K. Parekh

Changing pattern of gene expression is associated with ventricular myocyte dysfunction and altered mechanisms of Ca2+ signalling in young type 2 Zucker diabetic fatty rat heart

The association between type 2 diabetes and obesity is very strong, and cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. The aim of this study was to investigate early changes in the pattern of genes encoding cardiac muscle regulatory proteins and associated changes in ventricular myocyte contraction and Ca2+ transport in young (9‐ to 13‐week‐old) type 2 Zucker diabetic fatty (ZDF) rats. The amplitude of myocyte shortening was unaltered; however, time‐to‐peak shortening and time to half‐relaxation of shortening were prolonged in ZDF myocytes (163 ± 5 and 127 ± 7 ms, respectively) compared with age‐matched control rats (136 ± 5 and 103 ± 4 ms, respectively). The amplitude of the Ca2+ transient was unaltered; however, time‐to‐peak Ca2+ transient was prolonged in ZDF myocytes (66.9 ± 2.6 ms) compared with control myocytes (57.6 ± 2.3 ms). The L‐type Ca2+ current was reduced, and inactivation was prolonged over a range of test potentials in ZDF myocytes. At 0 mV, the density of L‐type Ca2+ current was 1.19 ± 0.28 pA pF−1 in ZDF myocytes compared with 2.42 ± 0.40 pA pF−1 in control myocytes. Sarcoplasmic reticulum Ca2+ content, release and uptake and myofilament sensitivity to Ca2+ were unaltered in ZDF myocytes compared with control myocytes. Expression of genes encoding various L‐type Ca2+ channel proteins (Cacna1c, Cacna1g, Cacna1h and Cacna2d1) and cardiac muscle proteins (Myh7) were upregulated, and genes encoding intracellular Ca2+ transport regulatory proteins (Atp2a2 and Calm1) and some cardiac muscle proteins (Myh6, Myl2, Actc1, Tnni3, Tnn2, and Tnnc1) were downregulated in ZDF heart compared with control heart. A change in the expression of genes encoding myosin heavy chain and L‐type Ca2+ channel proteins might partly underlie alterations in the time course of contraction and Ca2+ transients in ventricular myocytes from ZDF rats.

Inhibitory Effects of Salinomycin on Cell Survival, Colony Growth, Migration, and Invasion of Human Non-Small Cell Lung Cancer A549 and LNM35: Involvement of NAG-1.

A major challenge for oncologists and pharmacologists is to develop more potent and less toxic drugs that will decrease the tumor growth and improve the survival of lung cancer patients. Salinomycin is a polyether antibiotic used to kill gram-positive bacteria including mycobacteria, protozoans such as plasmodium falciparum, and the parasites responsible for the poultry disease coccidiosis. This old agent is now a serious anti-cancer drug candidate that selectively inhibits the growth of cancer stem cells. We investigated the impact of salinomycin on survival, colony growth, migration and invasion of the differentiated human non-small cell lung cancer lines LNM35 and A549. Salinomycin caused concentration- and time-dependent reduction in viability of LNM35 and A549 cells through a caspase 3/7-associated cell death pathway. Similarly, salinomycin (2.5–5 µM for 7 days) significantly decreased the growth of LNM35 and A549 colonies in soft agar. Metastasis is the main cause of death related to lung cancer. In this context, salinomycin induced a time- and concentration-dependent inhibition of cell migration and invasion. We also demonstrated for the first time that salinomycin induced a marked increase in the expression of the pro-apoptotic protein NAG-1 leading to the inhibition of lung cancer cell invasion but not cell survival. These findings identify salinomycin as a promising novel therapeutic agent for lung cancer.

Frondoside A enhances the antiproliferative effects of gemcitabine in pancreatic cancer

Pancreatic cancer has a very poor prognosis. While gemcitabine is the mainstay of therapy and improves quality of life, it has little impact on survival. More effective treatments are desperately needed for this disease. Frondoside A is a triterpenoid glycoside isolated from the Atlantic sea cucumber, Cucumaria frondosa. Frondoside A potently inhibits pancreatic cancer cell growth and induces apoptosis in vitro and in vivo. The aim of the present study was to investigate whether frondoside A could enhance the anti-cancer effects of gemcitabine. Effects of frondoside A and gemcitabine alone and in combination on proliferation were investigated in two human pancreatic cancer cell lines, AsPC-1 and S2013. To investigate possible synergistic effects, combinations of low concentrations of the two drugs were used for a 72 h treatment period in vitro. Growth inhibition was significantly greater with the drug combinations than their additive effects. Combinations of frondoside A and gemcitabine were tested in vivo using the athymic mouse model. Xenografts of AsPC-1 and S2013 cells were allowed to form tumours prior to treatment with the drugs alone or in combination for 30 days. Tumours grew rapidly in placebo-treated animals. Tumour growth was significantly reduced in all treatment groups. At the lowest dose tested, gemcitabine (4 mg/kg/dose), combined with frondoside A (100 μg/kg/day) was significantly more effective than with either drug alone. To conclude: The present data suggest that combinations of frondoside A and gemcitabine may provide clinical benefit for patients with pancreatic cancer.

The Effects of Different Repetitive Transcranial Magnetic Stimulation (rTMS) Protocols on Cortical Gene Expression in a Rat Model of Cerebral Ischemic-Reperfusion Injury.

Although repetitive Transcranial Magnetic Stimulation (rTMS) in treatment of stroke in humans has been explored over the past decade the data remain controversial in terms of optimal stimulation parameters and the mechanisms of rTMS long-term effects. This study aimed to explore the potential of different rTMS protocols to induce changes in gene expression in rat cortices after acute ischemic-reperfusion brain injury. The stroke was induced by middle cerebral artery occlusion (MCAO) with subsequent reperfusion. Changes in the expression of 96 genes were examined using low-density expression arrays after MCAO alone and after MCAO combined with 1Hz, 5Hz, continuous (cTBS) and intermittent (iTBS) theta-burst rTMS. rTMS over the lesioned hemisphere was given for two weeks (with a 2-day pause) in a single daily session and a total of 2400 pulses. MCAO alone induced significant upregulation in the expression of 44 genes and downregulation in 10. Two weeks of iTBS induced significant increase in the expression of 52 genes. There were no downregulated genes. 1Hz and 5Hz had no significant effects on gene expression, while cTBS effects were negligible. Upregulated genes included those involved in angiogenesis, inflammation, injury response and cellular repair, structural remodeling, neuroprotection, neurotransmission and neuronal plasticity. The results show that long-term rTMS in acute ischemic-reperfusion brain injury induces complex changes in gene expression that span multiple pathways, which generally promote the recovery. They also demonstrate that induced changes primarily depend on the rTMS frequency (1Hz and 5Hz vs. iTBS) and pattern (cTBS vs. iTBS). The results further underlines the premise that one of the benefits of rTMS application in stroke may be to prime the brain, enhancing its potential to cope with the injury and to rewire. This could further augment its potential to favorably respond to rehabilitation, and to restore some of the loss functions.

Functional capacity of macrophages determines the induction of type 1 diabetes.

Abstract:  Macrophages are potent immune regulators and are critical in the development and pathogenesis of autoimmune diabetes. They are said to be the first cell type to infiltrate the pancreatic islet, serve as antigen‐presenting cells, and are important as effector cells during diabetogenesis. The article examines the role of macrophages in autoimmune diabetes with particular emphasis on the role of galectin‐3, a β‐galactoside‐binding lectin, and T1/ST2, an IL‐1 receptor‐like protein, both of which play significant roles in the immunomodulatory functions of macrophages. Multiple low‐dose streptozotocin (MLD‐STZ) induces infiltration of mononuclear cells in the islets of susceptible strains leading to insulitis. Deletion of the galectin‐3 gene from C57BL/6 mice significantly attenuates this effect as evaluated by quantitative histology of mononuclear cells and loss of insulin‐producing β cells. In contrast, deletion of the ST2 gene enhanced insulitis after MLD‐STZ treatment when compared with relatively resistant wild‐type BALB/c mice. Thus, it appears that functional capacity of macrophages influences their participation in T helper (Th) 1‐mediated autoimmunity and the development of autoimmune diabetogenesis.

Effects of a sucrose‐enriched diet on the pattern of gene expression, contraction and Ca2+ transport in Goto–Kakizaki type 2 diabetic rat heart

What is the central question of this study? Poor diet is a risk factor for development of type 2 diabetes mellitus and its associated complications. In this study, the effects of sucrose‐enriched diet on the pattern of gene expression, contraction and Ca2+ transport in type 2 diabetic heart are explored. What is the main finding and its importance? The altered pattern of gene expression in type 2 diabetic hearts was further altered in diabetic and control rats that received a sucrose‐enriched diet, and these alterations were associated with changes in ventricular myocyte shortening and Ca2+ transport.

Shortening and intracellular Ca2+ in ventricular myocytes and expression of genes encoding cardiac muscle proteins in early onset type 2 diabetic Goto–Kakizaki rats

There has been a spectacular rise in the global prevalence of type 2 diabetes mellitus. Cardiovascular complications are the major cause of morbidity and mortality in diabetic patients. Contractile dysfunction, associated with disturbances in excitation–contraction coupling, has been widely demonstrated in the diabetic heart. The aim of this study was to investigate the pattern of cardiac muscle genes that are involved in the process of excitation–contraction coupling in the hearts of early onset (8–10 weeks of age) type 2 diabetic Goto–Kakizaki (GK) rats. Gene expression was assessed in ventricular muscle with real‐time RT‐PCR; shortening and intracellular Ca2+ were measured in ventricular myocytes with video edge detection and fluorescence photometry, respectively. The general characteristics of the GK rats included elevated fasting and non‐fasting blood glucose and blood glucose at 120 min following a glucose challenge. Expression of genes encoding cardiac muscle proteins (Myh6/7, Mybpc3, Myl1/3, Actc1, Tnni3, Tnn2, Tpm1/2/4 and Dbi) and intercellular proteins (Gja1/4/5/7, Dsp and Cav1/3) were unaltered in GK ventricle compared with control ventricle. The expression of genes encoding some membrane pumps and exchange proteins was unaltered (Atp1a1/2, Atp1b1 and Slc8a1), whilst others were either upregulated (Atp1a3, relative expression 2.61 ± 0.69 versus 0.84 ± 0.23) or downregulated (Slc9a1, 0.62 ± 0.07 versus 1.08 ± 0.08) in GK ventricle compared with control ventricle. The expression of genes encoding some calcium (Cacna1c/1g, Cacna2d1/2d2 and Cacnb1/b2), sodium (Scn5a) and potassium channels (Kcna3/5, Kcnj3/5/8/11/12, Kchip2, Kcnab1, Kcnb1, Kcnd1/2/3, Kcne1/4, Kcnq1, Kcng2, Kcnh2, Kcnk3 and Kcnn2) were unaltered, whilst others were either upregulated (Cacna1h, 0.95 ± 0.16 versus 0.47 ± 0.09; Scn1b, 1.84 ± 0.16 versus 1.11 ± 0.11; and Hcn2, 1.55 ± 0.15 versus 1.03 ± 0.08) or downregulated (Hcn4, 0.16 ± 0.03 versus 0.37 ± 0.08; Kcna2, 0.35 ± 0.03 versus 0.80 ± 0.11; Kcna4, 0.79 ± 0.25 versus 1.90 ± 0.26; and Kcnj2, 0.52 ± 0.07 versus 0.78 ± 0.08) in GK ventricle compared with control ventricle. The amplitude of ventricular myocyte shortening and the intracellular Ca2+ transient were unaltered; however, the time‐to‐peak shortening was prolonged and time‐to‐half decay of the Ca2+ transient was shortened in GK myocytes compared with control myocytes. The results of this study demonstrate changes in expression of genes encoding various excitation–contraction coupling proteins that are associated with disturbances in myocyte shortening and intracellular Ca2+ transport.

Pharmacokinetics in Mouse and Comparative Effects of Frondosides in Pancreatic Cancer.

The frondosides are triterpenoid glycosides from the Atlantic sea cucumber Cucumaria frondosa. Frondoside A inhibits growth, invasion, metastases and angiogenesis and induces apoptosis in diverse cancer types, including pancreatic cancer. We compared the growth inhibitory effects of three frondosides and their aglycone and related this to the pharmocokinetics and route of administration. Frondoside A potently inhibited growth of pancreatic cancer cells with an EC50 of ~1 µM. Frondoside B was less potent (EC50 ~2.5 µM). Frondoside C and the aglycone had no effect. At 100 µg/kg, frondoside A administered to CD2F1 mice as an i.v. bolus, the Cpmax was 129 nM, Cltb was 6.35 mL/min/m2, and half-life was 510 min. With i.p. administration the Cpmax was 18.3 nM, Cltb was 127 mL/min/m2 and half-life was 840 min. Oral dosing was ineffective. Frondoside A (100 µg/kg/day i.p.) markedly inhibited growth cancer xenografts in nude mice. The same dose delivered by oral gavage had no effect. No evidence of acute toxicity was seen with frondoside A. Frondoside A is more potent inhibitor of cancer growth than other frondosides. The glycoside component is essential for bioactivity. Frondoside A is only effective when administered systemically. Based on the current and previous studies, frondoside A appears safe and may be valuable in the treatment of cancer.

Structural lesions and changing pattern of expression of genes encoding cardiac muscle proteins are associated with ventricular myocyte dysfunction in type 2 diabetic Goto-Kakizaki rats fed a high-fat diet.

Given the clinical prevalence of type 2 diabetes and obesity and their association with high mortality linked to cardiovascular disease, the aim of the study was to investigate the effects of feeding type 2 diabetic Goto–Kakizaki (GK) rats either high‐ or low‐fat diets on cardiomyocyte structure and function. The GK rats were fed either a high‐fat diet (HFD) or a low‐fat diet (LFD) from the age of 2 months for a period of 7 months. The GK‐HFD rats gained more weight, ate less food and drank less water compared with GK‐LFD rats. At 7 months, non‐fasting blood glucose was higher in GK‐LFD (334 ± 35 mg dl−1) compared with GK‐HFD rats (235 ± 26 mg dl−1). Feeding GK rats with a HFD had no significant effect on glucose clearance following a glucose challenge. Time‐to‐peak (tpeak) shortening was reduced in myocytes from GK‐HFD (131.8 ± 2.1 ms) compared with GK‐LFD rats (144.5 ± 3.0 ms), and time‐to‐half (t1/2) relaxation of shortening was also reduced in myocytes from GK‐HFD (71.7 ± 6.9 ms) compared with GK‐LFD rats (86.1 ± 3.6 ms). The HFD had no significant effect on the amplitude of shortening. The HFD had no significant effect on tpeak, t1/2 decay, amplitude of the Ca2+ transient, myofilament sensitivity to Ca2+, sarcoplasmic reticulum Ca2+ content, fractional release of Ca2+ and the rate of Ca2+ uptake. Structurally, ventricular myocytes from GK‐HFD rats showed extensive mitochondrial lesions, including swelling, loss of cristae, and loss of inner and outer membranes, resulting in gross vacuolarization and deformation of ventricular mitochondria with a subsequent reduction in mitochondrial density. Expression of genes encoding various L‐type Ca2+ channel proteins (Cacnb2) and cardiac muscle proteins (Myl2 and Atp2a1) were downregulated in GK‐HFD compared with GK‐LFD rats. Structural lesions and changed expression of genes encoding various cardiac muscle proteins might partly underlie the altered time course of myocyte shortening and relaxation in myocytes from GK‐HFD compared with GK‐LFD rats.

Different Profile of mRNA Expression in Sinoatrial Node from Streptozotocin-Induced Diabetic Rat

Background Experiments in isolated perfused heart have shown that heart rate is lower and sinoatrial node (SAN) action potential duration is longer in streptozotocin (STZ)–induced diabetic rat compared to controls. In sino-atrial preparations the pacemaker cycle length and sino-atrial conduction time are prolonged in STZ heart. To further clarify the molecular basis of electrical disturbances in the diabetic heart the profile of mRNA encoding a wide variety of proteins associated with the generation and transmission of electrical activity has been evaluated in the SAN of STZ-induced diabetic rat heart. Methodology/Principal Findings Heart rate was measured in isolated perfused heart with an extracellular suction electrode. Expression of mRNA encoding a variety of intercellular proteins, intracellular Ca2+-transport and regulatory proteins, cell membrane transport proteins and calcium, sodium and potassium channel proteins were measured in SAN and right atrial (RA) biopsies using real-time reverse transcription polymerase chain reaction techniques. Heart rate was lower in STZ (203±7 bpm) compared to control (239±11 bpm) rat. Among many differences in the profile of mRNA there are some worthy of particular emphasis. Expression of genes encoding some proteins were significantly downregulated in STZ-SAN: calcium channel, Cacng4 (7-fold); potassium channel, Kcnd2 whilst genes encoding some other proteins were significantly upregulated in STZ-SAN: gap junction, Gjc1; cell membrane transport, Slc8a1, Trpc1, Trpc6 (4-fold); intracellular Ca2+-transport, Ryr3; calcium channel Cacna1g, Cacna1h, Cacnb3; potassium channels, Kcnj5, Kcnk3 and natriuretic peptides, Nppa (5-fold) and Nppb (7-fold). Conclusions/Significance Collectively, this study has demonstrated differences in the profile of mRNA encoding a variety of proteins that are associated with the generation, conduction and regulation of electrical signals in the SAN of STZ-induced diabetic rat heart. Data from this study will provide a basis for a substantial range of future studies to investigate whether these changes in mRNA translate into changes in electrophysiological function.