Myocardial injury is a common complication of sepsis. MicroRNA (miRNA) miR-214-3p is protective against myocardial injury caused by sepsis, but its mechanism in lipopolysaccharide (LPS)- induced cardiomyocyte injury is still unclear. An AC16 cell injury model was induced by LPS treatment. Cell Counting Kit-8 and flow cytometry assay showed decreased cell viability and increased apoptosis in LPS-treated AC16 cells. The levels of caspase- 3, Bax, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), myosin 6 (Myh6), myosin 7 (Myh7), reactive oxygen species (ROS), and malondialdehyde (MDA) were increased in LPS-treated AC16 cells, but the levels of Bcl-2 and superoxide dismutase (SOD) were decreased. MiR-214-3p was down-regulated and cathepsin B (CTSB) was upregulated in LPS-treated AC16 cells. At the same time, miR-214-3p could target CTSB and reduce its expression. We also found that a miR-214-3p mimic or CTSB silencing could significantly reduce LPSinduced apoptosis, decrease ROS, MDA, caspase-3, and Bax and increase SOD and Bcl-2. CTSB silencing could significantly reduce ANP, BNP, Myh6, and Myh7 in LPS-treated AC16 cells. The effects of CTSB silencing were reversed by a miR-214-3p inhibitor. In summary, miR-214-3p could inhibit LPSinduced myocardial injury by targeting CTSB, which provides a new idea for myocardial damage caused by sepsis.
- MeSH
- Atrial Natriuretic Factor metabolism MeSH
- Myocytes, Cardiac * pathology MeSH
- Cathepsin B * genetics metabolism MeSH
- Humans MeSH
- Lipopolysaccharides MeSH
- MicroRNAs * genetics metabolism MeSH
- bcl-2-Associated X Protein metabolism MeSH
- Reactive Oxygen Species metabolism MeSH
- Sepsis * MeSH
- Superoxide Dismutase metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
The cardiac excitation-contraction coupling is the cellular process through which the heart absolves its blood pumping function, and it is directly affected when cardiac pathologies occur. Cardiomyocytes are the functional units in which this complex biomolecular process takes place: they can be represented as a two-stage electro-chemo and chemo-mechanical transducer, along which each stage can be probed and monitored via appropriate micro/nanotechnology-based tools. Atomic force microscopy (AFM), with its unique nanoresolved force sensitivity and versatile modes of extracting sample properties, can represent a key instrument to study time-dependent heart mechanics and topography at the single cell level. In this work, we show how the integrative possibilities of AFM allowed us to implement an in vitro system which can monitor cardiac electrophysiology, intracellular calcium dynamics, and single cell mechanics. We believe this single cell-sensitive and integrated system will unlock improved, fast, and reliable cardiac in vitro tests in the future.
- MeSH
- Data Analysis MeSH
- Electrophysiological Phenomena * MeSH
- Myocytes, Cardiac cytology physiology MeSH
- Mechanical Phenomena * MeSH
- Microscopy, Atomic Force * instrumentation methods MeSH
- Molecular Imaging MeSH
- Excitation Contraction Coupling * MeSH
- Calcium Signaling MeSH
- Publication type
- Journal Article MeSH
Coronary heart disease (CHD) is one of the most commonly seen cardiovascular conditions across the globe. Junctional cadherin 5 associated (JCAD) protein is found in the intercellular junctions of endothelial cells and linked to cardiovascular diseases. Nonetheless, the influence of JCAD on cardiomyocyte injury caused by CHD is unclear. A model of H2O2-induced H9c2 cell injury was constructed, and JCAD mRNA and protein levels were assessed by qRT-PCR and Western blot. The impacts of JCAD on the proliferation or apoptosis of H9c2 cells were explored by CCK-8 assay, Western blot and TUNEL staining. The effect of JCAD on the inflammatory response and vascular endothelial function of H9c2 cells was detected using ELISA kits. The levels of Wnt/β-catenin pathway-related proteins were assessed by Western blot. H2O2 treatment led to a rise in the levels of JCAD in H9c2 cells. Over-expression of JCAD promoted H2O2-induced cellular injury, leading to notably elevated contents of inflammatory factors, along with vascular endothelial dysfunction. In contrast to over-expression of JCAD, silencing of JCAD attenuated H2O2-induced cellular injury and inhibited apoptosis, inflammatory response and vascular endothelial dysfunction. Notably, JCAD could regulate the Wnt/β-catenin pathway, while DKK-1, Wnt/β-catenin pathway antagonist, counteracted the enhancing impact of JCAD over-expression on H2O2-induced H9c2 cell injury, further confirming that JCAD acts by regulating the Wnt/β-catenin pathway. In summary, over-expression of JCAD promoted H2O2-induced H9c2 cell injury by activating the Wnt/β-catenin pathway, while silencing of JCAD attenuated the H2O2-induced cell injury.
- MeSH
- Apoptosis * drug effects MeSH
- beta Catenin metabolism MeSH
- Cell Line MeSH
- Down-Regulation * drug effects MeSH
- Cadherins metabolism MeSH
- Myocytes, Cardiac * metabolism drug effects MeSH
- Rats MeSH
- Hydrogen Peroxide * pharmacology MeSH
- Cell Proliferation drug effects MeSH
- Wnt Signaling Pathway * drug effects MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
The ratio of densities of Na-Ca exchanger current (INaCa) in the t-tubular and surface membranes (INaCa-ratio) computed from the values of INaCa and membrane capacitances (Cm) measured in adult rat ventricular cardiomyocytes before and after detubulation ranges between 1.7 and 25 (potentially even 40). Variations of action potential waveform and of calcium turnover within this span of the INaCa-ratio were simulated employing previously developed model of rat ventricular cell incorporating separate description of ion transport systems in the t-tubular and surface membranes. The increase of INaCa-ratio from 1.7 to 25 caused a prolongation of APD (duration of action potential at 90% repolarisation) by 12, 9, and 6% and an increase of peak intracellular Ca(2+) transient by 45, 19, and 6% at 0.1, 1, and 5 Hz, respectively. The prolonged APD resulted from the increase of INaCa due to the exposure of a larger fraction of Na-Ca exchangers to higher Ca(2+) transients under the t-tubular membrane. The accompanying rise of Ca(2+) transient was a consequence of a higher Ca(2+) load in sarcoplasmic reticulum induced by the increased Ca(2+) cycling between the surface and t-tubular membranes. However, the reason for large differences in the INaCa-ratio assessed from measurements in adult rat cardiomyocytes remains to be explained.
- MeSH
- Cell Membrane metabolism MeSH
- Myocytes, Cardiac metabolism MeSH
- Rats MeSH
- Membrane Potentials physiology MeSH
- Models, Cardiovascular * MeSH
- Sodium metabolism MeSH
- Heart Ventricles metabolism MeSH
- Calcium metabolism MeSH
- Animals MeSH
- Check Tag
- Rats MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Cardiac hypertrophy is the result of responses to various physiological or pathological stimuli. Recently, we showed that polycystin-1 participates in cardiomyocyte hypertrophy elicited by pressure overload and mechanical stress. Interestingly, polycystin-1 knockdown does not affect phenylephrine-induced cardiomyocyte hypertrophy, suggesting that the effects of polycystin-1 are stimulus-dependent. In this study, we aimed to identify the role of polycystin-1 in insulin-like growth factor-1 (IGF-1) signaling in cardiomyocytes. Polycystin-1 knockdown completely blunted IGF-1-induced cardiomyocyte hypertrophy. We then investigated the molecular mechanism underlying this result. We found that polycystin-1 silencing impaired the activation of the IGF-1 receptor, Akt, and ERK1/2 elicited by IGF-1. Remarkably, IGF-1-induced IGF-1 receptor, Akt, and ERK1/2 phosphorylations were restored when protein tyrosine phosphatase 1B was inhibited, suggesting that polycystin-1 knockdown deregulates this phosphatase in cardiomyocytes. Moreover, protein tyrosine phosphatase 1B inhibition also restored IGF-1-dependent cardiomyocyte hypertrophy in polycystin-1-deficient cells. Our findings provide the first evidence that polycystin-1 regulates IGF-1-induced cardiomyocyte hypertrophy through a mechanism involving protein tyrosine phosphatase 1B.
The functional significance of having two nuclei in a cell is unknown. Having two stores of genetic material may be advantageous for cell growth. Nuclear protein import is at a critical juncture in the cell to modify cell growth. This study addressed the potential for differential nuclear protein import in two nuclei of the same cell. Isolated adult rat cardiomyocytes were microinjected with an exogenous fluorescent protein conjugated with nuclear localization amino acid sequences to facilitate nuclear import and its detection. Our results demonstrate the rate of nuclear protein import was not significantly different between the two nuclei in the same cell. These data demonstrate that the two nuclei are functionally similar in a binucleated cardiomyocyte, at least as far as nucleocytoplasmic transport is concerned.
Cardiovascular diseases are associated with an altered cardiomyocyte metabolism. Because of a shortage of human heart tissue, experimental studies mostly rely on alternative approaches including animal and cell culture models. Since the use of isolated primary cardiomyocytes is limited, immortalized cardiomyocyte cell lines may represent a useful tool as they closely mimic human cardiomyocytes. This study is focused on the AC16 cell line generated from adult human ventricular cardiomyocytes. Despite an increasing number of studies employing AC16 cells, a comprehensive proteomic, bioenergetic, and oxygen-sensing characterization of proliferating vs. differentiated cells is still lacking. Here, we provide a comparison of these two stages, particularly emphasizing cell metabolism, mitochondrial function, and hypoxic signaling. Label-free quantitative mass spectrometry revealed a decrease in autophagy and cytoplasmic translation in differentiated AC16, confirming their phenotype. Cell differentiation led to global increase in mitochondrial proteins [e.g. oxidative phosphorylation (OXPHOS) proteins, TFAM, VWA8] reflected by elevated mitochondrial respiration. Fatty acid oxidation proteins were increased in differentiated cells, whereas the expression levels of proteins associated with fatty acid synthesis were unchanged and glycolytic proteins were decreased. There was a profound difference between proliferating and differentiated cells in their response to hypoxia and anoxia-reoxygenation. We conclude that AC16 differentiation leads to proteomic and metabolic shifts and altered cell response to oxygen deprivation. This underscores the requirement for proper selection of the particular differentiation state during experimental planning.NEW & NOTEWORTHY Proliferating and differentiated AC16 cell lines exhibit distinct proteomic and metabolic profiles with critical implications for experimental design. Proliferating cells predominantly utilize glycolysis and are highly sensitive to hypoxia, whereas differentiated cells display enhanced mitochondrial biogenesis, oxidative phosphorylation, and resistance to anoxia-reoxygenation. These findings provide novel insights into the metabolic adaptations during differentiation and highlight the necessity of selecting the appropriate cellular stage to ensure accurate experimental outcomes.
- MeSH
- Cell Differentiation * physiology MeSH
- Cell Line MeSH
- Energy Metabolism MeSH
- Cell Hypoxia physiology MeSH
- Myocytes, Cardiac * metabolism MeSH
- Humans MeSH
- Mitochondrial Proteins metabolism MeSH
- Mitochondria * metabolism MeSH
- Oxidative Phosphorylation MeSH
- Cell Proliferation MeSH
- Proteomics methods MeSH
- Signal Transduction * physiology MeSH
- Mitochondria, Heart * metabolism MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
This study was designed to measure nitrite/nitrate and cytokine levels of serum obtained from septic shock patients and to describe potential depressant effects of human septic serum on rat cardiomyocytes. Serum was prepared from 10 nonseptic patients and 10 patients with documented septic shock. Adult rat ventricular myocytes were exposed to 20 % serum in the medium. Cardiomyocyte contractility was assessed by measuring shortening fraction and shortening velocity. Serum levels of nitrite/nitrate, a marker of nitric oxide final metabolites, and cytokines (tumor necrosis factor (TNF)-?, interleukin (IL) 1ß, 6, 10, 8 and 12p70) were measured. Compared with serum from non-septic patients, serum of septic shock patients induced rapid reduction of the extent and velocity of shortening in isolated cardiomyocytes. Nitrite/nitrate, TNF-?, IL-1ß and IL-12p70 concentrations of tested serum for cardiomyocyte studies were not increased in septic serum compared with controls. In contrast, septic serum that induced a depression of in vitro contractility, had increased levels of IL-6, IL-8 and IL-10. We can conclude that the depression of in vitro contractility induced by septic serum is not directly dependent on elevated levels of nitric oxide metabolites, TNF-? or IL-1ß. Our results support the view that other cytokines, including IL-6, IL-8 and IL-10, are potent circulating mediators of myocardial depression in cardiomyocytes.
- MeSH
- Cytokines blood adverse effects MeSH
- Interleukins blood adverse effects MeSH
- Myocytes, Cardiac physiology drug effects MeSH
- Myocardial Contraction genetics radiation effects MeSH
- Humans MeSH
- Heart Diseases etiology complications physiopathology MeSH
- Rats, Sprague-Dawley MeSH
- Shock, Septic blood physiopathology MeSH
- Tumor Necrosis Factor-alpha blood adverse effects MeSH
- Tumor Necrosis Factors blood adverse effects MeSH
- Check Tag
- Humans MeSH
- MeSH
- Cell Nucleus physiology chemistry MeSH
- Financing, Organized MeSH
- Histone Deacetylases physiology genetics metabolism MeSH
- Ion Channels physiology physiopathology MeSH
- Calcineurin physiology genetics metabolism MeSH
- Calmodulin physiology genetics metabolism MeSH
- Cardiomegaly etiology metabolism physiopathology MeSH
- Myocytes, Cardiac enzymology physiology MeSH
- Humans MeSH
- Sarcolemma enzymology physiology metabolism MeSH
- Cardiac Electrophysiology MeSH
- Heart Failure etiology metabolism physiopathology MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
