P3, a three unit phrase  repeated with an inter-song interval (ISI) of 170–194 s; P2, a phrase consisting of only units II & III repeated every 84–96 s; and P1 with a phrase consisting of only unit II repeated every 45–49 s. The different ISI values were approximate multiples of each other within a season. When comparing data from each season, across seasons, the ISI value for each song increased significantly through time , at 0.30 s/Year (95%CI 0.217–0.383), 0.8 s/Year (95%CI 0.655–1.025) and 1.73 s/Year (95%CI 1.264–2.196) for the P1, P2 and P3 songs respectively. The proportions of each song-type averaged at 21.5, 24.2 and 56% for P1, P2 and P3 occurrence respectively and these ratios could vary by up to ± 8% (95% CI) amongst years. On some occasions animals changed the P3 ISI to be significantly shorter (120–160 s) or longer (220–280 s). Hybrid song patterns occurred where animals combined multiple phrase types into a repeated song. In recent years whales introduced further complexity by splitting song units. This variability of song-type and proportions implies abundance measure for this whale sub population based on song detection needs to factor in trends in song variability to make data comparable between seasons. Further, such variability in song production by a sub population of pygmy blue whales raises questions as to the stability of the song types that are used to delineate populations. The high level of song variability may be driven by an increasing number of background whale callers creating ‘noise’ and so forcing animals to alter song in order to ‘stand out’ amongst the crowd.Sea noise collected over 2003 to 2017 from the Perth Canyon, Western Australia was analysed for variation in the South Eastern Indian Ocean pygmy blue whale song structure. The primary song-types were:  Baleen whales commonly use low frequency, high intensity sounds to communicate over large distances –3. The pBalaenoptera musculus brevicauda) or termed here, the South Eastern Indian Ocean (SEIO) pygmy blue whale, a part of which traverses the Western Australia coast each year [Population specific vocalisations are useful in the monitoring and management of cryptic or offshore species such as the Australian pygmy blue whale population  of the vocal signal at the noise recorders of the passive acoustic observatory. Tracking analysis could only be carried out for 2010, 2011 and 2012 where data sets had been collected successfully from four recorders within the observatory.Localisation of pygmy blue whale vocalisations was based on the type II song unit as defined in . SpectroWhale localisation results were filtered to only include individual locations with errors of less than 0.5 km (an error ellipse was derived for each location). The filtered results were sorted into individual days and viewed one day at a time. Spectrograms of whale sounds for each localisation event were viewed, checked manually and classified based on song structure and song repetition interval. Each localised detection was compared with the previous localisation event and based on the signal characteristics from the spectrogram, as well as the location of the detected vocalisation, it was manually determined whether it was likely to be the same vocalising animal. It was assumed that the average swimming speed of a pygmy blue whale was less than 20 km per hour when comparing locations across longer time scales. A track consistent with a single whale combined with a common ISI amongst songs was used as a criterion for the likelihood of detections being from the same animal. Whale tracks were numbered chronologically and where possible were carried over from the previous sample. Where a consistent signal was lost for more than one sample (greater than 1800 s), or the location of the source did not fit the criterion above, the successive vocalisation was classified as a new vocal animal. The start and end time of each track, length of time calling, song-type, track direction and distance travelled was recorded for each whale track. While each song sequence was unique for an individual whale, many of the sequences were potentially produced by the same whale due to normal breaks in calling. Thus, the manual analysis considers events that occurred independently in time for an individual whale but which may have been replicated at a later time by the same individual.Quantitative analysis of population wide variability in song structure was carried out using the statistical program R . RepeateAcross the five years of manually analysed samples (2010 to 2016), 2,627 song events from SEIO pygmy blue whales were analysed and identified to song structure. No calls from pygmy blue whales of other Indian Ocean stocks were detected or noted. The highest number of song events analysed were in 2010 and 2011 with 509 and 598 records respectively . Peaks iP3) consists of a phrase of three units (sounds) repeated in a sequence with approximately 180–200 s  between the start of one phrase and the start of the next  or two  unit phrase and repeating the shortened phrase at a reduced ISI compared to that of the typical song. Combinations of different phrase structures were also identified and termed hybrid song-types. Hybrid song types were named based on the base phrase structure. In 2016 another variation was observed with different units of the song pulsed or broken. Temporal variations to the P3 song sequence in the form of long or short repetition ISI times were also observed. Details of the phrase and song variants are summarised in A number of variations to the pygmy blue whale P3 song consists of a repeated phrase with three temporal variants: a) the normal variant; b) a variant with a longer repetition interval than the typical song (P3L); and c) a variant with a shorter than normal ISI (P3S). The long variation of the P3 song followed the same basic structure but with 220 to 280 s intervals between the start of one sequence and the start of the next. The extra length of ISI in the song appeared in the length of time between the end of the previous phrase’s type III unit and the beginning of the type I unit of the next phrase sequence. The time between the successive units within the sequence remained consistent between P3L and P3 phrase. The P3L variation was identified on 11 (0.4%) occasions making it one of the least common song variants. P3L was only recorded in 2010 and 2011.The complete P3S variation followed the same structure as the P3 phrase but had only a 120 to 160 s interval between the start of one phrase sequence and the next. The phrase appears to be shortened in the type I unit and the time between the type I and type II units. The time between the end of the third and start of the first unit of the next sequence did not vary greatly from the repeated P3 phrase structure. The P3S song was uncommon and only identified 67 (2.6%) times, occurring in the 2010, 2011, 2012 and 2014 datasets. The production of P3S was limited to March, April and May, months with the highest number of song events analysed overall.The P1) was the simplest variation with the shortest ISI with the first and last units of the P3 phrase dropped leaving only the type II unit repeated in a sequence with ~ 50 s intervals. The P1 phrase was one of the least common variations in the manually processed data, identified on 91 (4.3%) occasions. Comparisons across the sample years revealed that the number of P1 song events appeared to increase between 2009 and 2011. In the manual data, after 2011 the number of whales producing the P1 song remained relatively constant. Analysis of song structure by month showed the highest number of P1 song events in March, April and May.The one unit phrase variation , P1 was P2) was the most common variant of the P3 phrase in the manual analysis. The subsequent P2 song sequence is repeated at ~ 80 to 100 s ISI. There were 1,220 P2 song events extracted from the manually analysed data, which represented 57.7% of sampled song events. The proportion of the P2 song events remained stable across sample years with peaks in 2010 and 2014 coinciding with peaks in the number of song events recorded.The two unit phrase  based on the provided song catalogue. The greatest disagreement between observers was in identifying temporal variations of the P3 phrase type, P3S where the P3 phrase was repeated with an unusually short ISI value and P3L where the ISI between consecutive phrases was unusually long. When temporal variations were ignored, and observers were asked to classify signals into one of the three phrase structures or two song sequence variations, the agreement between observers was almost perfect . Based on the results of the Fleiss Kappa Analysis, it can be said that the classifications assigned to phrase and song variations are consistent and appropriate.The appropriateness of song classification was tested with a Fleiss unweighted Kappa analysis on the classification of 22 spectrograms by five untrained observers. The analysis found substantial agreement between observers  producing this song variation. The P3A variant appeared to be increasing over years, with more than twice the number of occurrences in 2016 (189), compared with the other sample years combined (74). Instances of mixed P3A and P3 song sequences were observed but were rare.Aside from variations in the ern P3A,  is a comP2A song pattern (P2A) was first found in the 2014 data set and is a hybridisation combining the P2 (type II & III), followed by the P1 (type II only) phrase structure. The song repetition interval conforms to that of the P2 and P1 phrases with the time between type II units approximately 100 and 50 s respectively, resulting in a total song length of approximately 150 s. The P2A song variation is slightly less common than the P3A variant with 41 occurrences (1.6%) of this song variant over the sample years. Where followed in time the P2A song was consistently repeated (P2 & P1 repetitions).The  pattern , P2A wasP3B song pattern (P3B) was the rarest song variation detected thus far, only found in the 2010 and 2011 data sets. Along with the P1 phrase, the P3B song structure is the only other variant with the absent type III unit. The song consists of a complete P3 phrase followed by a two unit sequence consisting of only the type I and type II unit. The length of the song is approximately 280 s, with the P3 section lasting approximately 180 s and the following two units approximately 100 s. There have been very few occurrences of the P3B song variant (< 0.2%).The  pattern , P3B wasP3 and hybrid song events appeared to be increasing in the 2016 data set. The greatest diversity in song structure was found in the years with the largest sample sizes  are shown on P1, P2 and P3 unit songs were derived. The same analysis is shown for 2003–2017 on P2 and P1, ISI (P2/P1) was consistent at 1.932 (95%CI 1.9113–1.9527) while the ratio between the P3 and P2 ISI (P3/P2) was statistically the same, at 1.970 (95%CI 1.9401–1.9999). Thus, the ISI for the three songs  were multiples of each other, each ~ 1.951 times longer than the previous song-type according to the statistics. Given that the starting resolution was 0.1 s in the ISI-curve analysis technique, then the ISI of P2 is approximately twice the length of P1 while P3 is approximately twice that of P2.When a season’s data set was assembled and gridded using the time-level difference analysis, the technique discriminated ISI, as given by the peaks of time between repeat type II song units, for the three predominant pygmy blue whale song-types and multiples of the ISI of each. An example of the 2015 season’s analysis of the ISI, as measured by time differences between consecutive type II units, was shown on P1, P2 and P3 types were present (hybrid song-types were ignored) and correcting for additions of multiples of shorter song-types. The trends calculated across time are presented on P1 (type I) only song present on average across the seasons analysed 21.5% of the time , the P2 song (type I & II) present 24.2%  and the P3 song  present 56.0% of the time . Note the earlier years had relatively short sea noise sample lengths compared to the P2 and P3 song length plus fewer whales singing, so early years have been excluded from calculations of statistics of the proportions of P2 and P3 songs.To calculate the relative proportions of each song-type from the ISI-curve using equations 1–4  requiredP3), but with a further two common variations (P1 and P2) in which not all units are repeated per phrase, sections where phrases combining different combinations of units are repeated and recently, the alteration of units by splitting them into two sections.The song structure of SEIO pygmy blue whale has been shown to be variable and changing across time, while still retaining its uniqueness when compared with the song types of other Southern Hemisphere pygmy blue whale stocks. The fundamental song structure is three units repeated in a phrase  or where a consecutive song had a different arrangement of units to the previous phrase-type (hybrids); 3) the duration of a phrase cycle; and 4) the interval between consecutive phrases within a song, which increased yearly. In the context of this discussion, the P1, P2 and P3 phrase type refer to singular sequences of these phrases while the P1, P2 and P3 song variations refer to repeated sequences of the respective phrase types. Hybrid song types are those which alternate between two different phrase structures. There are a number of adaptive processes by which song changes can occur and a number of factors that may be driving these changes. Gradual changes to song structure over a long period of time may be attributed to slow evolutionary processes such as genetic or cultural drift [The observed changes in song structure were in the form of variations to: 1) the structure of units with broken or split units observed in recent years; 2) variability in phrase composition where different units were omitted , with terved by  relate tChanges to song repetition interval may result from an increase in ambient noise, primarily here from the chorus of other pygmy blue whales singing in the same area. In the most recent data sets the largest obstacle to identifying the song structure of vocalising animals in the manual analysis was other vocalising animals which flooded samples with pygmy blue whale signals. As population numbers increase . The ISI-curve analysis method was largely independent of biases, although: 1) the curves produced for each song-type will be slightly smeared due to different ISI values between song events; and 2) was sensitive to sample length pre 2009 (samples < 300 s). For the samples of shorter length the proportions of the longer song-types which could be detected in that sample length dropped significantly, thus lowering curve peak values and so increasing errors in determining curve peak values and baseline noise levels (since the peak level may have been close to that of background noise or the background noise was sloped downwards for longer ISI times as it approached the sample length). But, the trends in the ISI-curve analysis technique were largely consistent across seasons which were treated independently, the expected proportion of third multiples of the P1 ISI were identified correctly by the technique, and the equations used gave the correct ratio of song proportions within < 1% when comparing derived proportions from set proportions using a simulator  will not directly equate to the number of animals present at the time. The ISI-curve analysis shows that while across the 13 years of sampling available the relative proportions of each song-type does not change in any systematic way, suggesting phrase production per unit time may be comparable for relative abundance, the proportions have considerable variation in ranges amongst years of up to ± 8% for the 95% CI of any song-type  needs to be made. One technique utilised by the authors is to split a sea noise sample into time windows less than the P2, ISI , count the number of type II units within each window and use the maximum value in any window as an estimate of the number of vocalising whales. Using the manual ISI analysis song-type proportions suggest this is correct for 96% of songs, while using the ISI-curve analysis technique for proportions of song-types averaged across seasons suggests this is correct for 80% of songs.Either analysis technique indicates that song occurrence is not a direct reflection of calling whale abundance. The presence of shorter ISI values than the ong-type . Thus, cThe large degree of variability in ISI values demonstrates that differences exist in timing and phrase production even within similar song structures. This indicates the possibility that variability in phrase structure may be attributed to individual callers and reflects important social information such as identity, sex or size , 56. ThePatterns in the timing of song production and as seen in the ISI analysis are reminiscent of the phonology of speech and song in other species. Phonology, or the arrangement of sounds within a language, allows for the interpretation of different song elements based on a hierarchical context , 62. TheFor a number of cetaceans, it is nearly impossible to separate genetic linkages on the maternal side and cultural processes as the cause of particular behaviours that are passed from mother to calf , 14, 32.P1 and P2 variants of the typical, P3 phrase song. Increased complexity within songs is thought to be a reflective of cognitive fitness, which may be a favourable trait for sexual selection [Culture is recognised as a driver of behaviour in cetacean species. Culture relies on the social and familial networks of a species and in turn is a driver of social behaviours of a species such as the production of song , 67–69. election , 70, 71.election , 30, 41.election , 26, 58.election , 58, 68.election , 72, 73.P3, SEIO pygmy blue whale phrase-type is the least intense and thus in high levels of ambient noise is the hardest to detect [Whilst not energetically costly, singing represents a cost to the animals in terms of the time involved, as it is assumed to preclude other behaviours such as feeding , 75. As o detect , 47, 76.o detect , 76, 77.o detect , 78. Thuo detect , 70, 79 o detect , 53. Foro detect . Such a o detect , 78. FroOne of the most significant findings of this study is that variability exists in the characteristic song of the SEIO pygmy blue whale subpopulation. Song structure has previously been used as a diagnostic tool to separate populations of pygmy blue whale . Variability in what were previously considered to be static signals raises questions as to the validity of song structure as a diagnostic for sub populations. Consequently, it is recommended that a global study on variability within and between the vocalisations of all sub populations of pygmy blue whale be conducted.Through long term passive acoustic monitoring we found three distinct variations to the South Eastern Indian Ocean pygmy blue whale phrase structure in the Perth Canyon, Western Australia, and a further three song pattern variations. Within these phrase structures there exist variations in the inter-song interval resulting in two further temporal variations on the three unit phrase structure. Further, the most recent data sets include variations to the units where they are split, or ‘broken’, within the existing song structures, which adds an additional level of complexity. The mechanisms behind the increase in song diversity are unclear though the rapid appearance of new phrase variants that represent progressive changes to the original phrase structure is consistent with cultural evolution. Such rapid change, with new variants appearing within a migratory season, indicate that the levels of variability cannot be attributed to genetic processes. Variability in song and phrase structure is not prolific throughout the population with all the variations present within one year. This sets pygmy blue whales apart from the well-studied humpback whales where changes in song structure are generally propagated through the entire population and supersede earlier song-types. This raises the question as to whether physical environmental conditions may influence song production as has been documented for other baleen whale populations . Peaks iS1 File(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(TXT)Click here for additional data file.


Myocardial computed tomography perfusion (CTP) allows the assessment of the functional relevance of coronary artery stenosis. This study investigates to what extent the contour sharpness of sequences acquired by dynamic myocardial CTP is influenced by the following noise reduction methods: temporal averaging and adaptive iterative dose reduction 3D (AIDR 3D).Dynamic myocardial CT perfusion was conducted in 29 patients at a dose level of 9.5±2.0 mSv and was reconstructed with both filtered back projection (FBP) and strong levels of AIDR 3D. Temporal averaging to reduce noise was performed as a post-processing step by combining two, three, four, six and eight original consecutive 3D datasets. We evaluated the contour sharpness at four distinct edges of the left-ventricular myocardium based on two different approaches: the distance between 25% and 75% of the maximal grey value (d) and the slope in the contour (m).Iterative reconstruction reduced contour sharpness: both measures of contour sharpness performed better for FBP than for AIDR 3D . Increasing levels of temporal averaging degraded contour sharpness. When FBP reconstruction was applied, contour sharpness was best without temporal averaging  and poorest for the strongest levels of temporal averaging .The use of both temporal averaging and iterative reconstruction degrades objective contour sharpness parameters of dynamic myocardial CTP. Thus, further advances in image processing are needed to optimise contour sharpness of 4D myocardial CTP. While non-invasive computed tomography coronary angiography (CTA) is highly accurate in diagnosing coronary artery stenosis , 2 compaMyocardial CT perfusion (CTP) is a promising approach to detect myocardial ischaemia as a predictor of the functional relevance of a coronary stenosis diagnosed during CTA , 6. CompDespite the huge potential of 4D CTP there are important challenges that have to be addressed. Multiple acquisitions lead to potentially high radiation exposure , while lTemporal averaging of multiple datasets and iterative reconstruction techniques as means of noise reduction reduce beam hardening artefacts but may lead to poorer image sharpness from blurred edges. Two feasibility studies of temporal averaging have been published so far , 18 and The purpose of this study was to objectively analyse the effects of temporal averaging and iterative reconstruction on contour sharpness in myocardial 4D CTP.This is the contour sharpness sub-study of the prospective 4D CT perfusion pilot study . A detaiTo lower the heart rate and variability, patients with a heart rate of >60 bpm received beta blockers before the CT examination . Another three patients were administered 5 mg (1 patient) or 10 mg (2 patients) of ivabradin . Immediately before the CT scan all patients received sublingual nitroglycerine (1.2 mg). The patients were asked in advance not to ingest any xanthine-containing food and beverages for at least 12 hours before adenosine injection. The administration of xanthine-containing medicine (theophylline) was avoided for a 24-hour interval before the adenosine injection due to possible antagonistic effects of xanthine, which could degrade the effect of adenosine.Coronary artery calcium scoring (CACS) was performed without injection of contrast agent at 100 kV and 140 mA, covering 14 cm of the heart in z-direction. The CTA was planned based on the position of the coronary arteries on CACS plus 15 mm in each direction of the z-axis. The tube voltage was 120 kV for patients with a body mass index (BMI) < 36 und 135 kV for BMI ≥ 36. See the main manuscript for further details. For both, the CTA and CTP, the patients received non-ionic contrast agent  at a flow rate of 7 ml/s through an IV line in the right antecubital vein. For CTA and CTP, the patients were administered 58 ml and 42 ml of contrast agent, respectively, followed by a saline flush of 80 ml. CTP always followed the CTA and was initiated after continuous infusion of adenosine for 3 min.The CTP coverage in z-direction was calculated from the extent of the myocardium in z-direction on the CACS scan plus 10 mm in each direction in order to not miss parts of the myocardium during dynamic CTP, or plus 15 mm in the case of a large positional deviation in z-direction between CACS before CTA and CTP. To determine the CTP start point after contrast agent injection, the arterial input function of the CTA SureStart was used. In each case, scanning was performed at 70–80% of the RR interval. The dynamic CTP with one acquisition every heart beat was followed by three single late phases 10, 20 and 35 s after the dynamic scan. In order to cover 20 heart beats, the total scan time of the dynamic phase was adjusted to the individual heart rate. CTP was initiated after continuous adenosine infusion for 3 min. Images were acquired with a gantry rotation time of 350 ms, and we used a tube voltage of 100 kV and tube current of 140 mA for patients with BMI ≤ 30 and 200 mA for BMI > 30. The vasodilatory effective adenosine was adapted to the body weight (140 μg/kg/min) and administered continuously over a maximum period of 6 min by an IV infusion through the left cubital vein using a perfusion system.We performed the CT image reconstruction by using an imaging matrix of 512 x 512 pixels covering a field of view (FOV) of 180 mm in axial direction. The reconstructions were performed in 5% intervals of the available RR interval in all patients, and additionally of the visually chosen best phase. Cardiac volumes were reconstructed using two methods: filtered back projection (FBP) and Adaptive Iterative Dose Reduction 3D (AIDR-3D) . For thei centred around the reference time point t. The averaging level N  designates the number of additions during the temporal averaging. The averaged volumes are the arithmetic means of the input volumes in the time domain. The intensity I of a voxel at position x and time t is obtained from N equally weighted consecutive voxel intensities according to the formulaAssuming little cardiac motion and deformation, we applied temporal averaging of several consecutive 3D datasets for noise reduction. The cardiac phase detection algorithm  was usedWe combined the different original 4D datasets from consecutive heart beats into one new 3D dataset to test the different combination levels. This resulted in six temporally averaged 3D volumes with the different levels of temporal averaging for each of the 29 patients and a total of 174 volumetric averaged datasets for all patients. This process was performed before reading as a post-processing step. See the main manuscript for further details. Only the FBP 4D CTP dataset was taken into account for the temporal averaging analysis in order not to overlay AIDR3D-instrinsic spatially diffusive smoothing with the averaging along the time dimension, which would result in overly blurred images. For the same reason, the comparison between FBP and AIDR 3D was performed without applying temporal averaging.https://github.com/CardiacImagingCharite/CardiacPerfusion). For the analysis of the contour sharpness, a straight line was placed above the heart, displayed as 4-chamber view, to connect the myocardium of the left and the right ventricle, taking care to avoid the inclusion of any perfusion defects or blooming artefacts into the measurement. Thus, we integrated 4 different representative edge localisations of the myocardium between: 1) the right ventricle and the septal myocardium, 2) the septal myocardium and left ventricle, 3) the left ventricle and lateral left ventricular myocardium and 4) the lateral left ventricular myocardium and surrounding epicardial tissue  and high value of the slope in the contour  if not mentioned otherwise. We performed the statistical analysis using SPSS version 20. The analyses were performed as intraindividual comparisons. First, we used the Shapiro-Wilk test to check normal distribution. If the values were normally distributed, we used ANOVA as overall test for repeated measurements for the comparison of the different temporal averaging levels, and the t-test for dependent values was used for the respective single comparisons. If normal distribution was refused, we used the Friedman test as overall test and the Wilcoxon signed-rank test for the single comparisons. Since we included only two different reconstructions into this analysis (FBP and AIDR 3D), overall testing was not necessary for the comparison of the reconstructions. The normally used p value of <0.05 was adapted here yielding <0.003 according to Bonferroni corrections for the fifteen possible comparisons of the five temporal averaging levels: 1–2, 1–3, 1–4, 1–6, 1–8, 2–3, 2–4, 2–6, 2–8, 3–4, 3–6, 3–8, 4–6, 4–8, 6–8. For the comparison of the two reconstructions, a p value of <0.05 was considered to indicate statistically significant differences.The 29 included patients had a mean age of 64±11 years, and there was a male-to-female ratio of 9:1 . More thContour sharpness parameters were slightly inferior for AIDR 3D strong versus FBP at all 4 edges of the myocardium, as demonstrated by both methods: (d) higher values for the distance between 25% and 75% of the maximal grey value, and (m) lower values for the slope between 25% and 75% Figs  Tables. In detail, the comparison of the slope between the point of 25% and 75% (m) showed higher values for FBP than for strong levels of AIDR 3D . We used two different parameters for the quantitative evaluation of the contour sharpness: the distance between 25% and 75% of the maximal grey value (d) and the slope in the contour (m).Comparisons were performed between FBP and strong levels of AIDR 3D , as well as between the different levels of temporal averaging  for FBP reconstruction. Measurements were performed at 4 different edges of the myocardium.Contour sharpness parameters were slightly poorer for AIDR 3D strong versus FBP at all 4 edges of the myocardium and also for higher levels of temporal averaging, as demonstrated by lower values for the slope . The distance between 25% and 75% of the maximal grey value tended to be slightly increased, but the difference was not significant (p>0.059).Higher levels of temporal averaging led to slightly poorer values for both contour sharpness parameters, demonstrated by higher values for the distance between 25% and 75% of the maximal grey value and lower values for the slope Figs  Tables.Thus, with increasing combination levels, the slope in the contour was poorer at all 4 edges . The distance between 25% and 75% showed the same tendency, but without statistical significance . The best (lowest) values were achieved without temporal averaging, the maximal temporal averaging levels showed the highest values , indicating the strongest reduction of contour sharpness. This goes in line with the visual impression of a smooth edge which was characterised by less variation in HU at a single edge localisation with increasing levels of temporal averaging AIDR 3D .Both noise reduction methods investigated here, iterative reconstruction and temporal averaging, degraded contour sharpness. In our analysis, both objective contour sharpness parameters deteriorated for strong levels of AIDR 3D compared to FBP and for increasing levels of temporal averaging. The use of two different but complementary parameters for contour sharpness analysis is in agreement with established approaches that have been optimised for the analysis of myocardial contours . These qSince poorer contour sharpness leads to blurring and thus reduces the evaluability of the affected structures, contour sharpness is supposed to be a very valuable quantitative parameter of image quality. Morphological delineation is needed for generating precise perfusion maps. Because accurate and sharp contours aid the segmentation, contour sharpness is an important quantitative parameter for dynamic CT perfusion imaging.Published data to prove the feasibility of 4D CTP of the entire myocardium are still limited. A study conducted in 32 Asian patients  shows a In general, the diagnostic accuracy of dynamic myocardial CTP can further be improved if the results of the coronary CTA are additionally considered. CTA is highly accurate in detecting significant coronary stenosis compared with ICA as the reference standard , 2. SincAn important advantage of myocardial CTP instead of different functional tests  is the option to perform the examination in the same session as the coronary CTA which enables immediate functional information and facilitates the implementation in clinical routine. Special benefits of dynamic versus static CTP include the possibility of calculating the absolute myocardial blood flow and acquiring time-attenuation curves .A major limitation of our study is the small sample size of only 29 patients. Nevertheless, it is one of the largest whole-heart CT myocardial perfusion dataset studies to date. In addition, to statistically deal with the relatively small patient collective, we performed intraindividual comparisons and adapted the p value according to Bonferroni correction. In this analysis, we did not use post-processing motion correction, which would have improved the contour sharpness, especially for higher levels of temporal averaging. Even though we minimised motion during scans by very careful patient preparation, it is not possible to completely eliminate cardiac motion. In addition, in this analysis, we only used two objective parameters, which cannot cover all aspects of image quality. Therefore, we evaluated the results of this sub-study taking the additional objective and subjective image quality parameters discussed in the main manuscript into account. The comparison between CTA/CTP  and CTP/MRI (15 patients) is also published within the main study. The results of the main study and this sub-study need to be seen as a whole, since they are complementary. Our scanning parameters were relatively low at 103±8 kV and 148±21 mA, which is a reasonable approach in clinical practice in order to comply with the ALARA radiation safety principle. Such low scanning parameters have an influence on the performance and image quality of AIDR 3D and temporal averaging in general and on contour sharpness in particular. There are different approaches to deal with multiple datasets , 24. It In conclusion, our results show that AIDR 3D and temporal averaging slightly reduce the contour sharpness of the myocardium. Thus, further developments in image processing are needed to optimise contour sharpness of 4D myocardial CTP.S1 Appendix(PDF)Click here for additional data file.S1 TableWe used two different parameters for the quantitative evaluation of the contour sharpness: the distance between 25% and 75% of the maximal grey value (d) and the slope in the contour (m). Comparisons were performed between the different levels of temporal averaging . Measurements were performed at 4 representative edge localisations of the myocardium. Results were recorded for 3 different slice thicknesses.(DOCX)Click here for additional data file.S2 TableWe used two different parameters for the quantitative evaluation of the contour sharpness: the distance between 25% and 75% of the maximal grey value (d) and the slope in the contour (m). Comparisons were performed between the different levels of temporal averaging . Measurements were performed at 4 representative edge localisations of the myocardium. Results were recorded for 3 different slice thicknesses.(DOCX)Click here for additional data file.


Renalase plasma concentration was demonstrated to be highly and positively associated with left ventricle mass index in HFrEF (p = 0.029), as well as increased plasma concentration of BNP (p = 0.006). In the HFmrEF group of patients, body mass index was positively associated with LVMI (p = 0.05), while in the patients with HFpEF, diabetes mellitus was demonstrated to have a positive association with LVMI (p = 0.043). These findings suggest that renalase concentration may be measured in order to differentiate patients with reduced ejection fraction. Plasma renalase concentrations positively correlated with left ventricle hypertrophy in patients with reduced ejection fraction, being strongly associated with increased left ventricular mass index.Heart failure represents a growing health problem, with increasing morbidity and mortality globally. According to the mechanisms involved in the pathogenesis of heart failure, many biomarkers have been proposed for the timely diagnosis and prognostication of patients with heart failure, but other than natriuretic peptides, none of them has gained enough clinical significance. Renalase, a new protein derived from kidneys was demonstrated to metabolize catecholamines and to have a cardioprotective role. The aim of the study was to determine whether renalase and brain natriuretic peptide (BNP) concentration could be used to differentiate heart failure patients stratified to the category of the ejection fraction and whether plasma renalase could be used as a biomarker for left ventricle hypertrophy in all subgroups of heart failure patients. We included patients diagnosed with heart failure and stratified them to the three subgroups according to the ejection fraction. Regarding echocardiographic parameters, HFmrEF had an intermediate profile in between HFrEF and HFpEF, with statistical significance in most evaluated parameters. BNP concentration was significantly different in all three subgroups ( Heart failure (HF) represents a response to a previous cardiovascular injury presented with an abnormal cardiac structure or function, leading to increased intracardiac pressures or a decreased cardiac output , with inIn order to maintain cardiovascular and homeostasis in general, during the early stages of heart failure development, many physiological compensatory mechanisms are initiated, including the activation of the sympathetic nervous system (SNS) and cytokine secretion . The actIn order to test the hypothesis that the kidney has an important role in preserving cardiovascular health, a new protein, subsequently called renalase, was discovered in 2005 , predomiHypothesizing that renalase may reflect sympathetic activity, thus exerting cytokine properties, the aims of the study were to determine firstly whether renalase and brain natriuretic peptide (BNP) concentration could be used to differentiate heart failure patients stratified to the category of the ejection fraction and secondly whether plasma renalase could be used as a biomarker for left ventricle hypertrophy in all subgroups of heart failure patients.The investigation was designed as a single-center, cross-sectional study and was conducted between May and October 2018 in the Institute for Treatment and Rehabilitation Niska Banja, in Nis, Serbia. All study participants were over 18 years old and gave written informed consent prior to the investigation. The study was approved by the institutional ethics committees. The study cohort represented 75 patients with a current history of HF, independent of etiology, and 35 healthy volunteers. All heart failure patients were clinically stable or in compensated heart failure status and had been admitted to the institute for the purpose of rehabilitation. The patients received the standard recommended pharmacological therapy [The diagnosis of heart failure was established according to the presence of signs and symptoms of heart failure, BNP plasma concentration over 35 pg/mL), relevant structural heart changes , and/or diastolic abnormality (E/A ratio < 0.75 or ≥1.5 or deceleration time of E‐wave < 140 ms) [5 pg/mL, n = 27), HFmrEF  and HFpEF . Afterwards, their baseline characteristics and possible risk factors were compared.Two-dimensional echocardiography was performed on all participants, using a commercially available system  and was analyzed according to the current guidelines . Left veAll biochemical measurements were routinely obtained on the day of enrolment by using the apparatus Sysmex XS 1000, Europe GmbH. The serum concentrations of CRP were determined quantitatively, with a nephelometric test (Orion Diagnostica Turbox), and afterwards, plasma was stored and frozen at -80°C until all the samples were collected and prepared for biomarker quantification. Estimated glomerular filtration rate was assessed by the MDRD  equation, while the subjects' most recent height and weight measurements were used to calculate body mass index.The concentrations of evaluated biomarkers were measured in plasma samples using commercially available ELISA kits, according to the manufacturer's instructions. For renalase  the range of detection was 3.12-200 ng/mL, and for BNP , the minimum detectable dose was 14 pg/mL.p < 0.05, and all statistical analyses were performed using R software, version 3.0.3.  [Data are presented as mean ± standard deviation or as absolute and relative numbers. Data distribution was tested using a Shapiro-Wilk test, and normally distributed data were analyzed with one-way ANOVA and Tukey test. Nonparametric analysis of data was carried out with the Kruskal-Wallis and the Mann-Whitney test. Linear regression was used to establish the relationship between variables. The level of significance was set at Austria) .p = 0.445) or the gender (p = 0.744). With regard to the etiology of the heart failure, coronary artery disease was most prevalent in the HFrEF patients (80.8%), compared to the HFmrEF (69.6%) and HFpEF (48%), with statistical significance between all three subgroups (p = 0.042). However, there were no significant differences between the subgroups regarding the other etiological factors for the development of heart failure. Analysis of the clinical history showed that hyperlipidemia was a statistically significant parameter in all three subgroups (p = 0.033), being the most prevalent in HFpEF patients and least in HFrEF. However, family history of cardiovascular disease was similarly prevalent in HFrEF and HFpEF patients, compared to HFmrEF patients (p = 0.033). Regarding biochemical measurements, there were statistically significant differences in blood urea nitrogen (BUN) concentration (p = 0.031), acidum uricum (p < 0.001), and triglycerides (p = 0.016) between all three subgroups, as shown in p < 0.001) (data not shown in the tables). BNP concentration was significantly different in all three subgroups (p < 0.001), and renalase was statistically higher in HFrEF (p = 0.007) compared to the HFmrEF and HFpEF where its concentrations were similar, without statistical significance , left ventricle mass (p = 0.003), LV mass index (p = 0.005), LV end-systolic volume (p < 0.001), LV end-diastolic volume (p < 0.001), left atrium (p = 0.005), mitral regurgitation (p < 0.001), tricuspid regurgitation (p < 0.001), TAPSE (p < 0.001), and right ventricle (p = 0.003). However, the E/A and systolic pressure of RV were different between the subgroups, although this was not statistically significant.Regarding echocardiographic parameters, HFmrEF had an intermediate profile in between HFrEF and HFpEF, as shown in p = 0.029), as well as increased plasma concentration of BNP (p = 0.006). In the HFmrEF group of patients, the body mass index was positively associated with LVMI (p = 0.05), while in the patients with HFpEF, diabetes mellitus was demonstrated to have a positive association with LVMI (p = 0.043), as shown in Renalase plasma concentration was demonstrated to be highly and positively associated with the left ventricle mass index in HFrEF  [Additional research has hypothesized that the increase in plasma renalase is due to its behaviour as a cytokine. However, some very important cytoprotective properties of renalase have been documented in the animal models of renal and cardiac injury . In the α) , 24, butα) . The samα) . Furtherα) . Similarα) , 21, 27.α) , 27.p = 0.029, β = 0.041) in heart failure patients with reduced EF. Note that our cohort of HFrEF patients consisted mostly of patients with coronary artery disease (over 80%), with ischemia being the main underlying mechanism. Therefore, we may presume that the elevated plasma renalase may be associated with the risk of coronary artery disease. Similar research was conducted, but the investigators reported the opposite that the decrease in plasma renalase was a significant risk factor for cardiovascular disease [Subsequently, a strong association between functional polymorphism of the renalase gene (Glu37Asp) and cardiac hypertrophy, ventricular dysfunction, poor exercise capacity, and ischemia was demonstrated in patients with coronary disease , further disease . Some in disease , again s disease .All the evidence suggests an important role for renalase in the development of cardiovascular diseases. According to the proposed mechanism, an excess of renalase probably occurs as a result of an adrenergic overstimulation, aiming to protect the tissues, with renalase being upregulated following different kinds of stress conditions , 26. HowIn summary, plasma renalase concentration was higher in patients with heart failure than in the control group and was highest in patients with a reduced ejection fraction, compared to HFmrEF and HFpEF, where renalase concentration showed similar result. These findings suggest that plasma renalase levels may be measured in order to differentiate patients with ejection fraction below 40% serving as a potent biomarker for identification of heart failure patients with reduced ejection fraction. We also demonstrated that high plasma renalase concentrations positively correlate with left ventricle hypertrophy in patients with reduced ejection fraction, being strongly associated with increased left ventricular mass index.The exact pathophysiological link between renalase and heart failure and its potential use as a biomarker in identification or risk stratification in heart failure patients is yet to be established.


Cardiac troponins T and I are considered highly sensitive and specific markersfor the diagnosis of acute myocardial infarction. Currently, a series ofnonprimary cardiac abnormalities may manifest as an elevation in high-sensitiveassays. The reduction in their detection limits has allowed earlier diagnosisand the use of evidence-based therapeutic measures; however, this characteristichas increased the spectrum of detectable noncoronary heart diseases, which poseschallenges for characterizing acute coronary syndromes and creates a new rolefor these tests in known disorders in intensive care units, especially sepsis.Management of patients through a greater understanding of how these markersbehave should be re-evaluated to ensure their correct interpretation. These molecules, calledhigh-sensitive or high-sensitivity cTnT (hsTnT) and cTnI (hsTnI), are detectedearlier and at extremely low levels, thereby reducing the "blind" detection intervalof fourth-generation cTn assays and aiding not only in diagnosticconfirmation(7) but also in detecting patients at high risk forcardiovascular events.(8) According to the guidelines for non-ST-segmentelevation AMI (NSTEMI) and unstable angina, these tests have resulted in a 20%relative increase in type 1 AMI detection (due to spontaneous atherosclerotic plaquefissuring and dissection) and a reduction in cases of unstable angina. However,these characteristics should be treated with caution, since these markers arepresent in myocardial damage of diverse etiologies, sometimes without theinvolvement of clinically evident myocardial necrosis ((9)Biochemical analysis of myocardial necrosis markers, especially cardiac troponins(cTns), gained attention in the early 1990s for the diagnosis and prognosticevaluation of patients with acute myocardial infarction (AMI). Today, cTns areconsidered fundamental for the management of acute coronary syndromes (ACSs) andconstitute a critical step for their definition together with clinical andelectrocardiographic criteria. In addition to their high accuracy for infarctiondetection, the use of these markers has brought great utility for the choice ofdifferent therapeutic strategies.necrosis . An eval(10) According to the guideline, thecriterion used for AMI is the demonstration of cTn levels above the99th percentile or a drop in the marker and at least onecriterion involving clinical or complementary signs, such aselectrocardiographic changes indicative of new ischemia  and thedemonstration of a new infarct area or segmental contractility disorders inimaging examinations. After percutaneous coronary intervention and myocardialrevascularization, values three times the upper reference limit (URL) in the99th percentile are accepted. Based on the clinicalcircumstances, spontaneous AMI due to atherosclerotic plaque rupture, erosion,fissuring, or dissection is classified as type 1, AMI due to increased oxygendemand of the myocardium is type 2, AMI related to sudden cardiac death is type3, AMI associated with a percutaneous procedure is type 4a, AMI associated withintracoronary stent thrombosis is type 4b, and AMI associated with myocardialrevascularization surgery is type 5. The high-sensitive test also has theutility of excluding the AMI diagnosis at the initial presentation, since thenegative predictive value of the test is 97 to 99%.(7) With new tests,elevations are more commonly found in patients with structural heart disease,including those with obstructive coronary disease, kidney disease, and stableangina.(11) In situations of stable angina, values in the99th percentile have been found in 37% of cases with plaquesconsidered vulnerable and in up to 2% of the general population in otherstudies.(14) The latterpopulation may have heart failure, renal failure, or left ventricularhypertrophy in addition to coronary disease. In patients with compensated heartfailure, hsTnT can also be found very close to the clinical decision limit(14ng/L).(13) The clinical context should be considered forthe correct interpretation of these markers. Thus, the management of a criticalpatient deserves a detailed analysis of preexisting conditions when abnormalcurves from cTn assays are evaluated. The recommended flowcharts for patientswith suspected ACS include a strategy that involves serial collection of thebiomarker within a few hours. The diagnosis is confirmed in most cases usingclinical and electrocardiographic criteria and values more than five times theURL. Serial changes are more accurate when below or near the 99thpercentile, and very high values and a lack of significant variation in theinitial presentation are generally indicative of chronic myocardial injury,which considerably reduces their potential for the diagnosis of AMI type 1((15) Each hsTnT andhsTnI assay has its specific cutoff points, and very low values from both testscan accurately rule out a clinical presentation within a fewhours.(9) However, diverse clinical situations and thenontypical presentation of ACS in the intensive care unit (ICU) setting impairanalysis of the cTn levels combined with clinical data. Moreover, no parameteris available to evaluate the kinetics of these markers starting from the alreadyvery high values in the critical patient. Rigorous hemodynamic control,electrocardiogram, bedside echocardiography, or an imaging examination, whichmay demonstrate loss of viability in a new segment of the left ventricle, shouldbe essential for the differential diagnosis and confirmation of AMI. Givenelevated cTns, a chronic condition can be assumed only after the exclusion ofsymptoms suggestive of ischemia, a normal electrocardiogram and echocardiogram,and the absence of a rising and/or falling marker curve.The document created by the European Society of Cardiology (ESC), AmericanCollege of Cardiology Foundation (ACCF), American Heart Association (AHA), andWorld Heart Federation (WHF) for the universal definition of AMI considers anelevation and/or drop in cTns according to the associated clinical situation tobe essential for diagnostic purposes.I type 1.(15) Eac(16) Lower values found in the initial evaluation ofpatients for acute coronary syndrome may not be related to type 1 AMI , and transient elevations can be detected withoutclinical evidence of AMI in some situations.(17) Substantialincreases followed by falls should be considered for diagnosis  and have obtained the best positivepredictive value (90%).(9) This situation is observed in clinical practiceand was detailed in the study by Apple et al., which demonstrated that patientswith ACS had lower cTn levels and an earlier decline in the presence of unstableangina or the absence of persistent ST-segment elevation.(18) Patients withpersistent ST-segment elevation or progression to "Q" AMI have higher biomarkerpeaks and a late decline (7-10 days). The first situation refers to thepredominance of ischemic cTn release and the second to necrotic release. Anintermediate cTn release behavior probably exists depending on the nature of theinsult, time to reperfusion therapy, clinical course, and the patient'streatment success.An important conceptual theory for the interpretation of these tests is theformation of blebs containing cTns, of which approximately 2 to 6% are free inthe cytosol; these blebs release their material into the extracellular mediumdue to the reduced oxygen supply. When the tissue damage becomes prolonged, theblebs increase and rupture, triggering cell death. Loss of the structuralcontent of myocardial cells is reflected in the prolonged cTn release curve inAMI. However, if reperfusion occurs or if the injury is transient, these blebsare reabsorbed or may leak their cytoplasmic contents into the circulation withthe cell membrane still intact. This short detection period represents a pointrelease of the cytoplasmic content, which corresponds to the half-life of thedetected substances .(16) Low(19) Proposed mechanisms for the release of cTns inthe presence of septic shock also include focal ischemia and the effect ofendotoxins, cytokines, and reactive oxygen species on cardiomyocytes. Tumornecrosis factor (TNF) may modulate the activation or biosynthesis of proteases;for example, calpains and caspases may participate in the degradation ofcontractile proteins, including cTns.(21)Sepsis and other systemic disorders may lead to myocardial depression and celldamage caused by increased consumption and/or a reduced supply of oxygen to theheart.(24) Typically, cardiacoutput is elevated, leading to increased cardiac work and oxygen demand. Thepresence of tachycardia with a decreased diastolic filling time also increasesoxygen consumption. The coronary reserve flow becomes limited, and ischemia mayoccur. Finally, treatment with high doses of inotropic agents to increase theoxygen supply may increase the incidence of cardiovascular complications andaffect the outcome unfavorably in patients with adequate fluid resuscitation.Prolonged resuscitation may elevate ischemic damage with increased fillingpressures, parietal strain, and additional cardiomyocyte injury during septicshock.(25)Experimental studies suggest that generalized microvascular dysfunction is aprominent sign of septic shock and may indicate relative ischemia due to themicrovascular shunt effect or secondary flow heterogeneity resulting fromendothelial dysfunction, capillary plugs, interstitial edema, and free radicalproduction. Some studies suggest areas of reversible mismatch due toredistribution of the microcirculation in response to metabolicchanges.(26) Although some studies have shown an associationbetween several types of tachycardia and elevations in cTns, other factors mayconfound this association, such as the presence of coronary artery disease(CAD), other associated clinical conditions, and hemodynamicchanges.(28) Speculation alsoexists that the tachycardia present in sepsis and septic shock may cause cTnrelease in the absence of structural heart disease, CAD, and myocardialdepressing factors (sepsis) and thus may only represent a manifestation of theimbalance between oxygen supply and demand (type 2 AMI).Tachycardia during the hyperdynamic state is a striking finding. Stretching ofthe myocardial fiber and an increase in parietal tension are probable mechanismsdue to the parallel increase in natriuretic peptide and cTns in several types oftachycardia.(29) However, TNF values were not correlated withelevations in cTns in healthy humans tested with endotoxin, suggesting thatother mechanisms must be involved. The interleukin 6 (IL-6) level alsocontributes to increased cell permeability; the level is elevated significantlyin patients with septic shock and positive for cTns compared to the level inthose negative for cTns.(30) Additionally, cTn-positive patients have ahigher level of histological changes. Necrosis with contraction bands(coagulative myocytolysis) and fibrillar rupture is more commonly associatedwith calcium overload and typically is associated with reperfusion injuries andthe use of catecholaminergic drugs.(31)Mediators, such as TNF, are also implicated in the increased permeability of thecardiomyocyte membrane. As previously reported, this phenomenon could explainthe presence of cTns in the absence of irreversible celldamage.(34)When restricted to sepsis cases, elevated levels have been shown to beassociated with the disease severity. Generally, these results are very similarto those showing increased levels indicating worse left ventricular function anda consequent unfavorable outcome.(35) The study by Røsjøet al. indicated that hsTnT was an important early marker of circulatorydysfunction in what was then termed severe sepsis but was not added to theSimplified Acute Physiology Score (SAPS II) for the prediction of hospitalmortality. In this situation, cardiovascular dysfunction may not be the onlyfactor responsible for mortality in sepsis. hsTnT was present in all initialcases of severe sepsis, whereas 60% of patients had positive fourth-generationcTnT. The authors concluded that an initial hsTnT level of less than 14ng/L(99th percentile) in early severe sepsis might indicate a lowprobability of progression to septic shock.(34) A meta-analysispublished in 2013 showed that both cTnT and cTnI were independent factors ofmortality regardless of the causes or comorbidities involved in their elevationand corrected for confounding biases, such as renal failure or heart diseasepresent prior to inclusion in the study.(35) The field of cardiology shares thesame hypothesis, because evidence suggests that mortality increases in patientsadmitted with hsTnT levels above the 99th percentile regardless ofthe causal agent of cTn elevation. The SWEDEHEART registry noted that patientsadmitted with suspected acute coronary syndrome and hsTnT levels above 14ng/Lshowed higher mortality rates, although only 18.2% of the patients actually hadtype 1 AMI.(36)The prognosis of sepsis depends on the severity of organ dysfunctions, especiallycardiovascular dysfunction. Many studies have discussed the fact that cTns areindependent parameters of the outcome, with no distinction between the causes ofelevation of these biomarkers.(37) With the use of the high-sensitivetests, this correlation reached 60% in some cohort analyses. When studiesevaluated serial changes in the biomarker, 60% of the patients had stable cTnlevels, and the rest had a 20% rise and fall pattern.(38) The latter patternmay be related to a coronary event (type 1 AMI) or myocardial injury secondaryto stroke. Acute changes in autonomic control with exaggerated catecholaminerelease may be a noncoronary consequence of increased cTns instroke.(39) The insula is an important region for autonomicregulation and is frequently affected by involvement of the anteriorcirculation, and an association between stroke in this cortical area and anincrease in cTn values has been demonstrated.(40) However, otherstudies have not demonstrated a correlation between the cTn values and thelocation or volume of the infarction.(41) We add that exposure to excessivecatecholaminergic stimulation and myocardial ischemia due to coronaryvasoconstriction or pre-existing CAD may occur in type 2 AMI. The frequency ofassociated type 1 AMI is uncertain, but after adjusting for age and sex,patients with ischemic stroke are less likely to have associated coronarylesions despite elevations of the cTn levels similar to cases of NSTEMI;moreover, half of the patients in one study had no coronary lesions on theangiogram.(42) Hospital mortality is higher in patients withintracranial hemorrhage than in those with ischemic stroke, and the riskassociated with cTn elevation is higher in ischemic stroke than in hemorrhagicstroke.(43)After introduction of the sensitive myocardial necrosis markers, the associationbetween cTns and stroke was demonstrated extensively. A systematic review of 15studies prior to the use of high-sensitive cTns showed that 18.1% of patientshad elevated levels during the event.Thus, in patients with stroke and elevated cTns, an initial assessment of whetheran acute increase in the biomarker has occurred or whether it is elevated butstable is reasonable. Then, a careful assessment whether an associated ACSexists is essential. Once a high but stable value is detected, we should lookfor comorbidities associated with cTn elevation that may benefit from moreaggressive long-term management. A frequent situation is encountering abnormalcTn levels in stroke patients with a recent ACS or dilated cardiomyopathycomplicated by atrial fibrillation preceding the event.(44) However, because heart failure is a complexsyndrome, single biomarker assessment may not reflect all of itscharacteristics. The accumulated evidence emphasizes that cTns can addinformation to natriuretic peptides. After that description, subsequent studiescorrelated cTn levels with the disease severity. The cTn levels do not allow theetiology to be diagnosed but may reflect the increase in the left ventricularmass and provide data on the ejection fraction and diastolicdysfunction.(45) Elevation in the cTn levels was associated withthe symptom severity, increased need for supportive therapy with vasopressorsand inotropes, and worsening of the outcome at 30 days.(44)Biomarkers in the context of acutely decompensated heart failure have becomeimportant for diagnostic and prognostic assessments and are currently part ofthe standard clinical evaluation. Current recommendations indicate the use ofnatriuretic peptides.(46) These findingsalso apply to high-sensitive tests. Serial changes in the cTn concentrations arealso important, because the persistence of elevated cTn levels during or afterhospitalization is related to worse outcomes, whereas decreasing or stablevalues are associated with lower adverse outcome rates.(47) Despite thisfinding, the clinical usefulness is uncertain. The challenge persists in thedifferentiation between ACS-mediated release and noncoronary release. A Class Irecommendation with Level of Evidence C is included in the European guidelinesfor the use of cTns in acute heart failure.(44) However, the cTnconcentrations should be interpreted broadly. A careful evaluation with imagingand coronary angiography should be part of the investigation, especially incases of recent-onset heart failure, positive cTns, and high clinical suspicionof ischemia of coronary origin.A meta-analysis based on 77,297 patients concluded that detecting cTns in heartfailure patients increased their 1-year mortality and readmission rates,demonstrating a hazard ratio of 2.3 (95%CI 1.8 - 3.0).Epicardial involvement may be a striking finding in acute pericarditis, and cTnsare elevated in approximately 30 to 49% of cases. When they reflect myocardialinjury, the finding of cTns together with other findings compatible with globalor regional left ventricular dysfunction may indicate myopericarditis.(19)Elevation of the biomarker does not seem to be related to the prognosis ofmyopericarditis and is considered a weak marker of the extent of myocardialinvolvement.(48) The marker concentrations in that study showeddifferent behavior according to the clinical presentation. Patients presentingsymptoms for more than 13 days had substantially lower values. In contrast toother clinical conditions, cTn concentrations in myocarditis do not imply anadverse prognosis. Increased cTn levels were not useful to predict complicationsduring follow-up of 486 patients with acute pericarditis or myopericarditis, anda systematic review including eight studies concluded that cTns were not able topredict adverse events during clinical follow-up.(50) Nevertheless, the reduced rate of eventsand the small sample size in these studies should be considered.Although they appear to be useful for myocarditis diagnosis, cTns have limitedsensitivity, at least in studies using less sensitive markers. Better accuracywas gained with hsTnT in one study  with a sensitivity of 83% and specificity of 80% achieved using a cutoffof 50ng/L.(51)Takotsubo cardiomyopathy, which is also called stress-induced cardiomyopathy orapical ballooning syndrome, is reported in approximately 0.7 to 2.5% ofsuspected ACS cases.(52)Although we observe Takotsubo cardiomyopathy most commonly in women withemotional or physiological stress with a presentation of cardiomyopathy, it mayoccur in a broader clinical spectrum, including presentation in younger men andin those without a triggering event.(53) This elevation is disproportionate to thefinding of regional left ventricular dysfunction on imagingtests.(54)Most patients have a mild to moderate cTn increase within 24 hours ofpresentation.Some studies have attempted to differentiate patients with Takotsubocardiomyopathy from those with ACS using biomarker behavior, since diagnosticconfirmation is often performed only after finding no obstructive lesions oncoronary angiography and normal ventriculography. A prospective analysis of themagnitude of the elevations in cTnT and cTnI found that cases with the formerbelow 6ng/mL and the latter below 15ng/mL showed little probability of beingTakotsubo cardiomyopathy.(55)Because the presentation more closely involves regional left ventricular changesthan loss of viability due to necrosis, the ratio between markers has beenstudied at ACS patient admission. The ratio that best differentiated Takotsuboand ST-segment elevation AMI (STEMI) was the ratio between peaks of theN-terminal prohormone of brain natriuretic peptide (NT-proBNP) in ng/L to cTnTin µg/L. A cutoff value of 2,889 was able to distinguish Takotsubocardiomyopathy and STEMI (91% sensitivity and 95% specificity), and a ratio of5,000 was able to discriminate Takotsubo cardiomyopathy and NSTEMI (83%sensitivity and 95% specificity).(56)The incidence of myocardial contusion in patients with closed chest trauma rangesfrom 3% to 56% of cases depending on the diagnostic criteria used. The lack ofspecific signs and symptoms and the broad spectrum of clinical presentation makeits evaluation difficult.(57)Both cTnT and cTnI have an equivalent sensitivity profile and greater accuracyfor myocardial contusion detection. These indicators aid in the selection ofpatients who must remain under intensive cardiacmonitoring.(58) Patients with ECG changes,elevated cTn, or both must remain under observation in the ICU for at least 24hours, during which time most complications of myocardial contusiondevelop.(56)A study identified that patients with brain injury, thoracic injury, closed chesttrauma, and shock more frequently had higher cTnI values. In the same analysis,sustained (greater than 36 hours) and significant cTnI release (cTnI peak≥ 2µg/L) was more frequently associated with thoracic trauma (82%)and the presence of electrocardiographic changes. Based on electrocardiographicabnormalities, the sensitivity, specificity, and positive and negativepredictive values of cTnI release were 63%, 98%, 40%, and 98%, respectively.Mortality could not be discriminated according to the marker values, butpatients with a normal electrocardiogram and cTnI level 8 hours after chesttrauma had an almost null probability of complicated myocardial contusion andrequired no additional evaluation.(59) Intermediate-risk patients are consideredthose with signs of right ventricular dysfunction who are hemodynamically stableand cTn positive. Kucher et al. concluded that a normal echocardiogram andnegative cTns were useful for identifying patients with a lower risk of earlymortality.(60) However, the reason for the increase in thesemarkers in pulmonary thromboembolism is still unclear. In one study, 63% ofcases with right ventricular dilatation had elevated cTns, whereas 29% of caseswith positive cTns had a normal end-diastolic diameter.(61) cTnI was alsoassociated with more segmental defects on ventilation/perfusion scintigraphy.However, hypoxia secondary to the decrease in the ventilation/perfusion ratio,hypoperfusion due to shock and coronary flow decrease, and systemic vein tocoronary artery embolism by patent foramen ovale can also be considered originsof the cTn elevation. Transmural right ventricular infarction despite normalcoronary arteries has also been found in cases of massive pulmonarythromboembolism.(62) When evaluating kinetics, some studies haveconcluded that the cTnT peak is lower and shorter than that ofAMI.(63) Although this behavior suggests that cTns arereleased from the cytosolic pool during ischemia, given such release kinetics,this hypothesis still requires testing. A meta-analysis of 20 studies with 1,985participants demonstrated that cTn elevation was significantly associated withincreased short-term mortality resulting from thromboembolism, includingsituations with a preserved hemodynamic status.(64) Another recentmeta-analysis of 1,366 patients identified a four-fold greater risk ofshort-term death in 55 patients.(65)Pulmonary thromboembolism patients with signs of shock and hypotension presenthigh mortality. After careful risk assessment, the use of thrombolytics in thesepatients is generally accepted.(10)The pathophysiological mechanism by which the cTn levels are elevated in chronicrenal failure (CRF) remains uncertain. The most investigated associations arethe presence of diffuse obstructive CAD with microinfarcts and left ventricularhypertrophy. A strong association exists between the cTnT level and the presenceof multivessel CAD in patients with asymptomatic CRF undergoing coronaryangiography.th percentile derived from the healthy general population as acutoff. In contrast to patients with preserved renal function, those with CRFmore commonly have baseline cTn above the 99th percentile,particularly when the assay is highly sensitive.(66) Therefore, aserial cTn analysis with a relative increase of approximately 20%, asrecommended by the guidelines, should be performed in the appropriate clinicalcontext for the diagnosis of AMI.(9) Both cTnT and cTnI can be used, and there isno consensus in the guidelines on the advantages of cTnI over cTnT for AMIdiagnosis in patients with CRF.(10) Importantly, both cTns are markers ofdiagnostic choice, and no substitutes are acceptable, such as the MB fraction ofcreatine kinase, with the prerogative of loss of cTn specificity in thiscontext.A study underscored the challenges in interpreting AMI in CRF using the99th percentile of the URL,resulting in a sensitivity of 94% and specificity of 86%. However, this changemay adversely change the sensitivity of the assay, and use of the URL providedby the manufacturer and/or institution for the exclusion or confirmation of AMIis advisable in these cases.(11) The diagnostic accuracy of high-sensitivityassays is also compromised in hemodialysis patients, since almost all patientshave baseline levels above the 99th percentile. In a series of 670hemodialysis patients evaluated for dyspnea or chest pain, the receiveroperating characteristic (ROC) curve for the hsTnT test was only 0.68 butsignificantly improved to 0.9 with serial evaluation over 3 hours. The mostfavorable cutoff point for the relative variation was 24%.(67) However, clinicaljudgment is a critical component for assessing chest pain in hemodialysispatients. Although the dynamic changes in markers improve the specificity forthe diagnosis of AMI in patients with CRF, relying on this parameter alone maybe associated with a loss of up to 12% of STEMI cases.(68)A study suggested that URL elevation occurred in this group of patients. In ananalysis of 75 patients with CRF, hsTnT was used with high accuracy for thediagnosis of AMI at a level twice the 99(70)During evaluation of a patient with CRF, a baseline cTnT or cTnI value shouldnever be interpreted as part of the loss in glomerular filtration alone. Evenwithout the association with AMI, elevated levels indicate a worse prognosis andshould be valued.(71) The involvement of the cardiac musculaturemanifests transiently with falls in the systolic and diastolic functions; thisphenomenon is called "heart fatigue".(72)Cardiac troponins may be elevated immediately after strenuous physical exercise,which is a phenomenon that has been studied in long-distancerunners.(73) In fact, these assays may be elevated duringshort periods of exercise in both cardiopathic and noncardiopathicpatients.(74) Several studieshave demonstrated that transient elevations do not indicate myocardial damage,since the levels normalize within 24 to 48 hours.(19) These data alsoreinforce the theory of cTn release in the cytosol and not from the structuralcontent of the myocytes.High-sensitive cTns are detected in approximately 80-86% of marathon runnersafter exercise.(75)In a meta-analysis of 1,120 individuals, Shave et al. found that the exerciseduration was related to increased cTnT levels without correlation with theparticipants' ages. With the use of high-sensitive assays, a negativecorrelation was also found between the cTn levels and performance, and anonsignificant correlation was found with age. This finding can help theclinician evaluate symptomatic patients after competitive exercises inpractice.(76)Interestingly, transient alterations in right ventricular function were found inhigh-intensity athletes. However, this finding has not added prognosticinformation to date.(19)The clinical impact of exercise-induced cTn elevations is still unclear. Symptomsduring exercise are relatively common, and marathoners presenting withdizziness, chest pain, and sometimes circulatory collapse may pose a challengefor diagnosis in the presence of elevated cTns. Currently, there are no data todiscourage athletes from competitive activity due to elevation in cTns afterexertion. Moreover, D-dimers and natriuretic peptides as well as cTns may beelevated after exercise.Lack of attention to compatible findings after high-intensity efforts, includingbiomarker elevations and transient right ventricular dysfunction in the absenceof clinical signs indicative of ACS, heart failure, or pulmonary embolism, cantrigger a costly, invasive, and unnecessary investigation. Treating the patientand not the clinical assay is still a fundamental precept.(77) Similarly, anotherstudy reported that ST-segment elevation was present in 16% of obstructive CADand 11% of nonobstructive CAD cases, respectively.(78) The recommendationis that coronary angiography should be used as an investigation if nounequivocal factor is the cause of type 2 AMI, such as known hemorrhagic shockin CAD.(79)Since the last AMI definition, diagnostic choice markers have emerged to supportclinical and electrocardiographic data. However, one of the major challenges inthe ICU environment is the differentiation between increases in cTns due tohemodynamic instability, sepsis, and other disorders and type 1 AMI.Electrocardiographic abnormalities may not provide diagnostic help, since thepresence of ST-segment elevation can occur in cases of type 2 AMI. Spatz et al.found that ST-segment elevation was present in 17% or 15% of type 2 AMI caseswhere obstructive CAD was present or absent, respectively.th percentile, ina clinical situation consistent with myocardial ischemia, and in cases with anevident imbalance between oxygen supply and demand without atheroscleroticplaque rupture and with one more criterion according to the universaldefinition. (79)The absence of spontaneous rupture and dissection of atherosclerotic plaques is apurely clinical analysis and increases the ambiguity of the AMI diagnosis due toimbalance between oxygen supply and demand, contributing to disparities in itsincidence (1.6% to 26%). The highest incidence was obtained with morerestrictive criteria. In addition, several studies did not place supply ordemand thresholds for oxygen, since several anatomical and pathophysiologicalfactors could be blamed. Patients with sepsis are also sometimes included inanalyses with those with type 2 AMI. Sepsis and septic shock share other factorsbesides the imbalance in oxygen demand and supply in their pathophysiology. Type2 AMI is diagnosed when there is evidence of necrosis due to increased anddecreased cTns with at least one value above the 99(79)Since the term was created in 2007, no practical guidelines have been availablefor management of type 2 AMI. Observational studies show that such patients aremanaged less frequently with revascularization, beta-blockers,angiotensin-converting enzyme inhibitors, statins, and antiplatelet agents, evenwhen significant CAD is present.(80) However, for patients with type 2 AMI, therewas an increase in indications for specialized cardiovascular evaluation,echocardiograms, and coronary angiography, which had no impact on the treatmentor prognosis. No specific therapy was available in these cases, and a missedopportunity actually existed. In the acute phase, it is intuitive to carry outstrategies for adequate oxygen supply and reduced oxygen demand, includingvolemic state adjustment, pressure, and inotropic support, if necessary; the useof blood products; heart rate control; and ventilatory support with an activesearch for the causal factor. Depending on the clinical situation, coronaryangiography should be used to investigate the presence of CAD. If applicable,the guidelines should be followed. However, if obstructive CAD is absent,strategies for risk reduction are scarce. In general, despite the differentdefinitions used among studies, there is a very somber prognosis for type 2 AMI,with long-term mortality reaching 63% after 3 years offollow-up.(78) Studies have focused on all-cause mortality atthe expense of cardiovascular causes.Shah et al. examined the impact of lowering the threshold of a high-sensitivetroponin test based on the incidence, management, and outcomes of type 1 andtype 2 AMI and myocardial injury. These tests resulted in higher costs, greateruse of hospital resources, and a better prognosis for type 1AMI.With the advent of high-sensitive troponin assays, both the detection sensitivity ofacute myocardial infarction and the detection of elevated marker levels insituations unrelated to acute coronary syndromes have increased. The increase in theprevalence of noncoronary conditions, which are associated with increases in cardiactroponins, presents challenges for the diagnosis of acute myocardial infarction,especially in the elderly population, in which coronary and noncoronary heartdiseases are found. Several strategies for the use of these markers have beenstudied without a definitive opinion in the current guidelines. Considering elevatedcardiac troponin values at the initial presentation is important, since thepredictive value of the test increases for cardiovascular and noncardiovasculardiseases. The behavior of the serial test can also help identify several situations,including acute myocardial infarction. This evaluation, combined with a rigorousclinical analysis, should increase the use of earlier therapeutic strategies andexclude patients with greater safety.


Ischemic mitral regurgitation (IMR) occurs as an adverse consequence of left ventricle remodeling post-myocardial infarction. A change in mitral valve configuration with an imbalance between closing and tethering forces underlie this pathological condition. These abnormalities lead to impaired leaflet coaptation and a variable degree of mitral regurgitation, which can in turn influence the ventricular filling status, the heart rhythm and the afterload regardless of the residual ischemic insult. The IMR correction can be pursued through under-sizing mitral annuloplasty and papillary muscle approximation to restore the mitral valve and left ventricle physiological geometry to, consequently, achieve normalization of the engaged physical forces. Because the structures involved undergo extremely large deformations, a biomechanics model based on the Euler’s Elastica –the mitral leaflet– interlaced with nonlinear chordae tendineae anchored on papillary muscles has been constructed to elucidate the interactions between closing and tethering forces. The model takes into account the actual updated geometrical and mechanical features of the valvular and subvalvular apparatuses in physiological and IMR conditions, as well as in case of papillary muscle approximation, finally furnishing ad hoc geometry-based mathematical relations that could be utilised to support—and optimize—the relevant choices in cardiac surgery.  Ischemic mitral regurgitation (IMR) is an acquired geometric dysfunction of mitral valve motion secondary to myocardial infarction that is characterized by post-ischemic adverse left ventricular remodelling, left ventricular distortion and, finally, enlargement of left side cardiac chambers. The main pathophysiological mechanism behind the IMR is the displacement of papillary muscles along a posterior, apical or lateral vector. Several biomechanical features are involved in determining an imbalance between tethering and closing forces at level of valvular and subvalvular mitral valve apparatus. Tethering forces are identified in papillary muscles displacement, annular dilatation, left ventricle (LV) dilatation and LV sphericity while closing forces are composed by altered mitral systolic annular contraction, reduction of LV contractility, global LV dyssynchrony and papillary muscle dyssynchrony. Patients with moderate to severe IMR are destined for surgical treatment and the American College of Cardiology/American Heart Association (ACC/AHA) guidelines recommend consideration of mitral-valve repair or chordal-sparing replacement . HoweverPapillary muscle approximation (PMA) combined with restrictive annuloplasty (RA) has proved beneficial in reducing augmented leaflet tethering for both symmetric and asymmetric shapes, as illustrated in y-axis), the end point angle lθ  and the coefficient ω2 = P l2/B, B being the bending stiffness and the load P obeying the equation:lθ and α, determined by the slope of the chorda tendinea. Thus, the α results itself a function of both the angle lθ and the position of the papillary muscle, in the problem at hand, was subjected to a displacement vector u to simulate the PM approximation.We considered a planar problem, in which the flexural response and the deformed shape of the mitral valve leaflets were modelled as nonlinear cantilever beams subjected to end-point loads, whose directions and intensities accord the axial tensile forces in the chorda tendinea, guaranteeing geometric compatibility and equilibrium. Experimental observations have recently shown that the flexural response of aortic valves can be depicted in a satisfactorily manner by means of a beam bending test  Here, alint load ,16 were int load ,16):(1)xabscissa , while tε = (λ2−1)/2 is the Green strain, the stretch c/Lcλ = l being the ratio between current and reference chorda lengths. In our problem, the postoperative unknown length cl will explicitly depend upon both the angle lθ and the PM displacement u, while the unknown reference length cL is evaluated by means of a pull-back operation, starting from the observed pre-operative chorda. Utilizing material constants c1 and c2 from literature stress–strain tests [refT = P/A denotes the nominal stress .Chordae experience uniaxial tension in physiological conditions and are modelled here as one dimensional (incompressible) elements exhibiting a Fung-type behaviour, with strain energy density:in tests , the tena and p, respectively, indicating the components belonging to the anterior and the posterior structures, and the chordae angles aα and pα. The equilibrium at each leaflet free edge-chorda connection node requires:i = {a,p} and the additional subscript k denotes the configuration at which equilibrium is needed. Furthermore, to simulate the RA and PMA, additional unknowns are introduced   .Echocardiographic assessment of IMR plays an important role in evaluating the mitral regurgitation severity through the direct measurement of specific geometrical parameters indicating poor LV remodelling, which has been directly associated to the grade of the pathology. Specifically, different morphological unfavourable characteristics, obtainable through diagnostic tools such as 2D echography with 3D reconstruction 4 and cardiac MRI, can independently permit the IMR classification ,21,22. AML and δPML, respectively—as well as the effective regurgitant orifice Area  [pre. For convenience, i = {a,p}, the preoperative coordinates pred is the interpapillary muscle distance. Denoting the preoperative location of the PMs with mipre, their distance pred reads:The parameters by which the clinical practice mainly relies to assess the MV remodelling and IMR are the mitral valve diameter (MVD), the tenting area (TA) and the tenting height (TH), the anterior and posterior leaflet angles—δa width) ,23. To ea width) . The difD and TH . In partchorda nodes. One has:The constants ua and up. In terms of their components, these vectors were respectively written as aζ and pζ indicating the orientation of each papillary displacement towards the predicted postoperative position. Then, the nodes of the MV structure can be redefined in the current configuration in terms of the nine unknowns just described. One finds the posterior MV joint in position chordae orientation, in other words:To model the reconfiguration of the preoperative apparatus via restrictive annuloplasty combined with PMA, the unknown postoperative parameters optTH, and that the mitral annulus antero-posterior diameter reduces by r, in other words:a and ζp were treated as additional design variables, determined to optimize the PMA in such a way to minimize the chordae tendinea stress. This was achieved by implementing an ad hoc random iterative procedure, schematized in Furthermore, problem-specific constraints were introduced to ensure both mitral leaflets meet at a desired coaptation height, say The postoperative configuration is shown in The theoretical predictions also highlight the optimal positioning of the PMs, achieving an end-systolic IPD of about 27.5 mm, actually suggesting a 28% reduction of the ED-IPD (End- diastolic-interpapillary muscle distance), in accordance with the clinical PMA cases. Additionally, the mathematical model indicates the optimal subvalvular arrangement and the predicted PM positions in agreement with the displacement executed in surgery, as reported in r into two variables  and (20), while coaptation constraints (21) were slightly modified by decoupling the restriction terature . StartinValve anulus stress ratio, estimated as the ratio between the post-operative and pre-operative bending moments of the anterior and posterior elasticas predicted by the analytical model in correspondence of the valve roots, given by Chordae stress ratio Chordae stretch ratio chordae tendineae stretching and tethering forces with respect to the preoperative condition. This potentially compromises the mechanical seal of the leaflet coaptation and may be a prodromal signal to secondary reopening.As shown in optTH, in line with physiological values of tenting heights from healthy control groups and successful follow-ups, reported in au and pu were prescribed, leading to the increase of higher drag forces within both PM roots and ventricular walls: consequently, an over-estimated correction could potentially compromise the local ventricular wall motility and functionality, as well as the stability of the subvalvular implant because of the pull of the muscle wall reaction forces. Mitral repair for IMR is currently undergoing a paradigmatic shift in stating the need to address the mitral valve apparatus in its entirety . The current clinical evidence suggests positive results with PM surgery ,6,25,26,Elastica–based biomechanical model, by integrating geometrical data and mechanical stresses, could advantageously represent a complementary tool to orient the surgeon’s choices in PMA procedures, and provide a first step towards the definition of a biomechanical index able to predict effectiveness of MV repair, suggest optimal design protocols for LV surgical remodelling and plan PMA success.We propose a simple structural scheme, in which nonlinear elastic solutions are founded by incorporating all the key geometrical and mechanical factors governing the problem and are actually considered in the preoperative, operative and post-operative phases. Our results confirm the leading variables considered in the surgical procedure, including the pivotal role of the direction of PM displacement. The model highlighted how the morphological factors are directly involved in the estimation of postoperative outcomes, revealing how the whole process and the postoperative outcome depend on the synergistic action of initial geometry, deformed configurations and in situ evolving mechanical stresses. Preoperative evaluation of chordae and annular stresses can be extremely helpful in understanding the physical status of the valvular and subvalvular systems and to know how the severity of IMR compromises the health of the structure. An excessively stressed structure can in fact undergo local yielding phenomena, which in turn can compromise the elastic properties of the apparatus and, consequently, its functionality. It is felt that the proposed Authors acknowledge several limitations in this study. Firstly, the model was based on echocardiographic measure of TH, IPMD, mitral annulus diameter and LV remodelling and no 3D reconstructions were performed. This would have required the inclusion of normalization parameters of the mitral valve geometry following the surgical management of symmetric or asymmetric deformations. Indeed, the model introduces some simplifying hypotheses, the most important of which being the two-dimensional kinematics, which; thus, sacrifices more realistic representations of the MV, obtained for example by resorting to approaches based on finite elements ,28. In f


Myocardial slices, also known as “cardiac tissue slices” or “organotypic heart slices,” are ultrathin (100–400 μm) slices of living adult ventricular myocardium prepared using a high-precision vibratome. They are a model of intermediate complexity as they retain the native multicellularity, architecture, and physiology of the heart, while their thinness ensures adequate oxygen and metabolic substrate diffusion in vitro. Myocardial slices can be produced from a variety of animal models and human biopsies, thus providing a representative human in vitro platform for translational cardiovascular research. In this review, we compare myocardial slices to other in vitro models and highlight some of the unique advantages provided by this platform. Additionally, we discuss the work performed in our laboratory to optimize myocardial slice preparation methodology, which resulted in highly viable myocardial slices from both large and small mammalian hearts with only 2–3% cardiomyocyte damage and preserved structure and function. Applications of myocardial slices span both basic and translational cardiovascular science. Our laboratory has utilized myocardial slices for the investigation of cardiac multicellularity, visualizing 3D collagen distribution and micro/macrovascular networks using tissue clearing protocols and investigating the effects of novel conductive biomaterials on cardiac physiology. Myocardial slices have been widely used for pharmacological testing. Finally, the current challenges and future directions for the technology are discussed. Myocardial slices, also known as “cardiac tissue slices” or “organotypic heart slices,” are ultrathin slices of living adult ventricular myocardium prepared using a high-precision vibratome Fig. A. They aBasic cardiac research has been traditionally performed on biological models ranging from isolated cardiomyocytes to complex multicellular preparations. Isolated cardiomyocytes have provided the bedrock for research over the last few decades. However, during enzymatic isolation, connections between neighboring cardiac cells and the extracellular matrix are lost and a high proportion of cardiomyocytes is damaged, with significant implications for cardiomyocyte function –5. Multi2+ handling. A major advantage provided by myocardial slices is that several slices can be prepared from each heart/specimen, increasing experimental throughput while also reducing the number of animals used in research. Finally and most importantly, from a translational perspective, myocardial slices can be produced from human biopsies [The ultrathin nature of myocardial slices combines the benefits of a complex multicellular three-dimensional preparation with the simplicity of acquiring structural and functional data from a two-dimensional monolayer. Myocardial slices have allowed our group to study different cardiac cell populations within their physiological location in a representative manner avoiding enzymatic isolation . The maxbiopsies , 10. ManThere has been a slow but progressive increase in the number of laboratories preparing myocardial slices since they were first described . MyocardNature Protocols [Nature Protocols [2 [In the optimized protocol that our group published in rotocols , we descrotocols , we do nrotocols , 17–21. rotocols . In our rotocols , sheets As myocardial slices provide a clinically relevant and representative human model system in vitro, they are particularly useful for translational studies. A substantial amount of research has already demonstrated that myocardial slices are an appropriate model for acute pharmacological testing and in vitro safety screening , 13, 23 Techniques have been developed to culture myocardial slices in vitro. The current gold standard methodology is based on the protocol described by Brandenburger et al . In brie