Thesis (M.S.)--Brock University, 2009. / Includes bibliographical references (leaves 96-103).

Thesis (Ph. D.)--University of North Carolina at Greensboro, 2005. / Title from PDF title page screen. Includes bibliographical references (p. 88-100)

Thesis (Ph. D.)--University of Wisconsin--Madison, 1970. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Theses (M.S.)--Marshall University, 2007. / Title from document title page. Includes abstract. Document formatted into pages: contains vii, 96 p. Includes vitae. Bibliography: p. 87-92.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "August 2006" Includes bibliographical references.

Abstract Adrenomedullin (AM) is a 52-amino acid peptide which is produced in many tissues, including adrenal medulla, lung, kidney and heart. Intravenous administration of AM causes a long-lasting hypotensive effect, accompanied with an increase in the cardiac output in experimental animals. This study was aimed to examine whether AM has any direct effects on myocardial function. In addition to the myocardial contractility, the effects of AM on coronary vascular tone and A-type natriuretic peptide (ANP) release from atria and B-type natriuretic peptide (BNP) gene expression in the ventricles were studied in the perfused rat heart preparation. In spontaneously beating hearts, AM had no effects on the heart rate, but dose-dependently increased the developed tension (DT) with an EC50 of 7 x 10-11 nmol/l, reflecting a potent positive inotropic effect. The lower the initial resting tension, the higher was the elevation in DT. In paced hearts, a protein kinase A inhibitor, H-89, had no effect on AM-induced inotropic effect, and AM did not increase the cAMP content of the ventricular myocardium. In contrast, the inhibitors of sarcoplasmic reticulum Ca2+ stores, ryanodine and thapsigargin, as well as a protein kinase C inhibitor, staurosporine, significantly attenuated the inotropic response to AM. L-type Ca2+ channel blocker, diltiazem, also suppressed the AM-induced elevation in DT. Moreover, AM increased the duration of myocyte action potentials between 10 mV and - 50 mV in isolated rat atria, consistent with an increase in L-type Ca2+ channel current during the plateau. Inotropic effect of endothelin-1 (ET-1), another locally acting peptide, was enhanced by inhibiting the myocardial nitric oxide (NO) synthesis by Nω-nitro-L-arginine methyl ester (L-NAME) in perfused rat heart. The AM-induced inotropic action was unaltered by L-NAME treatment. When AM and ET-1 were administrated in combined infusion, the inotropic response was significantly smaller than that following the infusion of the peptides alone. This attenuated response was more than overcome by infusion of L-NAME, although the individual responses to AM and ET-1 were not modulated by L-NAME at the doses used in the combination. Consistent with its vasodilator action, AM dose-dependently dilated the coronary arteries of the perfused heart. The effect of AM was not dependent on NO under basal conditions or in coronary arteries constricted with ET-1. Furthermore, AM enhanced the stretch-induced release of ANP from the right atrium, but did not affect the ventricular BNP expression induced by ET-1. In conclusion, AM exerts regulatory actions on the heart by increasing cardiac contractility, dilating coronary arteries and modulating stretch-induced ANP release. The inotropic effect of AM was independent of cyclic AMP, but may involve activation of protein kinase C, Ca2+ influx through L-type Ca2+ channels and the release of Ca2+ from the sarcoplasmic reticulum. Endogenous NO production did not modulate the inotropic effect of AM, although the effect of ET-1 was suppressed. Combined administration of AM and ET-1 produced a weak inotropic response most likely because of a potentiated synthesis of NO. Finally, AM had a coronary vasodilator effect and augmented the stretch-induced ANP release in the right atrium.

Abstract Stretch of the cardiac muscle activates several physiological processes leading to changes in the function of the muscle. These changes include increase of the contraction force accompanied by changes in the intracellular calcium concentration. This phenomenon is known as Frank-Starling relation of the heart. In addition to this, stretch also influences the membrane voltage of individual myocytes predisposing the cardiac muscle to arrhythmias. In atrial muscle stretch augments the secretion of the atrial natriuretic peptide (ANP). Although several cellular components are known to be sensitive to mechanical stimulus the precise mechanisms participating to these stretch-induced changes are not known in detail. Further it is not known if these changes are causally related or if they share a common causal factor. This research was aimed to study the stretch-induced changes in the rat atrium. The possible interactive mechanisms were studied by recording intracellular action potentials, changes in the intracellular calcium concentration, contraction force and ANP secretion during stretch. The plausible mechanosensitive cellular components were incorporated into a mathematical model that was used to further study the mechanisms. The role of intracellular acidosis as a possible modulator of the mechanotransduction was of special interest. In isolated rat left atrium moderate stretch produced by increasing the intra-atrial pressure increased the contraction force in a biphasic manner. The immediate increase of the force was caused by altered properties of the contractile element, but the following slow increase was accompanied by an increase of the Ca2+ transient. These changes were followed by lengthening of the late phase of action potentials and augmented secretion of the ANP. Intensive sustained stretch was also found to induce delayed afterdepolarizations (DADs). Gadolinium (Gd3+), blocker of stretch-activated ion channels reduced the stretch-dependent activation of the contraction and inhibited the stretch-induced DADs. The mathematical model simulated the experimental findings at best when stretch-activated channel (SA-channel) activation and increased troponin-C affinity were used to mimic the stretch. The modelling data suggested that the SA-channel current increases the sarcoplasmic reticulum calcium content in a time dependent manner leading to Ca2+ transient augmentation during systole. Bigger Ca2+ transients induce a depolarizing current during the late phase of the action potential (AP) repolarization via the Na+/Ca2+ exchanger causing the lengthening of the action potentials. A small reduction of the intracellular pH (0.18 units) with 20 mM propionate was found to modulate the stretch-induced changes in the rat atrium. Acidosis leads to an increase in the diastolic [Ca2+]i during stretch, inhibits the stretch-induced changes in action potentials and slows down the contraction development during stretch by inhibiting the fast component of the force increase. These changes in E-C-coupling (excitation-contraction-coupling) were accompanied by a simultaneous augmentation of the ANP secretion. Furthermore, it was shown that contraction force and diastolic [Ca2+]i of the stretched tissue are more sensitive to acidosis than in non-stretched tissue. In conclusion, the stretch-induced changes in rat atrial myocytes are mediated by at least two mechanisms; stretch-activated Ca2+ influx and change in the properties of the contractile element. The action potential changes can be largely explained by modulation of the membrane voltage by intracellular calcium via Na+/Ca2+-exchanger. The co-occurrence of the changes in the [Ca2+]i and ANP secretion suggests that the stretch-induced ANP secretion can be mediated by [Ca2+]i.

There are two opposing schools of thought concerning the source of calcium ions for the initiation and maintenance of contractions of smooth muscle. Bohr (1964) and Woodward et al. (1970) believe that the calcium for the initiation of contraction is released from bound intracellular stores, whereas Somlyo et al. (1969) believe that most of the activator calcium comes from the extracellular fluid. It was felt that the determination of the source(s) and sink(s) of calcium ions in arteriolar smooth muscle would be required for an understanding of the control of peripheral blood pressure, and so experiments to obtain this information were carried out using the rabbit anterior mesenteric portal vein as a model of arteriolar smooth muscle. Spontaneous contractions of the vein stop within one minute after the addition of EGTA to the bath and can be returned by simply raising the extracellular calcium concentration. The addition of 1 mM MnC1₂, or raising the MgC1₂ concentration above 5 mM, or raising the CaC1₂ concentration above 10 mM all inhibit spontaneous activity; this inhibition can be rapidly reversed by the addition of appropriate amounts of EGTA. The removal of free extracellular calcium either by adding EGTA or placing the vein in calcium-free solution inhibits the responses to all agonists within five minutes. If the calcium concentration is reduced from 2.5 mM to 0.1 mM, the responses to agonists are greatly decreased. If one sets the response in normal Kreb's solution to each concentration of agonist equal to 100 per cent, then the relative reduction of responses in low calcium solution is inversely proportional to both the potency and concentration of the agonist used. If, however, one produces contractions by adding calcium to tissues bathed in calcium-free solution containing noradrenaline, then the curves of the relative response versus calcium concentrations are independent of the concentration of noradrenaline. The addition of either EGTA or manganese to a tissue already contracted in response to any agonist produces a rapid relaxation to a decreased, but sustained tension. The degree of relaxation is proportional to the concentration of manganese or EGTA added. The addition of manganese is also able to inhibit the initiation of responses to noradrenaline, KC1, serotonin, histamine and procaine. The inhibition by manganese of the responses to noradrenaline, KC1, and serotonin but not histamine and procaine can be reversed by increasing the extracellular calcium concentration. The addition of MnC1₂ or LaCl₃ does not selectively inhibit a slow phase of the contraction to noradrenaline in the mesenteric portal vein as it does in aorta (Van Breeman, 1969). The effect of adding MnCl₂ is similar to the effect of decreasing extracellular calcium, in that the relative inhibition of response is inversely proportional to the potency and concentration of agonist used. The effect of altering pH is the same on contractions produced by each agonist tested; lowering the pH below 7.4 inhibits the responses, raising it above pH 7.4 potentiates the responses. If the vein is placed in calcium-free solution containing EGTA for 10 minutes, the addition of CaC1₂ produces a contraction. The response to calcium is transient unless the final concentration of the added calcium is 5 mM or greater; at these higher calcium concentrations the response is biphasic; an initial transient response is followed by a slow tonic response. The addition of stock solutions to produce final concentrations of 5 mM Mg⁺⁺ or 0.5 mM Mn⁺⁺ or 0.1 mM Ca⁺⁺ in the bath after the EGTA treatment abolishes the transient responses to calcium but has little effect on the tonic portion of a contraction produced by 10 mM Ca⁺⁺. When these EGTA experiments are carried out in low sodium solution (17% of normal or less) the response to 2.5 mM Ca⁺⁺ which is normally transient, becomes instead a sustained contraction. It is concluded then that: 1. Calcium ions themselves are able to control the permeability of the membrane to calcium. 2. The initiation of responses to all agonists probably involves the release of membrane-bound calcium and the influx of extracellular calcium. Differences in efficacy are probably due to differences in ability to release the membrane-bound calcium. 3. A continued influx of calcium is required to maintain a contraction produced by any agonist. 4. Manganese competes with calcium at a membrane site to inhibit the initiation of contraction, and to relax a tissue which is already contracted. 5. Responses to all agonists are potentiated at a pH greater than 7.4, and decreased below pH 7.4. 6. The relaxation process in the mesenteric-portal vein seems to depend in some manner upon extracellular sodium ions. / Medicine, Faculty of / Anesthesiology, Pharmacology and Therapeutics, Department of / Graduate

This dissertation aimed to explore the S2 region with an attempt to modulate its elasticity in order to tune the contraction output. Two peptides, the stabilizer and destabilizer, showed high potential in modifying the S2 region at the cellular level, thus they were prepared for animal model testing. In this research, (i) S2 elasticity was studied, and the stabilizer and destabilizer peptides were built to tune contraction output through modulating S2 flexibility; (ii) the peptides were attached to heart homing adducts and the bond between them was confirmed; and (iii) it was shown that minor changes were imposed on the modulating peptides' functionality upon attaching to the heart homing adducts. S2 flexibility was confirmed through comparing it to other parts of myosin using simulated force spectroscopy. Modulatory peptides were built and computationally tested for their efficacy through interaction energy measurement, simulated force spectroscopy and molecular dynamics; these were attached to heart homing adducts for heart delivery. Interaction energy tests determined that tannic acid (TA) served well for this purpose. The stoichiometry of the bond between the TA and the modulating peptides was confirmed using mass spectroscopy. The functionality of the modulating peptides was shown to be unaltered through expansion microscopy where they located to the same position on the sarcomere with and without TA. They were also shown to cause the sarcomeres to contract similarly with and without the TA in contractility assay. Taken together, this work prepared the modulating peptides for animal model tests by attaching them to tannic acid.

No description available.