[Clinical and genetic analysis of a Chinese patient with Alström syndrome].

OBJECTIVE: To explore the genetic etiology for a patient with Alström syndrome (ALMS) presenting as dilated cardiomyopathy. METHODS: A 41-year-old male patient who had presented at the Sixth Medical Center of PLA General Hospital on October 20, 2021 was selected as the study subject. Clinical and laboratory examinations were carried out. Whole exome sequencing (WES) was employed for genetic testing, and candidate variants were validated by Sanger sequencing and pathogenicity analysis. RESULTS: The patient had a 14-year medical history characterized by dilated cardiomyopathy, complete atrioventricular block, visual impairment, sensorineural hearing loss, truncal obesity, insulin resistance, type 2 diabetes, hypertension, renal dysfunction, and paranoid delusions. Genetic testing revealed that he has harbored compound heterozygous variants of the ALMS1 gene, namely c.6823C>T (p.Arg2275Ter) and c.9442_9445dup (p.Ser3149LysfsTer2). Sanger sequencing confirmed that they were inherited from his father and mother, respectively. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), both variants were predicted to be pathogenic (PVS1_VeryStrong+PM2_Supporting+PM3+PP3, PVS1_VeryStrong+PM2_Supporting+PM3). Literature review indicated that the complete atrioventricular block in the patient was a phenotype unreported previously. CONCLUSION: The c.6823C>T (p.Arg2275Ter) and c.9442_9445dup (p.Ser3149LysfsTer2) compound heterozygous variants of the ALMS1 gene probably underlay the pathogenesis in this patient. Above findings have expanded the phenotypic spectrum of ALMS and provided insights for clinicians dealing with similar cases.

High Glucose Attenuates Cardioprotective Effects of Glucagon-Like Peptide-1 Through Induction of Mitochondria Dysfunction via Inhibition of ß-Arrestin-Signaling.

An increased vulnerability has been detected after ischemia/reperfusion injury in cardiomyocytes in diabetic patients. Glucagon-like peptide-1 (GLP-1) has been proven to have a notable cardioprotective effect in cardiomyocytes. However, in diabetic patients, the cardioprotective effects of GLP-1 are compromised, which is called GLP-1 resistance. ß-arrestin is one of the two main downstream effectors of GLP-1 and ß-arrestin signaling pathway exerts cardioprotective effects upon activation of GLP-1R. Our hypothesis is that the increased vulnerability of cardiomyocytes in diabetic patients is partly due to disruption of the ß-arrestin signaling pathway. To test this, we analyzed cardiomyocyte viability and survival in high glucose and normal glucose condition after hypoxia/reoxygenation injury in vitro, additional GLP-1 was used to determine whether ß-arrestin signaling pathway was involved. We also investigated the role of mitochondrial dysfunction in GLP-1 resistance. Our results showed that cardioprotective effects of GLP-1 were reduced in high glucose cultured H9C2 cells compared to normal glucose cultured H9C2, verifying the existence of GLP-1 resistance in high glucose cultured H9C2 cells. Further study suggested that ß-arrestin plays a key role in GLP-1 resistance: ß-arrestin expression is notably downregulated in high glucose condition and cardioprotective effects of GLP-1 can be diminished by downregulation of ß-arrestin in normal glucose condition while upregulation of ß-arrestin can restore cardioprotective effects of GLP-1 in high glucose condition. Then we explore how ß-arrestin affects the cardioprotective effects of GLP-1 and found that ß-arrestin exerts cardioprotective effects by improving mitochondria quality control via the PI3K/Akt signaling pathway. Thus, our study found out a new mechanism of GLP-1 resistance of cardiomyocytes in high glucose conditions that impaired ß-arrestin expression, caused mitochondria dysfunction and eventually cell death. Our study provided a new perspective in treating myocardial ischemia/reperfusion injury in diabetic patients.

Essential Role Of High Glucose-Induced Overexpression Of PKCß And PKCδ In GLP-1 Resistance In Rodent Cardiomyocytes.

PURPOSE: Myocardia in diabetic patients exhibit increased vulnerability after ischemia/reperfusion injury (IRI). It has been demonstrated that glucagon-like peptide-1 (GLP-1) has a protective effect on cardiomyocytes. Protein kinase C (PKC) acts as a key regulator of many signaling pathways including oxidative stress and apoptosis. Our hypothesis is that increased vulnerability of myocardia in diabetic patients is partly due to GLP-1 resistance. The aim of this study was to explore the role of PKC in GLP-1 resistance in diabetic cardiomyocytes. METHODS: Cardiac function of diabetic or non-diabetic mice after myocardial IRI was detected with or without administration of GLP-1 analog exendin-4. Impacts of diabetes mellitus on GLP-1R expression in myocardia after IRI were accessed by Western blot. By transfecting PKC isoforms siRNA, in vitro study helped to identify the exact PKC isoforms which contributed to the downregulatio n of GLP-1R or impaired post-receptor signaling pathways in rodent cardiomyocytes (H9C2 cells) cultured by high glucose. RESULTS: The cardioprotective effects of endogenous GLP-1 were impaired in diabetic mice after myocardial IRI and administration of exendin-4 had no significant effects in restoring cardiac function. GLP-1 receptor (GLP-1R) expression decreased in H9C2 cells cultured by high glucose and knockdown of PKCß partly restored GLP-1R expression. Overexpression of PKCδ induced by high glucose in H9C2 cells impaired GLP-1 post-receptor anti-apoptotic signaling pathways by inhibition of Akt phosphorylation. Knockdown of both PKCß and PKCδ significantly restored cardioprotective effects of GLP-1 in H9C2 cells cultured by high glucose. CONCLUSION: Our study found out a new mechanism of GLP-1 resistance that high glucose-induced overexpression of PKCß and PKCδ impaired cardioprotective effects of GLP-1 by downregulation of GLP-1R and inhibition of GLP-1 post-receptor anti-apoptotic signaling pathways, thus provided a new perspective in treating myocardial IRI in diabetic patients.

Preliminary evaluation of 18F­AlF­NOTA­MAL­Cys40­Exendin­4 in rodent heart after myocardial ischemia and reperfusion.

Glucagon­like peptide­1 (GLP­1) and its receptor (GLP­1R) exert cardioprotective effects after myocardial ischemia and reperfusion (MI/R) in animal models and human clinical trials. Receptor imaging with positron emission tomography (PET) provides a non­invasive method for monitoring GLP­1R expression. In the present study, a fluorine­18­labeled aluminum fluoride exendin­4 analog [18F­AlF conjugated with 1,4,7­triazacyclononanetriacetic acid (NOTA)­maleimide (MAL)­Cys40­exendin­4] was synthesized and evaluated in a rat MI/R model for GLP­1R imaging. NOTA­MAL­Cys40­exendin­4 was synthesized by coupling Cys40­exendin­4 with NOTA­MAL. NOTA­MAL­Cys40­exendin­4 was then conjugated with 18F­AlF to obtain 18F­AlF­NOTA­MAL­Cys40­exendin­4. The yield of 18F­AlF­NOTA­MAL­Cys40­exendin­4 was 18.5±3.4% (not decay corrected). The process was completed within ~30 min. In rat MI/R models, the tracer exhibited specific binding to GLP­1R and an appropriate signal­to­noise ratio. At 8 h post­MI/R, tracer uptake reached its peak [0.35±0.053% of injected dose (%ID)/g; n=6] in ischemic myocardium. Localized tracer uptake decreased 1 day (0.20±0.032 %ID/g; n=6) and 3 days (0.16±0.017 %ID/g; n=6) post­MI/R compared with 8 h post­MI/R, but still remained higher compared with sham­operated groups (0.06±0.012 %ID/g; n=6). Pre­injected unlabeled exendin­4 effectively blocked tracer accumulation (0.09±0.041 %ID/g; n=6). In conclusion, 18F­AlF­NOTA­MAL­Cys40­exendin­4 demonstrated favorable characteristics for GLP­1R imaging following MI/R. PET imaging using 18F­AlF­NOTA­MAL­Cys40­exendin­4 in rodent hearts after MI/R revealed a dynamic pattern of GLP­1R upregulation.

Mst1 participates in the atherosclerosis progression through macrophage autophagy inhibition and macrophage apoptosis enhancement.

Emerging evidence favors the notion that macrophage autophagy plays a prominent role in the pathogenesis of vulnerable plaque, suggesting the therapeutic potential of targeting autophagy in atherosclerosis. Here ApoE(-/-) mice were crossed with Mst1 knockout or Mst1 Tg mice to generate ApoE(-/-):Mst1(-/-) and ApoE(-/-):Mst1Tg mice. All animals were fed high-fat-diet for 4months to induce arterial atherosclerosis. Murine macrophage RAW264.7 cells were subjected to ox-LDL (50µg/mL) in an effort to examine the cellular mechanisms. A significant increase in the levels of Mst1 and p-Mst1 was observed in the aorta of ApoE(-/-) mice. Mst1 knockout significantly reduced atherosclerotic area, decreased lipid core area and macrophage accumulation as compared with ApoE(-/-) mice. Along the same line, Mst1 overexpression increased plaque area, lipid core and macrophage accumulation as compared with ApoE(-/-) mice. Mst1 deficiency significantly increased levels of Beclin1 and LC3II, while decreased that of p62 in aortic atherosclerosis. Moreover, in vitro data indicated that Mst1 knockdown prompted more typical autophagosomes upon ox-LDL challenge. Mst1 knockdown also enhanced autophagic flux as evidenced by GFP-mRFP-LC3 staining, increased LC3-II expression and decreased p62 expression in the presence of bafilomycin A1. Mst1 knockdown decreased, while Mst1 overexpression increased macrophage apoptosis upon ox-LDL exposure. In conclusion, Mst1 deficiency diminishes atherosclerosis and stabilizes atherosclerotic plaques in ApoE(-/-) mice. Mst1 may participate in atherosclerosis progression through inhibition of macrophage autophagy and promotion of macrophage apoptosis.

Notch3 Ameliorates Cardiac Fibrosis After Myocardial Infarction by Inhibiting the TGF-ß1/Smad3 Pathway.

Notch3 and TGF-ß1 signaling play a key role in the pathogenesis and progression of chronic cardiovascular disease. However, whether Notch3 protects against myocardial infarction (MI) and the underlying mechanisms remains unknown. C57BL/6 mice were randomized to be treated with Notch3 siRNA (siNotch3) or lentivirus carrying Notch3 cDNA (Notch3) before coronary artery ligation. Four weeks after constructing MI model, cardiac function and fibrosis were compared between groups. The cardiac fibroblast cells (CFs) were isolated from newborn C57BL/6 mice (1-3 days old) and transfected with lentivirus carrying Notch3 cDNA. TGF-ß1 (5 ng/ml), a well-known pro-fibrotic factor, was administered 72 h after Notch3 cDNA administration in CFs. The related proteins of fibrosis such as a-smooth muscle actin (a-SMA), Type I collagen, metalloprotease (MMP)-9 and the tissue inhibitor of metalloproteinases (TIMP)-2 were examined by western blot analysis. Notch3 cDNA treatment attenuated cardiac damage and inhibited fibrosis in mice with MI. Meanwhile, Notch3 siRNA administration aggravated cardiac function damage and markedly enhanced cardiac fibrosis in mice with MI. Overexpression of Notch3 inhibited TGF-ß1-induced fibroblast-myofibroblast transition of mouse cardiac fibroblast cells, as evidenced by down-regulating a-SMA and Type I collagen expression. Notch3 cDNA treatment also increased MMP-9 expression and decreased TIMP-2 expression in the TGF-ß1-stimulated cells. This study indicates that Notch3 is an important protective factor for cardiac fibrosis in a MI model, and the protective effect of Notch3 is attributable to its action on TGF-ß1/Smad3 signaling.

Lin28a protects against cardiac ischaemia/reperfusion injury in diabetic mice through the insulin-PI3K-mTOR pathway.

The insulin-PI3K-mTOR pathway exhibits a variety of cardiovascular activities including protection against I/R injury. Lin28a enhanced glucose uptake and insulin-sensitivity via insulin-PI3K-mTOR signalling pathway. However, the role of lin28a on experimental cardiac I/R injury in diabetic mice are not well understood. Diabetic mice underwent 30 min. of ischaemia followed by 3 hrs of reperfusion. Animals were randomized to be treated with lentivirus carrying lin28a siRNA (siLin28a) or lin28a cDNA (Lin28a) 72 hrs before coronary artery ligation. Myocardial infarct size (IS), cardiac function, cardiomyocyte apoptosis and mitochondria morphology in diabetic mice who underwent cardiac I/R injury were compared between groups. The target proteins of lin28a were examined by western blot analysis. Lin28a overexpression significantly reduced myocardial IS, improved LV ejection fraction (LVEF), decreased myocardial apoptotic index and alleviated mitochondria cristae destruction in diabetic mice underwent cardiac I/R injury. Lin28a knockdown exacerbated cardiac I/R injury as demonstrated by increased IS, decreased LVEF, increased apoptotic index and aggravated mitochondria cristae destruction. Interestingly, pre-treatment with rapamycin abolished the beneficial effects of lin28a overexpression. Lin28a overexpression increased, while Lin28a knockdown decreased the expression of IGF1R, p-Akt, p-mTOR and p-p70s6k after cardiac I/R injury in diabetic mice. Rapamycin pre-treatment abolished the effects of increased p-mTOR and p-p70s6k expression exerted by lin28a overexpression. This study indicates that lin28a overexpression reduces IS, improves cardiac function, decreases cardiomyocyte apoptosis index and alleviates cardiomyocyte mitochondria impairment after cardiac I/R injury in diabetic mice. The mechanism responsible for the effects of lin28a is associated with the insulin-PI3K-mTOR dependent pathway.