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Mineralocorticoid receptor phase separation modulates cardiac preservation

Nature Cardiovascular Research volume 4, pages 710–726 (2025)Cite this article

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Abstract

Heart transplantation is the gold standard treatment for patients with end-stage heart failure. However, there is a shortage of donor hearts available. The short tolerable cold ischemic time for delivering donor hearts to matching recipients is closely responsible for this shortage. Here we uncover the phenomenon of mineralocorticoid receptor (MR) phase separation, which exacerbates injury to the murine and human donor heart during cold storage and can be modulated with pharmacological inhibition to improve preservation quality. Interestingly, donor cardiomyocytes strongly expressed MR, which undergoes preservation-related phase separation. The phenomenon of macromolecular phase separation is not limited to the heart or MR during preservation. Cold preservation of the lung, liver and kidney also displays phase separation of other transcriptional regulators including histone deacetylase 1 (HDAC1), bromodomain-containing 4 (BRD4) and MR. Our results reveal an understudied area of preservation biology that may be further exploited to improve the preservation of multiple solid organs.

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Fig. 1: MR is preferentially expressed in human cardiomyocytes during cardiac preservation and negatively impacts donor heart function after reperfusion.
Fig. 2: MR undergoes LLPS into nuclear condensates under cold hypoxic preservation conditions.
Fig. 3: IDR of MR governs the capacity for phase separation and determines cardiac preservation quality.
Fig. 4: Canrenone treatment significantly improved porcine and human donor heart function after prolonged preservation and reduced cardiac injury.
Fig. 5: MR phase separation in human hearts was reduced by canrenone treatment, and this was associated with reversal of MR genome occupancy patterns as well as increased abundance of metabolites needed for energy production.
Fig. 6: Death process and cold organ preservation is associated with increased osmolality with molecular phase separation as a shared theme in cold static organ preservation.

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Data availability

Raw sequencing and processed data generated in this study are deposited in the NCBI Gene Expression Omnibus under accession number GSE261124. Raw and processed mass spectral data are available on the MassIVE server under accession number MSV000096745. Material derived from this work may be requested from P.C.T. (tang.paul2@mayo.edu), and human samples can be shared with the scientific community via a material transfer agreement. Source data are provided with this paper.

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Acknowledgements

We are very grateful for the generosity of donors and the ‘Gift of Life Michigan’ organ procurement organization, which provided hearts for research through the procurement facility. We also thank Essential Pharmaceuticals for the gift of the commercial HTK preservation solution for our studies. Key administrative and technical support by M. McCotter, A. Malis, S. Marshall and G. Rising at the University of Michigan was greatly appreciated. We thank A. Sarcon at Mayo Clinic for the artwork. This study was supported by the National Institutes of Health grants HL164416, HL166140 (P.C.T.), U01-AI132895 (J.S.P.), AI151588, AI173950 (J.L.P., M.C.), HL163672, HL139735 (Z.W.), HL159871, HL134569 and HL109946 (Y.E.C.), the Thoracic Surgery Foundation — Southern Thoracic Surgery Association (P.C.T.), a Gardner Surgical Investigator Award, American Association for Thoracic Surgery (P.C.T.), a McKay Research Grant (P.C.T.), the Frankel Cardiovascular Center, University of Michigan—Ann Arbor (to P.C.T.), Mayo Clinic (to P.C.T.) and an American Heart Association Career Development Award (930124 to I.L.). M.R.P. acknowledges the T32 Training Program in Translational Cardiovascular Science (T32 GM135119). Y.G. acknowledges NIH R01HL109810. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Author information

Authors and Affiliations

  1. Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA

    Ienglam Lei & Paul C. Tang

  2. Department of Cardiac Surgery, University of Michigan, Ann Arbor, MI, USA

    Ienglam Lei, Hüseyin Sicim, Wei Huang, Liu Liu, Ashraf Abou El Ela, Mulan Jiang, Francis D. Pagani, Y. Eugene Chen, Zhong Wang & Paul C. Tang

  3. Department of Cardiovascular Surgery, Mayo Clinic, Rochester, MN, USA

    Ienglam Lei, Hüseyin Sicim, Wenbin Gao, Aurora Lee, Sahar A. Saddoughi & Paul C. Tang

  4. Department of Cardiac Surgery, Université de Montréal, Montréal, Quebec, Canada

    Pierre Emmanuel Noly

  5. Department of Cell and Regenerative Biology, University of Wisconsin, Madison, WI, USA

    Melissa R. Pergande & Ying Ge

  6. Department of Chemistry, University of Wisconsin, Madison, WI, USA

    Mallory C. Wilson & Ying Ge

  7. Department of Pathology, Yale University, New Haven, CT, USA

    Jordan S. Pober

  8. Department of Surgery, University of Michigan, Ann Arbor, MI, USA

    Jeffrey L. Platt & Marilia Cascalho

  9. Department of Internal Medicine, Division of Cardiovascular Medicine, University of Michigan, Ann Arbor, MI, USA

    Bertram Pitt

  10. Department of Molecular and Integrative Physiology, Internal Medicine, Pharmacology, University of Michigan, Ann Arbor, MI, USA

    Richard M. Mortensen

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  1. Ienglam Lei
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Contributions

I.L., Z.W., Y.E.C. and P.C.T. were involved in conceptualization of the study. I.L., H.S., W.G., W.H., P.E.N., M.R.P., M.C.W., A.L., L.L. A.A.E.E., M.J., J.L.P., J.S.P., M.C., F.D.P., Y.E.C., B.P., Z.W., R.M.M. and Y.G. designed and implemented the methodology. I.L., H.S., W.G., W.H., P.E.N., M.R.P., M.C.W., A.L., A.A.E.E., M.J. and P.C.T. contributed to the investigations. I.L., H.S., W.G., W.H., P.E.N., M.R.P., M.C.W., A.L. and P.C.T. visualized the data. I.L. and P.C.T. acquired funding for the studies. I.L. and P.C.T. administered the project. J.L.P., J.S.P., Z.W., Y.E.C., R.M.M., Y.G. and P.C.T. provided supervision for the study. I.L. and P.C.T. wrote the original draft. All authors contributed to data interpretation and discussion of results and commented on the paper.

Corresponding author

Correspondence to Paul C. Tang.

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Competing interests

I.L., Z.W., F.D.P., B.P., Y.E.C. and P.C.T. have filed a US provisional patent (title: Histone-acetylation-modulating agents for the treatment and prevention of organ injury, no. 63/045,784, international application no. PCT/US2021/039650). A.A.E.E. has a consulting agreement with TransMedics. F.D.P. is an ad hoc, noncompensated scientific advisor for Medtronic, Abbott, FineHeart and CH Biomedical and a noncompensated medical monitor for Abiomed. F.D.P. is also a member of the data safety monitoring board for Carmat and the NHLBI PumpKIN clinical trial as well as the chair of data safety and management for the DCD heart national FDA clinical trial and the EXPAND heart-Continuous access protocol national FDA trial. P.C.T. is a noncompensated member of the data safety monitoring board for the XVIVO Perfusion PRESERVE trial. The other authors declare no competing interests.

Peer review information

Nature Cardiovascular Research thanks Jason P. Fine, Sarah L. Longnus, Lori West and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Single-cell transcriptomic expression of cell type markers and differential cardiomyocyte transcriptomes.

a, Violin plot constructed from integrated dataset displaying cell markers genes for the respective cell population clusters including endothelial cells (EC), fibroblasts (FB) smooth muscle cells (SMC), neuronal cells (NC), leukocytes (LK) as well as cardiomyocyte (CM) clusters 1 and 2. UMAP plot of single-cell transcriptomic analysis from cold (4 °C) preserved human hearts showing cell population expression of b, cardiac muscle action potential pathway genes and c, autophagy related pathway transcripts. UMAP plot of cell population expression of d, RYR2 and e, CACNA1C as well as f, PINK1 and g, FIS1.

Extended Data Fig. 2 Donor heart preservation is associated with increased MR protein expression.

Increased MR protein expression is seen with normalization to baseline preservation time in a, human donor hearts at baseline, 4 and 10 h of 4 °C static preservation with HTK without reperfusion (n = 3/group) and b, murine donor heart after baseline and 16 h of 4 °C HTK preservation followed by ex-vivo perfusion (n = 4/group). c, MR immunostaining (purple) with DAPI (blue) in MRfl/fl versus Myh6CreERT2;MRfl/fl hearts with tamoxifen injection. Representative fluorescence images of n = 4/group. d, QRT-PCR analysis of cytokine transcript expression in transplanted donor hearts preserved for 16 h normalized to control MRfl/fl hearts at baseline time (n = 3). e, Immunostaining of CD45 (red) and Ly6C (red) with DAPI (blue) in transplanted hearts. Representative fluorescence images of n = 4/group. f. Quantitation of positive staining cell counts expressed as percentage of total cells averaged over 4 high powered (200x) fields (n = 4). Data are presented as means ± SD. *P < 0.05 and **P < 0.01 by two-sided Mann-Whitney test, Tukey’s corrections were used for multiple comparisons using one-way ANOVA.

Source data

Extended Data Fig. 3 MR dynamics in living cells show propensity for LLPS and condensate formation that is driven by the intrinsic disordered protein region.

a, Neonatal rat cardiomyocytes (NRCM) expressing green fluorescent protein (GFP)-MR using adenovirus were bathed in cold (4°C) HTK preservation solution under hypoxic conditions (1% O2) for 10 h. A laser was used to perform fluorescence recovery after photobleaching (FRAP) within the nucleus. The dark region in the middle represents the nucleoli. Photobleached regions are circled and fluorescence recovery was quantified in adjoining graph. Data are presented as means ± SD (n = 3 biological replicates). b, We designed constructs that used lipofectamine to transfect Human Embryonic Kidney (HEK-293) cell cultures to express a mCherry-optoDrop (OptoDroplets) vector with either a full length MR (MR_FL) protein or a mutant MR without the IDR at the N-terminal domain (MRΔIDR). c. The HEK-293 cells were then immersed in histidine-tryptophan-ketoglutarate preservation solution under normoxic conditions and exposed to blue light for up to 60 s to induce MR condensate formation (white arrow). MR_FL is seen throughout the entire cell whereas MRΔIDR remained mostly within the cytoplasm thus outlining the nucleus. Data shown represent n = 4 biological replicates. d, Laser “photobleaching” of MR_FL condensates in live HEK-293 cells followed by fluorescence recovery. Photobleached region is circled. Image shown represent n = 4 biological replicates.

Source data

Extended Data Fig. 4 Cryopreservation is ligand independent.

Adeno-associated virus 9 (AAV) was administered to Myh6CreERT2;MRfl/fl mice 28 days prior to cardiac preservation. AAV9 was used to deliver cardiac-specific constructs containing MR with a deleted ligand binding domain (LBD, MRΔLBD). a, After 16 h of preservation followed by ex-vivo perfusion, construct expression in-vivo was verified by western blot. b, Murine donor hearts from wild type control, Myh6CreERT2;MRfl/fl mice infected with AAV-Luciferase (AAV-Luc) control, and Myh6CreERT2;MRfl/fl mice infected with AAV-MRΔLBD (MR missing the LBD) were cold (4 °C) preserved for 16 h followed by ex-vivo perfusion with Kreb buffer (n = 8). c, Immunostaining of MR condensates (red) with DAPI (blue). Representative fluorescence images of n = 4/group. d, Transcript expression of MR target genes ZFP219, CAMK2D, and PER1 in ex-vivo perfused murine hearts cold preserved for 16 h (n = 4 per group). Murine donor hearts were cold preservation with HTK only versus HTK + aldosterone (50 μM) (n = 8/group) for 12 h followed by ex-vivo perfusion. The cardiac functions e, contractility and f, relaxation were assessed by conduction catheter. Data are presented as means ± SD. *P < 0.05 and **P < 0.01 by two-sided Mann-Whitney test. Tukey’s corrections were used for multiple comparisons using one-way ANOVA for multiple group comparisons.

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Extended Data Fig. 5 Canrenone improves donor heart function with ex-vivo perfusion and transplantation after prolonged preservation.

a, Canrenone dose titration with murine donor hearts cold (4 °C) preserved for 16 h followed by ex-vivo perfusion with Kreb buffer (n = 6/group). Tested canrenone dosages of 12.5 μM, 50 μM, and 200 μM are indicated.Preservation time titration of canrenone (50 μM) versus vehicle control (n = 5/group) with ex-vivo perfusion and conductance catheter assessment of cardiac b, contractility and c, relaxation. Transplanted donor heart d, contractility and e, relaxation (n = 8 for HTK+Veh, n = 9 for HTK+Canr) as well as f, circulating cardiac troponin I levels were compared following 16 h of cardiac preservation with canrenone versus vehicle control (n = 8/group). Cold preservation with HTK only, HTK + finerenone (50 μM) versus HTK + spironolactone (50 μM) (n = 8/group) with ex-vivo perfusion and conductance catheter assessment of cardiac g, contractility and h, relaxation. Data are presented as means ± SD. *P < 0.05 and **P < 0.01 by two-sided Mann-Whitney test.

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Extended Data Fig. 6 Canrenone treatment improved porcine donor heart function after 4 h of preservation.

Ex-vivo assessment of pig hearts preserved with HTK ± Canrenone (Canr) treatment for 10 h. Hemodynamic and coronary measurements recorded in working mode after 1 h of perfusion include a, contractility (max dP/dt), b, relaxation (min dP/dt), c, cardiac output (ml/min/g) and d, coronary blood flow, e, Cardiac troponin I (cTnI) were quantified in ex-vivo perfusate for porcine heart (n = 8 per group). f. Western blot of BAX, Cleaved CASPASE 3 and BCL2 in ex-vivo perfused human heart. g. Quantification of western blots (n = 4). Data are presented as means ± SD. *P < 0.05 and **P < 0.01 by two-sided Mann-Whitney test.

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Extended Data Fig. 7 Canrenone reverses cold (4 °C) preservation-induced transcriptomic changes in Cardiomyocytes Cluster 1 (CM1) and Cardiomyocytes Cluster 2 (CM2) within human donor hearts during storage.

a. Cold preservation induced gene expression was identified by comparing human donor heart with 10 h of preservation compared to baseline. Red dots indicate upregulated genes in CM1 population during cold storage while blue dots show the effect of canrenone on genes represented by the red dots. Light blue dots indicate downregulated genes in CM1 population during cold storage while purple dots show the effect of canrenone on genes represented by the light blue dots. Volcano plot demonstrates that Canrenone reverses the cold preservation-induced global up- and downregulated genes in CM1. b. Heatmap of gene expression in CM1 of human donor hearts cold (4 °C) preserved for 10 h versus baseline (n = 3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 h of cold (4°C) preservation of human donor hearts (n = 3/group). c. Cold preservation induced gene expression was identified by comparing human donor heart with 10 h of preservation compared to baseline. Red dots indicate upregulated genes in CM2 population during cold storage while blue dots show the effect of canrenone on genes represented by the red dots. Light blue dots indicate downregulated genes in CM2 population during cold storage while purple dots show the effect of canrenone on genes represented by the light blue dots. Volcano plot demonstrates that Canrenone reverses the cold preservation-induced global up- and downregulated genes in CM2. d. Heatmap of gene expression in CM2 of human donor hearts cold (4 °C) preserved for 10 h versus baseline (n = 3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 h of cold (4 °C) preservation of human donor hearts (n = 3/group). Baseline is where samples were collected in the back table immediately following preservation solution administration and removal of the donor heart from the surgical field.

Extended Data Fig. 8 Canrenone reverses cold (4°C) preservation induced transcriptomic changes in fibroblasts (FB) and endothelial cells (EC) within human donor hearts during storage.

a. Cold preservation induced gene expression was identified by comparing human donor heart with 10 h of preservation compared to baseline. Red dots indicate upregulated genes in FB population during cold storage while blue dots show the effect of canrenone on genes represented by the red dots. Light blue dots indicate downregulated genes in FB population during cold storage while purple dots show the effect of canrenone on genes represented by the light blue dots. Volcano plot demonstrates that Canrenone reverses the cold preservation-induced global up- and downregulated genes in FB. b. Heatmap of gene expression in FB of human donor hearts cold (4 °C) preserved for 10 h versus baseline (n = 3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 h of cold (4 °C) preservation of human donor hearts (n = 3/group). c. Cold preservation induced gene expression was identified by comparing human donor heart with 10 h of preservation compared to baseline. Red dots indicate upregulated genes in EC population during cold storage while blue dots show the effect of canrenone on genes represented by the red dots. Light blue dots indicate downregulated genes in EC population during cold storage while purple dots show the effect of canrenone on genes represented by the light blue dots. Volcano plot demonstrates that Canrenone reverses the cold preservation-induced global up- and downregulated genes in EC. d. Heatmap of gene expression in EC of human donor hearts cold (4 °C) preserved for 10 h versus baseline (n = 3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 h of cold (4 °C) preservation of human donor hearts (n = 3/group). Baseline is where samples were collected in the back table immediately following preservation solution administration and removal of the donor heart from the surgical field.

Extended Data Fig. 9 Canrenone reverses cold (4 °C) preservation induced transcriptomic changes in smooth muscle cells (SMC) and leukocytes (LK) within human donor hearts during storage.

a. Cold preservation induced gene expression was identified by comparing human donor heart with 10 h of preservation compared to baseline. Red dots indicate upregulated genes in SMC population during cold storage while blue dots show the effect of canrenone on genes represented by the red dots. Light blue dots indicate downregulated genes in SMC population during cold storage while purple dots show the effect of canrenone on genes represented by the light blue dots. Volcano plot demonstrates that Canrenone reverses the cold preservation-induced global up- and downregulated genes in SMC. b. Heatmap of gene expression in SMC of human donor hearts cold (4 °C) preserved for 10 h versus baseline (n = 3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 h of cold (4 °C) preservation of human donor hearts (n = 3/group). c. Cold preservation induced gene expression was identified by comparing human donor heart with 10 h of preservation compared to baseline. Red dots indicate upregulated genes in LK population during cold storage while blue dots show the effect of canrenone on genes represented by the red dots. Light blue dots indicate downregulated genes in LK population during cold storage while purple dots show the effect of canrenone on genes represented by the light blue dots. Volcano plot demonstrates that Canrenone reverses the cold preservation-induced global up- and downregulated genes in LK. d. Heatmap of gene expression in LK of human donor hearts cold (4 °C) preserved for 10 h versus baseline (n = 3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 h of cold (4 °C) preservation of human donor hearts (n = 3/group).Baseline is where samples were collected in the back table immediately following preservation solution administration and removal of the donor heart from the surgical field.

Extended Data Fig. 10 Pseudotime analysis showing the modulatory effect of canrenone on cold donor heart preservation associated gene expression.

Density distribution plot showing the distribution of cells having differential transcriptome expression profile along pseudotime for cold (4 °C) preserved human hearts without reperfusion at a, baseline versus 10 h of preservation (n = 3/group) or b, for human hearts cold (4 °C) preserved for 10 h without reperfusion using HTK alone versus HTK + canrenone (CANR, n = 3/group). c, “Rolling wave” heatmap plot along pseudotime trajectory showing the differentially expressed genes ordered according to hierarchical clustering following 10 h of human heart preservation without reperfusion using HTK + VEH versus HTK + CANR (n = 4/group). P-values for pseudotime dependency using the Kolmogorov-Smirnov test are shown.

Supplementary information

Supplementary Information

Supplementary Figs. 1–3.

Reporting Summary

Supplementary Table 1

Supplemental data for metabolomic analysis of human perfused hearts.

Supplementary Video 1

Representative videos of pig hearts preserved with HTK ± canrenone for 4 and 10 h followed by ex situ perfusion.

Supplementary Video 2

Representative videos of human hearts preserved with HTK ± canrenone for 10 h followed by ex situ perfusion.

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Lei, I., Sicim, H., Gao, W. et al. Mineralocorticoid receptor phase separation modulates cardiac preservation. Nat Cardiovasc Res 4, 710–726 (2025). https://doi.org/10.1038/s44161-025-00653-x

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