Timely reperfusion after a myocardial infarction is necessary to salvage the ischemic region; however, reperfusion itself is a major contributor to the final tissue damage. Currently, there is no clinically relevant therapy available to reduce ischemia-reperfusion injury. While many drugs have shown promise in reducing ischemia-reperfusion injury in preclinical studies, none of these drugs have demonstrated benefit in large clinical trials. Part of the failure to translate therapies can be attributed to the reliance on small animal models for preclinical studies. While animal models encapsulate the complexity of the systemic in vivo environment, they do not fully recapitulate human cardiac physiology. In this thesis, we utilized cardiac tissue engineering methods in conjunction with cardiomyocytes derived from human induced pluripotent stem cells, to establish a biomimetic human tissue-engineered model of ischemia-reperfusion injury. The resulting cardiac constructs were subjected to simulated ischemia or ischemia-reperfusion injury in vitro. We demonstrated that the presence of reperfusion injury can be detected and distinguished from ischemic injury. Furthermore, we demonstrated that we were able to detect changes in reperfusion injury in our model following ischemic preconditioning, modification of reperfusion conditions, and addition of cardioprotective therapeutics. This work establishes the utility of the human tissue model in studying ischemia-reperfusion injury and the potential of the human tissue platform to help translate therapeutic strategies into the clinical setting.

Atherosclerosis is a multifactorial, chronic inflammatory condition characterised by endothelial dysfunction, hyperlipidaemia and the accumulation of fatty deposits within the tunica intima of medium-to-large sized muscular arteries. This disease can prove fatal with patients suffering lethal myocardial infarction or stroke. Recently, two studies investigating the role of G-protein-coupled receptor 55 (GPR55) in atherosclerosis reported conflicting results; one reported a pro-atherogenic role for GPR55 and the other, an anti-atherogenic role for this receptor. Interestingly, another study demonstrated that the activation of GPR55 by lysophosphatidylinositol (LPI) in cultured rat neonatal ventricular cardiomyocytes provokes distinct cellular functions that are dependent on the location of GPR55, leading to suggestions that GPR55 may regulate cardiomyocyte function at two cellular sites and be a potential therapeutic target for cardiac disorders. While it has been demonstrated that GPR55 is important in the maintenance of cardiac function of healthy mice, what is currently unknown is if GPR55 has a role in the cardiovascular remodelling and cardiac function of atherosclerosis prone mice. To address this, the present studies were conducted to investigate 1) the role of GPR55 in atherogenesis, 2) if GPR55 has a role in the cardiac function of mice suffering from atherosclerosis, 3) the signalling pathway by which LPI activates cardiomyocytes, 4) the impact of GPR55 activation on the outcome of myocardial ischaemia/reperfusion (I/R) injury and, 5) the signalling mechanisms by which GPR55 elicits any observed effects on the myocardium in response to such injury. Using C57BL/6 (wildtype; WT), apolipoprotein E knockout (ApoE-/-; mouse model of atherosclerosis), GPR55 knockout (GPR55-/-) and novel ApoE-/-/GPR55-/- mice, this study has established that in the presence of high fat feeding (to accelerate atherosclerosis), GPR55 has a complex role whereby it both regulates risk factors associated with atherosclerosis (i.e. body weight and fat mass) yet promotes the development of fatty streaks within the vasculature, via a lipid independent mechanism. In terms of cardiac function, GPR55 exerted a protective role by maintaining the systolic function of high fat fed ApoE-/- mice, yet negatively affected the contractile reserve of these mice. With regard to infarct size, the present study established that LPI-induced activation of GPR55 (pre-global ischaemia) exacerbates myocardial tissue injury via a Rho-associated protein kinase (ROCK) dependent mechanism. Finally, this study established that LPI signals through the same signalling pathway as it did in the isolated heart, in both mouse and human-induced pluripotent stem cell-derived cardiomyocytes thus suggesting a translational role for GPR55 in the human heart. In conclusion, despite further research being required, the data presented within this thesis provides evidence that GPR55 may have the potential to be targeted for therapeutic gains in atherosclerosis and myocardial I/R injury.

CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior / O objetivo do presente estudo foi verificar os efeitos da L-alanil-glutamina (Ala-Gln) na isquemia e reperfusÃo em cÃrebro de ratos. Foram utilizados 48 ratos machos, da linhagem Wistar, com idade mÃdia de 62 dias e peso mÃdio de 276,38g, distribuÃdos em quatro grupos: Sham 30 minutos, Isquemia, Sham 90 minutos e Isquemia/ ReperfusÃo. Foi utilizado um modelo de isquemia cerebral experimental global, com oclusÃo da artÃria carÃtida comum bilateral e administraÃÃo de soluÃÃo salina ou Ala-Gln. Os resultados do presente estudo mostraram elevaÃÃo estatisticamente significante no percentual de Ãrea de necrose do grupo Isquemia (13,24  8,82) em relaÃÃo ao grupo Sham 30 minutos (0,12  0,20, p= 0,01). O mesmo ocorreu em relaÃÃo à Ãrea de necrose do grupo Isquemia/ReperfusÃo (13,30  9,91) em relaÃÃo ao Sham 90 minutos (0,70  1,35, p= 0,01). Tais resultados demonstram a efetividade do modelo Isquemia e Isquemia/ReperfusÃo cerebrais utilizados. NÃo foi observada alteraÃÃo significante no percentual de Ãrea de necrose entre os grupos Isquemia Salina (13,24  8,82) e Isquemia Ala-Gln (15,35  6,80, p= 0,34). A mÃdia do percentual de Ãrea isquÃmica do grupo Isquemia/ReperfusÃo Ala-Gln (4,65  1,44) foi significantemente inferior Ãquela encontrada no grupo Isquemia/ReperfusÃo Salina (13,30  9,91, p= 0,03). A administraÃÃo prÃvia de Ala-Gln a ratos submetidos à Isquemia/reperfusÃo cerebral nÃo promoveu reduÃÃo no percentual de Ãrea de necrose na lesÃo isquÃmica. Por outro lado, esse dipeptÃdeo reduziu o percentual de necrose cerebral na lesÃo Isquemia/ReperfusÃo cerebral. / The aim of this study was to investigate the effects of L-alanyl-glutamine (Ala-Gln) in ischemia and reperfusion in rat brain. We used 48 male rats, Wistar, with a mean age of 62 days and average weight of 276.38 g, divided into four groups: Sham 30 minutes ischemia, 90 minutes and Sham Ischemia / Reperfusion. We used a model of experimental global cerebral ischemia with occlusion of bilateral common carotid artery and administration of saline or Ala-Gln. The results of this study showed a statistically significant increase in the percentage of necrotic area of the ischemia group (13.24  8.82) than in group Sham 30 minutes (0.12  0.20, p= 0.01). The same occurred in relation to the area of necrosis in ischemia-reperfusion group (13.30  9.91) compared to Sham 90 minutes (0.70  1.35, p= 0.01). These results demonstrate the effectiveness of the model Ischemia and Ischemia / Reperfusion brain used. There was no significant change in the percentage of necrotic area between Salina ischemia groups (13.24  8.82) and ischemia Ala-Gln (15.35  6.80, p= 0.34). The average percentage of ischemic area of group Ischemia / Reperfusion Ala-Gln (4.65  1.44) was significantly lower than that in group Ischemia / Reperfusion Salina (13.30  9.91, p= 0.03). The prior administration of Ala-Gln in rats subjected to ischemia / reperfusion did not cause reduction in the percentage of necrosis in ischemic injury. Moreover, this dipeptide reduced the percentage of necrosis in the cerebral injury cerebral ischemia / reperfusion.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / Investigar os efeitos da ornitina &#945;-cetoglutarato (OKG) no sangue e mÃsculo gastrocnÃmio de ratos submetidos à isquemia-reperfusÃo do membro pÃlvico. MÃtodo - Quarenta e dois ratos foram distribuÃdos aleatoriamente em trÃs grupos: Sham (S), Isquemia (I) e Isquemia-reperfusÃo (R). Estes grupos foram distribuÃdos em subgrupos de acordo com o tempo e com o composto utilizado na gavagem. Todos os animais receberam gavagem de caseinato de cÃlcio ou OKG em dose Ãnica, noventa minutos antes da primeira laparotomia exploradora (LE). Os subgrupos S receberam apenas caseinato, os subgrupos I e R receberam caseinato ou OKG na mesma dose, de 5g/kg de peso. As amostras foram colhidas em trÃs momentos: imediatamente apÃs a LE; apÃs 6h da LE com isquemia (6h de isquemia) e sem isquemia e apÃs 6,5h da LE com isquemia-reperfusÃo (0,5h de reperfusÃo) e sem isquemia-reperfusÃo. Para anÃlise dos resultados foram utilizados: mÃdia, desvio padrÃo e teste de normalidade de Korogorov-Smirnov. Caso os resultados fossem normalizÃveis, aplicou-se o teste ANOVA para avaliaÃÃo de diferenÃa significante, no caso dos resultados nÃo serem normalizÃveis utilizou-se o teste de Kurskal-Wallis com o mesmo fim. Em todos os testes fixou-se em 0,05 ou 5%, a significÃncia estatÃstica. Resultados - No grupo S, nos metabÃlitos plasmÃticos, apÃs 6h e 6,5h da LE, quando comparados ao momento da LE (0h), houve aumento de: CPK 6h [141,83  47,88 versus 67,17  21,58 â p<0,004], CPK 6,5h [180,67  70,19 versus 67,17  21,58 â p<0,001]; LDH 6h [248,96  80,62 versus 74,40  33,84 â p<0,001]. Nos metabÃlitos musculares houve aumento de: lactato 6,5h [ 3,52  1,27 versus 1,57  0,76 â p<0,008]. Houve reduÃÃo de: piruvato 6h [0,035  0,024 versus 0,087  0,041 â p<0,004]. No grupo I foram observadas, para o subgrupo submetido à isquemia + caseinato em relaÃÃo ao sham, no plasma, elevaÃÃes em: CPK [635,17  231,71 versus 141,83  47,88 â p<0,001], LDH [551,16  142,63 versus 248,96  80,62 â p<0,002], piruvato [0,390  0,069 versus 0,061  0,045 â p<0,001]. No mÃsculo houve elevaÃÃo de: piruvato [0,127  0,044 versus 0,035  0,024 â p<0,002], lactato [8,158  0,717 versus 2,737  0,499 â p<0,001] e TBARS [0,012  0,004 versus 0,002  0,001 â p<0,001. Neste mesmo grupo comparando-se o subgrupo isquemia + OKG ao subgrupo sham encontrou-se, no plasma, elevaÃÃo em: CPK [868,17  308,30 versus 141,83  47,88 â p<0,001], glutationa [19,545  2,088 versus 6,432  1,062 â p<0,001] e no mÃsculo elevaÃÃo da glutationa [101,85  16,45 versus 14,73  0,87 p<0,001]. Ainda no grupo I, foi observado, para o subgrupo isquemia + OKG versus isquemia + caseinato, no plasma, queda em: LDH [296,26  93,62 versus 551,16  142,62 â p<0,004], glicose [104,16  20,81 versus 160,33  27,47 â p<0,001], piruvato [0,046  0,012 versus 0,390  0,069 â p<0,001], e elevaÃÃo de glutationa [19,54  2,08 versus 5,52  0,92 â p<0,001]. No mÃsculo foi evidenciada diminuiÃÃo do piruvato [0,047  0,031 versus 0,127  0,045 â p<0,004], lactato [2,47  0,74 versus 8,15  0,71 â p<0,001], TBARS [0,004  0,004 versus 0,012  0,004 â p<0,013] e elevaÃÃo glutationa [101,85  16,45 versus 14,44  2,09 â p<0,001]. No grupo R. foi observada, quando comparado o subgrupo reperfusÃo + caseinato ao Sham, no plasma, elevaÃÃo de: CPK [606,33  79,84 versus 180,66  70,19 â p<0,001], No mÃsculo elevaÃÃo do piruvato [0,065  0,027 versus 0,030  0,033 â p<0,047] e lactato [7,16  2,33 versus 3,52  1,27 â p<0,013] alÃm de queda na G6PDH [0,462Â0,22 versus 0,207Â0,22 p<0,04] . No grupo R quando comparado o subgrupo reperfusÃo + OKG ao subgrupo Sham, no plasma, foi evidenciado aumento em: CPK [558,00  102,83 versus 180,66  70,19 â p<0,001] e glicose [232,16  59,76 versus 118,16  24,22 â p<0,001]. No mÃsculo houve diminuiÃÃo de G6PDH [0,182Â0,22 versus 0,462Â0,22]. Ainda no grupo R quando comparado o subgrupo reperfusÃo + OKG ao reperfusÃo + caseinato, no plasma, houve elevaÃÃo da glicose [232,16  59,76 versus 158,00  24,20 â p<0,013]. No mÃsculo foi notada queda no lactato [3,63  1,16 versus 7,16  2,33 â p<0,008] e elevaÃÃo da glutationa [63,18  18,98 versus 16,17  1,96 â p<0,001]. ConclusÃes - O trauma cirÃrgico desencadeou alteraÃÃes significativas em alguns metabÃlitos estudados. O modelo de isquemia-reperfusÃo demonstrou efetividade. A OKG, em dose Ãnica por gavagem, demonstrou aÃÃes prÃ-glicolÃticas aerÃbica a nÃvel muscular e sistÃmico; proteÃÃo contra lesÃo da cÃlula muscular, e efeito antioxidante muscular e sistÃmico durante a lesÃo de isquemia quanto apÃs lesÃo de isquemia/reperfusÃo. / To investigate the effects of the ornitine &#945;-ketoglutarate (OKG) upon metabolites in vivo concentrations in whole blood and gastrocnemic muscle tissue of rats submitted to ischemia-reperfusion of the pelvic limb. Methods - Forty two rats were randomly distributed into three groups: Sham (S), Ischemia (I) and Ischemia-reperfusion (R). These groups were redistributed into subgroups, according to time and to the substance used in the gavage. All animals received via gavage calcium caseinate or OKG as a single dose, ninety minutes before the first laparotomy (L). The subgroup S received only caseinate, whereas subgroups I and R received caseinate or OKG, at the same dose of 5g/kg body weight. Samples were collected at three moments: immediately after L; after 6h with ischemia (ischemia of 6h) or without ischemia; and after 6,5h of L with ischemia-reperfusion (reperfusion of 0,5h) or without ischemia-reperfusion. Data expressed as: mean  standard deviation, normality test of Korogorov-Smirnov. In case of the results went normalized, the test ANOVA was applied for evaluation significant difference, in case of the results was not normalized, the test of Kurskal-Wallis was used. Significant variations were considered when p <0,05.Results - In S group, at the plasmatic samples, after six hours (6h) or six hours and thirty minutes (6,5h) of L, when compared versus at the moment of L (0h), there was increase of: CPK 6h [141,83  47,88 versus 67,17  21,58 - p <0,004], CPK 6,5h [180,67  70,19 versus 67,17  21,58 - p <0,001]; LDH 6h [248,96  80,62 versus 74,40  33,84 - p <0,001]. In muscular samples there was increase of: lactate 6,5h [3,52  1,27 versus 1,57  0,76 - p <0,008]; there were reductions of: pyruvate 6h [0,035  0,024 versus 0,087  0,041 - p <0,004]. In group I it was observed, for the subgroup submitted to ischemia + caseinate in relation to sham, in plasma, elevations of: CPK [635,17  231,71 versus 141,83  47,88 - p <0,001], DHL [551,16  142,63 versus 248,96  80,62 - p <0,002], pyruvate [0,390  0,069 versus 0,061  0,045 - p <0,001]. In muscle there were elevations of: pyruvate [0,127  0,044 versus 0,035  0,024 - p <0,002], lactate [8,15  0,71 versus 2,73  0,49 - p <0,001] and TBARS [0,012  0,004 versus 0,002  0,001 - p <0,001]. In this same group when comparing subgroup ischemia + OKG versus subgroup sham there were, in the plasma, elevations of: CPK [868,17  308,30 versus 141,83  47,88 - p <0,001], glutathione [19,54  2,08 versus 6,43  1,06 - p <0,001]. In muscle there was elevation of glutathione [101,851  16,457 versus 14,737  0,874 p <0,001]. Still in the I group, it was observed, when subgroup ischemia + OKG was compared to ischemia + caseinate, in the plasma, a decrease of: LDH [296,26  93,62 versus 551,16  142,62 - p <0,004], glucose [104,16  20,81 versus 160,33  27,47 - p <0,001], pyruvate [0,046  0,012 versus 0,390  0,069 - p <0,001] and an elevation of glutathione [19,54  2,08 versus 5,52  0,92 - p <0,001]. In muscle there were decreases of pyruvate [0,047  0,031 versus 0,127  0,045 - p <0,004], lactate [2,47  0,74 versus 8,15  0,71 - p <0,001], TBARS [0,004  0,004 versus 0,012  0,004 - p <0,013]. It was observed elevation of glutathione [101,85  16,45 versus 14,44  2,09 - p <0,001]. In R group, it was observed, when comparing subgroup reperfusion + caseinate versus sham at the plasma, elevations of: CPK [606,33  79,84 versus 180,66  70,19 - p <0,001]. In muscle it was observed elevations of lactate [7,16  2,33 versus 3,52  1,27 - p <0,013] and decrease of G6PDH [0,462Â0,22 versus 0,207Â0,22 p<0,04]. In R group, when comparing subgroup reperfusion + OKG to subgroup sham, at the plasma, it was evidenced increase of: CPK [558,00  102,83 versus 180,66  70,19 - p <0,001], glucose [232,16  59,76 versus 118,16  24,22 - p <0,001]. In muscle there was observed decrease of G6PDH [0,182Â0,22 versus 0,462Â0,22]. Still in the group R when comparing the subgroup reperfusion + OKG versus the subgroup reperfusion + caseinate, at the plasma, there were elevations of glucose [232,16  59,76 versus 158,00  24,20 - p <0,013]. In muscle it was noticed a decrease of lactate [3,63  1,16 versus 7,16  2,33 - p <0,008] and elevation of glutathione [63,18  18,98 versus 16,17  1,96 - p <0,001]. Conclusions - Surgical trauma promoted significant alterations in some studied samples. The ischemia-reperfusion model demonstrated to be effective. OKG, as a single dose for gavage demonstrated pro glycolytic aerobic effect. Muscular and systemic protection against muscle cell lesion was also observed as well as an antioxidant effect at the end of ischemia and after ischemia-reperfusion injury

A long held hypothesis in mitochondrial biology holds that increases in mitochondrial Ca2+ levels stimulate the activity of matrix dehydrogenases that catalyze production of NADH and eventually donate electrons to electron transport in order to increase ATP formation. At the same time, mitochondrial Ca2+ overload is a deleterious event leading to opening of the mitochondrial permeability transition pore, increasing reactive oxygen species and initiating pathways that contribute to cell death. These fundamental hypotheses are best studied in the heart because of the critical energy supply-demand relationship in myocardium, but were untestable in vivo until the discovery of the mitochondrial Ca2+ uniporter (MCU). The molecular identity of the MCU pore forming subunit was recently discovered, which allowed me to study a transgenic mouse with myocardial delimited expression of a dominant negative MCU. My lab developed mice with myocardial-delimited transgenic expression of a dominant negative MCU to test these fundamental hypotheses and to determine how MCU controls physiological and pathological stress responses in vivo, ex vivo, and in situ. My studies provide new, unanticipated information that contributes to our understanding the relationship between mitochondrial Ca2+, oxygen utilization, cardiac pacemaking and pathologic stress responses in heart. Here, I show that mice with myocardial-targeted MCU inhibition have hearts with surprisingly high oxygen consumption rates due to elevated cytoplasmic Ca2+ in response to physiological stress. Loss of MCU effectively preserved inner mitochondrial membrane potential and prevented an oxidative burst thought to drive myocardial injury and death, but nevertheless failed to protect myocardium from ischemia-reperfusion injury. Increases in oxygen consumption, elevation in cytoplasmic Ca2+ and transcriptional reprogramming mitigate the protective actions of MCU inhibition in vivo. Mice with myocardial selective MCU inhibition have a reduced response to isoproterenol-induced heart rate increase but have normal baseline heart rates. My studies provide novel insight into how MCU contributes to myocardial Ca2+ homeostasis, metabolism, and transcription leading to surprising actions on physiological and pathophysiological responses in heart.

Die rechtzeitige Reperfusion eines ischämischen Gewebes kann einen zusätzlichen Schaden induzieren. Gezielte Interventionen in der frühen Reperfusionsphase können diesen sogenannten Reperfusionsschaden jedoch vermindern. Letzteres Phänomen beschreibt der Begriff Postkonditionierung. Volatile Anästhetika sind in der Lage, den genannten Mechanismus zu aktivieren. Dieser Vorgang wird Anästhetika-induzierte Postkonditionierung (APOST) genannt. Die vorliegende Arbeit hatte zum Ziel, die Rolle des mitochondrialen Kalzium-abhängigen Kalium-Kanals mit großer Leitfähigkeit (mBKCa) und der mitochondrialen permeability-transition-Pore (mPTP) in der Desfluran-induzierten Postkonditionierung zu beleuchten. Zur Untersuchung der genannten Phänomene wurde ein etabliertes in vivo-Mausmodell des akuten Myokardinfarkts verwendet. Hierbei reduzierte die Gabe von 1,0 MAC Desfluran in der frühen Reperfusionsphase die Infarktgröße signifikant. Somit konnte eine Desfluran-induzierte Postkonditionierung beobachtet werden. Die pharmakologische Aktivierung des mBKCa mittels NS1619 reduzierte die Infarktgröße in einem vergleichbaren Ausmaß wie Desfluran. Weiterhin zeigten sich keine additiven Effekte bei der Kombination beider Interventionen. Als Bestätigung dessen hob die Blockade des mBKCa mittels Iberiotoxin die APOST auf. Diese Ergebnisse lassen auf eine Beteiligung des mBKCa bei der Desfluran-induzierten Postkonditionierung schließen. Durch Pharmakologische Aktivierung der mPTP mittels Atractylosid wurde die APOST aufgehoben. Das Gegenexperiment zeigte keine additiven Effekte bei gleichzeitiger Desflurangabe und Inhibierung der mPTP mittels Cyclosporin A. Die alleinige mPTP-Inhibition resultierte in Infarktgrößen, welche mit denen bei Desflurangabe vergleichbar waren. Folglich erscheint eine Beteiligung der mPTP an der Desfluran-induzierten Postkonditionierung wahrscheinlich. Die Aktivierung der mPTP konnte die kardioprotektiven Auswirkungen der mBKCa-Öffnung nicht vollständig aufheben. Ebenso wurde durch Inhibition der mPTP der Effekt der mBKCa-Blockierung nur teilweise aufgehoben. Demzufolge scheint die Signalvermittlung der APOST über den mBKCa, zumindest teilweise, mPTP-unabhängig zu sein. Die Ergebnisse der vorliegenden Studie demonstrieren eine Beteiligung des mBKCa und der mPTP an der Desfluran-induzierten Postkonditionierung in Mäusen in vivo. Weiterhin legen sie die Vermutung nahe, dass die Signalvermittlung der APOST über den mBKCa teilweise unabhängig von der mPTP erfolgt. / Reperfusion of ischemic tissue may induce additional damage itself. Through interventions during early reperfusion this so-called reperfusion injury can be decreased. This phenomenon is called post-conditioning. Volatile anesthetics are capable of activating this mechanism which we call anesthetic-induced post-conditioning (APOST). The objective of this work was to elucidate the role of the mitochondrial large-conductance calcium-activated potassium channel and the mitochondrial permeability transition pore in desflurane-induced postconditioning. An established in-vivo mouse-model for myocardial infarction was used to investigate the mentioned phenomenon. The administration of 1.0 MAC desflurane during early reperfusion reduced infarct size significantly. Thus a desflurane-induced post-conditioning was observed. Pharmacological activation of mBKCa by NS1619 reduced infarct sizes to similar extend as desflurane. No additional effects of both interventions were found. Affirming this conclusion blockade of mBKCa by iberiotoxin abolished APOST. These results suggest mBKCA to be involved in desflurane-induced post-conditioning. Activation of mPTP by atractyloside abolished APOST. The control experiment demonstrated no additional effects by simultaneous administration of desflurane and inhibition of mPTP by cyclosporine A. Inhibition of mPTP alone resulted in infarct sizes similar to desflurane application alone. Consequently mPTP is likely to be involved in desflurane-induced post-conditioning. Activation of mPTP was not able to completely abolish the cardioprotective effect of mBKCa opening. Likewise the effect of mBKCa inhibition was only neutralized partially through inhibition of mPTP. Accordingly, signal transduction of APOST through mBKCa seems to be at least partially independent of mPTP. The results of this study demonstrate mBKCa and mPTP to be involved in desflurane-induced post-conditioning in mice in-vivo. Furthermore, these data suggest a partial mPTP-independent signaling pathway of APOST through mBKCa.

The overall aim of this Thesis was to characterise the energetic properties of the mouse papillary muscle as this preparation could become a useful model to study alterations of energetic aspects of cardiac pathologies and heart-focussed genetic changes. Measurements of resting and active metabolism of the papillary muscles were made in vitro using the myothermic technique. In the first study the mechanism underlying impaired contractility of post-ischaemic rat papillary muscle was investigated. The rat preparation is well established and was used to develop protocols and approaches that could later be used as the basis for studies with mouse papillary muscle. The muscles were exposed to simulated ischaemia for 60 min and change in energetics was studied 30 min into the reperfusion phase. The work output was reduced to 66 ± 3% of the pre-ischaemia value and the enthalpy output decreased to 71 ± 3% of pre-ischaemia value. However, there was no change in either initial, 19 ± 3%, or net mechanical efficiency, 9.0 ± 0.9%. These data, in combination with studies of Ca2+ handling, suggests that the reduced work output was caused by attachment of fewer cross-bridges in each twitch, but with no change in work generated by each cross-bridge. The following two studies involved characterisation of the energetics of the mouse papillary muscle and included measurements of resting and active metabolism. The resting metabolic rate varied with muscle size but the mean initial value was tilda 25 mW g-1 and the estimated steady value tilda 5 mW g-1 . The resting metabolic rate declined exponentially with time towards a steady value, with a time constant of 18 ± 2 min. There was no alteration in isometric force output during this time. The magnitude of resting metabolism depended inversely on muscle mass, more than doubled following a change in substrate from glucose to pyruvate and was increased 2.5-fold when the osmolarity of the bathing solution was increased by addition of 300 mM sucrose. Addition of 30 mM BDM affected neither the time course of the decline in metabolic rate nor the eventual steady value. The energy requirements associated with contractile activity were tilda7 mJ g-1 twitch-1 at a contraction frequency of 1 Hz. The enthalpy output was not affected by changing substrate from glucose to pyruvate but did decrease with an increase in temperature. The enthalpy output was partitioned into force-dependent and force-independent components using BDM to selectively inhibit cross-bridge cycling. The force-independent enthalpy output was 18.6 ± 1.9% of the initial enthalpy output. Muscle initial efficiency was &tilda32% and net efficiency tilda 17% when shortening at a realistic velocity. The enthalpy output decreased with increased contraction frequency but was independent of shortening velocity. On the basis of these values, it was calculated that the twitch energetics were consistent with ATP splitting by half the cross-bridges and the pumping of one Ca 2+ into the SR for every three cross-bridge cycles. The lack of influence of shortening velocity on energy cost supports the idea that the amount of energy to be used is determined early in a twitch and is not greatly influenced by events that occur during the contraction. The suitability of the mouse papillary muscle as a model to study ischaemia and reperfusion damage was also assessed. This preparation is excellent for studying muscle specific changes in work and enthalpy output; however, due to the long-term instability and variability amongst preparations, the suitability of this preparation in prolonged experiments remains uncertain.

Reperfusion injury may mediate renal dysfunction following ischaemia. A murine model was developed to investigate the role of the tissue factor-thrombin-protease activated receptor pathway in renal ischaemia reperfusion injury (IRI). In this model, mice received 25 minutes of ischaemia and subsequent periods of reperfusion. C57BL6, protease activated receptor-1 (PAR-1) knockout mice, and tissue factor (TF) deficient mice were used. Following 24 hours IRI, PAR-1 deficiency resulted in protection against severe renal failure compared to the C57BL6 mice (creatinine, 118.2 ?? 6.3 vs 203 ?? 12 ??mol/l, p&lt0.001). This was confirmed by lesser tubular injury. By 48 hours IRI, this resulted in a survival benefit (survival, 87.5% vs 0%, p&lt0.001). Treatment of C57BL6 mice with hirudin, a specific thrombin inhibitor, offered renoprotection at 24 hours IRI (creatinine, 107 ?? 10 ??mol/l, p&lt0.001), leading to a 60% survival rate at 48 hours IRI (p&lt0.001). TF deficient mice expressing less than 1% of C57BL6 mouse TF were also protected (creatinine, 113.6 ?? 7 ??mol/l, p&lt0.001), with a survival benefit of 75% (p&lt0.001). The PAR-1 knockout, hirudin treated C57BL6 and TF deficient mice had reduced myeloperoxidase activity and tissue neutrophil counts compared to the C57BL6 mice, along with reduced KC and MIP-2 chemokine mRNA and protein expression. Hirudin treatment of PAR-1 knockout mice had no additional benefit over PAR-1 absence alone, suggesting no further contribution by activation of other protease activated receptors (creatinine at 24 hours IRI, 106.5 ?? 10.5 ??mol/l, p&gt0.05). Furthermore, immunofluoresence staining for fibrin(ogen) showed no difference between C57BL6 and PAR-1 knockout mice, suggesting no major contribution by fibrin in this model. Renal IRI resulted in increased levels of TF mRNA expression in the C57BL6, PAR-1 knockout, and hirudin treated C57BL6 mice compared to normal controls, suggesting that TF mRNA expression was upregulated in this model. This resulted in increased TF functional activity in the C57BL6 and PAR-1 knockout mice, but TF activity was negligible in hirudin treated C57BL6 and TF deficient mice. The data therefore suggests that the TF-thrombin cascade contributes to renal IRI by signalling via PAR-1 that then regulates chemokine gene expression and subsequent neutrophil recruitment.

Se estudió la producción de calor (Ht) y evolución mecánica en ventrículos de rata estimulados electricamente a 1.5 Hz, perfundidos según la Técnica de Langendorff y sometidos a 45 min de isquemia (Isq) y 45 min de reperfusión (Rep). La influencia de la temperatura se evaluó a 25, 30 y 35 °C. Los 4 componentes diferenciables del calor activo (H1, H2, H3 y H4)(Ponce-Hornos y col, Pflugers Arch-Eur J Physiol 429; 1995) se analizaron en contracciones aisladas en los 10 primeros min de Isch. Los músculos se montaron en un calorímetro (Ponce-Hornos y col, Am. J. of Physiol. 243; 1982) que permitió medir Ht y simultaneamente la presión desarrollada en condiciones isovolúmicas (P) y la presión de reposo (PR). <br />A partir de los estudios realizados fue posible descartar procesos que fueron postulados como responsables de la falla cardíaca isquémica e involucrar en medida apropiada a otros. Entre los primeros, fue posible descartar la caída del pH y los cambios en la energía libre del ATP como responsables en etapas muy tempranas. También pudo disociarse la caída de la P respecto de la desaparición de un componente mitocondrial Ca++ dependiente (H4). Entre los segundos, se identificó un evento energético durante la Isch de alto valor predictivo de la disfunción mecánica y/o daño durante la Rep. También, los experimentos dan apoyo a los cambios en la PR como responsables de la abrupta caída en la P al inicio de la Isch. Además, fué posible vincular a la interacción actomiosínica con la mayor fracción de liberación de energía en el período inicial de la Rep. También se asoció la baja recuperación mecánica durante la Rep con altos valores relativos de calor independiente de tensión.

Matrix metalloproteinase2 (MMP2) was recently revealed to have targets and actions within the cardiac myocyte. In ischemia/reperfusion (I/R) injury, MMP2 is activated and degrades troponin I (TnI) and actinin. The regulation of intracellular MMP2 activity is relatively unknown and is thus the subject of this thesis. The localization of MMP2 in caveolae of endothelial cells suggests that caveolin1 (Cav1) may play a role in regulating MMP2. Whether Cav1 is responsible for regulating MMP2 in the heart is unknown. A Cav1 knockout mouse model was used to explore the role Cav1 may play in the regulation of MMP2 activity. The initial studies found that MMP2 and Cav1 were colocalized in cardiomyocytes and that MMP2 activity in Cav1/ hearts was markedly enhanced. Additionally, the caveolin scaffolding domain inhibited MMP2 activity in a concentrationdependent manner. To explore whether increased MMP2 in Cav1/ hearts translates to impaired cardiac function, Cav1+/+ and Cav1/ isolated working hearts were physiologically challenged with increasing increments of left atrial preload followed by increasing concentrations of isoproterenol. Cav1/ hearts show similar or better cardiac function compared to Cav1+/+ hearts following preload challenge or adrenergic stimulation in vitro, and this appears unrelated to changes in MMP2. Though the function of Cav1/ hearts appears similar to that of Cav1+/+ hearts during physiological situations, whether this is the case during I/R injury is not known. Cav1+/+ and Cav1/ isolated working mouse hearts exposed to global, noflow ischemia showed no functional differences. However, Cav1/ hearts had significantly higher levels of both TnI and actinin following I/R than Cav1+/+ hearts. Posttranslational modifications of the intracellular MMP2 substrates could alter susceptibility to MMP2 proteolysis. Isolated working mouse hearts were exposed to isoproterenol and/or I/R injury to examine the phosphorylation status of TnI. Isoproterenol and I/R both result in the phosphorylation of TnI, however, isoproterenol lead to a more highly phosphorylated form of TnI than that observed in hearts exposed I/R alone. These and subsequent studies will further reveal the molecular mechanisms that underlie the complex interactions between Cav1 and MMP2. This may eventually lead to a novel avenue of therapeutic intervention for heart diseases.