Mechanical properties of the lateral cortex of mammalian auditory outer hair cells.

Mammalian auditory outer hair cells generate high-frequency mechanical forces that enhance sound-induced displacements of the basilar membrane within the inner ear. It has been proposed that the resulting cell deformation is directed along the longitudinal axis of the cell by the cortical cytoskeleton. We have tested this proposal by making direct mechanical measurements on outer hair cells. The resultant stiffness modulus along the axis of whole dissociated cells was 3 x 10(-3) N/m, consistent with previously published values. The resultant axial and circumferential stiffness moduli for the cortical lattice were 5 x 10(-4) N/m and 3 x 10(-3) N/m, respectively. Thus the cortical lattice is a highly orthotropic structure. Its axial stiffness is small compared with that of the intact cell, but its circumferential stiffness is within the same order of magnitude. These measurements support the theory that the cortical cytoskeleton directs electrically driven length changes along the longitudinal axis of the cell. The Young's modulus of the circumferential filamentous components of the lattice were calculated to be 1 x 10(7) N/m2. The axial cross-links, believed to be a form of spectrin, were calculated to have a Young's modulus of 3 x 10(6) N/m2. Based on the measured values for the lattice and intact cell cortex, an estimate for the resultant stiffness modulus of the plasma membrane was estimated to be on the order of 10(-3) N/m. Thus, the plasma membrane appears to be relatively stiff and may be the dominant contributor to the axial stiffness of the intact cell.     

The cell-type specificity of mitochondrial dynamics

Recent advances in mitochondrial imaging have revealed that in many cells mitochondria can be highly dynamic. They can undergo fission/fusion processes modulated by various mitochondria-associated proteins and also by conformational transitions in the inner mitochondrial membrane. Moreover, precise mitochondrial distribution can be achieved by their movement along the cytoskeleton, recruiting various connector and motor proteins. Such movement is evident in various cell types ranging from yeast to mammalian cells and serves to direct mitochondria to cellular regions of high ATP demand or to transport mitochondria destined for elimination. Existing data also demonstrate that many aspects of mitochondrial dynamics, morphology, regulation and intracellular organization can be cell type-/tissue-specific. In many cells like neurons, pancreatic cells, HL-1 cells, etc., complex dynamics of mitochondria include fission, fusion, small oscillatory movements of mitochondria, larger movements like filament extension, retraction, fast branching in the mitochondrial network and rapid long-distance intracellular translocation of single mitochondria. Alternatively, mitochondria can be rather fixed in other cells and tissues like adult cardiomyocytes or skeletal muscles with a very regular organelle organization between myofibrils, providing the bioenergetic basis for contraction. Adult cardiac cells show no displacement of mitochondria with only very small-amplitude rapid vibrations, demonstrating remarkable, cell type-dependent differences in the dynamics and spatial arrangement of mitochondria. These variations and the cell-type specificity of mitochondrial dynamics could be related to specific cellular functions and demands, also indicating a significant role of integrations of mitochondria with other intracellular systems like the cytoskeleton, nucleus and endoplasmic reticulum (ER).     

Mitochondrial mechanism of heat stress-induced injury in rat cardiomyocyte

Heat stress results in cardiac dysfunction and even cardiac failure. To elucidate the cellular and molecular mechanism of cardiomyocyte injury induced by heat stress, the changes of structure and function in cardiac mitochondria of heat-exposed Wistar rats and its role in cardiomyocyte injury were investigated. Heat stress induced apoptosis and necrosis of cardiomyocytes in a time- and dose-dependent fashion. In the mitochondria of heat-stressed cardiomyocytes, the respiratory control rate and oxidative phosphorylation efficiency (P:O) were decreased gradually with the rise of rectal temperature. The Ca2+ -adenosine triphosphatase activity and Ca2+ content were also reduced. Exposing isolated mitochondria to the heat stress induced special internal environmental states including Ca2+ overload, oxidative stress, and altered mitochondrial membrane permeability transition (MPT). In vivo, the heat stress-induced mitochondrial MPT alteration was also found. The changes of mitochondrial MPT resulted in the release of cytochrome c from mitochondria into the cytosol, and in turn, caspase-3 was activated. Transfection of bcl-2 caused Bcl-2 overexpression in cardiomyocyte, which protected the mitochondria and reduced the heat stress-induced cardiomyocyte injury. In conclusion, it appears that the destruction of mitochondrial structure and function not only resulted in the impairment of physiological function of cardiomyocytes under heat stress but may also further lead to severe cellular injury and even cell death. These findings underline the contribution of mitochondria to the injury process in cardiomyocytes under heat stress.     

Ultrastructure of mitochondria apparatus of cardiomyocytes in apoptosis induced by long-term anoxia in rats

Apoptosis in cardiomyocytes was induced by incubation of pieces of cardiac tissues under condition of anoxia. Electronmicroscopic investigation detected previously unknown changes in mitochondrial ultrastructure. The mitochondrial population was characterized by morphological heterogeneity. In addition to a mitochondrial population characterized with irrigated cleared matrix, anoxia induced the appearance of an atypical and previously unknown population of small electron-dense cardiomyocyte mitochondria. They were characterized by unusual localization inside electron-light mitochondria ("mitochondria inside mitochondria"). The most part of mitochondria with the irrigated matrix are commonly characterized by unusual types of rearrangements of the inner mitochondrial membrane. Under anoxic conditions, the inner mitochondrial membrane formed electron-dense ordered structures. This is a spongy structure with cells of equal size. Results of our study are discussed in terms of conception of changes in mitochondrial reticulum ultrastructure during apoptosis.     

Ultrastructure and morphology of the mitochondriome of cardiomyocytes from invertebrates. I. The mitochondriome of cardiomyocytes from insects

The cardiomyocyte mitochondrial ultrastructure of two insect species (the American cockroach Periplaneta americana, and a dragonfly Aeschna sp.) has been studied. Mitochondria in cardiomyocytes of these insects are connected by intermitochondrial contacts, similar in morphology to vertebrate intermitochondrial contacts. The number of intermitochondrial contacts differs in cardiomyocytes of the studied insects, numbering 12 and 18 per 100 mitochondria in cardiomyocytes of the cockroach and dragonfly, respectively, which is due presumably to differences in activity of these insects. Cardiomyocytes of both species have several features in common. It was shown that cross-striated myofibrils oriented in different directions occupy 50-58% of the cytoplasmic volume, while mitochondria cover only 16-18%. The pattern of mitochondrial localization differs in cardiomyocytes of the two studied insects. In the cockroach, cardiomyocyte mitochondria are seen both in the center of the cell and on its periphery, in protrusions; whereas in the dragonfly, mitochondria of cardiomyocytes are confined to the protrusions of the abluminal cell side. Mitochondrial profiles are small, their packing is not dense. Mitochondria in cardiomyocytes of these insects have few plastic cristae and dense matrix.     

Mechanical anisotropy of adherent cells probed by a three-dimensional magnetic twisting device

We describe a three-dimensional magnetic twisting device that is useful in characterizing the mechanical properties of cells. With the use of three pairs of orthogonally aligned coils, oscillatory mechanical torque was applied to magnetic beads about any chosen axis. Frequencies up to 1 kHz could be attained. Cell deformation was measured in response to torque applied via an RGD-coated, surface-bound magnetic bead. In both unpatterned and micropatterned elongated cells on extracellular matrix, the mechanical stiffness transverse to the long axis of the cell was less than half that parallel to the long axis. Elongated cells on poly-L-lysine lost stress fibers and exhibited little mechanical anisotropy; disrupting the actin cytoskeleton or decreasing cytoskeletal tension substantially decreased the anisotropy. These results suggest that mechanical anisotropy originates from intrinsic cytoskeletal tension within the stress fibers. Deformation patterns of the cytoskeleton and the nucleolus were sensitive to loading direction, suggesting anisotropic mechanical signaling. This technology may be useful for elucidating the structural basis of mechanotransduction. cytoskeleton; prestress; stress fibers; mechanotransduction; mechanical deformation     

Prohibitin protects against oxidative stress-induced cell injury in cultured neonatal cardiomyocyte

Oxidative stress is one of the main causes of myocardial injury, which is associated with cardiomyocyte death. Mitochondria play a key role in triggering the necrosis and apoptosis pathway of cardiomyocytes under oxidative stress. Although prohibitin (PHB) has been acknowledged as a mitochondrial chaperone, its functions in cardiomyocytes are poorly characterized. The present research was designed to investigate the cardioprotective role of PHB in mitochondria. Oxidative stress can increase the PHB content in mitochondria in a time-dependent manner. Overexpression of PHB in cultured cardiomyocytes by transfection of recombinant adenovirus vector containing PHB sense cDNA resulted in an increase of PHB in mitochondria. Compared with the non-transfection cardiomyocytes, PHB overexpression could protect the mitochondria from oxidative stress-induced injury. The mitochondria-mediated apoptosis pathway was consistently suppressed in PHB-overexpressed cardiomyocytes after hydrogen peroxide (H₂O₂) treatment, including a reduced change in mitochondrial membrane permeability transition and an inhibited release of cytochrome c from mitochondria to cytoplasma. As a result, the oxidative stress-induced cardiomyocyte apoptosis was suppressed. These data indicated that PHB protected the cardiomyocytes from oxidative stress-induced damage, and that increasing PHB content in mitochondria constituted a new therapeutic target for myocardium injury. XiaoHua Liu and Zhe Ren contributed equally to this work. ● Prohibitin is an evolutionarily conserved and ubiquitously expressed protein involved in mitochondrial structure, function, and inheritance whose function in cardiomyocyte is not known. In this study, we found oxidative stress could induce increased expression in cardiomyocytes and mitochondrial translocation of PHB, and PHB can protect against oxidative stress in cultured neonatal cardiomyocyte.     

The mitochondrial ubiquitin ligase plays an anti‐apoptotic role in cardiomyocytes by regulating mitochondrial fission

Apoptosis plays a critical role in the development of myocardial infarction. Cardiomyocytes are enriched with mitochondria and excessive mitochondrial fission can trigger cellular apoptosis. Recently, the mitochondrial ubiquitin ligase (MITOL), localized in the mitochondrial outer membrane, was reported to play an important role in the regulation of mitochondrial dynamics and apoptosis. However, the underlying mechanism of its action remains uncertain. The present study was aimed at uncovering the role of MITOL in the regulation of cardiomyocyte apoptosis. Our results showed that MITOL expression was up‐regulated in cardiomyocytes in response to apoptotic stimulation. Mitochondrial ubiquitin ligase overexpression blocked dynamin‐related protein 1 accumulation in the mitochondria, and attenuated the mitochondrial fission induced by hydrogen peroxide. Conversely, MITOL knockdown sensitized cardiomyocytes to undergo mitochondrial fission, resulting in subsequent apoptosis. These findings suggest that MITOL plays a protective role against apoptosis in cardiomyocytes, and may serve as a potential therapeutic target for apoptosis‐related cardiac diseases.     

Dynamics of Mitochondrial DNA Nucleoids Regulated by Mitochondrial Fission Is Essential for Maintenance of Homogeneously Active Mitochondria during Neonatal Heart Development

Mitochondria are dynamic organelles, and their fusion and fission regulate cellular signaling, development, and mitochondrial homeostasis, including mitochondrial DNA (mtDNA) distribution. Cardiac myocytes have a specialized cytoplasmic structure where large mitochondria are aligned into tightly packed myofibril bundles; however, recent studies have revealed that mitochondrial dynamics also plays an important role in the formation and maintenance of cardiomyocytes. Here, we precisely analyzed the role of mitochondrial fission in vivo. The mitochondrial fission GTPase, Drp1, is highly expressed in the developing neonatal heart, and muscle-specific Drp1 knockout (Drp1-KO) mice showed neonatal lethality due to dilated cardiomyopathy. The Drp1 ablation in heart and primary cultured cardiomyocytes resulted in severe mtDNA nucleoid clustering and led to mosaic deficiency of mitochondrial respiration. The functional and structural alteration of mitochondria also led to immature myofibril assembly and defective cardiomyocyte hypertrophy. Thus, the dynamics of mtDNA nucleoids regulated by mitochondrial fission is required for neonatal cardiomyocyte development by promoting homogeneous distribution of active mitochondria throughout the cardiomyocytes.     

Quantitative ultrastructural criteria of myocardial damage in postischemic reperfusion

Swollen clarified mitochondria were found during postischemic reperfusion in cardiomyocytes with and without signs of myofibril relaxation. The values of morphometric parameters of mitochondria were similar in both groups of cells studied. Mean indexes characterizing the condition of cardiomyocyte mitochondria may serve as quantitative criteria of the severity of myocardial damage: surface to volume, ratio, visual index of mitochondrial condition and relative volume density, i.e. volume density with respect to control cell volume.     

Doxorubicin-induced cardiomyocyte apoptosis: Role of mitofusin 2

BackgroundDoxorubicin (DOX) is an anti-tumor agent that is widely used in clinical setting for cancer treatment. The application of the DOX, however, is limited by its cardiac toxicity which can induce heart failure through an undefined mechanism. Mitofusin 2 (Mfn2) is a mitochondrial GTPase fusion protein that is located on the outer membrane of mitochondria (OMM). The Mfn2 plays an important role in mitochondrial fusion and fission. The aim of this study is to identify the role of the Mfn2 in DOX-induced cardiomyocyte apoptosis.MethodsCultured neonatal rat cardiomyocytes were used in this study. Mfn2 expression in cardiomyocytes was determined after the cardiomyocytes were challenged with DOX. Cardiomyocyte mitochondrial fission, mitochondrial reactive oxygen species (ROS) production was assessed with mitochondrial fragmentation and MitoSOX fluorescence probe, respectively. Cardiomyocyte apoptosis was determined with caspase3 activity and TUNEL staining.ResultsChallenging of the cardiomyocytes with DOX resulted in increasing in cardiomyocyte oxidative stress and apoptosis. In addition, levels of Mfn2 in cardiomyocytes were decreased after the cells were challenged with DOX which was associated with increased mitochondrial fission (fragmentation) and mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 attenuated the DOX-induced increase in mitochondrial fission and prevented cardiomyocyte mitochondrial ROS production. An increase in cardiomyocyte levels of Mfn2 or pretreatment of cardiomyocytes with an anti-oxidant, Mito-tempo, also prevented the DOX-induced cardiomyocyte apoptosis.ConclusionOur results indicate that DOX results in a decreased cardiomyocyte Mfn2 expression which promotes mitochondrial fission and ROS production further leads to cardiomyocyte apoptosis.     

Ultrastructural and morphometric characteristics of cardiomyocyte mitochondriomes of several invertebrate species. II. Heart ventricle cardiomyocyte mitochondriome of several gastropod mollusks

The mitochondrial ultrastructure in ventricle cardiomyocytes of three gastropod molluscs (Clione limacina, Helix pomatia, Lymnaea stagnalis) has been studied. Mitochondria in cardiomyocytes of these molluscs are connected by intermitochondrial contacts of the same morphology as intermitochondrial contacts in vertebrate cardiomyocytes. Their numbers in cardiomyocytes of the above molluscs being, respectively, 61, 35.1 and 29.2 contacts per 100 mitochondria. In Clione limacina cardiomyocyte contractile elements located on the periphery of cell occupy 21.1% of the cytoplasm volume. Mitochondria form a core making large dense central accumulations taking up 54.9% of the cytoplasm volume. Numerous mitochondria have vesicular or tubular cristae and light matrix. Unlike cardiomyocytes of Clione limacina, in Helix pomatia and Lymnaea stagnalis contractile material predominates in cardiomyocytes occupying 43.7% and 49.2% of the cytoplasm volume, respectively. Mitochondria located on the periphery and in the center of cardiomyocytes in Lymnaea stagnalis and Helix pomatia occupy 31 and 32.5% of the cytoplasma volume, respectively. Mitochondria in cardiomyocytes of both these molluscs have plastic cristae and dense matrix. The differences in cardiomyocyte mitochondriom organization in the studied molluscs can be explained by different functional heart loading in these due to different levels of their locomotor activity.     

Inner mitochondrial membrane structure and fusion dynamics are altered in senescent human iPSC-derived and primary rat cardiomyocytes

Dysfunction of the aging heart is a major cause of death in the human population. Amongst other tasks, mitochondria are pivotal to supply the working heart with ATP. The mitochondrial inner membrane (IMM) ultrastructure is tailored to meet these demands and to provide nano-compartments for specific tasks. Thus, function and morphology are closely coupled. Senescent cardiomyocytes from the mouse heart display alterations of the inner mitochondrial membrane. To study the relation between inner mitochondrial membrane architecture, dynamics and function is hardly possible in living organisms. Here, we present two cardiomyocyte senescence cell models that allow in cellular studies of mitochondrial performance. We show that doxorubicin treatment transforms human iPSC-derived cardiomyocytes and rat neonatal cardiomyocytes in an aged phenotype. The treated cardiomyocytes display double-strand breaks in the nDNA, have β-galactosidase activity, possess enlarged nuclei, and show p21 upregulation. Most importantly, they also display a compromised inner mitochondrial structure. This prompted us to test whether the dynamics of the inner membrane was also altered. We found that the exchange of IMM components after organelle fusion was faster in doxorubicin-treated cells than in control cells, with no change in mitochondrial fusion dynamics at the meso-scale. Such altered IMM morphology and dynamics may have important implications for local OXPHOS protein organization, exchange of damaged components, and eventually the mitochondrial bioenergetics function of the aged cardiomyocyte.     

Tools for studying and modulating (cardiac muscle) cell mechanics and mechanosensing across the scales

Cardiomyocytes generate force for the contraction of the heart to pump blood into the lungs and body. At the same time, they are exquisitely tuned to the mechanical environment and react to e.g. changes in cell and extracellular matrix stiffness or altered stretching due to reduced ejection fraction in heart disease, by adapting their cytoskeleton, force generation and cell mechanics. Both mechanical sensing and cell mechanical adaptations are multiscale processes. Receptor interactions with the extracellular matrix at the nanoscale will lead to clustering of receptors and modification of the cytoskeleton. This in turn alters mechanosensing, force generation, cell and nuclear stiffness and viscoelasticity at the microscale. Further, this affects cell shape, orientation, maturation and tissue integration at the microscale to macroscale. A variety of tools have been developed and adapted to measure cardiomyocyte receptor-ligand interactions and forces or mechanics at the different ranges, resulting in a wealth of new information about cardiomyocyte mechanobiology. Here, we take stock at the different tools for exploring cardiomyocyte mechanosensing and cell mechanics at the different scales from the nanoscale to microscale and macroscale.     

C5a-blockade improves burn-induced cardiac dysfunction

We previously reported that generation of the anaphylatoxin C5a is linked to the development of cardiac dysfunction in sepsis due to C5a interaction with its receptor (C5aR) on cardiomyocytes. Burn injury involves inflammatory mechanisms that can lead to C5a generation as well. In this study, we investigated the effects of C5a blockade on burn-induced cardiac dysfunction. Using a standardized rat model of full thickness scald injury, left ventricular pressures were recorded in vivo followed by in vitro assessment of sarcomere contraction of single cardiomyocytes. Left ventricular pressures in vivo and cardiomyocyte sarcomere contractility in vitro were significantly reduced following burn injury. In the presence of anti-C5a Ab, these defects were greatly attenuated 1, 6, and 12 h after burn injury and completely abolished 24 h after burn. In vitro incubation of cardiomyocytes with bacterial LPS accentuated the impaired contractility, which was partially prevented in cardiomyocytes from burned rats that had received an anti-C5a Ab. Based on Western blot analyses, real-time PCR, and immunostaining of left ventricular heart tissue, there was a significant increase in cardiomyocyte expression of C5aR after burn injury. In conclusion, an in vivo blockade of C5a attenuates burn-induced cardiac dysfunction. Further deterioration of contractility due to the exposure of cardiomyocytes to LPS was partially prevented by C5a-blockade. These results suggest a linkage between C5a and burn-induced cardiac dysfunction and a possible contribution of LPS to these events.     

Effects of acute ventricular volume manipulation on in situ cardiomyocyte cell membrane configuration

Effects of mechanical stimulation on cardiac electrical activity, gene expression, protein synthesis, and tissue remodelling have received increasing attention in recent years, as reviewed in this issue of PBMB. Little is known, though, about how changes in ventricular filling affect the cell configuration of cardiomyocytes in the ventricular wall. Here, we present first electron-microscopic insight into changes in cardiomyocyte cell structure in situ during acute ventricular volume manipulation. Apart from confirming the anticipated ventricular volume-related changes in cardiomyocyte sarcomere length, there is evidence of (i) unfolding of ‘slack’ membrane, primarily from sarcolemmal invaginations near the Z-lines, and (ii) stretch-induced incorporation of sub-membrane caveolae into the surface membrane. The functional relevance of these changes in cardiomyocyte membrane configuration—other than to cater for the length-dependent modulation of the cell surface to cell volume ratio—remains to be elucidated.     

MFN1介导的线粒体融合在心肌细胞凋亡中的作用研究

目的:探讨线粒体融合关键蛋白MFN1介导的线粒体融合在调控心肌细胞凋亡中的作用。方法:通过si RNA降低体外培养H9C2心肌细胞中MFN1的表达后,采用Western blot检测线粒体细胞色素c(Cyto c)释放及其下游凋亡效应分子Caspase9与Caspase3活性,流式细胞术检测细胞内活性氧(ROS)的产生情况,流式细胞术检测细胞凋亡的情况。结果:干扰MFN1可显著促进H9C2心肌细胞内细胞色素c由线粒体释放至胞浆,促进Caspase9与Caspase3的激活,增加细胞内活性氧ROS产生并提高细胞凋亡率(均P0.05)。结论:MFN1介导的线粒体融合可保护心肌细胞凋亡,其机制可能与抑制ROS产生与细胞色素C释放有关。     

Mitochondrial effects with ceramide-induced cardiac apoptosis are different from those of palmitate

The objective of this study was to evaluate whether ceramide, palmitate, and inhibitors of mitochondrial electron transport chain shared similar effects on the mitochondria of intact cardiomyocytes in order to determine the likelihood that ceramide and palmitate utilize similar mitochondrial mechanisms or pathways to apoptosis. In embryonic chick cardiomyocytes, ceramide, 100 microM for 24h, induced a 42.9+/-5.8% increase in cell death assessed by the MTT assay, and a significant (P<0.01) 3.9+/-0.6-fold increase in apoptosis assessed by propidium iodide staining of permeabilized cells. Mitochondrial potential (delta psi (m)), as demonstrated microscopically and by flow cytometry of cardiomyocytes stained with a J-aggregate dye, was markedly and significantly reduced by ceramide, palmitate, and two different inhibitors of the mitochondrial electron transport chain-rotenone and antimycin A. In contrast, the effect on mitochondria as assessed by CMX-Ros oxidation was dramatically different, as palmitate, rotenone, and antimycin A each produced a reduction, while ceramide increased CMX-Ros fluorescence. Further ceramide-induced cardiomyocyte apoptosis and loss of delta psi (m) operated through a cyclosporine-insensitive pathway similar to rotenone and antimycin A but distinct from palmitate which induced apoptosis though a cyclosporine-sensitive mechanism in these cells. These data suggest that ceramide acts on the mitochondria of intact cells through a cyclosporine-insensitive mechanism likely from a combination of actions including production of mitochondrial oxidants. The discordant findings between ceramide and palmitate suggest that palmitate-induced cell death is not primarily mediated by de novo ceramide synthesis.     

Coordinated behavior of mitochondria in both space and time: a reactive oxygen species-activated wave of mitochondrial depolarization

Reactive oxygen species (ROS) can trigger a transient burst of mitochondrial ROS production via ROS activation of the mitochondrial permeability transition pore (MPTP), a phenomenon termed ROS-induced ROS release (RIRR). The goal of this study was to investigate if the generation of ROS in a discrete region of a cardiomyocyte could serve to propagate RIRR-mediated mitochondrial depolarizations throughout a cell. Our experiments revealed that localized RIRR activated either RIRR-mediated fluctuations in mitochondrial membrane potential (time period: 3-10 min) or a traveling wave of depolarization of the cell's mitochondria (velocity: approximately 5 microm/min). Both phenomena appeared to be mediated by the mitochondrial permeability transition pore and eventually encompassed the majority of the mitochondrial population of both isolated rat and rabbit cardiomyocytes. Furthermore, depolarization was often reversible; the waves of depolarization were then followed by a rapid (approximately 40 microm/min) repolarization wave of the mitochondria. We show that the RIRR can function to communicate the mitochondrial permeability transition from one mitochondrion to another in the isolated adult cardiomyocyte.