibited activation of JNK and p38(MAPK), but not ERK in response to acute oxidative and chemotoxic stress. Furthermore, DiOHF prevented stress-activation of the direct upstream regulators MKK4/7 (MAPK kinase 4/7) and MKK3/6 respectively. We utilized small-molecule affinity purification and identified CaMKII (Ca(2+)/calmodulin-dependent protein kinase II) as a kinase targeted by DiOHF and demonstrated potent CaMKII inhibition by DiOHF in vitro. Moreover, the specific inhibition of CaMKII with KN-93, but not KN-92, prevented oxidative stress-induced activation of JNK and p38(MAPK). The present study indicates DiOHF inhibition of CaMKII and attenuation of MKK3/6→p38(MAPK) and MKK4/7→JNK signalling as a requirement for the protective effects of DiOHF against stress stimuli and myocardial I/R injury.