Shey-Shing Sheu - US grants

Ca2+ is an essential messenger in numerous key biological processes. However, the cellular mechanisms of the control of intracellular free Ca2+ concentration (Ca2+ activity) are still incompletely understood. The present proposed studies will take the advantage of newly developed experimental techniques (e.g. ion-sensitive microelectrodes and fluorescent indicator quin 2) to explore the following questions: (1) What are the relative contributions of sarcolemmal Na-Ca exchange versus Ca-pump for the Ca2+ extrusion at physiologial resting state? Do Ca-pump, Na-Ca exchange and Na-K pump coordinate effectively to keep intracellular Ca2+ and Na+ low at unphysiological conditions (e.g. Na-K pump is inhibited completely)? (2) Is the Na-Ca exchange electrogenic? (3) What is the relative importance between sarcolemmal efflux systems and intracellular buffering systems for the Ca2+ homeostasis? (4) How does cAMP modulate ca2+ transport and metabolism? We will combine the use of ion-sensitive microelectrodes, voltage clamp and tension recording to measure simultaneously intracellular ion activities, membrane voltage, membrane current and muscle contraction in sheep cardiac Purkinje fibers and guinea pig ventricular papillary muscles. We will also measure cytosolic free Ca2+ concentration in isolated cardiac myocytes from rat and guinea pig ventricles. The Ca2+ transport systems in plasma membrane and intracellular organelles will be distinguished by chemical or pharmacological manipulations. For instance, Na-Ca exchange can be inhibited by removing all Na+ in external solution and sarcoplasmic reticulum can be blocked by caffeine. These proposed studies will allow us to analyze quantitatively the contribution of several Ca2+ controlling systems for the homeostasis of this ion. Furthermore, they will provide the information about the universal role of Ca2+ in many physiological and pathological states of the heart. Therefore, these studies will broaden our understanding on the fundamental principles of normal and abnormal cardiac excitation and contraction.

The long-term objective of this proposal is to understand intracellular Ca2+ homeostasis in cardiac muscle. Studies with fluorescence digital imaging microscope (FDIM) cellular Ca2+ concentration ([Ca2+]i) in numerous tissues. Moreover, the dynamic nature of intracellular Ca2+ is amplified by the activation of sarcolemmal receptors that are linked to phospholipid breakdown. Although receptor-mediated Ca2+ signal is the subject of intense research interest, little is known about its role in heart. Therefore, we will exploit several techniques to accomplish five specific aims: 1) to complete the experimental work already in progress. 2) To determine quantitatively the spatial distribution of [Ca2+]i in cardiac cells. 3) To improve the time resolution of our FDIM for recording the temporal distribution of [Ca2+]i. 4) To investigate the effects of alpha1- adrenergic receptor, low affinity muscarinic receptor and purinergic receptor activation of [Ca2+]i. 5) To assess the role of protein kinase C (PKC) in modulating L-type Ca2+ channels. Most of the experiments will use single cells from guinea pig and rat ventricles. The [Ca2+]i will be determined with FDIM. The L-type Ca2+ channels will be isolated electrophysiologically by the whole-cell patch-clamp. To correlated [Ca2+]i measurements with the functional aspects of the heart, intracellular Na+ activity and contractility will be measured in papillary muscles. The quantification of [Ca2+]i will be achieved by measuring the ratio values of fura-2 fluorescence at two wavelengths and referring to calibrations obtained from "in vitro" and "in vivo" conditions. The temporal resolution of imaging [Ca2+]i will be improved by a computer- controlled dual beam illumination system. To study the link between the receptors that are coupled to phosphoinostitide turnover and [Ca2+]i, agonists for the alpha1-adrenergic receptor (methoxamine), the low affinity muscarinic receptor (carbachol) and the purinergic receptor (ATP) will be used. To identify the sources of Ca2+ responsible for receptor-mediated [Ca2+]i changes, drugs (e.g. nitrendipine for L-type Ca2+ channels) that inhibit cellular Ca2+ - transport systems will be used. The modulation of L-type Ca2+ channels by PKC will be studied by using phorbol esters, synthetic diacylglycerols, and purified PKC isozymes. These proposed studies will provide information about intracellular Ca2+ homeostasis in cardiac muscle. Because Ca2+ is a key regulator for cardiac function in physiological and pathological states, these studies will broaden our understanding on the fundamental principles of normal and abnormal cardiac excitation and contraction.

neurotoxicology; NMDA receptors; calcium flux; mitochondria; BCL2 gene /protein; apoptosis; neural degeneration; membrane permeability; mitochondrial membrane; membrane potentials; transfection /expression vector; calcium indicator; tissue /cell culture; fluorescence microscopy;

The long-term objective of our research is to elucidate mechanisms of mitochondrial Ca2+ transport in cardiac muscle cells under both physiological and pathological conditions. Extensive studies have implicated that mitochondrial Ca2+ play a pivotal role in controlling cellular Ca2+ homeostasis, energy metabolism, and apoptosis. However, little is known about the molecular identities and functional diversities of mitochondrial Ca2+ transporters. Our Central hypothesis is that "cardiac mitochondria contain at least two Ca2+-activated influx mechanisms, a mitochondrial ryanodine receptor that operates most effectively in the lower ranges (<50 ^M) of Ca2+ and a Ca2+ uniporter that operates most effectively in higher ranges of Ca2+. These two Ca2+ transporters sequester Ca2+ proficiently and complementarity for regulating Ca2+ homeostasis, ATP production, and reactive oxygen species generation. These mitochondrial Ca2+- mediated functions are achieved physiologically by a concomitant increase in mitochondrial ADP, serving not only as a substrate for ATP production but also an inhibitor for mitochondrial permeability transition pores. In diseased states, this coordinated interaction between Ca2+ and ADP is disrupted and prone the cells to Ca2+- and oxidative stress-mediated injury and death". The four specific aims are: 1) to further characterize the molecular properties of mitochondrial ryanodine receptor, 2) to evaluate the distinct role of mitochondrial ryanodine receptor and Ca2+ uniporter in Ca2+ regulation, 3) to determine the modulation of mitochondrial Ca2+ uptake by redox environments, and 4) to elucidate the role of mitochondrial Ca2+ and ADP in balancing cellular ATP generation and Ca2+ homeostasis in healthy and cardiomyopathic hearts. Working closely with our collaborators, we will use multidisciplinary approaches encompassing cell biology, biochemistry, biophysics, and molecular biology, to elucidate the molecular and functional characteristicsof mitochondrial Ca2+ influx mechanisms. Recent studies of diseases caused by either mitochondrial DNA mutations or mitochondrial dysfunction all suggest that Ca2+ deregulation is most critical. Some examples of such diseases are cardiomyopthy in chronic heart failure, ischemic heart disease, neurodegenerative diseases, diabetics, obesity, and aging. Therefore, completion of our research aims will not only to have a significant impact on our understanding of basic mechanisms in the etiology of mitochondria-mediated diseases, but also on our strategies in developing the therapeutic means for treating these diseases.

Maintaining mitochondrial function is critical for everyday operation of the heart. Proper mitochondrial function requires maintaining regulated and selective permeability of the mitochondrial inner membrane to ions and metabolites. Mitochondrial permeability transition occurs when the inner membrane loses its selective permeability by opening of a large-conductance nonselective channel, the permeability transition pore (PTP). High concentrations of mitochondrial Ca2+ and reactive oxygen species (ROS) are known to open PTP. PTP opening depolarizes mitochondria and causes mitochondrial swelling; thus, sustained opening of PTP leads to mitochondrial dysfunction and cell death, which is associated with many cardiovascular diseases including ischemia-reperfusion (I-R) injury and heart failure. Therefore, understanding how PTP is regulated has significant clinical value. It has long been known that increasing mitochondrial Ca2+ concentration opens PTP. More recently, mitochondrial dynamics mediated by fission and fusion have also been suggested to be involved in regulating PTP. However, the mechanisms by which Ca2+ and mitochondrial dynamics regulate PTP remain unknown. Our new findings show that increasing mitochondrial Ca2+ induces phosphorylation of cyclophilin D (CypD) through GSK-3? activation in mitochondria. Furthermore, we have found a transient opening of PTP (tPTP) that is distinct from conventional PTP and is regulated by mitochondrial dynamics proteins. Inhibition of the fission protein Drp1 increases this novel tPTP. Importantly, the inner membrane fusion protein OPA1 was found to be a critical factor for the novel tPTP. Although Drp1 inhibition is known to decrease pathologic PTP opening and reduce myocardial infarction in I-R, the mechanism of this fission inhibition-mediated protection is unknown. We postulate that the mitochondrial dynamics-mediated novel tPTP is a structurally distinct entity from conventional PTP, and thus in pathological conditions, can serve as a relief valve for excess matrix Ca2+ and proton gradient that induces ROS overproduction; as such, it could thereby prevent pathologic opening of PTP. Supported by our findings, the Central Hypothesis is that CypD phosphorylation induced by matrix Ca2+ is a key event for PTP opening, while mitochondrial dynamics regulates novel tPTP, and their interplay determines cardiac pathology outcomes. We will test this hypothesis by three specific aims: (1) to determine the mechanism of Ca2+-induced PTP opening, (2) to determine the mechanism of mitochondrial dynamics-regulated novel tPTP opening, and (3) to investigate the interplay between conventional PTP and novel tPTP in the pathological setting. The proposed studies will utilize advanced in vitro and in vivo cell and molecular biological approaches along with new fluorescence-based assays. Completion of the proposed studies will generate a new paradigm for the regulatory mechanisms of different forms of PTP and their functional interplay. The new findings will provide mechanistic basis for a new therapeutic strategy to decrease heart I-R injury and other cardiac pathology associated with PTP.

DESCRIPTION (provided by applicant): Heart failure is a serious medical problem: over 5 million people in the US affected. The cause for chronic heart failure is multifaceted and includes bioenergetic deficiency, Ca2+ overload, and oxidative stress. ADP is the key substrate for ATP formation. Moreover, ADP is a potent inhibitor for the opening of mitochondrial permeability transition pore (mPTP). Although the molecular identity of mPTP is still unsolved, 2 different concepts stand out. One indicates that adenine nucleotide translocase (ANT) is not required for mPTP, whereas the other depicts mPTP as a multi-protein complex, the ANT and the mitochondrial peptidyl-prolyl cis-trans isomerase known as cyclophilin-D (Cyp-D), are the key components. Interestingly, binding of ADP to ANT facilitates cardiolipin to stabilize respiratory chain supercomplexes so that the efficiency of ATP generation is enhanced. Oxidation of cardiolipin destabilizes these supercomplexes and has been linked to mitochondrial dysfunction associated with aging, diabetic cardiomyopathy, ischemia-reperfusion injury, and heart failure. Furthermore, ADP inhibits efflux of inorganic phosphate Pi, the key mitochondrial Ca2+ buffer that forms Ca2+-Pi complex. Finally, activation of the mitochondrial fission protein, DLP1 causes mitochondrial fission, ROS generation, and mPTP opening. Taken together, these results lead us to hypothesize that binding of ADP to ANT serves two fundamental roles in mitochondrial function: enhancing ATP generation efficiency by stabilizing cardiolipin-ETC complexes and inhibiting mPTP by decreasing ROS generation and DLP1 activation. Physiologically, ADP- mediated mPTP inhibition minimizes excessive mitochondrial ROS generation and Ca2+ release from mitochondria in order to optimize the effectiveness of excitation-contraction-metabolism (ECM) coupling. Pathologically, defects of this ADP regulatory mechanism lead to energetic failure, oxidative stress, and Ca2+ dysregulation that enhance cardiac vulnerability to injury. We propose 2 Specific Aims: Specific Aim 1: To determine the mechanisms for ADP inhibition of mPTP. Hypothesis: ADP stabilizes cardiolipin integrity and inhibits DLP1 activity in the mitochondria and thus protects against Cyp-D-independent mPTP opening. Moreover, ADP decreases ROS generation via mPTP inhibition, and thus minimizes feedback activation of mPTP by ROS. Specific Aim 2: To assess the role of ADP in cardiac protection. Hypothesis: The mPTP in hearts of diseased models exhibits increased sensitivity to Ca2+-induced opening due to its predisposition to the first hit stresses including oxidative stress, high DLP1 activity, and/or diminished Ca2+ buffering capacity. Maintenance of optimal matrix ADP levels alleviates this increased mPTP vulnerability. Disturbances in the interaction between metabolic signaling and Ca2+/redox/cell death signaling are fundamental in disease pathogenesis including cardiac diseases. The exploratory and high-risk ideas in this application, if validated, will break new ground in a wide spectrum of disease mechanisms and treatments.

DESCRIPTION (provided by applicant): In cardiac muscle, uptake of Ca2+ by mitochondria during the excitation-contraction (EC) coupling is important for synchronizing ATP production with the needs of contraction (excitation-bioenergetics (EB) coupling). However, an integrative mechanism to describe the EB coupling is missing mainly due to the lack of information about the molecular identities of several key proteins involved in this process. Recent ground-breaking studies have shown that mitofusin 2 (Mfn2) is responsible for tethering endoplasmic reticulum to mitochondria. Moreover, several components of the mitochondrial Ca2+ uniporter (mtCU) including its pore unit (MCU) have been uncovered. These progresses open up a new opportunity for applying molecular tools to elucidate the mechanisms of mitochondria-sarcoplasmic reticulum (MITO-SR) tethering in controlling bioenergetics and Ca2+ dynamics. Our labs were the first to show a privileged transport of Ca2+ from SR to mitochondria in cardiomyocytes due to their juxtaposition, secured by tethering with Mfn2 family proteins. Dr. Sheu has a long standing expertise in using genetic and physiological tools to study in and ex vivo the cardiac mitochondrial Ca2+ and reactive oxygen species (ROS) regulation and Dr. Csordas has a strong track record in using biochemical and imaging techniques to investigate MITO-SR tethering and local Ca2+ crosstalk. Together, we will combine these interdisciplinary approaches to test the hypothesis that MITO-SR tethering via Mfn2 family proteins creates a micro-domain of high Ca2+ between these two organelles during EC coupling. Moreover, mtCUs are clustered in the region of inner mitochondrial membrane (IMM) that is in proximity with SR. Losses of this juxtaposition decrease EB coupling efficiency that leads to energy deficiency and oxidative stress and subsequent heart failure (HF). Three specific aims are: 1) to identify the tethering components that bridge MITO-SR associations. Hypothesis: Mfn2, possibly a truncated form, aligns SR with mitochondrial contact points. 2) To determine the distribution of mtCU in the IMM. Hypothesis: mtCU is preferentially localized in the areas where mitochondria and SR are in contact. 3) To elucidate the mechanisms by which the disrupted MITO-SR association leads to HF. Hypothesis: The loss of MITO-SR association leads to the inefficiency of EB coupling, as a result, electron transport chain activities and matrix NADPH levels decrease, which cause ROS to increase. The increase in ROS together with the decrease in ATP enhances the susceptibility of mitochondrial permeability transition pore for opening, especially under the energy-demanding stresses, which leads to cardiac injury and failure. The destruction of mitochondrial Ca2+ homeostasis is a key element for leading to mitochondrial dysfunction-associated clinical phenotypes including heart diseases (e.g. HF), neurodegenerative diseases, metabolic diseases (diabetes), and aging. Because MITO-SR juxtaposition is a critical factor in controlling mitochondrial Ca2+ dynamics, it is of scientific importance and clinical relevance that the present proposal will bring forth the molecular mechanism underlying the cardiac MITO-SR tethering and translate this unique structure to the physiological regulation of mitochondrial Ca2+ influx in bioenergetics and to the pathological implication of energy deficiency and oxidative stress in HF.

Project Summary/Abstract Mitochondrial dynamics, including fission, fusion, and movement, is a fundamental mechanism in regulating mitochondrial function. Dynamin-related protein 1 (Drp1) is the major GTP hydrolyzing protein that is responsible for fission. Studies have shown that Drp1 is abundantly expressed in adult cardiac myocytes. Paradoxically, compared to numerous cell types, adult cardiac myocytes exhibit very low frequency in mitochondrial fission. This dichotomy between the abundance of Drp1 and scarcity of mitochondrial fission has prompted us to investigate the non-canonical roles of Drp1 in cardiac muscle cells. We hypothesize that Drp1 is strategically accumulated in the mitochondria associated sarcoplasmic reticulum (SR) membrane (MAM). During excitation- contraction coupling, the localized high Ca2+ in the SR-mitochondria junctions further increases the translocation of nearby cytosolic Drp1 to mitochondria. Then, Drp1 is anchored firmly in the MAM by actin. The activation of Drp1 leads to enhanced mitochondrial respiration for ATP generation as such the heart can work most effectively and sustainably. However, excessive Drp1 activation leads to persistent mitochondrial transition pore opening and excessive reactive oxygen species (ROS) generation that causes cell injury and death. The following three specific aims are proposed to test this hypothesis. Aim 1: To determine whether and how Drp1 is preferentially localized in the SR-mitochondria junctions. Aim 2: To determine the molecular mechanisms by which Drp1 regulates excitation-contraction-bioenergetics coupling. Aim 3: To determine how chronic over activation of Drp1 leads to dysfunction in the stressed heart. We will employ multiple techniques, including biochemistry (from in vitro to in situ assays), molecular biology (gene knock in or knock out, overexpression, RNA interference), cell biology (confocal, fluorescence resonance energy transfer, super-resolution microscopy, electron microscopy), cardiac physiology (echocardiogram, NMR spectroscopy), and isoproterenol infusion mouse model of cardiac hypertrophy and failure, to obtain experimental results that will lead to mechanistic insights. Successful completion of the proposed aims will allow us to introduce a new paradigm that describes the regulation of excitation-contraction-bioenergetics-ROS nexus by Drp1 through localized Ca2+ activation and actin anchoring. This fundamental signaling mechanism will describe not only how a healthy heart can perform perpetually in face of enormous workload but also why the over activation of this unique process can lead to heart failure. Finally, this new knowledge will provide new insights in developing and discovering therapeutic agents targeting Drp1-mediated signaling pathways for treating heart failure.

Mitochondrial impairment is a main contributor and potential therapeutic target in the development of heart failure (HF). During excitation-contraction coupling, Ca2+ is released from the sarcoplasmic reticulum of dyadic junction (jSR) to initiate muscle contraction. jSR is often tethered to mitochondria, where a high [Ca2+] nanodomain is created to facilitate Ca2+ propagation to the mitochondrial matrix to stimulate ATP production (excitation-energetics coupling). The beating heart consumes much energy; thus, cardiomyocytes must be efficient in dynamically balancing energy demands and supplies while avoiding potential Ca2+ mediated toxicity. We find that mitochondrial Ca2+ uniporter (mtCU), responsible for Ca2+ uptake, concentrate to hotspots at the interface with jSR whereas the robust Na+-dependent Ca2+ extrusion (mitochondrial Na+/Ca2+ exchanger, NCLX) is mostly excluded from these segments. In this proposal, we put forward the overarching theme that the mitochondria, which associate with jSR, remodel their membrane structure and protein distribution asymmetrically into two zones proximal and distal from jSR, to protect their long-term integrity while serving the excitation-energetics coupling. Imbalance in this adaptation leads to HF. Based on our preliminary data and published literature; we hypothesize that mitochondrial Ca2+ influx and efflux are uniquely distanced, and so a [Ca2+] gradient is created in the matrix to ensure an effective Ca2+ mediated energy production without toxicity by minimizing the amount of Ca2+ required to cycle through the matrix for a given [Ca2+] rise. The mitochondrial zone proximal to the jSR forms a Ca2+ receptacle with enhanced Ca2+ entry but limited exit and less membrane barriers for diffusion, while the mitochondrial zone distal to jSR has dense cristae membrane for vigorous ATP generation without subjecting to Ca2+ toxicity. Finally, the constant high Ca2+ in Ca2+ receptacle zone renders it more susceptible for physiological mitophagy via mitochondrial fission. However, prolonged stress turns this physiological defense mechanism maladaptive, with excess of fragmented mitochondria without jSR Ca2+ input (no excitation-energetics coupling) due to the loss of juxtaposition, as such leads to HF etiology. Three specific aims are: 1) Investigate the physiological implications of differential submitochondrial distribution of mitochondrial Ca2+ uptake and extrusion mechanisms in excitation-energetics coupling. 2. To establish submitochondrial structural zoning associated with the zonal Ca2+ transport. 3. To assess the impact of zoning on mitochondrial maintenance/quality control and how excessive fragmentations associated with stresses could turn zoning maladaptive and lead to HF.