C1QTNF9 Antibody

What is the molecular structure of C1QTNF9/CTRP9?

C1QTNF9 belongs to the highly conserved CTRP family of secreted proteins. Like most CTRPs, it consists of four distinct domains: an N-terminal signal peptide, a domain with conserved cysteine residues, a collagen-like domain containing variable Gly-X-Y repeats, and a C-terminal globular C1q domain. CTRP9 has the highest structural similarity to adiponectin among all CTRPs, with approximately 54% amino acid identity in the globular domain. The protein is mainly secreted as a trimer and can be detected in serum .

Where is C1QTNF9/CTRP9 primarily expressed in the body?

CTRP9 is predominantly expressed in the heart, particularly in cardiac endothelial cells where it acts in a paracrine manner on cardiomyocytes. Cardiomyocytes themselves express very small amounts of CTRP9. It is also expressed in smaller amounts in adipose tissue . The expression level of CTRP9 in the heart has been found to be 100-fold higher than that of adiponectin, highlighting its significant role in cardiac function .

What are the primary signaling pathways activated by C1QTNF9/CTRP9?

CTRP9 activates several important signaling pathways in target cells. It primarily activates AMPK (AMP-activated protein kinase), AKT (protein kinase B), and p44/42 MAPK (mitogen-activated protein kinase) signaling pathways . Additionally, CTRP9 can activate PKA (protein kinase A) to inhibit cardiomyocyte apoptosis . In particular, the globular subtype of CTRP9 (gCTRP9) formed by post-translational proteolysis modification activates AMPK, Akt, and eNOS to stimulate protective mechanisms for cardiac survival .

How does C1QTNF9/CTRP9 affect inflammatory pathways?

CTRP9 exerts anti-inflammatory effects through multiple mechanisms. It reduces the secretion of proinflammatory cytokines from macrophages, inhibiting NFκB signaling pathways. In vascular endothelial cells treated with TNF-α to induce inflammation, CTRP9 significantly prevents the activation of NFκB and subsequently increases the phosphorylation of AMPK to reduce inflammatory cytokines . CTRP9 also demonstrates an anti-inflammatory effect on macrophages by inducing an increase in iNOS expression in a time- and dose-dependent manner via activation of JAK2/STAT3 signaling .

What are the optimal conditions for detecting C1QTNF9/CTRP9 by Western blot?

For Western blot detection of human CTRP9/C1qTNF9, researchers should prepare samples under reducing conditions and use Immunoblot Buffer Group 1. A PVDF membrane can be probed with 2 μg/mL of sheep anti-human CTRP9/C1qTNF9 antigen affinity-purified polyclonal antibody followed by HRP-conjugated anti-sheep IgG secondary antibody. Under these conditions, CTRP9/C1qTNF9 typically appears as a specific band at approximately 42 kDa .

How should CTRP9 antibodies be stored for optimal performance?

CTRP9 antibodies should be stored in a manual defrost freezer to avoid repeated freeze-thaw cycles, which can compromise antibody quality. Unopened antibodies remain stable for 12 months from the date of receipt when stored at -20 to -70°C. After reconstitution, antibodies maintain stability for 1 month when stored at 2 to 8°C under sterile conditions, or for 6 months when stored at -20 to -70°C under sterile conditions .

How does C1QTNF9/CTRP9 protect against diabetic cardiomyopathy?

CTRP9 plays a crucial protective role against diabetic cardiomyopathy through multiple mechanisms. CTRP9 knockout mice exhibit left ventricular diastolic dysfunction after 12 weeks of high-fat diet feeding, while overexpression of CTRP9 in the heart rescues this HFD-induced diastolic dysfunction. CTRP9 prevents cardiac insulin resistance and mediates myocardial glucose uptake, as demonstrated through dynamic 18F-fluoro-deoxyglucose (FDG) positron-emission tomography (PET) imaging . Additionally, CTRP9 counteracts inflammation in the heart, with RNA sequencing revealing that CTRP9 knockout mice show upregulation of genes related to immune response activation and increased leukocyte count in the heart .

What metabolic effects does CTRP9 exert on the diabetic heart?

CTRP9 neutralizes myocardial lipotoxicity in the diabetic heart. CTRP9-deficient mice show exaggerated cardiac hypertrophy, fibrosis, endoplasmic reticulum stress (ERS)-initiated apoptosis, and oxidative stress compared with HFD-fed wild-type mice . In an ischemia/reperfusion model of rats fed with HFD, CTRP9 treatment significantly increased the expression of myocardial disulfide bond oxidase-like protein, reduced ERS, and alleviated diabetes mellitus-induced heart injury .

How does CTRP9 protect against myocardial ischemic injury?

CTRP9 provides substantial protection against myocardial ischemic injury. Systemic administration of CTRP9 significantly reduces infarct size and myocardial apoptosis by activating the AMPK signaling pathway . Overexpression of CTRP9 activates PKA to inhibit cardiomyocyte apoptosis, increases the survival rate of mice, restores cardiac function, and reduces myocardial apoptosis and fibrosis following myocardial injury .

What role does CTRP9 play in post-myocardial infarction complications?

CTRP9 plays a pivotal role in preventing complications following myocardial infarction (MI). Systemic administration of CTRP9 attenuates atrial inflammation, fibrosis, and vulnerability to atrial fibrillation in post-MI rats . CTRP9 administration improves post-MI early cardiac function by regulating M1/M2 macrophage polarization, suggesting its potential in modulating the inflammatory response after MI .

How does CTRP9 protect against atherosclerosis development?

CTRP9 protects against atherosclerosis through multiple mechanisms. Overexpression of CTRP9 enhances the stability of plaques by reducing the secretion of proinflammatory cytokines from macrophages and inhibits the formation of atherosclerotic plaques in ApoE knockout mice . CTRP9 overexpression substantially attenuates atherosclerotic lesion size in ApoE knockout mice fed with HFD and reduces the proportion of macrophages in atherosclerotic regions, indicating that CTRP9 exerts a protective role in early atherosclerotic lesions .

What cellular mechanisms underlie CTRP9's anti-atherosclerotic effects?

At the cellular level, CTRP9 exhibits athero-protective functions through several mechanisms. First, it reduces the number of lipid droplets, lowers the levels of cholesteryl ester, and promotes cholesterol efflux in oxidized low-density lipoprotein (ox-LDL)-induced THP1 macrophages, thereby reducing foam cell formation . Second, CTRP9 inhibits ox-LDL-impaired endothelial dysfunction, including migration, proliferation, reactive oxygen species (ROS) production, apoptosis, angiogenesis, and nitric oxide generation in human umbilical vein endothelial cells (HUVECs) . Third, CTRP9 inhibits the production of ROS and enhances mitochondrial biogenesis in human aortic vascular endothelial cells (HAECs) .

What are the most informative animal models for studying CTRP9 function?

Several animal models have proven valuable for studying CTRP9 function. CTRP9 knockout mice provide crucial insights into the protein's physiological role, particularly in the context of high-fat diet feeding, which reveals its importance in maintaining cardiac function . Transgenic mice with cardiac-specific CTRP9 overexpression allow researchers to examine the protective effects of enhanced CTRP9 signaling . Disease-specific models, such as transverse aortic coarctation to induce pressure overload, help elucidate CTRP9's role in cardiac remodeling , while arteriovenous shunt models reveal its function in pulmonary arterial hypertension .

How do CTRP9 knockout models compare with wild-type in metabolic disease studies?

CTRP9 knockout mice show exacerbated pathology in metabolic disease studies compared to wild-type mice. Under high-fat diet conditions, CTRP9-deficient mice exhibit left ventricular diastolic dysfunction, upregulation of genes related to immune response activation, and increased cardiac leukocyte infiltration . These mice also show exaggerated cardiac hypertrophy, fibrosis, endoplasmic reticulum stress-initiated apoptosis, and oxidative stress compared to HFD-fed wild-type mice , highlighting CTRP9's protective role against metabolic stress.

What techniques are most reliable for measuring CTRP9 levels in experimental samples?

For reliable measurement of CTRP9 levels, Western blotting is an established technique. Using a PVDF membrane probed with sheep anti-human CTRP9/C1qTNF9 antibody (2 μg/mL) followed by HRP-conjugated secondary antibody allows detection of CTRP9 at approximately 42 kDa under reducing conditions . RNA sequencing has also been effectively employed to investigate CTRP9-related gene expression changes, particularly in identifying immune response genes upregulated in CTRP9 knockout mice . Additionally, animal studies have used ELISA techniques to measure circulating CTRP9 levels in serum samples.

How can researchers effectively study CTRP9 signaling pathways?

To study CTRP9 signaling pathways, researchers should focus on the phosphorylation status of key signaling molecules. Analysis of AMPK, AKT, and p44/42 MAPK phosphorylation provides crucial information about CTRP9 activity . For inflammatory pathway assessment, monitoring NFκB activation and JAK2/STAT3 signaling is important . Cell-based assays using macrophages (RAW 264.7 or THP1) or endothelial cells (HUVECs or HAECs) treated with CTRP9 can reveal pathway-specific effects on inflammation, cholesterol efflux, reactive oxygen species production, and nitric oxide generation .

What imaging techniques are most valuable for assessing CTRP9's cardiac effects?

Dynamic 18F-fluoro-deoxyglucose (FDG) positron-emission tomography (PET) imaging has proven valuable for assessing CTRP9's effects on cardiac glucose metabolism. This technique has demonstrated that CTRP9 is necessary to prevent cardiac insulin resistance and mediate myocardial glucose uptake . Echocardiography is essential for evaluating cardiac function parameters, particularly left ventricular diastolic function, which is compromised in CTRP9 knockout mice after high-fat diet feeding . For atherosclerosis studies, imaging techniques that quantify atherosclerotic plaque size and stability are crucial for evaluating CTRP9's protective effects .

What evidence supports CTRP9 as a therapeutic target for cardiovascular diseases?

Multiple lines of evidence support CTRP9 as a therapeutic target for cardiovascular diseases. Systemic administration of CTRP9 significantly reduces infarct size and myocardial apoptosis by activating the AMPK signaling pathway in models of myocardial ischemia . CTRP9 administration attenuates atrial inflammation, fibrosis, and vulnerability to atrial fibrillation in post-MI rats . In diabetic cardiomyopathy models, overexpression of CTRP9 in the heart rescues HFD-induced diastolic dysfunction . CTRP9 also demonstrates potent anti-atherosclerotic effects, including enhanced plaque stability and reduced atherosclerotic lesion size in ApoE knockout mice .

How might CTRP9-based therapies be developed and optimized?

Development of CTRP9-based therapies could take several approaches. Direct administration of recombinant CTRP9 protein has shown efficacy in animal models and could be optimized for human applications. Alternatively, development of small molecule agonists that mimic CTRP9 signaling through AMPK, AKT, or PKA pathways might provide more targeted therapeutic options. Gene therapy approaches to increase cardiac CTRP9 expression represent another potential strategy, as cardiac-specific overexpression of CTRP9 has demonstrated protective effects in animal models . For optimal therapeutic outcomes, treatment protocols should consider the timing of intervention relative to disease progression, dosage requirements, and potential combination with existing cardiovascular therapies.

How do CTRP9 levels correlate with cardiovascular disease in clinical studies?

Clinical studies have revealed complex correlations between CTRP9 levels and cardiovascular diseases. CTRP9 levels are significantly reduced in female patients with coronary artery disease (CAD), type 2 diabetes mellitus (T2DM), and CAD secondary to T2DM compared to healthy subjects. In these patients, CTRP9 expression negatively correlates with monocyte chemoattractant protein 1 secretion in serum . Conversely, other studies have found that levels of circulating CTRP9 are significantly increased in patients with T2DM and CAD, suggesting a possible compensatory response to insulin resistance, inflammatory milieu, and endothelial dysfunction . These variations may result from sampling differences and age variations across study cohorts.

What genetic factors influence CTRP9 function in cardiovascular disease?

Genetic factors significantly impact CTRP9 function in cardiovascular disease. Polymorphisms of the CTRP9 gene have been associated with increased susceptibility and pathogenesis of CAD. Two single nucleotide polymorphisms (SNPs) have been successfully genotyped in CAD patients, with the frequency of the AA genotype lower in CAD patients than in healthy controls, while the CC genotype frequency is higher. These findings suggest that the CC genotype of CTRP9 correlates with an increased risk of CAD, though the specific mechanism requires further investigation .

How does CTRP9 function in pulmonary vascular diseases?

CTRP9 demonstrates significant protective effects in pulmonary vascular diseases. In vitro and in vivo experiments have confirmed that CTRP9 improves pulmonary arterial pressure by reducing inflammation and enhancing endothelial cell survival and function . CTRP9 also promotes hypoxia-mediated cell apoptosis and prevents cell migration in pulmonary arterial smooth muscle cells, potentially limiting vascular remodeling . In models of right ventricular hypertrophy induced by pulmonary artery banding, CTRP9 mediates cardio-protective effects by inhibiting reactive oxygen species production through AMPK-mediated activation of antioxidant enzymes .

What is the relationship between CTRP9 and other metabolic regulators?

CTRP9 interacts with several other metabolic regulators. It has the highest structural similarity to adiponectin (54% amino acid identity in the globular domain) among all CTRPs , suggesting potential functional overlap. Interestingly, CTRP9 is upregulated in adiponectin knockout mice, indicating possible compensatory mechanisms . The relationship between CTRP9 and insulin signaling is also significant, as CTRP9 prevents cardiac insulin resistance and mediates myocardial glucose uptake . Future research should further explore these interactions to better understand CTRP9's role in the broader metabolic regulatory network and its potential as a therapeutic target for metabolic disorders.