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. 2022 Nov 28;11(23):3809.
doi: 10.3390/cells11233809.

Peculiarities of the Acetylcholine Action on the Contractile Function of Cardiomyocytes from the Left and Right Atria in Rats

Xenia Butova  1 , Tatiana Myachina  1 , Raisa Simonova  1 , Anastasia Kochurova  1 , Yakov Bozhko  2 , Michael Arkhipov  2 , Olga Solovyova  1   3 , Galina Kopylova  1 , Daniil Shchepkin  1 , Anastasia Khokhlova  1   3
Affiliations

Peculiarities of the Acetylcholine Action on the Contractile Function of Cardiomyocytes from the Left and Right Atria in Rats

Xenia Butova et al. Cells. .

Abstract

Acetylcholine (ACh) is the neurotransmitter of the parasympathetic nervous system that modulates cardiac function, and its high concentrations may induce atrial fibrillation. We compared the ACh action on the mechanical function of single cardiomyocytes from the left atria (LA) and the right atria (RA). We exposed single rat LA and RA cardiomyocytes to 1, 10, and 100 µM ACh for 10-15 min and measured the parameters of sarcomere shortening-relengthening and cytosolic calcium ([Ca2+]i) transients during cell contractions. We also studied the effects of ACh on cardiac myosin function using an in vitro motility assay and analyzed the phosphorylation level of sarcomeric proteins. In LA cardiomyocytes, ACh decreased the time to peak sarcomere shortening, time to 50% relengthening, and time to peak [Ca2+]i transients. In RA cardiomyocytes, ACh affected the time of shortening and relengthening only at 10 µM. In the in vitro motility assay, ACh reduced to a greater extent the sliding velocity of F-actin over myosin from LA cardiomyocytes, which was accompanied by a more pronounced decrease in phosphorylation of the myosin regulatory light chain (RLC) in LA cardiomyocytes than in RA cardiomyocytes. Our findings indicate that ACh plays an important role in modulating the contractile function of LA and RA, provoking more pronounced changes in the time course of sarcomere shortening-relengthening and the kinetics of actin-myosin interaction in LA cardiomyocytes.

Keywords: [Ca2+]i transients; acetylcholine; actin–myosin interaction; left and right atria; phosphorylation of sarcomeric proteins; sarcomere shortening; single cardiomyocytes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Dose–response curves of the amplitude and time course parameters of sarcomere shortening–relengthening in single mechanically non-loaded LA and RA cardiomyocytes to 1–100 µM ACh. (A) Representative recordings of time-dependent sarcomere length (SL) changes in LA and RA cardiomyocytes from the control group (0 µM) and after incubation with ACh for 10–15 min. (B). Analyzed parameters derived from SL change signal. (C) End-diastolic SL (EDSL). (D) Fractional SL shortening amplitude (FSLS = SLS/EDSL × 100%). (E) Time to peak SL shortening (TTPS). (F) Time from peak shortening to 50% sarcomere relengthening (TTR50). The number of n cells from N hearts (n/N) is shown in square brackets (LA) or parentheses (RA) in the (C) panel. Values are plotted as medians with interquartile ranges. * p < 0.05: 1, 10, and 100 µM ACh compared to the control group (0 µM ACh); one-way ANOVA (EDSL), Brown–Forsythe and Welch ANOVA tests (FSLS), and Kruskal–Wallis test (TTPS and TTR50).
Figure 2
Figure 2
Dose–response curves of the amplitude and time course parameters of [Ca2+]i transients in single mechanically non-loaded LA and RA cardiomyocytes to ACh (1–100 µM). (A) Representative recordings of [Ca2+]i transients in LA and RA cardiomyocytes from the control group (0 µM) and after incubation with 1, 10, and 100 µM ACh for 10–15 min. (B) Analyzed parameters derived from F/F0 ([Ca2+]i changes) signal. (C) Amplitude of [Ca2+]i transient (CaT). (D) Time to peak [Ca2+]i transient (TTPCa). (E) Time to 50% decay of [Ca2+]i transients (TTD50). The number of n cells from N hearts (n/N) is shown in square brackets [LA] or parentheses (RA) in the (C) panel. Values are plotted as medians with interquartile ranges. * p < 0.05: 1, 10, and 100 µM ACh compared to the control group (0 µM ACh); Kruskal–Wallis test for all parameters.
Figure 3
Figure 3
The acute effects of ACh on the sliding velocity of F-actin (vF-actin) over myosin from LA and RA cardiomyocytes in an in vitro motility assay. The number of n samples from N hearts (n/N) is shown in square brackets [LA] or parentheses (RA). Data are presented as dots (values) and medians (boxes) with interquartile range (bars). The same hearts were used for ACh groups and individual controls. * p < 0.05: 1, 10, and 100 µM ACh compared to the individual control group (0 µM ACh), Mann–Whitney U-test.
Figure 4
Figure 4
The example of gel electrophoresis of the sarcomeric protein extraction from RA cardiomyocytes of the control group (0 µM) and after incubation with 100 µM acetylcholine (ACh) for 10–15 min. cMyBP-C–cardiac myosin binding protein-C; TnT–troponin T; Tpm–tropomyosin; TnI–troponin I; RLC–myosin regulatory light chain. Phosphorylation was assessed using Pro-Q Diamond and SYPRO Ruby (Invitrogen, Eugene, OR, USA). Precision Plus Protein™ Unstained Standards (Bio-Rad, Hercules, CA, USA) was used as molecular weight markers for protein.
Figure 5
Figure 5
The acute effects of ACh on sarcomeric protein phosphorylation in LA and RA cardiomyocytes. cMyBP-C–cardiac myosin binding protein-C; RLC–myosin regulatory light chain; TnT–troponin T; TnI–troponin I; Tpm–tropomyosin. (A) Phosphorylation levels of cMyBP-C and RLC. (B) Phosphorylation levels of TnT, TnI, and Tpm. The same hearts were used for ACh groups and individual controls. Phosphorylation is expressed as the ratio of the intensities of protein bands stained with Pro-Q Diamond and SYPRO Ruby, and then calculated as the % change related to the individual control values. The number of n samples from N hearts is shown in square brackets [LA] or parentheses (RA). Data are presented in box and whisker plots, where the boxes are drawn from Q1 to Q3, horizontal lines represent median values and whiskers give the 100% range of the values. * p < 0.05: 1, 10, and 100 µM ACh compared to the individual control group (0 µM ACh), Mann–Whitney U-test.
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