The heart pumps blood throughout the body to provide oxygen and nourishment. To do so, proteins in the heart create electrical signals that tell the heart muscles to contract in a coordinated manner. Heart disease can cause cells to lose control of the production or activity of these proteins, creating disorganized electrical signals called arrhythmias that interfere with the heart’s ability to pump. Sometimes these arrhythmias lead to sudden death.

Researchers do not know exactly what triggers these changes in the heart’s normal electrical rhythms. This has made it difficult to develop strategies to prevent these disruptions or to fix them when they occur.

By studying rat and human heart cells, Nassal et al. now show that a protein called KChIP2 stops working properly during heart disease. Most importantly, because of the decreased level of KChIP2 in heart disease, KChIP2 loses the ability to restrict the production of two microRNA molecules – a role that KChIP2 was not previously known to perform. This loss of activity sets off a cascade of signals that worsens the balance of electrical activity in the heart cells, creating arrhythmias.

Treatments that restored proper levels of the fully working KChIP2 protein to the heart cells or that blocked the signals set off by a lack of KChIP2 returned the electrical activity of the cells back to normal. This also stopped the development of arrhythmias. Further studies are now needed to investigate whether these treatments have the same effects in living mammals. If effective, this could ultimately lead to new treatments for heart diseases and arrhythmias.

Cardiac excitability is controlled by a combination of depolarizing and repolarizing currents, whose dysregulation during heart failure (HF) or myocardial infarction (MI) play a significant role in clinically relevant arrhythmias (Tomaselli and Marbán, 1999; Wang and Hill, 2010). Aberrant remodeling culminates in altered Ca²⁺ current (I_Ca) (Houser et al., 2000; Wang et al., 2008), Na⁺ current (I_Na) (Pu and Boyden, 1997; Maltsev et al., 2002), and a host of outward K⁺ currents (I_K) (Näbauer and Kääb, 1998), creating impaired cardiac excitability and performance, accounting for high rates of mortality in HF patients (Nattel et al., 2007; Tomaselli and Zipes, 2004). However, large variability and breadth of electrical changes present challenges in determining which mechanisms are critical in driving arrhythmias and disease progression. Intriguingly, loss of the Potassium Channel Interacting Protein 2 (KChIP2) has proven to be a consistent event following cardiac stress, sparking interest into understanding its contribution in disease remodeling (Näbauer and Kääb, 1998; Nass et al., 2008; Jin et al., 2010).

It is well described that KChIP2 associates with and modulates the Kv4 family of potassium channels, which together define the fast transient outward potassium current (I_to,f), maintaining early cardiac repolarization (An et al., 2000; Niwa and Nerbonne, 2010). However, emerging evidence suggests KChIP2 may not be limited to this role (Thomsen et al., 2009; Li et al., 2005; Deschênes et al., 2008). Investigations following KChIP2 knock-down show reduced transcript expression for the cardiac sodium channel gene, SCN5A, and its accessory subunit SCN1B, in addition to Kv4.3 protein, prompting the loss of both I_to,f and I_Na (Deschênes et al., 2008). Considerably, these changes reflect conditions observed in the diseased heart, but more importantly implicate potential transcriptional significance for KChIP2 at the center of that remodeling. Indeed, other members of the KChIP family not expressed in the myocardium behave as transcriptional repressors, while also maintaining the ability to interact with Kv4 channels (An et al., 2000; Carrión et al., 1999; Savignac et al., 2005; Gomez-Villafuertes et al., 2005; Ronkainen et al., 2011). Therefore, we sought to identify the existence of cardiac KChIP2 transcriptional activity and its significance in electrical remodeling and arrhythmia susceptibility. Here, we find KChIP2 transcriptionally represses a set of miRNAs known as miR-34b and-34c. Through KChIP2 loss, miR-34b/c are elevated, subsequently targeting other ion channel genes defining I_Na and I_to densities. Either restoring KChIP2 expression or blocking miR-34b/c activity during cardiac stress reverses this remodeling and completely negates the occurrence of re-entrant arrhythmias. Together, this work unveils a novel, transcriptional mechanism for KChIP2, and defines it as a central mediator of cardiac electrical activity.

This study established a novel transcriptional role for cardiac KChIP2, whereby it maintains a repressive influence over the miR-34b/c promoter. KChIP2 loss either by direct silencing or pathologic means, removes repression over miR-34b/c expression. Consequentially, reductions in transcript and protein expression for Nav1.5, Navβ1, and Kv4.3 are observed as an outcome of miR-34b/c targeting to seed regions present in the 3’-UTR of these genes, allowing KChIP2 to manipulate functional expression of a host of critical cardiac ion channel genes, ultimately acting as a key regulator of cardiac excitability and arrhythmia susceptibility.

While we evaluated a discrete pathway targeted by KChIP2 transcriptional activity, there are doubtless many other gene targets. To begin to address this discussion, a gene expression array was performed on NRVM following 48 hr of KChIP2 silencing. Evaluation of genes that experienced at least a two fold change revealed an increase in expression for 293 genes and a decrease in expression for 407 genes in response to KChIP2 silencing (Supplementary file 1). Notably, of the genes experiencing increased expression, 192 of them (65.5%) were predicted to contain a DRE motif within promoter elements, implicating the potential of KChIP2 transcriptional activity directly mediating these changes. Additionally, of the genes responding with reduced expression, 71 of them (17.4%) were predicted to contain a miR-34b/c target site within their 3’-UTR. Importantly, we see significant reduction to SCN5A and SCN1B, consistent with previous data and the mechanisms investigated here. Considerably, whether these changes are the direct consequence of KChIP2 transcriptional repression, or more indirect KChIP2 dependent mechanisms, including prolonged APD from loss in I_to density contributing to altered Ca²⁺ handling, the results are still relevant to cardiac remodeling, particularly given the associated loss of KChIP2 in cardiac disease states. Notably, a diverse range of gene pathways were implicated in response to KChIP2 loss, including cardiovascular signaling, regulation to G-protein coupled receptor pathways, relaxation and contraction, TGFβ signaling, apoptotic, and NFκB dependent signaling mechanisms, all of which have a relevance in disease remodeling (Supplementary file 1). Importantly, these data are supportive of our conclusion that KChIP2 is a key regulator of cardiac pathology. While our study focused on the transcriptional pathway by which KChIP2 could exert a concerted regulation of I_Na and I_to, further investigations pursuing some of the targets highlighted from this gene expression array will likely reveal a broader role for KChIP2 as a transcriptional regulator of cardiac physiology much like is seen for KChIP3 (DREAM) in the brain.

Indeed, the role of KChIP2 as a multimodal regulator of cardiac ion channels has been an emerging topic. Recent work has identified KChIP2 regulation of Ca_v1.2 through direct interaction with an inhibitory N-terminal domain on the channel, effectively reducing I_Ca,L in the absence of KChIP2 (Thomsen et al., 2009; Foeger et al., 2013). Our previous work has also suggested that KChIP2 is part of a larger macromolecular complex that includes subunits for both Na_v1.5 and Kv4 channels that lead to functional increases in both currents when coexpressed with KChIP2 (DeschenesDeschênes et al., 2008). In the same study we also identified transcriptional changes in SCN5A and SCN1B following acute knockdown of KChIP2 in NRVM, which provided the motivational basis for the work presented here. Taken together, these observations suggest a highly promiscuous nature for KChIP2.

Notably, another member of the KChIP family, KChIP3, has also been discovered to interact with multiple membrane proteins, including regulation of Kv4 channels (Mellström et al., 2008; Buxbaum et al., 1998), while also displaying Ca²⁺ regulated transcriptional repression (Carrión et al., 1999). Even the role of KChIP3 as a transcriptional repressor is multimodal, including direct binding to DRE motifs, in addition to interacting with and suppressing the activity of the cAMP response element-binding protein (CREB), an established transcriptional activator (Ledo et al., 2002). Both of these processes are Ca²⁺ regulated, due to three functional high affinity EF-hand motifs residing in the protein (Carrión et al., 1999). Occupancy of these sites upon increases in intracellular Ca²⁺ lead to conformational changes that cause DNA binding release (Carrión et al., 1999) or dissociation from CREB (Ledo et al., 2002), causing de-repression of downstream gene targets. Given that the entirety of the KChIP gene family displays strong conservation around these EF-hand residues, suggests conservation of these Ca²⁺ regulated responses. Indeed, we observed that caffeine stimulation produced increased activity of the miR-34b/c promoter (Figure 2D) in the presence of KChIP2. Additionally, when we deleted the DRE element in the miR-34b/c promoter, we observed an incomplete removal of suppression (Figure 2C). However, as KChIP3 represses gene expression through alternative CREB dependent regulation, the partially retained repressive activity may be attributed to this secondary function. Further analysis of the promoter by MatInpsector revealed several potential sites of predicted CREB binding that may have allowed for partially maintained KChIP2 suppression, even in the absence of the DRE site. Given that KChIP2 and KChIP3 share a high degree of homology only reinforces the observation of multiple activities for KChIP2 as well.

The physiologic implications of KChIP2 targeting miR-34b/c expression is one of tremendous significance for many cardiac pathologic states. Rapid depletion of KChIP2 protein is a widespread event that underlies remodeling in many cardiac diseases, including chronic HF, MI, and atrial fibrillation (Nattel et al., 2007). Considerably, these diseases also present with reductions in I_to and I_Na. The relationship between KChIP2 and I_to has been heavily studied, frequently identifying that KChIP2 loss induces the destabilization of Kv4.2/4.3 channels and mediates the decline in current density (Foeger et al., 2013). However, the work presented here offers the unique alternative that translation block through miRNA interaction mediates the decline in Kv4.3. Given that Kv4.2 does not contain a miR-34 target region in its 3’-UTR, but still experiences degradation following KChIP2 loss, it is likely that both mechanisms contribute to the resulting loss in I_to,f. However, it is also observed that reduced KChIP2 expression stimulated by phenylephrine + propranolol in in vitro cultures of NRVM experienced increased Kv4.2 protein while KChIP2 and Kv4.3 levels were reduced (Panama et al., 2011), supporting the opportunity for miRNA dependent translational block targeting Kv4.3, rather than just destabilization of all Kv4 channels.

In the same settings of cardiac disease where KChIP2 is down, there are also observations of I_Na depletion (Valdivia et al., 2005; Zicha et al., 2004). Our data of miR-34 targeting Na_v1.5 provides a means for describing this loss in activity. Notably, others have shown a loss in the full length transcript for Nav1.5 mRNA and a corresponding increase in a truncated isoform without the miR-34b/c target region present (Shang et al., 2007), reinforcing the observations for miR-34b/c mediating the decline of SCN5A. Overall, the consequential loss of both I_Na, and I_to, suggests KChIP2 loss during cardiac stress may be a nodal event in a cascade of gene regulation defining electrical remodeling in the stressed myocardium. Indeed, earlier work was done that sought to determine the significance of KChIP2 in the development of hypertrophic remodeling. In a rat TAC banding model, it was observed that maintaining KChIP2 expression attenuated hypertrophy and pathogenic remodeling that otherwise lead to a worsening myocardium during pressure overload (Jin et al., 2010). This reverse in remodeling was attributed to changes in intracellular Ca²⁺ signaling brought on by restoration of an abbreviated APD. Yet, we were able to observe that inhibition of miR-34b/c could also attenuate adverse remodeling without influencing APD (Figure 7) implicating multiple pathways of KChIP2 intervention. Indeed, the miR-34 family has recently been implicated in the development and progression of hypertrophy and heart failure, in rodent models of both MI and pressure overload (Bernardo et al., 2012). Critically, these studies, combined with our own data, show that blockade of the miR-34 family can attenuate pathologic remodeling, expanding the significance of KChIP2 and miR-34 in cardiac pathogenesis.

There are still some challenges in understanding the role of KChIP2 in the progression of hypertrophy and heart failure. Investigations conducted in KChIP2 null mice have shown that when submitted to TAC banding, there is no worsened phenotype when compared to wild type mice (Speerschneider et al., 2013). In fact, arrhythmia susceptibility was lowered in the KChIP2 null mice during heart failure, believed to be the result of reduced dispersion of repolarization. At the same time, there were no observed changes to I_Na. While our current understanding is unable to account for this disparity, it may be that compensatory regulation exists in these mice as a consequence of constitutive KChIP2 absence during development, fundamentally changing its regulatory significance. Evidence for this is observed when restoring KChIP2 expression in myocytes isolated from KChIP2 null mice, which resulted in no rescue of Kv4.2 protein expression or recovery of I_to,f (Foeger et al., 2013). However, restoration of KChIP2 following acute loss from pathologic consequences in a rat model was able to rescue I_to,f (Jin et al., 2010), consistent with what we see in our own maintenance of KChIP2 following prolonged PE exposure (Figure 4E). The significance of this begins to suggest deviations in KChIP2 regulatory impact depending on acute versus constitutive loss.

Ultimately, our endpoint was to determine whether electrical dysregulation brought on by KChIP2 loss was able to influence arrhythmia susceptibility through the activity of miR-34b/c. Despite only rescuing I_Na and not I_to in the NRVMs, as evidenced by the shortened ERP with sustained APD prolongation (Figure 7D and F), we found this was sufficient to rescue arrhythmia induction following PE treatment (Figure 7C). Indeed, previous studies have revealed the relationship between changes in Na⁺ channel density and arrhythmia induction. As I_Na becomes compromised, it begins to resolve an expanding interval of premature stimuli declared the vulnerability period. Within this interval, reentry is more likely to occur as a result of non-uniform conduction block surrounding the point of excitation (Starmer et al., 2003). Both theoretical (Starmer et al., 1991, 1993) and experimental (Nesterenko et al., 1992; Starmer et al., 1992) studies show that when Na⁺ channel availability is reduced, the vulnerable period increases. Therefore, by restoring Na⁺ channel through miR-34b/c inhibition, we are effectively minimizing the vulnerable period and making unidirectional conduction block less likely to occur.

Care must still be taken before translating these mechanisms to the clinical setting. Our investigated pathway was developed using cultured rodent myocytes, differing from human electrophysiology in its APD and the impact of underlying currents. We must also understand the electrical impact of miR-34 inhibition in vivo. However, we know from this investigation that miR-34b/c are elevated in native human HF tissue (Figure 5A), and that functionally, the inhibition of miR-34b/c in human derived cardiomyocytes following stress can achieve restoration of both I_Na and I_to (Figure 6C and D), reinforcing species dependent conservation. At the same time, conduction block due to compromised cellular excitability has long been understood to be important for clinically relevant arrhythmias (Shah et al., 2005). These observations together suggest strong therapeutic potential for targeting miR-34 in the treatment of electrical instabilities. Currently, the use of locked nucleic acids and related technologies have been used to successfully target miRNA activity in vivo (Olson, 2014). While miR-34b/c is also expressed outside the heart, it is unclear what long-term consequences its inhibition will have as a therapeutic. However, these outcomes will have to be weighed against the potential therapeutic advantage it will have in alleviating cardiac events.

Overall, this newly identified KChIP2/miR-34 pathway reflects electrical remodeling observed within multiple cardiac pathologies. Moreover, the events brought on by KChIP2 loss are critical in initiating electrical instabilities and arrhythmias implicated in sudden cardiac death. The identification of KChIP2 transcriptional capacity significantly reshapes its role in cardiac biology as a core mediator of cardiac electrical activity and reveals KChIP2 and miR-34 as therapeutic targets for managing arrhythmogenesis in heart disease.

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Author details

1. Drew M Nassal

   1. Heart and Vascular Research Center, Department of Medicine, Case Western Reserve University, Cleveland, United States
   2. Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, United States

   Contribution

   DMN, Conceptualization, Data curation, Formal analysis, Funding acquisition, Validation, Investigation, Visualization, Methodology, Writing—original draft, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-8018-3913

2. Xiaoping Wan

   Heart and Vascular Research Center, Department of Medicine, Case Western Reserve University, Cleveland, United States

   Contribution

   XW, Data curation, Formal analysis, Investigation, Methodology, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

3. Haiyan Liu

   Heart and Vascular Research Center, Department of Medicine, Case Western Reserve University, Cleveland, United States

   Contribution

   HL, Investigation

   Competing interests

   The authors declare that no competing interests exist.

4. Danielle Maleski

   Heart and Vascular Research Center, Department of Medicine, Case Western Reserve University, Cleveland, United States

   Contribution

   DM, Data curation, Formal analysis, Investigation, Methodology

   Competing interests

   The authors declare that no competing interests exist.

5. Angelina Ramirez-Navarro

   Heart and Vascular Research Center, Department of Medicine, Case Western Reserve University, Cleveland, United States

   Contribution

   AR-N, Investigation, Methodology

   Competing interests

   The authors declare that no competing interests exist.

6. Christine S Moravec

   Department of Molecular Cardiology, Cleveland Clinic, Cleveland, United States

   Contribution

   CSM, Resources, Supervision, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

7. Eckhard Ficker

   Heart and Vascular Research Center, Department of Medicine, Case Western Reserve University, Cleveland, United States

   Contribution

   EF, Conceptualization, Supervision, Methodology, Project administration

   Competing interests

   The authors declare that no competing interests exist.

8. Kenneth R Laurita

   Heart and Vascular Research Center, Department of Medicine, Case Western Reserve University, Cleveland, United States

   Contribution

   KRL, Conceptualization, Resources, Formal analysis, Supervision, Methodology, Project administration, Writing—review and editing

   Competing interests

   The authors declare that no competing interests exist.

9. Isabelle Deschênes

   1. Heart and Vascular Research Center, Department of Medicine, Case Western Reserve University, Cleveland, United States
   2. Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, United States

   Contribution

   ID, Conceptualization, Resources, Supervision, Funding acquisition, Validation, Investigation, Methodology, Writing—original draft, Project administration, Writing—review and editing

   For correspondence

   isabelle.deschenes@case.edu

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0002-1812-7267

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