Mark A. Sussman

Cardiac Stem Cell and Myocyte Aging, Heart Failure, and Insulin-Like Growth Factor-1 Overexpression

Abstract— To determine whether cellular aging leads to a cardiomyopathy and heart failure, markers of cellular senescence, cell death, telomerase activity, telomere integrity, and cell regeneration were measured in myocytes of aging wild-type mice (WT). These parameters were similarly studied in insulin-like growth factor-1 (IGF-1) transgenic mice (TG) because IGF-1 promotes cell growth and survival and may delay cellular aging. Importantly, the consequences of aging on cardiac stem cell (CSC) growth and senescence were evaluated. Gene products implicated in growth arrest and senescence, such as p27Kip1, p53, p16INK4a, and p19ARF, were detected in myocytes of young WT mice, and their expression increased with age. IGF-1 attenuated the levels of these proteins at all ages. Telomerase activity decreased in aging WT myocytes but increased in TG, paralleling the changes in Akt phosphorylation. Reduction in nuclear phospho-Akt and telomerase resulted in telomere shortening and uncapping in WT myocytes. Senescence and death of CSCs increased with age in WT impairing the growth and turnover of cells in the heart. DNA damage and myocyte death exceeded cell formation in old WT, leading to a decreased number of myocytes and heart failure. This did not occur in TG in which CSC-mediated myocyte regeneration compensated for the extent of cell death preventing ventricular dysfunction. IGF-1 enhanced nuclear phospho-Akt and telomerase delaying cellular aging and death. The differential response of TG mice to chronological age may result from preservation of functional CSCs undergoing myocyte commitment. In conclusion, senescence of CSCs and myocytes conditions the development of an aging myopathy.

Bone marrow cells adopt the cardiomyogenic fate in vivo

The possibility that adult bone marrow cells (BMCs) retain a remarkable degree of developmental plasticity and acquire the cardiomyocyte lineage after infarction has been challenged, and the notion of BMC transdifferentiation has been questioned. The center of the controversy is the lack of unequivocal evidence in favor of myocardial regeneration by the injection of BMCs in the infarcted heart. Because of the interest in cell-based therapy for heart failure, several approaches including gene reporter assay, genetic tagging, cell genotyping, PCR-based detection of donor genes, and direct immunofluorescence with quantum dots were used to prove or disprove BMC transdifferentiation. Our results indicate that BMCs engraft, survive, and grow within the spared myocardium after infarction by forming junctional complexes with resident myocytes. BMCs and myocytes express at their interface connexin 43 and N-cadherin, and this interaction may be critical for BMCs to adopt the cardiomyogenic fate. With time, a large number of myocytes and coronary vessels are generated. Myocytes show a diploid DNA content and carry, at most, two sex chromosomes. Old and new myocytes show synchronicity in calcium transients, providing strong evidence in favor of the functional coupling of these two cell populations. Thus, BMCs transdifferentiate and acquire the cardiomyogenic and vascular phenotypes restoring the infarcted heart. Together, our studies reveal that locally delivered BMCs generate de novo myocardium composed of integrated cardiomyocytes and coronary vessels. This process occurs independently of cell fusion and ameliorates structurally and functionally the outcome of the heart after infarction.

The Rac and Rho Hall of Fame: A Decade of Hypertrophic Signaling Hits

Over the last decade, the Rho family GTPases have gained considerable recognition as powerful regulators of actin cytoskeletal organization. As with many high profile signal transducers, these molecules soon attracted the attention of the cardiovascular research community. Shortly thereafter, two prominent members known as RhoA and Rac1 were linked to agonist-induced gene expression and myofilament organization using the isolated cardiomyocyte cell model. Subsequent creation of transgenic mouse lines provided evidence for more complex roles of RhoA and Rac1 signaling. Clues from in vitro and in vivo studies suggest the involvement of numerous downstream targets of RhoA and Rac1 signaling including serum response factor, NF-&kgr;B, and other transcription factors, myofilament proteins, ion channels, and reactive oxygen species generation. Which of these contribute to the observed phenotypic effects of enhanced RhoA and Rac activation in vivo remain to be determined. Current research efforts with a more translational focus have used statins or Rho kinase blockers to assess RhoA and Rac1 as targets for interventional approaches to blunt hypertrophy or heart failure. Generally, salutary effects on remodeling and ischemic damage are observed, but the broad specificity and multiple cellular targets for these drugs within the myocardium demands caution in interpretation. In this review, we assess the evolution of knowledge related to Rac1 and RhoA in the context of hypertrophy and heart failure and highlight the direction that future exploration will lead.

Animal models of heart failure: a scientific statement from the American Heart Association.

Heart failure (HF) is a leading cause of morbidity and mortality in the United States. Despite a number of important therapeutic advances for the treatment of symptomatic HF,1 the prevalence, mortality, and cost associated with HF continue to grow in the United States and other developed countries. Given the aging of our population and the prevalence of diseases such as diabetes mellitus and hypertension that predispose patients to this syndrome, it is possible that HF prevalence will increase in the next decade. Current treatments primarily slow the progression of this syndrome, and there is a need to develop novel preventative and reparative therapies. Development of these novel HF therapies requires testing of the putative therapeutic strategies in appropriate HF animal models. The purposes of this scientific statement are to define the distinctive clinical features of the major causes of HF in humans and to recommend those distinctive pathological features of HF in humans that should be present in an animal model being used to identify fundamental causes of HF or to test preventative or reparative therapies that could reduce HF morbidity and mortality. HF is a clinical syndrome with primary symptoms including dyspnea, fatigue, exercise intolerance, and retention of fluid in the lungs and peripheral tissues. The causes of HF are myriad, but the common fundamental defect is a decreased ability of the heart to provide sufficient cardiac output to support the normal functions of the tissues because of impaired filling and/or ejection of blood. HF is a significant health burden in both the developed world and in emerging nations. In the United States, over a half million new diagnoses of HF occur each year, and the prevalence is 5.8 million individuals >20 years of age.1 HF has a substantial societal burden, with yearly costs in the United …

Activation of the Unfolded Protein Response in Infarcted Mouse Heart and Hypoxic Cultured Cardiac Myocytes

Endoplasmic reticulum (ER) stresses that reduce ER protein folding activate the unfolded protein response (UPR). One effector of the UPR is the transcription factor X-box binding protein-1 (XBP1), which is expressed on ER stress–mediated splicing of the XBP1 mRNA. XBP1 induces certain ER-targeted proteins, eg, glucose-regulated protein 78 (GRP78), that help resolve the ER stress and foster cell survival. In this study, we determined whether hypoxia can activate the UPR in the cardiac context. Neonatal rat ventricular myocyte cultures subjected to hypoxia (16 hours) exhibited increased XBP1 mRNA splicing, XBP1 protein expression, GRP78 promoter activation, and GRP78 protein levels; however, the levels of these UPR markers declined during reoxygenation, suggesting that the UPR is activated during hypoxia but not during reoxygenation. When cells were infected with a recombinant adenovirus (AdV) encoding dominant-negative XBP1 (AdV-XBP1dn), UPR markers were reduced; however, hypoxia/reoxygenation-induced apoptosis increased. Confocal immunocytofluorescence demonstrated that hypoxia induced GRP78 in neonatal rat and isolated adult mouse ventricular myocytes. Moreover, mouse hearts subjected to in vivo myocardial infarction exhibited increased GRP78 expression in cardiac myocytes near the infarct, but not in healthy cells distal to the infarct. These results indicate that hypoxia activates the UPR in cardiac myocytes and that XBP1-inducible proteins may contribute to protecting the myocardium during hypoxic stress.

Endoplasmic Reticulum Stress Gene Induction and Protection From Ischemia/Reperfusion Injury in the Hearts of Transgenic Mice With a Tamoxifen-Regulated Form of ATF6

Ischemia/reperfusion (I/R) affects the integrity of the endoplasmic reticulum (ER), the site of synthesis and folding of numerous proteins. Therefore, I/R may activate the unfolded protein response (UPR), resulting in the induction of a collection of ER stress proteins, many of which are protective and function to resolve the ER stress. In this study, we showed that when mouse hearts were subjected to ex vivo I/R, the levels of 2 ER stress-inducible markers of the UPR, the ER-targeted cytoprotective chaperones glucose-regulated proteins 78 and 94 (GRP78 and GRP94), were increased, consistent with I/R-mediated UPR activation in the heart. The UPR-mediated activation of ATF6 (Activation of Transcription Factor 6) induces cytoprotective ER stress proteins, including GRP78 and GRP94. To examine whether ATF6 protects the myocardium from I/R injury in the heart, we generated transgenic (TG) mice featuring cardiac-restricted expression of a novel tamoxifen-activated form of ATF6, ATF6-MER. When NTG and ATF6-MER TG mice were treated with or without tamoxifen for 5 days, only the hearts from the tamoxifen-treated TG mice exhibited increased levels of many ER stress–inducible mRNAs and proteins; for example, GRP78 and GRP94 transcript levels were increased by 8- and 15-fold, respectively. The tamoxifen-treated TG mouse hearts also exhibited better functional recovery from ex vivo I/R, as well as significantly reduced necrosis and apoptosis. These results suggest that the UPR is activated in the heart during I/R and that, as a result, the ATF6 branch of the UPR may induce expression of proteins that can function to reduce I/R injury.

Nuclear Targeting of Akt Enhances Kinase Activity and Survival of Cardiomyocytes

Abstract— Heart failure is associated with death of cardiomyocytes leading to loss of contractility. Previous studies using membrane-targeted Akt (myristolated-Akt), an enzyme involved in antiapoptotic signaling, showed inhibition of cell death and prevention of pathogenesis induced by cardiomyopathic stimuli. However, recent studies by our group have found accumulation of activated Akt in the nucleus, suggesting that biologically relevant target(s) of Akt activity may be located there. To test this hypothesis, a targeted Akt construct was created to determine the antiapoptotic action of nuclear Akt accumulation. Nuclear localization of the adenovirally encoded Akt construct was confirmed by confocal microscopy. Cardiomyocytes expressing nuclear-targeted Akt showed no evidence of morphological remodeling such as altered myofibril density or hypertrophy. Nuclear-targeted Akt significantly elevated levels of phospho-Akt and kinase activity and inhibited apoptosis as effectively as myristolated-Akt in hypoxia-induced cell death. Transgenic overexpression of nuclear-targeted Akt did not result in hypertrophic remodeling, altered cardiomyocyte DNA content or nucleation, or enhanced phosphorylation of typical cytoplasmic Akt substrates, yet transgenic hearts were protected from ischemia-reperfusion injury. Gene array analyses demonstrated changes in the transcriptional profile of Akt/nuc hearts compared with nontransgenic controls distinct from prior characterizations of Akt expression in transgenic hearts. Collectively, these experiments show that targeting of Akt to the nucleus mediates inhibition of apoptosis without hypertrophic remodeling, opening new possibilities for therapeutic applications of nuclear-targeted Akt to inhibit cell death associated with heart disease.

Pim-1 regulates cardiomyocyte survival downstream of Akt

The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the myocardium in Pim-1–deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury, and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-XL protein levels, as well as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim-1–overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1–deficient hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.

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.

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.