Abstract-The 4.1 proteins are a family of multifunctional adaptor proteins. They promote the mechanical stability of plasma membranes by interaction with the cytoskeletal proteins spectrin and actin and are required for the cell surface expression of a number of transmembrane proteins. Protein 4.1R is expressed in heart and upregulated in deteriorating human heart failure, but its functional role in myocardium is unknown. To investigate the role of protein 4.1R on myocardial contractility and electrophysiology, we studied 4.1R-deficient (knockout) mice (4.1R KO). ECG analysis revealed reduced heart rate with prolonged Q-T interval in 4.1R KO. No changes in ejection fraction and fractional shortening, assessed by echocardiography, were found. The action potential duration in isolated ventricular myocytes was prolonged in 4.1R KO. Ca 2ϩ transients were larger and slower to decay in 4.1R KO. The sarcoplasmic reticulum Ca 2ϩ content and Ca 2ϩ sparks frequency were increased. The Na ϩ /Ca 2ϩ exchanger current density was reduced in 4.1R KO. The transient inward current inactivation was faster and the persistent Na ϩ current density was increased in the 4.1R KO group, with possible effects on action potential duration. Although no major morphological changes were noted, 4.1R KO hearts showed reduced expression of NaV1.5␣ and increased expression of protein 4.1G. Our data indicate an unexpected and novel role for the cytoskeletal protein 4.1R in modulating the functional properties of several cardiac ion transporters with consequences on cardiac electrophysiology and with possible significant roles during normal cardiac function and disease. Key Words: cardiac cytoskeleton Ⅲ ion transporter regulation Ⅲ EC coupling T he cardiac cytoskeleton is important in conferring stability to the myocardium, in sensing the mechanical stretch, and in coordinating the assembly of cellular structures and intercellular signaling. 1 One group of cytoskeletal proteins, the spectrin-and ankyrin-associated system, is involved in the complex interplay between actin, spectrin, and various ion transporters in relation to the regulation of intracellular [Ca 2ϩ ]. 2 Beta II spectrin, muscle LIM-only protein, and ankyrin B and G have all been associated with cardiac function and regulation of the electrophysiological properties of the myocardium. 3 The 4.1 protein family is also part of the spectrin-associated cytoskeleton. It promotes the interaction between spectrin and F-actin and, thus, membrane stability. 2 In mammals, the 4 genes, EPB41, EPB41L1, EPB41L2, and EPB41L3, encode proteins 4.1R, 4.1G, 4.1N, and 4.1B. mRNA transcripts from all 4 genes are found in mouse myocardium. 4 All 4.1 proteins have a FERM membranebinding domain, a spectrin-actin binding (SAB) domain, and a C-terminal domain. 2 A FERM-adjacent domain regulates the activities of both FERM and SAB domains. 5 The FERM and C-terminal domains bind to membrane proteins, 6 whereas the SAB domain binds the spectrin-actin cytoskeleton. 7 Multiple ion channels, pumps, and exc...

The 4.1 proteins are a family of multifunctional adaptor proteins that organise signalling/transport/cell adhesion molecules. They are capable of interaction with the spectrin-actin network thereby conferring mechanical stability to the cell membrane, with several ion transporters associated to this macromolecular complex. Protein 4.1R is expressed in the heart and upregulated in deteriorating human heart failure. However, no data exists on the roles of protein 4.1R in myocardial regulation and function. In particular, it is unknown whether this protein can influence cardiac contractility and/or electrophysiology. 4.1R-deficient mice (KO) were studied using echocardiography and ECG monitoring with radiotelemetry. Left ventricular dimensions were increased in KO mice (LV diameter - Dia (cm): WT = 0.43 ± 0.01 [6] (mean ± SEM [n]); KO = 0.49 ± 0.01 [6]; p < 0.01) - Sys cm): WT = 0.29 ± 0.01 [6]; KO = 0.34 ± 0.02 [6]; p < 0.05) with no changes in ejection fraction and fractional shortening. ECG analysis revealed reduced heart rate (RR interval (ms): WT = 113±5 [6]; KO = 139 ± 601 [6]; p < 0.01) accompanied by prolonged QT interval (corrected (ms): WT = 46 ± 2 [6]; KO = 52 ± 1 [6]; p < 0.05). The action potential duration (APD) measured in isolated ventricular myocytes was prolonged in KO (APD 90% at 1Hz (ms) WT = 146 ± 20 [21]; KO = 231 ± 29 [28]; p < 0.05). Ca transients, elicited by 1Hz field-stimulation and measured using the fluorescent indicator indo-1, were larger (amplitude (ratio units r.u.): WT = 0.07 ± 0.006 [22]; KO = 0.1 ± 0.006 [33]; p<0.05) and slower to decay in the KO group (time to 50% decline (ms): WT = 78 ± 3 [22]; KO = 91 ± 3 [33]; p < 0.05). This was associated with increased SR Ca content, (20 mM caffeine-induced indo-1 transient amplitude (r.u.): WT = 0.09 ± 0.01 [7]; KO = 0.13 ± 0.01 [16]; p < 0.05) and increased frequency of Ca sparks, measured by confocal microscopy using Fluo-4 (sparks/100μm/s. WT = 0.52 ± 0.08 [111]; KO = 1.21 ± 0.14 [124]; p < 0.001). We conclude that protein 4.1 R affects repolarisation of cardiac myocytes. This may have a role in bringing about QT prolongation in KO mice. The prolonged APD, together with effects on Ca handling proteins, may alter Ca regulation and cell contractility. The specific mechanisms controlling these effects are under investigation.