Contribution of caveolin protein abundance to augmented nitric oxide signaling in conscious dogs with pacing-induced heart failure.

Myocardial NO signaling appears elevated in heart failure (HF). Whether this results from increased NO production, induction of the high-output NO synthase (NOS)2 isoform, or changes in NOS regulatory pathways (such as caveolae) remains controversial. We tested the hypothesis that increased abundance of caveolin-3 and/or sarcolemmal caveolae contribute to increased NO signaling in pacing-induced HF. Abundance of caveolin-3 (0.59+/-0.08 versus 0.29+/-0.08 arbitrary units, P = 0.01) but not caveolin-1 was increased in HF compared with control conditions, assessed by Western blot. Additionally, transmission electron microscopy revealed increased caveolae (2. 7+/-0.4 versus 1.3+/-0.3 per micrometer myocyte membrane, P<0.005). The association between caveolin-3 and NOS3 at the sarcolemma and T tubules was unchanged in HF compared with control myocytes. The impact of NOS inhibition with L-N(G)-methylarginine hydrochloride (L-NMMA) on beta-adrenergic inotropy was assessed in conscious dogs before and after HF. In control dogs, dobutamine (5 microg. kg(-1) x min(-1)) increased +dP/dt by 36+/-7%, and this was augmented to 66+/-24% by 20 mg/kg L-NMMA (P = 0.04 versus without L-NMMA, n = 8) but not affected by 10 mg/kg L-NMMA (34+/-10%, P = NS; n = 8). In HF, dobutamine +dP/dt response was depressed (P<0.001 versus control), and increased concentrations were required to match control inotropic responses (10 to 15 microg. kg(-1) x min(-1), 48+/-7%). L-NMMA enhanced +dP/dt responses similarly at 10 mg/kg (61+/-17%, P = 0.02; n = 4) and 20 mg/kg (54+/-7%, P = 0.04; n = 7). Caveolin-3 abundance positively correlated with L-NMMA augmentation of dobutamine inotropic responses in HF (r = 0.9, P = 0.03; n = 4). Thus, in canine pacing-induced HF, expression of caveolin-3 and of sarcolemmal caveolae is increased. This increase is associated with augmented agonist-stimulated NO signaling, likely via a compartmentation effect.

Intravenous allopurinol decreases myocardial oxygen consumption and increases mechanical efficiency in dogs with pacing-induced heart failure.

Allopurinol, an inhibitor of xanthine oxidase, increases myofilament calcium responsiveness and blunts calcium cycling in isolated cardiac muscle. We sought to extend these observations to conscious dogs with and without pacing-induced heart failure and tested the prediction that allopurinol would have a positive inotropic effect without increasing energy expenditure, thereby increasing mechanical efficiency. In control dogs (n=10), allopurinol (200 mg IV) caused a small positive inotropic effect; (dP/dt)(max) increased from 3103+/-162 to 3373+/-225 mm Hg/s (+8.3+/-3.2%; P=0.01), but preload-recruitable stroke work and ventricular elastance did not change. In heart failure (n=5), this effect was larger; (dP/dt)(max) rose from 1602+/-190 to 1988+/-251 mm Hg/s (+24.4+/-8.7%; P=0.03), preload-recruitable stroke work increased from 55.8+/-9.1 to 84. 9+/-12.2 mm Hg (+28.1+/-5.3%; P=0.02), and ventricular elastance rose from 6.0+/-1.6 to 10.5+/-2.2 mm Hg/mm (P=0.03). Allopurinol did not affect myocardial lusitropic properties either in control or heart failure dogs. In heart failure dogs, but not controls, allopurinol decreased myocardial oxygen consumption (-49+/-4.6%; P=0. 002) and substantially increased mechanical efficiency (stroke work/myocardial oxygen consumption; +122+/-42%; P=0.04). Moreover, xanthine oxidase activity was approximately 4-fold increased in failing versus control dog hearts (387+/-125 versus 78+/-72 pmol/min. mg(-1); P=0.04) but was not detectable in plasma. These data indicate that allopurinol possesses unique inotropic properties, increasing myocardial contractility while simultaneously reducing cardiac energy requirements. The resultant boost in myocardial contractile efficiency may prove beneficial in the treatment of congestive heart failure.

Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies.

Pacing-induced heart failure in the dog recapitulates many of the electrophysiological and hemodynamic abnormalities of the human disease; however, the mechanisms underlying altered Ca2+ handling have not been investigated in this model. We now show that left ventricular midmyocardial myocytes isolated from dogs subjected to 3 to 4 weeks of rapid pacing have prolonged action potentials and Ca2+ transients with reduced peaks, but durations approximately 3-fold longer than controls. To discriminate between action potential effects on Ca2+ kinetics and direct changes in Ca2+ regulatory processes, voltage-clamp steps were used to examine the time constant for cytosolic Ca2+ removal (tauCa). tauCa was prolonged by just 35% in myocytes from failing hearts after fixed voltage steps in physiological solutions (tauCa control, 216+/-25 ms, n=17; tauCa failing, 292+/-23 ms, n=22; P<0.05), but this difference was markedly accentuated when Na+/Ca2+ exchange was eliminated (tauCa control, 282+/-30 ms, n=13; tauCa failing, 576+/-83 ms, n=11; P<0. 005). Impaired sarcoplasmic reticular (SR) Ca2+ uptake and a greater dependence on Na+/Ca2+ exchange for cytosolic Ca2+ removal was confirmed by inhibiting SR Ca2+ ATPase with cyclopiazonic acid, which slowed Ca2+ removal more in control than in failing myocytes. beta-Adrenergic stimulation of SR Ca2+ uptake in cells from failing hearts sufficed only to accelerate tauCa to the range of unstimulated controls. Protein levels of SERCA2a, phospholamban, and Na+/Ca2+ exchanger revealed a pattern of changes qualitatively similar to the functional measurements; SERCA2a and phospholamban were both reduced in failing hearts by 28%, and Na+/Ca2+ exchange protein was increased 104% relative to controls. Thus, SR Ca2+ uptake is markedly downregulated in failing hearts, but this defect is partially compensated by enhanced Na+/Ca2+ exchange. The alterations are similar to those reported in human heart failure, which reinforces the utility of the pacing-induced dog model as a surrogate for the human disease.

Loss of cardiac magnesium in experimental heart failure prolongs and destabilizes repolarization in dogs.

OBJECTIVES: We sought to determine whether heart failure results in loss of cardiac magnesium sufficient to alter cellular electrophysiology. BACKGROUND: Free magnesium has numerous intracellular roles affecting metabolism, excitability and RNA synthesis. Total cardiac magnesium content is reduced in heart failure, but it is unclear whether magnesium loss is primary or iatrogenic. Furthermore, it is unknown whether free magnesium levels are affected or whether a change in free magnesium would alter cellular electrophysiology. METHODS: Eight mongrel dogs underwent demand ventricular pacing (VVI) at 250 beats/min for 3 weeks to induce heart failure. Sublingual epithelial magnesium was measured before pacing and at death. Left ventricular myocytes were isolated and loaded with Mag-Indo-1 to measure free magnesium ([Mg2+]i); myocytes from eight normal dogs served as controls. To test whether changes in [Mg2+]i in this range could alter cellular repolarization, current-clamped myocytes were dialyzed with 0.5 or 1.0 mmol/liter MgCl2. RESULTS: Mean sublingual epithelial magnesium fell significantly in the paced animals, from 36.9 +/- 0.5 to 33.9 +/- 0.7 mEq/liter (p < 0.01). Mean cardiac [Mg2+]i was significantly lower in the dogs with heart failure--0.49 +/- 0.06 versus 1.06 +/- 0.15 mmol/liter (p < 0.003). Time to 90% repolarization was significantly shorter in cells dialyzed with 1.0 mmol/liter compared with 0.5 mmol/liter MgCl2 in myocytes from normal dogs or dogs with heart failure (596 +/- 34 vs. 760 +/- 58 ms in normal dogs and 586 +/- 29 vs. 838 +/- 98 ms in dogs with heart failure; p < 0.05 for each). CONCLUSIONS: Experimental heart failure results in both tissue and cardiac magnesium loss in the absence of drug therapy. Free cardiac magnesium is significantly reduced, possibly contributing to abnormal repolarization in heart failure.

Repolarization abnormalities, arrhythmia and sudden death in canine tachycardia-induced cardiomyopathy.

OBJECTIVES: This study sought to determine whether the canine model of tachycardia-induced heart failure (HF) is an effective model for sudden cardiac death (SCD) in HF. BACKGROUND: Such a well established HF model that also exhibits arrhythmias and SCD, along with repolarization abnormalities that could trigger them, may facilitate the study of SCD in HF, which still eludes effective treatment. METHODS: Twenty-five dogs were VVI-paced at 250 beats/min for 3 to 5 weeks. Electrocardiograms were obtained, and left ventricular endocardial monophasic action potentials (MAPs) were recorded at six sites at baseline and after HF. Weekly Holter recordings were made with pacing suspended for 24 h. RESULTS: Six animals (24%) died suddenly, one with Holter-documented polymorphic ventricular tachycardia (VT). Holter recordings revealed an increased incidence of VT as HF progressed. Repolarization was significantly (p < 0.05) prolonged, as indexed by a corrected QT interval (mean [+/-SD] 311 +/- 25 to 338 +/- 25 ms) and MAP duration measured at 90% repolarization (MAPD90) (181 +/- 19 to 209 +/- 28 ms), and spatial MAPD90 dispersion rose by 40%. We further tested whether CsCl inhibition of repolarizing K+ currents, which are reportedly downregulated in HF, might preferentially prolong the MAPD90 in HF. With 1 mEq/kg body weight of CsCl, MAPD90 rose by 86 +/- 100 ms in dogs with HF versus only 28 +/- 16 ms in control animals (p = 0.002). Similar disparities in CsCl sensitivity were observed in myocytes isolated from normal and failing hearts. CONCLUSIONS: Tachycardia-induced HF exhibits malignant arrhythmia and SCD, along with prolonged, heterogeneous repolarization and heightened sensitivity to CsCl at chamber and cellular levels. Thus, it appears to be a useful model for studying mechanisms and therapy of SCD in HF.

Adverse influence of systemic vascular stiffening on cardiac dysfunction and adaptation to acute coronary occlusion.

BACKGROUND: [corrected] Age is an independent risk factor for increased mortality from ischemic heart disease. Arterial stiffening with widening of the pulse pressure may contribute to this risk by exacerbating cardiac dysfunction after total coronary artery occlusion. METHODS AND RESULTS: To test the above hypothesis, 14 open-chest dogs underwent surgery in which the intrathoracic aorta was bypassed with a stiff plastic tube. Directing ventricular outflow through the bypass widened the arterial pulse pressure from 41 to 115 mm Hg at similar mean pressure and flow. Hearts ejecting into the native aorta (NA) exhibited only modest dysfunction after two minutes of mid-left anterior descending coronary artery occlusion. However, the same occlusion applied during ejection into the bypass tube (BT) induced far more severe cardiodepression (ie, systolic pressure fell by -41+/-10 mm Hg for BT versus -15+/-3 mm Hg for NA, and end-systolic volume rose by 15+/-3 versus 6+/-2 mL), with a threefold greater decline in ejection fraction. This disparity was not due to higher baseline work loads because total pressure-volume area was similar in both cases. Furthermore, marked increases in basal work load and wall stress induced by angiotensin II infusion (in four additional studies) did not reproduce this behavior. Although peak systolic chamber stress was greater with the BT, this did not increase systolic dyskinesis as measured in the central ischemic zone. However, the total mass of myocardium that was rendered severely ischemic (ie, flow reduced by > or = 80%) was twice as large with BT ejection, likely expanding the region of dyskinesis. This disparity may relate to altered phasic coronary flow during BT ejection, which displays marked enhancement of systolic flow and renders the heart more vulnerable to diminished mean and systolic perfusion pressures. CONCLUSIONS: Cardiac ejection into a stiff systemic vasculature augments cardiac dysfunction and ischemia due to coronary occlusion by tightening the link between cardiac systolic performance and myocardial perfusion. This may contribute to the higher mortality risk from ischemic heart disease due to age.

Endomyocardial gene expression during development of pacing tachycardia-induced heart failure in the dog.

Selective and specific changes in gene expression characterize the end-stage failing heart. However, the pattern and relation of these changes to evolving systolic and diastolic dysfunction during development of heart failure remains undefined. In the present study, we assessed steady-state levels of mRNAs encoding a group of cardiac proteins during the early development of left ventricular dysfunction in dogs with pacing-induced cardiomyopathy. Corresponding hemodynamic assessments were made in the conscious state in the same animals and at the same time points at baseline, after 1 week of ventricular pacing, and at the onset of clinical heart failure. Systolic dysfunction dominated after 1 week of pacing, whereas diastolic dysfunction was far more pronounced with the onset of heart failure. Atrial natriuretic factor mRNA was undetectable in 7 of 12 hearts at baseline but was expressed in all hearts at 1 week (P < .01 by chi 2 test), and it increased markedly with progression to failure (P = .05). Creatine kinase-B mRNA also rose markedly with heart failure (P < .01). Levels of mRNA encoding beta-myosin heavy chain, mitochondrial creatine kinase, phospholamban, and sarcoplasmic reticulum Ca(2+)-ATPase did not significantly change from baseline, despite development of heart failure. Additional analysis to determine if these mRNA changes were related to the severity of diastolic or systolic dysfunction revealed that phospholamban mRNA decreased in hearts with larger net increases in end-diastolic pressure (+19.2 +/- 1.9 mm Hg) compared with those hearts in which it did not change (+4.0 +/- 4.9, P < .02).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of reduced aortic compliance on cardiac efficiency and contractile function of in situ canine left ventricle.

This study tests the hypothesis that arterial vascular stiffening adversely influences in situ left ventricular contractile function and energetic efficiency. Ten reflex-blocked anesthetized dogs underwent a bypass operation in which a Dacron graft was sewn to the ascending aorta and connected to the infrarenal abdominal aorta via a plastic conduit. Flow was directed through either native aorta or plastic conduit by placement of vascular clamps. Arterial properties were measured from aortic pressure-flow data, and ventricular function was assessed by pressure-volume (PV) relations. Coronary sinus blood was drained via an extracorporeal circuit for direct measurement of myocardial O2 consumption (MVO2). Data at multiple steady-state preload volumes were combined to derive chamber function and energetics relations. Energetic efficiency was assessed by the inverse slope of the MVO2-PV area relation. Directing flow through plastic versus native aorta resulted in a 60-80% reduction in compliance but little change in mean resistance. Arterial pulse pressure rose from 34 to 99 mm Hg (p less than 0.001). Contractile function assessed by the end-systolic PV relation, stroke work-end-diastolic volume relation, and dP/dtmax at matched end-diastolic volume did not significantly change. However, MVO2 increased by 32% (p less than 0.01) and was matched by a rise in PV area, such that the MVO2-PV area relation and efficiency was unaltered. The MVO2 required to sustain a given stroke volume, however, increased from 20% to 40%, depending on the baseline level (p less than 0.001). Thus, whereas the contractile function and efficiency of normal hearts are not altered by ejection into a stiff vascular system, the energetic cost to the heart for maintaining adequate flow is increased. This suggests a mechanism whereby human vascular stiffening may yield little functional decrement at rest but limit reserve capacity under conditions of increased demand.

Mean circulatory filling pressure: potential problems with measurement.

Three experimental series using 22 acutely splenectomized mongrel dogs were completed to 1) compare fibrillation (Fib) and acetylcholine (ACh) injection as methods to stop the heart for the mean circulatory filling pressure (Pmcf) maneuver, and 2) test whether Pmcf equals portal venous pressure 7 s after heart stoppage (Pportal7s). Blood volume changes of -10, -20, +10, or +20 ml/kg were imposed and Pmcf and Pportal measurements were obtained. Pportal7s and Pmcf were significantly different with volume depletion but were similar under control conditions. Pmcf with ACh and Pmcf with Fib were significantly different only after a volume change of -20 ml/kg. However, severe pulmonary congestion and atelectasis were detected in animals where Ach was used to stop the heart. In some cases (with injection directly into the pulmonary artery) the damage was severe enough to cause irreversible arterial hypoxia. Thus we conclude that the repeated use of ACh may exert a detrimental influence on pulmonary function, changing the physiological status of the experimental animal. Also, the central venous pressure at 7 s of heart stoppage (Pcv7s) is not a fully accurate estimate of the true mean circulatory filling pressure during the Pmcf maneuver, because Pcv7s did not equal the Pportal7s under all experimental conditions.