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Background: Loss of brain-derived neurotrophic factor (BDNF)/TrkB (tropomyosin kinase receptor B) signaling accounts for brain and cardiac disorders. In neurons, β-adrenergic receptor stimulation enhances local BDNF expression. It is unclear if this occurs in a pathophysiological relevant manner in the heart, especially in the β-adrenergic receptor-desensitized postischemic myocardium. Nor is it fully understood whether and how TrkB agonists counter chronic postischemic left ventricle (LV) decompensation, a significant unmet clinical milestone. Methods: We conducted in vitro studies using neonatal rat and adult murine cardiomyocytes, SH-SY5Y neuronal cells, and umbilical vein endothelial cells. We assessed myocardial ischemia (MI) impact in wild type, β3AR knockout, or myocyte-selective BDNF knockout (myoBDNF KO) mice in vivo (via coronary ligation [MI]) or in isolated hearts with global ischemia-reperfusion (I/R). Results: In wild type hearts, BDNF levels rose early after MI ( Conclusions: BDNF loss underscores chronic postischemic heart failure. TrkB agonists can improve ischemic LV dysfunction via replenished myocardial BDNF content. Direct cardiac β3AR stimulation, or β-blockers (via upregulated β3AR), is another BDNF-based means to fend off chronic postischemic heart failure.

The heart is a functional syncytium controlled by a delicate and sophisticated balance ensured by the tight coordination of its several cell subpopulations. Accordingly, cardiomyocytes together with the surrounding microenvironment participate in the heart tissue homeostasis. In the right atrium, the sinoatrial nodal cells regulate the cardiac impulse propagation through cardiomyocytes, thus ensuring the maintenance of the electric network in the heart tissue. Notably, the central nervous system (CNS) modulates the cardiac rhythm through the two limbs of the autonomic nervous system (ANS): the parasympathetic and sympathetic compartments. The autonomic nervous system exerts non-voluntary effects on different peripheral organs. The main neuromodulator of the Sympathetic Nervous System (SNS) is norepinephrine, while the principal neurotransmitter of the Parasympathetic Nervous System (PNS) is acetylcholine. Through these two main neurohormones, the ANS can gradually regulate cardiac, vascular, visceral, and glandular functions by turning on one of its two branches (adrenergic and/or cholinergic), which exert opposite effects on targeted organs. Besides these neuromodulators, the cardiac nervous system is ruled by specific neuropeptides (neurotrophic factors) that help to preserve innervation homeostasis through the myocardial layers (from epicardium to endocardium). Interestingly, the dysregulation of this neuro-signaling pathway may expose the cardiac tissue to severe disorders of different etiology and nature. Specifically, a maladaptive remodeling of the cardiac nervous system may culminate in a progressive loss of neurotrophins, thus leading to severe myocardial denervation, as observed in different cardiometabolic and neurodegenerative diseases (myocardial infarction, heart failure, Alzheimer’s disease). This review analyzes the current knowledge on the pathophysiological processes involved in cardiac nervous system impairment from the perspectives of both cardiac disorders and a widely diffused and devastating neurodegenerative disorder, Alzheimer’s disease, proposing a relationship between neurodegeneration, loss of neurotrophic factors, and cardiac nervous system impairment. This overview is conducive to a more comprehensive understanding of the process of cardiac neuro-signaling dysfunction, while bringing to light potential therapeutic scenarios to correct or delay the adverse cardiovascular remodeling, thus improving the cardiac prognosis and quality of life in patients with heart or neurodegenerative disorders.

Developmental cardiac tissue is regenerative while operating under low oxygen. After birth, ambient oxygen is associated with cardiomyocyte cell cycle exit and regeneration. Likewise, cardiac metabolism undergoes a shift with cardiac maturation. Whether there are common regulators of cardiomyocyte cell cycle linking metabolism to oxygen tension remains unknown. The objective of the study is to determine whether mitochondrial UCP2 is a metabolic oxygen sensor regulating cardiomyocyte cell cycle. Neonatal rat ventricular myocytes (NRVMs) under moderate hypoxia showed increased cell cycle activity and UCP2 expression. NRVMs exhibited a metabolic shift toward glycolysis, reducing citrate synthase, mtDNA, mitochondrial membrane potential (ΔΨm), and DNA damage/oxidative stress, while loss of UCP2 reversed this phenotype. Next, WT and mice from a global UCP2-KO mouse line (UCP2KO) kept under hypoxia for 4 weeks showed significant decline in cardiac function that was more pronounced in UCP2KO animals. Cardiomyocyte cell cycle activity was reduced, while fibrosis and DNA damage was significantly increased in UCP2KO animals compared with WT under hypoxia. Mechanistically, UCP2 increased acetyl-CoA levels and histone acetylation, and it altered chromatin modifiers linking metabolism to cardiomyocyte cell cycle under hypoxia. Here, we show a potentially novel role for mitochondrial UCP2 as an oxygen sensor regulating cardiomyocyte cell cycle activity, acetyl-CoA levels, and histone acetylation in response to moderate hypoxia.

The extra-cellular-matrix (ECM) composition of scar tissue after myocardial infarction (MI)has been largely investigated. Although fibronectin and collagen are favorable for newmyocyte formation, other components that may increase the scar stiffness and reducethe remodeling of the ischemic area, remain to be identified. Our preliminary studiesidentified primary Serum Amyloid A3 (SAA3) extracellular accumulation that maycontribute to the chronic alteration of the ischemic myocardium’s scar. Specifichistological staining such as thioflavin and Congo red, showed amyloid deposition inmouse hearts 1 month after MI; furthermore, immunohistochemistry for SAA3 detectedthe deposition of the misfolded protein alongside fibronectin and collagen. Serum amyloidamyloidosis (AA) is characterized by deposition of hepatic misfolded protein and SAA3 isthe only amyloid protein that is released locally after inflammation, mostly bymesenchymal progenitor cells. We have reported earlier that two days after MI, a cohortof mesenchymal cells begin to de novo express Podoplanin (PDPN), a plateletaggregation-inducing type I transmembrane glycoprotein, as a signal of activation.PDPN+ cells, in addition to cytokines, release extracellular vesicles including exosomes(Exo) as major paracrine entities driving intercellular communications in homeostasis anddisease. Exo derived from activated PDPN+ cells isolated from MI hearts highly expressSAA3 and injection of activated PDPN+ cell Exo in uninjured healthy mouse hearts leadsto recruitment of immune cells, an extended epicardial fibrosis and amyloidosis with asubsequent impairment in the contractility and increase of the end systolic volumes anddiameters. SAA3 binds Toll-like receptors, and in vitro treatment of bone marrow derivedmonocytes either with PDPN+ cells derived Exo or recombinant SAA3, activated themtowards pro-inflammatory phenotype on contrary these stimuli failed to activate TLR2knocked out monocytes showing an impairment in the expression of major cytokine,chemokine and pro inflammatory markers. Thus, PDPN+ cells in the ischemic heartrelease SAA3 through Exo prolonging inflammation and macrophage recruitment viaTLR2 and contribute to amyloidosis after MI.

Fibrosis and blood hypoperfusion stimulated by paracrine signals enhances the ventricular dysfunctionafter myocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN) positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells could independentlyalter healthy heart physiology and structure. PDPN+ cells were isolated 2 days after MI, culture expandedand activated with TNFα and Angiotensin II. Exosomes derived from activated PDPN+cells conditionedmedia (PDPN+exo) were used in vitro for the treatment of mouse cardiac endothelial cells (mCECs) andmouse fibroblast (3T3) and in vivo for the treatment of healthy mouse hearts. In vitro, PDPN+exoinfluenced the phenotype of mCECs, stimulating their lineage into lymphatic endothelial cells andfacilitated fibroblasts transition to myofibroblast. Characterization of the protein content of PDPN+exoshowed high concentration of Notch receptors and γ-Secretase, suggesting these cellular transitions maydepend on exosome-mediated Notch translocation and cleavage. In fact, after exosomes treatmentcleaved notch (NICD) translocated in the nuclei of mCECs and 3T3 as early as 1h of treatment and eitherHes-1 or Hey-1, major transcription factors activated by NICD were enhanced within 2d of treatment.Using DAPT, a γSecretase inhibitor, notch cleavage was inhibited, and no phenotype switching in responseto exosome treatment was observed. In vivo, PDPN+exo were injected into the left ventricle of healthymouse hearts followed by boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase of the enddiastolic and systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Podoplanin positive cellsrepresent 80% of the scar’s cells of a chronic infarcted myocardium and the specific exosomes cargo highlyinfluence the lineage of cardiac cells altering the biology of endothelial cells and fibroblasts which mayfacilitate adverse remodeling.

Aging is a multifactorial process associated with gradual loss of function and decay involving several neurohormonal systems, such as the autonomic nervous system (ANS). Progressive remodeling of ANS, induces a circulating catecholamines spillover and cardiac autonomic fibers depletion with raising both morbidities and mortality risk. Neurotrophic factors (NF) play a pivotal role in modulating neuronal function and are impaired in cardiovascular disorders. Whether and how physiological aging impacts these neurobiomarkers and cardiac innervation remains still unclear. Therefore, we investigated the impact of aging on neurotrophins (such as BDNF and NGF) production and secretion and its consequences, on cardiac nervous system homeostasis. In vivo, we used young (age: 3 months; n=10) and old (age: 24 months; n=11) male Fisher rats. In vitro, human neuroblastoma cells (SH-SY5Y) were stimulated with serum withdrawn from both experimental groups. Old rats showed a significant reduction in overall ANS fiber density, sympathetic (marked by dopamine β-hydroxylase, dβh) and cholinergic compartment (evidenced by vesicular acetylcholine transporter, VaChT) compared to the young group, assessed by immunohistochemical staining. In addition, we observed a marked downregulation of GAP-43 and BDNF protein levels in left ventricle total lysates via immunoblot analysis, in aged hearts as opposed to young ones. Conversely, no changes were observed in NGF protein expression. To further investigate the autocrine effect of aging on autonomic nerve fibers, we treated SH-SY5Y cells in vitro, with blood serum obtained by young or old rats. Both stimuli induced a remarkable increase in neuronal sprouting, as evidenced via crystal violet assay. Nevertheless, we found a bulky drop in the neuronal function of cells stimulated with old rat serum. Interestingly, this effect was accompanied by a sizeable blunt in GAP-43 and BDNF protein levels, compared to cells treated with young rat serum. Taken together, our data suggest that neuronal function impairment aging-induced associated with significant BDNF impoverishment, might favor maladaptive remodeling of cardiac ANS.

Background Aging is a multifactorial process associated with an impairment of autonomic nervous system (ANS) function. Progressive ANS remodeling includes upregulation of expression of circulating catecholamines and depletion of cardiac autonomic nerve fibers, and it is responsible, in part, for the increased susceptibility to cardiac diseases observed in elderly subjects. Neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), are involved in synaptogenesis and neurite outgrowth processes, supporting neuronal cell differentiation and maturation. However, whether and how these factors and their downstream signaling are involved in cardiac aging remains unclear. Here, we tested whether, in the aged heart, the overall extent of autonomic fibers is reduced, owing to lower production of trophic factors such as BDNF and NGF. Methods In vivo, we used young (age: 3 months; n = 10) and old (age: 24 months; n = 11) male Fisher rats, whereas, we used human neuroblastoma (SH-SY5Y) cells in vitro. Results Compared to the young rats, old rats displayed a marked reduction in the overall ANS fiber density, affecting both sympathetic and cholinergic compartments, as indicated by dopamine β-hydroxylase (dβh) and vesicular acetylcholine transporter (VaChT) immunohistochemical staining. In addition, a marked downregulation of GAP-43 and BDNF protein was observed in the left ventricular lysates of old rats compared to those of young rats. Interestingly, we did not find any significant difference in cardiac NGF levels between the young and old groups. To further explore the impact of aging on ANS fibers, we treated SH-SY5Y cells in vitro with serum obtained from young and old rats. Sera from both groups induced a remarkable increase in neuronal sprouting, as evidenced by a crystal violet assay. However, this effect was blunted in cells cultured with old rat serum and was accompanied by a marked reduction in GAP-43 and BDNF protein levels. Conclusions Our data indicate that physiological aging is associated with an impairment of ANS structure and function and that reduced BDNF levels are responsible, at least in part, for these phenomena.

Superseding fibrosis through paracrine signals enhances the ventricular dysfunction aftermyocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN), a platelet aggregation-inducing type I transmembrane glycoprotein,positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells couldindependently alter healthy heart physiology and structure. PDPN+ cells were isolated 2 daysafter MI, cultured expanded and activated with TNFα and AngiotensinII. Exosomes derived fromactivated PDPN+ cells conditioned media were used in vitro treatment of mouse cardiacendothelial cells (mCECs), mouse embryonic fibroblast (MEF) and monocytes and in vivo forthe treatment of healthy mouse hearts. PDPN+ cells derived exosomes (PDPN-exo)reprogramed mCECs to the lymphatic phenotype enhancing the expression of the majorlymphatic lineage markers and upregulated the expression of fibrotic markers suggesting anendothelial-mesenchymal transition. Furthermore, PDPN-exo drove the MEF to myo-fibroblastphenotype and monocytes toward pro-inflammatory phenotype. Proteomic analysis of PDPN-exo suggest these transitions may depend on NOTCH cleavage trough β-γSecretase. In vivo,PDPN-exo were initially injected into the left ventricle of healthy mouse hearts followed withexosomes boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase ofthe end diastolic and systolic volumes. The fibrotic area was characterized by vessels doublepositive to endothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Inconclusion these data suggest that PDPN-exo alter the biology of mCECs, fibroblast andmonocytes and participate in adverse remodeling after MI; their specific cargo may representa cohort of targets for the treatment of cardiac fibrosis.

The extra-cellular-matrix (ECM) composition of scar tissue after myocardial infarction (MI)has been largely investigated; although fibronectin and collagen are favorable for newmyocyte formation there is a missing component that increase the stiffness and reducethe remodeling of the ischemic area. We identified a primary Serum Amyloid A3 (Saa3)extracellular accumulation that contribute to the chronic alteration of the tissue. Serumamyloid amyloidosis (AA) are characterized by deposition of hepatic misfolded protein,Saa3 is the only amyloid protein that is released locally after inflammation, mostly bymesenchymal progenitor cells. We already described that two days after MI,mesenchymal and endothelial progenitor cells express Podoplanin (PDPN) a plateletaggregation-inducing type I transmembrane glycoprotein as a signal of activation. Exosomes derived from this cohort of cells actively release Saa3 in the ECM affecting thebiology of fibrosis beyond the inflammation. Specific histological staining such asthioflavin t and Congo red, showed amyloid deposition in mouse hearts 1 month after MI;furthermore, immunohistochemistry for Saa3 detected the deposition of the misfoldedprotein alongside fibronectin and collagen. PDPN+ cells were isolated 2 days after MI,cultured expanded and activated with TNFα and AngiotensinII. Activated PDPN positivecells highly expressed Saa3 and exosomes derived from activated PDPN+ cellsconditioned media were used in vivo for the treatment of healthy mouse hearts. Treatedmice developed an extended epicardial fibrosis and amyloidosis with a subsequentimpairment in the contractility and increase of the end diastolic and systolic volumes. Thefibrotic area was characterized by infiltrating CD45+ cells. Novel therapies aimed atpromoting clearance of existing amyloid deposits may be an effective approach in thenear future in the treatment of scar remodeling after MI.

Superseding fibrosis through paracrine signals enhances the ventricular dysfunction aftermyocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN), a platelet aggregation-inducing type I transmembrane glycoprotein,positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells couldindependently alter healthy heart physiology and structure. PDPN+ cells were isolated 2 daysafter MI, cultured expanded and activated with TNFα and AngiotensinII. Exosomes derivedfrom activated PDPN+ cells conditioned media were used in vitro treatment of mouse cardiacendothelial cells (mCECs), mouse embryonic fibroblast (MEF) and monocytes and in vivo forthe treatment of healthy mouse hearts. PDPN+ cells derived exosomes (PDPN-exo)reprogramed mCECs to the lymphatic phenotype enhancing the expression of the majorlymphatic lineage markers and upregulated the expression of fibrotic markers suggesting anendothelial-mesenchymal transition. Furthermore, PDPN-exo drove the MEF to myo-fibroblastphenotype and monocytes toward pro-inflammatory phenotype. Proteomic analysis of PDPN-exo suggest these transitions may depend on NOTCH cleavage trough β-γSecretase andSerum Amyloid A3 protein accumulation/mis-folding. In vivo, PDPN-exo were initially injectedinto the left ventricle of healthy mouse hearts followed with exosomes boosters delivered byretro-orbital vein injection. Treated mice developed an extended epicardial fibrosis andamyloidosis with a subsequent impairment in the contractility and increase of the end diastolicand systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. In conclusionthese data suggest that PDPN-exo alter the biology of mCECs, fibroblast and monocytes andparticipate in adverse remodeling after MI; their specific cargo may represent a cohort oftargets for the treatment of cardiac fibrosis.

Left ventricular hypertrophy (LVH) is a well established cardiovascular risk factor, accounting for an increase in cardiovascular morbid-mortality, although how much the magnitude and the kind of LVH could affect cardiovascular outcomes is in large part unknown. We speculate that mild LVH in absence of left ventricular (LV) chamber dilation, could play a protective role towards functional capacity, clinical outcome, cardiovascular and total morbi-mortality in conditions in which LV systolic function is generally reduced. Accordingly to many epidemiological observations, the availability of extra-quote of systolic function could lead to a significative improvement in the final outcome of some kinds of heart patients, as those undergoing bypass-grafting, where the stress for heart and cardiovascular system is always high. We suppose that the functional reserve available for patients with LVH could make the difference with respect to other patients undergoing myocardial revascularization. Similarly, the availability of a contractile reserve warranted by LVH could ensure a little gain in the outcome for patients after other major cardiovascular events (such as myocardial infarction or other heart surgery as surgical valve replacement). However, our hypothesis only involves mild LVH without LV chamber dilation, that is the initial stage of "non-dilated concentric" LVH and "non-dilated eccentric" LVH according to the new four-tiered classification of LVH based on relative wall thickness and LV dilation. Support for our hypothesis derives from the well-known protective role of systolic function that is a major factor in almost all cardiovascular diseases, where LV ejection fraction (LVEF) has shown to significantly improve quality of life, as well as morbidity and mortality. The knowledge that mild LVH in absence of LV chamber dilation is not as harmful in such conditions as believed at present could make avoidable some drugs prescription in some stages of the disease. Furthermore, it may allow a better evaluation of the risk profile of patients with LVH undergoing some cardiovascular major events like bypass grafting, myocardial infarction or surgical heart valve replacement.

Aldosterone (Aldo) has been intensively studied for years since its isolation by Simpson and Tait in the 1950s. Interestingly, although most early research around Aldo's actions focused primarily on its interaction with the kidney, it was soon evident that this hormone is able to exert unexplained extra-renal effects, on various target organs including the heart. Importantly, over the course of the last decade a number of studies in pre-clinical models (in vitro and in vivo) and in humans have clearly demonstrated that Aldo, following the interaction with its receptor, the mineralocorticoid receptor (MR), is able to activate specific intracellular genomic and non-genomic pathways thus, regulating the homeostasis of the entire cardiovascular system. In this regard, Aldo has been shown to influence the function and growth of different type of cells including cardiomyocytes, fibroblasts and vascular cells (vascular smooth muscle cells and endothelial cells). Importantly, when present at high levels, Aldo have been associated with the onset of disparate cardiovascular disorders including heart failure (HF). For this reason, several pre-clinical study and clinical trials have been designed in order to test the effects of Aldo antagonism in HF patients. In this chapter, we will guide the readers through the current knowledge around Aldo activity in cardiovascular system and the relevant therapies acting in blocking the noxious Aldo/MR signaling pathway.

Aldosterone (Aldo) has been intensively studied for years since its isolation by Simpson and Tait in the 1950s. Interestingly, although most early research around Aldo's actions focused primarily on its interaction with the kidney, it was soon evident that this hormone is able to exert unexplained extra-renal effects, on various target organs including the heart. Importantly, over the course of the last decade a number of studies in pre-clinical models (in vitro and in vivo) and in humans have clearly demonstrated that Aldo, following the interaction with its receptor, the mineralocorticoid receptor (MR), is able to activate specific intracellular genomic and non-genomic pathways thus, regulating the homeostasis of the entire cardiovascular system. In this regard, Aldo has been shown to influence the function and growth of different type of cells including cardiomyocytes, fibroblasts and vascular cells (vascular smooth muscle cells and endothelial cells). Importantly, when present at high levels, Aldo have been associated with the onset of disparate cardiovascular disorders including heart failure (HF). For this reason, several pre-clinical study and clinical trials have been designed in order to test the effects of Aldo antagonism in HF patients. In this chapter, we will guide the readers through the current knowledge around Aldo activity in cardiovascular system and the relevant therapies acting in blocking the noxious Aldo/MR signaling pathway.

Background: Caloric restriction (CR) has been described to have cardioprotective effects and improve functional outcomes in animal models and humans. Chronic ischemic heart failure (HF) is associated with reduced cardiac sympathetic innervation, dysfunctional β-adrenergic receptor signaling, and decreased cardiac inotropic reserve. We tested the effects of a long-term CR diet, started late after myocardial infarction on cardiac function, sympathetic innervation, and β-adrenergic receptor responsiveness in a rat model of postischemic HF. Methods and Results: Adult male rats were randomly assigned to myocardial infarction or sham operation and 4 weeks later were further randomized to a 1-year CR or normal diet. One year of CR resulted in a significant reduction in body weight, heart weight, and heart weight/tibia length ratio when compared with normal diet in HF groups. At the end of the study period, echocardiography and histology revealed that HF animals under the CR diet had ameliorated left ventricular remodeling compared with HF rats fed with normal diet. Invasive hemodynamic showed a significant improvement of cardiac inotropic reserve in CR HF rats compared with HF-normal diet animals. Importantly, CR dietary regimen was associated with a significant increase of cardiac sympathetic innervation and with normalized cardiac β-adrenergic receptor levels in HF rats when compared with HF rats on the standard diet. Conclusions: We demonstrate, for the first time, that chronic CR, when started after HF established, can ameliorate cardiac dysfunction and improve inotropic reserve. At the molecular level, we find that chronic CR diet significantly improves sympathetic cardiac innervation and β-adrenergic receptor levels in failing myocardium.

The mineralocorticoid hormone aldosterone (Aldo) has been intensively studied for its ability to influence both the physiology and pathophysiology of the cardiovascular system. Indeed, although research on Aldo actions for decades has mainly focused on its effects in the kidney, several lines of evidence have now demonstrated that this hormone exerts disparate extrarenal adverse effects, especially in the circulatory system. Accordingly, in the last lusters, a number of studies in preclinical models (in vitroandin vivo) and in humans have established that Aldo, following the interaction with its receptor—the mineralocorticoid receptor (MR)—is able to activate specific intracellular genomic and nongenomic pathways, thus regulating the homeostasis of the cardiovascular system. Importantly, through this mechanism of action, this hormone becomes a crucial regulator of the function and growth of different types of cells, including fibroblasts, cardiomyocytes, and vascular cells. For this main reason, it is plausible that when Aldo is present at high levels in the blood, it profoundly modifies the physiology of these cells, therefore being at the foundation of several cardiovascular disorders, such as heart failure (HF). On these grounds, in this review, we will provide an updated account on the current knowledge concerning Aldo activity in the cardiovascular system and the most recent preclinical studies and clinical trials designed to test better approaches able to counter the hyperactivity of the Aldo/MR signaling pathway in the setting of cardiovascular diseases.

The study aimed at investigating the correlations among the physical and bulk properties of carrier based dry powder inhaler formulations and the performance of the powder inhaler device estimated by in-vitro tests for a specific active pharmaceutical ingredient (API), and at obtaining predictive models for the in-vitro performance. Samples from scale-up process batches having different formulations, process settings and bulk size, were characterized by rheological, density and particle size tests. In vitro performance was evaluated by several parameters obtained by a dosage unit sampling apparatus (DUSA) and a next generation impactor (NGI). Correlations between powder properties and performance properties were established using partial least square (PLS) regression analysis. Variable importance in projection (VIP) was used in order to assess the most influential powder characterization variables to estimate the analytical ones. Particle size, density and rate of flowability are significant for modeling the Delivered Dose of the API and the total quantity of powder related to each dose. Powder characterization variables, describing the degree of cohesiveness and the flow properties of powder, are related to the total amount of the active ingredient for different formulations. DUSA test variables were satisfactory predicted on the basis of powder characterization variables, while NGI performance variables were predicted with higher error.

Introduction and Hypothesis: Restricted diets are effective interventions to enhance cardiovascular function and metabolic profile and are known to improve life spam. IF (Intemittent fasting) dietary regimen has a cardioprotective effect in a rat model of myocardial infarction (MI) when diet is started before MI induction. In heart failure (HF), upregulation of G protein-coupled receptor kinase 2 (GRK2) contributes to dysfunctional beta-adrenergic receptor (βAR) signaling and to decrease cardiac inotropic reserve. Moreover, it has been shown that caloric restriction has positive effects on the development of left ventricular hypertrophy and improves ischemic tolerance. Hence, we will test whether a long-term restricted diet, started late after MI, is beneficial in HF and how it could affect cardiac adrenergic signaling. Methods: forty rats were randomly assigned to MI or sham operation. Four weeks later, a time point when post-ischemic HF was established, HF and sham rats were further randomized to a one year IF dietary restriction or ad libitum diet. Thus, our final animal population consisted in 4 groups: Sham normal diet, Sham IF diet, HF normal diet and HF IF diet. Results: One year of IF diet induced a robust decrease in body weight. In HF groups, restricted diet resulted in improved cardiac systolic function and reduced left ventricle end diastolic diameter compared to HF rats fed with normal diet, as measured by echocardiography at the end of the study period. No differences in cardiac function and dimension were observed between sham groups treated with different diets. Consistently, invasive hemodynamic showed that both LV contractility and relaxation in response to βAR stimulation were significantly increased in IF HF rats compared to HF normal diet animals. IF diet resulted in improved cardiac βAR density and adenylyl cyclase activity in HF rats when compared to HF rats treated with standard diet. Restoration of βAR signaling was associated to a dramatic reduction in cardiac GRK2 protein levels. Conclusions: We have demonstrated for the first time that IF, started when HF is already established, ameliorates cardiac function and inotropic reserve in an experimental model of HF. At the molecular level, IF diet significantly improves βAR signaling in HF.

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