Chronic verapamil treatment remodelsI_Ca,Lin mouse ventricle

Abstract

(41), although in late-stage heart failure the consensus finding is that I Ca,L declines (47). The unchanged level of I Ca,L in all but late-stage heart failure suggests that ventricular myocytes may have the capacity to homeostatically regulate I Ca,L .At the plasma membrane the Ca 2ϩ channel complex exists as a heteromultimer consisting of the ␣ 1 -subunit (Ca V 1.2), a cytosolic ␤-subunit (Ca V ␤ 2 ), and a covalently linked ␣ 2 ␦-subunit. Ca V 1.2, the pore-forming subunit, determines ion selectivity and voltage sensitivity and is a binding site for clinically used Ca 2ϩ channel blockers. Ca V ␤ coexpression primarily increases Ca V 1.2 current density in heterologous expression systems (4,8,21,48) and in myocytes that overexpress Ca V ␤ (45). In addition to increasing functional expression, Ca V ␤s alter channel kinetics (29,36,44) and voltage dependence (5, 28, 39). Among the accessory subunits, the Ca V ␤s are the only accessory proteins known to increase current density. In contrast to Ca V ␤, the ␣ 2 ␦-subunit has modest effects on channel current density and has variable effects on channel voltage dependence and kinetics (6,25,26,31). Overall, Ca V ␤ 2 in the heart serves as a positive regulator of I Ca,L (11). We recently showed (13,19) channel function. Thus both positive and negative regulators, in the form of auxiliary proteins, influence ultimate I Ca,L density. Ca 2ϩ channel blockers (CCBs) are frequently used to regulate blood pressure in hypertensive patients (22,32,38); however, little is known about the long-term cardiac effects of these drugs on cardiac myocytes. The primary objective of this study was to test the hypothesis that cardiac I Ca,L is homeostatically regulated. We postulated that Ca 2ϩ entering through the L-type channel provides feedback regulation. According to our hypothesis, decreasing I Ca,L , such as via chronic CCB, would elicit upregulation of I Ca,L . Conversely, stressors that acutely increase cytosolic Ca 2ϩ cause long-term decreases of I Ca,L as in heart failure (7, 46). Our results demonstrate that long-term Ca 2ϩ channel blockade induces an increase of Ca V 1.2 mRNA and protein and significantly increases basal I Ca,L . In contrast, chronic ␤-adrenergic stimulation induces upregulation of positive and negative regulators of I Ca,L with no significant change in basal I Ca,L . Together, these results suggest that the main pore-forming subunit plays a greater role in determining I Ca,L than auxiliary subunits. MATERIALS AND METHODSMini-osmotic pumps. Female or male ICR mice (12-14 wk of age) weighing between 25 and 30 g were anesthetized with a ketaminexylazine mixture (ip), allowing the subcutaneous implantation of mini-osmotic pumps (Alzet, model 2001). The pumps were filled with verapamil (Ver), isoproterenol (Iso), isoproterenol and verapamil (Iso ϩ Ver), or vehicle alone (0.02% ascorbic acid). Mice carrying drug-loaded minipumps are abbreviated as Iso, Ver, and Iso ϩ Ver for pumps containing isoproterenol, verapamil, or the two drugs together. Control animals c...

The cardiomyocyte molecular clock, regulation of Scn5a, and arrhythmia susceptibility

“…Adult ventricular myocytes were isolated from the mouse heart as described previously at 6 -8 wk following injections to avoid any confounding effects of acute tamoxifen toxicity (8,20,24,45). Mice were anesthetized and hearts were rapidly excised and retrogradely perfused at 3 ml/min for 4 -8 min at 37°C with a Ca 2ϩ -free bicarbonate-based perfusion buffer containing (in mM) 113 NaCl, 4.7 KCl, 0.6 KH 2PO4, 1.2 MgSO4, 0.6 NaH2PO4, 5.5 glucose, 12 NaHCO 3, 10 KHCO3, 10 HEPES, 0.032 phenol red, 10 2,3-butanedione monoxime, and 30 taurine.…”

The RGK family of GTP-binding proteins: Regulators of voltage-dependent calcium channels and cytoskeleton remodeling

“…Among the most unique features of RGK proteins is their ability to be transcriptionally regulated [3,5,6,11,[20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] (Table 2). Indeed, Gem/Kir was discovered as a gene upregulated in human T cells after stimulation with mitogens [5] or in BCR-Abl-transformed B cells [11].…”

“…The other RGK subfamily members also demonstrate tissue and molecule-specific transcriptional regulation [3,4,6,20,21,24,25,27,[30][31][32][33][34][35]. Rem is expressed predominantly in cardiac muscle, but also at more modest levels in lung, kidney, and skeletal muscle [3]; Rem2 is highly expressed in the brain and kidney, but also in neuroendocrine tissues [4]; Rad is found in abundance in cardiac and skeletal muscle [6]; while Gem/Kir is found in a diverse set of tissues, including myeloid cells, kidney, liver, and lung [5].…”

“…Since Rem2 is the only RGK protein abundantly expressed in neuronal tissues [4], Rem2 signaling appears crucial for synapse development and maturation. Finally, Rem expression is increased in cardiomyocytes following treatment with isoproterenol, a β-adrenergic receptor agonist [32], but decreased in cardiac muscle upon injection of mice with lipopolysaccharide [3]. While these results are intriguing, the physiological relevance of RGK transcriptional regulation is still unclear.…”

“…While these results are intriguing, the physiological relevance of RGK transcriptional regulation is still unclear. However, it should be noted that RGK signaling may control a previously unappreciated negative feedback cascade operating to protect both pancreatic β-cells (Rem2) and cardiomyocytes (Rem) from uncontrolled Ca 2+ signaling in the presence of persistent hyperglycemia or chronic β-adrenergic stimulation [23,32]. Since disruption of intracellular Ca 2+ homeostasis readily induces cellular dysfunction, this putative pathway may play a significant protective role.…”