CDN1163

The small molecule SERCA activator CDN1163 increases energy metabolism in human skeletal muscle cells

Background and objective: A number of studies have highlighted muscle-specific mechanisms of thermogenesis involving futile cycling of Ca2+ driven by sarco (endo)plasmic reticulum Ca2+-ATPase (SERCA) and generating heat from ATP hydrolysis to be a promising strategy to counteract obesity and metabolic dysfunction. However, to the best of our knowledge, no experimental studies concerning the metabolic effects of pharmacologically targeting SERCA in human skeletal muscle cells have been reported. Thus, in the present study, we aimed to explore the effects of SERCA-activating compound, CDN1163, on energy metabolism in differentiated human skeletal muscle cells (myotubes). Methods: In this study, we used primary myotube cultures derived from muscle biopsies of the musculus vastus lateralis and musculi interspinales from lean, healthy male donors. Energy metabolism in myotubes was studied using radioactive substrates. Oxygen consumption rate was assessed with the Seahorse XF24 bioanalyzer, whereas metabolic genes and protein expressions were determined by qPCR and immunoblotting, respectively. Results: Both acute (4 ?h) and chronic (5 days) treatment of myotubes with CDN1163 showed increased uptake and oxidation of glucose, as well as complete fatty acid oxidation in the presence of carbonyl cyanide 4-(trifluromethoxy)phenylhydrazone (FCCP). These effects were supported by measurement of oxygen consumption rate, in which the oxidative spare capacity and maximal respiration were enhanced after CDN1163-treatment. In addition, chronic treatment with CDN1163 improved cellular uptake of oleic acid (OA) and fatty acid β-oxidation. The increased OA metabolism was accompanied by enhanced mRNA-expression of carnitine palmitoyl transferase (CPT) 1B, pyruvate dehydrogenase kinase (PDK) 4, as well as increased AMP-activated protein kinase (AMPK)Thr172 phosphorylation. Moreover, following chronic CDN1163 treatment, the expression levels of stearoyl-CoA desaturase (SCD) 1 was decreased together with de novo lipogenesis from acetic acid and formation of diacylglycerol (DAG) from OA. Conclusion: Altogether, these results suggest that SERCA activation by CDN1163 enhances energy metabolism in human myotubes, which might be favourable in relation to disorders that are related to metabolic dysfunction such as obesity and type 2 diabetes mellitus.

Suppression of Ca2+ oscillations by SERCA inhibition in human alveolar type 2 A549 cells: rescue by ochratoxin A but not CDN1163

Aims: Lung type 2 alveolar cells, by secreting surfactant to lower surface tension, contribute to enhance lung compliance. Stretching, as a result of lung expansion, triggers type 1 alveolar cell to release ATP, which in turn stimulates Ca²⁺-dependent surfactant secretion by neighboring type 2 cells. In this report, we studied ATP-triggered Ca²⁺ signaling in human alveolar type 2 A549 cells.
Main methods: Ca²⁺ signaling was examined using microfluorimetric measurement with fura-2 as fluorescent dye.
Key findings: Ca²⁺ oscillations triggered by ATP relied on inositol 1,4,5-trisphosphate-induced Ca²⁺ release and store-operated Ca²⁺ entry. Pathological conditions such as influenza virus infection and diabetes reportedly inhibit sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA). We found that a very mild inhibition of SERCA by cyclopiazonic acid (CPA) sufficed to decrease Ca²⁺ oscillation frequency and the percentage of cells exhibiting Ca²⁺ oscillations. Ochratoxin A (OTA), an activator of SERCA, could prevent the suppressive effects by CPA. Inhibition of SERCA by hydrogen peroxide also suppressed Ca²⁺ oscillations. Interestingly, hydrogen peroxide-induced inhibition was prevented by OTA but aggravated by CDN1163, an allosteric activator of SERCA. CDN1163 also had an untoward effect of releasing intracellular Ca²⁺.
Significance: Different modes of activation of SERCA may determine the outcome of rescue of Ca²⁺ oscillations in case of SERCA inhibition in alveolar type 2 cells.

Stimulation of Ca2+ -ATPase Transport Activity by a Small-Molecule Drug

The sarco(endo)plasmic reticulum Ca²⁺ -ATPase (SERCA) hydrolyzes ATP to transport Ca²⁺ from the cytoplasm to the sarcoplasmic reticulum (SR) lumen, thereby inducing muscle relaxation. Dysfunctional SERCA has been related to various diseases. The identification of small-molecule drugs that can activate SERCA may offer a therapeutic approach to treat pathologies connected with SERCA malfunction. Herein, we propose a method to study the mechanism of interaction between SERCA and novel SERCA activators, i. e. CDN1163, using a solid supported membrane (SSM) biosensing approach. Native SR vesicles or reconstituted proteoliposomes containing SERCA were adsorbed on the SSM and activated by ATP concentration jumps. We observed that CDN1163 reversibly interacts with SERCA and enhances ATP-dependent Ca²⁺ translocation. The concentration dependence of the CDN1163 effect provided an EC₅₀ =6.0±0.3 μM. CDN1163 was shown to act directly on SERCA and to exert its stimulatory effect under physiological Ca²⁺ concentrations. These results suggest that CDN1163 interaction with SERCA can promote a protein conformational state that favors Ca²⁺ release into the SR lumen.

Sarco/Endoplasmic Reticulum Ca2+ ATPase 2 Activator Ameliorates Endothelial Dysfunction; Insulin Resistance in Diabetic Mice

Background: Sarco/endoplasmic reticulum Ca²⁺-ATPase2 (SERCA2) is impaired in various organs in animal models of diabetes. The purpose of this study was to test the effects of an allosteric SERCA2 activator (CDN1163) on glucose intolerance, hepatosteatosis, skeletal muscle function, and endothelial dysfunction in diabetic (db/db) mice. Methods: Either CDN1163 or vehicle was injected intraperitoneally into 16-week-old male control and db/db mice for 5 consecutive days. Results: SERCA2 protein expression was decreased in the aorta of db/db mice. In isometric tension measurements of aortic rings from db/db mice treated with CDN1163, acetylcholine (ACh)-induced relaxation was improved. In vivo intraperitoneal administrations of CDN 1163 also increased ACh-induced relaxation. Moreover, CDN1163 significantly decreased blood glucose in db/db mice at 60 and 120 min during a glucose tolerance test; it also decreased serum insulin levels, hepatosteatosis, and oxygen consumption in skeletal muscle during the early period of exercise in db/db mice. Conclusions: CDN1163 directly improved aortic endothelial dysfunction in db/db mice. Moreover, CDN1163 improved hepatosteatosis, skeletal muscle function, and insulin resistance in db/db mice. The activation of SERCA2 might be a strategy for the all the tissue expressed SERCA2a improvement of endothelial dysfunction and the target for the organs related to insulin resistance.

Pharmacological activation of SERCA ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice

Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscular weakness because of the loss of dystrophin. Extracellular Ca2+ flows into the cytoplasm through membrane tears in dystrophin-deficient myofibers, which leads to muscle contracture and necrosis. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) takes up cytosolic Ca2+ into the sarcoplasmic reticulum, but its activity is decreased in dystrophic muscle. Here, we show that an allosteric SERCA activator, CDN1163, ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice. The administration of CDN1163 prevented exercise-induced muscular damage and restored mitochondrial function. In addition, treatment with CDN1163 for 7 weeks enhanced muscular strength and reduced muscular degeneration and fibrosis in mdx mice. Our findings provide preclinical proof-of-concept evidence that pharmacological activation of SERCA could be a promising therapeutic strategy for DMD. Moreover, CDN1163 improved muscular strength surprisingly in wild-type mice, which may pave the new way for the treatment of muscular dysfunction.