Abstract Body (Do not enter title and authors here): Background: Hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) is a marker of the first heart field (FHF) but is absent in the second heart field (SHF) during early development. While its role in the cardiac conduction system is well-established, the function of HCN4 in left ventricle development remains largely unexplored.

Methods: Human pluripotent stem cells (hPSC) were differentiated into ventricular cardiomyocytes (vCM), with day 5 (d5) marking the FHF stage post-WNT inhibition. HCN4 was knocked down using siRNA, and effects were measured by qPCR and ATP CellTiter-Glo® assay.

Results: By d8, a 72-hour siRNA knockdown of HCN4 led to a significant reduction in its mRNA expression, decreasing from 11.2±0.3 in controls to 4.3±0.1 (p<0.05, n=3), corresponding to a 51.3% knockdown. Expression levels of key FHF or vCM markers (TBX5, HAND1, IRX4, CTNT) were not affected, except for NKX2-5, which showed a modest increase from 50.7±0.6 to 60.3±4.6 (p<0.05, n=3). Spontaneous beating rates remained comparable between treatment and control groups (50.0±0.7 vs. 48.3±2.7 bpm, n=3, p>0.05), suggesting that HCN4 knockdown does not impair the automaticity of d8 vCM. Treatment with ZD7288, a non-specific blocker of HCN channels led to a dose-dependent (10, 50, 100 µM) decrease in FHF cellular ATP production (p<0.05, n=8). Ivabradine, a more specific but use-dependent HCN blocker, had no effect, likely due to limited channel activity in FHF progenitors, which do not exhibit rhythmic firing. This differential response suggests that FHF mitochondrial function may be modulated by ZD7288, potentially due to inhibition of multiple subunits of HCN channels and/or off-target effects on Na⁺ or Ca²⁺ channels. Furthermore, to assess specificity of the response, 50µM ZD7288 treatment decreased ATP levels more in FHF progenitors than in SHF progenitors (81±2.8% vs. 91±2.0%, p<0.05, n=4), suggesting a greater reliance of FHF progenitors on HCN channel-mediated energy production.

Conclusion: HCN4 is actively transcribed in a hPSC model of FHF development. Its knockdown did not affect expression of most FHF and vCM markers. Pharmacological inhibition of HCN channels significantly reduced ATP levels in the progenitors with greater sensitivity in FHF, a development timepoint known to exhibit a shift toward oxidative metabolism. The data warrants further investigation of non-canonical roles of HCN channels as a regulator of ventricular chamber development.