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BackgroundAcute decompensation is associated with increased mortality in heart failure (HF) patients, though the underlying etiology remains unclear. Extracellular vesicles (EVs) and their cargo may mark specific cardiovascular physiologic states. We hypothesized that EV transcriptomic cargo, including long non-coding RNAs (lncRNAs) and mRNAs, is dynamic from the decompensated to recompensated HF state, reflecting molecular pathways relevant to adverse remodeling.MethodsWe examined differential RNA expression from circulating plasma extracellular RNA in acute HF patients at hospital admission and discharge alongside healthy controls. We leveraged different exRNA carrier isolation methods, publicly available tissue banks, and single nuclear deconvolution of human cardiac tissue to identify cell and compartment specificity of the topmost significantly differentially expressed targets. EV-derived transcript fragments were prioritized by fold change (−1.5 to + 1.5) and significance (ResultsWe identified 138 lncRNAs and 147 mRNAs (present mostly as fragments in EVs) differentially expressed between HF and control. Differentially expressed transcripts between HFrEF vs. control were primarily cardiomyocyte derived, while those between HFpEF vs. control originated from multiple organs and different (non-cardiomyocyte) cell types within the myocardium. We validated 5 lncRNAs and 6 mRNAs to differentiate between HF and control. Of those, 4 lncRNAs (AC092656.1, lnc-CALML5-7, LINC00989, RMRP) were altered by decongestion, with their levels independent of weight changes during hospitalization. Further, these 4 lncRNAs dynamically responded to stress in cardiomyocytes and pericytesin vitro, with a directionality mirroring the acute congested state.ConclusionCirculating EV transcriptome is significantly altered during acute HF, with distinct cell and organ specificity in HFpEF vs. HFrEF consistent with a multi-organ vs. cardiac origin, respectively. Plasma EV-derived lncRNA fragments were more dynamically regulated with acute HF therapy independent of weight change (relative to mRNAs). This dynamicity was further demonstrated with cellular stressin vitro. Prioritizing transcriptional changes in plasma circulating EVs with HF therapy may be a fruitful approach to HF subtype-specific mechanistic discovery.CLINICAL PERSPECTIVEWhat is new?We performed extracellular transcriptomic analysis on the plasma of patients with acute decompensated heart failure (HFrEF and HFpEF) before and after decongestive efforts.Long non-coding RNAs (lncRNAs) within extracellular vesicles (EVs) changed dynamically upon decongestion in concordance with changes within human iPSC-derived cardiomyocytes under stress.In acute decompensated HFrEF, EV RNAs are mainly derived from cardiomyocytes, whereas in HFpEF, EV RNAs appear to have broader, non-cardiomyocyte origins.What are the clinical implications?Given their concordance between human expression profiles and dynamicin vitroresponses, lncRNAs within EVs during acute HF may provide insight into potential therapeutic targets and mechanistically relevant pathways.These findings provide a “liquid biopsy” support for the burgeoning concept of HFpEF as a systemic disorder extending beyond the heart, as opposed to a more cardiac-focused physiology in HFrEF.

BackgroundCardiorenal syndrome (CRS)—renal injury during heart failure (HF)—is linked to higher morbidity. Whether circulating extracellular vesicles (EVs) and their RNA cargo directly impact its pathogenesis remains unclear.MethodsUsing a microfluidic kidney chip model (KC), we investigated transcriptional effects of circulating EVs from patients with CRS on renal epithelial/endothelial cells. We used small RNA-seq on circulating EVs and regression to prioritize subsets of EV miRNAs associated with serum creatinine, a biomarker of renal function.In silicopathway analysis, human genetics, and interrogation of expression of miRNA target genes in the KC model and in a separate cohort of individuals post-renal transplant with microarray-based gene expression was performed for validation.ResultsRenal epithelial and endothelial cells in the KC model exhibited uptake of EVs. EVs from patients with CRS led to higher expression of renal injury markers (IL18,NGAL,KIM1) a greater cystatin C secretion relative to non-CRS EVs. Small RNA-seq and regression identified 15 miRNAs related to creatinine, targeting 1143 gene targets specifying pathways relevant to renal injury, including TGF-b and AMPK signaling. We observed directionally consistent changes in expression of TGF-b pathway members (BMP6, FST, TIMP3) in KC model exposed to CRS EVs, as well as in renal tissue after transplant rejection. Mendelian randomization suggested a role for FST in renal function.ConclusionEVs from patients with CRS directly elicit adverse transcriptional and phenotypic responses in a KC model by regulating biologically relevant pathways, suggesting a novel role for EVs in CRS.Trial RegistrationClinicalTrials.govNCT 03345446.FundingAHA (SFRN16SFRN31280008), NHLBI (1R35HL150807-01) and NCATS (UH3 TR002878).

Increasing evidence shows that endothelial cells play critical roles in maintaining vascular homeostasis, regulating vascular tone, inhibiting inflammatory response, suppressing lipid leakage, and preventing thrombosis. The damage or injury of endothelial cells induced by physical, chemical, and biological risk factors is a leading contributor to the development of mortal cardiovascular and cerebrovascular diseases. However, the underlying mechanism of endothelial injury remains to be elucidated. Notably, no drugs effectively targeting and mending injured vascular endothelial cells have been approved for clinical practice. There is an urgent need to understand pathways important for repairing injured vasculature that can be targeted with novel therapies. Exercise training-induced protection to endothelial injury has been well documented in clinical trials, and the underlying mechanism has been explored in animal models. This review mainly summarizes the protective effects of exercise on vascular endothelium and the recently identified potential therapeutic targets for endothelial dysfunction.

Metabolic remodeling of cardiac muscles during pathological hypertrophy is characterized by downregulation of fatty acid oxidation (FAO) regulator, peroxisome proliferator-activated receptor alpha (PPARα). Thereby, we hypothesized that a cardiac-specific induction of PPARα might restore the FAO-related protein expression and resultant energy deficit. In the present study, consequences of PPARα augmentation were evaluated for amelioration of chronic oxidative stress, myocyte apoptosis, and cardiac function during pathological cardiac hypertrophy.Nanotized PPARα overexpression targeted to myocardium was done by a stearic acid-modified carboxymethyl-chitosan (CMC) conjugated to a 20-mer myocyte-targeted peptide (CMCP). Overexpression of PPARα ameliorated pathological hypertrophy and improved cardiac function. Augmented PPARα in hypertrophied myocytes revealed downregulated p53 acetylation (lys 382), leading to reduced apoptosis. Such cells showed increased binding of PPARα with p53 that in turn reduced interaction of p53 with glycogen synthase kinase-3β (GSK3β), which upregulated inactive phospho-GSK3β (serine [Ser]9) expression within mitochondrial protein fraction. Altogether, the altered molecular milieu in PPARα-overexpressed hypertrophy groups restored mitochondrial structure and function both in vitro and in vivo.Cardiomyocyte-targeted overexpression of a protein of interest (PPARα) by nanotized plasmid has been described for the first time in this study. Our data provide a novel insight towards regression of pathological hypertrophy by ameliorating mitochondrial oxidative stress in targeted PPARα-overexpressed myocardium.PPARα-overexpression during pathological hypertrophy showed substantial betterment of mitochondrial structure and function, along with downregulated apoptosis. Myocardium-targeted overexpression of PPARα during pathological cardiac hypertrophy led to an overall improvement of cardiac energy deficit and subsequent cardiac function, thereby, opening up a potential avenue for cardiac tissue engineering during hypertrophic cardiac pathophysiology.

Cardiac hypertrophy and associated heart fibrosis remain a major cause of death worldwide. Phytochemicals have gained attention as alternative therapeutics for managing cardiovascular diseases. These include the extract from the plant Terminalia arjuna, which is a popular cardioprotectant and may prevent or slow progression of pathological hypertrophy to heart failure. Here, we investigated the mode of action of a principal bioactive T. arjuna compound, arjunolic acid (AA), in ameliorating hemodynamic load-induced cardiac fibrosis and identified its intracellular target. Our data revealed that AA significantly represses collagen expression and improves cardiac function during hypertrophy. We found that AA binds to and stabilizes the ligand-binding domain of peroxisome proliferator-activated receptor α (PPARα) and increases its expression during cardiac hypertrophy. PPARα knockdown during AA treatment in hypertrophy samples, including angiotensin II-treated adult cardiac fibroblasts and renal artery-ligated rat heart, suggests that AA-driven cardioprotection primarily arises from PPARα agonism. Moreover, AA-induced PPARα up-regulation leads to repression of TGF-β signaling, specifically by inhibiting TGF-β-activated kinase1 (TAK1) phosphorylation. We observed that PPARα directly interacts with TAK1, predominantly via PPARα N-terminal transactivation domain (AF-1) thereby masking the TAK1 kinase domain. The AA-induced PPARα-bound TAK1 level thereby shows inverse correlation with the phosphorylation level of TAK1 and subsequent reduction in p38 MAPK and NF-κBp65 activation, ultimately culminating in amelioration of excess collagen synthesis in cardiac hypertrophy. In conclusion, our findings unravel the mechanism of AA action in regressing hypertrophy-associated cardiac fibrosis by assigning a role of AA as a PPARα agonist that inactivates non-canonical TGF-β signaling.

Altered cardiac adaptation of physiologically hypertrophied heart during detraining remained obscure for long time. We had previously reported the switching of protein kinase C (PKC) isoforms (-α to -δ) associated with functional deterioration of heart at detraining in mice undergone swim exercise. Here we report that, myocardium targeted overexpression of insulin-like growth factor 1 (IGF1) and knockdown of insulin-like growth factor 1 receptor (IGF1R) during detraining and exercise respectively altered the activation of PKCs and eventual cardiac condition. Moreover, downregulation of mammalian target of rapamycin complex 2 (mTORC2) was recorded in both IGF1R knockdown or detraining groups. Additionally, knocking down of mTORC2 during exercise exhibited impaired cardiac condition. Interestingly, significantly increased interactions of mTORC2 with both PKCα and δ was recorded exclusively in exercise group. This interaction resulted into hydrophobic motif phosphorylation of both PKCs (Serine657-PKCα; Serine662-PKCδ). Serine phosphorylation on one hand activated PKCα mediated cell survival and on the other hand alleviated the apoptotic activity of PKCδ during exercise. Mutation of Serine662 of PKCδ in exercised mice showed higher Tyrosine311 phosphorylation with increased apoptotic load similar to that in detrained animals. These observations confirmed that differential and conditional activation of PKCs depend upon IGF1 induced mTORC2 activation. Furthermore, blocking of PKCα resulted in activated p53 which in turn repressed IGF1 expression during swim, mimicking the condition of detrained heart. In conclusion, this is the first report to unravel the intricate molecular mechanism of switching a physiologically hypertrophied heart to a pathologically hypertrophied heart during exercise withdrawal.

Diverse array of therapeutic regimens, drugs or siRNA, are commonly used to regress cardiac hypertrophy, although, bystander effect and lower retention of bioactive molecules significantly reduce their functional clinical efficacy. Carvedilol, a widely used and effective anti-hypertrophic drug, simultaneously blocks β-adrenergic receptors non-specifically in various organs. Likewise, non-specific genome-wide downregulation of p53 expression by specific siRNA efficiently abrogates cardiac hypertrophy but results in extensive tumorigenesis affecting bystander organs. Therefore, delivery of such therapeutics had been a challenge in treating cardiovascular dysfunction. Cardiac tissue engineering was successfully accomplished in this study, by encapsulating such bioactive molecules with a stearic acid modified Carboxymethyl chitosan (CMC) nanopolymer conjugated to a homing peptide for delivery to hypertrophied cardiomyocytes in vivo. The peptide precisely targeted cardiomyocytes while CMC served as the vector mediator to pathological myocardium. Controlled delivery of active therapeutic payloads within cardiomyocytes resulted in effective regression of cardiac hypertrophy. Thus, this novel nano-construct as a spatio-temporal vector would be a potential tool for developing effective therapeutic strategies within cardiac micro-environment via targeted knockdown of causal genes.