Fig. 2. Arterial myocyte NADH:NAD⁺ is sensitive to changes in proximal cardiomyocyte beating frequency.

A Schematic illustration depicting the preparation of arterial myocyte/induced pluripotent stem cell-derived cardiomyocyte (iPSC-CM) co-cultures. Primary aortic vascular smooth muscle cells (passage 0–2) were treated with adenovirus to express the NADH:NAD⁺-sensitive fluorescent biosensor peredox-mCherry (i.). Arterial myocytes were then seeded and allowed to integrate (48 h) onto a two-dimensional monolayer of iPSC-CMs (ii.). Imaging was performed on either arterial/cardiac co-cultured myocytes or arterial myocytes alone during electrical stimulation (1–3 Hz; iii.) while monitoring peredox-mCherry green:red fluorescence. B Exemplary brightfield and fluorescence images showing red (mCherry) and green (T-sapphire) fluorescence of peredox-mCherry expressing arterial myocytes in the presence of non-fluorescent cardiac myocytes in an arterial + cardiac myocyte co-culture. Scale bars represent 100 μm. Experiment was repeated six times with similar results. C Exemplary time series of arterial myocyte NADH:NAD⁺ in either an arterial/cardiac myocyte co-culture (green trace) or arterial myocytes alone (– cardiac myocytes; black trace) at baseline (0 Hz) and during electrical stimulation (1–3 Hz). D Box and whiskers plots (line: median, box: 25th to 75th percentile, whiskers: min and max) summarizing fold-change in NADH:NAD⁺ in arterial myocytes in arterial/cardiac myocyte co-cultures or arterial myocytes alone. Αrterial/cardiac myocytes, n = 50 cells from 5 independent experiments, 2 vs. 1 Hz, *p = 0.0047; 3 vs. 1 Hz, *p = 0.0012; Arterial myocytes only, n = 52 cells from 6 independent experiments, 2 vs. 1 Hz, *p = 0.0029; 3 vs. 1 Hz, ns: p = 0.6574 (Linear mixed models, log-transformed NADH:NAD⁺). Source data are provided as a Source Data file.