Modulation of the cardiac sodium channel NaV1.5 peak and late currents by NAD+ precursors.

RATIONALE: The cardiac sodium channel NaV1.5, encoded by SCN5A, produces the rapidly inactivating depolarizing current INa that is responsible for the initiation and propagation of the cardiac action potential. Acquired and inherited dysfunction of NaV1.5 results in either decreased peak INa or increased residual late INa (INa,L), leading to tachy/bradyarrhythmias and sudden cardiac death. Previous studies have shown that increased cellular NAD+ and NAD+/NADH ratio increase INa through suppression of mitochondrial reactive oxygen species and PKC-mediated NaV1.5 phosphorylation. In addition, NAD+-dependent deacetylation of NaV1.5 at K1479 by Sirtuin 1 increases NaV1.5 membrane trafficking and INa. The role of NAD+ precursors in modulating INa remains unknown. OBJECTIVE: To determine whether and by which mechanisms the NAD+ precursors nicotinamide riboside (NR) and nicotinamide (NAM) affect peak INa and INa,Lin vitro and cardiac electrophysiology in vivo. METHODS AND RESULTS: The effects of NAD+ precursors on the NAD+ metabolome and electrophysiology were studied using HEK293 cells expressing wild-type and mutant NaV1.5, rat neonatal cardiomyocytes (RNCMs), and mice. NR increased INa in HEK293 cells expressing NaV1.5 (500 µM: 51 ± 18%, p = .02, 5 mM: 59 ± 22%, p = .03) and RNCMs (500 µM: 60 ± 26%, p = .02, 5 mM: 74 ± 39%, p = .03) while reducing INa,L at the higher concentration (RNCMs, 5 mM: -45 ± 11%, p = .04). NR (5 mM) decreased NaV1.5 K1479 acetylation but increased INa in HEK293 cells expressing a mutant form of NaV1.5 with disruption of the acetylation site (NaV1.5-K1479A). Disruption of the PKC phosphorylation site abolished the effect of NR on INa. Furthermore, NAM (5 mM) had no effect on INa in RNCMs or in HEK293 cells expressing wild-type NaV1.5, but increased INa in HEK293 cells expressing NaV1.5-K1479A. Dietary supplementation with NR for 10-12 weeks decreased QTc in C57BL/6 J mice (0.35% NR: -4.9 ± 2.0%, p = .14; 1.0% NR: -9.5 ± 2.8%, p = .01). CONCLUSIONS: NAD+ precursors differentially regulate NaV1.5 via multiple mechanisms. NR increases INa, decreases INa,L, and warrants further investigation as a potential therapy for arrhythmic disorders caused by NaV1.5 deficiency and/or dysfunction.

Elucidation of transcriptome-wide microRNA binding sites in human cardiac tissues by Ago2 HITS-CLIP.

MicroRNAs (miRs) have emerged as key biological effectors in human health and disease. These small noncoding RNAs are incorporated into Argonaute (Ago) proteins, where they direct post-transcriptional gene silencing via base-pairing with target transcripts. Although miRs have become intriguing biological entities and attractive therapeutic targets, the translational impacts of miR research remain limited by a paucity of empirical miR targeting data, particularly in human primary tissues. Here, to improve our understanding of the diverse roles miRs play in cardiovascular function and disease, we applied high-throughput methods to globally profile miR:target interactions in human heart tissues. We deciphered Ago2:RNA interactions using crosslinking immunoprecipitation coupled with high-throughput sequencing (HITS-CLIP) to generate the first transcriptome-wide map of miR targeting events in human myocardium, detecting 4000 cardiac Ago2 binding sites across >2200 target transcripts. Our initial exploration of this interactome revealed an abundance of miR target sites in gene coding regions, including several sites pointing to new miR-29 functions in regulating cardiomyocyte calcium, growth and metabolism. Also, we uncovered several clinically-relevant interactions involving common genetic variants that alter miR targeting events in cardiomyopathy-associated genes. Overall, these data provide a critical resource for bolstering translational miR research in heart, and likely beyond.

Inhibition of MCU forces extramitochondrial adaptations governing physiological and pathological stress responses in heart.

Myocardial mitochondrial Ca(2+) entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca(2+) are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca(2+) uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca(2+) entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca(2+) were not increased compared with WT, suggesting that DN-MCU expression increased OCR by enhanced energetic demands related to extramitochondrial Ca(2+) homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca(2+)] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca(2+) homeostasis. Mitochondrial Ca(2+) overload is thought to dissipate the inner mitochondrial membrane potential (ΔΨm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved ΔΨm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared with WT. Taken together, our findings show that chronic myocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca(2+) homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent anticipated therapeutic responses to ischemia-reperfusion injury.

A microRNA processing defect in smokers' macrophages is linked to SUMOylation of the endonuclease DICER.

Despite the fact that alveolar macrophages play an important role in smoking-related disease, little is known about what regulates their pathophysiologic phenotype. Evaluating smoker macrophages, we found significant down-regulation of multiple microRNAs (miRNAs). This work investigates the hypothesis that cigarette smoke alters mature miRNA expression in lung macrophages by inhibiting processing of primary miRNA transcripts. Studies on smoker alveolar macrophages showed a defect in miRNA maturation. Studies on the miRNA biogenesis machinery led us to focus on the cytosolic RNA endonuclease, DICER. DICER cleaves the stem-loop structure from pre-miRNAs, allowing them to dissociate into their mature 20-22-nucleotide single-stranded form. DICER activity assays confirmed impaired DICER activity following cigarette smoke exposure. Further protein studies demonstrated a decreased expression of the native 217-kDa form of DICER and an accumulation of high molecular weight forms with cigarette smoke exposure. This molecular mass shift was shown to contain SUMO moieties and could be blocked by silencing RNA directed at the primary SUMOylating ligase, Ubc9. In determining the cigarette smoke components responsible for changes in DICER, we found that N-acetylcysteine, an antioxidant and anti-aldehyde, protected DICER protein and activity from cigarette smoke extract. This massive down-regulation of miRNAs (driven in part by alterations in DICER) may be an important regulator of the disease-promoting macrophage phenotype found in the lungs of smokers.

Broad therapeutic benefit after RNAi expression vector delivery to deep cerebellar nuclei: implications for spinocerebellar ataxia type 1 therapy.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal dominant, late-onset neurodegenerative disease caused by a polyglutamine (polyQ) expansion in the ataxin-1 protein, which causes progressive neurodegeneration in cerebellar Purkinje cells and brainstem nuclei. Here, we tested if reducing mutant ataxin-1 expression would significantly improve phenotypes in a knock-in (KI) mouse model that recapitulates spatial and temporal aspects of SCA1. Adeno-associated viruses (AAVs), expressing inhibitory RNAs targeting ataxin-1, were injected into the deep cerebellar nuclei (DCN) of KI mice. This approach induced ataxin-1 suppression in the cerebellar cortex and in brainstem neurons. RNA interference (RNAi) of ataxin-1 preserved cerebellar lobule integrity and prevented disease-related transcriptional changes for over a year. Notably, RNAi therapy also preserved rotarod performance and neurohistology. These data suggest that delivery of AAVs encoding RNAi sequences against ataxin-1, to DCN alone, may be sufficient for SCA1 therapy.

Nonallele specific silencing of ataxin-7 improves disease phenotypes in a mouse model of SCA7.

Spinocerebellar ataxia type 7 (SCA7) is a late-onset neurodegenerative disease characterized by ataxia and vision loss with no effective treatments in the clinic. The most striking feature is the degeneration of Purkinje neurons of the cerebellum caused by the presence of polyglutamine-expanded ataxin-7. Ataxin-7 is part of a transcriptional complex, and, in the setting of mutant ataxin-7, there is misregulation of target genes. Here, we designed RNAi sequences to reduce the expression of both wildtype and mutant ataxin-7 to test if reducing ataxin-7 in Purkinje cells is both tolerated and beneficial in an animal model of SCA7. We observed sustained reduction of both wildtype and mutant ataxin-7 as well as a significant improvement of ataxia phenotypes. Furthermore, we observed a reduction in cerebellar molecular layer thinning and nuclear inclusions, a hallmark of SCA7. In addition, we observed recovery of cerebellar transcripts whose expression is disrupted in the presence of mutant ataxin-7. These data demonstrate that reduction of both wildtype and mutant ataxin-7 by RNAi is well tolerated, and contrary to what may be expected from reducing a component of the Spt-Taf9-Gcn5 acetyltransferase complex, is efficacious in the SCA7 mouse.

siSPOTR: a tool for designing highly specific and potent siRNAs for human and mouse.

RNA interference (RNAi) serves as a powerful and widely used gene silencing tool for basic biological research and is being developed as a therapeutic avenue to suppress disease-causing genes. However, the specificity and safety of RNAi strategies remains under scrutiny because small inhibitory RNAs (siRNAs) induce off-target silencing. Currently, the tools available for designing siRNAs are biased toward efficacy as opposed to specificity. Prior work from our laboratory and others' supports the potential to design highly specific siRNAs by limiting the promiscuity of their seed sequences (positions 2-8 of the small RNA), the primary determinant of off-targeting. Here, a bioinformatic approach to predict off-targeting potentials was established using publically available siRNA data from more than 50 microarray experiments. With this, we developed a specificity-focused siRNA design algorithm and accompanying online tool which, upon validation, identifies candidate sequences with minimal off-targeting potentials and potent silencing capacities. This tool offers researchers unique functionality and output compared with currently available siRNA design programs. Furthermore, this approach can greatly improve genome-wide RNAi libraries and, most notably, provides the only broadly applicable means to limit off-targeting from RNAi expression vectors.

Silencing mutant ATXN3 expression resolves molecular phenotypes in SCA3 transgenic mice.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disease caused by a polyglutamine expansion in the deubiquitinating enzyme, Ataxin-3. Currently, there are no effective treatments for this fatal disorder but studies support the hypothesis that reducing mutant Ataxin-3 protein levels might reverse or halt the progression of disease in SCA3. Here, we sought to modulate ATXN3 expression in vivo using RNA interference. We developed artificial microRNA mimics targeting the 3'-untranslated region (3'UTR) of human ATXN3 and then used recombinant adeno-associated virus to deliver them to the cerebellum of transgenic mice expressing the full human disease gene (SCA3/MJD84.2 mice). Anti-ATXN3 microRNA mimics effectively suppressed human ATXN3 expression in SCA3/MJD84.2 mice. Short-term treatment cleared the abnormal nuclear accumulation of mutant Ataxin-3 throughout the transduced SCA3/MJD84.2 cerebellum. Analysis also revealed changes in the steady-state levels of specific microRNAs in the cerebellum of SCA3/MJD84.2 mice, a previously uncharacterized molecular phenotype of SCA3 that appears to be dependent on mutant Ataxin-3 expression. Our findings support the preclinical development of molecular therapies aimed at halting the expression of ATXN3 as a viable approach to SCA3 and point to microRNA deregulation as a potential surrogate marker of SCA3 pathogenesis.

Fndc5 is translated from an upstream ATG start codon and cleaved to produce irisin myokine precursor protein in humans and mice.

Witmer et al. provide genomic and molecular evidence to demonstrate that Fndc5 (irisin myokine precursor protein) is translated in humans from an overlooked upstream ATG codon.

Mitoregulin supports mitochondrial membrane integrity and protects against cardiac ischemia-reperfusion injury.

We and others discovered a highly-conserved mitochondrial transmembrane microprotein, named Mitoregulin (Mtln), that supports lipid metabolism. We reported that Mtln strongly binds cardiolipin (CL), increases mitochondrial respiration and Ca 2+ retention capacities, and reduces reactive oxygen species (ROS). Here we extend our observation of Mtln-CL binding and examine Mtln influence on cristae structure and mitochondrial membrane integrity during stress. We demonstrate that mitochondria from constitutive- and inducible Mtln-knockout (KO) mice are susceptible to membrane freeze-damage and that this can be rescued by acute Mtln re-expression. In mitochondrial-simulated lipid monolayers, we show that synthetic Mtln decreases lipid packing and monolayer elasticity. Lipidomics revealed that Mtln-KO heart tissues show broad decreases in 22:6-containing lipids and increased cardiolipin damage/remodeling. Lastly, we demonstrate that Mtln-KO mice suffer worse myocardial ischemia-reperfusion injury, hinting at a translationally-relevant role for Mtln in cardioprotection. Our work supports a model in which Mtln binds cardiolipin and stabilizes mitochondrial membranes to broadly influence diverse mitochondrial functions, including lipid metabolism, while also protecting against stress.

A Specialized Centrosome-Proteasome Axis Mediates Proteostasis and Influences Cardiac Stress through Txlnb.

Background: Centrosomes localize to perinuclear foci where they serve multifunctional roles, arranging the microtubule organizing center (MTOC) and anchoring ubiquitin-proteasome system (UPS) machinery. In mature cardiomyocytes, centrosomal proteins redistribute into a specialized perinuclear cage-like structure, and a potential centrosome-UPS interface has not been studied. Taxilin-beta (Txlnb), a cardiomyocyte-enriched protein, belongs to a family of centrosome adapter proteins implicated in protein quality control. We hypothesize that Txlnb plays a key role in centrosomal-proteasomal crosstalk in cardiomyocytes. Methods: Integrative bioinformatics assessed centrosomal gene dysregulation in failing hearts. Txlnb gain/loss-of-function studies were conducted in cultured cardiomyocytes and mice. Txlnb's role in cardiac proteotoxicity and hypertrophy was examined using CryAB-R120G mice and transverse aortic constriction (TAC), respectively. Molecular modeling investigated Txlnb structure/function. Results: Human failing hearts show consistent dysregulation of many centrosome-associated genes, alongside UPS-related genes. Txlnb emerged as a candidate regulator of cardiomyocyte proteostasis that localizes to the perinuclear centrosomal compartment. Txlnb's interactome strongly supports its involvement in cytoskeletal, microtubule, and UPS processes, particularly centrosome-related functions. Overexpressing Txlnb in cardiomyocytes reduced ubiquitinated protein accumulation and enhanced proteasome activity during hypertrophy. Txlnb-knockout (KO) mouse hearts exhibit proteasomal insufficiency and altered cardiac growth, evidenced by ubiquitinated protein accumulation, decreased 26Sß5 proteasome activity, and lower mass with age. In Cryab-R120G mice, Txlnb loss worsened heart failure, causing lower ejection fractions. After TAC, Txlnb-KO mice also showed reduced ejection fraction, increased heart mass, and elevated ubiquitinated protein accumulation. Investigations into the molecular mechanisms revealed that Txlnb-KO did not affect proteasomal subunit expression but led to the upregulation of Txlna and several centrosomal proteins (Cep63, Ofd1, and Tubg) suggesting altered centrosomal dynamics. Structural predictions support Txlnb's role as a specialized centrosomal-adapter protein bridging centrosomes with proteasomes, confirmed by microtubule-dependent perinuclear localization. Conclusions: Together, these data provide initial evidence connecting Txlnb to cardiac proteostasis, hinting at the potential importance of functional bridging between specialized centrosomes and UPS in cardiomyocytes.

RNAi or overexpression: alternative therapies for Spinocerebellar Ataxia Type 1.

Spinocerebellar Ataxia Type 1 (SCA1) is an autosomal dominant late onset neurodegenerative disease caused by an expanded polyglutamine tract in ataxin-1. Here, we compared the protective effects of overexpressing ataxin-1-like using recombinant AAVs, or reducing expression of mutant ataxin-1 using virally delivered RNA interference (RNAi), in a transgenic mouse model of SCA1. For the latter, we used an artificial microRNA (miR) design that optimizes potency, efficacy and safety to suppress ataxin-1 expression (miS1). Delivery of either ataxin-1-like or miS1 viral vectors to SCA1 mice cerebella resulted in widespread cerebellar Purkinje cell transduction and improved behavioral and histological phenotypes. Our data indicate the utility of either approach as a possible therapy for SCA1 patients.

RNAi medicine for the brain: progresses and challenges.

RNAi interference (RNAi) is a powerful gene silencing technology that has immense potential for treating a vast array of human ailments, for which suppressing disease-associated genes may provide clinical benefit. Here, we review the development of RNAi as a therapeutic modality for neurodegenerative diseases affecting the central nervous system (CNS). We overview promising preclinical data for the application of RNAi in the CNS and discuss key challenges (e.g. delivery and specificity) that remain as these approaches transition to the clinic.

Modulation of miR-29 influences myocardial compliance likely through coordinated regulation of calcium handling and extracellular matrix.

MicroRNAs (miRNAs) control the expression of diverse subsets of target mRNAs, and studies have found miRNA dysregulation in failing hearts. Expression of miR-29 is abundant in heart, increases with aging, and is altered in cardiomyopathies. Prior studies demonstrate that miR-29 reduction via genetic knockout or pharmacologic blockade can blunt cardiac hypertrophy and fibrosis in mice. Surprisingly, this depended on specifically blunting miR-29 actions in cardiomyocytes versus fibroblasts. To begin developing more translationally relevant vectors, we generated a novel transgene-encoded miR-29 inhibitor (TuD-29) that can be incorporated into a viral-mediated gene therapy for cardioprotection. Here, we corroborate that miR-29 expression and activity is higher in cardiomyocytes versus fibroblasts and demonstrate that TuD-29 effectively blunts hypertrophic responses in cultured cardiomyocytes and mouse hearts. Furthermore, we found that adeno-associated virus (AAV)-mediated miR-29 overexpression in mouse hearts induces early diastolic dysfunction, whereas AAV:TuD-29 treatment improves cardiac output by increasing end-diastolic and stroke volumes. The integration of RNA sequencing and miRNA-target interactomes reveals that miR-29 regulates genes involved in calcium handling, cell stress and hypertrophy, metabolism, ion transport, and extracellular matrix remodeling. These investigations support a likely versatile role for miR-29 in influencing myocardial compliance and relaxation, potentially providing a unique therapeutic avenue to improve diastolic function in heart failure patients.

Dengue Virus Infection Alters Inter-Endothelial Junctions and Promotes Endothelial-Mesenchymal-Transition-Like Changes in Human Microvascular Endothelial Cells.

Dengue virus (DENV) is a pathogenic arbovirus that causes human disease. The most severe stage of the disease (severe dengue) is characterized by vascular leakage, hypovolemic shock, and organ failure. Endothelial dysfunction underlies these phenomena, but the causal mechanisms of endothelial dysfunction are poorly characterized. This study investigated the role of c-ABL kinase in DENV-induced endothelial dysfunction. Silencing c-ABL with artificial miRNA or targeting its catalytic activity with imatinib revealed that c-ABL is required for the early steps of DENV infection. DENV-2 infection and conditioned media from DENV-infected cells increased endothelial expression of c-ABL and CRKII phosphorylation, promoted expression of mesenchymal markers, e.g., vimentin and N-cadherin, and decreased the levels of endothelial-specific proteins, e.g., VE-cadherin and ZO-1. These effects were reverted by silencing or inhibiting c-ABL. As part of the acquisition of a mesenchymal phenotype, DENV infection and treatment with conditioned media from DENV-infected cells increased endothelial cell motility in a c-ABL-dependent manner. In conclusion, DENV infection promotes a c-ABL-dependent endothelial phenotypic change that leads to the loss of intercellular junctions and acquisition of motility.

Correction: Escudero-Flórez et al. Dengue Virus Infection Alters Inter-Endothelial Junctions and Promotes Endothelial-Mesenchymal-Transition-like Changes in Human Microvascular Endothelial Cells. Viruses 2023, 15, 1437.

Ryan L [...].

Control of hepatic nuclear superoxide production by glucose 6-phosphate dehydrogenase and NADPH oxidase-4.

Redox-regulated signal transduction is coordinated by spatially controlled production of reactive oxygen species within subcellular compartments. The nucleus has long been known to produce superoxide (O(2)(·-)); however, the mechanisms that control this function remain largely unknown. We have characterized molecular features of a nuclear superoxide-producing system in the mouse liver. Using electron paramagnetic resonance, we investigated whether several NADPH oxidases (NOX1, 2, and 4) and known activators of NOX (Rac1, Rac2, p22(phox), and p47(phox)) contribute to nuclear O(2)(·-) production in isolated hepatic nuclei. Our findings demonstrate that NOX4 most significantly contributes to hepatic nuclear O(2)(·-) production that utilizes NADPH as an electron donor. Although NOX4 protein immunolocalized to both nuclear membranes and intranuclear inclusions, fluorescent detection of NADPH-dependent nuclear O(2)(·-) predominantly localized to the perinuclear space. Interestingly, NADP(+) and G6P also induced nuclear O(2)(·-) production, suggesting that intranuclear glucose-6-phosphate dehydrogenase (G6PD) can control NOX4 activity through nuclear NADPH production. Using G6PD mutant mice and G6PD shRNA, we confirmed that reductions in nuclear G6PD enzyme decrease the ability of hepatic nuclei to generate O(2)(·-) in response to NADP(+) and G6P. NOX4 and G6PD protein were also observed in overlapping microdomains within the nucleus. These findings provide new insights on the metabolic pathways for substrate regulation of nuclear O(2)(·-) production by NOX4.

Rational design of therapeutic siRNAs: minimizing off-targeting potential to improve the safety of RNAi therapy for Huntington's disease.

RNA interference (RNAi) provides an approach for the treatment of many human diseases. However, the safety of RNAi-based therapies can be hampered by the ability of small inhibitory RNAs (siRNAs) to bind to unintended mRNAs and reduce their expression, an effect known as off-target gene silencing. Off-targeting primarily occurs when the seed region (nucleotides 2-8 of the small RNA) pairs with sequences in 3'-UTRs of unintended mRNAs and directs translational repression and destabilization of those transcripts. To date, most therapeutic RNAi sequences are selected primarily for gene silencing efficacy, and later evaluated for safety. Here, in designing siRNAs to treat Huntington's disease (HD), a dominant neurodegenerative disorder, we prioritized selection of sequences with minimal off-targeting potentials (i.e., those with a scarcity of seed complements within all known human 3'-UTRs). We identified new promising therapeutic candidate sequences which show potent silencing in cell culture and mouse brain. Furthermore, we present microarray data demonstrating that off-targeting is significantly minimized by using siRNAs that contain "safe" seeds, an important strategy to consider during preclinical development of RNAi-based therapeutics.

Preclinical safety of RNAi-mediated HTT suppression in the rhesus macaque as a potential therapy for Huntington's disease.

To date, a therapy for Huntington's disease (HD), a genetic, neurodegenerative disorder, remains elusive. HD is characterized by cell loss in the basal ganglia, with particular damage to the putamen, an area of the brain responsible for initiating and refining motor movements. Consequently, patients exhibit a hyperkinetic movement disorder. RNA interference (RNAi) offers therapeutic potential for this disorder by reducing the expression of HTT, the disease-causing gene. We have previously demonstrated that partial suppression of both wild-type and mutant HTT in the striatum prevents behavioral and neuropathological abnormalities in rodent models of HD. However, given the role of HTT in various cellular processes, it remains unknown whether a partial suppression of both alleles will be safe in mammals whose neurophysiology, basal ganglia anatomy, and behavioral repertoire more closely resembles that of a human. Here, we investigate whether a partial reduction of HTT in the normal non-human primate putamen is safe. We demonstrate that a 45% reduction of rhesus HTT expression in the mid- and caudal putamen does not induce motor deficits, neuronal degeneration, astrogliosis, or an immune response. Together, these data suggest that partial suppression of wild-type HTT expression is well tolerated in the primate putamen and further supports RNAi as a therapy for HD.