Cardiotoxicity is a feared side effect that may limit the clinical use of anthracyclines. It may indeed affect the quality of life and survival of patients with cancer, regardless of oncological prognosis. This paper provides an overview of anthracycline-induced cardiotoxicity in terms of definition, classification, incidence, risk factors, possible mechanisms, diagnosis, and treatment. We also report effective strategies for preventing cardiotoxicity. In addition, we discuss limiting current approaches, the need for a new classification, and early cardiotoxicity detection and treatment. Probably, anthracycline-induced cardiotoxicity is a continuous phenomenon that starts from myocardial cell injury; it is followed by left ventricular ejection fraction (LVEF) and, if not diagnosed and cured early, progressively leads to symptomatic heart failure. Anthracycline-induced cardiotoxicity can be detected at a preclinical phase. The role of biomarkers, in particular troponins, in identifying subclinical cardiotoxicity and its therapy with angiotensin-converting enzyme inhibitors  to prevent LVEF reduction is a recognized and effective strategy. If cardiac dysfunction has already occurred, partial or complete LVEF recovery may still be obtained in case of early detection of cardiotoxicity and prompt heart failure treatment. Anthracyclines are cytostatic antibiotics , introduUnfortunately, anthracyclines are considered the principal culprit drugs behind chemotherapy-induced cardiotoxicity –5. The p2 and as high as 18–48% at 700 mg/m2 . , 7. 4, 7The specific mechanisms of anthracycline cardiotoxicity still remain unclear. A potential mechanism is the generation of reactive oxygen species (ROS), changes in iron metabolism, and Ca2þ signaling. In 2014, topoisomerase (Top) 2β was indicated as the critical mediator of anthracycline's cardiac toxic effect . Top2 caThe cardiomyocyte has always been considered the main cellular target of anthracycline toxic effect in the heart, as their destruction results in the progressive development of cardiac dysfunction. More recently, however, other cell types—such as cardiac progenitor cells, cardiac fibroblasts, and endothelial cells—have been identified as potential additional targets, creating a more complex and intriguing scenario in the pathogenesis of anthracycline-induced cardiomyopathy  11). So. So11). A previous and more dated classification identified three distinct types of anthracycline-induced cardiotoxicity : acute, In particular, recent findings challenge this old classification, suggesting that anthracycline-induced cardiotoxicity is potentially a continuous phenomenon, starting at the myocardial cell level, followed by progressive functional decline, progressively leading to overt heart failure.  5, 8, 1, 18, 15.2) or standard dose followed by trastuzumab-based regimens—cardiac monitoring is not suggested by the American Society of Clinical Oncology guidelines. Moreover, they suggest a diagnosis of cardiotoxicity based on clinical symptoms  , 18. A fsymptoms . Reasonssymptoms , 21. Othsymptoms . Moreovesymptoms , 18.n = 2,625) population scheduled for anthracycline therapy showed that close monitoring of LVEF after chemotherapy allowed nearly all (98%) cases of cardiotoxicity to be identified within the first 12 months of follow-up , or on studies with small populations, retrospective design, short follow-up, or on case reports –30.Up until 2010, the response to heart failure therapy of patients with anthracycline-induced cardiotoxicity hadn't been thoroughly investigated. Moreover, these kind of patients have been excluded from large randomized trials evaluating the impact of current heart failure therapies .The effectiveness of ACE-inhibitors and beta-blockers has been prospectively assessed in two extensive papers , 31. In On the contrary, close monitoring and timely treatment with HF therapies have reported that they are critical for functional recovery in a non-selected population treated with anthracycline, allowing early detection of cardiotoxicity in the vast majority of cases during the first year after chemotherapy, with normalization of LVEF  in 82% of cases . HoweverToday, at an early preclinical stage, we can detect cardiotoxicity long before symptoms of heart failure occur and before an asymptomatic drop in LVEF. Most data relate to cardiac biochemical markers: mainly troponins and echocardiography of tissue Doppler and strain , 7, 8.Troponin may be considered the gold standard biomarker for myocardial injury and cardiotoxicity from different causes/etiologies . Troponip < 0.001). Based on the high negative predictive value (99%), TnI has been able to safely identify low-risk patients, limiting the need and subsequent costs of close long-term cardiac monitoring  was assessed before and during the first 72 h after chemotherapy (early TnI), and after 1 month (late TnI) . Three dnitoring , 35, 38.In summary, we can assert that troponin evaluation in patients treated with anthracyclines allows for:Prediction of the development of future left ventricular dysfunction;Prediction of left ventricular dysfunction severity, because the peak value of troponin is closely related to the extent of LVEF reduction;Stratification of cardiac risk after anthracyclines and tailoring of the schedule of post-chemotherapy monitoring of cardiac function;Identification of cardiotoxicity prone patients, in whom a cardioprotective therapy can be considered; andExclusion of most patients from prolonged cardiologic surveillance.On the other hand, the identified weakness points are:Repeated assessments of troponins are needed to detect positivity;The ideal timing for troponin detection must still be defined;Standardization of routine troponin use in this clinical setting is a current need; andTiming in which a single sampling of troponin could be obtained.Although patients with pre-treatment levels of natriuretic peptides  tend to experience cardiac events (including cardiac dysfunction), the results are sparse , 57, 58.More recently , BNP levActually, studies of other biomarkers, including microRNAs (miRNAs), C-reactive protein (CRP), growth differentiation factor-15 (GDF-15), myeloperoxidase (MPO), and galectin-3  have not demonstrated an association between pretreatment biomarker levels and cardiovascular outcomes , 61.Regarding monitoring for cardiovascular toxicity during therapy, CRP has shown conflicting findings .More recently, some reports emerged in the field of microRNAs, in particular for miR-1, showing a trend to earlier detection of cardiotoxicity respective to troponin , 63.Another study of patients during a period of 10 years after anthracycline therapy did not find an association with Gal-3 and LV dysfunction.A separate study that included Gal-3 and ST2 found no association with these biomarkers and LVEF 1 year after therapy , 65.Novel echocardiographic methods have emerged as sensitive parameters in the early identification of cardiotoxicity. In particular, introduction of tissue Doppler and strain imaging techniques can detect early subclinical changes in cardiac function, before LVEF falls , 19, 51.The recent ASE/EACVI consensus defined a relative decrease in GLS of >15% from baseline as an indicator of subclinical LV dysfunction and appropriate use criteria for multi-modality imaging include strain for the evaluation of patient candidates for chemotherapy , 70. FinHowever, these methodologies are not always readily available in all laboratories and seldom used in the routine evaluation of patients receiving anthracyclines .Breakthroughs in laboratory technology have allowed for the introduction of more specific and sensitive troponin assay methods , which aThe first HS troponin trial enrolled 45 breast cancer patients who were treated with anthracyclines, taxanes, and trastuzumab . InternaPresent oncologic guidelines recommend limiting the total cumulative dose of anthracyclines to 450–550 mg/ml . HoweverEpirubicin, idarubicin, and mitoxantrone are analogs of anthracyclines that are less cardiotoxic than conventional anthracyclines. Epirubicin cardiotoxicity occurs after higher doses of doxorubicin. However, to obtain the same clinical response, higher doses must be given. In preclinical studies and animal models, idarubicin and mitoxantrone also showed a less cardiotoxic profile than doxorubicin , 7.In the heart, liposomes cannot get out from the vascular space because capillaries have tight junctions. As the tendency to accumulate in the heart cells is limited, this may reduce the risk of cardiotoxicity. On the contrary, the liposomes reach high concentrations in the tumor site, leaving the circulatory system where tumor growth damages the capillaries itself , 74.Before pharmacologic strategies, primary prevention starts indeed with lifestyle corrective measures.Since a strong link exists between cancer and cardiovascular risk factor, addressing smoking and sedentary habits , as well as high alcohol intake, is pivotal. A healthy diet has been associated with a protective effect in terms of cancer relapses and cardiovascular disease, while smoking has an ominously detrimental effect. While light to moderate alcohol intake has shown a protective impact in terms of cardiovascular disease, the results in terms of risk of developing cardiotoxicity are conflicting –77.Of notice, several pieces of evidence emerged on the protective role of exercise training  against cardiotoxicity .The use of cardioprotective drugs to reduce the direct cardiotoxic effect is a potential alternative to anthracycline treatment modifications, dosage limitations, or interruptions , 5, 8.The hypothesis that iron chelators may reduce the cardiotoxicity induced by anthracyclines suggests that dexrazoxane may be a clinically useful cardioprotective agent , 79. DoxMacedo et al. recently published a systematic review and meta-analysis of nine trials  on the efficacy of dexrazoxane in patients with breast cancer treated with anthracyclines (with or without trastuzumab). Despite the quality of available evidence remaining low, dexrazoxane was shown to reduce the risk of heart failure and cardiac events, independently from previous exposure to anthracyclines. The oncological response and survival rates were not affected by dexrazoxane .Other potentially cardioprotective agents have been studied in animal models and small clinical studies. Preliminary data are promising, but they need to be ratified by further extensive studies , 5, 7, 8Several heart failure drugs have been shown to be effective in terms of cardioprotection against anthracylines  86–98).–98.86–98Overall, a recent meta-analysis of randomized clinical trials of adult patients that underwent chemotherapy and cardiovascular therapies vs. placebo with follow-up  showed higher  LVEF values at follow-up in cancer patients receiving neurohormonal therapies .In vitro studies and a small randomized clinical trial, the drug was able to prevent the development of ventricular dysfunction  in the development and progression of cardiomyopathy induced by anthracyclines . ValsartTelmisartan, started before epirubicin, was able to prevent strain reduction and inflammatory markers increase because of its RAS blocking action, but also because of its anti-inflammatory and anti-oxidant properties .In the PRADA (Prevention of Cardiac Dysfunction during Adjuvant Breast Cancer Therapy) trial candesartan—but not metoprolol—administrated with adjuvant chemotherapy including anthracyclines, with or without trastuzumab, can protect against an early decline in LVEF, assessed with cardiac MRI .The MANTICORE-101 study (Multi-disciplinary Approach to Novel Therapies in Cardiology Oncology Research) tested the use of perindopril vs. bisoprolol in the prevention of left ventricular remodeling, defined as an increase in end-diastolic diameters and primary study point, and of left ventricular dysfunction in HER2+ breast cancer patients treated with trastuzumab prior to anthracycline . NeitherThe combination of enalapril and carvedilol have been tested in the OVERCOME study . The study involved 90 patients treated with anthracyclines, with malignant hemopathies. LVEF didn't change in the intervention group after 6 months, but decreased significantly in controls. In addition, the intervention group had a lower rate of combined death or heart failure or death, heart failure, and a final LVEF of <45 % .A recent randomized trial, including 43 breast cancer patients, evaluated the use of spironolactone vs. placebo. Spironolactone was started 1 week before anthracyclines. Three weeks after the end of chemotherapy, the treated group did not show relevant variations in LVEF and rise in troponin I and NT-proBNP . In ELEVThe effect of statins on cardiotoxicity of anthracyclines is most likely due to their pleiotropic effect, and in particular, to their antioxidant properties . Forty hConversely, the LVEF value in the control group resulted in a significant reduction from the baseline. The protective effect of statins also emerged when chemotherapy was started in patients already receiving statins for the prevention of cardiovascular disease . In a reA recent study identified the molecular and cellular signature of dose-dependent, doxorubicin-mediated cardiotoxicity and provided evidence that prokineticin receptor (PKR-1)-1, acting at myocardial and vascular level, is a promising target to combat cardiotoxicity of cancer treatments .Since G protein-coupled receptors (GPCRs) are a target of 40% of clinically used drugs and newly identified cardioprotective agents that bind GPCRs of adrenalin, adenosine, melatonin, ghrelin, galanin, gpelin, prokineticin, and cannabidiol may further aid in the cardioprotective task .Prevention may be an option for all patients who are candidates for cardiotoxic therapy (primary prevention) or restricted to patients with preclinical symptoms of cardiotoxicity, with the advantage of limiting prophylactic therapy to a small number of patients  .A randomized trial has tested the cardioprotective capacity of enalapril, involving 473 patients with different types of cancers treated with high-dose chemotherapy . 114 patTwo studies are currently evaluating the efficacy of carvedilol as a preventive therapy in a selected patient with a deterioration in the strain parameter. For Research NCT02177175 , the primary endpoint is the identification of a reduced LVEF value during the 1-year follow-up. At Northwestern University of Chicago, the research is still hiring.Enalapril, which began early after the increase in troponin during anthracycline chemotherapy and continued for 12 months, is an effective therapy to avoid left ventricular dysfunction and subsequent heart events . RepeateIn brief, the main result of the study was that the two approaches appear to be similarly effective in preventing left ventricular dysfunction and adverse cardiac events, endorsing the use of enalapril in averting anthracycline-induced cardiotoxicity, irrespectively from the strategy used.Which strategy is best? Secondary prevention  has the limitation of repeated blood samplings. Nevertheless, considering the high negative predictive value of troponin , 37, 38,Anthracycline-induced cardiotoxicity is still a significant problem that compromises the quality of life and overall survival of cancer patients. However, recent findings demonstrate that this form of cardiomyopathy is mostly reversible with early detection and prompt therapeutic introduction strategy. Probably, anthracycline-induced cardiotoxicity is a single and continuous phenomenon, from cellular to clinical stage, starting with myocardial cell injury, followed by progressive LVEF decline and, potentially, overt heart failure. The current standard for monitoring cardiac function (periodic assessment of LVEF), detects cardiotoxicity at a late stage when a significant impairment has already occurred, precluding the chance of effectively prevent and treat its development.The use of troponins to identify patients with subclinical cardiotoxicity combined with early treatment with ACE-inhibitors occurrence appears to be an effective method to prevent anthracycline-related left ventricular dysfunction and cardiac events.Finally, adoption of internal procedures, shared in a multi-disciplinary team, may actively aid in optimizing patient management. In this respect, a direct relationship with the laboratory medicine service for the assessment of troponin values during chemotherapy and the availability of a cardiologist and a dedicated nurse staff should always mix with an active collaboration with the referral oncologist/hematologist  for updates and remains of pivotal importance , 108.DC, FI, and CC contributed conception, design of the review, and wrote the first draft. All authors contributed to manuscript revision, read, and approved the submitted version.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.


E), relative area change (RAC), diastolic diameter (D), regurgitant flow, and time-averaged wall shear stress (TAWSS) of the proximal PA, as well as the total arterial resistance (Rt) and compliance (Ct) at the distal pulmonary vasculature. Results found that the average proximal PA was stiffer  with a larger diameter  and a reduced RAC  in PAH compared to our control group. Also, higher total resistance  and lower total compliance  were observed in the PAH group. Furthermore, lower TAWSS values were seen at the main PA arteries (MPAs) of PAH patients  compared to controls. Correlation analysis within the PAH group found that E was directly correlated to the PA regurgitant flow  and inversely related to TAWSS . Results suggest that the estimated elastic modulus E may be closely related to PAH hemodynamic changes in pulmonary arteries.Vascular wall stiffness and hemodynamic parameters are potential biomechanical markers for detecting pulmonary arterial hypertension (PAH). Previous computational analyses, however, have not considered the interaction between blood flow and wall deformation. Here, we applied an established computational framework that utilizes patient-specific measurements of hemodynamics and wall deformation to analyze the coupled fluid–vessel wall interaction in the proximal pulmonary arteries (PA) of six PAH patients and five control subjects. Specifically, we quantified the linearized stiffness ( Pulmonary arterial hypertension (PAH) is a complex cardiovascular disease characterized by a progressive remodeling of the pulmonary arteries. This ongoing process, which is promoted by an increase in pulmonary arterial (PA) pressure, leads to right atrial dysfunction  and RV functions, and hemodynamics in the PA can serve as potential biomechanical markers for detection of PAH and prediction of its progression  magnetic resonance (MR) images were acquired using a 3-Tesla Philips scanner with ECG gating from six patients diagnosed with PAH based on catheterization. Blood velocities and volumetric flow rates at the main pulmonary artery (MPA) were post-processed from the PC-MR images using Q-Flow software . Right heart catheterization (RHC) was also performed at rest (within 1 week of imaging examination) on the PAH patients using standard techniques. Five human subjects with no known cardiovascular disease or other co-morbidities served as control in this study and also underwent MR imaging from which cine MR images were acquired. Because invasive hemodynamics measurements and PC-MR images were not acquired in the control group, the MPA pressure and volumetric flow waveforms measured in healthy human subjects from a previous study was used /Amin  using a protocol described in details in our previous study , proximal resistances  and distal resistances (Equation 2):Model parameters were calibrated with patient-specific data using a protocol that was previously described , and h denote the fluid density, pulse wave propagation speed, MPA cross sectional area at the end-diastolic, and main PA thickness, respectively.where ρ, Ci for each outlet i were then derived from an equivalent total arterial resistance (RT) and compliance (CT) by assuming an equal flow split between left and right PA and according their equivalent outlet area .RT) and compliance (CT) for each subject (Equation 5) were iteratively adjusted along with the elastic modulus (E) in two nested loops until measured RAC, pulse ( ΔP = Psys − Pdias), and diastolic (Pdias) pressures were achieved.Initial estimated values of total arterial resistance  was computed from all subjects (PAH and controls).Once calibrated, E, Rt, and Ct), TAWSS, as well as geometrical quantities (RAC and diameter) were estimated and used to perform a non-parametric Mann–Whitney U-test. Also, Spearman's rank correlation tests were performed to quantify the relationships between hemodynamic quantities and the calibrated model parameters in the PAH (n = 6) and the control (n = 5) groups. All statistical analyses were performed using the MATLAB software  for which a statistical significance of P ≤ 0.050 was defined.Median and interquartile range (IQR) of the calibrated model parameters  in the PAH group compared to that in the control group  than that in the control group  in the PAH group compared to the control group  computed using the calibrated model parameters was lower in the PAH group in comparison to the control group. Maximum surface displacement (over the cardiac cycle) was generally found to decrease with increasing linearized stiffness patients . AdditioE. A strong linear correlation  showed a regurgitant (back) flow after the systolic phase in all PAH patients  were considered . When only PAH patients were considered, however, TAWSS averaged over the entire PA was strongly correlated with E  E is directly correlated with the total regurgitant flow in the PA, (3) TAWSS is inversely associated with the increase in the linearized elastic modulus E in PAH patients, and (4) distal resistances Rt and compliance Ct are, respectively, higher and lower with PAH.We have applied a patient-specific computational framework to investigate the interactions between vessel wall mechanics and hemodynamics in the PA of six PAH patients and five control subjects. The key findings are as follows: (1) the linearized elastic modulus E of the proximal PA is approximately four times stiffer in the PAH group  since our threshold was set to 0.050, given the small number of cases, the correlation in six PAH patients can be indicative of a potential for the significant correlation if tested in a larger cohort. Arguably, this borderline significant results could be attributed to differences in using nominal values such as volumetric inflow and PA diameters to determine WSS . A comparison of the estimated TAWSS values between PAH and control groups shows an interesting trend. Besides finding that TAWSS is lower at the proximal PA arteries in PAH patients than the control subjects ; which iThere are some limitations associated with this study, some which are already summarized in a previous study (Zambrano et al., Pulmonary arterial stiffness and hemodynamic metrics measured at the proximal PA are believed to be potential biomechanical markers for the identification of PAH disease. Our study, using a novel FSI computational framework and clinical patient data, analyzed these aspects and their relationship in six PAH patients and five control subjects. Our results showed that proximal PA stiffness is associated to PA hemodynamic parameters such as TAWSS and back regurgitant flow. Although study with a larger cohort of patients would need to be conducted to reach stronger conclusions, these associations highlight the potential of hemodynamic parameters of becoming important biomarkers to stratify PAH severity in terms of PA stiffness.The original contributions presented in the study are included in the article/The study was approved by the SingHealth Centralised Institutional Review Board and written informed consent was obtained from all participants.LL, LZ, and SB designated and directed the overall project. BZ and NM implemented all the analysis and were involved in the evaluation of the results and preparation of the manuscript. XZ, J-LT, and LZ contributed to patient and image acquisition. BZ, XZ, J-LT, and LZ contributed to data analysis and statistics. CF contributed to model development and model parameter estimation. All the authors contributed to the evaluation of the results, discussion, and the overall 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.


Koalas are infected with the koala retrovirus (KoRV) that exists as exogenous or endogenous viruses. KoRV is genetically diverse with co-infection with up to ten envelope subtypes (A-J) possible; KoRV-A is the prototype endogenous form. KoRV-B, first found in a small number of koalas with an increased leukemia prevalence at one US zoo, has been associated with other cancers and increased chlamydial disease. To better understand the molecular epidemiology of KoRV variants and the effect of increased viral loads (VLs) on transmissibility and pathogenicity we developed subtype-specific quantitative PCR (qPCR) assays and tested blood and tissue samples from koalas at US zoos (n = 78), two Australian zoos (n = 27) and wild-caught (n = 21) in Australia. We analyzed PCR results with available clinical, demographic, and pedigree data.All koalas were KoRV-A-infected. A small number of koalas (10.3%) at one US zoo were also infected with non-A subtypes, while a higher non-A subtype prevalence (59.3%) was found in koalas at Australian zoos. Wild koalas from one location were only infected with KoRV-A. We observed a significant association of infection and plasma VLs of non-A subtypes in koalas that died of leukemia/lymphoma and other neoplasias and report cancer diagnoses in KoRV-A-positive animals. Infection and VLs of non-A subtypes was not associated with age or sex. Transmission of non-A subtypes occurred from dam-to-offspring and likely following adult-to-adult contact, but associations with contact type were not evaluated. Brief antiretroviral treatment of one leukemic koala infected with high plasma levels of KoRV-A, -B, and -F did not affect VL or disease progression.Our results show a significant association of non-A KoRV infection and plasma VLs with leukemia and other cancers. Although we confirm dam-to-offspring transmission of these variants, we also show other routes are possible. Our validated qPCR assays will be useful to further understand KoRV epidemiology and its zoonotic transmission potential for humans exposed to koalas because KoRV can infect human cells. Phascolarctos cinereus) are infected with a gammaretrovirus  that relatively recently became endogenous and is fixed in the genome of every koala cell, including germ cells, and is transmitted vertically in a Mendelian fashion [The majority of koalas  of the envelope protein (Env) involved in retrovirus receptor recognition and binding , 8. Sinceletions , 20, 21.eletions –25. One eletions .Similarly, very little is known about viral loads (VLs) of KoRV variants and how they may affect pathogenicity and transmissibility. Increased retroviral levels, like those reported for HIV and human T-lymphotropic virus type 1 (HTLV-1), are associated with increased pathogenicity and transmissibility –29. StudLess is known about the transmissibility of KoRV subtypes among koalas. The original study that identified KoRV-B reported dam-to-joey transmission of KoRV-B but only small numbers of captive koalas were studied at one zoo . While tTo better understand the distribution, transmissibility, and pathogenicity of KoRV variants, we developed and validated KoRV generic and subtype-specific qPCR assays to detect and quantify these novel subtypes. We applied these new tools to many zoo and wild koalas to determine their geographic and inter-koala distribution and VLs. We also tested biospecimens from zoo koalas with and without disease to evaluate potential associations of KoRV genotype and titers with disease. By using koala pedigree records we evaluated transmission of KoRV subtypes from infected parents to their offspring. Our molecular epidemiology findings will have importance for koala management and for biosafety practices in persons exposed to koalas to reduce zoonotic risks.P. c. adustus). Koala pedigrees were available from the 2017 North American Regional Studbook prepared by the San Diego Zoo and from breeding records kept at the Taronga Zoo. The pedigrees were used to investigate KoRV transmissibility in parent–offspring groups. Longitudinal blood specimens were available from eight koalas at six zoos to assess changes in VLs and subtypes over time. Our study followed the Policy Guide for Conducting Scientific Studies and was approved by the Institutional Animal Care and Use Committee (IACUC) at each zoo.EDTA-treated blood specimens for koalas at U.S zoos were opportunistically collected and processed within 24 h. Tissue specimens from U.S. zoo koalas, including spleen, lymph node, liver, heart, kidney, brain, thymus, intestine, lungs, muscle, skin, nodules, and/or bone marrow were obtained at necropsy following recent death or from specimen biobank archives. Archived plasma, and peripheral blood mononuclear cells (PBMCs) or buffy coats, were also available from three koala populations in Australia: (1) Taronga Zoo in Sydney, New South Wales, (2) a Queensland zoo that wished to remain anonymous, and (3) wild koalas from St. Bees Island in Australia that were available from a San Diego Zoo study in collaboration with the University of Queensland. Animal records indicate all koalas in this study are Queensland koalas . Genomic DNA (gDNA) from PBMCs or tissue specimens were extracted using a QIAamp DNA Mini kit  following manufacturer’s instructions and stored at − 20 °C until testing. RNA was extracted from plasma and tissues using a QIAamp Viral RNA Mini kit  following the manufacturer’s instructions but modified by replacing tRNA in the kit with 10 ug of Dextran. Dextran as a nucleic acid carrier, in contrast to tRNA, does not affect spectrophotometric measurements of nucleic acids at ODs of 260 and 280 nm. RNA samples were treated with DNase I using a DNA-free kit  to remove any contaminating DNA and tested immediately or stored at − 80 °C. Nucleic acid quality and concentrations were determined using a NanoDrop One microvolume spectrophotometer (ThermoFisher Scientific) that is calibrated at least annually.env nucleotide sequences at the time were aligned using Clustal W in MEGA v7 to identify regions of identity and dissimilarity for design of generic and type-specific primers, respectively. The KoRV type-specific primers and probes are located in variable region A in env , which shares 44–70% intersubtype nucleotide identity , B (2001-bp), E (2010-bp) and F (1998-bp) were initially used in developing the generic and specific qPCRs. For better quantification and comparison of KoRV copy numbers between assays, and for simplifying the qPCR systems, a mosaic plasmid was synthesized by Integrated DNA Technologies  to include tandem koala β-actin (GenBank # DQ058212) and generic and subtype-specific  env sequences  as a unique standard control for all seven qPCR assays. KoRV-E and -F env sequences were kindly provided by Xu et al. prior to publication [Available complete and near complete KoRV bp), E 20-bp and FOptimal conditions for the qPCR assays included 95 °C for 9 min, 95 °C for 15 s, 62 °C  or 68 °C  for 30 s for 55 cycles using a Bio-Rad CFX96 instrument  and the AmpliTaq Gold polymerase . Ten microliters of RNA extract equivalent to 50 ul plasma or 200 ng tissue was used for KoRV viral and β-actin RNA testing, and 200 ng gDNA was used for proviral and β-actin DNA testing. Koala β-actin DNA testing was used to confirm the quality of the extracted DNA and was not used to normalize DNA pVLs since it is not a single-copy housekeeping gene and studies have shown β-actin levels can vary widely in animals depending on various factors, including cellular activation , 35. Prohttps://igraph.org/r/).Log10 VLs for KoRV-A, -B, -E, -F and -J in both PBMCs and plasma from whole blood samples, and various tissues were analyzed. Descriptive analysis included the mean, median, standard deviation and range of each KoRV subtype VL. Age at testing, final health status classification for each koala , sex  and location  were included in our analysis for investigating potential associations between KoRV VLs as a continuous outcome and these koala characteristics. As KoRV VLs are subject to an assay detection limit, the Tobit model with random effects was applied to perform the analysis, adjusting for the repeated VL records from animals with multiple specimens. The Tobit model refers to a class of regression models in which the observed range of the dependent variable is censored in some way . In furtIn total, 126 koalas were available for our study, of which 27 (21.4%) were from two Australian zoos (24 were from Taronga Zoo and 3 from a Queensland zoo), 78 (61.9%) were from ten US zoos, and 21 (16.7%) were wild-caught at St. Bees Island  and from 8 joeys with 118 total blood specimens tested when including multiple specimens from eight koalas collected longitudinally. Only tissues  were available for two joeys that died while in the pouch and only liver and spleen tissues were available for a joey that died from anemia. Multiple tissues  were also available from 28 adults at two US zoos for a total of 77 tissue specimens tested. Of these 28 adult koalas, five also had blood specimens available.The KoRV generic and type-specific and the koala β–actin assay could each reliably detect 10 copies of DNA or RNA targets/reaction in each replicate. Each assay could detect five copies of DNA or RNA target/reaction except the KoRV-B and KoRV-E RNA specific tests that only detected 90% and 70%, respectively, of the replicates containing five DNA or RNA copies per/reaction. Hence, the assay cutoff for generic and KoRV subtype-specific detection was between 5–10 DNA and RNA copies/reaction. The koala β–actin assay could detect 90% of the DNA and RNA replicates with one copy/reaction and 100% of the DNA and RNA replicates at five copies/reaction, and we thus set the cutoff for that assay at 1–5 copies/reaction.0 to 107) of the KoRV-mosaic plasmid control. Each assay could detect between 100–107 copies/reaction. Testing of the different KoRV subtype controls gave quantification cycle (Cq) assay values that averaged 20 for 107 copies, 23 for 106 copies, 26 for 105 copies, 29 for 104 copies, 32 for 103 copies, 35 for 102 copies, 39 for 101 copies, and 42 for 100 copies with an average correlation coefficient (R2) of 0.995 for the replicates. Each assay also showed 100% specificity for detection of the KoRV-specific  env sequences, except for the KoRV-B test which also detects KoRV-J. Nonetheless, the KoRV-J-specific assay did not detect KoRV-B env sequences and can be used to distinguish these two genotypes.The linear dynamic range and specificity of each qPCR assay was measured by testing ten replicates of ten-fold dilutions  Table . A smallThe distribution of VLs for each detected KoRV subtype is shown in the box plots in Figs. Although the KoRV-A median log10 pVLs 7.43) were higher for blood PBMCs than non-A subtypes range 2.26–6.36) for all koala characteristics, the KoRV-A median log10 blood plasma VLs (4.62) were lower than the median log10 plasma VLs for KoRV-B, -E, and -F  . The mean log10 VLs for KoRV-E was significantly lower than that for subtype B . Compared to other causes, koalas that died from leukemia-lymphoma or other cancers had significantly higher VLs compared to those that died from other causes  and compared to those alive .Overall, we found that different subtypes showed significantly different VLs compared to subtype B. For example, for plasma RNA the mean log10 VLs for KoRV-A and KoRV-F were significantly higher than that for KoRV-B  or that were alive .When controlling for other covariates including subtype, koalas at the Australian zoos were found to have the highest mean VLs. When compared to wild koalas the mean difference was 1.87 , while compared to koalas in US zoos the mean difference was 1.31 . Analysis of the pVLs gave results similar to the plasma RNA VLs with koalas with leukemia-lymphoma or other cancers again having significantly higher pVLs compared to those that died from other causes  at six zoos. Of these eight, five adult koalas were infected with only KoRV-A, which showed fluctuating KoRV levels over time. For example, plasma VLs decreased 0.8 and 1.2 logs over 8 months and about 0.4 logs over 2 years for two KoRV-A-infected male koalas at US zoos. In contrast, plasma KoRV-A VLs increased 0.7 logs over 19 days for a female at the Taronga Zoo. The one joey was infected with low levels of KoRV-B in the PBMCs and KoRV-E in the plasma at 9 months of age but both KoRV-B and -E were undetectable 19 days later. Two adults with longitudinal specimens died from leukemia/lymphoma. The male housed at the Taronga Zoo, had two specimens collected 19 days apart and was infected with KoRV-A, -B, -F and –E at both time points and had DNA and RNA levels within 0.5 logs, except for KoRV-E DNA levels that increased by almost 2.5 logs at the later time point. The female (named Brooklyn) housed at the LA Zoo had 12 samples collected every 3–4 days over a month. KoRV-A, -B, -F were detected at each time point, while KoRV-J was detected intermittently with 3/12 plasma RNA specimens testing positive while all matching PBMC DNA specimens were KoRV-J-negative.Brooklyn received antiretroviral treatment with integrase  and nucleotide reverse transcriptase (tenofovir) inhibitors for 33 days for experimental treatment of her leukemia/lymphoma. While KoRV-A levels in Brooklyn were relatively stable over time averaging 7.10 and 7.59 log10 copies for plasma and PBMCs, respectively, the KoRV-B and -F averages were 1.24 (8.05 vs 6.81) and 0.67 (7.54 vs 6.87) logs higher in plasma than in PBMC DNA  or from the dam (n = 15) or sire (n = 11) only. To evaluate dam-to-offspring transmission we examined the KoRV profiles and disease status of 55 offspring and their dams (Table p < 0.0001). Within the group of dams infected with subtype B and other non-A subtypes, 11/14 (78.6%) of the offspring were also infected with subtype B and other non-A subtypes. Within the subtype B-negative dams, 5/41 (12.2%) of the offspring were at least subtype B-positive. These results indicate that dams with KoRV-B alone or with other non-A subtypes were more likely to transmit their non-A KoRV subtypes to their offspring, but that discordant transmission profiles exist.We found within the 55 complete pairs of dam and offspring data, that 36 pairs were negative for subtype B. For three pairs, the dam was infected with subtype B but not the offspring and for five offspring with subtype B infection, their dam was only infected with KoRV-A. For 11 dam-offspring pairs, both koalas were infected with at least subtype B. Overall, we found that the dam KoRV subtype was significantly associated with the offspring subtypes  with discordant KoRV infections from the dam, including one KoRV-A, -B and -E-infected dam that birthed three KoRV-A-only infected offspring . The sire of two of these offspring tested KoRV-A-positive only. One female offspring with KoRV-B, -F, and -E tested at 72 months was born to a dam with only KoRV-A; the sire’s name was not recorded. Of the four other offspring  with discordant KoRV infections, the dams were all KoRV-A-positive only while the one male sire of three of these was infected with KoRV-B and other non-A variants. The sire’s name for the fourth offspring was not recorded.p = 0.01) though the 95% CI is large.We also evaluated if subtype transmission was associated with disease in the offspring. Of the 55 dam/offspring pairs, neoplasia was identified in 10 (18.2%) pairs with leukemia/lymphoma in 7 pairs and other cancers in three Table . Of thesWe developed highly sensitive and specific qPCR assays to further characterize KoRV molecular epidemiology, disease potential, and transmissibility in many koalas from the US and Australia. We show that non-A KoRV subtypes are present in zoo koalas in the US and Australia but were absent from all 21 wild koalas from St. Bees Island. The prevalence and levels of non-A subtypes in both plasma RNA and PBMC DNA was significantly higher in koalas at Australian zoos than in those at US zoos. Our results indicate that unlike KoRV-A, non-A KoRV subtypes are likely replication active, evolving exogenous retroviruses that are not yet widely distributed. These results are supported by analysis of integrated KoRV-A, -B, -D and -E in a wild koala using long-read genome sequence assembly, the greater genetic KoRV envelope diversity seen in non-A subtypes and by recent KoRV variant prevalence studies of wild koala populations , 19, 20.Our findings also suggest that the breeding and animal management programs at each international zoo likely reflects the current level of local KoRV diversity. For example, following the original importation from Australia the majority of the initial US koala population were born at the San Diego Zoo, which in our study were infected by only KoRV-A and perhaps by chance, a founder effect, or animal management strategies, have not become infected with non-A KoRVs. All non-A KoRVs in the US were found in koalas that were housed at or originated from the LA Zoo where KoRV-B was first identified . Some koWe did not observe a significant difference in the distribution and tissue levels of KoRV subtypes by gender or age for both RNA and DNA detection and levels similar to those reported for a large population of wild koalas infected with KoRV-A and -B . In contet. al who reported higher pVLs and plasma VLs in koalas with neoplasia compared to other disease categories in wild koalas by using a generic KoRV polymerase qPCR assay [The first study that identified KoRV-B reported finding an association of lymphoma with infection though this determination was based on small numbers of koalas . To furtCR assay . These dCR assay , 30.. [. reported a significant association of KoRV-B infection and both chlamydial disease and neoplasia but then reported higher KoRV-D levels in healthy koalas [Since retroviral disease can be associated with increased VLs, we also examined KoRV subtype levels in animals with disease by using the Tobit model, which included all detectable measurements and those that were BLD providing a more robust analysis of the data compared to an analysis of just the detectable VLs like those shown in Figs. . . Quigleyy koalas , 15. How. who followed a cohort of 16 adult koalas for a period of 4 years with sampling about every 6 months and found variable KoRV-A, -B, -D, and -F expression profiles over time [. reported variations of plasma VLs in ten koalas and observed an increase in VLs over 18 months as animals progressed to neoplasia, but subtyping was not conducted then [Limited data exist on the longitudinal follow-up of KoRV infections. We were able to measure VLs in eight koalas with longitudinal specimens, including two adults with leukemia/lymphoma. We found that proviral load and/or plasma VLs fluctuated over time by as much as 2.5-fold and even the disappearance of non-A subtypes in as little as 19 days. Our results are comparable to those of Quigley et alver time . In 2005ted then . The lon. using generic polymerase qPCR reported an association of increasing severity of chlamydial disease with increasing KoRV VLs in koalas from South Australia but not from those in Queensland [Although chlamydial infection was not present in our study population, it remains to be determined whether a specific KoRV subtype contributes to co-infection with chlamydia and associated chlamydial disease progression. One study identified an increase of chlamydial disease in a population of koalas infected with KoRV-B from northern Australia, but total KoRV levels were not associated with chlamydial disease . Howevereensland . They dieensland . While i. who used an age of ≤ 2 years [. report [. reported finding four new KoRV-B infections in three adult females and one adult male over a period of almost three years. While they were unable to determine the partner in these wild koalas nor did they report aggressive behavior, the results could suggest sexual transmission since the majority were females, but additional studies are needed to further evaluate KoRV transmission between adults.By testing many zoo koalas with known pedigrees, we were able to evaluate potential KoRV transmission routes, which is key for understanding the epidemiology and prevention of these infections. Overall, our results are consistent with the major route of transmission being from dam-to-offspring for KoRV-B, supporting previous findings in a small number of zoo koalas , 22. In  2 years , whereas. also reported transmission of KoRV-B between adult koalas but mostly in females more suggestive of transmission during mating [Interestingly, in our study we identified eight offspring (7 adults and 1 joey) with discordant KoRV infections from the dam, supporting results from other recent studies , 33. Theg mating . AdditioVaccinations and antiretroviral drugs have each been proposed as measures to prevent or treat KoRV infection. We show in one leukemic animal with a high viral burden of KoRV-A, -B, and -F that treatment with both raltegravir and tenofovir for 33 days had little effect on reducing VLs and disease outcome. While 33 days is a short period to draw meaningful conclusions on the efficacy of raltegravir and tenofovir in this case, it is noteworthy that both drugs, if active on KoRV, may likely inhibit infection of the exogenously expressed KoRVs because they have been shown to strongly inhibit other gammaretroviruses in vitro –40. It iTesting and subsequent physical separation of animals infected with KoRV-B from those with only KoRV-A infection or KoRV-negative animals has also been suggested to prevent transmission of these variants and is the current koala management practice in the U.S. However, quarantining non-A KoRV-infected koalas may be impractical and costly, especially for wild populations. Separating koalas infected with specific subtypes could also reduce the koala genetic diversity in zoo-based populations like those in St. Bees Island . A KoRV Our findings also have implications for the biosafety of persons working with koalas. KoRVs are pathogenic and can infect human cell lines in vitro raising questions for potential zoonotic transfer to humans following exposure to zoo or wild koalas. The presence of high levels of the various KoRV-subtypes in multiple tissues, including blood, and the endogenous source of KoRV that cannot be eliminated through breeding, all provide multiple exposure opportunities. While the stability of infectious KoRV in the environment is currently unknown, other gammaretroviruses like feline leukemia virus can survive for at least two days in culture medium but are inactivated after only a few hours if dried . BiosafeThere are some limitations of our study. First, our study design was cross-sectional using convenience and archived specimens. Second, we did not test for all known KoRV subtypes or use NGS of PCR products to identify KoRVs. These additional KoRV subtypes were discovered after our study concluded and enough materials and resources were not available to design new assays and re-test koalas for these additional variants. Although NGS is clearly useful for determining the viral composition of a sample, it has not been validated for measuring viral loads and its use with PCR products can lead to overestimation of quasispecies unless corrective measures are taken like the use of unique molecular identifiers . ThirdlyOur results show a significant association of non-A KoRV infection and increased plasma VLs with leukemia and other cancers in infected koalas. We also document carcinoma in koalas infected with only KoRV-A. Although we confirm dam-to-offspring transmission of these non-A KoRV variants, we also show other routes are possible. Our validated qPCR assays will be useful to further characterize KoRV epidemiology and its zoonotic potential.Additional file 1: Additional table and figure.


Pericardial fat (PF) has been suggested to directly act on cardiomyocytes, leading to diastolic dysfunction. The aim of this study was to investigate whether a higher PF volume is associated with a lower diastolic function in healthy subjects.2, normal left ventricular ejection fraction), with (a)typical chest pain  from the cardiology outpatient clinic were retrospectively included in this study. All patients underwent a coronary computed tomographic angiography for the measurement of pericardial fat volume, as well as a transthoracic echocardiography for the assessment of diastolic function parameters. To assess the independent association of PF and diastolic function parameters, multivariable linear regression analysis was performed. To maximize differences in PF volume, the group was divided in low (lowest quartile of both sexes) and high (highest quartile of both sexes) PF volume. Multivariable binary logistic analysis was used to study the associations within the groups between PF and diastolic function, adjusted for age, BMI, and sex.254 adults , lower E/e (p < 0.01) and E/A (p = 0.01), reduced e′ lateral (p < 0.01), reduced e′ septal p = 0.03), compared to subjects with low pericardial fat.These findings confirm that pericardial fat volume, even in healthy subjects with normal cardiac function, is associated with diastolic function. Our results suggest that the mechanical effects of PF may limit the distensibility of the heart and thereby directly contribute to diastolic dysfunction.Trial registration NCT01671930 Diastolic heart failure is a major cause of morbidity and mortality  and is pDespite the clear definition, the understanding of the pathological mechanism of diastolic dysfunction remains poor. Various potential mechanisms have been suggested, but none of them can adequately explain the pathological process. Since increased pericardial fat (PF) volume is associated with adverse cardiovascular disease (CVD) outcomes, interest has peaked into this relationship and the potential effects of PF on cardiac dysfunction , 4.PF is divided into two fat components: the Epicardial Adipose Tissue (EAT) and the Cardiac Adipose tissue (CAT). It is presumed that the EAT, due to its anatomical proximity to the myocardium, has the most effects on the myocardium. In normal physiology, EAT may have positive metabolic effects as it has an important function in lipid storing, and it also secretes endocrine factors . It demoMoreover, Ng et al. found an association between EAT volume index and interstitial myocardial fibrosis in an overweight to obese population . This asIn summary, it is unknown whether PF and/or EAT influences diastolic cardiac function in healthy subjects before any symptoms of diastolic failure occur. Most studies looking into the associations between PF or EAT with diastolic function have been performed in subjects with heart failure, CVD, overweight, or (pre-)diabetes –20, 25. This study was approved by the Institutional Review Board (IRB) and Ethics Committee. Involved data were collected on a routine basis from within the Maastricht biomarker CT study  and analysed anonymously in accordance with Institutional Review Board guidelines. The study complies with the ethical principles of the Helsinki Declaration.This study cohort is comprised of patients from the cardiology outpatient clinic presenting with (a)typical chest pain, who were according to the standard care protocol referred for coronary computed tomographic angiography (CCTA) for the evaluation of stable CVD, in accordance with the current guidelines , 27. Inc2 without history or diagnosis of acute coronary syndrome at the time of CCTA were included. Exclusion criteria for this subgroup study were: left ventricular ejection fraction (LVEF) < 45%, diastolic dysfunction, atrial fibrillation, and diabetes mellitus.254 patients enrolled in the echocardiography subgroup of the Maastricht Biomarker CT study were retrospectively included in this study , 29. A fSerum samples were collected just before CCTA, processed within 2 h and directly stored at − 80 °C until analysis. Total cholesterol (CV 2.0%), triglycerides (CV 2.5%), high-density (CV 3.0%) and low-density lipoprotein concentrations were measured as previously described  . Serum cAll 254 patients had undergone a standardized non-enhanced scan to determine the calcium score using the Agatston method  at our cSemi-automatic segmentation determined the PF volume by dedicated software  using a threshold from − 150 to − 50 Hounsfield Units to distinguish visceral adipose tissue, as set by the software . BecauseEchocardiography was performed within a period of 3 months from the CCTA by an experienced echocardiographist. Transthoracic images of the left ventricle (LV) were acquired to assess morphology, function and mass . LV function and -mass were calculated by off-line analysis using Xcelera software package (Philips), according to current ESC/AHA guidelines .Only four diastolic parameters are decisive in the evaluation of diastolic function according to the American Society of Echocardiography (ASE)/European Association of Cardiovascular Imaging (EACVI) guidelines, namely, left atrial volume index (LAVI), e′ septal, e′ lateral , and peak velocity of tricuspid regurgitation (TR) . TherefoBaseline characteristics of the sample were summarized using mean and standard deviation or median and interquartile range (IQR) for normally distributed and skewed continuous variables, respectively. Categorical data were presented as absolute number and percentage. To assess the independent association of PF and diastolic function parameters in these 254 patients, linear regression analysis was performed with either LAVI or e′ septal or e′ lateral or TR as the dependent variable. These models were adjusted for BMI, age, sex, and their interaction terms with PF, since it is known that these parameters are strongly associated with PF , 35, 36.t test for continuous variables with a normal distribution, or the Mann–Whitney U-test for non-normal distributed continuous variables. Pearson’s Chi square test was used for categorical variables. Data are presented as proportions, mean ± standard deviations, and data with a non-normal distribution are presented as the median .This study is based on a sample of healthy participants without diastolic dysfunction, therefore, only mild differences in diastolic function were expected. To maximize the differences in PF volume, the group was divided into low PF (lowest quartile of both sexes separately) and high PF (highest quartile of both sexes separately). The lowest and highest quartile groups were matched for cardiovascular risk factors, i.e., sex, systolic and diastolic blood pressure, total and LDL cholesterol, and kidney function. Differences in other baseline characteristics across these extreme quartiles of PF volume were investigated using the independent-samples To assess the independent association of PF and diastolic function parameters in these extreme quartiles (n = 130), also multivariable linear regression analysis was performed with either LAVI, or e′ septal, or e′ lateral, or E/e′, or TR as the dependent variable. These models were adjusted for BMI, age, and sex. Results are presented as regression coefficient with 95% confidence interval (95% CI).To investigate the association of EAT with the total PF and EAT with diastolic function, Pearson’s correlation coefficient was computed. Because only in 10% of the subjects an EAT volume was known, this subgroup was considered too small to perform regression analysis. All statistical analyses were performed with IBM SPSS Statistics Version 25.0 . Two-sided p-values of ≤ 0.05 were considered statistically significant.The baseline characteristics for the total sample and the lowest and highest quartile groups of PF volume are presented in Table Median (IQR) PF volume in the total cohort were 1.411 IQR 1.035, 2.057) dl. Since males have a higher PF volume than females , the upper and lower PF volume quartiles of males and females were combined for the analysis , BMI , glucose , HDL cholesterol (1.5 ± 0.4 versus 1.2 ± 0.4) and triglycerides , see Table The association between diastolic function and PF volume was investigated, as some of the diastolic parameters are expected to deteriorate during the development of diastolic dysfunction before clinical criteria for diastolic dysfunction are met Fig. .Fig. 4PFAlthough still in the normal range, significant differences in the diastolic function parameters were found between the lowest and highest PF quartiles. As shown in Table In the total sample n = 254), significant associations for all four diastolic parameters with the PF volume were found after adjusting for BMI, age, and sex. These data are depicted in Table , signifiIn 10% of the total sample (n = 24), the EAT volume was studied by manually dividing the PF into the different CAT and EAT volumes. This random selection of 6 patients per PF quartile was made since the manual subdivision of the PF is extremely laborious, and to ascertain that the sample reflects the entire population. The data showed that with an increasing PF, no similar increase in the relative volume of EAT and CAT can be expected, as the relationship with the relative amount of EAT and CAT is lacking (p > 0.7). These data are illustrated in Fig. To gain further insight into whether EAT is the major culprit in hampering diastolic function as suggested because of its anatomic proximity to the myocardium, separate correlations of EAT were made with the different diastolic parameters. Despite the small number, a direct correlation of the percentage of EAT and e′ lateral was found. There was no correlation with EAT and the other diastolic function parameters , insights in the compartmental distribution of PF and its consequences on diastolic function can be gathered. We found that at increased PF volumes, the EAT and CAT compartments increased at a same amount relatively to the whole fat depot. This is surprising as Wu et al. reported that subjects after bariatric surgery showed a great loss of CAT and only a small decrease in EAT . TherefoThe association of high PF with e′ lateral suggests that in a healthy population the mechanical effects of PF limit the distensibility of the heart first, which subsequently contributes to diastolic dysfunction. This study suggests that secondly, after progression of this relaxation problem of the lateral wall, the LAVI might increase despite the compression of the PF mass, as seen in diastolic dysfunction. But this remains speculative, as we did not measure the mobility of the lateral wall of the left ventricle during the systolic phase. However, during systole the PF mass will be less restrictive than during diastole, which is in line with our hypothesis. Most notably, a mechanically limited heart is accompanied by pressure changes within the cardiomyocytes, which in turn can affect the metabolism of these cells, and thereby, negatively influence diastolic function.Most of the research on PF so far focused on adipokine release and a potentially causal role in the formation of fibroses. Pressure changes due to increased PF leading to an altered metabolism are an alternative pathway how PF can influence cardiac function. Thus, although the underlying mechanism remains unknown, the idea that mechanical effects of high PF cause a diminished mobility of the myocardium is supported by the current data. As others already suggested, this diminished mobility may provoke fibrosis, which has been associated with diastolic dysfunction, however this remains to be elucidated. In our population changes in diastolic function parameters were associated with an increase in PF, however, the diastolic function was within normal range; hence no causality with fibrosis could be made.As we performed a cross-sectional retrospective study, our study has some limitations by design. Due to the retrospective design, the low and high PF groups were not matched on all relevant characteristics. However, we did adjust our analyses for age, BMI, and sex. Although we corrected for age, BMI, and sex, some of metabolic characteristics such as glucose, HDL-cholesterol, and triglycerides, may confound the associations, although these metabolic characteristics were within normal range. Also, because of the retrospective design, there was timeframe of a maximum of three months between the CCTA and TTE, this may have influenced our association. In addition, since the manual subdivision of the PF is extremely laborious, we only separated the PF components in 10% of the total cohort, following random selection. Thus, the power was limited for exploring the metabolic effects of EAT, independently of PF, on diastolic function. The cross-sectional outline of this study does not allow any conclusions regarding possible causality. Future work should therefore include a prospective approach to evaluate causal relationships.Finally, it is important to bear in mind that our study population consisted of relatively healthy subjects, whose cardiac diastolic function was considered to be good. We only studied the associations between PF and small variations in normal diastolic function, which also explains why we did not find correlations between the diastolic parameters and age, BMI, or sex, in our sample (Additional file The purpose of the current study was to determine the association of PF and cardiac diastolic function in a healthy population. Linear regression analysis revealed that PF, independently of age, BMI, and sex, is associated with the four diastolic ultrasound parameters which are decisive in the evaluation of diastolic function. A potential underlying mechanism of this may be that increased PF may compress the heart, leading to a limited distensibility in the diastole and fibrosis as seen in cardiac remodeling, and thus, may lead to diastolic dysfunction. This study adds to the growing body of research that explores possible mechanisms in the development of diastolic failure. Concluding, we confirm that PF, even in healthy subjects with normal cardiac function and without diabetes, does hinder diastolic function. The exact causality of this effect and the relationship with fibrosis remains to be determined.Additional file 1. Supplementary figures.


Secondary mitral regurgitation (sMR) is characterized by left ventricular (LV) dilatation or dysfunction, resulting in failure of mitral leaflet coaptation. sMR complicates up to 35% of ischaemic cardiomyopathies  and 57%  Mitral regurgitation (MR) is the most common valvular disease, and prevalence is increasing with improved life expectancy in the Western world . PrimaryNormal function of the mitral valve (MV) relies on leaflet coaptation during ventricular systole. This requires undistorted LV geometry, intact MV apparatus, synchronous contraction of papillary muscles and a balance between opening and closing forces generated by LV contraction and relaxation. Failure of any of these processes results in sMR, although the mechanism depends on the insult to the LV. For example, an inferolateral myocardial infarction may cause focal myocardial remodeling and an akinetic superolateral papillary muscle, leading to asymmetric chordal traction on posterior and anterior leaflets, apical displacement of the coaptation point and ultimately eccentric, posteriorly directed MR (Carpentier type IIIb mechanism). Less frequently, symmetric dilatation of the LV occurs due to dilated cardiomyopathy (DCM) or severe 3-vessel coronary disease, leading to symmetric annular dilatation, equal papillary muscle displacement, and tenting of both leaflets resulting in a central jet of regurgitation (Carpentier type Ia mechanism) .A vicious cycle ensues in which the resultant sMR causes left atrial and ventricular volume overload and dilatation, which in turn promotes further sMR . ChronicReliance on clinical examination to detect sMR lacks sensitivity, and the main diagnostic tool is transthoracic echocardiography (TTE) with color flow doppler. On finding MR, the role of the imager is to determine the mechanism of MR, through the assessment of the mitral leaflets, annulus, chordae and papillary muscles, along with associated changes in the ventricle and atrium.2 on TTE and > 1.6 cm2 on TOE and posterior leaflet angle > 45°. A full summary of these factors are categorized in Although the classical definition of sMR requires that the MV leaflets are normal, it is now known that changes occur in response to the stresses exerted upon the tented leaflets that counteract the loss of leaflet coaptation. These compensatory changes include progressive leaflet lengthening, thickening and fibrosis , 11. 2D R2 = 0.97; P < 001), closed mitral leaflet area , tenting area and volume (R2 = 0.84), and papillary muscle tethering distances   . CT is a = 0.84) , which c = 0.84) .2/m2  despite less severe LV remodeling with the former. This is due to localized inferobasal LV remodeling following inferior MI, resulting in displacement of the contiguous papillary muscle , 41, 42.Direct papillary muscle infarction (PMI) also contributes to sMR in up to a third of patients following acute MI. Late gadolinium enhancement (LGE) of the papillary muscles on CMR can diagnose PMI but the prognostic significance of these findings are unclear , 46. FolThe presence of sMR increases the poor prognosis of reduced ventricular function, and the frequency and severity of adverse outcomes increase as the MR worsens. Heart failure affects the majority of patients with sMR within 5 years, including those who have normal LVEF . The int2 are independent predictors of pulmonary oedema (Transthoracic echocardiography (TTE) is usually the first line investigation for the investigation of LV size and function by 2D Simpson's biplane method. 3D volumetric assessment is more accurate and reproducible  but undey oedema  and cardy oedema , respectCMR is not only the gold standard for measuring LV structure  and function  but also quantifies pathophysiological changes in the myocardium. CMR measures the presence, extent and location of post-infarction scar using LGE and thereby identifies those with greater risk of sMR  and high2D and 3D assessment by TTE supplemented by TOE are the investigations of choice for imaging the pathognomonic changes to the mitral leaflets, annulus, chordae and papillary muscles in ischaemic sMR.CMR or CT assessment of the valve is useful if adequate imaging is not obtained with echocardiography .LV volumetric assessment by 3D echo is superior to 2D echo, however, CMR is the gold-standard technique to assess the LV due to superior spatial resolution, allowing accurate measurement of LV volume, geometry, fibrosis and extent of scar.2 or > 50% of the LA area is associated with increased mortality at 1 year (OR 4.47 cf mild or absent MR)  , this issent MR) .2, RVol < 30 ml were identified as having low mortality risk while those with EROA > 0.3 cm2 and Rvol > 45 ml were high risk. For the intermediate group (EROA 0.2–0.29 cm2 and RVol 30–44 ml), a regurgitant fraction above and below 50% was found to be a threshold which could divide patients into the high and low risk categories, respectively . The problem of non-hemispherical shape can be surmounted by using biplane to measure VCW in perpendicular views or by using multiplanar reconstruction to measure vena contracta area (VCA). While the threshold for VCA severity remains to be clearly defined, 98% of sMR patients with an EROA > 0.2 cmgitation . 3D cologitation . 3D valugitation .r = 0.6–0.92) in only 26–66% patients when categorizing MR as severe  and aortic stroke volume from phase-contrast velocity flow mapping in the aortic sinus  63). As. As63). s severe –69. Of ts severe , but thes severe –72. Secos severe . Thirdlys severe  and of ns severe . More daMultiparametric TTE is the mainstay for quantifying sMR;There are major limitations of 2D TTE and TOE quantification of sMR;3D echo overcomes many of the limitations of 2D but reproducibility is an issue;Quantification of sMR by CMR is more reproducible and may be a better arbiter of outcome but further research is needed.Multimodality imaging plays a central role in predicting benefit from intervention, including accurate quantification of sMR relative to the degree of LV remodeling, and identification of mitral valve characteristics that are more amenable to successful intervention.Although mitral annuloplasty produces excellent long-term results in pMR , this is2) and greater tethering (assessed by measurement of the posterior leaflet angle > 40.2°) as strongly associated with high likelihood of sMR recurrence (Pre-operative characteristics that are associated with increased failure rates on echo include more extreme LV remodeling (LV end-diastolic diameter > 65 mm) and greater distortion of the MV apparatus (leaflet tenting height > 6 mm) . Similarcurrence . The potcurrence . A full While a number of devices are in development, transcatheter edge-to-edge repair using MitraClip is currently the most widely used device. Other devices such as Cardioband and the Carillon mitral contour system have CE approval and have shown promising results in reducing mitral regurgitant volume , 81. In Patient selection is one of the most important factors determining success of TMVR as evidenced by the contrasting outcomes of the COAPT  and MITR2), and smaller transmitral pressure gradient (< 4 mmHg) have better outcomes following TMVR (2)   and greaTMVR (2) .Pre-procedural assessment by CT is expected to increase with growth of TMVR. The major advantages of CT are accurate measurement of mitral annular dimensions, which has implications for device sizing, landing zone, determining risk of LV outflow tract (LVOT) obstruction, and delineation of mitral annular calcification, in whom TMVR carries higher risk . 3D echoIntraprocedural 3D TOE is essential during all steps of TMVR and is superior to 2D TOE . Post-prThere are multiple validated pre-operative characteristics on echo identifying likely outcome from surgical repair in sMR;Echo and CT both have an important role in the pre-procedure work up for TMVR;Intraprocedural 3D TOE is critical to the success of TMVR and superior to 2D TOE.The conflicting MITRA-FR and COAPT studies have re-ignited debate regarding patient selection in sMR. At the forefront of this discussion are the imaging parameters used to assess the severity of MR, LV size, and function and their relationship to one another. Several have suggested that this is a consequence of population heterogeneity, with two cohorts: those in whom chronic heart failure and global LV remodeling is the primary pathology and in whom there is a linear relationship between LV dilatation and EROA for any given LVEF. Accordingly, the degree of sMR is “proportionate” to the LV remodeling (with a lower RVol), and these patients are most likely to benefit from heart failure therapies. The contrasting cohort are those with predominant MV valve abnormalities, in whom there is greater regurgitation which is “disproportionate” to LV remodeling. These patients are expected to respond more favorably to mitral valve intervention –94. In ssMR is common and mortality is high with and without treatment. Medical and surgical therapies do not significantly impact survival but TMVR may reduce mortality and heart failure hospitalization. Assessment of the sMR jet and MV apparatus by 2D and 3D TTE and TOE using a multiparametric approach remain the foundation for assessment. There are however limitations and CMR has the potential to revolutionize the assessment of patients with sMR but is currently underutilized in this field due to lack of research. The role of CT is growing in sMR due to pre-procedural assessment of the patient under consideration for TMVR.All authors contributed to the writing and proofreading of the article.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. The handling editor declared a past co-authorship with the authors RS and SM.


Heart Failure (HF) is the most common cause of death in Friedreich’s ataxia (FRDA), an inherited mitochondrial disease. Myocardial fibrosis and myocardial hypertrophy are well-documented autopsy features among FRDA patients with HF.To leverage the unique tissue characterization features of cardiac magnetic resonance (CMR) for characterizing myocardial remodeling in patients with genetically confirmed FRDA without HF and preserved left ventricular ejection fraction (LVEF > 55%).ic), a marker of cardiomyocyte size.Twenty-seven FRDA’s patients  and 10 healthy controls  underwent a CMR for assessment of LV function, myocardial T1, late gadolinium enhancement (LGE), extracellular volume fraction (ECV), and intracellular water-lifetime , and decreased LV end-diastolic volume index , compared with controls. Additionally, ECV and cardiomyocyte size  were larger in FRDA patients . ECV and τic were positively associated with LV mass-to-volume ratio . LVMASSi and cardiomyocyte mass-index [(1−ECV)·LVMASSi] declined with age at the CMR exam, independent of the age at initial diagnosis.As compared to controls, FRDA patients had a preserved LVEF , larger LV mass index (LVMASSi: 61±21.7 vs. 45±4.2g/mic is associated with more severe cardiomyopathy classification, suggesting a role for these markers in tracking disease progression.LV hypertrophy and concentric LV remodeling in FRDA are associated at the tissue level with an expansion of the ECV and an increase in cardiomyocyte size. The adverse tissue remodeling assessed by ECV and τ FXN gene on chromosome 9q13 [Friedreich’s ataxia (FRDA) is the most frequent autosomal recessive ataxia worldwide with a prevalence of approximately 1 in 50,000 [ome 9q13 . The mutome 9q13 . Thus, mCardiac involvement is found in up to 60% of all FRDA patients and it is considered the leading cause of death . It consic), using T1-weighted CMR imaging, in a cohort of patients with FRDA without signs of HF. We also investigated whether myocardial tissue phenotyping by CMR can highlight particular characteristics of LV remodeling in FRDA’s cardiomyopathy, beyond those currently assessed with Weidemann’s imaging-based classification [Cardiac magnetic resonance (CMR) imaging has emerged as a promising diagnostic technique in FRDA patients, not only to determine the severity of LV remodeling ,11, but fication  of diseaFXN. As a control population, 10 volunteers were prospectively recruited under an IRB-approved protocol and reported elsewhere [We recruited 27 consecutive patients with genetic confirmation of FRDA regularly followed at the University Hospital of the State University of Campinas (UNICAMP), Brazil between 2014 and 2018. All enrolled patients were homozygous for GAA expansions at lsewhere ,17. The lsewhere  to defin0; 192 x 128 matrix; 8-mm slice), covering approximately 2 cardiac cycles  in two short-axis slices at the level of the mid segments of the LV. In the same locations, T1 imaging was done before, and 4 to 5 times after the injection of gadolinium to cover an approximately 30-min period of contrast clearance. Volunteers were also scanned using a 3-Tesla system  using the same techniques as for FRDA patients.All subjects underwent CMR imaging in supine position in a 3-Tesla magnet . Electrocardiographically gated cine imaging with steady state free-precession (SSFP) using repetition time TR = 3.4 ms, echo time TE = 1.5 ms, and in-plane spatial resolution = 1.5–2 mm was performed to assess left ventricular (LV) volumes, function and mass. An inversion-recovery-prepared, gradient-echo sequence with segmented acquisition was applied for late gadolinium enhancement (LGE) imaging at 10 minutes after the end of a cumulative administration of 0.2 mmol/kg of gadoterate meglumine . Slice locations for LGE imaging (8-mm slice thickness) matched those for cine imaging. For the T1 quantification we used Look-Locker imaging  with a nic, a cell size-dependent parameter, were adjustable parameters of this model. Blood hematocrit was a fixed parameter of the model.Standard methods were applied to measure LV mass, volumes and function . For each Look-Locker image series, the endocardial and epicardial borders of the LV were traced, excluding papillary muscles , and theχ2 test otherwise. Bivariate correlation was used to assess associations . Multi-variate linear regression analysis was used to build a model for τic, with LV mass index and age at disease onset as simultaneous predictors. A variance inflation factor < 3 was used as criterion as guard against multicollinearity. The Cochran Armitage test was used to test for a trend in the proportion of patients with ECV > median across the levels of the Weidemann classification of FRDA cardiac phenotype. Based on the previously described variability of ECV in healthy volunteers [http://www.Rproject.org). A two-sided p-value less than 0.05 was considered statistically significant.Results are summarized as mean±standard deviation. T-tests were used to compare continuous variables between groups. Binary data was compared using Fisher exact test when appropriate, or a lunteers , the preDemographic, clinical and laboratory data of all recruited patients and controls are illustrated in Regarding neurological impairment, median FARS.3 score was 60 (range: 23–97). In this cohort, neurological function, expressed by FARS.3 scores, was strongly associated with time since disease onset . Male patients had a significantly worse neurological status than females . Patients with diabetes mellitus had a worse (i.e. higher) FARS.3 score than non-diabetic patients . The difference in FARS.3 between diabetic and non-diabetic subjects remained significant with simultaneous adjustment by age.ic  were significantly higher in the FRDA group compared to healthy controls. In the FRDA group, τic correlated positively with age , and τic was significantly lower in patients with diabetes mellitus (DM) compared to non-diabetic subjects . This difference of τic with respect to DM remained significant (P = 0.004) with simultaneous adjustment of τic by age . Four out of 27 patients (15%) had positive LGE with an atypical pattern for coronary artery disease, comprising the mid inferolateral (n = 2) and the apical lateral segment (n = 2), whereas none of the controls presented such finding. Mid inferolateral segments in two patients showing LGE and matching with segments in T1 imaging were excluded from T1 mapping analysis.All FRDA patients completed the CMR studies without any adverse reactions. The images in CMR-derived global parameters of myocardial remodeling, such as LV end systolic and diastolic volumes, mass, and mass-to-volume ratios, were noticeably different between FRDA patients and controls . LV massAccording to Weidemann’s criteria for the FRDA-CM cardiac phenotype , 8 (30%)ic), reveals a tissue phenotype in FRDA characterized by interstitial fibrosis and cardiomyocyte hypertrophy, consistent with previous histological studies [In this study, FRDA patients with preserved LVEF and without HF had a cardiac phenotype characterized by concentric LV hypertrophy in accordance with previous reports ,22. The  studies . Novel m6. There are few published studies; most of them assessed small cohorts and solely employed echocardiographic data [A cardiomyopathy was the underlying cause of death in 59% of patients with FRDA in a large retrospective study . Despitehic data ,25. Recehic data ,27. Henchic data ; secondlhic data . Taking ic, has been validated by histological analyses as a non-invasive measure directly related to cardiomyocyte size [ic indicates that cardiomyocyte hypertrophy is prevalent in FRDA-CM. τic was approximately twice as long in FRDA-CM compared to controls, suggesting a doubling of cardiomyocyte size, and a 4 x larger cross-sectional area. The latter estimation for cardiomyocyte cross-sectional area is in excellent agreement with histological results from a previous study [2 vs. 249 μm2 in controls). Patients with later onset, i.e. shorter disease duration when presenting for the CMR study, had higher τic values. FRDA-CM generally first presents with cardiomyocyte hypertrophy, and in a later phase—to borrow a term from HCM—manifests itself by a regression of cardiomyocyte hypertrophy or possibly LV atrophy and LV dilation. This could potentially explain why patients diagnosed earlier, and with longer disease duration, have a lower τic [The intracellular water-lifetime, τyte size . It depeyte size . A higheus study  of FRDA ower τic ,28. In mower τic , myofibrFRDA is associated with an increased risk of diabetes mellitus (DM). It has been previously established that DM is independently associated with worse functional status in FRDA , indepenECV correlated with the classification of cardiac phenotype based on the criteria of Weidemann et al . This cl2 x 250μm2 in controls). The authors hypothesized that such hypertrophy could be an adaptive response to fiber loss.Our results are consistent with pathological analyses of the heart in FRDA ,34. Koepic. Moreover, standard T2-weighted images have not been acquired, despite proven useful for other cardiomyopathies. Fourthly, while healthy volunteers and FRDA subjects have been imaged using the same filed strength (3 Tesla) and the exact same imaging parameters, two different systems . Our reference values for healthy volunteers are in broad agreement with published reference values [ic) has been validated as a consistent marker of cardiomyocyte size [The current study has several limitations. Firstly, the observational cross-sectional design of this study did not allow a longitudinal analysis. While cross-sectional observations such as the association of CMR parameter with FARS3 scores and the age at the MRI exam are of interest and hypothesis-generating, only a longitudinal analysis can provide further insights on disease progression in FA. Secondly, is its limited sample size. Although we detected significant differences between FDRA patients and healthy controls, further investigations should be performed to further elucidate myocardial tissue remodeling during the progression to heart failure. Thirdly, we chose to use a Look-Locker approach rather than standard sequences  since this approach has been validated for quantification of τe values . In addiyte size  and subsyte size ,36, confic were notably elevated in FRDA patients even in the absence of LGE, consistent with a cardiac hypertrophy phenotype. CMR biomarkers of tissue remodeling from T1 mapping data provide novel insights into the myocardial tissue remodeling and cardiac disease severity in FRDA.LV mass index, ECV and τ


Increased wave reflection is an independent predictor of cardiovascular events, possibly due to effects on left ventricular (LV) function. We investigated the relationship between reflected waves in early systole, the forward decompression wave in mid-late systole and LV mechanical behavior. Invasively acquired ascending aortic velocity, pressure, and LV long and minor axes’ dimensions were measured simultaneously in 11 anesthetized dogs during both control conditions and aortic occlusion to cause additional early wave reflection. Wave intensity analysis (WIA) was used to identify the arrival of the reflected wave and the onset of a forward decompression wave in mid-late systole. The arrival time of the reflected wave coincided with the time when minor axis shortening began to decline from its peak, even during aortic occlusion when this time is 12 ms earlier. The initial decline in long axis shortening corresponded to the time of the peak of the reflected wave. The forward decompression wave was consistently observed to have a slow and then rapid phase. The slow phase onset coincided with time of maximum shortening velocity of the long axis. The onset of the later larger rapid phase consistently coincided with an increased rate of deceleration of both axes during late systole. Forward decompression waves are generated by the LV when the long axis shortening velocity falls. Reflected wave arrival has a detrimental effect on LV function, particularly the minor axis. These observations lend support to suggestions that therapies directed toward reducing wave reflection may be of value in hypertension and cardiovascular disease. Increased wave reflection is an independent predictor of cardiovascular events in hypertension  and has LV motion undergoes a complex pattern of change during the cardiac cycle  however,Wave intensity analysis (WIA) has proved a useful method for studying pressure-flow dynamics in conjunction with LV wall motion patterns in health and disease . Rapid sWe hypothesized that wave reflections would influence LV minor (M) and long (L) axis function. Further, to establish mechanistic links between waves and LV function, we used aortic occlusion as means of inducing earlier and larger wave reflections and examined their effect on LV function.The experimental protocol was approved by the animal care committee of the University Of Calgary, Faculty of Medicine and conforms to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Eleven open chest mongrel dogs were anesthetized using 30 mg/kg of sodium pentobarbital administered intravenously, in addition to a maintenance dose of 75 mg/h for the length of the experiment. The dogs were intubated and mechanically ventilated . The forelegs and left hind leg of the dogs were used for recording the ECG.U) was derived using the post mortem diameter and assuming a circular cross-sectional area. Pressure at the aortic root (P) and LV (Plv) was measured using high-fidelity pressure catheters . The aortic root catheter was placed approximately 1 cm distal to the aortic valve and was introduced into the aorta via the brachial or the carotid arteries. The LV pressure catheter was also inserted into either a brachial artery or into the LV directly through the myocardium of the LV apex. A mercury manometer was used to calibrate the pressure catheters before every experiment, and all data were digitally recorded at a sampling rate of 200 Hz. LV long and minor axes’ dimensions were measured throughout the cardiac cycle. Two pairs of ultrasound sonomicrometer crystals  were implanted in the mid-wall of the LV myocardium to measure the movement of the long (base–apex) and short  axes throughout the cardiac cycle. The septum and free wall crystals were implanted at the mid-level, between the base and apex of the LV.An ultrasonic flow probe  was snug-fitted to the ascending aorta to measure blood flow rate, from which velocity  = SBP − DBP, where SBP is the systolic blood pressure and DBP is the diastolic blood pressure. Mean arterial pressure (MAP) = DBP + (PP/3) and Stroke volume (SV) = (EDV − ESV), where EDV and ESV are end diastolic and systolic volume. Cardiac output (CO) = SV × heart rate (HR), where HR is the heart rate. Total peripheral resistance (TPR) = MAP/CO, and total arterial compliance (TAC) = SV/PP. Effective arterial elastance and compliance are, respectively, (EEA) = ESBP/SV, where ESBP is end systolic blood pressure, and (C) = SV/PP.I) is the rate of energy flux per unit area (W/m2) and can be writtenWave intensity analysis is based on the solution of the one-dimensional conservation equations of mass and momentum. Wave intensity (dThe water hammer equation for forward (+) and backward (−) waves is3 and c is the wave speed, which we calculated using the PU loop, as previously described  were used to analyze the data. ow meter . Up to tt = 0 and the following hemodynamic events and their timings were identified: peak aortic pressure (Pmax); peak LV pressure (Plvmax) and the derivative of Plv with respect to time (Umax); an inflection point on the descending limb of the aortic velocity waveform before aortic valve closure (Ui) was determined from the first derivative of Ui with respect to t. LV volume was calculated by considering the LV as an ellipsoid using the following equationThe R wave of the QRS complex was taken as time r1 = LV long axis radius (base–apex) and r2 = septum to free wall radius whose dimensions were acquired using the sonomicrometer crystals. Time to peak volume decline (Vmax) was determined by plotting the first derivative of volume with respect to time.Where 2) was calculated by integrating the area under the peak of the wave with respect to time.The onset of ejection was identified from the onset of the upstroke of the aortic flow waveform, which also indicated the onset of the FCW. The FDW wave observed in mid-to-late systole was always seen to have a “slow” (D1) then “rapid” (D2) phase as shown in L) and minor (M) axis shortening was determined by differentiating the axial displacement with respect to time. As reported previously  and phase III  for each axis were determined from the second derivative of axial velocity of shortening. Minor and long axes rate of shortening (acceleration and deceleration) at each phase were also determined.Velocity of the LV long  was estimated using the equations derived by Average fiber myocardial stress in the mid-wall  was recorded.Where MmaxU was 2 ± 2 ms (within-observer coefficient of variation (CV) = 2%). The time to LmaxU was 2 ± 4 ms (CV = 1%). The time to the onset of the FCW wave was 1 ± 2 ms (CV = 1.78%), the onset of the BCW was 1 ± 1 ms (CV = 1%), the onset of the FDW (D1) was 2 ± 3 ms (CV = 1%), and the onset of the FDW (D2) was 1 ± 4 ms (CV = 1%).Measurement of the time intervals from the peak of the R wave of the QRS complex to the peak shortening velocity of both the minor and long axes as well as to the onset of all three WIA waves was repeated on three separate occasions by a single observer. The mean difference ± SD to the time of p-value)  . All anaThe baseline characteristics of the dogs during control are presented in MmaxU, at 110 ± 4 ms. The time of MmaxU agreed very closely with the arrival time of the reflected waves  . The long axis reached its maximum velocity of shortening, LmaxU, approximately 40 ms after the minor axis (147 ± 20 ms) and after the time of arrival of the reflected wave. However, the time of LmaxU corresponded very closely with the time of the peak of the BCW  .The LV minor axis reached its maximum velocity of shortening, < 0.001)  and was < 0.001) . The onsdiff = 0 ± 3 ms, CCC = 0.86, p < 0.001; minor axis diff = −1 ± 1 ms, CCC = 0.93, p < 0.001) (Pmax)  and the occurrence of an inflection point (Ui) on the descending limb of the aortic flow wave . As expected, D2 was also seen to coincide with a sharp decline in LV pressure  . This was just prior to closure of the aortic valve (phase III) and corresponded very closely with the onset of D2 (long axis < 0.001) . This ti< 0.001) .To allow mechanistic links between wave reflection and LV shortening velocity to be elucidated, aortic clamping was used to alter the magnitude and timing of the reflected wave. During occlusion, there was a significant increase in systolic, diastolic, mean, and pulse pressure , and thiMmaxU was reduced by 20% (from 1.5 ± 0.4 to 1.2 ± 0.2 m/s) and the time of MmaxU also occurred earlier (97 ± 36 ms); hence, the relationship between the arrival time of the reflected waves and the time of MmaxU was preserved  , LmaxU reduced by 11% (from 0.9 ± 0.5 to 0.8 ± 0.2 m/s), and time of the peak reflected wave and LmaxU was in good agreement . This time also corresponded with the onset of FDW (D1) .Proximal aortic occlusion had a marked effect on wave reflection  increasi< 0.001) . During FDW (D1) . Aortic ame time , and σmaWaves carry important information about LV function and the interaction between LV and arterial circulation . Our aimThe reflected wave is believed to play a detrimental role in the pathophysiology of cardiovascular disease in renal failure , hyperteThe strength of this study is the direct, invasive measurement of the reflected wave and left ventricular velocities. No assumptions about the timing and magnitude of waves were made, and measuring pressure and flow simultaneously to calculate wave intensity is superior to pressure waveform analysis alone. This work provides insight into how wave reflection influences LV function and wave generation. Wave reflection causes a deceleration in LV shortening and the function of the minor axis is particularly sensitive to the arrival of reflected waves. These observations lend support to previous suggestions that therapies directed toward reducing wave reflection may be of value in hypertension and cardiovascular disease.A limitation of the study was that only longitudinal and radial motion were studied . Changes in LV geometry over the cardiac cycle are intrinsically complex, particularly during ejection, and possible effects of rotation could not be accounted for in our study. However, the crystals used for measuring the axial movement were embedded into the myocardium at the mid-level where rotation is minimal, and thus, this should not invalidate our principal conclusions. Nevertheless, further studies resolving the full three-dimensional strain patterns in the ventricle would be valuable. A further limitation is the lack of wall thickness measurements to investigate the effect of wave reflection on myocardial stress in more detail. A final limitation is the assumption that the aortic area is uniform throughout the cardiac cycle, and we cannot rule out the possibility of aortic distortion during the cross-clamping in or variability during the cardiac cycle .In conclusion, This is the first study to simultaneously measure the arrival of reflected waves during systole with LV long and minor axes function. These measurements provided strong evidence that the arrival of the reflected wave initiates a deceleration in LV minor axis shortening. The data have also shown that the forward decompression wave generated by the LV has a slow and then rapid phase that are generated by two distinct changes in the rate of shortening of the LV axes. The results of this work are physiologically and clinically relevant as increased wave reflection causes a reduction in the contraction velocity of the LV. Reducing wave reflection may be of value in hypertension and cardiovascular disease patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by animal care committee, Calgary University.CP: data analysis, manuscript drafting, figures, and table production. AH: statistical analysis, data interpretation, and manuscript editing. MH: data interpretation and clinical implication and manuscript editing. AK: manuscript concept, data collection and interpretation and manuscript editing. All authors contributed to the article and approved the submitted version.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.


The aim of this study was to assess intra-observer and interobserver variability of biventricular function, volumes and mass in a heterogeneous group of patients with congenital heart disease (CHD) using cardiovascular magnetic resonance (CMR) imaging.CMR imaging provides highly accurate measurements of biventricular volumes and mass and is frequently used in the follow-up of patients with acquired and congenital heart disease. Data on reproducibility are limited in patients with CHD, while measurements should be reproducible, since CMR imaging has a main contribution to decision making and timing of (re)interventions.Thirty-five patients with CHD  were included in this study. A short axis set of contiguous slices was acquired with CMR imaging using a steady-state free precession (SSFP) pulse sequence. Intra-observer and interobserver variability was assessed for left ventricular (LV) and right ventricular (RV) volumes, function and mass by calculating the coefficient of variability.Intra-observer variability was between 2.9% and 6.8%, with the smallest variations in LV and RV end-diastolic volume (EDV) and the highest variations in LV end-systolic volume (ESV) and RV mass. Interobserver variability was between 3.9% and 10.2%, with the smallest variations in LV ejection fraction (EF) and RV EDV and the highest variations in biventricular ESV and mass.Intra-observer and interobserver variability of biventricular parameters assessed by CMR imaging is good for a heterogeneous group of patients with CHD. CMR imaging is an accurate and reliable method for follow-up of biventricular function, volumes and mass and should allow adequate assessment of changes in ventricular size and global ventricular function.Supported by NHF grant 2006B095