Giulia Borile

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.

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.

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.

Content of mitochondrial calcium uniporter (MCU) in cardiomyocytes is regulated by microRNA-1 in physiologic and pathologic hypertrophy

Significance Mitochondrial calcium uniporter (MCU) is the core channel subunit of the mitochondrial Ca2+ uniporter complex (MCUC) and contributes to the regulation of ATP production. Here, we identify that the adaptive and maladaptive remodeling occurring in rodent and human cardiomyocytes is associated with changes in MCU content, which are inversely correlated with those of its regulator microRNA-1 (miR-1). The present study thus defines the molecular mechanism by which MCU content and, consequently, mitochondrial Ca2+ uptake are directly modified by alteration of miR-1, a key regulator affecting physiological and pathological heart growth. These findings provide evidence for the miR-1/MCU axis as a potential target for the development of novel therapeutic approaches. The mitochondrial Ca2+ uniporter complex (MCUC) is a multimeric ion channel which, by tuning Ca2+ influx into the mitochondrial matrix, finely regulates metabolic energy production. In the heart, this dynamic control of mitochondrial Ca2+ uptake is fundamental for cardiomyocytes to adapt to either physiologic or pathologic stresses. Mitochondrial calcium uniporter (MCU), which is the core channel subunit of MCUC, has been shown to play a critical role in the response to β-adrenoreceptor stimulation occurring during acute exercise. The molecular mechanisms underlying the regulation of MCU, in conditions requiring chronic increase in energy production, such as physiologic or pathologic cardiac growth, remain elusive. Here, we show that microRNA-1 (miR-1), a member of the muscle-specific microRNA (myomiR) family, is responsible for direct and selective targeting of MCU and inhibition of its translation, thereby affecting the capacity of the mitochondrial Ca2+ uptake machinery. Consistent with the role of miR-1 in heart development and cardiomyocyte hypertrophic remodeling, we additionally found that MCU levels are inversely related with the myomiR content, in murine and, remarkably, human hearts from both physiologic (i.e., postnatal development and exercise) and pathologic (i.e., pressure overload) myocardial hypertrophy. Interestingly, the persistent activation of β-adrenoreceptors is likely one of the upstream repressors of miR-1 as treatment with β-blockers in pressure-overloaded mouse hearts prevented its down-regulation and the consequent increase in MCU content. Altogether, these findings identify the miR-1/MCU axis as a factor in the dynamic adaptation of cardiac cells to hypertrophy.

Multispot multiphoton Ca2+ imaging in acute myocardial slices

Abstract. Multiphoton microscopy has become essential for dynamic imaging in thick living tissues. High-rate, full-field image acquisition in multiphoton microscopy is achievable by parallelization of the excitation and detection pathways. We developed our approach via a diffractive optical element which splits a pulsed laser into 16 beamlets and exploits a descanned detection system consisting of an array of beamlet-associated photomultiplier tubes. The optical performance of the multiphoton multispot system (MCube) has been characterized in cardiac tissue sections and subsequently used for the first time for fluorescence imaging of cardiomyocyte Ca2+ dynamics in viable acute cardiac slices. Multispot multiphoton microscopy (MMM) has never been used before to monitor Ca2+ dynamics in thick, viable tissue samples. Acute heart slices are a powerful close-to-in vivo model of Ca2+ imaging allowing the simultaneous observation of several cells in their own tissue environment, exploiting the multiphoton excitation ability to penetrate scattering tissues. Moreover, we show that the concurrent high spatial and temporal resolutions afforded by the parallel scanning in MMM can be exploited to simultaneously assess subcellular Ca2+ dynamics in different cells in the tissue. We recorded local Ca2+ release events including macrosparks, travelling waves, and rotors.

MiR-133 Modulates the β1Adrenergic 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.

Grating-Coupled Surface Plasmon Resonance (GC-SPR) Optimization for Phase-Interrogation Biosensing in a Microfluidic Chamber

Surface Plasmon Resonance (SPR)-based sensors have the advantage of being label-free, enzyme-free and real-time. However, their spreading in multidisciplinary research is still mostly limited to prism-coupled devices. Plasmonic gratings, combined with a simple and cost-effective instrumentation, have been poorly developed compared to prism-coupled system mainly due to their lower sensitivity. Here we describe the optimization and signal enhancement of a sensing platform based on phase-interrogation method, which entails the exploitation of a nanostructured sensor. This technique is particularly suitable for integration of the plasmonic sensor in a lab-on-a-chip platform and can be used in a microfluidic chamber to ease the sensing procedures and limit the injected volume. The careful optimization of most suitable experimental parameters by numerical simulations leads to a 30–50% enhancement of SPR response, opening new possibilities for applications in the biomedical research field while maintaining the ease and versatility of the configuration.

Multimodal label-free ex vivo imaging using a dual-wavelength microscope with axial chromatic aberration compensation

Abstract. Label-free microscopy is a very powerful technique that can be applied to study samples with no need for exogenous fluorescent probes, keeping the main benefits of multiphoton microscopy, such as longer penetration depths and intrinsic optical sectioning while enabling serial multitechniques examinations on the same specimen. Among the many label-free microscopy methods, harmonic generation (HG) is one of the most intriguing methods due to its generally low photo-toxicity and relative ease of implementation. Today, HG and common two-photon microscopy (TPM) are well-established techniques, and are routinely used in several research fields. However, they require a significant amount of fine-tuning to be fully exploited, making them quite difficult to perform in parallel. Here, we present our designed multimodal microscope, capable of performing simultaneously TPM and HG without any kind of compromise thanks to two, separate, individually optimized laser sources with axial chromatic aberration compensation. We also apply our setup to the examination of a plethora of ex vivo samples to prove its capabilities and the significant advantages of a multimodal approach.

High speed microscopy techniques for signaling detection in live cells

Alterations in intracellular cardiomyocyte calcium handling have a key role in initiating and sustaining arrhythmias. Arrhythmogenic calcium leak from sarcoplasmic reticulum (SR) can be attributed to all means by which calcium exits the SR store in an abnormal fashion. Abnormal SR calcium exit maymanifest as intracellular Ca2+ sparks and/or Ca2+ waves. Ca2+ signaling in arrhythmogenesis has been mainly studied in isolated cardiomyocytes and given that the extracellular matrix influences both Ca2+ and membrane potential dynamics in the intact heart and underlies environmentally mediated changes, understanding how Ca2+ and voltage are regulated in the intact heart will represent a tremendous advancement in the understanding of arrhythmogenic mechanisms. Using novel high-speed multiphoton microscopy techinques, such as multispot and random access, we investigated animal models with inherited and acquired arrhythmias to assess the role of Ca2+ and voltage signals as arrhythmia triggers in cell and subcellular components of the intact heart and correlate these with electrophysiology.

Calcium and voltage imaging in arrhythmia models by high-speed microscopy

Alterations in intracellular cardiomyocyte calcium handling have a key role in initiating and sustaining arrhythmias. Arrhythmogenic calcium leak from sarcoplasmic reticulum (SR) can be attributed to all means by which calcium exits the SR store in an abnormal fashion. Abnormal SR calcium exit maymanifest as intracellular Ca2+ sparks and/or Ca2+ waves. Ca2+ signaling in arrhythmogenesis has been mainly studied in isolated cardiomyocytes and given that the extracellular matrix influences both Ca2+ and membrane potential dynamics in the intact heart and underlies environmentally mediated changes, understanding how Ca2+ and voltage are regulated in the intact heart will represent a tremendous advancement in the understanding of arrhythmogenic mechanisms. Using novel high-speed multiphoton microscopy techinques, such as multispot and random access, we investigated animal models with inherited and acquired arrhythmias to assess the role of Ca2+ and voltage signals as arrhythmia triggers in cell and subcellular components of the intact heart and correlate these with electrophysiology.