Heat shock protein inducer GGA*-59 reverses contractile and structural remodeling via restoration of the microtubule network in experimental Atrial Fibrillation.

BACKGROUND: Atrial Fibrillation (AF) is the most common progressive tachyarrhythmia. AF progression is driven by abnormalities in electrical impulse formation and contractile function due to structural remodeling of cardiac tissue. Previous reports indicate that structural remodeling is rooted in derailment of protein homeostasis (proteostasis). Heat shock proteins (HSPs) play a critical role in facilitating proteostasis. Hence, the HSP-inducing compound geranylgeranylacetone (GGA) and its derivatives protect against proteostasis derailment in experimental models for AF. Whether these compounds also accelerate reversibility from structural remodeling in tachypaced cardiomyocytes is unknown. OBJECTIVE: To investigate whether the potent HSP inducer GGA*-59 restores structural remodeling and contractile dysfunction in tachypaced cardiomyocytes and explore the underlying mechanisms. MATERIALS AND RESULTS: HL-1 cardiomyocytes post-treated with GGA*-59 or recombinant HSPB1 (rcHSPB1) revealed increased levels of HSPB1 expression and accelerated recovery from tachypacing (TP)-induced calcium transient (CaT) loss compared to non-treated cardiomyocytes. In addition, protein levels of the microtubule protein (acetylated) α-tubulin, and contractile proteins cardiac troponin I (cTnI) and troponin T (cTnT) were reduced after TP and significantly recovered by GGA*-59 or rcHSPB1 post-treatment. The mRNA levels of α-tubulin encoding genes, but not cardiac troponin genes, were reduced upon TP and during recovery, but significantly enhanced by GGA*-59 and rcHSPB1 post-treatment. In addition, TP increased calpain activity, which remained increased during recovery and GGA*-59 post-treatment. However, HDAC6 activity, which deacetylates α-tubulin resulting in microtubule disruption, was significantly increased after TP and during recovery, but normalized to control levels by GGA*-59 or rcHSPB1 post-treatment in HL-1 cardiomyocytes. CONCLUSIONS: Our results imply that the HSP inducer GGA*-59 and recombinant HSPB1 accelerate recovery from TP-induced structural remodeling and contractile dysfunction in HL-1 cardiomyocytes. GGA*-59 increases HSPB1 levels, represses HDAC6 activity and restores contractile protein and microtubule levels after TP, indicating that HSP-induction is an interesting target to accelerate recovery from AF-induced remodeling.

HALT & REVERSE: Hsf1 activators lower cardiomyocyt damage; towards a novel approach to REVERSE atrial fibrillation.

BACKGROUND: Atrial fibrillation is a progressive arrhythmia, the exact mechanism underlying the progressive nature of recurrent AF episodes is still unknown. Recently, it was found that key players of the protein quality control system of the cardiomyocyte, i.e. Heat Shock Proteins, protect against atrial fibrillation progression by attenuating atrial electrical and structural remodeling (electropathology). HALT & REVERSE aims to investigate the correlation between electropathology, as defined by endo- or epicardial mapping, Heat Shock Protein levels and development or recurrence of atrial fibrillation following pulmonary vein isolation, or electrical cardioversion or cardiothoracic surgery. STUDY DESIGN: This study is a prospective observational study. Three separate study groups are defined: (1) cardiothoracic surgery, (2) pulmonary vein isolation and (3) electrical cardioversion. An intra-operative high-resolution epicardial (group 1) or endocardial (group 2) mapping procedure of the atria is performed to study atrial electropathology. Blood samples for Heat Shock Protein determination are obtained at baseline and during the follow-up period at 3 months (group 2), 6 months (groups 1 and 2) and 1 year (group 1 and 2). Tissue samples of the right and left atrial appendages in patients in group 1 are analysed for Heat Shock Protein levels and for tissue characteristics. Early post procedural atrial fibrillation is detected by continuous rhythm monitoring, whereas late post procedural atrial fibrillation is documented by either electrocardiogram or 24-h Holter registration. CONCLUSION: HALT & REVERSE aims to identify the correlation between Heat Shock Protein levels and degree of electropathology. The study outcome will contribute to novel diagnostic tools for the early recognition of clinical atrial fibrillation. TRIAL REGISTRATIONS: Rotterdam Medical Ethical Committee MEC-2014-393, Dutch Trial Registration NTR4658.

Atrial heat shock protein levels are associated with early postoperative and persistence of atrial fibrillation.

BACKGROUND: Early detection and staging of atrial fibrillation (AF) is of importance for clinical management. Serum (bio)markers, such as heat shock proteins (HSP), may enable AF staging and identify patients at risk for AF recurrence and postoperative AF (PoAF). OBJECTIVE: This study evaluates the relation between serum and atrial tissue HSP levels, stages of AF, AF recurrence after treatment, and PoAF from patients undergoing cardiothoracic surgery. METHODS: Patients without (control) and with paroxysmal, persistent (PerAF), or longstanding persistent (LSPerAF) AF were included. HSPB1, HSPA1, HSPB7, and HSPD1 levels were measured in serum obtained prior to and post intervention. HSPB1, HSPA1, HSPA5, HSPD1, HSPB5, and pHSF1 levels were measured in left and/or right atrial appendages (respectively, LAA and RAA). RESULTS: In RAA, HSPA5 levels were significantly lower in LSPerAF and HSPD1 levels significantly higher in PerAF patients compared to controls. In RAA of controls who developed PoAF, HSPA1 and HSPA5 levels were significantly higher compared to those without PoAF. Also, HSPB1 RAA levels were lower and HSPA5 LAA levels higher in patients undergoing arrhythmia surgery who developed AF recurrence within 1 week after surgery compared to patients who did not. CONCLUSION: HSPA5 RAA and HSPD1 RAA and LAA levels are altered in persistent stages of AF. RAA HSPA1 and HSPA5 levels associate with development of PoAF. Additionally, HSPB1 RAA and HSPA5 LAA levels can predict AF recurrence in patients who underwent arrhythmia surgery. Nevertheless, HSP levels in serum cannot discriminate AF stages from controls, nor predict PoAF or AF recurrence after treatment.

Oral geranylgeranylacetone treatment increases heat shock protein expression in human atrial tissue.

BACKGROUND: Heat shock proteins (HSPs) are important chaperones that regulate the maintenance of healthy protein quality control in the cell. Impairment of HSPs is associated with aging-related neurodegenerative and cardiac diseases. Geranylgeranylacetone (GGA) is a compound well known to increase HSPs through activation of heat shock factor-1 (HSF1). GGA increases HSPs in various tissues, but whether GGA can increase HSP expression in human heart tissue is unknown. OBJECTIVE: The purpose of this study was to test whether oral GGA treatment increases HSP expression in the atrial appendages of patients undergoing cardiac surgery. METHODS: HSPB1, HSPA1, HSPD1, HSPA5, HSF1, and phosphorylated HSF1 levels were measured by western blot analysis in right and left atrial appendages (RAAs and LAAs, respectively) collected from patients undergoing coronary artery bypass grafting (CABG) who were treated with placebo (n = 13) or GGA 400 mg/da(n = 13) 3 days before surgery. Myofilament fractions were isolated from LAAs to determine the levels of HSPB1 and HSPA1 present in these fractions. RESULTS: GGA treatment significantly increased HSPB1 and HSPA1 expression levels in RAA and LAA compared to the placebo group, whereas HSF1, phosphorylated HSF1, HSPD1, and HSPA5 were unchanged. In addition, GGA treatment significantly enhanced HSPB1 levels at the myofilaments compared to placebo. CONCLUSION: Three days of GGA treatment is associated with higher HSPB1 and HSPA1 expression levels in RAA and LAA of patients undergoing CABG surgery and higher HSPB1 levels at the myofilaments. These findings pave the way to study the role of GGA as a protective compound against other cardiac diseases, including postoperative atrial fibrillation.

Cell-Free Circulating Mitochondrial DNA: A Potential Blood-Based Marker for Atrial Fibrillation.

Atrial fibrillation (AF), the most common, progressive tachyarrhythmia is associated with serious complications, such as stroke and heart failure. Early recognition of AF, essential to prevent disease progression and therapy failure, is hampered by the lack of accurate diagnostic serum biomarkers to identify the AF stage. As we previously showed mitochondrial dysfunction to drive experimental and human AF, we evaluated whether cell-free circulating mitochondrial DNA (cfc-mtDNA) represents a potential serum marker. Therefore, the levels of two mtDNA genes, COX3 and ND1, were measured in 84 control patients (C), 59 patients undergoing cardiac surgery without a history of AF (SR), 100 paroxysmal (PAF), 116 persistent (PeAF), and 20 longstanding-persistent (LS-PeAF) AF patients undergoing either cardiac surgery or AF treatment (electrical cardioversion or pulmonary vein isolation). Cfc-mtDNA levels were significantly increased in PAF patients undergoing AF treatment, especially in males and patients with AF recurrence after AF treatment. In PeAF and LS-PeAF, cfc-mtDNA levels gradually decreased. Importantly, cfc-mtDNA in serum may originate from cardiomyocytes, as in vitro tachypaced cardiomyocytes release mtDNA in the medium. The findings suggest that cfc-mtDNA is associated with AF stage, especially in males, and with patients at risk for AF recurrence after treatment.

Mitochondrial Dysfunction Underlies Cardiomyocyte Remodeling in Experimental and Clinical Atrial Fibrillation.

Atrial fibrillation (AF), the most common progressive tachyarrhythmia, results in structural remodeling which impairs electrical activation of the atria, rendering them increasingly permissive to the arrhythmia. Previously, we reported on endoplasmic reticulum stress and NAD+ depletion in AF, suggesting a role for mitochondrial dysfunction in AF progression. Here, we examined mitochondrial function in experimental model systems for AF (tachypaced HL-1 atrial cardiomyocytes and Drosophila melanogaster) and validated findings in clinical AF. Tachypacing of HL-1 cardiomyocytes progressively induces mitochondrial dysfunction, evidenced by impairment of mitochondrial Ca2+-handling, upregulation of mitochondrial stress chaperones and a decrease in the mitochondrial membrane potential, respiration and ATP production. Atrial biopsies from AF patients display mitochondrial dysfunction, evidenced by aberrant ATP levels, upregulation of a mitochondrial stress chaperone and fragmentation of the mitochondrial network. The pathophysiological role of mitochondrial dysfunction is substantiated by the attenuation of AF remodeling by preventing an increased mitochondrial Ca2+-influx through partial blocking or downregulation of the mitochondrial calcium uniporter, and by SS31, a compound that improves bioenergetics in mitochondria. Together, these results show that conservation of the mitochondrial function protects against tachypacing-induced cardiomyocyte remodeling and identify this organelle as a potential novel therapeutic target.

DNA damage-induced PARP1 activation confers cardiomyocyte dysfunction through NAD+ depletion in experimental atrial fibrillation.

Atrial fibrillation (AF) is the most common clinical tachyarrhythmia with a strong tendency to progress in time. AF progression is driven by derailment of protein homeostasis, which ultimately causes contractile dysfunction of the atria. Here we report that tachypacing-induced functional loss of atrial cardiomyocytes is precipitated by excessive poly(ADP)-ribose polymerase 1 (PARP1) activation in response to oxidative DNA damage. PARP1-mediated synthesis of ADP-ribose chains in turn depletes nicotinamide adenine dinucleotide (NAD+), induces further DNA damage and contractile dysfunction. Accordingly, NAD+ replenishment or PARP1 depletion precludes functional loss. Moreover, inhibition of PARP1 protects against tachypacing-induced NAD+ depletion, oxidative stress, DNA damage and contractile dysfunction in atrial cardiomyocytes and Drosophila. Consistently, cardiomyocytes of persistent AF patients show significant DNA damage, which correlates with PARP1 activity. The findings uncover a mechanism by which tachypacing impairs cardiomyocyte function and implicates PARP1 as a possible therapeutic target that may preserve cardiomyocyte function in clinical AF.

The protective role of small heat shock proteins in cardiac diseases: key role in atrial fibrillation.

Atrial fibrillation (AF) is the most common tachyarrhythmia which is associated with increased morbidity and mortality. AF usually progresses from a self-terminating paroxysmal to persistent disease. It has been recognized that AF progression is driven by structural remodeling of cardiomyocytes, which results in electrical and contractile dysfunction of the atria. We recently showed that structural remodeling is rooted in derailment of proteostasis, i.e., homeostasis of protein production, function, and degradation. Since heat shock proteins (HSPs) play an important role in maintaining a healthy proteostasis, the role of HSPs was investigated in AF. It was found that especially small heat shock protein (HSPB) levels get exhausted in atrial tissue of patients with persistent AF and that genetic or pharmacological induction of HSPB protects against cardiomyocyte remodeling in experimental models for AF. In this review, we provide an overview of HSPBs as a potential therapeutic target for normalizing proteostasis and suppressing the substrates for AF progression in experimental and clinical AF and discuss HSP activators as a promising therapy to prevent AF onset and progression.

Spatial distribution of conduction disorders during sinus rhythm.

BACKGROUND: Length of lines of conduction block (CB) during sinus rhythm (SR) at Bachmann's bundle (BB) is associated with atrial fibrillation (AF). However, it is unknown whether extensiveness of CB at BB represents CB elsewhere in the atria. We aim to investigate during SR 1) the spatial distribution and extensiveness of CB 2) whether there is a predilection site for CB and 3) the association between CB and incidence of post-operative AF. METHODS: During SR, epicardial mapping of the right atrium (RA), BB and left atrium was performed in 209 patients with coronary artery disease. The amount of conduction delay (CD, Δlocal activation time ≥7ms) and CB (Δ≥12ms) was quantified as % of the mapping area. Atrial regions were compared to identify potential predilection sites for CD/CB. Correlations between CD/CB and clinical characteristics were tested. RESULTS: Areas with CD or CB were present in all patients, overall prevalence was respectively 1.4(0.2-4.0) % and 1.3(0.1-4.3) %. Extensiveness and spatial distribution of CD/CB varied considerably, however occurred mainly at the superior intercaval RA. Of all clinicalcharacteristics, CD/CB only correlated weakly with age and diabetes (P<0.05). A 1% increase in CD or CB caused a 1.1-1.5ms prolongation of the activation time (P<0.001). There was no correlation between CD/CB and post-operative AF. CONCLUSION: CD/CB during SR in CABG patients with electrically non-remodeled atria show considerable intra-atrial, but also inter-individual variation. Despite these differences, a predilection site is present at the superior intercaval RA. Extensiveness of CB at the superior intercaval RA or BB does not reflect CB elsewhere in the atria and is not associated with post-operative AF.

Studying cancer metastasis: Existing models, challenges and future perspectives.

Cancer metastasis causes most cancer-related deaths. Several model systems to study the complex and multi step process of metastasis exist, including in vitro systems, ex-vivo organ slices, Drosophila Melanogaster and zebrafish models and the use of the chorio allantoic membrane (CAM) of fertilized chicken eggs. These models are relatively easy and cheap but often lack the opportunity to study the complete metastasis cascade. More complex but also more expensive is the use of animal models including the more recently developed patient derived tumor xenografts (PDTX). In this review, we give an overview of the existing metastatic models, discuss the challenges of improving current models to enhance translation from the preclinical to the clinical setting and consider future perspectives.

Difference in CXCR4 expression between sporadic and VHL-related hemangioblastoma.

Central nervous system hemangioblastomas occur sporadically and in patients with von Hippel-Lindau (VHL) disease due to a VHL germline mutation. This mutation leads to enhanced transcription of chemokine receptor 4 (CXCR4), its ligand (CXCL12) and vascular endothelial growth factor A (VEGFA). We aimed to determine in VHL-related and sporadic hemangioblastomas CXCR4, CXCL12, and VEGFA protein expression and to correlate this to hemangioblastoma size and expression in normal surrounding tissue. 27 patients with a hemangioblastoma were included for analysis of immunohistochemistry of tissue, MRI and DNA. Hemangioblastomas overexpress CXCR4, CXCL12, and VEGFA compared to normal surrounding tissue. In sporadic hemangioblastomas the mean percentage of CXCR4 positive hemangioblastoma cells was 16 %, SD 8.4, in VHL-related hemangioblastomas 8 %, SD 4.4 (P = 0.002). There was no relation between preoperative tumor size and CXCR4 or CXCL12 expression. Compared to normal surrounding tissue CXCR4, CXCL12, and VEGFA were overexpressed in hemangioblastomas. Most interestingly, sporadic hemangioblastomas overexpress CXCR4 compared to VHL-related hemangioblastoma.

Diagnosis and Therapy of Atrial Fibrillation: The Past, The Present and The Future.

Atrial fibrillation (AF) is the most common age-related cardiac arrhythmia. It is a progressive disease, which makes treatment difficult. The progression of AF is caused by the accumulation of damage in cardiomyocytes which makes the atria more vulnerable for AF. Especially structural remodeling and electrical remodeling, together called electropathology are sustainable in the atria and impair functional recovery to sinus rhythm after cardioversion. The exact electropathological mechanisms underlying persistence of AF are at present unknown. High resolution wavemapping studies in patients with different types of AF showed that longitudinal dissociation in conduction and epicardial breakthrough were the key elements of the substrate of longstanding persistent AF. A double layer of electrically dissociated waves propagating transmurally can explain persistence of AF (Double Layer Hypothesis) but the molecular mechanism is unknown. Derailment of proteasis -defined as the homeostasis in protein synthesis, folding, assembly, trafficking, guided by chaperones, and clearance by protein degradation systems - may play an important role in remodeling of the cardiomyocyte. As current therapies are not effective in attenuating AF progression, step-by-step analysis of this process, in order to identify potential targets for drug therapy, is essential. In addition, novel mapping approaches enabling assessment of the degree of electropathology in the individual patient are mandatory to develop patient-tailored therapies. The aims of this review are to 1) summarize current knowledge of the electrical and molecular mechanisms underlying AF 2) discuss the shortcomings of present diagnostic instruments and therapeutic options and 3) to present potential novel diagnostic tools and therapeutic targets.

RhoA Activation Sensitizes Cells to Proteotoxic Stimuli by Abrogating the HSF1-Dependent Heat Shock Response.

BACKGROUND: The heat shock response (HSR) is an ancient and highly conserved program of stress-induced gene expression, aimed at reestablishing protein homeostasis to preserve cellular fitness. Cells that fail to activate or maintain this protective response are hypersensitive to proteotoxic stress. The HSR is mediated by the heat shock transcription factor 1 (HSF1), which binds to conserved heat shock elements (HSE) in the promoter region of heat shock genes, resulting in the expression of heat shock proteins (HSP). Recently, we observed that hyperactivation of RhoA conditions cardiomyocytes for the cardiac arrhythmia atrial fibrillation. Also, the HSR is annihilated in atrial fibrillation, and induction of HSR mitigates sensitization of cells to this disease. Therefore, we hypothesized active RhoA to suppress the HSR resulting in sensitization of cells for proteotoxic stimuli. METHODS AND RESULTS: Stimulation of RhoA activity significantly suppressed the proteotoxic stress-induced HSR in HL-1 atrial cardiomyocytes as determined with a luciferase reporter construct driven by the HSF1 regulated human HSP70 (HSPA1A) promoter and HSP protein expression by Western Blot analysis. Inversely, RhoA inhibition boosted the proteotoxic stress-induced HSR. While active RhoA did not preclude HSF1 nuclear accumulation, phosphorylation, acetylation, or sumoylation, it did impair binding of HSF1 to the hsp genes promoter element HSE. Impaired binding results in suppression of HSP expression and sensitized cells to proteotoxic stress. CONCLUSION: These results reveal that active RhoA negatively regulates the HSR via attenuation of the HSF1-HSE binding and thus may play a role in sensitizing cells to proteotoxic stimuli.