In heart failure with reduced ejection fraction, edema and congestion are related to reduced cardiac function. Edema and congestion are further aggravated by chronic kidney failure and pulmonary abnormalities. Furthermore, together with edema/congestion, sodium/water retention is an important sign of the progression of heart failure. Edema/congestion often anticipates clinical symptoms, such as dyspnea and hospitalization; it is associated with a reduced quality of life and a major risk of mortality. It is very important for clinicians to predict the signs of congestion with biomarkers and, mainly, to understand the pathophysiological findings that underlie edema. Not all congestions are secondary to heart failure, as in nephrotic syndrome. This review summarizes the principal evidence on the possible roles of the old and new congestion biomarkers in HFrEF patients . Furthermore, we provide a description of conditions other than congestion with increased congestion biomarkers, in order to aid in reaching a differential diagnosis. To conclude, the review focuses on how congestion biomarkers may be affected by new HF drugs  approved for HFrEF. Heart failure (HF) is a complex syndrome consisting of symptoms  usually accompanied by signs . It is due to a structural and/or functional abnormality of the heart that results in elevated intracardiac pressures and/or inadequate cardiac output at rest and/or during exercise .We can distinguish specific phenotypes based on the assessment of left ventricular ejection fraction (LVEF). Reduced LVEF  is defined as ≤40%; those with HF symptoms and signs in the presence of reduced LVEF are designated as HF with reduced ejection fraction patients (HFrEF).Despite recent advances in HFrEF management , HFrEF remains a highly prevalent disorder with a high mortality rate and is burdened by several hospitalizations for acute decompensated heart failure (ADHF) .ADHF hospitalizations are generally associated with signs and symptoms of congestion . ADHF reSystemic congestion is not exclusively related to cardiac congestion, and the simple absence of clinical signs of fluid overload cannot exclude increased left ventricular filling pressures (LVEDP) .We can distinguish two different forms of congestion: the intravascular one if congestion is predominantly present in the vascular system, and the tissue one if it is interstitial. Although most HF patients have a combination of both forms, one phenotype can prevail. Each of these kinds of congestion has a specific pathophysiology and requires a different diagnostic approach.Peripheral congestion occurs in the form of peripheral edema, with a palpable swelling in the body tissues caused by an expanded interstitial fluid volume. This occurs if the capillary filtration exceeds the amount of fluid conducted out by lymphatic drainage. It is observed in different clinical conditions like HF, renal failure, liver failure, or pathologies affecting the lymphatic system. Edema usually becomes clinically apparent when the interstitial volume exceeds 2.5–3 L.Assessment of congestion by a simple clinical evaluation remains unsatisfactory. The most commonly used and traditional strategy is clinical evaluation, associated with chest radiography and natriuretic peptide (NP) measurement ,10.It is very important for clinicians to predict the presence of congestion with biomarkers and to understand the pathophysiological mechanisms that underlie edema.There is increased attention not only on the role of congestion biomarkers in recognizing patients at risk of developing HF who are potential candidates for targeted treatments for its prevention, but also on their role in formulating an initial diagnosis and a prognostic stratification.This review summarizes the principal evidence on the possible roles  of old and new congestion biomarkers in HFrEF patients. Furthermore, we describe conditions other than congestion with increased congestion biomarkers in order to facilitate a differential diagnosis. To conclude, the review focuses on how congestion biomarkers may be affected by new HF drugs  approved for HFrEF.A biomarker is a biological molecule found in blood, other body fluids, or tissue and may relate to data obtained from vital parameters , or from imaging/instrumental tests .Biomarkers can help in identifying disease peculiarities (risk factor or risk marker), disease state , or disease outcome (rate of progression) . BiomarkIdeal biomarkers should be precise, standardized, patient-acceptable, easily interpretable by clinicians, sensitive, and highly outcome-specific.Many researchers have sought out cardiac biomarkers to improve the prediction, diagnosis, and prognosis of HF. Several biomarkers related to different findings in HF pathophysiology have been studied  15,16].,16.15,16Natriuretic peptides (NPs) are the most known and widely used biomarkers. Brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) are neurohormones produced and stored in the heart, both in the atria and ventricles, and released from the heart due to increased end-diastolic wall-stress in response to pressure changes and volume overload.NPs are synthesized as prohormones and subsequently cleaved into the active hormones BNP and ANP, and into the inactive NT-proBNP and mid-regional proANP (MR-proANP). Circulating BNP and ANP half-lives are relatively short (about 20 min). Plasma endopeptidases and NP receptors remove them from the circulation. NT-proBNP and MR-proANP have longer circulating half-lives (around 90 min), and their clearance is mainly renal ,18.Cardiac troponins (cTn) are usually evaluated in patients with acute HF (AHF) to rule out myocardial infarction. However, congestion at the inlet was significantly associated with cTn levels at the time of discharge, implying that the high intracardiac filling pressure and the increased wall stress associated with HF decompensation can induce subclinical myocardial injury .The primary function of troponin levels in HF is to stratify risks. Higher troponin I or T levels at the hospital admission of patients with AHF were associated with lower EF and a higher rate of in-hospital mortality ,21.Recently accumulating data suggest cancer antigen-125 (CA-125) as a marker of congestion in HF. The increase in circulating CA-125 concentrations is due to at least two pathophysiological mechanisms that partially overlap . On the CA125 has been demonstrated to be a tool for risk stratification in patients admitted with ADHF and CA125 <23 U/mL identifies a subgroup of patients at low risk of short-term adverse events .CA125 and no NT-proBNP seem to identify patients with AHF, and congestive intrarenal venous flow (IRVF) patterns. The IRVF measured by Doppler ultrasound may be a potential surrogate marker of renal congestion and adverse outcomes in heart failure .Soluble suppressor of tumorigenesis-2 , the circulating form of the interleukin-33 membrane receptor, is secreted by myocardial cells in response to cardiac overload. Vascular congestion, mechanical stretch, and inflammation stimulate the expression of sST2 . Lungs hThe protein galectin-3 is currently gaining interest as an eligible biomarker in cardiac disease. Galectin-3 is a biomarker of fibrosis, inflammation, and oxidative stress. Normally, Gal-3 expression in the heart is low. In the failing heart, Gal-3 is released by activated cardiac macrophages and cardiac fibroblasts , taking Cholestatic liver injury can be measured by bilirubin, alkaline phosphatase, and gamma-glutamyl transpeptidase levels , which hLaboratory anomalies of liver function may predict the prognosis of patients with advanced HF, and the assessment of both cardiac and liver function is very important in the management of these patients .Hemoconcentration could be another sign of congestion. Plasma volume may be indirectly estimated by several formulas using hemoglobin and/or hematocrit levels, which seem useful for monitoring congestion and decongestion both in acute and chronic settings . SeveralUnimportant differences in the prognostic value of biomarkers in HFpEF vs. HFrEF were found in previous studies except for cystatin-C and NT-proBNP; in fact, these two markers showed a less evident prognostic value in HFpEF .Patients with HFpEF and HFrEF had higher median levels of hs-TnT, GDF-15 (growth differentiation factor 15), and NT-pro-BNP compared with controls, but not ST2; NT-proBNP and hs-TnT levels were higher in HFrEF in comparison with HfpEF . In a foNPs are usually only moderately elevated in HFpEF patients, and levels may decrease in the asymptomatic phase. The reason for this is that NPs are liberated and produced in response to augmented myocardial wall stress. Hypertrophic hearts  are not characterized by severely elevated end-diastolic wall stress. In contrast, in the case of supraventricular tachycardia or excess water (as in HFrEF), levels of NPs may become very high. In the first scenario, the lack of chronically increased wall stress causes lower NP production and lower circulating levels when compared to HFrEF. NP plasma levels increase in parallel with the severity of diastolic abnormalities .Congestion in HF can be defined as fluid accumulation inside the intravascular compartment and the interstitial space because of increased cardiac filling pressures determined by maladaptive sodium and water retention by the kidneys . It is tThe fluid accumulation leading to ADHF starts in the intravascular compartment. Continuously increased hydrostatic pressures in the capillary vessels subsequently lead to tissue congestion. Most patients with ADHF have a combination of both intravascular and tissue congestion .Currently, the gold standard for assessing intravascular congestion is to measure right atrial pressure  and pulmonary artery systolic pressure  by right heart catheterization. However, right heart catheterization is invasive, and, therefore, routine performance of this procedure is not attractive. To assess changes in congestion on a day-to-day basis, non-invasive measurements are needed, such as changes in plasma levels of congestion biomarkers between admission to and discharge from the hospital.Congestion biomarkers could be useful to identify subclinical congestion in hospitalized patients, with prognostic implications. In fact, incomplete decongestion at discharge from the hospital is associated with higher rates of both death and re-admission to the hospital for heart failure. Patients with residual congestion on day 7 of hospitalization have a more than two-fold increase in 180-day mortality and an almost two-fold increase in the risk of re-hospitalization for HF compared with patients without congestion . It is tMulti-biomarker diagnostic approaches could be useful to predict outcomes in patients at risk or with overt HF in many settings. In the past, multi-biomarker scores have been evaluated for risk stratification in ADHF  and chroIn recent years, diagnostic strategies based on multi-biomarkers reflecting different pathophysiological pathways have been proposed, with the aim of better understanding the phenotype of the disease in each individual patient and possibly designing an adapted therapy. For example, demonstrating an increase in a specific biomarker could lead to the initiation or titration of a therapy that neutralizes the mechanisms leading to such an elevation. Importantly, these strategies have never been tested in randomized controlled trials .Further research is required to explore the use of a multi-biomarker approach for improving the diagnostic and prognostic precision of models, as well as to better understand disease phenotypes and direct the patient toward the best therapeutic strategy. The role of congestion biomarkers in the prognosis of ADHF would provide useful information about patients who may respond to more aggressive treatments and those who will progress and/or may become candidates for cardiac transplantation in the future. In addition, more research is needed to determine if new biomarkers can be used as a substitute for clinical trial results.NPs may also guide therapy in HF, as serial NP measurements provide data about medication adjustments to achieve targets independently of symptoms. However, in this regard, the data seem quite conflicting. In patients with HFrEF, NP-guided therapy seems to be helpful. The studies STARS-BNP  and PROTNPs represent the gold standard for biomarkers in HF, and all major societies, including AHA and ESC, recommend the dosage of BNP and NT-proBNP for diagnosing HF in their guidelines. Nevertheless, there are determinants influencing the clinical interpretation of their levels. While obesity lowers BNP and NT-proBNP serum levels  , factorsCardiac troponins are traditionally used for the diagnosis of acute myocardial infarction (AMI), but they are also elevated in HF because of coronary causes, cytotoxicity, apoptosis, and inflammation. Troponin kinetics help distinguish between AMI and HF.Other pathologies are characterized by cardiac troponin elevation: tachyarrhythmias, hypertensive emergencies, critical illnesses (shock/sepsis/burns), myocarditis, Takotsubo syndrome, valvular heart diseases, aortic dissection, pulmonary embolism, pulmonary hypertension, renal dysfunction, acute neurological events, cardiac contusion or cardiac procedures, thyroid disorders, infiltrative diseases, drug toxicity, extreme endurance efforts, or rhabdomyolysis .In patients with COVID-19 pneumonia, there are high troponin and liver enzymes levels in many patients regardless of HF, which means that their diagnostic value in this context is low for separating these two entities . On the In all these cases, an increase in cardiac troponin concentration in blood serum indicates reversible or irreversible damage to cardiomyocytes, and in some cases , elevation can occur even in the absence of cardiomyocyte damage .Recent evidence about CA-125 suggests that plasma levels of this glycoprotein could be useful as biomarkers in HF, reflecting mesothelium activation due to hydrostatic pressure and inflammation. Apart from its common use as a tumor marker in ovarian cancer, an abnormally high expression of serum CA-125 is also present in other neoplasms  , in non-Galectin-3 is widely expressed in human tissues, including all types of immune cells, epithelial cells, endothelial cells, and sensory neurons. It contributes to cell survival, cell–cell interaction, and cell-extracellular matrix interaction; thus, it is involved in cell differentiation, inflammation, fibrogenesis, angiogenesis, and host defense .Its serum levels are elevated in many other conditions: atrial fibrillation (particularly in persistent AF), chronic coronary heart disease, AMI, chronic kidney disease, cirrhosis, renal cell carcinoma and other malignant lesions such as hepatocellular carcinoma, prostate cancer, pancreatic cancer, colorectal cancer, bladder cancer and lymphoma.sST2 has gained interest as a potential biomarker for inflammatory diseases like asthma, rheumatoid arthritis, and inflammatory bowel disease, as well as in cardiovascular pathophysiology. It can be used for predicting and monitoring HF; in fact, it was included in the 2017 ACC/AHA update of HF guidelines. Elevated levels of serum sST2 have been reported in AMI, acute aortic syndromes, arterial hypertension, atrial fibrillation, and in heart transplant recipients with acute allograft rejection . Lastly,Mid-regional pro-adrenomedullin (MR-proADM) represents the level and activity of adrenomedullin. Its synthesis is widely distributed in tissues, including bone, adrenal cortex, kidney, lung, blood vessels, heart, adipose tissue, anterior pituitary gland, thalamus, and hypothalamus; among its effects are vasodilatation, positive inotropy, diuresis, bronchodilation, and anti-inflammatory/anti-microbial action. High levels of this biomarker have been reported in HF, hypertension, pneumoniae, ARDS, cirrhosis, and cancer. Moreover, it has shown effectiveness as a diagnostic marker for differentiating between septic and non-septic patients with Systemic Inflammatory Response Syndrome (SIRS) and as a prognostic marker for stratifying the mortality risk in patients with sepsis ; recentlCopeptin, a surrogate marker of vasopressin (AVP), has an application in the diagnosis of diabetes insipidus and other diseases with altered vasopressin secretion. High copeptin serum levels could have a diagnostic and prognostic role in acute coronary syndromes, a prognostic value in pneumoniae, chronic obstructive pulmonary disease, diabetes mellitus, stroke, and sepsis , and AVPGDF-15 is a stress-responsive cytokine, widely present in mammalian tissues and plays multiple roles in various pathologies, performing anti-inflammatory and immunosuppressive actions. Elevated circulating levels of GDF-15 have been reported in patients with acute pulmonary embolism, idiopathic pulmonary arterial hypertension, chronic kidney disease, metastatic prostate, breast, and colorectal carcinoma, with a strong association between serum levels, histological malignant grade, and metastatic progression. As regards CV diseases, GDF-15 is related to cardiometabolic risk, and its synthesis increases during tissue injuries and inflammatory states. High levels of GDF-15 are associated with the development and progression of pathologies such as HF, atherosclerosis, atrial fibrillation, obesity, and diabetes .P3P serum type III procollagen peptide is released by fibroblasts during collagen synthesis and indicates a rapid collagen turnover. Therefore, its serum levels are elevated in viral and alcoholic hepatitis, lung fibrosis , CHF 8585, post The neutrophil gelatinase-associated lipocalin (NGAL), among the members of the adipokines superfamily, has emerged as a pleiotropic acute phase protein that takes part in many inflammatory, immune, and metabolic processes; thus, it has a potential role as a biomarker in these fields. Its serum level is upregulated in several acute and chronic inflammatory/autoimmune conditions  , as wellIn clinical medicine, the BUN/Cr ratio has been extensively used to differentiate pre-renal kidney disfunction from intrinsic renal parenchymal disease. The BUN/Cr ratio is often used to determine dehydration in patients presenting to emergency departments, and a high ratio has a prognostic value in many diseases, including stroke , gastroiCongestion is the principal cause of HF decompensation and hospitalization, leading to a worst prognosis. Therefore, the early detection of subclinical congestion with new biomarkers is very important for the management of these patients.Most new biomarkers are not used daily for the diagnosis of HF or for differential diagnosis in patients with new dyspnea. New biomarkers are usually used for prognostic stratification in HF. They may add prognostic information to common cardiac biomarkers.Cardiac interstitial fibrosis markers seem to have the biggest prognostic value in HF patients . Among tAs an HF biomarker, Galectin-3 may have a diagnostic and prognostic role (prognostic in both acute and chronic HF), since its expression is upregulated, especially in severe HF patients and in those with renal dysfunction. To date, Galectin-3 measurement is recommended by the 2017 Guidelines of the American Heart Association for risk stratification and prognosis evaluation of patients with HF .GDF-15 levels are slowly increasing over time in patients with CHF, and these increases are associated with worsening functional status and adverse outcomes . MoreoveFibroblast growth factor-21 (FGF21) appears to be a promising biomarker candidate for HF. Pre-clinical studies have shown that FGF21 is implicated in the physiopathology of HF . However, in the clinical setting, FGF21 levels seem to rise in HF. It is unclear whether FGF21 might have a causal role in HF, and whether circulating FGF21 can be used as a biomarker to improve the prediction, diagnosis, and prognosis of HF .Fibroblast growth factor-23 is a hormone dominantly secreted by osteocytes and osteoblasts in bones; however, it can also be produced by liver and heart muscle under stress . It showCopeptin has been found to be an excellent marker of HF development in patients with ACS, in the risk stratification of patients with HF symptoms presenting acutely to an emergency unit, as well as an accurate marker of risk in both hospitalized and ambulatory patients with CHF . As regaNeutrophil gelatinase-associated lipocalin (NGAL) is a promising diagnostic biomarker. It is a human protein belonging to the lipocalin superfamily. Both plasma and urinary NGAL levels are increased in HF patients. The elevation is dependent on the clinical stage of HF, together with the presence of renal failure and consequent congestion .Widely recognized as a major marker of neuroendocrine tumor (NET), chromogranin A (CgA) has been found to be related to the clinical deterioration and higher risk of mortality in patients with AHF and CHF . High leAnother molecule, fatty acid-binding protein 4 (FABP4), has recently been shown to play a role in atherosclerosis and coronary artery disease, and it has been directly related to cardiac alterations such as left ventricular hypertrophy (LVH) and both systolic and diastolic cardiac dysfunction . Some reThe P3P has been defined as a marker for fibrosis in patients with hepatic disease ,107. A hElevated soluble neprilysin (sNEP) levels have been suggested as a prognostic biomarker in HFrEF. Levels of sNEP were significantly reduced in HFrEF when compared to the controls .Recently, four key genes  were identified as potential biomarkers for HF, providing a new research idea for the treatment of HF .N-acetylglucosamine/galactosamine (GlycA) and sialic acid (GlycB) correlate with inflammation status and with prognostic value in long-term outcomes of patients with HF .Researchers are searching for new biomarkers with possible implications for diagnosis, risk stratification, and treatment effect monitoring. These biomarkers could potentially be used to better define study inclusion criteria and enable the enrolment of patients who are more likely to respond to a specific drug. In the case of a close relationship between biomarker levels and clinical endpoints, changes in biomarker levels after a treatment may be a surrogate endpoint, potentially reducing the duration and cost of a clinical trial. With omics-based tools, biomarkers might be more precisely selected for use in clinical trials to identify responses that closely reflect the biological effects of the new drug. The future role of biomarkers will be linked to providing a distinct phenotype for HF patients in order to choose the right therapy. Old and new HF biomarkers could be integrated into validated scores for prognostic stratification  and linkHowever, the new biomarkers still need to be validated for clinical implications in multicentric studies with large populations. Further studies are needed prior to formalizing individual patient care algorithms guided by fibrosis biomarkers. Therapy guidance in chronic HF using galectin-3 and sST2, was suggested by several post hoc analyses .There are emerging data about sST2, galectin-3, and procollagen as potential uses for guiding therapies. Mortality benefits from statin use, as well as anti-fibrotic therapies, such as aldosterone antagonism, may vary based on levels of these fibrosis markers .We propose an algorithm for the diagnosis and risk stratification of patients with suspected HF . In patiIn the future, it will be very interesting to see how new drugs for HF affect congestion biomarkers. Among the new drugs, gliflozins promote diuresis and natriuresis . In 60 oTo date, BNP is a substrate of neprilysin; BNP levels increase with neprilysin inhibition. So, the clinical validity of measuring BNP in sacubitril/valsartan-treated patients has been questioned, and the use of NT-proBNP has been preferred and recommended because it is not a substrate of neprilysin inhibition. NT-proBNP should be measured within 8 to 10 weeks of drug initiation .Finally, new trials should be designed to combine different classes of diuretics/drugs to treat therapy-resistant edema in patients with HFrEF, and new biomarkers could play a role in the phenotyping of patients most sensitive to specific diuretics/drugs.The search for cardiac biomarkers to enhance the prediction, diagnosis, and prognosis of HF is a major challenge in cardiology. Several biomarkers correlated with different findings of HF pathophysiology were studied. However, they still need to be validated for clinical implications. The most interesting role of biomarkers may be in helping the choice of the right therapy in HF patients by HF score and providing specific phenotypes of HF patients.


A one-step sandwich chemiluminescence immunometric assay (LIA) was developed for the quantification of bifunctional peptidylglycine-α-amidating monooxygenase (PAM) in human plasma (PAM-LIA). PAM is responsible for the activation of more than half of known peptide hormones through C-terminal α-amidation. The assay employed antibodies targeting specific catalytic PAM-subunits, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), to ensure detection of full-length PAM. The PAM-LIA assay was calibrated with a human recombinant PAM enzyme and achieved a detection limit of 189 pg/mL and a quantification limit of 250 pg/mL. The assay demonstrated good inter-assay (6.7%) and intra-assay (2.2%) variabilities. It exhibited linearity when accessed by gradual dilution or random mixing of plasma samples. The accuracy of the PAM-LIA was determined to be 94.7% through spiking recovery experiments, and the signal recovery after substance interference was 94–96%. The analyte showed 96% stability after six freeze–thaw cycles. The assay showed strong correlation with matched EDTA and serum samples, as well as matched EDTA and Li-Heparin samples. Additionally, a high correlation was observed between α-amidating activity and PAM-LIA. Finally, the PAM-LIA assay was successfully applied to a sub-cohort of a Swedish population-based study, comprising 4850 individuals, confirming its suitability for routine high throughput screening. PAM is a monopeptide, comprising two functional subunits with distinct catalytic activities, a peptidylglycine alpha-hydroxylating monooxygenase (PHM) and a peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL) one Fig. .Figure 18. Along with alternative splicing, the latter can also be attributed to a tissue-specific endoproteolytic cleavage of protease sensitive linker between two catalytic domains. Initially isolated from the bovine neurointermediate pituitary lobe10, the majority of the PAM activity was associated with separate PHM and PAL catalytic domains. In contrast, in bovine atrium and rat medullary thyroid carcinoma purified PAM was present as an intact bifunctional enzyme of a 113-kDa and 75-kDa, respectively13.Encoded by a single copy gene with more than 160 kb of DNA comprising 25 exons, human PAM exists in at least six different isoforms, including membrane bound and soluble variants, as well as single monofunctional PHM and PAL enzymes4. It was demonstrated, that PAM double knock-out in mice and fruits flies is lethal in the first week of gestation or early larval stage, respectively, leading to a complete depletion of amidated peptide hormones15. Furthermore, the Drosophila nemy mutants, which encode defective redox enzymes (cytochrome B561 homologue), a required cofactor for PHM activity, showed decreased levels of amidated peptides and learning and memory deficits16. PAM is expressed in most mammalian cell types with the highest PAM activity in pituitary and hypothalamus17. In human, the modulation in PAM activity has long been linked to the presence or potential development of the diverse human pathologies18. The increased α-amidating activity was found in several tumors, including the medullary thyroid carcinoma, neuroendocrine and pancreatic endocrine tumors, and also was associated with multiple sclerosis, post-polio syndrome and many others22. Multiple PAM mutations resulting in reduced activity were shown to be associated with an increased risk of type 2 diabetes, possibly by affecting insulin granule packaging and release from β-cells25.When measured in CSF of the Alzheimer’s patients, the PAM activity was significantly reduced in comparison to the healthy individuals26. According to the recent findings, also in plasma the reduced PAM activity was associated with higher risk of AD development 7 years prior the diagnosis27. The elevated α-amidating activity was correlated with higher incidence of heart failure and heart fibrillation, as well as untreated systolic and diastolic hypertension28. In addition to its primary function of catalyzing the C-terminal amidation of numerous peptide hormones, Bäck et al. has demonstrated that PAM is also necessary for the formation of atrial secretory granules29. Studies using knockout mice that lack cardiomyocyte PAM have shown a decrease in the content of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) despite the fact that these peptides do not require α-amidation. Thus, due to its essential role in regulation of versatile physiological and pathophysiological functions, PAM was suggested to be significant therapeutic target and biomarker for various human clinical conditions4.PAM is indispensable for life and is the only known enzyme to catalyze the C-terminal α-amidation34. One other drawback is the utilization of the synthetic radiolabeled tri- or quadropeptides as a substrate34. To our knowledge, there are only few activity assays, which utilize the unmodified naturally occurring full-length peptide hormone precursor as a substrate35. Finally, the quantification of PAM activity is usually accessed under optimal co-factor concentration, such as copper and ascorbate, which may differ from their actual concentrations in vivo and possibly artificially elevate the determined PAM activity. While the activity-based quantification of PAM has been explored, the information regarding the direct measurement of PAM enzyme concentration is scarce in the existing literature36. Commercially available ELISA assays for PAM quantification lack information regarding the specificity of the capturing and detecting antibodies. This limitation becomes particularly disadvantageous when attempting to distinguish between the full-length enzyme and single subunits. Another drawback is the lack of information addressing the correlation between PAM enzyme activity and its concentration, which might hinder the transferability of findings gained from activity-based measurements into quantitative estimations of enzyme load.To this day the most common way to quantify PAM enzyme is by measuring its activity. Most of these methods are unapplicable for the routine large-scale applications, since the quantification of the α-amidated products require time-consuming analytical chromatography or other substrate/product separation methods (e.g. with ethyl-acetate) for each sampleHere we present the one-step chemiluminescence immunometric assay, which allows to determine the concentration of bifunctional PAM enzyme in human plasma (PAM-LIA). Conformational capture and developing antibodies were directed against PHM and PAL subunit, respectively, enabling the detection of the full-length enzyme variant. The PAM-LIA assay is designed in 96-well microtiter plate-based format, requires a small sample volume of 20 µL for single determination, short incubation time and operates within a wide calibration range of 0.25–723 ng/mL. Our immunoassay enables high-throughput screening of a large number of samples within a short timeframe. To demonstrate the efficiency of PAM-LIA assay as well as possible application of PAM as a predictive biomarker for several cardiovascular events, we measured PAM concentration in a sub-cohort of 4850 study subjects from a Swedish population-based study, the Malmo Preventive Project.PAM-LIA is a one-step high throughput microtiter plate-based chemiluminescence immunometric assay for the quantitative detection of full-length PAM in human with the capture and detection antibodies being specifically directed against PAL and PHM subunits, respectively Fig. . No crosTechnical performance of the PAM-LIA was summarized in Table Confounding factors used for the substance interference studies are listed in the Table The ex vivo analyte stability in six native EDTA plasma samples, collected from healthy reported individuals, was determined Fig. A. The avThe accepted operational temperature range of PAM-LIA assay was determined between 18 °C and 32 °C Fig. B. The PAp < 0.0001) and 0.84 (p < 0.0001) between matched Li-Heparin and EDTA plasma    Fig. D. This c01) Fig. B.In addition to measuring PAM concentrations in human plasma and serum, the assay was found to be suitable for use with rat, porcine and ape plasma. The average PAM concentrations in these samples were determined to be 7.1 ng/mL , 8.42 ng/mL (n = 3) and 143.0 ng/mL (n = 11), respectively.The distribution of PAM concentration in serum samples from a sub MPP cohort of n = 4850 individuals is presented in Fig. p < 0.0001), BMI (p < 0.0001), systolic (p = 0.0002) and diastolic blood pressure (p = 0.0019), and triglyceride levels (p = 0.0008). However, there were no significant correlations found between gender, glucose, LDL, HDL, and total cholesterol levels and PAM protein.Furthermore, PAM concentration was divided into quartiles and correlated with clinical baseline parameters such as age, BMI, cholesterol, glucose levels, etc. Table . The resp = 0.024) and AF , while those with PAM concentrations below 79.5 ng/mL had a lower incidence of MACE  , atrial fibrillation (AF), and major adverse cardiovascular events (MACE) in a sub cohort of MPP, excluding individuals with pre-existing coronary event and stroke. The results indicated that a PAM concentration above the median value was associated with a higher incidence for HF, AF and MACE. Specifically, individuals with PAM concentrations below 93 ng/mL had a lower incidence of HF  offers significant advantages over currently applied methods for measuring the level of PAM in bodily fluids. Firstly, most validated methods for measuring PAM levels involve time-consuming and non-scalable methods, which employ the quantification of product formation. Secondly, measuring PAM activity often requires optimal substrate conditions, which may not accurately reflect in vivo conditions. In contrast, the PAM-LIA assay directly measures the protein concentration, which may offer more accurate insights into the in vivo status of the enzyme.The current assay set-up enables the measurement of the full-length PAM by targeting the PAL subunit through capturing antibodies and detecting the PHM subunit. PAM protein is expressed in various isoforms, including different variants of the full-length protein as well as single subunits, depending on the tissue. To obtain a more comprehensive view of PAM’s expression profile, different permutation of capture and tracer antibodies could be used to target either PHM or PAL alone, either as a single subunit or as part of the full-length enzyme. By combining different assay setups, important insights into the total PAM load in different tissue extracts or bodily fluids can be obtained.The strong correlation between PAM concentrations measured using the PAM-LIA assay and the amidating activity supports the assay's reliability as a proxy for PAM activity. The assay’s compatibility with various sample matrices, including EDTA plasma, serum, and Li-Heparin plasma, increases its versatility and utility in clinical testing. The broad operational temperature range (18–32 °C) of the PAM-LIA assay indicates its robustness and adaptability to varying laboratory conditions without significant impact on accuracy. The high ex vivo stability of the PAM analyte in plasma samples, with only a 4.3% reduction in concentration after six freeze–thaw cycles, suggests that the assay can be used with stored samples without substantial loss of accuracy.Furthermore, the PAM-LIA assay demonstrated successful application in measuring PAM concentrations in plasma samples from rat, porcine and ape species, highlighting its potential for use in comparative and translational research involving different mammalian models. The detection of PAM in various tissue extracts, including porcine pituitary extract, soluble and solubilized membrane liver fraction from rat, suggests that the assay could detect different PAM isoforms expressed in various tissues.36. However, it is important to note that although rat and human PAM share a high sequence similarity, the use of such an assay setup may not be the most suitable for detecting human PAM. Our results demonstrate a significant decline in antibody sensitivity when plasmatic rat PAM is used as an analyte.Among the immunoassay-based methods for direct quantification of PAM, the PAM-LIA assay has shown several advantages when compared to previously reported methods. To our knowledge, Sturmer et al. was the first to report the direct quantification of PAM in an immunoassay-based setup, utilizing chicken antibodies to detect rat PAMUnlike PAM-LIA assay, the commercially available immunoassay-based kits for human PAM detection lack information regarding the correlation between PAM concentration and PAM activity , which raises questions about the validity of these assays for accurate PAM quantification. Additionally, the lack of information regarding the target of the solid- and liquid phase antibodies makes it challenging to distinguish whether the detected product is a single subunit or the full-length PAM. This ambiguity adds further uncertainty to the quantification process.When comparing the analytical performance characteristics of different assay setups, it becomes evident that the PAM-LIA assay outperforms the other reported assay set-ups with respect to calibration range and range of operational temperature, demonstrates higher precision (intra- and inter-CV), reproducibility, linearity, spiking recovery and correlation with PAM α-amidating activity (Tab. S2). The wide calibration range of the PAM-LIA eliminates the need for sample dilution, which is required for previously reported assays. This advantage becomes especially significant when a large number of samples need to be analyzed. Another advantage of PAM-LIA assay is its one-step protocol, which enables the simultaneous incubation of the target analyte with detection and capture antibodies. In contrast, alternative immunoassays, such as the one described by Sturmer et al. or commercially available ELISA kits, involve more intricate time-consuming procedures that require preliminary antibody-antigen formation steps, sequential incubation of the analyte with capturing antibodies, followed by the washing steps, and subsequent incubation with detection antibodies.The compatibility of the one-step PAM-LIA assay with high-throughput screening processes, such as 96-well format plates, makes it suitable for large-scale clinical automated testing and research. In a sub-cohort of 4850 individuals, we observed the correlation between PAM concentration and various clinical baseline parameters, including SBP and DBP, suggesting its potential for identifying subpopulations at risk for specific health conditions or monitoring intervention effectiveness.28. Our data supports the previously proposed hypothesis that the elevated PAM levels could have several origins. Firstly, the co-secretion of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), with PAM being involved in secretory vesicles formation in the heart atrium29. Secondly, the increased levels of bioactive adrenomedullin secretion, a peptide hormone that is known to be elevated in patients with HF, AF, and other cardiovascular pathologies, and which requires PAM for its activation. We have also observed significant correlation between PAM-LIA and bioactive adrenomedullin concentration in MPP sub cohort  and 494–817 residues (PAL494–817) of human PAM (UniProtKB: P19021-1), respectively, were synthesized and separately expressed in transiently transfected human kidney 293 (HEK293) cell. Both constructs were C-terminally truncated with decahistidine tag. The overexpressed recombinant constructs PHM31–377 and PAL494–817 were purified by nickel affinity chromatography with > 95% sample purity as measured by capillary gel electrophoresis, and used for immunization or screening of hybridoma cells.Soluble PHM and PAL subunits, comprising 31–377 residues   for 30 min at 20 °C in the dark. After quenching the reaction with 5% 1M Tris/HCl, pH 7.0, the MACN-labeled α-PHM antibodies were separated from the unbound MACN by gel filtration via CentriPure P10 column (emp BIOTECH GmbH) and further purified using HPLC column . After supplementation with 5% BSA, the labeled antibodies were stored at − 20 °C.200 µL of 10 µg/mL α-PAL antibodies (2 µg/well) were dissolved in 50 mM Tris/HCl, 100 mM NaCl, pH 7.8 and applied to high-binding polystryrene microtiter plates per well and incubated for 18 h at 4–6 °C. Unbound α-PAL antibodies were discarded. After blocking with 6.5 mmol/L of KHThe assay was calibrated with defined concentrations of commercially obtained recombinant PAM  in a range of 0–723.4 ng/mL, dissolved in 100 mM Tris/HCl, pH 8.0, 3% protease free BSA.2PO4, 185 mM K2HPO4, 100 mM NaCl, 10 mM EDTA, 0.1% w/v unspecified bovine IgG, 0.02% w/v unspecified murine IgG, 50 mM amastatin HCl, 100 µM leupeptin hemisulfate, 0,5% protease free BSA, pH 7,0), containing 50 ng/mL liquid phase antibodies (10 ng/well), were applied to a microtiter 96-well plate , coated with solid phase antibodies. The incubation for 3 h at 22 °C under agitation of 600 rpm followed . Unbound labeled antibodies were removed by washing 5 times with 350 µL standard washing buffer (sphingotec GmbH). Chemiluminescence emission was detected for 1 s with Centro LB 960 microtiter plate luminescence reader (Berthold Technologies).20 µL of EDTA plasma sample/calibrator in duplicates and 200 µL assay buffer  and five samples spiked with recombinant PAM (range 51.1–305.9 ng/mL). Prior the measurements samples were subdivided into aliquots and stored at − 80 °C. The measurements were performed over the period of 5 days with three runs per day in a total of 15 independent assays, conducted by three operators. For one run one microtiter plate was used for calibrators and samples, each in duplicates. The limit of quantification (LoQ), defined as the concentration of PAM that can be reliably measured with a coefficient of variation (CV) of 20%, was determined using GraphPad Prism 8.4.3. To this end, twelve EDTA plasma samples were selected, covering a concentration range of 75.3 pg/mL to 305.9 ng/mL. Each sample was measured in duplicates by three different operators in 15 independent assays. The CV (%) was calculated for each measurement and plotted against the corresponding PAM concentration. The LoQ value was then determined by interpolating from dose–response curve and represented the PAM concentration at which the CV reached 20%. The limit of detection (LoD) was determined by Analyse-it software, utilizing 55 independent measurements of plasma samples depleted from PAM , and 32 samples with a low analyte concentration ranging between 114 and 308 pg/mL. Nonparametric estimation of normal quantile with an error of 5% was used Fig. .The intra-assay precision was determined by concentration measurement of three EDTA samples with PAM concentration of 1.4 ng/mL, 66 ng/mL and 117 ng/mL. PAM concentration in each sample was determined as 16-times iteration within of a single assay.Spiking recovery (accuracy) was assessed by adding known concentrations  of synthetic full-length PAM  to the analyte-depleted EDTA plasma (zero-matrix) and comparing the detected PAM level to expected concentrations. Each concentration was measured in quadruplicate. To generate zero-matrix, EDTA plasma was incubated with α-PAL and α-PHM antibodies, coupled to a ultralink hydrazine resin (ThermoFischer Scientific), for 4 h at 4–6 °C. The remaining PAM concertation in the analyte free matrix was < 250 pg/mL, when measured in PAM-LIA assay under standard protocol conditions.For dilution recovery (linearity), three pools of EDTA plasma were 10% stepwise diluted with analyte-low matrix. Plasma pool concentrations were 674.7 ng/mL and 323.5 ng/mL, when spiked with recombinant PAM, and 91.2 ng/mL, containing the endogenous PAM of several self-reported healthy individuals. The zero-matrix was generated as described in spiking recovery experiments.For mixing recovery, eight EDTA plasma samples with predetermined PAM concentration in a range between 1.7 and 481.1 ng/mL were mixed in a 1 to 1 ratio in random manner, resulting in total of 19 combined pools. The deviation between the expected and measured PAM concentration was determined.To access the substrate interference, two EDTA plasma pools  were mixed with 32 substances in a significant molar excess in respect to analyte (Tab. S1), incubated for 1 h at RT and measured according to a standard assay protocol. The samples were measured in quadruplicate. Recovery calculations were corrected for the effect of volume increase after supplementation and deviations to the not supplemented samples were calculated.To determine the freeze–thaw stability under ex vivo conditions, the six EDTA-plasma samples from self-reported healthy individuals underwent up to 6 freeze–thaw cycles. The samples were stored at − 80 °C for at least 24 h prior the following freeze–thaw cycle to occur.To access the operative temperature range, eight EDTA plasma samples in a range between 1,8 ng/mL to 466.4 ng/mL were measured in duplicate according to a standard assay protocol at 18 °C, 20 °C, 22 °C, 25 °C, 28 °C, 32 °C. The PAM concentration determined at 22 °C was set as a reference point.The acceptance criterion for all technical assay parameters was ± 20% CV from the original or expected concentration.28. Correlations were calculated as Spearman rank correlation. p values < 0.05 were considered significant.To determine the matrix effect on PAM concentration, PAM-LIA was measured in matched EDTA plasma and serum samples, as well as in matched EDTA plasma and Li-Heparin samples from 31 and 70 healthy self-reported individuals, respectively. Additionally, PAM amidating activity were measured in Li-Heparin samples, in order to investigate the correlation between the soluble PAM activity and PAM concertation. PAM activity was measured in house as described in Kaufmann et al.47) were measured in serum. Previously, detailed descriptions were provided for the methods used to assess baseline cardiovascular risk factors and for linking records with the Swedish national inpatient and cause of death registries to retrieve endpoints related to the incidence of major adverse cardiovascular events (MACE), atrial fibrillation (AF) and congestive heart failure (HF)50. Written informed consent was obtained from all participants and/or their legal guardians before entering MPP-RES. The study was approved by The Regional Ethical Review board at Lund University, Sweden (LU 244-02) and complied with the Helsinki Declaration (IRB = 2009/633).For normal distribution and other baseline characteristics of PAM concentration in healthy population, the sub cohort of randomly selected 4850 individuals from the Swedish single-center prospective population based study  according to PAM quartiles were performed using one-way ANOVA. In order to assess the incidence of HF, AF, and MACE during the 16-year follow-up period with respect to PAM concentration, the Cox Proportional Hazards Model was used. The individuals with prevalent coronary event and stroke were excluded. The model was adjusted for baseline risk factors including age, gender, BMI, systolic blood pressure, diastolic blood pressure, glucose, diabetes, LDL, HDL, total cholesterol, and triglycerides. The incidence of Alzheimer´s disease was accessed during the 7-year follow-up period in MPP sub-cohort, comprising n = 3886 individuals . The individuals with prevalent AD were excluded. The cut-off value of 88.2 ng/mL were used. The model was adjusted for baseline risk factors as described above.51. Distribution was tested with the D'Agostino-Pearson omnibus test.All statistical analysis were carried out with GraphPrism 8.4.3 and Analyse-it software. Graphics were prepared using ChimeraSupplementary Information.


To determine the role of magnetic resonance imaging (MRI)-based metrics to quantify myocardial toxicity following radiotherapy (RT) in human subjects through review of current literature.Twenty-one MRI studies published between 2011-2022 were identified from available databases. Patients received chest irradiation with/without other treatments for various malignancies including breast, lung, esophageal cancer, Hodgkin’s, and non-Hodgkin’s lymphoma. In 11 longitudinal studies, the sample size, mean heart dose, and follow-up times ranged from 10-81 patients, 2.0-13.9 Gy, and 0-24 months after RT (in addition to a pre-RT assessment), respectively. In 10 cross-sectional studies, the sample size, mean heart dose, and follow-up times ranged from 5-80 patients, 2.1-22.9 Gy, and 2-24 years from RT completion, respectively. Global metrics of left ventricle ejection fraction (LVEF) and mass/dimensions of cardiac chambers were recorded, along with global/regional values of T1/T2 signal, extracellular volume (ECV), late gadolinium enhancement (LGE), and circumferential/radial/longitudinal strain.LVEF tended to decline at >20 years follow-up and in patients treated with older RT techniques. Changes in global strain were observed after shorter follow-up (13±2 months) for concurrent chemoradiotherapy. In concurrent treatments with longer follow-up (8.3 years), increases in left ventricle (LV) mass index were correlated with LV mean dose. In pediatric patients, increases in LV diastolic volume were correlated with heart/LV dose at 2 years post-RT.Regional changes were observed earlier post-RT. Dose-dependent responses were reported for several parameters, including: increased T1 signal in high-dose regions, a 0.136% increase of ECV per Gy, progressive increase of LGE with increasing dose at regions receiving >30 Gy, and correlation between increases in LV scarring volume and LV mean/V10/V25 Gy dose.Global metrics only detected changes over longer follow-up, in older RT techniques, in concurrent treatments, and in pediatric patients. In contrast, regional measurements detected myocardial damage at shorter follow-up and in RT treatments without concurrent treatment and had greater potential for dose-dependent response. The early detection of regional changes suggests the importance of regional quantification of RT-induced myocardial toxicity at early stages, before damage becomes irreversible. Further works with homogeneous cohorts are required to examine this matter. Cancer and cardiovascular disease are the two most common causes of death globally, taking an estimated 9.6 million and 17.9 million lives each year, respectively . The conIn this review, we focus our primary attention on RT-induced myocardial toxicity (MCT) evaluated by cardiac magnetic resonance (CMR) imaging. To provide background, a brief description of the basic pathophysiology and risk factors for developing RT-induced myocardial injury is presented first, followed by a brief review of currently available imaging modalities for evaluating cardiovascular toxicity. We then discuss in detail key CMR-based quantitative metrics to assess MCT and conclude with a discussion on the benefits of each metric, the need for high quality image registration, and suggestions for future work.RT-induced MCT results from diverse mechanisms leading to multiscale effects on cardiac structure and function. In brief, on the subcellular level, oxidative stress and the inflammatory response within endothelial cells are responsible for the production of numerous cytokines ) associated with RT-induced cardiotoxicity  , 11. HigOn the cellular and matrix level, histological evaluation of MCT is frequently associated with fibrosis within the myocardium . Though The cumulative effects of these subcellular, cellular, and matrix changes are responsible for early  and latIn addition to the length of time since RT, several other risk factors have been shown to increase the risk of developing RT-induced MCT . First, Due to the clinical significance of developing cardiovascular complications and the extended period of time over which they may occur, careful and repeated monitoring for evidence of MCT is essential. The variably progressive nature of RT-induced MCT, which can lead to irrevocable pathological remodeling if unmitigated, creates a specific clinical need for detecting cardiovascular dysfunction at its earliest  stages when medical interventions might provide the greatest benefit. Early complications like constrictive pericarditis (preceded by shortness of breath symptom) may appear within one year after RT  with potBoth highly available and generally affordable, echocardiography is commonly used to measure cardiac left ventricular ejection fraction (LVEF), strain, and strain rate, as well as diastolic function, valvular function, vascular flow, and large vessel pathology . DespiteA second imaging technique for assessing MCT by measuring LVEF is through multiple-gated acquisition (MUGA) nuclear scanning . MUGA isCardiac positron emission tomography (PET) is another form of nuclear imaging which has been proven to be an effective modality for assessment of myocardial perfusion, fibrosis, and inflammation with higher sensitivity to early changes compared to echocardiography . HoweverIn general, nuclear imaging has the drawback of requiring radiation exposure to the patient, though the added risk to cancer patients  is less clear. Thus, nuclear imaging can be a beneficial tool in the diagnosis of MCT after a thoughtful risk-benefit analysis for a given patient.Cardiac computed tomography (CT) is a third modality used for structural and functional cardiac assessments and has particular strength in evaluating coronary artery disease . Other pFinally, a fourth imaging modality, and the focus of this review, is cardiac magnetic resonance. CMR has been described as the gold standard method to assess LV function . CMR canSome of the cardiovascular metrics capable of evaluation by CMR include: the dimensions/volume/mass of the heart and relevant substructures , left atrium (LA), and right atrium (RA)), tissue characterization  using T1/T2 parameters and LGE, regional and global quantification of myocardial contraction using various 2D/3D strain techniques  over circumferential, radial and longitudinal directions, and quantification of flow in terms of velocity, pressure drop, and shear stress on the LV endocardium and aortic wall , 34, 39.The focus of this review will be on studies that have used CMR techniques to quantify MCT metrics in patients who have undergone RT. Though clearly important, a full review of chemotherapy-induced cardiotoxicity (without RT) and evaluation of cardiotoxicity metrics using other imaging modalities or biomarkers is beyond the scope of this article. We point the interested reader to numerous informative prior reviews , 40–42. Figure LVEF is the most common screening tool to quantify global cardiac function and is defined as the stroke volume divided by the end diastolic volume. Normal LVEF ranges between 50-70% . In termP<0.001) at a median of 20 years post-treatment [A decrease in LVEF was reported in a few studies. In 31 patients with Hodgkin’s disease who were treated with mediastinal RT, LVEF dropped below 55% in 23% of patients at 24 years post-RT treatment . Also, lreatment . Finallyreatment . No measp=0.059); however, patients who underwent 3DCRT with/without chemotherapy demonstrated a temporary decrease of LVEF which was resolved at 12- and 24-months. Interestingly, for the IMRT group, the LVEF increased at 24-months follow up  [LVEF stayed within normal limits in multiple studies. In 20 breast cancer patients who received 3D conformal RT (3DCRT), a median LVEF of 63% was reported at 8.3 years . SimilarRecent RT-induced MCT studies showed no significant changes of LVEF and/or relations between LVEF and dose mostly due to short follow-ups , 55, lowA limited number of studies investigated the dimensions and mass of different cardiac chambers to find possible early changes of subclinical myocardial dysfunction and associated dose-dependency.p=0.01) [Increase of cardiac volumes and dose-dependency were noted in a few studies. In 3/11 patients with esophageal cancer, LV systolic volume increased at 3-5 months following neoadjuvant chemoradiotherapy . An incrp=0.01) .2, normalized to body surface area) at 2 years was correlated with the radiation dose to 98% of the heart volume  and LV  [p=0.012), V10 , and V25  at a mean of 8.3 years in 20 breast cancer patients who underwent anthracycline-based chemotherapy and 3DCRT [Dose-dependency response was found in two studies. In 81 pediatric cancer patients  who were treated with chemotherapy and irradiation, increase in indexed LV end-diastolic volume (2.1±6.5 ml/m P<0.05) . In addind 3DCRT .The dimensions of cardiac chambers stayed within normal range in two studies with breast cancer populations. In the first study with 49 patients who underwent IMRT, 3DCRT with/without chemotherapy, LV and RV volumes did not change over 24 months follow-up . In the P<0.06) compared to healthy controls [P<0.05), while only RV systolic volume and RA diastolic volume dropped significantly at 13±2 months (P<0.05). For those who underwent RT treatment, RV and LV volumes were reduced at both therapy completion and 13±2 months (P<0.05) without affecting the atrial dimensions [Decreases of cardiac chambers were shown in two studies. At 20-year follow-up of 80 HL/NHL survivors who were treated by mediastinal RT with/without chemotherapy, a drop in RV volumes and LV diastolic volume/mass were noted  .T1, or longitudinal relaxation time, is a biological parameter that quantifies the time required for nuclei of hydrogen atoms to recover towards thermodynamic equilibrium along the main magnetic field. It can be measured either globally or locally, which allows for quantification of heterogenous T1 distribution. T1 values differ based on local molecular environment , as well as sex, age, and other parameters. Normal native T1 values of the myocardium acquired using a modified Look-Locker inversion recovery (MOLLI) method at 1.5T and 3T scanners were reported as 950±21 ms and 1052±23 ms, respectively . NotablyP <0.01) and 1.5 years  compared to the baseline (1183±46 ms) at the basal septum (a highly irradiated area (43±4 Gy)). However, no correlation was found between regional radiation dose and percent change of T1 at the basal septum [p=0.01) [Increase of T1 signal was reported in a few studies. In 24 survivors of esophageal cancer who were treated with chemoradiotherapy, increase of T1 value was noted at 0.5 years  .p=0.054) with no dose-dependent response at 46.6 months following chemoradiotherapy [p=0.4) at 67.6 months [The majority of studies found T1 values within normal range. During RT of 10 patients with different thoracic malignancies, no T1 signal changes were noted at half-therapy (956±14 ms) and at therapy completion (968±72 ms), compared to baseline (966±39 ms) . A similotherapy . T1 sign6 months .Fluctuation of T1 values over different follow-up times was noted in a single study of 66 patients with breast cancer who were treated by epirubicin-based chemotherapy followed by RT or with left-sided RT alone. T1 value increased at therapy completion in the group receiving epirubicin-based chemotherapy followed by RT and returned to baseline at 13±2 months, while the group receiving left-sided RT only demonstrated no significant changes in T1 at both follow-ups .p=0.022 and at 3 months, -23 ms, P<0.001) [Decrease of T1 signal was found in 51 patients with breast cancer post-RT treatment  . A summaDifferent behaviors of T1 signal  were reported following cancer treatment. Increase of T1 was noted in longer follow-ups , regionaSimilar to T1, T2 (or transverse relaxation time) is a biological parameter that is tissue-specific. Normal T2 values of myocardium acquired from steady-state free precession (SSFP) technique ranged between 52.18±3.4 ms and 45.1 ms at 1.5T and 3T scanners, respectively. T2 changes are mostly associated with change of water content in the tissue. The main pathology associated with longer T2 values is myocardial edema .p=0.13) [In RT-involved treatments, T2 values were reported to be within normal range. At 24 years follow up of 80 HL and NHL survivors who were treated with mediastinal RT with/without chemotherapy, T2 values did not differ from healthy controls  . Similarp=0.13) .P<0.001) and returned to baseline at 13±2 months (46 ms) [In 66 breast cancer patients with two different treatments, T2 stayed unchanged in the left-sided RT group at the end of therapy and 13±2 months post-RT (46-47 ms); however, in patients who underwent epirubicin-based chemotherapy followed by RT , T2 increased at therapy completion  . Table 3T2 signal stayed unchanged over longer follow-ups   component of the myocardium. One of the distinct features of myocardial pathologies  is the expansion of this extracellular space. Quantitative evaluation of extracellular expansion is now possible by acquisition of the hematocrit and pre- and post-T1 values of myocardium and blood pool ). Equation Normal myocardial ECV values of 25±4% and 26±4% at 1.5T and 3T, respectively, have been reported , 61. It P<0.01) was noted at 6 months post chemoradiotherapy in 24 patients with esophageal cancer in high dose regions (43±4 Gy) [P<0.001)) at 67.6 months [Increase of ECV and its dose-dependency have been reported in a few recent studies. Increase of ECV  . Segment6 months . Lastly,6 months .p=0.24) [No significant change and/or relation between ECV and dose were also reported in a few studies. In 80 HL and NHL survivors who were treated by mediastinal RT with/without chemotherapy, ECV did not differ compared to healthy controls in patients with 20-year follow-up  . Similarp=0.24) . Over shp=0.24)  and amonp=0.24) . A 6-monp=0.24) . Table 4P <.001) [Global analysis of ECV  and shorP <.001) . FurtherLGE MRI is a standard non-invasive method for assessing ischemic and nonischemic myopathic processes with high spatial resolution . NotablyP<0.01) among 24 esophageal cancer patients [Enhanced LGE volumes were reported in a few studies. In a study of patients with Hodgkin’s disease survival of at least 20 years, 29% of 31 patients demonstrated late enhancement 24 years following mediastinal RT . Similarpatients . Also, ipatients .P<0.05) was correlated with the dose to 20% of LV volume  [A few studies showed dose-dependent LGE response. In 12 out of 24 esophageal patients, LGE was detected in 15.38% of AHA segments receiving 40 Gy and in 21.2% of AHA segments receiving 60 Gy at 23.5 months post-RT . In anot P<0.05) .p=0.03) and between the ratio of LA scar-to-wall volume and dose (P<0.01) at 3.1 years [Lastly, one study was done on evaluation of RT-induced LA chamber enhancement at 3.1 years among 7 patients with lymphoma and esophageal cancer who were treated with RT. They found that there is a linear relationship between the LA mean dose (25.9 Gy) and its scar volume (2.5 cm3)  and 1.5 year  post chemoradiotherapy in 19 patients with esophageal cancer . A summaGlobal changes of LGE were mostly noted over longer follow-ups (>20 years) , particuSeveral studies did not find any signs of LGE, potentially due to small sample size  , low radCardiac function is a combination of contraction, twist, and expansion of myocardium in multiple axes. Regional and global quantification of myocardial deformation  in any of these axes may serve as a potential metric to detect sub-clinical myocardial changes and assess myocardial function. Strain can quantify spatial components of contractile function over multiple directions . With regards to the LV, MRI-derived strain measurements in healthy subjects have reported pooled mean values of global longitudinal strain (GLS), global circumferential strain (GCS), and global radial strain (GRS) as -18.6% (-19.5% to -17.6%), -21.0% (−22.4% to -19.6%), and 38.7% (30.5% to 46.9%), respectively . NotablyP<0.05) of the 16 segment AHA model, as well as a negative correlation between the magnitude of longitudinal strain and dose in segment 6 (P<0.01) [In 22 breast cancer patients who underwent RT, decrease in the magnitude of circumferential, radial, and longitudinal strains were noted in segmental regions 6 weeks post-treatment, with a negative correlation between radial strain and maximum dose in segments 6 and 14 ((P<0.01)  , no correlation was found between GLS/GCS/GRS and dose , 50, 55.To evaluate the effects of radiation to the heart and its substructures, it is important to accurately align the planning CT  with magnetic resonance images that are more sensitive in detecting MCT across different time points, patient positions, and respiratory and cardiac phases. Without accurate registration of the dose and the MRI, accurate analyses of dose-effect correlations are not feasible. Lacking quantitative registrations, the uncertainties associated with simple visual comparisons or simplistic registrations between dose and MRI likely contribute to the overall variation between different studies, time points, tumors, and measurement techniques of the relevant parameters.Recent studies have shown that mono- and multi-modality image registration of the heart, LV and thoracic aorta on CT and MR datasets in the axial plane at various time points, breathing phases, and contrast levels can comply with American Association of Physicists in Medicine (AAPM) Task Group (TG) 132 criteria (mean distance to agreement (MDA) < 3 mm and Dice > 0.8) . It shouImage registration is a transformation process in which the features from one coordinate space are mapped onto another space. One image is considered as reference (static) while the other images move/deform based on the reference image with the following strategic approaches: spatial transformation , interpolation (intensity-based or object-based), similarity measurements , and finally optimization . Proper selection of the image registration workflow depends on the modalities, dimensions, and degree of deformation. Accurate image registration can provide complementary information from two different datasets to improve therapeutic decision-making and to examine spatially registered cross-modality correlations –79.Despite the importance of image registration, particularly in the quantitative mapping of CT-derived RT dose onto MRI datasets, few studies have incorporated image registration in their work . As a reMRI is considered a ‘gold standard’ modality for left ventricular volume quantification  and has Global metrics  have been shown to detect myocardial changes at longer follow-ups >20 years)  when dam years  wRegional metrics have been shown to have more potentials to detect early changes and/or dose-dependencies. For example, segmental strain analysis over short follow-ups  has shown a dose-dependent response  since hiImportantly, unlike systemic therapy , where a more homogeneous distribution of the drug is assumed over the entire heart, radiation dose distribution is clearly spatially heterogeneous, with local cardiac doses depending on the proximity of the tumor to the heart . Accurately accounting for these dose variations requires careful regional assessments of the heart for each unique patient and treatment plan. As a result, patient-specific multimodal image registration (from planning CT to MRI) will play a key role in accurately quantifying cumulative regional doses across the beating heart and determining local correlations between clinically useful MRI-based metrics and RT dose. In summary, a compact list of important findings is provided in Fig. The findings in this review suggest that global MRI-based metrics primarily detect RT-induced MCT only after long follow-ups , with the exception of pediatric patients that may show changes in relevant metrics over shorter follow-ups within 1-2 years. Since the radiation delivered during RT is patient-specific and highly spatially heterogeneous depending on the particular type and location of the tumor, the dose to regional cardiac structures is also heterogeneous. Thus, regional quantifications of both dose and relevant MRI-based metrics may be required for an accurate assessment of potential MCT. Furthermore, since the likelihood of recovery or mitigation of the effects of MCT decrease with time since undergoing RT, regional assessments of cardiac structure and function  offer the potential of detecting early subclinical changes before changes in global metrics are measurable.Some of the major regional myocardial findings on MRI currently reported in the literature include an increase of T1 signal at high-irradiated regions , decreasTo optimally detect these regional changes and quantify potential dose–effect relationships locally requires accurate image registration between planning CT  and various MR sequences that have the ability to detect local subclinical abnormalities. In conclusion, the continual advancement of quantitative cardiovascular MR techniques, the ability to plan and control precise spatiotemporal delivery of therapeutic radiation, and our growing understanding of the evolving effect of localized RT doses on regional and global cardiovascular dysfunction offers significant potential to improve the cardiovascular outcomes of the myriad patients undergoing radiotherapy each year by aiding in optimal therapeutic planning, the early diagnosis of impending cardiovascular toxicity, and the clinical evaluation/monitoring of novel interventions or alternate treatment regimens.


LVNC patients had higher NT-proBNP [237 (156–489) vs. 156 (139–257) pg/mL] and Galectin-3 [7.3 (6.0–11.5) vs. 5.6 (4.8–8.3) ng/mL], and lower ADAMTS13 (767.3 ± 335.5 vs. 962.3 ± 253.7 ng/mL) and ADAMTS13/vWF ratio . Conclusion: LVNC patients with HFpEF have diffuse fibrosis, which is more extensive at the apical level, explaining the decrease in apical deformation and overexpression of Galectin-3. Lower transmural and base-to-apex deformation gradients underpin the sequence of myocardial maturation failure. Endothelial dysfunction, expressed by the lower ADAMTS13 and ADAMTS13/vWF ratio, may play an important role in the mechanism of HFpEF in patients with LVNC.Left ventricular non-compaction (LVNC) with preserved ejection fraction (EF) is still a controverted entity. We aimed to characterize structural and functional changes in LVNC with heart failure with preserved EF (HFpEF). Methods: We enrolled 21 patients with LVNC and HFpEF and 21 HFpEF controls. For all patients, we performed CMR, speckle tracking echocardiography (STE), and biomarker assessment for HFpEF (NT-proBNP), for myocardial fibrosis , and for endothelial dysfunction . By CMR, we assessed native T1 and extracellular volume (ECV) for each LV level . By STE, we assessed longitudinal strain (LS), globally and at each LV level, base-to-apex gradient, LS layer by layer, from epicardium to endocardium, and transmural deformation gradient. Results: In the LVNC group, mean NC/C ratio was 2.9 ± 0.4 and the percentage of NC myocardium mass was 24.4 ± 8.7%. LVNC patients, by comparison with controls, had higher apical native T1 (1061 ± 72 vs. 1008 ± 40 ms), diffusely increased ECV (27.2 ± 2.9 vs. 24.4 ± 2.5%), with higher values at the apical level (29.6 ± 3.8 vs. 25.2 ± 2.8%) (all  Left ventricular non-compaction (LVNC) is characterized by the morphologic findings of a bilayer myocardium, with hypertrabeculation of the endocardial layer, a usually thin compacted epicardial layer, and the presence of deep intertrabecular recesses that communicate with the left ventricular (LV) cavity . LVNC caMyocardial compaction gradually progresses from epicardium to endocardium and from base to apex ,8. The mWe aimed to characterize structural and functional changes in patients with LVNC and HF with preserved ejection fraction (HFpEF), by comparison with patients with HFpEF but without LVNC, by using cardiac magnetic resonance (CMR), echocardiography with speckle tracking, and biomarkers.In this prospective, single-center study, we enrolled 21 ambulatory patients over 18 years of age and in sinus rhythm, who fulfilled diagnostic criteria for LVNC and HFpEF . We defi2 in females and >115 g/m2 in males, relative wall thickness (RWT) > 0.42, left atrial volume index (LAVI) > 34 mL/m2)] and/or functional abnormalities  consistent with the presence of LV diastolic dysfunction/raised LV filling pressures, including raised natriuretic peptides [N-terminal pro-B-type natriuretic peptide (NT-proBNP) > 125 pg/mL). We used as a control group 21 patients with similar age and sex, with diagnostic criteria for HFpEF but no echocardiographic and CMR criteria for LVNC. For all patients, we performed clinical examination, 12-lead electrocardiogram, and 2D echocardiography (2DE) with STE, CMR, and blood tests. Exclusion criteria were: recent hospitalization (<4 weeks) for acute HF, acute or chronic coronary syndromes, pericardial diseases, previous history of myocarditis, significant valvular heart diseases, hypertrophic cardiomyopathy, Fabry disease, amyloidosis, renal failure with hemodialysis, moderate or severe anemia, sustained atrial arrhythmias, inappropriate quality of echocardiographic images for STE analysis, and contraindication for CMR. Informed consent was obtained in all subjects prior to enrolment. The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committee of the University and Emergency Hospital .HFpEF was defined according to the current guidelines’ diagnostic criteria : presenc-mean value of native T1 for all segments , for basal , mid (T1 mid), and apical  segments, and the gradient between apical and basal T1 (T1 base-to-apex gradient);-mean value of ECV for all segments , for basal , mid (ECV mid) and apical  segments, and the gradient between apical and basal ECV (ECV base-to-apex gradient).CMR was performed using a 1.5-T MR scanner . CMR analysis was performed by a single cardiac expert radiologist (A.I.N), using the syngo.MR Cardiology VB20A post-processing software . Steady-state free precession cine images and late gadolinium enhancement (LGE) images were acquired according to the current recommendations . Left veConventional transthoracic echocardiography (TTE) was performed using a commercially available ultrasound system  with a 3.5 MHz transducer. Standard images were acquired and digitally stored for offline analysis using a vendor-specific software . The quantification of the cardiac chamber size and function was performed based on the current guidelines’ recommendations . LVEF waSTE analysis was performed by two observers  according to the current recommendations . The echBlood samples were collected into syringes pre-loaded with EDTA. Two blood test tubes were obtained from each patient. They were immediately centrifuged for 15 min at 4000 rpms to separate serum (2.5–3 mL of serum/patient). Serum was stored at −80 °C before being analyzed for the measurement of specific biomarkers for HF (NT-proBNP), myocardial fibrosis , and endothelial dysfunction . Plasma vWF, ADAMTS13, and Galectin-3 levels were determined using the sandwich ELISA method with available commercial kits, according to the manufacturer’s instructions.t-tests. Non-normally distributed continuous variables were presented as median and interquartile range (IQR) and compared using the Mann–Whitney U test. Categorical variables were expressed as percentages and compared using Pearson’s Chi-square test. Correlation between continuous variables was performed using Pearson’s or Spearman’s correlation coefficient, as appropriate. A p-value < 0.05 was considered statistically significant.All statistical analyses were performed using the SPSS version 21.0 . The normal distribution of the continuous variables was tested by the Shapiro–Wilk test. Normally distributed continuous variables were reported as mean ± SD and compared for statistical significance using independent sample We screened 47 patients potentially having LVNC, with 26 patients being excluded: 5 patients with <4 prominent trabeculations along the LV endocardial border, 6 patients without HF, 11 patients with LVEF < 50%, 2 patients with poor image quality, 1 patient with severe aortic regurgitation, and 1 patient who refused to undergo CMR. The characteristics of the study groups are summarized in p = 0.63), being found in 4 LVNC patients (19%) and in 3 control patients (14%), in both groups in the mid-basal segments.In the LVNC group, native T1 values were significantly higher only at the apical level, whereas ECV was globally expanded, mainly at the apical level . Both T1LS apical was lower in the LVNC group, by comparison with the control group , due to In the LVNC patients, NT-proBNP and Galectin-3 were higher, whereas ADAMTS13 and the ADAMTS13/vWF ratio were lower .p = 0.01) and ECV base-to-apex gradient , while compact myocardium mass correlated negatively with the ECV base-to-apex gradient . These correlations suggest that fibrosis is higher when the degree of hypertrabeculation increases and the compact myocardium mass decreases.In the LVNC patients, the percent of NC myocardium mass correlated positively with T1 base-to-apex gradient  and NT-proBNP . Endothelial dysfunction biomarkers correlated positively with compact myocardium mass . These findings indicate that endothelial dysfunction is greater when compacted myocardium mass decreases in LVNC patients.In the LVNC patients, Galectin-3 correlated positively with native apical T1 , with similar age, sex, and comorbidities. By using a multimodality approach, we showed that patients with LVNC and HFpEF have endothelial dysfunction, myocardial fibrosis, and significantly decreased longitudinal strain in the apical non-compacted LV segments, associated with increased ECV and T1 myocardial times in CMR . Our finThis is the first study that showed lower levels of ADAMTS13 in LVNC patients, with a reduced ADAMTS13/vWF ratio, suggesting that endothelial dysfunction may play an important role in the pathogenesis of LVNC. Endothelial cells make a major contribution to ADAMTS13 production ; thus, lEndothelial dysfunction may also play a role in the mechanism of LVNC thromboembolic complications, since low ADAMTS13 levels were associated with an increased risk of arterial thrombosis and ischemic stroke .We found higher levels of Galectin-3 in patients with LVNC and HFpEF. To our knowledge, this is the first study that has evaluated the level of Galectin-3 in LVNC. Galectin-3 plays a dominant role in the fibrotic processes, being released into extracellular space, where it activates the fibroblasts . It has Our results are in accordance with previous CMR studies in LVNC ,33 that Although we found diffusely expanded ECV, LGE was present in only four patients with LVNC (19%), and surprisingly in the medio-basal compacted segments. Compared to other cardiomyopathies, LGE seems to be less frequent in LVNC ,36, and Mechanisms of fibrosis in LVNC are unclear and may be multifactorial. Our data showed the presence of endothelial dysfunction in LVNC patients, which can cause CMD with myocardial ischaemia, promoting diffuse fibrosis. Diminished coronary flow reserve, supported by subendocardial perfusion defects , has beeIn our study, LVNC patients had lower values of LS in the apical segments. CMR findings suggested that diffuse apical fibrosis may be the substrate of lower LS values in the apical non-compacted segments. We also found a compensatory increase in LS in basal and mid-inferior segments, with lower values of base-to-apex LS gradient. In agreement with our findings, previous studies reported reduced LS in LVNC patients with preserved EF, when compared with normal subjects ,42. BellLVNC is also different from other cardiomyopathies. Thus, patients with LVNC with reduced EF showed lower apical LS values than patients with HCM, with a reduced base-to-apex deformation gradient . By compIn LVNC, trabeculations are commonly localized in the endocardial layers, as the myocardial compaction progresses from epicardium to endocardium. By assessing multilayer LS, we found lower values of LS in the endocardial layers, with significantly lower transmural LS.Our findings suggest that subclinical myocardial fibrosis is the substrate of decreased apical myocardial deformation. Subclinical myocardial fibrosis might be monitored by Galectin-3. Thus, reducing Galectin-3 level might be a new therapeutical target to prevent myocardial fibrosis. Furthermore, if endothelial dysfunction is validated in future large-scale studies, ADAMTS13 level and the ADAMTS13/VWF ratio might be used in clinical practice to monitor the progression of endothelial dysfunction, and these biomarkers might become other therapeutical targets in patients with LVNC. Finally, the assessment of longitudinal strain by speckle tracking in patients with LVNC should become mandatory for diagnosis and for monitoring treatment.There are several limitations of our study. First, this is a single-center study with a rather limited number of patients. However, LVNC is a rare disease, and we included symptomatic patients with LVNC and HFpEF, carefully diagnosed by strict criteria, and compared them with matched patients with HFpEF. Considering the reduced number of patients, this study is hypothesis-generating research that needs validation in a large-scale study, ideally by an international multicenter collaboration. Second, there is a limited number of men in both groups, due to the mandatory presence of a diagnosis of HFpEF (by protocol), which is more frequent in women than men. Third, asymptomatic coronary artery disease was not ruled out by either coronary angiography or computed tomography. Finally, genetic testing was unavailable; we consider that it would have been important to corelate our findings with a genetic diagnosis.Patients with LVNC and HFpEF had endothelial dysfunction, expressed by a lower ADAMTS13 level and ADAMTS13/vWF ratio, and the overexpression of Galectin-3. These lead to diffuse myocardial fibrosis, more extensive at the apical level, explaining the decrease in apical deformation. The positive correlation of NC myocardium with ECV base-to-apex gradient, together with the decrease in transmural and base-to-apex deformation gradients, reinforces the hypothesis of myocardial maturation failure and its sequence. All these findings support the idea that LVNC with HFpEF may be a distinct phenotype, rather than an adaptative hypertrabeculation process.


Diabetes predisposes affected individuals to impaired myocardial perfusion and ischemia, leading to cardiac dysfunction. Increased myocardial stiffness is an independent and significant risk factor in diastolic dysfunction. This study sought to estimate myocardial stiffness in Type 2 diabetes (T2DM) patients using the intrinsic wave velocity propagation (IVP) along the longitudinal wall motion during late diastole and evaluate the value of IVP in assessing cardiac function and structure.87 and 53 participants with and without T2DM (control group) were enrolled. Of the 87 T2DM patients (DM group), 43 were complicated with hypertension (DM + H group), and 44 were not (DM-H group). Ultrasound parameters were measured and analyzed, including color M-mode flow propagation velocity, global longitudinal systolic strain (GLS), and IVP.P < 0.001). After stratification for hypertension, IVP in both DM + H (1.71 ± 0.25 m/s) and DM-H (1.53 ± 0.20 m/s) groups were found to be significantly higher than that in the control group (1.40 ± 0.19 m/s); also, the difference of IVP between DM + H and DM-H group reached statistical significance. Moreover, IVP was significantly correlated with flow propagation velocity during early diastole (Pve) , flow propagation velocity during late diastole (Pva) , GLS , interventricular septal thickness at end-diastole (IVSd) , blood glucose , systolic blood pressure  and diastolic blood pressure .IVP was higher in the DM group than in the control group (1.62 ± 0.25 m/s and 1.40 ± 0.19 m/s, The results indicated the application potential of IVP in assessing the early detection of cardiac function changes noninvasively and sensitively. The correlation with myocardial stiffness warrants further studies to substantiate its potential clinical utility. Diabetes is a severe global health problem associated with the increased risk of heart failure . DiabeteNow, the possibility of imaging the heart at very high frame rates allows minute tracking of many wave-like phenomena, such as shear wave elastography technique using acoustic radiation force to mechanically stimulate tissue and monitor the response , 12, natIt was hypothesized that during left ventricular (LV) filling after atrial contraction, the fast traction on the mitral annulus by the atrial contraction generates a wave into the LV, which travels from base to apex with a speed proportional to myocardial elasticity. The propagating wave velocity along longitudinal tissue motion direction is referred to as the Intrinsic Velocity Propagation (IVP) , 17. It This work aimed to explore whether there is a relationship between IVP and diastolic dysfunction and discuss the potential of IVP in assessing MS in T2DM patients.This pilot study was conducted between April 2019 and March 2021 in Tongji Hospital, the most widely circulating hospital in its region. One hundred forty participants were enrolled, with 87 patients diagnosed as T2DM (DM group) and 53 healthy controls (control group). Among the T2DM patients, 43 were complicated with hypertension (DM + H group), and 44 were exempt from other complications (DM-H group). The diagnosis criteria of T2DM were promulgated by WHO: fasting blood glucose (FPG) ≥ 7.0 mmol/L or 2-hour postprandial blood glucose (2hPG) ≥ 11.1 mmol/L. Patients were excluded from the study if they were complicated with valvular heart disease, congenital heart disease, coronary heart disease, cardiomyopathy, and history of cardiac surgery in case of possible confounding; those with poor echocardiographic image quality were also excluded. The control group was chosen from healthy people undergoing a physical examination, including biochemical routines, electrocardiogram, and echocardiography. They had no history of diabetes, hypertension, cardiovascular disease, or cardiac surgery. All experiments were carried out in accordance with the ethical standards put forward by the Declaration of Helsinki and approved by a local institutional review board. Each participant signed written informed consent.Echocardiography was acquired using Vivid E9 commercial scanners  equipped with an M5S transducer at a frequency of 1.7–3.4 MHz. The rate of conventional two-dimensional ultrasound images is ≥70 frames/s. Three consecutive cardiac cycles are stored in each section. Several conventional echocardiographic parameters were measured, including interventricular septal thickness at end-diastole (IVSd), left ventricular posterior wall thickness at end-diastole (LVPWd), left ventricular internal diameter at end-diastole (LVIDd), left ventricular ejection fraction (LVEF), Mitral E velocity, Mitral A velocity, and Mitral annular septal e' velocity. E/A and E/e' ratios were later calculated.The speed scale and scanning speed (100 mm/s) were adjusted to obtain the first-color M-mode aliasing on the apical four-chamber view. Early and late diastolic filling waves on the M-mode map, including flow propagation velocity during early diastole (Vpe) and flow propagation velocity during late diastole (Vpa), were then recorded.EchoPac  analysis software was applied to calculate GLS offline. The movement trajectory of each point in the cardiac tissue during the cardiac cycle was automatically tracked by two-dimensional speckle tracing in apical long-axis, two-chamber, and four-chamber standard view, respectively. The myocardial deformation of each ventricular wall segment in the region of interest was recorded.The tissue Doppler frame rate was adjusted to 350–450 frames/s. To minimize the bias caused by the Doppler Angle, the imaging field of view was adjusted carefully to align each left ventricular wall with the incident ultrasound beam. The ultrasound images of each LV wall were stored in apical long-axis, two-chamber, and four-chamber standard views, respectively. Data were recorded and analyzed using the EchoPac workstation . Q analysis was first entered, then the left ventricular myocardium was traced using the curved anatomy M type (CAMM) to reconstruct the axial tissue velocity map. The ratio of the tissue velocity map was adjusted to create velocity aliasing; the scanning speed was adjusted to the maximum. The velocity aliasing was used to identify the onset of motion. The wave speed, IVP, was the slope of isovelocity wavefront propagating in time and space during late diastole . The meaBland-Altman analysis was applied to test Intra- and inter-observer variability of IVP in 20 randomly selected data sets. Two observers were blind to both results made by each other and the medical history and examination data of each subject.Post hoc analysis was achieved through the Bonferroni test. The correlation between IVP and echocardiographic parameters was tested by Pearson correlation analysis. Bland-Altman analysis was used on 20 randomly selected data sets to analyze inter and intra-observer variability and consistency. P < 0.05 was considered statistically significant.All statistical parameters were performed using IBM SPSS . Continuous data were expressed as mean ± SD; categorical data were expressed as percentages. The normality of data was tested using the Shapiro-Wilk test. Between-group comparison of the mean was made by student's t-test; Among-groups comparisons of normally distributed continuous variables was achieved by One-way ANOVA; comparisons of skewedly distributed variables was done by Kruskal Wallis test. When significant at the 0.05 level, pairwise comparisons were performed, and As was shown in P < 0.001). Such phenomenon was also observed in IVSd , Mitral A velocity , E/A ratio , Mitral annular septal e' velocity , E/e' ratio, Vpe  and GLS(%). After stratification for hypertension (P < 0.001). Compared with the control group, IVSd , Mitral A velocity, E/e' ratio , Vpa , and GLS(%)  were significantly higher, while E/A ratio , mitral annular septal e' velocity , and Vpe  were significantly lower in DM + H group patients. Only Vpe in the DM-H group was significantly lower than in the control group . The differences in IVSd, LVIDd, Mitral E velocity, E/A ratio, E/e' ratio, Vpa, and GLS were observed between the DM-H group and control group. However, they did not reach statistically significant.Overall, the IVP of the DM group was higher than that of the control group , Pva , IVSd , LVIDd , systolic blood pressure , diastolic blood pressure , and GLS . No significant correlation was found between IVP and HbA1c, blood glucose, LVPWd, LVEF, Mitral E velocity, Mitral A velocity, E/A ratio, e' velocity, and E/e’ ratio.The results of correlation analyses were as follows : Pve  The IVP of the DM + H group was higher than that of the DM-H group and control group; (2) Mitral A velocity, E/e’ ratio, and Vpa were higher in the T2DM patients, while E/A ratio, Mitral annular septal e ‘ velocity, and Vpe were higher in the control group; (3) The association between IVP and part of the above-mentioned conventional ultrasound parameters showcased weak to moderate correlation.Myocardial relaxation is critical to the diastolic function that allows adequate filling of the ventricles prior to the next cardiac cycle. The early stages of diabetic cardiomyopathy have subtle changes in cardiac function, one of which is left ventricular (LV) diastolic dysfunction that includes impaired early diastolic filling, increased atrial filling, prolonged isovolumic relaxation, and cardiomyocyte disarray and interstitial myocardial fibrosis through histologic examination, the embodiment of altered myocardial stiffness , 7.The generation of IVP is considered at the beginning of ventricular filling after atrial contraction when the base of the LV is stretched, and a pulse-like wave is generated that propagates to the apex along the longitudinal wall motion during late diastole , 22 PrevMore than two-thirds of T2DM patients are complicated by hypertension . HyperteThe blood flow from the LA to the LV through the mitral valve can be embodied as the wave; the speed produced under the pressure gradient in the ventricle is referred to as the flow propagation velocity (FPV). FPV is a recognized indicator of changes in myocardial mechanical properties. Reduced flow propagation during early diastole is commonly seen in patients with impaired LV relaxation , 28. WheE/e' ratio is one of the well-recognized parameters in evaluating LV filling pressure diastolic function . In thisHere, this work explored the application prospect of IVP in assessing LV diastolic dysfunction in T2DM patients. The results implied that IVP might be potential to evaluate diastolic dysfunction and reflect the change in myocardial stiffness. Different from the generation of acoustic radiation force pulse in shear wave elastography, this novel ultrasound technique detects the wave velocity generated by myocardial stretch from the base to the apex level through high frame rate Doppler ultrasound imaging, which means the external stimulation is not required , 34. TheSeveral limitations deserve to be addressed: The data quality of IVP is affected by time resolution and low frame frequency. The application of this technique is currently unsuitable for patients with arrhythmias or tachycardia. Limited by experimental conditions, in this pilot study, the association of IVP with myocardial stiffness was relatively subjective, for there is currently no direct reference standard. Limited by the small sample size, one possibility is that the group variability may be due to inter/intra-observer variability. However, the trend of IVP value changes could not be ignored and was worthy of further exploration. Myocardial stiffness also can be acutely modulated by proteins such as titin, including post-translational modifications  and isoform switch . The atrThe study showed that IVP was higher in T2DM patients when compared to the control group. This novel index might be a promising echocardiographic parameter in assessing early change in cardiac function and structure, implying the possibility of evaluating myocardial stiffness directly and noninvasively. Further clinical studies with larger sample sizes are warranted to explore the clinically relevant.