Mitochondrial Calcium Handling Research Articles

Abstract Tu079: The Calcium Handling Machinery and Electrophysiology is Remodeled in Friedreich’s Ataxia

Background: Friedreich's ataxia (FA) is a progressive neurodegenerative disorder. FA is caused by a deficiency in the frataxin protein due to an expansion of GAA repeats in the first intron of the frataxin gene. Reduced frataxin protein expression is thought to negatively affect mitochondrial function, leading to increased oxidative damage. Although FA is considered a neurodegenerative disorder, it is not completely understood why most patients present with left ventricular systolic dysfunction and die from cardiac events. Objective: The objective of our study is to determine whether abnormal calcium handling is a molecular mechanism of cardiac dysfunction in FA. Methods: We used the frataxin knock-out (KO) mouse model of FA as well as human heart samples from donors with FA and from unaffected donors. The expression of calcium-handling proteins was assessed using proteomics and westernblot. We used echocardiography and telemetric ECG to assess cardiac function in the mice. We also performed in-vivo programmed electrical stimulation (PES) to assess ventricular arrhythmogenesis in the mouse heart. Results: Proteomics and western blot showed that SERCA2, Ryr2, PLN, CASQ2, and CaMKII were downregulated and that the phosphorylation of Ryr2 and PLN (Ser16 and Thr17) were also significantly decreased in left ventricular tissue from humans with FA and from KO mice. The mitochondrial calcium handling proteins VDAC and MCU were upregulated in left ventricular tissue from humans with FA and from KO mice. On telemetric ECG, the RR tended to prolong in the KO animals compared to wild types (p=0.053), and heart rate variability analysis indicated heightened cardiac parasympathetic input. Echocardiography showed that the ejection fraction and fractional shortening were significantly decreased and left ventricular wall thickness and diameter were significantly increased in the KO mice. In PES, ventricular arrhythmogenesis was greater in KO mice compared to wild types. Conclusions: The reduced expression of calcium handling proteins may contribute to cardiac contractile and electrophysiological abnormalities in FA. Keywords: Friedreich's ataxia, SERCA2, Ryr2, PLN, CASQ2, and CaMKII.

Diabetes cardiomyopathy: targeted regulation of mitochondrial dysfunction and therapeutic potential of plant secondary metabolites.

Diabetic cardiomyopathy (DCM) is a specific heart condition in diabetic patients, which is a major cause of heart failure and significantly affects quality of life. DCM is manifested as abnormal cardiac structure and function in the absence of ischaemic or hypertensive heart disease in individuals with diabetes. Although the development of DCM involves multiple pathological mechanisms, mitochondrial dysfunction is considered to play a crucial role. The regulatory mechanisms of mitochondrial dysfunction mainly include mitochondrial dynamics, oxidative stress, calcium handling, uncoupling, biogenesis, mitophagy, and insulin signaling. Targeting mitochondrial function in the treatment of DCM has attracted increasing attention. Studies have shown that plant secondary metabolites contribute to improving mitochondrial function and alleviating the development of DCM. This review outlines the role of mitochondrial dysfunction in the pathogenesis of DCM and discusses the regulatory mechanism for mitochondrial dysfunction. In addition, it also summarizes treatment strategies based on plant secondary metabolites. These strategies targeting the treatment of mitochondrial dysfunction may help prevent and treat DCM.

Unraveling the nexus of age, epilepsy, and mitochondria: exploring the dynamics of cellular energy and excitability.

Epilepsy, a complex neurological condition marked by recurring seizures, is increasingly recognized for its intricate relationship with mitochondria, the cellular powerhouses responsible for energy production and calcium regulation. This review offers an in-depth examination of the interplay between epilepsy, mitochondrial function, and aging. Many factors might account for the correlation between epilepsy and aging. Mitochondria, integral to cellular energy dynamics and neuronal excitability, perform a critical role in the pathophysiology of epilepsy. The mechanisms linking epilepsy and mitochondria are multifaceted, involving mitochondrial dysfunction, reactive oxygen species (ROS), and mitochondrial dynamics. Mitochondrial dysfunction can trigger seizures by compromising ATP production, increasing glutamate release, and altering ion channel function. ROS, natural byproducts of mitochondrial respiration, contribute to oxidative stress and neuroinflammation, critical factors in epileptogenesis. Mitochondrial dynamics govern fusion and fission processes, influence seizure threshold and calcium buffering, and impact seizure propagation. Energy demands during seizures highlight the critical role of mitochondrial ATP generation in maintaining neuronal membrane potential. Mitochondrial calcium handling dynamically modulates neuronal excitability, affecting synaptic transmission and action potential generation. Dysregulated mitochondrial calcium handling is a hallmark of epilepsy, contributing to excitotoxicity. Epigenetic modifications in epilepsy influence mitochondrial function through histone modifications, DNA methylation, and non-coding RNA expression. Potential therapeutic avenues targeting mitochondria in epilepsy include mitochondria-targeted antioxidants, ketogenic diets, and metabolic therapies. The review concludes by outlining future directions in epilepsy research, emphasizing integrative approaches, advancements in mitochondrial research, and ethical considerations. Mitochondria emerge as central players in the complex narrative of epilepsy, offering profound insights and therapeutic potential for this challenging neurological disorder.

Empagliflozin ameliorates diabetic cardiomyopathy probably via activating AMPK/PGC-1α and inhibiting the RhoA/ROCK pathway.

Diabetic cardiomyopathy (DCM) increases the risk of hospitalization for heart failure (HF) and mortality in patients with diabetes mellitus. However, no specific therapy to delay the progression of DCM has been identified. Mitochondrial dysfunction, oxidative stress, inflammation, and calcium handling imbalance play a crucial role in the pathological processes of DCM, ultimately leading to cardiomyocyte apoptosis and cardiac dysfunctions. Empagliflozin, a novel glucose-lowering agent, has been confirmed to reduce the risk of hospitalization for HF in diabetic patients. Nevertheless, the molecular mechanisms by which this agent provides cardioprotection remain unclear. To investigate the effects of empagliflozin on high glucose (HG)-induced oxidative stress and cardiomyocyte apoptosis and the underlying molecular mechanism. Twelve-week-old db/db mice and primary cardiomyocytes from neonatal rats stimulated with HG (30 mmol/L) were separately employed as in vivo and in vitro models. Echocardiography was used to evaluate cardiac function. Flow cytometry and TdT-mediated dUTP-biotin nick end labeling staining were used to assess apoptosis in myocardial cells. Mitochondrial function was assessed by cellular ATP levels and changes in mitochondrial membrane potential. Furthermore, intracellular reactive oxygen species production and superoxide dismutase activity were analyzed. Real-time quantitative PCR was used to analyze Bax and Bcl-2 mRNA expression. Western blot analysis was used to measure the phosphorylation of AMP-activated protein kinase (AMPK) and myosin phosphatase target subunit 1 (MYPT1), as well as the peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) and active caspase-3 protein levels. In the in vivo experiment, db/db mice developed DCM. However, the treatment of db/db mice with empagliflozin (10 mg/kg/d) for 8 wk substantially enhanced cardiac function and significantly reduced myocardial apoptosis, accompanied by an increase in the phosphorylation of AMPK and PGC-1α protein levels, as well as a decrease in the phosphorylation of MYPT1 in the heart. In the in vitro experiment, the findings indicate that treatment of cardiomyocytes with empagliflozin (10 μM) or fasudil (FA) (a ROCK inhibitor, 100 μM) or overexpression of PGC-1α significantly attenuated HG-induced mitochondrial injury, oxidative stress, and cardiomyocyte apoptosis. However, the above effects were partly reversed by the addition of compound C (CC). In cells exposed to HG, empagliflozin treatment increased the protein levels of p-AMPK and PGC-1α protein while decreasing phosphorylated MYPT1 levels, and these changes were mitigated by the addition of CC. Adding FA and overexpressing PGC-1α in cells exposed to HG substantially increased PGC-1α protein levels. In addition, no sodium-glucose cotransporter (SGLT)2 protein expression was detected in cardiomyocytes. Empagliflozin partially achieves anti-oxidative stress and anti-apoptotic effects on cardiomyocytes under HG conditions by activating AMPK/PGC-1α and suppressing of the RhoA/ROCK pathway independent of SGLT2.

An Arabidopsis mutant line lacking the mitochondrial calcium transport regulator MICU shows an altered metabolite profile

ABSTRACT Plant metabolism is constantly changing and requires input signals for efficient regulation. The mitochondrial calcium uniporter (MCU) couples organellar and cytoplasmic calcium oscillations leading to oxidative metabolism regulation in a vast array of species. In Arabidopsis thaliana, genetic deletion of AtMICU leads to altered mitochondrial calcium handling and ultrastructure. Here we aimed to further assess the consequences upon genetic deletion of AtMICU. Our results confirm that AtMICU safeguards intracellular calcium transport associated with carbohydrate, amino acid, and phytol metabolism modifications. The implications of such alterations are discussed.

The NADase CD38 may not dictate NAD levels in brain mitochondria of aged mice but regulates hydrogen peroxide generation

Aging is a time-related functional decline that affects many species. One of the hallmarks of aging is mitochondrial dysfunction, which leads to metabolic decline. The NAD decline during aging, in several tissues, correlates with increase in NADase activity of CD38. Knock out or pharmacological inhibition of CD38 activity can rescue mitochondrial function in several tissues, however, the role of CD38 in controlling NAD levels and metabolic function in the aging brain is unknown. In this work, we investigated CD38 NADase activity controlling NAD levels and mitochondrial function in mice brain with aging. We demonstrate that NADase activity of CD38 does not dictate NAD total levels in brain of aging mice and does not control mitochondrial oxygen consumption nor other oxygen parameters markers of mitochondrial dysfunction. However, for the first time we show that CD38 regulates hydrogen peroxide (H2O2) generation, one of the reactive oxygen species (ROS) in aging brain, through regulation of pyruvate dehydrogenase and alfa-ketoglutarate dehydrogenase, as mitochondria H2O2 leakage sites. The effect may be related to mitochondrial calcium handling differences in CD38 absence. Our study highlights a novel role of CD38 in brain energy metabolism and aging.

Lithium isotopes differentially modify mitochondrial amorphous calcium phosphate cluster size distribution and calcium capacity.

Lithium is commonly prescribed as a mood stabilizer in a variety of mental health conditions, yet its molecular mode of action is incompletely understood. Many cellular events associated with lithium appear tied to mitochondrial function. Further, recent evidence suggests that lithium bioactivities are isotope specific. Here we focus on lithium effects related to mitochondrial calcium handling. Lithium protected against calcium-induced permeability transition and decreased the calcium capacity of liver mitochondria at a clinically relevant concentration. In contrast, brain mitochondrial calcium capacity was increased by lithium. Surprisingly, 7Li acted more potently than 6Li on calcium capacity, yet 6Li was more effective at delaying permeability transition. The size distribution of amorphous calcium phosphate colloids formed in vitro was differentially affected by lithium isotopes, providing a mechanistic basis for the observed isotope specific effects on mitochondrial calcium handling. This work highlights a need to better understand how mitochondrial calcium stores are structurally regulated and provides key considerations for future formulations of lithium-based therapeutics.

PO-01-124 THE CALCIUM HANDLING MACHINERY IS REMODELED IN FRIEDREICH’S ATAXIA

Friedreich's ataxia (FA) is an inherited neurodegenerative disorder that causes progressive nervous system damage. FA is caused by a deficiency in the frataxin protein due to an expansion of GAA repeats in the first intron of the frataxin gene. Reduced frataxin protein expression is thought to negatively affect mitochondrial function, leading to increased oxidative damage. Although FA is considered a neurodegenerative disorder, it is not completely understood why most patients present with left ventricular systolic dysfunction and die from cardiac events. The objective of our study is to determine whether the abnormal calcium handling machinery is a molecular mechanism that perpetuates cardiac dysfunction in FA. We used the frataxin knock-out (KO) mouse model of FA as well as human heart samples from donors with FA and from unaffected donors. The expression of calcium handling machinery proteins was assessed with proteomics and western blot. In left ventricular myocytes from KO and WT mice, the IonOptix system was used for calcium imaging, the seahorse assay was utilized to measure oxygen consumption rate (OCR) and confocal imaging was used to quantify the mitochondrial membrane potential (Δψm), and reactive oxygen species (ROS). ECG and echocardiography were used to assess cardiac function in the mice. In the proteomic analysis and western blot, SERCA2, Ryr2 and CaMKII were significantly downregulated, and the mitochondrial calcium handling proteins voltage-dependent anion channel and mitochondrial calcium uniporter were significantly upregulated in left ventricular tissue from humans with FA and from KO mice. On the ECG, the RR, PR, QRS, and QTc intervals were significantly longer in the KO mice. Echocardiography showed that the ejection fraction and fractional shortening were significantly decreased and left ventricular wall thickness and diameter were significantly increased in the KO mice. Moreover, the calcium transient decay was significantly prolonged in ventricular myocytes from KO mice compared to WT. Additionally, the Δψm was significantly depolarized, the OCR was significantly decreased, and ROS levels were significantly elevated in left ventricular myocytes from KO compared to WT. The development of left ventricular contractile dysfunction in FA is associated with reduced expression of calcium handling proteins, abnormal calcium cycling, and mitochondrial dysfunction.

454 A Human 3D Model of Duchenne Muscular Dystrophy Cardiomyopathy to Investigate Calcium Regulation and Mitochondrial Dysfunction

OBJECTIVES/GOALS: We will use control- and DMD-engineered heart tissues to better model and investigate DMD cardiomyopathy. We will primarily assess cardiac calcium handling, mitochondrial function, and mitochondrial calcium handling, as calcium regulation and mitochondrial function are known to be affected in DMD. METHODS/STUDY POPULATION: We will use patient-derived stem cells, differentiated into cardiomyocytes in bioprinted 3D heart tissue muscle chambers to better model DMD cardiomyopathy. We will look at calcium handling and general mitochondrial function, as well as mitochondrial calcium handling, using a novel multifunctional genetic probe I previously developed allowing for simultaneous observation of cytosolic and mitochondrial calcium in real time. Optical mapping will also be used for tissue-level analysis. We will establish the functional differences at baseline, and then progress heart failure in the tissues to see how the abnormalities seen in the DMD tissues may get worse. Finally, we will investigate the effects of early restoration of dystrophin function on the effects of DMD cardiomyopathy development. RESULTS/ANTICIPATED RESULTS: We anticipate that DMD tissues will show more irregular/abnormal calcium handling, as seen in 2D hiPSC-CMs, as well as disruptions to mitochondrial function and ultrastructural development, as well as a decreased synchronization between cytosolic and mitochondrial calcium dynamics. We anticipate that these abnormalities will be exacerbated as the disease state progresses, but at least partially ameliorated with the restoration of dystrophin function. DISCUSSION/SIGNIFICANCE: DMD is a fatal disease with no known cure. Patients develop heart failure in their teens and die in their 20s, so any new insight that may prolong life and improve quality of life for patients is drastically needed. This would be the most accurate preclinical model of DMD cardiomyopathy to date and would investigate yet-untapped aspects of the disease state.

The effect of regulating MCU expression on experimental ischemic brain injury

Mitochondrial calcium uniporter (MCU) is a critical channel for Ca2+ influx into mitochondria. The present study aimed to determine if MCU knockdown has beneficial effects on ischemic brain injury and to explore the underlying mechanisms. The present study demonstrated that MCU knockdown but not total knockout (KO) attenuated ischemia infarction volume and primary cortical neuronal cells' ischemic damage. MCU knockdown maintained mitochondrial ultrastructure, alleviated calcium overload, and reduced mitochondrial apoptosis. Moreover, MCU knockdown regulated the changes of MICU1 and MICU2 after cerebral infarction, while no changes were observed in other mitochondrial calcium handling proteins. Based on metabolomics, MCU knockdown reversed middle cerebral artery occlusion (MCAO)-induced up-regulated phosphoenolpyruvate and down-regulated GDP to protect energy metabolism after cerebral infarction. Furthermore, a total of 87 and 245 differentially expressed genes (DEGs) were detected by transcriptome sequencing among WT mice, MCU KO mice and MCU knockdown mice in the MCAO model, respectively. Then, NR4A1 was identified as one of the DEGs in different MCU expressions in vivo ischemia stroke model via transcriptomic screening and genetic validation. Furthermore, MCU knockdown downregulated the ischemia-induced upregulation of NR4A1 expression. Together, this is the further evidence that the MCU knockdown exerts a protective role after cerebral infarction by promoting calcium homeostasis, inhibiting mitochondrial apoptosis and protecting energy metabolism.

The BBSome regulates mitochondria dynamics and function

ObjectiveThe essential role of mitochondria in regulation of metabolic function and other physiological processes has garnered enormous interest in understanding the mechanisms controlling the function of this organelle. We assessed the role of the BBSome, a protein complex composed of eight Bardet-Biedl syndrome (BBS) proteins, in the control of mitochondria dynamic and function. MethodsWe used a multidisciplinary approach that include CRISPR/Cas9 technology-mediated generation of a stable Bbs1 gene knockout hypothalamic N39 neuronal cell line. We also analyzed the phenotype of BBSome deficient mice in presence or absence of the gene encoding A-kinase anchoring protein 1 (AKAP1). ResultsOur data show that the BBSome play an important role in the regulation of mitochondria dynamics and function. Disruption of the BBSome cause mitochondria hyperfusion in cell lines, fibroblasts derived from patients as well as in hypothalamic neurons and brown adipocytes of mice. The morphological changes in mitochondria translate into functional abnormalities as indicated by the reduced oxygen consumption rate and altered mitochondrial distribution and calcium handling. Mechanistically, we demonstrate that the BBSome modulates the activity of dynamin-like protein 1 (DRP1), a key regulator of mitochondrial fission, by regulating its phosphorylation and translocation to the mitochondria. Notably, rescuing the decrease in DRP1 activity through deletion of one copy of the gene encoding AKAP1 was effective to normalize the defects in mitochondrial morphology and activity induced by BBSome deficiency. Importantly, this was associated with improvement in several of the phenotypes caused by loss of the BBSome such as the neuroanatomical abnormalities, metabolic alterations and obesity highlighting the importance of mitochondria defects in the pathophysiology of BBS. ConclusionsThese findings demonstrate a critical role of the BBSome in the modulation of mitochondria function and point to mitochondrial defects as a key disease mechanism in BBS.

Protein aggregation and calcium dysregulation are hallmarks of familial Parkinson’s disease in midbrain dopaminergic neurons

Mutations in the SNCA gene cause autosomal dominant Parkinson’s disease (PD), with loss of dopaminergic neurons in the substantia nigra, and aggregation of α-synuclein. The sequence of molecular events that proceed from an SNCA mutation during development, to end-stage pathology is unknown. Utilising human-induced pluripotent stem cells (hiPSCs), we resolved the temporal sequence of SNCA-induced pathophysiological events in order to discover early, and likely causative, events. Our small molecule-based protocol generates highly enriched midbrain dopaminergic (mDA) neurons: molecular identity was confirmed using single-cell RNA sequencing and proteomics, and functional identity was established through dopamine synthesis, and measures of electrophysiological activity. At the earliest stage of differentiation, prior to maturation to mDA neurons, we demonstrate the formation of small β-sheet-rich oligomeric aggregates, in SNCA-mutant cultures. Aggregation persists and progresses, ultimately resulting in the accumulation of phosphorylated α-synuclein aggregates. Impaired intracellular calcium signalling, increased basal calcium, and impairments in mitochondrial calcium handling occurred early at day 34–41 post differentiation. Once midbrain identity fully developed, at day 48–62 post differentiation, SNCA-mutant neurons exhibited mitochondrial dysfunction, oxidative stress, lysosomal swelling and increased autophagy. Ultimately these multiple cellular stresses lead to abnormal excitability, altered neuronal activity, and cell death. Our differentiation paradigm generates an efficient model for studying disease mechanisms in PD and highlights that protein misfolding to generate intraneuronal oligomers is one of the earliest critical events driving disease in human neurons, rather than a late-stage hallmark of the disease.

Computational Model of the Effect of Mitochondrial Dysfunction on Excitation–Contraction Coupling in Skeletal Muscle

It has become well established that mitochondria not only regulate myoplasmic calcium in skeletal muscle, but also use that calcium to stimulate oxidative phosphorylation (OXPHOS). While experimental approaches have allowed for imaging of mitochondrial calcium and membrane potentials in isolated fibers, capturing the role of mitochondria and the impact of mitochondrial impairments on excitation–contraction coupling (ECC) remains difficult to explore in intact muscle. Computational models have been widely used to examine the structure and function of skeletal muscle contraction; however, models of ECC to date lack communication between the myoplasm and mitochondria for regulating calcium and ATP during sustained contractions. To address this, a mathematical model of mitochondrial calcium handling and OXPHOS was integrated into a physiological model of ECC incorporating action potential propagation, calcium handling between the sarcoplasmic reticulum (SR) and the myoplasm, and crossbridge cycling. The model was used to examine the protective role of mitochondria during repeated stimulation and the impact of mitochondrial dysfunction on ECC resulting from progressive OXPHOS inhibition. Pathological myoplasmic calcium accumulation occurred through distinct mechanisms in the model in the case of either electron transport chain, F1F0 ATP synthase, or adenine nucleotide transporter impairments. To investigate the effect of each impairment on force, a model of calcium-stimulated apoptosis was utilized to capture dysfunction-induced reductions in muscle mass, driving whole muscle force loss. The model presented in this study can be used to examine the role of mitochondria in the regulation of calcium, ATP, and force generation during voluntary contraction.

Deregulation of Mitochondrial Calcium Handling Due to Presenilin Loss Disrupts Redox Homeostasis and Promotes Neuronal Dysfunction.

Mitochondrial dysfunction and oxidative stress are major contributors to the pathophysiology of neurodegenerative diseases, including Alzheimer’s disease (AD). However, the mechanisms driving mitochondrial dysfunction and oxidative stress are unclear. Familial AD (fAD) is an early onset form of AD caused primarily by mutations in the presenilin-encoding genes. Previously, using Caenorhabditis elegans as a model system to study presenilin function, we found that loss of C. elegans presenilin orthologue SEL-12 results in elevated mitochondrial and cytosolic calcium levels. Here, we provide evidence that elevated neuronal mitochondrial generated reactive oxygen species (ROS) and subsequent neurodegeneration in sel-12 mutants are a consequence of the increase of mitochondrial calcium levels and not cytosolic calcium levels. We also identify mTORC1 signaling as a critical factor in sustaining high ROS in sel-12 mutants in part through its repression of the ROS scavenging system SKN-1/Nrf. Our study reveals that SEL-12/presenilin loss disrupts neuronal ROS homeostasis by increasing mitochondrial ROS generation and elevating mTORC1 signaling, which exacerbates this imbalance by suppressing SKN-1/Nrf antioxidant activity.

Long non-coding RNA Tug1 modulates mitochondrial and myogenic responses to exercise in skeletal muscle

BackgroundMitochondria have an essential role in regulating metabolism and integrate environmental and physiological signals to affect processes such as cellular bioenergetics and response to stress. In the metabolically active skeletal muscle, mitochondrial biogenesis is one important component contributing to a broad set of mitochondrial adaptations occurring in response to signals, which converge on the biogenesis transcriptional regulator peroxisome proliferator-activated receptor coactivator 1-alpha (PGC-1α), and is central to the beneficial effects of exercise in skeletal muscle. We investigated the role of long non-coding RNA (lncRNA) taurine-upregulated gene 1 (TUG1), which interacts with PGC-1α in regulating transcriptional responses to exercise in skeletal muscle.ResultsIn human skeletal muscle, TUG1 gene expression was upregulated post-exercise and was also positively correlated with the increase in PGC-1α gene expression (PPARGC1A). Tug1 knockdown (KD) in differentiating mouse myotubes led to decreased Ppargc1a gene expression, impaired mitochondrial respiration and morphology, and enhanced myosin heavy chain slow isoform protein expression. In response to a Ca2+-mediated stimulus, Tug1 KD prevented an increase in Ppargc1a expression. RNA sequencing revealed that Tug1 KD impacted mitochondrial Ca2+ transport genes and several downstream PGC-1α targets. Finally, Tug1 KD modulated the expression of ~300 genes that were upregulated in response to an in vitro model of exercise in myotubes, including genes involved in regulating myogenesis.ConclusionsWe found that TUG1 is upregulated in human skeletal muscle after a single session of exercise, and mechanistically, Tug1 regulates transcriptional networks associated with mitochondrial calcium handling, muscle differentiation and myogenesis. These data demonstrate that lncRNA Tug1 exerts regulation over fundamental aspects of skeletal muscle biology and response to exercise stimuli.

Mitochondrial Damage in Myocardial Ischemia/Reperfusion Injury and Application of Natural Plant Products.

Ischemic heart disease (IHD) is currently one of the leading causes of death among cardiovascular diseases worldwide. In addition, blood reflow and reperfusion paradoxically also lead to further death of cardiomyocytes and increase the infarct size. Multiple evidences indicated that mitochondrial function and structural disorders were the basic driving force of IHD. We summed up the latest evidence of the basic associations and underlying mechanisms of mitochondrial damage in the event of ischemia/reperfusion (I/R) injury. This review then reviewed natural plant products (NPPs) which have been demonstrated to mitochondria-targeted therapeutic effects during I/R injury and the potential pathways involved. We realized that NPPs mainly maintained the integrality of mitochondria membrane and ameliorated dysfunction, such as improving abnormal mitochondrial calcium handling and inhibiting oxidative stress, so as to protect cardiomyocytes during I/R injury. This information will improve our knowledge of mitochondrial biology and I/R-induced injury's pathogenesis and exhibit that NPPs hold promise for translation into potential therapies that target mitochondria.

Metabolically driven maturation of human-induced-pluripotent-stem-cell-derived cardiac microtissues on microfluidic chips.

The immature physiology of cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) limits their utility for drug screening and disease modelling. Here we show that suitable combinations of mechanical stimuli and metabolic cues can enhance the maturation of hiPSC-derived cardiomyocytes, and that the maturation-inducing cues have phenotype-dependent effects on the cells' action-potential morphology and calcium handling. By using microfluidic chips that enhanced the alignment and extracellular-matrix production of cardiac microtissues derived from genetically distinct sources of hiPSC-derived cardiomyocytes, we identified fatty-acid-enriched maturation media that improved the cells' mitochondrial structure and calcium handling, and observed divergent cell-source-dependent effects on action-potential duration (APD). Specifically, in the presence of maturation media, tissues with abnormally prolonged APDs exhibited shorter APDs, and tissues with aberrantly short APDs displayed prolonged APDs. Regardless of cell source, tissue maturation reduced variabilities in spontaneous beat rate and in APD, and led to converging cell phenotypes (with APDs within the 300-450 ms range characteristic of human left ventricular cardiomyocytes) that improved the modelling of the effects of pro-arrhythmic drugs on cardiac tissue.

Interaction of Mitochondrial Calcium and ROS in Neurodegeneration.

Neurodegenerative disorders are currently incurable devastating diseases which are characterized by the slow and progressive loss of neurons in specific brain regions. Progress in the investigation of the mechanisms of these disorders helped to identify a number of genes associated with familial forms of these diseases and a number of toxins and risk factors which trigger sporadic and toxic forms of these diseases. Recently, some similarities in the mechanisms of neurodegenerative diseases were identified, including the involvement of mitochondria, oxidative stress, and the abnormality of Ca2+ signaling in neurons and astrocytes. Thus, mitochondria produce reactive oxygen species during metabolism which play a further role in redox signaling, but this may also act as an additional trigger for abnormal mitochondrial calcium handling, resulting in mitochondrial calcium overload. Combinations of these factors can be the trigger of neuronal cell death in some pathologies. Here, we review the latest literature on the crosstalk of reactive oxygen species and Ca2+ in brain mitochondria in physiology and beyond, considering how changes in mitochondrial metabolism or redox signaling can convert this interaction into a pathological event.

Adrenergic Receptor Regulation of Mitochondrial Function in Cardiomyocytes.

Adrenergic receptors (ARs) are G protein-coupled receptors that are stimulated by catecholamines to induce a wide array of physiological effects across tissue types. Both α1- and β-ARs are found on cardiomyocytes and regulate cardiac contractility and hypertrophy through diverse molecular pathways. Acute activation of cardiomyocyte β-ARs increases heart rate and contractility as an adaptive stress response. However, chronic β-AR stimulation contributes to the pathobiology of heart failure. By contrast, mounting evidence suggests that α1-ARs serve protective functions that may mitigate the deleterious effects of chronic β-AR activation. Here, we will review recent studies demonstrating that α1- and β-ARs differentially regulate mitochondrial biogenesis and dynamics, mitochondrial calcium handling, and oxidative phosphorylation in cardiomyocytes. We will identify potential mechanisms of these actions and focus on the implications of these findings for the modulation of contractile function in the uninjured and failing heart. Collectively, we hope to elucidate important physiological processes through which these well-studied and clinically relevant receptors stimulate and fuel cardiac contraction to contribute to myocardial health and disease.

Electrophysiological characterization of mitochondrial currents at the contact site of the inner and outer membrane

Mitochondrial ionic homeostasis is critical for maintaining mitochondrial morphology, membrane potential, bioenergetics, and calcium handling in the regulation of cell survival and death. Ion fluxes in and out of mitochondria are facilitated by ion channels and transporters in the outer and the inner mitochondrial membranes (OMM and IMM, respectively). We characterized the ionic current at the contact sites between the OMM and IMM using the patch clamp technique. Mitoplasts, mitochondria devoid of the OMM, were prepared by incubating isolated mitochondria in hypotonic solution.