Alain Lacampagne

Remodeling of ryanodine receptor complex causes “leaky” channels: A molecular mechanism for decreased exercise capacity

During exercise, defects in calcium (Ca2+) release have been proposed to impair muscle function. Here, we show that during exercise in mice and humans, the major Ca2+ release channel required for excitation–contraction coupling (ECC) in skeletal muscle, the ryanodine receptor (RyR1), is progressively PKA-hyperphosphorylated, S-nitrosylated, and depleted of the phosphodiesterase PDE4D3 and the RyR1 stabilizing subunit calstabin1 (FKBP12), resulting in “leaky” channels that cause decreased exercise tolerance in mice. Mice with skeletal muscle-specific calstabin1 deletion or PDE4D deficiency exhibited significantly impaired exercise capacity. A small molecule (S107) that prevents depletion of calstabin1 from the RyR1 complex improved force generation and exercise capacity, reduced Ca2+-dependent neutral protease calpain activity and plasma creatine kinase levels. Taken together, these data suggest a possible mechanism by which Ca2+ leak via calstabin1-depleted RyR1 channels leads to defective Ca2+ signaling, muscle damage, and impaired exercise capacity.

AMPK Activation Stimulates Autophagy and Ameliorates Muscular Dystrophy in the mdx Mouse Diaphragm

Duchenne muscular dystrophy (DMD) is characterized by myofiber death from apoptosis or necrosis, leading in many patients to fatal respiratory muscle weakness. Among other pathological features, DMD muscles show severely deranged metabolic gene regulation and mitochondrial dysfunction. Defective mitochondria not only cause energetic deficiency, but also play roles in promoting myofiber atrophy and injury via opening of the mitochondrial permeability transition pore. Autophagy is a bulk degradative mechanism that serves to augment energy production and eliminate defective mitochondria (mitophagy). We hypothesized that pharmacological activation of AMP-activated protein kinase (AMPK), a master metabolic sensor in cells and on-switch for the autophagy-mitophagy pathway, would be beneficial in the mdx mouse model of DMD. Treatment of mdx mice for 4 weeks with an established AMPK agonist, AICAR (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside), potently triggered autophagy in the mdx diaphragm without inducing muscle fiber atrophy. In AICAR-treated mdx mice, the exaggerated sensitivity of mdx diaphragm mitochondria to calcium-induced permeability transition pore opening was restored to normal levels. There were associated improvements in mdx diaphragm histopathology and in maximal force-generating capacity, which were not linked to increased mitochondrial biogenesis or up-regulated utrophin expression. These findings suggest that agonists of AMPK and other inducers of the autophagy-mitophagy pathway can help to promote the elimination of defective mitochondria and may thus serve as useful therapeutic agents in DMD.

Defects in ryanodine receptor calcium release in skeletal muscle from post-myocardial infarct rats

Defective calcium (Ca2+) signaling and impaired contractile function have been observed in skeletal muscle secondary to impaired myocardial function. However, the molecular basis for these muscle defects have not been identified. In this study, we evaluated the alterations of the ryanodine‐sensitive Ca2+ release channels (RyR1) by analyzing global and local Ca2+ signaling in a rat postmyocardial infarction (PMI) model of myocardial overload. Ca2+ transients, measured with multiphoton imaging in individual fibers within a whole extensor digitorum longus (EDL) muscle, exhibited significantly reduced amplitude and a prolonged time course in PMI. Spatiotemporal properties of spontaneous Ca2+ sparks in fibers isolated from PMI EDL muscles were also significantly altered. In addition, RyR1 from PMI skeletal muscles were PKA‐hyperphosphorylated and depleted of the FK506 binding protein (FKBP12). These data show that PMI skeletal muscles exhibit altered local Ca2+ signaling, associated with hyperphosphorylation of RyR1. The observed changes in Ca2+ signaling may contribute to defective excitation‐contraction coupling in muscle that can contribute to the reduced exercise capacity in PMI, out of proportion to the degree of cardiac dysfunction.

Mitochondrial production of reactive oxygen species contributes to the β-adrenergic stimulation of mouse cardiomycytes

Non‐technical summary  When under stress, the heart beat becomes stronger, in part due to enhanced fluxes of Ca2+ at the level of the cardiac cell. It is known that this effect is mediated by activation of β‐receptors on the cardiac cell surface. This leads to modifications of intracellular proteins that in turn increase the flux of Ca2+ within the cell. In this study we show that activation of β‐receptors increases the production of reactive oxygen species (ROS) in the heart cell. These ROS generate enhanced Ca2+ fluxes and more vigorous contraction. This finding shows a new cellular signalling route for regulating the power of the heart beat and might contribute to our understanding of diseases with defective cardiac contraction, such as heart failure.

Transmural stretch-dependent regulation of contractile properties in rat heart and its alteration after myocardial infarction

The “stretch‐sensitization” response is essential to the regulation of heart contractility. An increase in diastolic volume improves systolic contraction. The cellular mechanisms of this modulation, the Frank‐Starling law, are still uncertain. Moreover, their alterations in heart failure remains controversial. Here, using left ventricular skinned rat myocytes, we show a nonuniform stretch‐sensitization of myofilament activation across the ventricular wall. Stretch‐dependent Ca2+ sensitization of myofilaments increases from sub‐epicardium to sub‐endocardium and is correlated with an increase in passive tension. This passive tension‐dependent component of myofibrillar activation is not associated with expression of titin isoforms, changes in troponin I level, and phosphorylation status. Instead, we observe that stretch induces phosphorylation of ventricular myosin light chain 2 isoform (VLC2b) in sub‐endocardium specifically. Thus, VLC2b phosphorylation could act as a stretch‐dependent modulator of activation tuned within normal heart. Moreover, in postmyocardial infarcted rat, the gradient of stretch‐dependent Ca2+ sensitization disappears associated with a lack of VLC2b phosphorylation in sub‐endocardium. In conclusion, nonuniformity is a major characteristic of the normal adult left ventricle (LV). The heterogeneous myocardial deformation pattern might be caused not only by the morphological heterogeneity of the tissue in the LV wall, but also by the nonuniform contractile properties of the myocytes across the wall. The loss of a contractile transmural gradient after myocardial infarction should contribute to the impaired LV function.

Hidden face of the anterior pituitary.

The traditional view holds that the anterior pituitary is an endocrine gland with a complex and heterogeneous distribution of cells throughout the parenchyma. Thus, a long-distance mode of intraorgan communication is not usually taken into account in our understanding of pituitary functioning. However, recent in situ pituitary studies have begun to unveil a hitherto unknown route of large-scale information transfer within the pituitary. Agranular folliculostellate cells - the sixth type of pituitary cell initially discovered almost half a century ago - are the functional units of a dynamically active cell network wiring the whole gland. Because folliculostellate cells communicate with their endocrine neighbors, this opens the door to considering the pituitary as a cellular puzzle more ordered than was first thought. Hence, cell networking within the pituitary gland could have a privileged role in coordinating the activities of distant cells in both physiological and pathological conditions.

SR33805, a Ca2+ antagonist with length-dependent Ca2+-sensitizing properties in cardiac myocytes

This study examined the effects of SR33805, a fantofarone derivative with reported strong Ca2+ ‐antagonistic properties, on the contractile properties of intact and skinned rat ventricular myocytes. On intact cells loaded with the Ca2+‐fluorescent indicator Indo‐1, the application of low concentrations of SR33805 enhanced the amplitude of unloaded cell shortening and decreased the duration of cell shortening. Amplitude of the Ca2+ transient was also decreased. These effects were accompanied with a shortening of the action potential and a dose‐dependent blockade of L‐type calcium current (IC50=2.4 × 10−8 M). On skinned cardiac cells, the application of a low SR33805 concentration (10−8 M) induced a significant increase in maximal Ca2+‐activated force at the two‐tested sarcomere lengths (SLs), 1.9 and 2.3 μm. The application of a larger dose of SR33805 (10−6–10−5 M) induced a significant leftward shift of the tension–pCa relation that accounts for Ca2+‐sensitization of the myofilaments, particularly at 2.3 μm SL. In conclusion, despite its strong Ca2+‐antagonistic properties SR33805 increases cardiac cell contractile activity as a consequence of its Ca2+‐sensitizing effects. These effects are attributable to both an increase in the maximal Ca2+‐activated force and a length‐dependent Ca2+‐sensitization.

0006 : Non-enzymatic lipid mediators, neuroprostanes exerts the anti-arrhythmic properties of docosahexaenoic acid

The cardioprotective effects through prevention of cardiac arrhythmias of long-chain polyunsaturated fatty acids of the n-3 series (PUFAs) have been demonstrated over the last 40 years. The main n-3 PUFAs are eicosapentaenoic acid (C20:5 n-3, EPA) and docosahexaenoic acid (C22:6 n-3, DHA) and both are highly peroxidable due to the presence of skipped dienes. The effects of n-3 PUFAs on cardiac function are controversial, notably due to lack of information on the mechanisms involved. Particularly, it is not well understood which is the active lipid: the PUFA or one of its oxygenated metabolites. Neuroprostanes are lipid mediators produced by non-enzymatic free radical peroxidation of DHA. Plasmatic 4(RS)-4-F4t-NeuroP concentration is negatively correlated with the risk of atherosclerosis suggesting a beneficial active role in some cardiovascular disease. In this study, we show that oxidized DHA and more specifically the isomers 4(RS)-4-F4t-NeuroP possess strong anti-arrhythmic properties (AAP) in isolated ventricular cardiomyocytes and in vivo in post-myocardial infarcted mice. Calcium imaging and biochemical experiments indicate that arrhythmias are associated with Ca2+ leak from the sarcoplasmic reticulum following oxidation and phosphorylation of the type 2 ryanodine receptor (RyR2) leading to dissociation of the FKBP12.6/ RyR2 complex. DHA per se has no AAP. Oxidized DHA as well as 4(RS)-4- F4t-NeuroP prevented posttranslational modifications of the RyR2 and stabilized the complex FKBP12.6/RyR2 to normalize Ca2+. This effect of 4(RS)- 4-F4t-NeuroP was further associated with the suppression in vitro and in vivo of cardiac arrhythmias. Our findings, demonstrates 4(RS)-4-F4t-NeuroP as a mediator of the AAP that exerts cardioprotective characteristics of DHA.

Amyloid β production is regulated by β2-adrenergic signaling-mediated post-translational modifications of the ryanodine receptor

Alteration of ryanodine receptor (RyR)-mediated calcium (Ca2+) signaling has been reported in Alzheimer disease (AD) models. However, the molecular mechanisms underlying altered RyR-mediated intracellular Ca2+ release in AD remain to be fully elucidated. We report here that RyR2 undergoes post-translational modifications (phosphorylation, oxidation, and nitrosylation) in SH-SY5Y neuroblastoma cells expressing the β-amyloid precursor protein (βAPP) harboring the familial double Swedish mutations (APPswe). RyR2 macromolecular complex remodeling, characterized by depletion of the regulatory protein calstabin2, resulted in increased cytosolic Ca2+ levels and mitochondrial oxidative stress. We also report a functional interplay between amyloid β (Aβ), β-adrenergic signaling, and altered Ca2+ signaling via leaky RyR2 channels. Thus, post-translational modifications of RyR occur downstream of Aβ through a β2-adrenergic signaling cascade that activates PKA. RyR2 remodeling in turn enhances βAPP processing. Importantly, pharmacological stabilization of the binding of calstabin2 to RyR2 channels, which prevents Ca2+ leakage, or blocking the β2-adrenergic signaling cascade reduced βAPP processing and the production of Aβ in APPswe-expressing SH-SY5Y cells. We conclude that targeting RyR-mediated Ca2+ leakage may be a therapeutic approach to treat AD.

0330: Cardiac p11 expression is related to 5-HT4 receptor pathway in failing and non-failing rat left ventricular cardiomyocytes

Aim Heart failure is the inability to maintain a sufficient cardiac output necessary to meet metabolic demand. Cellular compensatory mechanisms, such as the serotonin 4 receptor (5-HT4R) pathway, take place to improve the defective cardiac excitation-contraction coupling (CEC). However, little is known about the regulation of this pathway. Our objective was to investigate the potential involvement of the 5-HT4R partner p11 in the activation of the pathway during heart failure. Methods and results Wistar rats underwent ligation of the left coronary artery to mimic infarction. Control Sham-operated animals underwent the same surgery without ligation. Seven weeks post myocardial infarction (PMI) hearts were collected and/or enzymatically digested in order to perform biochemical studies or to assess intracellular calcium (Ca2+) handling at the single cell level. p11 mRNA expression in the left posterior wall was significantly increased at 7 weeks PMI compared to Sham (272±40 vs.141±27 A.U, P Conclusion Cardiac expression of p11 seems to play a role in the modulation of the response to 5-HT4R pathway in both failing and non-failing hearts.