Cardiomyocyte survival and DNA reparation in myocardium of C57Bl/6 and mdx mice after dynamical stress

The cardiomyocytes of mdx mice are the prospective model of research on the survival of terminally differentiated cardiomyocytes and the formation of cardiomyopathy in conditions of oxidative stress. Previously, it was observed that dynamical stress induces the formation of low-molecular fragments of DNA. It is beyond questioning that DNA fragmentation develops through the formation of double-strand breaks in DNA (DNA DSB). To record the appearance and disappearance of DSB DNA in the cardiomyocytes of mdx mice after dynamic stress, the antibodies were applied to the phosphorylated form of an H2Ax histon (γ-H2Ax). In the absence of stress, DSB DNA were detected in the nuclei of cells of the myocardium for C57Bl/6 mice (0.05%) and for mdx mice (6.7%), accordingly. For C57Bl mice, 1 h after stress, the share of marked cardiomyocyte nuclei increased up to 1% and, for mdx mice, up to 41.7%. 24 h after stress, in the myocardium of mdx mice, 5.2% of cardiomyocytes in the nuclei were stained, while, for C57BL/6 mice, marked cardiomyocytes in the nuclei were not determined. 24 h after stress, the cell loss of cardiomyocytes for mdx mice was 2.39–2.50%. For C57Bl mice, the general level of cell loss did not exceed a threshold of 0.38%. The obtained data allow us to suspect that, during the survival of cardiomyocytes in mdx mice, a mechanism of DNA reparation is involved.     

Soluble factors released by dedifferentiated fat cells reduce the functional activity of iPS cell-derived cardiomyocytes

Interactions between tissues such as epicardial adipose (EAT), and myocardial tissues is important in the pathogenesis of heart failure. Changes in adipose tissues in obesity or diabetes impair preadipocyte differentiation. Furthermore, proinflammatory cytokine secretion is higher in preadipocytes than in mature adipocytes in diabetes and obesity. However, how undifferentiated cells committed to the adipose lineage directly influence cardiomyocytes is not yet understood. We used human-derived dedifferentiated fat (DFAT) cells as models of undifferentiated cells committed to an adipose lineage. Here, we evaluated the effects of soluble factor interactions in indirect cocultures of DFAT cells and induced pluripotent stem cell-derived cardiomyocytes. Our RNA sequencing findings showed that these interactions were predominantly inflammatory responses. Furthermore, proinflammatory cytokines secreted by DFAT cells reduced myocardial functions such as contraction frequency and catecholamine sensitivity, and simultaneously increased apoptosis, decreased antioxidative stress tolerance, and reduced oxygen consumption rates in cardiomyocytes. These adverse effects might be attributable to monocyte chemoattractant protein-1, chemokine (C-X-C motif) ligands 1 (CXCL1), and 12, granulocyte colony-stimulating factor, interleukins 6 and 8, macrophage migration inhibitory factor (MIF), and plasminogen activator inhibitor 1-A among the proinflammatory mediators secreted by DFAT cells. Our results could be useful for understanding the pathogenesis of EAT-related heart failure in terms of the involvement of undifferentiated cells committed to the adipose lineage. Furthermore, we suggest the importance of focusing on surrounding adipose tissues as a strategy with which to maximize the survival and function of transplanted stem cell-derived cardiomyocytes.     

Cover Image,Volume 116, Number 5, May 2019

Human pluripotent stem cell-derived endothelial cells (hPSC-ECs) present an attractive alternative to primary EC sources for vascular grafting. However, there is a need to mature them towards either an arterial or venous subtype. A vital environmental factor involved in the arteriovenous specification of ECs during early embryonic development is fluid shear stress; therefore, there have been attempts to employ adult arterial shear stress conditions to mature hPSC-ECs. However, hPSC-ECs are naïve to fluid shear stress, and their shear responses are still not well understood. Here, we used a multiplex microfluidic platform to systematically investigate the dose-time shear responses on hPSC-EC morphology and arterial-venous phenotypes over a range of magnitudes coincidental with physiological levels of embryonic and adult vasculatures. The device comprised of six parallel cell culture chambers that were individually linked to flow-setting resistance channels, allowing us to simultaneously apply shear stress ranging from 0.4 to 15 dyne/cm ². We found that hPSC-ECs required up to 40 hr of shear exposure to elicit a stable phenotypic change. Cell alignment was visible at shear stress <1 dyne/cm ², which was independent of shear stress magnitude and duration of exposure. We discovered that the arterial markers NOTCH1 and EphrinB2 exhibited a dose-dependent increase in a similar manner beyond a threshold level of 3.8 dyne/cm ², whereas the venous markers COUP-TFII and EphB4 expression remained relatively constant across different magnitudes. These findings indicated that hPSC-ECs were sensitive to relatively low magnitudes of shear stress, and a critical level of ~4 dyne/cm ² was sufficient to preferentially enhance their maturation into an arterial phenotype for future vascular tissue engineering applications.     

Decreased Bioenergetic Health Index in monocytes isolated from the pericardial fluid and blood of post-operative cardiac surgery patients

Monitoring the bioenergetics of leucocytes is now emerging as an important approach in translational research to detect mitochondrial dysfunction in blood or other patient samples. Using the mitochondrial stress test, which involves the sequential addition of mitochondrial inhibitors to adherent leucocytes, we have calculated a single value, the Bioenergetic Health Index (BHI), which represents the mitochondrial function in cells isolated from patients. In the present report, we assess the BHI of monocytes isolated from the post-operative blood and post-operative pericardial fluid (PO-PCF) from patients undergoing cardiac surgery. Analysis of the bioenergetics of monocytes isolated from patients’ PO-PCF revealed a profound decrease in mitochondrial function compared with monocytes isolated from their blood or from healthy controls. Further, patient blood monocytes showed no significant difference in the individual energetic parameters from the mitochondrial stress test but, when integrated into the BHI evaluation, there was a significant decrease in BHI compared with healthy control monocytes. These data support the utility of BHI measurements in integrating the individual parameters from the mitochondrial stress test into a single value. Supporting our previous finding that the PO-PCF is pro-oxidant, we found that exposure of rat cardiomyocytes to PO-PCF caused a significant loss of mitochondrial membrane potential and increased reactive oxygen species (ROS). These findings support the hypothesis that integrated measures of bioenergetic health could have prognostic and diagnostic value in translational bioenergetics.     

Glucose is essential for the initiation of fatty acid oxidation in ATP-depleted cultured ventricular myocytes

Cultured cardiac myocytes were depleted of ATP by incubation with oligomycin (1 mg/ml). Then the ability of the cells to oxidize various substrates and to restore ATP levels was studied. Following ATP depletion, the cells were found to be able to oxidize glucose given alone, but not palmitate. However, with both substrates, palmitate was oxidized in the presence of glucose and ATP recovery was enhanced. Pyruvate had a minor effect on palmitate oxidation, while acetate given alone was oxidized, but did not enhance cellular ATP content. These results show that glucose is essential for restoration of mitochondrial function and the coupling between oxidation and ATP synthesis.