Inhibition of Ferroptosis Enables Safe Rewarming of HEK293 Cells following Cooling in University of Wisconsin Cold Storage Solution.

The prolonged cooling of cells results in cell death, in which both apoptosis and ferroptosis have been implicated. Preservation solutions such as the University of Wisconsin Cold Storage Solution (UW) encompass approaches addressing both. The use of UW improves survival and thus extends preservation limits, yet it remains unclear how exactly organ preservation solutions exert their cold protection. Thus, we explored cooling effects on lipid peroxidation and adenosine triphosphate (ATP) levels and the actions of blockers of apoptosis and ferroptosis, and of compounds enhancing mitochondrial function. Cooling and rewarming experiments were performed in a cellular transplantation model using Human Embryonic Kidney (HEK) 293 cells. Cell viability was assessed by neutral red assay. Lipid peroxidation levels were measured by Western blot against 4-Hydroxy-Nonenal (4HNE) and the determination of Malondialdehyde (MDA). ATP was measured by luciferase assay. Cooling beyond 5 h in Dulbecco's Modified Eagle Medium (DMEM) induced complete cell death in HEK293, whereas cooling in UW preserved ~60% of the cells, with a gradual decline afterwards. Cooling-induced cell death was not precluded by inhibiting apoptosis. In contrast, the blocking of ferroptosis by Ferrostatin-1 or maintaining of mitochondrial function by the 6-chromanol SUL150 completely inhibited cell death both in DMEM- and UW-cooled cells. Cooling for 24 h in UW followed by rewarming for 15 min induced a ~50% increase in MDA, while concomitantly lowering ATP by >90%. Treatment with SUL150 of cooled and rewarmed HEK293 effectively precluded the increase in MDA and preserved normal ATP in both DMEM- and UW-cooled cells. Likewise, treatment with Ferrostatin-1 blocked the MDA increase and preserved the ATP of rewarmed UW HEK293 cells. Cooling-induced HEK293 cell death from hypothermia and/or rewarming was caused by ferroptosis rather than apoptosis. UW slowed down ferroptosis during hypothermia, but lipid peroxidation and ATP depletion rapidly ensued upon rewarming, ultimately resulting in complete cell death. Treatment throughout UW cooling with small-molecule Ferrostatin-1 or the 6-chromanol SUL150 effectively prevented ferroptosis, maintained ATP, and limited lipid peroxidation in UW-cooled cells. Counteracting ferroptosis during cooling in UW-based preservation solutions may provide a simple method to improve graft survival following cold static cooling.

Peri-Operative Kinetics of Plasma Mitochondrial DNA Levels during Living Donor Kidney Transplantation.

During ischemia and reperfusion injury (IRI), mitochondria may release mitochondrial DNA (mtDNA). mtDNA can serve as a propagator of further injury but in specific settings has anti-inflammatory capacities as well. Therefore, the aim of this study was to study the perioperative dynamics of plasma mtDNA during living donor kidney transplantation (LDKT) and its potential as a marker of graft outcome. Fifty-six donor-recipient couples from the Volatile Anesthetic Protection of Renal Transplants-1 (VAPOR-1) trial were included. Systemic venous, systemic arterial, and renal venous samples were taken at multiple timepoints during and after LDKT. Levels of mtDNA genes changed over time and between vascular compartments. Several donor, recipient, and transplantation-related variables significantly explained the course of mtDNA genes over time. mtDNA genes predicted 1-month and 24-month estimated glomerular filtration rate (eGFR) and acute rejection episodes in the two-year follow-up period. To conclude, mtDNA is released in plasma during the process of LDKT, either from the kidney or from the whole body in response to transplantation. While circulating mtDNA levels positively and negatively predict post-transplantation outcomes, the exact mechanisms and difference between mtDNA genes are not yet understood and need further exploration.

Hibernator-Derived Cells Show Superior Protection and Survival in Hypothermia Compared to Non-Hibernator Cells.

Mitochondrial failure is recognized to play an important role in a variety of diseases. We previously showed hibernating species to have cell-autonomous protective mechanisms to resist cellular stress and sustain mitochondrial function. Here, we set out to detail these mitochondrial features of hibernators. We compared two hibernator-derived cell lines (HaK and DDT1MF2) with two non-hibernating cell lines (HEK293 and NRK) during hypothermia (4 °C) and rewarming (37 °C). Although all cell lines showed a strong decrease in oxygen consumption upon cooling, hibernator cells maintained functional mitochondria during hypothermia, without mitochondrial permeability transition pore (mPTP) opening, mitochondrial membrane potential decline or decreased adenosine triphosphate (ATP) levels, which were all observed in both non-hibernator cell lines. In addition, hibernator cells survived hypothermia in the absence of extracellular energy sources, suggesting their use of an endogenous substrate to maintain ATP levels. Moreover, hibernator-derived cells did not accumulate reactive oxygen species (ROS) damage and showed normal cell viability even after 48 h of cold-exposure. In contrast, non-hibernator cells accumulated ROS and showed extensive cell death through ferroptosis. Understanding the mechanisms that hibernators use to sustain mitochondrial activity and counteract damage in hypothermic circumstances may help to define novel preservation techniques with relevance to a variety of fields, such as organ transplantation and cardiac arrest.

Converse role of class I and class IIa HDACs in the progression of atrial fibrillation.

Atrial fibrillation (AF), the most common persistent clinical tachyarrhythmia, is associated with altered gene transcription which underlies cardiomyocyte dysfunction, AF susceptibility and progression. Recent research showed class I and class IIa histone deacetylases (HDACs) to regulate pathological and fetal gene expression, and thereby induce hypertrophy and cardiac contractile dysfunction. Whether class I and class IIa HDACs are involved in AF promotion is unknown. We aim to elucidate the role of class I and class IIa HDACs in tachypacing-induced contractile dysfunction in experimental model systems for AF and clinical AF. METHODS AND RESULTS: Class I and IIa HDACs were overexpressed in HL-1 cardiomyocytes followed by calcium transient (CaT) measurements. Overexpression of class I HDACs, HDAC1 or HDAC3, significantly reduced CaT amplitude in control normal-paced (1 Hz) cardiomyocytes, which was further reduced by tachypacing (5 Hz) in HDAC3 overexpressing cardiomyocytes. HDAC3 inhibition by shRNA or by the specific inhibitor, RGFP966, prevented contractile dysfunction in both tachypaced HL-1 cardiomyocytes and Drosophila prepupae. Conversely, overexpression of class IIa HDACs (HDAC4, HDAC5, HDAC7 or HDAC9) did not affect CaT in controls, with HDAC5 and HDAC7 overexpression even protecting against tachypacing-induced CaT loss. Notably, the protective effect of HDAC5 and HDAC7 was abolished in cardiomyocytes overexpressing a dominant negative HDAC5 or HDAC7 mutant, bearing a mutation in the binding domain for myosin enhancer factor 2 (MEF2). Furthermore, tachypacing induced phosphorylation of HDAC5 and promoted its translocation from the nucleus to cytoplasm, leading to up-regulation of MEF2-related fetal gene expression (ß-MHC, BNP). In accord, boosting nuclear localization of HDAC5 by MC1568 or Go6983 attenuated CaT loss in tachypaced HL-1 cardiomyocytes and preserved contractile function in Drosophila prepupae. Findings were expanded to clinical AF. Here, patients with AF showed a significant increase in expression levels and activity of HDAC3, phosphorylated HDAC5 and fetal genes (ß-MHC, BNP) in atrial tissue compared to controls in sinus rhythm. CONCLUSION: Class I and class IIa HDACs display converse roles in AF progression. Whereas overexpression of Class I HDAC3 induces cardiomyocyte dysfunction, class IIa HDAC5 overexpression reveals protective properties. Accordingly, HDAC3 inhibitors and HDAC5 nuclear boosters show protection from tachypacing-induced changes and therefore may represent interesting therapeutic options in clinical AF.

Activation of histone deacetylase-6 induces contractile dysfunction through derailment of α-tubulin proteostasis in experimental and human atrial fibrillation.

BACKGROUND: Atrial fibrillation (AF) is characterized by structural remodeling, contractile dysfunction, and AF progression. Histone deacetylases (HDACs) influence acetylation of both histones and cytosolic proteins, thereby mediating epigenetic regulation and influencing cell proteostasis. Because the exact function of HDACs in AF is unknown, we investigated their role in experimental and clinical AF models. METHODS AND RESULTS: Tachypacing of HL-1 atrial cardiomyocytes and Drosophila pupae hearts significantly impaired contractile function (amplitude of Ca(2+) transients and heart wall contractions). This dysfunction was prevented by inhibition of HDAC6 (tubacin) and sirtuins (nicotinamide). Tachypacing induced specific activation of HDAC6, resulting in α-tubulin deacetylation, depolymerization, and degradation by calpain. Tachypacing-induced contractile dysfunction was completely rescued by dominant-negative HDAC6 mutants with loss of deacetylase activity in the second catalytic domain, which bears α-tubulin deacetylase activity. Furthermore, in vivo treatment with the HDAC6 inhibitor tubastatin A protected atrial tachypaced dogs from electric remodeling (action potential duration shortening, L-type Ca(2+) current reduction, AF promotion) and cellular Ca(2+)-handling/contractile dysfunction (loss of Ca(2+) transient amplitude, sarcomere contractility). Finally, atrial tissue from patients with AF also showed a significant increase in HDAC6 activity and reduction in the expression of both acetylated and total α-tubulin. CONCLUSIONS: AF induces remodeling and loss of contractile function, at least in part through HDAC6 activation and subsequent derailment of α-tubulin proteostasis and disruption of the cardiomyocyte microtubule structure. In vivo inhibition of HDAC6 protects against AF-related atrial remodeling, disclosing the potential of HDAC6 as a therapeutic target in clinical AF.

Cooling of Cells and Organs Confers Extensive DNA Strand Breaks Through Oxidative Stress and ATP Depletion.

Cooling at 4°C is routinely used to lower metabolism and preserve cell and tissue integrity in laboratory and clinical settings, including organ transplantation. However, cooling and rewarming produce cell damage, attributed primarily to a burst of reactive oxygen species (ROS) upon rewarming. While DNA represents a highly vulnerable target of ROS, it is unknown whether cooling and/or rewarming produces DNA damage. Here, we show that cooling alone suffices to produce extensive DNA damage in cultured primary cells and cell lines, including double-strand breaks (DSBs), as shown by comet assay and pulsed-field gel electrophoresis. Cooling-induced DSB formation is time- and temperature-dependent and coincides with an excess production of ROS, rather than a decrease in ATP levels. Immunohistochemistry confirmed that DNA damage activates the DNA damage response marked by the formation of nuclear foci of proteins involved in DSB repair, γ-H2Ax, and 53BP1. Subsequent rewarming for 24 h fails to recover ATP levels and only marginally lowers DSB amounts and nuclear foci. Precluding ROS formation by dopamine and the hydroxychromanol, Sul-121, dose-dependently reduces DSBs. Finally, a standard clinical kidney transplant procedure, using cold static storage in UW preservation solution up to 24 h in porcine kidney, lowered ATP, increased ROS, and produced increasing amounts of DSBs with recruitment of 53BP1. Given that DNA repair is erroneous by nature, cooling-inflicted DNA damage may affect cell survival, proliferation, and genomic stability, significantly impacting cellular and organ function, with relevance in stem cell and transplantation procedures.

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.

Screening of novel HSP-inducing compounds to conserve cardiomyocyte function in experimental atrial fibrillation.

BACKGROUND: The heat shock protein (HSP) inducer, geranylgeranylacetone (GGA), was previously found to protect against atrial fibrillation (AF) remodeling in experimental model systems. Clinical application of GGA in AF is limited, due to low systemic concentrations owing to the hydrophobic character of GGA. OBJECTIVES: To identify novel HSP-inducing compounds, with improved physicochemical properties, that prevent contractile dysfunction in experimental model systems for AF. METHODS: Eighty-one GGA-derivatives were synthesized and explored for their HSP-inducing properties by assessment of HSP expression in HL-1 cardiomyocytes pretreated with or without a mild heat shock (HS), followed by incubation with 10 µM GGA or GGA-derivative. Subsequently, the most potent HSP-inducers were tested for preservation of calcium transient (CaT) amplitudes or heart wall contraction in pretreated tachypaced HL-1 cardiomyocytes (with or without HSPB1 siRNA) and Drosophilas, respectively. Finally, CaT recovery in tachypaced HL-1 cardiomyocytes posttreated with GGA or protective GGA-derivatives was determined. RESULTS: Thirty GGA-derivatives significantly induced HSPA1A expression after HS, and seven showed exceeding HSPA1A expression compared to GGA. GGA and nine GGA-derivatives protected significantly from tachypacing (TP)-induced CaT loss, which was abrogated by HSPB1 suppression. GGA and four potent GGA-derivatives protected against heart wall dysfunction after TP compared to non-paced control Drosophilas. Of these compounds, GGA and three GGA-derivatives induced a significant restoration from CaT loss after TP of HL-1 cardiomyocytes. CONCLUSION: We identified novel GGA-derivatives with improved physicochemical properties compared to GGA. GGA-derivatives, particularly GGA*-59, boost HSP expression resulting in prevention and restoration from TP-induced remodeling, substantiating their role as novel therapeutics in clinical AF.

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.

Application of kinomic array analysis to screen for altered kinases in atrial fibrillation remodeling.

BACKGROUND: Dysregulation of protein kinase-mediated signaling is an early event in many diseases, including the most common clinical cardiac arrhythmia, atrial fibrillation (AF). Kinomic profiling represents a promising technique to identify candidate kinases. OBJECTIVE: In this study we used kinomic profiling to identify kinases altered in AF remodeling using atrial tissue from a canine model of AF (atrial tachypacing). METHODS: Left atrial tissue obtained in a previous canine study was used for kinomic array (containing 1024 kinase pseudosubstrates) analysis. Three groups of dogs were included: nonpaced controls and atrial tachypaced dogs, which were contrasted with geranylgeranylacetone-treated dogs with AF, which are protected from AF promotion, to enhance specificity of detection of putative kinases. RESULTS: While tachypacing changed activity of 50 kinases, 40 of these were prevented by geranylgeranylacetone and involved in differentiation and proliferation (SRC), contraction, metabolism, immunity, development, cell cycle (CDK4), and survival (Akt). Inhibitors of Akt (MK2206) and CDK4 (PD0332991) and overexpression of a dominant-negative CDK4 phosphorylation mutant protected against tachypacing-induced contractile dysfunction in HL-1 cardiomyocytes. Moreover, patients with AF show down- and upregulation of SRC and Akt phosphorylation, respectively, similar to findings of the kinome array. CONCLUSION: Contrasting kinomic array analyses of controls and treated subjects offer a versatile tool to identify kinases altered in atrial remodeling owing to tachypacing, which include Akt, CDK4, and SRC. Ultimately, pharmacological targeting of altered kinases may offer novel therapeutic possibilities to treat clinical AF.

Differences in mitochondrial function and morphology during cooling and rewarming between hibernator and non-hibernator derived kidney epithelial cells.

Hibernators show superior resistance to ischemia and hypothermia, also outside the hibernation season. Therefore, hibernation is a promising strategy to decrease cellular damage in a variety of fields, such as organ transplantation. Here, we explored the role of mitochondria herein, by comparing epithelial cell lines from a hibernator (hamster kidney cells, HaK) and a non-hibernator (human embryonic kidney cells, HEK293) during cold preservation at 4 °C and rewarming. Cell survival (Neutral Red), ATP and MDA levels, mitochondrial membrane potential (MMP), mitochondrial morphology (using fluorescent probes) and metabolism (seahorse XF) were assessed. Hypothermia induced dispersion of the tubular mitochondrial network, a loss of MMP, increased oxygen radical (MDA) and decreased ATP production in HEK293. In contrast, HaK maintained MMP and ATP production without an increase in oxygen radicals during cooling and rewarming, resulting in superior cell survival compared to HEK293. Further, normothermic HaK showed a dispersed mitochondrial network and higher respiratory and glycolysis capacity compared to HEK293. Disclosing the mechanisms that hibernators use to counteract cell death in hypothermic and ischemic circumstances may help to eventually improve organ preservation in a variety of fields, including organ transplantation.

Endoplasmic Reticulum Stress Is Associated With Autophagy and Cardiomyocyte Remodeling in Experimental and Human Atrial Fibrillation.

BACKGROUND: Derailment of proteostasis, the homeostasis of production, function, and breakdown of proteins, contributes importantly to the self-perpetuating nature of atrial fibrillation (AF), the most common heart rhythm disorder in humans. Autophagy plays an important role in proteostasis by degrading aberrant proteins and organelles. Herein, we investigated the role of autophagy and its activation pathway in experimental and clinical AF. METHODS AND RESULTS: Tachypacing of HL-1 atrial cardiomyocytes causes a gradual and significant activation of autophagy, as evidenced by enhanced LC3B-II expression, autophagic flux and autophagosome formation, and degradation of p62, resulting in reduction of Ca2+ amplitude. Autophagy is activated downstream of endoplasmic reticulum (ER) stress: blocking ER stress by the chemical chaperone 4-phenyl butyrate, overexpression of the ER chaperone-protein heat shock protein A5, or overexpression of a phosphorylation-blocked mutant of eukaryotic initiation factor 2α (eIF2α) prevents autophagy activation and Ca2+-transient loss in tachypaced HL-1 cardiomyocytes. Moreover, pharmacological inhibition of ER stress in tachypaced Drosophila confirms its role in derailing cardiomyocyte function. In vivo treatment with sodium salt of phenyl butyrate protected atrial-tachypaced dog cardiomyocytes from electrical remodeling (action potential duration shortening, L-type Ca2+-current reduction), cellular Ca2+-handling/contractile dysfunction, and ER stress and autophagy; it also attenuated AF progression. Finally, atrial tissue from patients with persistent AF reveals activation of autophagy and induction of ER stress, which correlates with markers of cardiomyocyte damage. CONCLUSIONS: These results identify ER stress-associated autophagy as an important pathway in AF progression and demonstrate the potential therapeutic action of the ER-stress inhibitor 4-phenyl butyrate.

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.

Hydroximic acid derivatives: pleiotropic HSP co-inducers restoring homeostasis and robustness.

According to the "membrane sensor" hypothesis, the membrane's physical properties and microdomain organization play an initiating role in the heat shock response. Clinical conditions such as cancer, diabetes and neurodegenerative diseases are all coupled with specific changes in the physical state and lipid composition of cellular membranes and characterized by altered heat shock protein levels in cells suggesting that these "membrane defects" can cause suboptimal hsp-gene expression. Such observations provide a new rationale for the introduction of novel, heat shock protein modulating drug candidates. Intercalating compounds can be used to alter membrane properties and by doing so normalize dysregulated expression of heat shock proteins, resulting in a beneficial therapeutic effect for reversing the pathological impact of disease. The membrane (and lipid) interacting hydroximic acid (HA) derivatives discussed in this review physiologically restore the heat shock protein stress response, creating a new class of "membrane-lipid therapy" pharmaceuticals. The diseases that HA derivatives potentially target are diverse and include, among others, insulin resistance and diabetes, neuropathy, atrial fibrillation, and amyotrophic lateral sclerosis. At a molecular level HA derivatives are broad spectrum, multi-target compounds as they fluidize yet stabilize membranes and remodel their lipid rafts while otherwise acting as PARP inhibitors. The HA derivatives have the potential to ameliorate disparate conditions, whether of acute or chronic nature. Many of these diseases presently are either untreatable or inadequately treated with currently available pharmaceuticals. Ultimately, the HA derivatives promise to play a major role in future pharmacotherapy.

Loss of proteostatic control as a substrate for atrial fibrillation: a novel target for upstream therapy by heat shock proteins.

Atrial fibrillation (AF) is the most common, sustained clinical tachyarrhythmia associated with significant morbidity and mortality. AF is a persistent condition with progressive structural remodeling of the atrial cardiomyocytes due to the AF itself, resulting in cellular changes commonly observed in aging and in other heart diseases. While rhythm control by electrocardioversion or drug treatment is the treatment of choice in symptomatic AF patients, its efficacy is still limited. Current research is directed at preventing first-onset AF by limiting the development of substrates underlying AF progression and resembles mechanism-based therapy. Upstream therapy refers to the use of non-ion channel anti-arrhythmic drugs that modify the atrial substrate- or target-specific mechanisms of AF, with the ultimate aim to prevent the occurrence (primary prevention) or recurrence of the arrhythmia following (spontaneous) conversion (secondary prevention). Heat shock proteins (HSPs) are molecular chaperones and comprise a large family of proteins involved in the protection against various forms of cellular stress. Their classical function is the conservation of proteostasis via prevention of toxic protein aggregation by binding to (partially) unfolded proteins. Our recent data reveal that HSPs prevent electrical, contractile, and structural remodeling of cardiomyocytes, thus attenuating the AF substrate in cellular, Drosophila melanogaster, and animal experimental models. Furthermore, studies in humans suggest a protective role for HSPs against the progression from paroxysmal AF to persistent AF and in recurrence of AF. In this review, we discuss upregulation of the heat shock response system as a novel target for upstream therapy to prevent derailment of proteostasis and consequently progression and recurrence of AF.

Heat shock protein-inducing compounds as therapeutics to restore proteostasis in atrial fibrillation.

Atrial fibrillation (AF) is the most common clinical tachyarrhythmia associated with significant morbidity and mortality and is expected to affect approximately 30 million North Americans and Europeans by 2050. AF is a persistent disease, caused by progressive, often age-related, derailment of proteostasis resulting in structural remodeling of the atrial cardiomyocytes. It has been widely acknowledged that the progressive nature of the disease hampers the effective functional conversion to sinus rhythm in patients and explains the limited effect of current drug therapies. Therefore, research is directed at preventing new-onset AF by limiting the development of substrates underlying AF promotion. Upstream therapy refers to the use of drugs that modify the atrial substrate- or target-specific mechanisms of AF, with the ultimate aim to prevent the occurrence (primary prevention) and recurrence of the arrhythmia following (spontaneous) conversion and to prevent the progression of AF (secondary prevention). Recently, we observed that heat shock protein (HSP)-inducing drugs, such as geranylgeranylacetone, prevent derailment of proteostasis and remodeling of cardiomyocytes and thereby attenuate the AF substrate in cellular, Drosophila melanogaster, and animal experimental models. Also, correlative data from human studies were consistent with a protective role of HSPs in preventing the progression from paroxysmal AF to permanent AF and in the recurrence of AF. In this review, we discuss novel HSP-inducing compounds as emerging therapeutics for the primary and secondary prevention of AF.

HSPB1, HSPB6, HSPB7 and HSPB8 protect against RhoA GTPase-induced remodeling in tachypaced atrial myocytes.

BACKGROUND: We previously demonstrated the small heat shock protein, HSPB1, to prevent tachycardia remodeling in in vitro and in vivo models for Atrial Fibrillation (AF). To gain insight into its mechanism of action, we examined the protective effect of all 10 members of the HSPB family on tachycardia remodeling. Furthermore, modulating effects of HSPB on RhoA GTPase activity and F-actin stress fiber formation were examined, as this pathway was found of prime importance in tachycardia remodeling events and the initiation of AF. METHODS AND RESULTS: Tachypacing (4 Hz) of HL-1 atrial myocytes significantly and progressively reduced the amplitude of Ca²âº transients (CaT). In addition to HSPB1, also overexpression of HSPB6, HSPB7 and HSPB8 protected against tachypacing-induced CaT reduction. The protective effect was independent of HSPB1. Moreover, tachypacing induced RhoA GTPase activity and caused F-actin stress fiber formation. The ROCK inhibitor Y27632 significantly prevented tachypacing-induced F-actin formation and CaT reductions, showing that RhoA activation is required for remodeling. Although all protective HSPB members prevented the formation of F-actin stress fibers, their mode of action differs. Whilst HSPB1, HSPB6 and HSPB7 acted via direct prevention of F-actin formation, HSPB8-protection was mediated via inhibition of RhoA GTPase activity. CONCLUSION: Overexpression of HSPB1, as well as HSPB6, HSPB7 and HSPB8 independently protect against tachycardia remodeling by attenuation of the RhoA GTPase pathway at different levels. The cardioprotective role for multiple HSPB members indicate a possible therapeutic benefit of compounds able to boost the expression of single or multiple members of the HSPB family.