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.

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.

Evidence for cardiac atrophic remodeling in cancer-induced cachexia in mice.

Cachexia is a common complication in cancer patients, which dramatically reduces quality of life and survival. In contrast to the well-studied feature of skeletal muscle loss, alterations in cardiac muscle are unclear. Recently, we reported that heart contractile function was significantly impaired in mice with colon-26 (C26) tumors, a widely used rodent model of cancer cachexia. In the present study, we investigated the potential underlying mechanisms for decreased heart function, specifically related to cardiac remodeling and atrophy. In cachectic mice bearing C26 tumors compared to mice without tumors, there was a gene expression pattern for cardiac remodeling, including increased BNP and c-fos, decreased PPARα and its responsive gene CPT1ß, and a switch from 'adult' isoforms (MHCα, GLUT4) to 'fetal' isoforms (MHCß and GLUT1). Echocardiography identified a decreased cardiac wall thickness. RT-PCR and Western blotting revealed a decreased amount of cardiac myofibrillar proteins MHC and troponin I, induced expression of E-3 ligases (MuRF-1 and Atrogin-1) and increased protein ubiquitination, providing evidence for cardiac atrophy in mice with cancer cachexia. Regulatory signaling pathways mediating these changes may include p44/42 MAPK. Together, these data provide evidence that pathways leading to cardiac remodeling and atrophy occur in mice with C26 cachexia.

Cardiac alterations in cancer-induced cachexia in mice.

Cachexia is a common syndrome in advanced cancer patients and causes up to 22% of cancer-related deaths. It remains elusive whether cancer cachexia causes heart failure. We investigated the effect of cancer cachexia on heart function and cardiac muscle structure in a mouse model. Male CD2F1 mice were inoculated with either colon-26 adenocarcinoma cells (Tumor group) or vehicle (PBS) (No Tumor group and Pair-fed group). Heart function as measured by fractional shortening in vivo using transthoracic echocardiography was performed on day 14 after tumor or PBS inoculation. At necropsy (day 17), hearts were collected for histology, transmission electron microscopy, RT-PCR and SDS-PAGE analysis. Mice from the Tumor group displayed a significantly reduced fractional shortening compared to mice in the No Tumor and Pair-fed groups. In hearts of the Tumor mice compared to the other groups, there was marked fibrosis and transmission electron microscopy revealed disrupted myocardial ultrastructure. Gene expression of troponin I, a regulator of cardiac muscle contraction, was reduced. Moreover, both mRNA and protein levels of myosin heavy chain (MHC) were altered whereby MHCalpha (adult isoform) was decreased and MHCbeta (fetal isoform) was increased indicating reactivation of the fetal gene expression pattern. In conclusion, heart function was diminished in mice with tumor-induced cachexia, and this impaired function was associated with increased fibrosis, disrupted myocardial structure and altered composition of contractile proteins of cardiac muscle.

Role of heat shock factor-1 activation in the doxorubicin-induced heart failure in mice.

Treating cancer patients with chemotherapeutics, such as doxorubicin (Dox), cause dilated cardiomyopathy and congestive heart failure because of oxidative stress. On the other hand, heat shock factor-1 (HSF-1), a transcription factor for heat shock proteins (Hsps), is also known to be activated in response to oxidative stress. However, the possible role of HSF-1 activation and the resultant Hsp25 in chemotherapeutic-induced heart failure has not been investigated. Using HSF-1 wild-type (HSF-1(+/+)) and knock-out (HSF-1(-/-)) mice, we tested the hypothesis that activation of HSF-1 plays a role in the development of Dox-induced heart failure. Higher levels of Hsp25 and its phosphorylated forms were found in the failing hearts of Dox-treated HSF-1(+/+) mice. More than twofold increase in Hsp25 mRNA level was found in Dox-treated hearts. Proteomic analysis showed that there is accumulation and aggregation of Hsp25 in Dox-treated failing hearts. Additionally, Hsp25 was found to coimmunoprecipitate with p53 and vice versa. Further studies indicated that the Dox-induced higher levels of Hsp25 transactivated p53 leading to higher levels of the pro-apoptotic protein Bax, but other p53-related proteins remained unaltered. Moreover, HSF-1(-/-) mice showed significantly reduced Dox-induced heart failure and higher survival rate, and there was no change in Bax upon treating with Dox in HSF-1(-/-) mice. From these results we propose a novel mechanism for Dox-induced heart failure: increased expression of Hsp25 because of oxidant-induced activation of HSF-1 transactivates p53 to increase Bax levels, which leads to heart failure.

Chronic heart failure and the substrate for atrial fibrillation.

AIMS: We sought to define the underlying mechanisms for atrial fibrillation (AF) during chronic heart failure (HF). METHODS AND RESULTS: Preliminary studies showed that 4 months of HF resulted in irreversible systolic dysfunction (n = 9) and a substrate for sustained inducible AF (>3 months, n = 3). We used a chronic (4-month) canine model of tachypacing-induced HF (n = 10) to assess atrial electrophysiological remodelling, relative to controls (n = 5). Left ventricular fractional shortening was reduced from 37.2 +/- 0.83 to 13.44 +/- 2.63% (P < 0.05). Left atrial (LA) contractility (fractional area change) was reduced from 34.9 +/- 7.9 to 27.9 +/- 4.23% (P < 0.05). Action potential durations (APDs) at 50 and 90% repolarization were shortened by approximately 60 and 40%, respectively, during HF (P < 0.05). HF-induced atrial remodelling included increased fibrosis, increased I(to), and decreased I(K1), I(Kur), and I(Ks) (P < 0.05). HF induced increases in LA Kv channel interacting protein 2 (P < 0.05), no change in Kv4.3, Kv1.5, or Kir2.3, and reduced Kir2.1 (P < 0.05). When I(Ca-L) was elicited by action potential (AP) clamp, HF APs reduced the integral of I(Ca) in control myocytes, with a larger reduction in HF myocytes (P < 0.05). I(CaL) measured with standard voltage clamp was unchanged by HF. Incubation of myocytes with N-acetylcysteine (a glutathione precursor) attenuated HF-induced electrophysiological alterations. LA angiotensin-1 receptor expression was increased in HF. CONCLUSION: Chronic HF causes alterations in ion channel expression and ion currents, resulting in attenuation of the APD and atrial contractility and a substrate for persistent AF.