Tomoregulin-1 Inhibits Cardiac Hypertrophy Caused By Negative Pressure In The TAK1-JNK Pathway

Background Hypertrophic cardiomyopathy (HCM) is an inherited heart muscle disease characterized by left ventricle and/or right ventricle hypertrophy. It is one of the most important cause of sudden death, heart failure and stroke disability. There remain little in our basic understanding of the molecular pathophysiology of HCM and therefore, the identification of important modifier genes involved in the pathogenesis of HCM is necessary, which may lead to the discovery of new therapeutic strategies. Tomoregulin-1 is growth factor that is primarily involved in the mid to late stages of embryonic development and in maintenance of adult central nervous system (CNS) function, and it is expressed abnormally in a variety of CNS pathologies. Tomoregulin-1 is also expressed in the heart tissue and can directly bind to the Cripto which is involved in cardiac development. Gene microarray of our laboratory shows the expression of Tomoregulin-1 is increased significantly in the heart tissue from the inherited HCM mice model (cTnTR92Q transgenic mice). However, the effect of Tomoregulin-1 on the heart, particularly on HCM, remains unknown. In the present study, we propose a possible mechanism by which Tomoregulin-1 regulates the development of HCM by establishing the heart-specific Tomoregulin-1 knockdown and heart-specific Tomoregulin-1 overexpression mice.Methods (1) The location and expression of Tomoregulin-1 in the heart of wild-type (WT) mice and HCM mice:The location of Tomoregulin-1 in the heart of WT mice was detected by immunofluorescence staining.The temporal expression pattern of Tomoregulin-1 in the heart of WT mice and in both inherited HCM (cTnTR92Q transgenic mice) and pressure overload (thoracic aorta constriction, TAC) induced HCM mice was analyzed by Western blot.(2) The establishment of heart-specific Tomoregulin-1 knockdown and overexpression mice:The vector was constructed by inserting the siRNA targeting on Tomoregulin-1 and full-length mouse Tomoregulin-1 gene into the down steam of a-MHC promoter respectively. The mice were created by the method of microinjection. The genotype of mice of Tomoregulin-1 knockdown and Tomoregulin-1 overexpression mice was detectedBackground With the improvement of people’s living standard and material conditions, the prevalence and incidence of obesity has rapidly increased globally and has reached epidemic proportions. Obesity is associated with many deleterious outcomes such as type 2 diabetes, hypercholesterolemia, hypertension or heart disease, and is directly related to increased mortality and reduced life expectancy. With the completion of the human genome project, increasing numbers of risk alleles associated with obesity have been identified. Leptin receptor gene (Lepr) is one of the commonly studied candidate genes for obesity. Lepr, which is encoded by the diabetes (db) gene and is highly expressed in the choroid plexus, regulates energy homeostasis and the balance between food intake and energy expenditure by binding to its ligand leptin. Lepr deficiency or leptin deficiency both can lead to obesity and diabetes clinical manifestations of different levels. Various animal models associated to Lepr have been established to study human obesity and type 2 diabetes. However, these models do not develop the full phenotype of type 2 diabetes, such as the transient hyperglycemia of dbldb mice in the C57BL/6J genetic background and the delayed onset of glucose intolerance in the Zucker rats. Thus, establishment of another animal model associated to Lepr should be helpful for biomedical and pharmacological research on obesity and type 2 diabetes.Methods Establishment of a Lepr knockout rat(Lepr-/-) by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system: two gRNAs targetingLepr exon 4 were transcribed in vitro, a mixture of Cas9 mRNA (20ng/μl) and gRNA (10ng/μl per gRNA) were microinjected into zygotes of Sprague Dawley (SD) rats, and 8 pups were born. The genotype of Lepr-/- was detected by PCR, the knockout of Lepr was determined by TA clone and sequence analysis, the expression of LEPR was determined by Western blot to choose Lepr-/- founder to establish a colony. Rats from offspring generation were used in the present study. The body weight, daily food consumption, fasting and random glucose levels were measured in Lepr-/- rats and their wild-type (WT) littermates from 1 to 8 months of age in both genders. The glucose tolerance test and serum insulin level were performed in Lepr-/- rats and their wild-type (WT) littermates at 2,4 and 8 months of age in both genders. Diabetes-associated lipid metabolism parameters, such as circulating triglycerides, total cholesterol, high density lipoprotein and low density lipoprotein, were measured in Lepr-/- rats and their wild-type (WT) littermates at 2,4 and 8 months of age in both genders. The Lepr-/- rats and their wild-type (WT) littermates were chosen for observation of the pathological changes of pancreas, liver, adipose tissue and kidney induced by Lepr knockout at 2,4 and 8 months of age in both genders. The Lepr-/- rats and their WT littermates were euthanized at 8 months of age. Their femurs were dissected and the distal femur were analyzed by μCT. The bone volume/total (tissue) volume (BV/TV), the trabecular number (Tb.N), the trabecular separation (Tb.Sp) and the bone mineral density (BMD) of the distal femur were recorded.Results Using CRISPR/Cas 9 system,8 pups were born. After PCR amplification, TA clone and further sequencing, Founder 2, carrying a 298-bp deletion and 4-bp insertion, was chosen to establish a colony. Western blot analysis of total protein from liver tissue of the Lepr-/- rats confirmed the absence of LEPR protein. The Lepr-/- rats emerged with severe early-onset obesity as early as 1 month of age. The body weight of the male and female Lepr-/- rats were 1.6 and 2.6-fold respectively compared with the WT littermates at 8 months of age. The increased body weight of Lepr-/- rats was associated with significantly elevated daily food consumption in both genders. The male Lepr-/- rats emerged with higher fasting glucose level at 4 months of age and this hyperglycemia continued to 8 months of age. Significantly higher random glucose levels occurred as early as 2 months of age in male Lepr-/- rats and reached peak glucose at 4 months of age, which is 2.64-fold compared with those of their WT littermates. However, persistent hyperglycemia was not observed in the female Lepr-/- rats. Glucose intolerance appeared in male Lepr-/- rats at 2 months of age and deteriorated with aging. In female Lepr-/- rats, glucose intolerance only was observed at 4 months of age. The Lepr-/- rats showed significant hyperinsulinemia at 2,4 and 8 months of age in both genders after the glucose loading. The Lepr-/- rats also showed significant dyslipidemia at 2,4 and 8 months of age in both genders. The pathological observation at 8 months of age showed, compared with their WT littermates, the pancreatic islets of the Lepr-/- rats exhibited obviously severe vacuolation, hypertrophy, fibrosis and hemorrhage. Severe hepatic steatosis was obvious in the Lepr-/- rats. The adipocytes of the Lepr-/- rats were obviously larger. The kidney tissues of the Lepr-/- rats exhibited expansion of glomerular matrix, segmental glomerulosclerosis and tubular damage such as tubular expansion and regeneration. The μCT analysis indicated that BV/TV, Tb.N and the BMD of the distal femur were significantly decreased, whereas Tb.Sp was increased in the Lepr-/- rats compared with their WT littermates at 8 months of age.Conclusions The Lepr-/- rats were successfully created using CRISPR/Cas 9 system. Homozygous Lepr null rats were characterized by obesity, hyperphagia, blood glucose elevation, glucose intolerance, hyperinsulinemia and dyslipidemia, and also developed some diabetic complications such as pancreatic, hepatic and renal lesions. Our model had rescued some deficiency of the existing rodent models, and it was proven to be a more useful and better animal model for biomedical and pharmacological research on obesity and diabetes. by PCR, the expression level of Tomoregulin-1 was determined by Western blot to choose Tomoregulin-1 knockdown and Tomoregulin-1 overexpression founder to establish a colony. Offsprings were used in present study.(3) The phenotype analysis of cardiac structure and function of heart-specific Tomoregulin-1 knockdown and overexpression mice in the physiological conditions:The cardiac structure and function of the non-transgenic littermates (NTG), Tomoregulin-1 knockdown and Tomoregulin-1 overexpression mice at 1,3,5 and 7 months of age were analyzed by M-mode echocardiography.(4) The phenotype analysis of heart-specific Tomoregulin-1 knockdown and overexpression mice after pressure overload:TAC operation was performed on the NTG, Tomoregulin-1 knockdown and Tomoregulin-1 overexpression mice at 8-10 weeks of age. At 4 weeks after sham and TAC operation, survival data of the experimental mice were recorded; the cardiac structure and function were analyzed by M-mode echocardiography; hearts were weighed and the heart weight-to-body weight ratio was calculated; pathologic changes were observed by light microscopy; procollagen type Ⅲα1 (Col3α1) mRNA was detected by real-time PCR. The expression of TGFβ type 2 receptor (TβR2), TGFβ type 1 receptor (TβR1), TGFβ-activated kinase 1(TAK1) and c-Jun N-terminal kinase (JNK), which were involved in cardiac hypertrophy, in the heart tissues from the NTG, Tomoregulin-1 knockdown and Tomoregulin-1 overexpression mice of sham and TAC group were analyzed by Western blot.Results (1) Tomoregulin-1 was primarily expressed in cardiomyocytes, but not cardiac fibroblasts. The expression of Tomoregulin-1 was low in the neonatal heart and increased in the adult heart from the WT mice. Its expression was increased significantly in in two HCM models, the cTnTR92Q transgenic mice and the thoracic aorta constriction (TAC)-induced HCM mice.(2) Two lines of the heart-specific Tomoregulin-1 knockdown and heart-specific Tomoregulin-1 overexpression mice were both established.(3) In physiological conditions, the Tomoregulin-1 knockdown mice presented thin-walled ventricles, larger left ventricular diameters and decreased cardiac function at 1,3,5 and 7 months of age compared with the NTG mice. The Tomoregulin-1 overexpression mice showed thick-walled ventricles, smaller left ventricular diameters and increased cardiac function before 5 months of age. The thick-walled ventricle phenotype caused by Tomoregulin-1 overexpression was not obvious after 5 months of age compared with NTG mice.(4) After pressure overload, the survival rate was all 100% in the NTG, Tomoregulin-1 knockdown and Tomoregulin-1 overexpression mice of sham group. The survival rate of the Tomoregulin-1 knockdown mice with TAC was significantly reduced to 66.7% compared to the NTG mice with TAC, whereas the survival rate of the Tomoregulin-1 overexpression mice with TAC was 84.6%. Furthermore, the gradient of the cardiac echocardiography left ventricular wall thickness parameters were all significantly increased in Tomoregulin-1 knockdown mice, Tomoregulin-1 overexpression significantly reduced the geometrical changes of HCM induced by TAC. The increased cardiac function to compensate for the heart dysfunction was also improved in Tomoregulin-1 overexpression mice. Compared with the NTG mice with TAC, the Tomoregulin-1 knockdown showed a significantly increased heart weight-to-body weight ratio, and the Tomoregulin-1 overexpression significantly decreased the heart weight-to-body weight ratio. Myocardial disarray and fibrosis were clearly observed in the heart tissues from the Tomoregulin-1 knockdown mice in both sham and TAC group. However, the pathological changes of TAC operation was significantly inhibited by Tomoregulin-1 overexpression. Moreover, the expression of Col3α1, which is responsible for abnormal myocardial stiffness and impaired pumping capacity of the heart, was significantly increased in Tomoregulin-1 knockdown mice in the sham and TAC group, the pathological increase of the Col3α1 in Tomoregulin-1 overexpression mice with TAC was significantly decreased. The expression of TβR1, TAK1 and JNK, which were involved in cardiac hypertrophy, were all activated in Tomoregulin-1 knockdown mice and deteriorated after pressure overload, whereas Tomoregulin-1 overexpression significantly inhibited the activation of TβR1, TAK1 and JNK in both the physiological and pathological conditions.Conclusions Tomoregulin-1 overexpression significantly improved cardiac hypertrophy after pressure overload; knockdown of the Tomoregulin-1 expression accelerated the pathological progress. TAK1-JNK pathways in the myocardium involved in the protective regulatory effect of Tomoregulin-1 on the cardiac hypertrophy induced by pressure overload. Tomoregulin-1 maybe an important modifier gene of HCM and could provide a new strategy to treat the HCM in clinical.