Immediate and delayed effects of a brief period of calcium loading,induced by the calcium channel agonist Bayer K 8644, on the ultrastructure and sarcolemmal integrity of murine diaphragm in vitro

Exposure of in vitro mouse diaphragm to the dihydropyridine Ca²⁺ channel agonist Bayer K 8644 (Bay) and partial depolarisation for 120 min induced severe structural damage within 30–60 min and membrane permeabilisation (60–120 min). Exposure to Bay and depolarisation for 30 min (brief Ca²⁺ loading) followed by washout for 90 min produced similar effects, even though significant membrane damage does not occur until the second hour of incubation. Development/expression of necrosis during the washout period was not inhibited by manoeuvres designed to combat the effects of any remaining Bay, or by use of a Ca²⁺-free, 1 mM EGTA medium for washout. Exposure to Bay for 15 min followed by washout did not induce damage. Electron microscopic examination revealed that the mitochondria of cells fixed after 30 or 120 min exposure to Bay and depolarisation were in the orthodox (swollen) configuration, but that mitochondria in muscles that had undergone washout with Ca²⁺-free-EGTA saline were electron dense. Further incubation of these muscles in Ca²⁺-containing medium caused readoption of the swollen configuration. We conclude that membrane permeabilisation can occur after removal of the Ca²⁺ load, and that mitochondrial Ca²⁺ overload is not necessary for Ca²⁺-induced damage/death to occur.This work was supported by the Wellcome Trust, Grant no. 030661/Z/89     

The biochemical and clinical consequences of 2'-deoxycoformycin in refractory lymphoproliferative malignancy

A deficiency of adenosine deaminase, an enzyme important in purine nucleoside catabolism, is associated with a severe combined immunodeficiency disease in children. Inhibition of this enzyme in vitro and in vivo results in an impairment in lymphoblast proliferation. We have investigated the pharmacologic inhibition of this enzyme by 2'-deoxycoformycin in 15 patients with hematologic malignancies. Biochemical consequences of the administration of this agent were closely monitored in erythrocytes, nucleated peripheral blood and bone marrow cells, serum, and urine. A marked rise in erythrocyte dATP was accompanied by a depletion of ATP in those patients exhibiting toxicity. Most patients excreted large amounts of deoxyadenosine but not adenosine in the urine. Serum deoxyadenosine rose in patients demonstrating a marked decrease in cell mass. The biochemical disturbances and clinical toxicity, including hepatic, renal, and conjunctival abnormalities, were usually reversible. Central nervous system toxicity, which potentially was the most serious consequence, was associated with high erythrocyte dATP/ATP ratios and high levels of cerebrospinal fluid deoxyadenosine. In patients with lymphoma and leukemia, objective responses were observed but were short- lived. Patients with chronic lymphocytic leukemia receiving weekly low doses of the drug demonstrated minimal toxicity and some efficacy. The chemotherapeutic potential o 2'-deoxycoformycin, as either a single agent or in combination with Ara-A, merits further exploration.     

Depressed pacemaker activity of sinoatrial node myocytes contributes to the age-dependent decline in maximum heart rate

An inexorable decline in maximum heart rate (mHR) progressively limits human aerobic capacity with advancing age. This decrease in mHR results from an age-dependent reduction in “intrinsic heart rate” (iHR), which is measured during autonomic blockade. The reduced iHR indicates, by definition, that pacemaker function of the sinoatrial node is compromised during aging. However, little is known about the properties of pacemaker myocytes in the aged sinoatrial node. Here, we show that depressed excitability of individual sinoatrial node myocytes (SAMs) contributes to reductions in heart rate with advancing age. We found that age-dependent declines in mHR and iHR in ECG recordings from mice were paralleled by declines in spontaneous action potential (AP) firing rates (FRs) in patch-clamp recordings from acutely isolated SAMs. The slower FR of aged SAMs resulted from changes in the AP waveform that were limited to hyperpolarization of the maximum diastolic potential and slowing of the early part of the diastolic depolarization. These AP waveform changes were associated with cellular hypertrophy, reduced current densities for L- and T-type Ca²⁺ currents and the “funny current” (I_f), and a hyperpolarizing shift in the voltage dependence of I_f. The age-dependent reduction in sinoatrial node function was not associated with changes in β-adrenergic responsiveness, which was preserved during aging for heart rate, SAM FR, L- and T-type Ca²⁺ currents, and I_f. Our results indicate that depressed excitability of individual SAMs due to altered ion channel activity contributes to the decline in mHR, and thus aerobic capacity, during normal aging.One of the most insidious aspects of growing older is an inevitable decline in maximum heart rate (mHR), which limits maximum aerobic capacity with advancing age (1–3). The decline in mHR proceeds at approximately the same rate for all individuals, without regard for lifestyle or physical fitness (4–8). For many otherwise healthy elderly people, it is the factor that ultimately restricts the ability to live independently (9, 10).The decrease in mHR with age results primarily from a parallel age-dependent decline in “intrinsic heart rate” (iHR) (11–13), which is measured during autonomic blockade, and thus reflects the spontaneous pacemaker activity of the sinoatrial node of the heart. Although it is known that the intact sinoatrial node from aged animals contracts more slowly (14, 15) and contains fewer pacemaker myocytes (16), little is known about the functional properties of individual myocytes from the sinoatrial node of the aged heart.Sinoatrial myocytes (SAMs) are highly specialized cells that serve a primarily electrical function as cardiac pacemakers via their production of spontaneous action potentials (APs). Sinoatrial APs are characterized by a spontaneous depolarization during diastole that drives the membrane potential to threshold, thereby triggering the subsequent AP. This “diastolic depolarization” (DD) phase of the sinoatrial AP results from the coordinated activity of numerous membrane conductances, including L- and T-type Ca²⁺ currents (I_Ca,L and I_Ca,T, respectively) and the “funny current” (I_f), all of which contribute directly to the DD by conducting inward current at diastolic potentials (17–23). I_Ca,L also contributes indirectly to the DD by stimulating Ca²⁺ efflux from the sarcoplasmic reticulum of SAMs (24), thereby activating the Na⁺-Ca²⁺ exchange current (I_NCX), which is also known to be critical for normal pacemaker activity (25–29).In this study, we determined the effects of aging on heart rates (HRs) and on spontaneous APs and membrane currents in acutely isolated SAMs. We observed age-dependent decreases in AP firing rates (FRs) in SAMs that corresponded to the age-dependent reductions in iHRs and mHRs. The slower AP FRs resulted from changes in the AP waveform that were associated with an increase in cell size and with alterations in I_Ca,L, I_Ca,T, and I_f. These findings indicate that changes in expression and/or regulation of ion channels in SAMs comprise part of the molecular program that limits mHR, and thus aerobic capacity, during normal aging.