Redox modification of ryanodine receptors by mitochondria‐derived reactive oxygen species contributes to aberrant Ca2+ handling in ageing rabbit hearts

Ageing is associated with increased risk of sudden cardiac death due to malignant arrhythmias. Shortened refractoriness of Ca2+ release due to increased activity of Ca2+ release channels (RyRs) is recognized as an important contributor to cardiac-triggered arrhythmias. However, molecular mechanisms of RyR dysfunction and its contribution to arrhythmias in ageing remain to be examined. Using ventricular myocytes isolated from old rabbit hearts we demonstrate that age-associated increase in rate of production of reactive oxygen species (ROS) by mitochondria leads to the thiol-oxidation of RyRs, which underlies the hyperactivity of the channels and thus shortened refractoriness of Ca2+ release in cardiomyocytes from the ageing heart. Mitochondria-specific scavenging of ROS in old myocytes restored the redox status of RyRs, reducing SR Ca2+ leak and arrhythmogenic spontaneous Ca2+ waves. We conclude that increased ROS production by mitochondria contributes to age-associated increased risk of stress-induced arrhythmia and sudden cardiac death through thiol-modifications of RyRs. Abstract Ageing is associated with a blunted response to sympathetic stimulation and an increased risk of arrhythmia and sudden cardiac death. Aberrant calcium (Ca2+) handling is an important contributor to the electrical and contractile dysfunction associated with ageing. Yet, the specific molecular mechanisms underlying abnormal Ca2+ handling in ageing heart remain poorly understood. In this study, we used ventricular myocytes isolated from young (5–9 months) and old (4–6 years) rabbit hearts to test the hypothesis that changes in Ca2+ homeostasis are caused by post-translational modification of ryanodine receptors (RyRs) by mitochondria-derived reactive oxygen species (ROS) generated in the ageing heart. Changes in parameters of Ca2+ handling were determined by measuring cytosolic and intra-sarcoplasmic reticulum (SR) Ca2+ dynamics in intact and permeabilized ventricular myocytes using confocal microscopy. We also measured age-related changes in ROS production and mitochondria membrane potential using a ROS-sensitive dye and a mitochondrial voltage-sensitive fluorescent indicator, respectively. In permeablized myocytes, ageing did not change SERCA activity and spark frequency but decreased spark amplitude and SR Ca2+ load suggesting increased RyR activity. Treatment with the antioxidant dithiothreitol reduced RyR-mediated SR Ca2+ leak in permeabilized myocytes from old rabbit hearts to the level comparable to young. Moreover, myocytes from old rabbits had more depolarized mitochondria membrane potential and increased rate of ROS production. Under β-adrenergic stimulation, Ca2+ transient amplitude, SR Ca2+ load, and latency of pro-arrhythmic spontaneous Ca2+ waves (SCWs) were decreased while RyR-mediated SR Ca2+ leak was increased in cardiomyocytes from old rabbits. Additionally, with β-adrenergic stimulation, scavenging of mitochondrial ROS in myocytes from old rabbit hearts restored redox status of RyRs, which reduced SR Ca2+ leak, ablated most SCWs, and increased latency to levels comparable to young. These data indicate that an age-associated increase of ROS production by mitochondria leads to the thiol-oxidation of RyRs, which underlies the hyperactivity of RyRs and thereby shortened refractoriness of Ca2+ release in cardiomyocytes from the ageing heart. This mechanism probably plays an important role in the increased incidence of arrhythmia and sudden death in the ageing population. Ageing is associated with increased incidence of cardiac arrhythmia and sudden cardiac death (Kannel et al. 1987; Lakatta, 1993). Previously, in a rabbit model, we have shown that ageing alters the structure and the electrical and mechanical activity of the heart causing both systolic and diastolic dysfunction, slowing conduction velocity, and altering conduction anisotropy, which provides a pro-arrhythmic substrate that increases the risk of malignant arrhythmia (Cooper et al. 2012). Although much is known regarding the substrate that predisposes and sustains arrhythmias in the ageing heart, the underlying age-associated molecular trigger remains to be thoroughly examined. At the cellular level, aberrant Ca2+ handling is recognized as an important contributor to the electrical dysfunction associated with ageing (Xiao et al. 1994; Lakatta Dibb et al. 2004; Zhu et al. 2005; Howlett, 2010; Janczewski Lakatta Chaudhary et al. 2011). In aged rat and rabbit ventricles, Morita et al. showed that exposure to oxidative stress led to early afterdepolarizations (EADs) in myocytes and EAD-mediated tachyarrhythmias at the tissue level (Morita et al. 2009). Recent studies identified the SR Ca2+ release channel, the ryanodine receptor (RyR), as a protein target that is sensitive to oxidation, and post-translational modifications of RyRs by reactive oxygen species (ROS) that destabilize interdomain interactions within RyRs (Mochizuki et al. 2007) have been implicated in alterations of Ca2+ homeostasis in conditions accompanied by oxidative stress, such as heart failure or myocardial infarct (Giordano, 2005; Zima Györke Terentyev et al. 2008; Belevych et al. 2009, 2011b, 2012). Specifically, accelerated leak of Ca2+SR via oxidized RyRs was shown to result in diminished systolic Ca2+ release that determines strength of contraction and an enhanced propensity to generate pro-arrhythmic, diastolic Ca2+ waves during β-adrenergic stimulation (Terentyev et al. 2008; Belevych et al. 2011b, 2012). Yet, the role of redox-mediated alterations of Ca2+ handling (RyRs in particular) with regard to ageing and Ca2+-triggered arrhythmias remains to be investigated. Moreover, it is well established that mitochondria, one of the main sources of ROS in myocytes, if damaged or dysfunctional, could increase ROS production rate up to 10-fold (Grivennikova et al. 2010). Dysfunctional mitochondria have been implicated as a mechanism for ventricular arrhythmia by altering ion channel function, action potential heterogeneity, and cell excitability (O’Rourke et al. 1994; Brown et al. 2010; Brown Bovo et al. 2012). The goal of this study was to test the hypothesis that changes in Ca2+ homeostasis associated with ageing are caused by post-translational modification of RyRs by mitochondria-derived ROS generated in the old heart. Importantly, we sought to examine the mechanisms and effects of ROS generation and to determine whether exposure of cardiomyocytes from old rabbits to a reducing agent and a mitochondria-specific ROS scavenger reverses the age-related pro-arrhythmogenic cellular phenotype. Our results show that the age-associated increase in rate of ROS production by mitochondria leads to the thiol-oxidation of RyRs, which underlies the hyperactivity of RyRs and thereby shortened refractoriness of Ca2+ release in cardiomyocytes from the ageing heart. We conclude that this mechanism contributes to age-associated increased risk of stress-induced arrhythmia and sudden death. All animal work was performed in accordance with the local guidelines of the institutions and only after approval by the Institutional Animal Care and Use Committee in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Ventricular myocytes were isolated from young (5–9 months) and old (4–6 years) female, New Zealand White rabbits (RSI Farms, as et al. The rabbits were with and and were after and a with at and action were at using with an was and of the were and Ca2+ were in and were using at Ca2+ were using and from a potential of and were measured and using and as in et al. were at and at are to the cell and as Ca2+ Ca2+ and Ca2+ spark activity in isolated rabbit ventricular myocytes were by a confocal with a in and using and respectively. Ca2+ were with for and after the dye was with the of an was at were in and at and with a β-adrenergic receptor were via stimulation at using the SR Ca2+ load and was at the of the activity was by measuring the rate of of Ca2+ and SR activity was via a rate of by the rate of of Ca2+ from the rate of of Ca2+ et al. 2004; Belevych et al. test the propensity for myocytes were at for and the latency the in the and the was the of mitochondria-derived ROS cardiomyocytes from old myocytes were with the mitochondria-specific ROS scavenger for The Ca2+ were performed β-adrenergic stimulation with Ca2+ spark myocytes were permeabilized with for The and the SR Ca2+ load, was at the of the The dye was with the of an was at were with for and were permeabilized with for and the dye was with the of an in was at SERCA activity was in permeabilized myocytes with in the SR as in Belevych et al. In in myocytes, SR Ca2+ was via the of was with and the SR Ca2+ was measured by Ca2+ in the of the RyR et al. the of a reducing agent were performed in permeabilized myocytes in the or of dithiothreitol The was to the and The was to using the was as et al. Belevych et al. RyR-mediated SR Ca2+ leak in intact myocytes with were with the Ca2+ to SERCA after of stimulation at and from in the SR was stimulation using confocal microscopy. The of of was used as a of the leak et al. ROS production was measured in isolated rabbit ventricular myocytes in using the ROS-sensitive dye for as (Terentyev et al. The dye was with the of an in and was at The rate of ROS production was measured at and in the of after in cardiomyocytes from young and old rabbits and cardiomyocytes with from old rabbits. membrane potential was with a voltage-sensitive fluorescent indicator, as (Ho et al. 2011). In isolated rabbit ventricular cardiomyocytes were with and was measured in was at with a and the were at was to the with the mitochondrial membrane potential was at and after of with cardiomyocytes from young rabbits and with cardiomyocytes from old rabbits with and The of in RyRs was determined with the as et al. Terentyev et al. 2008; et al. 2011). In myocytes were with for in the at and the from cell were and via oxidation RyR oxidation was to and RyR oxidation with reducing or respectively. The levels of SERCA calcium channel RyR and were determined by levels of at its protein and protein were using from and respectively. levels of RyRs were determined using and as et al. The levels of of RyRs and at specific in myocytes at conditions and after with were to by of for with an of protein and or cell were a via and with or rabbit specific for and with a or were with and and and by using and National Institutes of are as of the of measured or rabbits. were performed with test and and that are not are as were at we the of ageing cellular and Ca2+ homeostasis The Ca2+ in cardiomyocytes isolated from young and old rabbits was in the of a confocal using the Ca2+ The effects of ageing cytosolic Ca2+ at conditions are in and in Ageing did not change Ca2+ transient and transient did it change transient that ageing not and SERCA at Importantly, ageing not cellular action potential Ca2+ and in cardiomyocytes in Moreover, with we did not changes in levels of important RyRs, SERCA and Ageing to maintain SR Ca2+ by RyR-mediated Ca2+ leak confocal and at data from Ca2+ transient amplitude transient amplitude and rate and Ca2+ from heart and confocal with of spark activity in cardiomyocytes from young and old rabbits. of Ca2+ in permeabilized cardiomyocytes from young and old rabbits. show data for spark frequency spark amplitude and transient amplitude respectively. and for spark from heart All are the of ageing in a animal RyR activity we a to SR Ca2+ and parameters of spontaneous local Ca2+ release through RyR Ca2+ Ageing reduced spark amplitude and spark at but had spark of SR Ca2+ load via the amplitude of the Ca2+ in myocytes but of SR Ca2+ in old myocytes in with myocytes from young rabbits and spark frequency conditions of reduced SR Ca2+ load that RyRs are more in cardiomyocytes from old rabbits et al. oxidation underlies increased RyR-mediated SR Ca2+ leak in cardiomyocytes from old rabbits and of RyRs from cardiomyocytes from young and old rabbits and data for by and were from heart of changes in in permeabilized cardiomyocytes from young and old rabbits after the of are to the and data for Ca2+SR and after in cardiomyocytes from young and old rabbits. was to using the was Ca2+SR were as young and old for young old for old old and for young and old from heart All are of changes in in permeabilized cardiomyocytes from young and old rabbits and from permeabilized cardiomyocytes from old rabbits in the of after of SR and cytosolic Ca2+ of RyRs, and of of from of SR Ca2+ for permeabilized and old for from heart are We the of RyR modification in myocytes from young and old rabbits with an as in and in The of RyR was decreased in cardiomyocytes from old rabbits that ageing leads to increased RyR modification by reactive species the role of redox status in the age-related increase of RyR activity and of SR Ca2+ load, we the of antioxidant with a capacity to and oxidation, SR Ca2+ in myocytes from young and old rabbits. The results are in and in with the spark myocytes from old rabbits SR Ca2+ load, and of restored the SR Ca2+ in myocytes from old rabbits to the levels comparable to myocytes from young of to or to both and (Terentyev et al. effects SR Ca2+ These data indicate that oxidation and not or enhanced RyR activity in ageing enhanced RyR-mediated SR Ca2+ leak changes in SERCA function, Ca2+ was measured in the of RyR-mediated Ca2+ leak as et al. and that ageing not change SERCA which is with the data from Ca2+ in intact Importantly, of had the rate of in old results that of the of SR to Ca2+ in ageing is caused by an activity of RyRs while SERCA function remains examine the mechanism of RyR oxidation in ageing myocytes, we performed mitochondrial membrane potential with and ROS production rate with the ROS-sensitive of mitochondrial membrane potential in young and old myocytes as measured by at in and in myocytes from old rabbits had more depolarized mitochondrial membrane at and after β-adrenergic Additionally, β-adrenergic stimulation but depolarized mitochondrial membrane potential in both young and old myocytes by and respectively. ROS via mitochondrial dysfunction has been shown to be a of protein oxidation in the heart (Giordano, 2005; et al. 2011; Bovo et al. we the of ageing ROS production using as shown in and in and We that cardiomyocytes from old rabbits show a rate of ROS production to young myocytes at which is with β-adrenergic Importantly, myocytes from old rabbit hearts that were with the mitochondria-specific ROS scavenger a reduced rate of ROS production to old myocytes at and with β-adrenergic Additionally, β-adrenergic stimulation, we in the rate of ROS production young myocytes and old myocytes with These data the important role of age-associated mitochondrial dysfunction increased ROS production and protein redox status in the heart. cardiomyocytes have mitochondrial which is in the of of mitochondrial membrane potential at measured by using and and data of at and are to the in the of young and old for young old and for young old for young young and for old old from heart are of ROS production is in old cardiomyocytes and is with a mitochondrial ROS scavenger changes in at and with with cardiomyocytes from young and old rabbits and from cardiomyocytes with for from old rabbits. the data for rate of ROS production at and old for young old and for young old and for old old the data for rate of ROS production with and old for young old and for old old and for young old from heart are the role of ageing aberrant Ca2+ dynamics and mitochondrial we Ca2+ in myocytes from young and old rabbits and myocytes from old rabbits that were with myocytes were with the β-adrenergic The effects of ageing Ca2+ β-adrenergic stimulation are in and in from old rabbit hearts had Ca2+ transient amplitude and transient amplitude to young myocytes reduced for both Moreover, myocytes from old rabbit hearts in the of the mitochondria-specific ROS scavenger transient to old This increase transient amplitude to levels comparable to young cardiomyocytes that with SR Ca2+ load of of Ca2+ SERCA activity. the of Ca2+ transient that activity was in old myocytes young This was by in old ROS scavenger reverses the effects of ageing with regard to SR Ca2+ release β-adrenergic stimulation confocal and at from cardiomyocytes from young and old and from cardiomyocytes from old rabbits with for All were performed in the of data for Ca2+ transient and old for young data for transient and old for old old for young data for rate and old for young for old old data for rate old from heart are SERCA function and reduced activity for the diminished SR Ca2+ load in old with test if of Ca2+ channels to β-adrenergic stimulation plays a role in age-related changes in we performed to that of amplitude by is in old myocytes in to young with (Xiao et al. 1994; et al. However, with did not amplitude in old myocytes which that a increase in SR Ca2+ in old myocytes not and RyRs as the most of RyR-mediated leak was performed in intact shown in of intact old myocytes with for age-related of SR Ca2+ leak, while of ageing in young results the role of RyRs, which are oxidized by mitochondria-derived ROS in the of SR Ca2+ in ageing β-adrenergic of in old cardiomyocytes Ca2+ and are shown for and after from young and old were to cell were with membrane and from a potential of increase of at in response to was in young myocytes in old Ca2+ and from old cardiomyocytes with and after of All are Ageing increases SR Ca2+ leak, which is with mitochondrial ROS scavenging with of from intact cardiomyocytes from young and old rabbits as well as from cardiomyocytes from old rabbits with for and from cardiomyocytes from young rabbits in the of was to the with from of SR Ca2+ leak from intact old and young for young young for young old and old young for old old from heart are The results of the in that of old myocytes with the of oxidized RyRs, redox status to that of RyRs from young rabbit ventricular changes in levels of RyR and in ageing and the effects of the of we performed using RyR and and and and that ageing not levels of RyRs and at ageing is associated with a increase in the level of at and at in the of of from old myocytes with did not effects of mitochondria-specific ROS scavenger status of RyRs or SR Ca2+ by reducing oxidized RyRs and not by altering RyR and in ageing of mitochondrial ROS decreased RyR in ageing myocytes with the β-adrenergic of RyRs from and old myocytes with and to in the and of the agent and in the of the reducing agent of RyRs from young myocytes, old myocytes, and old myocytes with heart are of mitochondrial ROS not change status of RyRs and in ageing myocytes β-adrenergic stimulation of RyRs at and and and in old and old myocytes with for conditions and after with for data for levels of of RyRs at and and at from with were to RyR or levels measured in in and to levels by of myocytes with and for heart are in the arrhythmogenic potential associated with ageing was by measuring spontaneous Ca2+ waves (SCWs) in myocytes in the of after of for shown in and we a increase in the rate of of as well as an in latency in old myocytes to young was in young myocytes in the of which RyRs. with this age-related not only did the of old myocytes that to the young but increased refractoriness of RyR-mediated Ca2+ release in old myocytes as by an increase in latency the ageing cellular in cardiomyocytes the role of mitochondrial ROS in aberrant Ca2+ dynamics and in the ageing heart. ROS scavenger reverses the effects of ageing with regard to refractoriness of RyR-mediated Ca2+ release β-adrenergic stimulation confocal and at the of with cardiomyocytes from young and old rabbits and from cardiomyocytes with for from old rabbits with Ca2+ waves (SCWs) are by an data for of old and young for young for old old from heart data for old and young for young old and old young for old old from heart All are we a a mechanism that is with and its to alterations in Ca2+ as it to increased arrhythmogenic potential in the ageing heart. We showed that ageing increased SR Ca2+ leak via post-translational oxidation of RyRs caused by production of mitochondrial This led to diminished systolic Ca2+ accelerated RyR-mediated of Ca2+SR as well as the of in the of the β-adrenergic the age-related in Ca2+ homeostasis at the level of the SR and at as in systolic Ca2+ release due to decreased SR Ca2+ as well as malignant β-adrenergic This mechanism probably underlies the enhanced molecular trigger leading to arrhythmias. Although it has been shown that ageing is associated with aberrant RyR function et al. the molecular mechanism underlying the increase in RyR activity has yet to be thoroughly investigated. an important of this study is that ageing increases ROS levels in rabbit cardiomyocytes leading to the oxidation of RyRs, is known that the RyR is a protein et al. and to oxidation et al. Terentyev et al. 2008; Belevych et al. 2011b, et al. 2011). of reactive oxygen or species have also been shown to increase the of RyRs et al. et al. Zima Terentyev et al. 2008; et al. 2011; et al. modifications be by the reducing agent et al. et al. et al. Terentyev et al. we of an age-associated increase in RyR oxidation and we that the exposure of permeabilized myocytes to reduced SR Ca2+ leak SR Ca2+ effects SERCA function in old myocytes indicate that RyR oxidation via increased ROS is the mechanism of SR Ca2+ leak in cardiomyocytes in ageing. studies specific animal or the amplitude of Ca2+ conditions be increased et al. reduced or (Xiao et al. 1994; Howlett, 2010). We show that age-related changes in RyR activity in myocytes isolated from ageing rabbit hearts not changes in parameters of Ca2+ which to conditions associated with RyR hyperactivity infarct or early of heart failure et al. 2006; Belevych et al. 2009, 2011b, 2012). Additionally, studies that the function of important and be by ROS et al. 2004; Zima et al. 2008; et al. 2010; et al. 2010). In study, we show a of age-associated changes in parameters conditions for SR Ca2+ and and Ca2+ by which that the age-associated increase in levels of ROS in is not to in old rabbit data RyRs as a sensitive target for by ROS in the ageing heart within the of Ca2+ has been well established by et al. and that mitochondria are a for age-related increase in ROS production and the of ROS in cardiomyocytes et al. Chaudhary et al. 2011). studies have that the mitochondria and RyR SR Ca2+ release are in cardiomyocytes to local Ca2+ et al. et al. et al. 2011). be that this be for of from mitochondria in the of ROS that RyR function et al. 2011). Although of mitochondrial membrane potential leads to the of oxidative and ROS it has been that of the mitochondria leads to increased and increased levels of such as and et al. Györke Belevych et al. We show in myocytes from old rabbits that mitochondrial ROS increases oxidation of RyRs and RyR activity as by increased rate of SR Ca2+ leak reduced RyR refractoriness results in increased potential which is in increased frequency of in the of These data are in with studies that show that redox modification of RyRs by mitochondrial ROS RyR activity that results in increased frequency of (Ho et al. 2011; Bovo et al. 2012). Importantly, the cellular of old myocytes was by of young myocytes with to increase RyR activity and Additionally, scavenging mitochondrial ROS during β-adrenergic stimulation in a of rate of ROS generation and in myocytes, RyR oxidation was restored the Ca2+ transient amplitude was increased the SR Ca2+ leak and the frequency of were reduced and and the SR Ca2+ by of was to that comparable to young myocytes The effects of ageing Ca2+ homeostasis β-adrenergic stimulation are in the of Ca2+ in intact and effects Ca2+ in permeabilized myocytes conditions This is probably due to the reduced of RyRs to at increased levels of Ca2+ in conditions action of SERCA is by ROS modification results in increased of RyRs to Ca2+ (Terentyev et al. increased SR Ca2+ load in to in SR Ca2+ leak via oxidized RyRs in old cardiomyocytes in SR Ca2+ and Ca2+ transient Our results are in with that increased activity of RyRs is to changes in Ca2+ spark frequency et al. or Ca2+ transient amplitude, or et al. and enhanced SR Ca2+ is to effects of RyRs Ca2+ et al. In to oxidation, post-translational of RyR activity has been to at RyR and and et al. et al. et al. et al. 2004; et al. 2005; et al. 2006; et al. 2006; Zima et al. et al. 2008; et al. 2011; et al. We did not effects of that and in ageing suggesting was role for modifications in increase in RyR activity We also did not in levels of RyR at of the or of to β-adrenergic we a increase in RyR at and from ageing myocytes with in with identified as a (Xiao et al. is also sensitive to by et al. stress has been shown to increase activity et al. thereby RyR function via enhanced at However, of old myocytes with that RyR oxidation and aberrant Ca2+ handling in old myocytes did not RyR suggesting changes in activity levels of that RyR with ageing. results indicate that the of oxidation status of RyRs with mitochondria-specific ROS scavenger is to RyR function in ageing of also a in the frequency of in old with that oxidation and of RyRs much effects RyR activity of modifications et al. 2012). These data a mechanism underlying the of age-associated Ca2+ waves and decreased RyR refractoriness during β-adrenergic rate of mitochondrial ROS production with with sympathetic stimulation is in altering RyR redox leading to SR Ca2+ leak, and causing the of spontaneous Ca2+ waves. an age-related increase of mitochondrial ROS the ROS balance a of ROS production and mitochondrial are important and that are in redox that leads to age-related cardiac dysfunction and arrhythmias. and to the and of the and of data and the and performed and the All and the All were in of and were by The is not for the or of by the be to the for the