Chronological and biological aging of the human left ventricular myocardium: Analysis of microRNAs contribution.

Aging is the main risk factor for cardiovascular diseases. In humans, cardiac aging remains poorly characterized. Most studies are based on chronological age (CA) and disregard biological age (BA), the actual physiological age (result of the aging rate on the organ structure and function), thus yielding potentially imperfect outcomes. Deciphering the molecular basis of ventricular aging, especially by BA, could lead to major progresses in cardiac research. We aim to describe the transcriptome dynamics of the aging left ventricle (LV) in humans according to both CA and BA and characterize the contribution of microRNAs, key transcriptional regulators. BA is measured using two CA-associated transcriptional markers: CDKN2A expression, a cell senescence marker, and apparent age (AppAge), a highly complex transcriptional index. Bioinformatics analysis of 132 LV samples shows that CDKN2A expression and AppAge represent transcriptomic changes better than CA. Both BA markers are biologically validated in relation to an aging phenotype associated with heart dysfunction, the amount of cardiac fibrosis. BA-based analyses uncover depleted cardiac-specific processes, among other relevant functions, that are undetected by CA. Twenty BA-related microRNAs are identified, and two of them highly heart-enriched that are present in plasma. We describe a microRNA-gene regulatory network related to cardiac processes that are partially validated in vitro and in LV samples from living donors. We prove the higher sensitivity of BA over CA to explain transcriptomic changes in the aging myocardium and report novel molecular insights into human LV biological aging. Our results can find application in future therapeutic and biomarker research.

Minimally invasive system to reliably characterize ventricular electrophysiology from living donors.

Cardiac tissue slices preserve the heterogeneous structure and multicellularity of the myocardium and allow its functional characterization. However, access to human ventricular samples is scarce. We aim to demonstrate that slices from small transmural core biopsies collected from living donors during routine cardiac surgery preserve structural and functional properties of larger myocardial specimens, allowing accurate electrophysiological characterization. In pigs, we compared left ventricular transmural core biopsies with transmural tissue blocks from the same ventricular region. In humans, we analyzed transmural biopsies and papillary muscles from living donors. All tissues were vibratome-sliced. By histological analysis of the transmural biopsies, we showed that tissue architecture and cellular organization were preserved. Enzymatic and vital staining methods verified viability. Optically mapped transmembrane potentials confirmed that action potential duration and morphology were similar in pig biopsies and tissue blocks. Action potential morphology and duration in human biopsies and papillary muscles agreed with published ranges. In both pigs and humans, responses to increasing pacing frequencies and ß-adrenergic stimulation were similar in transmural biopsies and larger tissues. We show that it is possible to successfully collect and characterize tissue slices from human myocardial biopsies routinely extracted from living donors, whose behavior mimics that of larger myocardial preparations both structurally and electrophysiologically.

Single-cell analyses reveal an attenuated NF-κB response in the Salmonella-infected fibroblast.

The eukaryotic transcriptional regulator Nuclear Factor kappa B (NF-κB) plays a central role in the defense to pathogens. Despite this, few studies have analyzed NF-κB activity in single cells during infection. Here, we investigated at the single cell level how NF-κB nuclear localization - a proxy for NF-κB activity - oscillates in infected and uninfected fibroblasts co-existing in cultures exposed to Salmonella enterica serovar Typhimurium. Fibroblasts were used due to the capacity of S. Typhimurium to persist in this cell type. Real-time dynamics of NF-κB was examined in microfluidics, which prevents cytokine accumulation. In this condition, infected (ST+) cells translocate NF-κB to the nucleus at higher rate than the uninfected (ST-) cells. Surprisingly, in non-flow (static) culture conditions, ST- fibroblasts exhibited higher NF-κB nuclear translocation than the ST+ population, with these latter cells turning refractory to external stimuli such as TNF-α or a second infection. Sorting of ST+ and ST- cell populations confirmed enhanced expression of NF-κB target genes such as IL1B, NFKBIA, TNFAIP3, and TRAF1 in uninfected (ST-) fibroblasts. These observations proved that S. Typhimurium dampens the NF-κB response in the infected fibroblast. Higher expression of SOCS3, encoding a "suppressor of cytokine signaling," was also observed in the ST+ population. Intracellular S. Typhimurium subverts NF-κB activity using protein effectors translocated by the secretion systems encoded by pathogenicity islands 1 (T1) and 2 (T2). T1 is required for regulating expression of SOCS3 and all NF-κB target genes analyzed whereas T2 displayed no role in the control of SOCS3 and IL1B expression. Collectively, these data demonstrate that S. Typhimurium attenuates NF-κB signaling in fibroblasts, an effect only perceptible when ST+ and ST- populations are analyzed separately. This tune-down in a central host defense might be instrumental for S. Typhimurium to establish intracellular persistent infections.

Intracellular Salmonella induces aggrephagy of host endomembranes in persistent infections.

Xenophagy has been studied in epithelial cells infected with Salmonella enterica serovar Typhimurium (S. Typhimurium). Distinct autophagy receptors target this pathogen to degradation after interacting with ubiquitin on the surface of cytosolic bacteria, and the phagophore- and autophagosome-associated protein MAP1LC3/LC3. Glycans exposed in damaged phagosomal membranes and diacylglycerol accumulation in the phagosomal membrane also trigger S. Typhimurium xenophagy. How these responses control intraphagosomal and cytosolic bacteria remains poorly understood. Here, we examined S. Typhimurium interaction with autophagy in fibroblasts, in which the pathogen displays limited growth and does not escape into the cytosol. Live-cell imaging microscopy revealed that S. Typhimurium recruits late endosomal or lysosomal compartments that evolve into a membranous aggregate connected to the phagosome. Active dynamics and integrity of the phagosomal membrane are requisite to induce such aggregates. This membranous structure increases over time to become an aggresome that engages autophagy machinery at late infection times (> 6 h postentry). The newly formed autophagosome harbors LC3 and the autophagy receptor SQSTM1/p62 but is devoid of ubiquitin and the receptor CALCOCO2/NDP52. Live-cell imaging showed that this autophagosome captures and digests within the same vacuole the aggresome and some apposed intraphagosomal bacteria. Other phagosomes move away from the aggresome and avoid destruction. Thus, host endomembrane accumulation resulting from activity of intracellular S. Typhimurium stimulates a novel type of aggrephagy that acts independently of ubiquitin and CALCOCO2, and destroys only a few bacteria. Such selective degradation might allow the pathogen to reduce its progeny and, as a consequence, to establish persistent infections.