Resting Membrane Potential Regulates Na⁺–Ca²⁺ Exchange‐Mediated Ca²⁺ Overload during Hypoxia–Reoxygenation in Rat Ventricular Myocytes

Abstract

In the heart, reperfusion following an ischaemic episode can result in a marked increase in [Ca2+]i and cause myocyte dysfunction and death. Although the Na+–Ca2+ exchanger has been implicated in this response, the ionic mechanisms that are responsible have not been identified. In this study, the hypothesis that the diastolic membrane potential can influence Na+–Ca2+ exchange and Ca2+ homeostasis during chemically induced hypoxia–reoxygenation has been tested using right ventricular myocytes isolated from adult rat hearts. Superfusion with selected [K+]o of 0.5, 2.5, 5, 7, 10 and 15 mm yielded the following resting membrane potentials: −27.6 ± 1.63 mV, −102.2 ± 1.89, −86.5 ± 1.03, −80.1 ± 1.25, −73.6 ± 1.02 and −66.4 ± 1.03, respectively. In a second set of experiments myocytes were subjected to chemically induced hypoxia–reoxygenation at these different [K+]o, while [Ca2+]i was monitored using fura‐2. These results demonstrated that after chemically induced hypoxia–reoxygenation had caused a marked increase in [Ca2+]i, hyperpolarization of myocytes with 2.5 mm[K+]o significantly reduced [Ca2+]i (7.5 ± 0.32 vs. 16.9 ± 0.55 %); while depolarization (with either 0.5 or 15 mm[K+]o) significantly increased [Ca2+]i (31.8 ± 3.21 and 20.8 ± 0.36 vs. 16.9 ± 0.55 %, respectively). As expected, at depolarized membrane potentials myocyte hypercontracture and death increased in parallel with Ca2+ overload. The involvement of the Na+–Ca2+ exchanger in Ca2+ homeostasis was evaluated using the Na+–Ca2+ exchanger inhibitor KB‐R7943. During reoxygenation KB‐R7943 (5 μm) almost completely prevented the increase in [Ca2+]i both in control conditions (in 5 mm[K+]o: 2.2 ± 0.40 vs. 10.8 ± 0.14 %) and in depolarized myocytes (in 15 mm[K+]o: −2.1 ± 0.51 vs. 11.3 ± 0.05 %). These findings demonstrate that the resting membrane potential of ventricular myocytes is a critical determinant of [Ca2+]i during hypoxia–reoxygenation. This appears to be due mainly to an effect of diastolic membrane potential on the Na+–Ca2+ exchanger, since at depolarized potentials this exchanger mechanism operates in the reverse mode, causing a significant Ca2+ influx.

The protective effect of ursodeoxycholic acid in an in vitro model of the human fetal heart occurs via targeting cardiac fibroblasts

“…For instance, inwardly rectifying K + (Kir) channels are thought to be the principal K + channel subtype responsible for maintaining RMP (Baczkó et al, 2003). Chilton et al (2005) reported K + conductance in adult rat cardiac myofibroblasts to be carried by several K + channel subtypes, namely K ir and delayed rectifier voltage-gated K Shibukawa et al, 2005).…”

Efficacy of selective NCX inhibition by ORM-10103 during simulated ischemia/reperfusion

“…The reduction in [K þ ] i depolarizes the sarcolemma (Baczko et al, 2003;Tanaka et al, 2002), further facilitating reverse mode NCX activity and leads to gradual [Ca 2 þ ] i accumulation. During the early phase of ischemia the major source of the [Ca 2 þ ] i increase is the leaky SR (Zucchi and Ronca-Testoni, 1997), while during the late phase, [Ca 2 þ ] i accumulation via NCX is accelerated (Bourdillon and Poole-Wilson, 1981;Haigney et al, 1992).…”

The Na+/Ca2+Exchange Inhibitor 2-(2-(4-(4-Nitrobenzyloxy)phenyl)ethyl)isothiourea Methanesulfonate (KB-R7943) Also Blocks Ryanodine Receptors Type 1 (RyR1) and Type 2 (RyR2) Channels

“…Concentrations ranging from 1 to 5 M produce a half-maximal block (IC 50 ) of the NCX reverse mode (inhibition of Ca 2ϩ entry), and inhibition is not selective among the three NCX isoforms (Iwamoto, 2004). KB-R7943 has been used to selectively inhibit the reverse mode of NCX to understand its contribution to cardiac ischemia and injury (Schröder et al, 1999;Seki et al, 2002;Amran et al, 2003;Baczkó et al, 2003;Iwamoto, 2004;Stys, 2004;Dietz et al, 2007;MacDonald and Howlett, 2008;O'Rourke, 2008). However, besides its effect on NCX, KB-R7943 also has off-target effects, including binding to the norepinephrine transporter, (IC 50 Ͻ 3 M) (Matsuda et al, 2001) and block of Ba 2ϩ currents through Ca v 1.2 with inhibitory constants similar to those needed to inhibit NCX (IC 50 ϭ 6.8 M) (Ouardouz et al, 2005).…”