Non-alcoholic fatty liver disease and thrombocytopenia IV: its association with granulocytopenia

ABSTRACT Introduction: We have previously shown that some patients present thrombocytopenia (less than 100 × 109/L platelets) in non-alcoholic fatty liver disease (NAFLD). To further explore the nature of this association, we have now analyzed the association of thrombocytopenia with neutropenia (less than 0.5 × 109/L granulocytes) in NAFLD. Material and methods: Persons with NAFLD were prospectively accrued in the study after February 2018. The presence of NAFLD was defined by both serologic determinations (Fibromax ®) and liver transient elastography (TE/Fibroscan ®). Results: In 123 consecutive patients with NAFLD without cirrhosis, thrombocytopenia was identified in 20 (16%), whereas neutropenia was identified in 9 (7%). In the subset of 20 patients with NAFLD and thrombocytopenia, granulocytopenia was identified in 5 (25%), whereas in the subset of 9 patients with granulocytopenia, thrombocytopenia was identified in 5 (55%). We found a significant association between thrombocytopenia and both leukopenia and granulocytopenia (OR 8.25, 95% CI 1.9-34.2, p = 0.004). Conclusions: Both thrombocytopenia and neutropenia were identified in persons with NAFLD and, as there is a significant relationship between these two variables, we speculate that this finding may support the possibility of hypersplenism being involved in the cytopenias found in NAFLD without cirrhosis.

Non-alcoholic fatty liver disease and thrombocytopenia IV: its association with granulocytopenia.

INTRODUCTION: We have previously shown that some patients present thrombocytopenia (less than 100 × 109/L platelets) in non-alcoholic fatty liver disease (NAFLD). To further explore the nature of this association, we have now analyzed the association of thrombocytopenia with neutropenia (less than 0.5 × 109/L granulocytes) in NAFLD. MATERIAL AND METHODS: Persons with NAFLD were prospectively accrued in the study after February 2018. The presence of NAFLD was defined by both serologic determinations (Fibromax ®) and liver transient elastography (TE/Fibroscan ®). RESULTS: In 123 consecutive patients with NAFLD without cirrhosis, thrombocytopenia was identified in 20 (16%), whereas neutropenia was identified in 9 (7%). In the subset of 20 patients with NAFLD and thrombocytopenia, granulocytopenia was identified in 5 (25%), whereas in the subset of 9 patients with granulocytopenia, thrombocytopenia was identified in 5 (55%). We found a significant association between thrombocytopenia and both leukopenia and granulocytopenia (OR 8.25, 95% CI 1.9-34.2, p = 0.004). CONCLUSIONS: Both thrombocytopenia and neutropenia were identified in persons with NAFLD and, as there is a significant relationship between these two variables, we speculate that this finding may support the possibility of hypersplenism being involved in the cytopenias found in NAFLD without cirrhosis.

Chronic Collection of Cerebrospinal Fluid from Rhesus Macaques (Macaca mulatta) with Cisterna Magna Ports: Update on Refinements.

More than 20 y ago, we developed an animal model for chronic and continuous collection of cerebrospinal fluid (CSF) from conscious rhesus macaques. Since our previous publication in 2003, we have successfully implanted 168 rhesus macaques using this approach. Our experience enables us to provide up-to-date information regarding the model, including refine- ments to our implant design, reductions in maintenance, and new procedures for dealing with contamination. The results of our experiences have reduced the number of surgeries required and helped to increase the longevity of the implant, with some functioning for more than 18 y. Building on our success in rhesus macaques, we attempted to develop similar animal models in the African green monkeys and dogs but have been unable to develop reliable chronic models for CSF collection in these species.

Decreased complexity of glucose dynamics in diabetes in rhesus monkeys.

Until recently, preclinical and clinical work on diabetes has focused on the understanding of blood glucose elevation and its detrimental metabolic sequelae. The advent of continuous glucose monitoring (CGM) technology now allows real time monitoring of blood glucose levels as a time series, and thus the exploration of glucose dynamics at short time scales. Previous work has shown decreases in the complexity of glucose dynamics, as measured by multiscale entropy (MSE) analysis, in diabetes in humans, mice, and rats. Analyses for non-human primates (NHP) have not been reported, nor is it known if anti-diabetes compounds affect complexity of glucose dynamics. We instrumented four healthy and six diabetic rhesus monkeys with CGM probes in the carotid artery and collected glucose values at a frequency of one data point per second for the duration of the sensors' life span. Sensors lasted between 45 and 78 days. Five of the diabetic rhesus monkeys were also administered the anti-diabetic drug liraglutide daily beginning at day 39 of the CGM monitoring period. Glucose levels fluctuated during the day in both healthy and diabetic rhesus monkeys, peaking between 12 noon - 6 pm. MSE analysis showed reduced complexity of glucose dynamics in diabetic monkeys compared to healthy animals. Although liraglutide decreased glucose levels, it did not restore complexity in diabetic monkeys consistently. Complexity varied by time of day, more strongly for healthy animals than for diabetic animals. And by dividing the monitoring period into 3-day or 1-week subperiods, we were able to estimate within-animal variability of MSE curves. Our data reveal that decreased complexity of glucose dynamics is a conserved feature of diabetes from rodents to NHPs to man.

The organization of frequency and binaural cues in the gerbil inferior colliculus.

The inferior colliculus (IC) is the common target of separate pathways that transmit different types of auditory information. Beyond tonotopy, little is known about the organization of response properties within the 3-dimensional layout of the auditory midbrain in most species. Through study of interaural time difference (ITD) processing, the functional properties of neurons can be readily characterized and related to specific pathways. To characterize the representation of ITDs relative to the frequency and hodological organization of the IC, the properties of neurons were recorded and the sites recovered histologically. Subdivisions of the IC were identified based on cytochrome oxidase (CO) histochemistry. The results were plotted within a framework formed by an MRI atlas of the gerbil brain. The central nucleus was composed of two parts, and lateral and dorsal cortical areas were identified. The lateral part of the central nucleus had the highest CO activity in the IC and a high proportion of neurons sensitive to ITDs. The medial portion had lower CO activity and fewer ITD-sensitive neurons. A common tonotopy with a dorsolateral to ventromedial gradient of low to high frequencies spanned the two regions. The distribution of physiological responses was in close agreement with known patterns of ascending inputs. An understanding of the 3-dimensional organization of the IC is needed to specify how the single tonotopic representation in the IC central nucleus leads to the multiple tonotopic representations in core areas of the auditory cortex.

Characterization of an investigative safety pharmacology model to assess comprehensive cardiac function and structure in chronically instrumented conscious beagle dogs.

INTRODUCTION: There has been an increasing need to conduct investigative safety pharmacology studies to complement regulatory-required studies, particularly as it applies to a comprehensive assessment of cardiovascular (CV) risk. METHODS: We describe refined methodology using a combination of telemetry and direct signal acquisition to record concomitant peripheral hemodynamics, ECG, and left ventricular (LV) structure (LV chamber size and LV wall thickness) and function, including LV pressure-volume (PV) loops to determine load independent measures of contractility (end systolic elastance, Ees, and preload recruitable stroke work, PRSW) in conscious beagle dogs. Following baseline characterization, 28days of chronic rapid ventricular pacing (RVP) was performed and cardiac function monitored: both as a way to compare measures during development of dysfunction and to characterize feasibility of a model to assess CV safety in animals with underlying cardiac dysfunction. RESULTS: While ±dP/dT decreased within a few days of RVP and remained stable, more comprehensive cardiac function measurements, including Ees and PRSW, provided a more sensitive assessment confirming the value of such endpoints for a more clear functional assessment. After 28days of RVP, the inodilator pimobendan was administered to further demonstrate the ability to detect changes in cardiac function. Expectedly pimobendan caused a leftward shift in the PV loop, improved ejection fraction (EF) and significantly improved Ees and PRSW. DISCUSSION: In summary, the data show the feasibility and importance in measuring enhanced cardiac functional parameters in conscious normal beagle dogs and further describe a relatively stable cardiac dysfunction model that could be used as an investigative safety pharmacology risk assessment tool.

Odanacatib increases mineralized callus during fracture healing in a rabbit ulnar osteotomy model.

The effects of the cathepsin K inhibitor odanacatib (ODN) on fracture healing were monitored for ~6 and 15 weeks post-fracture in two separate studies using the unilateral transverse mid-ulnar osteotomy model in skeletally mature female rabbits. Rabbits were pre-treated for 3-4 weeks with vehicle (Veh), ODN (2 mg/kg, po, daily), or alendronate (ALN) (0.3 mg/kg, sc, twice-weekly) prior to osteotomy. In Study 1, the animals were maintained on the same respective treatment for ~6 weeks. In Study 2, the animals were also continued on the same therapy or switched from Veh to ODN or ODN to Veh for 15 weeks. No treatment-related impairment of fracture union was seen by qualitative histological assessments in the first study. Cartilage retention was detected in the calluses of ALN-treated rabbits at week-6, while calluses in the ODN and Veh groups contained bony tissue with significantly less residual cartilage. ODN treatment also markedly increased the number of cathepsin K-(+) osteoclasts in the callus, indicating enhanced callus remodeling. From the second study, ex vivo DXA and pQCT confirmed that ODN treatment pre- and post-osteotomy increased callus bone mineral content and bone mineral density (BMD) versus Veh (p < 0.001) and discontinuation of ODN post-surgery returned callus BMD to Veh. Peak load of ODN- or ALN-treated calluses were comparable to Veh. ODN increased callus yield load (20%, p = 0.056) and stiffness (26%, p < 0.05) versus Veh. These studies demonstrated that ODN increased mineralized callus during the early phase of fracture repair without impairing callus formation or biomechanical integrity at the fracture site.

CNS amyloid-ß, soluble APP-α and -ß kinetics during BACE inhibition.

BACE, a ß-secretase, is an attractive potential disease-modifying therapeutic strategy for Alzheimer's disease (AD) as it results directly in the decrease of amyloid precursor protein (APP) processing through the ß-secretase pathway and a lowering of CNS amyloid-ß (Aß) levels. The interaction of the ß-secretase and α-secretase pathway-mediated processing of APP in the rhesus monkey (nonhuman primate; NHP) CNS is not understood. We hypothesized that CNS inhibition of BACE would result in decreased newly generated Aß and soluble APPß (sAPPß), with increased newly generated sAPPα. A stable isotope labeling kinetics experiment in NHPs was performed with a (13)C6-leucine infusion protocol to evaluate effects of BACE inhibition on CNS APP processing by measuring the kinetics of sAPPα, sAPPß, and Aß in CSF. Each NHP received a low, medium, or high dose of MBI-5 (BACE inhibitor) or vehicle in a four-way crossover design. CSF sAPPα, sAPPß, and Aß were measured by ELISA and newly incorporated label following immunoprecipitation and liquid chromatography-mass spectrometry. Concentrations, kinetics, and amount of newly generated APP fragments were calculated. sAPPß and sAPPα kinetics were similar, but both significantly slower than Aß. BACE inhibition resulted in decreased labeled sAPPß and Aß in CSF, without observable changes in labeled CSF sAPPα. ELISA concentrations of sAPPß and Aß both decreased and sAPPα increased. sAPPα increased by ELISA, with no difference by labeled sAPPα kinetics indicating increases in product may be due to APP shunting from the ß-secretase to the α-secretase pathway. These results provide a quantitative understanding of pharmacodynamic effects of BACE inhibition on NHP CNS, which can inform about target development.

Acute gamma-secretase inhibition of nonhuman primate CNS shifts amyloid precursor protein (APP) metabolism from amyloid-beta production to alternative APP fragments without amyloid-beta rebound.

The accumulation of amyloid beta (Abeta) in Alzheimer's disease is caused by an imbalance of production and clearance, which leads to increased soluble Abeta species and extracellular plaque formation in the brain. Multiple Abeta-lowering therapies are currently in development: an important goal is to characterize the molecular mechanisms of action and effects on physiological processing of Abeta, as well as other amyloid precursor protein (APP) metabolites, in models which approximate human Abeta physiology. To this end, we report the translation of the human in vivo stable-isotope-labeling kinetics (SILK) method to a rhesus monkey cisterna magna ported (CMP) nonhuman primate model, and use the model to test the mechanisms of action of a gamma-secretase inhibitor (GSI). A major concern of inhibiting the enzymes which produce Abeta (beta- and gamma-secretase) is that precursors of Abeta may accumulate and cause a rapid increase in Abeta production when enzyme inhibition discontinues. In this study, the GSI MK-0752 was administered to conscious CMP rhesus monkeys in conjunction with in vivo stable-isotope-labeling, and dose-dependently reduced newly generated CNS Abeta. In contrast to systemic Abeta metabolism, CNS Abeta production was not increased after the GSI was cleared. These results indicate that most of the CNS APP was metabolized to products other than Abeta, including C-terminal truncated forms of Abeta: 1-14, 1-15 and 1-16; this demonstrates an alternative degradation pathway for CNS amyloid precursor protein during gamma-secretase inhibition.

Analyzing variability in neural responses to complex natural sounds in the awake songbird.

Studies of auditory processing in awake, behaving songbirds allow for the possibility of new classes of experiments, including those involving attention and plasticity. Detecting and determining the significance of plasticity, however, requires assessing the intrinsic variability in neural responses. Effects such as rapid plasticity have been investigated in the auditory system through the use of the spectrotemporal receptive field (STRF), a characterization of the properties of sounds to which a neuron best responds. Here we investigated neural response variability in awake recordings obtained from zebra finch field L, the analog of the primary auditory cortex. To quantify the level of variability in the neural recordings, we used three similarity measures: an STRF-based metric, a spike-train correlation-based metric, and a spike-train discrimination-based metric. We then extracted a number of parameters from these measures, quantifying how they fluctuated over time. Our results indicate that 1) awake responses are quite stable over time; 2) the different measures of response are complementary-specifically, the spike-train-based measures yield new information complementary to the STRF; and 3) different STRF parameters show distinct levels of variability. These results provide critical constraints for the design of robust decoding strategies and novel experiments on attention and plasticity in the awake songbird.

Hemodynamic and cardiac neurotransmitter-releasing effects in conscious dogs of attention- and wake-promoting agents: a comparison of d-amphetamine, atomoxetine, modafinil, and a novel quinazolinone H3 inverse agonist.

Conscious coronary sinus-cannulated dogs were used to assess the hemodynamic effects and local cardiac norepinephrine (NE) and histamine (HA) release of 4 mechanistically diverse agents either clinically approved or representing a potential novel mechanism for the promotion of wakefulness or attention. Dosing regimens were based on reported or concurrently determined wake-promoting activities in canine models. The central nervous system stimulant, d-amphetamine [0.1 mg x kg(-1) x 10 min intravenous (IV)], significantly elevated mean arterial pressure (+30%) and increased coronary sinus and peripheral venous NE concentrations, indicative of cardiac neurotransmitter release. The selective NE reuptake inhibitor atomoxetine (2.0 mg x kg(-1) x 10 min(-1) IV) and modafinil (30.0 mg x kg(-1) x 10 min(-1) IV) also significantly elevated mean arterial pressure (+15% and +30%, respectively), but with no effect on coronary sinus or peripheral NE concentration, suggesting central mechanisms underlying the hemodynamic effects. The preclinical demonstrations of pressor effects with d-amphetamine, atomoxetine, and modafinil are consistent with clinically reported hemodynamic effects with these agents. The quinazolinone HA receptor subtype H3 inverse agonist 5r (0.3 mg x kg(-1) x 10 min(-1) IV) displayed no effect on hemodynamics or on coronary sinus or peripheral NE and HA concentrations. These data suggest the potential for therapeutic effect with the latter mechanism in the absence of peripheral cardiac neurotransmitter release or obvious changes in cardiovascular function.

Enhanced hindlimb collateralization induced by acidic fibroblast growth factor is dependent upon femoral artery extraction.

UNLABELLED: Recent investigations have established the feasibility of using exogenously delivered angiogenic growth factors to increase collateral artery development in animal models of myocardial and hindlimb ischemia. OBJECTIVE: Our aim was to evaluate the ability of a stabilized form of acidic fibroblast growth factor (aFGF-S117) to stimulate collateralization and arteriogenesis in the rabbit hindlimb following the surgical induction of ischemia by femoral artery extraction. A secondary objective was to examine angiogenic and arteriogenic effects of aFGF-S117 in the absence of a peripheral blood flow deficit. METHODS AND RESULTS: Five days after femoral artery removal, aFGF-S117 (1, 3, or 30 microg/kg) was intramuscularly delivered into the hindlimb, three times per week for 2 consecutive weeks. End-point measurements performed on day 20 found that hindlimb reserve blood flow was significantly improved in rabbits that received 3 or 30 microg/kg of aFGF-S117, with no difference in efficacy between these two doses. These hemodynamic results were supported by angiographic evidence showing enhanced density of collateral vessels in the medial thigh region and histological findings of increased capillary density within the gastrocnemius muscle from rabbits treated with aFGF-S117. When an efficacious dose of 3 microg/kg of aFGF-S117 was administered to sham-operated rabbits with intact femoral arteries, there was no change in any of the blood flow, angiographic or histological parameters measured. CONCLUSIONS: These findings demonstrate that a stabilized form of aFGF stimulated the development of functional collateral arteries in the rabbit hindlimb, an effect which was dependent upon removal of the femoral artery. These results suggest that aFGF-S117 may have therapeutic potential for the treatment of arterial occlusive disorders.

Effects of inhibition of alpha-CGRP receptors on cardiac and peripheral vascular dynamics in conscious dogs with chronic heart failure.

Whether endogenous calcitonin gene-related peptide (CGRP) plays a role in heart failure is unclear. Seven dogs were instrumented with left ventricular (LV) pressure gauges, pacers, coronary occluder and aortic, atrial, and coronary sinus catheters. Hemodynamic recordings and response to alpha-CGRP challenge were obtained for baseline in the conscious state. Rapid pacing (240 beats/min) was then initiated. The coronary artery was occluded for 90 minutes followed by reperfusion after 2 weeks of pacing. After 6 weeks of pacing, LV pressure (-11 +/- 6%), LV dP/dt (-53 +/- 5%), and mean arterial pressure (-15 +/- 4%) decreased (P < 0.01), while left atrial pressure (+19 +/- 3 mm Hg from 7 +/- 1 mm Hg) and heart rate (+53 +/- 16%) increased (P < 0.01). Infusion of the alpha-CGRP receptor antagonist alpha-CGRP[8-37] (30 microg/kg/min, i.v.), which blocked the exogenous alpha-CGRP challenge, did not affect any of these indices. Regional blood flow, as measured by the microsphere technique, in the nonischemic myocardium, as well as cerebral and renal vasculatures were unaltered during the infusion of alpha-CGRP[8-37]. Plasma concentrations of CGRP from both arterial and coronary sinus samples were unchanged after 6 weeks of pacing as compared with control. Thus, we conclude that endogenous alpha-CGRP does not appear to play a major role in the regulation of cardiac and peripheral vascular dynamics in the late stage of heart failure.

Vascular endothelial growth factor stimulates angiogenesis without improving collateral blood flow following hindlimb ischemia in rabbits.

This study was designed to test the ability of adenovirus-delivered vascular endothelial growth factor (Ad-VEGF) to stimulate angiogenesis and arteriogenesis in the rabbit hindlimb following the induction of ischemia and to evaluate the functional changes in the collateral circulation. Ten days after the surgical induction of hindlimb ischemia, either a control virus (1 x 10(9) pfu) or an adenovirus containing the gene for VEGF(165) (1 x 10(6), 1 x 10(7), 1 x 10(8), or 1 x 10(9) pfu) was administered intramuscularly into the ischemic limb. Thirty days after administration of the adenoviral vectors, skeletal muscle capillary density was assessed and angiography was performed as markers of angiogenesis and arteriogenesis, respectively. Hindlimb blood flow was directly measured and hyperemic tests were performed to evaluate the functional improvements in collateral blood flow. Animals treated with Ad-VEGF at 1 x 10(8) and 1 x 10(9) pfu showed elevated levels of circulating VEGF and dose-dependent hindlimb edema. These doses also led to a robust angiogenic response (i.e., increase in capillary density), but failed to improve collateral blood flow. Consistent with the lack of a functional response, there was no angiographic evidence of enhanced arteriogenesis with any dose of Ad-VEGF. Following the induction of hindlimb ischemia, administration of Ad-VEGF stimulated capillary sprouting (i.e., angiogenesis), but did not increase the growth and development of larger conduit vessels (i.e., arteriogenesis) or improve collateral blood flow. These results support the concept that VEGF may not be expected to have therapeutic utility for the treatment of peripheral or myocardial ischemia.

An alternative method of chronic cerebrospinal fluid collection via the cisterna magna in conscious rhesus monkeys.

Models of chronic cerebrospinal fluid (CSF) collection previously have been established for nonhuman primates and canines; many of these methods implement stainless-steel cannulas into the lateral or 4th ventricles or catheters into the cerebral or spinal subarachnoid space. These models have proved successful and reliable but unfortunately require invasive techniques to pass through the skull or require a laminectomy to enter the spinal subarachnoid space, involve the use of expensive and highly specialized stereotaxic equipment for the precise placement of the implants, and may require exteriorized hardware which is cumbersome to maintain and unaesthetic. The model we developed for the rhesus monkey allows for direct access to CSF outflow from the cisterna magna by using a 3.5-French fenestrated silicone catheter which was placed 1.0 cm into the cisterna. The catheter was attached to a titanium port placed subcutaneously between the scapulae to permit easy access for sampling CSF in a conscious, chaired rhesus monkey. We currently have instrumented animals from which we have consistently collected CSF for over 18 months. This novel, economical, less-invasive method permits chronic, reliable collection of CSF in conscious rhesus monkeys and has the additional advantages that the model is easier to maintain and more aesthetic.

Use of three infusion pumps for postoperative administration of buprenorphine or morphine in dogs.

Results of using an implantable osmotic pump, a preset disposable infusion pump, or a reusable programmable infusion pump for postoperative administration of buprenorphine or morphine in dogs undergoing abdominal surgery are described. Ten dogs underwent abdominal surgery for implantation of vascular access ports. Dogs were given buprenorphine s.c. by use of an implantable osmotic pump (4 dogs), morphine s.c. by use of a preset infusion pump (4), or buprenorphine intra-arterially by use of a programmable infusion pump (2). Dogs were monitored, and serum buprenorphine or morphine concentration was measured for 72 hours after surgery; pumps were removed 48 hours after surgery. Severity of pain was determined by assigning a pain score. The preset infusion pump and the programmable infusion pump resulted in comparable pain relief and sustained serum analgesic concentrations throughout the recovery period. However, the cost of the pumps and other associated factors may limit their use to dogs undergoing invasive surgical procedures expected to result in substantial postoperative pain. The level of analgesia obtained with the implantable osmotic pumps was inconsistent.

Antiarrhythmic efficacy of combined I(Ks) and beta-adrenergic receptor blockade.

Suppression of malignant ventricular arrhythmias by selective blockade of the cardiac slowly activating delayed rectifier current (I(Ks)) has been demonstrated with the benzodiazepine L-768673 [(R)-2-(2,4-trifluoromethyl-phenyl)-N-[2-oxo-5-phenyl-1-(2,2,2-trifluoro-ethyl)-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl]acetamide] in canine models of recent and healed myocardial infarction. The present study extends the initial antiarrhythmic assessment of I(Ks) blockade by demonstrating prevention of ischemic malignant arrhythmias in dogs with recent (8.0 +/- 0.4 days) anterior myocardial infarction with the coadministration of a subeffective dose of L-768673 and a subeffective, minimally beta-adrenergic blocking dose of timolol. Administered individually, neither 0.3 microg/kg i.v. L-768673 nor 1.0 microg/kg i.v. timolol prevented the induction of ventricular tachyarrhythmia (VT) by programmed ventricular stimulation (PVS) or the development of malignant ventricular arrhythmia in response to acute coronary artery thrombosis. In contrast, coadministration of 0.3 microg/kg i.v. L-768673 + 1.0 microg/kg i.v. timolol suppressed the induction of VT by PVS (8/10, 80% rendered noninducible versus 1/10, 10% noninducible in vehicle group; p < 0.01) and prevented the development of acute ischemic lethal arrhythmias (3/10, 30% incidence versus 8/10, 80% incidence in vehicle group; p < 0.05). Concomitant administration of low-dose L-768673 + timolol produced modest increases in QTc and paced QT intervals (4.5 +/- 1.2 and 5.5 +/- 1.4%; both p < 0.01), increases in noninfarct zone relative and effective refractory periods (7.0 +/- 1.7 and 12.3 +/- 3.0%; both p < 0.01), and lesser increases in infarct zone relative and effective refractory periods (5.3 +/- 1.6 and 5.8 +/- 1.4%; both p < 0.01). These findings suggest that concomitant low-dose I(Ks) and beta-adrenergic blockade may constitute a potential pharmacologic strategy for prevention of malignant ischemic ventricular arrhythmias.