Are functional assessments in rehabilitation settings valid measures?In this cross-sectional study of 1036 Medicare beneficiaries, the correlation of functional assessments from rehabilitation services with National Health and Aging Trends Study assessments was 0.63 when assessments were performed no more than 90 days apart and 0.66 when performed no more than 30 days apart. Differences in assessment scores were generally small; however, assessments from the National Health and Aging Trends Study tended to be lower than those from rehabilitation services in a small sample of older adults with less function.These findings suggest that Medicare rehabilitation functional assessments may be sufficiently associated with functional outcomes for use in some research applications. This cross-sectional study evaluates the validity of individual-level routine care functional assessments in Medicare rehabilitation settings compared with criterion-standard National Health and Aging Trends Study research assessments obtained no more than 90 days later. Assessment of functional outcomes is currently limited by a lack of large data sets. Functional assessments are included in Medicare rehabilitation assessment files, yet the validity of these measures in routine care is unknown.To evaluate the validity of individual-level routine care functional assessments in Medicare rehabilitation settings compared with criterion-standard National Health and Aging Trends Study (NHATS) research assessments obtained no more than 90 days later.This cross-sectional study of individuals aged 65 years and older used the 2011 to 2015 NHATS linked with Medicare assessment files. Individuals with a discharge assessment from inpatient rehabilitation facilities, skilled nursing facilities, or home health agencies and a criterion-standard NHATS assessment were included. Data analysis was performed June 2019 to November 2019.Summary functional assessment based on independence with eating, toilet hygiene, bathing, dressing, transfers, and mobility or walking. Linear regression was used to assess agreement between the 2 scales, adjusting for time between assessments and rehabilitation setting.R2 = 0.42). Differences in scores were generally small , and only 59 assessments (5.7%) differed by more than 2 SDs of the mean difference. Rehabilitation service scores were typically higher than NHATS scores in individuals with lower mean scores; however, the population with lower mean scores was small (156 [15.1%]).A total of 1036 adults aged 65 years and older  met the study criteria. The correlation of the assessments was 0.63 . The correlation increased to 0.66  for assessments no more than 30 days apart. The linear regression model adjusting for rehabilitation setting and days between evaluations found the assessments were strongly correlated (β = 1.00 ; intercept, 0.72 ; In this large sample of older US adults, routine care rehabilitation facility functional assessments had overall moderate correlation with criterion-standard research assessments. Function is an important patient-centered outcome, but it is difficult to assess on a national scale. Medicare beneficiaries undergo functional assessments while receiving rehabilitation services from inpatient rehabilitation facilities (IRFs), skilling nursing facilities (SNFs), and home health agencies (HHAs). The assessments are based on the individual’s ability to perform specific activities, as measured by rehabilitation staff. While the purpose of the assessments is to estimate resource needs and determine payment amounts, these data are also available for use in research studies. However, the validity of the assessments performed in the rehabilitation setting is not known.7 However, prior studies were generally performed in small samples of patients and with a limited number of evaluators who received specific training. To our knowledge, nationally representative validation of routine care assessments has not been performed. Because of the lack of large-scale validation, concerns have been raised about the appropriateness of using the data for research purposes.7 These concerns are based on the large number of staff who perform the assessments, the lack of required standardized training on evaluation and scoring, and a potential bias because of facility performance and associated payments.The assessment instruments used in rehabilitation settings are the Functional Independence Measure (FIM), Minimum Data Set, and the Outcome and Assessment Information Set , a large population-based survey of health and functional ability trends among Medicare beneficiaries aged 65 years and older.10 The NHATS is also linked to Medicare, creating the opportunity to compare functional assessments from rehabilitation settings with research-based functional assessments. The advantages of this comparison are that the data are drawn from a large, nationally representative sample in routine practice, masked to any knowledge of a subsequent comparison with a criterion standard that is complete and rigorous. Disadvantages of this comparison are the variable time interval between Medicare and NHATS assessments as well as differences in the instruments used by the various rehabilitation settings.The validity of Medicare rehabilitation assessments can be explored by comparing them with criterion-standard functional assessments with known reliability and validity,In this study, we aimed to compare the functional assessments performed in routine care Medicare rehabilitation settings with the NHATS assessments considered to be the criterion standard. To enable the comparison, we crosswalked overlapping functional instruments and limited the comparisons to NHATS assessments performed no more than 90 days after an assessment in a rehabilitation setting. If the correlation and agreement between the assessments are satisfactory, they would support the use of Medicare rehabilitation functional assessments in some research contexts.STROBE) reporting guideline.The design was a cross-sectional validation study using retrospective data comparing functional assessments in Medicare rehabilitation settings with similar criterion-standard assessments from the NHATS. The setting was Medicare rehabilitation facilities (IRFs and SNFs), and older adults’ homes for HHA and NHATS assessments. The study was approved by the University of Michigan institutional review board, and written informed consent was obtained from NHATS participants at the time of enrollment in NHATS. This study followed the Strengthening the Reporting of Observational Studies in Epidemiology  for the rehabilitation setting and NHATS assessment, adjusting for days between the assessments. Next, we added a variable of rehabilitation facility setting to assess differences explained by rehabilitation setting. We calculated the difference in assessment scores (NHATS score − rehabilitation service score) and used Bland-Altman difference against the mean plots to assess agreement between the 2 assessments and to visually inspect for variation in the differences in scores across the range of mean scores.14 Items with missing data were infrequent  and therefore excluded. All analyses were performed using Stata version 15.1 (StataCorp) and SAS statistical software version 9.4 (SAS Institute). Data analyses were performed from June 2019 to November 2019. Statistical significance was set at α < .05, and all tests were 2-tailed.We used descriptive statistics to summarize the population, days between assessments, and the function scores. We compared the correlation of the overall function score between Medicare functional assessments and NHATS assessments using the Pearson correlation coefficient for eligible assessments within 90 days and separately for assessments within 30 and 15 days. We used linear regression to examine the association of the rehabilitation facility score with the NHATS score, the intercept, and the squared value of the correlation coefficient  rehabilitation service functional score was 27.5 (7.2) compared with 30.5 (10.1) for the NHATS assessment. The median (interquartile range) time between assessments was 37 (18-61) days. The correlation coefficient of the 2 assessments was 0.63  . The corR2 was 0.40 . Days since rehabilitation setting assessment was associated with a small increase in the NHATS assessment score . The model intercept was 5.73 , and the overall was 0.40 . In the The mean (SD) difference of the rehabilitation service scores (NHATS score − rehabilitation score) was 2.96 (7.91). Plots of the differences in the scores are displayed in R2 = 0.47; 95% CI, 0.42-0.52; bathing: R2 = 0.46; 95% CI, 0.41-0.51; dressing: R2 = 0.52; 95% CI, 0.43-0.62). The correlations either remained stable or slightly increased as the days between assessments narrowed . The eating assessment consistently had the lowest correlation, ranging from 0.34 to 0.37 .Mean scores and correlations of the 6 individual disability components by assessment time difference are displayed in Our study of more than 1000 individuals found that functional assessments in rehabilitation settings are correlated with criterion-standard research assessments. The assessments also had good overall agreement. These findings provide important new evidence to support the use of routine care assessments as a functional outcome measure in some contexts. Given that these measures can be linked to Medicare claims, the range of potential applications of these measures is broad.17 The magnitude of correlation suggests that the 2 assessments measure a similar underlying construct of function, which ranges from an independent to a dependent status. We also found that the correlation of the assessments increased slightly as the time between assessments narrowed from 90 days to 30 days. This finding supports construct validity, given that we expected function to change somewhat after rehabilitation, with some people continuing to improve while others experience some worsening.The correlation of the 2 assessments performed within 90 days was in the range of what is considered moderate by some and substantial by others.12 Therefore, a fully independent person who does not use devices would score a 42 in NHATS but a 37 in SNF or HHA. Another potential reason for differences in scores is that the NHATS functional scores were obtained by self-report whereas function in rehabilitation services were rated by staff observing the patient. We were not able to determine with our data which approach was a more accurate overall measure of function.The analysis of the differences of the scores found a small bias for higher NHATS scores overall and a normal distribution of differences with few outliers. The slightly higher scores in NHATS could be in part because of a difference in the scaling of items in NHATS compared with SNF and HHA, considering that the NHATS scale for each functional item was up to 7 points for individuals who are independent and do not use devices, whereas the functional scales were mostly truncated at 6 points for SNF and HHA.While the overall NHATS scores were slightly higher than rehabilitation scores, we found that most individuals with lower mean function scores had NHATS assessments lower than rehabilitation assessments, particularly when compared with HHA assessments. The reason for this finding is not certain. First, only 156 individuals (64 from HHA) had lower mean function, so it is possible that the differences at the lower mean range were random selection error. Other possibilities are that individuals with lower mean scores may have had conditions that rendered them more susceptible to functional worsening after rehabilitation  or conditions associated with lower self-perception of dependence  compared with independent staff ratings. Future studies are needed to evaluate how reasons for rehabilitation and comorbidities influence agreement in the scales.Functional assessments, a highly patient-centered measure, are challenging to incorporate into longitudinal studies. Therefore, the Medicare data makes it feasible to better understand function and the factors associated with it among older adults. The assessments could be used in observational studies linked to Medicare or potentially as a clinical trial outcome. Based on our findings, it would be appropriate to use data from rehabilitation settings in large studies like ours with either a similar distribution of function or populations with moderate to high function. We recommend caution using this data in populations with low function until we have a better understanding of why rehabilitation scores are typically higher than NHATS scores in individuals with lower mean function scores.The primary strengths of our study were rehabilitation assessments from routine care, a large sample, and a rigorous criterion standard. Including data from all 3 rehabilitation settings was another strength. Correlations for each setting were in the moderate range. By including all settings, we were able to derive an equation that can be used to standardize functional scores across sites.Our study has important limitations in addition those already noted. We were only able to select FIM components that overlap in topics and scoring scales with the NHATS assessment. We had relatively few assessments from IRF after applying our exclusions. Because NHATS dates are limited to month and year, our difference in days could be off by up to 30 days. Our analysis was limited to discharge assessments from rehabilitation services. Therefore, we were not able to make conclusions about the validity of admission and interim assessments.It is important to note that, as of fiscal year 2020, the FIM assessments will no longer be performed in IRFs. The assessment was replaced by the quality indicators in the Quality Reporting Program (QRP). Based on the considerable overlap of FIM and the QRP items, the FIM was dropped to reduce administrative burden. Although a separate validation of the NHATS with QRP-graded function would be preferred, it seems likely that that QRP measures will also generally correlate with criterion-standard measures of function given the similarity of FIM and QRP items. The major difference in FIM and QRP items is in the scaling of the items. Compared with the 7-point scales on the FIM, the QRP has 6-point scales because it does not consider the use of a device. The QRP categories also have a slight difference in what is considered supervision and the percentage of effort a helper provides to perform the activity. The QRP items are specifically used as quality metrics and therefore bias in scoring is also a concern, as it was with the FIM.This study extends our knowledge of the validity of rehabilitation functional assessments in the routine care setting. Our results demonstrated that these data were correlated and agree with a rigorous criterion-standard functional assessment. These findings suggest that rehabilitation facility assessments provide sufficiently accurate estimates of function for application in many research contexts.


However, the exact localization of these receptors to fine membrane structures and subcellular compartmentation of β3-AR/cGMP signals underpinning this protection in health and disease remain elusive. Here, we used a Förster Resonance Energy Transfer (FRET)-based cGMP biosensor combined with scanning ion conductance microscopy (SICM) to show that functional β3-ARs are mostly confined to the T-tubules of healthy rat cardiomyocytes. Heart failure, induced via myocardial infarction, causes a decrease of the cGMP levels generated by these receptors and a change of subcellular cGMP compartmentation. Furthermore, attenuated cGMP signals led to impaired phosphodiesterase two dependent negative cGMP-to-cAMP cross-talk. In conclusion, topographic and functional reorganization of the β3-AR/cGMP signalosome happens in heart failure and should be considered when designing new therapies acting via this receptor.Cardiomyocyte β In human and rodent ventricular myocardium, catecholamine binding to β3-ARs elicits negative inotropic and positive lusitropic effects by signalling via Gi and the second messenger 3’,5’-cyclic guanosine monophosphate (cGMP) /cGMP-dependent signalling pathway. The same study showed localization of β3-ARs together with eNOS in caveolae-enriched membrane fractions, which had been separated via ultracentrifugation  . Unlike ization,  and its ization, . It was  failure . Endothehe heart . It has  2 and 5 . Recentlfugation . Another KO mice . Despitealosomes . However3-ARs are localized exclusively within the transverse (T)-tubules and stimulate a cGMP pool which is predominantly regulated by phosphodiesterases (PDEs) 2 and 5. Furthermore, by using the cAMP specific FRET-based biosensor Epac1-camps we show that β3-AR stimulation can decrease overall adenylate cyclase dependent cAMP levels in healthy cardiomyocytes by a PDE2-mediated cGMP-to-cAMP cross-talk. This cross-talk appears to be disrupted in heart failure, where β3-AR stimulation no longer has a significant effect on overall cAMP levels. In failing cells, β3-AR/cGMP signals decrease in the T-tubules. Heart failure leads to altered co-localization of sGC and caveolin-3, as shown via immunocytochemical staining. Together, these alterations result in the impairment of the β3-AR-dependent cGMP signalling pathway and of a PDE2-mediated β3-AR induced decrease of local cAMP.In this study, we employ a highly sensitive Förster Resonance Energy Transfer (FRET)-based biosensor, red cGES-DE5, in combination with scanning ion conductance microscopy (SICM). We demonstrate that in healthy rat cardiomyocytes, functional β3-AR-dependent cGMP dynamics, we used ventricular cardiomyocytes isolated from healthy and failing rat hearts transduced with an adenovirus to express a highly sensitive cytosolic FRET biosensor red cGES-DE5. As a model of heart failure, we used rats which underwent left coronary artery ligation for 16 weeks  in control cells did not abolish this cGMP production  of healthy control rat ventricular cardiomyocytes expressing the cGMP biosensor red cGES-DE5, led to the production of substantial amounts of cGMP  in aboutoduction . The sig3-AR/cGMP dynamics. Following the application of selective PDE blockers we applied the non-selective PDE inhibitor IBMX. We found that β3-AR/cGMP levels are under the control of multiple PDEs. PDE1 inhibition has a minimal effect on β3-AR/cGMP production, whereas PDE2 and PDE5 represent the most prominent β3-AR/cGMP degrading families   . In cont3-AR signalosome in control cells and failing cardiomyocytes, we performed immunocytochemical staining of sGC subunits together with caveolin-3 or α-actinin. We detected partial co-localization of sGCα1 with caveolin-3 , taurine , creatine monohydrate , penicillin/streptomycin , carnitine hydrochloride  BSA , laminin (Sigma Aldrich L2020), isoproterenol hydrochloride , ICI118551 , CGP 20712A , SR 59230A hydrochloride , L-NAME hydrochloride , Vinpocetine , EHNA hydrochloride , Cilostamide , Tadalafil , IBMX (Santa Cruz sc-201188), self-made rabbit sGCα and β subunit antibodies  according to the standards for the care and use of animal subjects determined by the UK Home Office (ASPA1986 Amendments Regulations 2012) incorporating the EU directive 2010/63/EU. The Animal Welfare and Ethical Review Body Committee of Imperial College London approved all protocols.The parts of the investigation  which were performed in Germany, conformed to the guide for the care and use of laboratory animals published by the National Institutes of Health . The experimental procedures were in accordance with the German Law for the Protection of Animals and with the guidelines of the European Community.The following procedure was exclusively performed at Imperial College London in the UK: Left descending coronary artery ligation was performed as described . Rats weYGRKKRRQRRRGGGGDGVWRVSYTTFTVSKYWCYR) or with a scrambled control peptide without any cellular targets in cardiomyocytes (sequence: YGRKKRRQRRRGGGGYWTVYTKVDFCGSRYVRTSW) as described previously   for PDE2, 10 µmol/L cilostamide for PDE3 or 100 nmol/L Tadalafil for PDE5, and 100 µmol/L of the unspecific PDE inhibitor 3-Isobutyl-1-methylxanthine (IBMX). To study β3-AR/cGMP localization on cardiomyocyte surface structures, cells were continuously superfused with FRET buffer containing 100 nmol/L of the β1-AR blocker CGP 20712A and 50 nmol/L of the β2-AR blocker ICI 118,551. SICM was used to scan and visualize cardiomyocyte T-tubule openings and crest surface structures. Next, the scanning nanopipette was lowered onto either T-tubule openings or crest structures and receptor ligand was locally applied from the nanopipette filled with 50 µmol/L Isoproterenol (ISO) and 50 µmol/L CGP 20712A and 25 µmol/L ICI 118,551 by switching SICM pipette holding potential from −200 to +500 mV as described .Thank you for submitting your article "βThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:3-adrenergic receptor and associated signaling molecules in signaling domains of adult rat cardiomyocytes and its redistribution in a model of myocardial ischemia. They use FRET-based cGMP and cAMP sensors and scanning ion conductance (SICM) and confocal microscopy. The authors show Isoproterenol-evoked cGMP generation in control cardiomyocytes, its contribution by β3-adrenoceptors using β1 and β2 antagonists and its dependence on NO generation. The authors can demonstrate that ISO-evoked GMP production is reduced in failing cardiomyocytes. Using a large set of phosphodiesterase inhibitors, they elaborate the contribution of PDE2 and PDE5 isoforms for ISO-evoked cGMP generation. Combined FRET and SICM surface imaging reveals that β3-adrenoceptor-mediated cGMP generation occurs predominantly in the T tubular cleft in healthy myocytes, and that in failing myocytes the cGMP response is similar in T tubules and at the crest. Modulation of caveolin binding similarly equalizes T-tubule and crest signalling in healthy myocytes. Using confocal microscopy, the authors then show data suggesting that the β3-adrenergic receptor-associated signaling molecules sGCα1 and sGβ1 dissociate from caveolin 3-containing domains in failing cardiomyocytes. In a final set of experiments the authors demonstrate that β3-adrenoceptor activation attenuates cAMP generation  in myocytes from healthy animals but that this control is lost in failing cells.Schobesberger et al. analyze the localization of βOpinion:1- and β2-ARs, β3-ARs are not desensitized in HF, and their activator is currently undergoing a Phase IIb clinical trial for treating heart failure. The combined SICM and FRET methods are unique to this investigative group and are valuable for the question at hand. There are a number of intriguing results presented, and the presentation is generally quite clear. However, the conclusions of the study are somewhat overstated, based on the data presented . This must be addressed.The study provides some interesting new data and is relevant because unlike the βEssential revisions:3-AR does not exist in murine cardiac myocytes. The authors should examine their data in view of these observations or argue against.1) First and foremost, recent work of PC Simpson showed clearly that β2) Evidence that the myocytes are from a state of heart failure should be included in the Results (echo data).1-AR, β2-AR antagonists, PDE inhibitors) must be justified – selectivity of drugs for the targets is key to the interpretation of most data in this manuscript.3) The choice of drug concentrations (β4) There are issues with use of statistical tests. MW or t-tests are used to compare multiple groups . This should be re-done with ANOVA or non-parametric equivalent. Please notice also other comments about statistical analysis of the data.5) Interpretation of t-tubule/crest responses  does not appear justified by the data. In Figure 3, comparison of E and J suggest that T-tubule responses are lost but not that they are redistributed to other areas of the sarcolemma. By contrast, comparison of the impact of the C3SD peptide and its scrambled control in healthy cells suggests a gain of crest signalling, rather than a loss of t-tubular signals.6) Care must be taken with interpretation of work using the C3SD peptide. The sequence replicates the so-called caveolin scaffolding domain (CSD) which for many years was considered to mediate the regulatory role of caveolins through interaction with a complementary caveolin binding motif (CBM) in target proteins. This central tenet has been questioned within the last decade by work from the Parton and Dart laboratories which show that most CBM in caveolin's 'binding partners' are inaccessible for interaction with the CSD. This does not necessarily weaken the evidence for a role for CSD, which may exert effects via alternative mechanisms. However, we are not aware of any evidence that the C3SD peptide leads to 'deletion of intact caveolae'. The cartoons  imply that Cav3  is a caveolar neck protein, which is not accurate. Furthermore, these cartoons suggest that with the C3SD peptide, the t-tubular cGMP signal is reduced which is not borne out by the data in part A of this figure. The legend states 'in cardiomyocytes treated with..C3SD..the cGMP signal in…crest areas is even stronger than when stimulated in t-tubules' but the comparison between these 2 datasets is not made .7) It is important to acknowledge the limitation of confocal imaging to provide robust support for the location of proteins in caveolae . Could more meaningful information be gained from confocal images  by a separation of interior (t-tubular) and surface (crest) staining?8) The increased co-localisation of sGC and α-actinin is not explained  – this seems to suggest increased sCG at z discs, i.e., t-tubules in failing cells which is not consistent with the overall interpretation of data.3-AR stimulation on cAMP production (indexed with the Epac-based sensor) is small even in control cells. If this significant difference is retained when the data are analysed using ANOVA, the authors should comment on the relevance of such a small change. Perhaps measurement of cAMP signals in different subcellular compartments might reveal profound differences.9) Figure 5 – the magnitude of the impact of β3-AR are caveolae-based (presumably because of lack of an appropriate antibody) yet some unsubstantiated statements are made e.g. 'β3 -AR dependent cAMP pools are formed in caveolae'. Some evidence (referenced in the manuscript) supports the location of some sGC subunits in buoyant caveolae-containing fractions, and the evidence for caveolar location of eNOS is robust. However, comprehensive evidence to support statements regarding the location of the β3 signalosome in caveolae is lacking.10) No direct evidence is presented here that β3-AR dependent reduction in cAMP seen in control cells is absent in failing cells (i.e. cAMP lower in control cells in the presence of β3-AR stimulation). Yet comparison of panel A and B in Figure 6 suggests that cAMP is lower in failing cells in the presence of β3-stimulation. Even given an increase in PDE2 expression/activity in failing cells (not shown explicitly) which could increase degradation of cAMP, how can the data in Figure 5 be reconciled with the cartoons in Figure 6?11) Figure 6. There seems to be some lack of accuracy in depicting current (and past) data from this group. Why are β1-AR only in crest in healthy myocytes . Are the PDE2/5 symbols misplaced at the bottom of panel A . The data in Figure 5C suggest that the normal β3-AR stimuli applied to T-tubules vs. Crest membrane regions were performed in different cells, with comparisons between them analyzed with non-paired statistics. This is not explicitly stated but is apparent by the statistical test used (Mann Whitney U). This seems problematic given that the level of FRET response varies from cell to cell, and even accepting IBMX-based normalization between cells, the statistical power to identify quantitative changes would be challenging. If getting both stimuli in the same cell is impossible and unpaired analysis is required, then the ability to properly normalize between cells is critical. The concern with it is raise by the key data shown in Figure 3. While the extent to which β3-AR-mediates cGMP at the Crest appears similar between healthy and failing cardiomyocytes, the FRET response at T-tubule declines in the latter group. If this was due to a redistribution of β3-AR to the Crest region, would you not expect a rise in the% FRET response there? Is it possible the results can be explained by a decline in β3-R levels or signal-coupling in the T-tubules after MI? Data in Figure 3E and 3J should be combined into a 2WANOVA, and a statistical test made for the interaction between heart failure (or not) and location where the agonist was applied.12) It appears that the differently targeted β3 signaling in plasma membrane compartments or its modulation by heart failure , however there are differences in their findings to the current ones that deserve comment. They too found β3R expressed in both caveolin enriched and heavy (non-lipid raft) regions shown by direct Western Blot. Their results differ however, in that the distribution in each region did not appear to change in the canine HF model , and signaling seemed only observed if the β3R was stimulated in the non-lipid raft compartment. The methods were clearly different, but results should be discussed, particularly in light of Comment 12. Ideally, using a membrane fraction, then Cav3 pull down, and β3-AR protein analysis would help confirm if there is indeed a redistribution in this model that was not observed in the canine model reported previously. If there is a real difference, the question then becomes what goes on in human – is it more like rat or dog?13) This study is not the first to examine β14) There are a number of figures shown where the FRET tracings themselves do not appear to represent the summary data. Examples are in Figure 2 and 5. There are also concerns that for some analysis, very few animals were used, , and this raises concerns about reproducibility.3-AR-stimulated cGMP and cAMP via PDE2 regulation. The modest decline from β3-AR agonist in controls seems to be reproduced in the post MI cells too, though the statistical variance is slightly different so one is borderline significant and the other not. The proper test would be a 2WANOVA, where the impact of heart condition is one variable, and the β3-AR application the other. This could be done for the β3-AR response, and then for +/- PDE2-I response where β3-AR stimulation is present. To support the authors' conclusions, there would need to be a significant interaction.15) Figure 5 is another key figure in the study, looking at potential crosstalk between βThe authors choose forskolin to stimulate cAMP and PDE2-dependent hydrolysis, but this should be contrasted to a β-AR agonist to better support their conclusion .16) The novelty of the immunofluorescent microscopy data is questioned by prior reports showing similar co-distribution of sGC and Caveolin 3 in normal and failing hearts . There is also a concern that the microscopy is based on freshly isolated cells, whereas the functional data were obtained 48 hours post isolation (time needed to introduce the FRET probes). What is the immunohistology distribution after this 48 hours? Myocytes are known to change the localization of ion channels and various GPCRs during this time period.17) The MI model is not described, so it is difficult to know the severity of dysfunction generated. Equally important, the location in the heart from which myocytes cells were isolated post MI – is not noted. This is important; e.g. was this peri-infarct, or from the remote territory? How was that determined? How reduced was the EF generated and what other evidence was there supporting this as heart failure – e.g. elevated filling pressures, pulmonary edema, etc.. What was the sex of the animals?18) The scheme shown in Figure 6 is interesting but not really confirmed by the data shown and so remains speculative. This is because Figure 5 does not test the relative signaling involved in the two different compartments (TT vs. Crest), and with cAMP being stimulated coupled to a Cav3/localizing β-AR, versus not.3 receptors. The authors should do the similar experiments in the presence of CGP plus ICI (β1 and β2 adrenoceptor blockers).19) In Figure 1B and f the authors show a reduced cGMP generation after Isoproterenol stimulation. However, it is not clear whether this is entirely mediated via β20) In Figure 2E it is not entirely clear whether the FRET response by IBMX (about 4%) represents the cGMP generation on top of Isoproterenol evoked cGMP generation. If not the IBMX effect together with Isoproterenol is not larger than with ISO alone . This should be clarified.3-ARs are localized in the T-tubules of healthy cells and redistribute to the non166 tubular sarcolemma in heart failure”, the authors state that in failing hearts cGMP generation can be detected across the sarcolemma. The authors should more specifically state that in healthy cardiomyocytes there is no crest response which becomes now obvious in failing hearts. At least this is demonstrated in Figure 3D and I.21) In the first paragraph of the subsection “Functional β22) The immunostainings shown in Figure 4 and Figure 5—figure supplement 1 entirely depend on the specificity of the antibodies directed against sGCα1, caveolin3 and sGCβ1. The authors should comment in the manuscript about what is known about the specificity of these antibodies. Have they been used previously in knockout control cells or what other efforts have been made to demonstrate their specificity?23) Figure 4—figure supplement 1: In the model in figure B and C, it is suggested that cGMP formation is reduced in the T tubules after induction of heart failure. However, if there is no statistical significance Figure 4—figure supplement 1A, B between the first bar (T tubule scramble) and the third bar (T tubular C3SD). Thus, this model is not supported entirely by the data.3-agonists. It seems to be in the range of 10-15%?24) In Figure 5 the authors should somehow indicate more clearly at which time points the changes in cAMP generation are quantified in C. Is this at the time point just before IBMX application? The authors should clearly state in the manuscript the percentage of inhibition in cAMP generation by the β25) The model in Figure 6 in healthy cardiomyocytes indicates that cGMP act on PDE2 to limit cAMP generation. The authors should elaborate in a little bit more detail how this is achieved for readers not entirely familiar with the previous literature. Essential revisions:3-AR does not exist in murine cardiac myocytes. The authors should examine their data in view of these observations or argue against.1) First and foremost, recent work of PC Simpson showed clearly that βWe are aware of the recent publication from the PC Simpson group describing single cell PCR analysis of all adrenergic receptor types in wildtype mice : 1103-1115). However, in this manuscript we deal with rat myocytes only. Therefore, we can’t comment on the mouse cells.3-AR are indeed expressed in rat cardiomyocytes. Also, it is proven that they are overexpressed in various disease states in the rats :455-61; The Trend of β3-Adrenergic Receptor in the Development of Septic Myocardial Depression: A Lipopolysaccharide-Induced Rat Septic Shock Model. Yang, N., Shi, X. L., Zhang, B. L., Rong, J., Zhang, T. N., Xu, W., and Liu, C. F., Cardiology, (2018), 139(4), 234–244; Cardiac effects of long-term active immunization with the second extracellular loop of human β1- and/or β3-adrenoceptors in Lewis rats. Montaudon E, Dubreil L, Lalanne V, Vermot Des Roches M, Toumaniantz G, Fusellier M, Desfontis JC, Martignat L, Mallem MY. Pharmacol Res. (2015), 100:210-9).Different publications have clearly shown that there are species differences in the expression profiles of adrenergic receptors and thatβ3-AR specific agonists and antagonists have been reported on the rat cardiomyocytes , 62:8-17.; Nebivolol: a multifaceted antioxidant and cardioprotectant in hypertensive heart disease. Khan MU, Zhao W, Zhao T, Al Darazi F, Ahokas RA, Sun Y, Bhattacharya SK, Gerling IC, Weber KT. J Cardiovasc Pharmacol. (2013), 62(5):445-51).Additionally, in many publications the effects of β3-AR correctly, particularly not in disease states, where they are upregulated: https://doi.org/10.1161/CIRCRESAHA.117.310942).Moreover, we have already expressed our criticism of the PC Simpson group publication in an open letter, stating that their methodological approach may not represent the presence of βNevertheless, in our experiments we also noticed that not every cell responded to Isoproterenol treatment with a distinct cGMP FRET signal, therefore we added “β-adrenergic stimulation  of healthy control rat ventricular cardiomyocytes expressing the cGMP biosensor red cGES-DE5, led to the production of substantial amounts of cGMP  in about 2/3 of all tested cells” into our Results. Additionally, we changed the graphs containing Isoproterenol stimulation of cGMP into scatter plots, to make the non-responders visually discernible from the responding cells .2) Evidence that the myocytes are from a state of heart failure should be included in the Results (echo data).We agree with the reviewer and have added the respective echo data as Figure 1.Additionally, we added the following passage into the Results section:“Echocardiographic and biometric data show heart failure phenotype. Echocardiographic and biometric data were collected and are summarised in Figure 1.”And the following sentence into the Materials and methods section: “Echocardiography and biometric data are summarised in Figure 1.”1-AR, β2-AR antagonists, PDE inhibitors) must be justified – selectivity of drugs for the targets is key to the interpretation of most data in this manuscript.3) The choice of drug concentrations  There are issues with use of statistical tests. MW or t-tests are used to compare multiple groups . This should be re-done with ANOVA or non-parametric equivalent. Please notice also other comments about statistical analysis of the data.3-AR-dependent cGMP and both figures show a potential trend of altered cGMP regulation.We thank the reviewer for the observation and have now performed renewed statistical tests in most figures using either non-parametric Mann Whitney U or a mixed ANOVA followed by χ2-test with cells assigned to the respective rats they were isolated from. As a result, Figure 2 and Figure 5E lost their statistical significance, but are retained in the manuscript as Figure 3 shows the main PDEs degrading β5) Interpretation of t-tubule/crest responses  does not appear justified by the data. In Figure 3, comparison of E and J suggest that T-tubule responses are lost but not that they are redistributed to other areas of the sarcolemma. By contrast, comparison of the impact of the C3SD peptide and its scrambled control in healthy cells suggests a gain of crest signalling, rather than a loss of t-tubular signals.We thank the reviewer for the suggestion and have reworded the text accordingly as follows:3-ARs in non-tubulated surface areas in failing cells might be linked to a disrupted association of β3-AR with caveolar signalosomes. […] However, this difference can be abolished by treating cells with the TAT-C3SD peptide, but not with a scrambled peptide, with the level the response on the crest areas increasing to the level of T-tubules .”“The increased activity of β3-AR stimulation we changed the presentation of Figures 1,2 and 4.In addition, to better visualise the cell responses to β6) Care must be taken with interpretation of work using the C3SD peptide. The sequence replicates the so-called caveolin scaffolding domain (CSD) which for many years was considered to mediate the regulatory role of caveolins through interaction with a complementary caveolin binding motif (CBM) in target proteins. This central tenet has been questioned within the last decade by work from the Parton and Dart laboratories which show that most CBM in caveolin's 'binding partners' are inaccessible for interaction with the CSD. This does not necessarily weaken the evidence for a role for CSD, which may exert effects via alternative mechanisms. However, we are not aware of any evidence that the C3SD peptide leads to 'deletion of intact caveolae'.We thank the reviewer for this correction and have reworded this part in the manuscript to “We investigated this hypothesis by using the cell-permeable peptide disruptor of caveolar signalling TAT-C3SD. The addition of this peptide leads to the dissociation of caveolar signalosomes by inhibiting signalling which is dependent upon the binding to the caveolin-3 specific scaffolding domain (C3SD)”, while citing the MacDougall et al., 2012 publication from the Calaghan S. group.The cartoons  imply that Cav3  is a caveolar neck protein, which is not accurate. Furthermore, these cartoons suggest that with the C3SD peptide, the t-tubular cGMP signal is reduced which is not borne out by the data in part A of this figure. The legend states 'in cardiomyocytes treated with..C3SD..the cGMP signal in…crest areas is even stronger than when stimulated in t-tubules' but the comparison between these 2 datasets is not made .We thank the reviewer for this observation and have changed the cartoon to portray Cav 3 more accurately. We also performed additional C3SD peptide measurements for the Figure 5—figure supplement 1.7) It is important to acknowledge the limitation of confocal imaging to provide robust support for the location of proteins in caveolae . Could more meaningful information be gained from confocal images  by a separation of interior (t-tubular) and surface (crest) staining?We thank the reviewer for this observation and the suggestion to look at different cell areas in our confocal images. We have performed further Pearson Correlation Coefficient measurements by drawing the regions of interest so they either measured the membranes or the cell interior. We could however detect no significant differences in the cell areas. We added a remark about the limitation of confocal microscopy to detect structures underneath the diffraction limit in the Discussion, as follows: “Though the immunocytochemical method is limited in its spatial resolution and can therefore not resolve the caveolae structures themselves, it allows us to detect an alteration in sGC localisation in heart failure and, which could potentially be indicative of dysregulated caveolar signalosomes as reported previously in a pressure overload induced heart failure model using mice ”.8) The increased co-localisation of sGC and α-actinin is not explained  – this seems to suggest increased sCG at z discs, i.e., t-tubules in failing cells which is not consistent with the overall interpretation of data.We understand the point the reviewer is making, however after more stringent statistical testing Figure 5F has lost its significance. Nonetheless, we opted to leave the images and graph in the manuscript as it might give a potential trend of where the sGCα subunit might be relocating to in heart failure we and have added the following sentence in the Discussion section: “We have observed a trend to an increased overlap between our sGCα and α-actinin staining in our confocal imaging, which could potentially represent an increased redistribution of sGCα to the areas of the Z-disc not directly associated with the T-tubules or caveolar signalosomes.”3-AR stimulation on cAMP production (indexed with the Epac-based sensor) is small even in control cells. If this significant difference is retained when the data are analysed using ANOVA, the authors should comment on the relevance of such a small change. Perhaps measurement of cAMP signals in different subcellular compartments might reveal profound differences.9) Figure 5 – the magnitude of the impact of βWe have acquired new data that and added to the figure, this effect is statistically significant .However, we appreciate the reviewer’s comment and have added the following paragraph into the Discussion:3-AR stimulation of about 10 percent on overall cAMP levels, as measured via the cytosolic FRET sensor Epac1-camps, could be of physiological relevance when brought into the context of lowering pathological cAMP signalling levels on a whole or in confined signalling compartments.”“The observed response of β3-AR are caveolae-based (presumably because of lack of an appropriate antibody) yet some unsubstantiated statements are made e.g. 'β3 -AR dependent cAMP pools are formed in caveolae'. Some evidence (referenced in the manuscript) supports the location of some sGC subunits in buoyant caveolae-containing fractions, and the evidence for caveolar location of eNOS is robust. However, comprehensive evidence to support statements regarding the location of the β3 signalosome in caveolae is lacking.10) No direct evidence is presented here that β3-AR association with caveolae in myocytes transgenically expressing the human β3-AR, which allowed us to perform membrane fractionation protein assays as well as a proximity ligation assay using a human specific β3-AR and Caveolin 3 antibody. We also currently have a paper in press  containing further evidence of this association via a PLA colocalization signal between the endogenous rat β3-AR and AMPK (itself in caveolae) from rat neonatal cardiac myocytes. In any case we added a remark in the Introduction to mention our previous work on the caveolae and β3-AR association: “Recently, overexpression of β3-AR in transgenic mice has been shown to protect the heart from catecholamine-induced hypertrophy and remodelling via an eNOS)/soluble guanylyl cyclase (sGC)/cGMP-dependent signalling pathway. The same study showed localization of β3-ARs together with eNOS in caveolae-enriched membrane fractions, which had been separated via ultracentrifugation ”.We thank the reviewer for this comment. In the past we have shown β3-AR dependent reduction in cAMP seen in control cells is absent in failing cells (i.e. cAMP lower in control cells in the presence of β3-AR stimulation). Yet comparison of panel A and B in Figure 6 suggests that cAMP is lower in failing cells in the presence of β3-stimulation. Even given an increase in PDE2 expression/activity in failing cells (not shown explicitly) which could increase degradation of cAMP, how can the data in Figure 5 be reconciled with the cartoons in Figure 6?11) Figure 6. There seems to be some lack of accuracy in depicting current (and past) data from this group. Why are β1-AR only in crest in healthy myocytes . Are the PDE2/5 symbols misplaced at the bottom of panel A . The data in Figure 5C suggest that the normal βWe thank the reviewer for spotting the misplaced PDE symbols and have changed the overall schematic to better reflect current and old data .3-AR stimuli applied to T-tubules vs. Crest membrane regions were performed in different cells, with comparisons between them analyzed with non-paired statistics. This is not explicitly stated but is apparent by the statistical test used (Mann Whitney U). This seems problematic given that the level of FRET response varies from cell to cell, and even accepting IBMX-based normalization between cells, the statistical power to identify quantitative changes would be challenging. If getting both stimuli in the same cell is impossible and unpaired analysis is required, then the ability to properly normalize between cells is critical. The concern with it is raise by the key data shown in Figure 3. While the extent to which β3-AR-mediates cGMP at the Crest appears similar between healthy and failing cardiomyocytes, the FRET response at T-tubule declines in the latter group. If this was due to a redistribution of β3-AR to the Crest region, would you not expect a rise in the% FRET response there? Is it possible the results can be explained by a decline in β3-R levels or signal-coupling in the T-tubules after MI? Data in Figure 3E and 3J should be combined into a 2WANOVA, and a statistical test made for the interaction between heart failure (or not) and location where the agonist was applied.12) It appears that the differently targeted βUnfortunately, a proper normalization with the red-DE5 FRET sensor is not yet established and performing both measurements of crest and T-tubule in one cell could lead to potential desensitization of receptors and alterations in the FRET sensor. We appreciate the reviewer’s suggestion that we use a more stringent statistical test and have now used a mixed ANOVA followed by χ2-test. We also updated the text to better reflect the data of an increased crest signal in heart failure.3 signaling in plasma membrane compartments or its modulation by heart failure , however there are differences in their findings to the current ones that deserve comment. They too found β3R expressed in both caveolin enriched and heavy (non-lipid raft) regions shown by direct Western Blot. Their results differ however, in that the distribution in each region did not appear to change in the canine HF model , and signaling seemed only observed if the β3R was stimulated in the non-lipid raft compartment. The methods were clearly different, but results should be discussed, particularly in light of Comment 12. Ideally, using a membrane fraction, then Cav3 pull down, and β3-AR protein analysis would help confirm if there is indeed a redistribution in this model that was not observed in the canine model reported previously. If there is a real difference, the question then becomes what goes on in human – is it more like rat or dog?13) This study is not the first to examine β3-ARs and the current lack of a suitable antibody, which could detect rat β3-ARs with appropriate specificity, we have refrained from performing this experiment. We agree that the data from Trappanese et al., 2015, does not match our data exactly, however we assume that this can at least in part be explained by the different pathophysiological courses of heart failure in the two different models. We have shown in the past, that in human a residual cGMP increase was detectable after β3-AR agonist application on biopsies from patients with different types of heart disease. .We thank the reviewer and believe the suggested experiment could be of potential value if it were feasible. However, due to the relatively low expression of β14) There are a number of figures shown where the FRET tracings themselves do not appear to represent the summary data. Examples are in Figure 2 and 5. There are also concerns that for some analysis, very few animals were used, , and this raises concerns about reproducibility.The reviewers concern about low animal numbers was noted and additional experiments were performed for figures in which only two animals were used, with the exception of Figure 4—figure supplement 1A, B) in order to increase n numbers and alleviate the concerns about reproducibility. We kept the current traces of FRET experiments, since we believe that the traces are representative of the summary data.3-AR-stimulated cGMP and cAMP via PDE2 regulation. The modest decline from β3-AR agonist in controls seems to be reproduced in the post MI cells too, though the statistical variance is slightly different so one is borderline significant and the other not. The proper test would be a 2WANOVA, where the impact of heart condition is one variable, and the β3-AR application the other. This could be done for the β3-AR response, and then for +/- PDE2-I response where β3-AR stimulation is present. To support the authors' conclusions, there would need to be a significant interaction.15) Figure 5 is another key figure in the study, looking at potential crosstalk between βThe authors choose forskolin to stimulate cAMP and PDE2-dependent hydrolysis, but this should be contrasted to a β-AR agonist to better support their conclusion .1-AR desensitization in heart failure such a direct comparison would have been complicated, if we had opted to use a direct β-AR agonist.We thank the reviewer for the suggestions and have performed further experiments to increase n-number and for statistics we used a mixed ANOVA followed by χ2-test to incorporate the variance introduced by the different animals. The differences are still significant. We chose forskolin as our cAMP stimulant as we wished to have a direct comparison between control and heart failure cells. Due to β16) The novelty of the immunofluorescent microscopy data is questioned by prior reports showing similar co-distribution of sGC and Caveolin 3 in normal and failing hearts . There is also a concern that the microscopy is based on freshly isolated cells, whereas the functional data were obtained 48 hours post isolation (time needed to introduce the FRET probes). What is the immunohistology distribution after this 48 hours? Myocytes are known to change the localization of ion channels and various GPCRs during this time period.We thank the Editor and the reviewers for raising this important point. Cardiomyocytes do indeed remodel but the remodelling of rat cells is not so extensive as to preclude useful experiments. Papers from our group  and others  have demonstrated that factors such as the organization of the surface of the cardiomyocytes (z-groove ratio) and t-tubule density (rat cells lose only 25% of their t-tubule area over the first 48 hrs). Functional factors are not markedly altered, neither is the capacitance of the membrane or the staining of tubular structures after 48 hours despite the cells looking morphologically quite different due to the rounding of the myocytes ends. Rat cells compare favourably with those isolated from mouse which completely lose their form and function over 48 hrs .17) The MI model is not described, so it is difficult to know the severity of dysfunction generated. Equally important, the location in the heart from which myocytes cells were isolated post MI – is not noted. This is important; e.g. was this peri-infarct, or from the remote territory? How was that determined? How reduced was the EF generated and what other evidence was there supporting this as heart failure – e.g. elevated filling pressures, pulmonary edema, etc.. What was the sex of the animals?We thank the reviewer for their observation and have added the respective echocardiography and biometric data as Figure 1.Additionally, we added the following passage into the Results section:“Echocardiographic and biometric data were collected and are summarised in Figure 1.”And the following into the Materials and methods section: “Echocardiographic and biometric data are summarised in Figure 1.”Animals were all male, we have added this information in the Materials and methods section as follows: “All animals were male.”18) The scheme shown in Figure 6 is interesting but not really confirmed by the data shown and so remains speculative. This is because Figure 5 does not test the relative signaling involved in the two different compartments (TT vs. Crest), and with cAMP being stimulated coupled to a Cav3/localizing β-AR, versus not.3-AR activation to lower and thereby attenuate cAMP levels in control and heart failure. We have slightly changed Figure 6  to be less speculative. See Figure 7.We appreciate the reviewer’s point, but with the experiment shown in Figure 5  we wished to detect if there was an overall potential of β3 receptors. The authors should do the similar experiments in the presence of CGP plus ICI (β1 and β2 adrenoceptor blockers).19) In Figure 1B and f the authors show a reduced cGMP generation after Isoproterenol stimulation. However, it is not clear whether this is entirely mediated via β3-AR response only in the MI cells (with inhibiting β1 and β2 receptors); however, we were unable to perform the suggested experiment as we did not have any more MI cells (in the 2 months of time that we were given for the revision).We agree with the reviewer that it would benefit our work if we could show the β20) In Figure 2E it is not entirely clear whether the FRET response by IBMX (about 4%) represents the cGMP generation on top of Isoproterenol evoked cGMP generation. If not the IBMX effect together with Isoproterenol is not larger than with ISO alone . This should be clarified.We thank the reviewer for this point and have tried to make the data description clearer by adding the following passage into the figure legend: “The scatter plot/histograms present the average whole cell cGMP-FRET responses evoked by PDE inhibition further to the isoproterenol responses in% from (A-D) (E).”, that means i.e. if the ratio after ISO application was at 0.98 and the following ratio after single PDE inhibition was at 0.97 and the ratio after IBMX application at 0.96, then the values would be 1 for the single PDE and 2 for the total PDE inhibition.3-ARs are localized in the T-tubules of healthy cells and redistribute to the non166 tubular sarcolemma in heart failure”, the authors state that in failing hearts cGMP generation can be detected across the sarcolemma. The authors should more specifically state that in healthy cardiomyocytes there is no crest response which becomes now obvious in failing hearts. At least this is demonstrated in Figure 3D and I.21) In the first paragraph of the subsection “Functional β3-ARs reside mainly in the T-tubules with very few responses being detectable outside of T-tubules , whereas in failing cells, they β3-ARs responses after localised stimulation can be detected in both tubulated and non-tubulated areas across the sarcolemma .”We thank the reviewer for this comment and have changed the manuscript text accordingly to “In healthy cardiomyocytes, we observed that functional β22) The immunostainings shown in Figure 4 and Figure 5—figure supplement 1 entirely depend on the specificity of the antibodies directed against sGCα1, caveolin3 and sGCβ1. The authors should comment in the manuscript about what is known about the specificity of these antibodies. Have they been used previously in knockout control cells or what other efforts have been made to demonstrate their specificity?We agree with the reviewer that the specificity of antibodies is essential for the experiments shown in our manuscript. Therefore, we used the best available antibodies and added the specificity info into the text. We have commented on the specificity of the antibodies we employed in our manuscript in the Materials and methods section as follows: “rabbit sGCα and β subunit antibodies , mouse α-actinin , mouse Caveolin 3 ”.Additionally, we tested the specificity of sGCα. Freshly isolated adult mouse cardiomyocytes  were fixed with ice-cold methanol and co-stained with sGCb1 and Cav3 antibodies as described in the Materials and methods and Figure 5—figure supplement 1) Confocal images were using the small laser intensities in all conditions similar to those used in this manuscript.23) Figure 4—figure supplement 1: In the model in figure B and C, it is suggested that cGMP formation is reduced in the T tubules after induction of heart failure. However, if there is no statistical significance in Figure 4—figure supplement 1A, B between the first bar (T tubule scramble) and the third bar (T tubular C3SD). Thus, this model is not supported entirely by the data.We thank the reviewer for the observation and have altered the cartoon accordingly, so that the cGMP formation in HF tubules does not appear smaller. See figure 4—figure supplement 1.3-agonists. It seems to be in the range of 10-15%?24) In Figure 5 the authors should somehow indicate more clearly at which time points the changes in cAMP generation are quantified in C. Is this at the time point just before IBMX application? The authors should clearly state in the manuscript the percentage of inhibition in cAMP generation by the β3-AR led to a significant reduction of approximately 10.3% of the forskolin stimulated cAMP production ”.We thank the reviewer for the suggestion and have attempted to clarify the measurement steps in the manuscript with the following explanation in the Figure 5 figure legend: “Scatter plot/histogram presenting whole cell cAMP-FRET responses depicted as the percentage of the maximal possible cAMP FRET response (= Forskolin followed by IBMX). The measured Forskolin or IBMX responses were the respective maximal responses, equalling the lowest FRET ratio value, achieved after each stimulus.” Additionally, we added the average response rate of 10.3% into the manuscript text as follows: “In healthy cells, stimulation of β25) The model in Figure 6 in healthy cardiomyocytes indicates that cGMP act on PDE2 to limit cAMP generation. The authors should elaborate in a little bit more detail how this is achieved for readers not entirely familiar with the previous literature.We thank the reviewer for the suggestion and have elaborated more on this point in the Discussion section as follows: “At the same time as being degraded by PDE2, cGMP can also increase PDE2 activity on cAMP more strongly by binding to it, thereby triggering the so-called cGMP-to-cAMP crosstalk.This crosstalk between the second messengers of which one (cAMP) has increasingly been associated with detrimental signalling pathways in the context of heart failure, which the other (cGMP) could potentially attenuate ”, as well as in the figure legend .


Asparagus officinalis stalk end was extracted and synthesized to carboxymethyl cellulose (CMCas) using monochloroacetic acid (MCA) via carboxymethylation reaction with various sodium hydroxide (NaOH) concentrations starting from 20% to 60%. The cellulose and CMCas were characterized by the physical properties, Fourier Transform Infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), Scanning electron microscopy (SEM) and X-ray diffraction (XRD). In addition, mechanical properties of CMCas films were also investigated. The optimum condition for producing CMCas was found to be 30% of NaOH concentration for the carboxymethylation reaction, which provided the highest percent yield of CMCas at 44.04% with the highest degree of substitution (DS) at 0.98. The melting point of CMCas decreased with increasing NaOH concentrations. Crystallinity of CMCas was significantly deformed (p < 0.05) after synthesis at a high concentration. The L* value of the CMCas was significantly lower at a high NaOH concentration compared to the cellulose. The highest tensile strength (44.59 MPa) was found in CMCas film synthesized with 40% of NaOH concentration and the highest percent elongation at break (24.99%) was obtained in CMCas film treated with 30% of NaOH concentration. The applications of asparagus stalk end are as biomaterials in drug delivery system, tissue engineering, coating, and food packaging.Cellulose from  Asparagus officinalis L.) is a nutritious and perennial vegetable continually used as antifungal activities, anticancer and anti-inflammatory herbal medicine in Asia. In the processing of asparagus, the spears which are 2–3% of total weight of the asparagus are typically processed into three types of products: canned, fresh, and frozen [Asparagus  and its application such as packaging film so far.Thus, CMC can be versatile applied in various field such as drug delivery carrier , wound has. The effect of NaOH concentration mechanical properties (tensile strength and percentage elongation at break) and morphology of CMCas films was also carried out.Therefore, this study aimed to determine the effect of NaOH concentration at 20–60% on percent yield, the degree of substitution (DS), thermal properties, chemical structure, viscosity, crystallinity, morphology of CMCTM , sodium hydroxide and glacial acetic acid from Lab-scan, isopropyl alcohol (IPA), Monochloroacetic acid from Sigma-Aldrich .Asparagus stalk end was purchased from Nong Ngulueam Sub-District . All chemicals used in the preparation and analysis of synthesized CMC were analytical reagent (AR) grade or the equivalent. Absolute methanol, ethanol, and sodium silicate from Union science Co., Ltd. , hydrogen peroxide from QReCTwo grams of asparagus stalk end was cut into small pieces and dried in an oven 105 °C for 6 h. Then dried asparagus was weighed to calculate the percent dryness as described in Equation (1). Dried asparagus stalk end was grounded by using a hammer mill  to less than 1 mm. The dried powder was then stored in polypropylene (PE) bags at ambient temperature until used. The percent dryness was calculated by the following Equation (1)w/v) NaOH solution at 1:20 (w/v) treated at 100 °C for 3 h. The black slurry was filtered and rinsed with cold water until a neutral pH of rinsed water was obtained. To obtain the cellulose fiber, the washed fiber residue was dried in an oven at 55 °C for 24 h. Hydrogen peroxide and sodium silicate were used to bleach the cellulose fiber. The bleached cellulose was then grounded by using the hammer mill size 70 mash . The bleached cellulose powder was kept in PE bags at ambient temperature until used.Cellulose from dried asparagus stalk end powder was extracted according to the method of ,16. Briew/v) and 350 mL of isopropanol (IPA) were blended with 15 g of cellulose powder extracted from asparagus stalk end and this was mixed for 30 min. Subsequently, the carboxymethylation reaction was synthesized according to the method of [as product was in powder form. The percent yield was calculated by the following Equation (2) [50 mL of various concentrations of NaOH at 20, 30, 40, 50, and 60% (ethod of . The fintion (2) :(2)YielDS) of CMCas presents the average number of hydroxyl group replaced by carboxymethyl and sodium carboxymethyl groups at C2, 3, and 6 in the cellulose structure. The DS of CMCas was evaluated by the USP XXIII method for Crosscarmellose sodium which are titration and residue on combustion [DS was showed in the following Equation (3);A is the DS of carboxymethyl acid and S is the DS of sodium carboxymethyl which A and S were calculated using Equations (4) and (5):M is consumption of the titration to end point (mEq) and C is the amount of ash remained after ignition (%).The degree of substitution  for approximately l to l.5 h and placed into the desiccator to obtain black residue. Sufficient sulfuric acid was added to moisten the entire residues and heated up until the gas fumes (white smoke) was utterly disappeared. The crucible with the black residue was ignited at 800 ± 25 °C to produce white residue and placed in the desiccators to get a constant weight. The residue on ignition was presented in the percentage and calculated using Equation (6).A crucible was dried in an oven at 100 °C for 1 h and transferred to a desiccator until the weight reached at an accurate value  . CMCas was were determined by using FTIR spectra . 2 mg of dry sample was pressed into a pellet with KBr. Transmission level measured at the wavenumber range of 4000–400 cm−1 [The aldehyde groups of the cellulose from stalk asparagus and CMC400 cm−1 .as sample was weight and dissolved in 25 mL of distilled water with stirring at 80 °C for 10 min—automatic stirring action set at 960 rpm for 10 s. The temperature of the sample was varied from 30, 40, 50 and 60 °C at 5 min intervals and held speed at 160 rpm until end of the test [A Rapid Visco Analyzer  was used to determine the viscosity of samples. 1 g of cellulose or CMCthe test .as were determined using Differential Scanning Calorimeter (DSC)  according to the melting temperature determined from the thermogram. The determination was performed under a nitrogen atmosphere at a flow rate of 50 mL min−1. Samples (10 mg) contained in aluminum pans were heated from 40 to 450 °C with a heating rate of 10 °C min−1 [The thermal characterization of cellulose from asparagus stalk end and CMC°C min−1 .The crystallinity of the samples was determined using X-ray diffraction patterns carried out by X-ray powder diffractometer  in the reflection mode. Prior to the test, the samples were dried in a hot air oven  at 105 °C for 3 h to produce a powder form. Scans were carried out in the range of scattering angle (2θ) from 10 to 60° with at a scan rate of 5°/min .as. The samples were analyzed through a large field detector. The acceleration voltage was used at 15 kV with 1500× original magnification [Scanning Electron Microscopy (SEM)  was used to analyze the surface morphology of cellulose from asparagus stalk end and CMCfication .as was evaluated by using a Color Quest XE Spectrocolorimeter  in order to express the CIELAB color as three values: L* for the lightness from blackness (0) to whiteness (100), a* from greenness (−) to redness (+), and b* from blueness (−) to yellowness (+). The total color differences (E) take into account the comparisons between the L*, a* and b* value of the sample and standard and calculated by the following Equation (7) [The color characteristic of cellulose form asparagus stalk end and CMCtion (7) (7)∆E=(LWI) was also calculated by the following Equation (8) to represent the degree of whiteness of samples [The whiteness index ( samples .(8)WI=(as in 180 mL of distilled water with a constantly stirred at 80w/w) glycerol was added to the solution and stirred for additional 5 min. The solution was poured on acrylic plates (20 cm × 15 cm) and the dried at room temperature for 72 h. After that, the film was peeled from the plates and stored in polyethylene bags at ambient temperature [The film-forming solutions were prepared by dissolving 4 g of CMCperature .as films was determined using a micrometer model GT-313-A . The examined mechanical properties which were tensile strength (TS) and percent elongation at break (EB) of the CMCas film were tested (10 measurement each) using a universal texturometer  according to the standard procedure of ASTM D882-80a [as films were cut into 15 × 140 mm as test specimens. The specimens were carried out by using an initial grip separation distance of 100 mm and crosshead speed at 20 mm/min. The TS and EB were calculated by following Equations (9) and (10), respectively.The thickness of CMCp < 0.05). All measurements were analyzed in triplicated. The results were represented the mean values ± standard deviation. The figures present the standard deviation as the appropriate values. The error bars for some data points overlap the mean values.Statistic data were analyzed by a one-way analysis of variance (ANOVA) using SPSS software version 16.0 0 . Duncan’s multiple range test was employed to evaluate significant differences among the treatments  is shown in as increased when increasing of NaOH concentration from 20% to 30% (w/v) and then decreased with further increasing NaOH concentration at over 30% (w/v). The resulted phenomenon might be occurred due to a limitation of sodium monochloroacetate (NaMCA) as etherifying agents for substituting cellulose. Moreover, the NaOH concentration was not high enough to complete for converting the cellulose into alkali cellulose [The percent yield of CMCellulose . This reellulose .as was carried out shown in as was in between 0.49 and 0.98 which reached the highest value at the concentration of NaOH at 30% (w/v). However, the DS of CMCas was reduced at higher concentration of NaOH at 40–60% (w/v) ranged from 0.83–0.49. This phenomenon can be explained by investigating the carboxymethylation process, where two reaction occur concurrently. The first reaction involved a cellulose hydroxyl reacting with sodium monochloroacetate (NaMCA) in the presence of NaOH to obtain CMCn shown in Equation (11) and Equation (12).The effect of various concentrations of NaOH on DS of CMCThe second reaction involves NaOH reacting with NaMCA to form sodium glycolate as by-product shown in Equation (13) .NaOH + Mimosa pigra peel. The degradation was taken place due to a high concentration of NaOH [The second reaction overwhelms the first reaction together with a strong alkaline concentration. If the alkaline level in the second reaction is too high, a side reaction will form a high level of sodium glycolate as a by-product, thus lowering the DS. This result was agreed with the result found by  who stud of NaOH . Moreove of NaOH . The maxas structures. The substitution reaction of CMCas during the carboxymethylation was confirmed by FTIR [as have similar functional groups with same absorption bands such as hydroxyl group (–OH stretching) at 3200–3600 cm−1, C–H stretching vibration at 3000 cm−1, carbonyl group (C=O stretching) at 1600 cm−1, hydrocarbon groups (–CH2 scissoring) at 1450 cm−1, and ether groups (–O– stretching) at 1000–1200 cm−1 [as synthesized with various NaOH concentrations are shown in 2) and ether group (–O–) notably increased in the CMCas sample; however, the absorption band of hydroxyl group (–OH) reduced when compared to those cellulose samples  reduced with increase in the temperature. Increasing temperature plays a role in reducing the cohesive forces while simultaneously increasing the rate of molecular interchange, causing the lower viscosity [as solution affected by NaOH concentrations was changed in accordance with DS value at the same temperature [as synthesized with 20% (w/v) NaOH concentration was the highest in viscosity due to the swelling and gelatinization of cellulose comparable to previous studies [The relationship between different NaOH concentrations and viscosity of CMCiscosity . Accordiperature ,35. The  studies . As far  studies ,36. An i studies , pH valu studies .as are shown in m) of cellulose and CMCas synthesized with 20, 30, 40, 50, and 60 % (w/v) NaOH concentrations were 178.79, 206.61, 193.13, 182.34, 178.34 and 161.21 °C, respectively. The highest Tm of CMCas increased when 20% (w/v) NaOH concentration was employed. The Tm at 20–30% (w/v) of NaOH concentrations were higher than Tm of cellulose due to the substituent of carboxymethyl groups influenced on increasing ionic character and intermolecular bonds between the polymer chains [m at 40–50% (w/v) of NaOH concentrations decreased due to the melting temperature of CMCas slightly decreased as increasing the concentration of NaOH because of the increase in alkalization reaction together with a high substitution of carboxymethyl group. Moreover, the decrease in Tm was caused by an increase in number of the carbon atoms producing carbon skeleton, after a rapid quenching cooling with rate of 80 °C/min from the isotropic state at 200 °C [m of 60% (w/v) of NaOH concentration decreased, side reaction was in the majority with sodium glycolate forming as a by-product and chain breaking of CMCas polymer similar to the results presented in Mimosa pigra peel [The effect of various NaOH concentrations on the thermal property of cellulose from asparagus stalk end and CMCr chains . The Tm t 200 °C . As the gra peel .as were evaluated by using XRD shown in as material. All CMCas samples were less value in a peak of intensity (au) compared to cellulose from asparagus stalk end. Decrease in crystallinity index of cellulose may be due to the reorganization or cleavage of molecular according to alkalization of NaOH solution [The changes on the structure of cellulose from asparagus stalk end and CMCsolution . The incsolution .w/v) NaOH [as decreased by the effect of alkali solution prior to the carboxymethylation reaction on cellulose structure.These results are accord with results of various studies which were tested in cavendish banana cellulose  and duri/v) NaOH . Thus, tas powder with different NaOH concentrations were characterized by SEM with an acceleration voltage of 15 kV and 1500× original magnification , the surface uniformity decreased. The surface of CMCas powder mixed with NaOH concentration at 30% started to peel without cracking as shown in as powder has been begun to crack and deform when the powder treated with 40% of NaOH concentration  of NaOH concentration. The a* and b* values showed the highest increased in CMCas treated with 40% (w/v) of NaOH concentration. Moreover, an increase in NaOH concentration up to 40% decreased the yellowness of CMCas probably due to the first reaction of carboxymethylation (Equation (12)) to produce CMC or sodium glycolate [w/v), the decreasing of all color values was occurred. Therefore, the carboxymethylation reaction might be the reason the color values of cellulose and CMCas has been changed [as had similar trend compared to a* and b* values. These results are akin to the results from the previous studies [as had same trend with L* value  of NaOH. The TS value was correlated with an increasing DS value due to the substituted carboxymethyl groups in anhydroglucopyranose unit affecting an increase in intermolecular force among the polymer chains and the ionic character [as films started to decline at a high NaOH concentration due to the polymer degradation and formation of by-product generated from sodium glycolate increased the reduction of CMC content and thus decreasing the intermolecular forces.The tensile strength (TS) and percentage of elongation at break (EB) of CMCD882-80a . The TS haracter . Neverthas films also increased with increasing NaOH concentrations up to 30% (w/v) of NaOH and drop down after that. At high NaOH concentrations, the crystallinity of the cellulose structure decreased with increasing a flexibility. In addition, CMCas films has been obtained the lower flexibility at 60% (w/v) of NaOH concentrations because of an occurrence of hydrolysis reaction in cellulose chain [The EB of CMCse chain .Asparagus officinalis stalk end could be favorably used as a raw material to produce carboxymethyl cellulose. The alkali solution is the main parameter that affected all characteristics of CMCas synthesized with various NaOH concentrations. The percent yield of CMCas initially increased with NaOH concentrations increase up to 30% (w/v) and then gradually decreased with further NaOH concentrations increase related with the DS value of CMCas. The lightness (L*) of CMCas has been changed after treated with increasing NaOH concentration. The crystallinity of CMCas was found to decrease after synthesized with alkali solution. The highest tensile strength and elongation at break values of CMCas films were found in 40% (w/v) NaOH-synthesized CMCas films and 30% (w/v) NaOH-synthesized CMCas films, respectively.This study revealed that


Background and objectives: Previous studies consistently found no significant difference between supervised and home-based rehabilitation after anterior cruciate ligament reconstruction (ACLR). However, the function of the nonoperative knee, hamstring strength at deep flexion, and neuromuscular control have been overlooked. This prospective observational study was performed to investigate the outcomes after ACLR in operative and nonoperative knees between supervised and home-based rehabilitations. Materials and Methods: After surgery, instructional videos demonstrating the rehabilitation process and exercises were provided for the home-based rehabilitation group. The supervised rehabilitation group visited our sports medicine center and physical therapists followed up all patients during the entire duration of the study. Isokinetic muscle strength and neuromuscular control (acceleration time (AT) and overall stability index (OSI)) of both operative and nonoperative knees, as well as patient-reported knee function (Lysholm score), were measured and compared between the two groups 6 months and 1 year postoperatively. Results: The supervised rehabilitation group showed higher muscle strength of hamstring and quadriceps in nonoperative knees at 6 months  and higher hamstring strength in operative and nonoperative knees at 1 year  than the home-based rehabilitation group. At 6 months and 1 year, OSIs in operative and nonoperative knees were significantly better in the supervised rehabilitation group than in the home-based rehabilitation group . At 1 year, the supervised rehabilitation group also demonstrated faster AT of the hamstrings . Lysholm scores gradually improved in both groups over 1 year; however, the supervised rehabilitation group showed higher scores at 1 year . Conclusions: This study demonstrated that supervised rehabilitation may offer additional benefits in improving muscle strength, neuromuscular control, and patient-reported knee function compared with home-based rehabilitation up to 1 year after ACLR. Anterior cruciate ligament (ACL) injury is common in sports involving cutting, jumping, and pivoting movements, and often requires ACL reconstruction (ACLR) followed by postoperative rehabilitation ,2,3. TheOwing to the importance of postoperative rehabilitation following ACLR, previous studies investigated whether rehabilitation supervised by physical therapists would yield a superior outcome compared with home-based rehabilitation ,13,14,15The purpose of this study was to investigate and compare the outcomes, including neuromuscular control and muscle strength, of both operative and nonoperative knees between a group of patients undergoing supervised physical therapy and a group of patients undergoing unsupervised home-based rehabilitation up to postoperative 1 year following ACLR. Lysholm scores were also compared between these two groups. We hypothesized that although there may be no differences in the muscle strength of the operative knees and the Lysholm scores between the two groups, there would be a difference in the muscle strength of the nonoperative knees, hamstring strength at deep flexion, and neuromuscular control.This prospective observational study aimed to compare functional outcomes between two groups of patients, a group undergoing supervised rehabilitation and a group undergoing home-based rehabilitation, at 6 months and 12 months. The study was approved by the institutional review board of the hospital to which the corresponding author is affiliated (IRB No.2018AN0261).Participants who were scheduled for ACLR at our institution from August 2018 to May 2019 and who could attend follow-up visits at 2 weeks, 6 weeks, 3 months, 6 months, and 1 year were candidates for the study. Patients with previous knee surgeries, with concomitant neurological diseases, who could not understand Korean, and who had visual impairment were not eligible. Patients with previous knee surgeries and concomitant neurological diseases were excluded because these conditions may affect the baseline and postoperative quadriceps and hamstring strength and neuromuscular control. Additionally, because physical therapists and instructional videos communicated in the Korean language, patients who could not understand Korean or had visual impairment were excluded. Patients who were willing to participate in the supervised rehabilitation program visited our sports medicine center once a week for 3 months. If the patients were unable to attend the supervised program, instructional videos were provided. Informed consent was acquired from all individual participants included in the study.® RT  for the femoral side.All operations were performed using the same technique  under a spinal or general anesthesia by a single knee arthroscopic specialist. After the patient was positioned supine, a tourniquet was applied on proximal thigh and the limb was supported by a leg holder with the knee joint at 90° of flexion. Standard knee arthroscopic examination was performed through anterolateral and anteromedial portals using a 30°, 4.0-mm arthroscope. After a full intra-articular examination, any concomitant pathology of the articular cartilage and meniscus that required attention was corrected before ACLR. An approximately 3-cm skin incision was made anteromedially to the tibial tuberosity to harvest the semitendinosus and gracilis tendons, which were prepared using a standard tendon stripper. The harvested tendons were prepared on a back table and the ends of the grafts were sutured to form a closed loop. After a central mid-patellar portal was made, the femoral tunnel was created using the ACL RetroConstruction System and FlipCutter drill  through anatomic outside-in retrograde-reaming technique ,19. ThenInstructional videos demonstrating the rehabilitation process and exercises were provided. These videos were divided into goals and instructions over five stages, as described by Hohmann et al. and Cavanaugh and Powers ,20. A toPatients who chose to attend the supervised rehabilitation program visited our sports medicine center once a week for 3 months. The same physical therapist followed up all patients during the entire duration of the study. After 3 months, the patients were followed up bi-weekly or at longer intervals at the discretion of the physical therapist. The same-stage rehabilitation protocol for the home-based group was used for the supervised rehabilitation group.During the first two weeks, the goal was to control postoperative pain and swelling and to gain ROM up to 90°. Then, the patients aimed to gradually gain ROM up to 130° and attain normal gait by postoperative 6 weeks. After this period, the patients focused on restoring muscle strength and proprioception. All the patients were braced up to 3 months postoperatively. Once the patients achieved enough strength and proprioception by 3 months, they could work on improving functional performance. After postoperative 6 months, the patients were permitted for gradual participation in sports activities if they achieved >85% of quadriceps and hamstring strengths and ROM >130 degrees. However, the types, frequency, and intensity of sports were tailored to each patient according to his or her physical and psychological conditions assessed by the physical therapists and orthopedic surgeon. Specific details of the rehabilitation protocol were based on the orthopedic textbook, “Campbell’s Operative Orthopedics” .Isokinetic quadriceps and hamstring strength (concentric/concentric muscle contraction for extension/flexion) was measured using Biodex multi-joint system 4 . Each patient was seated on the device with the hips and knees flexed to 90° and the trunk perpendicular to the floor. A strap was used to restrict each thigh, and the lateral femoral condyle of the knee joint was aligned with the rotational axis of the isokinetic machine. Each patient then performed a set that consisted of five isokinetic knee flexions and extensions  of both operative and nonoperative legs at 180°/s, with a rest time of 30 s between sets. Flexion and extension strengths were measured by peak torque normalized to body weight , and deep flexion strength was evaluated as the peak torque/body weight at 100° of knee flexion, which is an important factor in assessing the recovery of hamstring muscle strength after hamstring-harvesting ACLR ,22. FlexPrevious studies showed that the acceleration time (AT) can be a meaningful index of muscle activation and neuromuscular readiness to produce maximal muscular contraction ,24,25. IAs another measure of neuromuscular control, we also assessed dynamic postural stability using the Biodex Stability System . Because a torn ACL can lead to compromised postural control and reduced knee stability ,27, the The Lysholm score was used to evaluate the patient-reported knee function . The Lysholm score ranges from 0 to 100, and is a valid outcome measure for patients with ACL injuries .On the basis of previous studies on quadriceps muscle strength in patients with knee joint injuries ,3, a difp < 0.05 was considered statistically significant.Repeated measures analysis of variance was used to compare three time points  for the hamstring-to-quadriceps (H-Q) ratio, quadriceps and hamstring strength, AT, and dynamic postural stability. If a significant difference was found, a Bonferroni post hoc test was used to determine the differences in all variables, between the time points and between the groups. The Shapiro-Wilk test was used to determine whether a continuous variable followed a normal distribution. Statistical analysis was performed using the SPSS software version 21.0 , and a value of No significant differences were found in age, height, weight, or body mass index (BMI) between the two groups. The detailed demographic data are presented in p < 0.001), quadriceps, 114.9 ± 68.2, 178.5 ± 37.7, and 227.7 ± 41.8 (p < 0.001), at baseline, 6 months, and 1 year, respectively; home-based rehabilitation: hamstring, 54.8 ± 26.3, 89.7 ± 30.6, and 93.4 ± 23.7 (p < 0.001), quadriceps, 106.6 ± 53.8, 144.8 ± 64.8, and 179.9 ± 77.2 (p < 0.001), at baseline, 6 months, and 1 year, respectively; In both the supervised and home-based rehabilitation groups, the H-Q ratio did not significantly change from the preoperative period to postoperative 6 months and 1 year; however, the muscle strength of hamstring and quadriceps in operative knees gradually improved over 6 months and 1 year  at baseline, 6 months, and 1 year, respectively; nonoperative knees: 93.4 ± 24.7, 103.1 ± 18.6, and 115.7 ± 24.7 (p = 0.010), at baseline, 6 months, and 1 year, respectively; p < 0.001), at baseline, 6 months, and 1 year, respectively; In the supervised rehabilitation group, the hamstring muscle strength at 100° of knee flexion gradually improved over 6 months and 1 year in both operative and nonoperative knees ; quadriceps, 269.8 ± 60.5 vs. 225.5 ± 44.8 (p = 0.045); p = 0.035); nonoperative knees, 128.9 ± 29.9 vs. 100 ± 22.8 (p = 0.010); p > 0.05, The supervised rehabilitation group showed higher muscle strength of hamstring and quadriceps in nonoperative knees at 6 months , 98.9 ± 20.1 vs. 70.4 ± 19.2 (p = 0.001); nonoperative knees, 103.1 ± 18.6 vs. 67.1 ± 19.4 (p < 0.001), 115.7 ± 24.7 vs. 78.8 ± 17.7 (p < 0.001), at 6 months and 1 year, respectively; The hamstring strength at deep flexion was significantly higher in both operative and nonoperative knees at 6 months and 1 year in the supervised rehabilitation group , quadriceps, 47.7 ± 13, 40.8 ± 11, and 33.1 ± 10 (p = 0.002); nonoperative knees: hamstring, 55.4 ± 13, 49.2 ± 7, and 30.8 ± 11 (p < 0.001), quadriceps, 48.5 ± 12, 31.5 ± 6, and 26.2 ± 5 (p < 0.001), at baseline, 6 months, 1 year, respectively; p = 0.003); nonoperative knees: hamstring, 60.8 ± 24, 44.6 ± 11, and 43.8 ± 18 (p < 0.001), at baseline, 6 months, 1 year, respectively; p > 0.05, In the supervised rehabilitation group, the AT of the hamstring and quadriceps and OSI in both operative and nonoperative knees gradually improved over 6 months and 1 year ; nonoperative knees, 30.8 ± 11 vs. 43.8 ± 18 (p = 0.036); p > 0.05); however, the OSI of the nonoperative knees in the supervised rehabilitation group was significantly lower than that in the home-based group , 0.7 ± 0.2 vs. 2.0 ± 0.6 (p < 0.001); nonoperative knees, 0.9 ± 0.3 vs. 2.1 ± 0.6 (p = 0.000), 0.6 ± 0.2 vs. 1.9 ± 0.9 (p < 0.001), at 6 months and 1 year, respectively; No significant difference in the AT of the hamstring and quadriceps was found between the two groups at baseline and 6 months (p < 0.001); home-based rehabilitation group, 46.3 ± 17.4, 66.3 ± 16.8, 75.6 ± 15.1 (p < 0.001), at baseline, 6 months, and 1 year, respectively; p = 0.016; In both groups, the Lysholm score gradually improved over 6 months and 1 year (supervised rehabilitation group, 44.3 ± 8.7, 71.3 ± 7.0, and 87.3 ± 5.8 (The most important finding of this study was that the supervised rehabilitation group demonstrated greater muscle strength, better neuromuscular control, and higher patient-reported knee function than the home-based rehabilitation group. Except for one study which found some benefits from the home-based rehabilitation in flexion and extension ROM at postoperative 3 months , previouOne of the main outcome measures investigated in previous studies comparing supervised and home-based rehabilitation was quadriceps and hamstring strength ,10,12,34Despite the importance of neuromuscular control in maintaining muscle activation and dynamic knee joint stability , there iPrevious studies comparing the Lysholm scores between the supervised and home-based rehabilitation groups reported no significant difference between the two groups at 6 months and 1 year ,9,12. OuTo date, multiple studies have demonstrated contralateral muscle strength and neuromuscular control deficits after ACLR ,39,40,41Another possible explanation for the difference between the two groups may be derived from the interaction with physical therapists. A previous systematic review identified that self-confidence, optimism, self-motivation, stress, social support, and athletic self-identity are predictors that affect ACLR outcomes . ConstanThe present study had some limitations. First, as this was a nonrandomized, prospective observational study, a selection bias may have been present. Even though the demographic data showed no significant difference in sex, age, height, weight, and BMI between the two groups, patients with different psychological factors may have self-selected one rehabilitation program over the other. Second, preoperative OSI of nonoperative knees in the supervised rehabilitation group was significantly lower than that in the home-based group. Thus, the better neuromuscular control in the supervised rehabilitation group at 6 months and 1 year may be attributed to this baseline difference. Moreover, the home-based rehabilitation group had two more patients with concurrent meniscus tear, which might explain the worse neuromuscular control in this group . Third, Unlike previous studies that showed no significant difference between supervised and home-based rehabilitation, the current study suggests that supervision may lead to better outcomes in muscle strength, neuromuscular control, and patient-reported knee function up to 1 year after ACLR. The supervised rehabilitation group showed higher muscle strength of hamstring and quadriceps in nonoperative knees at 6 months and higher hamstring strength in operative and nonoperative knees at 1 year. The hamstring strength at deep flexion was also higher in both operative and nonoperative knees at 6 months and 1 year in the supervised rehabilitation group. Furthermore, the supervised rehabilitation group had better neuromuscular control assessed by AT and OSI in 6 months and 1 year. Lastly, the supervised rehabilitation group showed greater Lysholm score than the home-based rehabilitation group.Although patients undergoing home-based rehabilitation may achieve an acceptable level of knee function and stability over the year, given the potential deficits and increased risk of ACL injury in the contralateral limbs, sufficient education may be needed for the patients who do not choose to undergo supervised rehabilitation.