In heart failure (HF), Ca 2+ /calmodulin kinase II (CaMKII) expression is increased. Altered Na + channel gating is linked to and may promote ventricular tachyarrhythmias (VTs) in HF. Calmodulin regulates Na + channel gating, in part perhaps via CaMKII. We investigated effects of adenovirus-mediated (acute) and Tg (chronic) overexpression of cytosolic CaMKIIδ C on Na + current (I Na ) in rabbit and mouse ventricular myocytes, respectively (in whole-cell patch clamp). Both acute and chronic CaMKIIδ C overexpression shifted voltage dependence of Na + channel availability by -6 mV (P < 0.05), and the shift was Ca 2+ dependent. CaMKII also enhanced intermediate inactivation and slowed recovery from inactivation (prevented by CaMKII inhibitors autocamtide 2-related inhibitory peptide [AIP] or KN93). CaMKIIδ C markedly increased persistent (late) inward I Na and intracellular Na + concentration (as measured by the Na + indicator sodium-binding benzofuran isophthalate [SBFI]), which was prevented by CaMKII inhibition in the case of acute CaMKIIδ C overexpression. CaMKII coimmunoprecipitates with and phosphorylates Na + channels. In vivo, transgenic CaMKIIδ C overexpression prolonged QRS duration and repolarization (QT intervals), decreased effective refractory periods, and increased the propensity to develop VT. We conclude that CaMKII associates with and phosphorylates cardiac Na + channels. This alters I Na gating to reduce availability at high heart rate, while enhancing late I Na (which could prolong action potential duration). In mice, enhanced CaMKIIδ C activity predisposed to VT. Thus, CaMKIIdependent regulation of Na + channel function may contribute to arrhythmogenesis in HF.

Cardiac pacemaker cells create rhythmic pulses that control heart rate; pacemaker dysfunction is a prevalent disorder in the elderly, but little is known about the underlying molecular causes. Popeye domain containing (Popdc) genes encode membrane proteins with high expression levels in cardiac myocytes and specifically in the cardiac pacemaking and conduction system. Here, we report the phenotypic analysis of mice deficient in Popdc1 or Popdc2. ECG analysis revealed severe sinus node dysfunction when freely roaming mutant animals were subjected to physical or mental stress. In both mutants, bradyarrhythmia developed in an age-dependent manner. Furthermore, we found that the conserved Popeye domain functioned as a high-affinity cAMP-binding site. Popdc proteins interacted with the potassium channel TREK-1, which led to increased cell surface expression and enhanced current density, both of which were negatively modulated by cAMP. These data indicate that Popdc proteins have an important regulatory function in heart rate dynamics that is mediated, at least in part, through cAMP binding. Mice with mutant Popdc1 and Popdc2 alleles are therefore useful models for the dissection of the mechanisms causing pacemaker dysfunction and could aid in the development of strategies for therapeutic intervention.

Voltage-gated sodium channels composed of pore-forming ␣ and auxiliary ␤ subunits are responsible for the rising phase of the action potential in cardiac muscle, but the functional roles of distinct sodium channel subtypes have not been clearly defined. Immunocytochemical studies show that the principal cardiac poreforming ␣ subunit isoform Nav1.5 is preferentially localized in intercalated disks, whereas the brain ␣ subunit isoforms Nav1.1, Nav1.3, and Nav1.6 are localized in the transverse tubules. Sodium currents due to the highly tetrodotoxin (TTX)-sensitive brain isoforms in the transverse tubules are small and are detectable only after activation with ␤ scorpion toxin. Nevertheless, they play an important role in coupling depolarization of the cell surface membrane to contraction, because low TTX concentrations reduce left ventricular function. Our results suggest that the principal cardiac isoform in the intercalated disks is primarily responsible for action potential conduction between cells and reveal an unexpected role for brain sodium channel isoforms in the transverse tubules in coupling electrical excitation to contraction in cardiac muscle.

Background-Voltage-gated sodium channels composed of pore-forming ␣ and auxiliary ␤ subunits are responsible for the rising phase of the action potential in cardiac muscle, but their localizations have not yet been clearly defined. Methods and Results-Immunocytochemical studies show that the principal cardiac ␣ subunit isoform Na v 1.5 and the ␤2 subunit are preferentially localized in intercalated disks, identified by immunostaining of connexin 43, the major protein of cardiac gap junctions. The brain ␣ subunit isoforms Na v 1.1, Na v 1.3, and Na v 1.6 are preferentially localized with ␤1 and ␤3 subunits in the transverse tubules, identified by immunostaining of ␣-actinin, a cardiac z-line protein. The ␤1 subunit is also present in a small fraction of intercalated disks. The recently cloned ␤4 subunit, which closely resembles ␤2 in amino acid sequence, is also expressed in ventricular myocytes and is localized in intercalated disks as are ␤2 and Na v 1.5. Conclusions-Our results suggest that the primary sodium channels present in ventricular myocytes are composed of Na v 1.5 plus ␤2 and/or ␤4 subunits in intercalated disks and Na v 1.1, Na v 1.3, and Na v 1.6 plus ␤1 and/or ␤3 subunits in the transverse tubules. Key Words: ion channels Ⅲ sodium Ⅲ myocytes Ⅲ ventricles Ⅲ immunohistochemistry V oltage-gated sodium channels are responsible for the initiation of action potentials in most excitable cells. They are composed of a pore-forming ␣ subunit and 1 or 2 auxiliary ␤ subunits. 1 Ten genes encoding ␣ subunits have been identified, and 9 have been functionally expressed. 2 The different ␣ subunit isoforms have distinct patterns of development and localization in the nervous system and skeletal and cardiac muscle, and they have different physiological and pharmacological properties. Isoforms preferentially expressed in the central nervous system (Na v 1.1, Na v 1.2, Na v 1.3, Na v 1.6) are inhibited by nanomolar concentrations of tetrodotoxin, as is the isoform present in adult skeletal muscle (Na v 1.4). In contrast, the primary cardiac isoform (Na v 1.5) requires micromolar concentrations of tetrodotoxin for inhibition because of the presence of a cysteine instead of an aromatic residue in the pore region of domain I. 3 Recent studies show that the brain-type isoforms Na v 1.1, Na v 1.3, and Na v 1.6 are expressed in ventricular myocytes and have distinct subcellular localization and function. 4 The 4 known ␤ subunits of sodium channels divide into 2 groups: ␤1 and ␤3 are most similar in sequence and are noncovalently associated with ␣ subunits, 5,6 while the ␤2 and ␤4 subunits are also closely related in amino acid sequence and are disulfide-linked to ␣ subunits. 7,8 The ␤ subunits are multifunctional because they modulate channel gating, regulate the level of expression at the plasma membrane, and function as cell adhesion molecules through interaction with the cytoskeleton, extracellular matrix, and other cell adhesion molecules that regulate cell migration and aggregation. 9 mRNA for the ␤1 subunit has been...

The majority of Na + channels in the heart are composed of the tetrodotoxin (TTX)-resistant (K D , 2-6 µM) Na v 1.5 isoform; however, recently it has been shown that TTX-sensitive (K D , 1-10 nM) neuronal Na + channel isoforms (Na v 1.1, Na v 1.3 and Na v 1.6) are also present and functionally important in the myocytes of the ventricles and the sinoatrial (SA) node. In the present study, in mouse SA node pacemaker cells, we investigated Na + currents under physiological conditions and the expression of cardiac and neuronal Na + channel isoforms. We identified two distinct Na + current components, TTX resistant and TTX sensitive. At 37• C, TTX-resistant i Na and TTX-sensitive i Na started to activate at ∼ −70 and ∼ −60 mV, and peaked at −30 and −10 mV, with a current density of 22 ± 3 and 18 ± 1 pA pF −1 , respectively. TTX-sensitive i Na inactivated at more positive potentials as compared to TTX-resistant i Na . Using action potential clamp, TTX-sensitive i Na was observed to activate late during the pacemaker potential. Using immunocytochemistry and confocal microscopy, different distributions of the TTX-resistant cardiac isoform, Na v 1.5, and the TTX-sensitive neuronal isoform, Na v 1.1, were observed: Na v 1.5 was absent from the centre of the SA node, but present in the periphery of the SA node, whereas Na v 1.1 was present throughout the SA node. Nanomolar concentrations (10 or 100 nM) of TTX, which block TTX-sensitive i Na , slowed pacemaking in both intact SA node preparations and isolated SA node cells without a significant effect on SA node conduction. In contrast, micromolar concentrations (1-30 µM) of TTX, which block TTX-resistant i Na as well as TTX-sensitive i Na , slowed both pacemaking and SA node conduction. It is concluded that two Na + channel isoforms are important for the functioning of the SA node: neuronal (putative Na v 1.1) and cardiac Na v 1.5 isoforms are involved in pacemaking, although the cardiac Na v 1.5 isoform alone is involved in the propagation of the action potential from the SA node to the surrounding atrial muscle.