Elsevier

Heart Rhythm

Volume 12, Issue 6, June 2015, Pages 1306-1314
Heart Rhythm

The cardiomyocyte molecular clock regulates the circadian expression of Kcnh2 and contributes to ventricular repolarization

https://doi.org/10.1016/j.hrthm.2015.02.019Get rights and content

Background

Sudden cardiac death (SCD) follows a diurnal variation. Data suggest the timing of SCD is influenced by circadian (~24-hour) changes in neurohumoral and cardiomyocyte-specific regulation of the heart’s electrical properties. The basic helix-loop-helix transcription factors brain muscle arnt-like1 (BMAL1) and circadian locomotor output control kaput (CLOCK) coordinate the circadian expression of select genes.

Objective

We sought to test whether Bmal1 expression in cardiomyocytes contributes to K+ channel expression and diurnal changes in ventricular repolarization.

Methods

We used transgenic mice that allow for the inducible cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1−/−). We used quantitative polymerase chain reaction, voltage clamping, promoter-reporter bioluminescence assays, and electrocardiographic telemetry.

Results

Although several K+ channel gene transcripts were downregulated in iCSΔBmal1−/−mouse hearts, only Kcnh2 exhibited a robust circadian pattern of expression that was disrupted in iCSΔBmal1−/− hearts. Kcnh2 underlies the rapidly activating delayed-rectifier K+ current, and the rapidly activating delayed-rectifier K+ current recorded from iCSΔBmal1−/− ventricular cardiomyocytes was ~50% smaller than control ventricular myocytes. Promoter-reporter assays demonstrated that the human Kcnh2 promoter is transactivated by the coexpression of BMAL1 and CLOCK. Electrocardiographic analysis showed that iCSΔBmal1−/− mice developed a prolongation in the heart rate–corrected QT interval during the light (resting) phase. This was secondary to an augmented circadian rhythm in the uncorrected QT interval without a corresponding change in the RR interval.

Conclusion

The molecular clock in the heart regulates the circadian expression of Kcnh2, modifies K+ channel gene expression, and is important for normal ventricular repolarization. Disruption of the cardiomyocyte circadian clock mechanism likely unmasks diurnal changes in ventricular repolarization that could contribute to an increased risk of cardiac arrhythmias/SCD.

Introduction

The discovery that the incidence of sudden cardiac death (SCD) follows a time-of-day dependence suggests that circadian factors participate in the initiation of lethal arrhythmias.1, 2 Biological circadian rhythms are evolutionarily conserved cycles that repeat every ~24 hours and synchronize behavior and physiology with the daily environment.3, 4 The cardiovascular system shows robust circadian rhythms in blood pressure, heart rate, electrocardiographic (ECG) properties, and gene expression.5 Historically, an increase in SCD in early morning rise hours has been linked to the circadian variations in myocardial ischemic events and autonomic signaling; however, more recent studies also support a cardiomyocyte-specific circadian or molecular clock mechanism in the heart.6, 7, 8

The cellular mechanism responsible for generating biological circadian rhythms is a conserved gene regulatory network composed of a transcriptional-translational feedback loop called the molecular clock, which is expressed in almost all cells including cardiomyocytes.9, 10 The positive limb of the molecular clock is formed by the transcription factors BMAL1 (brain muscle arnt-like1) and CLOCK (circadian locomotor output control kaput), and the negative limb is regulated by periods 1, 2, and 3 and cryptochromes 1 and 2. Circadian changes in gene expression and cardiac function are linked to the cardiomyocyte molecular clock mechanism.11, 12 Bray et al13 showed that transgenic mice that selectively overexpress a dominant-negative CLOCK mutation in cardiomyocytes alter gene expression in both atrial and ventricular myocytes, heart rate, contractile function, and metabolism. We recently found that the inducible cardiomyocyte-specific deletion of Bmal1 (iCSΔBmal1−/−) in adult mice disrupts molecular clock signaling in the heart, causes a loss in the circadian expression of the Na+ channel gene Scn5a (Nav1.5), decreases macroscopic Na+ current recorded from isolated ventricular cardiomyocytes, slows the heart rate, and increases the frequency of cardiac arrhythmias.8 Emerging evidence suggests that the cardiomyocyte molecular clock might regulate the expression of several cardiac K+ channels and ventricular repolarization as well.6, 7 However, these studies do not distinguish the relative contribution of the cardiomyocyte molecular clock from circadian clock signaling in other tissues. The purpose of this study was to determine how Bmal1 expression in cardiomyocytes contributes to changes in K+ channel expression and ventricular repolarization.

Section snippets

Inducible deletion of Bmal1 in adult cardiomyocytes

All animal procedures were conducted in compliance with the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care and were approved by the Institutional Animal Care and Use Committee of the University of Kentucky. The inducible cardiac-specific ΔBmal1 (iCSΔBmal1) mouse model used for these studies was described previously.8 Cre-recombination was activated once the mice reached 12 weeks of age by intraperitoneal injections of tamoxifen (2 mg/d) for 5

Kcnh2 is a candidate clock-controlled gene

We investigated the role of cardiomyocyte molecular clock in the regulation of cardiac K+ channel gene expression important in mouse and human ventricular repolarization (Online Supplemental Table 1). To identify K+ channel gene candidates that are expressed in a circadian pattern, we used the high-resolution CircaDB microarray data set for the heart (http://circadb), which analyzed gene expression every 2 hours for 48 hours in combination JTK_CYCLE statistical analysis.21 We then confirmed

Discussion

The circadian molecular clock is a highly conserved, cell-autonomous, transcriptionally mediated mechanism that provides an evolutionary advantage by optimizing an organism’s physiology to anticipate the daily variation in the environment.24 To date, there is only tangential and circumstantial evidence linking the cardiomyocyte molecular clock to circadian changes in K+ channel expression and cardiac electrophysiology. In 2003, Yamashita et al6 hypothesized that the expression of certain

Conclusion

This is the first work to clearly demonstrate that intrinsic circadian mediators, including the cardiomyocyte molecular clock, are modulators of the electrical properties in the heart, which possibly contributes to the daily variation in SCD.

Is there a diurnal rhythm in the corrected QT (QTc) interval? For more than 25 years, clinician scientists have been working to answer this question. It is an important question because if there is a circadian rhythm in the QTc interval, then the time of

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  • Cited by (0)

    This work was supported by the National Institutes of Health (grant nos RC1ES018636 and AR55246 [to Dr Esser] and R01 HL087039 [to Dr Delisle]).

    Dr Delisle has a research contract with Gilead Scientific.

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