Restoring Atrial T-Tubules Augments Systolic Ca Upon Recovery From Heart Failure.

BACKGROUND: Transverse (t)-tubules drive the rapid and synchronous Ca2+ rise in cardiac myocytes. The virtual complete atrial t-tubule loss in heart failure (HF) decreases Ca2+ release. It is unknown if or how atrial t-tubules can be restored and how this affects systolic Ca2+. METHODS: HF was induced in sheep by rapid ventricular pacing and recovered following termination of rapid pacing. Serial block-face scanning electron microscopy and confocal imaging were used to study t-tubule ultrastructure. Function was assessed using patchclamp, Ca2+, and confocal imaging. Candidate proteins involved in atrial t-tubule recovery were identified by western blot and expressed in rat neonatal ventricular myocytes to determine if they altered t-tubule structure. RESULTS: Atrial t-tubules were lost in HF but reappeared following recovery from HF. Recovered t-tubules were disordered, adopting distinct morphologies with increased t-tubule length and branching. T-tubule disorder was associated with mitochondrial disorder. Recovered t-tubules were functional, triggering Ca2+ release in the cell interior. Systolic Ca2+, ICa-L, sarcoplasmic reticulum Ca2+ content, and SERCA function were restored following recovery from HF. Confocal microscopy showed fragmentation of ryanodine receptor staining and movement away from the z-line in HF, which was reversed following recovery from HF. Acute detubulation, to remove recovered t-tubules, confirmed their key role in restoration of the systolic Ca2+ transient, the rate of Ca2+ removal, and the peak L-type Ca2+ current. The abundance of telethonin and myotubularin decreased during HF and increased during recovery. Transfection with these proteins altered the density and structure of tubules in neonatal myocytes. Myotubularin had a greater effect, increasing tubule length and branching, replicating that seen in the recovery atria. CONCLUSIONS: We show that recovery from HF restores atrial t-tubules, and this promotes recovery of ICa-L, sarcoplasmic reticulum Ca2+ content, and systolic Ca2+. We demonstrate an important role for myotubularin in t-tubule restoration. Our findings reveal a new and viable therapeutic strategy.

Serial block face scanning electron microscopy reveals region-dependent remodelling of transverse tubules post-myocardial infarction.

The highly organized transverse tubule (t-tubule) network facilitates cardiac excitation-contraction coupling and synchronous cardiac myocyte contraction. In cardiac failure secondary to myocardial infarction (MI), changes in the structure and organization of t-tubules result in impaired cardiac contractility. However, there is still little knowledge on the regional variation of t-tubule remodelling in cardiac failure post-MI. Here, we investigate post-MI t-tubule remodelling in infarct border and remote regions, using serial block face scanning electron microscopy (SBF-SEM) applied to a translationally relevant sheep ischaemia reperfusion MI model and matched controls. We performed minimally invasive coronary angioplasty of the left anterior descending artery, followed by reperfusion after 90 min to establish the MI model. Left ventricular tissues obtained from control and MI hearts eight weeks post-MI were imaged using SBF-SEM. Image analysis generated three-dimensional reconstructions of the t-tubular network in control, MI border and remote regions. Quantitative analysis revealed that the MI border region was characterized by t-tubule depletion and fragmentation, dilation of surviving t-tubules and t-tubule elongation. This study highlights region-dependent remodelling of the tubular network post-MI and may provide novel localized therapeutic targets aimed at preservation or restoration of the t-tubules to manage cardiac contractility post-MI. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Postnatal cardiovascular morbidity following preterm pre-eclampsia: An observational study.

OBJECTIVE: To explore the nature of postnatal cardiovascular morbidity following pregnancies complicated by preterm pre-eclampsia and investigate associations between pregnancy characteristics and maternal postnatal cardiovascular function. STUDY DESIGN: This was an observational sub-study of a single-centre feasibility randomised double-blind placebo-controlled trial (https://www. CLINICALTRIALS: gov; NCT03466333), involving women with preterm pre-eclampsia, delivering before 37 weeks. Eligible women underwent echocardiography, arteriography and blood pressure monitoring within three days of birth, six weeks and six months postpartum. Correlations between pregnancy and cardiovascular characteristics were assessed using Spearman's correlation. MAIN OUTCOME MEASURES: The prevalence of cardiovascular dysfunction and remodelling six months following preterm pre-eclampsia. RESULTS: Forty-four women completed the study. At six months, 27 (61 %) had diastolic dysfunction, 33 (75 %) had raised total vascular resistance (TVR) and 18 (41 %) had left ventricular remodelling. Sixteen (46 %) women had de novo hypertension by six months and only two (5 %) women had a completely normal echocardiogram. Echocardiography did not change significantly from six weeks to six months. Earlier gestation at delivery and lower birthweight centile were associated with worse six-month diastolic dysfunction (E/E': rho = -0.39, p = 0.001 & rho = -0.42, p = 0.005) and TVR (rho = -0.34, p = 0.02 & rho = -0.37, p = 0.01). CONCLUSIONS: Preterm pre-eclampsia is associated with persistent cardiovascular morbidity-six months postpartum in the majority of women. These cardiovascular changes have significant implications for long-term cardiovascular health. The graded severity of diastolic dysfunction and TVR with worsening pre-eclampsia phenotype suggests a dose-effect. However, the mechanistic link remains uncertain.

Enhanced calcium release at specialised surface sites compensates for reduced t-tubule density in neonatal sheep atrial myocytes.

Cardiac myocytes rely on transverse (t)-tubules to facilitate a rapid rise in calcium throughout the cell. However, despite their importance in triggering synchronous Ca2+ release, t-tubules are highly labile structures. They develop postnatally, increase in density during exercise training and are lost in diseases such as heart failure (HF). In the majority of settings, an absence of t-tubules decreases function. Here we show that despite reduced t-tubule density due to immature t-tubules, the newborn atrium is highly specialised to maintain Ca2+ release. To compensate for fewer t-tubules triggering a central rise in Ca2+, Ca2+ release at sites on the cell surface is enhanced in the newborn, exceeding that at all Ca2+ release sites in the adult. Using electron and super resolution microscopy to investigate myocyte ultrastructure, we found that newborn atrial cells had enlarged surface sarcoplasmic reticulum and larger, more closely spaced surface and central ryanodine receptor clusters. We suggest that these adaptations mediate enhanced Ca2+ release at the sarcolemma and aid propagation to compensate for reduced t-tubule density in the neonatal atrium.

Chronic developmental hypoxia alters mitochondrial oxidative capacity and reactive oxygen species production in the fetal rat heart in a sex-dependent manner.

Insufficient oxygen supply (hypoxia) during fetal development leads to cardiac remodeling and a predisposition to cardiovascular disease in later life. Previous work has shown hypoxia causes oxidative stress in the fetal heart and alters the activity and expression of mitochondrial proteins in a sex-dependent manner. However, the functional effects of these modifications on mitochondrial respiration remain unknown. Furthermore, while maternal antioxidant treatments are emerging as a promising new strategy to protect the hypoxic fetus, whether these treatments convey similar protection to cardiac mitochondria in the male or female fetus has not been investigated. Therefore, using an established rat model, we measured the sex-dependent effects of gestational hypoxia and maternal melatonin treatment on fetal cardiac mitochondrial respiration, reactive oxygen species (ROS) production, and lipid peroxidation. Pregnant Wistar rats were subjected to normoxia or hypoxia (13% oxygen) during gestational days (GDs) 6-20 (term ~22 days) with or without melatonin treatment (5 µg/ml in maternal drinking water). On GD 20, mitochondrial aerobic respiration and H2 O2 production were measured in fetal heart tissue, together with lipid peroxidation and citrate synthase (CS) activity. Gestational hypoxia reduced maternal body weight gain (p < .01) and increased placental weight (p < .05) but had no effect on fetal weight or litter size. Cardiac mitochondria from male but not female fetuses of hypoxic pregnancy had reduced respiratory capacity at Complex II (CII) (p < .05), and an increase in H2 O2 production/O2 consumption (p < .05) without any changes in lipid peroxidation. CS activity was also unchanged in both sexes. Despite maternal melatonin treatment increasing maternal and fetal plasma melatonin concentration (p < .001), melatonin treatment had no effect on any of the mitochondrial parameters investigated. To conclude, we show that gestational hypoxia leads to ROS generation from the mitochondrial electron transport chain and affects fetal cardiac mitochondrial respiration in a sex-dependent manner. We also show that maternal melatonin treatment had no effect on these relationships, which has implications for the development of future therapies for hypoxic pregnancies.

Cardiomyocyte-specific loss of plasma membrane calcium ATPase 1 impacts cardiac rhythm and is associated with ventricular repolarisation dysfunction.

Plasma membrane calcium ATPase 1 (PMCA1, Atp2b1) is emerging as a key contributor to cardiac physiology, involved in calcium handling and myocardial signalling. In addition, genome wide association studies have associated PMCA1 in several areas of cardiovascular disease including hypertension and myocardial infarction. Here, we investigated the role of PMCA1 in basal cardiac function and heart rhythm stability. Cardiac structure, heart rhythm and arrhythmia susceptibility were assessed in a cardiomyocyte-specific PMCA1 deletion (PMCA1CKO) mouse model. PMCA1CKO mice developed abnormal heart rhythms related to ventricular repolarisation dysfunction and displayed an increased susceptibility to ventricular arrhythmias. We further assessed the levels of cardiac ion channels using qPCR and found a downregulation of the voltage-dependent potassium channels, Kv4.2, with a corresponding reduction in the transient outward potassium current which underlies ventricular repolarisation in the murine heart. The changes in heart rhythm were found to occur in the absence of any structural cardiomyopathy. To further assess the molecular changes occurring in PMCA1CKO hearts, we performed proteomic analysis. Functional characterisation of differentially expressed proteins suggested changes in pathways related to metabolism, protein-binding, and pathways associated cardiac function including ß-adrenergic signalling. Together, these data suggest an important role for PMCA1 in basal cardiac function in relation to heart rhythm control, with reduced cardiac PMCA1 expression resulting in an increased risk of arrhythmia development.

Distributed acoustic sensing for active offshore shear wave profiling.

The long-term sustainability of the offshore wind industry requires the development of appropriate investigative methods to enable less conservative and more cost-effective geotechnical engineering design. Here we describe the novel use of distributed acoustic sensing (DAS) as part of an integrated approach for the geophysical and geotechnical assessment of the shallow subsurface for offshore construction. DAS was used to acquire active Scholte-wave seismic data at several locations in the vicinity of a planned windfarm development near Dundalk Bay, Irish Sea. Complimentary additional datasets include high-resolution sparker seismic reflection, cone penetration test (CPT) data and gravity coring. In terms of fibre optic cable selection, a CST armoured cable provided a reasonable compromise between performance and reliability in the offshore environment. Also, when used as a seismic source, a gravity corer enabled the fundamental mode Scholte-wave to be better resolved than an airgun, and may be more suitable in environmentally sensitive areas. Overall, the DAS approach was found to be effective at rapidly determining shear wave velocity profiles in areas of differing geological context, with metre scale spatial sampling, over multi-kilometre scale distances. The application of this approach has the potential to considerably reduce design uncertainty and ultimately reduce levelised costs of offshore wind power generation.

Peptide location fingerprinting identifies species- and tissue-conserved structural remodelling of proteins as a consequence of ageing and disease.

Extracellular matrices (ECMs) in the intervertebral disc (IVD), lung and artery are thought to undergo age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and bioinformatically identified novel target proteins alongside common age-associated differences within protein structures which were conserved between three ECM-rich organs, two species, three IVD tissue regions, sexes and in an age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI), enzyme/inhibitor activity (alpha-2 macroglobulin), activation states (complement C3) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin and collagen XIV exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across both species, irrespective of differences in lifespan and tissue function.

Alkaline nucleoplasm facilitates contractile gene expression in the mammalian heart.

Cardiac contractile strength is recognised as being highly pH-sensitive, but less is known about the influence of pH on cardiac gene expression, which may become relevant in response to changes in myocardial metabolism or vascularization during development or disease. We sought evidence for pH-responsive cardiac genes, and a physiological context for this form of transcriptional regulation. pHLIP, a peptide-based reporter of acidity, revealed a non-uniform pH landscape in early-postnatal myocardium, dissipating in later life. pH-responsive differentially expressed genes (pH-DEGs) were identified by transcriptomics of neonatal cardiomyocytes cultured over a range of pH. Enrichment analysis indicated "striated muscle contraction" as a pH-responsive biological process. Label-free proteomics verified fifty-four pH-responsive gene-products, including contractile elements and the adaptor protein CRIP2. Using transcriptional assays, acidity was found to reduce p300/CBP acetylase activity and, its a functional readout, inhibit myocardin, a co-activator of cardiac gene expression. In cultured myocytes, acid-inhibition of p300/CBP reduced H3K27 acetylation, as demonstrated by chromatin immunoprecipitation. H3K27ac levels were more strongly reduced at promoters of acid-downregulated DEGs, implicating an epigenetic mechanism of pH-sensitive gene expression. By tandem cytoplasmic/nuclear pH imaging, the cardiac nucleus was found to exercise a degree of control over its pH through Na+/H+ exchangers at the nuclear envelope. Thus, we describe how extracellular pH signals gain access to the nucleus and regulate the expression of a subset of cardiac genes, notably those coding for contractile proteins and CRIP2. Acting as a proxy of a well-perfused myocardium, alkaline conditions are permissive for expressing genes related to the contractile apparatus.

Interaction of background Ca2+ influx, sarcoplasmic reticulum threshold and heart failure in determining propensity for Ca2+ waves in sheep heart.

Ventricular arrhythmias can cause death in heart failure (HF). A trigger is the occurrence of Ca2+ waves which activate a Na+ -Ca2+ exchange (NCX) current, leading to delayed after-depolarisations and triggered action potentials. Waves arise when sarcoplasmic reticulum (SR) Ca2+ content reaches a threshold and are commonly induced experimentally by raising external Ca2+ , although the mechanism by which this causes waves is unclear and was the focus of this study. Intracellular Ca2+ was measured in voltage-clamped ventricular myocytes from both control sheep and those subjected to rapid pacing to produce HF. Threshold SR Ca2+ content was determined by applying caffeine (10  mM) following a wave and integrating wave and caffeine-induced NCX currents. Raising external Ca2+ induced waves in a greater proportion of HF cells than control. The associated increase of SR Ca2+ content was smaller in HF due to a lower threshold. Raising external Ca2+ had no effect on total influx via the L-type Ca2+ current, ICa-L , and increased efflux on NCX. Analysis of sarcolemmal fluxes revealed substantial background Ca2+ entry which sustains Ca2+ efflux during waves in the steady state. Wave frequency and background Ca2+ entry were decreased by Gd3+ or the TRPC6 inhibitor BI 749327. These agents also blocked Mn2+ entry. Inhibiting connexin hemi-channels, TRPC1/4/5, L-type channels or NCX had no effect on background entry. In conclusion, raising external Ca2+ induces waves via a background Ca2+ influx through TRPC6 channels. The greater propensity to waves in HF results from increased background entry and decreased threshold SR content. KEY POINTS: Heart failure is a pro-arrhythmic state and arrhythmias are a major cause of death. At the cellular level, Ca2+ waves resulting in delayed after-depolarisations are a key trigger of arrhythmias. Ca2+ waves arise when the sarcoplasmic reticulum (SR) becomes overloaded with Ca2+ . We investigate the mechanism by which raising external Ca2+ causes waves, and how this is modified in heart failure. We demonstrate that a novel sarcolemmal background Ca2+ influx via the TRPC6 channel is responsible for SR Ca2+ overload and Ca2+ waves. The increased propensity for Ca2+ waves in heart failure results from an increase of background influx, and a lower threshold SR content. The results of the present study highlight a novel mechanism by which Ca2+ waves may arise in heart failure, providing a basis for future work and novel therapeutic targets.

Cardiac Transverse Tubules in Physiology and Heart Failure.

In mammalian cardiac myocytes, the plasma membrane includes the surface sarcolemma but also a network of membrane invaginations called transverse (t-) tubules. These structures carry the action potential deep into the cell interior, allowing efficient triggering of Ca2+ release and initiation of contraction. Once thought to serve as rather static enablers of excitation-contraction coupling, recent work has provided a newfound appreciation of the plasticity of the t-tubule network's structure and function. Indeed, t-tubules are now understood to support dynamic regulation of the heartbeat across a range of timescales, during all stages of life, in both health and disease. This review article aims to summarize these concepts, with consideration given to emerging t-tubule regulators and their targeting in future therapies.

Maternal melatonin: Effective intervention against developmental programming of cardiovascular dysfunction in adult offspring of complicated pregnancy.

Adopting an integrative approach, by combining studies of cardiovascular function with those at cellular and molecular levels, this study investigated whether maternal treatment with melatonin protects against programmed cardiovascular dysfunction in the offspring using an established rodent model of hypoxic pregnancy. Wistar rats were divided into normoxic (N) or hypoxic (H, 10% O2 ) pregnancy ± melatonin (M) treatment (5 µg·ml-1 .day-1 ) in the maternal drinking water. Hypoxia ± melatonin treatment was from day 15-20 of gestation (term is ca. 22 days). To control for possible effects of maternal hypoxia-induced reductions in maternal food intake, additional dams underwent pregnancy under normoxic conditions but were pair-fed (PF) to the daily amount consumed by hypoxic dams from day 15 of gestation. In one cohort of animals from each experimental group (N, NM, H, HM, PF, PFM), measurements were made at the end of gestation. In another, following delivery of the offspring, investigations were made at adulthood. In both fetal and adult offspring, fixed aorta and hearts were studied stereologically and frozen hearts were processed for molecular studies. In adult offspring, mesenteric vessels were isolated and vascular reactivity determined by in-vitro wire myography. Melatonin treatment during normoxic, hypoxic or pair-fed pregnancy elevated circulating plasma melatonin in the pregnant dam and fetus. Relative to normoxic pregnancy, hypoxic pregnancy increased fetal haematocrit, promoted asymmetric fetal growth restriction and resulted in accelerated postnatal catch-up growth. Whilst fetal offspring of hypoxic pregnancy showed aortic wall thickening, adult offspring of hypoxic pregnancy showed dilated cardiomyopathy. Similarly, whilst cardiac protein expression of eNOS was downregulated in the fetal heart, eNOS protein expression was elevated in the heart of adult offspring of hypoxic pregnancy. Adult offspring of hypoxic pregnancy further showed enhanced mesenteric vasoconstrictor reactivity to phenylephrine and the thromboxane mimetic U46619. The effects of hypoxic pregnancy on cardiovascular remodelling and function in the fetal and adult offspring were independent of hypoxia-induced reductions in maternal food intake. Conversely, the effects of hypoxic pregnancy on fetal and postanal growth were similar in pair-fed pregnancies. Whilst maternal treatment of normoxic or pair-fed pregnancies with melatonin on the offspring cardiovascular system was unremarkable, treatment of hypoxic pregnancies with melatonin in doses lower than those recommended for overcoming jet lag in humans enhanced fetal cardiac eNOS expression and prevented all alterations in cardiovascular structure and function in fetal and adult offspring. Therefore, the data support that melatonin is a potential therapeutic target for clinical intervention against developmental origins of cardiovascular dysfunction in pregnancy complicated by chronic fetal hypoxia.

Electrophysiological and Proarrhythmic Effects of Hydroxychloroquine Challenge in Guinea-Pig Hearts.

Hydroxychloroquine (HCQ), clinically established in antimalarial and autoimmune therapy, recently raised cardiac arrhythmogenic concerns when used alone or with azithromycin (HCQ+AZM) in Covid-19. We report complementary, experimental, studies of its electrophysiological effects. In patch clamped HEK293 cells expressing human cardiac ion channels, HCQ inhibited IKr and IK1 at a therapeutic concentrations (IC50s: 10 ± 0.6 and 34 ± 5.0 µM). INa and ICaL showed higher IC50s; Ito and IKs were unaffected. AZM slightly inhibited INa, ICaL, IKs, and IKr, sparing IK1 and Ito. (HCQ+AZM) inhibited IKr and IK1 (IC50s: 7.7 ± 0.8 and 30.4 ± 3.0 µM), sparing INa, ICaL, and Ito. Molecular induced-fit docking modeling confirmed potential HCQ-hERG but weak AZM-hERG binding. Effects of µM-HCQ were studied in isolated perfused guinea-pig hearts by multielectrode, optical RH237 voltage, and Rhod-2 mapping. These revealed reversibly reduced left atrial and ventricular action potential (AP) conduction velocities increasing their heterogeneities, increased AP durations (APDs), and increased durations and dispersions of intracellular [Ca2+] transients, respectively. Hearts also became bradycardic with increased electrocardiographic PR and QRS durations. The (HCQ+AZM) combination accentuated these effects. Contrastingly, (HCQ+AZM) and not HCQ alone disrupted AP propagation, inducing alternans and torsadogenic-like episodes on voltage mapping during forced pacing. O'Hara-Rudy modeling showed that the observed IKr and IK1 effects explained the APD alterations and the consequently prolonged Ca2+ transients. The latter might then downregulate INa, reducing AP conduction velocity through recently reported INa downregulation by cytosolic [Ca2+] in a novel scheme for drug action. The findings may thus prompt future investigations of HCQ's cardiac safety under particular, chronic and acute, clinical situations.

Peptide Location Fingerprinting Reveals Tissue Region-Specific Differences in Protein Structures in an Ageing Human Organ.

In ageing tissues, long-lived extracellular matrix (ECM) proteins are susceptible to the accumulation of structural damage due to diverse mechanisms including glycation, oxidation and protease cleavage. Peptide location fingerprinting (PLF) is a new mass spectrometry (MS) analysis technique capable of identifying proteins exhibiting structural differences in complex proteomes. PLF applied to published young and aged intervertebral disc (IVD) MS datasets (posterior, lateral and anterior regions of the annulus fibrosus) identified 268 proteins with age-associated structural differences. For several ECM assemblies (collagens I, II and V and aggrecan), these differences were markedly conserved between degeneration-prone (posterior and lateral) and -resistant (anterior) regions. Significant differences in peptide yields, observed within collagen I α2, collagen II α1 and collagen V α1, were located within their triple-helical regions and/or cleaved C-terminal propeptides, indicating potential accumulation of damage and impaired maintenance. Several proteins (collagen V α1, collagen II α1 and aggrecan) also exhibited tissue region (lateral)-specific differences in structure between aged and young samples, suggesting that some ageing mechanisms may act locally within tissues. This study not only reveals possible age-associated differences in ECM protein structures which are tissue-region specific, but also highlights the ability of PLF as a proteomic tool to aid in biomarker discovery.

PDE5 Inhibition Suppresses Ventricular Arrhythmias by Reducing SR Ca2+ Content.

[Figure: see text].

Optimising Large Animal Models of Sustained Atrial Fibrillation: Relevance of the Critical Mass Hypothesis.

BACKGROUND: Large animal models play an important role in our understanding of the pathophysiology of atrial fibrillation (AF). Our aim was to determine whether prospectively collected baseline variables could predict the development of sustained AF in sheep, thereby reducing the number of animals required in future studies. Our hypothesis was that the relationship between atrial dimensions, refractory periods and conduction velocity (otherwise known as the critical mass hypothesis) could be used for the first time to predict the development of sustained AF. METHODS: Healthy adult Welsh mountain sheep underwent a baseline electrophysiology study followed by implantation of a neurostimulator connected via an endocardial pacing lead to the right atrial appendage. The device was programmed to deliver intermittent 50 Hz bursts of 30 s duration over an 8-week period whilst sheep were monitored for AF. RESULTS: Eighteen sheep completed the protocol, of which 28% developed sustained AF. Logistic regression analysis showed only fibrillation number (calculated using the critical mass hypothesis as the left atrial diameter divided by the product of atrial conduction velocity and effective refractory period) was associated with an increased likelihood of developing sustained AF (Ln Odds Ratio 26.1 [95% confidence intervals 0.2-52.0] p = 0.048). A receiver-operator characteristic curve showed this could be used to predict which sheep developed sustained AF (C-statistic 0.82 [95% confidence intervals 0.59-1.04] p = 0.04). CONCLUSION: The critical mass hypothesis can be used to predict sustained AF in a tachypaced ovine model. These findings can be used to optimise the design of future studies involving large animals.

Distinct circadian mechanisms govern cardiac rhythms and susceptibility to arrhythmia.

Electrical activity in the heart exhibits 24-hour rhythmicity, and potentially fatal arrhythmias are more likely to occur at specific times of day. Here, we demonstrate that circadian clocks within the brain and heart set daily rhythms in sinoatrial (SA) and atrioventricular (AV) node activity, and impose a time-of-day dependent susceptibility to ventricular arrhythmia. Critically, the balance of circadian inputs from the autonomic nervous system and cardiomyocyte clock to the SA and AV nodes differ, and this renders the cardiac conduction system sensitive to decoupling during abrupt shifts in behavioural routine and sleep-wake timing. Our findings reveal a functional segregation of circadian control across the heart's conduction system and inherent susceptibility to arrhythmia.

Substrate for the Myocardial Inflammation-Heart Failure Hypothesis Identified Using Novel USPIO Methodology.

OBJECTIVES: The purpose of this study was to identify where ultrasmall superparamagnetic particles of iron oxide (USPIO) locate to in myocardium, develop a methodology that differentiates active macrophage uptake of USPIO from passive tissue distribution; and investigate myocardial inflammation in cardiovascular diseases. BACKGROUND: Myocardial inflammation is hypothesized to be a key pathophysiological mechanism of heart failure (HF), but human evidence is limited, partly because evaluation is challenging. USPIO-magnetic resonance imaging (MRI) potentially allows specific identification of myocardial inflammation but it remains unclear what the USPIO-MRI signal represents. METHODS: Histological validation was performed using a murine acute myocardial infarction (MI) model. A multiparametric, multi-time-point MRI methodology was developed, which was applied in patients with acute MI (n = 12), chronic ischemic cardiomyopathy (n = 7), myocarditis (n = 6), dilated cardiomyopathy (n = 5), and chronic sarcoidosis (n = 5). RESULTS: USPIO were identified in myocardial macrophages and myocardial interstitium. R1 time-course reflected passive interstitial distribution whereas multi-time-point R2* was also sensitive to active macrophage uptake. R2*/R1 ratio provided a quantitative measurement of myocardial macrophage infiltration. R2* behavior and R2*/R1 ratio were higher in infarcted (p = 0.001) and remote (p = 0.033) myocardium in acute MI and in chronic ischemic cardiomyopathy (infarct: p = 0.008; remote p = 0.010), and were borderline higher in DCM (p = 0.096), in comparison to healthy controls, but were no different in myocarditis or sarcoidosis. An R2*/R1 threshold of 25 had a sensitivity and specificity of 90% and 83%, respectively, for detecting active USPIO uptake. CONCLUSIONS: USPIO are phagocytized by cardiac macrophages but are also passively present in myocardial interstitium. A multiparametric multi-time-point MRI methodology specifically identifies active myocardial macrophage infiltration. Persistent active macrophage infiltration is present in infarcted and remote myocardium in chronic ischemic cardiomyopathy, providing a substrate for HF.

Sex-dependent effects of developmental hypoxia on cardiac mitochondria from adult murine offspring.

Insufficient oxygen supply (hypoxia) during fetal and embryonic development can lead to latent phenotypical changes in the adult cardiovascular system, including altered cardiac function and increased susceptibility to ischemia reperfusion injury. While the cellular mechanisms underlying this phenomenon are largely unknown, several studies have pointed towards metabolic disturbances in the heart of offspring from hypoxic pregnancies. To this end, we investigated mitochondrial function in the offspring of a mouse model of prenatal hypoxia. Pregnant C57 mice were subjected to either normoxia (21%) or hypoxia (14%) during gestational days 6-18. Offspring were reared in normoxia for up to 8 months and mitochondrial biology was assessed with electron microscopy (ultrastructure), spectrophotometry (enzymatic activity of electron transport chain complexes), microrespirometry (oxidative phosphorylation and H202 production) and Western Blot (protein expression). Our data showed that male adult offspring from hypoxic pregnancies possessed mitochondria with increased H202 production and lower respiratory capacity that was associated with reduced protein expression of complex I, II and IV. In contrast, females from hypoxic pregnancies had a higher respiratory capacity and lower H202 production that was associated with increased enzymatic activity of complex IV. From these results, we speculate that early exposure to hypoxia has long term, sex-dependent effects on cardiac metabolic function, which may have implications for cardiovascular health and disease in adulthood.