. Cell death and the subsequent post-mortem changes, called necrosis, are integral parts of normal development and maturation cycle. Despite the importance of this process, the mechanisms underlying cell death are still poorly understood. In the recent literature, cell death is said to occur by two alternative, opposite modes: apoptosis, a programmed, managed form of cell death, and necrosis, an unordered and accidental form of cellular dying. The incorrect consequence is the overlapping of: a) the process whereby cells die, cell death; and b) the changes that the cells and tissues undergo after the cells die. Only the latter process can be referred to as necrosis and represents a 'no return' process in cell life. In this review, we discuss the excellent basic research developed in this field during last decades and problems that remain to be resolved in defining both experimentally and mechanicistically the events that lead to and characterize cell death. IntroductionCell death is part of normal development and maturation cycle, and is the component of many response patterns of living tissues to xenobiotic agents (i.e. micro organisms and chemicals) and to endogenous modulations, such as inflammation and disturbed blood supply (1,2). Cell death is an important variable in cancer development, cancer prevention and cancer therapy (3-5). In the treatment of cancer, the major approach is the removal, by surgery, of the neoplasm and/or the induction of cell death in neoplastic cells by radiation, toxic chemicals, antibodies and/or cells of the immune system (6-9). On the other hand, this pathobiological process remains poorly understood and the physiological and biochemical factors that lead to cell death are still not clear. One main factor is the existing confusion between 'apoptosis' process, as compared and contrasted with 'necrosis', leading to the overlapping of the ante mortem changes, i.e. the process of cell death, and the post-mortem changes, i.e. the necrosis process. The pathobiology of cell deathThe elegant scientific exploration of sub-cellular molecular anatomy of the last decades have reinforced the cell concept as 'the smallest integrating unit in biology: a pseudo-intelligent computer that receives, screens, changes, reacts to and adapts to a host of environmental signals, all of this activity apparently

Mitochondrial Calcium (mCa 2+) overload is a central event in myocardial-ischemia reperfusion (IR) injury that leads to metabolic derangement as well as activation of the mitochondrial permeability transition pore (mPTP). mPTP activation results in necrosis and loss of cardiomyocytes which results in acute death in some individuals while survivors are prone to developing heart failure and are predisposed to recurrent infarction events. mCa 2+ has also long been known to activate cellular bioenergetics implicating mCa 2+ in the highly metabolically demanding state of cardiac contractility. The mitochondrial calcium uniporter channel (mtCU) is a multi-subunit complex that resides in the inner mitochondrial membrane and is required for mitochondrial Ca 2+ (mCa 2+) uptake. Mitochondrial Calcium Uniporter B (MCUB, CCDC109B gene), a recently identified paralog of MCU, is reported to negatively regulate mCa 2+ uptake; however, its precise regulation of uniporter function and contribution to cardiac physiology remain unresolved. Size exclusion chromatography of mitochondria isolated from ventricular tissue revealed MCUB was undetectable in the high-molecular weight (MW) fraction of sham animals (~700kD, size of functional mtCU), but 24 hours following myocardial ischemia-reperfusion injury (IR) MCUB was clearly observed in the high-MW fraction. To investigate how MCUB contributes to mtCU regulation we created a stable MCUB-/-HeLa cell line using CRISPR-Cas9n. MCUB deletion increased histamine-mediated mCa 2+ transient amplitude by ~50% versus Wild-Type (WT) controls (mito-R-GECO1). Further, MCUB deletion increased mtCU capacitance (patch-clamp) and rate of [mCa 2+ ] uptake. Fast

Rationale: Embryonic heart is characterized of rapidly dividing cardiomyocytes required to build a working myocardium. Cardiomyocytes retain some proliferative capacity in the neonates but lose it in adulthood. Consequently, a number of signaling hubs including microRNAs are altered during cardiac development that adversely impacts regenerative potential of cardiac tissue. Embryonic stem cell cycle miRs are a class of microRNAs exclusively expressed during developmental stages; however, their effect on cardiomyocyte proliferation and heart function in adult myocardium has not been studied previously. Objective: To determine whether transient reintroduction of embryonic stem cell cycle miR-294 promotes cardiomyocyte cell cycle reentry enhancing cardiac repair after myocardial injury. Methods and Results: miR-294 is expressed in the heart during development, prenatal stages, lost in the neonate, and adult heart confirmed by qRT-PCR and in situ hybridization. Neonatal ventricular myocytes treated with miR-294 showed elevated expression of Ki67, p-histone H3, and Aurora B confirmed by immunocytochemistry compared with control cells. miR-294 enhanced oxidative phosphorylation and glycolysis in Neonatal ventricular myocytes measured by seahorse assay. Mechanistically, miR-294 represses Wee1 leading to increased activity of the cyclin B1/CDK1 complex confirmed by qRT-PCR and immunoblot analysis. Next, a doxycycline-inducible AAV9-miR-294 vector was delivered to mice for activating miR-294 in myocytes for 14 days continuously after myocardial infarction. miR-294–treated mice significantly improved left ventricular functions together with decreased infarct size and apoptosis 8 weeks after MI. Myocyte cell cycle reentry increased in miR-294 hearts analyzed by Ki67, pH3, and AurB (Aurora B kinase) expression parallel to increased small myocyte number in the heart. Isolated adult myocytes from miR-294 hearts showed increased 5-ethynyl-2′-deoxyuridine+ cells and upregulation of cell cycle markers and miR-294 targets 8 weeks after MI. Conclusions: Ectopic transient expression of miR-294 recapitulates developmental signaling and phenotype in cardiomyocytes promoting cell cycle reentry that leads to augmented cardiac function in mice after myocardial infarction.

Rationale: Diabetic cardiomyopathy (DbCM) is a major complication in type-1 diabetes (T1D), accompanied by altered cardiac energetics, impaired mitochondrial function and oxidative stress. Previous studies indicate that T1D is associated with increased cardiac expression of Krüppel-like factor-5 (KLF5) and Peroxisome Proliferator Activated Receptor (PPAR)α that regulate cardiac lipid metabolism. Objective: In this study, we investigated the involvement of KLF5 in DbCM and its transcriptional regulation. Methods and Results: KLF5 mRNA levels were assessed in isolated cardiomyocytes from cardiovascular patients with diabetes and was higher compared with non-diabetic individuals. Analyses in human cells and diabetic mice with cardiomyocyte-specific FOXO1 deletion showed that FOXO1 bound directly on the KLF5 promoter and increased KLF5 expression. Diabetic mice with cardiomyocyte-specific FOXO1 deletion had lower cardiac KLF5 expression and were protected from DbCM. Genetic, pharmacologic gain and loss of KLF5 function approaches and AAV-mediated Klf5 delivery in mice showed that KLF5 induces DbCM. Accordingly, the protective effect of cardiomyocyte FOXO1 ablation in DbCM was abolished when KLF5 expression was rescued. Similarly, constitutive cardiomyocyte-specific KLF5 overexpression caused cardiac dysfunction. KLF5 caused oxidative stress via direct binding on NADPH oxidase (NOX)4 promoter and induction of NOX4 expression. This was accompanied by accumulation of cardiac ceramides. Pharmacologic or genetic KLF5 inhibition alleviated superoxide formation, prevented ceramide accumulation and improved cardiac function in diabetic mice. Conclusions: Diabetes-mediated activation of cardiomyocyte FOXO1 increases KLF5 expression, which stimulates NOX4 expression, ceramide accumulation and causes DbCM.

Background Sepsis is the overwhelming host response to infection leading to shock and multiple organ dysfunction. Cardiovascular complications greatly increase sepsis‐associated mortality. Although murine models are routinely used for preclinical studies, the benefit of using genetically engineered mice in sepsis is countered by discrepancies between human and mouse sepsis pathophysiology. Therefore, recent guidelines have called for standardization of preclinical methods to document organ dysfunction. We investigated the course of cardiac dysfunction and myocardial load in different mouse models of sepsis to identify the optimal measurements for early systolic and diastolic dysfunction. Methods and Results We performed speckle‐tracking echocardiography and assessed blood pressure, plasma inflammatory cytokines, lactate, B‐type natriuretic peptide, and survival in mouse models of endotoxemia or polymicrobial infection (cecal ligation and puncture, [ CLP ]) of moderate and high severity. We observed that myocardial strain and cardiac output were consistently impaired early in both sepsis models. Suppression of cardiac output was associated with systolic dysfunction in endotoxemia or combined systolic dysfunction and reduced preload in the CLP model. We found that cardiac output at 2 hours post‐ CLP is a negative prognostic indicator with high sensitivity and specificity that predicts mortality at 48 hours. Using a known antibiotic (ertapenem) treatment, we confirmed that this approach can document recovery. Conclusions We propose a non‐invasive approach for assessment of cardiac function in sepsis and myocardial strain and strain rate as preferable measures for monitoring cardiovascular function in sepsis mouse models. We further show that the magnitude of cardiac output suppression 2 hours post‐ CLP can be used to predict mortality.

Increased abundance of GRK2 [G protein–coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser670 is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.

Sepsis is the overwhelming systemic immune response to infection, which can result in multiple organ dysfunction and septic shock. Myocardial dysfunction during sepsis is associated with advanced disease and significantly increased in-hospital mortality. Our group has shown that energetic failure and excess reactive oxygen species (ROS) generation constitute major components of myocardial dysfunction in sepsis. Because ROS production is central to cellular *

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