In the United States, routine middle cerebral artery peak systolic velocity (MCA-PSV) Doppler screening for the detection of antenatal twin anemia-polycythemia sequence (TAPS) is not recommended. The current and only national clinical guideline from the highly-influential Society for Maternal-Fetal Medicine states that, “There is no evidence that monitoring for TAPS with MCA PSV Doppler at any time, including > 26 weeks, improves outcomes, so that this additional screening cannot be recommended at this time.” We argue this recommendation has disproportionate influence on patients and the care they are offered and receive. We use current evidence to highlight and dispel pervasive myths surrounding antenatal TAPS and the value of routine MCA-PSV screening. An ethical framework that illustrates the importance of giving patients the opportunity for routine screening is presented. Findings demonstrate that: (1) both spontaneous and post-laser TAPS is a serious, potentially life-threatening complication, (2) treatment for TAPS is effective and includes expectant management, intrauterine transfusion (IUT), or surgery, (3) and routine MCA-PSV, which has satisfactory diagnostic accuracy, is currently the only way to provide early detection of TAPS. We conclude that routine TAPS screening is a medically proven valuable resource that should be offered to patients in need and to the clinicians who are trying to act toward their benefit. Monochorionic (MC) twin pregnancies have long been fraught with a mortality rate that exceeds dichorionic twins by over seven times . In addiTwin anemia-polycythemia sequence was first described in 2006 by Robyr et al. . For thoHowever, many providers and clinical organizations have chosen not to embrace and utilize current-and-growing TAPS knowledge for reasons we discuss below.Antenatal TAPS can only be treated if it is first diagnosed using middle cerebral artery peak systolic velocity (MCA-PSV) Doppler ultrasound ,8,9,10. Such refusal can be seen in published clinical guidelines put forth by the Society for Maternal-Fetal Medicine (SMFM), wherein their recommendation states, “There is no evidence that monitoring for TAPS with MCA PSV Doppler at any time, including > 26 weeks, improves outcomes; so that this additional screening cannot be recommended at this time” . AdditioThis paper seeks to use current information to dispel these myths while discussing inherent ethical issues borne from withholding the opportunity for routine MCA-PSV screening for MC patients. What we have provided is an argumentative review to refute deeply embedded assumptions and information, which have led to poorly examined guidelines and practices that negatively impact the welfare of our patient populations. The approaches to this paper follow the philosophical traditions of theoretical analysis, which is an accepted methodology in bioethics and the social sciences that intersect with medical research methods. The postulates, theoretical support, and argumentative framework substantiate our descriptive claims to achieve valid and sound logical argumentation to be considered for the advancement of future research and discussion in this area. TAPS, a complication arising from intertwin anastomoses connecting the fetal circulations, has been recognized, studied, and received considerable publication for over 12 years . TAPS isThis slow transfusion promotes anemia in one fetus and polycythemia in the other, while no serious difference occurs in the amounts of amniotic fluid . AbsenceTAPS can occur spontaneously in 3% to 6% of otherwise uncomplicated MC twin pregnancies ,18 or foAntenatal TAPS is diagnosed based on measurement of the middle cerebral artery peak systolic velocity (MCA-PSV) . The besLike TTTS, antenatal TAPS is represented by stages with progression through stages culminating in fetal death. Also similar to TTTS, the progression of TAPS stages are not always linear, do not always progress, and may even regress . The folStage 1: Delta MCA-PSV > 0.5 MoM, without signs of fetal compromiseStage 2: Delta MCA-PSV > 0.7 MoM, without signs of fetal compromiseStage 3: As Stage 1 or 2, with cardiac compromise of donor *Stage 4: Hydrops of donorStage 5: Intrauterine demise of one or both fetuses preceded by TAPS* Defined as critically abnormal flow: Doppler shows absent or reversed end-diastolic flow in umbilical artery, pulsatile flow in umbilical vein and/or increased pulsatility index or reversed flow in ductus venosus.Other typical ultrasound TAPS findings include placental discordance; the anemic donor’s region of the placenta is thickened and hyperechoic, and the plethoric recipient’s region of the placenta appears normal  and starGiven the rareness of MCDA pregnancies, much of the research regarding TAPS, including short and long-term outcomes, has been based on findings from case series and case studies . FurtherDetection of antenatal TAPS is crucial, as it affects management of the pregnancy ,30 and gTo obtain MCA-PSV measurement, an axial section of the brain, including thalami, cavum septi pellucidi and greater wing of sphenoid, with the circle of Willis identified by color Doppler, should be obtained. The MCA closest to the probe is sampled at or near its origin from the internal carotid artery. The waveform peak is measured, with the angle of insonation as close as possible to 0°, and always < 30°. Higher inter- and intra- observer variability results from angle correction and sampling of more distal regions of the MCA. The fetus should be quiescent, as heart-rate accelerations and movement can alter measurements . A step-p < 0.001) and sensitivity and specificity of MCA-PSV ≥ 1.5 MoM to predict severe anemia in TAPS donors was 94% and 74%, respectively. The sensitivity of MCA-PSV ≤ 1.0 MoM to predict polycythemia in TAPS recipients was 97% and specificity was 96% [While MCA-PSV serves as a gold standard for diagnosis of anemia in singletons, the diagnostic accuracy and utility of MCA-PSV measurement for TAPS has been considered uncertain . More sp was 96% . The autA 2018 analysis of 45 uncomplicated MC and 35 TAPS twins by Tollenaar et al. showed that the sensitivity and specificity of the cut-off MCA-PSV values (> 1.5 MoM in the donor and < 1.0 MoM in the recipient) to predict postnatal TAPS was 46% and 100%, respectively . Delta MFindings in p = 0.039), as more transfusions are required [In addition to cut-off values, whether or not a fetus has been transfused can also affect the accuracy of MCA-PSV Doppler measurements for TAPS. A 2019 meta-analysis by Martinez-Portilla et al. examined observational studies evaluating the performance of MCA-PSV using a 1.5 MoM threshold for the prediction of fetal anemia . The refrequired . Other factors that may influence MCA-PSV include: gender, cardiac status, uterine contractions, fetal behavioral state , and advHowever, prediction may be improved through the use of serial MCA-PSV measurements to track trends (versus a single measurement), resulting in a reduction of the false-positive rate to less than 5% .In summary, MCA-PSV Doppler is a non-invasive and accurate test for detecting fetal anemia, polycythemia, and TAPS. Sensitivity and specificity levels are high and acceptable. False positives and negatives can be reduced by adhering to the proper scanning technique, obtaining intra- and inter-rater consensuses, performing serial MCA-PSV readings, and remaining mindful of additional conditions that may alter readings. Finally, the recommended use of the improved antenatal classification system by Tollenaar et al. , which iAmongst the most systemic myths surrounding TAPS is that other abnormal ultrasound findings will always appear and/or that TAPS will always coexist with other fetal disorders that are detectable without MCA-PSV screening. It is this myth that inspires some health care providers to start screening for TAPS only when other complications such as TTTS or IUGR are presenting. While we applaud governing bodies such as NICE for recognizing and endorsing routine MCA-PSV screening to mitigate the dangers of TAPS to MC pregnancies already complicated by other syndromes , such reThe primary perspective about which abnormal findings “should be” observed alongside TAPS is discordant amniotic fluid levels. How this myth was developed, and why it still exists, is perplexing. The earliest publications describing TAPS and its natural history, and all of those since, are quick and clear to define TAPS as notable due to its non-presence of TOPS ; for thiWhile ‘post-laser’ TAPS provides a very clear demarcation of when to begin MCA-PSV screening based on the premise of ‘other symptoms or complications,’ spontaneous TAPS is a notable threat, occurring at a rate of up to 6% in otherwise ‘uncomplicated’ MC pregnancies and will not always be accompanied by such qualifying symptoms . It is important to note we are not arguing that other complications cannot accompany TAPS—they certainly can. The argument is that other complications or indications are not common nor required and TAPS screening should not be predicated upon their development. To expand on possible comorbidities, on rare occasions, TAPS may precede TTTS . Some obn = 14) were spontaneous [A 2016 literature review, which assessed 14 studies containing 29 pregnancies complicated by TAPS, showed that 48% of TAPS cases  present is no longer state-of-the-art in modern obstetrics. There is no rationale to support deferring TAPS screening until additional unsupported and unnecessary criteria have been met. Just as deferring routine bi-weekly ultrasounds and refusing to acknowledge inherent MC risks was detrimental to 2010 MC care, deferring routine TAPS screening and failure to acknowledge its inherent stand-alone risk is now detrimental to 2019 MC care. There are a host of effective treatments for TAPS, including fetoscopic laser surgery , intrauA 2018 systematic review of morbidity and mortality associated with intrauterine interventions for the treatment of TAPS concluded that there is no mortality difference between any of the treatment modalities. Though expectant management may be associated with an increase in adverse perinatal outcomes when compared laser therapy and IUT .In a retrospective study, where laser treatment for antenatally detected TAPS is compared to IUT or expectant management; laser therapy appeared to improve perinatal outcome by prolonging pregnancy and reducing respiratory distress syndrome . The medTreatment with IUT in the donor can be performed either intravascularly or intraperitoneal. Intraperitoneal IUT is preferred, since intraperitoneal transfusion may allow slower absorption of red blood cells into the fetal circulation, preventing rapid loss of transfused blood in the circulation of the recipient twin . It is iWith the first RCT underway, there is no current consensus on an optimal TAPS treatment protocol; to what extent individual interventions for TAPS improve fetal outcome is not fully understood; and what is deemed optimal will vary based on gestational age and other clinical observations. However, TAPS is associated with significant pre- and post-natal morbidity and mortality ,26 and tIn the absence of evidence on optimal management, Tollenaar et al.  suggest If MCA-PSV Dopplers are being performed correctly, something that does not exist should not be found. As demonstrated in the discussion of Myth 2 see , the senFalse positives can be further reduced by adhering to proper scanning technique, obtaining intra- and inter-rater consensuses and remaining mindful of additional conditions that may alter readings, such as previous transfusions. Importantly, performing serial MCA-PSV readings can reduce the false positive rate to only 5% .There may be apprehension on behalf of some providers regarding MCA-PSV screening and its interpretation for diagnosing TAPS ; howeverWomen experiencing pregnancies with obstetric complications face increased rates of stress and anxiety ,52 and MAdditionally, steps can be taken to mitigate maternal anxiety with regards to screening and treatment for TAPS. Specifically, reducing parental uncertainty has been demonstrated to help alleviate maternal anxiety during medically complicated pregnancies ,59. ThisThe monozygotic twinning rate is roughly 3.5 per 1000 pregnancies, with approximately 70% being monochorionic , thus clNonetheless, six-percent represents a roughly 1 in 16 risk of being affected by a disorder that can result in serious fetal injury and/or death . When coWe have already established that other complications preceding TAPS are not necessarily present , emphasizing the need to routinely check for the presence of TAPS. Risks associated with premature birth are well-established and should not be undervalued. This reality forces us to revisit the argument that TAPS diagnosis, and resulting conversations regarding treatment options, should always include a careful explanation of benefits, risks, and unknowns.In the end, rarely will the best decision be abundantly clear. The primary argument of this paper, however, is that the decision ultimately rests with the patient. The patient must receive all of the evidence available—predicated upon MCA-PSV Doppler measurement values—so that she can manage her care and make informed decisions.A 2016 literature review showed that amongst patients with spontaneous TAPS, average gestational age at birth was 32 weeks (23.6–38 weeks) . When anAs of 2019, the only current United States clinical recommendations regarding MCA PSV Doppler screening can be found in the Society for Maternal Fetal Medicine (SMFM) Publication Committee’s 2013 publication in the American Journal of Obstetrics and Gynecology, titled “Twin-twin Transfusion Syndrome” . This reTwin anemia-polycythemia sequence: diagnostic criteria, classification, perinatal management and outcome by Slaghekke et al. [The citation used to support this recommendation is based on the 2010 paper e et al. . Howevere et al. . Additionally, Table 4 in the 2010 Slaghekke et al. paper reveals that when TAPS was detected antenatally using MCA-PSV Doppler and interventions were performed , the result of those interventions was a 100% survival rate . Again, While some fetal treatment centers have developed and disseminated their own screening standards, which include routine MCA-PSV screening starting at gestational week 16  , our resBecause clinicians may have concerns regarding the validity of screening and diagnostic techniques as well as the risks associated with treatment, information related to MCA-PSV for routine TAPS screening is not consistently offered to patients. Providing patients with this information fits within a normative framework for reproductive health, involving three unified elements: (1) a dialectical, therapeutic relationship that balances the duties of providers and the rights of patients,  and preserves the fiduciary role of clinicians; (2) considerations of justice that afford patients with fair and reasonable opportunities to secure their values and interests , and; (3) ethical acts that promote the welfare of patients (beneficence), while avoiding unnecessary harms . Such frameworks that address challenges in prenatal screening and prenatal decision-making are presented in the literature ,64,65. TOne of the most critical ethical considerations for TAPS screening is the development and preservation of the therapeutic, fiduciary relationship between clinician and patient, established through intimacy, trust, and care and requiring clinicians to prudently care for and act toward the benefit of their patients. In the context of routine TAPS screening, the therapeutic, fiduciary relationship requires clinicians to fully inform and disclose available screening options, recognizing the needs of patients as individual persons and not as statistics or probabilities. As part of their fiduciary responsibilities, clinicians are expected to learn new technologies and practices supported by evidence-based medicine , implement current policies and recommendations into clinical practice, and address any concerns  with colleagues and experts in the field. Fulfilling these obligations strengthens clinicians’ confidence and, more importantly, patient care. Goals of justice require that patients are afforded fair and reasonable opportunities to secure their values and interests, including opportunities to achieve health regardless of clinicians’ preconceived beliefs of their interests and socioeconomic status. Non-directive counseling for routine TAPS screening is essential without clinician bias, criticism, or personal opinion. Withholding information is a violation of reproductive freedom and parental rights in securing the interests and the rights of their fetuses   TAPS is a serious, potentially life-threatening complication, (2) treatment for antenatal TAPS is effective and includes either expectant management, IUT, or surgery, (3) and routine MCA-PSV, which has satisfactory sensitivity and specificity, is currently the only way to provide early detection of antenatal TAPS ,71. The Monochorionic twin pregnancies are inherently ethically complex pregnancies with the potential for difficult decisions , thus itWe recognize MCA-PSV screening for antenatal TAPS is currently not a standard of care for U.S. women pregnant with monochorionic twins. However, because of the information we present above, which not only dispels myths about MCA-PSV screening but also provides ethical arguments for screening, we present a call to action for U.S. organizations and clinicians to establish routine MCA-PSV screening as a standard of care for this high-risk population. We recognize the potential burdens this technology may pose for organizations and clinicians in terms of the time and effort it takes to establish fair guidelines and institutional policies and promote a standard of care unique to this population. However, the magnitude of life-altering burdens a patient may experience by not having the opportunity to be properly screened is much greater and can ultimately diminish the relationships patients have with their clinicians, and communities have with their health organizations. The patient and our future children require positive changes motivated by care and compassion rather than nocuous myths.


Primary graft dysfunction (PGD) remains the leading cause of early mortality post-heart transplantation. Despite improvements in mechanical circulatory support and critical care measures, the rate of PGD remains significant. A recent consensus statement by the International Society of Heart and Lung Transplantation (ISHLT) has formulated a definition for PGD. Five years on, we look at current concepts and future directions of PGD in the current era of transplantation. Heart transplantation remains the closest resemblance to a ‘cure’ for end-stage heart failure. Worldwide, more than 4000 adults undergo heart transplantation annually . Whilst PGD presents as severe ventricular dysfunction of the donor graft which fails to meet the circulatory requirements of the recipient in the immediate post-transplant period. It may manifest as either single or biventricular dysfunction with low cardiac output and hypotension despite adequate filling pressures –5. In 202) lasting at least 1 h. The requirement of an intra-aortic balloon pump signifies moderate PGD-LV, whereas requirement of extracorporeal short-term mechanical circulatory support in the form of extracorporeal membrane oxygenation (ECMO) or ventricular assist devices (VADs) in any form  is diagnostic of severe PGD-LV [PGD is defined as being a separate entity from secondary graft dysfunction, which is when a discernible cause for allograft dysfunction is identified. Such causes can include hyperacute rejection, graft dysfunction secondary to pulmonary hypertension or a recognised intraoperative complication. The diagnosis of PGD is made within 24 h post-transplantation and is separated into PGD-LV, for PGD affecting the left ventricle or biventricular failure; and PGD-RV for isolated right ventricular involvement . A severe PGD-LV .2, TPG < 15 mmHg and/or pulmonary artery systolic pressure < 50 mmHg) [PGD-RV does not have a severity scale and is diagnosed based on the requirement of a right-sided short-term VAD (RVAD) or right heart catheter measured haemodynamics in keeping with isolated right-sided dysfunction  .Previous studies had used the need for mechanical circulatory support as a criterion for diagnosis of primary graft dysfunction; however, the timing of initiation of therapy and endpoints differs from these studies, leading to significant variation in incidence reporting 2.3–32.4%) [% 4, 8–1–18–11. WThe donor heart is exposed to a multitude of physiological insults at four specific time points: brainstem death, hypothermic ischaemia, warm ischaemia and ischaemia-reperfusion injury.The first insult occurs during declaration of brainstem death in donation after brainstem death (DBD). Raised intracranial pressure (ICP) invariably is the final pathway in DBD donors in which case the aetiology of death is usually a result of an intracerebral haemorrhage, hypoxia leading to oedema, inflammation or a space occupying lesion in the cranium. The cerebral perfusion pressure is usually maintained by homeostatic mechanisms which result in an increased arterial pressure. Due to the limited plasticity of the brain within the confines of the cranium, brain herniation ensues resulting in pontine ischaemia . This caVasomotor tone is reduced to the loss of spinal cord sympathetic activity resulting in unopposed vasodilatation, further reducing preload and indirectly afterload, affecting coronary blood flow and reducing myocardial perfusion. As a response, there is an intense release of myocardial noradrenaline immediately after brain death that results in mitochondrial and cytosolic calcium overload to increase contractility to counteract this unopposed vasodilatation . PituitaThese patients are also prone to metabolic disturbances due to acidosis, hypomagnesaemia or hypokalemia secondary to mannitol initiation to reduce ICP, catecholamine administration, all of which contribute to increased myocardial oxygen demand or reduced myocardial perfusion . This in3) administration only in depleted donors [Early donor management may reduce the deleterious effects of the abovementioned phenomenon. Early administration of vasopressors is aimed at reducing the unopposed vasodilatation . Methylpd donors , 21, 22.Despite advancements in normothermic ex vivo perfusion devices, cold storage remains the mainstay of transportation after organ retrieval. Organ retrieval often has two distinct phases, a warm phase and a cold phase. The warm phase involves dissection of the heart and exposure of the innominate artery and the two cavae with variations in the degree of dissection depending on whether the lungs are to be retrieved as well. The retrieval surgeon also palpates the coronary arteries for potential disease. The aorta is prepared for administration of cold cardioplegia for commencement of the cold phase.+ ions [+/H+ exchanger is activated resulting in an increase in intracellular Na+ which activates the Na+/Ca2+ exchanger [2+. This will play a major role in the pathogenesis of ischaemic-reperfusion injury.Depending on the size of the patient and type of cardioplegic solution administered, the volume of administration may differ. The retrieval surgeon then performs a left atriotomy by lifting the heart. Once the aortic cross-clamp is applied, cold cardioplegia is infused via the aortic root at approximately 4 °C. The retrieval process is completed with the heart placed in a cold storage container. Cold storage induces hypothermic arrest of metabolism and maintains viability during this reduced metabolic state, therefore abating cellular swelling and minimalizing reperfusion injury . At thes+ ions . The Na+xchanger , 28. TheOn arrival at the recipient centre, the heart is removed from cold storage and inspected. The period in which the heart is removed from cold storage or from ex vivo normothermic perfusion to the final release of the recipient aortic cross-clamp is termed warm ischaemic time. The heart is exposed to warmer temperatures which slowly increases its metabolic rate resulting in increased formation of oxygen free radicals. In a study by Marasco et al., warm ischaemic time and increasing donor age were independent predictors of early survival, suggesting an acceleration of the deleterious effects described during the cold ischaemic phase . SimilarIschaemia-reperfusion injury (IRI) is defined as cardiomyocyte damage secondary to myocardial restoration of blood flow . On releAnimal studies do however highlight attenuation of IRI using preconditioning in diabetics –48, femaAnother mechanism of IRI is hypercontracture-mediated sarcolemmal rupture (HMSR). During the ischaemic phase, the low cytosolic ATP concentrations are quickly exhausted resulting in myofibrillar shortening that remains fixed as all cross-bridges between actin and myosin remain in an attached state . This ca2+ overload which develops during ischaemia and is rapidly re-energised, as it would on release of the cross-clamp [+/Ca2+ exchanger pumps. Sarcolemmal rupture also results in increasing Na+ influx into cardiomyocytes via gap junctions and may propagate to adjacent cells [Reperfusion-induced hypercontracture occurs after prolonged ischaemia. There is greater myofibrillar shortening and cytoskeletal damage when compared to the ischaemic phase alone. It causes a marked rise in end-diastolic pressure with increased ventricular wall stiffness. It is shown to be due to Cass-clamp . In cellss-clamp . Sarcoless-clamp  and ankyss-clamp , impairint cells .Donation after circulatory death (DCD) provides a different set of insults to the donor heart. The heart is placed in a hypoxemic and hypercarbic environment following withdrawal of life support . This ca+/H+ exchanger in a pathway similar to the warm ischaemic period described above, with a net result of an accumulation of intracellular Ca2+ and follows the ischaemic-reperfusion pathway on reperfusion.This prolonged ischaemic period causes intracellular acidosis which causes activation of the NaATP channels). Endogenous nitric oxide, a potent vasodilator, is also implicated in the pathogenesis alongside vasopressin deficiency [Some recipient factors may also predispose a patient to PGD. Recipients, especially patients who have been on inotropic support and mechanical devices, have activation of their systemic inflammatory response, which causes intense vasodilation and therefore lowering of the systemic vascular resistance. The net result is a hypotensive state despite adequate filling and a high cardiac output that is refractory to vasopressor support . The exaficiency .Prolonged ischaemic time has been shown in previous studies to correlate with complications occurring after cardiopulmonary bypass . This asAnother pathogenic mechanism that has been suggested in the past is recipient pulmonary hypertension. The donor heart in its ischaemic state is exposed to a relatively high pulmonary vascular resistance, giving rise to right-sided heart failure. Due to the poor adaptability of the right ventricle to a sudden change in vascular resistance, biventricular dysfunction ensues due to a reduced left-sided preload, thereby reducing coronary perfusion and end organ perfusion in the systemic circulation . This hoThere are several risk factors that have been suggested that may contribute to PGD. These include donor factors, recipient factors and procedural factors.Avtaar Singh et al. (2018) highlighted donor age to be a significant risk factor on multivariate analysis. This may have been due to the poorer tolerance for long ischaemic times in hearts from older donors. They highlighted an odds risk of 20% for each decade increment in donor age. A similar finding was noted by Russo and colleagues . They noFemale-donor to male-recipient gender mismatch is a risk factor for PGD. The exact mechanism is poorly understood as this persisted despite organ size matching. Male-donor to female-recipients did not result in an increased PGD rate. Gender-mismatched organs also had a poorer survival at up to 5 years. In liver transplantation, recipients had poorer graft survival in female-donor/male-recipient cohorts compared with other donor-recipient gender groups despite adjustment for donor risk factors and recipient variables . The rolIntracranial haemorrhage is the most common cause of death for DBD donors in the UK. One study showed increased PGD rates in organs retrieved from donors with intracranial haemorrhage compared to donors with traumatic cause of death . The exaDonors with hypertension often have a degree of left ventricular hypertrophy (LVH). This is more apparent across Europe where the average age of the donor is older compared to North America . The useInotropic support is sometimes needed during the donor organ retrieval process due to the depletion of catecholamine and/or a systemic inflammatory phenomenon as described above. Noradrenaline use can cause left ventricular dysfunction in the absence of brain death . It alsoPre-operative recipient ECMO/VAD usage has also been linked to PGD post-operatively , 96, 97.A recent study by Truby and colleagues highlighted the role of continuous flow LVAD (CF-LVAD) in recipients as a risk factor for severe PGD . This isThe role of recipient diabetes as a predictor of PGD is evident in multiple studies . The resSeveral studies have associated advancing recipient age with PGD and mortality , 92. A rA recipient re-sternotomy indicates previous surgery, most commonly for durable ventricular assist device implantation, previous congenital cardiac surgery or previous coronary bypass or valvular surgery . The disAmiodarone is a class III antiarrhythmic used for a variety of arrhythmias including both ventricular, supraventricular and atrial tachyarrhythmias. Its mechanism of action is primarily by prolonging the refractory period myocardium. Arrhythmias are highly prevalent in advanced heart failure patients . The eviIn DBD donations, the ischaemic time consists of the placement of the donor aortic cross-clamp till the release of the recipient aortic cross-clamp . For DCDCold ischaemic time consists of the time spent in cold storage and warm ischaemia ensues once the heart is removed from cold storage or the OCS rig till release of the aortic cross-clamp. Prolonged ischaemic time is associated with an increased rate of PGD , 92, 119Prolonged cardiopulmonary bypass (CPB) duration independently predicts post-operative morbidity and mortality after general cardiac surgery . KirklinThe size of the donor graft has many implications on the recipient. It is a powerful predictor of survival recipients receiving undersized grafts having an increased 1-year mortality, and a 36% increased mortality within the first 30 days . Smits sThe current definition for PGD is based on the treatment options utilised. Several biomarkers have been suggested as potential predictors of PGD, although to date none are used in routine care.The pro-inflammatory state accompanying PGD supports the use of inflammatory markers as potential predictive biomarkers. Tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), neutrophils and procalcitonin (PCT) have all been suggested as potential biomarkers.Tumour necrosis factors are produced by lymphocytes and macrophages that cause cell lysis . TNF-α hBirks and colleagues noted an increased expression of TNF-α in unused donor hearts due to poor function and compared them with donors with good ventricular function (used donors) and patients with advanced heart failure (HF) . In lungA Brazilian group showed recipients receiving grafts from donors expressing lower concentrations of plasma soluble tumour necrosis factor receptors 2 (sTNFR2) and IL-6, required more inotropic support post-transplantation .Interleukin 6 (IL-6) is produced in response to infections and tissue injuries by stimulation of acute phase responses. It bridges the adaptive and innate immune responses and plays a major role in autoimmune conditions , 147. BiIL-6 was also shown to be associated with PGD post-lung transplantation. The serum and bronchioalveolar lavage concentrations of IL-6 were higher in transplant recipients with PGD than those without .Animal models have shown that ischaemic cardiac myocytes in watershed viable zones of a myocardial infarction exhibited reperfusion-dependent expression of IL-6 mRNA after reperfusion , 151.Procalcitonin (PCT) is a 116-amino acid peptide that has an approximate molecular weight of 14.5 kDa and belongs to the calcitonin (CT) superfamily of peptides . It is a−1 was a potentially useful tool in predicting the end-management heart usability for transplantation. Similar findings were noted by Wagner and colleagues with increased 30-day mortality and early graft dysfunction in hearts utilised from donors with elevated PCT levels [In transplantation, PCT was used to differentiate bacterial infection from organ rejection. PCT levels are elevated in both conditions but significantly higher in the presence of infections , 154. ItT levels . The incT levels , but notT levels  or lung T levels .Both NLR and platelet to lymphocyte ratio (PLR) have been used as markers of inflammation with prognostic value in coronary artery disease , 161 andn = 111). A Polish single-centre study noted similar findings in their renal transplant cohort, with NLR showing good predictive value of early graft dysfunction [A group in Argentina studied the relationship between the two ratios and survival after heart transplantation . They nofunction . Anotherfunction . HoweverCardiac troponins are regulatory proteins that control the calcium-mediated interaction between actin and myosin. The measurements of serum cardiac troponin I (cTnI) and cardiac troponin T (cTnT) have shown to be sensitive and specific markers of myocardial damage .The pathophysiology of brainstem death results in a catecholamine storm that is believed to cause transient myocardial ischaemia and injury . CirculaP = 0.28). Vijay et al. conducted a similar study, using endomyocardial biopsy proven rejection at 1 year as the primary endpoint. They noted a linear correlation between grade of rejection at 1-year and donor cTnT levels [Several studies however have shown that elevated serum troponin levels in donors may predict adverse outcomes post-transplantation. Increasing cTnT levels have been shown to be associated with a reduction in left ventricular ejection fraction in the donor . PotapovT levels . AnotherT levels .In donors with subarachnoid haemorrhages, cTnI was a good indicator for left ventricular dysfunction, however this was reversible, and did not affect outcomes post-transplant , 174. BoA recent study of cTnI in the preservation solution (University of Wisconsin solution and Custodiol) during transportation of heart grafts however showed that elevated cTnI  was predictive of post-transplant PGD . They al2). The authors however could not show a correlation between post-operative inotropic support and BNP levels.Brain natriuretic peptide (BNP) and its amino-terminal pro-fragment, N-terminal pro–B-type natriuretic peptide (NT-proBNP) are commonly used for diagnosis and prognostication in heart failure , 180. ThSWItch/sucrose non-fermentable, a matrix-associated, actin-dependent regulator of chromatin subfamily a-like 1 (SMARCAL1) is an intracellular protein that acts as a DNA-dependent ATPase involved in transcription, DNA repair and chromatin dynamics . AharineHypoxia-inducible factor (HIF)-1 is a heterodimeric α, β transcription factor that mediates tissue responses to hypoxia . AharineSeveral published abstracts have highlighted the role of serum exosome proteomic analysis in the recipient as a potential biomarkers for PGD , 192. GiA single administration of cold flush preservation fluid remains the gold standard for myocardial protection during transplantation. It confers reliable protection for a limited amount of ischaemic time in young donor hearts . The incTreatment of PGD thus far is still primarily supportive care. In a consensus statement by Kobashigawa et al., the treatment and management of PGD across five high volume transplant centres were evaluated. PGD is initially managed by using inotropic support using catecholamines and phosphodiesterase inhibitors. The most common escalation therapy follow inotropic use is using an intra-aortic balloon pump. Following this, advanced mechanical support is initiated which is usually directed by the expertise of the transplant units themselves. The most common mode of support is extracorporeal membranous oxygenation with both central and peripheral cannulation strategies utilised. The heart should be allowed to eject to prevent stasis and thromboembolic complications.For primary RV-PGD, nitric oxide may be used to reduce the pulmonary vascular resistance. Following a period of support on ECMO, a short-term ventricular assist device is then implanted depending on the level of support needed . Where possible, the patient is then re-evaluated for a redo-orthotopic heart transplantation.Levosimendan is a calcium sensitising agent and inodilator which increases cardiac contractility. Its primary mode of action is by increasing the sensitivity of troponin-C to calcium during systole thereby increasing cardiac performance without increasing myocardial oxygen consumption. It also reduces peripheral vascular resistances by opening of adenosine triphosphate–dependent K+ channels . It was The inotropic effects of levosimendan may often take hours to develop, which may be offset by its potent vasodilation capacity . It may Plasmapheresis is a process by which whole blood is passed through a filter to separate the plasma components from the larger cellular components of red blood cells, white blood cells and platelets . Its usePGD is the leading cause of early morbidity and mortality following heart transplantation. It is thought to be multifactorial in origin and several risk factors implicated. The search for an accurate biomarker however remains elusive. Treatment options to date remain supportive with no definitive pharmacological agents identified as of yet. Plasmapheresis may have a role in depletion of inflammatory chemokines and cytokines for treatment of PGD.


C1-continuous meshes are automatically builtfrom the anatomy and deformation captured from dynamic MRI sequences. Diastolicdeformation is simulated using a mechanical model that combines passive andactive material properties. The problem of non-uniqueness of constitutiveparameter estimation using the well known Guccione law is characterized byreformulation of this law. Using this reformulated form, and by constraining theconstitutive parameters to be constant across time points during diastole, weseparate the effects of passive constitutive properties and the residual ATduring diastolic relaxation. Finally, the method is applied to two clinicalcases and one control, demonstrating that increased residual AT during diastoleprovides a potential novel index for delineating healthy and pathologicalcases.An unresolved issue in patients with diastolic dysfunction is that theestimation of myocardial stiffness cannot be decoupled from diastolic residualactive tension (AT) because of the impaired ventricular relaxation duringdiastole. To address this problem, this paper presents a method for estimatingdiastolic mechanical parameters of the left ventricle (LV) from cine and taggedMRI measurements and LV cavity pressure recordings, separating the passivemyocardial constitutive properties and diastolic residual AT. Dynamic PatientParameters in organ-level cardiac mechanical models can be broadly classifiedas passive and active. Typically within computational models, passive constitutiveparameters have been used to characterize the diastolic function, and with theaddition of active contraction models simulate systole . VariousHowever, an unsolved problem in patients with diastolic dysfunction is thatthe estimation of myocardial stiffness cannot be decoupled from impaired ventricularrelaxation  is simulated using patient-specific geometry, with the loadingcondition determined from LV cavity pressure recordings. The geometry are obtainedwith an automatic dynamic meshing process, which captures the LV anatomy using oneframe of the cine MRI data. 2.1The data used in this study were acquired from two patients selected forCardiac Resynchronization Therapy (CRT) in St Thomas’ Hospital, Londonand one healthy subject for control. This study conforms to the principlesoutlined in the Declaration of Helsinki and was carried out as part of a localethics committee-approved protocol with informed consent obtained from thepatients. Patient case 1 is a 74-year-old female with NYHA Class II heartfailure despite optimal medical treatment. There was significant LV systolicdysfunction with an LV ejection fraction of 16% and QRS duration of 168 ms. TheLV is significantly dilated with an end systolic volume (ESV) of 335 ml. Patientcase 2, a 78-year-old male, has the same disease classification as case 1, withejection fraction of 17% and an ESV of 186 ml. The control case used in thisstudy is a healthy 36-year-old male.For each of these three data set, cardiac deformation is characterized byspatially aligned cine MRI  and 3D tagged MRI . The LV cavity pressure transient is obtained from the cardiaccatheterization procedure (separately from the MR scan), when the rate of changeof LV pressure is measured. 2.2N tracked myocardialpoints, or a time series of the Lagrangian displacement vectors from framej to framei{jZi∈ ℝN3 | i = 1, 2,…, T} where T is the number of MRIframes. The automatic extraction of these displacements from the combinedshort-axis cine and 3D tagged MRI is performed with the Image RegistrationToolkit,1 which uses anon-rigid registration method based on free-form deformations developed by Critical information for the guidance of mechanical parameter estimationare the 3-dimensional displacements of 2.32.3.1C1-continuous representationof the geometry. The fiber field, representing the dominant orientation oftissue microstructure within the LV, is embedded in the geometric model withtransmural heterogeneity  frame ofcine MRI, the LV mechanical mesh at ED is built with cubic-Hermite (CH)elements using methods developed by 2.3.2Uj (in our specific casethis is at ED) and a time-series of N ~ 6000,regular grid points inside the LV myocardium, 3N ≫8M material points’ displacementjZi ∈ℝN3  from time point j to i, wefind, for each of the time points i , the nodal positions and derivativesUi  that define a M-node cubic-Hermitemesh. This mesh is found by minimizing the error in the mesh approximationto the observed positions of data points, formulated as a standard linearleast-squared minimization problem.Given the constructed geometrical model at end-diastole and the 4Dmyocardial displacement field from ie isdefined as the L2 norm of the residual vector– the difference between the observed positionszi of material points attime point iyi in the fitted meshHξ ∈𝕄N×8M3is the shape matrix related to the CH basis functions ,iP (LV cavity pressure at time point i),zT (i) ,yi). That is,−1, takes thedeformed position xi as its input andretrieves the unloaded initial positionx0 x0=𝕄2.4.1The model developed in this study only represents the LV, and doesnot include representations of the right ventricle (RV), great vessels,pericardium and organs around the heart. Thus the effects of thesestructures on the LV mechanics are not explicitly modeled. To account forthese physical constraints on the heart, we prescribe the kinematic movementof the LV model at its base plane and apex node  to match2.4.2W is defined as C1,C2, C3 andC4 are the constitutive parameters to beestimated, ffE, ssEand nnE are the Green–Lagrange strains infiber (f), sheet (s) and sheet normal(n) directions, and snE,fnE and fsEare the Green–Lagrange shear strains in the fs, fnand fs planes. The f, s andn directions correspond to the fiber axes aligned withthe microstructure of the myocardium.Consistent with existing literature and based on the experimentalresults of uniaxial and biaxial tests in isolated cardiac muscle , the myo2.4.3α andr2–r4 asα andr2–r4(non-negative) are the scale factor and anisotropies ofC2–C4,respectively. The underlined parameters  andr4 on the left side of the above equationsare those to be actually estimated in the following section. As we outlinebelow, this reformulation uncouplesC1–C4into C1-α  andr3–r4(anisotropy stiffness ratios), which clearly reveals the parametercorrelation of the original Guccione’s law in theC1-α space. Themotivation for using this formulated version of the law is thatC1-α assists in theinterpretation of parameter estimation results in terms of myocardialhomogeneous stiffness, and r2 indicates therelative stiffness along the myofiber compared to other material directions.This coupling relationship was explained in detail in our previous work, constantβ is the coefficient for the linear lengthdependence of AT, Eff is theLagrangian–Green strain along fiber direction, andHowever, in our study, we found that the pure passive mechanicalmodel could not fully explain the deformation presented in the earlydiastole  is estimated at each time point of diastolic MR images, using apre-estimated constant passive material parameter set (explained in detailbelow). At the end-diastole, the heart is assumed to be completely relaxed and thus zT is zero.The activation level n models , is assu2.5i during diastole . The first diastolic frame is definedas the minimum/zero pressure frame, while the last diastolic frame isdefined as the frame synchronous to the R wave. The LV reference state , defined as the state at which both the cavitypressure and myocardial AT are zero, is unknown. The state measured by the firstdiastolic frame is unlikely to be the reference state because while the pressurefor this frame is assumed to be zero, the AT, particularly in the diseasedcases, is likely to exist, and thus the the LV measured by the first diastolicframe is expected to be smaller than its reference state in terms of cavityvolume.As outlined above, model parameters are estimated by matching thesimulated LV deformation with that observed in each of the diastolic MRI frameiJ}  by the LV shape measured in one of the diastolic MRI frames , where LV inflating pressure and contractingresidual tension are assumed to be roughly balanced. k∈  is defined as the referenceframe number. Secondly, the unloaded state2.5.2kbefore the ED frame, and the reference state k (line 3 of Ck (line 4), which arechosen as the optimal parameters with which the reference state can be bestdeformed to the ED state. These estimated constitutive parameters are inturn used in the third step to refine the reference stateCk (line 7). Finally acriterion based on the physiological constraints of estimated AT, which isexplained below, is devised to retrospectively choose the most sensiblereference frame number (line 8) and the most plausible estimation ofconstitutive parameters, reference state, and active tensions (line 9).Applying this approach, iJ} forestimating constitutive parameters and active tensions  and fitted(yig) meshes at time pointof ith MRI frame, i.e. i ∈  is the diastolic MRI frame number, g enumerates theindex of Gauss points embedded inside each mesh volume , andxig andyig denote the spatialcoordinates of a Gauss point g at time point ofith MRI frame in the simulated and fitted meshrespectively. Gauss points are the sample points used in the standardGauss–Legendre quadrature scheme for computing numerical integration. That is, In line 8 k = 1, the first frame satisfyingthis criterion is chosen. We have tested and demonstrated the validity ofthis criterion using synthetic data where the ground-truth is known Ui)in 3.23.3In C1, we also estimated the tensionprofiles for each case using C1 = 0.5 andC1 = 2.0, based on the variability ofC1 in the literature. The right panel of C1 is much smaller than the difference acrosspatient cases. In addition, the estimated AT increases with the increase ofC1, which is a result of the decreasednon-linearity in the Guccione constitutive law due to the compensatory decreaseof α.To test the sensitivity of the residual AT profile against theassumption of 3.44In this study, we present a method for estimating diastolic active andpassive myocardium parameters – namely the myocardial constitutive propertiesand diastolic residual AT – from combined cine and tagged MR images andcavity pressure measurements. Applying this method to three clinical cases, thediastolic AT estimation results show a significant difference between healthy andtwo diseased cases, which may provide an interesting starting point for furtherclinical research. Below we discuss the issues related to the estimation ofmyocardial constitutive parameters, and the sensitivity of key steps in our methodson AT estimation results.4.14.1.1C1 and the ratio ofC2 : C3. C1 and α. Ourprevious study (C1 and αa andb are constant).Clinically, tagged MRI data covering the whole of diastole isdifficult to record, while the end-diastolic frame  is always available. Thus,it is desirable to characterize myocardial stiffness using displacements(relative to the reference state) extracted from the end-diastolic frame oftagged MRI. However, the issues associated with the identification of theGuccione parameters using displacements extracted from only one MRI framehas been already reported by several previous studies . For thius study further C1-α curvesestimated from different MRI frames intersect at the ground-truth parameterpoint. In order to obtain the complete set of unique parameter values,multiple tagged MRI frames during diastole can be used. Each frame canprovide information from which a distinct curve of the formappendix  show thaC1is one, based on the average value reported in literature k andstep 1 – the choice of reference frame number(2)step 3 – the deflation step of estimating referencestate.In k = 1 and the deflation stepis omitted. This scenario is effectively equivalent to directly using the statemeasured by the first diastolic MRI frame as the unloaded reference state, asituation that is only true if the residual AT and LV cavity pressure are bothzero. In scenario 2, the deflation step is included on top of scenario 1.Similarly, the frame number k determined by the criterion ofk is the most important step, because this affectsboth the estimation of constitutive parameters and reference state. Inparticular, if k = 1 and the deflation step is omitted(scenario 1), the influence of AT is not considered  iszero, based on the data reported by Limitations in the measurements include the pressure data recordingswhich have a level of uncertainty due to the calibration error, in partbecause only the 4.3.2a and b, such as SQP , in order to furtherinvetigate and correlate our new indices with clinical diagnosis.5Our methods of integrating the clinical MRI and LV cavity pressure dataacross multiple measurement points in the diastole enabled us to provide, to ourknowledge, the first attempt to estimate the diastolic residual active tensionprofile in human subjects, which has significant potential to provide an importantmetric characterizing diastolic heart failure. The results from our preliminaryapplication of this method indicates that early diastolic residual AT in the twodiseased cases are significantly higher than the normal cases, which may wellindicate that myocardial relaxation  is impaired in those twopatient cases.Appendices


Dear Editor,A 53-year-old man presented to our neurology department with a progressive, throbbingheadache accompanied by focal neurologic deficits. His known medical history includedcongenital heart disease with dextrocardia, a repaired ventricular septal defect, and aright ventricular-left atrial fistula which had been surgically corrected as well. Healso had a long history of unexplained dyspnea. A computed tomography (CT) scan of thehead revealed a brain abscess. The patient was admitted to initiate specific therapeuticinterventions. However, after admission, he experienced significant worsening ofdyspnea, low peripheral oxygen saturation, and cyanosis of the extremities. A chestX-ray showed dextrocardia, an increased cardiothoracic index, and enlargement of theproximal pulmonary arteries , whichr. Patients with HS present with organarrangement variations other than the typical asymmetry expected in normal anatomy(situs solitus) or its exact mirror image (situs inversus). Normal visceral arrangement depends on a series ofintricate processes that take place during early mesoderm development, such as adequateexpression and leftward flow of growth signals,3. Impairment in any ofthese factors during organogenesis may lead to abnormal organ positioning and HS.HS is a rare condition that occurs in approximately 1 in 10,000 live births-4: congenital spleenabsence and duplication of right-sided structures characterize the asplenia syndrome,whereas the presence of multiple accessory spleens and duplication of left-sidedstructures illustrate the polysplenia syndrome. Classical findings in HS include cardiacmalpositioning, septal defects, bilateral bilobed or trilobed lungs, midline liver,intestinal malrotation, and abnormal spleen development. Intestinal malrotation can leadto gut volvulus and ischemia,6, whereas complete asplenia predisposesto bacterial infections and sepsis,2. Up to 75% of patients with polyspleniahave significant cardiac malformations, namely endocardial cushion defects,double-outlet right ventricle, left heart obstruction, and anomalous venousreturn. The severity ofcongenital heart disease remains a main determinant of the long-term prognosis of HSpatients—even after surgical repair of congenital heart disease, patients are prone todeveloping arrhythmias, thromboembolism due to right atrium enlargement, and progressive systolicdysfunction.Patients with HS have historically been classified as having either asplenia (rightisomerism) or polysplenia (left isomerism) syndromesIn conclusion, HS is a complex syndrome that has remarkable phenotypic variability and isa challenge to manage. Patients with HS are prone to develop potentiallylife-threatening complications, which should be promptly diagnosed and managed.Therefore, imaging studies are critical in evaluating these patients, because theydelineate the spectrum of possible cardiac and extracardiac involvement in HS andassociated complications.


Peripheral artery disease (PAD) is a major cause of acute and chronic illness, with extremely poor prognosis that remains underdiagnosed and undertreated. Trimethylamine-N-Oxide (TMAO), a gut derived metabolite, has been associated with atherosclerotic burden. We determined plasma levels of TMAO by mass spectrometry and evaluated their association with PAD severity and prognosis. 262 symptomatic PAD patients  categorized in intermittent claudication  and critical limb ischemia  were followed-up for a mean average of 4 years (min 1-max 102 months). TMAO levels were increased in CLI compared to IC (P < 0.001). Receiver operating characteristic (ROC) curves for severity (CLI) rendered a cutoff of 2.26 µmol/L for TMAO . Patients with TMAO > 2.26 µmol/L exhibited higher risk of cardiovascular death  that remained significant after adjustment for confounding factors. TMAO levels were associated to disease severity and CV-mortality in our cohort, suggesting an improvement of PAD prognosis with the measurement of TMAO. Overall, our results indicate that the intestinal bacterial function, together with the activity of key hepatic enzymes for TMA oxidation (FMO3) and renal function, should be considered when designing therapeutic strategies to control gut-derived metabolites in vascular patients. Despite this prospect and its extremely poor prognosis, PAD remains undiagnosed and undertreated, highlighting the need for new markers that may provide insight into the underlying pathophysiology, improve long-term clinical risk prediction, and suggest novel therapeutic targets.Peripheral artery disease (PAD) is a largely unrecognized manifestation of atherosclerotic pathologies and a major cause of acute and chronic illness. Its prevalence in Western societies increases with age; 20% of the patients over 65 years are diagnosed with PAD, and is associated with exceptionally high risk for myocardial infarction (MI), ischemic stroke and death. Given that PAD typically presents in later life, its prevalence will undoubtedly escalate in response to changing population demographics and lifestyle4. In this regard, several human and animal studies have identified trimethylamine N-oxide (TMAO), metabolite of the gut microbe-derived trimethylamine (TMA), as a potential promoter of chronic diseases including atherosclerosis in humans7. TMA is generated by the action of specific microbial enzymes on dietary nutrients , then absorbed by the host and converted into TMAO by hepatic flavin monooxygenases (FMO3). Subsequently, TMAO is either transported to the tissues for accumulation as an osmolyte or, more commonly, cleared by the kidney8. In humans circulating TMAO and its precursors have been associated with atherosclerotic burden10, increased risk for cardiovascular (CV) disease, and Major adverse CV events (MACE) and death in coronary and peripheral arterial diseases14. In preclinical models TMAO accelerated atherosclerosis development15, and induced thrombosis16, supporting a role for gut microbiota in the pathogenesis of atherosclerosis and its associated complications.Recent studies have shown a direct link between dietary nutrients, intestinal microbiota, and cardiometabolic diseasesTaking in consideration the need of new markers for CV risk evaluation in PAD and the association of TMAO with vascular pathologies and worse outcome, we hypothesized that the measurement of TMAO could help to assess outcome evaluation in symptomatic PAD patients.Table Logistic regression analysis showed and association between TMAO and PAD severity alone  curves were plotted to assess disease severity (IC vs. CLI) for TMAO [AUC: 0.731 ± 0.032 (95% CI: 0.669–0.792) p < 0.001] rendering a cut-off value of >2.26 µmol/L , that was latter used to performed similar multivariate analysis. High TMAO levels  presented a significant association with PAD severity alone and after adjusting by other covariates  and mortality, either all-cause (n = 101) or cardiovascular (n = 39) during the follow-up .Cox regression analyses for overall mortality were performed before and after covariate adjustment. High TMAO concentrations were associated to all-cause mortality in the unadjusted model, but not after correcting by traditional risk factors . TMAO showed no independent association with this outcome in the multivariate Cox analysis  and continuous NRI analyses were performed. The addition of the categorical variable TMAO (>2.26 µmol/L) to the considered models including the previously mentioned relevant covariates, improved risk prediction for CV mortality in symptomatic PAD patients as assessed by NRI in models 2, 3 and 4 19. Nonetheless, some authors found no predictive value of high TMAO levels for CV events or mortality in smaller cohorts of patients with suspected coronary artery disease20 or receiving dialysis21, and the contribution of TMAO to early atherosclerosis development in healthy-middle-aged adults is unclear22. Symptomatic PAD patients present a complex pathophysiology, frequently associated with comorbidities such as diabetes, hypertension, or CKD, that are suspected to greatly interfere with the metabolism of microbial derived products8. Indeed, TMAO homeostasis considerably depends on liver FMO3 activity16 and renal clearance23. We found increased levels of TMAO in CLI patients compared to IC, and also determined hepatic enzymes for liver function assessment, finding no differences in transaminases between IC and CLI. Despite these results, we cannot exclude increased FMO3 activity in our cohort as no direct measurement of the enzymatic activity was performed. Moreover, other factors such as sex hormones, bile acids or insulin have been also described to regulate FMO3 activity and increase TMA oxidation25.The analysis of circulating gut-microbiome derived products has identified TMAO, a secondary product after TMA is metabolized by the liver enzyme FMO3, as a possible prognostic marker in cardiometabolic diseases23, since it has been described that patients with decreased kidney function present elevated levels of TMAO in circulation compared to those without CKD28. A trend for reduced eGFR according to TMAO quartiles was reported in the KarMeN study29, while no association of TMAO with creatinine was observed in the EPIC-Heidelberg study30, suggesting an irrelevant role of the kidney function for TMAO homeostasis in healthy subjects. In contrast, we found a negative correlation between TMAO and eGFR in a small cohort of people with no manifest cardiovascular disease, but with more than two cardiovascular risk factors, and older than those in the studies by Krüger et al. and Kühn et al.30. These data suggest that individuals with mildly impaired kidney function might be more susceptible to the detrimental effects of TMAO accumulation. In line with these observations and taking in consideration the high percentage of CKD patients in our PAD population (39%), we found a lineal-inverse association between TMAO and eGFR, and tested the possible interaction between the two according to disease severity. No interaction between TMAO and eGFR was found, however, considering the important role of the kidney on TMAO homeostasis8, eGFR was included as covariate for further regression analysis. To assess the accuracy of TMAO levels for PAD patient stratification we established the association between TMAO and PAD severity after adjustment for traditional risk factors and described a cutoff value with diagnostic purposes.TMAO accumulation might be also related to worse kidney clearance31 and a role for gut derived metabolites in thrombosis has been described in vivo, showing that intestinal microbes can directly modulate platelet hyperresponsiveness and clot formation rate via TMAO generation16. Even if most studies point towards TMAO as marker of worse outcome in CV pathologies, controversy remains when specific patient cohorts are evaluated. For example, no correlation between TMAO and increased risk for CV disease or MACE was observed in a group of end-stage renal disease patients21 and in subject with suspected CAD undergoing coronary angiography20. Mueller et al. speculated that those results could be confounded at least in part by impaired kidney function or poor metabolic control20 and encouraged the consideration of these parameters when interpreting the results. In addition, TMAO has been studied mainly in coronary pathologies, while it is prognostic value in other arterial localization, such as PAD, has been little investigated. We report an association between TMAO and CV-mortality, but not with all-cause death or MACE. Our results differ in part from previous data, describing an association of TMAO levels and global death in PAD14. However, differences between the studied populations regarding PAD definition and severity status should be considered when interpreting the data. Senthong et al. included by the term PAD the majority of non-coronary arterial territories14, while our cohort is restricted to symptomatic lower limb artery disease (mean ABI 0.55 ± 0.19). TMAO appears to be better for CV-mortality prognosis than for all-cause death, which might be important for patient evaluation.Increased levels of plasma TMAO have been shown to predict future major adverse cardiac events including myocardial infarction, stroke, and death in different CV pathologies16 and converts TMA in TMAO24, was not measured.The current study including 262 patients could be considered as small, however the high percentage of deaths  provides the statistical power required to support our conclusions. Events were recorded during a mean follow up of 4 years, reasonable to estimate early to medium-term mortality. Longer term studies should be designed to confirm the involvement of TMAO in PAD mortality. No causal relationship between high TMAO levels and CV-mortality can be inferred from our prospective study. Death cause in some patients was unknown and we might have lost some cases related to CV events. Finally, the influence of two important variables for TMAO production, the use of antibiotics and modifications in dietary habits were not recorded and could not be included as confounding variables in our population. FMO3 activity, which has been implicated in thrombosis riskWe show increased TMAO levels according to PAD severity and an independent association between TMAO and elevated risk for CV-mortality. The design of novel therapeutic strategies towards gut-derived metabolite control in vascular patients will need to consider not only intestinal bacterial function, but also the activity of key hepatic enzymes for TMA oxidation (FMO3), and renal function.et al.33. As the cohort has included new cases we include the complete description in supplemental material and methods. Control subjects were previously described by Marcos-Jubilar M et al.34.The clinical and demographic characteristics of the PAD cohort were previously described by Martinez-Aguilar PAD Patients  were prospectively enrolled at the outpatient service of the Department of Vascular Surgery of Complejo Hospitalario de Navarra between 2010 and 2017 . Blood samples were collected at the time of clinical evaluation and tested for biochemical parameters. Ankle brachial index (ABI) was measured at rest, in both lower limbs.Fontaine classification was used for severity assessment as follows: intermittent claudication  diagnosed by hemodynamic study (Doppler ultrasound), and critical limb ischemia  with lower limb rest pain and/or trophic lesions (Fontaine class III-IV) confirmed by imaging studies . Exclusion criteria were established as follows: patients with Fontaine class IV and infected-lesions, individuals with evidence of neoplastic disease, generalized or localized inflammatory disease (moderate or severe), severe chronic kidney disease, on haemodialysis, or receiving antinflammatory drugs.Control subjects (n = 45) were enrolled at the outpatient service of the Department of Internal Medicine, Clínica Universidad de Navarra (April 2016-December 2017). Blood samples were collected at the time of clinical evaluation. Patients were included if older than 45 years, with ≥2 cardiovascular risk factors and no manifested cardiovascular disease at recruitment. Exclusion criteria included active neoplastic disease, acute or chronic inflammatory disease of any aetiology, and intake of nonsteroidal anti-inflammatory or steroid drugs 2 weeks before blood withdrawal. Samples and data from control patients were provided by the Biobank of the University of Navarra and were processed following standard operating procedures approved by the Ethical and Scientific Committees.The study was approved by the Institutional Review Boards of Complejo Hospitalario de Navarra and Clínica Universidad de Navarra, according to the standards of the Declaration of Helsinki on medical research, and written informed consent was obtained from all patients who were enrolled in this study.PAD patients were followed up for a mean period of 4 years (min 1 max 102 months) at the outpatient service of the Department of Vascular Surgery every 3, 6 or 12 months, depending on the severity of PAD. Death, either all-cause or cardiovascular, and MACE including amputation, stroke, myocardial infarction and all-cause death were recorded.et al.35, a precise and reliable UHPLC-MS/MS method has been implemented in our laboratory for the quantification of TMAO in human plasma. Frozen citrate plasma samples, in which the corresponding stabilized TMA salt was formed, were utilized to perform these analyses. Concentrations of the analyte in plasma samples was determined from calibration curves, which indicated a good linearity (r2 ≥ 0.999) within the studied concentration range (100 nM–10 μM), using peak area ratio of the analyte to its isotope. The implemented UHPLC-MS/MS method has good analytical accuracy, precision and recovery; in fact, the recoveries ranged between 97% and 104% and, accuracy and precision of the assays were between 3.7% and 7.3%  and hs-CRP32. Multicollinearity was evaluated by the variance inflation factor and models calibrated by the Homer-Lemeshow goodness-of-fit test. Patients without outcome were censored at the date of their last follow-up. Hazard ratios (HR) and their 95% CI for death  and MACE were estimated using Cox regression models after adjusting for relevant covariates. Fine-Gray competing risk models were used to obtain sub-hazard ratios for CV death, considering non-CV death as a competing event. Due to the low number of CV death events, the following basal models were considered for adjustment for relevant covariables: model 1, unadjusted; model 2, sex and hsCRP (log); model 3, diabetes mellitus, hypertension and eGFR (<60 mL/min/1.73 m2); model 4, smoking, dyslipidemia and HDL-C. The additional value of TMAO for risk prediction of CV death was assessed with Harrell’s C statistics and the continuous net reclassification index (NRI) index. Values are expressed as mean ± SD or median (interquartile range), and categorical variables as numbers and percentages. Analyses were performed with STATA version 12  and SPSS version 15. All p-values are two-tailed, and statistical significance was set at p < 0.05.Normality was demonstrated by the Kolmogorov-Smirnov test. Non-normally distributed variables were log-transformed. Differences between two groups of subjects were tested by Student’s t test  or Mann-Whitney U test (nonparametric test). χ2 or Fisher’s exact test was used for categorical variables. Association studies were performed by Pearson correlation test for continuous variables. Receiver Operating Characteristic (ROC) curves were plotted to assess disease severity (IC vs. CLI), and the cut-off value for TMAO established with the Youden Index. Multivariable logistic regression models were adjusted for relevant covariates: age, sex, cigarette smoking, diabetes mellitus, hypertension, dyslipidemia, HDL-C, eGFR (<60 mL/min/1.73 mSupplemental material SREP-19-23703A


Background: Severe aortic stenosis (AS) complicated by pulmonary hypertension (PH) is associated with poor outcomes after surgical aortic valve replacement (AVR). There is still scarce information about predictors of secondary PH in this group of patients. Objectives: The aim of this study was to investigate the prognostic impact of biomarkers together with conventional Doppler echocardiographic parameters of left ventricular diastolic function on elevated pulmonary systolic pressure (PSP) in severe AS patients before surgical AVR. Methods: Sixty patients with severe isolated AS  underwent echocardiography, N-terminal pro B-type natriuretic peptide (NT-proBNP) and growth differentiation factor-15 (GDF-15) measurements before AVR. PSP, left ventricular ejection fraction (LV EF), parameters of LV diastolic function  and left atrial (LA) volume) were evaluated. PH was defined as an estimated PSP ≥ 45 mmHg. Results: Of the 60 patients, 21.7% with severe isolated AS had PH with PSP ≥ 45 mmHg (58.5 ± 11.2 mmHg). LV EF did not differ between groups and was not related to an elevated PSP . Parameters of LV diastolic dysfunction , MV DT ≤ 177.5 ms , LA volume > 100 mL ) and biomarkers  and GDF-15 > 3393 pg/mL ) were significantly associated with elevated PSP in severe AS. Conclusions: Left ventricular diastolic dysfunction and elevated biomarkers levels could predict the development of pulmonary hypertension in patients with severe aortic stenosis. Elevation of biomarkers paired with worsening of LV diastolic dysfunction could help to stratify patients for earlier surgical treatment before the development of pulmonary hypertension. Degenerative aortic stenosis (AS) is the most common acquired valvular heart disease and its prevalence is expected to increase due to the aging population ,2. PulmoSeveral biomarkers, reflecting structural and biological changes of myocardium in response to chronic pressure overload in AS patients, were found to predict presence of symptoms and cardiovascular death or hospitalization in this group of patients ,9,10. HoThe purpose of our study was to investigate the prognostic impact of biomarkers together with conventional echocardiographic parameters of LV diastolic function on elevated pulmonary systolic pressure (PSP) in severe isolated AS patients.2, who underwent two-dimensional Doppler echocardiographic evaluation, N-terminal pro-brain natriuretic peptide (NT-proBNP) and growth differentiation factor 15 (GDF-15) measurement before AVR. Patients with documented coronary heart disease or chronic pulmonary disease, atrial fibrillation and moderate–severe mitral regurgitation were excluded from the study.The prospective study (performed between November 2014 and May 2016) included 60 patients with symptomatic severe AS, defined as an aortic valve area less than 1 cmThe study was approved by Kaunas Regional Biomedical Research Ethics Committee, No. BE-2–8, issued on 29 October 2014. All patients gave their informed consent.Two-dimensional echocardiography was performed using a GE Vivid 7 system . Echocardiographic studies were performed by an experienced independent echocardiographer, blinded to the patient’s clinical data. Digital loops were stored and analyzed offline .Anatomic and Doppler examinations and measurements were performed according to American Society of Echocardiography recommendations . The aorContinuous wave Doppler was used to assess maximal tricuspid regurgitation flow velocity to estimate the systolic pressure gradient between the right ventricle (RV) and right atrium (RA). RV systolic pressure was calculated by adding an estimated RA pressure. In our study PH was defined as an estimated PSP ≥ 45 mmHg, based on tricuspid regurgitation flow velocity ≥ 3.0 m/s and presence of other echo PH signs .Plasma was sampled 24 h before AVR, processed immediately and NT-proBNP was calculated. Blood samples were stored at −80 °C and GDF-15 was measured with a solid-phase enzyme-linked immunosorbent assay (ELISA) technique. For the evaluation of renal function, serum creatinine levels were determined and glomerular filtration rate was calculated using the CKD-EPI creatinine equation .p value of <0.05 was used for declaring statistical significance.Continuous variables distribution was verified using the Kolmogorov–Smirnov test. Continuous variables are presented as means (±SD) if normally distributed, and NT-proBNP are expressed as median and interquartile range . Categorical variables are presented as frequencies and percentages. Characteristics of patients with and without PH were compared using the Student’s t-test for continuous variables and chi-square test for categorical variables. For comparison of two groups the Mann–Whitney test was applied and for three groups the Kruskal–Wallis test. Correlations were computed using Pearson’s method. A two-sided Logistic regression analysis was performed using all the independent variables to assess their relative contribution odds ratio (OR) to the development of PSP ≥ 45 mmHg. The incremental value of each clinical and Doppler echocardiography variable in predicting the development of PSP ≥ 45 mmHg was assessed in terms of the construction of receiver-operating characteristic (ROC) curves. All statistical analyses were performed using SPSS 21.0 software .2) were prospectively enrolled in the study. Patients were divided into two groups according to the presence or absence of PH, with PSP cut-off value of 45 mmHg. Thirteen patients (21.7%) had PH (mean systolic PSP 58.5 ± 11.2 mmHg). Clinical, demographic, echocardiographic and biomarker parameters of the overall cohort and those with and without PH characterized by echocardiographic assessment are shown in Sixty symptomatic patients (mean age 69 ± 9 years) with severe AS . There were no group differences in New York Heart Association (NYHA) functional class and in the prevalence of comorbidities such as arterial hypertension, diabetes mellitus and renal insufficiency (p > 0.05). Aortic valve area did not differ between groups; nor did LV EF  and LV EF was not related to elevated PSP. Patients with PH had larger LV dimensions (p = 0.05), though LV myocardial mass index did not differ between the groups (p = 0.374). Significant differences in parameters of LV diastolic function were found. Increased PSP (≥45 mmHg) was associated with higher E/E’ ratio (p = 0.047), shorter MV DT (p = 0.014) and larger LA volume (p = 0.010). Associations of PSP with parameters of LVDD divided in tertiles, i.e., E/E’ ratio, MV DT and increased LA volume, are presented in Patients in both groups were similar in terms of age, sex and body mass index . Moderate correlation was found between GDF-15 , NT-proBNP  and elevated PSP. The combination of both biomarkers (GDF-15 and NT-proBNP) strengthened the correlation with PSP .Biomarker concentrations were higher in patients with aortic stenosis (AS) and elevated PSP  vs. 602 (366–1818) ng/L, p = 0.270).In univariate logistic regression analysis, LVDD parameters and levels of two biomarkers were highly significant in predicting elevated PSP in severe AS patients. The strongest determinants were E/E’ ratio, MV DT, LA volume, NT-proBNP and GDF-15 values . In contThe parameters of worsening LVDD together with elevated levels of two biomarkers were found to be robust predictors of PH in severe AS patients .ROC curve analysis of echocardiographic parameters and biomarker levels in prediction of PSP ≥ 45 mmHg is presented in Our study evaluated the factors for the development of PH in patients with severe AS. The main finding of our data was that concomitant PH in patients with severe AS was associated with worsening of LVDD , as well as with elevated levels of biomarkers (NT-proBNP and GDF-15). Several studies reported that in patients with AS the prevalence of PH is high and development of post-capillary PH is significantly associated with worse clinical outcomes and poor prognosis following both transcatheter and surgical AVR ,5,7,14. We found that LV diastolic dysfunction, but not LV systolic function, was a predictor of elevated PSP in this group of patients. Several studies concluded that LV systolic function was not associated with PH in AS ,16, althSeveral authors have demonstrated association of PH with LVDD and higher LV filling pressures ,19. The LVDD parameters were found to be of great importance for prognosis of increased PSP levels before aortic valve surgery for severe AS. In the present study, worsening of Doppler-derived LVDD parameters  were shown to be independent predictors of PH in severe AS before AVR.This prospective study demonstrated that not only echocardiographic parameters of LVDD, but also elevated numbers of biomarkers (NT-proBNP and GDF-15) had significant association with PH. Just recently, the measurements of NT-proBNP have been shown to be a useful tool in the identification or exclusion of HF . NT-proBGrowth differentiation factor 15 (GDF-15) is a stress-responsive cytokine, the expression of which is strongly up-regulated in cardiac myocytes by various stressors, including increased pressure overload conditions . This biOur findings suggest that in patients with severe isolated AS, LVDD—and not the severity of AS or LV EF—determines PH in symptomatic patients. Further studies are required to assess whether the association of LVDD parameters  together with biomarkers (GDF-15 and NT-proBNP) can be used to search high risk asymptomatic patients with severe AS and whether these findings could help in the selection of patients, who would benefit from earlier surgery.A limitation of this study might be a relatively small sample size. Secondly, LV filling pressures and pulmonary pressures were not measured by invasive cardiac catheterization. Although right-heart catheterization (RHC) is the best method to measure the pulmonary artery pressure, echocardiographic estimation of PSP correlates well with RHC measurements  and is wLeft ventricular diastolic dysfunction and elevated biomarkers levels are factors for the development of pulmonary hypertension in patients with severe aortic stenosis. Elevation of biomarkers paired with worsening of left ventricular diastolic dysfunction could help to stratify patients for earlier surgical treatment before the development of pulmonary hypertension.


Introduction: In the chronic AV-block (CAVB) dog model, structural, contractile, and electrical remodeling occur, which predispose the heart to dofetilide-induced Torsade de Pointes (TdP) arrhythmias. Previous studies found a relation between electrical remodeling and inducibility of TdP, while structural remodeling is not a prerequisite for arrhythmogenesis. In this study, we prospectively assessed the relation between in vivo markers of contractile remodeling and TdP inducibility.Methods: In 18 anesthetized dogs, the maximal first derivative of left ventricular pressure (LV dP/dtmax) was assessed at acute AV-block (AAVB) and after 2 weeks of chronic AV-block (CAVB2). Using pacing protocols, three markers of contractile remodeling, i.e., force-frequency relationship (FFR), mechanical restitution (MR), and post-extrasystolic potentiation (PESP) were determined. Infusion of dofetilide (0.025 mg/kg in 5 min) was used to test for TdP inducibility.Results: After infusion of dofetilide, 1/18 dogs and 12/18 were susceptible to TdP-arrhythmias at AAVB and CAVB2, respectively (p = 0.001). The inducible dogs at CAVB2 showed augmented contractility at a CL of 1200 ms  with a negative FFR, while the non-inducible dogs retained their positive FFR. The time constant (TC) of the MR curve was significantly higher in the inducible dogs . Furthermore, a linear correlation was found between a weighted score of the number and severity of arrhythmias and contractile parameters, i.e., contractility at CL of 1200 ms , the slope of the FFR  and the TC of MR . Thus, more severe arrhythmias were seen in dogs with the most pronounced contractile remodeling.Conclusion: Contractile remodeling is concomitantly observed with susceptibility to dofetilide-induced TdP-arrhythmias. The inducible dogs show augmented contractile remodeling compared to non-inducible dogs, as seen by a negative FFR, higher maximal response of MR and PESP and slowed MR kinetics. These altered contractility parameters could reflect disrupted Ca2+ handling and Ca2+-overload, which predispose the heart to delayed- and early afterdepolarizations that could trigger TdP-arrhythmias. Despite advances in treatment and prevention strategies, sudden cardiac death caused by ventricular arrhythmias remains a common cause of death in patients with heart failure or compensated hypertrophy , 2004. IThe chronic AV-block (CAVB) dog model is a widely used animal model to study ventricular remodeling and its relation with ventricular arrhythmias . In thisVentricular remodeling is a complex process and can be divided into multiple components, such as structural, electrical, and contractile remodeling. However, the contribution of each of the three different components to arrhythmogenesis is not fully elucidated. Previous studies have shown that electrical remodeling is an important contributor to the susceptibility of TdP,  while st2+ handling, can be measured in vivo by three physiological mechanisms of the heart: force-frequency relationship (FFR), mechanical restitution (MR) and post-extrasystolic potentiation (PESP). The FFR accounts for potentiation of contractility when heart rate is increased. MR and PESP relate to changes in contractile force of the extrasystolic beat and post-extrasystolic beat, respectively, when a basic stimulation train is interrupted with extrastimuli. MR represents the restoration of contractile force of the extrasystolic beat when the CI of the extrastimulus is lengthened. PESP displays the opposite behavior: when CI is shortened, the contractility of the post-extrasystolic beat is enhanced. Thus, the earlier an extrasystole occurs, the lower the contractility of the extrasystolic beat and the higher the contractility of the post-extraystolic beat.Yet, the time course of contractile remodeling and its relation to TdP-inducibility is less well described. Contractile adaptations, as a result of changes in Cain vivo. Furthermore, in isolated cardiomyocytes from CAVB-dogs, Ca2+-overload of the sarcoplasmic reticulum (SR) can result in spontaneous Ca2+ release, which triggers EADs/DADs  also reflect the propensity for sustained TdP-arrhythmias. If contractility and arrhythmogenesis are intertwined, contractile parameters could function as markers of pro-arrhythmia and might eventually help to identify the patient at risk for life-threatening arrhythmias. Therefore, we aimed to investigate the relation between in vivo contractility measures and susceptibility to TdP-arrhythmias in the CAVB dog. We assessed these measures after 2 weeks of remodeling, since CAVB-dogs are susceptible to TdP-arrhythmias from that moment onward.ADs/DADs . HoweverAnimal handling was in accordance with the “Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 on the protection of animals used for scientific purposes” and the Dutch law, laid down in the Experiments on Animals Act. All experiments were performed with approval of the Central Authority for Scientific Procedures on Animals (CCD), registered as project AVD115002016531.2 and N2O (1:2). Periprocedurally antibiotics (ampicillin 1000 mg i.v. preoperatively and ampicillin 1000 mg i.m. postoperatively) and analgesics (metacam 0.2 mg/kg s.c. preoperatively and buprenorphine 0.3 mg i.m. postoperatively) were administered.Eighteen adult purpose-bred mongrel dogs of either sex  were included. Experiments were performed under general anesthesia with mechanical ventilation at 12 breaths/min. The dogs were premedicated with a mixture of 0.5 mg/kg methadone, 0.5 mg/kg vetranquil and 0.02 mg/kg atropine i.m.. General anesthesia was induced with 25 mg/kg pentobarbital i.v. and maintained by isoflurane (1.5%) in a mixture of OTen surface-ECG leads  were recorded throughout the experiment and stored on hard disk. Under aseptic conditions, the femoral artery and vein and carotid artery were dissected and sheaths were placed by Seldinger technique. Right and left ventricular monophasic action potential (RV and LV MAP) catheters  and a left ventricular 7F pressure catheter  were positioned under fluoroscopic guidance.Kr blocker dofetilide . TdP was defined as a run of five or more ectopic beats, with twisting morphology of the QRS-complexes. When ≥3 TdP occurred in the first 10 min after start of dofetilide administration, the dog was considered inducible.Two serial experiments were performed. In the first experiment, baseline surface ECG, LV, and RV MAPD and left ventricular pressure during SR were recorded. Subsequently, His bundle ablation was done as described previously . When thDuring the second experiment, after 2 weeks of remodeling at CAVB2, baseline ECG, LV and RV MAPD and left ventricular pressure were recorded and the pacing protocol and susceptibility test with dofetilide were repeated.FFR, MR, and PESP were measured during a pacing protocol from the LV MAP catheter. The FFR protocol consisted of 3 min of steady-state pacing at three different CLs of 300 ms, 750 ms, and 1200 ms. For the MR and PESP protocol, the LV was paced with a basic CL of 600 ms, which was interrupted every 20th beat by an extrastimulus with an incremental CI ranging from 250 ms up to 800 ms, with steps of 50 ms.max) was calculated offline with computer software . For the FFR, the mean LV dP/dtmax of five consecutive beats was used at the three stimulation frequencies. A straight line was fitted through these points and its slope was calculated to quantify the orientation of the FFR. MR and PESP were defined as the LV dP/dtmax of the extrasystolic and post-extrasystolic beat, respectively . In addition, normalized MR and PESP were calculated by taking the ratio of LV dP/dtmax of the extrasystolic and post-extrasystolic beat, respectively, to the mean LV dP/dtmax of the five beats immediately preceding the extrasystole. Both MR and PESP were fitted to monoexponential functions using non-linear least squares regression, with the equation: As a measure of contractility, the maximal slope of left ventricular pressure rise (LV dP/dtc) with the van der Water formula . QT-interval was corrected for heart rate  with post hoc Bonferroni correction. Group analysis was performed with an unpaired Student’s t-test. Correlation analysis was done with Pearson’s correlation coefficient. All statistical analyses were performed with GraphPad Prism 6.0 . A p-value < 0.05 was considered as statistically significant.Data are expressed as mean ± standard error of the mean (SEM). Serial data were analyzed with a paired Student’s Figure 2. Contractile remodeling was present after 2 weeks of CAVB, as seen by a significant increase in LV dP/dtmax during IVR compared to AAVB . At AAVB, a positive FFR was observed with augmentation of LV dP/dtmax at higher frequencies . This relationship was blunted at CAVB2. At AAVB, the MR curve demonstrated a monoexponential rise in LV dP/dtmax toward a plateau with lengthening of the CI. The PESP curve displayed the opposite behavior. . Both these parameters were increased at CAVB2.Contractile parameters at AAVB and CAVB are shown in Table 1. At AAVB, all electrophysiological parameters have adapted to the sudden drop in heart rate. When challenged with dofetilide, a further significant increase was seen in QT, LV and RV MAPD, and STV. After 2 weeks of CAVB, electrical remodeling was present, as seen by a significant increase in QT, QTc, and LV MAPD under baseline conditions compared to AAVB. RV MAPD did not increase to the same extent as LV MAPD, thus creating a high interventricular dispersion (ΔMAPD). Finally, also STV showed a significant increase, which was further augmented after infusion of dofetilide.The course of electrical remodeling is depicted in p = 0.002 compared to AAVB) were inducible after dofetilide infusion. Separate measurement of contractile parameters of the inducible and non-inducible dogs are depicted in Figure 3. At AAVB, no differences were found in contractile measures between the two groups. However, at CAVB2, the inducible dogs demonstrated a completely different pattern of contractile remodeling compared to the non-inducible dogs. At low stimulation frequency with a CL of 1200 ms, the inducible subjects had augmentation of contractility compared to the non-inducible dogs . Furthermore, the slope of the FFR-curve was inverted in the inducible dogs, while the non-inducible dogs retained their positively oriented FFR . As seen from Table 2, these differences in LV dP/dtmax between inducible and non-inducible dogs are not due to differences in end-diastolic pressure (EDP). A significantly higher end-systolic pressure (ESP) is seen in the inducible dogs at low stimulation rate.Of the 18 dogs tested, one was susceptible to dofetilide-induced TdP at AAVB. After 2 weeks of CAVB, 12/18 dogs . PESP, on the other hand, was increased in the inducible dogs at whole range of CIs . When MR was normalized to the LV dP/dtmax of the preceding beats, the TC of MR appeared significantly higher in the inducible dogs . Normalized PESP was similar between the groups and had equal TC .At small CIs, MR was similar in both groups, but diverged when the CI of the extrasystole rised above 450 ms, after which the susceptible dogs showed a significantly higher LV dP/dtTable 3). Only STV could distinguish between inducible and non-inducible dogs both at baseline and when challenged with dofetilide. Dogs that were inducible to drug-induced TdP had significantly higher STV at baseline  which increased even more when challenged with dofetilide .Electrically, no differences were found between the two groups at AAVB. At CAVB, the RR-interval, QT- and QTc-interval, LV MAPD, RV MAPD, and ΔMAPD were similar between the inducible and non-inducible dogs (Table 4). This weighted score was correlated with both contractile and electrical remodeling. As illustrated in Figure 5, a linear correlation was found between this weighted score and LV dP/dtmax at CL of 1200 ms , the slope of FFR  and TC of MR . Of the electrical parameters, STV at baseline was also correlated with the weighted score of arrhythmic severity .In order to quantify the severity of arrhythmias, we developed a new weighted scoring system, which takes into account both the number of arrhythmias in 10 min and whether the arrhythmias were sustained or defibrillations were needed  prior to administration of dofetilide. In Figure 6, the effects of ouabain administration on FFR are depicted. In all dogs, ouabain resulted in a rise in contractility. Ouabain itself did not induce any arrhythmias. Unfortunately, in three out of four dogs, contractility at a CL of 1200 ms could not reach a level comparable to that of the inducible dogs and retained a positive FFR. As expected, these three dogs remained non-inducible after additional dofetilide infusion. Nevertheless, in one dog, contractility after ouabain administration increased to a level similar to that of the inducible dogs, with a more blunted FFR. Interestingly, this dog did become inducible to TdP-arrhythmias after infusion of dofetilide .As a proof of principle of the importance of contractile remodeling and Ca2+-homeostasis may be the common denominator that explains the observed relation between contractile remodeling and arrhythmogenesis in the CAVB-dog model.The results of this study demonstrate that contractile remodeling is already present after 2 weeks of CAVB and that this remodeling process differs substantially between TdP-susceptible and TdP-resistant dogs. Moreover, the more contractile or electrical remodeling have occurred, the higher the number and severity of arrhythmias after dofetilide challenge. Alterations in Cain vivo measurements of contractility and inducibility of TdP-arrhythmias. Nevertheless, the relation between disturbances of Ca2+-homeostasis and arrhythmogenesis on a cellular level is well established. Previous in vitro studies have demonstrated altered Ca2+ handling in isolated cardiomyocytes of CAVB-dogs with increased amplitude and duration of Ca2+-transients along with a higher Ca2+-content of the SR   . Furtherhythmias .max postpacing, which reflects an increased SR-load, was associated with the occurrence of DADs on MAP recordings. Since DADs and EADs share a common molecular mechanism, we expected to find the same relation between enhanced contractility and susceptibility of TdP-arrhythmias.Our results are consistent with the study by A blunted or inverted FFR has been observed in isolated muscle preparations of failing myocardium , 2002 asFigure 3A). There is strong evidence that increased [Na]i during diastole plays a central role in the inversion of the FFR   . Howeveri in combination with enhanced NCX activity results in Ca2+-overload, spontaneous Ca2+ release, and subsequent EADs and TdP-arrhythmias. Supporting this hypothesis, it has been shown that lowering [Na]i by administration of the selective late Na+-current blocker ranolazine could reduce the number of EADs in vitro and TdP-arrhythmias in vivo  resulted in increased cellular Ca2+ load and spontaneous Ca2+ release, which was associated with increased STV and the occurrence of DADs and EADs  have also been shown to exhibit disrupted Caurvivors . The cau2+ transients or SR function have been done, therefore hypotheses on mechanism of contractile remodeling and the relation with TdP-arrhythmias are based on assumptions derived from previous molecular work. Explanations other than related to Ca2+-handling may also be possible, such as mechano-electrical feedback, i.e., direct effects of altered loading conditions on repolarization. Secondly, LV dP/dtmax has important limitations as a measure of contractility, since it is also dependent on ventricular loading. While we did not measure ventricular volumes, we have shown that EDP are not different between inducible and non-inducible dogs. Therefore, we can assume the differences found in LV dP/dtmax are predominantly caused by changes in contractility. Finally, the relation between contractile remodeling and TdP was only measured at CAVB2, therefore no conclusion can be made on this association later in the remodeling process.No direct measurements of CaIn the CAVB dog model, contractile and electrical remodeling are already present after 2 weeks of AV-block and develop concomitantly with susceptibility to dofetilide-induced TdP. Furthermore, contractile parameters are altered to a far larger extent in inducible dogs, as seen by development of an augmented negative FFR, higher maximal response of MR and PESP and slowed MR kinetics.DS did experimental work, analysis of data, and wrote the manuscript. JB delivered technical support during experiments. MS contributed to experimental concepts and design of the study. MV was responsible for organization of experiments and revised the manuscript thoroughly. All authors approved the submitted version.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.