Cardiac Regeneration: New Hope for an Old Dream.

The regenerative capacity of the heart has long fascinated scientists. In contrast to other organs such as liver, skin, and skeletal muscle, the heart possesses only a minimal regenerative capacity. It lacks a progenitor cell population, and cardiomyocytes exit the cell cycle shortly after birth and do not re-enter after injury. Thus, any loss of cardiomyocytes is essentially irreversible and can lead to or exaggerate heart failure, which represents a major public health problem. New therapeutic options are urgently needed, but regenerative therapies have remained an unfulfilled promise in cardiovascular medicine until today. Yet, through a clearer comprehension of signaling pathways that regulate the cardiomyocyte cell cycle and advances in stem cell technology, strategies have evolved that demonstrate the potential to generate new myocytes and thereby fulfill an essential central criterion for heart repair.

Impact of persistently high sea surface temperatures on the rhizobiomes of Zostera marina in a Baltic Sea benthocosms.

Persistently high marine temperatures are escalating and threating marine biodiversity. The Baltic Sea, warming faster than other seas, is a good model to study the impact of increasing sea surface temperatures. Zostera marina, a key player in the Baltic ecosystem, faces susceptibility to disturbances, especially under chronic high temperatures. Despite the increasing number of studies on the impact of global warming on seagrasses, little attention has been paid to the role of the holobiont. Using an outdoor benthocosm to replicate near-natural conditions, this study explores the repercussions of persistent warming on the microbiome of Z. marina and its implications for holobiont function. Results show that both seasonal warming and chronic warming, impact Z. marina roots and sediment microbiome. Compared with roots, sediments demonstrate higher diversity and stability throughout the study, but temperature effects manifest earlier in both compartments, possibly linked to premature Z. marina die-offs under chronic warming. Shifts in microbial composition, such as an increase in organic matter-degrading and sulfur-related bacteria, accompany chronic warming. A higher ratio of sulfate-reducing bacteria compared to sulfide oxidizers was found in the warming treatment which may result in the collapse of the seagrasses, due to toxic levels of sulfide. Differentiating predicted pathways for warmest temperatures were related to sulfur and nitrogen cycles, suggest an increase of the microbial metabolism, and possible seagrass protection strategies through the production of isoprene. These structural and compositional variations in the associated microbiome offer early insights into the ecological status of seagrasses. Certain taxa/genes/pathways may serve as markers for specific stresses. Monitoring programs should integrate this aspect to identify early indicators of seagrass health. Understanding microbiome changes under stress is crucial for the use of potential probiotic taxa to mitigate climate change effects. Broader-scale examination of seagrass-microorganism interactions is needed to leverage knowledge on host-microbe interactions in seagrasses.

Contractile Force of Transplanted Cardiomyocytes Actively Supports Heart Function After Injury.

BACKGROUND: Transplantation of pluripotent stem cell-derived cardiomyocytes represents a promising therapeutic strategy for cardiac regeneration, and the first clinical studies in patients with heart failure have commenced. Yet, little is known about the mechanism of action underlying graft-induced benefits. Here, we explored whether transplanted cardiomyocytes actively contribute to heart function. METHODS: We injected cardiomyocytes with an optogenetic off-on switch in a guinea pig cardiac injury model. RESULTS: Light-induced inhibition of engrafted cardiomyocyte contractility resulted in a rapid decrease of left ventricular function in ≈50% (7/13) animals that was fully reversible with the offset of photostimulation. CONCLUSIONS: Our optogenetic approach demonstrates that transplanted cardiomyocytes can actively participate in heart function, supporting the hypothesis that the delivery of new force-generating myocardium can serve as a regenerative therapeutic strategy.

Modulating cardiac physiology in engineered heart tissue with the bidirectional optogenetic tool BiPOLES.

Optogenetic actuators are rapidly advancing tools used to control physiology in excitable cells, such as neurons and cardiomyocytes. In neuroscience, these tools have been used to either excite or inhibit neuronal activity. Cell type-targeted actuators have allowed to study the function of distinct cell populations. Whereas the first described cation channelrhodopsins allowed to excite specific neuronal cell populations, anion channelrhodopsins were used to inhibit neuronal activity. To allow for simultaneous excitation and inhibition, opsin combinations with low spectral overlap were introduced. BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing) is a bidirectional optogenetic tool consisting of the anion channel Guillardia theta anion-conducting channelrhodopsin 2 (GtACR2 with a blue excitation spectrum and the red-shifted cation channel Chrimson. Here, we studied the effects of BiPOLES activation in cardiomyocytes. For this, we knocked in BiPOLES into the adeno-associated virus integration site 1 (AAVS1) locus of human-induced pluripotent stem cells (hiPSC), subjected these to cardiac differentiation, and generated BiPOLES expressing engineered heart tissue (EHT) for physiological characterization. Continuous light application activating either GtACR2 or Chrimson resulted in cardiomyocyte depolarization and thus stopped EHT contractility. In contrast, short light pulses, with red as well as with blue light, triggered action potentials (AP) up to a rate of 240 bpm. In summary, we demonstrate that cation, as well as anion channelrhodopsins, can be used to activate stem cell-derived cardiomyocytes with pulsed photostimulation but also to silence cardiac contractility with prolonged photostimulation.

Salinity and host drive Ulva-associated bacterial communities across the Atlantic-Baltic Sea gradient.

The green seaweed Ulva is a model system to study seaweed-bacteria interactions, but the impact of environmental drivers on the dynamics of these interactions is little understood. In this study, we investigated the stability and variability of the seaweed-associated bacteria across the Atlantic-Baltic Sea salinity gradient. We characterized the bacterial communities of 15 Ulva sensu lato species along 2,000 km of coastline in a total of 481 samples. Our results demonstrate that the Ulva-associated bacterial composition was strongly structured by both salinity and host species (together explaining between 34% and 91% of the variation in the abundance of the different bacterial genera). The largest shift in the bacterial consortia coincided with the horohalinicum (5-8 PSU, known as the transition zone from freshwater to marine conditions). Low-salinity communities especially contained high relative abundances of Luteolibacter, Cyanobium, Pirellula, Lacihabitans and an uncultured Spirosomaceae, whereas high-salinity communities were predominantly enriched in Litorimonas, Leucothrix, Sulfurovum, Algibacter and Dokdonia. We identified a small taxonomic core community (consisting of Paracoccus, Sulfitobacter and an uncultured Rhodobacteraceae), which together contributed to 14% of the reads per sample, on average. Additional core taxa followed a gradient model, as more core taxa were shared between neighbouring salinity ranges than between ranges at opposite ends of the Atlantic-Baltic Sea gradient. Our results contradict earlier statements that Ulva-associated bacterial communities are taxonomically highly variable across individuals and largely stochastically defined. Characteristic bacterial communities associated with distinct salinity regions may therefore facilitate the host's adaptation across the environmental gradient.

Genome-scale signatures of adaptive gene expression changes in an invasive seaweed Gracilaria vermiculophylla.

Invasive species can successfully and rapidly colonize new niches and expand ranges via founder effects and enhanced tolerance towards environmental stresses. However, the underpinning molecular mechanisms (i.e., gene expression changes) facilitating rapid adaptation to harsh environments are still poorly understood. The red seaweed Gracilaria vermiculophylla, which is native to the northwest Pacific but invaded North American and European coastal habitats over the last 100 years, provides an excellent model to examine whether enhanced tolerance at the level of gene expression contributed to its invasion success. We collected G. vermiculophylla from its native range in Japan and from two non-native regions along the Delmarva Peninsula (Eastern United States) and in Germany. Thalli were reared in a common garden for 4 months at which time we performed comparative transcriptome (mRNA) and microRNA (miRNA) sequencing. MRNA-expression profiling identified 59 genes that were differently expressed between native and non-native thalli. Of these genes, most were involved in metabolic pathways, including photosynthesis, abiotic stress, and biosynthesis of products and hormones in all four non-native sites. MiRNA-based target-gene correlation analysis in native/non-native pairs revealed that some target genes are positively or negatively regulated via epigenetic mechanisms. Importantly, these genes are mostly associated with metabolism and defence capability (e.g., metal transporter Nramp5, senescence-associated protein, cell wall-associated hydrolase, ycf68 protein and cytochrome P450-like TBP). Thus, our gene expression results indicate that resource reallocation to metabolic processes is most likely a predominant mechanism contributing to the range-wide persistence and adaptation of G. vermiculophylla in the invaded range. This study, therefore, provides molecular insight into the speed and nature of invasion-mediated rapid adaption.

How to repair a broken heart with pluripotent stem cell-derived cardiomyocytes.

Heart regeneration addresses a central problem in cardiology, the irreversibility of the loss of myocardium that eventually leads to heart failure. True restoration of heart function can only be achieved by remuscularization, i.e. replacement of lost myocardium by new, force-developing heart muscle. With the availability of principally unlimited human cardiomyocytes from pluripotent stem cells, one option to remuscularize the injured heart is to produce large numbers of cardiomyocytes plus/minus other cardiovascular cell types or progenitors ex vivo and apply them to the heart, either by injection or application as a patch. Exciting progress over the past decade has led to the first clinical applications, but important questions remain. Academic and increasingly corporate activity is ongoing to answer them and optimize the approach to finally develop a true regenerative therapy of heart failure.

Human engineered heart tissue transplantation in a guinea pig chronic injury model.

Myocardial injury leads to an irreversible loss of cardiomyocytes (CM). The implantation of human engineered heart tissue (EHT) has become a promising regenerative approach. Previous studies exhibited beneficial, dose-dependent effects of human induced pluripotent stem cell (hiPSC)-derived EHT patch transplantation in a guinea pig model in the subacute phase of myocardial injury. Yet, advanced heart failure often results from a chronic remodeling process. Therefore, from a clinical standpoint it is worthwhile to explore the ability to repair the chronically injured heart. In this study human EHT patches were generated from hiPSC-derived CMs (15 × 106 cells) and implanted epicardially four weeks after injury in a guinea pig cryo-injury model. Cardiac function was evaluated by echocardiography after a follow-up period of four weeks. Hearts revealed large transmural myocardial injuries amounting to 27% of the left ventricle. EHT recipient hearts demonstrated compact muscle islands of human origin in the scar region, as indicated by a positive staining for human Ku80 and dystrophin, remuscularizing 5% of the scar area. Echocardiographic analysis demonstrated no significant functional difference between animals that received EHT patches and animals in the cell-free control group (fractional area change 36% vs. 34%). Thus, EHT patches engrafted in the chronically injured heart but in contrast to the subacute model, grafts were smaller and EHT patch transplantation did not improve left ventricular function, highlighting the difficulties for a regenerative approach.

Human Engineered Heart Tissue Patches Remuscularize the Injured Heart in a Dose-Dependent Manner.

BACKGROUND: Human engineered heart tissue (EHT) transplantation represents a potential regenerative strategy for patients with heart failure and has been successful in preclinical models. Clinical application requires upscaling, adaptation to good manufacturing practices, and determination of the effective dose. METHODS: Cardiomyocytes were differentiated from 3 different human induced pluripotent stem cell lines including one reprogrammed under good manufacturing practice conditions. Protocols for human induced pluripotent stem cell expansion, cardiomyocyte differentiation, and EHT generation were adapted to substances available in good manufacturing practice quality. EHT geometry was modified to generate patches suitable for transplantation in a small-animal model and perspectively humans. Repair efficacy was evaluated at 3 doses in a cryo-injury guinea pig model. Human-scale patches were epicardially transplanted onto healthy hearts in pigs to assess technical feasibility. RESULTS: We created mesh-structured tissue patches for transplantation in guinea pigs (1.5×2.5 cm, 9-15×106 cardiomyocytes) and pigs (5×7 cm, 450×106 cardiomyocytes). EHT patches coherently beat in culture and developed high force (mean 4.6 mN). Cardiomyocytes matured, aligned along the force lines, and demonstrated advanced sarcomeric structure and action potential characteristics closely resembling human ventricular tissue. EHT patches containing ≈4.5, 8.5, 12×106, or no cells were transplanted 7 days after cryo-injury (n=18-19 per group). EHT transplantation resulted in a dose-dependent remuscularization (graft size: 0%-12% of the scar). Only high-dose patches improved left ventricular function (+8% absolute, +24% relative increase). The grafts showed time-dependent cardiomyocyte proliferation. Although standard EHT patches did not withstand transplantation in pigs, the human-scale patch enabled successful patch transplantation. CONCLUSIONS: EHT patch transplantation resulted in a partial remuscularization of the injured heart and improved left ventricular function in a dose-dependent manner in a guinea pig injury model. Human-scale patches were successfully transplanted in pigs in a proof-of-principle study.

Cross-Host Protection of Marine Bacteria Against Macroalgal Disease.

Despite an increasing awareness of disease impacts on both cultivated and native seaweed populations, the development of marine probiotics has been limited and predominately focused on farmed animals. Bleaching (loss of thallus pigmentation) is one of the most prevalent diseases observed in marine macroalgae. Endemic probiotic bacteria have been characterized to prevent bleaching disease in red macroalgae Agarophyton vermiculophyllum and Delisea pulchra; however, the extent to which probiotic strains provide cross-protection to non-endemic hosts and the influence of native microbiota remain unknown. Using A. vermiculophyllum as a model, we demonstrate that co-inoculation with the pathogen Pseudoalteromonas arctica G-MAN6 and D. pulchra probiotic strain Phaeobacter sp. BS52 or Pseudoalteromonas sp. PB2-1 reduced the disease risks compared to the pathogen only treatment. Moreover, non-endemic probiotics outperformed the endemic probiotic strain Ralstonia sp. G-NY6 in the presence of the host natural microbiota. This study highlights how the native microbiota can impact the effectiveness of marine probiotics and illustrates the potential of harnessing probiotics that can function across different hosts to mitigate the impact of emerging marine diseases.

Intraspecific diversity and genetic structure in the widespread macroalga Agarophyton vermiculophyllum.

Single-gene markers, such as the mitochondrial cox1, microsatellites, and single-nucleotide polymorphisms are powerful methods to describe diversity within and among taxonomic groups and characterize phylogeographic patterns. Large repositories of publicly-available, molecular data can be combined to generate and evaluate evolutionary hypotheses for many species, including algae. In the case of biological invasions, the combination of different molecular markers has enabled the description of the geographic distribution of invasive lineages. Here, we review the phylogeography of the widespread invasive red macroalga Agarophyton vermiculophyllum (synonym Gracilaria vermiculophylla). The cox1 barcoding provided the first description of the invasion history and hinted at a strong genetic bottleneck during the invasion. Yet, more recent microsatellite and SNP genotyping has not found evidence for bottlenecks and instead suggested that genetically diverse inocula arose from a highly diverse source population, multiple invasions, or some mix of these processes. The bottleneck evident from cox1 barcoding likely reflects the dominance of one mitochondrial lineage, and one haplotype in particular, in the northern source populations in Japan. Recent cox1 sequencing of A. vermiculophyllum has illuminated the complexity of phylogeographic structure in its native range of the northwest Pacific Ocean. For example, the western coast of Honshu in the Sea of Japan displays spatial patterns of haplotypic diversity with multiple lineages found together at the same geographic site. By consolidating the genetic data of this species, we clarify the phylogenetic relationships of a well-studied macroalga introduced to virtually every temperate estuary of the Northern Hemisphere.

Draft genome and description of Waterburya agarophytonicola gen. nov. sp. nov. (Pleurocapsales, Cyanobacteria): a seaweed symbiont.

This work introduces Waterburya agarophytonicola Bonthond and Shalygin gen. nov., sp. nov, a baeocyte producing cyanobacterium that was isolated from the rhodophyte Agarophyton vermiculophyllum (Ohmi) Gurgel et al., an invasive seaweed that has spread across the northern hemisphere. The new species genome reveals a diverse repertoire of chemotaxis and adhesion related genes, including genes coding for type IV pili assembly proteins and a high number of genes coding for filamentous hemagglutinin family (FHA) proteins. Among a genetic basis for the synthesis of siderophores, carotenoids and numerous vitamins, W. agarophytonicola is potentially capable of producing cobalamin (vitamin B12), for which A. vermiculophyllum is an auxotroph. With a taxonomic description of the genus and species and a draft genome, this study provides as a basis for future research, to uncover the nature of this geographically independent association between seaweed and cyanobiont.

How do microbiota associated with an invasive seaweed vary across scales?

Communities are shaped by scale dependent processes. To study the diversity and variation of microbial communities across scales, the invasive and widespread seaweed Agarophyton vermiculophyllum presents a unique opportunity. We characterized pro- and eukaryotic communities associated with this holobiont across its known distribution range, which stretches over the northern hemisphere. Our data reveal that community composition and diversity in the holobiont vary at local but also larger geographic scales. While processes acting at the local scale (i.e., within population) are the main structuring drivers of associated microbial communities, changes in community composition also depend on processes acting at larger geographic scales. Interestingly, the largest analysed scale (i.e., native and non-native ranges) explained variation in the prevalence of predicted functional groups, which could suggest a functional shift in microbiota occurred over the course of the invasion process. While high variability in microbiota at the local scale supports A. vermiculophyllum to be a generalist host, we also identified a number of core taxa. These geographically independent holobiont members imply that cointroduction of specific microbiota may have additionally promoted the invasion process.

Response of foundation macrophytes to near-natural simulated marine heatwaves.

Marine heatwaves have been observed worldwide and are expected to increase in both frequency and intensity due to climate change. Such events may cause ecosystem reconfigurations arising from species range contraction or redistribution, with ecological, economic and social implications. Macrophytes such as the brown seaweed Fucus vesiculosus and the seagrass Zostera marina are foundation species in many coastal ecosystems of the temperate northern hemisphere. Hence, their response to extreme events can potentially determine the fate of associated ecosystems. Macrophyte functioning is intimately linked to the maintenance of photosynthesis, growth and reproduction, and resistance against pathogens, epibionts and grazers. We investigated morphological, physiological, pathological and chemical defence responses of western Baltic Sea F. vesiculosus and Z. marina populations to simulated near-natural marine heatwaves. Along with (a) the control, which constituted no heatwave but natural stochastic temperature variability (0HW), two treatments were applied: (b) two late-spring heatwaves (June, July) followed by a summer heatwave (August; 3HW) and (c) a summer heatwave only (1HW). The 3HW treatment was applied to test whether preconditioning events can modulate the potential sensitivity to the summer heatwave. Despite the variety of responses measured in both species, only Z. marina growth was impaired by the accumulative heat stress imposed by the 3HW treatment. Photosynthetic rate, however, remained high after the last heatwave indicating potential for recovery. Only epibacterial abundance was significantly affected in F. vesiculosus. Hence both macrophytes, and in particular F. vesiculosus, seem to be fairly tolerant to short-term marine heatwaves at least at the intensities applied in this experiment (up to 5°C above mean temperature over a period of 9 days). This may partly be due to the fact that F. vesiculosus grows in a highly variable environment, and may have a high phenotypic plasticity.

Polaribacter septentrionalilitoris sp. nov., isolated from the biofilm of a stone from the North Sea.

A new member of the family Flavobacteriaceae was isolated from the biofilm of a stone at Nordstrand, a peninsula at the German North Sea shore. Phylogenetic analysis of the 16S rRNA gene sequence showed that strain ANORD1T was most closely related to the validly described type strains Polaribacter porphyrae LNM-20T (97.0 %) and Polaribacter reichenbachii KMM 6386T (96.9 % 16S rRNA gene sequence similarity) and clustered with Polaribacter gangjinensis K17-16T (96.0 %). Strain ANORD1T was determined to be mesophilic, Gram-negative, non-motile and strictly aerobic. Optimal growth was observed at 20-30 °C, within a salinity range of 2-7 % sea salt and from pH 7-10. Like other type strains of the genus Polaribacter, ANORD1T was tested negative for flexirubin-type pigments, while carotenoid-type pigments were detected. The DNA G+C content of strain ANORD1T was 30.6 mol%. The sole respiratory quinone detected was menaquinone 6 (MK-6). The major fatty acids identified were C15 : 0, iso-C15 : 0, C15 : 1 ω6c and iso-C15 : 0 3-OH. Based on the polyphasic approach, strain ANORD1T represents a novel species in the genus Polaribacter, with the name Polaribacter septentrionalilitoris sp. nov. being proposed. The type strain is ANORD1T (=DSM 110039T=NCIMB 15081T=MTCC 12685T).

Engineering Cardiac Muscle Tissue: A Maturating Field of Research.

Twenty years after the initial description of a tissue engineered construct, 3-dimensional human cardiac tissues of different kinds are now generated routinely in many laboratories. Advances in stem cell biology and engineering allow for the generation of constructs that come close to recapitulating the complex structure of heart muscle and might, therefore, be amenable to industrial (eg, drug screening) and clinical (eg, cardiac repair) applications. Whether the more physiological structure of 3-dimensional constructs provides a relevant advantage over standard 2-dimensional cell culture has yet to be shown in head-to-head-comparisons. The present article gives an overview on current strategies of cardiac tissue engineering with a focus on different hydrogel methods and discusses perspectives and challenges for necessary steps toward the real-life application of cardiac tissue engineering for disease modeling, drug development, and cardiac repair.

Conspecificity of the model organism Ulva mutabilis and Ulva compressa (Ulvophyceae, Chlorophyta).

As one of the most abundant and ubiquitous representatives of marine and brackish coastal macrophytobenthos communities, the genus Ulva is not only an important primary producer but also of ecological and morphogenetic interest to many scientists. Ulva mutabilis became an important model organism to study morphogenesis and mutualistic interactions of macroalgae and microorganisms. Here, we report that our collections of Ulva compressa Linnaeus (1753) from Germany are conspecific with the type strains of the model organism U. mutabilis Føyn (1958), which were originally collected at Olhão on the south coast of Portugal and have from that time on been maintained in culture as gametophytic and parthenogenetic lab strains. Different approaches were used to test conspecificity: (i) comparisons of vegetative and reproductive features of cultured material of U. mutabilis and German U. compressa demonstrated a shared morphological pattern; (ii) gametes of U. compressa and U. mutabilis successfully mated and developed into fertile sporophytic first-generation offspring; (iii) molecular phylogenetics and species delimitation analyses based on the Generalized Mixed Yule-Coalescent method showed that U. mutabilis isolates (sl-G[mt+]) and (wt-G[mt-]) and U. compressa belong to a unique Molecular Operational Taxonomic Unit. According to these findings, there is sufficient evidence that U. mutabilis and U. compressa should be regarded as conspecific.

Heat waves and their significance for a temperate benthic community: A near-natural experimental approach.

Climate change will not only shift environmental means but will also increase the intensity of extreme events, exerting additional stress on ecosystems. While field observations on the ecological consequences of heat waves are emerging, experimental evidence is rare, and lacking at the community level. Using a novel "near-natural" outdoor mesocosms approach, this study tested whether marine summer heat waves have detrimental consequences for macrofauna of a temperate coastal community, and whether sequential heat waves provoke an increase or decrease of sensitivity to thermal stress. Three treatments were applied, defined and characterized through a statistical analysis of 15 years of temperature records from the experimental site: (1) no heat wave, (2) two heat waves in June and July followed by a summer heat wave in August and (3) the summer heat wave only. Overall, 50% of the species showed positive, negative or positive/negative responses in either abundance and/or biomass. We highlight four possible ways in which single species responded to either three subsequent heat waves or one summer heat wave: (1) absence of a response (tolerance, 50% of species), (2) negative accumulative effects by three subsequent heat waves (tellinid bivalve), (3) buffering by proceeding heat waves due to acclimation and/or shifts in phenology (spionid polychaete) and (4) an accumulative positive effect by subsequent heat waves (amphipod). The differential responses to single or sequential heat waves at the species level entailed shifts at the community level. Community-level differences between single and triple heat waves were more pronounced than those between regimes with vs. without heat waves. Detritivory was reduced by the single heat wave while suspension feeding was less common in the triple heat wave regime. Critical extreme events occur already today and will occur more frequently in a changing climate, thus, leading to detrimental impacts on coastal marine systems.

Vicingus serpentipes gen. nov., sp. nov., a new member of the Flavobacteriales from the North Sea.

A new member of the Flavobacteriales was isolated from the surface of a stone collected on the German North Sea shore. The bacterium, strain ANORD5T, is a mesophilic, chemoheterotrophic aerobic, typical marine bacterium. Optimal growth was observed at 20-30 °C, pH 7.0-8.5 and 1-2 % sea salt. The 16S rRNA gene sequence revealed a distant relationship with the representatives of the Cryomorphaceae, with less than 90 % sequence similarity. Strain ANORD5T forms a cluster together with Owenweeksia hongkongensis UST20020801T (89.9 %), Cryomorpha ignava 1-22T (87.9 %), Luteibaculum oceani CC-AMWY-103BT (88.1 %) and Phaeocystidibacter luteus PG2S01T (87.3 %). Strain ANORD5T has a low DNA G+C content (31 mol%). Based on morphological, physiological and phylogenetic data, strain ANORD5T is considered a type strain of a new species and a new genus of the family Cryomorphaceae for which the name Vicingus serpentipes is proposed. The type strain is ANORD5T (=NCIMB 15042T=DSM 103558T=MTCC 12686T).

Diltiazem prevents stress-induced contractile deficits in cardiomyocytes, but does not reverse the cardiomyopathy phenotype in Mybpc3-knock-in mice.

KEY POINTS: Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac illness and can lead to diastolic dysfunction, sudden cardiac death and heart failure. Treatment of HCM patients is empirical and current pharmacological treatments are unable to stop disease progression or reverse hypertrophy. In this study, we tested if the non-dihydropyridine Ca2+ channel blocker diltiazem, which previously showed potential to stop disease progression, can improve the phenotype of a HCM mouse model (Mybpc3-targeted knock-in), which is based on a mutation commonly found in patients. Diltiazem improved contractile function of isolated ventricular cardiomyocytes acutely, but chronic application did not improve the phenotype of adult mice with a fully developed HCM. Our study shows that diltiazem has beneficial effects in HCM, but long-term treatment success is likely to depend on characteristics and cause of HCM and onset of treatment. ABSTRACT: Left ventricular hypertrophy, diastolic dysfunction and fibrosis are the main features of hypertrophic cardiomyopathy (HCM). Guidelines recommend ß-adrenoceptor or Ca2+ channel antagonists as pharmacological treatment. The Ca2+ channel blocker diltiazem recently showed promising beneficial effects in pre-clinical HCM, particularly in patients carrying MYBPC3 mutations. In the present study we evaluated whether diltiazem could ameliorate or reverse the disease phenotype in cells and in vivo in an Mybpc3-targeted knock-in (KI) mouse model of HCM. Sarcomere shortening and Ca2+ transients were measured in KI and wild-type (WT) cardiomyocytes in basal conditions (1-Hz pacing) and under stress conditions (30 nm isoprenaline, 5-Hz pacing) with or without pre-treatment with 1 µm diltiazem. KI cardiomyocytes exhibited lower diastolic sarcomere length (dSL) at baseline, a tendency to a stronger positive inotropic response to isoprenaline than WT, a marked reduction of dSL and a tendency towards arrhythmias under stress conditions. Pre-treatment of cardiomyocytes with 1 µm diltiazem reduced the drop in dSL and arrhythmia frequency in KI, and attenuated the positive inotropic effect of isoprenaline. Furthermore, diltiazem reduced the contraction amplitude at 5 Hz but did not affect diastolic Ca2+ load and Ca2+ transient amplitude. Six months of diltiazem treatment of KI mice did not reverse cardiac hypertrophy and dysfunction, activation of the fetal gene program or fibrosis. In conclusion, diltiazem blunted the response to isoprenaline in WT and KI cardiomyocytes and improved diastolic relaxation under stress conditions in KI cardiomyocytes. This beneficial effect of diltiazem in cells did not translate in therapeutic efficacy when applied chronically in KI mice.