Micro-encapsulation of heart explant-derived stem cells (EDCs) within protective nanoporous gel (NPG) cocoons improves cardiac function and long-term retention of transplanted cells after ischemic injury by limiting detachment induced cell death and vascular clearance of intramyocardial injected cells. Although cocooned EDCs boost cardiac function, the fundamental mechanism is unclear. Here, we investigate the effects of altering cocoon stiffness and size on human EDC mediated repair of damaged myocardium using an immunodeficient mouse model of ischemic cardiomyopathy. First, we found that increasing cocoon stiffness by altering NPG content boosted cell viability and migration; effectively forcing cocooned cells to adopt a migratory, invasive phenotype. Although cocooning improved retention of transplanted cells, increasing cocoon stiffness had no additional effects on long-term engraftment despite markedly improving cardiac function and fibrosis after myocardial infarction. Given increased cocoon stiffness boosted the production and microRNA cargo within EDC nanovesicles, the observed benefits in post-ischemic function are likely dependent more on paracrine production of transplanted cells rather than simply increasing the number of cells retained. The effect of cocoon diameter on EDC phenotype and cell mediated repair of ischemic myocardium was evaluated using microfluidic-based cocooning enabling deterministic encapsulation within defined cocoon size and intracapsular cell number while maintaining a fixed cocoon stiffness. Increased cocoon size enhanced post-ischemic cardiac function by reducing clearance of transplanted cells and increased paracrine stimulation of endogenous repair. The latter being attributable to microfluidic cocooning closely following the expected Poisson distribution with smaller cocoons having a greater proportion of single cells while larger cocoons contained greater proportions of multicellular aggregates which enhanced cell-cell interactions to increase the amount and breadth of cytokines/nanoparticles delivered to injured myocardium. In conclusion, altering the biophysical properties of NPG surrounding cocooned cells provides a straightforward means of boosting the regenerative potential of heart EDCs for repair of injured myocardium.

Introdução: A cardiomiopatia dilatada é uma das principais causas de insuficiência cardíaca com alta morbidade e mortalidade. Alterações genéticas em mais de 29 genes já foram relacionadas com a doença, contudo, elas explicam apenas uma pequena porcentagem dos casos sugerindo haver então outros genes relacionados com a doença. Foram relacionados para o presente projeto quatro genes candidatos, previamente relacionados com outras doenças cardíacas, para verificação de uma possível associação com a cardiomiopatia dilata idiopática. Objetivos: Avaliar a presença e frequência de mutações nos genes ACTC1, CSRP3, FKBP1A e FKBP1B de pacientes com cardiomiopatia dilatada do Instituto do Coração de São Paulo (InCor, FMUSP), investigar se há correlações entre o genótipo e o fenótipo e estudar as alterações funcionais desencadeadas pelas mutações encontradas. Métodos: Amostras de DNA de 186 pacientes com cardiomiopatia dilatada idiopática foram selecionados de um banco de dados do Instituto do Coração e triados geneticamente para alterações nos genes selecionados. Resultados e Discussão: Foram encontradas nove novas variantes genéticas. Cinco delas também estavam presentes no grupo controle, sendo excluídas como causativas da doença. Três delas não estavam presentes no grupo controle, contudo, dados de bioinformática avaliaram as alterações com um risco baixo de serem causativas. Uma alteração no gene CSRP3, que levava a troca de aminoácido, não estava presente no grupo controle e apresentou dados indicativos de mutação causativa da doença. Lâminas cardíacas foram avaliadas para verificação de possíveis dos mecanismos de ação, contudo, houve apenas a exclusão de alguns mecanismos previamente descritos na literatura. Conclusões: Não há evidências de que as alterações nos genes ACTC1, FKBP1A e FKBP1B estariam associadas com o desenvolvimento da doença. A alteração no gene CSRP3 também é capaz de causar cardiomiopatia dilatada idiopática / Introduction: Dilated Cardiomyopathy is one of the leading causes of heart failure with high morbidity e mortality. Already known genetic alterations explain little percentage of cases suggesting that other genes would be related with the disease. Four candidates genes previously related with other cardiac diseases were selected for the project to verify a possible relationship with dilated cardiomyopathy. Objectives: Evaluate the presence and frequency of genetic alterations in ACTC1, FKBP1A, FKBP1B and CSRP3 genes in dilated cardiomyopathy patients of São Paulo Heart Institute (Incor, FMUSP). To investigate for genotype/phenotype correlations and to study functional alterations triggered by the found mutations. Methods: DNA samples from 186 patients with dilated cardiomyopathy were selected from the Incor DNA bank and genetically screened for alterations in the selected genes. Results and Discussion: Nine new genetic variants were found. Five of them were present in the control population, thus being excluded as disease causative. Three of them were not present in the control population; however, bioinformatics analysis evaluated the alterations having a low risk of being causatives. One alteration in CSRP3 gene that resulted in amino-acid change was not present in control population and exhibit indicative data of disease causing mutation. Analysis of patient heart showed no difference from some disease mechanisms described in literature. Conclusions: There are no evidences that alterations in ACTC1, FKBP1A and FKBP1B genes would associated with disease development. Alteration in CSRP3 gene is also capable to cause dilated cardiomyopathy s

Background: Hypertrophic Cardiomyopathy (HCM) and Long QT Syndrome (LQTS) are genetic cardiovascular diseases that cause sudden cardiac death. When an individual is diagnosed with an inherited disease such as HCM/LQTS it is critical that their biological relatives are notified of their increased risk. Newly diagnosed individuals in turn notify other at-risk family members through a successive process called cascade screening. This facilitates screening of at-risk biological relatives through genetic testing and/or clinical testing, and treatment for HCM/LQTS prior to development of life-threatening complications. However, for cascade screening to detect all potential cases the disease risk must be effectively communicated to all at-risk relatives. The responsibility for notifying family members of this risk largely falls to the first person diagnosed in the family (proband). Empiric evidence suggests that around half of at-risk relatives are not screened in accordance with cascade screening recommendations, potentially due to information about HCM/LQTS risk not being communicated effectively in their families. Factors have been identified that influence communication about genetic risk in families with non-cardiac disease; however, it is not known if or how these factors apply in families with genetic cardiac disease. These include network factors, which describe characteristics of relationships between family members and non-network factors, which describe characteristics of individuals including individual factors, disease factors, and sociocultural factors. There is a critical need to understand communication in families with HCM/LQTS in order to facilitate effective genetic risk communication in families, improve adherence to cascade screening recommendations, and prevent death and complications from cardiovascular diseases. Objectives: The purpose of this study was to improve our understanding of the relationships among network and non-network factors and communication of genetic risk for HCM/LQTS between probands and their relatives. I proposed the following aims: Aim 1: Describe family social network structures and communication paths about risk for HCM/LQTS from probands to their relatives. Aim 2: Identify which network and non-network factors are associated with who is told about risk for HCM/LQTS. Methods: The sample for this study included individuals with HCM or LQTS recruited through the University of Iowa Cardiology Clinics (UI) and the University of Wisconsin Inherited Arrhythmia Clinic (UW). Data were collected using a structured interview, family pedigree, and survey. Analysis included egocentric social network analysis, descriptive, bivariate, and multilevel logit regression modeling. Results: Participants in this study had an average of 24 living at-risk relatives in their families. Overall, just over half (52%) of these at-risk relatives had been reported to have been told about their risk. However, within families, the percentage of relatives told about their risk ranged from 0%-100%. Ninety percent of first-degree relatives were told about their risk, 61% of second-degree relatives were told and 33% of third-degree relatives were told. Recruitment site affiliation was determined to be a confounder and so analyses were calculated separately for UI and UW. In both the UI and UW samples, network factors including closer geographic distance, increased emotional closeness, increased relationship quality, increased frequency of communication, higher betweenness centrality, and closer degree of biological relation were independently associated with increased odds of communication of risk. In the UI sample, non-network factors that were independently associated with increased odds of communication of risk included younger age at diagnosis; having LQTS; having positive genetic test results; having an ICD; younger current age; being female; having increased role limitations due to physical functioning; feeling anxious about telling family members about risk; feeling communication was a burden; feeling that communication was a responsibility or duty; being happy to be able to share important information; and identifying financial issues, pregnancies, or upcoming marriages as playing a role in communication. In a multivariate model, increased frequency of communication, closer degree of biological relation, having an ICD, and identifying financial issues and pregnancies as contributors to communication were significantly associated with communication of genetic risk information. In the UW sample, non-network factors that were independently associated with increased odds of communication of risk included younger age, decreased emotional wellbeing, increased role limitations due to emotional wellbeing, and decreased energy and fatigue. In a multivariate model, increased frequency of communication and closer degree of biological relation were significantly associated with communication. Although over half of at-risk relatives were told about their risk, just over half of those (53.8%) were reported to have screened for disease, which represents 27% of all at-risk relatives. Of those tested, 35% were reported as diagnosed with HCM/LQTS. Conclusion: Communication of genetic risk for HCM/LQTS in families is inadequate and contributes to the problem of relatives not being screened for disease. Insight on the factors that influence communication in families at risk of sudden cardiac death can guide development of interventions, policies, and future research aimed at improving genetic risk communication and cascade screening, and preventing death and complications from inherited cardiac diseases. This research is applicable for genetic conditions where population based screening methods are not effective and rely on families to communicate risk and need for screening.

Diabetic cardiomyopathy characterized by left ventricular hypertrophy predisposes diabetic and obese individuals to development of cardiac dysfunction and subsequently to heart failure. Whether hyperinsulinemia has an underlying role in development and or progression of diabetic heart disease is not well understood. We therefore studied the effects of acute hyperinsulinemia on cardiac function in euglycemic states. Acute hyperinsulinemia neither affected baseline nor inotropic response to β-adrenergic stimulation. Previous studies from our laboratory have indicated a potential role for GRK2, a serine threonine kinase in development of cardiac dysfunction in diabetic states in humans as well as in mice. To assess whether GRK2 mediates the detrimental effects of chronic hyperinsulinemia on cardiac dysfunction in mouse model of diet induced obesity, we utilized cardiomyocyte knockout of GRK2. Our results suggested lack of cardiac functional impairments in high fat fed wildtype mice, which hindered our attempts to ascertain the role of GRK2 in diabetic cardiomyopathy. Mouse models of diet induced obesity have been routinely used to study the effects of obesity and diabetes on cardiac dysfunction but recent evidence from multiple research groups has emphasized the need for evaluation of the utility and relevance of the murine diet induced obesity model for studying cardiovascular abnormalities associated with hyperinsulinemic states, including T2DM and obesity. We therefore studied the effect of chronic fat feeding (>20 weeks) alone or in combination with concomitant hypertension on cardiac function in C57BL/6J mice. Different diets were formulated with either lard (32% saturated fat, 68% unsaturated fat) or hydrogenated coconut oil (95% saturated fat) as the source of fat and fatty acids, which contributed 60% of total calories. Insulin resistance and glucose intolerance were readily observed in mice fed a high fat diet in each of the studies. HFD resulted in the development of cardiac hypertrophy; however cardiac function as measured by B-mode echocardiography and LV catheterization was unaffected in high fat diet groups compared to their respective control diet groups. Further, dietary fat feeding regardless of the source of fat modestly altered the gene expression of a few pathological hypertrophic markers or of fibrosis related genes. However, there was an increase in expression of PPARa target genes such as Pdk4 and fatty acid metabolism genes including CD36, AcadL and Cpt1b. Cardiac mitochondrial function as assessed by oxygen consumption rates, ATP synthesis rates and reactive oxygen species production rates were unaltered in high fat diet fed mice. These results suggest that while chronic fat feeding in mice causes cardiac hypertrophy and potentially cardiometabolic remodeling, it might not be sufficient to activate pathological hypertrophic mechanisms that impair cardiac function and cause cardiac fibrosis.

Lipotoxic cardiomyopathy increases the risk of heart failure in obese patients by adversely altering heart structure and function via toxic lipid specie mediated cellular stress and cell death. Increased fatty acid uptake and esterification in cardiomyocytes increases toxic lipid intermediates. These cardiotoxic lipid species such as diacylglycerol have recently been shown to deacidify lysosomes in cardiomyocytes by activating protein kinase C βII mediated NADPH oxidase 2 generation of superoxide that inhibits proton pumps on lysosomal membranes by S-nitrosylation. Autophagy, a lysosome dependent cellular survival process, is impaired upon cardiomyocyte lipid-overload due to inhibition of pH-dependent proteolytic autophagosome degradation in the lysosome. Subsequent accumulation of autophagic vesicles heightens cardiomyocyte sensitization to additional stresses of ischemia-reperfusion or ER dysfunction, culminating in impaired cardiac metabolic flexibility leading to cell death. Low cardiomyocyte regenerative capacity calls for strategies to preserve cell number in states of increased stress, such as lipid-induced impairment of autophagy. Lysosome-targeted reacidifying devices can provide an effective means to restore autophagic flux. In this thesis, a therapeutic strategy utilizing poly(DL-lactide-co-glycolide) (PLGA) nanoparticle degradation to reacidify lysosomes and revert cardiotoxic lipid specie induced blockade in autophagic flux in cardiomyocytes is presented. Endocytosed PLGA acidic nanoparticles were designed to rapidly degrade and release acidic monomers in lysosomes to restore pH dependent phosphatase and cathepsin L activity in cardiomyocytes with acute lipotoxicity. Optimized pre-palmitate treatment periods demonstrated that PLGA nanoparticles with polyethylenimine cationic surface coatings provide an effective restoration of autophagic flux in the presence of lipid-overload modeled by acute palmitate treatment in cardiomyocytes.

No description available.

Systemic amyloidoses are a group of lethal diseases where proteins aggregate into fibrillar structures, called amyloid fibrils, that deposits throughout the body. Transthyretin (TTR) causes one type of amyloidosis, in which the aggregates mainly infiltrate nervous and cardiac tissue. Almost a hundred different mutations in the TTR gene are known to trigger the disease, but wild-type (wt) TTR is also incorporated into the fibrils, and may alone form amyloid. Patients with the TTRV30M mutation show, for unknown reasons, two clinical phenotypes. Some have an early onset of disease without cardiomyopathy while others have a late onset and cardiomyopathy. It has previously been described that amyloid fibrils formed from TTRV30M can have two different compositions; either with truncated molecules beside full-length TTR (type A) or only-full-length molecules (type B).  In this thesis, the clinical importance of the two types of amyloid fibrils was investigated. We found that the fibril composition types are correlated to the two clinical phenotypes seen among TTRV30M patients, with type A fibrils present in late onset patients and type B fibrils in early onset patients. The only treatment for hereditary TTR amyloidosis has been liver transplantation, whereby the liver producing the mutant TTR is replaced by an organ only producing wt protein. However, in some patients, cardiac symptoms progress post-transplantationally. We demonstrated that the propensity to incorporate wtTTR differs between fibril types and tissue types in TTRV30M patients, with cardiac amyloid of type A having the highest tendency. This offers an explanation to why particularly cardiac amyloidosis develops after transplantation, and suggests which patients that are at risk for such development. By examining patients with other mutations than TTRV30M, we showed that, in contrast to the general belief, a fibril composition with truncated TTR is very common and might even be the general rule. This may explain why TTRV30M patients often have a better outcome after liver transplantation than patients with other mutations. In conclusion, this thesis has contributed with one piece to the puzzle of understanding the differences in clinical phenotype and treatment response between TTR amyloidosis patients, by demonstrating corresponding differences at a molecular level.

An accumulation of sudden unexpected cardiac deaths (SUCD) occurred in young Swedish orienteers, most of whom were elite athletes. From 1979 to 1992 the incidence in 18 to 34 year old male elite orienteers ranked on the national level the same year as death was calculated to 30 (per 100,000), which represents a 20 to 40 fold increase from the expected rate. From 1989 to 1992, the incidence was 50. There were, however, no indications on any similar clusters of SUCD in other sports. A special program to alter behaviour in orienteers was implemented in 1992-1993, after which there have been no more cases of SUCD in orienteers below 35 years of age. A histopathological re-evaluation of 16 cases of SUCD revealed myocarditis in 75% of these cases. In parallel, four of those cases also had changes mimicing arrhythmogenic right ventricular cardiomyopathy (ARVC). The combination of an increased incidence and myocarditis suggested that infection may be a pathogenetic factor. A broad search for different microorganisms in archival sera from five cases and tissues from the autopsies in two of those cases revealed the only common finding that all had antibodies to Chlamydia pneumoniae. DNA from C. pneumoniae was detected in the lung and heart in one of two cases. The intimate contact with nature of orienteers suggested possible zoonotic/vectorborne pathogens. Bartonella is such a pathogen and known to cross-react with C. pneumoniae. The use of PCR to test for DNA from the gltA gene of Bartonella in the two formerly mentioned cases of SUCD, and in three additional cases, gave positive bands from the hearts in four cases and the lung in a fifth case. The PCR products were sequenced and found to be identical to B. henselae in three cases and almost identical to B. quintana in the remaining two cases. Four of the five cases had antibodies to Bartonella when using micro immunofluorescence test with the antigens B. henselae, B. quintana, and B. elizabethae. The total prevalence of antibodies to Bartonella was 31% in 1,136 elite orienteers vs. 6.8% in 322 healthy blood donors (p<0.001), suggesting widespread exposure in the elite. It is hypothesized that subacute or reactivated Bartonella infection has a pathogenetic role in SUCD in orienteers, and may be involved in the development of ARVC-like disease.

Structural barriers can promote discordant action potential (AP) duration alternans, T wave alternans (TWA) and tachyarrhythmia in animal hearts and simulation studies. We hypothesized that heterogeneous scar (gray zone) and dense midwall scar (midwall core) would promote TWA in patients with cardiomyopathy by slowing conduction and uncoupling transmural APs, respectively. Scar core and gray zone were quantified in 40 cardiomyopathy patients using late gadolinium enhanced cardiac magnetic resonance imaging and related to the results of a clinically validated TWA test. The percentages of gray zone, epicardial core and midwall core were greater in the +TWA group, correlated with TWA magnitude and related to a lower heart rate onset for TWA. These specific scar patterns contribute to the genesis and severity of TWA in cardiomyopathy. Greater knowledge of the substrates that promote TWA in cardiomyopathy patients is valuable in determining those at risk of lethal ventricular arrhythmias.

Immunoglobulin-mediated inhibition of amyloid fibril formation in vivo is a promising strategy for the treatment of protein misfolding diseases such as the amyloidoses. Here we focus on transthyretin amyloidoses, a group of protein conformation diseases caused by the misfolding of the serum protein transthyretin into fibrillar structures that deposit in specific organs and tissues—often with serious pathological consequences. Using a structure-guided immunological approach, we report a novel antibody that selectively recognizes monomeric, misfolded conformations of transthyretin in vitro. Raised to an epitope normally buried in the native form of transthyretin, this antibody was found to suppress transthyretin fibrillogenesis at substoichiometric concentrations in vitro. Overall, the selectivity and inhibitory nature of the antibody signals the potential use of conformation specific antibodies in the diagnosis and treatment of transthyretin amyloidoses, conditions which remain difficult to treat and are widely under/misdiagnosed at the current time.