Joy McCarthy

Ryanodine and caffeine prevent ventricular arrhythmias during acute myocardial ischemia and reperfusion in rat heart.

This study investigates the possible role of oscillatory release of calcium from sarcoplasmic reticulum in the genesis of ventricular arrhythmias during acute myocardial ischemia and reperfusion in isolated rat hearts. We used ryanodine and caffeine, which are known to modulate the oscillatory release of calcium from sarcoplasmic reticulum. During 30 minutes of left main coronary artery ligation, all 13 control hearts developed ventricular premature beats (number of beats, 225 ± 51) and ventricular tachycardia (duration, 123 ±21 seconds); five hearts developed ventricular fibrillation. In a separate series of experiments, reperfusion after 15 minutes of coronary artery ligation caused ventricular fibrillation to occur within 15 seconds in all 12 hearts. Ryanodine (10−9 to 10−7 M) abolished ventricular arrhythmias during coronary artery ligation and prevented reperfusion ventricular fibrillation. Ryanodine (10−9, 10−8, and 10−7 M) caused 15%, 23%, and 74% decreases in the maximal rate of rise of left ventricular pressure development and 20%, 32%, and 85% decreases in the maximal rate of fall of left ventricular pressure development, respectively, prior to coronary artery ligation. During acute myocardial ischemia, ryanodine 10−9 M maintained and 10−5 M impaired left ventricular function; 10−7 M caused left ventricular failure. Coronary perfusion rate did not increase during ischemia. Antiarrhythmic activity occurred independent of preservation of high energy phosphates, reduction in tissue lactate, or tissue cyclic adenosine monophosphate in the ischemic myocardium. Caffeine 10−2 M decreased the incidence of ventricular arrhythmias during ischemia and upon reperfusion; protection occurred coincident with development of diastolic contracture. Caffeine increased ischemic tissue cyclic adenosine monophosphate content and worsened tissue energy status. Our findings suggest that oscillatory release of calcium from the sarcoplasmic reticulum may play an important role in ventricular arrhythmogenesis during acute myocardial ischemia and reperfusion. Impairment of left ventricular mechanical function appears likely to preclude the use of ryanodine and caffeine in vivo as ventricular antiarrhythmic agents.

Effect of nitrovasodilators and inhibitors of nitric oxide synthase on ischaemic and reperfusion function of rat isolated hearts

1 The functional role of the nitric oxide (NO)/guanosine 3′:5′‐cyclic monophosphate (cyclic GMP) pathway in experimental myocardial ischaemia and reperfusion was studied in rat isolated hearts. 2 Rat isolated hearts were perfused at constant pressure with Krebs‐Henseleit buffer for 25 min (baseline), then made ischaemic by reducing coronary flow to 0.2 ml min−1 for 25 or 40 min, and reperfused at constant pressure for 25 min. Drugs inhibiting or stimulating the NO/cyclic GMP pathway were infused during the ischaemic phase only. Ischaemic contracture, myocardial cyclic GMP and cyclic AMP levels during ischaemia, and recovery of reperfusion mechanical function were monitored. 3 At baseline, heart rate was 287±12 beats min−1, coronary flow was 12.8±0.6 ml min−1, left ventricular developed pressure (LVDevP) was 105±4 mmHg and left ventricular end‐diastolic pressure 4.6±0.2 mmHg in vehicle‐treated hearts (control; n=12). Baseline values were similar in all treatment groups (P>0.05). 4 In normoxic perfused hearts, 1 μM NG‐nitro‐L‐arginine (L‐NOARG) significantly reduced coronary flow from 13.5±0.2 to 12.1±0.1 ml min−1 (10%) and LVDevP from 97±1 to 92±1 mmHg (5%; P<0.05, n=5). 5 Ischaemic contracture was 46±2 mmHg, i.e. 44% of LVDevP in control hearts (n=12), unaffected by low concentrations of nitroprusside (1 and 10 μM) but reduced to ∼30 mmHg (∼25%) at higher concentrations (100 or 1000 μM; P<0.05 vs control, n=6). Conversely, the NO synthase inhibitor L‐NOARG reduced contracture at 1 μM to 26±3 mmHg (23%), but increased it to 63±4 mmHg (59%) at 1000 μM (n=6). Dobutamine (10 μM) exacerbated ischaemic contracture (81±3 mmHg; n=7) and the cyclic GMP analogue Sp‐8‐(4‐p‐chlorophenylthio)‐3′,5′‐monophosphorothioate (Sp‐8‐pCPT‐cGMPS; 10 μM) blocked this effect (63±1 mmHg; P<0.05 vs dobutamine alone, n=5). 6 At the end of reperfusion, LVDevP was 58±5 mmHg, i.e. 55% of pre‐ischaemic value in control hearts, significantly increased to ∼80% by high concentrations of nitroprusside (100 or 1000 μM) or L‐NOARG at 1 μM, while a high concentration of L‐NOARG (1000 μM) reduced LVDevP to ∼35% (P<0.05 vs control; n=6). 7 Ischaemia increased tissue cyclic GMP levels 1.8 fold in control hearts (P<0.05; n=12); nitroprusside at 1 μM had no sustained effect, but increased cyclic GMP ∼6 fold at 1000 μM; L‐NOARG (1 or 1000 μM) was without effect (n=6). Nitroprusside (1 or 1000 μM) marginally increased cyclic AMP levels whereas NO synthase inhibitors had no effect (n=6). 8 In conclusion, the cardioprotective effect of NO donors, but not of low concentrations of NO synthase inhibitors may be due to their ability to elevate cyclic GMP levels. Because myocardial cyclic GMP levels were not affected by low concentrations of NO synthase inhibitors, their beneficial effect on ischaemic and reperfusion function is probably not accompanied by reduced formation of NO and peroxynitrite in this model.

Effect of levosimendan on myocardial contractility, coronary and peripheral blood flow, and arrhythmias during coronary artery ligation and reperfusion in the in vivo pig model

OBJECTIVE To determine whether levosimendan, a calcium sensitiser that facilitates the activation of the contractile apparatus by calcium, improves myocardial contractile function during severe ischaemia and reperfusion without exacerbating the incidence of arrhythmias. DESIGN Pigs were pretreated orally twice daily for 10 days with 0.08 mg/kg levosimendan or placebo. On day 11 the left main coronary artery was ligated for 30 minutes, followed by 30 minutes of reperfusion. A bolus dose of levosimendan, 11.2 μg/kg intravenously, or placebo was given 30 minutes before coronary ligation, followed by a continuous infusion of 0.2 μg/kg/min levosimendan or placebo for the remainder of the experiment. RESULTS During the ischaemic period, cardiac output was higher in the levosimendan group than in the placebo group (mean (SD): 2.6 (0.5) v 2.0 (0.2) l/min, p < 0.05) and systemic vascular resistance was lower (2024 (188) v 2669 (424) dyne.s−1.cm−5, p < 0.005). During reperfusion, cardiac output and contractility (LVmaxdP/dt (pos), 956 (118) v 784 (130) mm Hg/s, p < 0.05) were increased by levosimendan. The incidence of ischaemic ventricular fibrillation and tachycardia was similar in the two groups but there were more arrhythmic events (ventricular tachycardia and ventricular fibrillation) in the levosimendan treated group (8/12 levosimendan v 1/9 control p = 0.05). CONCLUSIONS Levosimendan improved cardiac output and myocardial contractility during coronary artery ligation and reperfusion. However, it increased the number of arrhythmic events during ischaemia in this model of in vivo regional ischaemia.

TNF alpha is required for hypoxia-mediated right ventricular hypertrophy

Hypoxia has been shown to activate the pleiotropic cytokine TNFα in the lung. TNFα in turn, is known to induce pulmonary vasoconstriction. Additional effects of this cytokine in hypoxia mediated cardiopulmonary remodeling are poorly understood. To further evaluate the role of TNFα in chronic hypoxia we exposed TNFα null (TNFα–/–) and wild-type mice to three weeks of hypobaric hypoxia (10% O2). Equivalent erythocytosis (Hematocrit increased by ≥ 40%) developed in both genetic backgrounds. In contrast, right ventricular systolic pressure increased in response to three weeks of hypoxia in the wild-type mice (≥ 75%), yet was unaltered in the TNFα–/– mice. Concomitantly right ventricular hypertrophy was attenuated in the TNFα–/– mice (35 ± 5% increase) when compared to wild-type mice (124 ± 6% increase p < 0.001, n ≥ 20). Interestingly in both strains the lung wet weights increased to a similar degree in response to hypoxia. In conclusion, our data demonstrate that TNFα is an integral autocoid in chronic hypoxia mediated right ventricular hypertrophy. Moreover, additional components of cardiopulmonary remodeling may be regulated by TNFα signaling as suggested by the negligible right ventricular systolic pressure response to hypoxia in the absence of TNFα.

PKCε Promotes Cardiac Mitochondrial and Metabolic Adaptation to Chronic Hypobaric Hypoxia by GSK3β Inhibition

PKCε is central to cardioprotection. Sub‐proteome analysis demonstrated co‐localization of activated cardiac PKCε (aPKCε) with metabolic, mitochondrial, and cardioprotective modulators like hypoxia‐inducible factor 1α (HIF‐1α). aPKCε relocates to the mitochondrion, inactivating glycogen synthase kinase 3β (GSK3β) to modulate glycogen metabolism, hypertrophy and HIF‐1α. However, there is no established mechanistic link between PKCε, p‐GSK3β and HIF1‐α. Here we hypothesized that cardiac‐restricted aPKCε improves mitochondrial response to hypobaric hypoxia by altered substrate fuel selection via a GSK3β/HIF‐1α‐dependent mechanism. aPKCε and wild‐type (WT) mice were exposed to 14 days of hypobaric hypoxia (45 kPa, 11% O2) and cardiac metabolism, functional parameters, p‐GSK3β/HIF‐1α expression, mitochondrial function and ultrastructure analyzed versus normoxic controls. Mitochondrial ADP‐dependent respiration, ATP production and membrane potential were attenuated in hypoxic WT but maintained in hypoxic aPKCε mitochondria (P < 0.005, n = 8). Electron microscopy revealed a hypoxia‐associated increase in mitochondrial number with ultrastructural disarray in WT versus aPKCε hearts. Concordantly, left ventricular work was diminished in hypoxic WT but not aPKCε mice (glucose only perfusions). However, addition of palmitate abrogated this (P < 0.05 vs. WT). aPKCε hearts displayed increased glucose utilization at baseline and with hypoxia. In parallel, p‐GSK3β and HIF1‐α peptide levels were increased in hypoxic aPKCε hearts versus WT. Our study demonstrates that modest, sustained PKCε activation blunts cardiac pathophysiologic responses usually observed in response to chronic hypoxia. Moreover, we propose that preferential glucose utilization by PKCε hearts is orchestrated by a p‐GSK3β/HIF‐1α‐mediated mechanism, playing a crucial role to sustain contractile function in response to chronic hypobaric hypoxia. J. Cell. Physiol. 226: 2457–2468, 2011.

Effects of mibefradil, a novel calcium channel blocking agent with T-type activity, in acute experimental myocardial ischemia: maintenance of ventricular fibrillation threshold without inotropic compromise☆

OBJECTIVES We tested whether mibefradil, a selective T-type calcium channel blocking agent, could differentially inhibit experimental ventricular arrhythmogenesis more than contractility during acute regional ischemia and reperfusion compared with that during L-channel blockade by verapamil. BACKGROUND T-type calcium channels are found in nodal and conduction tissue and in vascular smooth muscle, but in much lower density in contractile myocardium. The potential role of mibefradil in ventricular arrhythmogenesis remains unclear. METHODS Mibefradil (Ro 40-5967, 1 mg/kg body weight intravenously [i.v.]) was given as a bolus 30 min before anterior descending coronary artery ligation, followed by 2 mg/kg per h i.v. during 20 min of ischemia and 25 min of reperfusion in open chest pigs. In a second group, mibefradil was given in a dose twice as high. A third group received verapamil (0.3 mg/kg i.v.), followed by an infusion of 0.6 mg/kg per h. RESULTS During the ischemic period, the low (clinically relevant) dose of mibefradil prevented the fall of the ventricular fibrillation threshold, without depressing the maximal rate of pressure development of the left ventricle (LVmax dP/dt). This low dose increased left ventricular blood flow, whereas peripheral arterial pressure remained unchanged. The higher dose of both mibefradil and verapamil was antiarrhythmic during ischemia, at the cost of depressed contractile activity. During reperfusion, only the higher dose of mibefradil and verapamil was antiarrhythmic but both depressed contractile activity. CONCLUSIONS Mibefradil is antiarrhythmic, without inotropic compromise. Speculatively, both T-type and L-type calcium channel blockade are involved in these effects.

Effects of Sphingosine-1-Phosphate on Acute Contractile Heart Failure (ACHF)

Reperfusion is the optimal way to salvage a heart after an ischemic insult [1, 2]. To protect the heart from lethal reperfusion injury, two pathways have been discovered called RISK (Reperfusion Injury Salvage Kinases) [3] and SAFE (Survivor Activating factor Enhancement) [4] which reduce cell death. Though the effects of SAFE and its protective components have been examined in myocardial ischemic-reperfusion injury, no work has been done in heart failure (HF). Acute heart failure can be defined as acute impairment of the heart’s ability to fill or empty the left ventricle properly [5]. While acute diastolic dysfunction affects filling, acute systolic function impairs ejection. We developed an isolated rat heart model which was underperfused to simulate the systolic failure and was subject to high levels of circulating free fatty acids (FFA), known to have deleterious effects in a failing heart as shown from the previous models of heart disease [6]. Sphingosine-1-phosphate (S-1-P) is a component of the lipid bilayer of the high density lipoprotein (HDL) molecule, which protects against myocardial ischemic-reperfusion injury via the SAFE pathway. HDL is involved in beneficial cardiovascular functions including reverse cholesterol transport system, anti-inflammatory, anti-oxidant and antiapoptotic effects [7]. We hypothesize that activation of the SAFE pathway via S-1-P could improve contractile function in ACHF. Underperfused failing hearts were treated with S1-P. Functional recovery was the percentage of the baseline rate-pressure product. Acute contractile heart failure (ACHF) was induced by lowering the perfusion pressure to 20 mmHg and increasing the free fatty acid concentration of the perfusate, while lowering the glucose concentration. From these preliminary findings we propose that S-1-P given to an acutely failing heart improves the rate pressure product by increasing the heart rate but not the developed pressure. This is the first study to show that a preconditioning agent (S-1-P) used in heart failure protects the heart rate rather than left ventricular developed pressure (LVDP) in an acute contractile failing heart. (Table 1, Fig. 1)

Combination of a Calcium Antagonist, Verapamil, with an Angiotensin Converting Enzyme Inhibitor, Trandolapril, in Experimental Myocardial Ischemia and Reperfusion: Antiarrhythmic and Hemodynamic Effects of Chronic Oral Pretreatment

The combination of a calcium antagonist with an angiotensin-converting enzyme (ACE) inhibitor is increasingly used in the therapy of hypertension, but there are no experimental data supporting the use of this combination in acute myocardial ischemia and reperfusion. We tested the effects of oral pretreatment in a pig model, paying special attention to arrhythmias and adverse hemodynamic effects. Pigs received verapamil 240 mg + trandolapril 4 mg, verapamil 240 mg, or placebo orally once daily for 10 days, after which a coronary artery was ligated for 20 minutes and then allowed to reperfuse. The ventricular fibrillation threshold (VFT) was measured during ischemia to assess the vulnerability of the heart to ventricular fibrillation, whereas spontaneous tachyarrhythmias were monitored during reperfusion. Regional left ventricular (LV) blood flow was measured with radioactive microspheres. During the ischemic period, both the combination of verapamil plus trandolapril, and verapamil alone, prevented a fall in the VFT, indicating antiarrhythmic activity. The combination maintained LV contractile activity and cardiac output (CO) at preligation levels, whereas verapamil alone decreased cardiac output. During reperfusion, verapamil plus trandolapril prevented spontaneous ventricular tachyarrhythmias and increased blood flow in the reperfused zone. In contrast, verapamil was not antiarrhythmic and decreased CO. Thus the addition of the ACE inhibitor trandolapril to the calcium antagonist verapamil resulted in antiarrhythmic activity during ischemia and reperfusion, and produced a better hemodynamic profile.