NADPH oxidase 4 contributes to TRPV4-mediated endothelium-dependent vasodilation in human arterioles by regulating protein phosphorylation of TRPV4 channels.

Impaired endothelium-dependent vasodilation has been suggested to be a key component of coronary microvascular dysfunction (CMD). A better understanding of endothelial pathways involved in vasodilation in human arterioles may provide new insight into the mechanisms of CMD. The goal of this study is to investigate the role of TRPV4, NOX4, and their interaction in human arterioles and examine the underlying mechanisms. Arterioles were freshly isolated from adipose and heart tissues obtained from 71 patients without coronary artery disease, and vascular reactivity was studied by videomicroscopy. In human adipose arterioles (HAA), ACh-induced dilation was significantly reduced by TRPV4 inhibitor HC067047 and by NOX 1/4 inhibitor GKT137831, but GKT137831 did not further affect the dilation in the presence of TRPV4 inhibitors. GKT137831 also inhibited TRPV4 agonist GSK1016790A-induced dilation in HAA and human coronary arterioles (HCA). NOX4 transcripts and proteins were detected in endothelial cells of HAA and HCA. Using fura-2 imaging, GKT137831 significantly reduced GSK1016790A-induced Ca2+ influx in the primary culture of endothelial cells and TRPV4-WT-overexpressing human coronary artery endothelial cells (HCAEC). However, GKT137831 did not affect TRPV4-mediated Ca2+ influx in non-phosphorylatable TRPV4-S823A/S824A-overexpressing HCAEC. In addition, treatment of HCAEC with GKT137831 decreased the phosphorylation level of Ser824 in TRPV4. Finally, proximity ligation assay (PLA) revealed co-localization of NOX4 and TRPV4 proteins. In conclusion, both TRPV4 and NOX4 contribute to ACh-induced dilation in human arterioles from patients without coronary artery disease. NOX4 increases TRPV4 phosphorylation in endothelial cells, which in turn enhances TRPV4-mediated Ca2+ entry and subsequent endothelium-dependent dilation in human arterioles.

Corrigendum: Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium.

[This corrects the article DOI: 10.3389/fcell.2021.687598.].

Distinct Signaling Functions of Rap1 Isoforms in NO Release From Endothelium.

Small GTPase Rap1 plays a prominent role in endothelial cell (EC) homeostasis by promoting NO release. Endothelial deletion of the two highly homologous Rap1 isoforms, Rap1A and Rap1B, leads to endothelial dysfunction ex vivo and hypertension in vivo. Mechanistically, we showed that Rap1B promotes NO release in response to shear flow by promoting mechanosensing complex formation involving VEGFR2 and Akt activation. However, the specific contribution of the Rap1A isoform to NO release and the underlying molecular mechanisms through which the two Rap1 isoforms control endothelial function are unknown. Here, we demonstrate that endothelial dysfunction resulting from knockout of both Rap1A and Rap1B isoforms is ameliorated by exogenous L-Arg administration to rescue NO-dependent vasorelaxation and blood pressure. We confirmed that Rap1B is rapidly activated in response to agonists that trigger eNOS activation, and its deletion in ECs attenuates eNOS activation, as detected by decreased Ser1177 phosphorylation. Somewhat surprising was the finding that EC deletion of Rap1A does not lead to impaired agonist-induced vasorelaxation ex vivo. Mechanistically, the deletion of Rap1A led to elevated eNOS phosphorylation both at the inhibitory, T495, and the activating Ser1177 residues. These findings indicate that the two Rap1 isoforms act via distinct signaling pathways: while Rap1B directly positively regulates eNOS activation, Rap1A prevents negative regulation of eNOS. Notably, the combined deficiency of Rap1A and Rap1B has a severe effect on eNOS activity and NO release with an in vivo impact on endothelial function and vascular homeostasis.

Endothelin-1 potentiates TRPV1-mediated vasoconstriction of human adipose arterioles in a protein kinase C-dependent manner.

BACKGROUND AND PURPOSE: The TRPV cation channels have emerged as important regulators of vascular tone. TRPV1 channels and endothelin-1 are independently associated with the pathophysiology of coronary vasospasm, but the relationship between their vasomotor functions remains unclear. We characterized the vasomotor function of TRPV1 channels in human arterioles and investigated regulation of their vasomotor function by endothelin-1. EXPERIMENTAL APPROACH: Human arterioles (mainly from adipose tissue) were threaded on two metal wires, equilibrated in a physiological buffer at 37°C and exposed to increasing concentrations of capsaicin, with or without SB366791 (TRPV1-selective inhibitor) or GF109203X (PKC-selective inhibitor). Some arterioles were pre-constricted with endothelin-1 or phenylephrine or high potassium buffer. TRPV1 mRNA and protein expression in human arteries were also assessed. KEY RESULTS: TRPV1 transcripts and proteins were detected in human resistance arteries. Capsaicin (1 µM) induced concentration-dependent constriction of endothelium-intact and endothelium-denuded human adipose arterioles (HAA), which was significantly inhibited by SB366791. Pre-constriction of HAA with endothelin-1, but not high potassium buffer or phenylephrine, significantly potentiated capsaicin (0.1 µM)-induced constriction. GF109203X significantly inhibited potentiation of capsaicin-induced constriction by endothelin-1. CONCLUSION AND IMPLICATIONS: TRPV1 channels are expressed in the human vasculature and affect vascular tone of human arterioles on activation. Their vasomotor function is modulated by endothelin-1, mediated in part by PKC. These findings reveal a novel interplay between endothelin-1 signalling and TRPV1 channels in human VSMC, adding to our understanding of the ion channel mechanisms that regulate human arteriolar tone and may also contribute to the pathophysiology of coronary vasospasm.

Endothelin receptor A and p66Shc regulate spontaneous Ca2+ oscillations in smooth muscle cells controlling renal arterial spontaneous motion.

Adaptor protein p66Shc is overexpressed in smooth muscle cells of renal resistance vessels of hypertensive salt-sensitive rats and is involved in the regulation of renal vascular tone. We applied 2-photon laser scanning fluorescence microscopy to analyze spontaneous dynamic fluctuations in intracellular calcium concentrations ([Ca2+]i) in smooth muscle cells embedded in the walls of freshly isolated renal resistance arteries. The amplitude, number of events, and frequency of spontaneous [Ca2+]i oscillations triggered by endogenously released endothelin-1 were recorded in smooth muscle cells of the renal arteries. Endothelin receptor A antagonist BQ123 dramatically reduced the amplitude and frequency of spontaneous Ca2+ events, producing marked inhibition of renal vessels spontaneous motion. Spontaneous Ca2+ fluctuations in smooth muscle cells of p66Shc knockout (p66ShcKO) rats had significantly higher amplitude than in control rats. The frequency of spontaneous [Ca2+]i oscillations did not change in p66ShcKO rats, suggesting that p66Shc expression did not affect endothelin-1 release from resident endothelial cells. Acute application of endothelin-1 revealed significantly elevated production of the total [Ca2+]i in p66ShcKO rats. Spontaneous cytosolic Ca2+ oscillations in smooth muscle cells of renal vessels mediate their spontaneous motion via the endothelin-1/endothelin receptor A pathway. p66Shc decreases the amplitude of individual changes in [Ca2+]i, which mitigates the spontaneous motion of renal vessels.-Palygin, O., Miller, B. S., Nishijima, Y., Zhang, D. X., Staruschenko, A., Sorokin, A. Endothelin receptor A and p66Shc regulate spontaneous Ca2+ oscillations in smooth muscle cells controlling renal arterial spontaneous motion.

Transient receptor potential vanilloid 4 (TRPV4) activation by arachidonic acid requires protein kinase A-mediated phosphorylation.

Transient receptor potential vanilloid 4 (TRPV4) is a Ca2+-permeable channel of the transient receptor potential (TRP) superfamily activated by diverse stimuli, including warm temperature, mechanical forces, and lipid mediators such as arachidonic acid (AA) and its metabolites. This activation is tightly regulated by protein phosphorylation carried out by various serine/threonine or tyrosine kinases. It remains poorly understood how phosphorylation differentially regulates TRPV4 activation in response to different stimuli. We investigated how TRPV4 activation by AA, an important signaling process in the dilation of coronary arterioles, is affected by protein kinase A (PKA)-mediated phosphorylation at Ser-824. Wildtype and mutant TRPV4 channels were expressed in human coronary artery endothelial cells (HCAECs). AA-induced TRPV4 activation was blunted in the S824A mutant but was enhanced in the phosphomimetic S824E mutant, whereas the channel activation by the synthetic agonist GSK1016790A was not affected. The low level of basal phosphorylation at Ser-824 was robustly increased by the redox signaling molecule hydrogen peroxide (H2O2). The H2O2-induced phosphorylation was accompanied by an enhanced channel activation by AA, and this enhanced response was largely abolished by PKA inhibition or S824A mutation. We further identified a potential structural context dependence of Ser-824 phosphorylation-mediated TRPV4 regulation involving an interplay between AA binding and the possible phosphorylation-induced rearrangements of the C-terminal helix bearing Ser-824. These results provide insight into how phosphorylation specifically regulates TRPV4 activation. Redox-mediated TRPV4 phosphorylation may contribute to pathologies associated with enhanced TRPV4 activity in endothelial and other systems.

Shaker-related voltage-gated K+ channel expression and vasomotor function in human coronary resistance arteries.

OBJECTIVES: KV channels are important regulators of vascular tone, but the identity of specific KV channels involved and their regulation in disease remain less well understood. We determined the expression of KV 1 channel subunits and their role in cAMP-mediated dilation in coronary resistance arteries from subjects with and without CAD. METHODS: HCAs from patients with and without CAD were assessed for mRNA and protein expression of KV 1 channel subunits with molecular techniques and for vasodilator response with isolated arterial myography. RESULTS: Assays of mRNA transcripts, membrane protein expression, and vascular cell-specific localization revealed abundant expression of KV 1.5 in vascular smooth muscle cells of non-CAD HCAs. Isoproterenol and forskolin, two distinct cAMP-mediated vasodilators, induced potent dilation of non-CAD arterioles, which was inhibited by both the general KV blocker 4-AP and the selective KV 1.5 blocker DPO-1. The cAMP-mediated dilation was reduced in CAD and was accompanied by a loss of or reduced contribution of 4-AP-sensitive KV channels. CONCLUSIONS: KV 1.5, as a major 4-AP-sensitive KV 1 channel expressed in coronary VSMCs, mediates cAMP-mediated dilation in non-CAD arterioles. The cAMP-mediated dilation is reduced in CAD coronary arterioles, which is associated with impaired 4-AP-sensitive KV channel function.

Contribution of KV1.5 Channel to Hydrogen Peroxide-Induced Human Arteriolar Dilation and Its Modulation by Coronary Artery Disease.

RATIONALE: Hydrogen peroxide (H2O2) regulates vascular tone in the human microcirculation under physiological and pathophysiological conditions. It dilates arterioles by activating large-conductance Ca2+-activated K+ channels in subjects with coronary artery disease (CAD), but its mechanisms of action in subjects without CAD (non-CAD) when compared with those with CAD remain unknown. OBJECTIVE: We hypothesize that H2O2-elicited dilation involves different K+ channels in non-CAD versus CAD, resulting in an altered capacity for vasodilation during disease. METHODS AND RESULTS: H2O2 induced endothelium-independent vasodilation in non-CAD adipose arterioles, which was reduced by paxilline, a large-conductance Ca2+-activated K+ channel blocker, and by 4-aminopyridine, a voltage-gated K+ (KV) channel blocker. Assays of mRNA transcripts, protein expression, and subcellular localization revealed that KV1.5 is the major KV1 channel expressed in vascular smooth muscle cells and is abundantly localized on the plasma membrane. The selective KV1.5 blocker diphenylphosphine oxide-1 and the KV1.3/1.5 blocker 5-(4-phenylbutoxy)psoralen reduced H2O2-elicited dilation to a similar extent as 4-aminopyridine, but the selective KV1.3 blocker phenoxyalkoxypsoralen-1 was without effect. In arterioles from CAD subjects, H2O2-induced dilation was significantly reduced, and this dilation was inhibited by paxilline but not by 4-aminopyridine, diphenylphosphine oxide-1, or 5-(4-phenylbutoxy)psoralen. KV1.5 cell membrane localization and diphenylphosphine oxide-1-sensitive K+ currents were markedly reduced in isolated vascular smooth muscle cells from CAD arterioles, although mRNA or total cellular protein expression was largely unchanged. CONCLUSIONS: In human arterioles, H2O2-induced dilation is impaired in CAD, which is associated with a transition from a combined large-conductance Ca2+-activated K+- and KV (KV1.5)-mediated vasodilation toward a large-conductance Ca2+-activated K+-predominant mechanism of dilation. Loss of KV1.5 vasomotor function may play an important role in microvascular dysfunction in CAD or other vascular diseases.

Chronic Omega-3 Polyunsaturated Fatty Acid Treatment Variably Affects Cellular Repolarization in a Healed Post-MI Arrhythmia Model.

INTRODUCTION: Over the last 40 years omega-3 polyunsaturated fatty acids (PUFAs) have been shown to be anti-arrhythmic or pro-arrhythmic depending on the method and duration of administration and model studied. We previously reported that omega-3 PUFAs do not confer anti-arrhythmic properties and are pro-arrhythmic in canine model of sudden cardiac death (SCD). Here, we evaluated the effects of chronic omega-3 PUFA treatment in post-MI animals susceptible (VF+) or resistant (VF-) to ventricular tachyarrhythmias. METHODS: Perforated patch clamp techniques were used to measure cardiomyocyte action potential durations (APD) at 50 and 90% repolarization and short term variability of repolarization. The early repolarizing transient outward potassium current Ito was also studied. RESULTS: Omega-3 PUFAs prolonged the action potential in VF- myocytes at both 50 and 90% repolarization. Short term variability of repolarization was increased in both untreated and treated VF- myocytes vs. CONTROLS: Ito was unaffected by omega-3 PUFA treatment. Omega-3 PUFA treatment attenuated the action potential prolongation in VF+ myocytes, but did not return repolarization to control values. CONCLUSIONS: Omega-3 PUFAs do not confer anti-arrhythmic properties in the setting of healed myocardial infarction in a canine model of SCD. In canines previously resistant to ventricular fibrillation (VF-), omega-3 PUFA treatment prolonged the action potential in VF- myocytes, and may contribute to pro-arrhythmic responses.

Rap1 promotes endothelial mechanosensing complex formation, NO release and normal endothelial function.

Decreased nitric oxide (NO) bioavailability underlies a number of cardiovascular pathologies, including hypertension. The shear stress exerted by flowing blood is the main determinant of NO release. Rap1 promotes integrin- and cadherin-mediated signaling. Here, we show that Rap1 is a critical regulator of NO production and endothelial function. Rap1 deficiency in murine endothelium attenuates NO production and diminishes NO-dependent vasodilation, leading to endothelial dysfunction and hypertension, without deleterious effects on vessel integrity. Mechanistically, Rap1 is activated by shear stress, promotes the formation of the endothelial mechanosensing complex-comprised of PECAM-1, VE-cadherin and VEGFR2- and downstream signaling to NO production. Our study establishes a novel paradigm for Rap1 as a regulator of mechanotransduction.

Heart failure duration progressively modulates the arrhythmia substrate through structural and electrical remodeling.

AIMS: Ventricular arrhythmias are a common cause of death in patients with heart failure (HF). Structural and electrical abnormalities in the heart provide a substrate for such arrhythmias. Canine tachypacing-induced HF models of 4-6 weeks duration are often used to study pathophysiology and therapies for HF. We hypothesized that a chronic canine model of HF would result in greater electrical and structural remodeling than a short term model, leading to a more arrhythmogenic substrate. MAIN METHODS: HF was induced by ventricular tachypacing for one (short-term) or four (chronic) months to study remodeling. KEY FINDINGS: Left ventricular contractility was progressively reduced, while ventricular hypertrophy and interstitial fibrosis were evident at 4 month but not 1 month of HF. Left ventricular myocyte action potentials were prolonged after 4 (p<0.05) but not 1 month of HF. Repolarization instability and early afterdepolarizations were evident only after 4 months of HF (p<0.05), coinciding with a prolonged QTc interval (p<0.05). The transient outward potassium current was reduced in both HF groups (p<0.05). The outward component of the inward rectifier potassium current was reduced only in the 4 month HF group (p<0.05). The delayed rectifier potassium currents were reduced in 4 (p<0.05) but not 1 month of HF. Reactive oxygen species were increased at both 1 and 4 months of HF (p<0.05). SIGNIFICANCE: Reduced Ito, outward IK1, IKs, and IKr in HF contribute to EAD formation. Chronic, but not short term canine HF, results in the altered electrophysiology and repolarization instability characteristic of end-stage human HF.

Characterization of blood pressure and endothelial function in TRPV4-deficient mice with l-NAME- and angiotensin II-induced hypertension.

Transient receptor potential vanilloid type 4 (TRPV4) is an endothelial Ca(2+) entry channel contributing to endothelium-mediated dilation in conduit and resistance arteries. We investigated the role of TRPV4 in the regulation of blood pressure and endothelial function under hypertensive conditions. TRPV4-deficient (TRPV4(-/-)) and wild-type (WT) control mice were given l-NAME (0.5 g/L) in drinking water for 7 days or subcutaneously infused with angiotensin (Ang) II (600 ng/kg per minute) for 14 days, and blood pressure measured by radiotelemetry. TRPV4(-/-) mice had a lower baseline mean arterial pressure (MAP) (12-h daytime MAP, 94 ± 2 vs. 99 ± 2 mmHg in WT controls). l-NAME treatment induced a slightly greater increase in MAP in TRPV4(-/-) mice (day 7, 13 ± 4%) compared to WT controls (6 ± 2%), but Ang II-induced increases in MAP were similar in TRPV4(-/-) and WT mice (day 14, 53 ± 6% and 37 ± 11%, respectively, P < 0.05). Chronic infusion of WT mice with Ang II reduced both acetylcholine (ACh)-induced dilation (dilation to 10(-5) mol/L ACh, 71 ± 5% vs. 92 ± 2% of controls) and the TRPV4 agonist GSK1016790A-induced dilation of small mesenteric arteries (10(-8) mol/L GSK1016790A, 14 ± 5% vs. 77 ± 7% of controls). However, Ang II treatment did not affect ACh dilation in TRPV4(-/-) mice. Mechanistically, Ang II did not significantly alter either TRPV4 total protein expression in mesenteric arteries or TRPV4 agonist-induced Ca(2+) response in mesenteric endothelial cells in situ. These results suggest that TRPV4 channels play a minor role in blood pressure regulation in l-NAME- but not Ang II-induced hypertension, but may be importantly involved in Ang II-induced endothelial dysfunction.

Ibandronate and ventricular arrhythmia risk.

INTRODUCTION: Bisphosphonates, including ibandronate, are used in the prevention and treatment of osteoporosis. METHODS AND RESULTS: We report a case of suspected ibandronate-associated arrhythmia, following a single dose of ibandronate in a 55-year-old female. ECG at presentation revealed frequent ectopy and QT/QTc interval prolongation; at follow-up 9 months later the QT/QTc intervals were normalized. Proarrhythmic potential of ibandronate was assessed with a combination of in vivo and in vitro approaches in canines and canine ventricular myocytes. We observed late onset in vivo repolarization instability after ibandronate treatment. Myocytes superfused with ibandronate exhibited action potential duration (APD) prolongation and variability, increased early afterdepolarizations (EADs) and reduced Ito (P < 0.05), with no change in IKr . Ibandronate-induced APD changes and EADs were prevented by inhibition of intracellular calcium cycling. Ibandronate increased sarcoplasmic reticulum calcium load; during washout there was an increase in calcium spark frequency and spontaneous calcium waves. Computational modeling was used to examine the observed effects of ibandronate. While reductions in Ito alone had modest effects on APD, when combined with altered RyR inactivation kinetics, the model predicted effects on APD and SR Ca(2+) load consistent with observed experimental results. CONCLUSION: Ibandronate may increase the susceptibility to ventricular ectopy and arrhythmias. Collectively these data suggest that reduced Ito combined with abnormal RyR calcium handling may result in a previously unrecognized form of drug-induced proarrhythmia.

Arachidonic acid-induced dilation in human coronary arterioles: convergence of signaling mechanisms on endothelial TRPV4-mediated Ca2+ entry.

BACKGROUND: Arachidonic acid (AA) and/or its enzymatic metabolites are important lipid mediators contributing to endothelium-derived hyperpolarizing factor (EDHF)-mediated dilation in multiple vascular beds, including human coronary arterioles (HCAs). However, the mechanisms of action of these lipid mediators in endothelial cells (ECs) remain incompletely defined. In this study, we investigated the role of the transient receptor potential vanilloid 4 (TRPV4) channel in AA-induced endothelial Ca(2+) response and dilation of HCAs. METHODS AND RESULTS: AA induced concentration-dependent dilation in isolated HCAs. The dilation was largely abolished by the TRPV4 antagonist RN-1734 and by inhibition of endothelial Ca(2+)-activated K(+) channels. In native and TRPV4-overexpressing human coronary artery ECs (HCAECs), AA increased intracellular Ca(2+) concentration ([Ca(2+)]i), which was mediated by TRPV4-dependent Ca(2+) entry. The AA-induced [Ca(2+)]i increase was inhibited by cytochrome P450 (CYP) inhibitors. Surprisingly, the CYP metabolites of AA, epoxyeicosatrienoic acids (EETs), were much less potent activators of TRPV4, and CYP inhibitors did not affect EET production in HCAECs. Apart from its effect on [Ca(2+)]i, AA induced endothelial hyperpolarization, and this effect was required for Ca(2+) entry through TRPV4. AA-induced and TRPV4-mediated Ca(2+) entry was also inhibited by the protein kinase A inhibitor PKI. TRPV4 exhibited a basal level of phosphorylation, which was inhibited by PKI. Patch-clamp studies indicated that AA activated TRPV4 single-channel currents in cell-attached and inside-out patches of HCAECs. CONCLUSIONS: AA dilates HCAs through a novel mechanism involving endothelial TRPV4 channel-dependent Ca(2+) entry that requires endothelial hyperpolarization, PKA-mediated basal phosphorylation of TRPV4, and direct activation of TRPV4 channels by AA.

Differential effects of the peroxynitrite donor, SIN-1, on atrial and ventricular myocyte electrophysiology.

Oxidative stress has been implicated in the pathogenesis of heart failure and atrial fibrillation and can result in increased peroxynitrite production in the myocardium. Atrial and ventricular canine cardiac myocytes were superfused with 3-morpholinosydnonimine-N-ethylcarbamide (SIN-1), a peroxynitrite donor, to evaluate the acute electrophysiologic effects of peroxynitrite. Perforated whole-cell patch clamp techniques were used to record action potentials. SIN-1 (200 µM) increased the action potential duration (APD) in atrial and ventricular myocytes; however, in the atria, APD prolongation was rate independent, whereas in the ventricle APD, prolongation was rate dependent. In addition to prolongation of the action potential, beat-to-beat variability of repolarization was significantly increased in ventricular but not in atrial myocytes. We examined the contribution of intracellular calcium cycling to the effects of SIN-1 by treating myocytes with the SERCA blocker, thapsigargin (5-10 µM). Inhibition of calcium cycling prevented APD prolongation in the atrial and ventricular myocytes, and prevented the SIN-1-induced increase in ventricular beat-to-beat APD variability. Collectively, these data demonstrate that peroxynitrite affects atrial and ventricular electrophysiology differentially. A detailed understanding of oxidative modulation of electrophysiology in specific chambers is critical to optimize therapeutic approaches for cardiac diseases.

Cardiomyocyte-specific overexpression of an active form of Rac predisposes the heart to increased myocardial stunning and ischemia-reperfusion injury.

The GTP-binding protein Rac regulates diverse cellular functions including activation of NADPH oxidase, a major source of superoxide production (O(2)(·-)). Rac1-mediated NADPH oxidase activation is increased after myocardial infarction (MI) and heart failure both in animals and humans; however, the impact of increased myocardial Rac on impending ischemia-reperfusion (I/R) is unknown. A novel transgenic mouse model with cardiac-specific overexpression of constitutively active mutant form of Zea maize Rac D (ZmRacD) gene has been reported with increased myocardial Rac-GTPase activity and O(2)(·-) generation. The goal of the present study was to determine signaling pathways related to increased myocardial ZmRacD and to what extent hearts with increased ZmRacD proteins are susceptible to I/R injury. The effect of myocardial I/R was examined in young adult wild-type (WT) and ZmRacD transgenic (TG) mice. In vitro reversible myocardial I/R for postischemic cardiac function and in vivo regional myocardial I/R for MI were performed. Following 20-min global ischemia and 45-min reperfusion, postischemic cardiac contractile function and heart rate were significantly reduced in TG hearts compared with WT hearts. Importantly, acute regional myocardial I/R (30-min ischemia and 24-h reperfusion) caused significantly larger MI in TG mice compared with WT mice. Western blot analysis of cardiac homogenates revealed that increased myocardial ZmRacD gene expression is associated with concomitant increased levels of NADPH oxidase subunit gp91(phox), O(2)(·-), and P(21)-activated kinase. Thus these findings provide direct evidence that increased levels of active myocardial Rac renders the heart susceptible to increased postischemic contractile dysfunction and MI following acute I/R.

Endurance exercise training normalizes repolarization and calcium-handling abnormalities, preventing ventricular fibrillation in a model of sudden cardiac death.

The risk of sudden cardiac death is increased following myocardial infarction. Exercise training reduces arrhythmia susceptibility, but the mechanism is unknown. We used a canine model of sudden cardiac death (healed infarction, with ventricular tachyarrhythmias induced by an exercise plus ischemia test, VF+); we previously reported that endurance exercise training was antiarrhythmic in this model (Billman GE. Am J Physiol Heart Circ Physiol 297: H1171-H1193, 2009). A total of 41 VF+ animals were studied, after random assignment to 10 wk of endurance exercise training (EET; n = 21) or a matched sedentary period (n = 20). Following (>1 wk) the final attempted arrhythmia induction, isolated myocytes were used to test the hypotheses that the endurance exercise-induced antiarrhythmic effects resulted from normalization of cellular electrophysiology and/or normalization of calcium handling. EET prevented VF and shortened in vivo repolarization (P < 0.05). EET normalized action potential duration and variability compared with the sedentary group. EET resulted in a further decrement in transient outward current compared with the sedentary VF+ group (P < 0.05). Sedentary VF+ dogs had a significant reduction in repolarizing K(+) current, which was restored by exercise training (P < 0.05). Compared with controls, myocytes from the sedentary VF+ group displayed calcium alternans, increased calcium spark frequency, and increased phosphorylation of S2814 on ryanodine receptor 2. These abnormalities in intracellular calcium handling were attenuated by exercise training (P < 0.05). Exercise training prevented ischemically induced VF, in association with a combination of beneficial effects on cellular electrophysiology and calcium handling.

MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex.

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.