Atypical ALPK2 kinase is not essential for cardiac development and function

Abstract

Protein kinases play an integral role in cardiac development, function, and disease. Recent experimental and clinical data have implied that protein kinases belonging to a family of atypical α-protein kinases, including α-protein kinase 2 (ALPK2), are important for regulating cardiac development and maintaining function via regulation of WNT signaling. A recent study in zebrafish reported that loss of ALPK2 leads to severe cardiac defects; however, the relevance of ALPK2 has not been studied in a mammalian animal model. To assess the role of ALPK2 in the mammalian heart, we generated two independent global Alpk2-knockout (Alpk2-gKO) mouse lines, using CRISPR/Cas9 technology. We performed physiological and biochemical analyses of Alpk2-gKO mice to determine the functional, morphological, and molecular consequences of Alpk2 deletion at the organismal level. We found that Alpk2-gKO mice exhibited normal cardiac function and morphology up to one year of age. Moreover, we did not observe altered WNT signaling in neonatal Alpk2-gKO mouse hearts. In conclusion, Alpk2 is dispensable for cardiac development and function in the murine model. Our results suggest that Alpk2 is a rapidly evolving gene that lost its essential cardiac functions in mammals.NEW & NOTEWORTHY Several studies indicated the importance of ALPK2 for cardiac function and development. A recent study in zebrafish report that loss of ALPK2 leads to severe cardiac defects. In contrast, murine Alpk2-gKO models developed in this work display no overt cardiac phenotype. Our results suggest ALPK2, as a rapidly evolving gene, lost its essential cardiac functions in mammals.

Fig. 1.

Generation and characterization of α-protein kinase 2-global knockout (Alpk2-gKO) mouse lines. A: targeting strategy. B: sequencing result of flanking regions surrounding the crRNA targeting for generated mouse lines. C: outcome of generated deletions at the protein level. D: results of RT-PCR reaction over the targeted region confirms deletion and do not indicate alternative splicing events. E: quantitative RT-PCR analysis of other α-protein kinase transcript levels indicate no compensation at the transcript level in Alpk2-KO animals. Data are normalized to corresponding GAPDH levels, and KO groups are expressed as fold changes relative to wild-type (+/+) control. Four homozygous knockout (Δ/Δ) and 4 littermate wild-type (+/+) control animals for each line were used to assess levels of α-protein kinases. No statistically significant effect of either the Alpk2^Δ31 [F(1, 6) = 2.079, P = 0.199, Wilks' Λ = 0.743, partial η² = 0.257] or Alpk2^Δ17 [F(2, 5) = 1.001, P = 0.431, Wilks' Λ = 0.741 partial η² = 0.286] genotype was observed on Alpk1 or Alpk3 transcripts. PAM, protospacer adjacent motif; bp, base pair.

Fig. 2.

WNT signaling is not altered in P1 hearts of α-protein kinase 2-global knockout (Alpk2-gKO) mice. A–C: sections of P1 mouse hearts. Nuclei are in gray (DAPI), WNT positive nuclei are in green. Scale bar = 50 μm. No difference in WNT positive nuclei fraction is seen between Alpk2^+/+;WNT⁺(A), Alpk2 ^Δ17/Δ17;WNT⁺(B), and Alpk2 ^Δ31/Δ31;WNT⁺(C) animal hearts. D: quantification of WNT⁺ nuclei. Each point represents data from a single microscopy field. Homozygous Alpk2-KO (Δ/Δ) and wild-type (+/+) control animals heterozygous for the WNT reporter were used to study effect of Alpk2 on WNT signaling. Animal genotype had no effect on these numbers of WNT positive nuclei (Wald χ² (2) = 0.232, P = 0.89). E: quantitative RT-PCR analysis of the BAX-to-Bcl2 ratio (Bax/Bcl2), a marker of tissue apoptotic propensity. Four homozygous knockout (Δ/Δ) and 4 littermate wild-type (+/+) control animals for each studied line were used to assess Bax/Bcl2 ratio. Bax/Bcl2 transcript ratio was not different between Alpk2^Δ31 (P = 0.143)- or Alpk2^Δ17 (P = 0.859)-KO animals compared with littermate wild-type controls.

Fig. 3.

α-Protein kinase 2-global knockout (Alpk2-gKO) mice display normal cardiac function and morphology. A–E: echocardiographic analysis showing fractional shortening (A), left ventricular internal diameter during diastole (LVIDd; B), left ventricular internal diameter during systole (LVIDs; C), interventricular septum diameter at diastole (IVSd; D), and left ventricular posterior wall thickness during diastole (LVPWd; E). Animals (n = 4–12) of both sexes were measured per timepoint. Alpk2^Δ17 genotype alone [F(4, 75) = 1.129, P = 0.35, Wilks' Λ = 0.943, partial η² = 0.057] or over time [F(12, 198.723) = 0.462, P = 0.935, Wilks' Λ = 0.93, partial η² = 0.024], genotype of Alpk2^Δ31 had no effect either [F(8, 30) = 0.904, P = 0.526, Wilks' Λ = 0.649, partial η² = 0.194] on echocardiographic parameters. Heart weight-to-tibia length ratio (F) is not changed for both studied Alpk2-gKO mouse lines at 12 mo of age. Alpk2^Δ31 [F(2, 18) = 0.682, P = 0.518, partial η² = 0.07] and Alpk2^Δ17 [F(1, 15) = 0.444, P = 0.515, partial η² = 0.029]. For Alpk2^Δ17, homozygous knockouts (Δ/Δ) were compared with littermate wild-type animals (+/+). For Alpk2^Δ31, 3 genotypes were compared: homozygous knockouts (Δ/Δ), heterozyogus knockouts (Δ/+), and wild-type (+/+) animals.

Fig. 4.

Quantitative RT-PCR analysis of cardiac stress markers atrial natriuretic factor (Anf) and B-type natriuretic peptide (Bnp) and fibrotic markers collagen-α1 type I (Coll3a1), collagen-α1 type III in Alpk2^Δ17 mouse line at 2 mo of age, and α-protein kinase 2 (Alpk2)^Δ31 line animals 12 mo of age. Data are normalized to corresponding Gapdh levels, and knockout groups are expressed as fold changes relative to wild-type (+/+) control. No statistically significant effect of Alpk2^Δ31 [F(8, 28) = 0.154, P = 0.995, Wilks' Λ = 0.917, partial η² = 0.042] and Alpk2^Δ17 [F(12, 13.52) = 0.203, P = 0.996, Wilks' Λ = 0.645 partial η² = 0.136] genotype on levels of cardiac stress and fibrosis markers. Three genotypes were compared for each line: homozygous knockouts (Δ/Δ), heterozygous knockouts (Δ/+), and wild-type (+/+) animals.