Comparison of sarcoplasmic reticulum Ca2+-ATPase function in human,dog, rabbit,and mouse ventricular myocytes

It has been reported that sarcoplasmic reticulum (SR) Ca(2+) uptake is more rapid in rat than rabbit ventricular myocytes, but little information is available on the relative SR Ca(2+) uptake activity in others species, including humans. We induced Ca(2+) transients with a short caffeine pulse protocol (rapid solution switcher, 10 mM caffeine, 100 ms) in single ventricular myocytes voltage clamped (-80 mV) with pipettes containing 100 microM fluo-3 and nominal 0 Ca(2+), in 0 Na(+)(o)/0 Ca(2+)(o) solution to inhibit Na/Ca exchange. SR in non-paced human, dog, rabbit, and mouse ventricular myocytes could be readily loaded with Ca(2+) under our experimental conditions with a pipette [Ca(2+)] = 100 nM. Resting [Ca(2+)](i) was similar in four types of ventricular myocytes. Activation of the Ca(2+)-release channel with a 100-ms caffeine pulse produced a rise in [caffeine](i) to slightly above 2 mM, the threshold for caffeine activation of Ca(2+) release. This caused a similar initial rate of rise and peak [Ca(2+)](i) in the four types of ventricular myocytes. However, there were significant differences in the duration of the plateau (top 10%) [Ca(2+)](i) transients and the time constant of the [Ca(2+)](i) decline (reflecting activity of the SR Ca(2+)-ATPase), with values for human > dog > rabbit > mouse. In paced myocytes under physiologic conditions, SR Ca(2+) content was greater in mouse than in rabbit myocytes, while peak I(Ca,L) was smaller in mouse. These findings confirm substantial species difference in SR Ca(2+)-ATPase activity, and suggest that the smaller the animal and the more rapid the heart rate, greater the activity of the SR Ca(2+)-ATPase. In addition, it appears that substantial species differences exist in the degree of SR Ca(2+) loading and I(Ca,L) under physiologic conditions.     

Channel microband electrodes: a complete working surface for potential step transients

The strongly implicit procedure is used to simulate the chronoamperometric transient resulting from a potential step at a channel microband electrode. A working surface is presented which permits the analysis of data recorded at any flow rate, including cases where axial diffusion effects are significant, and for cells of arbitrary geometry.     

Defective intracellular Ca2+ homeostasis contributes to myocyte dysfunction during ventricular remodelling induced by chronic volume overload in rats

1. Previous studies have demonstrated progressive ventricular hypertrophy, dilatation and contractile depression in response to chronic volume overload. Whether this decompensation was related to intrinsic myocyte dysfunction was not clear. The present study evaluated ventricular myocyte function at critical times during the progression of ventricular remodelling induced by volume overload. 2. Chronic volume overload was induced with an infrarenal aortocaval fistula in rats. Myocyte contraction and intracellular Ca(2+) concentrations ([Ca(2+)](i)) were evaluated using a fura-2 fluorescence and edge detection system. Protein levels of sarcoplasmic reticulum (SR) Ca(2+) transporters were determined by western blots. Progressive ventricular dilatation developed following creation of the fistula. Although myocyte function in 5 week fistula rats was comparable to that of the control group, myocytes from rats 10 weeks post-fistula demonstrated significant depression of cell shortening and peak [Ca(2+)](i). Application of isoproterenol (0.1 micromol/L) was not able to compensate for the functional deficiency in myocytes from 10 week fistula rats. Caffeine (10 mmol/L) induced SR Ca(2+) release, as well as protein expression of SR Ca(2+)-ATPase, and ryanodine receptors were reduced in myocytes obtained from the same group of 10 week fistula rats. 3. These data indicate that the transition to heart failure secondary to chronic volume overload is related to depressed myocyte contractility secondary to altered intracellular Ca(2+) homeostasis.     

Reaction times to chromatic stimuli

The relative contributions of chromatic and achromatic activity to reaction time were investigated under conditions in which spatial and temporal transients were manipulated. Simple reaction times (RT) were obtained to eight photometrically matched (1 cd/m2) wavelengths between 448 and 658 nm. These stimuli were incrementally presented on a spatially coextensive 1.2 degrees white adapting field. RT was wavelength dependent for 5 or 10 cd/m2 steady backgrounds or a 5 Hz 1.2 cd/m2 flickering background. RT varied as a trichromatic saturation-like function (slowest RT at 572 nm). In comparison, RT was wavelength independent when no adapting field was present and when the 1.2 cd/m2 field was flickered at 15 Hz.     

Effects of adenosine on Ca2+ transients and tension in aequorin-injected ferret papillary muscles

The effects of adenosine on Ca²⁺ transients and tension in ferret papillary muscles were investigated using the aequorin method. Adenosine (0.01–1 mM) reduced the peak of Ca²⁺ transients and caused a slight concentration-dependent decrease in tension. Adenosine prolonged the decay time of Ca²⁺ transients but did not alter the time course of tension. In isoproterenol (0.1 M)-treated preparations, adenosine decreased the peak of Ca²⁺ transients but did not alter the peak of tension. Adenosine prolonged the isoproterenol-shortened decay time of Ca²⁺ transients. The effects of adenosine on Ca²⁺ transients were antagonized by the selective A₁ receptor antagonist 8-cyclopentyl-1,3,-dipropylxanthine. In the presence of isoproterenol, adenosine (0.1 mM) shifted the intracellular [Ca²⁺]/tension relation to the left. These results can be explained by the hypothesis that adenosine inhibits the activity of adenylate cyclase via stimulation of the A₁ receptor, other mechanisms however cannot be overlooked. The prolongation of the decay time of Ca²⁺ transients and the increase in the Ca²⁺ sensitivity of the contractile elements are the underlying mechanisms of adenosine which maintain developed tension in twitch response, although adenosine decreases the peak of Ca²⁺ transients.     

Functional characterization of cardiac progenitor cells and their derivatives in the embryonic heart post‐chamber formation

There is scant information on the fate of cardiac progenitor cells (CPC) in the embryonic heart after chamber specification. Here we simultaneously tracked three lineage‐specific markers (Nkx2.5, MLC2v, and ANF) and confirmed that CPCs with an Nkx2.5⁺MLC2v^?ANF^? phenotype are present in the embryonic (E) day 11.5 mouse ventricular myocardium. We demonstrated that these CPCs could give rise to working cardiomyocytes and conduction system cells. Using a two‐photon imaging analysis, we found that the majority of CPCs are not capable of developing Ca²⁺ transients in response to β‐adrenergic receptor stimulation. In contrast, Nkx2.5⁺ cells expressing MLC2v but not ANF are capable of developing functional Ca²⁺ transients. We showed that Ca²⁺ transients could be invoked in Nkx2.5⁺MLC2v⁺ANF⁺ cells only upon inhibition of Gi, muscarinic receptors, or nitric oxide synthase (NOS) signaling pathways. Our data suggest that these inhibitory pathways may delay functional specification in a subset of developing ventricular cells. Developmental Dynamics 238:2787–2799, 2009. © 2009 Wiley‐Liss, Inc.     

Automated detection of intercellular signaling in astrocyte networks using the converging squares algorithm

Intercellular calcium waves in central nervous system astrocyte networks underline the principle mechanism of cell signaling in astrocyte syncsytiums, which putatively contribute to the modulation of neuronal signaling and metabolic regulation. In support of carrying out systems level analyses of astrocyte networks, we have optimized and validated the converging squares image segmentation algorithm to automatically detect the relative spatial locations of all cells in a visible network as a preliminary step towards analyzing the dynamics of astrocyte intracellular calcium transients, which are the signals that mediate intercellular calcium waves. We used the temporal derivatives of pixel intensities as the data source for the algorithm. The method works by converging progressively smaller squares until the signal peak is reached. It is robust to noise and performs comparably to manual cell signal identification, but is much faster and efficient. This is the first reported application of this algorithm to glial networks that we are aware of.     

Ca2+‐dependent ion channels underlying spontaneous activity in insect circadian pacemaker neurons

Electrical activity in the gamma frequency range is instrumental for temporal encoding on the millisecond scale in attentive vertebrate brains. Surprisingly, also circadian pacemaker neurons in the cockroach Rhyparobia maderae (Leucophaea maderae) employ fast spontaneous rhythmic activity in the gamma band frequency range (20–70 Hz) together with slow rhythmic activity. The ionic conductances controlling this fast spontaneous activity are still unknown. Here, Ca²⁺ imaging combined with pharmacology was employed to analyse ion channels underlying spontaneous activity in dispersed circadian pacemakers of the adult accessory medulla, which controls circadian locomotor activity rhythms. Fast spontaneous Ca²⁺ transients in circadian pacemakers accompany tetrodotoxin (TTX)‐blockable spontaneous action potentials. In contrast to vertebrate pacemakers, the spontaneous depolarisations from rest appear to be rarely initiated via TTX‐sensitive sustained Na⁺ channels. Instead, they are predominantly driven by mibefradil‐sensitive, low‐voltage‐activated Ca²⁺ channels and DK‐AH269‐sensitive hyperpolarisation‐activated, cyclic nucleotide‐gated cation channels. Rhythmic depolarisations activate voltage‐gated Na⁺ channels and nifedipine‐sensitive high‐voltage‐activated Ca²⁺ channels. Together with Ca²⁺ rises, the depolarisations open repolarising small‐conductance but not large‐conductance Ca²⁺‐dependent K⁺ channels. In contrast, we hypothesise that P/Q‐type Ca²⁺ channels coupled to large‐conductance Ca²⁺‐dependent K⁺ channels are involved in input‐dependent activity.