Paul M. L. Janssen

Accumulation of autophagic vacuoles and cardiomyopathy in LAMP-2-deficientmice

Lysosome-associated membrane protein-2 (LAMP-2) is a highly glycosylated protein and an important constituent of the lysosomal membrane. Here we show that LAMP-2 deficiency in mice increases mortality between 20 and 40 days of age. The surviving mice are fertile and have an almost normal life span. Ultrastructurally, there is extensive accumulation of autophagic vacuoles in many tissues including liver, pancreas, spleen, kidney and skeletal and heart muscle. In hepatocytes, the autophagic degradation of long-lived proteins is severely impaired. Cardiac myocytes are ultrastructurally abnormal and heart contractility is severely reduced. These findings indicate that LAMP-2 is critical for autophagy. This theory is further substantiated by the finding that human LAMP-2 deficiency causing Danons disease is associated with the accumulation of autophagic material in striated myocytes.

Interplay of IKK/NF-κB signaling in macrophages and myofibers promotes muscle degeneration in Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a lethal X-linked disorder associated with dystrophin deficiency that results in chronic inflammation and severe skeletal muscle degeneration. In DMD mouse models and patients, we find that IkappaB kinase/NF-kappaB (IKK/NF-kappaB) signaling is persistently elevated in immune cells and regenerative muscle fibers. Ablation of 1 allele of the p65 subunit of NF-kappaB was sufficient to improve pathology in mdx mice, a model of DMD. In addition, conditional deletion of IKKbeta in mdx mice elucidated that NF-kappaB functions in activated macrophages to promote inflammation and muscle necrosis and in skeletal muscle fibers to limit regeneration through the inhibition of muscle progenitor cells. Furthermore, specific pharmacological inhibition of IKK resulted in improved pathology and muscle function in mdx mice. Collectively, these results underscore the critical role of NF-kappaB in the progression of muscular dystrophy and suggest the IKK/NF-kappaB signaling pathway as a potential therapeutic target for DMD.

Cardiac adenoviral S100A1 gene delivery rescues failing myocardium

Cardiac-restricted overexpression of the Ca2+-binding protein S100A1 has been shown to lead to increased myocardial contractile performance in vitro and in vivo. Since decreased cardiac expression of S100A1 is a characteristic of heart failure, we tested the hypothesis that S100A1 gene transfer could restore contractile function of failing myocardium. Adenoviral S100A1 gene delivery normalized S100A1 protein expression in a postinfarction rat heart failure model and reversed contractile dysfunction of failing myocardium in vivo and in vitro. S100A1 gene transfer to failing cardiomyocytes restored diminished intracellular Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load mechanistically due to increased SR Ca2+ uptake and reduced SR Ca2+ leak. Moreover, S100A1 gene transfer decreased elevated intracellular Na+ concentrations to levels detected in nonfailing cardiomyocytes, reversed reactivated fetal gene expression, and restored energy supply in failing cardiomyocytes. Intracoronary adenovirus-mediated S100A1 gene delivery in vivo to the postinfarcted failing rat heart normalized myocardial contractile function and Ca2+ handling, which provided support in a physiological context for results found in myocytes. Thus, the present study demonstrates that restoration of S100A1 protein levels in failing myocardium by gene transfer may be a novel therapeutic strategy for the treatment of heart failure.

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.

S100A1: a regulator of myocardial contractility.

S100A1, a Ca2+ binding protein of the EF-hand type, is preferentially expressed in myocardial tissue and has been found to colocalize with the sarcoplasmic reticulum (SR) and the contractile filaments in cardiac tissue. Because S100A1 is known to modulate SR Ca2+ handling in skeletal muscle, we sought to investigate the specific role of S100A1 in the regulation of myocardial contractility. To address this issue, we investigated contractile properties of adult cardiomyocytes as well as of engineered heart tissue after S100A1 adenoviral gene transfer. S100A1 gene transfer resulted in a significant increase of unloaded shortening and isometric contraction in isolated cardiomyocytes and engineered heart tissues, respectively. Analysis of intracellular Ca2+ cycling in S100A1-overexpressing cardiomyocytes revealed a significant increase in cytosolic Ca2+ transients, whereas in functional studies on saponin-permeabilized adult cardiomyocytes, the addition of S100A1 protein significantly enhanced SR Ca2+ uptake. Moreover, in Triton-skinned ventricular trabeculae, S100A1 protein significantly decreased myofibrillar Ca2+ sensitivity ([EC50%]) and Ca2+ cooperativity, whereas maximal isometric force remained unchanged. Our data suggest that S100A1 effects are cAMP independent because cellular cAMP levels and protein kinase A-dependent phosphorylation of phospholamban were not altered, and carbachol failed to suppress S100A1 actions. These results show that S100A1 overexpression enhances cardiac contractile performance and establish the concept of S100A1 as a regulator of myocardial contractility. S100A1 thus improves cardiac contractile performance both by regulating SR Ca2+ handling and myofibrillar Ca2+ responsiveness.

Bleeding and stent thrombosis on P2Y12-inhibitors: collaborative analysis on the role of platelet reactivity for risk stratification after percutaneous coronary intervention

AIMS Although platelet reactivity during P2Y12-inhibitors is associated with stent thrombosis (ST) and bleeding, standardized and clinically validated thresholds for accurate risk stratification after percutaneous coronary intervention (PCI) are lacking. We sought to determine the prognostic value of low platelet reactivity (LPR), optimal platelet reactivity (OPR), or high platelet reactivity (HPR) by applying uniform cut-off values for standardized devices. METHODS AND RESULTS Authors of studies published before January 2015, reporting associations between platelet reactivity, ST, and major bleeding were contacted for a collaborative analysis using consensus-defined, uniform cut-offs for standardized platelet function assays. Based on best available evidence for each device (exploratory studies), LPR-OPR-HPR categories were defined as <95, 95-208, and >208 PRU for VerifyNow, <19, 19-46, and >46 U for the Multiplate analyser and <16, 16-50, and >50% for VASP assay. Seventeen studies including 20 839 patients were used for the analysis; 97% were treated with clopidogrel and 3% with prasugrel. Patients with HPR had significantly higher risk for ST [risk ratio (RR) and 95% CI: 2.73 (2.03-3.69), P < 0.00001], yet a slight reduction in bleeding [RR: 0.84 (0.71-0.99), P = 0.04] compared with those with OPR. In contrast, patients with LPR had a higher risk for bleeding [RR: 1.74 (1.47-2.06), P < 0.00001], without any further benefit in ST [RR: 1.06 (0.68-1.65), P = 0.78] in contrast to OPR. Mortality was significantly higher in patients with HPR compared with other categories (P < 0.05). Validation cohorts (n = 14) confirmed all results of exploratory studies (n = 3). CONCLUSIONS Platelet reactivity assessment during thienopyridine-type P2Y12-inhibitors identifies PCI-treated patients at higher risk for mortality and ST (HPR) or at an elevated risk for bleeding (LPR).

The effect of myosin light chain 2 dephosphorylation on Ca2+-sensitivity of force is enhanced in failing human hearts

OBJECTIVE Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1+/-2.0%) compared to donor (31.9+/-4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. METHODS Cooperative effects in isometric force and rate of force redevelopment (K(tr)) were studied in single Triton-skinned human cardiomyocytes at various [Ca(2+)] before and after protein phosphatase-1 (PP-1) incubation. RESULTS Maximum force and K(tr) values did not differ between failing and donor hearts, but Ca(2+)-sensitivity of force (pCa(50)) was significantly higher in failing myocardium (Deltap Ca(50)=0.17). K(tr) decreased with decreasing [Ca(2+)], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa(50) to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute K(tr) values was found after PP-1 in failing and donor myocytes, while the shape of the K(tr)-Ca(2+) relationships remained unaltered. CONCLUSIONS Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca(2+)-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca(2+)-sensitivity and impaired Ca(2+)-handling on diastolic function in human heart failure.

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.

Follistatin Gene Delivery Enhances Muscle Growth and Strength in Nonhuman Primates

A vector delivered into muscles of monkeys generates a natural regulatory molecule, which increases muscle size and strength and may be useful therapeutically. Beyond Mighty Mouse: Building Muscle Mass Strength in Monkeys Patients with progressive neuromuscular disorders all experience the foreboding of the severe disability that awaits them and from which there is little to no relief. Although this class of disorders has multiple genetic and physiological origins, a therapy that directly addresses the debilitating muscle weakness that is the hallmark of these maladies would enhance the lives of millions. Now, in an extension of their previous work in dystrophic mice, Kota et al. describe such a therapeutic approach in preclinical studies performed in nonhuman primates. This treatment mode is applicable to several progressive neuromuscular disorders whether or not scientists have defined their precise genetic defects. The authors used a gene therapy approach to introduce a version of the human gene encoding follistatin into the muscles of the femurs of healthy cynomolgus macaques. Follistatin is a potent inhibitor of myostatin, a signaling molecule that regulates skeletal muscle mass. Follistatin blocks myostatin signaling and augments muscle size and strength safely in mice but, until now, has not been tested in primates. Kota et al. injected a follistatin-producing gene therapy vector into the leg muscles of the monkeys and measured increases in muscle mass and strength. Sustained follistatin expression caused no aberrations in the structures or functions of a variety of organs. This promising progress comes with some caveats. Because healthy monkeys served as subjects for this therapeutic protocol, these findings are not predictive of the outcome in a clinical setting with patients suffering from muscle disorders. In certain genetic neuromuscular diseases, the muscles undergo a repeated cycle of degeneration and regeneration. The vector used in this study does not integrate into the muscle cell genome and thus can be lost from the cells during the degeneration-regeneration cycles. However, the authors point out that the enhancement of muscle size and strength observed in similarly treated dystrophic mice persisted for more than a year even though there was appreciable muscle turnover. More study is needed before follistatin enters the clinic, such as a molecular assessment of gene and vector sequences in multiple tissues. Nonetheless, the work of Kota et al. constitutes proof of principle for the use of myostatin inhibitors to build muscle in primates. Antagonists of myostatin, a blood-borne negative regulator of muscle growth produced in muscle cells, have shown considerable promise for enhancing muscle mass and strength in rodent studies and could serve as potential therapeutic agents for human muscle diseases. One of the most potent of these agents, follistatin, is both safe and effective in mice, but similar tests have not been performed in nonhuman primates. To assess this important criterion for clinical translation, we tested an alternatively spliced form of human follistatin that affects skeletal muscle but that has only minimal effects on nonmuscle cells. When injected into the quadriceps of cynomolgus macaque monkeys, a follistatin isoform expressed from an adeno-associated virus serotype 1 vector, AAV1-FS344, induced pronounced and durable increases in muscle size and strength. Long-term expression of the transgene did not produce any abnormal changes in the morphology or function of key organs, indicating the safety of gene delivery by intramuscular injection of an AAV1 vector. Our results, together with the findings in mice, suggest that therapy with AAV1-FS344 may improve muscle mass and function in patients with certain degenerative muscle disorders.

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.