Mitochondrial Respiratory Capacity is Restored in Hibernating Cardiomyocytes Following Co-Culture with Mesenchymal Stem Cells

Introduction

Although the mortality rate associated with cardiac disease has decreased in recent years, it remains one of the leading causes of death globally ¹ . Ischemic cardiac disease presents as a large clinical spectrum, yet pre-clinical research in this area focuses largely on myocardial infarction (MI). MI is characterized by the presence of infarct tissue that cannot be rescued. At the other end of the spectrum is hibernating myocardium (HM), a subtype of cardiovascular disease that has potential for full or partial recovery. HM is defined as chronic ischemia due to reduced coronary artery blood flow that does not result in cell death, yet has decreased regional function, impaired ability to respond to increased work demands, and increases risk of arrhythmia ² . Although HM tissue is viable, as noted by increased glucose uptake relative to oxygen consumption, standard treatment with coronary artery bypass graft (CABG) does not result in full functional recovery ³ , but does reduce the risk for future cardiac events and decrease mortality ⁴ .

Our group has a well-established swine model of HM that is amenable to CABG treatment, providing an animal model that closely mimics clinical HM cases ⁵ . This model of HM has allowed us to characterize proteomic changes in HM, identifying that the mitochondrial proteome is dysregulated in HM tissue, as exhibited by decreased expression of electron transport chain (ETC) complexes, mitochondrial fusion proteins, and regulators of mitochondrial biogenesis ^{6 –8} . These indicators of mitochondrial dysfunction are not restored to normal following restoration of blood flow with CABG ^9,10 . In addition, electron micrographs of this tissue show the mitochondria are small, segmented, and disorganized ¹⁰ . These data suggest the persistent mitochondrial dysfunction in HM despite revascularization inhibits full functional recovery.

To address this gap in treatment for HM, additional therapies are needed to address mitochondrial dysfunction and optimize myocardial recovery. HM presents a unique target for stem cell therapy as it is fully viable but dysfunctional cardiac tissue with the potential to be rescued. Mesenchymal stem cells (MSCs) are an ideal candidate for treatment, as previous studies have shown they have the potential to increase in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) signaling, which mediates mitochondrial biogenesis ¹¹ , suggesting a possible therapeutic mechanism for this mitochondrial-driven disease.

This study presents a novel in vitro model of HM in cardiomyocytes that exhibits preserved viability and decreased mitochondrial function in response to chronic, low-grade ischemia, mimicking the functional phenotype of HM hearts. This model allows, for the first time, an investigation of mitochondrial respiratory dynamics in HM as well as their response to mesenchymal stem cell therapy.

Materials and Methods

Results

Cardiomyocytes exposed to 24 hours of hypoxia (1% oxygen) maintain viability but have impaired mitochondrial function. Hypoxia was confirmed by measurement of fluorescence intensity with Image IT Hypoxia dye (52.79±8.2 vs 0.006±0.004) (Figure 1(a)-(c)), and viability was quantified by trypan blue exclusion (Figure 1(d)) showing no increase in cell death under the hypoxic protocol. Following 24 hours of hypoxia, cardiomyocytes exhibited decreased basal (40.67±1.4 vs 34.52±2.24) and maximal respiration (121.06±4.75 vs 82.95±5.38) (Figure 1(e)-(g)). Following 24 hours of reoxygenation, measurements of basal (37.53±2.78 vs 30±2.41) and maximal (104.77±7.9 vs 80.52±8.21) respiration remain impaired. These characteristics of impaired function and maintained viability following the outlined hypoxic conditions provide an in vitro model consistent with the mitochondrial adaptations seen in HM.

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Figure 1.

Cardiomyocyte exposure to 24 hours of 1% oxygen mimics the cellular phenotype of hibernating myocardium (HM). Measurement of cellular hypoxia at (a) baseline and (b) 24 hours following exposure to hypoxic conditions. (c) Quantification of hypoxia by measurement of fluorescent intensity (n=4; p=0.0076; nd=not detectable). (d) Cell viability was unchanged following exposure to 24 hours of hypoxia (n=4). (e) Measurement of oxygen consumption rate (OCR) indicated impaired respiratory capacity following hypoxia that was not restored following reoxygenation. Basal (f) and maximal (g) respiration was significantly reduced following hypoxia and was not restored following reoxygenation (p=0.0005 and p<0.0001, respectively).

Hibernating cardiomyocytes co-cultured with bone marrow-derived MSCs in a transwell system to prevent cell-cell contact and facilitate paracrine signaling during the reoxygenation period exhibited increased basal (30±2.41 vs 101.1±2.6) and maximal (80.52±8.21 vs 314.4±9.4) respiration, as well as increased ATP production (24.56±2.08 vs 83.2±2.8) (Figure 2(a)-(d)). Quantitative real-time polymerase chain reaction of cardiomyocytes show decreased expression of electron transport chain proteins following exposure to hypoxia. Complex 4 is increased following reoxygenation (0.8228±0.037 vs 1.114±0.049) and treatment with MSC co-culture (0.8228±0.037 vs 0.9821±0.028) (Figure 2(e)). Complex 5—ATP synthase—is increased following reoxygenation (1.035±0.022 vs 1.124±0.031) and is further increased following co-culture with MSCs (1.035±0.022 vs 1.235±0.039) (Figure 2(f)). Complexes 1, 2, and 3 of the ETC were measured, but no significant changes were measured (data not shown). Nuclear-bound PGC1α protein expression is increased following reoxygenation (0.013±0.0024 vs 0.026±0.0024) and is further increased following treatment with MSC co-culture (0.013±0.0024 vs 0.0379±0.0045) (Figure (g)).

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Figure 2.

Transwell co-culture of mesenchymal stem cells (MSCs) during reoxygenation restores mitochondrial respiratory function in hibernating myocardium (HM) cardiomyocytes. (a) Measurement of oxygen consumption rate (OCR) in cardiomyocytes following transwell co-culture with MSCs resulted in an overall increase in mitochondrial respiratory activity. (b) Basal and (c) maximal respiration were significantly increased following co-culture with MSCs (p=0.0032 and p<0.0001, respectively). (d) ATP production was significantly increased following MSC co-culture (p<0.001). Measurement of RNA expression of electron transport chain (ETC) complex 4 (e) and 5 (f) showed significant increases following co-culture with MSCs. (g) Measurement of nuclear-bound peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) protein was significantly increased following MSC co-culture (p<0.001).

Discussion

This study presents the first in vitro model of HM that exhibits maintained cellular viability and impaired function following chronic, low-grade hypoxia. We have also shown that the mitochondrial adaptations to HM in this model are not restored to normal function following reoxygenation alone (Figure 1(d)). This mimics the clinical experience in which CABG, the standard treatment of HM, may not fully restore cardiac function. Our previous work with a large animal model of HM has shown that hibernating cardiac tissue is characterized by decreased expression of ETC complexes, which is replicated in this model (Figure 2(e)-(f)), suggesting the mechanism of disease is related to mitochondrial function ^{6 –8} .

We have previously identified the hallmark adaptations in HM tissue, which center around dysregulation of mitochondrial morphology, proteome, and function. Heart tissue has high energetic demand, making it critically dependent on effective mitochondrial function. The recovery of heart mitochondria in response to HM is dependent upon a dynamic process involving fission, fusion, and autophagy ^{14 –16} . A critical balance between these variables is necessary to preserve mitochondrial energetics and contractile function in the ischemic heart ¹⁷ . We have previously demonstrated that inadequate mitochondrial bioenergetic capacity is present in HM pre- and post-CABG, as manifested by reduced expression of ETC proteins ¹⁷ . These observations suggest the process of mitochondrial dynamism is incomplete with CABG alone and that recovery of the mitochondrial proteome remains inadequate, despite evidence that cardiac tissue is no longer ischemic following CABG, as indicated by full restoration of regional blood flow in the HM region ^8,18 . Fusion protein (OPA-1, mitofusins 1 and 2) expression is necessary to re-establish normal mitochondrial energetics and function in the adult heart ¹⁹ . We have also demonstrated that PGC1α, a driver of mitochondrial biogenesis, is significantly downregulated in HM and is not restored following CABG ¹⁷ . Using transmission electron microscopy, we have observed in our HM model that mitochondria appear smaller and more variable in size despite CABG, which could be explained by the dysregulation of mitochondrial fusion, fission, and autophagy. This mitochondrial morphology does not normalize following CABG ¹⁷ . These observations identify the mitochondrial basis of HM, but clinical studies and animal models of HM are limited in their ability to allow the direct study of mitochondrial respiration. The development of this in vitro model aims to bridge this gap in appropriate research models of HM by creating a model that can allow for direct manipulation and measurement of mitochondrial respiration.

Stem cells provide a unique opportunity for addressing this underlying cause of cardiac dysfunction. Several recent clinical studies have advanced the notion that a cell-based therapeutic approach can either improve global function and/or reduce scar in patients with an ischemic cardiomyopathy. Studies in MI have shown that stem cell treatment results in increased PGC1α signaling, which mediates mitochondrial biogenesis ¹¹ . The results of this study of in vitro HM indicate an increase in PGC1α following reoxygenation and treatment with MSCs, which may indicate an increase in mitochondrial biogenesis in HM cells, resulting in increased mitochondrial function and respiration. By improving mitochondrial respiration, the ischemic heart is better equipped to respond to increased workload and can improve functional recovery following CABG.

There is a clinical need for an adjunct therapy to CABG that addresses the impairment of mitochondrial function seen in patients and animal models of this disease. Co-culture treatment of HM cardiomyocytes with MSCs shows improvement in both mitochondrial function and ATP production, both of which are critical for effectively functioning cardiac tissue. These data suggest a novel mechanism for the therapeutic potential of MSCs in heart disease that is independent of VEGF-mediated angiogenesis, which has previously been thought to be a primary mechanism of MSC therapy in ischemic tissue ^20,21 . As shown by our previous in vivo work in HM animals, as well as these in vitro findings, re-oxygenation alone is not enough to fully restore the function of HM tissue ²² . It follows that a therapeutic mechanism of angiogenesis alone would not be sufficient to explain the therapeutic benefit that is seen with MSC therapy in ischemic conditions. These results suggest that MSC therapy may act upon mitochondrial machinery by way of an increase in mitochondrial biogenesis to restore function. These results suggest that MSC therapy as an adjunct treatment to revascularization may address the current gap in treatment for HM patients. Identification of this therapeutic mechanism also suggests there may be a potential for MSCs to provide a benefit to impaired mitochondrial function in the absence of reoxygenation, which could be critical in instances of chronic ischemia that cannot be revascularized.

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