However, the role of AVP in HF-induced AF still remains unclear. Pulmonary veins (PVs) are central in the genesis of AF. The purpose of this study was to determine if AVP is directly involved in the regulation of PV electrophysiological properties and calcium , OPC 21268 , or OPC 41061  for 4–6 h.Patch clamp, confocal microscopy with Fluo-3 fluorescence, and Western blot analyses were used to evaluate the electrophysiological characteristics, Ca+) and Na+/Ca2+ exchanger (NCX) currents than control PV cardiomyocytes. AVP (1 μM) treated PV cardiomyocytes had smaller Ca2+i transients, and sarcoplasmic reticulum (SR) Ca2+ content as well as higher Ca2+ leak. However, combined AVP (1 μM) and OPC 21268 (0.1 μM) treated PV cardiomyocytes had a slower PV beating rate, larger Ca2+i transients and SR Ca2+ content, smaller late Na+ and NCX currents than AVP (1 μM)-treated PV cardiomyocytes. Western blot experiments showed that AVP (1 μM) treated PV cardiomyocytes had higher expression of NCX and p-CaMKII, and a higher ratio of p-CaMKII/CaMKII.AVP (0.1 and 1 μM)-treated PV cardiomyocytes had a faster beating rate (108 to 152%) than the control cells. AVP (1 μM) treated PV cardiomyocytes had higher late sodium (Na2+ homeostasis through vasopressin V1 signaling.AVP increases PV arrhythmogenesis with dysregulated Ca AVP binds to its receptor to promote vasoconstriction through modulation of adenosine triphosphate-sensitive potassium channel function and nitric oxide (NO) production, and enhances the vascular response to catecholamines . AVP is lipase C . IP3 indlum (SR) , 11, wheHF and atrial fibrillation (AF) are closely interrelated with sharing similar risk factors and pathophysiology . Patient2+ regulation plays a pivotal role in PV electrical activity. Late sodium currents (INa-late) and intracellular Ca2+ (Ca2+i) dynamics play an important role in PV arrhythmogenesis and HF. Dysregulated Ca2+ homeostasis, and increased Ca2+ spark frequency promotes arrhythmogenesis of PV cardiomyocytes in HF, which may play an important role in the development of AF [2+ homeostasis, we suggest that enhanced AVP might contribute to PV arrhythmogenesis. The aim of this study was an evaluation of the effects of AVP on the PV electrophysiological characteristics and Ca2+ handling and an investigation of the potential mechanisms.Pulmonary vein (PV) myocardium consists of a mixture of working cardiomyocytes and pacemaker cells. It plays a critical role in the genesis and maintenance of AF . PVs arent of AF . Interesnt of AF , 21. How2 1.8, MgCl2 0.5, HEPES 10 and glucose 11; with pH adjusted to 7.4 by titration with 1 N NaOH. The perfusate was replaced with oxygenated Ca2+-free Tyrode’s solution containing 300 units/ml collagenase (Sigma Type I) and 0.25 units/ml of protease  for 8~12 min. Proximal PVs were cut away from the atrium and lung, and were gently shaken in 5~10 ml of Ca2+-free oxygenated Tyrode’s solution until single cardiomyocytes were obtained. The solution was then gradually changed to oxygenated normal Tyrode’s solution. Cells were allowed to stabilize in the bath for at least 30 min before the experiments were started. Single cardiomyocytes with spontaneous activity were identified by the presence of constant beating during perfusion with Tyrode’s solution. PV cardiomyocytes were incubated without (control) and with AVP (0.1 and 1 μM), AVP (1 μM) combined with or without OPC 21268 , OPC 41061  [The investigation was approved by the local ethics review board and conformed to the institutional Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. Male rabbits (2.0~3.0 kg) were euthanized using intramuscular injections of a mixture of Zoletil 50 (10 mg/kg) and xylazine (5 mg/kg) with an overdose of isoflurane (5% in oxygen) from a precision vaporizer. Single cardiomyocytes from rabbit PVs were enzymatically dissociated through a previously described procedure . In brie, 10 nM) , 23 for 2+ current, ICa-L) at an approximately similar period, as previously described [75, APD50, and APD20) [A whole-cell patch-clamp was used to record ionic currents and action potentials (APs) in isolated PV cardiomyocytes with an Axopatch 1D amplifier  at 35 °C ± 1 after rupture or perforation  .2 1, MgATP 5, HEPES 10, Na2GTP 0.1, and Na2 phosphocreatine 5, titrated to a pH of 7.2 with CsOH for the experiments on the ICa-L; with a solution containing (in mM) NaCl 20, CsCl 110, MgCl2 0.4, CaCl2 1.75, tetraethylammonium chloride (TEA-Cl) 20, BAPTA 5, glucose 5, MgATP 5, and HEPES 10, titrated to a pH of 7.25 with CsOH for experiments on the Na+/ Ca2+ exchanger (NCX) current; and with a solution containing (in mM) CsCl 130, Na2ATP 4, MgCl2 1, EGTA 10, and HEPES 5 at a pH of 7.3 with NaOH for the INa-Late; with a solution containing (in mM) KCl 20, K aspartate 110, MgCl2 1, MgATP 5, HEPES 10, EGTA 0.5, Na2GTP 0.1, and Na2 phosphocreatine 5, titrated to a pH of 7.2 with KOH for experiments on the AP.Micropipettes were filled with a solution containing (in mM) CsCl 130, MgClNa-Late was recorded at room temperature with an external solution containing (in mM): NaCl 130, CsCl 5, MgCl2 1, CaCl2 1, HEPES 10, and glucose 10 at a pH of 7.4 with NaOH by a step/ramp protocol  to enhance INa-Late since the non-equilibrium gating gives rise to a fast recovery of the Na+ channel from inactivated state and reactivation during repolarization ramp [2 has been shown to block all movement of monovalent ions through Na+ channels, which may avoid persistent Ca2+ current through Na+ channels [+ current, which has been shown to dissect the full measure of INa-Late [Na-late was measured as the tetrodotoxin (30 μM)-sensitive part of the current traces obtained when the voltage was ramped back to − 100 Mv [The Iion ramp . The extINa-Late . The INa− 100 Mv .Ca-L was measured as an inward current during depolarization from a holding potential of − 50 mV to test potentials ranging from − 40 to + 60 mV in 10 mV steps for 300 ms at a frequency of 0.1 Hz using a perforated patch clamp with amphotericin B. NaCl and KCl in the external solution were replaced with TEA-Cl and CsCl. To avoid run-down effect  [Ca-L was measured at 5~15 min after perforating the membrane patch in each PV cardiomyocyte.The Iolution) , the ICa2 2, MgCl2 1, HEPES 5, and glucose 10 with a pH of 7.4, and contained strophanthidin (10 μM), nitrendipine (10 μM), and niflumic acid (100 μM).The NCX current was elicited by test pulses of between − 100 and + 100 mV from a holding potential of − 40 mV for 300 ms at a frequency of 0.1 Hz. The amplitudes of the NCX current were measured as 10 mM nickel-sensitive currents. The external solution (in mM) consisted of NaCl 140, CaCl2+  for 30 min at room temperature [0), to obtain reliable information about transient intracellular Ca2+ (Ca2+i) changes from baseline values (F - F0 (ΔF)/F0) and to exclude variations in the fluorescence intensity by different volumes of injected dye. The Ca2+i transient, peak systolic Ca2+i, and diastolic Ca2+i were measured during spontaneous beating and pacing with a 2-Hz field-stimulation with 10-ms twice-threshold strength square-wave pulses. The SR Ca2+ content was estimated by the rapid addition of 20 mM caffeine after electric stimulation at 2 Hz for at least 30 s. The total SR Ca2+ content was determined from the peak amplitude of the caffeine-induced Ca2+i transients.As described previously, PV cardiomyocytes were loaded with fluorescent Caperature . The Flu2+ leak, PV cardiomyocytes were incubated with AVP (1 μM) or AVP (1 μM) plus KN-93  for 4~6 h before experiments. PV cardiomyocytes were stimulated at 2 Hz in normal Tyrode’s solution to bring the cellular Ca2+ content to a steady state. In the control condition, Ca2+i was monitored while 0 Na+, 0 Ca2+ Tyrode’s solution (without tetracaine) was perfused for a minimum of 20 s. In the test condition, the superfusate was rapidly switched to 0 Na+, 0 Ca2+ Tyrode’s solution with 1 mM tetracaine after the last pulse for a minimum of 20 s. Under this condition, the ryanodine receptor (RyR) is abruptly blocked and the Ca2+ leak can be measured as a drop in Ca2+i.For the measurement of SR Ca+ deoxycholate, 0.1% Na+ dodecyl sulfate (SDS), and protease inhibitor cocktails . The protein concentration was determined using Bio-Rad protein assay reagent . Proteins were separated on 4%~ 12% SDS-polyacrylamide gel by electrophoresis (PAGE) under reducing conditions and electrophoretically transferred to an equilibrated polyvinylidene difluoride membrane . All blots were probed with primary antibodies against NCX , Ca2+/calmodulin-dependent protein kinase II , pCaMKII at Thr 286, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) , AVP V1a receptor , AVP V2 receptor , and all secondary antibodies conjugated with horseradish peroxidase. All bound antibodies were detected with an enhanced chemiluminescence detection system  and analyzed with AlphaEaseFC software. All targeted bands were normalized to GAPDH to confirm equal protein loading.Control and AVP (1 μM)-treated PV cardiomyocytes were centrifuged and washed with cold PBS, and lysed on ice for 30 min in RIPA buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40, 0.5% NaP value of < 0.05 was considered statistically significant.All continuous parameters were expressed as mean ± the standard error of the mean (SEM). An unpaired t-test, one-way or two-way analysis of variance (ANOVA) with a Duncan post-hoc test was used to compare differences between the groups. A As shown in Fig. The beating rate in OPC 21268 (0.1 μM) or OPC-41061 (10 nM)-treated PV cardiomyocytes was similar to that in control PV cardiomyocytes. However, combined OPC 21268 (0.1 μM) and AVP (1 μM)-treated PV cardiomyocytes had similar beating rate and the slope of late diastolic depolarization as compared to the control  did not change the current density of INa-Late and NCX of PV cardiomyocytes. However, OPC 21268 (0.1 μM) can reverse the effects of AVP (1 μM) on ILate-Na and NCX of PV cardiomyocytes.Figure 2+ transients and caffeine-induced Ca2+ transients than the control by 59 and 60%, which suggests they had stored less Ca2+. Similarly, spontaneous Ca2+ transients in AVP (1 μM)-treated PV cardiomyocytes (n = 20) were smaller than those in the control (n = 22) by 67% (P < 0.001). OPC 21268 (0.1 μM)-treated PV cardiomyocytes combined with or without AVP (1 μM) had similar Ca2+ transients and caffeine-induced Ca2+ transients to the control, suggesting that the effects of AVP (1 μM) on Ca2+ transients and SR Ca2+ content can be attenuated by OPC 21258. Moreover, AVP (1 μM)-treated PV cardiomyocytes had greater Ca2+ leak than the control, which was attenuated by the presence of KN-93 (1 μM).As can be seen in Fig. AVP (1 μM)-treated PV cardiomyocytes had larger protein expressions of NCX and p-CaMKII, but the protein expression of CaMKII was similar between the control and AVP (1 μM)-treated PV cardiomyocytes Fig. . Compare2+ homeostasis in PV cardiomyocytes. AVP increased the PV beating rate with a dose-dependent response. The increased PV beating rate caused by AVP can be ameliorated by V1 receptor antagonist OPC 21268. OPC 21268 did not change PV electrophysiological properties or Ca2+ homeostasis, suggesting that AVP directly regulates PV spontaneous activity via V1 receptor signaling. Previous study had shown that OPC 41061 displayed an inhibitory effect on AVP-induced cAMP increasing and modulated intracellular Ca2+ at the concentration of 10 nM [50 values for OPC 41061-mediated inhibition of voltage-gated potassium currents ranged between 2 and 7 μM in GH3 cells derived from rat pituitary tumors [2+ and decreased protein synthesis by 50% within 20–30 min [In this study, it was found for the first time, that AVP has direct effects on electrical activity and Caof 10 nM . The IC5y tumors . Therefoy tumors –38. Simi0–30 min . Since t0–30 min , our finNa-Late increases PV arrhythmogenesis [Na-Late and NCX than control PV cardiomyocytes. There are different methods of measuring the INa-Late [Na-Late to a greater degree, we applied high concentration of tetrodotoxin (30 μM) to block Na+ current during a repolarizing voltage ramp [Na-Late. Increasing INa-Late plays an important role in PV arrhythmogenesis by reducing the repolarization reserve. This opposes the repolarizing potassium currents and delays repolarization [Na-Late is more likely to develop early afterdepolarization in response to triggers [Na-Late would result in an increase of intracellular Na+ concentration, which would activate reversed NCX [Na-Late in addition to the increasing effect of AVP on NCX protein.We previously found that enhanced Iogenesis . In the INa-Late , 41. To age ramp , 31, whirization . A reductriggers . INa-Latrsed NCX , subseqursed NCX . We foun2+ homeostasis plays a crucial role in the maintenance of cardiac excitation and contraction, and significantly regulates cardiac contractility. Inhomeostasis of intracellular Ca2+i is common in a number of pathological conditions and contributes to arrhythmogenicity [2+ removal function in forward mode at membrane voltages less than the equilibrium potential [2+ efflux, and resulted in a decrease of Ca2+i transients. This decrease in Ca2+i transients by AVP may contribute to the known adverse cardiac effects after AVP treatment [2+i transients and SR Ca2+ content in PV cardiomyocytes by the antagonist OPC 21268, suggests that this effect is mainly V1 signal dependent. Previous study has found that AVP can elicit Ca2+ entry through a receptor-mediated Ca2+-membrane non-selective cation channel in aortic smooth muscle cells, which regulates smooth muscle contractility and enhances vascular tone. OPC 21268 was noted to reverse AVP-induced activation of nonselective cation currents in aortic smooth muscle cells [2+ influx through vasopressin-induced nonselective cation currents of PV smooth muscle cells may increase vascular stretch, further increasing PV arrhytmogenesis in vivo. In addition, the activity of vascular smooth muscle cells may influence the membrane potential of PV cardiomyocytes via intercellular transfer of electrical signals occurring between PV cardiomyocytes and vascular smooth myocytes of PVs, thereby exacerbating the propensity of PV arrhythmogenesis or cardiac dysrhythmias. Moreover, several previous studies have demonstrated the ability of caffeine to activate intermediate-conductance Ca2+-activated potassium channels [2+ transient could be secondarily attributed to its activation of these channels.Cardiac Cagenicity , 45. Theotential . AVP enhreatment . Counterle cells . PVs conle cells . Therefochannels –52, whicchannels . Therefo2+ leak, the release of small amounts of Ca2+, occurs when altered RyR spontaneously opens in diastole [2+ release activates the forward mode of NCX current on the late diastolic depolarization. This late diastolic depolarization acceleration by NCX is required for the subsequent timely rapid AP upstroke [2+ leak resulting steepness of late diastolic potential, which could be responsible for increasing automaticity [2+ leak and depletion of SR Ca2+ content, contributing to impaired contractility and HF progression [2+ leak than control cells. Clinically, greater SR Ca2+ leak was well demonstrated in atrial myocytes in AF patients [2+ leak may also contribute to a decrease in Ca2+i transients and SR Ca2+ content as well. CaMKII regulates several Ca2+-handling proteins and has been shown to be a central regulator of excitation-contraction coupling [2+ leak [2+ leak can be amplified by NCX, triggering ectopic focal discharges or facilitating microreentry circuits promoting AF maintenance [2+ leak. The attenuation effects of KN-93 on AVP-induced Ca2+ leak in PV cardiomyocytes also suggests that activation of CaMKII is important for the effects of AVP on PV cardiomyocytes. In addition, the greater INa-Late in AVP-treated PV cardiomyocytes may also arise from the higher pCaMKII because activation of CaMKII is an important activator of INa-Late. Enhanced INa-Late synergistically increases the risk of cardiac arrhythmias by the activation of CaMKII [SR Cadiastole . Diastolupstroke , 56. In maticity . Blockingression , 59. We patients . Greatercoupling , and inccoupling . CaMKII-[2+ leak , 62, and[2+ leak . Diastolntenance . In thisf CaMKII . AccordiOur study may be limited in some respects. First, the PV cardiomyocytes received AVP for a relatively short time and AVP treatment of different duration may not have the same effects. Furthermore, AVP is usually associated with stress and pathological conditions. We only studied the effects of AVP on healthy PV cardiomyocytes, it is unclear whether our findings or theory applies to pathological settings such as HF. Finally, the details of the molecular regulation responsible for the effects of AVP in PV cardiomyocytes has not been fully elucidated.2+ homeostasis through vasopressin V1 signaling.AVP increases PV arrhythmogenesis with dysregulated Ca


Nature Communications; 10.1038/s41467-018-06785-y published online 16 Oct 2018.Correction to: F1,297 = 6.57, P = 0.02, partial η2 = 0.17; stimulus × trial × group, F1,297 = 1.32, P = 0.23; n = 35)”. The correct version states “F1,33=6.58” instead of “F1,297 = 6.57”, and “F9,297 = 1.32” instead of “F1,297 = 1.32”. These errors have now been corrected in both the PDF and HTML versions of the Article. The correct values were used in the statistical analysis and the errors do not affect the conclusions.In the original version of this Article, the fourth sentence of the legend to Figure 1b incorrectly read “Note, that the group difference stemmed from significantly smaller CS+ evoked SCRs averaged across the whole test phase, but the speed of re-extinction did not differ significantly between drug groups  and trial (1–10) as within-, and group  as between-subject factor: stimulus × group,


This review discusses how mutations affect CaM signaling function and how this may relate to the distinct arrhythmia phenotypes/mechanisms observed in patients; this involves mechanistic interpretation of negative dominance and mutation-specific CaM-target interactions. Knowledge of the mechanisms involved may allow critical approach to clinical manifestations and aid in the development of therapeutic strategies for “calmodulinopathies,” a recently identified nosological entity.In spite of the widespread role of calmodulin (CaM) in cellular signaling, CaM mutations lead specifically to cardiac manifestations, characterized by remarkable electrical instability and a high incidence of sudden death at young age. Penetrance of the mutations is surprisingly high, thus postulating a high degree of functional dominance. According to the clinical patterns, arrhythmogenesis in CaM mutations can be attributed, in the majority of cases, to either prolonged repolarization , or to instability of the intracellular Ca While some of these are clearly complementary in achieving a functional goal  and can coexist, others are devoted to apparently competing aims  and need to be separated. This requires mechanisms allowing intracellular Ca2+ to act on its targets with high specificity. Several strategies are employed by the cell to achieve this goal. Ca2+ buffering by intracellular proteins and small molecules, leads to a strictly controlled mobility of the ion. Active Ca2+ compartmentalization within organelles allows to keep “resting” Ca2+ concentration in the general (or “bulk”) cytosolic compartment at very low levels (around 10−7 M), i.e., below the threshold required to activate downstream effectors; at the same time, structural organization  allows very small Ca2+ fluxes to achieve high Ca2+ concentration in the specific subcellular compartment hosting the target effector , a protein present in all cell types and highly conserved throughout evolution , or as a sensor intrinsic to a given effector (bound pool), thus affording either diffuse or highly confined signaling. Furthermore, in various cells types, the cytosolic CaM pool can be redistributed to the nuclear compartment upon a rise in Cats range , 4. Alsorocesses .In keeping with its central and evolutionarily conserved function, CaM is generated in a highly redundant mode. Indeed, an identical amino acid sequence is encoded by 3 CaM genes  . Such re2+ at the same time contributes as a charge carrier to electrical excitation  and triggers the development of mechanical force; therefore, Ca2+ is crucial to excitation-contraction coupling (ECC)  . Severaland fate . This miand fate .Our objective is to revise the information available on the various aspects of CaM structure/function that we see as potentially relevant to a mechanistic interpretation of CaM mutations phenotypes, with a focus on cardiac ones.CaM is composed of 149 amino acid residues to form a 17 kDa protein. The protein is ubiquitous and expressed in all eukaryotic cells, with 100% identity in its amino acid sequence among vertebrates. Three genes  encode CaM with an identical amino acid sequence, thus resulting in potential redundancy (2+ binding site each (2 Ca2+ binding sites per lobe). All EF hand domains can bind Ca2+; however, while the N-lobe (EF I and EF II) has higher affinity for Mg2+, the C-lobe (EF III and EF IV) binds Ca2+ with ten times higher affinity  Figure  12); th; th12); affinity . Anothere C-lobe .2+ , EF I and EF II (N-lobe) are in a packed conformation, thus with low affinity for Ca2+; EF III and EF IV (C-lobe) are instead in a partially open conformation, more prone to Ca2+ binding. At resting Ca2+ levels (about 10−7 M), all CaM binding sites are typically unoccupied. When Ca2+ rises, its binding to the C-lobe sites triggers a conformational change leading the N-lobe to increase its affinity for Ca2+. In other words, Ca2+ binding to CaM is “cooperative,” i.e., the overall affinity increases when Ca2+ concentration exceeds the threshold for C-lobe occupation. CaM can potentially bind 1–4 Ca2+ ions; activation of downstream targets requires CaM being loaded with 3 Ca2+ ions at least  are characterized by the presence of a typical basic amino acid sequence called the “IQ motif.” The IQ motif is closely preceded by a “preIQ” region, which is a common site of permanent CaM binding ; the detailed mechanism and Ca2+-dependency of this specific interaction will be described in paragraph Voltage-gated Ca2+ channels (Cav1.2).Also of interest are the structural aspects of CaM's interaction with target proteins, which are necessary for modulation of their activity. “Anchor” domains on target proteins are characterized by hydrophobic residue sequences flanked by negatively charged ones; the latter provide electrostatic interaction that may orient CaM binding . Many Ca binding . CaM int binding , 14; indhibition . A well-2+ sensing, CaM domains mainly involved in target binding are the lobes. Both CaM lobes contain nine methionine residues, playing a key role in target recognition, whose high flexibility provides plasticity crucial for this function  sites, either at the N-lobe or at the C-lobe, suitable to interact with hydrophobic residues in the anchor . As a general interpretational scheme, CaM binding may stabilize an otherwise short-lived configuration, spontaneously assumed by the target protein and associated with a specific functional state , tumor progression proteins (in cancerous cells) , protein2+  and “pre-bound” pools. As mentioned previously, such a distribution may vary according to CaM's occupancy by Ca2+ .2+ concentration, 99% of total intracellular CaM is bound to cellular proteins, leaving about 50-100 nM of free CaM (1%) in the cell  , but valthe cell . Nonethethe cell ; its fun2+ concentrations , pre-bound CaM may largely exist as apo-CaM. Pre-bound apo-CaM may even be released to the free CaM pool in response to Ca2+ elevation, thus representing a local diffusible CaM store  is the most abundant type in cardiomyocytes (2+ ones): large conductance, activation at larger depolarizations and long lasting openings. The activation of this channel is driven by the action potential upstroke, with ICaL reaching a peak in 2–7 ms. Thereafter, channels inactivate with time constants in the order of 50–100 ms at plateau potential, due to both Ca2+ and voltage-dependent gating  membranes . This is responsible for the opening of ryanodine receptors (RyRs), Ca2+-activated Ca2+channels clustered in the SR membrane facing Cav1.2 channels.Camyocytes . The symt gating . Ca2+ inCaL is modulated by two feedback signals that, albeit of opposite sign, are both dependent on the rise of Ca2+ concentration close to cytosolic mouth of the channel and involve CaM. Ca2+-dependent inactivation (CDI) is responsible for most of the rapid ICaL decay occurring during sustained depolarization (2+-dependent facilitation (CDF), a weaker phenomenon, reflects instead the increase in ICaL peak conductance that may be observed during repetitive activation at high rates  requires Ca2+ binding to the C-lobe, whose affinity for the target IQ motif is thus increased; C-lobe interaction with the IQ motif stabilizes the channel conformational state associated with CDI , identifies their respective role in sensing Ca2+ at the channel mouth  vs. global cell Ca2+ (sensitive to even weak Ca2+ buffering). Unlike in neuronal channel isoforms, Cav1.2 channels retain robust CDI even in the presence of strong Ca2+ buffering; such form of CDI is entirely triggered by Ca2+ association with the C-lobe  may best support selective sensing of a smaller but sustained Ca2+ signal  to a pre-IQ motif present on the C-terminus of the αwith CDI  , which then phosphorylates the Cav1.2 channel at two serine residues close to the EF-hand motif . IKs gating is positively regulated by cytosolic Ca2+ through a CaM-dependent process . Interpretation of the effect of a helix B Kv7.1 mutant (K526E) and of interference of Ca2+-CaM with Kv7.1 pull-down by PIP2 has led to the following model: at diastolic Ca2+ levels, CaM is bound to a non-activating Kv7.1 site (helix A) by its apo-C-lobe; the N-lobe is displaced from the helix B site. As cytosolic Ca2+ increases, calcification of the C-lobe causes its dissociation from helix A and the N-lobe then interacts with its site on the helix B; this results in stabilization of the channel open state  and limit its open probability; when Ca2+ increases, N-lobe binding is reinforced and C-lobe is released thereby relieving the inhibitory effect on channel gating , IKs enhancement requires Ca2+ elevation. Furthermore, both CaM lobes are involved and a preserved C-lobe Ca2+ affinity is essential for the signaling function.Both models imply that CaM binding to Kq proteins. Receptor activation may result in PIP2 depletion, which would reduce IKs; however, IP3-induced Ca2+ release from the sarcoplasmic reticulum may compensate PIP2 reduction by activating positive IKs regulation by Ca2+-CaM. This may represent the main physiological role of IKs modulation by Ca2+-CaM, which would therefore be of particular relevance during activation of the PLC-IP3 signaling pathway.PIP2 is a membrane phospholipid degraded by phospholipase C (PLC) to produce inositol 3-phosphate (IP3) in response to activation of a number of membrane receptors associated with Gv7.1 channel trafficking; indeed, mutations disrupting N- and C-lobe integrity reduce channel membrane expression  inhibits IKs . While tKs regulation also occurs indirectly by CaMKII-mediated phosphorylation of the channel at serine 484; contrary to direct CaM modulation, phosphorylative modulation is inhibitory and may account for IKs downregulation upon sustained β-adrenergic receptor activation  leads to the enhancement of persistent Na+ current, thus suggesting a role of CaM in stabilizing the inactivated state , containing ~5,035 amino acid residues in total, sharing the general structure of the six-transmembrane ion channel superfamily . Since Rtrations . This sttrations . The RyR species .2+ in CaM-independent ways. At the same time, they are regulated by CaM in both Ca2+-dependent and -independent ways. This makes investigation of CaM's role in RyRs' regulation very complex.RyRs are strongly regulated by Ca2+ on both sides of the SR membrane in a CaM-independent way. Although not the focus of the present review, a brief discussion of such “direct” regulation by Ca2+ is required to understand the potential difficulty in isolating the CaM-dependent one.RyR gating is highly sensitive to Ca2+ binding motifs  mechanism  and is disrupted by increases in luminal Ca2+  for both apo-CaM and holo-CaM, thus resulting in a pre-bound CaM pool (2+ concentrations in the μM range (2+-independent. Increasing Ca2+ up to 100 μM has been reported to increase the number of CaM molecules bound to RyR2 (from 1 to 7.5 per RyR2 tetramer)  ; however2+-dependent discrepancy of CaM effects on RyR opening between RyR1 and RyR2 channels?Given that CaM binding domains are highly homologous between RyR1 and RyR2 , what maMutations in both the N-terminal and central RyR2 regions similarly destabilize the channel closed state; this suggests that the latter may require interaction between these two regions (“zipping” model of RyR gating). This view is confirmed by the effect of peptides  interfering with such interaction . On the 2+, the two channel domains interact with each other in the folded protein; such Ca2+-dependent interaction is required for channel opening. Binding of holo-CaM to the channel may disrupt the activating interdomain interaction, thus explaining CaM-induced RyR1 inhibition at high Ca2+ concentrations (2+ concentrations (favoring apo-CaM) may produce CaM-dependent RyR1 activation, which remains an open question.A different gating model has been proposed for RyR1 channels, in which the channel CaM binding domain is followed, within about 450 residues, by a CaM-like sequence. FRET data indicate that, at high Catrations . However2+ shifts CaM binding to the channel by about 3 nM, corresponding in the 3D structure to two different protein domains. In view of the fact that CaM activates RyR1 at low Ca2+ and inhibits it at high Ca2+, the two domains may be seen as activator and inhibitory sites, respectively. Importantly, these studies indicate that the Ca2+-dependent shift in CaM binding site is a consequence of a rearrangement of the binding surface of CaM, rather than of RyR1 conformation (2+-dependent). The same technique shows that in RyR2 CaM binds to the “inhibitory site” (as identified in RyR1) already at low Ca2+; this might explain why RyR2 is inhibited by CaM in a Ca2+-independent way  in RyR1 and Ca2+-independent  in RyR2. Formation of the Ca2+-CaM complex does affect RyR2 gating significantly, but this occurs through an indirect mechanism, involving CaMKII activation.In conclusion, even if the detailed mechanism remains to be resolved, it is now accepted that direct regulation of RyR gating by CaM is Ca2+ with a Kd of 20-100 nM, which dramatically decreases (to 60 pM) after enzyme autophosphorylation  and, additional serine residues  or “Ca2+ spark” (synchronous release from a RyR2 cluster), a function pivotal to both contractile and electrical function of cardiomyocytes  Camyocytes .Mutations in one of the three CALM genes, even in the heterozygous form, have been described in patients with a severe cardiac phenotype, characterized by a high propensity to ventricular arrhythmias, syncopal episodes and sudden death at a young age .Despite sharing strong electrical instability, two distinct phenotypes can be identified in carriers of CaM mutations: the long QT syndrome (LQTS) , 73, chaA point of interest in the interpretation of CALM mutations is their extremely high penetrance: 1 mutant allele in 6 encoding for the same amino acid sequence (as in heterozygous mutations) is sufficient to result in marked functional derangements.Also in view of the multiplicity of functions exerted by CaM in many cell types, all this suggests that, for one reason or another, mutant CaMs must interact with their target with high specificity. In the following paragraphs we will describe the cellular functional derangements associated with the LQTS and CPVT phenotypes and address, as much as current knowledge allows, the mechanisms underlying target specificity of CaM mutations.v7.1 (IKs) and Cav1.2 (ICaL) might theoretically be involved in prolongation of repolarization. Enhancement of the “window” (INaW) or “late” (INaL) components of the Na+ current INa (Nav1.5 channel) might represent a further potential mechanism.Prolongation of action potential duration (APD), reflected as QT interval prolongation on the ECG, can result from loss of function of outward currents, or gain of function of inward ones; therefore, CaM abnormalities affecting modulation of Kv1.2 function (ICaL enhancement) has emerged as the dominant mechanism in CALM gene mutations associated with delayed repolarization.Nevertheless, gain of Cade novo heterozygous missense CALM gene mutations in LQT-infants with recurrent cardiac arrest  involved in Ca2+ binding. The CALM1-p.F142L (next to C-lobe EF IV), albeit outside the EF-hand, is expected to alter the energetics of the conformational change associated to Ca2+ binding  of 7 (compatible with heterozygosity) was enough to impair CDI (v1.2 channel function can be explained by the fact that modulation of this target requires a pre-bound apo-CaM pool (containing mixtures of WT and mutant CaMs) and subsequent Ca2+ binding to this pool (impaired in mutants by loss of Ca2+ affinity). This interpretation would explain sparing of RyR2, whose modulation may not require Ca2+ binding, and of CaMKII, which binds CaM directly in its holo-form (not represented if Ca2+ affinity is reduced). Selective CDI impairment by additional mutations reducing C-lobe Ca2+ affinity (CALM2-p.D132H and CALM1-p.D132V) has also been reported in transfection studies on human induced pluripotent stem cell-derived cardiomyocytes  and implies lack of functional dominance of the mutation for this target. However, at least according to a current model of CaM-Nav1.5 interaction  , it is surprising that CaM mutations with loss of C-lobe Ca2+ affinity may not affect IKs. One tentative explanation is compensation of the loss of CaM-dependent regulation by PIP2 signaling; it would be therefore interesting to evaluate the effect of known CaM mutations on IKs under conditions of PIP2 depletion.CaM mutations resulting in downregulation of Kp.F142L) . Nonetherization . Conside2+ waves,” i.e., macroscopic surges of cytosolic Ca2+ resulting from spontaneous RyR opening at a point site, followed by auto-regenerative propagation (by Ca2+-induced Ca2+ release) of the ionic perturbation to the whole cell , which results in a depolarizing current, also referred to as “transient inward current” (ITI). While “Ca2+ overload” facilitates Ca2+ waves (by increasing RyRs open probability), it is neither necessary nor sufficient to induce them; indeed some degree of intrinsic RyR2 instability may be involved even in the prototypical case of digitalis toxicity , typically induced by exercise, or other conditions associated with enhanced adrenergic stimulation . The proole cell . The mectoxicity .2+ binding  (CALM3-p.A103V)  , 85, whi protein , that deaffinity .2+-independent, the mechanism must conceivably reside in a change of 3D protein conformations involved in CaM-RyR2 complexing; nonetheless, the nature of this change remains to be clarified.In conclusion, the features of CPVT mutations may explain why they are not generally associated with an LQTS phenotype (however see below), but a general mechanism by which they induce RyR2 instability cannot be clearly envisioned. Since CaM interaction with RyR2 is essentially Cav1.2 and RyR2 channels, several mutations have been associated with both LQTS and CPVT phenotypes. CaM mutations were found in five subjects with QT prolongation; nonetheless, two of them (CALM2-p.D132E and –p.Q136P) were associated with arrhythmia features strongly suggestive of SR instability and thus were assigned to the CPVT phenotype  occurring in genes CALM2 . Nonetheless, it should be considered that impairment of IQTS case , SR instCALM1-p.F90L mutation in a patient with IVF, i.e., VF episodes without the features of either LQTS or CPVT (mild QT prolongation only during exercise recovery)   . The muteraction . In hetehannels) ; nonetheCALM2-p.N98S mutation and obtained reversal of the phenotype by knocking out the mutant allele by gene editing  provide the means to test mutation effects in the context of each patient's genetic background. Recent studies have exploited this cellular model to test the effect of CaM mutations. Yamamoto et al. reported a typical LQTS phenotype for the heterozygous  editing , thus sumutation .CALM1-p.F142L mutation  and its failure to shorten adequately at high pacing rates. As expected from the increase in Ca2+ influx, the amplitude of V-triggered Ca2+ transients was significantly increased; nonetheless, SR Ca2+ content was normal and no spontaneous Ca2+ release events were observed, thus suggesting preserved homeostatsis of intracellular Ca2+. This argues against SR instability as the arrhythmogenic mechanism in this case and suggests a primary role of prolonged and “stiff” (non-rate-adaptive) repolarization instead (Ks was found to be unaffected and a persistent component of INa (likely contributed by INaW) was significantly reduced   , hiPSC-C2+ sensor to maintain physiological Ca2+ levels in cells. In addition to this homeostatic role, CaM signal targeting is required to transduce fundamental cell processes, for which Ca2+-CaM complexing is not necessarily involved, but may still have a modulatory effect. This is possible because of the presence of CaM-binding sequences suitable to allow CaM to bind multiple targets even in its “apo” form; this generates a quantitatively prevailing “pre-bound” CaM pool. CaM binding to targets occurs with very high specificity, which is required to explain restriction of CaM mutations phenotype to the myocardium and, within it, to specific subcellular targets.CaM functions as a Ca2+ sensing function is crucial in impairing CDI of sarcolemmal Ca2+ channels (carrying ICaL), it is not required for mutations associated with RyR2 instability. For the latter, changes in CaM affinity for RyR2 channels are apparently more important; however, the direction and even the need for such changes are still unclear. Possibly, mutations induce complex (3D) modifications in the protein-protein binding interface, of which changes in CaM affinity for the target are just a gross readout. This is a field in which new information is strongly required.Whereas mutation-induced loss of CaCaL and RyR2, respectively, we hypothesize that coexistence of QT prolongation and SR instability (mixed phenotypes) might be accounted for by impaired ICaL CDI, possibly with the complement of (very common) conditions weakening homeostatic control of intracellular Ca2+.Whereas, “pure” LQTS and CPVT phenotypes suggest abnormal modulation of IBased on the information reviewed above, mechanism-guided therapeutic approaches to calmodulinopathies should ideally address the interaction of mutant CaM with its targets. Particularly in the case of LQTS-type mutations, this approach is justified by the role of the high target affinity of mutant CaMs in causing negative dominance of the mutation. Tools for this purpose are not available yet, but possibilities exist and are currently explored.CaL blockade seems a logical approach in the case of ICaL gain of function, resulting from loss of CDI (LQTS phenotype); indeed, verapamil did shorten the QT interval in hiPSC-CMs from CALM1-p.F142L carriers  and SR instability (CPVT phenotype) (The role and mechanism of Naenotype) .Ks suggests that factors affecting membrane PIP2 levels  potentially influence CaM mutation penetrance. If this were the case, such factors might represent easily accessible therapeutic targets.The interplay between CaM- and PIP2-dependent modulation of ICaL CDI, which seems to diverge from what would be expected, has thus far been only superficially addressed.The ultimate mechanism of arrhythmia facilitation by loss of IFinally, the molecular basis of RyR2 dysfunction in the context of CaM mutations remains largely unresolved, thus preventing identification of mechanism-specific targets.BB wrote a general manuscript draft. CR focused on the section about CaM modulation of voltage-gated channels. M-CK, LS, and AG provided text and discussion for integration with clinical and genetic aspects of calmodulinopathies. LC and AZ supervised the process and edited the manuscript to its final version.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.


Structural variants (SVs) are known to play important roles in a variety of cancers, but their origins and functional consequences are still poorly understood. Many SVs are thought to emerge from errors in the repair processes following DNA double strand breaks (DSBs).We used experimentally quantified DSB frequencies in cell lines with matched chromatin and sequence features to derive the first quantitative genome-wide models of DSB susceptibility. These models are accurate and provide novel insights into the mutational mechanisms generating DSBs. Models trained in one cell type can be successfully applied to others, but a substantial proportion of DSBs appear to reflect cell type-specific processes. Using model predictions as a proxy for susceptibility to DSBs in tumors, many SV-enriched regions appear to be poorly explained by selectively neutral mutational bias alone. A substantial number of these regions show unexpectedly high SV breakpoint frequencies given their predicted susceptibility to mutation and are therefore credible targets of positive selection in tumors. These putatively positively selected SV hotspots are enriched for genes previously shown to be oncogenic. In contrast, several hundred regions across the genome show unexpectedly low levels of SVs, given their relatively high susceptibility to mutation. These novel coldspot regions appear to be subject to purifying selection in tumors and are enriched for active promoters and enhancers.We conclude that models of DSB susceptibility offer a rigorous approach to the inference of SVs putatively subject to selection in tumors.The online version of this article (10.1186/s13059-019-1635-1) contains supplementary material, which is available to authorized users. Structural variation (SV) in tumor genomes is known to play important roles in disease progression and may be critical in driving the development of certain cancer types –3. HowevA recent study of whole genome sequencing (WGS) data from breast tumors identified SV hotspots and putative driver SVs, but could not discern the relative contributions of mutational bias and selection underlying these hotspots . ResolviVariable rates of SVs observed across the genome are likely to be affected by differences in the efficiency of repair of DNA double strand breaks (DSBs). DSBs can be repaired by homologous recombination (HR) at the G2 and S stages of the cell cycle and, more commonly, by canonical non-homologous end joining (c-NHEJ) which operates throughout the cell cycle . The c-NPrevious studies have also shown DSB formation to be influenced by underlying chromatin structures and genomic sequences. It has long been known that certain cytogenetically mapped loci, termed “fragile sites” undergo recurrent DSBs in cells under replicative stress and in cancer . More reRandom forests have been used to model a variety of biological phenomena because they perform well in the presence of inter-correlated input variables showing non-linear relationships. For example, they have been used to predict nuclear compartments , cancer r = 0.905 to 0.992, p < 2.2e−16) and were combined to reduce noise, although random forest models generated from any single one of the replicates yielded very similar results , shown in Additional file We uniformly processed four DSB datasets from three related platforms (DSBCapture and BLISS are both based upon modifications to the BLESS protocol) and covering three different cell types, collating matched chromatin data for each. These datasets include two novel DSB mapping datasets derived from the K562 erythroleukemia and MCF7 breast cancer cell lines using the recently developed BLISS method  see the” sectionsee the “” sectionr = 0.918, 21 feature model r = 0.922). We conclude that models constructed using the 11 selected genomic features  between NHEK and K562 observed DSB profiles. We measured the correlation of observed and predicted DSB frequencies across all nine model and feature combinations and always found correlations (r = 0.58 to 0.85) that improved on the base correlations (r = 0.38 to 0.63) seen between the observed DSB datasets  collections have been established for a variety of tumor types, and each possesses advantages and shortcomings. The International Cancer Genome Consortium (ICGC) provides high-resolution SV calls based upon whole genome sequencing (WGS) for 2146 patients across 17 cohorts , but samAnalogously to the DSB datasets, we determined the number of tumor SV breakpoints per 50 kb region for each of the ICGC and TCGA SV datasets see the “” sectionCertain classes of relatively simple SVs  are often the product of one or two DSBs, while more complex intrachromosomal rearrangements can be difficult to classify accurately, and may have origins in poorly understood phenomena such as chromothripsis . Indeed,d-score, to measure this divergence between expectations given a DSB model and observed SV breakpoint rates in tumors. In brief, this metric relies on fitting known distributions to the observed SV breakpoint dataset and to the predicted DSB dataset. Based upon the known distributions we then transform the observed SV counts and predicted DSB values to p values, reflecting the probability that each value is drawn from the fitted distribution  have long been studied for their unusual properties of generating SVs, both in normal cells and in cancer . These rd-scores show a significant shift above the distribution for all genes and above the driver gene sets as well  DSB frequencies at 294 50 kb regions coinciding with annotated CFS gene loci across the genome, in comparison to regions associated with all annotated genes, and regions associated with putative cancer driver genes Fig. c. Althouell Fig. d. This rd-score metric. We call regions with significantly more SV breakpoints than expected, or SV hotspots, cancHpredL , and regions with fewer SV breakpoints than expected, or SV coldspots, cancLpredH  , corresponding to regions showing unusually high SV frequencies on the background of high susceptibility to DSBs. Finally, we define a fourth class of regions that have predicted DSB rates close to zero but high SV breakpoint frequencies (cancHpredL2). In principle, these regions are a class of SV hotspots but, as shown in Fig. We examined a range of functional annotation enrichments in the four classes of regions using circular permutation to assess significance see the “. The annp < 2.2e−16), and the pancancer ESBs from either set do not significantly overlap , demonstrating that a given model can be used to detect regions of interest in various cell types.We again wanted to test the utility of DSB random forest models applied to different cell types by testing the accuracy of predictions made by a model trained in one cell type given features for a different cell type, as in Fig. d-scores to uncover genes that might be reclassified as oncogenic due to a higher than expected SV breakpoint frequency in cancer. Likewise, we investigated the ten regions with the lowest d-scores (cancLpredH), which we predict to be under purifying selection, for signals of potential functionality. For this analysis, we used the NHEK model predictions paired with ICGC carcinoma SV breakpoints.We closely examined the ten 50 kb regions with the highest (cancHpredL) d-scores overlap a gene, and four overlap COSMIC genes. CHEK2 and CDKN2A are known tumor suppressors, and TMPRSS2 and ERG are frequently involved in translocation events forming fusion oncogenes in certain cancers. For example, it fuses with TMPRSS2 in most prostate cancers, with EWS in Ewing’s sarcoma, and with FUS in AML. Two adjacent 50 kb regions on chr17q12 overlap GRB7 and IKZF3. GRB7 encodes a protein that interacts with epidermal growth factor receptor (EGFR), a well-known proto-oncogene, and IKZF3 is a zinc finger protein and transcription factor involved in B lymphocyte regulation and differentiation as well as chromatin remodeling. This region also corresponds to a known fragile site FRA17A [d-scores, seven overlap a known gene and two known oncogenes. The oncogene, CDC27, or cell division cycle 27, encodes a component of the APC and has been shown to interact with other mitotic checkpoint proteins. It is highly conserved and may be necessary for cell survival. There is also a non-coding RNA found on chr2 in the centromeric region, LOC654342, which overlaps an H3K27ac peak, and may be acting as a regulatory element.Nine out of ten regions with the highest e FRA17A . Of the Recent in vitro studies of DSB frequency in cell lines have suggested that a variety of underlying genomic features are associated with DSB susceptibility. We have shown that accurate models of genome-wide DSB frequency can be built from a modest number of such features, with replication timing, open chromatin, and marks of active promoter or enhancer regions associated with increased DSBs. Although active regulatory regions often harbor actively transcribed genes, it appears that chromatin accessibility at these sites rather than transcription itself determines DSB propensity. The variable importance metrics also show certain features to be more influential in particular cell types, with CTCF and H3K36me3 having more predictive power in MCF7 than in NHEK or K562. Not only are DSB patterns cell type specific, but the factors influencing those patterns also depend on cell type, suggesting different mutational mechanisms at play. As a matter of course, our models’ accuracies decline when applied to cell lines other than the training set, but they still generate reasonable DSB frequency predictions, with correlations between 0.57 and 0.83 to the observed data, which are large improvements over a simple inference. Since chromatin features influence mutation patterns and are cell type-specific, it will be important to use mutational propensity profiles for matched cell types in future cancer studies. In general, caution should be exercised in extrapolating from the currently small number of models to more poorly characterized cell types lacking experimentally determined DSB data.Our models of genome-wide DSB susceptibility predict DSB frequencies for all 50 kb loci and reflect the established correlations between replication timing and DSB frequency , 10. A rWe used our genome-wide models of DSB susceptibility to interrogate the largest tumor SV breakpoint collections and found surprising levels of agreement, such that SV breakpoint enriched regions often show shifts to higher predicted DSB susceptibility. In spite of variable sample sizes, the classes of simple SV likely to arise by one or two DSBs  showed significant increases in predicted DSB susceptibility. The NHEK model best predicted the patterns of DSB susceptibility in tumors, showing genome-wide elevations of predicted DSBs for all of these SV classes relative to control regions. Thus, the chromatin-mediated DSB susceptibility captured in the model may shape the landscape of SV recurrence in these classes.There are many reasons why one might expect a much poorer agreement between the predictions of in vitro DSB frequency models and the patterns of SV breakpoints observed in tumor sequencing studies. The available collections of SV breakpoints in tumors are far from perfect, and even the best ICGC data suffer large variations in sample size, sample heterogeneity, sequencing depths, and SV calling methods across tumor cohorts. In addition, fundamental aspects of tumor biology  are expected to place distinct limits on the agreement we can see with the DSB patterns seen in cell lines. Evidence is also emerging that there are important properties of the mutational landscape in tumors that are unlikely to be captured by in vitro model systems. For example, a recent study of intra-tumor diversification in colorectal cancer suggests that most mutations occur during the final clonal expansion of these tumors, resulting from mutational processes that are absent from normal colorectal cells . EnhanceThere is great interest in “hotspot” genomic regions harboring recurrent SVs in tumors, on the basis that such regions may be under positive selection, conferring a proliferative or survival advantage to tumor cells. However, a rigorous inference of selection requires a proxy for the expected rate of recurrence within such regions. Using model predictions as this proxy we have produced refined hotspot predictions, reflecting SV breakpoint frequencies relative to the predicted susceptibility of each region. Since our predictions of DSB susceptibility are genome-wide it was also possible to predict coldspot regions, regions possessing unexpectedly low SV breakpoint rates given model predictions, and putatively subject to negative or purifying selection in tumors. If selection in tumors is prominent in driving SV breakpoint frequencies away from DSB model predictions, we might expect hotspot and coldspot regions to show unusual functional enrichments. Multiple caveats apply to the annotations examined but analysis using the NHEK model shows that ICGC carcinoma hotspots are enriched for putative oncogenes. Coldspots occupy gene-rich neighborhoods but and are also enriched in active promoters and strong enhancers, and insulators, indicating regulatory regions that may have experienced purifying selection in tumors. Methods for the detection of DSBs in human cells, as well as the causes and consequences of these mutations, remain active areas of fundamental research. Further development of both in vitro and in silico model systems will undoubtedly provide new insights into somatic mutation and repair processes and their significance to tumorigenesis.When inferring selection on single nucleotide variants it is standard practice to make comparisons between the observed variant frequencies and the frequencies expected, according to a model of single nucleotide mutation rates. We have developed models of DSB mutation rates that can be used to generate expected SV breakpoint frequencies and illuminate regions with significant deviations from these expectations. This approach provides statistically rigorous protocols to prioritize novel loci putatively under selection in tumors, generating testable hypotheses for further experimental studies.scan for matches, allowing at most one mismatch per barcode. Trimmed reads were then aligned to GRCh37 using bwa mem, and reads with mapping scores below 30 were discarded. Next, PCR duplicates were identified by searching for proximal reads (within 30 bp of the reference genome) with at most two mismatches in the UMI sequence, which were then grouped and collapsed into a single break location. Finally, we generated .bed files with DSB locations and the number of unique UMIs indicating that location.DSB profiles were generated with an adapted version of the Breaks labeling in situ and sequencing protocol , in whicWe downloaded ten tracks from ENCODE for multiple chromatin marks, replication timing, open chromatin, several DNA binding proteins, and nucleosome pull-downs from the UCSC genome browser . We usedFor the extended model in Additional file We generated DSB frequency scores from each of the four HTS DSB profiling datasets: two in NHEK cells, one for K562, unpublished, and one for MCF7, unpublished. As mentioned in the results, two replicates for each of two DSB HTS profiling methods, DSBCapture and BLESS, were available from Lensing et al. . We tookhttps://cran.r-project.org/web/packages/randomForest/index.html), which calculates the average prediction error rate (MSE) for each datapoint (50 kb bin) across all trees in the random forest. Then, for each feature variable, the values are randomly permuted and the MSE for each 50 kb bin is calculated again. The final variable importance score is the average difference in MSE before and after the permutation, normalized by the standard deviation of these differences. Because many features are inter-correlated, their importance measures were very similar. Therefore, in order to determine a consistent ranking of features’ importance values, we generated ten random forest models per dataset and calculated the average and standard deviation of importance across the ten models.We used the randomForest package in R to generate random forest models with 500 trees and five OOB permutations per tree . To calculate variable importance, we used the importance command within the randomForest package . DNAcopy generates a set of continuous segments, outputting regions with little or no copy number change, so we filtered these, defining segments with a CN ratio > 1 as amplifications and ratios < − 1 as deletions. The segments were lifted from hg38 to hg19 using UCSC’s liftOver tool. For each CNV, we counted a single DSB to occur in a 50 kb bin if either or both ends of the segment overlapped the bin. The TCGA-BLOOD group includes the two blood cancer cohorts: acute myeloid leukemia (LAML) and lymphoid neoplasm diffuse large B cell lymphoma (DLBC), while the TCGA-SCCA group includes three squamous cell carcinomas: cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), head and neck squamous cell carcinoma (HNSC), and lung squamous cell carcinoma (LUSC). The BRCA group includes only the TCGA breast cancer cohort (BRCA), and the PANC group includes all 33 cancer types, shown in Additional file To determine SV DSB rates from TCGA data, we downloaded CNV data from TCGA , which chttps://dcc.icgc.org/projects). As with the TCGA CNV, a single DSB was counted per 50 kb bin if either one or two ends of a SV overlapped the region. The ICGC pancancer group contains SVs from 17 cancer studies, shown in Additional file We downloaded available WGS SV calls from the ICGC Data Portal (We determined enriched SV breakpoint regions (ESBs) per cohort or SV type grouping by ranking the 50 kb bins by the number of DSB, excluding regions with no DSB in the group, and using the number of DSB in the top 5% as the cutoff. All 50 kb regions with a DSB count greater than or equal to the cutoff were designated ESBs. We used a Wilcoxon ranked sum test (R wilcox.test command) to test for a significant increase in the predicted DSB values for ESBs compared to all other regions, and we excluded regions in which no DSB were found in any cancer study since these are likely to be unmappable or blacklisted regions.q = 177, m = 2839, n = 61,903–2839, and k = 3072).The correlation between TCGA and ICGC pancancer SV breakpoint counts was calculated using Spearman’s rho and excluding 50 kb regions with no SV breakpoints in either the TCGA or ICGC datasets. The top 5% ESBs were found for each dataset, with 2839 regions found in TCGA and 3072 in ICGC, and the significance of the overlap was calculated using a hypergeometric test  which represent the probability of seeing a given breakpoint frequency or DSB prediction or greater in the known distribution. The actual and fitted distributions and quantile-quantile plots are shown in Additional file We used the R package fistdistrplus  to deterp values between the predicted DSB and the actual SV breakpoints, called d-scores. Using the fistdistrplus R package again, we determined the best-fit distribution for the d-scores, choosing between a t-distribution, a normal, and a Cauchy distribution. Again, we used a maximum likelihood method and the BIC measurement and excluded extreme outliers. In all cases, a t-distribution with four degrees of freedom (df = 4) was the best fit, so each 50 kb bin was assigned a p value from this distribution according to its d-score. The histograms and quantile-quantile plots of the d-scores and fitted distributions are shown in Additional file Next, for each 50 kb bin, we calculated the difference in log d-score p values to categorize regions into informative subsets, using the R command qt  to determine the d-score cutoffs. The cancHpredL class of regions have d-scores in the upper one percentile (> 3.75), and the cancLpredH have d-scores in the lower one percentile (< − 3.75). The cancHpredH class has d-scores in the 40th to 70th percentiles and SV breakpoint frequencies or DSB predictions with p values less than 0.01, so these regions have significantly  high SV breakpoints or DSB predictions but insignificant d-scores . The cancHpredL2 class consists of regions with SV breakpoint p values less than 0.01, and DSB predictions less than 0.5 for the NHEK models and less than 0.001 for the MCF7 model.We used the We used a binomial test to measure the significance of overlaps between sets when comparing results from the MCF7 model and the NHEK model applied to ICGC breast cancer data and MCF7 cell line features (R command binom.test).d-score classes and the HMEC track, from primary mammary epithelial cells, with the MCF7 model’s d-score classes. The regioneR package performs random circular permutation of regions of interest and then computes the number of overlaps between the permutated set and a second set of regions. The p value represents how often, over the course of the permutations, the two sets overlap to the same extent that they do without any permutation. We used 1000 iterations to achieve a maximum p value of 0.001.We used the R package regioneR  to compuAdditional file 1:Figure S1. Correlations between DSB profiles from three DSB HTS experiments from three different cell types at 50 kb resolution. Figure S2. Chromatin and genomic features are intercorrelated. Figure S3. Correlations between individual genomic features and DSB frequency from NHEK DSBCapture data. Figure S4. Hi-C eigenvector data improves the random forest model for DSB frequency. Figure S5. Predicted versus observed values for one third of the NHEK DSBCapture data where the random forest model was trained on the other two thirds of the data. Figure S6. ICGC SV types and cohorts. Figure S7. TCGA CNV data across cancer types. Figure S8. Correlation between predicted DSB values and observed SV breaks. Figure S9. Correspondence between SV and DSB shown for NHEK DSBCapture data and ICGC pancancer SV calls. Figure S10. TCGA CNV breakpoint enriched regions have elevated DSB frequencies in blood cancers. Figure S11. Inference of positively and negatively selected regions in ICGC breast cancers. Figure S12. Chromatin state enrichments for d-score classes. Figure S13. Finding the best-fit distribution for SV breakpoint frequencies. Figure S14. Finding the best-fit distribution for predicted DSB values. Figure S15. Finding the best-fit distribution for d-score frequencies. (DOCX 12760 kb)Additional file 2:Tables S1. bigWig files from http://hgdownload.soe.ucsc.edu/gbdb/hg19/bbi/ used to create feature sets. Table S2. Extended NHEK Feature Set. Table S3. Sources of annotation files and their names. (XLSX 62 kb)


Double strand breaks (DSBs) are induced in the DNA following exposure of cells to ionizing radiation (IR) and are highly consequential for genome integrity, requiring highly specialized modes of processing. Erroneous processing of DSBs is a cause of cell death or its transformation to a cancer cell. Four mechanistically distinct pathways have evolved in cells of higher eukaryotes to process DSBs, providing thus multiple options for the damaged cells. The homologous recombination repair (HRR) dependent subway of gene conversion (GC) removes IR-induced DSBs from the genome in an error-free manner. Classical non-homologous end joining (c-NHEJ) removes DSBs with very high speed but is unable to restore the sequence at the generated junction and can catalyze the formation of translocations. Alternative end-joining  operates on similar principles as c-NHEJ but is slower and more error-prone regarding both sequence preservation and translocation formation. Finally, single strand annealing (SSA) is associated with large deletions and may also form translocations. Thus, the four pathways available for the processing of DSBs are not alternative options producing equivalent outcomes. We discuss the rationale for the evolution of pathways with such divergent properties and fidelities and outline the logic and necessities that govern their engagement. We reason that cells are not free to choose one specific pathway for the processing of a DSB but rather that they engage a pathway by applying the logic of highest fidelity selection, adapted to necessities imposed by the character of the DSB being processed. We introduce DSB clusters as a particularly consequential form of chromatin breakage and review findings suggesting that this form of damage underpins the increased efficacy of high linear energy transfer (LET) radiation modalities. The concepts developed have implications for the protection of humans from radon-induced cancer, as well as the treatment of cancer with radiations of high LET. Cancer is intricately linked to the formation of chromosomal translocations, one of the most prominent manifestations of genomic instability that underpin the evolution of this disease ,2,3. ChrIt is widely recognized that in vertebrates, DSBs represent a particularly dangerous DNA lesion because they directly disrupt the integrity of the DNA molecule and generate a condition where fundamental concepts evolved to repair other forms of DNA lesions fail ,5,6,7. TThis characteristic of DSBs imposes the expectation that their processing will be problematic and therefore inefficient and slow. Strikingly, however, work carried out in the last 30 years documents an impressive DSB-processing system with an extraordinary total repair capacity, comprising multiple repair pathways, some of which operate with very high speed ,5,6,7. TDespite these apparently impressive characteristics of the DSB-processing system, DSB induction remains a high-risk event as amply documented by the long-described detrimental effects of IR: Point mutations, as well as genomic rearrangements, causing cell death and most prominently cancer . As a coA highly consequential recognition from recent in-depth analysis of DDR and DSB repair is that the efficient overall DSB processing mentioned above is achieved by the coordination of multiple pathways, which have very distinct operational requirements and which surprisingly produce results that are not equivalent ,5,6,7. TFor DSB processing, in contrast, while all characterized pathways remove the physical rupture from the DNA, they do so with widely different accuracy in terms of restoring the original sequence in the vicinity of the DSB, and occasionally they even join the wrong DNA ends, causing translocations. Four different pathways evolved to process DSBs. The homologous recombination (HR) repair-based pathway, gene conversion (GC), is the only error-free pathway capable of restoring both the DNA integrity and the original DNA sequence. Classical non-homologous end joining (c-NHEJ), alternative end joining , and single-strand annealing (SSA), another HR-dependent pathway, remove the break from the genome, but can cause mutations, deletions, and translocations owing to functional characteristics that are inherent to their mechanistic base and which are described in the following section ,5,6,7. AA working hypothesis guiding our research activities is that a key parameter in DSB repair pathway engagement is the degree of chromatin destabilization generated in the genome by a DSB. We postulate that this chromatin destabilization affects each of the DSB processing pathways in a different manner.Several parameters that may determine the degree of chromatin destabilization generated by a DSB have been identified and are presently under intensive investigation to establish their contribution to DSB processing. Thus, a DSB generated in euchromatin is likely to have different consequences than a DSB generated in heterochromatin. Even within euchromatin, a DSB generated in an actively transcribed region of the genome is likely to be highly destabilizing. Also, DSBs generated in regions of active DNA replication will generate pronounced chromatin destabilization and possibly also a disruption of the replication fork. 1 phase. DSBs induced before or during mitosis are therefore likely to generate severe chromatin destabilization, as amply documented by the observation that mitosis is by far the most radiosensitive phase of the cell cycle [Furthermore, chromatin is highly dynamic in each phase of the cell cycle and can undergo dramatic changes when cells transit from one phase of the cell cycle to the next. Perhaps the most dramatic such changes in chromatin organization occur when cells enter mitosis, as well as after completion of mitosis when cells enter Gll cycle . In the case of IR, the form of DSBs generated adds another level of complexity and a whole new set of parameters to the above-described aspects of chromatin organization and activities . In starWe postulate that the degree of chromatin destabilization by a DSB will be determined by the above outlined physical and biological parameters and that the degree of chromatin destabilization will, in-turn, determine the pathway that will engage in the processing of this DSB. All available information suggests that the prime goal of this integrated processing system is to leave no DSB unprocessed and that this feat is achieved by joining together, even genomically incongruent, DNA ends. Although this goal is achieved at the cost of events that change and destabilize the entire genome, such as DNA sequence alterations, nucleotide losses, and translocations, such an outcome is clearly preferable to the broken genome that would be left behind when high fidelity processing mechanisms fail in the chromatin environment of the DSB. Along this line of reasoning, unrepaired DSBs, or DNA ends detected after completion of bulk DSB processing, reflect a failure to bring two DNA ends in proximity to each other and join them together. This concept also explains why higher eukaryotes rely heavily on pathways that simply join DNA ends together independently of their origin  and less so, at least apparently, on more complex but error-free processes, such as GC—as yeast and bacteria actually do! Indeed, end-joining pathways may primarily and more appropriately be considered as DNA-end management/scavenging systems rather than genuine repair mechanisms, designed to remove DNA ends from the genome by bringing them together and joining them to each other. They may actually reflect late events in evolution that contribute to the management of integrity of increasing-size genomes. Fatal events causing cell death and cancer may thus be accidents that are “taken into account” during engagement of a particular pathway for the processing of a specific DSB. Considering all the above information, we propose that the engagement of a particular DSB processing pathway is dictated by necessity rather than by choice. Our working hypothesis is that cells must in principle be evolutionarily programmed to use first the DSB processing pathway with the highest probability of restoring an intact genome, but that they will also adaptively recruit lower fidelity options if the state of chromatin at the rupture site generates conditions that compromise this pathway. The concept of necessity suggests a hierarchy in DSB processing, with the highest fidelity pathway interrogated first. Pathways of decreasing fidelity will be interrogated, sequentially, only when the previous higher fidelity pathway fails in the context of existing chromatin organization and activity. The “last resort” pathways will put together any DNA ends including those belonging to different chromatin regions and even to different chromosomes. Such hierarchical interrogation of existing DSB processing pathways in the removal of DSBs is hypothesized to reflect an inherent logic in their evolution aiming at maximizing the preservation of genetic information after adaptation to realities. It also implies that the cell is not “choosing” among one of the four repair pathways, but rather that it applies the above “logic” to every DSB and always ends up using the best pathway under the circumstances.Below, we summarize key characteristics of the DSB processing pathways to illustrate the basis for the above concepts and review results of experiments that describe how these concepts and hypotheses can be applied to understand the role of DSB complexity in the engagement of a processing pathway. We will illustrate how radiation modalities of increasing LET induce DSBs of increasing complexity and will show that increased DSB complexity compromises DSB processing by c-NHEJ for an as of yet undefined subset of DSBs. Finally, we will use this property to validate in defined biological models a type of complexity, DSB-clusters, as one of the key lesions underpinning the increased efficacy of high LET radiation.As outlined above, the most intriguing characteristic of the DSB processing apparatus is that it integrates four pathways, each of which processes DSBs using different molecular mechanisms . Seen unNormally, DSBs generated by IR have blunt ends or contain very short protruding single stranded DNA ends . C-NHEJ Before joining by the LIG4/XRCC4/XLF/PAXX complex, each of the DNA ends needs to be “cleaned” to enable ligation. This is because IR breaks DNA by damaging the sugar moiety of terminal nucleotides, remnants of which need to be removed. This processing utilizes Artemis, Aprataxin and Aprataxin, and PNK-like factor (APLF). DNA synthesis by members of the X family DNA polymerases, Pol λ and Pol µ, fills gaps in the DNA and adds new nucleotides in a non-complementary manner, ultimately completing the restoration of the DNA molecule ,14. The c-NHEJ molecular apparatus functions highly efficiently; assisted by the simplicity of the catalyzed reaction and the high numbers of KU and DNA-PKcs molecules in the cell, it removes DNA ends from the genome with unparalleled speed in a cell cycle-independent manner . Indeed,In conclusion, c-NHEJ features as a surveillance mechanism, swiftly scavenging DNA ends from the genome by joining them pairwise as soon as they happen to be close to each other in space. It thus keeps the genome free of DNA ends, which improves its stability. A conundrum generated by the above outlined molecular characteristics of c-NHEJ is how the three remaining DSB processing pathways engage the DSB in a c-NHEJ-proficient background and why the cell apparently favors a fundamentally error-prone pathway. Below, we present forms of DSBs that compromise c-NHEJ and facilitate the utilization of the remaining DSB processing pathways.The ends of DSBs that are not removed from the genome by c-NHEJ are prepared for processing by three alternative pathways all of which begin, albeit to a different extent, with the exonucleolytic degradation of the 5′-strands of each DNA end to generate 3′-single-stranded DNA (ssDNA), in a reaction known as DNA-end resection . DNA-endThe most salient feature of GC is that it requires a homologous template in the form of the sister chromatid ,20,21,222-phase of the cell cycle [The constraints of the above described mechanism ensure that exclusively, the original ends of DSB will be joined together. Furthermore, the templated DNA synthesis on the identical sister chromatid enables GC to faithfully restore DNA sequence in the vicinity of the DSB. As a result, GC operates in an error-free manner without causing translocations or sequence modifications in the cellular genome . On the ll cycle . In addiSSA is also a homology dependent pathway of DSB processing ,23,24,252-phase.In contrast to HRR, SSA does not require RAD51 but uses instead the strand annealing protein RAD52 to anneal the DSB-flanking homologous DNA sequences, as they become exposed after DNA-end resection ,23,24,25Alt-EJ operates on similar principles as c-NHEJ and simply joins two DNA ends together ,12,26,27POLQ gene [2-phase and reduced activity in G1, as expected from its dependence on DNA-end resection [0 cells [Proteins implicated in alt-EJ, in addition to the above-mentioned resection proteins, are often not dedicated to this pathway and are involved in other aspects of the DNA metabolism as well. For example, Poly (ADP-ribose) Polymerase 1 (PARP-1) facilitates alt-EJ but also has many additional functions in the cell ,12,26,27OLQ gene ,31,32. POLQ gene . AlthougOLQ gene ,26,27, i[0 cells ,34,35. Similarly to c-NHEJ, alt-EJ joins any DNA ends independently of origin. However, because it operates with slower kinetics than c-NHEJ and is carried out by a much less coordinated apparatus, a drifting apart of the DNA ends is more likely and as a consequence also the probability of translocation formation. Alt-EJ has also no built-in mechanisms to restore DNA sequence at the DSB junction. This fact, together with the often-required DNA-end resection, increases the frequency of deletions and other sequence alterations at the junction; indeed, these changes in the genome are much more extensive than those generated by c-NHEJ. Collectively, the above outline indicates that only GC operates in an error-free manner. All remaining DSB processing pathways are error prone and all can, in principle, contribute to the formation of chromosomal translocations and sequence alterations at the DSB junction. Below we present results rationalizing the evolution of repair pathways with such diversity.As mentioned above, DSBs form in irradiated cells as minority lesions ,37 when Because increased ionization clustering increases the probability of DSB induction, it was at first hypothesized that the increased efficacy of high LET radiations derives from increased induction of DSBs. Surprisingly, however, extensive work testing this hypothesis demonstrated that per unit of dose, radiation modalities of different LET produce similar numbers of DSBs ,46,47,48These results led to the follow-up hypothesis that high LET radiations are more efficient biologically because they generate more complex DSBs that are difficult for the cell to process ,51,52,53Another form of complexity that has been frequently considered to explain the increased efficacy of high LET radiation is that of DSB clusters . DSB cluNotably, for both forms of DSB complexity, the supportive evidence for their relevance to high LET effects derives largely from mathematical modeling. For DSB complexity owing to the presence of non-DSB lesions at the DSB site, biochemical experimental evidence exists but is indirect. As a result, the relative contribution of the two forms of complexity in the increased effectiveness of high LET radiation remains unknown. Below, we will describe a biological model system we recently developed to generate in cells simple DSBs and DSB clusters using the I-SceI homing meganuclease . The sysDespite increased complexity, DSBs induced by high LET IR, such as 56Fe+ ions, are actively processed, albeit with a somewhat reduced efficiency compared to DSBs induced by low LET IR, such as x-rays  Figure b. SimilaA striking observation with high LET IR is that when cell survival is analyzed in c-NHEJ mutants, the LET-dependent enhancement of cell killing disappears . This isA possible interpretation for this interesting observation is that wild-type cells gain their radioresistance to low LET IR, as compared to high LET IR, by repairing, using c-NHEJ, a subset of DSBs, which after exposure to high LET IR change form and become refractory to processing by c-NHEJ. As a consequence, and because high and low LET IR have similar yields of DSBs , c-NHEJ-We hypothesized that the mega-molecular apparatus of c-NHEJ, consisting of proteins the size of DNA-PK and coordinated by several additional interacting components, must operate precisely, like a “Swiss watch”, to remove DSBs with the unparalleled speed measured experimentally. This overall operation accuracy and speed are likely to be particularly sensitive to the generation of DSB clusters. We postulated that, particularly, DSB clusters in which the individual DSBs are located close together are likely to destabilize its function. In a cell exposed to IR, only a small subset of DSBs (chromatin rupture events more precisely) will be caused by a cluster of DSBs. However, their yields will increase and the distance between the individual DSBs in a cluster will decrease with increasing LET of radiation.It is not possible to test the hypothesis of DSB cluster formation as a key cause, both for the high LET radiation action as well as for the associated suppression of c-NHEJ, by exposing cells to IR. This is because IR induces a daunting spectrum of DNA lesions distributed throughout chromatin at locations that are different for each cell, thus making molecular analysis of their consequences impossible. To address this problem, we engaged in the development of a molecularly defined biological model of DSB clusters in order to examine their suppressive consequences on c-NHEJ and analyze their processing by GC, alt-EJ, and possibly also SSA. Below, we briefly describe this model and summarize results confirming that complex DSB clusters suppress the function of c-NHEJ while allowing processing by the remaining repair pathways. A relevant observation worth mentioning in the context of the present section is the generation of DSBs in cells exposed to protons, a low LET radiation modality, that are preferably processed by GC. Specifically, it was observed that cells lacking proteins of the homologous recombination DSB repair pathway are more sensitive to proton than to photon irradiation. The chromatin context and the specifics of DSB induction that generate this dependence on GC remains to be elucidated. However, the observation is relevant for the application of protons in the treatment of cancer as they may be particularly indicated for cancers that share molecular features of BRCA-mutant tumors, “BRCAness” ,66.Saccharomyces cerevisiae), but which can be introduced, typically as a single copy, in the form of a construct designed according to the question addressed [A RE frequently used to induce DSBs in the human genome and study its processing requirements is the I-SceI homing endonuclease. I-SceI recognizes an 18 base-pair sequence that is not present in the vertebrate genome , it has been successfully used to elucidate key requirements of DSB-processing pathways. We opted therefore for this system and designed constructs allowing study of the processing of DSB clusters by available DSB-repair pathways, focusing first on the elucidation of the above described unique characteristics of high LET radiation. For this purpose and in deviation from available I-SceI cell systems, we generated CHO cell lines containing multiple I-SceI integration sites  to mimic the multiple DSBs typically induced in irradiated cells. Furthermore, we designed constructs harboring the I-SceI site either as a single copy to generate single DSBs, or as pairs and quadruplets to generate DSB clusters. The distances between the DSBs vary between approximately 50 and 200 (average nucleosome distance) base pairs. This model system is designed on the assumption that transient expression of I-SceI will rupture chromatin by generating either single DSBs or DSB clusters. Each such event is considered as a single chromatin rupture of “complexity” proportional to the number of DSBs involved. Indeed, single DSBs and DSB clusters initiate qualitatively and quantitatively similar initial γ-H2AX foci formation at numbers equivalent to the integration sites, although 53BP1 accretion appears enhanced in regions of chromatin-harboring DSB clusters. Chromatin restitution may occur by ligation of proximal or apical DNA ends .The first test of DSB clusters as a putative cause for the increased efficacy of high LET IR is summarized in Defects in c-NHEJ are associated with an increase in the formation of translocations. Analysis of translocations forming during processing of single DSBs and DSB clusters is shown in We developed a rationale for the evolution in vertebrate cells of four mechanistically distinct pathways for the processing of DSBs, which surprisingly have very different capacities to reconstitute the original genome. We reasoned that in cells, these pathways must be wired in ways allowing first the engagement of the highest fidelity pathway—following an inherent logic aiming at maximizing genomic integrity. We further argued that when cells use a pathway of lower fidelity to process a specific DSB, they do so to address necessities that arise from the suppression of a high-fidelity pathway during processing in the context of chromatin. Such necessities arise as a consequence of the DSB location in chromatin, as well as a consequence of chromatin activities in the affected chromatin domain that may interfere with the function of a repair pathway. Similar necessities also arise as a consequence of increasing DSB complexity. Results obtained using a defined biological model system demonstrate that DSB clusters compromise c-NHEJ. By comparing the responses to high-LET IR of cells deficient in c-NHEJ, we developed the hypothesis that a significant proportion of the increased killing generated by high-LET IR derives from the generation of DSB clusters. The results support a model according to which cells are not freely choosing one pathway for the processing of a DSB, but rather apply logic, adapted to necessities generated by realities deriving from the character of the DSB being processed. Among the four processing pathways available to vertebrate cells for the removal of DSBs, only GC faithfully restores the genome to its original state. All remaining pathways, but c-NHEJ in particular, operate as DNA-end scavenging mechanisms, ultimately aiming at leaving no DNA ends open in the genome. The extremely high efficiency of c-NHEJ in higher eukaryotes begs the question regarding the benefits of its evolution. The large differences in the karyotypes between otherwise genetically related species and the newly discovered phenomenon of chromothripsis in cancer support the hypothesis that c-NHEJ may function as an accelerator of evolution.


Amylin and adrenomedullin are two peptides structurally related to calcitonin gene-related peptide (CGRP). We studied the occurrence of amylin in trigeminal ganglia and cerebral blood vessels of the cat with immunocytochemistry and evaluated the role of amylin and adrenomedullin in the cerebral circulation by in vitro and in vivo pharmacology. Immunocytochemistry revealed that numerous nerve cell bodies in the trigeminal ganglion contained CGRP immunoreactivity (-ir); some of these also expressed amylin-ir but none adrenomedullin-ir. There were numerous nerve fibres surrounding cerebral blood vessels that contained CGRP-ir. Occasional fibres contained amylin-ir while we observed no adrenomedullin-ir in the vessel walls. With RT-PCR and Real-Time-PCR we revealed the presence of mRNA for calcitonin receptor-like receptor (CLRL) and receptor-activity-modifying proteins (RAMPs) in cat cerebral arteries. In vitro studies revealed that amylin, adrenomedullin, and CGRP relaxed ring segments of the cat middle cerebral artery. CGRP and amylin caused concentration-dependent relaxations at low concentrations of PGF2a-precontracted segment (with or without endothelium) whereas only at high concentration did adrenomedullin cause relaxation. CGRP8-37 blocked the CGRP and amylin induced relaxations in a parallel fashion. In vivo studies of amylin, adrenomedullin, and CGRP showed a brisk reproducible increase in local cerebral blood flow as examined using laser Doppler flowmetry applied to the cerebral cortex of the alpha-chloralose-anesthetized cat. The responses to amylin and CGRP were blocked by CGRP8-37. The studies suggest that there is a functional sub-set of amylin-containing trigeminal neurons which probably act via CGRP receptors.