Heart Failure Induced by Perinatal Ablation of Cardiac Myosin Light Chain Kinase

Abstract

Background: Germline knockout mice are invaluable in understanding the function of the targeted genes. Sometimes, however, unexpected phenotypes are encountered, due in part to the activation of compensatory mechanisms. Germline ablation of cardiac myosin light chain kinase (cMLCK) causes mild cardiac dysfunction with cardiomyocyte hypertrophy, whereas ablation in adult hearts results in acute heart failure with cardiomyocyte atrophy. We hypothesized that compensation after ablation of cMLCK is dependent on developmental staging and perinatal-onset of cMLCK ablation will result in more evident heart failure than germline ablation, but less profound when compared to adult-onset ablation.Methods and Results: The floxed-Mylk3 gene was ablated at the beginning of the perinatal stage using a single intra-peritoneal tamoxifen injection of 50 mg/kg into pregnant mice on the 19th day of gestation, this being the final day of gestation. The level of cMLCK protein level could no longer be detected 3 days after the injection, with these mice hereafter denoted as the perinatal Mylk3-KO. At postnatal day 19, shortly before weaning age, these mice showed reduced cardiac contractility with a fractional shortening 22.8 ± 1.0% (n = 7) as opposed to 31.4 ± 1.0% (n = 11) in controls. The ratio of the heart weight relative to body weight was significantly increased at 6.68 ± 0.28 mg/g (n = 12) relative to the two control groups, 5.90 ± 0.16 (flox/flox, n = 11) and 5.81 ± 0.33 (wild/wild/Cre, n = 5), accompanied by reduced body weight. Furthermore, their cardiomyocytes were elongated without thickening, with a long-axis of 101.8 ± 2.4 μm (n = 320) as opposed to 87.1 ± 1.6 μm (n = 360) in the controls.Conclusion: Perinatal ablation of cMLCK produces an increase of heart weight/body weight ratio, a reduction of contractility, and an increase in the expression of fetal genes. The perinatal Mylk3-KO cardiomyocytes were elongated in the absence of thickening, differing from the compensatory hypertrophy shown in the germline knockout, and the cardomyocyte thinning shown in adult-inducible knockout.

Mylk3null C57BL/6N mice develop cardiomyopathy, whereasNntnull C57BL/6J mice do not

“…Compared with the Mus musculus reference genome (B6J), 43 variants amongst the B6N and B6J substrains were identified, 38 in the B6N and 5 in the B6J, potentially affecting 63 genes (Table S5). A variant in Mylk3, Chr8:85365179A>T, c-5T>A, SNP ID rs245783224, was considered a good candidate because it is almost exclusively expressed in the heart, with ablation resulting in cardiac dysfunction, which is modest in the germ line knockout (28,29,30). In humans, it is highly expressed in heart and~10-fold lower in skeletal muscle, with two recent articles showing an association of variants in MYLK3 with DCM (31,32).…”

Impact of cardiac myosin light chain kinase gene mutation on development of dilated cardiomyopathy

“…[11][12][13] Mice overexpressing myosin phosphatase exhibited MLC2v dephosphorylation and DCM phenotypes. [8][9][10] Ding et al [8][9][10] Ding et al…”

“…30 In fact, a conditional MYLK3 knockout mouse showed reduced MLC2v phosphorylation and DCM phenotype like LV dilation, wall thinning, and decreased fractional shortening. [8][9][10] Ding et al…”

“…Indeed, over 40 genes have been reported as a cause of DCM, resulting in the identification of diseasecausing mutations in 20% of DCM probands. [7][8][9][10] In humans, reduced MLC2v phosphorylation was observed in LV biopsy samples from end-stage heart failure including DCM. 4 Recently, cardiac myosin light chain kinase (cMLCK), which phosphorylates ventricular myosin regulatory light chain 2 (MLC2v) and regulates sarcomere and cardiomyocyte organization, 5,6 was cloned from the myocardia of patients with heart failure.…”

“…5 Interestingly, in animal models, knockout mice show reduced MLC2v phosphorylation and DCM-like phenotypes such as reduced left ventricular (LV) systolic function and LV dilation. [7][8][9][10] In humans, reduced MLC2v phosphorylation was observed in LV biopsy samples from end-stage heart failure including DCM. [11][12][13] However, few data exist regarding the information for the relationship between cMLCK mutations and MLC2v phosphorylation, despite the fact that cMLCK gene (MYLK3) mutations have been identified in DCM pedigrees.…”