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Altered intracellular Ca2+dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+concentrations. This study describes methodology to directly measure intracellular Ca2+levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+concentration standards. Titration of Ca2+concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+concentrations. These standard plots can be used to convert transient Ca2+fluorescence obtained with experimental cells to Ca2+concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

Tissue Doppler imaging (TDI) and color M-mode Doppler flow propagation velocity (Vp) are used to assess cardiac function in humans, but the feasibility and applicability of these measurements to murine cardiomyopathic models of heart failure remain unclear. Left ventricular (LV) function was measured by TDI and Vp among mice exhibiting severe dilated cardiomyopathy (TOT), pressure-overload hypertrophy (TAC), and normal controls (NTG). Transmitral flow pattern in TACs and TOTs showed a restrictive filling pattern, but early diastolic mitral annulus velocity was comparable among the three studied groups. Propagation velocity in an anesthetized state was comparable in all three groups. However, while Vp increased in all three groups in the conscious state, the increase in NTGs was statistically greater than in TACs and TOTs. Collectively, results indicate that color M-mode Doppler echocardiography can be used to assess LV function in mice. Furthermore, Vp is depressed by anesthesia, a complication that can lead to misinterpretation of LV function in normal hearts.

Altered cellular adhesion and apoptotic signaling in cardiac remodeling requires coordinated regulation of multiple constituent proteins that comprise cytoskeletal focal adhesions. One such protein activated by cardiac remodeling is related adhesion focal tyrosine kinase (RAFTK, also known as pyk2). Adenoviral-mediated expression of RAFTK in neonatal rat cardiomyocytes involves concurrent increases in phosphorylation of Src, c-Jun N-terminal kinase, and p38 leading to characteristic apoptotic changes including cleavage of poly(ADP-ribose) polymerase, caspase-3 activation, and increased DNA laddering. DNA laddering was decreased by mutation of the Tyr(402) Src-binding site in RAFTK, suggesting a central role for Src activity in apoptotic cell death that was confirmed by adenoviral-mediated Src expression. Multiple apoptotic signaling cascades are recruited by RAFTK as demonstrated by prevention of apoptosis using caspase-3 inhibitor IV (caspase-3 specific inhibitor), PP2 (Src-specific kinase inhibitor), or Csk (cellular negative regulator for Src), as well as dominant negative constructs for p38beta or MKP-1. These RAFTK-mediated phenotypic characteristics are prevented by concurrent expression of wild-type or a phosphorylation-deficient paxillin mutated at Tyr(31) and Tyr(118). Wild-type or mutant paxillin protein accumulation in the cytoplasm has no overt effect upon cell structure, but paxillin accumulation prevents losses of myofibril organization as well as focal adhesion kinase, vinculin, and paxillin protein levels mediated by RAFTK. Apoptotic signaling cascade inhibition by paxillin indicates interruption of signaling proximal to but downstream of RAFTK activity. Chronic RAFTK activation in cardiac remodeling may represent a maladaptive reactive response that can be modulated by paxillin, opening up novel possibilities for inhibition of cardiomyocyte apoptosis and structural degeneration in heart failure.

Stem cell homing and engraftment to the heart and new tissue formation capable of replacing the lost myocardium and improving the functional performance of the damaged heart represent the goals of regenerative medicine in cardiac pathophysiology. Theoretically, this outcome can be achieved by using any source of stem cells, embryonic or adult, or other cell types. In fact, several interventions have been used in an attempt to promote myocardial regeneration. These therapeutic strategies have used fetal cardiomyocytes; skeletal myoblasts; embryonic-derived endothelial cells; bone marrow–derived immature myocytes; fibroblasts; smooth muscle cells; endothelial progenitor cells; and hematopoietic, mesenchymal, and embryonic stem cells.1–3 In all cases, promising results have been obtained, and the amelioration in cardiac performance was, at times, coupled with the structural and functional integration of the graft with the host myocardium.4,5 Despite all these efforts, however, the most appropriate form of cellular therapy for myocardial injury remains to be identified, leaving unanswered the key question: What is/are the optimal cell/cells for cardiac repair? The difficulty encountered in identifying the ideal cell to rebuild the heart is certainly due to the many variables that have to be taken into account, including easy access to the source; the possibility of obtaining a significant number of cells; the time required for the cells to home, grow, and differentiate; and their long-term efficacy. The use of multipotent cells that are not resident in the heart but are derived from other organs such as the bone marrow has raised the problem of transdifferentiation and stem cell plasticity. In addition to the complexity of this issue, it is inevitable that when a strongly ingrained and sincerely believed theory is challenged, a variety of reactions are evoked, ranging from enthusiasm and hope to awe, suspicion, and hostility. Despite the evidence for adult stem cell plasticity, …

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.

Introduction of the constitutively active calcineurin gene into neonatal rat cardiomyocytes by adenovirus resulted in decreased mitochondrial membrane potential ( P < 0.05). Infection of H9c2 cells with calcineurin adenovirus resulted in increased superoxide production ( P < 0.001). Transgenic mice with cardiac-specific expression of a constitutively active calcineurin cDNA (CalTG mice) exhibit a two- to threefold increase in heart size that progresses to heart failure. We prepared mitochondria enriched for the subsarcolemmal population from the hearts of CalTG mice and transgene negative littermates (control). Intact, well-coupled mitochondria prepared from one to two mouse hearts at a time yielded sufficient material for functional studies. Mitochondrial oxygen consumption was measured with a Clark-type oxygen electrode with substrates for mitochondrial complex II (succinate) and complex IV [tetramethylpentadecane (TMPD)/ascorbate]. CalTG mice exhibited a maximal rate of electron transfer in heart mitochondria that was reduced by ∼50% ( P < 0.002) without a loss of respiratory control. Mitochondrial respiration was unaffected in tropomodulin-overexpressing transgenic mice, another model of cardiomyopathy. Western blotting for mitochondrial electron transfer subunits from mitochondria of CalTG mice revealed a 20–30% reduction in subunit 3 of complex I (ND3) and subunits I and IV of cytochrome oxidase (CO-I, CO-IV) when normalized to total mitochondrial protein or to the adenine nucleotide transporter (ANT) and compared with littermate controls ( P < 0.002). Impaired mitochondrial electron transport was associated with high levels of superoxide production in the CalTG mice. Taken together, these data indicate that calcineurin signaling affects mitochondrial energetics and superoxide production. The excessive production of superoxide may contribute to the development of cardiac failure.

Cellular remodeling during progression of dilation involves focal adhesion contact reorganization. However, the signaling mechanisms and structural consequences leading to impaired cardiomyocyte adhesion are poorly defined. These events were studied in tropomodulin-overexpressing transgenic mice that develop dilated cardiomyopathy associated with chronic elevation of intracellular calcium. Analysis of tropomodulin-overexpressing transgenic hearts by immunoblot and confocal microscopy revealed activation and redistribution of signaling molecules known to regulate adhesion. Calcium-dependent pyk2/related focal adhesion tyrosine kinase (RAFTK) showed changes in expression and phosphorylation state, similar to changes observed for a related downstream target molecule of pyk2/RAFTK termed focal adhesion kinase. Paxillin, the target substrate molecule for focal adhesion kinase phosphorylation, was redistributed in tropomodulin-overexpressing transgenic hearts with enhanced paxillin phosphorylation and cleavage. Certain aspects of the in vivo signaling phenotype including increased paxillin phosphorylation could be recapitulated in vitro using neonatal rat cardiomyocytes infected with recombinant adenovirus to overexpress tropomodulin. In addition, increasing intracellular calcium levels with ionomycin induced pyk2/RAFTK phosphorylation, and adenovirally mediated expression of wild-type pyk2/RAFTK resulted in increased phospho-pyk2/RAFTK levels and concomitant paxillin phosphorylation. Collectively, these results delineate a cardiomyocyte signaling pathway associated with dilation that has potential relevance for cardiac remodeling, focal adhesion reorganization, and loss of contractility.

The JHM strain of mouse hepatitis virus (JHMV) elicits an I-A-restricted delayed-type hypersensitivity (DTH) response mediated by a Thy-1+, Lyt-1+, and CD4+ T cell. Adoptive transfer of these polyclonal CD4+ T cells from immunized mice prevents death in lethally infected recipients without significantly reducing virus titer in the central nervous system (CNS). These observations raise the possibility that the recruitment of mononuclear cells into the CNS may play a critical role in survival from a lethal CNS infection. Transient DTH response to nonviral antigens induced an accumulation of monocytes in the CNS that was maximal at 48 h post-challenge and virtually resolved by 5 days post-challenge. By contrast the induction of prolonged DTH responses resulted in the accumulation of a large number of monocytes that persisted in the CNS for at least 5 days post-challenge. Neither type of DTH reaction suppressed virus replication or prevented death from concomitant lethal JHMV infection.

To test the hypothesis that early interventional treatment with insulin-like growth factor-1 (IGF-1) alleviates subsequent development of dilated cardiomyopathy, cardiac-specific IGF-1 expression was introduced by selective cross-breeding into a transgenic mouse model of heart failure that displays phenotypic characteristics of severe dilation. Hemodynamic, structural, and cellular parameters of the heart were compared between nontransgenic, tropomodulin-overexpressing cardiomyopathic, and the hybrid tropomodulin/IGF-1-overexpressing mice. Beneficial effects of IGF-1 were apparent by multiple indices of cardiac structure and function, including normalization of heart mass, anatomy, hemodynamics, and apoptosis. IGF-1 expression also acted as a proliferative stimulus as evidenced by calculated increases in myocyte number as well as expression of Ki67, a nuclear marker of cellular replication. Cellular analyses revealed that IGF-1 inhibited characteristic cardiomyocyte elongation in dilated hearts and restored calcium dynamics comparable to that observed in normal cells. Collectively, these results provide novel information regarding the ability of IGF-1 to inhibit progression of cardiomyopathic disease in a defined model system and suggest that heart failure may benefit from early interventional IGF-1 treatment.

Transfer of cardiac progenitor cells (CPCs) improves cardiac function in heart failure patients. However, CPC function is reduced with age, limiting their regenerative potential. Aging is associated with numerous changes in cells including accumulation of mitochondrial DNA (mtDNA) mutations, but it is unknown how this impacts CPC function. Here, we demonstrate that acquisition of mtDNA mutations disrupts mitochondrial function, enhances mitophagy, and reduces the replicative and regenerative capacities of the CPCs. We show that activation of differentiation in CPCs is associated with expansion of the mitochondrial network and increased mitochondrial oxidative phosphorylation. Interestingly, mutant CPCs are deficient in mitochondrial respiration and rely on glycolysis for energy. In response to differentiation, these cells fail to activate mitochondrial respiration. This inability to meet the increased energy demand leads to activation of cell death. These findings demonstrate the consequences of accumulating mtDNA mutations and the importance of mtDNA integrity in CPC homeostasis and regenerative potential.

Introduction: Diabetes is a multi organ disease and diabetic cardiomyopathy can result in heart failure, which is a leading cause of morbidity and mortality in diabetic patients. Defects in mTOR signaling are believed to contribute to metabolic dysfunctions in diabetic liver and hearts, but evidence is missing that mTOR activation is causal to the development of diabetic cardiomyopathy. PRAS40 (Proline Rich Akt Substrate of 40kDa) is a specific component of mTORC1 that interacts with RAPTOR to inhibit mTORC1 kinase activity Methods and Results: The db/db mouse exhibits key characteristics of T2DM, namely hyperinsulinemia, insulin resistance, hyperglycemia, and develops diabetic cardiomyopathy with decreased cardiac function. Selective mTORC1 inhibition in cardiomyocytes from db/db mice in vivo was achieved using PRAS40 delivered via recombinant cardiotropic adeno-associated vector serotype 9 (AAV9) driven by a cardiomyocyte-specific myosin light chain (MLC2v) promoter construct. Myocardial dysfunction was prevented in AAV-PRAS40 treated db/db mice compared to the AAV-Control group, as seen by significantly higher cardiac ejection fraction (EF%), fractional shortening (FS%) measured by serial echocardiography, despite similar increases in body weight, serum glucose levels, and fat deposition in the liver. The effects of PRAS40 were tested in a model of T2DM induced by high fat diet (HFD). AAV-PRAS40 or AAV-Control was injected at 7 weeks of age and mice were fed HFD chow of or standard for an additional 25 weeks. Decreased cardiac function was observed in the HFD control group measured by echocardiography. This preservation of function was associated with decreased left ventricular diastolic dimension (LVID) and improved diastolic function. This phenotype was associated with improved metabolic function, blunted hypertrophic growth, and preserved insulin sensitivity. Conclusions: mTORC1 inhibition with PRAS40 prevents diabetic cardiomyopathy and improves metabolic function in diabetic mice. These findings may open novel avenues for therapeutic strategies using PRAS40 directed against diabetic-related diseases.

> For good ideas and true innovation, you need human interaction, conflict, argument, debate. > > Margaret Heffernan Over the years, I have been asked several times whether there would ever be a sequel to the inaugural Point/Counterpoint JMCC editorial in which I squared off against my colleague Dr Murry,1 whom I feel privileged to call my friend as well as Chuck. In that treatise penned at the outset of growing controversy surrounding bone marrow stem cell therapy, we were asked to adopt opposing viewpoints for the sake of spirited debate, elevating scientific discourse, and demonstrating that difference of professional opinion can be held while preserving mutual respect and personal integrity. I agreed to join Chuck in that original meeting of the minds recognizing the importance of presenting multiple distinct interpretations of findings as long as the asserted positions remain consistent with the facts. Scientific research evolves and grows stronger through open and honest dialog, and I also believe that the field of regenerative medicine will gain strength from educated differences of opinion. With full awareness of the disturbingly acrimonious tone recently adopted by some researchers in the pursuit of a prevailing opinion to silence their critics, I sincerely hope and trust that Chuck et al will take the following rejoinder in the same spirit …

Despite the increasing use of stem cells for regenerative-based cardiac therapy, the optimal stem cell population(s) remains in a cloud of uncertainty. In the past decade, the field has witnessed a surge of researchers discovering stem cell populations reported to directly and/or indirectly contribute to cardiac regeneration through processes of cardiomyogenic commitment and/or release of cardioprotective paracrine factors. This review centers upon defining basic biological characteristics of stem cells used for sustaining cardiac integrity during disease and maintenance of communication between the cardiac environment and stem cells. Given the limited successes achieved so far in regenerative therapy, the future requires development of unprecedented concepts involving combinatorial approaches to create and deliver the optimal stem cell(s) that will enhance myocardial healing.

Coxsackievirus B (CVB) is an enterovirus that most commonly causes a self-limited febrile illness in infants, but cases of severe infection can manifest in acute myocarditis. Chronic consequences of mild CVB infection are unknown, though there is an epidemiologic association between early subclinical infections and late heart failure, raising the possibility of subtle damage leading to late-onset dysfunction, or chronic ongoing injury due to inflammatory reactions during latent infection. Here we describe a mouse model of juvenile infection with a subclinical dose of coxsackievirus B3 (CVB3) which showed no evident symptoms, either immediately following infection or in adult mice. However following physiological or pharmacologically-induced cardiac stress, juvenile-infected adult mice underwent cardiac hypertrophy and dilation indicative of progression to heart failure. Evaluation of the vasculature in the hearts of adult mice subjected to cardiac stress showed a compensatory increase in CD31+ blood vessel formation, although this effect was suppressed in juvenile-infected mice. Moreover, CVB3 efficiently infected juvenile c-kit+ cells, and cardiac progenitor cell numbers were reduced in the hearts of juvenile-infected adult mice. These results suggest that the exhausted cardiac progenitor cell pool following juvenile CVB3 infection may impair the heart's ability to increase capillary density to adapt to increased load., Author Summary Coxsackievirus B (CVB) is a significant human pathogen, causing myocarditis, aseptic meningitis and encephalitis. The lasting consequences of juvenile CVB infection on the developing host have yet to be adequately inspected. Here, we show that CVB efficiently infected juvenile cardiac progenitor cells both in culture and the young heart. Furthermore, we describe a mouse model of juvenile infection with a subclinical dose of CVB which showed no symptoms of disease into adulthood. However following physiological or pharmacologically-induced cardiac stress, juvenile-infected mice underwent cardiac hypertrophy and dilation indicative of progression to heart failure. These results suggest that mild CVB infection in the young host may impair the ability of the heart to adapt to increased load leading to pathological remodeling later in adult life.

Pin1 is an evolutionarily conserved peptidyl-prolyl isomerase that binds and changes the three-dimensional conformation of specific phospho-proteins. By regulating protein structure and folding, Pin1 affects the stability, interaction, and activity of a broad spectrum of target proteins, thus impacting upon diverse cellular processes. This review discusses the pivotal role Pin1 plays in regulating cardiac pathophysiology by functioning as a "molecular orchestrator" of a myriad of signal transduction pathways in the heart.

The concept of myocyte division and myocyte-mediated regeneration has re-emerged in the past five 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 myocytes resistance to pro-proliferative stimuli. Previous attempts have indeed provided a platform for basic knowledge on molecular players and signaling 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. The current review will provide an overview of previous and current literature on myocytes cell cycle and division. Furthermore, this overview will point-out the limitations of current approaches and focus on re-igniting basic questions that may be essential in understand myocardial resistance to division.

Abstract —Cardiovascular disease risk is higher in men than women, but the basis for this discrepancy remains controversial. Estrogenic stimulation of the myocardium or isolated cardiomyocytes has been purported to exert multiple beneficial effects associated with inhibition of maladaptive responses to pathogenic insults. This report describes a significant difference between the sexes in myocardial activation of Akt, a protein kinase that regulates a broad range of physiological responses including metabolism, gene transcription, and cell survival. We find that young women possess higher levels of nuclear-localized phospho-Akt 473 relative to comparably aged men or postmenopausal women. Both localization of phospho-Akt 473 in myocardial nuclei of sexually mature female mice versus males and Akt kinase activity in nuclear extracts of hearts from female mice versus males are elevated. Cytosolic localization of phospho-forkhead, a downstream nuclear target of Akt, is also increased in female relative to male mice, suggesting a potential mechanism for cardioprotective nuclear signaling resulting from Akt activation. Phospho-Akt 473 levels and localization at cardiac nuclei are similarly increased in transgenic mice with myocardium-specific expression of insulin-like growth factor I, a proven stimulus for Akt activation. Phospho-Akt 473 is also localized to the nucleus of cultured cardiomyocytes after exposure to 17β-estradiol or genistein (a phytoestrogen in soy protein–based diets), and neonatal exposure of litters to genistein elevated nuclear phospho-Akt 473 localization. The activation of Akt in a gender-dependent manner may help explain differences observed in cardiovascular disease risk between the sexes and supports the potential beneficial effects of estrogenic stimulation.

In this study, we investigated cardiomyocyte cytoarchitecture in a mouse model for dilated cardiomyopathy (DCM), the muscle LIM protein (MLP) knockout mouse and substantiated several observations in a second DCM model, the tropomodulin-overexpressing transgenic (TOT) mouse. Freshly isolated cardiomyocytes from both strains are characterized by a more irregular shape compared with wild-type cells. Alterations are observed at the intercalated disks, the specialized areas of mechanical coupling between cardiomyocytes, whereas the subcellular organization of contractile proteins in the sarcomeres of MLP knockout mice appears unchanged. Distinct parts of the intercalated disks are affected differently. Components from the adherens junctions are upregulated, desmosomal proteins are unchanged, and gap junction proteins are downregulated. In addition, the expression of N-RAP, a LIM domain– containing protein located at the intercalated disks, is upregulated in MLP knockout as well as in TOT mice. Detailed analysis of intercalated disk composition during postnatal development reveals that an upregulation of N-RAP expression might serve as an early marker for the development of DCM. Altered expression levels of cytoskeletal proteins (either the lack of MLP or an increased expression of tropomodulin) apparently lead to impaired function of the myofibrillar apparatus and to physiological stress that ultimately results in DCM and is accompanied by an altered appearance and composition of the intercalated disks.

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.

Centromere protein A (CENP-A) is a homolog of histone H3 that epigenetically marks the heterochromatin of chromosomes. CENP-A is a critical component of the cell cycle machinery that is necessary for proper assembly of the mitotic spindle. However, the role of CENP-A in the heart and cardiac progenitor cells (CPCs) has not been previously studied. This study shows that CENP-A is expressed in CPCs and declines with age. Silencing CENP-A results in a decreased CPC growth rate, reduced cell number in phase G 2/M of the cell cycle, and increased senescence associated β-galactosidase activity. Lineage commitment is not affected by CENP-A silencing, suggesting that cell cycle arrest induced by loss of CENP-A is a consequence of senescence and not differentiation. CENP-A knockdown does not exacerbate cell death in undifferentiated CPCs, but increases apoptosis upon lineage commitment. Taken together, these results indicate that CPCs maintain relatively high levels of CENP-A early in life, which is necessary for sustaining proliferation, inhibiting senescence, and promoting survival following differentiation of CPCs.

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.

Background —Failing human hearts are characterized by altered cytoskeletal and myofibrillar organization, impaired signal transduction, abnormal protein turnover, and impaired energy metabolism. Thus, expression of multiple classes of genes is likely to be altered in human heart failure. Methods and Results —We used high-density oligonucleotide arrays to explore changes in expression of ≈7000 genes in 2 nonfailing and 2 failing human hearts with diagnoses of end-stage ischemic and dilated cardiomyopathy, respectively. We report altered expression of (1) cytoskeletal and myofibrillar genes (striated muscle LIM protein-1 [SLIM1], myomesin, nonsarcomeric myosin regulatory light chain-2 [MLC 2 ], and β-actin); (2) genes responsible for degradation and disassembly of myocardial proteins (α 1 -antichymotrypsin, ubiquitin, and gelsolin); (3) genes involved in metabolism (ATP synthase α-subunit, succinate dehydrogenase flavoprotein [SDH Fp] subunit, aldose reductase, and TIM17 preprotein translocase); (4) genes responsible for protein synthesis (elongation factor-2 [EF-2], eukaryotic initiation factor-4AII, and transcription factor homologue-HBZ17); and (5) genes encoding stress proteins (αB-crystallin and μ-crystallin). In 5 additional failing hearts and 4 additional nonfailing controls, we then compared expression of proteins encoded by the differentially expressed genes, αB-crystallin, SLIM1, gelsolin, α 1 -antichymotrypsin, and ubiquitin. In each case, changes in protein expression were consistent with changes in transcript measured by microarray analysis. Gelsolin protein expression was also increased in cardiomyopathic hearts from tropomodulin-overexpressing (TOT) mice and rac1-expressing (racET) mice. Conclusions —Altered expression of the genes identified in this study may contribute to development of the heart failure phenotype and/or represent compensatory mechanisms to sustain cardiac function in failing human hearts.

Heart disease remains one of the leading causes of morbidity and mortality in the industrialized nations of the world. Intense investigation has centered around identifying and manipulating intracellular signaling pathways that direct hypertrophic and myopathic responses in an attempt to intervene in the progression or reverse certain forms of heart disease. We show here that cyclosporin A-mediated inhibition of the calcium-regulated phosphatase, calcineurin (PP2B), reverses cardiac hypertrophy and myopathic dilation in two transgenic mouse models of cardiomyopathy. Reversal was demonstrated by gravimetric analysis, echocardiography, histological analysis, and molecular analysis of hypertrophy-associated gene expression. In contrast, a third mouse model of hypertrophic cardiomyopathy due to activated NFAT3 cardiac-specific expression was not affected by cyclosporin A. These results suggest that calcineurin may function in the long-term maintenance of cardiac hypertrophy or myopathic disease states.

Objective: Dilation and hypertrophy often occur concurrently in cardiomyopathy, yet the interaction between these two functionally distinct conditions remains unknown. Methods: Combinatorial effects of hypertrophy and dilation were investigated by cross-breeding of two cardiomyopathic transgenic mouse lines which develop either hypertrophy (calcineurin-mediated) or dilation (tropomodulin-mediated). Results: Altering the intensity of signals driving hypertrophy and dilation in cross-bred litters resulted in novel disease phenotypes different from either parental line. Augmenting the calcineurin-dependent hypertrophic stimulus in tropomodulin overexpressing transgenics elevated heart:body weight ratios, increased ventricular wall thickness, and significantly accelerated mortality. These effects were evident in calcineurin cross-breeding to tropomodulin backgrounds of transgene homozygosity (severe dilation) or heterozygosity (mild dilation to asymptomatic). Molecular analyses indicated that tropomodulin and calcineurin signaling events in the first week after birth were critical for determination of disease outcome, substantiated by demonstration that temporary neonatal inhibition of tropomodulin expression prevents dilation. Conclusions: This study shows that postnatal timing of altered signaling in cardiomyopathic transgenic mouse models is a pivotal part of determining outcome. In addition, intensifying hypertrophic stimulation exacerbates dilated cardiomyopathy, supporting the concept of shared molecular signaling between hypertrophy and dilation.

Dilated cardiomyopathy is characterized by decreased contractile function and loss of myofibril organization. Previously unexplored structural and molecular events that precede and initiate dilation can now be studied in tropomodulin-overexpressing transgenic (TOT) mice exhibiting progressive dilated cardiomyopathy. Onset of dilation did not correspond to a change in transgene expression levels, which were more than threefold above normal at birth and remained elevated throughout postnatal life. Similarly, mitogen-activated protein kinase activation (p38, ERK1/ERK2, JNK1/JNK2) was not associated with dilation. In contrast, calcineurin was activated before dilation, presumably due to doubling of intracellular diastolic calcium levels in TOT cardiomyocytes. Amplitude of systolic calcium transients was greatly increased as well, demonstrating the novel and unique calcium handling profile of TOT cardiomyocytes. Loss of myofibril organization was not apparent by confocal microscopy until over 1 week after birth, although neonatal sarcomeric abnormalities were revealed by ultrastructural analysis. Rapid postnatal increases in heart:body weight ratio at 1.5 weeks were followed by two waves of mortality between 2 and 3 weeks after birth coincident with maturational stress. Ultimately, TOT pathogenesis is a compensatory response to altered sarcomeric structure driven by calcineurin activation within days after birth, making TOTs an excellent paradigm for studying the role of calcium overload in dilated cardiomyopathy.

Objective: Methylation of the promoter region of the estrogen receptor gene alpha ( ER α ) occurs as a function of age in human colon, and results in inactivation of gene transcription. In this study, we sought to determine whether such age-related methylation occurs in the cardiovascular system, and whether it is associated with atherosclerotic disease. Methods: We used Southern blot analysis to determine the methylation state of the ER α gene in human right atrium, aorta, internal mammary artery, saphenous vein, coronary atherectomy samples, as well as cultured aortic endothelial cells and smooth muscle cells. Results: An age related increase in ER α gene methylation occurs in the right atrium (range 6 to 19%, R =0.36, P

Hypertrophic cardiomyopathy (HCM) is an inherited form of heart disease that affects 1 in 500 individuals. Here it is shown that calcineurin, a calcium-regulated phosphatase, plays a critical role in the pathogenesis of HCM. Administration of the calcineurin inhibitors cyclosporin and FK506 prevented disease in mice that were genetically predisposed to develop HCM as a result of aberrant expression of tropomodulin, myosin light chain–2, or fetal β-tropomyosin in the heart. Cyclosporin had a similar effect in a rat model of pressure-overload hypertrophy. These results suggest that calcineurin inhibitors merit investigation as potential therapeutics for certain forms of human heart disease.

Loss of myofilaments has been observed in both adaptive cardiac responses (ie, hypertrophy) as well as in chemotheraputic use of antineoplastic drugs with cardiotoxic side effects (ie, doxorubicin). An understanding of the degenerative process is a prerequisite for determining approaches to limit the cardiomyopathic changes associated with chronic heart disease or long-term chemotheraputic treatments. However, little is known about the specific events and molecular changes that initiate the degenerative process. To study this process, neonatal rat cardiomyocytes were treated with doxorubicin, which induced rapid and widespread thin-filament degeneration as observed by fluorescence confocal microscopy. which demonstrated deterioration of sarcomeric thin-filament structure. Changes in the spontaneous beating of cardiomyocytes corresponding with myofibrillar degeneration were apparent using differential interference contrast video microscopy. After finding induction of kinase activity by doxorubicin in cultured cardiomyocytes, the protective effects of specific inhibitors of kinase activity were assessed for their ability to inhibit doxorubicin-induced myofibrillar breakdown. Doxorubicin-induced changes appeared similar to the degeneration observed after treatment with a protein kinase activator (phorbol 12-myristate 13-acetate) or a serine-threonine protein phosphatase inhibitor (okadaic acid). Collectively, these results indicate that activation of protein kinase is an important event in the initiation of myofibrillar degeneration by doxorubicin. Further analyses of myofibrillar proteins with respect to biochemical modifications will be necessary to determine if phosphorylation events transmit signal(s) to initiate degeneration.

Myofibril degeneration is a common hallmark of cardiomyopathies leading to cardiac failure. However, information regarding myofibrillar organization in histologic analysis of sectioned tissue is limited by both large size and variable orientation of the cardiomyocytes. In order to observe individual cardiomyocytes from a single mouse heart, we have developed a new procedure for ‘trapping’ dissociated cardiomyocytes on a polyester mesh. Cells can then be visualized by fluorescence confocal microscopy and three-dimensional reconstruction of cells can be performed without distortion of intracellular organization resulting from cytofugation. Since the cells are trapped between the mesh and coverslip, the mesh is not seen in reconstructed images. Sarcomeric protein organization in cardiomyocytes can now be conveniently studied using this procedure. Furthermore, individual atrial or ventricular cardiomyocytes are easily identified in the preparation and decreased expression of α-myosin heavy chain protein expression is apparent in propythiouracil (PTU) treated mice. This procedure will be a valuable asset in the analysis of intracellular changes occurring within the hearts of cardiomyopathic mice.

See related article, pages 748–755 Pressure overload stimulation of the heart stimulates a medley of signaling leading to hypertrophic remodeling. This compensatory effort to increase cardiac output depends on harmonious blending of kinase activities. Participation of protein kinase C (PKC) isoforms in hypertrophic signaling following agonist stimulation or pressure overload is unequivocal, but teasing out the specific roles for various isoform subtypes has been a frustrating endeavor. Researchers have approached this issue by tinkering with PKC isoform kinase activities via overexpression, activating/interfering peptides, or creation of knockout mice.1 Information gleaned from these studies has made 1 point abundantly clear: PKC isoforms have a propensity for shared activation stimuli and common substrate specificities. As such, altering the activity of 1 PKC isoform almost invariably has consequences for the expression/activation/localization of other family members that normally lead to coordinated PKC responses when the heart is subjected to stress. In the case of a study by Klein et al2 in this issue, losing PKCe prompts changes in PKCδ that spell bad news for cardiac structure and function. Klein et al identify cross-regulation between the novel PKCδ and PKCe isoforms in the heart. They demonstrate that PKCδ protein expression is similar in normal and knockout PKCe−/− mice under basal conditions and in normal hearts subjected to pressure overload, but that PKCδ protein expression and phosphorylation levels are increased in PKCe−/− hearts following pressure overload. Prior studies by other groups show that pressure overload-induced hypertrophy leaves PKCe unaffected, whereas PKCδ phosphorylation as well as protein expression level are significantly increased.3,4 Isoform-specific activation studies using selective peptides that show induction of either PKCe or PKCδ promote similar prohypertrophic effects.5 Klein et al found hypertrophic stimulation in the absence of PKCe provokes a compensatory rise in PKCδ activity, reinforcing the …

> The best way to predict future is to invent it. > > —Alan Kay The efficacy of bypass surgery in patients with ischemic cardiomyopathy remains impossible to forecast accurately. In particular, individual variability in factors such as disease comorbidities, genetic background, aging, disease progression, lifestyle, and medications makes the prediction of the outcome of bypass surgery1 a herculean task. Identifying readily available patient-specific information to prognosticate the likelihood of benefit with highly invasive procedures such as bypass surgery not only could minimize patient suffering but also could significantly reduce the cost and resource burdens on a healthcare system that is already stretched to the limit. Article see p 157 Over the last several years, the cardiovascular field has embraced (with varying degrees of enthusiasm) regenerative medicine. Indeed, expert opinions in the field have ranged from beliefs that stem cells can do virtually anything (eg, repair and replace damaged myocardium) to the notion that they are essentially useless (eg, whatever observed biological effects are functionally meaningless). Despite the ongoing debate, most available evidence now suggests that the heart is a self-renewing organ to some (yet to be clarified) extent and that such reparative processes involve a resident pool of self-replicating and renewing c-kit–positive cardiac stem cells (CSCs).2,3 CSCs have been shown to repair damaged heart after myocardial infarction,4 to contribute to neomyogenesis and angiogenesis,5 and to augment heart function.6 If the functional outcome of bypass surgery depends substantially on the functional competency of CSCs, then forecasting the potential benefit of major surgical interventions such as coronary bypass grafting may be accomplished by preemptively assessing the regenerative potential of the CSC pool. …

Decline in stem cell functionality and regenerative capability is a crucial contributor to aging, upon which the c-kit+ cardiac progenitor cell (CPC) pool with enhanced regenerative potential decreases and CPCs with limited proliferative and differentiation potential progressively accumulate. With cardiac stem cells currently being used in the clinic, especially in the elderly population, there is a dire need to understand the phenotypic, molecular and functional characteristics of senescent stem cells to effectively manipulate them for therapy. Nucleostemin (NS), a nucleolar stress sensor protein associated with stem cell pluripotency and regeneration declines upon cardiac aging. We hypothesize that NS is a critical antagonizer of senescence in CPCs. NS expression decreases significantly (-59%, p

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/EBPβ (Ccaat-enhanced binding protein β), 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.

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.

Rationale: Short-term β-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 β-adrenergic drive on CPC survival and proliferation are unknown. Objective: We sought to determine the relationship between β-adrenergic activity and regulation of CPC function. Methods and Results: Mouse and human CPCs express only β2 adrenergic receptor (β2-AR) in conjunction with stem cell marker c-kit. Activation of β2-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 β2-AR expression or treatment with β2-antagonist ICI 118, 551 impairs CPC proliferation and survival. β1-AR expression in CPC is induced by differentiation stimuli, sensitizing CPC to isoproterenol-induced cell death that is abrogated by metoprolol. Efficacy of β1-AR blockade by metoprolol to increase CPC survival and proliferation was confirmed in vivo by adoptive transfer of CPC into failing mouse myocardium. Conclusions: β-adrenergic stimulation promotes expansion and survival of CPCs through β2-AR, but acquisition of β1-AR on commitment to the myocyte lineage results in loss of CPCs and early myocyte precursors.

Lens epithelial cells undergo a dramatic transformation during the process of differentiation into elongated fiber cells. The membrane-associated actin cytoskeleton is likely to play a critical role in the stabilization and maintenance of the highly elongated fiber cell shape. Tropomodulin is a tropomyosin-binding protein associated with actin filaments in a variety of terminally differentiated cell types where stable actin filament organization is required for cell function. We now present results of studies to determine the temporal expression of tropomodulin in the developing lens. In situ hybridization experiments detected expression of tropomodulin mRNA in the developing mouse lens in elongating cells with a pattern similar to that of the fiber specific beta- and gamma-crystallins. Tropomodulin mRNA expression first appeared around 11.5 days post-coitum in elongating cells in the posterior part of the lens vesicle. At later stages the signal for tropomodulin was present in the elongating cells at the lens equator and in cortical fiber cells; signal was absent from the epithelium. To investigate the possible link between tropomodulin expression and fiber differentiation we used a well-established lens epithelial explant culture system in which fiber differentiation is induced by fibroblast growth factor (FGF). Tropomodulin expression was only observed in FGF-treated explants in conjunction with morphologic changes characteristic of lens fiber cell differentiation. The appearance of tropomodulin during the process of fiber cell differentiation suggests that tropomodulin may be important for stabilization and/or determination of actin filament length.

Background Brief episodes of ischemia protect or “precondition” the heart and reduce the size of infarcts caused by subsequent sustained coronary artery occlusion, yet the mechanisms responsible for this cardioprotection remain unresolved. We tested the theory that translocation of protein kinase C (PKC) to the myocyte membranes, initiated in response to brief preconditioning ischemia and manifest during the initial minutes of the sustained occlusion, mediates this phenomenon by attempting to (1) blunt the cardioprotective effects of preconditioning by administration of the PKC inhibitors H-7 and polymyxin B, (2) visualize by fluorescence staining and confocal microscopy changes in the amount or location of PKC, and (3) quantify by incorporation of 32 P into PKC-specific peptide changes in the subcellular distribution of PKC in preconditioned versus control hearts. Methods and Results In the first three limbs of this study, anesthetized open-chest dogs underwent four 5-minute episodes of preconditioning ischemia or a comparable control period before 1 hour of sustained occlusion and 4 to 5 hours of reperfusion. Collateral blood flow was assessed with radioactive microspheres; area at risk (AR) was delineated by injection of blue dye; and the area of necrosis (AN) was measured by tetrazolium staining. AN/AR was smaller in preconditioned versus control dogs that received no treatment (6±2% versus 19±3%, P P P 32 P into PKC-specific peptide revealed no quantitative difference in the subcellular distribution of PKC between control and preconditioned cohorts. Conclusions H-7 and polymyxin B did not blunt the reduction in infarct size achieved with ischemic preconditioning. Neither fluorescence staining and confocal microscopy nor biochemical quantification revealed evidence of preconditioning-induced translocation of PKC to the cell membranes. These results fail to support the hypothesis that translocation of PKC, triggered by preconditioning ischemia, is an important mechanism for the reduction in infarct size seen with preconditioning in the dog model.

Tropomodulin (Tmod) is a tropomyosin-binding protein involved in the structuring of actin filaments. This report describes Tmod expression in distinct patterns during embryonic development in a wider variety of adult and embryonic vertebrate tissues than previously reported. Identical Tmod cDNAs were cloned from mouse brain, skeletal muscle, heart, and hematopoeitic cells. Genomic blotting demonstrates that Tmod is encoded by a single gene, which has a 1077-bp open reading frame that is highly homologous to that of the human erythrocyte. The spatial and temporal expression of the Tmod gene was examined during mouse embryogenesis using in situ hybridization. Tmod mRNA is present by 9.5 days postcoitum (p.c.) in the developing rostral somites, coincident with expression of contractile protein genes in myotomes, suggesting that Tmod may play an important role in sarcomeric thin filament organization in skeletal muscle. While the expression of Tmod mRNA in cardiac muscle is earlier than that in skeletal muscle, its appearance in the heart also coincides with the expression of genes for thin filament proteins and correlates with initial myocardial contractions at 8.0 days p.c. Tmod mRNA is not detected in developing smooth muscle of the gut, but Tmod mRNA is expressed in hematopoeitic cells in yolk sac and developing liver. The sensory ganglia and epithelia of the inner ear express Tmod mRNA as do other sensory neurons such as those in the olfactory epithelium. Expression levels in the brain are much lower prenatally than postnatally. These findings show that Tmod expression in many cell types is developmentally regulated, suggesting that the interaction of actin filaments with this tropomyosin binding protein is an important process in tissue and cell differentiation.

K oyaanisquatsi : ko · yaa · nis · katsi [from the Hopi language], n. 1 crazy life. 2. life in turmoil. 3. life disintegrating. 4. life out of balance. 5. a state of life that calls for another way of living. In the complex interconnected world of myocardial cell signaling, structure and function depend on balancing of positive and negative regulatory stimuli, sequentially or in tandem, to promote homeostatic conditions. Transient variations in balance of signal transduction are the norm as the myocardium dynamically adjusts to physiological demands and conditions. Fortunately, a finely tuned array of antagonistic signaling molecules normally keeps each other from running amok. However, when myocardial signaling suffers from koyaanisquatsi , the resultant cellular alterations lead to cardiomyopathic disease. A healthy heart is highly responsive to catecholamines that stimulate adrenergic receptor signaling to increase contractile force.1 β-Adrenergic receptors transduce signals through G proteins that stimulate the classic G(s)-adenyl cyclase-3′-5′-adenosine monophosphate (cAMP) signaling cascade. The cAMP pathway senses and amplifies β-adrenergic receptor activation, subsequently altering gene expression in conjunction with cAMP-response element binding (CREB) and cAMP-response element modulator (CREM) proteins.2,3 cAMP-response elements (CREs) are found in the promoters of numerous genes regulating multiple facets of cell function.2–4 CRE-mediated transactivation in cardiomyocytes undergoes a rapid “burst,” peaking within 8 hours after induction followed by an attenuation phase characterized by a decrease in CRE-dependent transcription and refractoriness of transactivation by CREB, even in the presence of cAMP-elevating agonist.5 Several mechanisms have been explored to account …

Rationale: Short term β-adrenergic stimulation promotes contractility in response to stress, but is ultimately detrimental in the failing heart due to accrual of cardiomyocyte death. Endogenous myocardial repair may partially offset cardiomyocyte losses, but consequences of long term β-adrenergic drive upon myocardial repair and regeneration are unknown. Objective: Modest recovery of cardiac contractility following long term β-adrenergic blockade in the clinical setting may depend, in part, upon restoration of endogenous repair therefore we sought to determine the relationship between β-adrenergic activity and regulation of cardiac progenitor cell (CPC) function and influence upon myocardial repair. Methods and results: Mouse and human CPCs express only β2 adrenergic receptor (β2-AR) in conjunction with stem cell marker c-kit. Activation of β2-AR signaling promotes proliferation associated with increased Akt phosphorylation, up-regulation of eNOS and cyclin D1, and decreased levels of GRK2. Conversely, silencing of β2-AR expression or treatment with β2-antagonist ICI 118, 551 impairs proliferation and survival. β1-AR expression in CPC is induced by differentiation stimuli, sensitizing CPC to isoproterenol-induced cell death that is abrogated by the β1-AR specific antagonist metoprolol. Efficacy of β1-AR blockade by metoprolol to increase CPC survival and proliferation was confirmed in vivo by adoptive transfer of CPC into failing mouse myocardium, concomitant with increased ejection fraction, fractional shortening and hemodynamic performance. Conclusions: β-adrenergic stimulation promotes expansion and survival of CPCs through β2-AR, but acquisition of β1-AR upon commitment to the myocyte lineage results in loss of early myocyte precursors and ineffective myocardial repair. Thus, β1-AR-specific blockade is likely to provide for enhanced CPC participation in recovery of function in the failing heart.

Myocardial regeneration and repair in response to injury are governed in part by cell survival, proliferation and pluripotency. Proliferation and survival in cardiac progenitor cells (CPCs) are mediated by Pim-1, a serine threonine kinase, and nucleostemin (NS), a nucleolar stress sensor protein. The role of NS in regulating CPC pluripotency and the molecular mechanism of NS induction and action is largely unknown. The hypothesis of the study is that NS, induced by Pim-1 mediated stabilization of transcription factor c-Myc is critical to maintain CPC pluripotency and inhibits senescence. NS and c-Myc protein levels are increased in cultured CPCs overexpressing Pim-1 (3.1 and 5.5 fold, p

Rationale Cardiac stem cells (CSC) regulate cardiomyogenesis and support regenerative processes in the heart, but aging adversely affects stem cell repair capacity. Aging is a primary cause of impaired cardiac function characterized by accumulation of senescent cells. CSC senescence is associated with permanent growth arrest that decreases survival signaling and cellular replacement, inevitably diminishing the capacity of the heart to maintain tissue homeostasis. Therefore, promoting CSC growth may improve cardiac performance with age. Pim-1 kinase exhibits protective and proliferative effects in the myocardium but the role of Pim-1 in cardiac aging has not been thoroughly studied. Objective Demonstrate that Pim-1 promotes stem cell growth in the aged myocardium correlating with increased expression of centromere protein A (CENP-A), a kinetochore-associated protein known to support cell proliferation in numerous species and cell types. Methods & Results CENP-A expression levels were evaluated from murine myocardial tissue samples ranging in age from 11 days post coitum to 4 months of age with analysis by immunoblot as well as quantitative PCR. CENP-A expression was colocalized with c-kit as a marker of CSC by immunohistochemical labeling, revealing a decline in CENP-A expression over the time course of postnatal myocardial maturation. The impact of Pim-1 upon CENP-A level was assessed by comparative analysis of non-transgenic mice versus genetically modified transgenic mouse lines expressing either Pim-1 (wild type) or a dominant negative functionally dead Pim-1 mutant. Pim-1 overexpression increases persistence of CENP-A in CSCs with age, as well as the prevalence of cycling CSCs as marked by phosph-H3 expression, while the functionally dead mutant accelerates CENP-A diminution and decreases CSC proliferation. Conclusion CENP-A decline in c-kit positive cells with age provides intriguing evidence of a potential mechanism for the diminished capacity of CSCs to maintain tissue homeostasis. Pim-1 mitigates CENP-A diminution, demonstrating the promising potential of Pim-1 to promote cardiac growth and repair with age.

Rationale: Mitochondrial morphological dynamics affect the outcome of ischemic heart damage. Mitochondrial fission protein Dynamin Related Protein 1 (Drp1) is a mediator of mitochondrial morphological changes and cell death during ischemic injury. Mitochondrial integrity is maintained by cardioprotective kinase Pim1, which enhances resistance to apoptotic challenge and ischemia reperfusion injury. In this study we examine the relationship between Pim1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemia. Objective: To demonstrate that Pim1 inhibits Drp1 translocation to the mitochondria in response to ischemic injury. Methods and Results: Simulated ischemia and simulated ischemia reperfusion (sI & sI/R) induced mitochondrial fragmentation and cell death in neonatal rat cardiac myocytes (NRCM), respectively. Mitochondrial fragmentation accompanied Drp1 translocation to the mitochondria in NRCM. Inhibition of Drp1 by mdivi1 preserved mitochondrial reticular morphology and inhibited apoptotic cell death. Mice subjected to sI/R injury displayed Drp1 mitochondrial localization, while exposure to mdivi1 led to reduced infarct size. Interestingly, transgenic hearts overexpressing Pim1 decreased total Drp1 levels, increased phosphorylation of Drp1 at serine 637, and inhibited Drp1 localization to mitochondria while preserving reticular morphology after sI. In contrast, Pim1 dominant negative (PDN) transgenic hearts and NRCM exhibit increased Drp1 translocation to mitochondria and fragmented mitochondria. PDN hearts exhibit decreased phosphorylation of serine 637 and upregulation of BH3 protein PUMA, inducing Drp1 accumulation at mitochondria and increased sensitivity to apoptotic stimuli. In PDN NRCMs, overexpression of Puma dominant negative (PumaDN) attenuated localization of Drp1 to mitochondria and inhibited cell death during sI. Conclusion: Pim1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to simulated ischemia. Therefore, selective manipulation of Pim1 should be pursued as a therapeutic target to maintain mitochondrial morphology for cardioprotection.

The endoplasmic reticulum (ER) is essential for protein homeostasis, or proteostasis, which governs the balance of the proteome. In addition to secreted and membrane proteins, proteins bound for many other cellular locations are also made on ER-bound ribosomes, emphasizing the importance of protein quality and quantity control in the ER. Unlike cytosolic E3 ubiquitin ligases studied in the heart, synoviolin/Hrd1, which has not been studied in the heart, is an ER transmembrane E3 ubiquitin ligase, which we found to be upregulated upon protein misfolding in cardiac myocytes. Given the strategic location of synoviolin in the ER membrane, we addressed the hypothesis that synoviolin is critical for regulating the balance of the proteome, and accordingly, myocyte size. We showed that in vitro, adenovirus-mediated overexpression of synoviolin decreased cardiac myocyte size and protein synthesis, but unlike atrophy-related ubiquitin ligases, synoviolin did not increase global protein degradation. Furthermore, targeted gene therapy using adeno-associated virus 9 (AAV9) showed that overexpression of synoviolin in the left ventricle attenuated maladaptive cardiac hypertrophy and preserved cardiac function in mice subjected to trans-aortic constriction (AAV9-control TAC = 22.5 ± 6.2% decrease in EF vs. AAV9-synoviolin TAC at 6 weeks post TAC; P

Rationale: Bone marrow–derived cells to treat myocardial injury improve cardiac function and support beneficial cardiac remodeling. However, survival of stem cells is limited due to low proliferation of transferred cells. Objective: To demonstrate long-term potential of c-kit + bone marrow stem cells (BMCs) enhanced with Pim-1 kinase to promote positive cardiac remodeling. Methods and Results: Lentiviral modification of c-kit + BMCs to express Pim-1 (BMCeP) increases proliferation and expression of prosurvival proteins relative to BMCs expressing green fluorescent protein (BMCe). Intramyocardial delivery of BMCeP at time of infarction supports improvements in anterior wall dimensions and prevents left ventricle dilation compared with hearts treated with vehicle alone. Reduction of the akinetic left ventricular wall was observed in BMCeP-treated hearts at 4 and 12 weeks after infarction. Early recovery of cardiac function in BMCeP-injected hearts facilitated modest improvements in hemodynamic function up to 12 weeks after infarction between cell-treated groups. Persistence of BMCeP is improved relative to BMCe within the infarct together with increased recruitment of endogenous c-kit + cells. Delivery of BMC populations promotes cellular hypertrophy in the border and infarcted regions coupled with an upregulation of hypertrophic genes. Thus, BMCeP treatment yields improved structural remodeling of infarcted myocardium compared with control BMCs. Conclusions: Genetic modification of BMCs with Pim-1 may serve as a therapeutic approach to promote recovery of myocardial structure. Future approaches may take advantage of salutary BMC actions in conjunction with other stem cell types to increase efficacy of cellular therapy and improve myocardial performance in the injured myocardium.

> “Life is about rhythm. We vibrate, our hearts are pumping blood, we are a rhythm machine, that's what we are.” > > —Mickey Hart As you read these words, an intrinsic rhythm of life ripples through your heart. Your personal “metronome” is required to begin at the earliest stages of embryonic development and continues unceasingly as life unfolds. Understanding the genesis of our rhythm continues to be refined by intense investigation from developmental biologists who are unraveling the intertwined orchestration of myocardial organization, timing, and cardiac precursor cell incorporation. Almost a decade ago, a remarkable publication identified an embryonic stem cell subpopulation essential for cardiac formation expressing a LIM homeodomain transcription factor called Isl1.1 Isl1-null mice fail to develop major cardiac segments resulting in embryonic lethality, convincingly demonstrating an essential role of Isl1+ stem cells in the initial stages of cardiogenesis. Potential significance of these cellular developmental biology observations did not go unnoticed by those committed to the comparatively nascent field of myocardial regeneration who long for a specific cardiac stem cell marker. Following closely on the heels of initial Isl1-related revelations, a spate of high-impact publications touted the potential of Isl1 as a marker for pluripotent cardiac precursors capable of generating all cardiovascular lineages.2–5 Thereafter, as the proverbial saying goes, “then all hell broke loose” and researchers became embroiled in yet another controversy about cardiac stem cell biology.6–9 Fortunately, time discovers truth and a clearer vision emerges, as presented in this issue of Circulation Research by Weinberger et al10 that helps reconcile speculation with reality and dispels some of the hyperbole about Isl1 in the adult heart. Article, see p 1303 At the core of the controversy are the questions of whether Isl1+ cells persist in the adult heart, what types of …