Disruption of mitochondria-sarcoplasmic reticulum microdomain connectomics contributes to sinus node dysfunction in heart failure.

The sinoatrial node (SAN), the leading pacemaker region, generates electrical impulses that propagate throughout the heart. SAN dysfunction with bradyarrhythmia is well documented in heart failure (HF). However, the underlying mechanisms are not completely understood. Mitochondria are critical to cellular processes that determine the life or death of the cell. The release of Ca2+ from the ryanodine receptors 2 (RyR2) on the sarcoplasmic reticulum (SR) at mitochondria-SR microdomains serves as the critical communication to match energy production to meet metabolic demands. Therefore, we tested the hypothesis that alterations in the mitochondria-SR connectomics contribute to SAN dysfunction in HF. We took advantage of a mouse model of chronic pressure overload-induced HF by transverse aortic constriction (TAC) and a SAN-specific CRISPR-Cas9-mediated knockdown of mitofusin-2 (Mfn2), the mitochondria-SR tethering GTPase protein. TAC mice exhibited impaired cardiac function with HF, cardiac fibrosis, and profound SAN dysfunction. Ultrastructural imaging using electron microscope (EM) tomography revealed abnormal mitochondrial structure with increased mitochondria-SR distance. The expression of Mfn2 was significantly down-regulated and showed reduced colocalization with RyR2 in HF SAN cells. Indeed, SAN-specific Mfn2 knockdown led to alterations in the mitochondria-SR microdomains and SAN dysfunction. Finally, disruptions in the mitochondria-SR microdomains resulted in abnormal mitochondrial Ca2+ handling, alterations in localized protein kinase A (PKA) activity, and impaired mitochondrial function in HF SAN cells. The current study provides insights into the role of mitochondria-SR microdomains in SAN automaticity and possible therapeutic targets for SAN dysfunction in HF patients.

Dimethyl fumarate dose-dependently increases mitochondrial gene expression and function in muscle and brain of Friedreich's ataxia model mice.

Previously we showed that dimethyl fumarate (DMF) dose-dependently increased mitochondrial gene expression and function in cells and might be considered as a therapeutic for inherited mitochondrial disease, including Friedreich's ataxia (FA). Here we tested DMF's ability to dose-dependently increase mitochondrial function, mitochondrial gene expression (frataxin and cytochrome oxidase protein) and mitochondrial copy number in C57BL6 wild-type mice and the FXNKD mouse model of FA. We first dosed DMF at 0-320 mg/kg in C57BL6 mice and observed significant toxicity above 160 mg/kg orally, defining the maximum tolerated dose. Oral dosing of C57BL6 mice in the range 0-160 mg/kg identified a maximum increase in aconitase activity and mitochondrial gene expression in brain and quadriceps at 110 mg/kg DMF, thus defining the maximum effective dose (MED). The MED of DMF in mice overlaps the currently approved human-equivalent doses of DMF prescribed for multiple sclerosis (480 mg/day) and psoriasis (720 mg/day). In the FXNKD mouse model of FA, which has a doxycycline-induced deficit of frataxin protein, we observed significant decreases of multiple mitochondrial parameters, including deficits in brain mitochondrial Complex 2, Complex 4 and aconitase activity, supporting the idea that frataxin deficiency reduces mitochondrial gene expression, mitochondrial functions and biogenesis. About 110 mg/kg of oral DMF rescued these enzyme activities in brain and rescued frataxin and cytochrome oxidase expression in brain, cerebellum and quadriceps muscle of the FXNKD mouse model. Taken together, these results support the idea of using fumarate-based molecules to treat FA or other mitochondrial diseases.

Dual role of inorganic polyphosphate in cardiac myocytes: The importance of polyP chain length for energy metabolism and mPTP activation.

We have previously demonstrated that inorganic polyphosphate (polyP) is a potent activator of the mitochondrial permeability transition pore (mPTP) in cardiac myocytes. PolyP depletion protected against Ca2+-induced mPTP opening, however it did not prevent and even exacerbated cell death during ischemia-reperfusion (I/R). The central goal of this study was to investigate potential molecular mechanisms underlying these dichotomous effects of polyP on mitochondrial function. We utilized a Langendorff-perfused heart model of I/R to monitor changes in polyP size and chain length at baseline, 20 min no-flow ischemia, and 15 min reperfusion. Freshly isolated cardiac myocytes and mitochondria from C57BL/6J (WT) and cyclophilin D knock-out (CypD KO) mice were used to measure polyP uptake, mPTP activity, mitochondrial membrane potential, respiration and ATP generation. We found that I/R induced a significant decrease in polyP chain length. We, therefore, tested, the ability of synthetic polyPs with different chain length to accumulate in mitochondria and induce mPTP. Both short and long chain polyPs accumulated in mitochondria in oligomycin-sensitive manner implicating potential involvement of mitochondrial ATP synthase in polyP transport. Notably, only short-chain polyP activated mPTP in WT myocytes, and this effect was prevented by mPTP inhibitor cyclosprorin A and absent in CypD KO myocytes. To the contrary, long-chain polyP suppressed mPTP activation, and enhanced ADP-linked respiration and ATP production. Our data indicate that 1) effect of polyP on cardiac function strongly depends on polymer chain length; and 2) short-chain polyPs (as increased in ischemia-reperfusion) induce mPTP and mitochondrial uncoupling, while long-chain polyPs contribute to energy generation and cell metabolism.

Inorganic polyphosphate in cardiac myocytes: from bioenergetics to the permeability transition pore and cell survival.

Inorganic polyphosphate (polyP) is a linear polymer of Pi residues linked together by high-energy phosphoanhydride bonds as in ATP. PolyP is present in all living organisms ranging from bacteria to human and possibly even predating life of this planet. The length of polyP chain can vary from just a few phosphates to several thousand phosphate units long, depending on the organism and the tissue in which it is synthesized. PolyP was extensively studied in prokaryotes and unicellular eukaryotes by Kulaev's group in the Russian Academy of Sciences and by the Nobel Prize Laureate Arthur Kornberg at Stanford University. Recently, we reported that mitochondria of cardiac ventricular myocytes contain significant amounts (280±60 pmol/mg of protein) of polyP with an average length of 25 Pi and that polyP is involved in Ca(2+)-dependent activation of the mitochondrial permeability transition pore (mPTP). Enzymatic polyP depletion prevented Ca(2+)-induced mPTP opening during ischaemia; however, it did not affect reactive oxygen species (ROS)-mediated mPTP opening during reperfusion and even enhanced cell death in cardiac myocytes. We found that ROS generation was actually enhanced in polyP-depleted cells demonstrating that polyP protects cardiac myocytes against enhanced ROS formation. Furthermore, polyP concentration was dynamically changed during activation of the mitochondrial respiratory chain and stress conditions such as ischaemia/reperfusion (I/R) and heart failure (HF) indicating that polyP is required for the normal heart metabolism. This review discusses the current literature on the roles of polyP in cardiovascular health and disease.

Calcium signaling in cardiac mitochondria.

Mitochondrial Ca signaling contributes to the regulation of cellular energy metabolism, and mitochondria participate in cardiac excitation-contraction coupling (ECC) through their ability to store Ca, shape the cytosolic Ca signals and generate ATP required for contraction. The mitochondrial inner membrane is equipped with an elaborate system of channels and transporters for Ca uptake and extrusion that allows for the decoding of cytosolic Ca signals, and the storage of Ca in the mitochondrial matrix compartment. Controversy, however remains whether the fast cytosolic Ca transients underlying ECC in the beating heart are transmitted rapidly into the matrix compartment or slowly integrated by the mitochondrial Ca transport machinery. This review summarizes established and novel findings on cardiac mitochondrial Ca transport and buffering, and discusses the evidence either supporting or arguing against the idea that Ca can be taken up rapidly by mitochondria during ECC.

Mitochondria-mediated cardioprotection by trimetazidine in rabbit heart failure.

Trimetazidine (TMZ) is used successfully for treatment of ischemic cardiomyopathy, however its therapeutic potential in heart failure (HF) remains to be established. While the cardioprotective action of TMZ has been linked to inhibition of free fatty acid oxidation (FAO) via 3-ketoacyl CoA thiolase (3-KAT), additional mechanisms have been suggested. The aim of this study was to evaluate systematically the effects of TMZ on calcium signaling and mitochondrial function in a rabbit model of non-ischemic HF and to determine the cellular mechanisms of the cardioprotective action of TMZ. TMZ protected HF ventricular myocytes from cytosolic Ca(2+) overload and subsequent hypercontracture, induced by electrical and ß-adrenergic (isoproterenol) stimulation. This effect was mediated by the ability of TMZ to protect HF myocytes against mitochondrial permeability transition pore (mPTP) opening via attenuation of reactive oxygen species (ROS) generation by the mitochondrial electron transport chain (ETC) and uncoupled mitochondrial nitric oxide synthase (mtNOS). The majority of ROS generated by the ETC in HF arose from enhanced complex II-mediated electron leak. TMZ inhibited the elevated electron leak at the level of mitochondrial ETC complex II and improved impaired activity of mitochondrial complex I, thereby restoring redox balance and mitochondrial membrane potential in HF. While TMZ decreased FAO by ~15%, the 3-KAT inhibitor 4-bromotiglic acid did not provide protection against palmitic acid-induced mPTP opening, indicating that TMZ effects were 3-KAT independent. Thus, the beneficial effect of TMZ in rabbit HF was not linked to FAO inhibition, but rather associated with reduced complex II- and uncoupled mtNOS-mediated oxidative stress and decreased propensity for mPTP opening.

Measuring mitochondrial function in intact cardiac myocytes.

Mitochondria are involved in cellular functions that go beyond the traditional role of these organelles as the power plants of the cell. Mitochondria have been implicated in several human diseases, including cardiac dysfunction, and play a role in the aging process. Many aspects of our knowledge of mitochondria stem from studies performed on the isolated organelle. Their relative inaccessibility imposes experimental difficulties to study mitochondria in their natural environment-the cytosol of intact cells-and has hampered a comprehensive understanding of the plethora of mitochondrial functions. Here we review currently available methods to study mitochondrial function in intact cardiomyocytes. These methods primarily use different flavors of fluorescent dyes and genetically encoded fluorescent proteins in conjunction with high-resolution imaging techniques. We review methods to study mitochondrial morphology, mitochondrial membrane potential, Ca(2+) and Na(+) signaling, mitochondrial pH regulation, redox state and ROS production, NO signaling, oxygen consumption, ATP generation and the activity of the mitochondrial permeability transition pore. Where appropriate we complement this review on intact myocytes with seminal studies that were performed on isolated mitochondria, permeabilized cells, and in whole hearts.

Functional remodelling of perinuclear mitochondria alters nucleoplasmic Ca2+ signalling in heart failure.

Mitochondrial dysfunction in cardiomyocytes is a hallmark of heart failure development. Although initial studies recognized the importance of different mitochondrial subpopulations, there is a striking lack of direct comparison of intrafibrillar (IF) versus perinuclear (PN) mitochondria during the development of HF. Here, we use multiple approaches to examine the morphology and functional properties of IF versus PN mitochondria in pressure overload-induced cardiac remodelling in mice, and in non-failing and failing human cardiomyocytes. We demonstrate that PN mitochondria from failing cardiomyocytes are more susceptible to depolarization of mitochondrial membrane potential, reactive oxygen species generation and impairment in Ca2+ uptake compared with IF mitochondria at baseline and under physiological stress protocol. We also demonstrate, for the first time to our knowledge, that under normal conditions PN mitochondrial Ca2+ uptake shapes nucleoplasmic Ca2+ transients (CaTs) and limits nucleoplasmic Ca2+ loading. The loss of PN mitochondrial Ca2+ buffering capacity translates into increased nucleoplasmic CaTs and may explain disproportionate rise in nucleoplasmic [Ca2+] in failing cardiomyocytes at increased stimulation frequencies. Therefore, a previously unidentified benefit of restoring the mitochondrial Ca2+ uptake may be normalization of nuclear Ca2+ signalling and alleviation of altered excitation-transcription, which could be an important therapeutic approach to prevent adverse cardiac remodelling. This article is part of the theme issue 'The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease'.

T lymphocytes from malignant hyperthermia-susceptible mice display aberrations in intracellular calcium signaling and mitochondrial function.

Gain-of-function RyR1-p.R163C mutation in ryanodine receptors type 1 (RyR1) deregulates Ca2+ signaling and mitochondrial function in skeletal muscle and causes malignant hyperthermia in humans and mice under triggering conditions. We investigated whether T lymphocytes from heterozygous RyR1-p.R163C knock-in mutant mice (HET T cells) display measurable aberrations in resting cytosolic Ca2+ concentration ([Ca2+]i), Ca2+ release from the store, store-operated Ca2+ entry (SOCE), and mitochondrial inner membrane potential (ΔΨm) compared with T lymphocytes from wild-type mice (WT T cells). We explored whether these variables can be used to distinguish between T cells with normal and altered RyR1 genotype. HET and WT T cells were isolated from spleen and lymph nodes and activated in vitro using phytohemagglutinin P. [Ca2+]i and ΔΨm dynamics were examined using Fura 2 and tetramethylrhodamine methyl ester fluorescent dyes, respectively. Activated HET T cells displayed elevated resting [Ca2+]i, diminished responses to Ca2+ mobilization with thapsigargin, and decreased rate of [Ca2+]i elevation in response to SOCE compared with WT T cells. Pretreatment of HET T cells with ryanodine or dantrolene sodium reduced disparities in the resting [Ca2+]i and ability of thapsigargin to mobilize Ca2+ between HET and WT T cells. While SOCE elicited dissipation of the ΔΨm in WT T cells, it produced ΔΨm hyperpolarization in HET T cells. When used as the classification variable, the amplitude of thapsigargin-induced Ca2+ transient showed the best promise in predicting the presence of RyR1-p.R163C mutation. Other significant variables identified by machine learning analysis were the ratio of resting cytosolic Ca2+ level to the amplitude of thapsigargin-induced Ca2+ transient and an integral of changes in ΔΨm in response to SOCE. Our study demonstrated that gain-of-function mutation in RyR1 significantly affects Ca2+ signaling and mitochondrial fiction in T lymphocytes, which suggests that this mutation may cause altered immune responses in its carrier. Our data link the RyR1-p.R163C mutation, which causes inherited skeletal muscle diseases, to deregulation of Ca2+ signaling and mitochondrial function in immune T cells and establish proof-of-principle for in vitro T cell-based diagnostic assay for hereditary RyR1 hyperfunction.

PK11195 Protects From Cell Death Only When Applied During Reperfusion: Succinate-Mediated Mechanism of Action.

Aim: Reperfusion after myocardial ischemia causes cellular injury, in part due to changes in mitochondrial Ca2+ handling, oxidative stress, and myocyte energetics. We have previously shown that the 18-kDa translocator protein of the outer mitochondrial membrane (TSPO) can modulate Ca2+ handling. Here, we aim to evaluate the role of the TSPO in ischemia/reperfusion (I/R) injury. Methods: Rabbit ventricular myocytes underwent simulated acute ischemia (20 min) and reperfusion (at 15 min, 1 h, and 3 h) in the absence and presence of 50 µM PK11195, a TSPO inhibitor. Cell death was measured by lactate dehydrogenase (LDH) assay, while changes in mitochondrial Ca2+, membrane potential (ΔΨm), and reactive oxygen species (ROS) generation were monitored using confocal microscopy in combination with fluorescent indicators. Substrate utilization was measured with Biolog mitochondrial plates. Results: Cell death was increased by ~200% following I/R compared to control untreated ventricular myocytes. Incubation with 50 µM PK11195 during both ischemia and reperfusion did not reduce cell death but increased mitochondrial Ca2+ uptake and ROS generation. However, application of 50 µM PK11195 only at the onset and during reperfusion effectively protected against cell death. The large-scale oscillations in ΔΨm observed after ~1 h of reperfusion were significantly delayed by 1 µM cyclosporin A and almost completely prevented by 50 µM PK11195 applied during 3 h of reperfusion. After an initial increase, mitochondrial Ca2+, measured with Myticam, rapidly declined during 3 h of reperfusion after the initial transient increase. This decline was prevented by application of PK11195 at the onset and during reperfusion. PK11195 prevented a significant increase in succinate utilization following I/R and succinate-induced forward-mode ROS generation. Treatment with PK11195 was also associated with a significant increase in glutamate and a decrease in leucine utilization. Conclusion: PK11195 administered specifically at the moment of reperfusion limited ROS-induced ROS release and cell death, likely in part, by a shift from succinate to glutamate utilization. These data demonstrate a unique mechanism to limit cardiac injury after I/R.

Ketone Ester D-ß-Hydroxybutyrate-(R)-1,3 Butanediol Prevents Decline in Cardiac Function in Type 2 Diabetic Mice.

Background Heart failure is responsible for approximately 65% of deaths in patients with type 2 diabetes mellitus. However, existing therapeutics for type 2 diabetes mellitus have limited success on the prevention of diabetic cardiomyopathy. The aim of this study was to determine whether moderate elevation in D-ß-hydroxybutyrate improves cardiac function in animals with type 2 diabetes mellitus. Methods and Results Type 2 diabetic (db/db) and their corresponding wild-type mice were fed a control diet or a diet where carbohydrates were equicalorically replaced by D-ß-hydroxybutyrate-(R)-1,3 butanediol monoester (ketone ester [KE]). After 4 weeks, echocardiography demonstrated that a KE diet improved systolic and diastolic function in db/db mice. A KE diet increased expression of mitochondrial succinyl-CoA:3-oxoacid-CoA transferase and restored decreased expression of mitochondrial ß-hydroxybutyrate dehydrogenase, key enzymes in cardiac ketone metabolism. A KE diet significantly enhanced both basal and ADP-mediated oxygen consumption in cardiac mitochondria from both wild-type and db/db animals; however, it did not result in the increased mitochondrial respiratory control ratio. Additionally, db/db mice on a KE diet had increased resistance to oxidative and redox stress, with evidence of restoration of decreased expression of thioredoxin and glutathione peroxidase 4 and less permeability transition pore activity in mitochondria. Mitochondrial biogenesis, quality control, and elimination of dysfunctional mitochondria via mitophagy were significantly increased in cardiomyocytes from db/db mice on a KE diet. The increase in mitophagy was correlated with restoration of mitofusin 2 expression, which contributed to improved coupling between cytosolic E3 ubiquitin ligase translocation into mitochondria and microtubule-associated protein 1 light chain 3-mediated autophagosome formation. Conclusions Moderate elevation in circulating D-ß-hydroxybutyrate levels via KE supplementation enhances mitochondrial biogenesis, quality control, and oxygen consumption and increases resistance to oxidative/redox stress and mPTP opening, thus resulting in improvement of cardiac function in animals with type 2 diabetes mellitus.

Characteristics and function of cardiac mitochondrial nitric oxide synthase.

We used laser scanning confocal microscopy in combination with the nitric oxide (NO)-sensitive fluorescent dye DAF-2 and the reactive oxygen species (ROS)-sensitive dyes CM-H(2)DCF and MitoSOX Red to characterize NO and ROS production by mitochondrial NO synthase (mtNOS) in permeabilized cat ventricular myocytes. Stimulation of mitochondrial Ca(2+) uptake by exposure to different cytoplasmic Ca(2+) concentrations ([Ca(2+)](i) = 1, 2 and 5 microm) resulted in a dose-dependent increase of NO production by mitochondria when L-arginine, a substrate for mtNOS, was present. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca(2+) uniporter with Ru360 as well as blocking the respiratory chain with rotenone or antimycin A in combination with oligomycin inhibited mitochondrial NO production. In the absence of L-arginine, mitochondrial NO production during stimulation of Ca(2+) uptake was significantly decreased, but accompanied by increase in mitochondrial ROS production. Inhibition of mitochondrial arginase to limit L-arginine availability resulted in 50% inhibition of Ca(2+)-induced ROS production. Both mitochondrial NO and ROS production were blocked by the nNOS inhibitor (4S)-N-(4-amino-5[aminoethyl]aminopentyl)-N'-nitroguanidine and the calmodulin antagonist W-7, while the eNOS inhibitor L-N(5)-(1-iminoethyl)ornithine (L-NIO) or iNOS inhibitor N-(3-aminomethyl)benzylacetamidine, 2HCl (1400W) had no effect. The superoxide dismutase mimetic and peroxynitrite scavenger MnTBAP abolished Ca(2+)-induced ROS generation and increased NO production threefold, suggesting that in the absence of MnTBAP either formation of superoxide radicals suppressed NO production or part of the formed NO was transformed quickly to peroxynitrite. In the absence of L-arginine, mitochondrial Ca(2+) uptake induced opening of the mitochondrial permeability transition pore (PTP), which was blocked by the PTP inhibitor cyclosporin A and MnTBAP, and reversed by L-arginine supplementation. In the presence of the mtNOS cofactor (6R)-5,6,7,8,-tetrahydrobiopterin (BH(4); 100 microm) mitochondrial ROS generation and PTP opening decreased while mitochondrial NO generation slightly increased. These data demonstrate that mitochondrial Ca(2+) uptake activates mtNOS and leads to NO-mediated protection against opening of the mitochondrial PTP, provided sufficient availability of l-arginine and BH(4). In conclusion, our data show the importance of L-arginine and BH(4) for cardioprotection via regulation of mitochondrial oxidative stress and modulation of PTP opening by mtNOS.

Mitochondrial Quality Control in Aging and Heart Failure: Influence of Ketone Bodies and Mitofusin-Stabilizing Peptides.

Aim: Aging and heart failure (HF) are each characterized by increased mitochondrial damage, which may contribute to further cardiac dysfunction. Mitophagy in response to mitochondrial damage can improve cardiovascular health. HF is also characterized by increased formation and consumption of ketone bodies (KBs), which may activate mitophagy and provide an endogenous mechanism to limit the adverse effects of mitochondrial damage. However, the role of KBs in activation of mitophagy in aging and HF has not been evaluated. Methods: We assessed mitophagy by measuring mitochondrial Parkin accumulation and LC3-mediated autophagosome formation in cardiomyocytes from young (2.5 months), aged (2.5 years), and aged rabbits with HF (2.5 years) induced by aortic insufficiency and stenosis. Levels of reactive oxygen species (ROS) generation and redox balance were monitored using genetically encoded sensors ORP1-roGFP2 and GRX1-roGFP2, targeted to mitochondrial or cytosolic compartments, respectively. Results: Young rabbits exhibited limited mitochondrial Parkin accumulation with small (~1 µm2) puncta. Those small Parkin puncta increased four-fold in aged rabbit hearts, accompanied by elevated LC3-mediated autophagosome formation. HF hearts exhibited fewer small puncta, but many very large Parkin-rich regions (4-5 µm2) with completely depolarized mitochondria. Parkin protein expression was barely detectable in young animals and was much higher in aged and maximal in HF hearts. Expression of mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1) was reduced by almost 50% in HF, consistent with improper fusion-fission, contributing to mitochondrial Parkin build-up. The KB ß-hydroxybutyrate (ß-OHB) enhanced mitophagy in young and aging myocytes, but not in HF where ß-OHB further increased the number of cells with giant Parkin-rich regions. This ß-OHB effect on Parkin-rich areas was prevented by cell-permeable TAT-MP1Gly peptide (thought to promote MFN2-dependent fusion). Basal levels of mitochondrial ROS were highest in HF, while cytosolic ROS was highest in aged compared to HF myocytes, suggesting that cytosolic ROS promotes Parkin recruitment to the mitochondria. Conclusion: We conclude that elevated KB levels were beneficial for mitochondrial repair in the aging heart. However, an impaired MFN2-DRP1-mediated fusion-fission process in HF reduced this benefit, as well as Parkin degradation and mitophagic signaling cascade.

Mitofusin 2 Is Essential for IP3-Mediated SR/Mitochondria Metabolic Feedback in Ventricular Myocytes.

Aim: Endothelin-1 (ET-1) and angiotensin II (Ang II) are multifunctional peptide hormones that regulate the function of the cardiovascular and renal systems. Both hormones increase the intracellular production of inositol-1,4,5-trisphosphate (IP3) by activating their membrane-bound receptors. We have previously demonstrated that IP3-mediated sarcoplasmic reticulum (SR) Ca2+ release results in mitochondrial Ca2+ uptake and activation of ATP production. In this study, we tested the hypothesis that intact SR/mitochondria microdomains are required for metabolic IP3-mediated SR/mitochondrial feedback in ventricular myocytes. Methods: As a model for disrupted mitochondrial/SR microdomains, cardio-specific tamoxifen-inducible mitofusin 2 (Mfn2) knock out (KO) mice were used. Mitochondrial Ca2+ uptake, membrane potential, redox state, and ATP generation were monitored in freshly isolated ventricular myocytes from Mfn2 KO mice and their control wild-type (WT) littermates. Results: Stimulation of ET-1 receptors in healthy control myocytes increases mitochondrial Ca2+ uptake, maintains mitochondrial membrane potential and redox balance leading to the enhanced ATP generation. Mitochondrial Ca2+ uptake upon ET-1 stimulation was significantly higher in interfibrillar (IFM) and perinuclear (PNM) mitochondria compared to subsarcolemmal mitochondria (SSM) in WT myocytes. Mfn2 KO completely abolished mitochondrial Ca2+ uptake in IFM and PNM mitochondria but not in SSM. However, mitochondrial Ca2+ uptake induced by beta-adrenergic receptors activation with isoproterenol (ISO) was highest in SSM, intermediate in IFM, and smallest in PNM regions. Furthermore, Mfn2 KO did not affect ISO-induced mitochondrial Ca2+ uptake in SSM and IFM mitochondria; however, enhanced mitochondrial Ca2+ uptake in PNM. In contrast to ET-1, ISO induced a decrease in ATP levels in WT myocytes. Mfn2 KO abolished ATP generation upon ET-1 stimulation but increased ATP levels upon ISO application with highest levels observed in PNM regions. Conclusion: When the physical link between SR and mitochondria by Mfn2 was disrupted, the SR/mitochondrial metabolic feedback mechanism was impaired resulting in the inability of the IP3-mediated SR Ca2+ release to induce ATP production in ventricular myocytes from Mfn2 KO mice. Furthermore, we revealed the difference in Mfn2-mediated SR-mitochondrial communication depending on mitochondrial location and type of communication (IP3R-mRyR1 vs. ryanodine receptor type 2-mitochondrial calcium uniporter).

Mitochondrial Ca2+ and the heart.

It is now well established that mitochondria accumulate Ca(2+) ions during cytosolic Ca(2+) ([Ca(2+)](i)) elevations in a variety of cell types including cardiomyocytes. Elevations in intramitochondrial Ca(2+) ([Ca(2+)](m)) activate several key enzymes in the mitochondrial matrix to enhance ATP production, alter the spatial and temporal profile of intracellular Ca(2+) signaling, and play an important role in the initiation of cell death pathways. Moreover, mitochondrial Ca(2+) uptake stimulates nitric oxide (NO) production by mitochondria, which modulates oxygen consumption, ATP production, reactive oxygen species (ROS) generation, and in turn provides negative feedback for the regulation of mitochondrial Ca(2+) accumulation. Controversy remains, however, whether in cardiac myocytes mitochondrial Ca(2+) transport mechanisms allow beat-to-beat transmission of fast cytosolic [Ca(2+)](i) oscillations into oscillatory changes in mitochondrial matrix [Ca(2+)](m). This review critically summarizes the recent experimental work in this field.

Cardiac-specific Conditional Knockout of the 18-kDa Mitochondrial Translocator Protein Protects from Pressure Overload Induced Heart Failure.

Heart failure (HF) is characterized by abnormal mitochondrial calcium (Ca2+) handling, energy failure and impaired mitophagy resulting in contractile dysfunction and myocyte death. We have previously shown that the 18-kDa mitochondrial translocator protein of the outer mitochondrial membrane (TSPO) can modulate mitochondrial Ca2+ uptake. Experiments were designed to test the role of the TSPO in a murine pressure-overload model of HF induced by transverse aortic constriction (TAC). Conditional, cardiac-specific TSPO knockout (KO) mice were generated using the Cre-loxP system. TSPO-KO and wild-type (WT) mice underwent TAC for 8 weeks. TAC-induced HF significantly increased TSPO expression in WT mice, associated with a marked reduction in systolic function, mitochondrial Ca2+ uptake, complex I activity and energetics. In contrast, TSPO-KO mice undergoing TAC had preserved ejection fraction, and exhibited fewer clinical signs of HF and fibrosis. Mitochondrial Ca2+ uptake and energetics were restored in TSPO KO mice, associated with decreased ROS, improved complex I activity and preserved mitophagy. Thus, HF increases TSPO expression, while preventing this increase limits the progression of HF, preserves ATP production and decreases oxidative stress, thereby preventing metabolic failure. These findings suggest that pharmacological interventions directed at TSPO may provide novel therapeutics to prevent or treat HF.

Signaling mechanisms that mediate nitric oxide production induced by acetylcholine exposure and withdrawal in cat atrial myocytes.

Fluorescence microscopy and the NO-sensitive indicator 4,5-diaminofluorescein were used to determine the effects of acetylcholine (ACh) on intracellular NO (NOi) in cat atrial myocytes. Field stimulation (1 Hz) of cells or exposure of quiescent cells to ACh (1 to 10 micromol/L) had no effect on NOi. However, in field-stimulated cells, ACh exposure increased NOi, and ACh withdrawal elicited an additional, prominent increase in NOi production. During ACh exposure, addition of 1 micromol/L atropine increased NOi production similar to ACh withdrawal. ACh-induced increases in NOi were reduced by prior exposure to 1 mmol/L extracellular Ca2+ ([Ca2+]o) and prevented by 0.5 mmol/L [Ca2+]o, 1 micromol/L verapamil, 1 micromol/L atropine, 10 micromol/L L-N5-(1-iminoethyl)ornithine, 10 micromol/L W-7, or incubating cells in pertussis toxin or 10 micromol/L LY294002 (inhibits phosphatidylinositol 3-kinase). Switching to 0.5 mmol/L [Ca2+]o during ACh withdrawal prevented the additional increase in NOi. ACh exposure increased phosphorylation (Ser473) of protein kinase B (Akt), and this effect was blocked by LY294002 and unaffected in low (0.5 mmol/L) [Ca2+]o. Confocal microscopy revealed that ACh exposure increased NOi at local subsarcolemmal sites, and ACh withdrawal additionally increased NOi by recruiting additional subsarcolemmal release sites. Disruption of caveolae by 2 mmol/L methyl-beta-cyclodextrin abolished ACh-induced NOi production. We conclude that in cat atrial myocytes, ACh stimulates NOi release from local subsarcolemmal sites. ACh-induced increases in NOi requires both muscarinic receptor-mediated Gi protein/phosphatidylinositol 3-kinase/Akt signaling and voltage-activated Ca2+ influx for stimulation of calmodulin-dependent endothelial NO synthase activity. Increases in NOi elicited by ACh withdrawal result from the recovery of Ca2+ influx after ACh inhibition. NO signaling elicited by ACh withdrawal stimulates rapid recovery from cholinergic atrial inhibition.

Inositol 1,4,5-trisphosphate-mediated sarcoplasmic reticulum-mitochondrial crosstalk influences adenosine triphosphate production via mitochondrial Ca2+ uptake through the mitochondrial ryanodine receptor in cardiac myocytes.

AIMS: Elevated levels of inositol 1,4,5-trisphosphate (IP3) in adult cardiac myocytes are typically associated with the development of cardiac hypertrophy, arrhythmias, and heart failure. IP3 enhances intracellular Ca(2+ )release via IP3 receptors (IP3Rs) located at the sarcoplasmic reticulum (SR). We aimed to determine whether IP3-induced Ca(2+ )release affects mitochondrial function and determine the underlying mechanisms. METHODS AND RESULTS: We compared the effects of IP3Rs- and ryanodine receptors (RyRs)-mediated cytosolic Ca(2+ )elevation achieved by endothelin-1 (ET-1) and isoproterenol (ISO) stimulation, respectively, on mitochondrial Ca(2+ )uptake and adenosine triphosphate (ATP) generation. Both ET-1 and isoproterenol induced an increase in mitochondrial Ca(2+ )(Ca(2 +) m) but only ET-1 led to an increase in ATP concentration. ET-1-induced effects were prevented by cell treatment with the IP3 antagonist 2-aminoethoxydiphenyl borate and absent in myocytes from transgenic mice expressing an IP3 chelating protein (IP3 sponge). Furthermore, ET-1-induced mitochondrial Ca(2+) uptake was insensitive to the mitochondrial Ca(2+ )uniporter inhibitor Ru360, however was attenuated by RyRs type 1 inhibitor dantrolene. Using real-time polymerase chain reaction, we detected the presence of all three isoforms of IP3Rs and RyRs in murine ventricular myocytes with a dominant presence of type 2 isoform for both receptors. CONCLUSIONS: Stimulation of IP3Rs with ET-1 induces Ca(2+ )release from the SR which is tunnelled to mitochondria via mitochondrial RyR leading to stimulation of mitochondrial ATP production.

Beta 2-adrenergic receptor signaling acts via NO release to mediate ACh-induced activation of ATP-sensitive K+ current in cat atrial myocytes.

In atrial myocytes, an initial exposure to isoproterenol (ISO) acts via cAMP to mediate a subsequent acetylcholine (ACh)-induced activation of ATP-sensitive K(+) current (I(K,ATP)). In addition, beta-adrenergic receptor (beta-AR) stimulation activates nitric oxide (NO) release. The present study determined whether the conditioning effect of beta-AR stimulation acts via beta(1)- and/or beta(2)-ARs and whether it is mediated via NO signaling. 0.1 microM ISO plus ICI 118,551 (ISO-beta(1)-AR stimulation) or ISO plus atenolol (ISO-beta(2)-AR stimulation) both increased L-type Ca(2+) current (I(Ca,L)) markedly, but only ISO-beta(2)-AR stimulation mediated ACh-induced activation of I(K,ATP). 1 microM zinterol (beta(2)-AR agonist) also increased I(Ca,L) and mediated ACh-activated I(K,ATP). Inhibition of NO synthase (10 microM L-NIO), guanylate cyclase (10 microM ODQ), or cAMP-PKA (50 microM Rp-cAMPs) attenuated zinterol-induced stimulation of I(Ca,L) and abolished ACh-activated I(K,ATP). Spermine-NO (100 microM; an NO donor) mimicked beta(2)-AR stimulation, and its effects were abolished by Rp-cAMPs. Intracellular dialysis of 20 microM protein kinase inhibitory peptide (PKI) abolished zinterol-induced stimulation of I(Ca,L). Measurements of intracellular NO ([NO](i)) using the fluorescent indicator DAF-2 showed that ISO-beta(2)-AR stimulation or zinterol increased [NO](i). L-NIO (10 microM) blocked ISO- and zinterol-induced increases in [NO](i). ISO-beta(1)-AR stimulation failed to increase [NO](i). Inhibition of G(i)-protein by pertussis toxin significantly inhibited zinterol-mediated increases in [NO](i). Wortmannin (0.2 microM) or LY294002 (10 microM), inhibitors of phosphatidylinositol 3'-kinase (PI-3K), abolished the effects of zinterol to both mediate ACh-activated I(K,ATP) and stimulate [NO](i). We conclude that both beta(1)- and beta(2)-ARs stimulate cAMP. beta(2)-ARs act via two signaling pathways to stimulate cAMP, one of which is mediated via G(i)-protein and PI-3K coupled to NO-cGMP signaling. Only beta(2)-ARs acting exclusively via NO signaling mediate ACh-induced activation of I(K,ATP). NO signaling also contributes to beta(2)-AR stimulation of I(Ca,L). The differential effects of beta(1)- and beta(2)-ARs can be explained by the coupling of these two beta-ARs to different effector signaling pathways.