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. 2019 Sep 16;9:13307. doi: 10.1038/s41598-019-49791-w

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Statistical analysis of the recorded data. Threshold voltage V₅₀ and energy expenditure E₅₀ obtained for the stimulation sequences with the multiple pulses (n = 2 or 3) were normalized to the respective values obtained for the same pulse width w (shown in ms) and inter-pulse interval Δ (shown in ms) for a single pulse stimulation (n = 1). (a–c) show threshold voltage V₅₀ dependency on stimulation pulse width w, the number of pulses used in the sequence n, and the inter-pulse interval Δ. In (a), at the same interval Δ, the influence of pulse width w and the number of pulses n is demonstrated. In (b,c), note that multiple pulses with smaller w could lead to a significant reduction in the threshold voltage (V₅₀/V_50n1 < 1). In (d), energy expenditure E₅₀ required to activate cardiomyocyte is shown for studied pulse sequences. For most of the sequences examined, E₅₀ was larger when the cardiomyocyte was stimulated using multiple pulses (n > 1). An exception to this rule was the double pulse with w = 1 ms and Δ = 0.5 or 1 ms.