Non-technical summary When under stress, the heart beat becomes stronger, in part due to enhanced fluxes of Ca 2+ 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 Ca 2+ 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 Ca 2+ 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.Abstract The sympathetic adrenergic system plays a central role in stress signalling and stress is often associated with increased production of reactive oxygen species (ROS). Furthermore, the sympathetic adrenergic system is intimately involved in the regulation of cardiomyocyte Ca 2+ handling and contractility. In this study we hypothesize that endogenously produced ROS contribute to the inotropic mechanism of β-adrenergic stimulation in mouse cardiomyocytes. Cytoplasmic Ca 2+ transients, cell shortening and ROS production were measured in freshly isolated cardiomyocytes using confocal microscopy and fluorescent indicators. As a marker of oxidative stress, malondialdehyde (MDA) modification of proteins was detected with Western blotting. Isoproterenol (ISO), a β-adrenergic agonist, increased mitochondrial ROS production in cardiomyocytes in a concentration-and cAMP-protein kinase A-dependent but Ca 2+ -independent manner. Hearts perfused with ISO showed a twofold increase in MDA protein adducts relative to control. ISO increased Ca 2+ transient amplitude, contraction and L-type Ca 2+ current densities (measured with whole-cell patch-clamp) in cardiomyocytes and these increases were diminished by application of the general antioxidant N -acetylcysteine (NAC) or the mitochondria-targeted antioxidant SS31. In conclusion, increased mitochondrial ROS production plays an integral role in the acute inotropic response of cardiomyocytes to β-adrenergic stimulation. On the other hand, chronically sustained adrenergic stress is associated with the development of heart failure and cardiac arrhythmias and prolonged increases in ROS may contribute to these defects.

Obesity and insulin resistance are associated with enhanced fatty acid utilization, which may play a central role in diabetic cardiomyopathy. We now assess the effect of the saturated fatty acid palmitate (1.2 mmol/l) on Ca 2؉ handling, cell shortening, and mitochondrial production of reactive oxygen species (ROS) in freshly isolated ventricular cardiomyocytes from normal (wild-type) and obese, insulin-resistant ob/ob mice. Cardiomyocytes were electrically stimulated at 1 Hz, and the signal of fluorescent indicators was measured with confocal microscopy. Palmitate decreased the amplitude of cytosolic Ca 2؉ transients (measured with fluo-3), the sarcoplasmic reticulum Ca 2؉ load, and cell shortening by ϳ20% in wild-type cardiomyocytes; these decreases were prevented by the general antioxidant N-acetylcysteine. In contrast, palmitate accelerated Ca 2؉ transients and increased cell shortening in ob/ob cardiomyocytes. Application of palmitate rapidly dissipated the mitochondrial membrane potential (measured with tetra-methyl rhodamine-ethyl ester) and increased the mitochondrial ROS production (measured with MitoSOX Red) in wild-type but not in ob/ob cardiomyocytes. In conclusion, increased saturated fatty acid levels impair cellular Ca 2؉ handling and contraction in a ROSdependent manner in normal cardiomyocytes. Conversely, high fatty acid levels may be vital to sustain cardiac Ca 2؉ handling and contraction in obesity and insulin-resistant conditions. Diabetes 56: 1136 -1142, 2007 C ardiac muscle cells generate ATP at a high rate to support the continuous contractile function of the beating heart. Cardiac cells use various substrates to generate ATP, and the extent of substrates utilized depends on the substrate availability, the energy demand, and the physiological or pathological condition (1). In humans as well as in different animal models, obesity, insulin resistance, and type 2 diabetes are associated with an altered cardiac metabolism characterized by an enhanced reliance on fatty acids and a decreased glucose utilization. These changes play a central role in the development of diabetic cardiomyopathy (2). For instance, application of the saturated fatty acid palmitate had markedly different effects on power output and oxygen consumption in hearts of control mice and ob/ob mice, which are obese, insulin resistant, and have increased serum free fatty acid concentrations (3). Moreover, ob/ob hearts displayed a decreased mitochondrial oxidative capacity and an increased fatty acid-induced mitochondrial uncoupling (4).Cellular Ca 2ϩ handling is altered in type 2 diabetes (5), and diabetic cardiomyopathy is characterized by defective sarcoplasmic reticulum (SR) function, which results in smaller and slower cytoplasmic Ca 2ϩ transients (6,7). Mitochondria play a central role in the development of diabetes complications, and the mitochondrial dysfunction is characterized by decreased mitochondrial Ca 2ϩ loading capacity and increased production of reactive oxygen species (ROS) (8 -10). Increased ROS production...

Mammals exposed to a cold environment initially generate heat by repetitive muscle activity (shivering).

The involvement of Ca2+ in insulin‐mediated glucose uptake in skeletal muscle is uncertain. Here we study the possible role of Ca2+ influx via canonical transient receptor potential 3 (TRPC3) channels in insulin‐mediated glucose uptake. Experiments were performed on adult skeletal mouse muscle fibers. Ca2+ influx and glucose uptake were measured with fluorescent indicators and confocal microscopy. TRPC3 protein expression was knocked down using a novel technique where functionalized carbon nanotubes were used to transfect cells with small interfering RNA. The interaction between TRPC3 and the glucose transporter 4 (GLUT4) was studied with immunoprecipitation and immunofluorescence staining. Knock down of TRPC3 resulted in ∼ 80% decrease in insulin‐mediated glucose uptake. TRPC3 can be activated by diacylglycerol (DAG) and knock down of TRPC3 inhibited the DAG‐induced Ca2+ influx. TRPC3 and GLUT4 co‐immunoprecipitated and showed co‐localization in the proximity of the t‐tubular system, which is the major site of insulin‐mediated glucose transport. In conclusion, TRPC3 interacts functionally and physically with GLUT4 and Ca2+ influx through TRPC3 has a large impact on insulin‐mediated glucose uptake.

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 mM lactic acid nor increasing the [K + ] of the bath solution from 5 to 10 mM had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 mM [K + ]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca 2+ ] ([Ca 2+ ] i ). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca 2+ ] i , compared with an increase of ∼20% without cyanide. In conclusion, lactic acid or [K + ] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca 2+release from the sarcoplasmic reticulum.