Molecular evidence for an intrinsic circadian pacemaker in the cardiac ganglion of the American lobster, Homarus americanus - Is diel cycling of heartbeat frequency controlled by a peripheral clock system?

Authors: CHRISTIE, ANDREW - University Of Hawaii; YU, ANDY - University Of Hawaii; RONCALLI, VITTORIA - University Of Hawaii; PASCUAL, MICHA - University Of Hawaii; CIESLAK, MATTHEW - University Of Hawaii; Warner, Amanda; LAMEYER, TESS - Bowdoin College; STANHOPE, MEREDITH - Bowdoin College; DICKINSON, PATSY - Bowdoin College; Hull, Joe

Submitted to: Marine Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/7/2018
Publication Date: 7/19/2018
Citation: Christie, A.E., Yu, A., Roncalli, V., Pascual, M.G., Cieslak, M.C., Warner, A.N., Lameyer, T.J., Stanhope, M.E., Dickinson, P.S., Hull, J.J. 2018. Molecular evidence for an intrinsic circadian pacemaker in the cardiac ganglion of the American lobster, Homarus americanus - Is diel cycling of heartbeat frequency controlled by a peripheral clock system?. Marine Genomics. 41:19-30.

Interpretive Summary: Most organisms exhibit recurring patterns in physiology and/or behavior in relation to a 24-hr day. These circadian oscillations are typically regulated by a core group of conserved clock genes that function in keeping track of time. In crustaceans, cardiac control has been suggested to be circadian in nature. If so, the necessary regulatory clock would be expected to reside in the cardiac ganglion. To examine the molecular basis of cardiac circadian control, a high-throughput sequencing approach was used to generate a cardiac ganglion specific transcriptome using tissue isolated from the American lobster. Sequences with high similarity to known clock gene products were identified in the transcriptomic dataset and subsequently confirmed using PCR. The data generated in this study confirm the presence of a cardiac ganglion clock and provide a foundation for assessing the physiological role of circadian signaling in maintaining crustacean cardiac activity.

Technical Abstract: Whether cardiac output in decapod crustaceans is under circadian control has long been debated, with mixed evidence for and against the hypothesis. Moreover, the locus of the clock system controlling cardiac activity, if it is under circadian control, is unknown. However, a report that the crayfish heart in organ culture maintains a circadian oscillation in heartbeat frequency suggests the presence of a peripheral pacemaker within the cardiac neuromuscular system itself. Because the decapod heart is neurogenic, with contractions controlled by the five motor and four premotor neurons that make up the cardiac ganglion (CG), a likely locus for a circadian clock is the CG itself. Here, a CG-specific transcriptome was generated for the lobster, Homarus americanus, and was used to assess the presence/absence of transcripts encoding putative clock-related proteins in the ganglion. Using known Homarus brain/eyestalk ganglia clock-related proteins as queries, BLAST searches of the CG tran- scriptome were conducted for the five proteins that form the core clock, i.e., clock, cryptochrome 2, cycle, period and timeless, as well as for a variety of clock-associated, clock input pathway and clock output pathway proteins. With the exception of pigment dispersing hormone receptor [PDHR], a putative clock output pathway protein, one or more transcripts encoding each of the proteins searched for were identified from the CG assembly; no PDHR-encoding transcripts were found. RT-PCR confirmed the expression of all core clock transcripts in multiple independent CG cDNAs; RNA-Seq data suggest that both the motor and premotor neurons could contribute to the cellular locus of a pacemaker. These data provide support for the possible existence of an intrinsic circadian clock in the H. americanus CG, and form a foundation for guiding future anatomical, molecular and physiological investigations of circadian signaling in the lobster cardiac neuromuscular system.