Hypokalemia Promotes Arrhythmia by Distinct Mechanisms in Atrial and Ventricular Myocytes

Figure 1.. Hypokalemia promotes a steady-state increase in Ca2+ transients and Ca2+ waves in ventricular myocytes and a subpopulation of atrial myocytes. A, In field-stimulated ventricular cells, rapidly lowering [K+]o from 5.0 to 2.7 mmol/L produced an initial depression of Ca2+ transient magnitude. A secondary rising phase followed which ultimately yielded larger Ca2+ transients compared with control conditions (right, n=15 cells, 8 hearts). A similar biphasic response to hypokalemia was observed in some atrial cardiomyocytes (13 of 31 cells, 10 hearts), with associated over-activity (arrow). Other atrial cells exhibited only a monophasic reduction in Ca2+ transient amplitude. B, Ca2+ waves were assessed during pauses in the electrical stimulus. Ventricular cells and those atrial cells which exhibited a biphasic response demonstrated an increased frequency of Ca2+ waves during hypokalemia. For Ca2+ wave measurements, ncells=17, 7, 12; nhearts=10, 5, 11 in ventricular, biphasic atrial, and monophasic atrial populations. Statistics: Wilcoxon signed-rank test.

Figure 2.. The biphasic Ca2+ transient response to hypokalemia requires t-tubules. A, T-tubule staining in intact tissue and isolated cells (caveolin-3 and di-8-ANEPPS, respectively) revealed a dense and well-organized t-tubule network in ventricular cells (scale bars=10 µm). T-tubules were only observed in approximately one-third of atrial cells (B, 21 of 56 cells, 4 hearts), and when present, these tubules were less well organized and at lower density (C) than in ventricular cells (40 cells, 3 hearts). D, Paired imaging of t-tubules and Ca2+ revealed that only tubulated atrial cells exhibited a biphasic response to hypokalemia (n=12 cells, 10 hearts), while untubulated cells exhibited a monophasic decline in Ca2+ transient amplitude (n=7 cells, 5 hearts). A similar monophasic decline in Ca2+ transients was reproduced in experimentally detubulated ventricular myocytes (n=10 cells, 3 hearts). Statistics: (B): z-test; (C): Mann-Whitney rank-sum test.

Figure 3.. The NKA (Na+, K+-ATPase) and Na+-Ca2+ exchanger (NCX) are robustly expressed within the t-tubules of both ventricular and atrial cardiomyocytes. A, Immunolabeling in intact tissue and isolated ventricular cells showed a robust expression of the α1 NKA isoform in both the surface membrane and t-tubules, while the α2 isoform was preferentially expressed in t-tubules. B, In atrial cells, NKA α1 was expressed in both the surface membrane and, when present, the t-tubules. NKA α2 expression was low in atrial cells, never present in t-tubules, and appeared limited to the intercalated disks. NCX (right) was robustly expressed in both the surface membrane and t-tubules of ventricular and atrial cells. C, In ventricular cells, preferential blockade of NKA α2 with 0.3 µmol/L ouabain19 inhibited the biphasic response of Ca2+ transients to hypokalemia (5 of 5 cells). In atrial cells, both biphasic (7 cells) and monophasic responses (3 cells) continued to be observed in the presence of low-dose ouabain, in agreement with limited α2 expression in these cells. Scale bars in (A) and (B)=10 µm in zoomed-out images, 2 µm in enlargements.

Figure 4.. Decreased Na+, K+-ATPase (NKA) activity during hypokalemia inhibits Ca2+ extrusion by t-tubular Na+-Ca2+ exchanger (NCX), elevating sarcoplasmic reticulum (SR) Ca2+ content. A, NKA activity was measured during hyperpolarizing voltage ramps and calculated as the K+-sensitive current (see Online Figure I for protocol and representative traces). Hypokalemia induced a modest and similar reduction in NKA current in ventricular myocytes and atrial cells, regardless of the presence of t-tubules (ncells=10, 7, 9; nhearts=4, 4, 5 in ventricular, tubulated atrial, untubulated atrial cells). B, SBFI experiments revealed no change in global cytosolic [Na+] during the protocol (ncells=12, 8; nhearts=4, 4 for ventricular, atrial cells). C, However, tubulated cells showed slowed Ca2+ removal by NCX during hypokalemia, as indicated by the declining phase of Ca2+ transients stimulated in the continuous presence of 10 mmol/L caffeine (change in tau values shown at right, ncells=8, 16; nhearts=3, 5, in ventricular, tubulated atrial cells, P=0.016, 0.011 vs Ko=5.0 by Wilcoxon signed-rank test). No change in Ca2+ removal rate was observed in untubulated atrial cells (ncells=16; nhearts=5; P=0.912).…

Figure 5.. Close Na+, K+-ATPase (NKA)-Na+-Ca2+ exchanger (NCX) crosstalk drives the biphasic Ca2+ transient response to hypokalemia. A mathematical model of the rat ventricular cardiomyocyte was employed to investigate the contribution of various ion channels and transporters to changes in cellular [Na+], SR Ca2+ content, and Ca2+ transients. A, With baseline model settings (blue lines), reducing [K+]o from 5.0 to 2.7 mmol/L triggered a modest accumulation of intracellular [Na+], and a biphasic response of SR Ca2+ content and Ca2+ transients which occurred over a markedly longer time course than that observed experimentally. To simulate greater crosstalk between NCX and NKA, the fraction of NCX operating in reverse mode (NCXrev) was increased in a step-wise manner, which produced an augmented and accelerated biphasic response (see also enlargement of the first 1000 s in [B], presented normalized to [K+]o=5.0). Removing the NCXrev contribution prevented any secondary rise in Ca2+ transients, in resemblance to experiments in untubulated cells. C, Sensitivity analyses were performed to examine the effects of modulating individual ion fluxes±5%. Reducing t-tubule-associated fluxes such as INa…

Figure 6.. Early afterdepolarization (EAD) generation during hypokalemia: mechanisms in ventricular cells. A, In electrically stimulated ventricular and atrial myocytes, Ca2+ oscillations were commonly observed during the late phase of the Ca2+ transient, consistent with EAD generation (arrows, ncells=24, 16, 10 in ventricular, tubulated, untubulated atrial cells during 3-min hypokalemia). B, Patch-clamp recordings of action potentials (APs) confirmed the occurrence of EADs in ventricular cells during hypokalemia (7 of 9 cells from 5 hearts), which were prevented when Ca2+ was buffered by EGTA in the patch pipette (lower, 8 of 8 cells from 4 hearts). C, Mean changes in resting membrane potential (RMP) and delayed afterdepolarization (DAD) incidence (ncells=9 from 5 hearts). D, Proportion of cells exhibiting EADs. E, Steady-state K+ currents were reduced at negative potentials during hypokalemia (ncells=8, 7 in normo-, hypokalemia from 4, 4 hearts). F, Pairing AP recordings with Ca2+ imaging (fluo-4, confocal line-scans) revealed EADs associated with spontaneous Ca2+ release (left) or reactivation of L-type Ca2+ current (right). Statistics: (A): z-test; (C): paired t-test for RMP, Wilcoxon…

Figure 7.. IKur maintains brief action potential (AP) configuration in untubulated atrial myocytes, promoting early afterdepolarization (EADs) driven by nonequilibrium INa during hypokalemia. A, In tubulated atrial myocytes, hypokalemia-induced EADs were frequently associated with Ca2+ waves. B, Buffering of intracellular Ca2+ with patch pipettes containing 60 µmol/L EGTA reduced EAD incidence in tubulated cells. However, in untubulated cells, EADs remained present and were observed to be triggered during rapid AP repolarization from negative potentials (arrow). C, Incidence of EADs in tubulated and untubulated atrial myocytes (−EGTA: ncells=10, 14 from 7, 8 hearts; +EGTA: ncells=10, 11 from 6, 9 hearts). D, AP configuration in tubulated and untubulated atrial cells (ncells=21, 48 from 12, 20 hearts). E, EADs observed in hypokalemic, untubulated atrial myocytes were inhibited by rapid application of 1 µmol/L TTX (top) or 10 mmol/L caffeine (bottom). F, Short AP configuration in untubulated atrial myocytes was associated with larger outward steady-state K+ current than in tubulated cells (protocol described in Online Figure IIA; ncells=11, 22 in tubulated, untubulated from 6, 11 hearts). G,…

Figure 8.. Schematic overview. Distinct mechanisms were observed to promote arrhythmogenesis in ventricular and atrial cardiomyocytes. DAD indicates delayed afterdepolarization; EAD, early afterdepolarization; NCX, Na+-Ca2+ exchanger; and NKA, Na+, K+-ATPase.