1. The roles of the sarcoplasmic reticulum (SR) Ca2+-ATPase and Na+-Ca2+ exchange in Ca2" removal from cytosol were compared in isolated rabbit and rat ventricular myocytes during caffeine contractures and electrically stimulated twitches. Cell shortening and intracellular calcium concentration (ICa2`]j) were measured in indo-1-loaded cells.2. Na'-Ca2" exchange was inhibited by replacement of external Na+ by Li'. To avoid net changes in cell or SR Ca2" load during a twitch in 0 Na+ solution, intracellular Na+ (Na{) was depleted using a long pre-perfusion with 0 Na+, 0 Ca2+ solution. SR Ca21 accumulation was inhibited by caffeine or thapsigargin (TG). 3. Relaxation of steady-state twitches was 2-fold faster in rat than in rabbit (before and after Na{ depletion). In contrast, caffeine contractures (where SR Ca2+ accumulation is inhibited), relaxed faster in rabbit cells. Removal of external Na+ increased the half-time for relaxation of caffeine contractures 15-and 5-fold in rabbit and rat myocytes respectively (and increased contracture amplitude in rabbit cells only). The time course of relaxation in 0 Na+, 0 Ca2+ solution was similar in the two species. [Ca2+]i decline, so that T was virtually the same in both species and comparable to that during caffeine application in 0 Na+, 0 Ca2+ solution. Thus, the combined participation of slow Ca21 transport mechanisms (mitochondrial Ca21 uptake and sarcolemmal Ca2+-ATPase) is similar in these species.7. We conclude that during the decline of the [Ca2+]i transient, the Na+-Ca2+ exchange is about 2-to 3-fold faster in rabbit than in rat, whereas the SR Ca2+-ATPase is 2-to 3-fold faster in the rat. While the SR Ca2+-ATPase is more powerful than the Na+-Ca2+ exchange in both cell types the dominance is much more marked in rat (-% 13-fold vs. 2-5-fold in rabbit). Finally we estimate that the fraction of Ca2+ transported by the SR, Na+-Ca2+ exchange and slow systems during a twitch are 70, 28 and 2 % respectively in rabbit myocytes and 92, 7 and 1 % respectively in rat myocytes.

Diabetes mellitus (DM) encompasses a multitude of secondary disorders, including heart disease. One of the most frequent and potentially life threatening disorders of DM-induced heart disease is ventricular tachycardia (VT). Here we show that toll-like receptor 2 (TLR2) and NLRP3 inflammasome activation in cardiac macrophages mediate the production of IL-1β in DM mice. IL-1β causes prolongation of the action potential duration, induces a decrease in potassium current and an increase in calcium sparks in cardiomyocytes, which are changes that underlie arrhythmia propensity. IL-1β-induced spontaneous contractile events are associated with CaMKII oxidation and phosphorylation. We further show that DM-induced arrhythmias can be successfully treated by inhibiting the IL-1β axis with either IL-1 receptor antagonist or by inhibiting the NLRP3 inflammasome. Our results establish IL-1β as an inflammatory connection between metabolic dysfunction and arrhythmias in DM.

SUMMARY1. Contraction and intracellular Ca2+ (Ca2+i) transients were measured in isolated rabbit ventricular myocytes during twitches and contractures induced by rapid application of 10 mM-caffeine.2. The amplitude of caffeine-induced contractures and the accompanying CaO+ transients were larger than during normal twitches and also declined more slowly.This may be because only a fraction of sarcoplasmic reticulum (SR) Ca2+ is released during a normal twitch, or because of a temporal overlap of SR Ca2+ release and uptake during the twitch.3. When a caffeine contracture was initiated in Na+-free, Ca2+-free medium (to prevent sarcolemmal Na+-Ca2+ exchange) the contracture and CaO+ transient were larger and decreased much more slowly. Thus, Ca2+ extrusion via Na+-Ca2+ exchange may limit the amplitude of caffeine-induced contractures.4. Relaxation half-time (th) for the twitch (0 17 + 003 s) was increased to 0 54 + 0-07 s for caffeine contractures in control solution and 88 + 1 s for caffeineinduced contractures in Na+-free, Ca2+-free solution. These results confirm that the SR Ca2+ pump and Na+-Ca2+ exchange are the predominant mechanisms for cytoplasmic Ca2`removal during relaxation. However slower mechanisms can still reduce intracellular [Ca2+].5. Relaxation of caffeine contractures in Na+-free solution was further slowed when (a) mitochondrial Ca2+ uptake was inhibited with the oxidative phosphorylation uncoupler, FCCP (t, = 19 7 + 3-2 s), or (b) the sarcolemmal Ca2+-ATPase pumping ability was depressed by a large transmembrane [Ca2+] gradient (ti= 27-5+69 s).6. When the four Ca2+ transport systems were simultaneously inhibited (i.e. SR Ca2+ pump, Na+-Ca2+ exchange, mitochondrial Ca2+ uptake and sarcolemmal Ca2+ pump), relaxation was practically abolished, but the cell could recover quickly when Na+ was reintroduced and caffeine removed.7. We conclude that, under our experimental conditions, the sarcolemmal Ca2+ pump and mitochondria are -37-and 50-fold slower than the Na+-Ca2+ exchange at removing Ca2+ from the cytoplasm. Additionally, the SR Ca2+ pump is about 3-4 times faster than Na+-Ca2+ exchange.MS 9636

Fluorescent Ca indicators have been extremely valuable in understanding intracellular [Ca] ([Ca]i) regulation in many cell types. The calibration of these indicators in the intracellular environment, however, has been a continuous challenge. We performed in vivo calibrations of indo-1 in isolated rabbit ventricular myocytes loaded with the acetoxymethylester form of indo-1 and used the perforated patch variation of whole cell voltage clamp. Voltage, [Na], and [K] gradients were eliminated to approach equilibrium. We also took advantage of the powerful Na/Ca exchange in cardiac myocytes so that [Ca]i would be equilibrated with [Ca]o (because there was no [Na] or voltage gradient). The equilibration of [Na] and [Ca] across the membrane was tested by measuring the reversal potential of Na current and poking the cell to test for changes in [Ca]i-dependent fluorescence ratio. The apparent dissociation constant, Kd for indo-1 in the cellular environment was 844 nM, which is approximately 2-3 times higher than that in aqueous solutions. In a separate series of experiments, a null point approach was used to determine the [Ca]i in intact cells at rest for very long periods (82 +/- 6 nM). This is lower than that measured 15 s after a train of steady-state twitches ([Ca]i = 294 +/- 53 nM). These experiments also allowed the direct assessment of the shortening versus [Ca]i relationship in intact cells.

We investigated the role of Ca/calmodulin-dependent protein kinase (CaMKII) in relaxation and cytosolic free [Ca] ([Ca]i) decline during steady-state (SS) and postrest (PR) twitches in intact rat ventricular myocytes. Half-time of mechanical relaxation and time constant of [Ca]i decline (tau) were twofold greater during PR than with SS at 1 Hz. This difference was 1) abolished by inhibition of sarcoplasmic reticulum (SR) Ca accumulation by thapsigargin or caffeine; 2) greater at higher stimulation frequency and extracellular [Ca], which affected only SS tau; 3) abolished by the protein phosphatase inhibitor okadaic acid (10 microM, which selectively accelerated [Ca]i decline during PR); 4) still present during stimulation or inhibition of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA) by 10 microM forskolin or 1 microM H-89, respectively (SS and PR tau values were abbreviated and prolonged, respectively); and 5) suppressed by 10 microM KN-62, a selective inhibitor of CaMKII, which selectively prolonged [Ca]i decline during SS twitches. Both protein kinase inhibitors were also shown to decrease the SR Ca-uptake rate in digitonin-permeabilized rat myocytes. We conclude that CaMKII plays a major role in modulation of relaxation in rat ventricular myocytes, enhancing SR Ca uptake in a activity-dependent fashion. Our results are also compatible with a background, activity-independent stimulation of SR Ca uptake by PKA in intact rat myocytes.