Expression of Circadian Clock and Melatonin Receptors within Cultured Rat Cardiomyocytes

Abstract

Melatonin, the pineal gland hormone, provides entrainment of many circadian rhythms to the ambient light/dark cycle. Recently, cardiovascular studies have demonstrated melatonin interactions with many physiological processes and diseases, such as hypertension and cardiopathologies. Although membrane melatonin receptors (MT1, MT2) and the transcriptional factor RORα have been reported to be expressed in the heart, there is no evidence of the cell-type expressing receptors as well as the possible role of melatonin on the expression of the circadian clock of cardiomyocytes, which play an important role in cardiac metabolism and function. Therefore, the aim of this study was to evaluate the mRNA and protein expressions of MT1, MT2, and RORα and to determine whether melatonin directly influences expression of circadian clocks within cultured rat cardiomyocytes. Adult rat cardiomyocyte cultures were created, and the cells were stimulated with 1 nM melatonin or vehicle. Gene expressions were assayed by real-time polymerase chain reaction (PCR). The mRNA and protein expressions of membrane melatonin receptors and RORα were established within adult rat cardiomyocytes. Two hours of melatonin stimulation did not alter the expression pattern of the analyzed genes. However, given at the proper time, melatonin kept Rev-erbα expression chronically high, specifically 12 h after melatonin treatment, avoiding the rhythmic decline of Rev-erbα mRNA. The blockage of MT1 and MT2 by luzindole did not alter the observed melatonin-induced expression of Rev-erbα mRNA, suggesting the nonparticipation of MT1 and MT2 on the melatonin effect within cardiomyocytes. It is possible to speculate that melatonin, in adult rat cardiomyocytes, may play an important role in the light signal transduction to peripheral organs, such as the heart, modulating its intrinsic rhythmicity.

Developmental and light-entrained expression of melatonin and its relationship to the circadian clock in the sea anemone Nematostella vectensis

“…Previous studies in mammals showed that a short melatonin pulse (2 hours) is not enough to alter the expression of clock-related genes [63]. We observed that the addition of melatonin in 12-hour pulses results in oscillations in expression of N. vectensis circadian clock genes, supporting a hypothesis that the interaction of melatonin and clock genes appeared early in the evolution of the animals.…”

Interrelationship between 3,5,3´-triiodothyronine and the circadian clock in the rodent heart

“…Quantitative RT-PCR was performed using specific RNA standards and TaqMan assays. Oligonucleotides sequences are reported previously (rat- Bmal1, Clock, Per1,2,3, Cry1 and Dbp (Young et al, 2001b), Slc2a4, Pdk4 and Ucp3 (Young et al, 2001a), Rora (Peliciari-Garcia et al, 2011); mice- Pdk4 and Ucp3 (Tsai et al, 2010)) or were newly designed for each gene from rat sequences available in GenBank; primer and probe sequences for newly designed assays are listed in Table 1. All quantitative RT-PCR data are presented as the number of transcripts/constitutive gene molecules.…”

Non-Ultradian Cardiac Rhythms: Circadian Regulation of the Heart

“…Indeed, functional clocks have been described in endothelia (Dermot et al, 2007;Takeda et al, 2007), adipose tissue (Zvonik et al, 2006), and fibroblasts (Welsh et al, 2004). However, rhythmic clock gene expression persists in vitro in isolated rat cardiomyocyte cultures (Durgan et al, 2005;Peliciari-Garcia, 2011), demonstrating the presence of a functional cardiomyocyte clock. Intrerestingly, exogenously administered melatonin alters circadian expression of rev-erb mRNA in rat cardiomyocyte cultures (Peliciari-Garcia et al, 2011).…”

“…However, rhythmic clock gene expression persists in vitro in isolated rat cardiomyocyte cultures (Durgan et al, 2005;Peliciari-Garcia, 2011), demonstrating the presence of a functional cardiomyocyte clock. Intrerestingly, exogenously administered melatonin alters circadian expression of rev-erb mRNA in rat cardiomyocyte cultures (Peliciari-Garcia et al, 2011). In addition to the membrane bound melatonin receptors, RORA has been described as a functional nuclear melatonin receptor, and may provide an important link between melatonin and regulation of peripheral oscillators, including the heart (Peliciari-Garcia et al, 2011).…”

“…Numerous beneficial cardioprotective effects have been reported for melatonin (Reiter and Tan, 2009). Cardiovascular tissues express both MT1 and MT2 melatonin receptors throughout the body, and cardiomyocytes express membrane bound melatonin receptors both in vivo and in vitro (Pang et al, 1993;Peliciari-Garcia et al, 2011). The nuclear melatonin receptor RORa has been localized to heart as well (Naji et al, 2004), and cardiovascular modulation of heart rate in primates may involve activation of intracellular MT3 receptor quinone reductase 2 (QR2) (Inui and Hazeki, 2010).…”