Effects of late, repetitive remote ischaemic conditioning on myocardial strain in patients with acute myocardial infarction.

Late, repetitive or chronic remote ischaemic conditioning (CRIC) is a potential cardioprotective strategy against adverse remodelling following ST-segment elevation myocardial infarction (STEMI). In the randomised Daily Remote Ischaemic Conditioning Following Acute Myocardial Infarction (DREAM) trial, CRIC following primary percutaneous coronary intervention (P-PCI) did not improve global left ventricular (LV) systolic function. A post-hoc analysis was performed to determine whether CRIC improved regional strain. All 73 patients completing the original trial were studied (38 receiving 4 weeks' daily CRIC, 35 controls receiving sham conditioning). Patients underwent cardiovascular magnetic resonance at baseline (5-7 days post-STEMI) and after 4 months, with assessment of LV systolic function, infarct size and strain (longitudinal/circumferential, in infarct-related and remote territories). At both timepoints, there were no significant between-group differences in global indices (LV ejection fraction, infarct size, longitudinal/circumferential strain). However, regional analysis revealed a significant improvement in longitudinal strain in the infarcted segments of the CRIC group (from - 16.2 ± 5.2 at baseline to - 18.7 ± 6.3 at follow up, p = 0.0006) but not in corresponding segments of the control group (from - 15.5 ± 4.0 to - 15.2 ± 4.7, p = 0.81; for change: - 2.5 ± 3.6 versus + 0.3 ± 5.6, respectively, p = 0.027). In remote territories, there was a lower increment in subendocardial circumferential strain in the CRIC group than in controls (- 1.2 ± 4.4 versus - 2.5 ± 4.0, p = 0.038). In summary, CRIC following P-PCI for STEMI is associated with improved longitudinal strain in infarct-related segments, and an attenuated increase in circumferential strain in remote segments. Further work is needed to establish whether these changes may translate into a reduced incidence of adverse remodelling and clinical events. Clinical Trial Registration: http://clinicaltrials.gov/show/NCT01664611 .

Functional cardiac orexin receptors: role of orexin-B/orexin 2 receptor in myocardial protection.

Orexins/hypocretins exert cardiovascular effects which are centrally mediated. In the present study, we tested whether orexins and their receptors may also act in an autocrine/paracrine manner in the heart exerting direct effects. Quantitative reverse transcription-PCR (RT-PCR), immunohistochemical and Western blot analyses revealed that the rat heart expresses orexins and orexin receptors (OXR). In isolated rat cardiomyocytes, only orexin-B (OR-B) caused an increase in contractile shortening, independent of diastolic or systolic calcium levels. A specific orexin receptor-2 (OX2R) agonist ([Ala11, d-Leu15]-Orexin B) exerted similar effects as OR-B, whereas a specific orexin receptor-1 (OX1R) antagonist (SB-408124) did not alter the responsiveness of OR-B. Treatment of the same model with OR-B resulted in a dose-dependent increase in myosin light chain and troponin-I (TnI) phosphorylation. Following ischaemia/reperfusion in the isolated Langendorff perfused rat heart model, OR-B, but not OR-A, exerts a cardioprotective effect; mirrored in an in vivo model as well. Unlike OR-A, OR-B was also able to induce extracellular signal-regulated kinase (ERK) 1/2 (ERK1/2) and Akt phosphorylation in rat myocardial tissue and ERK1/2 phosphorylation in human heart samples. These findings were further corroborated in an in vivo rat model. In human subjects with heart failure, there is a significant negative correlation between the expression of OX2R and the severity of the disease clinical symptoms, as assessed by the New York Heart Association (NYHA) functional classification. Collectively, we provide evidence of a distinct orexin system in the heart that exerts a cardioprotective role via an OR-B/OX2R pathway.

Daily remote ischaemic conditioning following acute myocardial infarction: a randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning (rIC) is a cardioprotective tool which has shown promise in preclinical and clinical trials in the context of acute ischaemia. Repeated rIC post myocardial infarction may provide additional benefits which have not previously been tested clinically. METHODS: The trial assessed the role of daily rIC in enhancing left ventricular ejection fraction (LVEF) recovery in patients with impaired LVEF (<45%) after ST segment elevation myocardial infarction (STEMI) treated with primary percutaneous coronary intervention (P-PCI). Patients were recruited from four UK hospitals and randomised to receive either 4 weeks of daily rIC or sham conditioning using the autoRIC Device (CellAegis) starting on day 3 post P-PCI. The primary endpoint was the improvement in LVEF over 4 months assessed by cardiac MRI (CMR). Seventy-three patients (38 cases, 35 controls) completed the study. RESULTS: The treatment and control groups were well matched at baseline including for mean LVEF (42.8% vs 44.3% respectively, p=0.952). There was no difference in the improvement in LVEF over 4 months between the treatment and control groups (4.8%±7.8% vs 4.6%±5.9% respectively, p=0.924). No differences were seen in the secondary outcome measures including changes in infarct size and left ventricular end-diastolic and systolic volumes, major adverse cardiac and cerebral event, mean Kansas City Cardiomyopathy Questionnaire score and change in N-terminal pro-brain natriuretic peptide levels. CONCLUSIONS: Daily rIC starting on day 3 and continued for 4 weeks following successful P-PCI for STEMI did not improve LVEF as assessed by CMR after 4 months when compared with a matched control group. TRIAL REGISTRATION NUMBER: NCT0166461.

Regulation of vascular function and blood pressure by circadian variation in redox signalling.

There is accumulating evidence that makes the link between the circadian variation in blood pressure and circadian variations in vascular contraction. The importance of vascular endothelium-derived redox-active and redox-derived species in the signalling pathways involved in controlling vascular smooth muscle contraction are well known, and when linked to the circadian variations in the processes involved in generating these species, suggests a cellular mechanism for the circadian variations in blood pressure that links directly to the peripheral circadian clock. Relaxation of vascular smooth muscle cells involves endothelial-derived relaxing factor (EDRF) which is nitric oxide (NO) produced by endothelial NO synthase (eNOS), and endothelial-derived hyperpolarising factor (EDHF) which includes hydrogen peroxide (H2O2) produced by NADPH oxidase (Nox). Both of these enzymes appear to be under the direct control of the circadian clock mechanism in the endothelial cells, and disruption to the clock results in endothelial and vascular dysfunction. In this review, we focus on EDRF and EDHF and summarise the recent findings on the influence of the peripheral circadian clock mechanism on processes involved in generating the redox species involved and how this influences vascular contractility, which may account for some of the circadian variations in blood pressure and peripheral resistance. Moreover, the direct link between the peripheral circadian clock and redox-signalling pathways in the vasculature, has a bearing on vascular endothelial dysfunction in disease and aging, which are both known to lead to dysfunction of the circadian clock.

Remote ischemic preconditioning of cardiomyocytes inhibits the mitochondrial permeability transition pore independently of reduced calcium-loading or sarcKATP channel activation.

Ischemic preconditioning (IPC) inhibits Ca(2+)-loading during ischemia which contributes to cardioprotection by inhibiting mechanical injury due to hypercontracture and biochemical injury through mitochondrial permeability transition (MPT) pores during reperfusion. However, whether remote-IPC reduced Ca(2+)-loading during ischemia and its subsequent involvement in inhibiting MPT pore formation during reperfusion has not been directly shown. We have developed a cellular model of remote IPC to look at the impact of remote conditioning on Ca(2+)-regulation and MPT pore opening during simulated ischemia and reperfusion, using fluorescence microscopy. Ventricular cardiomyocytes were isolated from control rat hearts, hearts preconditioned with three cycles of ischemia/reperfusion or naïve myocytes remotely conditioned with effluent collected from preconditioned hearts. Both conventional-IPC and remote-IPC reduced the loss of Ca(2+)-homeostasis and contractile function following reenergization of metabolically inhibited cells and protected myocytes against ischemia/reperfusion injury. However, only conventional-IPC reduced the Ca(2+)-loading during metabolic inhibition and this was independent of any change in sarcKATP channel activity but was associated with a reduction in Na(+)-loading, reflecting a decrease in Na/H exchanger activity. Remote-IPC delayed opening of the MPT pores in response to ROS, which was dependent on PKCε and NOS-signaling. These data show that remote-IPC inhibits MPT pore opening to a similar degree as conventional IPC, however, the contribution of MPT pore inhibition to protection against reperfusion injury is independent of Ca(2+)-loading in remote IPC. We suggest that inhibition of the MPT pore and not Ca(2+)-loading is the common link in cardioprotection between conventional and remote IPC.

The time-of-day variation in vascular smooth muscle contractility depends on a nitric oxide signalling pathway.

The dip in blood pressure during the resting-period is paradoxically associated with an increase in total peripheral resistance and occurs at a time when the vascular response to vasoconstrictor compounds is heightened, and to vasodilators reduced. However, the cellular mechanisms responsible for this time-of-day variation are not well defined. We have investigated the role of nitric oxide synthase (NOS) signalling in the control of contraction in mesenteric resistance arteries using wire myography, combined with quantitative PCR analysis of gene transcription and western blot analysis of protein. Small rings of mesenteric arteries, isolated from rats at two opposing time-points corresponding to the animal's active and resting-period, were mounted in a wire myograph. Vessels exhibited a time-of-day variation in their contractile-response to phenylephrine, with a reduced maximal contraction during the active- versus the resting-period (11.8±0.8 versus 18.6±1.2 mN P<0.001). Vessels preconstricted with phenylephrine were also more responsive to vasodilation with acetylcholine during the active-period, with an EC50 of 58.6±11 versus 232±31 nM in resting-period vessels (P<0.0001). These differences were abolished in the presence of l-NAME. Quantitative RT-PCR reveals a functioning peripheral circadian clock in mesenteric arteries and a 3.3-fold increase in endothelial NO synthase mRNA levels in active- versus resting-period vessels (P<0.001), which translated to a 1.7-fold increase in total eNOS protein (P<0.05). The time-of-day variation in the response of mesenteric resistance vessels to phenylephrine and acetylcholine is dependent on NOS signalling.

Diurnal variation in excitation-contraction coupling is lost in the adult spontaneously hypertensive rat heart.

BACKGROUND: Excitation-contraction coupling of the normotensive rat heart exhibits a time-of-day variation in its response to isoproterenol (ISO), with a decrease during the animal's active period. Pressure-induced hypertrophy is known to adversely affect the circadian clock in the heart and this study sets out to determine whether this alters the time-of-day variation in E-C coupling. METHOD AND RESULTS: Hearts from juvenile (6-8 week) and adult (24-28 week) spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto (WKY) rats were isolated during the animals active and resting periods. Left ventricular developed pressure (LVDP) recorded from isolated perfused adult SHR hearts did not show the night-time dip in response to ISO that was present in normotensive hearts. Left ventricular myocytes isolated from juvenile WKY and SHRs during the resting period had a higher systolic [Ca]i and faster rate of decay of the Ca- transient, under basal conditions and in response to 10 nmol/l ISO, than the active period. LV-myocytes isolated from adult WKYs had a similar time-of-day variation in their Ca-transient. However, LV-myocytes from adult SHRs had lost this diurnal variation in both basal systolic [Ca]i and in response to ISO. Adult SHR hearts were hypertrophic in comparison to age-matched WKYs, had disrupted cycling of the circadian genes CLOCK and Per2, and this was matched by depressed nNOS cycling. CONCLUSION: The dip in response of the heart to ISO stimulation during the animal's active period is absent in adult SHRs. This may result from disruption to the circadian clock mechanism, which depresses the cycling of nNOS expression.

Phenylephrine preconditioning involves modulation of cardiac sarcolemmal K(ATP) current by PKC delta, AMPK and p38 MAPK.

Preconditioning of hearts with the α(1)-adrenoceptor agonist phenylephrine decreases infarct size and increases the functional recovery of the heart following ischaemia-reperfusion. However, the cellular mechanisms responsible for this protection are not known. We investigated the role of protein kinase C ε and δ (PKCε and PKCδ), AMP-activated protein kinase (AMPK), p38 MAPK (p38) and sarcolemmal ATP-sensitive potassium (sarcK(ATP)) channels in phenylephrine preconditioning using isolated rat ventricular myocytes. Preconditioning of ventricular myocytes with phenylephrine increased the recovery of contractile activity following metabolic inhibition and re-energisation from 30.1±1.9% to 66.5±5.2% (P<0.01) and increased the peak sarcK(ATP) current activated during metabolic inhibition from 32.1±1.8 pA/pF to 46.0±5.0 pA/pF (P<0.05), which was required for protection. Phenylephrine preconditioning resulted in a sustained activation of PKCε and PKCδ, and transient activation of AMPK, which was dependent upon activation of PKCδ but not PKCε. P38 was also activated by phenylephrine preconditioning and this was blocked by inhibitors of PKCε, PKCδ or AMPK. Inhibition of PKCδ, AMPK or p38 was sufficient to prevent the increase in current, suggesting that these kinases are involved in modulation of sarcK(ATP) channel current by phenylephrine preconditioning. However, whilst inhibition of AMPK and p38 prevented the protection from phenylephrine preconditioning, PKCδ inhibition paradoxically had no effect. The increase in sarcK(ATP) current induced by phenylephrine preconditioning requires PKCδ, AMPK and p38 and may contribute to the observed improvement in contractile recovery.

Inotropic response of cardiac ventricular myocytes to beta-adrenergic stimulation with isoproterenol exhibits diurnal variation: involvement of nitric oxide.

RATIONALE: Although >10% of cardiac gene expression displays diurnal variations, little is known of their impact on excitation-contraction coupling. OBJECTIVE: To determine whether the time of day affects excitation-contraction coupling in rat ventricles. METHODS AND RESULTS: Left ventricular myocytes were isolated from rat hearts at 2 opposing time points, corresponding to the animals resting or active periods. Basal contraction and [Ca(2+)](i) was significantly greater in myocytes isolated during the resting versus active periods (cell shortening 12.4+/-0.3 versus 11.0+/-0.2%; P<0.05 and systolic [Ca(2+)](i) 422+/-12 versus 341+/-9 nmol/L; P<0.01. This corresponded to a greater sarcoplasmic reticulum (SR) Ca(2+) load (672+/-20 versus 551+/-13 nmol/L P<0.001). The increase in systolic [Ca(2+)](i) in response to isoproterenol (>3 nmol/L) was also significantly greater in resting versus active period myocytes, reflecting a greater SR Ca(2+) load at this time. This diurnal variation in response of Ca(2+)-homeostasis to isoproterenol translated to a greater incidence of arrhythmic activity in resting period myocytes. Inhibition of neuronal NO synthase during stimulation with isoproterenol, further increased systolic [Ca(2+)](i) and the percentage of arrhythmic myocytes, but this effect was significantly greater in active period versus resting period myocytes. Quantitative RT-PCR analysis revealed a 2.65-fold increase in neuronal NO synthase mRNA levels in active over resting period myocytes (P<0.05). CONCLUSIONS: The threshold for the development of arrhythmic activity in response to isoproterenol is higher during the active period of the rat. We suggest this reflects a reduction in SR Ca(2+) loading and a diurnal variation in neuronal NO synthase signaling.

Ischemic preconditioning of the whole heart confers protection on subsequently isolated ventricular myocytes.

Current cellular models of ischemic preconditioning (IPC) rely on inducing preconditioning in vitro and may not accurately represent complex pathways triggered by IPC in the intact heart. Here, we show that it is possible to precondition the intact heart and to subsequently isolate individual ventricular myocytes that retain the protection triggered by IPC. Myocytes isolated from Langendorff-perfused hearts preconditioned with three cycles of ischemia-reperfusion were exposed to metabolic inhibition and reenergization. Injury was assessed from induction of hypercontracture and loss of Ca(2+) homeostasis and contractile function. IPC induced an immediate window of protection in isolated myocytes, with 64.3 +/- 7.6% of IPC myocytes recovering Ca(2+) homeostasis compared with 16.9 +/- 2.4% of control myocytes (P < 0.01). Similarly, 64.1 +/- 5.9% of IPC myocytes recovered contractile function compared with 15.3 +/- 2.2% of control myocytes (P < 0.01). Protection was prevented by the presence of 0.5 mM 5-hydroxydecanoate during the preconditioning stimulus. This early protection disappeared after 6 h, but a second window of protection developed 24 h after preconditioning, with 54.9 +/- 4.7% of preconditioned myocytes recovering Ca(2+) homeostasis compared with 12.6 +/- 2.9% of control myocytes (P < 0.01). These data show that "true" IPC of the heart confers both windows of protection in the isolated myocytes, with a similar temporal relationship to in vivo preconditioning of the whole heart. The model should allow future studies in isolated cells of the protective mechanisms induced by true ischemia.

Role of mitochondrial re-energization and Ca2+ influx in reperfusion injury of metabolically inhibited cardiac myocytes.

OBJECTIVE: We used isolated myocytes to investigate the role of mitochondrial re-energization and Ca2+ influx during reperfusion on hypercontracture, loss of Ca2+ homeostasis and contractile function. METHODS: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion injury was assessed from hypercontracture, loss of Ca2+ homeostasis ([Ca2+]i measured with fura-2) and failure of contraction in response to electrical stimulation. Mitochondrial membrane potential was followed using the potentiometric dye tetramethylrhodamine ethyl ester. RESULTS: Metabolic inhibition led to contractile failure and rigor accompanied by a sustained increase in [Ca2+]i. Reperfusion after 10 min metabolic inhibition led to an abrupt repolarization of the mitochondrial membrane potential (after 25.5+/-1.2 s), a transient fall in [Ca(2+]i followed by an abrupt hypercontracture (37.1+/-1.8 s) in 84% of myocytes. Ca2+ homeostasis (diastolic [Ca2+]i < 250 nM) recovered in only 23.3+/-5.1% of cells and contractions recovered in 15.3+/-2.2%. Oligomycin abolished the hypercontracture on reperfusion, but mitochondrial repolarization was unaffected. Preventing Ca2+ influx during reperfusion with [Ca2+]i-free Tyrode or with an inhibitor of Na(+)/Ca2+ exchange did not prevent the hypercontracture, but increased the percentage of cells recovering Ca2+ homeostasis and contractile function. The presence of 0.5 microM cyclosporin A did not prevent hypercontracture but increased the percentage of cells recovering Ca2+ homeostasis to 56.2+/-3.6% and contractile function to 52+/-4.3%. CONCLUSIONS: Reperfusion-induced hypercontracture, and loss of Ca2+ homeostasis and contractile function are initiated following mitochondrial re-energization. The hypercontracture requires the production of oxidative ATP but not Ca2+ influx during reperfusion. Loss of Ca2+ homeostasis and contractile function are linked to Ca2+ influx during reperfusion, probably via opening of mitochondrial permeability transition pores.

Diazoxide causes early activation of cardiac sarcolemmal KATP channels during metabolic inhibition by an indirect mechanism.

OBJECTIVE: We have used isolated myocytes to investigate the effects of diazoxide on sarcolemmal KATP channel (sarcoKATP) activity and action potential failure during metabolic inhibition, and the role of these channels in protection of functional recovery on reperfusion. MATERIALS AND METHODS: Isolated adult rat ventricular myocytes were exposed to metabolic inhibition (NaCN and iodoacetate) and reperfusion. Functional recovery was assessed from the ability of cells to contract on electrical stimulation and to recover calcium homeostasis, measured with fura-2. Action potentials and KATP currents were measured using patch clamp. RESULTS: Pretreatment with diazoxide (100 microM, 5 min) increased the proportion of cells that recovered contractile function after MI and reperfusion from 16.8 +/- 2.4% to 65.0 +/- 2.2% (p<0.001) and the proportion of cells in which [Ca2+]i recovered to <250 nM. Pretreatment also accelerated action potential and contractile failure during MI. In cell-attached patches, MI activated sarcoKATP channels after 224 +/- 11 s, and diazoxide pretreatment decreased this to 145 +/- 24 s (p<0.01). However, diazoxide present in the patch pipette did not accelerate sarcoKATP channel activation. Intracellular Mg2+ rose earlier in diazoxide-pretreated cells. The sarcoKATP blocker HMR 1883 delayed action potential failure and reduced diazoxide protection. CONCLUSIONS: Diazoxide pretreatment increases recovery of function and [Ca2+]i following reperfusion. Protection is coupled with early action potential failure, due to early activation of sarcoKATP channels during metabolic inhibition (MI), which is likely to involve an indirect effect of diazoxide.