HCN channelopathy and cardiac electrophysiologic dysfunction in genetic and acquired rat epilepsy models.

OBJECTIVE: Evidence from animal and human studies indicates that epilepsy can affect cardiac function, although the molecular basis of this remains poorly understood. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels generate pacemaker activity and modulate cellular excitability in the brain and heart, with altered expression and function associated with epilepsy and cardiomyopathies. Whether HCN expression is altered in the heart in association with epilepsy has not been investigated previously. We studied cardiac electrophysiologic properties and HCN channel subunit expression in rat models of genetic generalized epilepsy (Genetic Absence Epilepsy Rats from Strasbourg, GAERS) and acquired temporal lobe epilepsy (post-status epilepticus SE). We hypothesized that the development of epilepsy is associated with altered cardiac electrophysiologic function and altered cardiac HCN channel expression. METHODS: Electrocardiography studies were recorded in vivo in rats and in vitro in isolated hearts. Cardiac HCN channel messenger RNA (mRNA) and protein expression were measured using quantitative PCR and Western blotting respectively. RESULTS: Cardiac electrophysiology was significantly altered in adult GAERS, with slower heart rate, shorter QRS duration, longer QTc interval, and greater standard deviation of RR intervals compared to control rats. In the post-SE model, we observed similar interictal changes in several of these parameters, and we also observed consistent and striking bradycardia associated with the onset of ictal activity. Molecular analysis demonstrated significant reductions in cardiac HCN2 mRNA and protein expression in both models, providing a molecular correlate of these electrophysiologic abnormalities. SIGNIFICANCE: These results demonstrate that ion channelopathies and cardiac dysfunction can develop as a secondary consequence of chronic epilepsy, which may have relevance for the pathophysiology of cardiac dysfunction in patients with epilepsy.

Enhanced extravasation, stability and in vivo cardiac gene silencing via in situ siRNA-albumin conjugation.

A potential barrier to progression of siRNA therapeutics to the clinic is the ability of these agents to cross the vascular endothelium to reach target cells. This study aimed to bypass the endothelial barrier by harnessing the extravasation capability of the serum protein albumin to allow siRNA to reach cardiomyocytes. A strategy for conjugating siRNA to albumin in vivo was developed that involved activating 3'-amine, 2'-O-methyl, phosphorothioate modified siRNA with succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to yield maleimide-functionalized siRNA ("activated siRNA"); this thiol-reactive species can then irreversibly link to the single surface-exposed cysteine residue of endogenous albumin following intravenous administration. An IGF-I-receptor (IGF-IR) siRNA sequence which was effective in vitro was used to test the ability of the siRNA-albumin conjugate to bypass the endothelial barrier in Balb/C mice and produce silencing. In situ conjugation of maleimide-functionalized siRNA to albumin in mouse serum occurred within minutes of addition, and the resulting conjugate was found to be nuclease stable by SDS-PAGE analysis. In Sprague-Dawley rats, activated siRNA showed a significantly enhanced elimination half-life (75.9 ± 18.2 min) compared to unactivated siRNA (5.1 ± 0.2 min). Intravenous injection of this activated siRNA (1 mg/kg daily for four days) significantly reduced left ventricle IGF-IR mRNA to 64.1 ± 4.1% of that in vehicle-treated animals (mean ± SEM), while the control siRNA (unconjugated) had no effect (n = 4, P > 0.05). Imaging of microvessels from mice treated with fluorescein-labeled activated siRNA showed clear evidence of extravasation and cellular uptake in capillary endothelial cells, cardiomyocytes and vascular smooth muscle cells for mice treated with the activated siRNA but not mice treated with the unactivated siRNA. siRNA-albumin constructs are therefore capable of extravasation to the myocardium resulting in silencing in this otherwise silencing-resistant organ.

Loss of the long non-coding RNA OIP5-AS1 exacerbates heart failure in a sex-specific manner.

Long non-coding RNAs (lncRNAs) have been demonstrated to influence numerous biological processes, being strongly implicated in the maintenance and physiological function of various tissues including the heart. The lncRNA OIP5-AS1 (1700020I14Rik/Cyrano) has been studied in several settings; however its role in cardiac pathologies remains mostly uncharacterized. Using a series of in vitro and ex vivo methods, we demonstrate that OIP5-AS1 is regulated during cardiac development in rodent and human models and in disease settings in mice. Using CRISPR, we engineered a global OIP5-AS1 knockout (KO) mouse and demonstrated that female KO mice develop exacerbated heart failure following cardiac pressure overload (transverse aortic constriction [TAC]) but male mice do not. RNA-sequencing of wild-type and KO hearts suggest that OIP5-AS1 regulates pathways that impact mitochondrial function. Thus, these findings highlight OIP5-AS1 as a gene of interest in sex-specific differences in mitochondrial function and development of heart failure.

Coexpressed Class B G Protein-Coupled Secretin and GLP-1 Receptors Self- and Cross-Associate: Impact on Pancreatic Islets.

Class B guanine nucleotide-binding protein (G protein)-coupled receptors form symmetrical homodimeric complexes along the lipid face of transmembrane segment 4 (TM4) and can form heterodimeric complexes, although their structure is unknown. The current study demonstrates that the lipid face of TM4 is also the predominant determinant for formation of heteroreceptor complexes between two class B receptors, secretin receptor (SecR) and glucagonlike peptide-1 receptor (GLP-1R), which are expressed on pancreatic islet cells. Because these receptors use the same interface for formation of homo- and heteroreceptor complexes, competitive forces may affect expression of different complexes. Assessment of SecR and GLP-1R dimeric complexes via recombinant expression in Chinese hamster ovary cells revealed that homodimeric receptor complexes were more stable than the heterodimeric complexes, and the homodimeric SecR/SecR is more stable than the GLP-1R/GLP-1R complex. Given the greater tendency for homodimeric compared with heterodimeric complex formation, the heteroreceptor complexes lacked the expression that might have been predicted by geometry alone. Nevertheless, cells coexpressing these receptors formed heterodimeric complexes that correlated with reduced intracellular calcium responses to secretin, but no change in the cyclic adenosine monophosphate responses to each natural agonist. This functional effect was confirmed in pancreatic islets isolated from wild-type and GLP-1R knockout mice. In these cells, the increased calcium response mediated by secretin in the absence of GLP-1R was paralleled by an increased glucose-dependent insulin response, indicating that the heterodimeric receptor complexes modulate secretin responses. Furthermore, the heterodimeric receptor complexes also mediated agonist-induced cross-receptor internalization, a process that could have broad functional significance in sites of natural receptor coexpression.

Commercially Supplied Amine-Modified siRNAs May Require Ultrafiltration prior to Conjugation with Amine-Reactive Compounds.

Conjugation of siRNA to macromolecules such as serum albumin has multiple potential benefits, including enhanced extravasation via albumin-mediated transcytosis across endothelial cells and reduced renal clearance. In attempting to conjugate siRNA to albumin, we used commercially sourced amine-modified siRNA and reacted it with the heterobifunctional linker succinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate (SMCC) to introduce a maleimide group suitable for conjugation to the thiol group of the surface-exposed cysteine residue (Cys 34) within albumin. We found the conjugation of the SMCC-treated siRNA to bovine serum albumin (BSA) to be very inefficient and investigated the cause of the low yield of conjugate. Ultrafiltration with phosphate-buffered saline prior to activation with SMCC dramatically increased the yield of siRNA-albumin conjugate (~15-fold). Communication with the commercial supplier revealed that ammonium acetate buffer was used in a desalting step as part of the siRNA purification process prior to supply, likely resulting in ammonium counterions to the siRNA polyanion, which would interfere with conjugation by consuming the SMCC. After ultrafiltration, a greatly reduced amount of SMCC could be used to affect conjugation, without significant reduction in yield. These data indicate that amine-modified siRNA sourced commercially may require ultrafiltration or dialysis prior to use in conjugation reactions.

Characterization of the acute cardiovascular effects of intravenously administered insulin-like growth factor-I in conscious Sprague-Dawley rats.

1. Insulin-like growth factor (IGF)-I has acute effects on cardiovascular function, including a well-characterized vasodilator response in isolated arteries. In addition to increasing the release of nitric oxide, IGF-I also has effects on a variety of other signalling pathways that affect vascular tone, in particular interactions with the sympathetic nervous system and the renin-angiotensin-aldosterone system. We sought to characterize the effects of intravenous IGF-I on blood pressure and on responses to noradrenaline (NA), angiotensin II, acetylcholine and dobutamine. 2. Administration of IGF-I administration caused small decreases in mean arterial pressure (5.4 +/- 1.5%) and responsiveness to the prazosin-sensitive vasoconstrictor effects of NA (a 2.1 +/- 0.6-fold increase in ED(50); n = 40; P < 0.01) and both effects were maximal at 200 microg/kg IGF-I. In addition, IGF-I significantly increased pulse pressure increases induced by low doses of dobutamine (from an increase in pulse pressure of 9.9 +/- 1.2 to 13.4 +/- 1.9 mmHg; n = 39; P < 0.05). Administration of IGF-I had no significant effect on responses to AngII or ACh. 3. Intravenous administration of IGF-I receptor antisense oligonucleotides (400 microg/kg) abolished the effects of IGF-I on NA-induced vasoconstriction (n = 11; P < 0.05), whereas administration of a mismatch oligonucleotide did not. 4. These data indicate that the maximal effects of exogenously administered IGF-I include modest direct vasodilation and inhibition of constrictor responses to NA and an increase in the effect of dobutamine on pulse pressure. The magnitude of these effects was less than what previous in vitro studies and those performed in anaesthetized animals may have indicated likely. 5. The modest magnitude of the dilator effects of IGF-I observed in conscious rats in vivo in the present study suggests that IGF-I is unlikely to be a major player in regulating vascular tone in normotensive animals.