The small, basic, and cysteine-rich antifungal protein PAF is abundantly secreted into the supernatant by the ␤-lactam producer Penicillium chrysogenum. PAF inhibits the growth of various important plant and zoopathogenic filamentous fungi. Previous studies revealed the active internalization of the antifungal protein and the induction of multifactorial detrimental effects, which finally resulted in morphological changes and growth inhibition in target fungi. In the present study, we offer detailed insights into the mechanism of action of PAF and give evidence for the induction of a programmed cell death-like phenotype. We proved the hyperpolarization of the plasma membrane in PAF-treated Aspergillus nidulans hyphae by using the aminonaphtylethenylpyridinium dye di-8-ANEPPS. The exposure of phosphatidylserine on the surface of A. nidulans protoplasts by Annexin V staining and the detection of DNA strand breaks by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) gave evidence for a PAF-induced apoptotic-like mechanism in A. nidulans. The localization of reactive oxygen species (ROS) by dichlorodihydrofluorescein diacetate and the abnormal cellular ultrastructure analyzed by transmission electron microscopy suggested that ROS-elicited membrane damage and the disintegration of mitochondria played a major role in the cytotoxicity of PAF. Finally, the reduced PAF sensitivity of A. nidulans strain FGSC1053, which carries a dominant-interfering mutation in fadA, supported our assumption that G-protein signaling was involved in PAF-mediated toxicity.A large number of small, basic, cysteine-rich antimicrobial proteins are produced by organisms throughout all kingdoms. They display a great variety in their primary structure, in species specificity, and in the mechanism of action.Few ascomycetes secrete strongly related antifungal proteins, which do not show any sequence homology with other antimicrobial proteins, but most of these proteins exhibit structural similarities (46): a net positive charge and numerous cysteine residues that are involved in disulfide bond formation. These properties contribute to a compact tertiary structure and a high stability against environmental impact and finally support the model of a membrane-disturbing nature. The antifungal protein PAF from P. chrysogenum and AFP from A. giganteus are the two most intensively studied peptides in the group of antifungals from ascomycetes, but the information available on their exact mechanism of action is still rather limited (32,55,68,69). PAF inhibits the growth of various important plant pathogenic and zoopathogenic filamentous fungi, e.g., Aspergillus fumigatus, A. niger, A. nidulans, and Botrytis cinerea. Previous studies revealed the induction of multifactorial detrimental effects on target organisms that include growth inhibition, reduction of cellular metabolism, severe changes in hyphal morphology, increased K ϩ efflux, and the generation of intracellular reactive oxygen species (ROS) (32, 48). PAF was found to...

Key pointsr Genetic mutations in cardiac troponin I (cTnI) are associated with development of hypertrophic cardiomyopathy characterized by myocyte remodelling, disorganization of cytoskeletal proteins and altered energy metabolism.r The L-type Ca 2+ channel is the main route for calcium influx and is crucial to cardiac excitation and contraction. The channel also regulates mitochondrial function in the heart by a functional communication between the channel and mitochondria via the cytoskeletal network.r We find that L-type Ca 2+ channel kinetics are altered in cTnI-G203S cardiac myocytes and that activation of the channel causes a significantly greater increase in mitochondrial membrane potential and metabolic activity in cTnI-G203S cardiac myocytes. r We propose that L-type Ca 2+ channel antagonists, such as diltiazem, might be effective in reducing the cardiomyopathy by normalizing mitochondrial metabolic activity.Abstract Genetic mutations in cardiac troponin I (cTnI) account for 5% of families with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy is associated with disorganization of cytoskeletal proteins and altered energy metabolism. The L-type Ca 2+ channel (I Ca-L ) plays an important role in regulating mitochondrial function. This involves a functional communication between the channel and mitochondria via the cytoskeletal network. We investigate the role of I Ca-L in regulating mitochondrial function in 25-to 30-week-old cardiomyopathic mice expressing the human disease-causing mutation Gly203Ser in cTnI (cTnI-G203S). The inactivation rate of I Ca-L is significantly faster in cTnI-G203S myocytes [cTnI-G203S: τ 1 = 40.68 ± 3.22, n = 10 vs. wild-type (wt): τ 1 = 59.05 ± 6.40, n = 6, P < 0.05]. Activation of I Ca-L caused a greater increase in mitochondrial membrane potential ( m , 29.19 ± 1.85%, n = 15 vs. wt: 18.84 ± 2.01%, n = 10, P < 0.05) and metabolic activity (24.40 ± 6.46%, n = 8 vs. wt: 9.98 ± 1.57%, n = 9, P < 0.05). The responses occurred because of impaired communication between I Ca-L and F-actin, involving lack of dynamic movement of actin-myosin and block of the mitochondrial voltage-dependent anion channel. Similar responses were observed in precardiomyopathic mice. I Ca-L antagonists nisoldipine and diltiazem decreased m to basal levels. We conclude that the Gly203Ser mutation

Increased reactive oxygen species (ROS) production and elevated intracellular Ca(2+) following cardiac ischemia-reperfusion injury are key mediators of cell death and the development of cardiac hypertrophy. The L-type Ca(2+) channel is the main route for calcium influx in cardiac myocytes. Activation of the L-type Ca(2+) channel leads to a further increase in mitochondrial ROS production and metabolism. We have previously shown that the application of a peptide derived against the alpha-interacting domain of the L-type Ca(2+) channel (AID) decreases myocardial injury post reperfusion. Herein, we examine the efficacy of simultaneous delivery of the AID peptide in combination with the potent antioxidants curcumin or resveratrol using multifunctional poly(glycidyl methacrylate) (PGMA) nanoparticles. We highlight that drug loading and dissolution are important parameters that have to be taken into account when designing novel combinatorial therapies following cardiac ischemia-reperfusion injury. In the case of resveratrol low loading capacity and fast release rates hinder its applicability as an effective candidate for simultaneous therapy. However, in the case of curcumin, high loading capacity and sustained release rates enable its effective simultaneous delivery in combination with the AID peptide. Simultaneous delivery of the AID peptide with curcumin allowed for effective attenuation of the L-type Ca(2+) channel-activated increases in superoxide (assessed as changes in DHE fluorescence; Empty NP = 53.1 ± 7.6%; NP-C-AID = 7.32 ± 3.57%) and mitochondrial membrane potential (assessed as changes in JC-1 fluorescence; Empty NP = 19.8 ± 2.8%; NP-C-AID=13.05 ± 1.78%). We demonstrate in isolated rat hearts exposed to ischemia followed by reperfusion, that curcumin and the AID peptide in combination effectively reduce muscle damage, decrease oxidative stress and superoxide production in cardiac myocytes.

The effects of adenosine 5'-triphosphate (ATP) on human and mouse skeletal muscle fibres in primary culture were investigated. ATP-evoked changes in intracellular calcium concentration ([Ca(2+)](i)) were measured and compared with those induced by agonists of the nicotinic acetylcholine (Ach)- and P2X purinoreceptors. While ATP was effective on both myoblasts and multi-nucleated myotubes in the micromolar range, Ach failed to induce any change in [Ca(2+)](i) at early stages of development. In contrast, myofibres with peripheral nuclei showed little response to ATP but responded to Ach with a large change in [Ca(2+)](i). The responsiveness of the myotubes to Ach paralleled that to potassium. The removal of external calcium abolished the response to ATP. P2X receptor agonists mimicked the response to ATP with the order of potency being ATP>2',3'- O-(4-benzoyl)-benzoyl-ATP>beta,gamma-methylene-ATP>alpha,beta-methylene-ATP. Under voltage-clamp conditions ATP induced an inward current that showed little inactivation. These results are consistent with the existence of P2X receptor-mediated signal transduction pathway in cultured mammalian skeletal muscle cells.

Translation fidelity is crucial for prokaryotes and eukaryotic nuclear‐encoded proteins; however, little is known about the role of mistranslation in mitochondria and its potential effects on metabolism. We generated yeast and mouse models with error‐prone and hyper‐accurate mitochondrial translation, and found that translation rate is more important than translational accuracy for cell function in mammals. Specifically, we found that mitochondrial mistranslation causes reduced overall mitochondrial translation and respiratory complex assembly rates. In mammals, this effect is compensated for by increased mitochondrial protein stability and upregulation of the citric acid cycle. Moreover, this induced mitochondrial stress signaling, which enables the recovery of mitochondrial translation via mitochondrial biogenesis, telomerase expression, and cell proliferation, and thereby normalizes metabolism. Conversely, we show that increased fidelity of mitochondrial translation reduces the rate of protein synthesis without eliciting a mitochondrial stress response. Consequently, the rate of translation cannot be recovered and this leads to dilated cardiomyopathy in mice. In summary, our findings reveal mammalian‐specific signaling pathways that respond to changes in the fidelity of mitochondrial protein synthesis and affect metabolism.

The effects of thymol on steps of excitation-contraction coupling were studied on fast-twitch muscles of rodents. Thymol was found to increase the depolarization-induced release of calcium from the sarcoplasmic reticulum, which could not be attributed to a decreased calcium-dependent inactivation of calcium release channels/ryanodine receptors or altered intramembrane charge movement, but rather to a more efficient coupling of depolarization to channel opening. Thymol increased ryanodine binding to heavy sarcoplasmic reticulum vesicles, with a half-activating concentration of 144 micro M and a Hill coefficient of 1.89, and the open probability of the isolated and reconstituted ryanodine receptors, from 0.09 +/- 0.03 to 0.22 +/- 0.04 at 30 micro M. At higher concentrations the drug induced long-lasting open events on a full conducting state. Elementary calcium release events imaged using laser scanning confocal microscopy in the line-scan mode were reduced in size, 0.92 +/- 0.01 vs. 0.70 +/- 0.01, but increased in duration, 56 +/- 1 vs. 79 +/- 1 ms, by 30 micro M thymol, with an increase in the relative proportion of lone embers. Higher concentrations favored long events, resembling embers in control, with duration often exceeding 500 ms. These findings provide direct experimental evidence that the opening of a single release channel will generate an ember, rather than a spark, in mammalian skeletal muscle.

Certain filamentous fungi, such as the penicillin-producing strain Penicillium chrysogenum, secrete small, highly basic and cysteine-rich proteins with antifungal effects. Affected fungi include a number of important zoopathogens, including those infecting humans. Recent studies, however, have pointed to a membrane-perturbing effect of these antifungal compounds, apparent as a potassium efflux from affected fungal cells. If present on mammalian cells, this would severely hinder the potential therapeutic use of these molecules. Here we studied the effects of the P. chrysogenum-derived antifungal peptide (PAF) on a number of mammalian cells to establish whether the protein has any cytotoxic effects, alters transmembrane currents on excitable cells or activates the immune system. PAF, in a concentration range of 2-100 mug/ml, did not cause any cytotoxicity on human endothelial cells from the umbilical vein. Applied at 10 mug/ml, it also failed to modify voltage-gated potassium channels of neurones, skeletal muscle fibers, and astrocytes. PAF also left the hyperpolarization-activated non-specific cationic current (I(h)) and the L-type calcium current unaffected. Finally, up to 2 mug/ml, PAF did not induce the production of pro-inflammatory cytokines such as IL-6, IL-8, and TNF-alpha. These results suggest that PAF should have only minor, if any, effects on mammalian cells in the intended therapeutic concentration range.

SummaryHeterozygous mice (αMHC403/+) expressing the human disease-causing mutation Arg403Gln exhibit cardinal features of hypertrophic cardiomyopathy (HCM) including hypertrophy, myocyte disarray, and increased myocardial fibrosis. Treatment of αMHC403/+mice with the L-type calcium channel (ICa-L) antagonist diltiazem has been shown to decrease left ventricular anterior wall thickness, cardiac myocyte hypertrophy, disarray, and fibrosis. However, the role of the ICa-L in the development of HCM is not known. In addition to maintaining cardiac excitation and contraction in myocytes, the ICa-L also regulates mitochondrial function through transmission of movement of ICa-L via cytoskeletal proteins to mitochondrial voltage-dependent anion channel. Here, the authors investigated the role of ICa-L in regulating mitochondrial function in αMHC403/+mice. Whole-cell patch clamp studies showed that ICa-L current inactivation kinetics were significantly increased in αMHC403/+cardiac myocytes, but that current density and channel expression were similar to wild-type cardiac myocytes. Activation of ICa-L caused a significantly greater increase in mitochondrial membrane potential and metabolic activity in αMHC403/+. These increases were attenuated with ICa-L antagonists and following F-actin or β-tubulin depolymerization. The authors observed increased levels of fibroblast growth factor-21 in αMHC403/+mice, and altered mitochondrial DNA copy number consistent with altered mitochondrial activity and the development of cardiomyopathy. These studies suggest that the Arg403Gln mutation leads to altered functional communication between ICa-L and mitochondria that is associated with increased metabolic activity, which may contribute to the development of cardiomyopathy. ICa-L antagonists may be effective in reducing the cardiomyopathy in HCM by altering metabolic activity.