MicroRNA therapy stimulates uncontrolled cardiac repair after myocardial infarction in pigs.

Prompt coronary catheterization and revascularization have markedly improved the outcomes of myocardial infarction, but have also resulted in a growing number of surviving patients with permanent structural damage of the heart, which frequently leads to heart failure. There is an unmet clinical need for treatments for this condition1, particularly given the inability of cardiomyocytes to replicate and thereby regenerate the lost contractile tissue2. Here we show that expression of human microRNA-199a in infarcted pig hearts can stimulate cardiac repair. One month after myocardial infarction and delivery of this microRNA through an adeno-associated viral vector, treated animals showed marked improvements in both global and regional contractility, increased muscle mass and reduced scar size. These functional and morphological findings correlated with cardiomyocyte de-differentiation and proliferation. However, subsequent persistent and uncontrolled expression of the microRNA resulted in sudden arrhythmic death of most of the treated pigs. Such events were concurrent with myocardial infiltration of proliferating cells displaying a poorly differentiated myoblastic phenotype. These results show that achieving cardiac repair through the stimulation of endogenous cardiomyocyte proliferation is attainable in large mammals, however dosage of this therapy needs to be tightly controlled.

Cardiovascular Response to Intraneural Right Vagus Nerve Stimulation in Adult Minipig.

OBJECTIVE: This study explored intraneural stimulation of the right thoracic vagus nerve (VN) in sexually mature male minipigs to modulate safe heart rate and blood pressure response. MATERIAL AND METHODS: We employed an intraneural electrode designed for the VN of pigs to perform VN stimulation (VNS). This was delivered using different numbers of contacts on the electrode and different stimulation parameters (amplitude, frequency, and pulse width), identifying the most suitable stimulation configuration. All the parameter ranges had been selected from a computational cardiovascular system model. RESULTS: Clinically relevant responses were observed when stimulating with low current intensities and relatively low frequencies delivered with a single contact. Selecting a biphasic, charge-balanced square wave for VNS with a current amplitude of 500 µA, frequency of 10 Hz, and pulse width of 200 µs, we obtained heart rate reduction of 7.67 ± 5.19 beats per minute, systolic pressure reduction of 5.75 ± 2.59 mmHg, and diastolic pressure reduction of 3.39 ± 1.44 mmHg. CONCLUSION: Heart rate modulation was obtained without inducing any observable adverse effects, underlining the high selectivity of the intraneural approach.

Extracellular vesicle-mediated bidirectional communication between heart and other organs.

In recent years, a wealth of studies has identified various molecular species released by cardiac muscle under physiological and pathological conditions that exert local paracrine and/or remote endocrine effects. Conversely, humoral factors, principally produced by organs such as skeletal muscle, kidney, or adipose tissue, may affect the function and metabolism of normal and diseased hearts. Although this cross communication within cardiac tissue and between the heart and other organs is supported by mounting evidence, research on the role of molecular mediators carried by exosomes, microvesicles, and apoptotic bodies, collectively defined as extracellular vesicles (EVs), is at an early stage of investigation. Once released in the circulation, EVs can potentially reach any organ where they transfer their cargo of proteins, lipids, and nucleic acids that exert potent biological effects on recipient cells. Although there are a few cases where such signaling was clearly demonstrated, the results from many other studies can only be tentatively inferred based on indirect evidence obtained by infusing exogenous EVs in experimental animals or by adding them to cell cultures. This area of research is in rapid expansion and most mechanistic interpretations may change in the near future; hence, the present review on the role played by EV-carried mediators in the two-way communication between heart and skeletal muscle, kidneys, bone marrow, lungs, liver, adipose tissue, and brain is necessarily limited. Nonetheless, the available data are already unveiling new, intriguing, and ample scenarios in cardiac physiology and pathophysiology.

Proteome Dynamics and Bioinformatics Reveal Major Alterations in the Turnover Rate of Functionally Related Cardiac and Plasma Proteins in a Dog Model of Congestive Heart Failure.

Protein pool turnover is a critically important cellular homeostatic component, yet it has been little explored in the context of heart failure (HF) pathophysiology. We used in vivo 2H labeling/proteome dynamics for the nonbiased discovery of turnover alterations involving functionally linked cardiac and plasma proteins in canine tachypacing-induced HF, an established preclinical model of dilated cardiomyopathy. Compared with controls, dogs with congestive HF displayed bidirectional turnover changes of 28 cardiac proteins, that is, a reduced half-life of several key enzymes involved in glycolysis, homocysteine metabolism and glycogenesis, and increased half-life of proteins involved in proteolysis. Changes in plasma proteins were more modest: only 5 proteins, involved in various functions including proteolysis inhibition, hemoglobin, calcium and ferric iron binding, displayed increased or decreased turnover rates. In other dogs undergoing cardiac tachypacing, we infused for 2 weeks the myokine Follistatin-like protein 1, known for its ameliorative effects on HF-induced alterations. Proteome dynamics proved very sensitive in detecting the partial or complete prevention, by Follistatin-like protein 1, of cardiac and plasma protein turnover alterations. In conclusion, our study unveiled, for the first time in a large mammal, numerous HF-related alterations that may serve as the basis for future mechanistic research and/or as conceptually new molecular markers.

Gene Therapy for Heart Failure: New Perspectives.

PURPOSE OF REVIEW: The current knowledge of pathophysiological and molecular mechanisms responsible for the genesis and development of heart failure (HF) is absolutely vast. Nonetheless, the hiatus between experimental findings and therapeutic options remains too deep, while the available pharmacological treatments are mostly seasoned and display limited efficacy. The necessity to identify new, non-pharmacological strategies to target molecular alterations led investigators, already many years ago, to propose gene therapy for HF. Here, we will review some of the strategies proposed over the past years to target major pathogenic mechanisms/factors responsible for severe cardiac injury developing into HF and will provide arguments in favor of the necessity to keep alive research on this topic. RECENT FINDINGS: After decades of preclinical research and phases of enthusiasm and disappointment, clinical trials were finally launched in recent years. The first one to reach phase II and testing gene delivery of sarcoendoplasmic reticulum calcium ATPase did not yield encouraging results; however, other trials are ongoing, more efficient viral vectors are being developed, and promising new potential targets have been identified. For instance, recent research is focused on gene repair, in vivo, to treat heritable forms of HF, while strong experimental evidence indicates that specific microRNAs can be delivered to post-ischemic hearts to induce regeneration, a result that was previously thought possible only by using stem cell therapy. Gene therapy for HF is aging, but exciting perspectives are still very open.

New insight on porcine carboxylesterases expression and activity in lung tissues.

Over the course of the last twenty years, there has been a growing recognition of the pig's potential as a valuable model for studying human drug metabolism. This study aimed to investigate the expression, enzymatic activity, inhibitory susceptibility, and cellular localization of carboxylesterases (CES) in porcine lung tissue not yet explored. Our results showed that CESs hydrolysis activity followed Michaelis-Menten kinetics in both cytosolic and microsomal fractions of porcine lung tissues (N = 8), with comparable hydrolysis rates for tested substrates, namely 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD). We also determined the CESs hydrolysis activity in a representative sample of the porcine liver that, as expected, displayed higher activity than the lung ones. The study demonstrated variable levels of enzyme activities and interindividual variability in both porcine lung fractions. Inhibition studies used to assess the CESs' involvement in the hydrolysis of pNPA, 4-MUA, and FD suggested that CESs may be the enzymes primarily involved in the metabolism of ester compounds in the pig lung tissue. Overall, this study provides insight into the distribution and diversity of CES isoforms involved in substrate hydrolysis across different cellular fractions (cytosol and microsomes) in porcine lungs.

A multi-channel stimulator with an active electrode array implant for vagal-cardiac neuromodulation studies.

BACKGROUND: Implantable vagus nerve stimulation is a promising approach for restoring autonomic cardiovascular functions after heart transplantation. For successful treatment a system should have multiple electrodes to deliver precise stimulation and complex neuromodulation patterns. METHODS: This paper presents an implantable multi-channel stimulation system for vagal-cardiac neuromodulation studies in swine species. The system comprises an active electrode array implant percutaneously connected to an external wearable controller. The active electrode array implant has an integrated stimulator ASIC mounted on a ceramic substrate connected to an intraneural electrode array via micro-rivet bonding. The implant is silicone encapsulated for biocompatibility and implanted lifetime. The stimulation parameters are remotely transmitted via a Bluetooth telemetry link. RESULTS: The size of the encapsulated active electrode array implant is 8 mm × 10 mm × 3 mm. The stimulator ASIC has 10-bit current amplitude resolution and 16 independent output channels, each capable of delivering up to 550 µA stimulus current and a maximum voltage of 20 V. The active electrode array implant was subjected to in vitro accelerated lifetime testing at 70 °C for 7 days with no degradation in performance. After over 2 h continuous stimulation, the surface temperature change of the implant was less than 0.5 °C. In addition, in vivo testing on the sciatic nerve of a male Göttingen minipig demonstrated that the implant could effectively elicit an EMG response that grew progressively stronger on increasing the amplitude of the stimulation. CONCLUSIONS: The multi-channel stimulator is suitable for long term implantation. It shows potential as a useful tool in vagal-cardiac neuromodulation studies in animal models for restoring autonomic cardiovascular functions after heart transplantation.

Simultaneous decoding of cardiovascular and respiratory functional changes from pig intraneural vagus nerve signals.

Objective. Bioelectronic medicine is opening new perspectives for the treatment of some major chronic diseases through the physical modulation of autonomic nervous system activity. Being the main peripheral route for electrical signals between central nervous system and visceral organs, the vagus nerve (VN) is one of the most promising targets. Closed-loop VN stimulation (VNS) would be crucial to increase effectiveness of this approach. Therefore, the extrapolation of useful physiological information from VN electrical activity would represent an invaluable source for single-target applications. Here, we present an advanced decoding algorithm novel to VN studies and properly detecting different functional changes from VN signals.Approach. VN signals were recorded using intraneural electrodes in anaesthetized pigs during cardiovascular and respiratory challenges mimicking increases in arterial blood pressure, tidal volume and respiratory rate. We developed a decoding algorithm that combines discrete wavelet transformation, principal component analysis, and ensemble learning made of classification trees.Main results. The new decoding algorithm robustly achieved high accuracy levels in identifying different functional changes and discriminating among them. Interestingly our findings suggest that electrodes positioning plays an important role on decoding performances. We also introduced a new index for the characterization of recording and decoding performance of neural interfaces. Finally, by combining an anatomically validated hybrid neural model and discrimination analysis, we provided new evidence suggesting a functional topographical organization of VN fascicles.Significance. This study represents an important step towards the comprehension of VN signaling, paving the way for the development of effective closed-loop VNS systems.

Q-PINE: A quick to implant peripheral intraneural electrode.

Objective. The implantation of intraneural electrodes in amputees has been observed to be effective in providing subjects with sensory feedback. However, this implantation is challenging and time consuming. Surgeons must be especially trained to execute the implantation. Therefore, we aimed at developing a novel peripheral intraneural electrode and insertion mechanism, which could drastically reduce the overall implantation time while achieving a high neural selectivity.Approach.A new insertion method based on hollow microneedles was developed to realize the prompt and effective simultaneous implantation of up to 14 active sites in a transversal manner. Each needle guided two Pt/Ir microwires through the nervous tissue. After the insertion, the microneedles were released, leaving behind the microwires. Each microwire had one active site, which was coated with poly-3,4-ethylenedioxythiophene (PEDOT) to enhance the electrochemical properties. The active sites were characterized by evaluating the impedance, charge storage capacity, and maximum injectable charge. Twelve quick to implant peripheral intraneural electrodes (Q-PINEs) were implanted in four pig sciatic nerves to evaluate the implantation time and neural selectivity. We compared the stimulation of the sciatic nerve with that of its branches.Main results. The average surgical access time was 23 min. The insertion time for 12 electrodes was 6.7 min (std. ±1.6 min). The overall implantation time was reduced by 40.3 min compared to the previously reported values. The Q-PINE system demonstrated a satisfactory performance duringin vitroandin vivocharacterization. The electrochemical results showed that the PEDOT coating successfully increased the electrochemical parameters of the active sites.Significance.With an average impedance of 1.7 kΩ, a maximum charge level of 76.2 nC could be achieved per active site. EMG recruitment curves showed that 46% of the active sites exhibited selective stimulation of four out of six muscles. The histological analysis indicated that the microwires successfully penetrated the nerve and single fascicles.

Magnetic resonance imaging of infarct-induced canonical wingless/integrated (Wnt)/ß-catenin/T-cell factor pathway activation, in vivo.

AIMS: Combined magnetic resonance imaging (MRI) of molecular and morpho-functional changes might prove highly valuable for the elucidation of pathological processes involved in the development of cardiac diseases. Our aim was to test a novel MRI reporter gene for in vivo assessment of the canonical Wnt/ß-catenin/TCF pathway activation, an important regulator of post-ischaemic cardiac remodelling. METHODS AND RESULTS: We designed and developed a chimeric construct encoding for both of iron-binding human ferritin heavy chain (hFTH) controlled by the ß-catenin-responsive TCF/lymphoid-enhancer binding factor (Lef) promoter and constitutively expressed green fluorescent protein (GFP). It was carried by adeno-associated virus serotype 9 (rAAV9) vectors and delivered to the peri-infarct myocardium of rats subjected to coronary ligation (n = 11). By 1.5 T MRI and a multiecho T2* gradient echo sequence, we detected iron accumulation only in the border zone of the transduced infarcted hearts. In the same cardiac area, post-mortem histological analysis confirmed the co-existence of iron accumulation and GFP. The iron signal was absent when rats (n = 6) were chronically treated with SEN195 (10 mg/kg/day), a small-molecular inhibitor of ß-catenin/TCF-dependent gene transcription. Canonical Wnt pathway inhibition attenuated the post-ischaemic remodelling process, as demonstrated by the significant preservation of cardiac function, the 42 ± 1% increase of peri-infarct arteriolar density and 43 ± 3% reduction in infarct scar size compared with untreated animals. CONCLUSIONS: The TCF/Lef promoter-hFTH construct is a novel and reliable MRI reporter gene for in vivo detection of the canonical Wnt/ß-catenin/TCF activation state in response to cardiac injury and therapeutic interventions.