Effects of Dynasore on cellular ATP content in unstressed Hela cells.

<p>Low dose Dynasore does not change cellular ATP content in unstressed Hela cells, whereas high dose of Dynasore (>10 µM) increases ATP content. Cellular ATP content were calculated and compared among the different treatment groups. (*<i>P</i><0.05, **<i>P</i><0.01 when compared to control cells without Dynasore treatment).</p

The cardiac lusitropic effect of Dynasore.

<p>Effect of 1 µM Dynasore pretreatment on ventricular function was studied in Langendorff perfused mouse hearts subjected to 30 min no-flow global ischemia followed by 60 min reperfusion. Hearts were paced at 360 bpm during the whole experimental protocol except the ischemia period and pacing was reinitiated at 2 min into the reperfusion period. A. Representative Left ventricular pressure tracing in a control heart and a Dynasore treated heart. B, C; Left ventricular end diastolic pressure (LVEDP, B) and left ventricular developed pressure (LVDP, C) are summarized and compared between control group and Dynasore group. (* <i>P</i><0.05).</p

Dynasore preserves cellular ATP content in stressed cardiomyocytes.

<p>A. Dynasore preserves cardiomyocyte ATP content. Adult mouse cardiomyocytes were exposed to 30µM H₂O₂ for 35 min in the absence and presence of Dynasore. Cellular ATP content (total ATP normalized to amount of surviving cardiomyocytes) were calculated and compared among the different treatment groups. B. Direct supplement of ATP (3 mM in culture medium) increases cardiomyocyte survival after H₂O₂ exposure. (**<i>P</i><0.01, ***<i>P</i><0.001 when compared to H₂O₂ stressed cardiomyocytes without Dynasore treatment; †<i>P</i><0.05, †††<i>P</i><0.001 when compared to control cardiomyocytes).</p

Dynasore prevents oxidative stress-induced mitochondrial fission.

<p>Cultured human Hela cells were used for mitochondrial morphology study. Top, Hela cells have elongated connected mitochondrial network (left), which was fragmented after oxidative stress (right). Bottom, 1 µM Dynasore pretreatment prevents oxidative stress-induced mitochondrial fragmentation.</p

Dynasore decreases cardiomyocyte death in I/R injured mouse hearts.

<p>A. Myocardial infarct size was analyzed by Propidium Iodide (PI) perfusion. Left, representative fluorescence images of PI staining in a control and Dynasore treated hearts subjected to I/R injury. Right, average infarct size is presented as percentage over total left ventricular area and compared between the two treatment groups. B. Dynasore decreases cardiac troponin I (cTnI) efflux. Myocardial damage was evaluated by measurement of the release of cTnI in the coronary effluent during the 60 min reperfusion period. (* <i>P</i><0.05, ***<i>P</i><0.001).</p

Effect of 1 µM Dynasore pretreatment on ventricular function in Langendorff perfused mouse hearts during ischemia/reperfusion injury.

*<p>, ***indicate <i>P</i><0.05, <i>P</i><0.001 when compared between the two treatment groups.</p><p>LVEDP, left ventricular end diastolic pressure; LVESP, left ventricular end systolic pressure; BL, baseline.</p

Cellular Targets of Dynasore.

<p>Dynasore is a specific small molecular GTPase inhibitor that targets Dynamin1 and Dynamin2 which are responsible for pinching off endocytic vesicles, and Drp1 which is responsible for mitochondrial fission.</p

Microtubule plus-end-tracking proteins target gap junctions directly from the cell interior to adherens junctions.

Gap junctions are intercellular channels that connect the cytoplasms of adjacent cells. For gap junctions to properly control organ formation and electrical synchronization in the heart and the brain, connexin-based hemichannels must be correctly targeted to cell-cell borders. While it is generally accepted that gap junctions form via lateral diffusion of hemichannels following microtubule-mediated delivery to the plasma membrane, we provide evidence for direct targeting of hemichannels to cell-cell junctions through a pathway that is dependent on microtubules; through the adherens-junction proteins N-cadherin and beta-catenin; through the microtubule plus-end-tracking protein (+TIP) EB1; and through its interacting protein p150(Glued). Based on live cell microscopy that includes fluorescence recovery after photobleaching (FRAP), total internal reflection fluorescence (TIRF), deconvolution, and siRNA knockdown, we propose that preferential tethering of microtubule plus ends at the adherens junction promotes delivery of connexin hemichannels directly to the cell-cell border. These findings support an unanticipated mechanism for protein delivery to points of cell-cell contact.</p

The ESCRT-III pathway facilitates cardiomyocyte release of cBIN1-containing microparticles

<div><p>Microparticles (MPs) are cell–cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous <i>Bin1</i> deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.</p></div

Cardiac bridging integrator 1 (cBIN1) is present in human plasma and reduced in heart failure.

<p>(A) Western blot (WB) analysis of protein lysates and immunoprecipitation products from normal human heart lysates and plasma. (B) Mass spectrometry analysis of a cBIN1 protein band from human heart lysate immunoprecipitation (IP). (C) Hypotonic shock induced by double-distilled water increases plasma cBIN1 measured by a cBIN1-specific enzyme-linked immunosorbent assay (ELISA), allowing detection of full blood content. Left: final plasma cBIN1 measured by ELISA following increasing amount of water dilution. Red arrow: Maximal concentration was reached at 25% dilution of plasma (1 volume of plasma with 3 volumes of water). Right: final plasma cBIN1 concentration in 25% plasma diluted in physiological buffered saline (PBS), triton detergent buffer (TB), and double-distilled water. (D) Plasma cBIN1 quantified in healthy control patients and patients with heart failure (HF) (<i>n</i> = 10). *** indicates <i>p</i> < 0.001 using a Student <i>t</i> test. Con., control; IgG, immunoglobulin G; SH3, SRC homology 3.</p