Oliver Zeitz

Overexpression of FK506-Binding Protein FKBP12.6 in Cardiomyocytes Reduces Ryanodine Receptor–Mediated Ca2+ Leak From the Sarcoplasmic Reticulum and Increases Contractility

Abstract — The FK506-binding protein FKBP12.6 is tightly associated with the cardiac sarcoplasmic reticulum (SR) Ca2+-release channel (ryanodine receptor type 2 [RyR2]), but the physiological function of FKBP12.6 is unclear. We used adenovirus (Ad)-mediated gene transfer to overexpress FKBP12.6 in adult rabbit cardiomyocytes. Western immunoblot and reverse transcriptase–polymerase chain reaction analysis revealed specific overexpression of FKBP12.6, with unchanged expression of endogenous FKBP12. FKBP12.6-transfected myocytes displayed a significantly higher (21%) fractional shortening (FS) at 48 hours after transfection compared with Ad-GFP–infected control cells (4.8±0.2% FS versus 4±0.2% FS, respectively; n=79 each;P =0.001). SR-Ca2+ uptake rates were monitored in &bgr;-escin–permeabilized myocytes using Fura-2. Ad-FKBP12.6–infected cells showed a statistically significant higher rate of Ca2+ uptake of 0.8±0.09 nmol/s−1/106 cells (n=8, P <0.05) compared with 0.52±0.1 nmol/s−1/106 cells in sham-infected cells (n=8) at a [Ca2+] of 1 &mgr;mol/L. In the presence of 5 &mgr;mol/L ruthenium red to block Ca2+ efflux via RyR2, SR-Ca2+ uptake rates were not significantly different between groups. From these measurements, we calculate that SR-Ca2+ leak through RyR2 is reduced by 53% in FKBP12.6-overexpressing cells. Caffeine-induced contractures were significantly larger in Ad-FKBP12.6–infected myocytes compared with Ad-GFP–infected control cells, indicating a higher SR-Ca2+ load. Taken together, these data suggest that FKBP12.6 stabilizes the closed conformation state of RyR2. This may reduce diastolic SR-Ca2+ leak and consequently increase SR-Ca2+ release and myocyte shortening.

Impaired Contractile Performance of Cultured Rabbit Ventricular Myocytes After Adenoviral Gene Transfer of Na+-Ca2+ Exchanger

Na+-Ca2+ exchanger (NCX) gene expression is increased in the failing human heart. We investigated the hypothesis that upregulation of NCX can induce depressed contractile performance. Overexpression of NCX was achieved in isolated rabbit ventricular myocytes through adenoviral gene transfer (Ad-NCX). After 48 hours, immunoblots revealed a virus dose-dependent increase in NCX protein. Adenoviral &bgr;-galactosidase transfection served as a control. The fractional shortening (FS) of electrically stimulated myocytes was analyzed. At 60 min−1, FS was depressed by 15.6% in the Ad-NCX group (n=143) versus the control group (n=163, P <0.05). Analysis of the shortening-frequency relationship showed a steady increase in FS in the control myocytes (n=26) from 0.027±0.002 at 30 min−1 to 0.037±0.002 at 120 min−1 (P <0.05 versus 30 min−1) and to 0.040±0.002 at 180 min−1 (P <0.05 versus 30 min−1). Frequency potentiation of shortening was blunted in NCX-transfected myocytes (n=27). The FS was 0.024±0.002 at 30 min−1, 0.029±0.002 at 120 min−1 (P <0.05 versus 30 min−1, P <0.05 versus control), and 0.026±0.002 at 180 min−1 (NS versus 30 min−1, P <0.05 versus control). Caffeine contractures, which indicate sarcoplasmic reticulum Ca2+ load, were significantly reduced at 120 min−1 in NCX-transfected cells. An analysis of postrest behavior showed a decay of FS with longer rest intervals in control cells. Rest decay was significantly higher in the Ad-NCX group; after 120 seconds of rest, FS was 78±4% in control and 65±3% in the Ad-NCX group (P <0.05) relative to steady-state FS before rest (100%). In conclusion, the overexpression of NCX in rabbit cardiomyocytes results in the depression of contractile function. This supports the hypothesis that upregulation of NCX can result in systolic myocardial failure.

Hydroxyl Radical-Induced Acute Diastolic Dysfunction Is Due to Calcium Overload via Reverse-Mode Na+-Ca2+ Exchange

Hydroxyl radicals (OH) are involved in the development of reperfusion injury and myocardial failure. In the acute phase of the OH-mediated diastolic dysfunction, increased intracellular Ca2+ levels and alterations of myofilaments may play a role, but the relative contribution of these systems to myocardial dysfunction is unknown. Intact contracting cardiac trabeculae from rabbits were exposed to OH, resulting in an increase in diastolic force (Fdia) by 540%. Skinned fiber experiments revealed that OH-exposed preparations were sensitized for Ca2+ (EC50: 3.27±0.24×10−6 versus 2.69±0.15×10−6 mol/L;P <0.05), whereas maximal force development was unaltered. Western blots showed a proteolytic degradation of troponin T (TnT) with intact troponin I (TnI). Blocking of calpain I by MDL-28.170 inhibited both TnT-proteolysis and Ca2+ sensitization, but failed to prevent the acute diastolic dysfunction in the intact preparation. The OH-induced diastolic dysfunction was similar in preparations with intact (540±93%) and pharmacologically blocked sarcoplasmic reticulum (539±77%), and was also similar in presence of the L-type Ca2+-channel antagonist verapamil. In sharp contrast, inhibition of the reverse-mode sodium-calcium exchange by KB-R7943 preserved diastolic function completely. Additional experiments were performed in rat myocardium; the rise in diastolic force was comparable to rabbit myocardium, but Ca2+ sensitivity was unchanged and maximal force development was reduced. This was associated with a degradation of TnI, but not TnT. Electron microscopic analysis revealed that OH did not cause irreversible membrane damage. We conclude that OH-induced acute diastolic dysfunction is caused by Ca2+ influx via reverse mode of the sodium-calcium exchanger. Degradation of troponins appears to be species-dependent but does not contribute to the acute diastolic dysfunction.

Levosimendan improves diastolic and systolic function in failing human myocardium

Ca(2+)-sensitizers increase myocardial contractility, but may worsen diastolic dysfunction. Levosimendan, through its unique troponin-C interaction, may preserve diastolic function. We investigated the effects of levosimendan (10(-7)-10(-5) M) on diastolic and systolic function in multicellular cardiac muscle preparations from end-stage failing human hearts (1 and 2.5 Hz, 37 degrees C, 1.25 mM [Ca(2+)], pH 7.4). Levosimendan improved systolic function: at 1 Hz, developed force (F(dev)) increased from 13.84+/-3.27 to 16.40+/-3.57 (10(-7) M, P<0.05), while diastolic force (F(dia)) decreased from 5.32+/-0.67 to 4.94+/-0.61 mN/mm(2) (P<0.05). Under control conditions, the increase in stimulation frequency from 1 to 2.5 Hz resulted in a decrease in F(dev) of -0.51+/-1.80 mN/mm(2) (negative force-frequency relationship). Levosimendan improved this relationship: at 10(-7) M, this change became positive (+1.81+/-2.06 mN/mm(2), P<0.05). Diastolic function was markedly improved in the presence of levosimendan; the increase in F(dia) of 1.56+/-0.42 mN/mm(2) (control) was attenuated to 0.70+/-0.19 mN/mm(2) (P<0.05). To allow for a more detailed analysis, preparations were sometimes divided into two groups, based on their force-frequency behavior. Twitch timing parameters were accelerated by levosimendan in preparations with a negative force-frequency relationship. Levosimendan improves both systolic and diastolic function in failing human myocardium. Effects are even more pronounced at higher heart rates and under prevailing diastolic dysfunction.

Transient and sustained impacts of hydroxyl radicals on sarcoplasmic reticulum function : protective effects of nebivolol

The hydroxyl radical (*OH) is a very reactive oxygen-free radical species that has profound effects on myocardial contractility. We investigated the impact of *OH on free radical induced injury in right ventricular rabbit cardiac trabeculae. Additionally, we investigated the protective properties of the beta-adrenoceptor antagonist nebivolol. The contractile response to a brief, 2 min exposure to *OH consisted of a severe but transient rigor-like contracture, followed by a new steady state in which diastolic force (Fdia) remained increased and developed force (Fdev) remained decreased. In the new steady state sarcoplasmic reticulum function only partly recovered, reflected by a > 50% blunted force-frequency relationship. In the presence of nebivolol (10(-6) M), during the early phase the increase in Fdia was significantly smaller, and recovered better while Fdev was higher during peak. Moreover, nebivolol completely abolished blunting of the force-frequency relationship, which was observed in the sustained *OH injury phase. The results indicate that hydroxyl radical injury induces systolic and diastolic dysfunction, and that nebivolol can effectively prevent a large part of this *OH injury.

Influence of cyclosporine A on contractile function, calcium handling, and energetics in isolated human and rabbit myocardium

OBJECTIVEnThe immunosuppressive drug Cyclosporine A (CsA) is a key substance in pharmacological therapy following solid organ transplantation and has been suggested to prevent cardiac hypertrophy. We investigated the direct effects of CsA on myocardial function, because these are largely unknown.nnnMETHODSnIn multicellular cardiac muscle preparations from end-stage failing and non-failing human hearts as well as from non-failing rabbit hearts we investigated the effects of CsA on contractile performance, sarcoplasmic reticulum (SR) Ca2+-load, cytosolic calcium transients, calcium sensitivity of the myofilaments, and myocardial oxygen consumption.nnnRESULTSnIn failing human muscle preparations there was a concentration dependent decrease in contractile force; the maximal effect amounted to 55.6+/-6.4% of control while EC50 was reached at 1.0+/-0.3 nM (n=6). These concentrations are at and even below the therapeutic plasma levels. CsA decreased the aequorin light signal in human failing trabeculae to 71.5+/-5.9% (n=5), indicating decreased calcium transients. Estimation of the SR calcium load via measurement of rapid cooling contractures revealed a decrease to 84.4+/-6.5% in failing human preparations (n=6). Measurements of both decreased SR calcium load and force development in presence of CsA were also observed in four non-failing human muscle preparations. In rabbit muscle preparations (n=8), developed force decreased to 50.2+/-7.7% (n=8, EC50: 1.9+/-0.4 nM) and rapid cooling contractures to 74.0+/-7.4% of control at 100 nmol/l CsA. No direct effects were observed on myofilament calcium sensitivity nor on maximal force development of permeabilized preparations from the rabbit (n=7). Oxygen consumption measurements showed that CsA decreased the economy of contraction to 76.4+/-7.9% in rabbit preparations (n=8).nnnCONCLUSIONSnCsA causes a direct cardio-depressive effect at clinically relevant concentrations, most likely due to altered handling of Ca2+ by the SR.

Potentiation of beta-adrenergic inotropic response by pyruvate in failing human myocardium

BACKGROUNDnPyruvate has been shown to increase contractile function in isolated myocardium and to improve hemodynamics in patients with congestive heart failure. We tested the hypothesis that pyruvate potentiates the inotropic response to beta-adrenergic stimulation and to elevated extracellular calcium, since this may be of potential therapeutic value in the clinical setting of acute heart failure in order to circumvent deleterious effects on energy demand as can occur during catecholamine therapy.nnnMETHODS AND RESULTSnWe investigated isometrically contracting isolated multicellular muscle preparations from terminal failing human hearts at 37 degrees C, pH 7.4, and a stimulation frequency of 1 Hz. At an extracellular calcium concentration of 1.25 mM, pyruvate (10 mM) alone increased developed force (F(dev)) from 9.0+/-2.3 to 21.1+/-4.3 mN/mm(2) (n=9, P<0.001) and isoproterenol (1 microM) alone increased F(dev) from 9.5+/-2.0 to 31.3+/-5.4 mN/mm(2) (P<0.001), whereas the combination of pyruvate and isoproterenol increased F(dev) over-proportionally from 9.0+/-2.3 to 47.4+/-6.4 mN/mm(2) (P<0.01). In a separate series we assessed the combination of pyruvate and calcium. Although F(dev) did not increase from 12 to 16 mM [Ca(2+)](o), 10 mM pyruvate further increased F(dev) from 25.8+/-5.0 to 30.6+/-4.7 mN/mm(2) (P<0.01). Rapid cooling contractures revealed that altered myofilament responsiveness and/or sarcoplasmic reticulum (SR) calcium load must underlie the positive inotropic effect of pyruvate.nnnCONCLUSIONnA combination of pyruvate and beta-adrenergic stimulation may be of therapeutic value in acute heart failure by reducing the concentrations of potential deleterious catecholamines that are currently necessary to maintain adequate tissue perfusion.

Intracellular β-blockade: overexpression of Gαi2 depresses the β-adrenergic response in intact myocardium

Objective: Increased levels of inhibitory G proteins have been observed in heart failure, but their physiological relevance in mediating the reduced β-adrenergic response is largely unknown. Methods: To evaluate the functional consequences of Gαi2 overexpression, we studied myocardial contraction in intact isometric contracting cardiac rabbit trabeculae and isolated myocytes after adenovirus-mediated gene transfer of Gαi2. Results: Neither Gαi2 nor lacZ (control) overexpression altered baseline contractile force. After 72 h of continuous contractions, developed force ( F dev) increased after addition of 1 μM isoproterenol by 28.5±9.7 mN/mm2 in the control group, which was unchanged from the initial response at t = 0 h (23.7±3.8 mN/mm2). In sharp contrast, in preparations transfected with AdGαi2, the response to isoproterenol was significantly attenuated (5.9±2.0 vs. 27.6±4.2 mN/mm2, t = 72 vs. 0 h, respectively, P <0.01). In a primary culture of transfected isolated myocytes from a nearly identical baseline, isoproterenol increased cell shortening by 3.1±0.6% in the lacZ transfected myocytes, but only by 1.3±0.5% in Gαi2 transfected myocytes ( t = 72 h, P <0.01). In Gαi2 transfected myocytes, pertussis toxin restored β-adrenergic responsiveness, indicating specificity of attenuation by the transgene. Conclusions: Overexpression of Gαi2 attenuates the positive inotropic effects of β-adrenergic stimulation in myocardium. In addition, the method we developed allows investigation of a causal link between altered protein expression and subsequent alterations in contractile function in a physiological relevant in vitro manner.

Protective role of nebivolol in hydroxyl radical induced injury

Summary: Increased oxidative stress has been postulated as one of the main mechanisms underlying stunned myocardium, and may play an important role in and during development of heart failure. Pharmacological interventions may attenuate or prevent detrimental effects of oxygen free radicals on the myocardium. Nebivolol has been shown to attenuate contractile dysfunction in hydroxyl radical mediated injury, but the mechanism(s) remain unknown. It was investigated whether nebivolol could partly attenuate the contractile dysfunction through a direct effect on the myofilaments. In demembranized muscles from explanted human hearts, nebivolol induced a slight desensitization of the myofilaments to calcium. Therefore, during the calcium overload that occurs during reperfusion after an ischemic event, the contractile dysfunction is less severe in the presence of nebivolol. We conclude that the protection of nebivolol in hydroxyl radical induced contractile dysfunction is mediated in part through a direct effect on the myofilaments, in addition to the previously shown preservation of sarcoplasmic reticulum function.

High intracellular Na+ preserves myocardial function at low heart rates in isolated myocardium from failing hearts.

We investigated the hypothesis that increased intracellular [Na+]i in heart failure contributes to preservation of SR Ca2+ load which may become particularly evident at slow heart rates.