Correction: Establishing standardized transthoracic echocardiography reference ranges for mouse models: insights into the impact of anesthesia, sex, and age

Transthoracic echocardiography (TTE) is a vital non-invasive tool for diagnosing, monitoring, and guiding treatment in a wide range of heart conditions. TTE reference ranges from large healthy populations have become an increasingly common and valuable tool for diagnostic decision-making in clinical medicine 1; 2; 3; 4; 5.Mouse models play a critical role in cardiology research, offering valuable insights into the molecular mechanisms, genetics, and potential treatments for cardiovascular diseases 6; 7; 8. Their widespread use in cardiology stems from the ease of genetic manipulation in mice, their relatively short generation times and lifespans, and the similarity of many physiological and pathological processes between mice and humans 9.Advancements in echocardiographic equipment and transducers have enabled its successful adaptation from humans to rodents 10; 11. Hence, the establishment of TTE reference ranges healthy mice create a benchmark for normal cardiac physiology and will improve the understanding of normal values and variation across sex, age or strains. TTE reference ranges are not only fundamental to evaluate disease models by revealing how diseases or interventions impact the model, but they also benchmark animal welfare by signaling health issues when values deviate -which can aid in assessment of drug safety, revealing potential toxic or adverse effects from normal values. TTE reference ranges allow comparison across studies by standardizing data, ensuring consistency and reproducibility of research findings. In mouse models, they have the potential to improve data interpretation, animal welfare, and research reliability towards translatability 12; 13. However, reference ranges are not very common in mice, often highly specific in their nature for the mouse model and typically based on very small numbers of mice 14 15 16.Building on the recent ESC position paper outlining requirements for murine echocardiography 9, we expanded these foundations using data from the International Mouse Phenotyping Consortium (IMPC). From more than 15,000 conscious and anesthetized C57BL/6N wildtype mice, we derived standardized transthoracic echocardiography (TTE) reference ranges stratified by sex and age. This large-scale, quantitative analysis establishes a generalizable framework for defining normal cardiac morphology and function in mice and provides the first validated, multicenter resource for reproducible echocardiographic phenotyping in preclinical cardiovascular research.The International Mouse Phenotyping ConsortiumThe International Mouse Phenotyping Consortium (IMPC) represents a multi-institutional and collaborative research initiative encompassing twenty-four major research organizations and funding agencies, distributed globally 17. The IMPC seeks to generate and phenotype a knockout mouse line for every protein-coding gene in the orthologous mouse genome (www.mousephenotype.org) 18. Phenotyping is carried out under the uniform operating procedures detailed in IMPReSS (International Mouse Phenotyping Resource of Standardized Screens; www.mousephenotype.org/impress/index), which were developed and validated during the pilot programs EUMORPHIA and EUMODIC 19. In brief, bodyweights were taken shortly before transthoracic echocardiography. For anesthetized TTE recordings, the animal was placed in an induction chamber and anesthetized with 1.5-3% isoflurane or injected with tribromoethanol as an injectable anesthetic. While sedated, either as part of the TTE session or as a separate preparatory procedure, the animal undergoes hair removal of the chest. With the hair removed, the animal was placed on the imaging platform with its paws taped to ECG surface electrodes and a rectal probe inserted to monitor body temperature which was maintained at 36-37°C.IMPCDuring imaging, anesthesia was adjusted to maintain proper heart rate and keep the animal from waking up.For awake TTE examinations, the animal was firmly held by the nape (in the supine position)in the palm of one hand with the tail held tightly between the last two fingers.To facilitate ultrasound imaging, pre-warmed ultrasound gel was placed on the chest at the area of imaging, and transthoracic echocardiography recordings captured. For short-axis mode papillary muscles were used as an anatomic point of reference. TTE measurements were performed identically across all IMPC centers following a standardized protocol. The parasternal long-axis (PLAX) view served as the starting point for high-throughput TTE diagnostics in mice, with the transducer positioned vertically and the notch oriented toward the animal's head. The probe was then rotated approximately 35° counterclockwise to visualize the aortic root and apex, ensuring clear imaging of the left ventricular (LV) anterior and posterior walls. From this position, B-mode images of the LV in PLAX were recorded. Subsequently, the parasternal short-axis (PSAX) view was obtained by rotating the transducer approximately 90° clockwise to achieve a cross-sectional view of the LV, using the papillary muscles as an anatomic landmark. Both B-mode and M-mode images were acquired, with at least three M-mode recordings per view 21.For M-mode imaging, the acquisition axis was placed centrally through the LV at the level of the papillary muscles, following system guidelines. After imaging, animals were removed from the platform and allowed to recover on a heating pad. All TTE measurements were analyzed offline using VisualSonics analytical software, Vevolab (VisualSonics Inc.).Quantitative parameters included left ventricular internal diameter in systole (LVIDs) and diastole (LVIDd), as well as anterior and posterior wall thicknesses in systole and diastole (LVAWs, LVPWs, LVAWd, LVPWd). In mice, we refer to the parameters as LVIDd and LVIDs, which correspond to the clinical measurements LVEDD (left ventricular end-diastolic diameter) and LVESD (left ventricular end-systolic diameter) in patients, respectively.Papillary muscles were excluded from the traced LV boundaries 21. Derived parameters were calculated from two-dimensional M-mode images using the Bespoke methods were developed to assess TTE reference ranges and are independent of the methodologies implemented on the IMPC portal.Data and statistical analysis was conducted using R (version 4.2.2, R Core Team 2022 23 with figures and tables produced in ggplot2 and ggpubr. Variability of all the data was assessed by the metric coefficient of variation (COV). Visual methods (histograms and qqplots), as well as a formal statistical test (Shapiro-Wilks-test) were conducted to test whether the scores of the individual parameters were normally distributed. Data were separated by age, sex and anesthesia regime and histograms for each parameter were plotted. Reference ranges were calculated based on median, 2.5th percentile and 97.5th percentile. In addition, the mean, standard deviation, and parameter sample size were provided to reflect the distribution of each parameter. Based on this, the 95% confidence intervals can be calculated by mean±1.96*standard deviation for each parameter.To investigate the relative importance of different regressors on the outcome variable in linear models, we applied the R package "relaimpo" developed by Groemping 24 and calculated the relative importance (based on the metric 'lmg') of four predictors, namely anesthesia (conscious, isoflurane and tribromoethanol), sex (males and females), body weight and age (EA and LA) in all 15,765 mice. Adjusted R 2 depicted the total proportion of variance explained by the model with all four predictors and the relative proportion of contribution for each predictor was shown by relative (%) of adjusted R 2 .To investigate the effect of anesthesia on the different parameters in EA mice, we calculated a one-way Analysis of Variance (ANOVA) with planned comparisons of "Conscious versus Isoflurane" and "Conscious versus Tribromoethanol", separated by sex whereas "Isoflurane versus Tribromoethanol" was not tested. These planned comparisons were used to compare conscious vs unconscious states. The null hypothesis tested whether the two datasets originate from distributions with the same mean. P-values and F-values with degrees of freedom were calculated.The effects of sex (female vs male) and age (EA vs LA) were compared using the identical statistical analyses. In each case a simple two-tailed t-test was performed and the Cohen's d effect size calculated from the "effsize" package (R library). Due to the central limit theorem (CLT) 25, the large sample sizes allowed parametric statistical testing of these effects. The biological relevance of large samples may be overstated, which is why we also calculated the effect sizes in order to be able to estimate this factor. 1). In addition, the body weight (BW) of each mouse is weighed before the TTE measurement, and the body temperature of anesthetized mice is measured with a rectal probe to monitor physiological functions (Supplemental Figure 1 with histograms, mean ±SD, median and 95% reference ranges, separated by sex). LVAWd and LVAWs were inconsistently collected in the IMPC and not further processed in this analysis.In multi-center, large-scale, high-throughput programs such as the IMPC, variability in the measured values was to be expected. However, the extent of this variability dictates the sensitivity and robustness of each parameter. Variability testing was performed on all DR 21 TTE data from the IMPC, independently of anesthetic agent in this analysis. For each sex, individual TTE parameters were tested for variability in EA and LA populations. The coefficient of variation (COV) (100*standard deviation/mean) assumes a parametric distribution and normalizes the variability to the most typical score (mean) but is sensitive to outliers 26. Based on this analysis, exclusion criteria were defined as any parameter with ≥30 for COV, based upon Eurachem guidelines 27. Figure 1 shows that the retained parameters are all clustered closely together, however the excluded parameter shows a wide range of variability.Specifically, one TTE-parameter, respiration rate, exceeded the variability criteria in both sexes (male and female) and LA age but not in EA and was excluded from further analysis (Figure 1). The variability threshold was partially exceeded for LVIDs in females of EA age, however, in females of LA and males of both ages (EA and LA) the threshold was not exceeded and LVIDs was retained, whereas LVAWd and LVAWs values are exclusively shown in supplementals.The remaining 9 TTE-parameters (CO, EF, LVIDd, LVIDs, FS, HR, LVPWd, LVPWs and SV) consistently presented with low variability across the whole IMPC dataset thereby giving high confidence to establish robust, generalizable reference ranges for EA and LA populations on the C57BL/6N inbred genetic background.The distribution of data was assessed via histograms for the nine selected TTE parameters stratified by sex, age, and anesthetic regime (Figure 2). Under tribromoethanol anesthesia, data points were only captured in EA from five (FS, HR, LVIDd, LVIDs and LVPWd) of the nine TTE parameters.This visual representation of the frequency of occurrence per value in the data was useful for revealing conformity to and deviations from a normal distribution for each parameter.Visual inspection of the histograms showed that the data appeared practically normal in EA for parameters CO, EF, LVIDd, LVIDs, FS, LVPWd, LVPWs and SV and modestly skewed for HR in conscious mice but not under isoflurane anesthesia. Under tribromoethanol anesthesia, FS, HR, LVIDd, LVIDs and LVPWd appeared practically normally distributed whereas HR was modestly skewed. To assess normality mathematically, we applied the Shapiro-Wilk test which revealed statistically significant deviation from a normal distribution for some, but not all, TTE parameters. 3.If there are several predictors, the question naturally arises as to which predictor is more important or useful for predicting the outcome variable. For correlated predictors, the standardized coefficients may not indicate which predictor is more important. Here, the calculation of the 'relative importance' of the predictors 24 is applied for the predictors sex (males and females), anesthesia (conscious, isoflurane and tribromoethanol), age (EA and LA) and body weight across the grand total of 15,765 mice. Anesthesia showed highest proportion of contribution with a range of 32 (SV) to 99% (EF and FS) relative % of adjusted R 2 for all 9 TTE parameters wherein sex, weight and age covered rather low proportions of variance (Table 3).Interestingly, male, and data showed distributions by visual inspection (Figure 2). To test the hypothesis that there is between each sex, a simple two-tailed t-test was performed independently for each anesthetic regime and age and Cohen's d was calculated as an effect size (Supplemental Figure and a and stratified by In the EA for for we of a However, for all parameters the Cohen's d value revealed small to effect the that the large sizes be the biological between the sexes for parameters. In the LA for most of the parameters and for we of a In the Cohen's d value revealed small to effect sizes for the of however, large effect sizes were in EF, LVIDd, LVIDs and in mice under isoflurane and for Here, with a relatively size than EA the biological between the sexes in the LA are both significant and of or large effect sizes for some, TTE parameters. there is a between investigate the effect of different anesthetic on cardiac function and TTE conscious data stratified by sex and age are for comparison with of isoflurane or tribromoethanol data (Figure data are placed for ease of Figure shows distribution for isoflurane and tribromoethanol by EA (Figure to and LA (Figure to data were for tribromoethanol anesthesia in LA the physiological benchmark of highest heart rate in conscious mice compared to anesthetized animals was in EA and LA populations (Figure and To assess the between EA anesthetic we tested conscious versus isoflurane and conscious versus tribromoethanol by a one-way with planned and highly significant between for LVPWd in EA females conscious versus (Table 2). These data in TTE parameters that can be to the anesthetic is to establish reference ranges by or and by anesthetic or between these two age independent of sex and the linear of and each TTE we calculated the coefficient R stratified by sex, age and anesthesia. of the linear between TTE and were defined based on guidelines and very data are placed for ease of by the IMPC are all of one used inbred genetic To test the of the reference ranges C57BL/6N inbred mice, we used data from inbred mouse from TTE data from a collaborative the with inbred of and four inbred of the dataset was included using wildtype animals from studies conducted at the Mouse data is upon we used TTE reference ranges from the ESC position paper on the echocardiographic acquisition and analysis of left ventricular function in healthy mice each we presented the data by sex and with the 95% reference range calculated for conscious and studies at or isoflurane anesthetized mice and ESC In the ESC we used mean to 95% reference stratified by HR or HR and compared with the 95% reference range calculated stratified to the of the ESC Figure shows the data from the collaborative with the reference ranges by sex whereas Figure data from the Figure from and Figure for studies conducted at the Mouse and for all TTE most values reference is a of outliers that of the reference ranges which is to be with of small size and between inbred mouse strains. Figure 9 shows the ESC reference ranges with the reference ranges calculated for and for all TTE most values reference This visual representation of the reference ranges per value in the data was useful for revealing conformity deviations from the reference ranges calculated for each parameter. In both HR or HR there are that the reference ranges which is to be with the of the ESC multicenter of small mouse numbers and between inbred mouse echocardiography (TTE) is the imaging most used in cardiology While TTE is an important role in the of cardiovascular diseases valuable and quantitative insights into and processes The of and the an in cardiology guidelines for the assessment and of echocardiography. 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To this we a of the data analysis and with an assessment of the variability of TTE parameters in the nine TTE parameters that were highly and low for the respiration rate with high variability was excluded but LVAWd and LVAWs are the These parameters were only collected by a of centers and not included in the but the values are in in the the nature of the IMPC there are several the the sex, age, body and impact on TTE is for ensuring reference values. 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This variability in data acquisition and across IMPC as tribromoethanol was used in a of and during of data the dataset for this anesthetic is and to mice the sample size for reference range and the IMPC its this dataset provides that the of anesthesia echocardiographic thereby one of the of this These the of anesthetic and in echocardiography and to of anesthesia as a critical variable cardiac assessment in preclinical research.The reference ranges are to the and and are not for imaging such as or anesthetic such as of from mouse models to often arises from the of standardized procedures and reference to and assessment of cardiac function in mice. The reference ranges this by for cardiac morphology and function based on more than 15,000 C57BL/6N mice. These values can be used to typical ranges for of a sex and age and as a tool for preclinical echocardiographic this on mice to establish a physiological the standardized framework provides the for in disease or these reference ranges a critical resource for echocardiographic from or genetic a and to that have the of anesthesia, sex, and age on murine cardiac the a multicenter reference framework derived from more than 15,000 wildtype C57BL/6N mice. These data a physiological from animals on a standard and establish reference ranges stratified by sex, age, and body anesthesia as the of variability in echocardiographic effects were but across independent datasets and the robustness and of these reference the research with a standardized for and cardiac phenotyping in preclinical isoflurane and tribromoethanol anesthesia. Reference ranges reference ranges of the ESC