Cardiomyocytes with disrupted CFTR function require CaMKII and Ca(2+)-activated Cl(-) channel activity to maintain contraction rate.

The physiological role of the cystic fibrosis transmembrane conductance regulator (CFTR) in cardiomyocytes remains unclear. Using spontaneously beating neonatal ventricular cardiomyocytes from wild-type (WT) or CFTR knockout (KO) mice, we examined the role of CFTR in the modulation of cardiomyocyte contraction rate. Contraction rates of spontaneously beating myocytes were captured by video imaging. Real-time changes in intracellular ([Ca(2+)](i)) and protein kinase A (PKA) activity were measured by fura-2 and fluorescence resonance energy transfer, respectively. Acute inhibition of CFTR in WT cardiomyocytes using the CFTR inhibitor CFTR(inh)-172 transiently inhibited the contraction rate. By contrast, cardiomyocytes from CFTR KO mice displayed normal contraction rates. Further investigation revealed that acute inhibition of CFTR activity in WT cardiomyocytes activated L-type Ca(2+) channels, leading to a transient increase of [Ca(2+)](i) and inhibition of PKA activity. Additionally, we found that contraction rate normalization following acute CFTR inhibition in WT cardiomyocytes or chronic deletion in cardiomyocytes from CFTR KO mice requires the activation of Ca(2+)/calmodulin-dependent kinase II (CaMKII) and Ca(2+)-activated Cl(-) channels (CaCC) because simultaneous addition of myristoylated-autocamtide-2-related inhibitory peptide or niflumic acid and CFTR(inh)-172 to WT cardiomyocytes or treatment of cardiomyoctes from CFTR KO mice with these agents caused sustained attenuation of contraction rates. Our results demonstrate that regulation of cardiomyocyte contraction involves CFTR. They also reveal that activation of CaMKII and CaCC compensates for loss of CFTR function. Increased dependence on CaMKII upon loss of CFTR function might leave cystic fibrosis patients at increased risk of heart dysfunction and disease.

A small peptide inhibitor of the low voltage-activated calcium channel Cav3.1.

The calcium channel gamma(6) subunit modulates low voltage-activated (LVA) calcium current in both human embryonic kidney (HEK) cells and cardiomyocytes, although the mechanism of modulation is unknown. We recently showed that gamma(6) contains a critical GxxxA motif in the first transmembrane domain (TM1) that is essential for its inhibition of the Cav3.1 (LVA) calcium current. In this study, we tested the hypothesis that an eight-amino acid peptide that contains the GxxxA motif from gamma(6) TM1 can act as a novel pharmacological inhibitor of the Cav3.1 calcium current by performing whole-cell electrophysiology. Our results demonstrate that the peptide inhibits Cav3.1 current by dynamically binding and dissociating from the Cav3.1 channel in a concentration-dependent but largely voltage-independent manner. By selectively substituting residues within the peptide, we show that both the GxxxA framework and surrounding aliphatic side-chains contribute to the presumably interhelical interactions between gamma(6) TM1 and the Cav3.1 channel. The fast kinetics of the interaction supports the view that gamma(6) acts as an endogenous LVA channel antagonist within the plasma membrane, suggesting a mechanism other than regulation of surface expression or membrane trafficking of the pore-forming subunit of the channel. We also demonstrate that the peptide has different affinities for Cav3.1 and Cav1.2 calcium currents, which is consistent with the selective effect of gamma(6) on LVA and high voltage-activated calcium currents in vivo.

A critical GxxxA motif in the gamma6 calcium channel subunit mediates its inhibitory effect on Cav3.1 calcium current.

The eight members of the calcium channel gamma subunit family are integral membrane proteins that regulate the expression and behaviour of voltage and ligand gated ion channels. While a subgroup consisting of gamma(2), gamma(3), gamma(4) and gamma(8) (the TARPs) modulate AMPA receptor localization and function, the gamma(1) and gamma(6) subunits conform to the original description of these proteins as regulators of voltage gated calcium channels. We have previously shown that the gamma(6) subunit is highly expressed in atrial myocytes and that it is capable of acting as a negative modulator of low voltage activated calcium current. In this study we extend our understanding of gamma(6) subunit modulation of low voltage activated calcium current. Using engineered chimeric constructs, we demonstrate that the first transmembrane domain (TM1) of gamma(6) is necessary for its inhibitory effect on Cav3.1 current. Mutational analysis is then used to identify a unique GxxxA motif within TM1 that is required for the function of the subunit strongly suggesting the involvement of helix-helix interactions in its effects. Results from co-immunoprecipitation experiments confirm a physical association of gamma(6) with the Cav3.1 channel in both HEK cells and atrial myocytes. Single channel analysis reveals that binding of gamma(6) reduces channel availability for activation. Taken together, the results of this study provide both a molecular and a mechanistic framework for understanding the unique ability of the gamma(6) calcium channel subunit to modulate low voltage activated (Cav3.1) calcium current density.

Calcium channel gamma subunits: a functionally diverse protein family.

The calcium channel gamma subunits comprise an eight-member protein family that share a common topology consisting of four transmembrane domains and intracellular N- and C-termini. Although the first gamma subunit was identified as an auxiliary subunit of a voltage-dependent calcium channel, a review of phylogenetic, bioinformatic, and functional studies indicates that they are a functionally diverse protein family. A cluster containing gamma1 and gamma6 conforms to the original description of the protein family as they seem to act primarily as subunits of calcium channels expressed in muscle. Members of a second cluster (gamma2, gamma3, gamma4, gamma8) function as regulators of AMPA receptor localization and function in the brain and are collectively known as TARPs. The function of members of the third cluster (gamma5, gamma7) remains unclear. Our analysis shows that the members of each cluster contain conserved regulatory motifs that help to differentiate the groups. However, the physiological significance of these motifs in many cases remains to be demonstrated.

Identification of the calcium channel alpha 1E (Ca(v)2.3) isoform expressed in atrial myocytes.

Antisense oligonucleotides targeting the calcium channel alpha 1E (Ca(v)2.3) subunit significantly inhibit the insulin-like growth factor-1 (IGF-1)-stimulated increase in low voltage-activated (LVA) (T-type) calcium current in cultured rat atrial myocytes [Proc. Natl. Acad. Sci. U.S.A. 94(1997) 14936]. As part of a continuing effort to understand the regulation of LVA current expression in the heart, we have identified the specific alpha 1E isoform that is expressed in atrial tissue. Through reverse transcription-polymerase chain reaction (RT-PCR), nine overlapping partial clones spanning the entire coding region of the cardiac alpha 1E mRNA were obtained. The predominate isoform in atrial tissue was identified and found to be highly homologous to the alpha 1E isoform previously isolated from kidney and the islets of Langerhans [Eur. J. Biochem. 257(1998) 274]. The expression of alpha 1E in the heart occurs specifically in cardiac myocytes and not in smooth muscle or fibroblasts as demonstrated by RT-PCR performed on isolated atrial myocytes and by in situ hybridization.

Left ventricular and aortic dysfunction in cystic fibrosis mice.

BACKGROUND: Left ventricular (LV) abnormalities have been reported in cystic fibrosis (CF); however, it remains unclear if loss of cystic fibrosis transmembrane conductance regulator (CFTR) function causes heart defects independent of lung disease. METHODS: Using gut-corrected F508del CFTR mutant mice (ΔF508), which do not develop human lung disease, we examined in vivo heart and aortic function via 2D transthoracic echocardiography and LV catheterization. RESULTS: ΔF508 mouse hearts showed LV concentric remodeling along with enhanced inotropy (increased +dP/dt, fractional shortening, decreased isovolumetric contraction time) and greater lusitropy (-dP/dt, Tau). Aortas displayed increased stiffness and altered diastolic flow. ß-adrenergic stimulation revealed diminished cardiac reserve (attenuated +dP/dt,-dP/dt, LV pressure). CONCLUSIONS: In a mouse model of CF, CFTR mutation leads to LV remodeling with alteration of cardiac and aortic functions in the absence of lung disease. As CF patients live longer, more active lives, their risk for cardiovascular disease should be considered.

MRP4 and CFTR in the regulation of cAMP and ß-adrenergic contraction in cardiac myocytes.

Spatiotemporal regulation of cAMP in cardiac myocytes is integral to regulating the diverse functions downstream of ß-adrenergic stimulation. The activities of cAMP phosphodiesterases modulate critical and well-studied cellular processes. Recently, in epithelial and smooth muscle cells, it was found that the multi-drug resistant protein 4 (MRP4) acts as a cAMP efflux pump to regulate intracellular cAMP levels and alter effector function, including activation of the cAMP-stimulated Cl(-) channel, CFTR (cystic fibrosis transmembrane conductance regulator). In the current study we investigated the potential role of MRP4 in regulating intracellular cAMP and ß-adrenergic stimulated contraction rate in cardiac myocytes. Cultured neonatal ventricular myocytes were used for all experiments. In addition to wildtype mice, ß(1)-, ß(2)-, and ß(1)/ß(2)-adrenoceptor, and CFTR knockout mice were used. MRP4 expression was probed via Western blot, intracellular cAMP was measured by fluorescence resonance energy transfer, while the functional role of MRP4 was assayed via monitoring of isoproterenol-stimulated contraction rate. We found that MRP4 is expressed in mouse neonatal ventricular myocytes. A pharmacological inhibitor of MRP4, MK571, potentiated submaximal isoproterenol-stimulated cAMP accumulation and cardiomyocyte contraction rate via ß(1)-adrenoceptors. CFTR expression was critical for submaximal isoproterenol-stimulated contraction rate. Interestingly, MRP4-dependent changes in contraction rate were CFTR-dependent, however, PDE4-dependent potentiation of contraction rate was CFTR-independent. We have shown, for the first time, a role for MRP4 in the regulation of cAMP in cardiac myocytes and involvement of CFTR in ß-adrenergic stimulated contraction. Together with phosphodiesterases, MRP4 must be considered when examining cAMP regulation in cardiac myocytes.

Disruption of growth hormone secretion alters Ca2+ current density and expression of Ca2+ channel and insulin-like growth factor genes in rat atria.

The influence of the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis on expression of low-voltage-activated (LVA) Ca2+ current in atrial tissue was investigated using spontaneous dwarf (SpDwf) rats, a mutant strain that lacks GH. Atrial myocytes from SpDwf rats express LVA and high-voltage-activated (HVA) Ca2+ currents and the Ca2+ channel alpha1-subunit genes CaV1.2, CaV2.3, CaV3.1, and CaV3.2. LVA current density decreases significantly beginning at, or shortly after, birth in normal animals; however, its density is maintained in SpDwf rats at 1 pA/pF for > or =12 wk after birth. The abundance of mRNAs encoding CaV2.3 and CaV3.2 declines with advancing age in normal atrial development, yet expression of CaV2.3 mRNA remains significantly elevated in older SpDwf animals. Quantitation of local transcript levels for mRNAs encoding IGF-I and IGF-I receptor (IGF-IR) also reveals significant differences in expression of these transcripts in atrial tissue of SpDwf animals compared with controls. In SpDwf rats, the abundance of IGF-IR mRNA remains elevated at many postnatal ages, whereas mRNA encoding IGF-I is maintained only in older animals. Physiological concentrations of IGF-I cause two- to threefold increases in LVA current density in primary cultures of atrial myocytes, and this effect is blocked by an antisense oligonucleotide targeting the IGF-IR. Thus disruption of GH production in SpDwf animals alters expression of atrial LVA Ca2+ channel and IGF genes as well as postnatal regulation of LVA Ca2+ current density, most likely acting through compensatory mechanisms via the local IGF-IR.

Molecular cloning of calcium channel alpha(2)delta-subunits from rat atria and the differential regulation of their expression by IGF-1.

Calcium channels are multimeric proteins consisting of pore-forming (alpha(1)) and auxiliary (alpha(2)delta, beta, gamma) subunits. The auxiliary alpha(2)delta-subunit regulates calcium current density and activation/inactivation kinetics when co-expressed with some, but not all, alpha(1)-subunits. Here we report the differential expression of three alpha(2)delta-subunit cDNAs in rat atria, atrial myocytes and ventricle, and demonstrate that IGF-1 selectively increases the expression of the alpha(2)delta-3 mRNA in the atria. mRNA encoding the alpha(2)delta-1- and alpha(2)delta-2-subunits, but not the alpha(2)delta-3-subunit, is detected in the rat ventricle whereas all three transcripts are found in atrial tissue. Analysis of the rat alpha(2)delta-1 cDNA sequence indicates that the atria express the alpha(2)delta-1e alternatively spliced isoform of this gene. The complete cDNA sequences of the alpha(2)delta-2- and alpha(2)delta-3-subunits from rat atria were determined and found to share 96% and 95% identity, respectively, with their counterparts in mouse. Treatment of acutely cultured atrial myocytes with IGF-1 caused a significant increase of the amount of alpha(2)delta-3, but not alpha(2)delta-1 or alpha(2)delta-2, mRNA. Both L-type and T-type calcium currents are recorded from cardiac tissue although their expression is regionally specific and changes with age and physiological state. Differential regulation of the expression of alpha(2)delta-subunit genes is likely to contribute to alterations in the expression of calcium current in the mammalian heart.

Quantitative analysis of the expression and distribution of calcium channel alpha 1 subunit mRNA in the atria and ventricles of the rat heart.

Two distinct calcium currents are present in mammalian cardiac myocytes. Utilizing quantitative RT-PCR methods, we have analysed the expression patterns and abundance of four calcium channel alpha 1 subunit mRNAs in different regions of the rat heart and compared them to the known density of calcium currents recorded from rat atria. Our results show that Ca(V)1.2 is the most abundant of the four alpha 1 subunit transcripts in the rat heart. The Ca(V)1.2 message is more abundant in ventricle than in atria and does not vary in expression as a function of developmental age. Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 mRNAs are 10-100 times less abundant than Ca(V)1.2. Interestingly, Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 are expressed in both atria and ventricle. The abundance of atrial Ca(V)3.1 mRNA does not change significantly during development and remains high in older animals. In contrast, levels of atrial Ca(V)3.2 mRNA are high in embryonic tissue and at 3- and 4-weeks postnatal but become undetectable at 5 weeks. Expression of atrial Ca(V)2.3 mRNA is highest at 4-weeks postnatal and then declines gradually. We have previously documented that the LVA calcium current density is highest within 4-5 weeks after birth and then declines gradually reaching less than 30% of its maximal value at 12-14 weeks. The complex relationship between atrial LVA current density and the abundance of Ca(V)2.3, Ca(V)3.1 and Ca(V)3.2 mRNA suggests that their contribution to the cardiac LVA current may vary as a function of postnatal age.

Distribution and relative expression levels of calcium channel beta subunits within the chambers of the rat heart.

Calcium channel beta subunits expressed in rat atria and atrial myocytes are identified and their expression quantified and compared to beta subunit expression in the ventricle. mRNAs encoding all four know beta subunits are expressed in atrial myocytes including the following splice variants: beta1a, beta2b, beta2c, beta2e and beta4d. The specific beta2 splice variants expressed in the atria (beta2b, beta2c, beta2e) differ from those previously reported from rat ventricle. Beta2 isoform is the most abundant beta mRNA expressed in the heart and the amount of both beta2 subunit mRNA and beta2 subunit protein is significantly greater in the ventricles than in the atria. The expression of individual beta2 splice variants varies with age and within different chambers of the heart. The beta2b splice variant appears in both atria and ventricle in both young (4.5 week) and old (16 week) animals, the beta2c isoform is more highly expressed in young as compared to old animals and the beta2e splice variant is robustly expressed only in 4.5 week ventricle. Beta4 mRNA expression is higher in atrial tissue than in ventricles and its expression decreases in older animals. In contrast, the abundance of the beta3 mRNA does not significantly change as a function of postnatal age. Antisense oligonucleotides targeting sequence common to all the beta isoforms as well as that specific for beta2 significantly reduced HVA calcium current density in isolated atrial cells confirming that the beta2 subunits contribute to the regulation of HVA calcium current expression in the rat atria. The complexity of beta isoform expression within the heart may provide a mechanism for functional fine-tuning of the cardiac HVA current.

Calcium channel gamma6 subunits are unique modulators of low voltage-activated (Cav3.1) calcium current.

The calcium channel gamma (gamma) subunit family consists of eight members whose functions include modulation of high voltage-activated (HVA) calcium currents in skeletal muscle and neurons, and regulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propanoic acid (AMPA) receptor targeting. Cardiac myocytes express at least three gamma subunits, gamma(4), gamma(6) and gamma(7), whose function(s) in the heart are unknown. Here we compare the effects of the previously uncharacterized gamma(6) subunit with that of gamma(4) and gamma(7) on a low voltage-activated calcium channel (Cav3.1) that is expressed in cardiac myocytes. Co-expression of both the long and short gamma(6) subunit isoforms, gamma(6L) and gamma(6S), with Cav3.1 in HEK-293 cells significantly decreases current density by 49% and 69%, respectively. Two other gamma subunits expressed in cardiac myocytes, gamma(4) and gamma(7), have no significant effect on Cav3.1 current. Neither gamma(6L), gamma(6S), gamma(4) nor gamma(7) significantly affect the voltage dependency of activation or inactivation or the kinetics of Cav3.1 current. Transient expression of gamma(6L) in an immortalized atrial cell line (HL-1) significantly reduces the endogenous low voltage-activated current in these cells by 63%. Green fluorescent protein tagged gamma(6L) is localized primarily in HEK-293 cell surface membranes where it is evenly distributed. Expression of gamma(6L) does not affect the level of Cav3.1 mRNA or the amount of total Cav3.1 protein in transfected HEK-293 cells. These results demonstrate that the gamma(6) subunit has a unique ability to inhibit Cav3.1 dependent calcium current that is not shared with the gamma(4) and gamma(7) isoforms and is thus a potential regulator of cardiac low voltage-activated calcium current.