The BKCa (slo) channel regulates the cardiac function of Drosophila

Abstract

The large conductance, calcium, and voltage-active potassium channels (BK_Ca) were originally discovered in Drosophila melanogaster as slowpoke (slo). They are extensively characterized in fly models as ion channels for their roles in neurological and muscular function, as well as aging. BK_Ca is known to modulate cardiac rhythm and is localized to the mitochondria. Activation of mitochondrial BK_Ca causes cardioprotection from ischemia-reperfusion injury, possibly via modulating mitochondrial function in adult animal models. However, the role of BK_Ca in cardiac function is not well-characterized, partially due to its localization to the plasma membrane as well as intracellular membranes and the wide array of cells present in mammalian hearts. Here we demonstrate for the first time a direct role for BK_Ca in cardiac function and cardioprotection from IR injury using the Drosophila model system. We have also discovered that the BK_Ca channel plays a role in the functioning of aging hearts. Our study establishes the presence of BK_Ca in the fly heart and ascertains its role in aging heart function.

Keywords: BK channels; antioxidants; life span; mitochondria; potassium channel; reactive oxygen species.

FIGURE 1

Cardiac function of wild‐type and slo mutant flies. (a) Cardiac function of Drosophila was evaluated by optical coherence tomography (OCT). Continuous images were obtained for wild‐type and slo mutant flies at week 2, week 4, and week 6. Note irregular beating in week 6 in slo mutant flies. (b) Fractional shortening analysis of wild‐type and slo mutant flies at week 2, week 4, and week 6. (c) Analysis of heart rate obtained from OCT images for wild‐type and slo mutant flies at week 2, week 4, and week 6. (n = 25–30 flies in each group). p Values for the data reported were calculated using a two‐tailed t‐test.

FIGURE 2

Localization of BK_Ca in the Drosophila heart. (a–d) Cardiac tubes were isolated and labeled with anti‐BK_Ca (a) and anti‐ATP synthase (for mitochondria, b). (c) Merge images of (a) and (b, d) enlarged image from a boxed region in (c). (e–i). Cardiac tubes labeled with anti‐BK_Ca and wheat germ agglutinin (WGA, plasma membrane marker) isolated from wild‐type flies. (e) Cardiac tube labeled with WGA. (f) Cardiac tube labeled with anti‐BK_Ca. (g) Cardiac tube stained for nucleus with DAPI. (h) Merged image of (e, f) and (g, i) Enlarged region from a white box in (h) BK_Ca does not localize to the WGA labeled plasma membrane. (j–n) Cardiac tubes labeled with anti‐BK_Ca and WGA isolated from slo mutant flies. (j) Cardiac tube labeled with WGA. (k) Cardiac tube labeled with anti‐BK_Ca showed no significant labeling for BK_Ca. (l) Cardiac tube stained for nucleus with DAPI. (m) Merged image of (j, k) and (l, n) Enlarged region from a white box in m.

FIGURE 3

ROS production in the Drosophila heart. ROS production was estimated by staining freshly isolated cardiac tubes from wild‐type and slo mutant flies with the DHE dye and imaging them at the same settings. The slo mutant showed a higher amount of staining of ROS as compared to the wild‐type flies (n = 3); the fluorescence intensity was quantified using Image J (FIJI) software. Bar graphs represent the quantification of ROS production from the fly heart.

FIGURE 4

Slo mutants have abnormal cardiac fibers. Cardiac tubes from larvae were isolated and stained with phalloidin to label Actin filaments in wild‐type (a) and slo mutant (b). Stained Actin filaments were measured using Image J. (c) Fiber thickness was obtained, and a frequency histogram was plotted for wild‐type and slo mutant flies. The slo mutant flies showed increased fiber thickness. (d) Bar graphs indicate that slo mutant flies had thick Actin filaments as compared to the wild‐type flies (n = 5 flies).

FIGURE 5

Cardiac functional evaluation after hypoxia and reoxygenation. Adult wild‐type and slo mutant flies were subjected to hypoxia and reoxygenation. (a–d) Cardiac function of male wild‐type and slo mutant flies. Fractional shortening (a), heart rate (b), end‐diastolic diameter (c), and end‐systolic diameter (d) were calculated for male wild‐type (black) and slo mutant (gray) flies. (e–h) Cardiac function of female wild‐type and slo mutant flies. Fractional shortening (e), heart rate (f), end‐diastolic diameter (g), and end‐systolic diameter (h) were calculated for male wild‐type (black) and slo mutant (gray) flies. p Values for the data reported were calculated using two‐tailed t‐tests.

FIGURE 6

Slo in Drosophila cardiac physiology. (a) Schematic of role of Slo/ BK_Ca in Drosophila. Slo is known to play a direct role in cardioprotection and cardiac function. These cardiac outcomes are directly impacted by heart rate, circadian rhythm, and aging, where BK_Ca is known to play a significant role. The circulatory system in Drosophila consists of an open system with an aorta in the thorax and a cardiac tube along the dorsal aspect of the A1 abdominal segment. The cardiac tube function is controlled by transverse and bipolar neurons. (b) Schematic representation of impact of ablation of removal of slo on cardiac function upon IR stress. The up arrows indicate increased function, the down arrows indicate decreased function and the two‐sided arrows indicate no change as compared to the baseline function. Blue arrows are for the wild‐type and red arrows are for the slo mutant flies.