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The mitochondrial network continually undergoes events of fission and fusion. Under physiologic conditions, the network is in equilibrium and is characterized by the presence of both elongated and punctate mitochondria. However, this balanced, homeostatic mitochondrial profile can change morphologic distribution in response to various stressors. Therefore, it is imperative to develop a method that robustly measures mitochondrial morphology with high accuracy. Here, we developed a semi-automated image analysis pipeline for the quantitation of mitochondrial morphology for both in vitro and in vivo applications. The image analysis pipeline was generated and validated utilizing images of primary cortical neurons from transgenic mice, allowing genetic ablation of key components of mitochondrial dynamics. This analysis pipeline was further extended to evaluate mitochondrial morphology in vivo through immunolabeling of brain sections as well as serial block-face scanning electron microscopy. These data demonstrate a highly specific and sensitive method that accurately classifies distinct physiological and pathological mitochondrial morphologies. Furthermore, this workflow employs the use of readily available, free open-source software designed for high throughput image processing, segmentation, and analysis that is customizable to various biological models.

Impaired relaxation is a prevalent form of diastolic dysfunction, present in nearly all cases of heart failure and in many asymptomatic adults. To date, there are no accepted treatments for impaired relaxation, despite its biochemical control by calcium reuptake, thin filament deactivation, and crossbridge kinetics. Mechanical modification of relaxation was previously theorized to occur through afterload; we, however, have recently shown that relaxation was actually modified by the strain rate of myocardial lengthening. We termed this Mechanical Control of Relaxation, or the sensitivity of the relaxation rate to the strain rate. The mechanisms underlying Mechanical Control of Relaxation are unknown, but computational models and our preliminary data suggest a dependence on myosin detachment. The objective of this study was to evaluate whether myosin was a modifying factor of Mechanical Control of Relaxation. Intact cardiac trabeculae and cardiomyocytes were obtained from rats (Sprague Dawley both treated and untreated with propylthiouracil, Spontaneously Hypertensive, and Wistar Kyoto strains) and underwent load-clamp studies. Mechanical Control of Relaxation could be improved by reducing preload (length) 5%, increasing the sensitivity of the relaxation rate to strain rate by 28±16%. Treatment with 400μM Omecamtiv Mecarbil, a myosin-ATPase specific drug, induced similar increases. Myosin isoform differences were also studied. Collagenase treatment of intact trabeculae increased Mechanical Control by 21±3%; intact myocyte studies show no collagen-dependence. These data provide evidence that the properties of myosin’s response to strain rate are major factors that modify Mechanical Control of myocardial Relaxation.

Impaired cardiac relaxation is present in nearly all cases of heart failure and possibly in up to 25% of the asymptomatic population. Myocardial relaxation is known to be biochemically modified by the calcium reuptake rate, thin filament calcium sensitivity, and crossbridge kinetics. Mechanical regulation of relaxation was thought to be regulated via afterload, but we have recently shown that a lengthening strain was sufficient to modify relaxation. Further, the relaxation rate is actually dependent on the strain rate, a relationship that we termed Mechanical Control of Relaxation. Computational modeling suggests that myosin detachment is a key mechanism underlying Mechanical Control of Regulation, but to date, no experimental evidence for this was available. The objective of this study was to determine if myosin head position changed in response to lengthening strains during relaxation. Intact cardiac trabeculae were mounted within the beamline of the Biophysical Collaborative Access Team (BioCAT) beamline at the Advanced Photon Source at Argonne National Laboratories. The trabeculae were paced and load-clamps were performed during time-resolved imaging of the equatorial axis, which primarily reflects myosin head positioning. Activation (pacing) caused the myosin head localization to shift from the thick filament to near the thin filament (increased I 1,1 /I 1,0 ratio). During stretch, there was a transient decline of the I 1,1 /I 1,0 ratio which recovered until relaxation was complete, when the ratio again reduced indicating myosin returned to the thick filament. These preliminary data suggest that Mechanical Control of Relaxation is caused by perturbations in myosin, but the late-diastolic kinetics suggests that the strain-rate dependent detachment does not lead to immediate deactivation of myosin heads. Modifications of myosin ATPase properties may reveal more specific regulatory targets, which may provide new insight and targets for treating impaired myocardial relaxation.

Rapid myocardial relaxation is essential in maintaining cardiac output, and impaired relaxation is an early indicator of diastolic dysfunction. While the biochemical modifiers of relaxation are well known to include calcium handling, thin filament activation, and myosin kinetics, biophysical and biomechanical modifiers can also alter relaxation. We have previously shown that the relaxation rate is increased by an increasing strain rate, not a reduction in afterload. The slope of the relaxation rate to strain rate relationship defines Mechanical Control of Relaxation (MCR). To investigate MCR further, we performed in vitro experiments and computational modeling of preload-adjustment using intact rat cardiac trabeculae. Trabeculae studies are often performed using isometric (fixed-end) muscles at optimal length (Lo, length producing maximal developed force). We determined that reducing muscle length from Lo increased MCR by 20%, meaning that reducing preload could substantially increase the sensitivity of the relaxation rate to the strain rate. We subsequently used computational modeling to predict mechanisms that might underlie this preload-dependence. Computational modeling was not able to fully replicate experimental data, but suggested that thin-filament properties are not sufficient to explain preload-dependence of MCR because the model required the thin-filament to become more activated at reduced preloads. The models suggested that myosin kinetics may underlie the increase in MCR at reduced preload, an effect that can be enhanced by force-dependence. Relaxation can be modified and enhanced by reduced preload. Computational modeling implicates myosin-based targets for treatment of diastolic dysfunction, but further model refinements are needed to fully replicate experimental data.

Background: Heart failure and especially heart failure with preserved ejection fraction are significant burdens on health. Intact cardiac trabeculae may reveal functional changes that cannot be seen in other ex vivo (skinned, single molecule) preparations. For example, using intact trabeculae, we have shown that relaxation is mechanically controlled by the lengthening strain rate at end systole, not afterload. Objective: We sought to evaluate the effect of myosin activator Omecamtiv Mercarbil (OM) and inhibitor Mavacamten (Mav) on physiologic function in intact trabeculae. Methods/Results: Afterload-clamp protocols were applied to intact cardiac trabeculae from Sprague Dawley rats to simulate physiologic work-loops and evaluate mechanical control of relaxation. Both OM and Mav reduced stroke work (force x length) by >50% and power (force x velocity) by ~50% at doses reducing developed force by 50%. These were mediated by dose-dependent reductions in both force and shortening length. We have recently reported preliminary results that OM improves contraction-relaxation coupling and makes the relaxation rate more sensitive to strain rate in a dose-dependent manner. Mav does not lead to significant changes in contraction-relaxation coupling at any dose; Mav alters the sensitivity of relaxation rate to strain rate only at doses that reduce developed force by 50%. Summary/Perspective: Intact rat cardiac trabeculae reveal function mimicking physiology. OM and Mav show remarkable similarities in force, work, and power, which may be due to the high expression of alpha-myosin heavy chain. OM treatment not only modified contractility but increased sensitivity of the relaxation rate to strain rate in a dose dependent manner, which may explain why diastolic dysfunction is not more prevalent in clinical studies. Mav appears to only modify the attachment of crossbridges as expected.

Global brain ischemia/reperfusion induces neuronal damage in vulnerable brain regions, leading to mitochondrial dysfunction and subsequent neuronal death. Induction of neuronal death is mediated by release of cytochrome c (cyt c) from the mitochondria though a well-characterized increase in outer mitochondrial membrane permeability. However, for cyt c to be released it is first necessary for cyt c to be liberated from the cristae junctions which are gated by Opa1 oligomers. Opa1 has two known functions: maintenance of the cristae junction and mitochondrial fusion. These roles suggest that Opa1 could play a central role in both controlling cyt c release and mitochondrial fusion/fission processes during ischemia/reperfusion. To investigate this concept, we first utilized in vitro real-time imaging to visualize dynamic changes in mitochondria. Oxygen-glucose deprivation (OGD) of neurons grown in culture induced a dual-phase mitochondrial fragmentation profile: (i) fragmentation during OGD with no apoptosis activation, followed by fusion of mitochondrial networks after reoxygenation and a (ii) subsequent extensive fragmentation and apoptosis activation that preceded cell death. We next evaluated changes in mitochondrial dynamic state during reperfusion in a rat model of global brain ischemia. Evaluation of mitochondrial morphology with confocal and electron microscopy revealed a similar induction of fragmentation following global brain ischemia. Mitochondrial fragmentation aligned temporally with specific apoptotic events, including cyt c release, caspase 3/7 activation, and interestingly, release of the fusion protein Opa1. Moreover, we uncovered evidence of loss of Opa1 complexes during the progression of reperfusion, and electron microscopy micrographs revealed a loss of cristae architecture following global brain ischemia. These data provide novel evidence implicating a temporal connection between Opa1 alterations and dysfunctional mitochondrial dynamics following global brain ischemia.

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The interaction of light with biological tissue has been successfully utilized for multiple therapeutic purposes. Previous studies have suggested that near infrared light (NIR) enhances the activity of mitochondria by increasing cytochrome c oxidase (COX) activity, which we confirmed for 810 nm NIR. In contrast, scanning the NIR spectrum between 700 nm and 1000 nm revealed two NIR wavelengths (750 nm and 950 nm) that reduced the activity of isolated COX. COX-inhibitory wavelengths reduced mitochondrial respiration, reduced the mitochondrial membrane potential (ΔΨm), attenuated mitochondrial superoxide production, and attenuated neuronal death following oxygen glucose deprivation, whereas NIR that activates COX provided no benefit. We evaluated COX-inhibitory NIR as a potential therapy for cerebral reperfusion injury using a rat model of global brain ischemia. Untreated animals demonstrated an 86% loss of neurons in the CA1 hippocampus post-reperfusion whereas inhibitory NIR groups were robustly protected, with neuronal loss ranging from 11% to 35%. Moreover, neurologic function, assessed by radial arm maze performance, was preserved at control levels in rats treated with a combination of both COX-inhibitory NIR wavelengths. Taken together, our data suggest that COX-inhibitory NIR may be a viable non-pharmacologic and noninvasive therapy for the treatment of cerebral reperfusion injury.

Background: Global brain ischemia/reperfusion induces neuronal damage in vulnerable brain regions. A key event leading to neuronal death is mitochondrial cytochrome c (cyt c) release which we have demonstrated is accompanied by release of mitochondrial Opa1, a key mitochondrial fusion protein. We propose that cyt c release is dependent on disruption of Opa1, thus inadvertently promoting fission. Methods: Mitochondrial dynamics in vitro were analyzed with real-time imaging of neurons transfected with mito-localized GFP and MR-DEVD to detect caspase activation. Ischemia (OGD) was achieved with a media-gas exchange system and confocal microscopy. Mitochondrial dynamics in vivo was assessed with (i) 3D-electron microscopy to quantify mitochondrial volume, shape, and length and (ii) immunofluorescence for ATP synthase and Opa1. Results: Real-time imaging of cells during OGD (A, n=3/group) demonstrated that mitochondria transition from a tubular morphology to a highly fragmented morphology during ischemia. Tubular morphology was rapidly regained in the initial stages of reoxygenation, suggesting fusion remains intact following ischemia. However, mitochondria progressively undergo fragmentation culminating in cell death. Next, we utilized a rat model of cerebral ischemia to evaluate mitochondrial dynamics in vivo. Immunofluorescence (B, n=5/group) and 3D TEM (C, n=3/group) of control brains both exhibit mitochondria with long tubular morphology and Opa1 fluorescence in CA1 neurons was consistent with mitochondrial localization. In contrast, ischemia followed by 6 or 24 hrs of reperfusion resulted in extensive mitochondrial fragmentation (i.e. reduced mitochondrial length Conclusion: These data provide novel evidence that Opa1 may play a role in cell death during brain reperfusion.

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ∼50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulin-mediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.