A novel mechanism for myocardial stunning involving impaired Ca(2+) handling.

The mechanism of myocardial stunning has been studied extensively in rodents and is thought to involve a decrease in Ca(2+) responsiveness of the myofilaments, degradation of Troponin I (TnI), and no change in Ca(2+) handling. We studied the mechanism of stunning in isolated myocytes from chronically instrumented pigs. Myocytes were isolated from the ischemic (stunned) and nonischemic (normal) regions after 90-minute coronary stenosis followed by 60-minute reperfusion. Baseline myocyte contraction was reduced, P<0.01, in stunned myocytes (6.3+/-0.4%) compared with normal myocytes (8.8+/-0.4%). The time for 70% relaxation was prolonged, P<0.01, in stunned myocytes (131+/-8 ms) compared with normal myocytes (105+/-5 ms). The impaired contractile function was associated with decreased Ca(2+) transients (stunned, 0.33+/-0.04 versus normal, 0.49+/-0.05, P<0.01). Action potential measurements in stunned myocytes demonstrated a decrease in plateau potential without a change in resting membrane potential. These changes were associated with decreased L-type Ca(2+)-current density (stunned, -4.8+/-0.4 versus normal, -6.6+/-0.4 pA/pF, P<0.01). There were no differences in TnI, sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a), and phospholamban protein quantities. However, the fraction of phosphorylated phospholamban monomer was reduced in stunned myocardium. In rats, stunned myocytes demonstrated reduced systolic contraction but actually accelerated relaxation and no change in Ca(2+) transients. Thus, mechanisms of stunning in the pig are radically different from the widely held concepts derived from studies in rodents and involve impaired Ca(2+) handling and dephosphorylation of phospholamban, but not TnI degradation.

Cyclosporine reduces left ventricular mass with chronic aortic banding in mice, which could be due to apoptosis and fibrosis.

A tacit assumption in studies of left ventricular (LV) hypertrophy is that left ventricular/body weight (LV/BW) reflects the extent of myocyte hypertrophy. The goal of the current investigation was to determine if there was another explanation for the reduced LV/BW observed after inhibiting calcineurin with cyclosporine during the development of pressure overload LV hypertrophy as compared with animals that did not receive cyclosporine. Accordingly, we examined the prevalence of fibrosis and apoptosis and measured cell size in the hearts from mice at 1 and 3 weeks after transverse aortic banding with and without chronic cyclosporine. Although LV/BW, compared to aortic banded vehicle treated mice, was reduced by 30% in aortic banded cyclosporine treated mice, myocyte cross sectional area was similar in both banded groups (346+/-9 microm2 v 336+/-13 microm2). The volume percent interstitial fibrosis was greater in aortic banded cyclosporine treated animals (1.4+/-0.2%) compared with aortic banded vehicle treated animals (0.9+/-0.2%, P<0.05) or in sham animals (0.6+/-0.1%). Surprisingly, lesions including myocytes containing iron were observed and were most prominent in aortic banded cyclosporine treated animals. Apoptosis, quantitated with TUNEL staining as percent of myocytes, was increased in aortic banded cyclosporine treated animals at 7 days (1.6+/-0.4%) compared with aortic banded vehicle treated animals (0.4+/-0.1%, P<0.01) and was still increased at 21 days. Immunoblotting demonstrated a decrease in the phosphorylation of Akt and Bad, and also Bcl-2 levels were reduced in aortic banded cyclosporine treated animals at 7 days compared with aortic banded vehicle treated animals. These proteins protect against apoptosis, and support the concept that cyclosporine inhibited the calcineurin pathway, resulting in enhanced apoptosis. Thus, the decrease in LV/BW in the aortic banded cyclosporine treated animals actually may be due, at least in part, to cell loss and death, as reflected by the enhanced fibrosis and apoptosis and the focal iron deposits in myocytes.

Paradoxically enhanced endothelin-B receptor-mediated vasoconstriction in conscious old monkeys.

BACKGROUND: We investigated the effects of aging on the responses to endothelin (ET) in conscious old (19.8+/-0.6 years) and young adult (6.8+/-0.3 years) monkeys and compared these results with those of other vasoconstrictors, eg, phenylephrine (PE) and angiotensin II (Ang II). METHODS AND RESULTS: The monkeys (Macaca fascicularis) were chronically instrumented. Baseline total peripheral resistance (TPR) was not different between the 2 groups. As expected, TPR rose less (P<0.05) with PE (5 microgram/kg) in old monkeys (34+/-3%) than in young monkeys (57+/-6%); TPR also rose less with Ang II. Surprisingly, TPR rose more (P<0.05) with endothelin-1 (ET-1, 25 ng. kg(-1). min(-1)) in old monkeys (36+/-6%) than in young monkeys (10+/-2%). An ET(B) receptor agonist, sarafotoxin (S6c, 30 ng. kg(-1). min(-1)) was administered in the presence of an ET(A) receptor antagonist, BQ-123 (1 mg/kg). Under these conditions, TPR increased more (P<0.05) in old monkeys (59+/-10%) than in young monkeys (31+/-4%). In the presence of nitric oxide synthase (NOS) inhibition with N(W)-nitro-L-arginine methyl ester (60 mg/kg), vasoconstriction induced by S6c no longer differed with age, because it was enhanced in young monkeys (P<0.05) (68+/-9% versus 31+/-4%) but not in old monkeys (58+/-6% versus 59+/-10%). Thus, after NOS inhibition, vasoconstrictor responses to ET were no longer enhanced in old monkeys. CONCLUSIONS: Peripheral vasoconstriction (PE and Ang II) is reduced in old monkeys, as expected. Paradoxically, vasoconstriction induced by ET-1 was actually enhanced in old monkeys, which appears to be a result of impaired endothelium-dependent vasodilation, which with ET-1 should involve the ET(B) receptor.

Alpha-human atrial natriuretic peptide, carperitide, reduces infarct size but not arrhythmias after coronary occlusion/reperfusion in dogs.

Carperitide, a recombinant form of alpha-hANP, possesses potent diuretic, natriuretic, and vasodilatory activity, and inhibits the renin-aldosterone system and sympathetic nervous activity. However, its beneficial effects on ischemic myocardium have not been studied fully. We examined carperitide's effects on infarct size, hemodynamics, and arrhythmia frequency in anesthetized dogs (n = 20) subjected to a 90-min coronary artery occlusion/6-h reperfusion protocol. Intravenous infusion of carperitide (0.2 microg/kg/min) commenced 15 min after occlusion and continued during occlusion/reperfusion. Ventricular fibrillation developed in two of 10 control versus three of 10 treated dogs (p = NS). Hemodynamics, collateral blood flow to the ischemic wall measured 10 min after occlusion, and extent of area at risk were comparable for the two groups. Infarct size/area at risk was smaller in treated than in control dogs (4.5 +/- 2.1% vs. 27.8 +/- 7.8%, respectively; p < 0.05). During occlusion, carperitide tended to increase collateral blood flow (+39%) and significantly decreased left ventricular systolic pressure (-13%) and end-diastolic pressure (-40%) compared with baseline. In control dogs, collateral blood flow tended to decrease (-8.3%), whereas most hemodynamic parameters did not change significantly with respect to baseline. The number of arrhythmias recorded during occlusion/reperfusion was similar in the two groups. Intravenous administration of carperitide limited infarct size, but did not reduce incidence of ventricular arrhythmias after 90-min coronary occlusion/6-h reperfusion in anesthetized dogs. Although the beneficial effects of carperitide may be attributable to concomitant changes in hemodynamics and collateral blood flow, the precise mechanisms require further investigation.

Peripheral vascular endothelial dysfunction and apoptosis in old monkeys.

To determine the effects of aging on vasoactivity in a primate model (Macaca fascicularis), 13 young male monkeys (aged 7.1+/-0.4 years) and 9 old male monkeys (aged 19.8+/-0.6 years) were chronically instrumented for measurement of left ventricular and aortic pressures and cardiac output. Total cholesterol, triglyceride, and fasting blood sugar levels were not different between the 2 groups. There were no significant differences in baseline mean aortic pressure and total peripheral resistance (TPR) in the young monkeys versus the old monkeys. TPR fell less (P<0.05) with acetylcholine (1 microg/kg) in old monkeys (-25+/-1%) than in young monkeys (-34+/-2%), whereas decreases in TPR with sodium nitroprusside were similar in old and young monkeys. There was no evidence of atherosclerosis, but apoptosis of endothelial cells was enhanced (P<0.05) in the aortas and femoral arteries, but not in the media, of the old monkeys. There was a relationship (r=0.62, P=0.013) between the incidence of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive endothelial cells and endothelial cell density in the femoral artery. The reduced endothelial cell density was also correlated (r=0.82, P<0.01) with depressed TPR responses to acetylcholine. Thus, vascular endothelial dysfunction was present in old monkeys without evidence of atherosclerosis, which may be due to endothelial apoptosis and reduced endothelial cell density.

Beta-adrenergic receptor blockade arrests myocyte damage and preserves cardiac function in the transgenic G(salpha) mouse.

Transgenic (TG) mice with cardiac G(salpha) overexpression exhibit enhanced inotropic and chronotropic responses to sympathetic stimulation, but develop cardiomyopathy with age. We tested the hypothesis that cardiomyopathy in TG mice with G(salpha) overexpression could be averted with chronic beta-adrenergic receptor (beta-AR) blockade. TG mice and age-matched wild-type littermates were treated with the beta-AR blocker propranolol for 6-7 months, starting at a time when the cardiomyopathy was developing but was not yet severe enough to induce significant cardiac depression (9.5 months of age), and ending at a time when cardiac depression and cardiomyopathy would have been clearly manifest (16 months of age). Propranolol treatment, which can induce cardiac depression in the normal heart, actually prevented cardiac dilation and the depressed left ventricular function characteristic of older TG mice, and abolished premature mortality. Propranolol also prevented the increase in myocyte cross-sectional area and myocardial fibrosis. Myocyte apoptosis, already apparent in 9-month-old TG mice, was actually eliminated by chronic propranolol. This study indicates that chronic sympathetic stimulation over an extended period is deleterious and results in cardiomyopathy. Conversely, beta-AR blockade is salutary in this situation and can prevent the development of cardiomyopathy.

Mechanisms of desensitization to a PDE inhibitor (milrinone) in conscious dogs with heart failure.

The goal of this study was to determine the extent to which the effects of milrinone were desensitized in heart failure (HF) and to determine the mechanisms, i.e., whether these effects could be ascribed to changes in cAMP or phosphodiesterase (PDE) activity in HF. Accordingly, we examined the effects of milrinone in seven conscious dogs before and after HF was induced by rapid ventricular pacing at 240 beats/min. The dogs were chronically instrumented for measurements of left ventricular (LV) pressure and first derivative of LV pressure (dP/dt), arterial pressure, LV internal diameter, and wall thickness. Milrinone (10 micrograms . kg-1. min-1 iv) increased LV dP/dt by 1,854 +/- 157 from 2,701 +/- 105 mmHg/s (P < 0.05) before HF. After HF the increase in LV dP/dt in response to milrinone was attenuated significantly (P < 0.05); it increased by 615 +/- 67 from 1,550 +/- 107 mmHg/s, indicating marked desensitization. In the presence of ganglionic blockade the increases in LV dP/dt (+445 +/- 65 mmHg/s) in response to milrinone were markedly less (P < 0.01), and milrinone increased LV dP/dt even less in HF (+240 +/- 65 mmHg/s). cAMP and PDE activity were measured in endocardial and epicardial layers in normal and failing myocardium. cAMP was decreased significantly (P < 0.05) in LV endocardium (-26%) but not significantly in LV epicardium (-14%). PDE activity was also decreased significantly (P < 0.05) in LV endocardium (-18%) but not in LV epicardium (-4%). Thus significant desensitization to milrinone was observed in conscious dogs with HF. The major effect was autonomically mediated. The biochemical mechanism appears to be due in part to the modest reductions in PDE activity in failing myocardium, which, in turn, may be a compensatory mechanism to maintain cAMP levels in HF. Reductions in cAMP and PDE levels were restricted to the subendocardium, suggesting that the increased wall stress and reduced coronary reserve play a role in mediating these changes.

Differential regulation of inotropy and lusitropy in overexpressed Gsalpha myocytes through cAMP and Ca2+ channel pathways.

We investigated the mechanisms responsible for altered contractile and relaxation function in overexpressed Gsalpha myocytes. Although baseline contractile function (percent contraction) in Gsalpha mice was similar to that of wild-type (WT) mice, left ventricular myocyte contraction, fura-2 Ca2+transients, and Ca2+ channel currents (ICa) were greater in Gsalpha mice in response to 10(-8) M isoproterenol (ISO) compared with WT mice. The late phase of relaxation of the isolated myocytes and fura-2 Ca2+ transients was accelerated at baseline in Gsalpha but did not increase further with ISO. In vivo measurements using echocardiography also demonstrated enhanced relaxation at baseline in Gsalpha mice. Forskolin and CaCl2 increased contraction similarly in WT and Gsalpha mice. Rp-cAMP, an inhibitor of protein kinase, blocked the increases in contractile response and Ca2+ currents to ISO in WT and to forskolin in both WT and Gsalpha. It also blocked the accelerated relaxation in Gsalpha at baseline but not the contractile response to ISO in Gsalpha myocytes. Baseline measurements of cAMP and phospholambation phosphorylation were enhanced in Gsalpha compared with WT. These data indicate that overexpression of Gsalpha accelerates relaxation at end diastolic but does not affect baseline systolic function in isolated myocytes. However, the enhanced responses to sympathetic stimulation partly reflect increased Ca2+ channel activity; i.e the cellular mechanisms mediating these effects appear to involve a cAMP-independent as well as a cAMP-dependent pathway.

Cyclosporine attenuates pressure-overload hypertrophy in mice while enhancing susceptibility to decompensation and heart failure.

Left ventricular hypertrophy (LVH) is a compensatory mechanism to cope with pressure overload. Recently, a calcineurin pathway mediating LVH and its prevention by cyclosporine was reported. We examined whether calcineurin mediates LVH due to pressure overload in mice. Pressure overload was induced by aortic banding in 53 mice (32 treated with cyclosporine [25 mg. kg-1. d-1], 21 treated with vehicle). There were 17 sham-operated mice (9 treated with vehicle, 8 treated with cyclosporine). At 3 weeks after surgery, LV weight to body weight was greater in the nontreatment banded group (4.39+/-0. 16 mg/g) than in the cyclosporine-treated banded group (3.95+/-0.14 mg/g, P<0.05), with both groups being greater compared with the entire group of sham-operated mice (3.02+/-0.04 mg/g). The pressure gradient between the ascending and abdominal aorta was not different between the cyclosporine-treated (49.6+/-6.1 mm Hg) and nontreatment groups (48.7+/-4.6 mm Hg). Although LV systolic pressure was lower in the cyclosporine-treated banded animals, LV systolic wall stress was similar in the nontreatment banded group and in the cyclosporine-treated group. However, LV dP/dt was lower (P=0.05) in the cyclosporine-treated banded group (4774+/-656 mm Hg/s) than in the nontreatment banded group (6604+/-516 mm Hg/s). During the protocol, 23 of 32 mice in the cyclosporine-treated group and 9 of 21 mice in the nontreatment group died. All deaths occurred within 10 days after surgery. Deaths caused by heart failure were 7.2-fold higher (P<0.05) in the cyclosporine-treated group, whereas deaths due to other causes were not different between the 2 groups. In addition, LV function of mice was assessed at 48 hours after banding; LV ejection fraction measured with echocardiography was lower (P<0.05) in the cyclosporine-treated banded group (66+/-3.0%) than in the nontreatment banded group (79+/-1.5%), whereas LV systolic wall stresses were similar. Calcineurin phosphatase activity was depressed similarly in both cyclosporine-treated groups compared with both nontreatment groups. Thus, cyclosporine could attenuate, but not prevent, LVH at the expense of inhibiting an important compensatory mechanism in response to pressure overload, resulting in reduced LV wall stress and function and increased susceptibility to decompensation and heart failure.

Does inhibition of coronary nitric oxide synthesis alter coronary vascular tone in normal dogs?

To examine whether endothelial nitric oxide formation contributes to the vascular tone of resistance coronary vessels in vivo, we administered NG-nitro-L-arginine methyl ester (L-NAME) (10 and 100 micrograms/kg/min), a nitric oxide synthase inhibitor, as well as D-enantiomer into the left circumflex artery in normal dogs. Intracoronary L-NAME, which was associated with dose-related reductions in acetylcholine-induced coronary vasodilation, significantly reduced the baseline left circumflex blood flow by 6% and increased coronary vascular resistance of the left circumflex artery by 6%. D-enantiomer was ineffective in altering baseline coronary blood flow and vascular resistance of the left circumflex artery. These results indicate that continuous nitric oxide formation in the vasculature is important in the regulation of the coronary vascular tone of resistance vessels in vivo, and serves to maintain the vessels in a dilated state.