Therapeutic Target For Cardioprotection Research Articles

Non-coding RNAs and their potential exploitation in cancer therapy-related cardiotoxicity.

Life expectancy in cancer patients has been extended in recent years, thanks to major breakthroughs in therapeutic developments. However, this also unmasked an increased incidence of cardiovascular diseases in cancer survivors, which is in part attributable to cancer therapy-related cardiovascular toxicity. Non-coding RNAs (ncRNAs) have received much appreciation due to their impact on gene expression. NcRNAs, which include microRNAs, long ncRNAs and circular RNAs, are non-protein-coding transcripts that are involved in the regulation of various biological processes, hence shaping cell identity and behaviour. They have also been implicated in disease development, including cardiovascular diseases, cancer and, more recently, cancer therapy-associated cardiotoxicity. This review outlines key features of cancer therapy-associated cardiotoxicity, what is known about the roles of ncRNAs in these processes and how ncRNAs could be exploited as therapeutic targets for cardioprotection.

Promoting mitochondrial fusion in doxorubicin-induced cardiotoxicity: a novel therapeutic target for cardioprotection.

Changes in mitochondrial dynamics have been recognized as being one of the mechanisms related to cardiotoxicity following a high cumulative dose of doxorubicin (DOX). A mitochondrial division inhibitor-1 (Mdivi-1) and fusion promoter (M1) have been shown to be cardioprotective in a variety of cardiovascular settings, however, their anticardiotoxic efficacy against DOX therapy remains unclear. We therefore investigated whether treatment with Mdivi-1 and M1 protects the heart against DOX-induced cardiotoxicity via mitochondria-targeted pathways. Male Wistar rats (n=40) received DOX (3 mg/kg, six doses, n=32) or 3% dimethylsulfoxide (DMSO) in the normal saline solution (NSS) (n=8) as a control. DOX-injected rats were given one of four treatments beginning with the first DOX injection via intraperitoneal injection: 1) 3% DMSO in NSS (n=8), 2) Mdivi-1 (1.2 mg/kg per day, n=8), 3) M1 (2 mg/kg per day, n=8), and 4) Mdivi-1+M1 (n=8) for 30 days. Cardiac function, mitochondrial function, oxidative stress, myocardial injury, and protein expression associated with inflammation, autophagy, mitophagy, apoptosis, and mitochondrial dynamics were determined. DOX caused a significant deterioration in mitochondrial function and dynamic regulation, and an increase in markers of oxidative stress, inflammation, myocardial injury, apoptosis, autophagy, and mitophagy, resulting in impaired cardiac function. Cotreatment of DOX with Mdivi-1, M1, or a combination of the two mitigated these detrimental effects of DOX. These findings imply that either inhibiting fission or promoting fusion of mitochondria protects the heart from DOX-induced myocardial damage. Modulation of mitochondrial dynamics could be a novel therapeutic target in alleviating DOX-induced cytotoxic effects without compromising its anticancer efficacy.

Trpc6 Promotes Doxorubicin-Induced Cardiomyopathy in Male Mice With Pleiotropic Differences Between Males and Females.

Background: Doxorubicin is a widely used and effective chemotherapy, but the major limiting side effect is cardiomyopathy which in some patients leads to congestive heart failure. Genetic variants in TRPC6 have been associated with the development of doxorubicin-induced cardiotoxicity, suggesting that TRPC6 may be a therapeutic target for cardioprotection in cancer patients.Methods: Assessment of Trpc6 deficiency to prevent doxorubicin-induced cardiac damage and function was conducted in male and female B6.129 and Trpc6 knock-out mice. Mice were treated with doxorubicin intraperitoneally every other day for a total of 6 injections (4 mg/kg/dose, cumulative dose 24 mg/kg). Cardiac damage was measured in heart sections by quantification of vacuolation and fibrosis, and in heart tissue by gene expression of Tnni3 and Myh7. Cardiac function was determined by echocardiography.Results: When treated with doxorubicin, male Trpc6-deficient mice showed improvement in markers of cardiac damage with significantly reduced vacuolation, fibrosis and Myh7 expression and increased Tnni3 expression in the heart compared to wild-type controls. Similarly, male Trpc6-deficient mice treated with doxorubicin had improved LVEF, fractional shortening, cardiac output and stroke volume. Female mice were less susceptible to doxorubicin-induced cardiac damage and functional changes than males, but Trpc6-deficient females had improved vacuolation with doxorubicin treatment. Sex differences were observed in wild-type and Trpc6-deficient mice in body-weight and expression of Trpc1, Trpc3 and Rcan1 in response to doxorubicin.Conclusions: Trpc6 promotes cardiac damage following treatment with doxorubicin resulting in cardiomyopathy in male mice. Female mice are less susceptible to cardiotoxicity with more robust ability to modulate other Trpc channels and Rcan1 expression.

Abstract 9481: Inhibition of Apoptosis and Ferroptosis Signaling Pathways Alleviates Myocardial Ischemia-Reperfusion Injury in Rats Through Modulation of Mitochondrial Function

Introduction: Apoptosis and ferroptosis have been implicated in cardiac ischemia/reperfusion (I/R) injury. Inhibitors of apoptosis (Z-VAD) and ferroptosis (FER-1) have been shown to protect the heart in several cardiovascular disorders. However, the impacts of Z-VAD, FER-1, and combined therapy during cardiac I/R injury have not been elucidated. Hypothesis: Z-VAD and FER-1 provide cardioprotection by reducing myocardial cell injury and improving cardiac function. The combined treatment exerts better benefits than either single regimen. Methods: Male rats (n=25) were equally divided into 5 groups: sham, vehicle, Z-VAD at 3.3 mg/kg, FER-1 at 2 mg/kg, and combined FER-1 and Z-VAD. All treatments were intravenously injected to the rats 15 min before ischemia. Left anterior descending coronary was ligated for 30 min, followed by 120-min reperfusion in all animals except the sham group. Left ventricular ejection fraction (LVEF), infarct size, mitochondrial reactive oxygen species (ROS) production, and protein expressions of apoptosis and ferroptosis were determined. Results: Cardiac I/R injury led to cardiac mitochondrial dysfunction and cell death, contributed to infarct size expansion and LV dysfunction (Fig 1A-D). Z-VAD, FER-1 and combined treatments markedly increased %LVEF, reduced ROS levels and infarct size (the %reduction is 42.9%, 41.4%, and 47.7%, respectively, compared with vehicle). Cleaved caspase 3 (apoptosis) and ACSL4 (ferroptosis) expression levels were also reduced in those rats. The combined treatment did not show a significant advantage, compared with either single regimen (Fig 1A-D). Conclusions: Inhibitors of either apoptosis or ferroptosis similarly reduced cardiac mitochondrial dysfunction, apoptosis and ferroptosis, resulting in decreased infarct size, and finally leading to improved LV function. These findings indicate that ferroptosis could be a novel therapeutic target for cardioprotection in cardiac I/R injury.

266Loss of pumilio 1 phosphorylation turns off the regulation of miRNA-221 on gene p27kip1 in the heart

Abstract Background MicroRNAs regulate gene expression post-transcriptionally via complementary sequence pairing of mRNA 3'UTR and miRNA seed region. MiR-221 downregulates p27kip1 Cyclin-dependent kinase inhibitor 1B (p27), a cell cycle inhibitor, through direct targeting. The down-regulation of p27 caused by miR-221 upregulation induces cancer cell proliferation and has been reported to be closely associated with human cancer metastasis. This regulation is dependent on the existence of pumilio 1 (PUM1). Meanwhile miR-221 mimics protect cardiac myocytes against ischemic injury. It is a potential therapeutic target for cardioprotection. We investigated whether miR-221 targeted p27 in the heart. Purpose To test the hypothesis that the regulation of miR-221 on p27 is abolished in the heart due to lack of RNA binding protein (RBP) PMU1 phosphorylation but not due to p27 3'UTR shortening. Methods Primary cardiac fibroblasts (cFB) were isolated and cultured under normoxia and hypoxia. After transfections of miR-221 mimics and mimic control (MC), cell proliferation was assessed by cell number, CCk-8 and Ki67 staining. In vivo rats undergoing left coronary artery ligation (MI) and Sham were treated with intravenous miR-221 mimics. Cells or cardiac tissue were collected to measure p27 and PUM1 by RT-qPCR and Western blot (WB). Full length p27 (coding plus 3'UTR sequence) and 3'UTR p27 sequence were quantified using specific primers. The ratio of 3'UTR/Full length was compared to indicate changes between groups. The ratios of 3'UTR/Full length were compared to indicate changes between groups. Results MiR-221 mimics significantly downregulated the expression of p27 mRNA and protein in cFB in both normoxia and hypoxia. P27 downregulation led to cFB proliferation, which could be abrogated by p27 overexpression. miR-221 mimic treatment increased cardiac miR-221 by ∼2–5 fold in Sham and ∼15 fold in MI. Whilst p27 expression was not affected in either Sham or MI hearts. Simultaneously α-SMA+ cells, an indication of cFB number and activation, were reduced by miR-221 mimic in Sham and MI heart. The ratios of p27 3'UTR/full length did not vary significantly between cultured cFB and heart (Sham and MI), indicating the 3'UTR length did not vary between cFB and the whole heart. PUM1 mRNA expression was stable in cFB and the heart. However, phosphorylated PUM1 was highly expressed in cFB but completely diminished in Sham and MI hearts. Conclusion MiR-221 downregulated p27 in primary cFB and induced cell proliferation in vitro. miR-221 could not regulate cardiac p27 expression nor increase cFB proliferation in vivo. MiR-221 induced p27 regulation was correlated with the presence or absence of phosphorylated PUM1. For the first time, we demonstrated that loss of phosphorylated PUM1 turns off the regulation of miRNA-221 on gene p27 in the heart. The mechanism of PUM1 phosphorylation warrant future investigation.

Ablation of the calpain-targeted site in cardiac myosin binding protein-C is cardioprotective during ischemia-reperfusion injury

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is essential for normal heart function and protects the heart from ischemia-reperfusion (I/R) injury. It is known that protein kinase-A (PKA)-mediated phosphorylation of cMyBP-C prevents I/R-dependent proteolysis, whereas dephosphorylation of cMyBP-C at PKA sites correlates with its degradation. While sites on cMyBP-C associated with phosphorylation and proteolysis co-localize, the mechanisms that link cMyBP-C phosphorylation and proteolysis during cardioprotection are not well understood. Therefore, we aimed to determine if abrogation of cMyBP-C proteolysis in association with calpain, a calcium-activated protease, confers cardioprotection during I/R injury. Calpain is activated in both human ischemic heart samples and ischemic mouse myocardium where cMyBP-C is dephosphorylated and undergoes proteolysis. Moreover, cMyBP-C is a substrate for calpain proteolysis and cleaved by calpain at residues 272-TSLAGAGRR-280, a domain termed as the calpain-target site (CTS). Cardiac-specific transgenic (Tg) mice in which the CTS motif was ablated were bred into a cMyBP-C null background. These Tg mice were conclusively shown to possess a normal basal structure and function by analysis of histology, electron microscopy, immunofluorescence microscopy, Q-space MRI of tissue architecture, echocardiography, and hemodynamics. However, the genetic ablation of the CTS motif conferred resistance to calpain-mediated proteolysis of cMyBP-C. Following I/R injury, the loss of the CTS reduced infarct size compared to non-transgenic controls. Collectively, these findings demonstrate the physiological significance of calpain-targeted cMyBP-C proteolysis and provide a rationale for studying inhibition of calpain-mediated proteolysis of cMyBP-C as a therapeutic target for cardioprotection.

Developing LRP1 Agonists into a Therapeutic Strategy in Acute Myocardial Infarction.

Cardioprotection refers to a strategy aimed at enhancing survival pathways in the injured yet salvageable myocardium following ischemia-reperfusion. Low-density lipoprotein receptor-related protein 1 (LRP1) is a multifunctional receptor that can be targeted following reperfusion, to induce a cardioprotective signaling through the activation of the reperfusion injury salvage kinase (RISK) pathway. The data from preclinical studies with non-selective and selective LRP1 agonists are promising, showing a large therapeutic window for intervention to reduce infarct size after ischemia-reperfusion. A pilot clinical trial with plasma derived α1-antitrypsin (AAT), a naturally occurring LRP1 agonist, supports the translational value of LRP1 as a novel therapeutic target for cardioprotection. A phase I study with a selective LRP1 agonist has been completed showing no toxicity. These findings may open the way to early phase clinical studies with pharmacologic LRP1 activation in patients with acute myocardial infarction (AMI).

Abstract 16883: Ablation of Slc26a6 Protects the Heart From Ischemia/Reperfusion Injuries

Introduction: Ischemic heart disease is the most common type of heart disease, accompanied by a major decline in local myocardial pH, resulting in abnormal cardiac function. However, the mechanisms of pH regulation and the homeostasis of H + neutralizing buffers, such as HCO 3 - and Cl - in cardiomyocytes remain incompletely understood. We have recently identified a key molecule, Slc26a6, in mouse and human hearts, which mediates electrogenic Cl - /HCO 3 - exchange and plays key roles in the regulation of cardiac pH i , excitability and contractility. Because Slc26a6 is an acid loader in cardiomyocytes, we hypothesize that ablation of Slc26a6 may protect the heart from ischemia/reperfusion (I/R) injury. Methods: We generated I/R model using wild type (WT) and Slc26a6 knockout ( Slc26a6 -/- ) mice. Multidisciplinary in vivo and in vitro approaches were used, including echocardiography, electrophysiology, hemodynamic monitoring, electrocardiography (ECG), and histochemistry. In addition, cellular experiments were performed to test the Cl - /HCO 3 - exchange currents, Ca 2+ transient, sarcoplasmic reticulum Ca 2+ load, and sarcomere shortening. Results: ECG recordings were performed during the I/R surgery to monitor the I/R injury. 24 hours after reperfusion, troponin I level was lower in Slc26a6 -/- I/R mice relative to WT I/R mice, suggesting less cardiac injuries in Slc26a6 -/- mice. This was validated 4 weeks after I/R. Slc26a6 -/- I/R mice showed better systolic and diastolic function than WT I/R mice, assessed by echocardiography and hemodynamics monitoring. Moreover, histological analyses revealed lower collagen formation and smaller infarct size in Slc26a6 -/- compared to WT mice. In vitro analyses of sarcomere shortening, Ca 2+ transients, and sarcoplasmic reticulum Ca 2+ load of the cardiomyocytes from infarct zone corroborated the in vivo findings, showing more preserved cellular function in Slc26a6 -/- compared to WT mice. Conclusion: Ablation of Slc26a6 is cardioprotective in a murine model of I/R, which may result from the elevated pH i and the reduction of acidification during I/R. Our findings suggest critical roles of Slc26a6 in I/R injury. Slc26a6 may represent a novel therapeutic target for cardioprotection in ischemia through pH regulation.

Uncovering novel landscape of cardiovascular diseases and therapeutic targets for cardioprotection via long noncoding RNA-miRNA-mRNA axes.

Protein coding sequences account for around 3% of the human genome, the rest are noncoding RNA (ncRNA) including long ncRNA(lncRNA) and miRNA. Accumulating evidence indicates that lncRNAs and miRNAs are candidate biomarkers for diagnosis, prognosis and therapy of cardiovascular diseases. The lncRNAs act as sponge-like effects on numerous miRNAs, subsequently regulating miRNAs and their targets, mRNA functions. The role of lncRNA-miRNA-mRNA axis in pathogenesis of cardiovascular diseases has been recently reported and highlighted. Herein, this review discusses emerging roles of lncRNA-miRNA-mRNA axis in cardiovascular pathophysiology and regulation, with a novel focus on cardioprotective network activities of the two subgroup ncRNAs.

Tyrosine phosphorylation of eNOS regulates myocardial survival after an ischaemic insult: role of PYK2.

Endothelial nitric oxide (NO) synthase (eNOS) is known to play a cardioprotective protective. However, the molecular mechanisms regulating eNOS activity during ischaemia/reperfusion (I/R) injury are incompletely understood. eNOS is a substrate for several kinases that positively or negatively affect its enzymatic activity. Herein, we sought to correlate eNOS phosphorylation status with cardiomyocyte survival and we investigated the contribution of the proline-rich tyrosine kinase 2 (PYK2)/eNOS axis to the regulation of myocardial infarct size in vivo. Exposure of H9c2 cardiomyocytes to H2O2 lead to PYK2 phosphorylation on its activator site (Y402) and eNOS phosphorylation on the inhibitor site Y656 and the activator site S1176. Both H2O2-induced eNOS phosphorylation events were abolished by PYK2 pharmacological inhibition or gene knockdown. Activity assays demonstrated that phosphorylation of the tyrosine inhibitory site exerts a dominant effect over S1176. In cardiomyocytes subjected to oxidative stress or oxygen-glucose deprivation, inhibition of PYK2 limited cell injury; this effect was prevented by inhibition of NO production. In vivo, ischaemia-reperfusion induced an early activation of PYK2, leading to eNOS phosphorylation on Y656, which, in turn, reduced NO output, as judged by the low tissue levels of its downstream effector cGMP. Moreover, pharmacological blockade of PYK2 alleviated eNOS inhibition and prevented cardiac damage following I/R injury in wild-type, but not in eNOS KO mice. The current studies demonstrate that PYK2 is a pivotal regulator of eNOS function in myocardial infarction and identify PYK2 as a novel therapeutic target for cardioprotection.

Parkinson's disease proteins: Novel mitochondrial targets for cardioprotection.

Ischemic heart disease (IHD) is the leading cause of death and disability worldwide. Therefore, novel therapeutic targets for protecting the heart against acute ischemia/reperfusion injury (IRI) are required to attenuate cardiomyocyte death, preserve myocardial function, and prevent the onset of heart failure. In this regard, a specific group of mitochondrial proteins, which have been linked to familial forms of Parkinson's disease (PD), may provide novel therapeutic targets for cardioprotection. In dopaminergic neurons of the substantia nigra, these PD proteins, which include Parkin, PINK1, DJ-1, LRRK2, and α-synuclein, play essential roles in preventing cell death—through maintaining normal mitochondrial function, protecting against oxidative stress, mediating mitophagy, and preventing apoptosis. These rare familial forms of PD may therefore provide important insights into the pathophysiology underlying mitochondrial dysfunction and the development of PD. Interestingly, these PD proteins are also present in the heart, but their role in myocardial health and disease is not clear. In this article, we review the role of these PD proteins in the heart and explore their potential as novel mitochondrial targets for cardioprotection.

THE ADULT MURINE HEART IS PROTECTED AGAINST ISCHEMIA-REPERFUSION INJURY IN THE ABSENCE OF BOTH MITOFUSIN (MFN) PROTEINS

BackgroundCardiac-specific ablation of both Mfn1 and Mfn2 in the adult heart results in mitochondrial fragmentation and a lethal cardiomyopathy after about 6 weeks. The effect of combined Mfn1 and Mfn2...

THE DIFFERENTIAL EFFECTS OF SIRTUIN-3 IN CARDIO-PROTECTION

Background The mitochondrial specific deacetylase, Sirtuin-3 has been reported to regulate oxidative phosphorylation, the activity of Cyclophilin D (a key component of the mitochondrial permeability transition pore, MPTP), and the ROS scavenger MnSOD. We hypothesise Sirtuin-3 to be a potential therapeutic target for cardio-protection based its ability to prevent MPTP formation and inhibit ROS generation. Methods and Results In HL-1 cells (a murine cardiac cell line), Sirtuin-3 over-expression reduced cell death following simulated ischemia-reperfusion injury. Futhermore, Sirtuin-3 over-expression reduced MPTP formation, and induced mitochondrial fusion. To investigate the role of endogenous Sirtuin-3 in the adult heart, Sirtuin-3 (whole body) KO mice and WT littermates were subjected to in vivo cardiac ischemia (30 min) followed by 24 hrs reperfusion. Myocardial infarct (MI) size was determined as a percentage of area at risk. Interestingly, no differences in MI size were observed between WT and KO mice under fed conditions. However, overnight fasting (to induce Sirtuin-3 expression and activity) resulted in a smaller MI size in the Sirtuin-3 KO when compared to WT mice. Conclusions We report that the role of Sirtuin-3 in cardio-protection differs between the HL-1 cardiac cell line and the adult heart. In HL-1 cells, Sirtuin-3 over-expression protects against acute IRI, suggesting that activating Sirtuin-3 in this cell-line may be cardio-protective. In contrast, fasted mice deficient in Sirtuin-3 had smaller MI following IRI, suggesting that inhibiting Sirtuin-3 in the fasted adult heart may be cardio-protective. This finding may have clinical implications in patients who are fasted before surgery.

DJ-1 protects against cell death following acute cardiac ischemia–reperfusion injury

Novel therapeutic targets are required to protect the heart against cell death from acute ischemia–reperfusion injury (IRI). Mutations in the DJ-1 (PARK7) gene in dopaminergic neurons induce mitochondrial dysfunction and a genetic form of Parkinson's disease. Genetic ablation of DJ-1 renders the brain more susceptible to cell death following ischemia–reperfusion in a model of stroke. Although DJ-1 is present in the heart, its role there is currently unclear. We sought to investigate whether mitochondrial DJ-1 may protect the heart against cell death from acute IRI by preventing mitochondrial dysfunction. Overexpression of DJ-1 in HL-1 cardiac cells conferred the following beneficial effects: reduced cell death following simulated IRI (30.4±4.7% with DJ-1 versus 52.9±4.7% in control; n=5, P<0.05); delayed mitochondrial permeability transition pore (MPTP) opening (a critical mediator of cell death) (260±33 s with DJ-1 versus 121±12 s in control; n=6, P<0.05); and induction of mitochondrial elongation (81.3±2.5% with DJ-1 versus 62.0±2.8% in control; n=6 cells, P<0.05). These beneficial effects of DJ-1 were absent in cells expressing the non-functional DJ-1L166P and DJ-1Cys106A mutants. Adult mice devoid of DJ-1 (KO) were found to be more susceptible to cell death from in vivo IRI with larger myocardial infarct sizes (50.9±3.5% DJ-1 KO versus 41.1±2.5% in DJ-1 WT; n≥7, P<0.05) and resistant to cardioprotection by ischemic preconditioning. DJ-1 KO hearts showed increased mitochondrial fragmentation on electron microscopy, although there were no differences in calcium-induced MPTP opening, mitochondrial respiratory function or myocardial ATP levels. We demonstrate that loss of DJ-1 protects the heart from acute IRI cell death by preventing mitochondrial dysfunction. We propose that DJ-1 may represent a novel therapeutic target for cardioprotection.

Mitochondrial Dynamics in the Heart as a Novel Therapeutic Target for Cardioprotection

Traditionally, mitochondria have been regarded solely as energy generators for cells; however, accumulating data have demonstrated that these complex organelles play a variety of roles within the cardiomyocyte that extend beyond this classic function. Mitochondrial dynamics involves mitochondrial movements and morphologic alterations by tethering, fusion, and fission, which depend on cellular energy requirements and metabolic status. Many studies have indicated that mitochondrial dynamics may be a fundamental component of the maintenance of normal cellular homeostasis and cardiac function. Mitochondrial dynamics is controlled by the protein machinery responsible for mitochondrial fusion and fission, but cardiomyocytes are densely packed as part of an intricate cytoarchitecture for efficient and imbalanced contraction; thus, mitochondrial dynamics in the adult heart are restricted and occur more slowly than in other organs. Cardiac mitochondrial dynamics is important for cardiac physiology in diseased conditions such as ischemia-reperfusion (IR) injury. Changes in mitochondrial morphology through modulation of the expression of proteins regulating mitochondrial dynamics demonstrates the beneficial effects on cardiac performance after IR injury. Thus, accurately defining the roles of mitochondrial dynamics in the adult heart can guide the identification and development of novel therapeutic targets for cardioprotection. Further studies should be performed to establish the exact mechanisms of mitochondrial dynamics.

35 Protective action of synthetic diarylamines on mitochondrial permeability transition pore

Mitochondria can undergo a generalised increase of the permeability of the inner membrane, called mitochondrial permeability transition (MPT), due to the formation of non-specific pores (MPTP), which is an early...

Sustained intracellular acidosis activates the myocardial Na +/H + exchanger independent of amino acid Ser 703 and p90 rsk

The mammalian Na +/H + exchanger isoform 1 (NHE1) is a ubiquitously expressed pH-regulatory membrane protein that functions in the myocardium and other tissues. It is an important mediator of the myocardial damage that occurs after ischemia–reperfusion injury and is implicated in heart hypertrophy. Regulation of NHE1 has been proposed as a therapeutic target for cardioprotection. We therefore examined mechanisms of control of NHE1 in the myocardium. Several different amino acids have been implicated as a being critical to NHE1 regulation in a number of tissues including Ser 703, Ser 770, and Ser 771. In the myocardium, NHE1 is activated in response to a variety of stimuli including activation by an ERK-dependent sustained intracellular acidosis. In this study, we determined whether Ser 703 and p90 rsk activity are critical in activation of NHE1 by sustained intracellular acidosis. In vitro phosphorylation of NHE1 C-terminal fusion proteins determined that ERK-dependent phosphorylation of the cytoplasmic region was not dependent on Ser 703; however, phosphorylation by p90 rsk required Ser 703. A Ser703Ala mutation decreased basal NHE1 activity in CHO cells but not in cardiomyocytes. NHE1 with a Ser703Ala mutation was activated in response to sustained intracellular acidosis in CHO cells. In addition, sustained intracellular acidosis also activated the Ser703Ala mutant protein in isolated cardiomyocytes and phosphorylation levels were also increased by acidosis. The presence of a dominant-negative p90 rsk kinase also did not prevent activation and phosphorylation of NHE1 by sustained intracellular acidosis in isolated cardiomyocytes. We conclude that Ser 703 and p90 rsk are not required for activation by sustained intracellular acidosis and that p90 rsk phosphorylation of Ser 703 is independent of this type of activation.