Inhibition of cyclin-dependent kinase 7 mitigates doxorubicin cardiotoxicity and enhances anticancer efficacy.

AIMS: The anthracycline family of anticancer agents such as doxorubicin (DOX) can induce apoptotic death of cardiomyocytes and cause cardiotoxicity. We previously reported that DOX-induced apoptosis is accompanied by cardiomyocyte cell cycle re-entry. Cell cycle progression requires cyclin-dependent kinase 7 (CDK7)-mediated activation of downstream cell cycle CDKs. This study aims to determine whether CDK7 can be targeted for cardioprotection during anthracycline chemotherapy. METHODS AND RESULTS: DOX exposure induced CDK7 activation in mouse heart and isolated cardiomyocytes. Cardiac-specific ablation of Cdk7 attenuated DOX-induced cardiac dysfunction and fibrosis. Treatment with the covalent CDK7 inhibitor THZ1 also protected against DOX-induced cardiomyopathy and apoptosis. DOX treatment induced activation of the proapoptotic CDK2-FOXO1-Bim axis in a CDK7-dependent manner. In response to DOX, endogenous CDK7 directly bound and phosphorylated CDK2 at Thr160 in cardiomyocytes, leading to full CDK2 kinase activation. Importantly, inhibition of CDK7 further suppressed tumour growth when used in combination with DOX in an immunocompetent mouse model of breast cancer. CONCLUSION: Activation of CDK7 is necessary for DOX-induced cardiomyocyte apoptosis and cardiomyopathy. Our findings uncover a novel proapoptotic role for CDK7 in cardiomyocytes. Moreover, this study suggests that inhibition of CDK7 attenuates DOX-induced cardiotoxicity but augments the anticancer efficacy of DOX. Therefore, combined administration of CDK7 inhibitor and DOX may exhibit diminished cardiotoxicity but superior anticancer activity.

Tetracycline and sulfadiazine toxicity in human liver cells Huh-7.

As typical antibiotics, tetracycline (TC) and sulfadiazine (SDZ) enter the human body through the food chain. Therefore, it is necessary to understand their individual and combined toxicity. In this study, the effects of TC, SDZ, and their mixture on cell viability, cell membrane damage, liver cell damage, and oxidative damage were evaluated in in vitro assays with human liver cells Huh-7. The results showed cytotoxicity of TC, SDZ, and their mixture, which induced oxidative stress and caused membrane and cell damage. The effect of antibiotics on Huh-7 cells increased with increasing concentration, except for lactate dehydrogenase (LDH) activity that commonly showed a threshold concentration response and cell viability, which commonly showed a biphasic trend, suggesting the possibility of hormetic responses where proper doses are included. The toxicity of TC was commonly higher than that of SDZ when applied at the same concentration. These findings shed light on the individual and joint effects of these major antibiotics on liver cells, providing a scientific basis for the evaluation of antibiotic toxicity and associated risks.

RBL2 Regulates Cardiac Sensitivity to Anthracycline Chemotherapy.

Background: Anthracycline chemotherapies cause heart failure in a subset of cancer patients. We previously reported that the anthracycline doxorubicin (DOX) induces cardiotoxicity through the activation of cyclin-dependent kinase 2 (CDK2). Objectives: The aim of this study was to determine whether retinoblastoma-like 2 (RBL2/p130), an emerging CDK2 inhibitor, regulates anthracycline sensitivity in the heart. Methods: Rbl2-/- mice and Rbl2+/+ littermates received DOX (5 mg/kg/wk for 4 weeks intraperitoneally, 20 mg/kg cumulative). Heart function was monitored with echocardiography. The association of RBL2 genetic variants with anthracycline cardiomyopathy was evaluated in the SJLIFE (St. Jude Lifetime Cohort Study) and CPNDS (Canadian Pharmacogenomics Network for Drug Safety) studies. Results: The loss of endogenous Rbl2 increased basal CDK2 activity in the mouse heart. Mice lacking Rbl2 were more sensitive to DOX-induced cardiotoxicity, as evidenced by rapid deterioration of heart function and loss of heart mass. The disruption of Rbl2 exacerbated DOX-induced mitochondrial damage and cardiomyocyte apoptosis. Mechanistically, Rbl2 deficiency enhanced CDK2-dependent activation of forkhead box O1 (FOXO1), leading to up-regulation of the proapoptotic protein Bim. The inhibition of CDK2 desensitized Rbl2-depleted cardiomyocytes to DOX. In wild-type cardiomyocytes, DOX exposure induced Rbl2 expression in a FOXO1-dependent manner. Importantly, the rs17800727 G allele of the human RBL2 gene was associated with reduced anthracycline cardiotoxicity in childhood cancer survivors. Conclusions: Rbl2 is an endogenous CDK2 inhibitor in the heart and represses FOXO1-mediated proapoptotic gene expression. The loss of Rbl2 increases sensitivity to DOX-induced cardiotoxicity. Our findings suggest that RBL2 could be used as a biomarker to predict the risk of cardiotoxicity before the initiation of anthracycline-based chemotherapy.

Loss of Rbl2 (Retinoblastoma-Like 2) Exacerbates Myocardial Ischemia/Reperfusion Injury.

Background The postmitotic state of adult cardiomyocytes, maintained by the cell cycle repressor Rbl2 (retinoblastoma-like 2), is associated with considerable resistance to apoptosis. However, whether Rbl2 regulates cardiomyocyte apoptosis remains unknown. Methods and Results Here, we show that ablation of Rbl2 increased cardiomyocyte apoptosis following acute myocardial ischemia/reperfusion injury, leading to diminished cardiac function and exaggerated ventricular remodeling in the long term. Mechanistically, ischemia/reperfusion induced expression of the proapoptotic protein BCL2 interacting protein 3 (Bnip3), which was augmented by deletion of Rbl2. Because the Bnip3 promoter contains an adenoviral early region 2 binding factor (E2F)-binding site, we further showed that loss of Rbl2 upregulated the transcriptional activator E2F1 but downregulated the transcriptional repressor E2F4. In cultured cardiomyocytes, treatment with H2O2 markedly increased the levels of E2F1 and Bnip3, resulting in mitochondrial depolarization and apoptosis. Depletion of Rbl2 significantly augmented H2O2-induced mitochondrial damage and apoptosis in vitro. Conclusions Rbl2 deficiency enhanced E2F1-mediated Bnip3 expression, resulting in aggravated cardiomyocyte apoptosis and ischemia/reperfusion injury. Our results uncover a novel antiapoptotic role for Rbl2 in cardiomyocytes, suggesting that the cell cycle machinery may directly regulate apoptosis in postmitotic cardiomyocytes. These findings may be exploited to develop new strategies to limit ischemia/reperfusion injury in the treatment of acute myocardial infarction.

Physiological and pathological roles of protein kinase A in the heart.

Protein kinase A (PKA) is a central regulator of cardiac performance and morphology. Myocardial PKA activation is induced by a variety of hormones, neurotransmitters, and stress signals, most notably catecholamines secreted by the sympathetic nervous system. Catecholamines bind ß-adrenergic receptors to stimulate cAMP-dependent PKA activation in cardiomyocytes. Elevated PKA activity enhances Ca2+ cycling and increases cardiac muscle contractility. Dynamic control of PKA is essential for cardiac homeostasis, as dysregulation of PKA signalling is associated with a broad range of heart diseases. Specifically, abnormal PKA activation or inactivation contributes to the pathogenesis of myocardial ischaemia, hypertrophy, heart failure, as well as diabetic, takotsubo, or anthracycline cardiomyopathies. PKA may also determine sex-dependent differences in contractile function and heart disease predisposition. Here, we describe the recent advances regarding the roles of PKA in cardiac physiology and pathology, highlighting previous study limitations and future research directions. Moreover, we discuss the therapeutic strategies and molecular mechanisms associated with cardiac PKA biology. In summary, PKA could serve as a promising drug target for cardioprotection. Depending on disease types and mechanisms, therapeutic intervention may require either inhibition or activation of PKA. Therefore, specific PKA inhibitors or activators may represent valuable drug candidates for the treatment of heart diseases.

Loss of PKA regulatory subunit 1α aggravates cardiomyocyte necrosis and myocardial ischemia/reperfusion injury.

Reperfusion therapy, the standard treatment for acute myocardial infarction, can trigger necrotic death of cardiomyocytes and provoke ischemia/reperfusion (I/R) injury. However, signaling pathways that regulate cardiomyocyte necrosis remain largely unknown. Our recent genome-wide RNAi screen has identified a potential necrosis suppressor gene PRKAR1A, which encodes PKA regulatory subunit 1α (R1α). R1α is primarily known for regulating PKA activity by sequestering PKA catalytic subunits in the absence of cAMP. Here, we showed that depletion of R1α augmented cardiomyocyte necrosis in vitro and in vivo, resulting in exaggerated myocardial I/R injury and contractile dysfunction. Mechanistically, R1α loss downregulated the Nrf2 antioxidant transcription factor and aggravated oxidative stress following I/R. Degradation of the endogenous Nrf2 inhibitor Keap1 through p62-dependent selective autophagy was blocked by R1α depletion. Phosphorylation of p62 at Ser349 by mammalian target of rapamycin complex 1 (mTORC1), a critical step in p62-Keap1 interaction, was induced by I/R, but diminished by R1α loss. Activation of PKA by forskolin or isoproterenol almost completely abolished hydrogen-peroxide-induced p62 phosphorylation. In conclusion, R1α loss induces unrestrained PKA activation and impairs the mTORC1-p62-Keap1-Nrf2 antioxidant defense system, leading to aggravated oxidative stress, necrosis, and myocardial I/R injury. Our findings uncover a novel role of PKA in oxidative stress and necrosis, which may be exploited to develop new cardioprotective therapies.

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Atmospheric haze pollution is a popular environmental issue in recent years. The aerosols reduce solar radiation reaching land surface, with consequences on the growth of crops. In order to examine the effects of low solar radiation intensity on the physiological characteristics and mineral nutrition of grain crops, the random designed field experiment of rice cultivar 'Nanjing 5055' planted under different shading degrees (CK, natural sunlight control; Y1 and Y2 were treatments with shading rates of 19% and 45%, respectively) were conducted. The response of chlorophyll content (SPAD), leaf area index (LAI), net photosynthetic rate of leaves, grain yields and secondary/micro element contents (Ca, Mg, Fe, Zn, Mn, Cu) in rice were measured during key growth stages (jointing, heading, and grain filling stages). Results showed that, shading treatments inhibited the synthesis of photosynthetic products and reduced the LAI during the whole growing period, but at the early stage it did not affect the chlorophyll content, which was significantly increased at the late growth stage. Compared with CK, the 1000-grain weight of rice was decreased by 14.4% and 18.4%, and seed setting rate was decreased by 4.3% and 12.9%, which resulted in rice yield reduction. With the increases of shading rates, rice yield was decreased by 58.5% and 66.4%, respectively. The nutrient concentrations, especially for the micro-elements, in brown rice and glume were increased. Shading had a negative effect on rice growth, which would eventually reduce the crop production. The higher contents of heavy metals such as Cu and Mn would be a pollution risk for human health. Therefore, the impacts of weakened solar radiation on quantity and quality of crops need comprehensive evaluation.

Novel Mechanisms of Anthracycline-Induced Cardiovascular Toxicity: A Focus on Thrombosis, Cardiac Atrophy, and Programmed Cell Death.

Anthracycline antineoplastic agents such as doxorubicin are widely used and highly effective component of adjuvant chemotherapy for breast cancer and curative regimens for lymphomas, leukemias, and sarcomas. The primary dose-limiting adverse effect of anthracyclines is cardiotoxicity that typically manifests as cardiomyopathy and can progress to the potentially fatal clinical syndrome of heart failure. Decades of pre-clinical research have explicated the complex and multifaceted mechanisms of anthracycline-induced cardiotoxicity. It is well-established that oxidative stress contributes to the pathobiology and recent work has elucidated important central roles for direct mitochondrial injury and iron overload. Here we focus instead on emerging aspects of anthracycline-induced cardiotoxicity that may have received less attention in other recent reviews: thrombosis, myocardial atrophy, and non-apoptotic programmed cell death.

PRKAR1A deficiency impedes hypertrophy and reduces heart size.

Protein kinase A (PKA) activity is pivotal for proper functioning of the human heart, and its dysregulation has been implicated in a variety of cardiac pathologies. PKA regulatory subunit 1α (R1α, encoded by the PRKAR1A gene) is highly expressed in the heart, and controls PKA kinase activity by sequestering PKA catalytic subunits. Patients with PRKAR1A mutations are often diagnosed with Carney complex (CNC) in early adulthood, and may die later in life from cardiac complications such as heart failure. However, it remains unknown whether PRKAR1A deficiency interferes with normal heart development. Here, we showed that left ventricular mass was reduced in young CNC patients with PRKAR1A mutations or deletions. Cardiac-specific heterozygous ablation of PRKAR1A in mice increased cardiac PKA activity, and reduced heart weight and cardiomyocyte size without altering contractile function at 3 months of age. Silencing of PRKAR1A, or stimulation with the PKA activator forskolin completely abolished α1-adrenergic receptor-mediated cardiomyocyte hypertrophy. Mechanistically, depletion of PRKAR1A provoked PKA-dependent inactivating phosphorylation of Drp1 at S637, leading to impaired mitochondrial fission. Pharmacologic inhibition of Drp1 with Mdivi 1 diminished hypertrophic growth of cardiomyocytes. In conclusion, PRKAR1A deficiency suppresses cardiomyocyte hypertrophy and impedes heart growth, likely through inhibiting Drp1-mediated mitochondrial fission. These findings provide a potential novel mechanism for the cardiac manifestations associated with CNC.

Doxorubicin induces cardiomyocyte apoptosis and atrophy through cyclin-dependent kinase 2-mediated activation of forkhead box O1.

Recent clinical investigations indicate that anthracycline-based chemotherapies induce early decline in heart mass in cancer patients. Heart mass decline may be caused by a decrease in cardiac cell number because of increased cell death or by a reduction in cell size because of atrophy. We previously reported that an anthracycline, doxorubicin (DOX), induces apoptotic death of cardiomyocytes by activating cyclin-dependent kinase 2 (CDK2). However, the signaling pathway downstream of CDK2 remains to be characterized, and it is also unclear whether the same pathway mediates cardiac atrophy. Here we demonstrate that DOX exposure induces CDK2-dependent phosphorylation of the transcription factor forkhead box O1 (FOXO1) at Ser-249, leading to transcription of its proapoptotic target gene, Bcl-2-interacting mediator of cell death (Bim). In cultured cardiomyocytes, treatment with the FOXO1 inhibitor AS1842856 or transfection with FOXO1-specific siRNAs protected against DOX-induced apoptosis and mitochondrial damage. Oral administration of AS1842856 in mice abrogated apoptosis and prevented DOX-induced cardiac dysfunction. Intriguingly, pharmacological FOXO1 inhibition also attenuated DOX-induced cardiac atrophy, likely because of repression of muscle RING finger 1 (MuRF1), a proatrophic FOXO1 target gene. In conclusion, DOX exposure induces CDK2-dependent FOXO1 activation, resulting in cardiomyocyte apoptosis and atrophy. Our results identify FOXO1 as a promising drug target for managing DOX-induced cardiotoxicity. We propose that FOXO1 inhibitors may have potential as cardioprotective therapeutic agents during cancer chemotherapy.

The circadian clock protects against ionizing radiation-induced cardiotoxicity.

Radiation therapy (RT) is commonly used to treat solid tumors of the breast, lung, and esophagus; however, the heart is an unintentional target of ionizing radiation (IR). IR exposure to the heart results in chronic toxicities including heart failure. We hypothesize that the circadian system plays regulatory roles in minimizing the IR-induced cardiotoxicity. We treated mice in control (Day Shift), environmentally disrupted (Rotating Shift), and genetically disrupted (Per 1/2 mutant) circadian conditions with 18 Gy of IR to the heart. Compared to control mice, circadian clock disruption significantly exacerbated post-IR systolic dysfunction (by ultrasound echocardiography) and increased fibrosis in mice. At the cellular level, Bmal1 protein bound to Atm, Brca1, and Brca2 promoter regions and its expression level was inversely correlated with the DNA damage levels based on the state of the clock. Further studies with circadian synchronized cardiomyocytes revealed that Bmal1 depletion increased the IR-induced DNA damage and apoptosis. Collectively, these findings suggest that the circadian clock protects from IR-induced toxicity and potentially impacts RT treatment outcome in cancer patients through IR-induced DNA damage responses.

Cell Cycle Proteins as Key Regulators of Postmitotic Cell Death.

Cell cycle progression in dividing cells, characterized by faithful replication of the genomic materials and duplication of the original cell, is fundamental for growth and reproduction of all mammalian organisms. Functional maturation of postmitotic cells, however, requires cell cycle exit and terminal differentiation. In mature postmitotic cells, many cell cycle proteins remain to be expressed, or can be induced and reactivated in pathological conditions such as traumatic injury and degenerative diseases. Interestingly, elevated levels of cell cycle proteins in postmitotic cells often do not induce proliferation, but result in aberrant cell cycle reentry and cell death. At present, the cell cycle machinery is known predominantly for regulating cell cycle progression and cell proliferation, albeit accumulating evidence indicates that cell cycle proteins may also control cell death, especially in postmitotic tissues. Herein, we provide a brief summary of these findings and hope to highlight the connection between cell cycle reentry and postmitotic cell death. In addition, we also outline the signaling pathways that have been identified in cell cycle-related cell death. Advanced understanding of the molecular mechanisms underlying cell cycle-related death is of paramount importance because this knowledge can be applied to develop protective strategies against pathologies in postmitotic tissues. Moreover, a full-scope understanding of the cell cycle machinery will allow fine tuning to favor cell proliferation over cell death, thereby potentially promoting tissue regeneration.

Genome-Wide RNAi Screen Identifies Regulators of Cardiomyocyte Necrosis.

Regulation of cellular death is central to nearly all physiological routines and is dysregulated in virtually all diseases. Cell death occurs by two major processes, necrosis which culminates in a pervasive inflammatory response and apoptosis which is largely immunologically inert. As necrosis has long been considered an accidental, unregulated form of cellular death that occurred in response to a harsh environmental stimulus, it was largely ignored as a clinical target. However, recent elegant studies suggest that certain forms of necrosis can be reprogrammed. However, scant little is known about the molecules and pathways that orchestrate calcium-overload-induced necrosis, a main mediator of ischemia/reperfusion (IR)-induced cardiomyocyte cell death. To rectify this critical gap in our knowledge, we performed a novel genome-wide siRNA screen to identify modulators of calcium-induced necrosis in human muscle cells. Our screen identified multiple molecular circuitries that either enhance or inhibit this process, including lysosomal calcium channel TPCN1, mitophagy mediatorTOMM7, Ran-binding protein RanBP9, Histone deacetylase HDAC2, chemokine CCL11, and the Arp2/3 complex regulator glia maturation factor-γ (GMFG). Notably, a number of druggable enzymes were identified, including the proteasome ß5 subunit (encoded by PSMB5 gene), which controls the proteasomal chymotrypsin-like peptidase activity. Such findings open up the possibility for the discovery of pharmacological interventions that could provide therapeutic benefits to patients affected by myriad disorders characterized by excessive (or too little) necrotic cell loss, including but not limited to IR injury in the heart and kidney, chronic neurodegenerative disorders, muscular dystrophies, sepsis, and cancers.

Inhibition of cyclin-dependent kinase 2 protects against doxorubicin-induced cardiomyocyte apoptosis and cardiomyopathy.

With the rapid increase in cancer survival because of improved diagnosis and therapy in the past decades, cancer treatment-related cardiotoxicity is becoming an urgent healthcare concern. The anthracycline doxorubicin (DOX), one of the most effective chemotherapeutic agents to date, causes cardiomyopathy by inducing cardiomyocyte apoptosis. We demonstrated previously that overexpression of the cyclin-dependent kinase (CDK) inhibitor p21 promotes resistance against DOX-induced cardiomyocyte apoptosis. Here we show that DOX exposure provokes cardiac CDK2 activation and cardiomyocyte cell cycle S phase reentry, resulting in enhanced cellular sensitivity to DOX. Genetic or pharmacological inhibition of CDK2 markedly suppressed DOX-induced cardiomyocyte apoptosis. Conversely, CDK2 overexpression augmented DOX-induced apoptosis. We also found that DOX-induced CDK2 activation in the mouse heart is associated with up-regulation of the pro-apoptotic BCL2 family member BCL2-like 11 (Bim), a BH3-only protein essential for triggering Bax/Bak-dependent mitochondrial outer membrane permeabilization. Further experiments revealed that DOX induces cardiomyocyte apoptosis through CDK2-dependent expression of Bim. Inhibition of CDK2 with roscovitine robustly repressed DOX-induced mitochondrial depolarization. In a cardiotoxicity model of chronic DOX exposure (5 mg/kg weekly for 4 weeks), roscovitine administration significantly attenuated DOX-induced contractile dysfunction and ventricular remodeling. These findings identify CDK2 as a key determinant of DOX-induced cardiotoxicity. CDK2 activation is necessary for DOX-induced Bim expression and mitochondrial damage. Our results suggest that pharmacological inhibition of CDK2 may be a cardioprotective strategy for preventing anthracycline-induced heart damage.

Focal Adhesion Kinase-mediated Phosphorylation of Beclin1 Protein Suppresses Cardiomyocyte Autophagy and Initiates Hypertrophic Growth.

Autophagy is an evolutionarily conserved intracellular degradation/recycling system that is essential for cellular homeostasis but is dysregulated in a number of diseases, including myocardial hypertrophy. Although it is clear that limiting or accelerating autophagic flux can result in pathological cardiac remodeling, the physiological signaling pathways that fine-tune cardiac autophagy are poorly understood. Herein, we demonstrated that stimulation of cardiomyocytes with phenylephrine (PE), a well known hypertrophic agonist, suppresses autophagy and that activation of focal adhesion kinase (FAK) is necessary for PE-stimulated autophagy suppression and subsequent initiation of hypertrophic growth. Mechanistically, we showed that FAK phosphorylates Beclin1, a core autophagy protein, on Tyr-233 and that this post-translational modification limits Beclin1 association with Atg14L and reduces Beclin1-dependent autophagosome formation. Remarkably, although ectopic expression of wild-type Beclin1 promoted cardiomyocyte atrophy, expression of a Y233E phosphomimetic variant of Beclin1 failed to affect cardiomyocyte size. Moreover, genetic depletion of Beclin1 attenuated PE-mediated/FAK-dependent initiation of myocyte hypertrophy in vivo Collectively, these findings identify FAK as a novel negative regulator of Beclin1-mediated autophagy and indicate that this pathway can facilitate the promotion of compensatory hypertrophic growth. This novel mechanism to limit Beclin1 activity has important implications for treating a variety of pathologies associated with altered autophagic flux.

Signaling Pathways in Cardiac Myocyte Apoptosis.

Cardiovascular diseases, the number 1 cause of death worldwide, are frequently associated with apoptotic death of cardiac myocytes. Since cardiomyocyte apoptosis is a highly regulated process, pharmacological intervention of apoptosis pathways may represent a promising therapeutic strategy for a number of cardiovascular diseases and disorders including myocardial infarction, ischemia/reperfusion injury, chemotherapy cardiotoxicity, and end-stage heart failure. Despite rapid growth of our knowledge in apoptosis signaling pathways, a clinically applicable treatment targeting this cellular process is currently unavailable. To help identify potential innovative directions for future research, it is necessary to have a full understanding of the apoptotic pathways currently known to be functional in cardiac myocytes. Here, we summarize recent progress in the regulation of cardiomyocyte apoptosis by multiple signaling molecules and pathways, with a focus on the involvement of these pathways in the pathogenesis of heart disease. In addition, we provide an update regarding bench to bedside translation of this knowledge and discuss unanswered questions that need further investigation.

GRAF1 deficiency blunts sarcolemmal injury repair and exacerbates cardiac and skeletal muscle pathology in dystrophin-deficient mice.

BACKGROUND: The plasma membranes of striated muscle cells are particularly susceptible to rupture as they endure significant mechanical stress and strain during muscle contraction, and studies have shown that defects in membrane repair can contribute to the progression of muscular dystrophy. The synaptotagmin-related protein, dysferlin, has been implicated in mediating rapid membrane repair through its ability to direct intracellular vesicles to sites of membrane injury. However, further work is required to identify the precise molecular mechanisms that govern dysferlin targeting and membrane repair. We previously showed that the bin-amphiphysin-Rvs (BAR)-pleckstrin homology (PH) domain containing Rho-GAP GTPase regulator associated with focal adhesion kinase-1 (GRAF1) was dynamically recruited to the tips of fusing myoblasts wherein it promoted membrane merging by facilitating ferlin-dependent capturing of intracellular vesicles. Because acute membrane repair responses involve similar vesicle trafficking complexes/events and because our prior studies in GRAF1-deficient tadpoles revealed a putative role for GRAF1 in maintaining muscle membrane integrity, we postulated that GRAF1 might also play an important role in facilitating dysferlin-dependent plasma membrane repair. METHODS: We used an in vitro laser-injury model to test whether GRAF1 was necessary for efficient muscle membrane repair. We also generated dystrophin/GRAF1 doubledeficient mice by breeding mdx mice with GRAF1 hypomorphic mice. Evans blue dye uptake and extensive morphometric analyses were used to assess sarcolemmal integrity and related pathologies in cardiac and skeletal muscles isolated from these mice. RESULTS: Herein, we show that GRAF1 is dynamically recruited to damaged skeletal and cardiac muscle plasma membranes and that GRAF1-depleted muscle cells have reduced membrane healing abilities. Moreover, we show that dystrophin depletion exacerbated muscle damage in GRAF1-deficient mice and that mice with dystrophin/GRAF1 double deficiency phenocopied the severe muscle pathologies observed in dystrophin/dysferlin-double null mice. Consistent with a model that GRAF1 facilitates dysferlin-dependent membrane patching, we found that GRAF1 associates with and regulates plasma membrane deposition of dysferlin. CONCLUSIONS: Overall, our work indicates that GRAF1 facilitates dysferlin-dependent membrane repair following acute muscle injury. These findings indicate that GRAF1 might play a role in the phenotypic variation and pathological progression of cardiac and skeletal muscle degeneration in muscular dystrophy patients.

Focal adhesion kinase antagonizes doxorubicin cardiotoxicity via p21(Cip1.).

Clinical application of potent anthracycline anticancer drugs, especially doxorubicin (DOX), is limited by a toxic cardiac side effect that is not fully understood and preventive strategies are yet to be established. Studies in genetically modified mice have demonstrated that focal adhesion kinase (FAK) plays a key role in regulating adaptive responses of the adult myocardium to pathological stimuli through activation of intracellular signaling cascades that facilitate cardiomyocyte growth and survival. The objective of this study was to determine if targeted myocardial FAK activation could protect the heart from DOX-induced de-compensation and to characterize the underlying mechanisms. To this end, mice with myocyte-restricted FAK knock-out (MFKO) or myocyte-specific expression of an active FAK variant (termed SuperFAK) were subjected to DOX treatment. FAK depletion enhanced susceptibility to DOX-induced myocyte apoptosis and cardiac dysfunction, while elevated FAK activity provided remarkable cardioprotection. Our mec6hanistic studies reveal a heretofore unappreciated role for the protective cyclin-dependent kinase inhibitor p21 in the repression of the pro-apoptotic BH3-only protein Bim and the maintenance of mitochondrial integrity and myocyte survival. DOX treatment induced proteasomal degradation of p21, which exacerbated mitochondrial dysfunction and cardiomyocyte apoptosis. FAK was both necessary and sufficient for maintaining p21 levels following DOX treatment and depletion of p21 compromised FAK-dependent protection from DOX. These findings identify p21 as a key determinant of DOX resistance downstream of FAK in cardiomyocytes and indicate that cardiac-restricted enhancement of the FAK/p21 signaling axis might be an effective strategy to preserve myocardial function in patients receiving anthracycline chemotherapy.

Transplantation of parthenogenetic embryonic stem cells ameliorates cardiac dysfunction and remodelling after myocardial infarction.

AIMS: Parthenogenetic embryonic stem cells (pESCs) derived from artificially activated oocytes without fertilization presumably raise minimal ethical concerns and may serve as attractive candidates for regenerative medicine. Here we investigated whether pESCs could repair myocardial infarction (MI), in comparison to embryonic stem cells (ESCs). METHODS AND RESULTS: A total of 89 mice that survived coronary artery ligation randomly received an intramyocardial injection of undifferentiated pESCs, ESCs, or saline. Sham-operated mice (n = 21) that received no treatment served as control animals. After 7 days, transplantation of pESCs increased expression of pro-angiogenic factors and reduced leucocyte infiltration. By 14 and 30 days post-MI, similar to treatment with ESCs, treatment with pESCs efficiently prevented cardiac remodelling and enhanced angiogenesis, in contrast to saline-treated hearts. Improved heart contractile function was also notable 30 days following transplantation of pESCs. Immunofluorescence staining revealed that tissues regenerated from pESCs in the infarcted myocardium were positive for markers of cardiomyocytes, endothelial cells, and smooth muscle cells. Unlike ESC-treated mice, which exhibited a high incidence of teratoma (6 of 34), the pESC-treated mice showed no teratomas (0 of 30) 30 days following transplantation. CONCLUSION: Transplantation of pESCs could attenuate cardiac dysfunction and adverse ventricular remodelling post-MI, suggesting that pESCs may provide promising therapeutic sources for MI in females.

Targeted focal adhesion kinase activation in cardiomyocytes protects the heart from ischemia/reperfusion injury.

OBJECTIVE: We previously reported that cardiac-restricted deletion of focal adhesion kinase (FAK) exacerbated myocyte death following ischemia/reperfusion (I/R). Here, we interrogated whether targeted elevation of myocardial FAK activity could protect the heart from I/R injury. METHODS AND RESULTS: Transgenic mice were generated with myocyte-specific expression of a FAK variant (termed SuperFAK) that conferred elevated allosteric activation. FAK activity in unstressed transgenic hearts was modestly elevated, but this had no discernable effect on anabolic heart growth or cardiac function. Importantly, SuperFAK hearts exhibited a dramatic increase in FAK activity and a reduction in myocyte apoptosis and infarct size 24 to 72 hours following I/R. Moreover, serial echocardiography revealed that the transgenic mice were protected from cardiac decompensation for up to 8 weeks following surgery. Mechanistic studies revealed that elevated FAK activity protected cardiomyocytes from I/R-induced apoptosis by enhancing nuclear factor-κB (NF-κB)-dependent survival signaling during the early period of reperfusion (30 and 60 minutes). Moreover, adenoviral-mediated expression of SuperFAK in cultured cardiomyocytes attenuated H(2)O(2) or hypoxia/reoxygenation-induced apoptosis, whereas blockade of the NF-κB pathway using a pharmacological inhibitor or small interfering RNAs completely abolished the beneficial effect of SuperFAK. CONCLUSIONS: Enhancing cardiac FAK activity attenuates I/R-induced myocyte apoptosis through activation of the prosurvival NF-κB pathway and may represent a novel therapeutic strategy for ischemic heart diseases.