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.
Highlights
The incidence of congestive heart failure in childhood ranges from 2.95 to 23.2 in each 1000 patients, of which just over 80% have congenital heart disease, followed by the population with cardiomyopathies (7%) and arrhythmias (2%) (Schmaltz, 2015)
We found that perinatal knockout, beginning at embryonic day 19, did produce mice exhibiting moderate heart failure accompanied by elongated cardiomyocytes, but in the absence of any hypertrophy
Conditional deletion of Mylk3 gene in Mylk3flox/flox/CAGGS-Cre-ERTM mice with tamoxifen injection at embryonic day 19, will be described as perinatal Mylk3-KO. This model produces a general knockout of the Mylk3 gene, the expression of cardiac myosin light chain kinase (cMLCK) is known to be restricted to the myocardium (Chan et al, 2008)
Summary
The incidence of congestive heart failure in childhood ranges from 2.95 to 23.2 in each 1000 patients, of which just over 80% have congenital heart disease, followed by the population with cardiomyopathies (7%) and arrhythmias (2%) (Schmaltz, 2015). Complex malformations can induce both volume- and pressure-overloaded heart failure (Hsu and Pearson, 2009a,b; Schmaltz, 2015). The brain natriuretic peptide (BNP)/N-terminal prohormone BNP is used as a biomarker heart failure in childhood, despite the knowledge that there is an age-dependent variation in normal individuals from ∼3000 pg/ml in 0–2 days of age, which drops to ∼100 pg/ml between 1 month and 1-year of age (Nir et al, 2009; Schmaltz, 2015). 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
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