The 4.1 proteins are a family of multifunctional adaptor proteins part of the spectrin-associated cytoskeleton. They promote the mechanical stability of plasma membranes by interaction with spectrin and actin, and they are required for the cell surface expression of a number of ion transporters (Bennett & Baines, 2001). Protein 4.1R is expressed in the heart and upregulated in deteriorating human heart failure but its functional role in the myocardium is unknown. To investigate the role of protein 4.1R on myocardial contractility and electrophysiology, we studied 4.1R-deficient mice (4.1R KO) (Shi et al., 1999). All procedures were performed according to the UK Animals (Scientific Procedures) Act 1986. ECG were measured using radiotelemetry. For the implantation of ECG transmitters the animals were anesthetised by 1.5% isoflurane in 100% oxygen. ECG recording, performed 1 week after surgery, showed that heart rate was reduced (R-R interval (ms): wild type (WT) = 113 ± 5 (6) mean ± SEM (n); 4.1R KO = 139 ± 6 (6); unpaired t test: p < 0.01) with prolonged Q-T interval in 4.1R KO (QTc (ms): WT = 46 ± 2 (6); 4.1R KO = 52 ± 1 (6); p<0.05). No changes in ejection fraction and fractional shortening, assessed by echocardiography, were found. The action potential duration (APD) in isolated ventricular myocytes was prolonged in 4.1R KO (APD₇₅ at 5 Hz (ms): WT = 57 ± 7 (20); 4.1R KO = 81 ± 7 (19); p<0.05). Ca²⁺ transients were larger (indo-1 ratio: WT = 0.072 ± 0.01 (22); 4.1R KO = 0.096 ± 0.01 (33); p=0.01) and slower to decay in 4.1R KO (time-to-50% decline (ms): WT = 78 ± 3 (22); 4.1R KO = 91 ± 3 (33); p=0.01). The sarcoplasmic reticulum (SR) Ca²⁺ content (assessed by the application of 20 mM caffeine), and Ca²⁺ sparks frequency were increased (caffeine-induced indo-1 transient (ratio units): WT = 0.09 ± 0.01 (7); 4.1R KO = 0.12 ± 0.01 (16); p<0.05); sparks frequency (events/µm/s): WT = 0.52 ± 0.08 (111); 4.1R KO = 1.21 ± 0.1 (124); p<0.001). The Na⁺/Ca²⁺ exchanger current density was reduced in 4.1R KO (Ni²⁺-sensitive current at + 80 mV (pA/pF): WT = 3.37 ± 0.1 (14); 4.1R KO = 2.05 ± 0.2 (16); p<0.05). The transient inward current inactivation was faster (I_to fast time constant (ms): WT = 71 ± 10 (34); 4.1R KO = 40 ± 6 (32); p<0.05) and the persistent Na⁺ current density was increased in 4.1R KO (integral of 30 µM tetradotoxin-sensitive current at -20 mV measured between 50-300ms from onset (pC/pF): WT = -57 ± 9 (16); 4.1R KO = -98 ± 9 (25); p<0.01), with possible effects on APD. Our data indicate that the cytoskeletal protein 4.1R modulates the functional properties of several cardiac ion transporters with consequences on cardiac electrophysiology and with possible significant roles during normal cardiac function and disease.