Conservation of cardiac L-type Ca2+ channels and their regulation in Drosophila: A novel genetically-pliable channelopathic model

Abstract

Dysregulation of L-type Ca²⁺ channels (LTCCs) underlies numerous cardiac pathologies. Understanding their modulation with high fidelity relies on investigating LTCCs in their native environment with intact interacting proteins. Such studies benefit from genetic manipulation of endogenous channels in cardiomyocytes, which often proves cumbersome in mammalian models. Drosophila melanogaster, however, offers a potentially efficient alternative as it possesses a relatively simple heart, is genetically pliable, and expresses well-conserved genes. Fluorescence in situ hybridization confirmed an abundance of Ca-α1D and Ca-α1T mRNA in fly myocardium, which encode subunits that specify hetero-oligomeric channels homologous to mammalian LTCCs and T-type Ca²⁺ channels, respectively. Cardiac-specific knockdown of Ca-α1D via interfering RNA abolished cardiac contraction, suggesting Ca-α1D (i.e. A1D) represents the primary functioning Ca²⁺ channel in Drosophila hearts. Moreover, we successfully isolated viable single cardiomyocytes and recorded Ca²⁺ currents via patch clamping, a feat never before accomplished with the fly model. The profile of Ca²⁺ currents recorded in individual cells when Ca²⁺ channels were hypomorphic, absent, or under selective LTCC blockage by nifedipine, additionally confirmed the predominance of A1D current across all activation voltages. T-type current, activated at more negative voltages, was also detected. Lastly, A1D channels displayed Ca²⁺-dependent inactivation, a critical negative feedback mechanism of LTCCs, and the current through them was augmented by forskolin, an activator of the protein kinase A pathway. In sum, the Drosophila heart possesses a conserved compendium of Ca²⁺ channels, suggesting that the fly may serve as a robust and effective platform for studying cardiac channelopathies.

Figure 1. Ca-α1D and Ca-α1T transcripts are abundant in Drosophila heart tubes

A) Confocal micrograph of a semi-intact wild-type w¹¹¹⁸ Drosophila heart tube extending along the dorsal side of the abdomen. The anterior conical chamber is outlined and displayed in (Figure 1B). Note the non-cardiac alary muscles (AM) and the retractors of tergite muscles (RT). Scale bar = 100 μm. B) Micrograph of the conical chamber after Ca-α1D (red) and GAPDH (white) mRNA molecules were labeled with FISH probes. A representative small region of a single cardiomyocyte used for mRNA quantitation is outlined in white. PC, pericardial cell. Scale bar = 25 μm. C) Examples of Ca_v α₁-subunit mRNA particle densities in cardiomyocyte areas of interest (e.g. white box in Figure 1B) and the quantitative determination of the number of subunit messages normalized to the number of GAPDH messages within the same regions of interest. There were significantly more Ca-α1D and Ca-α1T transcripts compared to cac transcripts. *** p < 0.001. (n = 11, 10, 11 animals for Ca-α1D, cac, and Ca-α1T).

Figure 2. Cardiac contraction and Ca²⁺ transients are suppressed by Ca-α1D knockdown

A) The Drosophila UAS-GAL4 bipartite expression system. Cardiac-specific TinCΔ4-Gal4 drives expression of UAS-transgenes including the simultaneous expression of a Ca²⁺ biosensor (UAS-GCaMP3) and UAS-RNAi. B) Exemplar M-mode tracings of heart tubes following Ca-α1D, cacophony, or Ca-α1T knockdown (Ca-α1D(−), cac(−), and Ca-α1T(−)). The progenies of w¹¹¹⁸ or the injection lines (KK) crossed with the driver line served as controls. Ca-α1D knockdown completely suppressed contraction. C) Quantitative measurements of cardiac physiological parameters. Population data confirmed substantially altered contraction following Ca-α1D knockdown. *** p < 0.0001 compared to w¹¹¹⁸; ## p < 0.01 compared to KK. There were no significant differences in cardiac variables between the controls. (n=25-31 animals) D) Representative Ca²⁺ transient recordings from individuals of the same population of heart tubes examined in Figure 2B–C. Ca-α1D silencing effectively abolished Ca²⁺ transients in the heart tubes. E) Measurements of Ca²⁺ transients confirmed a significant reduction in Ca²⁺ transient frequency and magnitude of the peak Ca²⁺ transient upon Ca-α1D RNAi expression. The time required to reach the peak Ca²⁺ transient magnitude (time to peak) and the time constant for the Ca²⁺ transient decay (tau decay) are not shown for Ca-α1D(−)#1 and Ca-α1D(−)#2 because of inaccurate measurements due to minimal Ca²⁺ activity in these hearts. Knockdown of cacophony or Ca-α1T produced no significant change in Ca²⁺ transient frequency, Ca²⁺ transient magnitude, time to peak, or tau decay. *** p < 0.001 compared to w¹¹¹⁸. (n=21-30 animals).

Figure 3. Morphological characterization of isolated Drosophila cardiomyocytes

A) GFP-ZASP52 cardiomyocytes after dissociation from detached heart tubes maintained their curved morphology. Note, the left-most cardiomyocyte originated from the conical chamber of the heart. The remaining cells most likely came from the middle one third of the cardiac tube and are representative of those commonly isolated. Scale bar = 20 μm. B) Sarcomere lengths along myofibrils within cardiomyocytes of semi-intact and detached whole hearts were not significantly different under low Ca²⁺ conditions or following exposure to DMSO or 100 μM blebbistatin. After cardiomyocyte dissociation, the average sarcomere length of isolated cells under all three conditions did not significantly differ; however, the sarcomeres were significantly shorter than those of semi-intact and detached whole hearts. *** p < 0.0001. (n=22-31 cells) C) Enlarged views of myofibrils demonstrating the effect of cardiomyocyte isolation on resting sarcomere length.

Figure 4. Voltage clamp recordings confirm A1D channels conduct the predominant transsarcolemmal Ca²⁺ current in Drosophila cardiomyocytes

A) Ca²⁺ current recording of a dissociated fly myocyte. The current in control cardiomyocytes was suppressed by a dihydropyridine, nifedipine, a hallmark of Ca_V1 channels. The average capacitance of the Drosophila cardiomyocyte membrane was 125±11 pF. B) Mean peak current density in control, hypomorphic cacophony (cac^s), and T-type (Ca-α1T) null cardiomyocytes across test potentials. At high voltages, comparable to the plateau phase of mammalian cardiac action potentials, peak currents of cac^s hypomorphic and T-type null cardiomyocytes were similar to that observed in control, indicating that A1D is the major high-voltage-activated Ca²⁺ channel isoform in the Drosophila heart. At low voltages, T-type null cardiomyocytes showed reduced current densities, suggesting a contribution of T-type channels at low activation voltages. * p < 0.5 compared to control. (n=4, 6, and 4 cells for control, cac^s, and T-type null). C) Population data of the Ca²⁺ current response to 10 μM nifedipine as compared to untreated control myocytes. (n=5 cells).

Figure 5. Ca²⁺-dependent inactivation is a conserved feature of Drosophila A1D

A) Illustration of the alpha subunit of the vertebrate LTCC with CDI interface regions NSCaTE (yellow), two EF hands (rose, green), and the IQ domain (blue). Sequence comparison of Oryctolagus’s CACNA1C and Drosophila’s A1D with CDI components highlighted in the same color as in the diagram on the left. The binding sites of Ca²⁺-free CaM (apoCaM) are highlighted in grey with key amino acid residues interacting with N-, C-, and both lobes of apoCaM bolded in blue, red, and black. B) Ca²⁺ current in fly cardiomyocytes decayed more rapidly than Ba²⁺ current in the same cell, demonstrating CDI (shaded rose) as Ba²⁺ cannot effectively bind calmodulin. C) Population data showing the fraction of current that remained after 300 ms of activation (r₃₀₀). The different degree of decay between Ca²⁺ and Ba²⁺ currents represents the extent of CDI (shaded rose). (n=7 cells).

Figure 6. Ca²⁺ current augmentation by protein kinase A is conserved in Drosophila cardiomyocytes

A) Depiction of the β-adrenergic pathway. Epinephrine binds to a G-protein coupled receptor and activates the enzyme adenyl cyclase receptor, which converts ATP to cyclic AMP (cAMP). This small signaling molecule activates protein kinase A (PKA), which phosphorylates LTCCs, augmenting their current size. Phosphodiesterase (PDE) deactivates cAMP. Forskolin bypasses this signaling cascade by directly activating adenyl cyclase. Drosophila homologs of the β-adrenergic pathway elements are in parentheses. Oct, octopamine. B) Ca²⁺ current in Drosophila cardiomyocytes increased in amplitude after application of 10 μM forskolin. C) Population data confirm augmentation of Drosophila A1D current by PKA. (n=4 cells).