Robotic major lung resection for lung cancer carries a risk for intraoperative hemodynamic instability. Systolic anterior motion (SAM) of the mitral valve is a rare and often misrecognized cause of intraoperative hemodynamic instability. If not promptly recognized, SAM leads to a complicated perioperative course. Here, we report for the first time a case of a patient with SAM with a severe degree of left ventricular outflow obstruction (LVOTO) undergoing robotic lung lobectomy and its challenging intraoperative management. A 70-year-old man undergoing robotic left upper lobectomy developed immediately after the induction of general anesthesia hemodynamic instability due to SAM-related LVOTO. The diagnosis was possible, thanks to the use of transesophageal echocardiography (TEE). The treatment strategies applied were preload optimization without fluid overload, ultra-short-acting beta-blockers, and vasopressors. Peripheral nerve blockades were preferred over epidural analgesia to avoid vasodilatation. The patient reported a good quality of recovery and no pain the day after surgery. The management of patients with higher risk of SAM and LVOTO development during robotic thoracic surgery requires a dedicated and skilled team together with high-impact treatment strategies driven by TEE. Since current guidelines do not recommend the use of TEE, even for patients with higher cardiac risk undergoing noncardiac surgery, the present case report may stimulate interest in future recommendations. In fact, in contrast with VATS in which the intrapleural space is open to the atmospheric pressure, RATS often needs CO2, insufflated through sealed ports, to maximize the intrapleural space by lung deflation and diaphragm flattening [2 insufflation, increasing the intrathoracic pressure, may cause both hypotension due to pressure-induced compression of the mediastinal vessels and gas exchange imbalance due to CO2 embolization or suboptimal pulmonary ventilation [Systolic anterior motion (SAM) describes the dynamic movement of the mitral valve (MV) anterior leaflet during systole towards the left ventricular outflow tract (LVOT). SAM could lead to MV regurgitation, LVOT obstruction (LVOTO), and consequent hemodynamic instability, potentially followed by hemodynamic instability. Clinical manifestations are hypotension, tachycardia, pulmonary hypertension, high wedge pressure, and in the worst scenario, pulmonary edema and shock unresponsive to inotropic support. Although SAM usually occurs in patients with hypertrophic cardiomyopathy (HCM) and after cardiac surgery for MV repair , this coattening . Hence, tilation ,8. Addittilation . All theA 70-year-old man  was referred to the thoracic surgery department to undergo robotic left upper lobectomy for stage IIA squamous cell carcinoma. On arrival, the vital signs were: respiratory rate, 17 breaths/min; temperature, 36.4 °C; blood pressure, 110/60 mmHg; heart rate, 45 beats/min; oxygen saturation level, 99% at room air; Glasgow Coma Scale score, 15. A 12-lead electrocardiogram showed sinus bradycardia, right bundle branch block, and left ventricular hypertrophy signs. His past medical history included chronic kidney disease stage IV, hypertension, hypertrophic ischemic cardiomyopathy NYHA class I treated with angioplasty in 2019, alcoholic liver disease with thrombocytopenia, metabolic syndrome, peripheral vascular disease, depressive disorder, and benign prostatic hyperplasia. Echocardiographic findings revealed severe concentric hypertrophy of LV more localized to the apical region and septum. The basal IVS thickness was 19 mm. The ejection fraction was 70% with no regional motion abnormalities and no significant valvular defects. Surgical intervention was performed the day after the admission. A low dose of propofol (1 mg/kg) was used for induction, and sevoflurane was used to maintain anesthesia. During induction, the pressure dropped to 50 mmHg, and desaturation occurred. This complication was managed with fluids and pure alpha-agonist administration along with a reduction of the volume of insufflation during manual ventilation to decrease intrathoracic pressure. After induction, paravertebral block with ropivacaine 7.5% 20 mL was performed with the patient positioned in lateral position to allow opioid-free anesthesia and postoperative analgesia. In the operating room, the patient was monitored with TEE throughout the procedure. During the surgery, TEE revealed a gradient of obstruction ranging between 60 and 180 mmHg . PreloadSAM with LVOTO is a rare cardiac event that may occur during robotic thoracic surgery, which requires prompt diagnosis and tailored hemodynamic, ventilatory, and pain management to avoid perioperative shock and other serious hemodynamic complications. SAM induces an impediment in the LV outflow tract, leading to variable degrees of mitral valve regurgitation, reduction in stroke volume, and hypotension. Early detection of SAM requires the use intraoperative TEE. The hemodynamic management of SAM is based on three main pillars: increasing preload, decreasing heart rate, and increasing afterload . SAM inc2 can cause adverse effects on hemodynamics, which are amplified by the positive pressure ventilation. Jones et al. [2 insufflation pressure [2 insufflation pressure in patients at higher risk of SAM or LVOTO. Diminished LV afterload increases SAM-related LVOTO. The increase in aortic pressure leads to higher LV end-diastolic pressure with the net effect of increasing the distance between the MV coaptation point and the septum, leaving LVOT open during the systole. This is the rationale for administering α-1 agonists  to increase the afterload  and to as et al.  observedpressure ,23,24,252 in RATS may exacerbate gas exchange impairment during OLV. Moreover, in patients undergoing RATS, there is an intrinsic risk of CO2 embolization and contralateral pneumothorax when the contralateral pleura is damaged. This risk should not be underestimated as it can influence LV preload, decreasing the venous return and increasing the venous pulmonary pressure, which in the end may elicit LVOTO in patients with predisposing factors for SAM development. Heart–lung interaction during mechanical ventilation is well known. OLV is particularly challenging for RV. Indeed, the combination of the decrease in venous return secondary to high intrathoracic pressure and the increase in PAPs are associated with a drop in right ventricular output and consequent right ventricle failure. Positive pressure ventilation decreases venous return to the RV and increases pulmonary pressure, leading to a dilation of the RV, leftward shift of the interventricular septum, and limitation in LV filling (ventricular interdependence) . The addConsequently, hypovolemic conditions, especially during mechanical ventilation, shift to the left the pressure–volume curve, increasing the risk of SAM, which is a preload-dependent phenomenon . The altAnalgesic techniques that mostly reduce systemic vascular resistances and induce vasodilation should be used with caution in patients with hypertrophic cardiomyopathy. The most effective technique for acute pain management after thoracic surgery is thoracic epidural analgesia (TEA). Several surveys have demonstrated that more than 50% (54%–85.6%) of thoracic anesthesiologists use TEA as the first-line treatment option for pain management ,30,31,32The management of patients with SAM in RATS includes peculiar treatment strategies, which, if not promptly and properly applied, may lead to catastrophic consequences. Notably, cases of SAM-related LVOTO are already reported in noncardiac surgery ,37 and tOn this basis, RATS still deserves specific considerations with respect to other noncardiac procedures. Thus, the present paper, which reports the first case of a patient with intraoperative SAM-related LVOTO during robotic lung major resection, may be useful in increasing knowledge in this field, highlighting the importance of being aware of this relatively neglected complication. The management of hemodynamic shock related to LVOTO and SAM requires a counterintuitive use of drugs and ventilation and a close cooperation between thoracic surgeons and anesthesiologists. Unfortunately, the current guidelines do not clearly recommend either the preoperative echocardiogram (TTE) or the intraoperative TEE for the assessment and management of patients with predisposing factors of SAM undergoing thoracic surgery. In fact, being SAM a dynamic condition, we strongly think that, at least in this highly selected population, TTE and TEE are complementary tools able to guarantee an uneventful course.


Carcinoid heart disease is a rare condition that occurs in less than half of patients with carcinoid syndrome. The disease mainly affects right‐sided heart valves; however, in 5%–10%, it can also involve left‐sided valves. This case illustrates the most complicated form of the disease involving four heart valves. The use of multimodality imaging in diagnosing and managing patients with systemic diseases involving the heart is of great importance and may improve the patients' survival and quality of life. The disease usually affects the right heart, and the left heart remains spared, as the lungs metabolize the vasoactive substances causing the disease. However, several conditions may lead to both right‐ and left‐sided involvement. These conditions include primary lung neoplasms, any intracardiac shunts, that is, atrial septal defect (ASD), patent foramen ovale (PFO), etc. and the presence of large burden of neoplastic tissue overwhelming the lung inactivation capacity by releasing notable amount of vasoactive substances.Transthoracic echocardiography (TTE) is the first imaging modality used in most cardiac patients. Screening for heart disease in those patients with carcinoid syndrome is of great importance since the patient may have no significant signs or symptoms in the early stages. Based on current evidence, TTE is the recommended modality to screen and monitor carcinoid heart disease.Cardiovascular magnetic resonance imaging (CMR) has excellent benefits in the diagnosis and monitoring of carcinoid heart disease. As the involvement of the pulmonary valve may be underestimated by echocardiography, CMR may have a leading role. Furthermore, the cardiac chamber sizes, the degree of valvular regurgitation and the right ventricular function are best appreciated in CMR. Magnetic resonance imaging (MRI) also has the unique capacity of tissue characterization technique, which may aid in the diagnosis of fibrosis and cardiac metastases.Cardiac CT (computed tomography) is rarely used in patients with carcinoid heart disease, and its central role remains to evaluate the coronary arteries and their relationship with myocardial metastases.Nuclear imaging for routine assessment of carcinoid heart disease is not established; even though PET (Positron emission tomography)/CT has a significant role in detecting cardiac metastases.The aim of this report was to point out the importance and the necessity of multimodality imaging and multidisciplinary approach in patients with carcinoid heart disease, especially complicated cases.2We describe a 56‐year‐old man who presented with lower extremity edema. He mentioned paroxysmal flushing, episodes of diarrhea accompanied by significant weight loss (24 kg in 18 months) during the past 5 years. He was managed with the impression of inflammatory bowel disease, irritable bowel syndrome and chronic malabsorption before referral to our center without any significant improvement. A cardiology consult was requested to evaluate right‐sided heart disease after developing exertional dyspnea (NYHA class III), abdominal distension, and lower extremity edema within the past 6 months.2 saturation of 95% in room air. His physical examination on presentation was significant for right‐sided S3, holosystolic murmur grade III/VI over the right lower sternal border, and an early diastolic decrescendo murmur grade III/VI over the left upper sternal border. He also had elevated jugular venous pulse (JVP), ascites and 2+ pitting edema in both lower extremities.He had a blood pressure of 130/70 mmHg, respiratory rate of 20 breaths/min, heart rate of 80 beats/min, temperature of 37°C (axillary), and O2.1His past medical history was significant for medically controlled hypertension and type II diabetes mellitus on Metformin.2.2Lower extremity edema accompanied with ascites are suggestive of right‐sided heart failure, liver cirrhosis, or portal hypertension. In this scenario, it is necessary to acknowledge the cause of right ventricular failure. The patient's physical examination raised the possibility of secondary failure due to valvular involvement. Besides, the patient had no history in favor of pulmonary thromboembolism or acute right ventricular myocardial infarction. Another item to consider is the cause of valvular involvement, which ranges from trauma, degenerative changes, rheumatic heart disease, tumor involvement, etc., to iatrogenic causes. A thorough history and comprehensive cardiac imaging including echocardiography and MR imaging, would lead to the diagnosis.2.32) with normal ejection fraction (Video The transthoracic echocardiography (TTE) showed normal left ventricular systolic function, moderate to severely enlarged and mildly dysfunctional right ventricle. Thickened aortic and mitral valve leaflets with moderate to severe regurgitation were noted. Both tricuspid and pulmonic valves were thickened, immobile and retracted with a “drum‐stick” appearance resulting in very severe regurgitations Figure , Video 1on Video . Volumeton Video . Color Don Video .2.4Octreotide, diuretics, and angiotensin receptor blockers were prescribed. However, despite the improvement of carcinoid syndrome symptoms, the right‐sided heart failure symptoms persisted. After multidisciplinary heart team discussion and regarding the pre‐operative assessment with EuroSCORE of 2.09%,2.5At the most recent clinical visit (6 months after the surgery), he had improved functional capacity and regained much of his lost body weight. In the follow‐up echocardiography, all four valves were optimally functional without complications.3Carcinoid heart disease is a rare condition and occurs in nearly half of patients with carcinoid syndrome and might be the initial manifestation of this syndrome in 20% of patients.The vasoactive substances secreted by the metastatic carcinoid tumor are usually metabolized and deactivated in the pulmonary circulation. Therefore, the substances that reach left heat are usually deactivated. Left‐sided heart valve involvement is infrequent and can occur in about 5%–10% of patients in certain circumstances, including intracardiac shunts , bronchial carcinoid tumors and a high burden of tumoral mass.In literature, there are reports of carcinoid heart disease involving the left and right heart, which were managed by tumor debulking and repair/replacement of the affected valves in certain cases.In a long‐term follow‐up study of 240 patients in the Mayo Clinic, valve replacement surgery revealed acceptable short‐term mortality. Besides, the earlier the intervention made, the more improved survival was noted.4In patients with multivalvular involvement, a multidisciplinary approach and thorough imaging assessment may aid in early diagnosis and guide necessary cardiac interventions. Thus, the patients' survival and quality of life can be improved.SHK had contact with the patient and wrote the manuscript. MP involved in patient's diagnosis and image analysis. NS involved in patient's management and manuscript preparation. MH performed pathological analysis and was involved in manuscript preparation. FN involved in data requisition and manuscript preparation. SH performed surgery and co‐wrote the manuscript. GH conceptualized the case and was involved in all the writing steps.Nothing to Disclose.Written informed consent was obtained from the patient to publish this report in accordance with the journal‘s patient consent policy.Video 1Click here for additional data file.Video 2Click here for additional data file.Video 3Click here for additional data file.


Pulmonary hypertension (PH) is an established risk factor in patients with heart failure (HF). However, right heart catheterisation (RHC) and vasoreactivity testing (VRT) are not routinely recommended in these patients.The primary objective of the present study was to explore the impact of VRT using sublingual glyceryl trinitrate (GTN) on transplant/ventricular assist device-free survival in HF patients with post-capillary PH. RHC parameters were correlated retrospectively with the primary outcome.p = 0.004), PA compliance , and total pulmonary resistance . Forest plot analysis including these three variables as well as PAC at baseline, delta PAC, and the presence of combined post- and pre-capillary PH revealed prognostic superiority of PAC-GTN, which was confirmed by Kaplan-Meier analysis.The cohort comprised 154 HF patients with post-capillary PH undergoing RHC with GTN-VRT at a tertiary heart failure centre. Multiple parameters were associated with survival. After adjustment for established prognosis-relevant clinical variables from the MAGGIC Score, variables with the most relevant odds ratios (OR) obtained after GTN-VRT were: calculated effective pulmonary arterial (PA) elastance (adjusted OR 2.26, 95%CI 1.30–3.92; In our cohort of symptomatic HF patients with post-capillary PH, improved PAC after administration of GTN was associated with survival independent of established hemodynamic and clinical risk factors. VRT using GTN may be better described as unloading test due to GTN's complex effects on the circulation. This could be used for advanced prognostication and should be investigated in further studies.  Post-capillary pulmonary hypertension (PH) is an established risk factor in patients with left heart failure (LHF), and those with advanced pulmonary vascular remodeling are known to have worse prognosis than those without. Increased vascular stiffness as a consequence of specific changes in the pulmonary vasculature leads to enhanced right ventricular afterload and right heart failure, which drives mortality in these patients . HoweverIsolated hemodynamic measurements are subject to significant spontaneous variations. The use of serial measurements, e.g., after acute vasoreactivity testing (VRT), likely improves hemodynamic prognostication , 9. HoweThe purpose of the present study was to explore the association of VRT results using sublingual GTN with outcomes in LHF patients with post-capillary PH. We hypothesized that application of GTN could provide incremental prognostic information by unmasking substantial pulmonary vascular disease.n = 85, 55%) were hospitalized because of worsening heart failure (31.8%), acutely decompensated heart failure (28.2%), diagnostic workup for evaluation of dyspnea (23.5%), and suspected pulmonary hypertension (16.5%). Inclusion criteria were a diagnosis of LHF with preserved or reduced left ventricular (LV) function according to current guidelines, availability of sufficient hemodynamic data, mean pulmonary artery pressure (mPAP) >20 mmHg and pulmonary artery wedge pressure (PAWP) >15 mmHg. PAWP between 10 and 15 mmHg at rest was accepted in a few cases (n = 12) if PAWP increased >25 mmHg during exercise or if clear features of left heart disease such as LV hypertrophy, reduced LV function, and/or significant left atrial enlargement were present. All patients included underwent guideline-compliant treatment for HF excluding PH targeted drugs. Exclusion criteria were loss to follow-up , severe heart valve stenosis, congenital heart defects, and constrictive pericarditis  that included vasoreactivity testing with GTN. Inpatients .The primary outcome measure was defined as survival free from heart transplantation (HTX) and left ventricular assist device (LVAD) implantation. Survival data were obtained through clinically indicated follow-up visits or telephone contact.All patients underwent transthoracic echocardiography according to recommendations of the respective guidelines as part of the clinical work-up with determination of left ventricular ejection fraction (LVEF), tricuspid annular plane systolic excursion (TAPSE), estimated systolic pulmonary artery pressure (sPAP), and valve assessment. Baseline laboratory examinations including N-terminal brain natriuretic peptide (NT-proBNP) and estimated glomerular filtration rate (eGFR), based on serum creatinine, were carried out by the respective in-house central laboratory as part of the clinical routine care.n = 90, 58%) were measured again during exercise. Instead of bicycle exercise, volume challenge (passive leg raise) was performed in 17 patients (11%) for additional measurements. After exercise / volume challenge, return to resting values was required for continuation of the examination. If mPAP was >20 mmHg and systolic blood pressure >100 mmHg, GTN was administered sublingually at an initial standard dose of 1.2 mg. GTN administration was repeated according to in-house standard operating procedures. The waiting time for repetition of measurements was a minimum of 5 min. A definition of positive response to GTN challenge was not determined in advance.RHC was performed in recompensated, stable patients under local anesthesia with insertion of a Swan-Ganz catheter  via the internal jugular vein or a cubital vein as described previously . The zerCardiac output (CO) was determined by the thermodilution (TD) technique. The calculated parameters were: stroke volume (SV = CO/heart rate); total pulmonary resistance (TPR = mPAP/CO); pulmonary arterial (PA) pulse pressure (PP = sPAP–dPAP); PA compliance (PAC = SV/PP); effective PA elastance (Ea = (1.65 x mPAP−7.79)/SV) ; PA pulsData are expressed as mean ± standard deviation or median [interquartile range] for normally or non-normally distributed parameters, respectively. Adherence to a Gaussian distribution was determined using the Shapiro test.t test for normally distributed parameters, and Fisher's exact test for categorical parameters, as appropriate. For dependent samples, the paired t test was used for normally distributed parameters, and otherwise the Wilcoxon signed rank test.For independent samples, comparison was made with the Independent-Samples Kruskal-Wallis test for non-normally distributed parameters, the Student P <0.05 was considered statistically significant. Statistical analyses were performed using either R version 3.6.0  or GraphPad Prism version 8.4.3 (471).We selected variables with the best predictive value for transplant/LVAD-free survival based on their ability to improve the predictive value of the MAGGIC score variables, an established score for risk prediction in patients with heart failure –18. OddsThis mono-centric analysis included 154 patients . Median age was 71 (IQR 62–76) years and 75% of the patients presented with symptoms according to NYHA class III. NT-proBNP levels [median 1890 (IQR 973-4182) pg/ml] were markedly elevated. Median LVEF was 45 [25–55]%, and the median TAPSE was 15 mm [12–19]. Classification according to heart failure type was as follows: 74 patients with preserved EF (≥50%), HFpEF; 12 with mid range reduced EF (40–49%), HFmrEF; and 68 with reduced EF (<40%), HFrEF). Duration of heart failure ≥ 18 months was present in 27% of HFpEF, 75% of HFmrEF and 79% of HFrEF patients. In HFpEF patients, 84% had a history of hypertension, 36% had coronary artery disease, 28% suffered from diabetes, 81% had atrial fibrillation, and 2 patients had hypertrophic cardiomyopathy. In HFmrEF patients, the etiology of HF was hypertensive in 33% and ischemic in 25%; 2 patients had dilated and 3 valvular cardiomyopathy. In HFrEF patients, 54% had ischemic etiology, and 34% had dilated cardiomyopathy, 7% valvular cardiomyopathy, 3% hypertensive cardiomyopathy; 1 patient had congenital heart disease.Nearly all patients (94%) were treated with diuretics; guideline-directed medical therapy was present as indicated. Patients had a high frequency of atrial fibrillation/flutter (73%) and device therapy (51%). Baseline characteristics, also stratified by our PAC-GTN cutoff, are provided in ). There were no significant side effects of GTN, especially no serious hypotension. Hemodynamics before and after GTN administration, stratified by our PAC-GTN cutoff, are provided in Median administered GTN dose was 2.4 (IQR 1.6-3.2) mg. GTN vasodilator challenge led to a significant change in most hemodynamic parameters, except heart rate and the PVR/SVR ratio . There wThe median follow-up was 30 [8–57] months in our cohort. Within this period 62 (40.3%) patients died, 3 (1.9%) underwent HTX, and 1 (0.6%) underwent LVAD implantation. Overall survival free from HTX and LVAD implantation was 57.2%.p-values in combination with highest AUC) with outcome were Ea-GTN, TPR-GTN, and PAC-GTN, all after GTN challenge.Univariate regression analysis revealed multiple associations between hemodynamic parameters and survival, both at baseline and post-GTN testing. Variables with the best ability to improve the predictive value of the MAGGIC score variables were selected. The components of the MAGGIC-Score are tagged within p = 0.004) for Ea-GTN, 2.29 (1.34–3.93) per SD increase (p = 0.003) for TPR-GTN, and 0.45 (0.25–0.80) per SD increase (p = 0.006) for PAC-GTN.The areas under the curve (AUC) in the ROC analysis of these parameters (adjusted for the MAGGIC score variables) to discriminate patients with poor outcome were 0.89 (95% CI 0.83-0.95) for Ea-GTN, 0.89 (0.84–0.95) for TPR-GTN, and 0.89 (0.84–0.95) for PAC-GTN. The respective odds ratios (OR) adjusted for the MAGGIC score variables were 2.26 (1.30–3.92) per SD increase , and established parameters such as PAC (baseline) and the presence of CpcPH (defined by PVR >3 WU and/or DPG ≥ 7 mmHg). In this multivariable analysis, considering several clinical important factors, PAC-GTN was the only independent significant factor associated with survival .r = −0.10; vs. LVEF: r = 0.08; vs. TAPSE: r = 0.17 ; and in patients with PAC-GTN ≤ 2.55 ml/mmHg: vs. NT-pro BNP: r = −0.16; vs. LVEF: r = −0.09; vs. TAPSE: r = 0.22; all p-values > 0.05.Correlation analyses were performed additionally to demonstrate independence of PAC-GTN related to known risk markers. In patients with PAC-GTN >2.55 ml/mmHg, there were the following correlations of PAC-GTN: vs. NT-pro BNP: r = −0.14; p = 0.09, and r = −0.04; p = 0.60). Kaplan-Meier survival analyses confirmed the prognostic power of PAC-GTN. Although presence of CpcPH and baseline PAC were both associated with survival, PAC-GTN was superior in prognostication , whereas the extent of PVR reduction was even smaller in PAC-GTN >2.55 than ≤ 2.55 ml/mmHg .Interestingly, reduction of RV oscillatory power and thus oscillatory load was more pronounced in PAC-GTN >2.55 than ≤ 2.55 ml/mmHg , and 24 patients with PVR ≤ 3 WU had PAC-GTN ≤ 2.55 ml/mmHg (thus changing from low to high risk). All in all, 38 patients (25%) had different hemodynamic prognostication by either PVR (CpcPH) or PAC-GTN (cut-off).Here we present a comprehensive analysis of the prognostic value of invasive hemodynamics at baseline and after challenge with sublingual GTN in HF patients with post-capillary PH. The relevant findings of our study are as follows: (i) three hemodynamic parameters  obtained after administration of GTN, all of them derived from pressure/flow relationships, showed significant prognostic value; (ii) PAC-GTN was the best prognostic marker, which was superior to established parameters such as PAC  and the There are few reports available concerning prognostic implications of VRT in pre-capillary PH , 21. In Three factors may have contributed to these new findings: the vasodilator used and its dosage, the measurement methods, and the most suitable hemodynamic parameter. Most drugs used for VRT are more or less selective pulmonary vasodilators that cause a small decrease or even an increase in PAWP, which is undesired in LHF (but nevertheless may result in PVR reduction). GTN decreases PAP and PAWP markedly by provoking venous and also arterial vasodilation, thus lowering pre- and afterload and indirectly increasing subendocardial blood flow , 27. TheSingle hemodynamic measurements may be subject to the bias of situational influences such as vasoconstriction and may mitigate the prognostic power of established hemodynamic indices such as CpcPH. Repeated measurements after vasodilatory challenge and thus ventricular unloading may be advantageous in this context. If unloading does not lead to markedly improved pressure-flow relationships , structural pulmonary vasculopathy may be present.Our analysis took multiple established prognostic hemodynamic factors into account, and Ea, TPR, and PAC as indicators of RV afterload  measuredWe included patients with different types of heart failure , which may be a source of bias. However, the effects of all types are elevated left-sided filling pressures, resulting in post-capillary PH . Our anaA hemodynamic unloading test using GTN may improve the prognostic power of PAC in patients with post-capillary PH and should be investigated in further prospective studies. Implications for therapeutic options of patients defined as high risk by this method remain elusive.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by Ethics Committee of the Faculty of Medicine at the University of Giessen. The patients/participants provided their written informed consent to participate in this study.AR, DG, and TK: conception and design, statistical analysis, interpretation of data, and drafting of the manuscript, GZ: acquisition of data. SK, SW, MR, KT, UK, VM, CH, and SR: analysis and interpretation of data and revising the manuscript critically for important intellectual content. All authors: final approval of the manuscript submitted.This research project uses a cohort that is part of the Kerckhoff Biomarker Registry (BioReg), which is financially supported by the Kerckhoff Heart Research Institute (KHFI) and the German Center for Cardiovascular Research e.V. (DZHK). The sponsors had no influence on the study design, statistical analyses, or draft of the article.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The renin-angiotensin-aldosterone system (RAAS) plays a vital role in cardiovascular homeostasis by regulating blood pressure, salt, and water balance. The kidneys produce renin which converts angiotensinogen to angiotensin-1 (AT-I) and angiotensin-converting enzyme (ACE) to angiotensin-II (AT-II). AT-II binds to receptors in the adrenal cortex to release aldosterone. AT-II and aldosterone promote water and salt retention, vascular tone, and myocardial contractility. These physiological changes raise blood pressure and circulation. Reduced renal perfusion pressure sensed by baroreceptors and the sympathetic nervous system’s β-adrenergic receptors trigger renin release and RAAS activation. RAAS restores hemodynamic stability in pathological states associated with low perfusion. This adaptive response is important for restoring perfusion and hemodynamic stability, but prolonged RAAS activation has deleterious effects on the cardiovascular system. Long-term mineralocorticoid exposure has been linked to left ventricular hypertrophy (LVH) and remodeling. AT-II activates fibroblasts and cardiac myocytes to promote cardiac remodeling. Blocking RAAS can eliminate the long-term negative effects of RAAS activation. Direct renin inhibitors, ACE inhibitors, angiotensin receptor blockers, and aldosterone antagonists are RAAS blockers.RAAS blockade improves mortality and hospitalization in systolic heart failure and acute myocardial infarction. RAAS blockade has not demonstrated the same benefits in other cardiac populations, such as those with preserved ejection fraction. Hypertrophic cardiomyopathy (HCM) causes LVH and asymmetric septal hypertrophy. When the outflow tract gradient exceeds 30 mmHg and is associated with septal hypertrophy, it is known as obstructive HCM. Dyspnea on exertion, syncope, and exertional angina are symptoms of HCM. RAAS activation worsens LVH by increasing blood pressure and by directly affecting cardiac myocytes with AT-II and aldosterone. RAAS blockade reverses myocardial fibrosis and slows HCM progression in animal models. We performed a meta-analysis of randomized clinical trials to further investigate the potential benefit of RAAS blockade in HCM patients. Although our findings included significant results for some of the RAAS blockade agents, these findings were not consistent throughout all the studies. Mavacamten, one of the newest treatments, has shown promising outcomes. The renin-angiotensin-aldosterone system (RAAS) is a hormonal cascade that plays a vital role in cardiovascular homeostasis through the regulation of blood pressure, salt, and water balance. The pathway begins with the production of renin from the kidneys which leads to the conversion of angiotensinogen to angiotensin-I (AT-I) which is subsequently converted to the active peptide angiotensin-II (AT-II) by the enzyme angiotensin-converting enzyme (ACE). Among the targets of AT-II are the adrenal glands where it binds to receptors in the cortex and leads to the release of aldosterone. AT-II and aldosterone are the major end products of this pathway which promote water and salt retention and increase systemic vascular tone and myocardial contractility. The cumulative effect of these physiological changes is an increase in arterial blood pressure and effective circulating volume . Major tWhile this adaptive response is important in restoring perfusion and hemodynamic stability, it is now well recognized that prolonged activation of RAAS is associated with several deleterious effects on the cardiovascular system. Mineralocorticoid receptors are found outside the adrenal cortex such as on cardiac myocytes and long-term exposure has been associated with left ventricular hypertrophy (LVH) and remodeling . SimilarHypertrophic cardiomyopathy (HCM) is common genetic cardiomyopathy characterized by LVH with or without asymmetric septal hypertrophy. An obstructive form is recognized when the outflow tract gradient exceeds 30 mmHg and is associated with pronounced septal hypertrophy. Symptoms of HCM overlap with those of heart failure and coronary artery disease and include dyspnea on exertion, syncope, and exertional angina. Activation of RAAS leads to a worsening of LVH both through increased blood pressure and through the direct effects of AT-II and aldosterone on cardiac myocytes. RAAS blockade in animal models has been shown to reverse myocardial fibrosis and potentially slow down the progression of HCM . To furtMethodsSearch StrategyA comprehensive literature search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) to identify all relevant publications reporting the effects of RAAS blockade in patients with HCM. Candidate studies were identified by conducting a literature search of PubMed and Cochrane Central Register of Controlled Trials in the Cochrane Library databases from inception to May 2020 . The actEligibility Criteria and SelectionTitles and abstracts were screened independently by two reviewers (H.A and K.L.B). Any discrepancies were resolved by consensus between the reviewing authors, or by consulting a third author (J.R) when an agreement could not be reached. The full text was reviewed when the articles did not provide enough information in the title and abstract. All screened articles were assessed for inclusion based on the following inclusion criteria: (1) study design was interventional; (2) presence of a control group; (3) study evaluated the effect of a RAAS blocker in patients with HCM; and (4) study reported relevant outcomes such as measures of LVH, diastolic function, and/or functional status.Data ExtractionData extraction was performed independently by two authors to compile information regarding patient and study characteristics as well as outcomes. Specific data items extracted for patient characteristics included age, sex, medications, and functional status. With regards to studying characteristics, extracted data items included authors, year, location, study design, intervention, sample size, and follow-up. To assess outcomes, we extracted data for LVH, diastolic function, and functional status. Data items extracted for LVH included left ventricular mass, left ventricular mass index, left ventricular fibrosis, N-terminal pro-peptide of collagen type I (PINP), and N-terminal pro-peptide of collagen type II (PIINP). Variables extracted to assess diastolic function included left atrial diameter, left atrial volume, N-terminal pro-brain natriuretic peptide (NT-proBNP), E/A, and E/E’. Finally, data for New York Heart Association (NYHA) class, peak oxygen consumption, bicycle ergometry, and six-minute walk test (6MWT) were extracted from included studies to assess functional status.ResultsA total of 109 articles were identified utilizing our literature search; of these, 102 did not meet our inclusion criteria. The final number of included studies was seven  in patients receiving losartan -13. Of tLeft ventricular fibrosis as assessed by late gadolinium enhancement (LGE) with CMR was reported by a total of three studies ,12,15. ATwo studies measured PINP and PIINP as markers of increased collagen synthesis and myocardial fibrosis ,16. KawaDiastolic FunctionTwo of the three trials that evaluated the effect of losartan did not observe a significant improvement in measures of diastolic function. Furthermore, Axelsson et al. conducted a subgroup analysis of patients with an observed decrease in left ventricular mass and found no significant improvement in the parameters of diastolic function. The authors observed a significant increase in left atrial volume as assessed by CMR in study participants over 12 months  . CandesaIn patients with the genetic predisposition to develop non-hypertrophic cardiomyopathy (NHCM) but no structural changes or symptoms, it has been suggested that the use of RAAS blockade may be more beneficial than when it is used in patients with left ventricular fibrosis and hypertrophy who have the genetic predisposition to HCM, which supports the hypothesis that ARBs can inhibit gene expression and reduce left ventricular mass in NHCMs ,13,18. IWe conclude through our study that HCM trials that investigated the effects of RAAS inhibitors on reducing LVH and fibrosis have demonstrated findings to be significant in some instances when using losartan or candesartan but not with valsartan; however, endpoints such as morbidity and mortality remain unknown. In addition, the mineralocorticoid receptor blocker represented by spironolactone did not show any evidence of playing a beneficial role in reducing left ventricular mass, improving left ventricular diastolic function, or relieving patient symptoms.According to the findings of our study, some of the RAAS blockade therapies have the potential to produce beneficial effects, such as the reduction of LVH, the improvement of left ventricular diastolic function, and the alleviation of symptoms in patients with HCM. However, the endpoints of morbidity and mortality are not yet known, and these beneficial effects were inconsistent throughout the studies.


Metabolite profiles showed altered branched-chain amino acid and acylcarnitine levels, decreased fatty acid uptake and oxidation, increased activity of the pentose phosphate pathway, dysregulation of the TCA cycle, and alterations in critical cellular antioxidant systems. These findings suggest processes of energy deficit, alterations in substrate availability, and enhanced production of reactive oxygen species in the affected myocardium. This profile potentially contributes to the development and maintenance of a chronic inflammatory state that leads to progression and severity of CCC. Further studies involving larger sample sizes and comparisons with heart failure patients without CCC are needed to validate these results, opening an avenue to investigate new therapeutic approaches for the treatment and prevention of progression of this unique and severe cardiomyopathy.Chronic Chagas cardiomyopathy (CCC) is the most frequent and severe clinical form of chronic Chagas disease, representing one of the leading causes of morbidity and mortality in Latin America, and a growing global public health problem. There is currently no approved treatment for CCC; however, omics technologies have enabled significant progress to be made in the search for new therapeutic targets. The metabolic alterations associated with pathogenic mechanisms of CCC and their relationship to cellular and immunopathogenic processes in cardiac tissue remain largely unknown. This exploratory study aimed to evaluate the potential underlying pathogenic mechanisms in the failing myocardium of patients with end-stage heart failure (ESHF) secondary to CCC by applying an untargeted metabolomic profiling approach. Cardiac tissue samples from the left ventricle of patients with ESHF of CCC etiology ( Trypanosoma cruzi (T. cruzi) that affects 6–7 million people in Latin America and more than 300,000 individuals in the United States and Europe; thus, it has been recognized as the parasitic disease with the highest disease burden in the world [Chagas disease (CD) is a neglected tropical disease caused by the protozoan parasite he world ,2. Chronhe world . CCC is he world . At preshe world ,6. Amonghe world ,9.The pathogenesis of CCC is complex and not yet fully understood , but invMass spectrometry-based metabolomics provides a useful approach for the identification of metabolites in biofluids or tissues and allows the characterization of molecular phenotypes in health and disease. Findings in humans and animal models provide clear evidence indicating severe metabolic alterations in the failing heart . Therefon = 7) and healthy control donors (n = 7) were included in the analysis. All participants were of mestizo ethnicity, with a large predominance of males in both groups (85.7% in both). Patients in the CCC group were significantly older compared to healthy controls  vs. 30  years, respectively, p < 0.001). All patients with CCC exhibited severely reduced ejection fractions (<20%) ) and a deteriorated New York Heart Association (NYHA) functional class. Regarding pharmacological treatment, all CCC patients were receiving beta-blockers, diuretics, intravenous inotropic agents, and immunosuppressive therapy before heart transplantation, while four were receiving anticoagulants and one statins. Moreover, three patients from the CCC group had previously been implanted with a left ventricular assist device. Finally, all healthy donors died from head trauma in the absence of concomitant thoracoabdominal trauma.A total of fourteen cardiac tissue samples from patients with ESHF of CCC etiology  analysis showed significant differences in the metabolic profiles from CCC and healthy tissue samples, in which 56.3% of the variance was explained by principal components 1 and 2 .Among the features, the abundance of 222 metabolites was significantly different between the groups, of which 40 metabolites matched known standards, being categorized as “MSI category 1 .” The diMetabolite Set Enrichment Analysis (MSEA) was used to explore, identify and interpret the differential patterns of metabolite abundances in CCC patients and healthy controls. This approach showed that the most significantly enriched pathways were the ones related to the metabolism of aspartate, the urea cycle, arginine and proline metabolism, carnitine synthesis, and the Warburg effect A. CompleCCC is a prevalent and complex disease governed by multiple molecular mechanisms for which no effective therapy is currently available ,20,21,22In this exploratory study, we applied an untargeted metabolomic profiling approach to investigate potential functional changes in ESHF of CCC etiology. This represents the first metabolomics study performed on human tissue samples from patients with CCC. Our analysis revealed significant biochemical alterations in critical metabolic pathways of hearts with CCC compared to healthy controls, highlighting a significant dysregulation in amino acid metabolism, glycerophospholipid metabolism, and antioxidant mechanisms, among others. Notably, the metabolic profiles observed in this study were characterized by decreased abundance of most metabolites, which could indicate imbalances in the metabolic pathways evaluated. Although diverse in function, these pathways are closely related due to shared substrates and dependence on the products of some pathways to feed alternative pathways for energy production . FinallyDespite contributing to a much lesser degree to ATP generation compared with fatty acids and glucose, amino acids (AAs) play critical roles in myocardial processes such as protein synthesis, signaling and metabolic intermediates, redox biology, and calcium homeostasis; however, under conditions of prolonged stress or ischemia, amino acids can become anaplerotic substrates . ParticuCurrently, evidence from metabolomic analyses in heart failure  has yielded conflicting results. Studies using high-performance liquid chromatography in plasma samples from heart failure (HF) patients have reported significantly higher levels of most altered AAs compared to healthy controls . These fRegarding the clinical significance of AAs as biomarkers, the study of Hakuno et al. observed a significant association between monoethanolamine, methionine, tyrosine, 1-methylhistidine, and histidine concentrations in serum with echocardiographic parameters such as the left ventricle (LV) ejection fraction, LV end-diastolic volume index, inferior vena cava diameter, and the mean mitral E/e′ in HF patients . FurtherFinally, amino acid supplementation has been proposed as a promising therapeutic option in heart failure patients considering the significant alterations in the availability of AAs and their metabolic impact. A recently published systematic review and meta-analysis showed that essential AA supplementation was associated with significant improvements in relevant parameters such as six-minute walk test distance, muscle mass, and quality of life. Nevertheless, these findings should be interpreted with caution due to the limitations of those studies, such as the high risk of bias of the included trials and the high heterogeneity, which prevented performing a meta-analysis . FurtherAcylcarnitines (ACs) facilitate the uptake and transport of fatty acids (FA) (C2-C26), which can be utilized for energy production in the mitochondria . The trap-adj = 0.0078). These findings are compatible with what has been observed in other studies performed in tissue samples of patients with ESHF of other etiologies and highlight the critical role of ACs and mitochondrial fatty acid oxidation in CCC [In the current study, we detected significantly decreased levels of myocardial ACs  and long-chain fatty acids in tissue samples from CCC patients compared to healthy controls. On the other hand, a single class of ACs, methyl malonyl carnitine, was found to be increased in the CCC group  and fatal outcomes two weeks after infection [In the setting of CD, evidence regarding the role of acylcarnitines and glycerophospholipids has been derived from acute and chronic mouse models of CD, our study being the first one to show similar results in humans ,24,46. Mnfection . Despitenfection ,49. Thernfection . On the Finally, our metabolic profiling detected reduced levels of unsaturated  and saturated (PC 15:0/0:0) FAs in cardiac tissue from CCC patients compared to healthy controls. These findings concur with previous data showing a downregulated expression of first- and third-step myocardial enzymes of FA β-oxidation in patients with ESHF and those undergoing cardiac transplantation . AdditioIt is known that a feature common to most etiologies of HF is the switching in myocardial substrate preference from FAs to glucose . In thisNevertheless, recent evidence has suggested a dual role of PPP in the pathophysiology of HF. The study of Badolia R et al. evaluated the activity of glucose metabolism accessory pathways in HF patients before and after LVAD-induced mechanical unloading. The authors of this study observed higher activities of glucose-6-phosphate-dehydrogenase (G6PD), transketolase (TKT), and transaldolase (TALDO) in the failing heart compared to healthy donors; however, significantly higher levels of the G6PD, TKT, and TALDO, along with significant reductions of sedoheptulose-7-phosphate and fructose-6-phosphate were observed in HF patients who responded positively after mechanical unloading (LVEF > 40% and a left ventricular end-diastolic diameter of <6 cm) compared to those who did not respond . The reaAs expected, the metabolic alterations present in CCC are also involved the TCA cycle, as five intermediates -malate, fumarate, and 2- methyl citrate) were found to be diminished in CCC samples compared to healthy controls . These fAging is associated with multiple changes in the cardiac tissue, highlighting pathological myocardial remodeling and microvascular dysfunction, which has been linked to multiple mechanisms, including mitochondrial dysfunction, aberrant signaling of the mechanistic target of rapamycin (mTOR), oxidative stress, autophagy, neurohormonal activation, dysregulation of miRNAs, among others ,66. DespT. cruzi infection, which must be validated in future studies using matched cohorts.At present, studies that have evaluated age-related changes in metabolomic profiles in cardiac tissue have been limited to murine models. These have reported significant age-related changes such as alterations in beta-oxidation mechanisms, with significantly lower levels of acylcarnitines, dysregulation of the TCA cycle, and a higher abundance of proteins involved in the process of glycolysis/gluconeogenesis such as glucose-6-phosphate and fructose-6-phosphate ,68. NeveThis study represents the first metabolomic profiling analysis performed on human tissue samples from patients with CCC, a relevant achievement considering the multiple limitations in the availability and logistics for obtaining human heart tissue samples. Availability of the infrastructure and equipment necessary for the immediate collection and optimal storage of these clinical samples ensures the quality of the primary material, and thus the fidelity of the derived metabolic profiles. Finally, we highlight the inclusion of only identified metabolites (MSI category 1) in the pathways analyses, which increased the robustness of our data interpretation.p-values observed, which may suggest a relevant role of these metabolites in the pathophysiology of the disease. Moreover, we used samples from deceased patients as a control group, given the absence of discarded hearts from potential donors who did not meet the criteria required for use in cardiac transplantation. However, we emphasize that sample collection was as rapid as possible, being performed within the first hour after death. On the other hand, previous studies suggest that it is possible to use this type of sample in the absence of sources equivalent to the case group [On the other hand, several limitations of our study deserve further discussion, highlighting the small number of patients that were enrolled in each group; therefore, we cannot rule out the presence of spectrum bias, and we highlight the need for larger, prospective validation studies to verify our results. The metabolites and associated pathways identified in this exploratory study serve as baseline for designing targeted metabolomic analyses, considering the highly significant se group ,75. FurtLC-MS grade solvents and mobile phase additives were obtained from Sigma Aldrich . The ZIC-pHILIC column was obtained from VWR .n = 7) at the Fundación Cardiovascular de Colombia (FCV), a fourth-level cardiovascular referral center located in Floridablanca, Colombia. All patients were classified according to the New York Heart Association (NYHA) and the American Heart Association (AHA) criteria and were receiving medical therapy following the guidelines of the European Society of Cardiology.Tissue from the left ventricular free wall was obtained from explanted hearts of patients with end-stage heart failure (ESHF) secondary to CCC , hemodynamic monitoring, electrocardiogram, and doppler echocardiography. Echocardiograms were routinely performed according to the FCV management algorithms for patients listed for heart transplantation. Left ventricular size and function were quantified by visual inspection, tricuspid annular plane systolic excursion (TAPSE), the myocardial performance index (MPI), and the fractional area change (FAC) on the last echocardiogram performed before heart explantation.n = 7) who had died because of noncardiac causes (mainly stroke and traffic accidents) was used as the control group. These samples were obtained through the Institute of Legal Medicine and Forensic Sciences of Bucaramanga . Briefly, sample collection was carried out by two professional teams: a forensic team and a medically trained team. They were on call 24 h a day. As soon as the teams were informed of a potential donor, they moved immediately to collect the samples and transport them to the Central Research Laboratory (CRL).Tissue from healthy hearts from unmatched organ donors , or explant (diseased group) and organ harvesting was no more than one hour. Once the samples arrived at the laboratory, they were processed under the same conditions. None of these donors showed evidence of heart disease and all presented normal left ventricular function and no history of myocardial disease or active infection at the time of death.Tissue explants were collected immediately following resection. Both CCC and control hearts were flushed with ice-cold before explant. At the time of explantation, two sections of the myocardial left ventricle free wall were collected from each participant. Approximately 100 mg of the frozen tissue per sample was mechanically homogenized and stored at −80 °C for further processing. Another tissue section was collected and fixed in 10% neutral buffered formalin and embedded in paraffin for histopathology studies.Metabolites were extracted using the methanol/chloroform method described by Le Belle et al. (2002) . BrieflyHydrophilic interaction liquid chromatography (HILIC) was carried out on a Dionex UltiMate 3000 UHPLC system  using a ZIC-pHILIC column . Chromatography solvents consisted of 20 mM ammonium carbonate in water (A) and acetonitrile (B). The column was maintained at 30 °C and a gradient program was used as follows: time 0 min: 20% A and 80% B, 30 min: 80% A and 20% B, 31–36 min: 92% A and 8% B, 37 min: 20% A and 80% B. The flow rate was 0.3 mL/min, the injection volume was 10 μL and samples were maintained at 5 °C before injection.m/z range 70–1050, electrospray probe temperature 150 °C, and capillary temperature 320 °C.For the MS analysis, samples were analyzed on a Q-Exactive mass spectrometer (MS) (Thermo Fisher Scientific) at Glasgow Polyomics, University of Glasgow. The MS was operated in polarity switching mode and the settings were as follows: resolution 70,000, AGC 1e6 (full scan mode), m/z 138), butylamine (C4H11N1) for positive ion electrospray ionization (PIESI) mode (m/z 74.096426) and COF3 for negative ion electrospray ionization (NIESI) mode (m/z 84.9906726). A standard mix containing approximately 200 metabolites was run at the start of every analysis batch to aid metabolite identification as described by Creek DJ, et al. [The calibration mass range was extended to cover small metabolites by the inclusion of low-mass calibrants with the standard Thermo calmix masses (below  m/z 74.06426 and http://polyomics.mvls.gla.ac.uk (accessed on 5 June 2017) software platform [m/z value, retention time, and intensities across samples. A pooled quality control (QC) sample was analyzed to correct signal drifts and remove batch effects, as the basis for performing drift correction.All raw files were converted into mzXML format, thereby centroiding the mass spectra and separating positive and negative ionization mode spectra into two different mzXML files using the command line version of MS convert (ProteoWizard). Accurate masses of standards were obtained within 3 ppm accuracy and intensities of the quality control samples were within specifications. The raw data were preprocessed and analyzed using Polyomics integrated Metabolomics Pipeline (PiMP) (platform . The prewww.hmdb.ca (accessed on 5 June 2017) [p < 0.05), a metabolite set enrichment analysis (MSEA) and metabolic pathway analysis (PA) were performed using the MetaboAnalyst® web portal and the KEGG database [The chemical structures of the candidate metabolites were determined by comparing the retention times and mass spectra to the commercial standards. The accurate mass and structure information of candidate metabolites were also matched with those of metabolites obtained from HMDB (ne 2017) , and METne 2017) . Metabolne 2017) . For thedatabase ,80.For the MSEA an Over Representation Analysis (ORA) algorithm was applied using a hypergeometric test . MSEA isp values from t-test and log2 fold change (FC) values using PiMP software: differential abundance threshold log2FC > 1 < and a p-value < 0.05. Whenever a compound level was below the level of detection in some samples  and not in others, an imputed value equal to the minimum observed among all the samples of the study was included. The heatmap of different metabolites was performed using a MetaboAnalyst platform  and the normalization procedure consisted of mean-centering and division by the standard deviation of each variable  using PiMP and R software. Differentially abundant metabolites were defined based on both variable .The Ethics Committees of Universidad Industrial de Santander (UIS) and the Fundación Cardiovascular de Colombia (FCV) approved the study protocol . In all cases, CCC patients or relatives of the healthy donors provided written informed consent. All clinical investigations were conducted according to the principles expressed in the Helsinki Declaration and its most recent amendment .The results of the present exploratory study suggest an important role of metabolic alterations in the pathophysiology of CCC, highlighting altered branched-chain amino acid and acylcarnitine levels, reduced fatty acid uptake and oxidation, activation of other glycolysis-coupled pathways such as PPP, and dysregulation of the TCA cycle. Further studies involving larger sample sizes and comparisons with age-matched heart failure patients without CCC are needed to validate these results, potentially opening an avenue to investigate new therapeutic approaches for the treatment and prevention of the progression of this unique and severe cardiomyopathy. One of the areas of interest for future studies should include improving the availability of energy substrates through the use of metabolic modulators, which could potentially be beneficial for patients with CCC, even in cases of advanced heart failure.


We have aimed at exposing left ventricular diastolic functions and the presence of known genetic mutations for familial erythrocytosis, in patients who exhibit idiopathic erythrocytosis.Sixty-four patients with idiopathic erythrocytosis  and 30 age-matched healthy subjects were prospectively evaluated. The regions of interest of the erythropoietin receptor, hemoglobin beta-globin, von Hippel-Lindau, hypoxia-inducible factor 2 alpha, and Egl-9 family hypoxia-inducible factor genes were amplified by PCR. Left ventricular (LV) mass was measured by M-mode and 2-dimensional echocardiography. LV diastolic functions were assessed by conventional echocardiography and tissue Doppler imaging.As a result of genetic analyses, genetic mutations for familial erythrocytosis were detected in 5 patients. It has been observed in our study that the risk of cardiovascular disorders is higher in patients. Interventricular septum thickness, left atrial diameter, and some diastolic function parameters such as deceleration time and isovolumetric relaxation time have been found to be significantly higher in idiopathic erythrocytosis group than in the controls.This study has shown that LV diastolic functions were impaired in patients with idiopathic erythrocytosis. In this patient group with increased risk of cardiovascular disorders, the frequent genetic mutations have been detected in 5 patients only. Therefore, further clinical investigations are needed as novel genetic mutations may be discovered in patients with idiopathic erythrocytosis because of cardiovascular risk. Primary-acquired erythrocytosis involves polycythemia vera (PV), which is characterized by the presence of JAK2 mutations, although these markers are not found in about 1% of patients with PV.–4 Secondary-acquired erythrocytosis, however, occurs when erythropoietin (EPO) drives the production of red cells. This situation can develop in various cardiac, pulmonary, and renal diseases or from external hypoxic conditions. For familial polycythemias, a third subgroup defined as a mixed polycythemia has been identified other than its primary and secondary subgroups; this group includes erythrocytosis associated with the Chuvash polycythemia and other von Hippel-Lindau (VHL) gene mutations. Genes carrying mutations associated with familial polycythemias have been identified as erythropoietin receptor (EPOR), VHL, prolyl hydroxylase domain protein 2, and hypoxia-inducible factors 2 alpha (HIF2a) genes in the online Mendelian inheritance in man classification.,8 Other polycythemias, defined by molecular lesions, are congenital methemoglobinemia, polycythemias caused by high oxygen and 2,3-bisphosphoglycerate deficiency.,10 Once all the known causes of erythrocytosis have been ruled out, there remains a group of patients classified as having idiopathic erythrocytosis (IE). IE is characterized by an increase in red blood cell mass of unknown etiology. The frequency of IE is considered to be 1.1 for 1000 subjects. Diagnosing IE requires the exclusion of PV, secondary-acquired polycythemia, and various congenital primary and secondary polycythemias. It has also been reported that the risk of thrombosis is low in IE, and there may be spontaneous progress to acute leukemia or myelofibrosis.Erythrocytosis or polycythemia as commonly known refers to an increase in erythrocyte mass. Both acquired and familial erythrocytosis (FE), except relative erythrocytosis, are examined by subgrouping as primary and secondary. Increased left ventricular mass (LVM) and deterioration in diastolic function parameters are risk factors for future heart failure development. In people with risk factors for the development of heart failure, asymptomatic LV dysfunction progresses to symptomatic heart failure over time. Approximately half of the cases of left heart failure are in the form of diastolic LV dysfunction.,16 Heart failure that results from impairment of myocardial relaxation and compliance is called diastolic heart failure. LV diastolic dysfunction refers to increased cardiac filling pressures as a result of decreased LV relaxation and increased stiffness. It is important to monitor LV diastolic function parameters before diastolic heart failure develops. Measurement of transmitral blood flow with pulsed-wave Doppler and measurement of myocardial velocities from mitral annulus, septal, and lateral regions by tissue Doppler have become the preferred tool for noninvasively evaluating diastolic functions. Although there are studies investigating the relationship between LV diastolic functions in anemia and polycythemia vera, no similar studies have been found in IE.,21In patients with PV, it has been shown that blood rheology is associated with cardiovascular risk, and the mechanism of this is associated with impaired left ventricular (LV) function.In our study, we have aimed at investigating and understanding whether isolated erythrocytosis would present as a risk factor for cardiovascular diseases. For this purpose, first, the primary- and secondary-acquired erythrocytoses were excluded, in which factors other than erythrocytosis could increase the risk of cardiovascular disease. Mutations in genes associated with FE have been investigated in all the cases. We have attempted to determine the presence of known mutations in proposed regions and tried to reveal new mutations related to erythrocytosis where possible. In addition, we have tried to determine the differences between the control group and the risk group with reference to the LV diastolic functions.The study was conducted in the hematology and cardiology clinics of Tekirdag Namik Kemal University between January 2014 and January 2017. The inclusion criteria were as follows: isolated erythrocytosis , absence of JAK2 V617F and exon 12 mutations, and absence of any defined causes of acquired secondary erythrocytosis except smoking. Exclusion criteria were as follows: the patients under 18 years of age and patients with known coronary artery disease, cerebrovascular disease, or peripheral arterial disease. Furthermore, patients with chronic obstructive pulmonary disease, congestive heart failure, congenital heart disease, history of cardiac operation, atrial fibrillation, atrial flutter, and left bundle branch block were excluded from the study.Sixty-four patients evaluated to have IE, and 30 healthy subjects that made up the control group were included in the study. Comprehensive clinical evaluations of the patients and controls who agreed to participate in the study were performed, and the patients and their families were questioned for erythrocytosis and related features. Moreover, routine laboratory findings (including blood count and lipid parameters) were recorded. Cardiovascular risk factors  were designated/assessed in the patients and controls. EPO levels were measured in all patients. Written informed consent was obtained from all subjects. This study was approved by the Institutional Review Board of Tekirdağ Namik Kemal University.All of the cases included in the study group were assessed for FE-related genomic alterations by studying the mostly reported genes and mutations in the literature. Exon 8 of the EPOR gene , entire coding and regulatory sequences of the hemoglobin beta-globin (HBB) gene , the genomic region encompassing the Arg200Trp mutation that constitutes more than 90% of mutations of the VHL gene , the genomic region including the most frequent mutations Ile533Val and Phe540Leu of the HIF2a gene , and the genomic sequences containing the Cys127Ser and the Gln157His mutations in the first exon of the Egl-9 family hypoxia-inducible factor (EGLN1) gene  were screened by direct sequencing. For this purpose, 5-cc peripheral blood samples were collected from the patients, and the samples were taken into K3-EDTA tubes, and genomic DNA isolations were carried out through a commercial kit  according to the manufacturer’s directives. The amount and purity of the isolated genomic DNA samples were determined. The regions of interest of the EPOR, HBB, VHL, HIF2a, and EGLN1 genes were amplified by PCR. The primer sequences used in PCR are given in Figure After the PCR, the products were checked by agarose gel electrophoresis. Then, PCR products were purified, and DNA sequence analysis reactions were performed. Repurified reaction products were subjected to capillary electrophoresis in an automated DNA sequencing device  to determine the sequence of DNA fragments. Tekirdağ Namik Kemal University, the Scientific Technological Research and Application Center, performed the processes starting from DNA isolation to obtaining the sequence results. The results of the DNA sequence analyses were evaluated by comparing with the reference sequences, and genomic changes that could be related to FE were determined.Echocardiographic evaluation was performed using high-resolution B-mode ultrasound with a 2.5-MHz transducer by 2 experienced cardiologists who did not know the diagnosis of patients . While taking echocardiographic recordings, attention was paid to the appropriate room temperature and dim lighting. During the procedure, patients were asked to breathe normally. The interobserver variability for all measurements was found to be between 8.5% and 9.8%. Measurements of 20 patients were repeated 2 days later to evaluate intraobserver variability, and intraobserver variability for all measurements was between 6.8% and 7.8%. The modified biplane Simpson method was used for LV ejection fraction measurements. The LVM was calculated with the Devereux formula as follows: 0.8 × {[(1.04 × [LVDD + IVST + PWT]3 − LVDD3)]} + 0.6.Two-dimensional, M-mode, pulse-wave, and tissue Doppler echocardiography was performed for the measurement of left heart cavity dimensions, wall thicknesses, LVM, systolic, and diastolic function parameters. Left atrium diameters, LV systolic diameters, left ventricular diastolic diameters (LVDD), interventricular septum thicknesses (IVST), and posterior wall thicknesses (PWT) were measured according to the recommendations of the American Society of Echocardiography by 2-dimensional and M-mode echocardiography.In order to evaluate the conventional diastolic functions, diastolic transmitral flow was measured by pulsed-wave Doppler between the mitral leaflet tips at a level of 1 cm below the plane of the mitral annulus, in the apical 4-chamber view. The early (E) and late (A) phase peak flow velocities and their ratio (E/A), E wave deceleration time (DT), isovolumetric relaxation time (IVRT), isovolumetric contraction time (IVCT), and ejection time were measured.Tissue Doppler measurements were made using a 2.5-MHz variable frequency phased array transducer. To obtain LV tissue Doppler recordings, Doppler sample volume was placed in the septal and lateral corners of the mitral annulus from the apical 4-chamber view. The Nyquist limit was set to a velocity range from −20 to 20 cm/s. The monitor weep speed was set at 100 mm/s. Diastolic early (e), late (a), and systolic (s) waves were measured from septal and lateral regions of the mitral annulus, septal e/a, lateral e/a, E/septal e, and E/lateral e ratios.t test. Chi-square tests were used for comparison of categorical variables. Pearson correlation analysis was performed to investigate the relationship between Hb values and diastolic echocardiographic parameters. A P value less than .05 was accepted as genetic statistically significant.Statistical analysis of the data was carried out by using SPSS for Windows 18.0 software. The Shapiro-Wilk test was used to examine whether the data were normally distributed. All variables were found to be normally distributed. Continuous variables were expressed as mean ± standard deviation, and categorical variables as numbers and percentages. In statistical analysis, continuous variables between control and patient groups were compared with Student As a result of genetic analyses, genetic mutations related to FE were detected in 5 patients Fig. . EPO serP > .05 for all). Hb  and hematocrit  values were significantly higher in the IE group compared with the control group (Table HBB: c.328 G>A (Hb San Diego) in heterozygous form was detected in a patient and his mother . IVST , left atrial diameter , DT , IVRT , and LV myocardial performance index  values were significantly higher in the patient group compared with the control group. Mitral E , E/A , septal e , septal e/a , lateral e , and lateral e/a  values were significantly lower in the patient group compared with the control group , DT , there was a negative correlation between Hb values and E , E/A , septal e , septal e/a , and lateral e/a    was detected in heterozygous form in 1 individual. It has been reported that HBB: c. + + 96 T> C is a silent mutation that does not cause any change in the Hb level in neither heterozygous nor homozygous states, but its compound heterozygosity with beta-thalassemia mutations increases the severity of beta-thalassemia. We detected EPOR exon 8 Arg437 His mutation (c.1310G>A) in 1 case and 3 family members. Pathological mutations in EPOR exon 8 lead to familial congenital polycythemia (PFCP). The diagnosis of PFCP has been made in a patient with IE in the presence of low or normal EPO serum level and a family history compatible with autosomal transition. The diagnosis of PFCP can be confirmed by detection of heterozygote EPOR mutation in only 12%–15% of cases. To date, mostly, mutations of EPOR, which cause PFCP, within exon 8 have been described. Until now, 2 cases with c.1310G>A variant have been described in the literature. One of them was a male 24 years of age who required regular phlebotomy. The other one was a 52 years old patient with a clinical history of recurrent venous thrombosis with normal Hb and hematocrit levels and no familial history of hematologic disorder. Our case represents the third subject in literature detected with this variant.Except for those newly defined HBB mutations, Hb San Diego was also detected in 1 case and her son. Hb San Diego has been described in subjects of various origins, and only 1 case had been previously reported from Turkey so far. The EGLN1 mutations cause secondary FE of the familial erythrocytosis type 3 online Mendelian inheritance in man classification. Already, EGLN1 encodes PHD2, which is the principal negative regulator of (HIFs) and EPAS1 encoding HIF-2a. It has been reported that EGLN1 c.380G>C together with EGLN1 c.12C>G as a haplotype can be a component of a complex array of genetic adaptations involving both PHD2 and HIF-2a that results in lower Hb in Tibetan highlanders.We detected EGLN1:c.380G>C (p.Cys127Ser) missense mutation in 1 case, which had been first detected by Lorenzo et al. The mechanism of the acquired thrombophilic state in these diseases is multifaceted. Prothrombotic features of myeloproliferative neoplasm clone–derived blood cells, not only erythrocytes but also platelets and leukocytes and procoagulant changes of normal vascular cells, in response to inflammatory stimuli, can be considered as mechanisms of this thrombophilic state. In fact, hyperviscosity due to erythrocytosis does not seem as an important factor for thrombosis in PV. Chuvash polycythemia causes thrombotic and hemorrhagic vascular complications that lead to early mortality usually before the age of 40 years.–38 Apart from Chuvash polycythemia, the association of FE and IE with thrombosis and cardiovascular complications are not clear.In PV and other Ph-negative myeloproliferative neoplasms, there is a high incidence of thrombosis due to an acquired thrombophilic state. Aging, coronary artery disease, diabetes, hypertension, and atrial fibrillation are important factors contributing to diastolic dysfunction. To clearly assess the contribution of erythrocytosis in diastolic dysfunction, we have excluded individuals with coronary artery disease and atrial fibrillation. Also, there was no difference between the groups in terms of age, gender, hypertension, diabetes, and smoking habits. In this study, we found some parameters such as decreased mitral E wave, transmitral E/A ratio, septal e, septal e/a, lateral e, and lateral e/a ratio and increased DT and IVRT values, as evidences of decreased LV relaxation. In the presence of mild diastolic dysfunction, relaxation of the left ventricle is impaired; early filling of the left ventricle in diastole becomes difficult; accordingly, mitral E wave, and septal e and lateral e wave amplitudes decrease, and E wave DT and IVRT get prolonged. These findings show that stage 1 diastolic dysfunction exists in patients with IE. In addition, the atrial contribution increases exaggeratedly due to late filling of the left ventricle in diastole, thereby increasing the mitral A wave amplitude. As a result, mitral E/A, septal e/a, and lateral e/a ratios decrease.In previous publications, it had been demonstrated that PV and some types of anemia had impaired LV diastolic functions. LV hypertrophy is an important risk factor for diastolic dysfunction. Therefore, we have performed M-mode and Doppler echocardiography for our patients and healthy controls. M-mode echocardiography showed a higher thickness of interventricular septum in our cases than the control subjects. This was thought to have been caused by increased workload of heart due to increased viscosity in the patient group. In the study of Kayrak et al., LVM had been found to have increased in patients with polycythemia vera. Although the septum thicknesses increased, the LVM data of patients were not different from those in the control group in our study. We think this is due to the fact that viscosity is lower in patients with IE compared with polycythemia vera and that the LV workload is less.We have not found any difference between the cases with erythrocytosis and healthy controls for cardiovascular risk factors such as hypertension, lipid parameters, diabetes, and smoking. Furthermore, we assessed cardiovascular status of all subjects with transthoracic echocardiography. It has been suggested that transthoracic echocardiography can be used a detection method for LV hypertrophy, which is considered as a cardiovascular risk factor in asymptomatic adults who have hypertension.Mitral E/A ratio has been found to be lower in the patients with pulsed-wave Doppler echocardiography. Additionally, a prolongation of IVRT and IVCT and the low value of mitral annulus septal e, in tissue Doppler echocardiography, have been detected, which were thought to be associated with slowing of the blood flow rate due to increased hematocrit levels. As a result, increased cardiac workload may lead to deterioration of myocardial relaxation over time, resulting in diastolic dysfunction. In our study, we have found an increase in MPI in the IE patients. This suggests that the LV systolic and diastolic functions begin to deteriorate together in these patients due to increased viscosity.Increased MPI is an indicator, showing that both LV diastolic and systolic functions are impaired together. MPI has been shown to have increased in PV.In conclusion, mutations that are known to cause familial erythrocytosis have been shown, and beta-globulin gene changes that are likely to cause secondary familial erythrocytosis have been identified in our study. In addition, it has been determined that echocardiographic findings related to cardiovascular workload and cardiovascular morbidity and mortality were present prevalently in IE cases.Even though FE patients are diagnosed rather prevalently in internal medicine/hematology clinics, no underlying pathogenetic cause is studied due to lack of established algorithms. FE is only differentially diagnosed from PV, with no follow-up procedures with reference to genetic aspects and cardiovascular risks involved. This means that such a relatively prevalently seen and important disease is being overlooked at the expense of patients. FE patients are under serious cardiovascular risks apparently with the AD genetic transfer perpetuating the risks for the next generations and the society, unless awareness is augmented and due care is given to such patients.We thank Orhan Cem Deniz and Yasemin Gundogdu who provided medical writing supports.


This study aimed to identify the stiffness and natural length of the human plantar aponeurosis (PA) during quiet standing using ultrasound shear wave elastography. The shear wave velocity (SWV) of the PA in young healthy males and females (10 participants each) was measured by placing a probe in a hole in the floor plate. The change in the SWV with the passive dorsiflexion of the metatarsophalangeal (MP) joint was measured. The Young’s modulus of the PA was estimated to be 64.7 ± 9.4 kPa, which exponentially increased with MP joint dorsiflexion. The PA was estimated to have the natural length when the MP joint was plantarflexed by 13.8°, indicating that the PA is stretched by arch compression during standing. However, the present study demonstrated that the estimated stiffness for the natural length in quiet standing was significantly larger than that in the unloaded condition, revealing that the PA during standing is stiffened by elongation and through the possible activation of intrinsic muscles. Such quantitative information possibly contributes to the detailed biomechanical modeling of the human foot, facilitating an improved understanding of the mechanical functions and pathogenetic mechanisms of the PA during movements. The PA consists of mostly type 1 collagen fibers arranged in the longitudinal direction, spans the entire plantar side of the longitudinal arch of the foot, and is thought to be closely connected to the paratenon of the Achilles tendon throught the periosteum of the calcaneus5, allowing mechanical energy to be stored and released in each contact of the foot during walking and running8. In addition, the PA enables the foot to become a rigid lever during the push-off phase of walking and running by the so-called windlass mechanism. In particular, the PA is stretched to generate tension as the metatarsophalangeal (MP) joint is dorsiflexed in the late stance phase. This causes the stiffening of the midtarsal joint and entire foot during the push-off phase of walking and running for effective generation of propulsive forces10. For the detailed understanding of the biomechanics and motor control of the human foot, and to clarify the pathogenetic mechanism of foot disorders, it is important to accurately identify the biomechanical function of the PA in vivo during movements. To the best of our knowledge, the in vivo kinematic and kinetic PA properties during human movement has not been quantified due to the difficulty of capturing this information non-invasively.The human foot is known to be unique among primates in possessing a well-developed plantar aponeurosis (PA)13. For this, previous studies attempted to capture the mechanical behaviors of the human PA in vitro using a tensile testing machine16, while recent studies captured the mechanical behaviors of the PA in vivo using ultrasound elasticity imaging29. Based on the measured mechanical properties, the PA is modeled as a single or a bundle of linear springs31.Consequently, efforts have been made to computationally predict the kinematics and kinetics of the PA during walking and running using motion capture and biomechanical modelingIn these studies, the mechanical properties of the PA were measured in an unloaded condition (in the prone or supine position). However, soft tissues surrounding the PA may mechanically interact with the PA. This possibly affected the mechanical properties in a physiological loading condition, such as walking and running. For the accurate estimation of the PA tensile force, and other internal forces and moments generated in the foot using a foot model, the quantification of the mechanical properties of the PA in a physiological loading condition is necessary.24. Such quantitative information is expected to clarify the mechanical functions of the PA during movements and the pathogenetic mechanism of foot disorders.In this study, we aimed to identify the stiffness, linearity, and natural length of the PA spring in the human foot during a quiet standing condition by quantifying the change in the shear wave velocity (SWV) of the PA while passively increasing the dorsiflexion of the MP joint using ultrasound shear wave elastography. In addition, we investigated if there exist gender differences in the mechanical properties of the PA, because previous studies suggested that there were considerable sex differences in the stiffness of the PATwenty healthy young participants  were enrolled in this study. No participant had any history of any lower limb injuries or clinical symptoms, and did not exhibit any neurological disorders. This study was reviewed and approved by the Office for Life Science Research Ethics and Safety, the University of Tokyo, and it was performed according to the Declaration of Helsinki. Written informed consent was obtained from all the participants before data collection.An Aplio i700 ultrasound scanner  with a linear array probe (effective length: 45 mm) was used to acquire the ultrasonic B-mode and shear wave images of the PA. The scanner was set at the musculoskeletal preset and high frequency (Res) mode. All measurements were performed by the same examiner, and the probe was manually operated.24. During standing, the participants were instructed to equally distribute their weight on the feet in the mediolateral and anteroposterior directions, and to maintain their line of sight horizontal. Unloaded measurements were also conducted in the sitting posture with the leg horizontally placed on a platform, to ensure that no external forces were applied to the sole of the foot.To scan the middle site of the PA in the longitudinal direction, the probe was placed on the plantar surface of the foot parallel to the line connecting the calcaneus and the second metatarsal head Fig. ; the cenOne ultrasound B-mode image and five shear wave images of the local SWV distribution of the aponeurosis in the square region of interest  were obtained for each posture. The shear wave image was recorded as a color-coded map Fig. . ThereafTo estimate the SWV and MP joint angle when the PA had natural length, SWV was plotted against different MP joint angles, and the following exponential function was fitted to the plots using the least-squares method:33:334 and 0.533, respectively.The Young’s modulus can be calculated based on SWV using the following equationt-tests or the Wilcoxon rank sum tests. The differences in the SWV among the unloaded and loaded  conditions were analyzed using a one-way repeated measure ANOVA (within-subject factor: angle). If one-way ANOVA was significant, the post-hoc Tukey’s HSD test was performed to test if the SWV was influenced by the weight-bearing and MP joint angles. The statistical significance level was set at P < 0.05. Friedman test with a post-hoc Wilcoxon signed rank test for multiple comparisons, followed by Bonferroni correction with the adjusted P-value set at P < 0.005 (0.05/28), was used if the normality or homogeneity of variance was violated. The estimated SWV for the natural length of the PA was compared with the SWV in the unloaded condition using paired t-test or Wilcoxon signed rank test. Data processing and analyses were implemented in R software, version 3.5.235.The possible gender differences in the SWV for each condition and the coefficients of the exponential function (Eq. ) were inThere were no significant gender-associated differences in the SWV of the PA Fig.  in all cx2 = 140, P < 0.0001). The SWV of the PA significantly increased as the MP joint was dorsiflexed by 10° (P < 0.0001)  Fig. . The pre01) Fig. , possibl01) Fig. . The mea36.In this study, we quantified the stiffness of the human PA under a physiological loading condition. Such information on the mechanical properties of the PA contributes to the detailed biomechanical modeling of the human foot, leading to an improved understanding of the mechanical functions of the PA during movements, such as walking, running, and jump landing, and of the pathogenesis of the plantar fasciitis and plantar fibromatosis29. Although the exact cause of this discrepancy remains unclear, it might be attributable to the difference in the leg posture during measurement. In previous studies, SWV measurements were performed when participants were in a supine or prone position on a bed, and therefore, the knee was completely extended. This complete extension of the knee might have stretched the gastrocnemius muscle37 and generated passive tensile force to pull up the calcaneal tuberosity, possibly leading to the stretching and stiffening of the PA even though the foot was unloaded. In contrast, the present study measured SWV when the knee was approximately 90° flexed in the unloading condition. Even in the standing posture, the knee was slightly flexed; it was not completely extended during measurements. Therefore, differences in the leg posture, particularly the knee joint angle, might considerably influence the SWV measurements. This possibility must be further investigated for an accurate and more consistent quantification of the mechanical properties of the PA.Our value of PA Young’s modulus (SWV = 4.6 m/s) was lower than previously reported (SWV = 5.1–8.6 m/s)16 and ultrasound elasticity imaging studies26. In the construction of the musculoskeletal models of the human foot incorporating the PA, the PA is often modeled as a single or a bundle of linear springs31. However, both the current and previous studies demonstrated the mechanical property of the PA to be not linear elastic. Therefore, caution is required when computationally predicting the kinetics of the PA during movements using a foot musculoskeletal model.The SWV exponentially increased with the dorsiflexion of the MP joint Fig. , indicat11, the plantarflexion angle of 13.8° approximately corresponds to a 2 mm change in the PA length. This is consistent with a recent cadaver study that showed that the medial arch was elongated by approximately 2 mm when the foot was axially loaded by approximately half body weight38. Therefore, the natural length of the PA can be approximately estimated as the length of PA during quiet standing (when MP angle = 0°) subtracted by 2 mm.The current study estimated that the MP joint was plantarflexed by 13.8° for the natural length of the PA. Assuming the radius of the metatarsal head to be ca. 9 mm5. An electromyographic study has demonstrated that the abductor hallucis and flexor digitorum brevis were activated in the standing posture39. Therefore, the activation of the intrinsic foot muscles might increase the stiffness of the PA during quiet standing, in addition to the elongation of the PA. During human walking, the intrinsic foot muscles reportedly contribute to the efficient generation of the propulsion force in the late stance phase40. Our results suggested that the interaction between the stiffness of PA and the activation of the intrinsic muscles of the foot should be considered for the accurate modeling of the mechanics of the PA during locomotion.However, the estimated SWV for the natural length of the PA is significantly larger than that in the unloaded condition Fig. , althoug24. However, in the current study, no significant gender-associated differences in the SWV of the PA were found in any condition. This is in agreement with Taş (2018)41—in that the SWV of the PA does not differ between males and females—but in disagreement with Shiotani et al. (2019)24—in that the SWV of the PA in females is significantly larger than that in males. However, tendinous connective tissues are usually considered mechanically weaker in females than in males42; the Achilles45 and patellar47 tendons are reportedly softer in females than in males, although some studies did not observe such gender-based differences53. Therefore, we believe that the stiffness is not larger in females than in males, and there exist no gender-associated differences in the SWV of the PA; however, this must be confirmed through further studies.It has been previously reported that there were considerable gender differences in the stiffness of the PAThe present study has some limitations. First, the number of the participants were relatively small, and the only young participants (range 19–33 years) were enrolled. Although we believe that the present study successively quantified the mechanical properties of the PA during quiet standing, they should be investigated with larger sample size and more wide range of ages in future study. Second, although the present study investigated the increase in the PA stiffness during the passive dorsiflexion of the MP joint during quiet standing, this experimental condition was certainly not identical to that of the foot at the time of toe off during walking. To directly infer the mechanical characteristics of the PA during the functioning of the windlass mechanism in vivo, further technical innovations are certainly necessary to directly capture the PA material property without using a probe, as placing a probe on the plantar surface of the foot certainly renders normal gait impossible.In conclusion, the Young’s modulus of the PA was estimated to be 64.7 ± 9.4 kPa during quiet standing and 45.30 ± 3.68 kPa in the unloaded condition, which exponentially increased with the dorsiflexion of the MP joint, suggesting that the PA was a nonlinear spring. The PA was estimated to have natural length when the MP joint was plantarflexed by 13.8°, indicating that the PA was elongated by approximately 2 mm during quiet standing owing to the compression of longitudinal arch. Furthermore, our study showed that the estimated SWV for the natural length of the PA was significantly larger than that in the unloaded condition, suggesting that the SWV of the PA during quiet standing was stiffened by elongation and through the possible activation of the intrinsic foot muscles located along and possibly attached to the PA. Such quantitative information possibly contributes to the detailed biomechanical modeling of the human foot, facilitating an improved understanding of the mechanical functions and pathogenetic mechanisms of the PA during movements.