Despite the success of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines, there remains a need for more prevention and treatment options for individuals remaining at risk of coronavirus disease 2019 (COVID-19). Monoclonal antibodies (mAbs) against the viral spike protein have potential to both prevent and treat COVID-19, and reduce the risk of severe disease and death. Here, we describe AZD7442, a combination of two mAbs, AZD8895 (tixagevimab) and AZD1061 (cilgavimab), that simultaneously bind to distinct, nonoverlapping epitopes on the spike protein receptor binding domain to neutralize SARS-CoV-2. Initially isolated from individuals with prior SARS-CoV-2 infection, the two mAbs were designed to extend their half-lives and reduce effector functions. The AZD7442 mAbs individually prevent the spike protein from binding to angiotensin-converting enzyme 2 receptor, blocking virus cell entry, and neutralize all tested SARS-CoV-2 variants of concern. In a nonhuman primate model of SARS-CoV-2 infection, prophylactic AZD7442 administration prevented infection, whereas therapeutic administration accelerated virus clearance from lung. In an ongoing phase 1 study in healthy participants (NCT04507256), a 300 mg intramuscular injection of AZD7442 provided SARS-CoV-2 serum geometric mean neutralizing titers greater than 10-fold above those of convalescent serum for at least 3 months, which remained 3-fold above those of convalescent serum at 9 months post-AZD7442 administration. Approximately 1 to 2% of serum AZD7442 was detected in nasal mucosa, a site of SARS-CoV-2 infection. Extrapolation of the time course of serum AZD7442 concentration suggests AZD7442 may provide up to 12 months of protection and benefit individuals at high-risk of COVID-19. AZD7442 neutralizes SARS-CoV-2, has prophylactic and therapeutic efficacy in NHPs, and is projected to contribute to long-term human protection. The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to cause substantial morbidity and mortality worldwide. Although rollout of effective COVID-19 vaccines has reduced hospitalizations and death in several countries  represent an approach for both the prevention and treatment of COVID-19  and AZD1061 (cilgavimab), in clinical development for the prevention of symptomatic COVID-19 and the treatment of mild-to-moderate and severe COVID-19 (. Hydrogen-deuterium exchange mass spectrometry confirmed that AZD8895 and AZD1061 associate with distinct peptide residues on opposing faces of the RBD (fig. S1). AZD8895, AZD1061, and AZD7442 bound to the SARS-CoV-2 spike protein with high affinity . AZD7442 had a greater than 3,000-fold higher binding affinity compared to the binding affinity of the human ACE2 receptor  for the spike protein . AZD8895 and AZD1061 potently blocked RBD binding to ACE2 individually , demonstrating that the two mAbs can independently block RBD binding to ACE2.Co-crystal structures of AZD8895 and AZD1061 showed simultaneous binding to the RBD at distinct, noncontiguous, and nonoverlapping epitopes 50 9, 32, and 10 ng/ml, respectively; ). Importantly, as a result of a combination of two mAbs with nonoverlapping epitopes, AZD7442 retained potent neutralizing activity (fold-change IC50 <3.0) against SARS-CoV-2 Alpha, Beta, Gamma, and Delta VOCs compared with the USA-WA/1/2020 or AUS/IC01/2020 reference strains in the SARS-CoV-2 neutralization assays .Since the COV2-2196 and COV2-2130 progenitor mAbs were re-engineered as IgG1κ formatted antibodies with Fc variant amino acids designed to confer extended antibody half-life (YTE modifications) and reduction of Fc receptor (FcR) interactions (TM modifications) to create AZD8895 and AZD1061, these were tested in neutralization assays to ensure that they retained their potency against the USA-WA/1/2020 strain and to evaluate their potency against emergent VOCs. The high binding affinities of the AZD7442 antibodies for the viral spike protein translated into potent SARS-CoV-2 neutralization activity. AZD8895, AZD1061, and AZD7442 potently neutralized the USA-WA1/2020 strain of SARS-CoV-2 . This increased affinity for FcRn translated into extended half-lives (t1/2) for AZD8895 and AZD1061 in NHPs compared with mAbs without YTE modifications following intravenous (IV) administration of 600 mg/kg AZD7442. AZD1061 and AZD8895 had a median t1/2 of 19 days based on pharmacokinetic data collected over 8 weeks compared with t1/2 of 8 to 10 days for IgG in NHPs  Fc . In vitro assays further confirmed that the AZD7442 antibodies displayed little or no Fc effector function, including antibody-dependent cellular phagocytosis, antibody-dependent cellular cytotoxicity, antibody-dependent complement deposition, or antibody-dependent natural killer cell activation. AZD7442, AZD8895, and AZD1061 also did not mediate antibody-dependent enhancement of infection (and fig. S2).AZD7442 includes YTE and TM modifications in the antibody Fc regions. In vitro, AZD8895 and AZD1061 exhibited approximately 9-fold greater affinities for the human neonatal Fc receptor (huFcRn) than AZD8895-TM and AZD1061-TM  and one in cynomolgus macaques . For each study, three to four NHPs were administered either a 40 mg/kg dose of isotype control antibody or AZD7442 at a dose ranging from 0.04 to 40 mg/kg by IV infusion 3 days prior to viral challenge (prophylaxis) or a 40 mg/kg dose of AZD7442 24 hours after viral challenge . To evaluate the contribution of Fc effector function to viral clearance, one group from each study received AZD7442-YTE (with YTE but not TM modification) as either prophylaxis  or treatment . In the prophylaxis studies, AZD7442 serum concentrations increased proportionally with dose across the 0.04 mg/kg to 40 mg/kg dose range. AZD7442 concentrations in the serum translated into high concentrations of serum neutralizing antibody titers in rhesus macaques and in cynomolgus macaques. Geometric mean pseudovirus neutralization (Neut50) titers peaked at 4.5 and 6.4 log10 for rhesus macaques administered 4 and 40 mg/kg AZD7442, respectively ; geometric mean SARS-CoV-2 plaque reduction neutralization test (PRNT50) titers of 3.4 and 4.6 log10 were measured for cynomolgus macaques administered 4 and 40 mg/kg AZD7442, respectively . Low neutralizing titers were detected in the isotype control-treated rhesus macaques beginning day 10 post-infection consistent with the development of an adaptive humoral immune response .AZD7442 was evaluated in NHP models of SARS-CoV-2 infection in prophylaxis or treatment settings in two separate studies, one in rhesus macaques (5 plaque forming units (PFUs) of SARS-CoV-2 strain USA-WA1/2020 . SARS-CoV-2 viral subgenomic messenger RNA (sgmRNA) was measured by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) in bronchoalveolar lavage (BAL) and nasal swab samples up to 14 days after virus challenge. In rhesus macaques treated prophylactically with isotype control, peak concentrations of viral sgmRNA were detected on day 2 post-infection, with geometric means of 4.66 log10 copies/ml (BAL) and 4.73 log10 copies/swab  of sgmRNA detected . In contrast, sgmRNA was undetectable in the BAL samples of rhesus macaques receiving AZD7442 or AZD7442-YTE ; low concentrations of sgmRNA were detected transiently (day 2 only) in nasal swab samples from two of four rhesus macaques receiving 4 mg/kg AZD7442 (3.32–3.60 log10 copies/swab) and from one of four animals that received 4 mg/kg AZD7442-YTE (4.94 log10 copies/swab) . The undetectable concentrations of sgmRNA in the BAL samples from rhesus macaques that received as little as 4 mg/kg AZD7442 (comparable to the human 300 mg dose) indicates that prophylactic administration of AZD7442 can protect against SARS-CoV-2 lower respiratory tract infection in rhesus macaques. The lack of Fc effector function and complement binding did not impact efficacy in this model as the non-TM (AZ7442-YTE) and the TM-modified AZD7442 showed similar in vivo efficacies (3C). Rhesus macaques treated with AZD7442 24 hours after inoculation showed a reduction in viral sgmRNA 24 hours later (day 2 post-infection) in BAL  of SARS-CoV-2 strain USA-WA1/2020 . SARS-CoV-2 burden was measured in BAL and nasal swab samples up to 5 days after virus challenge. Prophylactic AZD7442 administration demonstrated a dose-dependent reduction of infectious virus titers in BAL samples , and a reduction in viral sgmRNA concentrations in BAL and nasal swab samples  compared with isotype control antibody. Importantly, the 4 mg/kg dose (comparable to the human 300 mg dose) was fully protective in both NHP studies. Consistent with observations from the rhesus macaque study, faster virus clearance was observed in cynomolgus macaques administered 40 mg/kg AZD7442 therapeutically compared with isotype control . Of note, AZD7442 treatment reduced viral titers similarly to AZD7442-YTE (no TM modification) at an equivalent 40 mg/kg dose , suggesting that Fc effector function and complement binding are not required for treating SARS-CoV-2 infection at this dose.In the second study, cynomolgus macaques were challenged on day 0 with 10. In sections of lung parenchyma, there was mild to marked perivascular cuffing of medium-caliber blood vessels by lymphocytes, alveolar wall thickening, and mixed inflammatory cell infiltrates comprising lymphocytes, neutrophils, and macrophages within alveolar spaces. There was mild to marked bronchial/bronchiolar inflammation with loss or blunting of bronchial/bronchiolar epithelium and infiltrates of lymphocytes and neutrophils within the lamina propria and submucosa. Although there was some variability between sections consistent with focal SARS-CoV-2 infection, lung sections from animals administered AZD7442 either prophylactically or therapeutically showed an overall reduction in inflammation compared with those treated with isotype control . This study is ongoing, and the data presented herein includes only the initial time points for which analyses have been completed. Participants in the active arm received an intramuscular (IM) dose of 300 mg AZD7442 , IV AZD7442 at doses of 300 mg, 1,000 mg, or 3,000 mg , or 3,000 mg AZD7442 co-administered by IV ; all groups included 10 healthy participants.1/2 of approximately 90 days for both antibodies in each dose cohort following either IV or IM administration . After a single IM dose of 300 mg, the geometric mean Cmax of AZD8895 (16.5 μg/ml) and AZD1061 (15.3 μg/ml) were similar and reached at a median Tmax after 14 days. SARS-CoV-2 neutralizing antibody titers in serum conferred by AZD7442 were considerably higher than titers associated with convalescent plasma . Geometric mean neutralizing titers afforded by AZD7442 300 mg IM and IV were approximately 22- and 41-fold higher, respectively, 7 days after dosing than those of convalescent serum samples. At day 270 post AZD7442 administration, the geometric mean neutralizing titers remained 3-fold higher than those of convalescent plasma samples (n = 28). To better understand the relative concentration of SARS-CoV-2 neutralizing antibodies conferred by AZD7442, we compared the ratio of neutralizing antibodies in serum following a single IM dose of 300 mg AZD7442 to neutralizing antibodies in convalescent serum samples. This analysis revealed that, within 1 week following administration, AZD7442 confers neutralizing antibody concentrations that were approximately 25-fold greater than those associated with convalescent serum . The median concentrations of AZD8895 and AZD1061 in NLF following an IM dose of 300 mg were 200 ng/ml through 30 days post-dose Antibody combinations comprising mAbs targeting distinct epitopes on the SARS-CoV-2 spike protein provide a higher threshold against virus escape from neutralization than single antibodies  at day 270 post-dose that was approximately 3-fold greater than those detected in convalescent plasma and similar to titers following vaccination. Given the high efficacy of vaccines in preventing COVID-19, these data for AZD7442 at this dose are encouraging. In addition, pharmacologically active concentrations of AZD7442 in the nasal mucosa at 7 and 30 days post-dose were, on average, 40-fold above the IC50 of AZD7442. These data support the potential of an IM dose of 300 mg AZD7442 to provide protection against COVID-19 for up to 12 months, but this requires confirmation in clinical studies.Although NHP models do not mimic severe COVID-19 pathology in humans, the findings reported here contributed to the further clinical evaluation of AZD7442 in COVID-19 prevention and treatment settings. The 4 mg/kg AZD7442 IV dose that demonstrated prevention of SARS-CoV-2 infection in NHPs resulted in serum concentrations comparable to those achieved with an IM dose of 300 mg AZD7442 delivered to healthy adult human participants while animals that received 40 mg/kg AZD7442 as treatment showed near maximal serum concentrations within 1 day of dosing. The IM 300 mg dose of AZD7442 conferred an anti5 PFU or 105 TCID50 is required to infect the animals, and peak viral load occurs earlier than in humans (day 1 to 2 versus day 4 to 7) , and potency to neutralize SARS-CoV-2 and variants. The mAbs were also assessed for binding to Fc receptors and effector function in comparison with control mAbs that lack the TM or YTE modifications in the Fc region. Assay details are provided in the Supplementary materials.Pharmacokinetics of AZD7442 were assessed in NHPs (cynomolgus macaques) as part of an 8-week good laboratory practice toxicology study conducted at Charles River Laboratories, under protocols approved by the Charles River – Nevada Institutional Animal Care and Use Committee (IACUC). Animal numbers were determined taking into account ethical considerations, and the ability to detect potential toxicities in the different groups given the timepoints for necropsy. Animals were randomized and assigned to groups using a computer-based procedure prior to transfer to study; males and females were randomized separately. Personnel who performed ex vivo analyses for quantitation of AZD7442 were blinded to treatment groups at time of assay.th edition of the Guide for the Care and Use of Laboratory Animals; the Animal Welfare Act; and the 2015 reprint of the Public Health Service Policy on Human Care and Use of Laboratory Animals. The total number of animals used, group sizes and number of groups were considered the minimum required to properly characterize the effects of AZD7442, AZD7442-YTE and the isotope control mAb R347-TM-YTE, and the study was designed so that it did not need an unnecessary number of animals to accomplish its objectives. Animals were randomly assigned to groups based on weight and gender. Personnel who performed ex vivo analyses were blinded to treatment groups at time of assay. The assigned veterinary pathologist was blinded to treatment groups at time of slide reading. Animal Research: Reporting of In Vivo Experiments  guidelines were also followed in regard to the performance of all animal studies. Interventions to reduce pain, suffering and distress were taken whenever possible in accordance with these protocols.Prophylactic and therapeutic efficacy of AZD7442 was assessed in SARS-CoV-2 challenge studies in NHPs (rhesus macaques and cynomolgus macaques). These studies were approved by the IACUC (protocol numbers: 20-035 and 21-018P) and conducted at BIOQUAL, Inc., in adherence of the following standards of the Association for Assessment and Accreditation of Laboratory Animal Care: the 8Clinical results are reported from an ongoing phase 1, randomized, double-blind placebo-controlled, dose-escalation study of the safety, tolerability, and pharmacokinetics of AZD7442 in healthy adult participants with no prior history of COVID-19 and no prior receipt of a COVID-19 vaccine (NCT04507256). Participants, investigators, clinical staff, and the study monitor were all blinded from the assigned intervention. The study was conducted in compliance with the ethical principles originating in or derived from the Declaration of Helsinki and in compliance with the International Confederation on Harmonization Good Clinical Practice Guidelines. All participants provided written informed consent before entering the study. The study protocol and informed consent documentation were reviewed and approved by London – Riverside Ethics Committee, UK (reference: 20/LO/1010). Sixty healthy participants aged 18 to 55 years at a single site in the United Kingdom were randomized 5:1 to one of five dose cohorts to receive one of four AZD7442 doses  or placebo.Cynomolgus macaques were received from Orient BioResource and were between 2.2 and 5.1 years old; males weighing between 1.9 and 2.6 kg and females weighing between 2.2 and 2.5 kg at the time of dosing. AZD7442 was administered to cynomolgus macaques by an IV infusion at 600 mg/kg (n = 10) or by an IM injection at 300 mg/kg (n = 4); control animals were administered vehicle alone at volumes equivalent to AZD7442 by a single IV infusion (n = 12) or a single IM injection (n = 4). For the IV administered animals, all toxicology and safety assessment endpoints were evaluated at day 15  and day 57  necropsy timepoints.The IM animals had only general in-life procedures, observations, and measurements collected. IV infusion was by a suitable peripheral vein using an infusion pump connected to a temporary indwelling catheter once for 15 min. AZD8895 and AZD1061 were administered by separate 15-min infusions, for a total infusion time of 30 min. A 1 ml saline flush was administered following each 15 min infusion. IM injection to the anterior thigh was performed on temporarily restrained but not sedated animals. AZD8895 and AZD1061 were administered separately by two injections, one injection per thigh. In-life measurements included post dose and detailed clinical observations, injection site dermal scoring, body weights, food consumption, veterinary physical examinations, ophthalmic examinations, electrocardiography exams, neurologic examination, blood pressure and heart rate, respiratory rate and body temperature. Blood was collected by venipuncture at various times before AZD7442 administration and up to 8 weeks post dose to measure AZD8895 and AZD1061 serum concentrations and ex vivo pharmacodynamic evaluations. At study termination, animals were euthanized by IV injection of a commercially available veterinary euthanasia solution, followed by exsanguination.NHPs were quarantined and allowed to acclimatize for at least 7 days at BIOQUAL, Inc. Upon initiation of SARS-CoV-2 challenge procedures, the NHPs were housed in HEPA-filtered microisolator caging units. Potential confounders were not controlled for. Cage-side observations were performed at least twice a day. Physical assessments of anesthetized NHPs included heart rate, body weight, and rectal temperature at the time of sedation. No NHPs were excluded a priori after none showed signs of illness or poor health. Veterinary care was provided by full-time veterinarians. NHPs received a primate diet  ad libitum and were provided environmental enrichment by BIOQUAL, Inc, in accordance with the requirements of the Office of Laboratory Animal Welfare, the USA Animal Welfare Act, and the Guide for the Care and Use of Laboratory Animals.Macaca mulatta) of Indian origin, aged 5 to 6 years and weighing between 3.6 and 7.3 kg, received prophylactic 10 ml IV infusions of the following mAbs 3 days prior to SARS-CoV-2 challenge: 40 mg/kg isotype control mAb R347-TM-YTE ; 40 mg/kg AZD7442 ; 4 mg/kg AZD7442 ; or 4 mg/kg AZD7442-YTE . Four additional rhesus macaques weighing between 4.6 and 6.6 kg received a therapeutic 10 ml IV infusion of 40 mg/kg AZD7442 1 day after SARS-CoV-2 challenge .For the first NHP SARS-CoV-2 challenge study, 14 rhesus macaques (Macaca fascicularis) of Cambodian origin, aged 4 to 5 years old and weighing between 3.2 and 5.6 kg, received prophylactic 10 ml IV infusions of the following mAbs 3 days prior to SARS-CoV-2 challenge: 40 mg/kg isotype control mAb R347-TM-YTE ; 4 mg/kg AZD7442 ; 0.4 mg/kg AZD7442 ; or 0.04 mg/kg AZD7442 . Six additional cynomolgus macaques weighing between 3.4 and 6.3 kg received therapeutic 10 ml IV infusions of 40 mg/kg AZD7442  or 40 mg/kg AZD7442-YTE  1 day after SARS-CoV-2 challenge.In the second NHP SARS-CoV-2 challenge study, 18 cynomolgus macaques (5 PFU (rhesus macaques) or 105 TCID50 (cynomolgus macaques). Rhesus macaques were challenged with an in-house generated stock, lot no. 022320-1100, expanded in Vero E6 cells from BEI Resources   for virologic analyses. One rhesus macaque from each of the four groups  was euthanized on day 2 post-infection for histopathology analyses. Cynomolgus macaques from all groups were euthanized on day 5 post-infection for histopathology analyses. The design for each study is summarized in RNA was extracted from BAL and nasal swab samples using a QIAcube HT (Qiagen) and the Cador pathogen HT kit per manufacturer’s instructions and eluted in nuclease-free water. RNA was reverse transcribed using superscript VILO (Thermo Fisher Scientific) per manufacturer’s instructions and tested in duplicate with QuantStudio 6 and 7 Flex RTPCR System (Thermo Fisher Scientific). Viral titers were calculated to give copies/ml using a viral RNA standard curve and primers and probes  that target the SARS-CoV-2 E gene sub-genomic mRNA (sgmRNA): genomic RNA - 2019-nCoV_N1-F:5′-GAC CCC AAA ATC AGC GAA AT-3′ ; 2019-nCoV_N1-R: 5′-TCT GGT TAC TGC CAG TTG AAT CTG-3′; and 2019-nCoV_N1-P: 5′-FAM-ACC CCG CAT TAC GTT TGG ACC-BHQ1-3′: sgmRNA SG-F: CGATCTTGTAGATCTGTTCCTCAAACGAAC; SG-R: ATATTGCAGCAGTACGCACACACA; and FAM-ACACTAGCCATCCTTACTGCGCTTCG-BHQ. To generate a standard curve for the SARS-CoV-2 E gene sgmRNA assay, the SARS-CoV-2 E gene sgmRNA was cloned into a pcDNA3.1 expression plasmid. This insert was transcribed using the AmpliCap-Max-T7 High Yield Message Marker Kit (Cellscript) to obtain RNA. The limit of detection was 50 copies/ml.The concentrations of AZD7442, AZD7442-YTE, and control R347-TM-YTE in NHP serum samples were determined using an in-house enzyme-linked immunosorbent assay (ELISA) for human IgG against a known standard curve. Absorbance at 450 nm was recorded using a VersaMax or Omega microplate reader. Background-subtracted absorbance values for each standard was fit to a nonlinear, sigmoidal (4PL) curve using GraphPad Prism, and the ensuing curve was used to calculate concentrations of human IgG in each serum sample. Detailed ELISA methodology is provided in the Supplementary materials.50) was calculated as described previously , during infusion and after infusion (IV cohorts only), at 8 hours post-dose and 1-, 3-, 5-, 7-, 14-, 30-, 60-, 90-, 150-, and 210 days post dose , and 270 days post dose (300 mg dosing cohorts only). To determine the SARS-CoV-2 neutralizing activity of AZD7442, serum samples were collected and tested pre-dose and 7-, 30-, 60-, 90-, 150-, and 210 days post dose  and 270 days post dose (300 mg dosing cohorts only). To determine AZD7442 concentrations in NLF, Nasosorption Fx·i  samples were collected 7 and 30 days post dose. Preliminary interim results of serum and NLF antibody concentrations, and neutralizing antibody titers are reported to confirm preclinical information for AZD7442. AZD7442 concentrations and neutralizing antibody titers in human serum were predicted beyond month 9 using a pharmacokinetic model. This consisted of a central blood and peripheral distribution compartment with first-order absorption from the injection site into the central compartment, and first-order elimination from the central compartment.80) was calculated as described previously  at Viroclinics Biosciences. A standard number of SARS-CoV-2 infectious units were incubated with serial dilutions of serum. After a 1 hour pre-incubation period of the virus, 100 μl of the mixture was added to Vero E6 cells for 16 to 24 hours. After incubation, cells were formalin-fixed followed by incubation with a mAb targeting the viral nucleocapsid protein, followed by a secondary anti-human IgG peroxidase conjugate (Thermo Fisher Scientific) and KPL TrueBlue substrate. Images of all wells were acquired by a CTL ImmunoSpot analyzer, equipped with software to quantitate the nucleocapsid-positive cells . The 80% neutralization titer  was added to each synthetic absorptive matrix. Immediately upon elution, up to 60 μl of NLF eluant was used to measure the urea concentration with an enzymatic-colorimetric assay. Next, 180 μl of NLF eluant was subjected to immunocapture and enzymatic digestion treatments identical to the method described for serum sample analysis. The ratio of urea concentrations in NLF eluant and in serum were used for normalization of AZD7442 concentrations in the NLF to correct for differences in NLF sample volumes collected, and extraction efficiency.2 weighted, least-squares regression algorithm was used to quantify unknown samples.Separate validated assays with similar methodologies were used to measure the concentrations of AZD7442 in cynomolgus macaque and human serum. A 20 μl sample was diluted and extracted with streptavidin magnetic beads coated with biotinylated RBD of SARS-CoV-2. The isolated analytes were subjected to denaturation, reduction, alkylation, and trypsin digestion. After digestion, the extract was fortified with stable isotope labeled peptide internal standard working solution. The final extract was analyzed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS) with positive electrospray. A linear, 1/concentration50 values for binding of RBD to human ACE2 (for ≥2 independent assays). Percent neutralization is shown as regression dose-response curves, with means (SD) from two technical replicates, and mean IC50 values calculated from three independent experiments. In vivo, half-life and effector function parameters for AZD8895, AZD1061, and AZD7442 were assigned based on normalized area under the concentration curve (AUC) values. In vitro, Fc effector function versus mAbs with TM substitutions are reported as mean response with background subtracted from 2 or more independent experiments, and as AUC. Human IgG serum concentrations and serum neutralizing antibody titers in NHPs are provided as geometric mean (SD) as are viral burden in bronchial lavage and nasal swab samples in NHPs. All histological evaluation was performed by a board-certified veterinary pathologist with individual pathology scores reported as mean (SD). In human serum, antibody concentrations following IV and IM administration are shown as mean (SD), predicted mean, and 90% prediction interval, with neutralizing antibody titers reported as geometric mean PRNT80 titer  or GMT . Fold difference for AZD8895 and AZD1061 concentrations in NLF are shown as individual values and as mean (95% CI). GraphPad Prism software (versions 8.4.3 or higher) was used for data analysis and graph production.Descriptive statistics were used to present data. Peptide uptake during hydrogen-deuterium exchange is shown as mean (standard deviation [SD]) and measures of affinity for binding to spike trimer protein were based on at least two independent experiments. Nonlinear regression curves were used to calculate IC


Os experimentos de contratilidade foram realizados em átrio esquerdo isolado de cobaia exposto às concentrações crescentes da droga (1 a 5.000μM). O AE reduziu a força de contração atrial  de modo dependente de concentração. O efeito do AE sobre a ICa,L também foi avaliado em cardiomiócitos ventriculares isolados de camundongos, utilizando-se a técnica de “patch-clamp”. O AE apresentou um efeito inibitório (IC50=1.337±221μM) sobre os canais de Ca2+ sensíveis à voltagem (CaV1.2). Em conclusão, o AE apesenta efeito cardiodepressor que se deve, pelo menos em parte, à diminuição da entrada de Ca2+ nos cardiomiócitos.No presente trabalho investigou-se o efeito inotrópico do acetato de eugenil (AE), bem como sua ação sobre a corrente de Ca As doenças cardiovasculares são um problema de saúde pública e estão entre as principais causas de óbitos no mundo.Syzygium aromaticum (L.) Merr. & L.M. Perry, popularmente conhecida como cravo-da-índia, é constituída por diversos compostos químicos que apresentam uma ampla gama de efeitos farmacológicos. O eugenol . Além disso, o eugenol atua como cardioprotetor.Estudos demonstraram que o eugenol apresenta atividade cardiodepressora em ratosCa,L.Apesar de diversos estudos abordarem as propriedades farmacológicas do eugenol sobre o coração, até o momento não existem informações sobre a ação do AE no miocárdio. Dessa forma, o presente estudo descreve, pela primeira vez, os efeitos do AE sobre a contratilidade cardíaca e sua ação inibitória sobre a ICavia porcellus, 400-600g). Para os estudos eletrofisiológicos foram utilizados camundongos machos adultos C57Bl/6J. Todos os procedimentos foram aprovados pela Comissão de Ética para Uso de Animais (CEUA) da Universidade do Estado da Bahia (licença: 03/2017).Para os experimentos de contratilidade foram utilizadas cobaias machos e fêmeas , aerado com oxigênio , estirado para uma tensão de 1gF e estimulado eletricamente . A força contrátil foi captada por um transdutor isométrico, sendo os sinais digitalizados (512Hz) e armazenados em um computador. Os átrios esquerdos foram submetidos às concentrações crescentes de AE .O átrio esquerdo foi mantido em solução de Tyrode modificada  com a seguinte composição (em mM): 140 NaCl; 5,4 KCl; 0,5 MgClPara fazer a solução-estoque de AE (obtido da Sigma-Aldrich) foi usado dimetil sulfóxido (DMSO).2+ do tipo L  foi utilizada a técnica de patch-clamp,whole-cell voltage-clamp. A composição da solução interna (em mM) foi: 120 CsCl, 10 HEPES, 5 EGTA, 20 TEA-Cl e 5 NaCl . O Tyrode foi usado como solução externa. As células foram mantidas em um potencial de membrara de −80 mV, e depois foram submetidas a um pré-pulso que despolarizou a membrana para −40mV (50ms). Em seguida a membrana foi despolarizada para 0 mV . A amplitude da ICa,L foi medida pela diferença entre o final do pulso teste (0mV) e o pico. As células foram expostas ao AE . Os sinais foram digitalizados (5kHz) e armazenados em computador.Cardiomiócitos ventriculares de camundongos C57Bl/6J foram enzimaticamente isolados.Os resultados são expressos com média ± erro padrão da média e foram analisados estatisticamente empregando-se o teste “t” pareado na sua forma bicaudal .μM) reduziu em aproximadamente 60% a amplitude da contração atrial quando comparado com o controle. Tal efeito foi parcialmente revertido (aproximadamente 75%) após a remoção da droga, quando comparado com o controle. Na 50 (concentração que induz metade do efeito máximo) de 558±24,06μM e efeito máximo=100%.Os traçados da Ca,L medida experimentalmente. Na Ca,L. Na Ca,L na situação controle (ausência da droga) e na presença de 10, 700 e 3.000μM de AE. Na Ca,L de modo dependente de concentração (IC50=1.337±221μM).A 2+ do tipo L (CaV1.2) em cardiomiócitos.Os resultados aqui apresentados demonstram que o AE reduz a força de contração do músculo atrial de cobaia de modo dependente de concentração. Também foi observado que o AE inibe os canais de CaO AE apresentou um efeito cardiodepressor sobre o inotropismo atrial. Apesar de não existir dados na literatura que demonstrem o efeito cardiodepressor do AE, dados de seus análogos, como o eugenol, estão disponíveis. O eugenol, de modo semelhante à droga aqui investigada reduz a força de contração do miocárdio ventricular de cobaia2+ que é determinada pelo influxo de Ca2+ através dos canais de Ca2+ presentes no sarcolema, bem como pela quantidade de Ca2+ liberada pelo retículo sarcoplasmático (RS) no processo denominado de acoplamento excitação-contração. A despolarização sarcolemal leva a abertura dos canais de Ca2+ do tipo L durante a fase do platô do potencial de ação, o que leva a uma corrente de entrada de Ca2+. Esse influxo de Ca2+ estimula a liberação do Ca2+ armazenado no RS, processo conhecido como liberação de Ca2+ induzida por Ca2+, que induz na contratilidade cardíaca.2+ estão envolvidos na regulação da contratilidade no músculo cardíaco.A força da contração do miocárdio correlaciona-se com alterações da amplitude do transiente de CaCa,L em cardiomiócitos isolados. Os achados indicam que o AE reduz a amplitude da ICa,L, efeito que pode estar associado à ativação de receptores que modulam a ICa,L e/ou ao bloqueio direto desses canais. Este mecanismo pode ser o responsável pela redução da força induzida pelo AE, uma vez que leva a redução da liberação de Ca2+ pelo RS.Para tentar explicar o inotropismo negativo do AE sobre o músculo cardíaco foi verificado sua ação sobre a amplitude da I2+ nos cardiomiócitos. Nesses experimentos, foi observado que o eugenol apresenta uma IC50 de 127μM, valor menor do que a IC50 do AE (1.337μM). Esses dados sugerem que o eugenol é mais potente em bloquear a corrente de Ca2+ do que o AE.Sensch et al.,V1.2.O AE apresenta efeito cardiodepressor que pode ser explicado, pelo menos em parte, pela inibição do Ca Cardiovascular diseases are a public health problem being among the leading causes of death worldwide.Syzygium aromaticum (L.) Merr. & L.M. Perry, popularly known as clove, produces several chemical compounds that have a wide range of biological effects. Eugenol . In addition, eugenol acts as cardioprotector.Studies have shown that eugenol has cardiodepressor activity in ratsCa,L.Although several studies have adressed the pharmacological properties of eugenol on mammalian heart, so far there is no information on the action of EA on the cardiomyocytes. Thus, this study describes the effects of EA on atrial contractility and its inhibitory action on ICavia porcellus, both sexes, 400-600g) were used. For electrophysiological studies, adult male C57Bl/6J mice were used. All procedures were approved by the Ethics Committee for the Use of Animals (CEUA) of the University of the State of Bahia (license: 03/2017).For contractility experiments guinea pigs , aerated with oxygen (99.9%). The atrium was stretched to attain a resting tension of 1gF and it was electrically stimulated . The contractile force was captured by an isometric transducer. Signals were digitized (512Hz) and stored on a computer. The left atria were subjected to increasing concentrations of EA .The left atrium from guinea pig was maintained in a modified Tyrode's solution  with the following composition (in mM): 140 NaCl; 5.4 KCl; 0.5 MgClDimethyl sulfoxide (DMSO) was used to make the stock solution of EA (obtained from Sigma-Aldrich).2+ current  patch-clamp technique in whole-cell voltage-clamp mode was used.7,8 Cells were maintained at a resting membrane potential of −80mV, then the sarcolemma was subjected to a pre-pulse to −40mV (50ms) followed by a pulse to 0 mV . The ICa,L amplitude was measured by the difference between the current at the end of the test pulse (0mV) and the peak current. The cells were exposed to EA . The signals were digitized (5kHz) and stored on the computer.Ventricular cardiomyocytes from C57Bl/6J mice were isolated as previously described.The results are expressed as mean ± standard error of the mean and were statistically analyzed using the two-tailed “t” test (significance level: p<0.05).μM) reduced the amplitude of atrial contraction by approximately 60% when compared to the control. This effect was partially reversed (approximately 75%) after removing the drug (washout). In 50  of 558±24.06μM and a maximum effect = 100%.Representative traces in Ca,L measured in healthy isolated cardiomyocytes. Ca,L. In Ca,L in the control (absence of the drug), and in the presence of 10, 700, and 3,000 μM of EA. Ca,L in a concentration-dependent manner .2+ channels (CaV1.2) in isolated cardiomyocytes from mice.In our study we show that EA reduces the contraction force of guinea pig left atrial myocardium in a concentration-dependent manner. It was also observed that EA inhibits L-type CaThe EA showed a cardiodepressor effect on atrial inotropism. Although there are no data in the literature that demonstrate the negative inotropic effect of AE, information from its analogues, such as eugenol, is found. Similarly to the drug investigated here, Eugenol reduced the contraction force of ventricular cardiomyocyte from guinea pig2+ concentration which is determined by the Ca2+ influx through Ca2+ channels present in the sarcolemma, as well by the amount of Ca2+ released by the sarcoplasmic reticulum (SR) in the process called excitation-contraction coupling. The membrane depolarization leads to the opening of the L-type Ca2+ channels mainly during the plateau phase of action potential leading to an inward Ca2+ current. This Ca2+ entry stimulates the release of Ca2+ stored in SR, a process known as Ca2+-induced Ca2+ release, which contributes to cardiac contractility.2+ handling are involved in the control of cardiac contractility.The cardiomyocyte contraction correlates with the increase in cytosolic CaCa,L amplitude was investigated in isolated cardiomyocytes. The findings described here show that EA reduces the peak of ICa,L. This effect may be associated with the activation of receptors that modulate ICa,L, and/or direct action of EA on CaV1.2. The reduction of ICa,L is able to explain the decrease of contractility induced by EA, since it would lead to a reduction in the release of Ca2+ from SR. Although additional mechanism may also be involved.In order to explain the negative inotropic effect of EA on the cardiac muscle, the drug effect on the I2+ influx in cardiomyocytes. In these experiments, it was observed that eugenol has an IC50 of 127μM, a value lower than the IC50 observed for AE . These data suggest that blocking effect of eugenol on Ca2+ channels is more potent than EA.Sensch et al.,V1.2.Eugenyl acetate has a cardiodepressor effect that can be explained, at least in part, by the inhibition of Ca


Pi influx via PiT-1/-2 increases the abundance of PiT-1/-2 and depolarization-activated Ca2+ entry due to its electrogenic properties, which may lead to Ca2+ and Pi overload and oxidative stress. At least four mitochondrial Pi transporters are suggested, among which the phosphate carrier (PiC) is known to be mainly involved in mitochondrial Pi uptake. Pi transport via PiC may induce hyperpolarization and superoxide generation, which may lead to mitochondrial dysfunction and endoplasmic reticulum stress, together with generation of cytosolic ROS. Increase in net influx of Ca2+ and Pi and their accumulation in the cytosol and mitochondrial matrix synergistically increases oxidative stress and osteogenic differentiation, which could be prevented by suppressing either Ca2+ or Pi overload. Therapeutic strategies targeting plasmalemmal and mitochondrial Pi transports can protect against Pi-induced oxidative stress and vascular calcification.Inorganic phosphate (Pi) is essential for maintaining cellular function but excess of Pi leads to serious complications, including vascular calcification. Accumulating evidence suggests that oxidative stress contributes to the pathogenic progression of calcific changes. However, the molecular mechanism underlying Pi-induced reactive oxygen species (ROS) generation and its detrimental consequences remain unclear. Type III Na Vascular calcification includes intimal and medial calcification, which occur via two different pathological mechanisms. Intimal calcification involves the formation of atherosclerosis plaques with necrotic lipid core and activation of macrophages, which leads to endothelial cell inflammation. Blood flow is obstructed when the plaques rupture, causing ischemia. Medial calcification is an active and complex cell-mediated process characterized by the absence of inflammatory cells and is associated with inorganic phosphate (Pi) and calcium overload in vascular smooth muscle cells (VSMCs). It is also known as Mönckeberg’s sclerosis and causes arterial stiffness and decreases the compliance of vessels, which is observed in aging individuals and those with chronic kidney disease and diabetes mellitus. Medial calcification can happen in the small arteries of the toes to large arteries such as the tibial, popliteal, and femoral arteries of the legs or the carotid arteries ,2,3.2+ and Pi occur simultaneously in VSMCs. Under hyper-phosphatemic conditions, VSMCs are trans-differentiated into bone-like cells that express high levels of osteogenic markers, including Runt-related transcription factor 2 (Runx2), osteopontin (OPN), osteocalcin, alkaline phosphatase (ALP), SRY-Box transcription factor 9 (Sox9), and type II and X collagen (Col II and Col X), and low levels of α-smooth muscle actin (αSMA) and smooth muscle 22α (SM22α). Osteogenic trans-differentiation can be associated with other phenomena, including loss of inhibitors such as matrix Gla protein, pyrophosphate, and fetuin A, or enhancement of cell senescence features, such as DNA damage and oxidative stress.Research shows that several key events mediated by CaSo far, studies have described the pathogenesis of Pi-induced calcification related to osteogenic differentiation and apoptotic body formation in VSMCs. Some of them have emphasized the role of cytosolic and mitochondrial oxidative stress upstream of the upregulation of osteogenic genes and changes in calcification; however, the molecular mechanisms connecting high Pi levels with oxidative stress have not been elucidated. To understand the pathogenic processes underlying cellular Pi overload, research on how Pi enters the cytosol and is taken up by the mitochondrial matrix must be conducted.31P NMR and LC/MS/MS has been reported i), which was dependent on extracellular Ca2+ concentration and was blocked by VGCC inhibitors, verapamil or nimodipine. Furthermore, Pi-induced elevation in [Ca2+]i was abolished in cells lacking PiT-1/2 or by extracellular Na+ free condition i, the level of Ca2+ in the mitochondrial matrix increases due to more Ca2+ uptake from the cytosol. This elevation in matrix Ca2+ level stimulates the key mitochondrial enzymes of the tricarboxylic acid cycle, including pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, isocitrate dehydrogenase [2 and induces excessive ETC activation, subsequently accelerating mitochondrial superoxide generation from ETCs. Moreover, Ca2+ activates ATP synthase (complex V) [2+ accumulation promotes oxidative stress by activating nitric oxide synthase (NOS), which blocks complex IV by synthesizing NO, leading to excess ROS formation [2O2 production and mitochondrial swelling [As described above, high Pi activates depolarization-triggered Cain VSMCs . It has in VSMCs , leadingignaling . As anotrogenase ,110, whimplex V) ,112, α-gmplex V) , and ANTmplex V) , which eormation ,116,117.swelling .2+ uptake via MCU is driven by an electrical gradient [2+ uptake. This process is also accelerated by the presence of anions, such as Pi, acetate, β−hydroxybutyrate, glutamate, and bicarbonate, which can provide a H+ source for the H+ pumps of the respiratory chain. In the absence of adequate Pi, mitochondrial Ca2+ uptake, mitochondrial membrane potential, and O2 consumption rates are suppressed [2+-Pi salts in the matrix [2+, thereby maintaining active mitochondrial Ca2+ uptake by decreasing the uphill limitation of the Ca2+ gradient [2+ levels and oxidative stress generation by prolonged Ca2+ and Pi overload.Mitochondrial Cagradient ; thus, Eppressed . Moreovee matrix  and keepgradient ,123. Thu2+, the oxidative stress generated by excess Pi activates ERK/mTOR/p70S6K signaling. In turn, mTORC1 potentially suppresses the expression of antioxidant genes such as CAT, SOD1, and SOD2, causing an increase in oxidative stress [2+ concentration or by chelating intracellular Ca2+ [2+ load using voltage-gated Ca2+ channel blockers also significantly alleviated ROS generation and calcific changes in VSMCs [2+ in oxidative stress-triggered vascular calcification /AKT pathway also contributes considerably to high Pi-induced increase in VSMC osteo-induction ,141,142.radation . Indeed,radation .RUNX2 promoter, eliciting VSMC differentiation and calcification [A recent work has been shown that ER stress intensified the calcification of VSMCs by stimulating the release of Grp78-loaded extracellular vesicles . Additiofication . More cl2+ accumulation due to depolarization-activated Ca2+ influx. Mitochondrial Pi transport also increases net Ca2+ uptake due to augmented electrical driving force and free Ca2+ gradients. Oxidative stress due to Ca2+ and Pi overload induces mitochondrial dysfunction, ER stress, and lysosomal dysfunction, all of which are caused or aggravated by perturbations in Ca2+ homeostasis . Cytosolic alkalinization due to Pi uptake also accelerates mitochondrial Pi influx and facilitates PT pore opening. All these mechanisms amplify Pi overload-induced detrimental changes in the cytosol and mitochondria after oxidative stress. On the contrary, this positive feedback mechanism might prevent disease progression by inhibiting any one component of the signal amplification loop. Further investigations to elucidate the mechanism via which Pi accumulates with Ca


In this study, we tested the hypothesis that remodeling of 3′,5′-cyclic adenosine monophosphate (cAMP)-dependent protein kinase A (PKA) signaling is linked to these compartment-specific changes (up- or down-regulation) in Ca2+-handling. Perforated patch-clamp experiments were performed in atrial myocytes from 53 patients with AF and 104 patients in sinus rhythm (Ctl). A significantly higher frequency of transient inward currents (ITI) activated by spontaneous Ca2+ release was confirmed in myocytes from AF patients. Next, inhibition of PKA by H-89 promoted a stronger effect on the ITI frequency in these myocytes compared to myocytes from Ctl patients (7.6-fold vs. 2.5-fold reduction), while the β-agonist isoproterenol (ISO) caused a greater increase in Ctl patients (5.5-fold vs. 2.1-fold). ICa,L density was larger in myocytes from Ctl patients at baseline (p < 0.05). However, the effect of ISO on ICa,L density was only slightly stronger in AF than in Ctl myocytes (3.6-fold vs. 2.7-fold). Interestingly, a significant reduction of ICa,L and Ca2+ sparks was observed upon Ca2+/Calmodulin-dependent protein kinase II inhibition by KN-93, but this inhibition had no effect on ITI. Fluorescence resonance energy transfer (FRET) experiments showed that although AF promoted cytosolic desensitization to β-adrenergic stimulation, ISO increased cAMP to similar levels in both groups of patients in the L-type Ca2+ channel and ryanodine receptor compartments. Basal cAMP signaling also showed compartment-specific regulation by phosphodiesterases in atrial myocytes from 44 Ctl and 43 AF patients. Our results suggest that AF is associated with opposite changes in compartmentalized PKA/cAMP-dependent regulation of ICa,L (down-regulation) and ITI (up-regulation).Both, the decreased L-type Ca The increased Ca2+ waves then resulted in a rise in concurrent transient inward current (ITI), due to Ca2+ extrusion by the Na+-Ca2+ exchanger (NCX) [Atrial fibrillation (AF) has been commonly associated with profound remodeling of calcium  , and the2+-handling remodeling is a change in the phosphorylation state of the related proteins [Ca,L density has been shown to be in part linked to a phosphatase-dependent reduction in the phosphorylation state of the L-type Ca2+ channel [2+/Calmodulin-dependent protein kinase II (CaMKII) [2+, calmodulin and phosphatases. Furthermore, intracellular increase in cAMP levels can also activate a guanine nucleotide exchange factor, the exchange protein activated by cAMP (Epac) [A common mechanism proposed to explain Caproteins ,8,9,10. proteins ,5,6, hasproteins . Additio channel . The secP (Epac) , and phoP (Epac) .2+ regulatory proteins is emerging as an important mechanism modulating abnormal Ca2+ handling in AF. However, these findings suggest opposite modulatory phosphorylation states between the RyR2 [2+ channel [Therefore, remodeling of the phosphorylation state of Cathe RyR2 ,5 and th channel , making 2+ current and SR Ca2+ channel activity in AF is due to changes in cAMP-dependent PKA signaling, the key pathway for β-adrenergic receptor-mediated regulation of the phosphorylation state of these channels. Hence, the effect of the pharmacological manipulation of cAMP-dependent signaling on ICa,L density, frequency of SR Ca2+ release events large enough to produce ITI and cAMP levels was investigated in atrial myocytes from patients in sinus rhythm (Ctl) and with AF. Specifically, PKA and CaMKII were inhibited to eliminate PKA and CaMKII-dependent Ca2+ channel phosphorylation, and β-adrenergic signaling was stimulated to maximize Ca2+ channel phosphorylation.This study, therefore, tested the hypothesis that the remodeling of L-type CaCa,L amplitude, but strongly reduced spontaneous Ca2+ release large enough to produce spontaneous transient inward currents. These results suggest that, opposite to sinus rhythm, PKA-dependent phosphorylation is minimal for the L-type Ca2+ channel but eminent for the RyR2 at baseline in AF patients. However, β-adrenergic stimulation with isoprenaline increased ICa,L amplitude, ITI and cAMP levels in all compartments in both groups of patients. Therefore, changes in Ca2+ handling seen in patients with AF are likely due to modulations in cAMP-dependent regulation.Our data show that in AF, PKA-inhibition with H-89 had little effect on the IA total of 244 atrial human tissue samples were collected from patients undergoing cardiac surgery at the University Heart and Vascular Center Hamburg-Eppendorf (UKE). Atrial tissues were taken from patients in sinus rhythm (Ctl) or with atrial fibrillation (AF), subjected to atrial cannulation for extracorporeal circulation. All samples were taken with informed consent of the donors. The study was conducted in accordance with the Declaration of Helsinki principles and approved by the Ethical Committees of the Ärztekammer Hamburg .2+ antagonists were excluded from the study. Overall patient characteristics, including diagnostics and treatment were summarized in Patients treated with Ca2+ free solution containing 0.5 mg/mL collagenase , 0.5 mg/mL proteinase  and 2% bovine serum albumin . After 30 min, the tissue was removed from the enzyme solution, and cells were disaggregated with a Pasteur pipette in Ca2+-free solution containing 5% BSA and 2 µM blebbistatin . The remaining tissue was digested for 3 × 15 min in a fresh Ca2+ free solution containing 0.4 mg/mL collagenase and 2% BSA. Freshly isolated cells were suspended in Ca2+-free solution containing 5% BSA for patch-clamp experiments (duration ≈ 8 h) or in minimal essential medium  containing 1.2 mmol/L Ca2+, 2.5% fetal bovine serum , 1% penicillin-streptomycin, 2% HEPES and 2 µM blebbistatin (pH 7.6) and plated on 35 mm, laminin-coated culture dishes  for fluorescence resonance energy transfer (FRET) experiments. After 2 h of culture, the medium was replaced by 2 mL of FBS-free MEM with 2 µM blebbistatin. All experiments were performed at room temperature. Only elongated cells with clear cross striations and without granulation were used for experiments.A total of 428 human atrial myocytes from 148 patients in sinus rhythm and 96 patients with AF were isolated as previously described  and usedTI, caffeine-induced NCX currents and ICa,L were measured using a perforated patch-clamp technique in freshly isolated myocytes as previously described [Ca,L elicited by a 200 ms depolarization from −80 to 0 mV and the tail currents activated upon repolarization to −80 mV at a pacing frequency of 2Hz. The ICa,L amplitude was determined as the difference between the peak inward current and the current level at the end of a 200 ms depolarization pulse. Na+ currents were eliminated by a 50 ms pre-pulse to −50 mV. ITI currents were measured at −80 mV and the spontaneous ITI frequency was used to measure Ca2+ waves induced by spontaneous SR Ca2+ release [2+ load was measured using the NCX current time integral elicited by a rapid application of 10 mmol/L caffeine [2+:3Na+ for the NCX. Series resistance compensation was not performed. The extracellular medium (pH = 7.4) contained: NaCl 127 mM, TEA 5 mM, HEPES 10 mM, NaHCO3 4 mM, NaH2PO4 0.33 mM, glucose 10 mM, pyruvic acid 5 mM, CaCl2 2 mM, and MgCl2 1.8 mM. The intracellular medium (pH = 7.2 with CsOH) contained: aspartic acid 109 mM, CsCl 47 mM, Mg2ATP 3 mM, MgCl2 1 mM, Na2phosphocreatine 5 mM, Li2GTP 0.42 mM, HEPES 10 mM and 250 μg/mL amphotericin B.Spontaneous Iescribed . A HEKA  release . SR Ca2+caffeine , assumin2+ dye (2.5 μM fluo-4 AM) for 20 min under control Tyrode perfusion (in mmol/L): 140 NaCl, 4 KCl, 1.1 MgCl2, 10 HEPES, 10 glucose, 1.8 CaCl2; pH = 7.4, with NaOH). Ca2+ imaging was carried out with and a 63x glycerol-immersion objective and a resonance scanning Leica TCS SP8 X microscope  in the frame scanning mode at a frame rate of 90 Hz, with images being acquired every 30 s. Fluo-4  was excited at 488 nm and fluorescence emission measured between 500 and 650 nm. Image analysis was performed using IDL software . Images were corrected for the background fluorescence. The fluorescence values (F) were normalized by the basal fluorescence (F0) in order to obtain the fluorescence ratio (F/F0). Analysis to identify Ca2+ sparks was carried out using an automated detection system and a criterion that limited the detection of false events while detecting most Ca2+ sparks [Freshly isolated intact myocytes were loaded with a fluorescent Ca+ sparks .+-Ringer solution containing: 144 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 10 mM HEPES, adjusted to 7.4 pH with NaOH at room temperature (RT) was used to maintain cells, and images were taken every 5 s. The FRET system used consisted of a standard inverted microscope  with a 63×/1.4 oil immersion objective, an LED light source , a beam splitter , and a camera . A single-wavelength light-emitting diode  was used to excite the donor fluorophore (CFP), which was controlled by an Arduino digital-to-analog input-output board (Arduino). A dual view beam splitter  was used to split the emission light into donor (CFP) and acceptor (YFP) channels.FRET measurements were performed on human atrial myocytes 48 h after transduction with the adenovirus encoding Epac1-camps , pm-EpacThe fluorescence emitted was then measured at 475/30 nm and 527/40 nm, for CFP and YFP respectively and expressed the percent of 475/527 nm. To acquire time-lapse images the software Micro-Manager 1.4.5  was usedp < 0.05. ANOVA was used for multiple effects comparison, whereas the Student–Newman–Keuls post-test was used to evaluate specific effects. Experimenters were blinded to clinical information.Values were expressed as means ± SEM, and a two-tailed student’s t-test used to assess significant differences between data pairs with a significance value of Ca,L density and a higher ITI frequency at baseline. Furthermore, SR Ca2+ loading was similar for both patient groups. Next, the effects of the PKA-inhibitor H-89 (10 μM) at baseline were tested to determine if the changes in ICa,L density or ITI frequency could be associated with changes in cAMP-dependent PKA signaling. In AF-myocytes ICa,L density was comparable before and after exposure to H-89, yet notably in Ctl myocytes ICa,L density was significantly reduced to levels seen in AF myocytes , than Ctl ones (2.5-fold), indicating that the activation of RyR2 is more dependent on PKA at baseline in myocytes from AF patients. Jointly, these results suggest cAMP compartmentation which promoted a PKA-dependent ICa,L activation at baseline in Ctl patients, which is not seen in patients with AF, and a PKA-dependent RyR2 activation at baseline in AF patients, which could contribute, together with other factors, to the enhanced RyR2 activity in AF.In agreement with previous studies ,2,6, myomyocytes a. Inhibimyocytes b. The IT by H-89 c, althou density d. Indeed2+-handling were also modulated by CaMKII-dependent regulation, the effects of the CaMKII inhibitor KN-93 (1 μM) and its inactive analog KN-92 (1 μM) were investigated. ICa,L density was significantly reduced in response to KN-93 in myocytes from AF and Ctl patients (p = 0.05 and p < 0.005 respectively) (TI frequency in the same myocytes (2+ spark density and frequency in AF (p < 0.0001) (2+ load (To assess whether the observed changes in intracellular Cactively) a withoutmyocytes b. Howeve 0.0001) c, as wel. However, superimposed current traces before and after ISO exposure shown in Ca,L amplitude in patients with AF remained smaller in absolute terms after β-adrenergic stimulation with ISO compared to Ctl (right panel). Additionally, ISO caused a significant negative shift in the I-V relationship in both groups (p < 0.001 in both Ctl and AF)  (100 nM). As shown in  and AF) b.TI frequency  than from Ctl patients (5.5-fold).ISO eliminated the observed differences in Irequency c and ITImplitude  among Ct2+ loading or Na+-Ca2+ exchanger (NCX) activity, a protocol for caffeine-induced Ca2+ release from the SR was used to elicit the Ca2+ load in basal and upon ISO stimulation. Thus, the SR Ca2+ content was cleared with a brief caffeine pulse before reloading the SR during 30 stimulation pulses. Representative traces of caffeine-induced NCX-currents recorded before and after ISO exposure can be seen in 2+ into the SR before ISO treatment , sarcolemmal cAMP (pm-Epac1) and cAMP in the vicinity of the RyR2 (Epac1-JNC). Application of ISO demonstrated that AF promotes desensitization on the β-adrenergic pathway. p < 0.0001). Interestingly, this deregulation on cAMP levels is differentially regulated in the different cellular compartments. Contrary to the reaction in the cytosol, the increase of cAMP levels upon ISO stimulation was similar between Ctl and AF myocytes in the sarcolemma and the RyR2 compartments  by inhibiting all PDEs expressed in the atria  using IB2+-handling proteins, such as RyR2, in AF.These results indicate that β-adrenergic signaling is compartmentalized by PDEs in human atrial myocytes and suggest that cAMP-dependent PKA signaling might have a prominent role regulating the phosphorylation of specific CaTI frequency in myocytes from these patients. To further evaluate this, the effect of patients treated with β-blockers (BBs) on ICa,L density, ITI frequency and SR Ca2+ load was explored. Comparing atrial myocytes from Ctl or AF patients who were treated with BBs and from patients who did not receive this treatment (no BBs)  a–c, no s density a, SR Ca2 the upregulation of cAMP/PKA-dependent stimulation of SR Ca2+ release events large enough to produce ITI, and 3) PDE-dependent specific cAMP signaling in each relevant compartment for Ca2+-handling regulation. While inhibition of CaMKII reduced Ca2+ sparks frequency, it did not reduce ITI frequency in myocytes from patients with AF. Furthermore, Carvedilol, but not β1-blockers, strongly reduced the ITI frequency. Together our findings suggest that cAMP/PKA-dependent compartmentalized signaling has a potential to prevent large proarrhythmic SR Ca2+ release events and/or increase ICa,L density in AF and that Carvedilol, but not β1-blockers, may be a new therapeutic strategy to reduce Ca2+ release-induced atrial arrhythmias.Our study shows that AF is associated with (1) a cAMP/PKA-dependent reduction of the ICa,L density. Although different mechanisms have been proposed to explain this reduction, i.e., reduced channel expression [Ca,L density in AF myocytes while significantly reducing ICa,L density in Ctl myocytes , in incidence of spontaneous Ca2+ events and in the corresponding delayed afterdepolarizations when PKA was inhibited. Our findings demonstrate that a reduction of Ca2+ sparks or passive SR Ca2+ leak that had no effect on the resting membrane potential does not necessarily imply a reduction of large Ca2+ waves which induce a transient inward current and membrane depolarizations , heart failure (HF), and AF, have been linked to abnormal SR Cathe RyR2 ,5,27,28.the RyR2 . Here we2+ waves .2+ release in AF, and that Carvedilol treatment reduces the incidence of spontaneous Ca2+ release events and ITI in myocytes from patients with AF.In conclusion, our results show that cAMP-dependent PKA signaling is highly compartmentalized and regulated by PDEs, that PKA inhibition effectively reduces spontaneous SR Ca2+ sparks frequency and diminishing Ca2+ sparks amplitude, while decreasing SR Ca2+ load [2+ waves and RyR2 open duration but increasing Ca2+ sparks frequency [2+-handling [2+-handling to AF remodeling progression. CaMKII would enhance RyR2 phosphorylation at the dyadic clefts during the first episodes of AF or at the beginning of the progression of the arrhythmia increasing SR Ca2+ leak as an attempt to reduce SR Ca2+ load. A similar mechanism was proposed by Zhou et al. [2+ sensitivity [2+ release from the SR, and triggering arrhythmias.Furthermore, increasing evidence indicates that β-adrenergic signaling can activate different pathways other than PKA. Thus, β-adrenergic stimulation can also activate CaMKII via cAMP in a PKA-independent  ,55,56 paa2+ load . In linerequency . Interesrequency , the Epahandling . In thisu et al. , and Díau et al. , as wellu et al. ,61,62,63sitivity ,65, duri2+ channels (LTCC) are equally distributed in and out of the tubular system [2+-induced Ca2+-release events, RyR2 coupling to LTCC, PDE microdomains, RyR2 Ca2+ sensitivity and CaMKII activity. Further investigation regarding how structural changes in AF remodeling affect PKA, CaMKII and PDE interactions in basal and during β-adrenergic stimulation would provide valuable insights into AF pathophysiology.Although in atrial myocytes, L-type Car system , AF-assor system ,68 may a2+ handling in atrial myocytes. However, the exclusion of patients who received concomitant treatment that theoretically could mask any BB effects did not alter our results.Due to the isolation process potentially washing out acute effects of BB treatment, this study could only confirm long-term effects of BBs on the remodeling of CaCa,L during perforated-patch recordings [2+ concentration (2 mM) rather than a high Ca2+ concentration (5 mM), commonly used to increase SR Ca2+ release events, and which would produce an overestimation of CaMKII-dependent signaling.Several reasons, such as differences in patient cohort or in experimental conditions, could account for the discrepancies between this and previous studies. Usage of the perforated patch-clamp technique in the present study, preserving cAMP-dependent signaling, compared to using the ruptured patch-clamp technique which has been reported to attenuate cAMP-dependent signaling and removes the effects observed on Icordings , is like


This article presents a brief overview of the main biochemical and cellular processes involved in regulation of cyclic GMP production in photoreceptors. The main focus is on how the fluctuations of free calcium concentrations in photoreceptors between light and dark regulate the activity of retinal membrane guanylyl cyclase (RetGC) via calcium sensor proteins. The emphasis of the review is on the structure of RetGC and guanylyl cyclase activating proteins (GCAPs) in relation to their functional role in photoreceptors and congenital diseases of photoreceptors. In addition to that, the structure and function of retinal degeneration-3 protein (RD3), which regulates RetGC in a calcium-independent manner, is discussed in detail in connections with its role in photoreceptor biology and inherited retinal blindness. Light activates cGMP hydrolysis by cGMP phosphodiesterase-6 (PDE6) through GDP/GTP exchange in the Gt protein, transducin, catalyzed by photoactivated rhodopsin or cone pigments. As GTP transducin activates PDE6, the rapid decline in cGMP concentration closes the CNG channels and stops the influx of Na+ and Ca2+ in the outer segment, thus causing hyperpolarization of rods and cones. The recovery of photoreceptors from excitation includes multiple steps, regulating all components of the phototransduction cascade. G-protein-coupled receptor kinases GRK1 and GRK7 phosphorylate photoactivated pigments to allow them to bind arrestins, which blocks activation of Gt; the intrinsic Gt GTPase activity accelerated by RGS9 protein and PDE6 rapidly deactivates Gt and thus returns its effector enzyme PDE6 to its inactive self-inhibited state, which halts cGMP decay. Quenching the rhodopsin-transducin-PDE6 cascade is a necessary, but not the only, step in photoreceptor recovery: timely re-opening of the CNG channels also requires acceleration of cGMP synthesis by RetGC via a negative Ca2+ feedback mechanism [Synthesis of cGMP is an essential part of the fundamental phototransduction process that defines the function of vertebrate rods and cones. Many excellent review articles describing the details of the phototransduction pathway have been published, to name a few, references , 106. Foechanism , 101.Fig2+ feedback with an emphasis on its role in regulation of cGMP synthesis as a part of normal photoreceptor physiology and pathological processes underlying congenital blindness. In addition to that, we will review a recently emerged role of a non-Ca2+ sensor protein, retinal degeneration 3 (RD3), as a potent regulator of RetGC function in photoreceptors and its role in protecting rods and cones from degeneration. The authors do not intend to provide a comprehensive detailed review of Ca2+ and guanylyl cyclase regulation in photoreceptors, but choose instead to present their opinion on only some key aspects of such regulation in the physiology and disease of photoreceptors.The main purpose of this short review is to discuss the molecular mechanisms of the Ca2+ feedback originates from fluctuations of intracellular Ca2+ in the outer segment between light and dark. Although the levels of free Ca2+ concentrations reported in the literature slightly vary between different measurements and different animal species, they all indicate a steep, up to tenfold, decline of free Ca2+ in response to light. Ca2+ continuously extruded from the outer segment via Na+/Ca2+, K+ exchangers, NCKX1 in rods, and NCKX2/NCKX4 in cones [2+ concentrations in dark-adapted photoreceptors in the range of ~250–560 nM [2+ into the outer segment stops and its free concentrations rapidly decline to the range of ~20–50 nM [2+-binding proteins become affected, thus contributing to the shaping of photoresponses in rods and cones through negative Ca2+ feedback [2+ sensor proteins plays the major role in Ca2+ feedback [Cain cones , 117, re0–560 nM , 121. Wh20–50 nM , 121. Cofeedback , 101. Re2+ feedback belong to a distinct group of recoverin-like proteins in the EF-hand protein superfamily [2+-sensor proteins have been identified: recoverin (or S-modulin in lower vertebrate species), visinin, and guanylyl cyclase activating proteins (GCAPs) [2+ feedback, but later found not to be involved in shaping photoresponses [2+ sensor proteins have a number of common features: they have similar size (~21–25 kDa), are fatty acylated at the N-terminus, and have four helix-loop-helix EF-hand motifs, from EF-hand 1  to EF-hand 4  [2+ [Proteins that mediate Caerfamily , 14. The (GCAPs) , 14, 57. (GCAPs) , 44, 81. (GCAPs) . A subpo (GCAPs) . Another (GCAPs) , has beeesponses , 72. Theerminus) , 14, 57 erminus) , 57, 62.2+-sensor proteins. The N-fatty acyl groups in recoverin and GCAPs expressed in photoreceptors include saturated and non-saturated C12 and C14 residues, among which the saturated C14:0 myristoyl chain represents a relatively minor component [2+ [The N-fatty acyl residue plays different structural roles in the function of photoreceptor Caomponent , 81. Desnent [2+ , 33, 128nent [2+ , has beenent [2+ , 33. Thenent [2+ , evidentnent [2+ , so both2+ and the target enzyme [In contrast to recoverin, the myristoyl fatty chain in GCAPs remains buried inside the N-terminal semi-globule of the protein Figs. , 3, regat enzyme  as will 2+ sensor proteins is that not all four EF-hand motifs in their primary structure are the actual metal binding domains [2+ [2+-binding EF-hands (EF-hand 2 and-3), whereas three EF-hands  in GCAPs bind Ca2+ or Mg2+ [Another common feature shared by the photoreceptor Ca domains . The N-tains [2+ , 14, 62  or Mg2+  and play2+ recoverin [2+ buffer in photoreceptors, although its contribution to the overall Ca2+-buffering capacity of photoreceptors has been estimated to be relatively modest [A possible function of recoverin is to help rhodopsin, by extending the life of its active state, to more effectively propagate activation of the phototransduction cascade in dim light, before the receptor is quenched by phosphorylation and arrestin binding , 47, 48.ecoverin , 129 altecoverin  ecoverin . Recovery modest .2+-sensitive manner by activating RetGC in the light, when Ca2+ concentrations fall, and decelerating the cyclase in the dark, when Ca2+ influx through the open CNG channels elevates free Ca2+ levels in the outer segment [2+ feedback, contributing to the feedback much stronger than other regulatory components of the photoresponse affected by Ca2+ [2+ [2+ in GCAPs are also very close to the physiological levels of free Ca2+ in the outer segment [Kd values of the EF-hands for Ca2+ vary rather widely depending on the methods used for their evaluation. Below, we examine in more detail how the metal binding properties of individual EF-hands in GCAP1 relate to the sensor function of GCAPs as the RetGC regulators. We will also address possible reasons for the variability between different estimated EF-hand affinities for Ca2+ in GCAP1.Although the details of the functional role of GCAPs in regulation of the photoresponse require further study, it has been generally well established. GCAPs control the activity of RetGC in a Ca segment , 106  and the highest in EF4 (the lower-affinity site). Another important observation from studying the Ca2+ sensitivity of individual EF-hands was that although binding of Ca2+ in EF2 does not require Ca2+ to occupy EF-hands 3 or 4, binding of the metal in EF4 strictly requires EF3 being liganded by Ca2+ [2+-sensor parts of GCAP1: one operates through EF2 and the other through a pair of EF-hands 3 and 4 acting in concert [2+-bound state established by Stephen et al. [KdCa values for Ca2+-binding in EF-hands to the structural organization of GCAP1  , 60, 96 AP1 Fig. . In addi2+-sensitive regulation of RetGC by GCAPs, the effect of Ca2+ is highly cooperative (the Hill coefficient ≥ 2) [2+ binding to a purified GCAP1 shows that the binding occurs within the same physiological range of Ca2+ as the regulation of RetGC, but surprisingly does not show cooperativity (the Hill coefficient of 1) [As an important characteristic of the Caent ≥ 2) , 82, 89.nt of 1) . It is t2+-sensitive regulation of retinal guanylyl cyclase is that it is also highly sensitive to Mg2+[2+ with submillimolar affinities [2+ binding proteins, undergo conformational changes between different functional states, these conformational changes are rather subtle when compared to calmodulin or even to recoverin [2+ almost fully substitutes Ca2+ in at least two and probably all three metal binding EF-hands [2+ is ~1000-fold lower than for Ca2+ [2+ in the outer segment, near 1 mM [2+ concentration in illuminated photoreceptors [2+ concentrations in the outer segment fall after illumination, Mg2+ can easily displace Ca2+ from EF-hands 3 and 4  [2+ or Ca2+, must occupy both EF-hands 2 and -3 [2+ by Mg2+ in the light enables GCAPs to stay in complex with RetGC even after the CNG channels become closed and the concentration of intracellular free Ca2+ in light-adapted photoreceptors falls. Mg2+ binding also plays a crucial role in adjusting the Ca2+ sensitivity of GCAPs to the physiological range of free Ca2+ in the outer segment. The exact concentrations of Mg2+ in mammalian photoreceptors have never been directly measured, but the Ca2+ sensitivity of RetGC controlled by GCAP1 and GCAP2 in mouse photoreceptors best fits into the physiological range of free Ca2+ at the free Mg2+ concentrations near 1 mM [2+ measured in rods of lower vertebrates [2+, it would effectively undercut the dynamic range of RetGC regulation: when Mg2+ concentrations are reduced, the inhibition of the RetGC/GCAP complex by Ca2+ becomes too sensitive, and the cyclase activity decelerates well before the free Ca2+ reaches its normal dark-adapted level. Conversely, increase of GCAP affinity for Mg2+ versus Ca2+ would require much higher concentrations of Ca2+ to decelerate the cyclase. As we will further discuss in this article, such a low sensitivity of the cyclase to the inhibition by Ca2+ abnormally elevates Ca2+ influx through CNG channels and provokes photoreceptor death [2+ versus Ca2+ and thus to optimize the dynamic range of the cyclase regulation within the physiological ranges of the two cations in photoreceptors [Another important aspect of GCAP-mediated Cae to Mg2+, 82, becfinities , 84. Altecoverin . These cecoverin , 62. In EF-hands , 84. Thefor Ca2+ , but theear 1 mM , vastly eceptors , 121. Thf GCAP1) . To pres2 and -3 . Hence, ear 1 mM , close ttebrates . If GCAPor death . Evidenteceptors .2+ or Ca2+ in EF2 and EF3 allows GCAPs to always bind its target enzyme [2+ in EF4 are most critical for switching GCAPs between its RetGC-activator and RetGC-inhibitor states. Inactivation of the cation binding in EF4 locks GCAPs in a perpetual activator state, accelerating RetGC activity even at such free Ca2+ concentrations that by far exceed the normal physiological levels in the dark [2+ binding in EF4 is not essential for the ability of GCAPs to activate RetGC, but helps to adjust its Ca2+ sensitivity to the physiological range of Ca2+ [2+ [Whereas holding Mgt enzyme , 87, binthe dark , 83, 87. of Ca2+ . Structu of Ca2+ . This ha of Ca2+   of Ca2+ , 64. Ext of Ca2+ , 78 and  of Ca2+  identifi of Ca2+  Fig. 5)2+ or Ca2Ca2+ [2+ , 57, 61.Ca2+ [2+ , 42, 92.2+ sensor for the change of its functional state  [50 ~50–60 nM) occurs at free Ca2+ concentrations lower than in GCAP1 [2+ begins to drop following the activation of the phototransduction cascade. RetGC activity stimulated by GCAP1 restricts the amplitude of the dim flash response, by not allowing rods to close an excessive number of channels after absorbing a small number of photons, which helps to extend the dynamic range of rod responses. The free Ca2+ levels have to fall deeper in mid-phase of the response, before GCAP2-stimulated RetGC activity can kick in and accelerate the recovery. The amplitudes of dim-flash rod responses in GCAP1-/- mice, where upregulated GCAP2 takes over the RetGC activation, grow larger than normal, albeit not as large as in GCAP1/GCAP2 double-knockouts, and reach the peak later than normal, yet their recovery from the excitation occurs with a near-normal kinetics [-/- mice, the amplitude and the time to peak of rod responses remain similar to normal, but they recover more slowly [Rods express both GCAP1 and GCAP2, whereas cones almost exclusively express GCAP1 , 69, 123 to Ca2+ , 89. Und~130 nM) , 69, 89.in GCAP1 , 69, 89.in GCAP1 , 69 indie slowly .Fig. 6Se2+ falls to the levels sufficient for activation by GCAP2. Contrary to the “relay” fashion, the two GCAPs do not replace each other; rather, the fraction of the cyclase activity stimulated by GCAP2 in mid-phase of the response adds to the activity of the cyclase pool stimulated by GCAP1 in the earlier phase of the response . This is why we hereafter prefer, for the sake of clarity, to keep the original designations for the two isozymes, RetGC1 and RetGC2 [Retinal membrane guanylyl cyclase (RetGC) regulated in photoreceptors by GCAPs exists as two isozymes, RetGC1 and RetGC2 , 66, 125d RetGC2 . The sug2+-sensitive cGMP production is undetectable in mice lacking both isozymes, and their retinas do not respond to light [The joined activity of RetGC1 and RetGC2 presents the only source of cGMP in phototransduction. Cato light , 89. Howto light , 89, 123to light , 25, 123to light . At the to light .In their functional state, RetGC1 and RetGC2 are homodimers , 124. Th2+ GTP substrate molecules at the same time, with each substrate molecule simultaneously held by two opposing subunits in the homodimer [The three-dimensional structure of RetGC has not been determined, but the modeled structure of its catalytic domain, built based on the close homology to that of the established structure of adenylyl cyclase , gives aomodimer , 114 , no recessive loss-of-function mutations in GCAP1 (or GCAP2) affecting human vision were identified to date, in contrast to the multiple recessive mutations in RetGC1, causing GUCY2D retinal blindness. This may indirectly indicate that the both GCAPs activate human RetGC1 in vivo; otherwise, a loss of GCAP1 function alone would have likely resulted in finding vision disorders similar to the recessive GUCY2D blindness.Considering the specificity of GCAPs in the regulation of RetGC isozymes, it is important to emphasize that the physiological data assessing the interactions between the endogenous RetGC1 isozyme and GCAP isoforms in vivo using gene-knockout and transgenic mouse models unambiguously demonstrate that GCAP2 can effectively stimulate RetGC1, whereas GCAP1  does not significantly stimulate RetGC2 in vivo . A contrin vitro , whereasin vitro . Obviousin vitro , but notin vitro . The sam2+ sensor protein comes from site-directed mutagenesis and functional studies in vitro. The location of the binding interface for the cyclase on GCAP1 has been determined by probing the entire surface of GCAP1 by point mutations  [GUCY2D LCA [GUCY2D [GUCY2D LCA. It remains unknown at this point why these mutations cause CSNB rather than LCA, but the cause of GUCY2D-linked CSNB and LCA evidently relates to the lack of the RetGC1 activity rather than abnormality in its Ca2+ sensitive regulation.Multiple mutations in RetGC1 cause various forms of congenital blindness (reviewed in a great detail in a recent publication by Sharon and colleagues ). Most ois (LCA) , 112. This (LCA) . Althougis (LCA) , to someCY2D LCA . One of CY2D LCA . This opCY2D LCA , 46. Mor [GUCY2D . In this [GUCY2D  in these [GUCY2D , much asGUCY2D mutations that cause deregulation of the cyclase by Ca2+ feedback do not disable photoreceptor function, but rather cause premature death of well-responsive photoreceptors. Multiple mutations in RetGC1 and GCAPs causing this type of blindness have been excellently summarized in some comprehensive reviews [2+  typically cause dominant cone- or cone-rod degenerations [2+-liganded conformation at normal Ca2+ concentrations in the dark. Different molecular mechanisms can underpin this effect in the case of GCAP1: (1) The disease-causing mutations  Some mutations in GCAP1 can also affect its affinity for Ca2+ indirectly by changing the intermolecular interactions outside the immediate helix-loop-helix EF-hand domains. For example, the Leu176Phe substitution, originally used to identify and probe the calcium-myristoyl tug in GCAP1 in vitro [2+ in EF4 via a push-pull action between the exiting helix of EF4 and the myristoyl residue inside the N-terminal semi-globule of GCAP1 [2+ GCAP1 affinity for RetGC1 [2+-liganded GCAP1 produced by the normal GUCA1A allele to compete with the Leu176Phe GCAP1. (4) Another example of the indirect effect of mutations in GCAP1 on its Ca2+ sensitivity was reported recently in a case of a dominant retinopathy caused by Gly86Arg substitution in a “hinge” region connecting the two lobes of GCAP1 [2+ [2+ and higher affinity for RetGC [Mutations in GCAP1  can dirr” state , 87. (2)nds Ca2+ . ConsequGlu11Val , 71, 108in vitro , was subin vitro . This suof GCAP1 , 119. Itr RetGC1 , making of GCAP1 . FluoresCAP1 [2+ , 83, 109or RetGC , most li2+. A large fraction of cGMP-gated channels open, and free Ca2+ concentrations rise, in rod outer segments of transgenic mice that express Tyr99Cys or Glu155Gly GCAP1 under control of a rhodopsin promoter [2+ sensitivity of cGMP production in photoreceptors preferentially through the abnormal activation of the RetGC1 isozyme [2+-insensitive GCAP1 mutants leads to photoreceptor death by affecting the photoreceptors in their dark-adapted state, when the suppressed activity of PDE6 cannot effectively compensate for the accelerated activity of the cyclase. Artificial activation of PDE6 in the dark by expressing constitutively activated Gly90Asp rhodopsin [2+ in the dark.Much as in the case of the Arg838 substitutions in RetGC1, the net result of mutations in GCAP1 linked to the dominant retinopathies is the rise in intracellular free Capromoter , 122. Th isozyme . Consequ isozyme . The abnhodopsin , 107 draIt also worth noting that unlike mutations in RetGC1 that cause various recessive and dominant forms of blindness, all mutations identified in GCAP1 are, to our knowledge, dominant gain-of-function mutations , 106. A 2+ sensor, has emerged from more recent studies [rd3) in mice, hence the name of the protein. The loss of photoreceptors in rd3 mice is distinct from various types of retinal degenerations in other mouse lines [rd3 photoreceptors display near-normal morphology and abundance in the retina, but soon after completing their differentiation, they begin to degenerate. The exact rate of the degeneration varies depending on the particular background strain, but typically rd3 rods and cones disappear within several months [rd3 rods and cones are strongly suppressed [Adding more complexity to the control of RetGC activity in photoreceptors, the role of RD3, another regulator protein but not a Ca studies . RD3 wasse lines , 19. At l months . ERG phoppressed , even prppressed . The cauppressed . In a sippressed . A long ppressed .rd3 mice declines at least tenfold [rd3 mice, resulting in a dramatic reduction of rod and cone ERG responses [rd3 photoreceptors from degeneration and dysfunction [RD3 has a dual function in photoreceptor physiology. Firstly, photoreceptors require RD3 in order to properly accumulate RetGC in the outer segment. There are indications that RD3 can be directly involved in the process of RetGC relocation from the photoreceptor inner segment to the outer segment , 131. Th tenfold , 95, 99.function .rd3 photoreceptors per se is not the actual reason why they undergo rapid degeneration. Despite being significantly reduced, RetGC activity in rd3 retinas remains detectable and retains regulation by Ca2+ and GCAPs [rd3 mice still respond to light, albeit with much lower amplitudes of the ERG a-wave [rd3 photoreceptors still produce enough cGMP to support at least their rudimentary physiological responses. In a sharp contrast to that, in mice devoid of all RetGC activity by deletion of both RetGC1 and RetGC2 genes, rods and cones are completely unable to produce cGMP for phototransduction [rd3 mice degenerate much faster than in the RetGC-deficient mice [rd3 and RetGC knockout mice in those studies were congenic with the C57B6 strain [However, the lower guanylyl cyclase activity in nd GCAPs . FurtherG a-wave , 95. Evisduction , 89 and sduction , 95. Yet2+ GCAPs [2+ sensitivity of the RetGC:GCAP complex, but rather inactivates the complex entirely [rd3 degeneration. After deletion of GCAP1 and GCAP2, the cyclase activity in rd3 retinas declines even further, because now the cyclase is no longer activated by GCAPs at low Ca2+. Consequently, their ERG photoresponses are further suppressed as well [rd3 photoreceptors remain preserved at the ages when photoreceptors in rd3 mice expressing GCAPs have already died out [2+ feedback regulating phototransduction in the outer segment (Fig. In addition to the support of the normal content of RetGC in photoreceptor outer segment, RD3 also has no less important second role in the physiology of photoreceptors, which is protecting them from degeneration. This is directly related to another main property of RD3, which is inhibition of RetGC activity . RD3 str2+ GCAPs , 95. RD32+ GCAPs , 95. Impentirely . Recent  as well . Yet, qudied out , 99. Thedied out , 99—furtent Fig. .Fig. 8A Three-dimensional NMR structure of RD3  shows a 2+ GCAP and reduces its sensitivity to deceleration by Ca2+, but also makes the Mg2+ GCAP1: RetGC1 complex more resistant to inhibition by RD3 [2+ sensitivity of RetGC deceleration to a higher Ca2+ range, but also makes RetGC better resist the inhibition by RD3, again, most likely because of the increased affinity of the Gly86Arg GCAP1 for RetGC1 [Recent studies also indicate that a CORD6-related substitution in the cyclase dimerization domain, Arg838Ser, not only increases RetGC1 affinity for Mgn by RD3 . The mosr RetGC1 . If the


The purpose of this study was to compare femtosecond laser-assisted in situ keratomileusis with prophylactic cross linking (FS-LASIK Xtra) and small-incision lenticule extraction with prophylactic cross linking (SMILE Xtra) in terms of their postoperative clinical outcomes.  In this retrospective study, 24 patients (48 eyes) with myopia and myopia astigmatism were recruited from 2017 to 2018. All patients underwent comprehensive ophthalmic examinations preoperatively and follow-up visits at one and three months. Four patients (eight eyes) in each group were followed up for 12 months. The following were assessed at each visit: uncorrected distance visual acuity (UDVA), manifest refractive spherical equivalent (MRSE), keratometry values, biomechanical properties, anterior and posterior curvature, and corneal pachymetry. P=0.006, 0.01), and the SMILE Xtra group had significantly higher K1 and K2 values than the FS-LASIK group at one month after surgery . Corneal thickness decreased significantly at one month postoperatively and started to increase over the next 12 months in both groups (P=0.001). The biomechanical properties showed no significant intergroup differences at the 12-month follow-up.  The FS-LASIK Xtra and SMILE Xtra groups both included 24 eyes of 12 patients. At 1, 3, and 12 months after surgery, a UDVA of 20/20 or better was achieved for 91.7%, 91.7%, and 87.5% of individuals, respectively, in the FS-LASIK group and 95.8%, 100%, and 100% in the SMILE group, respectively. For 75% of eyes in the FS-LASIK Xtra group and 100% of eyes in the SMILE Xtra group, the achieved MRSE was within ±1.00D of attempted correction. The LASIK Xtra group had a significantly higher mean MRSE than the SMILE Xtra group at 3 and 12 months after surgery ( FS-LASIK Xtra and SMILE Xtra are safe and effective in the correction of myopia and myopia astigmatism, and both procedures have the same effect on postoperative corneal morphology and biomechanics. Femtosecond laser-assisted in situ keratomileusis (FS-LASIK) is a common refractive surgical procedure. It has excellent refractive correction ability and offers predictable and stable refractive results , 2. HoweCorneal cross linking (CXL) was first introduced by Wollensak et al. as a promising technique to slow or stop the progression of keratoconus . It alsoHowever, only one study has compared FS-LASIK Xtra with SMILE Xtra and found that the two procedures are comparable in their postoperative outcomes. However, it did not include corneal biomechanical properties . The preμm, predicted postoperative residual stroma bed thickness less than 280 μm, significant ocular diseases, systematic diseases, or medications that could affect wound healing, pregnancy, or breastfeeding.This was a retrospective interventional comparative study that included 48 eyes of 24 patients. Data were collected between May 2017 and November 2018. The study was approved by the Institutional Ethics Committee and was conducted in accordance with the tenets of the Declaration of Helsinki. The inclusion criteria included age ≥18 years, preoperative spherical error from −1.00D to −11.00D, refractive cylinder < −5.00D, stable refractive error for more than 1 year, and willingness and ability to comply with postoperative care. Exclusion criteria included a central corneal thickness (CCT) less than 480 All patients underwent comprehensive ophthalmic examinations preoperatively and follow-up visits at one and three months. Four patients (eight eyes) in each group were followed up for 12 months. The following were assessed at each visit: uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), intraocular pressure (IOP), manifest refractive spherical equivalent (MRSE), anterior segment slit-lamp examination, fundus examination, and corneal tomography . An ocular response analyzer  was used to measure the corneal compensated intraocular pressure (IOPcc), Goldmann correlated IOP value (IOPg), corneal resistance factor (CRF), and corneal hysteresis (CH). A Galilei Dual-Scheimpflug analyzer  assessed the SimK values, anterior instantaneous curvature, posterior axial curvature, and corneal pachymetry.μm; cap diameter, 7.0–7.5 mm; lenticule diameter, 6.0–6.5 mm with a transition zone of 0.1 mm; side cut incision, 2 mm at the 10 o'clock position of the cornea; and cut energy, 135 nJ. After femtolaser application, a blunt spatula was used to loosen the stromal lenticule, and then, it was removed using forceps. Immediately after removal of the lenticule, 0.22% riboflavin  was instilled into the intrastromal pocket and allowed to have a soak time of 90 seconds. The riboflavin was then completely washed out from the pocket using balanced saline solution. This was followed by ultraviolet A irradiation using Avedro's (Avedro Inc.) corneal cross-linking system at 30 mW/cm2 for 90 seconds, and the total energy was 2.7 J/cm2.All surgeries were performed by the same surgeon (YL) using the same reproducible technique. The SMILE procedure was performed under topical anesthesia using the VisuMax 500 kHz femtosecond laser . The following parameters were used: cap thickness, 110–120 μm or 110 μm; flap diameter, 8.5 mm; hinge position, 12 o'clock of the cornea; and side cut angle, 120 degrees. After excimer laser treatment, 0.22% riboflavin  was instilled onto the exposed stromal bed for 90 seconds. The balanced salt solution was then used to flush the remaining riboflavin from the stromal bed. The flap was repositioned followed by ultraviolet A irradiation using Avedro's (Avedro Inc.) corneal cross-linking system at 30 mW/cm2 for 90 seconds, and the total energy was 2.7 J/cm2.FS-LASIK was performed using the VisuMax 500 kHz femtosecond laser  for flap creation and a WaveLight EX500 excimer laser (Alcon Laboratories Inc.) for refractive correction. The following parameters were used: flap thickness, 90 After ultraviolet A irradiation, the regimens were prescribed for both eyes as follows: 0.5% levofloxacin eye drops , four times a day for two weeks, 0.5% Loteprednol etabonate ophthalmic suspension  in tapering dosages for four weeks (starting with four times per day), and sodium hyaluronate eye drops, four times a day for four weeks.t-tests were used to compare data between the two groups. If the data were not normally distributed, the Wilcoxon rank-sum test was performed. One-way analysis of variance for repeated measures and paired t-tests were used to analyze the data from preoperative to postoperative examinations and between consecutive postoperative visits. The differences in data are reported with 95% confidence intervals (CIs). A two-tailed P value ≤0.05 was considered statistically significant for all analyses.Statistical analyses were conducted using SPSS statistical software version 23.0 . UDVA and CDVA were converted to the logarithm of the minimum angle of resolution (logMAR) equivalents. The Kolmogorov–Smirnov test was used to check for the normal distribution of quantitative data, provided here as the mean ± standard deviation (SD). Independent two-sample P=0.935). There was no statistically significant difference in preoperative parameters between the groups  women and five (42%) men. The mean age was 25.25 ± 4.61 years and 25.08 ± 5.26 in the FS-LASIK Xtra group and SMILE group, respectively (e groups . In the P < 0.001), but not between groups    values were −0.099 ± 0.53, −0.609 ± 0.65, and −0.91 ± 0.17 at 1, 3, and 12 months after surgery, respectively, in the FS-LASIK group and −0.12 ± 0.47, −0.16 ± 0.37, and −0.05 ± 0.04, respectively, in the SMILE Xtra group . The cha6, 0.01) . In the K1 and K2 values increased at three months after surgery, but the change from 3 months to 12 months was not statistically significant in either group . In addition, the K1 and K2 values at the one-month visit after SMILE Xtra were higher than those after FS-LASIK Xtra . However, there were no significant differences after the 3-month and 12-month visits between the two groups , but there were no significant differences at 12 months postoperatively  (Tables P=1.000) (The CCT decreased significantly at one month postoperatively and started to increase over the next 12 months in both groups  Tables . There w) Tables . CompareP=1.000) . In termStudies have shown that SMILE is a safe surgery with comparable outcome results to FS-LASIK –18. HoweFor the biomechanical properties, Konstantopoulos et al. performed an animal experimental study, and the result revealed that LASIK Xtra had significantly lower maximum posterior elevation (MPE) than LASIK and SMILE at week six. However, there was no significant MPE difference between the LASIK Xtra and SMILE Xtra groups at any visit points . This su2 [2 proved to be safe and well tolerated. Wu et al. [2 for 90 seconds  to perform FS-LASIK Xtra. The results showed that FS-LASIK Xtra could effectively correct refractive error in patients with myopia with no significant complications during the 6-month and 12-month follow-up, indicating stability and morphologic changes similar to those with LASIK treatment. Liu et al. [2 to perform SMILE Xtra; only three eyes showed mild haze, and it disappeared at the six-month visit. In our study, we also used the same protocol to perform prophylactic CXL. The patients not only had good clinical outcomes but also had no postoperative complications.Currently, there are no established prophylactic CXL regimens or patient selection criteria. Too much UVA energy could cause haze, but too little UVA energy would be insufficient to provide the required corneal strength. Some studies reported a different total UVA energy range from 0.8 to 5.4 J/cm2 –26, but u et al.  and Kohnu et al.  also useIn terms of visual acuity, Kanellopoulos et al. , 27 founRegarding refractive error, Kanellopoulos et al.  found thK1 and K2 decreased significantly after the FS-LASIK Xtra and SMILE Xtra surgeries in our study. At the one-month follow-up, the K1 and K2 values were higher in the SMILE Xtra group than those in the FS-LASIK Xtra group, which might be related to the different flap or cap thickness between the two groups. However, the keratometry values remained stable from three months postoperatively in both groups. For corneal pachymetry, Kohnen et al. [P=0.001). The steady keratometry values and increased corneal thickness indicated that both surgical methods can maintain the stability of corneal morphology in the 12-month postoperative period.n et al.  found thn et al.  proposedTo the best of our knowledge, this is the first study comparing the corneal biomechanics between the two procedures used for the correction of myopia and myopia astigmatism. Nevertheless, our study also has limitations. First, we did not examine the endothelial cell count because it has been proven that there was no significant effect in the endothelial cell count before and after FS-LASIK Xtra or SMILE Xtra. Second, the study needs a larger group and longer follow-up duration to observe regression and presence of ectasia because the ectasia appears after three years in most cases. The last limitation is the study's retrospective nature. Therefore, prospective and multicenter studies are needed to address this limitation.In conclusion, FS-LASIK Xtra and SMILE Xtra are safe and effective in the correction of myopia and myopia astigmatism, and both procedures have the same effect on postoperative corneal morphology and biomechanics.