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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 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.

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.

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.