Synergistic effects of nicotine and hyperlipidemia induce cardiac damage via dynamic cardiomyocyte-based biosensing

Figures

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  Fig. 1. Diagram illustrating the ongoing and evolving biosensing system for cardiomyocytes, designed for the immediate tracking of nicotine levels in the culture setup. Cardiomyocytes were seeded onto a microelectrode array (MEA) device and exposed to either nicotine alone or a hyperlipidemic state simulated by PA with nicotine present. An electrophysiological biosensing system was used to monitor the real-time electrophysiological state of the cardiomyocytes. The results indicated that arrhythmias caused by nicotine alone could be reversible through conditioned culture. However, arrhythmias resulting from the synergistic action of nicotine in the hyperlipidemic state were irreversible

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  Fig. 2. Development and characterization of a cardiomyocyte-based biosensing platform. a Microfabrication processes of MEA device manufacture, including photolithography, Ti/Au deposition, liftoff and SU-8 insulation. b Schematic of an electrophysiological sensing platform based on cardiomyocytes. The orange box indicates the MEA device, the red box shows the optical micrograph of multiple electrodes, and the purple box displays the magnified image of an electrode. c Fluorescence micrograph of calcein-AM/PI staining: live cells (green, calcein-AM), dead/apoptotic cells (red, PI), and nuclei (blue, DAPI). Scale bar = 200 μm. d Representative electrophysiological signals from cardiomyocytes cultured on MEA devices (days 1–4). Statistical analysis of (e) signal amplitudes and (f) firing rates recorded by multiple electrodes over 4 days (N ≥ 24). Data in e, f are expressed as mean ± Standard Error of Mean. *p < 0.05; **p < 0.01; ***p < 0.001

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  Fig. 3. Extracellular recording of the effects of nicotine on cardiomyocytes. a Schematic of the experimental setup for assessing the electrophysiological effects of nicotine on cardiomyocytes. b Representative electrophysiological traces from cardiomyocytes treated with different nicotine concentrations for 24 h. c, d Normalized amplitude and firing rate curves of cardiomyocytes exposed to various nicotine concentrations over 60 min. e, f Bar graphs showing the changes in signal amplitude and firing rate after 24 h nicotine treatment (N ≥ 24). Poincaré plots of cardiomyocytes treated with (g) 1 mM and (h) 10 mM nicotine. The horizontal axis (ISIn) indicates the interval between current and previous extracellular action potentials; the vertical axis (ISIn+1) shows the interval between current and subsequent potentials. The time point for analysis was 24 h after nicotine treatment, with an analysis duration of 45 s. i Live/dead staining of cardiomyocytes. Scale bar = 100 μm. j Representative transmission electron microscopy (TEM) images of cellular and organellar morphology in cardiomyocytes after nicotine exposure. Blue arrows indicate mitochondrial structures. k Western blot analysis of…

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  Fig. 4. Effects of nicotine on cardiomyocytes under hyperlipidemic conditions. a Schematic of the combined influence of PA and nicotine on cardiomyocyte electrophysiology. b Electrophysiological recordings of primary cardiomyocytes treated with varying nicotine concentrations in 200 μM PA medium. c, d Bar graphs showing the changes in signal amplitude and firing rate following 24 h of PA and nicotine treatment (N ≥ 15). e Cell viability assessed by CCK-8 assay (N ≥ 4). f Live/dead staining of cardiomyocytes. Scale bar = 100 μm. Data in c–e are expressed as mean ± Standard Error of Mean. **p < 0.01; ***p < 0.001;****p < 0.0001

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  Fig. 5. Rapid nicotine withdrawal facilitates the restoration of cardiomyocyte electrophysiological activity. a Schematic of the experimental design to assess recovery in cardiomyocytes after short-term nicotine treatment. b Impact of nicotine exposure versus withdrawal on cardiomyocyte action potential amplitude. c Representative electrophysiological traces from cardiomyocytes with continued nicotine exposure or following withdrawal. d, e Bar graph and heat map showing the signal amplitude changes with nicotine exposure versus withdrawal(N ≥ 15). f, g Bar graph and heat map illustrating the alterations in firing frequency with nicotine exposure versus withdrawal. h Normalized amplitude heat maps of 32-channel electrophysiological signals in cardiomyocytes after 3 h nicotine exposure versus 12 h withdrawal. Data d, e are expressed as mean ± Standard Error of Mean. **p < 0.01; ***p < 0.001