Click chemistry-modified cardiomyocytes enhance cardiac contractility and prevent remodeling in ischemia-reperfusion injured hearts

Figures

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  Scheme 1. Schematic illustration of click chemistry-based fast cell surface modification to increase the retention of cardiomyocytes for cardiac repair. Created using BioRender.com.

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  Fig. 1. Optimization of lipid insertion and click reactions. (A) Optimization schematic for lipid insertion and click chemistry reactions in the hPSC-CM cell membrane. (B–D) Flow cytometric analyses comparing lipid insertion efficiencies under various conditions: (B) optimization of the lipid treatment solutions. (C) Optimization of lipid treatment time. (D) Optimization of the lipid concentration. (E, F) Flow cytometric analysis to refine the TCO reaction parameters in hPSC-CMs following lipid insertion with DSPE-PEG-Tz. (E) Optimization of TCO treatment time. (F) Optimization of the TCO ratio. The two graphs on the right side of the flow cytometry histogram represent the mean fluorescence intensity and proportion of cells in the positive gate, respectively. (G, H) Cell viability of hPSC-CMs post-modification: (G) Following lipid insertion. (H) After the two-step modification. Data are presented as means ± S.D.

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  Fig. 2. Assessment of cardiomyocyte phenotypic changes following click chemistry–based surface modification. (A) Immunofluorescence staining to evaluate structural maturation of cultured hPSC-CMs (scale bars = 10 μm). (B) Representative output from sarcomere organization texture analysis (SOTA). (C, D) Quantification of sarcomere length and organization score (n = 12; three independent experiments). (E) Immunofluorescence staining to assess gap junction formation in cultured hPSC-CMs (scale bars = 20 μm). (F) Quantification of connexin 43 expression (n = 12; three independent experiments). (G) ATP production rate analysis based on OCR and ECAR measurements. (H) Relative contributions of glycolysis and oxidative phosphorylation to total ATP production (three independent experiments). ns, not significant.

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  Fig. 3. Click chemistry–based surface modification does not alter the electrophysiological properties of cardiomyocytes. (A) Schematic overview of the analyzed action potential (AP) parameters and representative morphologies of nodal-like (N-like), atrial-like (A-like), and ventricular-like (V-like) APs. (B) Representative AP traces and 5-s segments of spontaneous beating activity recorded from hPSC-CMs under vehicle, Tz, or Tz + CHP conditions. (C) Pie charts summarizing the distribution of AP subtypes (N-like, A-like, and V-like) in each group (n = 30). (D) Quantitative comparison of AP parameters, including AP amplitude, maximum upstroke velocity (Vmax), AP duration at 80% repolarization (APD80), spontaneous beating frequency, and maximum diastolic potential (MDP), across the three culture conditions. Group differences were assessed using one-way ANOVA (APD80 and MDP) or the Kruskal–Wallis test (amplitude, Vmax, and frequency), followed by appropriate post hoc analyses. ns, not significant. (E) Field potential (FP) recordings obtained by multielectrode array (MEA) analysis. Spike amplitudes were quantified within active regions exhibiting detectable electrophysiological signals. (F)…

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  Fig. 4. CHP-mCMs adhesion test in decellularized and denatured heart tissues. (A) Schematic representation of the CHP-mCMs adhesion test in decellularized and denatured heart tissues. (B) Schematic of CHP adhesion test in decellularized and denatured rat heart tissues. (C) Fluorescence imaging of CHP in denatured rat heart tissue. (D) Mean fluorescence intensity of Fig. 4C inner white dash line. (E) Fluorescence imaging of nCMs and CHP-mCMs in denatured heart tissue. nCMs and CHP-mCMs were stained with calcein deep red. (F) Mean fluorescence intensity in Fig. 4E. (G) Confocal microscopy of nCMs and CHP-mCMs in decellularized heart tissue. Green, Denatured collagen. Blue, hPSC-CM. (H) Cell count data for nCMs and CHP-mCMs in denatured hearts following PBS washing. Data are presented as means ± S.D. Statistical significance was determined using unpaired t-test (two-tailed, **p < 0.01; ***p < 0.001; ****p < 0.0001).

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  Fig. 5. Effects of CHP-modified hPSC-CMs (CHP-mCMs) on cardiac function after myocardial infarction. (A) Representative echocardiography M-mode images in four groups at Base (1 week after myocardial infarction) and 6 weeks post-transplantation. Quantification data of (B) left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), (C) left ventricular internal diameter end-diastole (LVIDd) and left ventricular internal diameter end-systole (LVIDs), (D) septal wall thickness (SWT) and posterior wall thickness (PWT). (N = 11 for Control and CHP-mCM, and N = 10 for nCM and Tz-mCM, biological replicates, *p < 0.05, **p < 0.01, and ***p < 0.001, ****p < 0.0001). Data are presented as means ± S.D. Statistical significance was determined using two-way ANOVA with Tukey's multiple-comparisons test. (E) Representative graph of the hemodynamic pressure and volume (PV) curve during steady state. Hemodynamic measurements were performed at 6 weeks post-transplantation. Quantitative graphs of (F) cardiac output and stroke volume, maximum volume (Vmax) at end-diastole, maximal rate of pressure changes during systole (dP/dtmax) and minimal rate of pressure changes during…

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  Fig. 6. Early engraftment and tissue responses following CHP-mCM transplantation in infarcted hearts. (A) Representative left ventricular (LV) pressure tracings obtained by ex vivo Langendorff perfusion 1 week after cell transplantation, and quantitative analysis of LV developed pressure, heart rate, and the maximal and minimal rates of LV pressure change (dP/dtmax and dP/dtmin) in each group. (N = 6, biological replicates, **p < 0.01, ***p < 0.001, and ****p < 0.0001). (B) Representative images of transplanted hPSC-CMs in the infarct region visualized by RFP fluorescence and human cTnT immunostaining and quantification of graft area within the infarcted myocardium. (scale bars = 200 μm; high-magnification image scale bars = 100 μm, N = 6, biological replicates, ****p < 0.0001). (C) High-magnification images showing preferential localization of human cTnT-positive CHP-mCMs along denatured collagen within the infarct area. (D) Representative TUNEL and human cTnT co-staining of grafted hPSC-CMs and quantification of the proportion of TUNEL-positive hPSC-CMs (scale bars = 50 μm; N = 6, biological replicates, ****p < 0.0001). (E) Representative images of sarcomeric organization in engrafted…

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  Fig. 7. Enhanced engraftment and cardiac repair by CHP-modified CMs in infarcted hearts. (A) Representative immunofluorescent images comparing retention rates of transplanted human pluripotent stem cell (hPSC)-derived cardiomyocytes (hPSC-CMs) in each group at 6 weeks post-transplantation (hPSC-CMs expressing RFP and total CMs stained with total cTnT [green] and human CMs stained with human cTnT [white]), and quantification of engrafted hPSC-CMs (RFP+ and human cTnT+ cells) in ischemic hearts, expressed as the percentage of RFP+/human cTnT+ cells among total cTnT+ CMs within the infarct area (scale bars = 200 μm, N = 7, biological replicates, ****p < 0.0001). Representative immunofluorescent images of gap junction expression (gap junctions stained with CX43 [white] and total CMs stained with total cTnT [green]) at 6 weeks post-transplantation. The quantification graph shows the lateralized gap junctions to localized CX43 areas (scale bars = 200 μm, N = 7, biological replicates, ***p < 0.001 and ****p < 0.0001). (B) Representative images of CMs stained with total cTnT (green) on the border zone and infarct zone at 6 weeks and their quantification data (scale bars = 200 μm, N = 7,…