Dystrophin and calcium current are decreased in cardiomyocytes expressing Cre enzyme driven by αMHC but not TNT promoter

Abstract

The Cre/lox system is a potent technology to control gene expression in mouse tissues. However, cardiac-specific Cre recombinase expression alone can lead to cardiac alterations when no loxP sites are present, which is not well understood. Many loxP-like sites have been identified in the mouse genome that might be Cre sensitive. One of them is located in the Dmd gene encoding dystrophin, a protein important for the function and stabilization of voltage-gated calcium (Cav1.2) and sodium (Nav1.5) channels, respectively. Here, we investigate whether Cre affects dystrophin expression and function in hearts without loxP sites in the genome. In mice expressing Cre under the alpha-myosin heavy chain (MHC-Cre) or Troponin T (TNT-Cre) promoter, we investigated dystrophin expression, Nav1.5 expression, and Cav1.2 function. Compared to age-matched MHC-Cre− mice, dystrophin protein level was significantly decreased in hearts from MHC-Cre+ mice of more than 12-weeks-old. Quantitative RT-PCR revealed decreased mRNA levels of Dmd gene. Unexpectedly, calcium current (ICaL), but not Nav1.5 protein expression was altered in those mice. Surprisingly, in hearts from 12-week-old and older TNT-Cre+ mice, neither ICaL nor dystrophin and Nav1.5 protein content were altered compared to TNT-Cre−. Cre recombinase unpredictably affects cardiac phenotype, and Cre-expressing mouse models should be carefully investigated before experimental use.

circHIPK3 prevents cardiac senescence by acting as a scaffold to recruit ubiquitin ligase to degrade HuR

“…Moreover, one report showed that dystrophin and calcium current decreased in cardiomyocytes expressing Cre enzyme driven by αMHC but not TNT promoter (rat troponin T2 cardiac promoter) [26]. The differences in phenotype between αMHC-Cre and TNT-Cre mice may be due to genetic background and the "time window" of Cre activity.…”

“…The mouse genotype was identified by PCR (Figure S4B-E). Previous studies reported that Cre enzyme driven by αMHC might impact cardiac cell biology without crossing to a LoxP mouse [26]. To determine the potential effect of Cre enzyme, we compared the circHIPK3 expression in the heart and the cardiac function of Cre mice (αMHC MerCreMer/Wt mice with tamoxifen treatment) and CKO mice.…”

Myh6-driven Cre-recombinase activates the DNA damage response and the cell-cycle in the myocardium in the absence of loxP sites

“…Although these defects, which followed tamoxifen-induced activation of merCremer, were transient, this observation argues for caution when using constitutively active Cre recombinase models, because, as we previously observed ( Fisher et al, 2016 ), constitutively active Cre recombinase appears to be retained within myocyte nuclei. In this regard, it was recently reported that Myh6 -driven expression of constitutive (tamoxifen-independent) Cre recombinase in the hearts of wild-type mice disrupts the Dmd gene encoding dystrophin ( Gillet et al, 2019 ), which is interesting because the Dmd gene contains a degenerate loxP site ( Pugach et al, 2015 ). The study by Pugach et al (2015) is noteworthy because it describes degenerate loxP sites in 227 genes (including Dmd ) of the C57Bl/6 genome, among which 55 are expressed in cardiac muscle.…”

“…Although most defects caused by this transgene are transient, it was recently reported that mice expressing a constitutively active (i.e. tamoxifen-independent) Myh6-Cre transgene exhibit cardiac defects that are apparently permanent ( Pugach et al, 2015 ; Gillet et al, 2019 ). It remains of concern that, despite the potential for off-target effects of Cre recombinase to obfuscate experimental results when using Cre/loxP genetic models, many published reports continue to neglect to include necessary controls.…”