Heart transplantation is often an unrealizable therapeutic option for end-stage heart failure, which is why mechanical left ventricular assist devices (LVADs) become an increasingly important therapeutic alternative. Currently, there is a lack of information about molecular mechanisms which are influenced by LVADs, particularly regarding the pathophysiologically critical renin angiotensin system (RAS). We, therefore, determined regulation patterns of key components of the RAS and the β-arrestin signaling pathways in left ventricular (LV) tissue specimens from 8 patients with end-stage ischemic cardiomyopathy (ICM) and 12 patients with terminal dilated cardiomyopathy (DCM) before and after LVAD implantation and compared them with non-failing (NF) left ventricular tissue samples: AT1R, AT2R, ACE, ACE2, MasR, and ADAM17 were analyzed by polymerase chain reaction. ERK, phosphorylated ERK, p38, phosphorylated p38, JNK, phosphorylated JNK, GRK2, β-arrestin 2, PI3K, Akt, and phosphorylated Akt were determined by Western blot analysis. Angiotensin I and Angiotensin II were quantified by mass spectrometry. Patients were predominantly middle-aged (53 ± 10 years) men with severely impaired LV function (LVEF 19 ± 8%), when receiving LVAD therapy for a mean duration of 331 ± 317 days. Baseline characteristics did not differ significantly between ICM and DCM patients. By comparing failing with non-failing left ventricles, i.e., before LVAD implantation, a downregulation of AT1R, AT2R, and MasR and an upregulation of ACE, ACE2, GRK, β-arrestin, ERK, PI3K, and Akt were seen. Following LVAD support, then angiotensin I, ACE2, GRK, and β-arrestin were downregulated and AT2R, JNK, and p38 were upregulated. ACE, angiotensin II, AT1R, ADAM17, MasR, ERK, PI3K, and Akt remained unchanged. Some regulation patterns were influenced by the underlying etiology of heart failure, the severity of LV dysfunction at baseline, and the duration of LVAD therapy. Key components of the RAS and β-arrestin signaling pathways were divergently altered in failing left ventricles both before and after LVAD implantation, whereas a remarkable fraction remained unchanged. This indicates a rather incomplete molecular reverse remodeling, whose functional relevance has to be further evaluated. Due to a further increasing life expectancy and as a clear consequence of improving therapeutic options, the prevalence of patients suffering from end-stage heart failure will continue to increase . Even thWe, therefore, comprehensively analyzed adaptations of key mediators of the RAS and the known downstream effectors of the β-arrestin signaling pathway in failing left ventricles before and after cardiac unloading by LVAD therapy. We furthermore strove to delineate whether these adaptations are influenced by other determinants such as the heart failure etiology or the duration of LVAD therapy.This study was conducted according to the declaration of Helsinki and was approved by the local ethics committee. All participants gave a written informed consent.Eight patients with end-stage ischemic cardiomyopathy (ICM) and twelve patients with end-stage dilated cardiomyopathy (DCM) who were supported with a LVAD in a bridge to transplant (BTT) intention were included in this study. Both at LVAD implantation and at receiver heart explantation during the heart transplantation procedure, tissue specimens were acquired from left ventricles, respectively, ensuring not to sample scar or fibrotic tissue. Tissue specimens were then instantaneously deep-frozen in liquid nitrogen and stored at − 80 °C.Human heart tissue pieces were transferred to a Precellys Lysing Kit Cup  with TriFast  and shredded with a FastPrep-24 homogenizer . After the addition of chloroform, the uppermost aqueous phase contains the RNA, which was added to a new cup containing isopropanol to precipitate a pellet.The pellet was washed with − 20 °C ethanol (70%), dried, and dissolved in RNAse-free water.The RNasy Mini Kit and RNase-Free DNase Set  were used for the DNAse digestion following the manufacturer’s instructions.Nucleic Acid Concentration measurements were taken with the NanoDrop 2000c Spectrophotometer  at a wavelength of 260 nm.The cDNA synthesis was performed with the M-MLV Reverse Transcriptase  according to the manufacturer’s instructions.Primers were ordered by ThermoFisher Scientific (USA) using the TaqMan assay and the quantitative real-time PCR was performed on the ViiA 7 real-time PCR system  using the ViiA 7 Software . There was a threefold determination for each target:AT1R (Hs00258938_m1), AT2R (Hs02621316_s1), ACE (Hs00174179_m1), ACE2 (Hs01085333_m1), MasR (Hs00267157_s1), ADAM17 (Hs01041915_m1), and HPRT (HS02800695_m1).The relative quantification of gene expression was performed using the standard curve method. When using the standard curve method, the quantity of each experimental sample is first determined using a standard curve and is then expressed relative to a calibrator sample. In order to use this quantification method, all samples of interest were pooled and from this four tenfold serial dilutions of cDNA template known to express the gene of interest were prepared. Each serial dilution was used in separate real-time reactions, and their threshold cycle (Ct) values were determined. The Ct values were plotted versus the dilution factor and the data fitted to a straight line. This plot was then used as a standard or calibration curve for extrapolating relative expression level information for the same gene of interest in unknown experimental samples. The relative quantification calibration curve result for the gene of interest was normalized to that of a housekeeping gene in the same sample, and then the normalized numbers are compared between samples to get a fold change in expression. A standard or calibration curve was generated separately for each gene of interest and the used housekeeping gene HPRT.The tissue samples were pulverized, mixed with lysis buffer, mechanically crushed, and repeatedly cooled in liquid nitrogen. The samples were incubated on ice for 30 min, vortexed for 5–10 min, and the supernatant was transferred to a new cup after centrifugation. The protein concentration was determined by the Pierce BCA Protein Assay Kit  according to the manual instructions. The absorbance was measured by the Infinite M200 Pro Plate Reader  at 540 nm.The protein concentration of each sample was set to 1 μg /μl, consisting of 20% Blue Buffer (+ 10% β-mercaptoethanol), the lysate ,and Dulbecco’s phosphate buffered saline (PBS) . To specifically detect membrane proteins, the samples were denatured at 37 °C for 30 min and cytosolic proteins were denatured at 95 °C for 5 min.The polymerized gels consist of an 8% SDS-separating gel and a 5% SDS-stacking gel. Electrophoresis took place at 60 mA for about 2 h and a wet transfer at 400 mA for 2 h.To block unspecific proteins, the membranes were incubated with 5% milk in TBST for 1 h. The primary antibody was diluted in 5% milk in TBST and incubated at 4 °C overnight.The antibodies were ordered as follows:ThermoFisher, USA: Anti-PIK3CA (MA5-14870), Anti-ERK1/2 (MA5-15134), Anti-pERK1/2 (700012), Anti-p38 (702273), Anti- pp38 (MA5-15218), Anti-JNK3 (MA5-15403), Anti-pJNK1/2/3 (PA5-36753), Anti-GRK2 (PA5-27480), Anti-β-Arrestin2 (PA1-732) and BD Biosciences, USA: Anti-Akt (610876), Anti-Akt pS473 (560397), and Sigma-Aldrich, Germany: Anti-GAPDH (G8795).The secondary antibody was diluted in 5% milk in TBST and incubated for 1 h at room temperature. Antibodies were purchased from GE Healthcare, UK: Anti-Rabbit ECL IgG (whole Ab) HRP-linked (sheep) (NA934V) and Anti-Mouse ECL IgG (whole Ab) HRP-linked (sheep) (NA931VS).The membranes were visualized with the WesternBright ECL , according to the provided protocol. Development was performed with the Super-XR FuJi X-ray films  and the M35 X-OMAT Processor . The exposed X-ray films were scanned with the ChemiDoc MP Imaging System  and the degrees of blackening of the bands were determined with the ImageJ program.The blackening of the background was subtracted from the band of the sample and normalized to GAPDH. Normalization to GAPDH acts as a protein loading control.Angiotensin metabolites in cardiac tissue were quantified by Attoquant Diagnostics  as described previously . Frozen t-test was used to compare healthy NF patients with heart sick patients. In this case, the variance equality of the groups was first checked via the Levene test. If the variances were equal, a two-sample t-test was used; otherwise, with unequal variance, the Welch test was used instead.The statistical analysis was performed via GraphPad Prism and SPSS. The diagrams were created with GraphPad PRISM. The paired t-test was used to investigate whether LVAD therapy led to changes in the expression of the individual targets. The unpaired The 2-way ANOVA test was used to investigate whether the groups formed by disease, duration of treatment, and ejection fraction showed a difference in treatment between groups and whether there was an interaction between group effect and time.α-error of less than 5% was defined as statistically significant (p < 0.05).An Most patients were middle-aged men age 53 ± 10 years) with a severely impaired left ventricular systolic function (LVEF 19 ± 8%) receiving the guideline-recommended pharmacological and device therapy, when a LVAD system was implanted. The mean duration of mechanical unloading then was 331 ± 317 days, before heart transplantation could be performed. There were no significant differences between patients suffering from DCM as compared to those with ICM (see Table 0 years wP = 0.084) irrespective of the heart failure etiology  or the time on LVAD. In contrast, Angiotensin II was unaffected by LVAD therapy , which reached statistical significance only when comparing CHF patients with lower vs. higher than median LVEF at baseline (P = 0.028) indicating a more pronounced ACE upregulation in more severely impaired left ventricles. In contrast, ACE regulation was not influenced by LVAD therapy  tended to be upregulated in CHF vs. NF . In contrast to ACE, upregulation of ACE2 was more pronounced in less severely impaired left ventricles . Even though LVAD therapy seemed to moderately reduce ACE2 expression, this only reached statistical significance in DCM patients and in patients with shorter time on LVAD therapy .P < 0.001), but was further on not altered by LVAD therapy  predominantly in DCM patients, LVAD therapy caused an upregulation , which did not differ between DCM and ICM. Interestingly, this upregulation was more pronounced in left ventricles with a more reduced ejection fraction at baseline .Even though AT2R tended to be likewise downregulated in CHF as compared to NF  and without any alteration upon LVAD therapy . LVAD therapy significantly lowered these increased GRK2 expression levels  predominantly in DCM patients and in those treated by LVAD for a longer time  was significantly higher expressed in CHF as compared to NF , whereas a shorter LVAD duration did not have any impact  and in those treated for a shorter time with LVAD  and with a higher LVEF at baseline  and were analyzed as important downstream targets of GRK2/β-arrestin 2 signaling. ERK tended to be upregulated in CHF patients and after LVAD therapy meeting statistical significance in ICM patients  and was not influenced by baseline LVEF and duration of LVAD therapy  especially in those with a longer time on LVAD  and a lower LVEF at baseline , which was otherwise irrespective of heart failure etiology. Phosphorylated Akt in turn was not significantly altered in heart failure and after LVAD therapy  and an upregulation of many other upstream and downstream located pathway components .LVAD therapy then had a complex and highly heterogeneous effect with a down- , up- , or unchanged regulation pattern .Some of these expression changes or regulation patterns depend on the etiology of heart failure , the severity of heart failure before LVAD implantation, and the duration of LVAD therapy.Our work analyzed adaptations of key mediators of the cardiac renin angiotensin system and the associated β-arrestin signaling pathway in failing left ventricles before and after LVAD therapy and yielded the following main results:Our finding of a downregulation of RAS receptors in end-stage heart failure well corroborates the available literature –17, evenIn contrast, however, important up-  and downstream targets  of the RAS were higher expressed or upregulated in terminal heart failure. It would be tempting to speculate that this regulation pattern aims at compensating or overcoming the downregulation of RAS receptors at both the up- and downstream sites in order to maintain the overall signaling capacity. This would particularly apply to the protective ACE2/Ang1-7/MasR pathway, since only ACE2, but not ACE expression was significantly altered in the complete patient cohort of our work. A similar finding was reported by Ferrario et al. in their animal study, where an angiotensin receptor blockade using losartan augmented ACE2, but not ACE mRNA expression . Having The impact of LVAD therapy is well described for structural  or the Furthermore, genomic profiling revealed a LVAD-mediated alteration of several gene sets, such as genes involved in cell growth, apoptosis and cell signaling or genes controlling for the formation of vascular networks and the expression of diverse transcription factors , 24. IntThis was partly done by Baba et al., who focused on phosphorylation patterns of mitogen-activated protein kinases  and of the anti-apoptotic kinase Akt . In thisBeyond these single targets and in consideration of the many other parameters, which we determined in our study, we generally found a rather heterogenous LVAD-mediated effect on the RAS and β-arrestin signaling pathway with a downregulation of angiotensin I, ACE2, GRK, β-arrestin, an upregulation of AT2R, JNK, p38 and an unchanged regulation pattern of angiotensin II, ACE, ADAM17, AT1R, MasR, ERK, PI3K and Akt. Notably, most of these expression changes at least partly occurred in a direction, which aimed at restoring the expression patterns of non-failing left ventricles. This supports and extends the existing evidence, which clearly demonstrates a positive effect of cardiac unloading on many pathophysiologically relevant pathways in heart failure, as it was excellently gathered by Birks . But on A considerable number of RAS-/ β-arrestin pathway components was significantly influenced by the underlying etiology of heart failure , the extent of left ventricular impairment before LVAD implantation , and the duration of LVAD therapy: for example, ACE2 was significantly downregulated after LVAD therapy only in DCM, but not in ICM patients and in those with a shorter than a longer duration of LVAD therapy. This means that several factors exist—beyond cardiac unloading per se—which determine the molecular setup of LVAD-treated hearts, and against this background, it would be most important to investigate, whether this proves true also for clinical effects, i.e., the probability of cardiac recovery.Our study might have some limitations: Firstly, our patient cohort comprising 20 individuals seems to be rather small. But otherwise this compares well to other studies, which partly investigated even smaller numbers of patients , 30, 31,By investigating key components of the RAS and β-arrestin signaling pathways both before and after LVAD implantation, we found complex and remarkably different molecular adaptation patterns, which were additionally influenced by factors such as the etiology of heart failure, the duration of LVAD therapy, or the severity of left ventricular impairment. Future work is necessary to delineate the functional relevance of our findings.


Transthyretin‐related amyloidosis (ATTR) is a subgroup of amyloidosis that results from extracellular misassembled and toxic amyloid deposits affecting multiple organ systems, and cardiac tissues in particular. Because ATTR often presents as heart failure with preserved ejection fraction (HFpEF), it has been largely underdiagnosed. Once considered incurable with a grave prognosis, ATTR cardiomyopathy has seen the development of promising alternatives for diagnosis and treatment, with early diagnosis and treatment of ATTR cardiomyopathy highly beneficial due to its high mortality rate. For instance, diagnosing ATTR cardiomyopathy previously required a cardiac biopsy, but new modalities, such as cardiac magnetic resonance imaging and radionuclide bone scans, show promise in accurately diagnosing ATTR cardiomyopathy. Ongoing research and clinical trials have focused on identifying new treatments which primarily target amyloid fiber formation by inhibiting TTR gene expression, stabilizing the TTR tetramer, preventing oligomer aggregation, or affecting degradation of amyloid fibers. In this review, we describe the advances made in the diagnosis and treatment of ATTR in order to increase awareness of the disease and encourage a lower threshold for ATTR workup. Our review also highlights the need for improving the screening, diagnosis, and treatment guidelines for ATTR cardiomyopathy. Patients can present with only cardiac involvement, only peripheral or autonomic neuropathy, or a combination based on the type of ATTR and mutation.Patients with ATTR cardiomyopathy present with classic signs of heart failure, including shortness of breath, orthopnea, fatigue, and peripheral edema. In addition to symptoms of HFpEF, ATTR can be accompanied by atrial fibrillation, syncope, conduction system disease, and stroke; these are thought to result from the infiltration of ATTR into cardiac tissue and the peripheral nervous system. Peripheral neuropathy such as numbness, paresthesia, and pain, and autonomic dysfunction such as orthostatic hypotension, and bowel/bladder dysfunction are also seen in these patients.5The diagnosis of ATTR cardiomyopathy is challenging because patients present with a constellation of symptoms seen in more common conditions, such as hypertensive left ventricular hypertrophy, carpal tunnel syndrome, or idiopathic HFpEF. Clinicians are trained to attribute these nonspecific manifestations to more common diagnoses. In addition, the lack of consensus in screening criteria poses another barrier to routinely considering this condition in a diagnostic workup.Clinicians have now recognized that a high index of suspicion for recognizing the disease is required to diagnose ATTR cardiomyopathy.5.1A diagnosis of ATTR cardiomyopathy has previously required a cardiac biopsy. Given the perceived rarity of the disease and the low positive predictive value of this invasive diagnostic approach, cardiac biopsy has not been frequently used, which has perhaps contributed to the underdiagnosis of ATTR. When the decision is made to perform a biopsy, the most minimally invasive approach is often used. Common biopsy sites are the salivary gland, the subcutaneous fatty tissue of the abdominal wall, or the rectal, kidney, or gastric mucosa.5.2Echocardiography remains an excellent screening tool for ATTR cardiomyopathy given its cost‐effectiveness, accessibility, and lack of radiation. It is the initial diagnostic test of choice when cardiac amyloidosis is suspected.Early findings on echocardiogram usually include a nondilated left ventricle with a concentrically thickened myocardium characterized by increased echogenicity, thickening of the right ventricular free wall, and visibly dilated atria and interatrial septum.5.3Cardiac magnetic resonance imaging (MRI) is a sensitive modality in diagnosing cardiac amyloidosis. The key features of amyloidosis cardiomyopathy on cardiac MRI are increased left ventricular wall thickness, transmural or subendocardial late gadolinium enhancement, and elevated native T1 and extracellular volume fraction.5.499mTc‐PYP) as a cardiac imaging tracer, the sensitivity was 97%, with 100% specificity.99mTc‐MDP) was noted to localize ATTR deposits by the Perugini grading system, as 99mTc‐MDP preferentially binds to ATTR.99mTc‐DPD) was more accurate in identifying the amyloid deposit burden than was MRI. In the United States, given that 99mTc‐MDP is widely available in routine bone scans, some experts argue that it would be a more feasible option than cardiac MRI in clinical practice.Some data suggests that cardiac uptake on a radionuclide bone scan is >99% sensitive but not specific for ATTR cardiomyopathy.6Once considered untreatable, ATTR amyloidosis now can be managed with contemporary therapies that improve survival rate and halt disease progression. Currently, multiple drugs are in various stages of clinical trials, and these new treatments may improve the prognosis of patients with a confirmed diagnosis of ATTR amyloidosis Table .6.1Most medications being developed or tested in ongoing clinical trials have focused on either preventing the formation of the TTR‐related amyloid protein or inhibiting the degradation of the TTR tetramer. The tetramer is the four oligomer unit that normally forms the binding site for thyroxine and vitamin A in plasma and cerebrospinal fluid. The mutation or aging of TTR can result in instability of the tetramer, which, in turn, may cause the tetramer to breakdown and form an insoluble beta‐pleated sheet, which frequently infiltrates the myocardium.6.1.1Patisiran and revusiran are small interfering ribonucleic acids (siRNAs) that work to block the expression of both variant and wild‐type TTR.6.1.2Tafamidis, a selective tetramer stabilizer, is a small molecule that blocks the rate‐limiting step in TTR genesis by binding to the T4 binding sites to inhibit the dissociation of TTR tetramers.Evidence suggests that tafamidis treatment may be more effective in patients with New York Heart Association (NYHA) class I/II heart failure than in those with class III disease.Tafamidis is currently under postmarketing surveillance. As it is a new medication, most physicians have yet to familiarize themselves with its general indications, doses, and side effects. In Japan, as a general rule, only institutions with a multidisciplinary team that performs more than 15 myocardial biopsies annually in suspected cases are permitted to start administering tafamidis.Diflunisal, a nonsteroidal anti‐inflammatory drug (NSAID), has been shown to nonselectively bind to the T4 binding site of TTR and stabilize TTR tetramers to prevent amyloid fibril formation in vitro.AG10 is a potent selective kinetic stabilizer of TTR.6.1.3EGCG is a catechin in green tea that was found to bind to soluble TTR and decrease the frequency of tetramer dissociation in vitro.6.1.4Doxy/TUDCA appears to be effective in vitro in causing complete disaggregation of amyloid fibers and generating nontoxic molecular species.Miridesap is an antibody that targets serum amyloid P protein (SAP) or amyloid fibrils. SAP, a normal plasma glycoprotein synthesized by the liver, stabilizes and protects amyloid fibrils from proteolytic degradation. Miridesap binds SAP and promotes its hepatic clearance.PRX004 is a monoclonal antibody designed to specifically target TTR amyloid deposits and is currently being tested in a phase 1 trial.Dezamizumab is a humanized monoclonal IgG1 anti‐SAP antibody that is being studied for treating amyloidosis in a phase II trial.6.26.2.1Once a diagnosis of ATTR is confirmed, the patient should be evaluated for liver transplantation, as the source of amyloidogenic protein is the liver. However, Dubrey and colleagues found that nearly half of patients with family amyloidosis still developed evidence of amyloid cardiomyopathy after liver transplantation.6.36.3.1Traditional drug therapy for congestive heart failure is generally used for patients with ATTR, although there is no evidence that it affects the overall prognosis of amyloidosis. Patients with ATTR typically respond better to heart failure therapy than do those with AL amyloidosis.Left ventricular assist device and cardiac transplantation are options for patients with ATTR cardiomyopathy‐related end‐stage heart failure with no significant extracardiac involvement, based on United Network for Organ Sharing (UNOS) status. However, evidence of survival benefit is lacking given the limited number of cardiac transplants performed due to the scarcity of donor organs. Davis et al at Stanford University Hospital conducted a study in 19 patients undergoing heart transplantation for amyloid cardiomyopathy between 2008 and 2013.Some patients with cardiac amyloidosis have received anticoagulats because of the increased risk of intracardiac thrombosis, especially those with AL amyloidosis.Previous studies have demonstrated that life‐threatening arrhythmia‐related sudden cardiac death (SCD) is a common cause of death in patients with cardiac amyloidosis.The infiltration of myocardial tissue into the conduction system often leads to lethal arrhythmias. Electrocardiogram abnormalities among patients with amyloidosis warrant an electrophysiological workup. Pacing is primarily used for significant bradycardia and certain types of atrial‐ventricular blocks, as a result of myocardial tissue infiltration. The data are limited on the indication for pacemaker placement and the clinical outcomes among this subgroup of patients.7The prognosis of patients with ATTR is determined primarily by the presence and extent of cardiac involvement.In a retrospective study, Escher et al followed and assessed 5‐year mortality in 325 patients with confirmed cardiac amyloidosis.8Once considered incurable, ATTR cardiomyopathy, a subtype of amyloidosis‐associated cardiomyopathy, now has promising treatment alternatives. Currently, robust ongoing research and clinical trials are focused on identifying new treatments for ATTR cardiomyopathy. Most of the novel agents target amyloid fiber formation by inhibiting TTR gene expression, stabilizing the TTR tetramer, preventing oligomer aggregation, or affecting degradation of amyloid fibers. More research is needed to verify the mortality benefits and side effects of the medications. It is unclear if the indication for heart failure treatment, anticoagulation, ICD placement, and transplant are the same as in those without ATTR. In addition, there is no consensus in screening criteria for ATTR, and the diagnosis of ATTR cardiomyopathy remains challenging because its nonspecific manifestations can be easily categorized as HFpEF. With the use of new treatments, this significantly underdiagnosed disease will likely be revealed as more prevalent than once thought. Therefore, further study is warranted to clarify the screening, diagnosis, and treatment guidelines for ATTR patients with cardiomyopathy.The authors have no actual or potential conflict of interest in relation to this manuscript.


Patients with type 2 diabetes mellitus (T2DM) are susceptible to coexisted with chronic kidney disease (CKD), which may increase cardiovascular mortality in these patients. The present study aimed to verify whether CKD aggravates the deterioration of left ventricular (LV) myocardial strain in T2DM patients and to explore the risk factors associated with LV strain. In total, 105 T2DM patients and 52 healthy individuals were included and underwent cardiac magnetic resonance examination. Patients were divided into the following two groups: T2DM with CKD (n = 33) and T2DM without CKD (n = 72). The baseline clinical and biochemical indices were obtained from hospital records before the cardiac magnetic resonance scan. Cine sequences, including long-axis views (2-chamber and 4-chamber) and short-axis views, were acquired. LV function and global strain parameters were measured based on cine sequences and compared among three groups. Pearson’s analysis was performed to investigate the correlation between LV strain parameters and clinical indices. Multiple linear regression analysis was used to identify the independent indicators of LV strain.P < 0.05). The eGFR was positively associated with the magnitude of PS , while uric acid was negatively associated with the magnitude of PS  . Multivariable linear regression indicated that the magnitude of PS was independently associated with eGFR  and uric acid  .Compared with normal controls, T2DM patients without CKD had a significantly decreased magnitude of peak strain , peak systolic strain rate , and peak diastolic strain rate  . Furthermore, T2DM patients with CKD displayed markedly lower magnitudes of PS  and peak diastolic strain rate  than both normal controls and T2DM patients without CKD (all Kidney dysfunction may aggravate the deterioration of LV strain in T2DM patients. LV strain is positively associated with the estimated glomerular filtration rate and negatively associated with uric acid, which may be independent risk factors for predicting reduction of LV strain. Diabetes mellitus (DM) has caused a severe public health burden with an estimated 451 million people living with diabetes worldwide , 3. In DCardiac magnetic resonance (CMR) have served as the gold standard modality to assess cardiac structure and function. CMR derived strain based on cine sequence and tissue-tracking techniques has been proven to provide quantitative assessment of myocardial deformation based on every voxel of cine sequence –8. Relat2)  decreased and LV mass (124.02 ± 49.32 g vs. 96.14 ± 34.18 g) increased in the T2DM with CKD group .The T2DM with CKD group showed significantly higher values for LVEDV, LVESV, LV mass, and LV remodelling index but lower values for LVEF compared to normal controls . Furthermore, compared to T2DM patients without CKD, T2DM patients with CKD had markedly lower magnitudes of radial (20.79 ± 12.02% vs. 30.82 ± 10.48%), circumferential (– 13.48 ± 6.16% vs. – 19.37 ± 5.48%), and longitudinal (– 8.35 ± 3.39% vs. – 13.49 ± 3.78%) PS . Concurrently, the circumferential and longitudinal PDSR in T2DM patients with CKD were also lower than that in normal individuals and T2DM patients without CKD , circumferential (– 13.48 ± 6.16% vs. – 19.94 ± 2.71%), and longitudinal (– 8.35 ± 3.39% vs. 14.82 ± 3.33%) PS in T2DM with CKD group were remarkably lower than that in normal group  and that uric acid had a negative correlation with the magnitude of PS   . There was no significant correlation between other clinical parameters and PS . Multivariable stepwise linear regression analysis indicated that eGFR  and uric acid    Fig. . In addiThe reproducibility of LV PS was considered excellent. The ICC values in the intraobserver analysis were 0.942 , 0.950 (95% CI 0.920–0.970), and 0.978 (95% CI 0.965–0.987) for radial, circumferential, and longitudinal PS, respectively. The ICC values in the interobserver analysis were 0.845 , 0.863 (95% CI 0.920–0.970), and 0.937 (95% CI 0.901–0.960) for radial, circumferential, and longitudinal PS, respectively.The present study investigated the characteristics of LV function and strain in T2DM patients with or without CKD using CMR imaging. We verified the occurrence of decreased LV strain in T2DM patients compared to normal individuals. Furthermore, kidney dysfunction was proven to aggravate the deterioration of LV strain in T2DM patients with CKD. Finally, the eGFR and uric acid were independently associated with LV PS in radial, circumferential, and longitudinal directions.In T2DM patients without CKD, our data revealed a reduction in radial PS, radial PSSR, radial PDSR, and circumferential PDSR compared to normal individuals, but no significant difference in LVEF was observed between these two groups, which was consistent with previous strain data regarding myocardial mechanics in DM –22. The The underlying mechanism of reduced strain is still incompletely understood. It was demonstrated that hyperglycemia enhances fatty acid metabolism, suppresses glucose oxidation, and modifies intracellular signaling and leads to interstitial and perivascular fibrosis, contributing to reduction in ventricular compliance , 23. CaoAlthough CKD is also a common comorbidity of DM, studies on the additive effect of kidney dysfunction on cardiac function and deformation in T2DM are scarce. Thus, we conducted the present study and revealed that the magnitude of radial, circumferential, and longitudinal PS was markedly lower in T2DM patients with CKD than in both normal individuals and T2DM patients without CKD. Moreover, the LVEDV, LVESV, LV mass and LV remodelling index were markedly enlarged in T2DM patients with CKD compared to both normal individuals and T2DM patients without CKD. Taken together, these observations suggested that LV function progressively decreases in diabetic patients with CKD and is accompanied by left ventricle enlargement and hypertrophy.In addition, we identified that the eGFR and uric acid were independently associated with the magnitude of LV PS in the radial, circumferential, and longitudinal directions, indicating that the magnitude of PS may gradually decrease with the deterioration of eGFR and the accumulation of uric acid. Thus, T2DM patients with high uric acid and low eGFR levels have an increased risk of myocardial strain deterioration. In CKD patients, mechanisms including hemodynamic instability, activation of the neuroendocrine system, oxidative stress, and anaemia, contribute to myocardial injury and resulting in poor cardiac outcomes during the process of chronic kidney dysfunction –27. It cThere are several potential limitations in our study. First, this was a single centre study. The sample size is relevant small but adequately powered to demonstrate the additive effects of kidney dysfunction on left ventricular function and strain in type 2 diabetes mellitus patients. A larger cohort or multicentre study with pooled data is required in the future. Second, long-term follow-up data related to the impact of LV strain changes on cardiovascular events was not obtained, and further longitudinal studies are required to investigate the potential of LV strain for predicting cardiovascular outcomes in T2DM patients with CKD. Thirdly, subclinical coronary ischemic disease may not be excluded because the stress test was not performed. However, the clinical coronary artery disease was considered to be unlikely according to the evaluation of patients by clinical history, laboratory results, echocardiography and electrocardiography. Results could still reflect the additive effect of kidney dysfunction on diabetic cardiomyopathy after adjustment for other confounders including age, gender, BMI, systolic blood pressure, rest heart rate. Last, the fasting plasma glucose and lipid status parameters of the controls were not measured. However, we carefully checked the detailed medical history and medical examination reports of enrolment for the controls to ensure meeting the inclusion criteria.LV global strain is significantly compromised in T2DM patients, and kidney dysfunction may aggravate the deterioration of LV strain in diabetic patients. LV strain is positively associated with the estimated glomerular filtration rate and negatively associated with uric acid, which may be independent risk factors for predicting reduction of LV strain. Therefore, more attention should be paid to LV strain evaluation in T2DM patients with CKD.


Intensive endurance exercise may induce a broad spectrum of right ventricular (RV) adaptation/remodelling patterns. Late gadolinium enhancement (LGE) has also been described in cardiovascular magnetic resonance (CMR) of some endurance athletes and its clinical meaning remains controversial. Our aim was to characterize the features of contrast CMR and the observed patterns of the LGE distribution in a cohort of highly trained endurance athletes.Ninety-three highly trained endurance athletes  and 72 age and gender-matched controls underwent a resting contrast CMR. In a subgroup of 28 athletes, T1 mapping was also performed.p < 0.05). Focal LGE was significantly more prevalent in athletes than in healthy subjects , with a typical pattern in the RV insertion points. In T1 mapping, those athletes who had focal LGE had higher extracellular volume (ECV) at the remote myocardium than those without .High endurance training load was associated with larger bi-ventricular and bi-atrial sizes and a slight reduction of biventricular ejection fraction, as compared to controls in both genders (Highly trained endurance athletes showed a ten-fold increase in the prevalence of focal LGE as compared to control subjects, always confined to the hinge points. Additionally, those athletes with focal LGE demonstrated globally higher myocardial ECV values. This matrix remodelling and potential presence of myocardial fibrosis may be another feature of the athlete’s heart, of which the clinical and prognostic significance remains to be determined. Highly trained endurance athletes showed a ten-fold increase in the prevalence of focal LGE as compared to control subjects, always confined to the hinge points.Those athletes with focal LGE demonstrated globally higher myocardial ECV values.This matrix remodelling and potential presence of myocardial fibrosis may be another feature of the athlete’s heart, of which the clinical and prognostic significance remains to be determined.Long-lasting athletic training leads to structural, functional and electric cardiac adaptations known as the ‘athlete’s heart’ –3. This Late gadolinium enhancement (LGE) in the myocardium as assessed by contrast cardiovascular magnetic resonance (CMR) is associated with a poor prognosis in patients with cardiomyopathy –7. NeverCMR imaging is useful to assess cardiac morphology and function, and contrast enhanced CMR imaging with LGE correlates with histological indications of myocardial fibrosis in patients with previous myocardial infarction , 18. In The relationship between long-term endurance exercise and the presence of LGE is controversial. Current data are limited since a number of series that reported higher LGE in asymptomatic athletes , 23, 24,Therefore, the purpose of this study was to evaluate the prevalence and the distribution pattern of LGE and ECV as potential indicators of myocardial fibrosis in highly-trained endurance athletes of both gender and to compare them to those observed in age- and gender-matched control subjects.We recruited 93 triathletes from local triathlon clubs by invitation to participate in our study, between March 2015 and December 2017. Inclusion criteria were: (a) highly-trained endurance athletes (20–45 years) who had trained for a minimum of 12 h per week in the previous 5 years, (b) no cardiac symptoms or cardiovascular disease risk factors and (c) no family history of sudden cardiac death. The control group comprised 72 age- and gender-matched individuals. They were eligible if they only performed recreational sports of mild-moderate intensity for less than 3 h per week, and if they had no known history of cardiovascular disease.The subjects were evaluated through the following tests: detailed anamnesis, physical examination, blood test and 12-lead surface electrocardiograms (ECG), and also a maximal cardiopulmonary exercise test in an upright cycloergometer  following an incremental ramp protocol. Study exclusion criteria were the presence of heart disease, renal failure , claustrophobia or any other contraindication for CMR. Lifetime training history and current training load were registered by direct reporting of the athletes. The study protocol complied with the declaration of Helsinki, institutional review board and ethics committee approval were obtained for this study. All subjects provided written informed consent.n = 65) or a 1.5 T (n = 28) CMR scanner , using a dedicated cardiac coil and ECG gating. Control subjects were scanned with the same 3 T scanner. After standardized CMR planning [All athletes underwent CMR imaging with either a 3 T ]  . Areas op-value of less than 0.05 was considered to indicate statistical significance.Data were analysed with SPSS for Windows . A Gaussian distribution of all continuous variables was confirmed using a Kolmogorov–Smirnov test and values were reported as mean ± standard deviation (SD). To compare qualitative variables, a chi-squared test was used. An unpaired Student t-test was used to compare differences between athletes with and without LGE. Intraclass correlation coefficient for absolute agreement was used to evaluate the concordance between CMR measures performed by the two independent observers. A p < 0.05). As expected, athletes showed a lower heart rate and higher maximal oxygen consumption (VO2max) as compared with the control group. All controls had a normal ECG and athletes presented with exercise-induced adaptive ECG changes such as incomplete right bundle branch block  and mild or moderate sinus bradycardia . No ECG pathological abnormalities were found in controls or in the athlete’s population.Characteristics of the male and female triathletes and control subjects are shown in Table p < 0.001). Male athletes presented larger biventricular and right atrial cavities as compared to female athletes (p < 0.05). LV mass and bi-atrial size were also larger in athletes as compared to controls.Table + triathletes had a similar pattern consisting of a small volume of focal LGE confined to the inferior interventricular septum, commonly where the RV attaches to the septum – insertion point or hinge point  showed LGE [17 of 49 males (34.7%) and 18 of 44 females (40.9%)], while only two of 72 (2.8%) controls had evidence of LGE. Thus, the presence of LGE was markedly increased in highly trained endurance athletes (in both genders) as compared to age- and gender-matched control subjects. All LGETable + had a trend toward more hours of training per week . Nevertheless, LGE+ athletes (both genders) showed no differences in LV and RV volumes and ejection fraction as compared to LGE− athletes.The exercise-induced increase in systolic blood pressure was equivalent in LGE+ and LGE- athletes. Among men, their competition history revealed that those athletes with LGEIn a subgroup of 28 athletes , that were studied in the 1.5 T scanner, T1 mapping sequences were also included in the contrast CMR protocol. Globally, ECV mean values were 26.0 ± 2.3%, and within the range of previously reported normal intervals (ECV 25.3 ± 3.5%) , 30.p < 0.05), despite still within the reference limits of normality. These data are shown in Fig. p = 0.82), training load (p = 0.59), or with structural  or functional (considering left ventricular ejection fraction (LVEF); p = 0.11) ventricular changes. Concordance was high between the two observers for ECV values (intraclass correlation coefficient -absolute agreement- > 0.85).Additionally, 13 of those 28 athletes (46.4%) had LGE confined to the interventricular septum. Those LGE+ athletes, had higher ECV at remote LV myocardium  as compared to LGE- athletes  as expected, highly trained endurance athletes showed an increase of biventricular and biatrial cavity sizes and a slight reduction of biventricular ejection fraction; 2) the presence of LGE, as a potential marker of focal fibrosis, was ten-fold more prevalent in athletes (both genders) as compared to controls, and it was always located in the inferior insertion point; 3) the presence of focal LGE did not result in significant differences in ventricular volumes and ejection fraction or in parameters of maximum aerobic capacity; and 4) athletes with LGEAs expected, our results are in line with previous data proving that long-term endurance training promotes cardiac remodelling that consists of significant biventricular and biatrial dilatation in both genders and a slight reduction of biventricular ejection fraction (at rest). These changes are part of a common adaptation in trained athletes, known as the athlete’s heart , 31, 32.p > 0.05), as also previously described in other reports [Our study provides additional evidence that highly trained endurance athletes show a significantly higher prevalence of focal LGE than age- and gender-matched control subjects , 11, 23. reports ; this co reports , who notIn our study, all athletes with LGE showed a similar pattern, consisting of a small volume of focal LGE confined to the interventricular septum, commonly where the RV attaches to the septum (the inferior insertion point or hinge point). This pattern corresponds to a typical non-ischemic substrate. Other specific LGE patterns, such as subendocardial, transmural or subepicardial, were not identified in our population.This observation may be because our population was constituted by completely asymptomatic, otherwise healthy, young athletes, while in previous studies the athlete’s cohort was older , 34, 35 Regarding the presence of focal fibrosis in the insertion point, La Gerche et al.  has sugg+ athletes showed no significant differences in LV and RV volumes or function as compared to LGE− athletes. However, other authors [LGE authors , 24, 34 Our data demonstrates balanced biventricular and biatrial dilatation with a slight reduction of biventricular ejection fraction in athletes with and without LGE; this may arise, given the inclusion criteria, from the fact that our athletes practise the same endurance sport discipline and have similar training load. Thus, all this data potentially suggests that this LGE pattern might be another feature of the athlete’s heart, related to local mechanical stress due to the exercise-induced cardiac overload. While LGE has been correlated to focal fibrosis in chronic infarction, LGE dynamics are still controversial and not fully understood in other settings. The presence of LGE indicates that the local matrix and fibre structure has changed, similarly to what happens indeed in histologic fibrosis and scar; however, LGE could also be present due to localized structural remodelling regardless of the presence of real fibrosis. Experimental models of endurance training might shed some light in the real clinical significance of the observed LGE in athletes.p = 0.02) [Considering that the increased LV mass, observed in athletes, is due to an expansion of the cellular compartment, a decreased ECV is to be expected . In fact = 0.02) .Nevertheless, in our study we did not find a decrease in ECV in athletes. And when we separately analysed those athletes with focal LGE confined to the inferior insertion point, we observed that they had slightly higher ECV at remote LV myocardium than those without LGE, despite still being within the limits of normality , 30. TheFurther studies with long-term follow-up are clearly warranted to understand if these slightly higher ECV values are another feature of the athlete’s heart or if this may be able to early identify athletes who are starting to potentially develop diffuse interstitial fibrosis and have indeed an abnormal adaptation to training.The quantification of the training load by assessing self-reported training load represents a potential limitation, because these parameters depend on the individual perception and accuracy of athletes. Currently, no long-term follow-up data on outcomes is available to assess the prognostic and clinical implications of the observed LGE. The T1 mapping sequence was available in only 30% of athletes, among whom 71% were female; thus, findings need to be confirmed in larger populations.Highly trained endurance athletes showed a ten-fold increase in the prevalence of focal LGE as compared to age-matched control subjects, always with a constant pattern confined to the hinge point of the inferior interventricular septum. This was observed together with balanced biventricular and biatrial dilatation. These findings suggest that this distribution pattern of LGE might be another feature of the athlete’s heart, with a clinical and prognostic significance that remains to be determined. In addition, those athletes with focal LGE demonstrated globally higher myocardial ECV values. The underlying mechanisms leading to these findings are unknown and beyond the target of the present study but might be related to local mechanical stress due to the exercise-induced cardiac (pressure) overload.


Athletes had significant greater LV mass indexed for BSA . There was no significant difference in LV function or diameters. Recent studies showed contrasting findings in morphological changes due to competitive soccer in adolescent players (SP). We present a prospective study in 315 consecutive adolescent (10–14 years) male elite SP and 53 healthy matched active controls (CON). All participants underwent a complete transthoracic two-dimensional echocardiography (TTE). The mean age in SP was 12.8 ± 0.65 years compared to 12.6 ± 0.8 years in CON. For all left ventricular (LV) dimensions, mean Z-score values were higher in SP. There was a significant Z-score increase in interventricular septum diastolic diameter . If in SP Z-scores, any LV dimension above + 2.5 is measured, primary or secondary cardiomyopathies should be excluded. The Association for European Pediatric Cardiology recommends that cardiovascular screening should be performed on athletes when they start competitive activity.  NowadaysMorphological cardiac adaptation in response to sport depends on the type, duration, and intensity of training. Endurance exercise  results in an increased cardiac output leading to increased volume load and consecutively to an eccentric LV hypertrophy. Strength exercise  is characterized by an increased peripheral vascular resistance, which results in increased LV after load and therefore to a concentric LV hypertrophy.  There arThe aim of our prospective study was to measure left cardiac dimensions and function in elite non-symptomatic male adolescent SP and healthy matched controls using conventional TTE.This prospective study was performed over 2 years at a tertiary children’s university hospital. A total of 359 consecutive adolescent male elite SP of the Bavarian Football Association participated in our study. As controls (CON), we selected 53 age- and sex-matched inpatients from the pediatric surgery department of our hospital.2, no history of cardiovascular disease, Caucasian ethnicity, and field player. Two participants with a minor cardiovascular disease and 42 goalkeepers had to be excluded because of different training load. The SP training profiles were as follows: training load of 8.5 ± 3.5 h per week, one competitive match per week, 11 ± 1 months per year training, and 3.5 ± 1.5 years of training. CON were all recreationally active but were not taking part in any regular training scheme. All participants underwent a standardized cardiovascular screening protocol with a medical history, a physical examination, a 12-lead resting electrocardiogram, and a TTE at rest.Inclusion criteria were male sex, age 10–14 years, BMI 13–25 kg/mThe study is in compliance with the Declaration of Helsinki and was approved by the institutional review board of the University of Regensburg (15-101-0079). Informed consent was obtained of all children and/or their legal guardians.Echocardiography (TTE) was performed using a portable cardiovascular ultrasound system  with a 6S-RS (2.7–8.0 MHz) or a 3S-RS 1.5–3.6 MHz) transducer. The recent guideline for chamber quantification from the American Society of Echocardiography and the European Association of Cardiovascular Imaging were followed for echocardiographic measurements. [–3.6 MHz All studies were digitally stored for off-line analysis, retrospective re-evaluation, and follow-up examinations.t test was used. The body surface area (BSA) was calculated with Haycock’s formula. [p < 0.05. All analyses were conducted using IBM SPSS Statistics, version 25.Normality of data was assessed using Shapiro-Wilk. For normally distributed data, a Student’s independent formula.  In TTE eformula.  Statisti2). Both groups had regular mean systolic and mean diastolic blood pressure values at rest. Mean values (mm) for LV dimensions (SP vs. CON): IVSD 8.9 vs. 7.92 mm, IVSS 12.96 vs. 12.32 mm, LVPWD 8.38 vs. 7.63 mm, LVPWS 12.54 vs. 11.50 mm, LVIDD 44.90 vs. 44.42 mm, LVIDS 28.38 vs. 28.09 mm. For all LV dimensions, mean Z-scores were higher in SP than in CON. There was a significant increase in IVSD , LVPWD , and LVPWS . The mean Z-score of both the diastolic and the systolic diameter of the interventricular septum was above 2.0  in SP. A small subgroup of SP did develop supernormal LV wall Z-score values, but did not exceed + 2.5z. Athletes had significant greater LV mass indexed for BSA . There was no significant difference in conventional LV function  and LV systolic or diastolic internal diameter  and body surface area  suggesting cardiac adaption characteristic for resistance sports. This is in contrast with the traditional view published by Mitchell et al. showing soccer as a low-static and high-dynamic type of sport therefore predisposing more to endurance remodeling according [The main findings from this study were that Z-score values for LV wall thicknesses and indexed LVM were significantly higher in SP , including two cases of hypertrophic cardiomyopathy, eight of mild left ventricular hypertrophy, and six of mild left ventricular dilation.  ZdravkovThese contrasting findings may be a product of differences in training load, genetic background, imaging mode, ethnicity, or some combination of factors. Normalizing LVM for inter-individual variation in body size is a central issue in human biology. During the adolescent growth spurt, variability in body size descriptors needs to be interpreted in combination with biological maturation.  ThereforThere are several limitations of the present study. First of all, we did not test interobserver variability. Secondly, the size of our control group was relatively smaller in comparison to SP. Finally, our study was based on a male Caucasian population. Therefore, our results should not be extended to the general population.Our findings suggest that German elite soccer training in adolescent male is a type of sport predominantly related to cardiac resistance remodeling. A small subgroup of SP did even develop supernormal LV wall Z-score values, but did not exceed + 2.5z. We therefore recommend to rule out a primary or secondary cardiomyopathy  if there is a Z-score value above + 2.5 in any LV dimension.


The measurement of cardiac iron is important for preventing heart failure and monitoring iron-chelating treatment in thalassemia patients. An MRI T2* technique has been developed and clinically validated for this purpose –3. A recA recent study  has suggTo compare in vivo and ex vivo myocardial T2* measurements at a comparable temperature and using the correct model.3.A 23-year-old Thai female thalassemia majorpatient with longstanding cardiac and liver iron overload was studied. The patient underwent MRI scan using the bright blood T2* sequence . The patFigure This study has demonstrated the good reproducibility of the myocardial T2* measurement using the truncation model, where an in vivo T2* measurement from the patient data agrees with ex vivo one of the fixed heart. These data, along with the calibration study , support


Owing to its ease of application, noninvasive nature, and safety, echocardiography is an essential imaging modality to assess cardiac function in patients affected by ischemic heart disease (IHD). Over the past few decades, we have witnessed a continuous series of evolutions in the ultrasound field that have led to the introduction of innovative echocardiographic modalities which allowed to better understand the morphofunctional abnormalities occurring in cardiovascular diseases. This article offers an overview of some of the newest echocardiographic modalities and their promising application in IHD diagnosis, risk stratification, management, and monitoring after cardiac rehabilitation. Ischemic heart disease (IHD) is the primary global cause of death. Mortality from IHD in Western countries has, however, dramatically decreased during the last decades owing to greater focus on primary prevention and improved diagnosis and treatment . IHD diaOver the last decades, several technological advancements were made in the echocardiographic field, aiming to better understand the morphofunctional abnormalities occurring in cardiovascular diseases. These technologies are still struggling to enter the daily and routine use. This review wants to turn the spotlight on some of the newest cardiac ultrasound imaging technologies highlighting their promising application to the IHD diagnosis, management, and monitoring during cardiac rehabilitation.During ejection, left ventricular (LV) myocardium undergoes a three-dimensional 3D) deformation, characterized by longitudinal and circumferential shortening, and radial thickening. Added to this is the rotation of the LV around its long axis, i.e., viewed from apex, the apex rotates in anticlockwise direction, whereas the base rotates in the clockwise direction. The movement caused by this opposite rotation is called twist and is crucial for the LV ejection performance. The untwist, which occurs during diastole, generates a suction force that drives the early, rapid diastolic filling of the LV [D deformaTwo-dimensional (2D) speckle tracking echocardiography (STE) is a gray-scale based technique of myocardial deformation imaging, independent from insonation angle, which allows to assess all the above-mentioned components of LV contractile function. The myocardial deformation is quantified as strain or strain rate (SR). Strain is the percentage change in the length of a myocardial segment compared to its resting length; SR is the rate at which this deformation takes place, expressed as 1/s. Normally, strain and SR have negative values in systole when myocardium shortens and positive values in diastole when myocardium lengthens. The STE software identifies a number of bright speckles generated by the scatter of the ultrasound beam after its interaction with the myocardium and follows them frame-by-frame. Then, through an algorithm, it calculates the magnitude of myocardial deformation in each direction and generates strain and SR curves. The strain analysis is performed on a workstation where gray-scale images are transferred as digital media. Anyway, some of the currently available ultrasound systems offer the possibility of online STE analysis, on the echocardiographic machine itself, even on portable devices.In order to calculate the LV longitudinal strain, images of high quality from the apical 4-chamber (4CH), 2-chamber (2CH), and long-axis (LAX) view should be taken, so that the LV occupies most of the sector and is not foreshortened. The optimal gray-scale frame rate should be kept between 30 and 70 frames/s. Three cardiac cycles should be acquired for each view (ECG-gating is mandatory). The images can be transferred to the workstation. It is recommended to start the analysis from the apical long-axis view, where the aortic valve leaflets motion helps to identify the timing of valve closure, that is essential in the deformation study. The endocardial borders are manually traced in the end-systolic frame automatically brought up by the software. A region of interest (ROI), including the entire myocardial thickness, is generated by the software and can be manually modified in width. The software then tracks the myocardial speckles frame-by-frame, producing a moving image displaying the tracking during the cardiac cycle. If the tracking seems to be not adequate, the operator can go back and adjust the ROI or select a new ROI. Once the tracking is approved by the operator, the software divides the LV myocardium of the view in analysis in 6 segments, providing segmental and global longitudinal strain, myocardial velocities, and strain curves. The same process must be repeated for the apical 4-chamber and 2-chamber views to obtain strain values for all myocardial segments, their average, and the LV global longitudinal strain (GLS). Some ultrasound systems also provide the bull’s eye, which intuitively displays segmental and global peak-systolic longitudinal strain.A right ventricular (RV) focused 4-chamber view is required to analyze RV strain, whereas short-axis views are required to obtain information about circumferential and radial deformation  and LV twist .Recently, 3D STE has been introduced by applying speckle tracking technologies to 3D echocardiographic images. Images are usually acquired using a matrix-array transducer, from the apical position in a wide-angled acquisition ‘‘full-volume’’ mode. In this mode, a number of wedge-shaped subvolumes are acquired over consecutive cardiac cycles during a single breath hold and stitched together to create one pyramidal volume sample. A major limitation of 3D STE to date is the temporal resolution of the volumetric pyramidal data sets. Usually the rate of acquisition does not exceed 20–30 volumes/s, and, in most cases, to obtain higher temporal resolution, the field of view needs to be considerably narrowed. By fusing 2D speckle tracking information obtained from standard apical 4CH, 2CH, and LAX views, XStrain™ four-dimensional (4D) aims to make myocardial quantification imaging interpretation easier by the 3D/4D reconstruction of the LV. The user can freely rotate and zoom the beutel and superimpose the echographic scanning planes to better evaluate the contractility properties of the LV, using a physiological tool to analyze the complex multi-dimensional LV mechanics [STE is able to detect subclinical LV systolic dysfunction in an early-stage cardiovascular diseases, when LV ejection fraction (LVEF) is still normal. Actually, an isolated impairment of one myocardial layer can be present, compensated by the augmentation of function of the other layers, so that LVEF and overall LV performance remain preserved . For exaStrain and SR parameters are significantly reduced in ischemic-segments in patients with acute myocardial infarction (AMI) . RegionaMultilayer longitudinal strain assessed by 2DSTE, in particular endocardial strain, is useful to identify patients with AMI and significant/complex CAD ,9,10.As for the LV, RV peak systolic longitudinal strain is able to predict RV scar and well correlates with scar extent .STE adds a prognostic value to the traditional indexes of LV systolic function in both stable and intensive care setting . LongituPeak systolic GLS was also identified as independent predictor of LV function recovery during the follow-up of 12 months after AMI . A low Gp < 0.001), and was an excellent independent predictor of response to resynchronization therapy  [Finally, in a population of patients with ischemic dilated cardiomyopathy, D’Andrea et al. showed that GLS was strongly correlated with the total scar burden assessed by CMR  .The main role of left atrium (LA) is to modulate LV filling and cardiovascular performance. For this purpose, LA works as reservoir during ventricular systole, when it receives blood from pulmonary veins and its volume increases, as conduit during early ventricular diastole, passively allowing the passage of blood into the ventricle, and as booster pump during late ventricular diastole, when it actively contracts to complete LV filling. During last years the application of 2DSTE to the LA has been largely investigated in several studies ,26,27,28LA reservoir and conduit functions, respectively, evaluated during systole and early diastole by 2DSTE, are impaired in patients with CAD, even in absence of LA enlargement . LA reseLA strain and SR could be useful in predicting the severity of CAD. Actually, a strong inverse correlation between atrial deformation parameters and severity of coronary stenosis was found . MoreoveTDI is an echocardiographic technique which allows to measure myocardial velocities using Doppler principles. TDI can be performed in three different modalities: pulsed wave (PW), color, and 3D mode . Pulsed PWTDI represents the cardiac cycle throughout three waveforms: systolic myocardial velocity (S’) above the baseline, early diastolic myocardial relaxation velocity (e’) below the baseline, and late diastolic myocardial velocity associated to atrial contraction (a’) below the baseline. Systolic and diastolic time intervals can be obtained using TDI, such as isovolumic contraction time (IVCT), ejection time (ET), and isovolumic relaxation time (IVRT). Finally, the myocardial performance index (MPI) can be calculated as the sum of IVCT and IVRT divided by the ET.A significant decrease of S’, e’, and a’ waves has been observed in patients with IHD, even if cutoff values for maximum systolic and diastolic velocities which detect CAD are still unclear .S’ velocity can be used to quantify the entity of regional motion impairment. In a systematic review by Agarwal et al., the authors suggest that S’ velocity may be of help in detecting CAD especially in patients with poor 2D endocardial definition, where the qualitative wall motion analysis can be difficult.During both acute and chronic ischemia, the phenomenon of postsystolic shortening (PSS) can be quantified by TDI or STE. It is a myocardial shortening that takes place after aortic valve closure and therefore does not contribute to LV ejection. This phenomenon is probably due to the passive recoil from the interaction between ischemic and surrounding nonischemic myocardial segments. Several studies proved that PSS indicates myocardial viability and it is a predictor of systolic recovery in patients with IHD ,35,36. AAnalysis of intracardiac flows represents a way to approach the study of LV function . NormallEven if phase-contrast magnetic resonance imaging is the gold standard for measuring blood velocities in heart cavities, some echocardiographic techniques have been developed in order to visualize intracardiac flow, among which is the color Doppler flow mapping (CDFM). The HyperDoppler software is a recent CDFM-based technology that provides different possibilities to represent intracardiac flow data: a flow velocity vector map where velocity vectors are displayed as arrows superimposed on the traditional color Doppler flow images which can be followed frame by frame; a circulation parametric map where vortices are represented as compacted regions in blue (clockwise rotation) or in red (counterclockwise rotation); and a kinetic energy map where the highest level of kinetic energy is depicted in red. Normally, intracardiac vortices are visualized on apical long-axis view images . The LV vortex formation is the result of an optimal interaction between LV chamber geometry, morphology of the mitral valve apparatus, and normal electrical conduction system, which allows the harmonic contraction of the cardiac walls . If one In patients with STEMI, kinetic energy dissipation within the LV increases linearly with the increase of LVEF, as the flow turbulence into the LV is higher ,41. For Finally, in patients with anterior myocardial infarction, LV vortex flow analysis was useful to identify patients at risk for LV apical thrombus formation . The evaluation of coronary flow reserve (CFR) is performed by combining transthoracic Doppler echocardiography with vasodilator stress, such as adenosine or dipyridamole. An index of CFR is obtained from the variation between coronary blood flow velocity (CBFV) at baseline and CBFV at the peak of effect of vasodilator. A value of CFR less than 2 is generally considered abnormal. CBFV profile is recorded by PW Doppler, and it is represented by a biphasic wave, with a lower peak during systole and a higher peak during diastole. This pattern derives from the fact that myocardial extravascular resistance is higher in systole, due to the effect of myocardial contraction. The most used parameter in evaluation of CFR is peak diastolic flow, because it is easy to measure, reproducible, and has a closer correlation with CFR measured by Doppler flow wire and positron emission tomography (PET) . The LADStress echocardiography was recently reshaped with the ABCDE protocol: A for asynergy, B for thoracic ultrasound B-lines, C for contractile reserve, D for Doppler-based CFR (in LAD), and E for electrocardiogram-based heart rate reserve  . ABCDE pCFR is a composite measure of coronary macrovascular and microvascular status. A reduced CFR is associated to a negative prognosis in several groups of patients, including those with diabetes mellitus, arterial hypertension, ischemic cardiomyopathy, normal coronaries, and CAD . In patiRecently, the assessment of CFR on TTE has been proposed as method to detect cardiac allograft macro- and microvasculopathy in heart transplant . A recenVascular aging in large arteries is characterized by structural and functional changes, such as intima-media thickening and “stiffening” . ArteriaThe recently introduced radiofrequency (RF) data technology allows to measure arterial intima-media wall thickness (IMT) and stiffness. All measurements are taken in a selected area of common carotid artery. The blood vessel wall stiffness is expressed in pulse wave velocity (PWV) in meter per second, and is obtained from the brachial blood pressure and the accurate measurements of vessel diameter and distension (change in diameter). For both IMT and stiffness, the operator gets real-time feedback on measurement quality via quality indicators overlaid on the ultrasound image. This real-time feedback gives the operator the possibility to optimize his probe position, in order to obtain a scan plane perpendicular to the wall of the common carotid artery .Vascular intima-media thickening and stiffening are correlated to cardiovascular morbidity and mortality . ArteriaPWV is closely linked to the presence of angiographically documented CAD . MoreoveIn the EPHESUS study, performed on patients with heart failure and reduced LVEF following AMI, it was shown that an increased aortic stiffness, assessed by PWV, was associated with a negative prognosis and significantly contributed to cardiovascular death .All the above-mentioned echocardiographic technologies have the advantage of being safe, noninvasive, and relatively low cost, and they can be performed bedside. Moreover, they allow to better understand the morphofunctional abnormalities occurring in cardiovascular diseases . Table 1Exercise-based cardiac rehabilitation (CR) in patients affected by CAD increases functional capacity and reduces total and cardiovascular morbidity and mortality ,60,61,62Data regarding the clinical application of intracardiac flow analysis to patients who undergo CR after MI are still missing in literature. It would be attractive to evaluate LV vortex features and kinetic energy dissipation before and after a CR program, in order to obtain a further index of LV remodeling and function.13N-ammonia PET. For the combined TM group, CFR increased when compared to the combined non-TM group [An interesting recent pilot study explored the effects of standard CR with and without transcendental meditation (TM) on CFR assessed by TM group . These rEven arterial stiffness evaluation finds an interesting application in CR field. In particular, PWV was shown to be decreased in absence of changes of peripheric arterial blood pressure in patients with IHD undergoing a CR program ,71,72. MStandard echocardiography is an essential imaging modality for the assessment of patients affected by IHD, due to its unmatched ability to combine safety and ease of application with depth of diagnostic and prognostic information. The newer ultrasound technologies, which were developed over the past few decades, allowed to better understand the morphofunctional abnormalities occurring in cardiovascular diseases. These technological advancements revealed to be promising to further expand the role of echocardiography as modality of choice in IHD diagnosis, risk stratification, management, and monitoring after cardiac rehabilitation.


Anti-fibrotic therapies are of increasing interest to combat cardiac remodeling and heart failure progression. Recently, anti-fibrotic circular RNAs (circRNAs) have been identified in human and rodent cardiac tissue. In vivo (rodent) experiments proved cardiac anti-fibrotic effects of the natural compounds bufalin and lycorine by downregulating miRNA-671-5p, associated with a theoretic increase in the tissue level of circRNA CDR1as. Accordingly, we hypothesized that both anti-fibrotic drugs may inhibit focal myocardial fibrosis of the remodeled left ventricle (LV) also in a translational large animal model of heart failure (HF). Domestic pigs were repeatedly treated with subcutaneous injections of either bufalin, lycorine, or saline, (n = 5/group) between days 7–21 post acute myocardial infarction (AMI). At the 2-month follow-up, both bufalin and lycorine led to significantly reduced cardiac fibrosis. Bufalin treatment additionally led to smaller end-diastolic volumes, higher LV ejection fraction (EF), and increased expression of CDR1as of the AMI region. Elevated tissue levels of the circRNA CDR1as in the AMI region of the pig heart correlated significantly with LV and right ventricular EF, LV stroke volume, and negatively with infarct size. In conclusion, we successfully identified the circRNA CDR1as in pig hearts and show a significant association with improved LV and RV function by anti-fibrotic therapies in a translational animal model of HF. A myocardial infarction inevitably results in a considerable tissue loss. The consequence is an immediate increase in the contractile workload of the remaining ischemia-untouched myocardium. To maintain the left ventricular (LV) function and systemic circulation, series of initially adaptive and subsequently maladaptive changes occur in the chronic phase of ischemic heart disease ,2,3. TheBufalin (BUF) a was oriAmaryllidaceae family. It was previously shown to possess anti-inflammatory, anti-tumor [Lycorine (LYC) b is a plti-tumor , and antti-tumor .Both BUF and LYC downregulate miRNA-671-5p, a key player in the fibrotic response of murine fibroblasts and human cardiomyocytes . miRNA-6Thus, our study aimed to examine the effect of BUF and LYC on LV adverse remodeling and fibrosis in relation with the deregulation of anti-fibrotic circular RNA CDR1as in a translational model of ischemic heart failure in a pig heart.We induced closed-chest reperfused acute myocardial infarction in domestic pigs, mimicking human primary percutaneous coronary intervention in ST-elevation myocardial infarction, and administered repeated subcutaneous injections of LYC and BUF or saline solution (SAL), and investigated (1) the LV and right ventricular (RV) function and remodeling parameter and infarct size, (2) fibrosis of the remodeled ventricular wall, and (3) the expression of miR-671-5p, miR-7 and its target CDR1as. Since non-coding RNAs, such as miR-29a, exert an inhibitory effect on excessive fibrotic and collagen gene expression in cardiac fibroblasts , we alsoAll animal experiments were carried out in accordance with the “Guide for the Care and Use of Laboratory Animals”  and wereAfter overnight fasting, the animals were sedated with 12 mg/kg ketamine hydrochloride, 1 mg/kg xylazine and 0.04 mg/kg atropine, administered by an intramuscular injection. After surgical preparation of the right arteria femoralis, a 6F introducer was placed, and the animals received 200 IU/kg unfractionated heparin intraarterial. Coronary angiography was performed by using 6F guiding catheter. A balloon catheter  was inserted into the left anterior descending coronary artery (LAD), after the origin of the second diagonal branch. Reperfused acute myocardial infarction was induced by balloon occlusion of the mid LAD for 90 min, with a balloon inflation pressure of 5 atm, followed by the deflation of the balloon, inducing reperfusion. Control angiography confirmed the patency of the LAD. After hemodynamic stabilization, the balloon, guiding wire, catheter, and the introducer were removed, and the artery was sutured. The skin wound was closed in 3 layers, and the animals were allowed to recover.One day before AMI, the animals received a loading dose of clopidogrel (300 mg) and 500 mg aspirin per os. During the 2 months follow-up, a daily dose of 75 mg clopidogrel and 100 mg aspirin was administered per os. To quantify the left ventricular function (LVF) after reperfused AMI, and to measure the size of infarction and microvascular obstruction, cMRI was performed at day 3 post-AMI and at the final 57 days follow-up. The animals were then euthanized with 10 mL intravenous saturated KCl (10%) under general anesthesia.n = 5, 0.02 mg/kg), LYC , or saline . The randomization was done according to the computed randomization generator. Treatments were administered every other day for the following 21st days by subcutaneous injection. One animal showed a local inflammatory reaction on the site of bufalin injection; therefore, we reduced the doses to 0.01 mg/kg BUF and 0.1 mg/kg LYC 14 days after AMI. To reduce the psychical stress of the animals due to the repeated injections, we terminated the treatment at day 21 post AMI.Since we intended to inhibit fibrogensis and adverse remodeling, we started the treatments with BUF and LYC seven days after AMI, the animals being in the downstream phase of the main inflammatory processes. The animals were then randomized into three groups: BUF  , NT-proBNP , and liver enzymes  . The porcine normal range for serum Troponin I levels is 0–40 pg/mL, NT-proBNP is 0–180 pg/mL, and 5–40 mU/mL for ALT, 10–45 mU/mL for AST, and 0–380 mU/mL for ALP. The porcine TnI , NT-proBNP , ALT , AST (Sigma Aldrich), and ALP (Abcam) were assesses by commercially available ELISA.Tissue samples of approximately 150–250 mg were collected from the infarcted and remote  regions of the pig hearts following the autopsy and stored in 5% formaldehyde and RNA Later . For histological proof of LV remodeling, we examined cardiac fibrosis and compensatory cardiomyocyte hypertrophy as evidenced by the increase in cardiomyocyte diameter of the remote lateral wall. The fibrosis was quantified as the percentage fibrotic area in a standard Picrosirius Red stain. The cell diameter was calculated using a standard Haematoxylin–Eosin stain .cMRI+LE was performed at day 3 and 2-month after infarction under general anesthesia. The acquisition method and methods for calculation of the left and right ventricular parameter (end-diastolic and end-systolic volumes (EDV and ESV), stroke volume (SV), ejection fraction (EF), area at risk, infarct size, and microvascular obstruction) were published elsewhere .These methods are described in the To our knowledge, no publication has described a successful detection of CDR1as in the porcine heart. The putative sequence was derived from the NCBI database, by comparing the CDR1as sequence of humans and mice with the potential sequence of CDR1as in pigs. We then designed divergent primers to ensure amplification of the backsplice junction, which can only be found in circular transcripts. Sanger sequencing was performed by splitting the putative CDR1as in smaller parts. In the end, the sequencing results were put together to obtain the full sequence of porcine CDR1as.To determine if the previously amplified qPCR product possesses a backsplice junction and to check the sequence of porcine CDR1as Sanger sequencing was performed. Therefore, the presumed sequence of CDR1as was split in several parts and qPCR product, and divergent primers were sent to Sanger sequencing (Microsynth). In the end, the sequencing results were assembled to obtain the entire porcine CDR1as sequence.p-value < 0.05 was considered significant. One-way ANOVA on trimmed means was used for post-hoc analysis (R functions t1way and lincon). Spearman’s rank correlation coefficient was used as measure of association between expression levels of different transcripts. Here, we used robust methods instead of the classic ANOVA because of the existence of (often quite severe) single outliers in many groups. While there is no general optimal solution, in our case, the common recommendation of a 20% trimmed mean seems sensible, because in several groups there is exactly one severe outlier. Graphs were made using R studio (ggplot2) or Rhinoceros 3D and the Grasshopper plug-in , respectively. All analyses were performed blinded to the treatment groups, including also cMRI.Since no previous data regarding bufalin and lycorine treatment in porcine ischemic heart failure existed, no sample size calculation was done. Statistical analyses were performed using R (version 3.6.2). Data are presented as mean ± standard deviation. Two-way mixed ANOVA was conducted to assess the effect of treatment (between-group independent variable) on transcript expression in two heart regions (repeated-measures independent variable) and the effect of treatment on toxicology marker expression at two time points (repeated-measures independent variable in this case). Tukey’s method detected outliers for several combinations of treatment groups, heart regions, and outcome. Moreover, the normal distribution assumption, which was tested with the Shapiro–Wilks test and is needed for a classical ANOVA, was significantly violated in these groups. Therefore, a robust version on the 20% trimmed means was used, as implemented in the bwtrim function of the R package WRS2 (version 1.0.0) by Wilcox . A p-valThe animals tolerated the treatment well. Toxicology investigations including the liver enzymes ALT, AST, and ALP activity showed no signs of liver damage . With thThere was no difference between the groups regarding baseline or follow-up TnI or NT-proBNP . The chap = 0.125)  .There was no difference in microvascular obstruction  or LV enA trend towards higher LV EF was found in the BUF group at 2 months after AMI, as compared with the slight decrease in EF in SAL group . There wTwo-month cMRI-LE revealed no difference between the groups regarding RV EDV, ESV, and SV, but a trend towards better RV EF was found in the bufalin group . ChangesPicrosirius red staining (PSR) showed significantly less fibrosis in both bufalin and lycorine groups as compared with the SAL group in the remote myocardial area A,B. TherReal-time qPCR was performed to determine the relative gene expression of COL1A1, COL3A1, MMP-9, and miR-29a in the infarcted region and remote region of the MI and between the treatment groups.Except for miR-29, no significant differences between treatment groups were detected by two-way mixed ANOVA. p = 0.011).A previous study showed that bufalin and lycorine repress miR-671, which is crucial in the fibrotic response of murine fibroblasts and cardiac tissue of aortic stenosis patients . This miFurthermore, we explored whether miR-671 and miR-7 interact with CDR1as in the AMI region. Scatter plots show miR-671 and miR-7 versus CDR1as expression .Additionally, we investigated the association between the tissue expression level of CDR1as and the LV and RV function and found a significant positive correlation between the CDR1as and LV and RV EF and LV SV and a negative correlation regarding infarct size, suggesting a direct beneficial effect of CDR1as on the heart function post-myocardial infarction .In our study, we demonstrated the successful detection of circRNA CDR1as in the infarcted myocardial area in pigs for the first time. Here, we show a moderate beneficial effect of the two natural compounds bufalin and lycorine for prevention of infarct-related LV remodeling, significantly less myocardial fibrosis in the remodeled ventricular wall and the increase in CDR1as expression. In the early phase of myocardial infarction, inflammation is the dominant myocardial process, and we did not want to interfere with the inflammation. The aim of the treatment was to inhibit fibrogenesis and adverse ventricular remodeling. As the fibrotic process starts later, we decided to start the treatments 7 days after the AMI onset. Interestingly, even in a small sample, we could show significant correlations between the infarct tissue upregulated CDR1as and improved LV and RV function and decreased infarct size.The currently proposed mode of action for CDR1as is via binding of miR-7, thus acting as a miRNA sponge ,29. miR-The assumption that CDR1as, acting as a miR-7 sponge, could potentially have great clinical significance is an important research target largely spurred by the high degree of sequence conservation between species. An almost perfect conservation was shown for miR-7 between species as evolutionarily distinct as annelids and humans . Most ofIn line with previous in vitro and in vivo experiments , bufalinIn this study, we did not investigate the myocardial expression levels of CDR1as, miR-7, and miR-671 of the healthy heart, because it should have required either a myocardial biopsy before AMI induction (which would influence the outcome of AMI) or including an additional normal healthy animal group.Here, we demonstrated for the first time the successful detection of circRNA CDR1as in the infarcted myocardial area in pigs and showed a moderate beneficial effect of the two natural compounds bufalin and lycorine for prevention of infarct-related LV remodeling, less myocardial fibrosis in the remodeled ventricular wall, and the increase in CDR1as expression. The upregulated CDR1as in the infarcted tissue correlates significantly with the better LV and RV function and decreased infarct size.