ApoA-I mimetic does not improve left ventricular diastolic dysfunction in rabbits without aortic valve stenosis.

BACKGROUND: We previously demonstrated that high-density lipoprotein (HDL) infusions may improve left ventricular diastolic dysfunction (LVDD) in an aortic valve stenosis (AVS) model. Whether the benefit was direct or mediated by the observed reduction in AVS severity is not clear. Here, we aimed to test the direct effect of an ApoA-I mimetic on LVDD in the absence of AVS. METHODS: Rabbits were exposed to three different protocols to develop LVDD. First, rabbits were exposed to 0.5% cholesterol-rich diet for an average of 17 weeks. Second, rabbits were subjected to surgical ascending aortic constriction (AAC), to mimic the effect of fixed reduced aortic valve area, and studied after 10 weeks. The third model combined both cholesterol-enriched diet (for 12 weeks) and surgical AAC. The control group consisted of age-matched rabbits under normal diet. After development of LVDD, rabbits were randomized to receive infusions of saline or apoA-I mimetic (25 mg/kg) 3 times per week for 4 weeks. Detailed cardiac structure and function measurements were assessed at baseline and weekly during treatment period. Histological and molecular analyses were performed on LV samples. RESULTS: In the three models, echocardiographic results showed development of LVDD over time, with preserved LV systolic and aortic valve functions versus controls. ApoA-I mimetic infusions did not significantly improve echocardiographic parameters nor molecular markers of cardiac inflammation, oxidative stress and fibrosis. CONCLUSION: ApoA-I mimetic therapy did not directly improve LVDD. These results indicate that previously observed changes of LVDD were caused by AVS improvement induced by this treatment.

Novel Pathogenesis of Hypertension and Diastolic Dysfunction Caused by M3R (Muscarinic Cholinergic 3 Receptor) Signaling.

Multiple quantitative trait loci for blood pressure (BP) are localized in humans and rodent models. Model studies have not only produced human quantitative trait loci homologues but also provided unforeseen mechanistic insights into the function modality of quantitative trait loci actions. Presently, congenic knockins, gene-specific knockout, and in vitro and in vivo function studies were used in a rat model of polygenic hypertension, DSS (Dahl salt sensitive) rats. One gene previously unknown in regulating BP was detected with 1 structural mutation(s) for each of 2 quantitative trait loci classified into 2 separate epistatic modules 1 and 3. C17QTL1 in epistatic module 2 was identified to be the gene Chrm3 encoding the M3R (muscarinic cholinergic 3 receptor), since a single function-enhancing M3RT556M conversion correlated with elevated BP. To definitively prove that the enhanced M3R function is responsible for BP changes by the DSS alleles of C17QTL1, we generated a Chrm3 gene-specific rat knockout. We observed a reduction in BP without tachycardia in both sexes, regardless of the amount of dietary salt, and an improvement in diastolic and kidney dysfunctions. All occurred in spite of a significant reduction in M3R-dependent vasodilation. The previously seen sexual dimorphism for C17QTL1 on BP disappeared in the absence of M3R. A Chrm3-coding variation increased M3R signaling, correlating with higher BP. Removing the M3R signaling led to a decrease in BP and improvements in cardiac and renal malfunctions. A novel pathogenic pathway accounted for a portion of polygenic hypertension and has implications in applying new diagnostic and therapeutic uses against hypertension and diastolic dysfunction.

Unique quantitative trait loci in synergy permanently improve diastolic dysfunction.

BACKGROUND: Diastolic dysfunction often precedes the onset of diastolic heart failure. We previously demonstrated that diastolic dysfunction and left ventricular hypertrophy (LVH) in Dahl salt-sensitive rats can be ameliorated by quantitative trait loci (QTLs). METHODS: We analyzed cardiac phenotypes of 2 "single" congenic strains, C10S.L33 and C10S.L28, by echocardiography, in which a specific Dahl salt-sensitive rat chromosome segment was replaced by its Lewis homologue. C10S.L33 improves diastolic function (DF) and LVH only in rats aged 10 weeks, not aged 15 weeks. C10S.L28 alleviated LVH, but not diastolic dysfunction. Thus, the QTLs captured by C10S.L33 and C10S.L28 are designated as DF/LVH C10QTL7 and LVH C10QTL4, respectively. We then combined multiple single strains to form 2 congenic combinations. One of the 2 congenic combinations included the chromosome segments covered by C10S.L33 and C10S.L28. RESULTS: Diastolic dysfunction was either completely or partially reversed by 15 weeks in the 2 congenic combinations. LVH was permanently improved from 10 to 15 weeks. CONCLUSIONS: Distinct QTLs exist that regulate diastolic function and/or LVH in the short term when acting alone, but durably when combined. The Ccl2 chemokine (C-C motif) ligand 1 (Ccl2) gene is the prime candidate for DF/LVH C10QTL7, owing to a nonconserved coding mutation. Schlafen genes are candidates for LVH C10QTL4. Since CCL2 and Schlafens are not known for influencing diastolic function and left ventricular mass, novel long-term strategies of prognosis, diagnosis, and therapy for diastolic heart failure and LVH appear from this work.

Combining distinctive and novel loci doubles BP reduction, reverses diastolic dysfunction and mitigates LV hypertrophy.

OBJECTIVES: Diastolic dysfunction often represents the onset of diastolic heart failure (DHF). We previously showed in principle that diastolic function in Dahl salt-sensitive rats (DSS) can be genetically determined by quantitative trait loci (QTLs) that also modulate blood pressure (BP). METHODS: We analyzed cardiac phenotypes of four 'single' congenic strains by echocardiography, in which a specific DSS chromosome segment was replaced by its normotensive Lewis homologue. RESULTS: Two of the strains permanently lowered BP, and but attenuated diastolic dysfunction only in rats at 10 weeks of age, not at 15 weeks fed on a 2% NaCl diet starting from 8 weeks of age. We then combined multiple QTLs by integrating several 'single' congenic strains. As a result, BP was greatly reduced. Cardiac dysfunction and LV hypertrophy were continuously improved from 10 to 15 weeks, although the degree and timing of the improvement varied among different congenic combinations. CONCLUSION: Distinct QTLs exist that simultaneously modulate BP and diastolic function. These QTLs, in combination, synergistically lowered BP and permanently alleviated or reversed diastolic dysfunction. The genes that are contained in the congenic strains affecting diastolic function are not known for their specific influence on BP. Novel long-term strategies of prognosis, diagnosis and therapy for hypertensive DHF appear from this work.

Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein.

Alternative splicing is a key process underlying the evolution of increased proteomic and functional complexity and is especially prevalent in the mammalian nervous system. However, the factors and mechanisms governing nervous system-specific alternative splicing are not well understood. Through a genome-wide computational and expression profiling strategy, we have identified a tissue- and vertebrate-restricted Ser/Arg (SR) repeat splicing factor, the neural-specific SR-related protein of 100 kDa (nSR100). We show that nSR100 regulates an extensive network of brain-specific alternative exons enriched in genes that function in neural cell differentiation. nSR100 acts by increasing the levels of the neural/brain-enriched polypyrimidine tract binding protein and by interacting with its target transcripts. Disruption of nSR100 prevents neural cell differentiation in cell culture and in the developing zebrafish. Our results thus reveal a critical neural-specific alternative splicing regulator, the evolution of which has contributed to increased complexity in the vertebrate nervous system.

A fully automated robotic system for microinjection of zebrafish embryos.

As an important embodiment of biomanipulation, injection of foreign materials (e.g., DNA, RNAi, sperm, protein, and drug compounds) into individual cells has significant implications in genetics, transgenics, assisted reproduction, and drug discovery. This paper presents a microrobotic system for fully automated zebrafish embryo injection, which overcomes the problems inherent in manual operation, such as human fatigue and large variations in success rates due to poor reproducibility. Based on computer vision and motion control, the microrobotic system performs injection at a speed of 15 zebrafish embryos (chorion unremoved) per minute, with a survival rate of 98% (n = 350 embryos), a success rate of 99% (n = 350 embryos), and a phenotypic rate of 98.5% (n = 210 embryos). The sample immobilization technique and microrobotic control method are applicable to other biological injection applications such as the injection of mouse oocytes/embryos and Drosophila embryos to enable high-throughput biological and pharmaceutical research.