Anya Joyo

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.

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.Background —The mechanistic target of rapamycin (mTOR) is comprised of two structurally distinct multiprotein complexes, mTOR complexes 1 and 2 (mTORC1 and 2). 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 (MI). Predominant mTORC1 signaling mediated by suppression of mTORC2 with Rictor similarly increased cardiomyocyte apoptosis and tissue damage after MI. In comparison, preferentially shifting toward mTORC2 signaling by inhibition of mTORC1 with PRAS40 led to decreased cardiomyocyte apoptosis and tissue damage after MI. Conclusions —These results suggest that selectively increasing mTORC2 while concurrently inhibting of mTORC1 signaling is a novel therapeutic approach for the treatment of ischemic heart disease.

mTORC2 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.Background —The mechanistic target of rapamycin (mTOR) is comprised of two structurally distinct multiprotein complexes, mTOR complexes 1 and 2 (mTORC1 and 2). 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 (MI). Predominant mTORC1 signaling mediated by suppression of mTORC2 with Rictor similarly increased cardiomyocyte apoptosis and tissue damage after MI. In comparison, preferentially shifting toward mTORC2 signaling by inhibition of mTORC1 with PRAS40 led to decreased cardiomyocyte apoptosis and tissue damage after MI. Conclusions —These results suggest that selectively increasing mTORC2 while concurrently inhibting of mTORC1 signaling is a novel therapeutic approach for the treatment of ischemic heart disease.

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.

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.

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. STEM CELLS2012;30:2512–2522

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.

Differential Regulation of Cellular Senescence and Differentiation by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells

Background: Pin1 is a prolyl isomerase that modulates the structure of phosphoproteins. Results: Loss of Pin1 causes cell cycle arrest and senescence, whereas Pin1 overexpression increases differentiation and inhibits senescence of cardiac progenitor cells (CPCs). Conclusion: Pin1 has pleiotropic roles in CPCs. Significance: Pin1 may be a molecular target to enhance repair, survival, and differentiation and antagonize senescence of CPCs. 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.

Notch activation enhances lineage commitment and protective signaling in cardiac progenitor cells

Phase I clinical trials applying autologous progenitor cells to treat heart failure have yielded promising results; however, improvement in function is modest, indicating a need to enhance cardiac stem cell reparative capacity. Notch signaling plays a crucial role in cardiac development, guiding cell fate decisions that underlie myocyte and vessel differentiation. The Notch pathway is retained in the adult cardiac stem cell niche, where level and duration of Notch signal influence proliferation and differentiation of cardiac progenitors. In this study, Notch signaling promotes growth, survival and differentiation of cardiac progenitor cells into smooth muscle lineages in vitro. Cardiac progenitor cells expressing tamoxifen-regulated intracellular Notch1 (CPCeK) are significantly larger and proliferate more slowly than control cells, exhibit elevated mTORC1 and Akt signaling, and are resistant to oxidative stress. Vascular smooth muscle and cardiomyocyte markers increase in CPCeK and are augmented further upon ligand-mediated induction of Notch signal. Paracrine signals indicative of growth, survival and differentiation increase with Notch activity, while markers of senescence are decreased. Adoptive transfer of CPCeK into infarcted mouse myocardium enhances preservation of cardiac function and reduces infarct size relative to hearts receiving control cells. Greater capillary density and proportion of vascular smooth muscle tissue in CPCeK-treated hearts indicate improved vascularization. Finally, we report a previously undescribed signaling mechanism whereby Notch activation stimulates CPC growth, survival and differentiation via mTORC1 and paracrine factor expression. Taken together, these findings suggest that regulated Notch activation potentiates the reparative capacity of CPCs in the treatment of cardiac disease.

CENP-A is essential for cardiac progenitor cell proliferation.

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 G2/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.