Pulmonary arterial hypertension (PAH) manifests by increased proliferation and survival of pulmonary vascular cells in small pulmonary arteries (PAs), PA remodeling and unresolved increase of PA pressure. PA smooth muscle cells (PASMCs) in PAH undergo metabolic shift to glycolysis resulting in over-production of lactate, hyper-proliferation, and apoptosis resistance, but the mechanisms are not completely understood. By using lung tissues and pulmonary vascular cells from PAH and non-diseased human lungs, unbiased proteomics, network analysis, and gain-and-loss of function approaches, we here report that up-regulation of lactate dehydrogenase A (LDHA)-lactate axis promotes PASMC-specific over-lactylation and consequent over-accumulation of DNA topoisomerase 1 (TOP1) in small remodeled PAs from PAH lungs, leading to the up-regulation of Akt-mechanistic target of rapamycin 1 (mTORC1) signaling, hyper-proliferation, and reduced apoptosis. Smooth muscle-specific LDHA knockdown prevented, and Ldha inhibitor oxamate reversed SU5416/hypoxia-induced TOP1 accumulation, pulmonary vascular remodeling, and pulmonary hypertension (PH) in mice. Pharmacological inhibition of TOP1 with indotecan suppressed Akt-mTORC1, decreased proliferation, induced apoptosis in human PAH, but not control PASMCs, and reversed PA remodeling, PH, and RV dysfunction in rats. Collectively, these data provide a novel mechanistic link from LDHA-driven lactate over-production through lactylation and overaccumulation of TOP1, to the up-regulation of Akt-mTORC1, hyper-proliferation and apoptosis resistance of PASMCs, pulmonary vascular remodeling, and PH, and identify TOP1 as a new potentially attractive molecular target for the remodeling-focused therapeutic intervention. Take-home message:LDHA-lactate-induced over-lactylation and overaccumulation of Topoisomerase 1 (TOP1) promotes pulmonary artery smooth muscle cell hyper-proliferation, remodeling, and pulmonary arterial hypertension, which are reversed by TOP1 inhibitor indotecan.

Pulmonary arterial hypertension (PAH) is a progressive and potentially a rapidly fatal disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PA) leading to increased pulmonary vascular resistance and right heart failure. Central to the remodeling process is a switch of the smooth muscle cells in small PAs (PASMC) to a proliferative, apoptosis-resistant phenotype. There is reason to suspect that the plasminogen activator system may play an important role in the remodeling program in PAH based on its roles in vascular post-injury restenosis, fibrosis, angiogenesis and tumorigenesis. Plasminogen activator inhibitor-1 (PAI-1) is the primary physiological inhibitor of the plasminogen activators - urokinase-type and tissue-type (uPA and tPA, respectively). Immunohisto- chemical and immunoblot analyses revealed that PAI-1 was deficient in smooth muscle areas of small remodeled PAs and early-passage PASMC from subjects with PAH compared to non-PAH controls. PAI1-/- male and female mice developed spontaneous pulmonary vascular remodeling and pulmonary hypertension (PH) as evidenced by significant increase in PA medial thickness, systolic right ventricular pressure, and right ventricular hypertrophy. Lastly, the uPA inhibitors upamostat (WX-671) and amiloride analog BB2-30F down-regulated mTORC1 and SMAD3, restored PAI-1 levels, reduced proliferation, and induced apoptosis in human PAH PASMC. We examined the effect of inhibition of uPA catalytic activity by BB2-30F on the development of SU5416/Hypoxia (SuHx)-induced PH in mice. Vehicletreated SuHx-exposed mice had up-regulated mTORC1 in small PAs, developed pulmonary vascular remodeling and PH, as evidenced by significant increase of PA MT, sRVP, RV hypertrophy, and a significant decrease in the pulmonary artery acceleration time/pulmonary ejection time (PAAT/PET) ratio compared to age- and sex-matched normoxia controls, whereas BB2-30F-treated group was protected from all these pathological changes. Taken together, our data strongly suggest that PAI-1 down- regulation in PASMC from human PAH lungs promotes PASMC hyper-proliferation, remodeling, and spontaneous PH due to unopposed uPA activation. Further studies are needed to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate the progression and/or reverse pulmonary vascular remodeling and PH.

Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by a progressive increase in pulmonary vascular resistance leading to right ventricular failure and often death. Here we report that deficiency of transcription factor GATA6 is a shared pathological feature of PA endothelial (PAEC) and smooth muscle cells (PASMC) in human PAH and experimental PH, which is responsible for maintenance of hyper-proliferative cellular phenotypes, pulmonary vascular remodeling and pulmonary hypertension. We further show that GATA6 acts as a transcription factor and direct positive regulator of anti-oxidant enzymes, and its deficiency in PAH/PH pulmonary vascular cells induces oxidative stress and mitochondrial dysfunction. We demonstrate that GATA6 is regulated by the BMP10/BMP receptors axis and its loss in PAECs and PASMC in PAH supports BMPR deficiency. In addition, we have established that GATA6-deficient PAEC, acting in a paracrine manner, increase proliferation and induce other pathological changes in PASMC, supporting the importance of GATA6 in pulmonary vascular cell communication. Treatment with dimethyl fumarate resolved oxidative stress and BMPR deficiency, reversed hemodynamic changes caused by endothelial Gata6 loss in mice, and inhibited proliferation and induced apoptosis in human PAH PASMC, strongly suggesting that targeting GATA6 deficiency may provide a therapeutic advance for patients with PAH.

Background: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by remodeling of small pulmonary arteries (PA) due to hyper-proliferation of resident PA cells, leading to elevated right ventricular (RV) afterload and heart failure. PA smooth muscle (SM) cells (PASMC) in PAH have metabolic shift to glycolysis and lactate over-production. Goals: To investigate the role of lactate over-production in PA remodeling and PAH. Methods: RT-PCR, immunohistochemical, immunoblot, proteomic analyses; proliferation (Ki67), lactate, apoptosis assays; SU5416/Hypoxia (SuHx) rat and mouse models of PH. Results: Lactate dehydrogenase A (LDHA) was over-expressed in SMα-actin-positive areas of small muscular PAs from PAH subjects and rodents with SuHx PH, and distal human PAH PASMC compared to controls, promoting lactate over-production, consequent up-regulation of Yap/Taz, Akt-mTOR, increased proliferation and survival. SM22α-Ldha-/- mice were protected from SuHx-induced PH and RV hypertrophy. Lysine lactylation (Kla) was increased in small PAs and PASMC from human PAH lungs. Proteomic analysis identified 12 non-histone proteins that were hyper-lactylated in PAH PASMC. Following validation revealed that hyper-lactylation induced over-accumulation of TOP1 and deficiency of EMILIN1 in small PAs and PASMC from PAH lungs, leading to up-regulation of Yap/Taz, Akt-mTOR, TGFβ1, and increased proliferation. Lactate self-supported its over-production by up-regulating glycolysis and LDHA overexpression through EMILIN1-TGFβ1-HIF1α loop. Human PAH PASMC had elevated lactate secretion; exogenous lactate induced proliferation of control PAEC and PAAF. Pharmacological inhibition of LDHA-lactate reduced proliferation and selectively promoted apoptosis in human PAH PASMC, reversed SuHx-induced PA remodeling, PH, and RV hypertrophy in mice. Conclusions: LDHA-driven lactate over-production promotes proliferative, apoptosis-resistant PASMC phenotype, pulmonary vascular remodeling, and PH via hyper-lactylation and over-accumulation of TOP1, deficiency of EMILIN1, and consequent activation of Yap/Taz, Akt/mTOR, and TGFβ1. Targeting LDHA-lactate network could represent potentially attractive strategy to treat PAH.

Abnormal regulation of the repolarization process in cardiac myocytes is related to the induction of certain arrhythmias and ventricular dysfunction. Regulation of the repolarizing delayed outward rectifier current (IK) can be modulated by inotropic and inotrophic hormones, such as IGF‐1. The aim of this work was to assess the regulation of IK by the cell‐survival pathway, IGF‐1/PI3 K/Akt, in freshly isolated neonatal rat ventricular myocytes, using patch‐clamp technique in whole‐cell configuration. Cells were stimulated from holding potential of ‐40mV, by 500msec, 10‐mV step voltages to +40mV. IGF‐1 reduced the functional density of IK from 4.1 ± 0.5 pA/pF to 2.6 ± 0.3 pA/pF. IGF‐1 effect was eliminated by the PI3 Kinase inhibitor, LY294002 (3μM). Adenoviral transfection of cardiocytes with a constitutively activated PI3 K, BD110, mimicked the IGF‐1 effect on IK (2.5 ± 0.4 pA/pF), which was abrogated by LY 294002. Similar to BD110, transfection of the cardiac myocytes with wild‐type Akt reduced IK (2.4 ± 0.3 pA/pF), which was completely reversed by LY 294002. We conclude that IK is negatively regulated by IGF‐1 through downstream activation of PI3 Kinase and Akt. Such a mechanism could play an anti‐arrhythmic role during cardiac events that are associated with the activation of the PI3 K/Akt signaling, such as cardiac hypertrophy.

Angiotensin II (ANG II)-dependent signaling, namely MAP and PI3Kinase, play a role in the propagation of trophic signals during cardiac hypertrophy; and may affect the activity of ICa,L. To elucidate the role of MAPK and PI3K in the transduction of ANG II effects on ICa,L during hypertrophy, we used specific inhibitors for ERK1/2 (PD98059), p38 (SB203580) and PI3K/Akt (LY294002) during perforated patch-clamp on sham and shunt-induced eccentric hypertrophied cardiocytes from adult rats. ICa,L density did not change with hypertrophy. ANG II did not affect ICa,L in sham, but increased it by 21% in shunt cardiocytes. These effects of ANG II on ICa,L were not affected by Akt inhibition in both groups. However, LY 294002 alone diminished ICa,L density in sham (17%), but increased it in shunted animals (34%). In contrast, inhibition of p38 and ERK1/2 increased ICa,L density in both groups. Administration of ANG II with the MAPK inhibitors did not affect ICa,L density. From these data we conclude that MAP kinases inhibitors are able to increase ICa,L in both preparations. ANG II does not affect ICa,L activity in the sham, but increases it in the shunt through a MAP kinase- and not Akt-dependent pathway. Supported in part by grants GM08016-38 NIGMS/NIH and 2G12 RR003048 RCMI, Division of Research Infrastructure, NCRR/NIH.

Bradykinin (BK) and angiotensin II (ANG II) improve myocardial contractility. While ANG II induces cardiac hypertrophy through activation of MAPK and PI-3K, BK plays a major role in its regression. The purpose of this study was to elucidate the role of MAPK and PI-3K in the transduction of BK and ANG II signaling during the development of eccentric cardiac hypertrophy, induced by aorto-caval shunt in adult rats. Hearts were retrogradely superfused (20 min) with either Tyrode alone, or with ANG II (10−7M) or BK (10−6M). Activation of ERK1/2, p38 and Akt were assessed by western blot analysis for the relative level of phosphorylation versus total protein. As compared to sham, hypertrophied hearts showed higher activation of ERK1 (56%), ERK2 (227%), p38 (75%) and Akt (67%). In normal hearts, ANG II did not affect Akt activity but increased that of ERK1, ERK2 and p38 by 57%, 196%, 94%, respectively. However, ANG II had no major effect in the hypertrophied hearts. On the other hand, BK induced a minor activation of ERK1 (27%), ERK2 (31%), p38 (29%) and Akt (11%), in the normal hearts. However in the hypertrophied hearts, BK activation induced activation of ERK2 (42%) and p38 (57%). Thus, eccentric cardiac hypertrophy is associated with dysfunctional ANG II-MAPK activation but an enhanced BK-induced one. Supported in part by grants GM08016-38 NIGMS/NIH and 2G12 RR003048 RCMI, Division of Research Infrastructure, NCRR/NIH.

The outward rectifier (IK) and the inward rectifier (IK1) K+ channels, responsible for the action potential repolarization and maintenance of the resting potential respectively, are altered during cardiac hypertrophy. Activation of insulin-like growth factor-1 (IGF-1) during hypertrophy may affect channel activity. The aim was to study the modulatory effect of IGF-1 on IK and IK1 through EK1/2 and PI3K pathways during hypertrophy. Using specific inhibitors for ERK1/2 (PD98059) and PI3K/Akt (LY294002), whole-cell patch-clamp was conducted on sham and aortocaval shunt-induced eccentric cardiac hypertrophy myocytes from adult rats. In sham and shunt cardiocytes respectively: ERK1/2 inhibition caused reduction by 33% and 40% in IK density and 38% and 53% in that of IK1; Akt inhibition caused decrease by 29% and 23% in IK density and 47% and 17% in that of IK1. In the presence of LY294002, IGF-1 reduced IK density by 36% in sham and 23% in hypertrophied myocytes; whereas it had no effect on IK1 in both groups. In the presence of PD98059, IGF-1 had no affect on IK or IK1 in sham with slight 7% decrease in shunt. Thus, IK and IK1 are positively modulated by basal ERK and PI3K activities. ERK and PI3K are both sufficient and necessary for IK1 modulation by IGF-1; whereas only ERK is for IK. Cardiac hypertrophy is associated with a shift towards a larger role of PI3K in the modulation of both K+ channel. Supported in part by grants GM08016-38 NIGMS/NIH and 2G12 RR003048 RCMI, Division of Research Infrastructure, NCRR/NIH.

Angiotensin II (ANG II) and insulin‐like growth factor‐1 (IGF‐1) are activated early in the development of cardiac hypertrophy. We have shown that angiotensin‐converting enzymes (ACE) inhibitors regress eccentric hypertrophy and improve iontropy of the heart. The objective is to evaluate the role of MAPK and PI 3K in the transduction of ANG II and IGF‐1 signaling during the development and regression (by ACE inhibitor, Captopril 500mg/l) of eccentric cardiac hypertrophy. Western blot analysis revealed that in hypertrophied heart there is an increase in the activation of Akt (73%), p38 (124%), ERK1 (32%) and ERK2 (41%). In sham, ANG II (10−7M) increased Akt, p38, ERK1 and ERK2 phosphorylation by 37%, 165%, 28% and 73%, respectively. In hypertrophied hearts ANG II mainly increased p38 and ERK2 activation. IGF‐1 (10−8M) induced activation of Akt (160%) and p38 (139%) with no change in ERK1 and ERK2 activity in the sham hearts. However in the hypertrophied hearts, IGF‐1 induce activation of Akt (125%) and p38 (150%), with a significant activation of ERK2 (100%). Two‐week treatment with Captopril did not alter MAPK and PI 3K in the normal hearts. However, in shunt animals it decreased the activation level of Akt, with a minor effect on the activation level of ERK1; concomitant with 50% regression of cardiac hypertrophy.

The activities of the inward rectifier K+ channel (IK1), responsible for the maintenance of the resting potential, and the outward rectifier K+ channel (IK), controlling the repolarization of the action potential, are crucial for myocardial contraction; which is altered during eccentric cardiac hypertrophy. Angiotensin II (ANG II) has been shown to be a key element in the development of cardiac hypertrophy. The aim was to study the intracellular regulation of IK1 and IK by ANG II in sham and aortocaval shunt-induced eccentric cardiac hypertrophy in the adult rat. Whole-cell patch-clamp was conducted on freshly isolated ventricular myocytes from sham and shunt rat hearts in the presence of 1μM of either of the specific inhibitors for p38 (SB203580), ERK1/2 (PD98059) and PI 3K (LY294002). In the sham, the effect of ANG II on IK1 was completely abrogated upon ERK1/ERK2 inhibition, and it was reduced by more than 50% when p38 kinase activity was inhibited. In the hypertrophied myocytes, ANG II-induced effects on IK1 were completely prevented by either ERK1/ERK2 or Akt inhibition; whereas p38 inhibition reversed ANG II inhibition of IK1 into activation. On the other hand, neither p38 nor Akt inhibition affected the ANG II-induced decrement in IK; but it was prevented by ERK1/ERK2 inhibition in the sham. However, ANG II effect on IK was abrogated by ERK1/2, p38 or Akt inhibition in the hypertrophied ventricular myocytes. Thus, ANG II induced reductions in IK and IK1 seem to be mainly ERK1/2-dependent in the sham; but ERK1/2- and Akt-dependent in the shunt, in addition to p38 for IK. This project was supported by NIH/NIGMS SCORE S06GM08016-32

Given the central role of calcium in excitation-contraction coupling, changes in calcium homeostasis maybe expected to precede any loss of pump function. Cardiac hypertrophy has been shown to develop as a response to secreted humoral factors such as IGF-1and ANG II; thus their effect on calcium homeostasis may well be integral to cardiac performance during hypertrophy. The aim was to determine how increased levels of humoral factors present during cardiac hypertrophy affect calcium handling. Using perforated patch clamp techniques and fluorescence microscopy, we assessed the changes in membrane calcium currents and intracellular free calcium concentration in sham and volume-overload-induced hypertrophied (3-week) rat cardiomyocytes with acute administration of IGF-1 and ANG II. Results indicate that in normal cardiomyocytes, ICa,L decreased dose dependently when ANG II was administered, whereas IGF-1 caused a dose dependent increase in ICa,L. In hypertrophied myocytes, the response of ICa,L to IGF-1 (10−8 M) was significantly increased, while ANG II (10−6 M) served to augment ICa,L. Intracellular free calcium responses to ANG II (10-6 M) in normal myocytes showed dose-dependent increases, whereas in hypertrophied myocytes there was no significant change in [Ca2+]i. These data suggest that despite enhanced membrane calcium entry in hypertrophied cardiomyocytes due to ANG II and IGF-1, release of calcium from the sarcoplasmic reticulum is not correspondingly increased, perhaps revealing a defect in calcium handling during eccentric hypertrophy that may ultimately lead to decreased cardiac performance. Funded by NIH/NIGMS SCORE S06GM08016-32