Reactive Oxidative Species-Modulated Ca2+ Release Regulates ß2 Integrin Activation on CD4+ CD28null T Cells of Acute Coronary Syndrome Patients.

The number and activity of T cell subsets in the atherosclerotic plaques are critical for the prognosis of patients with acute coronary syndrome. ß2 Integrin activation is pivotal for T cell recruitment and correlates with future cardiac events. Despite this knowledge, differential regulation of adhesiveness in T cell subsets has not been explored yet. In this study, we show that in human T cells, SDF-1α-mediated ß2 integrin activation is driven by a, so far, not-described reactive oxidative species (ROS)-regulated calcium influx. Furthermore, we show that CD4+CD28null T cells represent a highly reactive subset showing 25-fold stronger ß2 integrin activation upon SDF-1α stimulation compared with CD28+ T cells. Interestingly, ROS-dependent Ca release was much more prevalent in the pathogenetically pivotal CD28null subset compared with the CD28+ T cells, whereas the established mediators of the classical pathways for ß2 integrin activation (PKC, PI3K, and PLC) were similarly activated in both T cell subsets. Thus, interference with the calcium flux attenuates spontaneous adhesion of CD28null T cells from acute coronary syndrome patients, and calcium ionophores abolished the observed differences in the adhesion properties between CD28+ and CD28null T cells. Likewise, the adhesion of these T cell subsets was indistinguishable in the presence of exogenous ROS/H2O2 Together, these data provide a molecular explanation of the role of ROS in pathogenesis of plaque destabilization.

Effectiveness of an Unexpected Disturbance Program in the Early Stage of Rehabilitation in Athletes With Unilateral Knee Ligament Injury.

CONTEXT: Sensorimotor, proprioceptive, and neuromuscular programs are critical for the successful rehabilitation of injured athletes, and these decrease reinjury rates. OBJECTIVE: To investigate the effects of an unexpected disturbance program (UDP) on balance and unilateral strength metrics in athletes with unilateral knee ligament injury. DESIGN: A 3-week parallel-group experimental design consisting of 9 rehabilitation sessions. SETTING: National Sports Institute. PARTICIPANTS: Twenty-one national-level athletes (age 21.4 [4.4] y, body mass 63.9 [10.8] kg, height 169.0 [10.2] cm) who had sustained a unilateral knee ligament injury. INTERVENTION: An UDP program designed to evoke rapid sensorimotor responses was compared with traditional training and a nonexercise control group. MAIN OUTCOME MEASURES: Unilateral total, anteroposterior, and mediolateral sway with eyes open and closed and unilateral isometric strength. RESULTS: Traditional exercises tended to outperform the UDP when unilateral balance testing was performed with eyes open; however, balance improvement following UDP tended to be greater in the eyes-closed condition. Significant strength gains in both the injured and uninjured legs were only observed following the UDP. This increase in unilateral isometric strength was 23.4 and 35.1 kg greater than the strength improvements seen in the traditional rehabilitation and control groups (P < .05). CONCLUSIONS: UDP could improve neural aspects of rehabilitation to improve rehabilitation outcomes by improving strength, sensorimotor function, and proprioception. Given the complementary adaptations, an UDP could provide an effective adjunct to traditional rehabilitation protocols and improve return-to-play outcomes.

Myocardial AKT: the omnipresent nexus.

One of the greatest examples of integrated signal transduction is revealed by examination of effects mediated by AKT kinase in myocardial biology. Positioned at the intersection of multiple afferent and efferent signals, AKT exemplifies a molecular sensing node that coordinates dynamic responses of the cell in literally every aspect of biological responses. The balanced and nuanced nature of homeostatic signaling is particularly essential within the myocardial context, where regulation of survival, energy production, contractility, and response to pathological stress all flow through the nexus of AKT activation or repression. Equally important, the loss of regulated AKT activity is primarily the cause or consequence of pathological conditions leading to remodeling of the heart and eventual decompensation. This review presents an overview compendium of the complex world of myocardial AKT biology gleaned from more than a decade of research. Summarization of the widespread influence that AKT exerts upon myocardial responses leaves no doubt that the participation of AKT in molecular signaling will need to be reckoned with as a seemingly omnipresent regulator of myocardial molecular biological responses.

Functional Effect of Pim1 Depends upon Intracellular Localization in Human Cardiac Progenitor Cells.

Human cardiac progenitor cells (hCPC) improve heart function after autologous transfer in heart failure patients. Regenerative potential of hCPCs is severely limited with age, requiring genetic modification to enhance therapeutic potential. A legacy of work from our laboratory with Pim1 kinase reveals effects on proliferation, survival, metabolism, and rejuvenation of hCPCs in vitro and in vivo. We demonstrate that subcellular targeting of Pim1 bolsters the distinct cardioprotective effects of this kinase in hCPCs to increase proliferation and survival, and antagonize cellular senescence. Adult hCPCs isolated from patients undergoing left ventricular assist device implantation were engineered to overexpress Pim1 throughout the cell (PimWT) or targeted to either mitochondrial (Mito-Pim1) or nuclear (Nuc-Pim1) compartments. Nuc-Pim1 enhances stem cell youthfulness associated with decreased senescence-associated ß-galactosidase activity, preserved telomere length, reduced expression of p16 and p53, and up-regulation of nucleostemin relative to PimWT hCPCs. Alternately, Mito-Pim1 enhances survival by increasing expression of Bcl-2 and Bcl-XL and decreasing cell death after H2O2 treatment, thereby preserving mitochondrial integrity superior to PimWT. Mito-Pim1 increases the proliferation rate by up-regulation of cell cycle modulators Cyclin D, CDK4, and phospho-Rb. Optimal stem cell traits such as proliferation, survival, and increased youthful properties of aged hCPCs are enhanced after targeted Pim1 localization to mitochondrial or nuclear compartments. Targeted Pim1 overexpression in hCPCs allows for selection of the desired phenotypic properties to overcome patient variability and improve specific stem cell characteristics.

Metabolic dysfunction consistent with premature aging results from deletion of Pim kinases.

RATIONALE: The senescent cardiac phenotype is accompanied by changes in mitochondrial function and biogenesis causing impairment in energy provision. The relationship between myocardial senescence and Pim kinases deserves attention because Pim-1 kinase is cardioprotective, in part, by preservation of mitochondrial integrity. Study of the pathological effects resulting from genetic deletion of all Pim kinase family members could provide important insight about cardiac mitochondrial biology and the aging phenotype. OBJECTIVE: To demonstrate that myocardial senescence is promoted by loss of Pim leading to premature aging and aberrant mitochondrial function. METHODS AND RESULTS: Cardiac myocyte senescence was evident at 3 months in Pim triple knockout mice, where all 3 isoforms of Pim kinase family members are genetically deleted. Cellular hypertrophic remodeling and fetal gene program activation were followed by heart failure at 6 months in Pim triple knockout mice. Metabolic dysfunction is an underlying cause of cardiac senescence and instigates a decline in cardiac function. Altered mitochondrial morphology is evident consequential to Pim deletion together with decreased ATP levels and increased phosphorylated AMP-activated protein kinase, exposing an energy deficiency in Pim triple knockout mice. Expression of the genes encoding master regulators of mitochondrial biogenesis, PPARγ (peroxisome proliferator-activated receptor gamma) coactivator-1 α and ß, was diminished in Pim triple knockout hearts, as were downstream targets included in mitochondrial energy transduction, including fatty acid oxidation. Reversal of the dysregulated metabolic phenotype was observed by overexpressing c-Myc (Myc proto-oncogene protein), a downstream target of Pim kinases. CONCLUSIONS: Pim kinases prevent premature cardiac aging and maintain a healthy pool of functional mitochondria leading to efficient cellular energetics.

Pim-1 preserves mitochondrial morphology by inhibiting dynamin-related protein 1 translocation.

Mitochondrial morphological dynamics affect the outcome of ischemic heart damage and pathogenesis. Recently, mitochondrial fission protein dynamin-related protein 1 (Drp1) has been identified as a mediator of mitochondrial morphological changes and cell death during cardiac ischemic injury. In this study, we report a unique relationship between Pim-1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemic stress. Transgenic hearts overexpressing cardiac Pim-1 display reduction of total Drp1 protein levels, increased phosphorylation of Drp1-(S637), and inhibition of Drp1 localization to the mitochondria. Consistent with these findings, adenoviral-induced Pim-1 neonatal rat cardiomyocytes (NRCMs) retain a reticular mitochondrial phenotype after simulated ischemia (sI) and decreased Drp1 mitochondrial sequestration. Interestingly, adenovirus Pim-dominant negative NRCMs show increased expression of Bcl-2 homology 3 (BH3)-only protein p53 up-regulated modulator of apoptosis (PUMA), which has been previously shown to induce Drp1 accumulation at mitochondria and increase sensitivity to apoptotic stimuli. Overexpression of the p53 up-regulated modulator of apoptosis-dominant negative adenovirus attenuates localization of Drp1 to mitochondria in adenovirus Pim-dominant negative NRCMs promotes reticular mitochondrial morphology and inhibits cell death during sI. Therefore, Pim-1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to sI.

Pathological hypertrophy amelioration by PRAS40-mediated inhibition of mTORC1.

Mechanistic target of rapamycin complex 1 (mTORC1), necessary for cellular growth, is regulated by intracellular signaling mediating inhibition of mTORC1 activation. Among mTORC1 regulatory binding partners, the role of Proline Rich AKT Substrate of 40 kDa (PRAS40) in controlling mTORC1 activity and cellular growth in response to pathological and physiological stress in the heart has never been addressed. This report shows PRAS40 is regulated by AKT in cardiomyocytes and that AKT-driven phosphorylation relieves the inhibitory function of PRAS40. PRAS40 overexpression in vitro blocks mTORC1 in cardiomyocytes and decreases pathological growth. Cardiomyocyte-specific overexpression in vivo blunts pathological remodeling after pressure overload and preserves cardiac function. Inhibition of mTORC1 by PRAS40 preferentially promotes protective mTORC2 signaling in chronic diseased myocardium. In contrast, strong PRAS40 phosphorylation by AKT allows for physiological hypertrophy both in vitro and in vivo, whereas cardiomyocyte-specific overexpression of a PRAS40 mutant lacking capacity for AKT-phosphorylation inhibits physiological growth in vivo, demonstrating that AKT-mediated PRAS40 phosphorylation is necessary for induction of physiological hypertrophy. Therefore, PRAS40 phosphorylation acts as a molecular switch allowing mTORC1 activation during physiological growth, opening up unique possibilities for therapeutic regulation of the mTORC1 complex to mitigate pathologic myocardial hypertrophy by PRAS40.

Differential regulation of cellular senescence and differentiation by prolyl isomerase Pin1 in cardiac progenitor cells.

Autologous c-kit(+) cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing differentiation. The prolyl isomerase Pin1 regulates multiple signaling cascades by modulating protein folding and thereby activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 is expressed in CPCs in vitro and in vivo and is associated with increased proliferation. Pin1 is required for cell cycle progression and loss of Pin1 causes cell cycle arrest in the G1 phase in CPCs, concomitantly associated with decreased expression of Cyclins D and B and increased expression of cell cycle inhibitors p53 and retinoblastoma (Rb). Pin1 deletion increases cellular senescence but not differentiation or cell death of CPCs. Pin1 is required for endogenous CPC response as Pin1 knock-out mice have a reduced number of proliferating CPCs after ischemic challenge. Pin1 overexpression also impairs proliferation and causes G2/M phase cell cycle arrest with concurrent down-regulation of Cyclin B, p53, and Rb. Additionally, Pin1 overexpression inhibits replicative senescence, increases differentiation, and inhibits cell death of CPCs, indicating that cell cycle arrest caused by Pin1 overexpression is a consequence of differentiation and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve differentiation, and antagonize senescence.

Fibronectin is essential for reparative cardiac progenitor cell response after myocardial infarction.

RATIONALE: Adoptive transfer of cardiac progenitor cells (CPCs) has entered clinical application, despite limited mechanistic understanding of the endogenous response after myocardial infarction (MI). Extracellular matrix undergoes dramatic changes after MI and therefore might be linked to CPC-mediated repair. OBJECTIVE: To demonstrate the significance of fibronectin (Fn), a component of the extracellular matrix, for induction of the endogenous CPC response to MI. METHODS AND RESULTS: This report shows that presence of CPCs correlates with the expression of Fn during cardiac development and after MI. In vivo, genetic conditional ablation of Fn blunts CPC response measured 7 days after MI through reduced proliferation and diminished survival. Attenuated vasculogenesis and cardiogenesis during recovery were evident at the end of a 12-week follow-up period. Impaired CPC-dependent reparative remodeling ultimately leads to continuous decline of cardiac function in Fn knockout animals. In vitro, Fn protects and induces proliferation of CPCs via ß1-integrin-focal adhesion kinase-signal transducer and activator of transcription 3-Pim1 independent of Akt. CONCLUSIONS: Fn is essential for endogenous CPC expansion and repair required for stabilization of cardiac function after MI.

Regulation of cardiac hypertrophic signaling by prolyl isomerase Pin1.

RATIONALE: Cardiac hypertrophy results from the complex interplay of differentially regulated cascades based on the phosphorylation status of involved signaling molecules. Although numerous critical regulatory kinases and phosphatases have been identified in the myocardium, the intracellular mechanism for temporal regulation of signaling duration and intensity remains obscure. In the nonmyocyte context, control of folding, activity, and stability of proteins is mediated by the prolyl isomerase Pin1, but the role of Pin1 in the heart is unknown. OBJECTIVE: To establish the role of Pin1 in the heart. METHODS AND RESULTS: Here, we show that either genetic deletion or cardiac overexpression of Pin1 blunts hypertrophic responses induced by transaortic constriction and consequent cardiac failure in vivo. Mechanistically, we find that Pin1 directly binds to Akt, mitogen activated protein kinase (MEK), and Raf-1 in cultured cardiomyocytes after hypertrophic stimulation. Furthermore, loss of Pin1 leads to diminished hypertrophic signaling of Akt and MEK, whereas overexpression of Pin1 increases Raf-1 phosphorylation on the autoinhibitory site Ser259, leading to reduced MEK activation. CONCLUSIONS: Collectively, these data support a role for Pin1 as a central modulator of the intensity and duration of 2 major hypertrophic signaling pathways, thereby providing a novel target for regulation and control of cardiac hypertrophy.

Mechanistic target of rapamycin complex 2 protects the heart from ischemic damage.

BACKGROUND: The mechanistic target of rapamycin (mTOR) comprises 2 structurally distinct multiprotein complexes, mTOR complexes 1 and 2 (mTORC1 and mTORC2). Deregulation of mTOR signaling occurs during and contributes to the severity of myocardial damage from ischemic heart disease. However, the relative roles of mTORC1 versus mTORC2 in the pathogenesis of ischemic damage are unknown. METHODS AND RESULTS: Combined pharmacological and molecular approaches were used to alter the balance of mTORC1 and mTORC2 signaling in cultured cardiac myocytes and in mouse hearts subjected to conditions that mimic ischemic heart disease. The importance of mTOR signaling in cardiac protection was demonstrated by pharmacological inhibition of both mTORC1 and mTORC2 with Torin1, which led to increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. Predominant mTORC1 signaling mediated by suppression of mTORC2 with Rictor similarly increased cardiomyocyte apoptosis and tissue damage after myocardial infarction. In comparison, preferentially shifting toward mTORC2 signaling by inhibition of mTORC1 with PRAS40 led to decreased cardiomyocyte apoptosis and tissue damage after myocardial infarction. CONCLUSIONS: These results suggest that selectively increasing mTORC2 while concurrently inhibiting mTORC1 signaling is a novel therapeutic approach for the treatment of ischemic heart disease.

Nucleolar stress is an early response to myocardial damage involving nucleolar proteins nucleostemin and nucleophosmin.

Nucleolar stress, characterized by loss of nucleolar integrity, has not been described in the cardiac context. In addition to ribosome biogenesis, nucleoli are critical for control of cell proliferation and stress responses. Our group previously demonstrated induction of the nucleolar protein nucleostemin (NS) in response to cardiac pathological insult. NS interacts with nucleophosmin (NPM), a marker of nucleolar stress with cytoprotective properties. The dynamic behavior of NS and NPM reveal that nucleolar disruption is an early event associated with stress response in cardiac cells. Rapid translocation of NS and NPM to the nucleoplasm and suppression of new preribosomal RNA synthesis occurs in both neonatal rat cardiomyocytes (NRCM) and cardiac progenitor cells (CPC) upon exposure to doxorubicin or actinomycin D. Silencing of NS significantly increases cell death resulting from doxorubicin treatment in CPC, whereas NPM knockdown alone induces cell death. Overexpression of either NS or NPM significantly decreases caspase 8 activity in cultured cardiomyocytes challenged with doxorubicin. The presence of altered nucleolar structures resulting from myocardial infarction in mice supports the model of nucleolar stress as a general response to pathological injury. Collectively, these findings serve as the initial description of myocardial nucleolar stress and establish the postulate that nucleoli acts as sensors of stress, regulating the cellular response to pathological insults.

Fibronectin contributes to pathological cardiac hypertrophy but not physiological growth.

Ability of the heart to undergo pathological or physiological hypertrophy upon increased wall stress is critical for long-term compensatory function in response to increased workload demand. While substantial information has been published on the nature of the fundamental molecular signaling involved in hypertrophy, the role of extracellular matrix protein Fibronectin (Fn) in hypertrophic signaling is unclear. The objective of the study was to delineate the role of Fn during pressure overload-induced pathological cardiac hypertrophy and physiological growth prompted by exercise. Genetic conditional ablation of Fn in adulthood blunts cardiomyocyte hypertrophy upon pressure overload via attenuated activation of nuclear factor of activated T cells (NFAT). Loss of Fn delays development of heart failure and improves survival. In contrast, genetic deletion of Fn has no impact on physiological cardiac growth induced by voluntary wheel running. Down-regulation of the transcription factor c/EBPß (Ccaat-enhanced binding protein ß), which is essential for induction of the physiological growth program, is unaffected by Fn deletion. Nuclear NFAT translocation is triggered by Fn in conjunction with up-regulation of the fetal gene program and hypertrophy of cardiomyocytes in vitro. Furthermore, activation of the physiological gene program induced by insulin stimulation in vitro is attenuated by Fn, whereas insulin had no impact on Fn-induced pathological growth program. Fn contributes to pathological cardiomyocyte hypertrophy in vitro and in vivo via NFAT activation. Fn is dispensable for physiological growth in vivo, and Fn attenuates the activation of the physiological growth program in vitro.

Increased mitotic rate coincident with transient telomere lengthening resulting from pim-1 overexpression in cardiac progenitor cells.

Cardiac regeneration following myocardial infarction rests with the potential of c-kit+ cardiac progenitor cells (CPCs) to repopulate damaged myocardium. The ability of CPCs to reconstitute the heart is restricted by patient age and disease progression. Increasing CPC proliferation, telomere length, and survival will improve the ability of autologous CPCs to be successful in myocardial regeneration. Prior studies have demonstrated enhancement of myocardial regeneration by engineering CPCs to express Pim-1 kinase, but cellular and molecular mechanisms for Pim-1-mediated effects on CPCs remain obscure. We find CPCs rapidly expand following overexpression of cardioprotective kinase Pim-1 (CPCeP), however, increases in mitotic rate are short-lived as late passage CPCePs proliferate similar to control CPCs. Telomere elongation consistent with a young phenotype is observed following Pim-1 modification of CPCeP; in addition, telomere elongation coincides with increased telomerase expression and activity. Interestingly, telomere length and telomerase activity normalize after several rounds of passaging, consistent with the ability of Pim-1 to transiently increase mitosis without resultant oncogenic transformation. Accelerating mitosis in CPCeP without immortalization represents a novel strategy to expand the CPC population in order to improve their therapeutic efficacy.

Cardiac progenitor cell commitment is inhibited by nuclear Akt expression.

RATIONALE: Stem cell therapies to regenerate damaged cardiac tissue represent a novel approach to treat heart disease. However, the majority of adoptively transferred stem cells delivered to damaged myocardium do not survive long enough to impart protective benefits, resulting in modest functional improvements. Strategies to improve survival and proliferation of stem cells show promise for significantly enhancing cardiac function and regeneration. OBJECTIVE: To determine whether injected cardiac progenitor cells (CPCs) genetically modified to overexpress nuclear Akt (CPCeA) increase structural and functional benefits to infarcted myocardium relative to control CPCs. METHODS AND RESULTS: CPCeA exhibit significantly increased proliferation and secretion of paracrine factors compared with CPCs. However, CPCeA exhibit impaired capacity for lineage commitment in vitro. Infarcted hearts receiving intramyocardial injection of CPCeA have increased recruitment of endogenous c-kit cells compared with CPCs, but neither population provides long-term functional and structural improvements compared with saline-injected controls. Pharmacological inhibition of Akt alleviated blockade of lineage commitment in CPCeA. CONCLUSIONS: Although overexpression of nuclear Akt promotes rapid proliferation and secretion of protective paracrine factors, the inability of CPCeA to undergo lineage commitment hinders their capacity to provide functional or structural benefits to infarcted hearts. Despite enhanced recruitment of endogenous CPCs, lack of functional improvement in CPCeA-treated hearts demonstrates CPC lineage commitment is essential to the regenerative response. Effective stem cell therapies must promote cellular survival and proliferation without inhibiting lineage commitment. Because CPCeA exhibit remarkable proliferative potential, an inducible system mediating nuclear Akt expression could be useful to augment cell therapy approaches.

Pim-1 kinase protects mitochondrial integrity in cardiomyocytes.

RATIONALE: Cardioprotective signaling mediates antiapoptotic actions through multiple mechanisms including maintenance of mitochondrial integrity. Pim-1 kinase is an essential downstream effector of AKT-mediated cardioprotection but the mechanistic basis for maintenance of mitochondrial integrity by Pim-1 remains unexplored. This study details antiapoptotic actions responsible for enhanced cell survival in cardiomyocytes with elevated Pim-1 activity. OBJECTIVE: The purpose of this study is to demonstrate that the cardioprotective kinase Pim-1 acts to inhibit cell death by preserving mitochondrial integrity in cardiomyocytes. METHODS AND RESULTS: A combination of biochemical, molecular, and microscopic analyses demonstrate beneficial effects of Pim-1 on mitochondrial integrity. Pim-1 protein level increases in the mitochondrial fraction with a corresponding decrease in the cytosolic fraction of myocardial lysates from hearts subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Cardiac-specific overexpression of Pim-1 results in higher levels of antiapoptotic Bcl-X(L) and Bcl-2 compared to samples from normal hearts. In response to oxidative stress challenge, Pim-1 preserves the inner mitochondrial membrane potential. Ultrastructure of the mitochondria is maintained by Pim-1 activity, which prevents swelling induced by calcium overload. Finally, mitochondria isolated from hearts created with cardiac-specific overexpression of Pim-1 show inhibition of cytochrome c release triggered by a truncated form of proapoptotic Bid. CONCLUSION: Cardioprotective action of Pim-1 kinase includes preservation of mitochondrial integrity during cardiomyopathic challenge conditions, thereby raising the potential for Pim-1 kinase activation as a therapeutic interventional approach to inhibit cell death by antagonizing proapoptotic Bcl-2 family members that regulate the intrinsic apoptotic pathway.

Mitochondrial translocation of Nur77 mediates cardiomyocyte apoptosis.

AIMS: The cascade of events leading to compromised mitochondrial integrity in response to stress is mediated by various combinatorial interactions of pro- and anti-apoptotic molecules. Nur77, an immediate early gene that encodes a nuclear orphan receptor, translocates from the nucleus to mitochondria to induce cytochrome c release and apoptosis in cancer cells in response to various pro-apoptotic treatments. However, the role of Nur77 in the cardiac setting is still unclear. The objective of this study is to determine the physiological relevance and pathophysiological importance of Nur77 in cardiomyocytes. METHODS AND RESULTS: Myocardial Nur77 is upregulated following cardiomyopathic injury and, while expressed in the postnatal myocardium, declines in level within weeks after birth. Nur77 is localized predominantly in cardiomyocyte nuclei under normal conditions where it is not apoptotic, but translocates to mitochondria in response to oxidative stress both in vitro and in vivo. Mitochondrial localization of Nur77 induces cytochrome c release and typical morphological features of apoptosis, including chromatin condensation and DNA fragmentation. Knockdown of Nur77 rescued hydrogen peroxide-induced cardiomyocyte apoptosis. CONCLUSION: Translocation of Nur77 from the nucleus to the mitochondria in cardiomyocytes results in the loss of mitochondrial integrity and subsequent apoptosis in response to ischaemia/reperfusion injury. Our findings identify Nur77 as a novel mediator of cardiomyocyte apoptosis and warrants further investigation of mitochondrial Nur77 translocation as a mechanism to control cell death in the treatment of ischaemic heart diseases.

Pim-1 kinase antagonizes aspects of myocardial hypertrophy and compensation to pathological pressure overload.

Pim-1 kinase exerts potent cardioprotective effects in the myocardium downstream of AKT, but the participation of Pim-1 in cardiac hypertrophy requires investigation. Cardiac-specific expression of Pim-1 (Pim-WT) or the dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice together with adenoviral-mediated overexpression of these Pim-1 constructs was used to delineate the role of Pim-1 in hypertrophy. Transgenic overexpression of Pim-1 protects mice from pressure-overload-induced hypertrophy relative to wild-type controls as evidenced by improved hemodynamic function, decreased apoptosis, increases in antihypertrophic proteins, smaller myocyte size, and inhibition of hypertrophic signaling after challenge. Similarly, Pim-1 overexpression in neonatal rat cardiomyocyte cultures inhibits hypertrophy induced by endothelin-1. On the cellular level, hearts of Pim-WT mice show enhanced incorporation of BrdU into myocytes and a hypercellular phenotype compared to wild-type controls after hypertrophic challenge. In comparison, transgenic overexpression of Pim-DN leads to dilated cardiomyopathy characterized by increased apoptosis, fibrosis, and severely depressed cardiac function. Furthermore, overexpression of Pim-DN leads to reduced contractility as evidenced by reduced Ca(2+) transient amplitude and decreased percentage of cell shortening in isolated myocytes. These data support a pivotal role for Pim-1 in modulation of hypertrophy by impacting responses on molecular, cellular, and organ levels.

C-MORE: A high-content single-cell morphology recognition methodology for liquid biopsies toward personalized cardiovascular medicine.

Cellular morphology has the capacity to serve as a surrogate for cellular state and functionality. However, primary cardiomyocytes, the standard model in cardiovascular research, are highly heterogeneous cells and therefore impose methodological challenges to analysis. Hence, we aimed to devise a robust methodology to deconvolute cardiomyocyte morphology on a single-cell level: C-MORE (cellular morphology recognition) is a workflow from bench to data analysis tailored for heterogeneous primary cells using our R package cmoRe. We demonstrate its utility in proof-of-principle applications such as modulation of canonical hypertrophy pathways and linkage of genotype-phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). In our pilot study, exposure of cardiomyocytes to blood plasma prior to versus after aortic valve replacement allows identification of a disease fingerprint and reflects partial reversibility following therapeutic intervention. C-MORE is a valuable tool for cardiovascular research with possible fields of application in basic research and personalized medicine.

Enhancement of myocardial regeneration through genetic engineering of cardiac progenitor cells expressing Pim-1 kinase.

BACKGROUND: Despite numerous studies demonstrating the efficacy of cellular adoptive transfer for therapeutic myocardial regeneration, problems remain for donated cells with regard to survival, persistence, engraftment, and long-term benefits. This study redresses these concerns by enhancing the regenerative potential of adoptively transferred cardiac progenitor cells (CPCs) via genetic engineering to overexpress Pim-1, a cardioprotective kinase that enhances cell survival and proliferation. METHODS AND RESULTS: Intramyocardial injections of CPCs overexpressing Pim-1 were given to infarcted female mice. Animals were monitored over 4, 12, and 32 weeks to assess cardiac function and engraftment of Pim-1 CPCs with echocardiography, in vivo hemodynamics, and confocal imagery. CPCs overexpressing Pim-1 showed increased proliferation and expression of markers consistent with cardiogenic lineage commitment after dexamethasone exposure in vitro. Animals that received CPCs overexpressing Pim-1 also produced greater levels of cellular engraftment, persistence, and functional improvement relative to control CPCs up to 32 weeks after delivery. Salutary effects include reduction of infarct size, greater number of c-kit(+) cells, and increased vasculature in the damaged region. CONCLUSIONS: Myocardial repair is significantly enhanced by genetic engineering of CPCs with Pim-1 kinase. Ex vivo gene delivery to enhance cellular survival, proliferation, and regeneration may overcome current limitations of stem cell-based therapeutic approaches.