Mineralocorticoid Receptor Phase Separation Modulates Cardiac Preservation

Ienglam Lei; Hüseyin Sicim; Wenbin Gao; Wei Huang; Pierre Emmanuel Noly; Melissa R Pergande; Mallory C Wilson; Aurora Lee; Liu Liu; Ashraf Abou El Ela; Mulan Jiang; Sahar A Saddoughi; Jordan S Pober; Jeffrey L Platt; Marilia Cascalho; Francis D Pagani; Y Eugene Chen; Bertram Pitt; Zhong Wang; Richard M Mortensen; Ying Ge; Paul C Tang

doi:10.1038/s44161-025-00653-x

. Author manuscript; available in PMC: 2026 Jun 1.

Published in final edited form as: Nat Cardiovasc Res. 2025 May 19;4(6):710–726. doi: 10.1038/s44161-025-00653-x

Mineralocorticoid Receptor Phase Separation Modulates Cardiac Preservation

Ienglam Lei ^1,^2,³, Hüseyin Sicim ^2,³, Wenbin Gao ³, Wei Huang ², Pierre Emmanuel Noly ⁴, Melissa R Pergande ⁵, Mallory C Wilson ⁶, Aurora Lee ³, Liu Liu ², Ashraf Abou El Ela ², Mulan Jiang ², Sahar A Saddoughi ³, Jordan S Pober ⁷, Jeffrey L Platt ⁸, Marilia Cascalho ⁸, Francis D Pagani ², Y Eugene Chen ², Bertram Pitt ⁹, Zhong Wang ², Richard M Mortensen ¹⁰, Ying Ge ^5,⁶, Paul C Tang ^1,^2,^3,^*

PMCID: PMC12239668 NIHMSID: NIHMS2090191 PMID: 40389663

Abstract

Heart transplantation is the gold standard treatment for end-stage heart failure patients. 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.

Heart failure affects about 6.5 million adults in the United States and constitutes a major health challenge. While heart transplantation is the most effective treatment for end-stage heart failure, the demand for donor hearts far exceeds supply. Donor hearts are under-utilized with less than 50% of potential donors becoming actual heart donors¹. Inefficient use of this life saving resource is contributed by the 4-hour time limit for cold static preservation where the heart is mechanically arrested but still experiences ischemia. This also limits the geographic allocation of hearts due to logistical constraints². Prolonged preservation periods increase the risk for primary graft dysfunction (PGD) where donor heart function is insufficient for end-organ perfusion. PGD occurs in ~10-20% of heart transplants².

Clinical use of ex-vivo normothermic human heart perfusion platforms over the past several years has shortened the period of cold ischemia³. However, cold preservation solution-induced mechanical arrest of donor hearts is still needed to place the organ on the perfusion system. On arrival to the recipient hospital, the heart is re-arrested to disengage it from the apparatus for transplantation which imposes obligate ischemic time. Indeed, a recent trial using an ex-vivo normothermic cardiac perfusion system for transport to the recipient hospital showed that severe left ventricle (LV) or right ventricle (RV) PGD was still high at 10.7%³.

The molecular pathophysiology that mediates PGD is poorly understood. Ischemic mechanical arrest from preservation solution-induced disruption of cardiac action potential under cold temperatures can be expected to cause changes in physical molecular interactions. Liquid-liquid phase separation (LLPS) in biological systems is a reversible thermodynamic process where molecularly dense (condensate) and dilute liquid phases can become separate under certain biophysical conditions. These events can induce proteins, nucleic acids and receptor-ligand pairs to signal more efficiently with each other than the surrounding environment thus forming membraneless organelles⁴. Of relevance to organ transplantation, changes in protein concentration and ambient temperature are determinants of molecular condensate through LLPS⁵.

Molecular LLPS can be contributed by a protein’s intrinsic disordered regions (IDR) consisting of polar and charged amino acids interspersed with aromatic residues. These sequence regions promote a mixture of specific proteins to coalesce via weak bonds to form dynamic flexible conformations without being locked into a fixed three-dimensional structure⁶. Multivalency of macromolecules can also promote intermolecular interactions that enhance phase separation^7,8. Furthermore, increased protein expression can reach a saturating concentration that drives phase separation under specific conditions^7,8. These summative forces all contribute to phase separation and condensate formation.

The NH₂-terminal domains of steroid hormone receptors including mineralocorticoid receptors (MR) all contain IDRs with multivalency consisting of ligand and DNA binding domains⁹. However, dynamic phase separation was previously demonstrated only for glucocorticoid, androgen, estrogen and progesterone receptors^10-13. Enrichment of biomolecules within condensates increases their spatial proximity, facilitating more efficient biochemical processes such as transcription¹⁴. We hypothesize that LLPS is an alternative biological mechanism that determines graft quality following preservation.

It is generally accepted based on numerous clinical trials and translational science studies that MR is important for mediating myocardial dysfunction and heart failure^15,16. However, little is known about its role in modulating donor heart function in transplantation. In this study, we discovered that MR signaling is magnified by LLPS with condensate formation during cold preservation and contributes to donor heart dysfunction. Indeed, MR activation results in oxidative stress, inflammation, cell death and endothelial dysfunction¹⁷. We also show that pharmacological inhibition of MR signaling can improve human donor heart function and increase ischemic tolerance. Using single-cell transcriptomics, we identified cardiomyocytes as a major source of deleterious MR signaling. We demonstrate that molecular LLPS with condensate formation is a widespread biophysical phenomenon in the preservation of many different solid organs and are expected to play a critical role in governing organ preservation quality.

RESULTS

MR mediates cardiac preservation quality

We performed single-cell transcriptomics on cold preserved human hearts to identify major cell populations based on respective markers such as cardiomyocytes (TNNT2), fibroblast (FBN1), endothelial cells (PECAM1), smooth muscle cells (ACTA2), Leukocytes (PTPRC (CD45)), and neuronal cells (NRNX1, Extended Data Fig. 1a). Cluster analysis further revealed two distinct cardiomyocyte populations (CM1 and CM2) based on transcriptomic profiles. Gene Set Enrichment Analysis (GSEA) showed that CM1 gene expression profile had a predilection for pathways mediating cardiac contractility while CM2 favored expression of mediators associated with autophagy and mitochondrial energetics (Fig. 1b, Extended Data Fig. 1b-c). This is exemplified by CM1 having a higher expression of RYR2, a calcium channel protein important for excitation-contraction coupling¹⁸ as well as CACNA1C, a calcium channel component¹⁹ (Extended Data Fig. 1d-e). Conversely, CM2 is distinguished by a prominent expression of PINK1²⁰ and FIS1²¹ which are associated with mitophagy (Extended Data Fig. 1f-g). Interestingly, MR expression was preferentially expressed in the CM1 population (Fig. 1c, padj=1.1E-48, log2FC= 1.52).

Fig 1. — a, Uniform Manifold Approximation and Projection (UMAP) plot of single-cell transcriptomic analysis from cold (4°C) preserved human hearts (9,708 cells) showing clustered cell types including endothelial cells (EC), smooth muscle cells (SMC), fibroblasts (FB), leukocytes (LK), neuronal cells (NC) as well as cardiomyocytes (CM) 1 and 2. 3 HTK preserved and 3 HTK+Canr preserved human donor hearts at baseline and 10 hours of storage. b, Gene Set Enrichment Analysis (GSEA) showing results of relative Gene Ontogeny (GO) enrichment between the cluster-specific overexpressed genes in CM1 versus CM2 (adjusted p <0.05). c, Depicts MR (NR3C2) expression embedded in UMAP plot. Murine donor hearts with cardiomyocyte-specific MR deficiency (Myh6^CreERT2;MR^fl/fl) versus MR^fl/fl control in the d, ex-vivo perfusion model assessing cardiac contractility and e, relaxation over a 20-minute measurement period (n=10 for MR^fl/fl,n=8 for Myh6^CreERT2;MR^fl/fl ). A syngeneic heterotopic heart transplant model where donor hearts from Myh6^CreERT2;MR^fl/fl versus MR^fl/fl control are transplanted into wild type recipient C57BL/6J mice. After 24 hours, the hearts were assessed for f, contractility and g, relaxation over a 1 minute in-vivo measurement period (n=7 for MR^fl/fl,n=8 for Myh6^CreERT2;MR^fl/fl). Representative h, and quantification i of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and 8-OHDG staining in perfused murine hearts quantified with imaging analysis (n = 8 per group). Circulating j, cardiac troponin I (cTnI) and k, tumor necrosis factor (TNFα) were quantified with enzyme-linked immunosorbent assay (ELISA, n=7 for MR^fl/fl,n=8 for Myh6^CreERT2;MR^fl/fl). Data are presented as means ± SD. *P<0.05, **P< 0.01 by two-sided Mann-Whitney test.

Given the known role of MR activation in mediating cardiac dysfunction^15,16, we examined MR protein expression and showed that it increased over time during cold preservation of human hearts and also increased in ex-vivo perfused murine hearts following prolonged preservation (Extended Data Fig. 2a-b). Therefore, we examined MR’s role in donor heart dysfunction using a transgenic murine model where MR deficiency is induced in Myh6⁺ cardiomyocytes using tamoxifen as confirmed on immunohistochemistry (Myh6^CreERT;MR^f/f, Extended Data Fig 2c). Cardiomyocyte-specific MR deletion improved cardiac contractility and relaxation in both ex-vivo and syngeneic heterotopic transplant models (Fig. 1d-g). MR deletion donor hearts had less oxidative stress and cell death after ex-vivo perfusion (Fig. 1h-i). Consistent with reduced cardiac injury, transplanted Myh6^CreERT;MR^f/f donor hearts following 16 hours of preservation had reduced cytokine expression (IL-1β, IL-6, CXCL9, CXCL10, Extended Data Fig. 2d). Recipients of MR deficient hearts also had less circulating cTnI and TNF (Fig. 1j-k) as well as reduced donor heart infiltration with CD45⁺ leukocytes and Ly6C⁺ macrophages (Extended Data Fig.2e-f).

Preservation induces dynamic MR LLPS

We noticed that prolonged preservation of murine (Fig. 2a) and human (Fig. 2b) donor hearts was associated with increasing localization of MR as “punctate” nuclear structures. This was reminiscent of phase separation and nuclear condensate formation by other steroid receptors (e.g. androgen, estrogen, progesterone, and glucocorticoid receptors) for the purpose of transcriptional regulation^10-12. Interestingly, MR nuclear translocation and puncta formation in cardiomyocytes only occurred in the setting of hypoxia while bathed in cold (4°C) HTK preservation solution. No MR puncta formation was observed under conditions of normoxia while cells were cultured in 37°C culture media (Fig. 2c).

Using the Predictor of Natural Disordered region (PONDR, pondr.com) online software, we confirmed a positively charged N-terminal domain (NTD) with IDR properties based on a PONDR score ≥0.5 using both VLXT and VSL2 meta-predictor models as described previously by others²² (Fig. 2d). LLPS phenomena is potentiated by increases in local molecular concentrations above a certain threshold as well as by other physicochemical conditions (e.g. temperature, pH). Under these settings, it becomes thermodynamically more favorable for key molecular components to interact through coalescence followed by de-mixing until the free energy of the medium stabilizes²³. We show that green fluorescent protein (GFP) tagged MR displays a concentration threshold at about 10 μM for condensate formation as quantified by turbidity studies as well as direct visualization of fluorescence within test tubes (Fig. 2e-f). Round MR condensates are also appreciated on fluorescence microscopy (Fig. 2g). Its dynamic nature is demonstrated by the capabilities of condensate fusion (Fig. 2h) and fluorescence recovery after photobleaching (FRAP, Fig. 2i). FRAP uses a laser to bleach a portion of the condensate but recovers as unbleached molecules move dynamically into the previously bleached area²⁴. We then cloned GFP-MR missing the NTD (GFP-MRΔIDR) and confirmed drastically reduced ability for LLPS by direct visualization and quantitative turbidity analysis (Fig. 2j-k).

To interrogate for the phenomenon of MR LLPS within living cells, we confirmed fluorescence recovery using FRAP of MR nuclear condensates within cardiomyocytes exposed to cold hypoxic preservation (Extended Data Fig. 3a). We also generated a protein chimera (optoDrop²⁵) by fusing MR with the mCherry red fluorescence protein and the Arabidopsis cytochrome 2 (Cry2) protein which is then expressed in Human Embryonic Kidney (HEK293) cells (Extended Data Fig. 3b). MR-Cry2 fusion promotes Cry2-Cry2 homo-oligomerization upon blue light (450nm) exposure but only if there is an intrinsic propensity for MR to phase separate²⁵. Light exposure confirmed a strong propensity for full length MR to form red condensates but not if the NTD is deleted (Extended Data Fig. 3c). MR clusters are verified to represent phase transition upon FRAP testing (Extended Data Fig. 3d).

Disruption of MR LLPS improves donor heart function

We used adeno-associated virus 9 (AAV9) to express full-length MR (AAV-MR) and MR missing the IDR (MRΔIDR) to reconstitute MR in the hearts of mice with tamoxifen-induced MR deficiency in cardiomyocytes (Myh6^CreERT;MR^f/f, Fig. 3a) with verification of construct expression by transcript and protein levels (Fig. 3b-c). Mutant MRΔIDR (40 KDa) was only detected in the murine hearts infected with AAV9-MRΔIDR. In ex-vivo perfused murine hearts after 16 hours of preservation, hearts expressing MRΔIDR had much less MR condensate formation on visual inspection and quantification (Fig. 3cd-e) than AAV-MR hearts thus confirming the importance of the IDR for MR phase separation in-vivo. Importantly, AAV-MRΔIDR hearts had much improved contractility and relaxation compared to AAV-MR hearts. AAV-MRΔIDR cardiac function was not different from AAV-Luc control hearts (Fig. 3f-g). This is consistent with higher cleaved caspase 3 and Bax abundance indicating more cell death in AAV-MR hearts compared with AAV-MRΔIDR hearts (Fig. 3h-ih). Nongenomic MR effects of mitogen-activated protein kinases (MAPK) signaling via JNK and Erk2 were not different between the groups suggesting MR phase separation does not influence MR-related MAPK signaling events (Fig. 3j-k). MR with IDR deletion also experienced less oxidative stress after ex-vivo perfusion as evidenced by the 8-OHDG staining (Fig. 3j-k). We used AAV9-MRΔLBD to express MRΔLBD (mutant MR with ligand binding domain deletion from a.a. 726-984) in Myh6^CreERT;MR^f/f murine heart following tamoxifen administration. Construct protein expression was confirmed (Extended Data Fig. 4a) with western blot. Ex-vivo cardiac perfusion showed that expression of MRΔLBD, which is unable to respond to ligand, does not protect the heart during cold preservation (Extended Data Fig. 4b). This confirms that the harmful effect of MR signaling during cold preservation is ligand-independent. MRΔLBD is also able to phase separate (Extended Data Fig. 4c) as well as induce the expression of well-known MR target genes such as ZFP219, CAMK2D, and PER1 (Extended Data Fig. 4d). While neither aldosterone nor cortisol is normally present in the preservation solution mixture, we have now experimentally added aldosterone. This demonstrates that MR ligands can also reduce cardiac preservation quality after 12 hours of cold storage (Extended Data Fig 4e-f).

Fig 3. — a, Adeno-associated virus 9 (AAV) was administered to Myh6^CreERT2;MR^fl/fl mice 14 days prior to cardiac preservation. AAV9 was used to deliver cardiac specific constructs containing full length MR (AAV-MR), MR missing the IDR (AAV-MRΔIDR) and the AAV-Luciferase (AAV-Luc) control. b, After 16 hours of preservation followed by ex-vivo perfusion, construct expression in-vivo was verified by b, PCR for transcript (n=4-9) and c, western blot for protein expression. d, Immunostaining of MR condensates (red) with DAPI (blue). Representative fluorescence images of n=4/group. e, Quantitation of MR condensates loci per cell with comparison by two-sided Mann-Whitney test (n= 13 nuclei, the nuclei were from 4 hearts as described in d). Ex-vivo perfusion model assessing cardiac f, contractility and g, relaxation over a 20 minute measurement period in wild type (WT) and AAV9 treated Myh6^CreERT2;MR^fl/fl hearts (n=8/group). h, Western blot of cleaved caspase 3 and Bax with tubulin control as well as phospho- and total JNK and Erk2 in ex-vivo perfused AAV-Luc, AAV-MR, and AAV-MRΔIDR expressing Myh6^CreERT2;MR^fl/fl hearts with i, associated quantification (n=5/group). j, 8-OHDG staining (green) as marker of DNA oxidative stress shown in AAV-Luc, AAV-MR, and AAV-MRΔIDR expressing Myh6^CreERT2;MR^fl/fl hearts (Representative fluorescence images of n=8/group ) with k, quantification of fluorescent intensity (n=8).. Data are presented as means ± SD. *P<0.05, **P< 0.01. One-way ANOVA followed by Tukey's multiple comparison test was used for comparisons of more than 2 groups.

Drug inhibition of MR improves donor heart function

To determine the therapeutic role of pharmacological MR inhibition on donor heart preservation, we administered the drug canrenone to donor hearts. Canrenone is the water-soluble active metabolite of spironolactone which itself is poorly water soluble and not suitable for intravascular administration. Canrenone is clinically utilized as a mineralocorticoid receptor antagonist for atrial fibrillation therapy²⁶, and to treat chronic heart failure²⁷. Following canrenone dose-response titrations (Extended Data Fig. 5a), we added canrenone (50 μM) versus vehicle control to HTK solution to preserve murine hearts and titrated the cold storage duration. We confirmed that canrenone steadily improved cardiac function as preservation duration increased out to 16 hours (Extended Data Fig. 5b-c). Cardiac functional improvement was again demonstrated in the murine syngeneic transplant model which was accompanied by reduced circulating cardiac troponin I indicating reduced cardiac injury with canrenone administration (Extended Data Fig. 5d-f). We also confirmed that other MRAs (finerenone (50 μM) and spironolactone (50 μM)) similarly improved cardiac contractility and relaxation after prolonged cold storage (Extended Data Fig. 5g-h).

To further assess clinical translational potential, we show that canrenone improved the ex-vivo contractility and relaxation of pig hearts preserved for 10 hours with 300% improvement from baseline (Fig. 4a-b, Video 1). Canrenone also increased cardiac output, improved coronary blood flow and reduced circulating cTnI (Fig. 4c-e). After 4 hours of porcine heart preservation, canrenone similarly improved cardiac contractility and relaxation by about 17% (Extended Data Fig. 6a-b, Video 1). Cardiac output and coronary blood flow were also improved while cTnI levels in the perfusate was lower (Extended Data Fig. 6c-e, Table 1). In porcine hearts treated with canrenone, staining for 8-OHDG confirmed reduced oxidative stress (Fig. 4f-g) while TUNEL staining showed reduced cell death (Fig. 4h-i). In human hearts preserved for 10 hours, canrenone also greatly improved ex-vivo cardiac function (Fig. 4j-k, Video 2) while accompanied by increased cardiac output, coronary blood flow and reduced circulating cTnI suggesting greater cardiac viability (Fig. 4l-n, Table 2). There is also consistent with reduced Cleaved CASPASE 3 and BAX abundance in the human donor with canrenone treatment (Extended Data Fig. 6f-g).

Fig 4. — Ex-vivo assessment of pig hearts preserved with HTK ± Canrenone (Canr) treatment for 10 hours. Hemodynamic and coronary measurements recorded in working mode after 1 hour of perfusion include a, contractility (max dP/dt), b, relaxation (min dP/dt), c, cardiac output (ml/min/g) and d, coronary blood flow (n = 8 per group). e, Cardiac troponin I (cTnI) was quantified in ex-vivo perfusate for porcine heart (n = 8 per group). Oxidative DNA damage in porcine hearts was f, visualized with 8-OHDG staining which was g, quantified with imaging analysis (n = 4 per group). Cell death in porcine hearts was h, visualized by staining with TUNEL with i, quantification of percentage of TUNEL positive cells (n = 8 per group). We also performed ex-vivo analysis of human hearts preserved with HTK alone versus HTK+Canrenone (Canr) treatment for 10 hours. Hemodynamic and coronary measurements recorded in working mode after 1 hour of perfusion include j, contractility (max dP/dt), k, relaxation (min dP/dt), l, cardiac output (ml/min/g) and m, coronary blood flow . n, Cardiac troponin I (cTnI) was quantified in ex-vivo perfusate for the human heart (n = 4 for HTK+Veh group; n=5 for HTK+Canr group). Data are presented as means ± SD. *P<0.05, **P< 0.01 by two-sided Mann-Whitney test.

Table 1.

Pig heart hemodynamic data from ex-vivo perfusion experiments, two-sided Mann-Whitney test

Pig Donor heart Groups	HTK 4h	HTK+Canr 4h	P value	HTK 10h	HTK+Canr 10h	P Value
Heart Rate (bpm)	109.6±26.4	130.7±39.8	n.s.	114.3±34.0	93.3±16.7	n.s.
LV Systolic Pressure (mmHg)	91.5±22.3	118.6±28.2	n.s.	52.9±27.6	127.0±21.8	< 0.01
LV Diastolic Pressure (mmHg)	5.0±5.4	10.0±9.8	n.s.	9.8±8.5	11.6±11.3	n.s.
Heart Weight before (g)	264.8±48.1	267.2±31.1	n.s.	249.6±17.6	250.6±25.7	n.s.
Heart Weigh after (g)	282.9±41.6	278.1±28.8	n.s.	261.9±20.7	267.9±5.0	n.s.

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Table 2.

Human heart hemodynamic data from ex-vivo perfusion experiments, two-sided Mann-Whitney test

Human Donor heart Groups	HTK 10h (n=5)	HTK+Canr 10h (n=4)	P Value
Heart Rate (bpm)	75.0±14.2	76.4±9.5	n.s.
LV Systolic Pressure (mmHg)	35.0±21.5	78.6±9.8	< 0.01
LV Diastolic Pressure (mmHg)	8.0±3.8	5.0±2.8	n.s.
Heart Weight before (g)	504.4±53.8	484.0±95.4	n.s.
Heart Weigh after (g)	576.4±71.5	559.2±102.7	n.s.

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Canrenone reversed the transcriptomic alterations associated with cold preservation in multiple individual human cardiac cell types including cardiomyocytes as determined by single-cell transcriptome sequencing. Pathway genes associated with the negative impact of cold preservation which were reversed by canrenone treatment include those related to mitochondrial energetics, cardiac contractility, cell death, and inflammatory mediators (Extended Data Fig. 7-9). Pseudo-time analysis of the density distribuvtion of differentially expressed genes also confirmed the ability of canrenone to mitigated the cold preservation associated cellular transcript expression and trajectory patterns related to muscle contraction and inflammation within human donor hearts (Extended Data Fig. 10). Besides MR, canrenone can bind androgen, estrogen and progesterone receptors which may lead to off-target effects²⁸. However, GSEA of preserved human hearts showed that canrenone treatment did not impact the expression of downstream targets of androgen, estrogen nor progesterone receptors (Supplementary Fig.1-3).

Inhibiting MR alters genome landscape

We performed CUT&RUN to determine MR binding sites to the DNA regions that correspond to downstream gene expression in human hearts (Fig. 5a). K means clustering revealed 4 major groupings of gene sets that correlate with preservation duration and response to canrenone treatment in human donor hearts during cold static preservation. By 10 hour with HTK preservation alone compared with baseline, there was increased MR binding to gene sites related to hypoxia and catecholamine responses (in cluster (C) 2) as well as apoptosis (C4) but a reduction in binding to vascular endothelial growth factor gene sites (C3). Bearing in mind that the donor heart is already exposed to canrenone for about 20 minutes at the baseline time point given procurement logistics, we already see a reduction of binding to genes related to response to steroid hormone (C1) as expected, response to hypoxia (C2), and vascular endothelial growth factor signaling (C3). While binding to these genes did increase by 10 hours of preservation with canrenone, the frequency is overall less than with HTK preservation alone. MR binding gene target related to apoptosis (C4) with canrenone treatment at 10 hours was much lower than HTK alone (Fig. 5a).

The peaks in C3 and C4 have the longest DNA distances from MR binding site to the transcriptional start site suggesting greater activation of enhancer elements in addition to promoter binding in the expression of mediators of vascular endothelial growth factor signaling, apoptotic and metabolic processes (Fig. 5b). These changes in genome architecture reflect altered MR binding patterns to DNA and correlate with reduced MR phase separation induced by canrenone treatment during cold preservation of human hearts (Fig. 5c). In-vitro droplet assay confirms that MR antagonism with canrenone increases the concentration threshold needed for MR phase separation as shown by imaging (Fig. 5d) and turbidity quantification (Fig. 5e).

MR inhibition improves heart metabolism

Canrenone treatment increased the abundance of metabolic substrates for energy production such as acetyl-L-carnitine which is a precursor to acetyl-CoA²⁹. Glutamine was also increased with canrenone treatment, and this was reported to be cardioprotective for ischemia-reperfusion by increasing flux through the hexosamine biosynthesis pathway and by increasing protein O-GlcNAc levels³⁰. Conversely, canrenone reduced the abundance of harmful L-tyrosine which has known direct cardiac effects of reducing heart rate and contractile force³¹ (Fig. 5f). A comparison table for metabolite abundance in human donor heart preserved with HTK±canrenone is shown in Supplementary table 1.

LLPS is a theme for organ preservation

Increased osmolality is known to reduce the threshold for LLPS through the promotion of molecular crowding. Condensate formation is also recognized as a mechanism for responding to cellular stress³². Comparing the serum osmolality from living human individuals to that of organ donors, we noted significantly elevated serum osmolality in organ donors (301.18± 8.23 vs 352.27± 23.31 mOsmol/kg, P=0.001, Fig. 6a). Preservation solution also underwent a modest osmolality increase while human donor hearts are under cold static storage (293.25± 1.28 vs 313.25± 3.89, P<0.001, Fig. 6b). To test the physiological relevance of these increases in osmolality, we confirmed that an increase of 100 mOsm above physiological levels was sufficient to induce robust MR LLPS in cardiomyocytes (Fig. 6c). We examined condensate formation in a variety of murine organs to determine if molecular LLPS is a shared theme in organ preservation biology. Indeed, MR condensate formation during preservation was also demonstrated for kidney, lungs, and hearts, but not in the liver (Fig. 6d). Phase separation for other transcriptional mediators also occurred in an organ specific manner. Increased HDAC1 condensates were seen in kidney, liver, and lungs but not in hearts. Interestingly, a preservation-related increase in BRD4 phase separation was appreciated in all organs examined (Fig. 6d). The biological significance of other type of molecular condensates have yet to be determined.

Fig 6. — Osmolality was examined a, in the serum of brain (n=8) and cardiac (n=3) death donors as well as in preoperative living patients scheduled to receive left ventricular assist device implants (n = 11 per group, non-transplant) serving as control. b, Measurement of the osmolality of preservation solution samples from human donor hearts preserved at baseline and after 10 hours of storage prior to reperfusion (n = 8 per group). c, H9C2 cardiomyocytes were transfected using AAV9-GFP-MR and subjected to culture with DMEM (300 mOsm) under normoxic conditions (20% O₂) as control. We then added sorbitol to increase osmolality to 400mOsm mimicking the serum osmolality of organ donors. Data shown represent n=6 biological replicates. d, HTK (for kidneys, livers, hearts) and Perfadex solution (for lungs) were used to preserve murine organs at 4°C. Expression pattern of mineralocorticoid receptors (MR, red), histone deacetylase 1 (HDAC1, red) and bromodomain containing 4 (Brd4, red) were compared at baseline and after 16 hours cold preservation under fluorescence microscopy. Data shown represent n=4 biological replicates. Data are presented as means ± SD. *P<0.05, **P< 0.01 by two-sided Mann-Whitney test (a) or two-sided paired t-test (b).

DISCUSSION

Heart transplants are often the preferred treatment option for patients with end stage heart failure given that they provides optimal quality of life and long-term survival¹. However, this option is limited since demand greatly exceeds supply of human donor hearts. Current principles of cardiac preservation center around hypothermia³³ and mechanical arrest³³ with more recent developments of machine perfusion with oxygenation for organ transport³⁴. Conventional static cold storage of human hearts has an obligate preservation duration limit of around 4 hours. While organ perfusion strategies have lengthened the out-of-body time by about 3 hours³⁴, our ability to further extend the temporal limits of cardiac preservation is limited by a knowledge gap in our understanding of cardiac preservation biology. In this study, we highlight the phenomenon of molecular phase separation as a critical theme in static hypothermic organ preservation with mechanistic implications for the occurrence of primary graft dysfunction.

This study demonstrates the importance of MR signaling via phase separation during cold cardiac preservation in mediating donor heart dysfunction following perfusion. The effector mechanisms are likely through MR mediated oxidative stress and MR is known to activate NADPH oxidase-dependent (Nox2, Nox4) superoxide production³⁵. Indeed, clinical studies have demonstrated the importance of MR antagonism for improving cardiac function in the settings of chronic heart failure and recovery from myocardial infarction³⁶. The importance of MR in determining organ preservation quality was not previously recognized. Through single-cell RNA sequencing of cold preserved human hearts, we unexpectedly found that MR transcripts were most strongly expressed in a subset of cardiomyocytes (CM1) which also preferentially expressed transcripts related to cardiac contraction and conduction. Concurrently, there is another cardiomyocyte subset (CM2) that preferentially expressed gene sets related to autophagy and mitophagy. Through both pharmacological (canrenone) and cardiomyocyte-specific genetic murine models, we demonstrate that MR antagonism or deletion was able to improve donor heart preservation quality, cardiac function and ischemic tolerance. Importantly, we were able to confirm using pseudotime analysis that canrenone treatment of human hearts attenuated the cellular transcriptomic trajectory in response to cold static storage.

During our studies, we noted the presence of MR “dots” within the cardiomyocyte nucleus which greatly increased in abundance with longer preservation duration. We then confirmed their identity as LLPS states via fluorescence recovery after photobleaching, turbidity studies, optogenetic techniques utilizing the Cry2 protein²⁵ as well as observation of condensate fusion events. The MR protein structure is multivalent with DNA and ligand binding domains as well as an identified IDR that is critical for its phase separation abilities. Weak interactions such as pi-pi, cation-pi, dipole-dipole, or cation-anion occurs through the IDR. This allows condensates to avoid forming a fixed three-dimensional structure but rather adopt a flexible conformation necessary for dynamic protein-protein interactions³⁷.

Beyond the multivalent nature of the MR protein itself, there appears to be external forces that promote LLPS specifically during cold cardiac preservation. We showed that MR protein expression progressively increases during cold preservation. This can lead to MR exceeding a concentration threshold that is known to thermodynamically favor aggregation leading to de-mixing until free energy stabilization within the medium²³. We also reveal that both the donor death process and organ preservation are associated with ambient hyperosmolar states. The degree of osmolality increase associated with these scenarios are also confirmed to be physiologically relevant for LLPS. This osmolality related phenomenon was attributed to an increased concentration of biomolecules in the nucleus due to reduced nuclear volume and exclusion of DNA bound proteins from chromatin compaction³⁸. Steroid receptors share a similar structure consisting of a C-terminal ligand binding domain (LBD), a central DNA-binding domain (DBD) as well as two activation function regions (AF1 and AF2) in the corresponding N-terminal domain (NTD) and LBD³⁹. While steroid receptor LLPS was previously shown for glucocorticoid¹⁰, androgen⁴⁰, estrogen³ and progesterone receptors⁴¹, the occurrence of MR condensates was not previously confirmed to the best of our knowledge.

Through murine cardiomyocyte-specific MR knock-out in-vivo rescue studies that reconstitute full-length MR versus MR missing the IDR, we showed that impaired MR phase separation was able to improve cardiac function after prolonged cold static preservation. Impaired MR condensate formation also reduced cardiac cell death consistent with enhanced cardiac function. Using canrenone in porcine hearts, we showed that MR antagonism improved cardiac function by about 17% at the clinically accepted 4 hour time point which is similar to the meaningful improvement in forward flow provided by an intra-aortic balloon pump used clinically⁴². At the 10 hour cold static preservation time point, we were able to show a clear improvement in both pig and human cardiac function by about 300% compared to standard HTK solution alone. This was accompanied by several corroborative parameters of cardiac viability including reduced oxidative stress, reduced cell death, increased cardiac output and more robust coronary flow. It is well recognized that increased oxidative stress contributes to contractile dysfunction and myocardial injury⁴³. While a significant beneficial effect of MRA is seen at the clinically accepted threshold of cold preservation for 4 hours, we used a 10 hour preservation time point to increase the likelihood of observing a larger effect size using canrenone. This demonstrates the clinical potential of significantly extending cold preservation time beyond currently accepted durations.

Canrenone treatment greatly reduced the abundance of MR condensates in human heart. This is not surprising as canrenone is a competitive inhibitor at MR’s aldosterone binding site²⁶ and inhibition of ligand-binding sites in multivalent molecules is known to inhibit phase separation⁴⁴. Thus, our CUT&RUN results showed how increased human heart preservation times promoted the binding of MR to DNA-regions for transcription of genes related to apoptosis and hypoxic responses. While this occurred in the setting of MR condensate formation, canrenone treatment attenuated MR phase separation and promptly reduced the binding of MR to its DNA binding sites early after canrenone administration. This suggests that a high concentration of MR inside phase separated condensates potentiates intracellular reactions such as binding of transcription factors to their target DNA promoter and enhancer sites. Transcription factor phase separation is thus a way of exercising transcriptional control to promote activation or repression of a wide array of genes by modulating interactions with DNA binding sites and other cofactors⁴⁵.

Metabolomic analysis revealed that canrenone treatment resulted in a favorable energetic profile with increase abundance acetyl-L-carnitine, an energy substrate²⁹ as well as glutamine which is known to improve cardiac ischemic tolerance³⁰. Canrenone also reduced the abundance of harmful L-tyrosine abundance, which has been shown to reduce contractility and contribute to coronary vasoconstriction³¹. While these metabolic changes are associated with differences in MR condensate formation induced by canrenone, the exact mechanisms of these differences in relation MR phase separation remain difficult to determine in human studies.

Perhaps unsurprisingly, LLPS is a shared theme during cold preservation of other organs including the liver, kidney and lungs. Furthermore, other components of the transcriptional machinery such as HDCA1 and BRD4 were also seen to undergo phase separation as a response to cold preservation. Thus, the relevance of our study likely extends to solid organ preservation in general. We anticipate that pharmacological or genetic interventions on other scaffold or client components of phase separated condensates would impact preservation quality. Furthermore, examining phase separation of other proteins in various compartment may also shed light on mechanisms of organ impairment during preservation. For example, phase separation of cyclic GMP–AMP synthase (cGAS) in the cytoplasm is a mechanism for innate immune activation⁴⁶.

Our study has several limitations. While we have identified the importance of MR phase separation in cardiomyocytes for determining cardiac preservation quality, we have not determined the precise mechanism of MR activation in this setting. The enzyme 11b-hydroxysteroid dehydrogenase type 2 (11HSD2) is expressed by specific cell types such as endothelial cells and converts endogenous glucocorticoids into inactive metabolites thus promoting MR activation by aldosterone. However, the expression of 11HSD2 is virtually absent in cardiomyocytes so glucocorticoids are expected to be the major MR ligand⁴⁷. It is unclear how MR signaling occurs in the absence of steroid hormones while the heart is bathed in a solution consisting of amino acids and electrolytes. It has been shown in castration resistant prostate cancer that association of androgen receptors within a phase separated condensate is sufficient to activate signaling in a ligand-independent manner⁴⁸. Reconstitution experiments expressing the MR construct lacking the ligand domain suggest that the ligand binding domain is not needed for MR activation in the cold cardiac preservation setting. However, it is possible that the worsening of cardiac function in the setting of AAV9-mediated MR mutant expression results from off-target effects. While our studies suggest the occurrence of ligand-independent activation of MR during cold cardiac preservation, the experimental addition of aldosterone may also further intensify the MR phase separation and condensate formation process to account for the further worsening of cardiac function. High dose aldosterone (50μM) in our study did further worsen donor heart function. Effect of lower physiological doses of aldosterone on heart function will need to be further characterized in future studies. An alternative mechanism includes reports that cardiomyocytes can produce low basal levels of aldosterone that increases with angiotensin II stimulation⁴⁹. It is also possible that circulating corticosteroids and/or mineralocorticoids in the deceased donor have already initiated deleterious MR activation in the donor heart prior to procurement. Furthermore, pharmacological MR antagonism of porcine and human hearts likely also has effects on the vasculature that has yet to be characterized. However, the improvement in coronary blood flow following treatment is encouraging and may reflect reduced MR mediated inflammation within the endothelial layer¹⁷. For our animal models, we did not simulate donor death which can be associated with hemodynamics instability and catecholamine surges that can cause cardiac injury. However, our human donors were exposed to clinical brain death processes.

In conclusion, our study reveals that LLPS of cellular components is likely a generalized mechanism that determines the quality of organ preservation for transplantation. Specifically, MR condensate formation can be mitigated by pharmacological antagonism to improve donor heart function following transplantation. It is known that small molecule therapeutics can partition selectively within nuclear condensates such as MED1⁵⁰ which suggests that LLPS may be exploited by targeting drugs to membraneless compartments of interest⁵⁰. The close relevance of murine findings to large animals and humans will promote a larger scale systematic approach to biological mechanistic and therapeutic screening for developing novel biotechnologies to improve organ preservation. Use of pharmacological MR antagonism may further increase the allowable preservation time of donor hearts preserved with existing normothermic or hypothermic ex-vivo perfusion technologies to advance transplantation outcomes.

Methods

Ethical Approval

Mice:

All rodent experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Michigan-Ann Arbor (PRO00010630) and at Mayo Clinic (A00007364-23). Both 12 weeks male and female wild-type C57BL/6J (stock number 000664) were obtained from the Jackson Laboratories. Cardiomyocyte-specific deletion of MR was achieved by breeding Myh6-CreErt2 mice (stock number 005657) and MR^f/f mice (Dr. Mortensen).

Pigs:

The pig experiments were approved by the IACUC at the University of Michigan-Ann Arbor (PRO00009611) and Mayo Clinic (A00007365-23). Male and female Yorkshire pigs weighing 40-50 kg were purchased from the South Campus Animal Farms of Michigan State University.

Human donor heart studies were approved by the University of Michigan Institutional Review Board (HUM00131275) and Mayo Clinic Institutional Review Board (23-006893).

Procurement and preparation of human and pig heart tissue

Heart procurement for both human and pigs was performed as a clinical standard. Briefly, the human or pig donor heart is retrieved per clinical protocol by incising the inferior vena cava to drain the right heart and then the left atrial appendage is incised to drain the left heart. Subsequently, the ascending aorta is cross-clamped distal to the cardioplegia catheter and 1 liter of cold (4°C) HTK solution is infused into the coronaries at a perfusion pressure of ~100 mmHg to induce mechanical arrest. The heart was excised and infused with 2 liters of HTK±Canrenone. The heart was then stored in the perfusion solution on ice for 4 or 10 hours following ex vivo perfusion. Donor hearts with LVEF >50% and donors <70 years of age were used for our experiments. There were no differences between human donor hearts preserved with HTK only (n=5) versus HTK+Canrenone (n=4) in terms of average age (57.8+8.9 years and 52.8+10.0 years respectively, P = 0.523 by Mann-Whitney test), left ventricular ejection fraction (60.0+0.7% versus 65.0+4.1% respectively, P = 0.404 by Mann-Whitney test) and female sex distribution (60% (3/5) versus 75% (3/4) respectively, P > 0.999 by Fisher’s exact test). There were no differences in past medical history between the HTK only versus HTK+canrenone group for hypertension (60% (3/5) versus 50% (2/4) respectively, P > 0.999), hypercholesterolemia (20% (1/5) versus 0% (0/5) respectively, P > 0.999), diabetes (20% (1/5) versus 25% (1/4) respectively, P > 0.999), and intravenous drug use (0% (0/5) versus 25% (1/4) respectively, P = 0.444) by Fisher’s exact test. There were no differences in the cause of death in the HTK only versus HTK+canrenone groups which were intracranial hemorrhage (40% (2/5) versus 25% (1/4) respectively, P = 0.206), anoxic brain injury (40% (2/5) versus 50% (2/4) respectively, P = 0.167), and trauma (20% (1/5) versus 20% (1/5), P > 0.999) by Fisher’s exact test.

Murine ex-vivo heart perfusion

3-month-old Male and female WT and Myh6-CreErt2;MR^f/f mice were i.p. injected with tamoxifen (80mg/kg) for 3 constituted days. The mice were then anesthetized with i.p. injection of 100 mg/kg ketamine and 5 mg/kg xylazine, and heparin (2000 U/kg). The mouse heart was rapidly exposed through a sternal incision. The aorta was cannulated and retrograde perfused with 5mL HTK±Canrenone. The heart was stored in HTK±Canrenone for 16 hours at 4°C and then perfused in Langendorff apparatus (ADInstruments Inc., Colorado Springs, CO) with Krebs-Henseleit buffer. A balloon was placed in the LV through the left atrium, and left ventricular diastolic pressure was set to ~8 mmHg at initiation to measure the hemodynamics measurements. LabChart software was used to record the hemodynamics. After 1-hour perfusion, the LV was collected and snap-frozen for analysis. We did not appreciate any visual differences in the size of the control and the MR knockout or AAV-infected murine hearts.

We examined for sex-based differences in ex-vivo murine cardiac function outlined in Fig. 1d-e. There were no female (n=4) versus male (n=4) differences in MR^f/f hearts preserved with HTK in terms of ex-vivo max dP/dt (344.4±575.3 vs 429.3±1280.0 mmHg/s respectively, P= 0.48) or min dP/dt (−240.6±300.0 vs −216.1±31.9 mmHg/s respectively, P= 0.48). Additionally, no sex difference was seen with Myh6^CreErt;MR^f/f hearts for max dP/dt (1793.6±764.0 vs 1510.2±1020.9 mmHg/s respectively, P= 0.69) or min dP/dt (−948.2±256.8 vs −855.2±519.5 mmHg/s respectively, P=0.49).

Murine heterotopic heart transplantation

Cardiac grafts procured from WT C57Bl/6J mice following arrest with cold HTK±Canrenone were transplanted into the right neck of sex-matched syngeneic recipient mice following 16 hours of cold preservation in corresponding HTK±Canrenone. The donor’s ascending aorta and pulmonary artery were connected to the recipient's right common carotid artery and right external jugular vein, respectively. At 24 hours after transplant, a conductance catheter was introduced into the LV to measure hemodynamics. The entire left ventricular free wall tissue was collected for analysis.

There were no female (n=2) versus male (n=5) differences in MR^f/f hearts preserved with HTK in terms of ex-vivo max dP/dt (854.8±54.6 vs 861.2±380.8 mmHg/s respectively, P= 0.86) or min dP/dt (−492.8±91.1 vs −455.3±251.1 mmHg/s respectively, P= 0.86). Additionally, no sex difference was seen with Myh6^CreErt;MR^f/f hearts for max dP/dt (1949.9±838.0 vs 1642.8±447.6mmHg/s respectively, P= 0.79) or min dP/dt (−1047.5±418.2 vs −1073.6±217.6 mmHg/s respectively, P= 0.49) by Mann-Whitney test.

Porcine and human ex-vivo heart perfusion

The porcine and human hearts were perfused in Radnoti perfusion apparatus(ADInstruments Inc., CO). Approximately 1 liter of autologous blood was mixed with ~3.5 liter KH buffers for perfusion. The perfusion pressure was kept constant at ~80 mmHg for the whole perfusion process. Shockable arrhythmias such as ventricular tachycardia, ventricular fibrillation, supraventricular tachycardia was electrically cardioverted to sinus rhythm by delivery of 10 or 20 joules of energy. Cardiac performance was assessed in “resting” and “working” modes as previously described by our group ⁵¹. The left atrial pressure in resting mode was typically 0-1 mmHg. To assess cardiac hemodynamics in “working mode”, perfusate was infused into the left atrium at 4-10mmHg at the 4-hour time point for LV loading and cardiac output assessment. Because cardiac performance was highly impaired as expected at the 10-hour time point, we loaded the left atrium to a pressure of up to 15mmHg to assess cardiac output. The Miller conductance catheter was introduced into the left ventricular cavity for measurement of ventricular performance parameters. The heart was perfused ex vivo for 3 hours and then arrested with 4°C HTK solution for specimen collection in formalin or snap frozen with liquid nitrogen.

We examined for sex-based differences in porcine cardiac function outlined in Extended Fig 5a-b. There were no female (n=3) versus male (n=5) differences following 4 hours porcine heart preservation with HTK only for ex-vivo max dP/dt (1714.3±376.3 vs 1711.6±150.1 mmHg/s respectively, P=0.57) or min dP/dt (−1080.7±120.3 vs −1099.7±379.6 mmHg/s respectively, P=0.79). Additionally, no sex difference was seen with HTK+canrenone preservation for max dP/dt (2113.7±251.1 vs 2108.7±410.0 mmHg/s respectively, P>0.99) or min dP/dt (−1390.1±52.1 vs −1360.1±129.2 mmHg/s respectively, P>0.99).

We also examined for sex-based differences in porcine cardiac function outlined in Fig 4a-b. There were no female (n=3) versus male (n=5) differences after 10 hours of porcine heart preservation with HTK only for ex-vivo max dP/dt (552.5±399.5 vs 611.5±442.1 mmHg/s respectively, P=0.79) or min dP/dt (−434.8±359.3 vs −471.5±282.2 mmHg/s respectively, P>0.99). Furthermore, no sex difference was seen with HTK+canrenone preservation for max dP/dt (1624.8±147.9 vs v 1648.8±269.0 mmHg/s respectively, P=0.79) or min dP/dt (−1352.7±253.0 vs −1232.9±213.0 mmHg/s respectively, P=0.57) by Mann-Whitney test.

Mouse serum assay and pig/human perfusate assay

Blood from recipient mice was centrifuged at 3000g in 4°C for 10 minutes. Serum cTnI, and TNFα concentrations were measured using Sigma-Aldrich MILLIPLEX kits (Cat. No.: MCYTMAG, MCVD2MAG). Pig and human arterial perfusate was centrifuged at 3000g in 4°C for 10 minutes. The abundance of cTnI was detected using Sigma MILLIPLEX kit (Cat. No.: HCVD1MAG). Blood from mice was obtained during the time of sacrifice at 24 hours after heterotopic syngeneic heart transplantation, Pig and human perfusate samples were collected at 3 hours just prior to end of ex-vivo perfusion.

Human blood collection for measurement of serum osmolality

Human blood was collected in left ventricular assist patients (LVAD) intraoperatively prior to making incision for LVAD implantation surgery. Human donor blood was collected prior to incision for organ procurement. Blood was spun down at 2000 xG for serum collection. Serum osmolality was measured using the Precision Systems^™ Touch Micro-Osmette^™ Osmometer (Model 6002; Natick, Massachusetts, United States) as per manufacturer instructions. There were no differences between LVAD patients (n=11) and human donor (n=11) in terms of average age (60.9+7.6 years and 54.5+12.6 years respectively, P = 0.133 by Mann-Whitney test), and female sex distribution (36.4% (4/11) versus 54.5% (6/11) respectively, P = 0.670 by Fisher’s exact test). Comorbidities between LVAD patients versus human donors were similar in terms of hypertension (36.4% (4/11) versus 45.5% (5/11) respectively, P = 1.000), hypercholesterolemia (27.3% (3/11) versus 9.1% (1/11) respectively, P = 0.586), and diabetes (9.1% (1/11) versus 9.1% (1/11) respectively, P = 1.000) by Fisher’s exact test.

Preservation solution collection for osmolality analysis during human donor heart preservation

Preservation solution at the baseline time point and after 10 hours of storage. Preservation solution osmolality was measured using the Precision Systems^™ Touch Micro-Osmette^™ Osmometer (Model 6002; United States) as per manufacturer instructions. Demographics and comorbidities information for the donors are as follows: Age 55.8±9.2 years; Sex 75% (6/8); Hypertension 50% (4/8); Hyperlipidemia 0% (0/8); Diabetes 12.5% (1/8).

Plasmid

pET30 expression vector was used as backbone of protein expression plasmids. GFP without stop codon at N-terminal , 14 amino acid linker sequence (GAPGSAGSAAGGSG) ⁵², in-frame full-length MR or MRΔIDR (a.a.1-599 of MR is deleted) was cloned into pET30 using NEB HIFI builder mix.

Protein purification

For protein expression, pET30 plasmids containing the GFP, GFP-MR or GFP-MRΔIDR were transformed into LOBSTR cells. A fresh colony was inoculated into LB media with kanamycin and chloramphenicol for 8 hours. The cells were added to 500 mL LB with 1mM IPTG for 18 hours of culture at 16 °C. Cells pellets were resuspended in 15mL of denaturing buffer (50mM Tris pH7.5, 300mM NaCl, 10mM imidazole, proteinase inhibitor), and sonicated at 10 cycles of 15s on and 60s off. The lysate was centrifuged at 12000 g for 30 minutes at 4°C.The supernatant was incubated with Ni-NTA agarose at 4°C for 1.5 hours. The agarose was centrifuged at 600xg for 10 minutes, and wash with 5mL of wash buffer (50mM Tris pH7.5, 300mM NaCl, 50mM imidazole, proteinase inhibitor). The protein was eluted with 3 times elution buffer (50mM Tris pH7.5, 300mM NaCl, 250mM imidazole, proteinase inhibitor). The fusion proteins were concentrated in 50mM Tris pH7.5, 125mM NaCl and 1mM DTT buffer using Amicon Ultra centrifugal filters (30K MWCO, Sigma) with 3 times centrifuge in the buffer. The purified proteins were then used for further examination.

Turbidity Measurements.

The turbidity of protein solutions was determined as the absorption at 340 nm using a Promega GloMax plate reader. The recombinant proteins were diluted to two times of the final concentration (1,3, 10,30 uM) in 50 mM Tris-HCl pH7.5, 10% Glycerol, and 1 mM DTT. The samples were then diluted with one volume of 20% PEG-8000 and 250 mM NaCl for turbidity measurement.

In vitro droplet imaging and Fluorescence recovery after photobleaching (FRAP)

Recombinant GFP, GFP-MR, GFP-MRΔIDR proteins were diluted to 20uM in in 50mM Tris-HCl pH7.5, 10% Glycerol, and 1mM DTT on ice. The proteins were then diluted with one volume of 20% PEG-8000 and 250 mM NaCl. The protein solution was added to a glass slide with an imager spacer and sealed with a coverslip. The slide was analyzed using Nikon A1 confocal after 5 minutes of incubation. For FRAP, specific droplets were bleached using 40% intensity, and images were collected every other second for 60 seconds. Fluorescence intensity at the bleached and unbleached spots was measured using the NIS element software. The values were then normalized to the intensity at the initial time before bleaching.

Fluorescence immunohistochemistry

The mice, pig or human hearts were embedded with Optimal Cutting Temperature compound at −70°C and sectioned into 7 μm slides using cryostat. The sections were fixed in cold methanol for 10 minutes and then boiled in 10mM citrate buffer pH6.0 for 10 minutes for antigen retrieval. The slides were blocked with 5% bovine serum albumin, and incubated with the following antibodies at 4°C overnight: anti-NR3C2 antibody (1:100 dilution, Sigma, AV45599 (targets the antigen located at the DNA binding domain of MR protein (aa651-691)); or CST, 58883S), anti-8OHdG (1:100, Santa Cruz, sc393871), anti-HDAC1 (1:100 dilution, CST 34589), anti-Brd4 (1:200, Abclonal, A20019). After three washes with phosphate buffered saline with Tween 20(PBST), a secondary antibody conjugate with Alexa Fluor 488/647 was applied for 1 h. The sections were mounted with anti-fade mounting medium (Thermofisher Scientific, P36930) for image evaluation using the BZ-X800 (Keyence)/LSM 980 microscope. The images were analyzed with ImageJ software for staining intensity quantification and enumeration of positively stained cells.

Western blotting

Proteins were extracted using lysis buffer with 1X complete EDTA-free proteinase inhibitor cocktail (Roche) followed by centrifugation at 4°C for 15 min at 15,000 g. Total protein concentration was measured by Bradford assay, and 40 μg of protein was then separated by SDS-PAGE for transfer to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% nonfat dry milk and then incubated with primary antibodies overnight at 4°C. After washing with PBST, the membranes were incubated with diluted secondary antibody at room temperature. The membranes were then quantified by Azure Image System. Primary antibodies utilized include α-tubulin (1:2000, Cell Signaling Technology, 3873), Bcl2 (1:2000, CST, 3498), Cleaved Caspase 3 (1:2000, Abclonal, A2156), phosphor-JNK (1:1000, CST, 9251), total JNK (1:1000,CST, 9252), phosphor-Erk1/2 (1:1000, CST, 4370), total Erk1/2 (1:1000, CST, 4696) and MR (1:1000, Sigma, AV45599). Secondary antibodies include: IRDye^® 680RD Donkey anti-Mouse IgG (1:2000, LI-COR, 926-68072), IRDye^® 800CW Donkey anti-Rabbit IgG (1:2000, LI-COR, 926-32213), Peroxidase AffiniPure Donkey Anti-Mouse IgG (1:3000, Jackson ImmunoResearch Laboratories, 715-035-150), Peroxidase AffiniPure Donkey Anti-Rabbit IgG (1:3000, Jackson ImmunoResearch Laboratories, 715-035-152).

Adeno-associated virus 9 packaging and delivery

The full-length MR, MRΔIDR (MR lacking a.a.1-599), and MRΔLBD (lacking a.a 726-984) coding sequences were amplified from mouse heart cDNA and cloned into the AAV9 plasmid (AAV9:cTNT:3Flag-hYAP S127A, Addgene, 86558). Gene expression was driven by cTnT promoter. The hYAP S127A was replaced by MR and MRΔIDR and the 3xFlag was kept for protein identification. Viral particles expressing MR, MRΔIDR, MRΔLBD and Luciferase were produced by the University of Michigan vector core or VectorBuilder. 1X10¹² viral particles were i.p. injected into 10-weeks-old Myh6-CreErt2;MR^f/f mice. After 2 weeks, the mice were treated with tamoxifen (80mg/kg) for 3 days, and then used ex vivo perfusion study.

NMCM and NRCM isolation

Neonatal mice pups were obtained by crossing C57BL/6J mice in our animal facility, and the neonatal rat pups were from the timed-pregnant rat purchased from Charles River Laboratories. Isolation of NMCM and NRCM followed a similar protocol except for neonates. The neonate heart was removed with a sterile tweezer and placed in cold PBS then cut into small pieces and incubated with Trypsin-EDTA at 4°C overnight. On the second day, Trypsin was removed, and heart tissues were washed with Hanks' Balanced Salt Solution (HBSS). The heart tissue was then incubated in 15mL 1mg/mL collagenase II/HBSS at 37°C with stirring for 15 minutes. The tissues were then centrifuged and resuspended in a culture medium and passed through a 70uM cell strainer. The cardiomyocytes in the culture medium were enriched via two rounds of 45-minute culture. Purified cardiomyocytes from the third supernatant were then counted and seeded at a density of 1 million cells per 10 cm². Once the cardiomyocytes were attached to the dish, the culture medium was changed to 5% Horse serum in DMEM.

Single nuclei RNA-seq

The human heart tissues were collected from basal free wall of the left ventricle in donor hearts stored at baseline and 10 hours stored in HTK±canrenone (n=3 per group). The single nuclei were isolated and followed by chromium 10x library preparation using fix RNA profiling platform in the Advanced Genomic Core at the University of Michigan. Libraries were then sequenced using NovaSeq 6000 (Illumina, California). The raw sequencing results were aligned to human genome GRCh 38 and aggregated with CellRanger v7.1.0⁵³. The count matrix was further processed with Seurat v4.4.0⁵⁴. The data was filtered with RNA count < 25000, RNA count > 1000, and % of mitochondrial gene < 5%, and then normalized with SCTransform⁵⁵. UMAP was used to visualize data⁵⁶ for dimension reduction. FindClusters function⁵⁷ was used for cluster detection with the default resolution (0.8). We then annotated the cell types of each cluster based on the highest positive fold changes and correlated with relevant specific cell markers described in the literature⁵⁸. In addition to the graphical display, we used the FindMarkers function⁵⁹ in the Seurat package to perform differential expression gene (DEG) analysis among phenotype-associated subpopulations. DEG changes between conditions including HTK versus HTK+canrenone preservation as well as preservation duration comparing baseline versus 10 hours were also analyzed using FindMarkers function (Wilcoxon rank-sum test (two-sided), adjust p < 0.05, log2 fold change > 1.5 or <0.7).

Pseudotime trajectory analysis

We used Slingshot⁶⁰ to order the pseudotime trajectories of cardiomyocytes and identified gene expression patterns that were altered by canrenone during preservation. Cells isolated from donor hearts at the baseline time point served as the cluster starting point. The cell density was plotted in pseudotime order to compare the progression distribution difference of cardiomyocytes in HTK+canrenone using Komogorov-Smirnov test. To assess the significant changes in gene expression between HTK and HTK+canrenone alongside the pseudotime, we used the conditionTest function in tradeseq package⁶¹. The DEG was identified with fold change >2 or <1/2 and fdr < 0.05 with Benjamini & Hochberg correction. The significant DEGs were then visualized using the smoothed expression patterns using the heatmap function.

CUT&RUN sequencing

The CUT&RUN-seq was performed using Epicypher kit (SKU: 14-1048). The nuclei were isolated from flash-frozen human heart tissues and fixed with 0.1% formaldehyde for 1 minutes. Antibody against MR (Sigma, AV45599) was performed to characterize MR binding sites. The released CUT&RUN DNA was used for sequencing library preparation with NEBNext Ultra II DNA Library Prep Kit (E7103, NEB) and sequencing with 2X150 bp paired-end mode. The sequencing results was trimmed using Trimmomatic ⁶²and aligned to the human genome using bowtie2 with dovetail function. The peaks were called with MACS3. The heatmaps were generated using Deeptools⁶³.

Metabolomic Analysis

One milligram (±0.1 mg) of each of the HTK±canrenone human left ventricle tissues was suspended in 500 μL of ice-cold methanol containing internal standards and homogenized using bead beating (S=0.8, T=5 min, D=0 min, cycles=1). One milliliter of ice-cold chloroform was added to each sample, vortexed and incubated for 10 minutes on ice. 200 μL of water were added to the sample and vortexed, then centrifuged at 2000 g for 2 minutes. The organic (containing primarily lipids) and aqueous (containing primarily metabolites) phases were removed and each dried in vacuo.

The organic phase was resuspended in 200 μL 1:5 chloroform:methanol and the aqueous phase was resuspended in 1:1 acetonitrile:water. Both phases were analyzed using an Acuity ultra-performance liquid chromatography (UPLC) system (Waters, MA) coupled with a MaXis II quadrupole-time-of-flight (Bruker, Germany) mass spectrometer⁶⁴. Mass spectral data in both positive and negative modes were collected at 3 Hz within a window ranging from m/z 50-1300. Organic phase extracts were separated via reversed-phase chromatography (RPLC) using an ACUITY UPLC HSS T3 (Waters, MA) column using a 30 min gradient (0-10’ 50-82% B, 10-20’ 82-99% B, 20-25’ held at 99% B, 25-25.1’ 99-50 % B, 25.1-30’ held at 50% B) at 30 μL/min flow. Aqueous phase extracts were separated via hydrophilic interaction liquid chromatography (HILIC) using an ACUITY UPLC BEH Amide column (Waters, MA) operating on a 30 min gradient (0-0.5’ held at 99% B, 0.5-25’ 99-30% B, 25-25.1’ 30-99% B, 25.1-30’ held at 99% B) at 30 μL/min.

Bucket lists were generated using the software’s T-ReX 3D algorithm for positive and negative modes individually, then merged after feature table assessment (≤5.0 ppm mass error, 30 sec retention time). Annotations of known metabolites were performed in MetaboScape by search-matching collected tandem MS data with public metabolite databases from MassBank (via the in-program MCube Spectral Library Annotation Method). Metabolite intensities were normalized using the Probabilistic Quotient Normalization function in MetaboScape after manual identification and annotation of spiked standards. Significantly altered metabolites were determined in MetaboScape using a T-test (adjusted p-value < 0.5).

Statistical Analysis

Statistical evaluation of experimental data was performed using Prism 10.0 (GraphPad Software). Comparisons for 2 groups will use Mann-Whitney U test while >3 groups will use one-way ANOVA with post hoc analysis as described in the figure legends. P values of <0.05 were deemed significant. Figure legends provide the exact P values, definition, and number of replicates corresponding to each dataset.

Sample size estimates account for the variability in multiple physiological and biomolecular endpoints. Given the parameter variability in human and animal cardiac hemodynamic and biological responses shown by other research groups^65-68, and our previous published studies^51,69,70, we expect sample sizes of about 7-8 animals per group to be adequate for detecting differences with significance (α)=0.05 and power=0.80.

Extended Data

Extended Data Fig. 3. — 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 hours. 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 seconds 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.

Extended Data Fig. 4. — 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 hours 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 hours 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 hours (n=4 per group). Murine donor hearts were cold preservation with HTK only versus HTK + aldosterone (50 μM) (n=8/group) for 12 hours 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.

Extended Data Fig. 5. — a, Canrenone dose titration with murine donor hearts cold (4°C) preserved for 16 hours 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 hours 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.

Extended Data Fig. 6. — Ex-vivo assessment of pig hearts preserved with HTK ± Canrenone (Canr) treatment for 10 hours. Hemodynamic and coronary measurements recorded in working mode after 1 hour 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.

Extended Data Fig. 7. — a. Cold preservation induced gene expression was identified by comparing human donor heart with 10 hours 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 hours versus baseline (n=3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 hours 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 hours 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 hours versus baseline (n=3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 hours 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. — a. Cold preservation induced gene expression was identified by comparing human donor heart with 10 hours 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 hours versus baseline (n=3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 hours 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 hours 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 hours versus baseline (n=3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 hours 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. — a. Cold preservation induced gene expression was identified by comparing human donor heart with 10 hours 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 hours versus baseline (n=3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 hours 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 hours 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 hours versus baseline (n=3/group). Juxtaposed to the right is the HTK+Canr versus HTK+Veh effect on gene expression following 10 hours 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. — 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 hours of preservation (n=3/group) or b, for human hearts cold (4°C) preserved for 10 hours 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 hours 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 Material

Video 1

Download video file^{(17.6MB, mov)}

Video 2

Download video file^{(16.8MB, mov)}

Supplementary Table 1

NIHMS2090191-supplement-Supplementary_Table_1.xlsx^{(61KB, xlsx)}

Supplementary Figures 1-3

NIHMS2090191-supplement-Supplementary_Figures_1-3.pdf^{(1.3MB, pdf)}

Acknowledgments:

We are very grateful for the generosity of donors and “Gift of Life Michigan” organ procurement organization who provided hearts for research through the procurement facility. We also thank Essential Pharmaceuticals LLC, for the gift of 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 Dr. Sarcon Aida 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, HL109946 (Y.E.C.), Thoracic Surgery Foundation – Southern Thoracic Surgery Association (P.C.T.), Gardner Surgical Investigator Award- American Association for Thoracic Surgery (P.C.T.), McKay Research Grant (P.C.T.), Frankel Cardiovascular Center, University of Michigan-Ann Arbor (to P.C.T.), Mayo Clinic (to P.C.T.), and 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. would like to acknowledge NIH R01HL109810. The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

Footnotes

Data and materials availability: Raw sequencing and processed data generated in this study are deposited into NCBI Gene Expression Omnibus (GEO) with accession number GSE261124. Raw and processed mass spectral data are available in MassIVE server with accession number MSV000096745. Source data are provided in the supplementary materials. 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.

Competing interests: I.L., Z.W., F.D.P., B.P., Y.E.C., and P.C.T. have filed a U.S. 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). Ashraf Abou El Ela has a consulting agreement with TransMedics, Inc. F.D.P. is an ad hoc, non-compensated scientific advisor for Medtronic, Abbott, FineHeart and CH Biomedical, a non-compensated 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 non-compensated member of the Data Safety Monitoring Board for the XVIVO Perfusion Inc., PRESERVE trial. The remaining authors declare no competing interests.

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Associated Data

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Supplementary Materials

Video 1

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Video 2

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Supplementary Table 1

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Supplementary Figures 1-3

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PERMALINK

Mineralocorticoid Receptor Phase Separation Modulates Cardiac Preservation

Ienglam Lei, PhD

Hüseyin Sicim, MD

Wenbin Gao, MD, PhD

Wei Huang, MD

Pierre Emmanuel Noly, MD

Melissa R Pergande, PhD

Mallory C Wilson, BS

Aurora Lee, MD

Liu Liu, PhD

Ashraf Abou El Ela, MD

Mulan Jiang, B.S.

Sahar A Saddoughi, MD, PhD

Jordan S Pober, MD, PhD

Jeffrey L Platt, PhD

Marilia Cascalho, PhD

Francis D Pagani, MD, PhD

Y Eugene Chen, MD, PhD

Bertram Pitt, MD

Zhong Wang, PhD

Richard M Mortensen, MD, PhD

Ying Ge, PhD

Paul C Tang, MD, PhD

Abstract

RESULTS

MR mediates cardiac preservation quality

Fig 1. MR is preferentially expressed in human cardiomyocytes during cardiac preservation and negatively impacts donor heart function after reperfusion.

Preservation induces dynamic MR LLPS

Fig. 2. MR undergoes liquid-liquid phase separation into nuclear condensates under cold hypoxic preservation conditions.

Disruption of MR LLPS improves donor heart function

Fig 3. IDR of MR governs the capacity for phase separation and determines cardiac preservation quality.

Drug inhibition of MR improves donor heart function

Fig 4. Canrenone treatment significantly improved porcine and human donor heart function after prolonged preservation and reduced cardiac injury.

Table 1.

Table 2.

Inhibiting MR alters genome landscape

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.

MR inhibition improves heart metabolism

LLPS is a theme for organ preservation

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.

DISCUSSION

Methods

Ethical Approval

Mice:

Pigs:

Procurement and preparation of human and pig heart tissue

Murine ex-vivo heart perfusion

Murine heterotopic heart transplantation

Porcine and human ex-vivo heart perfusion

Mouse serum assay and pig/human perfusate assay

Human blood collection for measurement of serum osmolality

Preservation solution collection for osmolality analysis during human donor heart preservation

Plasmid

Protein purification

Turbidity Measurements.

In vitro droplet imaging and Fluorescence recovery after photobleaching (FRAP)

Fluorescence immunohistochemistry

Western blotting

Adeno-associated virus 9 packaging and delivery

NMCM and NRCM isolation

Single nuclei RNA-seq

Pseudotime trajectory analysis

CUT&RUN sequencing

Metabolomic Analysis

Statistical Analysis

Extended Data

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

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

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.

Extended Data Fig. 4. Cryopreservation is ligand independent.

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

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

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.

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.

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.

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

Supplementary Material

Acknowledgments:

Footnotes