Understanding mechanisms underlying titin regulation in cardiac muscle function is of critical importance given recent compelling evidence that highlight titin mutations as major determinants of ...human cardiomyopathy. We previously identified a cardiac biomechanical stress-regulated complex at the cardiac-specific N2B region of titin that includes four-and-a-half LIM domain protein-1 (Fhl1) and components of the mitogen-activated protein signaling cascade, which impacted muscle compliance in Fhl1 knock-out cardiac muscle. However, direct regulation of these molecular components in mediating titin N2B function remained unresolved. Here we identify Fhl1 as a novel negative regulator of titin N2B levels and phosphorylation-mediated mechanics. We specifically identify titin N2B as a novel substrate of extracellular signal regulated-kinase-2 (Erk2) and demonstrate that Fhl1 directly interferes with Erk2-mediated titin-N2B phosphorylation. We highlight the critical region in titin-N2B that interacts with Fhl1 and residues that are dependent on Erk2-mediated phosphorylation in situ. We also propose a potential mechanism for a known titin-N2B cardiomyopathy-causing mutation that involves this regulatory complex. These studies shed light on a novel mechanism regulating titin-N2B mechano-signaling as well as suggest that dysfunction of these pathways could be important in cardiac disease states affecting muscle compliance.
Background: Titin is critical for cardiac muscle function; however, limited knowledge exists of mechanisms important for its regulation.
Results: A four-and-a-half LIM domain protein-1/extracellular signal-regulated kinase-2-associated complex modulates titin-N2B levels, phosphorylation, and mechanics.
Conclusion: We reveal new mechanisms underlying titin mechano-signaling.
Significance: We advance our understanding of how titin-associated complexes/mutations can impact cardiac muscle function and disease.
Departments of 1 Medicine and 2 Pharmacology and 3 Biomedical Sciences PhD Program, Univeristy of California, San Diego School of Medicine, La Jolla, California
Submitted 2 June 2008
; accepted in ...final form 3 March 2009
Rat cardiac fibroblasts (CF) express multiple adenosine (ADO) receptors. Pharmacological evidence suggests that activation of A 2 receptors may inhibit collagen synthesis via adenylyl cyclase-induced elevation of cellular cAMP. We have characterized the signaling pathways involved in ADO-mediated inhibition of collagen synthesis in primary cultures of adult rat CF. ANG II stimulates collagen production in these cells. Coincubation with agents that elevate cellular cAMP the ADO agonist, 5'- N -ethylcarboxamidoadensoine (NECA), and forskolin inhibited the stimulatory effects of ANG II. However, direct stimulators and inhibitors of protein kinase A (PKA) did not alter ANG II-induced collagen synthesis, indicating that PKA does not mediate the inhibitory effects of NECA. Inhibitors of AMP-kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2) do not alter NECA-inhibited collagen synthesis. However, activation of exchange factor directly activated by cAMP (Epac) mimicked the effects of NECA on ANG II-stimulated collagen synthesis. Inhibition of phosphoinositol-3 kinase (PI3K) reduced the inhibitory effects of NECA on ANG II-induced collagen synthesis, suggesting that NECA acts via PI3K. Furthermore, inhibition of PI3K also relieved the inhibitory effect of Epac activation on ANG II-stimulated collagen synthesis. Thus it appears that ADO activates the A 2 R-G s -adenylyl cyclase pathway and that the resultant cAMP reduces collagen synthesis via a PKA-independent, Epac-dependent pathway that feeds through PI3K.
A 2 receptors; Epac; phosphoinositol-3 kinase; cardiac collagen deposition
Address for reprint requests and other correspondence: F. Villarreal, 9500 Gilman Dr., 0613J, BSB 4028, La Jolla, CA 92093 (e-mail: fvillarr{at}ucsd.edu )
Department of Medicine and Bioengineering, University of California, San Diego, La Jolla, California
Submitted 1 February 2008
; accepted in final form 27 May 2008
Although previous studies report a ...reduction in myocardial volume during systole, myocardial volume changes during the cardiac cycle have not been quantitatively analyzed with high spatiotemporal resolution. We studied the time course of myocardial volume in the anterior mid-left ventricular (LV) wall of normal canine heart in vivo ( n = 14) during atrial or LV pacing using transmurally implanted markers and biplane cineradiography (8 ms/frame). During atrial pacing, there was a significant transmural gradient in maximum volume decrease (4.1, 6.8, and 10.3% at subepi, midwall, and subendo layer, respectively, P = 0.002). The rate of myocardial volume increase during diastole was 4.7 ± 5.8, 6.8 ± 6.1, and 10.8 ± 7.7 ml·min –1 ·g –1 , respectively, which is substantially larger than the average myocardial blood flow in the literature measured by the microsphere method (0.7–1.3 ml·min –1 ·g –1 ). In the early activated region during LV pacing, myocardial volume began to decrease before the LV pressure upstroke. We conclude that the volume change is greater than would be estimated from the known average transmural blood flow. This implies the existence of blood-filled spaces within the myocardium, which could communicate with the ventricular lumen. Our data in the early activated region also suggest that myocardial volume change is caused not by the intramyocardial tissue pressure but by direct impingement of the contracting myocytes on the microvasculature.
myocardial structure; ventricular pacing; mechanical dyssynchrony
Address for reprint requests and other correspondence: H. Ashikaga, Div. of Cardiology, Johns Hopkins Univ. School of Medicine, 720 Rutland Ave., Traylor 903, Baltimore, MD 21205 (e-mail: ha8000{at}gmail.com )
Abstract Background Targeting the mitochondria during ischemia/reperfusion (IR) can confer cardioprotection leading to improved clinical outcomes. The cardioprotective potential of (−)-epicatechin ...(EPI) during IR via modulation of mitochondrial function was evaluated. Methods and results Ischemia was induced in rats via a 45 min occlusion of the left anterior descending coronary artery followed by 1 h, 48 h, or 3 week reperfusion. EPI (10 mg/kg) was administered IV 15 min prior to reperfusion for the single dose group and again 12 h later for the double dose group. Controls received water. Experiments also utilized cultured neonatal rat ventricular myocytes (NRVM) and myoblasts. A single dose of EPI reduced infarct size by 27% at 48 h and 28% at 3 week. Double dose treatment further decreased infarct size by 80% at 48 h, and 52% by 3 weeks. The protective effect of EPI on mitochondrial function was evident after 1 h of reperfusion when mitochondria demonstrated less respiratory inhibition, lower mitochondrial Ca 2 + load, and a preserved pool of NADH that correlated with higher tissue ATP levels. Mechanistic studies in NRVM revealed that EPI acutely stimulated maximal rates of respiration, an effect that was blocked by inhibitors of the mitochondrial pyruvate carrier, nitric oxide synthase, or soluble guanylyl cyclase. In myoblasts, knockdown of components of the mitochondrial pyruvate carrier blocked EPI-induced respiratory stimulation. Conclusions IV EPI confers cardioprotection via preservation of mitochondrial function potentially through enhanced substrate provision. These provocative results document a novel mechanism of a natural product with potential clinical utility.
In humans, DOCK8 immunodeficiency syndrome is characterized by severe cutaneous viral infections. Thus, CD8 T cell function may be compromised in the absence of DOCK8. In this study, by analyzing ...mutant mice and humans, we demonstrate a critical, intrinsic role for DOCK8 in peripheral CD8 T cell survival and function. DOCK8 mutation selectively diminished the abundance of circulating naive CD8 T cells in both species, and in DOCK8-deficient humans, most CD8 T cells displayed an exhausted CD45RA(+)CCR7(-) phenotype. Analyses in mice revealed the CD8 T cell abnormalities to be cell autonomous and primarily postthymic. DOCK8 mutant naive CD8 T cells had a shorter lifespan and, upon encounter with antigen on dendritic cells, exhibited poor LFA-1 synaptic polarization and a delay in the first cell division. Although DOCK8 mutant T cells underwent near-normal primary clonal expansion after primary infection with recombinant influenza virus in vivo, they showed greatly reduced memory cell persistence and recall. These findings highlight a key role for DOCK8 in the survival and function of human and mouse CD8 T cells.
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