Studies of myocardial metabolism In vivo have been considerably advanced by the introduction of coronary sinus catheterization. By determining the coronary arterio-venous differences of various substrates, Sing and other workers (Goodale, Olson) have been able to describe the overall picture of myocardial metabollism in man and intact experimental animals. This technique dos not, however, allow adequate pinpointing of the precise pathways of cardiac metabolism in health and disease, and Bing concludes a recent review (1958) by advocating further studies using newer techniques such as radio-isotopes in a controlled in vitro system.

mariagraciapereda@gmail.com / Hemorragic dengue fever is a prevalent infection in many countries around the world. Myocarditis is a severe manifestation of dengue virus infection. With prompt intervention and an early diagnosis, the outcome of this condition can be improve. We report a adult patient with complicated dengue, myocarditis, cardiac and respiratory insufficiency with acute renal injury.

Η απουσία της μυοειδικής πρωτεΐνης ενδιάμεσων ινιδίων δεσμίνης οδηγεί σε διατατική μυοκαρδιοπάθεια, η οποία χαρακτηρίζεται από μιτοχονδριακές διαταραχές και κυτταρικό θάνατο καθώς και από εκτεταμένη ίνωση και ασβεστίωση του μυοκαρδίου. Προκειμένου να διασαφηνιστεί ο μηχανισμός που οδηγεί στο φαινότυπο απουσίας δεσμίνης, πραγματοποιήθηκε πρωτεωμική ανάλυση μιτοχονδρίων από το φυσιολογικό μυοκάρδιο και από το μυοκάρδιο απουσίας δεσμίνης. Σημαντικές διαφορές εντοπίστηκαν σε πρωτεΐνες που σχετίζονται με το μεταβολισμό ακετικού οξέος και κετονοσωμάτων, στο μεταφορέα μαλικού-ασπαρτικού, στο σύμπλοκο PDH, στον μεταβολισμό αμινοξέων, στην αναπνοή και στη μεταφορά ενέργειας. Αναζητήθηκε να ελεγχθεί αν οι μιτοχονδριακές διαταραχές και συνεπώς η μυοκαρδιοπάθεια απουσίας δεσμίνης μπορεί να βελτιωθεί από την υπερέκφραση των μικρών θερμεπαγόμενων πρωτεϊνών, αΒ-κρυσταλλίνης και HSP25. Πραγματοποιήθηκε υπερέκφραση των μικρών θερμεπαγόμενων πρωτεϊνών αΒ- κρυσταλλίνης και HSP25 ανεξάρτητα στο μυοκάρδιο χωρίς δεσμίνη υπό τον έλεγχο του υποκινητή της α βαριάς αλυσίδας της μυοσίνης (αMHC). Η υπερέκφραση τόσο της αΒ-κρυσταλλίνης, όσο και της HSP25 είχε ως αποτέλεσμα την υψηλού βαθμού βελτίωση της μορφολογίας του μυοκαρδίου, όπως προκύπτει από τη μείωση της ίνωσης και της ασβεστίωσης και την αναίρεση των διαταραχών υπερδομής, όπως η διόγκωση και η βλάβη των μιτοχονδρίων καθώς και η άτακτη διευθέτηση και καταστροφή των μυοϊνιδίων. Η καρδιακή λειτoυργία θεραπεύθηκε σε σημαντικό βαθμό στην περίπτωση της HSP25 και σε μεγαλύτερο βαθμό στην περίπτωση της αΒ- κρυσταλλίνης, όπως προκύπτει από τη βελτίωση της συστολικής λειτουργίας, την αύξηση του πάχους του οπίσθιου τοιχώματος της αριστερής κοιλίας και τη μείωση της καταπόνησης που αυτό υφίσταται. Επίσης, στην περίπτωση της αΒ- κρυσταλλίνης, τα πρότυπα παρουσιάζουν 100% βιωσιμότητα σε πρωτόκολλο υποχρεωτικής άσκησης υπό μορφής κολύμβησης διάρκειας 24 ημερών, ενώ στις ίδιες συνθήκες τα πρότυπα χωρίς δεσμίνη εμφανίζουν 50% βιωσιμότητα. Παράλληλα, η υπερέκφραση αΒ-κρυσταλλίνης προσδίδει σημαντική προστασία έναντι των ελεύθερων ριζών οξυγόνου και αζώτου, όπως προκύπτει από πειράματα καλλιεργειών ενήλικων μυοκαρδιοκυττάρων με σήμανση με τον ιχνηθέτη CM-H2DCFDA. Το ίδιο xi συμπέρασμα προκύπτει από τη μείωση του μιτοχονδριακού ανιόντος υπεροξειδίου μετά από σήμανση με MitoSox Red. Ομοίως, η υπερέκφραση της αΒ-κρυσταλλίνης προστατεύει τα κύτταρα από την επίδραση οξειδωτικών ερεθισμάτων, όπως η επίδραση H202 επί της καλλιέργειας, όπως αποδεικνύεται από τη σήμανση με τον ιχνηθέτη CM-H2DCFDA. Παράλληλα, η αΒ-κρυσταλλίνη προσδίδει προστασία έναντι της αύξησης της διαβατότητας του μιτοχονδριακού πόρου μεταβατικής διαβατότητας (MPTP) και της πτώσης του μιτοχονδριακού δυναμικού (Δψ), όπως προκύπτει από τη σήμανση ενήλικων μυοκαρδιοκυττάρων με τον ποτενσιομετρικό ιχνηθέτη TMRM. Τόσο η αΒ-κρυσταλλίνη όσο και η HSP25 ενδέχεται να προστατεύουν το μυοκάρδιο χωρίς δεσμίνη μέσω της δράσης τους ως μοριακών συνοδών, προσδίδοντας αντιοξειδωτική προστασία μέσω αύξησης των επιπέδων γλουταθειόνης και δρώντας ως πλειοτροπικά αποπτωτικά μόρια / The absence of the muscle-specific intermediate protein desmin leads to dilated cardiomyopathy, which is characterized by mitochondrial defects and significant cell death, as well as extensive fibrosis and calcification. In an attempt to elucidate the mechanism leading to the desmin deficient phenotype, we performed a proteomics analysis of mitochondria isolated from wild type and desmin null myocardium. Significant differences were found in acetate and ketone metabolism, in malate-aspartate shuttle, PDH complex, aminoacid metabolism, as well as respiration and energy shuttling. We sought to investigate whether the mitochondrial abnormalities and consequently the desmin null cardiomyopathy could be improved by the overexpression of the small heat shock proteins aB-crystallin and HSP25. We overexpressed the two proteins independently in the desmin deficient myocardium under the control of the αΜΗC promoter. The overexpression of the two proteins resulted in significant improvement of myocardial morphology, as demonstrated by the reduction of fibrosis and calcification and the correction of ultrastructural defects, such as mitochondrial swelling and destruction and irregular positioning and deterioration of myofibrils. Cardiac function was ameliorated to a significant degree by HSP25 overexpression and to even higher extent by aBcrystallin overexpression, as proven by the improvement of systolic function, the increase in posterior wall thickness and the reduction of r/h, an indicator of LV wall stress. In addition, in the case of αΒ-crystallin overexpression, desmin null mice demonstrate a rate of 100% survival in an obligatory exercice swimming protocol, while under the same conditions, only 50% of desmin null mice survive. Also, αΒ-crystallin overexpression provides significant protection against reactive oxygen and nitrogen species, and more specifically against mitochondrial superoxide ion, as proven by tissue culture experiments with adult cardiomyocytes and CM-H2DCFDA and MitoSox Red staining. Similarly, αΒ-crystallin overexpression protects desmin null cardiomyocytes from oxidative insults, such as hydrogen peroxide, as demonstrated by CMH2DCFDA staining. Moreover, αΒ-crystallin protects against the permeability increase of MPTP and the decrease of mitochondrial potential (Δψ), as demonstrated by staining of adult cardiomyocytes with TMRM. αΒ-crystallin and HSP25 may protect the desmin null myocardium by acting as molecular chaperons, by providing antioxidant protection through their capacity to increase glutathione levels and by acting as pleiotropic anti-apoptotic molecules.

Evolution of viral myocarditis to dilated cardiomyopathy (DCM)represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. RESULTS: IRF9-/- mice showed dramatically increased mortality compared to the wildtype littermates (0% WT vs 72% IRF-9-/- on day 14, P<0.0001). On day 42, there was less cardiac hypertrophy and inflammation in IRF-9-/- mice compared to WT controls (p<0.05). Onn day 42 there was a dramatic increase in the number of cytotoxic and helper T-Cells in the wild-type mice that was not observed in the IRF-9-/- spleens (p<0.05). CONCLUSIONS: These data suggest a novel dual role of IRF-9 in not only regulating interferon in acute stage of viral infection in myocarditis, but also late acquired immunity activation, including CD4/8 populations, contributing to the development of chronic cardiomyopathy.

Coxsackievirus B3 (CVB3) is the primary cause of viral myocarditis. The role of cystein-x-cystein (CXC) chemokine ligand 10 (CXCL10, formerly interferon-y-inducible protein 10) in CVB3-induced myocarditis is unknown. To explore the contribution of CXCL10 to CVB3-induced myocarditis, we performed functional analyses using newly generated transgenic mice with cardiac-specific CXCL10 overexpression (Tg) and CXCL10 knock out (KO) mice. The mRNA levels of CXCL10 peaked in the myocardium at day 3 post-infection prior to immune infiltration, suggesting that mainly resident cells of the heart are the sources of CXCL10. Indeed, we showed that CXCL10 can be induced by IFN-y but not by CVB3 infection in cultured cardiomyocytes. Further, a transgenic mouse model with cardiac-specific overexpression of CXCL10 was generated. CXCL10 Tg mice had spontaneous infiltrations of mononuclear cells with limited mRNA upregulation of IFN-y and IL-10, which were not sufficient to cause the impairment of cardiomyocyte or cardiac function. Following CVB3 infection, the viral titre in the mouse hearts inversely correlated with the levels of CXCL10 at day 3 post-infection. Further, the decreased virus titers in the CXCL10 Tg mouse hearts led to reduced cardiac damage indicated by low serum cTnI levels and improved cardiac functional performance and vice versa in the KO mice. This antiviral ability of CXCL10 may be through increased recruitment of natural killer (NK) cells to the heart and increased IFN-y expression early post-infection. At days 7 and day 10 post-infection with massive influx of mononuclear cells, the expression of CXCL10 enhanced the infiltration of CXCR3+ cells, CD4+, and CD8+ T cells as well as their associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines did not alter the viral clearance and mouse survival. Our data demonstrate for the first time that CXCL1 0 confers the protection to the heart during the early course of CVB3 infection, which may be primarily attributed to NK cell recruitment to the site of infection. This data suggest that CXCL10 is an important player in the orchestrated action of a group of cytokines and chemokines in combating against the CVB3-induced myocarditis in the early phase of infection.

Evolution of viral myocarditis to dilated cardiomyopathy (DCM)represents a delicate balance between host innate immunity and T-cell acquired immunity. IRF-9 is a key member of a transcription factor family that regulates type I interferon (IFN) production, critical for innate antiviral protection. RESULTS: IRF9-/- mice showed dramatically increased mortality compared to the wildtype littermates (0% WT vs 72% IRF-9-/- on day 14, P<0.0001). On day 42, there was less cardiac hypertrophy and inflammation in IRF-9-/- mice compared to WT controls (p<0.05). Onn day 42 there was a dramatic increase in the number of cytotoxic and helper T-Cells in the wild-type mice that was not observed in the IRF-9-/- spleens (p<0.05). CONCLUSIONS: These data suggest a novel dual role of IRF-9 in not only regulating interferon in acute stage of viral infection in myocarditis, but also late acquired immunity activation, including CD4/8 populations, contributing to the development of chronic cardiomyopathy.

Coxsackievirus B3 (CVB3) is the primary cause of viral myocarditis. The role of cystein-x-cystein (CXC) chemokine ligand 10 (CXCL10, formerly interferon-y-inducible protein 10) in CVB3-induced myocarditis is unknown. To explore the contribution of CXCL10 to CVB3-induced myocarditis, we performed functional analyses using newly generated transgenic mice with cardiac-specific CXCL10 overexpression (Tg) and CXCL10 knock out (KO) mice. The mRNA levels of CXCL10 peaked in the myocardium at day 3 post-infection prior to immune infiltration, suggesting that mainly resident cells of the heart are the sources of CXCL10. Indeed, we showed that CXCL10 can be induced by IFN-y but not by CVB3 infection in cultured cardiomyocytes. Further, a transgenic mouse model with cardiac-specific overexpression of CXCL10 was generated. CXCL10 Tg mice had spontaneous infiltrations of mononuclear cells with limited mRNA upregulation of IFN-y and IL-10, which were not sufficient to cause the impairment of cardiomyocyte or cardiac function. Following CVB3 infection, the viral titre in the mouse hearts inversely correlated with the levels of CXCL10 at day 3 post-infection. Further, the decreased virus titers in the CXCL10 Tg mouse hearts led to reduced cardiac damage indicated by low serum cTnI levels and improved cardiac functional performance and vice versa in the KO mice. This antiviral ability of CXCL10 may be through increased recruitment of natural killer (NK) cells to the heart and increased IFN-y expression early post-infection. At days 7 and day 10 post-infection with massive influx of mononuclear cells, the expression of CXCL10 enhanced the infiltration of CXCR3+ cells, CD4+, and CD8+ T cells as well as their associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines did not alter the viral clearance and mouse survival. Our data demonstrate for the first time that CXCL1 0 confers the protection to the heart during the early course of CVB3 infection, which may be primarily attributed to NK cell recruitment to the site of infection. This data suggest that CXCL10 is an important player in the orchestrated action of a group of cytokines and chemokines in combating against the CVB3-induced myocarditis in the early phase of infection.

Coxsackievirus B3 (CVB3) is the primary cause of viral myocarditis. The role of cystein-x-cystein (CXC) chemokine ligand 10 (CXCL10, formerly interferon-y-inducible protein 10) in CVB3-induced myocarditis is unknown. To explore the contribution of CXCL10 to CVB3-induced myocarditis, we performed functional analyses using newly generated transgenic mice with cardiac-specific CXCL10 overexpression (Tg) and CXCL10 knock out (KO) mice. The mRNA levels of CXCL10 peaked in the myocardium at day 3 post-infection prior to immune infiltration, suggesting that mainly resident cells of the heart are the sources of CXCL10. Indeed, we showed that CXCL10 can be induced by IFN-y but not by CVB3 infection in cultured cardiomyocytes. Further, a transgenic mouse model with cardiac-specific overexpression of CXCL10 was generated. CXCL10 Tg mice had spontaneous infiltrations of mononuclear cells with limited mRNA upregulation of IFN-y and IL-10, which were not sufficient to cause the impairment of cardiomyocyte or cardiac function. Following CVB3 infection, the viral titre in the mouse hearts inversely correlated with the levels of CXCL10 at day 3 post-infection. Further, the decreased virus titers in the CXCL10 Tg mouse hearts led to reduced cardiac damage indicated by low serum cTnI levels and improved cardiac functional performance and vice versa in the KO mice. This antiviral ability of CXCL10 may be through increased recruitment of natural killer (NK) cells to the heart and increased IFN-y expression early post-infection. At days 7 and day 10 post-infection with massive influx of mononuclear cells, the expression of CXCL10 enhanced the infiltration of CXCR3+ cells, CD4+, and CD8+ T cells as well as their associated inflammatory cytokines. However, the augmented accumulation of these immune cells and associated cytokines did not alter the viral clearance and mouse survival. Our data demonstrate for the first time that CXCL1 0 confers the protection to the heart during the early course of CVB3 infection, which may be primarily attributed to NK cell recruitment to the site of infection. This data suggest that CXCL10 is an important player in the orchestrated action of a group of cytokines and chemokines in combating against the CVB3-induced myocarditis in the early phase of infection. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate

No description available.

Adenoviruses are non-enveloped, dsDNA tumor viruses responsible for a breadth of pathogenesis including acute respiratory disease and viral myocarditis. Gap junctions, which are formed by connexin proteins, directly couple the cytoplasms of apposed cells enabling immunological, metabolic, and electrical intercellular communication. The gap junction protein connexin43 (Cx43; gene name – GJA1) is the most widely expressed human connexin protein and is the predominant connexin in the working myocardium. Given the immunological role for Cx43 gap junctions, we hypothesized that gap junctions would be targeted during adenoviral infection. We find reduced Cx43 protein due to suppression of GJA1 transcription dependent upon β-catenin during adenoviral infection, with viral protein E4orf1 sufficient to induce β-catenin phosphorylation. Loss of gap junction function occurs prior to reduced Cx43 protein levels with Ad5 infection rapidly inducing Cx43 phosphorylation at residues previously demonstrated to alter gap junction conductance. Direct Cx43 interaction with ZO-1 plays a critical role in gap junction regulation. We find loss of Cx43/ZO-1 complexing during Ad5 infection by co-immunoprecipitation, with complementary studies in human induced pluripotent stem cell derived-cardiomyocytes revealing Cx43 gap junction remodeling concomitant with reduced ZO-1 complexing. These findings demonstrate specific targeting of gap junction function by Ad5 leading to disruptions in intercellular communication which would contribute to dangerous pathological states including arrhythmias in infected hearts. Intercellular junction proteins belonging to classically defined unique junctions exhibit extensive cross-talk and interdependency for expression and localization. We find reduced connexin43 (Cx43) phosphorylation at a known internalization motif, leading us to hypothesize that gap junctions are maintained during adenoviral infection in order to stabilize intercellular junctions and adenoviral receptors therein. Utilizing immunofluorescence confocal microscopy, we demonstrate that Cx43 reductions are primarily cytosolic with Cx43 preservation at the plasma membrane. Click-IT chemistry, a non-radioactive pulse-chase technique, reveals that Cx43 ½ life is extended during adenoviral infection. In order to test if remaining Cx43 exists in de facto gap junctions (i.e. not undocked or cytosolic connexons) we utilized 1 % Triton X-100 solubility fractionation and find Cx43 is indeed primarily junctional during adenoviral infection. Having demonstrated increases in junctional Cx43, we next asked how tightly coupled cells were during adenoviral infection and by ECIS measurements of electrical resistance we demonstrate a transient increase in mechanical coupling during infection. Our future aims are to uncover changes in Coxsackievirus and adenovirus receptor (CAR) protein localization to determine if adenoviral-induced changes to subcellular architecture predisposes neighboring cells to infection and enhances viral spread. These findings will add to the existing model of adenoviral infection and more broadly, contribute to the therapeutic design of adenoviral vectors for cancer and gene therapy. / Doctor of Philosophy / The human heart will beat more than 3 billion times during the average lifetime. This is accomplished by billions of individual heart muscle cells, called cardiomyocytes, contracting in synchrony. Cardiomyocytes require direct cell to cell communication in order to receive the proper cues and work in concert. Outside of the heart, including the lining of the lungs which acts as a first line of defense against invading pathogens, direct cell to cell communication is important for mounting proper immune responses. A primary means by which cells communicate directly with neighboring cells is through gap junctions which are formed of proteins called connexins. Six connexin proteins form a channel in the cell surface that binds to a similar channel on an apposing cell to create a continuous gap junction channel, coupling the cell interiors directly. The most widely expressed human connexin, and the most abundant connexin in the heart, is connexin43 (Cx43; gene name – GJA1). Adenoviruses are pathogens commonly associated with respiratory illnesses in addition to more serious diseases including viral myocarditis, or infection of the heart. Given that Cx43 gap junctions enable direct intercellular communication important in initiating immune responses, we hypothesized that adenovirus would target Cx43 and gap junctions during infection. We find reduced Cx43 protein in cells infected with human adenovirus, and revel that the expression of the GJA1 gene is suppressed. We next focused on potential signaling pathways that are changed during adenoviral infection. β-catenin is a factor with several cellular roles including regulating expression of specific genes including GJA1 (Cx43). We demonstrate β-catenin is activated during adenoviral infection and that this is necessary for reducing Cx43 transcripts. A pathway that activates β-catenin in this manner is the PI3K/Akt signaling axis, which has previously been shown to be turned on during adenovirus infection by a viral protein called E4orf1. We find the adenoviral protein E4orf1 is sufficient to induce β-catenin activation revealing a potential therapeutic target for future studies. We next determined that direct cell to cell communication through gap junctions is reduced before loss of the gap junction protein Cx43 during infection. Gap junctions are modified by the cell to change their ability to couple cells independently of protein levels alone and we find gap junction modifications consistent with altered communication ability. Furthermore, the gap junction protein Cx43 interacts with the cellular skeleton protein Zonula Occludens-1 (ZO-1) during movement into and out of gap junction clusters. We determined alterations in Cx43/ZO-1 interactions consistent with gap junction remodeling. In complimentary studies we find the same gap junction remodeling in cardiomyocytes revealing arrhythmogenic potential during acute adenoviral infection in human heart cells. Localized with gap junctions are several other junction proteins including the Coxsackievirus and adenovirus receptor (CAR) which is critical in cardiac development and also the primary receptor for species C adenoviruses (used in our studies). CAR expression has been demonstrated to alter Cx43 levels and indeed, many junctional proteins influence the expression and/or localization of other junctional proteins. Interestingly, despite reduced Cx43 levels and reduced gap junction function (cell to cell communication), we detected decreases in a gap junction modification that is associated with gap junction degradation, suggesting that new gap junction protein Cx43 is not being made but already synthesized Cx43 is degraded more slowly. We hypothesized Cx43 is maintained during adenoviral infection in order to recruit other junctional components, principally CAR, on uninfected neighbor cells to predispose them to infection. We observed using microscopy that Cx43 reductions are primarily inside the cell but Cx43 is preserved on the cell surface and at junctions between cells. We next asked if the protein is being degraded more slowly and find Cx43 exists for longer in infected cells signifying that it is being degraded more slowly. Utilizing a fractionation technique to separate gap junction connexin from connexon that is non-junctional or inside the cell, we detect an increase in junctional Cx43, revealing maintenance of Cx43 gap junction structures. Having now identified adenoviral-mediated maintenance of Cx43 gap junction structures, we next wanted to test for changing in mechanical coupling (i.e. how tightly are the cells connected to one another) where we demonstrate an increase in mechanical coupling during adenoviral infection. Our future directions are to determine if this increase in Cx43 gap junction maintenance and mechanical coupling is concomitant with changes in CAR expression/localization on uninfected neighboring cells and if altered, does this predispose uninfected neighbors of infected cells to infection.