Browsing by Author "Sturek, Michael S."

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    ADF/Cofilin Activation Regulates Actin Polymerization and Tension Development in Canine Tracheal Smooth Muscle
    (2009-09-03T15:28:09Z) Zhao, Rong; Gunst, Susan J.; Atkinson, Simon J.; Elmendorf, Jeffrey S.; Sturek, Michael S.
    The contractile activation of airway smooth muscle tissues stimulates actin polymerization and the inhibition of actin polymerization inhibits tension development. Actin depolymerizing factor (ADF) and cofilin are members of a family of actin–binding proteins that mediate the severing of F–actin when activated by dephosphorylation at serine 3. The role of ADF/cofilin activation in the regulation of actin dynamics and tension development during the contractile activation of airway smooth was evaluated in intact canine tracheal smooth muscle tissues. Two–dimensional gel electrophoresis revealed that ADF and cofilin exist in similar proportions in the muscle tissues and that approximately 40% of the total ADF/cofilin in unstimulated tissues is phosphorylated (inactivated). Phospho–ADF/cofilin decreased concurrently with tension development in response to stimulation with acetylcholine (ACh) or potassium depolarization indicating the activation of ADF/cofilin. Expression of an inactive phospho–cofilin mimetic (cofilin S3E), but not WT cofilin in the smooth muscle tissues inhibited endogenous ADF/cofilin dephosphorylation and ACh–induced actin polymerization. Expression of cofilin S3E in the tissues depressed tension development in response to ACh, but it did not affect myosin light chain phosphorylation. The ACh–induced dephosphorylation of ADF/cofilin required the Ca2+–dependent activation of calcineurin (PP2B). Expression of Slingshot (SSH) inactive phosphatase (C393S) decreased force development and cofilin dephosphorylation. Activation of ADF/cofilin was also required for the relaxation of tracheal muscle tissues induced by forskolin and isoproterenol. Cofilin activation in response to forskolin was not Ca2+–dependent and was not inhibited by calcineurin inhibitors, suggesting it was regulated by a different mechanism. Cofilin activation is required for actin dynamics and tension development in response to the contractile stimulation of tracheal smooth muscle and is regulated by both contractile and relaxing stimuli. These concepts are critical to understanding the mechanisms of smooth muscle contraction and relaxation, which may provide novel targets for therapeutic intervention in the treatment of abnormal airway responsiveness.
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    Coronary artery disease in metabolic syndrome: a role for the sarcoplasmic reticulum Ca2+ ATPase
    (2016-05-10) Rodenbeck, Stacey Dineen; Sturek, Michael S.; Day, Richard N.; Evans-Molina, Carmella; Mather, Kieren; Tune, Johnathan D.
    Coronary artery disease (CAD) is a leading cause of death among Americans and is fueled by underlying metabolic syndrome (MetS). The prevalence and lethality of CAD necessitates rigorous investigations into its underlying mechanisms and to facilitate the development of effective treatment options. Coronary smooth muscle (CSM) phenotypic modulation from quiescent to synthetic, proliferative, and osteogenic phenotypes is a key area of investigation, with underlying mechanisms that remain poorly understood. Using a well-established pre-clinical model of CAD and MetS, the Ossabaw miniature swine, we established for the first time the time course of Ca2+ dysregulation during MetS-induced CAD progression. In particular, we used the fluorescent Ca2+ dye, fura-2, to examine alterations in CSM intracellular Ca2+ regulation during CAD progression, as perturbations in intracellular Ca2+ regulation are implicated in several cellular processes associated with CAD pathology, including CSM contractile responses and proliferative pathways. These studies revealed that the function of several CSM Ca2+ handling proteins is elevated in early CAD, followed by loss of function in severe atherosclerotic plaques. Decreased intracellular Ca2+ regulation occurred concurrently with reductions in CSM proliferation, measured with Ki-67 staining. In particular, alterations in sarcoplasmic reticulum (SR) Ca2+ store together with altered function of the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) were associated with induction of proliferation. Organ culture of coronary arterial segments revealed that culture-induced medial thickening was prevented by SERCA inhibition with cyclopiazonic acid (CPA). Activation of SERCA with the small molecule activator, CDN1163, increased CSM proliferation, which was attenuated by treatment with CPA, thus establishing upregulated SERCA function as a proximal inducer of CSM proliferation. Further, we demonstrated that in vitro treatment of CSM from lean Ossabaw swine with the glucagon-like peptide-1 (GLP-1) receptor agonist, exenatide, increased SERCA function. However, in vivo treatment of Ossabaw swine with MetS with the GLP-1 receptor agonist, AC3174, had no effect on CAD progression and in vitro examination revealed resistance of SERCA to GLP-1 receptor agonism in MetS. These findings further implicate SERCA in CAD progression. Collectively, these are the first data directly linking SERCA dysfunction to CSM proliferation and CAD progression, providing a key mechanistic step in CAD progression.
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    Coronary Smooth Muscle Cell Cytodifferentiation and Intracellular Ca2+ Handling in Coronary Artery Disease
    (2019-08) Badin, Jill Kimberly; Sturek, Michael S.; Evans-Molina, Carmella; Moe, Sharon; Tune, Jonathan D.
    Metabolic syndrome (MetS) affects 1/3 of all Americans and is the clustering of three or more of the following cardiometabolic risk factors: obesity, hypertension, dyslipidemia, glucose intolerance, and insulin resistance. MetS drastically increases the incidence of coronary artery disease (CAD), which is the leading cause of mortality globally. A cornerstone of CAD is arterial remodeling associated with coronary smooth muscle (CSM) cytodifferentiation from a contractile phenotype to proliferative and osteogenic phenotypes. This cytodifferentiation is tightly coupled to changes in intracellular Ca2+ handling that regulate several key cellular functions, including contraction, transcription, proliferation, and migration. Our group has recently elucidated the time course of Ca2+ dysregulation during MetS-induced CAD development. Ca2+ transport mechanisms, including voltage-gated calcium channels, sarcoplasmic reticulum (SR) Ca2+ store, and sarco-endoplasmic reticulum Ca2+ ATPase (SERCA), are enhanced in early, mild disease and diminished in late, severe disease in the Ossabaw miniature swine. Using this well-characterized large animal model, I tested the hypothesis that this Ca2+ dysregulation pattern occurs in multiple etiologies of CAD, including diabetes and aging. The fluorescent intracellular Ca2+ ([Ca2+]i) indicator fura-2 was utilized to measure [Ca2+]i handling in CSM from lean and diseased swine. I found that [Ca2+]i handling is enhanced in mild disease with minimal CSM phenotypic switching and diminished in severe disease with greater phenotypic switching, regardless of CAD etiology. We are confident of the translatability of this research, as the Ca2+ influx, SR Ca2+ store, and SERCA functional changes in CSM of humans with CAD are similar to those found in Ossabaw swine with MetS. Single-cell RNA sequencing revealed that CSM cells from an organ culture model of CAD exhibited many different phenotypes, indicating that phenotypic modulation is not a discreet event, but a continuum. Transcriptomic analysis revealed differential expression of many genes that are involved in the osteogenic signaling pathway and in cellular inflammatory responses across phenotypes. These genes may be another regulatory mechanism common to the different CAD etiologies. This study is the first to show that CSM Ca2+ dysregulation is common among different CAD etiologies in a clinically relevant animal model.
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    IUPUI Imaging Research Council
    (Office of the Vice Chancellor for Research, 2012-04-13) Hutchins, Gary D.; Wilson, Kathryn J.; Sturek, Michael S.; Du, Eliza Y.; Fletcher, James W.; Long, Eric C.; Molitoris, Bruce A.; Johnson, Daniel P.; Day, Richard N.; Barnett, William K.; Palakal, Mathew J.
    Abstract The IUPUI Imaging Research Council was created by the IUPUI Vice Chancellor for Research to provide guidance and direction for expansion of research imaging initiatives across all Schools and Departments within IUPUI. The specific goals of the council are: • To encourage and coordinate collaboration among IUPUI researchers from different disciplines • To provide advice and guidance in the realization of highly competitive large grant proposals that will support and grow the IUPUI imaging efforts into major nationally and internationally recognized programs • To develop a strategic plan that will enable IUPUI to become nationally and internationally known as the place for imaging research and its applications • To determine strategic areas of strength and growth • To determine available and needed resources • To determine strategic external partnerships Activities organized by the council to date include sponsoring an IUPUI Imaging Research Workshop on November 17, 2011. This workshop consisted of invited presentations, a poster session, and working group breakout sessions. Working groups explored research opportunities and needs in four priority areas (neuroscience, cancer, cardiovascular disease, and remote sensing). The council has recently initiated a monthly seminar series and is actively developing an IUPUI research imaging strategic plan. For more information visit the IUPUI Imaging Research Initiative website at www.imaging.iupui.edu.
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    Membrane cholesterol balance in exercise and insulin resistance
    (2009-10) Habegger, Kirk M.; Elmendorf, Jeffrey S.; Roach, Peter J.; Brozinick, Joseph T.; Sturek, Michael S.; Considine, Robert V.
    Study has shown that plasma membrane (PM) cholesterol and cortical filamentous actin (F-actin) influence skeletal muscle glucose transport. Of fundamental and clinical interest is whether diabetogenic insults promote membrane/cytoskeletal dysfunction amendable for therapy. As exposure to excess fatty acid (FA)s induce glucose intolerance by mechanisms imperfectly understood, we tested if PM cholesterol/F-actin changes could contribute to FA-induced glucose transporter GLUT4 dysregulation in skeletal muscle. High-fat fed, insulin-resistant animals displayed elevated levels of skeletal muscle PM cholesterol and a loss in cortical F-actin, compared to normal-chow fed animals. Consistent with a PM cholesterol component of glucose intolerance, human skeletal muscle biopsies revealed an inverse correlation between PM cholesterol and whole-body glucose disposal. Mechanistically, exposure of L6 myotubes to the saturated FA palmitate induced an increase in PM cholesterol that destabilized actin filaments and decreased insulin-stimulated PM GLUT4 and glucose transport, which could be reversed with cholesterol lowering. Next, study tested if the lipid-lowering action of the antidiabetic AMP-activated protein kinase (AMPK) had a beneficial influence on PM cholesterol balance. Consistent with AMPK inhibition of 3-hydroxy-3-methylglutaryl CoA reductase, a rate-limiting enzyme of cholesterol synthesis, we found that AMPK activation promoted a significant reduction in PM cholesterol and amplified basal and insulin-stimulated GLUT4 translocation. A similar loss of PM cholesterol induced by β-cyclodextrin caused an analogous enhancement of GLUT4 regulation. Interestingly, PM cholesterol replenishment abrogated the AMPK effect on insulin, but not basal, regulation of GLUT4 translocation. Conversely, AMPK knockdown prevented the enhancement of both basal and insulin-stimulated GLUT4 translocation. As a whole these studies show PM cholesterol accrual and cortical F-actin loss uniformly in skeletal muscle from glucose-intolerant mice, swine, and humans. In vivo and in vitro dissection demonstrated this membrane/cytoskeletal derangement induces insulin resistance and is promoted by excess FAs. Parallel studies unveiled that the action of AMPK entailed lowering PM cholesterol that enhanced the regulation of GLUT4/glucose transport by insulin. In conclusion, these data are consistent with PM cholesterol regulation being an unappreciated aspect of AMPK signaling that benefits insulin-stimulated GLUT4 translocation during states of nutrient excess promoting PM cholesterol accrual.
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    A portable fiberoptic ratiometric fluorescence analyzer provides rapid point-of-care determination of glomerular filtration rate in large animals
    (Nature Publishing group, 2012-01) Wang, Exing; Meier, Daniel J.; Sandoval, Ruben M.; Von Hendy-Willson, Vanessa E.; Pressler, Barrak M.; Bunch, Robert M.; Alloosh, Mouhamad; Sturek, Michael S.; Schwartz, George J.; Molitoris, Bruce A.; Medicine, School of Medicine
    Measurement of the glomerular filtration rate (GFR) is the gold standard for precise assessment of kidney function. A rapid, point-of-care determination of the GFR may provide advantages in the clinical setting over currently available assays. Here we demonstrate a proof of principle for such an approach in a pig and dogs, two species that approximate the vascular access and GFR results expected in humans. In both animal models, a sub-millimeter optical fiber that delivered excitation light and collected fluorescent emissions was inserted into a peripheral vein (dog) or central venous access (pig) by means of commercial intravenous catheters. A mixture of fluorescent chimeras of a small freely filterable reporter and large non-filterable plasma volume marker were infused as a bolus, excited by light-emitting diodes, and the in vivo signals detected and quantified by photomultiplier tubes in both species in less than 60 min. Concurrent standardized 6-h iohexol plasma kidney clearances validated the accuracy of our results for both physiologic and a chronic kidney disease setting. Thus, our ratiometric technique allows for both measurement of plasma vascular volume and highly accurate real-time GFR determinations, enabling clinical decision making in real time.
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    A retrospective descriptive study of pain scores in the pre-diabetic patients on metformin
    (2015-07-01) Moore, Michele Nakamura; Mushi, Christina R.; Ang, Dennis C.; Mac Kinnon, Joyce L.; Sturek, Michael S.; Arnold, Brent L.
    Objectives: The purpose was to evaluate pain scores (SF-36 BPS) among pre-diabetic patients on metformin or placebo to determine if patients on metformin therapy report less pain (higher SF-36 BPS) than patients on placebo. Study design: A descriptive retrospective review of pain scores was conducted using secondary data analyses of the Diabetes Prevention Program (DPP) and Diabetes Prevention Program Outcomes Study (DPPOS) conducted from 1996 to 2008. Patients were randomly assigned to placebo, low (850 mg/day) or high dose (1700 mg/day) metformin groups. Pain scores using the SF-36 BPS standard version were taken before randomization and annually (year one through four). Results: Out of 3,819 patients that participated in the original study, 1,056 patients met the current study criteria. The metformin group included 506 patients and the placebo group included 550 patients. With an alpha level of 0.05 for all analyses, baseline pain scores between the metformin group and placebo group showed no significant difference. Year two showed significance between placebo and metformin pain scores (75.2 vs 78.6). All other years were not significant. Comparing low and high dose metformin and placebo groups, years one, two and three displayed significant differences in pain scores. In years one and two, the high dose metformin group reported less pain than the placebo group (80.7 vs 77.7; 80.1 vs 75.2) and the low dose metformin group (80.7 vs 71.8; 80.1 vs 68.6). In year three, the high dose metformin group had less pain than the low dose metformin group (78.4 vs 70.5).