Cre recombinase affects calcium dynamics already in young mice

The Cre/LoxP system is widely used in cardiovascular research to generate mouse models with tissue-specific inactivation of target genes. Studies have reported that expression of Cre recombinase under the αMHC promoter leads to age-dependent cardiotoxicity with ventricular hypertrophy, fibrosis and ventricular dysfunction at 6 months of age. This study explores the impact of Cre expression on intracellular Ca²⁺ dynamics in ventricular myocytes of αMHC-Cre mice as early as 3 months old.

Mice expressing Cre under the αMHC promoter (CRE) were compared to wild-type (WT) controls. Ventricular cardiomyocytes (VCMs) were isolated by the Langendorff method. Ca²⁺ transients and sarcomere shortening were simultaneously recorded from VCMs. Ventricular and atrial weights were assessed, VCM dimensions analyzed, and protein and mRNA levels of key proteins involved in Ca²⁺ dynamics measured by immunoblot analysis and quantitative real-time RT-PCR.

At 3 months, CRE mice showed no evidence of cardiac hypertrophy. Ventricular or atrial weights and VCM size were not different between CRE and WT mice. The same applied to protein levels of SERCA2a, NCX1, Cav1.2, PLN and its phosphorylated form PLN pThr17. Nevertheless Ca²⁺ dynamics were significantly altered in CRE mice. Under basal conditions resting and peak Ca²⁺ were reduced and Ca²⁺ transient decay was delayed up to 30% in VCMs from CRE vs. WT mice. These differences persisted upon stimulation with 1 µM isoproterenol, whereas Ca²⁺ transient amplitude increased in CRE VCMs. We confirmed a previously reported reduction in dystrophin, potentially explaining the changes in Ca²⁺ dynamics. Despite these changes sarcomere shortening parameters were not different between groups.

As early as 3 months of age, Cre expression in VCMs leads to changes in Ca²⁺ dynamics that do not yet affect sarcomere shortening and cannot be attributed to the regulation of key proteins involved in Ca²⁺ dynamics. Because changes in intracellular Ca²⁺ dynamics can affect gene expression through altered excitation-transcription coupling, researchers should be aware of these subtle changes that precede the prominent phenotype at 6 months of age. Therefore, it is essential to use Cre-positive mice as controls when analyzing knockout models generated by the Cre/LoxP system.