Neuroradiological findings in GAA-FGF14 ataxia (SCA27B): more than cerebellar atrophy.

Background: GAA-FGF14 ataxia (SCA27B) is a recently reported late-onset ataxia caused by a GAA repeat expansion in intron 1 of the FGF14 gene. Initial studies revealed cerebellar atrophy in 74-97% of patients. A more detailed brain imaging characterization of GAA-FGF14 ataxia is now needed to provide supportive diagnostic features and earlier disease recognition. Methods: We performed a retrospective review of the brain MRIs of 35 patients (median age at MRI 63 years; range 28-88 years) from Quebec (n=27), Nancy (n=3), Perth (n=3) and Bengaluru (n=2) to assess the presence of atrophy in vermis, cerebellar hemispheres, brainstem, cerebral hemispheres, and corpus callosum, as well as white matter involvement. Following the identification of the superior cerebellar peduncles (SCPs) involvement, we verified its presence in 54 GAA-FGF14 ataxia patients from four independent cohorts (Tübingen n=29; Donostia n=12; Innsbruck n=7; Cantabria n=6). To assess lobular atrophy, we performed quantitative cerebellar segmentation in 5 affected subjects with available 3D T1-weighted images and matched controls. Results: Cerebellar atrophy was documented in 33 subjects (94.3%). We observed SCP involvement in 22 subjects (62.8%) and confirmed this finding in 30/54 (55.6%) subjects from the validation cohorts. Cerebellar segmentation showed reduced mean volumes of lobules X and IV in the 5 affected individuals. Conclusions: Cerebellar atrophy is a key feature of GAA-FGF14 ataxia. The frequent SCP involvement observed in different cohorts may facilitate the diagnosis. The predominant involvement of lobule X correlates with the frequently observed downbeat nystagmus.

Quaking regulates circular RNA production in cardiomyocytes.

Circular RNAs (circRNAs) are a class of non-coding RNA molecules that are gaining increasing attention for their roles in various pathophysiological processes. The RNA-binding protein quaking (QKI) has been identified as a regulator of circRNA formation. In this study, we investigate the role of QKI in the formation of circRNAs in the heart by performing RNA-sequencing on Qki-knockout mice. Loss of QKI resulted in the differential expression of 17% of the circRNAs in adult mouse hearts. Interestingly, the majority of the QKI-regulated circRNAs (58%) were derived from genes undergoing QKI-dependent splicing, indicating a relationship between back-splicing and linear splicing. We compared these QKI-dependent circRNAs with those regulated by RBM20, another cardiac splicing factor essential for circRNA formation. We found that QKI and RBM20 regulate the formation of a distinct, but partially overlapping set of circRNAs in the heart. Strikingly, many shared circRNAs were derived from the Ttn gene, and they were regulated in an opposite manner. Our findings indicate that QKI not only regulates alternative splicing in the heart but also the formation of circRNAs.

Splicing factors in the heart: Uncovering shared and unique targets.

Alternative splicing generates specialized protein isoforms that allow the heart to adapt during development and disease. The recent discovery that mutations in the splicing factor RNA-binding protein 20 (RBM20) cause a severe form of familial dilated cardiomyopathy has sparked a great interest in alternative splicing in the field of cardiology. Since then, identification of splicing factors controlling alternative splicing in the heart has grown at a rapid pace. Despite the intriguing observation that a certain overlap exists between the targets of some splicing factors, an integrated and systematic analysis of their splicing networks is missing. Here, we compared the splicing networks of individual splicing factors by re-analyzing original RNA-sequencing data from eight previously published mouse models, in which a single splicing factor has been genetically deleted (i.e. HNRNPU, MBNL1/2, QKI, RBM20, RBM24, RBPMS, SRSF3, SRSF4). We show that key splicing events in Camk2d, Ryr2, Tpm1, Tpm2 and Pdlim5 require the combined action of the majority of these splicing factors. Additionally, we identified common targets and pathways among splicing factors, with the largest overlap between the splicing networks of MBNL, QKI and RBM24. We also re-analyzed a large-scale RNA-sequencing study on hearts of 128 heart failure patients. Here, we observed that MBNL1, QKI and RBM24 expression varied greatly. This variation in expression correlated with differential splicing of their downstream targets as found in mice, suggesting that aberrant splicing by MBNL1, QKI and RBM24 might contribute to the disease mechanism in heart failure.

The RNA-binding protein QKI governs a muscle-specific alternative splicing program that shapes the contractile function of cardiomyocytes.

AIMS: In the heart, splicing factors orchestrate the functional properties of cardiomyocytes by regulating the alternative splicing of multiple genes. Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the in vivo function of QKI in the adult mouse heart. METHODS AND RESULTS: We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes. CONCLUSION: Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes.

Knockdown of Ift88 in fibroblasts causes extracellular matrix remodeling and decreases conduction velocity in cardiomyocyte monolayers.

Background: Atrial fibrosis plays an important role in the development and persistence of atrial fibrillation by promoting reentry. Primary cilia have been identified as a regulator of fibroblasts (FB) activation and extracellular matrix (ECM) deposition. We hypothesized that selective reduction of primary cilia causes increased fibrosis and facilitates reentry. Aim: The aim of this study was to disrupt the formation of primary cilia in FB and examine its consequences on ECM and conduction in a co-culture system of cardiomyocytes (CM) and FB. Materials: Using short interfering RNA (siRNA), we removed primary cilia in neonatal rat ventricular FB by reducing the expression of Ift88 gene required for ciliary assembly. We co-cultured neonatal rat ventricular cardiomyocytes (CM) with FB previously transfected with Ift88 siRNA (siIft88) or negative control siRNA (siNC) for 48 h. We examined the consequences of ciliated fibroblasts reduction on conduction and tissue remodeling by performing electrical mapping, microelectrode, and gene expression measurements. Results: Transfection of FB with siIft88 resulted in a significant 60% and 30% reduction of relative Ift88 expression in FB and CM-FB co-cultures, respectively, compared to siNC. Knockdown of Ift88 significantly increased the expression of ECM genes Fn1, Col1a1 and Ctgf by 38%, 30% and 18%, respectively, in comparison to transfection with siNC. Conduction velocity (CV) was significantly decreased in the siIft88 group in comparison to siNC [11.12 ± 4.27 cm/s (n = 10) vs. 17.00 ± 6.20 (n = 10) respectively, p < 0.05]. The fraction of sites with interelectrode activation block was larger in the siIft88 group than in the siNC group (6.59 × 10-2 ± 8.01 × 10-2 vs. 1.18 × 10-2 ± 3.72 × 10-2 respectively, p < 0.05). We documented spontaneous reentrant arrhythmias in two cultures in the siIft88 group and in none of the siNC group. Action potentials were not significantly different between siNC and siIft88 groups. Conclusion: Disruption of cilia formation by siIft88 causes ECM remodeling and conduction abnormalities. Prevention of cilia loss could be a target for prevention of arrhythmias.

Secretome of atrial epicardial adipose tissue facilitates reentrant arrhythmias by myocardial remodeling.

BACKGROUND: Epicardial adipose tissue (EAT) accumulation is associated with cardiac arrhythmias. The effect of EAT secretome (EATs) on cardiac electrophysiology remains largely unknown. OBJECTIVE: The purpose of this study was to investigate the arrhythmogenicity of EATs and its underlying molecular and electrophysiological mechanisms. METHODS: We collected atrial EAT and subcutaneous adipose tissue (SAT) from 30 patients with atrial fibrillation (AF), and EAT from 3 donors without AF. The secretome was collected after a 24-hour incubation of the adipose tissue explants. We cultured neonatal rat ventricular myocytes (NRVMs) with EATs, subcutaneous adipose tissue secretome (SATs), and cardiomyocytes conditioned medium (CCM) for 72 hours. We implemented the electrophysiological changes observed after EATs incubation into a model of human left atrium and tested arrhythmia inducibility. RESULTS: Incubation of NRVMs with EATs decreased expression of the potassium channel subunit Kcnj2 by 26% and correspondingly reduced the inward rectifier K+ current IK1 by 35% compared to incubation with CCM, resulting in a depolarized resting membrane of cardiomyocytes. EATs decreased expression of connexin43 (29% mRNA, 46% protein) in comparison to CCM. Cells incubated with SATs showed no significant differences in Kcnj2 or Gja1 expression in comparison to CCM, and their resting potential was not depolarized. Cardiomyocytes incubated with EATs showed reduced conduction velocity and increased conduction heterogeneity compared to SATs and CCM. Computer modeling of human left atrium revealed that the electrophysiological changes induced by EATs promote sustained reentrant arrhythmias if EAT partially covers the myocardium. CONCLUSION: EAT slows conduction, depolarizes the resting potential, alters electrical cell-cell coupling, and facilitates reentrant arrhythmias.

Inhibition of minor intron splicing reduces Na+ and Ca2+ channel expression and function in cardiomyocytes.

Eukaryotic genomes contain a tiny subset of 'minor class' introns with unique sequence elements that require their own splicing machinery. These minor introns are present in certain gene families with specific functions, such as voltage-gated Na+ and voltage-gated Ca2+ channels. Removal of minor introns by the minor spliceosome has been proposed as a post-transcriptional regulatory layer, which remains unexplored in the heart. Here, we investigate whether the minor spliceosome regulates electrophysiological properties of cardiomyocytes by knocking down the essential minor spliceosome small nuclear snRNA component U6atac in neonatal rat ventricular myocytes. Loss of U6atac led to robust minor intron retention within Scn5a and Cacna1c, resulting in reduced protein levels of Nav1.5 and Cav1.2 channels. Functional consequences were studied through patch-clamp analysis, and revealed reduced Na+ and L-type Ca2+ currents after loss of U6atac. In conclusion, minor intron splicing modulates voltage-dependent ion channel expression and function in cardiomyocytes. This may be of particular relevance in situations in which minor splicing activity changes, such as in genetic diseases affecting minor spliceosome components, or in acquired diseases in which minor spliceosome components are dysregulated, such as heart failure.

COL4A1 Mutation as a Cause of Familial Recurrent Intracerebral Hemorrhage.

The COL4A1 mutation is a very rare monogenic cause of small vessel disease related to recurrent intracerebral hemorrhage. We report a family in which the index case presented with two intracerebral hemorrhages in the basal ganglia with severe periventricular leukoaraiosis and a cataract and vascular tortuosity in the ophthalmological study. His twin brother also had severe leukoaraiosis and multiple subcortical microhemorrhages as well as a congenital cataract and vascular tortuosity in the retina. The older sister had a porencephalic cyst and involvement of the periventricular white matter and intracerebral hemorrhage. In single-gene testing, all three were found to have the same COL4A1 mutation. Intracerebral subcortical hemorrhages or microhemorrhages and severe subcortical leukoaraiosis in familial cases may be related to COL4 mutations.

Effects of phytochemicals on thyroid function and their possible role in thyroid disease.

About 1 of 10 women, particularly those older than 60 years of age, shows some degree of thyroid hormone deficiency. Thyroid diseases are generally characterized by perturbations of thyroid signaling homeostasis. The most common examples of thyroid diseases include hypothyroidism, hyperthyroidism, and several types of thyroid cancers. Phytochemicals have been shown to have either beneficial or detrimental effects on thyroid function. Some flavonoids have been reported to affect the expression and the activity of several thyroid-related enzymes and proteins, and for this reason some concerns have been raised about the possible thyroid-disruptive properties of foods enriched in these substances. On the other hand, the beneficial effects of some plant-derived compounds, such as myricetin, quercetin, apigenin, rutin, genistein, and curcumin, and their possible role as adjuvants for the treatment of thyroid cancers have been described. Here, the role of phytochemicals in thyroid signaling modulation and their possible beneficial or detrimental effects on thyroid disease risk are discussed.

Phytochemical Composition and Cytotoxic Effects on Liver Hepatocellular Carcinoma Cells of Different Berries Following a Simulated In Vitro Gastrointestinal Digestion.

Berry fruits are rich in nutrients and polyphenols, providing potential health benefits. Understanding the factors that affect their bioavailability is becoming of utmost importance for evaluating their biological significance and efficacy as functional food. In this study, the phytochemical composition and the total antioxidant capacity of different varieties of five berries (blackberry, blackcurrant, blueberry, raspberry, and strawberry) were evaluated after an in vitro gastrointestinal digestion process. The cultivar of each berry that showed the higher content of total phenols and flavonoids was selected to study its cytotoxic effect on human hepatoma cells. Digestion resulted in a high reduction (p ˂ 0.05) of total phenolic, flavonoid and anthocyanin contents and total antioxidant capacity, in the "IN" samples compared to the "OUT" extracts, which represent the "serum-available" and the "colon-available" fractions, respectively. Incubation of the digested fraction for 24 h didn't exert any effect on cellular viability, while a dose- and time-dependent cytotoxicity was observed after 48 h and 72 h of incubation for all the berries analyzed. Our results suggest that the approach proposed in this work may represent a rapid tool for evaluating and identifying new berries with increased phytochemical bioavailability, highlighting their antiproliferative agents after an in vitro digestion.