No abstract

Epitranscriptomic events such as adenosine‐to‐inosine (A‐to‐I) RNA editing by ADAR can recode mRNAs to translate novel proteins. Editing of the mRNA that encodes actin crosslinking protein Filamin A (FLNA) mediates a Q‐to‐R transition in the interactive C‐terminal region. While FLNA editing is conserved among vertebrates, its physiological function remains unclear. Here, we show that cardiovascular tissues in humans and mice show massive editing and that FLNA RNA is the most prominent substrate. Patient‐derived RNA‐Seq data demonstrate a significant drop in FLNA editing associated with cardiovascular diseases. Using mice with only impaired FLNA editing, we observed increased vascular contraction and diastolic hypertension accompanied by increased myosin light chain phosphorylation, arterial remodeling, and left ventricular wall thickening, which eventually causes cardiac remodeling and reduced systolic output. These results demonstrate a causal relationship between RNA editing and the development of cardiovascular disease indicating that a single epitranscriptomic RNA modification can maintain cardiovascular health.

Endothelial cell-derived lipase (EDL) is a new member of the lipase gene family with high sequence homology with lipoprotein lipase (LPL). EDL is a phospholipase with very little triacylglycerol lipase activity. To investigate the effects of EDL on binding and uptake of high-density lipoprotein (HDL), as well as on the selective uptake of HDL-derived cholesterol esters (CEs), HepG2 cells were infected with adenovirus coding for EDL. For comparison, cells were also infected with LPL and with lacZ as a control. Both HDL binding and particle uptake were increased 1.5-fold and selective HDL-CE uptake was increased 1.8-fold in EDL-infected HepG2 cells compared with controls. The effect of LPL was less pronounced, resulting in 1.1-fold increase in particle uptake and 1.3-fold increase in selective uptake. Inhibition of the enzymic activity with tetrahydrolipstatin (THL) significantly enhanced the effect of EDL, as reflected by a 5.2-fold increase in binding, a 2.6-fold increase in particle uptake and a 1.1-fold increase in CE selective uptake compared with incubations without THL. To elucidate the mechanism responsible for the effects of THL, we analysed the abundance of heparin-releasable EDL protein from infected HepG2 cells upon incubations with THL, HDL and free (non-esterified) fatty acids (FFAs). In the presence of THL, vastly more EDL protein remained bound to the cell surface. Additionally, HDL and FFAs reduced the amount of cell-surface-bound EDL, suggesting that fatty acids that are liberated from phospholipids in HDL release EDL from the cell surface. This was substantiated further by the finding that, in contrast with EDL, the amount of cell-surface-bound enzymically inactive mutant EDL (MUT-EDL) was not reduced in the presence of HDL and foetal calf serum. The increased amount of cell-surface-bound MUT-EDL in the presence of THL suggested that the enzymic inactivity of MUT-EDL, as well as an augmenting effect of THL that is independent of its ability to inactivate the enzyme, are responsible for the increased amount of cell-surface-bound EDL in the presence of THL. Furthermore, in cells expressing MUT-EDL, binding and holoparticle uptake were markedly higher compared with cells expressing the active EDL, and could be increased further in the presence of THL. Despite 1.7-fold higher binding and 1.8-fold higher holoparticle uptake, the selective CE uptake by MUT-EDL-expressing cells was comparable with EDL-expressing cells and was even decreased 1.3-fold with THL. Experiments in CLA-1 (CD-36 and LIMPII analogous 1, the human homologue of scavenger receptor class B type I)-deficient HEK-293 cells demonstrated that EDL alone has the ability to stimulate HDL-CE selective uptake independently of CLA-1. Thus our results demonstrate that EDL mediates both HDL binding and uptake, and the selective uptake of HDL-CE, independently of lipolysis and CLA-1.

It is clearly established that an ef®cient supply to the brain of a-tocopherol (aTocH), the most biologically active member of the vitamin E family, is of the utmost importance for proper neurological functioning. Although the mechanism of uptake of aTocH into cells constituting the blood±brain barrier (BBB) is obscure, we previously demonstrated that high-density lipoprotein (HDL) plays a major role in the supply of aTocH to porcine brain capillary endothelial cells (pBCECs). Here we studied whether a porcine analogue of human and rodent scavenger receptor class B, type I mediates selective (without concomitant lipoprotein particle internalization) uptake of HDL-associated aTocH in a similar manner to that described for HDL-associated cholesteryl esters (CEs). In agreement with this hypothesis we observed that a major proportion of aTocH uptake by pBCECs occurred by selective uptake, exceeding HDL 3 holoparticle uptake by up to 13-fold. The observation that selective uptake of HDL-associated CE exceeded HDL 3 holoparticle up to fourfold suggested that a porcine analogue of SR-BI (pSR-BI) may be involved in lipid uptake at the BBB. In line with the observation of selective lipid uptake, RT-PCR and northern and western blot analyses revealed the presence of pSR-BI in cells constituting the BBB. Adenovirus-mediated overexpression of the human analogue of SR-BI (hSR-BI) in pBCECs resulted in a fourfold increase in selective HDL-associated aTocH uptake. In accordance with the proposed function of SR-BI, selective HDL±CE uptake was increased sixfold in Chinese hamster ovary cells stably transfected with murine SR-BI (mSR-BI). Most importantly stable mSR-BI overexpression mediated a twofold increase in HDL-associated [14 C]aTocH selective uptake in comparison with control cells. In line with tracer experiments, mass transfer studies with unlabelled lipoproteins revealed that mSR-BI overexpression resulted in a twofold increase in endogenous HDL 3 -associated aTocH uptake. The results of this study indicate that SR-BI promotes the uptake of HDLassociated aTocH into cells constituting the BBB and plays an important role during the supply of the CNS with this indispensable micronutrient.

Heart failure with preserved ejection fraction (HFpEF) is a highly prevalent and intractable form of cardiac decompensation commonly associated with diastolic dysfunction. Here, we show that diastolic dysfunction in patients with HFpEF is associated with a cardiac deficit in nicotinamide adenine dinucleotide (NAD+). Elevating NAD+ by oral supplementation of its precursor, nicotinamide, improved diastolic dysfunction induced by aging (in 2-year-old C57BL/6J mice), hypertension (in Dahl salt-sensitive rats), or cardiometabolic syndrome (in ZSF1 obese rats). This effect was mediated partly through alleviated systemic comorbidities and enhanced myocardial bioenergetics. Simultaneously, nicotinamide directly improved cardiomyocyte passive stiffness and calcium-dependent active relaxation through increased deacetylation of titin and the sarcoplasmic reticulum calcium adenosine triphosphatase 2a, respectively. In a long-term human cohort study, high dietary intake of naturally occurring NAD+ precursors was associated with lower blood pressure and reduced risk of cardiac mortality. Collectively, these results suggest NAD+ precursors, and especially nicotinamide, as potential therapeutic agents to treat diastolic dysfunction and HFpEF in humans.