The popularization of genome-wide analyses and RNA sequencing led to the discovery that a large part of the human genome, while effectively transcribed, does not encode proteins. Long non-coding RNAs have emerged as critical regulators of gene expression in both normal and disease states. Studies of long non-coding RNAs expressed in the heart, in combination with gene association studies, revealed that these molecules are regulated during cardiovascular development and disease. Some long non-coding RNAs have been functionally implicated in cardiac pathophysiology and constitute potential therapeutic targets. Here, we review the current knowledge of the function of long non-coding RNAs in the cardiovascular system, with an emphasis on cardiovascular development and biology, focusing on hypertension, coronary artery disease, myocardial infarction, ischemia, and heart failure. We discuss potential therapeutic implications and the challenges of long non-coding RNA research, with directions for future research and translational focus.

Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) stimulates calcium release from acidic stores such as lysosomes and is a highly potent calcium-mobilising second messenger. NAADP plays an important role in calcium signalling in the heart under basal conditions and following β-adrenergic stress. Nevertheless, the spatial interaction of acidic stores with other parts of the calcium signalling apparatus in cardiac myocytes is unknown. We present evidence that lysosomes are intimately associated with the sarcoplasmic reticulum (SR) in ventricular myocytes; a median separation of 20 nm in 2D electron microscopy and 3.3 nm in 3D electron tomography indicates a genuine signalling microdomain between these organelles. Fourier analysis of immunolabelled lysosomes suggests a sarcomeric pattern (dominant wavelength 1.80 μm). Furthermore, we show that lysosomes form close associations with mitochondria (median separation 6.2 nm in 3D studies) which may provide a basis for the recently-discovered role of NAADP in reperfusion-induced cell death. The trigger hypothesis for NAADP action proposes that calcium release from acidic stores subsequently acts to enhance calcium release from the SR. This work provides structural evidence in cardiac myocytes to indicate the formation of microdomains between acidic and SR calcium stores, supporting emerging interpretations of NAADP physiology and pharmacology in heart.

3′,5′-Cyclic adenosine monophosphate (cAMP) signals in the heart are often confined to concentration microdomains shaped by cAMP diffusion and enzymatic degradation. While the importance of phosphodiesterases (degradative enzymes) in sculpting cAMP microdomains is well established in cardiomyocytes, less is known about cAMP diffusivity (D_cAMP) and factors affecting it. Many earlier studies have reported fast diffusivity, which argues against sharply defined microdomains. [cAMP] dynamics in the cytoplasm of adult rat ventricular myocytes were imaged using a fourth generation genetically encoded FRET-based sensor. The [cAMP]-response to the addition and removal of isoproterenol (β-adrenoceptor agonist) quantified the rates of cAMP synthesis and degradation. To obtain a read out of D_cAMP, a stable [cAMP] gradient was generated using a microfluidic device which delivered agonist to one half of the myocyte only. After accounting for phosphodiesterase activity, D_cAMP was calculated to be 32 µm²/s; an order of magnitude lower than in water. Diffusivity was independent of the amount of cAMP produced. Saturating cAMP-binding sites with the analogue 6-Bnz-cAMP did not accelerate D_cAMP, arguing against a role of buffering in restricting cAMP mobility. cAMP diffused at a comparable rate to chemically unrelated but similar sized molecules, arguing for a common physical cause of restricted diffusivity. Lower mitochondrial density and order in neonatal cardiac myocytes allowed for faster diffusion, demonstrating the importance of mitochondria as physical barriers to cAMP mobility. In adult cardiac myocytes, tortuosity due to physical barriers, notably mitochondria, restricts cAMP diffusion to levels that are more compatible with microdomain signalling.

Key points: Carbonic anhydrase enzymes are said to enhance membrane H⁺ and HCO₃⁻ transport, an idea tested almost exclusively in heterologous cell‐expression systems. In ventricular myocytes, inhibiting cytoplasmic enzyme activity slows membrane acid extrusion (HCO₃⁻ influx) on Na⁺–HCO₃⁻ co‐transporters, with no effect on Na⁺/H⁺ exchangers. Inhibiting exofacial enzyme activity has no effect on either transporter. Mathematical modelling simulates the influence of carbonic anhydrase on Na⁺–HCO₃⁻ co‐transport, provided the enzyme catalytically delivers H⁺ ions to the transporter (across a cytoplasmic nanodomain that is poorly accessible to intrinsic buffers), rather than catalysing protonation of the imported bicarbonate. Intrinsic cytoplasmic mobile buffers appear to deliver H⁺ to Na⁺/H⁺ exchangers, thus obviating the need for carbonic anhydrase. Conclusion: in native cardiac cells, intracellular carbonic anhydrase molecules partner Na⁺–HCO₃⁻ co‐transporters but not Na⁺/H⁺ exchangers, to enhance flux activity. The enzyme thus plays a key role in supporting bicarbonate transport and pH control in the heart. Carbonic anhydrase enzymes (CAs) catalyse the reversible hydration of CO₂ to H⁺ and HCO₃⁻ ions. This catalysis is proposed to be harnessed by acid/base transporters, to facilitate their transmembrane flux activity, either through direct protein–protein binding (a ‘transport metabolon’) or local functional interaction. Flux facilitation has previously been investigated by heterologous co‐expression of relevant proteins in host cell lines/oocytes. Here, we examine the influence of intrinsic CA activity on membrane HCO₃⁻ or H⁺ transport via the native acid‐extruding proteins, Na⁺–HCO₃⁻ cotransport (NBC) and Na⁺/H⁺ exchange (NHE), expressed in enzymically isolated mammalian ventricular myocytes. Effects of intracellular and extracellular (exofacial) CA (CA_i and CA_e) are distinguished using membrane‐permeant and ‑impermeant pharmacological CA inhibitors, while measuring transporter activity in the intact cell using pH and Na⁺ fluorophores. We find that NBC, but not NHE flux is enhanced by catalytic CA activity, with facilitation being confined to CA_i activity alone. Results are quantitatively consistent with a model where CA_i catalyses local H⁺ ion delivery to the NBC protein, assisting the subsequent (uncatalysed) protonation and removal of imported HCO₃⁻ ions. In well‐superfused myocytes, exofacial CA activity is superfluous, most likely because extracellular CO₂/HCO₃⁻ buffer is clamped at equilibrium. The CA_i insensitivity of NHE flux suggests that, in the native cell, intrinsic mobile buffer‐shuttles supply sufficient intracellular H⁺ ions to this transporter, while intrinsic buffer access to NBC proteins is restricted. Our results demonstrate a selective CA facilitation of acid/base transporters in the ventricular myocyte, implying a specific role for the intracellular enzyme in HCO₃⁻ transport, and hence pH_i regulation in the heart.

MicroRNAs (miRs) regulate complex processes, including angiogenesis, by targeting multiple mRNAs. miR-24-3p-3p directly represses eNOS, GATA2, and PAK4 in endothelial cells (ECs), thus inhibiting angiogenesis during development and in the infarcted heart. miR-24-3p is widely expressed in cardiovascular cells, suggesting that it could additionally regulate angiogenesis by acting on vascular mural cells. Here, we have investigated: (1) new miR-24-3p targets; (2) the expression and the function of miR-24-3p in human vascular ECs; (3) the impact of miR-24-3p inhibition in the angiogenesis reparative response to limb ischemia in mice. Using bioinformatics target prediction platforms and 3′-UTR luciferase assays, we newly identified Notch1 and its Delta-like ligand 1 (Dll1) to be directly targeted by miR-24-3p. miR-24-3p was expressed in human ECs and pericytes cultured under normal conditions. Exposure to hypoxia increased miR-24-3p in ECs but not in pericytes. Transfection with a miR-24-3p precursor (pre-miR-24-3p) increased miR-24-3p expression in ECs, reducing the cell survival, proliferation, and angiogenic capacity. Opposite effects were caused by miR-24-3p inhibition. The anti-angiogenic action of miR-24-3p overexpression could be prevented by simultaneous adenovirus (Ad)-mediated delivery of constitutively active Notch intracellular domain (NICD) into cultured ECs. We next demonstrated that reduced Notch signalling contributes to the anti-angiogenic effect of miR-24-3p in vitro. In a mouse unilateral limb ischemia model, local miR-24-3p inhibition (by adenovirus-mediated miR-24-3p decoy delivery) restored endothelial Notch signalling and increased capillary density. However, the new vessels appeared disorganised and twisted, worsening post-ischemic blood perfusion recovery. To better understand the underpinning mechanisms, we widened the search for miR-24-3p target genes, identifying several contributors to vascular morphogenesis, such as several members of the Wingless (Wnt) signalling pathway, β-catenin signalling components, and VE-cadherin, which synergise to regulate angiogenesis, pericytes recruitment to neoformed capillaries, maturation, and stabilization of newly formed vessels. Among those, we next focussed on β-catenin to demonstrate that miR-24-3p inhibition reduces β-catenin expression in hypoxic ECs, which is accompanied by reduced adhesion of pericytes to ECs. In summary, miR-24-3p differentially targets several angiogenesis modulators and contributes to autonomous and non-autonomous EC crosstalk. In ischemic limbs, miR-24-3p inhibition increases the production of dysfunctional microvessels, impairing perfusion. Caution should be observed in therapeutic targeting of miR-24-3p.