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Mitochondrial Dynamics and Cardiovascular Diseases

Posted in Cardiovascular Pharmacogenomics, Cell Biology, Signaling & Cell Circuits, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Origins of Cardiovascular Disease, Pharmacotherapy of Cardiovascular Disease, tagged Aging, cardiomyocyte, cardiomyocyte differentiation, Cardiomyopathy, cardiovascular, DLP1, Drp1, filamentous network, GTP hydrolysis, GTPase, heart, Heart Failure, IF, interfibrillar filaments, Mfn1, Mfn2, mitochondria, mitochondrial dynamics, mitochondrial fission, mitochondrial fusion, myocytes, neurodegeneration, Opa1, type 2 diabtes, ventricular myocytes on November 14, 2012| 18 Comments »

Mitochondrial Dynamics and Cardiovascular Diseases

Author and Curator: Ritu Saxena, Ph.D.

Morphological changes in mitochondria have been observed in several human diseases including myopathies, diabetes mellitus, liver diseases, neurodegeneration, aging, and cancer. Ong et al (2010) studied neonatal rat ventricular myocytes as an experimental model of aging and concluded that the interplay between mitochondrial fission and autophagy controls the rate of mitochondrial turnover. A disturbance in the balance is observed in aging heart cells resulting in giant mitochondria. This observation is an indication that mitochondrial morphology is connected to pathogenesis of cardiac disease. http://www.ncbi.nlm.nih.gov/pubmed/20631158 Thus, it is important to understand the mechanism of mitochondrial dynamics in order to correlate it with the development of cardiovascular diseases.

Mitochondrial dynamics

The shape of mitochondria is very dynamic in living cells, constantly interchanging between thread-like and grain-like morphology through what we know now as the fusion and fission processes, respectively. The fusion and fission processes together with the mitochondrial movement have been termed “mitochondrial dynamics”. Nucleoids, the assemblies of mitochondrial DNA (mtDNA) with its associated proteins, are distributed during fission in such a way that each mitochondrion contains at least one nucleoid.

Mitochondrial fusion is a complex process that involves the fusing together of four lipid bilayers. Proteins involved in the mitochondrial fission and fusion have been discussed in an earlier post published on October 31, 2012. Mitochondrial fusion requires two 85kD-GTPase isoforms mitofusin1 (Mfn1) and mitofusin2 (Mfn2). Mfn1 and Mfn1 are both anchored to the outer mitochondrial membrane. They contain – two transmembrane domains connected by a small intermembrane-space loop, a cytosolic N-terminal GTPase domain and two cytosolic hydrophobic heptad-repeat coiled-coil domains. The coiled-coil domains of Mfn1 and Mfn2 help in tethering adjacent mitochondria in both homo-oligomeric and hetero-oligomeic fashion. The fusion process requires GTP hydrolysis and the cells where Mfn2 had a GTPase mutation; mitochondria were not able to undergo fusion even after tethering. Mitochondrial fission and fusion have been illustrated in Figure 1.

Mitochondrial fission is opposite of the fusion process. Mammalian mitochondria undergo fission by the interaction of two proteins: dynamin-like protein 1 or dynamin-related protein 1 (DLP1/Drp1), an 80–85-kD cytosolic GTPase, and human fission protein 1 (hFis1), a 17-kD outer mitochondrial membrane anchored protein. Mitochondrial fission too requires GTP hydrolysis. DLP1 mainly localizes in the cytosol and with the help of hFis1, DLP1 is recruited to the constriction sites of the membrane. DLP1 translocation depends on actin and microtubules and once inside, DLP1 oligomerizes into a ring around the mitochondrion. The self-assembly of DLP1 stimulates the final step of fission which is disassembly and it requires GTP hydrolysis.

Figure 1: Model of mammalian mitochondrial fission and fusion (Hom et al, J Mol Cell Cardiol, 2009)

http://www.ncbi.nlm.nih.gov/pubmed?term=19281816

Additional information on different aspects of mitochondria could be found articles published earlier in the Pharmaceutical Intelligence webpage.

Mitochondrial dynamics in the heart

In cultured cardiovascular cell line the mitochondria are arranged in a filamentous network and are highly dynamic, constantly undergoing fusion and fission. Similar mitochondrial network is observed in vascular smooth muscle cells, cardiac stem cells, and neonatal cardiomyocytes. Thus, these cell types have been used to study mitochondrial dynamics.

However, in the adult cardiomyocyte, there are three distinct populations of mitochondria:

(i) peri-nuclear mitochondria,

(ii) subsarcolemmal (SSC) mitochondria, and

(iii) interfibrillar (IF) mitochondria

Electron micrographs of adult cardiac muscle cells, especially ventricular myocytes, show that mitochondria are numerous, making up about 35% of the cell volume, and that mitochondria are highly organized and compacted between contractile filaments and next to T-tubules. This crystal-like pattern of mitochondria in adult ventricular myocytes raises an interesting question- Do the mitochondria in these cells also undergo physiological fission, fusion, and movement just like other cell types? Whether the crystal-like lattice arrangement restricts their movements and prevents them from undergoing fusion or fission is unclear. It has been speculated that the fission and fusion processes might occur at a slower rate because of the tight packing. A four-dimensional (x, y, z axis and time) live-cell imaging is needed to detect possible movements like mitochondria winding slowly through the myofibrils in the third dimension.

Figure 2. Representative electron micrograph of adult murine heart depicting the three subpopulations of mitochondria: perinuclear (PN) mitochondria; interfibrillar (IF) mitochondria; and subsarcolemmal mitochondria (SSM). Photo credit: Ong et al, Cardiovascular Research (2010).

Expression of fission/fusion proteins in adult heart: Interestingly, it has been observed that proteins required for mitochondrial dynamics including fission and fusion proteins is abundantly present in the adult heart and would have been active during cardiomyocyte differentiation to ensure the unique spatial organization of the three different subpopulations of cardiac mitochondria.

Several studies suggest the existence of fission and fusion proteins in the adult heart.

Mfn1 and Mfn2 fusion proteins have been found to be expressed in highest amounts in the heart compared to that in human tissues of pancreas, skeletal muscle, brain, liver, placenta, lung, and kidney using both Northern and Western blot analysis. Infact, Mfn2 mRNA was found to be abundantly expressed in heart and muscle tissue but expressed only at low levels in other tissues. Mfn1 and Mfn2 expression has also been confirmed in heart tissue of rat and mouse by RT-PCR.
hFis1, a fission protein, has been shown to be ubiquitously expressed in isolated rat mitochondria in heart tissue apart from several other tissues.
DLP1 mRNA, coding for a fusion protein, have been detected in high levels in several adult tissues including heart, skeletal muscle, kidney and brain.
OPA1 codes for another fusion protein and four transcripts of OPA1 have been detected in adult mouse hearts.

Mitochondria in cardiac diseases:

Abnormal mitochondrial morphology corresponding to various cardiac diseases has been listed as follows:

Abnormally small and disorganized mitochondria – observed in endstage dilated cardiomyopathy, myocardial hibernation, cardiac rhabdomyoma, and ventricular-associated congenital heart diseases.
Disorganized clusters of fragmented mitochondria – observed in Tetralogy of Fallot and are located away from contractile filaments, along with having a very small diameter measured to be 0.1 μm as observed in the electron micrographs.
Big and defective mitochondria – observed in senescent cardiomyocytes.

http://www.ncbi.nlm.nih.gov/pubmed?term=19281816

Condition	Cell type	Change in mitochondrial morphology	Other findings	Study
Ischemia-perfusion injury	HL-1 cells	Fission	P38 inhibition at reperfusion allows mitochondrial re-fusion	Brady et al
β – Adrenergic stimulation by isoproterenol or exercise	Adult murine heart	Not investigated	Phosphorylation and inhibiton of Drp1 at Ser656	Cribbs and Strack et al
Cardiac differentiation	Embryonic stem cells	Fusion	Fusion is required to support Oxidative phosphorylation	Chung et al
Hyperglycemia	H9C2 rat myoblast	Fission		Yu et al
Post-MI heart failure and dilated cardiomyopathy	Adult rat and human heart	Fragmentation	Decrease in OPA1	Chen et al
Diabetes	Murine coronary endothelial cell	Fission	Decreased OPA1, increased Drp1	Makino et al
Diabetes	Adult murine diabetic heart	Fission	Lower mitochondrial membrane potential	Williamson et al
Ischaemia-reperfusion injury and cardioprotection	HL-1 cells, adult heart	Fission	Inhibiting fission cardioprotective	Ong et al
Cytosolic calcium overload	Neonatal cardiomyocytes and adult heart	Fission		Hom et al

Table 1: Studies implicating changes in mitochondrial morphology in cardiovascular diseases, Adapted from Ong et al, Cardiovascular Research (2010).

Mitochondrial dynamics in heart failure

Fission and Fusion in Heart Failure

Mutation or abnormal expression of fission and fusion proteins have been implicated in several diseases including neuropathies, Parkinson’s disease, type 2 diabetes and so on. However, few studies have addressed the involvement of mitochondrial dynamics in heart failure. Research groups have used cardiac-like cell lines, neonatal and adult cardiomyocytes, and animal models to demonstrate the importance of fission and fusion proteins. Observations from some studies have been listed below:

Mitochondria are highly organized and compacted between contractile filaments (interfibrillar) or adjacent to the sarcolemma (subsarcolemmal) in adult mammalian cardiomyocytes. However, during heart failure, interfibrillar mitochondria may lose their normal organization.
There is also a reduction in size and density of interfibrillar mitochondria in rodent models of heart failure.
It was recently reported that OPA1 is decreased in both human and rat heart failure.
Electron microscopic data showed an increase in the number and decrease in the size of the mitochondria in a coronary artery ligation rat heart failure model.
Inhibition of fission in cultured neonatal ventricular myocytes by overexpression of dominant negative mutant form of Drp1, Drp1-K38A, prevents overproduction of ROS, mitochondrial permeability transient pore formation and ultimately cell death under high glucose conditions.
In cultured neonatal and adult cardiomyocytes, cytosolic Ca2+ overload induced by thapsigargin (Tg) or potassium chloride (KCl) resulted in rapid mitochondrial fragmentation. Calcium overload is a common feature in heart failure, which might lead to increase in fission contributing to decrease in energy production in the failing heart.
In H9c2 cells, reduction in OPA1 increased apoptosis both at baseline and after simulated ischemia, via cytochrome c release from mitochondria.
Drosophila heart tube-specific silencing of OPA1 and mitochondrial assembly regulatory factor (MARF) increased mitochondrial morphometric heterogeneity and induced heart tube dilation with profound contractile impairment. In this model, human MFN1/2 was rescued MARF RNAi induced cardiomyopathy.
MFN-2-deficient mice have mild cardiac hypertrophy and mild depression of cardiac function. Also, mitochondria of cardiac myocytes lacking MFN-2 are pleiotropic and larger.
In rat hearts, decreased MFN2, increased Fis1 and no change in OPA1 expression was observed 12–18 weeks after myocardial infarction. http://www.ncbi.nlm.nih.gov/pubmed/22848903

However, further research is needed to accurately and fully define the role of abnormal mitochondrial morphology in heart failure. Those researches might lead to developing new interventions for treating abnormal mitochondrial function based diseases.

Reference:

Mitochondrial fission and fusion: potential therapeutic targets?

Posted in Autophagy, Autophagy-Modulating Proteins, Biochemical pathways, Biological Networks, Gene Regulation and Evolution, CANCER BIOLOGY & Innovations in Cancer Therapy, Cell Biology, Signaling & Cell Circuits, Disease Biology, Disease Biology, Small Molecules in Development of Therapeutic Drugs, Metabolic Immuno-Oncology, Metabolism, Metabolomics, Oxidative phosphorylation, Warburg effect, tagged Apoptosis, Autophagy, calcineurin, Cancer - General, cardiomyocytes, Cdk1, cell division, cell proliferation, CyclinB, cytosolic, Drp-1, Drp1, dynamin, Dynamin related protein, E3 ubiquitin ligase, energetics, GTPases, hFis1, lung adenocarcinoma, Lung cancer, membrane, Mfn-1, Mfn-2, Mfn1, Mfn2, MicroRNA, miR-30, miR-484, miR-499, mitochondria, Mitochondrial DNA, mitochondrial fission, mitochondrial fusion, mitofusion 1, mitofusion 2, mtDNA, nucleoids, Opa1, p53, PKA, Reactive oxygen species, ROS, sumoylation, ubiquitination on October 31, 2012| 7 Comments »

Author and Curator: Ritu Saxena, Ph.D.

Introduction: Mitochondrial fission & fusion

Mitochondria, double membranous and semi-autonomous organelles, are known to convert energy into forms that are usable to the cell. Apart from being sites of cellular respiration, multiple roles of mitochondria have been emphasized in processes such as cell division, growth and cell death. Mitochondria are semi-autonomous in that they are only partially dependent on the cell to replicate and grow. They have their own DNA, ribosomes, and can make their own proteins. Mitochondria have been discussed in several posts published in the Pharmaceutical Intelligence blog.

Mitochondria do not exist as lone organelles, but are part of a dynamic network that continuously undergoes fusion and fission in response to various metabolic and environmental stimuli. Nucleoids, the assemblies of mitochondrial DNA (mtDNA) with its associated proteins, are distributed during fission in such a way that each mitochondrion contains at least one nucleoid. Mitochondrial fusion and fission within a cell is speculated to be involved in several functions including mtDNA DNA protection, alteration of cellular energetics, and regulation of cell division.

Proteins involved in mitochondrial fission & fusion

Multiple mitochondrial membrane GTPases that regulate mitochondrial networking have recently been identified. They are classified as fission and fusion proteins:

Fusion proteins: Members of dynamin family of protein, mitofusin 1 (Mfn-1) and mitofusin 2 (Mfn-2), are involved in fusion between mitochondria by tethering adjacent mitochondria. These proteins have two transmembrane segments that anchor them in the mitochondrial outer membrane. Mutations in Mitofusin proteins gives rise to fragmented mitochondria, but this can be reversed by mutations in mammalian Drp1. Mitochondrial inner membranes are fused by dynamin family members called Opa1.

Fission proteins: Another member of the dynamin family of proteins, dynamin-related protein 1 (Drp-1) mediates fission of mitochondria. Drp-1 is activated by phosphorylation. Drp-1 proteins are largely cytosolic, but cycle on and off of mitochondria as needed for fission. Fission is a complex process and involves a series of well-defined stages and proteins. Cytosolic Drp-1 is activated by calcineurin or other cytosolic signaling proteins after which it translocates to the mitochondrial tubules where it assembles into foci through its interaction with another protein, hFis1. Once Drp-1 rings assemble on the constricted spots, outer membrane of mitochondria undergoes fission through GTP hydrolysis. Drp-1 is now left bound to one of the newly formed mitochondrial ends after which it slowly disassembles before returning to the cytoplasm.

Control of mitochondrial fission & fusion

Mitochondrial fission and fusion are controlled by several regulatory mechanisms. Few of which are mentioned as follows:
Drp-1 activation by Cdk1/Cyclin B mediated phosphorylation during mitosis – triggers fission
Drp-1 inactivation by cAMP-dependent protein kinase (PKA) in quiescent cells- prevents fission
Drp-1 activation after reversal of PKA phosphorylation by Calcineurin- triggers fission
Ubiquination of fission and fusion proteins by E3 ubiquitin ligase- alters fission
Sumoylation of fission proteins – regulates fission

Imparied mitochondrial fission leads to loss of mtDNA

Mitochondrial fission plays an important role in mitochondrial and cellular homeostasis. It was reported by Parone et al (2008) that preventing mitochondrial fission by down-regulating expression of Drp-1 lead to loss of mtDNA and mitochondrial dysfunction. An increase in cellular reactive oxygen species (ROS) was observed. Other cellular implications included depletion of cellular ATP, inhibition of cell proliferation and autophagy. The observations were made in HeLa cells.

MicroRNA regulation of mitochondrial fission

Although several factors have been attributed to the regulation of mitochondrial fission, the mechanism still remains poorly understood. Recently, regulation of mitochondrial fission via miRNAs has become a topic of interest. Following miRNAs have been found to be involved in mitochondrial fission:

miR-484: Wang et al (2012) demonstrated that miR-484 was able to regulate mitochondrial fission by suppressing the translation of a fission protein Fis1, leading to inhibition of Fis1-mediated fission and apoptosis in cardiomyocytes and in the adrenocortical cancer cells. The authors showed that Fis1 is necessary for mitochondrial fission and apoptosis, and is upregulated during anoxia, whereas miR-484 is downregulated. Underlying mechanism involved transactivation of miR-484 by a transcription factor, Foxo3a and miR-484 is able to attenuate Fis1 upregulation and mitochondrial fission, by binding to the amino acid coding sequence of Fis1 and inhibiting its translation.
miR-499: miR-499 was reported by Wang et al (2011) to be able to directly target both the α- and β-isoforms of the calcineurin catalytic subunit. Suppression of calcineurin-mediated dephosphorylation of Drp-1 lead to inhibition of the fission machinery ultimately resulting in the inhibition of cardiomyocyte apoptosis. miR-499 levels, by altering mitochondrial fusion were able affect the severity of myocardial infarction and cardiac dysfunction induced by ischemia-reperfusion. Modulation of miR-499 expression could provide a therapeutic approach for myocardial infarction treatment.
miR-30: It was reported by Li et al (2010) that miR-30 family members were able to inhibit mitochondrial fission and also the resulting apoptosis. While exploring the underlying molecular mechanism, the authors identified that miR-30 family members can suppress p53 expression. When cell received apoptotic stimulation, p53 was found to transcriptionally activate the fission protein, Drp-1. Drp-1 was able to induce mitochondrial fission. miR-30 family members were observed to inhibit mitochondrial fission through attenuation of p53 expression and its downstream target Drp-1.

Mitochondrial fission & fusion as a therapeutic target

Since alteration of mitochondrial fission and fusion have been reported to affect various cellular processes including apoptosis, proliferation, ATP consumption, the proteins involved in the process of fission and fusion might be harnessed as therapeutic target.

Mentioned below is a description of research where dynamics of the mitochondrial organelle has been utilized as a therapeutic target:

Inhibition of mitochondrial fission prevents cell cycle progression in lung cancer

A recent article published by Rehman et al (2012) in the FASEB journal drew much attention after interesting observations were made in the mitochondria of lung adenocarcinoma cells. The mitochondrial network of these cells exhibited both impaired fusion and enhanced fission. It was also found that the fragmented phenotype in multiple lung adenocarcinoma cell lines was associated with both a down-regulation of the fusion protein, Mfn-2 and an upregulation of expression of fission protein, Drp-1. The imbalance of Drp-1/Mfn-2 expression in human lung cancer cell lines was reported to promote a state of mitochondrial fission. Similar increase in Drp-1 and decrease in Mfn-2 was observed in the tissue samples from patients compared to adjacent healthy lung. Authors used complementary approaches of Mfn-2 overexpression, Drp-1 inhibition, or Drp-1 knockdown and were able to observe reduction of cancer cell proliferation and an increase spontaneous apoptosis. Thus, the study identified mitochondrial fission and Drp-1 activation as a novel therapeutic target in lung cancer.