Mitochondria in Control of Cell Fate

Abstract

The behavior of the mitochondrial permeability transition pore has been linked to mitochondrial maturation underlying cardiomyocyte differentiation in the embryo. Mitochondrial signaling in heart development has direct implications for cardiogenesis and stem cell lineage specification.

Heart formation requires maturation and integration of multiple systems to support development of the contractile apparatus in the nascent cardiomyocyte. Although a number of transcriptional networks that facilitate cardiogenesis have been mapped, master regulators of heart development remain elusive. A recent report highlights mitochondria, and more specifically the mitochondrial permeability transition pore (mPTP), as a gating mechanism underlying differentiation in the developing heart,¹ implicating cross-talk between genetic and metabolic signaling.

Immature mitochondria of early embryonic hearts must transition into more complex structures to ensure proficient and energetically competent cardiac development.^2–4 Developmental restructuring is recapitulated during spontaneous differentiation of stem cells, where pluripotent gene downregulation accelerates mitochondria DNA replication to promote mitochondrial biogenesis associated with lineage specification.^2–6 Cardiomyocytes isolated from day 9.5 embryos (e9.5) harbor few fragmented mitochondria, with poorly defined and unorganized cristae, which undergo elaborate maturation and by day e13.5 evolve into filamentous networks of elongated and branched mitochondria with abundant and organized cristae.¹ Moreover, mitochondria expansion from a predominately perinuclear localization to an extensive configuration across the cell facilitates energy supply and transfer between cellular compartments.^{1, 2, 4, 7–9} Mitochondrial structure and function are markers of differentiation capacity. Cell with less perinuclear mitochondria have greater spontaneous differentiation,¹⁰ and those with low mitochondrial membrane potential show greater propensity for mesodermal differentiation.¹¹ Remodeling of the mitochondrial infrastructure matches the evolving bioenergetic demands, with contractile function driving the requirement for efficient oxidative ATP generation in the developing heart.^{2, 7, 12}

Hom et al. now demonstrate that modulators of mPTP closure promote mitochondrial maturation and cardiomyocyte differentiation in the embryo (Figure).¹ A non-selective conduit with a molecular cut-off of 1.5 kDa, mPTP resides within the inner mitochondrial membrane.¹³ Transitional pore opening promotes mitochondrial permeability, dissipating ion and metabolite gradients and uncoupling oxidative metabolism from ATP generation.¹³ Excessive mitochondrial swelling triggers release of pro-apoptotic proteins, including cytochrome c, precipitating cell death via necrosis or apoptosis during extreme stress, such as ischemia/reperfusion injury.¹³ A physiological role for the mPTP opening-closing dynamics has however remained unclear as knockout of an essential pore component (cyclophilin-D) produces apparently healthy offspring,^{14, 15} albeit with abnormal cardiac response to stress.¹⁶ In wild-type embryos, developing e9.5 cardiogenic fields display lower mitochondrial membrane potential and greater reactive oxygen species (ROS) generation compared with older e13.5 cardiomyocytes, consistent with early mPTP involvement.¹ Genetic or pharmacologic closure of the mPTP at e9.5 induces structural and functional mitochondrial maturation and cardiomyocyte differentiation, while increased mPTP open probability would impair development.¹ Consistent with the proposed role of mPTP in heart development is an increased level of mitofusin-2, a dynamin-like protein involved in the rearrangement of mitochondrial membranes and permeability transition during stem cell cardiac differentiation.^{2, 17} Through influences on vital downstream processes, including ROS production, energy metabolism and calcium signaling, mPTP behavior could profoundly impact cardiac differentiation.

Figure.

Mitochondrial structure and energy metabolism supports the differentiation potential of pluripotent stem cells and early cardiac progenitors. Frequent opening of the mPTP maintains immature mitochondrial morphology and low oxidative capacity, requiring a high glycolytic capacity in early embryonic cardiomyocytes. Subsequent differentiation requires mPTP closure supporting the maturation of mitochondrial oxidative metabolism and reducing the reliance on glycolysis. ADP: adenosine diphosphate, ATP: adenosine triphosphate, FADH: flavin adenine dinucleotide, FADH₂: reduced flavin adenine dinucleotide,NAD+: nicotinamide adenine dinucleotide, NADH: reduced nicotinamide adenine dinucleotide, NADP: nicotinamide adenine dinucleotide phosphate, NADPH: reduced nicotinamide adenine dinucleotide phosphate, TCA: tricarboxylic acid, Δψ_m: mitochondrial membrane potential.

Immature cardiomyocytes exhibit underdeveloped electron transport chain and elevated ROS load, reduced with natural or stimulated mPTP closure promoting differentiation.¹ Mitochondrial ROS is positively correlated with mitochondrial membrane potential; yet permeability transition via mPTP can further promote ROS production by depleting mitochondrial antioxidants and impairing physiological electron transfer due to loss of electron transport chain components and conformational rearrangement of complex I.^{18, 19} Antioxidant-induced reduction of ROS at e9.5 promotes cardiomyocyte differentiation, while addition of stable oxidants impairs differentiation.¹ Indeed, low levels of ROS have been implicated in stimulating expression of cardiac genes and transcription factors, and promoting stem cell cardiac differentiation.^20–22 Such cardiogenic effects appear to be concentration-dependent, as high levels of ROS can delay cardiac differentiation.^{23, 24} The work of Hom et al. indicates that the redox status might control cardiogenesis in a temporal fashion, with early commitment of cardiac progenitors occurring in a highly oxidized environment, while subsequent cardiomyocyte differentiation proceeding under reduced ROS load.^{1, 25} The intimate effects of permeability transition on mitochondrial function and downstream pathways involved in differentiation, including ROS as well as associated energy metabolism and calcium signaling, would require further examination.

Transient mPTP openings may control mitochondrial ionic status to match oxidative metabolism with myocardial workload. In fact, mitochondria-dependent energetic circuits are critical regulators of de novo cardiogenesis.² Transient mPTP opening directly regulates cellular energy metabolism as it uncouples oxidative metabolism from ATP synthesis, a mechanism that operates in concert with ROS flashes to promote cardiomyocyte differentiation.^{12, 13} Knockout of the mPTP component cyclophilin D results in elevated mitochondrial matrix calcium, which enhances the activation of Ca²⁺-dependent dehydrogenases reducing metabolic flexibility.¹⁶ The early embryonic heart is primarily dependent upon anaerobic glycolysis for ATP generation, as a potential consequence of low substrate supply and oxygen availability.^{3, 26} With growing oxygen supply following the e10 stage, oxidation of substrates, in particular lactate, increases as the heart requires more oxygen to maintain contraction.^{27, 28} Hom et al. demonstrate a reliance on complex II for electron entry at day e9.5 with increasing importance of complex I at e13.5, supporting bioenergetic remodeling during differentiation.¹ Much of the evidence for metabolic remodeling during cardiogenesis is derived from in vitro differentiation of stem cells.^{2, 4, 5, 7–9} Like the early embryonic heart, pluripotent stem cells rely on glycolytic metabolism, with increased oxygen consumption and cellular respiration concomitant to the upregulation of tricarboxylic acid cycle and electron transport chain components associated with mitochondrial maturation during differentiation.^{2, 5–7, 12, 29, 30} Disruption of mitochondrial respiration impairs the ability of pluripotent stem cells to differentiate into cardiomyocytes and maintain stemness.^{2, 31} A growing body of literature has implicated a deterministic role for energy metabolism in driving cellular fate.^{6, 12, 32} Indeed, the differentiation potential of cardiac progenitors into cardiomyocytes relies upon mitochondrial content and the capacity for oxidative metabolism.³³ The necessary shift from glycolytic to oxidative metabolism during differentiation of pluripotent stem cells is dependent upon the regulation of mitochondrial substrate entry, including by downregulation of uncoupling protein 2 and changes in hexokinase isoforms.^{6, 34–36} The reverse of this process, dedifferentiation of somatic cells back to the pluripotent state also requires metabolic remodeling, which precedes the expression of pluripotency markers.^{6, 12} Thus, beyond matching bioenergetic supply and demand, the regulation of energy metabolism is central to fueling specification of cell fate.^{6, 12, 32–36}

The interconnectivity of mPTP, mitochondria maturation and embryonic development has significant implications for the understanding of normal cardiac differentiation, and heart vulnerability to injury. Opening of the mPTP is largely associated with dissipation of mitochondrial membrane potential, release of pro-apoptotic stimuli and induction of cell death, associated with pathologic conditions.³⁷ Inhibition of pore opening using a variety of techniques, including pre-and post-conditioning, promotes cardioprotection.^{13, 37} Therefore, future studies to identify mechanisms regulating mPTP behavior during embryogenesis may provide novel targets against cardiac injury. In addition, embryonic hearts tolerate transient mPTP openings, suggesting the presence of pro-survival pathways early in development, offering potential avenues for targeted protection. In contrast, sustained mPTP opening and the associated impairment in mitochondrial and cardiomyocyte maturation may impede the developing heart from matching the energetic demands of the mature embryo and could ultimately lead to congenital defects or embryonic lethality. Beyond disease pathogenesis, regulation of mitochondrial maturation and myocyte differentiation by permeability transition has the potential to impact lineage specification. Indeed, targeting master regulators of cell fate plasticity offers an innovative technology for purposes of tissue regeneration.³⁸ Case in point, cyclosporine A, a mPTP inhibitor, can augment the in vitro production of cardiac progenitor cells capable of integrating into the infarcted heart.³⁹ Moreover, the mPTP associated peripheral benzodiazepine receptor (PBR) has been implicated in cell proliferation and differentiation with PBR ligands affecting stem cell fate.⁴⁰ Manipulation of mPTP and its downstream signaling pathways may thus be considered to promote differentiation of resident cardiac stem cells for facilitated heart repair. Deciphering mechanisms underlying the role for mPTP and mitochondrial signaling in heart development offers implications for cardiac embryology and pathology, and more broadly may refine stem cell specification for regenerative applications.