The process of mitochondrial fission is under the control of the mitochondrial fission proteins Drp1 and Fis1. Drp1 is located mainly in the cytosol and comprises a GTPase, a central region, and a GTPase effector domain (GED) or assembly domain. Fis1 is localized in the outer mitochondrial membrane with most of the protein facing into the cytosol, acting as a docking station for Drp1. On activation, Drp1 translocates to the mitochondria (a process which is regulated by phosphorylation and sumoylation), oligomerizes, and constricts the mitochondrial scission site, a process which requires GTPase, thereby resulting in mitochondrial fission.
The process of mitochondrial fusion is under the control of the mitochondrial fusion proteins Mfn1 and 2 and OPA-1. Mitochondrial membrane fusion has been shown to be a distinct two-step process which occurs separately for the inner and outer membrane, but in chronology. Both the outer and inner membranes of the mitochondria must fuse properly in order for the matrix contents to mix properly. (A) The mitochondrial fusion proteins Mfn1 and Mfn2 are located on the outer mitochondrial membrane with a cytosolic GTPase domain and two hydrophobic heptad repeat (HR) regions separated by a transmembrane repeat. The C-terminal HR region (HR2) mediates oligomerization between Mfn molecules on adjacent mitochondria, allowing the membranes to fuse. GTP hydrolysis facilitates the fusion process. (B) The mitochondrial fusion protein OPA1 comprises an N-terminal mitochondrial import sequence (MIS), hydrophobic heptad repeat (HR) segments, coiled-coil domain (C C), a GTPase domain, a central domain, and a GTPase effector domain (GED) at the C-terminus. OPA1 mediates the fusion of the inner mitochondrial membranes.
During myocardial ischaemia and reperfusion (I/R) there is accumulation of cytosolic Ca2+ due to defects in multiple Ca2+ handling proteins such as SERCA2a, NCX, LTCC, and RyR2. Importantly, the resultant cytosolic Ca2+ overload with myocardial reperfusion causes an increased influx of Ca2+ into mitochondria and results in the opening of the mitochondrial permeability transition pore (mPTP). In addition to Ca2+ overload, I/R is associated with increased generation of myocardial reactive oxygen species (ROS). While small amounts of Ca2+ and oxygen are necessary for optimal cardiac function, cytosolic free Ca2+ overload and increased oxidative stress are thought to be major contributors to myocardial I/R-induced injury and myocyte death. The potential targets to modulate Ca2+ overload and reduce myocardial ischaemia-reperfusion injury are: (1) Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), (2) Na+/Ca2+ exchanger (NCX), (3) SR Ca2+ release channel RyR, (4), L-type Ca2+ channel (LTCC), (5) Na+-H+ exchanger (NHE), and (6) Ca2+ and calmodulin-dependent protein kinase II (CaMKII).
Activation of the survivor activating factor enhancement (SAFE) pathway, as represented by the binding of a low concentration of endogenous or exogenous tumour necrosis factor alpha (TNFα) to its TNF receptor 2 (TNFR2) at the onset of reperfusion with the subsequent activation of the transcription factor signal transducer and activator of transcription-3 (STAT-3), initiates a cardioprotective signalling cascade in both ischaemic pre- and postconditioning that is activated independently of the well-known reperfusion injury salvage kinases (RISK) pathway. The delineation of the SAFE pathway further emphasizes the importance of RISK-independent pathways in cardioprotection, which may have potential therapeutic application in the mitigation of ischaemic-reperfusion injury.
Abbreviations: RISK: Reperfusion Injury Salvage Kinases; SAFE: Survivor Activating Factor Enhancement; S1P: sphingosine-1-phosphate; TNFα: tumour necrosis factor alpha; GPCR: green protein coupled receptors; S1P R1/R3: sphingosine-1-phosphate receptors 1 or 3; TNFR2: tumour necrosis factor alpha receptor 2; MEK: mitogen-activated protein kinase; PI3K: phosphoinositide 3- kinase; Erk1/2: extracellular regulated kinases 1/2; Akt: protein kinase B; GSK-3β: glycogen synthase kinase-3 beta; JAK: janus kinase; STAT-3: signal transducer and activator of transcription-3; mPTP: mitochondrial permeability transition pore; P: phosphorylation
This schematic provides a simplified overview of the intracellular transduction pathways underlying cardioprotection elicited by the growth factors: transforming growth factor-β1 (TGF-β1), cardiotrophin-1 (CT-1), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), insulin, insulin-like growth factor (IGF), and urocortin. Ligand binding to their respective cell-surface receptors on the cardiomyocyte activates intracellular signalling kinase cascades including Raf-Ras-Mek1/2-Erk1/2 and PI3K-Akt of the reperfusion injury salvage kinase (RISK) pathway, the JAK-STAT pathway, and various anti-apoptotic mechanisms (including the phosphorylation and inhibition of Bax and BAD as well as the inhibition of cytochrome C release).
Many of the acute cardioprotective mechanisms manifested at the time of reperfusion converge on the mitochondria and include the inhibition of the mitochondrial permeability transition pore (mPTP), which can be achieved through several different mechanisms including the phosphorylation and inhibition of GSK3β; the opening of the ATP-sensitive mitochondrial potassium (Mito KATPM) channel by the eNOS-NO-PKG-PKC-ε cascade which produces mitochondrial ROS, which inhibits mitochondrial permeability transition pore opening; and the intracellular calcium modulation due to augmented SERCA uptake of calcium into the sarcoplasmic reticulum. More long-term cardioprotection may be achieved through the genetic transcription of various cardioprotective mediators such as iNOS, NFκB, MMP-1, phospholipase-1, and so on (not shown on diagram, see text for details).
The core structure of the mPTP remains unresolved. Known mPTP regulatory elements are depicted on the left side of the figure, whereas the right side indicates symbolically the threshold for mPTP-induction by oxidant stress. The middle row (horizontally) depicts the basal state of ANT and CyP-D as they relate to the basal threshold for mPTP induction by oxidant stress. The top row reflects factors that facilitate mPTP induction: atractyloside, Ca2+, and indirect effects of Pi. The bottom row includes factors that are known to inhibit mPTP induction: genetic deletion of ANT (ANT is dispensable for mPTP formation per se; inhibition of CyP-D by CsA remains protective), ADP, or bongkrekic acid (requirement/role of CyP-D under these conditions is unknown), CsA and genetic deletion of CyP-D in the presence of Pi (atractyloside, CsA and Ca2+ are no longer effective when compared with WT). Note the opposing mechanisms of Pi in mPTP induction: (i) Pi as a direct mPTP desensitizer (bottom row) is opposed by CyP-D binding (top row), whereas (ii) Pi may also act as an indirect mPTP sensitizer (through regulation of Mg2+ and/or polyphosphate levels; top row). Note that Ca2+ is not a major factor in mPTP induction in intact cardiomyocytes and neurons.
mPTP mitochondrial permeability transition pore
ANT adenine nucleotide translocator
BKA bongkrekic acid
CyP-D cyclophilin D
Pi inorganic phosphate
CsA cyclosporin A
ADP adenosine diphosphate
Ppif gene encoding CyP-D in mouse
There are three major signalling cascades of protein kinase activation in cardioprotection: (A) the GPCR/NPR-AKT-eNOS-PKG pathway, (B) the reperfusion-injury salvage kinase (RISK) pathway, and (C) the survival activating factor enhancement (SAFE) pathway, which centrally involves gp130-JAK-STAT signalling. In each system, there are molecules that are decreased in expression and/or activity with advancing age (marked in yellow) and possibly contribute to the loss of cardioprotection with aging. Such loss of cardioprotection with aging is one major problem in the translation of experimental data from (usually young and healthy) animals to the clinical situation in elderly humans.
Abbreviations: AMPK, AMP-activated kinase; ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; CB-R, cannabinoid receptor; Cx43, connexin 43; eNOS, endothelial NO synthase; ERK, extracellular regulated kinase; FGF-2, fibroblast growth factor 2; gp130, glycoprotein 130; GPCR, G-protein-coupled receptor; GSK3β, glycogen synthase kinase 3 β; H11K, H11 kinase; IGF, insulin-like growth factor 1; IL-6, interleukin 6; iNOS, inducible NO synthase; JAK, janus kinase; KATP, ATP-dependent potassium channel, MnSOD, manganese superoxide dismutase; MPTP, mitochondrial permeability transition pore; NO, nitric oxide; NPR, natriuretic peptide receptor; p38, p38 mitogen activated protein kinase; P70S6K, p70 ribsosomal S6 protein kinase; pGC, particulate guanylyl cyclase; PI3K, phosphoinositide 3-kinase; PKC, protein kinase C; PKG, protein kinase G; ROS, reactive oxygen species; sGC, soluble guanylyl cyclase; SIRT1, sirtuin 1; STAT3, signal transducer and activator of transcription 3; TNF-R, tumour necrosis factor receptor; UCN, urocortins.
Parathyroid hormone is a DPP-IV inhibitor and increases SDF-1-driven homing of CXCR4+ stem cells into the ischaemic heart
Mechanism of PTH-mediated cardioprotection. PTH administration after MI induces mobilization of stem cells from the BM to the peripheral blood. These stem cells circulate to the damaged heart, where they are incorporated by interaction of intact myocardial SDF-1 and the homing receptor CXCR4. PTH inhibits DPP-IV activity and thereby prevents the degradation of intact SDF-1. Thus, an increased amount of SDF-1 improves homing of mobilized CXCR4+ cells. Altogether, PTH reduced cardiac remodelling after MI and enhanced cardiac function by attenuating the development of ischaemic cardiomyopathy.
Mechanisms and consequences of altered Ca2+ handling in cardiomyocytes during initial reperfusion. Main events are connected through black lines, whereas red lines indicate important modulating factors. GCPR, G-coupled protein receptors; IP3, inositol trisphosphate; NOS, nitric oxide synthase; ROS, reactive oxygen species.
Pathophysiological role of SR–mitochondria functional units on lethal reperfusion injury. Calcium overload and re-energization cause calcium oscillations. ROS favour oscillations and trigger MPT. mNCX, mitochondrial Na/Ca exchanger; MCU: mitochondrial calcium uniporter.
Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia–reperfusion injury
Scheme summarizing the potential roles of Cx43 in the pathophysiology of ischaemia–reperfusion. Solid lines indicate roles for which there is experimental evidence. Broken lines indicate phenomena for which available evidence has been obtained under conditions other than ischaemia–reperfusion. PK, protein kinases; Src, Src tyrosine kinase.
Mitochondrial connexin43 as a new player in the pathophysiology of myocardial ischaemia–reperfusion injury
Potential mechanisms by which mitochondrial Cx43 could participate in ischaemic pharmacological (diazoxide) preconditioning. Monomeric Cx43 (in blue) could modulate mitochondrial K+ATP channels (in brown), but also the effects of diazoxide on the respiratory chain (in dark gray).103 Cx43 hemichannels could favor H+ and K+ leak resulting in protective mild uncoupling104 and swelling.105,106
Scheme of the pathogenesis of acute reperfusion injury. Reperfusion reactivates ATP production in mitochondria (Mito). Recovering energy production (High ATP) activates the Ca2+ pump (SERCA) of the sarcoplasmic reticulum (SR), which clears the cytosol from Ca2+ overload accumulated during ischemia. Repetitive release of Ca2+ through the ryanodine receptor Ca2+ release channel (RyR) and reuptake into the SR leads to Ca2+ oscillations with high cytosolic peak Ca2+ concentrations. This high Ca2+ together with ATP provokes myofibrillar hypercontracture (Ca2+ contracture) and subsequent disruption of cells (Necrosis). Ca2+ uptake through the uniporter into mitochondria causes the opening of mitochondrial permeability transition pores (mPTP) and cytochrome c (Cyt c) release. The former leads to failure of energy production (low ATP), and the latter activates apoptosis. Low ATP induces rigor contracture of the myofibrils, again leading to cell disruption. Protection by reperfusion injury salvage kinase pathways (RISK) may interfere favourably at the SR or at mitochondria.