A Role for the Sarcolemmal Membrane Associated Protein Isoform 3 in Development

Abstract

Sarcolemmal membrane associated proteins (SLMAPs) belong to a superfamily of tail anchored membrane proteins. The SLMAP gene alternatively splices to generate numerous isoforms broadly defined as SLMAP1 (~35 kDa), SLMAP2 (~45 kDa) and SLMAP3 (~80-95kDa). Notably, SLMAP3, the largest isoform is ubiquitously expressed and developmentally regulated, and has been linked to muscle formation, heart failure, and sudden cardiac death. SLMAP3 possesses a Forkhead-Associated (FHA) domain and regulates Hippo signaling via the STRIPAK complex, influencing cellular growth, death, and organogenesis. Employing a cre-lox mediated knockout approach, we targeted the SLMAP3 isoform in prenatal and postnatal mouse tissues to unravel its functional significance. Utilizing αMHC-MerCreMer, αMHC-cre, Nkx2.5-cre, and CMV-cre mouse lines, we investigated SLMAP3's role in postnatal, perinatal, prenatal hearts, and in a global context.

Using αMHC-MerCreMer and αMHC-cre mice we knockout (KO) SLMAP3 in postnatal and perinatal mouse hearts. However, in-vivo analyses revealed no significant alterations in cardiac structure or function in aged SLMAP3-deficient mice under normal or stressed conditions. Furthermore, the absence of SLMAP3 did not affect Hippo signaling. Interestingly, during longevity studies we discovered that the αMHC-cre mice were developing a lethal dilated cardiomyopathy (DCM) by 7 months of age due to robust cre activity within the myocardium. Cre caused a DNA damage response through the downregulation of activated p38 and increased expression of JNK, p53, and Bax, known inducers of cardiomyocyte death resulting in fibrosis. Thus, our data urges caution when employing this model and to use appropriate cre only controls.

To examine role of SLMAP3 during cardiac development we nullified SLMAP3 using the Nkx2.5-cre, ablating SLMAP3 during early cardiogenesis and up to adulthood. Hearts from SLMAP3- KO mouse embryos were noticeably smaller, with all four chambers displaying thinner myocardium. This phenotype was not linked to Hippo signaling or cell proliferation as SLMAP3-KO Embryonic day (E)12.5 hearts showed no changes in Hippo signaling or changes in proliferative events using pH3 or Ki67 markers. Microscopy analysis indicated that SLMAP3-KO cardiomyocytes were significantly smaller than Wt. Mediators of the cardiomyocyte hypertrophic response AKT1 and MTOR1 were not significantly altered in KO hearts, thus cardiac size was regulated through alternate mechanisms. Remarkably, the SLMAP3-KO hearts recovered with normal physiology after the postnatal period. We noted a marked increase of SLMAP1 and SLMAP2 expression spanning from early cardiogenesis into adulthood equally in Wt and SLMAP3-KO hearts. The dynamic regulation of these isoforms may underscore their potential compensatory role in cardiac development in the absence of SLMAP3.

The global KO of SLMAP3 using CMV-cre was embryonic lethal, presenting with stunted growth and craniorachischisis. These phenotypes were not associated with dysfunctional Hippo signaling or cell proliferation. E8.5 and E9.5 SLMAP3-KO neural plates displayed reduced length and width signifying defective convergent extension. Planar cell polarity (PCP) components, disheveled-2 and -3, and the activity of PCP targets, phospho-ROCK2, phospho-cofilin and phospho-JNK1/2 were dysregulated in SLMAP3-KO E12.5 brain lysates. Cytoskeletal proteins, actin, Nestin, and apical marker PKC zeta, were not polarized to their specified apical or basal domains nor were adherens and tight junctions in SLMAP3-deficient neural tubes and neuroepithelial cells (NEPCs) implicating perturbed apical-basal polarity (ABP). Proteomic analysis of SLMAP3 revealed associations with centrosomal proteins (Pcm1), Golgi (Golgb1), transport proteins (Kif5c) and the polarity protein, Scribble. Loss of SLMAP3 resulted in abnormal centrosomal localization, fragmented Golgi apparatus and mislocalized Scribble in neural tubes and NEPCs. Our data define a critical role for SLMAP3 in neurulation through mechanism that impacts centrosomal and Golgi dynamics.

Overall, this data defines the role of SLMAP3 as a critical component that regulates embryonic development, particularly during neurulation and cardiogenesis without impacting Hippo signaling. This research advances our understanding of the role of SLMAP3 in normal development and function with potential involvement in various diseases states, including, cardiogenesis, neural tube defects, and stunted growth.