Dysregulated Autophagy and Sarcomere Dysfunction in Patients With Heart Failure With Co-Occurrence of P63A and P380S BAG3 Variants.

BACKGROUND: Mutations to the co-chaperone protein BAG3 (B-cell lymphoma-2-associated athanogene-3) are a leading cause of dilated cardiomyopathy (DCM). These mutations often impact the C-terminal BAG domain (residues 420-499), which regulates heat shock protein 70-dependent protein turnover via autophagy. While mutations in other regions are less common, previous studies in patients with DCM found that co-occurrence of 2 BAG3 variants (P63A, P380S) led to worse prognosis. However, the underlying mechanism for dysfunction is not fully understood. METHODS AND RESULTS: In this study, we used proteomics, Western blots, and myofilament functional assays on left ventricular tissue from patients with nonfailing, DCM, and DCM with BAG363/380 to determine how these mutations impact protein quality control and cardiomyocyte contractile function. We found dysregulated autophagy and increased protein ubiquitination in patients with BAG363/380 compared with nonfailing and DCM, suggesting impaired protein turnover. Expression and myofilament localization of BAG3-binding proteins were also uniquely altered in the BAG3,63/380 including abolished localization of the small heat shock protein CRYAB (alpha-crystallin B chain) to the sarcomere. To determine whether these variants impacted sarcomere function, we used cardiomyocyte force-calcium assays and found reduced maximal calcium-activated force in DCM and BAG363/380. Interestingly, myofilament calcium sensitivity was increased in DCM but not with BAG363/380, which was not explained by differences in troponin I phosphorylation. CONCLUSIONS: Together, our data support that the disease-enhancing mechanism for BAG3 variants outside of the BAG domain is through disrupted protein turnover leading to compromised sarcomere function. These findings suggest a shared mechanism of disease among pathogenic BAG3 variants, regardless of location.

Bag3 Regulates Mitochondrial Function and the Inflammasome Through Canonical and Noncanonical Pathways in the Heart.

B-cell lymphoma 2-associated athanogene-3 (Bag3) is expressed in all animal species, with Bag3 levels being most prominent in the heart, the skeletal muscle, the central nervous system, and in many cancers. Preclinical studies of Bag3 biology have focused on animals that have developed compromised cardiac function; however, the present studies were performed to identify the pathways perturbed in the heart even before the occurrence of clinical signs of dilatation and failure of the heart. These studies show that hearts carrying variants that knockout one allele of BAG3 have significant alterations in multiple cellular pathways including apoptosis, autophagy, mitochondrial homeostasis, and the inflammasome.

Circadian Pattern of Ion Channel Gene Expression in Failing Human Hearts.

BACKGROUND: Ventricular tachyarrhythmias and sudden cardiac death show a circadian pattern of occurrence in patients with heart failure. In the rodent ventricle, a significant portion of genes, including some ion channels, shows a circadian pattern of expression. However, genes that define electrophysiological properties in failing human heart ventricles have not been examined for a circadian expression pattern. METHODS: Ventricular tissue samples were collected from patients at the time of cardiac transplantation. Two sets of samples (n=37 and 46, one set with a greater arrhythmic history) were selected to generate pseudo-time series according to their collection time. A third set (n=27) of samples was acquired from the nonfailing ventricles of brain-dead donors. The expression of 5 known circadian clock genes and 19 additional ion channel genes plausibly important to electrophysiological properties were analyzed by real-time polymerase chain reaction and then analyzed for the percentage of expression variation attributed to a 24-hour circadian pattern. RESULTS: The 5 known circadian clock gene transcripts showed a strong circadian expression pattern. Compared with rodent hearts, the human circadian clock gene transcripts showed a similar temporal order of acrophases but with a ≈7.6 hours phase shift. Five of the ion channel genes also showed strong circadian expression. Comparable studies of circadian clock gene expression in samples recovered from nonheart failure brain-dead donors showed acrophase shifts, or weak or complete loss of circadian rhythmicity, suggesting alterations in circadian gene expression. CONCLUSIONS: Ventricular tissue from failing human hearts display a circadian pattern of circadian clock gene expression but phase-shifted relative to rodent hearts. At least 5 ion channels show a circadian expression pattern in the ventricles of failing human hearts, which may underlie a circadian pattern of ventricular tachyarrhythmia/sudden cardiac death. Nonfailing hearts from brain-dead donors show marked differences in circadian clock gene expression patterns, suggesting fundamental deviations from circadian expression.

Association of Variants in BAG3 With Cardiomyopathy Outcomes in African American Individuals.

Importance: The prevalence of nonischemic dilated cardiomyopathy (DCM) is greater in individuals of African ancestry than in individuals of European ancestry. However, little is known about whether the difference in prevalence or outcomes is associated with functional genetic variants. Objective: We hypothesized that Bcl2-associated anthanogene 3 (BAG3) genetic variants were associated with outcomes in individuals of African ancestry with DCM. Design: This multicohort study of the BAG3 genotype in patients of African ancestry with dilated cardiomyopathy uses DNA obtained from African American individuals enrolled in 3 clinical studies: the Genetic Risk Assessment of African Americans With Heart Failure (GRAHF) study; the Intervention in Myocarditis and Acute Cardiomyopathy Trial-2 (IMAC-2) study; and the Genetic Risk Assessment of Cardiac Events (GRACE) study. Samples of DNA were also acquired from the left ventricular myocardium of patients of African ancestry who underwent heart transplant at the University of Colorado and University of Pittsburgh. Main Outcomes and Measures: The primary end points were the prevalence of BAG3 mutations in African American individuals and event-free survival in participants harboring functional BAG3 mutations. Results: Four BAG3 genetic variants were identified; these were expressed in 42 of 402 African American individuals (10.4%) with nonischemic heart failure and 9 of 107 African American individuals (8.4%) with ischemic heart failure but were not present in a reference population of European ancestry (P < .001). The variants included 2 nonsynonymous single-nucleotide variants; 1 three-nucleotide in-frame insertion; and 2 single-nucleotide variants that were linked in cis. The presence of BAG3 variants was associated with a nearly 2-fold (hazard ratio, 1.97 [95% CI, 1.19-3.24]; P = .01) increase in cardiac events in carriers compared with noncarriers. Transfection of transformed adult human ventricular myocytes with plasmids expressing the 4 variants demonstrated that each variant caused an increase in apoptosis and a decrease in autophagy when samples were subjected to the stress of hypoxia-reoxygenation. Conclusions and Relevance: This study demonstrates that genetic variants in BAG3 found almost exclusively in individuals of African ancestry were not causative of disease but were associated with a negative outcome in patients with a dilated cardiomyopathy through modulation of the function of BAG3. The results emphasize the importance of biological differences in causing phenotypic variance across diverse patient populations, the need to include diverse populations in genetic cohorts, and the importance of determining the pathogenicity of genetic variants.

A micro-ribonucleic acid signature associated with recovery from assist device support in 2 groups of patients with severe heart failure.

OBJECTIVES: This study was conducted to test the hypothesis that cardiac micro-ribonucleic acid (miR) profiling in severe heart failure patients at the time of ventricular assist device (VAD) placement would differentiate those who remained VAD-dependent from those with subsequent left ventricular (LV) recovery. BACKGROUND: The relationship of myocardial miR expression to ventricular recovery is unknown. METHODS: We studied 28 patients with nonischemic cardiomyopathy requiring VAD support consisting of test and validation cohorts from 2 institutions: 14 with subsequent LV recovery and VAD removal and 14 clinically matched VAD-dependent patients. Apical core myocardium was studied for expression of 376 miRs by polymerase chain reaction (PCR) array and real-time-PCR methods. Samples from 7 nonfailing hearts were used in confirmatory studies. RESULTS: By PCR array, 10 miRs were differentially expressed between LV recovery and VAD-dependent patients in the test cohort. The real-time PCR confirmed lower expression in LV recovery patients for 4 miRs (15b, -1.5-fold; 23a, -2.2-fold; 26a, -1.4-fold; and 195, -1.8-fold; all p < 0.04 vs. VAD dependent). The validation cohort similarly showed lower miRs expression in LV recovery patients (23a, -1.8-fold; and 195, -1.5-fold; both p < 0.03). Furthermore, miR 23a and 195 expression in nonfailing hearts was similar to LV recovery patients (both p < 0.04 vs. VAD dependent). The LV recovery patients also had significantly smaller cardiomyocytes by quantitative histology in both cohorts. CONCLUSIONS: Lower cardiac expression of miRs 23a and 195 and smaller cardiomyocyte size at the time of VAD placement were associated with subsequent LV functional recovery. Differential expression of miRs at VAD placement may provide markers to assess recovery potential.

Resistance to age-dependent thymic atrophy in long-lived mice that are deficient in pregnancy-associated plasma protein A.

Pregnancy-associated plasma protein A (PAPPA) is a metalloproteinase that controls the tissue availability of insulin-like growth factor (IGF). Homozygous deletion of PAPPA in mice leads to lifespan extension. Since immune function is an important determinant of individual fitness, we examined the natural immune ecology of PAPPA(-/-) mice and their wild-type littermates reared under specific pathogen-free condition with aging. Whereas wild-type mice exhibit classic age-dependent thymic atrophy, 18-month-old PAPPA(-/-) mice maintain discrete thymic cortex and medulla densely populated by CD4(+)CD8(+) thymocytes that are capable of differentiating into single-positive CD4 and CD8 T cells. Old PAPPA(-/-) mice have high levels of T cell receptor excision circles, and have bone marrows enriched for subsets of thymus-seeding progenitors. PAPPA(-/-) mice have an overall larger pool of naive T cells, and also exhibit an age-dependent accumulation of CD44(+)CD43(+) memory T cells similar to wild-type mice. However, CD43(+) T cell subsets of old PAPPA(-/-) mice have significantly lower prevalence of 1B11 and S7, glycosylation isoforms known to inhibit T cell activation with normal aging. In bioassays of cell activation, splenic T cells of old PAPPA(-/-) mice have high levels of activation antigens and cytokine production, and also elicit Ig production by autologous B cells at levels equivalent to young wild-type mice. These data suggest an IGF-immune axis of healthy longevity. Controlling the availability of IGF in the thymus by targeted manipulation of PAPPA could be a way to maintain immune homeostasis during postnatal development and aging.

Induction of CD56 and TCR-independent activation of T cells with aging.

Degeneration of the thymus and severe contraction of the T cell repertoire with aging suggest that immune homeostasis in old age could be mediated by distinct effectors. Therefore, receptors expressed on T cells as they undergo senescence in vitro, as well as those displayed by circulating T cells during normal chronologic aging, were examined. Monitoring of T cells driven to senescence showed de novo induction of CD56, the prototypic receptor of NK cells. Analysis of fresh T cells in peripheral blood showed an age-dependent induction of CD56. These unusual T cells expressed high levels of Bcl2, p16, and p53, and had limited, or completely lost, ability to undergo cell division, properties consistent with senescence. CD56 cross-linking without TCR ligation on CD56(+) T cells resulted in extensive protein phosphorylation, NF-kappaB activation, and Bax down-regulation. CD56 cross-linking was also sufficient to drive production of various humoral factors. These data suggest that the immunologic environment in old age is functionally distinct, rather than being a dysfunctional version of that seen at a young age. CD56(+) T cells are unique effectors capable of mediating TCR-independent immune cascades that could be harnessed to enhance protective immunity in the elderly.

CD56-expressing T cells that have features of senescence are expanded in rheumatoid arthritis.

OBJECTIVE: T cells deficient in CD28 expression have been implicated in the pathogenesis of rheumatoid arthritis (RA). Given that CD28-null T cells are functionally heterogeneous, we undertook this study to screen for novel receptors on these cells. METHODS: Seventy-two patients with RA (ages 35-84 years) and 53 healthy persons (32 young controls ages 19-34 years, 21 older controls ages 39-86 years) were recruited. Phenotypes and proliferative capacity of T cells from fresh leukocytes and of long-term cultures were monitored by flow cytometry. Lung biopsy specimens from patients with RA-associated interstitial pneumonitis (IP) were examined by immunohistochemistry. Receptor functionality was assessed by crosslinking bioassays. RESULTS: Chronic stimulation of CD28(+) T cells in vitro yielded progenies that lacked CD28 but that gained CD56. Ex vivo analysis of leukocytes from patients with extraarticular RA showed a higher frequency of CD56(+),CD28-null T cells than in patients with disease confined to the joints or in healthy controls. CD56(+),CD28-null T cells had nil capacity for proliferation, consistent with cellular senescence. CD56(+) T cells had skewed T cell receptor (TCR) alpha/beta-chain usage and restricted TCR third complementarity-determining region spectra. Histologic studies showed that CD56(+) T cells were components of cellular infiltrates in RA-associated IP. CD56 crosslinking on T cells sufficiently induced cytokine production, although CD56/TCR coligation induced higher production levels. CONCLUSION: Chronic activation of T cells induces counterregulation of CD28 and CD56 expression. The loss of CD28 is accompanied by the gain of CD56 that confers TCR-independent and TCR-dependent activation pathways. We propose that accumulation of CD56(+) T cells in RA contributes to maladaptive immune responses and that CD56(+) T cells are potential targets for therapy.

Diversity of NKR expression in aging T cells and in T cells of the aged: the new frontier into the exploration of protective immunity in the elderly.

Aging in the immune system is characterized by the contraction of the lymphocyte repertoire, exemplified by long-lived oligoclonal T cells that pervade the peripheral circulation. T-cell receptor (TCR) repertoire contraction likely explains the decline in immunity with chronological age as evidenced by the increased morbidity and mortality to common and new infections, and the low rates of protective responses to vaccination in the elderly. Interestingly, in vitro senescence models and cross sectional ex vivo studies have consistently demonstrated that senescent (or pre-senescent) T cells and T cells of the aged express unusually high densities of receptors that are normally found on natural killer (NK) cells, the killer cell immunoglobulin-like receptors (KIR) being the most diverse NK receptors (NKR). Molecular studies also show that T cells are programmed to express NKRs/KIRs, and T-cell clonal lineages express a variety of NKRs towards the end stages of their replicative lifespan. We propose that NKR/KIR induction in aging T cells is an adaptational diversification of the immune repertoire. We suggest that NKR/KIR expression in oligoclonal senescent and pre-senescent T cells is a compensatory adaptation to maintain immune competence despite the overall contraction in TCR diversity with aging. NKRs comprise a diverse superfamily of receptors. Mounting evidence for NKR/KIR signaling pathways in T cells divergent from those seen in NK cells indicate that senescent NKR(+)T cells are unique immune effectors. We suggest that appreciation of the functional diversity of these unusual NK-like T cells is central to the creative development of new strategies to enhance protective immunity in the aged.

Serum protein profile in systemic-onset juvenile idiopathic arthritis differentiates response versus nonresponse to therapy.

Systemic-onset juvenile idiopathic arthritis (SJIA) is a disease of unknown etiology with an unpredictable response to treatment. We examined two groups of patients to determine whether there are serum protein profiles reflective of active disease and predictive of response to therapy. The first group (n = 8) responded to conventional therapy. The second group (n = 15) responded to an experimental antibody to the IL-6 receptor (MRA). Paired sera from each patient were analyzed before and after treatment, using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS). Despite the small number of patients, highly significant and consistent differences were observed before and after response to therapy in all patients. Of 282 spectral peaks identified, 23 had mean signal intensities significantly different (P < 0.001) before treatment and after response to treatment. The majority of these differences were observed regardless of whether patients responded to conventional therapy or to MRA. These peaks represent potential biomarkers of active disease. One such peak was identified as serum amyloid A, a known acute-phase reactant in SJIA, validating the SELDI-TOF MS platform as a useful technology in this context. Finally, profiles from serum samples obtained at the time of active disease were compared between the two patient groups. Nine peaks had mean signal intensities significantly different (P < 0.001) between active disease in patients who responded to conventional therapy and in patients who failed to respond, suggesting a possible profile predictive of response. Collectively, these data demonstrate the presence of serum proteomic profiles in SJIA that are reflective of active disease and suggest the feasibility of using the SELDI-TOF MS platform used as a tool for proteomic profiling and discovery of novel biomarkers in autoimmune diseases.

Phospholamban gene ablation improves calcium transients but not cardiac function in a heart failure model.

UNLABELLED: Decreased amplitude and slower kinetics of cardiomyocyte intracellular calcium (Ca(i)(2+)) transients may underlie the diminished cardiac function observed in heart failure. These alterations occur in humans and animals with heart failure, including the TNF1.6 mouse model, in which heart failure arises from cardiac-specific overexpression of tumor necrosis factor alpha (TNF alpha). OBJECTIVE: Since ablation of phospholamban expression (PLBKO) removes inhibition of the sarcoplasmic reticulum (SR) Ca(2+) pump, enhances SR Ca(2+) uptake and increases contractility, we assessed whether ablation of phospholamban expression could improve cardiac function, limit remodeling, and improve survival in the TNF1.6 model of heart failure. METHODS: We bred PLBKO with TNF1.6 mice and characterized the progeny for survival, cardiac function (echocardiography), cardiac remodeling (hypertrophy, dilation, fibrosis), and Ca(2+)(i) transients and contractile function of isolated cardiomyocytes. RESULTS: PLB ablation did not improve survival, cardiac function, or limit cardiac chamber dilation and hypertrophy in TNF1.6 mice (TKO mice). However, contractile function and Ca(2+)(i) transients (amplitude and kinetics) of isolated TKO cardiomyocytes were markedly enhanced. This discordance between unimproved cardiac function, and enhanced Ca(2+)(i) cycling and cardiomyocyte contractile parameters may arise from a continued overexpression of collagen and decreased expression of gap junction proteins (connexin 43) in response to chronic TNF alpha stimulation. CONCLUSIONS: Enhancement of intrinsic cardiomyocyte Ca(2+)(i) cycling and contractile function may not be sufficient to overcome several parallel pathophysiologic processes present in the failing heart.

Morphological and functional changes in cardiac myocytes isolated from mice overexpressing TNF-alpha.

Transgenic (TG) TNF1.6 mice, which cardiac specifically overexpress tumor necrosis factor-alpha (TNF-alpha), exhibit heart failure (HF) and increased mortality, which is markedly higher in young (<20 wk) males (TG-M) than females (TG-F). HF in this model may be partly caused by remodeling of the extracellular matrix and/or structure/function alterations at the single myocyte level. We studied left ventricular (LV) structure and function using echocardiography and LV myocyte morphometry, contractile function, and intracellular Ca(2+) (Ca(i)(2+)) handling using cell edge detection and fura 2 fluorescence, respectively, in 12-wk-old TG-M and TG-F mice and their wild-type (WT) littermates. TG-F mice showed LV hypertrophy without dilatation and only a small reduction of basal fractional shortening (FS) and response to isoproterenol (Iso). TG-M mice showed a large LV dilatation, higher mRNA levels of beta-myosin heavy chain and atrial natriuretic factor versus TG-F mice, reduced FS relative to both WT and TG-F mice, and minimal response to Iso. TG-F and TG-M myocytes were similarly elongated (by approximately 20%). The amplitude of Ca(i)(2+) transients and contractions and the response to Iso were comparable in WT and TG-F myocytes, whereas the time to 50% decline (TD(50%)) of the Ca(i)(2+) transient, an index of the rate of sarcoplasmic reticulum Ca(2+) uptake, was prolonged in TG-F myocytes. In TG-M myocytes, the amplitudes of Ca(i)(2+) transients and contractions were reduced, TD(50%) of the Ca(i)(2+) transient was prolonged, and the inotropic effect of Iso on Ca(i)(2+) transients was reduced approximately twofold versus WT myocytes. Protein expression of sarco(endo)plasmic reticulum Ca(2+)-ATPase 2 and phospholamban was unaltered in TG versus WT hearts, suggesting functional origins of impaired Ca(2+) handling in the former. These results indicate that cardiac-specific overexpression of TNF-alpha induces myocyte hypertrophy and gender-dependent alterations in Ca(i)(2+) handling and contractile function, which may at least partly account for changes in LV geometry and in vivo cardiac function in this model.

Chronic exposure to interleukin 1beta induces a delayed and reversible alteration in excitation-contraction coupling of cultured cardiomyocytes.

While proinflammatory cytokines can depress cardiac contractility, the mechanism by which this occurs remains unclear. To clarify the cellular effects of interleukin (IL)-1beta, we assessed contractility, calcium homeostasis, and gene expression in cardiomyocytes exposed to this proinflammatory cytokine. Neonatal rat cardiomyocytes were exposed to IL-1beta in the presence or absence of an inhibitor of nitric oxide (NO) synthase. Videomicroscopy was used to follow calcium transients (Fura-2 fluorescence) and amplitude of contraction, both unstimulated and after isoproterenol challenge. Gene expression was assessed by Northern and Western blot analyses. Both basal contractility (amplitude of contraction, maximum speed of contraction and relaxation) and amplitude of calcium transients were decreased, respectively, ca. 60% ( P< or =0.05) and ca. 40% ( P< or =0.05) after 3 days of IL-1beta exposure. Contractile function and amplitude of calcium transients returned to control values when cells where cultured an additional 3 days in the absence of IL-1beta. IL-1beta-treated cells had reduced responses to isoproterenol as evidenced by a lack of enhanced amplitude of contraction and a reduction in cAMP production. IL-1beta decreased the expression of genes important to the regulation of calcium homeostasis (phospholamban, sarcoplasmic reticulum calcium ATPase) at both the transcript and protein level. Alterations in contractile function did not occur through NO-mediated pathways. These results support the hypothesis that IL-1beta may play an important role in contractile dysfunction through alterations in calcium homeostasis.