Restoring Atrial T-Tubules Augments Systolic Ca Upon Recovery From Heart Failure.

BACKGROUND: Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca2+. METHODS: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patch clamp, Ca2+, and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure. RESULTS: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca2+ release in the cell interior. Systolic Ca2+, ICa-L, sarcoplasmic reticulum Ca2+ content, and sarcoendoplasmic reticulum Ca2+ ATPase function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca2+ transient, the rate of Ca2+ removal, and the peak L-type Ca2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. CONCLUSIONS: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of ICa-L, sarcoplasmic reticulum Ca2+ content, and systolic Ca2+. We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.

Cardiac Transverse Tubules in Physiology and Heart Failure.

In mammalian cardiac myocytes, the plasma membrane includes the surface sarcolemma but also a network of membrane invaginations called transverse (t-) tubules. These structures carry the action potential deep into the cell interior, allowing efficient triggering of Ca2+ release and initiation of contraction. Once thought to serve as rather static enablers of excitation-contraction coupling, recent work has provided a newfound appreciation of the plasticity of the t-tubule network's structure and function. Indeed, t-tubules are now understood to support dynamic regulation of the heartbeat across a range of timescales, during all stages of life, in both health and disease. This review article aims to summarize these concepts, with consideration given to emerging t-tubule regulators and their targeting in future therapies.

PDE5 Inhibition Suppresses Ventricular Arrhythmias by Reducing SR Ca2+ Content.

[Figure: see text].

Enhanced calcium release at specialised surface sites compensates for reduced t-tubule density in neonatal sheep atrial myocytes.

Cardiac myocytes rely on transverse (t)-tubules to facilitate a rapid rise in calcium throughout the cell. However, despite their importance in triggering synchronous Ca2+ release, t-tubules are highly labile structures. They develop postnatally, increase in density during exercise training and are lost in diseases such as heart failure (HF). In the majority of settings, an absence of t-tubules decreases function. Here we show that despite reduced t-tubule density due to immature t-tubules, the newborn atrium is highly specialised to maintain Ca2+ release. To compensate for fewer t-tubules triggering a central rise in Ca2+, Ca2+ release at sites on the cell surface is enhanced in the newborn, exceeding that at all Ca2+ release sites in the adult. Using electron and super resolution microscopy to investigate myocyte ultrastructure, we found that newborn atrial cells had enlarged surface sarcoplasmic reticulum and larger, more closely spaced surface and central ryanodine receptor clusters. We suggest that these adaptations mediate enhanced Ca2+ release at the sarcolemma and aid propagation to compensate for reduced t-tubule density in the neonatal atrium.

Interaction of background Ca2+ influx, sarcoplasmic reticulum threshold and heart failure in determining propensity for Ca2+ waves in sheep heart.

Ventricular arrhythmias can cause death in heart failure (HF). A trigger is the occurrence of Ca2+ waves which activate a Na+ -Ca2+ exchange (NCX) current, leading to delayed after-depolarisations and triggered action potentials. Waves arise when sarcoplasmic reticulum (SR) Ca2+ content reaches a threshold and are commonly induced experimentally by raising external Ca2+ , although the mechanism by which this causes waves is unclear and was the focus of this study. Intracellular Ca2+ was measured in voltage-clamped ventricular myocytes from both control sheep and those subjected to rapid pacing to produce HF. Threshold SR Ca2+ content was determined by applying caffeine (10  mM) following a wave and integrating wave and caffeine-induced NCX currents. Raising external Ca2+ induced waves in a greater proportion of HF cells than control. The associated increase of SR Ca2+ content was smaller in HF due to a lower threshold. Raising external Ca2+ had no effect on total influx via the L-type Ca2+ current, ICa-L , and increased efflux on NCX. Analysis of sarcolemmal fluxes revealed substantial background Ca2+ entry which sustains Ca2+ efflux during waves in the steady state. Wave frequency and background Ca2+ entry were decreased by Gd3+ or the TRPC6 inhibitor BI 749327. These agents also blocked Mn2+ entry. Inhibiting connexin hemi-channels, TRPC1/4/5, L-type channels or NCX had no effect on background entry. In conclusion, raising external Ca2+ induces waves via a background Ca2+ influx through TRPC6 channels. The greater propensity to waves in HF results from increased background entry and decreased threshold SR content. KEY POINTS: Heart failure is a pro-arrhythmic state and arrhythmias are a major cause of death. At the cellular level, Ca2+ waves resulting in delayed after-depolarisations are a key trigger of arrhythmias. Ca2+ waves arise when the sarcoplasmic reticulum (SR) becomes overloaded with Ca2+ . We investigate the mechanism by which raising external Ca2+ causes waves, and how this is modified in heart failure. We demonstrate that a novel sarcolemmal background Ca2+ influx via the TRPC6 channel is responsible for SR Ca2+ overload and Ca2+ waves. The increased propensity for Ca2+ waves in heart failure results from an increase of background influx, and a lower threshold SR content. The results of the present study highlight a novel mechanism by which Ca2+ waves may arise in heart failure, providing a basis for future work and novel therapeutic targets.

Increased Ca buffering underpins remodelling of Ca2+ handling in old sheep atrial myocytes.

KEY POINTS: Ageing is associated with an increased risk of cardiovascular disease and arrhythmias, with the most common arrhythmia being found in the atria of the heart. Little is known about how the normal atria of the heart remodel with age and thus why dysfunction might occur. We report alterations to the atrial systolic Ca2+ transient that have implications for the function of the atrial in the elderly. We describe a novel mechanism by which increased Ca buffering can account for changes to systolic Ca2+ in the old atria. The present study helps us to understand how the processes regulating atrial contraction are remodelled during ageing and provides a basis for future work aiming to understand why dysfunction develops. ABSTRACT: Many cardiovascular diseases, including those affecting the atria, are associated with advancing age. Arrhythmias, including those in the atria, can arise as a result of electrical remodelling or alterations in Ca2+ homeostasis. In the atria, age-associated changes in the action potential have been documented. However, little is known about remodelling of intracellular Ca2+ homeostasis in the healthy aged atria. Using single atrial myocytes from young and old Welsh Mountain sheep, we show the free Ca2+ transient amplitude and rate of decay of systolic Ca2+ decrease with age, whereas sarcoplasmic reticulum (SR) Ca content increases. An increase in intracellular Ca buffering explains both the decrease in Ca2+ transient amplitude and decay kinetics in the absence of any change in sarcoendoplasmic reticulum calcium transport ATPase function. Ageing maintained the integrated Ca2+ influx via ICa-L but decreased peak ICa-L . Decreased peak ICa-L was found to be responsible for the age-associated increase in SR Ca content but not the decrease in Ca2+ transient amplitude. Instead, decreased peak ICa-L offsets increased SR load such that Ca2+ release from the SR was maintained during ageing. The results of the present study highlight a novel mechanism by which increased Ca buffering decreases systolic Ca2+ in old atria. Furthermore, for the first time, we have shown that SR Ca content is increased in old atrial myocytes.

Dependence of cardiac transverse tubules on the BAR domain protein amphiphysin II (BIN-1).

RATIONALE: Transverse tubules (t-tubules) regulate cardiac excitation-contraction coupling and exhibit interchamber and interspecies differences in expression. In cardiac disease, t-tubule loss occurs and affects the systolic calcium transient. However, the mechanisms controlling t-tubule maintenance and whether these factors differ between species, cardiac chambers, and in a disease setting remain unclear. OBJECTIVE: To determine the role of the Bin/Amphiphysin/Rvs domain protein amphiphysin II (AmpII) in regulating t-tubule maintenance and the systolic calcium transient. METHODS AND RESULTS: T-tubule density was assessed by di-4-ANEPPS, FM4-64 or WGA staining using confocal microscopy. In rat, ferret, and sheep hearts t-tubule density and AmpII protein levels were lower in the atrium than in the ventricle. Heart failure (HF) was induced in sheep using right ventricular tachypacing and ferrets by ascending aortic coarctation. In both HF models, AmpII protein and t-tubule density were decreased in the ventricles. In the sheep, atrial t-tubules were also lost in HF and AmpII levels decreased. Conversely, junctophilin 2 levels did not show interchamber differences in the rat and ferret nor did they change in HF in the sheep or ferret. In addition, in rat atrial and sheep HF atrial cells where t-tubules were absent, junctophilin 2 had sarcomeric intracellular distribution. Small interfering RNA-induced knockdown of AmpII protein reduced t-tubule density, calcium transient amplitude, and the synchrony of the systolic calcium transient. CONCLUSIONS: AmpII is intricately involved in t-tubule maintenance. Reducing AmpII protein decreases t-tubule density, reduces the amplitude, and increases the heterogeneity of the systolic calcium transient.

How cardiomyocyte excitation, calcium release and contraction become altered with age.

Cardiovascular disease is the main cause of death globally, accounting for over 17 million deaths each year. As the incidence of cardiovascular disease rises markedly with age, the overall risk of cardiovascular disease is expected to increase dramatically with the aging of the population such that by 2030 it could account for over 23 million deaths per year. It is therefore vitally important to understand how the heart remodels in response to normal aging for at least two reasons: i) to understand why the aged heart is increasingly susceptible to disease; and ii) since it may be possible to modify treatment of disease in older adults if the underlying substrate upon which the disease first develops is fully understood. It is well known that age modulates cardiac function at the level of the individual cardiomyocyte. Generally, in males, aging reduces cell shortening, which is associated with a decrease in the amplitude of the systolic Ca(2+) transient. This may arise due to a decrease in peak L-type Ca(2+) current. Sarcoplasmic reticulum (SR) Ca(2+) load appears to be maintained during normal aging but evidence suggests that SR function is disrupted, such that the rate of sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA)-mediated Ca(2+) removal is reduced and the properties of SR Ca(2+) release in terms of Ca(2+) sparks are altered. Interestingly, Ca(2+) handling is modulated by age to a lesser degree in females. Here we review how cellular contraction is altered as a result of the aging process by considering expression levels and functional properties of key proteins involved in controlling intracellular Ca(2+). We consider how changes in both electrical properties and intracellular Ca(2+) handling may interact to modulate cardiomyocyte contraction. We also reflect on why cardiovascular risk may differ between the sexes by highlighting sex-specific variation in the age-associated remodeling process. This article is part of a Special Issue entitled CV Aging.

Methods for isolating atrial cells from large mammals and humans.

The identification of disturbances in the cellular structure, electrophysiology and calcium handling of atrial cardiomyocytes is crucial to the understanding of common pathologies such as atrial fibrillation. Human right atrial specimens can be obtained during routine cardiac surgery and may be used for isolation of atrial myocytes. These samples provide the unique opportunity to directly investigate the effects of human disease on atrial myocytes. However, atrial myocytes vary greatly between patients, there is little if any access to truly healthy controls and the challenges associated with assessing the in vivo effects of drugs or devices in man are considerable. These issues highlight the need for animal models. Large mammalian models are particularly suitable for this purpose as their cardiac structure and electrophysiology are comparable with humans. Here, we review techniques for obtaining atrial cardiomyocytes. We start with background information on solution composition. Agents shown to increase viable cell yield will then be explored followed by a discussion of the use of tissue-dissociating enzymes. Protocols are detailed for the perfusion method of cell isolation in large mammals and the chunk digest methods of cell isolation in humans.

Perturbed atrial calcium handling in an ovine model of heart failure: potential roles for reductions in the L-type calcium current.

Heart failure (HF) is commonly associated with reduced cardiac output and an increased risk of atrial arrhythmias particularly during ß-adrenergic stimulation. The aim of the present study was to determine how HF alters systolic Ca(2+) and the response to ß-adrenergic (ß-AR) stimulation in atrial myocytes. HF was induced in sheep by ventricular tachypacing and changes in intracellular Ca(2+) concentration studied in single left atrial myocytes under voltage and current clamp conditions. The following were all reduced in HF atrial myocytes; Ca(2+) transient amplitude (by 46% in current clamped and 28% in voltage clamped cells), SR dependent rate of Ca(2+) removal (kSR, by 32%), L-type Ca(2+) current density (by 36%) and action potential duration (APD90 by 22%). However, in HF SR Ca(2+) content was increased (by 19%) when measured under voltage-clamp stimulation. Inhibiting the L-type Ca(2+) current (ICa-L) in control cells reproduced both the decrease in Ca(2+) transient amplitude and increase of SR Ca(2+) content observed in voltage-clamped HF cells. During ß-AR stimulation Ca(2+) transient amplitude was the same in control and HF cells. However, ICa-L remained less in HF than control cells whilst SR Ca(2+) content was highest in HF cells during ß-AR stimulation. The decrease in ICa-L that occurs in HF atrial myocytes appears to underpin the decreased Ca(2+) transient amplitude and increased SR Ca(2+) content observed in voltage-clamped cells.

A functional role for transverse (t-) tubules in the atria.

Mammalian ventricular myocytes are characterised by the presence of an extensive transverse (t-) tubule network which is responsible for the synchronous rise of intracellular Ca(2+) concentration ([Ca(2+)]i) during systole. Disruption to the ventricular t-tubule network occurs in various cardiac pathologies and leads to heterogeneous changes of [Ca(2+)]i which are thought to contribute to the reduced contractility and increased susceptibility to arrhythmias of the diseased ventricle. Here we review evidence that, despite the long-held dogma of atrial cells having no or very few t-tubules, there is indeed an extensive and functionally significant t-tubule network present in atrial myocytes of large mammals including human. Moreover, the atrial t-tubule network is highly plastic in nature and undergoes far more extensive remodelling in heart disease than is the case in the ventricle with profound consequences for the resulting systolic Ca(2+) transient. In addition to considering the functional role of the t-tubule network in the healthy and diseased atria we also provide an overview of recent data concerning the putative factors controlling the formation of t-tubules and conclude by posing some important questions that currently remain to be addressed and whether or not targeting t-tubules offers potential novel therapeutic possibilities for heart disease.

The origin of T-tubules.

Ring-like structures made up of caveolae appear to drive the development of membrane invaginations called T-tubules which are important for muscle contraction.

Age-related divergent remodeling of the cardiac extracellular matrix in heart failure: collagen accumulation in the young and loss in the aged.

The incidence of heart failure (HF) increases with age. This study sought to determine whether aging exacerbates structural and functional remodeling of the myocardium in HF. HF was induced in young (~18 months) and aged sheep (>8 years) by right ventricular tachypacing. In non-paced animals, aging was associated with increased left ventricular (LV) end diastolic internal dimensions (EDID, P<0.001), reduced fractional shortening (P<0.01) and an increase in myocardial collagen content (P<0.01). HF increased EDID and reduced fractional shortening in both young and aged animals, although these changes were more pronounced in the aged (P<0.05). Age-associated differences in cardiac extracellular matrix (ECM) remodeling occurred in HF with collagen accumulation in young HF (P<0.001) and depletion in aged HF (P<0.05). MMP-2 activity increased in the aged control and young HF groups (P<0.05). Reduced tissue inhibitor of metalloproteinase (TIMP) expression (TIMPs 3 and 4, P<0.05) was present only in the aged HF group. Secreted protein acidic and rich in cysteine (SPARC) was increased in aged hearts compared to young controls (P<0.05) while serum procollagen type I C-pro peptide (PICP) was increased in both young failing (P<0.05) and aged failing (P<0.01) animals. In conclusion, collagen content of the cardiac ECM changes in both aging and HF although; whether collagen accumulation or depletion occurs depends on age. Changes in TIMP expression in aged failing hearts alongside augmented collagen synthesis in HF provide a potential mechanism for the age-dependent ECM remodeling. Aging should therefore be considered an important factor when elucidating cardiac disease mechanisms.

Serial block face scanning electron microscopy reveals region-dependent remodelling of transverse tubules post-myocardial infarction.

The highly organized transverse tubule (t-tubule) network facilitates cardiac excitation-contraction coupling and synchronous cardiac myocyte contraction. In cardiac failure secondary to myocardial infarction (MI), changes in the structure and organization of t-tubules result in impaired cardiac contractility. However, there is still little knowledge on the regional variation of t-tubule remodelling in cardiac failure post-MI. Here, we investigate post-MI t-tubule remodelling in infarct border and remote regions, using serial block face scanning electron microscopy (SBF-SEM) applied to a translationally relevant sheep ischaemia reperfusion MI model and matched controls. We performed minimally invasive coronary angioplasty of the left anterior descending artery, followed by reperfusion after 90 min to establish the MI model. Left ventricular tissues obtained from control and MI hearts eight weeks post-MI were imaged using SBF-SEM. Image analysis generated three-dimensional reconstructions of the t-tubular network in control, MI border and remote regions. Quantitative analysis revealed that the MI border region was characterized by t-tubule depletion and fragmentation, dilation of surviving t-tubules and t-tubule elongation. This study highlights region-dependent remodelling of the tubular network post-MI and may provide novel localized therapeutic targets aimed at preservation or restoration of the t-tubules to manage cardiac contractility post-MI. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Impaired ß-adrenergic responsiveness accentuates dysfunctional excitation-contraction coupling in an ovine model of tachypacing-induced heart failure.

Reduced inotropic responsiveness is characteristic of heart failure (HF). This study determined the cellular Ca2+ homeostatic and molecular mechanisms causing the blunted ß-adrenergic (ß-AR) response in HF.We induced HF by tachypacing in sheep; intracellular Ca2+ concentration was measured in voltage-clamped ventricular myocytes. In HF, Ca2+ transient amplitude and peak L-type Ca2+ current (ICa-L) were reduced (to 70 ± 11% and 50 ± 3.7% of control, respectively, P <0.05) whereas sarcoplasmic reticulum (SR) Ca2+ content was unchanged. ß-AR stimulation with isoprenaline (ISO) increased Ca2+ transient amplitude, ICa-L and SRCa2+ content in both cell types; however, the response of HF cells was markedly diminished (P <0.05).Western blotting revealed an increase in protein phosphatase levels (PP1, 158 ± 17% and PP2A, 188 ± 34% of control, P <0.05) and reduced phosphorylation of phospholamban in HF (Ser16, 30 ± 10% and Thr17, 41 ± 15% of control, P <0.05). The ß-AR receptor kinase GRK-2 was also increased in HF (173 ± 38% of control, P <0.05). In HF, activation of adenylyl cyclase with forskolin rescued the Ca2+ transient, SR Ca2+ content and SR Ca2+ uptake rate to the same levels as control cells in ISO. In conclusion, the reduced responsiveness of the myocardium to ß-AR agonists in HF probably arises as a consequence of impaired phosphorylation of key intracellular proteins responsible for regulating the SR Ca2+ content and therefore failure of the systolic Ca2+ transient to increase appropriately during ß-AR stimulation.

Optimising Large Animal Models of Sustained Atrial Fibrillation: Relevance of the Critical Mass Hypothesis.

BACKGROUND: Large animal models play an important role in our understanding of the pathophysiology of atrial fibrillation (AF). Our aim was to determine whether prospectively collected baseline variables could predict the development of sustained AF in sheep, thereby reducing the number of animals required in future studies. Our hypothesis was that the relationship between atrial dimensions, refractory periods and conduction velocity (otherwise known as the critical mass hypothesis) could be used for the first time to predict the development of sustained AF. METHODS: Healthy adult Welsh mountain sheep underwent a baseline electrophysiology study followed by implantation of a neurostimulator connected via an endocardial pacing lead to the right atrial appendage. The device was programmed to deliver intermittent 50 Hz bursts of 30 s duration over an 8-week period whilst sheep were monitored for AF. RESULTS: Eighteen sheep completed the protocol, of which 28% developed sustained AF. Logistic regression analysis showed only fibrillation number (calculated using the critical mass hypothesis as the left atrial diameter divided by the product of atrial conduction velocity and effective refractory period) was associated with an increased likelihood of developing sustained AF (Ln Odds Ratio 26.1 [95% confidence intervals 0.2-52.0] p = 0.048). A receiver-operator characteristic curve showed this could be used to predict which sheep developed sustained AF (C-statistic 0.82 [95% confidence intervals 0.59-1.04] p = 0.04). CONCLUSION: The critical mass hypothesis can be used to predict sustained AF in a tachypaced ovine model. These findings can be used to optimise the design of future studies involving large animals.

Altered atrial cytosolic calcium handling contributes to the development of postoperative atrial fibrillation.

AIMS: Atrial fibrillation (AF) is a commonly occurring arrhythmia after cardiac surgery (postoperative AF, poAF) and is associated with poorer outcomes. Considering that reduced atrial contractile function is a predictor of poAF and that Ca2+ plays an important role in both excitation-contraction coupling and atrial arrhythmogenesis, this study aims to test whether alterations of intracellular Ca2+ handling contribute to impaired atrial contractility and to the arrhythmogenic substrate predisposing patients to poAF. METHODS AND RESULTS: Right atrial appendages were obtained from patients in sinus rhythm undergoing open-heart surgery. Cardiomyocytes were investigated by simultaneous measurement of [Ca2+]i and action potentials (APs, patch-clamp). Patients were followed-up for 6 days to identify those with and without poAF. Speckle-tracking analysis of preoperative echocardiography revealed reduced left atrial contraction strain in poAF patients. At the time of surgery, cellular Ca2+ transients (CaTs) and the sarcoplasmic reticulum (SR) Ca2+ content were smaller in the poAF group. CaT decay was slower in poAF, but the decay of caffeine-induced Ca2+ transients was unaltered, suggesting preserved sodium-calcium exchanger function. In agreement, western blots revealed reduced SERCA2a expression in poAF patients but unaltered phospholamban expression/phosphorylation. Computational modelling indicated that reduced SERCA activity promotes occurrence of CaT and AP alternans. Indeed, alternans of CaT and AP occurred more often and at lower stimulation frequencies in atrial myocytes from poAF patients. Resting membrane potential and AP duration were comparable between both groups at various pacing frequencies (0.25-8 Hz). CONCLUSIONS: Biochemical, functional, and modelling data implicate reduced SERCA-mediated Ca2+ reuptake into the SR as a major contributor to impaired preoperative atrial contractile function and to the pre-existing arrhythmogenic substrate in patients developing poAF.

Phosphodiesterase 5 inhibition improves contractile function and restores transverse tubule loss and catecholamine responsiveness in heart failure.

Heart failure (HF) is characterized by poor survival, a loss of catecholamine reserve and cellular structural remodeling in the form of disorganization and loss of the transverse tubule network. Indeed, survival rates for HF are worse than many common cancers and have not improved over time. Tadalafil is a clinically relevant drug that blocks phosphodiesterase 5 with high specificity and is used to treat erectile dysfunction. Using a sheep model of advanced HF, we show that tadalafil treatment improves contractile function, reverses transverse tubule loss, restores calcium transient amplitude and the heart's response to catecholamines. Accompanying these effects, tadalafil treatment normalized BNP mRNA and prevented development of subjective signs of HF. These effects were independent of changes in myocardial cGMP content and were associated with upregulation of both monomeric and dimerized forms of protein kinase G and of the cGMP hydrolyzing phosphodiesterases 2 and 3. We propose that the molecular switch for the loss of transverse tubules in HF and their restoration following tadalafil treatment involves the BAR domain protein Amphiphysin II (BIN1) and the restoration of catecholamine sensitivity is through reductions in G-protein receptor kinase 2, protein phosphatase 1 and protein phosphatase 2 A abundance following phosphodiesterase 5 inhibition.

Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation.

BACKGROUND: CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). RESULTS: We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. CONCLUSION: We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.