To determine morphological and functional cardiovascular magnetic resonance (CMR) patterns in histopathologically confirmed myocardial involvement in patients with systemic sclerosis (SSc).Twenty patients  with histopathologically proven cardiac involvement in SSc in the years 2008–2016 were retrospectively evaluated. Morphological, functional and late gadolinium enhancement (LGE) images were acquired in standard angulations at 1.5 T CMR. Pathologies were categorized: 1) Pericardial effusion; 2) pathologic left (LV) or right ventricular (RV) contractility ; 3) reduced left (LV-EF) and right ventricular ejection fraction (RV-EF); 4) fibrosis and/or inflammation (positive LGE); 5) RV dilatation. 95 % confidence intervals (CI) were calculated for appearance of pathologic EF and RV dilatation.Seven patients (35 %) had positive CMR findings in three categories, 9 patients (45 %) in four categories and 4 patients (20 %) in five categories. The distribution of pathologic findings was: minimal pericardial effusion in 7 patients (35 %), moderate pericardial effusion >5 mm in nine patients (45 %); abnormal LV or RV contractility in 19 patients (95 %), reduced LV or RV function in 14 patients , pathologic LGE in all patients, RV dilatation in 6 patients .CMR diagnosis of myocardial involvement in SSc requires increased attention to subtle findings. Pathologic findings in at least three of five categories indicate myocardial involvement in SSc.The online version of this article (doi:10.1186/s12968-016-0289-3) contains supplementary material, which is available to authorized users. Systemic sclerosis (SSc) is a rare chronic systemic autoimmune disease associated with different autoantibodies, the most common are anti-topoisomerase I (Anti-Scl-70) and anti-centromere antibodies (ACA). SSc is associated with high morbidity and mortality . The patIn these patients with cardiac involvement, non-segmental perfusion defects can be observed in myocardial stress perfusion cardiovascular magnetic resonance (CMR), suggesting resemblances in the pathogenesis of SSc associated Raynaud’s phenomenon and transient myocardial ischemia . CardiacSeveral CMR studies have pointed out characteristic findings in patients with SSc. These are: Pericardial effusion , left veStandard treatment of SSc includes cyclophosphamide, which can be cardiotoxic . In our Aim of this study was to systematically determine morphological and functional CMR imaging patterns in systemic sclerosis in patients with histopathologically confirmed myocardial involvement in SSc.Twenty patients  with a history of SSc for 3.0 years  were retrospectively included. One patient had lcSSc, 19 had dcSSc. The mean modified Rodnan skin score was 26 . Troponin I was elevated in 13 patients (65 %), brain natriuretic peptide in 6 patients (30 %). Anti-Scl-70 antibodies were positive in 15 patients (75 %), additionally anti-centromere antibodies were positive in two patients (10 %). Three patients (15 %) had pulmonary arterial hypertension, seven patients (35 %) had pathologic Holter ECG.This study retrospectively evaluated a subpopulation of patients with cardiac involvement in SSc as proven by endomyocardial biopsy (EMB) previously undergoing CMR in the years 2008–2016. Clinical findings in a part of the study group (16 patients) were published by Mueller et al. . All patEMB was sampled from the right ventricular septum. Histopathological and immunohistological analyses were performed by experienced cardiopathologists as described by Mueller et. al. : The degCMR was performed on a 1.5 T scanner  equipped with a gradient system with a maximum strength of 45 mT/m and a maximum slew rate of 200 T/m/s. A six channel body array coil and six coils of the spine coil were used for reception of MR-signals. All images were acquired in breath hold technique with ECG triggering.For functional imaging, Steady State Free Precession (SSFP) cine loops (repetition time (TR) 3 ms; echo time (TE) 1.5 ms; flip angle 60°, slice thickness 5 mm; matrix 256x192; 25 frames per cardiac cycle) were acquired in standard angulations: four-chamber view (4CV), two-chamber view (2CV) and a stack of short-axis slices (gap 5 mm) covering both entire ventricles from base to apex. For anatomical overview, a T2w Half-fourier Acquisition Single-shot Turbo Spin-Echo (HASTE) in 5 mm gapless axial slices covering the thorax was performed. Optimal inversion time (TI) was determined with an inversion time localizer (TI Scout) SSFP, magnetization preparation: slice selective inversion pulse, 20 ms increment for inversion recovery measurement, . The inversion time localizer was acquired 9 min after administration of 0.15 mmol Gadobutrol per kg body weight  in a mid-cavity short-axis slice. LGE imaging was performed 10–15 min after contrast agent application with a 2D T1-weighted inversion recovery spoiled gradient echo sequence  in standard angulations with corresponding slice positions to the functional imaging sequences. For all sequences, the field of view was 280–340 mm adapted to patient’s size, kept to a minimum, respectively.Image analysis for the retrospective study analysis was performed by two independent readers (eleven and five years of experience in cardiac imaging), aware of the histopathologic biopsy result, using an offline workstation CVI42 . Initial reading of the images in clinical routine had been performed by different readers, blinded to the result of the subsequent biopsy. The results of the initial reports were evaluated categorically in ‘cardiac SSc involvement’ and ‘no cardiac SSc involvement’.Contractility was evaluated according to the following criteria: Presence of hypokinesia, dyskinesia, intraventricular synchrony, and diastolic function for both ventricles respectively, in a visual qualitative analysis.2 for females, >100 ml/m2 for males; RV-EDVI >96 ml/m2 for females, >111 ml/m2 for males. The ventricular function was classified as reduced, if the ejection fraction was below the lower limit calculated as mean -2SD of the normal collective considering gender and age <60 years: LV-EF <58 % for females, <57 % for males; RV-EF <52 % for females, <50 % for males. LV-MM was classified as increased, if LV-MMI was above the upper limit determined as mean +2SD of the normal collective considering gender and age <60 years: LV-MMI >77 g/m2 for females, >91 g/m2 for males.Functional parameters were determined by volumetry of the left and right ventricle in a stack of short-axis slices according to the modified Simpson rule . End-diaAdditionally, the right ventricular diameter was measured in four-chamber view 4CV. RV dilatation was assumed if cross-section dimension in 4CV and basal short-axis was above two standard deviations of the normal value according to Hergan et al. .LV myocardial thickness was evaluated in a mid-ventricular short-axis slice in segment 9  and 11  and rated according to Kawel et al. : LV myocRV myocardial thickness was measured in the posterior wall in a mid-ventricular short-axis slice. RV hypertrophy was assumed if RV wall thickness was 5 mm or higher , 19. MaxInversion time localizer nulling patterns of the myocardium and blood pool were categorized by the nulling type patterns 1  and the abnormal patterns 2–4 as imposed by Pandey et al. : PhysiolLGE was evaluated qualitatively and semi-quantitatively. Datasets in which the Inversion Time (TI) Scout indicated an insufficient nulling of the myocardium in contrast to the blood pool were excluded from LGE quantification. For quantification of LGE contours in all short axis slices were drawn manually with care to exclude pericardial fat and blood pool at the myocardial borders. For visual reading and semi-automated thresholding, the recommendations of the Society for Cardiovascular Magnetic Resonance Task Force were adopted : signal Statistical analysis was performed using JMP . All continuous variables are expressed as mean value ± standard deviation and range in square brackets. Interobserver-variability for continuous variables was evaluated with Bland-Altman difference plots. Mean difference and the limits of agreement defined as ±1.96 SD were calculated for LV-EDV, LV-EF, RV-EDV and RV-EF. Over all categorical variables in visual analysis interobserver-agreement was measured with Cohen’s Kappa. Paired two-sided t-test was applied for null hypothesis if Kappa equals zero. Agreement in Kappa statistic was rated according to Landis and Koch : ‘moderapathologic sample/total sample for LV-EF, RV-EF, LV-EDVI, and RV-EDVI. Higher occurrence of pathologic values in the study cohort was assumed if lower 95 % CI was above 2.5 %. Normal distribution for each value was assessed visually in curves.To test if among the study cohort more than the 2.5 % of the normal collective had pathologic values, 95 % confidence interval (CI) was calculated for the quotient To determine how many pathologic findings occurred in one patient, CMR findings were categorized: 1) moderate pericardial effusion; 2) ventricular kinesic pattern ; 3) reduced ventricular function (reduced LV-EF or LV-EF); 4) suspect for fibrosis and inflammation (positive LGE); 5) right ventricular dilatation.Myocardial fibrosis was found in all patients. The mean degree of myocardial fibrosis over all patients was 12.3 % . Five patients (25 %) had an inflammation grade 1; ten patients (50 %) grade 2; five patients (25 %) grade 3. One patient had 300 copies/μg myocardial DNA of parvovirus B19 genotype 1, indicating virus persistence.In visual motion analysis, septal regional LV hypokinesia was found in 15 patients (75 %); LV intraventricular dyssynchrony in 13 patients (65 %); and LV diastolic dysfunction of a restrictive pattern with passive filling in 17 patients (85 %). Regional or global RV hypokinesia was found in 14 patients (70 %); RV dyskinesia in 2 patients (10 %); RV intraventricular dyssynchrony in 12 patients (60 %); and restrictive RV diastolic dysfunction in 13 patients (65 %). 19 patients (95 %) had pathologic LV and or RV ventricular kinesia pattern .Systemic Sclerosis.Additional file 1: SSFP loop of Fig. SSFP: Steady State Free Precession.Additional file 2: The SSFP loop depicts minimal pericardial effusion and right ventricular dilatation in a male patient in four-chamber view SSFP. The loop demonstrates intraventricular and interventricular dyssynchrony as well as restriction in diastole. The volumetric analysis indicated left ventricular dilatation in 4 patients  and right ventricular dilatation in 6 patients . Ventricular function was reduced in 14 patients: 14 patients  had reduced RV-EF; 10 of which  had both reduced LV-EF and RV-EF. Increased LV-MMI was found in two patients (10 %) by left ventricular myocardial mass volumetry  was 10.4 mm , in the inferolateral wall (segment 11) 8.1 mm . 7 patients (35 %) had septal myocardial hypertrophy (segment 9), 7 patients (35 %) had inferolateral myocardial hypertrophy (segment 11). Five of these patients had septal and inferolateral myocardial hypertrophy, 2 patients had asymmetric septal hypertrophy.RV Myocardial thickness was 3.3 mm , normal 1–4 mm in 18 patients (90 %), and pathologic 5 mm in two patients (10 %).Seven patients (35 %) had minimal pericardial effusion, nine patients (45 %) had moderate pericardial effusion >5 mm . Pathologic nulling patterns were found in nine patients 53 %): Type 2 in two patients (12 %); type 3 in five patients  had intermediate signal-intensity LGE in linear and patchy pattern Fig.  and 3, tIn the semi-quantitative analysis, pathologic LGE was found in all patients: full intensity LGE was found in 5.1 %  of the LV myocardial mass in patchy and linear patterns. Intermediate-signal intensity LGE was found in 25.9 %  of the LV myocardial mass.Inversion time for LGE imaging has been set to 283 ms  for the first image, for the last image to 312 ms . TI was elevated from first to last LGE image for 28 ms .All results are summarized by frequency in Table In evaluation of the initial clinical CMR reports blinded to subsequent EMB, the report was diagnosing ‘cardiac SSc involvement’ in 6 patients (30 %).The interobserver variability analysis indicated a mean difference for LV-EDV of -1.8 ml with limits of agreement -11.4 ml; 7.9 ml. The mean difference for LV-EF was 0.7 %, limits of agreement were -5.5 %; 7 %. The mean difference for RV-EDV was 1.7 ml, limits of agreement were -7.9 ml; 11.4 ml. The mean difference for RV-EF was 0.2 %, limits of agreement were -5.7 %; 6.1 %. Kappa statistic for interobserver-agreement in categorical variables was ‘almost perfect’ 0.913, p < 0.0001. LV-EF, RV-EF, LV-EDVI, and RV-EDVI were normally distributed.In categorical analysis of pathologic CMR findings, seven patients (35 %) had positive findings in three categories, nine patients (45 %) in four categories, and four patients (20 %) in five categories.In the present study we systematically evaluated CMR findings in a rare subgroup of SSc patients with histopathologically confirmed myocardial involvement.In CMR, pathologic findings were documented in all SSc patients, confirming a high occurrence of abnormal volumetric data as compared to normal healthy collectives 95%CI). Diastolic dysfunction is known to develop early in the course of myocardial involvement in SSc even before the appearance of clinical symptoms . The per5%CI. Dia1 evaluation with an ordinal scale by means of the inversion time localizer. Pathologic nulling patterns of inversion time localizer were common in our patient group, indicating severe diffuse myocardial fibrosis. Diffuse myocardial fibrosis frequently evades visual detection and is significantly underestimated in LGE quantification, for nulling of the myocardium suppresses general enhancement by shifting the threshold [1-mapping, the values remain unspecific for diffuse fibrosis [1- mapping for absolute quantification of fibrosis. None of our parameters is likely to correlate with fibrosis in biopsy: LGE does shift threshold of healthy towards pathologic when nulling myocardium, and functional parameters are subject to situational adaption. Extent of cardiac involvement in SSc is not necessarily dependent on other objectively verifiable parameters and subject to subclinical periods, primary or secondary involvement and influenced by cardiotoxic therapy [In visual reading and semi-quantitative evaluation, we found typical non-coronary distribution of full intensity LGE as described before: a linear enhancement ; as wellhreshold . As a refibrosis . We did fibrosis . In cons therapy , 12.CMR is accepted as one of the non-invasive modalities to investigate SSc involvement of the heart . Yet, to1-mapping should be a promising method to further investigate diffuse myocardial fibrosis in SSc involvement [As systolic dysfunction is a rare finding in SSc, the severity of the involvement tends to be underestimated . Based oolvement . T1-mappolvement .Our results might contribute to improve the non-invasive diagnosis of cardiac involvement in SSc, using CMR to identify and estimate cardiac involvement. Discrete CMR imaging patterns could be found concurrently, also in severe SSc myocardial involvement. In our retrospective evaluation, myocardial involvement in SSc was systematically underdiagnosed in the initial clinical report blinded to the subsequent histopathologic work-up with true positive diagnoses in only 30 % of our SSc cohort. The main reason may be underestimation of subtle morphologic and functional findings in SSc involvement, as this is a rare cardiac diagnosis. To date, mainly CMR findings in the maximum degree of occurrence have been published, potentially leading to a trend to underestimate subtle, yet SSc-typical, findings. Most CMR findings appear to be unspecific and can be found in a variety of diseases. However, the combination of certain findings as described in our work seems to be characteristic for cardiac SSc involvement. For the lack of correlation, CMR cannot replace EMB. EMB itself is rarely performed in a clinical setting. Application of the proposed criteria may improve assessment of clinically relevant SSc involvement. Although CMR findings in patients with cardiac involvement in SSc can easily go underdiagnosed and classified as unspecific, the combination of several CMR findings is frequent and therefore allows the diagnosis of cardiac involvement in patients with SSc by CMR: Besides distinct LGE patterns, most important findings are involvement of both ventricles, pathologic LV/RV contractility, dyssynchrony or restriction. Reading of inversion time localizer for diffuse LGE and can be easily implemented without additional technical requirements.This approach might be useful for risk stratification when considering therapeutic options, and may eventually reduce morbidity and mortality in a patient group at risk for major adverse cardiac events. Currently two studies are recruiting SSc patients eligible for autologous stem cell transplantation, one in Chicago, IL, USA and one in Tübingen, Germany  . These s2 imaging was performed, but non-quantitative T2 imaging cannot reliably depict diffuse edema [1 nor T2- mapping were available at the time patients were examined.The study population is comparably small and the study CMR image readers were biased by knowledge of histopathologically proven involvement in SSc in all patients. Only patients with severe disease were examined. Mild and limited cutaneous SSc might present with less pathologic CMR findings per individual. No Tse edema , 36 and se edema . NeitherCMR seems to be a valuable tool to identify and assess the extent of cardiac involvement and therefore might be useful for risk stratification and making a decision upon therapeutic strategies. Myocardial involvement in systemic sclerosis can be assumed if pathologic findings in three categories are found: 1) pericardial effusion; 2) ventricular kinesic pattern ; 3) reduced LV-EF or RV-EF; 4) positive LGE or pathologic inversion time localizer; 5) right ventricular dilatation.


Songs are distinct, patterned sounds produced by a variety of animals including baleen whales. Fin whale songs, which consist of short pulses repeated at regular interpulse intervals (IPIs), have been suggested as a tool to distinguish populations. Fin whale songs were analyzed from data collected from 2000–2012 in Southern California and from 2004–2010 in the Gulf of California using autonomous acoustic recorders. IPIs were measured for each identifiable song sequence during two random days of each month with recordings. Four distinct song types were identified: long doublet, short doublet, long triplet, and short triplet. Long and short doublets were the dominant songs in Southern California, while long and short triplets were dominant in the Gulf of California. An abrupt change in song type occurred in both areas during the monitoring period. We argue that each song type is unique to a population and these changes represent a shift in the primary population in the monitoring area. Occasional temporal and spatial song overlap indicated some exchange or visitation among populations. Fin whales appear to synchronize and gradually modify song rhythm over long time scales. A better understanding of the evolutionary and ecological importance of songs to fin whale populations is needed. They can also be defined as stereotyped signals that serve a reproductive function2. Songs have been documented in a variety of animals, including insects, birds, and mammals. Songs can be innate, whereby they do not need to be heard and learned through imitation, but when dialects exist they are typically acquired via vocal learning, which is the modification of sounds through experience with other individuals4. The function of song has been most extensively studied in oscine birds, which use songs for mate attraction or intrasexual competition2. As a reproductive display, therefore, song is primarily shaped by sexual selection5.Songs are distinctive sounds or sequences of sounds produced in a repeated pattern8. Complexity of baleen whale songs ranges from highly variable songs of humpback whales (Megaptera novaeangliae)6, to less complex blue whale  songs1. Fin whale  song consists of low frequency, 20 Hz pulses arranged in a regular sequence10. These songs are produced by males and are postulated to have a reproductive function7. In addition, there is evidence of regional specificity in songs that may be linked to population structure. The specificity can show itself in the presence of higher frequency pulse components12, or in the interpulse interval (IPI) durations of a song sequence, with all members in an area seemingly matching the same frequency and IPI features13.Songs are used by a number of baleen whale species14. Singlets are songs with only one distinct IPI. Doublet songs have two alternating IPIs of different lengths. Triplets also have at least two distinct IPIs, where one of them may be repeated two or more times.Based on their IPIs, fin whale songs can be classified into three broad categories: singlets, doublets, and triplets15. Additionally, in the Northeastern Pacific there is a resident population of fin whales in the Gulf of California16, and possibly another in Southern California17. Until recently, a single northern hemisphere fin whale subspecies was recognized, but mitogenomic evidence now points to two separate subspecies, one in the North Pacific and one in the North Atlantic18. It is unclear whether there will be any further subdivision to populations in the North Pacific, but song has been suggested as a possible tool for undertaking such delineation.The distribution and population structure of fin whales appears to be more complex than previously thought. Their migration patterns do not conform to the generally posited baleen whale seasonal migration from summer feeding grounds to winter breeding grounds as they have more varied movements24. Based on such analyses, we know fin whales produce 20 Hz calls year-round in Southern California, with a decrease in calling during the summer17. In contrast, these calls peak in the Gulf of California in the summer and fall25. In addition to patterned songs, fin whales also use 20 Hz pulses in irregular and call-counter call patterns that likely have a different social context than song26 and do not show obvious differences across populations. Thus analysis of simple presence of 20 Hz signals, even though useful for indication of fin whale presence, may not provide information on population structure or ecology of the species. A more detailed analysis of calling patterns is needed to reveal those elements.Previous analyses of fin whale acoustic records have generally focused on broad spatial and temporal patterns of call occurrence, or more detailed call descriptions, while grouping all 20 Hz calls into a single categoryWe used multi-year recordings from Southern California and the Gulf of California Fig.  to descrFour distinct song types were identified in the data and were termed long doublet, short doublet, long triplet, and short triplet based on the total IPI durations of the song and the patterns of intervals within the series. All four song types were recorded in the Gulf of California and three were heard in Southern California, though only two were common in each region: short and long triplets in the Gulf of California and short and long doublets in Southern California Fig. .Figure 210 as having seasonally variable IPIs. This monthly pattern of longer IPIs during the winter months and shorter IPIs in the summer months was also shown to be the dominant descriptor within the GAM framework , followed by a longer one  IPI and two longer IPIs of similar length ~10–11 s). The short triplet was the dominant call from winter 2004 to spring 2005 at the southern Gulf of California site, and it was the only triplet recorded at the northern Gulf of California sites Fig. . The IPI0–11 s. TThere was a qualitative difference in characteristics of the pulses that comprise songs in these two regions. Southern California doublet songs were comprised of two alternating pulses (notes and backbeats) with different frequency characteristics Fig. . The banAmong the four major song types there occasionally was substantial variability in the sequencing of the IPIs. Doublet songs also included singlets made up of one of the doublet IPIs. Of the days with short doublet song occurrence, 18% had singlets, while 71% of days with long doublet song had singlets. In long doublet song sequences, the longer IPI of the series was generally repeated as a singlet, whereas in the short doublet, the shorter IPI was more commonly repeated as a singlet. Notably, the long singlet (singlet variant of the long doublet) was more common than the long doublet at both the southern Gulf of California site and the later recording period in Southern California Fig. . TripletThere was some spatial separation and difference in occurrence among the four song types. For example, the long doublet and long triplet songs were never recorded in the northern Gulf of California, but occurred at the other locations Fig. . The shoFour distinct fin whale song types were present in Southern California and the Gulf of California between 2000 and 2012. Even though multiple song types were detected in both regions, in any given area during any one time, only one song type was dominant. In Southern California, the dominant song was always a doublet and in the Gulf of California it was a triplet. Within each region there was an abrupt switch between predominant song types. In Southern California there was a relatively sudden change in the dominant song type from the long doublet to the short doublet in 2006. Similarly, in 2005 in the southern Gulf of California, the dominant song switched from the short triplet to the long triplet. Additionally, our data provide an example of a gradual, consistent, long-term and population-wide slowing down of song beat (or an increase in IPI). Below, we address possible reasons for the observed song type switch and long-term song modification and discuss whether these patterns may indicate a change in population that is most common in the monitored area or if they imply song switching by a single population. Our data do not provide a definitive answer, but we argue that population change is the more likely reason due to a number of factors, including differences in seasonal and inter-annual patterns of each song type.10. It was replaced by a short doublet song with weak seasonal variability and a strong inter-annual increase in IPIs. Additionally, the seasonal occurrence of the doublet song types was quite different, with the long doublet song generally absent in summer whereas the short doublet song was common in that season. These changes indicate that fin whales in Southern California did not just change the characteristics of their song, but they also altered when they sing and how the song changes over time. Similarly, triplet songs in the Gulf of California changed from the long triplet that was more common during the winter and spring to the short triplet that was more common during the fall. A switch between song types on a seasonal basis could suggest different functions of the song within the same population. Our data indicate a major shift from one song type to another at a single point over the 12 year datasets, such that a change in song function seems an implausible explanation. Rather, it is more likely that the observed shift from one song type to another represents a shift in which population was using the monitored area, and that the shift persisted over several years.Our analysis of long-term trends revealed that each song type had distinct seasonality and inter-annual trends in IPIs. When the song type switched in an area, the temporal variability of the new dominant song type did not match patterns of the previous song type. For example, long doublet song changed seasonally, and reset annually3. If adults retain the ability to adapt their song after maturity, such as in semi-flightless passerine (Philesturnus carunculatus)27, they are engaging in open-ended vocal learning. Evidence for vocal learning in baleen whales is rare and is largely limited to changes in signal frequency28. One exception are humpback whales which synchronously change their songs across populations29, and include one documented occurrence of a population adopting the song of a new immigrant into the area30. Unlike the case of humpback whales where the whole population adopted the new song, we detected long doublet song for years after it was no longer the dominant song in Southern California. Several questions remain about the links between song and population identity in fin whales: how long are songs retained by an individual after moving into a new area; are excursions to new areas temporary or do individuals stay in new areas? To answer these questions, research matching an individual’s song characteristics to genetic or photo identifications over time is needed. Considering that occurrence of non-dominant songs in any region was a relatively rare event, however, this is likely to be a difficult and long-term task. In summary, along with the qualitative difference in characteristics of the pulses that comprise songs in these two regions, we argue all of the above factors point to a likely distinction in song types across populations.The link between song style and population identity is also supported by the geographic patterns in the detection of each song type. Each of the song types observed within our dataset was heard primarily in one region, but three of the four song types were heard less frequently in the other region, thus suggesting seasonal or other occasional movements of each group. The long triplet from the Gulf of California was recorded in Southern California, and both Southern California doublet songs were recorded in the Gulf of California. Those observations suggest that even when individuals are in an area where their song is not dominant, they do not adopt the new song but continue singing their own song. In oscine birds, songs are often learned during early development which ends in song crystallization10. The short doublet could represent a Southern California resident fin whale population. There is recent evidence for an increase in the fin whale population in this area31, as well as its residence in the region17. The short triplet song described here is consistent with song previously recorded from a resident population in the Gulf of California16. The long triplet is a new song and may indicate a population from a previously unmonitored area. The short doublet and short triplet song types are heard year-round in Southern California and the Gulf of California, respectively, and have similar seasonal peaks in occurrence . Such year-round presence of the dominant songs in each region supports the assertion that songs are produced by populations that are present year-round. Detection of these songs in other regions suggests some degree of movement among areas. The Gulf of California triplet song was heard in Southern California during the summer, while the doublet song from Southern California occurred in the Gulf of California during the winter and spring. In both cases, this pattern is consistent with the general baleen whale migration patterns: to higher latitudes during the summer and lower latitudes during the winter32. The occurrence of non-dominant songs, especially doublets in the Gulf of California, can persist in the non-dominant area for a couple of months. During that time, the non-dominant song did not show any change in pattern that would indicate adaptation to the dominant song type of the region. Observations of song types across a broader geographic region could help elucidate whether these song types truly represent populations that are primarily resident to a relatively smaller area.If we accept the hypothesis that songs indicate different populations, we could infer that four fin whale populations occurred in Southern California and the Gulf of California. One population singing the long doublet song could be an indicator of a pan-Pacific fin whale population, since that song was also recorded concurrently off Hawaii and in the Bering Sea, as well as Southern California18. Satellite tracking of fin whales from Southern California indicates that some animals may be more likely to move coastally while others travel further offshore33. Acoustic work from other locations in the Northeastern Pacific also may show separation of coastal and offshore groups. For example, fin whale song recorded along the Juan de Fuca Ridge from 2003 to 2006 consisted of pulses repeated at 25 and 30 s IPIs22, consistent with the pan-Pacific long doublet song10. The same song was also recorded off British Columbia in 2010/2011, in addition to a different song, with IPIs ranging from 11–13.4 s and 15–17.7 s and gradually increasing over time34. That latter song is not identical to the Southern California song, as the IPIs are shorter in songs off British Columbia, but it is more similar to the Southern California short doublet than the pan-Pacific long doublet song. These different songs may point to a possible distinction in songs between coastal and offshore fin whale populations across a broad swath of the Northeastern Pacific Ocean.The patterns of songs in these two regions may point to existence of fin whale populations with different ecological characteristics, with one group possibly occupying a more coastal niche and the other occurring more offshore. Mitogenomic analysis, while not conclusive, pointed to two possible lineages in the North Pacific fin whale subpopulation35. Examination of fin whale songs across inshore and offshore sites in Southern California might shed light on whether these seasonal movement patterns are reflected in song occurrence and whether they also indicate further population structure. Analysis of seven years of data (2006–2012) across Southern California showed generally higher presence of fin whale calls (not necessarily songs) in the southwestern offshore region and peak detections in the winter and spring17. Data analyzed for this paper were primarily recorded on what would be considered offshore locations and we detected songs year round. More detailed information on genetics and seasonal singing behavior across the Southern California region is needed to better understand regional population dynamics.On a finer scale, recent visual surveys in Southern California indicate seasonal movements of animals in this region. During the winter, fin whales are sighted inshore and during the spring and summer they appear to move offshore20. During that time period, there were two instances of a sudden change in IPIs in the spring, indicating this is likely a regular occurrence. Similarly, we noted a sudden decrease in IPIs in the long triplet song at the end of the recording at the southern site in the Gulf of California. Unfortunately we do not have additional data to evaluate whether this is a singular or regular occurrence in this area. Despite these challenges, it is clear that song types are reasonably consistent over long periods and may be reliable indicators of the occurrence of a population. Song with IPIs consistent with the long triplet is reported from recordings from the Gulf of California from 198714. This song appears to have changed little over that period. Multi-year data will be needed from multiple locations across a region to fully understand fin whale song dynamics.Even though we believe songs identify populations, modifications within some song types, either seasonally or long-term, may make it challenging to use song as a tool to monitor populations. Such changes within songs suggest it may be difficult to associate a song recorded during any short time period to a specific population unless there is a comprehensive catalog of song types and their characteristics. For example, one of the few papers that presented long-term time series of fin whale song patterns only included two years of data14. One possible explanation is a change in environmental conditions. Fin whales are thought to be opportunistic feeders36, but if population-specific food preferences exist they could drive such population movements. Prey changes, driven by events such as the El Niño Southern Oscillation, are known to affect the distribution of other baleen whale species37. More detailed information on the food preferences of different fin whale populations would be needed to evaluate if changing ocean conditions, such as for example the occurrence of a brief La Niña in 2005/0639, could have caused a shift in these populations.Assuming fin whales did not switch their song, but these sudden changes in 2005 in the Gulf of California and 2006 in Southern California indicate a shift in the occurrence of a population, why did the populations suddenly shift? We know from previous reports that abrupt disappearance of songs can occur. For example, in the Gulf of California, a fin whale song with 9–12 s IPIs was recorded in 1985, but it was not reported again in 198740, so the same may be true for other animals. For fin whales, that could mean different patterns in song complexity such as variable arrangements of singlets, doublets, and triplets throughout a song. While we observed significant variability in sequencing within each song type, we did not perform additional analysis on these features because we could not make confident assumptions on the individual producing consecutive sequences. Individual variability may also be apparent as variation in amplitude or frequency of the signal. We did not analyze these features for this work because autonomous recorder data are not ideally suited for such detailed analysis as it is not possible to know how much variability in those features results from propagation effects versus actual individual variability. Acoustic tags deployed on calling whales would be a better place to start teasing apart these finer scale patterns and answering individual-level questions.Beyond using song as a tool for population identification, it may also be possible to identify individual whales by evaluating differences in song sequencing, or variations in amplitude and frequency between song sequences. It has been shown that small deviations in song pattern can indicate individual identity in birdsSome of the challenge in interpreting our data comes from changes of recording location over time, both in Southern California and the Gulf of California. For example, it would have been informative to have concurrent recordings in the southern and northern parts of the Gulf of California. Also, more recent recordings from those areas could indicate whether the triplets recorded during 2000s have persisted or if they have changed. In Southern California, the gap in recordings during 2004 happened during a time of change in song types. Analysis of a more northerly site in 2005 and 2006, since those were the only available data from the region during that time, may have introduced some small-scale regional complexity to our data interpretation. However, preliminary analysis of data from the more northerly site since 2006 shows patterns that very closely match those at the southern site reported in this paper, therefore we believe the sites to be comparable. In any case, consistent recordings from a single site over a long time scale would reduce uncertainty in interpretation that occurs when time and space get confounded due to gaps in recordings.10. When rhythm synchronization was studied in other organisms, synchronization of faster rhythms (>1 Hz) was typically investigated. The ability to anticipate such rhythms has been observed only in humans and a few animal species, including a California sea lion 41. However, the neural mechanisms involved in subsecond and suprasecond timing are quite distinct42. Even though perception of rhythm is not common among animals43, some examples of broad synchrony in rhythmic communicative signals include songs of whales44 and indris (Indri indri)45 or footdrumming of kangaroo rats (Dipodomys spectabilis)46. We are not aware of examples of rhythm synchronization over long time periods or of long-term systemic drift in the song rhythm. However, synchronization typically indicates a strong social interaction and may show group unity, bonding, and increased cooperation as generation of a complicated rhythmic display is perceived as an honest signal of coordination and time together47. In addition, matching of changing songs indicates a level of cultural transmission48. Cultural transmission has been documented in other baleen whales; for example, elaborate songs of humpback whales are modified through horizontal cultural transmission29. More detailed data on the interactions between singing fin whales would be necessary to shed light on mechanisms for this synchronization, which in turn may lead to a better understanding of the ecology, reproductive biology, and social structure of these populations.Another intriguing question coming from these data is how do fin whale populations synchronize song over large scales and gradually modify the IPI. This synchrony was also noted for fin whale songs across an ocean basinFin whale songs exhibit more complexity than evident from cursory investigations and our understanding of their occurrence and meaning is still rudimentary. In the future, to move beyond description of the songs themselves, studies that incorporate a larger variety of data , as well as approaches from neuroscience, behavioral studies, and ecology, would be most likely to lead to an increased understanding of this complex system, how it evolved, and its importance to the survival of this endangered species.49 and they were analyzed following procedures described in Oleson et al.10. Since 2004, all data were collected at higher sample rates using High-frequency Acoustic Recording Packages50. While most recordings were collected continuously at 200 kHz, several data sets were recorded on a duty cycle and at a lower sample rate  were calculated for each picked sequence. Sequences with similar IPI patterns during each day were identified during this post-processing. First, the arrangement of pulses occurring together with similar timing  was assessed to create song categories. Then IPIs of songs in the same category were pooled and sorted to separate short and long IPIs, which typically did not overlap. The daily median, and first and third quartiles for each IPI of each distinct sequence type were calculated. Based on the medians, songs were further classified into short or long versions of the song.51. The link function of the GAM relates the mean of the response variable to the predictor variable. Two predictor variables used for the descriptive models were month and year during which the IPIs were recorded. Since the median IPIs were not normally distributed, we assumed a quasi-Poisson distribution in the response variable (IPI) and a logarithmic link function. The year was fit as a spline with an upper limit of three degrees of freedom and month as a cyclical cubic regression with a limit of four degrees of freedom. Model results for each individual variable and both of them combined were evaluated and the best model fit was determined based on the generalized cross validation (GCV) score, while also minimizing the number of variables used. Only data from the dominant geographic region where the song was detected were used for this analysis.Trends in IPIs for each song type were evaluated to examine how song patterns change across seasons and years. To describe this change, we developed generalized additive models (GAMs) of short and long IPIs of each song type using the mgcv library in RStudio statistical software package (Version 0.98.507). GAMs are extensions of generalized linear models; they allow the additive predictors to be described by nonparametric smooth functionsIn addition to temporal trends in IPIs, we investigated overall spatial and temporal patterns in song occurrence. We calculated the frequency of occurrence of each song type from pre- and post-2004 recordings in Southern California, and from southern and northern recording locations in the Gulf of California. The frequency was calculated as the number of days of occurrence of a particular song divided by the total number of days analyzed for that period and location. To evaluate seasonal patterns in song occurrence in both regions, we summarized the monthly occurrence of each song type at each location by calculating the percentage of days analyzed during which that song type was identified.Supplementary information


Moreover, longitudinal strain rate, circumferential strain and E/E′ ratio were significantly impaired in IBD. Pediatric patients with IBD feature subclinical signs of LV systolic and diastolic myocardial impairment early in the course of CD and UC. This may not be reversible even when IBD is clinically controlled. Patients with IBD should be regularly screened for signs of CVD.Inflammatory bowel disease (IBD) is an established risk factor for cardiovascular disease (CVD). However, whether cardiac consequences present early in IBD is unknown. This is the first study in children aiming to unmask altered myocardial mechanics in IBD. We enrolled 50 consecutive normotensive children with Crohn’s disease (CD) (n = 28) or ulcerative colitis (UC) (n = 22). The study groups consisted of 18 patients with active inflammatory disease (mean age 14.6 ± 2.5 years) and 32 children with IBD in remission (14.3 ± 2.3 years). 60 age- and gender-matched children served as healthy controls. Speckle tracking stress echocardiography (STE) was used to assess left ventricular (LV) myocardial strain and strain rate. Circumferential strain rate was significantly decreased in children with active IBD (−1.55 ± 0.26 s While CD and UC are characterized by specific, clinically different characteristics, both diseases have a chronically-remitting inflammatory process of the gastrointestinal tract as well as potential extra-intestinal manifestations in common6. IBD is an established risk factor for cardiovascular disease (CVD)7. Patients with IBD were shown to have a twofold to threefold increased risk for the development of venous thromboembolism8, specifically during acute disease flares when a disturbed homeostasis of anti- and procoagulants results in a hypofibrinolytic state9. Other known concomitant cardiovascular (CV) manifestations of CD and UC include Takayasu’s arteritis11, pericarditis and myocarditis13. The predominant underlying processes of CV pathology in IBD are currently thought to be the chronic exposure to the persistent/remittant inflammation and an altered lipid metabolism on the one hand as well as undesired adverse effects of long-term administered antiinflammatory drugs such as corticosteroids, TNF-α inhibitors, etc. on the other hand14. Furthermore, chronic inflammation has been demonstrated to be accompanied by pathological collagen disposition in affected organs16. Whether this pathologically altered collagen disposition process also affects other organs, e.g. the heart in IBD, yet remains to be illuminated. Interestingly, studies in both adult and pediatric patients have demonstrated an association of IBD and early signs of subclinical atherosclerosis18. Moreover, Doppler-based ultrasound studies have been utilized to reveal both increased arterial stiffness and carotid intima-media-thickness (IMT) in adult patients with CU19. Conventional echocardiographic studies focusing on left ventricular (LV) function, however, have yet failed to convincingly detect overt cardiac involvement in patients with IBD without other confounding CV risk factors.Inflammatory bowel diseases – essentially comprised of Crohn’s disease (CD)20. STE has been successfully used to identify early subclinical cardiac involvement in a variety of asymptomatic populations with chronic diseases and unremarkable conventional echocardiography. Representative examples include adult patients with arterial hypertension21 and children with uncomplicated type 1 diabetes mellitus22. Furthermore, STE is sensitive enough to detect discretely altered myocardial contractility due to transient changes in serum glucose levels23. Studies in adult patients have utilized STE to demonstrate subclinical cardiac impairment in both UC25 and CD26. However, whether myocardial function is already pathologically changed in pediatric patients with IBD is still unknown. This is the first study in children aiming to utilize STE in CD, UC and healthy controls to uncover subclinical myocardial impairment in an early state of IBD.Speckle tracking echocardiography (STE) is an advanced echocardiographic methodology for the quantification of myocardial function28. Study group patients were stratified into sub-groups of active disease and IBD in remission by two experienced pediatric gastroenterologists according to disease activity indices, endoscopy results, degree of mucosal healing, clinical course and individual well-being. Primary exclusion criteria were other past or present health conditions likely affecting the cardiovascular system such as LV dysfunction, acquired valvular disease, congenital heart disease, kidney disease, developmental delay, body mass index >30 kg/m2, pathologic ECG-changes at rest or during exercise as well as technical limitations such as poor echocardiographic image quality, submaximal effort during exercise testing or short leg length. In addition, all patients with unclear underlying disease were excluded from the study. Several primarily enrolled patients were excluded from the study during the echocardiographic examination due to valvular disease (n = 1), poor echocardiographic image quality (n = 12) or inadequate cycling effort (n = 2).110 children aged 6 to 17 years were enrolled for this study. The study group consisted of 18 consecutive patients with active IBD  and 32 patients with IBD in remission  who are being followed up at the Pediatric Gastroenterology Clinic at Helios University Medical Centre Wuppertal, Germany. 60 healthy age- and sex-matched volunteers  served as the control group. 22 patients had been diagnosed with CD  and 28 had UC . Inclusion criteria for the study group were the definite diagnosis of CD or UC, which had been based on standard clinical, radiological, endoscopic and histological criteria findings in accordance with the revised Porto criteria and Montreal classificationclinical trial number: 103/2014).Healthy controls had a completely negative medical history both regarding the cardiovascular and gastrointestinal as well as any other organ system. Initially, a thorough history and physical examination as well as both resting and exercise standard echocardiography and ECG were obtained in all study subjects. Every participant as well as their legal guardian signed a written informed consent prior to inclusion in the study. The study sample size was achieved by enrolling all patients from the hospital’s Pediatric Gastroenterology-Inflammatory Bowel Diseases Section who agreed to participate. A priori, a study design was established dividing the study population into subgroups of IBD patients with acute inflammation and clinical remission. The study was carried out in accordance with the declaration of Helsinki’s ethical principles for medical research involving human subjects and approved by the Witten/Herdecke University ethics committee  – excluding the endoscopic components30. In order to achieve a comprehensive characterization of the study group, disease activity for UC patients was furthermore assessed with the pediatric ulcerative colitis activity index (PUCAI)31.In 25 patients, a standard Mayo score was utilized32. Accordingly, it was utilized for all subjects, in which invasive PCDAI-required diagnostics have been obtained more than four weeks prior to the echocardiographic study (n = 22).The pediatric Crohn’s disease activity index (PCDAI) was used to assess the burden of disease for 19 CD patients. Similarly to the partial Mayo score in UC, there is a less invasive form of the PCDAI: the abbreviated PCDAI, which has been shown to correlate well with the complete PCDAI33. The commercially available ultrasound device iE33 by Phillips Ultrasound Inc., USA, with a S5-1 Sector Array transducer (Sector 1–5 MHz) was used. All images were digitally recorded and subsequently transferred to an offline workstation for analysis, using XCelera Version 3.1.1.422 by Phillips Ultrasound Inc., USA. Image acquisition was carried out in the apical 4-, 3- and 2-chamber views, the parasternal long axis view and in two short axis views at the mitral level and at the level of the papillary muscles. M-mode images were obtained at the level of the aortic valve and the LV for subsequent measurement of aortic root diameter, left atrial diameter, interventricular septum, LV cavity and LV posterior wall. Fractional shortening, LV mass, relative wall thickness, LV enddiastolic/endsystolic volume, EF, stroke volume and cardiac output were assessed. Utilizing pw-Doppler and pw-TDI E/A-ratio, E/E′-ratio and mitral deceleration time were detected for the assessment of LV diastolic function as described elsewhere34. All echocardiographic parameters were evaluated utilizing Z-scores35.All enrolled subjects underwent a comprehensive echocardiographic study including spectral and color flow Doppler examinations according to the standard guidelines of the American Heart Association36. Specifically, circumferential strain (CS) was measured in the standard parasternal short-axis at the mitral valve plane (SAXB) and the papillary muscle plane (SAXM). As recently suggested, five consecutive cardiac cycles synchronized to a continuous ECG were recorded with frame rate adjusted between 60 and 90 frames per second37. To achieve accurate deformation parameters, a special focus was set to avoid noise and minimize artifacts during the entire process of echocardiographic image acquisition. Data was anonymized, digitally stored in DICOM format and transferred to an off-line workstation for postprocessing utilizing the commercially available software QLAB Version 10. All echocardiographic examiners and interpreters were blinded to the study group status of the participants. Global and segmental strain and strain rate were assessed in seven segments per view for longitudinal strain (LS) and six segments for CS by manual tracing of the endocardial border line at end-systole. Tissue tracking quality was verified in real-time and full thickness coverage of the myocardium including the endocardial and epicardial contours was readjusted manually where necessary. Inter- and intraobserver variability was assessed by additional evaluation of resting and exercise echocardiographic images by a second independent interpreter, who was blinded to the study group status and the results of the first echocardiographic reader. The results were reproducible and inter-/intrarater variability was below 6%.Standard cross-sectional 2D grayscale LV images were acquired for myocardial deformation (strain and strain rate) analyses. Using conventional B-Mode imaging longitudinal strain and strain rate were measured in standard apical 4-chamber (AP4), 3-chamber (AP3) and 2-chamber (AP2) views as previously described in detailTo unmask potential abnormalities in myocardial performance that might remain undiscovered at rest, we additionally exposed the study population to bicycle ergometer stress testing and performed STE. The children were asked to peddle in a supine position on a standard bicycle ergometer at 60 rounds per minute against a ramp protocol with inclining resistance. Echocardiographic images were acquired at an intermediate (approximately 0.5–1 Watt per kilogram body weight) and at the maximum level of physical exhaustion (approximately 2 Watts per kilogram body weight). A standardized pattern of consecutive images was acquired at the above-mentioned viewing planes. Peripheral blood pressure measurements were obtained at 2-minute intervals and a 12-channel ECG was continuously monitored.Demographics, clinical parameters, hemodynamic and echocardiographic data were described as mean and standard deviation. Clinical, hemodynamic and echocardiographic data of the three groups were compared utilizing the ANOVA Test. P-values < 0.05 constituted statistical significance. The data distribution was graphically displayed using Box-Whisker-Plots. Linear regression analyses and Pearson’s correlation were performed to assess the potential association of echocardiographic variables and clinical parameters. GraphPad Prism Version 6  and Microsoft Excel Version 16.0 for PC were used for all statistical analyses.Baseline clinical characteristics are outlined in Table Most patients were on oral 5-ASA , corticosteroids  and azathioprine . Other IBD medications used included TNF-alpha inhibitors, 6-mercaptopurine, cyclosporine and probiotics Table . ConventMost conventional echocardiographic parameters including LV ejection fraction and estimated LV mass were similar in children with IBD and in healthy controls Table . Patient−1) and those with IBD in remission (−1.49 ± 0.26 s−1) in comparison to healthy volunteers (−1.8 ± 0.4) at rest (p = 0.001). A representative echocardiographic example is demonstrated in Fig. −1) and IBD in remission . Similarly, longitudinal strain rate was decreased in patients with IBD throughout all analyzed echocardiographic view planes both at rest and during stress testing without reaching statistical significance .Results from LV strain analyses showed an analogical tendency of continuously depressed circumferential myocardial performance in IBD versus healthy controls both at rest and during bicycle ergometry testing. However, these differences were not statistically significant. Peak LV longitudinal strain was unchanged in children with IBD and healthy controls in the resting (p = 0.807) and exercise state (p = 0.566). LV myocardial performance parameters did not differ significantly between children with CD and UC  in combination with ergometer stress testing in asymptomatic normotensive pediatric patients with IBD and healthy controls. To determine the significance of inflammatory disease activity, a priori, IBD patients were stratified into subgroups of children with active disease and IBD in remission. The reason for this subdivision was the underlying concept of inflammation as a potential impairing factor for myocardial contractility in these patients. Cardiac impairment has been demonstrated before in other autoimmune inflammatory diseases such as psoriasis24. In detail, the authors report of altered longitudinal deformation, while circumferential strain rate was not assessed. In our study, longitudinal strain rate was also impaired in IBD patients, without reaching a degree of statistical significance (p = 0.123). Our findings are further in line with a conventional two-dimensional echocardiographic study from 2016, in which subclinical cardiac involvement was demonstrated in adult patients with CD and UC25. Interestingly, while the study reports IBD patients to feature significantly lower left ventricular ejection fraction and altered enlarged LV morphologic parameters, there was no difference between CD and UC patients, which is also in accordance with findings from the present study (data not shown). As disease duration was short (on average ≤ 3 years) in our study cohort, it is not surprising that only subtle changes of LV performance were detectable and not all dimensions of myocardial deformation exhibited the same degree of changed cardiac dynamics. Importantly and without exception, LV peak circumferential strain rate was depressed in IBD patients both at rest and during exercise testing in all analyzed echocardiographic view planes representing the entirety of the LV , strain rate is considered the more robust index for the non-invasive quantification of LV contractility46. Therefore, firstly, strain values may have likely been more distorted and thus resulted in less pronounced differences between the analyzed groups. Secondly, as atrial electromechanical conduction has also been shown to be prolonged in IBD, and exposure to chronic inflammation may result in structural and electrophysiological changes in the atrial tissue that causes slow conduction47. A similar pathomechanism can likely take place in the ventricles as well. We hypothesize, that IBD-mediated alterations of cardiac mechanics may cause a delay in myocardial fiber deformation rather than overtly impaired total LV contractility . Delayed LV peak myocardial contractility can be measured using strain rate, but not with strain.The phenomenon of significantly altered LV peak strain rate and merely unchanged cardiac strain can be explained by two co-occurring mechanisms. Strain describes the percent change in systolic (and diastolic) myocardial length in relation to its end-diastolic state . Strain rate is the temporal integration of strain, measured in sCuriously, one patient was initially enrolled as an asymptomatic healthy control subject. 3 months later, she became symptomatic with abdominal pain and recurrent diarrhea. Upon diagnostic work-up she received the diagnosis of CD and was included into the study group with a disease duration of 5.5 months Figure . Interes48. Furthermore, CD was associated with impairment in LV global longitudinal myocardial function and Crohn’s Disease Activity Index was correlated with LV global longitudinal strain in in adult patients26. Strain rate was not assessed in that study. In the present study, strain and strain rate in patients with active inflammation and those with IBD in remission did not differ widely with the exception of a weak correlation between PUCAI and global circumferential strain rate under stress in UC patients; most probably due to the discrete nature of the cardiac alterations in these patients. Hence, myocardial impairment in this patient population may be too subtle to result in overt differences between inflamed IBD and patients in clinical remission. The key difference between this study and other studies investigating LV performance in IBD patients is firstly, that our study cohort consists of children and secondly the short disease duration of less than three years on average. Subsequently, in this short time span myocardial performance in IBD patients in remission may not have recuperated (yet) to a superior level when compared to patients with active IBD. Therefore, marked differences that have been observed in adult patients with longer disease duration may not be detectable in pediatric patients in the early disease phase. However, even though the observed correlation between disease activity and global circumferential strain rate in UC patients is discrete and should not be over-interpreted, it might constitute an early sign of this association. Follow-up studies must be performed to assess the dynamic evolvement of myocardial performance in relation to IBD activity.Interestingly, both pediatric patients with active IBD and those with IBD in clinical remission exhibited signs of depressed myocardial performance. While several viewing planes revealed lower LV deformation in inflamed IBD, overall, there were no marked differences between the two subgroups. At first, this might seem somewhat counterintuitive given the concept that inflammatory activity ought to be the most relevant causative factor for cardiac alterations in these patients – e.g. recent studies have demonstrated IBD activation to feature significantly depressed coronary flow reserve50. Similarly to the above outlined rather discrete differences in LV systolic performance indices between IBD patients and healthy volunteers, diastolic dysfunction also only manifests in a subtle manner. Therefore, E/A ratio was most probably not yet significantly changed in IBD patients as disease influence was not (yet) long-acting in this study population. Nevertheless, it is still both surprising and important that impaired myocardial relaxation can already be measured in patients with IBD duration of less than three years. This is further in agreement with another study from 2016 reporting of degenerated LA volume and mechanical functions and increased atrial electromechanical delay in adult patients with UC – all of which was related to duration of disease51.The E/E′ ratio was significantly increased in IBD patients (p = 0.031) as compared to healthy controls Table , represe52. Nevertheless, the biological relevance of this finding is unlikely to be high as there were no statistical differences of posterior wall diameter, end-systolic septal diameter or overall estimated LV mass between the study groups and all conventional echocardiographic parameters were within normal limits as evaluated by Z-scores.As expected, conventional echocardiography yielded only minor differences between the analyzed study groups. We observed a slightly increased statistically significant, yet clinically irrelevant interventricular septal end-diastolic diameter in IBD patients Table . The use19. Moreover, in a pediatric study, IBD patients showed signs of premature endothelial dysfunction, increased carotid intima-media thickness (IMT) and decreased flow-mediated dilation of brachial arteries17. In contrast, IMT was not significantly different between the study groups. This is most probably due to shorter disease duration in our study cohort and the use of a standard b-mode ultrasound device which may not be sensitive enough to assess subtle changes in carotid artery IMT .UC in adults has been associated with both increased arterial stiffness and carotid intima-media thicknessPediatric patients with CD and UC show subclinical signs of impaired LV systolic and diastolic myocardial performance early in the course of the disease. LV function is altered both in patients with active inflammation and in IBD in remission. Long-term studies are needed to verify these subtle findings and to build correlations to clinical outcome parameters. This underlines the importance of cardiovascular prevention in the day-to-day clinical care of patients with IBD.Tables S1–2 and Figure S1


These results contrast previous observations of body mass index as a significant predictor of incident diastolic dysfunction and increased left ventricular mass index among subjects without prevalent diabetes. We discuss potential explanations for the observed discrepancies and general difficulties associated with cardiovascular risk assessment based on body mass index and related metabolic factors.A recent study found that among individuals with a preserved left ventricular ejection fraction ≥ 55%, global longitudinal strain was significantly lower in overweight patients (i.e., body mass index ≥ 25 kg/m It was suggested that the development of heart failure with preserved EF may be prevented by controlling overweight in patients with T2DM. We believe that this study warrants further discussion.We have read with great interest the study by Suto et al. on how overweight might affect left ventricular (LV) function in subjects with and without type 2 diabetes mellitus (T2DM) . The autThe results contrast previous observations of BMI as a significant predictor of incident diastolic dysfunction and increased LV mass index among subjects without diabetes at baseline . AlthougNumerically, it may seem as if the presence of overweight reduced GLS to a greater extent among patients with T2DM as compared with non-diabetics, and this is certainly supported by the Cardiovascular Continuum concept, by which hyperglycemia accelerates the cardiovascular aging process . HoweverFinally, in the previous study, BMI was independently associated with incident echocardiographic abnormalities at the expense of insulin sensitivity . The fre


Sepsis could produce myocardial depression and typically it affects the left ventricle (LV). Sepsis could also affect right ventricle (RV), in addition to the interdependence with LV. RV pressure may be elevated secondary to pulmonary vasoconstriction, leading to RV dysfunction. Unlike LV, RV is poorly prepared to compensate for acute overload. Aggressive volume replacement may be vital to maintain RV function, but excess hydration can cause RV dilation, decreased LV diastolic filling, and reduced cardiac output. In patients having signs of inadequate cardiac output even after initial volume resuscitation, RV function should be assessed with echocardiogram. If RV dysfunction is noted, then fluid therapy should be guided by CVP measurements. If cardiac output increases with increasing CVP, maintaining higher filling pressures on the right side is indicated. On the other hand, increasing CVP with worsening of the cardiac output could worsen the RV dysfunction. In addition to the fluid management, treatment of other reversible causes like acidosis and hypoxia is also a key. There is growing evidence that sepsis-associated cardiac dysfunction is not limited to the Left Ventricle (LV) but often involves the Right Ventricle (RV) as well. Normally, the RV has little trouble keeping up with the LV; however, in the presence of decreased contractility, increased afterload, or insufficient filling, which are three conditions commonly associated with sepsis, RV output may be unable to keep pace with increased LV output. Persistent RV dysfunction in sepsis has been associated with worse outcome. We report a case of systemic sepsis induced right ventricular (RV) failure, pulmonary hypertension and eventual improvement.Fifty-one-year-old Caucasian female was admitted to our hospital with complaints of fever and chills for three days and low back pain for two days. Past medical history included recent spinal abscess followed by implantation of spinal nerve stimulator, mitral valve prolapse and GERD. Her medications included omeprazole and acetaminophen as needed for chronic back pain. On physical examination, patient appeared sick. Vital signs: BP 80/50 mmHg, Pulse 120/min, respiratory rate 24/min. No skin lesions; neck was supple. Jugular veins were distended up to angle of the ear with prominent ‘a’ and ‘v’ waves. Her P2 was loud; No murmurs or gallop; lung fields were clear with normal vesicular breath sounds. Abdomen was soft and non tender. She had increased warmth and tenderness at the level of D10 - D12 vertebrae. Neurological examination was normal. Her labs revealed WBC count of 15,000 with bandaemia of 20%, lactic acid of 45 mgs%. Her EKG revealed sinus tachycardia. Serial troponin levels were normal. She was treated with broad spectrum antibiotics, intravenous fluids and dopamine as infusion. Her blood culture was positive for Staphylococcus aureus. Her echocardiogram revealed small left ventricle with ejection fraction of 65%, flattened interventricular septum, normal left atrium (LA), bowing of the interatrial septum to the LA, right atrial (RA) and RV enlargement with severe hypokinesia of RV . EstimatEarly circulatory response to sepsis and septic shock is characterized by vasodilation and increased vascular permeability, which leads to a marked reduction in intravascular filling pressures. As a result, LV ejection fraction (LVEF) often decreases during sepsis . Unlike Right ventricular dysfunction is common in sepsis and septic shock because of decreased myocardial contractility and elevated PVR despite a concomitant decrease in systemic vascular resistance. Aggressive volume replacement may be vital in maintaining RV function, but excess hydration can cause RV dilation, decreased LV diastolic filling, and reduced cardiac output. The mainstay of treatment for acute right heart dysfunction in the setting of sepsis should concentrate on fluid repletion, monitoring for signs of RV overload, and correcting reversible causes of elevated PVR, such as hypoxia, acidosis, and lung hyperinflation.


Regeneration of the early postnatal heart following resection or ischemic infarction involves replacement of lost myocardium and vasculature with restoration of normal myocardial thickness and architecture, with long-term normalization of systolic function. Why would the heart permanently forego such a remarkable regenerative program shortly after birth? The answer may lie in within the fundamental principal of evolutionary tradeoff.Heart failure is a costly and deadly disease, affecting over 23 million patients worldwide, half of which die within 5 years of diagnosis. The pathophysiological basis of heart failure is the inability of the adult heart to regenerate lost or damaged myocardium. Although limited myocyte turnover does occur in the adult heart, it is insufficient for restoration of contractile function (Nadal-Ginard,  The Origin of Species stated that “The whole organism is so tied together that when slight variations in one part occur, and are accumulated through natural selection, other parts become modified. This is a very important subject, most imperfectly understood.” He captured the concept of evolutionary tradeoff perfectly by saying that “in order to spend on one side, nature is forced to economize on the other side.” This concept may hold the key to understanding why the adult mammalian heart does not regenerate, while the fetal and neonatal hearts can  estimates the divergence of penguins from other birds began approximately 61–62 million years ago  fossil from approximately 56.3 million years ago suggests evolutionary changes to became far more hydrodynamic  to unguligrade (hoofed) animals. The digits of ancestors of the horse allowed it to maneuver on complex surfaces, which translated into greater agility, while the later hoof was superior for running long distances on flat surfaces, which translated into greater speed and endurance. With the development of hoofs, horses lost much of their agility and their ability to browse and maneuver in confined spaces. As a tradeoff, however, they gained great speed and endurance, which was probably needed to escape predators Forsten, .To determine whether the loss of the endogenous cardiac regeneration ability of the mammalian heart is a form of evolutionary tradeoff, we must carefully consider certain aspects of mammalian heart evolution, the events that occur at the time of cell cycle arrest of postnatal mammals, and the mechanism of cardiomyocyte turnover in the adult heart.The vertebrate heart has undergone a significant degree of evolutionary changes. In the earliest vertebrates, the heart was a low-pressure contractile vessel, which later developed into a two-chamber heart in early fish. Further evolution in fish, amphibians, birds and mammals later resulted in septation, chamber separation and marked thickening of the ventricles with increased ventricular pressure. In addition to the morphological changes, cardiac evolution was associated with a several fold increase in heart rate, which is needed to sustain high cardiac output. Therefore, the mammalian heart evolved from a single low-pressure tube, to the complex, high-pressure, high heart rate, multi-chamber organ of adult mammals.In the evolution literature, the concept of space constraint is hypothesized as a major determinant of loss of the ability of cardiomyocytes to divide. While this may certainly be a factor, it does not explain the permanent loss of cardiomyocyte proliferative ability. In other words, space constraint may in fact stop cardiomyocyte proliferation, but why would it prevent any ability of cardiomyocytes to divide even after substantial cardiomyocyte loss? Another argument against that concept is the fact that the mammalian heart increases in size during postnatal development by several folds through hypertrophic cardiomyocyte growth. This occurs following the permanent cell cycle arrest of cardiomyocytes, and therefore argues against the space constraint hypothesis. In other words, if the heart still has to grow several folds during postnatal development, why does it do so through hypertrophic rather than hyperplastic growth?2 of 32–35 mmHg, the blood ejected from the left ventricle is only 65% saturated with a PaO2 of 25–28 mmHg  is critical for their maintenance and function. Therefore, we reasoned that stabilization of Hif-1α identifies cycling cardiomyocytes within the uninjured adult heart. We generated a transgenic mouse that expresses a fusion protein where the oxygen dependent degradation (ODD) domain of Hif-1α is fused to the tamoxifen inducible CreERT2 under the cardiomyocyte-specific MHC promoter, thereby fate mapping hypoxic cardiomyocytes and their progeny after tamoxifen administration. Shortly after the tamoxifen pulse, we identified a rare population of hypoxic cardiomyocytes that contributed widely to new cardiomyocyte formation in the adult heart. Intriguingly, these hypoxic cardiomyocytes displayed characteristics of fetal/neonatal cardiomyocytes such as smaller cell size, mononucleation and absence of oxidative DNA damage. These findings support the hypothesis that cardiomyocyte turnover in the postnatal mammalian heart is inhibited by oxidative metabolism and the resulting oxidative DNA damage.Evolutionary tradeoffs can manifest as a specific cost (loss or gain) of a functional trait. Current evidence suggests that loss of the regenerative ability of the postnatal mammalian heart might be a manifestation of a one-trait evolutionary tradeoff, where the cost of higher energy efficiency is loss of cardiomyocyte proliferation. Conceptually, this has important implications for cardiac regeneration therapies aimed at inducing adult cardiomyocyte proliferation. For example, it may not be possible to regenerate the adult mammalian heart without either profoundly altering the redox state of cardiomyocytes, or foregoing at least some of the energy advantage provided by mitochondrial respiration.This work is supported by a grant from the Hamon Center for Regenerative Science and Medicine, University of Texas Southwestern Medical Center. HS is supported by National Institute of Health grant 1R01HL115275-01. NL is supported by a Sir Keith Murdoch Australia to US Fellowship from the American Australian Association.WE, SX, MH, and NL researched the topic and helped write the manuscript. HS conceived the hypothesis and edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In current clinical routine, functional heart parameters are derived from breathhold CINE MRI. To enable free-breathing acquisitions and imaging e.g. in highly arrhythmic patients, realtime imaging is gaining interest. The increasing clinical availability of this new technique demands evaluation of functional parameters compared to the gold standard .7≍23.628°) [14 patients with different cardiac pathologies were randomly selected. All patients underwent our clinical routine imaging protocol and additionally real-time (RT) imaging in three short axis slices . RT imaging was performed with a radial Tiny Golden Angle Ψ7≍3.628° [23 and temporal resolution of about 60 ms (CS) and 30 ms (REF).Reconstruction from the retrospective gated CINE data (REF) were compared to compressed sensing realtime reconstruction via TyGRASP  (CS). ReESV, EDV, SV, and EF were determined for LV and RV in the different reconstructions from the slices CS and matching slices of REF. Functional parameters were compared via correlation  and Bland-Altman analysis. Further analysis was performed by pairwise t-tests .2 > 0.7; all p < 5%) can be appreciated for all correlations. Only small average biases with slight underestimation of parameters in CS compared to REF are present both for LV and RV. T-tests showed no significant difference between parameters determined from CS or REF.Figures The findings are in line with , where n


There is a persisting debate regarding indications and timing of pulmonary valve replacement in patients with pulmonary insufficiency after repair of Tetralogy of Fallot (ToF). The status of right ventricular function is important is this decision but remains elusive. 4D-flow CMR enables quantification of ventricular kinetic energy (KE) which may provide improved understanding of the pathophysiology of the dilated ventricle with impaired function. In this study we wanted to quantify ventricular KE in patients with pulmonary insufficiency after repair of ToF.Nine patients with pulmonary insufficiency after repair of ToF  underwent cardiovascular magnetic resonance (CMR) with a 1.5 T Philips scanner including four-dimensional phase-contrast flow sequence. Eight healthy volunteers  were used as controls. Ventricular segmentation was performed in cine ssfp images, 30 time frames per cardiac cycle covering the heart and imported to the 4D flow dataset. Ventricular KE calculated as KE=½mv² was summed over all voxels inside the right and left ventricle and calculated for each time frame.Peak systolic KE, as well as KE indexed for the stroke volume (SV) of the ventricle was lower in the left ventricle of ToF patients than in controls. Peak diastolic KE was higher in the right ventricle of patients than in controls, also when KE was indexed for SV, Figure ToF patients have decreased left ventricular systolic KE compared to controls, indicating left ventricular affection in ToF patients, possibly through septal dyssynchrony. During diastole, right ventricular KE is increased compared to controls, most likely caused by the pulmonary regurgitation. Thus, 4D-flow MRI offers new insights in ToF physiology and inter-ventricular interaction.


The myocardial lactate extraction ratio (LER) significantly decreased after the ACh test in both groups , significantly more so in the LGE-positive group (p = 0.017). Multivariate logistic regression analysis showed that a post-ACh LER < 0  was a significant predictor of LGE-positivity . In the LGE-positive group, an LGE volume greater than the median significantly predicted a post-ACh LER of <0 . ACh-provoked coronary vasomotor abnormality is closely correlated with myocardial fibrosis in patients with HF but without organic coronary stenosis. Coronary vasomotor abnormalities in fibrotic myocardium may worsen HF.Myocardial fibrosis and microvascular dysfunction are key determinants of outcome in heart failure (HF); we examined their relationship in patients with HF. Our study included 61 consecutive patients with HF but without coronary stenosis. All underwent gadolinium-enhanced cardiac magnetic resonance to evaluate late gadolinium enhancement (LGE) and an acetylcholine (ACh) provocation test to evaluate microvascular dysfunction. During the ACh provocation test, we sampled blood simultaneously from the coronary sinus and aortic root to compare lactate concentrations. We quantified coronary blood flow volume using an intracoronary Doppler-tipped guidewire. We detected LGE in 34 patients (LGE-positive); 27 were LGE-negative. Coronary blood flow volume increased significantly after the ACh provocation test only in LGE-negative patients (before vs. after ACh, 47.5 ± 36.8 vs. 69.2 ± 48.0 ml/min, respectively;  Myocardial fibrosis is a key component of heart failure (HF). There is a substantial body of evidence published over the past decade that there is a strong relationship between myocardial fibrosis and worsening of HF –5. The pThe arterioles are the true intra-myocardial regulatory component of the coronary circulation, and these vessels represent the largest proportion (approximately 55 %) of the total coronary vascular resistance . IncreasThe intracoronary acetylcholine (ACh) provocation test is a sensitive, safe test for the assessment of coronary vasomotor function in the catheterization laboratory. Administration of ACh causes vasodilation under normal conditions; however, in the presence of endothelial dysfunction it leads to vasoconstriction due to decreasing of nitric oxide synthesis –21.Coronary microvascular dysfunction affects the development of nonobstructive coronary disease. Microvascular dysfunction is also a significant and independent determinant of long-term cardiac events in patients with HF –31. HoweThis study included 61 consecutive patients with suspected non-ischemic HF who underwent both cardiac catheterization and contrast-enhanced CMR between January 2007 and December 2013 at Kumamoto University Hospital.Eligible patients were ≥20 years of age and met the criteria for the diagnosis of HF set by the American College of Cardiology/American Heart Association Stage B or C classification. The patients were clinically stable and under optimal medical therapy for HF, including stable doses of an angiotensin-converting enzyme (ACE) inhibitor or angiotensin II receptor blocker (ARB) and a β-blocker, if not contraindicated. Twenty-one of the 61 patients (37.7 %) were, however, unable to tolerate ACE inhibitor, ARB or beta blocker therapy because of hypotension even at low doses: seven could not tolerate an ACE inhibitor (or ARB) and beta blocker combination therapy, and seven could not tolerate either an ACE inhibitor, ARB or a beta blocker alone. Thirty-nine of 61 patients (63.9 %) had dilated cardiomyopathy (DCM) and eleven of the 61 patients (18.0 %) had hypertrophic cardiomyopathy (HCM) , significant coronary stenosis (>50 %), renal insufficiency with an estimated glomerular filtration rate of <30 ml/min per 1.73 mWritten informed consent was obtained from each patient before cardiac catheterization. This study was approved by the Human Ethics Committee of our institution.Left ventricular ejection fraction (LVEF), left atrial diameter, interventricular septal wall thickness (IVSd) and posterior wall thickness (PWd) were measured by two sonographers according to the recommendations for chamber quantification of the American Society of Echocardiology .E/e′ is the ratio between early mitral inflow velocity (E) and mitral annular early diastolic velocity (e′); we measured e′ at the septal mitral annulus. Left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) were calculated by either of the two methods described above; LVEF was calculated as follows:CMR was performed on a 3.0 T magnetic resonance instrument . Patients were scanned in the supine position using a dedicated 6- or 32-channel cardiac torso coil, between January 2007 and December 2013.Double-angulated scout images were obtained to plan the cardiac axis views. In all patients, images were acquired using T1- and T2-weighted black-blood, electrocardiogram-gated cine imaging by a segmented steady-state free-precession sequence in the three long cardiac axes with LGE imaging. Approximately 10 min after the injection of 0.1 mmol/kg of a gadolinium-based contrast agent , we acquired two-dimensional inversion-recovery sequences that included the left ventricle from the base to the apex. The imaging parameters were: time to repeat/time to echo = 4.5/2.2 ms; time of inversion = 250–350 ms; acquisition pixel size = 0.95 × 1.13 mm; and slice thickness = 8.0 mm. The acquisition time per slice was approximately 14 s.Hyperenhanced myocardial voxels resembling LGE were defined as signal intensity higher than 6 standard deviations above the mean intensity of the region of interest, which was placed over an area of the myocardium evaluated as normal .A board-certified cardiovascular radiologist blinded to the results of the cardiac catheterization study independently determined the dichotomous presence of LGE in the left ventricle. The patients were classified into LGE-positive or LGE-negative groups.Imaging data were analyzed using commercially available post-processing software . The endocardial and epicardial borders of the myocardium were manually traced on each short-axis slice. The left ventricular volume was calculated by subtracting the endocardial volume from the epicardial volume at end-diastole. Calculations were performed by two cardiologists blinded to the clinical data. We also calculated the  %LGE as the proportion of the LGE volume in the total left ventricular volume.All vasoactive drugs, including calcium antagonists, isosorbide dinitrate, isosorbide mononitrate and nicorandil were discontinued ≥72 h before the ACh test. We performed all examinations the morning after an overnight fast. A 5 Fr or 6 Fr arterial cannula was placed in the radial or femoral artery, and a 6 Fr venous sheath was placed in the right femoral vein , 28, 33.Blood was sampled from the CS and the Ao simultaneously to measure the plasma lactate concentration , 28. TheLER (%) = 100 × /arterial lactate concentration (mg/dl).We defined an LER < 0 % as indicative of myocardial lactate production.Additionally, we continuously monitored and evaluated the change of coronary blood supply by measuring the quantitative coronary blood flow volume (CBFV) using an intracoronary Doppler-tipped guidewire  . The quaWe compared the LER and CBFV before and after the ACh provocation test. We then diagnosed microvascular spasm according to the presence of myocardial lactate production and a decrease in the CBFV without epicardial vasospasm during the ACh provocation test.After the ACh test, adenosine triphosphate was administered into a central vein at 150 µg/kg/min until maximal hyperemia was reached as determined by the coronary flow reserve (CFR). The CFR was calculated as follows :CFR = average peak velocity at maximal hyperemia/average peak velocity at rest.All patients were diagnosed by cardiac catheterization as previously reported , 27. EpiPatients without epicardial coronary artery spasm who were positive for lactate production were diagnosed with microvascular spasm based on a decrease in the CBFV without epicardial vasospasm, but with the occurrence of chest symptoms and ischemic changes on an electrocardiogram.Patients who were positive for lactate production but had no decrease in the CBFV were diagnosed with microvascular dysfunction. Patients who were negative for lactate production were further assessed using the CFR. Patients with an abnormal CFR were diagnosed with microvascular coronary dysfunction and analyzed along with the patients in the microvascular dysfunction group .Patients without abnormal results were considered not to have ischemic heart disease (IHD).t test or the Mann–Whitney U test for unpaired data. Categorical values are presented as number (percentage) and were compared by the Chi-squared test or Fisher’s exact test, as appropriate. Variables with a skewed distribution were transformed logarithmically before Pearson’s correlation to fulfill the conditions required for the type of analysis performed.Normally distributed data are presented as mean ± standard deviation or median (interquartile range). Differences between groups were examined by Student’s Univariate logistic regression analysis was performed to identify significant parameters related to LGE-positivity. Multivariate logistic regression analysis was then performed using the forced inclusion model. The Hosmer–Lemeshow statistic was applied to assess model calibration.p value of <0.05 was considered statistically significant. All statistical analyses were performed with SPSS, version 20 .A two-tailed LGE was observed in 34 patients  Fig. a, b. Then = 34) and LGE-negative (n = 27) groups. The hemoglobin and serum potassium concentrations were significantly higher in the LGE-positive group . Two of the 13 patients who had epicardial coronary artery spasm (15.3 %) had trivial atherosclerosis (<25 %). A greater proportion of patients were diagnosed as having non-ischemic HF in the LGE-negative group     .The CBFV significantly increased after the ACh provocation test only in the LGE-negative group    was a significant and independent predictor of a post-ACh LER of <0 %  the patient with HF but without coronary stenosis had coronary vasomotor dysfunction Fig. d; (b) thLactate production occurred in myocardial tissue with LGE on CMR in HF without coronary stenosis, and microvascular dysfunction is therefore a potential cause of cardiac ischemia. In the LGE-positive group, however, lactate production increased without a decrease in blood flow during the ACh test.We and others have also previously examined the relationship between LGE and the transcardiac release of troponin T, and the relationship between LGE volume with LVEDP and transcardiac secretion of B-type natriuretic peptide (BNP) in patients with non-ischemic HF , 37. PulIntra-coronary ACh provocation test is the standard method for diagnosing epicardial coronary artery spasm in patients without coronary artery stenosis. The ACh test is an endothelium-dependent coronary reactivity test used to assess endothelial function and coronary artery spasm. We previously found that patients with coronary microvascular spasm exhibit vascular endothelial dysfunction .In this study, some patients exhibited lactate production after the ACh test without a concomitant reduction in CBFV. We propose that this may be a consequence of a blood flow imbalance that developed due to endothelium-dependent microvascular dysfunction at the LGE site in the coronary artery during the test. Indeed, 64.7 % of the patients in the LGE-positive group exhibited LGE in the LAD area Fig. c. MoreovBased on these study results, we speculate that myocardial fibrosis could indicate the presence of cardiac ischemia, which in turn induces further myocardial fibrosis. Therefore, a vicious circle involving cardiac fibrosis and ischemia may be occurring in HF.It has been reported that patients with HF have more extensive coronary microvascular rarefaction and myocardial fibrosis than normal patients, and that the progression of coronary microvascular rarefaction correlates with myocardial fibrosis . Therefon = 50) in this study, The CBFV significantly increased after the ACh provocation test in the LGE-negative group , but the CBFV did not increased after the ACh provocation test in the LGE-positive group . The LER in the ACh test significantly decreased in both groups , and lactate production significantly increased after ACh provocation only in the LGE-positive group (p = 0.021). LGE was significantly and independently correlated with an LER < 0 % after the ACh provocation test in the multivariate logistic regression analyses . These results in the patients without HCM (n = 50) were almost same as the results of all study patient (n = 61)  or without HCM (n = 50) indicated that LGE on CMR correlated with lactate production after the ACh provocation test and coronary microvasomotor abnormality.This study had some limitations. First, the etiology of HF in the cohort was heterogeneous, and the mechanisms of lactate production may have differed among cardiomyopathies. In particular, DCM or other cardiomyopathy differ pathophysiological mechanisms far from HCM. In addition, HCM rarely occur HF and are used different medication from other cardiomyopathy. To establish the extent of this possible limitation, we excluded HCM patients from this study and examined the relationship between LGE and microvascular dysfunction using the same methods. In the patients without HCM n = 0 in thisSecond, we enrolled a relatively small number of patients from a single center, limited to patients with HF but without coronary stenosis. Many patients declined to undergo cardiac catheterization, as they had no chest pain; consequently we were only able to recruit 61 patients in 7 years. Indeed, 35 patients rejected invasive cardiac catheterization because they were diagnosed as having non-ischemic heart disease before hospitalization.Third, 21 patients (34.4 %) could not tolerate ACE inhibitor, ARB or beta blocker therapy even at low doses because of hypotension. It has been reported that ACE inhibitors, ARBs and beta blockers influence microvascular function ; howeverFourth, we examined coronary microvascular function in the LAD alone, as in our opinion it would have been too time-consuming and invasive to perform an ACh provocation test in both coronary arteries. Indeed, 22 of the 34 LGE-positive patients (64.7 %) exhibited LGE in the LAD area Fig. c.Fifth, the quantitative analysis of myocardial fibrosis by LGE is that the fibrotic process is often diffuse in nonischemic cardiomyopathies, and the extent of LGE may be underestimated in such patients because of the lack of normal, non-fibrotic myocardium as a reference.Finally, we used LGE to evaluate myocardial fibrosis rather than extracellular volume fraction (ECV). Although ECV is now an extensively validated method of measuring myocardial fibrosis, this was not the case at the start of our study –54. To mLate gadolinium enhancement on cardiac magnetic resonance is correlated with lactate production in the acetylcholine provocation test. The presence of late gadolinium enhancement indicates ischemic damage due to coronary vasomotor abnormality. Late gadolinium enhancement in patients with HF may indicate worsening of HF. Further research is required to understand the role of myocardial fibrosis in HF more fully, and to identify new means of further improving patient outcomes.Translational Outlook: Patients with nonischemic HF exhibiting LGE on CMR likely have ischemia caused by vasomotor abnormality.