The AIN-76A defined rodent diet accelerates the development of heart failure in SHHF rats: a cautionary note on its use in cardiac studies.

Previous studies from our laboratory have shown positive benefits of linoleic acid (LA) feeding for attenuation of rat heart failure (HF). However, another research group concluded LA feeding was detrimental to cardiac function, using the American Institute of Nutrition 76A (AIN) diet as a background diet for the experimental animals only. To reconcile these conflicting results and determine whether (i) AIN has effects on cardiovascular function, and (ii) AIN reverses the positive effects of LA feeding, studies were performed using spontaneously hypertensive heart failure (SHHF) rats in both a survival study with lifetime feeding of AIN (control: Purina 5001) and a 2 × 2 factorial design for 6 weeks in young male SHHF rats with background diet and LA as variables. During a lifetime of AIN feeding, mortality from heart failure is significantly accelerated, cardiolipin altered and triglycerides increased. In young rats, 6 weeks on the AIN diet promoted increased systolic and diastolic blood pressure, increased fed and fasting blood glucose, increased serum inflammatory eicosanoids, decreased docosahexanoic acid, increased posterior wall thickness in diastole and an altered cardiolipin subspecies profile. The addition of LA to the AIN diet was able to rescue blood pressure. However, the combination increased retroperitoneal fat mass, body weight and fed blood glucose beyond the levels with the AIN diet alone. Because the AIN diet has wide ranging effects on cardiovascular parameters, our results suggest that it should not be used in animal studies involving the cardiovascular system unless induction of cardiac dysfunction is the desired outcome.

Decreased cardiolipin synthesis corresponds with cytochrome c release in palmitate-induced cardiomyocyte apoptosis.

Apoptosis has been identified recently as a component of many cardiac pathologies. However, the potential triggers of programmed cell death in the heart and the involvement of specific metabolic pathway(s) are less well characterized. Detachment of cytochrome c from the mitochondrial inner membrane is a necessary first step for cytochrome c release into the cytosol and initiation of apoptosis. The saturated long chain fatty acid, palmitate, induces apoptosis in rat neonatal cardiomyocytes and diminishes content of the mitochondrial anionic phospholipid, cardiolipin. These changes are accompanied by 1) acyl chain saturation of phosphatidic acid and phosphatidylglycerol, 2) large increases in the levels of these two phospholipids, and 3) a decline in cardiolipin synthesis. Although cardiolipin synthase activity is unchanged, saturated phosphatidylglycerol is a poor substrate for this enzyme. Under these conditions, decreased cardiolipin synthesis and release of cytochrome c are directly and significantly correlated. The results suggest that phosphatidylglycerol saturation and subsequent decreases in cardiolipin affect the association of cytochrome c with the inner mitochondrial membrane, directly influencing the pathway to cytochrome c release and subsequent apoptosis.

Fatty acid-induced apoptosis in neonatal cardiomyocytes: redox signaling.

Exposure of neonatal rat cardiac myocytes to palmitate and glucose produces apoptosis as seen by cytochrome c release, caspase 3-like activation, DNA laddering, and poly(ADP-ribose) polymerase cleavage. The purpose of this study was to understand the role of reactive oxygen species in the initiation of programmed cell death by palmitate. We found that palmitate (but not oleate) produces inhibition of carnitine palmitoyltransferase I, accumulation of ceramide, and inhibition of electron transport complex III. These events are subsequent to cytochrome c release and loss of the mitochondrial membrane potential. No differences in H2O2 production or N-terminal c-Jun kinase phosphorylation were detected between myocytes incubated in palmitate and control myocytes (nonapoptotic) incubated in oleate. These results suggest that the palmitate-induced loss of the mitochondrial membrane potential is not associated with H2O2 synthesis and that a membrane potential is required to generate reactive oxygen species following ceramide inhibition of complex III.

The rapid mode of calcium uptake into heart mitochondria (RaM): comparison to RaM in liver mitochondria.

A mechanism of Ca(2+) uptake, capable of sequestering significant amounts of Ca(2+) from cytosolic Ca(2+) pulses, has previously been identified in liver mitochondria. This mechanism, the Rapid Mode of Ca(2+) uptake (RaM), was shown to sequester Ca(2+) very rapidly at the beginning of each pulse in a sequence [Sparagna et al. (1995) J. Biol. Chem. 270, 27510-27515]. The existence and properties of RaM in heart mitochondria, however, are unknown and are the basis for this study. We show that RaM functions in heart mitochondria with some of the characteristics of RaM in liver, but its activation and inhibition are quite different. It is feasible that these differences represent different physiological adaptations in these two tissues. In both tissues, RaM is highly conductive at the beginning of a Ca(2+) pulse, but is inhibited by the rising [Ca(2+)] of the pulse itself. In heart mitochondria, the time required at low [Ca(2+)] to reestablish high Ca(2+) conductivity via RaM i.e. the 'resetting time' of RaM is much longer than in liver. RaM in liver mitochondria is strongly activated by spermine, activated by ATP or GTP and unaffected by ADP and AMP. In heart, RaM is activated much less strongly by spermine and unaffected by ATP or GTP. RaM in heart is strongly inhibited by AMP and has a biphasic response to ADP; it is activated at low concentrations and inhibited at high concentrations. Finally, an hypothesis consistent with the data and characteristics of liver and heart is presented to explain how RaM may function to control the rate of oxidative phosphorylation in each tissue. Under this hypothesis, RaM functions to create a brief, high free Ca(2+) concentration inside mitochondria which may activate intramitochondrial metabolic reactions with relatively small amounts of Ca(2+) uptake. This hypothesis is consistent with the view that intramitochondrial [Ca(2+)] may be used to control the rate of ADP phosphorylation in such a way as to minimize the probability of activating the Ca(2+)-induced mitochondrial membrane permeability transition (MPT).

A metabolic role for mitochondria in palmitate-induced cardiac myocyte apoptosis.

After cardiac ischemia, long-chain fatty acids, such as palmitate, increase in plasma and heart. Palmitate has previously been shown to cause apoptosis in cardiac myocytes. Cultured neonatal rat cardiac myocytes were studied to assess mitochondrial alterations during apoptosis. Phosphatidylserine translocation and caspase 3-like activity confirmed the apoptotic action of palmitate. Cytosolic cytochrome c was detected at 8 h and plateaued at 12 h. The mitochondrial membrane potential (DeltaPsi) in tetramethylrhodamine ethyl ester-loaded cardiac myocytes decreased significantly in individual mitochondria by 8 h. This loss was heterogeneous, with a few energized mitochondria per myocyte remaining at 24 h. Total ATP levels remained high at 16 h. The DeltaPsi loss was delayed by cyclosporin A, a mitochondrial permeability transition inhibitor. Mitochondrial swelling accompanied changes in DeltaPsi. Carnitine palmitoyltransferase I activity fell at 16 h; this decline was accompanied by ceramide increases that paralleled decreased complex III activity. We conclude that carnitine palmitoyltransferase I inhibition, ceramide accumulation, and complex III inhibition are downstream events in cardiac apoptosis mediated by palmitate and occur independent of events leading to caspase 3-like activation.

Cardiac fatty acid metabolism and the induction of apoptosis.

Fatty acids are the primary source of energy in the adult heart. Recently, however, it was discovered that certain saturated fatty acids, such as palmitate and stearate, cause cardiac and other types of cells to undergo programmed cell death (apoptosis). In cardiac ischemia/reperfusion injury, where blood flow is blocked and then restored to the heart, recovery of cardiac cells is inversely proportional to the concentration of fatty acids (largely composed of palmitate and stearate) in the reperfusate. The aim of this review is to summarize what is known about fatty acid induction of heart disease, the role of fatty acids in apoptosis, and apoptosis in the heart, including the role that mitochondria play in this process.

The Ca2+ transport mechanisms of mitochondria and Ca2+ uptake from physiological-type Ca2+ transients.

Mitochondria contain a sophisticated system for transporting Ca2+. The existence of a uniporter and of both Na+-dependent and -independent efflux mechanisms has been known for years. Recently, a new mechanism, called the RaM, which seems adapted for sequestering Ca2+ from physiological transients or pulses has been discovered. The RaM shows a conductivity at the beginning of a Ca2+ pulse that is much higher than the conductivity of the uniporter. This conductivity decreases very rapidly following the increase in [Ca2+] outside the mitochondria. This decrease in the Ca2+ conductivity of the RaM is associated with binding of Ca2+ to an external regulatory site. When liver mitochondria are exposed to a sequence of pulses, uptake of labeled Ca2+ via the RaM appears additive between pulses. Ruthenium red inhibits the RaM in liver mitochondria but much larger amounts are required than for inhibition of the mitochondrial Ca2+ uniporter. Spermine, ATP and GTP increase Ca2+ uptake via the RaM. Maximum uptake via the RaM from a single Ca2+ pulse in the physiological range has been observed to be approximately 7 nmole/mg protein, suggesting that Ca2+ uptake via the RaM and uniporter from physiological pulses may be sufficient to activate the Ca2+-sensitive metabolic reactions in the mitochondrial matrix which increase the rate of ATP production. RaM-mediated Ca2+ uptake has also been observed in heart mitochondria. Evidence for Ca2+ uptake into the mitochondria in a variety of tissues described in the literature is reviewed for evidence of participation of the RaM in this uptake. Possible ways in which the differences in transport via the RaM and the uniporter may be used to differentiate between metabolic and apoptotic signaling are discussed.

Mitochondrial calcium uptake from physiological-type pulses of calcium. A description of the rapid uptake mode.

A controversy in the field of bioenergetics has been whether mitochondria are capable of sequestering enough Ca2+ from cytosolic Ca2+ pulses to raise their intramitochondrial free Ca2+ level ([Ca2+]m). This is significant because an increase in [Ca2+]m has been linked to an increase in cellular metabolic rate through various mechanisms. To resolve this question, we exposed isolated liver mitochondria to physiological type pulses of Ca2+ produced using a pulse-generating system (Sparagna, G. C., Gunter, K. K., and Gunter, T. E. (1994) Anal. Biochem. 219, 96-103). We then measured the resulting mitochondrial Ca2+ uptake. The uniporter was previously thought to be the only specific Ca2+ uptake mechanism in mitochondria. Our studies have uncovered an additional uptake mechanism, the rapid mode of uptake or RaM, which functions at the beginning of each pulse and allows mitochondria to sequester a considerable amount of Ca2+ from short pulses. We have shown that the RaM is reset by decreasing the [Ca2+] between pulses for a very short time, making this uptake mode ideally suited for Ca2+ sequestration from Ca2+ pulse sequences. With rapid Ca2+ uptake occurring at the beginning of each pulse, liver mitochondria may be able to sequester sufficient Ca2+ from a short sequence of pulses to activate the cellular metabolic rate.

A system for producing and monitoring in vitro calcium pulses similar to those observed in vivo.

We have used a computer-controlled automatic pipettor system in conjunction with a fluorescence spectrometer to produce Ca2+ pulses and pulse sequences within a cylindrical fluorescence cuvette. These pulses or pulse sequences are similar to those observed in the cytosol of many types of cells in vivo and show good reproducibility. Their intensity, duration, shape, and periodicity can be controlled and determined at will. Pulses of other ions or of transmembrane potential can be produced by the same apparatus. As an example of the use of this apparatus, initial studies on uptake of pulses of Ca2+ by isolated liver mitochondria are described in which controls are used to verify that initial rapid mitochondrial Ca2+ uptake is actual net uptake, as opposed to binding or exchange.