Current Perspectives of Mitochondria in Sepsis-Induced Cardiomyopathy.

Sepsis-induced cardiomyopathy (SICM) is one of the leading indicators for poor prognosis associated with sepsis. Despite its reversibility, prognosis varies widely among patients. Mitochondria play a key role in cellular energy production by generating adenosine triphosphate (ATP), which is vital for myocardial energy metabolism. Over recent years, mounting evidence suggests that severe sepsis not only triggers mitochondrial structural abnormalities such as apoptosis, incomplete autophagy, and mitophagy in cardiomyocytes but also compromises their function, leading to ATP depletion. This metabolic disruption is recognized as a significant contributor to SICM, yet effective treatment options remain elusive. Sepsis cannot be effectively treated with inotropic drugs in failing myocardium due to excessive inflammatory factors that blunt ß-adrenergic receptors. This review will share the recent knowledge on myocardial cell death in sepsis and its molecular mechanisms, focusing on the role of mitochondria as an important metabolic regulator of SICM, and discuss the potential for developing therapies for sepsis-induced myocardial injury.

Inhibition of the mPTP and Lipid Peroxidation Is Additively Protective Against I/R Injury.

BACKGROUND: During myocardial ischemia/reperfusion (I/R) injury, high levels of matrix Ca2+ and reactive oxygen species (ROS) induce the opening of the mitochondrial permeability transition pore (mPTP), which causes mitochondrial dysfunction and ultimately necrotic death. However, the mechanisms of how these triggers individually or cooperatively open the pore have yet to be determined. METHODS: Here, we use a combination of isolated mitochondrial assays and in vivo I/R surgery in mice. We challenged isolated liver and heart mitochondria with Ca2+, ROS, and Fe2+ to induce mitochondrial swelling. Using inhibitors of the mPTP (cyclosporine A or ADP) lipid peroxidation (ferrostatin-1, MitoQ), we determined how the triggers elicit mitochondrial damage. Additionally, we used the combination of inhibitors during I/R injury in mice to determine if dual inhibition of these pathways is additivity protective. RESULTS: In the absence of Ca2+, we determined that ROS fails to trigger mPTP opening. Instead, high levels of ROS induce mitochondrial dysfunction and rupture independently of the mPTP through lipid peroxidation. As expected, Ca2+ in the absence of ROS induces mPTP-dependent mitochondrial swelling. Subtoxic levels of ROS and Ca2+ synergize to induce mPTP opening. Furthermore, this synergistic form of Ca2+- and ROS-induced mPTP opening persists in the absence of CypD (cyclophilin D), suggesting the existence of a CypD-independent mechanism for ROS sensitization of the mPTP. These ex vivo findings suggest that mitochondrial dysfunction may be achieved by multiple means during I/R injury. We determined that dual inhibition of the mPTP and lipid peroxidation is significantly more protective against I/R injury than individually targeting either pathway alone. CONCLUSIONS: In the present study, we have investigated the relationship between Ca2+ and ROS, and how they individually or synergistically induce mitochondrial swelling. Our findings suggest that Ca2+ mediates mitochondrial damage through the opening of the mPTP, although ROS mediates its damaging effects through lipid peroxidation. However, subtoxic levels both Ca2+ and ROS can induce mPTP-mediated mitochondrial damage. Targeting both of these triggers to preserve mitochondria viability unveils a highly effective therapeutic approach for mitigating I/R injury.

Reversing mitochondrial defects in aged hearts: role of mitochondrial calpain activation.

Aging chronically increases endoplasmic reticulum (ER) stress that contributes to mitochondrial dysfunction. Activation of calpain 1 (CPN1) impairs mitochondrial function during acute ER stress. We proposed that aging-induced ER stress led to mitochondrial dysfunction by activating CPN1. We posit that attenuation of the ER stress or direct inhibition of CPN1 in aged hearts can decrease cardiac injury during ischemia-reperfusion by improving mitochondrial function. Male young (3 mo) and aged mice (24 mo) were used in the present study, and 4-phenylbutyrate (4-PBA) was used to decrease the ER stress in aged mice. Subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) were isolated. Chronic 4-PBA treatment for 2 wk decreased CPN1 activation as shown by the decreased cleavage of spectrin in cytosol and apoptosis inducing factor (AIF) and the α1 subunit of pyruvate dehydrogenase (PDH) in mitochondria. Treatment improved oxidative phosphorylation in 24-mo-old SSM and IFM at baseline compared with vehicle. When 4-PBA-treated 24-mo-old hearts were subjected to ischemia-reperfusion, infarct size was decreased. These results support that attenuation of the ER stress decreased cardiac injury in aged hearts by improving mitochondrial function before ischemia. To challenge the role of CPN1 as an effector of the ER stress, aged mice were treated with MDL-28170 (MDL, an inhibitor of calpain 1). MDL treatment improved mitochondrial function in aged SSM and IFM. MDL-treated 24-mo-old hearts sustained less cardiac injury following ischemia-reperfusion. These results support that age-induced ER stress augments cardiac injury during ischemia-reperfusion by impairing mitochondrial function through activation of CPN1.

Calpain-mediated protein targets in cardiac mitochondria following ischemia-reperfusion.

Calpain 1 and 2 (CPN1/2) are calcium-dependent cysteine proteases that exist in cytosol and mitochondria. Pharmacologic inhibition of CPN1/2 decreases cardiac injury during ischemia (ISC)-reperfusion (REP) by improving mitochondrial function. However, the protein targets of CPN1/2 activation during ISC-REP are unclear. CPN1/2 include a large subunit and a small regulatory subunit 1 (CPNS1). Genetic deletion of CPNS1 eliminates the activities of both CPN1 and CPN2. Conditional cardiomyocyte specific CPNS1 deletion mice were used in the present study to clarify the role of CPN1/2 activation in mitochondrial damage during ISC-REP with an emphasis on identifying the potential protein targets of CPN1/2. Isolated hearts from wild type (WT) or CPNS1 deletion mice underwent 25 min in vitro global ISC and 30 min REP. Deletion of CPNS1 led to decreased cytosolic and mitochondrial calpain 1 activation compared to WT. Cardiac injury was decreased in CPNS1 deletion mice following ISC-REP as shown by the decreased infarct size compared to WT. Compared to WT, mitochondrial function was improved in CPNS1 deletion mice following ischemia-reperfusion as shown by the improved oxidative phosphorylation and decreased susceptibility to mitochondrial permeability transition pore opening. H2O2 generation was also decreased in mitochondria from deletion mice following ISC-REP compared to WT. Deletion of CPNS1 also resulted in less cytochrome c and truncated apoptosis inducing factor (tAIF) release from mitochondria. Proteomic analysis of the isolated mitochondria showed that deletion of CPNS1 increased the content of proteins functioning in regulation of mitochondrial calcium homeostasis (paraplegin and sarcalumenin) and complex III activity. These results suggest that activation of CPN1 increases cardiac injury during ischemia-reperfusion by impairing mitochondrial function and triggering cytochrome c and tAIF release from mitochondria into cytosol.

Cardioprotective Effects of Oroxylum indicum Extract Against Doxorubicin and Cyclophosphamide-Induced Cardiotoxicity.

Administration of Chemotherapeutics, especially doxorubicin (DOX) and cyclophosphamide (CPS), is commonly associated with adverse effects such as myelosuppression and cardiotoxicity. At this time, few approved therapeutic options are currently available for the management of chemotherapy-associated cardiotoxicity. Thus, identification of novel therapeutics with potent cardioprotective properties and minimal adverse effects are pertinent in treating Doxorubicin and Cyclophosphamide-induced cardiotoxicity. Oroxylum indicum extract (OIE, Sabroxy®) is a natural product known to possess several beneficial biological functions including antioxidant, anti-inflammatory and cytoprotective effects. We therefore set to investigate the cardioprotective effects of OIE against Doxorubicin and Cyclophosphamide-induced cardiotoxicity and explore the potential cardioprotective mechanisms involved. Adult male mice were treated with DOX and CPS in combination, OIE alone, or a combination of OIE and DOX & CPS. Swimming test was performed to assess cardiac function. Markers of oxidative stress were assessed by levels of reactive oxygen species (ROS), nitrite, hydrogen peroxide, catalase, and glutathione content. The activity of interleukin converting enzyme and cyclooxygenase was determined as markers of inflammation. Mitochondrial function was assessed by measuring Complex-I activity. Apoptosis was assessed by Caspase-3 and protease activity. Mice treated with DOX and CPS exhibited reduced swim rate, increased oxidative stress, increased inflammation, and apoptosis in the heart tissue. These cardiotoxic effects were significantly reduced by co-administration of OIE. Furthermore, computational molecular docking studies revealed potential binding of DOX and CPS to tyrosine hydroxylase which validated our in vivo findings regarding the inhibition of tyrosine hydroxylase activity. Our current findings indicated that OIE counteracts Doxorubicin and Cyclophosphamide-induced cardiotoxicity-through inhibition of ROS-mediated apoptosis and by blocking the effect on tyrosine hydroxylase. Taken together, our findings suggested that OIE possesses cardioprotective effects to counteract potentially fatal cardiac complications associated with chemotherapy treatment.

Mitochondrial redox regulation and myocardial ischemia-reperfusion injury.

Mitochondrial reactive oxygen species (ROS) have emerged as an important mechanism of disease and redox signaling in the cellular system. Under basal or pathological conditions, electron leakage for ROS production is primarily mediated by complexes I and III of the electron transport chain (ETC) and by the proton motive force (PMF), consisting of a membrane potential (ΔΨ) and a proton gradient (ΔpH). Several factors control redox status in mitochondria, including ROS, the PMF, oxidative posttranslational modifications (OPTM) of the ETC subunits, SOD2, and cytochrome c heme lyase (HCCS). In the mitochondrial PMF, increased ΔpH-supported backpressure due to diminishing electron transport and chemiosmosis promotes a more reductive mitochondrial physiological setting. OPTM by protein cysteine sulfonation in complex I and complex III has been shown to affect enzymatic catalysis, the proton gradient, redox status, and enzyme-mediated ROS production. Pathological conditions associated with oxidative or nitrosative stress, such as myocardial ischemia and reperfusion (I/R), increase mitochondrial ROS production and redox dysfunction via oxidative injury to complexes I and III, intensely enhancing protein cysteine sulfonation and impairing heme integrity. The physiological conditions of reductive stress induced by gains in SOD2 function normalize I/R-mediated ROS overproduction and redox dysfunction. Further insight into the cellular mechanisms by which HCCS, biogenesis of c-type cytochrome, and OPTM regulate PMF and ROS production in mitochondria will enrich our understanding of redox signal transduction and identify new therapeutic targets for cardiovascular diseases in which oxidative stress perturbs normal redox signaling.

Targeting Mediators of Inflammation in Heart Failure: A Short Synthesis of Experimental and Clinical Results.

Inflammation has emerged as an important contributor to heart failure (HF) development and progression. Current research data highlight the diversity of immune cells, proteins, and signaling pathways involved in the pathogenesis and perpetuation of heart failure. Chronic inflammation is a major cardiovascular risk factor. Proinflammatory signaling molecules in HF initiate vicious cycles altering mitochondrial function and perturbing calcium homeostasis, therefore affecting myocardial contractility. Specific anti-inflammatory treatment represents a novel approach to prevent and slow HF progression. This review provides an update on the putative roles of inflammatory mediators involved in heart failure (tumor necrosis factor-alpha; interleukin 1, 6, 17, 18, 33) and currently available biological and non-biological therapy options targeting the aforementioned mediators and signaling pathways. We also highlight new treatment approaches based on the latest clinical and experimental research.

Apelin-13 Reverses Bupivacaine-Induced Cardiotoxicity via the Adenosine Monophosphate-Activated Protein Kinase Pathway.

BACKGROUND: Cardiotoxicity can be induced by the commonly used amide local anesthetic, bupivacaine. Bupivacaine can inhibit protein kinase B (AKT) phosphorylation and activated adenosine monophosphate-activated protein kinase alpha (AMPKα). It can decouple mitochondrial oxidative phosphorylation and enhance reactive oxygen species (ROS) production. Apelin enhances the phosphatidylinositol 3-kinase (PI3K)/AKT and AMPK/acetyl-CoA carboxylase (ACC) pathways, promotes the complete fatty acid oxidation in the heart, and reduces the release of ROS. In this study, we examined whether exogenous (Pyr1) apelin-13 could reverse bupivacaine-induced cardiotoxicity. METHODS: We used the bupivacaine-induced inhibition model in adult male Sprague Dawley (SD) rats (n = 48) and H9c2 cardiomyocyte cell cultures to explore the role of apelin-13 in the reversal of bupivacaine cardiotoxicity, and its possible mechanism of action. AMPKα, ACC, carnitine palmitoyl transferase (CPT), PI3K, AKT, superoxide dismutase 1 (SOD1), and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (p47-phox) were quantified. Changes in mitochondrial ultrastructure were examined, and mitochondrial DNA, cell viability, ROS release, oxygen consumption rate (OCR) were determined. RESULTS: Apelin-13 reduced bupivacaine-induced mitochondrial DNA lesions in SD rats (P < .001), while increasing the expression of AMPKα (P = .007) and PI3K (P = .002). Furthermore, apelin-13 blocked bupivacaine-induced depolarization of the mitochondrial membrane potential (P = .019) and the bupivacaine-induced increases in ROS (P = .001). Also, the AMPK pathway was activated by bupivacaine as well as apelin-13 (P = .002) in H9c2 cardiomyocytes. Additionally, the reduction in the PI3K expression by bupivacaine was mitigated by apelin-13 in H9c2 cardiomyocytes (P = .001). While the aforementioned changes induced by bupivacaine were not abated by apelin-13 after pretreatment with AMPK inhibitor compound C; the bupivacaine-induced changes were still mitigated by apelin-13, even when pretreated with PI3K inhibitor-LY294002. CONCLUSIONS: Apelin-13 treatment reduced bupivacaine-induced oxidative stress, attenuated mitochondrial morphological changes and mitochondrial DNA damage, enhanced mitochondrial energy metabolism, and ultimately reversed bupivacaine-induced cardiotoxicity. Our results suggest a role for the AMPK in apelin-13 reversal of bupivacaine-induced cardiotoxicity.

Long-Chain Acyl-Carnitines Interfere with Mitochondrial ATP Production Leading to Cardiac Dysfunction in Zebrafish.

In the human heart, the energy supplied by the production of ATP is predominately accomplished by ß-oxidation in mitochondria, using fatty acids (FAs) as the primary fuel. Long-chain acylcarnitines (LCACs) are intermediate forms of FA transport that are essential for FA delivery from the cytosol into mitochondria. Here, we analyzed the impact of the LCACs C18 and C18:1 on mitochondrial function and, subsequently, on heart functionality in the in vivo vertebrate model system of zebrafish (Danio rerio). Since LCACs are formed and metabolized in mitochondria, we assessed mitochondrial morphology, structure and density in C18- and C18:1-treated zebrafish and found no mitochondrial alterations compared to control-treated (short-chain acylcarnitine, C3) zebrafish embryos. However, mitochondrial function and subsequently ATP production was severely impaired in C18- and C18:1-treated zebrafish embryos. Furthermore, we found that C18 and C18:1 treatment of zebrafish embryos led to significantly impaired cardiac contractile function, accompanied by reduced heart rate and diminished atrial and ventricular fractional shortening, without interfering with cardiomyocyte differentiation, specification and growth. In summary, our findings provide insights into the direct role of long-chain acylcarnitines on vertebrate heart function by interfering with regular mitochondrial function and thereby energy allocation in cardiomyocytes.

MCU overexpression evokes disparate dose-dependent effects on mito-ROS and spontaneous Ca2+ release in hypertrophic rat cardiomyocytes.

Cardiac dysfunction in heart failure (HF) and diabetic cardiomyopathy (DCM) is associated with aberrant intracellular Ca2+ handling and impaired mitochondrial function accompanied with reduced mitochondrial calcium concentration (mito-[Ca2+]). Pharmacological or genetic facilitation of mito-Ca2+ uptake was shown to restore Ca2+ transient amplitude in DCM and HF, improving contractility. However, recent reports suggest that pharmacological enhancement of mito-Ca2+ uptake can exacerbate ryanodine receptor-mediated spontaneous sarcoplasmic reticulum (SR) Ca2+ release in ventricular myocytes (VMs) from diseased animals, increasing propensity to stress-induced ventricular tachyarrhythmia. To test whether chronic recovery of mito-[Ca2+] restores systolic Ca2+ release without adverse effects in diastole, we overexpressed mitochondrial Ca2+ uniporter (MCU) in VMs from male rat hearts with hypertrophy induced by thoracic aortic banding (TAB). Measurement of mito-[Ca2+] using genetic probe mtRCamp1h revealed that mito-[Ca2+] in TAB VMs paced at 2 Hz under ß-adrenergic stimulation is lower compared with shams. Adenoviral 2.5-fold MCU overexpression in TAB VMs fully restored mito-[Ca2+]. However, it failed to improve cytosolic Ca2+ handling and reduce proarrhythmic spontaneous Ca2+ waves. Furthermore, mitochondrial-targeted genetic probes MLS-HyPer7 and OMM-HyPer revealed a significant increase in emission of reactive oxygen species (ROS) in TAB VMs with 2.5-fold MCU overexpression. Conversely, 1.5-fold MCU overexpression in TABs, that led to partial restoration of mito-[Ca2+], reduced mitochondria-derived reactive oxygen species (mito-ROS) and spontaneous Ca2+ waves. Our findings emphasize the key role of elevated mito-ROS in disease-related proarrhythmic Ca2+ mishandling. These data establish nonlinear mito-[Ca2+]/mito-ROS relationship, whereby partial restoration of mito-[Ca2+] in diseased VMs is protective, whereas further enhancement of MCU-mediated Ca2+ uptake exacerbates damaging mito-ROS emission.NEW & NOTEWORTHY Defective intracellular Ca2+ homeostasis and aberrant mitochondrial function are common features in cardiac disease. Here, we directly compared potential benefits of mito-ROS scavenging and restoration of mito-Ca2+ uptake by overexpressing MCU in ventricular myocytes from hypertrophic rat hearts. Experiments using novel mito-ROS and Ca2+ biosensors demonstrated that mito-ROS scavenging rescued both cytosolic and mito-Ca2+ homeostasis, whereas moderate and high MCU overexpression demonstrated disparate effects on mito-ROS emission, with only a moderate increase in MCU being beneficial.

Impact of Aldosterone on the Failing Myocardium: Insights from Mitochondria and Adrenergic Receptors Signaling and Function.

The mineralocorticoid aldosterone regulates electrolyte and blood volume homeostasis, but it also adversely modulates the structure and function of the chronically failing heart, through its elevated production in chronic human post-myocardial infarction (MI) heart failure (HF). By activating the mineralocorticoid receptor (MR), a ligand-regulated transcription factor, aldosterone promotes inflammation and fibrosis of the heart, while increasing oxidative stress, ultimately induding mitochondrial dysfunction in the failing myocardium. To reduce morbidity and mortality in advanced stage HF, MR antagonist drugs, such as spironolactone and eplerenone, are used. In addition to the MR, aldosterone can bind and stimulate other receptors, such as the plasma membrane-residing G protein-coupled estrogen receptor (GPER), further complicating it signaling properties in the myocardium. Given the salient role that adrenergic receptor (ARs)-particularly ßARs-play in cardiac physiology and pathology, unsurprisingly, that part of the impact of aldosterone on the failing heart is mediated by its effects on the signaling and function of these receptors. Aldosterone can significantly precipitate the well-documented derangement of cardiac AR signaling and impairment of AR function, critically underlying chronic human HF. One of the main consequences of HF in mammalian models at the cellular level is the presence of mitochondrial dysfunction. As such, preventing mitochondrial dysfunction could be a valid pharmacological target in this condition. This review summarizes the current experimental evidence for this aldosterone/AR crosstalk in both the healthy and failing heart, and the impact of mitochondrial dysfunction in HF. Recent findings from signaling studies focusing on MR and AR crosstalk via non-conventional signaling of molecules that normally terminate the signaling of ARs in the heart, i.e., the G protein-coupled receptor-kinases (GRKs), are also highlighted.

Growth differentiation factor 11 attenuates cardiac ischemia reperfusion injury via enhancing mitochondrial biogenesis and telomerase activity.

It has been reported that growth differentiation factor 11 (GDF11) protects against myocardial ischemia/reperfusion (IR) injury, but the underlying mechanisms have not been fully clarified. Considering that GDF11 plays a role in the aging/rejuvenation process and that aging is associated with telomere shortening and cardiac dysfunction, we hypothesized that GDF11 might protect against IR injury by activating telomerase. Human plasma GDF11 levels were significantly lower in acute coronary syndrome patients than in chronic coronary syndrome patients. IR mice with myocardial overexpression GDF11 (oe-GDF11) exhibited a significantly smaller myocardial infarct size, less cardiac remodeling and dysfunction, fewer apoptotic cardiomyocytes, higher telomerase activity, longer telomeres, and higher ATP generation than IR mice treated with an adenovirus carrying a negative control plasmid. Furthermore, mitochondrial biogenesis-related proteins and some antiapoptotic proteins were significantly upregulated by oe-GDF11. These cardioprotective effects of oe-GDF11 were significantly antagonized by BIBR1532, a specific telomerase inhibitor. Similar effects of oe-GDF11 on apoptosis and mitochondrial energy biogenesis were observed in cultured neonatal rat cardiomyocytes, whereas GDF11 silencing elicited the opposite effects to oe-GDF11 in mice. We concluded that telomerase activation by GDF11 contributes to the alleviation of myocardial IR injury through enhancing mitochondrial biogenesis and suppressing cardiomyocyte apoptosis.

Reperfusion mediates heme impairment with increased protein cysteine sulfonation of mitochondrial complex III in the post-ischemic heart.

A serious consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which causes mitochondrial dysfunction. The cascading ROS can propagate and potentially induce heme bleaching and protein cysteine sulfonation (PrSO3H) of the mitochondrial electron transport chain. Herein we studied the mechanism of I/R-mediated irreversible oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the catalytic activity of complex III was significantly impaired. Spectroscopic analysis indicated that I/R mediated the destruction of hemes b and c + c1 in the mitochondria, supporting I/R-mediated complex III impairment. However, no significant impairment of complex III activity and heme damage were observed in mitochondria from the risk region of rat hearts subjected only to 30-min ischemia, despite a decreased state 3 respiration. In the I/R mitochondria, carbamidomethylated C122/C125 of cytochrome c1 via alkylating complex III with a down regulation of HCCS was exclusively detected, supporting I/R-mediated thioether defect of heme c1. LC-MS/MS analysis showed that I/R mitochondria had intensely increased complex III PrSO3H levels at the C236 ligand of the [2Fe2S] cluster of the Rieske iron­sulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSO3H of the hinge protein at C65 and of cytochrome c1 at C140 and C220, which are confined in the intermembrane space. MS analysis also showed that I/R extensively enhanced the PrSO3H of the core 1 (uqcrc1) and core 2 (uqcrc2) subunits in the matrix compartment, thus supporting the conclusion that complex III releases ROS to both sides of the inner membrane during reperfusion. Analysis of ischemic mitochondria indicated a modest reduction from the basal level of complex III PrSO3H detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSO3H. In conclusion, reperfusion-mediated heme damage with increased PrSO3H controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart.

Preconditioning or Postconditioning with 8-Br-cAMP-AM Protects the Heart against Regional Ischemia and Reperfusion: A Role for Mitochondrial Permeability Transition.

The cAMP analogue 8-Br-cAMP-AM (8-Br) confers marked protection against global ischaemia/reperfusion of isolated perfused heart. We tested the hypothesis that 8-Br is also protective under clinically relevant conditions (regional ischaemia) when applied either before ischemia or at the beginning of reperfusion, and this effect is associated with the mitochondrial permeability transition pore (MPTP). 8-Br (10 µM) was administered to Langendorff-perfused rat hearts for 5 min either before or at the end of 30 min regional ischaemia. Ca2+-induced mitochondria swelling (a measure of MPTP opening) and binding of hexokinase II (HKII) to mitochondria were assessed following the drug treatment at preischaemia. Haemodynamic function and ventricular arrhythmias were monitored during ischaemia and 2 h reperfusion. Infarct size was evaluated at the end of reperfusion. 8-Br administered before ischaemia attenuated ventricular arrhythmias, improved haemodynamic function, and reduced infarct size during ischaemia/reperfusion. Application of 8-Br at the end of ischaemia protected the heart during reperfusion. 8-Br promoted binding of HKII to the mitochondria and reduced Ca2+-induced mitochondria swelling. Thus, 8-Br protects the heart when administered before regional ischaemia or at the beginning of reperfusion. This effect is associated with inhibition of MPTP via binding of HKII to mitochondria, which may underlie the protective mechanism.

Acylated Ghrelin Attenuates l-Thyroxin-induced Cardiac Damage in Rats by Antioxidant and Anti-inflammatory Effects and Downregulating Components of the Cardiac Renin-angiotensin System.

ABSTRACT: This study investigated the protective effect of acylated ghrelin (AG) against l-thyroxin (l-Thy)-induced cardiac damage in rats and examined possible mechanisms. Male rats were divided into five intervention groups of 12 rats/group: control, control + AG, l-Thy, l-Thy + AG, and l-Thy + AG + [D-Lys3]-GHRP-6 (AG antagonist). l-Thy significantly reduced the levels of AG and des-acyl ghrelin and the AG to des-acyl ghrelin ratio. Administration of AG to l-Thy-treated rats reduced cardiac weights and levels of reactive oxygen species and preserved the function and structure of the left ventricle. In addition, AG also reduced the protein levels of cleaved caspase-3 and cytochrome c and prevented mitochondrial permeability transition pore opening. In the left ventricle of both control + AG-treated and l-Thy + AG-treated rats, AG significantly increased left ventricular levels of manganese superoxide dismutase (SOD2), total glutathione (GSH), and Bcl2. It also reduced the levels of malondialdehyde, tumor necrosis factor-α (TNF-α), interleukin-6, and Bax and the nuclear activity of nuclear factor-kappa B. Concomitantly, in both treated groups, AG reduced the mRNA and protein levels of NADPH oxidase 1, angiotensin (Ang) II type 1 receptor, and Ang-converting enzyme 2. All the beneficial effects of AG in l-Thy-treated rats were prevented by the coadministration of [D-Lys3]-GHRP-6, a selective growth hormone secretagogue receptor subtype 1a antagonist. In conclusion, AG protects against hyperthyroidism-induced cardiac hypertrophy and damage, which is mainly due to its antioxidant and anti-inflammatory potentials and requires the activation of GHS-R1a.

Progress in cardiac research: from rebooting cardiac regeneration to a complete cell atlas of the heart.

We review some of the important discoveries and advances made in basic and translational cardiac research in 2020. For example, in the field of myocardial infarction (MI), new aspects of autophagy and the importance of eosinophils were described. Novel approaches, such as a glycocalyx mimetic, were used to improve cardiac recovery following MI. The strategy of 3D bio-printing was shown to allow the fabrication of a chambered cardiac organoid. The benefit of combining tissue engineering with paracrine therapy to heal injured myocardium is discussed. We highlight the importance of cell-to-cell communication, in particular, the relevance of extracellular vesicles, such as exosomes, which transport proteins, lipids, non-coding RNAs, and mRNAs and actively contribute to angiogenesis and myocardial regeneration. In this rapidly growing field, new strategies were developed to stimulate the release of reparative exosomes in ischaemic myocardium. Single-cell sequencing technology is causing a revolution in the study of transcriptional expression at cellular resolution, revealing unanticipated heterogeneity within cardiomyocytes, pericytes and fibroblasts, and revealing a unique subpopulation of cardiac fibroblasts. Several studies demonstrated that exosome- and non-coding RNA-mediated approaches can enhance human induced pluripotent stem cell (iPSC) viability and differentiation into mature cardiomyocytes. Important details of the mitochondrial Ca2+ uniporter and its relevance were elucidated. Novel aspects of cancer therapeutic-induced cardiotoxicity were described, such as the novel circular RNA circITCH, which may lead to novel treatments. Finally, we provide some insights into the effects of SARS-CoV-2 on the heart.

Thymoquinone reduces mitochondrial damage and death of cardiomyocytes induced by clozapine.

The generation of a reactive nitrenium ion by microsomal/mitochondrial cytochrome P450 (CYPs) from clozapine (CLZ) has been suggested as the main cause of cardiotoxicity by this drug. Previous studies indicated that thymoquinone (TQ) as an active constituent of Nigella sativa has pharmacological effects such as antioxidant, reactive oxygen species (ROS) scavenger, and inhibitory effect on CYPs enzymes. Therefore, we hypothesized that TQ with these pharmacological effects can reduce CLZ-induced toxicity in isolated cardiomyocytes and mitochondria. Rat left ventricular cardiomyocytes and mitochondria were isolated by collagenase perfusion and differential centrifugation respectively. Then, isolated cardiomyocytes and mitochondria were pretreated with different concentrations of TQ (1, 5, and 10 µmol/l) for 30 min and then followed by exposure to CLZ (50 µmol/l) for 6 h. After 6 h of incubation, using biochemical evaluations and flow cytometric analysis, the parameters of cellular toxicity including cytotoxicity, the level of oxidized/reduced glutathione (GSH/GSSG), malondialdehyde (MDA), reactive oxygen species (ROS) formation, lysosomal membrane integrity, mitochondria membrane potential (ΔΨm) collapse, and mitochondrial toxicity including succinate dehydrogenase (SDH) activity and mitochondrial swelling were analyzed. We observed a significant toxicity in isolated cardiomyocytes and mitochondria after exposure with CLZ which was related to ROS formation, oxidative stress, GSH depletion, lysosomal and mitochondrial damages, and mitochondrial dysfunction and swelling, while TQ pretreatment reverted the above toxic effect of CLZ on isolated cardiomyocytes and mitochondria. Our results indicate that TQ prevents and reverses CLZ-induced cytotoxicity and mitochondrial damages in isolated cardiomyocytes and mitochondria, providing an experimental basis for clinical treatment on CLZ-induced cardiotoxicity.

Mitochondrial Disruption Is Involved in the Effect of Nuclear Factor of Activated T cells, Cytoplasmic 4 on Aggravating Cardiomyocyte Hypertrophy.

ABSTRACT: Nuclear factor of activated T cells, cytoplasmic 4 (NFATc4), a nuclear transcription factor, has been implicated in cardiac hypertrophy through the enhancement of hypertrophic gene expression. However, the role of NFATc4 in mitochondrial modulation is mostly unknown. The current study aimed to investigate the role of NFATc4 in regulating mitochondrial function during phenylephrine (PE)-induced cardiac hypertrophy. Our results showed that overexpression of NFATc4 aggravated the PE-induced decrease in mitochondrial genesis, membrane potential, and mitochondrial gene expression as well as impaired mitochondrial respiration. However, knockdown of NFATc4 relieved PE-induced perturbations in mitochondria and cardiomyocyte hypertrophy. Mechanistically, by activating phosphoinositide-dependent kinase 1 and promoting a combination of AKT and phosphoinositide-dependent kinase 1, phosphorylation and sequential acetylation of PGC-1α were aggravated by NFATc4 and suppressed the activity of PGC-1α. In conclusion, NFATc4-regulated factors were shown to be associated with mitochondrial function and exacerbated PE-induced mitochondrial dysfunction. These findings revealed new roles of NFATc4 in cardiac hypertrophy.

Hyperglycaemia-Induced Contractile Dysfunction and Apoptosis in Cardiomyocyte-Like Pulsatile Cells Derived from Mouse Embryonic Stem Cells.

Hyperglycaemia, a key metabolic abnormality in diabetes mellitus, is implicated in pathological cardiogenesis during embryological development. However, the underlying mechanisms and potential therapeutic targets remain unknown. We, therefore, studied the effect of hyperglycaemia on mouse embryonic stem cell (mESC) cardiac differentiation. The mESCs were differentiated via embryoid body (EB) formation and cultured under conditions with baseline (25 mM) or high (50 mM) glucose. Time-lapse microscopy images of pulsatile mESCs and Ca2+ transients were recorded. Biomarkers of cellular changes were detected using immunocytochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) assay, and Western blot analyses. Differentiated, spontaneously beating mESCs stained positive for cardiac troponin T, α-actinin 2, myosin heavy chain, and connexin 43. Hyperglycaemia decreased the EB diameter and number of beating EBs as well as the cellular amplitude of contraction, the Ca2+ transient, and the contractile response to caffeine (1 mM), but had no effect on the expression of the sarco-endoplasmic reticulum calcium transport ATPase 2 (SERCA 2). Furthermore, hyperglycaemia decreased the expression of B cell lymphoma 2 (Bcl-2) and increased the expression of cytoplasmic cytochrome c and the number of TUNEL-positive cells, but had no effect on the expression of one of the mitochondrial fusion regulatory proteins, optic atrophy protein 1 (OPA1). Overall, hyperglycaemia suppressed the mESC cardiomyocyte-like differentiation and induced contractile dysfunction. The results are consistent with mechanisms involving abnormal Ca2+ handling and mitochondrial-dependent apoptosis, factors which represent potential therapeutic targets in developmental diabetic cardiac disease.

Allicin attenuates myocardial apoptosis, inflammation and mitochondrial injury during hypoxia-reoxygenation: an in vitro study.

BACKGROUND: Myocardial ischemia-reperfusion (IR) injury is a damage due to an initial reduction in blood flow to the heart, preventing it from receiving enough oxygen, and subsequent restoration of blood flow through the opening of an occluded coronary artery producing paradoxical harmful effects. The finding of new therapies to prevent IR is of utmost importance. Allicin is a compound isolated from garlic having the ability to prevent and cure different diseases, and a protective effect on the myocardium was also demonstrated. Therefore, the aim of this study was to evaluate the in vitro protective effect of Allicin against myocardial IR injury on cardiomyocytes. METHODS: We established an in vitro hypoxia-reoxygenation (HR) model of primary porcine cardiomyocytes to simulate myocardial IR injury. Primary porcine cardiomyocytes were extracted from Mini-musk swines (1 day old). After a period of adaptation of at least 2-3 days, cardiomyocytes in good condition were selected and randomly divided into control group (normal oxygen for 5 h), HR group (2 h of hypoxia/3 h of reoxygenation), and HR + Allicin group (hypoxia/reoxygenation + Allicin treatment). RESULTS: After the induction of hypoxia/reoxygenation, Allicin treatment enhanced the cell viability. Moreover, Allicin treatment resulted in a reduction of apoptosis from 13.5 ± 1.2% to 6.11 ± 0.15% compared with the HR group (p < 0.05), and the apoptosis related proteins were regulated as well, with a decreased expression of Bax, cleaved caspase-3 and cytosolic cytochrome C and an increase in Bcl-2 expression in the HR + Allicin group (all p < 0.01). Pro-inflammatory cytokines, such as interleukin-6 and tumor necrosis factor alpha were down-regulated by the treatment with Allicin (both p < 0.01). In addition, it significantly decreased intracellular reactive oxygen species generation (p < 0.01) and reduced the loss of mitochondrial membrane potential (p < 0.01). Furthermore, the expression of PPARγ coactivator-1α and endothelial nitric oxide synthase was up-regulated (both p < 0.01), while the expression of Endothelin-1, hypoxia inducing factor-1α and transforming growth factor beta was down-regulated (all p < 0.01) by Allicin treatment. CONCLUSIONS: These results suggested that Allicin protected the cardiomyocytes against HR damage by reducing apoptosis, inflammation and mitochondrial injury, thus providing a basis for its potential use in the treatment of myocardial IR.