Enhancement of Spontaneous Activity by HCN4 Overexpression in Mouse Embryonic Stem Cell-Derived Cardiomyocytes - A Possible Biological Pacemaker

<div><p>Background</p><p>Establishment of a biological pacemaker is expected to solve the persisting problems of a mechanical pacemaker including the problems of battery life and electromagnetic interference. Enhancement of the funny current (<i>I</i>_f) flowing through hyperpolarization-activated cyclic nucleotide-gated (HCN) channels and attenuation of the inward rectifier K⁺ current (<i>I</i>_K1) flowing through inward rectifier potassium (K_ir) channels are essential for generation of a biological pacemaker. Therefore, we generated HCN4-overexpressing mouse embryonic stem cells (mESCs) and induced cardiomyocytes that originally show poor <i>I</i>_K1 currents, and we investigated whether the HCN4-overexpressing mESC-derived cardiomyocytes (mESC-CMs) function as a biological pacemaker <i>in vitro</i>.</p><p>Methods and Results</p><p>The rabbit <i>Hcn4</i> gene was transfected into mESCs, and stable clones were selected. mESC-CMs were generated via embryoid bodies and purified under serum/glucose-free and lactate-supplemented conditions. Approximately 90% of the purified cells were troponin I-positive by immunostaining. In mESC-CMs, expression level of the <i>Kcnj2</i> gene encoding K_ir2.1, which is essential for generation of <i>I</i>_K1 currents that are responsible for stabilizing the resting membrane potential, was lower than that in an adult mouse ventricle. HCN4-overexpressing mESC-CMs expressed about a 3-times higher level of the <i>Hcn4</i> gene than did non-overexpressing mESC-CMs. Expression of the <i>Cacna1h</i> gene, which encodes T-type calcium channel and generates diastolic depolarization in the sinoatrial node, was also confirmed. Additionally, genes required for impulse conduction including <i>Connexin40</i>, <i>Connexin43</i>, and <i>Connexin45</i> genes, which encode connexins forming gap junctions, and the <i>Scn5a</i> gene, which encodes sodium channels, are expressed in the cells. HCN4-overexpressing mESC-CMs showed significantly larger <i>I</i>_f currents and more rapid spontaneous beating than did non-overexpressing mESC-CMs. The beating rate of HCN4-overexpressing mESC-CMs responded to ivabradine, an <i>I</i>_f inhibitor, and to isoproterenol, a beta-adrenergic receptor agonist. Co-culture of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with aggregates composed of mESC-CMs resulted in synchronized contraction of the cells. The beating rate of hiPSC-CMs co-cultured with aggregates of HCN4-overexpressing mESC-CMs was significantly higher than that of non-treated hiPSC-CMs and that of hiPSC-CMs co-cultured with aggregates of non-overexpressing mESC-CMs.</p><p>Conclusions</p><p>We generated HCN4-overexpresssing mESC-CMs expressing genes required for impulse conduction, showing rapid spontaneous beating, responding to an <i>I</i>_f inhibitor and beta-adrenergic receptor agonist, and having pacing ability in an <i>in vitr</i>o co-culture system with other excitable cells. The results indicated that these cells could be applied to a biological pacemaker.</p></div

Measurement of <i>I</i>_f currents in mESC-CMs.

<p>A. Representative <i>I</i>_f currents in <i>EGFP</i>-(left) or <i>Hcn4/EGFP-</i>(right) overexpressing mESC-CMs. HCN4-overexpressing mESC-CMs showed a larger <i>I</i>_f current than did non-overexpressing cells. <i>I</i>_f currents through activated HCN channels were obtained during hyperpolarizing test pulses of 5 seconds between -45 and -125 mV in 20 mV increments from a holding potential of -35 mV. B. <i>I</i>_f-V relationship curve in <i>EGFP</i>-(blue line) or <i>Hcn4/EGFP-</i>(red line) overexpressing mESC-CMs.</p

Spontaneous beating rates in mESC-CMs.

<p>A. Beating rates in control mESC-CMs, HCN4-overexpressing mESC-CMs (HCN4/EGFP mESC-CMs-2) and EGFP-stably-transfected mESC (EGFP mESC-CMs-2). HCN4-overexpressing mESC-CMs (HCN4/EGFP mESC-CMs-2) showed significantly more rapid beating than did non-overexpressing cells (Control and EGFP mEDC-CMs-2) (n = 8 per group). Data are expressed as mean ± SD. B. Representative action potentials in HCN4-overexpressing mESC-CMs (HCN4/EGFP mESC-CMs) (upper panel) and in non-overexpressing cells (EGFP mESC-CMs) (bottom panel). C and D. Beating rates of HCN4-overexpressing mESC-CMs decreased in response to ivabradine and increased in response to isoproterenol. (n = 6 in each group). Data are expressed as mean ± SD.</p

Establishment of purified HCN4-overexpressing mESC-CMs.

<p>A. (a) Representative immunofluorescence staining in <i>EGFP</i>-(left) or <i>Hcn4/EGFP-</i>(right) overexpressing mESC-CMs. α-actinin (red), EGFP (green) and Hoechst nuclear staining (blue) in the upper panel, troponin I (red) and Hoechst nuclear staining (blue) in the middle panel and myosin light chain (MLC)-2v (red), MLC-2a (green) and Hoechst nuclear staining (blue) in the bottom panel. (b) Percentages of immunofluorescence-positive cells for α-actinin (n = 7 in each group), EGFP, troponin I (n = 7 in each group), and MLC-2v and MLC-2a (n = 8 in EGFP mESC-CMs, n = 6 in HCN4/EGFP mESC-CMs). Data are expressed as mean ± SD. Bar = 50 μm. B. RT-PCR showed increases in mRNA expression for cardiac markers <i>Nkx 2</i>.<i>5</i>, <i>Tnnt2</i>, <i>connexin</i>, <i>Scn5a</i>, <i>Cacna1h</i>, and mouse endogenous <i>Hcn4</i> in mESC-CMs with or without HCN4 overexpression (lane 4 to 6). Rabbit exogenous <i>Hcn4</i> was expressed only in rabbit HCN4-transfected mESCs (lane 2) and mESC-CMs (lane 5).</p

Generation of mESC lines stably overexpressing rabbit <i>Hcn4</i>.

<p>A. A transfection construct bearing the rabbit <i>Hcn4</i>-IRES-<i>EGFP</i> cassette. B. Representative living mESCs observed by phase contrast microscopy (a to c) and fluorescence microscopy (d to f). <i>Hcn4/EGFP</i> or <i>EGFP</i>-stably-transfected mESCs (HCN4/EGFP mESC-2 and EGFP mESC-1 cell lines) were positive for EGFP (green) fluorescence (e and f). C. Immunofluorescence staining of a pluripotency marker, OCT4, in <i>Hcn4/EGFP</i>-stably-transfected mESCs (a to c) and <i>EGFP</i>-stably-transfected mESCs (d to f). OCT4 was expressed in both mESC lines. Bar = 50 μm. D. Measurement of <i>I</i>_f currents in <i>EGFP</i>-stably-transfected mESCs (a) and <i>Hcn4/EGFP</i>-stably-transfected mESCs (b). Activation of the <i>I</i>_f current was demonstrated in <i>Hcn4/EGFP</i>-stably-transfected mESCs. <i>I</i>_f currents through activated HCN channels were obtained during hyperpolarizing test pulses of 5 seconds between -45 and -125 mV in 20 mV increments from a holding potential of -35 mV.</p

Spontaneous beating in hiPSC-CMs synchronized with beating in HCN4-overexpressing mESC-CMs.

<p>A. Cell aggregates composed of EGFP-stably-transfected mESCs (EGFP mESC-CMs) (a and c) and HCN4-overexpressing mESC-CMs (HCN4/EGFP mESC-CMs) (b and d) observed by phase contrast microscopy (a and b) and fluorescence microscopy (green: EGFP, c and d). Bar = 50 μm. B. hiPSC-CMs (arrows) co-cultured with aggregates of HCN4/EGFP mESC-CMs (*). Bar = 50 μm. C. Beating rates of hiPSC-CMs co-cultured with aggregates of HCN4/EGFP mESC-CMs and aggregates of EGFP mESC-CMs. Data are expressed as mean ± SD.</p

Quantitative PCR for total <i>Hcn4</i> and <i>Kcnj2</i> genes.

<p>A. Relative expression of total <i>Hcn4</i> gene. HCN4-overexpressing mESC-CMs expressed about 3-times higher mRNA levels of total <i>Hcn4</i> than did EGFP mESC-CMs. B. Relative expression of <i>Kcnj2</i> gene. In mESC-CMs, the expression level of <i>Kcnj2</i> was lower than that in an adult mouse ventricle.</p

Representative CT images of thoracic and abdominal aortas in <i>klotho</i> mutant (<i>kl/kl)</i> and wild-type (WT) mice.

<p>A and B, a WT mouse (A) and a <i>kl/kl</i> mouse (B) before and after 4 weeks of control food (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181009#pone.0181009.s001" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181009#pone.0181009.s002" target="_blank">S2</a> Movies). C and D, a WT mouse (C) and a <i>kl/kl</i> mouse (D) before and after 4 weeks of EPA food (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181009#pone.0181009.s003" target="_blank">S3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181009#pone.0181009.s004" target="_blank">S4</a> Movies).</p

Eicosapentaenoic acid prevents arterial calcification in <i>klotho</i> mutant mice

<div><p>Background</p><p>The <i>klotho</i> gene was identified as an “aging-suppressor” gene that accelerates arterial calcification when disrupted. Serum and vascular klotho levels are reduced in patients with chronic kidney disease, and the reduced levels are associated with arterial calcification. Intake of eicosapentaenoic acid (EPA), an n-3 fatty acid, reduces the risk of fatal coronary artery disease. However, the effects of EPA on arterial calcification have not been fully elucidated. The aim of this study was to determine the effect of EPA on arterial calcification in <i>klotho</i> mutant mice.</p><p>Methods and results</p><p>Four-week-old <i>klotho</i> mutant mice and wild-type (WT) mice were given a diet containing 5% EPA (EPA food, <i>klotho</i> and WT: n = 12, each) or not containing EPA (control food, <i>klotho</i> and WT: n = 12, each) for 4 weeks. Calcium volume scores of thoracic and abdominal aortas assessed by computed tomography were significantly elevated in <i>klotho</i> mice after 4 weeks of control food, but they were not elevated in <i>klotho</i> mice after EPA food or in WT mice. Serum levels of EPA and resolvin E1, an active metabolite of EPA, in EPA food-fed mice were significantly increased compared to those in control food-fed mice. An oxidative stress PCR array followed by quantitative PCR revealed that NADPH oxidase-4 (<i>NOX4</i>), an enzyme that generates superoxide, gene expression was up-regulated in arterial smooth muscle cells (SMCs) of <i>klotho</i> mice. Activity of NOX was also significantly higher in SMCs of <i>klotho</i> mice than in those of WT mice. EPA decreased expression levels of the NOX4 gene and NOX activity. GPR120, a receptor of n-3 fatty acids, gene knockdown by siRNA canceled effects of EPA on NOX4 gene expression and NOX activity in arterial SMCs of <i>klotho</i> mice.</p><p>Conclusions</p><p>EPA prevents arterial calcification together with reduction of NOX gene expression and activity via GPR120 in <i>klotho</i> mutant mice.</p></div

Gene expression of cytoglobin (<i>Cygb</i>), glutathione peroxidase 3 (<i>GPX3</i>) and NADPH oxidase (<i>NOX</i>) and activity of NOX.

<p>A to C, Expression levels of <i>Cygb</i> (A), <i>GPX3</i> (B) and <i>NOX4</i> (C) genes in arterial smooth muscle cells (SMCs) of wild-type (WT) and <i>klotho</i> mutant (<i>kl/kl)</i> mice treated with EPA and not treated with EPA (control) (n = 6, each). D, NOX activity in arterial SMCs of WT and <i>kl/kl</i> mice treated with EPA and not treated with EPA (control) (n = 6, each).</p