Harald Kögler

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.

Calsarcin-2 deficiency increases exercise capacity in mice through calcineurin/NFAT activation

The composition of skeletal muscle, in terms of the relative number of slow- and fast-twitch fibers, is tightly regulated to enable an organism to respond and adapt to changing physical demands. The phosphatase calcineurin and its downstream targets, transcription factors of the nuclear factor of activated T cells (NFAT) family, play a critical role in this process by promoting the formation of slow-twitch, oxidative fibers. Calcineurin binds to calsarcins, a family of striated muscle-specific proteins of the sarcomeric Z-disc. We show here that mice deficient in calsarcin-2, which is expressed exclusively by fast-twitch muscle and encoded by the myozenin 1 (Myoz1) gene, have substantially reduced body weight and fast-twitch muscle mass in the absence of an overt myopathic phenotype. Additionally, Myoz1 KO mice displayed markedly improved performance and enhanced running distances in exercise studies. Analysis of fiber type composition of calsarcin-2-deficient skeletal muscles showed a switch toward slow-twitch, oxidative fibers. Reporter assays in cultured myoblasts indicated an inhibitory role for calsarcin-2 on calcineurin, and Myoz1 KO mice exhibited both an excess of NFAT activity and an increase in expression of regulator of calcineurin 1-4 (RCAN1-4), indicating enhanced calcineurin signaling in vivo. Taken together, these results suggest that calsarcin-2 modulates exercise performance in vivo through regulation of calcineurin/NFAT activity and subsequent alteration of the fiber type composition of skeletal muscle.

Insulin Causes [Ca2+]i-Dependent and [Ca2+]i-Independent Positive Inotropic Effects in Failing Human Myocardium

Background—Insulin has been shown to exert positive inotropic effects in several in vitro and in vivo models, but signal transduction and substrate dependency remain unclear. We examined inotropic responses and signal transduction mechanisms of insulin in human myocardium. Methods and Results—Experiments were performed in isolated trabeculae from end-stage failing hearts of 58 nondiabetic and 3 diabetic patients undergoing heart transplantation. The effect of insulin (0.3 and 3 IU/L) on isometric twitch force (37°C, 1 Hz) was tested in the presence of glucose or pyruvate as energetic substrate. Furthermore, intracellular Ca2+ transients (aequorin method), sarcoplasmic reticulum (SR) Ca2+ content (rapid cooling contractures), and myofilament Ca2+ sensitivity (semiskinned fibers) were assessed. In addition, potential signaling pathways were tested by blocking glycolysis, PI-3-kinase, protein kinase C, diacylglycerol kinase, insulin-like growth factor-1 receptors, or transsarcolemmal Ca2+ entry via the Na+/Ca2+ exchanger. Insulin exerted concentration-dependent and partially substrate-dependent positive inotropic effects. The phosphatidylinositol-3-kinase inhibitor wortmannin and the Na2+/Ca2+ exchanger reverse-mode inhibitor KB-R7943 completely or partially prevented the functional effects of insulin. In contrast, insulin-like growth factor-1 receptor blockade, protein kinase C inhibition, and diacylglycerol kinase blockade were without effect. The inotropic response was associated with increases in intracellular Ca2+ transients, SR Ca2+ content, and increased myofilament Ca2+ sensitivity. Conclusions—Insulin exerts Ca2+-dependent and -independent positive inotropic effects through a phosphatidylinositol-3-kinase–dependent pathway in failing human myocardium. The increased [Ca2+]i originates at least in part from enhanced reverse-mode Na+/Ca2+ exchange and consequently increased SR-Ca2+ load. These nongenomic functional effects of insulin may be of clinical relevance, eg, during insulin-glucose-potassium infusions.

Insulin-Like Growth Factor-1 Exerts Ca2+-Dependent Positive Inotropic Effects in Failing Human Myocardium

Abstract— Myocardial generation of insulin-like growth factor-1 (IGF-1) is altered in hypertrophy and heart failure, but there are no reports on acute functional effects of IGF-1 in human cardiac muscle. We examined inotropic responses and signal transduction mechanisms of IGF-1 in human myocardium. Experiments were performed in isolated trabeculae or cardiomyocytes from 46 end-stage failing hearts. The effect of IGF-1 (0.001 to 0.2 &mgr;mol/L) on isometric twitch force (37°C, 1 Hz), intracellular Ca2+ transients (aequorin method), sarcoplasmic reticulum (SR) Ca2+ content (rapid cooling contractures), L-type Ca2+ current (whole-cell voltage clamp), and cAMP concentrations was assessed. In addition, the effects of blocking IGF-1 receptors, phosphoinositide 3-kinase (PI3-kinase), protein kinase C (PKC), or transsarcolemmal Ca2+ entry were tested. IGF-1 exerted concentration-dependent positive inotropic effects (twitch force increased to maximally 133±4% of baseline values at 0.1 &mgr;mol/L;P <0.05). The IGF-1 receptor antibody &agr;IR3 or the PI3-kinase inhibitor wortmannin prevented the functional effects. The inotropic response was paralleled by increases in Ca2+ transients and SR Ca2+ content. IGF-1 (0.1 &mgr;mol/L) increased L-type Ca2+ current amplitude by 24±7% (P <0.05). Blockade of SR function did not affect the inotropic response to IGF-1. In contrast, L-type Ca2+ channel blockade with diltiazem partially prevented (≈50%) the inotropic response to IGF-1. Inhibition of PKC (GF109203X), Na+-H+ exchange (HOE642), or reverse-mode Na+-Ca2+ exchange (KB-R7943) reduced the response to IGF-1 by ≈60% to 70%. IGF-1 exerts Ca2+-dependent positive inotropic effects through activation of IGF-1 receptors and a PI3-kinase-dependent pathway in failing human myocardium. The increased [Ca2+]i with IGF-1 originates from both enhanced L-type Ca2+ currents and enhanced Na+-H+ exchange-dependent reverse-mode Na+-Ca2+ exchange. These nongenomic functional effects of IGF-1 may be of clinical relevance.

Effects of Adenovirus-Mediated Sorcin Overexpression on Excitation-Contraction Coupling in Isolated Rabbit Cardiomyocytes

&NA; To evaluate the effect of sorcin on cardiac excitation‐contraction coupling, adult rabbit ventricular myocytes were transfected with a recombinant adenovirus coding for human sorcin (Ad‐sorcin). A &bgr;‐galactosidase adenovirus (Ad‐LacZ) was used as a control. Fractional shortening in response to 1‐Hz field stimulation (at 37°C) was significantly reduced in Ad‐sorcin‐transfected myocytes compared with control myocytes (2.10±0.05% [n=311] versus 2.42±0.06% [n=312], respectively; P<0.001). Action potential duration (at 20°C) was significantly less in the Ad‐sorcin group (458±22 ms, n=11) compared with the control group (520±19 ms, n=10; P<0.05). In voltage‐clamped, fura 2‐loaded myocytes (20°C), a reduced peak‐systolic and end‐diastolic [Ca2+]i was observed after Ad‐sorcin transfection. L‐type Ca2+ current amplitude and time course were unaffected. Caffeine‐induced Ca2+ release from the sarcoplasmic reticulum (SR) and the accompanying inward Na+‐Ca2+ exchanger (NCX) current revealed a significantly lower SR Ca2+ content and faster Ca2+‐extrusion kinetics in Ad‐sorcin‐transfected cells. Higher NCX activity after Ad‐sorcin transfection was confirmed by measuring the NCX current‐voltage relationship. &bgr;‐Escin‐permeabilized rabbit cardiomyocytes were used to study the effects of sorcin overexpression on Ca2+ sparks imaged with fluo 3 at 145 to 160 nmol/L [Ca2+] using a confocal microscope. Under these conditions, caffeine‐mediated SR Ca2+ release was not different between the two groups. Spontaneous spark frequency, duration, width, and amplitude were lower in sorcin‐overexpressing myocytes. In summary, sorcin overexpression in rabbit cardiomyocytes decreased Ca2+‐transient amplitude predominately by lowering SR Ca2+ content via increased NCX activity. The effect of sorcin overexpression on Ca2+ sparks indicates an effect on the ryanodine receptor that may also influence excitation‐contraction coupling. (Circ Res. 2003;93:132‐139.)

Excessive Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPase Expression Causes Increased Sarcoplasmic Reticulum Ca2+ Uptake but Decreases Myocyte Shortening

Background—Increasing sarcoplasmic/endoplasmic reticulum (SR) Ca2+-ATPase (SERCA) uptake activity is a promising therapeutic approach for heart failure. We investigated the effects of different levels of SERCA1a expression on contractility and Ca2+ cycling. We tested whether increased SERCA1a expression levels enhance myocyte contractility in a gene-dose–dependent manner. Methods and Results—Rabbit isolated cardiomyocytes were transfected at different multiplicities of infection (MOIs) with adenoviruses encoding SERCA1a (or &bgr;-galactosidase as control). Myocyte relaxation half-time was decreased by 10% (P=0.052) at SERCA1a MOI 10 and by 28% at MOI 50 (P<0.05). Myocyte fractional shortening was increased by 12% at MOI 10 (P<0.05) but surprisingly decreased at MOI 50 (−22%, P<0.05) versus control. SR Ca2+ uptake (in permeabilized myocytes) demonstrated a gene-dose–dependent decrease in Km by 29% and 46% and an increase in Vmax by 37% and 72% at MOI 10 and MOI 50, respectively (all P<0.05 versus control). Ca2+ transient amplitude was increased in Ad-SERCA1a–infected myocytes at MOI 10 (by 121%, P<0.05), but at MOI 50, the Ca2+ transient amplitude was not significantly changed. Caffeine-induced Ca2+ transients indicated significantly increased SR Ca2+ content in Ad-SERCA1a–infected cells, by 72% at MOI 10 and by 87% at MOI 50. Mathematical simulations demonstrate that the functional increase in SR Ca2+-ATPase uptake activity at MOI 50 (and increased cytosolic Ca2+ buffering) is sufficient to curtail the Ca2+ transient amplitude and explain the reduced contraction. Conclusions—Moderate SERCA1a gene transfer and expression improve contractility and Ca2+ cycling. However, higher SERCA1a expression levels can impair myocyte shortening because of higher SERCA activity and Ca2+ buffering.

Relevance of brain natriuretic peptide in preload-dependent regulation of cardiac sarcoplasmic reticulum Ca2+ ATPase expression.

Background— In heart failure (HF), ventricular myocardium expresses brain natriuretic peptide (BNP). Despite the association of elevated serum levels with poor prognosis, BNP release is considered beneficial because of its antihypertrophic, vasodilating, and diuretic properties. However, there is evidence that BNP-mediated signaling may adversely influence cardiac remodeling, with further impairment of calcium homeostasis. Methods and Results— We studied the effects of BNP on preload-dependent myocardial sarcoplasmic reticulum Ca2+ ATPase (SERCA2a) expression. In rabbit isolated muscle strips stretched to high preload and shortening isotonically over 6 hours, the SERCA/glyceraldehyde phosphate dehydrogenase mRNA ratio was enhanced by 168% (n=8) compared with unloaded preparations (n=8; P<0.001). Recombinant human BNP at a concentration typically found in end-stage HF patients (350 pg/mL) abolished SERCA upregulation by stretch (n=9; P<0.0001 versus BNP free). Inhibition of cyclic guanosine 3′,5′ monophosphate (cGMP)–phosphodiesterase-5 mimicked this effect, whereas inhibition of cGMP-dependent protein kinase restored preload-dependent SERCA upregulation in the presence of recombinant human BNP. Furthermore, in myocardium from human end-stage HF patients undergoing cardiac transplantation (n=15), BNP expression was inversely correlated with SERCA levels. Moreover, among 23 patients treated with left ventricular assist devices, significant SERCA2a recovery occurred in those downregulating BNP. Conclusions— Our data indicate that preload stimulates SERCA expression. BNP antagonizes this mechanism via guanylyl cyclase-A, cGMP, and cGMP-dependent protein kinase. This novel action of BNP to uncouple preload-dependent SERCA expression may adversely affect contractility in patients with HF.

Exercise intolerance and systemic manifestations of pulmonary emphysema in a mouse model

BackgroundSystemic effects of chronic obstructive pulmonary disease (COPD) significantly contribute to severity and mortality of the disease. We aimed to develop a COPD/emphysema model exhibiting systemic manifestations of the disease.MethodsFemale NMRI mice were treated 5 times intratracheally with porcine pancreatic elastase (emphysema) or phosphate-buffered saline (control). Emphysema severity was quantified histologically by mean linear intercept, exercise tolerance by treadmill running distance, diaphragm dysfunction using isolated muscle strips, pulmonary hypertension by measuring right ventricular pressure, and neurohumoral activation by determining urinary norepinephrine concentration.ResultsMean linear intercept was higher in emphysema (260.7 ± 26.8 μm) than in control lungs (24.7 ± 1.7 μm). Emphysema mice lost body weight, controls gained weight. Running distance was shorter in emphysema than in controls. Diaphragm muscle length was shorter in controls compared to emphysema. Fatigue tests of muscle strips revealed impaired relaxation in emphysema diaphragms. Maximum right ventricular pressure and norepinephrine were elevated in emphysema compared to controls. Linear correlations were observed between running distance changes and intercept, right ventricular weight, norepinephrine, and diaphragm length.ConclusionThe elastase mouse model exhibited severe emphysema with consecutive exercise limitation, and neurohumoral activation. The model may deepen our understanding of systemic aspects of COPD.

Negative Inotropy of the Gastric Proton Pump Inhibitor Pantoprazole in Myocardium From Humans and Rabbits Evaluation of Mechanisms

Background— Proton pump inhibitors are used extensively for acid-related gastrointestinal diseases. Their effect on cardiac contractility has not been assessed directly. Methods and Results— Under physiological conditions (37°C, pH 7.35, 1.25 mmol/L Ca2+), there was a dose-dependent decrease in contractile force in ventricular trabeculae isolated from end-stage failing human hearts superfused with pantoprazole. The concentration leading to 50% maximal response was 17.3±1.3 &mgr;g/mL. Similar observations were made in trabeculae from human atria, normal rabbit ventricles, and isolated rabbit ventricular myocytes. Real-time polymerase chain reaction demonstrated the expression of gastric H+/K+–adenosine triphosphatase in human and rabbit myocardium. However, measurements with BCECF-loaded rabbit trabeculae did not reveal any significant pantoprazole-dependent changes of pHi. Ca2+ transients recorded from field-stimulated fluo 3–loaded myocytes (F/F0) were significantly depressed by 10.4±2.1% at 40 &mgr;g/mL. Intracellular Ca2+ fluxes were assessed in fura 2–loaded, voltage-clamped rabbit ventricular myocytes. Pantoprazole (40 &mgr;g/mL) caused an increase in diastolic [Ca2+]i by 33±12%, but peak systolic [Ca2+]i was unchanged, resulting in a decreased Ca2+ transient amplitude by 25±8%. The amplitude of the L-type Ca2+ current (ICa,L) was reduced by 35±5%, and sarcoplasmic reticulum Ca2+ content was reduced by 18±6%. Measurements of oxalate-supported sarcoplasmic reticulum Ca2+ uptake in permeabilized cardiomyocytes indicated that pantoprazole decreased Ca2+ sensitivity (Kd) of sarcoplasmic reticulum Ca2+ adenosine triphosphatase: control, Kd=358±15 nmol/L; 40 &mgr;g/mL pantoprazole, Kd=395±12 nmol/L (P<0.05). Pantoprazole also acted on cardiac myofilaments to reduced Ca2+-activated force. Conclusions— Pantoprazole depresses cardiac contractility in vitro by depression of Ca2+ signaling and myofilament activity. In view of the extensive use of this agent, the effects should be evaluated in vivo.

Improved systolic and diastolic myocardial function with intracoronary pyruvate in patients with congestive heart failure

Pyruvate increases myocardial performance in isolated myocardium and improves hemodynamics in patients with congestive heart failure.