Left ventricular noncompaction (LVNC) is a ventricular wall anomaly morphologically characterized by numerous, excessively prominent trabeculations and deep intertrabecular recesses. Accumulating data now suggest that LVNC is a distinct phenotype but must not constitute a pathological phenotype. Some individuals fulfill the morphologic criteria of LVNC and are without clinical manifestations. Most importantly, morphologic criteria for LVNC are insufficient to diagnose patients with an associated cardiomyopathy (CMP). Genetic testing has become relevant to establish a diagnosis associated with CMP, congenital heart disease, neuromuscular disease, inborn error of metabolism, or syndromic disorder. Genetic factors play a more decisive role in children than in adults and severe courses of LVNC tend to occur in childhood. We reviewed the current literature and highlight the difficulties in establishing the correct diagnosis for children with LVNC. Novel insights show that the interplay of genetics, morphology, and function determine the outcome in pediatric LVNC. Cardiomyopathies (CMP) are myocardial diseases of which dilated (DCM) and hypertrophic (HCM) CMP are the most common subtypes. Restrictive (RCM), left ventricular noncompaction (LVNC), and mixed cardiomyopathies occur less frequently. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare disorder . Accordiper se. Regional ventricular hypokinesia was not restricted to the non-compacted segments in the original description [isolated LVNC [isolated” in the original definition by Jenni et al. did not exclude the diagnosis of CMP, but was referring to the absence of CHD. Cardiac magnetic resonance (CMR) imaging was used in the following years to describe ratios between thickness, mass, or volume of NC/C [There are many difficulties with the definition of the phenotype of LVNC. It is beyond the scope and not the intention of this review to define the history of several decades of the different definitions the phenotype of LVNC and we refer to several excellent previous reviews ,8,11. Thcription . Coexistted LVNC . Of note of NC/C ,14. The  of NC/C . Normal  of NC/C . Differe of NC/C . Right vMany different terms and abbreviations used for noncompaction CMP and the noncompacted morphology are used and relate to the possible pathogenesis . In 1990Two developmental processes were described that govern the development of the normal myocardium. The first event is ventricular trabeculation which begins around week 4 of human development, the second is myocardial compaction during weeks 5 to 8 . As demoWhat is the function of trabeculae in the adult heart, if we understand that excessive trabeculations might be detrimental? There is actually evidence that trabeculae are determinants of cardiac performance in adult hearts. The physiological function of trabeculae in the human heart was studied, using the UK Biobank, MRI data with fractal analysis, and modeling. The relationship of trabeculae-associated genomic loci with the risk of cardiovascular disorders was studied . ReducedConsiderable evidence now favors the assignment of a mere phenotype to LVNC and genetic investigations are necessary to assign the possible pathogenicity of the phenotype ,26. TAFAZZIN [How could molecular genetic analysis help to determine the clinical outcome of the individuals affected with LVNC? The first genetic abnormality to cause LVNC without evidence of CHD was mutation in the X-linked gene TAFAZZIN . LinkageTAFAZZIN ,27. CandTAFAZZIN . In the TAFAZZIN ,30,31. CTAFAZZIN . In addition to genes coding for sarcomeric proteins, there are those coding for components of the nuclear envelope and Z-band, ion channels, sarcolemma proteins, and genes involved in signaling pathways or gene transcription. These genes associated with LVNC overlap with the genes causing CMP, therefore genetics suggest that LVNC-CMP may also be called HCM with LVNC or DCM with LVNC .MYH7 was reported first and most frequently in adults and in children with LVNC. ACTC1, TTNT2, and TPM1 were also described as LVNC genes [TTN became feasible [MYBPC3 was frequent [MYH7 [MYH7 and ACTC1 mutations had a lower risk for MACE than MYBPC3 and TTN [From the sarcomere genes, NC genes . Later sfeasible . In indifrequent ,33,34. Tfrequent . The mecfrequent . Single nt [MYH7 . MYH7 an and TTN .The spectrum of genetic variants identified in LVNC patients is heterogeneous because of the differences in enrolment criteria and site of investigation. The description of LVNC was first derived from a cohort of patients from tertiary centers with an obvious CMP. This would explain the relatively high yield in genetic testing in this preselected population ,37. The PRDM16 deletion or mutation, respectively [Especially in children, LVNC is often first detected as part of the clinical investigation into an unclear genetic syndrome, muscular dystrophy, or mitochondrial myopathy . Geneticectively . MYH7, ACTN2, and PRDM16). Non-truncating (transmembrane) variants in HCN4 and structural variants in RYR2, both variant classes in genes involved in arrhythmia phenotypes were specific for LVNC. LVNC-specific variants probably explain 5–10% of cases. Significant gene-disease associations were detected, but no significant excess of rare variation was observed for the majority of the analyzed genes. These findings were very similar to previous findings for HCM and DCM [https://gnomad.broadinstitute.org/; accessed on 18 April 2022) was observed for the variants in many of the sarcomere genes first reported to be associated with LVNC [MYH7 (TV) with an anticipated mechanism of haploinsufficiency have generally been considered to be non-pathogenic in DCM or HCM, but was proposed as a pathomechanism in LVNC. The most frequent MYH7 (TV) variant in LVNC is the splice site variant c.732+1G>A which does not seem to be a founder variant [https://clinicalgenome.org/; accessed on 18 April 2022) LVNC is not curated as a specific disease entity and it awaits further consideration if or how these specific LVNC variant classes will be assigned.In fact, to date more than hundred genes were described to be associated with LVNC ,20. From and DCM ,41. Inteith LVNC ,34. MYH7 variant . It is iPRDM16, MIB1, TBX20, NKX2-5, and TAFAZZIN. One of the CMP genes found by Mazzarotto et al. to be causative for LVNC only, and curated with limited evidence in ClinGen for DCM  has recently been investigated in more detail, namely PRDM16. Both, contractile dysfunction and partial uncoupling of cardiomyocytes were the result of modelling of PRDM16 haploinsufficiency and a human truncation mutant in zebrafish [PRDM16 in human iPSC-derived cardiomyocytes (iPSC-CMs) recapitulated the proliferative defects associated with LVNC at the single-cell level [Prdm16 knock out models recapitulated the LVNC phenotype with LV-specific dilatation and dysfunction [Several animal models were created to discover the underlying mechanisms of LVNC . Unfortuebrafish . There webrafish . Genome ll level . Varyingll level . Two murll level ,45. In oll level . Geneticll level . Impairell level . In a mofunction . In summMkl2), Myh7, and the transcription factor NK2 homeobox 5 (Nkx2-5) underlying familial LVNC. These triple compound heterozygous mice recapitulate signs of human LVNC [Murine models were also used to test the hypothesis that LVNC may result from a combination of genetic variants. Evidence for oligogenic inheritance of LVNC was shown for a combination of three inherited heterozygous missense variants in the MKL/myocardin-like protein 2 -Echocardiography with functional assessment of LV size and function-Holter-ECG (24 h)-Cardiopulmonary exercise testing (CET)-Cardiac magnetic resonance (CMR) imaging-Genetic testingFamily history should include a three-generation pedigree. ECG changes are usually unspecific and arrhythmias detected by Holter-ECG may include ventricular ectopic beats and sustained or non-sustained ventricular tachycardia. Echocardiography data show LV dilatation and/or systolic/diastolic dysfunction. An early marker of systolic impairment is a reduced global longitudinal strain (GLS). CET is a measure for cardiovascular performance and possible arrhythmias. CMR imaging is used to characterize LV size and function and markers such as late gadolinium enhancement (LGE) are able to provide noninvasive tissue characterization and measure of fibrosis. Genetic testing should only be performed, if a CMP is diagnosed clinically.With respect to subclinical CMP which can be detected by the newer imaging technologies there is still some uncertainty and no general agreement but the following is suggested. If strain is abnormal with normal LV-EF, the criteria for a CMP are not fulfilled according to the current AHA/ESC guidelines. These patients do not have CMP. LVNC with normal LVEF and without any other cardiovascular abnormalities, isolated LVNC, should be named LVET . SubclinBoth imaging modalities, echocardiography and CMR imaging, are essential to determine the morphology . The echSeveral studies have proposed CMR criteria to improve the diagnostic sensitivity and specificity . Most ofLV dilatation, systolic dysfunction, and late gadolinium enhancement (LGE) were independent predictors of poor outcome . InteresMyocardial extracellular volume (ECV) reflects diffuse myocardial fibrosis with CMR T1 mapping. Zhou et al. found elevated native T1 values in LVNC patients compared to normal controls . LGE negSpeckle-tracking echocardiography (STE) can detect subclinical changes in LV function, and may complement the echocardiographic assessment of LVNC patients in clinical routine. Although altered LV deformation in LVNC phenotype by STE does not enable the diagnosis of a definite CMP it can be helpful in clinical assessment. Particularly circumferential parameters are impaired in LVNC and can help to predict cardiovascular outcomes of LVNC . LV trabeculation was found to be correlated with reduced myocardial deformation indexes, even if left ventricular ejection fraction (LV-EF) was normal . ChildreWith regard to genetic testing the American Heart Association, the Heart Failure Society of America, and the American College of Medical Genetics and Genomics propose guidelines following a common line ,72,73: “Because existing diagnostic criteria lack specificity prognosis and outcome are difficult to determine, LVNC includes both, physiological conditions or adjustments and pathological disease . In cohoData on outcome of pediatric LVNC are scarce and therefore studies of adult patients are often taken into consideration. Overall survival was reduced in patients with LVNC compared with the expected survival of age- and sex-matched US population . In thisGenetic factors play a more important role in children than in adults with LVNC and severe forms of LVNC tend to be diagnosed in childhood ,38. LVNCMYBPC3, and TTN were associated with MACE in multivariate analysis [A systematic review by van Waning et al. in a large cohort of genetically confirmed 561 LVNC patients found worse clinical outcome in children compared to adults . The stuanalysis . The sevanalysis .Schultze-Berndt et al. detected a higher percentage of asymptomatic children than asymptomatic adult LVNC patients in their study cohort with primary CMP . ReducedIn summary, in pediatric LVNC lower BSA and younger age are considered risk factors for MACE ,39. CongWith respect to direct genotype-phenotype correlations in LVNC, several studies produced conflicting results. In a retrospective study of LVNC detected ≤21 years of age, it was less likely to find the genetic cause in isolated LVNC than in LVNC-CMP  . IsolateTAFAZZIN, RBM20, Lamin A/C, TTN-truncating variants and non-sarcomere genes are associated with worse outcome in LVNC [TAFAZZIN and MYH7 pathogenic variants were the most common. Patients with pathogenic variants showed poorer prognosis, especially those with multiple variants [MYH7, TTN, and MYBPC3 were most prevalent in the study, very comparable to other recent reports in adult and pediatric populations [Variants in specific genes such as in  in LVNC ,35,82,83 in LVNC . Sarcomevariants . In the ulations ,38. Riskulations . Van WanThe impact of LVNC subtypes was systematically reviewed in a study of Van Waning et al. . Adult aMYH7, LV systolic dysfunction, increased risk for MACE, and DCM without LVNC in relatives. Mutation in the head of MYH7, asymptomatic or mild disease, and isolated LVNC in relatives was associated with isolated LVNC. LVNC-HCM was associated with mutation of MYBPC3 and with HCM without LVNC in relatives [Genetic screening to identify family members at risk for developing CMP is useful and named “cascade screening” . From inelatives . These iA recent study addressed the role of ion channel gene variants and correlation with arrhythmias in a large cohort of pediatric LVNC . The LVNMYH7 mutation was found in Ebstein anomaly associated with LVNC [Patients with CHD and LVNC primarily present to the pediatric cardiologist and there are two possible etiologies. Pathologic remodeling induced by pressure or volume overload in CHD may lead to LVNC. Alternatively, genetic alteration may drive LVNC in CHD. In LVNC families, some relatives of affected members may have CHD or CMP with or without LVNC . MYH7 muith LVNC . In LVNCith LVNC . Van Wanith LVNC .LVET) seem to have the same outcome as the healthy population. The development of better imaging technologies, specifically CMR imaging, analyzes LVNC changes more easily and might lead to better diagnosis, if used with novel imaging biomarkers. We need larger systematic population-based studies and follow-up to identify individuals at risk for developing a CMP later in life specifically in the pediatric population.LVNC is a phenotype that awaits classification and can be found in a spectrum from physiologic state to mild or severe disease. Adult individuals with isolated LVNC (


However, the relative contribution of GABA and glycine to PKCγ+ interneuron inhibition remains unknown. We characterized inhibitory inputs onto PKCγ+ interneurons by combining electrophysiology to record spontaneous and miniature IPSCs  and immunohistochemical detection of GABAARα2 and GlyRα1 subunits in adult rat MDH. While GlyR-only- and GABAAR-only-mediated mIPSCs/sIPSCs are predominantly recorded from PKCγ+ interneurons, immunohistochemistry reveals that ~80% of their inhibitory synapses possess both GABAARα2 and GlyRα1. Moreover, nearly all inhibitory boutons at gephyrin-expressing synapses on these cells contain glutamate decarboxylase and are therefore GABAergic, with around half possessing the neuronal glycine transporter (GlyT2) and therefore being glycinergic. Thus, while GABA and glycine are presumably co-released and GABAARs and GlyRs are present at most inhibitory synapses on PKCγ+ interneurons, these interneurons exhibit almost exclusively GABAAR-only and GlyR-only quantal postsynaptic inhibitory currents, suggesting a pharmacological specialization of their inhibitory synapses.Mechanical allodynia  is a widespread symptom of inflammatory and neuropathic pain. Spinal or medullary dorsal horn (SDH or MDH) circuits mediating tactile sensation and pain need to interact in order to evoke mechanical allodynia. PKCγ-expressing (PKCγ W. W+ intei PKCγ+ interneurons in the adult rat MDH. According to decay time courses, three distinct populations of sIPSCs or mIPSCs can be recorded from these lamina IIi PKCγ+ interneurons: fast monoexponential, slow monoexponential, and biexponential sIPSCs or mIPSCs. All three types of sIPSCs or mIPSCs are present in all recorded PKCγ+ with rather similar frequencies. Moreover, the same quantal events are recorded from neighboring lamina IIi PKCγ− interneurons, suggesting that lamina IIi PKCγ+ and PKCγ− interneurons display similar types of inhibitory inputs. The sensitivity of fast and slow quantal events to GABAAR and GlyR antagonists suggests that monoexponential sIPSCs or mIPSCs are mediated by GlyR and GABAAR, respectively. Altogether, our electrophysiological results suggest that inhibitory inputs onto lamina IIi PKCγ+ or PKCγ− interneurons in the adult rat MDH are predominantly (at least 70%) mediated by either GlyR or GABAAR but not both. On the other hand, immunohistochemistry reveals that the vast majority (~80%) of inhibitory synapses onto PKCγ+ interneurons contain both GABAARs and GlyRs. We also show that the great majority (~90%) of inhibitory boutons presynaptic to the PKCγ+ cells possess GAD and can therefore presumably release GABA, while just under half of these also express GlyT2 and are therefore likely to co-release glycine.We performed electrophysiology and immunochemistry to characterize synaptic inhibition onto lamina IIi PKCγ+ interneurons in the adult rat MDH. Fast monoexponential sIPSCs or mIPSCs are mediated by GlyRs. Their decay τ is similar to that of bicuculline-resistant, strychnine-sensitive IPSCs (present results) and within the range of those of previously recorded GlyR-mediated IPSCs from SDH/MDH lamina I–II neurons [ARs mediated the slow monoexponential sIPSCs or mIPSCs as long-duration IPSCs were all suppressed by bath-applied bicuculline (present results). Moreover, their decay τ is within the range of those of previously recorded GABAAR-mediated IPSCs from SDH/MDH lamina I–II neurons [Two populations of monoexponential sIPSCs or mIPSCs  were recorded from lamina II neurons ,34,35,36 neurons ,34,35,36AR-mediated within the MDH [AR/GlyR IPSCs  in the SDH [i PKCγ+ interneurons at post-natal day (P) 21–35 are unlikely to be mixed GABAAR/GlyR IPSCs since these very IPSCs, in SDH lamina II neurons, are only present during early development and become virtually undetectable by approximately P23 [AR-mediated. First, similar biexponential GABAAR-mediated mIPSCs were already recorded in this very area in juvenile to adult rats [AR-mediated mIPSCs, biexponential mIPSCs exhibit larger amplitudes and longer decay τ of the slow component (here and in the work of [AR-mediated mIPSCs occur at a higher frequency than GlyR-mediated mIPSCs in MDH lamina II neurons [+ interneurons in the presence of bicuculline (~24%) is in the same range as that of kinetically isolated fast monoexponential IPSCs (~34%). Indeed, if our biexponential sIPSCs were mixed GABAAR/GlyR IPSCs, the proportion of GlyR-only sIPSCs in the presence of bicuculline should have been double; that is, the sum of the proportions of kinetically isolated fast monoexponential IPSCs and biexponential IPSCs. Therefore, our electrophysiological data strongly suggest that all quantal inhibition onto lamina IIi PKCγ+ interneurons in adult rat MDH is mediated by either GlyR or GABAAR but not by both types of receptors concurrently.Two types of biexponential-evoked IPSCs and mIPSCs were previously recorded from lamina II neurons: pure GABA the MDH  and mixe the SDH ,36. The tely P23 . They arult rats . Second, work of ). Finall neurons . The proi PKCγ− neurons as the same quantal events were recorded from PKCγ+ and PKCγ− interneurons. Therefore, whereas these two classes of lamina IIi interneurons exhibit differences in their membrane properties and excitatory inputs [This conclusion holds true for lamina IIy inputs ,39, theyAR, but not by both types of receptors concurrently. However, whereas GABAAR-only IPSCs in the SDH are exclusively monoexponentially decaying, those in the MDH can be mono- as well as biexponentially decaying . It is y cortex . Such fa subunit . Thus, wAR and GlyR are colocalized at the great majority (~80%) of inhibitory synapses on PKCγ+ cells and that nearly all (91%) of these cells have synapses at which the receptors are co-expressed. Nevertheless, some PKCγ+ interneurons (9%) also displayed synapses with only GABAARs or GlyRs, which may reflect the existence of different subpopulations of PKCγ+ interneurons [The present results establish that the subunits belonging to GABArneurons ,39.ARα2-IR. The GABAARα2 is also expressed by primary afferent axons, and so some of the immunostaining for this receptor might have been associated with primary afferent terminals. However, this is highly unlikely to account for any of the GABAARα2 puncta that we identified on PKCγ neurons because the vast majority of these puncta (98%) also contained the GlyRα1 subunit, which is not expressed by primary afferents [However, we have to consider certain technical issues. We have previously demonstrated with confocal microscopy that virtually all GlyRα1-IR puncta in the rat spinal dorsal horn are also immunoreactive for gephyrin . We havefferents ,57,58.AR or GlyR, so they may have had low levels of the other receptor that were below the detection threshold. Another possibility is that a synapse may only express other subunits for the receptor. We assessed the expression of GABAARα2 and GlyRα1 on lamina IIi PKCγ+ interneurons because these subunits are known to be abundant in SDH [ARα2 subunit, it is quite possible that they express another GABAA α subunit. It is thought that individual GABAA receptors may contain two different α subunits [AR and GlyR.Negative findings with immunohistochemistry  could result from low levels of the receptor (rather than its absence) at a synapse. Indeed, “single-labeled” puncta were often quite weakly stained for either GABAt in SDH ,60,61. Ft in SDH . Howeversubunits , and it + interneurons stained for GAD, while around 40% of them co-expressed GlyT2.Previous immunohistochemical studies showed that 30–50% of superficial SDH neurons are GABAergic, and about half of these are also enriched with glycine ,64,65. T+ cells.Detection of synaptic receptors required pepsin treatment, which presumably exposes epitopes that are normally masked by fixation ,66. ThisARα2 and GlyRα1 subunits and that >90% of presynaptic boutons were GAD+, whereas only around half were GlyT2+. Thus, many inhibitory synapses onto PKCγ+ interneurons may have both types of receptors but only involve GABA release. The few synapses that only express GABAARs should also operate as purely GABAergic. Similarly, inhibitory synapses that have only GlyRs should function as purely glycinergic.We can make certain predictions concerning the arrangement of neurotransmitters and receptors based on our anatomical finding that ~80% of inhibitory synapses had both GABA+ interneurons, GABA and glycine are co-released, and GlyR and GABAAR are both present on the postsynaptic membrane. One would therefore expect mixed GABAAR/GlyR-mediated mIPSCs to be recorded. However, our electrophysiological data show that, in normal conditions, lamina IIi PKCγ+ interneurons in adult rat MDH are exclusively bombarded by GABAAR-only- and GlyR-only-mediated IPSCs, similar to lamina II neurons in young rat SDH [i PKCγ+ interneurons in 6–8-week-old mice, Wang et al. [AR-GlyR-mediated IPSCs in the other five. However, in this report, dorsal roots were electrically stimulated, and therefore, many Aβ fibers were activated simultaneously: the few mixed GABAAR-GlyR-mediated IPSCs might thus result from summated Aβ fiber-evoked pure GABAAR- and GlyR-mediated IPSCs. Altogether, this suggests that some synapses can function as purely GABAergic or glycinergic even though presynaptic buttons co-release GABA/glycine and postsynaptic membranes contain both GABAA and glycine receptors. One possibility is that GABAAR and GlyR are differentially located within inhibitory synapses. Indeed, bath application of a benzodiazepine [AR-mediated component and the appearance of mixed GABAAR/GlyR mIPSCs in adult SDH lamina II neurons. This might stem from a different localization of GABAAR and GlyR in inhibitory synapses onto SDH neurons, with GABAARs being extrasynaptically distributed [ARα2 and GlyRα1 subunits at these synapses, one or another type of receptor is not functional, perhaps related to the differential expression of other subunits.However, GlyR-only inhibitory synapses, as revealed by immunohistochemistry, are unlikely to account for the 34% of GlyR-only-mediated sIPSCs or mIPSCs, even though such synapses would display high release probabilities. Moreover, our anatomical data suggest that, in numerous inhibitory synapses onto PKCγ rat SDH ,34,35,36g et al.  observediazepine  or inflaiazepine  leads totributed . HoweverAR-only- and GlyR-only-mediated sIPSCs or mIPSCs recorded from PKCγ+ interneurons suggests separate roles for these two inhibitory systems. There are two clinical forms of mechanical allodynia: dynamic and static. Whereas both types are impacted by modulating PKCγ activity [Aergic disinhibition, a static one [+ interneurons might be gated by specifically glycinergic and GABAAergic feedforward inhibitory microcircuits, respectively . There e injury ,26. It iAR and GlyR are colocalized at most inhibitory synapses on these cells and that GABA and glycine are often co-released, PKCγ+ interneurons exclusively displayed GABAAR-only- and GlyR-only-mediated spontaneous IPSCs. Behavioral evidence suggests that this specialization of inhibitory inputs onto PKCγ+ interneurons is functionally relevant.Although our anatomical data suggest that GABAData represent F-values for one or two-way ANOVA with corresponding degrees of freedom  and P-values. Factors for ANOVA were designed as follows: Cell type, for comparisons between PKCγ+ and PKCγ–; IPSC type for comparisons between fast monoexponential, slow monoexponential, and biexponential mIPSC; and Interaction, for effect between two factors on the dependent variable.


Notable changes in the wintertime Arctic atmospheric circulation have occurred over the last few decades. Despite its importance in understanding the recent changes in the Northern Hemisphere midlatitude climate, it remains unclear whether and how these changes are affected by recent Arctic sea ice loss. In this study, a regional scale model is used to separate the direct sea ice influence from the natural variability of large-scale atmospheric circulation. Results show that, in response to sea ice loss, the increase of geopotential height in the mid-to-upper troposphere is robust across the simulations, but the magnitude of the response is highly dependent on the background state of the atmosphere. In most cases the sea ice loss-induced atmospheric warming is trapped near the surface due to the high vertical stability of winter Arctic lower troposphere, accordingly, resulting in a small response of geopotential height. However, when a low-pressure system is located over the Barents Sea, the relatively weak stability allows an upward transport of the surface warming, causing a significantly larger geopotential height increase. This strong state-dependence of atmospheric response which is also found in recent studies using global-scale model experiments, highlights the importance of accurately representing the atmospheric background state for numerical model assessments of sea ice influence. For the winter season, the warming trend was relatively weak until the late 1990s, but it has become rapidly accelerated since then4. Meanwhile, the winter Arctic atmosphere has shown a notable increase in atmospheric pressure and associated anticyclonic circulation anomalies5. The change is particularly large and statistically significant over the Barents Sea region where winter sea ice has exhibited the strongest declining trend6. However, it is unclear whether the atmospheric circulation changes over the Barents Sea are a response to the underlying sea ice change or not8.Observations have revealed an ongoing rapid surface warming and sea ice loss over the Arctic region10, and also, an anomalous high-pressure near the Barents–Kara Sea is known to accelerate sea ice melting by increased horizontal heat and moisture transports into the Atlantic sector of the Arctic13. Because of the two-way interaction, the role of sea ice loss in the atmospheric circulation changes is difficult to identify in observational data. Moreover, not only Arctic sea ice, various factors that could affect the Arctic atmospheric circulation, such as ocean circulation and global-scale atmospheric circulation, also have experienced significant changes during the recent decades16.This is difficult to address because there exists positive feedback between the two processes: Arctic atmospheric circulation changes and Arctic sea ice loss. That is, these two processes can be induced by each other. For instance, excessive surface heat release due to sea ice loss results in a local increase of atmospheric pressure17, but whether it is caused by the Arctic sea ice reduction is still a subject of debate21. Recent studies have argued the statistical significance of the increasing winter extreme events, questioning the role of sea ice loss and examining the effects of internal variability and the amplified Arctic surface response to anthropogenic global warming24.This is an important issue in understanding the recent cooling trends of land surface temperature and the increased occurrence of extreme cold events in the Northern Hemisphere winter. It has been suggested that the Arctic warming from sea ice loss can elicit a large scale atmospheric circulation anomaly  that cools the midlatitude surface, forming the so-called “warm Arctic-cold continents pattern”26. Recent studies under the Polar Amplification Model Intercomparison Project (PAMIP) protocol27 have also reported that uncertainties could be attributed to other factors such as internal variability of the stratospheric polar vortex28, the ice-constraining method29, and an insufficient number of ensembles30. Also, note that the sea ice loss impact can be sensitive to experimental design because the additional turbulent heat flux from sea ice loss is known to vary with changing atmospheric conditions31. In Earth system model simulations, responses to the Arctic sea ice forcing can also develop outside the Arctic region. The tropics and midlatitudes responses eventually interact with the Arctic atmosphere, hence the interaction may blur signals of the direct response of the Arctic atmosphere to sea ice forcing. This is because the interaction with lower latitudes is dependent on the mean climate state which differs greatly from model to model. Consequently, in Earth system models, various factors can contaminate sea ice-forced signals, leading to large inter-model differences.Numerical models can be used to extract the contribution of recent sea ice changes by comparing two identical simulations but with different sea ice states. However, it has been pointed out that the uncertainty in Arctic climate modeling is high with Earth system models having different representations of dynamical and physical processes33 is used in this study to assess the direct influence of the wintertime Arctic sea ice loss on the Arctic atmospheric circulation. The main advantage of employing a regional model is that we can use a short model integration time (48 h in this study) so that the sea ice-forced signals in the atmosphere can be distinct before they are mixed with the interaction with large-scale atmospheric disturbances. This is difficult for a global-scale model simulation which requires a longer initial spin-up. Note that in some studies global-scale models have been utilized to examine the sea ice influence on a relative short-term scale (< 1 month)35, however, regional-scale models have rarely been used to conduct a sensitivity experiment with modified sea ice fields. Previous Polar WRF studies whose simulation setup is similar to that of this study have demonstrated that the model can successfully reproduce the winter Arctic climate37. Also, unlike global-scale models, regional simulations require lateral boundary conditions which are updated every 6 h in this study providing additional constraints to simulations. Therefore, interactions with lower latitudes can be controlled by observational information. Furthermore, the influence of initial conditions helps keep the simulated climatology more realistic which will prove to be important in this article.As an alternative, a regional-scale atmospheric model (Polar WRF)In the current study, we conducted two sets of simulations in which the simulation setups are identical to each other except for the mean sea ice concentration (SIC). The SIC difference at each grid point is based on observed mean SIC differences between two periods, 1979/80–1988/89 and 2006/07–2015/16. From the difference between the two sets of simulations, we expect that changes that are attributed only to the climatologically reduced sea ice can be obtained. Each simulation set consists of 903 realizations; each realization is a 48-h simulation initialized at 00 UTC every day during 10 winter seasons (December–February from 2006/2007 to 2015/2016). Note that the two simulation sets use the same sea ice albedo and thickness data which are temporally and spatially varying estimates from satellite observations. By generating the large ensemble of realizations this way, the simulated ensemble average can be representative of the winter Arctic climate, and the signal-to-noise ratio of atmospheric response to sea ice loss can be increased which is known to be low due to large internal variability.Figure Our analysis reveals that the magnitude of the atmospheric response is well explained by the geopotential height of the mid- to upper-troposphere. Accordingly, based on the simulated 500 hPa geopotential height over the Barents Sea region 70–80°N, 15–75°E), high-pressure and low-pressure cases are defined as the highest and lowest 5% of all the 903 realizations. For the high-pressure cases  heating. The positive buoyancy at the low-level troposphere produced by surface heating can elevate the geopotential heights of the atmosphere above the heating level. Accordingly, near and below that level, negative geopotential height anomalies are induced to compensate for the upper-level changes. This “anticyclonic above-cyclonic below” response has been rarely found in monthly or seasonally averaged data. This is because while the initial immediate atmospheric responses have a baroclinic structure, the synoptic eddy feedback tends to induce a barotropic anomaly structure by a potential eddy-vorticity fluxThe dependency on the atmospheric background state is also shown in Fig. 38, along with the decreased pressure response at the heating level  is only larger by 36% than that for the high-pressure cases (7.8 W m−2), which cannot fully account for the stronger response of low-pressure systems. Interestingly, ∆z500 is remarkably well explained by low-level atmospheric heatings, showing r = 0.76 with 850 hPa atmospheric temperature responses  simulations from 2006/2007 to 2015/2016 initialized every day at 00 UTC, consisting of 903 realizations. The sensitivity simulation set, hereinafter “High sea ice concentration (SIC)” run, is identical to the “control” run but with increased SICs. For each grid point, the mean winter SIC difference between two periods (1979/80–1988/89 minus 2006/07–2015/16) based on the European Centre for Medium-Range Weather Forecasts (ECMWF) next-generation reanalysis ERA546, was added if the difference is positive, and a SIC value was set so that it does not exceed 100%. Here the “sea ice increase-type” runs were used for sensitivity simulations, therefore, we can avoid any assumptions on surface temperature fields over the newly opened sea that would be needed for a “sea ice reduction-type” experiment. The responses in the atmosphere are obtained by extracting a “High SIC” simulation field from the corresponding control simulation field.The polar‐optimized version of the Weather Research and Forecasting model (Polar WRF) version 4.1.1The standard model simulation is a 48 h run which is in a conventional range for a mesoscale simulation. It is long enough for allowing model spin-up of the hydrologic cycle and the boundary layer processes and short enough for the simulated climate to benefit from the initial conditions. Simulations were extended to 288 h (12 days) only when there is a strong high-pressure or low-pressure anomaly over the Barents Sea region. The high-pressure and low-pressure cases are defined as the top and bottom 5% of the 903 realizations based on 500 hPa geopotential height over 70–80°N, 15–75°E region at 24–48 h of model integration time.47. The sea ice albedo, sea ice thickness, and snow depth over sea ice are from the Arctic System Reanalysis version 2 in which satellite retrievals of the quantities are assimilated48.Besides SIC, the “control” and “High SIC” runs use the same model configurations and background data. The simulations were run for a domain of 300 × 300 grids on a polar stereographic projection with a 24-km horizontal resolution, which encompasses the entire Arctic region north of 57°N. 70 vertical levels were defined on a terrain‐following sigma coordinate with 10 hPa top-level, corresponding to a resolution about 50 m near the surface and 440 m near the tropopause. The initial and boundary conditions for the simulations were taken from the 6 hourly atmospheric fields of the National Center for Environmental Prediction (NCEP) Final Operational Global Analysis dataSupplementary Figures.


Under-ice eddies are prevalent in the major circulation system in the western Arctic Ocean, the Beaufort Gyre. Theoretical studies hypothesize that the eddy-driven overturning and the ice-ocean drag are crucial mechanisms of the gyre equilibration in response to atmospheric winds. However, due to severe weather conditions and limitations of remote sensing instruments, there are only sparse eddy observations in the ice-covered Arctic Ocean. Hence, the evolution of the under-ice eddy field, its impact on the gyre variability, and their mutual response to the ongoing Arctic warming remain uncertain. Here, we infer the characteristics of the under-ice eddy field by establishing its tight connection to the angular velocities of isolated spinning sea ice floes in marginal ice zones. Using over two decades of satellite observations of marginal ice zones in the western Arctic Ocean, we identified and tracked thousands of floes and used idealized eddy modeling to infer the interannual evolution of the eddy energetics underneath the ice. We find that the eddy field is strongly correlated to the strength of the Beaufort Gyre on interannual timescales, which provides the major observational evidence consistent with the hypothesis of the gyre equilibration by eddies. The inferred trends over the past two decades signify that the gyre and its eddy field have been intensifying as the sea ice cover has been declining. Our results imply that with continuing sea ice decline, the eddy field and the Beaufort Gyre will keep intensifying and leading to enhanced transport of freshwater and biogeochemical tracers. On the other hand, the sea ice cover can act as the dominant gyre equilibration mechanism by slowing down the currents via ice-ocean drag, leaving a minor role for eddies5. While the gyre state is likely somewhere in between these two limiting cases, it is expected that as the sea ice continues to retreat in the Arctic Ocean, the gyre will shift towards the eddy-governed regime5. Indeed, bulk estimates of the BG energy budget suggest that since 2008 eddies have been considerably draining potential energy and eventually dissipating it as kinetic energy at the ice-ocean interface6. However, the presence of sea ice in the Arctic Ocean imposes significant limitations on in situ field campaigns and hinders the acquisition of satellite eddy measurements common in temperate oceans. Without comprehensive eddy observations, projections of the BG transition to a climate with a dramatically reduced sea ice cover remain uncertain.The mechanisms governing the variability of the basin-scale anticyclonic circulation in the western Arctic Ocean—the Beaufort Gyre (BG) Fig. a—have be8 due to repeating wind bursts and surface waves. As a result, vast BG areas are covered by ice floes behaving like brittle solid bodies floating over a turbulent ocean. The floes range dramatically in size, forming a power-law distribution that evolves seasonally and regionally between marginal ice zones and the pack ice11. Because of the overall thermodynamic melting that opens up ice-free ocean areas and the frequent atmospheric storms that pull the floes apart from each other, many floes at the edge of the BG marginal ice zone are weakly- or non-interacting. The BG region is unique since its broken-up floes are vast, Here, we infer the BG eddy characteristics by taking advantage of the unique seasonality of its sea ice field. While the BG ice pack remains consolidated for most of the year, it naturally breaks up in late spring and summer14. This component is dominant given the generally strong winds and low interactions between the floes in the BG marginal ice zone. Nonetheless, here we emphasize that the translation of isolated floes is accompanied by a considerable spin  of the BG marginal ice zone to track individual floes and retrieve their spinning rates  model . The simulated translational and spinning motion of floes is qualitatively consistent with the observations  and drifted to the BG interior as has been previously hypothesized30.By tuning the two-layer QG model parameters, we found the eddy field characteristics that lead to the best fit between the observed and simulated rotational statistics of floes, specifically matching the rotation rate variances across all floe length scales with an average error of 19% . Such large BG eddies have been recently identified in remote sensing imagery from their signatures on the sea ice distribution in marginal ice zones24 as well as from open-ocean altimetry25.Our novel method resulted in eddy field characteristics that are in many ways consistent with observations from various measurement platforms as well as with large-scale BG energy constraints. The surface eddies are abundant, ranging from 10 to 60 km in size with characteristic Rossby numbers of .1) Fig. a. While 6. During the 2008–2014 period, the rate of kinetic energy input to geostrophic BG currents was about 0.1 mW m6. The missing energy dissipation rate needed to close the BG energy budget in the 2007–2014 period was about 0.1-0.2 mW m6. Another way of gauging the importance of eddies is to compare the gyre equilibration timescale with the time eddies would take to drain the excess APE gained during the gyre spinup. The APE variation over the 2003–2014 period was estimated at 40 PJ6. At a rate of 0.7 mW m32, highlighting the major role of eddies in BG energy cycle and equilibration.The inclusion of the quadratic ice-ocean drag played a crucial role in finding the optimal eddy field. Consequently, in contrast with ice-free oceans, the dominant mechanism of eddy energy dissipation in ice-covered oceans is at the surface, not at the bottom. Our best-fit model allows to estimate that the dissipation of eddy kinetic energy (EKE) by the sea ice-ocean drag occurs at a rate of about 0.7 mW min situ observations from moorings A,C, and D that are away from bathymetric features . The surface and subsurface EKE in the QG model are both linearly proportional to the variance of the simulated floe rotation rates  matches well with the BG strength index and the magnitude of the regional currents; correlations between pairs of indices range from 0.83 to 0.9. The low energy years for the eddy field were 2005–2006 and 2013, while 2008–2010 and 2016–2018 were the high energy years. The tightly correlated interannual variability of the eddy field and the BG strength supports the eddy equilibration hypothesis. However, recent moderate-resolution numerical simulations of the wind-driven BG spinup suggest that its eddy field does not significantly change in response to the spinup of the gyre34, which contradicts our observations. This incongruence implies that even comprehensive general circulation models do not adequately simulate eddy formation processes in the BG, significantly underestimating EKE likely due to inaccurate representation of the stratification and vertical shear of the large-scale currents.The reconstruction of floe rotation rates over the 2003–2020 period allows us to identify the relation between the strength of eddies and large-scale BG currents and assess their interannual and decadal trends Fig. . In compThe presence of sea ice and its associated ice-ocean drag is crucial in constraining the evolution of the large-scale BG currents and its eddy field. On interannual timescales, the gyre indices and the floe rotation follow opposite trends with the sea ice concentration Fig. . Broad tThis study presents a unique method of inferring the under-ice eddy variability in the western Arctic Ocean by analyzing the motions of relatively isolated sea ice floes in marginal ice zones. Specifically, using theoretical arguments and idealized modeling, we deduced that the scale-dependency of the floe rotation rate is directly related to the characteristics of the underlying oceanic eddy field. Piecing together almost two decades of satellite floe observations with a physical model of eddy dynamics, we inferred the best-fit characteristics of the ocean eddy field, including eddy lengthscales, EKE, and dissipation rates.in situ observations of subsurface EKE at the four BG mooring locations. Since our estimated subsurface EKE levels agreed well with mooring observations, it is reasonable to conclude that the surface eddies driving floe rotation extend into the halocline layer, i.e., these are not predominantly mixed layer eddies. Note, marginal ice zones are known to have strong density gradients that support surface eddy formation via frontal instabilities36. However, most of our floe observations are taken shortly after the consolidated pack ice breaks up into smaller floes, and hence surface density gradients have not been fully developed. Since the eddy field requires some time to adjust to the relatively ice-free conditions after the sea ice breaks up in spring/summer, our estimated eddy characteristics are likely more attributable to winter-like eddy fields formed under consolidated sea ice. Highly concentrated winter sea ice can significantly suppress mixed layer instabilities and surface eddies38. Thus, the expected lack of mixed layer eddies implies that floes are rotated predominantly by eddies that have origins in the interior of the ocean, not at the surface. This rationalizes the good fit between the observed subsurface EKE and that estimated from floe rotation that depends on surface eddies.Our method was validated with independent in situ eddy observations because their spatial or temporal coverage is very sparse given the heterogeneity and interannual variability of the eddy field. We note that existing mesoscale and submesoscale eddy parameterizations were developed and calibrated based on ice-free theory and modeling40. They do not account for additional EKE dissipation at the ice-ocean interface and hence likely overestimate eddy diffusivities and associated overturning in ice-covered regions. As global warming drives the Arctic Ocean towards a seasonally ice-covered state, developing improved eddy parameterizations is particularly important to accurately capture changes in eddy dynamics, their impact on large-scale currents, and distribution of water masses in ice-covered regions.Our analysis unveiled the ongoing under-ice eddy response to climate change in the western Arctic Ocean and demonstrated its tight connection to the BG currents and sea ice cover. The critical finding of our study is that the eddy field has been responding rapidly to changes in sea ice, with decadal trends indicating that the floes and underlying ocean eddies are now spinning significantly faster than two decades ago. The presence of sea ice dramatically suppresses the eddy field as its EKE is dissipated by friction due to the ice-ocean drag. As a result, the large-scale currents and the eddy field in the region can be expected to become much more energetic given the observed trend in sea ice extent. To this day, it had not been possible to reach this conclusion using existing 6. It was estimated that from 2008 to 2014, the gyre was storing less potential energy relative to the kinetic energy it was receiving from combined wind and sea ice stress work. The apparent energy surplus can be explained by the intensifying energy transfer from the BG potential energy via interior baroclinic instabilities towards eddies and the eventual dissipation of EKE by sea ice-ocean drag. The conclusion that the increasing imbalance between the cumulative surface-stress work and the available potential energy is a signature of increasing eddy energy relied crucially on the hypothesis that the gyre is significantly equilibrated by eddies. However, the lack of comprehensive eddy observations questions the conclusion about the possible eddy intensification. Our study of floe rotation provided the much-needed independent estimate of the BG eddy field characteristics under sea ice, right where the dissipation of eddy kinetic energy occurs. Our comprehensive analysis spanning observations from 2003 to 2020 concludes that the BG eddy intensification is indeed ongoing. It is not simply a part of interannual gyre variability but rather represents a strong decadal trend in response to the continuing sea ice loss in the western Arctic Ocean.The hypothesis regarding the intensification of the BG eddy field with declining sea ice cover has been put forward based on the analysis of sources/sinks of gyre mechanical energy from 2003 to 2014Finally, we comment on the implications of our results for the ongoing debate on the mechanisms of gyre equilibration. We provided the first observational evidence linking the EKE to large-scale BG currents and established that the EKE dissipation at the ice-ocean boundary layer is an important term in the BG energy budget. These results are consistent with the eddy equilibration hypothesis. They imply that the sea ice-ocean drag on its own does not entirely equilibrate the gyre, even though it does significantly reduce the Ekman pumping and the kinetic energy input into the gyre. These conclusions come with a noteworthy caveat that our method does not distinguish the eddy formation mechanisms and reconstructs combined characteristics of all eddies with surface expressions capable of driving floe rotation. To better understand the role of mesoscale eddies in the BG equilibration and interannual variability, it is necessary to quantify the eddy formation mechanisms and the associated eddy overturning, focusing on eddies formed via baroclinic instabilities of the large-scale currents in the BG interior.https://worldview.earthdata.nasa.gov). In this study, both Corrected Reflectance True and False Color images spanning spring and summer from 2003 to 2020 are employed. The reader is referred to the work of Lopez-Acosta et al.15 and Wolfe et al.41 for specifications regarding satellite acquisitions.Satellite Imagery. Sea ice measurements are retrieved from Moderate Resolution Imaging Spectroradiometer (MODIS) optical imagery (Level 1B 250M). This dataset can be directly downloaded from the open-access Earth Observing System Data and Information System (EOSDIS) Worldview platform (42. These measurements combine SSH estimates from open and ice-covered waters derived from Envisat (2003–2011) and CryoSat-2 (2012–2014).Ocean SSH. We employ measurements of monthly dynamic ocean topography (DOT) and geostrophic velocity currents in the Arctic basin retrieved by Armitage et al.26.Atmosphere. The effect of atmospheric forcing is assessed using ERA5 10 m wind speed reanalysis data  and the Advanced Microwave Scanning Radiometer 2 (AMSR2) products.Sea Ice Concentration. We evaluate sea ice concentration to discard interactions between identified ice floes. It is obtained from the NSIDC product MASAM2https://www2.whoi.edu/site/beaufortgyre/data/mooring-data/.Beaufort Gyre Moorings. Mooring velocity measurements are analysed to estimate the strength of mean currents and the subsurface eddy kinetic energy. The data (and its description) from the four BG moorings named A, B, C, and D  can be downloaded from the Beaufort Gyre Exploration Project website: Regional mean flow velocity. We characterized the strength of oceanic and atmospheric forcing in the BG from kinetic energy (KE) estimates. The seasonal oceanic and the wind surface KE is calculated as BG SSH anomaly. To characterize the strength of the BG, we calculate the characteristic sea surface height difference between an area encompassing the center of the gyre and its periphery. The center of the gyre is defined as the region enclosing most of the gyre during the spring and summer seasons from 2003 to 2020 . The area surrounding the gyre excludes shallow waters that are below 100 m deep  are analyzed per satellite to extract sea ice features amid changing atmospheric conditions. The process consists of three main modules: image processing, feature identification, and sea ice tracking.We identified and tracked ice floes using a newly developed feature matching algorithm that analyzes Moderate Resolution Imaging Spectroradiometer (MODIS) imagery from the EOS-NASA satellites, Terra and AquaThe image processing module effectively minimizes noise due to uneven illumination and cloud coverage while increasing the contrast between ice floes and ocean water. Geometrical parameters are then extracted, including perimeter, major and minor axes of the ellipse that matches the second central moment of the floes, centroid position, and surface and convex areas. The convex area is defined as the area of the smallest convex polygon enclosing an ice floe. As a result, ice floes with length scales ranging from 4 to 75 km are readily identified in each image.Ice floes are tracked by (1) comparing the suite of geometrical parameters of the ice in successive images, (2) finding potential matches, and (3) selecting the best candidates based on the assessment of a similarity metric and the surface area difference between them. The rotation rates of tracked ice floes are calculated by superimposing the shapes of ice floe pairs, rotating one over its axis, and tracking the angle change until the overlapping area is minimal. Finally, the angular velocity of each ice floe is calculated by averaging Aqua and Terra outputs and taking into account the timestamp at which the MODIS images being analyzed were acquired.The last step in creating our library of floes and their rotation rates is identifying and retaining the relatively isolated floes, i.e., those that do not significantly interact with nearby floes. This ensures that ice floe rotation is primarily due to the effect of external atmospheric and oceanic stresses rather than collisions. To achieve this, we computed the bounding box of identified ice floes. Interacting floes were detected based on the proximity of their respective bounding box and the instantaneous value of their rotation rates. Finally, sea ice concentration was evaluated (from MASAM2) to discard any identifications in regions with concentrations higher than 90mentclass2pt{minimA its surface area, h its characteristic thickness, I its moment of inertia, g is the acceleration due to gravity, The statistical characteristics of ice floe rotation rates are obtained by evolving the floes with observed shapes as solid bodies subject to forces and torques due to ice-ocean and atmosphere-ice stresses. No collisions between the floes are simulated as the statistics are compared against those of observed non-colliding floes. The ocean- and atmosphere-ice stresses are parameterized using quadratic drag laws as:15. An Eulerian grid with a resolution of 250 m is defined in the coordinate system moving with the center of mass of a rotating ice floe and used to calculate the surface integrals of forces and torques. Floe thickness observations are not available in the BG, but floes are expected to be between 0.5 to 2 m thick during the breakup events of the winter ice pack. Even though the equilibration timescale is sensitive to the thickness of the floes, it ends up being relatively short (ranging from one to a few hours), implying negligible inertial terms in Eq. The ice floe area is estimated from the library of geometrical properties obtained from MODIS imageryt=1 d since the start of the simulations after the dependence on the initial floe conditions is completely lost. For a given oceanic flow corresponding to a snapshot of equilibrated mesoscale turbulence, floe rotation rates are recorded, from which the histogram of rotation rate variance is computed as a function of floe size for quantitative comparisons with observations.The oceanic mesoscale flow, 46 to identify an eddy field that reproduces the observed rotational statistics of ice floes. The QG model generates eddies from baroclinic instabilities of vertically-sheared horizontal currents, which is the most common eddy-formation mechanism in the ocean47. The simplicity of the QG model is matched by its flexibility to generate a wide range of eddy field characteristics by varying only a few of its parameters: the Rossby deformation radius, 46 within a square doubly-periodic domain of 400 km by side and 256 spectral modes (resolution of about 1.5 km). The spectral filtering is applied for the smallest (grid size) eddies in the domain to halt the forward enstrophy cascade and ensure numerical stability.We use an idealized two-layer quasi-geostrophic (QG) modelThe simulated background flow becomes unstable and generates mesoscale eddies that actively interact with each other, developing a homogeneous and isotropic turbulent eddy field with a clear inertial range of the kinetic energy spectrum. The production of eddy kinetic energy is balanced by its frictional dissipation, predominantly by the ice-ocean drag. A commonly used quadratic ice-ocean drag law , the rotation rate Err, defined asThe key adjustable parameters of the QG two-layer model include the bulk vertical shear of the background horizontal velocity Rd or decreasing Rd and U, while the effective velocity shear U is only a slight overestimate of The three QG model parameters dramatically affect the corresponding eddy field and hence the rotational statistics of the simulated floes. The magnitude of the vertical velocity shear affects the strength of the eddies and hence variances of floe rotations at each length scale, moving the variance–length-scale curve  vertically on a log scale. For a given velocity shear, changing the deformation radius and the ratio of layer depths affects the overall energy of eddies and, more importantly, the scales of the equilibrated eddies and hence the slope of the variance-lengthscale curve. Specifically, increasing 48.To validate our results that the statistics of floe rotation contain direct information about the oceanic eddy field, we compare the predictions of subsurface EKE from the floe statistics with the point EKE measurements from BG moorings. The EKE from floe rotation is calculated as nce Fig. . Most ofWhile the majority of predicted and observed EKE points are close to the 1:1 line, there are clear outliers (Fig. Supplementary Information 1.Supplementary Information 2.


In addition, the phosphorylation of dG and ETV followed Michaelis–Menten kinetics and each competitively inhibited the phosphorylation of the other. We observed that the kinetics of dA phosphorylation were strikingly different from those of dG phosphorylation, with an exponentially lower affinity for dGK and no effect of dA on dG or ETV phosphorylation. Finally, in an isolated heart perfusion model, we demonstrated that dG, dA, and ETV were phosphorylated and dG phosphorylation was inhibited by ETV. Taken together, these data demonstrate that dGK is inhibited by ETV and that the primary role of dGK is in the phosphorylation of dG rather than dA.Deoxyguanosine kinase (dGK) is reported responsible for the phosphorylation of deoxyadenosine (dA) and deoxyguanosine (dG) in the mitochondrial purine salvage pathway. Antiviral nucleoside analogs known as nucleoside reverse transcriptase inhibitors (NRTIs) must be phosphorylated by host enzymes for the analog to become active. We address the possibility that NRTI purine analogs may be competitive inhibitors of dGK. From a group of such analogs, we demonstrate that entecavir (ETV) competitively inhibited the phosphorylation of dG and dA in rat mitochondria. Mitochondria from the brain, heart, kidney, and liver showed a marked preference for phosphorylation of dG over dA (10–30-fold) and ETV over dA (2.5–4-fold). We found that ETV inhibited the phosphorylation of dG with an IC This type is characterized by childhood onset of muscle weakness associated with mtDNA depletion in skeletal muscle  that is associated with progressive external ophthalmoplegia and recurrent rhabdomyolysis as well as hepatic and neural deficits that present later in infancy or childhood  and deoxycytidine kinase (dCK) are both cytoplasmic , 3, 4. Il muscle . The mosl muscle , 12. Aut failure . Fourtee failure . Patient failure . There ihildhood . There afailure 6070 that  on NAD+ .via cytosolic supply of dNTPs irrespective of dGK activity, whereas mutations in DGUOK compromise cell cycle–independent mtDNA synthesis  are nucleoside analog drugs used to slow or stop multiplication of virus-infected cells. The prodrugs are phosphorylated through the salvage pathway in cells to their active forms , 23. PreAs ETV was inhibitory, we selected it for additional study. ETV is a drug used in the treatment of chronic hepatitis B and is a deoxyguanosine analog , 28, 29.ETV has been shown to be readily phosphorylated in hepatocyte cell culture; however, the deoxykinase(s) involved have never been investigated . Our res3H]-dG (0.5 μM) was incubated in isolated liver, kidney, heart, and brain mitochondria, while [3H]-dA (0.5 μM) was incubated only in liver mitochondria for 2 h in the presence of erythro-9-Amino-β-hexyl--α-methyl-9H-purine-9-ethanol hydrochloride, erythro-9-(2-Hydroxy-3-nonyl)-adenine hydrochloride (EHNA) (5 μM) and Immucillin H (I-H) (2.0 μM) as described in the dG and dA analogs including, ETV, adefovir, tenofovir, TDF, lobucavir, penciclovir, ganciclovir, and dideoxyguanosine were incubated at high concentration (100 μM) with respect to their natural analogs. [3H]-dG, [3H]-dA, or [3H]-ETV, all at 1 μM concentrations, in mitochondria freshly isolated from four postmitotic tissues, heart, liver, kidney, and brain. All four preparations of tissue mitochondria produced monophosphates (∼20%), diphosphates (∼< 5%), and triphosphates (∼75%) in the relative amounts shown. As noted in the 3H]-dG was sufficiently robust to measure in mitochondria isolated from any of the four tissues; however, further experiments on [3H]-dA phosphorylation were done only in isolated liver mitochondria.To detect potential tissue-specific effects, we studied the phosphorylation of -dA phosphorylation in liver mitochondria was potently inhibited by dG (A) or ETV, a guanosine analog (B), with very similar IC50 values of 0.034 ± 0.007 and 0.028 ± 0.006 μM, respectively. In contrast, [3H]-dG phosphorylation in heart mitochondria was not inhibited at all by the presence of up to 40 μM dA (A). ETV also inhibited [3H]-dG phosphorylation, but with a much higher IC50 value of 15.3 ± 2.2 μM (I). The IC50 of ETV inhibition of [3H]-dG phosphorylation in mitochondria from the brain, kidney, or liver , respectively) were quite similar. In contrast, the inhibition of [3H]-ETV phosphorylation in liver mitochondria by dG yielded a much more potent IC50 of 0.55 ± 0.10 μM (F), indicating the preference of dGK for its natural substrate, dG over ETV. As above for dG, [3H]-ETV phosphorylation in liver mitochondria was not inhibited at all by the presence of up to 40 μM dA (E).As dG, dA, and ETV are all similar compounds, it might be expected that they competitively inhibit each other’s phosphorylation. To compare the relative extent of this inhibition, we determined ICout 1 μM , we havech other . As showed by dG A or ETV,e analog B, with v40 μM dA A. ETV al± 2.2 μM I. The IC 0.10 μM F, indica40 μM dA E.Figure 3H]-dG and [3H]-ETV phosphorylation were studied in freshly isolated heart mitochondria. The Km values of dG and ETV were 0.72 ± 0.09 μM (B) and 0.77 ± 0.62 μM (B), with accompanying Vmax values of 101 ± 5 pmol/mg protein/hr (B) and 7.1 ± 2.2 pmol/mg protein/hr (B), respectively. These data indicate that dG and ETV bind with similar affinity to the enzyme but that phosphorylation of ETV is far less efficient than phosphorylation of dG. The effect of ETV on [3H]-dG phosphorylation was determined at four inhibitor concentrations. The rates obtained were analyzed for the best fit to three models of inhibition, competitive, noncompetitive, and uncompetitive using the kinetic wizard in Sigma Plot 14.0. As shown in Figure 4B, the best fit was obtained using a competitive model of inhibition with a Ki of 6.7 ± 0.8 μM. Given the similarity of structure between ETV and dG, competitive inhibition was expected. In turn, the effect of dG on [3H]-ETV phosphorylation at four different inhibitor concentrations was similarly determined as shown in i of 0.53 ± 0.38 μM.The kinetic properties of -dA was poorly phosphorylated in heart mitochondria, the phosphorylation of [3H]-dA was studied in the more robust liver mitochondrial preparation. Our initial experiments (A) clearly showed a major difference in phosphorylation of dA versus dG in that dA has an exponentially lower affinity for the enzyme such that up to 4 μM [dA], phosphorylation is still exhibiting linear kinetics. Thus, it was not possible from this experiment to establish a Km or Vmax for dA phosphorylation without going to significantly higher concentrations than would ever be expected in vivo. However, it was still quite clear that dG (B) and ETV (C) potently inhibited dA phosphorylation and that it is likely to represent competitive inhibition. Note that a dG concentration of 0.12 μΜ nearly completely inhibited the phosphorylation of the highest concentration of dA studied (4 μM). These data strongly suggest that the enzyme dGK functions under most conditions to phosphorylate dG rather than dA.As [eriments A clearly that dG B and ETV and ETV C potentl3H]-dGMP (1 μM) was quantitatively converted to [3H]-dGTP in heart mitochondria within 40 to 60 min of incubation. At the 30-min time point, the conversion of [3H]-dGMP to [3H]-dGTP (106 ± 4 pmol/mg protein) compared to the conversion of [3H]-dG to [3H]-dGTP (22.4 ± 0.8) was 4.6 times faster (p < 0.001). ETV had no effect on this pathway, indicating that dephosphorylation of dGMP to dG was not necessary and that the phosphorylation of dG is clearly a rate-limiting step in the phosphorylation pathway of dGTP. As shown in 3H]-dAMP in heart mitochondria was completely converted to [3H]-dATP within 10 min, far faster and more complete than the phosphorylation of [3H]-dA. The conversion was inhibited by P1,P5-di(adenosine-5) pentaphosphate pentasodium salt (Ap5A), an inhibitor of adenylate kinase, demonstrating that dAMP is a good substrate for this rapidly acting equilibrative enzyme. The phosphorylation of dGMP was not inhibited by Ap5A, indicating that the phosphorylation of dGMP was not catalyzed by adenylate kinase. Identical results for both [3H]-dAMP and [3H]-dGMP were obtained in liver mitochondria.In previous experiments studying thymidine phosphorylation in isolated mitochondria, we demonstrated that TMP could only be converted to TTP by first being dephosphorylated to thymidine . Thus, w3H]-dG, [3H]-dA, and [3H]-ETV in isolated mitochondria depends on the activity of the mitochondrial dGK. To determine the degree of phosphorylation in a whole tissue, which would include the activity of the cytosolic dCK, we utilized an isolated rat heart Langendorff retrograde perfusion model. The breakdown and phosphorylation of [3H]-ETV, [3H]-dG, and [3H]-dA (0.5 μM each) was measured in the perfusates and in the hearts after 60 min of perfusion (A and B). [3H]-ETV was completely resistant to breakdown during perfusion (A) and generated 0.25 ± 0.05 pmol/mg wet heart phosphorylated product (B) during the 60-min perfusion. Conversely, [3H]-dG was broken down rapidly in the perfusate from an initial level of 86.8 ± 0.7% to 25 ± 8.8% within just 3 min and was nearly completely converted to catabolites by 60 min of perfusion. However, the heart was still able to take up and maintain a small amount of dG to generate 0.18 ± 0.05 pmol/mg wet heart of phosphorylated dG during the 60-min perfusion. The addition of I-H (2 μM), which inhibits purine nucleoside phosphorylase (PNP), prevented most of the breakdown of dG in the perfusate (A + I-H), resulting in a three-fold increase in phosphorylated [3H]-dG to 0.57 ± 0.08 pmol/mg wet heart compared to [3H]-dG alone . Finally, adding unlabeled ETV (100 μM) to [3H]-dG + I-H inhibited [3H]-dG phosphorylation by 82.0 ± 0.7%, p < 0.0004 . [3H]-dA was even more susceptible to catabolism during perfusion, with the parent compound completely lost by 3 min even in the presence of I-H . The addition of EHNA to the perfusate provided only modest protection, as only 47.5% of the [3H]-dA remained by 3 min with complete loss by 60 min . Despite the loss of [3H]-dA from the perfusate, the heart took up and maintained enough dA to produce 0.48 ± 0.08 pmol/mg wet heart of phosphorylated [3H]-dA products at 60 min . This was similar to the amount of phosphorylated [3H]-dG observed in the presence of I-H. However, the further addition of ETV to the perfusate did not appear to inhibit [3H]-dA phosphorylation , suggesting that [3H]-dA phosphorylation in the perfused heart was not carried out by dGK, and perhaps was a function of dCK. Note that [3H]-dG and [3H]-dG catabolites equilibrate rapidly between the perfusion buffer and the heart, while the phosphorylated forms of [3H]-dG are retained solely in the heart tissue.The phosphorylation of -dG alone led to its rapid clearance from the perfusates into breakdown products (A). Addition of 2 μM I-H, a purine nucleotide phosphorylase inhibitor, largely prevented the breakdown of dG, but was not effective in preventing the breakdown of dA, which also required the addition of 5 μM EHNA to prevent deamination. Others have reported that deoxyadenosine is preferentially deaminated over phosphorylation in many tissues . A small amount of phosphorylated [3H]-dA was also observed in the heart, but this amount was not inhibited by ETV . While the effects of ETV on dCK are unknown, it seems likely that [3H]-dA phosphorylation was mediated by dCK rather than dGK. Future work focused specifically on the cytoplasmic dCK will be required to clearly demonstrate the importance of dCK in dA and ETV phosphorylation, as well as the extent dCK maybe inhibited by ETV.To measure phosphorylation of dA and dG under conditions in which both enzymes are present, we used an isolated perfused rat heart mode , A and Bproducts A. Additi tissues . Even inAs we have reported earlier , exogenoThe data in this paper demonstrate that intact functioning mitochondria from a variety of tissues phosphorylate dA, dG, and ETV. ETV competitively inhibits phosphorylation of dG, but not at levels that would be observed on treatment. However, ETV potently inhibits dA phosphorylation, which could affect the synthesis of dATP at ETV treatment levels and thus may be related to ETV toxicity. Further, dG itself is a potent inhibitor of dA phosphorylation, suggesting that dGK may play a more important role in dG phosphorylation than dA. Future work determining the role of the cytosolic dCK in phosphorylation of dG and dA should be undertaken.3H]-dG (3000 DPM/pmol), [3H]-dA (3000 DPM/pmol), and [3H]-ETV (3000 DPM/pmol) were purchased from Moravek Biochemicals. ETV, adefovir, tenofovir, TDF, lobucavir, penciclovir, ganciclovir, and dideoxyguanosine were purchased from Toronto Research Chemicals. I-H (forodesine hydrochloride) was purchased from MedChemExpress. All other chemicals including EHNA and Ap5A were purchased from Sigma-Aldrich.Radioactive compounds including -dG or [3H]-ETV as specified in figures in an incubation medium previously described  and spun in a microcentrifuge to remove precipitates. The supernatant was neutralized with AG 11 A8 resin and filtered through a 0.2-μM nylon syringe filter. The neutralized and filtered supernatants were analyzed by ultra high pressure liquid chromatography (UPLC). Radioactivity was determined from aliquots of each sample and processed through a Beckman Coulter multipurpose liquid scintillation counter (model 6500).Isolated tissue mitochondria were incubated at a concentration of 4 mg/ml when using [escribed . As the r kidney  and S2. r kidney )  with a reverse-phase C18 column  coupled with an inline diode array to detect UV signal at 254 nm and to an inline liquid scintillation counter (β-Ram) to quantitate radioactivity. Standards were used to identify the parent and phosphorylated peaks of [3H]-dG, [3H]-dA, or [3H]-ETV for 60 min under the conditions previously described , diphosphate (∼< 5%), and triphosphate (∼75%). The radioactivity in each of these peaks was quantitated and totaled to provide the total amount of radioactivity in the phosphorylated products for each labeled precursor. This total was divided by the specific radioactivity of the precursor and expressed as pmol/mg protein/hr. The Kms were calculated from Lineweaver–Burk plotsPhosphorylated products of [and Michaelis–Menten equationversus noncompetitive).using the enzyme kinetics program in SigmaPlot, version 14.0. Where appropriate, the kinetic wizard in SigmaPlot (version 14.0) was used to determine the best fit of data to specific models of inhibition (competitive All numeric results in the text and figure legends represent the mean and standard error of the mean for at least 3 independent experiments. Deviations from this are described in the figure legends.All data are contained within the manuscript.This article contains The authors declare that they have no conflicts of interest with the contents of this article.