MiR-21, MiR-29a, GATA4, and MEF2c Expression Changes in Endothelin-1 and Angiotensin II Cardiac Hypertrophy Stimulated Isl-1+Sca-1+c-kit+ Porcine Cardiac Progenitor Cells In Vitro

Abstract

Cost- and time-intensive porcine translational disease models offer great opportunities to test drugs and therapies for pathological cardiac hypertrophy and can be supported by porcine cell culture models that provide further insights into basic disease mechanisms. Cardiac progenitor cells (CPCs) residing in the adult heart have been shown to differentiate in vitro into cardiomyocytes and could contribute to cardiac regeneration. Therefore, it is important to evaluate their changes on the cellular level caused by disease. We successfully isolated Isl1+Sca1+cKit+ porcine CPCs (pCPCs) from pig hearts and stimulated them with endothelin-1 (ET-1) and angiotensin II (Ang II) in vitro. We also performed a cardiac reprogramming transfection and tested the same conditions. Our results show that undifferentiated Isl1+Sca1+cKit+ pCPCs were significantly upregulated in GATA4, MEF2c, and miR-29a gene expressions and in BNP and MCP-1 protein expressions with Ang II stimulation, but they showed no significant changes in miR-29a and MCP-1 when stimulated with ET-1. Differentiated Isl1+Sca1+cKit+ pCPCs exhibited significantly higher levels of MEF2c, GATA4, miR-29a, and miR-21 as well as Cx43 and BNP with Ang II stimulation. pMx-MGT-transfected Isl1+Sca1+cKit+ pCPCs showed significant elevations in MEF2c, GATA4, and BNP expressions when stimulated with ET-1. Our model demonstrates that in vitro stimulation leads to successful Isl1+Sca1+cKit+ pCPC hypertrophy with upregulation of cardiac remodeling associated genes and profibrotic miRNAs and offers great possibilities for further investigations of disease mechanisms and treatment.

Novel Identified Circular Transcript of RCAN2, circ-RCAN2, Shows Deviated Expression Pattern in Pig Reperfused Infarcted Myocardium and Hypoxic Porcine Cardiac Progenitor Cells In Vitro

“…We isolated pCPCs as previously described [ 27 ], and performed immunofluorescence staining. Our results showed positive expression of connexin-43 (Cx43) ( Figure 4 a, green), alpha smooth muscle actin (αSMA) ( Figure 4 b, green), islet-1 (Isl-1) ( Figure 4 c, green), pro-BNP ( Figure 4 d, green), and stem cells antigen-1 (Sca-1) ( Figure 4 e, green), confirming successful isolation of Sca-1 + , and Isl1 + pCPCs.…”

“…Porcine cardiac progenitor cells were isolated from pig hearts, and qPCR was performed to assess the relative gene expressions of ISL-1, SCA-1, c-Kit, and BNP, as previously described [ 27 ]. To further evaluate the expression of progenitor cell markers, such as islet-1 (Isl-1) and stem cell antigen (Sca-1), we also performed immunofluorescence staining.…”

New Insights and Current Approaches in Cardiac Hypertrophy Cell Culture, Tissue Engineering Models, and Novel Pathways Involving Non-Coding RNA

“…Pharmacological agents that are used for hypertrophy induction are for example phenylephrine ( Jain et al, 2018 ), angiotensin II ( Gelinas et al, 2018 ), noradrenaline ( Guven, 2018 ), endothelin-1 ( Zlabinger et al, 2019 ), and isoproterenol ( Zhang et al, 2016 ). These agents interfere with a known pathway that is crucial in remodeling and subsequent cardiac hypertrophy.…”

“…Induction of hypertrophy by all of these chemical agents show a similar modeling time of 24 h, which may be extended to 48 h depending on the used cell type (Zhang et al, 2016;Zlabinger et al, 2019;Zhao et al, 2020). The concentration needs to be optimized by testing different dilutions before the experiment, however the general used concentrations are 20 µM phenylephrine (Gelinas et al, 2018), 100 nM angiotensin II (Zlabinger et al, 2019), 10 mM isoproterenol (Zhang et al, 2016), and 10 nM endothelin-1 (Loonat et al, 2019).…”

“…MiR-21, MiR-29a, GATA4, and MEF2c Expression (Zlabinger et al, 2019) Ang II, Angiotensin II; PE, Phenylephrine; Iso, Isoproterenol; ET1, Endothelin-1; NA, Noradrenaline.…”