CRISPR/Cas9-mediated nexilin deficiency interferes with cardiac contractile function in zebrafish in vivo.

Nexilin (NEXN) plays a crucial role in stabilizing the sarcomeric Z-disk of striated muscle fibers and, when mutated, leads to dilated cardiomyopathy in humans. Due to its early neonatal lethality in mice, the detailed impact of the constitutive homozygous NEXN knockout on heart and skeletal muscle morphology and function is insufficiently investigated. Here, we characterized a constitutive homozygous CRISPR/Cas9-mediated nexn knockout zebrafish model. We found that Nexn deficient embryos developed significantly reduced cardiac contractility and under stressed conditions also impaired skeletal muscle organization whereas skeletal muscle function seemed not to be affected. Remarkably, in contrast to nexn morphants, CRISPR/Cas9 nexn-/- knockout embryos showed a milder phenotype without the development of a pronounced pericardial edema or blood congestion. nexn-specific expression analysis as well as whole transcriptome profiling suggest some degree of compensatory mechanisms. Transcripts of numerous essential sarcomeric proteins were massively induced and may mediate a sarcomere stabilizing function in nexn-/- knockout embryos. Our findings demonstrate the successful generation and characterization of a constitutive homozygous nexn knockout line enabling the detailed investigation of the role of nexn on heart and skeletal muscle development and function as well as to assess putative compensatory mechanisms induced by the loss of Nexn.

Zebrafish as model system for the biological characterization of CK1 inhibitors.

Introduction: The CK1 family is involved in a variety of physiological processes by regulating different signaling pathways, including the Wnt/ß-catenin, the Hedgehog and the p53 signaling pathways. Mutations or dysregulation of kinases in general and of CK1 in particular are known to promote the development of cancer, neurodegenerative diseases and inflammation. There is increasing evidence that CK1 isoform specific small molecule inhibitors, including CK1δ- and CK1ε-specific inhibitors of Wnt production (IWP)-based small molecules with structural similarity to benzimidazole compounds, have promising therapeutic potential. Methods: In this study, we investigated the suitability of the zebrafish model system for the evaluation of such CK1 inhibitors. To this end, the kinetic parameters of human CK1 isoforms were compared with those of zebrafish orthologues. Furthermore, the effects of selective CK1δ inhibition during zebrafish embryonic development were analyzed in vivo. Results: The results revealed that zebrafish CK1δA and CK1δB were inhibited as effectively as human CK1δ by compounds G2-2 with IC50 values of 345 and 270 nM for CK1δA and CK1δB versus 503 nM for human CK1δ and G2-3 exhibiting IC50 values of 514 and 561 nM for zebrafish CK1δA and B, and 562 nM for human CK1δ. Furthermore, the effects of selective CK1δ inhibition on zebrafish embryonic development in vivo revealed phenotypic abnormalities indicative of downregulation of CK1δ. Treatment of zebrafish embryos with selected inhibitors resulted in marked phenotypic changes including blood stasis, heart failure, and tail malformations. Conclusion: The results suggest that the zebrafish is a suitable in vivo assay model system for initial studies of the biological relevance of CK1δ inhibition.

An arrhythmogenic metabolite in atrial fibrillation.

BACKGROUND: Long-chain acyl-carnitines (ACs) are potential arrhythmogenic metabolites. Their role in atrial fibrillation (AF) remains incompletely understood. Using a systems medicine approach, we assessed the contribution of C18:1AC to AF by analysing its in vitro effects on cardiac electrophysiology and metabolism, and translated our findings into the human setting. METHODS AND RESULTS: Human iPSC-derived engineered heart tissue was exposed to C18:1AC. A biphasic effect on contractile force was observed: short exposure enhanced contractile force, but elicited spontaneous contractions and impaired Ca2+ handling. Continuous exposure provoked an impairment of contractile force. In human atrial mitochondria from AF individuals, C18:1AC inhibited respiration. In a population-based cohort as well as a cohort of patients, high C18:1AC serum concentrations were associated with the incidence and prevalence of AF. CONCLUSION: Our data provide evidence for an arrhythmogenic potential of the metabolite C18:1AC. The metabolite interferes with mitochondrial metabolism, thereby contributing to contractile dysfunction and shows predictive potential as novel circulating biomarker for risk of AF.

Hdac1-deficiency affects the cell cycle axis Cdc25-Cdk1 causing impaired G2/M phase progression and reduced cardiomyocyte proliferation in zebrafish.

Zebrafish have the ability to fully regenerate their hearts after injury since cardiomyocytes subsequently dedifferentiate, re-enter cell cycle, and proliferate to replace damaged myocardial tissue. Recent research identified the reactivation of dormant developmental pathways during cardiac regeneration in adult zebrafish, suggesting pro-proliferative pathways important for developmental heart growth to be also critical for regenerative heart growth after injury. Histone deacetylase 1 (Hdac1) was recently shown to control both, embryonic as well as adult regenerative cardiomyocyte proliferation in the zebrafish model. Nevertheless, regulatory pathways controlled by Hdac1 are not defined yet. By analyzing RNA-seq-derived transcriptional profiles of the Hdac1-deficient zebrafish mutant baldrian, we here identified DNA damage response (DDR) pathways activated in baldrian mutant embryos. Surprisingly, although the DDR signaling pathway was transcriptionally activated, we found the complete loss of protein expression of the known DDR effector and cell cycle inhibitor p21. Consequently, we observed an upregulation of the p21-downstream target Cdk2, implying elevated G1/S phase transition in Hdac1-deficient zebrafish hearts. Remarkably, Cdk1, another p21-but also Cdc25-downstream target was downregulated. Here, we found the significant downregulation of Cdc25 protein expression, explaining reduced Cdk1 levels and suggesting impaired G2/M phase progression in Hdac1-deficient zebrafish embryos. To finally prove defective cell cycle progression due to Hdac1 loss, we conducted Cytometer-based cell cycle analyses in HDAC1-deficient murine HL-1 cardiomyocytes and indeed found impaired G2/M phase transition resulting in defective cardiomyocyte proliferation. In conclusion, our results suggest a critical role of Hdac1 in maintaining both, regular G1/S and G2/M phase transition in cardiomyocytes by controlling the expression of essential cell cycle regulators such as p21 and Cdc25.

Association of blood lipids with onset and prognosis of amyotrophic lateral sclerosis: results from the ALS Swabia registry.

BACKGROUND: To date, the role of blood lipid levels and their association with the onset and prognosis of ALS is controversial. We explored these associations in a large, population-based case-control study. METHODS: Between October 2010 and June 2014, 336 ALS patients (mean age 65.7 ± 10.7; 57.7% male) and 487 sex- and age-matched controls from the same geographic region were recruited within the ALS registry in Southwest Germany. Triglycerides and cholesterol (high-density lipoprotein (HDL), low-density lipoprotein (LDL), total) were measured. The ALS cohort was followed up for vital status. Conditional logistic regression models were applied to calculate odds ratio (OR) for risk of ALS associated with serum lipid concentrations. In ALS patients only, survival models were used to appraise the prognostic value. RESULTS: High concentration of total cholesterol (OR 1.60, 95% confidence interval (CI) 1.03-2.49, top vs. bottom quartile), but not HDL, LDL, LDL-HDL ratio, or triglycerides, was positively associated with the risk of ALS. During the median follow-up time of 88.9 months, 291 deaths occurred among 336 ALS patients. In the adjusted survival analysis, higher HDL (HR 1.72, 95% CI 1.19-2.50) and LDL cholesterol levels (HR 1.58, 95% CI 1.11-2.26) were associated with higher mortality in ALS patients. In contrast, higher triglyceride levels were associated with lower mortality (HR 0.68, 95% CI 0.48-0.96). CONCLUSION: The results highlight the importance to distinguish cholesterol from triglycerides when considering the prognostic role of lipid metabolism in ALS. It further strengthens the rationale for a triglyceride-rich diet, while the negative impact of cholesterol must be further explored.

Hydroxylation of the NOTCH1 intracellular domain regulates Notch signaling dynamics.

Notch signaling plays a pivotal role in the development and, when dysregulated, it contributes to tumorigenesis. The amplitude and duration of the Notch response depend on the posttranslational modifications (PTMs) of the activated NOTCH receptor - the NOTCH intracellular domain (NICD). In normoxic conditions, the hydroxylase FIH (factor inhibiting HIF) catalyzes the hydroxylation of two asparagine residues of the NICD. Here, we investigate how Notch-dependent gene transcription is regulated by hypoxia in progenitor T cells. We show that the majority of Notch target genes are downregulated upon hypoxia. Using a hydroxyl-specific NOTCH1 antibody we demonstrate that FIH-mediated NICD1 hydroxylation is reduced upon hypoxia or treatment with the hydroxylase inhibitor dimethyloxalylglycine (DMOG). We find that a hydroxylation-resistant NICD1 mutant is functionally impaired and more ubiquitinated. Interestingly, we also observe that the NICD1-deubiquitinating enzyme USP10 is downregulated upon hypoxia. Moreover, the interaction between the hydroxylation-defective NICD1 mutant and USP10 is significantly reduced compared to the NICD1 wild-type counterpart. Together, our data suggest that FIH hydroxylates NICD1 in normoxic conditions, leading to the recruitment of USP10 and subsequent NICD1 deubiquitination and stabilization. In hypoxia, this regulatory loop is disrupted, causing a dampened Notch response.

CRISPR/Cas9-Mediated Constitutive Loss of VCP (Valosin-Containing Protein) Impairs Proteostasis and Leads to Defective Striated Muscle Structure and Function In Vivo.

Valosin-containing protein (VCP) acts as a key regulator of cellular protein homeostasis by coordinating protein turnover and quality control. Mutations in VCP lead to (cardio-)myopathy and neurodegenerative diseases such as inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD) or amyotrophic lateral sclerosis (ALS). To date, due to embryonic lethality, no constitutive VCP knockout animal model exists. Here, we generated a constitutive CRISPR/Cas9-induced vcp knockout zebrafish model. Similar to the phenotype of vcp morphant knockdown zebrafish embryos, we found that vcp-null embryos displayed significantly impaired cardiac and skeletal muscle function. By ultrastructural analysis of skeletal muscle cells and cardiomyocytes, we observed severely disrupted myofibrillar organization and accumulation of inclusion bodies as well as mitochondrial degeneration. vcp knockout was associated with a significant accumulation of ubiquitinated proteins, suggesting impaired proteasomal function. Additionally, markers of unfolded protein response (UPR)/ER-stress and autophagy-related mTOR signaling were elevated in vcp-deficient embryos, demonstrating impaired proteostasis in VCP-null zebrafish. In conclusion, our findings demonstrate the successful generation of a stable constitutive vcp knockout zebrafish line that will enable characterization of the detailed mechanistic underpinnings of vcp loss, particularly the impact of disturbed protein homeostasis on organ development and function in vivo.

Histone deacetylase 1 controls cardiomyocyte proliferation during embryonic heart development and cardiac regeneration in zebrafish.

In contrast to mammals, the zebrafish maintains its cardiomyocyte proliferation capacity throughout adulthood. However, neither the molecular mechanisms that orchestrate the proliferation of cardiomyocytes during developmental heart growth nor in the context of regeneration in the adult are sufficiently defined yet. We identified in a forward genetic N-ethyl-N-nitrosourea (ENU) mutagenesis screen the recessive, embryonic-lethal zebrafish mutant baldrian (bal), which shows severely impaired developmental heart growth due to diminished cardiomyocyte proliferation. By positional cloning, we identified a missense mutation in the zebrafish histone deacetylase 1 (hdac1) gene leading to severe protein instability and the loss of Hdac1 function in vivo. Hdac1 inhibition significantly reduces cardiomyocyte proliferation, indicating a role of Hdac1 during developmental heart growth in zebrafish. To evaluate whether developmental and regenerative Hdac1-associated mechanisms of cardiomyocyte proliferation are conserved, we analyzed regenerative cardiomyocyte proliferation after Hdac1 inhibition at the wound border zone in cryoinjured adult zebrafish hearts and we found that Hdac1 is also essential to orchestrate regenerative cardiomyocyte proliferation in the adult vertebrate heart. In summary, our findings suggest an important and conserved role of Histone deacetylase 1 (Hdac1) in developmental and adult regenerative cardiomyocyte proliferation in the vertebrate heart.

Long-Chain Acyl-Carnitines Interfere with Mitochondrial ATP Production Leading to Cardiac Dysfunction in Zebrafish.

In the human heart, the energy supplied by the production of ATP is predominately accomplished by ß-oxidation in mitochondria, using fatty acids (FAs) as the primary fuel. Long-chain acylcarnitines (LCACs) are intermediate forms of FA transport that are essential for FA delivery from the cytosol into mitochondria. Here, we analyzed the impact of the LCACs C18 and C18:1 on mitochondrial function and, subsequently, on heart functionality in the in vivo vertebrate model system of zebrafish (Danio rerio). Since LCACs are formed and metabolized in mitochondria, we assessed mitochondrial morphology, structure and density in C18- and C18:1-treated zebrafish and found no mitochondrial alterations compared to control-treated (short-chain acylcarnitine, C3) zebrafish embryos. However, mitochondrial function and subsequently ATP production was severely impaired in C18- and C18:1-treated zebrafish embryos. Furthermore, we found that C18 and C18:1 treatment of zebrafish embryos led to significantly impaired cardiac contractile function, accompanied by reduced heart rate and diminished atrial and ventricular fractional shortening, without interfering with cardiomyocyte differentiation, specification and growth. In summary, our findings provide insights into the direct role of long-chain acylcarnitines on vertebrate heart function by interfering with regular mitochondrial function and thereby energy allocation in cardiomyocytes.

IFITM proteins promote SARS-CoV-2 infection and are targets for virus inhibition in vitro.

Interferon-induced transmembrane proteins (IFITMs 1, 2 and 3) can restrict viral pathogens, but pro- and anti-viral activities have been reported for coronaviruses. Here, we show that artificial overexpression of IFITMs blocks SARS-CoV-2 infection. However, endogenous IFITM expression supports efficient infection of SARS-CoV-2 in human lung cells. Our results indicate that the SARS-CoV-2 Spike protein interacts with IFITMs and hijacks them for efficient viral infection. IFITM proteins were expressed and further induced by interferons in human lung, gut, heart and brain cells. IFITM-derived peptides and targeting antibodies inhibit SARS-CoV-2 entry and replication in human lung cells, cardiomyocytes and gut organoids. Our results show that IFITM proteins are cofactors for efficient SARS-CoV-2 infection of human cell types representing in vivo targets for viral transmission, dissemination and pathogenesis and are potential targets for therapeutic approaches.

Identification and Tissue-Specific Characterization of Novel SHOX-Regulated Genes in Zebrafish Highlights SOX Family Members Among Other Genes.

SHOX deficiency causes a spectrum of clinical phenotypes related to skeletal dysplasia and short stature, including Léri-Weill dyschondrosteosis, Langer mesomelic dysplasia, Turner syndrome, and idiopathic short stature. SHOX controls chondrocyte proliferation and differentiation, bone maturation, and cellular growth arrest and apoptosis via transcriptional regulation of its direct target genes NPPB, FGFR3, and CTGF. However, our understanding of SHOX-related pathways is still incomplete. To elucidate the underlying molecular mechanisms and to better understand the broad phenotypic spectrum of SHOX deficiency, we aimed to identify novel SHOX targets. We analyzed differentially expressed genes in SHOX-overexpressing human fibroblasts (NHDF), and confirmed the known SHOX target genes NPPB and FGFR among the most strongly regulated genes, together with 143 novel candidates. Altogether, 23 genes were selected for further validation, first by whole-body characterization in developing shox-deficient zebrafish embryos, followed by tissue-specific expression analysis in three shox-expressing zebrafish tissues: head (including brain, pharyngeal arches, eye, and olfactory epithelium), heart, and pectoral fins. Most genes were physiologically relevant in the pectoral fins, while only few genes were also significantly regulated in head and heart tissue. Interestingly, multiple sox family members (sox5, sox6, sox8, and sox18) were significantly dysregulated in shox-deficient pectoral fins together with other genes (nppa, nppc, cdkn1a, cdkn1ca, cyp26b1, and cy26c1), highlighting an important role for these genes in shox-related growth disorders. Network-based analysis integrating data from the Ingenuity pathways revealed that most of these genes act in a common network. Our results provide novel insights into the genetic pathways and molecular events leading to the clinical manifestation of SHOX deficiency.

Quantification of Biventricular Myocardial Strain Using CMR Feature Tracking: Reproducibility in Small Animals.

Myocardial strain is a well-validated parameter for evaluating myocardial contraction. Cardiovascular magnetic resonance myocardial feature tracking (CMR-FT) is a novel method for the quantitative measurements of myocardial strain from routine cine acquisitions. In this study, we investigated the influence of temporal resolution on tracking accuracy of CMR-FT and the intraobserver, interobserver, and interstudy reproducibilities for biventricular strain analysis in mice from self-gated CMR at 11.7 T. 12 constitutive nexilin knockout (Nexn-KO) mice, heterozygous (Het, N = 6) and wild-type (WT, N = 6), were measured with a well-established self-gating sequence twice within two weeks. CMR-FT measures of biventricular global and segmental strain parameters were derived. Interstudy, intraobserver, and interobserver reproducibilities were investigated. For the assessment of the impact of the temporal resolution for the outcome in CMR-FT, highly oversampled semi-4 chamber and midventricular short-axis data were acquired and reconstructed with 10 to 80 phases per cardiac cycle. A generally reduced biventricular myocardial strain was observed in Nexn-KO Het mice. Excellent intraobserver and interobserver reproducibility was achieved in all global strains (ICC range from 0.76 to 0.99), where global right ventricle circumferential strain (RCSSAX) showed an only good interobserver reproducibility (ICC 0.65, 0.11-0.89). For interstudy reproducibility, left ventricle longitudinal strain (LLSLAX) was the most reproducible measure of strain (ICC 0.90, 0.71-0.97). The left ventricle radial strain (LRSSAX) (ICC 0.50, 0.10-0.83) showed fair reproducibility and RCSSAX (ICC 0.36, 0.14-0.74) showed only poor reproducibility. In general, compared with global strains, the segmental strains showed relatively lower reproducibility. A minimal temporal resolution of 20 phases per cardiac cycle appeared sufficient for CMR-FT strain analysis. The analysis of myocardial strain from high-resolution self-gated cine images by CMR-FT provides a highly reproducible method for assessing myocardial contraction in small rodent animals. Especially, global LV longitudinal and circumferential strain revealed excellent reproducibility of intra- and interobserver and interstudy measurements.

Spen deficiency interferes with Connexin 43 expression and leads to heart failure in zebrafish.

Genome-wide association studies identified Spen as a putative modifier of cardiac function, however, the precise function of Spen in the cardiovascular system is not known yet. Here, we analyzed for the first time the in vivo role of Spen in zebrafish and found that targeted Spen inactivation led to progressive impairment of cardiac function in the zebrafish embryo. In addition to diminished cardiac contractile force, Spen-deficient zebrafish embryos developed bradycardia, atrioventricular block and heart chamber fibrillation. Assessment of cardiac-specific transcriptional profiles identified Connexin 43 (Cx43), a cardiac gap junction protein and crucial regulator of cardiomyocyte-to-cardiomyocyte communication, to be significantly diminished in Spen-deficient zebrafish embryos. Similar to the situation in Spen-deficient embryos, Morpholino-mediated knockdown of cx43 in zebrafish resulted in cardiac contractile dysfunction, bradycardia, atrioventricular block and fibrillation of the cardiac chambers. Furthermore, ectopic overexpression of cx43 in Spen deficient embryos led to the reconstitution of cardiac contractile function and suppression of cardiac arrhythmia. Additionally, sensitizing experiments by simultaneously injecting sub-phenotypic concentrations of spen- and cx43-Morpholinos into zebrafish embryos resulted in pathological supra-additive effects. In summary, our findings highlight a crucial role of Spen in controlling cx43 expression and demonstrate the Spen-Cx43 axis to be a vital regulatory cascade that is indispensable for proper heart function in vivo.

Genetic compensation prevents myopathy and heart failure in an in vivo model of Bag3 deficiency.

Mutations in the molecular co-chaperone Bcl2-associated athanogene 3 (BAG3) are found to cause dilated cardiomyopathy (DCM), resulting in systolic dysfunction and heart failure, as well as myofibrillar myopathy (MFM), which is characterized by protein aggregation and myofibrillar disintegration in skeletal muscle cells. Here, we generated a CRISPR/Cas9-induced Bag3 knockout zebrafish line and found the complete preservation of heart and skeletal muscle structure and function during embryonic development, in contrast to morpholino-mediated knockdown of Bag3. Intriguingly, genetic compensation, a process of transcriptional adaptation which acts independent of protein feedback loops, was found to prevent heart and skeletal muscle damage in our Bag3 knockout model. Proteomic profiling and quantitative real-time PCR analyses identified Bag2, another member of the Bag protein family, significantly upregulated on a transcript and protein level in bag3-/- mutants. This implied that the decay of bag3 mutant mRNA in homozygous bag3-/- embryos caused the transcriptional upregulation of bag2 expression. We further demonstrated that morpholino-mediated knockdown of Bag2 in bag3-/- embryos evoked severe functional and structural heart and skeletal muscle defects, which are similar to Bag3 morphants. However, Bag2 knockdown in bag3+/+ or bag3+/- embryos did not result in (cardio-)myopathy. Finally, we found that inhibition of the nonsense-mediated mRNA decay (NMD) machinery by knockdown of upf1, an essential NMD factor, caused severe heart and skeletal muscle defects in bag3-/- mutants due to the blockade of transcriptional adaptation of bag2 expression. Our findings provide evidence that genetic compensation might vitally influence the penetrance of disease-causing bag3 mutations in vivo.

Feasibility of real-time cardiac MRI in mice using tiny golden angle radial sparse.

Cardiovascular magnetic resonance imaging has proven valuable for the assessment of structural and functional cardiac abnormalities. Even although it is an established imaging method in small animals, the long acquisition times of gated or self-gated techniques still limit its widespread application. In this study, the application of tiny golden angle radial sparse MRI (tyGRASP) for real-time cardiac imaging was tested in 12 constitutive nexilin (Nexn) knock-out (KO) mice, both heterozygous (Het, N = 6) and wild-type (WT, N = 6), and the resulting functional parameters were compared with a well-established self-gating approach. Real-time images were reconstructed for different temporal resolutions of between 16.8 and 79.8 ms per image. The suggested approach was additionally tested for dobutamine stress and qualitative first-pass perfusion imaging. Measurements were repeated twice within 2 weeks for reproducibility assessment. In direct comparison with the high-quality, self-gated technique, the real-time approach did not show any significant differences in global function parameters for acquisition times below 50 ms (rest) and 31.5 ms (stress). Compared with WT, the end-diastolic volume (EDV) and end-systolic volume (ESV) were markedly higher (P < 0.05) and the ejection fraction (EF) was significantly lower in the Het Nexn-KO mice at rest (P < 0.001). For the stress investigation, a clear decrease of EDV and ESV, and an increase in EF, but maintained stroke volume, could be observed in both groups. Combined with ECG-triggering, tyGRASP provided first-pass perfusion data with a temporal resolution of one image per heartbeat, allowing the quantitative assessment of upslope curves in the blood-pool and myocardium. Excellent inter-study reproducibility was achieved in all the functional parameters. The tyGRASP is a valuable real-time MRI technique for mice, which significantly reduces the scan time in preclinical cardiac functional imaging, providing sufficient image quality for deriving accurate functional parameters, and has the potential to investigate real-time and beat-to-beat changes.

HDAC3 functions as a positive regulator in Notch signal transduction.

Aberrant Notch signaling plays a pivotal role in T-cell acute lymphoblastic leukemia (T-ALL) and chronic lymphocytic leukemia (CLL). Amplitude and duration of the Notch response is controlled by ubiquitin-dependent proteasomal degradation of the Notch1 intracellular domain (NICD1), a hallmark of the leukemogenic process. Here, we show that HDAC3 controls NICD1 acetylation levels directly affecting NICD1 protein stability. Either genetic loss-of-function of HDAC3 or nanomolar concentrations of HDAC inhibitor apicidin lead to downregulation of Notch target genes accompanied by a local reduction of histone acetylation. Importantly, an HDAC3-insensitive NICD1 mutant is more stable but biologically less active. Collectively, these data show a new HDAC3- and acetylation-dependent mechanism that may be exploited to treat Notch1-dependent leukemias.

Streamlining drug discovery assays for cardiovascular disease using zebrafish.

Introduction: In the last decade, our armamentarium of cardiovascular drug therapy has expanded significantly. Using innovative functional genomics strategies such as genome editing by CRISPR/Cas9 as well as high-throughput assays to identify bioactive small chemical compounds has significantly facilitated elaboration of the underlying pathomechanism in various cardiovascular diseases. However, despite scientific progress approvals for cardiovascular drugs has stagnated significantly compared to other fields of drug discovery and therapy during the past years.Areas covered: In this review, the authors discuss the aspects and pitfalls during the early phase of cardiovascular drug discovery and describe the advantages of zebrafish as an in vivo organism to model human cardiovascular diseases (CVD) as well as an in vivo platform for high-throughput chemical compound screening. They also highlight the emerging, promising techniques of automated read-out systems during high-throughput screening (HTS) for the evaluation of important cardiac functional parameters in zebrafish with the potential to streamline CVD drug discovery.Expert opinion: The successful identification of novel drugs to treat CVD is a major challenge in modern biomedical and clinical research. In this context, the definition of the etiologic fundamentals of human cardiovascular diseases is the prerequisite for an efficient and straightforward drug discovery.

Machine Learning Methods for Automated Quantification of Ventricular Dimensions.

Medaka (Oryzias latipes) and zebrafish (Danio rerio) contribute substantially to our understanding of the genetic and molecular etiology of human cardiovascular diseases. In this context, the quantification of important cardiac functional parameters is fundamental. We have developed a framework that segments the ventricle of a medaka hatchling from image sequences and subsequently quantifies ventricular dimensions.

Functional Characterization of Rare Variants in the SHOX2 Gene Identified in Sinus Node Dysfunction and Atrial Fibrillation.

Sinus node dysfunction (SND) and atrial fibrillation (AF) often coexist; however, the molecular mechanisms linking both conditions remain elusive. Mutations in the homeobox-containing SHOX2 gene have been recently associated with early-onset and familial AF. Shox2 is a key regulator of sinus node development, and its deficiency leads to bradycardia, as demonstrated in animal models. To provide an extended SHOX2 gene analysis in patients with distinct arrhythmias, we investigated SHOX2 as a susceptibility gene for SND and AF by screening 98 SND patients and 450 individuals with AF. The functional relevance of the novel mutations was investigated in vivo and in vitro, together with the previously reported p.H283Q variant. A heterozygous missense mutation (p.P33R) was identified in the SND cohort and four heterozygous variants (p.G77D, p.L129=, p.L130F, p.A293=) in the AF cohort. Overexpression of the pathogenic predicted mutations in zebrafish revealed pericardial edema for p.G77D and the positive control p.H283Q, whereas the p.P33R and p.A293= variants showed no effect. In addition, a dominant-negative effect with reduced heart rates was detected for p.G77D and p.H283Q. In vitro reporter assays demonstrated for both missense variants p.P33R and p.G77D significantly impaired transactivation activity, similar to the described p.H283Q variant. Also, a reduced Bmp4 target gene expression was revealed in zebrafish hearts upon overexpression of the p.P33R mutant. This study associates additional rare variants in the SHOX2 gene implicated in the susceptibility to distinct arrhythmias and allows frequency estimations in the AF cohort (3/990). We also demonstrate for the first time a genetic link between SND and AF involving SHOX2. Moreover, our data highlight the importance of functional investigations of rare variants.