Cellular and molecular mechanisms of zebrafish heart regeneration

Humans can, if they survived a cardiac injury such as heart infarction, heal this cardiac injury only by scarring and with minimal regeneration of some cardiac cells. Zebrafish, however, can fully regenerate cardiac tissue after surgical resection of up to 20% of the ventricle. Regenerating tissue includes cells of the three cardiac layers, i.e. myocardium, epicardium and endocardium. Thus, zebrafish, with its ability to regenerate damaged heart and as a model enabling genetic manipulations, provides the possibility to study cellular and molecular mechanisms of heart regeneration. Understanding these mechanisms may help develop new therapeutic approaches to improve the situation after a heart injury in humans. Since molecular mechanisms regulating heart regeneration are so far largely unknown, I aimed to identify and analyze molecular signals that are important for cardiac regeneration in zebrafish. Molecular signals that are crucial during heart development have been suggested to be reactivated during cardiac regeneration. Since Wnt/β-catenin signaling is crucial for vertebrate heart development, it is likely to be important for zebrafish cardiac regeneration as well. First, I focused on the functional role of Wnt/β-catenin signaling in cardiac regeneration mainly by using transgenic fish lines that allow inducible activation or inhibition of the pathway. By using in situ hybridization and expression profiling, I tested whether endogenous Wnt/β-catenin target genes are detectable in regenerating hearts and screened for activity of the β-catenin responsive reporter in TOPdGFP transgenic fish (Tg(TOP:GFP)w25) after ventricular resection. I could not identify endogenous Wnt/β-catenin targets during the early phase of regeneration up to 7 days post amputation (dpa) using oligoexpression microarrays or in situ hybridization. An injury specific activation of the β-catenin responsive TOPdGFP reporter was not detectable either, suggesting that Wnt/β-catenin signaling is not active during this early phase of regeneration. The manipulation of Wnt/β-catenin signaling using transgenic fish lines did not influence cell proliferation or the overall extent of zebrafish heart regeneration. These results suggested that Wnt/β-catenin signaling has no functional role during entire zebrafish heart regeneration. Second, I found the transcription factor Sox9a to be upregulated after ventricular resection during the early phase of heart regeneration. Using transgenic reporter fish lines, I detected Sox9a expression in cardiomyocytes and endothelial cells, part of which were proliferative. Furthermore Sox9a was expressed in some cells of the epicardial layer that activated the expression of developmental genes in the entire heart in response to injury. These results indicated that Sox9a is expressed in cells that were actively involved in the regenerative response. To gain insight into the functional role of Sox9a, I generated a transgenic fish line where a repressor construct is inducibly expressed, which then interferes with Sox9a target gene transcription. I detected a significant reduction in myocardial and endothelial regeneration after induction of the repressor. These results suggested that Sox9a function is important for regeneration of endothelial and myocardial cells after heart injury. Third, using oligoexpression microarrays, I performed systematic gene expression profiling of the zebrafish heart regeneration within the first 2 weeks following amputation. I found that known genes, which have previously been shown to be strongly expressed during heart regeneration, as well as novel genes were upregulated after ventricular resection. Some of these genes have been implicated in vertebrate heart development, supporting the idea that cardiac developmental genes are reactivated during heart regeneration. Hence, these results reveal a good starting point for further analysis of the cellular and molecular events occurring within the first days after cardiac injury. Finally, I developed a cryoinjury method that more closely resembled the injured tissue after human heart infarction. I induced tissue death by exposing the ventricle to dry ice and detected that the zebrafish heart can regenerate upon this cardiac injury similarly as in response to a ventricular resection injury. After cryoinjury, the entire epicardium activated the expression of developmental genes and started to proliferate. I detected also proliferating cardiomyocytes, indicating that similar cellular mechanisms are induced in the epicardium and the myocardium after cryoinjury and ventricular resection. Furthermore, activation of Sox9 expression early after cryoinjury suggested that molecular mechanisms of regeneration are also similar in both injury methods. Thus, cryoinjury provides a useful tool for future studies of zebrafish heart regeneration with more relevance to human cardiac infarction. I discuss all results with reference to vertebrate heart development and to the response after mammalian heart infarction. Furthermore, the results were put into the context of cellular mechanisms that are present in the process of zebrafish heart regeneration

Regeneration of Cryoinjury Induced Necrotic Heart Lesions in Zebrafish Is Associated with Epicardial Activation and Cardiomyocyte Proliferation

In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death

Measuring the predictability of life outcomes with a scientific mass collaboration.

How predictable are life trajectories? We investigated this question with a scientific mass collaboration using the common task method; 160 teams built predictive models for six life outcomes using data from the Fragile Families and Child Wellbeing Study, a high-quality birth cohort study. Despite using a rich dataset and applying machine-learning methods optimized for prediction, the best predictions were not very accurate and were only slightly better than those from a simple benchmark model. Within each outcome, prediction error was strongly associated with the family being predicted and weakly associated with the technique used to generate the prediction. Overall, these results suggest practical limits to the predictability of life outcomes in some settings and illustrate the value of mass collaborations in the social sciences

RANTES/CCL5 and Risk for Coronary Events: Results from the MONICA/KORA Augsburg Case-Cohort, Athero-Express and CARDIoGRAM Studies

BACKGROUND: The chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events. METHODS AND FINDINGS: We conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±4.8 years). Cox proportional hazard models adjusting for age, sex, body mass index, metabolic factors and lifestyle factors revealed no significant association between RANTES and incident coronary events (HR [95% CI] for increasing RANTES tertiles 1.0, 1.03 [0.75-1.42] and 1.11 [0.81-1.54]). None of six CCL5 single nucleotide polymorphisms and no common haplotype showed significant associations with coronary events. Also in the CARDIoGRAM study (>22,000 cases, >60,000 controls), none of these CCL5 SNPs was significantly associated with coronary artery disease. In the prospective Athero-Express biobank study, RANTES plaque levels were measured in 606 atherosclerotic lesions from patients who underwent carotid endarterectomy. RANTES content in atherosclerotic plaques was positively associated with macrophage infiltration and inversely associated with plaque calcification. However, there was no significant association between RANTES content in plaques and risk for coronary events (mean follow-up 2.8±0.8 years). CONCLUSIONS: High RANTES plaque levels were associated with an unstable plaque phenotype. However, the absence of associations between (i) RANTES serum levels, (ii) CCL5 genotypes and (iii) RANTES content in carotid plaques and either coronary artery disease or incident coronary events in our cohorts suggests that RANTES may not be a novel coronary risk biomarker. However, the potential relevance of RANTES levels in platelet-poor plasma needs to be investigated in further studies

RANTES/CCL5 and risk for coronary events: Results from the MONICA/KORA Augsburg case-cohort, Athero-express and CARDIoGRAM studies

Background: The chemokine RANTES (regulated on activation, normal T-cell expressed and secreted)/CCL5 is involved in the pathogenesis of cardiovascular disease in mice, whereas less is known in humans. We hypothesised that its relevance for atherosclerosis should be reflected by associations between CCL5 gene variants, RANTES serum concentrations and protein levels in atherosclerotic plaques and risk for coronary events. Methods and Findings: We conducted a case-cohort study within the population-based MONICA/KORA Augsburg studies. Baseline RANTES serum levels were measured in 363 individuals with incident coronary events and 1,908 non-cases (mean follow-up: 10.2±

Genetic associations at 53 loci highlight cell types and biological pathways relevant for kidney function.

Reduced glomerular filtration rate defines chronic kidney disease and is associated with cardiovascular and all-cause mortality. We conducted a meta-analysis of genome-wide association studies for estimated glomerular filtration rate (eGFR), combining data across 133,413 individuals with replication in up to 42,166 individuals. We identify 24 new and confirm 29 previously identified loci. Of these 53 loci, 19 associate with eGFR among individuals with diabetes. Using bioinformatics, we show that identified genes at eGFR loci are enriched for expression in kidney tissues and in pathways relevant for kidney development and transmembrane transporter activity, kidney structure, and regulation of glucose metabolism. Chromatin state mapping and DNase I hypersensitivity analyses across adult tissues demonstrate preferential mapping of associated variants to regulatory regions in kidney but not extra-renal tissues. These findings suggest that genetic determinants of eGFR are mediated largely through direct effects within the kidney and highlight important cell types and biological pathways

Cellular and molecular mechanisms of zebrafish heart regeneration

Humans can, if they survived a cardiac injury such as heart infarction, heal this cardiac injury only by scarring and with minimal regeneration of some cardiac cells. Zebrafish, however, can fully regenerate cardiac tissue after surgical resection of up to 20% of the ventricle. Regenerating tissue includes cells of the three cardiac layers, i.e. myocardium, epicardium and endocardium. Thus, zebrafish, with its ability to regenerate damaged heart and as a model enabling genetic manipulations, provides the possibility to study cellular and molecular mechanisms of heart regeneration. Understanding these mechanisms may help develop new therapeutic approaches to improve the situation after a heart injury in humans. Since molecular mechanisms regulating heart regeneration are so far largely unknown, I aimed to identify and analyze molecular signals that are important for cardiac regeneration in zebrafish. Molecular signals that are crucial during heart development have been suggested to be reactivated during cardiac regeneration. Since Wnt/β-catenin signaling is crucial for vertebrate heart development, it is likely to be important for zebrafish cardiac regeneration as well. First, I focused on the functional role of Wnt/β-catenin signaling in cardiac regeneration mainly by using transgenic fish lines that allow inducible activation or inhibition of the pathway. By using in situ hybridization and expression profiling, I tested whether endogenous Wnt/β-catenin target genes are detectable in regenerating hearts and screened for activity of the β-catenin responsive reporter in TOPdGFP transgenic fish (Tg(TOP:GFP)w25) after ventricular resection. I could not identify endogenous Wnt/β-catenin targets during the early phase of regeneration up to 7 days post amputation (dpa) using oligoexpression microarrays or in situ hybridization. An injury specific activation of the β-catenin responsive TOPdGFP reporter was not detectable either, suggesting that Wnt/β-catenin signaling is not active during this early phase of regeneration. The manipulation of Wnt/β-catenin signaling using transgenic fish lines did not influence cell proliferation or the overall extent of zebrafish heart regeneration. These results suggested that Wnt/β-catenin signaling has no functional role during entire zebrafish heart regeneration. Second, I found the transcription factor Sox9a to be upregulated after ventricular resection during the early phase of heart regeneration. Using transgenic reporter fish lines, I detected Sox9a expression in cardiomyocytes and endothelial cells, part of which were proliferative. Furthermore Sox9a was expressed in some cells of the epicardial layer that activated the expression of developmental genes in the entire heart in response to injury. These results indicated that Sox9a is expressed in cells that were actively involved in the regenerative response. To gain insight into the functional role of Sox9a, I generated a transgenic fish line where a repressor construct is inducibly expressed, which then interferes with Sox9a target gene transcription. I detected a significant reduction in myocardial and endothelial regeneration after induction of the repressor. These results suggested that Sox9a function is important for regeneration of endothelial and myocardial cells after heart injury. Third, using oligoexpression microarrays, I performed systematic gene expression profiling of the zebrafish heart regeneration within the first 2 weeks following amputation. I found that known genes, which have previously been shown to be strongly expressed during heart regeneration, as well as novel genes were upregulated after ventricular resection. Some of these genes have been implicated in vertebrate heart development, supporting the idea that cardiac developmental genes are reactivated during heart regeneration. Hence, these results reveal a good starting point for further analysis of the cellular and molecular events occurring within the first days after cardiac injury. Finally, I developed a cryoinjury method that more closely resembled the injured tissue after human heart infarction. I induced tissue death by exposing the ventricle to dry ice and detected that the zebrafish heart can regenerate upon this cardiac injury similarly as in response to a ventricular resection injury. After cryoinjury, the entire epicardium activated the expression of developmental genes and started to proliferate. I detected also proliferating cardiomyocytes, indicating that similar cellular mechanisms are induced in the epicardium and the myocardium after cryoinjury and ventricular resection. Furthermore, activation of Sox9 expression early after cryoinjury suggested that molecular mechanisms of regeneration are also similar in both injury methods. Thus, cryoinjury provides a useful tool for future studies of zebrafish heart regeneration with more relevance to human cardiac infarction. I discuss all results with reference to vertebrate heart development and to the response after mammalian heart infarction. Furthermore, the results were put into the context of cellular mechanisms that are present in the process of zebrafish heart regeneration

Cellular and molecular mechanisms of zebrafish heart regeneration

Humans can, if they survived a cardiac injury such as heart infarction, heal this cardiac injury only by scarring and with minimal regeneration of some cardiac cells. Zebrafish, however, can fully regenerate cardiac tissue after surgical resection of up to 20% of the ventricle. Regenerating tissue includes cells of the three cardiac layers, i.e. myocardium, epicardium and endocardium. Thus, zebrafish, with its ability to regenerate damaged heart and as a model enabling genetic manipulations, provides the possibility to study cellular and molecular mechanisms of heart regeneration. Understanding these mechanisms may help develop new therapeutic approaches to improve the situation after a heart injury in humans. Since molecular mechanisms regulating heart regeneration are so far largely unknown, I aimed to identify and analyze molecular signals that are important for cardiac regeneration in zebrafish. Molecular signals that are crucial during heart development have been suggested to be reactivated during cardiac regeneration. Since Wnt/β-catenin signaling is crucial for vertebrate heart development, it is likely to be important for zebrafish cardiac regeneration as well. First, I focused on the functional role of Wnt/β-catenin signaling in cardiac regeneration mainly by using transgenic fish lines that allow inducible activation or inhibition of the pathway. By using in situ hybridization and expression profiling, I tested whether endogenous Wnt/β-catenin target genes are detectable in regenerating hearts and screened for activity of the β-catenin responsive reporter in TOPdGFP transgenic fish (Tg(TOP:GFP)w25) after ventricular resection. I could not identify endogenous Wnt/β-catenin targets during the early phase of regeneration up to 7 days post amputation (dpa) using oligoexpression microarrays or in situ hybridization. An injury specific activation of the β-catenin responsive TOPdGFP reporter was not detectable either, suggesting that Wnt/β-catenin signaling is not active during this early phase of regeneration. The manipulation of Wnt/β-catenin signaling using transgenic fish lines did not influence cell proliferation or the overall extent of zebrafish heart regeneration. These results suggested that Wnt/β-catenin signaling has no functional role during entire zebrafish heart regeneration. Second, I found the transcription factor Sox9a to be upregulated after ventricular resection during the early phase of heart regeneration. Using transgenic reporter fish lines, I detected Sox9a expression in cardiomyocytes and endothelial cells, part of which were proliferative. Furthermore Sox9a was expressed in some cells of the epicardial layer that activated the expression of developmental genes in the entire heart in response to injury. These results indicated that Sox9a is expressed in cells that were actively involved in the regenerative response. To gain insight into the functional role of Sox9a, I generated a transgenic fish line where a repressor construct is inducibly expressed, which then interferes with Sox9a target gene transcription. I detected a significant reduction in myocardial and endothelial regeneration after induction of the repressor. These results suggested that Sox9a function is important for regeneration of endothelial and myocardial cells after heart injury. Third, using oligoexpression microarrays, I performed systematic gene expression profiling of the zebrafish heart regeneration within the first 2 weeks following amputation. I found that known genes, which have previously been shown to be strongly expressed during heart regeneration, as well as novel genes were upregulated after ventricular resection. Some of these genes have been implicated in vertebrate heart development, supporting the idea that cardiac developmental genes are reactivated during heart regeneration. Hence, these results reveal a good starting point for further analysis of the cellular and molecular events occurring within the first days after cardiac injury. Finally, I developed a cryoinjury method that more closely resembled the injured tissue after human heart infarction. I induced tissue death by exposing the ventricle to dry ice and detected that the zebrafish heart can regenerate upon this cardiac injury similarly as in response to a ventricular resection injury. After cryoinjury, the entire epicardium activated the expression of developmental genes and started to proliferate. I detected also proliferating cardiomyocytes, indicating that similar cellular mechanisms are induced in the epicardium and the myocardium after cryoinjury and ventricular resection. Furthermore, activation of Sox9 expression early after cryoinjury suggested that molecular mechanisms of regeneration are also similar in both injury methods. Thus, cryoinjury provides a useful tool for future studies of zebrafish heart regeneration with more relevance to human cardiac infarction. I discuss all results with reference to vertebrate heart development and to the response after mammalian heart infarction. Furthermore, the results were put into the context of cellular mechanisms that are present in the process of zebrafish heart regeneration

Causal factors in childhood and adolescence leading to anabolic-androgenic steroid use: A machine learning approach

Background: Prior research has demonstrated associations between anabolic-androgenic steroid (AAS) use and features from several childhood and adolescent psychosocial domains including body image concerns, antisocial traits, and low levels of parental care. However, prior approaches have been limited by their focus on individual features and lack of consideration of the relevant causal structure. Methods: We re-analyzed data from a previous cross-sectional cohort study of 232 male weightlifters aged 18–40, of whom 101 had used AAS. These men completed retrospective measures of features from their childhood and early adolescence, including body image concerns, eating disorder psychopathology, antisocial traits, substance use, and family relationships. Using an approach informed by principles of causal inference, we applied four machine-learning methods – lasso regression, elastic net regression, random forests, and gradient boosting – to predict AAS use. Results: The four methods yielded similar receiver operating curves, mean area under the curve (range 0.66 to 0.72), and sets of highly important features. Features related to adolescent body image concerns (especially muscle dysmorphia symptoms) were the strongest predictors. Other important features were adolescent rebellious behaviors; adolescent feelings of ineffectiveness and lack of interoceptive awareness; and low levels of paternal care. Conclusions: Applying machine learning within a causally informed approach to re-analyze data from a prior study of weightlifters, we identified six factors (most prominently those related to adolescent body image concerns) as proposed causal factors for the development of AAS use. Compared with the prior analyses, this approach achieved greater methodologic rigor and yielded stronger and broader findings