P < .0001). p62 accumulation was highest in the desmin (15.6%) and phospholamban (7.2%) groups. P62 accumulation was homogeneously distributed in the myocardium. Fibrosis was not associated with p62 accumulation in subgroup analysis of phospholamban hearts. In conclusion, accumulation of p62‐positive protein aggregates is homogeneously distributed in the myocardium independently of fibrosis distribution and associated with desmin and phospholamban cardiomyopathy. Proteotoxic protein accumulation is a diffuse process in the myocardium while a more localized second hit, such as local strain during exercise, might determine whether this leads to regional myocyte decay.Genetic cardiomyopathy is caused by mutations in various genes. The accumulation of potentially proteotoxic mutant protein aggregates due to insufficient autophagy is a possible mechanism of disease development. The objective of this study was to investigate the distribution in the myocardium of such aggregates in relation to specific pathogenic genetic mutations in cardiomyopathy hearts. Hearts from 32 genetic cardiomyopathy patients, 4 non‐genetic cardiomyopathy patients and 5 controls were studied. Microscopic slices from an entire midventricular heart slice were stained for p62 . The percentage of cardiomyocytes with p62 accumulation was higher in cardiomyopathy hearts (median 3.3%) than in healthy controls (0.3%;  In the normal situation, protein degradation pathways, like autophagy, prevent this accumulation of misfolded proteins. Recent studies suggest an important role for impaired autophagic processes in myocardial diseases such as myocardial infarction, pressure overload hypertrophy and various forms of cardiomyopathies.Autophagy regulates cellular homoeostasis by transporting intracellular cargo to the lysosomes for bulk degradation and recycling of macromolecules.P62 recognizes ubiquitinated proteins and promotes aggregation and sequestration (synonym sequestosome‐1) marking protein aggregates for degradation by autophagy or the ubiquitin proteasome system.PLN), we have recently demonstrated aggregates that are both p62 and phospholamban positive.DES, CRYAB, PLN, PKP2, DSP, LMNA, MYBPC3, MYH7, TNNI3 and TNNT2). In addition, we studied the distribution of these aggregates throughout the myocardium.In hearts of cardiomyopathy patients with a pathogenic variant in the gene encoding phospholamban . Written informed consent for collection and biobanking of tissue samples was obtained prior to transplantation or, in certain cases, approved by the ethics committee when obtaining informed consent was not possible because of death of the patient.DES) and the desmin‐related αB‐crystallin (CRYAB), phospholamban (PLN), desmosomal proteins (PKP2 and DSP), lamin A/C (LMNA), and sarcomeric proteins . Five control hearts were used (four donor hearts not used for transplantation and one heart acquired at autopsy after a non‐cardiac death). At the time of clinical presentation, the cardiomyopathy subtype of these patients had been classified by their cardiologist as arrhythmogenic [ACM] according to the revised task force criteria,Hearts from 32 patients with a genetic cardiomyopathy and 4 patients without known pathogenic mutations were included in the study that were obtained during heart transplantation or at autopsy. Genetic testing was performed in a clinical setting. Patients were classified in functional protein groups encoded by (likely) pathogenic variants (mutations) in genes encoding desmin  was used to stain for p62 using the Ventana BenchMark Ultraplatform (Roche). For p62 quantification, the heart slice was divided into five regions for the free wall of the left ventricle , one region for the septum and two regions for the right ventricle  as we have described previously Figure .8, 92.1Data are expressed as median [interquartile range]. A Mann‐Whitney test was used to compare percentages between two groups and a Kruskal‐Wallis test to compare multiple groups. Dunn's multiple comparison test was used to correct for multiple comparisons. Linear regression was used to test the association between two continues variables.33.1Patient characteristics are summarized in Table 3.2versus 3.3% [1.4‐6.4] in the cardiomyopathy hearts (P < .0001). In the cardiomyopathy hearts, p62 accumulation was highest in the desmin, phospholamban and desmosomal gene groups  followed by, lamin AC (1.3% [1.2‐2.5]) and sarcomeric gene (1.2% [0.3‐4.9]) groups . In the subgroup analysis of the other mutation groups, no significant difference was observed between RV, septum and LV , septum (3.1% [1.5‐6.8]) and LV (3.6%[1.4‐6.4]). In the desmosomal gene group, the percentage of p62‐positive cells in RV (2.1% [1.6‐3.1]) was significantly lower than the percentages in the septum (4.3% [2.8‐5.3]) and LV , RV (P = .9), LV (P = .9) and the three separate layers of the left ventricular wall .Given the relatively high numbers of phospholamban hearts, we decided to perform a subgroup analysis to study the association between p62 and fibrosis. The percentage of p62‐positive cardiomyocytes was not associated with the percentage fibrosis in the myocardium of the whole heart  gene. This observation is in line with the classical histopathological hallmark of many desmin‐related cardiomyopathies, that is abnormal cytoplasmic desmin aggregation in cardiomyocytes.DES p. Asn342Asp variant (15% p62‐positive cells in our study) induced cytoplasmic aggregates and small desmin fibrils without normal filament assembly. However, in a heterozygous co‐expression experiment, that is more comparable to the human situation, mutant and wild‐type desmin molecules were incorporated into the same filamentous structures without aggregate formation.CRYAB and 15% in DES cardiomyopathy replacing large parts of the sarcomeres, suggesting that these aggregates may play an important role in the impaired myocardial function of these cardiomyopathies. In addition, it has been demonstrated that aberrant protein aggregation in desmin cardiomyopathy leads to mitochondrial dysfunction, abnormal metabolism, and altered cardiomyocyte structure.The highest percentages of cardiomyocytes with aggresomes were observed in desmin cardiomyopathy patients, including one patient with a pathogenic mutation in the desmin‐related αB‐crystallin  mouse model where hearts not only showed focal fat infiltrations in the RV (consistent with arrhythmogenic cardiomyopathy), but also cytoplasmic aggregates consisting of intercalated disc proteins such as desmoplakin, plakoglobin and connexin 43. These aggregates coincided with disruption of the intercalated discs, intermediate filaments and microtubules.Also in desmosmal mutation‐positive arrhythmogenic cardiomyopathies, p62‐positive aggregates were observed. This is in line with observations in a recent desmoplakin (MYH7 and 1 TNNI3); therefore, the cardiomyopathy in these patients might have been affected by other variables that we have currently not yet identified. Also, in lamin A/C hearts we observed relatively low percentages of p62‐positive cardiomyocytes which is in concordance with a mouse model study where the increase of p62‐positive cells was only twofold compared to controls.The hearts of the other patient groups revealed the lowest percentages of p62‐positive cardiomyocytes that were comparable to the p62 levels of patients without known pathogenic mutations. Sarcomeric mutations are usually missense and nonsense variants which cause a dysfunctional protein that can still be integrated in the sarcomere of the cardiomyocyte. This might explain why in patients with sarcomeric mutations accumulation of mutated proteins and p62‐positivity appears to be limited. In the sarcomeric group, 3 patients had a variant of unknown significance  are urgently needed. Furthermore, we used explanted hearts, and therefore, our results only represent end‐stage disease.In conclusion, accumulation of p62‐positive protein aggregates is homogeneously distributed in the myocardium independently of fibrosis distribution and strongly associated with desmin and phospholamban cardiomyopathy. Proteotoxic protein accumulation probably is a diffuse process in the myocardium where a second hit, such as local strain during exercise, might determine whether this leads to regional myocyte decay and fibrosis.The authors confirm that there are no conflicts of interest.Joy van der Klooster: Conceptualization ; Data curation (lead); Formal analysis (lead); Methodology ; Project administration ; Resources ; Software ; Validation ; Visualization ; Writing‐original draft (lead). Shahrzad Sepehrkhouy: Data curation ; Methodology ; Software ; Writing‐review & editing . Dennis Dooijes: Data curation ; Resources ; Writing‐review & editing . Wouter te Rijdt: Data curation ; Methodology ; Resources ; Writing‐review & editing . Frederique Schuiringa: Data curation ; Investigation ; Writing‐review & editing . Jolanthe Lingeman: Data curation ; Investigation ; Writing‐review & editing . Peter van Tintelen: Resources ; Supervision ; Validation ; Writing‐review & editing . Magdalena Harakalova: Funding acquisition ; Investigation ; Software ; Writing‐review & editing . Roel Goldschmeding: Supervision ; Writing‐review & editing . Albert Suurmeijer: Conceptualization ; Methodology ; Supervision ; Writing‐review & editing . Folkert Asselbergs: Funding acquisition (lead); Resources ; Supervision ; Writing‐review & editing . Aryan Vink: Conceptualization (lead); Data curation ; Formal analysis ; Investigation ; Methodology ; Project administration ; Resources ; Software ; Supervision ; Validation ; Visualization ; Writing‐review & editing .Table S1Click here for additional data file.


Pulmonary artery (PA) enlargement, defined as pulmonary artery to ascending aorta diameter ratio (PA:A)>1 on computed tomography (CT), is a marker of pulmonary vascular disease in chronic lung diseases. PA enlargement is prevalent in cystic fibrosis (CF), but its relationship to hemodynamics and prognostic utility in severe CF are unknown. We hypothesized that the PA:A would have utility in identifying pulmonary hypertension (PH) in severe CF and that PA enlargement would be associated with reduced transplant-free survival.We conducted a retrospective study of adults with CF undergoing lung transplant evaluation at a single center between 2000 and 2015. CT, right heart catheterization (RHC), and clinical data were collected. The PA:A was measured from a single CT slice. We measured associations between PA:A and invasive hemodynamic parameters including PH defined as a mPAP ≥25mmHg using adjusted linear and logistic regression models. Kaplan-Meier and adjusted Cox regression models were used to measure associations between PA:A>1, RHC-defined PH, and transplant-free survival in severe CF.We analyzed 78 adults with CF that had CT scans available for review, including 44 that also had RHC. RHC-defined PH defined as a mPAP ≥25mmHg was present in 36% of patients with CF undergoing transplant evaluation. The PA:A correlated with mPAP  and PVR  and the PA:A>1 was an independent predictor of PH . PA:A>1 was independently associated with increased hazards for death or transplant . The presence of mPAP ≥25mmHg was independently associated with decreased survival in this cohort.PA enlargement is associated with pulmonary hemodynamics and PH in severe CF. PA enlargement is an independent prognostic indicator of PH and decreased survival in this population. Cystic fibrosis (CF) is an autosomal recessive genetic disorder that leads to functional impairment in the cystic fibrosis transmembrane conductance regulator (CFTR) channel, leading to impaired mucociliary transport, recurrent infections, airflow obstruction, bronchiectasis, and altered gas exchange. Lung function impairment is strongly associated with morbidity and mortality , 2. OtheRight heart catheterization (RHC) is the gold standard for confirming PH; however, the technique’s invasiveness precludes its use on a regular basis in chronic lung diseases including CF. Thus, there is evolving interest in using non-invasive tools for assessing the presence of PH, such as computed tomography (CT), Doppler echocardiography and cardiac magnetic resonance imaging , 9. CT-mThe University of Alabama at Birmingham (UAB) Institutional Review Board (X150128006) approved this study. The IRB waived requirement for informed consent based on the nature of the investigation. None of the data were obtained from a vulnerable population .Patients who underwent lung transplant evaluation at the University of Alabama at Birmingham (UAB) between January 1, 2000 and December 31, 2015 and carried a diagnosis of CF were identified using the UAB Lung Transplant Database. Subjects were enrolled if they had a diagnosis of CF with the presence of known CF-causing mutations and had measurable CT as part of lung transplantation work-up. Lung transplantation work-up was conducted on an outpatient basis in patients that were free from acute decompensation or exacerbations.1), forced vital capacity (FVC), diffusion lung capacity of carbon monoxide (DLCO), body mass index (BMI), use of supplemental oxygen, extra-pulmonary CF manifestations, and prior infectious history from culture data. Data regarding whether lung transplantation occurred and the date of transplant were also collected. All patients who had lung transplant underwent bilateral lung transplantation. The date of death was recorded as retrieved through medical record review. Transplant-free survival was calculated from date of transplant evaluation.Demographic and clinical data were collected from the medical records at the time of transplant evaluation and included age, sex, CF genotype, forced expiratory volume at 1 second  using previously published methods [CT scans were reviewed separately by two independent blinded investigators (ANZ and SG) to assess for inter-observer agreement of PA:A measurements. The first investigator measured the PA, A, and PA:A from the same scan twice to assess for intra-observer reproducibility. The mean PA, A, and PA:A values were used for all analysis and calculations. PA:A was calculated by taking the ratio of the PA measured at the level of the PA bifurcation and the ascending aortic diameter measured from the same CT slice  methods , 14, 15.Hemodynamic variables were calculated or measured from the RHC, including mean right atrial pressure (RAP), mean pulmonary artery pressure (mPAP), cardiac output (CO) by thermodilution, cardiac index (CI), pulmonary vascular resistance (PVR), and pulmonary capillary wedge pressure (PW). PH was defined as a mPAP ≥25 mmHg on resting RHC.Continuous variables are presented as mean ± standard deviation and categorical variables as number and percentage. We considered missing data to be “missing at random” due to the retrospective nature of the study. Intra-class correlation coefficient (ICC) and Bland Altman analyses were used to measure the level of intra-observer and inter-observer agreement for measurement of PA, A, and PA:A. Pearson’s coefficient was used determine the correlation between PA:A and mPAP, as well as FEV1, FVC, DLCO and other clinical or hemodynamic variables. Unpaired t-test analysis was performed to compare mean differences between two groups for continuous variables and Chi-square or Fisher’s exact testing were used to compare differences for categorical variables, as appropriate. Linear regression analyses were used to measure the association between a z-score PA:A as a continuous variable mPAP, adjusting for age, sex, FEV1 percent predicted, and LTOT use, covariates previously shown to be associated with mPAP. Logistic regression models were used to determine associations between demographic characteristics, PA enlargement, and the presence of PH (mPAP ≥25mmHg). Covariates were selected based on known associations with CF disease severity. Kaplan-Meier survival curves were used to describe transplant free survival according to PA enlargement or PH as a function of time from the initial lung transplant evaluation. Kaplan-Meier curves were compared using the log-rank test. We then used Cox regression analysis to measure associations between PA enlargement, RHC-defined PH, and transplant free survival adjusting for age, sex, FEV1 percent predicted, and LTOT use. Patients who received lung transplantation were censored, as were those who died while awaiting transplant. Patient who were alive and had not received transplantation were censored at the end of data collection. A p-value <0.05 was considered statistically significant. All analyses were performed using IBM SPSS version 26.0 .Fig 2). From these, spirometric data were missing in n = 27; DLCO in n = 35; 6-minute walk distance in n = 41; and RHC data in n = 34. The cohort was 47% male, 97% White, and had a mean age of 28±10 years as shown in Table 1. All individuals in the cohort had two severe disease-causing mutations, including 37 (48%) Delta F508 homozygotes  patients used supplemental oxygen at the time of evaluation, confirming very severe underlying lung disease. In the 44 patients that had available RHC data, the average mPAP was 24±9 mmHg corresponding to 16 (36%) with PH defined by mPAP ≥25 mmHg. Of patients with RHC data, 13 (30%) had a mPAP 20–24 mmHg, 15 (34%) had mPAP 25–34 mmHg, and 3 (7%) had mPAP ≥35 mmHg suggesting the majority have mild-to-moderate PH.There were 107 patients who carried a diagnosis of CF were referred for lung transplant evaluation between 2000 and 2015. Of those, 29 patients were excluded from analysis due to not having CT data, leaving a final cohort of 78 patients  as well as inter-observer . Bland Altman graphs representing inter and intra-observer variability are shown in Fig A in S1 File.In the 78 participants that had CT scan data available, the mean PA:A was 1.01±0.15, with 38 (48%) having PA enlargement (PA:A >1). The ICC for readings demonstrated excellent intra-observer agreement  and higher PVR  compared to the PA:A ≤1 group. Otherwise, hemodynamic parameters including PW or CO were not statistically different between groups. There were significant correlations between the PA:A and mPAP , PVR , but not PW . There were no statistically significant correlations between PA:A and FEV1, FVC, or DLCO. The PA:A was not associated with other clinical or hemodynamic variables including 6MWD or BMI. In linear regression models adjusted for age, sex, FEV1, and LTOT use, each standard deviation increase in PA:A was associated with a 6 (Standard Error 1) mmHg increase in mPAP (P<0.001).As shown in Fig 3.We then tested the utility of the PA:A in predicting PH defined as mPAP ≥25mmHg. PA enlargement was associated with PH in Fisher’s exact testing (p = 0.029). In logistic regression models adjusting for age, sex, FEV1 percent predicted, and LTOT use, the PA:A was associated with PH  as shown in Fig 4A. PA enlargement remained a significant independent predictor of death or lung transplant  in a Cox proportional hazard model adjusted for age, sex, FEV1 and LTOT (Table B in S1 File). In a separate Kaplan-Meier survival model shown in Fig 4B, the presence of PH defined as a mPAP ≥25 mmHg was associated with shorter median transplant-free survival  compared to CF patients without PH.In Kaplan-Meier analyses, patients with severe CF and PA enlargement had a 7-month shorter median survival time compared to those without PA enlargement  as shown in Among patients with severe CF undergoing lung transplant evaluation, the PA:A correlated with invasive hemodynamic variables obtained by RHC and PA enlargement was associated with the presence of PH as well as transplant-free survival. To our knowledge, this is the first study to examine the relationship between PA:A and invasive hemodynamics in this population of severe CF. These findings provide the basis for a novel, reproducible, and easily obtainable measurement for suggesting PH in CF, where other non-invasive tools such as echocardiography have been shown to be sub-optimal in assessing pulmonary arterial pressures in this subgroup of patients .1 becomes a less dynamic marker in end-stage CF as compared to FVC, which remains responsive in severe obstruction.In this cohort, the inter-observer and intra-observer reproducibility for measuring PA:A was excellent, consistent with findings in other lung diseases , 17, 18.Current recommendations for the evaluation of patients with suspected PH begin with an echocardiogram as an initial screening test to estimate the right ventricular systolic pressure and to evaluate for right heart chamber enlargement . While mThe presence of PH in CF is associated with increased morbidity and mortality , and worWe found that each standard deviation increase in PA:A corresponded to a 6±1 mmHg increase in mPAP and the presence of PA enlargement was associated with PH. These findings correspond to similar observations in patients with chronic obstructive lung disease (COPD) , 12 and While chronic hypoxia likely accounts for most of the vascular remodeling and altered hemodynamics, other factors could play a role in CF-relate PH pathogenesis. These include chronic inflammation and CFTR dysfunction. Giacchi and colleagues previously demonstrated correlations between PA pressures measured by echocardiography and erythrocyte sedimentation rate and c-reactive protein, suggesting that chronic inflammation may precede pulmonary vascular disease . CFTR coEfforts to define clinical characteristics associated with survival in patients with CF have been unsuccessful, with no single factor independently predictive of poor outcomes , 33. AltThe main limitations of our study are its retrospective nature, single center design, limited number of RHC for the cohort, and small sample size. Despite the small sample size, however, the relationships between PA:A and mPAP remained significant when controlling for other variables, emphasizing the strong role an enlarged PA:A plays in predicting PH. Another limitation is that our cohort was comprised of patients with severe obstructive lung disease as evidence by low FEV1 and high LTOT use, and we cannot therefore generalize our results to adults with mild or moderate CF. However, we have previously shown that PA enlargement is highly prevalent in CF with less severe lung disease . PA:A maWe demonstrated that PA:A is a widely-available, reproducible, non-invasive tool that can be used as an independent predictor of invasive hemodynamics and PH in CF patients undergoing lung transplant evaluation. The presence of PA enlargement is associated with reduced survival in this population. These findings suggest a potential role for PA:A as a tool for detection of PH and timely referral for transplant evaluation. These findings should be confirmed in future, multi-center studies to further assess the utility of PA enlargement as a screening tool for PH in CF.S1 FileFig A. Bland-Altman plot for PA:A reproducibility. Bland-Altman plots for intra-observer variability using two measurements from the same reader (Left Panel) and inter-observer variability using measurements from two independent readers (Right Panel).Table A. Gene mutation frequency in the CF cohort undergoing lung transplant evaluation.Table B. Multivariable Cox Proportional Hazards Model Results.(DOCX)Click here for additional data file.


In recent years, the efficacy of electroacupuncture (EA) pretreatment generating ischemic tolerance mimicking ischemic pretreatment (IP) has been continuously confirmed, which was first found in the brain and then in the heart. Furthermore, researchers have observed the intensive cardioprotection impact of EA pretreatment on patients undergoing percutaneous coronary intervention (PCI) and heart valve replacement, indicating that EA pretreatment tends to be a valuable and advantageous avenue for preventing acute myocardial ischemia/reperfusion (I/R) injury or treatment of ischemic heart disease (IHD). In reality, the heart protection mechanism of EA pretreatment is robust and pleiotropic, of which the regulatory molecular pathways are involved in multichannel, multilevel, and multitarget, including energy metabolism, inflammatory response, calcium overload, oxidative stress, autophagy, and apoptosis. Through a growing number of clinical tests and basic experiments with animal models, researchers progressively explored the optimal acupoints and parameters, where EA pretreatment induced acute and delayed ischemic tolerance for myocardial protection. Thereby, this article aims to collect the relevant evidence on EA pretreatment against myocardial ischemia/reperfusion injury (MIRI) and summarize the mechanism of cardioprotection of EA pretreatment to provide ideas and methods for further clinical applications. According to statistics, more than 8.5 million people throughout the world die from acute myocardial infarction (MI) each year , 2, of wIn numerous accumulating animal experiments and clinical trials, the phenomenon of ischemic tolerance induced by a series of pretreatment methods has inspired us with new ideas; that is, transient exposure to sublethal or nondestructive stimuli prior to the onset of illness can increase the tolerance of tissues or organs to the subsequent permanent and lethal injury. These pretreatment methods include ischemia , drug, hypoxia, and exercise, of which ischemic pretreatment (IP) has been confirmed to be one of the most effective endogenous protective mechanisms. However, in the view of applicability, the limitations and negative side effects of all the above pretreatment methods cannot be ignored, especially for weak or severely ill patients, which restricts the further clinical application.Electroacupuncture (EA) is a novel avenue improved on traditional acupuncture therapy combined with modern electric techniques, which reflects the ideas of “treating before sick” in traditional Chinese medicine (TCM) and is well accepted as a complementary therapy throughout several Asian countries –11. FurtSo far, the efficacy of EA pretreatment at Neiguan (PC6) inducing ischemic tolerance and protecting the injured myocardium has been continuously demonstrated . Since EWith the continuous development of medicine, modern medical needs are not only satisfied with the treatment of diseases but also require more active prevention. The definition of pretreatment was first proposed in the mid-1980s; Murry and colleagues observed the phenomenon of ischemic tolerance induced by IP in myocardial ischemia/reperfusion model of dogs; after several times of short-term repeated I/R treatment, myocardium enhanced the resistance to subsequent lethal MIRI stimulation, and myocardial damage caused by I/R such as necrosis, arrhythmia, myocardial systolic, and diastolic dysfunction was alleviated , which pAcupuncture therapy is based on “treating before sick” idea, one of the primary thinking in oriental medicine, which refers to piercing fine needle into the specific acupoints of the human body according to the meridian theory, used for the prevention, health care, and treatment of various diseases. In addition, a growing number of subhealth or sick people in Western society engaged in some form of complementary and alternative medicine (CAM) therapy, in which acupuncture therapy was the most frequently adopted. Moreover, acupuncture pretreatment consists of hand acupuncture pretreatment, moxibustion pretreatment, and EA pretreatment, and its great significance for the prevention and treatment of ischemic myocardial damage, such as arrhythmia, angina, or even sudden cardiac death in experimental studies and clinical tests has been verified –22.EA is a combination treatment of conventional acupuncture and electric current with certain parameters used to stimulate acupoints and tissues instead of manual operation. In 2003, the concept of “EA pretreatment” was first proposed, suggesting that EA stimulus manipulated in advance inherited the subsequent ischemic brain injury in rats with middle cerebral artery occlusion (MCAO) model . In the Although extensive experimental studies have proved the cardioprotective effect of EA pretreatment against MIRI, there is still doubt whether EA pretreatment can be used in clinical practice. Up till now, substantial evidence has shown the protective effect of acupuncture pretreatment for ischemic myocardium in accelerating the recovery of heart surgery, enhancing the left heart function, improving the symptoms of angina pectoris and palpitation, or reducing ischemia myocardial injury in patients , 25–27.In 2010, Yang and colleagues recruited 60 patients with acquired valvular heart disease requiring heart valve replacement, which were randomly assigned into the EA pretreatment group and the control group . Five daIn 2015, Wang and colleagues designed a 2-year follow-up randomized clinical trial and recruited 388 adult patients with stable angina, unstable angina, or asymptomatic myocardial ischemic to explore whether EA pretreatment played a role in cardioprotection for patients undergoing PCI . The plaThrough delivering stimulation at specific acupoints in the body, EA pretreatment enhances the afferent impulses and improves acupuncture outcome associated with the local and systemic regulation as a candidate for the prevention or rehabilitation of ischemic heart disease. To date, a large number of animal experiments have been performed to demonstrate that EA pretreatment improves the tolerance of myocardial cells to ischemia/reperfusion stimulation, combating myocardial ischemia/reperfusion injury.In terms of relevant evaluation parameters, the measurement of myocardial infarction area, evaluation of arrhythmia, and determination of biochemical markers in serum related to myocardial injury were often used to measure the effectiveness of electroacupuncture pretreatment against MIRI. 2+]i: free calcium concentration; Cx43: gap junction protein 43; SOD: superoxide dismutase; MDA: malondialdehyde; Nrf2: nuclear factor erythroid-2-related factor 2; HO-1: heme oxygenase-1; NO: nitric oxide; NOS: nitric oxide synthase.The effect evaluation parameters of EA pretreatment against MIRI in animal experiments are summarized. EA: electroacupuncture; MIRI: myocardial ischemia/reperfusion injury; LADCA: left anterior descending coronary artery; ECG: electrocardiogram; i: free calcium concentration; Cx43: gap junction protein 43; Nrf 2-ARE: NF-E2-related factor 2 antioxidant response element; ET: endothelia; CK: creatine kinase; HSP: heat shock protein.The electrical stimulation parameters, acupoints, and potential protective mechanisms of EA pretreatment against MIRI in animal experiments are summarized. EA: electroacupuncture; MIRI: myocardial ischemia/reperfusion injury; ESP: electrical stimulation parameters; ECM: extracellular matrix; MAPK: mitogen-activated protein kinase; SGIR: simulative global ischemia reperfusion; [CaAt present, the molecular mechanisms involved in MIRI include the adhesion of neutrophils and vascular endothelial cells, production of oxygen free radicals, calcium overload, abnormal energy metabolism, cardiomyocyte apoptosis, and vasospasm . In animOne leading reason inducing myocardial ischemia/reperfusion injury is the deficiency of myocardial energy synthesis in the ischemic area, of which the pathological basis includes dysfunction of ATP synthase F(0) complex subunit G1 (ATP5G1) . A largeκB) signal pathway in rats [Modern medical research proves that the inflammation reaction triggered by ischemia/reperfusion stimulation may cause secondary damage for ischemia tissues or organs , 38. One in rats .2+ in cardiomyocytes significantly influences the systolic function of the heart. Once the myocardial cell ischemia events occur, ATP supplying cell metabolism demand will be obviously insufficient, and Ca2+ transport in the endoplasmic reticulum can be abnormal, which contributes to calcium overload of cardiomyocytes and aggravates the myocardial cell injury [The dynamic balance of Cal injury , 43. Durl injury .When myocardial ischemia/reperfusion injury event occurs, oxidative stress will cause a large number of oxygen free radicals to be released, which is considered to be an important factor for the disruption of heart function, as well as the main factor leading to myocarditis and extracellular remodeling , 45. EviCardiomyocytes show strong autophagy characteristics and autophagy plays an important role in myocardial ischemia/reperfusion injury , 48. AutX receptor (FXR)/small heterodimer partner (SHP) cell apoptosis signal pathway and related gene expression [It has been demonstrated that apoptosis is the ultimate ending caused by myocardial ischemia/reperfusion injury, initiated at the time of myocardial ischemia beginning, amplified, and involved in the whole myocardial death during reperfusion . It was pression . Zhong apression .β1-adrenoceptor protein [It was reported that EA at Neiguan (PC6) alleviated the further injury of damaged myocardium after ischemia/reperfusion, and the mechanism was relative to the inhibition of cardiac sympathetic activation with protein kinase C (PKC) pathway, noradrenaline release, and the activity of cardiac sympathetic nerve partially , 63. In  protein . In anot protein . In addi protein , 65, 66.Acupuncture, one of the classical therapies in Chinese medicine throughout history, has been used to prevent and treat diseases for thousands of years. Currently, the existing evidence has fully demonstrated that EA pretreatment can alleviate ischemic myocardial injury and promote the recovery of heart function. Similar to other preconditioning procedures, EA pretreatment can induce ischemia tolerance against myocardial ischemia/reperfusion injury. However, different from other preconditioning procedures, the cardioprotection effect induced by EA pretreatment has to do with the specific electrical stimulation parameters and acupoint selected, of which multichannel, multilevel, and multitarget mechanisms are thought to be involved in the molecular mechanism.κB signal pathway and inhibit the overoccurrence of the inflammatory reaction, thereby combating the myocardial injury after I/R in rats. But, it is still not clear how EA treatment before ischemia brings about the change of inflammation-related factors after reperfusion. What occurs during the process of EA pretreatment still needs to be explained.In basic researches and clinical tests, the investigators have observed the intensive changes of biochemical, morphological, and gene targets in the myocardial cells after I/R injury, of which EA pretreatment is suggested to be the reason. Nevertheless, little work focuses on the reason and detailed information about the mechanisms of EA pretreatment has not yet fully understood. For example, EA pretreatment has been shown to regulate the TLR4/MyD88/NF-In addition, compared with other preconditioning procedures, EA pretreatment possesses some unmatched merits, such as low cost, safety, and easy operation, which is more easily accepted by patients in the clinic. For example, although IP is currently recognized as one of the most effective endogenous protective mechanisms, the protective effect is affected by factors such as age, hypertension, diabetes, and drugs (such as propofol). Moreover, acute cardiovascular events are usually unpredictable, and all these reasons limit the application of IP clinically. Similarly, there is still a gap between the potential benefits of EA and practical application in the prevention of myocardial ischemia/reperfusion injury. EA pretreatment has good repeatability and stability, of which the benefits are tightly associated with the determination of parameters and acupoints carried out. However, although Neiguan (PC6) acupoint is the first choice of selection in most studies currently, the parameters of EA pretreatment have not yet been unified completely, such as stimulation frequency, current intensity, pulse width, pulse interval, and intervention time. In the course of searching the literature, we found that in some experiments the specific scheme of EA pretreatment carried out was not full. Much more meticulous researches on EA pretreatment antagonizing myocardial ischemia/reperfusion injury need to be conducted for exploring the best parameters to achieve the best outcome. As for the possibility of clinical application of EA pretreatment in the future, it is necessary to develop preischemic markers indicating the effectiveness of EA pretreatment, with a hope for applying individual EA pretreatment according to genders, age, and pathological states such as hypertension. At last, we need more data for supporting the cardioprotective effect of EA pretreatment. Thus, large sample and multicenter randomized control trials need to be performed, which may provide more powerful evidence for making the cardioprotective mechanism of EA pretreatment and further clinical practice clear. Although there is still lots of work to be done, if we can successfully find satisfactory answers to the above questions, we can use EA pretreatment to turn the wish of patients with a high risk of acute myocardial ischemia/reperfusion injury to live a better life into reality.


Heart failure is a disease that increases the likelihood of morbidity and mortality with an increased direct and indirect cost to the healthcare system. The role of diet in the development, progression and treatment of heart failure is being studied with growing interest. The objective of this study is to assess the relationship between heart failure and selected heart failure risk factors: hyperlipidemia, hypertension, diabetes mellitus and plant-based diets as a clinical intervention. The search yielded 416 published articles, among them are three studies that used a plant-based diet as an intervention for heart failure. All interventional studies showed that plant-based diets have a positive impact on heart failure in terms of improved ejection fraction and positive cardiac muscle remodelling. Heart failure (HF) is a disease that increases the risk of morbidity and mortality alongside an increased direct and indirect costs to the healthcare system. The role of diet in the development, progression and treatment of heart failure is being studied with growing interest .The disease is defined by the heart’s inability to pump enough blood to provide the metabolic requirements of the body. It can present with preserved or reduced left ventricular ejection fraction . Risk faPlant‐based diets are a group of dietary choices that focus on increased intake of plant foods and reduce animal-based foods. Vegetarian diets, a type of plant‐based diet, emphasize the restriction of different types of animal-based food choices like meat, poultry, or fish, and have been associated with a reduced risk of cardiovascular risk factors, such as obesity, hypertension, diabetes mellitus, and ischemic heart disease ,6. EpideThe objective of this study is to assess the relationship between heart failure and plant-based diets as a clinical intervention. There is mounting evidence in the observational literature with respect to the health benefits of plant-based diets with limited emphasis on clinical interventions, this study focuses on clinical intervention as single or augmented therapy for heart failure. Moreover, this research focuses on summarizing the published evidence on the effects of plant-based diets on heart failure to provide a clearer understanding of the role nutrition can play in the treatment of the disease.This is a literature summary designed to qualitatively report relevant findings on the effects of a plant-based diet in heart failure patients or patients with selected risk factors of heart failure in clinical trials conducted from 2000 to March 2020. The objective of this study is to summarize the relevant interventional literature on plant-based diets and heart failure. All clinical trials published since 2000 until March 2020 were reviewed. Databases used for the search include PubMed, Medline, the Cochrane collaboration. Key words were: “Vegetarian”, “vegan”, “plant-based diet” and “heart failure”. The search yielded 416 articles. Limitations used were: Published after 2000, English language and clinical trials. Results were excluded if written before 2000, written in languages other than English and if the sample under study is cardiovascular disease other than heart failure. Studies that did not address the question directly were also excluded.The search yielded 416 published articles, among them, three studies used a plant-based diet as an intervention for heart failure. Findings are summarized in Table Pischke et al. in 2007 conducted an analysis on the effects of plant-based diets on ejection fraction in patients with heart disease. In 46 patients with left ventricular ejection fraction impairment, all patients were put on a plant-based diet for three months and were followed throughout. They found that all heart failure risk factors have improved during that period along with exercise tolerance. This finding shows that plant-based diets have a direct positive impact on clinical outcomes of heart failure in this sample group .In 2017, Choi et al. reported one heart failure case with ejection fraction of 35% and left ventricular systolic dysfunction. This patient was on plant-based diet for two months. The outcome of the intervention showed major changes in ejection fraction as it normalized to 50% and other heart failure risk factors improved significantly over that period .In 2019, Najjar and Montgomery reported three cases of heart failure patients who were put on plant-based diets for an average of 79 days. All patients showed a major improvement in heart failure risk factors and blood markers, and substantial changes in heart structure and function where ejection fraction improved by 92%, LV mass reduced by 21%, stroke-volume increased by 62% and overall, cardiac output increased by 17% in the patient group .Currently, the observational literature indicates that there might be a growing observed positive impact plant-based diets have on heart failure development and progression. Current research indicates that antioxidants, micronutrients and aversion to high fat nutritional choices are associated with reduction in heart failure incidence and morbidity. One potential explanation is the differing factors related to reduction of oxidative stress and inflammation . In termThis study has the following limitations: first, the small sample size of the case reports cannot be used to generalize the findings to all patients with heart failure and should be seen as a limited finding that points only to potential benefit. Larger studies are necessary to provide statistical power and more rigorous design to reduce the effects of potential confounders to a minimum. Second, plant-based diets incorporate diverse effective components such as varying micronutrients, different phytochemicals and antioxidants, along with reduction in saturated fat and dietary salt among other factors that have the potential to improve symptoms of heart failure. This means that such interventional studies included in this review although promising cannot establish the precise cause of improvement in heart failure patients under study. Finally, causation cannot be determined from the body of the literature whether observational or interventional due to the lack of rigorous interventional design with a sample size enough to establish statistical significance. Thus, a larger study with interventional design is needed.Plant-based diets show promising results in heart failure patients and should be viewed as an important adjunct to standard treatment. A number of small studies in the past two decades show a consistent positive clinical and risk factor improvements in patients with heart failure. These findings, although in small samples, can lead the way for more interventional studies with more rigorous design to shed more light on the effects of plant-based diet on heart failure as a clinical intervention.


Alternative methods of delivering cardiac resynchronization therapy (CRT), including left ventricular (LV) endocardial pacing, His bundle pacing, and left bundle branch pacing, have been developed in an effort to improve CRT. Noninvasive cardiac mapping using electrocardiographic imaging (ECGi) is a method of assessing ventricular activation and has been used in the CRT population to predict and assess response, guide LV lead placement, and optimize therapy.A 62-year-old man underwent an electrophysiology and ECGi study 21 weeks after the implantation of a St Jude Quadra Assura MP CRT-D system . This was implanted following a new diagnosis of heart failure during a hospital admission for breathlessness and syncope. Admission electrocardiogram showed sinus rhythm with left bundle branch block (LBBB) and a QRS duration of 170 ms. Transthoracic echocardiogram showed a dilated left ventricle with severely impaired systolic function (ejection fraction 25%), normal right ventricular (RV) function, and mild mitral regurgitation. He underwent a coronary angiogram, which showed unobstructed coronary arteries. He underwent cardiac magnetic resonance imaging, which confirmed the diagnosis of nonischemic cardiomyopathy with no evidence of scar on late gadolinium enhancement. The patient had a background of treated prostate cancer, hypertension, and hypercholesterolemia.ClinialTrials.gov identifier NCT04322877). The patient provided written consent for the study, which was conducted in accordance with the Declaration of Helsinki and approved by the local ethics committee. The patient was fitted with a 252-electrode CardioInsight Sensor Array Vest  before the procedure and underwent a low-dose computed tomography scan to obtain electrode and cardiac positions, as previously described.The electrophysiology and ECGi study were performed as part of a mechanistic cohort study ((1)Global right/left ventricular electrical synchrony (VVsync): the difference between the mean RV activation time and the mean LV activation time.(2)Global biventricular total activation time (VVtat): a measure of the total time required for both ventricles to activate.(3)Global left ventricular total activation time (LVtat): a measure of the total time required for the left ventricle to activate.(4)Global left ventricular dispersion time (LVdisp): the standard deviation of LV activation times; a measure of dyssynchrony within the left ventricle.Activation maps were created using the ECSYNC CardioInsight software  and the following activation parameters were calculated:,,These measures have been previously validated in patients undergoing ECGi assessment during CRT.The activation maps and activation parameters are displayed in As expected, intrinsic (LBBB) conduction showed dyssynchronous late activation of the left ventricle compared to the right ventricle, demonstrated by a very negative VVsync. All methods of CRT improved right/left ventricular synchrony, with VVsync having values closer to zero. In addition, all methods of CRT improved biventricular total activation (VVtat) and left ventricular activation (LVtat) times as well as dyssynchrony within the left ventricle (LVdisp). Interestingly, in this patient the improvement in VVtat, LVtat, and LVdisp was greater with endocardial CRT, His bundle pacing, and left bundle branch pacing, compared to conventional CRT.This case demonstrates the feasibility and utility of ECGi to compare different methods of delivering CRT. Although there has been great interest in alternatives to conventional CRT, to our knowledge this is the first report of the electrical effects of conventional CRT, endocardial CRT, His bundle pacing, and left bundle branch pacing in the same patient. In this patient, the alternative methods of CRT delivery seemed to result in superior ventricular resynchronization compared to conventional CRT. However, the cohort of patients in whom CRT is indicated is a heterogenous group and it is unlikely that a “one-size-fits-all” method of CRT delivery will be optimal in all patients. Differences in cardiac anatomy, scar, and conduction block within the His-Purkinje system are likely to affect response to different methods of CRT delivery. An electrophysiology study with ECGi assessment may therefore be a useful endeavor to find the optimal means of CRT delivery prior to implantation.Endocardial pacing results in more physiological activation of the myocardium and mechanistic studies have suggested an improvement in hemodynamics and activation times over conventional CRT.11,Biventricular pacing results in delivery of 2 nonphysiological wavefronts, which merge to activate the myocardium. Recruitment of the His-Purkinje system to activate the ventricles is an attractive concept to achieve synchrony in the heart failure population. It has been theorized that pacing the bundle of His with sufficient output can reverse bundle branch block. Observational studies have demonstrated the feasibility and effectiveness of His bundle pacing in delivering CRT to patients with heart failure, though thresholds required for capture and bundle-branch correction are high.•Electrocardiographic imaging (ECGi) is a noninvasive method for mapping electrical activation of the heart.•Endocardial pacing, His bundle pacing, and left bundle branch pacing are alternative methods of delivery of cardiac resynchronization therapy (CRT) and demonstrate different activation patterns using ECGi.•Comparison of alternative methods of CRT using an electrophysiology study and ECGi is a potential technique to tailor pacing strategy in patients who do not respond to conventional CRT.To our knowledge, this is the first report of a direct comparison of conventional CRT, endocardial CRT, His bundle pacing, and left bundle branch pacing in the same patient. Randomized controlled trials are required to assess the superiority of these newer techniques over conventional CRT. Given the heterogeneous cohort of patients who are indicated for CRT, it is unlikely that a single technique will be optimal in every patient. An electrophysiology study with ECGi is a potential method of assessment to help tailor therapy to individual patients.


The migration routes of wide-ranging species can be difficult to study, particularly at sea. In the western South Pacific, migratory routes of humpback whales between breeding and feeding areas are unclear. Male humpback whales sing a population-specific song, which can be used to match singers on migration to a breeding population. To investigate migratory routes and breeding area connections, passive acoustic recorders were deployed in the central New Zealand migratory corridor (2016); recorded humpback whale song was compared to song from the closest breeding populations of East Australia and New Caledonia (2015–2017). Singing northbound whales migrated past New Zealand from June to August via the east coast of the South Island and Cook Strait. Few song detections were made along the east coast of the North Island. New Zealand song matched New Caledonia song, suggesting a migratory destination, but connectivity to East Australia could not be ruled out. Two song types were present in New Zealand, illustrating the potential for easterly song transmission from East Australia to New Caledonia in this shared migratory corridor. This study enhances our understanding of western South Pacific humpback whale breeding population connectivity, and provides novel insights into the dynamic transmission of song culture. Files containing humpback song were recorded between 8 June and 9 August 2016 . It was During September and October 2016, when southbound migration might have been expected ,30, PAMG3.2.In total, 16 themes were present in the analysed songs, of which five themes contained multiple phrase types . In the New Zealand data, five themes (1–5) were included in all transcribed songs from STB . These five themes were also present in song recorded in Cook Strait on 10 and 22 July 2016 . The Ea3.3.3.3.1.a). Overall, clustering of song was similar when all variability among strings was considered  as when only median strings were compared (a). The ‘A’ song type from New Caledonia 2015 clustered separately to all other songs (a). The southbound song from East Australia 2015 (Z) clustered with the New Zealand 2016 song from the STB and Cook Strait (excepting the song from 9 August (Z)) (a). The 9 August 2016 (Z) song recorded in Cook Strait, New Zealand, matched most strongly with the northbound song from East Australia 2015 (X and Y), and these three song types were contained in a wider cluster that included the New Caledonia 2016 song (a). At a broader level, this cluster also featured the New Caledonia ‘B’ song type from 2015 (a). East Australia southbound 2016, East Australia northbound 2017 and New Caledonia 2017 clustered together (a), and these were all very similar songs sharing five themes ) a. The 9 016 song a. At a brom 2015 a. East Atogether a, and th3.3.2.b). Again, the ‘A’ song from New Caledonia 2015 clustered separately. The New Zealand 2016 song (except Cook Strait on 9 August (CS_Z)) clustered most strongly with itself, the southbound East Australian 2015 song (Z) and the 2016 New Caledonia song. This cluster was part of a wider group that included the Cook Strait 9 August song (CS_Z), the northbound East Australia 2015 song (X and Y) and the New Caledonia ‘B’ song from 2015. The 2017 song from New Caledonia and northbound East Australia clustered together with the 2016 southbound song from East Australia.When the DSI matrix was hierarchically clustered and bootstrapped, results were very similar to the LSI analysis b. Again,4.Humpback whales on their northbound migration to western South Pacific breeding grounds passed through central New Zealand during June, July and early August 2016, as demonstrated by song presence. The recorded whales migrated along the east coast of the South Island of New Zealand, and continued north through Cook Strait and into the STB, or along the east coast of the North Island, although the latter route was supported by fewer song recordings. Similarities in song themes suggested that these whales most likely continued onto the breeding ground of New Caledonia, although connectivity with the breeding ground of East Australia could not be ruled out due to a data gap for northbound East Australia 2016 song. Song similarities between 2015 and 2016, and 2016 and 2017, indicated that song was most likely transmitted from East Australia to New Caledonia along shared migration routes and/or on shared feeding grounds during the austral summer. Given the occurrence of two song types in the recordings from central New Zealand, there is strong potential for song transmission to occur within this migratory corridor.4.1.Northbound humpback whales have been observed in New Zealand waters between May and August, the austral winter ,30. HistThe location of the acoustic recorders in this study covered known historic and modern humpback whale migration paths through central New Zealand. The data suggest that the singing whales that passed the Kaikōura recorder on the east coast of the South Island during their northbound migration primarily travelled through Cook Strait and into the STB, rather than continuing along the east coast of the North Island, past the Wairarapa recording location. This conclusion is based on the low numbers of song detections made at Wairarapa. However, we cannot reject the hypothesis that whales detected at Kaikōura continued along the east coast of the North Island; these animals may have ceased singing, or travelled further offshore to an area that was outside of the detection range of the Wairarapa recorder. The song recorded in Cook Strait on 9 August 2016 was different to the song recorded at the same location earlier in the migratory period, demonstrating consistent use of Cook Strait by mature male whales, despite differences in song content. Song produced by humpback whales passing offshore, or to the west of New Zealand, outside of the STB, would not have been recorded in this study, and presents an area for future research.et al. [Humpback song can function as a proxy for the presence of mature male whales, but Dawbin  reportedet al.  hypotheset al. .There were no detections of song from humpback whales during the expected period of southbound migration (September–December), although acoustic contributions from seismic surveys meant that it was not possible to determine the absence of humpback whale song with certainty. However, humpback whale presence would not be expected in high quantities in the vicinity of the acoustic recorders during the southbound migration, as satellite tag studies have shown little evidence of southward travel near central New Zealand ,22,32. W4.2.Song recorded in central New Zealand during the northbound migration in 2016 matched song recorded on the New Caledonia breeding ground in 2016, and was dissimilar to song recorded in East Australia in 2016. The data, therefore, suggest that the singing humpback whales that passed through central New Zealand were on a migratory path towards New Caledonia. New Caledonian song is commonly similar to song heard at the breeding ground in Tonga within a given year ,62, and That said, sample sizes in this study are small and no song data were available from the northbound migration in East Australia in 2016 to compare with song recorded in New Zealand in 2016. In 2015, song evolved in East Australia during the breeding season, and the same could have occurred during 2016, meaning that the 2016 East Australia song presented here, from the southbound migration, would have been dissimilar to the northbound 2016 East Australia song and potentially the same as the song recorded in central New Zealand. The New Zealand migratory corridor has been connected to the East Australian breeding ground in recent times via song matching  and otheThe results presented here support the hypothesis that the singing humpback whales recorded during the northbound migration through central New Zealand travelled on to the breeding grounds of western Oceania, but in acknowledging the caveats of the data outlined above, there is also a possibility that some whales travelled to the breeding ground of East Australia. Whales that traversed other migration routes past New Zealand, outside of the recording area, or those that travelled through the recording area without detection, could have been travelling to different breeding areas.4.3.In order for song to be transmitted between whales, individuals must be within the limits of acoustic contact, which has been estimated to be approximately 20 km . This coet al. [In 2015, the ‘B’ song from New Caledonia was the same song produced by northbound East Australian whales. The ‘A’ song that was initially present in New Caledonia in 2015 was mostly replaced by the ‘B’ song . The Basensu ,32. Whalsensu ,19.A shared, or partially shared, southbound migratory route exists along the east coast of Australia as recently highlighted by a few New Caledonian whales that crossed the Coral Sea and migrated south with the East Australian migratory cohort ,67. ThisThe possible transmission points discussed above may also underpin the transmission of East Australian 2016 song to whales in New Caledonia for the 2017 breeding season. The data presented here provide strong support for song transmission during northbound migration. While this study does not provide explicit evidence for song transmission within the New Zealand migratory corridor, the presence of two song types illustrated a potential location for song sharing to occur. The recorded songs provided further evidence of an eastward transmission of song across the South Pacific over consecutive years, as has been widely reported ,39,62. D5.Autonomous passive acoustic instruments deployed in the marine environment of central New Zealand recorded singing humpback whales on their northward migration to western South Pacific breeding grounds from June until early August. Song detections revealed that the predominant migration route from the east coast of the South Island was through Cook Strait, rather than along the east coast of the North Island. The recorded song implied the most likely breeding ground destination of these whales was New Caledonia, although connectivity to East Australia could not be ruled out. Song themes from whales on their southbound migration past East Australia in 2015 were heard in New Caledonia in 2016, implying that song transmission occurred on summer feeding grounds or on a shared migratory route between the breeding seasons of 2015 and 2016. The presence of two song types in New Zealand recordings in 2016 demonstrated a potential for song sharing to occur in this migratory corridor. The results presented here greatly enhance our knowledge of humpback whale movements through central New Zealand, and provide information about song transmission and migration routes of western South Pacific humpback whales. While many questions remain unanswered, this study demonstrates the applicability of passive acoustic monitoring to provide a non-invasive, cost-effective methodology to study western South Pacific humpback whales, of which, some populations are still recovering from the effects of commercial whaling.


Atrioventricular plane displacement (AVPD) reflects longitudinal left ventricular (LV) systolic function, and wall thickening (WT) regional radial LV function. The temporal evolution of these measures after STEMI with CMR has not been evaluated. We aimed to investigate how AVPD and WT are affected globally and regionally from the sub-acute to the chronic phase after ST-elevation myocardial infarction (STEMI).n = 20) and patients from the CHILL-MI study (NCT01379261) prospectively underwent magnetic resonance imaging (MRI) 2–6 days and 6 months after STEMI (n = 77). CHILL-MI randomized STEMI-patients to cooling therapy initiated before reperfusion or standard of care. AVPD was measured at six points in three long axis cine images and wall thickening in short axis cine images. Infarction was quantified using late gadolinium enhancement (LGE) and used to define infarct and remote segments.Healthy volunteers without cardiovascular disease and medication  or WT (p = 0.85 and p = 0.99) between patients randomized to cooling therapy and standard of care. Therefore, the results are presented for the pooled cohort. Global AVPD was decreased in both the sub-acute  and the chronic phase  compared to controls (15 ± 2 mm) with a partial recovery of AVPD (p < 0.001) in the chronic phase. Patients with left anterior descending (LAD) and right coronary artery (RCA) infarcts had decreased AVPD in the chronic phase in both infarcted and remote segments. Mean WT was decreased in patients with LAD infarction both in the sub-acute and the chronic phase in both infarcted and remote segments. The decrease in WT in patients with RCA and left circumflex (LCx) infarcts was more affected in the infarcted segments, especially in the chronic phase.There were no difference in AVPD either at acute or chronic phase . The decrease in WT in remote myocardium even in the chronic phase needs to be taken into consideration when combining functional measurements with infarct quantification for diagnosis of post-ischemic stunning and hibernation. Myocardial infarction (MI) is a major cause of heart failure as well as death . InfarctWe have previously shown that AVPD is decreased in all LV segments within the first week of STEMI, even in segments remote from the infarcted area . This suRegional radial LV function is considered to be reduced mainly in infarcted regions after MI –18. HoweHowever, the evolution of LV longitudinal and radial function in patients after STEMI has not been fully explored with CMR. Thus, we aimed to investigate the evolution of longitudinal LV function, measured as AVPD, and radial function, measured as WT, globally and regionally from the sub-acute (2–6 days) to the chronic phase (6 months) after STEMI.NCT01379261) [sub-acute phase) and had follow-up CMR imaging after 6 months (chronic phase). Twenty healthy, age-matched controls from a previously published study [Patients from the international multicenter cardioprotection study CHILL-MI (1379261)  were coned study  were incThe imaging protocol has been published previously . In shorhttp://segment.heiberg.se) [Image analysis was performed using Segment version 2.0 (berg.se)  as previberg.se) , 23. In 2) as previously described and validated [The atrioventricular (AV) plane was identified in the anterior, anteroseptal, inferoseptal, inferior, inferolateral, and anterolateral segments of the LV at ED and ES using long-axis steady-state-free precession images Fig. . Mean AValidated .Fig. 1DTo determine WT for each segment we used mid-ventricular short-axis images. Wall thickening is the percent change of LV segment thickness between ED and ES calculated as WT =100* / ED wall thickness. We chose mid-ventricular images to reduce the effect of longitudinal movement in the images. Infarct size (IS) was quantified in late gadolinium enhancement images after manual delineation of the endocardial and epicardial borders using a validated semi-automatic algorithm with manual corrections .Infarcted segments were defined from the AHA 17-segment model and presence of infarction in these segments was verified in all patients using mid-ventricular late gadolinium enhancement images. Absence of infarction in remote segments was also verified in these late gadolinium enhancement images. Thus, anterior and anteroseptal segments were considered infarcted in patients with LAD occlusion, inferoseptal and anterolateral segments adjacent, and inferior and inferolateral segments remote. In patients with RCA occlusion, inferoseptal and inferior segments were considered infarcted, anteroseptal and inferolateral adjacent, and anterior and anterolateral remote. In patients with LCx infarct, inferolateral and anterolateral segments were considered infarcted, inferior and anterior adjacent, and anteroseptal and inferoseptal remote.p-value < 0.05 were considered statistically significant.Statistical analysis was performed using Microsoft Excel 2010 and Prism 5.0 (GraphPad Software Inc.). Continuous variables are presented as mean ± SD. Paired Student’s t test was used to compare the sub-acute and chronic phase measurements. Un-paired t-test was used to compare patient and control measurements. Pearson’s correlation analysis was used for assessing the relationship between IS and EF. Results with a Seventy-seven patients from CHILL-MI underwent sub-acute and chronic CMR imaging. Late gadolinium enhancement images from 3 patients in the chronic phase were missing, and they were excluded from IS analysis. Characteristics of patients and healthy controls are presented in Table There was a larger proportion of men in the patient group than in the control group, but ages were similar. The slightly increased heart rate seen in the sub-acute phase compared to controls was normalized in the chronic phase. End-diastolic volume was larger in the chronic phase than in the sub-acute phase showing post-MI remodeling. End-diastolic volume was also larger than in controls. Stroke volume increased from the sub-acute to the chronic phase to the same level as controls. The increased LV mass seen in the sub-acute phase decreased in the chronic phase and then did not differ from controls. Infarct size decreased from the sub-acute to the chronic phase for the entire patient population Table , and wheEF increased in patients from the sub-acute to the chronic phase but remained decreased compared to controls except in patients with LCx infarction. The negative correlation between infarct size and EF in the sub-acute phase (r = − 0.50) Fig.  was sligp = 0.9) nor in the chronic phase (p = 0.4). Mean AVPD was decreased in patients compared to controls both in the sub-acute and the chronic phases, and there was a partial recovery from the sub-acute to the chronic phase  and therefore had lower statistical power which may explain why these patients had decreased AVPD in fewer segments.Figure p < 0.05) and chronic phases  compared to controls  to stroke volume was decreased in patients in the sub-acute .There was no difference in WT in the patient group receiving cooling therapy compared to the group that did not . The decrease in WT in remote myocardium even in the chronic phase needs to be taken into consideration when combining functional measurements with infarct quantification for diagnosis of post-ischemic stunning and hibernation.


We used automated image analysis software to determine the proportion of collagen, fat, and myocytes across six histological regions of normal ventricular myocardium.Twenty-nine non-cardiac death cases referred to our national cardiac pathology center were included in this study. Whole hearts were macroscopically and microscopically normal following expert histopathological evaluation. Tissue sections from the right ventricular outflow tract, right ventricle (RV), anterior interventricular septum (IVS), posterior IVS, anterior left ventricle (LV), and posterior LV were stained with Picrosirius red for collagen and scanned using a high-resolution slide scanner. Quantification of collagen, fat, and myocyte proportions was performed using Visiopharm software after automated exclusion of perivascular collagen.The majority of decedents were male  with a mean age at death of 32.1 ± 9.9 (range 18-54) and mean BMI 28.7 ± 7.3. We report predicted values  for cardiac tissue composition within the RV, IVS, and LV . The proportion of collagen and fat were higher in the RV compared with the LV  and RV compared with the IVS . The ratio of epicardial versus endocardial fat was increased in both ventricles . In multivariable analysis, there was no significant association between collagen or fat proportion and sex , age at death , or BMI .Our findings provide location and sex-specific proportions of myocardial histological tissue composition that may aid quantitative evaluation of pathology in future studies. Excessive accumulation of collagen within the extracellular matrix, the hallmark of myocardial fibrosis, results in pathological structural remodeling of the heart muscle. Recently, histomorphometric analysis has been employed in the quantification of tissue components and fibrosis in arrhythmogenic cardiomyopathy 2We identified 29 noncardiac death cases with retained cardiac tissue referred to our national cardiac pathology center. Ethical approval for this study was obtained from the UK National Health Service Research Ethics Committee (ref. 10/H0724/38) and conformed to the principles outlined in the Declaration of Helsinki. Informed consent was provided by the next of kin at the time of referral. Demographics, clinical data, toxicological, and pathological findings were entered prospectively into a database. Whole hearts were macroscopically and microscopically normal following examination by expert cardiac pathologists. Tissue sections (5-μm thickness) were sampled at the mid-ventricular level or from the anterior aspect of the right ventricular outflow tract. In total, 6 regions were evaluated: right ventricular outflow tract; right ventricle (RV); anterior interventricular septum (IVS); posterior IVS; anterior left ventricle (LV); and posterior LV. The RV section included 3 sampled locations from the right anterior, lateral, and posterior walls. Right ventricular tissue areas were combined for the purpose of morphometric quantification. Sections were stained with Picrosirius Red (PSR) for collagen  and slid32, 243.0 ± 80.4 mm2, and 240.9 ± 65.3 mm2, respectively. P= .08) or fat (P = .06) proportion according to sex; predicted values for males (n = 25) and females (n = 4) are shown in The majority of decedents were male . The mean age at death was 32.1 ± 9.9 (range 18–54) and mean BMI 28.7 ± 7.3. Most sudden deaths were due to opioid toxicity  or noncardiac trauma . The mean heart weight was 359.6 ± 67.1 g. The mean sampled tissue areas for the RV, IVS, and LV were 192.8 ± 45.2 mmP< .001), RV compared with the IVS , and RV epicardium compared with endocardium . There was insufficient evidence to support differences in collagen proportion between the IVS and LV (P= .06). The LV epicardium and endocardium contained similar proportions of collagen (P= .34). Fatty tissue was increased in the RV compared with the LV  and RV compared with the IVS . The ratio of epicardial versus endocardial fat was increased in both ventricles . In multivariable analysis, there was no significant association between collagen or fat proportion and sex , age at death , or BMI .The proportion of collagen was higher in the RV compared with the LV . This is in line with earlier observations reporting higher concentrations of collagen within the right heart Overall, our study highlights the spectrum of normal variation of collagen and fat content within the RV, in contrast to the pathological changes seen in diseases such as arrhythmogenic cardiomyopathy 4This is the first study to report regional proportions of cardiac tissue composition in normal human ventricular myocardium. Previous work has demonstrated the validity of automated methods of histological image quantification in the assessment of myocardial fibrosis C.M., J.W., P.B., and A.J. were involved in the conception, design, and drafting of the article. C.M., A.A., M.P., S.S., E.R.B., and M.N.S. were involved in the interpretation of data and critical revision of content. I.C.S. supervised all the statistical aspects of this study including design; analyses were conducted by C.M. All authors have read and approved the final article.British Heart Foundation . Cardiac Risk in the Young UK (CRY) provides funding to support the CRY Centre for Cardiac Pathology.CM is funded by the None.


Hypoxia affects myocardial oxygen supply resulting in subclinical cardiac dysfunction in obstructive sleep apnea (OSA) patients, with cardiovascular complications being associated with increased oxidative burst (OB). The aims of our study were to assess left ventricular (LV) dynamic myocardial deformation and diastolic reserve at rest and upon exercise, along with OB determination in this patients subset.Conventional echocardiography, Doppler myocardial imaging and LV 2D speckle tracking echocardiography were performed in 55 OSA patients with preserved LV ejection fraction (EF) and 35 age and sex-comparable healthy controls. Peripheral OB levels were evaluated by flow cytometry.P <  0.001) and at peak exercise . Systolic pulmonary artery pressure (sPAP) and E/E′ ratios increase during effort were higher in OSA than in controls . The best correlate of E/E′ at peak stress was peak exertion capacity . OB was also increased in OSA patients (P = 0.001) but, unlike OSA severity, was not associated with LV diastolic dysfunction.Despite comparable LVEF, LV global longitudinal strain (GLS) was significantly reduced in OSA at rest (− 13.4 ± 3.8 vs − 18.4 ± 3.3 in controls, Evaluation of diastolic function and myocardial deformation during exercise is feasible through stress echocardiography. OSA patients with preserved LVEF show subclinical LV systolic dysfunction, impaired LV systolic and diastolic reserve, reduced exercise tolerance, and increased peripheral levels of OB. Therapy aimed at increasing LV diastolic function reserve might improve the quality of life and exercise tolerability in OSA patients. Obstructive sleep apnea (OSA) is characterized by repeated episodes of partial or complete upper airways collapse, resulting in apnea and hypopnea events, intermittent hypoxemia and frequent arousals . OSA is On this basis, we hypothesized that OSA patients with preserved LV ejection fraction (LVEF) would have dynamic abnormalities in LV myocardial deformation and/or increased dynamic diastolic stiffness. The aims of our study were to assess, by means of 2D speckle tracking echocardiography (2DSTE) and diastolic parameters, LV myocardial deformation and diastolic function indexes both at rest and during exercise, mainly focusing on the systolic and diastolic reserve. Reproducibility of 2DSTE measurements was also assessed. In addition, we evaluated peripheral levels of OB as we aimed to address any relationship with basal and dynamic heart function parameters.From October 2017 to May 2018, 55 consecutive patients affected by moderate-severe OSA, referred to our Respiratory Medicine Division, were enrolled. Patients with a clinical history of concomitant lung disease , coronary artery disease, valvular heart disease, congestive heart failure, arrhythmias, and pulmonary hypertension were excluded. All OSA patients were not being treated with continuous positive airway pressure (CPAP). Thirty-five age- and sex-comparable healthy subjects referred to our attention for a voluntary cardiovascular screening were enrolled as controls. The study was conducted in accordance with the amended Declaration of Helsinki. The local Ethics committee approved the study and all individuals gave written informed consent.2 ≤ 90% with a nadir of 85% during registration [2 < 90%.OSA diagnosis was performed with home overnight cardiorespiratory polygraphy using a VitalNight data acquisition and analysis system  according to reference guidelines 13]. Data were analysed by trained sleep physicians with more than 3-yr experience and OSA severity was graded with the apnea/hypopnea index (AHI) according to accepted criteria [. Data westration , 15. t90sb) were performed using a computer-assisted spirometer  according to international standards [Spirometry, lung volumes measurement and determination of the haemoglobin (Hb)-adjusted single-breath diffusing lung capacity of the carbon monoxide (DLCOtandards –18. The tandards . ArteriaRest echocardiography: LV ejection fraction (EF) and LV global longitudinal strain (GLS) were evaluated as systolic function indexes. LVEF was calculated using the Simpson biplane method, from the apical 4- and 2-chamber views. A biplane LVEF ≥52% for men and ≥ 54% for women were considered normal [2) + right atrial pressure (RAP). RAP was estimated by measuring the diameter of the inferior vena cava and its respiratory motion. Left atrial volume index (LAVI) was assessed through biplane area-length method, dividing the left atrial volume by the body surface area (BSA). Atrium acquisitions were made from the apical 4- and 2- chamber views. The presence of more than 2 between average E/e’ > 14, septal e’ < 7 cm/s or lateral e’ < 10 cm/s, TRV > 2.8 m/s (sPAP> 36 mmHg), and LAVI > 34 ml/m2, was considered expression of LV diastolic dysfunction in subjects with normal LVEF [Exercise stress echocardiography: Standard supine bicycle exercise stress echocardiography was performed with incremental steps of 25 W every 2 min [Standard trans-thoracic echocardiography, Doppler evaluation and strain analysis were performed at rest and at peak effort using market available equipment . All measurements were performed at our Cardiology Unit and independently assessed by two cardiologists expert in echocardiography, according to current recommendations –22. Restd normal . For strd normal , 21. Thery 2 min . Parametry 2 min .E.coli at 37 °C for 10 min. A sample without stimulus served as negative background control. Dihydrorhodamine (DHR) 123 was added for 10 min to allow the conversion to fluorescent rhodamine 123 upon reactive oxygen species (ROS) production. After erythrocytes were removed and washing, 200 μl of DNA staining solution was added for 10 min to exclude aggregation artifacts. Samples were acquired with a FACS CANTO flow cytometer . Analysis was performed with the FACS DIVA software.Oxidative burst of peripheral leukocytes was measured with the Phagoburst BURSTEST™ , according to the manufacturer’s instruction. Briefly, 100 μl of heparinized whole blood was incubated with opsonized t-test for paired and unpaired data was used to assess changes between groups.Statistical analyses were performed using a commercially available package . Variables are presented as mean ± standard deviation (SD). Two-tailed p <  0.05) associated with the explained variable by univariate analysis. Odds ratios (OR) were calculated using a logistic regression method, and Beta-coefficients were obtained by linear regression method. Values of p <  0.05 were considered significant for all analyses.Linear regression analyses and partial correlation test by Pearson’s method were used to assess univariate relations. The following variables were included into the analysis: clinical parameters , risk factors, co-morbidities); lung function, sleep-related and metabolic parameters; standard echocardiographic and 2DSTE parameters. To identify significant independent determinants of resting and dynamic LV diastolic function in OSA patients, their individual association with clinical, functional and echocardiographic variables was assessed by multivariate analysis, using a bidirectional stepwise regression. Variables included in our analysis were all the variables significantly (Receiver operating characteristic (ROC) curve analysis was performed to select optimal cut-off values of echocardiographic parameters. Reproducibility of GLS measurements was determined in all the subjects. Intra-observer variability and inter-observer variability were examined using the coefficient of variation (COV), defined as the ratio of the standard deviation (σ) to the mean (μ) (%), and by Bland-Altman analysis. COV, 95% confidence intervals (CIs), and percent errors were reported.Our study population was composed of 90 subjects, including 55 OSA patients and 35 healthy controls. Main demographic and clinical characteristics are summarized in Table LVEF was similar in OSA and control cohorts, and preserved in both Table . LV mass(Intra-observer variability: COV: LV GLS: 5.37 (ICC 0.73); Bland-Altman analysis: LV GLS . Inter-observer variability: COV: LV GLS: 7.22 (ICC 0.77); Bland-Altman analysis: LV GLS .Overall, we observed a good reproducibility of global LV strain measurements in our study population, similar to that reported for speckle-tracking-based strain measures performed in selected samples – including healthy young adults , 21. TheE/e’ ratio, sPAP and LAVI at rest were all significantly higher in patients affected by OSA as compared to healthy controls; moreover, both septal and lateral e’ velocities were significantly lower in the former Table , Fig. 2.E/e’ increase during exertion was significantly higher in patients affected by OSA when compared to controls; sPAP increase (difference between peak exertion and resting sPAP) also showed the same trend along with a three-fold decrease of oxygen saturation Table .p <  0.001) and oxygen saturation level  as compared to healthy controls , resting LV GLS > − 15%  and severe AHI  were independently associated with LV diastolic dysfunction at rest.Logistic regression analysis showed that arterial hypertension , to blood lactates at rest , to resting LV GLS , and to the AHI   . As TDI Previous studies show that the prevalence of LV diastolic dysfunction at rest among OSA patients varies from 23 to 56%, depending on the sample size and the method of diastolic function assessment –34. In o2 level. The E/e’ ratio at peak exercise also resulted significantly correlated to LV GLS, to AHI, which reflects OSA severity, and to blood lactates, which are suggestive of inadequate tissue perfusion. These findings can be explained by the mentioned pathological mechanisms responsible for myocardial remodelling and fibrosis. The latter provokes impairment in both systolic and diastolic function, which can worsen during exercise, leading to reduced functional capacity.To our knowledge this is the first study addressing the LV diastolic performance both at rest and at peak exercise in a selected population of OSA patients. In middle-aged healthy subjects, the E/e′ ratio does not change significantly with exercise because of proportional increases in both the mitral inflow and annular velocities –38. ConvOur study has some limitations. First, it is a single-centre study with a sample size too small to drive definitive general conclusions concerning subclinical LV functional impairment in OSA patients. Also, our findings cannot necessarily be extrapolated to patients with mild disease. Finally, despite we found no correlations between the diastolic impairment during exercise and patients age, OB, BMI, smoking and co-morbidities, we cannot definitely exclude their contribution. Indeed, while we excluded OSA patients with major heart and lung diseases, the prevalence of cardiovascular risk factors, such as smoke, obesity, dyslipidemia, diabetes, and hypertension, was still not negligible in our patient cohort. Further studies in larger populations will certainly help to address their impact by ad hoc sub-group analyses.In conclusion, evaluation of diastolic function and myocardial deformation during exercise is feasible through stress echocardiography and provides valuable information in predicting exercise capacity in a selected population of moderate-severe OSA patients with preserved LVEF. These patients show subclinical LV systolic dysfunction, impaired LV systolic and diastolic reserve, reduced exercise tolerance, and increased peripheral levels of OB. A CPAP treatment and a therapy aimed at increasing LV diastolic function reserve might improve the quality of life and exercise tolerability in this target population.