atrial fibrillation ( af ) represents the arrhythmia with the highest prevalence , accounting for one - third of hospitalisations related to cardiac rhythm disturbances . the main risk factors to develop af include age , cardiac surgery , valvular heart disease , congestive heart disease , ischaemic cardiomyopathy , obesity , hypertension and diabetes mellitus . the goal of therapy in af is , ideally , to abolish af episodes and to restore sinus rhythm . research , treatment of af remains difficult , which is rooted in the persistent and progressive nature of this arrhythmia . an initiator , trigger and substrate are mandatory for development of af . in this manuscript af persistence is caused by progressive changes in the electrophysiology and structure of atrial cardiomyocytes , as demonstrated in a goat model for af . in this model , af resulted in shortening of the atrial effective refractory period and reversion of the physiological rate adaptation ( shortening of the atrial refractory period at slower heart rates ) , facilitating induction and stability of af . the goat model of af also revealed that longstanding af is associated with changes in myocardial structure , including dedifferentiation , increase in cell size , perinuclear accumulation of glycogen , central loss of sarcomeres , changes in mitochondrial shape , fragmentation of the sarcoplasmatic reticulum and disorganisation of fibre orientation . importantly , structural changes are sustainable and impair electrical coupling and the functional recovery to sinus rhythm by pharmacological and electrical cardioversion . in addition , it was found that alterations of the myocardial structure underlie changes in electrophysiology as observed in af [ 2 , 5 ] . it should be noted that electropathology is already present when a patient enters the clinic for the first time with an episode of af . the currently available pharmacological therapy is directed at alleviation of electrical changes ( rhythm control ) and has limited effect on patient outcome . therapeutic approaches that halt the mechanisms conveying the af - induced structural remodelling may offer superior therapeutic perspectives . thus , from science to patients : reversal of electropathology of cardiomyocytes represents a key target to accomplish and maintain cardiac sinus rhythm after af . it is generally assumed that transition of short - lasting paroxysms of af to long - lasting episodes that no longer terminate spontaneously over time corresponds to a progression from a trigger - driven to a substrate - mediated arrhythmia due to a combination of electrical , structural and contractile atrial remodelling ( fig .  1 ) . previous studies have demonstrated that alterations in the atrial microscopic structure impair intra - atrial conduction . [ 7 , 8 ] were the first to provide evidence that two - dimensional propagation is discontinuous in nature at a microscopic level ( fig .  2 ) . in their experiments , variations in shape and amplitude of extracellular potential waveforms were studied when the direction of conduction was changed from longitudinal to transverse in relation to the fibre axis . they observed that in uniform anisotropic myocardium , the shape of the upstroke of the transmembrane action potential depended on the direction of propagation in relation to the fibre orientation . fast conduction in the longitudinal direction was associated with a slow upstroke and a long -foot ( time constant of the action potential representing voltage decay ) and slow conduction in the transverse direction with a fast upstroke and a short -foot . differences were explained by the anisotropic distribution of intercellular connections ( gap junctions , connexions ) . this was confirmed in a goat model for sustained ( 2 months ) af , where persistence of af was associated with a lower density and inhomogeneous distribution of gap junctions [ 9 , 10 ] . gap junctions along the fibre axis provide a low resistance to current flow that increases the space constant and hence the electrotonic current flow . subsequently , the upstroke of the transmembrane potential in this direction decreases and the -foot increases . due to the high resistance barriers in the transverse direction , the space constant is reduced and the electrotonic current is therefore smaller . this results in an increase in the upstroke of the transmembrane potential and a decrease in the -foot . consequently , as the safety factor is lower during longitudinal conduction than during transverse conduction , conduction block is more likely to occur during longitudinal conduction .fig . surface electrograms of paroxysms ( upper panel ) and persistent ( lower panel ) atrial fibrillation . a wave - map shows individual fibrillation waves represented by colours according to their sequence of appearance . a previously described mapping algorithm was used to classify them into peripheral waves ( entering the mapping area from outside the electrode array ) , breakthrough waves ( appearing at the epicardial surface inside the mapping area ) or discontinuous conduction waves ( fibrillation waves starting with a delay of 1340  ms from boundaries of other waves ) . sites of epicardial breakthroughs are indicated by white asterisks ; white arrows indicate direction(s ) of expansion of epicardial breakthrough or discontinuous fibrillation waves . a electrograms recorded in the longitudinal and transversal conduction direction in relation to fibre orientation in uniform anisotropic and non - uniform anisotropic tissue . the electrogram in the transverse conduction direction in non - uniform anisotropic tissue consists of multiple deflections indicating discontinuous conduction . these alterations may be caused by changes in myocardial structure , including increase in cell size , perinuclear accumulation of glycogen , central loss of sarcomeres ( myolysis ) , changes in mitochondrial shape , fragmentation of the sarcoplasmatic reticulum and disorganisation of fibre orientation as ( partly ) observed in panel b and c. b atrial myocardium from a patient in sinus rhythm ( sr ) and ( c ) a patient with permanent af ( peaf ) stained with toluidine blue . sr patients show normal structural cardiomyocytes , whereas the patient with peaf reveals cardiomyocytes with extensive perinuclear myolysis ( red asterisk ) persistence of atrial fibrillation . surface electrograms of paroxysms ( upper panel ) and persistent ( lower panel ) atrial fibrillation . a wave - map shows individual fibrillation waves represented by colours according to their sequence of appearance . a previously described mapping algorithm was used to classify them into peripheral waves ( entering the mapping area from outside the electrode array ) , breakthrough waves ( appearing at the epicardial surface inside the mapping area ) or discontinuous conduction waves ( fibrillation waves starting with a delay of 1340  ms from boundaries of other waves ) . sites of epicardial breakthroughs are indicated by white asterisks ; white arrows indicate direction(s ) of expansion of epicardial breakthrough or discontinuous fibrillation waves . a electrograms recorded in the longitudinal and transversal conduction direction in relation to fibre orientation in uniform anisotropic and non - uniform anisotropic tissue . the electrogram in the transverse conduction direction in non - uniform anisotropic tissue consists of multiple deflections indicating discontinuous conduction . these alterations may be caused by changes in myocardial structure , including increase in cell size , perinuclear accumulation of glycogen , central loss of sarcomeres ( myolysis ) , changes in mitochondrial shape , fragmentation of the sarcoplasmatic reticulum and disorganisation of fibre orientation as ( partly ) observed in panel b and c. b atrial myocardium from a patient in sinus rhythm ( sr ) and ( c ) a patient with permanent af ( peaf ) stained with toluidine blue . sr patients show normal structural cardiomyocytes , whereas the patient with peaf reveals cardiomyocytes with extensive perinuclear myolysis ( red asterisk ) next to gap junctional remodelling , also ion channel remodelling has been demonstrated to play a role in af progression . a marked reduction in ion ( preferably l - type ca and transient outward k ) channel protein expression , associated with a shortening of atrial effective refractory period , was observed in patients with af . this decrease in protein expression might be explained by increased ( calcium dependent ) calpain i activity , causing enhanced degradation of ion - channel proteins and contractile proteins in the myocardium [ 4 , 13 ] . modifications of the atrial architecture affecting cardiomyocyte geometry ( size and shape ) , gap junctions ( distribution and conductivity ) or the interstitial space ( size and distribution ) result in non - uniform anisotropic tissue . muscle bundles become electrically dissociated due to strands of fibrotic tissue giving rise to fractionation of extracellular potentials . fractionated electrograms are extracellular waveforms containing multiple , distinct deflections reflecting local asynchronous activation of myocardium surrounding the recording electrode . fractionation of fibrillation potentials can be functional and/or structural in nature as local asynchronous activation can be the result of a spatial dispersion in refractory periods ( functional ) or non - uniform tissue anisotropy ( structural , as explained above ) . during af , multiple , simultaneously circulating wavelets excite the atria . a fibrillation wave may encounter atrial tissue that is partially or totally refractory due to previous excitation by another wave . this results in slowing of conduction , conduction block and turning of fibrillation waves around lines of conduction block . all these conduction abnormalities give rise to local asynchronous activation of the myocardium and thus to multiple deflections in the extracellular potentials . high - density epicardial mapping during electrically induced af in humans without structural heart disease showed that there is a relation between the degree of fractionation in the right atrium and specific spatial patterns of activation . 3 ) , whereas electrograms with three or more deflections were recorded at pivot points or areas of slow conduction [ 15 , 16 ] . in older patients with structural heart disease and longstanding persistent af , it is likely that fractionation is more structurally determined due to dissociation of muscle bundles by fibrosis or fatty degeneration . recent studies suggested that ablation of areas with fractionated electrograms may eliminate af suggesting that fractionated electrograms are indeed indicative for arrhythmogenic regions perpetuating af .fig . the colour - coded activation map ( interelectrode distance , 2  mm ) shows two fibrillation waves ( its trajectories indicated by thick black lines ) separated by areas of conduction block . they are recorded along the line of conduction block reflecting activation on either side double potentials recorded along the lines of conduction block . the colour - coded activation map ( interelectrode distance , 2  mm ) shows two fibrillation waves ( its trajectories indicated by thick black lines ) separated by areas of conduction block . previous research has unequivocally demonstrated that the induction of the heat shock response provides protection against various cardiac diseases . sensing of cellular stress activates the heat shock transcription factor-1 ( hsf-1 ) and consequently results in the expression of heat shock proteins ( hsps ) . protein interactions such as folding and assisting in the establishment of proper protein conformation ( shape ) and prevention of unwanted protein aggregation , which occur during ageing of the cell and ( cardiac ) stress . because of their essential role in protein maintenance , some members of the hsp family are expressed at low to moderate levels in all organisms . in cardiomyocytes , hsps bind to damaged proteins , including sarcomeric proteins , and thereby prevent cardiomyocyte toxicity and further structural remodelling and functional loss [ 19 , 20 ] . hsps consist of five hsp families , including the hsp70 and hsp27 family , which have both been suggested to convey cardioprotective effects . therefore , impairment of hsp expression , especially hsp70 and hsp27 , has been related to the development of various cardiac diseases , including af , but also metabolic diseases , such as diabetes and obesity , which trigger the induction of af . these findings indicate that hsps represent an interesting target to treat cardiac diseases such as af . indeed , experimental and human studies revealed that hsps provide protection against af initiation . in dogs , pretreatment with the hsp inducer , geranylgeranylacetone ( gga ) , attenuates changes in electrophysiology and conduction velocity of atrial cardiomyocytes and prevents initiation of af in ( acute ) ischaemia . also protective effects of hsps were observed in a rabbit model for af induced by heart failure . a further indication for the protective effect of hsps is obtained from two studies in patients undergoing cardiac surgery . in both studies an inverse correlation between higher levels of hsp70 expression in atrial tissue and lower incidence of post - operative af was observed [ 24 , 25 ] . in addition , one study described a correlation between hsp70 and hsp27 levels in tissue and improved restoration of sinus rhythm after mitral valve surgery . in addition , high hsp27 levels in blood predict sinus rhythm maintenance after catheter ablation in patients with paroxysmal af . in an experimental study in mice , it was observed that induction of hsp70 prevents angiotensin ii - induced af and limits atrial fibrosis formation . together , these results indicate that induction of hsps mitigates the development of the atrial substrate for the induction of af . in addition to the cardioprotective effect of hsps against the induction of af , it is expected that induced expression of hsps also protects against af progression . for example , general hsp levels were induced by a heat shock or gga treatment in tachypaced hl-1 atrial cardiomyoyctes and these cardiomyocytes were compared with normal tachypaced hl-1 cardiomyocytes [ 20 , 28 ] . in these studies , dogs pretreated with gga showed attenuation of tachypacing - induced af promotion and limited recurrence of af after cardioversion , most likely by limiting the electrophysiological changes , including shortening of action potential duration and maintenance of l - type ca current . further studies revealed that specific members of the family of small hsps play a key role in cardioprotection against af . induction of hsp27 was found sufficient to prevent tachypacing - induced structural remodelling , while overexpressing hsp70 was not protective . other specific small hsp family members also show protection in cellular models , including hsp20 , cvhsp and hsp22 , but not the other small hsp family members . importantly , selective knockdown of hsp27 fully attenuates the protective effect of gga , demonstrating a main role for hsp27 in cardioprotection . a similar protective effect of the small hsp members as observed in hl-1 cardiomyocytes has been found in the drosophila melanogaster ( fruit fly ) model for tachypacing - induced contractile dysfunction . in the transparent fruit fly pupae this model also allows for general induction of hsps , either by heat shock or by hsp - inducing compounds such as gga , or specific induction of hsps via genetic manipulation specifically in the heart . indeed , induction of hsps , especially the small hsp , by these strategies prevents tachypacing - induced contractile dysfunction and structural remodelling in drosophila pupae . also in human af , there are indications that hsp27 may protect against cardiac remodelling and af progression . two independent studies report higher atrial expression levels of hsp27 to relate to a shorter duration of af and less extensive structural damage [ 20 , 31 ] . this suggests that in short - duration af , the hsp response gets activated , while it diminishes over time when af persists . as hsp27 conserves the cardiomyocyte structure , lower levels of hsp27 may trigger the progression of structural remodelling paving the way to longstanding and permanent af . therefore , securing hsp levels at an adequate level , for example , by treatment with hsp inducers , may limit the expansion of the af substrate during paroxysmal and short - term af . in agreement with this hypothesis , restoration of sinus rhythm in patients with permanent af after mitral valve surgery is related to the heat shock factor ( hsf ) -1 activity and induced hsp27 levels . taken together , induction of hsps attenuates electropathology of cardiomyocytes in af . in hsf1 activators lower cardiomyocyte damage : towards a novel therapeutic approach to reverse atrial fibrillation ( halt & reverse ) , we will focus on the electropathological substrate of af and dissect the mechanism of protection . pharmacological approaches to prevent mechanisms underlying atrial electropathology are being studied , with the hope that they might be useful therapeutic agents in treating af . it has been recognised that the efficacy of commonly used drugs on remodelling is limited . as the protective action of hsps depends on their temporary induction , drugs that boost the endogenous heat shock responses are of particular interest in the prevention of first onset , recurrence and progression of af . gga was originally used as an anti - ulcer agent and is a non - toxic acyclic isoprenoid compound with a retinoid skeleton that induces hsp synthesis in various tissues , including gastric mucosa , intestine , liver , heart , retina and the central nervous system [ 33 , 34 ] . the protective effect of gga - induced hsp expression on electropathology caused by tachycardia has been observed in various experimental models , including the dog model for af . these findings suggest that the induction of hsps by gga might have a potential value for clinical af . in in vivo tachypaced drosophila , gga treatment protected against contractile dysfunction of the heart wall and structural remodelling . also in the dog model for ( acute ) atrial ischaemia and tachypacing - induced af promotion , an hsp - inducing gga treatment revealed protective effects against cardiomyocyte remodelling , and consequently occurrence and recurrence of af . a direct protective effect of hsp on the earlier - mentioned gap junctional and ion channel remodelling has not been described ( yet ) . however , it has been shown that gga treatment attenuates the proteolytic activity of calpain   and prevents ion channel remodelling in af , suggesting a direct protective role of hsps . although protective effects with gga were observed , an important disadvantage of gga is its high logp value , which is around 9 , and therefore generally high dosages are required , as found in the dog studies for af [ 22 , 28 ] . to overcome this disadvantage , various derivatives of gga have been synthesised , with improved pharmaco - chemical and hsp - boosting properties ( fig . some of these derivatives also showed cardioprotective effects in tachypaced hl-1 cardiomyocytes ( fig . , we will test whether these derivatives reveal improved cardioprotective effects compared with gga and elucidate their mode of action . in addition , one of the selected gga derivatives will be further tested in an animal model for af . finally , gga has also been tested in patients undergoing cardiac surgery , and atrial tissue will be used to test whether gga induces hsp expression in patients and protects against post - surgery af.fig . 4geranylgeranylacetone ( gga ) derivatives induce hsp70 expression and reveal cardioprotective effects . a western blot showing induction of hsp70 levels in hl-1 cardiomyocytes treated with gga and some of the gga derivatives ( nyk compounds ) . b representative calcium transient tracers of non - treated and normally paced ( 1  hz ) hl-1 cardiomyocytes ( control ) or tachypaced cardiomyocytes ( tp , 5  hz ) without or with treatment of 10  m gga or gga derivatives ( nyk compounds ) as indicated . atrial fibrillation ( af ) naturally progresses in time ( red line ) , which is rooted in the underlying electropathology . by pharmacological induction of hsp levels ( blue arrow ) , we aim to halt or even reverse electropathology and consequently af progression ( green line ) geranylgeranylacetone ( gga ) derivatives induce hsp70 expression and reveal cardioprotective effects . a western blot showing induction of hsp70 levels in hl-1 cardiomyocytes treated with gga and some of the gga derivatives ( nyk compounds ) . b representative calcium transient tracers of non - treated and normally paced ( 1  hz ) hl-1 cardiomyocytes ( control ) or tachypaced cardiomyocytes ( tp , 5  hz ) without or with treatment of 10  m gga or gga derivatives ( nyk compounds ) as indicated . atrial fibrillation ( af ) naturally progresses in time ( red line ) , which is rooted in the underlying electropathology . by pharmacological induction of hsp levels ( blue arrow ) , we aim to halt or even reverse electropathology and consequently af progression ( green line ) ultimately , by inducing cardioprotective hsp expression , the halt&reverse consortium wants to halt or even reverse the structure of cardiomyocytes and therefore the electropathology in patients with af ( fig . hereby , we expect to significantly contribute to novel rational approaches , to enhance successful recovery of heart function after electrical cardioversion ( ecv ) and/or cardiac surgery for the first time , we exploit hsp levels to predict the electropathological outcome in patients , as measured by our unique mapping approaches . in addition , at the end of this project , we will know whether hsps are suitable biomarkers for af . our unique approach will ultimately result in reduced hospital time , induced quality of life for the patient and lower costs for society . the findings will be of benefit to other ( cardiac , kidney ) diseases , as many of those are characterised by sustainable structural remodelling . thus , the project is of major importance from a scientific , clinical and economical perspective .