Functional significance of myocardial activity at 18F-FAPI PET/CT in hypertrophic cardiomyopathy identified by cardiac magnetic resonance feature-tracking strain analysis.

PURPOSE: This study aimed to evaluate the functional significance of 18F-labeled fibroblast activation protein inhibitor (18F-FAPI) activity in hypertrophic cardiomyopathy (HCM) by comparison with cardiac magnetic resonance feature-tracking (CMR-FT) strain analysis. METHODS: A total of 49 HCM patients were included in this study. Two independent control groups of healthy participants with a matched age and sex to the HCM patients were also enrolled. Left ventricular (LV) 18F-FAPI activity was analyzed for extent (FAPI%) and intensity (maximum target-to-background ratio, TBRmax). The CMR tissue characterization parameters of the LV included late gadolinium enhancement, native T1 value, and extracellular volume fraction. LV strain analysis was performed in radial, circumferential, and longitudinal peak strains (PS). RESULTS: Intense LV myocardial 18F-FAPI uptake was observed in HCM patients, whereas no obvious uptake was detected in healthy participants (median TBRmax, 9.1 vs. 1.2, p < 0.001). The strain parameters of HCM patients, compared with healthy participants, were significantly impaired (mean radial PS, 23.5 vs. 36.0, mean circumferential PS, -14.5 vs. -20.0, and mean longitudinal PS, -9.9 vs. -16.0, all p < 0.001). At segmental levels, there was a moderate correlation between 18F-FAPI activity and strain parameters. The number of positive 18F-FAPI uptake segments (n = 653) was higher than that of hypertrophic segments (n = 190) and positive CMR tissue characterization segments (n = 525) (all p < 0.001). In segments with negative CMR tissue characterization findings, the strain capacity of positive 18F-FAPI uptake segments was lower than that of negative 18F-FAPI uptake segments (median radial PS, 30.5 vs. 36.1, p = 0.026 and median circumferential PS, -18.4 vs. -19.7, p = 0.041). CONCLUSION: 18F-FAPI imaging can partially reflect the potential strain reduction in HCM patients. 18F-FAPI imaging detects more involved myocardium than CMR tissue characterization techniques, and the additionally identified myocardium has impaired strain capacity.

Familial hypertrophic cardiomyopathy caused by a de novo Gly716Arg mutation of the ß-myosin heavy chain.

The present study was performed to identify the genotype of a hypertrophic cardiomyopathy family and investigate the clinicopathogenic characteristics and prognostic features of relevant genetic abnormalities. Target sequence capture sequencing was performed to screen for pathogenic alleles in a 32-year-old female patient (proband). Sanger sequencing was carried out to verify the results. Sanger sequencing was also performed on other family members to identify allele carriers. A survival analysis was carried out using published literature and our findings. We found that the proband and her son harboured a Gly716Arg sequence variant of the ß-myosin heavy chain. Neither the proband's father nor the mother were carriers of this sequence variant; thus, the mutation was classified as "de novo". Further survival analysis revealed that female patients appear to have a longer life expectancy compared with males. Our study may provide an effective approach for the genetic diagnosis of hypertrophic cardiomyopathy.

A genome-wide map of conserved microRNA targets in C. elegans.

BACKGROUND: Metazoan miRNAs regulate protein-coding genes by binding the 3' UTR of cognate mRNAs. Identifying targets for the 115 known C. elegans miRNAs is essential for understanding their function. RESULTS: By using a new version of PicTar and sequence alignments of three nematodes, we predict that miRNAs regulate at least 10% of C. elegans genes through conserved interactions. We have developed a new experimental pipeline to assay 3' UTR-mediated posttranscriptional gene regulation via an endogenous reporter expression system amenable to high-throughput cloning, demonstrating the utility of this system using one of the most intensely studied miRNAs, let-7. Our expression analyses uncover several new potential let-7 targets and suggest a new let-7 activity in head muscle and neurons. To explore genome-wide trends in miRNA function, we analyzed functional categories of predicted target genes, finding that one-third of C. elegans miRNAs target gene sets are enriched for specific functional annotations. We have also integrated miRNA target predictions with other functional genomic data from C. elegans. CONCLUSIONS: At least 10% of C. elegans genes are predicted miRNA targets, and a number of nematode miRNAs seem to regulate biological processes by targeting functionally related genes. We have also developed and successfully utilized an in vivo system for testing miRNA target predictions in likely endogenous expression domains. The thousands of genome-wide miRNA target predictions for nematodes, humans, and flies are available from the PicTar website and are linked to an accessible graphical network-browsing tool allowing exploration of miRNA target predictions in the context of various functional genomic data resources.

Protective effects of circulating microvesicles derived from myocardial ischemic rats on apoptosis of cardiomyocytes in myocardial ischemia/reperfusion injury.

OBJECTIVE: To investigate the effects of circulating microvesicles derived from myocardial ischemia (I-MVs) on apoptosis in myocardial ischemia/reperfusion (I/R) injury in rats. METHODS: I-MVs from rats undergoing myocardial left anterior descending (LAD) coronary artery ligation were isolated by ultracentrifugation from circulating blood and characterized by flow cytometry. I-MVs were administered intravenously (4.8 mg/kg) at 5 min before reperfusion procedure in I/R injury model which was induced by 30-min of ischemia and 120-min of reperfusion of LAD in rats. RESULTS: Treatment with I-MVssignificantly reduced the size of myocardial infarction, the activities of serum CK-MB and LDH, and the number of apoptotic cardiomyocytes. The activities of caspase 3, caspase 9 and caspase 12 in myocardium were also decreased significantly with I-MVs treatment. Moreover, the expression of Bax was decreased but Bcl-2 was increased. The expression of glucose regulated protein 78 (GRP78), sarco/endoplasmic reticulum Ca2+-ATPase 2 (SERCA2) and phosphorylated phospholamban (p-PLB) were increased after being treated with I-MVs. CONCLUSION: I-MVs could protect hearts from I/R injury in rats through SERCA2 and p-PLB of calcium regulatory proteins to alleviate intrinsic myocardial apoptosis including mitochondrial and endoplasmic reticulum pathways.

[Effects of circulating microvesicles derived from myocardial ischemic preconditioning on myocardial ischemia/reperfusion injury in rats].

OBJECTIVE: To investigate the effects of circulating microvesicles (MVs) derived from ischemic preconditioning (IPC) on myocardial ischemia/reperfusion (I/R) injury in rats and explore the underlying mechanism. METHODS: To establish the IPC model, the rats were subjected to brief cycles of left anterior descending (LAD) coronary occlusion and reperfusion. The blood was drawn from abdominal aorta once the operation was finished. IPC-MVs were isolated by ultracentrifugation from the peripheral blood and characterized by flow cytometry. The myocardial I/R model of rats was established in vivo. Rats were injected via the femoral vein with IPC-MVs at 7 mg/kg. Morphological changes of myocardium were observed microscopically after HE staining. Apoptosis of myocardial cells was detected with TUNEL assay. Myocardial infarct size was detected by TTC staining. Moreover, activity of plasma lactate dehydrogenase (LDH) was tested by colorimetry. The activity of caspase 3 in myocardium was assayed with spectrophotometry. Expression levels of Bcl-2 and Bax protein were examined with Western blot. RESULTS: The concentration of IPC-MVs, which was detected by flow cytometry, was 4380±745 cells/µl. Compared with I/R group, IPC-MVs alleviated the damage of tissues in I/R injured rats significantly. The myocardial infarct size and the cardiomyocyte apoptotic index were obviously decreased after IPC-MVs treatment (P<0.01, respectively). The activity of plasma LDH was significantly decreased in IPC-MVs treated rats (P<0.01). Moreover, the activity of caspase 3 was markedly decreased after IPC-MVs treatment (P<0.01). In addition, the expression of Bcl-2 was increased (P<0.01), the expression of Bax was decreased (P<0.01), the ratio of Bcl-2/Bax was significantly increased after IPC-MVs treatment (P<0.01). CONCLUSIONS: IPC-MVs protected myocardial against I/R injury by up-regulating the expression of Bcl-2 protein, down-regulating the expression of Bax protein, increasing the ratio of Bcl-2/Bax and decreasing cleavage of caspase 3.

Flow cytometric analysis of circulating microvesicles derived from myocardial Ischemic preconditioning and cardioprotection of Ischemia/reperfusion Injury in rats.

OBJECTIVE: To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles (MVs) from myocardial ischemic preconditioning (IPC) treated rats (IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion (I/R) injury in rats. METHODS: Myocardial IPC was elicited by three.cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending (LAD) coronary artery. Platelet-free plasma (PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFR PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 µm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure (MAP), heart rate (HR) and ST-segment of electro-cardiogram (ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin (HE) staining. The activity of plasma lactate dehydrogenase (LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining. RESULTS: Total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs (LMVs) and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (<1 Vm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats (P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR (P<0.01), decreased ST-segment and LDH activity (P < 0.05, P < 0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment (P < 0.01). CONCLUSION: The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.

Myocardial bridging as a common phenotype of hypertrophic cardiomyopathy has no effect on prognosis.

BACKGROUND: The prognostic significance of myocardial bridging in hypertrophic cardiomyopathy (HCM) remains controversial. This investigation sought to evaluate the impact of myocardial bridging on prognosis of patients with HCM. METHODS: A total of 298 adult patients (73% male, mean age, 53 ± 12 years) with HCM were retrospectively enrolled at Fuwai Hospital from 1999 to 2011. Myocardial bridging was evaluated by coronary angiography. Follow-up data were collected by telephone interviews and mailed questionnaires. RESULTS: Thirty-four (11%, 34/298) patients were determined with myocardial bridging and the middle of left anterior descending artery was the most frequently involved segment (77%, 26/34). Patients with myocardial bridging were younger than those without bridging (48 ± 9 versus 54 ± 12 years, P = 0.001). During the follow-up of 4.2 ± 2.3 years (range, 0.7-12.2 years), the presence of myocardial bridging was not evidently associated with increased risk for all-cause death (P = 0.54), cardiovascular death (P = 0.60), sudden cardiac death (P = 0.53) and deterioration of heart failure (P = 0.84). CONCLUSIONS: Myocardial bridging was a relatively common morphological component of HCM but not a predictor for adverse clinical outcomes.

Microvesicles derived from hypoxia/reoxygenation-treated human umbilical vein endothelial cells impair relaxation of rat thoracic aortic rings.

OBJECTIVE: To investigate the effects of microvesicles (MVs) derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs) on endothelium-dependent relaxation of rat thoracic aortic rings. METHODS: H/R injury model was established to induce HUVECs to release H/R-EMVs. H/R-EMVs from HUVECs were isolated by ultracentrifugation from the conditioned culture medium. H/R-EMVs were characterized using 1 µm latex beads and anti-PE-CD144 by flow cytometry. Thoracic aortic rings of rats were incubated with 2.5, 5, 10, 20 µg/ml H/R-EMVs derived from H/R-treated HUVECs for 4 hours, and their endothelium-dependent relaxation in response to acetylcholine (ACh) or endothelium-independent relaxation in response to sodium nitroprusside (SNP) was recorded in vitro. The nitric oxide (NO) production of ACh-treated thoracic aortic rings of rats was measured using Griess reagent. The expression of endothelial NO synthase (eNOS) and phosphorylated eNOS (p-eNOS, Ser-1177) in the thoracic aortic rings of rats was detected by Western blotting. Furthermore, the levels of SOD and MDA in H/R-EMVs-treated thoracic aortic rings of rats were measured using SOD and MDA kit. RESULTS: H/R-EMVs were induced by H/R-treated HUVECs and isolated by ultracentrifugation. The membrane vesicles (< 1 µm) induced by H/R were CD144 positive. ACh-induced relaxation and NO production of rat thoracic aortic rings were impaired by H/R-EMVs treatment in a concentration-dependent manner (P < 0.05, P < 0.01). The expression of total eNOS (t-eNOS) was not affected by H/R-EMVs. However, the expression of p-eNOS decreased after treated with H/R-EMVs. The activity of SOD decreased and the level of MDA increased in H/R-EMVs treated rat thoracic aortic rings (P < 0.01). CONCLUSION: ACh induced endothelium-dependent relaxation of thoracic aortic rings of rats was impaired by H/R-EMVs in a concentration-dependent manner. The mechanisms included a decrease in NO production, p-eNOS expression and an increase in oxidative stress.

Screening of pathogenic genes in Chinese patients with arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a heritable cardiac disease predominantly caused by mutations in desmosomal protein genes. Previous genetic analyses of the Chinese ARVC population are limited to small size and restriction to a single gene. This study was aimed to investigate the genotype in a large series of Chinese patients with ARVC through comprehensively screening nine ARVC-causing genes. METHODS: A total of 100 unrelated ARVC patients and 300 age, gender and ethnicity matched healthy controls were genetically tested with multiplexing targeted resequencing for nine previously reported ARVC-causing genes, including plakophilin-2, desmoplakin, desmoglein-2, desmocollin-2, plakoglobin, transforming growth factor beta-3, transmembrane protein 43, desmin and Lamin A/C. RESULTS: Fifty-nine mutations were identified in 64% of the patients, among which, 93% were located in desmosomal protein genes. Plakophilin-2 mutations accounted for 54% of the total and 58% of the desmosomal mutations, with a truncating mutation type making up about 2/3 of the plakophilin-2 mutations. Only four mutations were found in non-desmosomal genes; two in transmembrane protein 43 and two in transforming growth factor beta-3. Two of them (one of each gene) appeared as single missense mutations. No mutation was identified in desmin or Lamin A/C. Multiple mutations were found in 23% of the patients, with plakophilin-2 being found in 57% of the multi-mutation carriers. CONCLUSIONS: Plakophilin-2 was the most common gene mutation that was identified in Chinese ARVC patients. Non-desmosomal genes should be added to desmosomal protein genes when performing molecular genetic screening in patients with suspected ARVC.

microRNA target predictions across seven Drosophila species and comparison to mammalian targets.

microRNAs are small noncoding genes that regulate the protein production of genes by binding to partially complementary sites in the mRNAs of targeted genes. Here, using our algorithm PicTar, we exploit cross-species comparisons to predict, on average, 54 targeted genes per microRNA above noise in Drosophila melanogaster. Analysis of the functional annotation of target genes furthermore suggests specific biological functions for many microRNAs. We also predict combinatorial targets for clustered microRNAs and find that some clustered microRNAs are likely to coordinately regulate target genes. Furthermore, we compare microRNA regulation between insects and vertebrates. We find that the widespread extent of gene regulation by microRNAs is comparable between flies and mammals but that certain microRNAs may function in clade-specific modes of gene regulation. One of these microRNAs (miR-210) is predicted to contribute to the regulation of fly oogenesis. We also list specific regulatory relationships that appear to be conserved between flies and mammals. Our findings provide the most extensive microRNA target predictions in Drosophila to date, suggest specific functional roles for most microRNAs, indicate the existence of coordinate gene regulation executed by clustered microRNAs, and shed light on the evolution of microRNA function across large evolutionary distances. All predictions are freely accessible at our searchable Web site http://pictar.bio.nyu.edu.