SGLT2 inhibitors reduce infarct size in reperfused ischemic heart and improve cardiac function during ischemic episodes in preclinical models.

The sodium-glucose cotransporter 2 (SGLT2) inhibitors are a new class of effective drugs managing patients, who suffer from type 2 diabetes (T2D): Landmark clinical trials including EMPA-REG, CANVAS and Declare-TIMI have demonstrated that SGLT2 inhibitors reduce cardiovascular mortality and re-hospitalization for heart failure (HF) in patients with T2D. It is well established that there is a strong independent relationship among infarct size measured within 1 month after reperfusion and all-cause death and hospitalization for HF: The fact that cardiovascular mortality was significantly reduced with the SGLT2 inhibitors, fuels the assumption that this class of therapies may attenuate myocardial infarct size. Experimental evidence demonstrates that SGLT2 inhibitors exert cardioprotective effects in animal models of acute myocardial infarction through improved function during the ischemic episode, reduction of infarct size and a subsequent attenuation of heart failure development. The aim of the present review is to outline the current state of preclinical research in terms of myocardial ischemia/reperfusion injury (I/R) and infarct size for clinically available SGLT2 inhibitors and summarize some of the proposed mechanisms of action (lowering intracellular Na+ and Ca2+, NHE inhibition, STAT3 and AMPK activation, CamKII inhibition, reduced inflammation and oxidative stress) that may contribute to the unexpected beneficial cardiovascular effects of this class of compounds.

SGLT2 Inhibitor, Canagliflozin, Attenuates Myocardial Infarction in the Diabetic and Nondiabetic Heart.

The authors hypothesized that despite similar cardiovascular event rates, the improved cardiovascular survival from sodium glucose transporter 2 (SGLT2) inhibition, seen clinically, could be via a direct cytoprotective effect, including protection against myocardial ischemia/reperfusion injury. Langendorff-perfused hearts, from diabetic and nondiabetic rats, fed long-term for 4 weeks with canagliflozin, had lower infarct sizes; this being the first demonstration of canagliflozin's cardioprotective effect against ischemia/reperfusion injury in both diabetic and nondiabetic animals. By contrast, direct treatment of isolated nondiabetic rat hearts with canagliflozin, solubilized in the isolated Langendorff perfusion buffer, had no impact on infarct size. This latter study demonstrates that the infarct-sparing effect of long-term treatment with canagliflozin results from either a glucose-independent effect or up-regulation of cardiac prosurvival pathways. These results further suggest that SGLT2 inhibitors could be repurposed as novel cardioprotective interventions in high-risk cardiovascular patients irrespective of diabetic status.

The 10th Biennial Hatter Cardiovascular Institute workshop: cellular protection-evaluating new directions in the setting of myocardial infarction, ischaemic stroke, and cardio-oncology.

Due to its poor capacity for regeneration, the heart is particularly sensitive to the loss of contractile cardiomyocytes. The onslaught of damage caused by ischaemia and reperfusion, occurring during an acute myocardial infarction and the subsequent reperfusion therapy, can wipe out upwards of a billion cardiomyocytes. A similar program of cell death can cause the irreversible loss of neurons in ischaemic stroke. Similar pathways of lethal cell injury can contribute to other pathologies such as left ventricular dysfunction and heart failure caused by cancer therapy. Consequently, strategies designed to protect the heart from lethal cell injury have the potential to be applicable across all three pathologies. The investigators meeting at the 10th Hatter Cardiovascular Institute workshop examined the parallels between ST-segment elevation myocardial infarction (STEMI), ischaemic stroke, and other pathologies that cause the loss of cardiomyocytes including cancer therapeutic cardiotoxicity. They examined the prospects for protection by remote ischaemic conditioning (RIC) in each scenario, and evaluated impasses and novel opportunities for cellular protection, with the future landscape for RIC in the clinical setting to be determined by the outcome of the large ERIC-PPCI/CONDI2 study. It was agreed that the way forward must include measures to improve experimental methodologies, such that they better reflect the clinical scenario and to judiciously select combinations of therapies targeting specific pathways of cellular death and injury.

Cangrelor-Mediated Cardioprotection Requires Platelets and Sphingosine Phosphorylation.

In animal models platelet P2Y12 receptor antagonists put the heart into a protected state, not as a result of suppressed thrombosis but rather through protective signaling, similar to that for ischemic postconditioning. While both ischemic postconditioning and the P2Y12 blocker cangrelor protect blood-perfused hearts, only the former protects buffer-perfused hearts indicating that the blocker requires a blood-borne constituent or factor to protect. We used an anti-platelet antibody to make thrombocytopenic rats to test if that factor resides within the platelet. Infarct size was measured in open-chest rats subjected to 30-min ischemia/2-h reperfusion. Infarct size was not different in thrombocytopenic rats showing that preventing aggregation alone is not protective. While ischemic preconditioning could reduce infarct size in thrombocytopenic rats, the P2Y12 inhibitor cangrelor could not, indicating that it protects by interacting with some factor in the platelet. Ischemic preconditioning is known to require phosphorylation of sphingosine. In rats treated with dimethylsphingosine to block sphingosine kinase, cangrelor was no longer protective. Thus cangrelor's protective mechanism appears to also involve sphingosine kinase revealing yet another similarity to conditioning's mechanism.

Characterization of the Langendorff Perfused Isolated Mouse Heart Model of Global Ischemia-Reperfusion Injury: Impact of Ischemia and Reperfusion Length on Infarct Size and LDH Release.

INTRODUCTION: The Langendorff perfused isolated mouse heart model is commonly used to assess the efficacy of cardioprotective therapies, although the duration of ischemia and reperfusion vary considerably between different laboratories. We aimed to provide a thorough characterization of the model with different durations of ischemia and reperfusion by means of 2 different end points-infarct size (IS) using triphenyltetrazolium staining and lactate dehydrogenase (LDH) release. METHODS: C57/BL6 mice hearts were retrograde perfused on a Langendorff apparatus and allocated into 9 groups in a 3 × 3 factorial design-3 ischemic durations (25, 35, and 45 minutes) matched by 3 reperfusion durations (60, 120, and 180 minutes). A protocol of ischemic preconditioning (IPC) was applied to investigate IS and LDH kinetics with different ischemic durations. RESULTS: Infarct size progressively increased with the duration of both ischemia and reperfusion and was found to be independently associated with both determinants. In terms of LDH release kinetics, a peak was observed within the first 10 to 15 minutes of reperfusion and steadily declined thereafter, although a second smaller peak was observed in the 25-minute ischemia group. Only LDH peak release was associated with the ischemia length, with area under the curve (AUC) failing to follow ischemic duration. Interestingly, while IPC reduced IS in all ischemic durations investigated, a significant attenuation of LDH AUC was only observed in the 25-minute index ischemia group. Only a moderately positive correlation was observed between IS and LDH peak (R = .547, P = .006) and AUC (R = .664, P < .001). CONCLUSION: Myocardial IS measured by triphenyltetrazolium staining depends on both the duration of ischemia and the length of the reperfusion period. The LDH assessment may not be the most reliable tool to assess IS and/or to examine cardioprotective effectiveness at various times of ischemia.

Implications of Kidney Disease in the Cardiac Patient.

Traditional cardiovascular risk factors, particularly hypertension and diabetes, are common in the disease processes of both renal and cardiac pathology. Unfortunately the coexistence of renal impairment is not an innocent bystander in cardiovascular disease; it disorder not only increases the prevalence and severity of cardiovascular disease, but also negatively affects prognostic outcomes and the safety and efficacy of cardiac interventions. This article discusses the role and impact of kidney disease in the cardiac patient in 3 key common cardiovascular processes: coronary artery disease, arrhythmia, and heart failure.

Effect of remote ischaemic conditioning on contrast-induced nephropathy in patients undergoing elective coronary angiography (ERICCIN): rationale and study design of a randomised single-centre, double-blind placebo-controlled trial.

BACKGROUND: Contrast-induced nephropathy (CIN), an acute kidney injury resulting from the administration of intravascular iodinated contrast media, is an important cause of morbidity/mortality following coronary angiographic procedures in high-risk patients. Despite preventative measures intended to mitigate the risk of CIN, there remains a need for an effective intervention. Remote ischaemic conditioning (RIC), where non-injurious ischaemia is applied to an arm prior to the administration of contrast, has shown promise in attenuating CIN but its effectiveness in preserving long-term renal function is unknown, which will be studied as part of the effect of remote ischaemic conditioning against contrast-induced nephropathy (ERICCIN) trial. ( http://Controlled-trials.com Identifier: ISRCTN49645414.) METHODS: The ERICCIN trial is a single-centre, randomised double-blinded placebo-controlled trial which plans to recruit 362 patients who are at risk of CIN, defined by pre-existent renal impairment (estimated glomerular filtration rate <60 ml/min/1.73 m2), over a period of 2 years. Patients will be randomised to either control or RIC consisting of 4, 5 min 200 mmHg balloon-cuff inflation/deflations, to the upper arm. The primary endpoint will be the development of CIN (>25% of eGFR, or rise of creatinine of >44 µmol/l) at 48 h. A key secondary endpoint will be whether RIC impacts upon persistent renal impairment over the 3-month follow-up period. Additional secondary endpoints include the measurement of serum neutrophil gelatinase-associated lipocalin and urinary albumin at 6, 48 h and 3 months following administration of contrast. IMPLICATIONS: Findings from ERICCIN trial will potentially demonstrate that RIC attenuates contrast-induced acute and chronic kidney injury and influence future clinical practice guidelines in at-risk patients undergoing coronary angiographic procedures.

Matrix metalloproteinase inhibition protects CyPD knockout mice independently of RISK/mPTP signalling: a parallel pathway to protection.

The mitochondrial permeability transition pore (mPTP) is widely accepted as an end-effector mechanism of conditioning protection against injurious ischaemia/reperfusion. However, death can be initiated in cells without pre-requisite mPTP opening, implicating alternate targets for ischaemia/reperfusion injury amelioration. Matrix metalloproteinases (MMP) are known to activate extrinsic apoptotic cascades and therefore we hypothesised that MMP activity represents an mPTP-independent target for augmented attenuation of ischaemia/reperfusion injury. In ex vivo and in vivo mouse hearts, we investigated whether the MMP inhibitor, ilomastat (0.25 µmol/l), administered upon reperfusion could engender protection in the absence of cyclophilin-D (CyPD), a modulator of mPTP opening, against injurious ischaemia/reperfusion. Ilomastat attenuated infarct size in wild-type (WT) animals [37 ± 2.8 to 22 ± 4.3 %, equivalent to ischaemic postconditioning (iPostC), used as positive control, 27 ± 2.1 %, p < 0.05]. Control CyPD knockout (KO) hearts had smaller infarcts than control WT (28 ± 4.2 %) and iPostC failed to confer additional protection, yet ilomastat significantly attenuated infarct size in KO hearts (11 ± 3.0 %, p < 0.001), and similar protection was also seen in isolated cardiomyocytes. Moreover, ilomastat, unlike the cyclophilin inhibitor cyclosporine-A, had no impact upon reactive oxygen species-mediated mPTP opening. While MMP inhibition was associated with increased Akt and ERK phosphorylation, neither Wortmannin nor PD98059 abrogated ilomastat-mediated protection. We demonstrate that MMP inhibition is cardioprotective, independent of Akt/ERK/CyPD/mPTP activity and is additive to the protection observed following inhibition of mPTP opening, indicative of a parallel pathway to protection in ischaemic/reperfused heart that may have clinical applicability in attenuating injury in acute coronary syndromes and deserve further investigation.

Targeting reperfusion injury in acute myocardial infarction: a review of reperfusion injury pharmacotherapy.

INTRODUCTION: Acute myocardial infarction (AMI) (secondary to lethal ischemia-reperfusion [IR]) contributes to much of the mortality and morbidity from ischemic heart disease. Currently, the treatment for AMI is early reperfusion; however, this itself contributes to the final myocardial infarct size, in the form of what has been termed 'lethal reperfusion injury'. Over the last few decades, the discovery of the phenomena of ischemic preconditioning and postconditioning, as well as remote preconditioning and remote postconditioning, along with significant advances in our understanding of the cardioprotective pathways underlying these phenomena, have provided the possibility of successful mechanical and pharmacological interventions against reperfusion injury. AREAS COVERED: This review summarizes the evidence from clinical trials evaluating pharmacological agents as adjuncts to standard reperfusion therapy for ST-elevation AMI. EXPERT OPINION: Reperfusion injury pharmacotherapy has moved from bench to bedside, with clinical evaluation and ongoing clinical trials providing us with valuable insights into the shortcomings of current research in establishing successful treatments for reducing reperfusion injury. There is a need to address some key issues that may be leading to lack of translation of cardioprotection seen in basic models to the clinical setting. These issues are discussed in the Expert opinion section.

Conditioning the whole heart--not just the cardiomyocyte.

Conditioning, the recruitment of endogenous cytoprotective pathways that protect the myocardium against injurious ischaemia/reperfusion injury, has developed into a range of modalities that can be applied before (preconditioning), during (perconditioning) or after the injurious ischaemic insult (postconditioning), either directly to the heart or in a distal tissue (remote preconditioning). A wide range of triggers, signaling pathways and potential end-effector mechanisms have been identified, which appear common to all forms of conditioning. Interestingly, conditioning applies to not only the cardiac myocyte, but to all the constitutive cell types within the myocardium. As our understanding of conditioning mechanisms continue to develop and we start to realise some of the difficulties in translating these phenomena to clinical treatments, it may be time to take a more integrative approach to conditioning, considering the many cellular and tissue types within the heart, and how they contribute to cytoprotective adaptations. In this review, we shall look at the conditioning phenomena, how different cell types contribute to the conditioned phenotype, and where novel cardioprotective modalities may be developed.

Retrograde heart perfusion: the Langendorff technique of isolated heart perfusion.

In the late 19th century, a number of investigators were working on perfecting isolated heart model, but it was Oscar Langendorff who, in 1895, pioneered the isolated perfused mammalian heart. Since that time, the Langendorff preparation has evolved and provided a wealth of data underpinning our understanding of the fundamental physiology of the heart: its contractile function, coronary blood flow regulation and cardiac metabolism. In more recent times, the procedure has been used to probe pathophysiology of ischaemia/reperfusion and disease states, and with the dawn of molecular biology and genetic manipulation, the Langendorff perfused heart has remained a stalwart tool in the study of the impact upon the physiology of the heart by pharmacological inhibitors and targeted deletion or up-regulation of genes and their impact upon intracellular signalling and adaption to clinically relevant stressful stimuli. We present here the basic structure of the Langendorff system and the fundamental experimental rules which warrant a viable heart preparation. In addition, we discuss the use of the isolated retrograde perfused heart in the model of ischaemia-reperfusion injury ex-vivo, and its applicability to other areas of study. The Langendorff perfusion apparatus is highly adaptable and this is reflected not only in the procedure's longevity but also in the number of different applications to which it has been turned.

There is more to life than revascularization: therapeutic targeting of myocardial ischemia/reperfusion injury.

Ischemic heart disease is the world's leading cause of death, and while clinical advances have meant improved and more rapid revascularization, there remains a significant element of myocardial death that thus far has not been successfully targeted in clinical practice, namely lethal reperfusion injury. Ischemia-reperfusion injury has been the subject of intense research over the last 40 years and our appreciation of the mechanisms of cellular death and salvage have increased immensely over this time, to the extent that a number of clearly identifiable therapeutic targets can now be subjected to clinical trials, as the basic science translates into potentially effective therapies in patients presenting with acute coronary syndromes. In this review, we aim to provide an overview of current evidence regarding the mechanisms of lethal reperfusion injury and endogenous protection, how cardioprotective pharmacological manipulations have been approached to date, and to indicate where therapies may be targeted in the future.

Reperfusion kinase phosphorylation is essential but not sufficient in the mediation of pharmacological preconditioning: Characterisation in the bi-phasic profile of early and late protection.

OBJECTIVE: Pharmacological preconditioning (PPC) triggers early (ePPC) and delayed protection (dPPC), occurring within 1 h or after 24 h following the preconditioning stimulus, respectively, through recruitment of protein kinase signalling. Angiotensin II (ATII) is a recognised trigger of PPC, recruiting kinases and transcription factors known to be involved in both phases of protection. Our objectives were to determine whether ATII is capable of triggering dPPC and whether recruitment of pro-survival kinases, Akt and extracellular signal-regulated kinase (ERK), following the injurious ischaemic insult is essential for the mediation of PPC. METHODS: In a mouse Langendorff model of ischaemia/reperfusion injury, we undertook to determine whether ATII triggers both ePPC and dPPC. Western blot analysis was used to determine kinase phosphorylation at reperfusion, and kinase inhibitors wortmannin and PD98059 were used to ascertain the significance of kinase regulation. RESULTS: We demonstrated that ATII triggered PPC with attenuation of infarction at 1 and 24 h (19+/-4% and 25+/-4% versus control, 35+/-4% of risk zone, p < 0.05), consistent with the ePPC and dPPC time-course. This bi-phasic protection was associated with significant post-ischaemic phosphorylation of both Akt and ERK within the first 5 min of reperfusion. Akt and ERK phosphorylation was increased following ePPC by 4.5+/-0.5 and 1.9+/-0.6 fold, respectively (p < 0.001), and dPPC by 24+/-2.0 and 2.1+/-0.1 fold, respectively (p < 0.001). Both wortmannin and PD98059 administered during reperfusion ameliorated the phosphorylation of Akt and ERK and abrogated the resistance to infarction resulting from both ePPC and dPPC (33+/-3% and 35+/-4%, respectively, versus controls 33+/-4% and 33+/-5%, p = NS). There was no evidence of augmented phosphorylation of either p38 kinase or JNK at either time point. CONCLUSION: We demonstrate that PPC results in a clearly delineated time-course of bi-phasic protection against injurious ischemic injury that is correlated with reperfusion kinase phosphorylation of both Akt and ERK. These data indicate a novel mechanism of early and particularly delayed preconditioning.

Improved hypothesis testing for coefficients in generalized estimating equations with small samples of clusters.

The sandwich standard error estimator is commonly used for making inferences about parameter estimates found as solutions to generalized estimating equations (GEE) for clustered data. The sandwich tends to underestimate the variability in the parameter estimates when the number of clusters is small, and reference distributions commonly used for hypothesis testing poorly approximate the distribution of Wald test statistics. Consequently, tests have greater than nominal type I error rates. We propose tests that use bias-reduced linearization, BRL, to adjust the sandwich estimator and Satterthwaite or saddlepoint approximations for the reference distribution of resulting Wald t-tests. We conducted a large simulation study of tests using a variety of estimators (traditional sandwich, BRL, Mancl and DeRouen's BC estimator, and a modification of an estimator proposed by Kott) and approximations to reference distributions under diverse settings that varied the distribution of the explanatory variables, the values of coefficients, and the degree of intra-cluster correlation (ICC). Our new method generally worked well, providing accurate estimates of the variability of fitted coefficients and tests with near-nominal type I error rates when the ICC is small. Our method works less well when the ICC is large, but it continues to out-perform the traditional sandwich and other alternatives.