Spaceflight Activates Protein Kinase C Alpha Signaling and Modifies the Developmental Stage of Human Neonatal Cardiovascular Progenitor Cells
Spaceflight impacts cardiovascular function in astronauts; however, its impact on cardiac development and the stem cells that form the basis for cardiac repair is unknown. Accordingly, further research is needed to uncover the potential relevance of such changes to human health. Using simulated microgravity (SMG) generated by two-dimensional clinorotation and culture aboard the International Space Station (ISS), we assessed the effects of mechanical unloading on human neonatal cardiovascular progenitor cell (CPC) developmental properties and signaling. Following 6–7 days of SMG and 12 days of ISS culture, we analyzed changes in gene expression. Both environments induced the expression of genes that are typically associated with an earlier state of cardiovascular development. To understand the mechanism by which such changes occurred, we assessed the expression of mechanosensitive small RhoGTPases in SMG-cultured CPCs and observed decreased levels of RHOA and CDC42. Given the effect of these molecules on intracellular calcium levels, we evaluated changes in noncanonical Wnt/calcium signaling. After 6–7 days under SMG, CPCs exhibited elevated levels of WNT5A and PRKCA. Similarly, ISS-cultured CPCs exhibited elevated levels of calcium handling and signaling genes, which corresponded to protein kinase C alpha (PKCα), a calcium-dependent protein kinase, activation after 30 days. Akt was activated, whereas phosphorylated extracellular signal-regulated kinase levels were unchanged. To explore the effect of calcium induction in neonatal CPCs, we activated PKCα using hWnt5a treatment on Earth. Subsequently, early cardiovascular developmental marker levels were elevated. Transcripts induced by SMG and hWnt5a-treatment are expressed within the sinoatrial node, which may represent embryonic myocardium maintained in its primitive state. Calcium signaling is sensitive to mechanical unloading and directs CPC developmental properties. Further research both in space and on Earth may help refine the use of CPCs in stem cell-based therapies and highlight the molecular events of development.
As humankind prepares for an expanded presence in space, the molecular effects of microgravity (MG) have increasingly become the subject of investigation. As this research has evolved, the application of MG, whether real or simulated, has been found to have potential therapeutic use here on Earth [1–3]. Thus, efforts to characterize the biological response to reduced gravity conditions, including at a molecular level, will benefit society both in space and on Earth.
Research in our own laboratory has shown that simulated microgravity (SMG) impacts the developmental profile of human cardiovascular progenitor cells (CPCs) in an age-dependent manner . Interestingly, microarray analysis in those experiments identified small RhoGTPases and Wnt signaling as being some of the systems affected by the SMG environment in neonatal CPCs.
The effect of mechanical unloading on mouse embryonic stem cells (mESCs) has been shown to impact differentiation and stemness, with experiments by Blaber et al.  demonstrating that embryoid bodies retain markers of self-renewal and exhibit reduced definitive germ layer marker expression when flown in space. However, in the same studies, when mechanically unloaded embryoid bodies returned to Earth, they were able to differentiate more readily into contractile cardiomyocyte colonies. Similarly, Jha et al.  found that human induced pluripotent stem cells more readily differentiate into cardiomyocytes using three-dimensional culture coupled with transient, early exposure to SMG.
These separate experiments may represent a similar phenomenon in which a low gravity culture promotes an enhanced state of stemness under SMG or MG that results in increased differentiation ability when the cells are returned to normal gravity conditions. Thus, stem cell therapies relevant to cardiac repair may be improved by manipulating the mechanisms relevant to mechanical signaling in cardiovascular progenitors. In particular, inducing enhanced stemness in cardiovascular progenitors may facilitate a correspondingly enhanced clinical effect upon transplantation.
Alterations in mechanical sensing molecules, such as the small RhoGTPases, are believed to be involved in the molecular adaptation to MG [7,8]. Importantly, these molecules are also able to impact intracellular signaling pathways, such as calcium oscillations, which subsequently can activate AKT  and extracellular signal-regulated kinase (ERK) . In the context of cardiogenesis, these processes are critical to maintaining a balance between inductive and proliferative cues [11,12]. Therefore, manipulating the normal gravity environment of early CPCs may highlight important mechanisms by which early cardiac progenitors develop or expand. Such insights may be applied to further understand cardiovascular development and enhance the outcomes of stem cell-based regenerative therapies.
In the context of cardiac repair, early clinical trials of these types of therapies are promising [13–15], but are stymied by a failure of cell engraftment and controversy over the appropriate cell type . Therefore, the application of findings from MG experiments to Earth-based experiments may help overcome the shortcomings of current clinical trials involving the use of CPCs for cardiac repair.
In an effort to characterize both the effects of MG on a population of early CPCs as well as the potential use of these changes on Earth, we cultured neonatal human CPCs using a two-dimensional clinostat and in the National Laboratory aboard the International Space Station (ISS). We sought to identify changes in the transcription of genes involved in signaling in response to SMG and MG as well as the effects of such signaling changes on stemness. We then modeled features of these molecular changes in vitro using a small molecule under normal gravity conditions. In doing so, we present components of the adaptive cellular response to MG and their implications for enhancing the regenerative potential of neonatal CPCs.
Materials and Methods
Isolation and culture of early CPCs
The Institutional Review Board of Loma Linda University approved the protocol for use of tissue that was discarded during cardiovascular surgery, without identifiable private information, for this study with a waiver of informed consent. CPCs were isolated from cardiac tissue of neonates (1 day–1 month), as previously described . Briefly, atrial tissue was cut into small clumps (∼1.0 mm3) and then enzymatically digested using collagenase (Roche, Indianapolis, IN) at a working concentration of 1.0 mg/mL. The resulting solution was then passed through a 40-μm cell strainer. Cells were cloned in a 96-well plate by limiting dilution to a final concentration of 0.8 cells per well to create populations for expansion.
Then, clones were screened for the coexpression of Isl1 and c-Kit and supplemented with growth media comprising 10% fetal bovine serum (Thermo Scientific, Waltham, MA), 100 μg/mL penicillin–streptomycin (Life Technologies, Carlsbad, CA), 1.0% minimum essential medium nonessential amino acids solution (Life Technologies), and 22% endothelial cell growth media (Lonza, Basel, Switzerland) in Medium 199 (Life Technologies). The MycoAlert PLUS Mycoplasma Detection Kit (Lonza, Basel, Switzerland) was used to test for mycoplasma contamination
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