Diabetic cardiomyopathy is defined as changes in the structure and function of the myocardium that occur in diabetic patients in the absence of other cardiovascular risk factors. Our laboratory has shown that conjugated linoleic acid (CLA - a naturally-occurring polyunsaturated fatty acid with multiple health benefits) prevents endothelin-1-induced myocyte hypertrophy in vitro, as well as cardiac hypertrophy in vivo using a rodent model of spontaneously hypertensive heart failure. These cardioprotective effects of CLA were mediated through activation of peroxisome proliferator activated receptors (PPAR isomers α and γ) and stimulation of diacylglycerol kinase ζ (DGKζ). Thus, the aims of this study were to (i) determine the effect of CLA on hyperglycemia-induced structural and functional abnormalities of cardiomyocytes, and (ii) assess the role of PPAR-γ and DGK. High glucose treatment induced hypertrophy of primary adult cardiomyocytes, as indicated by augmented cell size and protein synthesis compared to untreated cardiomyocytes. The hyperglycemia-induced hypertrophy was attenuated by pretreatment with CLA (30 µM). The ability of CLA to prevent hyperglycemia-induced hypertrophy was suppressed by GW9662 (1 µM) and R59022 (10 μM), pharmacological inhibitors of PPAR-γ and DGK, respectively. In addition to structural abnormalities, high glucose impaired contractile function of adult cardiomyocytes as measured by maximal velocity of shortening, maximal velocity of relengthening, and peak shortening. Hyperglycemia-induced contractile dysfunction was likewise prevented by pretreatment with CLA (30 µM). Collectively, these findings support the idea that hyperglycemia is an independent risk factor for the development of diabetic cardiomyopathy. Hypertrophy and contractile dysfunction elicited by high glucose were prevented by CLA. The antihypertrophic actions of CLA are mediated, at least in part, by activation of PPAR-γ and DGK.

Le cœur est un organe central au fonctionnement du système cardiovasculaire. Il est physiologiquement compartimenté et est constitué de cellules spécialisées qui régulent les impulsions électriques ainsi que la contraction du myocarde. Le cœur adapte le flux sanguin en fonction des besoins du corps. En condition pathologique, le cœur recourt toutefois à des mécanismes compensatoires. Au niveau physiologique, la compensation s’observe par l’hypertrophie des cardiomyocytes qui, bien que bénéfique à court terme, exacerbe à long terme la fonction cardiaque. L’activation des « mitogen activated protein kinases » (MAPK) contribue autant au maintien de la fonction physiologique qu’à la détérioration pathologique du myocarde et serait également une cause de l’hypertrophie observée. Parmi les 5 groupes de MAPK connues, la MAPK p38 est formée de 4 isoformes dont les sérine/thréonine kinases p38α et p38γ sont exprimées de façon prédominante dans le cœur. Les p38 partagent les mêmes activateurs, mais leurs effecteurs diffèrent. Bien que le rôle de p38α semble impliqué dans l’aggravement des troubles cardiaques, celui de p38γ ne semble pas redondant à p38α et demeure incompris. Cette isoforme possède un motif de liaison aux domaines PDZ, unique chez les MAP kinases. Également, chez les cellules cardiaques, elle transloque au noyau en condition de stress. Le but de l’étude ici est de comprendre le rôle de p38γ et de ses motifs uniques sur la structure et la taille des cardiomyocytes. Afin de répondre au but de l’étude, plusieurs mutants adénoviraux de p38 ont été conçus. Un des mutants ne possède pas le motif de liaison aux domaines PDZ, deux autres contrôlent la localisation cellulaire soit au noyau, soit au cytoplasme, et un autre mutant est muté au site de phosphorylation. Des cardiomyocytes en culture ont été infectés par les différents mutants en présence de leur activateur en amont ou de la β-galactosidase. Les réseaux d’α-actinine, ainsi que la taille des cardiomyocytes, ont été observés par microscopie. Les observations effectuées montrent que p38γ entraîne une désorganisation des réseaux d’α-actinine lorsqu’il est phosphorylé. Également, il facilite l’hypertrophie des cardiomyocytes en présence de son activateur s’il est forcé hors du noyau ou en l’absence de son motif de liaison aux domaines PDZ. En conclusion, les résultats obtenus suggèrent que p38γ exerce bel et bien un rôle dans le maintien structural des cardiomyocytes par l’intermédiaire de l’α-actinine.

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In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures.

Cardiac hypertrophy is a fundamental response to an increased workload on the heart characterized by cardiac myocyte (CM) growth and left ventricular (LV) wall thickening. In a model of hypertension, e.g. chronic pressure overload, this process may become maladaptive, initially leading to impaired myocardial relaxation and LV filling, and subsequently to LV dilation, wall thinning, and contractile failure. Hemodynamic overload activates Gαq-mediated signaling responsible for transcriptional reactivation of fetal growth programs, activation of the mitogen-activated protein kinase (MAPK) cascade, and oxidative stress. The precise mechanism by which MAPK is activated in pressure overload, and the role oxidative stress plays in mediating this hypertrophic signaling are still under investigation. In CMs, the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA) maintains calcium stores, and thus may regulate calcium-dependent MAPK signaling. Our laboratory showed that SERCA is activated in CMs by reversible oxidative post-translational modification (OPTM) of its most reactive cysteine site (C674). We hypothesized that OPTMs mediate the effects of hypertrophic stimuli in CMs via reversible oxidation of SERCA at C674. To test this hypothesis, we employed a reductionist model: isolated adult rat ventricular myocytes (ARVM) overexpressing wild-type (WT) or mutant SERCA, in which C674 is substituted with a redox-insensitive serine (C674S). Using alpha-adrenergic receptor (αAR) stimulation as a model of Gq-mediated hypertrophy, we found that C674S expression decreased both CM growth and MAPK activation. Furthermore, biotin switch revealed that αAR stimulation induced a reversible OPTM of SERCA at C674. We generated a transgenic mouse expressing a single-allele C674S SERCA2 knock-in mutation (SKI) to explore this mechanism further in the setting of pressure overload, a disease model of Gq-activation in vivo. SKI mice subjected to ascending aortic constriction (AAC) had decreased hypertrophy compared to WT. Ventricular myocytes isolated from adult SKI mice also had diminished MAPK activation in response to hypertrophic stimulation in vitro and decreased SERCA function at baseline. These findings led us to the conclusion that redox-activation of SERCA via reversible modification of C674 is critical for the complete transduction of hypertrophic stimuli to MAPK signaling and CM hypertrophy.

Ankyrin-B (Ank-B) is a crucial scaffolding protein regulating expression and localization of contractile machinery in the cardiac muscle. Recent genetic investigations in the First Nations Community, the Gitxsan of Northern BC, identified a mutation in Ank-B (p.S646F c.1937 C>T) associated with a cardiac arrhythmia, Long QT Syndrome Type 4 (LQTS4). Distinct from other LQTS4 subtypes, individuals harbouring the p.S646F variant exhibit development deficits including cardiomyopathies and accessory electrical pathways. How p.S646F interferes with the development of the heart is unknown due to a fundamental lack of understanding regarding Ank-B proteostasis and its role in cardiac differentiation. Initial in silico analyses predicted the p.S646F mutant to be deleterious to the Ank-B protein. Using in vitro techniques, I determined p.S646F mutant reduced levels of Ank-B in H9c2 rat ventricular cardiomyoblasts. Furthermore, haploinsufficiency in mice was previously shown to result in developmental cardiac deficits. I, therefore, hypothesized that p.S646F interferes with Ank-B proteostasis, thereby affecting cardiomyocyte development. I showed that p.S646F destabilized Ank-B in cardiomyoblasts, due to increased degradation via the proteasome. Furthermore, overexpression of p.S646F Ank-B had a significant impact on cellular behaviour including reduced cell viability, and altered expression of cellular differentiation markers. Together these data address critical knowledge gaps with regards to Ank-B protein homeostasis and the role of Ank-B in cardiomyocyte viability and development. These findings inform the diagnosis and treatment of patients with the p.S646F variant, creating potential targeted pathways of intervention, and furthering our understanding of the role of the Ank-B in the development of the heart. / Graduate / 2019-04-26

In adult cardiomyocytes, the established mechanism of excitation-contraction coupling is calcium-induced calcium release (CICR) mediated by L-type Ca2+ channels (Cav1.2). Briefly, membrane depolarization opens voltage-gated Cav1.2 to allow for the influx of extracellular Ca2+ into the cytosol. This small sarcolemmal (SL) Ca2+ influx is necessary for triggering a larger release of Ca2+ from the intracellular Ca2+ storage site, the sarcoplasmic reticulum (SR), through the SR Ca2+ release channel also known as the ryanodine receptor (RyR). RyR-mediated release of SR Ca2+ effectively raises the cytosolic free Ca2+ concentration, allowing for Ca2+ binding to troponin C on the troponin-tropomysin complex, leading to cross-bridge formation and cell contraction. However, previous functional data suggests an additional CICR modality involving reverse mode Na+-Ca2+ exchanger (NCX) activity also exists in neonate cardiomyocytes. To further our understanding of how CICR changes occur during development, we investigated the spatial arrangement of caveolin-3 (cav-3), the principle structural protein of small membrane invaginations named caveolae, and NCX in developing rabbit ventricular myocytes. Using traditional as well as novel image processing and analysis techniques, both qualitative and quantitative findings firmly establish the highly robust and organized nature of NCX and cav-3 distributions during development. Specifically, our results show that NCX and cav-3 are distributed on the peripheral membrane as discrete clusters and are not highly colocalized throughout development. 3D distance analysis revealed that NCX and cav-3 clusters are organized with a distinct longitudinal and transverse periodicity of 1-1.5 μm and that NCX and cav-3 cluster have a pronounced tendency to be mutually exclusive on the cell periphery. Although these findings do not support the original hypothesis that caveolae is the structuring element for a restricted microdomain facilitating NCX-CICR, our results cannot rule out the existence of such microdomain organized by other anchoring proteins. The developmentally stable distributions of NCX and cav-3 imply that the observed developmental CICR changes are achieved by the spatial re-organization of other protein partners of NCX or non-spatial modifications. In addition, the newly developed image processing and analysis techniques can have wide applicability to the investigations on the spatial distribution of other proteins and cellular structures. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

INTRODUCTION: Space exploration is ultra-hazardous and unpredictably dangerous. One area of significant concern is the exposure of astronauts to deep space radiation and the development of deleterious health effects. Earth's magnetic field and atmosphere both act to deflect the majority of deep space radiation, protecting humans on the surface of earth as well as astronauts in low Earth orbit missions. Because this type of radiation is not encountered terrestrially, very limited evidence exists in regards to the effects on the well-being. Deep space radiation, which consists of high charge and energy (HZE) particles, may be experimentally reproduced for studies using a particle accelerator. The long-term degenerative effects of cosmic irradiation on the cardiovascular system are vastly unknown. Detailing the molecular response within cardiomyocytes after exposure to HZE irradiation will provide needed knowledge for scientists to accurately assess the cardiovascular risks associated with deep space radiation exposure. OBJECTIVE: The primary objective of this study is to characterize the molecular alterations of gene expression within murine cardiomyocytes following exposure to two different types of HZE. METHODS: Wild type C57B1/6NT (Taconic) mice were exposed to either 90 cGy, 1 GeV proton (1H) or 15 cGy, 1 GeV/nucleon iron (56Fe) HZE particle-radiation. Radiation exposure was performed at the NASA Space Radiation Laboratory located at the Brookhaven National Laboratory (Upton, NY). Biological samples were taken and transcriptome profiling was performed at various time points following irradiation (1, 3, 7, 14, and 28 days). RESULTS: Samples exposed to 56Fe-IR displayed significant levels of gene modulation, while proton-irradiation failed to elicit significant alterations in cardiomyocyte gene transcription compared to sham-irradiated samples. Network pathway analysis of iron-irradiated samples identified multiple biological pathways being significantly modulated. These biological pathways included cell death and survival, free radical scavenging, and inflammatory processes. Multiple points of upstream transcription regulation were identified as key nodes for regulating downstream expression. Validation of the signal transduction network by protein analysis showed that particle-IR clearly regulates a long lived signaling mechanism for p38 MAPK signaling and NFATc4 activation. Electrophoresis mobility shift assays supported the role of transcription factors GATA-4, STAT-3 and NF-𝜅B as key regulators. CONCLUSIONS: The molecular response to 56Fe-IR is unique and induces long-term modulations of gene expression in cardiomyocytes that last up to at least 28 days following radiation exposure. However, exposure to 1H-IR failed to elicit significantly robust alterations in gene expression in cardiomyocytes. Additionally, proteins involved in signal transduction and transcriptional activation via DNA binding play a significant role in the molecular response following HZE particle radiation. This study may have multiple implications for NASA's efforts to develop cardio-degenerative risk estimates for astronauts participating in future deep space missions. By identifying molecular mechanisms and potential molecular markers, scientists can begin to assess excess relative risks and develop strategies to mitigate risks of developing physiological changes which may compromise future missions. This study may also have major safety implications for the increasing number of patients receiving conventional and particle radiotherapy.

Résumé: Une des maladies cardiaques les plus sévères est l’insuffisance cardiaque (IC). Un facteur important à la base de l’IC est la mort des cardiomyocytes suite à un déséquilibre du métabolisme énergétique. Une meilleure compréhension des voies signalétiques à la base de ces déséquilibres aiderait donc à comprendre un des mécanismes à la base du développement de l’IC. La protéine ADAP1, reconnue pour être abondante au cerveau, est également présente dans d’autres organes. ADAP1 possède un domaine ArfGAP capable d’inactiver les protéines de la famille Arf et deux domaines PH de liaison aux phosphatidylinositols. ADAP1 est connue pour changer de localisation intracellulaire par l’action de facteurs de croissance. Son action entraîne l’ouverture du pore de transition de perméabilité mitochondriale (mPTP), menant directement à la mort cellulaire. Il est connu que la protéine AKT est sous le contrôle des mêmes facteurs de croissance qu’ADAP1 et qu'elle contrôle également l'ouverture des mPTPs. Nous avons émis l’hypothèse que dans les cardiomyocytes, ADAP1 transloque vers les mitochondries avec un effet conjoint d’AKT et que ce changement de localisation entraîne la mort cellulaire en stimulant l’ouverture des mPTPs. Afin de vérifier cette hypothèse, plusieurs expérimentations ont eu lieu. Tout d’abord, un immunobuvardage et une PCR quantitative ont révélé la forte expression d’ADAP1 au cœur de rat adulte. L’isolation des cellules cardiaques démontre qu’ADAP1 est majoritairement exprimée chez les cardiomyocytes. Contrairement à ce qui est rapporté chez les neurones, un immunobuvardage de fractions cellulaires a démontré que la forme humaine d’ADAP1 surexprimée est très peu localisée aux mitochondries des cardiomyocytes. Une analyse bio-informatique a permis de postuler que les sites de phosphorylation connus ADAP1 ont un grand potentiel pour être la cible d’AKT. Un essai MTT à démontrer que la double surexpression d’ADAP1 et d’un mutant constitutivement actif d’AKT (AKTca) diminue la viabilité des cardiomyocytes infectés, et ce de façon indépendante du domaine ArfGAP et dépendante de la présence de sérum. Un immunobuvardage sur des fractions cellulaires a démontré qu’AKTca ne modifie pas la localisation d’ADAP1 surexprimée et ne favorise pas sa translocation aux mitochondries. Un immunobuvardage a démontré que l’effet de la double surexpression n’est pas dépendant de la phosphorylation d’ADAP1 par AKTca. Bien qu’ADAP1 a le potentiel de mettre à jour un nouveau mécanisme moléculaire contribuant au développement de l’IC, notre hypothèse ne s’est pas vérifiée, car AKT n’influence pas la localisation d’ADAP1. Le rôle de la protéine ADAP1 chez les cardiomyocytes reste à déterminer et d'autres études sont ainsi requises. / Abstract: One of the most severe heart disease is heart failure. An important factor in the development of this disease is cardiomyocyte death cause d by an imbalance of energy metabolism. A better understanding of the signalling pathway at the base of these imbalances would therefore help to understand the mechanisms underlying the development of this disease. The ADAP1 protein, known to be abundant in the brain, is also present in other organs. This protein, with an enzymatic domain ArfGAP capable of inactivating proteins of the Arf family and two phosphatidylinositols binding domains, is known for changing its intracellular localization by the action of growth factor. ADAP1 mitochondrial localization leads to the formation of mPTP which directly lead to cell death. It is known that the AKT protein is under the control of the same growth factors than ADAP1 and is also control the opening of the mPTPs. We hypothesized that in cardiomyocytes, ADAP1 translocates to the mitochondria with a combined effect of AKT and that this change in localization leads to cell death by stimulating the opening of mPTPs. To test this hypothesis, many experiments were conducted. First of all, an immunoblotting and a quantitative PCR demonstrated that ADAP1 has a strong expression in the heart of adult rats. The isolation of cardiac cells demonstrates that ADAP1 is predominantly expressed in cardiomyocytes. Contrary to what is reported in neurons, an immunoblotting on cellular fractions demonstrated that the overexpression of the human form of ADAP1 is not localized to mitochondria in cardiomyocytes. A bioinformatic analysis, postulated that ADAP1 known phosphorylation sites have a great potential to be the target of AKT. An MTT assay demonstrated that a dual overexpression of ADAP1 and a constitutively active mutant of AKT (AKTca) decreases the viability of infected cardiomyocytes, and this, independently of the ArfGAP domain and dependent on the presence of serum. An immunoblotting on cellular fractions demonstrated that the presence of AKTca does not change the location of overexpressed ADAP1 and does not promotes its translocation to mitochondria. An immunoblotting demonstrated that the effect of the double overexpression is not dependent on the phosphorylation of AKT ca by ADAP1. Although ADAP1 has the potential to be implicated in a new molecular mechanism contributing to the development of heart failure, our hypothesis does not hold, because AKT does not influence the location of ADAP1. The role of ADAP1 in cardiomyoccyte is yet to be determined and so further study are needed

Les cellules dendritiques (DCs) possèdent des rôles clés dans la modulation de la réponse inflammatoire. Leur implication dans la réponse inflammatoire post-ischémie/reperfusion semble claire. Cependant, leurs rôles spécifiques restent encore à élucider. Nous avons émis l’hypothèse selon laquelle la modulation de la réponse des cellules dendritiques suite à la séquence d’ischémie/reperfusion pourrait diminuer les lésions du greffon cardiaque. L’objectif de ce travail a donc consisté en l’exploration et l’identification d’un mécanisme immunomodulateur dans la DC. Un modèle cellulaire d’hypoxie/réoxygénation (H/R) et un modèle de co-culture DCs / cardiomyocytes ont été utilisés. / Dendritic cells (DCs) play key roles during the inflammatory process. Although their involvement in ischemia/reperfusion (I/R)-related inflammation is known, their specific role in such a context remain to be elucidated.We hypothesized that the modulation of DC phenotype during I/R might decrease cardiac graft injuries. In this study, we aimed to explore and identify an immunomodulatory mechanism in DCs. An in vitro model of hypoxia/reoxygenation (H/R) and a co-culture model were used. Our results highlight that the purinergic receptor P2Y11 (P2Y11R) exhibits an immunosuppressive role in DCs. This effect was lost when cells were subjected to a H/R insult, due to P2Y11R downregulation during hypoxia.