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Arrhythmogenic right ventricular cardiomyopathy is a primary heart muscle disorder characterized by the early occurrence of arrhythmias often out of proportion to the extent of structural remodeling and contractile derangement. Approximately 40% of patients with arrhythmogenic right ventricular cardiomyopathy have one or more mutations in genes encoding proteins in desmosomes, intercellular adhesion junctions which, in cardiac myocytes, reside within intercalated disks. Some desmosomal proteins fulfill roles both as structural proteins in cell–cell adhesion junctions and as signaling molecules in pathways mediated by Wnt ligands. Evidence is increasing that mutations in desmosomal proteins can perturb the normal balance of critical proteins in junctions and the cytosol which, in turn, could alter gene expression by circumventing normal Wnt signaling pathways. This review highlights recent advances in understanding the pathogenesis of arrhythmogenic right ventricular cardiomyopathy and presents evidence suggesting that the disease is caused by a combination of altered cellular biomechanical behavior and altered signaling.

Background— Although cellular redox balance plays an important role in mechanically induced cardiac hypertrophy, the mechanisms of regulation are incompletely defined. Because thioredoxin is a major intracellular antioxidant and can also regulate redox-dependent transcription, we explored the role of thioredoxin activity in mechanically overloaded cardiomyocytes in vitro and in vivo. Methods and Results— Overexpression of thioredoxin induced protein synthesis in cardiomyocytes (127±5% of controls, P P P P P P P Conclusions— Thus, even though thioredoxin is an antioxidant, activation of thioredoxin participates in the development of pressure-overload cardiac hypertrophy, demonstrating the dual function of thioredoxin as both an antioxidant and a signaling protein. These results also support the emerging concept that the thioredoxin inhibitor Txnip is a critical regulator of biomechanical signaling.

Aims The majority of patients diagnosed with arrhythmogenic right ventricular cardiomyopathy (ARVC) have mutations in genes encoding desmosomal proteins, raising the possibility that abnormal intercellular adhesion plays an important role in disease pathogenesis. We characterize cell mechanical properties and molecular responses to oscillatory shear stress in cardiac myocytes expressing mutant forms of the desmosomal proteins plakoglobin and plakophilin, which are linked to ARVC in patients. Methods and Results Cells expressing mutant plakoglobin or plakophilin showed no differences in cell-cell adhesion relative to controls, while knocking down these proteins weakened cell-cell adhesion. However, cells expressing mutant plakoglobin failed to increase the amount of immunoreactive signal for plakoglobin or N-cadherin at cell-cell junctions in response to shear stress, as seen in control cells. Cells expressing mutant plakophilin exhibited a similar attenuation in the shear-induced increase in junctional plakoglobin immunoreactive signal in response to shear stress, suggesting the phenotype is independent of the type of mutant protein being expressed. Cells expressing mutant plakoglobin also showed greater myocyte apoptosis compared to controls. Apoptosis rates increased greatly in response to shear stress in cells expressing mutant plakoglobin, but not in controls. Abnormal responses to shear stress in cells expressing either mutant plakoglobin or plakophilin could be reversed by SB216763, a GSK3β inhibitor. Conclusions Desmosomal mutations linked to ARVC do not significantly affect cell mechanical properties, but cause myocytes to respond abnormally to mechanical stress through a mechanism involving GSK3β. These results may help explain why patients with ARVC experience disease exacerbations following strenuous exercise.

Germline loss‐of‐function BHD mutations cause cystic lung disease and hereditary pneumothorax, yet little is known about the impact of BHD mutations in the lung. Folliculin (FLCN), the product of the Birt–Hogg–Dube (BHD) gene, has been linked to altered cell–cell adhesion and to the AMPK and mTORC1 signaling pathways. We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho‐S6, a marker of mTORC1 activation, highlighting the cell type–dependent functions of FLCN. Cell–cell adhesion forces were significantly increased in FLCN‐deficient HBE cells, consistent with prior findings in FLCN‐deficient human kidney‐derived cells. To determine how these altered cell–cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd+/− mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg. Our results indicate that FLCN regulates the AMPK and mTORC1 pathways and cell–cell adhesion in a cell type–dependent manner. FLCN deficiency may impact the physiologic response to inflation‐induced mechanical stress, but further investigation is required. We hypothesize that FLCN‐dependent effects on signaling and cellular adhesion contribute to the pathogenesis of cystic lung disease in BHD patients., We found that downregulation of FLCN in human bronchial epithelial (HBE) cells decreased the phosphorylation of ACC, a marker of AMPK activation, while downregulation of FLCN in small airway epithelial (SAEC) cells increased the activity of phospho‐S6, a marker of mTORC1 activation, highlighting the cell type–dependent functions of FLCN. Cell–cell adhesion forces were significantly increased in FLCN‐deficient HBE cells, consistent with prior findings in FLCN‐deficient human kidney‐derived cells. To determine how these altered cell–cell adhesion forces impact the lung, we exposed mice with heterozygous inactivation of Bhd (similarly to humans with germline inactivation of one BHD allele) to mechanical ventilation at high tidal volumes. Bhd+/− mice exhibited a trend (P = 0.08) toward increased elastance after 6 h of ventilation at 24 cc/kg.

Currently, many diabetic cardiomyopathy (DC) studies focus on either in vitro molecular pathways or in vivo whole-heart properties such as ejection fraction. However, as DC is primarily a disease caused by changes in structural and functional properties, such studies may not precisely identify the influence of hyperglycemia or hyperlipidemia in producing specific cellular changes, such as increased myocardial stiffness or diastolic dysfunction. To address this need, we developed an in vitro approach to examine how structural and functional properties may change as a result of a diabetic environment. Particle-tracking microrheology was used to characterize the biomechanical properties of cardiac myocytes and fibroblasts under hyperglycemia or hyperlipidemic conditions. We showed that myocytes, but not fibroblasts, exhibited increased stiffness under diabetic conditions. Hyperlipidemia, but not hyperglycemia, led to increased cFos expression. Although direct application of reactive oxygen species had only limited effects that altered myocyte properties, the antioxidant N-acetylcysteine had broader effects in limiting glucose or fatty-acid alterations. Changes consistent with clinical DC alterations occur in cells cultured in elevated glucose or fatty acids. However, the individual roles of glucose, reactive oxygen species, and fatty acids are varied, suggesting multiple pathway involvement.

Background —Overloading the left ventricle in systole (pressure overload) is associated with a distinct morphological response compared with overload in diastole (volume overload). Methods and Results —We designed a novel computer-controlled experimental system that interfaces biaxially uniform strain with electrical pacing, so that cellular deformation can be imposed during a specified phase of the cardiac cycle. Cardiomyocytes were exposed to strain (4%) during either the first third (systolic phase) or last third (diastolic phase) of the cardiac cycle. Strain imposed during the systolic phase selectively activated p44/42 mitogen-activated protein kinase (MAPK) and MAPK/extracellular signal–regulated protein kinase kinase (MEK1/2, an activator of p44/42 MAPK) compared with strain imposed during the diastolic phase. In contrast, there was no difference in activation of p38 and c-Jun NH 2 -terminal kinases induced by strain imposed during the systolic phase (5.8- and 3.3-fold versus control, n=4) compared with the diastolic phase (5.5- and 3.1-fold). Induction of both brain natriuretic peptide (5.8-fold versus control, P P 3 H]leucine incorporation induced by strain imposed during the systolic phase (4.0-fold versus control) was greater than during the diastolic phase (2.7-fold, P Conclusions —Mechanical activation of p44/42 MAPK and MEK1/2, gene expression, and protein synthesis is regulated by the cardiac cycle, suggesting that mechanotransduction at the cellular level may underlie differences between pressure and volume overload of the heart.

Background —Increased biomechanical stresses in the fibrous cap of atherosclerotic plaques contribute to plaque rupture and, consequently, to thrombosis and myocardial infarction. Thin fibrous caps and large lipid pools are important determinants of increased plaque stresses. Although coronary calcification is associated with worse cardiovascular prognosis, the relationship between atheroma calcification and stresses is incompletely described. Methods and Results —To test the hypothesis that calcification impacts biomechanical stresses in human atherosclerotic lesions, we studied 20 human coronary lesions with techniques that have previously been shown to predict plaque rupture locations accurately. Ten ruptured and 10 stable lesions derived from post mortem coronary arteries were studied using large-strain finite element analysis. Maximum stress was not correlated with percentage of calcification, but it was positively correlated with the percentage of lipid ( P =0.024). When calcification was eliminated and replaced with fibrous plaque, stress changed insignificantly; the median increase in stress for all specimens was 0.1% (range, 0% to 8%; P =0.85). In contrast, stress decreased by a median of 26% (range, 1% to 78%; P =0.02) when lipid was replaced with fibrous plaque. Conclusions —Calcification does not increase fibrous cap stress in typical ruptured or stable human coronary atherosclerotic lesions. In contrast to lipid pools, which dramatically increase stresses, calcification does not seem to decrease the mechanical stability of the coronary atheroma.

The diagnosis of arrhythmogenic right ventricular cardiomyopathy (ARVC) can be challenging because the clinical presentation is highly variable and genetic penetrance is often low.To determine whether a change in the distribution of desmosomal proteins can be used as a sensitive and specific diagnostic test for ARVC, we performed immunohistochemical analysis of human myocardial samples.We first tested myocardium from 11 subjects with ARVC; of these samples, 8 had desmosomal gene mutations. We also tested myocardium obtained at autopsy from 10 subjects with no clinical or pathological evidence of heart disease as control samples. All ARVC samples but no control samples showed a marked reduction in immunoreactive signal levels for plakoglobin (also known as gamma-catenin), a protein that links adhesion molecules at the intercalated disk to the cytoskeleton. Other desmosomal proteins showed variable changes, but signal levels for the nondesmosomal adhesion molecule N-cadherin were normal in all subjects with ARVC. To determine whether a diminished plakoglobin signal level was specific for ARVC, we analyzed myocardium from 15 subjects with hypertrophic, dilated, or ischemic cardiomyopathies. In every sample, levels of N-cadherin and plakoglobin signals at junctions were indistinguishable from those in control samples. Finally, we performed blinded immunohistochemical analysis of heart-biopsy samples from the Johns Hopkins ARVC registry. We made the correct diagnosis in 10 of 11 subjects with definite ARVC on the basis of clinical criteria and correctly ruled out ARVC in 10 of 11 subjects without ARVC, for a sensitivity of 91%, a specificity of 82%, a positive predictive value of 83%, and a negative predictive value of 90%. The plakoglobin signal level was reduced diffusely in ARVC samples, including those obtained in the left ventricle and the interventricular septum.Routine immunohistochemical analysis of a conventional endomyocardial-biopsy sample appears to be a highly sensitive and specific diagnostic test for ARVC.

One of the challenges in labeling tissues for fluorescence microscopy is minimizing sample processing while maintaining or improving the information generated by the fluorescent label. Generally, tissues are extracted, fixed, and embedded in mounting media (such as paraffin), sectioned, and then postprocessed by removing the paraffin, blocking, labeling, and washing. Despite all of these steps, the consistency of labeling quality can vary as a result of several factors, including heterogeneity in labeling efficiency from slide to slide, the necessity of postprocessing to obtain information on sequential sections of tissue, interference from the mounting media, and loss of native three-dimensional structural information, especially in thicker sections. A method for embedding and processing tissues that have been labeled by intravital staining is described in this study. Intravital staining is the process in which live-cell dyes and other labels are injected into the bloodstream before fixation of the tissues. Tissues processed this way can be imaged upon sectioning without further staining and retain their native, three-dimensional information, thereby improving the information retained by the labels and speeding up sample processing.

Three-dimensional (3-D) tissue imaging offers substantial benefits to a wide range of biomedical investigations from cardiovascular biology, diabetes, Alzheimer's disease to cancer. Two-photon tissue cytometry is a novel technique based on high-speed multiphoton microscopy coupled with automated histological sectioning, which can quantify tissue morphology and physiology throughout entire organs with subcellular resolution. Furthermore, two-photon tissue cytometry offers all the benefits of fluorescence-based approaches including high specificity and sensitivity and appropriateness for molecular imaging of gene and protein expression. We use two-photon tissue cytometry to image an entire mouse heart at subcellular resolution to quantify the 3-D morphology of cardiac microvasculature and myocyte morphology spanning almost five orders of magnitude in length scales.

Background: Ex vivo T-cell depletion (TCD) by CD34+ selection has been used successfully for graft-versus-host disease prevention alleviating the need for additional pharmacologic immunosuppressants. TCD has been associated with higher rates of viral infections and cytomegalovirus (CMV) disease in particular compared to conventional (CONV) HCT. Modern methods of TCD achieve up to 5 log reduction of T-cells. At MSK, TCD HCT recipients (R) are routinely monitored for CMV, adenovirus (ADV), Epstein-Barr virus (EBV), and human herpes virus 6 (HHV6) in the blood by quantitative polymerase chain reaction (qPCR) assays. We report the incidence of viremia and end organ disease (EOD) by these viruses in TCD HCT recipients. Methods: Retrospective review of adults with acute leukemia (AL) and myelodysplastic syndrome (MDS) patients (pts) who received TCD HCT at MSK from June 2010 -December 2014. TCD was performed by the FDA approved CliniMACS CD34+ reagent system (Miltenyi Biotec, Gladbach, Germany). CMV was monitored at least weekly from day 14-100 and at least every 14 days until day 180. EBV was monitored through day 180. Monitoring for CMV and EBV was performed by the Clinical Microbiology Laboratory at MSK using Roche analyte specific reagents, and since March 2013, the Cobas Ampliprep/Cobas Taqman (CAP/CTM) CMV qPCR in plasma. Monitoring for ADV/HHV6 was performed by Viracor-IBT laboratories (Lenexa, KS). Preemptive treatment for CMV was started for ≥2 consecutive PCR >500copies/ml in whole blood or >300 IU/ml in plasma. Treatment of ADV/HHV6 viremia was at clinician's discretion. EBV post-transplant lymphoproliferative disorder (PTLD) was treated with rituximab as primary therapy. During the study, 11 pts were enrolled in CMV prevention trials of brincidofovir (BCV), and 5 pts received BCV for treatment of ADV. EOD was scored by standard criteria. Follow up was through June 2015. The cumulative incidence (CI) was estimated for viremia at 180 days and EOD at 365 days for CMV, ADV, HHV6, and EBV. Kaplan-Meier method and log-rank test were used to compare CI between groups. P Results: Of239 pts (median age 56.3 years, range 19.6-73.3), 161 (67%) pts had AL and 78 (33%) pts had MDS. Donors were matched-related 76 (32%), matched unrelated 116 (48%), or mismatched (related or unrelated) 47(20%); CMV R+ 148 (62%) or R- 91 (38%). CMV infection had the earliest onset post HCT at a median of 28 days (d) (IQR: 25-33), followed by HHV6 33d (25-52), ADV 71d (44-89), and EBV 85d (58-107). 188 (79%) pts had ≥1 dsDNA viral infection with 182 (76%) pts experiencing infection prior to d100. The CIs of viremia were: CMV 42% (68% in R+ vs 0% in R-, P Conclusions: 1) Viremias by dsDNA viruses occurred frequently and early post TCD HCT: 189 (79%) pts had at least 1 dsDNA viral infection, with onset of first infection before d100 in 183 (77%) pts. Around 58% of pts with CMV had coinfections with additional dsDNA virus(es). 2) Two thirds (67%) of CMV R+ and 41% of total pts received CMV antiviral treatment. 3) Overall, 18% of patients developed end organ disease by dsDNA viruses. 4) Viremias were managed effectively with intense monitoring and antiviral treatment, and rates of EOD were comparable to those of CONV HCT. Our data highlights the need for effective strategies to reduce the total burden of dsDNA viruses after TCD HCT. Figure 1. CIs of dsDNA viremia. Figure 1. CIs of dsDNA viremia. Figure 2. dsDNA viral co-infections Figure 2. dsDNA viral co-infections Figure 3. Coinfections among pts with CMV viremia Figure 3. Coinfections among pts with CMV viremia Disclosures Perales: Merck: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Giralt:TAKEDA: Consultancy, Honoraria, Research Funding; JAZZ: Consultancy, Honoraria, Research Funding, Speakers Bureau; AMGEN: Consultancy, Research Funding; SANOFI: Consultancy, Honoraria, Research Funding; CELGENE: Consultancy, Honoraria, Research Funding.

Background: Streptococcus pneumoniae is a major cause of sepsis, pneumonia and meningitis leading to significant morbidity and mortality. Invasive pneumococcal disease (IPD) and mortality rates in cancer patients (pts) are higher than in the general population. Since the introduction of routine childhood immunization with the 7-valent pneumococcal conjugate vaccine (PREVNAR) in 2000, the overall rate of invasive pneumococcal disease (IPD) decreased in adults in the US. The impact of PREVNAR on rates of IPD in cancer pts is not well studied. Methods: Retrospective review of pts treated at Memorial Sloan-Kettering Cancer Center from 1991-2012. Unique patient visits per year were obtained from the hospital database. Prevalence was calculated as number of cases per 1000 patient-visits per year. Pts with positive culture(s) for S. pneumoniae were identified from microbiology records. Serotype data is available through 2001. IPD was defined as a positive S. pneumoniae culture from a sterile site such as blood, CSF, or pleural fluid culture. Pneumococcal pneumonia was defined as a positive S. pneumoniae culture from thelower respiratory tract or a positive upper respiratory culture with either radiographic evidence for pneumonia and/or ICD-9 code "pneumococcal pneumonia" or "pneumonia" associated with the positive culture for S. pneumoniae. Colonization was defined as positive culture for S. pneumoniae without IPD or pneumonia. We compared prevalence between "early" (prior to PREVNAR, 1992-2001) and "late" (2002-2012) periods, in those with diagnoses of solid tumors and hematologic malignancies, and between ages ≤21 and >21 years. Pneumococcal vaccination rates were calculated as the number of patients who received any pneumococcal vaccine divided by the number of patient-visits per year. Results: The mean number of patient-visits per year was 46,394 and 96,954, for the early and late period, respectively. Of 1,033 patients with ≥1 culture positive for S. pneumoniae during the study, 329 (32%) had IPD, 394 (38%) had pneumococcal pneumonia and 310 (30%) had colonization. S. pneumoniae type 6 accounted for 39 (22%) isolates where serotype data was available. The annual prevalence of IPD, pneumonia and colonization gradually declined over the course of the study (Figure 1). Compared to the early period, the prevalence of IPD, pneumonia and colonization were lower in the late period (Table 1). The highest prevalence of IPD was observed in pts ≤21 years of age (2.8 and 1.3 for early and late periods, respectively). The prevalence of IPD in pts with hematologic malignancies was 5-fold higher than pts with solid tumors in the early period (1.8 vs 0.39 respectively; P Conclusions: 1) Over a 20 year period we observed a decline in the prevalence of IPD, pneumococcal pneumonia and colonization in cancer patients at a tertiary cancer center in NYC. 2) The prevalence of IPD, pneumococcal pneumonia and colonization was significantly lower (P Table 1. Prevalence for IPD, Pneumonia and Colonization by Period Prevalence Period IPD PNA Colonization 1992-2001 0.36 0.48 0.48 2002-2012 0.16 0.16 0.08 Prevalence ratio (Early/Late) 2.25 3.0 6.0 95% CI 2.0-2.7 2.6-3.4 4.8-7.7 P value Figure 1. Annual prevalence of IPD, pneumonia and colonization Figure 1. Annual prevalence of IPD, pneumonia and colonization Disclosures Kaltsas: Pfizer: Research Funding.

Background: Hematopoietic cell transplant (HCT) patients are at risk for serious infections by Herpes simplex1/2 (HSV) and Varicella zoster(VZV). Current guidelines recommend prophylaxis (PPX) with acyclovir (ACV) during the peri-transplant period and continuing throughout the period of immunosuppression. Brincidofovir (BCV, CMX001) is a broad-spectrum nucleotide analog with in vitro activity against double stranded DNA (dsDNA) viruses including HSV and VZV. BCV is in Phase 3 development for the prevention of cytomegalovirus (CMV) infection post-HCT. At present, there is no clinical data to support the efficacy of BCV PPX for HSV/VZV post-HCT. At Memorial Sloan-Kettering Cancer Center (MSK) from 2010 to June 2015, approximately 70 alloHCT recipients were treated with BCV for various indications. We examined rates of breakthrough HSV and VZV infections during administration of BCV. Methods: Retrospective review of alloHCT patients (pts) treated for ≥14 days with BCV, on protocols or emergency investigational new drug application (EIND) at MSK. Administration of concomitant (CON) ACV for HSV PPX during BCV treatment was at clinician discretion. CMX001-350 was an expanded access study of BCV for treatment of subjects with dsDNA viruses and limited or no therapeutic options. CMX001-202 was a placebo-controlled study of BCV as preemption of adenovirus (ADV) post-HCT. CMX001-304 is a currently ongoing open label study of BCV for preemption of ADV disease. After June 2011, all pts received BCV 200mg total weekly dose [100mg twice weekly (BIW) or 200mg weekly (QW)] for adult/adolescent pts and 4mg/kg total weekly dose (2mg/kg BIW or 4mg/kg QW) for pediatric pts. Patients enrolled on CMX-304 through June 16, 2015 are included in this analysis. Based on CON ACV PPX during BCV treatment, 3 patient groups were identified: 1) No CON ACV PPX (group A); 2) Partial CON ACV PPX (group B); and 3) CON ACV PPX (group 3). Diagnosis of HSV or VZV infection was based on viral culture or PCR and clinical symptoms. Data was extracted from medical, pharmacy, and microbiology records. Results: Of 55 alloHCT pts who received BCV ≥14 days, 27 (49%) were treated on CMX001-350, 1 (2%) on CMX001-202, 12 (22%) on CMX001-304 and 15 (27%) under EIND. The median age was 53 years (range, 3-69). Thirty nine (71%) pts received T-cell depleted allografts, 9 (16%) cord blood, and 7 (13%) conventional allografts. BCV started a median of 85 days [Interquartile range (IQR), 59.5-209.5] post-HCT. The primary indication for BCV was: ACV resistant HSV in 2 (4%) pts, disseminated VZV in 1 (2%) pts, CMV in 28 (51%) pts, ADV in 21 (38%) pts and other dsDNA viruses (BK polyoma virus, Human herpesvirus 6, John Cunningham virus) in 3 (5%) pts. Thirty one (56%) pts received BCV 100mg BIW. Among 35 pts in group A, duration of BCV was a median of 39 days (IQR, 29.5-78). One pt in group A received concomitant anti-CMV antivirals for 12 days. Among 13 pts in group B, the median duration of BCV monotherapy was 23 days (IQR, 13-40). Two pts in group B had brief concomitant exposure to anti-CMV antivirals. Among 7 patients in group C, the median duration of BCV treatment was 71 days (IQR, 40-132) (Table 1). Two pts developed HSV infections during BCV treatment. For one pt, the primary indication for BCV was ACV resistant HSV. The pt initially responded to BCV with resolution of oral lesions. During his treatment with BCV, he had intermittent oral ulcers and oral cultures positive for HSV. The second pt developed breakthrough HSV infection during interruption of BCV for severe diarrhea related to graft versus-host disease. There were no breakthrough VZV infections. For one pt, the primary indication for BCV was disseminated VZV. The pt had complete resolution of VZV and no further VZV reactivation during BCV treatment. Conclusions: 1) During 2,806 patient-days of BCV monotherapy in highly immunosuppressed HCT pts, there were no VZV infections and only 2 breakthrough HSV infections. 2) BCV treatment interruptions and impaired gastrointestinal absorption likely explain the breakthrough HSV infections. 3) Our retrospective analysis in this heterogeneous cohort of sick alloHCT recipients supports that BCV administration at doses currently studied for CMV prevention is likely effective for prophylaxis of HSV/VZV in HCT. Disclosures Off Label Use: Brincidofovir (CMX001) for the efficacy of prophylaxis for Herpes simples 1/2 and Varicella zoster..