Summary Age-related loss of muscle mass and force (sarcopenia) contributes to disability and increased mortality. Ryanodine receptor 1 (RyR1) is the skeletal muscle sarcoplasmic reticulum calcium release channel required for muscle contraction. RyR1 from aged (24 months) rodents were oxidized, cysteine-nitrosylated, and depleted of the channel stabilizing subunit calstabin1, compared to RyR1 from younger (3–6 months) adults. This RyR1 channel complex remodeling resulted in “leaky” channels with increased open probability leading to intracellular calcium leak in skeletal muscle. Similarly, six-month old mice harboring leaky RyR1-S2844D mutant channels exhibited skeletal muscle defects comparable to 24-month old wild type mice. Treating aged mice with S107, stabilized binding of calstabin1 to RyR1, reduced intracellular calcium leak, decreased reactive oxygen species (ROS), and enhanced tetanic Ca2+ release, muscle specific force and exercise capacity. Taken together these data indicate that leaky RyR1 contribute to age-related loss of muscle function.

Cancer-associated muscle weakness is poorly understood and there is no effective treatment. Here, we find that seven different mouse models of human osteolytic bone metastases, representing breast, lung and prostate cancers, as well as multiple myeloma exhibited impaired muscle function, implicating a role for the tumor-bone microenvironment in cancer-associated muscle weakness. We found that TGF-β, released from the bone surface as a result of metastasis-induced bone destruction upregulated NADPH oxidase 4 (Nox4), resulting in elevated oxidization of skeletal muscle proteins, including the ryanodine receptor/calcium (Ca2+) release channel (RyR1). The oxidized RyR1 channels leaked Ca2+, resulting in lower intracellular signaling required for proper muscle contraction. We found that inhibiting RyR1 leak, TGF-β signaling, TGF-β release from bone or Nox4 all improved muscle function in mice with MDA-MB-231 bone metastases. Humans with breast cancer- or lung cancer-associated bone metastases also had oxidized skeletal muscle RyR1 that is not seen in normal muscle. Similarly, skeletal muscle weakness, higher levels of Nox4 protein and Nox4 binding to RyR1, and oxidation of RyR1 were present in a mouse model of Camurati-Engelmann disease, a non-malignant metabolic bone disorder associated with increased TGF-β activity. Thus, metastasis-induced TGF-β release from bone contributes to muscle weakness by decreasing Ca2+-induced muscle force production.

Calcium (Ca 2+ ) released from the sarcoplasmic reticulum (SR) is crucial for excitation-contraction (E-C) coupling. Mitochondria, the major source of energy, in the form of ATP, required for cardiac contractility, are closely interconnected with the SR, and Ca 2+ is essential for optimal function of these organelles. However, Ca 2+ accumulation can impair mitochondrial function, leading to reduced ATP production and increased release of reactive oxygen species (ROS). Oxidative stress contributes to heart failure (HF), but whether mitochondrial Ca 2+ plays a mechanistic role in HF remains unresolved. Here, we show for the first time, to our knowledge, that diastolic SR Ca 2+ leak causes mitochondrial Ca 2+ overload and dysfunction in a murine model of postmyocardial infarction HF. There are two forms of Ca 2+ release channels on cardiac SR: type 2 ryanodine receptors (RyR2s) and type 2 inositol 1,4,5-trisphosphate receptors (IP3R2s). Using murine models harboring RyR2 mutations that either cause or inhibit SR Ca 2+ leak, we found that leaky RyR2 channels result in mitochondrial Ca 2+ overload, dysmorphology, and malfunction. In contrast, cardiacspecific deletion of IP3R2 had no major effect on mitochondrial fitness in HF. Moreover, genetic enhancement of mitochondrial antioxidant activity improved mitochondrial function and reduced posttranslational modifications of RyR2 macromolecular complex. Our data demonstrate that leaky RyR2, but not IP3R2, channels cause mitochondrial Ca 2+ overload and dysfunction in HF.ryanodine receptor | heart failure | mitochondria | calcium | IP3 receptor T ype 2 ryanodine receptor/Ca 2+ release channel (RyR2) and type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) are the major intracellular Ca 2+ release channels in the heart (1-3). RyR2 is essential for cardiac excitation-contraction (E-C) coupling (2), whereas the role of IP3R2 in cardiomyocytes is less well understood (3). E-C coupling requires energy in the form of ATP produced primarily by oxidative phosphorylation in mitochondria (4-8).Both increased and reduced mitochondrial Ca 2+ levels have been implicated in mitochondrial dysfunction and increased reactive oxygen species (ROS) production in heart failure (HF) (6,7,(9)(10)(11)(12)(13)(14)(15)(16)(17). Albeit Ca 2+ is required for activation of key enzymes (i.e., pyruvate dehydrogenase phosphatase, isocitrate dehydrogenase, and α-ketoglutarate dehydrogenase) in the tricarboxylic acid (also known as Krebs) cycle (18, 19), excessive mitochondrial Ca 2+ uptake has been associated with cellular dysfunction (14,20). Furthermore, the exact source of mitochondrial Ca 2+ has not been clearly established. Given the intimate anatomical and functional association between the sarcoplasmic reticulum (SR) and mitochondria (6, 21, 22), we hypothesized that SR Ca 2+ release via RyR2 and/or IP3R2 channels in cardiomyocytes could lead to mitochondrial Ca 2+ accumulation and dysfunction contributing to oxidative overload and energy depletion. Results and DiscussionIncreased Mitochondrial Ca...

Cell migration in a cultured neuronal network presents an obstacle to selectively measuring the activity of the same neuron over a long period of time. Here we report the use of nanopillar arrays to pin the position of neurons in a noninvasive manner. Vertical nanopillars protruding from the surface serve as geometrically better focal adhesion points for cell attachment than a flat surface. The cell body mobility is significantly reduced from 57.8 µm on flat surface to 3.9 µm on nanopillars over five day period. Yet, neurons growing on nanopillar arrays show a growth pattern that does not differ in any significant way from that seen on a flat substrate. Notably, while the cell bodies of neurons are efficiently anchored by the nanopillars, the axons and dendrites are free to grow and elongate into the surrounding area to develop a neuronal network, which opens up opportunities for long-term study of the same neurons in connected networks. KeywordsNanopillar; neuron; cell growth; cell migration; extracellular recording A fundamental understanding of neural network formation, transmission and remodeling requires measurements of individual neuron activities including firing threshold, firing rate and temporal sequence1. Extracellular approaches such as patterned multi-electrode arrays (MEA)2 -4 and planar field effect transistors4 have been successful in simultaneously measuring multi-cell activities over an extended period of time and thus have provided valuable information regarding the development and formation of neuronal networks. For example, the emergence of synchronous electrical firing pattern and developmental changes of the network activity have been observed in networks of cultured cortical neurons5 -7. In such studies, dissociated neurons obtained from fetal or neonatal brains are cultured atop the embedded electrodes or transistors. Electric signals generated by a neuron can be detected extracellularly if there is an electrode in close contact. However, it has been difficult to consistently measure the activity of the same neuron over a long term period. This difficulty is partly due to neuron mobility and partly due to lack of neuron-to-electrode specificity8. Neurons cultured on a flat substrate tend to migrate over time, especially in the first few weeks9 , 10. The migration range can be as long as hundreds of micrometers and well beyond the detection range of a single electrode or transistor. As a result, patterned electrodes or transistors are not always monitoring the activity of the same neuron as neurons move around. This presents a challenge to monitoring individual neuron activities in a neuronal network for an extended time (up to months), which demands stable and specific neuron-electrode correspondence.* To whom correspondence should be addressed. BC, bcui@stanford.edu; YC, yicui@stanford.edu. Considerable efforts have been put forth to control the migration of neurons and thus to improve the neuron-electrode interface11 -16. The first approach is to promote neuron-toelectrode atta...

Oxidative stress has been suggested to play a role in the pathogenesis of atrial fibrillation (AF). Indeed, the prevalence of AF increases with age as does oxidative stress. However, the mechanisms linking redox state to AF are not well understood. In this study we identify a link between oxidative stress and aberrant intracellular Ca2+ release via the type 2 ryanodine receptor (RyR2) that promotes AF. We show that RyR2 are oxidized in the atria of patients with chronic AF compared with individuals in sinus rhythm. To dissect the molecular mechanism linking RyR2 oxidation to AF we used two murine models harboring RyR2 mutations that cause intracellular Ca2+ leak. Mice with intracellular Ca2+ leak exhibited increased atrial RyR2 oxidation, mitochondrial dysfunction, reactive oxygen species (ROS) production and AF susceptibility. Both genetic inhibition of mitochondrial ROS production and pharmacological treatment of RyR2 leakage prevented AF. Collectively, our results indicate that alterations of RyR2 and mitochondrial ROS generation form a vicious cycle in the development of AF. Targeting this previously unrecognized mechanism could be useful in developing effective interventions to prevent and treat AF.

No abstract

Rationale: Unrepaired cardiomyocyte membrane injury causes irreplaceable cell loss, leading to myocardial fibrosis and eventually heart failure. However, the cellular and molecular mechanisms of cardiac membrane repair are largely unknown. MG53, a newly identified striated muscle-specific protein, is involved in skeletal muscle membrane repair. But the role of MG53 in the heart has not been determined. Objective: We sought to investigate whether MG53 mediates membrane repair in cardiomyocytes and, if so, the cellular and molecular mechanism underlying MG53-mediated membrane repair in cardiomyocytes. Moreover, we determined possible cardioprotective effect of MG53-mediated membrane repair. Methods and Results: We demonstrated that MG53 is crucial to the emergency membrane repair response in cardiomyocytes and protects the heart from stress-induced loss of cardiomyocytes. Disruption of the sarcolemmal membrane by mechanical, electric, chemical, or metabolic insults caused rapid and robust translocation of MG53 toward the injury sites. Ablation of MG53 prevented sarcolemmal resealing after infrared laser-induced membrane damage in intact heart, and exacerbated mitochondrial dysfunction and loss of cardiomyocytes during ischemia/reperfusion injury. Unexpectedly, the MG53-mediated cardiac membrane repair was mediated by a cholesterol-dependent mechanism: depletion of membrane cholesterol abolished, and its recovery restored injury-induced membrane translocation of MG53. The redox status of MG53 did not affect initiation of MG53 translocation, whereas MG53 oxidation conferred stability to the membrane repair patch. Conclusions: Thus, cholesterol-dependent MG53-mediated membrane repair is a vital, heretofore unappreciated cardioprotective mechanism against a multitude of insults and may bear important therapeutic implications. (Circ Res. 2010;107:76-83.)Key Words: membrane repair Ⅲ MG53 Ⅲ cholesterol Ⅲ ischemia/reperfusion injury Ⅲ heart I n eukaryotic cells, the plasma membrane partitions a Ϸ10 000-fold Ca 2ϩ gradient and prevents loss of vital intracellular constituents, thus representing the last line of defense for cell integrity, homeostasis, and function. Physical, chemical or metabolic disruption of the plasma membrane leads to a repairor-die emergency of the cell. Although the natural tendency to reseal the lipid biomembrane acts constitutively, recent studies indicate that plasma membrane disruption requires active emergency response mechanisms to mend the broken membrane. 1 In the heart, plasma membrane repair is of particular importance because cardiomyocytes are terminally differentiated cells, displaying only very limited self-renewal capability. 2 Cardiomyocytes undergo transient membrane injuries that occur as accidents under physiological conditions and can be exacerbated by various pathophysiological stresses. 3 Progressive necrotic and apoptotic cell death causes onset of myocardial fibrosis and undermines cardiac contractile and electrophysiological performance, ultimately leading to heart failure....

Rationale Atrial fibrillation (AF) is the most common cardiac arrhythmia, however the mechanism(s) causing AF remain poorly understood and therapy is suboptimal. The ryanodine receptor (RyR2) is the major calcium (Ca2+) release channel on the sarcoplasmic reticulum (SR) required for excitation-contraction coupling in cardiac muscle. Objective In the present study we sought to determine whether intracellular diastolic SR Ca2+ leak via RyR2 plays a role in triggering AF and whether inhibiting this leak can prevent AF. Methods and Results We generated three knock-in mice with mutations introduced into RyR2 that result in leaky channels and cause exercise induced polymorphic ventricular tachycardia in humans [catecholaminergic polymorphic ventricular tachycardia (CPVT)]. We examined AF susceptibility in these three CPVT mouse models harboring RyR2 mutations to explore the role of diastolic SR Ca2+ leak in AF. AF was stimulated with an intra-esophageal burst pacing protocol in the three CPVT mouse models (RyR2-R2474S+/−, 70%; RyR2-N2386I+/−, 60%; RyR2-L433P+/−, 35.71%), but not in wild type (WT) mice (P<0.05). Consistent with these in vivo results, there was a significant diastolic SR Ca2+ leak in atrial myocytes isolated from the CPVT mouse models. Calstabin2 (FKBP12.6) is an RyR2 subunit that stabilizes the closed state of RyR2 and prevents a Ca2+ leak through the channel. Atrial RyR2 from RyR2-R2474S+/− mice were oxidized and the RyR2 macromolecular complex was depleted of calstabin2. The Rycal drug S107 stabilizes the closed state of RyR2 by inhibiting the oxidation/phosphorylation induced dissociation of calstabin2 from the channel. S107 reduced the diastolic SR Ca2+ leak in atrial myocytes and decreased burst pacing-induced AF in vivo. S107 did not reduce the increased prevalence of burst pacing-induced AF in calstabin2-deficient mice, confirming that calstabin2 is required for the mechanism of action of the drug. Conclusions The present study demonstrates that RyR2-mediated diastolic SR Ca2+ leak in atrial myocytes is associated with AF in CPVT mice. Moreover, the Rycal S107 inhibited diastolic SR Ca2+ leak through RyR2 and pacing-induced AF associated with CPVT mutations.