Cardiovascular disease is one of the main causes of mortality and one of the significant burdens to society. Major cardiovascular diseases such as acute myocardial infarction (heart attack), heart failure and cardiac arrhythmia often result in the development of ischemia/reperfusion (I/R) injury. Untreated I/R injury is known to cause cardiac contractile dysfunction. It is established that matrix metalloproteinase-2 (MMP-2) is activated and degrades contractile proteins during I/R, and many other factors including metabolic enzymes, kinases and structural proteins are affected by I/R. However, the molecular mechanisms responsible for these changes are unclear. Since MMP-2 is known to its broad spectrum of action, I hypothesize that, in addition to contractile proteins, proteins related to regulation of energy metabolism are MMP-2 targets during I/R, and protein kinase such as myosin light chain kinase (MLCK) is also involved in this process. The use of proteomics in studying heart injury triggered by I/R will reveal new potential targets for pharmacological protection of heart from I/R induced contractile dysfunction. In addition, selective inhibition of MMP-2 using MMP-2 siRNA protects the heart from I/R injury. In this study, we investigated the protein modulation during I/R using proteomic approach. In order to study the effect of protein kinases (MLCK) and MMP-2, their selective inhibitors were used to inhibit those factors and evaluate the changes in energy metabolic proteins during I/R. Proteomic analysis revealed that six proteins are involved in energy metabolism: ATP synthase β subunit, cytochrome b-c1 complex subunit 1, 24-kDa mitochondrial NADH dehydrogenase, NADH dehydrogenase [ubiquinone] iron-sulfur protein 8, cytochrome c oxidase subunit, and succinyl-CoA ligase subunit, resulting in decreased levels in I/R hearts. The data suggests that energy metabolic proteins, especially the metabolic enzymes involved in the electron transport chain in the mitochondria may contribute to I/R injury. In addition, our data provides evidence that the right and left ventricles of the heart respond differently to I/R injury, in terms of the regulation of contractile proteins and energy metabolic enzymes. Studies using MLCK inhibitor, ML-7, and MMP-2 inhibitor, MMP-2 siRNA to investigate the effect of myosin light chain kinase (MLCK) and MMP-2 in energy metabolic proteins have shown that succinyl-CoA ligase and ATP synthase are affected by MLCK and MMP-2 respectively. These results demonstrate that the effect of inhibition of the MLCK and MMP-2 involves optimization of energy metabolism in I/R injury, likely resulting in increased energy production. Hence, the observed proteins increase in cardiac recovery after I/R. Also, inhibition of MLCK and MMP-2 by ML-7 and MMP-2 respectively shows cardio protective effect during I/R. In summary, this study provides a novel pathogenesis in the development of I/R-induced cardiac contractile dysfunction. Moreover, we suggest a new therapeutic approach whereby using MMP-2 siRNA can be a promising gene therapy in the development of new preventive or treatment strategies against I/R injury.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Also available in print.

Thesis (M. Phil.)--University of Hong Kong, 1999. / Includes bibliographical references.

Thesis (Ph. D.)--Cornell University, May, 2008. / Vita. Includes bibliographical references (leaves 104-122).

Abnormal homeostasis of intracellular Ca2+ plays a deleterious role in muscle pathologies by triggering processes that lead to dysfunction and necrotic or apoptotic cell death. One pathology where there is significant Ca2+ induced cell damage is ischemia, which initiates further damage (also mediated by Ca2+) generated by the required treatment process of revascularization; namely ischemia-reperfusion injury. Pharmacological agents used therapeutically for cell protection, especially for cardiac protection in ischemic heart diseases, have only directly targeted one of the elements regulating Ca2+ homeostasis, the L-type Ca2+ channels (calcium channel blockers). Other agents, like beta blockers, indirectly target various elements, including sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA) and ryanodine receptors (RyRs). However, there are no pharmacological agents that directly and specifically target these two crucial elements required for intracellular SR Ca2+ homeostasis. Dr. Julio A. Copello’s group has previously studied the cardioprotective agent CGP-37157 (CGP), a benzothiazepine (BZT) derivative of the benzodiazepine (BZD) clonazepam. CGP was previously thought to decrease intracellular SR Ca2+ by acting as a blocker of the mitochondrial Na+/Ca2+ exchanger (Omelchenko et al., 2003). They found, however, that CGP also activates RyRs and inhibits the SERCA, which could better explain the SR effects of the drug (Neumann et al., 2011). These results suggest that drugs inducing partial depletion of SR Ca2+ stores could provide cellular protection in stressful circumstances or processes. The aims of the dissertation were organized based on the two processes that cause damage to muscle cells during ischemia: ischemia and subsequent reperfusion (ischemia-reperfusion injury) (Ibanez et al., 2015). Aim one and two focused on drug-protective action during the ischemic event, while aim three focused on drug protective action in the reperfusion (early post-ischemia) process. In the first Aim, experiments were designed to test the hypotheses that RyRs and/or SERCA could also be the target of i) Drugs with structural similarities to CGP (i.e., other BZTs and some BZDs) and ii) Drug known to confer cellular protection under stressful cellular conditions such as antiepileptic agents. We found that some BZTs (K201, CGP analog) and antiepileptic agents (Sipatrigine and Pimozide) demonstrated potential to prevent SR Ca2+ overload by inhibition of SERCA and, in some cases also by inducing mild activation of RyR channels. These results provided potential mechanisms of action for agents with cell protective action: targeting SERCA and preventing Ca2+ overload in pre-ischemia process. From the results of the first aim, K201 had the most significant effects in both SERCA inhibition and RyRs activation. Therefore, Aim 2 experiments focused on exploring with greater detail the action of the compound K201 on RyRs, SERCA and Ca2+ signaling. We found that K201 is a more potent SERCA blocker than RyR agonist and that SERCA inhibition remains under acidosis mimicking ischemic conditions. In Aim 3, the focus was on testing drugs with potential to prevent the overloaded SR from leaking Ca2+ (via RyRs) upon reperfusion. For that, we have examined various classes of organic polycationic agents in their ability to act as fast and reversible RyRs blockers. Currently, no agent with these characteristics is availableas a therapeutic or has been well defined for use as an experimental drug. The membrane permeable cation DHBP was identified as a potent RyR inhibitor with potential for rapid and transient inhibition of spontaneous SR Ca2+ release during reperfusion. In summary, we have defined the ability of some BZTs and antiepileptic agents (K201, CGP analog, Sipatrigine and Pimozide) to prevent/slow down SR Ca2+ overload by inhibition of SERCA, which may play an important role in their mechanisms of cell protection in ischemic events. In the case of BZT, these drugs may help their cause by producing mild activation of RyR2 channels, In addition, we have identify DHBP as a reversible and fast acting RyR inhibitor with potential as template for development of transient inhibitors of spontaneous SR Ca2+ release which may have significant protective action against injury during early reperfusion of the heart.

CoordenaÃÃo de AperfeiÃoamento de NÃvel Superior / Os Ãcidos graxos insaturados Ãmega-3 (&#969;-3) e Ãmega-9 (&#969;-9) possuem aÃÃo anti-inflamatÃria e antioxidante, enquanto os Ãmega-6 (&#969;-6) sÃo prÃ-inflamatÃrios. Este estudo verificou os efeitos do prÃ-condicionamento com misturas de Ãleos contendo baixa relaÃÃo &#969;-6/&#969;-3 e elevada relaÃÃo &#969;-9/&#969;-6, em modelo experimental de isquemia-reperfusÃo cerebral. Foram utilizados 42 ratos Wistar, divididos em dois grupos: Controle (n=24) e Teste (n=18). O grupo Controle foi subdividido em 4 grupos de 6 animais, cada: Simulado - Ãgua (Sim-Ãgua), Isquemia-ReperfusÃo - Ãgua (IR-Ãgua), Simulado - IsolipÃdico (Sim-IsolipÃdico) e Isquemia-ReperfusÃo - IsolipÃdico (IR-IsolipÃdico). Os animais receberam Ãgua ou uma mistura isolipÃdica com relaÃÃes &#969;-6/&#969;-3 = 8:1 e &#969;-9/&#969;-6 = 0,4:1 por via orogÃstrica, durante sete dias, conforme seus grupos. O grupo Teste foi subdividido em 3 grupos de 6 animais: IR-Mix1, IR-Mix2 e IR-Mix3. Os animais do grupo Teste receberam misturas oleosas com relaÃÃes &#969;-6/&#969;-3 = 1,4:1 e &#969;-9/&#969;-6 = 3,4:1 , diferindo apenas na fonte de &#969;-3: Mix1, contendo o Ãcido &#969;-3 &#945;-linolÃnico; Mix2, contendo os Ãcidos &#969;-3 &#945;-linolÃnico, eicosapentaenÃico e docosaexaenoico, e Mix 3, contendo os Ãcidos &#969;-3 &#945;-linolÃnico e docosaexaenÃico, administradas por via orogÃstrica, durante sete dias. No sÃtimo dia, os animais dos grupos IR-Ãgua, IR-IsolipÃdico, IR-Mix1, IR-Mix2 e IR-Mix3 foram submetidos à isquemia cerebral com oclusÃo bilateral das artÃrias carÃtidas comuns por 1 hora, seguida de reperfusÃo por 3 horas. Os animais dos grupos Sim-Ãgua e Sim-IsolipÃdico foram submetidos à operaÃÃo simulada. Ao final do experimento, todos os animais foram decapitados e seus cÃrebros fatiados para anÃlise histopatolÃgica da Ãrea CA3 do hipocampo. A morte neuronal foi quantificada pela contagem de neurÃnios vermelhos (NV). Constatou-se que a quantidade de NV no grupo IR-Ãgua (36,83  9,79) foi maior (P = 0,0046) que a observada do grupo Sim-Ãgua (17,67  8,48), bem como a quantidade de NV no grupo IR-IsolipÃdico (29,83  12,19) foi maior (P = 0,0459) que a observada no grupo Sim-IsolipÃdico (14,17  11,62). NÃo foi constatada diferenÃa na quantidade de NV entre os grupos Sim-Ãgua (17,67  8,48) e Sim-IsolipÃdico (14,17  11,62), ou entre os grupos IR-Ãgua (36,83  9,79) e IR-IsolipÃdico (29,83  12,19). A quantidade de NV no grupo IR-Mix1 (12,33  6,31) foi menor que a verificada nos grupos IR-Ãgua (36,83  9,79; P < 0,01) e IR-IsolipÃdico (29,83  12,19; P < 0,05). As quantidades de NV nos grupos IR-Mix2 (10,67  2,81) e IR-Mix3 (7,33  6,47) tambÃm foram menores que as verificadas nos grupos IR-Ãgua (36,83  9,79; P < 0,001) e IR-IsolipÃdico (29,83  12,19; P < 0,01). NÃo foram constatadas diferenÃas nas quantidades de NV entre os grupos IR-Mix1 (12,33  6,31), IR-Mix2 (10,67  2,81) e IR-Mix3 (7,33  6,47), entre si. Conclui-se que, independentemente da fonte de &#969;-3, o prÃ-condicionamento com misturas de Ãleos contendo baixa relaÃÃo &#969;-6/&#969;-3 e elevada relaÃÃo &#969;-9/&#969;-6, protege os neurÃnios contra as lesÃes de isquemia-reperfusÃo cerebral em modelo experimental. / Omega-3 (&#969;-3) and omega-9 (&#969;-9) unsaturated fatty acids are anti-inflammatory and antioxidant, while omega-6 (&#969;-6) fatty acids are pro-inflammatory. This study investigated the preconditioning effects of fatty acids mixes with low ratio &#969;-6/&#969;-3 and high ratio &#969;-9/&#969;-6, in a brain ischemia-reperfusion experimental model. Forty-two Wistar rats were aleatory assigned to two groups: Control (n=24) and Test (n=18). Control group was divided into 4 groups, each with 6 animals: Water-Simulated (Water-Sim), Water - Ischemia-Reperfusion (Water-IR), Isolipid-Simulated (Isolipid-Sim) and Isolipid - Ischemia-Reperfusion (Isolipid-IR). The animals received water or a isolipid mix with &#969;-6/&#969;-3 ratio of 8:1 and &#969;-9/&#969;-6 ratio of 0,4:1 by gavage, for 7 days, according to their groups. Test group was divided into 3 groups of 6 animals: Mix1-IR, Mix2-IR, and Mix3-IR. All animals from Test group received oil mixes with &#969;-6/&#969;-3 ratio of 1,4:1 and &#969;-9/&#969;-6 ratio of 3,4:1 , differing only on the &#969;-3 source: Mix1, with &#969;-3 linolenic acid; Mix2, with &#969;-3 linolenic, eicosapentaenoic and docosahexaenoic acids, and Mix 3, with &#969;-3 linolenic and docosahexaenoic acids, by gavage, for 7 days. At the 7th day, animals from Water-IR, Isolipid-IR, Mix1-IR, Mix2-IR, and Mix3-IR groups were subjected to 1-hour brain ischemia by occlusion of both common carotid arteries, followed by a 3-hour reperfusion. Animals from Water-Sim and Isolipid-Sim groups were submitted to a simulated operation. At the end of the experiment, all animals were decapitated and their brains were sliced and sent to histological analysis of the CA3 hippocampal region. Neuronal death was quantified by the red neurons (RN) count. It was found that the number of RN in Water-IR group (36.83  9.79) was higher (P = 0.0046) than the number observed in Water-Sim group (17.67  8.48), and similarly, the number of RN in Isolipid-IR group (29.83  12.19) was higher (P = 0.0459) than the number observed in Isolipid-Sim group (14.17  11.62). There was no difference between the amount of RN from Water-Sim (17.67  8.48) and Isolipid-Sim (14.17  11.62) groups, nor between Water-IR (36.83  9.79) and Isolipid-IR (29.83  12.19) groups. The number of RN in Mix1-IR group (12.33  6.31) was lower than the number seen in Water-IR (36.83  9.79; P < 0.01) and Isolipid-IR (29.83  12.19; P < 0.05) groups. The amounts of RN in Mix2-IR (10.67  2.81) and Mix3-IR (7.33  6.47) groups were also lower than the amounts observed in IR-Water (36.83  9.79; P < 0.001) and IR-Isolipid (29.83  12.19; P < 0.01) groups. There were no differences between the Mix1-IR (12.33  6.31), Mix2-IR (10.67  2.81) and Mix3-IR (7.33  6.47) groups. In conclusion, regardless of the source of &#969;-3, preconditioning with fatty acids mixes with low ratio &#969;-6/&#969;-3 and high ratio &#969;-9/&#969;-6, protects the neurons against brain ischemia-reperfusion injuries in this experimental model.

No description available.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21960号 / 医博第4502号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 平井 豊博, 教授 松原 和夫, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Inhibitors of apoptosis proteins (IAPs) are key intrinsic regulators of caspases-3 and -7. During ischemia, IAP-2 is upregulated dramatically, while the other IAPs show little or no change. To test whether IAP-2 prevents cardiac apoptosis and injury following ischemia/reperfusion, we generated a line of transgenic mice that carried a mouse IAP-2 transgene. High levels of mouse IAP-2 transcripts and 70 kDa IAP-2 were expressed in the hearts of transgenic mice, whereas IAP-1 and XIAP levels remained the same. Immunohistochemical studies revealed more intense staining of IAP-2 in the myocytes of transgenic mouse hearts. To assess the role of IAP-2 in I/R injury, the transgenic mice were subjected to ligation of the left descending anterior coronary artery ligation followed by reperfusion. The infarct sizes, expressed as the percentage of the area at risk, were significantly smaller in the transgenic mice than in the non-transgenic mice (30 ± 2% vs. 44 ± 2%, respectively, P < 0.05). This protection was accompanied by a decrease of the serum level of troponin I in the transgenic mice. IAP-2 transgenic hearts had significantly fewer TUNEL-positive cardiac cells, which indicated an attenuation of apoptosis. Our results demonstrate that overexpression of IAP-2 renders the heart more resistant to apoptosis and I/R injury.

Generation of free radicals upon reperfusion has been cited as one of the major causes of ischaemia/reperfusion injury. The following series of experiments was designed to study the effect of manganese superoxide dismutase (MnSOD) overexpression in transgenic mice on ischemia/reperfusion injury. A species of 1.4 kb human MnSOD mRNA was expressed, and a 325% increase in MnSOD activity was detected in the hearts of transgenic mice with no changes in the other antioxidant enzymes or heat shock proteins. Immunocytochemical study indicated an increased labeling of MnSOD mainly in the heart mitochondria of the transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x left ventricular developed pressure, was 52 ± 4% in the transgenic hearts as compared to 31 ± 4% in the non-transgenic hearts. This protection was accompanied by a significant decrease in lactate dehydrogenase release from the transgenic hearts. Overexpression of MnSOD limited the infarct size in vivo in a left coronary artery ligation model. Our results demonstrate that overexpression of MnSOD renders the heart more resistant to ischemia/reperfusion injury.