Statins affect human iPSC-derived cardiomyocytes by interfering with mitochondrial function and intracellular acidification.

Statins are effective drugs in reducing cardiovascular morbidity and mortality by inhibiting cholesterol synthesis. These effects are primarily beneficial for the patient's vascular system. A significant number of statin users suffer from muscle complaints probably due to mitochondrial dysfunction, a mechanism that has recently been elucidated. This has raised our interest in exploring the effects of statins on cardiac muscle cells in an era where the elderly and patients with poorer functioning hearts and less metabolic spare capacity start dominating our patient population. Here, we investigated the effects of statins on human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-derived CMs). hiPSC-derived CMs were exposed to simvastatin, atorvastatin, rosuvastatin, and cerivastatin at increasing concentrations. Metabolic assays and fluorescent microscopy were employed to evaluate cellular viability, metabolic capacity, respiration, intracellular acidity, and mitochondrial membrane potential and morphology. Over a concentration range of 0.3-100 µM, simvastatin lactone and atorvastatin acid showed a significant reduction in cellular viability by 42-64%. Simvastatin lactone was the most potent inhibitor of basal and maximal respiration by 56% and 73%, respectively, whereas simvastatin acid and cerivastatin acid only reduced maximal respiration by 50% and 42%, respectively. Simvastatin acid and lactone and atorvastatin acid significantly decreased mitochondrial membrane potential by 20%, 6% and 3%, respectively. The more hydrophilic atorvastatin acid did not seem to affect cardiomyocyte metabolism. This calls for further research on the translatability to the clinical setting, in which a more conscientious approach to statin prescribing might be considered, especially regarding the current shift in population toward older patients with poor cardiac function.

Statins and Cardiomyocyte Metabolism, Friend or Foe?

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in cholesterol synthesis, and are the cornerstone of lipid-lowering treatment. They significantly reduce cardiovascular morbidity and mortality. However, musculoskeletal symptoms are observed in 7 to 29 percent of all users. The mechanism underlying these complaints has become increasingly clear, but less is known about the effect on cardiac muscle function. Here we discuss both adverse and beneficial effects of statins on the heart. Statins exert pleiotropic protective effects in the diseased heart that are independent of their cholesterol-lowering activity, including reduction in hypertrophy, fibrosis and infarct size. Adverse effects of statins seem to be associated with altered cardiomyocyte metabolism. In this review we explore the differences in the mechanism of action and potential side effects of statins in cardiac and skeletal muscle and how they present clinically. These insights may contribute to a more personalized treatment strategy.

Mitochondrial complex III activity: from invasive muscle biopsies to patient-friendly buccal swab analysis.

Drug-induced mitochondrial dysfunction is a common adverse effect, particularly in case of statins-the most prescribed drugs worldwide. These drugs have been shown to inhibit complex III (CIII) of the mitochondrial oxidative phosphorylation process, which is related to muscle pain. As muscle pain is the most common complaint of statin users, it is crucial to distinguish it from other causes of myalgia to prevent unnecessary cessation of drug therapy. However, diagnosing CIII inhibition currently requires muscle biopsies, which are invasive and not practical for routine testing. Less invasive alternatives for measurement of mitochondrial complex activities are only available yet for complex I and IV. Here, we describe a non-invasive spectrophotometric method to determine CIII catalytic activities using buccal swabs, which we validated in a cohort of statin and non-statin users. Our data indicate that CIII can be reliably measured in buccal swabs, as evidenced by reproducible results above the detection limit. Further validation on a large-scale clinical setting is recommended.

Isolation of multipotent progenitor cells from pleura and pericardium for tracheal tissue engineering purposes.

Tissue engineering (TE) of long tracheal segments is conceptually appealing for patients with inoperable tracheal pathology. In tracheal TE, stem cells isolated from bone marrow or adipose tissue have been employed, but the ideal cell source has yet to be determined. When considering the origin of stem cells, cells isolated from a source embryonically related to the trachea may be more similar. In this study, we investigated the feasibility of isolating progenitor cells from pleura and pericard as an alternative cells source for tracheal tissue engineering. Porcine progenitor cells were isolated from pleura, pericard, trachea and adipose tissue and expanded in culture. Isolated cells were characterized by PCR, RNA sequencing, differentiation assays and cell survival assays and were compared to trachea and adipose-derived progenitor cells. Progenitor-like cells were successfully isolated and expanded from pericard and pleura as indicated by gene expression and functional analyses. Gene expression analysis and RNA sequencing showed a stem cell signature indicating multipotency, albeit that subtle differences between different cell sources were visible. Functional analysis revealed that these cells were able to differentiate towards chondrogenic, osteogenic and adipogenic lineages. Isolation of progenitor cells from pericard and pleura with stem cell features is feasible. Although functional differences with adipose-derived stem cells were limited, based on their gene expression, pericard- and pleura-derived stem cells may represent a superior autologous cell source for cell seeding in tracheal tissue engineering.

A microRNA program regulates the balance between cardiomyocyte hyperplasia and hypertrophy and stimulates cardiac regeneration.

Myocardial regeneration is restricted to early postnatal life, when mammalian cardiomyocytes still retain the ability to proliferate. The molecular cues that induce cell cycle arrest of neonatal cardiomyocytes towards terminally differentiated adult heart muscle cells remain obscure. Here we report that the miR-106b~25 cluster is higher expressed in the early postnatal myocardium and decreases in expression towards adulthood, especially under conditions of overload, and orchestrates the transition of cardiomyocyte hyperplasia towards cell cycle arrest and hypertrophy by virtue of its targetome. In line, gene delivery of miR-106b~25 to the mouse heart provokes cardiomyocyte proliferation by targeting a network of negative cell cycle regulators including E2f5, Cdkn1c, Ccne1 and Wee1. Conversely, gene-targeted miR-106b~25 null mice display spontaneous hypertrophic remodeling and exaggerated remodeling to overload by derepression of the prohypertrophic transcription factors Hand2 and Mef2d. Taking advantage of the regulatory function of miR-106b~25 on cardiomyocyte hyperplasia and hypertrophy, viral gene delivery of miR-106b~25 provokes nearly complete regeneration of the adult myocardium after ischemic injury. Our data demonstrate that exploitation of conserved molecular programs can enhance the regenerative capacity of the injured heart.

Myocardial Disease and Long-Distance Space Travel: Solving the Radiation Problem.

Radiation-induced cardiovascular disease is a well-known complication of radiation exposure. Over the last few years, planning for deep space missions has increased interest in the effects of space radiation on the cardiovascular system, as an increasing number of astronauts will be exposed to space radiation for longer periods of time. Research has shown that exposure to different types of particles found in space radiation can lead to the development of diverse cardiovascular disease via fibrotic myocardial remodeling, accelerated atherosclerosis and microvascular damage. Several underlying mechanisms for radiation-induced cardiovascular disease have been identified, but many aspects of the pathophysiology remain unclear. Existing pharmacological compounds have been evaluated to protect the cardiovascular system from space radiation-induced damage, but currently no radioprotective compounds have been approved. This review critically analyzes the effects of space radiation on the cardiovascular system, the underlying mechanisms and potential countermeasures to space radiation-induced cardiovascular disease.

Cardiomyocyte cell cycle dynamics and proliferation revealed through cardiac-specific transgenesis of fluorescent ubiquitinated cell cycle indicator (FUCCI).

RATIONALE: Understanding and manipulating the cardiomyocyte cell cycle has been the focus of decades of research, however the ultimate goal of activating mitotic activity in adult mammalian cardiomyocytes remains elusive and controversial. The relentless pursuit of controlling cardiomyocyte mitosis has been complicated and obfuscated by a multitude of indices used as evidence of cardiomyocyte cell cycle activity that lack clear identification of cardiomyocyte "proliferation" versus cell cycle progression, endoreplication, endomitosis, and even DNA damage. Unambiguous appreciation of the complexity of cardiomyocyte replication that avoids oversimplification and misinterpretation is desperately needed. OBJECTIVE: Track cardiomyocyte cell cycle activity and authenticate fidelity of proliferation markers as indicators of de novo cardiomyogenesis in post-mitotic cardiomyocytes. METHODS AND RESULTS: Cardiomyocytes expressing the FUCCI construct driven by the α-myosin heavy chain promoter were readily and uniformly detected through the myocardium of transgenic mice. Cardiomyocyte cell cycle activity peaks at postnatal day 2 and rapidly declines thereafter with almost all cardiomyocytes arrested at the G1/S cell cycle transition. Myocardial infarction injury in adult hearts prompts transient small increases in myocytes progressing through cell cycle without concurrent mitotic activity, indicating lack of cardiomyogenesis. In comparison, cardiomyogenic activity during early postnatal development correlated with coincidence of FUCCI and cKit+ cells that were undetectable in the adult myocardium. CONCLUSIONS: Cardiomyocyte-specific expression of Fluorescence Ubiquitination-based Cell Cycle Indicators (FUCCI) reveals previously unappreciated aspects of cardiomyocyte cell cycle arrest and biological activity in postnatal development and in response to pathologic damage. Compared to many other methods and model systems, the FUCCI transgenic (FUCCI-Tg) mouse represents a valuable tool to unambiguously track cell cycle and proliferation of the entire cardiomyocyte population in the adult murine heart. FUCCI-Tg provides a desperately needed novel approach in the armamentarium of tools to validate cardiomyocyte proliferative activity that will reveal cell cycle progression, discriminate between cycle progression, DNA replication, and proliferation, and provide important insight for enhancing cardiomyocyte proliferation in the context of adult myocardial tissue.

Aortic allografts: final destination?-a summary of clinical tracheal substitutes.

The patient population in desperate need for an airway substitute are individuals with long segment tracheal defects that are considered, technically, inoperable. Regardless of the underlying etiology, benign or malignant growing processes, this patient category enters a palliative setting or require tracheal transplantation. Different airway substitutes have been categorized by Grillo as follows; tracheal transplantation, autogenous tissue, non-viable tissue, tissue-engineering and foreign materials. These fields have been explored in the past in animal models and in clinical patients. Research on airway replacement has been exposed to a level of controversies in the past years. The field has been turbulent and apocryphal. In particular, the area of tissue-engineering using stem cells has suffered from a major set-back leaving scientists, clinicians and ethical committees skeptical. Recently, a hopeful study emerged using aortic allografts as tracheal substitutes in patients with airway defects. The initial results seem promising and reliable. The developments of the field at this point seem striking and hopeful. The focus of this review is to shed light on developments in the field of aortic allografts as substitute for tracheal replacement.

Targeting chronic cardiac remodeling with cardiac progenitor cells in a murine model of ischemia/reperfusion injury.

BACKGROUND: Translational failure for cardiovascular disease is a substantial problem involving both high research costs and an ongoing lack of novel treatment modalities. Despite the progress already made, cell therapy for chronic heart failure in the clinical setting is still hampered by poor translation. We used a murine model of chronic ischemia/reperfusion injury to examine the effect of minimally invasive application of cardiac progenitor cells (CPC) in cardiac remodeling and to improve clinical translation. METHODS: 28 days after the induction of I/R injury, mice were randomized to receive either CPC (0.5 million) or vehicle by echo-guided intra-myocardial injection. To determine retention, CPC were localized in vivo by bioluminescence imaging (BLI) two days after injection. Cardiac function was assessed by 3D echocardiography and speckle tracking analysis to quantify left ventricular geometry and regional myocardial deformation. RESULTS: BLI demonstrated successful injection of CPC (18/23), which were mainly located along the needle track in the anterior/septal wall. Although CPC treatment did not result in overall restoration of cardiac function, a relative preservation of the left ventricular end-diastolic volume was observed at 4 weeks follow-up compared to vehicle control (+5.3 ± 2.1 µl vs. +10.8 ± 1.5 µl). This difference was reflected in an increased strain rate (+16%) in CPC treated mice. CONCLUSIONS: CPC transplantation can be adequately studied in chronic cardiac remodeling using this study set-up and by that provide a translatable murine model facilitating advances in research for new therapeutic approaches to ultimately improve therapy for chronic heart failure.

PIM1-minicircle as a therapeutic treatment for myocardial infarction.

PIM1, a pro-survival gene encoding a serine/ threonine kinase, influences cell proliferation and survival. Modification of cardiac progenitor cells (CPCs) or cardiomyocytes with PIM1 using a lentivirus-based delivery method showed long-term improved cardiac function after myocardial infarction (MI). However, lentivirus based delivery methods have stringent FDA regulation with respect to clinical trials. To provide an alternative and low risk PIM1 delivery method, this study examined the use of a non-viral modified plasmid-minicircle (MC) as a vehicle to deliver PIM1 into mouse CPCs (mCPCs) in vitro and the myocardium in vivo. MC containing a turbo gfp reporter gene (gfp-MC) was used as a transfection and injection control. PIM1 was subcloned into gfp-MC (PIM1-MC) and then transfected into mCPCs at an efficiency of 29.4±3.7%. PIM1-MC engineered mCPCs (PIM1-mCPCs) exhibit significantly (P<0.05) better survival rate under oxidative treatment. PIM1-mCPCs also exhibit 1.9±0.1 and 2.2±0.2 fold higher cell proliferation at 3 and 5 days post plating, respectively, as compared to gfp-MC transfected mCPCs control. PIM1-MC was injected directly into ten-week old adult FVB female mice hearts in the border zone immediately after MI. Delivery of PIM1 into myocardium was confirmed by GFP+ cardiomyocytes. Mice with PIM1-MC injection showed increased protection compared to gfp-MC injection groups measured by ejection fraction at 3 and 7 days post injury (P = 0.0379 and P = 0.0262 by t-test, respectively). Success of PIM1 delivery and integration into mCPCs in vitro and cardiomyocytes in vivo by MC highlights the possibility of a non-cell based therapeutic approach for treatment of ischemic heart disease and MI.

The heart: mostly postmitotic or mostly premitotic? Myocyte cell cycle, senescence, and quiescence.

The concept of myocyte division and myocyte-mediated regeneration has re-emerged in the past 5 years through development of sophisticated transgenic mice and carbon-dating of cells. Although recently, a couple of studies have been conducted as an attempt to intervene in myocyte division, the efficiency in adult animals remains discouragingly low. Re-enforcing myocyte division is a vision that has been desired for decades, leading to years of experience in myocyte resistance to proproliferative stimuli. Previous attempts have indeed provided a platform for basic knowledge on molecular players and signalling in myocytes. However, natural biological processes such as hypertrophy and binucleation provide layers of complexity in interpretation of previous and current findings. A major hurdle in mediating myocyte division is a lack of insight in the myocyte cell cycle. To date, no knowledge is gained on myoycte cell cycle progression and/or duration. This review will include an overview of previous and current literature on myocyte cell cycle and division. Furthermore, the limitations of current approaches and basic questions that might be essential in understanding myocardial resistance to division will be discussed.

Cardiac Hegemony of Senescence.

Cardiac senescence and age-related disease development have gained general attention and recognition in the past decades due to increased accessibility and quality of health care. The advancement in global civilization is complementary to concerns regarding population aging and development of chronic degenerative diseases. Cardiac degeneration has been rigorously studied. The molecular mechanisms of cardiac senescence are on multiple cellular levels and hold a multilayer complexity level, thereby hampering development of unambiguous treatment protocols. In particular, the synergistic exchange of the senescence phenotype through a senescence secretome between myocytes and stem cells appears complicated and is of great future therapeutic value. The current review article will highlight hallmarks of senescence, cardiac myocyte and stem cell senescence, and the mutual exchange of senescent secretome. Future cardiac cell therapy approaches require a comprehensive understanding of myocardial senescence to improve therapeutic efficiency as well as efficacy.

Rejuvenation of human cardiac progenitor cells with Pim-1 kinase.

RATIONALE: Myocardial function is enhanced by adoptive transfer of human cardiac progenitor cells (hCPCs) into a pathologically challenged heart. However, advanced age, comorbidities, and myocardial injury in patients with heart failure constrain the proliferation, survival, and regenerative capacity of hCPCs. Rejuvenation of senescent hCPCs will improve the outcome of regenerative therapy for a substantial patient population possessing functionally impaired stem cells. OBJECTIVE: Reverse phenotypic and functional senescence of hCPCs by ex vivo modification with Pim-1. METHODS AND RESULTS: C-kit-positive hCPCs were isolated from heart biopsy samples of patients undergoing left ventricular assist device implantation. Growth kinetics, telomere lengths, and expression of cell cycle regulators showed significant variation between hCPC isolated from multiple patients. Telomere length was significantly decreased in hCPC with slow-growth kinetics concomitant with decreased proliferation and upregulation of senescent markers compared with hCPC with fast-growth kinetics. Desirable youthful characteristics were conferred on hCPCs by genetic modification using Pim-1 kinase, including increases in proliferation, telomere length, survival, and decreased expression of senescence markers. CONCLUSIONS: Senescence characteristics of hCPCs are ameliorated by Pim-1 kinase resulting in rejuvenation of phenotypic and functional properties. hCPCs show improved cellular properties resulting from Pim-1 modification, but benefits were more pronounced in hCPC with slow-growth kinetics relative to hCPC with fast-growth kinetics. With the majority of patients with heart failure presenting advanced age, infirmity, and impaired regenerative capacity, the use of Pim-1 modification should be incorporated into cell-based therapeutic approaches to broaden inclusion criteria and address limitations associated with the senescent phenotype of aged hCPC.

Cell and gene therapy for severe heart failure patients: the time and place for Pim-1 kinase.

Regenerative therapy in severe heart failure patients presents a challenging set of circumstances including a damaged myocardial environment that accelerates senescence in myocytes and cardiac progenitor cells. Failing myocardium suffers from deterioration of contractile function coupled with impaired regenerative potential that drives the heart toward decompensation. Efficacious regenerative cell therapy for severe heart failure requires disruption of this vicious circle that can be accomplished by alteration of the compromised myocyte phenotype and rejuvenation of progenitor cells. This review focuses upon potential for Pim-1 kinase to mitigate chronic heart failure by improving myocyte quality through preservation of mitochondrial integrity, prevention of hypertrophy and inhibition of apoptosis. In addition, cardiac progenitors engineered with Pim-1 possess enhanced regenerative potential, making Pim-1 an important player in future treatment of severe heart failure.

ß-Adrenergic regulation of cardiac progenitor cell death versus survival and proliferation.

RATIONALE: Short-term ß-adrenergic stimulation promotes contractility in response to stress but is ultimately detrimental in the failing heart because of accrual of cardiomyocyte death. Endogenous cardiac progenitor cell (CPC) activation may partially offset cardiomyocyte losses, but consequences of long-term ß-adrenergic drive on CPC survival and proliferation are unknown. OBJECTIVE: We sought to determine the relationship between ß-adrenergic activity and regulation of CPC function. METHODS AND RESULTS: Mouse and human CPCs express only ß2 adrenergic receptor (ß2-AR) in conjunction with stem cell marker c-kit. Activation of ß2-AR signaling promotes proliferation associated with increased AKT, extracellular signal-regulated kinase 1/2, and endothelial NO synthase phosphorylation, upregulation of cyclin D1, and decreased levels of G protein-coupled receptor kinase 2. Conversely, silencing of ß2-AR expression or treatment with ß2-antagonist ICI 118, 551 impairs CPC proliferation and survival. ß1-AR expression in CPC is induced by differentiation stimuli, sensitizing CPC to isoproterenol-induced cell death that is abrogated by metoprolol. Efficacy of ß1-AR blockade by metoprolol to increase CPC survival and proliferation was confirmed in vivo by adoptive transfer of CPC into failing mouse myocardium. CONCLUSIONS: ß-adrenergic stimulation promotes expansion and survival of CPCs through ß2-AR, but acquisition of ß1-AR on commitment to the myocyte lineage results in loss of CPCs and early myocyte precursors.

Human cardiac progenitor cells engineered with Pim-I kinase enhance myocardial repair.

OBJECTIVES: The goal of this study was to demonstrate the enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for the treatment of myocardial infarction. BACKGROUND: Regenerative potential of stem cells to repair acute infarction is limited. Improved hCPC survival, proliferation, and differentiation into functional myocardium will increase efficacy and advance translational implementation of cardiac regeneration. METHODS: hCPCs isolated from the myocardium of heart failure patients undergoing left ventricular assist device implantation were engineered to express green fluorescent protein (hCPCe) or Pim-1-GFP (hCPCeP). Functional tests of hCPC regenerative potential were performed with immunocompromised mice by using intramyocardial adoptive transfer injection after infarction. Myocardial structure and function were monitored by echocardiographic and hemodynamic assessment for 20 weeks after delivery. hCPCe and hCPCeP expressing luciferase were observed by using bioluminescence imaging to noninvasively track persistence. RESULTS: hCPCeP exhibited augmentation of reparative potential relative to hCPCe control cells, as shown by significantly increased proliferation coupled with amelioration of infarction injury and increased hemodynamic performance at 20 weeks post-transplantation. Concurrent with enhanced cardiac structure and function, hCPCeP demonstrated increased cellular engraftment and differentiation with improved vasculature and reduced infarct size. Enhanced persistence of hCPCeP versus hCPCe was revealed by bioluminescence imaging at up to 8 weeks post-delivery. CONCLUSIONS: Genetic engineering of hCPCs with Pim-1 enhanced repair of damaged myocardium. Ex vivo gene delivery to modify stem cells has emerged as a viable option addressing current limitations in the field. This study demonstrates that efficacy of hCPCs from the failing myocardium can be safely and significantly enhanced through expression of Pim-1 kinase, setting the stage for use of engineered cells in pre-clinical settings.

MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16.

Undesired cell migration after targeted cell transplantation potentially limits beneficial effects for cardiac regeneration. MicroRNAs are known to be involved in several cellular processes, including cell migration. Here, we attempt to reduce human cardiomyocyte progenitor cell (hCMPC) migration via increasing microRNA-155 (miR-155) levels, and investigate the underlying mechanism. Human cardiomyocyte progenitor cells (hCMPCs) were transfected with pre-miR-155, anti-miR-155 or control-miR (ctrl-miR), followed by scratch- and transwell-assays. These functional assays displayed that miR-155 over-expression efficiently inhibited cell migration by 38 ± 3.6% and 59 ± 3.7% respectively. Conditioned medium from miR-155 transfected cells was collected and zymography analysis showed a significant decrease in MMP-2 and MMP-9 activities. The predicted 3'-UTR of MMP-16, an activator of MMP-2 and -9, was cloned into the pMIR-REPORT vector and luciferase assays were performed. Introduction of miR-155 significantly reduced luciferase activity which could be abolished by cotransfection with anti-miR-155 or target site mutagenesis. By using MMP-16 siRNA to reduce MMP-16 levels or by using an MMP-16 blocking antibody, hCMPC migration could be blocked as well. By directly targeting MMP-16, miR-155 efficiently inhibits cell migration via a reduction in MMP-2 and -9 activities. Our study shows that miR-155 might be used to improve local retention of hCMPCs after intramyocardial delivery.

Early and late outcome after aortic root replacement with a mechanical valve prosthesis in a series of 528 patients.

BACKGROUND: Aortic root replacement with a mechanical valve prosthesis is a widely accepted surgical technique. This study aims to evaluate short-term and long-term outcomes of this approach and to identify predictors of 30-day mortality. METHODS: We retrospectively analyzed a consecutive series of 528 patients (mean age, 54±13 years) who underwent aortic root replacement for aneurysm (83%), acute type A dissection (15%), or endocarditis (2%) in the period between 1974 and 2008. The mean time of follow-up was 9.0±7.0 years (range, 0 to 36 years). Concomitant aortic surgery was performed in 71%, coronary revascularization in 18%, and mitral valve surgery in 3%. Selective antegrade cerebral perfusion was applied in 25% and deep hypothermic circulatory arrest in 28% of patients. RESULTS: Overall 30-day mortality was 3.2% to 2.5% for elective surgery and 6.5% for urgent surgery. Morbidity included resternotomy for bleeding or tamponade (19%), pacemaker implantation (3.6%), myocardial infarction (4.0%), and neurologic damage (4.2%). Multivariate analysis revealed myocardial infarction (p<0.001) and the lack of glue use (p=0.018) as independent predictors of 30-day mortality. Subanalysis of the selective antegrade cerebral perfusion patients and the deep hypothermic circulatory arrest patients revealed infarction (p=0.005) and coronary artery disease (p=0.45) for selective antegrade cerebral perfusion and wrapping (p=0.035) for deep hypothermic circulatory arrest as independent risk factors. The survival rate was 87%, 73%, and 29% after 5, 10, and 25 years, respectively. CONCLUSIONS: Aortic root replacement with a mechanical valve prosthesis can be performed safely with low mortality and acceptable morbidity. Perioperative myocardial infarction is the strongest independent risk factor of 30-day mortality.