We have developed a mathematical model of the human atria myocyte based on averaged voltage-clamp data recorded from isolated single myocytes. Our model consists of a Hodgkin-Huxley-type equivalent circuit for the sarcolemma, coupled with a fluid compartment model, which accounts for changes in ionic concentrations in the cytoplasm as well as in the sarcoplasmic reticulum. This formulation can reconstruct action potential data that are representative of recordings from a majority of human atrial cells in our laboratory and therefore provides a biophysically based account of the underlying ionic currents. This work is based in part on a previous model of the rabbit atrial myocyte published by our group and was motivated by differences in some of the repolarizing currents between human and rabbit atrium. We have therefore given particular attention to the sustained outward K+ current (I[sus]), which putatively has a prominent role in determining the duration of the human atrial action potential. Our results demonstrate that the action potential shape during the peak and plateau phases is determined primarily by transient outward K+ current, I(sus) and L-type Ca2+ current (I[Ca,L]) and that the role of I(sus) in the human atrial action potential can be modulated by the baseline sizes of I(Ca,L), I(sus) and the rapid delayed rectifier K+ current. As a result, our simulations suggest that the functional role of I(sus) can depend on the physiological/disease state of the cell.

The mouse heart has become a widely used model for genetic studies of heart diseases. Thus, understanding gender differences in mouse cardiac repolarization is crucial to the interpretation of such studies. The objective of this study was to evaluate whether there are gender differences in cardiac repolarization in mouse ventricle and to gain insights into the ionic and molecular mechanisms underlying these differences. Action potential durations (APDs) and K(+) currents in male and female ventricular myocytes were compared using a patch-clamp technique. APD(20), APD(50), and APD(90) were found to be significantly longer in females than males. Examination of the different K(+) currents revealed that a significantly lower current density exists in female ventricular myocytes compared with male myocytes for the ultrarapid delayed rectifier K(+) current, I(Kur) (at +30 mV, male, 33.2+/-2.9 pA/pF [n= 22]; female, 20.9+/-1.73 pA/pF [n= 19], P<0.001). Consistent with these findings were the results of the ribonuclease protection assay, Western blots, and confocal analysis that showed a significantly lower expression level of Kv1.5 (coding for I(Kur)) in female compared with male ventricle. The additional K(+) currents present in mouse ventricle exhibited no gender differences. In agreement with these electrophysiological data, no differences in the expression levels for the K(+) channels underlying these currents were detected between both sexes. This study demonstrates that adult mice exhibit gender differences in cardiac repolarization. The expression of Kv1.5 and of its corresponding K(+) current, I(Kur), is significantly lower in female mouse ventricle, and as a result, the APD is lengthened.

Although the K + currents expressed in hearts of adult mice have been studied extensively, detailed information concerning their relative sizes and biophysical properties in ventricle and atrium is lacking. Here we describe and validate pharmacological and biophysical methods that can be used to isolate the three main time-and voltage-dependent outward K + currents which modulate action potential repolarization. A Ca 2+ -independent transient outward K + current, I to , can be separated from total outward current using an 'inactivating prepulse'. The rapidly activating, slowly inactivating delayed rectifier K + current, I Kur , can be isolated using submillimolar concentrations of 4-aminopyridine (4-AP). The remaining K + current, I ss , can be obtained by combining these two procedures: (i) inactivating I to and (ii) eliminating I Kur by application of low concentration of 4-AP. I ss activates relatively slowly and shows very little inactivation, even during depolarizations lasting several seconds. Our findings also show that the rate of reactivation of I to is more than 20-fold faster than that of I Kur . These results demonstrate that the outward K + currents in mouse ventricles can be separated based on their distinct time and voltage dependence, and different sensitivities to 4-AP. Data obtained at both 22 and 32• C demonstrate that although the duration of the inactivating prepulse has to be adapted for the recording temperature, this approach for separation of K + current components is also valid at more physiological temperatures. To demonstrate that these methods also allow separation of these K + currents in other cell types, we have applied this same approach to myocytes from mouse atria. Molecular approaches have been used to compare the expression levels of different K + channels in mouse atrium and ventricle. These findings provide new insights into the functional roles of I Kur , I to and I ss during action potential repolarization. The extensive efforts to develop transgenic mouse models of cardiovascular diseases have resulted in strong interest in defining the functional properties and molecular basis of the K + currents in mouse heart. Accordingly, a number of detailed studies focusing on K + currents in adult mouse ventricular myocytes have appeared (Benndorf et al. 1987;Wang & Duff, 1997;Barry et al. 1998;Babij et al. 1998;Zhou et al. 1998Zhou et al. , 2003 London et al. 1998a,b;Guo et al. 1999Guo et al. , 2000Wickenden et al. 1999; Xu et al. 1999a,b; DuBell et al. 2000;Jeron et al. 2000;Zaritsky et al. 2001;Kuo et al. 2001;Brunet et al. 2004;Bondarenko et al. 2004). In combination, these results provide a reasonably Judith Brouillette and Robert B. Clark contributed equally to this study consistent and quite detailed account of the number and type of time-and voltage-dependent K + currents expressed in adult mouse ventricle. Although a number of different strategies have been developed for separating the total K + current into individual K + conductance components, none of these approaches...

1. The K¤ currents which control repolarization in adult mouse ventricle, and the effects of changes in action potential duration on excitation-contraction coupling in this tissue, have been studied with electrophysiological methods using single cell preparations and by recording mechanical parameters from an in vitro working heart preparation. 2. Under conditions where Ca¥-dependent currents were eliminated by buffering intracellular Ca¥ with EGTA, depolarizing voltage steps elicited two rapidly activating outward K¤ currents: (i) a transient outward current, and (ii) a slowly inactivating or 'sustained' delayed rectifier. 3. These two currents were separated pharmacologically by the K¤ channel blocker 4-aminopyridine (4-AP). 4-AP at concentrations between 3 and 200 ìÒ resulted in (i) a marked increase in action potential duration and a large decrease in the sustained K¤ current at plateau potentials, as well as (ii) a significant increase in left ventricular systolic pressure in the working heart preparation. 4. The current-voltage (I-V) relation, kinetics, and block by low concentrations of 4-AP strongly suggest that the rapid delayed rectifier in adult mouse ventricle is the same K¤ current (Kv1.5) that has been characterized in detail in human and canine atria. 5. These results show that the 4-AP-sensitive rapid delayed rectifier is a very important repolarizing current in mouse ventricle. The enhanced contractility produced by 4-AP (50 ìÒ) in the working heart preparation demonstrates that modulation of the action potential duration, by blocking a K¤ current, is a very significant inotropic variable.

1. The hypothesis that Kv4.2 and Kv4.3 are two of the essential K+ channel isoforms underlying the Ca2+-independent transient outward K+ current (I) in rat ventricle has been tested using a combination of electrophysiological measurements and antisense technology in both native myocytes and a stably transfected mammalian cell line, mouse Ltk-cells (L-cells). 2. The transient outward currents generated by Kv4.2 channels in L-cells exhibit rapid activation and inactivation properties similar to those produced by It in rat ventricular cells.The current-voltage relationships and the voltage dependence of steady-state inactivation are also very similar in these two preparations. However, the recovery from inactivation of KV4.2 is much slower (time constant, 378 ms) than that of It in rat ventricular cells (58 ms).3. The K+ current due to K 4.2 can be blocked by millimolar concentrations of 4-aminopyridine in L-cells; a similar pharmacological response has been observed in rat ventricular myocytes. A Ca2+-independent transient outward potassium current potential. This current is characterized by its rapid (Ij) is expressed in most mammalian heart cells (Josephson, activation and inactivation, and its sensitivity to 4-amino-

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HF-1 b, an SP1 -related transcription factor, is preferentially expressed in the cardiac conduction system and ventricular myocytes in the heart. Mice deficient for HF-1 b survive to term and exhibit normal cardiac structure and function but display sudden cardiac death and a complete penetrance of conduction system defects, including spontaneous ventricular tachycardia and a high incidence of AV block. Continuous electrocardiographic recordings clearly documented cardiac arrhythmogenesis as the cause of death. Single-cell analysis revealed an anatomic substrate for arrhythmogenesis, including a decrease and mislocalization of connexins and a marked increase in action potential heterogeneity. Two independent markers reveal defects in the formation of ventricular Purkinje fibers. These studies identify a novel genetic pathway for sudden cardiac death via defects in the transition between ventricular and conduction system cell lineages.

Strain and gender differences observed in mouse cardiac repolarization can be explained by different androgen levels. As a consequence, androgens are major regulatory factors in cardiac repolarization and special attention should be paid to the hormonal status of the animal when studying hormonal regulation of cardiac repolarization.