The preclinical and phase 1 development of the novel oral cathepsin C inhibitor BI 1291583.

Preclinical and phase 1 study results indicate that BI 1291583 is a reversible, highly potent and highly selective CatC inhibitor that markedly inhibits active NSP production in a dose-dependent manner, supporting phase 2 trials in bronchiectasis patients https://bit.ly/47PZ8E5.

Novel cathepsin C inhibitor, BI 1291583, intended for treatment of bronchiectasis: Phase I characterization in healthy volunteers.

Novel treatments are needed to reduce inflammation, improve symptoms, address exacerbations, and slow disease progression in bronchiectasis. Cathepsin C (CatC) inhibition promises to achieve this through reduction of neutrophil-derived serine protease (including neutrophil elastase [NE] and proteinase 3 [PR3]) activation. Here, we present the phase I characterization of the novel CatC inhibitor, BI 1291583. Five phase I trials of BI 1291583 in healthy subjects are presented: a single-rising-dose study (NCT03414008) and two multiple-rising-dose studies (NCT03868540 and NCT04866160) assessing the safety, tolerability, pharmacodynamics, and pharmacokinetics of BI 1291583; a food effect study (NCT03837964); and a drug-drug interaction study (NCT03890887) of BI 1291583 and itraconazole. BI 1291583 was safe and well tolerated across the doses tested in these trials. Most adverse events (AEs) were mild or moderate in intensity, with no serious AEs, AEs of special interest or deaths reported in any trial. Drug-related skin exfoliation was not reported more frequently in subjects treated with BI 1291583 compared with placebo. BI 1291583 was readily absorbed, and pharmacokinetics were supra-proportional over the dose ranges assessed. Additionally, BI 1291583 inhibited CatC in a dose-dependent manner, inhibited downstream NE activity, and decreased PR3 levels. No food effect was observed. Co-administration of multiple doses of itraconazole increased BI 1291583 exposure approximately twofold. Due to these promising phase I results, a multinational phase II program of BI 1291583 in adults with bronchiectasis is ongoing (Airleaf™ [NCT05238675], Clairafly™ [NCT05865886], and Clairleaf™ [NCT05846230]).

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.

Local and systemic effects of angiotensin receptor blockade in an emphysema mouse model.

OBJECTIVES: COPD with emphysema causes marked neurohumoral activation. Angiotensin II receptors are highly expressed within the lung and interfere with mechanisms involved in the progression of emphysema. This study examined the effects of an angiotensin II receptor blocker (ARB) on pulmonary and systemic manifestations of emphysema in a mouse model. METHODS: Female NMRI mice received five intratracheal instillations of porcine pancreatic elastase (emphysema; n = 11) or phosphate-buffered saline (PBS; n = 4). Emphysema severity was quantified histologically by mean linear intercept, exercise tolerance by treadmill running distance, and lung biomechanics by compliance. Following emphysema induction, 6 mice were treated with the ARB irbesartan for 8 weeks, while 5 mice receiving standard food served as controls. RESULTS: Following emphysema induction, mean linear intercept was higher in elastase-treated than in PBS-treated lungs (103.0 ± 6.2 µm vs. 35.0 ± 0.6 µm; p = 0.043) while running distance was shorter in emphysema mice (418.6 ± 83.5 m vs. 906.6 ± 244.6 m, p = 0.028). Irbesartan-treated emphysema mice showed a lower mean linear intercept (90.8 ± 3.8 µm vs. 121.5 ± 8.1 µm; p = 0.005), improved compliance (163.6 ± 55.9 µl/cmH(2)O vs. 354.4 ± 72.5 µl/cmH(2)O; p = 0.063) and greater running distance (p ANOVA = 0.015) compared to emphysema mice receiving standard food. CONCLUSIONS: The ARB irbesartan elicits encouraging beneficial effects on emphysema severity, lung biomechanics and exercise capacity in an emphysema mouse model. These findings might help to understand the corresponding positive effects of angiotensin II receptor blockade noticed in patients with COPD.

Impaired Ca(2+)-handling in HIF-1alpha(+/-) mice as a consequence of pressure overload.

The hypoxia-inducible factor (HIF)-1 is critically involved in the cellular adaptation to a decrease in oxygen availability. The influence of HIF-1alpha for the development of cardiac hypertrophy and cardiac function that occurs in response to sustained pressure overload has been mainly attributed to a challenged cardiac angiogenesis and cardiac hypertrophy up to now. Hif-1alpha (+/+) and Hif-1alpha (+/-) mice were studied regarding left ventricular hypertrophy and cardiac function after being subjected to transverse aortic constriction (TAC). After TAC, both Hif-1alpha (+/+) and Hif-1alpha (+/-) mice developed left ventricular hypertrophy with increased posterior wall thickness, septum thickness and increased left ventricular weight to a similar extent. No significant difference in cardiac vessel density was observed between Hif-1alpha (+/+) and Hif-1alpha (+/-) mice. However, only the Hif-1alpha (+/-) mice developed severe heart failure as revealed by a significantly reduced fractional shortening mostly due to increased end-systolic left ventricular diameter. On the single cell level this correlated with reduced myocyte shortenings, decreased intracellular Ca(2+)-transients and SR-Ca(2+) content in myocytes of Hif-1a (+/-) mice. Thus, HIF-1alpha can be critically involved in the preservation of cardiac function after chronic pressure overload without affecting cardiac hypertrophy. This effect is mediated via HIF-dependent modulation of cardiac calcium handling and contractility.

BNP controls early load-dependent regulation of SERCA through calcineurin.

Heart failure is characterised by reduced expression of sarcoplasmic reticulum calcium-ATPase (SERCA) and increased expression of B-type natriuretic peptide (BNP). The present study was performed to investigate causality of this inverse relationship under in vivo conditions in the transversal aortic constriction mouse model (TAC). Left ventricular SERCA-mRNA expression was significantly upregulated in TAC by 32% after 6 h, but not different from sham after 24 h. Serum proANP and BNP levels were increased in TAC after 24 h (BNP +274%, p < 0.01; proANP +60%, p < 0.05), but only proANP levels were increased after 6 h (+182%, p < 0.01). cGMP levels were only increased 24 h after TAC (+307%, p < 0.01), but not 6 h after TAC. BNP infusion inhibited the increase in SERCA expression 6 h after TAC. In BNP-receptor-knockout animals (GC-A), the expression of SERCA was still significantly increased 24 h after TAC at the mRNA level by 35% (p < 0.05), as well as at the protein level by 25% (p < 0.05). MCIP expression as an indicator of calcineurin activity was regulated in parallel to SERCA after 6 and 24 h. MCIP-mRNA was increased by 333% 6 h after TAC, but not significantly different from sham after 24 h. In the GC-A-KO mice, MCIP-mRNA was significantly increased in TAC compared to WT after 24 h. In mice with BNP infusion, MCIP was significantly lower 6 h after TAC compared to control animals. In conclusion, mechanical load leads to an upregulation of SERCA expression. This is followed by upregulation of natriuretic peptides with subsequent suppression of SERCA upregulation. Elevated natriuretic peptides may suppress SERCA expression by inhibition of calcineurin activity via activation of GC-A.

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

BACKGROUND: Systemic 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. METHODS: Female 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. RESULTS: Mean linear intercept was higher in emphysema (260.7 +/- 26.8 microm) than in control lungs (24.7 +/- 1.7 microm). 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. CONCLUSION: The elastase mouse model exhibited severe emphysema with consecutive exercise limitation, and neurohumoral activation. The model may deepen our understanding of systemic aspects of COPD.

Overexpression of FK-506 binding protein 12.0 modulates excitation contraction coupling in adult rabbit ventricular cardiomyocytes.

The effect of the 12-kDa isoform of FK-506-binding protein (FKBP)12.0 on cardiac excitation-contraction coupling was studied in adult rabbit ventricular myocytes after transfection with a recombinant adenovirus coding for human FKBP12.0 (Ad-FKBP12.0). Western blots confirmed overexpression (by 2.6+/-0.4 fold, n=5). FKBP12.0 association with rabbit cardiac ryanodine receptor (RyR2) was not detected by immunoprecipitation. However, glutathione S-transferase pull-down experiments indicated FKBP12.0-RyR2 binding to proteins isolated from human and rabbit but not dog myocardium. Voltage-clamp experiments indicated no effects of FKBP12.0 overexpression on L-type Ca2+ current (I(Ca,L)) or Ca2+ efflux rates via the Na+/Ca2+ exchanger. Ca2+ transient amplitude was also not significantly different. However, sarcoplasmic reticulum Ca2+ load was approximately 25% higher in myocytes in the Ad-FKBP12.0 group. The reduced ability of I(Ca,L) to initiate sarcoplasmic reticulum Ca2+ release was observed over a range of values of sarcoplasmic reticulum Ca2+ content, indicating that overexpression of FKBP12.0 reduces the sensitivity of RyR2 to Ca2+. Ca2+ spark morphology was measured in beta-escin-permeabilized cardiomyocytes. Ca2+ spark amplitude and duration were significantly increased, whereas frequency was decreased in cells overexpressing FKBP12.0. These changes were accompanied by an increased sarcoplasmic reticulum Ca2+ content. In summary, the effects of FKBP12.0 overexpression on intact and permeabilized cells were similar to those of tetracaine, a drug known to reduce RyR2 Ca2+ sensitivity and distinctly different from the effects of overexpression of the FKBP12.6 isomer. In conclusion, FKBP12.0-RyR2 interaction can regulate the gain of excitation-contraction coupling.

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 degrees 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 microg/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 pH(i). Ca2+ transients recorded from field-stimulated fluo 3-loaded myocytes (F/F0) were significantly depressed by 10.4+/-2.1% at 40 microg/mL. Intracellular Ca2+ fluxes were assessed in fura 2-loaded, voltage-clamped rabbit ventricular myocytes. Pantoprazole (40 microg/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 (I(Ca,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 microg/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.

Celecoxib modulates hypertrophic signalling and prevents load-induced cardiac dysfunction.

In human hearts, the transition from cardiac hypertrophy to advanced heart failure (HF) is accompanied by a tremendous increase in Akt phosphorylation. In non-myocardial tissue, the cyclooxygenase (COX)-2 inhibitor celecoxib has been shown to COX-independently inhibit Akt signalling. We studied the effects of celecoxib on Akt signalling and hypertrophic response in myocardium. In rabbit isolated cardiac myocytes celecoxib concentration-dependently (10-100 micromol/L) inhibited the insulin-induced increase in phosphorylation of Akt and its downstream targets, GSK-3beta and p70 S6 kinase, by reducing the phosphorylation level of the upstream regulator PTEN. Inhibition of Akt signalling was accompanied by a significant suppression of characteristic features of cardiac hypertrophy: Celecoxib concentration-dependently suppressed the agonist-induced enhancement of total protein synthesis and BNP mRNA expression. In mice (C57BL/6NCrl) subjected to left ventricular (LV) pressure overload by aortic banding, celecoxib treatment (50mg x kg-1 x d-1) significantly attenuated LV dilation and contractile dysfunction compared with placebo-treated mice. Moreover, celecoxib significantly reduced mortality 8 weeks after banding. Thus, celecoxib can be used to titrate Akt signalling and hypertrophic response in myocardium. It reduces load-induced LV dilation, contractile dysfunction and mortality in vivo. This may have clinical implications for the prevention and treatment of maladaptive hypertrophy and its progression to HF in humans.

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.

Alpha1-adrenergic stress induces downregulation of Na+/Ca2+ exchanger in myocardial preparations from rabbits at physiological preload.

Alpha1-adrenergic stimulation and mechanical load are considered crucial for the expression of sarcolemmal Na+/Ca2+ exchanger (NCX1). However, the interaction between these processes is unknown. We investigated electrically stimulated (1 Hz, 1.75 mmol/L Ca2+) rabbit ventricular trabeculae at physiological preload under stimulation by the selective alpha1-agonist phenylephrine (PE, 10 micromol/L). Using quantitative real-time PCR, downregulation of mRNA to 76.5% (p<0.05) was found, while B-type natriuretic peptide (BNP) was increased to 569.5% (p<0.05) compared to control. These changes were abolished in the presence of both the alpha1-blocker prazosin (13 micromol/L) and the PKC inhibitor GF109203X (1 micromol/L). Furthermore, no changes in NCX mRNA levels under the influence of PE were found in unstretched trabeculae or in unstretched isolated rabbit myocytes (24 h), while BNP was increased in both preparations. In addition, since the alpha1-adrenergic effect could be Ca2+-dependent we tested increased extracellular Ca2+ (3.0 mmol/L) in stretched trabeculae and found downregulation of NCX1 to 75.2% (p<0.05). alpha1-stimulation decreases NCX1 mRNA in rabbit myocardium via PKC. This is critically load-dependent and may be mediated by changes in [Ca2+]. In hypertrophy and heart failure, distinct phenotypes with respect to NCX1 expression may result from the interaction between mechanical load and alpha1-adrenergic stimulation.

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 degrees 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.

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 micromol/L) on isometric twitch force (37 degrees 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 micromol/L; P<0.05). The IGF-1 receptor antibody alphaIR3 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 micromol/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 ( approximately 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 approximately 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.

Mechanical load-dependent regulation of gene expression in monocrotaline-induced right ventricular hypertrophy in the rat.

Rats treated with monocrotaline (MCT) develop pulmonary hypertension. Their right ventricles (RVs) exhibit severe pressure overload-induced hypertrophy, whereas the left ventricles (LVs) are normally loaded. In contrast, enhanced neuroendocrine stimulation during the transition to heart failure affects both ventricles. We assessed gene expression levels of Ca2+-regulating proteins in RVs and LVs of control and MCT rats in transition to heart failure to identify biomechanical load-regulated genes. In MCT RVs, both mRNA and protein levels of the Ca2+-ATPase of the sarcoplasmic/endoplasmic reticulum (SERCA2a) were reduced by 36% (P=0.001) and 17% (P=0.016), respectively, compared with control RVs. Phospholamban and ryanodine receptor mRNA levels likewise were reduced (by 27% [P=0.05] and 21% [P=0.011], respectively) in MCT RVs, whereas sarcolemmal Na+-Ca2+ exchanger expression was not altered. MCT LVs exhibited no significant expression changes compared with control LVs. Isometrically contracting MCT intact RV trabeculae showed enhanced baseline force development. Although control RV preparations exhibited a positive force-frequency relationship, MCT RVs showed a negative force-frequency relationship and blunted postrest potentiation. Contractile function of MCT LV trabeculae was normal. Maximum Ca2+-activated tension was enhanced by 64% in permeabilized RV MCT preparations (P=0.013). beta-Myosin heavy chain protein was upregulated in MCT RVs (P<0.001) but unaltered in MCT LVs. Degradation of troponin T was prominent in MCT RVs, a phenomenon not observed in the LV. Enhanced biomechanical load is necessary to induce the gene expression changes associated with the hypertrophic phenotype of the pressure-overloaded RV. Neuroendocrine factors, which equally affect both chambers, are not sufficient to alter the expression of Ca2+-cycling proteins.

Hydroxyl radical-induced acute diastolic dysfunction is due to calcium overload via reverse-mode Na(+)-Ca(2+) 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 Ca(2+) 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 (F(dia)) by 540%. Skinned fiber experiments revealed that OH-exposed preparations were sensitized for Ca(2+) (EC(50): 3.27+/-0.24 x 10(-6) versus 2.69+/-0.15 x 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 Ca(2+) 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 Ca(2+)-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 Ca(2+) 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 Ca(2+) 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.

Effects of adenovirus-mediated sorcin overexpression on excitation-contraction coupling in isolated rabbit cardiomyocytes.

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 beta-galactosidase adenovirus (Ad-LacZ) was used as a control. Fractional shortening in response to 1-Hz field stimulation (at 37 degrees 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 degrees 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 degrees 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. beta-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 predominantly 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.

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 beta-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 K(m) 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 Ca(2+) cycling. However, higher SERCA1a expression levels can impair myocyte shortening because of higher SERCA activity and Ca2+ buffering.

Disproportionate enhancement of myocardial contractility by the xanthine oxidase inhibitor oxypurinol in failing rat myocardium.

OBJECTIVE: Xanthine oxidase (XO) inhibitors enhance myofilament Ca(2+) responsiveness of normal rat myocardium. We examined whether this inotropic action is preserved or magnified in failing rat myocardium and whether the magnitude of this effect correlates with tissue xanthine-oxidoreductase (XOR) activity. METHODS: Hearts of 18-20 month-old SHHF (spontaneous hypertensive/heart failure) rats with end-stage heart failure, as well as of normal control rats, were perfused with the XO inhibitor oxypurinol. Afterwards, [Ca(2+)](i) and tension were measured simultaneously in fura-2-loaded intact isolated right ventricular trabeculae. XOR activity was determined fluorometrically in myocardial homogenates. RESULTS: In failing myocardium, 100 microM oxypurinol significantly increased systolic twitch tension (by 87 and 92% at 1.0 and 1.5 mM extracellular [Ca(2+)], respectively), without altering [Ca(2+)](i) transient amplitude. Oxypurinol did not alter the midpoint or cooperativity of the steady-state tension-[Ca(2+)](i) relationship, but significantly enhanced maximum Ca(2+)-activated tension by 75% in failing myocardium. Oxypurinol also exerted a positive inotropic effect in failing myocardium, which was, however, of significantly smaller relative magnitude. Failing rat myocardium exhibited higher XOR activity than nonfailing myocardium, and this activity was largely suppressed in oxypurinol-treated preparations. CONCLUSIONS: The magnitude of functional improvement with XOR inhibitors depends on the initial level of XOR activity. Specifically, the inotropic actions of oxypurinol are more pronounced in failing rat myocardium, a tissue that exhibits enhanced XOR activity. Our findings rationalize how XO inhibitors boost cardiac contractility and improve mechanoenergetic coupling, and why the effects might be relatively 'selective' for heart failure.

Na(+)-Ca(2+) exchanger overexpression predisposes to reactive oxygen species-induced injury.

OBJECTIVE: In heart failure (HF), the generation of reactive oxygen species (ROS) is enhanced. It was shown that failing cardiac myocytes are more susceptible to ROS-induced damage, possibly due to increased expression of the sarcolemmal Na-Ca exchanger (NCX). METHODS: We investigated the consequences of increased expression levels of NCX in adult rabbit ventricular cardiomyocytes (via adenovirus-mediated gene transfer, Ad-NCX1-GFP) with respect to tolerance towards ROS. After 48-h incubation, cells were monitored for morphological changes on an inverted microscope. ROS were generated via hydrogen peroxide (H(2)O(2)) (100 micromol/l) and Fe(3+)/nitrilotriacetate (Fe(3+)/NTA, 100/200 micromol/l) for 4 min and cell morphology was followed over 30 min. [Na(+)](i) and [Ca(2+)](i) in native cells were measured using SBFI-AM and Indo1-AM, respectively. RESULTS: In native myocytes, exposure to ROS induced hypercontracture. This was accompanied by a 1.3-fold increase in diastolic Indo1 fluorescence ratio (P<0.05). Overexpression of NCX significantly enhanced development of hypercontracture. After 15 min, the percentage of cells that had undergone hypercontracture (F(hyper)) was 85+/-4% vs. only 44+/-10% in control cells (P<0.05). Inhibition of NCX-mediated Ca(2+) entry with KB-R7943 (5 micromol/l) reduced F(hyper) to 33+/-11% (P<0.05). [Na(+)](i) was increased 2.9-fold 1 min prior to hypercontracture (P<0.05). CONCLUSIONS: ROS-induced hypercontracture is due to Ca(2+) entry via NCX which could be triggered by a concomitant substantial increase in [Na(+)](i). Elevated NCX levels predispose to ROS-induced injury, a mechanism likely contributing to myocyte dysfunction and death in heart failure.