Robert A. Haworth

The Ca2+-induced membrane transition in mitochondria: I. The protective mechanisms☆☆☆

Some physiological factors which control the rate of induction of the Ca2+-induced membrane transition (the term “Ca2+-induced membrane transition” is defined in the introduction) have been systematically investigated. To exclude the complicating factors of electron flow and energization, the transition was studied in mitochondria depleted of endogenous substrate, in the presence of uncoupler. In these mitochondria the transition could be induced by Ca2+, whether entry was mediated by ruthenium red-sensitive permeation or by A23187 facilitated diffusion. The rate of the transition was reduced fivefold by any agent which caused complete reduction of endogenous NAD. The rate of the transition was increased threefold by the exchange of endogenous ADP for phosphoenolpyruvate. A further increase was found on the addition of atractyloside, but bongkrekic acid caused inhibition. Addition of uncoupler to energized mitochondria when the endogenous NAD was already fully oxidized caused a stimulation of the transition. From these observations we conclude that mitochondria have a set of protective mechanisms (the term “protective mechanism” refers to the means by which these agents inhibit the Ca2+-induced transition; such a mechanism could be through allosteric interactions between the sites of binding of inhibitor and Ca2+; it would, however, be premature to conclude this on the basis of this paper) involving endogenous NADH, ADP, and energization which regulate the rate of the Ca2+-induced transition. ADP appears to work at two sites: one site which is internal, and another at the ADP/ATP translocase. In addition, we conclude that the transition requires neither electron flow nor energy, but rather the mere accessibility of some internal site to Ca2+. Finally, the key roles played by the protective agents in metabolism give the cell great potential flexibility in regulating the Ca2+-induced transition. This degree of control suggests that the transition has substantial physiological significance.

The Ca2+-induced membrane transition in mitochondria: II. Nature of the Ca2+ trigger site☆

The permeability of isolated mitochondria which have undergone the Ca2+-induced transition can be modulated over a wide range simply by adjusting the concentration of free Ca2+ in the medium. The effect varies sigmoidally with respect to Ca2+ concentration, with an apparent Km of 16 μm at pH 7.0. It is concluded that the trigger site (by “trigger site” we mean the site of binding of Ca2+ which, when Ca2+ is bound, will allow the transition in permeability to occur) is possibly also the site for high-affinity Ca2+ uptake. Added ADP, NADH and Mg2+ inhibit the Ca2+-induced permeability of mitochondria which have undergone the Ca2+-induced transition. Mg2+ and other ions, including H+, act like competitive inhibitors of the Ca2+ effect. In the presence of Ca2+, both neutral and charged molecules of molecular weight <1000 pass readily through the membrane. This response to Ca2+ is interpreted as a gating effect at the internal end of hydrophilic channels which span the inner membrane.

The Ca2+-induced membrane transition in mitochondria: III. Transitional Ca2+ release

The efflux of Ca2+ from mitochondria respiring at steady state, and much of uncoupler-induced Ca2+ efflux, is shown to be a consequence of the Ca2+-induced membrane transition (the Ca2+-induced transition is the Ca2+-dependent sudden increase in the nonspecific permeability of the mitochondrial inner membrane which occurs spontaneously when mitochondria are incubated under a variety of conditions (D. R. Hunter, R. A. Haworth, and J. H. Southard, 1976, J. Biol. Chem.251, 5069–5077)). Ca2+ release from mitochondria respiring at steady state is shown to be transitional by four criteria: (1) Ca2+ release is inhibited by Mg2+, ADP, and bovine serum albumin (BSA), all inhibitors of the transition; (2) release is selective for Ca2+ over Sr2+, a selectivity also found for the transition; (3) the time course of Ca2+ release is identical to the time course of the change in the mitochondrial population from the aggregated to the orthodox configuration; and (4) from kinetics, Ca2+ release from individual mitochondria is shown to occur suddenly, following a lag period during which no release occurs. Ca2+ release induced by uncoupler is shown to be mostly by a transitional mechanism, as judged by four criteria: (1) release of Ca2+ is ruthenium red-insensitive and is an order of magnitude faster than Sr2+ release which is ruthenium red-sensitive; (2) release of Ca2+ is strongly inhibited by keeping the mitochondrial NAD+ reduced; (3) the kinetics of Ca2+ release indicates a transitional release mechanism; and (4) uncoupler addition triggers the aggregated to orthodox configurational transition which, at higher levels of Ca2+ uptake, occurs in the whole mitochondrial population at a rate equal to the rate of Ca2+ release. Na2+-induced Ca2+ release was not accompanied by a configurational change; we therefore conclude that it is not mediated by the Ca2+-induced transition.

Reduction in density of transverse tubules and L-type Ca2+ channels in canine tachycardia-induced heart failure

OBJECTIVE Persistent supraventricular tachycardia leads to the development of a dilated cardiomyopathy with impairment of excitation-contraction (EC) coupling. Since the initial trigger for EC coupling in ventricular muscle is the influx of Ca(2+) through L-type Ca(2+) channels (I(Ca)) in the transverse tubules (T-tubules), we determined if the density of the T-tubule system and L-type Ca(2+) channels change in canine tachycardia pacing-induced cardiomyopathy. METHODS Confocal imaging of isolated ventricular myocytes stained with the membrane dye Di-8-ANEPPS was used to image the T-tubule system, and standard whole-cell patch clamp techniques were used to measure I(Ca) and intramembrane charge movement. RESULTS A complex staining pattern of interconnected tubules including prominent transverse components spaced every approximately 1.6 microm was present in control ventricular myocytes, but failing cells demonstrated a far less regular T-tubule system with a relative loss of T-tubules. In confocal optical slices, the average % of the total cell area staining for T-tubules decreased from 11.5+/-0.4 in control to 8.7+/-0.4% in failing cells (P<0.001). Whole-cell patch clamp studies revealed that I(Ca) density was unchanged. Since whole-cell I(Ca) is due to both the number of channels as well as the functional properties of those channels, we measured intramembrane charge movement as an assay for changes in channel number. The saturating amount of charge that moves due to gating of L-type Ca(2+) channels, Q(on,max), was decreased from 6.5+/-0.6 in control to 2.8+/-0.3 fC/pF in failing myocytes (P<0.001). CONCLUSIONS Cellular remodeling in heart failure results in decreased density of T-tubules and L-type Ca(2+) channels, which contribute to abnormal EC coupling.

Abnormal Ca2+ Release, but Normal Ryanodine Receptors, in Canine and Human Heart Failure

Abstract— Sarcoplasmic reticulum (SR) Ca2+ transport proteins, especially ryanodine receptors (RyR) and their accessory protein FKBP12.6, have been implicated as major players in the pathogenesis of heart failure (HF), but their role remain controversial. We used the tachycardia-induced canine model of HF and human failing hearts to investigate the density and major functional properties of RyRs, SERCA2a, and phospholamban (PLB), the main proteins regulating SR Ca2+ transport. Intracellular Ca2+ is likely to play a role in the contractile dysfunction of HF because the amplitude and kinetics of the [Ca2+]i transient were reduced in HF. Ca2+ uptake assays showed 44±8% reduction of Vmax in canine HF, and Western blots demonstrated that this reduction was due to decreased SERCA2a and PLB levels. Human HF showed a 30±5% reduction in SERCA2a, but PLB was unchanged. RyRs from canine and human HF displayed no major structural or functional differences compared with control. The Po of RyRs was the same for control and HF over the range of pCa 7 to 4. Subconductance states, which predominate in FKBP12.6-stripped RyRs, were equally frequent in control and HF channels. An antibody that recognizes phosphorylated RyRs yields equal intensity for control and HF channels. Further, phosphorylation of RyRs by PKA did not appear to change the RyR/FKBP12.6 association, suggesting minor &bgr;-adrenergic stimulation of Ca2+ release through this mechanism. These results support a role for SR in the pathogenesis of HF, with abnormal Ca2+ uptake, more than Ca2+ release, contributing to the depressed and slow Ca2+ transient characteristic of HF.

Depletion of T-tubules and specific subcellular changes in sarcolemmal proteins in tachycardia-induced heart failure

OBJECTIVE The T-tubule membrane network is integrally involved in excitation-contraction coupling in ventricular myocytes. Ventricular myocytes from canine hearts with tachycardia-induced dilated cardiomyopathy exhibit a decrease in accessible T-tubules to the membrane-impermeant dye, di8-ANNEPs. The present study investigated the mechanism of loss of T-tubule staining and examined for changes in the subcellular distribution of membrane proteins essential for excitation-contraction coupling. METHODS Isolated ventricular myocytes from canine hearts with and without tachycardia-induced heart failure were studied using fluorescence confocal microscopy and membrane fractionation techniques using a variety of markers specific for sarcolemmal and sarcoplasmic reticulum proteins. RESULTS Probes for surface glycoproteins, Na/K ATPase, Na/Ca exchanger and Ca(v)1.2 demonstrated a prominent but heterogeneous reduction in T-tubule labeling in both intact and permeabilised failing myocytes, indicating a true depletion of T-tubules and associated membrane proteins. Membrane fractionation studies showed reductions in L-type Ca(2+) channels and beta-adrenergic receptors but increased levels of Na/Ca exchanger protein in both surface sarcolemma and T-tubular sarcolemma-enriched fractions; however, the membrane fraction enriched in junctional complexes of sarcolemma and junctional sarcoplasmic reticulum demonstrated no significant changes in the density of any sarcolemmal protein or sarcoplasmic reticulum protein assayed. CONCLUSION Failing canine ventricular myocytes exhibit prominent depletion of T-tubules and changes in the density of a variety of proteins in both surface and T-tubular sarcolemma but with preservation of the protein composition of junctional complexes. This subcellular remodeling contributes to abnormal excitation-contraction coupling in heart failure.

Increased Nitration of Sarcoplasmic Reticulum Ca2+-ATPase in Human Heart Failure

Background—Reduced sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a isoform) activity is a major determinant of reduced contractility in heart failure. Ca2+-ATPase inactivation can occur through SERCA2a nitration. We therefore investigated the role of SERCA2a nitration in heart failure. Methods and Results—We measured SERCA2a levels and nitrotyrosine levels in tissue from normal and failing human hearts using Western blots. We found that nitrotyrosine levels in idiopathic dilated cardiomyopathic (DCM) hearts were almost double those of control hearts in age-matched groups. Nitrotyrosine was dominantly present in a single protein with the molecular weight of SERCA2a, and immunoprecipitation confirmed that the protein recognized by the nitrotyrosine antibody was SERCA2a. There was a positive correlation between the time to half relaxation and the nitrotyrosine/SERCA2a content (P<0.01) in myocytes isolated from control and DCM hearts. In experiments with isolated SR vesicles from porcine hearts, we also showed that the Ca pump is inactivated by peroxynitrite exposure, and inactivation was prevented by protein kinase A pretreatment. Conclusions—We conclude that SERCA2a inactivation by nitration may contribute to Ca pump failure and hence heart failure in DCM.

Contracture in isolated adult rat heart cells. Role of Ca2+, ATP, and compartmentation.

Isolated intact quiescent myocytes from the adult rat were used as a model system for investigating the determinants of contracture induced by metabolic deprivation. The model simulated the pattern of contracture and ATP decline seen in the intact heart during Ischemia. Three new insights into the contracture process were gained: (1) in the quiescent cell system, the rate of onset of contracture was independent of external Cn2+, supporting the view that the Ca2+ dependence of the rate of onset in the whole heart is related to beat-dependent substrate utilization; (2) the second phase of ATP decline was paralleled by a decline In the percentage of cells which had not undergone contracture, suggesting that–in any cell–contracture is immediately preceded by a total loss of ATP; and (3) oligomycin delayed the onset of contracture by 55 ± 12%, suggesting that mitochondria ATPase activity ia a significant drain on energy resources in the quiescent ischemic heart.

Control of the Mitochondrial Permeability Transition Pore by High-Affinity ADP Binding at the ADP/ATP Translocase in Permeabilized Mitochondria

Low levels of ADP binding at the ADP/ATP translocase caused inhibition of the Ca2+-inducedpermeability transition of the mitochondrial inner membrane, when measured using the shrinkage assay on mitochondria, which have already undergone a transition. Inhibition was preventedby carboxyatractyloside, but potentiated by bongkrekic acid, which increased the affinity forinhibition by ADP. This suggests that inhibition was related to the conformation of thetranslocase. Ca2+ addition was calculated to remove most of the free ADP. Ca2+ added after ADPinduced a slow decay of the inhibition, which probably reflected the dissociation of ADP fromthe translocator. We conclude that the probability of forming a permeability transition pore(PTP) is much greater when the translocase is in the CAT conformation than in the BKAconformation, and, in the absence of CAT and BKA, the translocator is shifted between theBKA and CAT conformations by ADP binding and removal, even in deenergized mitochondria with no nucleotide gradients.

The isolation of Ca2+-resistant myocytes from the adult rat.

Abstract A method is described for the preparation of Ca2+-resistant myocytes from the adult rat. Ca2+-resistant cells were obtained in yields of 6.3 ± 1.5 × 106 cells/gm wet weight tissue, with a purity of 67.5 ± 8.8% cells excluding trypan blue. Of the cells which excluded trypan blue, over 90% were rod-shaped. The rodshaped cells when isolated were quiescent, but beat in response to electric field stimulation in the presence of Ca2+. Incubation of the cells at 37° in the presence of 2 m m Ca2+ caused only a slow decline in the percentage of cells able to exclude trypan blue or to beat when stimulated. The rate of decline was comparable to that of cells incubated without Ca2+. Incubation of the cells in the presence of EGTA (ethylene glycol bis (β-aminoethyl ether)-N,N′-tetraacetic acid) for 30 min had little effect on the ability of the cells to withstand Ca2+. The key step in preparing Ca2+ resistant cells was found to be the inclusion of trypsin and Ca2+ at the final stage of incubation of tissue pieces. The trypsin appears to act not only by selective removal of cells susceptible to Ca2+ but also by conferring Ca2+ resistance on cells which otherwise would be Ca2+ susceptible. The properties displayed by this preparation indicate that the cells are both mechanically and electrochemically intact. They therefore provide an excellent model system for the study of excitation-contraction coupling at the cellular level, and also provide a more realistic starting point for studies of pathology than previous Ca2+-susceptible preparations.