Submicromolar [Ca2+] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca2+] exerts inhibitory effect during depolarization.SK channels are voltage independent and their gating is controlled by intracellular i.PKA phosphorylation attenuates This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive.2+‐activated K+ (SK) channels expressed in ventricular myocytes (VMs) are dormant in health, yet become functional in cardiac disease. We aimed to test the hypothesis that post‐translational modification of SK channels under conditions accompanied by enhanced adrenergic drive plays a central role in disease‐related activation of the channels. We investigated this phenomenon using a rat model of hypertrophy induced by thoracic aortic banding (TAB). Western blot analysis using anti‐pan‐serine/threonine antibodies demonstrated enhanced phosphorylation of immunoprecipitated SK2 channels in VMs from TAB rats vs. Shams, which was reversible by incubation of the VMs with PKA inhibitor H89 (1 μmol L–1). Patch clamped VMs under basal conditions from TABs but not Shams exhibited outward current sensitive to the specific SK inhibitor apamin (100 nmol L–1), which was eliminated by inhibition of PKA (1 μmol L–1). Beta‐adrenergic stimulation  evoked ISK in VMs from Shams, resulting in shortening of action potentials in VMs and ex vivo optically mapped Sham hearts. Using adenoviral gene transfer, wild‐type and mutant SK2 channels were overexpressed in adult rat VMs, revealing serine‐465 as the site that elicits PKA‐dependent phosphorylation effects on SK2 channel function. Concurrent confocal Ca2+ imaging experiments established that PKA phosphorylation lessens rectification of ISK via reduction Ca2+/voltage‐dependent inhibition of the channels at high [Ca2+] without affecting their sensitivity to activation by Ca2+ in the submicromolar range. In conclusion, upregulation of SK channels in diseased VMs is mediated by hyperadrenergic drive in cardiac hypertrophy, with functional effects on the channel conferred by PKA‐dependent phosphorylation at serine‐465.Small‐conductance Ca 2+‐activated K+ (SK) channels expressed in ventricular myocytes are dormant in health, yet become functional in cardiac disease.Small‐conductance Ca2+] in a biphasic manner. Submicromolar [Ca2+] activates the channel via constitutively‐bound calmodulin, whereas higher [Ca2+] exerts inhibitory effect during depolarization.SK channels are voltage independent and their gating is controlled by intracellular i.PKA phosphorylation attenuates This mechanism underlies the functional recruitment of SK channels not only in cardiac disease, but also in normal physiology, contributing to repolarization under conditions of enhanced adrenergic drive. During ad libitum. In total, 29 Sham rats and 30 TAB rats were used for the present study.Male Sham and TAB Sprague–Dawley rats  were purchased from Charles River Laboratories . Animals were shipped 5–7 days after surgery and acclimatized for 3–4 weeks in the Rhode Island Hospital animal facility. Experiments were performed 4–5 weeks after aortic banding procedure. Animals were fed Male Sham and TAB Sprague–Dawley rats were sedated with continuous isoflurane (1–3%) via induction chamber and nose cone and then the chest was shaved. Transthoracic M‐mode and two‐dimensional echocardiography was performed on a Vevo® 2100 Imaging System . The analysis included recording the dimensions of the left ventricle, as well as the total heart weight/body weight ratio. The entire procedure took 15–30 min and resulted in no pain to the animal. During the procedure, animals were closely monitored for any signs of distress, temperature and heart rate changes via an ECG. Rats were then monitored during recovery from anaesthesia for 1–2 h to ensure normal movement and activity before being returned to normal housing.–1 pentobarbital i.p. as a terminal procedure. The heart was removed from the rats via bilateral thoracotomy, mounted on optical mapping set up or a Langendorff apparatus and retrogradely perfused with Tyrode solution containing collagenase II  at 37°C. VMs were isolated as described previously . Unattached cells were removed after 1 h and the remaining VMs were infected with adenoviruses at a multiplicity of infection of 10 for SK channel and dominant‐negative mutant phospholamban virus (dnPLB) constructs. Myocytes were cultured at 37°C in 95% air/5% CO2 for 36–48 h before the analysis.Whole hearts and VMs were isolated from male Sham and TAB Sprague–Dawley rats. Rats were injected with 120 mg kget al. et al. PacI, before transfection into HEK293A cells (RRID:CVCL_6910) using Lipofectamine™ 2000 . Titre of amplified adenoviral stocks was determined using the Adeno‐X qPCR Titration Kit . To introduce S136D into rSK2 construct, we applied site‐directed mutagenesis using the Quik Change Site‐Directed Mutagenesis Kit . To introduce phosphomimetic mutation S465D and S465A into rat SK2 construct, we applied site‐directed mutagenesis using the Q5® Site‐Directed Mutagenesis Kit  in accordance with the manufacturer's instructions. dnPLB with K3E/R14E mutations was used to enhance SERCa2a function and restore Ca2+ transient amplitude   , phospho‐Ca2+/calmodulin‐dependent kinase II (phospho‐CaMKII) , phospho‐PKA substrate , phospho‐serine/threonine  and phospholamban  , GAPDH , calmodulin (CaM) , phospho‐calmodulin (phosphoCaM) , casein kinase 2a E‐2 (CK2) , protein phosphatase 2A C subunit (PP2A‐C) , Caet al. 2014). Samples (20–30 μg of proteins) were resolved on a 4–20% gel via SDS‐PAGE, transferred onto nitrocellulose membranes, and probed with antibodies specific for these proteins and subsequently probed with a goat anti‐mouse secondary , goat anti‐rabbit secondary  or donkey anti‐goat secondary antibodies . Blots were developed with ECL  and quantified and analysed using ImageJ  and Origin, version 8 .Freshly isolated or cultured rat VMs were lysed in lysis buffer from Cell Signaling , supplemented with phosphatase  and protease inhibitor cocktails  as described previously , supplemented with phosphatase  and protease inhibitor cocktails . A 2 h long immunoprecipitation of SK2 was performed at 4°C using a Catch and Release v2.0 Kit  in accordance with the manufacturer instructions using anti‐SK2 antibody  and a negative control antibody comprising normal mouse IgG . Samples were analysed by immunoblotting.–1 mannitol, 70 mmol L–1 sucrose, 10 mmol L–1 Hepes and 1 mmol L–1 EGTA (pH 7.4). The whole‐cell suspension was placed in a pre‐cooled 5 mL Wheaton™ Potter‐Elveheim Tissue Grinder . The cells were homogenized and homogenate was centrifuged at 700 g for 10 min. The pellet consisting of nuclei and cell debris was discarded and the supernatant was then centrifuged at 17,000 g for 15 min. The brown pellet was considered as mitochondrial fraction and discarded. The supernatant was used to precipitate membrane fraction at 100,000 g for 1 h. Samples were then solubilized using the NativePAGE™ Sample Prep Kit  in accordance with the manufacturer's instructions. Processed samples were resolved on NativePAGE™ 4–16% Bis‐Tris Protein Gels, 1.0 mm, 15‐well  by SDS‐PAGE at 150 V for 1 h and 250 V for 1.5 h. Samples were transferred onto nitrocellulose membranes before being probed with antibodies, as described above. The primary antibodies used were SK2 , Cav1.2α1c L‐type Ca2+ channel (LTCC) subunit , Na+/K+‐ATPase . The secondary antibodies used are described above.For the assessment of native protein complexes using blue native polyacrylamide gel electrophoresis (BN‐PAGE), we used freshly isolated VMs suspended in buffer containing 225 mmol L–1) and phosphatase inhibitor (1 μmol L–1) calyculin A for 15 min.In experiments utilizing site‐specific phospho‐SK2‐S465 antibodies (dilution 1:500), maximum phosphorylation for normalization was achieved by incubation of TAB and Sham VMs with β‐adrenergic agonist isoproterenol (ISO) (1 μmol LThe Duolink® proximity ligation assay (PLA)  allows for the detection of proteins that are colocalized <40 nm of each other. Oligonucleotide labelled secondary antibodies or PLA probes generate signal only when bound in close proximity to two primary antibodies that have bound to the sample in close proximity.Freshly isolated VMs were plated on laminin‐coated coverslips and prepared for the PLA and immunofluorescence by fixing with 4% paraformaldehyde and permeabilized with 0.2% Triton X‐100/PBS (pH 7.2) containing 1% BSA. The PLA probe protocol was followed in accordance with the manufacturer's instructions. Briefly, samples were blocked and washed before incubation with primary antibodies for 1 h at room temperature. Primary antibodies used were SK2  and Cav1.2α1c subunit  or RyR2 . Next, samples were washed and PLA probes added and for ligation they were incubated for 1 h at 37 °C. PLA probes used were Duolink® In Situ PLA® Probe Anti‐Goat PLUS  and Duolink® In Situ PLA® Probe Anti‐Rabbit MINUS . PLA probes were then amplified in accordance with the manufacturer's instructions using a DNA ligase at 37 °C, and then samples were immediately processed for imaging. Samples were also probed for RyR2. Primary antibody used was RyR2  or anti‐calsequestrin (CSQ) . Secondary antibody used was rabbit anti‐mouse IgG (H+L) cross‐adsorbed secondary antibody, Alexa Fluor 488 .–2 in ImageJ . Manders overlap coefficients were calculated to assess the level of colocalization between SK2‐LTCC PLA puncta and RyR2 in ImageJ , with an M1 coefficient of 1.0 indicating complete colocalization of red to green images and an M2 coefficient of 1.0 indicating complete colocalization of green to red images.Images were acquired using a SP5 II confocal system  equipped with a 63 × 1.4 numerical aperture oil objective using the 488 nm line of the argon ion laser and the 546 nm line of the HeNe laser for excitation. Emitted fluorescence was collected at the wavelengths 500–530 nm and 560–660 nm. The signal from each detected pair of PLA probes was visualized as a fluorescent red spot, and was quantified as PLA puncta μmet al. 2014). To record SK channel currents from rat VMs, depolarizing voltage steps from a holding potential of –40 mV at 10 mV intervals were applied at 2 s intervals under voltage clamp at room temperature. For recordings from rat VMs overexpression SK2, voltage steps were from a holding potential of –45 mV. Action potentials (APs) were elicited by short current pulses applied at 1.2× threshold under current clamp. Bath solution (pH 7.3) contained 140 mmol L–1 NaCl, 5.4 mmol L–1 KCl, 1 mmol L–1 MgCl2, 1 mmol L–1 CaCl2, 10 mmol L–1 Hepes and 5.6 mmol L–1 glucose. Recording electrodes were 2–4 MΩ containing nominally Ca2+ free pipette solution (pH 7.2): 90 mmol L–1 K‐aspartate, 50 mmol L–1 KCl, 5 mmol L–1 Mg‐ATP, 5 mmol L–1 NaCl, 1 mmol L–1 MgCl2, 0.1 mmol L–1 Tris‐GTP, 10 mmol L–1 Hepes and 0.1 mmol L–1 Rhod‐2 K+‐salt (Thermo Fisher Scientific). Free [Mg2+] was 1.37 mmol L–1 (Maxchelator)  was used to identify SK currents. UCL‐1684 is a non‐peptidic voltage independent blocker of SK channels (IC50 < 10 nmol L–1) . To inhibit PKA, synthetic peptide PKA Inhibitor 14–22 Amide (PKI)  was added to pipette solution (1 μmol L–1).Whole‐cell patch clamp recordings of currents and membrane potential were carried out using an Axopatch 200B amplifier  filtered at 2 kHz and digitized at a sampling rate of 5 kHz as described previously  equipped with 63× 1.4 numerical aperture oil objective in line‐scan mode at a rate of 5 ms per line, synchronized with the electrophysiological setup. Rhod‐2 was excited using the 543 nm line of the HeNe laser and fluorescence emission was collected at the wavelengths 560–660 nm. Dynamical Rhod‐2 fluorescence signal during Ca2+ transient was converted to [Ca2+]i /(Fmax – F), where Kd Rhod‐2 = 1.58 μmol L–1  was calculated based on , gassed with 95% O2 and 5% CO2. Constant flow perfusion was set to 10 mL min–1 with a peristaltic pump. Hearts were placed in a water‐heated chamber to maintain temperature at 37 ± 0.2°C and then 5 μmol L–1 blebbistatin was added to perfusate to reduce movement artefact. Hearts were stained with voltage sensitive indicator di‐4‐ANNEPS, using 20 μL of stock solution (1 mg mL–1 of DMSO) delivered through a bubble trap, above the aortic cannula. The ECGs were continuously monitored with a Powerlab system . The optical apparatus has been described previously . Hearts were stimulated with 150 ms cycle length and perfused with 50 nmol L–1 ISO and/or 10 nmol L–1 APA. Action potential durations (APDs) and conduction velocities were measured using dF/dt for activation and 75% of AP amplitude for repolarization by means of digital image analysis routines.Beating hearts were harvested from anaesthetized Sham and TAB rats via thoracotomy and were retrogradely perfused through the aorta in a Langendorff perfusion system  with 130 mmol L2+ imaging data was performed using Origin 8.0 . Data are presented as the mean ± SD. Statistical significance between groups were performed using Student's t test (paired and unpaired) and one‐way ANOVA with a Bonferroni post hoc test where appropriate. P < 0.05 was considered statistically significant.Statistical analysis of electrophysiological, biochemical and Caet al. et al. n = 29) and 4.61 ± 0.11* (n = 30) mg g–1 for Sham and TAB respectively , 100% of ex vivo TAB rat hearts develop ventricular tachycardia/ventricular fibrillation compared to 15% of Shams   Fig. C.2+ sensitivity of SK channels increases with dephosphorylation of associated CaM at the threonine‐79 site as a result of decreased levels of SK‐bound kinase CK2, or increased levels of phosphatase PP2A  and PKA, H89 (1 μmol L–1) for 30 min. Subsequent probing of immunoprecipitated SK2 channels with anti‐serine/threonine antibodies   was obtained by subtraction of traces before and after channel inhibition by APA. Figure ISK (blue), ICa  and ICa‐induced cytosolic Ca2+ transient derived from confocal line‐scan imaging recording of Rhod‐2 signal (in red) evoked by a depolarizing step to −20 mV. As seen in Fig. 2+]i still continues to rise, confirming biphasic regulation of SK channels by Ca2+ in native myocytes. Furthermore, the current–voltage (I–V) relationship data presented in Fig. ISK peaks at −20 mV  and declines at higher voltages, whereas amplitudes of both ICa and Ca2+ transients continue to rise  increased ISK amplitude and shifted the I–V curve to the right in parallel to an increase in ICa and Ca2+ transients  introduced into patch‐pipette solution diminished ISK in TAB VMs without significantly altering ICa and Ca2+ transient amplitudes   Fig. A. Theseons Fig. B. Unforet al. et al. et al. et al. et al. –1 for 3 min) increased ICa and Ca2+ transients and evoked ISK in VMs from Sham rat hearts, which was otherwise undetectable under basal conditions. The I–V curve in Fig. ISK from ISO‐stimulated TAB VMs . In line with previous studies in healthy VMs under basal conditions , APA significantly prolonged APD.It has remained unclear why APD and currents in healthy VMs from humans and various animal models are insensitive to APA, despite immunodetection of endogenous SK channels . A lower concentration of APA produces similar results to those when 100 nmol L–1 APA is used .Figure ex vivo hearts from Sham rats stained with the voltage sensitive indicator di‐4‐ANNEPS. Representative APD maps of Langendorff‐perfused Sham hearts under basal conditions and after incubation with 50 nmol L–1 ISO are shown in Fig. –1) has no effects under baseline but evokes significant prolongation of repolarization in the presence of ISO   . Manders coefficients of colocalization were also low, suggesting that the very few SK2‐RyR2 pairs visualized by the PLA technique do not reside in the dyad. Figure Previous studies using heterologous expression systems demonstrated that, in addition to being activated by Ca. et al.  demonstrPLA Fig. . To staiPLA Fig. B. The dABs Fig. C. ImporABs Fig. D, suggeABs Fig. . The ove2+ release channel clusters allowed us to employ mathematical apparatus developed by Trafford et al. . To estimate the decay of [Ca2+]sm, the [Ca2+]i + γ*d[Ca2+]i/dt trace was fit with a single exponential curve using the decay of [Ca2+]i as a guide to fitting  and TAB VMs (10 mV) was 7.6 ± 5.1 μmol L–1  which is within the previously reported activation range for SK channels , adenovirus‐mediated overexpression of rSK2 in cultured VMs produced a robust ISK between 36 and 48 h after infection with a multiplicity of infection of 10. As seen in Fig. ISK in parallel with the increase in Ca2+ transient amplitudes in voltage clamped VMs . This effect was more pronounced at higher depolarizations resulting in ISO‐mediated loss of rectification, as seen in respective current‐voltage relationships . To distinguish between the possible effect of increasing [Ca2+]i and the direct effect of PKA‐mediated phosphorylation of SK channels, rSK2 was coexpressed with dnPLB (K3E/R14E mutation)  and did not lessen the rectification of ISK as observed in the ISO‐treated group . These results are consistent with the hypothesis that PKA phosphorylation enhances ISK predominantly via reduction of voltage‐dependent inhibition of the channels by [Ca2+]i.To gain mechanistic insights into the roles of PKA in modulation of SK function, we used the well‐established experimental system of cultured rat VMs. In line with our previous report  and corresponding Ca2+ transients . To validate our approach, we performed similar analysis in VMs overexpressing an rSK2 mutant with enhanced sensitivity to [Ca2+]i   Fig. B. Pooleet al. et al.  as opposed to rSK2‐WT  but not CaMKII inhibitor KN93 (500 nmol L–1 for 30 min)  enhanced phosphorylation of S465.Next, we obtained custom antibodies that specifically recognize phospho‐S465. The western blot analysis presented in Fig. et al. et al. 2+. The results provide the first evidence that, under β‐adrenergic stimulation, PKA phosphorylation of SK channels evokes ISK that contributes to repolarization in healthy VMs and ex vivo hearts from Sham animals.SK channels are rapidly gaining recognition as a novel therapeutic target to treat cardiac arrhythmias  apams Figs A, whichams Figs C and exals Fig. ; (iii) atem Figs  and 18; 56A Fig. . In lineet al. et al. et al.  measured in voltage clamped VMs from TABs , the dynamics of the ISK response differs from the response of NCX1, another plasmamembrane Ca2+‐regulated transporter with similar EC50 ∼400 nmol L–1, which closely follows both rise and decay of [Ca2+]sm , suggesting that the role of [Mg2+]i in dynamic regulation of SK channels in cardiac myocytes is relatively small compared to [Ca2+]i. Unfortunately, we were unable to assess sensitivity of SK channels to inhibition by Ca2+ in native Sham rat VMs with preserved Ca2+ cycling in the absence of PKA activation. Under these conditions, we were unable to record discernible ISK despite of sufficient [Ca2+]sm for activation of the channels. Future single channel patch clamp studies from native VMs using an inside–out configuration are warranted to address this question.Notably, the abundant divalent cation MgMs Bers, . FurtherR2 Bers, . The fre2+‐dependent arrhythmia in diseased hearts ; National Heart, Lung, and Blood Institute at the National Institutes of Health (NIH) NIH RO1HL135236 (RTC); NIH R01HL110791 (GK); NIH R01HL096669 (BRC); and American Heart Association Grant in Aid 15GRNT25650002 and NIH R01HL121796 (DT).


CaM has an extraordinarily high level of evolutionary conservation, which led to the fundamental assumption that mutation would be lethal. However, in 2012, complete exome sequencing of infants suffering from recurrent cardiac arrest revealed de novo mutations in the three human CALM genes. The correlation between mutations and pathophysiology suggests defects in CaM-dependent ion channel functions. Here, we review the current state of the field for all reported CaM mutations associated with cardiac arrhythmias, including knowledge of their biochemical and structural characteristics, and progress towards understanding how these mutations affect cardiac ion channel function.Calmodulin (CaM) is a ubiquitous intracellular Ca Ca2+ activation of CaM modifies, activates, and de-activates enzymes and ion channels, as well as many other cellular processes. With notable roles in protein phosphorylation and dephosphorylation, cellular Ca2+ metabolism, cyclic nucleotide metabolism, gene expression, cell proliferation, muscle contraction, and proteolysis, CaM modification and regulation has implications for nearly every cell in the body  is low (~0.1 μM). In response to membrane depolarization (initiated by voltage-gated sodium channels); voltage-gated Ca2+ channels allow passage of Ca2+ ions into the cytosol in the near vicinity of RyR2. This activates a Ca2+ induced Ca2+ release mechanism in which the cardiac ryanodine receptor (RyR2) releases Ca2+ from the sarcoplasmic reticulum (SR) in quantities sufficient to elevate bulk cytosolic [Ca2+] from ~0.1 μM to ~1 μM. In cardiomyocytes, elevation of [Ca2+] is sensed by troponin C and this initiates muscle contraction via the sarcomeric proteins. Bulk cytosolic [Ca2+] is then reduced to lower levels (~0.1 μM) by the SR Ca2+-ATPase (SERCA), which pumps Ca2+ back into the SR where it is bound to the Ca2+ buffering protein calsquestrin  transient likely varies based on protein location within a cardiomyocyte. Cellular architecture and micro-domain structure are believed to influence and/or tune changes in [Ca2+] and this topic is the subject of much active investigation  into modified ion channel gating and in turn how this is integrated into overall cardiac function.In silico modeling has made limited progress in this arena, and extensive experimental data are necessary for evaluating each CaM-protein system . OverallGiven the diversity of cellular processes influenced by CaM, loss of, untimely, or improper function of a CaM-modified protein can also elicit consequences beyond cardiomyocyte function. For example, RyR2 calcium handling plays a role in other metabolic pathways. In pancreatic β cells, alterations to the activity of the RyR2 channel and its calcium release have been shown to have implications for insulin release and glucose homeostasis . Explora2+-CaM-protein interactions. All three types of data  are necessary for understanding CaM modification of a target protein, and how a CaM mutation can compromise the cellular process. Interdisciplinary investigations of this type can elucidate the mechanisms CaM uses to transduces changes in [Ca2+] into modified ion channel function. Such studies will aid understanding how changes in CaM protein sequence can be tolerated, cause or contribute to disease, or potentially even someday enhance physiology [Excitingly, the emergence of structural data describing more complete CaM-ion channel interactions is affording insight into the mechanisms of CaM channel modification. Moreover, atomic resolution data is allowing for structure-designed mutations that can interrogate each CaM interaction as an isolated variable with respect to ion channel function. In turn, these data provide a foundation that is necessary to interpret highly complex kinetic rate data of Caysiology .The discovery of mutations in the CaM protein was rather surprising to the many investigators who have been studying CaM structure and function over many decades. As is evident from the large number (now approaching 30) of mutations with clear disease association, the current paradigm has changed. At the most fundamental level, there is an urgent need to better understand the degree of intrinsic variation of gene sequence in all genes and the exact origins of having multiple copies of the same gene in a given organism. In the context of this review, one long-term objective for the field is to understand the extent of sequence variation and how it affects CaM function in cardiac physiology and disease pathology. In the meanwhile, efforts such as the proposed Calmodulinopathy registry are paving the way for research laboratories and physicians to connect to each other on a global scale and foster investigations aimed at improving treatment options for patients suffering from syndromes driven by CaM mutations.


I-motif DNAs have been widely employed as robust modulating components to construct reconfigurable DNA nanodevices that function well in acidic cellular environments. However, they generally display poor interactivity with fluorescent ligands under these complex conditions, illustrating a major difficulty in utilizing i-motifs as the light-up system for label-free DNA nanoassemblies and bioimaging. Towards addressing this challenge, here we devise new types of i-motif/miniduplex hybrid structures that display an unprecedentedly high interactivity with commonly-used benzothiazole dyes (e.g. thioflavin T). A well-chosen tetranucleotide, whose optimal sequence depends on the used ligand, is appended to the 5′-terminals of diverse i-motifs and forms a minimal parallel duplex thereby creating a preferential site for binding ligands, verified by molecular dynamics simulation. In this way, the fluorescence of ligands can be dramatically enhanced by the i-motif/miniduplex hybrids under complex physiological conditions. This provides a generic light-up system with a high signal-to-background ratio for programmable DNA nanoassemblies, illustrated through utilizing it for a pH-driven framework nucleic acid nanodevice manipulated in acidic cellular membrane microenvironments. It enables label-free fluorescence bioimaging in response to extracellular pH change. As two important four-stranded DNA structures, G-quadruplexes (G4s) and i-motifs have proven able to form in telomeric DNAs as well as genic regions of human cells ,2. They Among DNA ligands, molecular rotors generally show a high signal-to-background ratio for lighting up diverse DNA structures due to their weak fluorescence at the twisted intramolecular charge-transfer state . One com5T3C4 (iM9)  to the 5′ terminal of a bimolecular i-motif CC4 (iM9) , as depiC4 (iM9) . SurprisC4 (iM9) ,25,31–33C4 (iM9) . This fiC4 (iM9)  nanodeviC4 (iM9) ,37,38.1H-NMR  δ 8.30 , 8.08 , 8.02 , 7.92 , 7.77 , 7.67 , 7.6 , 6.82 , 4.22 , 3.10 . 13C-NMR  δ 171.77, 153.91, 150.52, 142.41, 133.33, 129.31, 127.87, 127.26, 124.30, 121.94, 116.42, 112.40, 106.77, 36.17. MS(ESI): m/z [M]+ = calcd. for C18H19N2S+ 295.12; found 295.16.Thioflavin T (ThT) and 3,3′-diethylthiacyanine (THIA) were purchased from Sigma-Aldrich (USA) and used without further purification. The concentration of aqueous ThT solution was quantified with UV-Vis spectroscopy as described previously . A recen2 were incubated for 10 min at 90°C and then cooled slowly, allowing them to fold at room temperature. To the DNA solution, 3 μM ThT was added and kept at 22°C for over 5 min before fluorescence measurement.Purified oligonucleotides were obtained as powders from Sangon Biotech , dissolved in TE buffer, and then quantified with UV–Vis spectroscopy. 1 μM i-motif DNAs in pH 5 TAE buffer containing 2 mM MgClThe FNA device  for 5 h. The gel was then stained in 1× GelRed solution and imaged by a Bio-Rad Gel Doc™ EZ imager (USA).2 were measured in an optical chamber (1-mm path length) with a JASCO J-1500 spectropolarimeter . Dry purified nitrogen was used to maintain a deoxygenation atmosphere. The solution background was subtracted from the CD signal.400 μl of 50 μM i-motif DNAs in pH 5 TAE buffer containing 2 mM MgCl2 were denatured at a rate of 0.5°C/min and monitored at 290 nm with the J-1500 spectropolarimeter. Normalized CD signal was adopted.400 μl of 50 μM DNA i-motifs in pH 5 TAE buffer containing 2 mM MgCl2. This FNA system was switched between pH 5 and pH 8 by alternate addition of 0.4% (v/v) 6 M HCl and 6 M NaOH, followed by a 30-min incubation at 60°C. It allows the i-motif DNA on the vertexes of two tetrahedrons to fold properly. The fluorescence spectra excited at 442 nm were recorded by a Hitachi F-4600 Fluorescence spectrometer . The buffer background was subtracted from the fluorescence spectra to reduce the light scattering influence especially in the case of measuring the weak fluorescence of ThT alone.The operation of the FNA device (0.4 μM) was monitored by the fluorescence of ThT (0.4 μM) in the TAE buffer (pH 5) containing 2 mM MgCl+ and Cl− were added as counter ions, then the system was solvated in TIP3P water molecules, which were extended up to 8.0 × 8.0 × 14.0 Å in a cubic box. Totally 6 ns of MDS in npt ensemble was run for the solvated system and the last 3 ns MDS was used as product, with coordinates saved for every 10 ps. The ground state of ThT was optimized by quantum chemical calculations using the Gaussian 16 program. Becke's three-parameter hybrid exchange function with the Lee-Yang-Parr gradient-corrected correlation functional  was used and 6-31G(d) basis set was chosen. The molecular interactions of TiM9 with ThT were studied by software AutoDock 4.2.6 with the Lamarckian genetic algorithm61. The grids number were 110 × 110 × 110 and a grid spacing was 0.375 Å.The model of TiM9 was built on the basis of an already known i-motif structure (PDB ID: 1YBL) from Protein Data Bank,59 then minimized and equilibrated by MDS using the Gromacs-5.1.4 suite with AMBER99SB force field60 under the solvents condition mimicking the experiments. HThe monomeric and dimeric DNA tetrahedrons were prepared with a total concentration of 100 nM. The prepared samples were scanned in scanasyst-air mode by a Multimode 8 Atomic Force Microscope (Bruker Inc.).2 was added and incubated for 1 h. Finally, 0.4 μM ThT was added and incubated for 5 min for fluorescence imaging. In the control experiments at pH 8, however, the ThT dye needed to be removed before cell imaging for an appropriate background. The fluorescence signals of ThT and LysoTracker were collected with the excitation wavelength of 405 and 590 nm, respectively. All the micrographs were taken on a confocal laser-scanning microscope .HepG2 cells were cultured in DMEM medium supplemented with 10% (v/v) FBS and 2% (v/v) 100× stock solution of penicillin-streptomycin mixture. 1 μM LysoTracker Red DND-99 was first added to HepG2 cell culture medium and incubated at 37°C for 20 min. After removing the LysoTracker dye by changing fresh cell culture medium, a mixture of cholesterolated Th46 and Th58 (each 1 μM) in pH 5 TAE buffer with 2 mM MgCl4T3C4A (AiM8A) to explore the ThT-interactive structures. Several nucleotide partners favorable for ThT binding in psDNAs , we, we40), n psDNAs  are heren psDNAs  whereas n psDNAs , indicatn psDNAs ,41. Howen psDNAs . This ten psDNAs . The resn psDNAs , comparan psDNAs . A similn psDNAs . Since tn psDNAs  and benen psDNAs ,25, the n psDNAs  and unimn psDNAs .4T3C3 (iM7) and iM9  even in the absence of ThT as compared to iM7 and iM9, accompanied by an increase in the CD signal  to show its pH-induced dimeric assembly Figure , lane 2.osensors ,30.Since dimeric DNA nanostructures can be visualized by atomic force microscopy (AFM) ,9,18,35,via resetting pH to 8. Figure We further tested the reconfigurability of the aforementioned FNA assembly in situ operated on the membrane surfaces of HepG2 cells, as depicted in Figure via endocytotic internalization  display an enhanced fluorescence emission when bound to the folded i-motifs ,17,41,563T3C4 and C4T3C5 for the 3′-overhang optimization, and demonstrated that 3′-GGGA works quite well (4T3C3 and C5T3C4 (Figure From Figure ite well , with a 4 Figure .We notice that the sequences of optimal overhangs vary with the ligands, as shown in Figures in situ on cellular membrane surface. This lays the foundation for label-free fluorescence bioimaging in response to extracellular pH changes.We have demonstrated a conceptually new strategy for general engineering of i-motif/miniduplex hybrids with high capacity for binding ThT and other molecular rotors. It allows the hybrids to function as a high signal-to-background light-up system as well as a modulating unit for a pH-driven dimeric FNA nanoswitch that can be manipulated It's known that functional biomolecular dimerization plays a crucial role in many physiological processes . Some ofgkaa020_Supplemental_FileClick here for additional data file.


IDH) 1/2–mutated AML, ivosidenib, an IDH1 inhibitor, and enasidenib, an IDH2 inhibitor, present well-tolerated options in the setting of refractory or relapsed (r/r) disease even in elderly and heavily pre-treated patients with response rates of 30–40%. Both substances have been approved by the U.S. Food and Drug Administration (FDA) for r/r AML patients with IDH1/2 mutations (but not yet by the European Medicines Agency (EMA)). For patients with FMS-like tyrosine kinase 3 (FLT3) mutations, treatment with the selective FLT3 inhibitor gilteritinib is well tolerated and leads to improved outcome compared with standard salvage therapy. The approval has been granted by the FDA and the EMA. Generally, we would recommend targeted therapy for IDH1/2- and FLT3-mutated AML if available. In order to improve outcome in relapsed AML, it will be important to intelligently combine novel substances with each other as well as chemotherapy in prospective clinical trials. The development of therapies with bispecific antibodies or chimeric antigen receptor T cells (CAR-T) are still in early development.Relapse is still a common scenario in acute myeloid leukemia (AML) treatment and occurs in 40–50% of younger and the great majority of elderly patients. The prognosis in relapsed AML patients is generally poor but depends largely on the timing of relapse (early versus late) and the possibility of allogeneic hematopoietic stem cell transplantation (HSCT). At the time of relapse, we again perform a mutational screening and cytogenetic analysis in all AML patients as clonal evolution of disease is frequent. Clinical trials should be first priority in all relapsed patients. In fit patients without prior transplant, we aim to perform HSCT after salvage therapy. In AML patients relapsing after HSCT and good performance status, intensive therapy can be considered with subsequent cellular therapy such as donor lymphocyte infusion (DLI) or a second HSCT. However, less than 20% of these patients are alive after 5 years. For those patients that are unfit, the therapeutic aim is to prolong life with acceptable quality of life. Here, hypomethylating agents (HMA), low-dose AraC (LDAC), and solely cytoreductive therapy with hydroxurea are options depending on first-line therapy. For those patients that have not been treated with venetoclax in first line, the combination therapy of venetoclax with demethylating agents achieves encouraging response rates. Venetoclax is currently also studied in combination with intensive salvage therapy. Importantly, for patients with isocitrate dehydrogenase ( FLT3, IDH1, and IDH2 offer novel therapeutic options. Due to next-generation sequencing (NGS) being widely available, mutational screening is frequently done in a targeted panel approach covering the most frequently mutated genes in AML. If NGS is not available, we would at least recommend sequencing the “targetable” mutations  as well as other prognostic mutations  [Despite improvements in acute myeloid leukemia (AML) therapy, relapse is still the most challenging aspect in AML. While 10–40% of younger AML patients are primarily refractory to AML induction therapy, the number is considerably higher for patients above 60 years (40–60%) . Thus, rFLT3- or IDH1/2-mutated patients), treatment with targeted therapies need to be integrated into therapeutic decision making (see below).AML patients relapsing after chemotherapy still have a curative treatment approach if they are fit enough to undergo HSCT . SalvageNearly all elderly patients become refractory or relapse after non-intensive therapy. While HMA treatment such as azacitidine has improved clinical outcome of this patient population, only few patients can be cured with these regimens . While tNPM1-mutated patients). Importantly, favorable remission rates were also observed in prognostically unfavorable subgroups . The combination of LDAC and venetoclax achieved CR/CRi in 54% of elderly previously untreated AML patients [TP53 or FLT3 mutations (30% and 44% respectively). These encouraging results led to the approval of venetoclax by the FDA in November 2018 in combination with azacitidine or decitabine or LDAC for the treatment of newly diagnosed AML in adults who are age 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy . Thus, the question comes up how venetoclax combination therapy works in r/r AML patients. In a multicenter historical study, 23 AML patients who were refractory to HMA treatment or relapsed after HMA therapy were treated with a combination of venetoclax and HMA. Forty-three percent achieved a CR or CRi in this cohort while OS was 74% at 6 months [Venetoclax is a highly selective, oral small-molecule B cell leukemia/lymphoma-2 (BCL2) inhibitor. Members of the BCL protein family regulate apoptosis in a pro- or anti-apoptotic manner. BCL2 shows anti-apoptotic activity, is highly expressed in leukemia stem cells, and maintains myeloblast survival , 12. Thupatients . Here, l6 months . Similar6 months . OverallCPX-351 is a liposomal formulation of cytarabine and daunorubicin mixed in a 5:1 M ratio. The liposomal drug delivery allows prolonged duration of higher plasma concentrations as well as enrichment of the substances in the bone marrow. CPX-351 was first studied in r/r AML patients and high-risk MDS patients in a dose-finding trial. Of 43 r/r AML patients, 9 patients showed a CR and 1 patient a CRi . In a suOther therapies have emerged in first-line therapy of AML. Glasdegib, a small molecular inhibitor of the sonic hedgehog pathway, has been approved by the FDA for newly diagnosed elderly AML patients in combination with LDAC therapy. The approval was based on phase II study with elderly AML or high-risk MDS patients being randomized to the combination therapy of glasdegib and LDAC (88 patients) versus LDAC alone (44 patients). The median OS was 8.8 months with combination therapy and 4.9 months with LDAC alone . WhetherFLT3-ITD and FLT3-TKD mutations are common mutations in AML with frequencies of 20–25% and 6–10%, respectively [FLT3-ITD specifically is a strong adverse prognostic marker at the time of relapse and only a minority of FLT3-ITD-positive patients can be cured at the time of relapse despite aggressive salvage therapy [FLT3-ITD mutation status has been integrated into the prognostic GOELAMS score [FLT3-mutated AML in first-line therapy in combination with intensive chemotherapy by the FDA and EMA. It is now standard treatment for newly diagnosed FLT3-mutated AML patients in combination with intensive therapy [FLT3 mutations had the best response with an ORR of 49% compared with 12% in FLT3 wild-type patients. These promising results in FLT3-mutated AML have led the way for a large randomized phase III trial in r/r FLT3-mutated AML. Here, monotherapy with gilteritinib was compared with chemotherapy  [FLT3-mutated AML by the FDA. We would recommend gilteritinib for patients with FLT3-mutated r/r AML if accessible. Quizartinib is also a next-generation TKI that is relatively selective for FLT3. It was tested in a dose-escalation phase I trial in r/r FLT3-ITD-positive AML patients [FLT3-ITD-positive AML [FLT3-ITD-positive AML patients. Crenolanib, another next-generation TKI with promising results in a phase II trial, is currently also studied in a phase III trial for r/r FLT3-mutated AML (NCT03250338). In summary, next-generation TKIs with specific inhibitory effects for mutated FLT3 are promising in r/r AML. While midostaurin has received approval for FLT3-mutated AML in first line, gilteritinib is now approved for r/r FLT3-mutated AML by the FDA and EMA. It will be interesting to await the results of combination therapy, e.g., gilteritinib with venetoclax, in r/r FLT3-mutated AML (NCT03625505).ectively . Importa therapy . Due to MS score . Thus, tMS score . The firMS score . Due to MS score , midosta therapy . However therapy , 31. How therapy . Gilteri therapy . PatientL trial) ••. Chemopatients . Here, mtive AML . SalvageIDH1 and IDH2 encode for enzymes that catalyze the conversion of isocitrate to a-ketoglutarate in the cytosol and peroxisomes (IDH1) and mitochondria (IDH2). IDH1 and IDH2 mutations are recurrent mutations in AML with a frequency of 7–14% and 8–15%, respectively [IDH1 and IDH2 is the production of the oncometablite 2-hydroxgluterate (2-HG) [IDH1/2 mutations do not have major prognostic relevance [ectively , 36, 37.e (2-HG) . While Ielevance , 20, theIDH1 mutations have been treated with ivosidenib [IDH1-mutant AML, enasidenib has been developed for IDH2-mutant AML. Enasidenib is an allosteric inhibitor that binds to the IDH2 dimer interface and blocks the production of 2-HG of IDH2 mutants. In a phase I/II trial dose-escalation and expansion trial, 239 patients with IDH2-mutant advanced myeloid malignancies were treated [IDH2. A rise in indirect bilirubin serum level was observed in 35% of patients. However, it was not a sign of intrinsic liver toxicity but instead due to off-target inhibition of the UGT1A1 enzyme. 9.6% of patients developed IDH-DS, with grade 3 or 4 in 6% of patients. The mechanisms underlying the evolving resistance to IDH inhibition after initial response have been studied. It was shown that resistance to evosidenib can emerge through the development of second-site IDH2 mutations in trans that inhibit the binding of the drug [Ivosidenib is an oral, targeted, small-molecule inhibitor of mutant IDH1. In a phase I dose-escalation and expansion trial, 258 AML patients with osidenib ••. In thosidenib ••. This  treated , 41••. Athe drug . IvosideFLT3-mutated r/r AML or ivosidenib/enasidenib in IDH1-/IDH2-mutated r/r AML). Cellular therapies such as CAR-T cells and bispecific antibody are less advanced in AML compared with ALL. CD33- and CD123-directed bispecific antibodies have been developed. These bispecific antibodies are also called dual-affinity retargeted antibodies. AMG 330 is anti-CD33 bispecific T cell engager antibody construct that was studied in r/r AML patients in a phase I trial. It was a dose-escalation study that intended to find the optimal dosing schedule. Of 40 patients, the majority of patients discontinued therapy (n = 35) with a median of one cycle being applied. Of the 35 patients who discontinued therapy, 27 patients (77%) discontinued due to disease progression. A total of 73% of patients experienced serious adverse events with cytokine release syndrome being very common (28% of patients) [Currently, we have little data about the efficacy and toxicity of combining novel substances with each other or with chemotherapy in r/r AML. It will be important to gain prospective data in randomized trials that study venetoclax with salvage chemotherapy or other targeted therapies  . SimilarFLT3- and IDH1-/IDH2-mutated AML patients, targeted therapies have shown results superior to standard therapy. The FDA has approved gilteritinib for FLT3-mutated AML as well as ivosidenib/enasidenib for IDH1-/IDH2-mutated r/r AML patients. Monotherapy with venetoclax, a bcl-2 inhibitor, has moderate efficacy in r/r AML. However, early results in combination with intensive chemotherapy or HMA are very encouraging. Trials combining novel substances with each are necessary. The development of cellular therapies such as CAR-T cells or bispecific T cell engager antibody constructs is very demanding in AML as it is difficult to identify the ideal target structure on AML blasts.R/R AML remains a common clinical scenario with poor outcome. HSCT is necessary for long-term remission and survival. Due to the limited treatment options in relapsed patients, clinical trials are the first choice of therapy. For


T-wave alternans (TWA) reflects every-other-beat alterations in the morphology of the electrocardiogram ST segment or T wave in the setting of a constant heart rate, hence, in the absence of heart rate variability. It is believed to be associated with the dispersion of repolarization and has been used as a non-invasive marker for predicting the risk of malignant cardiac arrhythmias and sudden cardiac death as numerous studies have shown. This review aims to provide up-to-date review on both experimental and simulation studies in elucidating possible mechanisms underlying the genesis of TWA at the cellular level, as well as the genesis of spatially concordant/discordant alternans at the tissue level, and their transition to cardiac arrhythmia. Recent progress and future perspectives in antiarrhythmic therapies associated with TWA are also discussed. Apart from TWA visible to the naked eye, several technologies to evaluate MTWA based on the ECG have been developed over the last two decades. Only two of these technologies have been studied in sizeable clinical studies, namely, the modified moving average (MMA) method and the spectral method. The MMA method allows for estimation of TWA during both exercise and in ambulatory ECG monitoring of daily activities  and calcium handling dynamics is bi-directional, however, it is hard if not impossible to identify which is the main and the secondary mechanism between the two. Under diseased conditions in which the feedback between membrane potential and intracellular calcium ([Ca2+]i) is positive, Hopf bifurcations occur, resulting in periodic oscillatory behaviors. When this feedback is negative, period-doubling bifurcation routes to chaos occur . The kinetics of restitution are described by the restitution curve, a plot of APD vs. previous DI. As shown in the n + DIn), longer DI allows for channels to fully recover by the next beat, resulting in longer APD, and the cycle of alternation begins.In 1968, the mechanism of alternans was first described using a graphical method, relating them to APD restitution . This re+ channels are inactivated mainly during the plateau of the AP and they have lesser direct effects on the APD, but incomplete recovery of these Na+ channels lowers the amplitude of AP which impacts the activation of other ion channels, influencing APD. The recovery of Na+ channels, for example, slows down under ischemic conditions  blocker ranolazine attenuated arrhythmogenic cardiac alternans by suppressing CaT alternans and decreasing the Ca2+-voltage coupling gain  and the two components of the delayed rectifier potassium current (IKr and IKs) will suppress alternans by reducing the APD and prolonging the DI  was blocked, suggesting that the alternans was associated with Ito. Ito-mediated spike-and-dome AP morphology is linked to Brugada syndrome (BrS). TWA is widely observed in BrS and alternans occurs at normal heart rates in which the DI is still very long , due to the slow recovery of ion channels and the gradual accumulation of ions on both sides of the cell membrane. The short-term memory effect is called APD accommodation (0 + a × expt/τ) of APD accommodation, and results showed that the mechanism associated with short-term memory can also be the basis for cardiac alternans formation in the heart  or no APD alternans is observed for a steep curve  to prolong the APD by increasing net inward current produced during the plateau of AP, but on the other hand it can also facilitate the inactivation of ICa,L by Ca2+-dependent inactivation and thus shorten the APD. Other calcium-sensitive currents in small quantities, such as calcium-activated non-selective cation currents and calcium-activated chlorine currents, can also affect the membrane voltage (n + DIn), a longer DI allows more time for recovery of ICa,L, leading to enhanced ICa,L, larger Ca2+ release, and longer APD during the following beat. In l971, Spear and Moore  of the SR nearly synchronously, which releases abundant Ca2+ stored in the SR into the cytosol through Ca-induced Ca-release mechanisms  working in “reverse mode.” After each contraction, the majority of cytoplasmic Ca2+ are taken back into the SR by SR-Ca-ATPase (SERCA) and, to a lesser extent, by NCX working in “forward mode.” In this case, cell Vm is determined by the consequences of [Ca2+]i dynamics, Ca2+-dependent ion currents, and Ca2+ fluxes and transporters .It may therefore be helpful to review the key processes involved in the uptake, buffering, and release of [Cachanisms . When IC2+]i probably did not cause electrical alternans. Rather it was the mechano-sensitive ion channels, such as volume- or stretch-activated channels (SACs), that were responsible. Indeed, these mechano-sensitive ion channels are also able to affect membrane potential on an every-other-beat basis and therefore may influence cardiac dynamics. Generally, the effects of MEF on the electrophysiology at high pacing rates are shown to be proarrhythmic ; and (3) regulation of the autonomic nervous system.At the tissue level, cellular alternans can be reflected as SCA, in which the whole tissue exhibits AP and CaT alternans of in-phase changes  to alternans (2:2) and from 2:2 alternans to 2:1 block can be observed, and more complex dynamics at even faster pacing rates. The cellular mechanism of alternans caused by a steep APD restitution curve has been mentioned earlier, but a steep APD restitution curve can further convert SCA to SDA at the tissue level . In restAfter Nolasco and Dahlen’s important early study , the APDThis restitution theory has also been further validated by modeling studies. Using a kinematic model to represent wavefront-waveback interactions and simulations with the Luo-Rudy model in a one-dimensional cable of cardiac cells, 2+ from SR into myocyte cytoplasm and their binding to troponin C is the final signal in myocardial contraction. Synchronous contraction of ventricular myocytes is critical for efficient cardiac pumping function. This requires both shuttling of Ca2+ between the cytoplasm and SR in individual myocytes, and myocardial layer synchronization of this process by electrical coupling among ventricular myocytes. Therefore, the instabilities of [Ca2+]i handling causes SDA and arrhythmias under diseased conditions, such as CPVT and diabetes (2+]i handling (discussion below: section “SDA and arrhythmogenesis arising from pre-existing tissue heterogeneities”), the bidirectional coupling of [Ca2+]i and Vm, and more complex instabilities of intracellular Ca2+ handling.Release of Cadiabetes . These m2+]i → Vm coupling is determined by the fact that [Ca2+]i feeds back on Vm. If the coupling is strong, such that large-amplitude CaT alternans results in large amplitude APD alternans and thus larger dispersion of refractoriness, the SDA and arrhythmogenic circumstances are potentiated. It has been reported that if Ca2+ → voltage coupling changed from positive to negative, it will lead to SDA  can create a situation where cytoplasmic Ca2+ cannot be fully reclaimed during each beat. 2+ uptake and the Ca2+ release on CaT alternans onset. However, 2+ release, and that RyRs rather than SERCA, is a key determinant of CaT alternans in intact working hearts. These experiments suggested that the alternation of systolic Ca2+ level depends on the alternation of SR Ca2+ content. On the contrary, later experiments have since shown that SR Ca2+ content remains constant before the occurrence of CaT alternans under some conditions, such as slow recovery from inactivation of RyRs and increased mitochondrial dysfunction. 2+]  during frequency-dependent CaT alternans in rabbit ventricular myocytes. Their measurements show that alternations in [Ca2+]SR are not required for CaT alternans to occur and it is rather likely that some other factor, such as the slow recovery from inactivation of RyRs, is of greater importance in initiating frequency-induced CaT alternans. Furthermore, the tendency of pacing-induced CaT alternans increases with increasing mitochondrial dysfunction due to inhibition of ATP synthesis or dissipation of the mitochondrial membrane potential  which suggested that CaT alternans continues even if the SR Ca content is held constant. They proposed a novel theory, the “3R” theory, in which CaT alternans emerges as collective behavior of Ca sparks, determined by three critical properties of the CRU network from which Ca sparks arise: randomness , refractoriness , and recruitment  Ca amplitude map]. Just as repolarization, regional differences in the spatial distribution of the APD restitution and [Ca2+]i handling characteristics are observed across the transmural ventricular walls (from epicardium to endocardium) and from apex to base  may be amplified, leading to SDA , excessive catecholamines binds to the receptors which then change the configuration of membrane ion channels through an enzymatic reaction and result in myocardial excitation and an increased heart rate. The parasympathetic and sympathetic efferent nerves reach the heart via the vagus nerve and via the L and R stellar ganglions, respectively. This gives the experimental opportunity to selectively denervate the heart. At present, simulation and experimental studies have shown that sympathetic activity can promote alternans. In early experimental studies, The concept that neural activity can exert a potential effect on arrhythmogenesis was first emphasized in 1976 by Lown and Verrier where they studied how electrical stability changes in the presence of acute myocardial ischemia. They used an animal model where there was 10 min of coronary artery occlusion followed by a sudden release, such that a consistent time process is shown in the emergence of arrhythmias and VF  and has 2+]i-Vm coupling provide the basis for a general hypothesis about the role of TWA-mediated arrhythmogenesis in various pathophysiological conditions. Although TWA mechanisms have been partially elucidated, a clear origin of cardiac alternans  and the spontaneous transition from spatially concordant or discordant alternans to arrhythmias remain ambiguous. Much like in HF, there is extensive ion current remodeling. HF increases the susceptibility to cardiac alternans, which is caused by HF-induced impairment in [Ca2+]i cycling  Furthermore, MEF indicates that alternating pulsus can contribute to electrical alternans. In the heart, numerous simulation and experimental studies have also highlighted SDA formation mechanisms including dynamic factors , pre-existing tissue heterogeneities, and regulation of the autonomic nervous system. SDA provides a substrate for unidirectional block and reentry and the manifestation of SDA is associated with increased spatial dispersion of refractoriness, wavefront fractionation, and the onset of reentrant arrhythmias.Currently potential mechanisms of alternans at the cellular level and SDA and their possible arrhythmogenic consequences at the tissue level have been reviewed. At the cellular level, many hypotheses have been proposed to explain the genesis of cardiac alternans: (1) Steep APD restitution curve is likely due to the actions of one or more ionic currents. Therefore, the maximum slope of the APD restitution curve can be flattened by changing the kinetics of one or more membrane currents, and this has been proved to prevent the development of cellular alternans ; (2) HowUp to date there are currently two techniques for evaluating TWA – the MMA method and the spectral method. The spectral method can be applied for TWA detection during exercise tests, and the MMA method can be applied always for evaluation of the TWA during both exercise and Holter monitoring during daily activities. But a large number of SCD occur unrelated to exercise, and the risk of arrhythmia may be also increased by other factors like mental stress . Thus, tBased on TWA electrophysiological mechanisms, there are two ways to achieve upstream antiarrhythmic effects. The first way is by suppressing cardiac alternans, which would depend on the ability to detect electrical alternans regardless of where it originates from the heart and then takes the form of adaptive pacing protocols. These protocols could be incorporated into an implantable device thereby inhibiting cardiac alternans and potentially suppressing the development of a proarrhythmic substrate, such as a premature beat. This has good clinical applicability as many implantable devices  have endocardial electrodes. Several studies have shown a close correlation between simultaneous measurements of cardiac alternans from the body surface ECGs and intracardiac EGMs, indicating that these methods can measure the same phenomena . Compare2+]i cycling dynamics are complex, depending on the nature of Ca2+ → voltage coupling. The differences in CaT between adjacent cells are magnified in negative coupling but decrease with positive coupling (in vitro Proarrhythmia Assay), the risk assessment of antiarrhythmic drugs using a mathematical model of the cardiac action potential is further regulated. Although CiPA initiatives incorporate cell action potentials as a component into the simulation evaluation, attempts to use mathematical simulations in drug safety evaluations are expanding, even using biophysical 3-dimensional models of the heart and torso to simulate the human ECG under drug action. Future efforts will require the combination of simulations with experimental studies so that the complex spatiotemporal properties of cardiac dynamics can be considered in different tissue-level models and provide new ideas for individualized treatment.Alternatively, use of antiarrhythmic drugs may provide another helpful clinical therapy for TWA treatment. Whatever the specific mechanisms for these arrhythmogenic alternans is, the ultimate common pathway for arrhythmogensis includes wave-break, conduction block, and the occurrence and maintenance of re-entrant arrhythmias . Numeroucoupling . Activatcoupling . HoweverAll authors participated in the conception and writing of the manuscript. KZ reviewed and suggested modifications to the content.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.


After complete activation of the adrenoceptors by agonists, CRP caused additional intracellular Ca2+ mobilization. We assume that CRP interacts with hitherto unknown structures on the surface of vital cells and thus interferes with the desensitization of adrenoceptors.Systemic diseases characterized by elevated levels of C-reactive protein (CRP), such as sepsis or systemic inflammatory response syndrome, are usually associated with hardly controllable haemodynamic instability. We therefore investigated whether CRP itself influences blood pressure and heart rate. Immediately after intravenous injection of purified human CRP (3.5 mg CRP/kg body weight) into anesthetized rabbits, blood pressure dropped critically in all animals, while control animals injected with bovine serum albumin showed no response. Heart rate did not change in either group. Approaching this impact on a cellular level, we investigated the effect of CRP in cell lines expressing adrenoceptors (CHO-α1A and DU-145). CRP caused a Ca It has a Caembranes . Thus, Cembranes –6. In adembranes .While it has been shown that blood pressure, heart rate and catecholamines as well as adrenergic receptors can affect circulating CRP concentrations, a systemic influence of CRP has hardly been investigated so far. The use of beta-adrenoceptor (β-AR) blockers has been found to correlate with reduced circulating CRP levels, presumably due to a reduction in shear stress on endothelial cells of the blood vessels and thus a reduced inflammatory response –10. In cIn the clinical situation, critically ill patients, suffering from e.g., sepsis or acute pancreatitis often develop systemic inflammatory response syndrome (SIRS) and are characterized by hardly controllable hemodynamic variables such as low mean arterial pressure (<70 mmHg) with subsequent reduced organ perfusion and additionally elevated CRP levels . Both pain vivo. We chose healthy rabbits, which express comparable endogenous CRP levels as humans  as animal model because the binding activity of rabbit CRP is comparable to that of human CRP and both play a similar biological role in the innate immune system  but may have different pathways of signal transduction. Activation of α1-adrenergic receptors results in a Ca2+ release from the intracellular Ca2+ storage in the endoplasmic reticulum. The activation of β1- and β2-ARs leads to the opening of voltage-gated Ca2+ channels and thus to an influx of external Ca2+ across the plasma membrane. Changes in the intracellular free Ca2+ concentration can be observed and quantified by ratiometric fluorescence  to a final concentration of 15 mg/mL. In order to keep possible endotoxin contamination as low as possible, we used a sterile CRP adsorber, treated the devices with NaOH or depyrogenated the glass devices at 220°C, prepared everything with sterile cell culture water and used sterile endotoxin free disposable material. Before elution we washed several times. After elution we concentrated the CRP by ultracentrifugation and collected it in fresh sterile buffer.Human plasma samples were collected from patients with high CRP concentrations, pooled and stored frozen at −20°C until the experiments began. Plasma pool was thawed, and CRP was purified by running an affinity chromatography column of a selective CRP-binding matrix  . ElutionSeven female New Zealand White rabbits were purchased from Harlan Winkelmann with either 3 kg or at 8–10 weeks age. At the time point of the experiment, 3 animals were ~24 weeks old (4.3–5.3 kg) and 4 animals were ~10 weeks old (1.85–1.95 kg). All rabbits were housed in groups of five and according to FELASA guidelines. Health monitoring at the commercial breeder followed the FELASA recommendations. The study protocol (Reg. No.: G0299/09) was approved by the University Animal Care Committee and the federal authorities for animal research in Berlin, Germany.On the day of the experiment, rabbits were anesthetized intramuscularly with a mixture of 35 mg/kg ketamine , 0.25 mg/kg medetomidine  and 0.1 mg/kg atropine , followed by inhalation of 1.1 Vol.% isoflurane  via mask. This was according to standard practice guidelines (Another catheter was inserted in an ear vein for administration of either CRP (3.5 mg/kg body weight corresponding to 50 μg/mL blood volume) or bovine serum albumin (BSA) as control. CRP or BSA, each diluted in application buffer (15 mg/mL), were slowly injected intravenously (volume ranging from 0.44 to 1.17 mL over 1 min) followed by a flush of 0.9% saline. Seven animals received treatment with CRP and five of these received treatment with BSA as a control protein either 1 h after or before CRP application. To rule out individual animal effects, control and CRP application was performed and compared in the same rabbits. Blood samples were taken before and after the experiment.The first animal died unexpectedly and immediately after the intravenous injection of CRP. It is to be assumed that the administration was too fast. The following rabbits were injected more slowly.Rabbit blood samples were collected in serum tubes. Serum was separated with centrifugation and frozen for later analysis. A 96-well-ELISA-plate  was coated with 9 μg/mL polyclonal rabbit-anti-human CRP-antibody . After incubation, wells were washed (0.1% Tween in PBS) and blocked with a 0.1% casein-solution. Samples were diluted in 5 mM EDTA-solution and incubated in coated wells. Horseradish-peroxidase-conjugated rabbit-anti-humanCRP antibodies (14 ng/mL) were added. After addition and incubation of peroxidase substrate  reaction was stopped by 1 M sulphuric acid. Finally, photometric measurement followed at 450 nm and 620 nm (reference) with an ELISA-reader . All measurements were performed at least twice with triplicates.2+ signaling we used two different cell lines. CHO-α1A is a clonal cell line derived from Chinese hamster ovaries stably exhibiting overexpression of the human α1A-adrenergic receptor (α1A-AR) with efficient coupling to intracellular calcium  and 1% penicillin/streptomycin solution at 37°C and 5% CO2 saturation. CHO-α1A and DU-145 cell lines were kindly provided by Dr. Gerd Wallukat, former Max Delbrueck Center for Molecular Medicine, Berlin.For the assays on Ca2+ alterations the fluorescent dye Fura-2 and a Victor X2 fluorescence multiplate reader  equipped with an injector unit were used. In light diminished conditions, cells were treated with 5 μM Fura-2  for 45 min at 37°C. Cells were washed and incubated in HBSS-HEPES  plus 2,5 mM Probenecid for another 30 min before transfer to the microplate reader , a selective α1-AR agonist and isoprenaline , a non-selective β-AR agonist. At the beginning, the baseline value was measured and after 30 s CRP, PE or ISO was added. All conditions were performed in technical duplicates.To determine whether hCRP has a direct effect on the resting heart rate and blood pressure of rabbits, animals were injected with hCRP (3.5 mg/kg) without further treatment and the HR and ABP were monitored over time. At the beginning and end of each experiment, blood samples were collected and analyzed by a specific human CRP ELISA. No human CRP was detectable in rabbits prior to CRP injection (data not shown). About 1 h after injection, the mean circulating human CRP concentration was 45 ± 9.1 mg/L .Immediately after intravenous injection of CRP, we observed a surprisingly drastic drop in ABP, while the HR stayed the same . ControlThe experimental setup as such, including the anesthesia and the catheters used, had no effect on the heart rate and lowered blood pressure only slightly over time .in vitro. As part of the group of GPCRs, these receptors have different pathways of signal transduction, which all use Ca2+ as second messenger. We therefore examined whether CRP affects calcium influx in vitro in two different cell lines expressing either exclusively α1A-AR (CHO-α1A) or both α and β-ARs (DU-145).Our next step was to investigate the influence of CRP on adrenoceptor signaling 2+]i) was investigated in comparison to the adrenoceptor stimulation by agonists. The maximum effective concentration of PE was 5 μM and of ISO 1 μM, respectively. CRP was applied to reach a final concentration of 50 μg/mL. As control protein, we used human serum IgG (50 μg/mL).In the first assay, the effect of CRP on the intracellular free calcium concentration . 2+]i in both cell lines was depending on the CRP concentration. A high CRP concentration caused a faster and higher [Ca2+]i increase.To investigate a dose-dependent effect of CRP on [Ca2+]i was investigated. Therefore, either CRP (50 μg/mL) was added 70 s after full adrenoceptor stimulation with PE or ISO or the agonists were added after CRP stimulation. This was performed in order to gain insight whether the CRP induced calcium influx is triggered via adrenoceptor activation or whether it uses independent mechanisms. Under the influence of an agonist, a receptor is desensitized in order to avoid overstimulation of a cell by signal molecules and no further [Ca2+]i increase was expected. The effect of sequential application of CRP following PE/ISO or vice versa is shown in 2+]i, but the decline of [Ca2+]i was stopped and stayed upregulated on a steady level, while the stimulation with PE after CRP application additionally increased [Ca2+]i  but a dramatic effect on the arterial blood pressure (ABP) as shown in Despite all caution in the first animal, the intravenous injection of CRP was probably too fast. The animal died immediately, we suspect due to a drastic drop in blood pressure caused by the effect of CRP. The administration of pure solution buffer containing bovine serum albumin (BSA) as a protein control showed no effect.in vivo recorded changes after a few minutes at the earliest. In rats, for example, the administration of 20 mg/kg CRP had no effect on blood pressure compared to control animals, but ABP was measured only every 15 min after administration  or in the plasma membrane of damaged or necrotic cells, which exhibit lysophosphatidylcholine , 36. We Further, CRP could interfere with their desensitization, perhaps it might even affect other GPCRs. The mechanism remains obscure and further research should follow to understand this effect in detail.in vitro studies on endothelial cells of different species suggest a damaging effect of CRP on the cardiovascular system by influencing the endothelial nitric oxide (NO) synthase (eNOS) in expression and function. CRP uncouples eNOS, resulting in increased superoxide production and reduced NO synthesis, which can lead to vasodilatation disorders  or with cells not yet precisely characterized in regard to GPCRs (DU-145). It remains to be investigated if the observations can also be reflected in humans.In order to obtain a first indication if CRP has a direct effect on the circulatory system of rabbits The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study protocol (Reg. No. G0299/09) was approved by the University Animal Care Committee and the federal authorities for animal research in Berlin, Germany.CB, BV, GY, JU, and AS designed and performed the experiments. CB, SM, PB, SF, and AS evaluated the data and created the manuscript. All authors contributed to the article and approved the submitted version.SF and PB were employed by Pentracor GmbH and iAdsorb GmbH, respectively. The remaining 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.