To establish the reference values of native T1 and extracellular volume (ECV) in patients without structural heart disease and had a negative adenosine stress 3T cardiac magnetic resonance.Short-axis T1 mapping images were acquired using a modified Look-Locker inversion recovery technique before and after administration of 0.15 mmol/kg gadobutrol to calculate both native T1 and ECV. To compare the agreement between measurement strategies, regions of interest (ROI) were drawn in all 16 segments then averaged to represent mean global native T1. Additionally, an ROI was drawn in the mid-ventricular septum on the same image to represent the mid-ventricular septal native T1.Fifty-one patients  were included. Mean global native T1 averaged from all 16 segments and a mid-ventricular septal native T1 were not significantly different . Men had lower mean global native T1  than women. Both mean global and mid-ventricular septal native T1 were not correlated with age . The calculated ECV was 26.6 ± 2.7 %, which was not influenced by either gender or age.We report the first study to validate the native T1 and ECV reference ranges, factors influencing T1, and the validation across measurement methods in older Asian patients without structural heart disease and had a negative adenosine stress test. These references allow for better detection of abnormal myocardial tissue characteristics in clinical practice. Extracellular volume (ECV) uses T1 values obtained pre- and post-contrast to calculate the extracellular component of the myocardium. The formula for estimation of ECV is derived from myocardial and blood T1 values before and after GBCA administration as well as the patient’s hematocrit. 22.1Adult patients with low-to-intermediate risk of cardiovascular disease (CVD) referred for clinical stress CMR scan due to suspected coronary artery disease were prospectively recruited at King Chulalongkorn Memorial Hospital, during July 2020 to January 2021. The study protocol was approved by the committees of Institutional Review Board, Faculty of Medicine, Chulalongkorn University. The need for consent was waived by an IRB (No. 302/64). All procedures performed in studies involving human participants were in accordance with the World Medical Association’s Declaration of Helsinki.The exclusion criteria were 1) patients under 18 years old, 2) any evidence of CVD as indicated by clinical history and physical exam, 3) history of diabetic mellitus, 4) pregnancy, 5) abnormal electrocardiographic (ECG) findings or presence of both atrial and ventricular arrhythmia at scan date, 6) abnormal structural heart conditions including abnormal ventricular function, chamber size, myocardial wall thickness and mass, moderate-to-severe valvular dysfunction or the presence of a scar on delayed enhancement imaging, and 7) artifact on pre-contrast T1 imaging, which compromise the accuracy of measuring the native T1 value. Baseline demographic data such as comorbidity and anthropometric parameters were collected as well as essential laboratory results including serum creatinine and hematocrit on the day of the CMR scan.2.2Supplementary table 1. The CMR stress test was performed by administering adenosine . Once adequate vasodilatory effect was achieved as confirmed by an increase in heart rate or patient attestation, a 0.10 mmol/kg of gadobutrol  was administered. During the gadobutrol infusion a first-pass perfusion sequence was run, which obtains base, mid, and apical single-shot fast gradient echo (GRE) images during each heartbeat. Seven minutes after the 1st gadobutrol injection, an additional 0.05 mmol/kg of gadobutrol was added then a 2D segmented inversion recovery (GRE) sequences were obtained to detect myocardial scar. Lastly, post-contrast T1 mapping images were acquired in identical slice locations as pre-contrast T1 mapping. Post-contrast T1 maps were acquired approximately 15 min after the 1st gadobutrol injection to approach steady-state conditions. The scan was performed using 3 Tesla  using an18-channel cardiac phased array receiver. The stress perfusion protocol included cines, pre-contrast T1 mapping, first pass perfusion, late gadolinium enhancement (LGE), and post-contrast T1 mapping. To assess left ventricular (LV) function and mass, two-, three-, and four-chamber long-axis images and 12 consecutive 8 mm short-axis cine images were acquired using a steady-state free precession (SSFP) sequence. Then, three short-axis images at basal, mid, and apical LV were acquired for pre-contrast T1 mapping using Modified look locker (3(3)3(3)5 MOLLI) technique. 2.3syngo.via, Siemens Healthineers, Erlangen, Germany), including LV chamber and function quantification, pre- and post-contrast T1 mappings, and ECV. For LV chamber assessment, the endocardial and epicardial contours were automatically delineated in diastole and systole in a stack of short-axis slices of the whole LV excluding papillary muscles and trabeculae, with manual adjustments when needed. The calculated parameters included LV end-diastolic volume, end-diastolic volume indexed to body size, ejection fraction, and mass index. Post-processing of native T1 mapping involved manually drawing regions of interest (ROIs) on grey scale short-axis images of T1 map in each of 16 segments of LV myocardium in accordance with standardized myocardial segmentation of the American Heart Association to obtain per-segment native T1 (A). The T1 values from all segments were averaged together to calculate a mean global native T1. In addition, we drew a single ROI in the mid-ventricular septum representing the mid-ventricular septal native T1 in order to compare agreement between measurement methods (B). We used the single ROI in the mid-ventricular septum and ROI placed in the center of blood pool on pre- and post- contrast T1 maps to calculate ECV using the formula ECV (%) = (100-Hct) * [Δ ∕ Δ(1/blood T1)].CMR images were checked to verify a diagnostic image quality. Two experienced observers blinded to clinical data analyzed the images. The image interpretation and post-processing were performed with post-processing software  were used to compare two different methods of native T1 measurement. Two experienced cardiologists independently tested inter-observer reproducibility. Statistical significance was indicated as p-value of less than 0.05.Statistical analysis was performed using SPSS . Continuous data were presented in mean ± standard deviation (SD). Categorical data were reported as frequencies and percentages. Ninety-five percent confident interval (95 % CI) of native T1 was used to define the normal value of native T1. To determine the difference between independent groups, an independent 32, p = 0.001), lower resting systolic blood pressure , and higher hematocrit  than women.Of 335 patients scheduled for CMR adenosine stress testing at 3T between July 2020 and January 2021, 204 patients had a negative stress result. Ninety-two patients were excluded due to presence of diabetes or known CVD, and 61 patients were excluded due to presence of abnormal ECG or structural heart conditions. The final population consisted of 51 patients. The majority of patients (92 %) were referred due to atypical chest discomfort with or without worsening dyspnea. The mean age was 65 ± 12 years, 65 % woman. The majority of patients were from age groups 61 to 70  followed by 51 to 60 , and 71 to 80 . The baseline characteristics of patients according to gender are shown in 2, p = 0.001, respectively). Overall, there was no significant difference between mean global and mid-ventricular septal native T1 values . The Bland-Altman plot also revealed good agreement between these measurement methods with 96 % of ICC . There was no significant correlation between ECV and age .The inter-observer reproducibility was 93 % for the per-segment native T1 measurement and 95 % for the mid-ventricular septal native T1 measurements. Mean global native T1 averaging from the value of all 16 segments provided the inter-observer reliability of 99 %. The intra-observer reproducibility was 99 % for the per-segment native T1 measurement, 95 % for the mid-ventricular septal native T1 measurements, and 99 % for the mean global native T1.4This is the first study to validate the native T1 and ECV reference ranges, factors influencing T1, and the validation across measurement methods in older Asian patients without structural heart disease and had a negative 3T CMR adenosine stress test.While several previous studies reported the normal reference of T1 and ECV in healthy subjects, a population-based reference values remain necessary. et.al demonstrated the positive correlation between age and native T1 value. et.al reported no significant influence of age for T1 values at 3T in an African-American population. In the literature the age dependence for normal T1 value has been conflicting to date. Roy Gender is another factor affecting native T1 value. There are multiple methods to measure native T1 from T1 mapping images. ROIs in abnormal areas on visual inspection should be drawn in focal myocardial disease. The study has some limitations. Firstly, this is a single-center and vendor-specific study; hence, this may limit its generalizability. Secondly, the results may not represent the true reference values in a “truly” healthy subject since a GBCA administration in asymptomatic healthy subjects is a major ethical concern. Instead, we recruited low-to-intermediate CVD risk patients referred for clinical scanning who had normal structural heart, including a negative adenosine stress CMR. Consequently, we decided to validate the normal references in this population, which are a majority of subjects come to the scanner in real-life practice. Lastly, ECV values were calculated after a 0.15 mmol/kg of gadobutrol injection, slightly different ECV values have been reported when different doses of contrast media were used. Despite the aforementioned limitations, this study shows the robustness of native T1 measurement demonstrated by high inter-and intra-observer reproducibility. This provides confidence in results when serially following patients with these sequences.5We provide normal native T1 and ECV reference ranges, factors influencing native T1, and the validation across measurement methods in patients without structural heart disease and who had a negative 3T CMR adenosine stress test. Females have higher native T1 than males without difference in ECV. These reference values allow for better detection of abnormal myocardial tissue characteristics in clinical practice.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


MyoT2* did not reach the threshold of clinically significant iron overload (<20 ms), in any of the patients. In 22 (52.4%) patients, at least one of the tissue parameters was reduced. Reduced myocardial T2* and/or T1 were found in 10 (23.8%) patients, including 4 pts with normal livT2*. LivT2* was reduced in 18 (42.9%) patients. MyoT1 and livT2* inversely correlated with ferritin . LivT2* by a dedicated sequence and livT2* by cardiac T2* mapping showed good agreement (ICC = 0.876 p < 0.001). Conclusions: In contemporary hemochromatosis, significant myocardial iron overload is rare. Low myocardial T2* and/or T1 values may warrant closer follow-up for accelerated myocardial iron overload even in patients without overt liver overload. Cardiac T2* mapping sequence allows for liver screening at the time of CMR.Background: Haemochromatosis (HCH), a common genetic disorder with variable penetrance, results in progressive but understudied iron overload. We prospectively evaluated organ iron loading and cardiac function in a tertiary center HCH cohort. Methods: 42 HCH patients (47 ± 14 years) and 36 controls underwent laboratory workup and cardiac magnetic resonance (CMR), including T1 and T2* mapping. Results: Myocardial T2* (myoT2*), myocardial T1 (myoT1) and liver T2* (livT2*) were lower in patients compared to controls (33 ± 4 ms vs. 36 ± 3 ms [ Hemochromatosis (HCH), a multisystemic disease resulting from iron overload, is one of the most common genetic disorders in Caucasians; 1–2 per 500 people are affected by one of the several disease types ,4,5. TheOxidative stress resulting from an accumulation of bioactive iron leads to tissue damage and organ dysfunction ,13. MostEchocardiography is an established noninvasive tool for cardiomyopathies, irrespective of their etiology. However, it has poor sensitivity for early myocardial involvement in HCH when standard techniques are used. More advanced techniques such as two-dimensional speckle tracking echocardiography (2D STE) have shown potential for early detection of myocardial alterations in HCH patients ,25,26. Liver iron loading has been traditionally assessed by liver biopsy ,26. The Myocardial T2* decreases as iron accumulates, with the normal myocardial T2* being approximately 35–40 ms. T2* values of <20 ms reflect significant myocardial iron overload , and valGiven the availability of appropriate noninvasive tools, myocardial assessment in the earlier stages of HCH, before significant damage to the LV is evident, seems achievable and may have significant diagnostic and prognostic implications. In this CMR study, we aimed to evaluate myocardial and liver iron status in a contemporary HCH cohort at a tertiary center and to compare these values in the treatment of naive and treated patients. We also assess the feasibility of cardiac parametric mapping for liver overload assessment during CMR. Additionally, inter-observer variability of the T2* mapping was evaluated.Forty-two patients of Caucasian descent aged 47 ± 14 years , with HCH without cardiovascular symptoms, were prospectively included between November 2014 and January 2019. The disease was diagnosed based on the presence of elevated serum iron parameters in the blood and the detection of the homozygous C282Y/C282Y HFE gene mutation. In carriers of other HFE genotypes (mixed heterozygotes C282Y/H63D or H63D/H63D homozygotes), excessive iron accumulation in hepatocytes was confirmed by a histopathological examination of the liver biopsy specimen or MR imaging. Other reasons for iron loading  were excluded . The labPatients were divided into two subgroups: Treatment-naive  and on treatment ; treatment according to clinical practice recommendations (14). The control group included 36 healthy age- and gender-matched volunteers. The study protocol was approved by the Institutional Ethics Committee (NKBBN/452/2016), and written informed consent was obtained from all subjects.ECG-gated CMR examinations were performed using a 1.5 T MR scanner  with an 18-element phased array cardiac coil and a 32-element spine coil with an auto-coil selection option, in line with SCMR/EuroCMR recommendations . T1 and These sequences were added to the routine protocol, which included cine and LGE short-axis CMR images prescribed every 10 mm (slice thickness of 8 mm) from base to apex. The in-plane resolution was typically 1.4 × 1.4 mm. Cine CMR was performed using a steady-state-free precession sequence. LGE images were acquired on average 8–15 min after contrast administration using segmented IR-GRE with inversion time adjusted as needed. The contrast (gadobutrol) dose was 0.1 mmol/kg.A dedicated module of the software Segment  for magnitude data analysis was used, which included error maps, showing pixel-wise fitting error levels. All parametric maps were generated by this software and then evaluated visually. Only areas with a fitting error level of 5% or less were analyzed, and preselected ROI placement was adjusted to meet this requirement to ensure the high quality of the data . An opti2 away from any visible vessels was analyzed in the liver. For myocardial T2* measurement, a full-thickness region of interest (ROI) was measured in the septum to avoid artifacts related to the proximity of the cardiac veins and lungs. For T1 measurement using a modified Look-Locker inversion recovery (MOLLI) sequence, a similar ROI was evaluated for comparisons and checked with LGE images to avoid areas of fibrosis/scar. An ROI of no less than 2 cm3. The LGE was assessed visually by two observers, and any discrepancy was solved by consensus with the 5SD method  ms vs. 981 [961–998] ms, p = 0.028). Both myocardial T2* (myoT2*) and myocardial T1 (myoT1) as well as liver T2* (livT2*) were significantly lower in HCH patients compared with controls . In nonep = 0.004). Myocardial and hepatic relaxometry parameters are summarized in Median liver T2* by cardiac mapping was also lower than in controls . Similarly, no correlation was found between ferritin and myoT2* . Conversely, myoT1 values as well as livT2* showed a rather weak but significant inverse correlation with ferritin concentration . Of note, there was no correlation between myoT1 and myoT2* .No correlations were found between LVEDVI and myoT2*  were significantly lower than in healthy controls. Of note, one or more of these parameters were below the reference range in over 50% of the study group. Low liver T2* (livT2*) was by far the most common abnormality , with features of significant liver iron loading  found in only 6 pts (10%). Ten patients (24%) showed abnormally low myocardial relaxation parameters . Hence, although myocardial iron overload was not clinically significant in any of the patients included in the study, it is likely that the pathological process may already be ongoing in a proportion of recently diagnosed treatment-naive individuals, while it is sufficiently controlled in the treated subgroup. This, given the high prevalence and variable clinical penetrance of the HFE gene mutations, suggests the need for early risk stratification and the potential for tailored preventive strategies. Importantly, the subgroup with low myocardial T1 and/or T2* included four patients with normal liver T2*, three of which were recently diagnosed, as treatment-naive patients. This points out the possibility that, in the presence of environmental or host-related modifiers, myocardial iron accumulation may occur in parallel or even precede significant hepatic overload . Of noteLow myocardial T1 (myoT1) has been found to be a useful indicator of iron overload in various settings, including thalassemias, other transfusion-dependent anemias, hemochromatosis, and myelodysplastic syndromes ,40,51. IIt should also be noted that myoT1 values in our cohort correlated significantly with ferritin values, as opposed to myoT2* values showing weak or no correlations with iron turnover parameters (which is concordant with a previously published study on HCH patients) . In thisPatients with myoT2* values > 20 ms are unlikely to have significant iron overload at the time of scanning, but those with borderline T2* and/or low T1 values might still deserve attention as a proportion of these patients will likely develop iron-overload cardiomyopathy in the future [Limited availability of CMR requires efficient use of scanner time, and measures should be taken to optimize exam protocols. Accordingly, we have shown that in patients with HCH, liver T2* values derived from the cardiac T2* mapping sequence did not differ from values acquired in a dedicated sequence with a broader range of echo times that represents a standard approach for liver assessment. It should be underlined that for robust T2* measurements in a wide range of T2* values, both liver and myocardial T2* times were measured with the combined signal model algorithm incorporated in the software Segment . Thus, cOur study has some limitations. Firstly, a relatively low number of subjects were included. However, this resulted from the strict inclusion criteria that included genetic testing, while additional genetic testing for rare mutations in HFE and other iron-related genes was not performed. Secondly, interobserver variability was assessed in a subgroup of our patients. Lastly, excess iron deposition begins within the epicardium and extends towards the mid-myocardium and endocardium. This was not specifically addressed in our methodology as best intramyocardial ROIs were selected to avoid partial volume effect and hence, the results may have been confounded by the degree of transmurality of iron deposits. Further research with a larger sample size and a more complex methodology could help to address this issue.In patients with hemochromatosis, standard LV volume and function parameters are not helpful for myocardial iron loading screening, irrespective of the time from diagnosis and the treatment status. Significant myocardial iron overload  is a rare finding in a contemporary HCH cohort at a tertiary center. However, abnormally low T2* and T1 values at the time of diagnosis could help identify patients at higher risk of cardiac iron overload even in the absence of liver overload and may warrant closer follow-up. Further research in larger cohorts is needed to demonstrate the clinical benefits of such an approach. Liver T2* measurement based on the cardiac T2* mapping sequence at the time of CMR can be useful for simultaneous liver screening.


Epicardial Adipose tissue (EAT) is related to low degree inflammation and oxidative damage in the adjacent tissue.Cardiac myofibrillary dysfunction, which can be measure by echocardiographical sstrain values.To explore whether EAT affects early myocardial dysfunction, as assessed Case–Control design. Patients lacking clinical significant heart failure, thyroid or renal disease or malignant abnormalities were included. Clinical-demographic and biochemical data were collected. EAT and myofibril deformation were measured by echocardiography.strain value  was associated with male sex and higher anthropometric and metabolic risk measures; particularly those with t2DM. Higher EAT was also associated higher strain value , and further correlation was evidenced , with significant influence of t2DM.A total of 71 patients were analyzed, and further subdivided according to type 2 Diabetes Mellitus (t2DM). Higher strain value, suggesting the influence of cardiac adipose tissue on the deformability of cardiac myofibril, with a more significant effect in the population with t2DM.EAT was related to  In the clinical practice, longitudinal strain (deformation in the base-apex direction) is useful for evaluating left ventricle contractile function [Cardiac myofibrillary dysfunction is an early subclinical manifestation of heart failure, and precedes a significant reduction of the left ventricle ejection fraction. Such myofibrillary dysfunction may be measure through an echocardiographical parameter known as the “ocardium ; whereasfunction . Some cafunction –5.Epicardial adipose tissue (EAT) is a type of visceral fat that shares similar embryonic origin than intra-abdominal visceral adipose tissue. EAT lays at the external wall of the myocardium and maintains intimal contact with the epicardial vessels, sharing the same microcirculation coming from branches of the coronary arteries .EAT shows compressibility and elasticity that are important to provide mechanical protection to the coronary arteries against the excessive distortion caused by the arterial pulse and myocardial contraction . EAT hasCurrently, it is not clear whether EAT may relate with cardiac myofibrillar dysfunction. Therefore, the aim of this study was to explore whether EAT impacts early myocardial dysfunction and the influence of comorbidities.The present study was carried out from June 2019 to February 2020 at Ticoman General Hospital, Mexico City. Subjects participating were adults older than 18 years old, with t2DM, who were attended at Internal Medicine outpatient Clinic; as well as healthy subjects, age- sex- matched. Participants were excluded if they presented additional co-morbidity, malignant disease, drug use or pregnancy.All cases were de-identified to comply with data protection recommendation for research. This project was registered and approved by the Institutional Boards of Ethics and Research, Ticoman General Hospital, SEDESA, Mexico City  and all the experiments were performed according to Health General Law—National Guideline, in accordance to the Good Clinical Practices Guidelines as well as the Ethics recommendation from Helsinki declaration. All participants, and/or their legal guardian(s), signed informed consent before have been recruited.Clinical-demographic data were collected during initial interview. Anthropometric data like weight, height and waist circumference were registered, and body mass index (BMI) was calculated. A blood sample was obtained by vein puncture after a 12-h fast, and plasma lipids, glycemia and HbA1c were determined by routine automatized laboratory analyzer.strain and strain rate were performed using tissue Ultrasound Doppler mode at 4 chamber, apical axis, using a 3.5 MHz transducer, Aloka alfa 6 equipment (Japan). Image captures were obtained from 3 cardiac cycles. Either EAT or Doppler determinations were simultaneously performed by two experienced cardiologist, unaware of the clinical data of the patients.Echocardiographic, long parasternal axis was used to measure EAT as follows. EAT was measure at free wall of the right ventricle. Determination was performed at the end of the systole, and 3 different determinations were recorded at 3 consecutive cardiac cycles, and calculating the EAT average, as described by Dr Iacobellis et al. . The meap-value ≤ 0.05 was considered as statistical significant.Kolmogórov-Smirnov test was used to determine normality of data distribution. Quantitative data were presented as mean and standard deviation or median and interquartile range appropriately. Qualitative data were expressed as n (%). Statistical comparisons included student T-test or Mann–Whitney test, as well as Pearson correlation. ROC analysis was used to estimate EAT cutoff value, then Odds Ratio (OR) risk and multiple regression analysis were performed. Statistical analysis was performed using the SPSS v.25.0. A The study population was constituted by 71 patients, mean aged 49.0 ± 9.3 years old, paired by gender ; mean HbA1c 7.4%, c-LDL 110.7 and mean EAT of 4.2 ± 1.2 mm.Strain value  and sub-analyses were performed by gender and T2DM . In general, a higher Strain value was related with higher values of weight, BMI and SBP; as well as higher EAT   Fig. ; while sp = 0.047), but not other well-known cardiovascular risk factors, such as gender (p = 0.20), BMI (p = 0.93), abdominal circumference (p = 0.85) or HbA1c (p = 0.89).Finally, logistic regression analysis showed that higher plasma LDL cholesterol tended to be associated with lower myocardial deformity , as well as with ventricular mass and high sensitivity C-reactive protein values [strain in subjects with metabolic syndrome, obesity and coronary artery disease [This finding let us speculate that EAT may limit the deformation capacity of the cardiac myofibril. Consistently, Cho et al. found that EAT was associated with the degree of subclinical myocardial dysfunction  mechanical impairment: the increase in the EAT would limit the movement of the myocardium, and 2) inflammation: epicardial fat has histological characteristics of brown fat, which is in close proximity with coronary arteries and has the potential ability to secrete pro-inflammatory adipokines and free fatty acids, which can produce atherosclerosis of the coronary microvasculature and ischemia of the myofibril , 14. In strain. In this regard, higher values of EAT in subjects with t2DM had previously been reported [To our knowledge, the present study is the first to evaluate the role of t2DM on the relation between EAT and longitudinal reported , 15, 16;Strain values in all spatial directions and controlled t2DM affected only longitudinal Strain [Similar observation was performed by Zhang et al. during comparison between patients with t2DM, where non-controlled t2DM preceded higher l Strain .Furthermore, deformity of myofibrill may be affected by several cardiometabolic risk factors, like weight, BMI and insulin resistance. Consistent with this statement, Liu et al., observed a reduction in left ventricle tension in hypercholesterolemic rabbits, in comparison with normal controls . LikewisStrain or HbA1c; but with t2DM. This is consistent with findings described by Xiaoling et al. where patients with t2DM had less myocardial deformity, being more evident in patients with HbA1c > 7% [Interestingly, multivariate analysis showed that EAT did not associate with A1c > 7% .Strain was considered as the cut-off value for our analyses, which was lower than the Strain values reported in other studies: − 15.80% to − 23.40% [Strain determination.In the present study, the median value of the − 23.40% . Such diStrain determination.Limitations of our study include the low number of patients, the lack of T2DM subgroup in male analysis and technological restrictions related with the available echocardiographic equipment, which lacks specific software for In conclusion, EAT significantly related to myofibril deformation, with additional influence of t2DM.


Symptomatic mitral valve (MV) regurgitation is a common cause of morbidity amongst cardiovascular disease patients, with a 1.3-fold increase in prevalence per decade of life past 60 years of age , 3. SurgConversely, the Cardiovascular Outcomes Assessment of the MitraClip Percutaneous Therapy for Heart Failure Patients with Functional Mitral Regurgitation (COAPT) trial showed that in carefully selected patients with moderate-to-severe SMR, MV transcatheter edge-to-edge repair (TEER) improves heart failure symptoms and survival, and decreases heart failure hospitalizations at 2-year follow-up when compared with medical therapy alone . In a reThe ventricular myocardium is composed of a surrounding basal loop with circumferential fibers in a transverse orientation, and an inner apical loop helix with oblique fibers oriented at approximately 60° angles . The intp = 0.02) at a median follow-up of 56 months. When compared with patients who had a GLS <−7%, those with GLS >−7% had more extensive LV remodeling with larger end-diastolic  and end-systolic volume  indices, and higher filling pressures  trial, which showed no benefit of TEER in 307 patients randomized to percutaneous MR correction vs. medical therapy  symptomatic heart failure and moderate to severe SMR despite optimal guideline-directed medical therapy; (2) LVEF 20–50%; (3) LV end-systolic diameter <70 mm; (4) pulmonary artery systolic pressure <70 mmHg; and, (5) no evidence of significant right ventricular dysfunction. The 614 patients enrolled in COAPT had a mean LV end-diastolic volume index, LVEF, and GLS of 101 ml/m therapy . The maip < 0.05) (p < 0.001)  . In pati< 0.001) . A fair < 0.001) , is thatWhile the aforementioned hypothesis regarding LV remodeling and GLS in patients with SMR undergoing TEER requires prospective and external validation, it provides important insights into appropriate patient selection and factors contributing to the success and application of this relatively new therapy. Pure SMR is primarily a ventricular disease with concomitant histopathologic mitral leaflet remodeling, and as such, it is advisable that our inclusion criteria when selecting candidates for TEER be modeled strictly after those applied in the COAPT trial. Significant GLS impairment may be considered as a risk factor for a lack of LV reverse remodeling and suboptimal outcomes after TEER, with specific cut-off values for clinical use to be determined. Additionally, maximally tolerated guideline-directed medical therapy, as well as cardiac resynchronization therapy when appropriate, are of paramount importance. Taking these factors into account may allow for appropriate risk stratification, expectant management, and a pathway to improved patient outcomes.The author confirms being the sole contributor of this work and has approved it for publication.


Recent advances in cardiac MRI (CMR) capabilities have truly transformed its potential for deep phenotyping of the diseased heart. Long known for its unparalleled soft tissue contrast and excellent depiction of three-dimensional (3D) structure, CMR now boasts a range of unique capabilities for probing disease at the tissue and molecular level. We can look beyond coronary vessel blockages and detect vessel disease not visible on a structural level. We can assess if early fibrotic tissue is being laid down in between viable cardiac muscle cells. We can measure deformation of the heart wall to determine early presentation of stiffening. We can even assess how cardiomyocytes are utilizing energy, where abnormalities are often precursors to overt structural and functional deficits. Finally, with artificial intelligence gaining traction due to the high computing power available today, deep learning has proven itself a viable contender with traditional acceleration techniques for real-time CMR. In this review, we will survey five key emerging MRI techniques that have the potential to transform the CMR clinic and permit early detection and intervention. The emerging areas are: (1) imaging microvascular dysfunction, (2) imaging fibrosis, (3) imaging strain, (4) imaging early metabolic changes, and (5) deep learning for acceleration. Through a concerted effort to develop and translate these areas into the CMR clinic, we are committing ourselves to actualizing early diagnostics for the most intractable heart disease phenotypes. Cardiovascular disease comes in many different forms, including ischemic disease associated with atherosclerosis, cardiomyopathies associated with aging and hypertension, arrhythmia, infection, valvular disease, and heart failure. Electrocardiography is a frontline diagnostic tool to detect abnormalities in heart rhythm, but deeper assessment always involves echocardiography to obtain real-time ultrasound images of the beating heart. From an echocardiogram, one can assess the structure and size of the heart wall and chambers, heart wall motion, valve morphology and function, and parameters related to cardiac output and ejection fraction. If echocardiography is performed in conjunction with a stress test , one can also probe how well the heart functions in response to stress.While echocardiography permits a deep assessment of heart morphology and function, the indices measurable are symptoms of disease that has progressed for some time. Much more challenging to uncover are early indicators of disease development, or information at the tissue or molecular level. To probe at these deeper levels, one can resort to complementary three-dimensional (3D) cross-sectional imaging modalities that have functional imaging capabilities, such as nuclear medicine and magnetic resonance imaging (MRI). Nuclear medicine provides sensitive assessment of metabolism; MRI can also assess energetics while offering exquisite delineation of 3D structure and unparalleled tissue contrast. Similar to echocardiography, cardiac MRI (CMR) can delineate heart structure and measure chamber volume and wall thickness, and it can accomplish this with greater robustness owing to its 3D nature and high spatial resolution. CMR also provides information on wall motion, valve morphology and function, and cardiac output and ejection fraction. A distinctive power of CMR, however, is its unique ability to inform on tissue composition and microstructure. For example, the position and extent of infarcts are readily visualized on delayed contrast-enhanced CMR as a bright region devoid of cardiomyocytes and filled with a gadolinium-based MR contrast agent. However, today’s CMR goes far beyond infarct identification and delineation. Over the past decade, CMR has seen considerable technological development, strengthening its role as an indispensable complement to echocardiography for difficult phenotypes such as non-ischemic disease and heart failure with preserved ejection fraction (HFpEF), phenotypes that present abnormalities much more subtle than changes in gross structure or motion.In this review, we will examine the latest CMR developments that enable us to probe at the microvascular, cellular, and molecular level. We begin with the most readily translatable and potentially most impactful technology, namely, new methods to interrogate microvascular dysfunction. While these are relatively nascent, they hold promise for a better understanding of certain cardiomyopathies and potentially early detection for at least half of all heart failure patients. Techniques to detect fibrosis are next discussed, given their value for detecting diffuse disease in non-ischemic settings, followed by strain CMR imaging of myocardial stiffness, another potential tool for early detection. We conclude with two technologies that are uniquely valuable but perhaps the furthest from clinical adoption: non-proton CMR to assess early metabolic abnormalities in heart failure, and artificial intelligence for accelerating CMR acquisitions  is a non-obstructive vessel disease . MicroveCurrent protocols for stress perfusion CMR involve administering a vasodilator, such as adenosine or regadenoson, to induce vasodilation of the coronary microvasculature and a double injection of a gadolinium-based contrast agent to measure perfusion both at stress and at rest . While g2) is well-known for its vasoactive effects on the microcirculation. When inspired CO2 is elevated, a state known as hypercapnia, different vascular responses are seen, depending on the CO2 level and the tissue bed. At low levels of elevated CO2 (<5% inspired CO2) blood vessels in most tissue beds dilate, while higher levels evoke vasoconstriction in many organs  can also be targeted to bring about hypoxic or hyperoxic effects on blood vessels, further bolstering one’s ability to assess both vasodilatory and vasoconstrictive response.Inspiring elevated levels of carbon dioxide , is introduced intravenously and allowed to reach a quasi-steady state concentration after 10 min. This steady state lasts approximately 40 min in rats, even longer in humans due to a slower elimination rate. Over this time interval, one can administer a vasoactive substance, such as adenosine or hypercapnia, or even multiple stimuli in succession, and measure how much the microvascular blood volume changes. It is noteworthy that blood-pool imaging of vasoreactivity was initially conceived to permit a more specific and direct measure of vasomodulation , an iron-based compound currently still approved in the U.S. as an iron supplement and used off-label as a blood-pool MRI contrast agent. Recently, the concept of blood-pool imaging of vasoreactivity was proposed to circumvent the limitations of conventional stress tests . A T1-redulation . By lookdulation , 13. InsBlood-pool MRI contrast agents are valuable beyond their intended utility for angiography. They enable lower dosing and a much longer window for image acquisition. They also provide an alternative approach to assessing microvascular reactivity that permits higher specificity, quantitative accuracy, and spatial resolution than is possible with conventional double first-pass perfusion assessment of MVD. However, at the time of this writing, there is no clinically approved MRI blood-pool contrast agent on the market. Perhaps when more clinical indications in which blood-pool agents provide a clear advantage come to light, or when new gadolinium-free versions become accessible, there will be an incentive for pharmaceuticals to bring MRI blood-pool agents back into the clinic.Fibrosis is a progressive disease that affects virtually all life-sustaining organs and is seen in diseases of the heart, lungs, liver, and kidneys. In the setting of heart disease, fibrosis presents differently depending on the disease phenotype. For instance, in myocardial infarction, dead cells in the ischemic region are eventually replaced with scar tissue. This type of fibrosis, known as replacement fibrosis, is straightforward to identify with late gadolinium enhancement (LGE), because the cell-free infarct has much more space than healthy myocardium into which the contrast agent can diffuse. However, this technique works only in a mature infarct and is non-specific to collagen deposition. A case in point is an acute myocardial infarct where substantial enhancement is seen, but only edema is present as scar has not yet developed. Only at much later times when edematous fluids have resorbed and only scar tissue remains is LGE more clearly correlated with fibrotic tissue. A different type of scarring, one much more difficult to detect, is interstitial fibrosis. Here, collagen deposits are found between cardiomyocytes and in the perivascular space. Interstitial fibrosis can be of the reactive kind  or the much rarer infiltrative kind (associated with amyloidosis and Anderson-Fabry disease) . The curIn 2007, the first type I collagen-targeted MRI contrast agent, EP-3533, was introduced by Caravan et al. . The agein vivo is highly unlikely, even in long retention applications such as fibrosis imaging. Development of manganese porphyrin-based fibrosis contrast agents is currently geared toward both young scar (fibrogenesis) and old scar (fibrosis).Non-gadolinium agents targeted to collagen have been reported, but these are relatively few compared to EP-3533. For example, c(RGDyC)-USPIO is an iron oxide nanoparticle that targets liver fibrogenesis by imaging activated hepatic stellate cells that specifically engulf the contrast agent . AnotherStrain imaging was introduced over a decade ago as a non-invasive echocardiographic method to assess myocardial stiffness and differentiate between passive and active movement of myocardial segments , 25. TheCardiac MRI has also developed its own version of strain imaging. Several strain CMR techniques currently exist, but none is fully developed and standardization is a distant goal . Strain Feature tracking, unlike the methods described above, is a strictly post-processing technique and is applicable to any modality, not only MRI. No additional imaging is necessary, and standard CINE images can be retrospectively analyzed using feature tracking of cavity/myocardial borders to calculate strain. It is already available clinically and has demonstrated both reproducibility  and accuDespite the lack of standardization for strain CMR, myocardial strain imaging has been investigated in different cardiac pathologies. Perhaps the most common application is ischemic heart disease. After a myocardial infarction, myocardial strain components are most impaired in the infarct  and straA more difficult phenotype to diagnose is non-ischemic heart disease, where late gadolinium enhancement typically does not reveal evident tissue abnormalities. In this setting, strain CMR can be invaluable. Strain measurements have shown impairment in patients relative to healthy control subjects. For example, in hypertrophic cardiomyopathy, mid-wall fibrosis underlies abnormal systolic strain measurements with or without late gadolinium enhancement . MyocardStrain echocardiography is a validated technique for assessing subtle myocardial changes. However, it is used strictly for larger muscular structures. It is unsuitable for thin structures of the right ventricle and the two atria. Strain CMR, on the other hand, is the gold-standard for assessment of the right ventricle and is the first modality to demonstrate right ventricular strain analysis . DiseaseHeart disease frequently involves early changes in cell metabolism before overt structural and functional alterations set in. The concept of using non-proton MR spectroscopy to assess the heart is not new. In fact, a study in 1979 already described the utility of 31-phosphorus (31P) MR spectroscopy in assessing adenosine triphosphate (ATP) levels in living hearts and those that had undergone potassium chloride-induced cardiac arrest . Fast-fo13P MR spectroscopy assesses muscle energy metabolism by monitoring the ratio of phosphocreatine (PCr) to ATP. Cardiac muscle is like any other muscle in our body in that it requires a substantial supply of ATP to power contractions. When an ATP molecule is hydrolyzed to release energy, it produces adenosine diphosphate (ADP) and an inorganic phosphate as by-products. To maintain a constant energy reservoir, ATP must be regenerated; the molecule PCr is required to transfer high-energy phosphate to convert ADP into ATP. During a period of low energy requirement, excess ATP is used to convert creatine back to PCr. Therefore, PCr acts as a high-energy reserve to maintain normal ATP levels regardless of fluxes in energy demands. Since ATP levels are relatively stable, assessment of the muscle cell’s energy store is achieved by monitoring the ratio of PCr and the three phosphate groups on ATP. Any decrease in the PCr/ATP ratio is indicative of a depleting PCr store.Heart failure is a prime example of a disease phenotype where 31P MR spectroscopy is informative. The healthy heart forms ATP by oxidizing both fats and glucose, and the proportion from each substrate is adapted to the environment—fatty acids are the major substrate in the healthy and trained heart, whereas glucose utilization increases in heart failure. Regardless, utilization of both substrates is reduced in the failing heart, resulting in a decline in the PCr/ATP ratio . This ob3 and 15 min scan time when imaging human calf muscle on a 3 Tesla scanner, with the spatial and temporal resolution improving to 1.5 × 1.5 × 1.6 mm3 and 30 frames/s, respectively, at 9.4 Tesla  to a neural network and train against ground-truth unaliased images from fully sampled datasets . The neuMost acceleration applications to date exploit the convolutional neural network architecture . This chAdaptions of the reconstruction techniques described above can involve, as examples, training on both the image domain and k-space or getting the neural network to uncover the optimal undersampling pattern in k-space. The first method, also known as dual-domain reconstruction, makes the reasonable and logical assumption that providing both object and frequency domain data for training should improve reconstruction quality , 68. WhiDespite the successes of different neural network architectures in achieving high quality reconstruction from undersampled acquisition data, there are quite a few technological shortcomings that should be recognized. Most obvious is the availability of large datasets for training. There are a handful of MRI databases currently available, such as the UK Biobank, which contains the largest collection to date of acquisitions in various body regions. For instance, it has an abundant repository of different types of brain and cardiac images. Assuming that access to a large database is not an issue, the next hurdle, one much more difficult to overcome, is the high dependency of training on the acquisition itself. If the tissue type changes, or any of the acquisition parameters  changes, the network must be re-trained for the new scenario. It is easy to appreciate that this limitation can severely curtail the clinical utility of deep learning-assisted acceleration.Recently, researchers have proposed so-called physics-based deep learning methods to partially address some of the limitations described above. These are dubbed physics-based, because the neural network is trained to solve a supervised learning task and, at the same time, adhere to any physical laws governing the system of interest. As an example, rather than treating each image as a new reconstruction problem, we can exploit known information in regard to the anatomy of interest or aliasing artifacts specific to different k-space trajectories. In the first physics-based reconstructions, the undersampling pattern and coil sensitivities, both with known effects on the reconstructed image, were taken into account to solve an inverse problem , 70. HowCardiac MRI is an excellent adjunct to echocardiography in providing information on heart anatomy and mechanical function. It also provides unique tissue characterization, such as delineating the extent of an infarct. Yet, in this review article, we have seen that emerging CMR techniques can explore cardiac tissue and function on much deeper levels, from structurally intact but functionally abnormal microvessels to pathological shifts in metabolic substrates. The question remains as to whether deeper phenotyping is necessary and beneficial. What types of heart disease can deeper phenotyping benefit, and how can it impact patient care? Heart failure with preserved ejection, which affects over half of all heart failure patients, is a difficult-to-treat disease that can benefit immensely. It is a heterogeneous disease with multiple subtypes spanning multiple comorbidities . Treatment will likely require a tailored approach, namely, fine-tuned characterization at the diagnostic level. For example, imaging microvascular dysfunction may provide early detection before the HFpEF heart takes on the classical signs of stiffness and hypertrophy. Alternatively, metabolic imaging with 31P MR spectroscopy may distinguish between different subtypes brought on by obesity vs. hypertension. A second heart disease phenotype that can benefit from deeper phenotyping is that involving diffuse fibrosis. For example, patients with hypertension are predisposed to developing myocardial fibrosis; yet, this tissue alteration is very difficult to detect on conventional cardiac imaging until the late stages of disease. If a fibrosis-targeted MRI scan can diagnose fibrogenesis at its early stages, then there is hope of intervening to slow or reverse progression. It is important to emphasize that while we can already envision what disease phenotypes will benefit from a deeper, more specific characterization of the heart, the full value of the technologies described in this review can only be appreciated when they are readily available, easily accessible to all heart patients.All the emerging techniques discussed in this article are well-established in both physics-based and preclinical studies. To facilitate their full clinical translation for early diagnosis of difficult heart disease phenotypes, we need to address outstanding impediments. In the following, we will examine each of the five emerging CMR techniques and identify both remaining hurdles and realistic solutions.Assessing coronary microvascular dysfunction is a decades-old practice, but it is also one in which the MRI version of stress testing is fraught with confounders that render accurate quantitation quite difficult. Obtaining two accurate absolute perfusion measurements  in quick succession by injecting two full doses of a gadolinium chelate is all but impossible. Residual contrast from the first dose, unless given sufficient time to clear from the body, will interfere with the pharmacokinetics of the second injection dose. The only practical solution is to employ a single injection that maintains a relatively stable blood profile over the tens of minutes of a stress test—only a blood-pool contrast agent can fulfill this role. Unfortunately, since 2017, there has not been a single MR blood-pool contrast agent approved clinically for imaging. The only one currently available on the market, ferumoxytol, is used off-label in the U.S. and is not approved in Europe or Canada. Low market demand was cited as the reason for stopping the production and distribution of Ablavar in 2017, despite the radiological benefits it provided to pediatric cardiac patients. Another possible reason for its retraction, although never confirmed, was potential concern for gadolinium demetalation from a long-circulating blood-pool chelate with an elimination half-life of 16 h. A possible solution is to develop blood-pool MR contrast agents that have a non-gadolinium MR-active metal. Manganese is a viable alternative, with five unpaired electrons as opposed to seven for gadolinium. By binding manganese with a porphyrin, a very stable structure can be exploited for blood-pool MR imaging , 73. WhiCurrent assessment of fibrosis is non-specific, because a measured increase in the distribution volume for gadolinium accumulation may or may not be related to collagen deposition. For nearly 20 years, researchers have described strategies to target gadolinium-based contrast agents to collagen, the protein that is greatly elevated in fibrotic tissue. Despite ample preclinical evidence of sensitivity and specificity, none of these molecular contrast agents have gained access to the clinical space. A plausible explanation is that with gadolinium-based agents, the chance for demetalation is simply too great for any application requiring prolonged retention in the body, and the risks to patients unacceptably high. Similar to blood-pool MR contrast agents, these targeted molecular agents would also benefit from utilizing a non-gadolinium MR-active metal. A fertile direction for further research is non-gadolinium MR contrast agent design and development, and this needs to happen in both the research lab and in industry.The landscape for strain CMR is competitive, as strain echocardiography has demonstrated immense power in recent years for early left ventricular disease diagnosis and even prognosis. An equivalent capability from any other modality, including strain CMR, would face stiff competition from a gold-standard that is portable, inexpensive, and widely available. However, because delineation of the atrial and right ventricular walls is challenging on echocardiography, these are the applications in which strain CMR can make a real impact. The greatest existing hurdle to widespread adoption of strain CMR is repeatability. Although high reproducibility of global strain measurements has been demonstrated for different types of strain CMR approaches –76, reprMetabolic imaging using 31P MR spectroscopy holds promise for very early detection of abnormal utilization of energy substrates in a variety of cardiac diseases, including heart failure, dilated and hypertrophic cardiomyopathies, hypertensive heart disease, coronary artery disease, and type I and II diabetes. The main technical limitation of 31P MR spectroscopy is its intrinsically low SNR. Moving to a higher field strength, such as 7 Tesla scanners, is one solution—in one report, the SNR gain was used to shorten to acquisition from 31 min on a 3 Tesla system to 6 min at 7 Tesla . These hReal-time or near real-time CMR using deep learning is a rapidly evolving field. If successful, we can image cardiac dynamics as it happens in real-time, akin to echocardiography. However, its main Achilles’ heel is 2-fold: need for large training datasets and the possibility of incorrectly reconstructing image features on which the network has not been trained. The first hurdle is surmountable for some types of MRI applications, including cardiac, but access to large databases will inevitably become a roadblock, unless existing algorithms can generalize beyond fixed acquisition parameter settings. The second hurdle is a much more difficult ethical question to address. How can one ensure that all disease phenotypes that can ever be encountered are available for training? Furthermore, since artifacts from reconstruction can bear remarkable resemblance to real pathology, how can radiologists be guided in distinguishing real from fake image features? The simple answer is that deep learning can never enter clinical decision making if it can lead to a false positive or, worse yet, false negative diagnosis. Notwithstanding this drawback, a better view of how deep learning can be clinically useful is to apply it to situations to which the patient would otherwise not have access. For example, if one could generate a deep-learning perfusion scan from a very low dose injection, then a diabetic heart failure patient who cannot take the full dose due to risks of gadolinium would not be deprived of the benefit of a contrast-enhanced exam. With creativity around how we use deep learning, it can become a very intelligent agent to the radiologist.H-LC drafted and edited the manuscript.


Vgll4 depletion allele and a cardiomyocyte-specific Vgll4 depletion allele. The whole-body deletion of Vgll4 caused defective embryo development and perinatal lethality. The analysis of the embryos at day 16.5 revealed that Vgll4 knockout embryos had reduced body size, malformed tricuspid valves, and normal myocardium. Few whole-body Vgll4 knockout pups could survive up to 10 days, and none of them showed body weight gain. In contrast to the whole-body Vgll4 knockout mutants, cardiomyocyte-specific Vgll4 knockout mice had no noticeable heart growth defects and had normal heart function. In summary, our data suggest that VGLL4 is required for embryo development but dispensable for myocardial growth.Congenital heart disease is one of the leading causes of pediatric morbidity and mortality, thus highlighting the importance of deciphering the molecular mechanisms that control heart development. As the terminal transcriptional effectors of the Hippo–YAP pathway, YAP and TEAD1 form a transcriptional complex that regulates the target gene expression and depletes either of these two genes in cardiomyocytes, thus resulting in cardiac hypoplasia. Vestigial-like 4 (VGLL4) is a transcriptional co-factor that interacts with TEAD and suppresses the YAP/TEAD complex by competing against YAP for TEAD binding. To understand the VGLL4 function in the heart, we generated two VGLL4 loss-of-function mouse lines: a germline  Congenital heart disease occurs in around 1% of live births . UnderstThe core canonical Hippo pathway in mammals comprises a serine/threonine kinase cascade consisting of sterile 20-like kinases MST1/2 (homologs of the Drosophila Hippo kinases), scaffold protein Salvador homolog 1 (SAV1), and LATS1/2 serine/threonine kinases. SAV1 directly interacts with and activates MST1/2, and the activated MST1/2 in turn phosphorylates and activates LATS1/2 . YAP andOne of the effects of the LATS1/2 phosphorylation of YAP/TAZ is to retain these transcriptional effectors in the cytoplasm . Once thDifferent from LATS1/2, Vestigial-like 4 (VGLL4) is another category of YAP suppressors, which does not interact with YAP and functions by competing against YAP for TEAD binding ,17. In mVgll4 loss-of-function mouse models to define VGLL4 function in the heart: one germline Vgll4 mutant allele and one cardiomyocyte-specific Vgll4 depletion mouse line. In both mouse lines, VGLL4 TDUs were targeted to abolish the VGLL4 function. Although the whole-body VGLL4 depletion resulted in small embryo size and perinatal lethality, the cardiomyocyte-specific depletion of VGLL4 did not cause heart growth defects. Our data, therefore, suggest that VGLL4 is required for embryo development but dispensable for myocardial development.We previously studied the relationship between YAP, TEAD1, and VGLL4 in the mouse heart, showing that the major interaction partner of TEAD1 switches from YAP in the neonatal heart to VGLL4 in the adult heart. Overexpressing a K225R-mutated VGLL4 in the neonatal heart disrupted the TEAD/YAP interaction and caused cardiac hypoplasia and heart failure . NeverthAll animal procedures were approved by the Institutional Animal Care and Use Committee of Boston Children’s Hospital and the Masonic Medical Research Institute.Gapdh. The primer sequences are provided in The total RNA was isolated using the Trizol reagent . For quantitative reverse transcription PCR (qRT-PCR), RNA was reverse-transcribed , and specific transcripts were measured using SYBR Green chemistry and normalized to t-test was used to test for statistical significance involving two groups.Values are expressed as mean ± SD. Student’s Vgll4 gene has seven annotated transcript variants (NCBI) that encode five VGLL4 protein isoforms (Vgll4 variant products have TDU domains (Vgll4 transcript variants into two groups: (I) Vgll4 variants containing exon 2  and (II) Vgll4 variants without exon 2 (X3 and X4).In mice, the isoforms . Among tisoforms B. Since  domains B. Based Vgll4 variants (variant 1) has been validated in the mouse heart [Vgll4 variants. Using Vgll4 variant 1 as a positive control, we examined the expression of Vgll4 X3 and X4 variants in 1-day-old (P1) and 28-day-old (P28) mouse hearts. In both P1 and P28 hearts, the expression of Vgll4 X4 was minimum, and the expression of Vgll4 X3 was significantly higher than that of Vgll4 variant 1  intercrosses yielded Vgll4d46/d46 (Vko) embryos at a Mendelian ratio at E16.5 and E18.5, but at postnatal day 1 (P0), only 8 of the 83 genotyped pups had the Vko genotype  and the other pair to the unaffected five-prime region. The TDU primer sets failed to detect signals in Vko transcripts; however, the primers amplifying Vgll4 five-prime regions did not detect a difference between Vhet and Vko, suggesting that the indel mutations disrupt the TDU-coding region without affecting the stability of Vgll4 mRNA. Together, these data show that VGLL4 is required for embryo development and normal postnatal growth.genotype B. The mapast P10 C. At E16controls D,E. Onlycontrols F,G. VgllVhet and Vko at P0  control pups. At P0, all these four parameters showed no significant differences between Vhet and WT pups , Cdk1, and Igf1r, was not altered in the Vko hearts  [Ccn1, also known as Cyr61) [Birc2, an apoptosis inhibitor gene) [Ccn2 was upregulated in the Vko heart, Ccn1 and Birc2 showed no difference between Vhet and Vko  , cellulas Cyr61) , and bacor gene) . Althoug and Vko I, suggesVko mutants is not due to heart dysfunction.These data show that VGLL4 is dispensable for myocardial growth and suggest that the perinatal lethality of Vko hearts. Vko hearts had preserved morphology, with the exception of the tricuspid valve, which appeared dysmorphic  proliferation rate by labeling the fetal heart with EdU for two hours. Compared with ificance C,D.Vgll4 d46 mutants die perinatally, we were unable to examine whether VGLL4 regulates postnatal heart growth. To systematically study VGLL4 function in the postnatal heart, we made a Vgll4 flox allele that targets the coding sequence of its last exon. The last exon (exon 5) of Vgll4 encodes TDU domains and has a long untranslated region (UTR). To perform recombinant genome targeting, we chose one 1167 bp intron fragment and one 1074 bp three-prime UTR as the left and right recombination arm, respectively  PAM sequence (Due to the fact that the ectively . In the tion arm . To incrsequence .Vgll4 flox founder lines. The germ-line transmission of the Vgll4 flox_neomycin (Vgll4fl_Neo) allele was successful. Since the presence of a selectable marker cassette may disrupt the expression of neighboring genes [Vgll4fl_Neo mouse line, in which the neomycin selection cassette was flanked with FLP recombinase-recognized FRT sequences. ROSA26-Flp is a transgenic mouse line that has the FLP recombinase gene knocked into the ROSA26 locus, thus constitutively expressing FLP in all tissues [Vgll4fl_Neo with ROSA26-Flp to remove the neomycin resistance cassette  cell targeting, neomycin-resistant clones were screened with PCR , and posng genes , we deci tissues . We thencassette A. We seqcassette .Vgll4 flox mice (fl/flVgll4) normally bred and had normal systolic heart function   A. The ad4cKO CMs B. Conseq4cKO CMs C.cKOVgll4 heart  caused pre-mature death and aortic valve defects [Vgll4 X3 variant that does not contain exon 2 is highly expressed in the mouse heart, suggesting that the deletion of Vgll4 exon 2 may not completely abolish the VGLL4 function. In this work, we generated a Vgll4 mutant allele that disrupted TDU domains (Vgll4d46). Compared with the published Vgll4dE2 mutants [Vgll4d46 is more severe, as reflected by the fact that a fraction of the Vgll4dE2 mutants could survive up to 8 weeks, but none of the Vgll4d46 mutants could survive over 10 days. Additionally, the Vgll4d46 pups that survived for 10 days could not gain body weight. These observations suggest that the deletion of exon 2 cannot completely disrupt the VGLL4 function.In mice, the  defects . Our cur mutants , the pheVgll4d46 mutants. The tricuspid valve growth defect was not observed in the Vgll4dE2 mutants, which displayed aortic valve and pulmonary valve malformation at both adult and neonatal stages [Vgll4dE2 mutants [d46Vgll4 tricuspid VICs had lower proliferation rates  mice. In addition to the fact that the d46Vgll4 embryos had normal myocardial thicknesses and CM proliferation rates, the data from the adult cKOVgll4 mice further suggest that VGLL4 is dispensable for myocardial growth. Together, the heart morphology data collected from the Vgll4dE2, d46Vgll4, and cKOVgll4 mutants highlight the importance of VGLL4 in heart valve development and suggest that VGLL4 is not required for myocardial development.The perinatal lethality of the As one of the essential terminal effectors of the Hippo–YAP integrative pathway, YAP senses developmental and stress signals and relays these signals to the nuclei to change the CMs’ gene expression, thereby controlling CM proliferation ,30, survCtgf (Ccn2); however, the cKOVgll4 CMs had no noticeable phenotypes. These data suggest that the endogenous TEAD1–VGLL4 complex does not maintain adult CM homeostasis under baseline conditions. One possibility is that the Vestigial-like (VGLL) protein family members have redundant functions, and the other VGLL proteins may compensate for the VGLL4 function in the KOVgll4 CMs. The other possibility is that the loss of the cardiomyocyte VGLL4 is not sufficient to increase the amount of the YAP–TEAD complex to a phenotype-causing extent, and this is because most YAP proteins are localized in the cytoplasm of healthy adult CMs. Notably, all our studies were carried out in unstressed mice, and it remains unknown whether VGLL4 has significant pathophysiological roles in disease-stressed hearts, in which the LATS1/2 suppression of YAP is attenuated, and the nuclei YAP is enriched [We previously found that the primary interaction partner of TEAD1 is YAP in the neonatal heart and that overexpressing a K225R-mutated VGLL4 in the neonatal heart disrupted the TEAD1–YAP interaction, suppressed CM proliferation, and caused heart failure . The curenriched ,35. As Yenriched ,36,37,38d46Vgll4 perinatal death was not clear. Since the deletion of Vgll4 exon 2 in skeletal muscle progenitor cells causes skeletal muscle growth defects [d46Vgll4 mutant pups. The other limit is that we were not able to provide cardiac functional results to explain the consequences of tricuspid valve malformation caused by VGLL4 depletion. Further tissue-specific VGLL4 knockout studies are needed to dissect how VGLL4 regulates tricuspid valve development and function.One of the limits of the current study is that the primary pathophysiological reason leading to the  defects , it is pVgll4 transcript variants, and our results indicate that disrupting the VGLL4 TDU-domain coding sequence is an efficient way to completely abolish the VGLL4 function. Lastly, we found that the CM-specific depletion of VGLL4 facilitated the formation of the YAP/TEAD1 complex and increased the YAP target gene expression without changing the YAP protein level and subcellular distribution, further corroborating the current knowledge that VGLL4 suppresses the YAP activity by reducing TEAD availability. In summary, to address whether VGLL4 regulates heart development, we generated two VGLL4 loss-of-function mouse models. Our results suggest that VGLL4 regulates tricuspid development and does not contribute to myocardial growth. In addition, we confirmed the presence of multiple In conclusion, our data suggest that endogenous VGLL4 minimally regulates myocardial growth at the fetal and postnatal heart development stages and that Hippo kinases are the primary YAP restrainers during myocardial development G. Future


We investigated the methodology and assessed its relationship with conventional parameters.Volumetric evaluation of 99mTc-PYP SPECT/CT scans of 25 patients who underwent endomyocardial biopsy and/or gene testing. Fourteen (56%) patients were diagnosed with ATTR-CA. SPECT/CT images were acquired at 3 hours after injection. Total volumes of the myocardial regions where uptakes were > 1.2 and 1.4 × aortic blood pool SUVmax were evaluated and defined as cardiac pyrophosphate volume (CPV1.2 and CPV1.4). The heart-to-contralateral lung (H/CL) ratio and myocardial SUVmax were also calculated. CPV1.2 achieved the highest sensitivity and specificity in diagnosing ATTR-CA. In patients diagnosed with ATTR-CA (n = 14), CPV1.2 negatively correlated with left ventricular ejection fraction and positively correlated with left ventricular posterior wall thickness and QRS duration. The correlation was stronger in CPV1.2 than in the H/CL ratio and SUVmax.We retrospectively evaluated 99mTc-PYP SPECT/CT may be superior to the H/CL ratio and SUVmax in assessing the disease burden of ATTR-CA. Larger studies are warranted to clarify whether volumetric measurement can assess prognosis and disease progression.Volumetric evaluation of The online version contains supplementary material available at 10.1007/s12350-021-02857-7. A large multicenter study demonstrated that in patients who had undergone serum and urine laboratory tests to exclude light-chain cardiac amyloidosis, bone scintigraphy enables the diagnosis of ATTR-CA with high specificity without invasive biopsy.3Transthyretin cardiac amyloidosis (ATTR-CA) is an increasingly recognized, progressive, and fatal cardiomyopathy.4 A multicenter study reported that planar heart-to-contralateral lung (H/CL) ratio of 99mTc-PYP uptakes had prognostic information.5 However, the H/CL ratio is based on two-dimensional imaging and has some inherent limitations, such as influences of blood pool and rib uptakes.6 Volumetric evaluation of bone scintigraphy with single-photon emission computed tomography/computed tomography (SPECT/CT) is an objective and quantitative method to overcome weaknesses of conventional methods.8 The volume of the myocardial region with abnormal uptake is expected to assess disease burden and has a close association with cardiac functional parameters. However, the methodology and characteristics of volumetric parameters have not been fully investigated in patients with ATTR-CA. The aim of this study was to investigate the methodology of volumetric evaluation of 99mTc-PYP SPECT/CT and to assess its correlation with cardiac functional parameters and other quantitative uptake parameters.Because of the recent introduction of life-prolonging drugs for ATTR-CA, there is a major unmet need for the early detection and precise quantitative assessment of disease burden, progression, and treatment response.99mTc-PYP SPECT/CT for suspected ATTR-CA at our hospital between October 2018 and June 2020. Among them, only patients who underwent endomyocardial biopsy (EMB) and/or TTR gene testing were included. Diagnosis criteria of ATTR-CA were based on one or more of the following: (1) EMB positive for ATTR or (2) documented TTR genetic mutation and evidence of cardiomyopathy without evidence of plasma cell dyscrasia (serum and urine immunofixation and a serum free light chain assay). For patients with multiple 99mTc-PYP studies, only the first study was included. The study was approved by the institutional ethics committee of our university (2019-218) with waiver of the requirement for informed consent as a retrospective patient selection.We retrospectively evaluated patients who underwent 99mTc-PYP intravenously, and thorax planar images were obtained at 3 hours after injection over a 2-minute duration on a hybrid SPECT/CT system  using a low-energy high-resolution collimator. SPECT/CT images of the thorax were acquired at 3 hours after injection with the following parameters: step-and-shoot acquisition with body contour, 120 steps at 15 s/step, and zoom 1.0.Patients received 740 MBq (20 mCi) of Images were reconstructed to a 128 × 128 matrix with a dedicated iterative algorithm (xSPECT QUANT) with 72 iterations and 1 subset, a 10-mm Gaussian filter. A low-dose non-contrast CT scan for attenuation correction was performed with the following parameters: 130 kV, 50 mAs with CARE Dose, pitch 1.5, rotation time 0.6 seconds, and collimation 16 × 1.2. The additional CT caused an estimated mean supplemental radiation dose of 1.0 mSv. Both the CT-based attenuation correction and a dual-energy scatter correction were automatically performed.99mTc-PYP activity in an aortic blood pool (ABP) using maximum standardized uptake value (SUVmax) and defined it as ABPmax. We placed a spherical volume of interest (VOI) in the center of the ascending aorta at the height of the pulmonary artery bifurcation using fused SPECT/CT images.9 The diameter of the VOI was half the size of the aortic diameter. Next, we placed a polygonal VOI manually to encompass the whole left and right ventricles, excluding adjacent rib and sternal uptakes. The total volumes of the myocardial regions where 99mTc-PYP uptakes were > 1.2 and 1.4 × ABPmax were automatically evaluated and defined as cardiac pyrophosphate volume .8 We used xSPECT Quant (Siemens) for calculating ABPmax and CPV. Because of the limitation of image resolution and the software, CPV less than 0.2 cm3 was rounded to 0.0 cm3. In patients with ATTR-CA, we also evaluated SUVmax within the whole left and right ventricular myocardium and the SUVmax was normalized to ABPmax (nSUVmax: SUVmax divided by ABPmax).10An example of SPECT/CT volumetric evaluation is shown in Figure 6 SPECT/CT images were also visually interpreted by experienced readers (SW and HW) blinded to clinical information and the results of the other imaging modality. The visual interpretation was graded qualitatively as positive  or negative within the whole left and right ventricular myocardium.11As a conventional parameter, the H/CL ratio was also calculated as the counts in a circular region of interest (ROI) drawn over the heart of a planar image, which was divided by background counts in an identical size ROI over the contralateral chest.n = 14) underwent echocardiography 4.6 ± 5.0 days before 99mTc-PYP imaging. Left ventricular ejection fraction (LVEF), left ventricular posterior wall thickness at end-diastole (LVPWTd), and E-to-early diastolic mitral annular tissue velocity ratio (E/e′) were assessed. The QRS duration on the electrocardiogram (ECG) was also measured 4.9 ± 2.9 days before 99mTc-PYP imaging. In addition, b-type natriuretic peptide (BNP) was measured by a blood examination test 4.9 ± 2.7 days before 99mTc-PYP imaging. All these parameters  were assessed in all 14 patients with ATTR-CA.All patients with ATTR-CA (E/e′), ECG (QRS), and blood examination (BNP). We also analyzed the correlation of CPV with other quantitative uptake parameters.The primary analysis was to optimize the method of volumetric evaluation for diagnosing ATTR-CA by comparing thresholds (1.2 and 1.4 × ABPmax). The secondary analysis was the correlation of CPV with cardiac functional parameters of echocardiography  and compared with a Chi-square or Fisher exact test as appropriate. Diagnostic accuracy for ATTR-CA was examined by receiver operating characteristic (ROC) analysis, and the area under the ROC curve (AUC) was calculated. Correlation of parameters was assessed with Pearson’s correlation coefficient. All statistical tests were two-sided, with P < .05 considered statistically significant. All analyses were performed using JMP version 12 .Continuous variables were summarized as mean ± standard deviation (SD) and compared with a student’s n = 9) and wild type . Among patients diagnosed with ATTRv-CA, 8 (89%) patients had Val30Met [p.V50M] mutation and 1 (11%) patient had Leu58Arg [p.L78A] mutation. Eleven (44%) patients were diagnosed with no ATTR-CA and used as control patients. All control patients underwent EMB and were negative for ATTR.In total, 25 patients were included with a mean age of 70.4 ± 12.8 years and 15 (60%) males. Population characteristics are summarized in Table P < .02) in patients with ATTR-CA than in those without ATTR-CA (Table n = 11), myocardial uptake was lower than ABPmax × 1.2 and CPV1.2 was 0.0 cm3. CPV1.2 showed no significant difference between patients with ATTRv-CA  and those with ATTRwt-CA .CPV and H/CL ratios were significantly higher P < .0 in patieROC curves for identifying ATTR-CA are summarized in Figure E/e′ (18.4 ± 5.1) was low. CPV1.2 significantly correlated with LVEF, LVPWTd, and QRS. The H/CL ratio did not significantly correlate with any of the values of LVEF, LVPWTd, E/e′, QRS, and BNP.The correlation between uptake parameters and cardiac functional parameters in patients with ATTR-CA is summarized in Table E/e′, QRS, and BNP).In one EMB-proven ATTRv-CA patient who did not show abnormal myocardial activity, we evaluated SUVmax in the interventricular septum using fused SPECT/CT images. Fourteen patients with ATTR-CA showed negative correlation between SUVmax and LVEF, but the SUVmax did not significantly correlate with other parameters  99mTc-PYP activity in SPECT, their method was based on previous studies of 18F-fluorodeoxyglucose positron emission tomography with suspected cardiac sarcoidosis patients.7 As shown by our results of CPV1.4, higher thresholds can cause false-negative diagnosis in patients with focal abnormal uptakes. Given the high diagnostic accuracy of visual SPECT/CT interpretation, it is unlikely that volumetric evaluation could improve diagnostic accuracy. However, it may have a role as a quantitative and objective marker.Our results demonstrated that setting the myocardial 12 N-terminal pro-BNP (NT-proBNP) and the estimated glomerular filtration rate were reported to stratify patients with both ATTRwt-CA and ATTRv-CA into prognostic categories.13 NT-proBNP and high-sensitivity cardiac troponin T were reported to assess prognosis in patients with ATTRwt-CA.12 Therefore, we expect CPV to play an important role in risk assessment. Miller et al also demonstrated that their volumetric parameters of 99mTc-PYP SPECT had prognostic value and low inter-observer variability.7Volumetric evaluation of bone scintigraphy can be an objective marker for disease burden of ATTR-CA and provide valuable prognostic information. We demonstrated that CPV1.2 significantly correlated with several cardiac functional parameters . In addition, the significant correlation was stronger in CPV1.2 than in the H/CL ratio and SUVmax. Although CPV1.2 did not significantly correlate with BNP, the correlation was stronger in CPV1.2 than in the H/CL ratio and SUVmax. The H/CL ratio is based on two-dimensional imaging and has some inherent limitations. Reduced LVEF < 50% was reported as a predictor of mortality in patients with ATTRwt-CA (hazard ratio: 1.85).15 However, some patients with ATTR-CA showed focal or absent 99mTc-PYP uptakes.17 We found several patients who had positive results of EMB and their myocardial 99mTc-PYP uptakes were focal (n = 2) or absent (n = 1). Sperry et al also reported that 1 of 33 (3%) patients with myocardial 99mTc-PYP uptakes had focal uptake.11 In addition, several causes of false-negative and false-positive results of bone scintigraphy in patients with suspected ATTR-CA have been reported, including Val30Met mutation.18We included only patients with negative EMB for ATTR as control patients, which is a unique and novel feature of our study. Several studies adopted positive standards for ATTR-CA as follows: (1) visual grade of 2 or 3 for myocardial uptake , (2) diffuse abnormal myocardial uptake in SPECT.9 Sperry et al also calculated mean blood pool counts at the ascending aorta.16 In addition, variation of radioactivity was reported to be relatively smaller in the aorta than in bone.9 Bone radioactivity has often been used as a reference for myocardial uptake.10 However, the method of placing a reference VOI in the LVBP7 can be subjective. In addition, in patients with small hearts, their LVBP activity can be contaminated by the spillover of myocardial uptake.The method of placing a reference VOI in the ascending aorta at the height of the pulmonary artery bifurcation is objective to evaluate background radiotracer activity.14 Due to cardiac motion from contraction and breathing, the position of the heart shown on a CT scan is not perfectly coregistered to the SPECT images. On a non-contrast low-dose CT scan, which is used in SPECT/CT, the myocardium is poorly visualized in normal patients; hence, defining a VOI which covers only myocardium is difficult in normal hearts. In addition, SUVmax represents only a single voxel value and can be contaminated by spillover from neighboring high bone activity.SUVmax has some inherent limitations and we did not evaluate it in patients without ATTR-CA. SUV depends on VOI definition, and given the poor spatial resolution of SPECT, it is often difficult to discriminate between relatively high blood pool radioactivity and uptake in the adjacent myocardial wall, especially in normal and equivocal patients.Our small population and single-center study design are weakness of this study. The thresholds and diagnostic accuracy of uptake parameters were based on our small datasets. The present findings require larger, multicenter, prospective validations. Our method needs a manual setting of a polygonal VOI which encompasses the left and right ventricles based on fused SPECT/CT images. However, if we use a proper uptake threshold, the influence of VOI variation is limited. We included only patients with negative EMB for ATTR as control patients, and the selection may have caused a bias. In daily practice, not all suspected ATTR-CA patients undergo EMB.99mTc-PYP SPECT/CT may be superior to the H/CL ratio and SUVmax in diagnosing patients with ATTR-CA and assessing the disease burden. Larger studies are warranted to clarify whether volumetric measurement of bone scintigraphy can identify patients with early-stage ATTR-CA and assess prognosis, disease progression, and treatment response.Optimized volumetric evaluation of 99mTc-PYP SPECT/CT was useful for detecting both focal and diffuse abnormal uptakes and showed a high diagnostic accuracy for ATTR-CA. Cardiac pyrophosphate volume was superior to the H/CL ratio and SUVmax in its correlation with cardiac functional parameters and may be a useful non-invasive marker for assessing the disease burden of ATTR-CA.Volumetric evaluation of 3 and their CPV1.4 were 0.0 cm3. The myocardial regions where 99mTechnetium-pyrophosphate uptakes were > 1.2 × aortic blood pool SUVmax are shown by green contours in two patients (A and B). ATTR-CA, transthyretin cardiac amyloidosis; CPV, cardiac pyrophosphate volume; EMB, Endomyocardial biopsy; SPECT, Single-photon emission computed tomography; SUV, standardized uptake value. Supplementary file1 (PDF 83 kb)SPECT/CT images of three EMB-proven ATTR-CA patients whose CPV were low. Their CPV1.2 were 3.9, 2.6, and 0.0 cmSupplementary file2 (PPTX 453 kb)Below is the link to the electronic supplementary material.