Ischemic heart disease (IHD) is the major underlying cause of myocardial infarction (MI), scarring, and hypertrophy leading to heart failure. Cardiac remodeling following myocardial infarction involves scar formation by synthesis and reorganization of ECM which is mediated by myofibroblasts. We have previously shown that the mechanosensitive ion channel TRPV4 (transient receptor potential vanilloid channel 4) regulates cardiac fibroblast differentiation into myofibroblasts via integration of soluble and mechanical signaling. However, the physiological or translational significance of TRPV4 in cardiac remodeling following MI is unknown. To determine this, we have subjected WT and TRPV4KO mice to MI (permanent LAD ligation). 2D‐echocardiography revealed that the cardiac function (ejection fraction and fractional shortening) is preserved post‐MI in TRPV4KO mice compared to WT mice. Further, we found reduced fibrosis at infarcted and remote zones in TRPV4KO‐MI hearts compared to WT‐MI and sham hearts. Furthermore, TRPV4KO hearts exhibited decreased cardiomyocyte apoptosis (TUNEL assay) and increased capillary density (CD31 staining) post‐MI compared to WT hearts. To explore the translational significance of these findings, in separate experiments, we have given an orally active TRPV4 antagonist GSK2193874, immediately after MI surgery and followed for 5 weeks. Cardiac function analysis revealed that both ejection fraction and fractional shortening were preserved in GSK2193874‐treated WT mice compared to either WT or vehicle treated mice. Our results thus suggest that targeting TRPV4 protects the heart from myocardial infarction‐induced damage by preserving cardiac structure and function via reduced myocyte apoptosis, diminished fibrosis and increased revascularization, and identifies TRPV4 as a novel therapeutic target for heart failure.Support or Funding InformationNIH (1RO1HL119705), NIH (1R15CA202847‐01).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

VEGF signaling via VEGF receptor-2 (VEGFR2) is a major regulator of endothelial cell (EC) functions, including angiogenesis. Although most studies of angiogenesis focus on soluble VEGF signaling, mechanical signaling also plays a critical role. Here, we examined the consequence of disruption of mechanical signaling on soluble signaling pathways. Specifically, we observed that small interfering RNA (siRNA) knockdown of a mechanosensitive ion channel, transient receptor potential vanilloid 4 (TRPV4), significantly reduced perinuclear (Golgi) VEGFR2 in human ECs with a concomitant increase in phosphorylation at Y1175 and membrane translocation. TRPV4 knockout (KO) ECs exhibited increased plasma membrane localization of phospho-VEGFR2 compared with normal ECs. The knockdown also increased phospho-VEGFR2 in whole cell lysates and membrane fractions compared with control siRNA-treated cells. siRNA knockdown of TRPV4 enhanced nuclear localization of mechanosensitive transcription factors, yes-associated protein/transcriptional coactivator with PDZ-binding motif via rho kinase, which were shown to increase VEGFR2 trafficking to the plasma membrane. Furthermore, TRPV4 deletion/knockdown enhanced VEGF-mediated migration in vitro and increased expression of VEGFR2 in vivo in the vasculature of TRPV4 KO tumors compared with wild-type tumors. Our results thus show that TRPV4 channels regulate VEGFR2 trafficking and activation to identify novel cross-talk between mechanical (TRPV4) and soluble (VEGF) signaling that controls EC migration and angiogenesis.-Kanugula, A. K., Adapala, R. K., Midha, P., Cappelli, H. C., Meszaros, J. G., Paruchuri, S., Chilian, W. M., Thodeti, C. K., Novel noncanonical regulation of soluble VEGF/VEGFR2 signaling by mechanosensitive ion channel TRPV4.

VEGF and VEGFR2 signaling is the major regulator of endothelial functions such as proliferation, migration, and angiogenesis. While many studies on the modulation of angiogenesis focus on targeting VEGF signaling, mechanical forces also play critical role. We have recently shown that the mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) negatively regulates angiogenesis via modulation of Rho/Rho kinase‐dependent endothelial mechanosensing. Nevertheless, it is not known if a cross‐talk exists between TRPV4 and VEGF signaling. To explore this nexus, we silenced TRPV4 in endothelial cells (EC) and measured VEGFR2 expression, localization, and phosphorylation. Immunofluorescence analysis revealed significant localization of total‐VEGFR2 around the perinuclear (Golgi) compartment in control EC. Small interfering RNA (siRNA) knockdown of TRPV4 significantly decreased perinuclear localization of VEGFR2. Interestingly, we found increased phosphorylation of VEGFR2 at Y1175 and its localization to the plasma membrane in TRPV4 knocked‐down cells. We also found increased plasma membrane localization of phospho‐VEGFR2 Y1175 in TRPV4 null EC. Western blot analysis further revealed a significant increase in phospho‐VEGFR2 at Y1175 in whole cell lysates and membrane fractions of TRPV4 knocked‐down cells compared to control siRNA‐treated cells. Finally, TRPV4 deletion/knockdown also increased VEGF‐mediated migration with localization of phospho‐VEGFR2 at the leading edge of migrating EC. Taken together, our results suggest that TRPV4 channels regulate VEGFR2 localization and activation to identify novel cross‐talk between mechanical (TRPV4) and soluble (VEGF) signaling in EC.Support or Funding InformationThis work was supported by National Institutes of Health (NIH) R01HL119705 and National Cancer Institute (NCI) R15CA202847This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

RationaleOur previous studies have shown that hearts of Col6−/− mice are protected from myocardial infarction (MI) injury, as these hearts exhibit reduced myocyte apoptosis, fibrosis and wall thinning leading to preserved long‐term cardiac function. The current project was designed to test whether knockdown of Col6 is feasible in the rat MI model and carries a similar cardioprotective effect during post‐MI remodeling.ObjectiveTo utilize a novel, non‐viral delivery method to deliver and determine whether siRNA‐mediated knockdown of Col6 is effective in the infarcted rat heart and affords cardioprotection in vivo in the weeks following MI injury.Methods and ResultsMI was induced via ligation of the left anterior descending artery (LAD) in adult Sprague‐Dawley rats and cardiac function was measured by transthoracic echocardiography. Calculated changes in endocardial volume, percent ejection fraction (%EF), and percent fractional shortening (%FAC) were performed at 1–6 weeks post‐MI. We witnessed a decrease in mean endocardial %EF of 44.7 ± 1.4 % in vehicle treated (control) rats, a parameter that was higher in siRNA treated hearts at 47.4 ± 1.8 %. A similar improvement was also seen in endocardial %FAC, which was 30.1 ± 1.4 % in vehicle treated rats versus 34.0 ± 1.3 % in siRNA treated rats. Knockdown of Col6 in vivo resulted in lower endocardial diastolic volume in siRNA treated rats (654.1 ± 81.3 μl in the vehicle group compared to 549.8 ± 8.4 μl for the siRNA treated group) and lower systolic volume in siRNA treated rats (362.7 ± 49.9 μl in the vehicle group compared to 291.6 ± 9.6 μl in the siRNA treated group).ConclusionssiRNA mediated Col6 knockdown using JET‐PEI as a novel delivery method was effective in vivo, reducing Col6 expression levels over 75% in the anterior wall of the LV. Echocardiography revealed that the siRNA injections preserved cardiac function and prevented left ventricular chamber dilation following MI, indicating that this approach may have a positive therapeutic benefit in the recovery from MI injury.Support or Funding InformationNIH R15HL132312This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abnormal angiogenesis is a hallmark of pathological conditions characterized by leaky immature vessels. Most of the studies on pathological angiogenesis including vascular normalization strategies are focused on the inhibition of pro‐angiogenic growth factor signaling, which has been met with limited success. However, there are few studies on endogenous regulators and/or mechanical signaling in the regulation of pathological angiogenesis. We have recently found mechanosensitive ion channel transient receptor potential vanilloid 4 (TRPV4) to be functionally low in tumor‐derived endothelial cells which exhibit aberrant mechanosensitivity and abnormal angiogenesis that may be imparted due to high basal Rho activity. Further, we demonstrated that tumors grown in TRPV4KO animals show enhanced growth as well as leaky immature vasculature. In the present study, we investigated the molecular mechanisms by which TRPV4 modulates vascular integrity. First, immunostaining revealed that normal EC exhibited distinct VE‐cadherin expression at the cell‐cell contacts in vitro which is significantly reduced in TRPV4KO EC. Next, we measured vascular growth in varying stiffness Matrigel plugs (700–900 Pa) implanted in WT and TRPV4KO mice. Interestingly, we found increased vascular growth with increasing stiffness from TRPV4KO mice. In contrast, Matrigel plugs from WT mice showed no significant vascular growth with increasing stiffness. Surprisingly, we found a significant stiffness‐dependent reduction in VE‐cadherin positive vessels in Matrigel plugs from TRPV4KO mice compared to WT mice, despite an increase in vascular growth. Further, using an in vivo tumor model with WT and TRPV4KO mice, we found a significant increase in VE‐cadherin negative vessels in TRPV4KO mice compared to their WT counterparts. Functionally, we found tumor vessels are leaky which enhanced Lewis lung carcinoma (LLC) tumor metastasis to lung in TRPV4KO mice. Taken together, our findings suggest mechanosensitive ion channel TRPV4 regulates vessel integrity by maintaining VE‐cadherin expression at cell‐cell contacts and identifies TRPV4 as a novel target for vascular normalization therapies. Support or Funding Information This work is supported by American Heart Association (AHA) Grant‐in‐aid (14GRNT20380935), NIH‐R15CA202847, and start‐up funds from NEOMED (CKT).

Heart failure is one of the leading causes of death which is often characterized by pathological fibrosis. Cardiac remodeling following myocadial infarction is a multiphase reparative process which involves replacement of damaged tissue with physiological (reparative) fibrosis to form scar that limit the expansion of the infarct. Myofibroblasts are critical mediator of this reparative fibrosis, however, hyperactivation of these cells can cause pathological fibrosis leading to heart failure. We have previously demonstrated that the mechanosensitive ion channel TRPV4 (transient receptor potential vanilloid channel 4) regulates cardiac fibroblast differentiation into myofibroblasts. However, the physiological or translational significance of TRPV4 in cardiac remodeling following MI is unknown. To explore this, we have induced MI (permanent LAD ligation) in WT and TRPV4KO mice and measured cardiac function for 8 weeks. Separately, WT mice were given an orally active TRPV4 antagonist GSK2193874, immediately after MI surgery and followed for 5 weeks. 2D-echocardiography revealed that the cardiac function (ejection fraction and fractional shortening) is preserved post-MI in both TRPV4KO and GSK2193874-treated WT mice compared to either WT or vehicle treated mice. Further, we found reduced cardiac fibrosis at infarcted and remote zones in TRPV4KO and GSK2193874-treated WT mice compared to their MI counter parts. Furthermore, TRPV4KO hearts exhibited decreased cardiomyocyte apoptosis (TUNEL assay) and increased capillary density (CD31 staining) post-MI compared to WT hearts. Our results thus suggest that targeting TRPV4 protects heart from myocardial infarction-induced damage by preserving cardiac structure and function via reduced myocyte apoptosis, diminished fibrosis and increased revascularization, and identifies TRPV4 as a novel therapeutic target for heart failure.

The cardiac remodeling following cardiac injury, such as a myocardial infarction or heart attack, involves the differentiation of cardiac fibroblasts (CFs) into highly contractile and hyper‐secretory myofibroblasts. However, over deposition of extracellular matrix (ECM) can often times lead to cardiac fibrosis. Although both soluble and mechanical factors mediate CF differentiation, the mechanotransduction mechanisms are poorly understood. Recently, our lab has shown that the mechanosensitive ion channel Transient Receptor Potential Vanilloid 4 (TRPV4) plays an indispensable role in regulating CF differentiation by integrating soluble and mechanical factors in vitro. Here, we investigated the physiological significance and molecular mechanism(s) by which TRPV4 regulates cardiac remodeling in vivo. Using trans‐aortic constriction (TAC) to induce pressure‐overload injury, we found that TRPV4 knockout (TRPV4KO) mice demonstrated preserved ejection fraction and decreased left ventricular mass 28 days post‐injury, when compared to their wild‐type (WT) counterparts. Immunohistological analysis revealed that absence of TRPV4 resulted in decreased cardiomyocyte hypertrophy, fibrosis, and CF differentiation as evidenced by wheat germ agglutinin, picrosirius red, and alpha‐SMA staining, respectively. To delineate the molecular mechanisms, we stimulated CFs isolated from WT and TRPV4KO mice with TGF‐b1 and found that CF differentiation was significantly attenuated in TRPV4KO mCF. Further, pre‐treatment with TRPV4 antagonist, AB159908, significantly inhibited TGF‐b1‐induced activation of RhoA in WT mCFs, suggesting that Rho is downstream of TRPV4 in CF differentiation. Further, we found a mechanosensitive transcription factor, MRTF‐A (myocardin‐related transcription factor‐A), which is downstream of Rho, was activated by TGF‐b1 or TRPV4 agonist, GSK1016790A. Finally, inhibition of MRTF‐A attenuated TGF‐β1‐induced CF differentiation. Altogether, these findings indicate that TRPV4 channels mediate cardiac fibrosis through Rho/MRTF‐A pathway, and TRPV4 could be a novel therapeutic target for the treatment of pressure‐overload‐induced cardiac hypertrophy and heart failure.

Cardiac fibroblast (CF) differentiation is a critical event in the remodeling of the myocardium following cardiac injury. We have recently shown that mechanosensitive ion channel Transient Receptor Potential Vanilloid 4 (TRPV4) plays a key role in regulating CF differentiation by integrating soluble and mechanical signaling in rat cardiac fibroblasts. Here, we investigated the physiological significance of TRPV4 and the molecular mechanisms involved following pressure-overload. We induced pressure-overload using Trans Aortic Constriction (TAC) in wild type (WT) and TRPV4 knockout (KO) mice and monitored cardiac function for 28 days. We found that TRPV4KO mice exhibited decreased myocardial cross sectional area and left ventricular mass when compared with WT. Next, we analyzed cardiac function using 2D echocardiography, which revealed that TRPV4KO mice had preserved cardiac function (ejection fraction and fractional shortening) 28 days post-TAC. Histological analysis demonstrated that TRPV4KO mice displaye...

During heightened cardiac work, O2 consumption by the heart benefits energy production via mitochondria. However, some electrons leak from the respiratory chain and yield superoxide, which is rapidly metabolized into H2O2 by SOD2. To understand the systemic effects of the metabolic dilator, H2O2, we studied mice with cardiac-specific SOD2 overexpression (SOD2-tg), which increases the H2O2 produced by cardiac mitochondria. Contrast echocardiography was employed to evaluate cardiac function, indicating that SOD2-tg had a significantly greater ejection fraction and a lower mean arterial pressure (MAP) that was partially normalized by intravenous injection of catalase. Norepinephrine-mediated myocardial blood flow (MBF) was significantly enhanced in SOD2-tg mice. Coupling of MBF to the double product (Heart Rate×MAP) was increased in SOD2-tg mice, indicating that the metabolic dilator, "spilled" over, inducing systemic vasodilation. The hypothesis that SOD2 overexpression effectively enhances mitochondrial function was further evaluated. Mitochondria of SOD2-tg mice had a decreased state 3 oxygen consumption rate, but maintained the same ATP production flux under the basal and L-NAME treatment conditions, indicating a higher bioenergetic efficiency. SOD2-tg mitochondria produced less superoxide, and had lower redox activity in converting cyclic hydroxylamine to stable nitroxide, and a lower GSSG concentration. EPR analysis of the isolated mitochondria showed a significant decrease in semiquinones at the SOD2-tg Qi site. These results support a more reductive physiological setting in the SOD2-tg murine heart. Cardiac mitochondria exhibited no significant differences in the respiratory control index between WT and SOD2-tg. We conclude that SOD2 overexpression in myocytes enhances mitochondrial function and metabolic vasodilation, leading to a phenotype of supernormal cardiac function.

Background: Small studies suggested circulating microRNAs (circmiRs) as biomarkers for Heart Failure (HF). However, standardized approaches and quality assessment are not established, and results have been inconsistent, with little replication between studies. We aimed to implement quality standards to enable comparison between cohorts and assess which circmiRs may add prognostic information in HF. Methods: We measured 15 circ- miRs in two independent cohorts totaling >2000 subjects. Cohort I (Barcelona) comprised of n=843 chronic HFrEF patients. Cohort II from Detroit comprised n= 1384 chronic HF patients (892 HFrEF, 492 HFpEF). Each sample was measured in duplicate, and normalized to an abundant and stable circmiR (miR-486-5p). Algorithms were installed to define each circmiR measurement as “valid”, “unmeasurable” or “invalid”. This allowed inclusion of valid low-level circmiR measurements while reducing noise from false amplification sig- nals. Results: In general, between 20–40% of measurements were “invalid”, while miR-499a_5p and -208a were “unmeasurable” in the majority of patients in both cohorts. Higher levels of circmiRs-133b, -1254, -622, -208a and -499a_5p were significantly associated with risk of death in both cohorts, with hazard ratios ranging from 1.103 to 1.365 per log increase (p-values 0.001 to 0.05). However, adding these circmiRs to established predictors (age, renal function and NTproBNP) did not further augment the c-stat beyond 0.71 (cohort I) or 0.78 (cohort II). Conclusion: We developed stringent quality assessment for circmiR testing, and for the first time robustly validate the association of circmiRs 208a, -499a_5p, -133b, -1254 and -622 with risk of death in HF patients. However, circmiR levels failed to incrementally improve prognostication offered by current biomarkers, possibly due to the relative high number of invalid measurements. This highlights the shortcomings of current PCR-based technology. Novel technologies under study that improve signal-noise ratios may enhance the prognostic performance of circmiRs. de Z. K. Zwinderman: S. Pinto-Sietsma: None. Y. Pinto: Ownership Interest; Modest; owns shares biomarker company with IP rights related to miRNA. hallmark of diabetes associated with heart diseases and TNF-alpha is a key factor involved in inflammation. We tested whether downregulation of TNF-alpha could prevent cardiac fibrosis in diabetic mice. Methods and Results: Diabetes was in- duced in C57BL/6 mice by injecting streptozotocin (STZ, 55 mg/kg/day) for 5 days. Citrate buffer was injected as control. One week after STZ injection, blood glucose levels sig- nificantly increased in diabetic C57BL/6 mice (336 ± 26 mg/dl) compared to control (108 ±21 mg/dl). The increased blood glucose was accompanied by increased cardiac fibrosis (MassionTricrime), and increased TNF-alpha mRNA and protein expression in heart tissue. genome clustered regularly interspaced short palindrom- ic repeats (CRISPR)/Cas9 system loss-of-function mutation into the endogenous TNF-alpha gene in vivo, we constructed lentivirus expressing CRISPR-Cas9 and a CRISPR guide RNA targeting TNF-alpha. The lentivirus particles were transplanted into the mouse myocardium by direct intramyocardial injection at the time of initial induc- tion of diabetes. Within 5 days of administration of the lentivirus, the mutagenesis rate of TNF-alpha in the heart was as high as >50% by surveyor assay. No off-target mutagenesis was detected in other tissues such as lung and liver. The CRISPR/Cas9 based knockdown approach resulted in decreased cardiac fibrosis (MassionTricrime), accompanied by re- duced expression of TNF -alpha in heart assessed by PCR, Western blot and immunostain-ing. No significant change of other inflammatory cytokines including IL-6 was observed in the hearts. Furthermore, the CRISPR/Cas9 approach did not alter cell proliferation (BrdU staining) and cell survival (TUNEL assay) in heart. Conclusion: This study demonstrates that inflammation contributes to cardiac fibrosis via TNF-alpha signaling pathway, and suggests that diabetes related cardiac fibrosis may be prevented by downregulation of TNF-alpha via novel CRISPR-Cas9 approach. Author Disclosures: Y. Li: B. Liu: B. Lan: L. L. Y. P. Q. F. Background: Global regulatory mechanisms controlling the complex metabolic remodeling which occurs during the onset of heart failure are still poorly understood. However, recent advances have suggested a synergistic link between epigenetic regulation and cellular me- tabolism, although how this interplay is carried out in the heart is largely unknown. Our recent analyses of chromatin binding proteins differentially regulated during cardiac hyper- trophy and failure identified the histone methyltransferase Smyd1. To determine the role of Smyd1 in the adult myocardium we generated inducible, cardiac-specific Smyd1 knockout (Smyd1-KO) mice, which exhibit cellular hypertrophy, chamber remodeling and cardiac dysfunction. In addition, bioinformatics analysis of transcripts differentially expressed in Smyd1-KO heart tissue, before heart function declined, showed that cellular metabolism was the most perturbed biological process in these animals and suggests that Smyd1 may be a key regulator of energy metabolism. Methods and Results: To investigate this hy-pothesis we carried out metabolomic and gene expression analysis of Smyd1-KO heart tissue to comprehensively characterize the abundance of energetics-related transcripts and metabolites in these animals. Interestingly, our results revealed systemic dysfunction in energy substrate metabolism characterized by downregulation of fatty acid ß -oxidation (observed as a decrease in PPAR- α , carnitine-palmitoyltransferase I, carnitine transporter OCTN2 and myocardial carnitine content [43% reduction]) and branched-chain amino acid (BCAA) oxidation (observed as a 2-fold accumulation of all BCAAs and the decreased expression of PP2Cm, an key activator of BCAA catabolism). In addition, our results identified a dramatic increase in myocardial lactate and alanine (340% and 170% respectively), concomitant with decreased expression of pyruvate dehydrogenase E1 β , indicative of glycolytic impairment. Conclusion: Overall, this study identifies a novel role for Smyd1 in regulating energy metabolism in the heart and provides key insights into the epigenetic mechanisms modulating metabolic disorders such as heart failure. Author Disclosures: J. Shibayama: None. D.W. Barton: None. L. Wang: None. T.N. Yuzyuk: None. J. Cox: None. A.V. Zaitsev: None. S. Franklin: None. chronically administered for several weeks and may lead to cardiomyopathy and heart failure. Recently, circulating microRNAs (c-miRNAs) have been suggested as potential biomarkers of myocardial injury and drug-induced cardiotoxicity. However, the potential of c-miRNAs as biomarkers of DOX-induced cardiotoxicity in a clinical setting was not assessed. Therefore, the aim of this study is to evaluate the cardiotoxic effects of DOX on the circulating levels of miR-1, miR-133b, miR-146a, miR-208a, miR-208b and miR- 423-5p in breast cancer patients. Methods: In brief, 59 female patients (50,02±8,64 age) received 4 cycles of chemotherapy with cumulative doses of 60 mg/m 2 DOX during 12 weeks. Cardiac troponin I (cTnI), LVEF and c-miRNAs were measured before the treatment and every 3 weeks after DOX administration. Results: miR-208a and miR-208b were undetectable in plasma even after the maximum dose of DOX. Circulating levels of miR-1, miR-133b, miR-146a and miR-423-5p were increased along the treatment reaching its peak at the third cycle (increase of 18,6-fold, 11,51-fold, 10,56-fold and 12,09-fold respectively; P<0,001) and earlier than cTnI (fourth cycle). For further analysis, patients with 2 SD above the mean were grouped as “High cTnI” and compared to the others (“control”). High cTnI group (n= 7) showed a cTnI increase from 6.0±1.6 to 134.0±10.52 whereas from 6.4±0.2 to 31±2.8 pg/ml in the control group (n= 52; P<0,001). No changes in LVEF were observed within or between the groups. Despite all miRNAs analyzed had been upregulated during the treatment, only miR-1 and miR- 133b (muscle-specific miRNAs) were differently expressed in high cTnI patients showing a positive correlation with cTnI (P<0,05). Respectively, miR-1 and miR-133b were increased by 5,05-fold and 4,46-fold (P<0,05) in high cTnI patients when compared with controls. Conclusion: Circulating miR-1 and miR-133b were upregulated by DOX treatment and correlate with subtle myocardial injury in a “normal heart”. These findings may lead to the development of biomarkers to monitor DOX-induced subclinical myo- cardial injury avoiding its progression to irreversible cardiomyopathy and heart failure. Cardiac fibroblast (CF) differentiation into highly contractile and hypersecretory myo- fibroblasts (myoFibs) is critical for reparative fibrosis following myocardial injury/insult. However, excessive and remote area fibrosis by myoFibs can lead to cardiac dysfunction and eventual heart failure. Recently, we have shown that a mechanosensitive ion channel TRPV4 mediates CF differentiation to myoFib in-vitro. In the present study, we investigated the underlying molecular mechanism and the physiological role of TRPV4 during cardiac remodeling following pressure overload (transverse aortic constriction, TAC), in wild type (WT) and TRPV4 knockout (TRPV4KO) mice. We found that TRPV4KO mice exhibited not only improved survival rates compared to WT, but cardiac function analysis showed pre- served ejection fraction and fractional shortening in TRPV4 null mice, post-TAC surgeries. Importantly, we found that TAC induced both interstitial and perivascular fibrosis in WT hearts, which was significantly reduced in TRPV4KO hearts. To understand the molecular mechanism, we isolated CFs from WT and TRPV4KO mouse hearts (mCFs). In vitro, we found that TGF- β 1-induced differentiation was comple

Endothelial cell proliferation is a critical event during angiogenesis, regulated by both soluble factors and mechanical forces. Although the proliferation of tumor cells is studied extensively, little is known about the proliferation of tumor endothelial cells (TEC) and its contribution to tumor angiogenesis. We have recently shown that reduced expression of the mechanosensitive ion channel TRPV4 in TEC causes aberrant mechanosensitivity that result in abnormal angiogenesis. Here, we show that TEC display increased proliferation compared to normal endothelial cells (NEC). Further, we found that TEC exhibit high basal ERK1/2 phosphorylation and increased expression of proliferative genes important in the G1/S phase of the cell cycle. Importantly, pharmacological activation of TRPV4, with a small molecular activator GSK1016790A (GSK), significantly inhibited TEC proliferation, but had no effect on the proliferation of NEC or the tumor cells (epithelial) themselves. This reduction in TEC proliferation by TRPV4 activation was correlated with a decrease in high basal ERK1/2 phosphorylation. Finally, using a syngeneic tumor model revealed that TRPV4 activation, with GSK, significantly reduced endothelial cell proliferation in vivo . Our findings suggest that TRPV4 channels regulate tumor angiogenesis by selectively inhibiting tumor endothelial cell proliferation.

The proliferation of endothelial cells (EC) is a critical event in angiogenesis, regulated by both soluble factors and mechanical forces. Although unregulated proliferation of tumor cells is well known, very little is understood about Tumor EC (TEC) proliferation and their role in tumor angiogenesis. We have recently found that TEC express low functional levels of the mechanosensitive ion channel TRPV4 which imparts aberrant mechanosensitivity leading to abnormal angiogenesis by TEC. Here, we investigated if TRPV4 plays a role in TEC proliferation and tumor angiogenesis. First, we found tumors implanted in TRPV4-/- mice exhibited enhanced angiogenesis and increased growth compared to wild-type (WT) tumors. Next, aortic ring explants from TRPV4-/- mice displayed significantly increased angiogenesis compared to the WT controls. In vitro studies showed TEC (and TRPV4-null ECs) proliferated at higher levels than NEC. Subsequently, we found TEC and TRPV4-/- EC to have high basal ERK1/2 activity compared to their normal counterparts. Importantly, either TRPV4 overexpression or TRPV4 activation with a small molecule activator, GSK1016790A, significantly inhibited elevated basal ERK1/2 phosphorylation levels and cell proliferation in TEC. Interestingly, TRPV4 activation also inhibited the increased expression of cell cycle associated genes, responsible for cell proliferation. Finally, we found that EC proliferation is higher in WT tumors which was significantly inhibited by GSK1016790A. Taken together, these findings suggest TRPV4 regulates tumor angiogenesis via modulation of ERK1/2-dependent TEC proliferation.

Background: Hydrogen sulfide (H 2 S) has been shown to protect against myocardial ischemic and inflammatory injury in part by preserving mitochondrial integrity. Since mitochondrial antiviral signaling (MAVS) protein has been implicated in attenuating Bax-mediated cytochrome c release from mitochondria caused by oxidative stress or ischemia, we sought to determine whether MAVS mediates the cardioprotective effects of H 2 S. Methods and Results: After baseline echocardiography, adult male wild type (WT) or MAVS KO mice underwent myocardial infarction (MI) by coronary artery ligation for 30 min. followed by 24 h reperfusion. Mice were pretreated with Na 2 S (100 μg/kg; ip) or saline 1 h before MI. Infarct size, measured with TTC staining, was reduced and LV fractional shortening (FS) was preserved with Na 2 S at 24h post MI in WT mice as compared to saline-treated mice, but not in MAVS KO mice (Figs. A and B). The risk area was not different between the groups. Western blot analysis revealed a significant decline in myocardial MAVS expression at 24h post MI, which was preserved with Na 2 S (Fig. C). Another subset of mice was subjected to permanent coronary artery occlusion and treated with Na 2 S or saline daily for 28 days. LVFS decreased significantly at 28 days post-MI in the saline group, but was significantly preserved with Na 2 S (Fig. D). Moreover, LV infarct scar size, assessed by trichrome staining, was smaller in Na 2 S group (22.4 ± 2.7%) as compared to control (33.5 ± 2.1%, P<0.05). Survival rate was 2 fold higher with Na 2 S compared to saline (P<0.05). Western blot analysis confirmed a significant decrease in MAVS at 28 days after MI, which was associated with increased Bax expression (Fig. E). These changes were blunted with Na 2 S treatment. Conclusion: Na 2 S protects against acute MI and prevents MI-induced heart failure in mice possibly through a mechanism involving MAVS. We propose that preserving MAVS with H 2 S donors can be a promising therapeutic tool for ischemic heart failure.

Ischemic heart disease (IHD) is the major underlying cause of myocardial infarction (MI), scarring, and hypertrophy leading to heart failure. The prevention of ischemic damage due to MI involves scar formation by synthesis and reorganization of ECM. Cardiac fibroblasts are the primary mediators of cardiac repair as they secrete and remodel extracellular matrix in the heart. We have recently demonstrated that mechanosensitive ion channel, TRPV4 (Transient Receptor Potential Vanilloid 4) mediates cardiac fibroblast differentiation into myofibroblasts in vitro. However, the physiological significance of TRPV4 in cardiac remodeling in vivo is not known. In the present study, we explored the role of TRPV4 in cardiac remodeling following MI. We subjected WT and TRPV4KO mice to MI (permanent occlusion of LAD) and measured cardiac function for 8 weeks by serial echocardiographic assessment. We found significant time‐dependent preservation of cardiac function (ejection fraction and fractional shortening) post‐MI in TRPV4KO mice compared to WT counterparts. Further, picro‐sirius red and Masson's trichrome staining revealed reduced collagen deposition in infarcted and remote zones of TRPV4KO hearts, 8 weeks post‐MI. Mechanistically, we found decreased cardiomyocyte apoptosis (TUNEL assay) and increased capillary density (CD31 staining) in TRPV4KO hearts compared to WT hearts, 1 week post‐MI. Taken together, these results suggest that absence of TRPV4 preserves cardiac function and structure post‐MI by reducing myocyte apoptosis and cardiac fibrosis probably by increasing revascularization.Support or Funding InformationNIH(1RO1HL119705)NIH(1R15CA202847‐01), AHA Grant in Aid(14GRNT20380935)

The impaired development of tumor vessels, attributed to their highly permeable, tortuous, and hyper‐dilatory properties make it a barrier for the efficient delivery of anti‐cancer agents. Although anti‐VEGF therapies have shown to induce transient normalization of the tumor vasculature and increase the efficacy of cancer drugs, they are met with limited success clinically. Here, we demonstrate that a mechanosenstive ion channel TRPV4 regulates tumor angiogenesis and tumor vessel maturation. Tumor xenograft experiments revealed that, compared to tumors in wild type (WT) mice, those in TRPV4 null mice exhibit greater vascular density and immaturity (increased vessel diameter and reduced pericyte coverage). Endothelial cells derived from the tumor (TEC) showed reduced TRPV4 expression and activity associated with high basal Rho activity as well as aberrant mechanosensitivity and abnormal angiogenesis. Activation of TRPV4 with a small‐molecule activator, GSK1016790A, or TRPV4 overexpression suppressed the endogenously high rho activity, normalized the aberrant capillary formation and abnormal migration exhibited by TEC in vitro. Finally, GSK1016790A in combination with anti‐cancer drug Cisplatin, but not alone, reduced tumor growth in WT mice by normalizing the tumor vessels. Together, these studies demonstrate that TRPV4 channels are critical regulators of tumor angiogenesis, and represent a potential novel therapeutic target for tumor vascular normalization and improving the efficacy of anti‐cancer drugs.

Diabetes is an independent risk factor for cardiovascular disease that can eventually cause cardiomyopathy and heart failure. Cardiac fibroblasts (CF) are the critical mediators of physiological and pathological cardiac remodeling; however, the effects of hyperglycemia on cardiac fibroblast function and differentiation is not well known. Here, we performed a comprehensive investigation on the effects of hyperglycemia on cardiac fibroblasts and show that hyperglycemia enhances cardiac fibroblast function and differentiation. We found that high glucose treatment increased collagen I, III, and VI gene expression in rat adult cardiac fibroblasts. Interestingly, hyperglycemia increased CF migration and proliferation that is augmented by collagen I and III. Surprisingly, we found that short term hyperglycemia transiently inhibited ERK1/2 activation but increased AKT phosphorylation. Finally, high glucose treatment increased spontaneous differentiation of cardiac fibroblasts to myofibroblasts with increasing passage compared with low glucose. Taken together, these findings suggest that hyperglycemia induces cardiac fibrosis by modulating collagen expression, migration, proliferation, and differentiation of cardiac fibroblasts.

Cardiac remodeling is triggered by a cascade of events following myocardial injury, including the differentiation of cardiac fibroblasts (CFs) into myofibroblasts. Myofibroblasts are hypersecretory and highly contractile cells, however, in excess, can lead to cardiac dysfunction and eventual heart failure. Recently, our lab has shown, in vitro, that TRPV4, a mechanosensitive ion channel, integrates mechanical and soluble signals to aid in the differentiation process of CFs. Our current study aims to investigate physiological role of TRPV4 during the remodeling process by subjecting wild type (WT) and TRPV4 knockout (KO) mice to myocardial infarction (MI) or transverse aortic constriction (TAC). Assessment of the post‐surgery survival and cardiac function found the TRPV4 KO mice to have improved survival rates and preserved ejection fraction. Cardiac fibrosis was examined using picrosirius red staining which revealed that the TRPV4 KO mice had significantly less fibrosis than their WT counterparts. Furthermore, a comparison of gene expression in the whole heart tissue allowed analysis of pro‐fibrotic genes, such as col1A2, α‐SMA, N‐FAT, TGF‐β1, and mechanosensitive transcription factor MRTF‐a, and found a reduction in the expression of these genes in the TRPV4 KO mice pre‐ and post‐cardiac injury. Altogether, our findings suggest that the absence of TRPV4 improves the cardiac function and remodeling following myocardial injury, and could lead to a novel therapeutic target for cardiac fibrosis.Grant Funding Source: Supported by NIH‐1R15HL106442‐01