Arvind Sood

Fluorescence Resonance Energy Transfer–Based Analysis of cAMP Dynamics in Live Neonatal Rat Cardiac Myocytes Reveals Distinct Functions of Compartmentalized Phosphodiesterases

Cardiac myocytes have provided a key paradigm for the concept of the compartmentalized cAMP generation sensed by AKAP-anchored PKA. Phosphodiesterases (PDEs) provide the sole route for degrading cAMP in cells and are thus poised to regulate intracellular cAMP gradients. PDE3 and PDE4 represent the major cAMP degrading activities in rat ventriculocytes. By performing real-time imaging of cAMP in situ, we establish the hierarchy of these PDEs in controlling cAMP levels in basal conditions and on stimulation with a β-adrenergic receptor agonist. PDE4, rather than PDE3, appears to be responsible for modulating the amplitude and duration of the cAMP response to beta-agonists. PDE3 and PDE4 localize to distinct compartments and this may underpin their different functional roles. Our findings indicate the importance of distinctly localized PDE isoenzymes in determining compartmentalized cAMP signaling.

β-Arrestin-mediated PDE4 cAMP phosphodiesterase recruitment regulates β-adrenoceptor switching from Gs to Gi

Phosphorylation of the β2 adrenoreceptor (β2AR) by cAMP-activated protein kinase A (PKA) switches its predominant coupling from stimulatory guanine nucleotide regulatory protein (Gs) to inhibitory guanine nucleotide regulatory protein (Gi). β-Arrestins recruit the cAMP-degrading PDE4 phosphodiesterases to the β2AR, thus controlling PKA activity at the membrane. Here we investigate a role for PDE4 recruitment in regulating G protein switching by the β2AR. In human embryonic kidney 293 cells overexpressing a recombinant β2AR, stimulation with isoprenaline recruits β-arrestins 1 and 2 as well as both PDE4D3 and PDE4D5 to the receptor and stimulates receptor phosphorylation by PKA. The PKA phosphorylation status of the β2AR is enhanced markedly when cells are treated with the selective PDE4-inhibitor rolipram or when they are transfected with a catalytically inactive PDE4D mutant (PDE4D5-D556A) that competitively inhibits isoprenaline-stimulated recruitment of native PDE4 to the β2AR. Rolipram and PDE4D5-D556A also enhance β2AR-mediated activation of extracellular signal-regulated kinases ERK1/2. This is consistent with a switch in coupling of the receptor from Gs to Gi, because the ERK1/2 activation is sensitive to both inhibitors of PKA (H89) and Gi (pertussis toxin). In cardiac myocytes, the β2AR also switches from Gs to Gi coupling. Treating primary cardiac myocytes with isoprenaline induces recruitment of PDE4D3 and PDE4D5 to membranes and activates ERK1/2. Rolipram robustly enhances this activation in a manner sensitive to both pertussis toxin and H89. Adenovirus-mediated expression of PDE4D5-D556A also potentiates ERK1/2 activation. Thus, receptor-stimulated β-arrestin-mediated recruitment of PDE4 plays a central role in the regulation of G protein switching by the β2AR in a physiological system, the cardiac myocyte.

Fractional flow reserve vs. angiography in guiding management to optimize outcomes in non-ST-segment elevation myocardial infarction: the British Heart Foundation FAMOUS–NSTEMI randomized trial

Aim We assessed the management and outcomes of non-ST segment elevation myocardial infarction (NSTEMI) patients randomly assigned to fractional flow reserve (FFR)-guided management or angiography-guided standard care. Methods and results We conducted a prospective, multicentre, parallel group, 1 : 1 randomized, controlled trial in 350 NSTEMI patients with ≥1 coronary stenosis ≥30% of the lumen diameter assessed visually (threshold for FFR measurement) (NCT01764334). Enrolment took place in six UK hospitals from October 2011 to May 2013. Fractional flow reserve was disclosed to the operator in the FFR-guided group (n = 176). Fractional flow reserve was measured but not disclosed in the angiography-guided group (n = 174). Fractional flow reserve ≤0.80 was an indication for revascularization by percutaneous coronary intervention (PCI) or coronary artery bypass surgery (CABG). The median (IQR) time from the index episode of myocardial ischaemia to angiography was 3 (2, 5) days. For the primary outcome, the proportion of patients treated initially by medical therapy was higher in the FFR-guided group than in the angiography-guided group [40 (22.7%) vs. 23 (13.2%), difference 95% (95% CI: 1.4%, 17.7%), P = 0.022]. Fractional flow reserve disclosure resulted in a change in treatment between medical therapy, PCI or CABG in 38 (21.6%) patients. At 12 months, revascularization remained lower in the FFR-guided group [79.0 vs. 86.8%, difference 7.8% (−0.2%, 15.8%), P = 0.054]. There were no statistically significant differences in health outcomes and quality of life between the groups. Conclusion In NSTEMI patients, angiography-guided management was associated with higher rates of coronary revascularization compared with FFR-guided management. A larger trial is necessary to assess health outcomes and cost-effectiveness.

A randomized trial of deferred stenting versus immediate stenting to prevent no- or slow-reflow in acute ST-segment elevation myocardial infarction (DEFER-STEMI).

Objectives The aim of this study was to assess whether deferred stenting might reduce no-reflow and salvage myocardium in primary percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI). Background No-reflow is associated with adverse outcomes in STEMI. Methods This was a prospective, single-center, randomized, controlled, proof-of-concept trial in reperfused STEMI patients with ≥1 risk factors for no-reflow. Randomization was to deferred stenting with an intention-to-stent 4 to 16 h later or conventional treatment with immediate stenting. The primary outcome was the incidence of no-/slow-reflow (Thrombolysis In Myocardial Infarction ≤2). Cardiac magnetic resonance imaging was performed 2 days and 6 months after myocardial infarction. Myocardial salvage was the final infarct size indexed to the initial area at risk. Results Of 411 STEMI patients (March 11, 2012 to November 21, 2012), 101 patients (mean age, 60 years; 69% male) were randomized (52 to the deferred stenting group, 49 to the immediate stenting). The median (interquartile range [IQR]) time to the second procedure in the deferred stenting group was 9 h (IQR: 6 to 12 h). Fewer patients in the deferred stenting group had no-/slow-reflow (14 [29%] vs. 3 [6%]; p = 0.006), no reflow (7 [14%] vs. 1 [2%]; p = 0.052) and intraprocedural thrombotic events (16 [33%] vs. 5 [10%]; p = 0.010). Thrombolysis In Myocardial Infarction coronary flow grades at the end of PCI were higher in the deferred stenting group (p = 0.018). Recurrent STEMI occurred in 2 patients in the deferred stenting group before the second procedure. Myocardial salvage index at 6 months was greater in the deferred stenting group (68 [IQR: 54% to 82%] vs. 56 [IQR: 31% to 72%]; p = 0.031]. Conclusions In high-risk STEMI patients, deferred stenting in primary PCI reduced no-reflow and increased myocardial salvage. (Deferred Stent Trial in STEMI; NCT01717573)

Vasodilatory Capacity of the Coronary Microcirculation is Preserved in Selected Patients With Non–ST-Segment–Elevation Myocardial Infarction

Background—The use of fractional flow reserve in patients with non–ST-segment–elevation myocardial infarction (NSTEMI) is a controversial issue. We undertook a study to assess the vasodilatory capacity of the coronary microcirculation in patients with NSTEMI when compared with a model of preserved microcirculation (stable angina [SA] cohort: culprit and nonculprit vessel) and acute microcirculatory dysfunction (ST-segment–elevation myocardial infarction [STEMI] cohort). We hypothesized that the vasodilatory response of the microcirculation would be preserved in NSTEMI. Methods and Results—A total of 140 patients undergoing single vessel percutaneous coronary intervention were included: 50 stable angina, 50 NSTEMI, and 40 STEMI. The index of microvascular resistance (IMR), fractional flow reserve, and coronary flow reserve were measured before stenting in the culprit vessel and in an angiographically normal nonculprit vessel in patients with SA. The resistive reserve ratio, a measure of the vasodilatory capacity of the microcirculation and calculated using the equation: baseline resistance index (TmnBase×PaBase[PdBase–Pw/PaBase–Pw])–IMR/IMR, where TmnBase referred to nonhyperemic transit time; PaBase and PdBase, the nonhyperemic aortic and distal coronary pressures, respectively; and Pw referred to the coronary wedge pressure, was also measured. Troponin was also measured ⩽24 hours after percutaneous coronary intervention. The resistive reserve ratio was significantly lower in the STEMI patients compared with the stable angina patients both culprit and nonculprit vessel (STEMI, 1.7 versus SA culprit, 2.8; P⩽0.001 and SA nonculprit, 2.9; P<0.0001) and compared with NSTEMI patients (NSTEMI, 2.46; P⩽0.001). The resistive reserve ratio was similar in stable angina and NSTEMI patients (P=0.6). IMR was significantly higher pre-PCI in STEMI compared with SA and NSTEMI (IMR STEMI, 36.51 versus IMR NSTEMI, 22.73 [P=0.01] versus IMR SA, 18.26 [P<0.0001]). However, there was no significant difference in IMR pre-PCI between NSTEMI and SA (IMR NSTEMI, 22.73; IMR SA, 18.26 [P=0.1]). Conclusions—The vasodilatory capacity of the microcirculation is preserved in selected patients with NSTEMI. The clinical use of fractional flow reserve in the culprit vessel may be preserved in selected patents with NSTEMI.

In cardiac myocytes, cAMP elevation triggers the down-regulation of transcripts and promoter activity for cyclic AMP phosphodiesterase-4A10 (PDE4A10)

Transcripts for the PDE4A10 cyclic AMP phosphodiesterase isoform are present in a wide variety of rat tissues including the heart. Sequence comparisons between the putative human and mouse promoters revealed a number of conserved regions including both an Sp1 and a CREB-binding site. The putative mouse PDE4A10 promoter was amplified from genomic DNA and sub-cloned into a luciferase reporter vector for investigation of activity in neonatal cardiac myocytes. Transfection with this construct identified a high level of luciferase expression in neonatal cardiac myocytes. Surprisingly, this activity was down-regulated by elevation of intracellular cAMP through a process involving PKA, but not EPAC, signalling. Such inhibition of the rodent PDE4A10 promoter activity in response to elevated cAMP levels is in contrast to the PDE4 promoters so far described. Site-directed mutagenesis revealed that the Sp1 binding site at promoter position -348 to -336 is responsible for the basal constitutive expression of murine PDE4A10. The conserved CREB-binding motif at position -370 to -363 also contributes to basal promoter activity but does not in itself confer cAMP inhibition upon the PDE4A10 promoter. EMSA analysis confirmed the authenticity of CREB and Sp1 binding sites. The transcriptional start site was identified to be an adenine residue at position -55 in the mouse PDE4A10 promoter. We present evidence that this novel down-regulation of PDE4A10 is mediated by the transcription factor ICER in a PKA dependent manner. The pool of cAMP in cardiac myocytes that down-regulates PDE4A10 is regulated by beta-adrenoceptor coupled adenylyl cyclase activity and via hydrolysis determined predominantly by the action of PDE4 (cAMP phosphodiesterase-4) and not PDE3 (cAMP phosphodiesterase-3). We suggest that increased cAMP may remodel cAMP-mediated signalling events by not only increasing the expression of specific PDE4 cAMP phosphodiesterases but also by down-regulating specific isoforms, such as is shown here for PDE4A10 in cardiac myocytes.

Fractional flow reserve versus angiography in guiding management to optimize outcomes in non–ST-elevation myocardial infarction (FAMOUS-NSTEMI): Rationale and design of a randomized controlled clinical trial

Background In patients with acute non–ST-elevation myocardial infarction (NSTEMI), coronary arteriography is usually recommended; but visual interpretation of the angiogram is subjective. We hypothesized that functional assessment of coronary stenosis severity with a pressure-sensitive guide wire (fractional flow reserve [FFR]) would have additive diagnostic, clinical, and health economic utility as compared with angiography-guided standard care. Methods and design A prospective multicenter parallel-group 1:1 randomized controlled superiority trial in 350 NSTEMI patients with ≥1 coronary stenosis ≥30% severity (threshold for FFR measurement) will be conducted. Patients will be randomized immediately after coronary angiography to the FFR-guided group or angiography-guided group. All patients will then undergo FFR measurement in all vessels with a coronary stenosis ≥30% severity including culprit and nonculprit lesions. Fractional flow reserve will be disclosed to guide treatment in the FFR-guided group but not disclosed in the “angiography-guided” group. In the FFR-guided group, an FFR ≤0.80 will be an indication for revascularization by percutaneous coronary intervention or coronary artery bypass surgery, as appropriate. The primary outcome is the between-group difference in the proportion of patients allocated to medical management only compared with revascularization. Secondary outcomes include the occurrence of cardiac death or hospitalization for myocardial infarction or heart failure, quality of life, and health care costs. The minimum and average follow-up periods for the primary analysis are 6 and 18 months, respectively. Conclusions Our developmental clinical trial will address the feasibility of FFR measurement in NSTEMI and the influence of FFR disclosure on treatment decisions and health and economic outcomes.

Assessment of Fractional Flow Reserve in Patients With Recent Non–ST-Segment–Elevation Myocardial Infarction Comparative Study With 3-T Stress Perfusion Cardiac Magnetic Resonance Imaging

Background—The use of fractional flow reserve (FFR) in acute coronary syndromes is controversial. The British Heart Foundation Fractional Flow Reserve Versus Angiography in Guiding Management to Optimize Outcomes in Non-ST-Elevation Myocardial Infarction (FAMOUS-NSTEMI) study (NCT01764334) has recently demonstrated the safety and feasibility of FFR measurement in patients with non–ST-segment–elevation myocardial infarction. We report the findings of the cardiac magnetic resonance (CMR) substudy to assess the diagnostic accuracy of FFR compared with 3.0-T stress CMR perfusion. Methods and Results—One hundred six patients with non–ST-segment–elevation myocardial infarction who had been referred for early invasive management were included from 2 centers. FFR was measured in all major patent epicardial coronary arteries with a visual stenosis estimated at ≥30%, and if percutaneous coronary intervention was performed, an FFR assessment was repeated. Myocardial perfusion was assessed with stress perfusion CMR at 3 T. The mean age was 56.7±9.8 years; 82.6% were men. Mean time from FFR evaluation to CMR was 6.1±3.1 days. The mean±SD left ventricular ejection fraction was 58.2±9.1%. Mean infarct size was 5.4±7.1%, and mean troponin concentration was 5.2±9.2 &mgr;g/L. There were 34 fixed and 160 inducible perfusion defects. There was a negative correlation between the number of segments with a perfusion abnormality and FFR (r=−0.77; P<0.0001). The overall sensitivity, specificity, positive predictive value, and negative predictive value for an FFR of ⩽0.8 were 91.4%, 92.2%, 76%, and 97%, respectively. Diagnostic accuracy was 92%. The positive and negative predictive values of FFR for flow-limiting coronary artery disease (FFR⩽0.8) in patients with non–ST-segment–elevation myocardial infarction (n=21) who underwent perfusion CMR before invasive angiography were 92% and 93%, respectively. Receiver operating characteristic analysis indicated that the optimal cutoff value of FFR for demonstrating reversible ischemia on CMR was ⩽0.805 (area under the receiver operating characteristic curve, 0.94 [0.9–0.99]; P<0.0001). Conclusions—FFR in patients with recent non–ST-segment–elevation myocardial infarction showed high concordance with myocardial perfusion in matched territories as revealed by 3.0-T stress perfusion CMR. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT02073422.

Diagnostic accuracy of 3.0-T magnetic resonance T1 and T2 mapping and T2-weighted dark-blood imaging for the infarct-related coronary artery in Non-ST-segment elevation myocardial infarction

Background Patients with recent non–ST‐segment elevation myocardial infarction commonly have heterogeneous characteristics that may be challenging to assess clinically. Methods and Results We prospectively studied the diagnostic accuracy of 2 novel (T1, T2 mapping) and 1 established (T2‐weighted short tau inversion recovery [T2W‐STIR]) magnetic resonance imaging methods for imaging the ischemic area at risk and myocardial salvage in 73 patients with non–ST‐segment elevation myocardial infarction (mean age 57±10 years, 78% male) at 3.0‐T magnetic resonance imaging within 6.5±3.5 days of invasive management. The infarct‐related territory was identified independently using a combination of angiographic, ECG, and clinical findings. The presence and extent of infarction was assessed with late gadolinium enhancement imaging (gadobutrol, 0.1 mmol/kg). The extent of acutely injured myocardium was independently assessed with native T1, T2, and T2W‐STIR methods. The mean infarct size was 5.9±8.0% of left ventricular mass. The infarct zone T1 and T2 times were 1323±68 and 57±5 ms, respectively. The diagnostic accuracies of T1 and T2 mapping for identification of the infarct‐related artery were similar (P=0.125), and both were superior to T2W‐STIR (P<0.001). The extent of myocardial injury (percentage of left ventricular volume) estimated with T1 (15.8±10.6%) and T2 maps (16.0±11.8%) was similar (P=0.838) and moderately well correlated (r=0.82, P<0.001). Mean extent of acute injury estimated with T2W‐STIR (7.8±11.6%) was lower than that estimated with T1 (P<0.001) or T2 maps (P<0.001). Conclusions In patients with non–ST‐segment elevation myocardial infarction, T1 and T2 magnetic resonance imaging mapping have higher diagnostic performance than T2W‐STIR for identifying the infarct‐related artery. Compared with conventional STIR, T1 and T2 maps have superior value to inform diagnosis and revascularization planning in non–ST‐segment elevation myocardial infarction. Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02073422.

FRACTIONAL FLOW RESERVE (FFR) VERSUS ANGIOGRAPHY IN GUIDING MANAGEMENT TO OPTIMIZE OUTCOMES IN NON-ST ELEVATION MYOCARDIAL INFARCTION (FAMOUS - NSTEMI) HEALTH ECONOMIC ANALYSIS

An economic model was developed to compare the medical resource cost and health outcome effects of physiology-guided management with FFR compared with standard angiography-guided management in patients with non-ST elevation myocardial infarction based on participants British Heart Foundation FAMOUS-