Elinor J. Griffiths

Mitochondria — potential role in cell life and death

See article by Mathur et al. [10] (pages 126–138) in this issue. nnThe 1990s saw a huge resurgence of interest in the role of mitochondria within cells, recognition that, as well as their traditional role as ‘powerhouses’ of the cell in generating ATP, mitochondria play an important role in other aspects of normal cell functioning, for example in cell calcium signalling. But possibly the greatest interest has been in the emerging role of mitochondria as regulators of the cell life–death transition, in both necrotic and apoptotic forms of cell death (for recent reviews see [1–3]). Changes in mitochondrial membrane potential (Δ Ψ m) are integral to the cell life–death transition [4], although whether as a primary cause or secondary event is as yet not known. Thus, measurements of Δ Ψ m can greatly facilitate our understanding of this process.nnIn physiological cell functioning, maintenance of Δ Ψ m is essential for ATP synthesis, as has been known for many years. Δ Ψ m is highly negative, approximately −180 mV, due to the chemiosmotic gradient of protons across the inner mitochondrial membrane, the energy of which is used to synthesise ATP by the respiratory chain. Δ Ψ m also provides the driving force for Ca2+ uptake into mitochondria by the Ca2+-uniporter, and it is now generally accepted that it is this Ca2+ signal in the mitochondria which stimulates ATP production in response to an increased demand energy by the cell (reviewed in [5]). However, under certain conditions, most notably oxidative stress and ATP depletion, excessive uptake of Ca2+ by mitochondria can trigger the mitochondrial permeability transition (MPT), where a non-specific pore opens in the mitochondrial membrane (reviewed in [1,2]). The MPT causes collapse of Δ Ψ m, so ATP cannot be resynthesised. Opening of … nn* Tel.: +44-117-928-3586; fax: +44-117-928-3581 elinor.griffiths{at}bristol.ac.uk

Reversal of mitochondrial Na/Ca exchange during metabolic inhibition in rat cardiomyocytes

During hypoxia of isolated cardiomyocytes, Ca2+ entry into mitochondria may occur via the Na/Ca exchanger, the normal efflux pathway, and not the Ca‐uniporter, the normal influx route. If this is the case, then depletion of myocyte Na+ should inhibit Ca2+ uptake, and collapse of the mitochondrial membrane potential (Δψm) would inhibit the uniporter. To test these hypotheses, isolated rat myocytes were exposed to metabolic inhibition, to mimic hypoxia, and [Ca2+]m and [Ca2+]c determined by selective loading of indo‐1 into these compartments. Δψm was determined using rhodamine 123. Following metabolic inhibition, [Ca2+]m was significantly lower in Na‐depleted cells than controls (P<0.001), [Ca2+]c was approximately the same in both groups, and mitochondria depolarised completely. Thus Na‐depletion inhibited mitochondrial Ca2+ uptake, suggesting that Ca2+ entry occurred via Na/Ca exchange, and the collapse of Δψm during metabolic inhibition is consistent with inactivity of the Ca‐uniporter.

Use of ruthenium red as an inhibitor of mitochondrial Ca2+ uptake in single rat cardiomyocytes

With the current resurgence of interest in the role of mitochondrial [Ca2+] in energy production and cellular Ca2+ signalling, ruthenium red (RR) is being increasingly used as an inhibitor of mitochondrial Ca2+ uptake. In the present study, the effects of RR on cell and mitochondrial [Ca2+], and on cell contractility were determined in isolated rat ventricular myocytes subjected to adrenergic and electrical stimulation. At low concentrations, 0–1 μM, RR inhibited mitochondrial Ca2+ uptake but this was a secondary effect due to a reduced total intracellular [Ca2+], a conclusion supported by the ability of RR to inhibit cell shortening. 5 μM RR completely inhibited cell contraction, whereas higher concentrations, 10–25 μM, induced spontaneous Ca2+ oscillations and contractile waves. These results indicate that great care must be taken when using RR in intact cells, and in interpreting any effects as resulting from a primary inhibition of mitochondrial Ca2+ uptake.

NADH Fluorescence in Isolated Guinea-Pig and Rat Cardiomyocytes Exposed to Low or High Stimulation Rates and Effect of Metabolic Inhibition with Cyanide

In this study we investigated whether NADH fluorescence levels changed in response to low or high rates of electrical stimulation in single ventricular myocytes isolated from rat and guinea-pig hearts, either during a single contraction or upon sustained electrical stimulation of cells. NADH levels were determined from cell autofluorescence and cell length monitored using an edge-tracking device. NADH/NAD+ was obtained by addition of cyanide, 100% NADH, and carbonylcyanide-p-trifluoromethoxy phenylhydrazone (FCCP), 100% NAD+. Rat myocytes exhibited slightly higher resting fluorescence levels than guinea-pig cells; however, NADH/NAD+ was higher in rat than guinea-pig cells (P < 0.05), 24.3+/-4.3 (N = 17) vs 14.6+/-1.6 (N = 17), respectively. There was no change in NADH fluorescence during a single contraction when cells were stimulated at either low (0.2 Hz) or high (3 Hz) rates in either species. Furthermore, NADH levels did not change upon sustained stimulation at 3 Hz in either species. Metabolic blockade with cyanide induced a dose dependent rise in NADH fluorescence which was similar for both rat and guinea-pig myocytes and reached a maximum at > or = 1 mM of cyanide. Although a full recovery of NADH fluorescence was seen in both types of cells after brief exposure to cyanide, the rate of recovery was significantly slower in rat myocytes; times to 90% recovery were 110+/-29 sec, N = 6, and 264+/-50 sec, N = 6, for guinea-pig and rat cells, respectively. This work demonstrates that although rat and guinea-pig myocytes have different resting NADH/NAD+, their response to electrical stimulation is the same, whereas in response to metabolic inhibition subtle differences are seen.

Human cardiac myosin autoantibodies impair myocyte contractility: a cause-and-effect relationship

The functional relevance of autoanti‐bodies (Abs) against cardiac myosin (CM) in clinical idiopathic dilated cardiomyopathy (DCM) remains controversial. The study sought to determine effects of human Abs affinity‐purified (AF) by immunoaffinity column chromotography on excitation‐contraction coupling in isolated myocytes. Effects of CM‐Abs from heart failure patients with DCM (n=19) and ischemic heart disease (IHD, n=19) on contractility, L‐type Ca2+ current, and Ca2+ transients in continuously perfused rat ventricular myocytes were studied. Immunofluorescence studies using confocal microscopy were carried out to determine whether Abs were internalized. AF‐Abs from either group did not differ in IgG titer but differed in their elution profiles. The IgG3 subclass response was higher in AF fractions from DCM (21%) than IHD (5%) patients. The Abs reduced the capacity of field‐stimulated myocytes to contract in a dose‐dependent manner. Inhibition of contraction, as a percentage of untreated cells, was greater with DCM than IHD‐Abs (P=0.004), and the effect was independent of Ab titer. An increase in frequency of the beating myocytes (0.2 to 3.0 Hz) raised peak systolic and diastolic levels of [Ca2+]i of cells treated with DCM but not IHD‐Abs (P<0.005). The AF‐Abs were not internalized by myocytes and had no effect on L‐type Ca2+ currents. The altered sensitivity of the myofilaments to [Ca2+]i by CM‐Abs may represent a potential mechanism of autoantibody‐mediated impairment in clinical DCM.‐Warraich, R. S., Griffiths, E., Falconar, A., Pabbathi, V., Bell, C., Angelini, G., Suleiman, M.‐S., Yacoub, M. H. Human cardiac myosin autoantibodies impair myocyte contractility: a cause‐and‐effect relationship. FASEB J. 20, 651–660 (2006)

Differences in the Calcium‐Handling Response of Isolated Rat and Guinea‐Pig Cardiomyocytes to Metabolic Inhibition: Implications for Cell Damage

Species differences in response to hypoxic damage have been observed in studies using whole hearts. The aims of this study were to determine whether (i) species differences in response to simulated hypoxia could be detected at the level of the single myocyte, and (ii) there were any interspecies differences in the Ca2+ handling properties of the cells. Ventricular myocytes were isolated from hearts of adult rats and guinea‐pigs and electrically stimulated on the stage of a fluorescence microscope. Cell length was measured using an edge‐tracking device, and total intracellular [Ca2+] ([Ca2+]i) determined using indo‐1. Cells were exposed to metabolic inhibition (MI) (2.5 mM NaCN and no glucose) to simulate hypoxia followed by washout of CN and re‐addition of glucose (‘reperfusion’). Following exposure to MI, rat cells underwent rigor contracture in 18.8 ± 0.8 min (n = 80 cells), whereas the time was longer for guinea‐pig cells (32.9 ± 1.2 min, n = 83) (P < 0.001). If cells were reperfused after 1‐5 min in rigor, then rat cells showed improved morphological recovery compared with guinea‐pig cells (P < 0.05); thereafter recovery decreased with increasing time spent in rigor, and was similar in both groups. In indo‐1 loaded cells, [Ca2+]i was significantly increased in cells from both species at the end of MI; however, the actual increase was much higher in guinea‐pig cells. Upon reperfusion, [Ca2+]i recovered fully in rat cells, but in guinea‐pig cells there was no significant decrease. The restoration of [Ca2+]i to normal levels in rat cells following MI was associated with improved contractile recovery compared with guinea‐pig cells. We conclude that rat cells are more resistant to effects of MI than are guinea‐pig cells; this may be related to species differences in Ca2+ handling during and following exposure to MI.

Mitochondrial Calcium Dysregulation during Hypoxic Injury to Cardiac Myocytes

The work discussed above provides evidence that dramatic alterations occur in mitochondrial Ca2+ transport pathways during hypoxia; Ca2+ entry occurs via Na+/Ca2+ exchange (the normal efflux pathway), whereas the Ca2+ uniporter, (the normal influx route) is largely inactive. Clonazepam, but not ruthenium red, provided protection against hypoxia/reoxygenation damage in this model. Clonazepam, however, though a useful tool for studying mitochondrial Ca2+ transport (at least in rat myocytes), cannot be used in whole animals because of its non-myocardial effects, mainly on the nervous system. More specific compounds are clearly needed, especially now it is apparent that Ca2+ transport pathways differ under normoxic and hypoxic conditions. A new ruthenium red derivative has recently been synthesized by Matlib and colleagues (1998), apparently with very few nonspecific effects in myocytes.