Mitochondrial fusion is thought to be important for supporting cardiac contractility, but is hardly detectable in cultured cardiomyocytes and is difficult to directly evaluate in the heart. We overcame this obstacle through in vivo adenoviral transduction with matrix-targeted photoactivatable GFP and confocal microscopy. Imaging in whole rat hearts indicated mitochondrial network formation and fusion activity in ventricular cardiomyocytes. Promptly after isolation, cardiomyocytes showed extensive mitochondrial connectivity and fusion, which decayed in culture (at 24-48 h). Fusion manifested both as rapid content mixing events between adjacent organelles and slower events between both neighboring and distant mitochondria. Loss of fusion in culture likely results from the decline in calcium oscillations/contractile activity and mitofusin 1 (Mfn1), because (i) verapamil suppressed both contraction and mitochondrial fusion, (ii) after spontaneous contraction or short-term field stimulation fusion activity increased in cardiomyocytes, and (iii) ryanodine receptor-2-mediated calcium oscillations increased fusion activity in HEK293 cells and complementing changes occurred in Mfn1. Weakened cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Thus, attenuated mitochondrial fusion might contribute to the pathogenesis of cardiomyopathy.ardiac contractions require a constant energy supply, which is provided by mitochondrial metabolism. ATP is needed for excitation-contraction coupling (ECC) for both contraction and relaxation in each cycle (1, 2). ECC-associated cytoplasmic Ca 2+ transients ([Ca 2+ ] c ) are propagated to the mitochondrial matrix (3) to regulate Ca 2+ -dependent mitochondrial dehydrogenases, ATP synthesis, and intracellular Ca 2+ homeostasis (4). Thus, cardiac mitochondria are forced to work permanently and likely require quality control mechanisms to keep them in a functioning state.In many tissues, mitochondria are permanently rebuilt through evolutionarily conserved cyclic processes of fusion and fission. Fusion involves content exchange, allowing complementation of mitochondrial solutes, proteins, and DNA. Fission allows segregation of damaged components (5, 6). Both fusion and fission are promoted by mitochondrial movements that can bring distant mitochondria close to one another and can also separate interacting structures (7). However, the spatial arrangements and mitochondrial morphology are determined by sarcomers in ventricular myocytes, the contractile units of the adult mammalian heart. The gaps among densely packed parallel myofibrils are inhabited by bullet-like mitochondria that run longitudinally, interacting intimately with the junctional sarcoplasmic reticulum (SR), in a conformation that facilitates Ca 2+ exchange and supports ECC (4,8). Despite the spatial restrictions intrinsic to adult cardiomyocyte mitochondria, interactions among these organelles have been proposed based on functional observations. Reactive oxygen species (ROS)...
Environmental stressors like ethanol (EtOH) commonly target mitochondria to influence the cell’s fate. Recent literature supports that chronic EtOH exposure suppresses mitochondrial dynamics, central to quality control, and sensitizes mitochondrial permeability transition pore opening to promote cell death. EtOH-induced tissue injury is primarily attributed to its toxic metabolic products but alcoholism also impairs tissues that poorly metabolize EtOH. We embarked on studies to determine the respective roles of EtOH and its metabolites in mitochondrial fusion and tBid-induced mitochondrial apoptosis. We used HepG2 cells that do not metabolize EtOH and its engineered clone that expresses EtOH-metabolizing Cytochrome P450 E2 and alcohol dehydrogenase (VL-17A cells). We found that fusion impairment by prolonged EtOH exposure was prominent in VL-17A cells, probably owing to reactive oxygen species increase in the mitochondrial matrix. There was no change in fusion protein abundance, mitochondrial membrane potential or Ca2+ uptake. By contrast, prolonged EtOH exposure promoted tBid-induced outer mitochondrial membrane permeabilization and cell death only in HepG2 cells, owing to enhanced Bak oligomerization. Thus, mitochondrial fusion inhibition by EtOH is dependent on its metabolites, whereas sensitization to tBid-induced death is mediated by EtOH itself. This difference is of pathophysiological relevance because of the tissue-specific differences in EtOH metabolism.
We have reported previously that millimeter waves (MMWs) protect T-cell functions from the toxic side effects of cyclophosphamide (CPA), an anticancer drug. Since the effect of MMWs has been reported to be mediated by endogenous opioids, the present study was undertaken to investigate the role of endogenous opioids in protection of T-cell functions by MMWs. The effect of MMWs (42.2 GHz, incident power density = 38 mW/cm²) was studied on CPA-induced suppression of cytokine release by T cells in the presence of selective opioid receptor antagonists (ORA). Production of cytokines was measured in CD4 T cells isolated from splenocytes. Treatment of mice with CPA suppressed the formation of Th1 cytokines (TNF-α, IFN-γ, and IL-2), shifting the overall balance toward Th2 (IL-4 and IL-5). MMW irradiation of CPA-treated groups up-regulated the production of Th1 cytokines suppressed by CPA. Treatment of the CPA+MMW group with selective kappa (κ) ORA further potentiated this effect of MMWs on Th1 cytokine production, whereas treatment with μ or δ ORA increased the imbalance of cytokine production in the Th2 direction. These results provide further evidence that endogenous opioids are involved in immunomodulation by MMWs.
Chronic alcohol consumption renders the lung more susceptible to infections, in large part, by disrupting essential alveolar macrophage functions. Emerging evidence suggests that these functional deficits could be due to a suppression of GM-CSF signaling, which is believed to compromise monocyte growth and maturation in the lung. However, in addition to controlling monocyte behaviors, GM-CSF is also important for regulating surfactant homeostasis in the lung. For example, mice with targeted deletion of the gene for GM-CSF accumulate large amounts of phospholipids in their lung. Moreover, decreased GM-CSF signaling in humans has been mechanistically linked to the development of pulmonary alveolar proteinosis (PAP), a rare disorder in which surfactant lipids and proteins accumulate in alveolar macrophages and the lung exhibits enhanced susceptibility to respiratory infections. Consistent with parallel mechanisms in the PAP and alcoholic lung, we recently reported that levels of intrapulmonary lipids, albeit triglycerides and free fatty acids, were markedly increased in BAL fluid, whole lung digests and alveolar macrophages of chronically alcohol exposed rats. Additionally, we showed that uptake of saturated fatty acids alone was capable of inducing phenotypic and functional changes in alveolar macrophages that mimicked those in the alcohol-exposed rat and human lung. Herein, we discuss the role of GM-CSF in surfactant homeostasis and highlight the evidence that links decreased GM-CSF signaling to alveolar macrophage dysfunction in both the PAP and alcohol-exposed lung. Moreover, we propose a mechanism by which lipid accumulation itself can contribute to altering alveolar macrophage behaviors and propose how targeting this mechanism could potentially reduce the susceptibility to pulmonary infections in alcoholics.
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