Circadian expressions of cardiac ion channel genes in mouse might be associated with the central clock in the SCN but not the peripheral clock in the heart

Figures & data

Figure 1. Confirmation of the successful suprachiasmatic nucleus (SCN) lesion. (A) Sham-operated mice: Substantial circadian rhythms in the heart rate and body temperature were observed. (B) SCN-lesioned mice: The arrow indicates the date that the SCN was lesioned. The SCN lesion was considered successful if the loss of circadian rhythms of heart rate and body temperature persisted for more than 14 days.

Table 1. Sequences of PCR primers and sequence-specific probes.

Target sequence	Accession no.	Assay ID of Primers and TaqMan^® Probe	Amplicon length (bp)
Per2	NM_011066	Mm00478113_m1	73
Bmal1	NM_007489	Mm00500226_m1	87
Dbp	NM_016974	Mm00497539_m1	57
KCNA5	NM_145983	Mm00524346_s1	61
KCND2	NM_019697	Mm00498065_m1	88
KCHIP2	NM_030716	Mm00518914_m1	74
KCNK3	NM_010608	Mm00807036_m1	128

Figure 2. Influence of SCNX and ANSB on the mRNA level of clock genes in the mouse heart (for each group, n = 3-6). Clock gene expressions in the atrium (A) and ventricle (B). All data were normalized against GAPDH and double-plotted. The solid line or dotted line indicates circadian rhythm fitted by least-squares cosine-curve fitting. Open diamonds, Bmal1 expression; open circles and closed triangles, Per2; closed squares, Dbp. The open and closed bars under the x-axis represent the light and dark periods, respectively, in one day. The expression of all of the clock genes lost circadian rhythm in the SCNX group and was dampened significantly in the ANSB group. ANSB, autonomic nervous system block; SCNX, suprachiasmatic nucleus lesioned.

Table 2. Clock gene expressions in the mouse heart.

Gene	Treatment	Site	MESOR	Amplitude	Acrophase (h)
Bmal1	Sham	Atrium	4.0 ± 0.1^§*	5.4 ± 0.1^§*^#	23.2 ± 0.3
		Ventricle	1.2 ± 0.1^§*^#	1.7 ± 0.1^§*^#	23.2 ± 0.2
	SCNX	Atrium	2.9 ± 0.3^§	1.5 ± 0.2^§	N/A
		Ventricle	0.7 ± 0.1^§	0.4 ± 0.1^§	N/A
	ANSB	Atrium	3.8 ± 0.2^§	2.2 ± 0.3^§	22.5 ± 0.1
		Ventricle	0.9 ± 0.1^§	0.4 ± 0.1^§	22.1 ± 0.4
Per2	Sham	Atrium	2.8 ± 0.3	2.5 ± 0.1^§*^#	12.8 ± 0.4
		Ventricle	0.6 ± 0.1	0.5 ± 0.1^§*^#	12.6 ± 0.3^#
	SCNX	Atrium	2.0 ± 0.2	1.3 ± 0.2^§	N/A
		Ventricle	0.6 ± 0.1	0.3 ± 0.1^§	N/A
	ANSB	Atrium	2.6 ± 0.4	0.9 ± 0.4^§	11.5 ± 1.2
		Ventricle	0.7 ± 0.1	0.04 ± 0.01^§	7.2 ± 2.3
Dbp	Sham	Atrium	5.8 ± 0.3^§*^#	7.2 ± 0.6^§*^#	9.9 ± 0.2
		Ventricle	1.9 ± 0.1^§*^#	2.2 ± 0.2^§*^#	9.9 ± 0.3^#
	SCNX	Atrium	3.6 ± 0.4^§	2.1 ± 0.4^§	N/A
		Ventricle	1.0 ± 0.1^§	0.5 ± 0.1^§ ^γ	N/A
	ANSB	Atrium	2.7 ± 0.2^§	2.4 ± 0.4^§	8.0 ± 0.4
		Ventricle	1.3 ± 0.1^§	1.5 ± 0.1^§	6.6 ± 0.1
Note: Data are mean ± SEM for 3–6 mice per group. MESOR, midline estimating statistic of rhythm; Sham, sham-operated; SCNX, suprachiasmatic nucleus lesioned; ANSB, autonomic nervous system block; N/A, not applicable. ^§One-way ANOVA p < 0.05, Bonferroni/Dunn, p < 0.05. *Sham vs. lesion; ^#Sham vs. ANSB; ^γLesion vs. ANSB.

Figure 3. Cardiac ion channel genes expressions were similarly altered in the SCNX and ANSB mice (for each group, n = 3–6). Cardiac ion channel genes expressions in the atrium (A) and ventricle (B). All data were normalized against GAPDH and double-plotted. The solid line or dotted line indicates circadian rhythm fitted by least-squares cosine-curve fitting. Open diamonds, KCNA5; closed triangles, KCND2; open squares, KCHIP2; open circles, KCNK3. The open and closed bars under the x-axis represent the light and dark periods, respectively, in one day. All four ion channel gene expressions lost circadian rhythm in the SCNX and ANSB mice.

Table 3. Cardiac ion channel gene expressions in the mouse heart.

Gene	Treatment	Site	MESOR	Amplitude	Acrophase (h)
KCNA5	Sham	Atrium	0.8 ± 0.1^§ ^* ^#	0.4 ± 0.1^§ ^*	15.6 ± 0.6
		Ventricle	1.0 ± 0.1^§ ^* ^#	0.3 ± 0.1	13.2 ± 0.2
	SCNX	Atrium	0.5 ± 0.1^§ ^γ	0.1 ± 0.1^§	N/A
		Ventricle	0.7 ± 0.1^§ ^γ	0.1 ± 0.1	N/A
	ANSB	Atrium	1.5 ± 0.2^§	0.3 ± 0.3^§	N/A
		Ventricle	1.5 ± 0.1^§	0.2 ± 0.1	N/A
KCND2	Sham	Atrium	2.8 ± 0.1^§ ^*	1.2 ± 0.2^§ ^*	5.4 ± 0.4
		Ventricle	1.1 ± 0.1^§	0.2 ± 0.1	3.0 ± 0.3
	SCNX	Atrium	1.8 ± 0.1^§ ^γ	0.6 ± 0.1^§ ^γ	N/A
		Ventricle	1.5 ± 0.1^§	0.2 ± 0.1	N/A
	ANSB	Atrium	11.7 ± 0.8^§	2.9 ± 0.4^§	N/A
		Ventricle	1.6 ± 0.1^§	0.1 ± 0.1	N/A
KCHIP2	Sham	Atrium	0.7 ± 0.1^§#	0.4 ± 0.1^§	11.1 ± 0.9
		Ventricle	0.8 ± 0.1^§ ^* ^#	0.3 ± 0.2	12.2 ± 1.1
	SCNX	Atrium	0.4 ± 0.1^§ ^γ	0.1 ± 0.1^§ ^γ	N/A
		Ventricle	0.6 ± 0.1^§ ^γ	0.2 ± 0.1	N/A
	ANSB	Atrium	2.4 ± 0.5^§	0.9 ± 0.6^§	N/A
		Ventricle	1.4 ± 0.1^§	0.1 ± 0.1	N/A
KCNK3	Sham	Atrium	3.1 ± 0.1^§#	1.2 ± 0.1^§#	1.7 ± 0.8
		Ventricle	2.2 ± 0.1^§#	0.4 ± 0.1^§	16.8 ± 2.5
	SCNX	Atrium	3.0 ± 0.1^§ ^γ	0.7 ± 0.1^§ ^γ	N/A
		Ventricle	2.0 ± 0.1^§ ^γ	0.5 ± 0.1^§ ^γ	N/A
	ANSB	Atrium	22.0 ± 3.2^§	10.5 ± 2.3^§	N/A
		Ventricle	3.2 ± 0.1^§	0.1 ± 0.1^§	N/A
Note: Data are mean ± SEM for 3–6 mice per group. Abbreviations are as in Table 1. ^§One-way ANOVA p < 0.05, Bonferroni/Dunn, p < 0.05. ^*Sham vs. lesion; ^#Sham vs. ANSB; ^γLesion vs. ANSB.

Figure 4. Heart rate and QT interval lost circadian rhythm in both SCNX and ANSB mice (for each group, n = 3–6). Heart rate (beats per min) and QT interval (ms) were recorded every 6 h and analysed by the least-squares cosine-curve fitting. The solid line indicates circadian rhythm fitted by least-squares cosine-curve fitting. Open circles, sham-operated mice; open triangles, ANSB mice; closed squares, SCNX mice. The open and closed bars under the x-axis represent the light and dark periods, respectively, in one day. The heart rate lost circadian rhythm in both SCNX and ANSB mice. The mean heart rate in the SCNX mice was significantly higher than that in the ANSB mice.

Table 4. Heart rate and QT interval.

Parameter	Treatment	MESOR	Amplitude	Acrophase (h)
Heart rate (bpm)	Sham	622 ± 9^§#	109 ± 21^§ ^* ^#	19 ± 1.4
	SCNX	669 ± 21^§	20 ± 4^§	N/A
	ANSB	466 ± 30^§ ^γ	9 ± 6^§	N/A
QT interval (ms)	Sham	40 ± 1^§ ^* ^#	8 ± 2^§ ^* ^#	5 ± 1.1
	SCNX	37 ± 1^§	3 ± 1^§	N/A
	ANSB	51 ± 2^§ ^γ	3 ± 1^§	N/A
Note: Data are mean ± SEM for 3–6 mice per group. Abbreviations are as in Table 1. ^§One-way ANOVA p < 0.05, Bonferroni/Dunn, p < 0.05. ^*Sham vs. lesion; ^#Sham vs. ANSB; ^γLesion vs. ANSB; N/A, not applicable (aperiodic).