Effect of a Lactobacilli-Based Direct-Fed Microbial Product on Gut Microbiota and Gastrointestinal Morphological Changes.

The calf's gastrointestinal tract (GIT) microbiome undergoes rapid shifts during early post-natal life, which can directly affect calf performance. The objectives of this study were to characterise and compare differences in the establishment and succession of GIT microbiota, GIT morphological changes, and the growth of dairy calves from birth until weaned. Forty-four newborn Holstein-Friesian calves were randomly selected and assigned to Treatment (TRT) and Control (CON) groups. The TRT group calves received a once-daily dose of a direct-fed microbial (DFM) liquid product containing Lacticaseibacillus paracasei, Lentilactobacillus buchneri, and Lacticaseibacillus casei, all formerly known as Lactobacillus. Fresh faecal samples were manually taken from the rectum of all calves, and gross necropsy was performed on the forestomachs and gastrointestinal tracts. Bacterial DNA was extracted from frozen faecal samples for 16S rRNA gene amplicon sequencing. Calves in the TRT group had greater live weights (p = 0.02) at weaning compared with calves in the CON group (mean = 69.18 kg, SD = 13.37 kg). The average daily live weight gain (ADG) and total feed intake were similar between the two groups. Calves in the TRT group had greater duodenum, abomasum, and reticulum weights (p = 0.05). Rumen and intestinal development (p < 0.05) and faecal microbial diversity (p < 0.05) were more pronounced in the TRT group. The relative abundances of eight genera differed (p < 0.001) between the groups. Supplementing calves with the LAB-based DFM increased live weight at weaning and had a more pronounced effect on the development of rumen and the gastrointestinal tract and on microbiota diversity and evenness. Future work is needed to better understand the potential association of LAB-DFM products on gut mucosa-associated microbiota.

Divergent maturational patterns of the infant bacterial and fungal gut microbiome in the first year of life are associated with inter-kingdom community dynamics and infant nutrition.

BACKGROUND: The gut microbiome undergoes primary ecological succession over the course of early life before achieving ecosystem stability around 3 years of age. These maturational patterns have been well-characterized for bacteria, but limited descriptions exist for other microbiota members, such as fungi. Further, our current understanding of the prevalence of different patterns of bacterial and fungal microbiome maturation and how inter-kingdom dynamics influence early-life microbiome establishment is limited. RESULTS: We examined individual shifts in bacterial and fungal alpha diversity from 3 to 12 months of age in 100 infants from the CHILD Cohort Study. We identified divergent patterns of gut bacterial or fungal microbiome maturation in over 40% of infants, which were characterized by differences in community composition, inter-kingdom dynamics, and microbe-derived metabolites in urine, suggestive of alterations in the timing of ecosystem transitions. Known microbiome-modifying factors, such as formula feeding and delivery by C-section, were associated with atypical bacterial, but not fungal, microbiome maturation patterns. Instead, fungal microbiome maturation was influenced by prenatal exposure to artificially sweetened beverages and the bacterial microbiome, emphasizing the importance of inter-kingdom dynamics in early-life colonization patterns. CONCLUSIONS: These findings highlight the ecological and environmental factors underlying atypical patterns of microbiome maturation in infants, and the need to incorporate multi-kingdom and individual-level perspectives in microbiome research to improve our understandings of gut microbiome maturation patterns in early life and how they relate to host health. Video Abstract.

Weighted Gene Co-Expression Network Analysis Identifies a Functional Guild and Metabolite Cluster Mediating the Relationship between Mucosal Inflammation and Adherence to the Mediterranean Diet in Ulcerative Colitis.

Diet influences the pathogenesis and clinical course of inflammatory bowel disease (IBD). The Mediterranean diet (MD) is linked to reductions in inflammatory biomarkers and alterations in microbial taxa and metabolites associated with health. We aimed to identify features of the gut microbiome that mediate the relationship between the MD and fecal calprotectin (FCP) in ulcerative colitis (UC). Weighted gene co-expression network analysis (WGCNA) was used to identify modules of co-abundant microbial taxa and metabolites correlated with the MD and FCP. The features considered were gut microbial taxa, serum metabolites, dietary components, short-chain fatty acid and bile acid profiles in participants that experienced an increase (n = 13) or decrease in FCP (n = 16) over eight weeks. WGCNA revealed ten modules containing sixteen key features that acted as key mediators between the MD and FCP. Three taxa (Faecalibacterium prausnitzii, Dorea longicatena, Roseburia inulinivorans) and a cluster of four metabolites (benzyl alcohol, 3-hydroxyphenylacetate, 3-4-hydroxyphenylacetate and phenylacetate) demonstrated a strong mediating effect (ACME: -1.23, p = 0.004). This study identified a novel association between diet, inflammation and the gut microbiome, providing new insights into the underlying mechanisms of how a MD may influence IBD. See clinicaltrials.gov (NCT04474561).

Isha yoga practices, vegan diet, and participation in Samyama meditation retreat: impact on the gut microbiome & metabolome - a non-randomized trial.

BACKGROUND: Growing evidence suggests a role for gut bacteria and their metabolites in host-signaling responses along the gut-brain axis which may impact mental health. Meditation is increasingly utilized to combat stress, anxiety, and depression symptoms. However, its impact on the microbiome remains unclear. This study observes the effects of preparation and participation in an advanced meditation program (Samyama) implemented with a vegan diet including 50% raw foods, on gut microbiome and metabolites profiles. METHODS: There were 288 subjects for this study. Stool samples were collected at 3-time points for meditators and household controls. Meditators prepared for 2 months for the Samyama, incorporating daily yoga and meditation practices with a vegan diet including 50% raw foods. Subjects were requested to submit stool samples for 3 time points - 2 months before Samyama (T1), right before Samyama (T2), and 3 months following Samyama (T3). 16 s rRNA sequencing was used to study participants' microbiome. Alpha and beta diversities along with short-chain fatty acid (SCFA) were assessed. Metabolomics were performed on a mass spectrometer coupled to a UHLPC system and analyzed by El-MAVEN software. RESULTS: Alpha diversity showed no significant differences between meditators and controls, while beta diversity showed significant changes (padj = 0.001) after Samyama in meditators' microbiota composition. After the preparation phase, changes in branched short-chain fatty acids, higher levels of iso-valerate (padj = 0.02) and iso-buytrate (padj = 0.019) were observed at T2 in meditators. Other metabolites were also observed to have changed in meditators at timepoint T2. CONCLUSION: This study examined the impact of an advanced meditation program combined with a vegan diet on the gut microbiome. There was an increase in beneficial bacteria even three months after the completion of the Samyama program. Further study is warranted to validate current observations and investigate the significance and mechanisms of action related to diet, meditation, and microbial composition and function, on psychological processes, including mood. TRIAL REGISTRATION: Registration number: NCT04366544 ; Registered on 29/04/2020.

The Pregnane X Receptor and Indole-3-Propionic Acid Shape the Intestinal Mesenchyme to Restrain Inflammation and Fibrosis.

BACKGROUND & AIMS: Fibrosis is a common complication of inflammatory bowel diseases (IBDs). The pregnane X receptor (PXR) (encoded by NR1I2) suppresses intestinal inflammation and has been shown to influence liver fibrosis. In the intestine, PXR signaling is influenced by microbiota-derived indole-3-propionic acid (IPA). Here, we sought to assess the role of the PXR in regulating intestinal inflammation and fibrosis. METHODS: Intestinal inflammation was induced using dextran sulfate sodium (DSS). Fibrosis was assessed in wild-type (WT), Nr1i2-/-, epithelial-specific Nr1i2-/-, and fibroblast-specific Nr1i2-/- mice. Immune cell influx was quantified by flow cytometry and cytokines by Luminex. Myofibroblasts isolated from WT and Nr1i2-/- mice were stimulated with cytomix or lipopolysaccharide, and mediator production was assessed by quantitative polymerase chain reaction and Luminex. RESULTS: After recovery from DSS-induced colitis, WT mice exhibited fibrosis, a response that was exacerbated in Nr1i2-/- mice. This was correlated with greater neutrophil infiltration and innate cytokine production. Deletion of the PXR in fibroblasts, but not the epithelium, recapitulated this phenotype. Inflammation and fibrosis were reduced by IPA administration, whereas depletion of the microbiota exaggerated intestinal fibrosis. Nr1i2-deficient myofibroblasts were hyperresponsive to stimulation, producing increased levels of inflammatory mediators compared with WT cells. In biopsies from patients with active Crohn's disease (CD) and ulcerative colitis (UC), expression of NR1I2 was reduced, correlating with increased expression of fibrotic and innate immune genes. Finally, both CD and UC patients exhibited reduced levels of fecal IPA. CONCLUSIONS: These data highlight a role for IPA and its interactions with the PXR in regulating the mesenchyme and the development of inflammation and fibrosis, suggesting microbiota metabolites may be a vital determinant in the progression of fibrotic complications in IBD.

Small intestinal resident eosinophils maintain gut homeostasis following microbial colonization.

The intestine harbors a large population of resident eosinophils, yet the function of intestinal eosinophils has not been explored. Flow cytometry and whole-mount imaging identified eosinophils residing in the lamina propria along the length of the intestine prior to postnatal microbial colonization. Microscopy, transcriptomic analysis, and mass spectrometry of intestinal tissue revealed villus blunting, altered extracellular matrix, decreased epithelial cell turnover, increased gastrointestinal motility, and decreased lipid absorption in eosinophil-deficient mice. Mechanistically, intestinal epithelial cells released IL-33 in a microbiota-dependent manner, which led to eosinophil activation. The colonization of germ-free mice demonstrated that eosinophil activation in response to microbes regulated villous size alterations, macrophage maturation, epithelial barrier integrity, and intestinal transit. Collectively, our findings demonstrate a critical role for eosinophils in facilitating the mutualistic interactions between the host and microbiota and provide a rationale for the functional significance of their early life recruitment in the small intestine.

Sequence characterisation and novel insights into bovine mastitis-associated Streptococcus uberis in dairy herds.

Streptococcus uberis is one of the most frequent mastitis-causing pathogens isolated from dairy cows. Further understanding of S. uberis genetics may help elucidate the disease pathogenesis. We compared the genomes of S. uberis isolates cultured from dairy cows located in distinctly different geographic regions of Australia. All isolates had novel multi locus sequence types (MLST) indicating a highly diverse population of S. uberis. Global clonal complexes (GCC) were more conserved. GCC ST86 and GCC ST143 represented 30% of the total isolates (n = 27) and were clustered within different geographic regions. Core genome phylogeny revealed low phylogenetic clustering by region, isolation source, and MLST. Identification of putative sortase (srtA) substrates and generation of a custom putative virulence factor database revealed genes which may explain the affinity of S. uberis for mammary tissue, evasion of antimicrobial efforts and disease pathogenesis. Of 27 isolates, four contained antibiotic resistance genes including an antimicrobial resistance cluster containing mel/mef(A), mrsE, vatD, lnuD, and transposon-mediated lnuC was also identified. These are novel genes for S. uberis, which suggests interspecies lateral gene transfer. The presence of resistance genes across the two geographic regions tested within one country supports the need for a careful, tailored, implementation and monitoring of antimicrobial stewardship.

Effect of a functional fibre supplement on glycemic control when added to a year-long medically supervised weight management program in adults with type 2 diabetes.

PURPOSE: Soluble fibre beneficially affects metabolism but whether it can augment the reductions in glycemia induced through intensive weight management has not been extensively studied. Our objective was to examine the adjunct effect of the soluble viscous fibre PGX® on glycemic control in adults with type 2 diabetes (T2D) enrolled in a year-long medically supervised weight management program. METHODS: In a placebo-controlled, double-blind study, 290 adults with overweight/obesity and T2D were randomized to receive PGX (15-20 g/day) or isocaloric placebo (rice flour, 6.4-8.6 g/day) as an adjunct to intensive weight management for 52 weeks. The primary outcome was change in glycemic control (HbA1c). Other outcome measures included weight loss, blood lipids, blood pressure, cytokines and fecal microbiota. RESULTS: Compared to baseline HbA1c in PGX (7.2 ± 1.1%) and placebo (7.0 ± 0.9%) groups, there was a significant reduction at 16 and 26 weeks, however, only PGX showed a significant absolute reduction of 0.23% at 52 weeks; there were no between-group differences in HbA1c. At 52 weeks, only PGX significantly decreased body weight compared to baseline and reduced waist circumference at all time points. Compared to baseline, only PGX showed a significant reduction in LDL cholesterol at 16 and 26 weeks. PGX significantly increased the relative abundance of Collinsella, Parabacteroides and Roseburia. CONCLUSION: Adding PGX to a weight management program for individuals with T2D provides a sustained reduction in HbA1c compared to placebo. Improvements in other metabolic outcomes suggest that PGX may be a promising adjunct to weight loss programs in patients with T2D. CLINICAL TRIAL: This trial was registered at ClinicalTrials.gov as NCT01644201.

Survey and Sequence Characterization of Bovine Mastitis-Associated Escherichia coli in Dairy Herds.

Escherichia coli is frequently associated with mastitis in cattle. "Pathogenic" and "commensal" isolates appear to be genetically similar. With a few exceptions, no notable genotypic differences have been found between commensal and mastitis-associated E. coli. In this study, 24 E. coli strains were isolated from dairy cows with clinical mastitis in three geographic regions of Australia (North Queensland, South Queensland, and Victoria), sequenced, then genomically surveyed. There was no observed relationship between sequence type (ST) and region (p = 0.51). The most common Multi Locus Sequence Type was ST10 (38%), then ST4429 (13%). Pangenomic analysis revealed a soft-core genome of 3,463 genes, including genes associated with antibiotic resistance, chemotaxis, motility, adhesion, biofilm formation, and pili. A total of 36 different plasmids were identified and generally found to have local distributions (p = 0.02). Only 2 plasmids contained antibiotic resistance genes, a p1303_5-like plasmid encoding multidrug-resistance (trimethoprim, quaternary ammonium, beta-lactam, streptomycin, sulfonamide, and kanamycin) from two North Queensland isolates on the same farm, while three Victorian isolates from the same farm contained a pCFSAN004177P_01-like plasmid encoding tetracycline-resistance. This pattern is consistent with a local spread of antibiotic resistance through plasmids of bovine mastitis cases. Notably, co-occurrence of plasmids containing virulence factors/antibiotic resistance with putative mobilization was rare, though the multidrug resistant p1303_5-like plasmid was predicted to be conjugative and is of some concern. This survey has provided greater understanding of antibiotic resistance within E. coli-associated bovine mastitis which will allow greater prediction and improved decision making in disease management.

Microbiome-derived inosine modulates response to checkpoint inhibitor immunotherapy.

Several species of intestinal bacteria have been associated with enhanced efficacy of checkpoint blockade immunotherapy, but the underlying mechanisms by which the microbiome enhances antitumor immunity are unclear. In this study, we isolated three bacterial species-Bifidobacterium pseudolongum, Lactobacillus johnsonii, and Olsenella species-that significantly enhanced efficacy of immune checkpoint inhibitors in four mouse models of cancer. We found that intestinal B. pseudolongum modulated enhanced immunotherapy response through production of the metabolite inosine. Decreased gut barrier function induced by immunotherapy increased systemic translocation of inosine and activated antitumor T cells. The effect of inosine was dependent on T cell expression of the adenosine A2A receptor and required costimulation. Collectively, our study identifies a previously unknown microbial metabolite immune pathway activated by immunotherapy that may be exploited to develop microbial-based adjuvant therapies.

Intestinal fungi are causally implicated in microbiome assembly and immune development in mice.

The gut microbiome consists of a multi-kingdom microbial community. Whilst the role of bacteria as causal contributors governing host physiological development is well established, the role of fungi remains to be determined. Here, we use germ-free mice colonized with defined species of bacteria, fungi, or both to differentiate the causal role of fungi on microbiome assembly, immune development, susceptibility to colitis, and airway inflammation. Fungal colonization promotes major shifts in bacterial microbiome ecology, and has an independent effect on innate and adaptive immune development in young mice. While exclusive fungal colonization is insufficient to elicit overt dextran sulfate sodium-induced colitis, bacterial and fungal co-colonization increase colonic inflammation. Ovalbumin-induced airway inflammation reveals that bacterial, but not fungal colonization is necessary to decrease airway inflammation, yet fungi selectively promotes macrophage infiltration in the airway. Together, our findings demonstrate a causal role for fungi in microbial ecology and host immune functionality, and therefore prompt the inclusion of fungi in therapeutic approaches aimed at modulating early life microbiomes.

Maternal low-dose aspartame and stevia consumption with an obesogenic diet alters metabolism, gut microbiota and mesolimbic reward system in rat dams and their offspring.

OBJECTIVE: We examined the impact of maternal low-dose aspartame and stevia consumption on adiposity, glucose tolerance, gut microbiota and mesolimbic pathway in obese dams and their offspring. DESIGN: Following obesity induction, female Sprague-Dawley rats were allocated during pregnancy and lactation to: (1) high fat/sucrose diet (HFS) +water (obese-WTR); (2) HFS +aspartame (obese-APM; 5-7 mg/kg/day); (3) HFS +stevia (obese-STV; 2-3 mg/kg/day). Offspring were weaned onto control diet and water and followed until 18 weeks. Gut microbiota and metabolic outcomes were measured in dams and offspring. Cecal matter from offspring at weaning was used for faecal microbiota transplant (FMT) into germ-free (GF) mice. RESULTS: Maternal APM and STV intake with a HFS diet increased body fat in offspring at weaning and body weight long-term with APM. Maternal APM/HFS consumption impaired glucose tolerance in male offspring at age 8 weeks and both APM and STV altered faecal microbiota in dams and offspring. Maternal obesity/HFS diet affected offspring adiposity and glucose tolerance more so than maternal LCS consumption at age 12 and 18 weeks. APM and STV altered expression of genes in the mesolimbic reward system that may promote consumption of a palatable diet. GF mice receiving an FMT from obese-APM and obese-STV offspring had greater weight gain and body fat and impaired glucose tolerance compared with obese-WTR. CONCLUSION: Maternal low-calorie sweetener consumption alongside HFS may disrupt weight regulation, glucose control and gut microbiota in dams and their offspring most notably in early life despite no direct low-calorie sweetener consumption by offspring.

Cooperation between Constitutive and Inducible Chemokines Enables T Cell Engraftment and Immune Attack in Solid Tumors.

We investigated the role of chemokines in regulating T cell accumulation in solid tumors. CCL5 and CXCL9 overexpression was associated with CD8+ T cell infiltration in solid tumors. T cell infiltration required tumor cell-derived CCL5 and was amplified by IFN-γ-inducible, myeloid cell-secreted CXCL9. CCL5 and CXCL9 coexpression revealed immunoreactive tumors with prolonged survival and response to checkpoint blockade. Loss of CCL5 expression in human tumors was associated with epigenetic silencing through DNA methylation. Reduction of CCL5 expression caused tumor-infiltrating lymphocyte (TIL) desertification, whereas forced CCL5 expression prevented Cxcl9 expression and TILs loss, and attenuated tumor growth in mice through IFN-γ. The cooperation between tumor-derived CCL5 and IFN-γ-inducible CXCR3 ligands secreted by myeloid cells is key for orchestrating T cell infiltration in immunoreactive and immunoresponsive tumors.

Histological improvement of non-alcoholic steatohepatitis with a prebiotic: a pilot clinical trial.

PURPOSE: In obesity and diabetes the liver is highly susceptible to abnormal uptake and storage of fat. In certain individuals hepatic steatosis predisposes to the development of non-alcoholic steatohepatitis (NASH), a disease marked by hepatic inflammation and fibrosis. Although the precise pathophysiology of NASH is unknown, it is believed that the gut microbiota-liver axis influences the development of this disease. With few treatment strategies available for NASH, exploration of gut microbiota-targeted interventions is warranted. We investigated the therapeutic potential of a prebiotic supplement to improve histological parameters of NASH. METHODS: In a placebo-controlled, randomized pilot trial, 14 individuals with liver-biopsy-confirmed NASH [non-alcoholic fatty liver activity score (NAS) ≥ 5] were randomized to receive oligofructose (8 g/day for 12 weeks followed by 16 g/day for 24 weeks) or isocaloric placebo for 9 months. The primary outcome measure was the change in liver biopsy NAS score and the secondary outcomes included changes in body weight, body composition, glucose tolerance, inflammatory markers, and gut microbiota. RESULTS: Independent of weight loss, oligofructose improved liver steatosis relative to placebo and improved overall NAS score (P = 0.016). Bifidobacterium was enhanced by oligofructose, whereas bacteria within Clostridium cluster XI and I were reduced with oligofructose (P < 0.05). There were no adverse side effects that deterred individuals from consuming oligofructose for treatment of this disease. CONCLUSIONS: Independent of other lifestyle changes, prebiotic supplementation reduced histologically-confirmed steatosis in patients with NASH. Larger follow-up studies are warranted. CLINICAL TRIAL: This trial was registered at Clinicaltrials.com as NCT03184376.

Iron Sequestration in Microbiota Biofilms As A Novel Strategy for Treating Inflammatory Bowel Disease.

Significant alterations of intestinal microbiota and anemia are hallmarks of inflammatory bowel disease (IBD). It is widely accepted that iron is a key nutrient for pathogenic bacteria, but little is known about its impact on microbiota associated with IBD. We used a model device to grow human mucosa-associated microbiota in its physiological anaerobic biofilm phenotype. Compared to microbiota from healthy donors, microbiota from IBD patients generate biofilms ex vivo that were larger in size and cell numbers, contained higher intracellular iron concentrations, and exhibited heightened virulence in a model of human intestinal epithelia in vitro and in the nematode Caenorhabditis elegans. We also describe an unexpected iron-scavenging property for an experimental hydrogen sulfide-releasing derivative of mesalamine. The findings demonstrate that this new drug reduces the virulence of IBD microbiota biofilms through a direct reduction of microbial iron intake and without affecting bacteria survival or species composition within the microbiota. Metabolomic analyses indicate that this drug reduces the intake of purine nucleosides (guanosine), increases the secretion of metabolite markers of purine catabolism (urate and hypoxanthine), and reduces the secretion of uracil (a pyrimidine nucleobase) in complex multispecies human biofilms. These findings demonstrate a new pathogenic mechanism for dysbiotic microbiota in IBD and characterize a novel mode of action for a class of mesalamine derivatives. Together, these observations pave the way towards a new therapeutic strategy for treatment of patients with IBD.

The consensus molecular subtypes of colorectal cancer.

Colorectal cancer (CRC) is a frequently lethal disease with heterogeneous outcomes and drug responses. To resolve inconsistencies among the reported gene expression-based CRC classifications and facilitate clinical translation, we formed an international consortium dedicated to large-scale data sharing and analytics across expert groups. We show marked interconnectivity between six independent classification systems coalescing into four consensus molecular subtypes (CMSs) with distinguishing features: CMS1 (microsatellite instability immune, 14%), hypermutated, microsatellite unstable and strong immune activation; CMS2 (canonical, 37%), epithelial, marked WNT and MYC signaling activation; CMS3 (metabolic, 13%), epithelial and evident metabolic dysregulation; and CMS4 (mesenchymal, 23%), prominent transforming growth factor-ß activation, stromal invasion and angiogenesis. Samples with mixed features (13%) possibly represent a transition phenotype or intratumoral heterogeneity. We consider the CMS groups the most robust classification system currently available for CRC-with clear biological interpretability-and the basis for future clinical stratification and subtype-based targeted interventions.

Usability on the p-medicine infrastructure: an extended usability concept.

Usability testing methods are nowadays integrated into the design and development of health-care software, and the need for usability in health-care information technology (IT) is widely accepted by clinicians and researchers. Usability assessment starts with the identification of specific objectives that need to be tested and continues with the definition of evaluation criteria and monitoring procedures before usability tests are performed to assess the quality of all services and tasks. Such a process is implemented in the p-medicine environment and gives feedback iteratively to all software developers in the project. GCP (good clinical practice) criteria require additional usability testing of the software. For the p-medicine project (www.p-medicine.eu), an extended usability concept (EUC) was developed. The EUC covers topics like ease of use, likeability, and usefulness, usability in trial centres characterised by a mixed care and research environment and by extreme time constraints, confidentiality, use of source documents, standard operating procedures (SOA), and quality control during data handling to ensure that all data are reliable and have been processed correctly in terms of accuracy, completeness, legibility, consistence, and timeliness. Here, we describe the p-medicine EUC, focusing on two of the many key tools: ObTiMA and the Ontology Annotator (OA).

Recovery of cardiac calcium release is controlled by sarcoplasmic reticulum refilling and ryanodine receptor sensitivity.

AIMS: In heart cells, the mechanisms underlying refractoriness of the elementary units of sarcoplasmic reticulum (SR) Ca(2+) release, Ca(2+) sparks, remain unclear. We investigated local recovery of SR Ca(2+) release using experimental measurements and mathematical modelling. METHODS AND RESULTS: Repeated Ca(2+) sparks were induced from individual clusters of ryanodine receptors (RyRs) in quiescent rat ventricular myocytes, and we examined how changes in RyR gating influenced the time-dependent recovery of Ca(2+) spark amplitude and triggering probability. Repeated Ca(2+) sparks from individual sites were analysed in the presence of 50 nM ryanodine with: (i) no additional agents (control); (ii) 50 µM caffeine to sensitize RyRs; (iii) 50 µM tetracaine to inhibit RyRs; or (iv) 100 nM isoproterenol to activate ß-adrenergic receptors. Sensitization and inhibition of RyR clusters shortened and lengthened, respectively, the median interval between consecutive Ca(2+) sparks (caffeine 239 ms; control 280 ms; tetracaine 453 ms). Recovery of Ca(2+) spark amplitude, however, was exponential with a time constant of ∼100 ms in all cases. Isoproterenol both accelerated the recovery of Ca(2+) spark amplitude (τ = 58 ms) and shortened the median interval between Ca(2+) sparks (192 ms). The results were recapitulated by a mathematical model in which SR [Ca(2+)] depletion terminates Ca(2+) sparks, but not by an alternative model based on limited depletion and Ca(2+)-dependent inactivation of RyRs. CONCLUSION: Together, the results strongly suggest that: (i) local SR refilling controls Ca(2+) spark amplitude recovery; (ii) Ca(2+) spark triggering depends on both refilling and RyR sensitivity; and (iii) ß-adrenergic stimulation influences both processes.

Predicting local SR Ca(2+) dynamics during Ca(2+) wave propagation in ventricular myocytes.

Of the many ongoing controversies regarding the workings of the sarcoplasmic reticulum (SR) in cardiac myocytes, two unresolved and interconnected topics are 1), mechanisms of calcium (Ca(2+)) wave propagation, and 2), speed of Ca(2+) diffusion within the SR. Ca(2+) waves are initiated when a spontaneous local SR Ca(2+) release event triggers additional release from neighboring clusters of SR release channels (ryanodine receptors (RyRs)). A lack of consensus regarding the effective Ca(2+) diffusion constant in the SR (D(Ca,SR)) severely complicates our understanding of whether dynamic local changes in SR [Ca(2+)] can influence wave propagation. To address this problem, we have implemented a computational model of cytosolic and SR [Ca(2+)] during Ca(2+) waves. Simulations have investigated how dynamic local changes in SR [Ca(2+)] are influenced by 1), D(Ca,SR); 2), the distance between RyR clusters; 3), partial inhibition or stimulation of SR Ca(2+) pumps; 4), SR Ca(2+) pump dependence on cytosolic [Ca(2+)]; and 5), the rate of transfer between network and junctional SR. Of these factors, D(Ca,SR) is the primary determinant of how release from one RyR cluster alters SR [Ca(2+)] in nearby regions. Specifically, our results show that local increases in SR [Ca(2+)] ahead of the wave can potentially facilitate Ca(2+) wave propagation, but only if SR diffusion is relatively slow. These simulations help to delineate what changes in [Ca(2+)] are possible during SR Ca(2+)release, and they broaden our understanding of the regulatory role played by dynamic changes in [Ca(2+)](SR).

Diffusion restrictions surrounding mitochondria: a mathematical model of heart muscle fibers.

Several experiments on permeabilized heart muscle fibers suggest the existence of diffusion restrictions grouping mitochondria and surrounding ATPases. The specific causes of these restrictions are not known, but intracellular structures are speculated to act as diffusion barriers. In this work, we assume that diffusion restrictions are induced by sarcoplasmic reticulum (SR), cytoskeleton proteins localized near SR, and crowding of cytosolic proteins. The aim of this work was to test whether such localization of diffusion restrictions would be consistent with the available experimental data and evaluate the extent of the restrictions. For that, a three-dimensional finite-element model was composed with the geometry based on mitochondrial and SR structural organization. Diffusion restrictions induced by SR and cytoskeleton proteins were varied with other model parameters to fit the set of experimental data obtained on permeabilized rat heart muscle fibers. There are many sets of model parameters that were able to reproduce all experiments considered in this work. However, in all the sets, <5-6% of the surface formed by SR and associated cytoskeleton proteins is permeable to metabolites. Such a low level of permeability indicates that the proteins should play a dominant part in formation of the diffusion restrictions.