Metabolic perturbations and inflammatory mediators playa fundamentalrole in both early and late adverse post-acute ischemic stroke outcomes.Using data from the observational MAGIC  study, we evaluated the effect of 130 serum metabolic features,using a nuclear magnetic spectroscopy approach, on the following outcomes:hemorrhagic transformation at 24 h after stroke, non-response to intravenousthrombolytic treatment with the recombinant tissue plasminogen activator(rt-PA), and the 3 month functional outcome. Blood circulating metabolites,lipoproteins, and inflammatory markers were assessed at the baselineand 24 h after rt-PA treatment. Adjusting for the major determinantsfor unfavorable outcomes , we found that acetone and 3-hydroxybutyrate were associatedwith symptomatic hemorrhagic transformation and with non-responseto rt-PA; while 24 h after rt-PA, levels of triglycerides high-densitylipoprotein (HDL) and triglycerides low-density lipoprotein (LDL)were associated with 3 month mortality. Cholesterol and phospholipidslevels, mainly related to smaller and denser very low-density lipoprotein(VLDL) and LDL subfractions were associated with 3 month poor functionaloutcomes. We also reported associations between baseline 24 h relativevariation (Δ) in VLDL subfractions and ΔC-reactive protein,Δinterleukin-10 levels with hemorrhagic transformation. Allobserved metabolic changes reflect a general condition of energy failure,oxidative stress, and systemic inflammation that characterize thedevelopment of adverse outcomes. There is an urgent need to find biomarkers usefulfor clinical practice and to better understand metabolic dysregulationon the basis of the pathophysiological mechanisms of the disease.In this light, metabolic perturbations are fundamental events thatcontribute to ischemic stroke, its progression, and the developmentof unfavorable outcomes.5 Regarding the knowledge about biomarkers associatedwith poor prognosis in the setting of stroke patients treated withthrombolysis, little evidence has been reported .6 Dyslipidaemia is a risk factor contributing tothe onset of ischemic stroke; high levels of total cholesterol andlow-density lipoprotein (LDL) cholesterol increase the risk for cerebralischemia.8 However, the effects of lipid levels onclinical outcomes after the ischemic attack are controversial: hightotal cholesterol and LDL levels have been associated with betterfunctional and vital outcomes after stroke,10 whilelow LDL levels increased the risk of early symptomatic intracranialhemorrhage11 and low total cholesterolwas related to a worse functional outcome in ischemic stroke patientsafter the thrombolytic treatment.12 Therefore,the effective contribution of lipid levels to stroke outcomes, particularlyafter thrombolysis, needs to be further investigated.Ischemic stroke is a leadingcause of death and disability continuouslyincreasing14 Here, using data of patients enrolled in the MAGIC  study,17 we aimed at providingmetabolic insights underlying susceptibility to early and late post-acuteischemic stroke (AIS) adverse outcomes.In thisframework, nuclear magnetic resonance (NMR)-based metabolomicscan provide crucial information, allowing a high-throughput analysisof various types of samples  and giving informationon various molecular features present in biological matrices.16We also studied theinterplay between statistically significantmetabolomic features and circulating inflammatory markers, such asΔ of metalloproteinase 9 (MMP9), alpha2 macroglobulin (A2M),serum amyloid protein (SAP), C reactive protein (CRP), interleukins(ILs), tumor necrosis factor alpha (TNFα), and monocyte chemo-attractantprotein I (MCPI), resulted to be statistically associated to secondaryintracerebral hemorrhage (sICH), 3 month mortality, and 3 month poorfunctional outcome, as previously described in the publication ofGori et al.An overview of thepresent study design is reported in 22.116 in which 327 patients were enrolled; only subjects for whom serumaliquots were available for metabolomics analysis are considered inthis study (n = 243).Thesubjects and the study samples considered are from the MAGIC studywww.sitsinternational.org), according to the SITS-Monitoring Study criteria,18 in the frame of the national, observational, and multicentricMAGIC study.16The study populationconsidered here is characterized by patients who had an AIS and wereadmitted for thrombolysis treatment with the recombinant tissue plasminogenactivator (rt-PA), in 14 different Italian centers, registered inthe Safe Implementation of Thrombolysis in Stroke International StrokeThrombolysis Register  and 24 h after (t2) the administrationof rt-PA. Adverse outcomes were defined as follows: (i) early outcomes(24 h) characterized by development of symptomatic intracerebral hemorrhage(sICH), according to the National Institute of Neurological Disordersand Stroke criteria,19 and the absenceof clinical response to systemic thrombolysis [<4 point decreaseon 24 h National Institutes of Health Stroke Scale (NIHSS)] and (ii)late outcomes (3 months) characterized by death and disability definedas the modified Rankin scale (mRS), dichotomized into a good  or poor  functional status.The whole study focuseson the analysis of serum samples collectedat two different time points: before . To stabilize approximately at the level of ±0.1K, the sample temperature (310 K), a BTO 2000 thermocouple was employedand each NMR tube was kept for at least 5 min inside the NMR probehead to equilibrate the acquisition temperature of 310 K.Serum samples wereanalyzed using a Bruker 600 MHz spectrometer working at a 600.13 MHzproton Larmor frequency equipped with a 5 mm PATXI 22 allowing the selective detection of different molecular components.Detailed procedures on parameters of NMR experiments are reportedelsewhere.13Foreach serum specimen, three one-dimensional proton NMR spectra  were acquired with different pulse sequences,Raw NMR data were multipliedby an exponential function of 0.3Hz line-broadening factor before the application of Fourier transform.Phase and baseline distortions were automatically corrected, and transformedspectra were calibrated to the glucose doublet at 5.24 ppm using TopSpin3.2 (BrukerBioSpin).2.51H 1D NOESY NMR spectra accordingto Bruker’s B.I.-LISA protocols.23 A complete list of the molecular features analyzed in this studyis presented in Table S1.18 metabolites and 112 lipoproteins wereidentified and estimated from 2.616Levels ofdifferent inflammatory markers have been measured, as previously reported in Gori et al.2.72.7.1t-testand a χ-square test were applied, respectively, for comparisonsincluding continuous variables and categorical variables.For demographics,clinical characteristics, and risk factors, a 2.7.225 was performed to exploredata patterns. Data were scaled to unit variance before analysis.Principalcomponent analysis (PCA)2.7.327 was used to study the potential associationbetween inflammatory markers and metabolomics data at t1 and t2. The significancethreshold has been imposed at a P-value of < 0.05.For completeness, adjusted P-values, using the Benjamini–Hochbergmethod,28 were also reported.Pearson correlationanalysis2.7.4t1), 24 h post (t2) rt-PA, and a single patient’s relative pre-24 h and post-rt-PAvariation [Δ(pre–post)rt-PA] of metabolitesand lipid concentrations.As main explanatory variables, we considered the baseline rt-PA on the four adverse outcomes was estimated bypenalized logistic regression analysis,29 including as covariates different patients’ characteristics,that is, age, sex, baseline, 24 h post-blood glucose , baseline NIHSS, time onset-to treatment, blood collectioncenter, risk factors, and comorbidities .The statistically robust effect ofeach variable at P-values, obtained using the Benjamini–Hochbergmethod,28 were also reported. Since correctionfor multiple testing increases the risk of false a negative, especiallyin the case where (possibly) weak associations are tested on a largenumber of variables, we presented both corrected and uncorrected P-values. We considered significant P-values< 0.05.Odd ratio (OR) valuesand 95% confidence interval (95% CI) werereported for each metabolite or lipoprotein analyzed. For completeness,adjusted 2.7.5t1 and t2 and considering specific patient’s relative metabolitesand lipids Δ(pre–post)rt-PA concentrations.Adverse outcomes were evaluated also by applyingreceiver operating characteristic (ROC) curve analysis on selectedanalytes at t1 and Δ(pre–post)rt-PA, and “Bas2” for t2] thatincluded only clinical and risk factors known to affect the outcomes. Second, we quantify howmuch the addition of a combination of metabolomic features increasesthe prediction for events when added to each “Bas” ROCcurve model. The metabolites and/or lipoproteins to be included arechosen among the top three statistically significant features basedon the results of the penalized logistic regression models.In detail, for each of the evaluated outcomes, we estimated thevalues of the area under the ROC curve (AUC-ROC) for two differentpenalized logistic regression models. First, we calculated AUC valuesfor models [hereafter referred as “Bas1” for t1 and Δ(pre–post)rt-PA ROC models, we combined statistically significantmetabolic features with specific blood circulating inflammatory markersstatistically associated with sICH, 3 month mortality, and 3 monthpoor functional outcome (mRS = 3–6), selecting them based onthe results reported in the original publication of Gori et al.16Third, for P-value, to highlight any significantchanges in the prediction of the adverse outcome after consideringthe association of metabolomics and clinical features.For all ROC models, 95% CIs of the AUCvalues have been calculated,together with a To avoidoverfitting, before performing any ROC analysis, all penalizedlogistic regression models were cross-validated using the leave-one-outscheme.2.7.630 Penalizedlogistic regression models were built using the logistf function ofthe R package “logistf”.31 To performROC analysis roc function of the R package, “pROC” wasused.32 To perform correlation analysis,the rcorr function of the R package “Hmisc” was used.The plot was generated using the R package “ggplot2”.33All statistical analyseswere performed using R (version 3.5.3), an open-source software forthe statistical management of data.33.116 Notwithstanding this,all demographics, clinical characteristics, and risk factors do notstatistically change  betweentheoriginal cohort and the sub-cohorts analyzed in this study (see Table S2). First, as a multivariate exploratoryapproach, PCA was performed using all 18 metabolites and 112 lipoproteinsdetected on n = 243 serum NMR spectra in order toobtain an overview of the variation in the data and to check the presenceof metabolic signatures among the different evaluated conditions.As shown in t1) and 24 h after (t2) the administration of rt-PA, and no outliers are highlighted, confirmingthe good quality of the data.All analyseshave been performed using data from 243 patients of the original cohortbelonging to the MAGIC study.t1) and24 h after (t2) rt-PA, Pearson correlationanalysis was also performed. In Tables S3 and S4, only the significant correlations  were reported, respectively, for t1 and t2 time points. Before theadministration of rt-PA (t1) we observed,in particular, correlations between lipoproteins and IL-8 and CRP;after the administration of rt-PA (t2),we observed, in particular, correlations between lipoproteins andIL-6, IL-12 and CRP. It is important to underline that no strong correlations, acetoneand 3-hydroxybutyrate were determined to be the only metabolites significantly associated with the developmentof symptomatic intracranial hemorrhage. At t2, 18 lipids out of 112 were associated with the evaluatedoutcome, especially lipids related to bigger and less dense very low-densitylipoprotein (VLDL) particles. Considering Δ(pre–post)rt-PA metabolite/lipid concentrations, we reported phenylalanine,pyruvate, and glucose to be the most statistically associated metabolites,while among lipid parameters, phospholipids related to VLDL particlesare the most statistically associated analytes rt-PA, we refer the reader to the Supporting Information(Tables S5–S7).Before startingthe thrombolytic therapy . Adding baseline levels of the interleukin I receptor antagonist(IL-IRa) and IL-10 to Bas1 + G model, the resulting model got worse.Considering these results, as reported in t2 time point,the additionof VLDL-2 cholesterol, phospholipids, and triglycerides to the Bas2model statistically increased the baseline AUC value of 0.556 to avalue of 0.636 .For the rt-PA metabolic feature concentrations, adding the values related to VLDLphospholipid main fraction and the ones related to the VLDL-2 cholesteroland phospholipid subfractions to the Bas 1 ROC model, we obtainedan improvement of the related AUC value, changing from 0.58 to a valueof 0.65 . Moreover,ROC analysis demonstrated that the addition of ΔCRP and ΔIL-10to the Bas1 + I model significantly improved the AUC for the predictionof sICH in AIS patients treated with thrombolysis .Finally, considering Δ(pre–post)3.2.2t1 time point,acetone and 3-hydroxybutyrate were determined to be associated withthe non-response to the intravenous thrombolysis. At t2, we estimated the association for phenylalanine, VLDL-5phospholipids, high-density lipoprotein (HDL)-4 triglycerides, HDL-2free cholesterol, acetone, and 3-hydroxybutyrate. About Δ(pre–post)rt-PA, alanine, acetone, the total particle number ofApo-B100, related LDL-3 sub-particles, and HDL-4 triglycerides werethe significant predictors of non-response to the therapeutic thrombolytictreatment (see S5–S7).At the t1, the addiction of acetone and 3-hydroxybutyrateto the baseline Bas1 ROC curve model did not statistically improvethe AUC (model Bas1: AUC = 0.54 and model Bas1 + J: AUC = 0.55). At t2, adding the same two ketone bodies, also reportingFDR values < 0.05, to the Bas2 ROC curve model, the AUC significantlyincreased to a value of 0.62 (model Bas2 + K). Regarding Δ(pre–post)rt-PA, the addition of acetone, the LDL-3 particle number,and LDL-3 apolipoprotein-B to the respective Bas1 ROC curve modelled to a slight and not significant improvement of the AUC, changingfrom 0.54 to 0.58 (model Bas1 + L).As reportedin 3.33.3.1t1. For t2, HDL triglycerides,LDL-6 related cholesterol, triglycerides and phospholipids, HDL-3,and HDL-4 triglycerides were determined associated with this lateoutcome. Considering Δ(pre-–post)rt-PA, triglycerides related to LDL and HDL particles and LDL-1, LDL-2,and HDL-4 sub-particles were determined associated with 3 month mortality(for more information see Tables S5–S7).As reported in t1, after adding triglyceridesrelated to the HDL-3 subfraction to the Bas1 ROC model, the AUC didnot improve (model Bas1 + A). Adding to this model levels of ΔMMP9,SAP, and A2M, a non-statistically significant improvement of the AUCwas obtained . At t2, after adding LDL-6, HDL-triglycerides,and LDL-6 cholesterol to the related baseline model, the AUC increased,without a statistical significance, from 0.76 to 0.80 (Bas2 + B).Lastly, considering Δ(pre–post)rt-PA, the addition of HDL, LDL, and LDL-2 triglycerides to the Bas1 ROCcurve model increased, without significance, the AUC value from 0.72to 0.74 (Bas1 + C). The Δ(pre–post) levels of IL-IRa,IL-10, and TNFα were added to the model Bas1 + C; the Bas1 +C + N model was determined to be worse  (see At .29) see 3.3.3.2t1, 15 different lipids, mainly related to smalldense VLDL, LDL, and HDL particles, were statistically associatedwith a 3 month poor functional outcome (mRS = 3–6). Considering t2 samples, acetic acid, 3-hydroxybutyrate, and16 different lipids were determined to be statistically related tothe development of poor functional outcomes (mRS = 3–6). Amongthe 16 lipids, triglycerides, total cholesterol, and free cholesterolestimated for LDL-6 subfractions appeared to be the most statisticallysignificant associated metabolic features. When relating Δ(pre–post)rt-PA metabolite and lipid concentrations to the 3 monthpoor functional outcome (mRS = 3–6), we found that glutamate,acetate, free cholesterol, phospholipids, and Apo A2, related mainlyto HDL-1 and HDL-2, were statistically associated with the outcome.As reportedin t1, the addition of the topthree statistically associated lipid fractions to the related baselineROC model built at t1 did not statisticallyimprove the AUC . For the ROC curve analysis, at t2, the addition of 24 h post rt-PA values ofVLDL-5 cholesterol, LDL-6 free cholesterol, and LDL-6 triglycerides,reporting also FDR values < 0.05 in the penalized logistic regression,to the Bas2 ROC model, led to a statically significant improvementin the already good value of the baseline AUC .Considering Δ(pre–post)rt-PA, addingglutamate and ApoA2, related to both HDL-1 and HDL-2, to the Bas1ROC curve model, the AUC remained the same .Lastly, adding to the Bas1 + F model Δ values of IL-6, IL-8,IL-10, IL-12, MCPI, TNFα, and CRP, the resulting Bas1 + F +O model, reported a similar value of the AUC .As reported in 4P-value < 0.05) witheach of the assessed post-AIS adverse outcomes. Since correction formultiple testing increases the risk of false negatives, especiallyin the case where (possibly) weak associations are tested on a largenumber of variables, in this work, we present both corrected and uncorrected P-values and discussing the biological implications of theresults for which P-values were significant beforecorrection, improving the value of baseline AUC models for each specificoutcome. Combining metabolomic features with inflammatory markersresulted in statistically significant associations among Δ levelsof VLDL-2 cholesterol, VLDL-2 phospholipids, the main fraction ofVLDL phospholipids, and ΔCRP, ΔIL-10 for the predictionof sICH.We observed that variousmetabolomic features  resulted tobe statistically associated .38 Our results also evidence the role of LDL andVLDL cholesterol, estimated at t2, inincreasing the risk of symptomatic intracranial hemorrhage developmentat 24 h.Wereported associations of LDL estimated at 39 which contributes to the development of adverseoutcomes, linked to the activity of rt-PA itself. Indeed, generatedplasmin after rt-PA activity on plasminogen can degrade non-targetproteins, including Apo A1, which represents the major protein constituentof HDL particles.Moreover, statistically significant associations betweenHDL-relatedparameters and adverse outcomes could reflect a general inflammationcondition that characterizes the post-stroke course. Inflammationmay alter the lipoprotein profile by modulating the HDL function


Introduction: traumatic brain injury (TBI) is very often associated with spasticity. Medical interventions may include medications such as baclofen, a Gamma-Aminobutyric Acid (GABA) -receptor agonist of poor lipid solubility. Intrathecal baclofen (ITB) administration is a contemporary treatment option which minimizes adverse effects in contrast with the oral form of the drug. Regarding low urinary tract dysfunction, TBI, as a suprapontine lesion, results in neurogenic detrusor overactivity. Frequency, urgency and urge incontinence are the predominant signs and symptoms of this condition. Our study aims to report the potential changes in bladder function in patients with spasticity, due to TBI, after the implantation of the baclofen pump and the control of spasticity. Material and Methods: We report three cases of TBI whose spasticity responded well to ITB. We evaluated our medical reports regarding bladder function retrospectively, before and after baclofen pump implantation. We compared the data of bladder diaries and urodynamic parameters. Results: Bladder function was improved in all patients. Regarding bladder diaries; the number of incontinence and micturition episodes was decreased and the volume per void was slightly increased. Regarding urodynamic parameters; bladder capacity and reflex volume increased, Pdetmax decreased, PVR was the same and DLPP was slightly decreased. Conclusions: Although the baclofen pump is implanted to treat spasticity, detrusor activity may be also affected. Therefore, patients’ urologic profiles should also be reevaluated after ITB. Further prospective studies are required to investigate the effect of ITB on bladder function in the clinical field and also at the basic science level. Spasticity presents as a common outcome of an upper motor neuron abnormality. Cerebral and spinal cord lesions may result in spasticity even though the pathophysiological mechanism may be different .Baclofen is a very potent agonist of GABA receptors and is an effective treatment modality for spasticity. The use of baclofen started as an oral agent in the early 1970s ,3,4. UnfTreating spasticity is very important for patients as their movements become more coordinated and functional, body formation becomes less contractive and more natural, quality of sleep gets better, discomfort or pain decreases and in general quality of life improves . BaclofeRegarding the lower urinary tract function, TBI results predominantly in neurogenic overactivity amongst others (pseudodussynergia and detrusor underactivity). TBI as a suprapontine lesion results in neurogenic detrusor overactivity in the majority of cases, by reducing, central inhibitory mechanisms ,14. If tOur study aims to report the potential changes in bladder function in patients with spasticity, due to TBI, after the implantation of a baclofen pump and the control of spasticity.We report three cases of TBI, whose spasticity responded well to intrathecal baclofen administration. All patients were males, aged 36, 29 and 27 years old and had been injured two, two-and-a-half, and three years respectively before baclofen pump implantation. We evaluated our medical reports referring to bladder function retrospectively, before and after baclofen pump implantation.Regarding the baclofen pump, it is reported in the medical records of the clinic where they had the pump implanted, that first a temporary catheter was placed into the lumbar region and propelled to the thoracic spinal cord; the bolus test was administered and after the positive reaction of the patients to baclofen they proceeded to the implantation. The ITB dosage was titrated in order to deteriorate the spasticity and allow the normal strength of the patients to manifest. Thus, one patient was titrated up to 200 μg/d, the other up to 300μg/d, and the 3rd one up to 350 μg/d.Bladder function was evaluated by urodynamics  , before In our study, we compared the bladder diaries and the urodynamic parameters before and after the baclofen pump implantation.Bladder function was improved in all patients. Regarding bladder diaries, incontinence episodes and micturitions were decreased, and the volume per void was slightly increased although the fluid intake was approximately the same. Data of the bladder diaries comparison are summarized in Our initial intention was to express our observation that baclofen pump implantation improved in the long term not only the degree of spasticity for patients with TBI but also their bladder function. We underline that this was accomplished with significantly lower doses of baclofen than orally  and therefore with almost no side effects. The quality of life was improved as there were fewer incontinence problems and the overnight sleep hours were augmented. Maybe it is worth bearing in mind that selecting patients for ITB therapy should depend  on whether it could result in a concomitant benefit on bladder function in addition to other therapies. It is strongly suggested that patients should be screened for response to the drug before implantation of the delivery pump.The pathophysiology of neurogenic detrusor overactivity (NDO) depends on several peripheral and central nervous system (CNS) factors. Different CNS disorders are associated with NDO via multifactorial pathological pathways.Normal micturition in humans and animals depends on afferent signals from the lower urinary tract (LUT), under the control of circuits in the brain and the spinal cord ,17,18. TDistension of the bladder wall is thought to be the main stimulus for initiation of the bladder reflux. Moreover, the urothelium itself is considered a sensor adjacent to afferent nerves . When thLesions emerging on the level below the pontine region may additionally result in detrusor–sphincter dyssynergia  while abIn patients with cortical lesions, voiding is generally coordinated and urine incontinence is attributed to damage of cerebral inhibitory centers, thus uninhibited detrusor contractions . Damage Symptoms are usually apparent during the storage phase  and during urodynamics, there is detrusor contraction in low volumes of bladder filling. During the voiding phase, hesitancy and weak urinary stream are often combined with the former ones. In men, the symptomatology could be confused with that of benign prostatic hyperplasia which could also exist.Different neurotransmitter systems are involved in micturition control and could be potential targets of therapies to control abnormal micturition. Glutamate is probably an excitatory transmitter supraspinally and is also involved in the pathway from the PMC to the preganglionic neuron ,32. AntaA, GABAB, and GABAC) and all of them are localized in both the brain and the spinal cord [A receptor agonist muscimol and GABAB receptor agonist baclofen [GABA in particular has been detected in both spinal and supraspinal synapses in mammalian CNS and more precisely in the supraspinal micturition pathway of some of them. Its role is of a rather tonic, inhibitory type ,19 and mnal cord ,36,37. Fbaclofen ,39,40,41baclofen . Baclofebaclofen . HoweverIntrathecal administration of the GABA-B agonist baclofen is nowadays generally accepted as a powerful treatment for spasticity of whatever etiology; improvement in mobility and function as well as relief of spastic pain is the most obvious benefit for the patient. The initial technical and methodical problems have been solved and today the procedure is generally assessed as safe .Selecting patients for ITB therapy depends partially on the local experience, but it generally concerns patients with severe spasticity lasting at least six months and having failed to respond to the maximum recommended doses of oral antispasmodic medications either because they are ineffective or because of too many side effects . It is aComplications, while rare, are most often related to the drug delivery catheter . ITB treAfter ITB implantation, close monitoring is essential to prevent complications and side effects. Either undertreatment or overdose can be life-threatening . The morThe current study leads to results that cannot be generalized or applied in everyday practice. The main reasons are the small sample size and the fact that it explains an observation of change in bladder behavior as a positive side effect of therapy used to treat other symptoms. Therefore, prospective studies are required to investigate the effect of ITB on bladder function in the clinical field and at the basic science level.ITB is effective, nondestructive, titratable, and reversible. In addition, it is associated with fewer CNS-related side effects than oral baclofen medication. ITB therapy may improve the range of motion, reduce the need for corrective orthopedic surgeries, facilitate movement, reduce the patient’s expenditure of energy, reduce difficulty in nursing interventions, avoid contractures, deteriorate spasticity related pain. It may ameliorate upper and lower extremities’ function or vocal mechanism’s difficulties and therefore improve daily living, and other aspects of self-care. It seems that there is also an effect on bladder function even though our data are not, till now, so strong to prove it, in the field of evidence-based medicine. Physicians conducting urodynamics should be alert and follow such cases in the long term to gather more data on the effect of baclofen pump implantation on lower urinary tract function. Furthermore, other issues arise such as: is it that baclofen affects immediately the detrusor muscle and suppresses its overactivity or is it that baclofen’s action on the urethral sphincter leads to relief, thus to a lower leak point pressure (LPP) and therefore to a lower maximum detrusor pressure (Pdetmax)? Prospective clinical studies have to be designed in order to identify and prove the effect of ITB on bladder function and more studies on basic science are needed to examine the role of GABA on urinary function.


In chronic heart failure, high intracardiac pressures induce a progressive remodeling of small pulmonary arteries up to pulmonary hypertension. At the end of left atrial conduit function, pulmonary and left heart end-systolic pressures equalization might affect left atrial systole. In this single-center prospective study, we aimed to investigate whether peak atrial contraction strain (PACS), measured by speckle tracking echocardiography, was independently associated with prognosis in heart failure with reduced ejection fraction (HFrEF).Outpatients with HFrEF and sinus rhythm referred to our echo-labs were enrolled. After clinical and echocardiographic evaluation, off-line speckle tracking echocardiography analysis was performed. Primary and secondary endpoint were cardiovascular death and heart failure hospitalization, respectively. Spline knotted survival model identified the optimal prognostic cut-off for PACS.n = 76) or PACS ≥8% (n = 76). Patients with PACS <8% had lower left ventricle and left atrial reservoir strain and higher New York Heart Association (NYHA) class and left atrial volume index (LAVI). Over a mean follow-up of 3.4 ± 2 years, 117 events  were collected. By univariate and multivariate Cox analysis, PACS emerged as a strong and independent predictor of cardiovascular death and heart failure hospitalization, after adjusting for age, sex, left ventricle strain, and E/e’, LAVI (HR 0.6 per 5 unit-decrease in PACS). Kaplan–Meier curves showed a sustained divergence in event-free survival rates for the two groups.The 152 patients were stratified based on PACS <8% (The reduction of PACS significantly and independently affects cardiovascular outcome in HFrEF. Therefore, its assessment, although limited to patients with sinus rhythm, could offer additive prognostic information for HFrEF patients. Chronic heart failure (HF) is a progressive cardiovascular disease with increasing incidence in the last years, parallel to an increasing medium age of the population worldwide. Despite therapeutic novelties, it is still characterized by a poor prognosis, particularly in the advanced stages of the disease , and a hIn this prospective single-center observational study, consecutive patients with HFrEF and sinus rhythm according to the 2016 ESC HF guidelines definition  referred to our HF ambulatories for a cardiologic visit including echocardiography between 2015 and 2017 were enrolled. Exclusion criteria were non-sinus rhythm, previous cardiac surgery or poor acoustic window.via phone calls and electronic medical records. All subjects gave their written informed consent for participation in this study. All work followed the 1975 Declaration of Helsinki. The study protocol was approved before the enrollment of the first patient by the local Ethics Committee .The patients were prospectively followed for a primary and a secondary endpoint, consisting in the occurrence of cardiovascular death and hospitalization for HF, respectively. Follow-up data were collected Echocardiographic examination was performed according to the EACVI/American Society of Echocardiography (ASE) recommendations for chamber quantification , using aLeft ventricular and left atrial dimensions were calculated using standard views. LV ejection fraction (LVEF) and LA volume and area were assessed using the biplane modified Simpson method from the apical 4- and 2-chamber views. LV dimensions and LA volume were indexed to body surface area obtaining LV mass index and LA volume index (LAVI). From the 4-chamber view, tricuspid annular plane systolic excursion (TAPSE) was measured by M-mode; maximum early diastolic (E) and late diastolic (A) velocities were assessed by trans-mitral pulsed wave doppler to calculate E/A ratio; then, peak systolic (S’), early diastolic (E’), and late diastolic (A’) annular velocities were obtained by tissue doppler imaging, E/E’ ratio was calculated and used as index of the LV filling pressure. Mitral and tricuspid regurgitation  were quantified by bidimensional (2D)-echocardiography according to EACVI/ASE recommendations . SystoliSpeckle tracking echocardiography analysis was performed on apical 2-, 3-, and 4- chamber images, obtained by 2D gray-scale echocardiography, with a stable electrocardiographic recording. Care was taken to obtain a good visualization of all chambers and a reliable delineation of the endocardial border. Measurements from three consecutive heart cycles were recorded and averaged. The frame rate was 60–80 frames/sec. Analysis was performed off-line by a single experienced and independent echocardiographer, who was not directly involved in the image acquisition and blinded to basic echocardiographic parameters, using a semiautomated 2D-strain software . The endocardial border was manually traced in apical views, delineating a region of interest (ROI) of six segments for each view. Then, necessary manual adjustments of the ROI were performed and the longitudinal strain curves for each segment were generated by the software. LV GLS was calculated as the average of 4-, 2-, and 3-chambers longitudinal strain curves. Both apical views were optimized in terms of orientation, depth, and gain to avoid LA foreshortening and to visualize the entire LA throughout the cardiac cycle. Global PALS and PACS were calculated at the end of the reservoir and the contraction phase, respectively, as the average of all LA segments in 4- and 2-chamber views, using QRS as starting point . In patiData are expressed as means ± SD (continuous variables) or as counts and percentages (binary variables).T-tests for continuous variables and Chi-squared analyses for categorical variables.Spline-knotted survival model was used to obtain optimal cutoff values of global PACS for the prediction of the primary endpoint . Using this cutoff, patients were divided into 2 groups based on the presence of PACS lower/higher than the cutoff. Differences between the groups were analyzed using Student Kaplan–Meier curves and Log-Rank test were used to assess the correlation of the two groups with events-free survival. Univariate and multivariate analysis were performed applying the Cox proportional hazard model to investigate the performance of global PACS as a predictor of primary and secondary endpoint; adjustment models were built using age, sex, LV strain, and E/e’, LAVI. The covariates were chosen based on their univariable association with the dependent variable as well as based on biological plausibility.P-values <0.05 were considered statistically significant.Analyses were performed using the Statistical Package for Social Sciences software, release 20.0 . n = 32) were female, mean LV EF was 30 ± 9%. All patients were receiving optimized HF therapy (according to current guidelines at the time of enrollment) including ACE inhibitors/angiotensin receptor blockers (91%), mineralcorticoid receptor antagonist (75%), betablockers (78%). Moreover, 81% of the patients had implantable cardiac device (ICD) or cardiac resynchronization therapy-defibrillator (CRT-D). Mean LA maximum volume indexed was 55 ± 18 ml/mq while LA minimum volume was 34 ± 20 ml.From an initial number of 168 patients, 152 patients were finally enrolled in this study. We excluded 8 patients for previous heart valve surgery, six because of atrial fibrillation during echocardiographic examination and two for poor acoustic window. Intra-operator reproducibility for LA strain analysis was already tested in our center. , 20 Meanr = 0.54, p < 0.001).Speckle tracking echocardiography analysis revealed severely reduced longitudinal deformation of both left ventricle and atrium . PACS value showed a moderate, statistically significant correlation with LA minimum volume  were registered. Spline-knotted curves showed a good association of global PACS <8% with risk of cardiovascular mortality .Therefore, this cut-off was used to stratify the population into two risk groups: group 1 with PACS <8% and group 2 with PACS >8%. Kaplan–Meier curves showed a sustained divergence in event-free survival rates for the two groups for both the primary and the secondary endpoint .The present study was the first to show an independent prognostic value of PACS in chronic HF, identifying <8% as the optimal cut-off value for risk stratification in HFrEF.Even though there currently is a lack of studies focused on this parameter alone, there are a lot of evidence on its association with outcome in HF and cardiomyopathies, if measured in adjunction to PALS –23. MoreThe rationale in considering PACS as an independent prognostic parameter in HFrEF could be deduced by the pathophysiology of chronic HF: first of all, the direct or indirect myocardial damage which causes overt LV systolic dysfunction in HFrEF and the chronic LV overload impairs myocardial contractility with severe LV GLS reduction accompanying LV EF reduction in the majority of cases. Therefore, although being important for the categorization of overall HF, none of these two parameters seems to be sensitive for risk stratification among patients with chronic systolic dysfunction. Then, the chronic increase of LV filling pressures reflects on the LA myocardium, which initially compensates for the high intracardiac pressures and eventually underwent LA wall ultrastructural changes, with remodeling and fibrosis, leading to LA dilatation and dysfunction. In these phases, LA evaluation is fundamental to assess the stage of the disease and whether the myocardial damage could be reversible or not. Therefore, PALS would be certainly reduced in a grade parallel to the age of the disease, and its value, reflecting the reservoir function in systole, would be probably influenced by LV contractile properties and longitudinal shortening. However, in the advanced phase, when the LA is not capable to face the elevated filling pressures, the transmission of hemodynamic overload on the pulmonary circulation takes place, with a chronic establishment of pulmonary hypertension, up to a point in which pulmonary and LA pressures equalized in diastole, thus affecting LA contraction and its contribution to LV filling. This represents the immediately preceding step before the transition to biventricular failure, due to severe and/or chronic pulmonary hypertension, often irreversible and requiring advance therapies.Hence, in patients with long-standing chronic HFrEF, the analysis of LA contractile function may offer additional information for risk stratification, identifying those patients who have completely lost the LA contribution to maintain LV filling, in all the cardiac cycle phases, and who are at higher risk of transition to advanced HF.In the light of the new therapies for HFrEF recommended in the latest ESC HF guidelines , there iConsidering the high feasibility and availability and low time-consumption of this parameter, the measurement of PACS could help clinicians to provide patients with HF and sinus rhythm a more tailored therapy and to decide whether to be more aggressive with medical therapy and to prescribe stricter follow up to HFrEF patients.Although this study shows global PACS promising results for the clinical risk stratification of HFrEF, some limitations should be encountered: first, it was a single center study conducted in a small cohort of patients with HFrEF, therefore, large-scale studies should confirm our findings.Moreover, PACS was investigated as an independent parameter and was not compared with PALS, which may be associated with it in some way. Finally, an intrinsic limitation of the parameter led to the exclusion of patients with atrial fibrillation, which is a common condition in HF, since PACS is not feasible in these patients.In conclusion, in patients with chronic HFrEF, LA contraction might be affected because of the chronic hemodynamics overload. The assessment of impaired LA contraction as a reduction of global PACS, acquired by speckle tracking echocardiography, significantly and independently affects CV outcome in HFrEF. Therefore, although limited to patients with sinus rhythm, the evaluation of global PACS could provide additive information for risk stratification of HFrEF patients.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Comitato Etico Regionale per la Sperimentazione Clinica della Regione Toscana Sezione: AREA VASTA SUD EST. The patients/participants provided their written informed consent to participate in this study.GM: conceptualization, methodology, software, formal analysis, writing—original draft, and supervision. MP: software, formal analysis, and investigation. GB: data curation and investigation. MS, LM, LC, and SV: data curation. ED: writing—review and editing, validation, and visualization. MF: investigation. FD’A: conceptualization and methodology. MC: conceptualization, methodology, software, and project administration. All authors have participated in the work and have reviewed and agreed with the content of the article.


Our data showed that a condition of ID was more frequently associated with impaired diastolic function, worse 6MWT and endothelial dysfunction, an effect that was accompanied by elevated MDA serum levels. Treatment with FCM, compared to the placebo, improved ferritin levels being associated with an improved 6MWT, enhanced cardiac diastolic function and endothelial reactivity associated with a significant reduction in MDA levels. In conclusion, this study confirmed that ID is a frequent comorbidity in patients with HFpEF and is associated with reduced exercise capacity and oxidative stress-related endothelial dysfunction. Supplementation with FCM determines a significant improvement in diastolic function and the exercise capacity of patients with HFpEF and is associated with an enhanced endothelial function and a reduced production of oxygen radical species.Both clinical and experimental evidence shows that iron deficiency (ID) correlates with an increased incidence of heart failure (HF). Moreover, data on iron supplementation demonstrating a beneficial effect in subjects with HF have mostly been collected in patients undergoing HF with reduced ejection fraction (HFrEF). Relatively poor data, however, exist on the potential of iron supplementation in patients with HF with preserved ejection fraction (HFpEF). Here, we report on data emerging from a multicentric, double-blind, randomized, placebo-controlled study investigating the effect of IV supplementation with a placebo or ferric carboxymaltose (FCM) on 64 subjects with HFpEF. ID was detected by the measurement of ferritin levels. These data were correlated with cardiac performance measurements derived from a 6 min walking test (6MWT) and with echocardiographic determinations of diastolic function. Moreover, an EndoPAT analysis was performed to correlate cardiac functionality with endothelial dysfunction. Finally, the determination of serum malondialdehyde (MDA) was performed to study oxidative stress biomarkers. These measurements were carried out before and 8 weeks after starting treatment with a placebo (100 mL of saline given i.v. in 10 min;  Iron deficiency (ID) represents an emerging issue for public health and is correlated with many disease states, including myocardial dysfunction ,2. To daThe development of ID with or without anemia occurs as a consequence of different pathophysiological conditions ,6, as weWhile the correlation between ID and myocardial dysfunction has clearly been shown, the potential for iron supplementation in counteracting myocardial damage and its consequences is still controversial.Evidence exists that IV iron supplementation produces benefits in HF ,11,12. IThese results have also been confirmed by results emerging from the CONFIRM-HF study , thus shSeveral studies have shown that iron-deficient HFpEF patients suffered a similar impact as the health-related QoL of HFrEF patients ,18. FurtIn this context, this study aimed to verify the possible impact of IV administration of FCM on cardiac performance and on reliable indicators of diastolic function in patients with HFpEF. This was correlated with oxidative stress and endothelial function, which represent reliable biomarkers of vascular impairment occurring at the early stages of myocardial dysfunction in subjects in which HFpEF and ID occur simultaneously.2), mitral E velocity > 90 cm/s, septal velocity < 9 cm/s, E/e’ ratio > 9, which have been shown to better correlate with an increased mortality rate , and oxidative stress serum biomarkers, respectively. In particular, patients were divided into three groups according to their diastolic function as assessed by the evaluation of the E/e’ ratio  and sex (m/f ratio in the 3 groups 34/30), with the presence of metabolic syndrome in 70% of the subjects enrolled and moderately overweight. A proportion of 44% had moderate arterial hypertension on treatment with ACE-i/ARB and/or beta-blockers. Patients with valvular pathology or atrial fibrillation were excluded from the study. None of the patients were smokers at the time of the study .In addition, the effect of IV administration of the placebo  or ferric carboxymaltose  infused at time 0 and after four weeks was evaluated. The outcome of the treatment was verified 8 weeks after starting the administration of FCM. All the patients completed the study. Moreover, the use of an iron-containing supplement was not allowed either before or during the study.The patients were enrolled at the Metabolic Diseases Outpatient Clinic of the IRC-FSH Center of the “Magna Graecia” University of Catanzaro, in collaboration with the UTIC B of the Umberto I Polyclinic in Rome and the Unit of Cardiology at IRCCS San Raffaele, Rome. All subjects provided written informed consent at the time of enrollment, and the protocol was approved by the local ethics committee and met all the principles of the Declaration of Helsinki. The protocol is registered on the ISRCTN registry (study ID. ISRCTN12833814). A condition of ID was defined on the basis of serum ferritin levels < 100 nanograms/mL or with levels between 100 and 299 µg/L in the presence of transferrin saturation (TSAT) < 20% . The degThese determinations were performed at time 0 and at 8 weeks after starting the study. Care was taken that patients maintained the same dietary regimen during the observation period. In analogy with the sample, on the day of the visit at time 0 and after 8 weeks, for the definition of the degree of diastolic function, the patients had an echocardiographic examination, took a vascular reactivity test using the EndoPAT method, and had an additional sample taken for the determination of the levels of serum malonyldialdehyde (MDA).The diastolic function parameters used for detecting HFpEF were represented by the mitral E velocity, E/e’ ratio, and left atrium volume. In particular, E velocity is detected transmitrally. The transmitral PW spectrum includes two velocity peaks generated by differences in the atrioventricular pressure during the diastolic phase of the cardiac cycle. The first peak (wave E) corresponds to rapid ventricular filling, the one observed when, following isovolumetric relaxation, the atrial pressure far exceeds the ventricular pressure, generating an acceleration in the flow in this sense. The extent of the E peak depends on ventricular relaxation, left atrial pressure, and ventricular protodiastolic elastic return; these parameters determine the faster or slower speed of rapid filling.The speed with which the atrio-ventricular pressure gradient tends to cancel itself following the first phase of diastole influences the speed and deceleration time (DT) of the E wave. The faster the ventricular pressure increase in protostole, the slower the deceleration time of the E wave.Tissue Doppler (TD) is a method that has been used to assess the speed of movement of the myocardial wall. The study of diastolic function using this technique is based on the assessment of the displacement speed of the mitral annulus, with image acquisitions through the apical projection in four chambers.The standard pattern of pulsed TD at the level of the mitral annulus includes three components: a positive (S) systolic wave, that is, directed toward the apex, and two diastolic waves, one early protodiastolic (e’) and the other late (a’) during atrial systole. Taken together, these rates and time intervals provide quantitative information on systolic longitudinal ventricular function and diastolic function. Compared to the transmitral flow Doppler, the pulsed annular TD is less dependent on variations in ventricular preload. For this reason, the calculation of the E/e’ ratio is a reliable parameter for the non-invasive estimation of filling pressures.An E/e’ ratio of less than 8 is often associated with a normal filling pressure, while a ratio above 15 is observed in the case of increased filling pressures. Values between 8 and 15 need further investigation to be interpreted correctly. 2.Left Atrial (LA) volume was measured from standard apical 2- and 4-chamber views at end-systole. LA borders were traced using planimetry. The borders consisted of the walls of the left atrium and a line drawn across the mitral annulus. Attention was given to bridge the ostia of pulmonary veins  to not include the veins in the measurement. If seen, the LA appendage was excluded from measurement. The biplane method of disks was used to calculate LA volume, and measurements were rounded to the nearest integer. Left Atrial Volume Index (LAVI) was calculated by dividing LA volume by body surface area and expressed as ml/mEndothelial function was assessed using the EndoPAT 2000 technique, which measures PAT using the reactive hyperemia index . Briefly, the examination was performed after 20 min of rest in a chair tilted at an angle of approximately 45° at room temperature, with a blood pressure cuff placed on the non-dominant upper arm (study arm), while the other arm served as a control. The hands were placed on the chair stands with the palms facing down so that the fingers hung freely.The EndoPAT probes were then placed on the tip of each index finger of both hands. The probes were prevented from touching any other fingers or objects and were then electronically inflated. The PAT signal was continuously recorded on a personal computer during the test. The baseline pulse width was measured from each fingertip for five minutes. After a five-minute baseline recording on each arm, the arterial flow was then stopped in the experimental arm by rapidly inflating the cuff to the occlusion pressure of 200 or 60 mmHg plus systolic blood pressure (whichever was greater). After five minutes of occlusion, the cuff pressure was rapidly reduced, and post-occlusion recording continued for an additional five minutes in the experimental and control arms. The pulse width response to hyperemia was automatically calculated from hyperemia in the finger of the experimental arm as a ratio of post-deflation mean pulse ingestion to obtain the RH–PAT ratio or PAT ratio. EndoPAT 2000 measured not only endothelial function with the RHI but also assessed arterial stiffness by measuring the peripheral augmentation index (PAIx) from the radial pulse wave analysis. PAIx is automatically calculated as the ratio of the difference between the early and late systolic peaks of the waveform to the early and late systolic peak of the waveform related to the systolic peak, expressed as a percentage.−1 cm−1 [Oxidative stress was assessed by quantifying the reactivity of thiobarbituric acid (TBA) as MDA in a spectrophotometer. To 0.5 mL of serum, 0.5 mL of 30% trichloroacetic acid (TCA)  was added and centrifuged at 3000 rpm for 5 min, and the supernatant was collected. Subsequently, 0.5 mL of supernatant was added to 0.5 mL of 1% TBA (Merck) in a boiling water bath for 30 min, after which the tubes were kept in an ice water bath for 10 min. The resulting absorbance of chromogen was determined at a wavelength of 532 nm at room temperature with respect to the blank reference. The concentration of MDA was read from the standard calibration curve plotted using 1, 1, 3, 3 “tetra-ethoxy propane (TEP). The amount of lipid peroxidation was expressed as MDA (µg/dL)”, using a molar extinction coefficient for MDA of 1.56 × 105 M−1 cm−1 .t-test, Fisher’s exact test, Pearson’s simple regression and logistic regression were used as appropriate. A two-tailed p-value < 0.05 indicated statistical significance.Data are presented as mean ± standard deviation (SD). StatView 5.0  and the Statistical Package for the Social Sciences (SPSS version 21) were used for the statistical analyses. Analysis of variance (ANOVA), followed by post hoc comparisons, Student’s unpaired The study, conducted on 64 patients with HfpEF, documented, at baseline, a condition of ID found in 38/64 patients corresponding to 53.1% of the total subjects entering the study . The popMoreover, the distribution of patients into three groups made according to the degree of diastolic dysfunction (as detected on the basis of E/e’ ratio) was, overall, consistent with the data of the most recent literature on this area of interest ,29. In fAccording to the degree of diastolic impairment, administration of FCB i.v. at time 0 and after 4 weeks from entering the study was able to improve ID levels, oxidative stress, and impaired endothelial function, compared to the placebo. Indeed, FCM restored the ferritin levels in patients with HFpEF associated with high degree diastolic dysfunction, an effect associated with improved cardiac performance as showed by the better response at the 6MWT . In addiOur data show, for the first time, that supplementation with FCM administered via i.v. infusion in such a way as to bring ferritin within physiological limits in a cohort of patients affected by HFpEF leads to an improvement in cardiac performance evaluated by means of a 6MWT. This improvement was mainly found to occur in patients in which diastolic function was particularly compromised (Group 1 of patients enrolled in the study), as assessed by the Echo cardio color Doppler study and in particular through reliable indicators such as the E/e’ ratio and LAVI.Moreover, the study showed that the improvement in cardiac performance, seen after supplementation with IV FCM, was associated with a marked improvement in endothelial function and in the degree of oxidative stress that characterized the basal conditions in patients at the time of their enrollment in the study.In particular, at 8 weeks after the administration of FCM , there was a marked improvement in vascular reactivity through the EndoPAT study, an effect accompanied by a significant reduction in MDA levels, a reliable index of the level of lipid peroxidation and, finally, of oxidative stress.Diastolic dysfunction is known to represent the pathophysiological mechanism characterizing the more severe forms of HFpEF ,30. In pConversely, diastolic dysfunction seems to have its pathophysiological origin in the progressive loss of the “lusitropic” properties of the myocardium, associated with progressive fibrosis rather than with the absolute or relative loss of the contractile myocardium .The pathophysiological bases of this condition are, to date, not fully clarified. In fact, the “phenotype” of the early stages of the pathology associated with HFpEF still remains to be defined, during which the development of myocardial compromise is resolved toward an impairment of the lusitropic properties of the myocardium rather than toward the cellular apoptosis of cardiomyocytes.primum movens” of this pathophysiological evolution in mitochondrial oxidative stress associated with impaired iron metabolism and endothelial dysfunction, leading, in the late stages, to myocardial impairment. In particular, Zhang et al. showed that in the heart tissue of patients with HF, myocardial iron concentration was lower compared to that detected in non-failing hearts [However, recent studies make it possible to identify the “g hearts . This efg hearts ,31. ThisOur data also show that endothelial functionality contributes to the pathophysiology of ID-associated disorders and HFpEF. Indeed, FCM administration was able to restore altered ferritin levels and exercise capacity in patients with HFpEF and produced an improvement in endothelial-dependent vasodilatation as investigated by EndoPAT analysis in our patients at time 0 and after FCM treatment. In fact, a lower capacity to respond to flow restriction with a vasodilatory response was found in Group 1, in which a significant correlation between ID and diastolic dysfunction was found. In this group of patients, restoring ferritin levels via FCM was accompanied by improvement in all the EndoPAT parameters of endothelial-mediated vasodilatation, showing that endothelial dysfunction has a crucial role in ID-related HFpEF. This correlates with evidence suggesting that endothelial impairment accompanies HFpEF ,31,32,33In conclusion, this study confirmed that ID is a frequent comorbidity in patients with HFpEF and is associated with reduced exercise capacity and oxidative stress-related endothelial dysfunction. Supplementation with FCM determines a significant improvement in the cardiac performance of patients with HFpEF enrolled in this study, an effect that was associated with enhanced endothelial function and reduced production of oxygen radical species. Further studies, however, based on a larger number of patients, are required in order to better clarify the potential for iron supplementation in patients with HFpEF.


MVP) and without (LV SVstandard) MVP left ventricular doming volume, using 4D flow (LV SV4DF) as the reference value. Significant differences were observed when comparing LV SVstandard and LV SVMVP (p < 0.001), and between LV SVstandard and LV SV4DF (p = 0.02). The Intraclass Correlation Coefficient (ICC) test demonstrated good repeatability between LV SVMVP and LV SV4DF  but only moderate repeatability between LV SVstandard and LV SV4DF . Calculating LV SV by including the MVP left ventricular doming volume has a higher consistency with LV SV derived from the 4DF assessment. In conclusion, LV SV short-axis cine assessment incorporating MVP dooming volume can significantly improve the precision of LV SV assessment compared to the reference 4DF method. Hence, in cases with bi-leaflet MVP, we recommend factoring in MVP dooming into the left ventricular end-systolic volume to improve the accuracy and precision of quantifying mitral regurgitation.There remains a debate whether the ventricular volume within prolapsing mitral valve (MV) leaflets should be included in the left ventricular (LV) end-systolic volume, and therefore factored in LV stroke volume (SV), in cardiac magnetic resonance (CMR) assessments. This study aims to compare LV volumes during end-systolic phases, with and without the inclusion of the volume of blood on the left atrial aspect of the atrioventricular groove but still within the MV prolapsing leaflets, against the reference LV SV by four-dimensional flow (4DF). A total of 15 patients with MV prolapse (MVP) were retrospectively enrolled in this study. We compared LV SV with (LV SV Valvular heart disease prevalence is expected to increase worldwide due to population ageing . Mitral Mitral regurgitation assessment is mainly conducted by echocardiography. An integrated approach of several parameters is the preferred approach advocated by the American College of Cardiology/American Heart Association Guidelines for the management of patients with valvular heart disease and also by the European Society of Cardiology/European Association of Cardiothoracic Surgery Guidelines ,9. Thesestandard), obtained by subtracting the aortic phase-contrast forward volume (AoPC) from the left ventricular (LV) stroke volume (SV), is the most widely researched CMR assessment [Cardiac magnetic resonance (CMR) imaging is the gold standard non-invasive modality for biventricular volume quantification ,13 and psessment . LV SV iIn patients with bi-leaflet mitral valve prolapse, LV SV assessment is challenging due to the significant doming of the mitral valve leaflets on the atrial side of the atrioventricular groove. It remains debated whether the blood volume trapped on the ventricular side of the doming leaflets during systole should be included in the left ventricular end-systolic volume and whether this will impact the quantification of MR.Four-dimensional flow (4DF) CMR allows quantifying transvalvular flow precisely using valve tracking procedures to generate a true reformatted plane through most mobile valves—mitral and tricuspid. Both mitral and aortic flows can quantify LV SV, applying the conservation of mass principle. Because of the technical advantage, 4DF CMR has become the new gold standard for flow quantification .This study aims to test the difference between the measurement of LV SV with and without the MVP left ventricular doming volume, compared with the reference method of 4DF for the quantification of LV SV.Fifteen cases with MVP from our routine CMR service from February 2021 to March 2022 were retrospectively enrolled in this study. All were adult patients, who were clinically stable as outpatients, and had baseline functional cine images and 4DF CMR assessment data. Exclusion criteria were limited to any CMR contraindication . No patient was consecutively enrolled.CMR study was conducted on a 3 Tesla Discovery MR750w GE system . The CMR protocol included baseline survey images and cines with 30 phases. Cine images were acquired during end-expiratory breath-hold with a Gated 2D FIESTA (Fast Imaging Employing Steady-state Acquisition) single-slice breath-hold sequence. Long-axis Gated 2D FIESTA cine in four-chamber, three-chamber, and two-chamber planes and short-axis Gated 2D FIESTA cines were also acquired. The number of left ventricular (LV) short-axis slices depended on each patient’s heart size. Cine imaging, gadolinium enhancement imaging, and 4DF acquisition methods have been previously published by our group ,20,21.LV volumes were quantified in a short-axis (SAX) stack using CVI42 version 5.14 . End-diastolic and end-systolic phases were manually defined, and contours were drawn automatically using artificial intelligence (AI), and then visually checked by an experienced operator. An expert with more than 10-year CMR analysis experience supervised and checked the quality of manual contour and independently interpreted the results.standard was obtained (LV SVstandard = LVEDV – LVESV) thus excluding MVP left ventricular doming volume. In the second group, MVP left ventricular doming volume was included in LV SV assessment (LV SVMVP) by manually drawing contours where AI failed to factor in MVP left ventricular doming volume  of 2.14 and TR (ms) of 4. Electrocardiogram gating was retrospective to avoid diastolic temporal blurring. Respiratory compensation was free-breathing. The acquired temporal resolution was 40 ms. The number of phases was kept consistent to cines at 30 cardiac phases.The initial VENC setting for 4DF CMR was 150–200 cm/s for all cases. This was optimised depending on beforehand available echocardiography data. 4DF CMR acquisition prescribed k-t adaptive Accelerated Cartesian MRI, namely kat-ARC or Hyperkat, a spatiotemporal-correlation-based autocalibrating parallel imaging method with cardiac motion adaptive temporal window selection . The k-t4DF) using the following equation of conservation of mass principle applied to blood flow haemodynamics: LV SV4DF = ABF + MVF.4DF analysis through the mitral valve and aortic valve was performed using CAAS MR Solutions , with automated velocity offset correction applied. An expert with more than 10-year 4DF and CMR analysis experience supervised and checked the quality of manual contour and independently interpreted the results. Automated valve tracking was done for two orthogonal views of the mitral and aortic valves, with manual correction applied on the region of interest contours if appropriate e,f. Aortstandard/LVSVMVP. An RF < 5% is defined as absent MR, 5–29% mild MR, 30–49% moderate MR, and ≥50% severe MR [MV regurgitant fraction (RF) was calculated by the ratio between mitral backward flow volume derived by 4DF and the respective LVSVevere MR .t-test or a Mann–Whitney U test as appropriate. Inter-observer correlation coefficient (ICC) estimates and associated 95% confidence intervals were calculated based on the absolute-agreement, 2-way mixed-effects model. Paired Student’s t-test was performed to compare LV SVstandard and LV SVMVP. A p value <0.05 is considered statistically significant.Data analyses were performed using SPSS statistical package . Continuous variables were recorded as mean ± standard deviation (SD). Shapiro–Wilk test was used to test the normality of parameters, followed by hypothesis testing with Student’s 2. All patients were in sinus rhythm, 14% had a history of hypertension and 33% were current or ex-smokers. More than half were in New York Heart Association (NYHA) class I (60%), four (27%) were in NYHA class II, two (13%) were in NYHA class III and one (7%) was in NYHA class IV. The most used long-term medications included beta-blockers (60%), angiotensin-converting enzyme inhibitors (40%), diuretics (27%), and calcium channel antagonists (7%).Patient characteristics: Patient demographics are summarised in standard was significantly greater than either LV SVMVP and LV SV4DF . The ejection fraction in the LV SVMVP group was significantly lower than the LV SVstandard group .Descriptive statistics for the recorded parameters are presented in LV SV4DF  with p <MVP with an average increase of 2.1% compared with using LV SVstandard. The MR severity of none/mild/moderate/severe was slightly different when calculated using LV SVMVP compared to LV SVstandard, changed from 4/8/2/0 to 3/9/2/0, respectively. The MR ejection fraction increased by an average of 0.3%, 2.3%, and 4.3% in none, mild, and moderate MR groups, respectively.MV ejection fraction was higher when calculated using mitral valve backward volume to LV SVstandard, and between the reference method and LV SVMVP. LV SV4DF and LV SVstandard achieved a moderate ICC score of 0.75 (p < 0.01) but was better with LV SVMVP where the agreement was good, ICC = 0.86 (p < 0.001).ICC test was used to assess the agreement between the reference method and LV SVIn this study, we highlight the importance of including the MPV left ventricular doming volume within the left ventricular end-systolic volume during routine clinical assessment by CMR. This volume is the LV volume which lies on the ventricular side of the mitral valve, but on the atrial aspect of the atrioventricular groove, during end-systole. The doming volume is not crossing through the mitral valve in systole and does not contribute to the regurgitant volume of the mitral valve. We demonstrated that by including the MVP doming volume, the LV SV shows better agreement with the reference 4DF measured LV SV. When the MVP doming volume is not included in the LV SV, this results in significantly higher LV SV compared to 4DF-derived LV SV, potentially resulting in over-estimation of the left ventricular ejection fraction. Our study demonstrated that including MVP doming volume within the left ventricular end-systolic volume is important for precisely quantifying LVSV in patients with MVP. If MVP doming volume is not accounted for, mitral regurgitation quantification may be overestimated, leading to inaccurate grading and potentially affecting clinical decision-making regarding timely intervention.AI auto-contouring algorithms in most commercial cardiac post-processing software rely on the detection of mitral and aortic valves and the apex of the left ventricle for the left ventricular volume assessment, which is proven to provide significant improvement in accuracy and reproducibility . HoweverTransthoracic echocardiography (TTE) is recommended as a first-line imaging test for valvular heart disease assessment by the European Society of Cardiology guidelines. However, TTE typically has methodological limitations due to dependency on flow convergence region geometric assumptions and doppler measurement angle dependency. When various echocardiographic methods give inconsistent MR grading, CMR is recommended for further precise assessment of not only MR severity but also volume assessment . FurtherMR is reported to have a prevalence in 36.9% of adult patients with MVP and 35.7% of teenagers with MVP ,30, wherThe management of MVP remains a challenge due to the lack of standardised risk-stratification models. A recent study presented a mechanistic approach to sudden death prevention in mitral valve prolapse patients and classified MVP patients with a history of ventricular fibrillation and ventricular scarring as a high-risk group. Implantable cardioverter defibrillators should be strongly considered for high-risk patients to prevent sudden death and conservative management with cardiac surveillance monitoring with or without medical therapy is recommended for low-risk MVP patients . Althougp < 0.001) [4DF CMR offers a three-directional velocity-encoded dataset enabling quantification of peak velocities and transvalvular blood flow with improved precision. This is crucial in assessing complex valvulopathy. Previous studies looked at the clinical utility of 4DF CMR against routine CMR and TTE for MR quantification in MVP . Spampin< 0.001) . Our stuOther studies examined the impact of factoring in the MVP doming volume to assess LV function and MR severity by CMR assessment. When accounting for the MVP doming volume, 66% of patients with bi-leaflet mitral valve prolapse were recategorised, suggesting that without factoring in the MVP doming volume, one could underestimate MR severity by 1 grade in two-thirds of patients . AnotherMitral annular disjunction (MAD) has a strong association with ventricular arrhythmias and sudden cardiac death , with a In routine MVP cases, the quantification of MR by CMR needs to be precise. In this paper, we used 4DF MRI as a method for further optimisation of LVSV by the most standard methods of short-axis cine stack segmentation which is routinely used across the globe in clinical practice. The presently described method could be incorporated into clinical practice to improve the routine practice of left ventricle segmentation of short-axis cine stack and MR quantification, requiring prospective studies to ascertain if outcomes are improved.This is a single-centre retrospective observational study. Future studies are needed to confirm our findings in multi-centre prospective studies. The data set in this study is relatively small in scale, potentially leading to insufficient study power and type II error, although the effect size was large and standard deviations consistent. More extensive prospective studies are needed to explore the clinical impact of improved precision of left ventricular segmentation and its impact on clinical management. There is a mixed male and female population in this study however with the majority of female participants (73.3%), which is in keeping with the prevalence of MVP. The gender differences could be further explored where a balanced number of participants from each gender and a larger scale of data are available. Only two MVP patients (13.3%) in this study are classified as moderate MR and no severe MR patient with MVP was in our retrospective CMR registry, in which factoring in MVP doming volume could have a greater difference in calculating LV SV and thus MR severity could be recategorised with a higher percentage in moderate or severe MVP patients. Finally, no direct MR grading comparison with 4DF assessment was performed in this study, as the aim of this study was not to compare the performance of two MR grading methods but to highlight the importance of factoring in MVP doming volume into LVESV during routine CMR assessment.This study shows that when LV SV short-axis cine assessment incorporates MVP dooming volume, it significantly improves the precision of LV SV assessment compared to the reference 4DF method. Hence in routine clinical CMR practice, in cases with bi-leaflet MVP, MVP dooming needs to be factored into the left ventricular end-systolic volume to improve the accuracy and precision of quantifying mitral regurgitation.


Neonates with congenital diaphragmatic hernia (CDH) have varying degrees of pulmonary hypoplasia, pulmonary hypertension (PH) and cardiac dysfunction. These neonates frequently require vasoactive support and are at high risk for mortality and morbidity, including prolonged ventilator support, need for extracorporeal membrane oxygenation (ECMO), prolonged length of stay, and need for tracheostomy. However, identifying which infants are at increased risk can be challenging. In this study, we sought to investigate the utility of the inotropic score (IS) and vasoactive inotropic score (VIS) as tools to predict significant clinical outcomes and overall survival in patients with CDH. Additionally, we evaluated the correlation between IS/VIS and postnatal echocardiographic variables.This was a retrospective chart review of 57 patients with CDH whose postnatal care was based on a standardized institutional protocol. We calculated the IS/VIS at 6-, 12-, 24-, 48 hours of life (HOL), on the day of CDH repair and 24- and 48 hours after surgical repair. The association of these scores with postnatal echocardiographic markers was analyzed using Pearson's correlation and linear regression, while logistic regression was used for binary outcomes, and Cox proportional hazards regression was used to assess associations with survival.p = 0.034) and 10.1% increase in risk of death (p = 0.021). An increase in IS/VIS at 12-, 24- and 48-HOL was associated with posterior septal bowing in the first postnatal echocardiogram . Additionally, we noted an inverse relationship between IS  and VIS  on the day of repair and left ventricle (LV) systolic function in first postnatal echocardiogram. Increase in IS  and VIS  on the day of repair was associated with decreased LV function on the post-repair echocardiogram.We found that every one-unit increase in IS/VIS at 6 HOL was associated with 13% increase in the odds of ECMO (This retrospective study showed a significant association between IS/VIS obtained at various time points with clinical outcomes and echocardiographic findings in CDH, which could be used to guide prognosis and management in this patient population. Congenital diaphragmatic hernia (CDH) is a congenital defect that presents with varying degrees of pulmonary hypoplasia, pulmonary hypertension and ventricular dysfunction, which play significant roles in the overall morbidity and mortality , 2. DespIn critically ill patients, utilizing scoring indices that accurately reflect severity of illness have been shown to be valuable by providing guidance to bedside clinical management and patient care research , 7. CongWith the interplay of complex physiology that exists in the CDH population, we hypothesized that the inotropic score and vasoactive inotropic score would be useful and effective bedside tools to predict clinical outcomes in CDH. To the best of our knowledge, no such studies have been conducted in the CDH population.Our primary objective with this study was to determine the correlation of the inotropic and vasoactive inotropic scores at 6-, 12-, 24-, 48-HOL, on the day of CDH repair and 24- and 48 hours following repair with various clinical outcomes and postnatal echocardiographic parameters. Inotropic score (IS) and vasoactive inotropic scores (VIS) are calculated as shown below , data was extracted from the medical records, and stored in encrypted folders on secure institutional servers.This retrospective cohort study included neonates ≤28 days of age admitted to the level IV NICU between January 2017 and December 2021 with a diagnosis of CDH, and who received one or more inotropic medications  during their hospital stay. Our exclusion criteria consisted of patients with critical congenital heart disease  or cardiac intervention within first few days of life), multiple congenital anomalies, and lethal genetic abnormalities who underwent redirection of care within 48 h of life.Baseline demographics, clinical, and outcomes data were obtained. Demographic data included gestational age, birthweight, sex, and APGAR scores at 1- and 5 min. Clinical data included fetal CDH measurements {Observed/Expected Total Fetal Lung Volume (O/E TFLV), Lung-Head Ratio (LHR) and percent liver herniation (%LH)}, severity of hernia  , side ofThe inotropic score and vasoactive inotropic scores were calculated (using the formulas above) for each of the following time points: 6-, 12-, 24-, 48-HOL, on the day of CDH repair and 24- and 48 hours.1.Associated IS/VIS at 6-, 12-, 24- and 48-HOL, on the day of repair and 24- and 48 hours following repair with primary and secondary outcomes measures.2.Associated 6-, 12-, 24- and 48-HOL IS/VIS with echocardiographic measures obtained in first postnatal echocardiogram.3.Associated IS/VIS on the day of repair and 24- and 48 hours following repair with first postnatal, post repair and 28-day echocardiographic measurements.Following data extraction, we analyzed IS/VIS at different time points with echocardiographic data and clinical outcomes. We performed the following analyses:Logistic regression and receiver operating characteristic (ROC) curve analyses were used to investigate associations of IS/VIS with binary outcomes, while Pearson's correlation and linear regression analysis were used for continuous outcomes and Cox proportional hazards regression was used to assess associations with survival time. Multiple logistic regression analysis was used to examine associations of IS/VIS with ECMO, after controlling for CDH severity. SAS version 9.4  was used for data analysis. A two-sided 5% significance level was used for hypothesis tests.After excluding 4 patients with lethal genetic syndrome who had redirection of care within the 24 h of birth and 1 patient with diaphragmatic eventration, 57 patients were included in the final study cohort .Patient characteristics are shown in p = 0.034) associated with ECMO (p = 0.045) associated with ECMO . Additionally, we found that every one-unit increase in IS and VIS at 6 HOL was associated with a 10.1% increase in risk of death  (IS and VIS at 6 HOL were significantly (ith ECMO . Every o= 0.021) .We correlated the IS/VIS obtained at various time points in the first 48 h of life  with echocardiographic markers of pulmonary hypertension and ventricular function seen in the first postnatal echocardiogram. We found that early increased VIS/IS was associated with worsening pulmonary hypertension.p = 0.038). We also noted that higher VIS at 12-HOL was significantly associated with bowing of interventricular septum (IVS) , higher IS at 12-HOL was significantly associated with IVS bowing  and higher IS and VIS at 24 hours were associated with bowing IVS . Higher VIS at 48 hours were significantly associated with bowing IVS . There was an inverse relationship between in IS/VIS at 6 HOL  12 HOL  and 24-HOL  with septal flattening, with higher scores associated with lower odds of septal flattening. The associations of IS/VIS obtained in first 48-HOL with first postnatal echocardiogram is shown in We found that IS/VIS obtained at 6-HOL was associated with increased odds of bidirectional atrial level shunting, with every one-unit increase in IS/VIS at 6-HOL associated with 13.9% increase in the odds of bidirectional atrial level shunting (odds ratio (OR) = 1.139, 95% CI: 1.007–1.289, r = −0.281, p = 0.036, r = −0.288, p = 0.031, r = −0.285, p = 0.037). Similarly, IS at day of repair explained 9.4% of the variation in LV function with every one-unit increase in IS predicting a 0.065-unit decrease in LV function in the post-repair echocardiogram  (p < 0.001) and 0.074 (p < 0.001), respectively  with the first post-natal, post-repair and 28-day echocardiogram , we note= 0.024) . Finallyectively . We founWe found IS and VIS to be a useful tool for assessing patients with CDH. We observed increased IS/VIS obtained at 6- HOL was associated with ECMO and mortality. IS/VIS obtained at different time points correlated well with various echocardiographic markers of PH and ventricular function, thus highlighting their potential utility as bedside tools for clinical assessment and guiding management.A thorough understanding of the pathophysiology of systemic hypotension in CDH is still evolving. Elevated pulmonary vascular resistance (PVR) and right ventricular (RV) afterload decreases pulmonary blood flow leading to decreased pulmonary venous return and net cardiac output. Recently, ventricular dysfunction has emerged as an important area of clinical significance . LV dysfEtiology of LV dysfunction in CDH is incompletely understood. The fetal and post-natal cardiac physiology contributing to LV dysfunction has been summarized in Various fetal and postnatal mechanisms for LV dysfunction in the CDH population have been hypothesized, such as underdeveloped left sided cardiac structures, increased SVR and ventricular interdependence –17. EarlIn our study, we observed a significant correlation between IS/VIS at 12-, 24- and 48-HOL with septal bowing seen in the first postnatal echocardiogram-with higher scores associated with septal bowing. We also found an inverse relationship between IS/VIS at 6-,12- and 24-HOL with septal flattening, which can be explained by preserved interventricular interaction with septal flattening, as opposed to septal bowing where the LV preload is decreased, and myocardial contractility is altered with geometric distortion of the left ventricle. This results ultimately in decreased stroke volume and cardiac output thus needing additional inotropic support and higher IS/VIS scores.Our results showed that an increase in IS/VIS on the day of repair was associated with decreased LV function in the first postnatal and post-repair echocardiogram which may be secondary to persistence of fetal cardiac maladaptation and delays in postnatal transition, resulting in increased need for vasoactive support during the repair and increased metabolic demands during the procedure. In addition, our results showed that an increase in IS/VIS correlated with increase in LV function in the 28-day echocardiogram. This can be explained by the fact that after CDH repair, the mediastinum and cardiac structures shift into anatomically appropriate positions, improving venous return and optimizing preload, permitting recovery of LV function and remodeling of myocardium, all of which contribute to the improved function noted on the echocardiogram obtained after 28 days of life.The IS/VIS have been validated as a clinical measure of cardioactive medication use in both the adult and pediatric population , 24, 25.We report that IS and VIS are useful prognostic variables in the CDH patient population, where there is an increased need for inotropic support and associated adverse outcomes, such as mortality and ECMO. We add a unique perspective by correlating these scoring modalities with specific echocardiographic markers in neonates with CDH. Additionally, we have taken both pre- and post-repair CDH physiologies into consideration by obtaining serial IS/VIS measurements up to 48 hours of life and 48 h following surgical repair. This provides a temporal understanding of changing CDH physiology from birth until after surgical repair, thus validating the contribution of ventricular remodeling and improvement in cardio-pulmonary physiology following repair in CDH.One of the major limitations of the study is the retrospective nature of the study due to which the specific timing of echocardiogram, timing, rate of escalation, dosing and type of vasopressor support was not standardized. Additionally, since the study focused specifically on inotropic use, we could not account for other management strategies for systemic hypotension such as use of fluid boluses, blood transfusions and/or use of hydrocortisone, which may have impacted the overall clinical picture of this patient population. We could also not account for contributors of systemic hypotension such as pulmonary vasodilators and/or sedation medications. VIS and IS ≥ 7.5 at 6-HOL had high specificity but low sensitivity in predicting ECMO. Thus, scores <7.5 at 6-HOL could be useful for identifying patients at low risk for ECMO, but other prognostic variables should be investigating to discriminate those who will versus will not receive ECMO among those with VIS and IS ≥ 7.5 at 6-HOL. Predictive accuracy for ECMO decreases after 6-HOL but it is fortunate that the highest predictive accuracy occurs early when it is most useful. Examining predictive accuracy at numerous time points increases the probability of Type I Error . Therefore, findings from this study should be considered exploratory and future prospective studies should focus on IS/VIS at 6-HOL and within the first day of repair. Despite these limitations, this is one of the first studies validating the use of these scoring tools in predicting outcomes and correlating with echocardiographic markers in the CDH population.Our goal with this study is to stimulate further research into the use of IS/VIS following its validation in the CDH population. This data has now laid the foundation for a potential pilot prospective model to further investigate these scoring tools in a standardized protocol-based setting and help predict various short-term and long-term clinical outcomes. This would further add to the evidence and encourage the use of these scoring modalities in the CDH population. Additionally, it would be beneficial to investigate the role of variables such as sex and ethnicity, to supplement these scoring tools, to further understand the impact of these factors on the overall clinical outcomes in CDH. In our study, IS and VIS ≥ 7.5 at 6 HOL was found to have high specificity but low sensitivity in predicting ECMO. These results suggest that IS and VIS < 7.5 at 6 HOL indicate low risk for needing ECMO, but future research should attempt to identify other prognostic variables to discriminate those who will versus will not need ECMO among CDH patients with IS and VIS ≥ 7.5 at 6 HOL. Finally, with the advent of fetal interventions, the impact of FETO on IS/VIS would be an indirect assessment of the impact of this fetal intervention on ventricular function in CDH, which is an area of ongoing research.The IS/VIS are objective markers of ventricular function and, with this study, we have correlated these scores at different time points with short term outcomes, and echocardiographic markers of PH/myocardial dysfunction in CDH. This study encourages the use of these scoring modalities in the CDH population and has opened avenues for further research.


The triglyceride glucose (TyG) index has been considered a new biomarker for the diagnosis of angiocardiopathy and insulin resistance. However, the association of the TyG index with subclinical left ventricular (LV) systolic dysfunction still lacks comprehensive exploration. This study was carried out to examine this relationship in patients with type 2 diabetes mellitus (T2DM).A total of 150 T2DM patients with preserved LV ejection fraction (LVEF ≥ 50%) from June 2021 to December 2021 were included in this study. The subclinical LV function was evaluated through global longitudinal strain (GLS), with the predefined GLS < 18% as the cutoff for subclinical LV systolic dysfunction. The TyG index calculation was obtained according to ln (fasting triglycerides (mg/dL) × fasting glucose (mg/dL)/2), which was then stratified into quartiles (TyG index—Q).n = 38, Q2 (8.89 < TyG index ≤ 9.44) n = 37, Q3 (9.44 < TyG index ≤ 9.83) n = 38, and Q4 (TyG index > 9.83) n = 37) were conducted. A negative correlation of the TyG index with GLS  was revealed according to correlation analysis. After gender and age were adjusted in multimodel logistic regression analysis, the higher TyG index  showed a significant association with GLS < 18%, which was still maintained after further adjustment for related clinical confounding factors . Receiver operator characteristic analysis indicated a diagnostic capacity of the TyG index for GLS < 18% .Analyses of clinical characteristics in the four TyG indexes-Q (Q1 (TyG index ≤ 8.89) A higher TyG index had a significant association with subclinical LV systolic dysfunction in T2DM patients with preserved ejection fraction, and the TyG index may have the potential to exert predictive value for myocardial damage. A causal relationship exists between diabetes mellitus and heart failure. For instance, diabetic cardiomyopathy as a diffuse cardiomyopathy resulting from glucose metabolism disorder has received increasing interest in recent years, which generally manifests as pathological cardiac remodeling and systolic and diastolic dysfunction and may eventually develop into overt heart failure , 2. StudA total of 165 hospitalized patients with T2DM categorized by World Health Organization criteria  at the D2 (kg/m2). Hypertension was described as systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg without antihypertensive medication or with a previous hypertension physician identification. After an at least 8-h overnight fast, an evaluation of total cholesterol, triglycerides, high- and low-density lipoprotein cholesterol, and uric acid was performed with an automatic biochemical analyzer. The fasting glucose index was detected with glycosylated hemoglobin (HbA1c) checked by high-performance liquid chromatography. Immunoturbidimetry depending on a COBAS INTEGRA 400 plus autoanalyzer (Germany) was carried out to determine the urinary albumin-to-creatinine ratio (UACR). The ln[fasting triglycerides (mg/dL) × fasting glucose (mg/dL)/2]   was usedThe ultrasound measurements of all subjects were collected according to the guidelines of the American Society of Echocardiography . Two-dimEchocardiography images for 2D speck-tracking echocardiography (STE) with a rate of 60–90 frames/s in three cardiac cycles were acquired. Then, the LV views of the apical four-chamber, apical two-chamber and apical three-chamber were analyzed using QLAB 8.1 2D imaging system. The trace of the endocardial border at end-diastole was performed in a manual manner to obtain the 2D strain‒time curve and bull's-eye plot of 17 segments of LV . One-way ANOVA or the Kruskal‒Wallis test was used to test for differences between groups. The Bonferroni test was adopted for post hoc comparisons. Least Pearson's Chi-square test was performed on categorical variable comparisons. The correlation between the TyG index and GLS was evaluated according to Pearson correlation coefficients. Three forced-entry logistic regression models were performed to determine the independent association of GLS < 18% with TyG index: model 1 (an unadjusted model), mode 2 (a multivariable model) adjusted for age and gender, and model 3 (a multivariable model) adjusted for age, gender, diabetes duration, systolic pressure, HbA1c, BMI, hypertension, heart rate, logarithmic microalbuminuria, LVEF and insulin therapy. To confirm an alternative index for identifying reduced GLS < 18%, models covering the TyG index and HbA1c were established, followed by a comparison of both according to the area under the receiver operating characteristic (ROC) curves (AUC). The measurement of GLS was performed by one professional physician to avoid interobserver and intraobserver variability. To prevent the ambiguity of negative size to a value, the GLS was given in the absolute value form. All statistical analyses were carried out on IBM SPSS statistics 26.0, with a P < 0.05). Moreover, insulin therapy statistically varied across the TyG index quartiles (P = 0.025) without obvious differences in sex, age, diabetes duration or BMI among groups (P > 0.05).A total of 150 T2DM patients ) were included, with the clinical features across quartiles of the TyG index listed in Table P > 0.05). However, the GLS exhibited a stepwise decrease in line with the increase in TyG index quartile . Pearson correlation analysis indicated strong and negative correlations of the TyG index  and HbA1c  with GLS.Table P = 0.003; Q4 vs Q1: OR 5.83, 95% CI 2.15 to 15.82, P < 0.001, respectively). After gender and age adjustment, the relation of the TyG index of the 3rd quartile and 4th quartile with GLS < 18% still existed . With further adjustment for confounders of age, gender, diabetes duration, systolic pressure, HbA1c, BMI, hypertension, heart rate, logarithmic microalbuminuria, LVEF and insulin therapy, the higher quartile of the TyG index remained an independent risk indicator related to GLS < 18% .Table P < 0.001) displayed a sensitivity of 73.8% with a specificity of 54.3% for predicting GLS < 18%. HbA1c also exhibited a high AUC of 0.742, a sensitivity of 62.9% and a specificity of 76.2% for reduced GLS < 18% (P < 0.001). Subsequently, the composite variable with the TyG index and HbA1c combined showed increased AUC and diagnostic values .The ROC curves depicted in Fig. Figure To the best of our knowledge, the present study was the first to explore the association of the TyG index with LV longitudinal myocardial function in patients with type 2 diabetes and preserved ejection fraction. The results demonstrated a close relationship of the increased TyG index with an elevated risk of LV longitudinal myocardial dysfunction. Moreover, it was found that the composite parameters of the TyG index and HbA1c exhibited a certain value for the identification of reduced GLS < 18%.The correlation between heart failure and diabetes has been notably confirmed by epidemiological and clinical studies –17. HoweTo date, the standard estimation of insulin resistance, the hyperinsulinemic-euglycemic clamp (HEGC) test, still requires diagnostic technology, which is expensive and is not available for basic-level hospital utilization. The TyG index, as an ideal surrogate of insulin resistance regardless of insulin treatment status, has been widely validated to be robustly related to cardiovascular events –26. HoweIn a study with a large cohort followed for 10 years, Sánchez-Íñigo et al. first proposed a positive correlation of the TyG index (AUC: 0.708) with heart events . SimilarPrevious studies have mostly focused on patients with existing symptoms of heart failure. In contrast, more emphasis was placed on early attention to the LV subclinical phase of T2DM patients in the present study. The relatively time-consuming requirement, high cost, and professional features of speckle tracking echocardiography and the accumulated training to perform effective measurement and analysis may limit its application in daily clinical practice for general diabetes physicians without enough experience with this technique. As stated above, the hyperglycemia elicited by insulin resistance activates the cascade of diabetic heart dysfunction, which induces metabolic disorders, followed by endothelial dysfunction, cardiac hypertrophy and fibrosis , 35. HEGThe limitations of this study should also be considered. First, as a result of the cross-sectional study, the specific causal relationship of the TyG index with reduced GLS remains unclear. Second, not all patients without coronary artery disease have undergone invasive coronary angiography. Third, despite the analysis on the medication of the patients, the underlying contributions of medications for lipid-lowering and LV function improvements failed to be controlled in this study. Fourth, insulin resistance parameters were not analyzed in this study. Last, covariates involved in the multivariable regression models were taken as potential confounders based on previous studies , 38 or tIn conclusion, the present study demonstrated a close relationship between an elevated TyG index and decreased GLS, which could be adopted as a sensitive and practical index to predict subclinical LV systolic dysfunction. Therefore, the TyG index should be punctually monitored for T2DM patients with preserved LVEF, which is expected to effectively identify the occurrence of diabetic heart disease and delay its development.