Empagliflozin reduces arrhythmogenic effects in rat neonatal and human iPSC-derived cardiomyocytes and improves cytosolic calcium handling at least partially independent of NHE1.

The antidiabetic agent class of sodium-glucose cotransporter 2 (SGLT2) inhibitors confer unprecedented cardiovascular benefits beyond glycemic control, including reducing the risk of fatal ventricular arrhythmias. However, the impact of SGLT2 inhibitors on the electrophysiological properties of cardiomyocytes exposed to stimuli other than hyperglycemia remains elusive. This investigation tested the hypothesis that the SGLT2 inhibitor empagliflozin (EMPA) affects cardiomyocyte electrical activity under hypoxic conditions. Rat neonatal and human induced pluripotent stem cell (iPSC)-derived cardiomyocytes incubated or not with the hypoxia-mimetic agent CoCl2 were treated with EMPA (1 µM) or vehicle for 24 h. Action potential records obtained using intracellular microelectrodes demonstrated that EMPA reduced the action potential duration at 30%, 50%, and 90% repolarization and arrhythmogenic events in rat and human cardiomyocytes under normoxia and hypoxia. Analysis of Ca2+ transients using Fura-2-AM and contractility kinetics showed that EMPA increased Ca2+ transient amplitude and decreased the half-time to recover Ca2+ transients and relaxation time in rat neonatal cardiomyocytes. We also observed that the combination of EMPA with the Na+/H+ exchanger isoform 1 (NHE1) inhibitor cariporide (10 µM) exerted a more pronounced effect on Ca2+ transients and contractility than either EMPA or cariporide alone. Besides, EMPA, but not cariporide, increased phospholamban phosphorylation at serine 16. Collectively, our data reveal that EMPA reduces arrhythmogenic events, decreases the action potential duration in rat neonatal and human cardiomyocytes under normoxic or hypoxic conditions, and improves cytosolic calcium handling at least partially independent of NHE1. Moreover, we provided further evidence that SGLT2 inhibitor-mediated cardioprotection may be partly attributed to its cardiomyocyte electrophysiological effects.

Time-regulated transcripts with the potential to modulate human pluripotent stem cell-derived cardiomyocyte differentiation.

BACKGROUND: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) are a promising disease model, even though hiPSC-CMs cultured for extended periods display an undifferentiated transcriptional landscape. MiRNA-target gene interactions contribute to fine-tuning the genetic program governing cardiac maturation and may uncover critical pathways to be targeted. METHODS: We analyzed a hiPSC-CM public dataset to identify time-regulated miRNA-target gene interactions based on three logical steps of filtering. We validated this process in silico using 14 human and mouse public datasets, and further confirmed the findings by sampling seven time points over a 30-day protocol with a hiPSC-CM clone developed in our laboratory. We then added miRNA mimics from the top eight miRNAs candidates in three cell clones in two different moments of cardiac specification and maturation to assess their impact on differentiation characteristics including proliferation, sarcomere structure, contractility, and calcium handling. RESULTS: We uncovered 324 interactions among 29 differentially expressed genes and 51 miRNAs from 20,543 transcripts through 120 days of hiPSC-CM differentiation and selected 16 genes and 25 miRNAs based on the inverse pattern of expression (Pearson R-values < - 0.5) and consistency in different datasets. We validated 16 inverse interactions among eight genes and 12 miRNAs (Person R-values < - 0.5) during hiPSC-CMs differentiation and used miRNAs mimics to verify proliferation, structural and functional features related to maturation. We also demonstrated that miR-124 affects Ca2+ handling altering features associated with hiPSC-CMs maturation. CONCLUSION: We uncovered time-regulated transcripts influencing pathways affecting cardiac differentiation/maturation axis and showed that the top-scoring miRNAs indeed affect primarily structural features highlighting their role in the hiPSC-CM maturation.

NOTCH1 is critical for fibroblast-mediated induction of cardiomyocyte specialization into ventricular conduction system-like cells in vitro.

Cardiac fibroblasts are present throughout the myocardium and are enriched in the microenvironment surrounding the ventricular conduction system (VCS). Several forms of arrhythmias are linked to VCS abnormalities, but it is still unclear whether VCS malformations are cardiomyocyte autonomous or could be linked to crosstalk between different cell types. We reasoned that fibroblasts influence cardiomyocyte specialization in VCS cells. We developed 2D and 3D culture models of neonatal rat cardiac cells to assess the influence of cardiac fibroblasts on cardiomyocytes. Cardiomyocytes adjacent to cardiac fibroblasts showed a two-fold increase in expression of VCS markers (NAV1.5 and CONTACTIN 2) and calcium transient duration, displaying a Purkinje-like profile. Fibroblast-conditioned media (fCM) was sufficient to activate VCS-related genes (Irx3, Scn5a, Connexin 40) and to induce action potential prolongation, a hallmark of Purkinge phenotype. fCM-mediated response seemed to be spatially-dependent as cardiomyocyte organoids treated with fCM had increased expression of connexin 40 and NAV1.5 primarily on its outer surface. Finally, NOTCH1 activation in both cardiomyocytes and fibroblasts was required for connexin 40 up-regulation (a proxy of VCS phenotype). Altogether, we provide evidence that cardiac fibroblasts influence cardiomyocyte specialization into VCS-like cells via NOTCH1 signaling in vitro.

Novel GAA mutations in patients with Pompe disease.

Pompe disease is an autosomal recessive disorder linked to GAA gene that leads to a multi-system intralysosomal accumulation of glycogen. Mutation identification in the GAA gene can be very important for early diagnosis, correlation between genotype-phenotype and therapeutic intervention. For this purpose, peripheral blood from 57 individuals susceptible to Pompe disease was collected and all exons of GAA gene were amplified; the sequences and the mutations were analyzed in silico to predict possible impact on the structure and function of the human protein. In this study, 46 individuals presented 33 alterations in the GAA gene sequence, among which five (c.547-67C>G, c.547-39T>G, p.R437H, p.L641V and p.L705P) have not been previously described in the literature. The alterations in the coding region included 15 missense mutations, three nonsense mutations and one deletion. One insertion and other 13 single base changes were found in the non-coding region. The mutation p.G611D was found in homozygosis in a one-year-old child, who presented low levels of GAA activity, hypotonia and hypertrophic cardiomyopathy. Two patients presented the new mutation p.L705P in association with c.-32-13T>G. They had low levels of GAA activity and developed late onset Pompe disease. In our study, we observed alterations in the GAA gene originating from Asians, African-Americans and Caucasians, highlighting the high heterogeneity of the Brazilian population. Considering that Pompe disease studies are not very common in Brazil, this study will help to better understand the potential pathogenic role of each change in the GAA gene. Furthermore, a precise and early molecular analysis improves genetic counseling besides allowing for a more efficient treatment in potential candidates.

Fibronectin expression is decreased in metastatic renal cell carcinoma following endostatin gene therapy.

Tumor cells induce the disruption of homeostasis between cellular and extracellular compartments to favor tumor progression. The expression of fibronectin (FN), a matrix glycoprotein, is increased in several carcinoma cell types, including renal cell carcinoma (RCC). RCC are highly vascularized tumors and are often amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the modulation of FN gene expression by ES gene therapy in a murine metastatic renal cell carcinoma (mRCC) model. Balb/C mice bearing Renca cells were treated with NIH/3T3-LXSN cells or NIH/3T3-LendSN cells. At the end of the experiment, the ES serum levels were measured, and the FN gene expression was assessed using real-time PCR. The tissue FN was evaluated by western blotting and by immunofluorescence analysis. The ES serum levels in treated mice were higher than those in the control group (P<0.05). ES treatment led to significant decreases at the FN mRNA (P<0.001) and protein levels (P<0.01). Here, we demonstrate the ES antitumor effect that is mediated by down-regulation of FN expression in mRCC.

New mutations in the GLA gene in Brazilian families with Fabry disease.

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the alpha-galactosidase A (GLA) gene. Evaluating the enzymatic activity in male individuals usually performs the diagnosis of the disease, but in female carriers the diagnosis based only on enzyme assays is often inconclusive. In this work, we analyzed 568 individuals from 102 families with suspect of FD. Overall, 51 families presented 38 alterations in the GLA gene, among which 19 were not previously reported in literature. The alterations included 17 missense mutations, 7 nonsense mutations, 7 deletions, 6 insertions and 1 in the splice site. Six alterations (R112C, R118C, R220X, R227X, R342Q and R356W) occurred at CpG dinucleotides. Five mutations not previously described in the literature (A156D, K237X, A292V, I317S, c.1177_1178insG) were correlated with low GLA enzyme activity and with prediction of molecular damages. From the 13 deletions and insertions, 7 occurred in exons 6 or 7 (54%) and 11 led to the formation of a stop codon. The present study highlights the detection of new genomic alterations in the GLA gene in the Brazilian population, facilitating the selection of patients for recombinant enzyme-replacement trials and offering the possibility to perform prenatal diagnosis.