Direct measurement of SR release flux by tracking ‘Ca²⁺ spikes’ in rat cardiac myocytes

Abstract

Ca2+ release flux across the sarcoplasmic reticulum (SR) during cardiac excitation‐contraction coupling was investigated using a novel fluorescence method. Under whole‐cell voltage‐clamp conditions, rat ventricular myocytes were dialysed with a high concentration of EGTA (4.0 mm, 150 nm free Ca2+), to minimize the residence time of released Ca2+ in the cytoplasm, and a low‐affinity, fast Ca2+ indicator, Oregon Green 488 BAPTA‐5N (OG‐5N; 1.0 mm, Kd≈ 31 μm), to optimize the detection of localized high [Ca2+] in release site microdomains. Confocal microscopy was employed to resolve intracellular [Ca2+] at high spatial and temporal resolution. Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [Ca2+] change is the sum of two terms: one major term proportional to the SR release flux/Ca2+ influx, and the other reflecting the running integral of the released Ca2+. Indeed, the OG‐5N transients in EGTA‐containing cells consisted of a prominent spike followed by a small pedestal. The OG‐5N spike closely resembled the first derivative (d[Ca2+]/dt) of the conventional Ca2+ transient (with no EGTA), and mimicked the model‐derived SR Ca2+ release function reported previously. In SR Ca2+‐depleted cells, the OG‐5N transient also closely followed the waveform of L‐type Ca2+ current (ICa). Using ICa as a known source of Ca2+ influx, SR flux can be calibrated in vivo by a linear extrapolation of the ICa‐elicited OG‐5N signal. The OG‐5N image signal was localized to discrete release sites at the Z‐line level of sarcomeres, indicating that the local OG‐5N spike arises from ‘Ca2+ spikes’ at transverse (T) tubule‐SR junctions (due to the imbalance between calcium ions entering the cytosol and the buffer molecules). Both peak SR release flux and total amount of released Ca2+ exhibited a bell‐shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca2+ release was most synchronized and produced maximal peak release flux (4.2 mm s−1) at 0 mV; in contrast, maximal total release occurred at −20 mV (71 versus 61 μm at 0 mV), but the localized release signals were partially asynchronous. Since the maximal conventional [Ca2+] transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca2+ released, but also the synchronization among release sites affects the whole‐cell Ca2+ transient and the Ca2+‐myofilament interaction.

Excitation-contraction coupling and mitochondrial energetics

“…This assumption is as weak as assuming that gain of CICR is determined by the bulk cytoplasmic Ca 2+ concentration. During CICR, bulk cytosolic Ca 2+ can be completely buffered with no effect of local EC coupling, as demonstrated in studies of "Ca 2+ spikes" [173,183]. Similarly, cytoplasmic high energy phosphate buffer systems (e.g., creatine kinase (CK) isoenzymes and the rapidly diffusable phosphocreatine, PCr) are present that can limit changes in bulk ATP while shuttling ADP to the mitochondrial adenine nucleotide transporter (ANT), effectively transferring the energetic signal from the site of ATP hydrolysis to the mitochondrion (reviewed in [164] [25,26,45,46,101,117,164] (see also further below in Bioenergetic consequences of mitochondrial Ca 2+ uptake).…”

Alternative Splicing as a Molecular Switch for Ca2+/Calmodulin-Dependent Facilitation of P/Q-Type Ca2+Channels

“…Regulation triggered by the C-terminal lobe of CaM appears invariably insensitive to internal Ca 2ϩ buffering, whereas that initiated by the N-terminal lobe is eliminated by elevated Ca 2ϩ buffering with EGTA or BAPTA. From Ca 2ϩ diffusion arguments (Naraghi and Neher, 1997;Song et al, 1998;DeMaria et al, 2001;Augustine et al, 2003), the implication is that the C-terminal lobe of CaM responds preferentially to the opening of its associated Ca 2ϩ channel (sensitive to local Ca 2ϩ influx), whereas the N-terminal lobe detects aggregate activity of more distant sources of Ca 2ϩ (sensitive to global Ca 2ϩ influx). For previously studied P/Q-type channels (all with EFa), this pattern has manifested as CDF driven by the C-terminal lobe of CaM and CDI induced by the N-terminal lobe of CaM (DeMaria et al, 2001).…”

Relationship between Ca2+sparklets and sarcoplasmic reticulum Ca2+load and release in rat cerebral arterial smooth muscle

“…As in previous studies (2,22,23), we used the relatively slow Ca 2ϩ buffer EGTA (10 mM) (on rate Ϸ 100-fold slower than fluo-5F or rhod-2) to restrict the fluorescence of the indicator to near the site of Ca 2ϩ entry (Յ1 m) (40). A similar strategy has been used to record Ca 2ϩ -release sites in ventricular myocytes using confocal microscopy (1,35). For further details about this protocol, readers are referred to Amberg et al (2).…”