Mitochondrial transplantation: From animal models to clinical use in humans

1. Introduction
The importance of the mitochondrion in the maintenance and preservation of cellular homeostasis and function is well established and there is a sufficient body of evidence to show that mitochondrial injury or loss of function is deleterious (Durhuus et al., 2015). The mechanisms leading to mitochondrial dysfunction are varied and include genetic changes occurring at the nuclear or the mitochondrial genome, environmental insult or alterations in homeosis. In all cases, the end result of mitochondrial dysfunction is cellular dysfunction that can limit or severely modulate organ function and ultimately increase morbidity and mortality.

In our research, we have focused on the myocardium, a highly aerobic organ in which mitochondria comprise 30% of cellular volume (Faulk et al., 1995; Faulk et al., 1995a; Toyoda et al., 2000; Toyoda et al., 2001; McCully et al., 2003). The mitochondria supply the energy requirements of the myocardium. This energy is derived through oxidative phosphorylation in the myocardium and is dependent upon the coronary circulation. Under equilibrium conditions the mitochondria within the heart extract greater than 79% of arterial oxygen from the coronary arteries (Fillmore et al., 2013). As heart rate increases or if myocardial workload is increased the oxygen demand is increased and is dependent upon increased coronary flow. Thus, any interruption or impedance in coronary blood flow will significantly limit oxygen delivery to the heart and significantly decrease function and hemostasis (Akhmedov et al., 2015; Doenst et al., 2013; Kolwicz et al., 2013). It is generally accepted that the cessation of coronary blood flow, and thus oxygen delivery, is the initial step in the process leading to myocardial ischemic injury. The sequence of events and the mechanisms associated with this injury are many and are reviewed elsewhere (Lesnefsky et al., 2003; Kalogeris et al., 2012; Ong et al., 2015 Kalogeris et al., 2016 ; Lesnefsky et al., 2017). The end result of ischemia is loss of high energy synthesis and the depletion of high energy stores such that the heart is unable to support hemostasis and maintain function (Rosca et al., 2013 ) .

This reduction of high energy synthesis and stores is rapid. 31Pnuclear magnetic resonance studies have shown that following regional or global ischemia wherein the blood flow to the heart is temporarily ceased, high energy phosphate synthesis and stores are rapidly decreased within 6 minutes and that this decrease continues for at least 60 180 minutes after the restoration of blood flow and is associated with significantly decreased myocardial cellular viability and myocardial function (Tsukube et al., 1997).

Mitochondrial modulations induced by ischemia in the myocardium are many. We and others have demonstrated that following ischemia there are changes in mitochondrial morphology and structure (Rousou et al., 2004; Lesnefsky et al., 2004; McCully et al., 2007). Transmission electron microscopy and lightscattering spectrophotometry have shown that ischemia significantly increases mitochondrial matrix and cristae area and mitochondrial matrix volume (McCully et al., 2007). In addition, there is a decrease in mitochondrial complex activity, cytochrome oxidase I Vmax and a decrease in oxygen consumption and an increase in mitochondrial calcium accumulation (Faulk et al.,1995a).

These changes occur in concert with changes in mitochondrial transcriptomics, with downregulation of annotation clusters for mitochondrion function and energy production and the downregulation of cofactor catabolism, generation of precursor metabolites of energy, cellular carbohydrate metabolism, regulation of biosynthesis, regulation of transcription, and mitochondrial structure and function (enrichment score >2.0, P<.05) ( Black et al., 2012; Masuzawa et al., 2013). In addition, there are changes evident in overall protein synthesis. Proteomic analysis has shown that ischemia significantly alters mitochondrial proteins involved in fatty acid and glucose metabolism, ATP biosynthesis, and oxidoreductase activity (fold change >1.4, P < .05) ( Black et al., 2012; Masuzawa et al., 2013). All these changes are associated with decreased myocardial cellular viability and decreased myocardial function and suggest that the mitochondrion plays a key role in myocardial viability and function following ischemia and repercussion. In total, these data have provided a basis for continued mitochondrial associated investigations into the rescue and preservation of myocardial tissue and myocardial function (Suleiman et al., 2001). The methodologies for these investigations have been many and varied. In general, the approach to cardioprotection has been either associative or indirect with emphasis on a single mechanistic route or complex or the use of an additive or inhibitor, used either as a single therapy or in combination with others. These include, but are not limited to, the use of pharmaceuticals either before or after ischemia, such as antioxidants, the use of calcium channel antagonists, adenosine, adenosine deaminase inhibitors, adenosine transport inhibitors, or a combination of both , adenosine receptor agonists, mitochondrial ATPsensitive potassium channel openers, phosphodiesterase inhibitors, 5' AMPactivated protein kinase activators, metabolic modulators, antiinflammatory agents and procedural approaches including, preischemia, postischemia and remote ischemic preconditioning (Hsiao et al., 2015 ; Madonna et al., 2015; Hausenloy et al., 2016; Laskowski et al., 2016; OrenesPiñero et al., 2015). In some methodologies, therapeutic intervention is required days or months prior to the ischemic event. Unfortunately, clinical trials using these approaches, either alone or in combination, have for the most part been unsuccessful. 2. Mitochondrial transplantation
We rationalized that the therapeutic approach to cardioprotection should be comprehensive and rather than involving a single or multiple mechanistic pathways, intervention should be specific. To this end, we speculated that the replacement or augmentation of mitochondria damaged during ischemia and reperfusion should be the target for therapeutic intervention. We hypothesized that viable mitochondria isolated form the patient’s own body, from a nonischemic area, and then delivered by direct injection into the ischemic organ would replace or augment damaged mitochondria; thus, allowing for the rescue of myocardial cells and restoration of myocardial function. We have termed this therapeutic intervention; mitochondrial transplantation (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016).
2.1 Mitochondrial isolation

Mitochondrial transplantation is based on the delivery of isolated, viable mitochondria to the target organ. The isolation of mitochondria can be performed using a variety of techniques and methodologies. In our initial studies, we used a standard procedure requiring consecutive low and high speed centrifugation to isolate purified mitochondria. This procedure required approximately 90120 minutes to complete. The repetitive centrifugation steps increases the time for mitochondrial isolation and ultimately reduce mitochondrial viability (Graham 2001; Frezza et al., 2007; Pallotti and Lenaz 2007; Wieckowski et al., 2009; FernándezVizarra et al., 2010; Schmitt S, et al., 2013).

In cardiac surgery, and in many other surgical interventions, the interventional time is 4560 minutes and therefore mitochondrial isolation times of 90120 minutes are inappropriate for clinical usage. Mitochondrial isolation time must be short and efficient so that the therapeutic use of mitochondrial transplantation would not extend the surgical time and possibly add to patient morbidity or mortality. To meet the demands and requirements for clinical application, we have developed a rapid methodology for the isolation of autologous mitochondria (Preble et al., 2014; Preble et al., 2014a).

Firstly, two small pieces of autologous tissue are obtained from the patient’s own body during the surgical procedure. The tissue is dissected out using a #6 biopsy punch. The amount of tissue is less than 0.1 gram. The source of tissue is dependent upon the surgical entry point and access. The only requirement being that the tissue source must be free from ischemia and be viable.

In our studies, we have used viable, nonischemic skeletal muscle tissue as a source for isolated mitochondria. The muscle tissue was obtained from the pectoralis major or the rectus abdominis based on standardized minithoracotomy or sternotomy, respectively. Other tissue sources can also be used and we have used liver tissue with excellent results. The tissue, once obtained is immediately used for mitochondria isolation.

The methodology for the isolation of mitochondria for use in mitochondrial transplantation is simple and rapid and can be performed in under 30 minutes. The freshly isolated tissue is homogenized using a commercial automated homogenizer. For our uses we have used the Miletenyi gentleMACS Dissociator (Miltenyi Biotec Inc., San Diego, CA).

This homogenizer was chosen over the more standardized PotterElvehjem homogenizer (glass with Teflon pestle) or bead based homogenizers as it provides a sterilized, disposable unit for homogenization with fixed plastic wings for homogenization and an automated programable homogenization protocol allowing for uniform and consistent homogenization of tissue that is not easily achieved with manual homogenization methods.

The other systems require cleaning and sterilization for each usage and allow for variance in homogenization based on the operator. Bead homogenization systems were not considered due to the possibility of contamination of the mitochondria by bead fragments. Once the tissue is obtained homogenization can be performed in 90120 seconds.

The homogenized tissue is then subjected to brief digestion (10 minutes on ice) with subtilisin A (Protease from Bacillus licheniformis, Type VIII, Sigma, Aldrich, St. Louis, MO) and the digested homogenate filtered through a series of disposable sterile mesh filters. The filtration can all be performed in 23 minutes.

The mitochondria can then be used for direct application or can be concentrated by centrifugation (9000 rpm at 4 oC for 10 minutes). The mitochondrial yield using this methodology is approximately 1 x 109 to 1 x 1010 mitochondria, using the two biopsy tissue samples (< 0.1 g) and provides sufficient mitochondria for quality assurance and quality control assessment ( Preble et al., 2014a). The isolated mitochondria are of the correct size and shape as assessed by electron microscopy and have normal cristae and membranes and show no damage or injury. The isolated mitochondria are pure and have no detectable cytosolic, nuclear or microsomal components. Functional analysis of isolated mitochondria shows that the isolated mitochondria maintain membrane potential and viability and that oxygen consumption and respiratory control index for malateinduced complex I and succinateinduced complex II are equal to that of mitochondria isolated by other methodologies (Rousou et al., 2004; McCully et al., 2009). Once isolated, the mitochondria are immediately used for mitochondrial transplantation. We have found that the isolated mitochondria can be stored on ice for approximately 1 hour but storage beyond this time point greatly reduces efficacy. This is in agreement with previous reports that have shown that mitochondrial bioenergetic function is decreased to <10–15% of normal after the mitochondria were frozen, even when preservatives are used (Wechsler, 1961; Olson et al., 1967). In all our studies, we have used total mitochondria for mitochondrial transplantation. The bioenergetic function of this population includes that of subsarcolemmal and intrafibrillar mitochondria. Previous studies have shown that these mitochondrial subpopulations have differing oxygen consumption and metabolism (Riva et al., 2005; Chen et al., 2008; Kurian et al., 2012). In our studies, we have examined the cardioprotective efficacy of intrafibrillar, subsarcolemmal and total mitochondria. We have found that total mitochondria provide for cardioprotection and that no added cardioprotection is provided using either subsarcolemmal or interfibrillar mitochondrial subpopulations (McCully et al., 2009). It is important that the isolated mitochondria be intact and viable. The use of dead, nonviable mitochondria, mitochondrial proteins or complexes, mitochondrial DNA/RNA or high energy phosphates alone or in combination do not provide cardioprotection (McCully et al., 2009). It has been previously demonstrated that exogenous ATP supplementation and/or ATP synthesis promoters do not restore highenergy phosphate stores and have no beneficial effects on postischemic functional recovery in the heart (McCully et al., 2009). Following determination of mitochondrial number and viability the isolated mitochondria are suspended in 1 ml of respiration buffer containing 250 mmol/l sucrose, 2 mmol/l KH2PO4, 10 mmol/l MgCl2, 20 mmol/l K+HEPES buffer, pH 7.2, 0.5 mmol/l K+EGTA, pH 8.0, 5 mmol/l glutamate, 5 mmol/l malate, 8 mmol/l succinate and 1 mmol/l ADP. Quality control and assurance parameters have been previously described by us (Preble et al., 2014). The isolated mitochondria are then directly injected into the ischemic zone of the heart just prior to reperfusion using a 1 mL tuberculin syringe with a 28or 32gauge needle. The injection volumes are 0.1 mL and contain approximately 1 x 107 mitochondria at each injection site. This volume is optimal and allows for mitochondrial uptake within the myocardium with no backflow leakage of the injected mitochondria. In our studies, we have found that 810 individual injections are sufficient to cover the areaat–risk, although the absolute number of injection sites can be increased. 2.2 Mitochondrial delivery
The direct injection of mitochondria is simple and allows for focal concentration of the injected mitochondria. In our studies the number of mitochondria used for direct injection is 13 x 107 mitochondria. The mitochondria are suspended in 1 mL respiration buffer and injected at 810 sites within the area at risk using a 1 mL tuberculin syringe with a 2832 gage needle (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016). Mitochondrial concentrations > 2 x 108 are not fully suspended in 1 mL of respiration buffer and are therefore not advised for use for mitochondrial transplantation by direct injection.

Fluorescence microscopy has demonstrated that transplanted mitochondria delivered by direct injection are present and viable for at least 28 days following injection into the myocardium, the end point of our animal experiments (Masuzawa et al., 2013, Kaza et al., 2016).

The transplanted mitochondria are widely distributed from the epicardium to the subendocardium >2–3 mm from the injection site (McCully et al., 2009; Masuzawa et al., 2013). The majority of injected mitochondria are found initially within the interstitial spaces between cardiomyocytes. Within 1 hour postdelivery, the transplanted mitochondria are detectable within cardiomyocytes residing near the sarcolemma between Zlines of the sarcomeres and in clusters around endogenous damaged mitochondria as well as near the nucleus. Enumeration of injected mitochondria has shown that 43.52% ± 4.46 (mean ± SEM) of the injected mitochondria are attached to or found within cardiomyocytes (Cowan et al., 2016).

We have speculated that the mechanisms through which the transplanted mitochondria distribute within the myocardium following direct injection into the myocardium may be associated with alterations in myocardial structure that occur after myocardial ischemia. Our studies have demonstrated that following ischemia there is a significant increase in myocardial interfibrillar space that provides for both longitudinal and transverse myocardial interfibrillar separations (Tansey et al., 2006).

These structural changes are not associated with alterations in conduction velocity anisotropy or in tissue edema but occur coincident with significant decreases in postischemic functional recovery and increased myocardial apoptosis and necrosis. We have hypothesized that these interfibrillar separations allow for the distribution of injected mitochondria within the myocardium.

While the delivery of mitochondria by direct injection is practical for many applications, it does not allow for global distribution of the transplanted mitochondria.

Multiple injections are needed for global distribution within the heart and require organ manipulation to access posterior and lateral aspects. To allow for global distribution of mitochondria we have recently demonstrated that mitochondria can also be delivered to the target organ by vascular infusion (Cowan et al., 2016).

In preliminary investigations, we have found that for optimal distribution 1 x 109 mitochondria in 5 mL respiration buffer is efficacious and multiple injections can be performed. Using this protocol we have demonstrated that vascular delivery of mitochondria through the coronary arteries results in the rapid and widespread distribution of exogenous mitochondria throughout the heart, within 10 minutes and provides for cardioprotection.

To demonstrate uptake and distribution of the transplanted mitochondria we have labeled isolated mitochondria with 18Frhodamine 6G and magnetic iron oxide nanoparticles. The use of these labels allowed for image analysis using positron emission tomography, computed tomography, and magnetic resonance imaging. Our results show that the transplantation of mitochondria by vascular delivery is rapid and effective Decaycorrected measurements of 18Frhodamine 6G using a dose calibrator revealed most of the 18Frhodamine 6G labeled mitochondria remained contained within the injected hearts throughout reperfusion (77.3% ± 5.5, mean ± SEM).

Positron emission tomography and computed tomography revealed that the transplanted mitochondria were distributed from the heart apex to the base. Quantitative assessment of perfused mitochondrial position in the heart tissue demonstrated that 24.76% ± 2.50 (mean ± SEM) and 23.64% ± 2.42 (mean ± SEM) of the transplanted mitochondria were associated with cardiomyocytes and blood vessels, respectively (Cowan et al., 2016).

To demonstrate the efficacy of vascular delivery of mitochondria to the heart, we delivered unlabeled mitochondria to the regionally ischemic heart. In these experiments we demonstrated that vascular delivery of mitochondria through the coronary arteries following transient ischemia and reperfusion significantly decreased myocardial infarct size and significantly enhanced postischemic functional recovery. The reductions in infarct size and the enhancement of regional functional recovery achieved with vascular delivery of mitochondria are not significantly different from that obtained by direct injection of mitochondria (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016). These data indicate that autologous mitochondrial transplantation is efficacious as a cardioprotective therapy whether these organelles ae delivered by directly injection or delivered by vascular infusion through the coronary arteries.

2.3 Localization to endorgan by vascular delivery
In all our studies, we have noted that the distribution of mitochondria following delivery by direct injection or by vascular infusion remains within the heart and is not detectable in other organs. This finding is important as the delivery of mitochondria by vascular infusion provides for localized therapy without crosscontamination to other endorgans. In the heart we deliver mitochondria by injection into the coronary arteries to avoid systemic distribution; however, we have also delivered mitochondria to the lung by vascular infusion through the pulmonary artery.

In these studies, the mitochondria were labeled with 18Frhodamine 6G as above. Positron emission tomography and computed tomography showed that the mitochondria were localized in the lung and were not detectable in any other areas of the body.

At present, we do not have a mechanism for this “endorgan homing”, where the transplanted mitochondria are retained by the immediate downstream organ, but suggest that this observation may play an important therapeutic role in future studies and applications using mitochondrial transplantation.

2.4 Mechanisms of mitochondrial uptake and internalization
In previous studies, we have investigated a variety of mechanisms that may be associated with mitochondrial uptake and internalization following mitochondrial. These studies were performed using well established pharmacological blockers of clathrin mediated endocytosis, actinmediated endocytosis, macropinocytosis, and tunneling nanotubes (Le et al., 2000; BereiterHahn Jet al., 2008; Lou et al., 2012; Islam et al., 2012; Huang et al., 2013; Kitani et al., 2014). Our studies demonstrated that autologous mitochondria delivered by direct injection are internalized by actindependent endocytosis (Pacak et al., 2015).

Mitochondrial uptake by vascular delivery appears to be more complex. The rapid and widespread uptake of mitochondria when delivered by vascular infusion would suggest that mechanisms allowing for the rapid passage of mitochondria through the vascular wall are involved. Previous studies support the concept that cells can routinely escape from the circulation. It has been previously shown that certain cardiac and mesenchymal stem cells appear to be actively expelled from the vasculature in a process different from diapedesis (Cheng et al., 2012; Allen et al., 2017).

Transmigration of stem cells through the vascular wall requires extensive remodeling of the endothelium (Allen et al., 2016). Cheng et al., 2012 have suggested that this occurs in three stages 1) adhesion of infused cells to the microvessel lining; 2) pocketing of infused cells by endothelial projections; 3) breakdown of the adjacent vascular wall, releasing cells into the interstitium.

The first two steps require integrindependent interactions between transplanted cells and host endothelium, while matrix metalloproteinases mediate the subsequent breakdown of the microvessel wall. These steps are unlikely to occur rapidly or to be associated with the mitochondrion.

Another possible mechanism for mitochondrial uptake may be diapedesislike. Previous studies have shown that some cells routinely escape from the circulation. For example, leukocyte extravasation (i.e. diapedesis) between venous endothelial cells is a wellunderstood process that involves cell adhesion proteins.

It is unlikely, however, that isolated, exogenous mitochondria use a similar mechanism as leukocytes to move through the wall of blood vessels as they do not express the array of proteins involved in diapedesis on their outer membrane (Pagliarini et al., 2008; Calvo et al., 2010).

Whether mitochondria pass between or through endothelial cells and the region of the vasculature at which this process occurs remain to be determined. The size of the isolated mitochondria and the rapidity of their uptake by vascular perfusion would not appear to support any currently defined mechanisms. At present, we are investigating several possible mechanisms of mitochondrial uptake by vascular infusion; but, have yet to conclusively identify a definitive mechanistic route.

3. Mitochondrial transplantation for cardioprotection

3.1 Animal models
In the isolated perfused rabbit heart model and subsequent studies in the in vivo rabbit and pig heart models, we have investigated the use of mitochondrial transplantation as a cardioprotective therapy (McCully et al., 2009; Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016). In these studies, the myocardium was made temporarily ischemic by ligating the left anterior descending artery with a snare. This temporary ligation results in the attenuation or cessation of coronary blood flow such that oxygen delivery to the myocardium is insufficient to meet oxygen demand. The resulting injury is termed ischemia/reperfusion injury and is characterized by the loss of myocardial cell viability and decreased contractile function within the area at risk. In general, we subject 2530% of the left ventricular mass to ischemia, resulting in a loss in cell viability of approximately 30% and a reduction in contractile force of approximately 25% based on regional systolic shortening. These decrements in cell viability and function are reproducible between large animal models and mimic events occurring in human acute myocardial infarction or surgical intervention during cardiopulmonary bypass. Following 30 minutes of temporary ligation the snare is released and coronary blood flow to the region is reestablished. To show the efficacy of mitochondrial transplantation, we typically deliver autologous mitochondria by direct injection or by vascular delivery, just prior to or at the very start of reperfusion. Control hearts receive vehicle alone (respiration buffer with no mitochondria).

3.2 Effects of mitochondrial transplantation on arrhythmogenicity
The transplantation of mitochondria has similarities in methodology with stem cell transplantation. In stem cell transplantation using skeletal muscle myoblasts, it has been reported that skeletal muscle myoblasts, clustering of the cells can occur, resulting in arrhythmia with postoperative episodes of sustained tachycardia due to alterations in electrical coupling (Macia et al., 2009). To demonstrate that mitochondrial transplantation is not proarrhythmic we have used serial 12 lead electrocardiogram (ECG) analysis and optical mapping (Masuzawa et al., 2013). Our results show that there is no proarrhythmogenicity associated with mitochondrial transplantation. We observed no ventricular tachycardia, bradycardia, fibrillation, or conduction system defects or repolarization heterogeneity associated with mitochondrial transplantation. In addition, there were no changes in serial ECG, QRS duration or corrected QT interval. There was no evidence of any changes associated with ventricular wall motion disturbances, left ventricle hypertrophy, valve dysfunction, fibrosis, or pericardial effusion either at the time of injection, or at any time up to 4 weeks following transplantation of autogeneic mitochondria (Masuzawa et al., 2013).
To confirm these findings, we also performed optical mapping (Masuzawa et al., 2013). In these studies, a 400fold increase in mitochondria, 8.4 x 107/gram tissue wet weight as compared to 2 x 105/gram tissue wet weight, was directly injected into the heart. The number of mitochondria injected per site was significantly greater than that used in the in situ heart (4.2 × 108 vs. 1.2 × 106), so that any acute arrhythmogenic responses could be observed. Our results showed that even with this highly increased mitochondrial load, sequential isopotential maps from the left ventricles injected with mitochondria showed no detectable abnormal impulse propagation on the myocardial surface associated with mitochondrial transplantation.

3.3 Effects of mitochondrial transplantation on immune and autoimmune response
In all our studies, we have used autologous mitochondria isolated from the patient’s own body for cardioprotection. The use of autologous tissue was based on the assumption that this approach would not cause an immune response and avoid the need for antirejection therapy required for nonautologous cellbased therapies (Kofidis et al., 2005). To confirm these expectations serial analysis immune and inflammatory markers was performed. The effects of mitochondrial transplantation were also investigated using multiplex (42plex) analysis of cytokines and chemokines (Masuzawa et al., 2013). The results from these studies confirmed that there was no immune or inflammatory response associated with autologous mitochondrial transplantation (Masuzawa et al., 2013). In addition there was no upregulation of cytokines associated with the immune response that is seen in patients with acute heart transplantation rejection (Rose, 2011).
The possibility of autoimmune response due to the presence of increased mitochondrial number in the myocardium was also investigated. The need for these studies was based on previous studies indicating that oxidative modification of E2 subunits of mitochondria pyruvate dehydrogenase, branched chain 2oxoacid dehydrogenase, and 2oxoglutarate dehydrogenase is a critical step leading to the induction of an autoimmune response in the liver as demonstrated by the presence of antimitochondrial antibodies (Leung et al., 2007). Indirect immunofluorescence was used to test for the presence of antimitochondrial antibodies (AMA) in serial blood samples. AMAs were not detected in the serum of any animals treated with autologous mitochondrial transplantation indicating that the transplantation of mitochondria does not induce an autoimmune response (Masuzawa et al., 2013).
3.4 Effects of mitochondrial transplantation on cellular viability and function

Our results have demonstrated that direct injection or vascular delivery of mitochondria to the heart rescues cell function and myocardial contractile function following ischemia and reperfusion. Creatine kinaseMB isoenzyme (CKMB) and cardiac troponin–I (cTnI) are specific and sensitive markers of myocardial injury, and elevated levels indicate myocardial injury (Pourafkari et al., 2015). Mitochondrial transplantation significantly decreased serum CKMB and cTnI indicating that myocardial injury following 30 minutes of transient ischemia was decreased (Masuzawa et al., 2013; Kaza et al., 2016). These effects were confirmed by infarct analysis using triphenyl tetrazolium chloride analysis to determine necrosis and terminal deoxynucleotidyl transferasemediated dUTP nickend labeling (TUNEL) and caspase activity to determine apoptosis in hearts treated with mitochondrial transplantation and those treated with vehicle alone (Masuzawa et al., 2013). Our results demonstrated that mitochondrial transplantation significantly decreased myocardial injury, including both necrosis and apoptosis, resulting from transient ischemia.
Increased myocardial cellular viability would be expected to correlate with enhanced myocardial function and our studies confirm this assumption. Our results show that 10 minutes following mitochondrial transplantation myocardial function is significantly enhanced as compared to hearts receiving injection of respiration media (vehicle) alone and that this function remains enhanced for at least 28 days – the end point of our studies (Masuzawa et al., 2013; Kaza et al., 2016). This is in contrast to hearts receiving vehicle alone that had persistent left ventricular hypokinesis.
3.5 Mechanisms of mitochondrial transplantation
The mechanisms through which mitochondrial transplantation provides cardioprotection have yet to be fully elucidated. At present, our studies have shown that the transplanted mitochondria act both extraand intracellularly. Once transplanted, the mitochondria increase total tissue ATP content and ATP synthesis. This increase in high energy acts rapidly, as early

as 10 minutes following delivery of the mitochondria to the heart, to enhance cardiac function as determined by echocardiography and pressure volume measurement. The mitochondria then act to upregulate proteomic pathways for the mitochondrion and the generation of precursor metabolites for energy and cellular respiration (P < 0.05, Enrichment Score > 2.0) (Masuzawa et al., 2013).
At 10 minutes to one hour following transplantation the transplanted mitochondrial are internalized into cardiomyocytes, by actindependent endocytosis (Pacak et al., 2015). Once internalized, the transplanted mitochondria further increase cardiomyocyte ATP content and upregulate cardioprotective cytokines (Masuzawa et al., 2013). These cytokines have been shown to be associated with enhanced cardiac function by stimulating cell growth, proliferation, and migration, enhancing vascularization, providing protection against cardiomyocyte apoptosis and improving functional cardiac recovery and cardiac remodeling independent of cardiac myocyte regeneration (Masuzawa et al., 2013).
We have recently shown that transplanted mitochondria also act at the mitochondrial genomic level. In previous experiments, we have shown that following ischemia there is damage to mitochondrial DNA resulting in the reduction of ATP synthesis (Levitsky et al., 2003). These alterations were associated with poor recovery following cardiac surgery in humans. Our studies demonstrate that mitochondrial transplantation replaces damaged mitochondrial DNA with intact mitochondrial DNA and rescues myocardial cell function (Pacak et al., 2015).
The transplanted mitochondria maintain viability and function for at least 28 days, the limit of our studies (Masuzawa et al., 2013; Cowan et al., 2016; Kaza et al., 2016). This is in contrast to xenoand allotransplanted cells that are rapidly rejected leading to the loss of transplanted cells, despite the use of antirejection pharmaceuticals (Yau et al., 2003; Hamamoto et al., 2009).

4 Human application
Premised upon these in vivo studies demonstrating the efficacy of mitochondrial transplantation for cardioprotection we have recently performed the first clinical application of mitochondrial transplantation (Emani et al., 2017). The study was performed in pediatric patients who suffered myocardial ischemiareperfusion injury. All procedures were performed under an Innovative Therapies process developed by the Boston Children’s Hospital Institutional Review Board. An individual review of the proposed therapy for each patient was provided by two independent physicians, not involved in the patient’s care. Families were extensively counseled regarding the experimental nature of the procedure and a separate Innovative Therapies consent form was signed.
Five pediatric patients in critical condition who were unable to be weaned off extracorporeal membrane oxygenation (ECMO) support due to myocardial dysfunction related to ischemia and reperfusion were treated with autologous mitochondria. Patient diagnosis included dextrotransposition of the great arteries (4 days and 25 days of age ), hypoplastic left heart syndrome (6 days), left ventricular outflow tract obstruction (6 months of age) and tricuspid atresia 1B (2 years of age) (Emani et al., 2017).
The cause of ischemia was coronary artery obstruction that was relieved in 4 patients, and LV distension with subendocardial ischemia in one patient. The autologous mitochondria were isolated from the patient’s rectus abdominis muscle. The patients received 10 mitochondrial injections, 100 uL each containing 1 x 107 ± 1 x 104 mitochondria. The mitochondria were delivered to the myocardium by direct injection with a 1 mL tuberculin syringe (28gauge needle). All injections were delivered to the area affected by ischemiareperfusion that was identified by epicardial echocardiography as being hypokinetic. Following mitochondrial transplantation, all 5 patients had significant improvement in their myocardial systolic function. Epicardial echocardiography showed moderate to severe systolic function ventricular dysfunction with regional hypokinesis prior to treatment (Emani et al., 2017). Ventricular function was improved to mildmoderate to normal systolic function at 46 days following autologous mitochondrial transplantation and improved to mild dysfunction in one patient and normal systolic function with no regional hypokinesia detected in any patient at 10 days following autologous mitochondrial transplantation (Emani et al., 2017).

All but one patient were successfully weaned off ECMO support by the 2nd day post mitochondrial transplantation. The single patient who was unable to wean off ECMO support suffered irreversible multiorgan failure despite the recovery of myocardial function following mitochondrial transplantation. This patient was on ECMO support for 15 days prior to treatment with mitochondrial transplantation. There were no adverse complications such as arrhythmia, intramyocardial hematoma or scarring with mitochondrial transplantation, in agreement with our animal studies. This case study demonstrates for the first time the potential role mitochondrial transplantation to improve ventricular dysfunction following ischemiareperfusion injury in humans.

While this is the first clinical usage of mitochondrial transplantation in humans, the same protocol can be used in adults and in other settings of ischemiareperfusion injury (Emani et al., 2017). The ability to use mitochondrial transplantation for clinical intervention in situations such as the stunned myocardium is enhanced by the fact that mitochondrial harvest and isolation can be performed within 2030 minutes during the same procedure and involves minimal manipulation of muscle tissue.

5 The timing of mitochondrial transplantation delivery
Studies by us and by others have shown that the mechanisms associated with ischemic myocardial injury converge on the mitochondrion. Rapidly following the onset of ischemia there are alterations in mitochondrial structure, complex activity, oxygen consumption, high energy synthesis and changes in mitochondrial transcriptomics and proteomics.

All these changes occur during ischemia and persist following reestablishment of coronary blood flow (reperfusion). For practical efficacy we therefore deliver the transplanted mitochondria just prior to reperfusion or during early reperfusion in order to limit the effects of ischemia on the transplanted mitochondria. We reasoned that if the mitochondria were transplanted at the start of ischemia they too would be damaged. This approach has been shown to be efficacious in our animal studies. In the human trial described above the delivery of mitochondria was many days after the ischemic insult. In these cases, the patient’s heart was unable to provide sufficient contractile force to be weaned from ECMO (Emani et al., 2017).

We believe that the role of mitochondrial transplantation is the rescue of cells and cellular function. This is premised on our animal studies. The results obtained in the clinical studies in humans are most likely the result of reversal of myocardial stunning. Stunning “describes the mechanical dysfunction that persists after reperfusion despite the absence of myocellular damage and despite the return of normal or nearnormal perfusion (Kloner et al., 1998). Mentzer, 2011, has noted that myocardial stunning, is a frequent consequence after heart surgery and is characterized by a requirement for postoperative inotropic support despite a technically satisfactory heart operation. In the stunned heart the myocardial cells remain alive but there is a prolonged depression of cardiac contractility after reperfusion. This depression in contractility can last days but with extended time results in patient mortality.
In the human cases in which we have used mitochondrial transplantation it is likely that we have rescued the stunned myocardium. The mechanisms most likely involve those we have demonstrated in our animal models, namely, restoration of high energy synthesis and replacement of damaged mitochondrial DNA. The veracity of these mechanisms remains to be elucidated.

6 Mitochondrial transplantation for the rescue of other tissues
While our studies have focussed on the heart and ischemia and reperfusion, other groups have shown that mitochondrial transplantation can be used to enhance drug sensitivity in human breast cancer cells (Elliott et al., 2012); to rescue cell function in cells harboring the mitochondrial DNA mutation (Chang et al., 2013); Parkinson’s disease (Chang et al., 2016); liver ischemia/reperfusion injury (Lin et al., 2013); in cellular studies demonstrating that isolated mitochondria rescue mitochondrial respiratory function and improved the cellular viability in cardiomyocytes (Kitani et al., 2014) and neurorecovery after stroke ( Hayakawa et al., 2016).

These studies demonstrate the potential of mitochondrial transplantation in a variety of diseases. The utility in other organ related diseases and syndromes remains to be investigated. However, diabetes, Alzheimer’s disease and dementia, posttraumatic stress disorder, concussion and others have all been shown to be associated with alterations occurring at or affecting mitochondrial function. Included in these pathological disorders are ischemiareperfusion events affecting pulmonary, renal, hepatic, cerebral, ocular and skeletal muscles. We expect that the application and usage of mitochondrial transplantation will provide for a simple and efficacious therapeutic approach to many of these disease and will significantly ameliorate morbidity and mortality.

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