Calcium/calmodulin-dependent protein kinase II causes atrial structural remodeling associated with atrial fibrillation and heart failure.

BACKGROUND: Atrial fibrillation (AF) is sustained by reentrant mechanisms that depend, in part, on atrial structural remodeling. Increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) activity occurs in persistent AF. A general consensus has been that electrophysiological actions of CaMKII must be the contributing factor, but electrical remodeling in AF differs considerably with electrophysiological effects of CaMKII. CaMKII has been associated with structural remodeling in several tissues, but not the cardiac atria. The role of CaMKII in sustaining AF remains undefined. OBJECTIVE: The purpose of this study was to assess the effects of CaMKII on AF-related structural remodeling. METHODS: We evaluated the objective in a porcine AF-heart failure model using atrial gene transfer of the CaMKII inhibitory peptide CaMKIIn. We used conventional methods including in vivo electrophysiological study, telemetry, western blot, echocardiography, and histology to quantify rhythm, function, microstructure, and signaling pathways relevant to CaMKII and structural remodeling. RESULTS: CaMKII levels and activity increased progressively in the early stages of AF-heart failure. Inhibiting CaMKII preserved atrial contractile function and attenuated atrial hypertrophy, fibrosis, and apoptosis but did not affect inflammation or myolysis. These effects were accompanied by significantly decreased phosphorylation of HDAC4, decreased expression of p38MAP-kinase, and alterations in the phosphorylation pattern and relative ratios of JNK isoforms. CONCLUSION: Our findings suggest that CaMKII mediates signaling pathways related to atrial contractile function and structural remodeling in AF. CaMKII inhibition is potentially a novel therapy for AF. These findings are of importance because no clinically relevant mediators of either atrial contractile function or structural remodeling have yet been identified.

BRG1 and BRM function antagonistically with c-MYC in adult cardiomyocytes to regulate conduction and contractility.

RATIONALE: The contractile dysfunction that underlies heart failure involves perturbations in multiple biological processes ranging from metabolism to electrophysiology. Yet the epigenetic mechanisms that are altered in this disease state have not been elucidated. SWI/SNF chromatin-remodeling complexes are plausible candidates based on mouse knockout studies demonstrating a combined requirement for the BRG1 and BRM catalytic subunits in adult cardiomyocytes. Brg1/Brm double mutants exhibit metabolic and mitochondrial defects and are not viable although their cause of death has not been ascertained. OBJECTIVE: To determine the cause of death of Brg1/Brm double-mutant mice, to test the hypothesis that BRG1 and BRM are required for cardiac contractility, and to identify relevant downstream target genes. METHODS AND RESULTS: A tamoxifen-inducible gene-targeting strategy utilizing αMHC-Cre-ERT was implemented to delete both SWI/SNF catalytic subunits in adult cardiomyocytes. Brg1/Brm double-mutant mice were monitored by echocardiography and electrocardiography, and they underwent rapidly progressive ventricular dysfunction including conduction defects and arrhythmias that culminated in heart failure and death within 3weeks. Mechanistically, BRG1/BRM repressed c-Myc expression, and enforced expression of a DOX-inducible c-MYC trangene in mouse cardiomyocytes phenocopied the ventricular conduction defects observed in Brg1/Brm double mutants. BRG1/BRM and c-MYC had opposite effects on the expression of cardiac conduction genes, and the directionality was consistent with their respective loss- and gain-of-function phenotypes. To support the clinical relevance of this mechanism, BRG1/BRM occupancy was diminished at the same target genes in human heart failure cases compared to controls, and this correlated with increased c-MYC expression and decreased CX43 and SCN5A expression. CONCLUSION: BRG1/BRM and c-MYC have an antagonistic relationship regulating the expression of cardiac conduction genes that maintain contractility, which is reminiscent of their antagonistic roles as a tumor suppressor and oncogene in cancer.

Impact of Insurance Precertification on Discharge of Stroke Patients to Acute Rehabilitation or Skilled Nursing Facility.

BACKGROUND AND PURPOSE: The purpose of this study is to determine if the common insurance practice of requiring precertification before a medically ready stroke patient can be discharged to a skilled nursing facility (SNF) or inpatient rehabilitation facility (IRF) causes a delay in discharge. Eliminating delays in discharge of stroke patients is important given the increasing demands for health-care efficiency after the passage of the Affordable Health Care Act. METHODS: A retrospective chart review of 1007 patients who were admitted to our comprehensive stroke center with the primary diagnosis of stroke over a 12-month period was performed. Out of the patient pool, 289 patients met the inclusion criterion of a primary diagnosis of stroke that required discharge to a SNF or IRF. All 289 patients were medically cleared for discharge to a SNF or IRF by a board-certified vascular neurologist. RESULTS: Of the 289 patients who met the inclusion criteria, 118 required insurance precertification and 171 did not require precertification before being discharged to a SNF or IRF. All 118 patients who required precertification had private health insurance. The patients who required insurance precertification had an average delay of discharge (DOD) of 1.5 days, and those patients who did not require precertification had an average DOD of .8 days (P value <.0001). After removing the outliers, the difference in the length of stay (LOS) between the 2 groups became statistically significant (P value < .04). CONCLUSION: The results of this study demonstrate that insurance precertification leads to delay in discharge, increased LOS, and increased hospital costs for stroke patients.

Accumulation of intraneuronal amyloid-ß is common in normal brain.

Intraneuronal amyloid-ß (iAß) accumulation has been demonstrated in Alzheimer disease (AD). Although extracellular amyloid plaques composed primarily of aggregated amyloid-ß are one of the main pathological features of AD, functional characterization of iAß is still lacking. In this study, we identified the normal distribution of iAß through an analysis of hippocampal sections from a series of over 90 subjects with diverse antemortem clinical findings. In addition to AD cases, iAß in pyramidal neurons was readily and reproducibly demonstrated in the majority of control cases. Similar findings for controls were made across all ages, spanning from infants to the elderly. There was no correlation of iAß between gender, postmortem interval, or age. While the possible pathophysiological significance of iAß accumulation in AD remains to be elucidated, careful examination of iAß found in the normal brain may be informative for determining the biological role of iAß and how this function changes during disease. Current findings support a physiological role for iAß in neuronal function over the entire lifespan.

Transgenic overexpression of keratinocyte-specific VEGF and Ang1 in combination promotes wound healing under nondiabetic but not diabetic conditions.

VEGF and Angiopoietin (Ang)1 are growth factors that independently improve wound healing outcomes. Using a tet-repressible mouse model coupled with streptozotocin-induced diabetes, we examined wound healing in diabetic and nondiabetic mice engineered to overexpress keratinocyte-specific (K5) VEGF, Ang1 or Ang1-VEGF combined. All nondiabetic mice healed more rapidly than their diabetic counterparts; however overexpression of VEGF, Ang1 or the combination failed to improve wound closure under diabetic conditions. Conversely, under nondiabetic conditions, combining Ang1 and VEGF resulted in rapid wound closure. Molecular analyses of diabetic and nondiabetic K5-Ang1-VEGF skin revealed no differences in VEGF expression but an 80% decrease in Ang1 under diabetic conditions, suggesting an integral role for Ang1. Nondiabetic K5-Ang1 mice healed more quickly and had significant increases in granulation tissue and a 60% decrease in re-epithelialization 7 days after wounding. Furthermore, Ang1 stimulated primary mouse keratinocytes showed significantly less migration into a wound bed in an in vitro wound healing bioassay and had decreased pMAPK, pNFκB, pAkt, and pStat3 signaling. These data suggest that combined Ang1-VEGF overexpression cannot overcome diabetes-induced delays in wound healing but is efficacious under nondiabetic conditions possibly via Ang1-mediated delays in re-epithelialization and enhancement of granulation tissue formation, thereby allowing more rapid secondary intention healing.

Therapeutic potential of c-Myc inhibition in the treatment of hypertrophic cardiomyopathy.

Investigating the pathophysiological importance of the molecular and mechanical development of cardiomyopathy is critical to find new and broader means of protection against this disease that is increasing in prevalence and impact. The current available treatment options for cardiomyopathy mainly focus on treating symptoms and strive to make the patient more comfortable while preventing progression of disease and sudden death. The proto-oncogene c-Myc (Myc) has been shown to be increased in many different types of heart disease, including hypertrophic cardiomyopathy, before any signs of the disease are present. As the mechanisms of action and multiple pathways of dependent actions of Myc are being dissected by many research groups, inhibition of Myc is becoming an attractive paradigm for prevention and treatment of cardiomyopathy and heart failure. Elucidating the role Myc plays in the development, propagation and perpetuation of cardiomyopathy and heart failure will one day translate into potential therapeutics for cardiomyopathy.

The role of E2F1 in the development of hypertrophic cardiomyopathy.

The overexpression of the transcription factor, E2F1, induces hypertrophy and apoptosis with cell cycle re-entry in cardiomyocytes in vitro and in vivo, suggesting that targeting E2F1 may have therapeutic potential. Accordingly, we tested the hypothesis that blocking the E2F1-mediated signal transduction pathway prevents cardiac hypertrophy by treating E2F1 knockout mice (E2F1-/-) with either isoproterenol (ISO) or Angiotensin II (ANG). Echocardi-ography was used to measure left ventricular mass index and myocardial performance index, a measure of combined systolic and diastolic left ventricular function. In control mice (E2F1+/+) both ISO and ANG treatments induced cardiac hypertrophy, and impaired ventricular function in ANG treated mice. In contrast to previously published work, E2F1-/- mice also demonstrated a similar pattern of cardiac hypertrophy and function after either treatment. Atrial natriuretic peptide, a molecular marker of hypertrophy and necropsy-determined body weight-normalized left ventricle mass were similarly increased in ISO and ANG treated E2F1+/+ and E2F-/- mice, supporting the echocardiographic data. These data indicate that E2F1 is not necessary for the development of cardiac hypertrophy although studies using an overexpression approach suggest a causal role of E2F1. The reason for this discrepancy is unclear, although it is possible that other E2F-family members (e.g., E2F2) may play a compensatory role. In conclusion, our data demonstrate that cardiac hypertrophy can be induced in an E2F1-independent fashion and suggest that in contrast to previous reports, targeting E2F1 may not be a good therapeutic approach.

Cutaneous manipulation of vascular growth factors leads to alterations in immunocytes, blood vessels and nerves: Evidence for a cutaneous neurovascular unit.

BACKGROUND: Skin cells produce soluble factors which influence keratinocyte proliferation, angiogenesis, nerve innervation and immunocyte response. OBJECTIVE: To test the hypothesis that epidermal-dermal interactions influence neural outgrowth, vascular survival, immunocyte recruitment and keratinocyte proliferation. METHODS: We genetically manipulated the epidermis to express excess vascular endothelial growth factor (VEGF) and/or angiopoietin-1 (Ang1) and then examined the epidermal and dermal phenotypes. We compared these findings with those occurring following overexpression of the Ang1 receptor Tie2 in endothelial cells or keratinocytes. RESULTS: Keratinocyte-overexpression of Ang1 resulted in increased epidermal thickness compared to control littermates. Keratinocyte-specific overexpression of Ang1 or VEGF increased dermal angiogenesis compared to control animals and combined Ang1-VEGF lead to further increases. Cutaneous leukocyte examination revealed increases in CD4(+) T cell infiltration in mice with keratinocyte-specific overexpression of Ang1, VEGF and Ang1-VEGF combined; in contrast only keratinocyte-specific Ang1 overexpression increased cutaneous F4/80(+) macrophage numbers. Interestingly, combined keratinocyte-derived Ang1-VEGF overexpression reduced significantly the number of F4/80(+) and Cd11c(+) cells compared to mice overexpressing epidermal Ang1 alone. Endothelial cell-specific Tie2 overexpression increased dermal angiogenesis but failed to influence the epidermal and immune cell phenotypes. Keratinocyte-specific Tie2 expressing mice had the highest levels of CD4(+), CD8(+) and CD11c(+) cell numbers and acanthosis compared to all animals. Finally, increases in the number of cutaneous nerves were found in all transgenic mice compared to littermate controls. CONCLUSION: These findings demonstrate that change to one system (vascular or epidermal) results in change to other cutaneous systems and suggest that individual molecules can exert effects on multiple systems.

Cell cycle re-entry and mitochondrial defects in myc-mediated hypertrophic cardiomyopathy and heart failure.

While considerable evidence supports the causal relationship between increases in c-Myc (Myc) and cardiomyopathy as a part of a "fetal re-expression" pattern, the functional role of Myc in mechanisms of cardiomyopathy remains unclear. To address this, we developed a bitransgenic mouse that inducibly expresses Myc under the control of the cardiomyocyte-specific MHC promoter. In adult mice the induction of Myc expression in cardiomyocytes in the heart led to the development of severe hypertrophic cardiomyopathy followed by ventricular dysfunction and ultimately death from congestive heart failure. Mechanistically, following Myc activation, cell cycle markers and other indices of DNA replication were significantly increased suggesting that cell cycle-related events might be a primary mechanism of cardiac dysfunction. Furthermore, pathological alterations at the cellular level included alterations in mitochondrial function with dysregulation of mitochondrial biogenesis and defects in electron transport chain complexes I and III. These data are consistent with the known role of Myc in several different pathways including cell cycle activation, mitochondrial proliferation, and apoptosis, and indicate that Myc activation in cardiomyocytes is an important regulator of downstream pathological sequelae. Moreover, our findings indicate that the induction of Myc in cardiomyocytes is sufficient to cause cardiomyopathy and heart failure, and that sustained induction of Myc, leading to cell cycle re-entry in adult cardiomyocytes, represents a maladaptive response for the mature heart.

Altered hypoxia-inducible factor-1 alpha expression levels correlate with coronary vessel anomalies.

The outflow tract myocardium and other regions corresponding to the location of the major coronary vessels of the developing chicken heart, display a high level of hypoxia as assessed by the hypoxia indicator EF5. The EF5-positive tissues were also specifically positive for nuclear-localized hypoxia inducible factor-1 alpha (HIF-1alpha), the oxygen-sensitive component of the hypoxia inducible factor-1 (HIF-1) heterodimer. This led to our hypothesis that there is a "template" of hypoxic tissue that determines the stereotyped pattern of the major coronary vessels. In this study, we disturbed this template by altering ambient oxygen levels (hypoxia 15%; hyperoxia 75-40%) during the early phases of avian coronary vessel development, in order to alter tissue hypoxia, HIF-1alpha protein expression, and its downstream target genes without high mortality. We also altered HIF-1alpha gene expression in the embryonic outflow tract cardiomyocytes by injecting an adenovirus containing a constitutively active form of HIF-1alpha (AdCA5). We assayed for coronary anomalies using anti-alpha-smooth muscle actin immunohistology. When incubated under abnormal oxygen levels or injected with a low titer of the AdCA5, coronary arteries displayed deviations from their normal proximal connections to the aorta. These deviations were similar to known clinical anomalies of coronary arteries. These findings indicated that developing coronary vessels may be subject to a level of regulation that is dependent on differential oxygen levels within cardiac tissues and subsequent HIF-1 regulation of gene expression.

Keratinocyte but not endothelial cell-specific overexpression of Tie2 leads to the development of psoriasis.

Psoriasis is initiated and maintained through a multifaceted interplay between keratinocytes, blood vessels, gene expression, and the immune system. One previous psoriasis model demonstrated that overexpression of the angiopoietin receptor Tie2 in endothelial cells and keratinocytes led to the development of a psoriasiform phenotype; however, the etiological significance of overexpression in each cell type alone was unclear. We have now engineered two new mouse models whereby Tie2 expression is confined to either endothelial cells or keratinocytes. Both lines of mice have significant increases in dermal vasculature but only the KC-Tie2-overexpressing mice developed a cutaneous psoriasiform phenotype. These mice spontaneously developed characteristic hallmarks of human psoriasis, including extensive acanthosis, increases in dermal CD4(+) T cells, infiltrating epidermal CD8(+) T cells, dermal dendritic cells and macrophages, and increased expression of cytokines and chemokines associated with psoriasis, including interferon-gamma, tumor necrosis factor-alpha, and interleukins 1alpha, 6, 12, 22, 23, and 17. Host-defense molecules, cathelicidin, beta-defensin, and S100A8/A9, were also up-regulated in the hyperproliferative skin. All of the phenotypic traits were completely reversed without any scarring following repression of the transgene and were significantly improved following treatment with the anti-psoriasis systemic therapeutic, cyclosporin A. Therefore, confining Tie2 overexpression solely to keratinocytes results in a mouse model that meets the clinical, histological, immunophenotypic, biochemical, and pharmacological criteria required for an animal model of human psoriasis.