Tania Zaglia

The Opa1-Dependent Mitochondrial Cristae Remodeling Pathway Controls Atrophic, Apoptotic, and Ischemic Tissue Damage

Summary Mitochondrial morphological and ultrastructural changes occur during apoptosis and autophagy, but whether they are relevant in vivo for tissue response to damage is unclear. Here we investigate the role of the optic atrophy 1 (OPA1)-dependent cristae remodeling pathway in vivo and provide evidence that it regulates the response of multiple tissues to apoptotic, necrotic, and atrophic stimuli. Genetic inhibition of the cristae remodeling pathway in vivo does not affect development, but protects mice from denervation-induced muscular atrophy, ischemic heart and brain damage, as well as hepatocellular apoptosis. Mechanistically, OPA1-dependent mitochondrial cristae stabilization increases mitochondrial respiratory efficiency and blunts mitochondrial dysfunction, cytochrome c release, and reactive oxygen species production. Our results indicate that the OPA1-dependent cristae remodeling pathway is a fundamental, targetable determinant of tissue damage in vivo.

Mutations in the area composita protein αT-catenin are associated with arrhythmogenic right ventricular cardiomyopathy

AIMS Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a major cause of juvenile sudden death and is characterized by fibro-fatty replacement of the right ventricle. Mutations in several genes encoding desmosomal proteins have been identified in ARVC. We speculated that αT-catenin, encoded by CTNNA3, might also carry mutations in ARVC patients. Alpha-T-catenin binds plakophilins and this binding contributes to the formation of the area composita, which strengthens cell-cell adhesion in contractile cardiomyocytes. METHODS AND RESULTS We used denaturing high-performance liquid chromatography and direct sequencing to screen CTNNA3 in 76 ARVC patients who did not carry any mutations in the desmosomal genes commonly mutated in ARVC. Mutations c.281T > A (p.V94D) and c.2293_2295delTTG (p.del765L) were identified in two probands. They are located in important domains of αT-catenin. Yeast two-hybrid and cell transfection studies showed that the interaction between the p.V94D mutant protein and β-catenin was affected, whereas the p.del765L mutant protein showed a much stronger dimerization potential and formed aggresomes in HEK293T cells. CONCLUSION These findings might point to a causal relationship between CTNNA3 mutations and ARVC. This first report on the involvement of an area composita gene in ARVC shows that the pathogenesis of this disease extends beyond desmosomes. Since the frequency of CTNNA3 mutations in ARVC patients is not rare, systematic screening for this gene should be considered to improve the clinical management of ARVC families.

Atrogin-1 deficiency promotes cardiomyopathy and premature death via impaired autophagy

Cardiomyocyte proteostasis is mediated by the ubiquitin/proteasome system (UPS) and autophagy/lysosome system and is fundamental for cardiac adaptation to both physiologic (e.g., exercise) and pathologic (e.g., pressure overload) stresses. Both the UPS and autophagy/lysosome system exhibit reduced efficiency as a consequence of aging, and dysfunction in these systems is associated with cardiomyopathies. The muscle-specific ubiquitin ligase atrogin-1 targets signaling proteins involved in cardiac hypertrophy for degradation. Here, using atrogin-1 KO mice in combination with in vivo pulsed stable isotope labeling of amino acids in cell culture proteomics and biochemical and cellular analyses, we identified charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex (ESCRT) required for autophagy, as a target of atrogin-1-mediated degradation. Mice lacking atrogin-1 failed to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation, and subsequent cardiomyocyte apoptosis, all of which increased progressively with age. Cellular proteostasis alterations resulted in cardiomyopathy characterized by myocardial remodeling with interstitial fibrosis, with reduced diastolic function and arrhythmias. CHMP2B downregulation in atrogin-1 KO mice restored autophagy and decreased proteotoxicity, thereby preventing cell death. These data indicate that atrogin-1 promotes cardiomyocyte health through mediating the interplay between UPS and autophagy/lysosome system and its alteration promotes development of cardiomyopathies.

MicroRNA-133 Modulates the β1-Adrenergic Receptor Transduction CascadeNovelty and Significance

Rationale: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate &bgr;-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of &bgr;-adrenergic receptors leads to impaired cardiac function, and &bgr;-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. Objective: To determine whether miR-133 affects &bgr;-adrenergic receptor signaling during progression to heart failure. Methods and Results: Based on bioinformatic analysis, &bgr;1-adrenergic receptor (&bgr;1AR) and other components of the &bgr;1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective &bgr;1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic &bgr;1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. Conclusions: miR-133 controls multiple components of the &bgr;1AR transduction cascade and is cardioprotective during heart failure.

Cardiac interstitial cells express GATA4 and control dedifferentiation and cell cycle re-entry of adult cardiomyocytes

Interstitial cells of the adult rat heart were characterized with respect to i) expression of cardiac markers of commitment and differentiation, ii) myogenic potential in vitro and iii) ability to modulate cardiomyocyte differentiation state. We demonstrate for the first time that fibroblasts and a proportion of pericytes in the adult rat heart express the transcription factor GATA4. This appears to be a peculiar property of the heart. Fibroblasts that are also derived from the splanchnopleuric mesoderm, such as those of the gut, or fibroblasts of different embryological origin, such as those of skin and skeletal muscle, lack this property. Of note, a nestin+/GATA4+ putative stem cell population is also detected in the adult heart. GATA4+ cardiac interstitial cells do not display myogenic potential in vitro. However, cardiac fibroblasts, but not skin fibroblasts, stimulate dedifferentiation of adult cardiomyocytes and their re-entry into the cell cycle in vitro, as demonstrated by the high number of cardiomyocytes expressing Ki67, phosphorylated histone H3 (H3P) and incorporating 5-bromodeoxiuridine (BrdU) in the co-cultures. In conclusion, cardiac fibroblasts have peculiar expression of myogenic transcription factors, a property that may have an impact for reprogramming these cells to the myogenic differentiation. In addition, they are able to modulate the behavior of adult cardiomyocytes, a property that may be used to promote dedifferentiation and proliferation of cardiac cells in the damaged myocardium.

MicroRNA-133 Modulates the β1-Adrenergic Receptor Transduction Cascade

Rationale: The sympathetic nervous system plays a fundamental role in the regulation of myocardial function. During chronic pressure overload, overactivation of the sympathetic nervous system induces the release of catecholamines, which activate &bgr;-adrenergic receptors in cardiomyocytes and lead to increased heart rate and cardiac contractility. However, chronic stimulation of &bgr;-adrenergic receptors leads to impaired cardiac function, and &bgr;-blockers are widely used as therapeutic agents for the treatment of cardiac disease. MicroRNA-133 (miR-133) is highly expressed in the myocardium and is involved in controlling cardiac function through regulation of messenger RNA translation/stability. Objective: To determine whether miR-133 affects &bgr;-adrenergic receptor signaling during progression to heart failure. Methods and Results: Based on bioinformatic analysis, &bgr;1-adrenergic receptor (&bgr;1AR) and other components of the &bgr;1AR signal transduction cascade, including adenylate cyclase VI and the catalytic subunit of the cAMP-dependent protein kinase A, were predicted as direct targets of miR-133 and subsequently validated by experimental studies. Consistently, cAMP accumulation and activation of downstream targets were repressed by miR-133 overexpression in both neonatal and adult cardiomyocytes following selective &bgr;1AR stimulation. Furthermore, gain-of-function and loss-of-function studies of miR-133 revealed its role in counteracting the deleterious apoptotic effects caused by chronic &bgr;1AR stimulation. This was confirmed in vivo using a novel cardiac-specific TetON-miR-133 inducible transgenic mouse model. When subjected to transaortic constriction, TetON-miR-133 inducible transgenic mice maintained cardiac performance and showed attenuated apoptosis and reduced fibrosis compared with control mice. Conclusions: miR-133 controls multiple components of the &bgr;1AR transduction cascade and is cardioprotective during heart failure.

Cardiac sympathetic neurons provide trophic signal to the heart via β2-adrenoceptor-dependent regulation of proteolysis

AIMS Increased cardiac sympathetic neuron (SN) activity has been associated with pathologies such as heart failure and hypertrophy, suggesting that cardiac innervation regulates cardiomyocyte trophism. Whether continuous input from the SNs is required for the maintenance of the cardiomyocyte size has not been determined thus far. METHODS AND RESULTS To address the role of cardiac innervation in cardiomyocyte size regulation, we monitored the effect of pharmacological sympathetic denervation in mice on cardiac structure, function, and signalling from 24 h to 30 days in the absence of other pathological stimuli. SN ablation caused an immediate reduction in the cardiomyocyte size with minimal consequences on the resting contractile function. Atrophic remodelling was mediated by the ubiquitin-proteasome system through FOXO-dependent early induction of the muscle-specific E3 ubiquitin ligases Atrogin-1/MAFbx and MuRF1, which was followed by activation of the autophagy-lysosome system. MuRF1 was found to be determinant in denervation atrophy as remodelling did not develop in denervated MuRF1 knock-out (KO) hearts. These effects were caused by decreased basal stimulation of cardiomyocyte β2-adrenoceptor (AR), as atrophy was prevented by treatment of denervated mice with the β2-AR agonist clenbuterol. Consistent with these data, we also observed that β2-AR KO mice showed cardiac atrophy at rest. CONCLUSION Cardiac SNs are strong regulators of the cardiomyocyte size via β2-AR-dependent repression of proteolysis, demonstrating that the neuro-cardiac axis operates constitutively for the determination of the physiological cardiomyocyte size. These results are of great clinical relevance given the role of β-AR in cardiovascular diseases and their modulation in therapy.

Combined inhibition of PI3Kβ and PI3Kγ reduces fat mass by enhancing α-MSH–dependent sympathetic drive

Blocking the activity of two PI3K isoforms in the brain may be a strategy for treating obesity. Making the Brain Promote Fat Loss Obesity is associated with diabetes and metabolic syndrome and is a growing global problem. Signals from the brain regulate whole body metabolism and can trigger adipose tissue to burn fat. Perino et al. found that mice expressing catalytically inactive forms of two phosphatidylinositol 3-kinases (PI3Kβ and PI3Kγ) were leaner, burned more fat, and expended more energy than their wild-type counterparts. Fat loss also occurred in mice that received inhibitors of PI3Kβ and PI3Kγ delivered specifically into the brain. Thus, drugs that block the activity of both PI3Kβ and PI3Kγ in the brain could be developed as a treatment to fight obesity. Obesity is defined as an abnormal increase in white adipose tissue and has become a major medical burden worldwide. Signals from the brain control not only appetite but also energy expenditure, both of which contribute to body weight. We showed that genetic or pharmacological inhibition of two phosphatidylinositol 3-kinases (PI3Kβ and PI3Kγ) in mice reduced fat mass by promoting increased energy expenditure. This effect was accompanied by stimulation of lipolysis and the acquisition of the energy-burning characteristics of brown adipocytes by white adipocytes, a process referred to as “browning.” The browning of the white adipocytes involved increased norepinephrine release from the sympathetic nervous system. We found that PI3Kβ and PI3Kγ together promoted a negative feedback loop downstream of the melanocortin 4 receptor in the central nervous system, which controls appetite and energy expenditure in the periphery. Analysis of mice with drug-induced sympathetic denervation suggested that these kinases controlled the sympathetic drive in the brain. Administration of inhibitors of both PI3Kβ and PI3Kγ to mice by intracerebroventricular delivery induced a 10% reduction in fat mass as quickly as 10 days. These results suggest that combined inhibition of PI3Kβ and PI3Kγ might represent a promising treatment for obesity.

Micro-Arrayed Human Embryonic Stem Cells-Derived Cardiomyocytes for In Vitro Functional Assay

Introduction The heart is one of the least regenerative organs in the body and any major insult can result in a significant loss of heart cells. The development of an in vitro-based cardiac tissue could be of paramount importance for many aspects of the cardiology research. In this context, we developed an in vitro assay based on human cardiomyocytes (hCMs) and ad hoc micro-technologies, suitable for several applications: from pharmacological analysis to physio-phatological studies on transplantable hCMs. We focused on the development of an assay able to analyze not only hCMs viability, but also their functionality. Methods hCMs were cultured onto a poly-acrylamide hydrogel with tunable tissue-like mechanical properties and organized through micropatterning in a 20×20 array. Arrayed hCMs were characterized by immunofluorescence, GAP-FRAP analyses and live and dead assay. Their functionality was evaluated monitoring the excitation-contraction coupling. Results Micropatterned hCMs maintained the expression of the major cardiac markers (cTnT, cTnI, Cx43, Nkx2.5, α-actinin) and functional properties. The spontaneous contraction frequency was (0.83±0.2) Hz, while exogenous electrical stimulation lead to an increase up to 2 Hz. As proof of concept that our device can be used for screening the effects of pathological conditions, hCMs were exposed to increasing levels of H2O2. Remarkably, hCMs viability was not compromised with exposure to 0.1 mM H2O2, but hCMs contractility was dramatically suppressed. As proof of concept, we also developed a microfluidic platform to selectively treat areas of the cell array, in the perspective of performing multi-parametric assay. Conclusions Such system could be a useful tool for testing the effects of multiple conditions on an in vitro cell model representative of human heart physiology, thus potentially helping the processes of therapy and drug development.

Optogenetic determination of the myocardial requirements for extrasystoles by cell type-specific targeting of ChannelRhodopsin-2

Significance Arrhythmias are potentially life-threatening electrical heart diseases that are difficult to predict and are mostly treated by empirical interventions. The mechanisms of arrhythmia initiation (triggers) are still poorly understood, mostly due to the technical limitations of conventional experimental methods in cardiac electrophysiology. Here, we use optogenetics, a technique based on the expression of photoactivated proteins such as ChannelRhodopsin-2 (ChR2), which allows for noninvasive control of the cell membrane potential through illumination with blue light. By developing mice with cell-specific expression of ChR2, we investigated the role of the different cardiomyocyte types present in the heart and determined the factors triggering arrhythmic beats in the normal heart and during myocardial ischemia, a condition frequently associated with lethal arrhythmias causing sudden cardiac death. Extrasystoles lead to several consequences, ranging from uneventful palpitations to lethal ventricular arrhythmias, in the presence of pathologies, such as myocardial ischemia. The role of working versus conducting cardiomyocytes, as well as the tissue requirements (minimal cell number) for the generation of extrasystoles, and the properties leading ectopies to become arrhythmia triggers (topology), in the normal and diseased heart, have not been determined directly in vivo. Here, we used optogenetics in transgenic mice expressing ChannelRhodopsin-2 selectively in either cardiomyocytes or the conduction system to achieve cell type-specific, noninvasive control of heart activity with high spatial and temporal resolution. By combining measurement of optogenetic tissue activation in vivo and epicardial voltage mapping in Langendorff-perfused hearts, we demonstrated that focal ectopies require, in the normal mouse heart, the simultaneous depolarization of at least 1,300–1,800 working cardiomyocytes or 90–160 Purkinje fibers. The optogenetic assay identified specific areas in the heart that were highly susceptible to forming extrasystolic foci, and such properties were correlated to the local organization of the Purkinje fiber network, which was imaged in three dimensions using optical projection tomography. Interestingly, during the acute phase of myocardial ischemia, focal ectopies arising from this location, and including both Purkinje fibers and the surrounding working cardiomyocytes, have the highest propensity to trigger sustained arrhythmias. In conclusion, we used cell-specific optogenetics to determine with high spatial resolution and cell type specificity the requirements for the generation of extrasystoles and the factors causing ectopies to be arrhythmia triggers during myocardial ischemia.