Impaired modulation of the cardiac L-type Ca2+ channel activity by ahnak-1 after myocardial infarction

Introduction: The L-type cardiac Ca2+ channel (Cav 1.2) is an important determinant of cardiac repolarization and the main source of activator Ca2+ during excitation-contraction coupling in cardiac cells. Its defective regulation is a major cause of arrhythmias and contractile dysfunction. We have recently shown that the cytoskeletal protein ahnak-1 modulates Ca2+ current through Cav 1.2 channels (ICaL) by interacting with the regulatory beta-subunit of the Cav 1.2 channel and that the genetic variant of ahnak-1 I5483T (previously Ile5236Thr), interferes with the beta-adrenergic stimulation of ICaL. Objective: To extend our study of the I5483T variant to ventricular cardiomyocytes dissociated from remodelled infarcted rat hearts (PMI). Methods: The patch-clamp technique was used to record ICaL from enzymatically dissociated ventricular cardiomyocytes from young (2-month-old) and six-month-old sham-operated and PMI rats. Results: Basal ICaL was increased from 11 ± 0.5 A/F in young cardiomyocytes to 14.6 ± 1.1 A/F and 15.7 ± 1 A/F in sham and PMI cardiomyocytes respectively, while isoprenaline (ISO, 1 µmol/L) further increased ICaL by 101 ± 6%, 109 ± 10% and 104 ± 12% respectively. When cells were intracellularly perfused with a peptide containing the mutated ahnak-1 sequence (10 µmol/L) basal ICaL was increased to 20 ± 1 A/F, 22 ± 2 A/F and 21 ± 2 A/F in young, sham and PMI cardiomyocytes respectively. In these cells ISO increased ICaL by 11 ± 4%, 33 ± 6% and 79 ± 12% respectively. Conclusion: Modulation of ICaL by ahnak-1is impaired by myocardial ischemia and remodelling. Since ahnak-1 and Cav 1.2 channels co-localize in the transverse T-tubule system, remodelling of T-tubules could affect the interaction of ahnak-1 with the regulatory beta subunit of these channels

Ahnak1 modulates L-type Ca(2+) channel inactivation of rodent cardiomyocytes

Ahnak1, a giant 700 kDa protein, has been implicated in Ca(2+) signalling in various cells. Previous work suggested that the interaction between ahnak1 and Cavbeta(2) subunit plays a role in L-type Ca(2+) current (I (CaL)) regulation. Here, we performed structure-function studies with the most C-terminal domain of ahnak1 (188 amino acids) containing a PxxP consensus motif (designated as 188-PSTP) using ventricular cardiomyocytes isolated from rats, wild-type (WT) mice and ahnak1-deficient mice. In vitro binding studies revealed that 188-PSTP conferred high-affinity binding to Cavbeta(2) (K (d) approximately 60 nM). Replacement of proline residues by alanines (188-ASTA) decreased Cavbeta(2) affinity about 20-fold. Both 188-PSTP and 188-ASTA were functional in ahnak1-expressing rat and mouse cardiomyocytes during whole-cell patch clamp. Upon intracellular application, they increased the net Ca(2+) influx by enhancing I (CaL) density and/or increasing I (CaL) inactivation time course without altering voltage dependency. Specifically, 188-ASTA, which failed to affect I (CaL) density, markedly slowed I (CaL) inactivation resulting in a 50-70% increase in transported Ca(2+) during a 0 mV depolarising pulse. Both ahnak1 fragments also slowed current inactivation with Ba(2+) as charge carrier. By contrast, neither 188-PSTP nor 188-ASTA affected any I (CaL) characteristics in ahnak1-deficient mouse cardiomyocytes. Our results indicate that the presence of endogenous ahnak1 is required for tuning the voltage-dependent component of I (CaL) inactivation by ahnak1 fragments. We suggest that ahnak1 modulates the accessibility of molecular determinants in Cavbeta(2) and/or scaffolds selectively different beta-subunit isoforms in the heart

PKA phosphorylation activates the calcium release channel (ryanodine receptor) in skeletal muscle: defective regulation in heart failure

The type 1 ryanodine receptor (RyR1) on the sarcoplasmic reticulum (SR) is the major calcium (Ca2+) release channel required for skeletal muscle excitation–contraction (EC) coupling. RyR1 function is modulated by proteins that bind to its large cytoplasmic scaffold domain, including the FK506 binding protein (FKBP12) and PKA. PKA is activated during sympathetic nervous system (SNS) stimulation. We show that PKA phosphorylation of RyR1 at Ser2843 activates the channel by releasing FKBP12. When FKB12 is bound to RyR1, it inhibits the channel by stabilizing its closed state. RyR1 in skeletal muscle from animals with heart failure (HF), a chronic hyperadrenergic state, were PKA hyperphosphorylated, depleted of FKBP12, and exhibited increased activity, suggesting that the channels are “leaky.” RyR1 PKA hyperphosphorylation correlated with impaired SR Ca2+ release and early fatigue in HF skeletal muscle. These findings identify a novel mechanism that regulates RyR1 function via PKA phosphorylation in response to SNS stimulation. PKA hyperphosphorylation of RyR1 may contribute to impaired skeletal muscle function in HF, suggesting that a generalized EC coupling myopathy may play a role in HF

Intravascular ADP and soluble nucleotidases contribute to acute prothrombotic state during vigorous exercise in humans

This article has been made available through the Brunel Open Access Publishing Fund and is available from the specified link - Copyright @ 2007 The Physiological Society .Extracellular ATP and ADP trigger vasodilatatory and prothrombotic signalling events in the vasculature. Here, we tested the hypothesis that nucleotide turnover is activated in the bloodstream of exercising humans thus contributing to the enhanced platelet reactivity and haemostasis. Right atrial, arterial and venous blood samples were collected from endurance-trained athletes at rest, during submaximal and maximal cycle ergometer exercise, and after early recovery. ATP-specific bioluminescent assay, together with high-performance liquid chromatographic analysis, revealed that plasma ATP and ADP concentrations increased up to 2.5-fold during maximal exercise. Subsequent flow cytometric analysis showed that plasma from exercising subjects significantly up-regulated the surface expression of P-selectin in human platelets and these prothrombotic effects were diminished after scavenging plasma nucleotides with exogenous apyrase. Next, using thin layer chromatographic assays with [gamma-P-32]ATP and H-3/C-14-labelled nucleotides, we showed that two soluble nucleotide-inactivating enzymes, nucleotide pyrophosphatase/phosphodiesterase and nucleoside triphosphate diphosphohydrolase, constitutively circulate in human bloodstream. Strikingly, serum nucleotide pyrophosphatase and hydrolase activities rose during maximal exercise by 20-25 and 80-100%, respectively, and then declined after 30 min recovery. Likewise, soluble nucleotidases were transiently up-regulated in the venous blood of sedentary subjects during exhaustive exercise. Human serum also contains 5'-nucleotidase, adenylate kinase and nucleoside diphosphate (NDP) kinase; however, these activities remain unchanged during exercise. In conclusion, intravascular ADP significantly augments platelet activity during strenuous exercise and these prothrombotic responses are counteracted by concurrent release of soluble nucleotide-inactivating enzymes. These findings provide a novel insight into the mechanisms underlying the enhanced risk of occlusive thrombus formation under exercising conditions

Antimicrobial susceptibility and molecular detection of chloramphenicol and florfenicol resistance among Escherichia coli isolates from diseased chickens

Seventy Escherichia coli isolates recovered from diseased chickens diagnosed with colibacillosis in Henan Province, China, between 2004 and 2005 were characterized for antimicrobial susceptibility profiles via a broth doubling dilution method. Overall, the isolates displayed resistance to trimethoprim-sulfamethoxazole (100%), oxytetracycline (100%), ampicillin (83%), enrofloxacin (83%), and ciprofloxacin (81%), respectively. Among the phenicols, resistance was approximately 79% and 29% for chloramphenicol and florfenicol, respectively. Molecular detection revealed that the incidence rates of the floR, cmlA, cat1, cat2 and cat3 were 29, 31, 16, 13, and 0%, respectively. Additionally, 10% of the isolates were positive for both floR and cmlA. As these antimicrobial agents may potentially induce cross-resistance between animal and human bacterial pathogens, their prudent use in veterinary medicine is highly recommended