Kate L. Weeks

PI3K(p110 alpha) Protects Against Myocardial Infarction-Induced Heart Failure Identification of PI3K-Regulated miRNA and mRNA

Objective—Myocardial infarction (MI) is a serious complication of atherosclerosis associated with increasing mortality attributable to heart failure. Activation of phosphoinositide 3-kinase [PI3K(p110&agr;)] is considered a new strategy for the treatment of heart failure. However, whether PI3K(p110&agr;) provides protection in a setting of MI is unknown, and PI3K(p110&agr;) is difficult to target because it has multiple actions in numerous cell types. The goal of this study was to assess whether PI3K(p110&agr;) is beneficial in a setting of MI and, if so, to identify cardiac-selective microRNA and mRNA that mediate the protective properties of PI3K(p110&agr;). Methods and Results—Cardiomyocyte-specific transgenic mice with increased or decreased PI3K(p110&agr;) activity (caPI3K-Tg and dnPI3K-Tg, respectively) were subjected to MI for 8 weeks. The caPI3K-Tg subjected to MI had better cardiac function than nontransgenic mice, whereas dnPI3K-Tg had worse function. Using microarray analysis, we identified PI3K-regulated miRNA and mRNA that were correlated with cardiac function, including growth factor receptor-bound 14. Growth factor receptor-bound 14 is highly expressed in the heart and positively correlated with PI3K(p110&agr;) activity and cardiac function. Mice deficient in growth factor receptor-bound 14 have cardiac dysfunction. Conclusion—Activation of PI3K(p110&agr;) protects the heart against MI-induced heart failure. Cardiac-selective targets that mediate the protective effects of PI3K(p110&agr;) represent new drug targets for heart failure.

Follistatin-mediated skeletal muscle hypertrophy is regulated by Smad3 and mTOR independently of myostatin

Smad3/Akt/mTOR/S6K/S6RP signaling plays a critical role in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.

The Athlete's Heart vs. the Failing Heart: Can Signaling Explain the Two Distinct Outcomes?

Cardiac remodeling is typically associated with disease and can lead to heart failure. In contrast, remodeling that occurs in the athletes heart is considered an adaptive physiological response. This review provides an overview of signaling mechanisms responsible for inducing left ventricular hypertrophy in the athletes heart and in settings of pathological hypertrophy and heart failure.

Phosphoinositide 3-Kinase p110α Is a Master Regulator of Exercise-Induced Cardioprotection and PI3K Gene Therapy Rescues Cardiac Dysfunction

Background—Numerous molecular and biochemical changes have been linked with the cardioprotective effects of exercise, including increases in antioxidant enzymes, heat shock proteins, and regulators of cardiac myocyte proliferation. However, a master regulator of exercise-induced protection has yet to be identified. Here, we assess whether phosphoinositide 3-kinase (PI3K) p110&agr; is essential for mediating exercise-induced cardioprotection, and if so, whether its activation independent of exercise can restore function of the failing heart. Methods and Results—Cardiac-specific transgenic (Tg) mice with elevated or reduced PI3K(p110&agr;) activity (constitutively active PI3K [caPI3K] and dominant negative PI3K, respectively) and non-Tg controls were subjected to 4 weeks of exercise training followed by 1 week of pressure overload (aortic-banding) to induce pathological remodeling. Aortic-banding in untrained non-Tg controls led to pathological cardiac hypertrophy, depressed systolic function, and lung congestion. This phenotype was attenuated in non-Tg controls that had undergone exercise before aortic-banding. Banded caPI3K mice were protected from pathological remodeling independent of exercise status, whereas exercise provided no protection in banded dominant negative PI3K mice, suggesting that PI3K is necessary for exercise-induced cardioprotection. Tg overexpression of heat shock protein 70 could not rescue the phenotype of banded dominant negative PI3K mice, and deletion of heat shock protein 70 from banded caPI3K mice had no effect. Next, we used a gene therapy approach (recombinant adeno-associated viral vector 6) to deliver caPI3K expression cassettes to hearts of mice with established cardiac dysfunction caused by aortic-banding. Mice treated with recombinant adeno-associated viral 6-caPI3K vectors had improved heart function after 10 weeks. Conclusions—PI3K(p110&agr;) is essential for exercise-induced cardioprotection and delivery of caPI3K vector can improve function of the failing heart.

Roles and post-translational regulation of cardiac class IIa histone deacetylase isoforms

Cardiomyocyte hypertrophy is an integral component of pathological cardiac remodelling in response to mechanical and chemical stresses in settings such as chronic hypertension or myocardial infarction. For hypertrophy to ensue, the pertinent mechanical and chemical signals need to be transmitted from membrane sensors (such as receptors for neurohormonal mediators) to the cardiomyocyte nucleus, leading to altered transcription of the genes that regulate cell growth. In recent years, nuclear histone deacetylases (HDACs) have attracted considerable attention as signal‐responsive, distal regulators of the transcriptional reprogramming that in turn precipitates cardiomyocyte hypertrophy, with particular focus on the role of members of the class IIa family, such as HDAC4 and HDAC5. These histone deacetylase isoforms appear to repress cardiomyocyte hypertrophy through mechanisms that involve protein interactions in the cardiomyocyte nucleus, particularly with pro‐hypertrophic transcription factors, rather than via histone deacetylation. In contrast, evidence indicates that class I HDACs promote cardiomyocyte hypertrophy through mechanisms that are dependent on their enzymatic activity and thus sensitive to pharmacological HDAC inhibitors. Although considerable progress has been made in understanding the roles of post‐translational modifications (PTMs) such as phosphorylation, oxidation and proteolytic cleavage in regulating class IIa HDAC localisation and function, more work is required to explore the contributions of other PTMs, such as ubiquitination and sumoylation, as well as potential cross‐regulatory interactions between distinct PTMs and between class IIa and class I HDAC isoforms.

Phosphoinositide 3-kinase (p110α) gene delivery limits diabetes-induced cardiac NADPH oxidase and cardiomyopathy in a mouse model with established diastolic dysfunction

Phosphoinositide 3-kinase [PI3K (p110α)] is able to negatively regulate the diabetes-induced increase in NADPH oxidase in the heart. Patients affected by diabetes exhibit significant cardiovascular morbidity and mortality, at least in part due to a cardiomyopathy characterized by oxidative stress and left ventricular (LV) dysfunction. Thus, PI3K (p110α) may represent a novel approach to protect the heart from diabetes-induced cardiac oxidative stress and dysfunction. In the present study, we investigated the therapeutic potential of a delayed intervention with cardiac-targeted PI3K gene therapy, administered to mice with established diabetes-induced LV diastolic dysfunction. Diabetes was induced in 6-week-old male mice by streptozotocin (STZ). After 8 weeks of untreated diabetes, LV diastolic dysfunction was confirmed by a reduction in echocardiography-derived transmitral E/A ratio. Diabetic and non-diabetic mice were randomly allocated to receive either recombinant adeno-associated viral vector-6 carrying a constitutively-active PI3K construct (recombinant adeno-associated-virus 6-constitutively active PI3K (p110α) (caPI3K) (rAAV6-caPI3K), single i.v. injection, 2 × 1011 vector genomes) or null vector, and were followed for a further 6 or 8 weeks. At study endpoint, diabetes-induced LV dysfunction was significantly attenuated by a single administration of rAAV6-caPI3K, administered 8 weeks after the induction of diabetes. Diabetes-induced impairments in each of LV NADPH oxidase, endoplasmic reticulum (ER) stress, apoptosis, cardiac fibrosis and cardiomyocyte hypertrophy, in addition to LV systolic dysfunction, were attenuated by delayed intervention with rAAV6-caPI3K. Hence, our demonstration that cardiac-targeted PI3K (p110α) gene therapy limits diabetes-induced up-regulation of NADPH oxidase and cardiac remodelling suggests new insights into promising approaches for the treatment of diabetic cardiomyopathy, at a clinically relevant time point (after diastolic dysfunction is manifested).

Gene delivery of medium chain acyl-coenzyme A dehydrogenase induces physiological cardiac hypertrophy and protects against pathological remodelling

We previously showed that medium chain acyl-coenzyme A dehydrogenase (MCAD, key regulator of fatty acid oxidation) is positively modulated in the heart by the cardioprotective kinase, phosphoinositide 3-kinase (PI3K(p110α)). Disturbances in cardiac metabolism are a feature of heart failure (HF) patients and targeting metabolic defects is considered a potential therapeutic approach. The specific role of MCAD in the adult heart is unknown. To examine the role of MCAD in the heart and to assess the therapeutic potential of increasing MCAD in the failing heart, we developed a gene therapy tool using recombinant adeno-associated viral vectors (rAAV) encoding MCAD. We hypothesised that increasing MCAD expression may recapitulate the cardioprotective properties of PI3K(p110α). rAAV6:MCAD or rAAV6:control was delivered to healthy adult mice and to mice with pre-existing pathological hypertrophy and cardiac dysfunction due to transverse aortic constriction (TAC). In healthy mice, rAAV6:MCAD induced physiological hypertrophy (increase in heart size, normal systolic function and increased capillary density). In response to TAC (~15 weeks), heart weight/tibia length increased by ~60% in control mice and ~45% in rAAV6:MCAD mice compared with sham. This was associated with an increase in cardiomyocyte cross-sectional area in both TAC groups which was similar. However, hypertrophy in TAC rAAV6:MCAD mice was associated with less fibrosis, a trend for increased capillary density and a more favourable molecular profile compared with TAC rAAV6:control mice. In summary, MCAD induced physiological cardiac hypertrophy in healthy adult mice and attenuated features of pathological remodelling in a cardiac disease model.

β‐Adrenergic Stimulation Induces Histone Deacetylase 5 (HDAC5) Nuclear Accumulation in Cardiomyocytes by B55α‐PP2A‐Mediated Dephosphorylation

Background Class IIa histone deacetylase (HDAC) isoforms such as HDAC5 are critical signal‐responsive repressors of maladaptive cardiomyocyte hypertrophy, through nuclear interactions with transcription factors including myocyte enhancer factor‐2. β‐Adrenoceptor (β‐AR) stimulation, a signal of fundamental importance in regulating cardiac function, has been proposed to induce both phosphorylation‐independent nuclear export and phosphorylation‐dependent nuclear accumulation of cardiomyocyte HDAC5. The relative importance of phosphorylation at Ser259/Ser498 versus Ser279 in HDAC5 regulation is also controversial. We aimed to determine the impact of β‐AR stimulation on the phosphorylation, localization, and function of cardiomyocyte HDAC5 and delineate underlying molecular mechanisms. Methods and Results A novel 3‐dimensional confocal microscopy method that objectively quantifies the whole‐cell nuclear/cytoplasmic distribution of green fluorescent protein tagged HDAC5 revealed the β‐AR agonist isoproterenol to induce β1‐AR‐mediated and protein kinase A‐dependent HDAC5 nuclear accumulation in adult rat cardiomyocytes, which was accompanied by dephosphorylation at Ser259/279/498. Mutation of Ser259/Ser498 to Ala promoted HDAC5 nuclear accumulation and myocyte enhancer factor‐2 inhibition, whereas Ser279 ablation had no such effect and did not block isoproterenol‐induced nuclear accumulation. Inhibition of the Ser/Thr phosphatase PP2A blocked isoproterenol‐induced HDAC5 dephosphorylation. Co‐immunoprecipitation revealed a specific interaction of HDAC5 with the PP2A targeting subunit B55α, as well as catalytic and scaffolding subunits, which increased >3‐fold with isoproterenol. Knockdown of B55α in neonatal cardiomyocytes attenuated isoproterenol‐induced HDAC5 dephosphorylation. Conclusions β‐AR stimulation induces HDAC5 nuclear accumulation in cardiomyocytes by a mechanism that is protein kinase A‐dependent but requires B55α‐PP2A‐mediated dephosphorylation of Ser259/Ser498 rather than protein kinase A‐mediated phosphorylation of Ser279.

Understanding Key Mechanisms of Exercise-Induced Cardiac Protection to Mitigate Disease: Current Knowledge and Emerging Concepts

The benefits of exercise on the heart are well recognized, and clinical studies have demonstrated that exercise is an intervention that can improve cardiac function in heart failure patients. This has led to significant research into understanding the key mechanisms responsible for exercise-induced cardiac protection. Here, we summarize molecular mechanisms that regulate exercise-induced cardiac myocyte growth and proliferation. We discuss in detail the effects of exercise on other cardiac cells, organelles, and systems that have received less or little attention and require further investigation. This includes cardiac excitation and contraction, mitochondrial adaptations, cellular stress responses to promote survival (heat shock response, ubiquitin-proteasome system, autophagy-lysosomal system, endoplasmic reticulum unfolded protein response, DNA damage response), extracellular matrix, inflammatory response, and organ-to-organ crosstalk. We summarize therapeutic strategies targeting known regulators of exercise-induced protection and the challenges translating findings from bench to bedside. We conclude that technological advancements that allow for in-depth profiling of the genome, transcriptome, proteome and metabolome, combined with animal and human studies, provide new opportunities for comprehensively defining the signaling and regulatory aspects of cell/organelle functions that underpin the protective properties of exercise. This is likely to lead to the identification of novel biomarkers and therapeutic targets for heart disease.

Nucleocytoplasmic shuttling: The ins and outs of quantitative imaging

Nucleocytoplasmic protein shuttling is integral to the transmission of signals between the nucleus and the cytoplasm. The nuclear/cytoplasmic distribution of proteins of interest can be determined via fluorescence microscopy, following labelling of the target protein with fluorophore‐conjugated antibodies (immunofluorescence) or by tagging the target protein with an autofluorescent protein, such as green fluorescent protein (GFP). The latter enables live cell imaging, a powerful approach that precludes many of the artefacts associated with indirect immunofluorescence in fixed cells. In this review, we discuss important considerations for the design and implementation of fluorescence microscopy experiments to quantify the nuclear/cytoplasmic distribution of a protein of interest. We summarise the pros and cons of detecting endogenous proteins in fixed cells by immunofluorescence and ectopically‐expressed fluorescent fusion proteins in living cells. We discuss the suitability of widefield fluorescence microscopy and of 2D, 3D and 4D imaging by confocal microscopy for different applications, and describe two different methods for quantifying the nuclear/cytoplasmic distribution of a protein of interest from the fluorescent signal. Finally, we discuss the importance of eliminating sources of bias and subjectivity during image acquisition and post‐imaging analyses. This is critical for the accurate and reliable quantification of nucleocytoplasmic shuttling.