Na'-and Ca 2 '-sensitive microelectrodes were used to measure intracellular Na' and Ca t+ activities (aiae and ace) of sheep ventricular muscle and Purkinje strands to study the interrelationship between Na' and Ca 2+ electrochemical gradients (4:t,N, and AAca) under various conditions . In ventricular muscle, aNe was 6 .4 t 1 .2 mM and ace was 87 f 20 nM ([Ca2+ ] = 272 nM) .A graded decrease of external Na'} activity (aNa) resulted in decrease of aNe, and increase of aca . There was increase of twitch tension in lowaN a solutions, and occasional increase of resting tension in 40% aNe . Increase of external Ca 2+(ac e ) resulted in increase of aba and decrease of aNe . Decrease of ace resulted in decrease of ace and increase of aN a . The apparent resting Na-Ca energy ratio (AAca/AANa) was between 2 .43 and 2 .63 . When the membrane potential (VQ,) was depolarized by 50 mM K+ in ventricular muscle, Vm depolarized by 50 mV, aNa decreased, and ac e increased, with the development of a contracture. The apparent energy coupling ratio did not change with depolarization . 5 X 10 -s M ouabain induced a large increase in aNa and aca, accompanied by an increase in twitch and resting tension . Under the conditions we have studied, AANa and AAca appeared to be coupled and n was nearly constant at 2 .5, as would be expected if the Na-Ca exchange system was able to set the steady level of ace . Tension threshold was about 230 nM ac . . The magnitude of twitch tension was directly related to ac a .

Changes in stimulation rate alter the electrical and mechanical characteristics of myocardial cells. We have investigated the possibility that intracellular sodium activity (aiNa) changes with stimulation and correlates with changes in contraction strength. Two kinds of liquid membrane Na+-selective microelectrodes were used to measure aiNa in guinea pig and sheep ventricular muscle and in sheep Purkinje strands. Stimulation produced a rate- and time-dependent elevation of aiNa. Small increases in aiNa were seen at stimulation rates as slow as 0.2 Hz, and faster rates of stimulation elevated aiNa by over 30%. The changes seen in Purkinje strands and ventricular muscle were similar. Following a period of stimulation, aiNa and Vm returned to their pre-stimulus levels with the same time courses. This is consistent with the suggestion that the post-stimulation hyperpolarization is the result of an increased rate of electrogenic Na+ extrusion. The effects of stimulation on aiNa and tension were compared with those of ouabain. The comparison suggests that rapid stimulation could produce increased contraction strength as the result of a substantial gain in intracellular calcium via a Na-Ca exchange mechanism, but that this is only one of several factors determining the force-frequency relationship.

Glycogen synthase kinase-3β (GSK3β) is a multifunctional kinase whose inhibition is known to limit myocardial ischemiareperfusion injury. However, the mechanism mediating this beneficial effect still remains unclear. Mitochondria and sarco/ endoplasmic reticulum (SR/ER) are key players in cell death signaling. Their involvement in myocardial ischemia-reperfusion injury has gained recognition recently, but the underlying mechanisms are not yet well understood. We questioned here whether GSK3β might have a role in the Ca 2+ transfer from SR/ER to mitochondria at reperfusion. We showed that a fraction of GSK3β protein is localized to the SR/ER and mitochondria-associated ER membranes (MAMs) in the heart, and that GSK3β specifically interacted with the inositol 1,4,5-trisphosphate receptors (IP 3 Rs) Ca 2+ channeling complex in MAMs. We demonstrated that both pharmacological and genetic inhibition of GSK3β decreased protein interaction of IP 3 R with the Ca 2+ channeling complex, impaired SR/ER Ca 2+ release and reduced the histamine-stimulated Ca 2+ exchange between SR/ER and mitochondria in cardiomyocytes. During hypoxia reoxygenation, cell death is associated with an increase of GSK3β activity and IP 3 R phosphorylation, which leads to enhanced transfer of Ca 2+ from SR/ER to mitochondria. Inhibition of GSK3β at reperfusion reduced both IP 3 R phosphorylation and SR/ER Ca 2+ release, which consequently diminished both cytosolic and mitochondrial Ca 2+ concentrations, as well as sensitivity to apoptosis. We conclude that inhibition of GSK3β at reperfusion diminishes Ca 2+ leak from IP 3 R at MAMs in the heart, which limits both cytosolic and mitochondrial Ca 2+ overload and subsequent cell death. Glycogen synthase kinase-3 (GSK3) was originally identified as a phosphorylating kinase for glycogen synthase. 1,2 It has two isoforms, α and β, that possess strong homology in their kinase domains with, however, distinct functions. 3 GSK3 is constitutively active but it can be inhibited by phosphorylation on serine 21 (Ser21) for GSK3α and Ser9 for GSK3β. 4 In the heart, GSK3β has several important roles in cardiac hypertrophy 5 and ischemia-reperfusion (IR) injury. 6 Accumulating evidence indicates that phospho-Ser9-GSK3β-mediated cytoprotection is achieved by an increased threshold for permeability transition pore (PTP) opening. [6][7][8][9] The mechanism by which GSK3β delays PTP opening still remains unclear. It has been reported that GSK3β could interact with ANT at the inner mitochondrial membrane in the heart 9 and/or to phosphorylate voltage-dependent anion channel (VDAC) and cyclophilin D (CypD) in cancer cells. 10,11 GSK3β also has other proposed mechanisms of action, including a poorly characterized role in calcium (Ca 2+ ) homeostasis regulation 12 and protein-protein interactions, 9 as well as functions in different subcellular fractions such as the nucleus, cytosol and mitochondria. 13 Reperfusion is the most powerful intervention to salvage ischemic myocardium. However, it can also paradoxically lead to ca...

We have measured the effects of changing tonicity of the bathing solution on intracellular sodium and calcium activities and tension of sheep cardiac Purkinje strands and ventricular muscle. For Purkinje strands in solutions of normal tonicity, resting membrane potential was -77.4 +/- 0.4 mV (mean +/- SE), sodium activity was 7.9 +/- 0.4 mM, and calcium activity was 98 +/- 9 nM. For ventricular muscle in solutions of normal tonicity, resting membrane potential was -86.4 +/- 1.2 mV, sodium activity was 6.9 +/- 0.5 mM, and calcium activity was 70 +/- 4 nM. Reduction of tonicity to 75% of normal in both tissues produced depolarization of a few millivolts, and sodium activity fell almost to the level predicted for simple osmotic dilution. In Purkinje strands, calcium activity fell much more than that predicted for simple osmotic dilution. Twitch contraction was reduced in the hypotonic solution. Increase of tonicity to 150% and 200% caused the resting membrane potential to become more negative. In both tissues, sodium activity increased somewhat less than predicted from simple water movement, and calcium activity increased proportionately much more than sodium activity. The much larger change of calcium activity in both hypo- and hypertonic solutions could be explained by water movement plus the effect of sodium-calcium exchange. In hypertonic solutions, tonic tension was increased, along with the rise in calcium activity; however, the twitch tension was reduced. This reduction of twitch tension may be due to a direct effect of hypertonicity on cross-bridge behavior, as has been reported for skeletal muscle.

SUMMARY1. The basis of the resting potential of chick embryo ventricular muscle was studied by use of ion-selective micro-electrodes. Membrane resting potential hyperpolarized from -65-4 + 1-1 mV (mean + s.E.) at age 4 day to ---75-8 + 0-6 mV at age 18 day. Action potential overshoot increased from + 19-8 + 0-9 at age 4 day to +33-1+0-6mVatage 18 day.2., Intracellular K+ activity measured with ion-selective micro-electrodes increased from 71-3 + 1-9 mm at age 4 day to 89-9 + 1-1 mM at age 18 day. Intracellular Na+ activity decreased from 12-5 + 0-4 to 7-0 + 0-3 mm during the same period. The difference between membrane resting potential and the calculated potassium equilibrium potential decreased with development. PNa/PK estimated from the constant field equation decreased from 0-012 at age 4 day to 0-005 at age 18 day.3. The hyperpolarization of resting potential and the increased action potential overshoot during development could be explained by a rise in intracellular K+ activity and a fall in intracellular Na+ activity, as if the Na-K exchange pump became more active.