Bridging the gap between in vitro and in vivo models: a way forward to clinical translation of mitochondrial transplantation in acute disease states.

Mitochondrial transplantation and transfer are being explored as therapeutic options in acute and chronic diseases to restore cellular function in injured tissues. To limit potential immune responses and rejection of donor mitochondria, current clinical applications have focused on delivery of autologous mitochondria. We recently convened a Mitochondrial Transplant Convergent Working Group (CWG), to explore three key issues that limit clinical translation: (1) storage of mitochondria, (2) biomaterials to enhance mitochondrial uptake, and (3) dynamic models to mimic the complex recipient tissue environment. In this review, we present a summary of CWG conclusions related to these three issues and provide an overview of pre-clinical studies aimed at building a more robust toolkit for translational trials.

Mitochondrial Transplantation's Role in Rodent Skeletal Muscle Bioenergetics: Recharging the Engine of Aging.

BACKGROUND: Mitochondria are the 'powerhouses of cells' and progressive mitochondrial dysfunction is a hallmark of aging in skeletal muscle. Although different forms of exercise modality appear to be beneficial to attenuate aging-induced mitochondrial dysfunction, it presupposes that the individual has a requisite level of mobility. Moreover, non-exercise alternatives (i.e., nutraceuticals or pharmacological agents) to improve skeletal muscle bioenergetics require time to be effective in the target tissue and have another limitation in that they act systemically and not locally where needed. Mitochondrial transplantation represents a novel directed therapy designed to enhance energy production of tissues impacted by defective mitochondria. To date, no studies have used mitochondrial transplantation as an intervention to attenuate aging-induced skeletal muscle mitochondrial dysfunction. The purpose of this investigation, therefore, was to determine whether mitochondrial transplantation can enhance skeletal muscle bioenergetics in an aging rodent model. We hypothesized that mitochondrial transplantation would result in sustained skeletal muscle bioenergetics leading to improved functional capacity. METHODS: Fifteen female mice (24 months old) were randomized into two groups (placebo or mitochondrial transplantation). Isolated mitochondria from a donor mouse of the same sex and age were transplanted into the hindlimb muscles of recipient mice (quadriceps femoris, tibialis anterior, and gastrocnemius complex). RESULTS: The results indicated significant increases (ranging between ~36% and ~65%) in basal cytochrome c oxidase and citrate synthase activity as well as ATP levels in mice receiving mitochondrial transplantation relative to the placebo. Moreover, there were significant increases (approx. two-fold) in protein expression of mitochondrial markers in both glycolytic and oxidative muscles. These enhancements in the muscle translated to significant improvements in exercise tolerance. CONCLUSIONS: This study provides initial evidence showing how mitochondrial transplantation can promote skeletal muscle bioenergetics in an aging rodent model.

Mitochondrial transplantation preserves myocardial function and viability in pediatric and neonatal pig hearts donated after circulatory death.

OBJECTIVE: Mitochondrial transplantation has been shown to preserve myocardial function and viability in adult porcine hearts donated after circulatory death (DCD) . Herein, we investigate the efficacy of mitochondrial transplantation for the preservation of myocardial function and viability in neonatal and pediatric porcine DCD heart donation. METHODS: Circulatory death was induced in neonatal and pediatric Yorkshire pigs by cessation of mechanical ventilation. Hearts underwent 20 or 36 minutes of warm ischemia time (WIT), 10 minutes of cold cardioplegic arrest, and then were harvested for ex situ heart perfusion (ESHP). Following 15 minutes of ESHP, hearts received either vehicle (VEH) or vehicle containing isolated autologous mitochondria (MITO). A sham nonischemic group (SHAM) did not undergo WIT, mimicking donation after brain death heart procurement. Hearts underwent 2 hours each of unloaded and loaded ESHP perfusion. RESULTS: Following 4 hours of ESHP perfusion, left ventricle developed pressure, dP/dt max, and fractional shortening were significantly decreased (P < .001) in DCD hearts receiving VEH compared with SHAM hearts. In contrast, DCD hearts receiving MITO exhibited significantly preserved left ventricle developed pressure, dP/dt max, and fractional shortening (P < .001 each vs VEH, not significant vs SHAM). Infarct size was significantly decreased in DCD hearts receiving MITO as compared with VEH (P < .001). Pediatric DCD hearts subjected to extended WIT demonstrated significantly preserved fractional shortening and significantly decreased infarct size with MITO (P < .01 each vs VEH). CONCLUSIONS: Mitochondrial transplantation in neonatal and pediatric pig DCD heart donation significantly enhances the preservation of myocardial function and viability and mitigates against damage secondary to extended WIT.

Model of Ischemia and Reperfusion Injury in Rabbits.

The protocol here provides a simple, highly replicable methodology to induce in situ acute regional myocardial ischemia in the rabbit for non-survival and survival experiments. New Zealand White adult rabbit is sedated with atropine, acepromazine, butorphanol, and isoflurane. The animal is intubated and placed on mechanical ventilation. An intravenous catheter is inserted into the marginal ear vein for the infusion of medications. The animal is pre-medicated with heparin, lidocaine, and lactated Ringer's solution. A carotid cut-down is performed to obtain arterial line access for blood pressure monitoring. Select physiologic and mechanical parameters are monitored and recorded by continuous real-time analysis. With the animal sedated and fully anesthetized, either a fourth intercostal space small left thoracotomy (survival) or midline sternotomy (non-survival) is performed. The pericardium is opened, and the left anterior descending (LAD) artery is located. A polypropylene suture is passed around the second or third diagonal branch of the LAD artery, and the polypropylene filament is threaded through a small vinyl tube, forming a snare. The animal is subjected to 30 min of regional ischemia, achieved by occluding the LAD by tightening the snare. Myocardial ischemia is confirmed visually by regional cyanosis of the epicardium. Following regional ischemia, the ligature is loosened, and the heart is allowed to re-perfuse. For both survival and non-survival experiments, the myocardial function can be assessed via an echocardiography (ECHO) measurement of the fractional shortening. For non-survival studies, data from sonomicrometry collected using three digital piezoelectric ultrasonic probes implanted within the ischemic area and the left ventricle developed pressure (LVDP) using an apically inserted left ventricle (LV) catheter can be continuously acquired for evaluating the regional and global myocardial function, respectively. For survival studies, the incision is closed, a left needle thoracentesis is performed for pleural air evacuation, and postoperative pain control is achieved.

Mitochondrial transplantation: Effects on chemotherapy in prostate and ovarian cancer cells in vitro and in vivo.

Prostate and ovarian cancers affect the male and female reproductive organs and are among the most common cancers in developing countries. Previous studies have demonstrated that cancer cells have a high rate of aerobic glycolysis that is present in nearly all invasive human cancers and persists even under normoxic conditions. Aerobic glycolysis has been correlated with chemotherapeutic resistance and tumor aggressiveness. These data suggest that mitochondrial dysfunction may confer a significant proliferative advantage during the somatic evolution of cancer. In this study we investigated the effect of direct mitochondria transplantation on cancer cell proliferation and chemotherapeutic sensitivity in prostate and ovarian cancer models, both in vitro and in vivo. Our results show that the transplantation of viable, respiration competent mitochondria has no effect on cancer cell proliferation but significantly decreases migration and alters cell cycle checkpoints. Our results further demonstrate that mitochondrial transplantation significantly increases chemotherapeutic sensitivity, providing similar apoptotic levels with low-dose chemotherapy as that achieved with high-dose chemotherapy. These results suggest that mitochondria transplantation provides a novel approach for early prostate and ovarian cancer therapy, significantly increasing chemotherapeutic sensitivity in in vitro and in vivo murine models.

Mitochondrial transplantation: the advance to therapeutic application and molecular modulation.

Mitochondrial transplantation provides a novel methodology for rescue of cell viability and cell function following ischemia-reperfusion injury and applications for other pathologies are expanding. In this review we present our methods and acquired data and evidence accumulated to support the use of mitochondrial transplantation.

Transcriptomic and proteomic pathways of diabetic and non-diabetic mitochondrial transplantation.

Reduced mitochondrial function increases myocardial susceptibility to ischemia-reperfusion injury (IRI) in diabetic hearts. Mitochondrial transplantation (MT) ameliorates IRI, however, the cardioprotective effects of MT may be limited using diabetic mitochondria. Zucker Diabetic Fatty (ZDF) rats were subjected to temporary myocardial RI and then received either vehicle alone or vehicle containing mitochondria isolated from either diabetic ZDF or non-diabetic Zucker lean (ZL) rats. The ZDF rats were allowed to recover for 2 h or 28 days. MT using either ZDF- or ZL-mitochondria provided sustained reduction in infarct size and was associated with overlapping upregulation of pathways associated with muscle contraction, development, organization, and anti-apoptosis. MT using either ZDF- or ZL-mitochondria also significantly preserved myocardial function, however, ZL- mitochondria provided a more robust long-term preservation of myocardial function through the mitochondria dependent upregulation of pathways for cardiac and muscle metabolism and development. MT using either diabetic or non-diabetic mitochondria decreased infarct size and preserved functional recovery, however, the cardioprotection afforded by MT was attenuated in hearts receiving diabetic compared to non-diabetic MT.

Mitochondrial remodeling and ischemic protection by G protein-coupled receptor 35 agonists.

Kynurenic acid (KynA) is tissue protective in cardiac, cerebral, renal, and retinal ischemia models, but the mechanism is unknown. KynA can bind to multiple receptors, including the aryl hydrocarbon receptor, the a7 nicotinic acetylcholine receptor (a7nAChR), multiple ionotropic glutamate receptors, and the orphan G protein-coupled receptor GPR35. Here, we show that GPR35 activation was necessary and sufficient for ischemic protection by KynA. When bound by KynA, GPR35 activated Gi- and G12/13-coupled signaling and trafficked to the outer mitochondria membrane, where it bound, apparantly indirectly, to ATP synthase inhibitory factor subunit 1 (ATPIF1). Activated GPR35, in an ATPIF1-dependent and pertussis toxin-sensitive manner, induced ATP synthase dimerization, which prevented ATP loss upon ischemia. These findings provide a rationale for the development of specific GPR35 agonists for the treatment of ischemic diseases.

Mitochondrial transplantation for organ rescue.

Mitochondrial transplantation involves the replacement or augmentation of native mitochondria damaged, by ischemia, with viable, respiration-competent mitochondria isolated from non-ischemic tissue obtained from the patient's own body. The uptake and cellular functional integration of the transplanted mitochondria appears to occur in all cell types. Efficacy and safety have been demonstrated in cell culture, isolated perfused organ, in vivo large animal studies and in a first-human clinical study. Herein, we review our findings and provide insight for use in the treatment of organ ischemia- reperfusion injury.

Mitochondrial Transplantation for Ischemia Reperfusion Injury.

Mitochondrial transplantation is a novel therapeutic intervention to treat ischemia-reperfusion-related disorders. This approach uses replacement of native mitochondria with viable, respiration-competent mitochondria isolated from non-ischemic tissue obtained from the patient's own body, to overcome the many deleterious effects of ischemia-reperfusion injury on native mitochondria. The safety and efficacy of this methodology has been demonstrated in cell culture, animal models and has been shown to be safe and efficacious in a phase I clinical trial in pediatric cardiac patients with ischemia-reperfusion injury. These studies have demonstrated that mitochondrial transplantation rescues myocardial cellular viability and significantly enhances postischemic myocardial function following ischemia-reperfusion injury. Herein, we describe methodologies for the delivery of isolated mitochondria.

A Large Animal Model for Acute Kidney Injury by Temporary Bilateral Renal Artery Occlusion.

Acute kidney injury (AKI) is associated with higher risk for morbidity and mortality post-operatively. Ischemia-reperfusion injury (IRI) is the most common cause of AKI. To mimic this clinical scenario, this study presents a highly reproducible large animal model of renal IRI in swine using temporary percutaneous bilateral balloon-catheter occlusion of the renal arteries. The renal arteries are occluded for 60 min by introducing the balloon-catheters through the femoral and carotid artery and advancing them into the proximal portion of the arteries. Iodinated contrast is injected in the aorta to assess any opacification of the kidney vessels and confirm the success of the artery occlusion. This is furtherly confirmed by the flattening of the pulse waveform at the tip of the balloon catheters. The balloons are deflated and removed after 60 min of bilateral renal artery occlusion, and the animals are allowed to recover for 24 h. At the end of the study, plasma creatinine and blood urea nitrogen significantly increase, while eGFR and urine output significantly decrease. The need for iodinated contrast is minimal and does not affect renal function. Bilateral renal artery occlusion better mimics the clinical scenario of perioperative renal hypoperfusion, and the percutaneous approach minimizes the impact of the inflammatory response and the risk of infection seen with an open approach, such as a laparotomy. The ability to create and reproduce this clinically relevant swine model eases the clinical translation to humans.

Autogenous mitochondria transplantation for treatment of right heart failure.

BACKGROUND: Right ventricular hypertrophy and failure are major causes of cardiac morbidity and mortality. A key event in the progression to right ventricular hypertrophy and failure is cardiomyocyte apoptosis due to mitochondrial dysfunction. We sought to determine whether localized intramyocardial injection of autologous mitochondria from healthy muscle treats heart failure. METHODS: Mitochondria transplanted from different sources were initially tested in cultured hypertrophic cardiomyocytes. A right ventricular hypertrophy/right ventricular failure model created through banding of the pulmonary artery in immature piglets was used for treatment with autologous mitochondria (pulmonary artery banded mitochondria injected/treated n = 6) from calf muscle, versus vehicle (pulmonary artery banded vehicle injected/treated n = 6) injected into the right ventricular free-wall, and compared with sham-operated controls (sham, n = 6). Animals were followed for 8 weeks by echocardiography (free-wall thickness, contractility), and dp/dt max was measured concomitantly with cardiomyocyte hypertrophy, fibrosis, and apoptosis at study end point. RESULTS: Internalization of mitochondria and adenosine triphosphate levels did not depend on the source of mitochondria. At 4 weeks, banded animals showed right ventricular hypertrophy (sham: 0.28 ± 0.01 cm vs pulmonary artery banding: 0.4 ± 0.02 cm wall thickness; P = .001), which further increased in pulmonary artery banded mitochondria injected/treated but declined in pulmonary artery banded vehicle injected/treated (0.47 ± 0.02 cm vs 0.348 ± 0.03 cm; P = .01). Baseline contractility was not different but was significantly reduced in pulmonary artery banded vehicle injected/treated compared with pulmonary artery banded mitochondria injected/treated and so was dp/dtmax. There was a significant difference in apoptotic cardiomyocyte loss and fibrosis in sham versus hypertrophied hearts with most apoptosis in pulmonary artery banded vehicle injected/treated hearts (sham: 1 ± 0.4 vs calf muscle vs vehicle: 13 ± 1.7; P = .001 and vs pulmonary artery banded mitochondria injected/treated: 8 ± 1.9, P = .01; pulmonary artery banded vehicle injected/treated vs pulmonary artery banded mitochondria injected/treated, P = .05). CONCLUSIONS: Mitochondrial transplantation allows for prolonged physiologic adaptation of the pressure-loaded right ventricular and preservation of contractility by reducing apoptotic cardiomyocyte loss.

Autologous mitochondrial transplantation for cardiogenic shock in pediatric patients following ischemia-reperfusion injury.

OBJECTIVES: To report outcomes in a pilot study of autologous mitochondrial transplantation (MT) in pediatric patients requiring postcardiotomy extracorporeal membrane oxygenation (ECMO) for severe refractory cardiogenic shock after ischemia-reperfusion injury (IRI). METHODS: A single-center retrospective study of patients requiring ECMO for postcardiotomy cardiogenic shock following IRI between May 2002 and December 2018 was performed. Postcardiotomy IRI was defined as coronary artery compromise followed by successful revascularization. Patients undergoing revascularization and subsequent MT were compared with those undergoing revascularization alone (Control). RESULTS: Twenty-four patients were included (MT, n = 10; Control, n = 14). Markers of systemic inflammatory response and organ function measured 1 day before and 7 days following revascularization did not differ between groups. Successful separation from ECMO-defined as freedom from ECMO reinstitution within 1 week after initial separation-was possible for 8 patients in the MT group (80%) and 4 in the Control group (29%) (P = .02). Median circumferential strain immediately following IRI but before therapy was not significantly different between groups. Immediately following separation from ECMO, ventricular strain was significantly better in the MT group (-23.0%; range, -20.0% to -28.8%) compared with the Control group (-16.8%; range, -13.0% to -18.4%) (P = .03). Median time to functional recovery after revascularization was significantly shorter in the MT group (2 days vs 9 days; P = .02). Cardiovascular events were lower in the MT group (20% vs 79%; P < .01). Cox regression analysis showed higher composite estimated risk of cardiovascular events in the Control group (hazard ratio, 4.6; 95% confidence interval, 1.0 to 20.9; P = .04) CONCLUSIONS: In this pilot study, MT was associated with successful separation from ECMO and enhanced ventricular strain in patients requiring postcardiotomy ECMO for severe refractory cardiogenic shock after IRI.

A Multi-Mode System for Myocardial Functional and Physiological Assessment during Ex Situ Heart Perfusion.

Ex situ heart perfusion (ESHP) has proven to be an important and valuable step toward better preservation of donor hearts for heart transplantation. Currently, few ESHP systems allow for a convenient functional and physiological evaluation of the heart. We sought to establish a simple system that provides functional and physiological assessment of the heart during ESHP. The ESHP circuit consists of an oxygenator, a heart-lung machine, a heater-cooler unit, an anesthesia gas blender, and a collection funnel. Female Yorkshire pig hearts (n = 10) had del Nido cardioplegia (4°C) administered, excised, and attached to the perfusion system. Hearts were perfused retrogradely into the aortic root for 2 hours before converting the system to an isovolumic mode or a working mode for further 2 hours. Blood samples were analyzed to measure metabolic parameters. During the isovolumic mode (n = 5), a balloon inserted in the left ventricular (LV) cavity was inflated so that an end-diastolic pressure of 6-8 mmHg was reached. During the working mode (n = 5), perfusion in the aortic root was redirected into left atrium (LA) using a compliance chamber which maintained an LA pressure of 6-8 mmHg. Another compliance chamber was used to provide an afterload of 40-50 mmHg. Hemodynamic and metabolic conditions remained stable and consistent for a period of 4 hours of ESHP in both isovolumic mode (LV developed pressure: 101.0 ± 3.5 vs. 99.7 ± 6.8 mmHg, p = .979, at 2 and 4 hours, respectively) and working mode (LV developed pressure: 91.0 ± 2.6 vs. 90.7 ± 2.5 mmHg, p = .942, at 2 and 4 hours, respectively). The present study proposed a novel ESHP system that enables comprehensive functional and metabolic assessment of large mammalian hearts. This system allowed for stable myocardial function for up to 4 hours of perfusion, which would offer great potential for the development of translational therapeutic protocols to improve dysfunctional donated hearts.

Mitochondrial transplantation by intra-arterial injection for acute kidney injury.

Acute kidney injury is a common clinical disorder and one of the major causes of morbidity and mortality in the postoperative period. In this study, the safety and efficacy of autologous mitochondrial transplantation by intra-arterial injection for renal protection in a swine model of bilateral renal ischemia-reperfusion injury were investigated. Female Yorkshire pigs underwent percutaneous bilateral temporary occlusion of the renal arteries with balloon catheters. Following 60 min of ischemia, the balloon catheters were deflated and animals received either autologous mitochondria suspended in vehicle or vehicle alone, delivered as a single bolus to the renal arteries. The injected mitochondria were rapidly taken up by the kidney and were distributed throughout the tubular epithelium of the cortex and medulla. There were no safety-related issues detected with mitochondrial transplantation. Following 24 h of reperfusion, estimated glomerular filtration rate and urine output were significantly increased while serum creatinine and blood urea nitrogen were significantly decreased in swine that received mitochondria compared with those that received vehicle. Gross anatomy, histopathological analysis, acute tubular necrosis scoring, and transmission electron microscopy showed that the renal cortex of the vehicle-treated group had extensive coagulative necrosis of primarily proximal tubules, while the mitochondrial transplanted kidney showed only patchy mild acute tubular injury. Renal cortex IL-6 expression was significantly increased in vehicle-treated kidneys compared with the kidneys that received mitochondrial transplantation. These results demonstrate that mitochondrial transplantation by intra-arterial injection provides renal protection from ischemia-reperfusion injury, significantly enhancing renal function and reducing renal damage.

Mitochondrial transplantation for myocardial protection in ex-situ‒perfused hearts donated after circulatory death.

BACKGROUND: Donation after circulatory death (DCD) offers an additional source of cardiac allografts, potentially allowing expansion of the donor pool, but is limited owing to the effects of ischemia. In this study, we investigated the efficacy of mitochondrial transplantation to enhance myocardial function of DCD hearts. METHODS: Circulatory death was induced in Yorkshire pigs (40-50 kg, n = 29) by a cessation of mechanical ventilation. After 20 minutes of warm ischemia, cardioplegia was administered. The hearts were then reperfused on an ex-situ blood perfusion system. After 15 minutes of reperfusion, hearts received either vehicle alone (vehicle [VEH], 10 ml; n = 8) or vehicle containing autologous mitochondria (vehicle with mitochondria as a single injection [MT], 5 × 109 in 10 ml, n = 8). Another group of hearts (serial injection of mitochondria [MTS]; n = 6) received a second injection of mitochondria (5 × 109 in 10 ml) after 2 hours of ex-situ heart perfusion and reperfused for an additional 2 hours. A Sham group (sham hearts; n = 6) did not undergo any warm ischemia. RESULTS: At the end of 4 hours of reperfusion, MT and MTS groups showed a significantly increased left ventricle/ventricular peak developed pressure (p = 0.002), maximal left ventricle/ventricular pressure rise (p < 0.001), fractional shortening (p < 0.001), and myocardial oxygen consumption (p = 0.004) compared with VEH. Infarct size was significantly decreased in MT and MTS groups compared with VEH (p < 0.001). No differences were found in arterial lactate levels among or within groups throughout reperfusion. CONCLUSIONS: Mitochondrial transplantation significantly preserves myocardial function and oxygen consumption in DCD hearts, thus providing a possible option for expanding the heart donor pool.

Preischemic autologous mitochondrial transplantation by intracoronary injection for myocardial protection.

OBJECTIVE: To investigate preischemic intracoronary autologous mitochondrial transplantation (MT) as a therapeutic strategy for prophylactic myocardial protection in a porcine model of regional ischemia-reperfusion injury (IRI). METHODS: The left coronary artery was cannulated in Yorkshire pigs (n = 26). Mitochondria (1 × 109) or buffer (vehicle [Veh]) were delivered as a single bolus (MTS) or serially (10 injections over 60 minutes; MTSS). At 15 minutes after injection, the heart was subjected to temporary regional ischemia (RI) by snaring the left anterior descending artery. After 30 minutes of RI, the snare was released, and the heart was reperfused for 120 minutes. RESULTS: Coronary blood flow (CBF) and myocardial function were increased temporarily during the pre-RI period. Following 30 minutes of RI, MTS and MTSS hearts had significantly increased CBF that persisted throughout reperfusion (Veh vs MTS and MTSS; P = .04). MTS and MTSS showed a significantly enhanced ejection fraction (Veh vs MTS, P < .001; Veh vs MTSS, P = .04) and developed pressure (Veh vs MTS, P < .001; Veh vs MTSS, P = .03). Regional function, assessed through segmental shortening (Veh vs MTS, P = .03; Veh vs MTSS, P < .001), fractional shortening (Veh vs MTS, P < .001; Veh vs MTSS, P = .04), and strain analysis (Veh vs MTS, P = .002; Veh vs MTSS, P = .003), was also significantly improved. Although there was no difference in the area at risk between treatment groups, infarct size was significantly reduced in both MT groups (Veh vs MTS and MTSS, P < .001). CONCLUSIONS: Preischemic MT by single or serial intracoronary injections provides prophylactic myocardial protection from IRI, significantly decreasing infarct size and enhancing global and regional function.