Trauma-induced secondary cardiac injury (TISCI) represents an under recognised complication of severe injury with haemorrhage. A limited number of clinical studies have supported the development of adverse cardiac events, such as arrhythmia, in association with biomarker proven TISCI. Pre-clinical studies using small animal models have provided insights into potential mechanisms and key effector molecules involved in the development of TISCI, but there remains a general lack of understanding regarding the in vivo functional implications of this indirect cardiac injury resulting from trauma-haemorrhage. This project aimed to investigate the implications of cardiac injury on myocardial systolic function. A robust, translatable model of TISCI was developed, which reflected the cardiac biomarker profile identified in clinical studies and, for the first time, demonstrated a significant, dose-dependent rise in Heart-type Fatty Acid Binding Protein (H-FABP) in response to trauma-haemorrhage. Non-invasive echocardiography was used to determine the acute myocardial response to injury and haemorrhage and also to assess the response of the left ventricle to resuscitation after an antecedent 60-minute period of trauma-haemorrhage. The functional studies presented here have enabled real time visualisation of the impact of trauma-haemorrhage upon systolic left ventricular function over 1 to 6 hours, both with and without resuscitation. Having established the trends in in vivo systolic function over time, further studies were then conducted to test the combination of adenosine, lidocaine and magnesium (ALM) as a cardiovascular rescue agent in TISCI. ALM, as an adjunct to fluid resuscitation, has shown great promise as a therapeutic agent which improves haemodynamic outcomes, reduces the volume of resuscitation fluid required and favours survival in the murine model of TISCI.

No description available.

Since 1933, studies have explored the concept of trauma induced secondary cardiac injury (TISCI), yet till 2012, it had not been defined as the incidence of cardiac events and rise in cardiac biomarkers following traumatic injury. Despite, improvements in early outcomes, trauma patients have reduced long-term mortality with cardiac disease being the major contributor. Although many putative mechanisms have been suggested for TISCI, the underpinning pathophysiology still remains unclear. In this thesis, a prospective study of 290 critically injured patients identifies a 13% incidence of adverse cardiac events (ACE) with consistently raised serum h-FABP levels in these patients. H-FABP was found to be a good predictor of ACE through ROC analysis and a h-FABP of 16.8 ng/ml used to define trauma induced secondary cardiac injury (TISCI). TISCI was associated with longer hospital stay and higher mortality. Patients who developed ACE had higher plasma levels of adrenaline and noradrenaline with a correlating increase in plasma h-FABP. On multivariate analysis, hypertension was the only independent risk factors for ACE. The increase in serum cardiac biomarkers was reflected by an increase in serum h- FABP in our group's trauma hemorrhage murine models. The hearts of these models were used in the experiments that form the last experimental chapter of this thesis. Protein expression studies confirm this increase in serum h-FABP by evidence of concurrent leaching in the cardiac tissue, along with Troponin I. Myocardial injury was evident on electron microscopy with evidence of interstitial and organelle oedema, myofibrillar degeneration, nuclear condensation and changes in mitochondrial morphology. Immunohistochemistry and western blotting protein studies demonstrate the translocation of the mitochondrial death-related protein AIF to the cytosol and nucleus, where it becomes its active pro-apoptotic form. This thesis propositions the utility of the cardiac biomarker h-FABP in predicting ACE and outcomes in critically injured patients. Although increasing serum noradrenaline and adrenaline levels are associated with higher incidence of ACE and biochemical evidence of cardiac injury with rising h-FABP levels, multivariate analysis negates their value as independent predictors of ACE. Leaching out of the proteins h-FABP and Troponin I in the murine cardiac tissue confirmed the value of serum measurements of these proteins as markers of cardiac injury. This was associated with widespread ultrastructural myocardial damage in the TH mice with changes in mitochondrial morphology. The mitochondrial damage seen is associated with the translocation of the mitochondrial death-related protein AIF to the cytosol and the nucleus where I propose its canonical signaling leading to nuclear degradation and cell death is the driver of cardiac dysfunction.

Introduction: Myocardial infarction is the most common form of acute cardiac injury attributed to heart failure. Despite advancements in prognosis and treatment, acute MI (AMI) still bears a considerable mortality rate within the initial year, with a significant portion of patients succumbing within the initial 30 days post-MI. The overall prognosis hinges on factors such as the extent of heart muscle damage, duration of the inflammatory response, and the efficacy of administered treatments in mitigating myocardial cell death and injury. This underscores the need for deeper mechanistic understanding and development of targeted therapies for cardiovascular diseases. In response to cardiac injury, macrophages are initially recruited to the infarcted myocardium and undergo phenotypic change from pro-inflammatory (M1) macrophages in the early stage to an anti-inflammatory (M2) macrophages phenotype in the later stage, orchestrating the initiation, maintenance, and eventual resolution of the inflammatory response. However, in chronic ischemia or severe infarction, continuous cardiomyocyte death prolongs pro-inflammatory macrophage activation resulting in robust secretion of pro-inflammatory cytokines perpetuating the inflammatory response and resulting in increased myocardial damage. Despite some understanding, further research is needed on the mechanisms and factors influencing macrophage function during injury. Circular RNAs are newly discovered non-coding RNA generated from protein-coding genes ubiquitously expressed in mammalian tissue, highly conserved among species, and recently implicated in the possible regulation of macrophage activation. However, their role in immunomodulation during cardiovascular injury remains unknown. Objective: This study focused on determining the specific role of circ-cdr1as in phenotypic switching between pro-, and anti-inflammatory macrophages and to determine whether functional regulation of circ-cdr1as modulates macrophage function post-cardiac injury. Methods and Results: We performed circular RNA microarray analyses to assess circular RNA transcriptome changes using RNA isolated from bone marrow derived macrophages (BMDM) polarized to a M1 phenotype by INFγ and TNFα or a M2 phenotype by IL-10, IL-4, and TGF-β. Following RNA isolation, samples are treated with RNaseR for enrichment of circular RNA and removal of linear RNA. We identified circRNAs differentially expressed in pro-and anti-inflammatory macrophages, including circRNA cdr1as (circ-cdr1as). RT-qPCR analysis revealed circ-cdr1as was one of the most downregulated in pro-inflammatory macrophages and significantly upregulated in anti-inflammatory macrophages in vitro. We established a circ-cdr1as overexpression system by generating a circ-cdr1as plasmid using pc3.1 plasmid with flanking regions that allows circularization of specified sequence for in vitro studies. For knockdown of circ-cdr1as, we used small hairpin RNA targeting the splicing junction found only in circular RNA. RT-qPCR and fluorescence activated cell sorting (FACS) analyses showed that overexpression of circ-Cdr1as increased transcription of anti-inflammatory markers and percentage of CD206+ (M2 macrophage marker) cells in naïve and pro-inflammatory macrophages in vitro. Meanwhile, knockdown decreased transcription of anti-inflammatory markers and increased the percentage of CD86+ (M1 macrophage marker) cells in naïve and anti-inflammatory macrophages in vitro. Disease enrichment analysis based on IPA system of the diseases associated with circular RNA involved in macrophage polarization indicated that cardiac fibrosis and cardiac enlargement as the top diseases. Therefore, we investigated the expression levels of circ-cdr1as in the heart after myocardial infarction (MI) injury in a mouse model. RT-qPCR analysis revealed downregulation of circ-cdr1as in the heart 3 days post MI, suggesting a possible physiological role for circ-cdr1as in MI pathophysiology. We isolated fibroblast, cardiomyocytes, CD31+ endothelial cells, and F4/80+ macrophages and investigated the transcriptional changes of circ-cdr1as to determine if it is cell-type specific. RT-qPCR analysis showed no significant difference in transcription of circ-cdr1as in fibroblast and endothelial cells. However, in cardiomyocytes and macrophages there was a significant downregulation of circ-cdr1as. To understand the role of circ-cdr1asmodulated macrophages in post-MI cardiac repair in vivo, we overexpressed circ-cdr1as in fluorescently labeled BMDMs and directly injected them into the ischemic myocardium immediately following MI surgery. FACS and immunohistochemistry analyses showed that these macrophages retained their anti-inflammatory phenotype during the initial stages of cardiac injury, and in the later stages improved cardiac left ventricular (LV) functions and reduced infarct size. Since circ-cdr1as was also decreased in cardiomyocytes post-MI, we additionally generated circ-cdr1as adeno associated virus 9 (circ-cdr1as-AAV-9) vectors to overexpress circ-cdr1as in mouse hearts. We performed tail vein injections of circ-cdr1as-AAV9 vectors 14 days prior to MI and conducted physiological and histological studies. Administration of circ-cdr1as-AAV9 significantly improved post-MI LV functions including ejection fraction (%EF) and fractional shortening (%FS) at 21-28D post MI, decreased infarct size, and improved angiogenesis. Interestingly, in the initial stages of cardiac injury, overexpression of circ-cdr1as reduced cardiomyocyte apoptosis and increased percentage of anti-inflammatory macrophages at injury site. Lastly, emerging evidence suggests that some circular RNAs function as microRNA (miR) sponges. Therefore, we investigated this mechanism to assess whether circular cdr1as invokes phenotypic changes in macrophages in both the steady-state and injured heart by acting as a sponge for miRNA to inhibit its function. Circ-cdr1as contains over 70 binding sites for miR-7, a microRNA known to exacerbate inflammation in lung related diseases through inhibition of KLF4. Pull-down assay indicated that circ-cdr1as directly interacts with miR-7. We found a reciprocal relationship between circ-cdr1as and miR-7 in macrophages and in the heart 3 days post-MI. Overexpression of miR-7 by miR-7-5p mimic increased the percentage of pro-inflammatory marker CD86 in naïve, pro-, and anti-inflammatory macrophages and upregulated transcription of pro-inflammatory markers. Meanwhile, inhibition of miR-7 had the opposite effect. We also found that expression of miR-7 target gene, KLF4, was reduced when macrophages were treated with miR-7 and increased when miR-7 was inhibited. Conclusions: In summary, this study suggests that circ-cdr1as plays a key role in regulating anti-inflammatory phenotype of macrophages through the modulation of miR-7 and its targets and exogenous delivery of circ-cdr1as may improve LV function over time. Therefore, circ-cdr1as may potentially be developed as an anti-inflammatory regulator in tissue inflammation after cardiac injury. / Biomedical Sciences

Esse estudo descreve casos de “morte súbita” em bovinos associados com a ingestão de Amorimia (Mascagnia) exotropica em seis propriedades rurais localizadas na região metropolitana de Porto Alegre e da serra gaúcha. Os bovinos intoxicados foram encontrados mortos sem história de sinais clínicos prévios, ou apresentaram tremores musculares, quedas bruscas, movimentos de pedalagem, opistótono, respiração ofegante e decúbito lateral, quando induzidos ao movimento poucos minutos antes da morte. Registrou-se maior número de casos entre os meses de maio e agosto. Nove bovinos foram necropsiados e os principais achados macroscópicos observados foram mucosa oral levemente cianótica (3/9), hidropericárdio leve a moderado (3/9), petéquias e equimoses no epicárdio (5/9), coágulo no interior do ventrículo esquerdo (4/9), edema pulmonar (5/9) e mucosas vermelhas no abomaso e no intestino delgado (6/9). Histologicamente havia necrose de coagulação no miocárdio (9/9) caracterizada por retração celular, aumento da eosinofilia do citoplasma com perda das estriações, núcleos em picnose e ocasionais núcleos em cariorrexia e cariólise. No coração, edema intersticial (3/9) e infiltrado inflamatório intersticial, predominantemente, mononuclear (7/9) também foram observados. Nos rins de três bovinos havia degeneração hidrópicovacuolar multifocal das células epiteliais dos túbulos contorcidos distais associada com núcleos picnóticos deslocados para periferia da célula. As lesões cardíacas desses bovinos foram demonstradas através da imuno-histoquímica para troponina cardíaca C (cTnC). Nos corações dos bovinos intoxicados ocorreu diminuição acentuada de cTnC no citoplasma de grupos dos cardiomiócitos com características microscópicas de degeneração e necrose e, em algumas áreas havia perda total de imunomarcação. Raras fibras musculares cardíacas sem alteração histológica apresentaram perda de imunomarcação. Nos demais cardiomiócitos dos bovinos intoxicados sem lesões histológicas observou-se intensa marcação citoplasmática. / This study describes cases of sudden death in cattle that were associated with the consumption of Amorimia (Mascagnia) exotropica and occurred in six ranches located in the mountainous region of Rio Grande do Sul and the metropolitan region of Porto Alegre, Brazil. Affected cattle were found dead with no history of previous clinical signs, or showed muscular tremors, falls, paddling, opistotonus, panting, and lateral recumbence after being induced to move, few minutes before death. Most cases were recorded between May and August. Nine cattle were necropsied and main gross findings were oral mucosa slightly cyanotic (3/9), mild to intermediate hydropericardium (3/9), epicardial petechiae and ecchymoses (5/9), clot within the left ventricle (4/9), lung edema (5/9), apart of abomasal and small intestinal reddened mucosa (6/9). Histologically, there was myocardial coagulation necrosis (9/9), which was characterized by cellular retraction, enhanced cytoplasmic eosinophilia, lack of cytoplasmic striations and occasional nuclear karyorrhexis and karyolysis. There also were interstitial edema (3/9) and interstitial inflammatory infiltrate (mainly mononuclear) (7/9) in the heart. Apart of multifocal vacuolar-hydropic degeneration in the epithelial cells of the distal convoluted tubules associated with pyknotic and eccentric nuclei in the kidneys of three cattle. The cardiac lesions of these cattle were demonstrated by immunohistochemistry for cardiac troponin C (cTnC). In the hearts of cattle intoxicated was severe reduction of the cTnC in the cytoplasm of cardiomyocytes groups with microscopic features of degeneration and necrosis, and in some areas with complete loss of immunoreactivity. Rare cardiac muscle fibers showed no histological abnormality loss of immune-marking. In the remaining cardiomyocytes in cattle poisoned histological lesions were absent, but an intense cytoplasmic staining in these cells was observed.

Esse estudo descreve casos de “morte súbita” em bovinos associados com a ingestão de Amorimia (Mascagnia) exotropica em seis propriedades rurais localizadas na região metropolitana de Porto Alegre e da serra gaúcha. Os bovinos intoxicados foram encontrados mortos sem história de sinais clínicos prévios, ou apresentaram tremores musculares, quedas bruscas, movimentos de pedalagem, opistótono, respiração ofegante e decúbito lateral, quando induzidos ao movimento poucos minutos antes da morte. Registrou-se maior número de casos entre os meses de maio e agosto. Nove bovinos foram necropsiados e os principais achados macroscópicos observados foram mucosa oral levemente cianótica (3/9), hidropericárdio leve a moderado (3/9), petéquias e equimoses no epicárdio (5/9), coágulo no interior do ventrículo esquerdo (4/9), edema pulmonar (5/9) e mucosas vermelhas no abomaso e no intestino delgado (6/9). Histologicamente havia necrose de coagulação no miocárdio (9/9) caracterizada por retração celular, aumento da eosinofilia do citoplasma com perda das estriações, núcleos em picnose e ocasionais núcleos em cariorrexia e cariólise. No coração, edema intersticial (3/9) e infiltrado inflamatório intersticial, predominantemente, mononuclear (7/9) também foram observados. Nos rins de três bovinos havia degeneração hidrópicovacuolar multifocal das células epiteliais dos túbulos contorcidos distais associada com núcleos picnóticos deslocados para periferia da célula. As lesões cardíacas desses bovinos foram demonstradas através da imuno-histoquímica para troponina cardíaca C (cTnC). Nos corações dos bovinos intoxicados ocorreu diminuição acentuada de cTnC no citoplasma de grupos dos cardiomiócitos com características microscópicas de degeneração e necrose e, em algumas áreas havia perda total de imunomarcação. Raras fibras musculares cardíacas sem alteração histológica apresentaram perda de imunomarcação. Nos demais cardiomiócitos dos bovinos intoxicados sem lesões histológicas observou-se intensa marcação citoplasmática. / This study describes cases of sudden death in cattle that were associated with the consumption of Amorimia (Mascagnia) exotropica and occurred in six ranches located in the mountainous region of Rio Grande do Sul and the metropolitan region of Porto Alegre, Brazil. Affected cattle were found dead with no history of previous clinical signs, or showed muscular tremors, falls, paddling, opistotonus, panting, and lateral recumbence after being induced to move, few minutes before death. Most cases were recorded between May and August. Nine cattle were necropsied and main gross findings were oral mucosa slightly cyanotic (3/9), mild to intermediate hydropericardium (3/9), epicardial petechiae and ecchymoses (5/9), clot within the left ventricle (4/9), lung edema (5/9), apart of abomasal and small intestinal reddened mucosa (6/9). Histologically, there was myocardial coagulation necrosis (9/9), which was characterized by cellular retraction, enhanced cytoplasmic eosinophilia, lack of cytoplasmic striations and occasional nuclear karyorrhexis and karyolysis. There also were interstitial edema (3/9) and interstitial inflammatory infiltrate (mainly mononuclear) (7/9) in the heart. Apart of multifocal vacuolar-hydropic degeneration in the epithelial cells of the distal convoluted tubules associated with pyknotic and eccentric nuclei in the kidneys of three cattle. The cardiac lesions of these cattle were demonstrated by immunohistochemistry for cardiac troponin C (cTnC). In the hearts of cattle intoxicated was severe reduction of the cTnC in the cytoplasm of cardiomyocytes groups with microscopic features of degeneration and necrosis, and in some areas with complete loss of immunoreactivity. Rare cardiac muscle fibers showed no histological abnormality loss of immune-marking. In the remaining cardiomyocytes in cattle poisoned histological lesions were absent, but an intense cytoplasmic staining in these cells was observed.

Esse estudo descreve casos de “morte súbita” em bovinos associados com a ingestão de Amorimia (Mascagnia) exotropica em seis propriedades rurais localizadas na região metropolitana de Porto Alegre e da serra gaúcha. Os bovinos intoxicados foram encontrados mortos sem história de sinais clínicos prévios, ou apresentaram tremores musculares, quedas bruscas, movimentos de pedalagem, opistótono, respiração ofegante e decúbito lateral, quando induzidos ao movimento poucos minutos antes da morte. Registrou-se maior número de casos entre os meses de maio e agosto. Nove bovinos foram necropsiados e os principais achados macroscópicos observados foram mucosa oral levemente cianótica (3/9), hidropericárdio leve a moderado (3/9), petéquias e equimoses no epicárdio (5/9), coágulo no interior do ventrículo esquerdo (4/9), edema pulmonar (5/9) e mucosas vermelhas no abomaso e no intestino delgado (6/9). Histologicamente havia necrose de coagulação no miocárdio (9/9) caracterizada por retração celular, aumento da eosinofilia do citoplasma com perda das estriações, núcleos em picnose e ocasionais núcleos em cariorrexia e cariólise. No coração, edema intersticial (3/9) e infiltrado inflamatório intersticial, predominantemente, mononuclear (7/9) também foram observados. Nos rins de três bovinos havia degeneração hidrópicovacuolar multifocal das células epiteliais dos túbulos contorcidos distais associada com núcleos picnóticos deslocados para periferia da célula. As lesões cardíacas desses bovinos foram demonstradas através da imuno-histoquímica para troponina cardíaca C (cTnC). Nos corações dos bovinos intoxicados ocorreu diminuição acentuada de cTnC no citoplasma de grupos dos cardiomiócitos com características microscópicas de degeneração e necrose e, em algumas áreas havia perda total de imunomarcação. Raras fibras musculares cardíacas sem alteração histológica apresentaram perda de imunomarcação. Nos demais cardiomiócitos dos bovinos intoxicados sem lesões histológicas observou-se intensa marcação citoplasmática. / This study describes cases of sudden death in cattle that were associated with the consumption of Amorimia (Mascagnia) exotropica and occurred in six ranches located in the mountainous region of Rio Grande do Sul and the metropolitan region of Porto Alegre, Brazil. Affected cattle were found dead with no history of previous clinical signs, or showed muscular tremors, falls, paddling, opistotonus, panting, and lateral recumbence after being induced to move, few minutes before death. Most cases were recorded between May and August. Nine cattle were necropsied and main gross findings were oral mucosa slightly cyanotic (3/9), mild to intermediate hydropericardium (3/9), epicardial petechiae and ecchymoses (5/9), clot within the left ventricle (4/9), lung edema (5/9), apart of abomasal and small intestinal reddened mucosa (6/9). Histologically, there was myocardial coagulation necrosis (9/9), which was characterized by cellular retraction, enhanced cytoplasmic eosinophilia, lack of cytoplasmic striations and occasional nuclear karyorrhexis and karyolysis. There also were interstitial edema (3/9) and interstitial inflammatory infiltrate (mainly mononuclear) (7/9) in the heart. Apart of multifocal vacuolar-hydropic degeneration in the epithelial cells of the distal convoluted tubules associated with pyknotic and eccentric nuclei in the kidneys of three cattle. The cardiac lesions of these cattle were demonstrated by immunohistochemistry for cardiac troponin C (cTnC). In the hearts of cattle intoxicated was severe reduction of the cTnC in the cytoplasm of cardiomyocytes groups with microscopic features of degeneration and necrosis, and in some areas with complete loss of immunoreactivity. Rare cardiac muscle fibers showed no histological abnormality loss of immune-marking. In the remaining cardiomyocytes in cattle poisoned histological lesions were absent, but an intense cytoplasmic staining in these cells was observed.