Development of a PFAS reaction library: identifying plausible transformation pathways in environmental and biological systems.

Perfluoroalkyl and polyfluoroalkyl substances (PFAS) are used in many consumer applications due to their stain repellency, surfactant properties, ability to form water-proof coatings and use in fire suppression. The production, application, transport, use and disposal of PFAS and PFAS-treated products have resulted in their wide-spread occurrence in environmental and biological systems. Concern over exposure to PFAS and their transformation products and metabolites has necessitated the development of tools to predict the transformation of PFAS in environmental systems and metabolism in biological systems. We have developed reaction libraries for predicting transformation products and metabolites in a variety of environmental and biological reaction systems. These reaction libraries are based on generalized reaction schemes that encode the process science of PFAS reported in the peer-reviewed literature. The PFAS reaction libraries will be executed through the Chemical Transformation Simulator, a web-based tool that is available to the public. These reaction libraries are intended for predicting the environmental transformation and metabolism of PFAS only.

Acute Consumption of Alcohol and Discrete Atrial Fibrillation Events.

BACKGROUND: Patients' self-reports suggest that acute alcohol consumption may trigger a discrete atrial fibrillation (AF) event. OBJECTIVE: To objectively ascertain whether alcohol consumption heightens risk for an AF episode. DESIGN: A prospective, case-crossover analysis. SETTING: Ambulatory persons in their natural environments. PARTICIPANTS: Consenting patients with paroxysmal AF. MEASUREMENTS: Participants were fitted with a continuous electrocardiogram (ECG) monitor and an ankle-worn transdermal ethanol sensor for 4 weeks. Real-time documentation of each alcoholic drink consumed was self-recorded using a button on the ECG recording device. Fingerstick blood tests for phosphatidylethanol (PEth) were used to corroborate ascertainments of drinking events. RESULTS: Of 100 participants (mean age, 64 years [SD, 15]; 79% male; 85% White), 56 had at least 1 episode of AF. Results of PEth testing correlated with the number of real-time recorded drinks and with events detected by the transdermal alcohol sensor. An AF episode was associated with 2-fold higher odds of 1 alcoholic drink (odds ratio [OR], 2.02 [95% CI, 1.38 to 3.17]) and greater than 3-fold higher odds of at least 2 drinks (OR, 3.58 [CI, 1.63 to 7.89]) in the preceding 4 hours. Episodes of AF were also associated with higher odds of peak blood alcohol concentration (OR, 1.38 [CI, 1.04 to 1.83] per 0.1% increase in blood alcohol concentration) and the total area under the curve of alcohol exposure (OR, 1.14 [CI, 1.06 to 1.22] per 4.7% increase in alcohol exposure) inferred from the transdermal ethanol sensor in the preceding 12 hours. LIMITATION: Confounding by other time-varying exposures that may accompany alcohol consumption cannot be excluded, and the findings from the current study of patients with AF consuming alcohol may not apply to the general population. CONCLUSION: Individual AF episodes were associated with higher odds of recent alcohol consumption, providing objective evidence that a modifiable behavior may influence the probability that a discrete AF event will occur. PRIMARY FUNDING SOURCE: National Institute on Alcohol Abuse and Alcoholism.

A Randomized, Double-Blind, Placebo-Controlled Trial of Intravenous Alcohol to Assess Changes in Atrial Electrophysiology.

OBJECTIVES: This study sought to identify acute changes in human atrial electrophysiology during alcohol exposure. BACKGROUND: The mechanism by which a discrete episode of atrial fibrillation (AF) occurs remains unknown. Alcohol appears to increase the risk for AF, providing an opportunity to study electrophysiologic effects that may render the heart prone to arrhythmia. METHODS: In this randomized, double-blinded, placebo-controlled trial, intravenous alcohol titrated to 0.08% blood alcohol concentration was compared with a volume and osmolarity-matched, masked, placebo in patients undergoing AF ablation procedures. Right, left, and pulmonary vein atrial effective refractory periods (AERPs) and conduction times were measured pre- and post-infusion. Isoproterenol infusions and burst atrial pacing were used to assess AF inducibility. RESULTS: Of 100 participants (50 in each group), placebo recipients were more likely to be diabetic (22% vs. 4%; p = 0.007) and to have undergone a prior AF ablation (36% vs. 22%; p = 0.005). Pulmonary vein AERPs decreased an average of 12 ms (95% confidence interval: 1 to 22 ms; p = 0.026) in the alcohol group, with no change in the placebo group (p = 0.98). Whereas no statistically significant differences in continuously assessed AERPs were observed, the proportion of AERP sites tested that decreased with alcohol (median: 0.5; interquartile range: 0.6 to 0.6) was larger than with placebo (median: 0.4; interquartile range: 0.2 to 0.6; p = 0.0043). No statistically significant differences in conduction times or in the proportion with inducible AF were observed. CONCLUSIONS: Acute exposure to alcohol reduces AERP, particularly in the pulmonary veins. These data demonstrate a direct mechanistic link between alcohol, a common lifestyle exposure, and immediate proarrhythmic effects in human atria. (How Alcohol Induces Atrial Tachyarrhythmias Study [HOLIDAY]; NCT01996943).

Referral to Cardiology Following Postpartum Cardiovascular Risk Screening at the Maternal Health Clinic in Kingston, Ontario.

BACKGROUND: Cardiovascular disease (CVD) is the leading cause of death globally among women, and certain pregnancy complications can be the earliest indicators of increased CVD risk. Nonetheless, there is no recommendation for follow-up of cardiovascular risk factors identified through postpartum screening programs. This study describes current referral practices and clinical course from the Maternal Health Clinic in Kingston, Ontario, for women deemed at high cardiovascular risk postpartum. METHODS: We investigated the cohort of women referred from the postpartum Maternal Health Clinic to cardiology for further assessment and management, specifically examining timing and recommended interventions to reduce CVD risk. RESULTS: Women referred to cardiology differed significantly from those not referred in history of hypertensive disorders of pregnancy (P < 0.05) and demonstrated a significantly worse CVD risk profile at 6 months postpartum (P < 0.0001). Life expectancy by the cardiometabolic model for women referred was 5 years shorter (P < 0.0001). Only half of the women referred to cardiology scheduled a visit; the median time to the scheduled appointment was 12 months. Of women seen by cardiology, 60% were deemed eligible for cardiac rehabilitation. CONCLUSIONS: Although women at highest risk for CVD are being identified and referred to cardiology, the existing system is not designed for this demographic. Too many women are missing their appointments or being seen beyond 1 year postpartum. To initiate lifestyle changes and/or therapeutic interventions, we suggest that CVD prevention programming begins within 1 year of delivery. Future studies should investigate the viability of traditional cardiac rehabilitation programs among this unique population.

Cardiovascular Risk Assessment and Follow-Up of Women After Hypertensive Disorders of Pregnancy:A Prospective Cohort Study.

OBJECTIVE: Hypertensive disorders of pregnancy (HDP) comprise an independent, sex-specific risk factor for cardiovascular disease (CVD) in women. This study examined the utility of CVD risk models proposed in the 2016 Canadian Cardiovascular Society (CCS) lipid guidelines to identify women requiring further screening or lipid treatment following HDP. METHODS: Using data collected from the postpartum Maternal Health Clinic (MHC) at Kingston General Hospital in Kingston, Ontario and the Preeclampsia New Emerging Team (PE-NET) cohort study, the study investigators used the models recommended by the CCS guidelines and the cardiometabolic model of life expectancy in each cohort to estimate CVD risk in women after HDP. (Canadian Task Force Classification II-2). RESULTS: Using the 10-Year Modified Framingham Risk Score, all women were classified by the 2016 CCS Guidelines as low risk, requiring no follow-up. The 30-Year and Lifetime Risk Scores resulted in significant reclassification of women at risk in the PE-NET control and HDP groups (P < 0.001 and P < 0.0001, respectively); 49.2% of women with HDP were classified as high risk, requiring follow-up, compared with 14.3% of control subjects. Using the cardiometabolic model, median life expectancy was significantly lower and expected onset of CVD was earlier in the HDP group compared with the control group (P < 0.0001). CONCLUSION: The 2016 CCS lipid guidelines' risk classification recommendations significantly underestimated lifelong CVD risk in the HDP group compared with the control group. Women with HDP also had a significant decrease in cardiometabolic life expectancy and an earlier predicted age at onset of CVD. Early primary prevention in this at-risk population may improve CVD outcomes and reduce the future burden on the health care system.

Passive Detection of Atrial Fibrillation Using a Commercially Available Smartwatch.

Importance: Atrial fibrillation (AF) affects 34 million people worldwide and is a leading cause of stroke. A readily accessible means to continuously monitor for AF could prevent large numbers of strokes and death. Objective: To develop and validate a deep neural network to detect AF using smartwatch data. Design, Setting, and Participants: In this multinational cardiovascular remote cohort study coordinated at the University of California, San Francisco, smartwatches were used to obtain heart rate and step count data for algorithm development. A total of 9750 participants enrolled in the Health eHeart Study and 51 patients undergoing cardioversion at the University of California, San Francisco, were enrolled between February 2016 and March 2017. A deep neural network was trained using a method called heuristic pretraining in which the network approximated representations of the R-R interval (ie, time between heartbeats) without manual labeling of training data. Validation was performed against the reference standard 12-lead electrocardiography (ECG) in a separate cohort of patients undergoing cardioversion. A second exploratory validation was performed using smartwatch data from ambulatory individuals against the reference standard of self-reported history of persistent AF. Data were analyzed from March 2017 to September 2017. Main Outcomes and Measures: The sensitivity, specificity, and receiver operating characteristic C statistic for the algorithm to detect AF were generated based on the reference standard of 12-lead ECG-diagnosed AF. Results: Of the 9750 participants enrolled in the remote cohort, including 347 participants with AF, 6143 (63.0%) were male, and the mean (SD) age was 42 (12) years. There were more than 139 million heart rate measurements on which the deep neural network was trained. The deep neural network exhibited a C statistic of 0.97 (95% CI, 0.94-1.00; P < .001) to detect AF against the reference standard 12-lead ECG-diagnosed AF in the external validation cohort of 51 patients undergoing cardioversion; sensitivity was 98.0% and specificity was 90.2%. In an exploratory analysis relying on self-report of persistent AF in ambulatory participants, the C statistic was 0.72 (95% CI, 0.64-0.78); sensitivity was 67.7% and specificity was 67.6%. Conclusions and Relevance: This proof-of-concept study found that smartwatch photoplethysmography coupled with a deep neural network can passively detect AF but with some loss of sensitivity and specificity against a criterion-standard ECG. Further studies will help identify the optimal role for smartwatch-guided rhythm assessment.

Smartphone-Based Geofencing to Ascertain Hospitalizations.

BACKGROUND: Ascertainment of hospitalizations is critical to assess quality of care and the effectiveness and adverse effects of various therapies. Smartphones, mobile geolocators that are ubiquitous, have not been leveraged to ascertain hospitalizations. Therefore, we evaluated the use of smartphone-based geofencing to track hospitalizations. METHODS AND RESULTS: Participants aged ≥18 years installed a mobile application programmed to geofence all hospitals using global positioning systems and cell phone tower triangulation and to trigger a smartphone-based questionnaire when located in a hospital for ≥4 hours. An in-person study included consecutive consenting patients scheduled for electrophysiology and cardiac catheterization procedures. A remote arm invited Health eHeart Study participants who consented and engaged with the study via the internet only. The accuracy of application-detected hospitalizations was confirmed by medical record review as the reference standard. Of 22 eligible in-person patients, 17 hospitalizations were detected (sensitivity 77%; 95% confidence interval, 55%-92%). The length of stay according to the application was positively correlated with the length of stay ascertained via the electronic medical record (r=0.53; P=0.03). In the remote arm, the application was downloaded by 3443 participants residing in all 50 US states; 243 hospital visits at 119 different hospitals were detected through the application. The positive predictive value for an application-reported hospitalization was 65% (95% confidence interval, 57%-72%). CONCLUSIONS: Mobile application-based ascertainment of hospitalizations can be achieved with modest accuracy. This first proof of concept may ultimately be applicable to geofencing other types of prespecified locations to facilitate healthcare research and patient care.

Brain Emboli After Left Ventricular Endocardial Ablation.

BACKGROUND: Catheter ablation for ventricular tachycardia and premature ventricular complexes (PVCs) is common. Catheter ablation of atrial fibrillation is associated with a risk of cerebral emboli attributed to cardioversions and numerous ablation lesions in the low-flow left atrium, but cerebral embolic risk in ventricular ablation has not been evaluated. METHODS: We enrolled 18 consecutive patients meeting study criteria scheduled for ventricular tachycardia or PVC ablation over a 9-month period. Patients undergoing left ventricular (LV) ablation were compared with a control group of those undergoing right ventricular ablation only. Patients were excluded if they had implantable cardioverter defibrillators or permanent pacemakers. Radiofrequency energy was used for ablation in all cases and heparin was administered with goal-activated clotting times of 300 to 400 seconds for all LV procedures. Pre- and postprocedural brain MRI was performed on each patient within a week of the ablation procedure. Embolic infarcts were defined as new foci of reduced diffusion and high signal intensity on fluid-attenuated inversion recovery brain MRI within a vascular distribution. RESULTS: The mean age was 58 years, half of the patients were men, half had a history of hypertension, and the majority had no known vascular disease or heart failure. LV ablation was performed in 12 patients (ventricular tachycardia, n=2; PVC, n=10) and right ventricular ablation was performed exclusively in 6 patients (ventricular tachycardia, n=1; PVC, n=5). Seven patients (58%) undergoing LV ablation experienced a total of 16 cerebral emboli, in comparison with zero patients undergoing right ventricular ablation (P=0.04). Seven of 11 patients (63%) undergoing a retrograde approach to the LV developed at least 1 new brain lesion. CONCLUSIONS: More than half of patients undergoing routine LV ablation procedures (predominately PVC ablations) experienced new brain emboli after the procedure. Future research is critical to understanding the long-term consequences of these lesions and to determining optimal strategies to avoid them.

The QT Interval as a Noninvasive Marker of Atrial Refractoriness.

BACKGROUND: Atrial refractoriness may be an important determinant of atrial fibrillation (AF) risk, but its measurement is not clinically accessible. Because the QT interval predicts incident AF and the atrium and ventricle share repolarizing ion currents, we investigated the association between an individual's QT interval and atrial effective refractory period (AERP). METHODS: In paroxysmal AF patients presenting for catheter ablation, the QT interval was measured from the surface 12-lead electrocardiogram. The AERP was defined as the longest S1-S2 coupling interval without atrial capture using a 600-ms drive cycle length. RESULTS: In 28 patients, there was a positive correlation between QTc and mean AERP. After multivariate adjustment, a 1-ms increase in QTc predicted a 0.70-ms increase in AERP. CONCLUSIONS: The QTc interval reflects the AERP, suggesting that the QTc interval may be used as a marker of atrial refractoriness relevant to assessing AF risk and mechanism-specific therapeutic strategies.

Alcohol Consumption, Left Atrial Diameter, and Atrial Fibrillation.

BACKGROUND: Alcohol consumption has been associated with atrial fibrillation (AF) in several epidemiologic studies, but the underlying mechanisms remain unknown. We sought to test the hypothesis that an atrial myopathy, manifested by echocardiographic left atrial enlargement, explains the association between chronic alcohol use and AF. METHODS AND RESULTS: We evaluated the relationship between cumulative alcohol consumption and risk of incident AF in 5220 Offspring and Original Framingham Heart Study participants (mean age 56.3 years, 54% women) with echocardiographic left atrial size measurements. The incidence of AF was 8.4 per 1000 person-years, with 1088 incident AF cases occurring over a median 6.0 years (25th-75th percentiles 4.0-8.7 years) of follow-up. After multivariable adjustment for potential confounders, every additional 10 g of alcohol per day (just under 1 drink per day) was associated with a 0.16 mm (95% CI, 0.10-0.21 mm) larger left atrial dimension. Also in multivariable adjusted analysis, every 10 g per day of alcohol consumed was associated with a 5% higher risk of developing new-onset AF (hazard ratio, 1.05; 95% CI, 1.01-1.09). An estimated 24% (95% CI, 8-75) of the association between alcohol and AF risk was explained by left atrial enlargement. CONCLUSIONS: Our study of a large, community-based sample identified alcohol consumption as a predictor of left atrial enlargement and subsequent incident AF. Left atrial enlargement may be an intermediate phenotype along the causal pathway linking long-term alcohol consumption to AF.

Atrial Fibrillation Associated Genetic Variants and Left Atrial Histology: Evaluation for Molecular Sub-Phenotypes.

INTRODUCTION: Genome wide association studies have identified several single nucleotide polymorphisms (SNPs) associated with atrial fibrillation (AF), but the mechanisms underlying these relationships have not yet been elucidated. Inflammation and fibrosis have been posited as important mechanisms responsible for AF. We sought to investigate the impact of SNP carrier status on inflammation and fibrosis in left atrial appendage tissue. METHODS AND RESULTS: Carrier status of 10 AF-associated SNPs was evaluated on DNA extracted from left atrial appendage tissue in 176 individuals (120 with AF). The presence of inflammation was evaluated through visual quantification of leukocyte infiltration following hematoxylin and eosin staining, while fibrosis was quantified using picrosirius red with fast green staining. Unadjusted and adjusted linear and logistic regression models were utilized to evaluate for an association between SNP carrier status and inflammation and fibrosis. On adjusted logistic regression analysis, the rs7164883 SNP (intronic within HCN4) was associated with a reduced odds of inflammation (odds ratio: 0.42; 95% CI: 0.22-0.81, P = 0.01), and was not associated with fibrosis on adjusted linear regression analysis (ß-coefficient: -0.31; 95% CI: -1.03-0.40, P = 0.40). None of the remaining SNPs exhibited significant associations with left atrial inflammation or fibrosis. CONCLUSIONS: Among 10 AF-associated SNPs, a single genetic variant was associated with reduced left atrial inflammation, while no histologic differences were observed in the remaining 9. The known AF-associated SNPs do not appear to predispose to the development of pro-inflammatory or pro-fibrotic AF sub-phenotypes.

Access to alcohol and heart disease among patients in hospital: observational cohort study using differences in alcohol sales laws.

OBJECTIVE:  To investigate the relation between alcohol consumption and heart disease by using differences in county level alcohol sales laws as a natural experiment. DESIGN:  Observational cohort study using differences in alcohol sales laws. SETTING:  Hospital based healthcare encounters in Texas, USA. POPULATION:  1 106 968 patients aged 21 or older who were residents of "wet" (no alcohol restrictions) and "dry" (complete prohibition of alcohol sales) counties and admitted to hospital between 2005 and 2010, identified using the Texas Inpatient Research Data File. OUTCOME MEASURES:  Prevalent and incident alcohol misuse and alcoholic liver disease were used for validation analyses. The main cardiovascular outcomes were atrial fibrillation, acute myocardial infarction, and congestive heart failure. RESULTS:  Residents of wet counties had a greater prevalence and incidence of alcohol misuse and alcoholic liver disease. After multivariable adjustment, wet county residents had a greater prevalence (odds ratio 1.05, 95% confidence interval 1.01 to 1.09; P=0.007) and incidence (hazard ratio 1.07, 1.01 to 1.13; P=0.014) of atrial fibrillation, a lower prevalence (odds ratio 0.83, 0.79 to 0.87; P<0.001) and incidence (hazard ratio 0.91, 0.87 to 0.99; P=0.019) of myocardial infarction, and a lower prevalence (odds ratio 0.87, 0.84 to 0.90; P<0.001) of congestive heart failure. Conversion of counties from dry to wet resulted in statistically significantly higher rates of alcohol misuse, alcoholic liver disease, atrial fibrillation, and congestive heart failure, with no detectable difference in myocardial infarction. CONCLUSIONS:  Greater access to alcohol was associated with more atrial fibrillation and less myocardial infarction and congestive heart failure, although an increased risk of congestive heart failure was seen shortly after alcohol sales were liberalized.

Transcriptional reversion of cardiac myocyte fate during mammalian cardiac regeneration.

RATIONALE: Neonatal mice have the capacity to regenerate their hearts in response to injury, but this potential is lost after the first week of life. The transcriptional changes that underpin mammalian cardiac regeneration have not been fully characterized at the molecular level. OBJECTIVE: The objectives of our study were to determine whether myocytes revert the transcriptional phenotype to a less differentiated state during regeneration and to systematically interrogate the transcriptional data to identify and validate potential regulators of this process. METHODS AND RESULTS: We derived a core transcriptional signature of injury-induced cardiac myocyte (CM) regeneration in mouse by comparing global transcriptional programs in a dynamic model of in vitro and in vivo CM differentiation, in vitro CM explant model, as well as a neonatal heart resection model. The regenerating mouse heart revealed a transcriptional reversion of CM differentiation processes, including reactivation of latent developmental programs similar to those observed during destabilization of a mature CM phenotype in the explant model. We identified potential upstream regulators of the core network, including interleukin 13, which induced CM cell cycle entry and STAT6/STAT3 signaling in vitro. We demonstrate that STAT3/periostin and STAT6 signaling are critical mediators of interleukin 13 signaling in CMs. These downstream signaling molecules are also modulated in the regenerating mouse heart. CONCLUSIONS: Our work reveals new insights into the transcriptional regulation of mammalian cardiac regeneration and provides the founding circuitry for identifying potential regulators for stimulating heart regeneration.

MicroRNA-144 is a circulating effector of remote ischemic preconditioning.

Remote ischemic preconditioning (rIPC) induced by cycles of transient limb ischemia and reperfusion is a powerful cardioprotective strategy with additional pleiotropic effects. However, our understanding of its underlying mediators and mechanisms remains incomplete. We examined the role of miR-144 in the cardioprotection induced by rIPC. Microarray studies first established that rIPC increases, and IR injury decreases miR-144 levels in mouse myocardium, the latter being rescued by both rIPC and intravenous administration of miR-144. Going along with this systemic treatment with miR-144 increased P-Akt, P-GSK3ß and P-p44/42 MAPK, decreased p-mTOR level and induced autophagy signaling, and induced early and delayed cardioprotection with improved functional recovery and reduction in infarct size similar to that achieved by rIPC. Conversely, systemic administration of a specific antisense oligonucleotide reduced myocardial levels of miR-144 and abrogated cardioprotection by rIPC. We then showed that rIPC increases plasma miR-144 levels in mice and humans, but there was no change in plasma microparticle (50-400 nM) numbers or their miR-144 content. However, there was an almost fourfold increase in miR-144 precursor in the exosome pellet, and a significant increase in miR-144 levels in exosome-poor serum which, in turn, was associated with increased levels of the miR carriage protein Argonaute-2. Systemic release of microRNA 144 plays a pivotal role in the cardioprotection induced by rIPC. Future studies should assess the potential for plasma miR-144 as a biomarker of the effectiveness of rIPC induced by limb ischemia, and whether miR-144 itself may represent a novel therapy to reduce clinical ischemia-reperfusion injury.

Transcutaneous electrical nerve stimulation as a novel method of remote preconditioning: in vitro validation in an animal model and first human observations.

Remote ischemic preconditioning (rIPC) induced by transient limb ischemia (li-rIPC) leads to neurally dependent release of blood-borne factors that provide potent cardioprotection. We hypothesized that transcutaneous electrical nerve stimulation (TENS) is a clinically relevant stimulus of rIPC. Study 1: seven rabbits were subjected to lower limb TENS; six to li-rIPC, and six to sham intervention. Blood was drawn and used to prepare a dialysate for subsequent analysis of cardioprotection in rabbit Langendorff preparation. Study 2: 14 healthy adults underwent upper limb TENS stimulation on one study day, 10 of whom also underwent li-rIPC on another study day. Blood was drawn before and after each stimulus, dialysate prepared, and cardioprotective activity assessed in mouse Langendorff preparation. The infarct size and myocardial recovery were measured after 30 min of global ischemia and 60 or 120 min of reperfusion. Animal validation: compared to control, TENS induced marked cardioprotection with significantly reduced infarct size (TENS vs. sham p < 0.01, rIPC vs. sham p < 0.01, TENS vs. rIPC p = ns) and improved functional recovery during reperfusion. Human study: compared to baseline, dialysate after rIPC (pre-rIPC vs. post-rIPC, p < 0.001) and TENS provided potent cardioprotection (pre-TENS vs. post-TENS p < 0.001) and improved myocardial recovery during reperfusion. The cardioprotective effects of TENS dialysates were blocked by pretreatment of the receptor heart with the opioid antagonist naloxone. TENS is a novel method for inducing cardioprotection and may provide an alternative to the limb ischemia stimulus for induction of rIPC clinically.

Electroacupuncture reduces myocardial infarct size and improves post-ischemic recovery by invoking release of humoral, dialyzable, cardioprotective factors.

Previous studies have shown that electroacupuncture (EA) can induce cardioprotection against ischemia-reperfusion (IR) injury, but its mechanisms are incompletely understood. We have previously shown that several other forms of remote preconditioning of the heart work, at least in part, via the release of circulating cardioprotective factors into the bloodstream, that can be dialyzed and subsequently shown to reduce IR injury in isolated hearts. We used the same methods to assess whether EA leads to similar humoral cardioprotection. EA rabbits were subjected to 60 min of bilateral stimulation at the Neiguan point, following which their blood was drawn, dialyzed, and used to perfuse hearts in Langendorff preparation and subsequently subjected to 60 min of global ischemia and 120 min of reperfusion. Compared to controls, dialysate from EA animals led to significant reduction in infarct size and improved functional recovery. The degree of cardioprotection was no different to that seen in animals randomized to receive remote preconditioning using transient limb ischemia (4 cycles of 5 min ischemia/5 min reperfusion). These results suggest that EA recapitulates the cardioprotection achieved by remote preconditioning, by similarly leading to release of circulating cardioprotective factors.

Remote cardioprotection by direct peripheral nerve stimulation and topical capsaicin is mediated by circulating humoral factors.

We have previously shown that remote ischemic preconditioning by limb ischemia (rIPC) or intra-arterial adenosine releases a dialyzable cardioprotective circulating factor(s), the release of which requires an intact neural connection to the limb and is blocked by pretreatment with S-nitroso-N-acetylpenicillamine (SNAP). Remote cardioprotection can be induced by other forms of peripheral stimulation including topical capsaicin, but the mechanisms of their signal transduction are incompletely understood. Rabbits were anesthetized by intravenous pentobarbital, intubated and ventilated, then randomized (4-7 animals in each group) to receive sham procedure, rIPC (4 cycles of 5 min lower limb ischemia, 5 min reperfusion), direct femoral nerve stimulation, topical capsaicin, pretreatment with intra-arterial SNAP + capsaicin, pretreatment with topical DMSO (a sensory nerve blocker) + topical capsaicin, or pretreatment with intra-arterial SNAP + femoral nerve stimulation, topical DMSO alone, or intra-arterial SNAP alone. Blood was then rapidly drawn from the carotid artery to produce the plasma dialysate which was used to perfuse a naïve heart from an untreated donor rabbit. The infarct size and recovery of LV-developed pressure and end-diastolic pressure were measured after 30 min of global ischemia and 120 min of reperfusion. Compared to sham, dialysate from rIPC, femoral nerve stimulation, and topical capsaicin groups all produced significant cardioprotection with significantly reduced infarct size, and improved the post-ischemic cardiac performance. Cardioprotection was not seen in the topical DMSO-capsaicin, SNAP + capsaicin, and SNAP + FNS groups. These results confirm the central role of peripheral nerves in the local signal transduction of remote cardioprotection. Direct electrical or peripheral neural stimulation evokes the release of cardioprotective substances into the bloodstream, with comparable effects to that of rIPC induced by limb ischemia.

Remote preconditioning provides potent cardioprotection via PI3K/Akt activation and is associated with nuclear accumulation of ß-catenin.

rIPC [remote IPC (ischaemic preconditioning)] has been shown to invoke potent myocardial protection in animal studies and recent clinical trials. Although the important role of PI3K (phosphoinositide 3-kinase)/Akt activation in the cardioprotection afforded by local IPC is well described, our understanding of the intracellular signalling of rIPC remains incomplete. We therefore examined the hypothesis that the myocardial protection afforded by rIPC is mediated via the PI3K/Akt/GSK3ß (glycogen synthase kinase 3ß) signalling pathway, activation of which is associated with nuclear accumulation of ß-catenin. rIPC was induced in mice using four cycles of 5 min of ischaemia and 5 min of reperfusion of the hindlimb using a torniquet. This led to reduced infarct size (19 ± 4% in rIPC compared with 39 ± 7% in sham; P<0.05), improved functional recovery and reduced apoptosis after global I/R (ischaemia/reperfusion) injury using a Langendorff-perfused mouse heart model. These effects were reversed by pre-treatment with an inhibitor of PI3K activity. Furthermore, Western blot analysis demonstrated that, compared with control, rIPC was associated with activation of the PI3K/Akt signalling pathway, resulting in phosphorylation and inactivation of GSK3ß, accumulation of ß-catenin in the cytosol and its translocation to the nucleus. Finally, rIPC increased the expression of ß-catenin target genes involved in cell-survival signalling, including E-cadherin and PPARδ (peroxisome-proliferator-activated receptor δ). In conclusion, we show for the first time that the myocardial protection afforded by rIPC is mediated via the PI3K/Akt/GSK3ß signalling pathway, activation of which is associated with nuclear accumulation of ß-catenin and the up-regulation of its downstream targets E-cadherin and PPARδ involved in cell survival.