Local Ca Release Events Research Articles

Spatio-Temporal Properties of IP3 Receptor-Mediated Ca Release in Cardiac Myocytes

In cardiac myocytes cytosolic Ca increase and subsequent cell contraction are mainly determined by ryanodine receptor (RyR) mediated Ca release. However atrial cells are also equipped with IP3 receptors (IP3Rs), a second type of Ca release channels. We investigated IP3R-mediated Ca release events and their subcellular spatio-temporal properties in single membrane-permeabilized rabbit atrial myocytes. Local Ca release events were detected by confocal microscopy (fluo-4, longitudinal line scan mode). Local IP3R-mediated Ca release events were evoked by exposure to inositol-1,4,5-triphosphate (IP3) in the presence of tetracaine to inhibit RyR-mediated Ca spark activity, and could be inhibited by the IP3R blocker 2-APB. We classified IP3R-mediated Ca release events as subsarcolemmal, perinuclear (events 5μm) or nuclear. Nuclear events were considered membrane associated when occurring at a distance <1μm from the nuclear envelope or membranes of the nucleoplasmic reticulum. With tetracaine the Ca spark frequency decreased to 0.7±0.3 (from 8.8±1.2 in control), whereas the frequency of events detected after subsequent addition of IP3 increased to 2.9±1.3 (events/100μm/s). Subsequent exposure to 2-APB reduced the frequency to 1.8±0.8. Compared to Ca sparks, local Ca release events in the presence of IP3 were lower in amplitude, prolonged in duration and had a slower rise time. The increase in frequency of local Ca release events was particularly pronounced in the perinucler regions compared to the cytosol or the subsarcolemmal space. Furthermore, IP3-mediated Ca release events could also be detected within the nucleus. These nuclear events occurred at a higher incidence at locations that were closely associated with membrane structures of the nuclear envelope or the nucleoplasmic reticulum. In conclusion, in atrial myocytes IP3R-mediated Ca release is spatially inhomogeneous and preferentially occurs in the nuclear and perinuclear regions.

Does Protein Kinase A–Mediated Phosphorylation of the Cardiac Ryanodine Receptor Play Any Role in Adrenergic Regulation of Calcium Handling in Health and Disease?

See related articles, pages 1743–1752 Cardiac myocyte ryanodine receptors (sarcoplasmic reticulum [SR] Ca release channel; cardiac ryanodine receptor [RyR2]) are localized in the junctional SR, in close proximity to L-type Ca channels (LTCCs) embedded in the membranes of the transverse (T)-tubules. This signaling microdomain has been termed the couplon,1 because it is here that excitation–contraction coupling takes place. As the heart fills with blood during diastole, RyR2 is stabilized in a closed state, allowing Ca uptake by the SR Ca ATPase to pump Ca from the cytoplasm into the SR, providing the primary source of Ca to activate the contractile apparatus during the next heart beat (systole). During systole, the cardiac action potential depolarizes the T-tubules and causes the opening of LTCCs. Ca influx through LTCCs elevates the [Ca] within the cytoplasmic space between the T-tubular and SR membrane, which promotes Ca binding to neighboring RyR2s, inducing some to open. Ca then moves out of the SR lumen into the subsarcolemmal, space and the additional elevation of [Ca] induces a regenerative opening of other RyRs in the couplon. The resulting localized Ca signal is called a Ca spark.2 The action potential synchronizes these local Ca release events throughout the cell (termed Ca-induced Ca release) to produce the global cardiac [Ca] transient.3 The stabilized closed state and Ca-dependent opening of RyR2 during diastole and systole are essential for normal cardiac diastolic and systolic function. Destabilizing the behavior of RyR2 would be expected to have a negative …

Loss of Intracellular and Intercellular Synchrony of Calcium Release in Systolic Heart Failure

Normal contraction of cardiac muscle requires a coordinated cellular mechanical response to the electric pacemaker signal that begins in the sinoatrial node and spreads through the conduction system and ultimately across the ventricular myocardium via gap junctions. In ventricular myocytes, depolarization by the action potential causes clusters of L-type calcium channels (LCCs) to open, allowing a small amount of Ca to enter the diadic cleft space between the sarcolemma and the sarcoplasmic reticulum (SR). When the Ca concentration in this space becomes sufficiently high to gate ryanodine receptors (RyRs), they open and release SR Ca into the cytoplasm, so that it can bind to and activate myofilaments, resulting in force development. Each cluster of LCCs on the sarcolemma, its opposing cluster of RyRs across the 10- to 12-nm diameter diadic cleft space, and associated SR regulatory proteins is a couplon.1 According to the local control theory of cardiac excitation-contraction (EC) coupling, couplons are activated independently. Independent activation of couplons depends on the extent of LCC activation, providing a mechanism for regulation of contractile force by stochastic recruitment of couplons.2 In systolic heart failure, numerous cellular defects in EC coupling and Ca regulation have been identified that contribute to contractile dysfunction (and ventricular arrhythmias), although there is a host of other major abnormalities leading to loss of force development, including fibrosis,3 defective energy metabolism,4 pH changes,5 heterogeneous conductance across connexin hemichannels,6 and defects in sarcomeric proteins.7 Thus, it is important to keep in mind that no single defect can explain the contractile dysfunction of systolic failure. Article see p 223 Recent studies of defective EC coupling in heart failure have addressed not only reductions in the extent and kinetics of Ca released in response to the action potential (ie, reduced recruitment of couplons and …

Increased leakage of sarcoplasmic reticulum Ca2+ contributes to abnormal myocyte Ca2+ handling and shortening in sepsis*

Changes in cardiac function due to sepsis have been widely reported. However, the underlying mechanisms remain poorly understood. In the mammalian heart, myocyte function and intracellular calcium homeostasis are closely coupled. In this study we tested the hypothesis that alterations in cardiac calcium homeostasis due to sepsis underlie the observed myocyte dysfunction. Randomized prospective animal study. Research laboratory. Male Sprague-Dawley rats weighing 250-275 g. We induced sepsis by cecal ligation and puncture in the rat, which mimics the type of infection caused by perforation of the intestine in humans. Forty-eight hours after cecal ligation and puncture, isolated cardiac ventricular cardiomyocytes demonstrated a 57% decreased peak systolic [Ca]. The time constant of the Ca transient increased 71% and 57% in myocytes obtained 24 hrs and 48 hrs after cecal ligation and puncture, respectively. The average shortening of cardiomyocytes 48 hrs after cecal ligation and puncture was significantly decreased. To investigate the cellular mechanisms of altered Ca transients and myocyte shortening, we measured Ca sparks, the spontaneous local Ca release events in cardiomyocytes at resting states. The Ca spark frequency progressively increased in myocytes 24 hrs and 48 hrs after cecal ligation and puncture. The total activity of sparks also increased compared with sham-operated animals. The overall leakage of sarcoplasmic reticulum Ca in resting states was increased in sepsis and resulted in reduced sarcoplasmic reticulum Ca content. Abnormal Ca leakage from the sarcoplasmic reticulum contributes significantly to the depressed myocyte shortening in sepsis. In the future, modalities that prevent this Ca leakage may prove beneficial in the treatment of sepsis-induced myocyte shortening.

Protein kinase C reduces the KCa current of rat tail artery smooth muscle cells.

The hypothesis that protein kinase C (PKC) is able to regulate the whole cell Ca-activated K (KCa) current independently of PKC effects on local Ca release events was tested using the patch-clamp technique and freshly isolated rat tail artery smooth muscle cells dialyzed with a strongly buffered low-Ca solution. The active diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (DOG) at 10 microM attenuated the current-voltage (I-V) relationship of the KCa current significantly and reduced the KCa current at +70 mV by 70 +/- 4% (n = 14). In contrast, 10 microM DOG after pretreatment of the cells with 1 microM calphostin C or 1 microM PKC inhibitor peptide, selective PKC inhibitors, and 10 microM 1,3-dioctanoyl-sn-glycerol, an inactive diacylglycerol analog, did not significantly alter the KCa current. Furthermore, the catalytic subunit of PKC (PKCC) at 0.1 U/ml attenuated the I-V relationship of the KCa current significantly, reduced the KCa current at +70 mV by 44 +/- 3% (n = 17), and inhibited the activity of single KCa channels at 0 mV by 79 +/- 9% (n = 6). In contrast, 0.1 U/ml heat-inactivated PKCC did not significantly alter the KCa current or the activity of single KCa channels. Thus these results suggest that PKC is able to considerably attenuate the KCa current of freshly isolated rat tail artery smooth muscle cells independently of effects of PKC on local Ca release events, most likely by a direct effect on the KCa channel.