Impact of maternal low-level cadmium exposure on glucose and lipid metabolism of the litter at different ages after weaning.

Cadmium (Cd) is a metal which may participate in the development of type II diabetes even if Cd exposure levels are mild. However, experimental studies focusing on daily environmentally relevant doses are scarce, particularly for glucose metabolism of the offspring of chronically exposed mothers. The aim is to measure the impact of maternal low level Cd exposure on glucose and lipid metabolism of offspring. Female rats were exposed to 0, 50 or 500 µg.kg-1.d-1 of CdCl2, 21 days before mating and during 21 days of gestation and 21 days of lactation. Pups exposure was organized in 3 groups (control, Cd1, Cd2) according to renal dams' Cd burden. Parameters of glucose and lipid metabolisms were measured for the pups on post-natal day 21, 26 and 60. Maternal Cd exposure led to significant amounts of Cd in the liver and kidney of pups. At weaning, insulin secretion upon glucose stimulation was unchanged, but the removal of circulating glucose was slower for pups born from the lowest impregnated dams (Cd1). Five days after, glucose tolerance of all groups was identical. Thus, this loss of insulin sensitivity was reversed, in part by increased adiponectin secretion for the Cd1 group. Furthermore, pups from dams accumulating the highest levels of Cd (Cd2) exhibited a compensatory increased insulin pancreatic secretion, together with increased circulating non-esterified fatty acids, indicating the establishment of insulin resistance, 2 months after birth. This study has demonstrated the influence of maternal exposure to low levels of Cd on glucose homeostasis in the offspring that might increase the risk of developing type II diabetes later in life.

Impact of chronic and low cadmium exposure of rats: sex specific disruption of glucose metabolism.

BACKGROUND: Several epidemiological and animal studies suggest a positive association between cadmium (Cd) exposure and incidence of type 2 diabetes, but the association remains controversial. Besides, the experimental data have mainly been obtained with relatively high levels of Cd, over various periods of time, and with artificial routes of administration. OBJECTIVES: Do environmental exposures to Cd induce significant disruption of glucose metabolism? METHODS: Adults Wistar rats were exposed for three months to 0, 5, 50 or 500 µg.kg-1.d-1 of CdCl2 in drinking water. Relevant parameters of glucose homeostasis were measured. RESULTS: Cd accumulated in plasma, kidney and liver of rats exposed to 50 and 500 µg.kg-1.d-1, without inducing signs of organ failure. In rats drinking 5 µg.kg-1.d-1 for 3 months, Cd exposure did not lead to any significant increase of Cd in these organs. At 50 and 500 µg.kg-1.d-1 of Cd, glucose and insulin tolerance were unchanged in both sexes. However, females exhibited a significant increase of both fasting and glucose-stimulated plasma insulin that was assigned to impaired hepatic insulin extraction as indicated by unaltered fasting C-peptide plasma levels. CONCLUSIONS: Glucose homeostasis is sensitive to chronic Cd exposure in a gender-specific way. Moreover, this study proves that an environmental pollutant such as Cd can have, at low concentrations, an impact on the glucose homeostatic system and it highlights the importance of a closer scrutiny of the underlying environmental causes to understand the increased incidence of type 2 diabetes.

Mitochondrial Morphology and Function of the Pancreatic ß-Cells INS-1 Model upon Chronic Exposure to Sub-Lethal Cadmium Doses.

The impact of chronic cadmium exposure and slow accumulation on the occurrence and development of diabetes is controversial for human populations. Islets of Langerhans play a prominent role in the etiology of the disease, including by their ability to secrete insulin. Conversion of glucose increase into insulin secretion involves mitochondria. A rat model of pancreatic ß-cells was exposed to largely sub-lethal levels of cadmium cations applied for the longest possible time. Cadmium entered cells at concentrations far below those inducing cell death and accumulated by factors reaching several hundred folds the basal level. The mitochondria reorganized in response to the challenge by favoring fission as measured by increased circularity at cadmium levels already ten-fold below the median lethal dose. However, the energy charge and respiratory flux devoted to adenosine triphosphate synthesis were only affected at the onset of cellular death. The present data indicate that mitochondria participate in the adaptation of ß-cells to even a moderate cadmium burden without losing functionality, but their impairment in the long run may contribute to cellular dysfunction, when viability and ß-cells mass are affected as observed in diabetes.

Mutation in lamin A/C sensitizes the myocardium to exercise-induced mechanical stress but has no effect on skeletal muscles in mouse.

LMNA gene encodes lamin A/C, ubiquitous proteins of the nuclear envelope. They play crucial role in maintaining nuclear shape and stiffness. When mutated, they essentially lead to dilated cardiomyopathy with conduction defects, associated or not with muscular diseases. Excessive mechanical stress sensitivity has been involved in the pathophysiology. We have previously reported the phenotype of Lmna(delK32) mice, reproducing a mutation found in LMNA-related congenital muscular dystrophy patients. Heterozygous Lmna(delK32/+) (Het) mice develop a progressive dilated cardiomyopathy leading to death between 35 and 70 weeks of age. To investigate the sensitivity of the skeletal muscles and myocardium to chronic exercise-induced stress, Het and wild-type (Wt) mice were subjected to strenuous running treadmill exercise for 5 weeks. Before exercise, the cardiac function of Het mice was similar to Wt-littermates. After the exercise-period, Het mice showed cardiac dysfunction and dilation without visible changes in cardiac morphology, molecular remodelling or nuclear structure compared to Wt exercised and Het sedentary mice. Contrary to myocardium, skeletal muscle ex vivo contractile function remained unaffected in Het exercised mice. In conclusion, the expression of the Lmna(delK32) mutation increased the susceptibility of the myocardium to cardiac stress and led to an earlier onset of the cardiac phenotype in Het mice.

Chronic Exposure to Low-Level Cadmium in Diabetes: Role of Oxidative Stress and Comparison with Polychlorinated Biphenyls.

Among the most important physiological functions, maintenance of the oxidation reduction equilibrium in cells stands out as a major homeostatic event. Many environmental contaminants efficiently trap cellular reducing compounds, but the actual importance of this mode of toxicity is far from being precisely known. This statement applies to cases of slowly developing chronic diseases, such as neurodegenerations, diabetes, and many others. The involvement of oxidative challenge in diabetes is considered in connection with chronic dietary exposure to low-level concentrations of cadmium. Comparison is made with polychlorobiphenyl molecules (PCB): they are structurally unrelated to cadmium, they preferentially distribute into different organs than cadmium, and they follow different metabolic pathways. Yet, they have also pro-oxidative properties, and they are associated with diabetes. Since neither cadmium nor PCB is a direct oxidant, they both follow indirect pathways to shift the redox equilibrium. Thus, a difference must be made between the adaptable response of the organism, i.e. the anti-oxidant response, and the irreversible damage generated by oxidizing species, i.e. oxidative damage, when exposure occurs at low concentrations. The approximate border between high and low levels of exposure is estimated in this review from the available relevant data, and the strengths and weaknesses of experimental models are delineated. Eventually, chronic low level exposure to these contaminants sparks cellular responses setting ground for dysfunction and disease, such as diabetes: oxidative damage is an accompanying phenomenon and not necessarily an early mechanism of toxicity.

Expression of sarco (endo) plasmic reticulum calcium ATPase (SERCA) system in normal mouse cardiovascular tissues, heart failure and atherosclerosis.

UNLABELLED: The sarco(endo)plasmic reticulum Ca(2+)ATPases (SERCA) system, a key regulator of calcium cycling and signaling, is composed of several isoforms. We aimed to characterize the expression of SERCA isoforms in mouse cardiovascular tissues and their modulation in cardiovascular pathologies (heart failure and/or atherosclerosis). Five isoforms (SERCA2a, 2b, 3a, 3b and 3c) were detected in the mouse heart and thoracic aorta. Absolute mRNA quantification revealed SERCA2a as the dominant isoform in the heart (~99%). Both SERCA2 isoforms co-localized in cardiomyocytes (CM) longitudinal sarcoplasmic reticulum (SR), SERCA3b was located at the junctional SR. In the aorta, SERCA2a accounted for ~91% of total SERCA and SERCA2b for ~5%. Among SERCA3, SERCA3b was the most expressed (~3.3%), mainly found in vascular smooth muscle cells (VSMC), along with SERCA2a and 2b. In failing CM, SERCA2a was down-regulated by 2-fold and re-localized from longitudinal to junctional SR. A strong down-regulation of SERCA2a was also observed in atherosclerotic vessels containing mainly synthetic VSMCs. The proportion of both SERCA2b and SERCA3b increased to 9.5% and 8.3%, respectively. IN CONCLUSION: 1) SERCA2a is the major isoform in both cardiac and vascular myocytes; 2) the expression of SERCA2a mRNA is ~30 fold higher in the heart compared to vascular tissues; and 3) nearly half the amount of SERCA2a mRNA is measured in both failing cardiomyocytes and synthetic VSMCs compared to healthy tissues, with a relocation of SERCA2a in failing cardiomyocytes. Thus, SERCA2a is the principal regulator of excitation-contraction coupling in both CMs and contractile VSMCs.

Efficient strategies for TALEN-mediated genome editing in mammalian cell lines.

TALEN is one of the most widely used tools in the field of genome editing. It enables gene integration and gene inactivation in a highly efficient and specific fashion. Although very attractive, the apparent simplicity and high success rate of TALEN could be misleading for novices in the field of gene editing. Depending on the application, specific TALEN designs, activity assessments and screening strategies need to be adopted. Here we report different methods to efficiently perform TALEN-mediated gene integration and inactivation in different mammalian cell systems including induced pluripotent stem cells and delineate experimental examples associated with these approaches.

miR-424/322 regulates vascular smooth muscle cell phenotype and neointimal formation in the rat.

AIMS: Our aim was to identify new microRNAs (miRNAs) implicated in pathological vascular smooth muscle cells (VSMCs) proliferation and characterize their mechanism of action. METHODS AND RESULTS: MicroRNAs microarray and qRT-PCR results lead us to focus on miR-424 or its rat ortholog miR-322 (miR-424/322). In vitro mir-424/322 level was decreased shortly after the induction of proliferation and increased in a time-dependent manner later on. In vivo its expression increased in the rat carotid artery from Day 4 up to Day 30 after injury. miR-424/322 overexpression in vitro inhibited proliferation and migration without affecting apoptosis and prevented VSMC dedifferentiation. Furthermore, miR-424/322 overexpression resulted in decreased expression of its predicted targets: cyclin D1 and Ca(2+)-regulating proteins calumenin and stromal-interacting molecule 1 (STIM1). Using reporter luciferase assays, we confirmed that cyclin D1 and calumenin mRNAs were direct targets of miR-322, whereas miR-322 effect on STIM1 was indirect. Nevertheless, consistent with the decreased STIM1 level, the store-operated Ca(2+) entry was reduced. We hypothesized that miR-424/322 could be a negative regulator of proliferation overridden in pathological situations. Thus, we overexpressed miR-424/322 in injured rat carotid arteries using an adenovirus, and demonstrated a protective effect against restenosis. CONCLUSION: Our results demonstrate that miR-424/322 is up-regulated after vascular injury. This is likely an adaptive response to counteract proliferation, although this mechanism is overwhelmed in pathological situations such as injury-induced restenosis.

Voluntary physical activity protects from susceptibility to skeletal muscle contraction-induced injury but worsens heart function in mdx mice.

It is well known that inactivity/activity influences skeletal muscle physiological characteristics. However, the effects of inactivity/activity on muscle weakness and increased susceptibility to muscle contraction-induced injury have not been extensively studied in mdx mice, a murine model of Duchenne muscular dystrophy with dystrophin deficiency. In the present study, we demonstrate that inactivity (ie, leg immobilization) worsened the muscle weakness and the susceptibility to contraction-induced injury in mdx mice. Inactivity also mimicked these two dystrophic features in wild-type mice. In contrast, we demonstrate that these parameters can be improved by activity (ie, voluntary wheel running) in mdx mice. Biochemical analyses indicate that the changes induced by inactivity/activity were not related to fiber-type transition but were associated with altered expression of different genes involved in fiber growth (GDF8), structure (Actg1), and calcium homeostasis (Stim1 and Jph1). However, activity reduced left ventricular function (ie, ejection and shortening fractions) in mdx, but not C57, mice. Altogether, our study suggests that muscle weakness and susceptibility to contraction-induced injury in dystrophic muscle could be attributable, at least in part, to inactivity. It also suggests that activity exerts a beneficial effect on dystrophic skeletal muscle but not on the heart.

Myoblasts and embryonic stem cells differentially engraft in a mouse model of genetic dilated cardiomyopathy.

The functional and architectural benefits of embryonic stem cells (ESC) and myoblasts (Mb) transplantations into infarcted myocardium have been investigated extensively. Whereas ESC repopulated fibrotic areas and contributed to myocardial regeneration, Mb exerted their effects through paracrine secretions and scar remodeling. This therapeutic perspective, however, has been less explored in the setting of nonischemic dilated cardiomyopathies (DCMs). Our aim was to compare the integration and functional efficacy of ESC committed to cardiac fate by bone morphogenic protein 2 (BMP-2) pretreatment and Mb used as gold standard following their transplantation into the myocardium of a mouse model of laminopathy exhibiting a progressive and lethal DCM. After 4 and 8 weeks of transplantation, stabilization was observed in Mb-transplanted mice (P = 0.008) but not in groups of ESC-transplanted or medium-injected animals, where the left ventricular fractional shortening (LVFS) decreased by 32 ± 8% and 41 ± 8% respectively. Engrafted differentiated cells were consistently detected in myocardia of mice receiving Mb, whereas few or no cells were detected in the hearts of mice receiving ESC, except in two cases where teratomas were formed. These data suggest that committed ESC fail to integrate in DCM where scar tissue is absent to provide the appropriate niche, whereas the functional benefits of Mb transplantation might extend to nonischemic cardiomyopathy.

A new method of ultrasonic nonviral gene delivery to the adult myocardium.

Cardiac gene transfer is a powerful molecular tool to improve our understanding of the role of new proteins and mutants in cardiac pathophysiology. There is a need for a simple efficient myocardial gene delivery technique in order to study the physiological role of proteins in their native environment. Here we tested a new method of myocardial nonviral gene delivery, by using the combination of ultrasound energy (USE), liposomes and high pressure injections to the rat heart. Wistar rats were subjected to intra-myocardial injections of liposomes-DNA or siRNA mix. The heart was exposed after an inter-costal incision, and then injections were conducted between two sets of USE heart exposure. Ultrasound application resulted in much higher transfection efficiency (2% of left ventricle) than the liposomes-DNA alone (0.12% of left ventricle) as shown by the beta-galactosidase staining. The ultrasonic based liposomes-DNA delivery resulted in low inflammatory response, as well as in low cardiac fibrosis as shown by total collagen staining. Quantitative real time polymerase chain reaction (PCR) showed that the ultrasonic delivery resulted in cardiac specific transduction. Moreover, 23,906±2197 and 71,883±4065 calcium tolerant transfected cardiac myocytes were isolated following the delivery of a GFP plasmid or tagged siRNA, respectively. This was sufficient to perform single cell physiological measurements and biochemical experiments on homogenates. We developed an interesting safe method for local gene transfer in the heart using ultrasound and liposomes gene delivery. This method is particularly useful to study the effect of gene transfer on cardiac myocytes maintained in their normal environment in animal models.