Epicardially Placed Bioengineered Cardiomyocyte Xenograft in Immune-Competent Rat Model of Heart Failure

Figure 1. Timeline of rat surgeries. Chronologic study timeline in weeks (W). Sham controls received a thoracotomy at the time of CHF induction but did not undergo permanent coronary occlusion. A portion of CHF control rats were randomly selected to receive the graft therapy. CHF control rats that were not selected for graft therapy still received a sham lateral thoracotomy. Plasma was obtained by removing the superior-most portion of the centrifuged whole blood for antibodies.

Figure 2. Rat diastolic pressure-volume relationship. Ex vivo pressure-volume (P/V) diastolic filling curves of sham (n = 4), CHF (n = 5), and graft-treated rats (n = 7). Left ventricular volumes were normalized to each rat's body weight. CHF results in a rightward shift in P/V filling curve and an increase in the operating volume. The graft-treated rats have a P/V filling curve shifted back towards sham and a lower operating volume, consistent with reversal of maladaptive remodeling related to compliance.

Figure 3. Cardiac histopathology. Representative left ventricular histopathology sections from 4 unique CHF rats (panels a–d) and 4 unique graft-treated rats (panels e–h). Masson's Trichrome stain was performed on 5 μm transverse sections at the level of the papillary muscles. Micrographs (0.6x) have a 2 mm scale bar for reference. CHF rats exhibit large transmural scars in addition to dilated ventricular cavities, as anticipated with the adverse remodeling from heart failure. Graft-treated hearts exhibit increased wall thickness and increased myocyte density in the previously infarcted region. They also exhibit smaller ventricular cavities compared to the CHF controls.

Figure 4. Rat immunologic titers. Total IgM (a) and IgG (b) values were evaluated in CHF (n = 5-10; black bars) rats and graft-treated rats (n = 5; gray bars) at baseline (prior to infarction), day 0 (immediately prior to graft therapy), and days 7, 21, and 49 post-graft therapy. Serum IgM and IgG values for the graft-treated rats were predominately equal to the values for CHF controls with the exception of IgM at baseline and at day 21. Values are mean ± standard error of the mean. ∗Unpaired t-test versus CHF, p < 0.05.

Figure 5. Rat-specific immune response: graft therapy. The graft-specific IgG antibody (donor-specific antibody (DSA)) response was evaluated in graft-treated rat serum (n = 7-8) at days 0, 4, 7, 21, and 49 post-graft implantation. A subphysiologic amount of DSA was detected on hiPSC-CMs and fibroblasts at days 0 and 4, with a stepwise increase between days 7 and 49 (1 μL plasma:100 μL cellular suspension). Despite the presence of these graft-specific antibodies, reversal of maladaptive remodeling was observed in graft-treated rats. ∗Unpaired t-test versus day 7, p < 0.05.

Figure 6. Unipolar electrograms from engineered graft. Superimposed colorized tracings depicting unipolar voltage electrograms (UVE) acquired utilizing a multielectrode array on one engineered cardiac graft. The black tracing exhibits the control depolarization (QS wave) and repolarization (T wave) waveforms via UVE collected without any pharmacologic manipulation. The blue tracing exhibits a UVE obtained from the same engineered graft exposed to isoproterenol (1E-2 M); a physiologic shortening of the QS-QS interval and QT interval is observed. The red tracing exhibits a UVE obtained from the same engineered graft exposed to timolol (1E-4 M); a physiologic lengthening of the QS-QS interval and QT interval is observed.

Figure 7. Intracellular calcium flux and mechanical contraction. Superimposed colorized tracings depicting the (a) height-normalized intracellular calcium flux waveforms from hiPSC-CMs and fibroblasts integrated into an engineered cardiac graft and the (b) height-normalized mechanical contraction waveforms from the same graft in milliseconds (ms). In black, the graft is beating at its spontaneous (spont) rate. The graft was then paced with at incrementally increasing rates between one Hertz (1 Hz) and three Hertz (3 Hz) to characterize the engineered cardiac graft's flux-frequency and force-frequency relationships. (c) The original intracellular calcium and mechanical contraction tracings, with arbitrary units on the y-axis and time in seconds on the x-axis. A decreasing calcium-flux and contraction amplitude is observed with increasing pacing frequency (negative flux-frequency and force-frequency relationships), suggesting functional deficit in the contractile machinery in this engineered cardiac graft.

Figure 8. Molecular phenotype of engineered cardiac grafts. Quantitative polymer chain reaction was utilized to assess the molecular phenotype of fully constituted, implantation-ready engineered cardiac grafts (n = 2). The phenotype is defined by expression of connexin 43 (Cxn43), kinase insert domain receptor (KDR), smooth muscle actin (SMA), sarcomeric alpha actinin (SAA), CD56, and platelet-derived growth factor receptor-α (PDGFRA). Grafts were composed of hiPSC-CMs and human fibroblasts on a bioabsorbable construct. Data are reported relative to a fibroblast-only cardiac graft as a control.