Regulation of cardiomyocyte t-tubule structure by preload and afterload: Roles in cardiac compensation and decompensation.

Mechanical load is a potent regulator of cardiac structure and function. Although high workload during heart failure is associated with disruption of cardiomyocyte t-tubules and Ca2+ homeostasis, it remains unclear whether changes in preload and afterload may promote adaptive t-tubule remodelling. We examined this issue by first investigating isolated effects of stepwise increases in load in cultured rat papillary muscles. Both preload and afterload increases produced a biphasic response, with the highest t-tubule densities observed at moderate loads, whereas excessively low and high loads resulted in low t-tubule levels. To determine the baseline position of the heart on this bell-shaped curve, mice were subjected to mildly elevated preload or afterload (1 week of aortic shunt or banding). Both interventions resulted in compensated cardiac function linked to increased t-tubule density, consistent with ascension up the rising limb of the curve. Similar t-tubule proliferation was observed in human patients with moderately increased preload or afterload (mitral valve regurgitation, aortic stenosis). T-tubule growth was associated with larger Ca2+ transients, linked to upregulation of L-type Ca2+ channels, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients advanced the heart down the declining limb of the t-tubule-load relationship. This bell-shaped relationship was lost in the absence of electrical stimulation, indicating a key role of systolic stress in controlling t-tubule plasticity. In conclusion, modest augmentation of workload promotes compensatory increases in t-tubule density and Ca2+ cycling, whereas this adaptation is reversed in overloaded hearts during heart failure progression. KEY POINTS: Excised papillary muscle experiments demonstrated a bell-shaped relationship between cardiomyocyte t-tubule density and workload (preload or afterload), which was only present when muscles were electrically stimulated. The in vivo heart at baseline is positioned on the rising phase of this curve because moderate increases in preload (mice with brief aortic shunt surgery, patients with mitral valve regurgitation) resulted in t-tubule growth. Moderate increases in afterload (mice and patients with mild aortic banding/stenosis) similarly increased t-tubule density. T-tubule proliferation was associated with larger Ca2+ transients, with upregulation of the L-type Ca2+ channel, Na+-Ca2+ exchanger, mechanosensors and regulators of t-tubule structure. By contrast, marked elevation of cardiac load in rodents and patients placed the heart on the declining phase of the t-tubule-load relationship, promoting heart failure progression. The dependence of t-tubule structure on preload and afterload thus enables both compensatory and maladaptive remodelling, in rodents and humans.

Persistent immune responses in the heart determine the outcome of cardiomyopathy syndrome in Atlantic salmon (Salmo salar).

Cardiomyopathy syndrome (CMS) caused by piscine myocarditis virus (PMCV) is a severe cardiac disease in Atlantic salmon (Salmo salar) and one of the leading causes of morbidity and mortality in the Norwegian aquaculture industry. Previous research suggest a variation in individual susceptibility to develop severe disease, however the role of the immune response in determining individual outcome of CMS is poorly understood particularly in cases where fish are also challenged by stress. The present study's aim was therefore to characterize cardiac transcriptional responses to PMCV infection in Atlantic salmon responding to infection under stressful conditions with a high versus low degree of histopathological damage. The study was performed as a large-scale controlled experiment of Atlantic salmon smolts from pre-challenge to 12 weeks post infection (wpi) with PMCV, during which fish were exposed to intermittent stressors. RNA sequencing (RNAseq) was used to compare the heart transcriptome of high responders (HR) with atrium histopathology score '4' and low responders (LR) with score '0.5' at 12 wpi. A high-throughput quantitative PCR (qPCR) analysis was used to compare immune gene transcription between individuals sampled at 6, 9 and 12 wpi. Based on RNAseq and qPCR results, RNAscope in situ hybridization (ISH) was performed for visualization of IFN-γ - and IFNb producing immune cells in affected heart tissue. Compared to LR, the transcription of 1592 genes was increased in HR at 12 wpi. Of these genes, around. 40 % were immune-related, including various chemokines, key antiviral response molecules, and genes. associated with a Th1 pro-inflammatory immune response. Further, the qPCR analysis confirmed. increased immune gene transcription in HR at both 9 and 12 wpi, despite a decrease in PMCV. transcription between these time points. Interestingly, increased IFNb transcription in HR suggests the. presence of high-quantity IFN secreting cells in the hearts of these individuals. Indeed, RNAscope. confirmed the presence of IFN-γ and IFNb-positive cells in the heart ventricle of HR but not LR. To conclude, our data indicate that in severe outcomes of PMCV infection various chemokines attract leucocytes to the salmon heart, including IFN-γ and IFNb-secreting cells, and that these cells play important roles in maintaining persistent antiviral responses and a sustained host immunopathology despite decreasing heart viral transcription.

Merging trait-based ecology and regime shift theory to anticipate community responses to warming.

Anthropogenic warming is altering species abundance, distribution, physiology, and more. How changes observed at the species level alter emergent community properties is an active and urgent area of research. Trait-based ecology and regime shift theory provide complementary ways to understand climate change impacts on communities, but these two bodies of work are only rarely integrated. Lack of integration handicaps our ability to understand community responses to warming, at a time when such understanding is critical. Therefore, we advocate for merging trait-based ecology with regime shift theory. We propose a general set of principles to guide this merger and apply these principles to research on marine communities in the rapidly warming North Atlantic. In our example, combining trait distribution and regime shift analyses at the community level yields greater insight than either alone. Looking forward, we identify a clear need for expanding quantitative approaches to collecting and merging trait-based and resilience metrics in order to advance our understanding of climate-driven community change.

Emerging human-shark conflicts in the New York Bight: A call for expansive science and management.

Recent spikes in interactions between humans and sharks in the New York Bight have sparked widespread reporting of possible causalities, many of which lack empirical support. Here we comment on the current state of knowledge regarding shark biology and management in New York waters emphasizing that the possible drivers of increased human-shark interactions are confounded by a lack of historical monitoring data. We outline several key research avenues that should be considered to ensure the safe and sustainable coexistence of humans, sharks, and their prey, in an era of accelerated environmental change.

DNA metabarcoding of cloacal swabs provides insight into diets of highly migratory sharks in the Mid-Atlantic Bight.

The abundances of migratory shark species observed throughout the Mid-Atlantic Bight (MAB) during productive summer months suggest that this region provides critical habitat and prey resources to these taxa. However, the principal prey assemblages sustaining migratory shark biomass in this region are poorly defined. We applied high-throughput DNA metabarcoding to shark feces derived from cloacal swabs across nine species of Carcharhinid and Lamnid sharks to (1) quantify the contribution of broad taxa (e.g., invertebrates, fishes) supporting shark biomass during seasonal residency in the MAB and (2) determine whether the species displayed distinct dietary preference indicative of resource partitioning. DNA metabarcoding resulted in high taxonomic (species-level) resolution of shark diets with actinopterygian and elasmobranch fishes as the dominant prey categories across the species. DNA metabarcoding identified several key prey groups consistent across shark taxa that are likely integral for sustaining their biomass in this region, including Atlantic menhaden (Brevoortia tyrannus), Atlantic mackerel (Scomber scombrus), and benthic elasmobranchs, including skates. Our results are consistent with previously published stomach content data for the shark species of similar size range in the Northwest Atlantic Ocean, supporting the efficacy of cloacal swab DNA metabarcoding as a minimally invasive diet reconstruction technique. The high reliance of several shark species on Atlantic menhaden could imply wasp-waist food-web conditions during the summer months, whereby high abundances of forage fishes sustain a diverse suite of migratory sharks within a complex, seasonal food web.

Stretch Harmonizes Sarcomere Strain Across the Cardiomyocyte.

BACKGROUND: Increasing cardiomyocyte contraction during myocardial stretch serves as the basis for the Frank-Starling mechanism in the heart. However, it remains unclear how this phenomenon occurs regionally within cardiomyocytes, at the level of individual sarcomeres. We investigated sarcomere contractile synchrony and how intersarcomere dynamics contribute to increasing contractility during cell lengthening. METHODS: Sarcomere strain and Ca2+ were simultaneously recorded in isolated left ventricular cardiomyocytes during 1 Hz field stimulation at 37 °C, at resting length and following stepwise stretch. RESULTS: We observed that in unstretched rat cardiomyocytes, differential sarcomere deformation occurred during each beat. Specifically, while most sarcomeres shortened during the stimulus, ≈10% to 20% of sarcomeres were stretched or remained stationary. This nonuniform strain was not traced to regional Ca2+ disparities but rather shorter resting lengths and lower force production in systolically stretched sarcomeres. Lengthening of the cell recruited additional shortening sarcomeres, which increased contractile efficiency as less negative, wasted work was performed by stretched sarcomeres. Given the known role of titin in setting sarcomere dimensions, we next hypothesized that modulating titin expression would alter intersarcomere dynamics. Indeed, in cardiomyocytes from mice with titin haploinsufficiency, we observed greater variability in resting sarcomere length, lower recruitment of shortening sarcomeres, and impaired work performance during cell lengthening. CONCLUSIONS: Graded sarcomere recruitment directs cardiomyocyte work performance, and harmonization of sarcomere strain increases contractility during cell stretch. By setting sarcomere dimensions, titin controls sarcomere recruitment, and its lowered expression in haploinsufficiency mutations impairs cardiomyocyte contractility.

BIN1, Myotubularin, and Dynamin-2 Coordinate T-Tubule Growth in Cardiomyocytes.

BACKGROUND: Transverse tubules (t-tubules) form gradually in the developing heart, critically enabling maturation of cardiomyocyte Ca2+ homeostasis. The membrane bending and scaffolding protein BIN1 (bridging integrator 1) has been implicated in this process. However, it is unclear which of the various reported BIN1 isoforms are involved, and whether BIN1 function is regulated by its putative binding partners MTM1 (myotubularin), a phosphoinositide 3'-phosphatase, and DNM2 (dynamin-2), a GTPase believed to mediate membrane fission. METHODS: We investigated the roles of BIN1, MTM1, and DNM2 in t-tubule formation in developing mouse cardiomyocytes, and in gene-modified HL-1 and human-induced pluripotent stem cell-derived cardiomyocytes. T-tubules and proteins of interest were imaged by confocal and Airyscan microscopy, and expression patterns were examined by RT-qPCR and Western blotting. Ca2+ release was recorded using Fluo-4. RESULTS: We observed that in the postnatal mouse heart, BIN1 localizes along Z-lines from early developmental stages, consistent with roles in initial budding and scaffolding of t-tubules. T-tubule proliferation and organization were linked to a progressive and parallel increase in 4 detected BIN1 isoforms. All isoforms were observed to induce tubulation in cardiomyocytes but produced t-tubules with differing geometries. BIN1-induced tubulations contained the L-type Ca2+ channel, were colocalized with caveolin-3 and the ryanodine receptor, and effectively triggered Ca2+ release. BIN1 upregulation during development was paralleled by increasing expression of MTM1. Despite no direct binding between MTM1 and murine cardiac BIN1 isoforms, which lack exon 11, high MTM1 levels were necessary for BIN1-induced tubulation, indicating a central role of phosphoinositide homeostasis. In contrast, the developing heart exhibited declining levels of DNM2. Indeed, we observed that high levels of DNM2 are inhibitory for t-tubule formation, although this protein colocalizes with BIN1 along Z-lines, and binds all 4 isoforms. CONCLUSIONS: These findings indicate that BIN1, MTM1, and DNM2 have balanced and collaborative roles in controlling t-tubule growth in cardiomyocytes.

Augmenting workload drives T-tubule assembly in developing cardiomyocytes.

Contraction of cardiomyocytes is initiated at subcellular elements called dyads, where L-type Ca2+ channels in t-tubules are located within close proximity to ryanodine receptors in the sarcoplasmic reticulum. While evidence from small rodents indicates that dyads are assembled gradually in the developing heart, it is unclear how this process occurs in large mammals. We presently examined dyadic formation in fetal and newborn sheep (Ovis aries), and the regulation of this process by fetal cardiac workload. By employing advanced imaging methods, we demonstrated that t-tubule growth and dyadic assembly proceed gradually during fetal sheep development, from 93 days of gestational age until birth (147 days). This process parallels progressive increases in fetal systolic blood pressure, and includes step-wise colocalization of L-type Ca2+ channels and the Na+ /Ca2+ exchanger with ryanodine receptors. These proteins are upregulated together with the dyadic anchor junctophilin-2 during development, alongside changes in the expression of amphiphysin-2 (BIN1) and its partner proteins myotubularin and dynamin-2. Increasing fetal systolic load by infusing plasma or occluding the post-ductal aorta accelerated t-tubule growth. Conversely, reducing fetal systolic load with infusion of enalaprilat, an angiotensin converting enzyme inhibitor, blunted t-tubule formation. Interestingly, altered t-tubule densities did not relate to changes in dyadic junctions, or marked changes in the expression of dyadic regulatory proteins, indicating that distinct signals are responsible for maturation of the sarcoplasmic reticulum. In conclusion, augmenting blood pressure and workload during normal fetal development critically promotes t-tubule growth, while additional signals contribute to dyadic assembly. KEY POINTS: T-tubule growth and dyadic assembly proceed gradually in cardiomyocytes during fetal sheep development, from 93 days of gestational age until the post-natal stage. Increasing fetal systolic load by infusing plasma or occluding the post-ductal aorta accelerated t-tubule growth and hypertrophy. In contrast, reducing fetal systolic load by enalaprilat infusion slowed t-tubule development and decreased cardiomyocyte size. Load-dependent modulation of t-tubule maturation was linked to altered expression patterns of the t-tubule regulatory proteins junctophilin-2 and amphiphysin-2 (BIN1) and its protein partners. Altered t-tubule densities did not influence dyadic formation, indicating that distinct signals are responsible for maturation of the sarcoplasmic reticulum.

Energetic connectivity of diverse elasmobranch populations - implications for ecological resilience.

Understanding the factors shaping patterns of ecological resilience is critical for mitigating the loss of global biodiversity. Throughout aquatic environments, highly mobile predators are thought to serve as important vectors of energy between ecosystems thereby promoting stability and resilience. However, the role these predators play in connecting food webs and promoting energy flow remains poorly understood in most contexts. Using carbon and nitrogen isotopes, we quantified the use of several prey resource pools (small oceanic forage, large oceanics, coral reef, and seagrass) by 17 species of elasmobranch fishes (n = 351 individuals) in The Bahamas to determine their functional diversity and roles as ecosystem links. We observed remarkable functional diversity across species and identified four major groups responsible for connecting discrete regions of the seascape. Elasmobranchs were responsible for promoting energetic connectivity between neritic, oceanic and deep-sea ecosystems. Our findings illustrate how mobile predators promote ecosystem connectivity, underscoring their functional significance and role in supporting ecological resilience. More broadly, strong predator conservation efforts in developing island nations, such as The Bahamas, are likely to yield ecological benefits that enhance the resilience of marine ecosystems to combat imminent threats such as habitat degradation and climate change.

Tubulator: an automated approach to analysis of t-tubule and dyadic organization in cardiomyocytes.

During cardiac disease, t-tubules and dyads are remodelled and disrupted within cardiomyocytes, thereby reducing cardiac performance. Given the pathological implications of such dyadic remodelling, robust and versatile tools for characterizing these sub-cellular structures are needed. While analysis programs for continuous and regular structures such as rodent ventricular t-tubules are available, at least in two dimensions, these approaches are less appropriate for assessment of more irregular structures, such as dyadic proteins and non-rodent t-tubules. Here, we demonstrate versatile, easy-to-use software that performs such analyses. This software, called Tubulator, enables automated analysis of t-tubules and dyadic proteins alike, in both tissue sections and isolated myocytes. The program measures densities of subcellular structures and proteins in individual cells, quantifies their distribution into transversely and longitudinally oriented elements, and supports detailed co-localization analyses. Importantly, Tubulator provides tools for three-dimensional assessment and rendering of image stacks, extending examinations from the single plane to the whole-myocyte level. To provide insight into the consequences of dyadic organization for synchrony of Ca2+ handling, Tubulator also creates 'distance maps', by calculating the distance from all cytosolic positions to the nearest t-tubule and/or dyad. In conclusion, this freely accessible program provides detailed automated analysis of the three-dimensional nature of dyadic and t-tubular structures. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

Energetic consequences of resource use diversity in a marine carnivore.

Understanding how intraspecific variation in the use of prey resources impacts energy metabolism has strong implications for predicting long-term fitness and is critical for predicting population-to-community level responses to environmental change. Here, we examine the energetic consequences of variable prey resource use in a widely distributed marine carnivore, juvenile sand tiger sharks (Carcharias taurus). We used carbon and nitrogen isotope analysis to identify three primary prey resource pools-demersal omnivores, pelagic forage, and benthic detritivores and estimated the proportional assimilation of each resource using Bayesian mixing models. We then quantified how the utilization of these resource pools impacted the concentrations of six plasma lipids and how this varied by ontogeny. Sharks exhibited variable reliance on two of three predominant prey resource pools: demersal omnivores and pelagic forage. Resource use variation was a strong predictor of energetic condition, whereby individuals more reliant upon pelagic forage exhibited higher blood plasma concentrations of very low-density lipoproteins, cholesterol, and triglycerides. These findings underscore how intraspecific variation in resource use may impact the energy metabolism of animals, and more broadly, that natural and anthropogenically driven fluctuations in prey resources could have longer term energetic consequences.

Risk assessment for seafood consumers exposed to mercury and other trace elements in fish from Long Island, New York, USA.

We determined concentrations of Hg, Pb, Cd, Cr, As, Ni, Ag, Se, Cu, and Zn in muscle tissue of six commonly consumed Long Island fish species (black seabass, bluefish, striped bass, summer flounder, tautog, and weakfish, total sample size = 1211) caught off Long Island, New York in 2018 and 2019. Long-term consumption of these coastal fish could pose health risks largely due to Hg exposure; concentrations of the other trace elements were well below levels considered toxic for humans. By combining the measured Hg concentrations in the fish (means ranging from 0.11 to 0.27 mg/kg among the fish species), the average seafood consumption rate, and the current US EPA Hg reference dose (0.0001 mg/kg/d), it was concluded that seafood consumption should be limited to four fish meals per month for adults for some fish (bluefish, tautog) and half that for young children. Molar ratios of Hg:Se exceeded 1 for some black seabass, bluefish, tautog, and weakfish.

Multimodal human thymic profiling reveals trajectories and cellular milieu for T agonist selection.

To prevent autoimmunity, thymocytes expressing self-reactive T cell receptors (TCRs) are negatively selected, however, divergence into tolerogenic, agonist selected lineages represent an alternative fate. As thymocyte development, selection, and lineage choices are dependent on spatial context and cell-to-cell interactions, we have performed Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) and spatial transcriptomics on paediatric human thymu​​s. Thymocytes expressing markers of strong TCR signalling diverged from the conventional developmental trajectory prior to CD4+ or CD8+ lineage commitment, while markers of different agonist selected T cell populations (CD8αα(I), CD8αα(II), T(agonist), Treg(diff), and Treg) exhibited variable timing of induction. Expression profiles of chemokines and co-stimulatory molecules, together with spatial localisation, supported that dendritic cells, B cells, and stromal cells contribute to agonist selection, with different subsets influencing thymocytes at specific developmental stages within distinct spatial niches. Understanding factors influencing agonist T cells is needed to benefit from their immunoregulatory effects in clinical use.

Corrigendum: The Physiology and Pathophysiology of T-Tubules in the Heart.

[This corrects the article DOI: 10.3389/fphys.2021.718404.].

The Physiology and Pathophysiology of T-Tubules in the Heart.

In cardiomyocytes, invaginations of the sarcolemmal membrane called t-tubules are critically important for triggering contraction by excitation-contraction (EC) coupling. These structures form functional junctions with the sarcoplasmic reticulum (SR), and thereby enable close contact between L-type Ca2+ channels (LTCCs) and Ryanodine Receptors (RyRs). This arrangement in turn ensures efficient triggering of Ca2+ release, and contraction. While new data indicate that t-tubules are capable of exhibiting compensatory remodeling, they are also widely reported to be structurally and functionally compromised during disease, resulting in disrupted Ca2+ homeostasis, impaired systolic and/or diastolic function, and arrhythmogenesis. This review summarizes these findings, while highlighting an emerging appreciation of the distinct roles of t-tubules in the pathophysiology of heart failure with reduced and preserved ejection fraction (HFrEF and HFpEF). In this context, we review current understanding of the processes underlying t-tubule growth, maintenance, and degradation, underscoring the involvement of a variety of regulatory proteins, including junctophilin-2 (JPH2), amphiphysin-2 (BIN1), caveolin-3 (Cav3), and newer candidate proteins. Upstream regulation of t-tubule structure/function by cardiac workload and specifically ventricular wall stress is also discussed, alongside perspectives for novel strategies which may therapeutically target these mechanisms.

Phospholamban antisense oligonucleotides improve cardiac function in murine cardiomyopathy.

Heart failure (HF) is a major cause of morbidity and mortality worldwide, highlighting an urgent need for novel treatment options, despite recent improvements. Aberrant Ca2+ handling is a key feature of HF pathophysiology. Restoring the Ca2+ regulating machinery is an attractive therapeutic strategy supported by genetic and pharmacological proof of concept studies. Here, we study antisense oligonucleotides (ASOs) as a therapeutic modality, interfering with the PLN/SERCA2a interaction by targeting Pln mRNA for downregulation in the heart of murine HF models. Mice harboring the PLN R14del pathogenic variant recapitulate the human dilated cardiomyopathy (DCM) phenotype; subcutaneous administration of PLN-ASO prevents PLN protein aggregation, cardiac dysfunction, and leads to a 3-fold increase in survival rate. In another genetic DCM mouse model, unrelated to PLN (Cspr3/Mlp-/-), PLN-ASO also reverses the HF phenotype. Finally, in rats with myocardial infarction, PLN-ASO treatment prevents progression of left ventricular dilatation and improves left ventricular contractility. Thus, our data establish that antisense inhibition of PLN is an effective strategy in preclinical models of genetic cardiomyopathy as well as ischemia driven HF.

CaMKII inhibition has dual effects on spontaneous Ca2+ release and Ca2+ alternans in ventricular cardiomyocytes from mice with a gain-of-function RyR2 mutation.

In conditions with abnormally increased activity of the cardiac ryanodine receptor (RyR2), Ca2+/calmodulin-dependent protein kinase II (CaMKII) can contribute to a further destabilization of RyR2 that results in triggered arrhythmias. Therefore, inhibition of CaMKII in such conditions has been suggested as a strategy to suppress RyR2 activity and arrhythmias. However, suppression of RyR2 activity can lead to the development of arrhythmogenic Ca2+ alternans. The aim of this study was to test whether the suppression of RyR2 activity caused by inhibition of CaMKII increases propensity for Ca2+ alternans. We studied spontaneous Ca2+ release events and Ca2+ alternans in isolated left ventricular cardiomyocytes from mice carrying the gain-of-function RyR2 mutation RyR2-R2474S and from wild-type mice. CaMKII inhibition by KN-93 effectively decreased the frequency of spontaneous Ca2+ release events in RyR2-R2474S cardiomyocytes exposed to the ß-adrenoceptor agonist isoprenaline. However, KN-93-treated RyR2-R2474S cardiomyocytes also showed increased propensity for Ca2+ alternans and increased Ca2+ alternans ratio compared with both an inactive analog of KN-93 and with vehicle-treated controls. This increased propensity for Ca2+ alternans was explained by prolongation of Ca2+ release refractoriness. Importantly, the increased propensity for Ca2+ alternans in KN-93-treated RyR2-R2474S cardiomyocytes did not surpass that of wild type. In conclusion, inhibition of CaMKII efficiently reduces spontaneous Ca2+ release but promotes Ca2+ alternans in RyR2-R2474S cardiomyocytes with a gain-of-function RyR2 mutation. The dominant effect in RyR2-R2474S is to reduce spontaneous Ca2+ release, which supports this intervention as a therapeutic strategy in this specific condition. However, future studies on CaMKII inhibition in conditions with increased propensity for Ca2+ alternans should include investigation of both phenomena.NEW & NOTEWORTHY Genetically increased RyR2 activity promotes arrhythmogenic Ca2+ release. Inhibition of CaMKII suppresses RyR2 activity and arrhythmogenic Ca2+ release. Suppression of RyR2 activity prolongs refractoriness of Ca2+ release. Prolonged refractoriness of Ca2+ release leads to arrhythmogenic Ca2+ alternans. CaMKII inhibition promotes Ca2+ alternans by prolonging Ca2+ release refractoriness.

Shark tooth collagen stable isotopes (δ15 N and δ13 C) as ecological proxies.

The isotopic composition of tooth-bound collagen has long been used to reconstruct dietary patterns of animals in extant and palaeoecological systems. For sharks that replace teeth rapidly in a conveyor-like system, stable isotopes of tooth collagen (δ13 CTeeth & δ15 NTeeth ) are poorly understood and lacking in ecological context relative to other non-lethally sampled tissues. This tissue holds promise, because shark jaws may preserve isotopic chronologies from which to infer individual-level ecological patterns across a range of temporal resolutions. Carbon and nitrogen stable isotope values were measured and compared between extracted tooth collagen and four other non-lethally sampled tissues of varying isotopic turnover rates: blood plasma, red blood cells, fin and muscle, from eight species of sharks. Individual-level isotopic variability of shark tooth collagen was evaluated by profiling teeth of different ages across whole jaws for the shortfin mako shark Isurus oxyrinchus and sandbar shark Carcharhinus plumbeus. Measurements of δ13 CTeeth and δ15 NTeeth were positively correlated with isotopic values from the four other tissues. Collagen δ13 C was consistently 13 C-enriched relative to all other tissues. Patterns for δ15 N were slightly less uniform; tooth collagen was generally 15 N-enriched relative to muscle and red blood cells, but congruent with fin and blood plasma (values clustered around a 1:1 relationship). Significant within-individual variability was observed across whole shortfin mako shark (δ13 C range = 1.4‰, δ15 N range = 3.6‰) and sandbar shark (δ13 C range = 1.2‰-2.4‰, δ15 N range = 1.7‰-2.4‰) jaws, which trended with tooth age. We conclude that amino acid composition and associated patterns of isotopic fractionation result in predictable isotopic offsets between tissues. Within-individual variability of tooth collagen stable isotope values suggests teeth of different ages may serve as ecological chronologies, that could be applied to studies on migration and individual-level diet variation across diverse time-scales. Greater understanding of tooth replacement rates, isotopic turnover and associated fractionation of tooth collagen will help refine potential ecological inferences, outlining clear goals for future scientific inquiry.

Regional variation in mercury bioaccumulation among NW Atlantic Golden (Lopholatilus chamaeleonticeps) and Blueline (Caulolatilus microps) Tilefish.

Mercury (Hg) concentrations in fishes from the NW Atlantic Ocean pose concern due to the importance of this region to U.S. fisheries harvest. In this study, total Hg (THg) concentrations and nitrogen stable isotope (δ15N) values were quantified in muscle tissues sampled from Golden (Lopholatilus chamaeleonticeps) and Blueline (Caulolatilus microps) Tilefish collected during a fishery-independent survey conducted in the NW Atlantic to compare bioaccumulation patterns between these species. Total Hg concentrations averaged (±SD) 0.4 ± 0.4 µg/g dry weight (d.w.) for L. chamaeleonticeps and 1.1 ± 0.7 µg/g d.w. for C. microps with <2% of all sampled fish, those >70 cm fork length, exceeding the most restrictive USEPA regulatory guidelines for human consumption (THg > 0.46 µg/g w.w.), when converted to wet weight concentrations. The THg concentrations reported here for individuals from the NW Atlantic stock are comparable to those reported for similarly sized individuals collected from the SW Atlantic stock but notably lower than those reported for Gulf of Mexico L. chamaeleonticeps, indicating different Hg exposure and assimilation kinetics for fish from the NW Atlantic, and highlights the broad geographic variability of Hg bioaccumulation among Tilefish stocks. Caulolatilus microps had higher δ15N values relative to L. chamaeleonticeps and a pattern of decreasing THg concentrations was also present from south to north across the study range. It is concluded that this trophic difference and spatial pattern in Tilefish THg concentrations emphasizes the habitat and resource partitioning mechanisms described for these sympatric species that permits their coexistence in the continental shelf environment. Importantly, regional variability in THg concentrations accentuate the possible roles of fine-scale biotic and abiotic processes that can act to regulate Hg bioaccumulation among individuals and species.

Role of t-tubule remodeling on mechanisms of abnormal calcium release during heart failure development in canine ventricle.

The goal of this work was to investigate the role of t-tubule (TT) remodeling in abnormal Ca2+ cycling in ventricular myocytes of failing dog hearts. Heart failure (HF) was induced using rapid right ventricular pacing. Extensive changes in echocardiographic parameters, including left and right ventricular dilation and systolic dysfunction, diastolic dysfunction, elevated left ventricular filling pressures, and abnormal cardiac mechanics, indicated that severe HF developed. TT loss was extensive when measured as the density of total cell volume, derived from three-dimensional confocal image analysis, and significantly increased the distances in the cell interior to closest cell membrane. Changes in Ca2+ transients indicated increases in heterogeneity of Ca2+ release along the cell length. When critical properties of Ca2+ release variability were plotted as a function of TT organization, there was a complex, nonlinear relationship between impaired calcium release and decreasing TT organization below a certain threshold of TT organization leading to increased sensitivity in Ca2+ release below a TT density threshold of 1.5%. The loss of TTs was also associated with a greater incidence of triggered Ca2+ waves during rapid pacing. Finally, virtually all of these observations were replicated by acute detubulation by formamide treatment, indicating an important role of TT remodeling in impaired Ca2+ cycling. We conclude that TT remodeling itself is a major contributor to abnormal Ca2+ cycling in HF, reducing myocardial performance. The loss of TTs is also responsible for a greater incidence of triggered Ca2+ waves that may play a role in ventricular arrhythmias arising in HF.NEW & NOTEWORTHY Three-dimensional analysis of t-tubule density showed t-tubule disruption throughout the whole myocyte in failing dog ventricle. A double-linear relationship between Ca2+ release and t-tubule density displays a steeper slope at t-tubule densities below a threshold value (∼1.5%) above which there is little effect on Ca2+ release (T-tubule reserve). T-tubule loss increases incidence of triggered Ca2+ waves. Chemically induced t-tubule disruption suggests that t-tubule loss alone is a critical component of abnormal Ca2+ cycling in heart failure.