Cardiac autonomic imbalance is associated with increased arrhythmia incidence and may contribute to mortality in chronic heart failure (CHF). Increased carotid chemoreflex (CC) sensitivity is also associated with higher mortality risk and is thought to play a role in autonomic imbalance. Chronic blood flow reduction plays an important role in enhanced CC sensitivity. We hypothesize that downregulation of the shear-sensitive transcription factor Kruppel-like Factor 2 (KLF2) mediates increased CC sensitivity in CHF and contributes to cardiac autonomic imbalance and increased arrhythmia incidence. Rabbits (male, 3-3.5kg, n=8-9 per group) were anaesthetised with isoflurane (2%) in oxygen and radiotelemetry transmitters and pacing wires were implanted. Rabbits were paced into CHF over the course of 3-4 weeks (320-380 bpm). Once in CHF animals were anaesthetised as previously described and an adenovirus (KLF2) solution was delivered to the carotid sinus (CHF KLF2). Ventilation, arterial pressure, and ECG was measured at rest and in response to CC activation with isocapnic hypoxia (IsoH) in pre-pace, CHF, and CHF KLF2 conditions. Heart rate variability (HRV) in the time domain was analyzed for the SD of interpulse intervals (SDNN). Power spectral analysis in the frequency domain was performed using the following frequency cutoffs: 0.0625-0.1875 Hz low-frequency (LF) band, 0.1875-0.5625 Hz high-frequency (HF) band. Cardiac function (CF) was quantified with echocardiography. Values are means ± S.E.M., compared by ANOVA or paired t-test. HRV in the time domain (pre-pace SDNN 15±1 ms, CHF SDNN 10±5 ms) and HF power (pre-pace 39±3 n.u., CHF 27±2 n.u.) were decreased in CHF. LF/HF ratio (pre-pace 0.5±0.06, CHF 0.8±0.1) and arrhythmia incidence (pre-pace 50±10/h, CHF 300±100/h) were increased in CHF (all p<0.05 vs. pre-pace), as were CC responses to IsoH. Transfection of KLF2 to the carotid bodies (CB) in CHF animals resulted in increased HRV (SDNN 15±2, HF 43±4 n.u.) and attenuation of arrhythmia incidence (46±13/h) (all p<0.05 vs. CHF). CC responses to IsoH were attenuated as well. CF was not different between CHF and CHF KLF2 conditions. Protein expression of KLF2 in the CB was decreased in CHF animals (0.5±0.1 sham vs. 0.3±0.1 CHF, n=8), and was restored with adenoviral transfection (0.6±0.1, n=8). Conversely, KLF2 knockdown with lentiviral KLF2 siRNA resulted in decreased CB KLF2 expression (0.4±0.1, n=8) and decreases in SDNN (12±1 ms) and HF power (30±6 n.u.) and increased LF/HF (1±3). Our findings indicate that increased CC sensitivity, cardiac autonomic imbalance, and arrhythmia incidence during CHF may be caused by down-regulation of KLF2 in the CB.