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

Abstract

Dysregulation of L-type Ca 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 Ca channels, respectively. Cardiac-specific knockdown of Ca-α1D via interfering RNA abolished cardiac contraction, suggesting Ca-α1D (i.e. A1D) represents the primary functioning Ca channel in Drosophila hearts. Moreover, we successfully isolated viable single cardiomyocytes and recorded Ca currents via patch clamping, a feat never before accomplished with the fly model. The profile of Ca currents recorded in individual cells when Ca 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 Ca-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 Ca channels, suggesting that the fly may serve as a robust and effective platform for studying cardiac channelopathies.

Straightjacket/α2δ3 deregulation is associated with cardiac conduction defects in myotonic dystrophy type 1

“…To link calcium transient to heart asynchrony, we first tested the propagation of calcium waves in normal and in asynchronously beating hearts. We applied the GCaMP3 fluorescent calcium sensor that allows detection of calcium waves in vivo (Limpitikul et al, 2018). As shown in Figure 4A, calcium waves perfectly shadow cardiac contractions in wild-type hearts, each calcium peak aligning with the beginning of contraction along the heart tube (here registered in segments A3 and A4).…”

As time flies by: Investigating cardiac aging in the short-lived Drosophila model

“…Old flies display altered intracellular cardiomyocyte calcium dynamics, with a prolonged transient decay, which promotes the extended periods of systole observed in senescent animals [23,44,59]. Recently, whole-cell patch clamp recordings of calcium currents across the membrane of isolated Drosophila cardiomyocytes confirmed the cells possess a conserved compendium of Land T-type calcium channels [60]. As in mammals, L-type (A1D in Drosophila) channels in flies serve as the main conduits for sarcolemmal calcium flux.…”

“…The root cause of these changes is not completely understood, but several lines of evidence indicate reduced myocardial β-adrenergic receptor density, their functional decline, and deficits in the β-adrenergic signaling cascade with advanced age [48,64,65]. Interestingly, Drosophila express components homologous to those of the vertebrate pathway, including adrenergic-like octopamine receptors (OctαRs, and OctβRs), adenylyl cyclase (rutabaga), phosphodiesterase (dunce), and both regulatory and catalytic subunits of protein kinase A (PKA), and their cardiac L-type calcium channels exhibit PKA-mediated current enhancement [60]. The expression of all aforementioned genes uniformly declines with age in Drosophila cardiomyocytes [15,24,66], suggesting old fly hearts may also exhibit blunted responses to adrenergic stimulation.…”

Straightjacket/α2δ3deregulation is associated with cardiac conduction defects in Myotonic Dystrophy type 1

“…To make a link between calcium transient and heart asynchrony, we first tested propagation of calcium waves in normal and in asynchronously beating hearts. We applied the GCaMP3 fluorescent calcium sensor that allows detection of calcium waves in vivo (Limpitikul et al, 2018). As shown in Figure 4A, calcium waves underlie perfectly cardiac contractions in wild type hearts so that each calcium peak aligns with the beginning of contraction along the heart tube (here registered in segments A3 and A4).…”

“…Ca V 1.2 mutations are known to lead to diverse heartbeat dysfunctions (Splawski et al, 2004), and Ca V 1.2 deregulation is associated with DM1 (Rau et al, 2011). Importantly, the Drosophila counterpart of Cav1.2 (Ca-1αD) is also ensuring cardiac contractions and calcium transients in the fly heart (Limpitikul et al, 2018) suggesting a conserved role of Cav1.2/Ca-1αD in heart beating. Here, by following fluorescent calcium sensor (GCaMP3), we found that calcium waves underlie synchronous cardiac contractions and are disrupted in asynchronously beating DM1 fly hearts, implying that aberrant calcium transient in cardiomyocytes is associated with heart asynchrony.…”