Calcium Channel α1C Research Articles

Novel cell lines derived from adult human ventricular cardiomyocytes

Background. – We have established proliferating human cardiomyocyte cell lines derived from non-proliferating primary cultures of adult ventricular heart tissue, using a novel method that may be applicable to many post-mitotic primary cultures. Methods and results. – Primary cells from human ventricular tissue, were fused with SV40 transformed, uridine auxotroph human fibroblasts, devoid of mitochondrial DNA. This was followed by selection in uridine-free medium to eliminate unfused fibroblasts. The fused cells were subcloned and screened for cell type-specific markers. Four clones (AC1, AC10, AC12, AC16) that express markers characteristic of cardiomyocytes were studied. Clones were homogeneous morphologically, and expressed transcription factors (GATA4, MYCD, NFATc4), contractile proteins such as α- and β-myosin heavy chain, α-cardiac actin, troponin I, desmoplakin, α actinin, the muscle-specific intermediate filament protein, desmin, the cardiomyocyte-specific peptide hormones, BNP, the L-type calcium channel α1C subunit and gap junction proteins, connexin-43 and connexin-40. Furthermore, dye-coupling studies confirmed the presence of functional gap junctions. EM ultra structural analysis revealed the presence of myofibrils in the subsarcolemmal region, indicating a precontractile developmental stage. When grown in mitogen-depleted medium, the AC cells stopped proliferating and formed a multinucleated syncytium. When the SV40 oncogene was silenced using the RNAi technique, AC16 cells switched from a proliferating to a more differentiated quiescent state, with the formation of multinucleated syncyntium. Concurrently, the cells expressed BMP2, an important signaling molecule for induction of cardiac-specific markers, that was not expressed by the proliferating cells. The presence of the combination of transcription factors in addition to muscle-specific markers is a good indication for the presence of a cardiac transcription program in these cells. Conclusions. – Based on the expression of myogenic markers and a fully functional respiratory chain, the AC cells have retained the nuclear DNA and the mitochondrial DNA of the primary cardiomyocytes. They can be frozen and thawed repeatedly and can differentiate when grown in mitogen-free medium. These cell lines are potentially useful in vitro models to study developmental regulation of cardiomyocytes in normal and pathological states.

The role of zinc (Zn) in lead (Pb)-induced reproductive toxicity

Objective: We here seek initial testicular gene expression changes conferring sensitivity/resistance to Pb-induced decreases in spermatogenesis. In 96 occupationally unexposed men, 15/21 post-IVF pregnancy failures were attributed to elevated Pb in the male tract, but 9/24 men with elevated Pb were successful, possibly due to normal Zn levels. Here, seminal plasma Pb and Zn levels correlated inversely (Spearman correlation [SC], n=67, r = -0.4, P < 0.001). Pb and Zn had opposing effects on sperm count (SC, respectively, r = -0.3, P < 0.02 and r = 0.3, P < 0.01). In obstructive azoopsermia, testis biopsy (TB) Pb was negatively related to sperm count (SC, n=33, r = -0.5, P < 0.002), apparently related to the correlation between Pb and % apoptosis (SC, n=17, r = 0.4, P < 0.08 [trend]). Expression of L-type voltage-dependent calcium channel (LVDCC) α1C subunit mRNA deletions in exons 6–9 (regulating heavy metal transport; Fisher’s exact test, n=13, P < 0.01) apparently protected against Pb. Consistently, in rodents, Pb (a calcium competitor) accumulates within testicular cell nuclei; while Zn co-administration with Pb prevents Pb accumulation and Pb-induced gene expression changes. Design: Adult (100 day) Pb-resistant Sprague-Dawley (SD) and Pb-sensitive Wistar male rats were given deionized water containing 0.0%, 0.025%, 0.05%, 0.15% or 0.3% lead acetate (PbAc) for 10 days. Materials and Methods: [1] Zn and Pb levels by atomic absorption; [2] total gene expression by microarrays (Rat Genome U34 Set; Affymetrix) with data mining via GeneSpring (Silicon Genetics); [3] LVDCC splicing variations in exons 6–9 by RT-PCR and amplicon sequence analysis; [4] percentage testicular apoptosis by TUNEL. Results: Highest-level PbAc-treated animals had blood leads >40 μg/dL, with <1 μg/dL in controls. The increase in testicular Pb was higher in Wistar rats. Testicular Zn increased in SD, but was unchanged in Wistars. In SD testes: [1] transcription factor modulators: CaMK IV increased and calcineurin decreased, altering CRE-containing (Ca regulated) gene expression gene dose-dependently [2] growth factors, xenobiotic detoxification genes (e.g. metallothioneins [MT]), DNA repair, and protein synthesis were all down-regulated; [3] undeleted LDCC α1C mRNA decreased, aberrant splicing increased, and [4] caspases were up-regulated. Apoptosis was increased. Only ∼10% of gene expression changes occured in Wistar testes, including both increased (transcription factors, ion channels) and decreased expression (ribosomal proteins). All magnitudes were greater in Wistar testes. Wistar-unique were increased expression (heat shock factors, cell cycle control proteins) and unchanged expression (e.g., MT). Undeleted LDCC α1C mRNA increased and aberrant splicing decreased. Conclusion: Levels of Pb that altered testicular gene expression are well below that causing widespread organ failure. In SD rats, increased testicular Zn associated with induction of MT, sequestering Pb. Altered Zn levels in Wistar testes with MT induction failure contributes to Pb sensitivity. Expression of deleted LVDCC α1C, reducing Pb transport, also contributes to Pb resistance. The data emphasize the importance of metal interactions to normal reproductive function. Supported by: NIH Grant Nos. ES06100 and ES10496 to SB and ES03749 to RZS.

Role of the C terminus of the α1C(CaV1.2) Subunit in Membrane Targeting of Cardiac L-type Calcium Channels

We have previously demonstrated that formation of a complex between L-type calcium (Ca(2+)) channel alpha(1C) (Ca(V)1.2) and beta subunits was necessary to target the channels to the plasma membrane when expressed in tsA201 cells. In the present study, we identified a region in the C terminus of the alpha(1C) subunit that was required for membrane targeting. Using a series of C-terminal deletion mutants of the alpha(1C) subunit, a domain consisting of amino acid residues 1623-1666 ("targeting domain") in the C terminus of the alpha(1C) subunit has been identified to be important for correct targeting of L-type Ca(2+) channel complexes to the plasma membrane. Although cells expressing the wild-type alpha(1C) and beta(2a) subunits exhibited punctate clusters of channel complexes along the plasma membrane with little intracellular staining, co-expression of deletion mutants of the alpha(1C) subunit that lack the targeting domain with the beta(2a) subunit resulted in an intracellular localization of the channels. In addition, three other regions in the C terminus of the alpha(1C) subunit that were downstream of residues 1623-1666 were found to contribute to membrane targeting of the L-type channels. Deletion of these domains in the alpha(1C) subunit resulted in a reduction of plasma membrane-localized channels, and a concomitant increase in channels localized intracellularly. Taken together, these results have demonstrated that a targeting domain in the C terminus of the alpha(1C) subunit was required for proper plasma membrane localization of the L-type Ca(2+) channels.