ORIGINAL PRE-CLINICAL SCIENCE
Methane supplementation improves graft function in experimental heart transplantation

https://doi.org/10.1016/j.healun.2020.11.003Get rights and content

BACKGROUND

Maintenance of cell viability during cold storage is a key issue in organ transplantation. Methane (CH4) bioactivity has recently been recognized in ischemia/reperfusion conditions; we therefore hypothesized that cold storage in CH4-enriched preservation solution can provide an increased defense against organ dysfunction during experimental heart transplantation (HTX).

METHODS

The hearts of donor Lewis rats were stored for 60 minutes in cold histidine-tryptophan-ketoglutarate (Custodiol [CS]) or CH4-saturated CS solution (CS-CH4) (n = 12 each). Standard heterotopic HTX was performed, and 60 minutes later, the left ventricular (LV) pressure-volume relationships LV systolic pressure (LVSP), systolic pressure increment (dP/dtmax), diastolic pressure decrement, and coronary blood flow (CBF) were measured. Tissue samples were taken to detect proinflammatory parameters, structural damage (by light microscopy), endoplasmic reticulum (ER) stress, and apoptosis markers (CCAAT/enhancer binding protein [C/EBP] homologous protein, GRP78, glycogen synthase kinase-3β, very low-density lipoprotein receptor, caspase 3 and 9, B-cell lymphoma 2, and bcl-2-like protein 4), whereas mitochondrial functional changes were analyzed by high-resolution respirometry.

RESULTS

LVSP and dP/dtmax increased significantly at the largest pre-load volumes in CS-CH4 grafts as compared with the CS group (114.5 ± 16.6 mm Hg vs 82.8 ± 4.6 mm Hg and 3,133 ± 430 mm Hg/s vs 1,739 ± 169 mm Hg/s, respectively); the diastolic function and CBF (2.4 ± 0.4 ml/min/g vs 1.3 ± 0.3 ml/min/g) also improved. Mitochondrial oxidative phosphorylation capacity was more preserved (58.5 ± 9.4 pmol/s/ml vs 27.7 ± 6.6 pmol/s/ml), and cytochrome c release was reduced in CS-CH4 storage. Signs of HTX-caused myocardial damage, level of ER stress, and the transcription of proapoptotic proteins were significantly lower in CS-CH4 grafts.

CONCLUSION

The addition of CH4 during 1 hour of cold storage improved early in vitro graft function and reduced mitochondrial dysfunction and activation of inflammation. Evidence shows that CH4 reduced ER stress–linked proapoptotic signaling.

Section snippets

Materials and methods

The experiments were carried out on male Lewis rats (250–350 g; Charles River, Sulzfeld, Germany) in accordance with EU Directive 2010/63 for the protection of animals used for scientific purposes and in compliance with criteria set down in the US National Institutes of Health Guidelines for the Care and Use of Laboratory Animals. The study was approved by the national competent authority of Hungary National Scientific Ethical Committee on Animal Experimentation (ATET) under license number

Hemodynamic parameters of the transplanted grafts

After transplantation, increasing LV balloon volumes (pre-load) resulted in elevated LVSP and dP/dtmax, which were both significantly increased at the largest pre-load values in the CS-CH4 group compared with CS alone (Figure 2A and B). A similar change in diastolic function was noted at higher pre-load volumes, bringing about significantly elevated dP/dtmin values (p < 0.05) compared with CS, reflecting better myocardial relaxation (Figure 2C). CBF was also significantly (p < 0.05) higher

Discussion

This study aimed to investigate whether adding CH4 to a cold preservation solution modifies the graft function in experimental HTX. The hemodynamic efficacy of CS-CH4 storage was evidenced by increased LVSP, cardiac contractility, and coronary circulation as compared with CS-treated grafts. The sum of biochemical data showed that the CH4-containing HTK solution effectively reduced the degree of oxidoreductive stress in myocardial samples and significantly influenced several components of ER

Disclosure statement

The authors declare no conflicts of interest. This study was supported by a National Research Development and Innovation Fund of Hungary (National Research Development and Innovation Fund Grant K120232 and NVKP_16-1-2016-0017 [National Heart Program]), an Economic Development and Innovation Operative Programme grant (GINOP-2.3.2-15-2016-00015), an Higher Educational Institutional Excellence Program grant (TUDFO/47138-1/2019-ITM), and a Human Resources Development Operational Programme grant (

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    These authors have contributed equally to this work.

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