4-Hydroxynonenal impairs miRNA maturation in heart failure via Dicer post-translational modification.

BACKGROUND AND AIMS: Developing novel therapies to battle the global public health burden of heart failure remains challenging. This study investigates the underlying mechanisms and potential treatment for 4-hydroxynonenal (4-HNE) deleterious effects in heart failure. METHODS: Biochemical, functional, and histochemical measurements were applied to identify 4-HNE adducts in rat and human failing hearts. In vitro studies were performed to validate 4-HNE targets. RESULTS: 4-HNE, a reactive aldehyde by-product of mitochondrial dysfunction in heart failure, covalently inhibits Dicer, an RNase III endonuclease essential for microRNA (miRNA) biogenesis. 4-HNE inhibition of Dicer impairs miRNA processing. Mechanistically, 4-HNE binds to recombinant human Dicer through an intermolecular interaction that disrupts both activity and stability of Dicer in a concentration- and time-dependent manner. Dithiothreitol neutralization of 4-HNE or replacing 4-HNE-targeted residues in Dicer prevents 4-HNE inhibition of Dicer in vitro. Interestingly, end-stage human failing hearts from three different heart failure aetiologies display defective 4-HNE clearance, decreased Dicer activity, and miRNA biogenesis impairment. Notably, boosting 4-HNE clearance through pharmacological re-activation of mitochondrial aldehyde dehydrogenase 2 (ALDH2) using Alda-1 or its improved orally bioavailable derivative AD-9308 restores Dicer activity. ALDH2 is a major enzyme responsible for 4-HNE removal. Importantly, this response is accompanied by improved miRNA maturation and cardiac function/remodelling in a pre-clinical model of heart failure. CONCLUSIONS: 4-HNE inhibition of Dicer directly impairs miRNA biogenesis in heart failure. Strikingly, decreasing cardiac 4-HNE levels through pharmacological ALDH2 activation is sufficient to re-establish Dicer activity and miRNA biogenesis; thereby representing potential treatment for patients with heart failure.

Ionizable Lipid Nanoparticle-Mediated Delivery of Plasmid DNA in Cardiomyocytes.

Introduction: Gene therapy is a promising approach to be applied in cardiac regeneration after myocardial infarction and gene correction for inherited cardiomyopathies. However, cardiomyocytes are crucial cell types that are considered hard-to-transfect. The entrapment of nucleic acids in non-viral vectors, such as lipid nanoparticles (LNPs), is an attractive approach for safe and effective delivery. Methods: Here, a mini-library of engineered LNPs was developed for pDNA delivery in cardiomyocytes. LNPs were characterized and screened for pDNA delivery in cardiomyocytes and identified a lead LNP formulation with enhanced transfection efficiency. Results: By varying lipid molar ratios, the LNP formulation was optimized to deliver pDNA in cardiomyocytes with enhanced gene expression in vitro and in vivo, with negligible toxicity. In vitro, our lead LNP was able to reach a gene expression greater than 80%. The in vivo treatment with lead LNPs induced a twofold increase in GFP expression in heart tissue compared to control. In addition, levels of circulating myeloid cells and inflammatory cytokines remained without significant changes in the heart after LNP treatment. It was also demonstrated that cardiac cell function was not affected after LNP treatment. Conclusion: Collectively, our results highlight the potential of LNPs as an efficient delivery vector for pDNA to cardiomyocytes. This study suggests that LNPs hold promise to improve gene therapy for treatment of cardiovascular disease.

Neuronal cholinergic signaling constrains norepinephrine activity in the heart.

It is well known that cholinergic hypofunction contributes to cardiac pathology, yet, the mechanisms involved remain unclear. Our previous study has shown that genetically engineered model of cholinergic deficit, the vesicular acetylcholine transporter knockdown homozygous (VAChT KDHOM) mice, exhibit pathological cardiac remodeling and a gradual increase in cardiac mass with aging. Given that an increase in cardiac mass is often caused by adrenergic hyperactivity, we hypothesized that VAChT KDHOM mice might have an increase in cardiac norepinephrine (NE) levels. We thus investigated the temporal changes in NE content in the heart from 3-, 6-, and 12-mo-old VAChT mutants. Interestingly, mice with cholinergic hypofunction showed a gradual elevation in cardiac NE content, which was already increased at 6 mo of age. Consistent with this finding, 6-mo-old VAChT KDHOM mice showed enhanced sympathetic activity and a greater abundance of tyrosine hydroxylase positive sympathetic nerves in the heart. VAChT mutants exhibited an increase in peak calcium transient, and mitochondrial oxidative stress in cardiomyocytes along with enhanced G protein-coupled receptor kinase 5 (GRK5) and nuclear factor of activated T-cells (NFAT) staining in the heart. These are known targets of adrenergic signaling in the cell. Moreover, vagotomized-mice displayed an increase in cardiac NE content confirming the data obtained in VAChT KDHOM mice. Establishing a causal relationship between acetylcholine and NE, VAChT KDHOM mice treated with pyridostigmine, a cholinesterase inhibitor, showed reduced cardiac NE content, rescuing the phenotype. Our findings unveil a yet unrecognized role of cholinergic signaling as a modulator of cardiac NE, providing novel insights into the mechanisms that drive autonomic imbalance.

Molecular basis of Period 1 regulation by adrenergic signaling in the heart.

The cardiac circadian clock is responsible for the modulation of different myocardial processes, and its dysregulation has been linked to disease development. How this clock machinery is regulated in the heart remains an open question. Because noradrenaline (NE) can act as a zeitgeber in cardiomyocytes, we tested the hypothesis that adrenergic signaling resets cardiac clock gene expression in vivo. In its anti-phase with Clock and Bmal1, cardiac Per1 abundance increased during the dark phase, concurrent with the rise in heart rate and preceded by an increase in NE levels. Sympathetic denervation altered Bmal1 and Clock amplitude, while Per1 was affected in both amplitude and oscillatory pattern. We next treated mice with a ß-adrenergic receptor (ß-AR) blocker. Strikingly, the ß-AR blockade during the day suppressed the nocturnal increase in Per1 mRNA, without altering Clock or Bmal1. In contrast, activating ß-AR with isoproterenol (ISO) promoted an increase in Per1 expression, demonstrating its responsiveness to adrenergic input. Inhibitors of ERK1/2 and CREB attenuated ISO-induced Per1 expression. Upstream of ERK1/2, PI3Kγ mediated ISO induction of Per1 transcription, while activation of ß2-AR, but not ß1-AR induced increases in ERK1/2 phosphorylation and Per1 expression. Consistent with the ß2-induction of Per1 mRNA, ISO failed to activate ERK1/2 and elevate Per1 in the heart of ß2-AR-/- mice, whereas a ß2-AR antagonist attenuated the nocturnal rise in Per1 expression. Our study established a link between NE/ß2-AR signaling and Per1 oscillation via the PI3Ky-ERK1/2-CREB pathway, providing a new framework for understanding the physiological mechanism involved in resetting cardiac clock genes.

Dense optical flow software to quantify cellular contractility.

Cell membrane deformation is an important feature that occurs during many physiological processes, and its study has been put to good use to investigate cardiomyocyte function. Several methods have been developed to extract information on cardiomyocyte contractility. However, no existing computational framework has provided, in a single platform, a straightforward approach to acquire, process, and quantify this type of cellular dynamics. For this reason, we develop CONTRACTIONWAVE, high-performance software written in Python programming language that allows the user to process large data image files and obtain contractility parameters by analyzing optical flow from images obtained with videomicroscopy. The software was validated by using neonatal, adult-, and human-induced pluripotent stem-cell-derived cardiomyocytes, treated or not with drugs known to affect contractility. Results presented indicate that CONTRACTIONWAVE is an excellent tool for examining changes to cardiac cellular contractility in animal models of disease and for pharmacological and toxicology screening during drug discovery.

Increased cholinergic activity under conditions of low estrogen leads to adverse cardiac remodeling.

Cholinesterase inhibitors are used in postmenopausal women for the treatment of neurodegenerative diseases. Despite their widespread use in the clinical practice, little is known about the impact of augmented cholinergic signaling on cardiac function under reduced estrogen conditions. To address this gap, we subjected a genetically engineered murine model of systemic vesicular acetylcholine transporter overexpression (Chat-ChR2) to ovariectomy and evaluated cardiac parameters. Left-ventricular function was similar between Chat-ChR2 and wild-type (WT) mice. Following ovariectomy, WT mice showed signs of cardiac hypertrophy. Conversely, ovariectomized (OVX) Chat-ChR2 mice evolved to cardiac dilation and failure. Transcript levels for cardiac stress markers atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) were similarly upregulated in WT/OVX and Chat-ChR2/OVX mice. 17ß-Estradiol (E2) treatment normalized cardiac parameters in Chat-ChR2/OVX to the Chat-ChR2/SHAM levels, providing a link between E2 status and the aggravated cardiac response in this model. To investigate the cellular basis underlying the cardiac alterations, ventricular myocytes were isolated and their cellular area and contractility were assessed. Myocytes from WT/OVX mice were wider than WT/SHAM, an indicative of concentric hypertrophy, but their fractional shortening was similar. Conversely, Chat-ChR2/OVX myocytes were elongated and presented contractile dysfunction. E2 treatment again prevented the structural and functional changes in Chat-ChR2/OVX myocytes. We conclude that hypercholinergic mice under reduced estrogen conditions do not develop concentric hypertrophy, a critical compensatory adaptation, evolving toward cardiac dilation and failure. This study emphasizes the importance of understanding the consequences of cholinesterase inhibition, used clinically to treat dementia, for cardiac function in postmenopausal women.

Moving Pieces in a Cellular Puzzle: A Cryptic Peptide from the Scorpion Toxin Ts14 Activates AKT and ERK Signaling and Decreases Cardiac Myocyte Contractility via Dephosphorylation of Phospholamban.

Cryptic peptides (cryptides) are biologically active peptides formed after proteolysis of native precursors present in animal venoms, for example. Proteolysis is an overlooked post-translational modification that increases venom complexity. The tripeptide KPP (Lys-Pro-Pro) is a peptide encrypted in the C-terminus of Ts14-a 25-mer peptide from the venom of the Tityus serrulatus scorpion that has a positive impact on the cardiovascular system, inducing vasodilation and reducing arterial blood pressure of hypertensive rats among other beneficial effects. A previous study reported that KPP and its native peptide Ts14 act via activation of the bradykinin receptor B2 (B2R). However, the cellular events underlying the activation of B2R by KPP are unknown. To study the cell signaling triggered by the Ts14 cryptide KPP, we incubated cardiac myocytes isolated from C57BL/6 mice with KPP (10-7 mol·L-1) for 0, 5, or 30 min and explored the proteome and phosphoproteome. Our results showed that KPP regulated cardiomyocyte proteins associated with, but not limited to, apoptosis, muscle contraction, protein turnover, and the respiratory chain. We also reported that KPP led to AKT phosphorylation, activating AKT and its downstream target nitric oxide synthase. We also observed that KPP led to dephosphorylation of phospholamban (PLN) at its activation sites (S16 and T17), leading to reduced contractility of treated cardiomyocytes. Some cellular targets reported here for KPP (e.g., AKT, PLN, and ERK) have already been reported to protect the cardiac tissue from hypoxia-induced injury. Hence, this study suggests potential beneficial effects of this scorpion cryptide that needs to be further investigated, for example, as a drug lead for cardiac infarction.

Redox-Active Drug, MnTE-2-PyP5+, Prevents and Treats Cardiac Arrhythmias Preserving Heart Contractile Function.

BACKGROUND: Cardiomyopathies remain among the leading causes of death worldwide, despite all efforts and important advances in the development of cardiovascular therapeutics, demonstrating the need for new solutions. Herein, we describe the effects of the redox-active therapeutic Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin, AEOL10113, BMX-010 (MnTE-2-PyP5+), on rat heart as an entry to new strategies to circumvent cardiomyopathies. METHODS: Wistar rats weighing 250-300 g were used in both in vitro and in vivo experiments, to analyze intracellular Ca2+ dynamics, L-type Ca2+ currents, Ca2+ spark frequency, intracellular reactive oxygen species (ROS) levels, and cardiomyocyte and cardiac contractility, in control and MnTE-2-PyP5+-treated cells, hearts, or animals. Cells and hearts were treated with 20 µM MnTE-2-PyP5+ and animals with 1 mg/kg, i.p. daily. Additionally, we performed electrocardiographic and echocardiographic analysis. RESULTS: Using isolated rat cardiomyocytes, we observed that MnTE-2-PyP5+ reduced intracellular Ca2+ transient amplitude, without altering cell contractility. Whereas MnTE-2-PyP5+ did not alter basal ROS levels, it was efficient in modulating cardiomyocyte redox state under stress conditions; MnTE-2-PyP5+ reduced Ca2+ spark frequency and increased sarcoplasmic reticulum (SR) Ca2+ load. Accordingly, analysis of isolated perfused rat hearts showed that MnTE-2-PyP5+ preserves cardiac function, increases SR Ca2+ load, and reduces arrhythmia index, indicating an antiarrhythmic effect. In vivo experiments showed that MnTE-2-PyP5+ treatment increased Ca2+ transient, preserved cardiac ejection fraction, and reduced arrhythmia index and duration. MnTE-2-PyP5+ was effective both to prevent and to treat cardiac arrhythmias. CONCLUSION: MnTE-2-PyP5+ prevents and treats cardiac arrhythmias in rats. In contrast to most antiarrhythmic drugs, MnTE-2-PyP5+ preserves cardiac contractile function, arising, thus, as a prospective therapeutic for improvement of cardiac arrhythmia treatment.

Neuromuscular synapse degeneration without muscle function loss in the diaphragm of a murine model for Huntington's Disease.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease characterized by chorea, incoordination and psychiatric and behavioral symptoms. The leading cause of death in HD patients is aspiration pneumonia, associated with respiratory dysfunction, decreased respiratory muscle strength and dysphagia. Although most of the motor symptoms are derived from alterations in the central nervous system, some might be associated with changes in the components of motor units (MU). To explore this hypothesis, we evaluated morphofunctional aspects of the diaphragm muscle in a mouse model for HD (BACHD). We showed that the axons of the phrenic nerves were not affected in 12-months-old BACHD mice, but the axon terminals that form the neuromuscular junctions (NMJs) were more fragmented in these animals in comparison with the wild-type mice. In BACHD mice, the synaptic vesicles of the diaphragm NMJs presented a decreased exocytosis rate. Quantal content and quantal size were smaller and there was less synaptic depression whereas the estimated size of the readily releasable vesicle pool was not changed. At the ultrastructure level, the diaphragm NMJs of these mice presented fewer synaptic vesicles with flattened and oval shapes, which might be associated with the reduced expression of the vesicular acetylcholine transporter protein. Furthermore, mitochondria of the diaphragm muscle presented signs of degeneration in BACHD mice. Interestingly, despite all these cellular alterations, BACHD diaphragmatic function was not compromised, suggesting a higher resistance threshold of this muscle. A putative resistance mechanism may be protecting this vital muscle. Our data contribute to expanding the current understanding of the effects of mutated huntingtin in the neuromuscular synapse and the diaphragm muscle function.

Vascular Kinin B1 and B2 Receptors Determine Endothelial Dysfunction through Neuronal Nitric Oxide Synthase.

B1- and B2-kinin receptors are G protein-coupled receptors that play an important role in the vascular function. Therefore, the present study was designed to evaluate the participation of kinin receptors in the acetylcholine (ACh)-induced vascular relaxation, focusing on the protein-protein interaction involving kinin receptors with endothelial and neuronal nitric oxide synthases (eNOS and nNOS). Vascular reactivity, nitric oxide (NO·) and reactive oxygen species (ROS) generation, co-immunoprecipitation were assessed in thoracic aorta from male wild-type (WT), B1- (B1R-/-), B2- (B2R-/-) knockout mice. Some vascular reactivity experiments were also performed in a double kinin receptors knockout mice (B1B2R-/-). For pharmacological studies, selective B1- and B2-kinin receptors antagonists, NOS inhibitors and superoxide dismutase (SOD) mimetic were used. First, we show that B1- and B2-kinin receptors form heteromers with nNOS and eNOS in thoracic aorta. To investigate the functionality of these protein-protein interactions, we took advantage of pharmacological tools and knockout mice. Importantly, our results show that kinin receptors regulate ACh-induced relaxation via nNOS signaling in thoracic aorta with no changes in NO· donor-induced relaxation. Interestingly, B1B2R-/- presented similar level of vascular dysfunction as found in B1R-/- or B2R-/- mice. In accordance, aortic rings from B1R-/- or B2R-/- mice exhibit decreased NO· bioavailability and increased superoxide generation compared to WT mice, suggesting the involvement of excessive ROS generation in the endothelial dysfunction of B1R-/- and B2R-/- mice. Alongside, we show that impaired endothelial vasorelaxation induced by ACh in B1R-/- or B2R-/- mice was rescued by the SOD mimetic compound. Taken together, our findings show that B1- and B2-kinin receptors regulate the endothelium-dependent vasodilation of ACh through nNOS activity and indicate that molecular disturbance of short-range interaction between B1- and B2-kinin receptors with nNOS might be involved in the oxidative pathogenesis of endothelial dysfunction.

Cardioprotective Action of Ginkgo biloba Extract against Sustained ß-Adrenergic Stimulation Occurs via Activation of M2/NO Pathway.

Ginkgo biloba is the most popular phytotherapic agent used worldwide for treatment of several human disorders. However, the mechanisms involved in the protective actions of Ginkgo biloba on cardiovascular diseases remain poorly elucidated. Taking into account recent studies showing beneficial actions of cholinergic signaling in the heart and the cholinergic hypothesis of Ginkgo biloba-mediated neuroprotection, we aimed to investigate whether Ginkgo biloba extract (GBE) promotes cardioprotection via activation of cholinergic signaling in a model of isoproterenol-induced cardiac hypertrophy. Here, we show that GBE treatment (100 mg/kg/day for 8 days, v.o.) reestablished the autonomic imbalance and baroreflex dysfunction caused by chronic ß-adrenergic receptor stimulation (ß-AR, 4.5 mg/kg/day for 8 days, i.p.). Moreover, GBE prevented the upregulation of muscarinic receptors (M2) and downregulation of ß1-AR in isoproterenol treated-hearts. Additionally, we demonstrated that GBE prevents the impaired endothelial nitric oxide synthase activity in the heart. GBE also prevented the pathological cardiac remodeling, electrocardiographic changes and impaired left ventricular contractility that are typical of cardiac hypertrophy. To further investigate the mechanisms involved in GBE cardioprotection in vivo, we performed in vitro studies. By using neonatal cardiomyocyte culture we demonstrated that the antihypertrophic action of GBE was fully abolished by muscarinic receptor antagonist or NOS inhibition. Altogether, our data support the notion that antihypertrophic effect of GBE occurs via activation of M2/NO pathway uncovering a new mechanism involved in the cardioprotective action of Ginkgo biloba.

Beneficial effects of angiotensin-(1-7) against deoxycorticosterone acetate-induced diastolic dysfunction occur independently of changes in blood pressure.

Mineralocorticoids have been implicated in the pathogenesis of diastolic heart failure. On the contrary, angiotensin (Ang)-(1-7) has emerged as a potential strategy for treatment of cardiac dysfunction induced by excessive mineralocorticoid receptor activation. A critical question about the cardioprotective effect of Ang-(1-7) in hypertensive models is its dependence on blood pressure (BP) reduction. Here, we addressed this question by investigating the mechanisms involved in Ang-(1-7) cardioprotection against mineralocorticoid receptor activation. Sprague-Dawley (SD) and transgenic (TG) rats that overexpress an Ang-(1-7) producing fusion protein (TG(A1-7)3292) were treated with deoxycorticosterone acetate (DOCA) for 6 weeks. After treatment, SD rats became hypertensive and developed ventricular hypertrophy. These parameters were attenuated in TG-DOCA. SD-DOCA rats developed diastolic dysfunction which was associated at the cellular level with reduced Ca(2+) transient. Oppositely, TG-DOCA myocytes presented enhanced Ca(2+) transient. Moreover, higher extracellular signal-regulated kinase phosphorylation, type 1 phosphatase, and protein kinase Cα levels were found in SD-DOCA cells. In vivo, pressor effects of DOCA can contribute to the diastolic dysfunction, raising the question of whether protection in TG was a consequence of reduced BP. To address this issue, BP in SD-DOCA was kept at TG-DOCA level by giving hydralazine or by reducing the DOCA amount given to rats (Low-DOCA). Under similar BP, diastolic dysfunction and molecular changes were still evident in DOCA-hydralazine and SD-low-DOCA, but not in TG-DOCA. In conclusion, Ang-(1-7) protective signaling against DOCA-induced diastolic dysfunction occurs independently of BP attenuation and is mediated by the activation of pathways involved in Ca(2+) handling, hypertrophy, and survival.