Histological findings of BMMNC retention 5 min after IC injection.

Recipient hearts were collected at 5 minutes after IC injection of 8x106 BMMNC that had been labeled with PKH67. A and B represent low- and high-magnification images of Isolectin-B4 staining, respectively (scale bar = 100 μm [A] and 30 μm [B]). Representative micro images from n = 4 hearts are presented. All donor BMMNC (green) retained in the heart were found to be entrapped in isolation and within the coronary microvasculature. Blue signals for nuclei (DAPI); red for endothelial cells (Isolectin-B4).</p

Different distribution of retained BMMNC between ventricular layers.

<p>At 5 minutes after IC injection of 8x10⁶ PKH67-labeled BMMNC, the number of donor cells within the endocardium-side myocardium, central myocardium and epicardium-side myocardium were counted in cross-section of immunohistolabelling samples. The concentration of cells progressively increased from the epicardium to the endocardium (n = 4, <i>p</i><0.0001, One-way ANOVA followed by Bonferroni post-hoc test).</p

Quantitative analysis of initial retention of BMMNC after IC injection.

(A) After IC injection of three different doses (1, 8, 40×106) of rat BMMNC in to a rat heart, the numbers of BMMNC in the coronary effluent in every minute were counted (Fig 1), from which their proportions to the total BMMNC exited in the coronary effluent over the first 5 minutes were calculated. It was observed that over 90% of the cells that were lost into the coronary effluent exited the heart within the first minute of injection. The proportion of cells lost did not differ between the doses (n = 8 hearts studied in each dose, p>0.99, Two-way repeated measures ANOVA). (B) Coronary flow rate was not altered by BMMNC injection using these three doses (n = 8 in each dose, p = 0.22, Two-way repeated measures ANOVA). The slight increase in volume during the first minute was due to injection of 3 ml of cell suspension. (C,D) Absolute numbers and rates of donor BMMNC retained in the heart were calculated. A larger number of injected cells resulted in a larger number of retained cells in the hearts (C, n = 8 in each dose, pp6 group, †p6 group). On the other hand, the proportion of retained cells of the total injected cell numbers was unchanged among the three doses (D; n = 8 in each dose, p = 0.67, One-Way ANOVA).</p

Flowchart of the study protocol.

CABG: Coronary artery bypass grafting; AFACS: Atrial fibrillation after cardiac surgery; K+: Serum potassium level.</p

Protocol for the Tight K Trial, Version 1 (April 2019).

Protocol for the Tight K Trial, Version 1 (April 2019).</p

Exit of BMMNC from the heart after IC injection.

<p>After IC injection of three different doses (1, 8, 40×10⁶) of rat BMMNC in to rat hearts using the Langendorff <i>ex-vivo</i> perfusion model, the numbers of BMMNC in the coronary effluent in every minute were counted. The majority of BMMNC injected exited from the heart into coronary effluent in 1 minute after IC injection of any cell dose studied. The pattern of cells exited from the heart did not differ between the doses (n = 8 different hearts studied in each dose, <i>p>0</i>.<i>99</i>, Two-way repeated measure ANOVA). Upper panel for 1×10⁶ BMMNC injection; Middle panel for 8×10⁶ BMMNC injection; Lower panel for 40×10⁶ BMMNC injection.</p

Histological findings of MSC retention after IC injection.

<p>Recipient rat hearts were collected at 60 minutes after injection of MSC which had been labeled with PKH67. <b>A</b> and <b>B</b> represent low- and high-magnification images of isolectin-B4 staining, respectively (scale bar = 100 μm [A] and 30 μm [B]). Representative micro images from n = 4 hearts are presented. All donor cells (green) were found within the coronary vasculature, with frequent formation of cell-clamps in the vasculature. The diameter of vasculatures having MSC appeared to be enlarged, compared to other intact ones. Blue signals for nuclei (DAPI); red for endothelial cells (Isolectin-B4).</p

Histological findings of BMMNC retention 60 min after IC injection.

<p>Recipient hearts were collected at 60 minutes after IC injection of 8x10⁶ BMMNC that had been labeled stained with PKH67. <b>A</b> and <b>B</b> represent low- and high-magnification micro images of Isolectin-B4 staining, respectively (scale bar = 100 μm [A] and 30 μm [B]). Representative micro images from n = 4 hearts are presented. Retained BMMNC continued to be localized in the microvasculature in isolation. No donor cell (green) was observed to have extravasated out of the coronary vasculature. Blue signals for nuclei (DAPI); red for endothelial cells (Isolectin-B4).</p

Relationship of cell-surface proteins and retention of BMMNC.

<p>The expression profiles of cell-surface proteins, including integrin and selectin ligand, on pre-injection BMMNC and BMMNC in the coronary effluent (collected over the five-minute duration post-IC injection of 8x10⁶ BMMNC) were compared by flow cytometric analysis. No difference was found in any of the surface proteins investigated (n = 6 hearts were studied, unpaired T-test), suggesting that these cell-surface proteins are not critical for retention in normal hearts.</p

Arbitration flowchart for determining if primary endpoint was met after site reported atrial fibrillation after cardiac surgery (AFACS).

Arbitration flowchart for determining if primary endpoint was met after site reported atrial fibrillation after cardiac surgery (AFACS).</p