Physiological and Pathophysiological Roles of Mitochondrial Na+-Ca2+ Exchanger, NCLX, in Hearts

It has been over 10 years since SLC24A6/SLC8B1, coding the Na+/Ca2+/Li+ exchanger (NCLX), was identified as the gene responsible for mitochondrial Na+-Ca2+ exchange, a major Ca2+ efflux system in cardiac mitochondria. This molecular identification enabled us to determine structure–function relationships, as well as physiological/pathophysiological contributions, and our understandings have dramatically increased. In this review, we provide an overview of the recent achievements in relation to NCLX, focusing especially on its heart-specific characteristics, biophysical properties, and spatial distribution in cardiomyocytes, as well as in cardiac mitochondria. In addition, we discuss the roles of NCLX in cardiac functions under physiological and pathophysiological conditions—the generation of rhythmicity, the energy metabolism, the production of reactive oxygen species, and the opening of mitochondrial permeability transition pores.

Hesperidin Extracted from Citrus reticulata Blanco Protects Cardiac Mitochondria Against Hypoxia/Reoxygenation Injury

This study was conducted to evaluate the protective effect of Hesperdin (Hes) extracted from Citrus reticulata Blanco on cardiac mitochondria in hypoxia/reoxygenation (HR) injury in vitro. Methods: H9C2 cardiomyocytes were cultured under normal (control), HR, and treatment conditions. The reactive oxygen species and calcium levels in experimental groups were analyzed by using suitable fluorescence kits. Results: The obtained results showed that the addition of Hes at dose of 0,01562 mg/mL sharply decreased the mitochondrial oxidative stress of H9C2 cells under HR conditions. In particular, Hes showed the remarkable efficiency in maintaing cellular calcium levels. In HR-exposed H9C2 cell group, the hydrogen peroxide and superoxide levels were highly increased compared to those in control group (1,54±0,06 and 1,74±0,38, p<0,05). HR also strongly induced the elevation of cytosolic Ca²⁺ and mitochondial Ca²⁺ of H9C2 cardiomyocytes with the values were 1,96±0,05% and 1,62±0,33 (ratio to control, p<0,05), respectively. Interestingly, post-hypoxic supplementation of Hes effectivelly abolished the negative incresement of these indicators with the lower levels of reactive oxygen species and the better modulation of Ca²⁺ homeostasis. Conclusion: The present results are pilot data on the effects of Hes in protecting cardiac mitochondria against HR injury.

EFHD1 ablation reduces cardiac mitoflash activation and protects cardiomyocytes from ischemia.

Altered levels of intracellular calcium (Ca2+) are a highly prevalent feature in different forms of cardiac injury, producing changes in contractility, arrhythmias, and mitochondrial dysfunction. In cardiac ischemia-reperfusion injury, mitochondrial Ca2+ overload leads to pathological production of reactive oxygen species (ROS), activates the permeability transition, and cardiomyocyte death. Here we investigated the cardiac phenotype caused by deletion of EF-hand domain-containing protein D1 (Efhd1-/-), a Ca2+-binding mitochondrial protein whose function is poorly understood. Efhd1-/- mice are viable and have no adverse cardiac phenotypes. They feature reductions in basal ROS levels and mitoflash events, both important precursors for mitochondrial injury, though cardiac mitochondria have normal susceptibility to Ca2+ overload. Notably, we also find that Efhd1-/- mice and their cardiomyocytes are resistant to hypoxic injury.

Kynurenic acid- a key metabolite protecting the heart from an ischemic damage

Background Myocardial ischemia is a major cause of death in patients with renal dysfunction. In order to identify a key metabolite which may protect cardiac function following renal injury, we have recently performed a metabolomics profiling analysis of LV lysates and plasma samples derived from animals that underwent an acute kidney injury (AKI) 1 or 7 days earlier, versus sham-operated controls. The analysis revealed that the kynurenic acid (kynurenate, KYNA) metabolite levels are highly elevated in all tested experimental samples relative to control. Purpose We wished to analyze whether KYNA may protect cardiomyocytes' survival and cardiac function upon an ischemic event and if so, to characterize whether the protecting effect may be linked to better preservation of the cardiac mitochondria. Methods Cellular viability of H9C2 rat cardiac myoblasts grown under normoxic or anoxic conditions with or without KYNA was determined by flow cytometry following Annexin-PI staining. The mitochondrial structure of the cells was determined by live cell staining with green (FITC) and deep red (Cy5) mito-tracker dyes. The potential effect of the metabolite on cardiac function following acute MI was tested in a murine model by echocardiography followed by histological staining of the cardiac sections with Picro Sirius Red. Results KYNA given at 10 mM concentration hardly affected the viability of H9C2 grown under normoxia, however the metabolite rescued the viability of the anoxic cells by 63% and largely improved their mitochondrial structure. Moreover, KYNA diluted in the drinking water of post-MI animals (250mg/ml), highly enhanced their cardiac recovery compared to untreated-animals as determined by echocardiography and collagen staining. Conclusions 1. KYNA may represent a key metabolite absorbed by the heart following AKI. 2. KYNA can enhance cardiac cell viability following an ischemic event both in vitro and in vivo in a mechanism which is mediated, at least in part, by protection of the cardiac mitochondria. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Weizmann Institute-Tel-Aviv Sourasky Medical Center joint research grant KYNA's protection of cardiac cells

Abstract P302: Alpha-1A Adrenergic Receptor Activation Increases Electron Transport Chain Activity In The Uninjured Mouse Heart

Decreased electron transport chain (ETC) activity in cardiac mitochondria is a hallmark of heart failure. Gain- and loss-of-function studies define the benefits of alpha-1A adrenergic receptor (α1A-AR) activation in the failing heart, such as increased cardiac contractility. However, the mechanisms behind these effects are unknown, and α1A-AR activation as a method of ETC regulation has not been studied. Here, we assessed the hypotheses that decreased α1A-AR activation reduces ETC enzyme activity, whereas increased α1A-AR activation enhances ETC enzyme activity. We profiled citrate synthase and ETC complex I-IV activities in isolated cardiac mitochondria from (1) wild-type (WT) CL57Bl/6J mice or global α1A-AR knockout mice (10-12 wks) and (2) WT mice (10-12 wks) treated with vehicle (0.9% saline) or the selective α1A-AR agonist A61603 (10 ng/kg/d, 3 d). Citrate synthase, a key enzyme in the citric acid cycle, fuels ETC activity and is a commonly used marker for mitochondrial mass. Global α1A-AR knockout increased citrate synthase activity in male mice compared to WT controls (5,292 ± 275 vs. 4,198 ± 339 nmol/min/mg, n = 5 each group, p = 0.04) (mean ± SEM) (Panel A). When normalized to citrate synthase activity, global α1A-AR knockout decreased complex I (37 ± 10% vs. 64 ± 5%, p = 0.02) (1,786 ± 421 vs. 2,766 ± 422 nmol/min/mg) and complex II (25 ± 9% vs. 50 ± 13%, p = 0.01) (1,332 ± 219 vs. 2,032 ± 213 nmol/min/mg) activities with a trend toward decreased complex IV activity (33 ± 13% vs. 49 ± 17%, p = 0.07) (1,707 ± 201 vs. 2,000 ± 238 nmol/min/mg) (Panel B). A61603 treatment led to a trend towards decreased citrate synthase activity in female mice compared to vehicle controls (6,662 ± 501 vs. 7,701 ± 421 nmol/min/mg, n = 3 each group, p = 0.09) (Panel C). When normalized to citrate synthase activity, A61603 increased complex I (27 ± 3% vs. 17 ± 2%, p = 0.03) (1,736 ± 92 vs. 1,326 ± 156 nmol/min/mg), complex III (61 ± 6% vs. 37 ± 5%, p = 0.02) (3,993 ± 258 vs. 2,894 ± 531 nmol/min/mg), and complex IV (70 ± 6% vs. 48 ± 6%, p = 0.03) (4,631 ± 100 vs. 3,676 ± 533 nmol/min/mg) activities (Panel D). In conclusion, we show that global α1A-AR knockout decreases ETC enzyme activity, while treatment with an α1A-AR agonist increases ETC enzyme activity. These findings may identify a novel mechanism through which α1A-AR activation protects the injured and failing heart.

Liquiritin from Radix Glycyrrhizae Protects Cardiac Mitochondria from Hypoxia/Reoxygenation Damage

Aims. The purpose of this study was to evaluate the protective effect of liquiritin (LIQ) from Radix Glycyrrhizae on cardiac mitochondria against hypoxia/reoxygenation (HR) injury. Methods. H9C2 cells were subject to the HR model. LIQ purified from Radix Glycyrrhizae (purity > 95%) was administrated to reoxygenation period. Cell viability, mitochondrial mass, mitochondrial membrane potential, reactive oxygen species, and mitochondrial Ca2⁺ level were then assessed by using Cell Counting kit-8 and suitable fluorescence probe kits. Results. LIQ administration remarkably reduced the rate of HR damage via increasing H9C2 cell viability level and preserving mitochondria after HR. Particularly, 60 μM of LIQ posthypoxic treatment markedly reduced cell death in HR-subjected H9C2 cell groups ( p < 0.05 ). Interestingly, posthypoxic treatment of LIQ significantly prevented the loss of mitochondrial membrane potential, the decrease in mitochondrial mass, the increase in reactive oxygen species production, and the elevation of mitochondrial Ca2⁺ level in HR-treated H9C2 cells. Conclusion. The present study provides for the first time the cardioprotective of LIQ posthypoxic treatment via reducing H9C2 cell death and protecting cardiac mitochondria against HR damage.

Aldosterone Excess Induced Mitochondria Decrease and Dysfunction via Mineralocorticoid Receptor and Oxidative Stress In Vitro and In Vivo

Aldosterone excess plays a major role in the progression of cardiac dysfunction and remodeling in clinical diseases such as primary aldosteronism and heart failure. However, the effect of aldosterone excess on cardiac mitochondria is unclear. In this study, we investigated the effect of aldosterone excess on cardiac mitochondrial dysfunction and its mechanisms in vitro and in vivo. We used H9c2 cardiomyocytes to investigate the effect and mechanism of aldosterone excess on cardiac mitochondria, and further investigated them in an aldosterone-infused ICR mice model. The results of the cell study showed that aldosterone excess decreased mitochondrial DNA, COX IV and SOD2 protein expressions, and mitochondria ATP production. These effects were abolished or attenuated by treatment with a mineralocorticoid receptor (MR) antagonist and antioxidant. With regard to the signal transduction pathway, aldosterone suppressed cardiac mitochondria through an MR/MAPK/p38/reactive oxygen species pathway. In the mouse model, aldosterone infusion decreased the amount of cardiac mitochondrial DNA and COX IV protein, and the effects were also attenuated by treatment with an MR antagonist and antioxidant. In conclusion, aldosterone excess induced a decrease in mitochondria and mitochondrial dysfunction via MRs and oxidative stress in vitro and in vivo.

The intra-mitochondrial O-GlcNAcylation system acutely regulates OXPHOS capacity and ROS dynamics in the heart

Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we used an in vitro reconstitution assay to characterize the intra-mitochondrial O- GlcNAc system without potential cytoplasmic confounding effects. We compared the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific O-GlcNAcylation inducer. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 displayed increased O-GlcNAcylation in response to NButGT. This was associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of CI substrates, and a striking reduction of mitochondrial ROS release, which could be related to O- GlcNAcylation of subunits within the NADH dehydrogenase module of CI. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.