Upon depletion of Ca2+ store, the stromal interaction molecule (STIM) senses Ca2+ level reduction and migrates from endoplasmic reticulum (ER)-like sites to the PM where it activates the channel proteins Orai and/or the transient receptor potential channels (TRPC) prompting Ca2+ refilling. Accumulating evidence suggests that SOCE dysregulation may trigger perturbation of intracellular Ca2+ signaling in neurons, glia or hematopoietic cells, thus participating to the pathogenesis of diverse neurodegenerative diseases. Under acute conditions, such as ischemic stroke, neuronal SOCE can either re-establish Ca2+ homeostasis or mediate Ca2+ overload, thus providing a non-excitotoxic mechanism of ischemic neuronal death. The dualistic role of SOCE in brain ischemia is further underscored by the evidence that it also participates to endothelial restoration and to the stabilization of intravascular thrombi. In Parkinson’s disease (PD) models, loss of SOCE triggers ER stress and dysfunction/degeneration of dopaminergic neurons. Disruption of neuronal SOCE also underlies Alzheimer’s disease (AD) pathogenesis, since both in genetic mouse models and in human sporadic AD brain samples, reduced SOCE contributes to synaptic loss and cognitive decline. Unlike the AD setting, in the striatum from Huntington’s disease (HD) transgenic mice, an increased STIM2 expression causes elevated synaptic SOCE that was suggested to underlie synaptic loss in medium spiny neurons. Thus, pharmacological inhibition of SOCE is beneficial to synapse maintenance in HD models, whereas the same approach may be anticipated to be detrimental to cortical and hippocampal pyramidal neurons. On the other hand, up-regulation of SOCE may be beneficial during AD. These intriguing findings highlight the importance of further mechanistic studies to dissect the molecular pathways, and their corresponding targets, involved in synaptic dysfunction and neuronal loss during aging and neurodegenerative diseases.In both excitable and non-excitable cells, calcium (Ca Ca2+ ions are extruded from the cell or sequestered in intracellular compartments (stores) by energy-driven pumps that maintain cytosolic Ca2+ concentrations approximately 100,000-fold the extracellular space. As a consequence of this large concentration gradient, a rapid influx of Ca2+ occurs upon opening of ion channels located on the plasma membrane (PM) or on the membranes of intracellular stores, including the endoplasmic reticulum (ER) that is considered the major intracellular store. In both excitable and non-excitable cells, including those involved in the immune response, Ca2+ signals are generated by store-operated Ca2+entry (SOCE), namely the opening of PM Ca2+ channels following the release of Ca2+ from intracellular stores. Upon ER leak, SOCE determines ER Ca2+ refilling through the activation of two currents: the Ca2+ release-activated Ca2+ current (ICRAC), a non-voltage activated and inwardly rectifying current, highly selective for Ca2+, and the store-operated Ca2+ current (ISOC), a non-selective outward current with different biophysical features 1 activity in mediating ISOC. Indeed, TRPC1 is recruited to the PM upon Orai/STIM complex formation from Rab4-vescicles , divided into two subfamilies, TRPC1/TRPC4/TRPC5 and TRPC3/TRPC6/TRPC7, based on biochemical and functional similarities. Search for SOCE partners led to the identification of several TRPCs, including TRPC1, TRPC3 and TRPC4 that are differently involved in this phenomenon. TRPC3 and TRPC4 can be activated by store depletion, while activation of TRPC5, TRPC6 and TRPC7 occurs via store-independent mechanisms , intraneuronal neurofibrillary tangles consisting of hyperphosphorylated and misfolded tau, and synaptic/neuronal loss leading to brain atrophy  oligomers play a more important role in neurotoxicity  is a neurodegenerative disorder that causes progressive memory loss, impairment of other cognitive functions and psychosis  pump  activation that is, in turn, essential for the stability of mushroom spines  or blockade of NMDA receptor overstimulation (with memantine) that provide some symptomatic benefits for a limited period of time, followed by a steep cognitive decline due to the fact that these are not disease modifying drugs and do not prevent cell death. The current drug development pipeline is largely based on the amyloid hypothesis; however, despite efficacy at reducing amyloid levels in patients, all the clinical trials to date have generated disappointing results with respect to cognition function. Given the compelling evidence that aberrant calcium signaling, particularly in the ER, is associated with familial AD mutations and sporadic AD risk factors, modulation of calcium channels or calcium regulating proteins is in varying stages of therapeutic validation —that mimics PD—decreases the level of TRPC1 and its interaction with STIM1, thus increasing neuronal death both in vitro and in vivo - and MPP+-induced toxicity through inhibition of SOCE-mediated Ca2+-overload  is a neurodegenerative disorder characterized by the selective loss of dopaminergic (DA) neurons in the Fleming, . The majFleming, . PathogeDrosophila thus affecting dopamine synthesis and release , an ER resident transmembrane protein that is regulated by ER CaThus, pharmacological inhibition of SOCE is beneficial to synapse maintenance in HD models, whereas the same approach may be anticipated to be detrimental to cortical and hippocampal pyramidal neurons Figure . Convers2+ homeostasis are implicated in ALS pathology, data on the involvement of SOCE and its molecular components are currently limited. Some evidence on SOCE dysregulation in ALS was provided only in glial cells that actively participate to the neurodegenerative process of the disease. In particular, astrocytes obtained from animal models of ALS or from human familial or sporadic ALS patients are able to release toxic factors that induce cell death in primary motor neurons  is a severe human progressive neurodegenerative disease affecting lower and upper motor neurons whose specific etiology is incompletely understood. Familial ALS (fALS) patients with known genetic mutations are relatively rare, and over 90% of cases occur sporadically. However, mutations in superoxide dismutase-1 (SOD1), TAR DNA-binding protein 43 (TARDBP/TDP-43) and C9orf72 have been identified in subsets of familial and sporadic patients  disease, one of the most frequent inherited neurological disorders characterized by either demyelinating or axonal neuropathy of motor and sensory peripheral nerves  contribute to the progression of neurodegenerative diseases , the Ca2+ channels  or the proteins that sense Ca2+ entry through SOCE and then activate the cellular response. Although some compounds that modulate SOCE have been developed and tested in pre-clinical models of neurodegenerative diseases, the validation of new drugs that specifically target SOCE components would result in successful treatment strategies for currently incurable neurodegenerative diseases, including AD, PD, HD and stroke.The experimental evidence reviewed in the present work clearly demonstrates that CaAS, GB and DA participated in the collection, review and analysis of the relevant literature, as well as in drafting and revising the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


PRKN , PINK1 , and DJ-1 , have often been associated to autosomal-recessive forms of EOPD , a homozygous missense mutation (c.835G > A) in the adenosine A1 receptor (A1R) gene (ADORA1) was found  within the transmembrane 7 (TM7) domain, but the functional consequences remain unknown. In contrast, it was recently determined that mutations affecting ADORA1 gene and more particularly the missense matution ADORA1 (p.G279S), are not a common risk factor for PD in the European population, arguing against ADORA1 as a candidate gene in PD  is a progressive, neurodegenerative disorder that affects ~1% of individuals over the age of 60, which turns to 5% in subjects up to 85 years , cannabinoid CB1 receptor (CB1R) and dopamine D2 and D4 receptors  modulate this tonic stimulation  mutation abolishes A1R function and whether this alteration depends on its heteromerization with A2AR receptor, specifically disrupting the function of the adenosine concentration-dependent switch. In the absence of experimental data, we can speculate that the mutation can be affecting the A1R/A2AR heteromer, resulting in a potential alteration of the fine-tuning modulation of striatal glutamatergic neurotransmission. Indeed, in such scenario, we can hypothesize that moderate concentrations of striatal adenosine would facilitate glutamate release and reduce the excitotoxicity threshold , may help to restore the normal functioning of the basal ganglia. In this sense, the A1R/A2AR heteromer could be considered as a potential therapeutic target for EOPD. Alternatively, the glutamatergic component of these forms of EOPD would represent an initial or master pathogenic event to dopamine denervation, as proposed in Hungtinton's disease pathophysiology . In this sense, the use of A2AR antagonists has been assessed for the treatment of PD  local A1R activation or (ii) pharmacological increase of the adenosine tone (below the threshold of A2AR activation) using adenosine transporters blockers and/or metabolizing enzymes, are expected to reach an effective treatment for EOPD.The dysregulation of this presynaptic module may lead to uncontrolled glutamate release which, in addition, might be potentiated by low dopamine innervation, which would not act upon inhibitory presynaptic DADORA1 presumably leading to EOPD supports the potential beneficial use of a multimodal approach for the pharmacological treatment of this neurodegenerative condition. This approach, based on the combination of pharmacological therapies  could be potentially extended to all forms of PD.Overall, the discovery of a novel mutation in VF-D, XA, SF: wrote the paper; AP-A: conceived the idea; FC: conceived the idea and wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The expression of JPH isoforms was examined in human and mouse pancreatic tissues, and JPH3 expression was found in both the beta cells. In mice, knockdown of Jph3 (si-Jph3) in islets decreased glucose-stimulated insulin secretion (GSIS) accompanied by mitochondrial function impairment. Si-Jph3 lowered the insulin secretory response to Ca2+ signaling in the presence of glucose, and reduced [Ca2+]c transient amplitude triggered by caffeine. Si-Jph3 also attenuated mitofusin 2 expression, thereby disturbing the spatial organization of ER–mitochondria contact in islets. These results suggest that the regulation of GSIS by the KATP channel-independent pathways is partly impaired due to decrease of JPH3 expression in mouse islets. JPH3 also binds to type 2 ryanodine receptors (RyR2) in mouse and human pancreatic tissues, which might contribute to Ca2+ release amplification in GSIS. This study demonstrates some previously unrecognized findings in pancreatic tissues: (1) JPH3 expresses in mouse and human beta cells; (2) si-Jph3 in mouse primary islets impairs GSIS in vitro; (3) impairment in GSIS in si-Jph3 islets is due to changes in RyR2-[Ca2+]c transient amplitude and ER-mitochondria contact.It is well accepted that junctophilin (JPHs) isoforms act as a physical bridge linking plasma membrane and endoplasmic reticulum (ER) for channel crosstalk in excitable cells. Our purpose is to investigate whether JPHs are involved in the proper communication between Ca Glucose-stimulated insulin secretion (GSIS) is the principal mechanism of insulin secretion. Loss or reduced GSIS are characteristic features of type 2 diabetes mellitus.9 The mechanism involved in triggering GSIS is well accepted as the KATP channel-dependent and -independent pathways,9 the mitochondrial metabolism is the essential upstream core shared by both the pathways. However, to date, the link between Ca2+ amplification handling and ATP production during GSIS is not fully understood.Insulin secretion is associated with alterations of intracellular glucose metabolism, electrical excitability and Ca16 Although pancreatic beta cells are electrically excitable,6 the role of JPHs in insulin release remains so far undefined. RyR2, inositol 1,4,5-trisphosphate receptor (IP3R) and sarco-endoplasmic reticulum Ca2+-ATPase 2b and 3  in endoplasmic reticulum (ER) are closely associated with insulin release.18 Given that Ca2+ release amplification depends on the organization of VGCC and RyR2 within JMCs, whether the impairment of JMCs affects insulin secretion in beta cells should be urgently clarified. Knockout mice lacking the JPH isoforms display related pathological phenotypes,22 indicating that JPHs are essential for the physiological communication. In vivo deletion of RyR2 reduces Ca2+, ATP and oxidative metabolism, thereby leading to metabolic reprogramming and cell death.23 Furthermore, sleep deprivation upregulates Jph3 transcription in mouse brain via stressful conditions through RyR-mediated intracellular calcium mobilization,24 suggesting that Jph3 might also be a functional gene under stress in addition to its structural contribution. Given that Ca2+ signaling in GSIS includes the amplifying features through KATP-independent pathway,26 JPHs probably contribute to maintaining mitochondria function in beta cells. Importantly, the relationships between abnormal JPH isoforms and human diseases have also been confirmed. JPH2 deficiency resulted in hypertrophied and failing myocardium,30 while Jph3 mutation caused Huntington's disease-like-2.32 Notably, strategies to maintain JPH2 level can prevent the progression from hypertrophy to heart failure,33 which highlights the potential therapeutic prospect of JPHs in beta cells in preventing the progress of type 2 diabetes. However, whether or not JPHs deficiency or mutation involves diabetic pathogenesis has so far not been proved.The family of junctophilin (JPHs) isoforms serves as a physical bridge and effectively contributes to the formation of JMCs for ion channel functional crosstalk in excitable cells.Jph3 deficiency caused the acute damage of GSIS, which was associated with the impairment of ER–mitochondria axis integrity. Therefore, our finding reveals a novel functional role for JPH3 in maintaining GSIS under physiological and probably pathological conditions.The present study demonstrated that JPH3 is the major pancreatic isoform, which expressed in human and mouse pancreatic beta cells. In mouse islets, in situ immunofluorescence double staining of four JPH isoforms or JPH3 and JPH4 alone with insulin was performed in mouse primary islets or human pancreatic tissues. JPH3 selectively coexpressed with insulin in mouse and human pancreatic beta cells  or 5.5 mmol/L (low) glucose, respectively  rise34 and ATP generation, we further examined the IP3-evoked mitochondrial Ca2+ transient amplitude in permeabilized si-Jph3 islets. Representative fluorescence recordings showed that the initial slope and peak amplitude of [Ca2+]m oscillations were considerably attenuated to 7.03±3.21 and 52.02±21.67% in si-Jph3 islets compared with si-Ctr islets  in si-Jph3 islet as compared with active mitochondria with high ΔΨ (red) in si-Ctr islets  islets. By contrast, caffeine lost triggering effect on insulin releases in si-Jph3 islets . Both contacts were well organized in a PM–ER–mitochondria or ER–mitochondria status in beta cell of mouse pancreatic tissue  and mitochondria (red) fluorescent proteins display yellow area, which indicates that organelles are closer than ~270 nm.35 Confocal microscope observation demonstrated the colocalization areas of two organelles reduced by 43% in si-Jph3 islets compared with si-Ctr islets  and MitoTracker (red) contact in a perpendicular position in the cell of si-Ctr islet. By contrast, ER and mitochondria separated from each other and their contact disappeared in a cell of si-Jph3 islet  plays a minor role in GSIS.41 Here, we show that acute knockdown of Jph3 directly leads to impaired GSIS accompanied by RyR2 Ca2+ insufficiency, indicating that JPH3 is critical for maintaining RyR2-Ca2+ signaling in GSIS.Impaired GSIS in islets is one of the characteristic features of type 2 diabetes mellitus.Jph3 islets. Mitochondrial ATP synthesis is initiated by Ca2+ and driven by ΔΨm.10 In the presence of glucose, Jph3 knockdown in islets affects IP3 evoked-Ca2+ rapidly shuttling through IP3R into the mitochondrial matrix, where Ca2+ activates Krebs cycle enzymes, leading to decreased ATP production. Therefore, JPH3 expression should be essential for triggering GSIS through KATP-independent pathways. Our findings suggest that decrease of JPH3 is responsible for impaired GSIS, and also imply a causal link between JPH3 defects and type 2 diabetes.Our study further demonstrates that impaired GSIS is associated with damaged mitochondrial function in si-Jph3 in islets also attenuated Mfn2 expression, thereby disturbing the spatial organization of ER–mitochondria contact, which probably also contributes to insufficient GSIS. The ER–mitochondria axis has been defined in muscle from obese or type 2 diabetic patients.42 Here, we further demonstrate that the spatial organized ultrastructural contacts of PM–ER–mitochondria do exist in mouse pancreatic beta cells, and that acute deficiency of JPH3 leads to alteration in ER–mitochondria contact morphology in islets. Moreover, IP3-evoked amplitude of [Ca2+]m oscillations are considerably attenuated in permeabilized si-Jph3 islets, which suggests that the organization of VGCC and RyR2 within JMCs are probably functionally associated with ER–mitochondria contact via RyR2-Ca2+ signaling. Notably, our findings showed that although human pancreatic tissue expresses JPH3 and a few JPH4, only JPH3 can directly bind to RyR2 in human pancreatic tissues, which is consistent with the binding and functional response of RyR2 by JPH3 in mouse si-Ctr islets. Homotypic and heterotypic interactions between Mfn2 on the ER and Mfn1 and 2 on mitochondria enable Ca2+ microdomains around open IP3R to propagate rapidly into the mitochondrial matrix, thereby increasing ATP production.43 Our findings further reveal that although both Mfn1 and Mfn2 abundantly express in beta cells, Jph3 knockdown in islets only selectively affects Mfn2 expression and Mfn2 transcription. Furthermore, acute knockdown of Mfn2 directly leads to impaired GSIS in islet, which highlights the possibility that decrease in Mfn2 expression due to JPH3 downregulation in beta cells might cause development of type 2 diabetes. Collectively, our findings, for the first time, suggest that the structural and functional ER–mitochondria axis integrity is of importance to maintain GSIS, and that Jph3 knockdown significantly affects Mfn2 expression and subsequently influences the mitochondria function in beta cells.The present results reveal that si-Jph3 knockdown in islets selectively affects Pgc-1α mRNA expression at the transcription level. Previous researches demonstrated that the raise of intracellular Ca2+ concentration contributes to Pgc-1α nuclear translocation and transcription in myocytes and skeletal muscle.44 PGC-1α takes part in shaping mitochondrial participation in calcium signalling that underlies its protective role against stress and proapoptotic stimuli in pathophysiological conditions.45 In addition, reduced RyR2 signaling can alter cardiac transcriptional programming, including Pgc-1α down-regulation, in a manner associated with cardiac pathology,23 and also cause abnormal Ca2+ handling in type 2 diabetic conditions.17 Our study further suggests that knockdown of Jph3 might contribute to RyR2 Ca2+ signaling-related Mfn2 reduction via weakened Pgc-1α signaling in pancreatic beta cells. Taken together, JPH3 takes an important role in maintaining the suitable distance and crosstalk between ER and mitochondria via Pgc-1α pathway in beta cells. Recently, it is revealed that a Pgc-1α-PPARα/δ partnership in nuclei can promote the target molecules transcription in skeletal muscle.46 PPARβ activation can also promote human and mouse embryonic stem cells differentiating into functional insulin-positive cells.47 Our findings imply that Pgc-1α and PPARβ in the nuclei of beta cells in human pancreatic tissue might act as co-activators, thereby contributing to regulating Mfn2 expression. Therefore, it is valuable to further explore whether JPH3 takes a role in human GSIS in the future.Our study demonstrates that, among the three examined Mfn2 modulators, Jph3 islets, indicating that Jph3 knockdown does not affect store-operated Ca2+ entry function. In addition, si-Jph3 caused Mfn2 attenuation or RyR2-Ca2+ insufficiency does not lead to ER stress and apoptosis-related protein expression, suggesting that GSIS insufficiency should not result from ER stress or apoptosis in si-Jph3 islets.The present study showed that the protein expression and redistribution kinetics of the STIM1 binding to the Orai1 are unchanged in si-33 Therefore, the dual functional roles for JPH3 in pancreatic beta cells, linking PM–ER and interaction with RyR2, may have implications for the development of new therapeutic strategies for type 2 diabetes.In conclusion, JPH3 is the major pancreatic isoform expressed in both human and mouse pancreatic beta cells. Our findings suggested the physiological roles of JPH3 in beta cells and implicated JPH3 as an important candidate involved in the impairment of GSIS in type 2 diabetic conditions. Strategies to maintain JPH2 level can prevent the progression from hypertrophy to heart failure.48 and cultured in RPMI 1640 containing 20% FBS for further investigation.Eight-week-old male Balb/c mice were obtained from the Experimental Animal Center, Zhejiang University, China . The experiments were conducted under the Guide for the care and use of laboratory animals (2011), and a protocol approved by the Institutional Animal Care and Use Committee of Zhejiang University (№ ZJU-2015-435-01), China. Islets were isolated by ductal collagenase V  injection from fully anesthetized miceμm consecutive sections were cut for immunocytochemistry analysis. Patient information could be found in Human pancreatic samples surrounding the cut tissues were obtained from surgical donors under prior informed consent procedure and with written consent from the Human Ethics Committee of the 2nd Affiliated Hospital (№ 2015-029), College of Medicine, Zhejiang University, China. Frozen 10 Jph3, Mfn2 or negative control siRNA (si-Ctr)  using Lipofectamine RNAiMAX (Life Technologies) transfection agent according to the manufacturer's instructions. Sequences for siRNA were shown in Isolated islets were transfected with 100 nM siRNA against 47 Samples were probed with the primary antibodies as follows: Insulin, GRP78, STIM1 ; JPH1, JPH2 ; JPH3, JPH4, Mfn1, Mfn2, PPAR, Pgc-1α ; Orai1 . Then islets were incubated with secondary antibodies and DAPI (Sigma Aldrich). Images were acquired under Leica DMI3000B microscope  or Olympus FV1000 confocal microscope . Colocalization ratio of GRP78 and MitoTracker was calculated by Metamorph software  automatically. Relative ratio of Pgc-1α nuclear translocation was analyzed by Imaris software .All immunostaining was conducted as previously described.47 Typically, sample lysates were resolved in SDS-PAGE and transferred onto PVDF membrane . The PVDF membranes were incubated with the following primary antibodies (in addition to the above-mentioned): Errα, Sp1, IP3R, LTCC, Lamin B ; RyR1, RyR2, RyR3 (Merck Millipore); SERCA2, GRP94, eIF2α, p-eIF2α, Bcl-2, Bax ; JPH1, JPH2, JPH3, JPH4, Mfn1, Mfn2, Pgc-1α ; GAPDH . Then samples were incubated with HRP-conjugated secondary antibodies . Blots were developed using enhanced chemiluminescence reagents (Pierce). Coimmunoprecipitates were analyzed by immunoblotting, using either anti-JPH3 or anti-RyR1, 2, 3 antibodies to detect the interaction between JPH3 and 3 RyR subtypes.Western blot was performed as previously described.Total RNA was isolated by Trizol (Life Technologies). For sqRT-PCR, reverse transcription was performed using M-MuLV first strand cDNA synthesis kit, and real-time PCR was performed using the PCR kit . For qRT-PCR, RNA was treated by RT reagent kit (RR047A), and amplifications were performed using SYBR Premix Ex Taq Kit . Primer sequences were displayed in μmol/L Cyclopiazonic acid  and 10 μmol/L D-myo-inositol-1,4,5-trisphosphate (IP3) (Tocris) dissolved in KRBH buffer containing 2.5 mmol/L glucose. The Krebs' Ringer Bicarbonate HEPES buffer consisted of 160 ml Buffer A, 200 ml Buffer B, 2.4 g HEPES, and 2 g BSA in a total volume of 1000 ml deionized water and filtered via 0.45 μm and 0.22 μm membranes. The Buffer A consisted of 34.6 g NaCl, 1.8 g KCl, 1.9 g CaCl2, 0.8 g KH2PO4, 1.5 g MgSO4•7H2O in 1000 ml deionized water. The Buffer B consisted of 13 g NaHCO3 in 1000 ml deionized water. Typically, for IP3 effect evaluation, islets were pretreated with 20 μg/ml digitonin (Sigma Aldrich) for 30 min.Islets were precultured in Krebs' Ringer Bicarbonate HEPES buffer containing 2.5 mmol/L glucose for 1 h. Cells were treated with different reagents for another hour. The supernatant was analyzed by Rat/Mouse Insulin ELISA Kit . The reagents were as follows: glucose 27.7 mmol/L or 5.5 mmol/L dissolved in KRBH buffer; 5 mmol/L caffeine , 50 μmol/L) and Rhod-2 (5 μmol/L) (Life Technologies) for [Ca2+]c and [Ca2+]m  indicator, respectively. Digitonin-permeabilized islets were used for IP3 experiment. Caffeine, CPA and IP3 were added to elicit Ca2+ release. Images were analyzed using software InVivo under live cell imaging system  by measuring fluorescence of individual islet at 549 nm and 576 nm, respectively. All analyses were processed as the relative fluorescence intensity. The slopes (from initiation to top) derived from the linear equation were calculated by Excel.Islets were loaded with X-rhod-1 AM  following the manufacturer's instructions.Islets were incubated with 2.5 mmol/L glucose for 60 min.μg/ml JC-1  for 30 min at 37 °C in the dark. Cells were then washed by PBS and observed under Leica DMI3000B microscope. The red fluorescent J-aggregate indicates normal ΔΨm, while the green monomer fluorescence demonstrates low ΔΨm.For the determination of ΔΨm, cells were incubated with 2 Islets were incubated with MitoTracker and ER-Tracker (Life Technologies) for 30 min following the manufacturer's instructions. Sections were photographed under Leica DMI3000B microscope  or Olympus FV1000 confocal microscope .t-test. Comparisons of multiple groups used ANOVA . The differences were considered significant at P<0.05.Data were reported as means±S.D. Experiments were performed three times as repeats, and each time the islets were obtained from different mice. Comparisons of two groups used two-tailed student's


Both second messengers are known to interact with each other, for example via Ca2+-dependent modulation of phosphodiesterase 1 (PDE1) and adenylyl cyclase 5/6 (AC 5/6) activities, which is supposed to occur especially at the local level in distinct subcellular microdomains. Currently, many studies analyze global and local cAMP signaling and its regulation in resting cardiomyocytes devoid of electrical stimulation. For example, Förster resonance energy transfer (FRET) microscopy is a popular approach for visualization of real time cAMP dynamics performed in resting cardiomyocytes to avoid potential contractility-related movement artifacts. However, it is unknown whether such data are comparable with the cell behavior under more physiologically relevant conditions during contraction. Here, we directly compare the cAMP-FRET responses to AC stimulation and PDE inhibition in resting vs. paced adult mouse ventricular cardiomyocytes for both cytosolic and subsarcolemmal microdomains. Interestingly, no significant differences in cAMP dynamics could be detected after β-adrenergic (isoproterenol) stimulation, suggesting low impact of rapidly changing contractile Ca2+ concentrations on cytosolic cAMP levels associated with AC activation. However, the contribution of the calcium-dependent PDE1, but not of the Ca2+-insensitive PDE4, to the regulation of cAMP levels after forskolin stimulation was significantly increased. This increase could be mimicked by pretreatment of resting cells with Ca2+ elevating agents. Ca2+ imaging demonstrated significantly higher amplitudes of Ca2+ transients in forskolin than in isoproterenol stimulated cells, suggesting that forskolin stimulation might lead to stronger activation of PDE1. In conclusion, changes in intracellular Ca2+ during cardiomyocyte contraction dynamically interact with cAMP levels, especially after strong AC stimulation. The use of resting cells for FRET-based measurements of cAMP can be justified under β-adrenergic stimulation, while the reliable analysis of PDE1 effects may require electric field stimulation.Calcium (Ca Upon stin vivo, mammalian heart is under constant contraction, which leads to rapid calcium (Ca2+) cycling in all cardiomyocytes during each contraction cycle. Upon depolarization, Ca2+ rapidly enters the cell via voltage-gated L-type calcium channels. This in turn triggers Ca2+ release from the sarcoplasmic reticulum (SR) via ryanodine receptors, providing a sufficient amount of this ion in the cytosol to activate contractile proteins. In the diastole, there is a Ca2+ reuptake back into SR via sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) [2+ is also known for dynamically regulating intracellular cAMP levels. This occurs either via the stimulation of the Ca2+/calmodulin-dependent PDE1 [2+ and cAMP tightly interact to regulate cardiac excitation contraction coupling (ECC). This interaction is supposed to occur especially in subcellular microdomains formed around calcium handling proteins which might directly impact on contractile function [+ levels during contraction are able to directly affect global and local cAMP levels in the cell. Thus, it remains unknown whether the cAMP imaging data obtained under such conditions truly represent actual cAMP dynamics occurring under more physiological conditions in beating cells.However, SERCA2) . Each of(SERCA2) ,12. Besient PDE1  or by inent PDE1 , all theent PDE1 ,16. Thus . Each ofunction ,17,18. Ffunction ,19. To afunction . Therefo2+ during contraction are not sufficient to affect overall PDE1 and AC activities. In sharp contrast, the contribution of PDE1 to cAMP degradation after forskolin stimulation was significantly increased in paced cardiomyocytes. This effect could be mimicked in resting cells by preincubation with Ca2+ elevating reagents such as thapsigargin and calcium ionophore. Thus, the cAMP dynamics under β-AR stimulation can be reliably studied in resting cells, while the exact analysis of PDE1-associated effects requires paced cardiomyocytes.Here, we sought to answer this question and performed FRET measurements in freshly isolated cardiomyocytes from transgenic Epac1-camps and pmEpac1 cAMP FRET sensor mice ,20 under2+ adaptation, cells were plated onto laminin coated coverslips and used for measurements during the next few hours after plating.All animal work was conducted according to relevant national and international guidelines. Institutional Committee “Tierschutzbüro UMG” and the national authorities LAVES and BGV Hamburg have approved this work. Transgenic mice were custom generated on FVB/NRj background  as previously described ,20 and h2, 5 mM HEPES, 10 mM glucose, 1 mM CaCl2, pH 7.5). The measuring chamber was then transferred onto a ZEISS AxioObserver A1 epifluorescence microscope equipped with an oil immersion 63x objective, polychrome V light source (TILL Photonics), DV2 DualView (Photometrics) and a CoolSNAP-HQ2 CCD-camera (Visitron Systems). Cells were treated with several cAMP stimulating substances . Resting cardiomyocytes were pretreated with Ca2+ elevating reagents thapsigargin and calcium ionophore A23187 (both from Sigma) for 3 min before starting the FRET measurements to mimic contractility driven increase in Ca2+.Coverslips with isolated cardiomyocytes were transferred into a measuring chamber and rinsed once with Tyrode’s solution  and detected at 535 ± 15 (YFP) and 480 ± 10 (CFP) nm. FRET changes were monitored using VisiView software (Visitron Systems) as the emission ratio of YFP over CFP, calculated as previously described  using Or2+ transients were recorded according to the corresponding FRET experiments. F340/F380 values in Fura2-AM experiments were calculated from the ratios of 510 nm emission light measured at 340 and 380 nm excitations. Data were analyzed using the IonWizard, GraphPad Prism and Origin 8.5 software.Freshly isolated wildtype cardiomyocytes were plated onto laminin coated glass coverslides, rinsed once and then loaded with 1 μM Fura2-AM (Life Technologies) for 15 min at 37°C. After washing the cells thrice with Tyrode solution, measuring chambers were transferred onto a Nikon Eclipse TE2000-U microscope equipped with a fluorescence detection system (IonOptix). CaThe values are expressed as means ± SEM. Normal distribution was tested by the Kolmogorov-Smirnov test. Differences between the paced and resting cells or between treated and untreated resting myocytes were analyzed by GraphPad Prism software using one-way ANOVA or Mann-Whitney tests with Bonferroni’s post-hoc test and considered significant at p<0.05. Calcium imaging data were analyzed using one-way ANOVA followed by the Greenhouse-Geisser correction which accounts for the sphericity of the data.2+ fluctuations associated with cardiomyocyte contraction impact on cAMP dynamics detected by FRET, we measured cAMP responses in resting and paced cardiomyocytes. To ensure that our pacing protocol leads to proper cardiomyocyte contraction and Ca2+ transients, we loaded freshly isolated wildtype cardiomyocytes with Fura2-AM. 2+ transients, while resting cells show no fluctuations in Ca2+ signal. Importantly, our pacing protocol altered neither the basal FRET nor the FRET ratio after full stimulation in Epac1-camps expressing cardiomyocytes  to increase the β-AR-dependent AC activity  or with 2+ during contraction can significantly affect neither basal nor stimulated AC activities detected by FRET.To ensure that the absence of any measurable effect of pacing was not due to very high degree of AC stimulation by saturating doses of ISO and forskolin or due to variable PDE activities, we analyzed cAMP signals also using non saturating ISO concentrations (1 nM instead of 100 nM) and after PDE inhibition by the unselective PDE inhibitor IBMX. Even in this case, there were no significant differences in the FRET responses when compared in resting and paced Epac1-camps cardiomyocytes . These d2+ via calmodulin [2+ elevation. Thus, it was important to investigate whether Ca2+ fluctuations associated with contraction would have any influence on the contribution of PDE1 into the cAMP signals detected in FRET experiments. Therefore, we performed FRET measurements similar to those shown in 2+ transients on the overall PDE1 activity after β-AR stimulation  levels or due to different compartmentation of forskolin- vs. β-AR-stimulated cAMP responses [2+ flux [2+ imaging data (2+ levels after forskolin stimulation, as compared to ISO stimulation. On the other hand, the absence of calcium ionophore and thapsigargin effects after ISO prestimulation (in vitro pacing protocol. In this case, FRET imaging represents a powerful approach for real-time cAMP monitoring in intact single cardiomyocytes.Since there were no major differences in cAMP dynamics after β-AR stimulation in resting vs. paced cells, resting cardiomyocytes can be used for reliable analysis of real-time AMP dynamics by FRET or other comparable approaches under such stimulation protocols see Figs , 2 and 4esponses ,26 which[2+ flux . Howevering data  stronglymulation , right amulation  indicatemulation . This mimulation . Althoug2+ during cardiomyocyte contraction can dynamically interact with cAMP levels under certain conditions. FRET-based imaging of cAMP dynamics in resting cells can be justified under β-adrenergic stimulation. However, under stronger global cAMP stimulation with forskolin and during the analysis of physiologically relevant effects of the Ca2+ activated PDE1, electric field stimulation is required to draw reliable conclusions.In summary, our results suggest that changes in intracellular Ca


Staphylococcus aureus bacteremia.Phosphatidylinositol 3-kinase δ (PIK3δ) is a tyrosine kinase essential for B cell survival, making it an important target in the treatment of chronic lymphocytic leukemia. Idelalisib is an inhibitor of PIK3δ demonstrating initial success in disease response, but is now shown to have a decreased overall survival and life-threatening serious adverse events. The following is an unfortunate case of a grade III adverse skin reaction secondary to idelalisib with the likely complication of methicillin-resistant  We provide a case of a male presenting with diffuse exfoliative rash secondary to idelalisib and complications of methicillin-resistant Staphylococcus aureus (MRSA) bacteremia.Important for B cell proliferation and homeostasis, phosphatidylinositol 3-kinase δ (PIK3δ) is a tyrosine kinase of interest in the treatment of chronic lymphocytic leukemia (CLL). As a specific PIK3δ inhibitor, the Gilead Sciences creation idelalisib (Zydelig), was approved by the US Food and Drug Administration (FDA) with breakthrough therapy designation on July 2014 for the treatment of relapsed CLL in combination with rituximab.A 65-year-old African American male was admitted to the hospital for a 6-day history of worsening generalized rash involving his mucous membranes. His medical history was significant for hepatitis B infection and CLL Rai Stage I. Prior treatment regimens included fludarabine, cyclophosphamide, and rituximab combination and ibrutinib alone. However, these were stopped due to varying adverse events. Consequently, the patient was started on idelalisib monotherapy 150 mg twice daily for 3 months. Off-label use of monotherapy was initiated as opposed to the FDA-approved combination therapy due to concerns of reactivation of hepatitis B with rituximab, which was discovered later in the course of his disease. Nevertheless, with the development of a desquamating rash starting at the soles of his feet, idelalisib was discontinued 2 weeks prior the current admission.3/µL [normal = 4.8-10.5 × 103/µL), and a mild normocytic anemia . At first, he was thought to have toxic epidermal necrolysis, supported by the temporal association with idelalisib. During his hospitalization, however, with its protracted course and clinical diagnosis of diffuse erythema and scaling involving ≥90% body surface area, the patient was classified as having idelalisib-induced exfoliative dermatitis secondary to a severe cutaneous adverse drug reaction.Examination revealed an exfoliative and erythematous rash throughout his torso, extremities , tongue,Unfortunately, a biopsy, which would have further classified this reaction, could not be completed due to the rapid decline of his hospital course. This included respiratory failure requiring intubation and MRSA-induced septic shock. A contrast computed tomography scan revealed axillary and mediastinal lymphadenopathy, with a large consolidation in the left upper lobe. He underwent a thoracotomy for decortication and assessment of the mass showed high-grade lymphoma. Ultimately, his family decided to pursue hospice care and the patient was terminally extubated.3 Idelalisib, a specific inhibitor of PIK3δ, was approved by the US FDA for the treatment of relapsed CLL.2 Initial registration trials, when combined with rituximab, an anti-CD20 monoclonal antibody, showed promising results including increased progression-free survival (PFS) with equal rates of adverse events in comparison to rituximab alone. Its overall response rate (ORR) in Phase III studies was 83% with a PFS of 24 months compared to an ORR of only 13% and a PFS of 7.3 months seen with rituximab and placebo.1 However, in May 2016, 7 clinical trials were discontinued due to decreased overall survival and higher rates of SAE with idelalisib. The most common SAE, which are now found as a black-box warning, include life-threatening hepatotoxicity, colitis, intestinal perforation, and pneumonitis.2 Interestingly, only 2% of patients in recent trials developed Grade ≥3 rash and only one case of toxic epidermal necrolysis. To our knowledge, this is one of the first few cases of idelalisib-induced severe cutaneous adverse drug reaction that can be found in the current literature. Treatment is mainly supportive with discontinuation of the drug.1 For more severe cases of other complications, like pneumonitis and colitis, systemic corticosteroids can be used.Primarily expressed in leukocytes as a tyrosine kinase, PIK3δ is essential in BCR signaling for the promotion of B cell survival and proliferation.2 Nevertheless, PIK3δ continues to be an important target for research in the treatment of CLL.This illustrates an uncommon, but severe case of adverse cutaneous reaction to idelalisib. Current evidence on the mechanism of this complication, and its more common SAE, remain limited and continued research to elucidate this pathophysiology is important not only for medical knowledge but also to help establish management strategies.


The inextricable interplay between activation of SK channels and Ca2+ dynamics suggests the pathology of one begets another. Yet, the exact mechanistic underpinning for the activation of cardiac SK channels remains unaddressed. Here, we investigated the intracellular Ca2+ microdomains necessary for SK channel activation. SK currents coupled with Ca2+ influx via L-type Ca2+ channels (LTCCs) continued to be elicited after application of caffeine, ryanodine or thapsigargin to deplete SR Ca2+ store, suggesting that LTCCs provide the immediate Ca2+ microdomain for the activation of SK channels in cardiomyocytes. Super-resolution imaging of SK2, Cav1.2 Ca2+ channel, and ryanodine receptor 2 (RyR2) was performed to quantify the nearest neighbor distances (NND) and localized the three molecules within hundreds of nanometers. The distribution of NND between SK2 and RyR2 as well as SK2 and Cav1.2 was bimodal, suggesting a spatial relationship between the channels. The activation mechanism revealed by our study paved the way for the understanding of the roles of SK channels on the feedback mechanism to regulate the activities of LTCCs and RyR2 to influence local and global Ca2+ signaling.Small-conductance Ca Recent studies have provided strong evidence for the existence and functional significance of SK channels in the heart7. All three isoforms of SK channels  are expressed in mouse and human cardiomyocytes8, and SK currents also exist in rabbit and rat ventricular myocytes10, canine pulmonary vein and left atrial myocytes11. Moreover, SK channels play significant roles in cardiac repolarization8, and are potential therapeutic targets against cardiac arrhythmias6.Small-conductance Ca2+ signaling cascade represents one of the most important signaling pathways that controls excitability, excitation-contraction coupling, and contractility of cardiomyocytes, as well as regulates mitochondrial function, cell death, and gene transcription12. Intracellular Ca2+ is tightly controlled on a beat-to-beat basis by multiple molecular complexes to mediate micro- and nano-domain Ca2+ concentrations critical for the precise regulation of diverse function of Ca2+. Main ion channels and transporters of Ca2+ into and out of the cells and intracellular Ca2+ stores include Ca2+ channels, Na+/Ca2+ exchanger, ryanodine receptor 2 (RyR2), and sarcoplasmic reticulum (SR) Ca2+-ATPase. The function of these molecules are orchestrated by a network of subcellular signaling molecules including calmodulin (CaM), Ca2+/CaM-dependent protein kinase II (CaMKII), phospholamban (PLB), cAMP, and protein kinase A (PKA).Ca2+ channels and transporters, gating of SK channels may be regulated by a network of proteins that participate in intracellular Ca2+ regulation. Among them, Ca2+ channels and RyR2 represent the two key molecules that may regulate SK channel gating because of their spatial proximity to SK channels in cardiomyocytes. Our previous studies revealed that cardiac SK2 channels coupled with L-type Ca2+ channels (LTCCs) through a physical bridge, α-actinin2, suggesting that LTCCs may functionally regulate SK2 channels by providing local Ca2+ domain to activate the SK channels13. However, a recent study suggests that RyR2-mediated SR Ca2+ release is both necessary and sufficient for SK channel activation, using SK2-overexpressed rat ventricular myocytes14. Another study reported that inhibition or knockdown of RyR2 or depletion of SR Ca2+ store significantly reduced SK currents in mouse atrial myocytes15. Both studies support the importance of RyR2 in the activation of cardiac SK channels.Similar to Ca2+-activated K+ (BK) channels in neuron. BK and SK channels are regulated by nano- vs. micro-domain intracellular Ca2+, respectively16. However, the precise mechanism for the activation of cardiac SK channels in cardiac myocytes remains incompletely understood. The lack of spatial localization of SK channels, Ca2+ channels, and RyR2 in cardiomyocytes further hinders the understanding of SK gating mechanisms in cardiomyocytes. Indeed, dysregulation of Ca2+ channels and RyR2 can result in life-threatening cardiac arrhythmias, both in hereditary cardiac arrhythmia syndrome or acquired heart diseases. New insights into the mechanistic links underlying the exquisite regulation of SK channels are critically important not only in normal but also in diseased hearts.In addition, previous studies have suggested the dichotomy in the regulation of SK compared to the large conductance Ca2+ channels and RyR2 in cardiomyocytes. We functionally test the source of local Ca2+ domain required for the activation of SK channels in rabbit ventricular myocytes. Our data provide high resolution quantification of a protein complex including SK2, LTCC, and RyR2 in cardiomyocytes. The distribution of nearest neighbor distances between SK2 and RyR2, as well as SK2 and LTCC, is bimodal, suggesting a spatial relationship between the channels. Our study reveals that Ca2+ influx through LTCCs and Ca2+ release via RyR2 provides the immediate and efficient Ca2+ microdomain for the activation of SK channels.In this study, we used stimulated emission depletion (STED) microscopy to acquire high resolution images to precisely localize SK channels in relation to Ca3. Recent studies further show that apamin-sensitive currents are expressed in rabbit ventricular myocytes17. Here, we document the functional expression of SK channels in rabbit ventricular myocytes by directly recording the apamin-sensitive currents and clamping the intracellular free Ca2+ concentration. Indeed, a recent study has shown that apamin is highly specific for SK channels18. Shown in Fig. Apamin-sensitive SK currents were previously identified in mouse and human hearts to play critical roles in atrial repolarization2+ influx through LTCCs, we designed a voltage-clamp protocol using a prepulse to progressively induce Ca2+ influx via LTCCs, followed immediately by a test pulse to monitor the SK currents. To avoid contaminations from other currents, specific inhibitors for transient outward K+ current (Ito), rapidly activating (IKr) and slowly activating delayed rectifier K+ currents (IKs), inward rectifier K+ currents (IK1), and Cl− currents were applied during the recordings. In addition, the test pulse was stepped to the observed reversal potential for Ca2+ currents (ECa) to minimize inward Ca2+ current (ICa) during the test pulse. Figure + currents progressively increased depending on the Ca2+ influx, suggesting intracellular Ca2+-dependence of the outward currents. For each cell, we first recorded the Ca2+ currents and measured ECa, then applied the same potential as the test pulse to monitor the SK currents. The protocol for ICa recordings and the typical current traces are shown in Fig. Ca in pC)  Fig. . The out2+ chelators with similar steady-state binding affinities but different binding rate constants with BAPTA being ~150 times faster than EGTA19. Therefore, the free Ca2+ concentration profile at the internal mouth of LTCCs is predicted to decline much more steeply with distance in BAPTA compared to EGTA16. In addition, previous studies have documented that processes that are sensitive to both BAPTA and EGTA are located within Ca2+ microdomains with distances ~50 nm to a few hundred nm. In contrast, processes that are affected by BAPTA, but not EGTA, are localized within “Ca2+ nanodomains” (within ∼20–50 nm of the Ca2+ source)19. We, therefore, compared the effects of BAPTA and EGTA on the activation of SK currents. Intracellular EGTA and BAPTA at a high concentration (10 mM) significantly reduced and completely abolished the Ca2+ influx-dependent, apamin-sensitive and progressively increasing K+ currents, respectively . We observed the Ca2+ influx-dependent, apamin-sensitive and progressive increase in K+ currents in both buffer conditions. However, the activation kinetics of the SK currents was markedly different, with much faster activation time course when BAPTA was used compared to EGTA. The half-activation time was ~3–4 times smaller in BAPTA compared to EGTA.Next, we tested the effects of intracellular BAPTA and EGTA at a lower concentration (50 µM) as shown in Fig. 2+ influx through Ca2+ channels may also trigger Ca2+ release from the SR. Therefore, Ca2+ sources for the activation of the SK currents may originate from both Ca2+ influx through LTCCs and Ca2+ release from RyR2. To identify whether Ca2+ influx through LTCCs is sufficient to activate the SK channels, we inhibited the RyR2 Ca2+ release using 30 µM ryanodine or depleted the SR Ca2+ store by using 10 mM caffeine or 1 µM thapsigargin. As shown in Fig. 2+ release, suggesting the critical role of Ca2+ influx through LTCCs in the activation of cardiac SK channels. However, the activation kinetics was significantly altered with shorter half-activation time when caffeine or thapsigargin was applied, suggesting the participation of SR Ca2+ release in the activation of SK channels .Figure X-Y) denote the nearest neighbor distances between channel X and channel Y. That is, NND(SK2-RyR2) will denote the NND between SK2 and RyR2 channels. Since NDD(SK2-RyR2) may differ from NND(RyR2-SK2), we will report both findings and determine if there is a significant difference between these two measurements.Results shown are obtained from first filtering and thresholding the STED images to detect channel clusters. Figure SK2-SK2) and NND(RyR2-RyR2) follow a unimodal Gamma distribution. NND(SK2-SK2) are 0.57 ± 0.51 μm while NND(RyR2-RyR2) are 0.83 ± 0.54 μm (data represent mean ± standard deviation). Indeed, the measurement for RyR2 channels from STED images closely agrees with the previous measurements using confocal imaging20 and super-resolution technique based on single-molecule localization21.The distributions pertaining to SK2 and RyR2 channels are summarized in Fig. RyR2-SK2 and SK2-RyR2) NNDs are summarized in Fig. RyR2-SK2) and NND(SK2-RyR2). This difference can be explained by the fact that we observed more RyR2 than SK2 channels. NND(SK2-RyR2) are 0.47 ± 0.46 μm while NND(RyR2-SK2) are 0.55 ± 0.65 μm. Importantly, as shown in Fig. SK2-RyR2) is bimodal with first and second modes at 0.09 μm and 0.41 μm, respectively. This bimodal distribution can be obtained from a finite mixture of two Gamma distributions. The bimodal aspect of NND(SK2-RyR2), as opposed to an exponential distribution, suggests a relationship  between the two channels.The inter-channel (SK2-SK2) are 0.50 ± 0.23 μm while NND(RyR2-RyR2) are 0.62 ± 0.27 μm. NND(RyR2-SK2) and NND (SK2-RyR2) are 0.29 ± 0.31 μm and 0.29 ± 0.28 μm, respectively (P = NS).Our analyses further suggest that there is a statistical difference in the distribution of the two channels localized on the surface Table  or in thSK2-SK2) are 0.68 ± 0.53 μm while NND(RyR2-RyR2) are 0.63 ± 0.28 μm. In contrast to the cell surface, there was a statistically significant difference between NND(RyR2-SK2) and NND(SK2-RyR2)  for channels localized within the cell interior (P < 0.05).For channels localized within the interior of the cells, NND to +1 (positive linear correlation).Another measure of colocalization that has the benefit of being an intuitive interpretation is the probability of the presence of one channel given that there is record of another. These probabilities simply show how likely we are to see one particular channel if we are certain (or there is a record) of the presence of another channel. We calculate the conditional probabilities based on the presence of signals rather than their intensities . On the cell surface, the probability of the presence of an RyR2 channel given that there is record of an SK2 channel (Prob (RyR2|SK2)) was 67.8% while the probability of the presence of an SK2 channel given that there is a record of an RyR2 channel (Prob (SK2|RyR2)) was 56.3%. In the cell interior, Prob (RyR2|SK2) and Prob (SK2|RyR2) were 54.1 and 53.6%, respectively. In summary, ~2/3 of RyR2 channels have associated SK2 channels while ~half of SK2 channels have associated RyR2 channels.v1.2 channel expression together with merged images in rabbit ventricular myocytes. NND(SK2-SK2) and NND(Cav1.2-Cav1.2) are summarized in Fig. SK2-SK2) and NND(Cav1.2-Cav1.2) follow a unimodal Gamma distribution. The inter-channel (SK2-Cav1.2 and Cav1.2-SK2) NNDs are summarized in Fig. SK2-Cav1.2) and NND(Cav1.2-RyR2). Notice that NND(SK2-Cav1.2) displays the characteristics of a bimodal distribution with first and second modes at 0.08 and 0.41 μm, respectively. This bimodal distribution can be obtained from a finite mixture of two Gamma distributions. Similar to SK2 and RyR2 channels, the bimodal distribution of NND  suggests that there is a non-random functional or spatial relationship between SK2 and Cav1.2 channels. Additionally, the Pearson’s correlation coefficient between SK2 and Cav1.2 was calculated to be 0.8. This is a strong indication that SK2 and Cav1.2 channels co-localize.Figure 7. A decade of studies of cardiac SK channels have paved the way for the mechanistic understanding of the critical roles of SK channels in the regulation of cardiac excitability4. However, the exact mechanistic underpinning for the activation of cardiac SK channels remains a key but unaddressed question in the field. The unique gating property of SK channels solely by intracellular Ca2+ highlights the significance of Ca2+ signaling in the regulation of SK channels. Two central molecules dominating the intracellular Ca2+ signaling in cardiomyocytes are LTCC and RyR2. Therefore, understanding the physical and functional relationships between these two molecules and SK channels is important not only from biophysical but also physiological and pathological points of view.SK channels contribute to cardiac repolarization, participate in electrical remodeling in heart failure and atrial fibrillation, and may serve as potential therapeutic targets against cardiac arrhythmias13. The interaction localizes SK channels to the entry of external Ca2+ source, suggesting the functional coupling between cardiac SK2 and LTCCs. SK channels are very sensitive to local subsarcolemmal Ca2+ and are activated by submicromolar concentrations of intracellular Ca2+ ions with apparent Kd of ~0.5 µM2. Our current study provides the estimation of the physical distance of LTCCs and RyR2 within hundreds of nanometers from SK2 channels in cardiomyocytes. The spatial proximity of the three molecules enables the optimal activation and precise control of the SK2 gating process on the beat-to-beat basis by integrating the local Ca2+ signaling. Moreover, SK channel activation is predicted to provide a feedback mechanism to regulate the activities of LTCCs and RyR2 to influence the local and global Ca2+ signaling through the effects of SK channels on membrane potentials.Our previous study reported that cardiac SK2 channels interact with LTCCs through a physical bridge, α-actinin2KCNN1, 2, and 3. The three subtypes of SK channels differ in their sensitivity to apamin1. All three subtypes are expressed in rabbit ventricular myocytes, as supported by the current recordings when three different concentrations of apamin were applied  suggests that there is a non-random functional or spatial relationship between SK2 and RyR2, as well as SK2 and Cav1.2 channels. Additionally, the Pearson’s correlation coefficient between SK2 and Cav1.2 was calculated to be 0.8. This is a strong indication that SK2 and Cav1.2 channels co-localize.Our analysis of the nearest neighbor distributions for SK2-CaSK2-RyR2) and NND(SK2-Cav1.2) is consistent with the well documented co-localization between Cav1.2 and RyR2 channels. Indeed, both the first and second modes of the two distributions are within 0.01 μm of one another.Figure 2+ content eliminated SK current in response to depolarization with intact Ca2+ influx through LTCCs, suggesting that SR Ca2+ release is both necessary and sufficient for the activation of SK channels in rat ventricular myocytes14. However, overexpression of SK2 channels in rat ventricular myocytes may significantly alter the SK2 localization in relations to LTCCs and RyR2. In contrast, our study explored the activation mechanism of endogenous SK channels in rabbit ventricular myocytes. Our findings revealed that Ca2+ influx through LTCCs provides the immediate and efficient Ca2+ microdomain for the activation of SK channels. Moreover, changes in SK channel activation kinetics following SR Ca2+ depletion suggest that both LTCC-mediated Ca2+ influx and SR Ca2+ release are necessary for the activation of cardiac SK channels. Indeed, our results are further supported by a recent study which shows that the inhibition of RyR2 or SERCA or knockdown of RyR2 by shRNA reduces but does not completely eliminate the SK currents in mouse atrial myocytes15.Using SK2 channel-overexpressing adult rat ventricular myocytes, it was reported that the complete depletion of SR Ca2+ uptake and release. Nonetheless, in our recording condition, SK channel activation kinetics was significantly altered after the inhibition of the SR Ca2+ release, suggesting the important role of SR Ca2+ release in the activation of SK channels  10, glucose 6, pyruvic acid 2.5, pH = 7.4. When blood was removed from the coronary circulation, the solution was supplemented with 1 mg/ml type II collagenase , 0.05 mg/ml protease type XIV  and 1 mg/ml bovine serum albumin. The heart was perfused for 30 min to enzymatically dissociate cells. Portions of the left ventricular wall were cut into small pieces and the cell suspension was washed with the above solution. Finally, the Ca2+ concentration was gradually restored to 1.2 mM.All animal care and procedures were approved by UC Davis Institutional Animal Care and Use Committee and was in accordance with NIH and institutional guidelines. For isolation of the rabbit ventricular myocytes, New Zealand White rabbits  were first injected with heparin (5000 U) and then anesthetized with isoflurane (5%). After achieving deep anesthesia, a standard enzymatic technique was used to isolate ventricular myocytes at 37 °C. Briefly, hearts were mounted on a Langendorff system and retrogradely perfused for 5 min with oxygenated solution containing (in mmol/l): NaCl 138, KCl 5.4, CaClv1.2 monoclonal antibody 30; or anti-RyR2 monoclonal antibody )31, incubated overnight at 4 °C, followed by application of secondary antibodies sequentially with tetramethylrhodamine (TMR) donkey-anti-goat secondary antibody (Thermo Fisher Scientific) followed by Oregon Green 488 goat-anti-mouse (Thermo Fisher Scientific) for 1 hour incubation at RT. For STED imaging, ProLong Gold Antifade Mountant (Thermo Fisher Scientific) was used to minimize photobleaching. Negative controls with secondary antibodies alone were performed for all experiments. For testing the SK2 antibody, Human Embryonic Kidney (HEK) 293 cells were cultured and transfected by human cardiac SK2 plasmid using the similar methods we reported before32.STED imaging was performed using Leica TCS SP8 STED 3× microscope. Isolated rabbit ventricular myocytes were fixed with 4% paraformaldehyde on No. 1.5-thickness/12-mm-round glass coverslips  for 20 minutes at room temperature (RT), and then washed 3 times with phosphate buffered saline (PBS) containing 0.05% Triton-X. Non-specific antibody binding was blocked with 10% bovine serum albumin (BSA) in 0.1% phosphate buffered saline with Tween 20 PBST for 1 hour at RT. For double labeling experiments, cells were first treated with a primary antibody , which was tested in HEK 293 cells expressing human cardiac SK2  implementing the standard Pearson’s colocalization analysis35.(A).Image filtering and channel cluster detection34 and can be summarized as follows. An image, I1, is generated from the raw image, I0, based on pixels with intensity lower than a criterion. The criterion is μ0 + ασ0, where μ0 and σ0 refer to the mean value and standard deviation of the raw image I0. A working and filtered image, Iw, is constructed by applying a mask M1 to the raw image I0. The mask is a binary image at a level of μ1 + βσ1, where μ1 and σ1 are the mean value and standard deviation of the image I1, respectively. A threshold, τi, is calculated by taking the ratio of the mean of the original image, μ0, to the mean, μ*, of the pixels with non-zero intensity of the working image.The channel clusters were detected by thresholding the pixel intensity of each image. The threshold value was obtained from a filtering process that is similar to the widely used spark detection methodτ, is calculated by taking the minimum value of all the thresholds, τi, generated from all raw images. The threshold, τ, is then used in the Volocity 3D image analysis software (see http://www.perkinelmer.com/ for more details) to identify three-dimensional connected regions. The filtering process and the statistical calculations were performed in Python. In this study, we used the parameters α = 1 and β = 1.The threshold, (B).Conditional probabilitiesv1.2 analysis.The conditional probabilities of SK2 given RyR2 and RyR2 given SK2 were calculated based on the presence of signals in the recordings as follows:(C).Distributions and finite mixturesThe analysis of STED images was based on the three-dimensional detection of SK2 channel and RyR2 clusters. The channel clusters were identified by thresholding the STED images consisting of 48 Z planes with a Z-step of 0.18 µm. The threshold value was obtained from an iterating process that is similar to the widely used spark detection methodX-Y) is used to denote the distribution of the nearest neighbor distances between channels X and Y. For example, NND (SK2-SK2) stands for the nearest neighbor distribution between SK2 clusters, while NND (SK2-RyR2) stands for the nearest neighbor distribution between SK2 and RyR2 clusters. All distributions were fitted using a finite mixture of Gamma distributions. The choice of a gamma distribution is based on the negative skewness of the data as well as its positive support. For a population of nearest neighbor distances, the following formula for finite mixture was used:αi and βi are the shape and scale parameters of the Gamma distribution. For NND (SK2-SK2), NND (RyR2-RyR2), and NND (Cav1.2-Cav1.2), a single Gamma distribution gave the best fit. For NND (SK2-RyR2) and NND (SK2-Cav1.2), a mixture of two Gamma distributions gave the best result. The use of two Gamma distributions validates the presence of functional coupling between SK2 and RyR2 as well as SK2 and Cav1.2. Additionally, one can test if the channels are randomly distributed by comparing their distribution to an exponential distribution. In the case of the Gamma distribution, this amounts to comparing αi to 1; the closer αi is to 1, the more exponential the distribution of NND becomes, and therefore the more randomly distributed the channels are. Randomly distributed channels will have no functional or spatial relationship between them.The following notation is used. NND: N-methylglucamine (NMG) 140, KCl 4, MgCl2 1, glucose 5, and HEPES 10, pH 7.4 with HCl. The internal solution contained (in mM): potassium gluconate 144, MgCl2 1.15, EGTA 5, HEPES 10, and CaCl2 yielding a free cytosol Ca2+ concentration of 500 nM using the software by C. Patton of Stanford University (http://maxchelator.stanford.edu/). The pH was adjusted to 7.25 using KOH. To isolate apamin-sensitive SK currents, extracellular solutions containing different concentrations of apamin were applied during the recordings, and the difference currents between the control and the apamin-containing solution were calculated to be the apamin-sensitive currents. The current was elicited from a holding potential of −55 mV using a family of voltage steps from −120 to + 60 mV with a 10-mV increment and 500-ms in duration.Whole-cell SK currents were recorded using conventional voltage-clamp technique. For recording of the apamin-sensitive currents under a fixed 500 nM free cytosol Ca2+ influx and Ca2+ released from SR, the extracellular solution contained (in mM): NMG 130, KCl 1, glucose 10, 4-amiopyridine 5, CaCl2 2, niflumic acid 0.05, E4031 0.001, chromanol 0.01, BaCl2 0.05, and HEPES 10, pH 7.4 with glutamic acid. The pipette solution consisted of (in mM): KCl 45, NMG 120, 10 HEPES, EGTA (or BAPTA) 0.05, pH 7.25 adjusted by glutamic acid. The voltage protocol was designed to isolate the SK currents as shown in Fig. For recording the SK currents activated by CaAll experiments were performed using 3 M KCl agar bridges. Cell capacitance was calculated as the ratio of total charge (the integrated area under the current transient) to the magnitude of the pulse (20 mV). Currents were normalized to cell capacitance to obtain the current density. The series resistance was compensated electronically. In all experiments, a series resistance compensation of ≥90% was obtained. Currents were recorded using Axopatch 200 A amplifier , filtered at 1 kHz using a 4-pole Bessel filter and digitized at a sampling frequency of 5 kHz. Data acquisition and analysis were carried out using pClamp 10 software (Molecular Devices) and Origin 6.1 software .t-test. P values of ≤0.05 were considered significant unless otherwise indicated.Where appropriate, pooled data are presented as mean ± S.E.M. Significant differences between the control and the experimental groups were tested using student’s Supplementary Information


Sentinel lymph node biopsy (SLNB) is well accepted to be a standard procedure in breast cancer surgery with clinically negative lymph nodes. Isosulfan blue is the first dye approved by the USA Food and Drug Administration for the localization of the lymphatic system. Few alternative tracers have been investigated. In this study, we aimed to compare the differences between 10 % fluorescein sodium and 1 % isosulfan blue in breast sentinel lymph node biopsy and to investigate the feasibility of using 10 % fluorescein sodium as a new dye for breast sentinel lymph node biopsy.A total of 30 New Zealand rabbits were randomly divided into the fluorescein sodium group and the isosulfan blue group (15 rabbits per group). Fluorescein sodium or isosulfan blue was injected subcutaneously into the second pair of mammary areolas.The average fading time of the second lymph nodes in the isosulfan blue group was significantly shorter than that in the fluorescein sodium group. Moreover, the detection rates of SLNs were higher in the fluorescein sodium group than in the isosulfan blue group. No significant differences between the fluorescein sodium group and isosulfan blue group were observed regarding the distances between the detected sentinel lymph nodes and second pair of mammary areolas, the distances between the second lymph nodes and second pair of mammary areolas, the number of detected sentinel lymph nodes and second lymph nodes, the average dyeing time of the sentinel and the second lymph nodes, and the average fading time of the second lymph nodes.In summary, we first reported that fluorescein sodium is a potential new tracer for breast sentinel lymph node biopsy. Breast cancer is the most common carcinoma in females and the second leading cause of cancer death in women . The senCurrently, different tracer materials have been reported in SLNB of breast cancer, such as blue dye, radioisotope, carbon nanoparticles, or a combination , 5. HoweFluorescein sodium (FS) is a fluorescent dye that is widely used in ophthalmic surgery and malignant brain tumor surgery , 10. TenThirty female New Zealand White rabbits weighing between 2.0 and 2.5 kg were randomly divided into the FS group and isosulfan blue group (15 rabbits each group).Solutions of 10 % FS  and 1 % isosulfan blue  were prepared using sterile water for injection as the solvent. Rabbits were anesthetized with pentobarbital intravenously . After anesthesia, rabbits were positioned supine and the planned injection sites were clipped. The skin flaps were elevated from the second mammary glands and extended to fossa axillaris bilaterally. Careful blunt dissection was performed to avoid the injury of blood vessels and lymphatic vessels. Intradermal injection of 0.3 ml 10 % FS or 1 % isosulfan blue was made at the bilateral sites of the second mammary gland of rabbits in FS group or isosulfan blue group, respectively.The operating room lights were adjusted to the dim mode, and a hand-held 485-nm wavelength blue light source  was used as exciting light for FS. Goggles with a blue light filter were equipped to search for the yellow-green FS-detected SLNs Fig. . The FS-After the observation of all of the data, animals were sacrificed with pentobarbital intravenously .p values were calculated by Student’s t test. Fisher’s exact test was used to compare the detection rates of SLNs between the two groups. All of the statistical analyses were carried out using STATA 11 . Differences with a p value less than 0.05 were considered statistically significant.The numerical data were presented as the means ± standard deviation (SD), and All rabbits in the FS group and isosulfan blue group were kept in the same cleaning cages and were fed with the same rabbit feed . No significant differences were observed between the FS group and isosulfan blue group regarding weight, the distances between the detected sentinel lymph nodes and second pair of mammary areolas, or the distances between the second lymph nodes and second pair of mammary areolas  and second lymph nodes . Furthermore, no significant differences were observed between the FS group and isosulfan blue group in the fading time of SLNs . However, the fading time of the second lymph nodes was much longer in the FS group than in the isosulfan blue group.p = 0.024). To further investigate the superiority of FS in SLN detection, we performed intradermal injection of 0.3 ml of 10 % FS into the second mammary gland of rabbits in which isosulfan blue was failed to detect the SLNs. All of the SLNs that could not be detected in the isosulfan blue group were successfully identified by the supplementary injection of FS.The detection rates of SLNs in the FS group were each 100 %. By contrast, the detection rates of SLNs in the isosulfan blue group (80 %) were significantly lower than those in the FS group  has been regarded as a novel method in SLNB in breast cancer. The detection rate of using ICG was reported to be 80 %, which is slightly lower than that of FS . FurtherIn summary, our study is the first report to compare the use of 10 % FS and 1 % isosulfan blue in SLN mapping of breast cancer. We found that it is feasible and safe to use FS in SLNB of breast cancer. Moreover, as a potential tracer, FS is superior to isosulfan blue regarding certain aspects of SLNB of breast cancer. However, our data are based on animal models; further studies are still required to evaluate the safety and feasibility of using FS in SLNB of breast cancer in patients.


DNMT3AR882 mutations act as dominant negative alleles in vitro1 and are associated with focal regions of DNA hypomethylation in primary acute myeloid leukemia (AML) samples and non-leukemic hematopoietic cells2. In primary AML cells, this hypomethylation manifests both as methylation loss and attenuated CpG island hypermethylation relative to normal hematopoietic stem/progenitor cells. Although DNMT3AR882 mutations have a clear effect on DNA methylation in AML cells, the functional consequences of these changes are not yet clear. Future study of the downstream effects of mutant DNMT3A-associated hypomethylation will require model systems to investigate the genomic targets that are affected, and to understand whether these changes alter gene regulation in ways that promote leukemogenesis. Examples of model systems include genetically modified mice, patient-derived xenografts, and human cell lines containing DNMT3AR882 mutations. The methylation phenotypes of mice lacking Dnmt3a, or expressing mutant Dnmt3a alleles, have been reported previously6, but much less is known about whether alterations in methylation caused by DNMT3AR882 alleles are retained in either patient-derived xenografts or human AML cell lines, and whether these models could therefore be used to accurately represent DNMT3AR882-dependent methylation changes in AML cells.DNMT3AR882C mutation7. We also evaluated four xenografts derived from a primary AML sample containing the DNMT3AR882H mutation. The OCI-AML3 line was obtained from the DSMZ cell collection and cultured via recommended conditions before DNA extraction at two independent passages for WGBS. The presence of the DNMT3AR882C allele in these cells was verified via targeted sequencing prior to methylation analysis  and the NRASQ61L mutation. No functional mutations were identified in other recurrently mutated AML genes with roles in epigenetic modification, such as IDH1, IDH2, ASXL1, EZH2, or TET2. Two missense variants of unknown significance were present in TET1 . We also extracted two replicate DNA samples from comparator AML cell lines that are wild-type for DNMT3A, including Kasumi-1 and NB4, which have a t translocation (creating the RUNX1-RUNX1T1 fusion gene) and a t translocation (resulting in a PML-RARA fusion), respectively. Patient-derived AML xenografts were generated in two independent humanized NSG mice (NSG-SGM3) from a primary AML sample with the DNMT3AR882H mutation  using DNA from OCI-AML3 cells, which is the only leukemia cell line currently known to have a native + cells, and primary AML samples with and without DNMT3AR882 mutations2. The primary AML samples demonstrated methylation patterns that were previously reported to be associated with DNMT3AR882 mutant AML samples, including lower methylation overall, and at CpG island-shores, compared to normal CD34 + cells. We also detected attenuated hypermethylation at CpG islands compared to AMLs with wild-type DNMT3A  in all genomic regions, including subtle hypomethylation at CpG island-shores, and attenuated hypermethylation of CpG islands  that were hypomethylated in primary AML cells with lls Fig. . Statistlls Fig. , and 90%pes Fig. . We perflls Fig. .DNMT3AR882 in this cell line. We verified that the mutant and wild-type alleles of DNMT3A were expressed equally in two replicate RNA-seq experiments  and other genes involved in DNA methylation were also similar between OCI-AML3 cells and a previously published set of 32 primary AML samples2, although expression of DNMT1 and BCAT113 were substantially higher in OCI-AML3 cells (Supplementary Figure 1 was significantly higher than Kasumi-1 cell lines (Figure DNMT3AR882.Given the virtual absence of the focal, canonical hypomethylation phenotype in the OCI-AML3 cell line, we performed additional experiments to assess the function of s Figure , even thDNMT3AR882 that accurately recapitulate the epigenetic phenotype of primary AML samples with this mutation will be critical to understand its functional consequences, and investigate targeted therapies. In this study, we found that DNMT3AR882-associated hypomethylation was preserved in patient-derived AML xenografts with DNMT3AR882, which displayed the same global and focal hypomethylation phenotypes as primary patient samples. The OCI-AML3 cell line, which harbors a DNMT3AR882C allele, showed none of these patterns, and were in fact hypermethylated at many of the DNMT3A-dependent loci. Although these cells have been used to represent AML samples with DNMT3AR882 mutations15, they are clearly not an appropriate model for understanding DNMT3AR882-dependent methylation phenotypes in AML cells, or for making inferences about specific genes or loci that may be dysregulated by DNMT3AR882. We have proposed that CpG island hypermethylation may be a normal response to abnormal proliferation in leukemic cells; these data suggest that the residual de novo methylation activity present in OCI-AML3 cells is adequate to methylate these DNMT3A-dependent regions during long periods of cell culture. It is also possible that these cells never possessed the DNMT3AR882 methylation signature, although previous analysis has shown that primary AML samples with DNMT3AR882 invariably display some level of focal hypomethylation at the loci examined here. Moreover, the similarities between OCI-AML3 and other AML cells lines with different initiating mutations suggests that the methylation patterns in these cells may be related to properties that are associated with immortalization. Regardless, the methylation patterns in OCI-AML3, cells are clearly very different from primary AML samples with DNMT3AR882 mutations, and therefore this cell line is not an appropriate model for understanding genomic patterns of DNA methylation that are caused by the DNMT3AR882 mutation.Models of Supplemental Figure LegendsSupplemental FiguresSupplemental Table S1Supplemental Table S2


Enhanced image contrast in biological second harmonic imaging microscopy (SHIM) has previously been reported via quantitative assessments of forward- to epi-generated signal intensity ratio and by polarization analysis. Here we demonstrate a new form of contrast: the material-specific, wavelength-dependence of epi-generated second harmonic generation (SHG) excitation efficiency, and discriminate collagen and myosin by ratiometric epi-generated SHG images at 920 nm and 860 nm. Collagen shows increased SHG intensity at 920 nm, while little difference is detected between the two for myosin; allowing SHIM to characterize different SHG-generating components within a complex biological sample. We propose that momentum-space mapping of the second-order non-linear structure factor is the source of this contrast and develop a model for the forward and epi-generated SHG wavelength-dependence. Our model demonstrates that even very small changes in the assumed material fibrillar structure can produce large changes in the wavelength-dependency of epi-generated SHG. However, in the case of forward SHG, although the same changes impact upon absolute intensity at a given wavelength, they have very little effect on wavelength-dependency beyond the expected monotonic fall. We also propose that this difference between forward and epi-generated SHG provides an explanation for many of the wavelength-dependency discrepancies in the published literature. Second harmonic generation (SHG) is mediated by the non-linear second-order susceptibility tensor χ(2), which is unusually high in collagen and myosin1. Using near-infrared excitation wavelengths, SHIM penetrates deep into tissues and constructs three-dimensional images of specimens, making it ideal for non-destructive tissue mapping. SHG intensity is influenced by the type2, quantity and organization3 of the collagen present. SHG polarimetry can determine the collagen fibre orientation5 and potentially discriminate myosin, type-I and type-II collagen6. Another potential contrast mechanism is the ratio of forward- to epi-generated signal7. However, wavelength-dependent SHG excitation efficiency has been little studied, with conflicting results and it has not been clear to date whether this signal carries any spectroscopic information.Second harmonic imaging microscopy (SHIM) involves illuminating non-centrosymmetric structures with an ultrashort pulse of focused laser radiation: the resulting transient power densities can then become so high (10(2) is independent of wavelength8. However, even in this case, wavelength-dependence will arise from factors including the variation in dipole oscillator radiated field amplitude with frequency and 1/λ2 fall in focal spot intensity due to diffraction. Consequently, this assumption predicts SHG efficiency falls monotonically with wavelength in a material-independent way. Shen et al.9 produced results that support this assumption when measuring 10 µm sections of mouse tail tendon, in both the forward- and epi- directions. Cox et al.10 have stated that there is no wavelength dependency when imaging thin sections of collagen tissue; Zipfel et al.11 measured SHG emission from collagen I gels and rat tail tendon sections using excitation wavelengths between 730 nm and 980 nm, describing an identical monotonic fall with wavelength for both materials, although this work appears to assess wavelength dependency for forward generated SHG only. However, some groups report more complex behaviours. For example, Zoumi et al.12 investigated epi-detected SHG efficiency from a synthetic skin model excited between 730 and 880 nm, reporting signals that peaked at 800 nm before plateauing at 760 nm. Theodossiou et al.13, meanwhile, observed that forward-detected SHG from rat tendon was very low at 800 nm reaching a maximum at 880 nm, while epi-detected SHG displayed multiple peaks of comparable amplitude. Deng et al.14 report epi-generated material dependent wavelength dependency, with an increase in intensity with wavelength for stromal tissue and an 850 nm maximum for hyperplastic parenchyma. Using a Zeiss LSM510 META system, we previously made epi-direction measurements on dermis, intervertebral disk, bovine tendon and various electrospun polymer scaffolds used in tissue-engineering and also found more complex behavior15. Notably, we reported SHG efficiency from type I collagen-containing samples appeared to increase at longer wavelengths, directly contradicting expectations based on the Kleinman symmetry.The full permutation symmetry (Kleinman symmetry) is widely assumed in biological SHIM and requires that χet al.16 expressed the far-field SHG intensity S2 under plane wave illumination as: -G is the second-order non-linear structure factor and is the 3-D Fourier Transform of the hyperpolarizability distribution β(r). In the forward-direction G changes, even if χ(2) remains constant.Freund n(λ) for water17 demonstrates the excitation wavelength range 0.76–1.0 µm corresponds to a momentum-space range of only 0.25 µm−1 in the forward-direction but 12 µm−1 in the epi-direction. Features in G due to the microstructure of β(r) are therefore much more likely to be resolved by a wavelength scan of epi-generated rather than forward-generated SHG.Literature data for r) and thus smears out features in G. The model used is based on Boyd’s paraxial solution to Gaussian beam propagation through a non-linear medium18. Whilst more accurate models have been recently developed19, Boyd’s approach results in a Fourier-transform relation between the axial distribution χ(2)(z) and the 1-D structure factor  inserted into the light path of a standard photomultiplier tube detection channel of our LSM510. The intensity of the image of fibroblast-generated type I collagen was determined with each filter using excitation wavelengths between 740 and 1000 nm  or detection efficiency , the system response was compared to external calibrations in two ways.22, and the ratio derived (z), representing fibres with 0.45 µm and 0.5 µm diameters and 0.23 µm period in χ(2) in the up/down polarity of the harmonophores. The models show that although this small change in the assumed fibrillar structure of χ(2)(z) impacts upon the absolute intensity at a given wavelength, it has very little effect on forward-generated SHG wavelength-dependency. For both 0.5 µm and 0.45 µm diameter fibres, there is a monotonic fall in forward-generated SHG intensity with increasing wavelength .It is however very important to note that the models presented here are intended primarily to illustrate the potential impact of such small changes on SHG wavelength-dependency in both epi- and forward directions; they are in no way intended to be predictive of the actual spatial structure of the collagen and myosin fibres in the samples. Further research is required to optimize the model parameters to facilitate fully accurate predictions. Potentially this would involve a combination of angle, polarization and wavelength-resolved measurements. However, collagen fibres do associate into bundles and so, in general, have a greater diameter than myosin fibres (e.g. 100–500 µm(2) is independent of wavelength. Conflicting accounts of wavelength dependency for epi-detected SHG may also be a result of variations in the proportions of epi-generated and backward-reflected forward-generated SHG within the signal detected, since, as we have shown, backward-reflected forward-generated SHG will have minimal wavelength dependency.We have demonstrated experimentally that SHG can carry spectroscopic information sufficient to discriminate different biological samples. Thus, by obtaining ratiometric SHG images at 920 nm and 860 nm, additional information on the nature of the SHG-generating components can be obtained. Furthermore, previous reports of strong wavelength-dependent SHG efficiency should not be discounted as system artefacts because we have shown that a theoretical rationale exists to explain such differences, particularly with epi-generated SHG, even when χ26.Our work shows the potential of this new source of contrast in SHIM; myosin within smooth muscle tissue and collagen from the surrounding connective tissue can be identified separately. Further work however is required to determine if these results can be reproduced with a range of collagen samples, including non-fibrous structures such as collagen gels. The technique might also be extended to allow SHIM to identify other SHG-generating biomolecules such as microtubules. Additionally, we hypothesize that with optimization of the model parameters, ratiometric information from SHIM at different wavelengths could provide additional structural information, in a similar manner to the ratio of forward- to epi-generated signalAll materials were purchased from Sigma Aldrich  unless otherwise stated.27, and cultured in fibroblast culture medium. . HDFs were used between passages 4 and 11.Normal HDFs were isolated from split thickness skin as described previously5 cells per well in a 6-well plate, in fibroblast culture medium supplemented with ascorbic acid-2-phosphate (50 µg/ml) and incubated for 21 days, replacing the supplemented medium every 2 or 3 days.HDF were seeded at 1 × 10Human tissue samples were obtained with informed consent and appropriate ethical approval (Human Tissue Bank licence no. 12179), anonymised and used in accordance with this licence.SHG images were obtained using an EC Plan Neofluar 40x oil objective (NA 1.3) on a Zeiss LSM510 META upright confocal microscope, equipped with a tuneable femtosecond pulsed Chameleon Ti:sapphire two-photon laser. SHG signal was collected in the backwards scattering, epi-direction (our system does not provide wavelength-resolved measurement in the forward-direction) using the HFT KP650 primary dichroic filter and META detector. Samples of bovine tendon, at least 1 cm thick, were imaged at depths of 10–40 µm using wavelengths between 760 and 1000 nm, in 20 nm increments. The laser power at the sample, measured by a thermal power meter below the objective, was kept constant throughout (10 mW). The pinhole was set to maximum and conditions were kept constant in terms of pixel number (512 × 512), amplifier gain (1) and offset (0.01), detector gain (604), pixel dwell time (6.39 µs) and averaging (x2). For each sample a spectral stack was generated from the signal between 362 and 704 nm, in 32 channels with 10.8 nm resolution.SHG images were obtained as described above except the SHG signal was collected, again in the epi- direction, using the commercial, turn-key LSM510 PMT detector and one of the following narrow band filters; ET390/20x, ET 420/20x or ET460/30x . All other settings were kept constant for each sample. The samples imaged were type I collagen generated by human dermal fibroblasts over 21 days in culture, bovine tendon, and myosin from chicken breast muscle. Combined samples of myosin and tendon were imaged using differing laser powers for the two regions (60 mW and 10 mW respectively), due to variations in the intensity of the SHG signal from the two components.20 to determine the mean fluorescent intensity for the whole field of view for each channel. The channels containing the SHG signal produced by 860 nm and 920 nm excitation were isolated and combined post-imaging. After image registration, ratio images were displayed by producing a composite RGB image. The 920-nm image was assigned to the red and blue channels, and the 860-nm image to the green channel. Thus, pixels producing a greater signal from 920 nm excitation appear purple and pixels with a greater signal from 860 nm appear green. Where there is no difference in intensity the pixels appear greyscale.Image analysis used ImageJBy making the slowly-varying amplitude approximation , Boyd describes the electric field associated with the second harmonic signal generated by a focused Gaussian beam as: -U0 defines the electric field amplitude at the Gaussian focus. For an infinite inhomogeneous medium, this equation can be integrated directly to yield:G(Δk). It can be shown that the cone angle of the SHG emission equals  (2)(z) distributions (5a and 5b). A unit-amplitude cosinusoidal oscillation of period 0.23 microns is apodized by a Hamming window of diameter 0.45 microns (5a) or a top-hat function of width 0.5 microns (5b). In both cases a unit-amplitude Hamming function is then added, to provide a consistent and numerically stable phase-matching peak near the origin.Figure −1 corresponds to a maximum unaliased Δk of 268 µm−1, with a momentum-space sampling interval of 0.065 µm−1. The maximum range is more than adequate to accommodate the quasi-phase-matching peak at 44 µm−1 when calculating the epi-generated spectrum, whilst the sampling rate provides four distinct calculated values of the forward-generated spectrum across our excitation wavelength range, with the full spectrum then being evaluated by interpolating between these values.To maintain an adequate momentum-space sampling rate, especially for forward-generated SHG, we zero-pad the spatial window out to 96 µm and discretize into N = 8192 pixels. Hence the spatial sampling rate of 85.33 µmhttps://orda.shef.ac.uk/.The datasets generated and/or analysed during the current study are available from the University of Sheffield online data repository