Deletion of Sarcolemmal Membrane-Associated Protein Isoform 3 (SLMAP3) in Cardiac Progenitors Delays Embryonic Growth of Myocardium without Affecting Hippo Pathway.

The slmap gene is alternatively spliced to generate many isoforms that are abundant in developing myocardium. The largest protein isoform SLMAP3 is ubiquitously expressed and has been linked to cardiomyopathy, Brugada syndrome and Hippo signaling. To examine any role in cardiogenesis, mice homozygous for floxed slmap allele were crossed with Nkx2.5-cre mice to nullify its expression in cardiac progenitors. Targeted deletion of the slmap gene resulted in the specific knockout (KO) of the SLMAP3 (~91 KDa) isoform without any changes in the expression of the SLMAP2 (~43 kDa) or the SLMAP1 (~35 kDa) isoforms which continued to accumulate to similar levels as seen in Wt embryonic hearts. The loss of SLMAP3 from cardiac progenitors resulted in decreased size of the developing embryonic hearts evident at E9.5 to E16.5 with four small chambers and significantly thinner left ventricles. The proliferative capacity assessed with the phosphorylation of histone 3 or with Ki67 in E12.5 hearts was not significantly altered due to SLMAP3 deficiency. The size of embryonic cardiomyocytes, marked with anti-Troponin C, revealed significantly smaller cells, but their hypertrophic response (AKT1 and MTOR1) was not significantly affected by the specific loss of SLMAP3 protein. Further, no changes in phosphorylation of MST1/2 or YAP were detected in SLMAP3-KO embryonic myocardium, ruling out any impact on Hippo signaling. Rat embryonic cardiomyocytes express the three SLMAP isoforms and their knockdown (KD) with sh-RNA, resulted in decreased proliferation and enhanced senescence but without any impact on Hippo signaling. Collectively, these data show that SLMAP is critical for normal cardiac development with potential for the various isoforms to serve compensatory roles. Our data imply novel mechanisms for SLMAP action in cardiac growth independent of Hippo signaling.

Cardiac-Specific Cre Induces Age-Dependent Dilated Cardiomyopathy (DCM) in Mice.

The genetic modification of the mouse genome using the cre-lox system has been an invaluable tool in deciphering gene and protein function in a temporal and/or spatial manner. However, it has its pitfalls, as researchers have shown that the unregulated expression of cre recombinase can cause DNA damage, the consequences of which can be very detrimental to mouse health. Previously published literature on the most utilized cardiac-specific cre, αMHC-cre, mouse model exhibited a nonlethal hypertrophic cardiomyopathy (HCM) with aging. However, using the same αMHC-cre mice, we observed a cardiac pathology, resulting in complete lethality by 11 months of age. Echocardiography and histology revealed that the αMHC-cre mice were displaying symptoms of dilated cardiomyopathy (DCM) by seven months of age, which ultimately led to their demise in the absence of any HCM at any age. Molecular analysis showed that this phenotype was associated with the DNA damage response through the downregulation of activated p38 and increased expression of JNK, p53, and Bax, known inducers of myocyte death resulting in fibrosis. Our data urges strong caution when interpreting the phenotypic impact of gene responses using αMHC-cre mice, since a lethal DCM was induced by the cre driver in an age-dependent manner in this commonly utilized model system.