Assessment of extreme hydrological conditions in the Bothnian Bay, Baltic Sea, and the impact of the nuclear power plant “Hanhikivi-1” on the local thermal regime

The results of the study aimed to assess the influence of future nuclear power plant “Hanhikivi-1” upon the local thermal conditions in the Bothnian Bay in the Baltic Sea are presented. A number of experiments with different numerical models were also carried out in order to estimate the extreme hydro-meteorological conditions in the area of the construction. The numerical experiments were fulfilled both with analytically specified external forcing and with real external forcing for 2 years: a cold year (2010) and a warm year (2014). The study has shown that the extreme values of sea level and water temperature and the characteristics of wind waves and sea ice in the vicinity of the future nuclear power plant can be significant and sometimes catastrophic. Permanent release of heat into the marine environment from an operating nuclear power plant will lead to a strong increase in temperature and the disappearance of ice cover within a 2 km vicinity of the station. These effects should be taken into account when assessing local climate changes in the future

Predicting self-terminating ventricular fibrillations in an isolated heart

Ventricular fibrillations (VFs) in isolated hearts induced by fast pacing are studied in a Langendorff preparation by measuring the electrical signals from the right atrium $(V_a)$ and the ventricle $(V_v)$ . We find that when there is a strong component of Vv detected in Va during VF, the induced VFs are usually not self-terminating. Criteria for the prediction of self-terminating VFs are developed based on the analysis of Vv and Va by the cross-wavelet power spectrum and cross-Fourier power spectrum methods. The success rate of our prediction criteria is about 80–90 %. Our findings suggest that a heart under VF can recover its sinus rhythm only when the sino-atrial node of the heart is not under strong influence of the VF from its ventricle

Restrictive Cardiomyopathy Troponin I R145W Mutation Does Not Perturb Myofilament Length-dependent Activation in Human Cardiac Sarcomeres*

The cardiac troponin I (cTnI) R145W mutation is associated with restrictive cardiomyopathy (RCM). Recent evidence suggests that this mutation induces perturbed myofilament length-dependent activation (LDA) under conditions of maximal protein kinase A (PKA) stimulation. Some cardiac disease-causing mutations, however, have been associated with a blunted response to PKA-mediated phosphorylation; whether this includes LDA is unknown. Endogenous troponin was exchanged in isolated skinned human myocardium for recombinant troponin containing either cTnI R145W, PKA/PKC phosphomimetic charge mutations (S23D/S24D and T143E), or various combinations thereof. Myofilament Ca2+ sensitivity of force, tension cost, LDA, and single myofibril activation/relaxation parameters were measured. Our results show that both R145W and T143E uncouple the impact of S23D/S24D phosphomimetic on myofilament function, including LDA. Molecular dynamics simulations revealed a marked reduction in interactions between helix C of cTnC (residues 56, 59, and 63), and cTnI (residue 145) in the presence of either cTnI RCM mutation or cTnI PKC phosphomimetic. These results suggest that the RCM-associated cTnI R145W mutation induces a permanent structural state that is similar to, but more extensive than, that induced by PKC-mediated phosphorylation of cTnI Thr-143. We suggest that this structural conformational change induces an increase in myofilament Ca2+ sensitivity and, moreover, uncoupling from the impact of phosphorylation of cTnI mediated by PKA at the Ser-23/Ser-24 target sites. The R145W RCM mutation by itself, however, does not impact LDA. These perturbed biophysical and biochemical myofilament properties are likely to significantly contribute to the diastolic cardiac pump dysfunction that is seen in patients suffering from a restrictive cardiomyopathy that is associated with the cTnI R145W mutation

A Langendorff-like system to quantify cardiac pump function in adult zebrafish

Zebrafish are increasingly used as a vertebrate model to study human cardiovascular disorders. Although heart structure and function are readily visualized in zebrafish embryos because of their optical transparency, the lack of effective tools for evaluating the hearts of older, nontransparent fish has been a major limiting factor. The recent development of high-frequency echocardiography has been an important advance for in vivo cardiac assessment, but it necessitates anesthesia and has limited ability to study acute interventions. We report the development of an alternative experimental ex vivo technique for quantifying heart size and function that resembles the Langendorff heart preparations that have been widely used in mammalian models. Dissected adult zebrafish hearts were perfused with a calcium-containing buffer, and a beat frequency was maintained with electrical stimulation. The impact of pacing frequency, flow rate and perfusate calcium concentration on ventricular performance (including end-diastolic and end-systolic volumes, ejection fraction, radial strain, and maximal velocities of shortening and relaxation) were evaluated and optimal conditions defined. We determined the effects of age on heart function in wild-type male and female zebrafish, and successfully detected hypercontractile and hypocontractile responses after adrenergic stimulation or doxorubicin treatment, respectively. Good correlations were found between indices of cardiac contractility obtained with high-frequency echocardiography and with the ex vivo technique in a subset of fish studied with both methods. The ex vivo beating heart preparation is a valuable addition to the cardiac function tool kit that will expand the use of adult zebrafish for cardiovascular research

Phenotyping an adult zebrafish lamp2 cardiomyopathy model identifies mTOR inhibition as a candidate therapy

Adult zebrafish is an emerging vertebrate model for studying genetic basis of cardiomyopathies; but whether the simple fish heart can model essential features of hypertrophic cardiomyopathy (HCM) remained unknown. Here, we report a comprehensive phenotyping of a lamp2 knockout (KO) mutant. LAMP2 encodes a lysosomal protein and is a causative gene of Danon disease that is characterized by HCM and massive autophagic vacuoles accumulation in the tissues. There is no effective therapy yet to treat this most lethal cardiomyopathy in the young. First, we did find the autophagic vacuoles accumulation in cardiac tissues from lamp2 KO. Next, through employing a set of emerging phenotyping tools, we revealed heart failure phenotypes in the lamp2 KO mutants, including decreased ventricular ejection fraction, reduced physical exercise capacity, blunted beta-adrenergic contractile response, and enlarged atrium. We also noted changes of the following indices suggesting cardiac hypertrophic remodeling in lamp2 KO: a rounded heart shape, increased end-systolic ventricular volume and density of ventricular myocardium, elevated actomyosin activation kinetics together with increased maximal isometric tension at the level of cardiac myofibrils. Lastly, we assessed the function of lysosomal-localized mTOR on the lamp2-associated Danon disease. We found that haploinsufficiency of mtor was able to normalize some characteristics of the lamp2 KO, including ejection fraction, beta-adrenergic response, and the actomyosin activation kinetics. In summary, we demonstrate the feasibility of modeling the inherited HCM in the adult zebrafish, which can be used to develop potential therapies.NIH [R01HL107304, HL81753, HL111437]; Mayo Foundation12 month embargo; published online: 20 June 2019This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Haploinsufficiency of mechanistic target of rapamycin ameliorates cardiomyopathy in adult zebrafish

The adult zebrafish is an emerging vertebrate model for studying human cardiomyopathies; however, whether the simple zebrafish heart can model different subtypes of cardiomyopathies, such as dilated cardiomyopathy (DCM), remains elusive. Here, we generated and characterized an inherited DCM model in adult zebrafish and used this model to search for therapeutic strategies. We employed transcription activator-like effector nuclease (TALEN) genome editing technology to generate frame-shift mutants for the zebrafish ortholog of human BCL2-associated athanogene 3 (BAG3), an established DCM-causative gene. As in mammals, the zebrafish bag3 homozygous mutant (bag3e2/e2 ) exhibited aberrant proteostasis, as indicated by impaired autophagy flux and elevated ubiquitinated protein aggregation. Through comprehensive phenotyping analysis of the mutant, we identified phenotypic traits that resembled DCM phenotypes in mammals, including cardiac chamber enlargement, reduced ejection fraction characterized by increased end-systolic volume/body weight (ESV/BW), and reduced contractile myofibril activation kinetics. Nonbiased transcriptome analysis identified the hyperactivation of the mechanistic target of rapamycin (mTOR) signaling in bag3e2/e2 mutant hearts. Further genetic studies showed that mtorxu015/+ , an mTOR haploinsufficiency mutant, repaired abnormal proteostasis, improved cardiac function and rescued the survival of the bag3e2/e2 mutant. This study established the bag3e2/e2 mutant as a DCM model in adult zebrafish and suggested mtor as a candidate therapeutic target gene for BAG3 cardiomyopathy.This work was supported in part by the Scientist Development Grant from the American Heart Association (14SDG18160021) to Y.D., the Ted and Loretta Rogers Cardiovascular Career Development Award Honoring Hugh C. Smith (from the Mayo Clinic) to Y.D., the National Institutes of Health (HL81753, HL107304, HL111437 and GM63904) to X.X., and the Mayo Foundation for Medical Education and Research to X.X.Open access journalThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

Robust activation of microhomology-mediated end joining for precision gene editing applications.

One key problem in precision genome editing is the unpredictable plurality of sequence outcomes at the site of targeted DNA double stranded breaks (DSBs). This is due to the typical activation of the versatile Non-homologous End Joining (NHEJ) pathway. Such unpredictability limits the utility of somatic gene editing for applications including gene therapy and functional genomics. For germline editing work, the accurate reproduction of the identical alleles using NHEJ is a labor intensive process. In this study, we propose Microhomology-mediated End Joining (MMEJ) as a viable solution for improving somatic sequence homogeneity in vivo, capable of generating a single predictable allele at high rates (56% ~ 86% of the entire mutant allele pool). Using a combined dataset from zebrafish (Danio rerio) in vivo and human HeLa cell in vitro, we identified specific contextual sequence determinants surrounding genomic DSBs for robust MMEJ pathway activation. We then applied our observation to prospectively design MMEJ-inducing sgRNAs against a variety of proof-of-principle genes and demonstrated high levels of mutant allele homogeneity. MMEJ-based DNA repair at these target loci successfully generated F0 mutant zebrafish embryos and larvae that faithfully recapitulated previously reported, recessive, loss-of-function phenotypes. We also tested the generalizability of our approach in cultured human cells. Finally, we provide a novel algorithm, MENTHU (http://genesculpt.org/menthu/), for improved and facile prediction of candidate MMEJ loci. We believe that this MMEJ-centric approach will have a broader impact on genome engineering and its applications. For example, whereas somatic mosaicism hinders efficient recreation of knockout mutant allele at base pair resolution via the standard NHEJ-based approach, we demonstrate that F0 founders transmitted the identical MMEJ allele of interest at high rates. Most importantly, the ability to directly dictate the reading frame of an endogenous target will have important implications for gene therapy applications in human genetic diseases