Pathologic hypertrophy of the cardiac muscle is a commonly encountered phenotype in clinical practice, associated with a variety of structural and non-structural diseases. Coronary microvascular disease is considered to play an important role in the natural history of this pathological phenotype. Non-invasive imaging modalities, most prominently positron emission tomography and cardiac magnetic resonance, have provided insights into the pathophysiological mechanisms of the interplay between hypertrophy and the coronary microvasculature. This article summarizes the current knowledge on coronary microvascular dysfunction in the most frequently encountered forms of pathologic hypertrophy.The online version contains supplementary material available at 10.1007/s12350-022-03040-2. LVH can be primary or secondary and characterizes a wide variety of cardiac structural and non-structural disease-entities such as ischemic heart disease, arterial hypertension, heart failure, valvular disease, and a range of genetical cardiomyopathies. The estimation of LV wall thickness and mass in routine clinical practice is derived from measurements obtained by echocardiography or cardiac magnetic resonance (CMR).Cardiac hypertrophy refers to an increase in the mass of the left or the right ventricle or both as an adaptive mechanism to pressure and/or volume stress, genetic mutations of several proteins, and/or loss of contractile mass as a result of prior infarction.2 This remodeling process commonly affects the entirety of the left ventricle, indicating a more diffuse nature of the disease.3 CMD in LVH is considered to confer important clinical and prognostic implications. Non-invasive imaging modalities allowing for assessment of myocardial blood flow (MBF) and myocardial flow reserve /rest MBF) have provided valuable insights on CMD in pathological LVH. Myocardial perfusion positron emission tomography (PET) which is considered the gold standard for routine clinical MBF assessment, but also CMR and contrast-enhanced transthoracic echocardiography (contrast-TTE) have been shown to be valuable tools in both clinical and research settings.Coronary microvascular disease (CMD), although not the hallmark and the primary mechanism for the pathogenesis of pathological hypertrophy, is considered to hold a critical role in the natural history of this pathological phenotype. Several pathophysiological mechanisms have been proposed and although our understanding remains incomplete, in most cases, CMD in LVH is multifactorial Table . PatientThis review aims to summarize the current knowledge on CMD in the most commonly encountered LVH-phenotypes in clinical practice.4 HCM is commonly morphologically characterized by asymmetrical hypertrophy of the left ventricle, with a predilection for the interventricular septum and histologically by hypertrophy of cardiac myocytes, myocyte disarray, interstitial fibrosis, and thickening and narrowing of the intramural coronary arteries.6 Given the several secondary etiologies of LVH, diagnosis of HCM should be established in the absence of other cardiac, systemic, or metabolic factors or diseases resulting in a hypertrophic phenocopy while at the same time, genetical testing if available is essential in the diagnostic process.7 Impaired MBF during stress and its sequelae, myocardial ischemia is considered an essential feature of HCM patients, and altered microvascular function is associated with poor outcomes.9 Compared to patients without pathological hypertrophy, HCM patients have significantly reduced hyperemic MBF and MFR while resting MBF is preserved.12 Clinically, a significant association has been reported between pathological MFR and symptoms (primarily angina), risk of sustained ventricular arrhythmia, progression to end-stage heart failure, and cardiovascular death.16Hypertrophic cardiomyopathy (HCM) is the most common monogenic cardiovascular disease, inherited through an autosomal dominant pattern.18 The latter contributes to endothelial dysfunction, which in turn leads to inadequate hyperemic MBF response predisposing for myocardial ischemia.10 Remodeling of intramural coronary arterioles increases coronary vascular resistance, and it seems to affect the entirety of the myocardium, even remote regions that are not hypertrophied.19 Increased loading conditions and wall stress in these left ventricles with severely impaired diastolic dysfunction as well as the increased metabolic demand of the hypertrophied myocardium further contribute to the presence and extent of CMD, particularly in the subendocardium.9 Several studies have shown that MBF is more blunted in the subendocardium and in the more hypertrophied areas, but without always sparing of the non-hypertrophied segments.19 , at CMR) has been shown to be present without CMD in some patients.21 In the majority of patients, myocardial segments without fibrosis have normal global MFR without regional heterogeneity of flow, whereas in the presence of LGE, the majority of symptomatic HCM patients exhibit impaired global MFR with significantly lower hyperemic MBF within fibrotic versus non-fibrotic segments.22The prevalence of CAD is no different in patients with versus without HCM.23 Genetical factors might also play a role, in view of the results from a 13N-ammonia PET and CMR study showing significantly lower peak MBF in patients with sarcomere myofilament mutations compared to matched genotype-negative patients.24Similar to the clinical nature of the disease and the phenotypical heterogeneity  a substantial heterogeneity in MFR reduction and CMD is observed in HCM patients, potentially owning also to the time-point of the diagnosis and the time of assessment of the microvasculature. While some patients present with normal MFR, others exhibit severely reduced MFR (consistent with significant CMD).25 Studies assessing the coronary microsiculation in patients with LVOT obstruction have shown contradictory results. A 13N-ammonia PET study comparing 11 non-obstructive patients, 12 obstructive patients, and 10 with latent-obstruction (defined as a provoked-LVOT gradient of > 30 mmHg), found no significant correlation between LVOT gradients and global parameters of ischemia or hyperemic MBF and MFR on PET, when comparing symptomatic patients with obstructive versus non-obstructive HCM.23 Other groups using 15O-water PET showed an inverse correlation between LVOT gradients and hyperemic MBF.26 These discrepant findings could potentially be explained by the labile nature of LVOT obstruction in HCM; a highly variable echocardiographic finding heavily dependent on a myriad of factors, including volume status, autonomic nervous system, physical position, and others.7HCM patients with obstructive physiology and elevated left ventricular outflow tract (LVOT) gradients appear to have a worse prognosis compared to those with non-obstructive disease.23 Petersen et al. demonstrated that hyperemic MBF decreased by .011 mL⋅min−1⋅g−1 for each millimeter increase of end-diastolic wall thickness.27 Similarly, pathology studies in HCM patients have demonstrated that the percentage of luminal narrowing of intramural coronary small arteries inversely correlated with heart weight and myocyte size and also that there was an inverse relationship between normalized coronary arteriolar lumen and degree of LV hypertrophy.28 CMR studies in HCM patients, utilizing cardiac diffusion tensor imaging for in vivo characterization of myocardial microstructure, demonstrated that even in myocardial segments with normal wall thickness, normal perfusion, and without scar, diffusion is more isotropic than in controls, suggesting the presence of underlying cardiomyocyte disarray.29 In the same population, hyperemic MBF and MFR were reduced, particularly within the subendocardium, not only in hypertrophied and scarred segments but also in segments with normal wall thickness. Transmural perfusion gradient (TPG) is a relative novel quantitative PET metric defined as the ratio of endocardial-to-epicardial MBF. In hypertrophic left ventricles, TPG is decreased during vasodilator-induced hyperemia, and in HCM patients, a reduction of TPG under stress indicates that the impairment of MBF affects primarily the endocardium.31 In HCM, impaired TPG is related to pathological LVEF stress response, potentially suggesting that the stress-induced subendocardial ischemia may also cause transient LV dysfunction.32It has further been shown that myocardial wall thickness is the strongest predictor for reduced global hyperemic MBF and MFR, while MBF decreased in proportion to the increase in end-diastolic wall thickness.33 The benefit of implementing an assessment of the microvasculature in the individual management of HCM patients remains to be elucidated by future studies.Being the most frequently encountered form of pathological LVH, with an estimated prevalence of 1 out of every 200 adults (0.5%), HCM represents a very heterogeneous population whereby CMD is present in several stages of the disease with important clinical and prognostic implications.34 The estimated prevalence of AFD in patients with unexplained LVH in adults is around .5% to 1%.37 Cardiac deposition of globotriaosylceramide in myocytes, vascular endothelium, and smooth muscle cells leads to myocardial ischemia, myocardial wall thickening, and progressively to interstitial replacement fibrosis.40 These alterations increase coronary vascular resistance and myocardial oxygen demand, which along with direct endothelial cell dysfunction due to globotriaosylceramide storage, nitric oxide pathway dysregulation, and microvascular remodeling are considered to mediate CMD in patients with AFD.42 In fact, CMD is considered to play an important role in AFD patients, in some representing the first manifestation of cardiac involvement, in others it is responsible for anginal symptoms.44  is caused by mutations in the gene encoding α-galactosidase A, resulting in inability to break down glycosphingolipids, which in turn leads to accumulation in multiple organs with subsequent renal, cardiac, and cerebrovascular injury.45 Compared to healthy controls, AFD patients seem to have reduced MFR and hyperemic MBF, irrespective of LVH or gender.42 Although AFD is an X-linked disease with males primarily being affected, most heterozygous females also express clinical manifestations, including cardiac involvement, but usually at a later age.47 CMD might be the earliest manifestation of cardiac involvement, seen even prior to any signs of LVH.42 Similarly, reduced hyperemic MBF is inversely related to the degree of LV wall thickness and to age in males, potentially corroborating the hypothesis that CMD precedes the development of LVH. Multiparametric CMR imaging has been used to describe the several stages of AFD, including the initial globotriaosylceramide storage phase characterized by low native T1 mapping values, and followed by the LVH stage with focal and global inflammation leading to fibrosis.51 At CMR, a reduction in hyperemic MBF has also been shown to be present early in the disease course and was demonstrated to be related with the severity of LVH, as well with regional inflammation and fibrosis.52 A CMR study in 114 AFD patients revealed that compared to matched, healthy controls, the patients had significantly lower MBF even prior to the occurrence of LVH and reduction of native T1 mapping values.53 Supporting the findings from PET studies, CMD was shown to be present not only prior to hypertrophic phenotype but is also preceding the storage phase of cardiac involvement.CMD in AFD has been investigated primarily in PET studies.54 This concept was assessed in PET-based studies with discrepant results, potentially owing to sample sizes too small to assess any ERT response effect.55 Technological advancements and increased availability of imaging modalities could allow for better evaluation of the effect of therapeutic options on the microvascular function and the potential long-term benefit.Assessment of the microvasculature function could have implications for the management of AFD patients: Early recognition of cardiac involvement could potentially prompt early initiation of disease-specific treatment that includes either intravenous enzyme replacement therapy  or oral chaperon therapy , or even cardiac supportive therapies.56 Primarily affecting the lungs and intrathoracic lymph nodes, cardiac involvement is present in a considerable amount of sarcoidosis patients, ranging from 5% to 10% of individuals with systemic manifestations, albeit the exact prevalence is most probably underestimated.58 Myocardial involvement in cardiac sarcoidosis (CS), phenotypically described as a chameleon-disease, is usually focal or scattered and commonly affects the free lateral LV wall or the basal intraventricular septum, that appears thinned, while in some cases it can result in LVH mimicking HCM.61 Atrioventricular conductance disturbances, ventricular arrhythmias, and progressive heart failure are the most common clinical manifestations and CS patients may present with angina symptoms even in the absence of significant epicardial CAD. The latter has been attributed to CMD, although this hypothesis up till recently was based on qualitative assessment of myocardial perfusion (with single-photon emission computed tomography) and not on quantification of MBF.65Sarcoidosis is a systemic inflammatory disease characterized by the formation of non-caseating granulomas upon an unknown trigger in genetically predisposed individuals.13N-ammonia and 18F-fluorodeoxyglucose (FDG) PET study has provided insights on the relationship between cardiac inflammation and MBF in CS patients.66 Sarcoid-mediated inflammation was shown to be associated with regional impairment of MFR. Although resting MBF were found to be within normal limits, irrespective of the presence of inflammation or not, hyperemic MBF and MFR were significantly lower in inflamed myocardium compared to non-inflamed regions. In a small portion of the study participants, assessment of treatment-response on the microcirculation was also evaluated, whereby those that did not respond to immunosuppressive treatment  had a further decline in hyperemic MBF and MFR. In contrast, response to steroid therapy was associated with preserved coronary circulatory function.67A retrospective, combined 68 Work utilizing contrast-TTE reported that in the absence of traditional risk factors for CAD, sarcoidosis patients without known cardiac involvement had a lower MFR as compared to healthy controls.69 The mechanism of CMD in CS patients is undoubtedly multifactorial. An early systematic inflammatory state affects the microvascular endothelium. It is considered to result in decreased nitric oxide production, which in turn impairs vasodilation response, particularly in actively inflamed myocardial regions.71 In advanced stages, a more global and diffuse impairment of MBF might also be present and potentially precedes the phase of structural myocardial changes, including fibrosis and scarring that leads to overt heart failure.66 Whether CMD in CS is a by-stander and to what extent it confers prognostic or therapeutic implications remains to be elucidated.Active inflammation in CS patients has also been associated with enhanced coronary vasoconstrictive reactivity at the epicardial and microvascular level, while medical treatment  was shown to reduce epicardial spasm effectively.72 Cardiac amyloidosis (CA) is caused by cardiac amyloid fibril deposition and the main forms clinically encountered (termed after the precursor protein of the amyloid deposit) include light-chain amyloidosis (AL) or transthyretin-types amyloidosis .74 Amyloid can be deposited in almost all cardiac structures from pericardium, epi-/endocardium to cardiac valves, myocardium and cardiac vasculature. Both left and right ventricular myocardium can be infiltrated, resulting in biventricular hypertrophy  that progresses to a restrictive form of cardiomyopathy.74 Deposition of amyloid leads to structural alterations predominantly within the subendocardial and midwall regions ultimately resulting in replacement fibrosis.75 Pathological hypertrophy of the ventricles may impede subendocardial perfusion due to capillary rarefaction and compression.76 Although significant luminal obstruction of the epicardial coronary arteries rarely occurs, direct vascular deposition of fibrils can further precipitate microvascular obstruction. Functional abnormalities, including autonomic dysfunction and endothelial dysfunction, that characterize amyloidosis are also considered to play a significant role in CMD in CA patients.Amyloidosis is a collection of systemic disorders characterized by the extracellular deposition of insoluble fibrils composed of misfolded proteins in multiple organs.80 Although most studies have focused on the non-bioptic identification of cardiac amyloid depositions, a few smaller studies have also assessed the microvasculature in CA using all available imaging modalities.85 In patients with both AL-CA and ATTR-CA and in the absence of epicardial CAD that were assessed with 13N-ammonia PET, rest MBF, hyperemic MBF and MFR were significantly lower when compared to a matched hypertensive patients’ group with LVH.76 , defined as the blood circulation time from the orifice of the coronary arteries to the pooling in the coronary sinus. This surrogate marker of CMD was significantly prolonged in CA patients compared with the HCM and the healthy controls.87 Real-time contrast TTE with flash bubble destruction has also been used for quantification of MBF in AL CA patients, whereby significant reduction of hyperemic MBF was observed.85 Even in patients with rare renal amyloid A (AA) amyloidosis, MFR was significantly lower at contrast-TTE compared to a group of individuals with non-amyloid chronic inflammatory disease and healthy controls.81 As cardiac involvement in AA amyloidosis usually occurs at later stages of the disease, assessment of microvascular function could potentially enable earlier identification of patients at risk.The last decade has heralded a paradigm shift in the field of CA, given the advent of disease-modifying therapeutic options (primarily for ATTR) such as stabilizing molecules (tafamidis) and genetic silencers (patisiran and inotersen) which have been shown to improve quality of life.Current knowledge indicates a close association and interplay between the coronary microvasculature and pathologic myocardial hypertrophy while the prevalence of patients with a hypertrophic phenotype is increasing in clinical practice. Multimodality imaging approaches for non-invasive quantification of MBF provide a distinctive opportunity to evaluate the function of the coronary microcirculation. Technical refinements and wider availability of said diagnostic tools hold the promise to allow for a better understanding of the pathophysiological mechanism and the related clinical and prognostic implications. Finally, integration of patient- and disease status-specific functional assessment of the microvasculature in the diagnostic approach may pave the way for better risk-stratification and patient-tailored therapeutic options.Supplementary file1 (PPTX 905 kb)Below is the link to the electronic supplementary material.


DSP genetic variants and the presence of a myocardial disease referred to patients with Carvajal syndrome. Since then, several reports have linked the DSP gene to familial forms of arrhythmogenic (ACM) and dilated cardiomyopathies. Left-dominant ACM is the most common phenotype in individuals carrying DSP variants. More recently, a new entity—“Desmoplakin cardiomyopathy”—was described as a distinct form of cardiomyopathy characterized by frequent left ventricular involvement with extensive fibrosis, high arrhythmic risk, and episodes of acute myocardial injury. The purpose of this review was to summarize the available evidence on DSP cardiomyopathy and to identify existing gaps in knowledge that need clarification from upcoming research.Desmoplakin (DSP) is a desmosomal protein that plays an essential role for cell-to-cell adhesion within the cardiomyocytes. The first association between  DSP gene, located on chromosome 6p24. DSP is expressed in both the skin and myocardium and plays an essential role in cell-to-cell adhesion within the cardiomyocytes by anchoring intermediate filaments to the desmosome . Th. Th20]. According to the published cohorts, DSP cardiomyopathy is a disease with a female predominance ,19, an oDSP carriers. Comprehensibly, the 2010 International Task Force (ITF) Criteria underperformed in patients with DSP cardiomyopathy, since the document was designed for “classical” ACM (with right-dominant or biventricular phenotypes), and ALVC variants were not contemplated [As previously mentioned, ALVC is the predominant phenotype in emplated .DSP carriers, half of the individuals with cardiac abnormalities failed to comply with diagnostic criteria for ACM [DSP and PKP2 genetic variants, the 2010 ITF criteria were insensitive for diagnosing ACM in DSP carriers , despite the presence of clinically confirmed cardiomyopathy [In a study of 38  for ACM . In a mumyopathy .DSP carriers, only 51% met the ITF diagnostic criteria at baseline. More importantly, the 2010 ITF criteria failed to identify individuals who presented with arrhythmic and heart failure events during follow-up, showing a limited prognostic value in this cohort [Wang et al.  also fous cohort .The recognition of the inadequacy of these criteria, which failed to include the entire phenotypic spectrum of the disease ,24, led DSP cohort [In our P cohort , by applP cohort , 67% of P cohort , in keepThe presence of inflammatory cell infiltrates in endomyocardial biopsies or autopsy samples has been reported in ACM patients since the first description of the disease in the early 1980s, thus suggesting a possible role of inflammation in its pathophysiology ,21.“hot phases” [DSP gene variant [In individuals with ACM, episodes of chest pain, accompanied by electrocardiographic changes and myocardial enzyme release, in the setting of normal coronary arteries, have been defined as  phases” ,15,22. Bhot phases” among 560 ACM patient probands followed at our reference center; in 58% of these subjects, CMR showed evidence of myocardial inflammation, according to the Revised Lake Louise criteria [Recently, Bariani et al. reported a 5% incidence of “criteria . Finallycriteria .“hot phases” consists of case reports. In a recent systematic review including 103 ACM patients with these myocarditis-like episodes, DSP was also the most frequently implicated gene (69%) [The majority of papers reporting the occurrence of ne (69%) . FurtherDSP truncating variants, were found in 3% [Interestingly, a recent European study was carried out, whereby genetic evaluations were performed on 336 consecutive patients with acute myocarditis—a P/LP variant in ACM- or DCM-associated genes was found in 8% of cases, and desmosomal mutations, mostly nd in 3% .p < 0.0001) [A recent multicenter study compared the outcomes of patients with acute myocarditis with and without desmosomal gene variants. In this report, genotype-positive patients (89% DSP variant carriers) had a significantly higher incidence of the main endpoint  than genotype-negative individuals  . Moreove 0.0001) .DSP carriers presented episodes of clinically suspected myocarditis during a mean follow-up of 11 years [DSP carriers [Across the reports on DSP cardiomyopathy, the prevalence of “hot phases” ranges from 14% to 22% ,18,19. SRegardless of their impact on disease progression, myocarditis-like episodes are part of the distinct phenotype of DSP cardiomyopathy, which warrant a careful differential diagnosis with other inflammatory myocardial diseases, such as myocarditis or sarcoidosis . Thus, gElectrocardiograms (ECG) of patients with DSP cardiomyopathy mostly reflect the presence of ALVC forms. Typical ECG abnormalities include low QRS voltages (peak to peak < 0.5 mV) in limb leads and T-wave inversion in lateral or inferior leads . These fDSP cohort [DSP carriers was previously reported by our group [In our P cohort , low QRSur group . Contrarur group .DSP truncating mutation, observed that individuals without signs of LV involvement already presented ECG abnormalities, pointing out that T-wave inversion in inferior leads may be a sensitive marker for ALVC in DSP carriers, even in the absence of overt disease [DSP probands, but when evident in leads V4–V6, an association with LV involvement was found [López-Ayala et al., in a cohort of patients with a founder  disease . In a muas found .The presence of low QRS voltages in limb leads and negative T waves in inferolateral leads have been pointed out as ECG markers that could aid in differential diagnosis in patients with overlapping features of ALVC and DCM ,13 Figu. The ECGDSP carriers showed a ventricular arrhythmia rate of 46% [DSP carriers.Predisposition to VAs is a characteristic feature of DSP cardiomyopathy since its first description ,9. One oe of 46% . FrequenALVC-related arrhythmic events comprise PVCs, non-sustained ventricular tachycardia (VT) and sustained VT , and ventricular fibrillation (VF) ,13.DSP cohort [Malignant VAs occurred in 29% of our P cohort  during aP cohort , corroboDSP carriers by Smith et al., with a particularly high incidence in individuals with proband status (43%) [Wang et al. also reported a high rate of malignant VAs (27%), including a substantial portion of patients with VF/aborted sudden death 17%) . The aut% . The aDSP variants [DSP carriers, in spite of a coexisting extensive scar. Wall motion abnormalities and LV dysfunctions detectable by echocardiography may not be present until advanced stages, since the subendocardial layer is usually spared [Hallmark cardiovascular imaging features in DSP cardiomyopathy are mostly obtained by CMR. ALVC is the most common phenotype in individuals carrying P/LP variants ,18,19. Cy spared .DSP carriers. In a multicenter study of DSP carriers, LGE was detected in a quarter of patients with LV-predominant disease, in the absence of overt dysfunction [Therefore, CMR is the most sensitive test for phenotype assessment in function . A seemifunction  with extfunction ,17,28.DSP patients, even in young carriers [Among the reviewed DSP cardiomyopathy cohorts, subepicardial LGE in ≥2 LV segments was present in 40% to 72% of patients ,18,19 T. NotablyDSP/FLNC variants [DSP carriers [DSP/FLNC carriers; the detection of myocardial fat infiltration was exclusive to the DSP/FLNC phenotypes [These findings are in line with a study by Augusto and colleagues, which established an association between a characteristic subepicardial, “ring-like” scar pattern and variants , among pcarriers . In theienotypes .DSP/FLNC carriers, in comparison to other DCM genotypes [p < 0.01) [They also noted a tendentially higher LVEF in enotypes . This is < 0.01) .Apart from the distinctive “ring-like” pattern, the preferential LGE distribution in ALVC, affecting predominantly the inferior wall-septal junction and inferolateral walls, has consistently been described since early reports ,29, and the cause, not the consequence, of the cardiomyopathic process, and is disproportionate to the level of LV dilation and dysfunction in DSP carriers.In DSP cardiomyopathy, LV dysfunction appears to be intimately related to the extent of LV scar ,12. Of nSince DSP cardiomyopathy is a recently described entity, extensive data regarding targeted therapy are not available. Current therapy reflects the management of the underlying phenotype (DCM/ALVC), and includes treatment of heart failure, arrhythmias, and the prevention of sudden cardiac death.DSP cardiomyopathy is accompanied by a high prevalence of VAs, often occurring in young individuals without HF or significant LV dysfunction ,17,18,19DSP cohort, regardless of RV involvement [DSP cohort, failing to identify 52% of DSP carriers with severe arrhythmias. Indeed, several events occurred in individuals with LVEF of 35–55%, and even with LVEF > 55% [LVEF < 55% was strongly associated with malignant VAs in a multicenter olvement . Smith eEF > 55% . Our stuEF > 55% . These oLMNA, FLNC, PLN, and RBM20 variants in their recommendations, do not include any consideration regarding DSP variant carriers [DSP) had rates of SCD/VT/VF comparable to those of LMNA patients [DSP patients, as a significant portion of events occur in patients with normal or nearly normal LVEF [It is noteworthy that the 2022 ESC Guidelines for the management of patients with ventricular arrhythmias and the prevention of SCD, while mentioning the increased risk of patients with carriers . For patcarriers . As demopatients ,32. In lmal LVEF ,18,19.p = 0.036), HF , and cardiac death  (18). However, the incidence of arrhythmias was not different between genders in two other large studies of DSP cardiomyopathy [The male gender has been considered an adverse prognostic marker, and was included as a minor risk factor in stratification algorithms in classic ACM . Accordimyopathy ,19.DSP carriers; proband status  and myocardial injury  were independent predictors of HF in this cohort [Wang et al. reported an incidence of HF of 6.6 (95% CI: 4.5–9.8) per 100 people per year among s cohort .DSP carriers.In summary, classical variables routinely used for risk estimation in DCM and ACM populations appear inadequate and fail to predict the arrhythmic risk in DSP cardiomyopathy. Dedicated tools to predict risk and guide ICD implantation remain an unmet need in the management of DSP genetic variants have been considered in published DSP cohorts. In the study by Wang et al., which included 51% of patients with normal ventricular function, family members had fewer symptoms, less lateral T-wave inversions, and less LGE on CMR compared to probands [Besides probands, family members carrying probands . Howeverprobands ,18,19. Iprobands .Several studies demonstrated that exercise plays a role in disease progression and increases the risk of malignant arrhythmias and SCD in “classical” ACM forms ,35,36,37Genotype–phenotype correlation studies demonstrated that, among patients carrying DSP pathogenic/likely pathogenic genetic variants, a wide clinical and instrumental spectrum is present. The main clinical findings include extensive subepicardial late gadolinium enhancement on cardiac magnetic resonance, which is often disproportionate to the degree of LV dysfunction, in association with frequent premature ventricular contractions and a propensity to life-threatening ventricular arrhythmias. Furthermore, patients can show recurrent myocarditis-like episodes of chest pain with documented myocardial injury, named “hotphase episodes”.Evidence supporting tailored risk stratification and clinical decision-making in these patients, particularly regarding primary prevention of sudden cardiac death, is currently lacking.


Although the treatment and prognosis of patients after heart transplantation have significantly improved, late graft dysfunction remains a critical problem. Two main subtypes of late graft dysfunction are currently described: acute allograft rejection and cardiac allograft vasculopathy, and microvascular dysfunction appears to be the first stage of both. Studies revealed that coronary microcirculation dysfunction, assessed by invasive methods in the early post-transplant period, correlates with a higher risk of late graft dysfunction and death during long-term follow-up. The index of microcirculatory resistance, measured early after heart transplantation, might identify the patients at higher risk of acute cellular rejection and major adverse cardiovascular events. It may also allow optimization and enhancement of post-transplantation management. Moreover, cardiac allograft vasculopathy is an independent prognostic factor for transplant rejection and survival rate. The studies showed that the index of microcirculatory resistance correlates with anatomic changes and reflects the deteriorating physiology of the epicardial arteries. In conclusion, invasive assessment of the coronary microcirculation, including the measurement of the microcirculatory resistance index, is a promising approach to predict graft dysfunction, especially the acute allograft rejection subtype, during the first year after heart transplantation. However, further advanced studies are needed to fully grasp the importance of microcirculatory dysfunction in patients after heart transplantation. Over the last 30 years, the treatment and prognosis of patients after heart transplantation (HTx) have significantly improved, despite the older age and higher morbidity of recipients . In addiEarly graft dysfunction occurs within the first 24 h after the transplant procedure. It is classified as primary or secondary, depending on the etiology. Primary graft dysfunction (PGD) is divided into PGD-left and PGD-right ventricle, leading to ventricular dysfunction requiring pharmacological or mechanical circulatory support. PGD risk factors can be donor-related , recipient-related , or surgical procedure-related  . In turnLate graft dysfunction includes two main subtypes: acute allograft rejection (AAR) and cardiac allograft vasculopathy (CAV). AAR-related deaths account for 11% of all deaths in the first three years following transplantation . The oveCMD is defined as a coronary microcirculation dysfunction resulting in myocardial ischemia ,7. Non-iAAR can be cell or antibody —mediated and known as Acute Cellular Rejection (ACR) and Antibody-Mediated Rejection (AMR), respectively. The risk factors include younger age, the female gender of the donor or recipient, and increased HLA mismatch ,22. ACR AMR is a less understood and more difficult-to-diagnose process, but it can potentially result in high morbidity ,25. AlmoCAV, also known as transplant coronary artery disease or cardiac transplant vasculopathy, is one of the three leading causes of death in HTx patients. The incidence of CAV concerns approximately 30% of patients five years after transplantation and 50% of patients ten years after transplantation . CAV typp = 0.002) [p = 0.011), and the optimal cutoff value of IMR to predict ACR was 15 [p = 0.02) and mild graft dysfunction assessed with the use of echocardiography-derived left and right myocardial performance indices . The authors unanimously concluded that microvascular dysfunction correlated with a higher probability of death, graft failure, or allograft vasculopathy five years after HTx . Increased IMR values in the first year after HTx were also associated with worse graft function and, simultaneously, less promising clinical outcomes [p < 0.001). In addition, the incidence of AAR in patients with an IMR ≥ 18 was 23.8%, whereas the incidence of AAR in those with an IMR < 18 was 6.3% . MACE occurred in 86 (36.3%) patients within a 10-year period, and its risk was significantly higher with IMR ≥ 18 . Considering the above, measuring the IMR early after HTx might identify patients at higher risk of ACR and MACE and optimize and improve post-transplant management. In addition, IMR has proven valuable in assessing the treatment response [p = 0.03) [p = 0.04) as an independent predictor of death or retransplantation. Okada et al. [p < 0.001) and long-term cardiac events  was significantly higher in IMR-high compared to IMR-low group. Patients treated with HTX in the high IMR group had better predictive efficacy than the low IMR group.Crea et al. showed that the patients with ACR had significantly higher IMR values at one month compared to those without ACR in observation  . In addiR was 15 . The autR was 15 . Haddad outcomes . Another = 0.03) . Moreovea et al.  measureda et al. . Both thp = 0.034). These findings were validated in an independent set of EBMs. The main conclusion was that the biopsy-based molecular examination of antibody-mediated microcirculation injury could improve the diagnosis of AMR in patients after HTx [As of this day, the data on the correlation between AMR and IMR are limited. So far, single reports have indicated a potential relationship between microcirculatory dysfunction and AMR. In their study, Afzali et al. assessed molecular diagnostics in human paraffin-embedded EMBs with AMR, classified according to ISHLT criteria. The gene set expression was subsequently compared between ISHLT diagnoses and correlated with donor-specific antibodies, endothelial injury by electron microscopy, and prognosis. In AMR patients (n = 17) with sequential biopsies, upregulated gene set expression was associated with a worse prognosis (fter HTx .p = 0.01) and microvascular  endothelium-dependent response to ACH, compared to patients who did not reach the endpoint. In conclusion, endothelial dysfunction, as reflected by abnormal ACH responses, preceded clinical endpoint development. Moreover, studies report that microcirculatory dysfunction can predict CAV and, when assessed regularly, can prevent graft dysfunction’s progression and improve patients’ survival after HTx [p = 0.004), while IMR significantly improved . Both FFR and IMR correlated with plaque volume measured using IVUS [CAV is an independent prognostic factor for allograft rejection and survival rate. The origin of CAV is multifactorial, including immune and non-immune factors. The abnormal immune processes appear to be of paramount importance, as allograft vasculopathy develops in the donor’s but not in the recipient’s arteries. CAV probably reflects an accelerated “normal” healing process following transplant-induced immune damage . Endothefter HTx . Lee et fter HTx . In the ing IVUS . They shLimitations of invasive assessment of microcirculation in heart transplant recipients should also be discussed. One is the risk of an atrioventricular block (AVB) in a denervated heart during intravenous infusion or intracoronary administration of adenosine. Studies have shown that adenosine induces AVB in healthy pediatric and young adult heart transplant recipients with minimal risk when low initial doses are used and therapy gradually escalates . Using rThe studies discussed above were mainly aimed at demonstrating the importance of early assessment of coronary microcirculation in patients after heart transplantation in predicting long-term complications, including AAR and CAV. The question remains about the clinical significance of the obtained results and how to use them to improve the prognosis of post-transplant patients in long-term follow-up. Optimal immunosuppressive therapy prevents rejection and damage to the donor organ while minimizing complications due to immunosuppressive treatment . Sub-theThe relationship between coronary microcirculation and late graft dysfunction is summarized in The last decade contributed to significant advancement in methods of microcirculatory evaluation. An invasive assessment of the coronary microcirculation, including the IMR measurement, is the most promising approach to predict subsequent graft dysfunction  during the first year after HTx. However, AAR is still a poorly understood and difficult-to-predict process that can result in high morbidity . Therefo


Renal denervation (RDN) protects against hypertension, hypertrophy, and heart failure (HF); however, it is not clear whether RDN preserves ejection fraction (EF) during heart failure (HFpEF). To test this hypothesis, we simulated a chronic congestive cardiopulmonary heart failure (CHF) phenotype by creating an aorta-vena cava fistula (AVF) in the C57BL/6J wild type (WT) mice. Briefly, there are four ways to create an experimental CHF: (1) myocardial infarction (MI), which is basically ligating the coronary artery by instrumenting and injuring the heart; (2) trans-aortic constriction (TAC) method, which mimics the systematic hypertension, but again constricts the aorta on top of the heart and, in fact, exposes the heart; (3) acquired CHF condition, promoted by dietary factors, diabetes, salt, diet, etc., but is multifactorial in nature; and finally, (4) the AVF, which remains the only one wherein AVF is created ~1 cm below the kidneys in which the aorta and vena cava share the common middle-wall. By creating the AVF fistula, the red blood contents enter the vena cava without an injury to the cardiac tissue. This model mimics or simulates the CHF phenotype, for example, during aging wherein with advancing age, the preload volume keeps increasing beyond the level that the aging heart can pump out due to the weakened cardiac myocytes. Furthermore, this procedure also involves the right ventricle to lung to left ventricle flow, thus creating an ideal condition for congestion. The heart in AVF transitions from preserved to reduced EF . In fact, there are more models of volume overload, such as the pacing-induced and mitral valve regurgitation, but these are also injurious models in nature. Our laboratory is one of the first laboratories to create and study the AVF phenotype in the animals. The RDN was created by treating the cleaned bilateral renal artery. After 6 weeks, blood, heart, and renal samples were analyzed for exosome, cardiac regeneration markers, and the renal cortex proteinases. Cardiac function was analyzed by echocardiogram (ECHO) procedure. The fibrosis was analyzed with a trichrome staining method. The results suggested that there was a robust increase in the exosomes’ level in AVF blood, suggesting a compensatory systemic response during AVF-CHF. During AVF, there was no change in the cardiac eNOS, Wnt1, or β-catenin; however, during RDN, there were robust increases in the levels of eNOS, Wnt1, and β-catenin compared to the sham group. As expected in HFpEF, there was perivascular fibrosis, hypertrophy, and pEF. Interestingly, increased levels of eNOS suggested that despite fibrosis, the NO generation was higher and that it most likely contributed to pEF during HF. The RDN intervention revealed an increase in renal cortical caspase 8 and a decrease in caspase 9. Since caspase 8 is protective and caspase 9 is apoptotic, we suggest that RDN protects against the renal stress and apoptosis. It should be noted that others have demonstrated a role of vascular endothelium in preserving the ejection by cell therapy intervention. In the light of foregoing evidence, our findings also suggest that RDN is cardioprotective during HFpEF via preservation of the eNOS and accompanied endocardial-endothelial function. After initial publications mentioning the HFpEF phenotype, starting in the 1980s, the studies on HFpEF were mainly of case series types or the comparative clinical descriptions from patients with HF. These studies revealed significant variability in the HFpEF prevalence. However, a report from the Helsinki Study in 1987 that had sampling from 501 people helped demonstrate the preserved left ventricle systolic function in more than 50% of the prevalent cases. Subsequently, numerous cohorts also reported on HFpEF prevalence in the patients including the Framingham Heart Study and the Strong Heart Study and the Cardiovascular Health Study. Almost every one of them confirmed HFpEF phenotype in large proportions of HF cases. These observations were also concordant with that of the data from the hospitalized patients and from large HF registries. Interestingly, the HFpEF confirmation in those studies was based on the combination of the HF—assessed based on clinical symptoms, signs, and the left ventricle ejection fraction (LVEF) threshold values without considering the data from atrial or ventricular structure or diastolic function ,7,8,9,10Clinical diagnosis and management of heart failure (HF) are currently linked to ejection fraction (EF). HF with preserved ejection fraction (HFpEF) is now considered a distinct disease from HF with reduced ejection fraction (HFrEF). Although the morbidity and mortality rates of HFpEF and HFrEF are comparable, therapeutic options for HFpEF patients remain elusive. The HFpEF was initially thought to be due to diastolic dysfunction but is now recognized as a complex interplay of impairment in cardiac reserve, as well as altered renal metabolism, vascular, pulmonary, and renal nerve activation ,12,13,14The reciprocal contribution by other organs to redistribute blood volume has been observed during myocardial infarction during hind-limb remote ischemic condition . The conTo determine whether the chronic volume overload causes increase in exosomes’ release in the blood of C57BL/6J experimental mice, we created AVF and at 16 weeks, measured the blood levels of exosomes by marking heat shock protein 70 (HSP70). The results revealed an increase in Exosomal amount in AVF mice group compared to WT mice. To mitigate AVF induced volume receptors we created renal denervation (RDN). The results suggested that RDN has tendency to increase exosomes in AVF mice compared with that of the RDN mice alone, but it was lower than the AVF group. Similarly, the levels of erythropoietin (EPO) were lower in the exosomes released during RDN intervention. Interestingly, the levels of exosomes secreted in AVF was significantly higher than the WT mice group. These results suggested that volume stress does cause an increase in the overall exosomes released independently to that of the renal denervation .To determine whether the AVF suppresses the regeneration and RDN procedure improves cardiac regeneration, we measured the levels of Wnt1 and β-catenin. The results demonstrated the increase in Wnt1 and β-catenin expression post RDN procedure in the mice, thus suggesting an improvement of cardiac regeneration by RDN during HFrEF to maintain HFpEF phenotype . InteresNext, we wanted to determine whether a putative increase in eNOS during RDN procedure will improve the endocardial-endothelial dependent cardiac function; hence, we measured the endocardial ring response (prepared from the cardiac left ventricle (LV) from C57BL/6J WT-control, AVF, RND, and AVF-RND mice groups) to acetylcholine (ACH). The results demonstrated an attenuation in endothelial dependent cardiac function in AVF group of mice; however, the RDN procedure was able to improve the cardiac endothelial function in experimental animals . FurtherHF is a serious medical emergency that encompasses multiple causes. In the clinic, it is usually diagnosed by a group of clinical symptoms and signs. Most of these presentations relate to the fact that the patients’ ventricles operate at relatively higher filling pressure, because this kind of congestive HF pathophysiology coincides with the spectrum of left ventricle ejection fraction (LVEF). It should be emphasized that regardless of the LVEF modus operandi, the clinical recognition of the symptoms and signs of HF are usually associated with major cardiac adverse events, thus affecting the quality and quantity of patients’ lives. The recognition and appreciation that approximately 50% of patients with this disease have almost normal or close to normal LVEF function makes this even more important, with the results stemming from the major clinical trials demonstrating differential responses to the HF therapies based on LVEF profile.Surprisingly, almost 50% of the patients tend to have distinct symptoms and related signs of HF with markedly normal left ventricular. This led to the recognition of the HF problem with pEF in the past 25 years that has truly spurred various clinical investigations coupled with informative outcome trials ,39. SkelOthers have shown an increase in eNOS levels and a concomitant decrease in hypertrophy by renal denervation procedure ,42. Our More recently, various clinical findings and animal experimentations have abundantly shown that RDN does improve the insulin sensitivity and endothelial function, even though the mechanism(s) involved remains unknown. When endothelial dysfunction in type 2 diabetes (T2D) in a rat model with insulin resistance was studied to explore the mechanisms and the effects of RDN, the RDN decreased the plasma and renal tissue norepinephrine levels significantly. In addition, the Von Willebrand (VW) factor levels were also decreased. Interestingly, the level of nitric oxide (NO) in plasma was also significantly increased after RDN. Side by side comparison with the T2D group and the sham group revealed that the endothelium-dependent and endothelium-independent diastolic function of the RDN group was also significantly improved. On further investigation, the relative expression levels of the molecules such as ACE2, Beclin1, LC3, and eNOS were found to be higher, but the levels of sequestome protein  were decreased. It was noteworthy that RDN could activate the p-AMPK expression and thus inhibit the expression of p-mTOR factor. In parallel in vitro experiments involving the cell culture, factors such as ACE2 helped activate the p-AMPK and that, in turn, was sufficient to inhibit the p-mTOR molecule, thereby inducing the autophagy process .It is known that renal denervation improves the vascular endothelial function, but it is unclear whether it also assists in the endocardial endothelial function during HFrEF phenotype . From ouIt is known that myocardial infarction (MI) increases the risk HF and atrial fibrillation (AF) in patients. It has been hypothesized that RDN may be able to suppress the atrial remodeling development. During the elucidation of the mechanism(s) of RDN towards the suppression of AF in the HF model after MI, it was demonstrated that RDN was able to reverse the atrial electrical as well as structural remodeling of the tissue and, thereby, could suppress the AF in an ischemic HF model. The authors revealed the beneficial effect of RDN might be, in fact, related to the prevention of phosphoinositide 3-kinase/AKT/endothelial nitric oxide synthase signaling pathway downregulation . In our vide supra). By creating the fistula, the red blood enters the vena cava without an injury to the heart. In fact, this model mimics the real CHF phenotype, such as the aging process, wherein we age and our preload keeps increasing beyond the level that our heart can help pump out, due to the weakened cardiomyocytes in the aging heart. This technique of creating the AVF also involves right ventricle to lung to left ventricle, thus enforcing the congestive heart failure (CHF) phenotype. After the AVF process, the heart transitions from the HFpEF to HFrEF over time. Again, our lab is one of the first labs that is instrumental in creating and perfecting the AVF procedure. To determine the protective role of renal nerve denervation (RDN), the RDN procedure was performed in one group of the AVF mice, and in a sham mouse group side by side. The mice were housed in the core animal care facility at the University of Louisville School of Medicine. To study the role(s) of renal nerve in volume sensing, experiments were carried out in renal “in-nerve and de-nerve” animals. The denervation procedure was performed by surgically removing all the nerves ending around renal artery and subsequently treated with phenol reagent, as described earlier [To create chronic/congestive (cardio-pulmonary) heart failure (CHF) in mice, an aorta-vena cava fistula (AVF) below the kidneys was performed, as mentioned previously, between the aorta and caudal vena cava approximately 0.5 cm below the kidneys using a 30-gauge sterile needle under aseptic surgical condition ,51,52,53 earlier ,54. ThesTo determine HFpEF and HFrEF phenotypes, serial and longitudinal ECHOs in mice at 1–6 weeks (HFpEF) and 12–16 weeks (HFrEF) were carried out. The concentric cardiac hypertrophy, a marker of HFpEF, was assessed via the long-axis ECHO as described earlier . After tIn addition, to focus on the exosomes containing the erythropoietin (EPO) regulated vascular density and cardiac function in HFpEF versus HFrEF, the role(s) of the circulating exosomal EPO in the context of the expression levels of molecules such as eNOS, ACE1, ACE2, SGLT1/2, and nephrin were assessed. To carry out the assessment, the renal and cardiac EPO, eNOS, Wnt1, and β-catenin, and respective levels of the renal  and medullary expression amounts of the caspases were also studied and quantified in sham, and AVF at 4–6 weeks following the AVF procedure. Circulating exosomes containing the EPO were analyzed by Western blotting (WB) experiments. The fibrosis was assessed by trichrome (collagen) and van-Giessen (elastin) staining procedure. A quantitative collagen estimation was carried out biochemically by the help of hydroxy proline measurements. Glomerular function rate (GFR) elicits overall function; therefore, reactivity to acetylcholine (ACH) was measured via the ex vivo preparation as described earlier . t-test for two groups, or two-way ANOVA  was used to compare the respective data with wild type, with RDN or without RDN according to the Bonferroni correction [p < 0.05.Data obtained from the same samples were used to express results in terms of percentage change(s) relative to the controls (prior to treatment/procedure). Experiments using commercial biochemical assays were performed in triplicate. Statistical significance was determined by student’s rrection . The dif


Background: Atrial fibrillation (AF) remains the most common arrhythmia following mitral valve surgery. Although numerous clinical and laboratory indicators and possible mechanisms of postoperative AF (PoAF) have been described, the role of microvascular dysfunction in pathogenesis has not been assessed. We aimed to evaluate the association between microvascular dysfunction and PoAF in patients undergoing isolated mitral valve replacement. Methods: 188 patients undergoing mitral valve replacement were included in this retrospective study. Demographic characteristics of the patients were recorded. Angiographic assessment of microvascular perfusion was performed using the myocardial blush grading technique for each patient. Univariate and multivariate logistic regression analyses were utilized to determine predictors of PoAF. Results: Of 188 patients  who underwent mitral valve replacement, 64 (34%) patients developed PoAF. In the PoAF group, a lower basal hemoglobin level , a higher left atrial diameter , and a lower total blush score (TBS)  were detected. Multivariate logistic regression analysis revealed that preoperative LA diameter , preoperative hemoglobin , and abnormal TBS  were independent predictors of PoAF. Conclusions: Our findings demonstrated that TBS at the preoperative period was an independent predictor of PoAF in patients undergoing isolated mitral valve replacement. Postoperative atrial fibrillation (PoAF) remains a well-known and frequently encountered arrhythmia following cardiac surgery ,2. The oThe mechanisms underlying PoAF have not been clearly identified yet, and its pathophysiology varies from classic atrial fibrillation (AF) in many aspects ,8. VarioThe myocardial blush grading (MBG) method has been well established as a basic angiographic technique to identify the efficacy of coronary microcirculation . To dateIn the current study, we aimed to investigate the effect of impaired microvascular perfusion on PoAF development following isolated mitral valve replacement, independent of other risk factors. We hypothesized that assessment of microvascular perfusion via MBG, a straightforward angiographic instrument, might provide incremental predictive data for risk estimation of PoAF beyond biological and clinical parameters.This study was designed as a double-center, non-randomized, observational, retrospective study. It was carried out to evaluate the relationship between preoperative MBG score and PoAF in patients undergoing isolated mitral valve surgery. The study was in compliance with the principles outlined in the Declaration of Helsinki and approved by the Local Ethics Committee of the Selçuk University Medical Faculty . In consideration of the acquired approval from the ethics committee, consent was granted by both hospitals involved in the study for its execution .The study population consisted of one hundred and eighty-eight patients scheduled for elective isolated mitral valve replacement for severe mitral valve pathology. Patients with a history or the presence of any atrioventricular conduction disorder or atrial dysrhythmia, hyperthyroidism, moderate-to-severe liver and renal failure, malignancy, pacemaker implantation, a contraindication to beta blockers, antiarrhythmic medication other than beta blockers, a history of previous cardiac operation, diabetes mellitus with late neurological complications, and more than 50% stenosis in any coronary territory, coronary spasm, coronary fistulae, and myocardial bridging evident on coronary angiogram were excluded. The study design is presented in the flowchart diagram .Arterial blood pressure > 140/90 mmHg or receiving antihypertensive treatment was defined as hypertension. Diabetes mellitus (DM) was described as fasting plasma glucose ≥ 126 mg/dL or random plasma glucose ≥ 200 mg/dL plus diabetic symptoms or 2 h plasma glucose ≥ 200 mg/dL in the oral glucose tolerance test or HbA1C level ≥ 6.5. Two-dimensional echocardiographic assessment and laboratory tests were performed before surgery. All demographic and baseline clinical features of the patients were recorded.Coronary angiography was consistently performed using the Judkins approach with 6F diagnostic catheters  in a standardized manner. Contrast injection was performed manually during the procedure. To be included in the study, the recordings had to be recorded until the venous phase, as required to evaluate MBG. In each patient, the best projection was preferred to determine the myocardial region of the coronary artery being examined. Two experienced interventional cardiologist who were blind to patients’ clinical data assessed the tissue-level perfusion using the MBG technique, as described: in  grade 0,PoAF has been described as a series of supraventricular beats with irregular R-R intervals that persist for 30 s or longer in the absence of P-waves. Continuous electrocardiography (ECG) monitoring in the intensive care unit was used to follow the subjects for the first 24 h postoperatively, while 12-lead ECG recording was checked regularly for rhythm follow-up on other days. When the subjects complained of angina, dyspnea, or palpitation, another 12-lead ECG was taken. Correction of electrolyte imbalance and appropriate fluid restitution were performed as required when PoAF occurred, followed by additional beta blockers and eventually amiodarone to provide medical conversion. p < 0.05) in the univariate analysis were included in a reverse multivariate regression model to determine the independent associates of PoAF. In this model, the least significant variable was progressively eliminated in each iteration until only the remaining variables exhibited statistical significance. The results of the model were reported as odds ratio (OR), 95% confidence interval, and p values. For all tests, a p value of <0.05 was considered statistically significant. Statistical analyses were performed using the SPSS software package . The Kolmogorov–Smirnov test was used to identify the distribution of variables. The data were expressed as mean (±SD) for normally distributed variables and median with interquartile range (IQR) for skewed continuous variables. Categorical variables were expressed as percentages. The Student t-test or Mann–Whitney U test were used to compare continuous variables as appropriate. Fisher’s exact and continuity correction (Yate’s correction) tests were used to compare categorical variables. In order to facilitate the regression analysis, the MBG and TBS values were converted into a dichotomous variable. For MBG, a score of 3 was classified as normal for each coronary territory, while values below 3 were categorized as abnormal. Similarly, for TBS, values of 8 and 9 were considered normal, while values below this range were categorized as abnormal. This transformation allowed for a more streamlined evaluation of these variables within the regression analysis. The baseline variables which were found significant (p = 0.007), left atrial (LA) diameter was found to be larger (5.9 (5.2–6.47) vs. 4.9 (4.5–5.8) cm; p < 0.001), and baseline hemoglobin level was found to be lower  in patients with PoAF (p = 0.003), cross-clamp time , and postoperative hospital stay  were found to be longer in patients with PoAF. One hundred eighty-eight patients undergoing mitral valve replacement  were included in the current study. Baseline demographic, clinical, laboratory, and echocardiographic parameters of the study population are given in ith PoAF . In addiA total of 192 coronary territories in patients with PoAF and a total of 372 coronary territories in the sinus rhythm group were assessed cautiously. Myocardial blush grades of each coronary territory are presented in p < 0.001) in patients with PoAF  in all coronary artery territories were significantly lower in patients with PoAF compared to the sinus rhythm group. Also, TBS was found to be lower , baseline hemoglobin , and abnormal TBS  were independent predictors of PoAF (To assess the predictors of PoAF, univariate and multivariate logistic regression analyses were performed. Univariate logistic regression analysis showed that age, preoperative LA diameter, baseline hemoglobin, CPB time, cross-clamp time, abnormal MBG scores for LAD, Cx, RCA territories, and abnormal TBS were risk factors for PoAF . Multiva of PoAF .Findings of the current study demonstrated that decreased TBS, increased left atrial diameter, and lower baseline hemoglobin levels were independent predictors of PoAF in patients undergoing isolated mitral valve replacement. These results established that assessment of microvascular perfusion preoperatively via the angiographic MBG method can be useful for prediction of PoAF in this patient group. To the best of our knowledge, this is the first study demonstrating the association between microvascular perfusion and PoAF following isolated mitral valve replacement. The risk of PoAF was documented to be higher in valvular and combined procedures involving coronary and valvular surgery than in coronary surgery alone. The PoAF rates were reported by Mariscalco et al.  as 22.9%The pathophysiology of AF development in the postoperative period is thought to be complex, consisting of several clinical and perioperative factors such as older age, male sex, past medical history of heart failure, chronic obstructive pulmonary disease, chronic kidney disease, diabetes mellitus, metabolic syndrome, obesity, high levels of brain natriuretic peptide, severe coronary artery disease, increased LA dimension, anemia, and blood transfusion preceding the surgery ,19. In t2 was used. In our study, although routine measurement of left atrial volume was not performed, the diameter of the left atrium was determined to be independently associated with PoAF. This finding adds to the notion that the left atrial structural remodeling had an impact on the occurrence of AF following mitral valve surgery.Left atrial diameter is an indirect measure of left ventricular filling pressure, which is frequently elevated in patients with mitral regurgitation. Indexed left atrial maximum volume was reported to be a robust, independent predictor of PoAF, even after adjustments for other biological and clinical parameters . OsranekOur findings revealed that decreased hemoglobin level was also independently associated with PoAF. Preoperative anemia has been demonstrated to independently increase postoperative mortality and morbidity along with the development of AF following heart surgery ,23. In tIf contrast injection is adequately performed and the duration of cine angiography is sufficient, tissue level vasculature filling exhibits a “ground-glass” or an angiographic “blush” appearance. This “ground-glass” view can be used in the catheterization laboratory to evaluate microvascular filling visually and is thus a sign of microvascular perfusion disorder . The MBGThe association between microvascular dysfunction and the occurrence of PoAF has not been fully elucidated and is likely to be complex. Patients with AF and without significant lesions in epicardial coronary arteries may still exhibit clinical signs of ischemia, such as angina, ST segment depression, or elevated troponin levels, particularly at the beginning of arrhythmia, indicating the presence of either microvascular perfusion disorder or arrhythmia-induced coronary spasm . MyocardIndeed, it is not clear whether coronary microvascular dysfunction is a pathophysiological substrate associated with AF or whether it is a consequence of this arrhythmia. A study evaluating coronary flow reserve in patients with lone recurrent atrial fibrillation confirmed impaired perfusion of atrial myocardium and deterioration of coronary flow reserve in this patient population, suggesting microvascular dysfunction as an important pathophysiological entity. In this research, several pathoanatomic changes such as atrial fibrosis, vascular degeneration of atrial cells, and perivascular and interstitial amyloid deposition accounted for the findings of normal baseline coronary flow and decreased coronary flow at peak hyperemia, as well as diminished coronary flow reserve, due to functional and/or structural changes in the microvascular network ,29,30. WIn agreement with the aforementioned studies, microvascular perfusion disorder might have played a critical role in the occurrence of PoAF, as a relevant pathophysiological mechanism, in our study. Additionally, considering the practicality and accessibility of the MBG score as a preoperative evaluation method for microvascular perfusion disturbance, which can occur in both the ventricles and atria, we believe it provides a viable approach for predicting postoperative atrial fibrillation. This is particularly relevant when compared to more sophisticated tests such as PET or cardiac magnetic resonance.While MBG has been well defined and validated in previous studies, direct visual assessment of coronary microvascular perfusion and not measuring coronary flow reserve via Doppler guide wire in angiography as an invasive technique are two of the most important limitations of our study. Nonetheless, we performed an intraobserver and interobserver variability analysis to validate the correct assessment of myocardial perfusion. More advanced echocardiographic parameters, such as left atrial strain and indexed left atrial maximal volume, were not measured in the present study. On the other hand, the left atrial diameter is a simple parameter, widely available and measurable with good reproducibility in daily clinical practice. There are no documented long-term clinical results for the patients. Furthermore, ECGs of the patients were recorded once daily after intensive care unit follow-up, which could have led to missing some of the paroxysmal AF attacks.Inflammation serves as a possible link between AF, endothelial dysfunction, and a decreased myocardial perfusion and perfusion reserve . Since tIn conclusion, our study demonstrates that reduced TBS at the preoperative period is associated with PoAF following isolated mitral valve replacement. The routine measurement of TBS preoperatively might be helpful for the prediction of PoAF in patients undergoing isolated mitral valve surgery enabling proactive measures to mitigate potential complications. During the postoperative follow-up of patients with low TBS values, diligent attention should be given to the occurrence of atrial fibrillation, as well as monitoring for hemodynamic and metabolic changes that may contribute to its facilitation. This comprehensive approach can provide timely recognition and appropriate management of atrial fibrillation, minimizing associated risks and optimizing patient outcomes. Whether the determination of this score can be added to routine evaluation for prediction of AF in patients undergoing mitral valve surgery is a matter that needs further investigation.


DICER1 syndrome is a tumor predisposition syndrome that is associated with up to 30 different neoplastic lesions, usually affecting children and adolescents. Here we identify a group of mesenchymal tumors which is highly associated with DICER1 syndrome, and molecularly distinct from other DICER1-associated tumors. This group of DICER1-associated mesenchymal tumors encompasses multiple well-established clinicopathological tumor entities and can be further divided into three clinically meaningful classes designated “low-grade mesenchymal tumor with DICER1 alteration” (LGMT DICER1), “sarcoma with DICER1 alteration” (SARC DICER1), and primary intracranial sarcoma with DICER1 alteration (PIS DICER1). Our study not only provides a combined approach to classify DICER1-associated neoplasms for improved clinical management but also suggests a role for global hypomethylation and other recurrent molecular events in sarcomatous differentiation in mesenchymal tumors with DICER1 alteration. Our results will facilitate future investigations into prognostication and therapeutic approaches for affected patients. DICER1 syndrome is associated with a predisposition to multiple tumor types. Here, the authors identify and characterize 3 molecular subgroups of mesenchymal tumors with DICER1 mutations. DICER1 utilizes its RNAse IIIa and IIIb domains to process pre-miRNAs, yielding a duplex containing either a mature 5p or 3p miRNA as well as a complementary ‘passenger strand’ that is ultimately degraded. The mature miRNA is then loaded onto an AGO protein to form an RNA-induced silencing complex, eventually resulting in down-regulation or silencing of mRNA targets. Specific metal-ion binding amino acids within the IIIa and IIIb domains are crucial for pre-miRNA cleavage. Failure of dicing of the pre-miRNA by RNase IIIb is a critical event in most DICER1-associated tumors. This failure arises because of the occurrence of tumor-confined missense variants that result in amino acid substitutions at these critical pre-miRNA-interacting residues within RNase IIIb4.DICER1 is a cytoplasmic endoribonuclease that is critical for the correct processing (cleavage) of precursor micro-RNA (pre-miRNA) double-stranded hairpins with 3’ and 5’ ends to their mature single-stranded formsDICER15. Typically, this is a loss of function (LOF) variant that is predicted to result in inactivation of the affected DICER1 allele. For the syndrome to occur, a second hit affecting exons encoding the RNase IIIb domain of DICER1 (a “hotspot” PV), as discussed above, is usually required4. The phenotypic spectrum of DICER1 syndrome is wide but some of the manifestations almost exclusively occur in persons with DICER1 syndrome. Both benign and malignant neoplasms are part of the syndrome. Pleuropulmonary blastoma (PPB), the most frequent primary lung malignancy in children, is highly characteristic of the syndrome. Other manifestations include ovarian Sertoli-Leydig cell tumor (SLCT), pediatric cystic nephroma, thyroid adenoma and carcinoma, embryonal rhabdomyosarcoma (ERMS)  and other rare entities9.DICER1 syndrome is a tumor susceptibility syndrome, characterized in 2009, determined by the occurrence of a germline pathogenic variant (PV) in DICER1 PVs, irrespective of the site of origin. These features comprise a subepithelial layer of malignant mesenchymal cells (cambium layer), areas of rhabdomyoblastic differentiation with positive staining for myogenin and myoD1, cellular/immature and occasionally malignant cartilage, foci of bone/osteoid and areas of anaplasia13. Furthermore, we have recently shown that both ERMS with DICER1 PVs and a tumor entity termed “primary intracranial sarcoma, DICER1-mutant” (PIS DICER1) are associated with DNA methylation signatures that are distinct from their morphological counterparts that are not DICER1-associated16. This finding, together with our recent speculations regarding the histomorphological similarities between DICER1-associated sarcomas13 arising at different sites led us to question whether in general, DICER1-associated tumors share common features such that they represent a distinct tumor entity, arising at various anatomical locations.Sarcomas are amongst the most common neoplasms in this syndrome and DICER1-associated sarcomas exhibit several characteristic morphological features, which can aid the pathologist in suspecting an association with DICER1 PVs, by DNA methylation profiling and identify three classes of mesenchymal tumors with DICER1 alteration, comprising tumors from various anatomical locations.Here, we address this hypothesis by analyzing 534 tumors, including a large number with DICER1 mutational status  of which 176 were reported to harbor DICER1 alterations . Detailed information on the tumor histotypes studied and DICER1 PVs of the study cohort is provided in Supplementary Table We analyzed whole genome DNA methylation data of 534 tumors including various histotypes associated with the DICER1 syndrome, as well as reference entities representing morphological counterparts of the tumors studied. The study set included a total of 431 tumors with known MYOD1-mutant spindle cell and sclerosing rhabdomyosarcoma (SRMS), ERMS, alveolar rhabdomyosarcoma (ARMS), low-grade endometrial stromal sarcoma (LGESS), high-grade endometrial stromal sarcoma (HGESS), Müllerian adenosarcoma (MAS), embryonal tumor with multilayered rosettes (ETMR), lung adenocarcinoma (LAC), clear cell renal cell carcinoma (RCC), ciliary body medulloepithelioma (MEPL), multinodular goiter (MG) and papillary thyroid carcinoma (PCA) each formed a distinct molecular cluster defined by diagnoses based on histology and established molecular testing, irrespective of DICER1 alteration status. For SLCT, we identified a subcluster that correlated with DICER1 PV status, which we termed “SLCT with DICER1 alteration” (SLCT DICER1). Furthermore, we identified three closely related methylation clusters of which one mostly resembled PIS DICER1 and two represented clusters combining multiple tumor types from several anatomically distinct locations  were mostly tumors previously diagnosed as MAS or SLCT, which clustered among the classes referred to here as LGMT DICER1 and SARC DICER1. These cases are discussed below in detail. Furthermore, tumors for which no consensus diagnosis of uterine ERMS or MAS was made during institutional workup and central review for a prior study, segregated into clusters of MAS or SARC DICER1. Cumulative copy number profiles of all molecular classes identified by DNA methylation analysis are shown in Supplementary Fig. n = 7), PPB type I/Ir , PPB type II , ERMS of the uterine cervix (n = 1) and the fallopian tube (n = 1), as well as nasal chondroid mesenchymal hamartoma . The SARC DICER1 class included ERMS of various locations , PPB type II , PPB type III , anaplastic sarcoma of the kidney  and pulmonary blastoma . The PIS DICER1 cluster almost exclusively consisted of PIS DICER1 (n = 26), but also encompassed one each of ASK and PPB III.The three clusters of LGMT DICER1, SARC DICER1 and PIS DICER1 identified by our methylation analyses combined multiple well established clinicopathological tumor entities which are known to be highly associated with the DICER1 syndrome and which may exhibit considerable morphological overlap  and female  patients in the SARC DICER1 class. This was due to the presence of uterine ERMS, which occurred in older patients as compared to other tumors of classes LGMT DICER1, SARC DICER1 and PIS DICER1  of 79/86 (92%) tumors which fell into clusters of LGMT DICER1, SARC DICER1 and PIS DICER1 identified morphological features shared across all three classes Fig. . These iIn detail, LGMT DICER1 were predominantly cystic or glandular with no or only sparse primitive, hypocellular mesenchyme Fig. . In a suConversely, SARC DICER1 predominantly consisted of primitive mesenchyme with variable cellularity and a mostly diffuse and patternless growth, although a fascicular growth pattern was present in some tumors Fig. . The mesPIS DICER1 were purely mesenchymal and were markedly cellular with a diffuse and/or fascicular growth pattern Fig. . Most tuDICER1 RNase IIIb hotspot PVs were identified in 83/86 (97%) of analyzed LGMT DICER1, SARC DICER1 and PIS DICER1, which were accompanied by DICER1 LOF PVs  in 57/83 tumors (69%). Furthermore, a second RNase IIIa/b mutation was identified in 4/83 (5%) tumors. Of the 22 tumors in which we did not identify a second DICER1 alteration 10/22 (46%) showed DICER1 RNase IIIb hotspot PVs with a variant allele frequency of > 0.5, suggesting loss of heterozygosity. Of the three tumors without DICER1 RNase IIIa/b hotspot PVs, DICER1 frameshift variants were present in 2/3 (66%) samples. Germline information was available for 53/86 (62%) patients, of which 28/53 (53%) showed a germline provenance of the DICER1 LOF PV identified in the tumor and therefore these tumors arose in the context of DICER1 syndrome. An overview of identified DICER1 PVs is provided in Fig. ense n = 2, framesTP53 , KRAS/NRAS , KMT2D , and NF1 . TP53, KRAS/NRAS, and NF1 PVs were only observed in SARC DICER1 and PIS DICER1, underlining shared oncogenic mechanisms of these neoplasms.Next, we performed panel-based next generation sequencing on a total of 80 tumors from all three classes Fig. . The oth2/number of involved chromosomes) of 14.7 (range 1–56), compared to SARC DICER1 and PIS DICER1, which showed complex genomes with broad and diverse copy-number variations (CNV) and a median GI of 92.9 (range 1–2532) and 314 (range 3 − 3216), respectively , MAS/ERMS  (n = 3), SLCT (n = 3), or MG (n = 1), which clustered stably with LGMT DICER1 or SARC DICER1 . Outliers in cluster SARC DICER1 showed frequent gains of chromosome 8, analogous to other SARC DICER1 tumors. In contrast, cumulative CNV profiles of MG, MAS and SLCT DICER1 showed CNV of chromosome 8 in only in a small subset of cases . LGMT DICER1 mostly represent cystic neoplasms of various organs that usually do not harbor additional molecular alterations and show an excellent clinical outcome18. In contrast, DICER1-associated sarcomas (SARC DICER1 and PIS DICER1) usually exhibit an overtly sarcomatous phenotype which is associated with frequent alterations in TP53, KRAS/NRAS and NF1, high genomic index, as well as a global hypomethylation signature. In contrast to LGMT DICER1, DICER1-associated sarcomas may show a more aggressive clinical course23. Although the difference in the global methylomic profiles of these three classes may reflect their cell type composition, such as dominant epithelium component in LGMT DICER1, dominant sarcoma component in the other two classes and no epithelium in PIS DICER1, the identification of these DICER1-associated tumor classes from various anatomical sites will enable meaningful clinical trial stratification in the future and suggests that rational drug development addressing the differing molecular foundations of mesenchymal tumors with DICER1 alteration may be a plausible goal.In this study, we identify a group of mesenchymal tumors with DICER1 alteration, which includes three classes termed LGMT DICER1, SARC DICER1 and PIS DICER1 Fig. . These tDICER1 alterations may induce or contribute to distinct tumor phenotypes dependent on the specific cellular context. While DICER1-associated mesenchymal tumor classes may share similar cellular backgrounds, other DICER1-associated neoplasms likely have different cellular origins depending on the tumor location. It remains unclear if the sub-clustering in DICER1-associated sarcomas into classes of SARC DICER1 and PIS DICER1 is due to a different cell of origin for CNS and non-CNS tumors, or if site-specific factors shaping the tumor microenvironment have influenced the molecular features.We also show that some other DICER1-associated lesions, such as SLCT DICER1, nodular thyroid lesions (MG and PCA), PINB, MEPL and WILMS correspond to molecular classes distinct from the classes of DICER1-associated mesenchymal tumors. This suggests that 24. In our study, DNA methylation profiling was able to classify tumors previously diagnosed as MAS, ERMS or MAS/ERMS  into specific clusters of MAS or SARC DICER1, highlighting the potential for molecular markers to aid tumor classification. Therefore, ancillary tests, such as DNA methylation profiling (in ensemble with the Heidelberg Sarcoma classifier25), CNV profiling or panel-based DNA sequencing may be helpful in correctly classifying a neoplasm as either one of the three classes of mesenchymal tumors with DICER1 alteration, or to exclude other DICER1-associated tumor entities, such as SLCT DICER1 or uterine MAS. A diagnostic algorithm for mesenchymal tumors with DICER1 alteration is proposed in Fig. A diagnosis of mesenchymal tumor with DICER1 alteration should always be taken into consideration when dealing with a mesenchymal neoplasm with areas of a subepithelial layer of malignant mesenchymal cells, areas of rhabdomyoblastic differentiation, cellular/immature and sometimes overtly malignant cartilage or foci of bone/osteoid. The presence of an overtly sarcomatous differentiation (LGMT DICER1 vs. SARC DICER1), anatomical location (SARC DICER1 vs. PIS DICER1) and the identification of a DICER1 hotspot PV, will usually be sufficient to classify DICER1-associated mesenchymal neoplasms. However, in some cases clinical presentation, histopathology and DICER1 hotspot sequencing could yield inconclusive or contradicting results and present a diagnostic challenge for pathologists, as is frequently the case in DICER1-associated tumors of the gynecologic tractWe consider that it would be desirable to implement the herein proposed nomenclature for mesenchymal tumors with DICER1 alteration. Nevertheless, we understand that in practice, nomenclature changes have the potential to result in confusion for clinicians and pathologists. Given that it may not be apparent that these “new” terms refer to neoplasms that already have established names in the literature rather than newly defined tumor types, this could potentially cause “loss” of important information when new terms are used. Adoption of our proposed nomenclature will require close cooperation between pathologists and clinicians dealing with these rare tumor types. Going forward, it may be rational to initially use both “new” and “old” terminology, for example “pulmonary sarcoma with DICER1 alteration (Pleuropulmonary blastoma type III)” or “cervical sarcoma with DICER1 alteration ”.28. Our results support this hypothesis by showing, for example, that PPB II is found in methylation clusters of both LGMT DICER1 and SARC DICER1, whereas PPB I is only found in LGMT DICER1 and PPB III is only found in SARC DICER1 or PIS DICER1. While disruption of DICER1 has been shown to lead to downregulation of miRNAs and enhance stemness and epithelial-to-mesenchymal transition in colorectal cancer cells29, as stated above, additional PVs in oncogenes and tumor suppressors other than DICER1 PVs, may contribute to this transition from a cystic to a mesenchymal phenotype.It is tempting to suggest that the development of DICER1-associated mesenchymal tumors at a specific anatomical location represents a disease continuum with transition from cystic to sarcomatous configuration. This hypothesis is based on the description of few patients in which a diagnosis of PPB I or CN was followed by occurrence of PPB II/III or ASK, respectively31. Similarly, we hypothesize that that the global hypomethylation signature identified in SARC DICER1 and PIS DICER1 is result of a continuing process that persists throughout tumor progression/sarcomatous differentiation, rather than selection for a precursor cell with a pre-existing hypomethylated state. Nevertheless, further studies into the cellular origins of mesenchymal tumors with DICER1 alteration are needed to expand on our hypothesis.Our study furthermore implicates global hypomethylation as a feature of sarcomatous differentiation in SARC DICER1 and PIS DICER1. Studies of Wilms tumor and glioblastoma, as well as their proposed cells of origin, have indicated that altered DNA methylation in tumors does not reflect methylation states of precursor cells, but rather represents demethylation during tumorigenesis32. In this context, our results raise the question of a potential link between disruption of the DICER1-associated miRNA machinery and global DNA methylation changes that warrants further investigation. In addition, there are other factors to consider that may contribute to demethylation. Characterization of DNA methylation associated proteins, as well as chromatin structure and composition may allow a better understanding on how DNA hypomethylation may be induced in DICER1-associated neoplasms.When compared to LGMT DICER1, the DMRs identified in both SARC DICER1 and PIS DICER1 were widely hypomethylated and enriched for genes associated with gene silencing through miRNAs. Growing evidence indicates that disruption of miRNAs signaling may be involved in the control of DNA methylation by targeting the DNA methylation machinery34. Our results similarly show that in mesenchymal tumors with DICER1 alterations DNA hypomethylation is correlated with chromosomal instability and, more specifically, that regions affected by chromosomal gains may especially be prone to be hypomethylated.Previous studies have demonstrated an association of hypomethylation and chromosomal instability in human and murine cancersIn conclusion, we demonstrate that mesenchymal tumors with DICER1 alteration comprise three distinct clinicopathological and molecular tumor classes, which may warrant a change in nomenclature. Our study provides a combined approach to classify DICER1-associated neoplasms, which not only has diagnostic implications but will also facilitate future investigations into prognostication and therapeutic approaches for patients with such tumors.64. Wherever available, H&E-stained formalin-fixed paraffin-embedded (FFPE) sections from tumors samples were reviewed by pathologists involved in the study . Clinical data were provided by collaborators. Raw DNA methylation and clinical data for SRMS and ARMS, as well as subsets of ERMS, LGESS and HGESS were downloaded from the DNA methylation sarcoma classifier25 (GEO accession number: GSE140686) for a subset of PINB and ETMR from the DNA methylation CNS classifier65 and Lambo et al. 66. (GEO accession numbers: GSE109381 and GSE122038), for LCA and a subset of PCA from the TCGA cohorts68 (https://tcga-data.nci.nih.gov/docs/publications/luad_2014/ and https://tcga-data.nci.nih.gov/docs/publications/thca_2014/), and for WILMS through the TARGET Data Matrix69 (https://ocg.cancer.gov/programs/target/data-matrix). An overview of all tumor samples included in various analyses is provided in Supplementary Data This study was approved by the institutional ethics committee  and performed in accordance with the Declaration of Helsinki. Tumor samples of AS, CN, ERMS, HGESS, LGESS, MAS, MAS/ERMS, MEPL, MG, NCH, PB, PCA, PINB, PIS DICER1, PPB and SLCT had all undergone histopathological evaluation by specialized pathologists, as well as molecular testing whenever applicable. Samples were collected at McGill University-affiliated hospitals in Montreal, Quebec, Canada and the Institute of Pathology, University Hospital Heidelberg, mainly from collaborating institutions in accordance with ethics review board regulations. Eligible participants signed an informed consent form. Clinical data, histological features and/or sequencing results of 403/534 tumors have previously been reported elsewhere in partrstatix70. Comparison of categorical data was performed using the Chi-squared Test. Differences between group averages were evaluated using one-way analysis of variance (ANOVA) with the Games–Howell post hoc test. All tests were two-sided. P-values were rounded to three decimal places. P-values less than 0.001 were stated as p <  0.001. A p-value of <0.05 was considered statistically significant. P-values of multiple comparisons were adjusted using the Bonferroni correction. Correlation was calculated using the Pearson correlation coefficient. Survival analyses (Kaplan–Meier estimates) were performed using the R-packages survival71 and survminer72.Statistical analyses were performed in R v.4.1.2,  using the package Genomic DNA was extracted from either fresh-frozen or FFPE tumor tissue. The Maxwell® 16 FFPE Plus LEV DNA Kit or the Maxwell 16 Tissue DNA Purification Kit (for frozen tissue) was applied on the automated Maxwell device  according to the manufacturer’s instructions. A minimum of 100 ng DNA was extracted in every case and provided for subsequent array-based DNA methylation analyses and DNA sequencing.DNA was subject to bisulfite conversion and processed on the Infinium HumanMethylation450K BeadChip or the Illumina Infinium EPIC (850k) BeadChip  according to the manufacturer’s instructions.minfi and Champ73. For clustering analyses, Illumina450k and 850k sample data were merged into a combined dataset by selecting the intersection of probes present on both arrays . DNA methylation data were normalized by applying background correction and dye bias correction. Probes targeting sex chromosomes, probes containing multiple single nucleotide polymorphisms, multiple hit probes and those which could not be uniquely mapped were removed. For unsupervised hierarchical clustering of DNA methylation data, 8000 and 20,000 probes with the most variably methylated probes across the dataset were selected. Distance between samples was calculated using Euclidean distance and average linkage was used to generate dendrograms. For unsupervised 2D representation of pairwise sample correlations, t-SNE dimensionality reduction was performed using the same distance metrics and default parameters. For group-wise comparisons, differentially methylated positions (DMPs) and regions (DMRs) were identified using Champ and the Probelasso method75. Only DMPs with a false-discovery rate (FDR) < 0.05 were considered. DMRs required a minimum of 5 significant probes per lasso . DMRs were only considered to be intersecting between classes if the signs (+/−) of difference in beta values were matching.Array data analysis was performed using R v.4.1.2, . Data normalization and preprocessing was performed using R packages copynumber76. Summary copy number profiles were created using the R-package conumee77. Gene amplifications and deletions were identified by manual inspection. The genomic index was computed from segmented copy number data as previously described 78. The upper and lower thresholds for segmental gains and losses were set at 0.1 and −0.1 (log2), respectively.Copy number alterations of genomic segments were calculated from the methylation array data and plotted using the R-packages 79. Reads were aligned to the reference genome hg19 and variants were annotated using ANNOVAR software80. Missense variants were classified as stop-loss variants, polymorphisms  or other variants that were assessed by two prediction algorithms: SIFT81 and PolyPhen82. Variants considered were SNVs with a damaging prediction by at least one algorithm or an unknown prediction by both algorithms, as well as all frameshift and nonsense mutations.DNA was sequenced using a customized SureSelect XT technology (Agilent) panel covering the coding regions of 201 genes  using R-packages Further information on research design is available in the Supplementary InformationPeer Review FileReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4


A 69-year-old man with persistent atrial fibrillation (AF) experiencing recurrences of suspected paroxysmal atrial tachycardia was referred for repeat ablation. He had previously undergone pulmonary vein isolation in 2016. He was suspected to be experiencing a tachycardia involving the mitral isthmus or left-sided veins initially; however, electroanatomic mapping did not reveal a circuit involving these structures. Instead, a focal arrhythmia with the earliest signal on the anterolateral mitral valve annulus was noted. Catheter manipulation in this region consistently terminated the tachycardia. A tachycardia involving the mitral isthmus or left-sided veins was suspected initially; however, EAM did not reveal a circuit involving these structures. Instead, a focal arrhythmia with the earliest signal on the anterolateral mitral valve annulus was noted . Catheter manipulation in this region consistently terminated the tachycardia. The Advisor™ HD Grid was then placed in the left ventricle just distal to this site, and although left ventricular (LV) pacing resulted in termination, eccentric activation was confirmed. Mapping with the ablation catheter during LV pacing revealed a pathway potential, which was eliminated instantly with ablation, restoring concentric activation .A 69-year-old man with persistent atrial fibrillation (AF) status post-ablation with pulmonary vein isolation in 2016 with recurrences of suspected paroxysmal atrial tachycardia was referred for repeat ablation. Electroanatomic mapping (EAM) in the left atrium spontaneously induced supraventricular tachycardia (SVT). The right pulmonary veins were ostially isolated and the left pulmonary veins had recovered at the anterior carina. The Advisor™ HD Grid Mapping Catheter, Sensor Enabled™  and EnSite Precision™ (Abbott) three-dimensional mapping system were used to map this 1:1 eccentric atrioventricular tachycardia with a cycle length of 430 ms 1 The mechanisms and pathogenesis of AF in patients with APs are poorly understood, complex, and often controversial. Even though therapeutic ablation of APs is associated with AF reduction, older age, male sex, and the presence of APs are independent risk factors for the presence and progression of clinical AF.2 Numerous additional risk factors like hypertension, diastolic heart failure, older age, complexity of coronary artery disease, structural changes in the atrium and ventricle, and the presence of renal disease all contribute to the development of AF in patients with CLAPs.3 Given the presence of these risk factors in our patient, it is likely that APs are only one of the many substrates for the development of AF, which was unlikely related to his AP.3 Additionally, previous studies have documented an AF recurrence rate of 10%–25% following AP ablation, and close monitoring of these patients is necessary for uncovering future recurrences.1 This case serves as a reminder to consider the differential of SVT in cases of redo AF ablations and revealed the likely therapeutic advantage of LV pacing near the pathway site to delineate a clear pathway potential.5AF is noted to occur in up to one-third of patients with accessory pathways (APs). There is a 15 times higher incidence of AF occurring in patients with manifest pathways compared to those with concealed accessory pathways (CLAPs), as the overall prevalence of AF in CLAP patients is thought to be <1%.