RESUMO
Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430 Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region. MyBP-C regulates contraction in a phosphorylation dependent fashion that may be through binding of its N-terminal domains with myosin or actin. Understanding the 3D organisation of MyBP-C in the sarcomere environment may provide new light on its function. We report here the fine structure of MyBP-C in relaxed rat cardiac muscle by cryo-electron tomography and subtomogram averaging of refrozen Tokuyasu cryosections. We find that on average MyBP-C connects via its distal end to actin across a disc perpendicular to the thick filament. The path of MyBP-C suggests that the central domains may interact with myosin heads. Surprisingly MyBP-C at Stripe 4 is different; it has weaker density than the other stripes which could result from a mainly axial or wavy path. Given that the same feature at Stripe 4 can also be found in several mammalian cardiac muscles and in some skeletal muscles, our finding may have broader implication and significance. In the D-zone, we show the first demonstration of myosin crowns arranged on a uniform 143 Å repeat.
Assuntos
Actinas , Tomografia com Microscopia Eletrônica , Ratos , Animais , Actinas/metabolismo , Miocárdio/metabolismo , Miosinas/metabolismo , Citoesqueleto de Actina/metabolismo , Mamíferos/metabolismoRESUMO
Myocardial remodeling in response to chronic myocardial infarction (CMI) progresses through two phases, hypertrophic "compensation" and congestive "decompensation." Nothing is known about the ability of uninfarcted myocardium to produce force, velocity, and power during these clinical phases, even though adaptation in these regions likely drives progression of compensation. We hypothesized that enhanced cross-bridge-level contractility underlies mechanical compensation and is controlled in part by changes in the phosphorylation states of myosin regulatory proteins. We induced CMI in rats by left anterior descending coronary artery ligation. We then measured mechanical performance in permeabilized ventricular trabecula taken distant from the infarct zone and assayed myosin regulatory protein phosphorylation in each individual trabecula. During full activation, the compensated myocardium produced twice as much power and 31% greater isometric force compared with noninfarcted controls. Isometric force during submaximal activations was raised >2.4-fold, while power was 2-fold greater. Electron and confocal microscopy demonstrated that these mechanical changes were not a result of increased density of contractile protein and therefore not an effect of tissue hypertrophy. Hence, sarcomere-level contractile adaptations are key determinants of enhanced trabecular mechanics and of the overall cardiac compensatory response. Phosphorylation of myosin regulatory light chain (RLC) increased and remained elevated post-MI, while phosphorylation of myosin binding protein-C (MyBP-C) was initially depressed but then increased as the hearts became decompensated. These sensitivities to CMI are in accordance with phosphorylation-dependent regulatory roles for RLC and MyBP-C in crossbridge function and with compensatory adaptation in force and power that we observed in post-CMI trabeculae.
Assuntos
Proteínas de Transporte/metabolismo , Contração Miocárdica/fisiologia , Infarto do Miocárdio/metabolismo , Miocárdio/metabolismo , Miócitos Cardíacos/metabolismo , Cadeias Leves de Miosina/metabolismo , Sarcômeros/metabolismo , Adaptação Fisiológica , Animais , Vasos Coronários/cirurgia , Ligadura , Masculino , Microscopia Confocal , Microscopia Eletrônica , Infarto do Miocárdio/fisiopatologia , Miócitos Cardíacos/fisiologia , Miócitos Cardíacos/ultraestrutura , Fosforilação , Ratos , Ratos Sprague-Dawley , Sarcômeros/fisiologia , Sarcômeros/ultraestruturaRESUMO
Myosin-binding protein-C (MyBP-C) is an accessory protein of the myosin filaments of vertebrate striated muscle. In the heart, it plays a key role in modulating contractility in response to ß-adrenergic stimulation. Mutations in the cardiac isoform (cMyBP-C) are a leading cause of inherited hypertrophic cardiomyopathy. Understanding cMyBP-C function and its role in disease requires knowledge of the structure of the molecule, its organization in the sarcomere, and its interactions with other sarcomeric proteins. Here we review the main structural features of this modular, elongated molecule and the properties of some of its key domains. We describe observations suggesting that the bulk of the molecule extends perpendicular to the thick filament, enabling it to reach neighboring thin filaments in the sarcomere. We review structural and functional evidence for interaction of its N-terminal domains with actin and how this may modulate thin filament activation. We also discuss the effects that phosphorylation of cMyBP-C has on some of these structural features and how this might relate to cMyBP-C function in the beating heart.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Sarcômeros/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Transporte/química , Humanos , Dados de Sequência Molecular , Ligação Proteica , Sarcômeros/ultraestruturaRESUMO
OBJECTIVE: The purpose of this study was to assess whether abnormal cardiac function in human fetuses with intrauterine growth restriction (IUGR) is associated with ultrastructural differences in the cardiomyocyte sarcomere. STUDY DESIGN: Nine severe early-onset IUGR fetuses and 9 normally grown fetuses (appropriate growth for gestational age) who died in the perinatal period were included prospectively. Cardiac function was assessed by echocardiography and levels of B-type natriuretic peptide and troponin-I. Heart sections were imaged by second harmonic generation microscopy, which allowed unstained visualization of cardiomyocyte's sarcomere length. RESULTS: Echocardiographic and biochemical markers showed signs of severe cardiac dysfunction in IUGR fetuses. Second harmonic generation microscopy demonstrated a significantly shorter sarcomere length in IUGR as compared with appropriate growth for gestational age fetuses. CONCLUSION: IUGR is associated with changes in the cardiomyocyte contractile machinery in the form of shorter sarcomere length, which could help to explain the cardiac dysfunction previously documented in IUGR.
Assuntos
Retardo do Crescimento Fetal/fisiopatologia , Feto/fisiopatologia , Miócitos Cardíacos/ultraestrutura , Peptídeo Natriurético Encefálico/análise , Sarcômeros/ultraestrutura , Troponina I/análise , Estudos de Casos e Controles , Ecocardiografia , Feminino , Retardo do Crescimento Fetal/diagnóstico por imagem , Retardo do Crescimento Fetal/patologia , Idade Gestacional , Humanos , Gravidez , Ultrassonografia Pré-NatalRESUMO
Intrauterine growth restriction (IUGR) affects 7-10% of pregnancies and is associated with cardiovascular remodeling and dysfunction, which persists into adulthood. The underlying subcellular remodeling and cardiovascular programming events are still poorly documented. Cardiac muscle is central in the fetal adaptive mechanism to IUGR given its high energetic demands. The energetic homeostasis depends on the correct interaction of several molecular pathways and the adequate arrangement of intracellular energetic units (ICEUs), where mitochondria interact with the contractile machinery and the main cardiac ATPases to enable a quick and efficient energy transfer. We studied subcellular cardiac adaptations to IUGR in an experimental rabbit model. We evaluated the ultrastructure of ICEUs with transmission electron microscopy and observed an altered spatial arrangement in IUGR, with significant increases in cytosolic space between mitochondria and myofilaments. A global decrease of mitochondrial density was also observed. In addition, we conducted a global gene expression profile by advanced bioinformatics tools to assess the expression of genes involved in the cardiomyocyte energetic metabolism and identified four gene modules with a coordinated over-representation in IUGR: oxygen homeostasis (GO: 0032364), mitochondrial respiratory chain complex I (GO:0005747), oxidative phosphorylation (GO: 0006119), and NADH dehydrogenase activity (GO:0003954). These findings might contribute to changes in energetic homeostasis in IUGR. The potential persistence and role of these changes in long-term cardiovascular programming deserves further investigation.
Assuntos
Metabolismo Energético/fisiologia , Retardo do Crescimento Fetal/metabolismo , Miocárdio/metabolismo , Placenta/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Retardo do Crescimento Fetal/genética , Retardo do Crescimento Fetal/fisiopatologia , Expressão Gênica , Mitocôndrias/genética , Mitocôndrias/metabolismo , Miocárdio/ultraestrutura , Fosforilação Oxidativa , Placenta/fisiopatologia , Gravidez , CoelhosRESUMO
Purpose: Raman spectroscopy allows molecular changes to be quantified in vivo from the tissues like the retina. Here we aimed to assess the metabolic changes in the retina of patients with multiple sclerosis (MS). Methods: We built a Raman spectroscopy prototype by connecting a scanning laser ophthalmoscope to a spectrophotometer. We defined the spectra of 10 molecules participating on energy supply, axon biology, or synaptic damage, which have been shown to be altered in the brain of patients with MS: cytochrome C, flavin adenine dinucleotide (FAD), nicotinamide adenine dinucleotide (NADH), N-acetyl-aspartate (NAA), excitotoxicity, glutamate, amyloid ß (Aß), τ and α-synuclein (SNCA), phosphatidyl-ethanolamine, and phosphatidyl-choline. We studied these molecules in a prospective cohort of patients with MS, either in the chronic phase or during relapses of acute optic neuritis (AON). Results: Significant changes to all these molecules were associated with age in healthy individuals. There was a significant decrease in NADH and a trend toward a decrease in NAA in patients with MS, as well as an increase in Aß compared with healthy controls. Moreover, NADH and FAD increased over time in a longitudinal analysis of patients with MS, whereas Aß diminished. In patients with acute retinal inflammation due to AON, there was a significant increase in FAD and a decrease in SNCA in the affected retina. Moreover, glutamate levels increased in the affected eyes after a 6-month follow-up. Conclusions: Alterations of molecules related to axonal degeneration are observed during neuroinflammation and show dynamic changes over time, suggesting progressive neurodegeneration.
Assuntos
Biomarcadores/metabolismo , Proteínas do Olho/metabolismo , Esclerose Múltipla/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Doenças Retinianas/metabolismo , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Análise Espectral Raman , Tomografia de Coerência ÓpticaRESUMO
Oligodendrocyte death and demyelination are hallmarks of multiple sclerosis (MS). Here we show that ATP signaling can trigger oligodendrocyte excitotoxicity via activation of calcium-permeable P2X(7) purinergic receptors expressed by these cells. Sustained activation of P2X(7) receptors in vivo causes lesions that are reminiscent of the major features of MS plaques, i.e., demyelination, oligodendrocyte death, and axonal damage. In addition, treatment with P2X(7) antagonists of chronic experimental autoimmune encephalomyelitis (EAE), a model of MS, reduces demyelination and ameliorates the associated neurological symptoms. Together, these results indicate that ATP can kill oligodendrocytes via P2X(7) activation and that this cell death process contributes to EAE. Importantly, P2X(7) expression is elevated in normal-appearing axon tracts in MS patients, suggesting that signaling through this receptor in oligodendrocytes may be enhanced in this disease. Thus, P2X(7) receptor antagonists may be beneficial for the treatment of MS.
Assuntos
Trifosfato de Adenosina/toxicidade , Encefalomielite Autoimune Experimental/terapia , Oligodendroglia/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2 , Receptores Purinérgicos P2/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Encefalomielite Autoimune Experimental/induzido quimicamente , Proteína Glial Fibrilar Ácida/metabolismo , Glicoproteínas , Humanos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Camundongos , Microscopia Imunoeletrônica/métodos , Proteína Básica da Mielina/metabolismo , Glicoproteína Mielina-Oligodendrócito , Oligodendroglia/metabolismo , Oligodendroglia/ultraestrutura , Nervo Óptico/citologia , Nervo Óptico/patologia , Nervo Óptico/ultraestrutura , Técnicas de Patch-Clamp/métodos , Fragmentos de Peptídeos , Inibidores da Agregação Plaquetária , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7RESUMO
Connexins form a diverse and ubiquitous family of integral membrane proteins. Characteristically, connexins are assembled into intercellular channels that aggregate into discrete cell-cell contact areas termed gap junctions (GJ), allowing intercellular chemical communication, and are essential for propagation of electrical impulses in excitable tissues, including, prominently, myocardium, where connexin 43 (Cx43) is the most important isoform. Previous studies have shown that GJ-mediated communication has an important role in the cellular response to stress or ischemia. However, recent evidence suggests that connexins, and in particular Cx43, may have additional effects that may be important in cell death and survival by mechanisms independent of cell to cell communication. Connexin hemichannels, located at the plasma membrane, may be important in paracrine signaling that could influence intracellular calcium and cell survival by releasing intracellular mediators as ATP, NAD(+), or glutamate. In addition, recent studies have shown the presence of connexins in cell structures other than the plasma membrane, including the cell nucleus, where it has been suggested that Cx43 influences cell growth and differentiation. In addition, translocation of Cx43 to mitochondria appears to be important for certain forms of cardioprotection. These findings open a new field of research of previously unsuspected roles of Cx43 intracellular signaling.
Assuntos
Adaptação Fisiológica/fisiologia , Apoptose/fisiologia , Núcleo Celular/fisiologia , Sobrevivência Celular/fisiologia , Junções Comunicantes/fisiologia , Mitocôndrias/fisiologia , Modelos Biológicos , Animais , Comunicação Celular/fisiologia , Humanos , Estresse Oxidativo/fisiologiaRESUMO
Glutamate excitotoxicity and complement attack have both been implicated separately in the generation of tissue damage in multiple sclerosis and in its animal model, experimental autoimmune encephalomyelitis. Here, we investigated whether glutamate receptor activation sensitizes oligodendrocytes to complement attack. We found that a brief incubation with glutamate followed by exposure to complement was lethal to oligodendrocytes in vitro and in freshly isolated optic nerves. Complement toxicity was induced by activation of kainate but not of AMPA receptors and was abolished by removing calcium from the medium during glutamate priming. Dose-response studies showed that sensitization to complement attack is induced by two distinct kainate receptor populations displaying high and low affinities for glutamate. Oligodendrocyte death by complement required the formation of the membrane attack complex, which in turn increased membrane conductance and induced calcium overload and mitochondrial depolarization as well as a rise in the level of reactive oxygen species. Treatment with the antioxidant Trolox and inhibition of poly(ADP-ribose) polymerase-1, but not of caspases, protected oligodendrocytes against damage induced by complement. These findings indicate that glutamate sensitization of oligodendrocytes to complement attack may contribute to white matter damage in acute and chronic neurological disorders.
Assuntos
Membrana Celular/imunologia , Proteínas do Sistema Complemento/imunologia , Ácido Glutâmico/metabolismo , Fibras Nervosas Mielinizadas/imunologia , Oligodendroglia/imunologia , Receptores de Ácido Caínico/metabolismo , Animais , Animais Recém-Nascidos , Antioxidantes/farmacologia , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/imunologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Proteínas do Sistema Complemento/metabolismo , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/imunologia , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/metabolismo , Doenças Autoimunes Desmielinizantes do Sistema Nervoso Central/fisiopatologia , Relação Dose-Resposta a Droga , Ácido Glutâmico/farmacologia , Masculino , Fibras Nervosas Mielinizadas/efeitos dos fármacos , Fibras Nervosas Mielinizadas/metabolismo , Neurotoxinas/metabolismo , Oligodendroglia/efeitos dos fármacos , Oligodendroglia/metabolismo , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/imunologia , Nervo Óptico/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/imunologia , Técnicas de Patch-Clamp , Polinucleotídeo Adenililtransferase/antagonistas & inibidores , Polinucleotídeo Adenililtransferase/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Ácido Caínico/agonistasRESUMO
Oligodendrocytes are vulnerable to excitotoxic signals mediated by AMPA receptors and by high- and low-affinity kainate receptors. Here we investigated the nature of the cell death triggered by activation of these receptors in primary cultures of oligodendrocytes from the rat optic nerve. Activation of AMPA receptors at both submaximal and maximal concentrations of the agonist induced massive calcium entry, mitochondrial depolarization, and a rise in the level of reactive oxygen species that correlated with a decrease in the levels of reduced glutathione. In addition, excitotoxicity initiated by submaximal, but not maximal, activation of AMPA receptors was prevented by caspase-3 blockade and by the concomitant blockade of caspases 8 and 9. In turn, maximal activation of high- or low-affinity kainate receptors induced mitochondrial events and toxicity levels similar to those observed with submaximal activation of AMPA receptors. In contrast to AMPA receptor-mediated insults, calcineurin inhibition or caspase-9 blockade was sufficient to prevent cell death triggered by both types of kainate receptors. Consistent with these results, prolonged glutamate receptor activation in freshly isolated optic nerves caused selective activation of caspase-3 and chromatin condensation in oligodendrocytes. Overall, the evidence presented here indicates that oligodendrocyte death by excitotoxicity is mediated by caspase-dependent and -independent mechanisms.
Assuntos
Caspases/metabolismo , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Receptores de AMPA/metabolismo , Receptores de Ácido Caínico/metabolismo , Animais , Inibidores de Calcineurina , Cálcio/metabolismo , Inibidores de Caspase , Morte Celular , Células Cultivadas , Grupo dos Citocromos c/metabolismo , Inibidores Enzimáticos/farmacologia , Glutationa/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurotoxinas/farmacologia , Oligodendroglia/enzimologia , Nervo Óptico/citologia , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Receptores de AMPA/efeitos dos fármacos , Receptores de Ácido Caínico/efeitos dos fármacos , Desacopladores/farmacologiaRESUMO
The influence of age, postmortem delay and freezing storage period on receptor-mediated G-protein activity was quantified in cortical membranes from 34 healthy subjects. Concentration-response curves of the [(35)S]GTPgammaS binding stimulation by agonists for alpha(2)-adrenoceptors (UK14304), mu-opioid (DAMGO), 5-HT(1A) (8-OH-DPAT), GABA(B) (baclofen) and muscarinic (carbachol) receptors were analyzed. Immunoreactivities of G(alpha)-protein subunits were also determined. Basal binding and UK14304, 8-OH-DPAT, and baclofen potency to stimulate [(35)S]GTPgammaS binding decreased with aging (1-88 years) without changes of efficacy. DAMGO-mediated stimulation increased both in potency and efficacy with aging. A negative correlation between age and immunoreactivity was observed for G(alphai1/2)-, but not for G(alphai3)-, G(alphao)-,and G(alphas)-proteins. Neither [(35)S]GTPgammaS binding nor G(alpha)-proteins changed with the postmortem delay (8-92 h). Basal [(35)S]GTPgammaS binding decreased with the sample storage period (1-85 months). A careful match between cases and controls should be taken into account when designing signal transduction studies in human disorders, specially for variables such as age and storage period.
Assuntos
Envelhecimento/fisiologia , Química Encefálica/fisiologia , Proteínas de Ligação ao GTP/fisiologia , Mudanças Depois da Morte , Preservação de Tecido , Adolescente , Adulto , Idoso , Envelhecimento/patologia , Encéfalo/patologia , Criança , Pré-Escolar , Criopreservação , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/agonistas , Proteínas Heterotriméricas de Ligação ao GTP/biossíntese , Proteínas Heterotriméricas de Ligação ao GTP/genética , Humanos , Lactente , Membranas/química , Pessoa de Meia-Idade , Fatores de TempoRESUMO
Automatic quantification of cardiac muscle properties in tissue sections might provide important information related to different types of diseases. Second harmonic generation (SHG) imaging provides a stain-free microscopy approach to image cardiac fibers that, combined with our methodology of the automated measurement of the ultrastructure of muscle fibers, computes a reliable set of quantitative image features (sarcomere length, A-band length, thick-thin interaction length, and fiber orientation). We evaluated the performance of our methodology in computer-generated muscle fibers modeling some artifacts that are present during the image acquisition. Then, we also evaluated it by comparing it to manual measurements in SHG images from cardiac tissue of fetal and adult rabbits. The results showed a good performance of our methodology at high signal-to-noise ratio of 20 dB. We conclude that our automated measurements enable reliable characterization of cardiac fiber tissues to systematically study cardiac tissue in a wide range of conditions.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Técnicas Fotoacústicas/métodos , Sarcômeros/química , Algoritmos , Animais , Simulação por Computador , Miocárdio/citologia , Coelhos , Razão Sinal-RuídoRESUMO
BACKGROUND: Intrauterine growth restriction (IUGR) induces fetal cardiac remodelling and dysfunction, which persists postnatally and may explain the link between low birth weight and increased cardiovascular mortality in adulthood. However, the cellular and molecular bases for these changes are still not well understood. We tested the hypothesis that IUGR is associated with structural and functional gene expression changes in the fetal sarcomere cytoarchitecture, which remain present in adulthood. METHODS AND RESULTS: IUGR was induced in New Zealand pregnant rabbits by selective ligation of the utero-placental vessels. Fetal echocardiography demonstrated more globular hearts and signs of cardiac dysfunction in IUGR. Second harmonic generation microscopy (SHGM) showed shorter sarcomere length and shorter A-band and thick-thin filament interaction lengths, that were already present in utero and persisted at 70 postnatal days (adulthood). Sarcomeric M-band (GO: 0031430) functional term was over-represented in IUGR fetal hearts. CONCLUSION: The results suggest that IUGR induces cardiac dysfunction and permanent changes on the sarcomere.
Assuntos
Modelos Animais de Doenças , Retardo do Crescimento Fetal/fisiopatologia , Coração Fetal/fisiopatologia , Feto/fisiopatologia , Sarcômeros/diagnóstico por imagem , Animais , Biomarcadores/análise , Pressão Sanguínea , Peso Corporal , Ecocardiografia , Feminino , Retardo do Crescimento Fetal/diagnóstico por imagem , Coração Fetal/diagnóstico por imagem , Perfilação da Expressão Gênica , Tamanho do Órgão , Placenta/metabolismo , Gravidez , CoelhosRESUMO
AIMS: Connexin43 is present at the inner membrane of cardiomyocyte mitochondria (mCx43), but its function remains unknown. METHODS AND RESULTS: In this study we verified the presence of mCx43 by a mass spectrometry-based proteomic approach in purified mitochondrial preparations from mouse myocardium and determined by western blot analysis that the C-terminus of mCx43 is oriented towards the intermembrane space. Cross-linking studies with dimethylsuberimidate indicated the presence of Cx43 hexamers in mitochondrial membranes. The contribution of Cx43 to both mitochondrial dye uptake and K(+) flux was assessed in wild-type mice using hemichannel blockers and Cx43KI32 mice in which Cx43 had been replaced by Cx32. Uptake of the Cx43 hemichannel-permeant dye Lucifer Yellow was reduced in mitochondria from wild-type mice by two hemichannel blockers (carbenoxolone and heptanol) and in Cx43KI32 compared with wild-type mice. Mitochondrial K(+) influx (PBFI fluorescence) was decreased in digitonin-permeabilized cardiomyocytes from Cx32 mutants compared with wild-type mice, and addition of the Cx43 hemichannel blocker 18alpha-glycyrrhetinic acid had an inhibitory effect on mitochondrial K(+) influx in wild-type cardiomyocytes, but not in cardiomyocytes from Cx32 mutants. CONCLUSION: These results indicate that mCx43 contributes to mitochondrial K(+) flux in cardiomyocytes, potentially by forming hemichannel-like structures.