Kenneth S. Ginsburg

Hypercontractile Female Hearts Exhibit Increased S-Nitrosylation of the L-Type Ca2+ Channel α1 Subunit and Reduced Ischemia/Reperfusion Injury

Mechanisms underlying gender differences in cardiovascular disease are poorly understood. We found previously that, under hypercontractile conditions, female hearts exhibit significantly less ischemia/reperfusion injury than males. Here we show that male wild-type (WT) mouse hearts pretreated with 10 nmol/L isoproterenol before ischemia exhibited increased injury versus female hearts, but this relative protection in females was absent in eNOS−/− and nNOS−/− hearts. In isoproterenol-treated female versus male hearts, there was also more endothelial NO synthase (eNOS) associated with cardiomyocyte caveolin-3, and more neuronal NOS (nNOS) translocation to caveolin-3 during ischemia/reperfusion. S-nitrosothiol (SNO) formation was increased in isoproterenol-treated ischemic/reperfused hearts in all mouse genotypes, but only in WT mice was SNO content significantly higher in females than males. Using the biotin switch method, we identified the L-type Ca2+ channel &agr;1 subunit as the predominant S-nitrosylated protein in membrane fractions, and following isoproterenol and ischemia/reperfusion male/female differences in SNO were seen only in WT hearts, but not in constitutive NOS−/− genotypes. The isoproterenol-induced increase in L-type Ca2+ current (ICa) was smaller in females versus in males, but NOS blockade increased ICa in females. This gender difference in ICa in isoproterenol-treated myocytes (and abolition on NOS inhibition) was mirrored exactly in Ca2+ transients and SR Ca2+ contents. In conclusion, these data suggest that eNOS and nNOS both play roles in the gender differences observed in ischemia/reperfusion injury under adrenergic stimulation, and also demonstrate increased S-nitrosylation of the L-type Ca2+ channels in female cardiomyocytes.

Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration.

Our aim was to measure the influence of sarcoplasmic reticulum (SR) calcium content ([Ca](SRT)) and free SR [Ca] ([Ca](SR)) on the fraction of SR calcium released during voltage clamp steps in isolated rabbit ventricular myocytes. [Ca](SRT), as measured by caffeine application, was progressively increased by conditioning pulses. Sodium was absent in both the intracellular and in the extracellular solutions to block sodium/calcium exchange. Total cytosolic calcium flux during the transient was inferred from I(Ca), [Ca](SRT), [Ca](i), and cellular buffering characteristics. Fluxes via the calcium current (I(Ca)), the SR calcium pump, and passive leak from the SR were evaluated to determine SR calcium release flux (J(rel)). Excitation-contraction (EC) coupling was characterized with respect to both gain (integral J(rel)/integral I(Ca)) and fractional SR calcium release. Both parameters were virtually zero for a small, but measurable [Ca](SRT). Gain and fractional SR calcium release increased steeply and nonlinearly with both [Ca](SRT) and [Ca](SR). We conclude that potentiation of EC coupling can be correlated with both [Ca](SRT) and [Ca](SR). While fractional SR calcium release was not linearly dependent upon [Ca](SR), intra-SR calcium may play a crucial role in regulating the SR calcium release process.

Quantitative Assessment of the SR Ca2+ Leak-Load Relationship

Abstract— Increased diastolic SR Ca2+ leak (Jleak) could depress contractility in heart failure, but there are conflicting reports regarding the Jleak magnitude even in normal, intact myocytes. We have developed a novel approach to measure SR Ca2+ leak in intact, isolated ventricular myocytes. After stimulation, myocytes were exposed to 0 Na+, 0 Ca2+ solution ±1 mmol/L tetracaine (to block resting leak). Total cell [Ca2+] does not change under these conditions with Na+-Ca2+ exchange inhibited. Resting [Ca2+]i declined 25% after tetracaine addition (126±6 versus 94±6 nmol/L;P <0.05). At the same time, SR [Ca2+] ([Ca2+]SRT) increased 20% (93±8 versus 108±6 &mgr;mol/L). From this Ca2+ shift, we calculate Jleak to be 12 &mgr;mol/L per second or 30% of the SR diastolic efflux. The remaining 70% is SR pump unidirectional reverse flux (backflux). The sum of these Ca2+ effluxes is counterbalanced by unidirectional forward Ca2+ pump flux. Jleak also increased nonlinearly with [Ca2+]SRT with a steeper increase at higher load. We conclude that Jleak is 4 to 15 &mgr;mol/L cytosol per second at physiological [Ca2+]SRT. The data suggest that the leak is steeply [Ca2+]SRT-dependent, perhaps because of increased [Ca2+]i sensitivity of the ryanodine receptor at higher [Ca2+]SRT. Key factors that determine [Ca2+]SRT in intact ventricular myocytes include (1) the thermodynamically limited Ca2+ gradient that the SR can develop (which depends on forward flux and backflux through the SR Ca2+ ATPase) and (2) diastolic SR Ca2+ leak (ryanodine receptor mediated).

The effect of Ca2+–calmodulin‐dependent protein kinase II on cardiac excitation–contraction coupling in ferret ventricular myocytes

1 The effect of Ca2+–calmodulin‐dependent protein kinase II (CaMKII) on excitation–contraction coupling (E–C coupling) was studied in intact ferret cardiac myocytes using the selective inhibitor KN‐93. KN‐93 decreased steady‐state (SS) twitch [Ca2+]i (by 51%), resting Ca2+ spark frequency (by 88%) and SS sarcoplasmic reticulum (SR) Ca2+ content evaluated by caffeine application (by 37.5%). 2 Increasing extracellular Ca2+ concentration ([Ca2+]o) to 5 mm in KN‐93 restored SR Ca2+ load and Ca2+ spark frequency towards that in control (2 mm Cao2+), but SS twitch [Ca2+]i was still significantly depressed by KN‐93. 3 KN‐93 decreased Ca2+ transient amplitude of SS twitches much more strongly than the amplitude of post‐rest (PR) twitches. In the control, the time constant (τ) of [Ca2+]i decline of SS twitches was faster than that for PR twitches. This stimulation‐dependent acceleration of [Ca2+]i decline was abolished by KN‐93. 4 Voltageclamp experiments demonstrated that KN‐93 significantly inhibited sarcolemmal L‐type Ca2+ current (ICa) during repetitive pulses by slowing recovery from inactivation. This may explain the preferential action of KN‐93 to suppress SS vs. PR twitches. 5 In KN‐93, even when both ICa and SR Ca2+ load were matched to the control levels by manipulation of conditioning voltage‐clamp pulses, contraction and twitch Ca2+ transients were still both significantly depressed (to 39 and 49% of control, respectively). 6 Since KN‐93 reduced SR Ca2+ release channel (RyR) activity during E–C coupling, even for matched SR Ca2+ load and trigger ICa, we infer that endogenous CaMKII is an important modulator of E–C coupling in intact cardiac myocytes. Effects of KN‐93 on ICa and SS twitch [Ca2+]i decline also indicate that endogenous CaMKII may have stimulatory effects on ICa and SR Ca2+ uptake.

Phospholemman-Phosphorylation Mediates the β-Adrenergic Effects on Na/K Pump Function in Cardiac Myocytes

Cardiac sympathetic stimulation activates β-adrenergic (β-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na]i), which in turn affects [Ca]i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na]i decline and current (IPump) to assess β-AR effects on NKA function in isolated myocytes. In WT myocytes, 1 &mgr;mol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (Km decreased from 18.8±1.4 to 13.6±1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na]i (from 12.5±1.8 to 10.5±1.4 mmol/L). In PLM-KO mice under control conditions Km (14.2±1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower Km(Na) in PLM-KO versus WT (12.9±0.9 versus 16.2±1.5). Thus, PLM inhibits NKA activity by decreasing its [Na]i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation).

KB-R7943 Block of Ca2+ Influx Via Na+/Ca2+ Exchange Does Not Alter Twitches or Glycoside Inotropy but Prevents Ca2+ Overload in Rat Ventricular Myocytes

BACKGROUND The Na(+)/Ca(2+) exchange (NCX) extrudes Ca(2+) from cardiac myocytes, but it can also mediate Ca(2+) influx, load the sarcoplasmic reticulum with Ca(2+), and trigger Ca(2+) release from the sarcoplasmic reticulum. In ischemia/reperfusion or digitalis toxicity, increased levels of intracellular [Na(+)] ([Na(+)](i)) may raise levels of intracellular [Ca(2+)] ([Ca(2+)](i)) via NCX, leading to cell injury and arrhythmia. METHODS AND RESULTS We used KB-R7943 (KBR) to selectively block Ca(2+) influx via NCX to study the role of NCX-mediated Ca(2+) influx in intact rat ventricular myocytes. Removing extracellular Na(+) caused [Ca(2+)](i) to rise, due to Ca(2+) influx via NCX, and this was blocked by 90% with 5 micromol/L KBR. However, KBR did not alter [Ca(2+)](i) decline due to NCX. Thus, we used 5 micromol/L KBR to selectively block Ca(2+) entry but not efflux via NCX. Under control conditions, 5 micromol/L KBR did not alter steady-state twitches, Ca(2+) transients, Ca(2+) load in the sarcoplasmic reticulum, or rest potentiation, but it did prolong the late low plateau of the rat action potential. When Na(+)/K(+) ATPase was inhibited by strophanthidin, KBR reduced diastolic [Ca(2+)](i) and abolished the spontaneous Ca(2+) oscillations, but it did not prevent inotropy. CONCLUSIONS In rat ventricular myocytes, Ca(2+) influx via NCX is not important for normal excitation-contraction coupling. Furthermore, the inhibition of Ca(2+) efflux alone (as [Na(+)](i) rises) may be sufficient to cause glycoside inotropy. In contrast, Ca(2+) overload and spontaneous activity at high [Na(+)](i) was blocked by KBR, suggesting that net Ca(2+) influx (not merely reduced efflux) via NCX is involved in potentially arrhythmogenic Ca(2+) overload.

Modulation of excitation–contraction coupling by isoproterenol in cardiomyocytes with controlled SR Ca2+ load and Ca2+ current trigger

Cardiac Ca2+ transients are enhanced by cAMP‐dependent protein kinase (PKA). However, PKA‐dependent modulation of ryanodine receptor (RyR) function in intact cells is difficult to measure, because PKA simultaneously increases Ca2+ current (ICa), SR Ca2+ uptake and SR Ca2+ loading (which independently increase SR Ca2+ release). We measured ICa and SR Ca2+ release ± 1 μm isoproterenol (ISO; isoprenaline) in voltage‐clamped ventricular myocytes of rabbits and transgenic mice (expressing only non‐phosphorylatable phospholamban). This mouse model helps control for any effect of ISO‐enhanced SR uptake on observed release, but the two species produced essentially identical results. SR Ca2+ load and ICa were adjusted by conditioning. We thus evaluated PKA effects on SR Ca2+ release at constant SR Ca2+ load and ICa trigger (with constant unitary ICa). The amount of SR Ca2+ release increased as a function of either ICa or SR Ca2+ load, but ISO did not alter the relationships (measured as gain or fractional release). This was true over a wide range of SR Ca2+ load and ICa. However, the maximal rate of SR Ca2+ release was ∼50% faster with ISO (at most loads and ICa levels). We conclude that the isolated effect of PKA on SR Ca2+ release is an increase in maximal rate of release and faster turn‐off of release (such that integrated SR Ca2+ release is unchanged). The increased amount of SR Ca2+ release normally seen with ISO depends primarily on increased ICa trigger and SR Ca2+ load, whereas faster release kinetics may be the main result of RyR phosphorylation.

Ca2+/calmodulin-dependent protein kinase II (CaMKII) regulates cardiac sodium channel NaV1.5 gating by multiple phosphorylation sites.

Background: CaMKII is up-regulated in heart failure and modulates Na+ current (INa), yet the mechanism is unclear. Result: CaMKII phosphorylates several sites in the first intracellular loop of NaV1.5, thereby altering INa gating properties. Conclusion: This multisite phosphorylation may contribute to acquired arrhythmogenesis. Significance: Identification of these regulatory sites is critical for potential therapeutic targeting of CaMKII and NaV1.5 in failing hearts. The cardiac Na+ channel NaV1.5 current (INa) is critical to cardiac excitability, and altered INa gating has been implicated in genetic and acquired arrhythmias. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is up-regulated in heart failure and has been shown to cause INa gating changes that mimic those induced by a point mutation in humans that is associated with combined long QT and Brugada syndromes. We sought to identify the site(s) on NaV1.5 that mediate(s) the CaMKII-induced alterations in INa gating. We analyzed both CaMKII binding and CaMKII-dependent phosphorylation of the intracellularly accessible regions of NaV1.5 using a series of GST fusion constructs, immobilized peptide arrays, and soluble peptides. A stable interaction between δC-CaMKII and the intracellular loop between domains 1 and 2 of NaV1.5 was observed. This region was also phosphorylated by δC-CaMKII, specifically at the Ser-516 and Thr-594 sites. Wild-type (WT) and phosphomutant hNaV1.5 were co-expressed with GFP-δC-CaMKII in HEK293 cells, and INa was recorded. As observed in myocytes, CaMKII shifted WT INa availability to a more negative membrane potential and enhanced accumulation of INa into an intermediate inactivated state, but these effects were abolished by mutating either of these sites to non-phosphorylatable Ala residues. Mutation of these sites to phosphomimetic Glu residues negatively shifted INa availability without the need for CaMKII. CaMKII-dependent phosphorylation of NaV1.5 at multiple sites (including Thr-594 and Ser-516) appears to be required to evoke loss-of-function changes in gating that could contribute to acquired Brugada syndrome-like effects in heart failure.

Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes.

Left ventricular hypertrophy (approximately 40%) was induced in rats by banding of the abdominal aorta. After 16 wk, ventricular homogenates were prepared for biochemical measurements and ventricular myocytes were isolated for functional studies. In myocytes, the effects of banding on intracellular Ca handling, contraction, and excitation-contraction (E-C) coupling were determined using indo 1 fluorescence and whole cell voltage clamp. After steady-state field or voltage-clamp stimulation to load the sarcoplasmic reticulum (SR), SR Ca content assessed by caffeine-induced Ca transients was the same in sham and banded groups. Despite this, cell shortening amplitudes were significantly depressed in the banded group, suggesting altered contractile properties. In banded rats, the SR Ca-adenosinetriphosphatase (Ca-ATPase) mRNA level was reduced, as was homogenate thapsigargin-sensitive SR Ca-ATPase, but cytosolic free Ca concentration ([Ca]i) decline attributed to SR Ca-ATPase activity in intact cells was not slowed. Banding also reduced Na/Ca exchange mRNA level but did not affect either Na-dependent sarcolemmal 45Ca transport in homogenate or the rate of [Ca]i decline in intact cells attributed to Na/Ca exchange (during caffeine-induced contractures). Banding also did not change the rate of [Ca]i decline mediated by the combined function of the mitochondrial Ca uptake and sarcolemmal Ca-ATPase in intact cells. Ca current (ICa) density and voltage dependence were the same in sham and banded groups. Ryanodine receptor mRNA, protein content, and ryanodine affinity were also unchanged in the banded group. At 1 mM extracellular Ca concentration ([Ca]o), banding did not affect E-C coupling efficacy in intact cells under voltage clamp (i.e., same contraction for given ICa and SR Ca load). However, when [Ca]o was reduced to 0.5 mM, the efficacy of E-C coupling was greatly depressed in the banded group (even though ICa and SR Ca content were matched). In summary, unloaded myocyte contraction was depressed in these hypertrophic hearts, but Ca transport was little altered, at 1 mM [Ca]o. However, reduction of [Ca]o to 0.5 mM appears to unmask a depressed fractional SR Ca release in response to a given ICa trigger and SR Ca load.

Comparison of sarcolemmal calcium channel current in rabbit and rat ventricular myocytes.

1. Fundamental properties of Ca2+ channel currents in rat and rabbit ventricular myocytes were measured using whole cell voltage clamp. 2. In rat, as compared with rabbit myocytes, Ca2+ channel current (ICa) was half‐activated at about 10 mV more negative potential, decayed slower, was half‐inactivated (in steady state) at about 5 mV more positive potential, and recovered faster from inactivation. 3. These features result in a larger steady‐state window current in rat, and also suggest that under comparable voltage clamp conditions, including action potential (AP) clamp, more Ca2+ influx would be expected in rat myocytes. 4. Ca2+ channel current carried by Na+ and Cs+ in the absence of divalent ions (Ins) also activated at more negative potential and decayed more slowly in rat. 5. The reversal potential for Ins was 6 mV more positive in rabbit, consistent with a larger permeability ratio (PNa/PCs) in rabbit than in rat. ICa also reversed at slightly more positive potentials in rabbit (such that PCa/PCs might also be higher). 6. Ca2+ influx was calculated by integration of ICa evoked by voltage clamp pulses (either square pulses or pulses based on recorded rabbit or rat APs). For a given clamp waveform, the Ca2+ influx was up to 25% greater in rat, as predicted from the fundamental properties of ICa and Ins. 7. However, the longer duration of the AP in rabbit myocytes compensated for the difference in influx, such that the integrated Ca2+ influx via ICa in response to the species‐appropriate waveform was about twice as large as that seen in rat.