Nucleoplasmic Ca²⁺

In the last severalyears,muchhasbeenlearnedaboutthe fundamental mechanisms underlying atrial fibrillation (AF). Most of the work in this area has focused on obtaining a better understanding of which ion channels lead to electrical remodeling in the fibrillating atrium and on deciphering the signaling pathways that lie upstream of both electrical and structural remodeling in AF. Both transcriptional regulation and posttranslational modifications (phosphorylation, oxidation, etc) have been shown to play an important role in the modulation of the electrophysiological effectors (ion channels, excitation-contraction (EC) coupling proteins, and gap junctions) and signaling proteins that underlie the genesis and maintenance of AF. Despite the clear importance of transcriptional regulation in both electrical and structural remodeling, few studies have attempted to understand the root cause underlying these transcriptional changes in the AF atrium.

Importantly, the last decade has also seen a significant increase in our understanding of the epigenetic mechanisms that underlie pathophysiological remodeling in the heart. Lately, a potential role for the involvement of epigenetic regulation mechanisms such as DNA methylation and histone modifications in the onset and progression of AF pathophysiology has been postulated. The misregulation of the addition or removal of acetyl groups on lysine residues of histones by histone acetyltransferases and HDACs (histone deacetylases), respectively, has been associated with cardiovascular diseases.¹ Although studies into their role in AF electrophysiology are relatively few in number, class IIa HDACs have been shown to participate in both the onset and perpetuation of AF by regulating deacetylation of cytoskeletal and contractile proteins and transcriptional reprogramming. One study demonstrated an association between HDAC6 and proteostasis in AF.² Furthermore, expression and activity of HDAC3 are increased in atrial tissue taken from patients with persistent AF, underscoring a likely contribution to progression of the AF disease state.¹ Another important regulatory mechanism for HDAC activity is posttranslational modification, such as oxidation and phosphorylation by kinases. In particular, CaMKII (Ca²⁺/calmodulin-dependent kinase type II) phosphorylation of HDAC4 has been shown to lead to upregulation of hypertrophic gene expression in the ventricle.³ In this issue of Circulation Research, Qi et al⁴ demonstrate that HDAC4 nuclear export by phosphorylation of CaMKIIδ plays a role in regulation of expression of L-type Ca²⁺ channels in the atria of dogs subjected to rapid atrial pacing. Even though some aspects of the study may appear to be an extension of findings previously made in the ventricle to the atria, there are important distinctions between the studies in the ventricle versus the atria. One important difference is that the current study clearly links HDAC4 signaling to ion channel remodeling in AF. Indeed, while several studies describe the involvement of HDAC in the regulation of NCX (Na⁺-Ca²⁺ exchanger)² and remodeling of Ca²⁺-activated K⁺ channels by HDAC2 in AF patients with concomitant HF has been reported, the precise role of HDACs in ion channel expression in the atria has not been well elucidated previously.²

A key aspect of the current study that merits discussion is the role of Ca²⁺ cycling in the activation of transcriptional processes in the AF atrium. The last several years have seen a burgeoning literature devoted to understanding the role of EC coupling in the creation of a vulnerable substrate for ventricular arrhythmias and more recently, AF. This includes studies from our own group that demonstrate the role of CaMKII-induced oxidation in the heart failure atrium in the genesis of arrhythmogenic triggered Ca²⁺ waves.^6,7 Much of this effect of Ca²⁺ on arrhythmogenesis is mediated by aberrant Ca²⁺ release by RyR2 (ryanodine receptor channel type-2), creating conditions for triggered and eventually reentrant activity.^6,8 However, it is also being increasingly understood, at least in the ventricle, that Ca²⁺ cycling can also contribute to arrhythmogenesis by a more subtle mechanism, by affecting transcription in the nucleus (ie, excitation-transcription coupling). Previous studies indicate that nucleoplasmic Ca²⁺ transients in the ventricle follow distinct kinetics and may be regulated quite differently from cytoplasmic Ca²⁺ during the progression from hypertrophy to heart failure.³ These alterations in nuclear Ca²⁺ likely lead to nuclear translocation of NFAT (nuclear factor of activated T-cells)–a mediator of calcineurindependent nuclear signaling. NFAT translocation has been shown to be an important mechanism underlying pathological remodeling in AF, including increased transcriptional regulation of ion channels such as I_CaL involved in electrical remodeling in AF.⁹ While this prior work strongly supports the role of nucleoplasmic Ca²⁺ cycling in the activation of transcriptional pathways, the signaling mechanisms that control the release and the kinetics of nuclear Ca²⁺ cycling are far from clear.

Intriguingly, it is recent work in atrial arrhythmogenesis that appears to provide a potential mechanism that mediates short- and long-term alterations in nuclear Ca²⁺ cycling. A Ca²⁺ release pathway speculated to contribute to arrhythmogenic substrate in the atrium is the ET-1 (endothelin 1)–Gα_q–IP₃R (inositol 1,4,5-trisphosphate receptor) pathway, with IP₃Rs shown to be preferentially located in the perinuclear region. ET-1–Gα_q signaling in particular activates phospholipase C and catalyzes the hydrolysis of PIP₂ (phosphatidylinositol 4,5-bisphosphate) into inositol 1,4,5-trisphosphate (IP₃) and DAG (diacylglycerol), in addition to production of cAMP.¹⁰ The rapid increase of IP₃ and DAG leads to multiple downstream signaling pathways affecting EC coupling and cardiomyocyte remodeling. Interestingly, the number of ET-1 binding sites and IP₃Rs in the atria far exceed binding sites in the ventricles,¹¹ suggestive of an important role of this pathway in atrial arrhythmogenesis.¹² By studying atrial myocytes from control and from IP₃R type 2 (IP₃R2)–deficient mice, Li et al¹³ determined that ET-1–dependent production of IP₃ promotes atrial arrhythmogenic Ca²⁺ signaling secondary to activation of IP₃R2. However, a further role of this pathway in AF beyond arrhythmogenic Ca²⁺ signaling in the cytosol was not explored. It is precisely this knowledge gap that the current study helps to address, by describing in detail how IP₃ signaling affects nucleoplasmic Ca²⁺ cycling, thereby setting up conditions for nuclear export of HDAC4 and resulting downstream transcriptional changes in the AF atrium. While a study by Xu at al¹⁴ did demonstrate a role for IP₃ signaling in nucleoplasmic Ca²⁺ signaling in the ventricle, that study did not systematically assess the effect of a pathological disease state (eg, heart failure) on nucleoplasmic Ca²⁺ cycling or the effect of systematic manipulation of different elements of ET-1–Gα_q–IP₃ signaling on Ca²⁺ transient behavior.

In the current study, Qi et al⁴ first compare nuclear Ca²⁺ transient characteristics in atrial myocytes in sinus rhythm with atrial myocytes subjected to atrial tachypacing (both in vivo and in vitro). After demonstrating clear alterations in diastolic and resting [Ca²⁺]_Nuc in AF and postulating a likely physiological mechanism for these changes, the investigators then go on to measure the effects of IP₃ manipulation on [Ca²⁺]_Nuc in the setting of AF. The study also links these changes in [Ca²⁺]_Nuc to enhanced nuclear export of HDAC4 via CaMKII phosphorylation of HDAC4. While the concept that nucleoplasmic Ca²⁺ can affect HDAC export from the nucleus via CaMKII signaling is not entirely novel, having been invoked in similar studies performed in ventricular myocytes, the systematic demonstration of a similar process in AF myocytes is an important advance in our understanding of the AF disease state.³ Notwithstanding the new signaling insights gleaned from this article, the true novelty of this study lies in its detailed assessment of Ca²⁺ cycling behavior in the nuclei of atrial myocytes, both in sinus rhythm and in the setting of AF. The study is timely, as it becomes increasingly clear that in addition to Ca²⁺ cycling in the cytosol, the dynamics of Ca²⁺ uptake and cycling behavior in other cellular compartments such as the nucleus and the mitochondria¹⁵ may also be important contributors to the creation of the AF disease state. In addition to its unique insights into the physiology of nuclear Ca²⁺ cycling, the study sheds additional mechanistic light on how IP₃R type 1 (IP₃R1) is upregulated in the nuclei of AF myocytes, invoking microRNA-26a as a likely regulator of IP₃R signaling and nuclear diastolic loading in the atrium.

Taken together, the article takes a number of established and emerging concepts in cardiac biology, all of which have been shown to have a role in the genesis of AF–Gα_q-IP₃R signaling, epigenetic control of transcription, microRNA control of translation, CaMKII signaling–and unites them under the common umbrella of nucleoplasmic Ca²⁺ cycling in the fibrillating atrium. What emerges is a new concept in atrial arrhythmogenesis that points toward compartmentalized Ca²⁺ cycling in the nucleus being a possible mastermind underlying the creation of a vulnerable substrate for AF (Figure).

Figure. Distinct roles of Ca2+ on arrhythmogenesis via excitation-contraction (EC) coupling and excitation-transcription coupling.

Schematic drawing of an adult atrial myocyte is showing parts of the sarcolemma, the nucleus, and the sarcoplasmic reticulum (SR). Cytosolic Ca²⁺ is the mediator for EC coupling. Ca²⁺-induced Ca²⁺ release from the SR through RyR2 (ryanodine receptor channel type 2) with subsequent activation of sarcolemmal NCX (Na⁺/Ca²⁺ exchanger) induces arrhythmias via generation of early and delayed afterdepolarizations and triggered activity. Nuclear Ca²⁺ is the mediator for excitation-transcription coupling. Nuclear Ca²⁺ transient through IP₃R (inositol 1,4,5-trisphosphate receptor) contributes to arrhythmogenesis via alteration of expression of NCX and L-type Ca²⁺ channel, leading to triggered activity and effective refractory period (ERP) shortening. AF indicates atrial fibrillation; DAD, delayed afterdepolarization; EAD, early afterdepolarization; and LTCC, L-type Ca²⁺ channel.