Abstract Background Hypertension is a major global health challenge with well-established cardiovascular risks, yet its relationship with bone mineral density and the skeletal relevance of antihypertensive-related targets remain unclear. Methods Based on individual-level data from 366,443 European-ancestry participants in the UK Biobank, this study adopted restricted cubic spline models to explore linear and nonlinear associations between systolic/diastolic blood pressure (SBP/DBP) and heel estimated bone mineral density (BMD). We stratified participants by median DBP to conduct systematic biomarker analyses covering renal, endocrine, inflammatory and metabolic indicators. Drug-target Mendelian randomization (MR) combined with colocalization and mediation analyses was further performed to identify and validate causal antihypertensive-related target genes associated with BMD. Results A significant inverted U-shaped association was identified between DBP and BMD (P-nonlinear=3.23×10 -9 ), with peak BMD observed at a DBP of 80–90 mmHg, while SBP showed a trend of nonlinear correlation. Biomarker analyses revealed that renal biomarker cystatin C and endocrine biomarker IGF-1 exhibited DBP-dependent associations with BMD, mediating the nonlinear DBP-bone density relationship. Drug-target MR demonstrated that genetically proxied MMP9 expression (ACE inhibitor-related) was negatively correlated with BMD (β=-0.036, P=2.59×10 -6 ), whereas CACNA1G expression (T-type calcium channel blocker target) was positively associated with BMD (β=0.042, P=1.57×10 -9 ). Conclusion The inverted U-shaped association between blood pressure and bone mass might partly reflected by renal dysfunction. Antihypertensive pathways mediated by MMP9 and CACNA1G exert opposing effects on bone mass, implying that skeletal health should be considered when selecting antihypertensive agents for vulnerable older populations. Novelty and Relevance What Is New? This large-scale population study identifies a significant inverted U-shaped association between diastolic blood pressure (DBP) and bone mineral density (BMD), with an optimal DBP range of 80–90 mmHg for preserving bone mass. The study reveals that renal biomarkers (cystatin C and IGF-1) exhibit DBP-dependent variation patterns that underpin the nonlinear blood pressure–bone density linkage. Using systematic drug-target Mendelian randomization and colocalization analyses, we further prioritize MMP9 and CACNA1G as antihypertensive-related targets associated with bone mass. What Is Relevant? This study demonstrates a nonlinear association and clarifies biomarker-mediated renal mechanisms linking blood pressure homeostasis to bone metabolism. The drug-target MR design minimizes confounding and provides reliable genetic evidence to explain why different antihypertensive pathways exert distinct skeletal outcomes. Clinical/Pathophysiological Implications? The optimal DBP range for bone preservation is 80–90 mmHg. Both excessively low and elevated DBP disrupt cystatin C and IGF-1 homeostasis, ultimately impairing bone formation and mineralization. Differential skeletal effects of MMP9 -related ACE inhibitor pathways and CACNA1G -related T-type calcium channel pathways suggest the need for individualized antihypertensive strategies.

BACKGROUND:Pharmacological inhibition of sclerostin (SOST) is clinically applied to treat osteoporosis. However, large-scale randomized controlled trials have reported conflicting findings regarding the cardiovascular effects of SOST inhibition. This study aimed to evaluate whether sustained SOST inhibition, mimicked by instrumental genetic variants, is associated with the altered risk of cardiovascular diseases (CVDs). METHODS:The individual-level genomic data were obtained from the UK Biobank, including 377,585 participants in the genome wide association study (GWAS) for estimated heel bone mineral density (eBMD). Summary-level genetic data for CVDs were retrieved from publicly available GWASs, with sample sizes ranging from 332,477 to 1,030,836 participants. The conditional quantile-quantile (QQ) plot was used to visualize the genetic pleiotropy between circulating SOST, eBMD and CVDs. By integrating genetic, transcriptomic, and proteomic data, Mendelian randomization was performed to assess the relationship between exposures and CVDs. RESULTS:We observed polygenic overlap between circulating SOST and eBMD as well as atrial fibrillation (AF). Trans-protein quantitative trait loci (pQTLs) around the B4GALNT3 gene, instead of the genetic variants across the genome, could be instrumental variables to proxy the therapeutic effect of SOST inhibition. Further, increasing level of B4GALNT3 gene expression in several tissues was associated with a decreased level of circulating SOST. Consistently, in vitro evidence validated that B4GALNT3 overexpression significantly decreased SOST protein secretion. Notably, increasing level of B4GALNT3 gene expression in the same tissues was also associated with increased eBMD and higher risk of AF. The proxied SOST inhibition (instrumented by B4GALNT3 genetic variants) had a significant causal effect on increased eBMD (β-coefficient=0.034, SE = 0.007, P = 2.81 × 10-7) and increased risk of AF (OR = 1.353, 95%CI = 1.077-1.701, P = 0.009). The results were replicated in the MR analyses with different instrumental variables based on different linkage disequilibrium (LD) thresholds. CONCLUSIONS:This multi-omics study suggested that reduced circulating SOST was associated with an increased risk of AF, which warrants attention in patients undergoing SOST inhibition treatment.

Compounds promoting anabolic effects on muscle and bone may offer an ideal treatment for osteosarcopenia while potentially impacting healthspan and lifespan. We previously demonstrated the anabolic effects of picolinic acid (PIC), a tryptophan metabolite, on bone both in vitro and in vivo. However, its effects on muscle and potential additional effects on lifespan and healthspan are not yet fully understood. This study aimed to investigate PIC's effects on muscle cells in vitro and its impact on mobility and lifespan in an animal model. Murine C2C12 and human myoblasts were treated with PIC (1, 50, and 100 µM) or vehicle for 5 days. Myogenic regulatory factors (MRFs) were evaluated, and the fusion index and myotubules' length were calculated at timed intervals (day 1, 3, and 5). In vivo, Caenorhabditis elegans were treated with increasing doses of PIC, and their lifespan and rate of movement (thrashing) were evaluated at timed intervals. PIC-treated myoblasts showed a higher and earlier expression of MRFs. On day 3, PIC-treated myotubes were significantly more fused and longer when treated with PIC than vehicle-treated controls. C. elegans treated with 1 mM of PIC showed a significantly longer lifespan. In addition, the mobility of PIC-treated C. elegans was significantly increased at all timed points. In conclusion, this study demonstrates that, besides its anabolic effect on bone, PIC has an anabolic effect on muscle, which is also associated with a longer lifespan in PIC-treated C. elegans. This evidence opens up promising avenues for further exploration of PIC as a novel therapy for osteosarcopenia with additional effects on healthspan and lifespan.

The ultimate goal of a genome-wide association study (GWAS) is to translate its discoveries into clinical practice. To explore the clinical use of GWAS findings in the bone field, we conducted a GWAS of dual-energy X-ray absorptiometry (DXA)-derived bone mineral density (BMD) traits at 11 skeletal sites, within over 30,000 European individuals from the UK Biobank. A total of 91 unique and independent loci were identified for 11 DXA-derived BMD traits and fractures, including 5 novel loci (harboring the genes ABCA1, CHSY1, CYP24A1, SWAP70, and PAX1) for 6 BMD traits. These loci exhibited evidence of association in both males and females, which could serve as independent replication. We demonstrated that each polygenic risk score (PRS) was independently associated with fracture risk. Although incorporating multiple PRSs (i.e., metaPRS) with clinical risk factors from the Fracture Risk Assessment Tool (FRAX) yielded the highest predictive performance, the improvement was modest in fracture prediction. Additionally, we uncovered genetic correlation and shared polygenicity between head BMD and intracranial aneurysm (IA). Finally, by integrating gene expression and GWAS datasets, we prioritized genes (e.g., ESR1 and SREBF1) encoding druggable human proteins along with their respective inhibitors/antagonists. In conclusion, this comprehensive investigation reveals a new genetic basis for BMD and its clinical relevance to fracture prediction. More importantly, it suggests that head BMD is genetically correlated with IA. The prioritization of genetically supported targets implies the potential repurposing of drugs [e.g., omega-3 polyunsaturated fatty acid (PUFA) supplements] for the prevention of osteoporosis.

Genome-wide association studies have identified multiple loci associated with bone mineral density, a major determinant of osteoporotic fracture risk. At one such locus, genetic, bioinformatic, and zebrafish knockout data strongly prioritize membrane palmitoylated protein 7 (MPP7) as a candidate gene, although its precise role in bone biology remains poorly defined. MPP7 encodes a member of the p55 Stardust family of membrane-associated guanylate kinase proteins, which are key regulators of epithelial cell polarity and junctional organization. Here, we investigated the functional role of MPP7 in bone biology. We found that MPP7 expression was significantly reduced—by approximately twofold—in bone tissue from osteoporotic patients compared with osteoarthritic patients and non-osteoporotic controls. Furthermore, we generated a CRISPR/Cas9-mediated MPP7 knockout in the human osteosarcoma HOS cell line and demonstrated that MPP7 deletion impairs osteogenic differentiation and completely abrogates mineralization through downregulation of ALPL expression. Knockout cells also displayed altered morphology, suggesting that MPP7 influences osteoblast function via effects on cell polarity and adhesion. Collectively, our findings, together with zebrafish genetic evidence, indicate that MPP7 plays a critical role in osteoblast differentiation and mineralization and may contribute to osteoporosis susceptibility in humans.

BACKGROUND:Lean body mass is a crucial physiological component of body composition. Although lean body mass has a high heritability, studies evaluating the genetic determinants of lean mass (LM) have to date been limited largely to genome-wide association studies (GWAS) and common variants. Using whole genome sequencing (WGS)-based studies, we aimed to discover novel genetic variants associated with LM in population-based cohorts with multiple ancestries. RESULTS:We describe the largest WGS-based meta-analysis of lean body mass to date, encompassing 10,729 WGS samples from six TOPMed cohorts and the Louisiana Osteoporosis Study (LOS) cohort, measured with dual-energy X-ray absorptiometry. We identify seven genome-wide loci significantly associated with LM not reported by previous GWAS. We partially replicate these associations in UK Biobank samples. In rare variant analysis, we discover one novel protein-coding gene, DMAC1, associated with both whole-body LM and appendicular LM in females, and a long non-coding RNA gene linked to appendicular LM in males. Both genes exhibit notably high expression levels in skeletal muscle tissue. We investigate the functional roles of two novel lean-mass-related genes, EMP2 and SSUH2, in animal models. EMP2 deficiency in Drosophila leads to significantly reduced mobility without altering muscle tissue or body fat morphology, whereas an SSUH2 gene mutation in zebrafish stimulates muscle fiber growth. CONCLUSIONS:Our comprehensive analysis, encompassing a large-scale WGS meta-analysis and functional investigations, reveals novel genomic loci and genes associated with lean mass traits, shedding new insights into pathways influencing muscle metabolism and muscle mass regulation.

This study aimed to evaluate the impact of picolinic acid (PIC), a metabolite derived from tryptophan, on age-related tissue regeneration and physical decline in zebrafish. Additionally, it examined changes in whole-body mass index (WB-BMI) as an indicator of musculoskeletal aging. Siblings born in August 2022 were randomly assigned to four groups at age 20 mo: (1) PIC in water (25 mg/kg/day), (2) PIC in water + 25 mg/kg oral gavage, (3) PIC in water + 75 mg/kg oral gavage, and (4) control (system water + gavage) for eight weeks. The treatment groups consisted of 15 fish. At week 7, swimming performance was recorded over a 30-minute period. Caudal fins were amputated for regeneration analysis. In week 8, fish were euthanized for whole-body micro-CT and for β-galactosidase (X-gal) staining to evaluate cellular senescence. The Mann-Whitney U test was used to compare WB-BMI between groups. Group 2 showed the highest swimming speed (44.5 m/min), followed by Group 1 (34.7 m/min) (p = 0.01 and p = 0.09, respectively) than the control group (31.7 m/min). WB-BMI was decreasing over the duration of the experiment in all 4 groups, with Group 1 maintaining the highest BMD compared to the control (non-significant, p > 0.5). Regeneration in Groups 2 and 3 was more advanced than in Group 1, with stronger staining by β-galactosidase. PIC at moderate doses supports locomotor function and overall health in aged zebrafish. It may influence regenerative outcomes through mechanisms involving cellular aging.

NGLY1 is a key enzyme in the process of misfolded protein deglycosylation. Bi-allelic pathogenic variants in NGLY1 cause N-glycanase deficiency, also known as congenital disorder of deglycosylation (NGLY1-CDDG). This rare and multisystem autosomal recessive disorder is linked to a variable phenotype of global developmental delay, neuromuscular abnormalities, and alacrima, and it lacks effective treatment. We have studied the possible underlying mechanisms for the neuromuscular and ophthalmic phenotypes in an ngly1-deficient zebrafish model carrying a similar genetic variant that has also been identified in previously reported patients. We investigated phenotypic, biochemical, and molecular details underlying ngly1 deficiency using a zebrafish model. ngly1-deficient zebrafish phenotypes were characterized using histological staining, transmission electron microscopy (TEM), and micro-CT imaging. Furthermore, fish brain molecular and biochemical characterization was performed by gene expression analysis and immunoblotting techniques. Impaired proteostasis was evident in the brain of the mutant zebrafish, including accumulation of poly-ubiquitinated proteins and amyloid fibril aggregation. The mutant fish featured neuromuscular abnormalities and significant aquaporin1-protein reduction in the eyes and brain. The zebrafish model of NGLY1 deficiency provides an ideal platform for studying the molecular and biochemical mechanisms underlying NGLY1-CDDG in humans. Our novel findings of impaired protein homeostasis encompassing amyloid fibril aggregation (folding) and poly-ubiquitinated protein accumulation (degradation) in the brains of mutant zebrafish offer new insights into the brain pathology associated with NGLY1 deficiency. These discoveries may also advance our understanding of other neurodegenerative disorders and facilitate the identification of potential therapeutic targets.

Understanding the early stages of human congenital myopathies is critical for proposing strategies for improving skeletal muscle performance by the functional integrity of cytoskeleton. SH3 and cysteine-rich domain 3 (Stac3) is a protein involved in nutrient sensing, and is an essential component of the excitation-contraction (EC) coupling machinery for Ca 2+ releasing. A mutation in STAC3 causes debilitating Native American myopathy (NAM) in humans, and loss of this gene in mice and zebrafish resulted in death in early life. Previously, NAM patients demonstrated increased lipids in skeletal muscle biopsy. However, elevated neutral lipids could alter muscle function in NAM disease via EC coupling apparatus is yet undiscovered in early development. Here, using a CRISPR/Cas9 induced stac3 knockout (KO) zebrafish model, we determined that loss of stac3 led to muscle weakness, as evidenced by delayed larval hatching. We observed decreased whole-body Ca 2+ level at 5 days post-fertilization (dpf) and defects in the skeletal muscle cytoskeleton, i.e., F-actin and slow muscle fibers at 5 and 7 dpf. Homozygous larvae exhibited elevated neutral lipid levels at 5 dpf, which persisted beyond 7 dpf. Myogenesis regulators such as myoD and myf5 , were significantly altered in stac3 -/- larvae at 5 dpf, thus a progressive death of the KO larva by 11 dpf. In summary, the presented findings suggest that stac3 -/- can serve as a non-mammalian model to identify lipid-lowering molecules for refining muscle function in NAM patients.

Background There is limited evidence suggesting that osteoporosis might exacerbate depressive symptoms, while more studies demonstrate that depression negatively affects bone density and increases fracture risk.Aims To explore the relationship between major depressive disorder (MDD) and fracture risk.Methods We conducted a nested case-control analysis (32 670 patients with fracture and 397 017 individuals without fracture) and a matched cohort analysis (16 496 patients with MDD and 435 492 individuals without MDD) in the same prospective UK Biobank data set. Further, we investigated the shared genetic architecture between MDD and fracture with linkage disequilibrium score regression and the MiXeR statistical tools. We used the conditional/conjunctional false discovery rate approach to identify the specific shared loci. We calculated the weighted genetic risk score for individuals in the UK Biobank and logistic regression was used to confirm the association observed in the prospective study.Results We found that MDD was associated with a 14% increase in fracture risk (hazard ratio (HR) 1.14, 95% CI 1.14 to 1.15, p<0.001) in the nested case-control analysis, while fracture was associated with a 72% increase in MDD risk (HR 1.72, 95% CI 1.64 to 1.79, p<0.001) in the matched cohort analysis, suggesting a longitudinal and bidirectional relationship. Further, genetic summary data suggested a genetic overlap between MDD and fracture. Specifically, we identified four shared genomic loci, with the top signal (rs7554101) near SGIP1. The protein encoded by SGIP1 is involved in cannabinoid receptor type 1 signalling. We found that genetically predicted MDD was associated with a higher risk of fracture and vice versa. In addition, we found that the higher expression level of SGIP1 in the spinal cord and muscle was associated with an increased risk of fracture and MDD.Conclusions The genetic pleiotropy between MDD and fracture highlights the bidirectional association observed in the epidemiological analysis. The shared genetic components (such as SGIP1) between the diseases suggest that modulating the endocannabinoid system could be a potential therapeutic strategy for both MDD and bone loss.

Human genetic studies have nominated Cadherin-like and PC-esterase Domain-containing 1 (CPED1) as a candidate target gene mediating bone mineral density (BMD) and fracture risk heritability. Recent efforts to define the role ofCPED1in bone in mouse and human models have revealed complex alternative splicing and inconsistent results arising from gene targeting, making its function in bone difficult to interpret. To better understand the role ofCPED1in adult bone mass and morphology, we conducted a comprehensive genetic and phenotypic analysis ofcped1in zebrafish, an emerging model for bone and mineral research. We analyzed two differentcped1mutant lines and performed deep phenotyping to characterize more than 200 measures of adult vertebral, craniofacial, and lean tissue morphology. We also examined alternative splicing of zebrafishcped1and gene expression in various cell/tissue types. Our studies fail to support an essential role ofcped1in adult zebrafish bone. Specifically, homozygous mutants for bothcped1mutant alleles, which are expected to result in loss-of-function and impact allcped1isoforms, exhibited no significant differences in the measures examined when compared to their respective wildtype controls, suggesting thatcped1does not significantly contribute to these traits. We identified sequence differences in critical residues of the catalytic triad between the zebrafish and mouse orthologs of CPED1, suggesting that differences in key residues, as well as distinct alternative splicing, could underlie different functions ofCPED1orthologs in the two species. Our studies fail to support a requirement ofcped1in zebrafish bone and lean tissue, adding to evidence that variants at 7q31.31 can act independently ofCPED1to influence BMD and fracture risk.

Here, we conducted genome-wide association studies (GWAS) of dual-energy X-ray absorptiometry (DXA)-derived bone mineral density (BMD) traits at 11 skeletal sites, within over 30,000 European individuals from the UK Biobank. A total of 92 unique and independent loci were identified for 11 DXA-derived BMD traits and fracture, including 5 novel loci (i.e., ABCA1, CHSY1, CYP24A1, SWAP70 and PAX1) and 2 sex-specific loci (i.e., CYP19A1 and CYP3A7). We demonstrated that polygenic risk scores (PRSs) were independently associated with fracture risk. Although incorporating multiple PRSs (metaPRS) with the clinical risk factors (i.e., the FRAX model) exhibited the highest predictive performance, the improvement was marginal in fracture prediction. The metaPRS were capable of stratifying individuals into different trajectories of fracture risk, but clinical risk factors played a more significant role in the stratification. Additionally, we uncovered genetic correlation and shared polygenicity between head BMD and intracranial aneurysm. And the joint associated genes such as PLCE1 might play important roles in the shared genetic basis. Finally, by integrating gene expression, and GWAS datasets, we prioritized genes (e.g. ESR1, SREBF1, CCR1 and NCOR1) encoding druggable human proteins along with their respective inhibitors/antagonists. In conclusion, this comprehensive investigation revealed new genetic basis for BMD and its clinical relevance on fracture prediction. More importantly, it was suggested that head BMD was genetically correlated with intracranial aneurysm. The prioritization of genetically supported targets implied the potential repurposing drugs (e.g. the n-3 PUFA supplement targeting SREBF1) for the prevention of osteoporosis. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by the National Natural Science Foundation of China (#82370887), the "Pioneer" and "Leading Goose" R&D Program of Zhejiang (#2023C03164), the Chinese National Key Technology R&D Program, Ministry of Science and Technology (#2021YFC2501702), and the funds from the Westlake Laboratory of Life Sciences and Biomedicine (#202208014). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Ethics approval for the UK Biobank research was obtained from the North West Multicentre Research Ethical Committee, and all participants provided informed consent (original ethics committee approval number: 21/NW/0157). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The summary statistics of the present GWAS on 11 DXA-BMD traits were deposited on the website (https://wbbc.westlake.edu.cn/downloads.html).

Musculoskeletal research should synergistically investigate bone and muscle to inform approaches for maintaining mobility and to avoid bone fractures. The relationship between sarcopenia and osteoporosis, integrated in the term ‘osteosarcopenia’, is underscored by the close association shown between these two conditions in many studies, whereby one entity emerges as a predictor of the other. In a recent workshop of Working Group (WG) 2 of the EU Cooperation in Science and Technology (COST) Action ‘Genomics of MusculoSkeletal traits Translational Network’ (GEMSTONE) consortium (CA18139), muscle characterization was highlighted as being important, but currently under-recognized in the musculoskeletal field. Here, we summarize the opinions of the Consortium and research questions around translational and clinical musculoskeletal research, discussing muscle phenotyping in human experimental research and in two animal models: zebrafish and mouse.

Zebrafish and other small laboratory fishes are emerging as important animal models for investigating human skeletal development and diseases. In recent years, there has been a notable increase in research publications employing X-ray radiography and micro-computed tomography to analyze the skeletal structures of these animals. However, evaluating bone morphology and mineral density in small laboratory fish poses unique challenges compared to well-established small rodent models. The varied approaches to image acquisition, analysis, and reporting across studies have led to substantial obstacles in interpreting and comparing research findings. This article addresses the urgent need for standardized reporting of parameters and methodologies related to image acquisition and analysis, as well as the adoption of harmonized nomenclature. Furthermore, it offers guidance on anatomical terminology, units of measurement, and the establishment of minimal parameters for reporting, along with comprehensive documentation of methods and algorithms used for acquisition and analysis. We anticipate that adherence to these guidelines will enhance the consistency, reproducibility, and interpretability of reported measurements of bone density and morphometry in small fish models. These advancements are vital for accurately interpreting phenotypes and gene functions, particularly in the context of multi-center studies.