Hydrophobic profiles of the tail anchors in SLMAP dictate subcellular targeting

<p>Abstract</p> <p>Background</p> <p>Tail anchored (TA) membrane proteins target subcellular structures via a C-terminal transmembrane domain and serve prominent roles in membrane fusion and vesicle transport. Sarcolemmal Membrane Associated Protein (SLMAP) possesses two alternatively spliced tail anchors (TA1 or TA2) but their specificity of subcellular targeting remains unknown.</p> <p>Results</p> <p>TA1 or TA2 can direct SLMAP to reticular structures including the endoplasmic reticulum (ER), whilst TA2 directs SLMAP additionally to the mitochondria. Despite the general structural similarity of SLMAP to other vesicle trafficking proteins, we found no evidence for its localization with the vesicle transport machinery or a role in vesicle transport. The predicted transmembrane region of TA2 is flanked on either side by a positively charged amino acid and is itself less hydrophobic than the transmembrane helix present in TA1. Substitution of the positively charged amino acids, in the regions flanking the transmembrane helix of TA2, with leucine did not alter its subcellular targeting. The targeting of SLMAP to the mitochondria was dependent on the hydrophobic nature of TA2 since targeting of SLMAP-TA2 was prevented by the substitution of leucine (L) for moderately hydrophobic amino acid residues within the transmembrane region. The SLMAP-TA2-4L mutant had a hydrophobic profile that was comparable to that of SLMAP-TA1 and had identical targeting properties to SLMAP-TA1.</p> <p>Conclusion</p> <p>Thus the overall hydrophobicity of the two alternatively spliced TAs in SLMAP determines its subcellular targeting and TA2 predominantly directs SLMAP to the mitochondira where it may serve roles in the function of this organelle.</p

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, &#945;MHC-cre, mouse model exhibited a nonlethal hypertrophic cardiomyopathy (HCM) with aging. However, using the same &#945;MHC-cre mice, we observed a cardiac pathology, resulting in complete lethality by 11 months of age. Echocardiography and histology revealed that the &#945;MHC-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 &#945;MHC-cre mice, since a lethal DCM was induced by the cre driver in an age-dependent manner in this commonly utilized model system

Specific Deletion of the FHA Domain Containing SLMAP3 Isoform in Postnatal Myocardium Has No Impact on Structure or Function

Sarcolemmal membrane-associated proteins (SLMAPs) belong to the superfamily of tail-anchored membrane proteins known to regulate diverse biological processes, including protein trafficking and signal transduction. Mutations in SLMAP have been linked to Brugada and defective sodium channel Nav1.5 shuttling. The SLMAP gene is alternatively spliced to generate numerous isoforms, broadly defined as SLMAP1 (~35 kDa), SLMAP2 (~45 kDa) and SLMAP3 (~80–95 kDa), which are highly expressed in the myocardium. The SLMAP3 isoform exhibits ubiquitous expression carrying an FHA domain and is believed to negatively regulate Hippo signaling to dictate cell growth/death and differentiation. Using the αMHC-MerCreMer-flox system to target the SLMAP gene, we specifically deleted the SLMAP3 isoform in postnatal mouse hearts without any changes in the expression of SLMAP1/SLMAP2 isoforms. The in vivo analysis of mice with SLMAP3 cardiac deficiency revealed no significant changes to heart structure or function in young or aged mice without or with isoproterenol-induced stress. SLMAP3-deficient hearts revealed no obvious differences in cardiac size, function or hypertrophic response. Further, the molecular analysis indicated that SLMAP3 loss had a minor impact on sodium channel (Nav1.5) expression without affecting cardiac electrophysiology in postnatal myocardium. Surprisingly, the loss of SLMAP3 did not impact Hippo signaling in postnatal myocardium. We conclude that the FHA domain-containing SLMAP3 isoform has no impact on Hippo signaling or sodium channels in postnatal myocardium, which is able to function and respond normally to stress in its absence. Whether SLMAP1/SMAP2 isoforms can compensate for the loss of SLMAP3 in the affairs of the postnatal heart remains to be determined

Specific Deletion of the FHA Domain Containing SLMAP3 Isoform in Postnatal Myocardium Has No Impact on Structure or Function

Sarcolemmal membrane-associated proteins (SLMAPs) belong to the superfamily of tail-anchored membrane proteins known to regulate diverse biological processes, including protein trafficking and signal transduction. Mutations in SLMAP have been linked to Brugada and defective sodium channel Nav1.5 shuttling. The SLMAP gene is alternatively spliced to generate numerous isoforms, broadly defined as SLMAP1 (~35 kDa), SLMAP2 (~45 kDa) and SLMAP3 (~80–95 kDa), which are highly expressed in the myocardium. The SLMAP3 isoform exhibits ubiquitous expression carrying an FHA domain and is believed to negatively regulate Hippo signaling to dictate cell growth/death and differentiation. Using the αMHC-MerCreMer-flox system to target the SLMAP gene, we specifically deleted the SLMAP3 isoform in postnatal mouse hearts without any changes in the expression of SLMAP1/SLMAP2 isoforms. The in vivo analysis of mice with SLMAP3 cardiac deficiency revealed no significant changes to heart structure or function in young or aged mice without or with isoproterenol-induced stress. SLMAP3-deficient hearts revealed no obvious differences in cardiac size, function or hypertrophic response. Further, the molecular analysis indicated that SLMAP3 loss had a minor impact on sodium channel (Nav1.5) expression without affecting cardiac electrophysiology in postnatal myocardium. Surprisingly, the loss of SLMAP3 did not impact Hippo signaling in postnatal myocardium. We conclude that the FHA domain-containing SLMAP3 isoform has no impact on Hippo signaling or sodium channels in postnatal myocardium, which is able to function and respond normally to stress in its absence. Whether SLMAP1/SMAP2 isoforms can compensate for the loss of SLMAP3 in the affairs of the postnatal heart remains to be determined

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

E2F6 Impairs Glycolysis and Activates BDH1 Expression Prior to Dilated Cardiomyopathy

<div><p>Rationale</p><p>The E2F pathway plays a critical role in cardiac growth and development, yet its role in cardiac metabolism remains to be defined. Metabolic changes play important roles in human heart failure and studies imply the ketogenic enzyme β-hydroxybutyrate dehydrogenase I (BDH1) is a potential biomarker.</p><p>Objective</p><p>To define the role of the E2F pathway in cardiac metabolism and dilated cardiomyopathy (DCM) with a focus on BDH1.</p><p>Methods and Results</p><p>We previously developed transgenic (Tg) mice expressing the transcriptional repressor, E2F6, to interfere with the E2F/Rb pathway in post-natal myocardium. These Tg mice present with an E2F6 dose dependent DCM and deregulated connexin-43 (CX-43) levels in myocardium. Using the Seahorse platform, a 22% decrease in glycolysis was noted in neonatal cardiomyocytes isolated from E2F6-Tg hearts. This was associated with a 39% reduction in the glucose transporter GLUT4 and 50% less activation of the regulator of glucose metabolism AKT2. The specific reduction of cyclin B1 (70%) in Tg myocardium implicates its importance in supporting glycolysis in the postnatal heart. No changes in cyclin D expression (known to regulate mitochondrial activity) were noted and lipid metabolism remained unchanged in neonatal cardiomyocytes from Tg hearts. However, E2F6 induced a 40-fold increase of the <i>Bdh1</i> transcript and 890% increase in its protein levels in hearts from Tg pups implying a potential impact on ketolysis. By contrast, BDH1 expression is not activated until adulthood in normal myocardium. Neonatal cardiomyocytes from Wt hearts incubated with the ketone β-hydroxybutyrate (β-OHB) showed a 100% increase in CX-43 protein levels, implying a role for ketone signaling in gap junction biology. Neonatal cardiomyocyte cultures from Tg hearts exhibited enhanced levels of BDH1 and CX-43 and were not responsive to β-OHB.</p><p>Conclusions</p><p>The data reveal a novel role for the E2F pathway in regulating glycolysis in the developing myocardium through a mechanism involving cyclin B1. We reveal BDH1 expression as an early biomarker of heart failure and its potential impact, through ketone signaling, on CX-43 levels in E2F6-induced DCM.</p></div

SLMAP3 isoform modulates cardiac gene expression and function.

The sarcolemmal membrane associated proteins (SLMAPs) belong to the super family of tail anchored membrane proteins which serve diverse roles in biology including cell growth, protein trafficking and ion channel regulation. Mutations in human SLMAP have been linked to Brugada syndrome with putative deficits in trafficking of the sodium channel (Nav1.5) to the cell membrane resulting in aberrant electrical activity and heart function. Three main SLMAP isoforms (SLMAP1 (35 kDa), SLMAP2 (45 kDa), and SLMAP3 (91 kDa)) are expressed in myocardium but their precise role remains to be defined. Here we generated transgenic (Tg) mice with cardiac-specific expression of the SLMAP3 isoform during postnatal development which present with a significant decrease (20%) in fractional shortening and (11%) in cardiac output at 5 weeks of age. There was a lack of any notable cardiac remodeling (hypertrophy, fibrosis or fetal gene activation) in Tg hearts but the electrocardiogram indicated a significant increase (14%) in the PR interval and a decrease (43%) in the R amplitude. Western blot analysis indicated a selective and significant decrease (55%) in protein levels of Nav1.5 while 45% drop in its transcript levels were detectable by qRT-PCR. Significant decreases in the protein and transcript levels of the calcium transport system of the sarcoplasmic reticulum (SERCA2a/PLN) were also evident in Tg hearts. These data reveal a novel role for SLMAP3 in the selective regulation of important ion transport proteins at the level of gene expression and suggest that it may be a unique target in cardiovascular function and disease

β-OHB Regulates CX-43 Protein Expression in Neonatal Cardiomyocytes.

<p>(<b>A)</b> Representative immunoblot of protein isolated from Wt and Tg myocardium at post-natal day 1 with anti-CX-43. (<b>B)</b> Densitometric quantification of CX-43 immunoblot. Expression is normalized against GAPDH. Results represent mean±SEM values (n = 4). (<b>C)</b> Representative immunoblot analyses of protein from Wt and Tg neonatal cardiomyocytes following incubation with or without β-OHB (β-hydroxybutyrate). (<b>D)</b> Densitometric quantification of CX-43 in treated and non-treated neonatal cardiomyocytes. Expression is normalized against GAPDH. Results represent the mean±SEM values (n = 3–5). A two-way ANOVA was used to measure statistical significance. (<b>E</b>) Representative immunoblot analysis of BDH1 in Wt and Tg neonatal cardiomyocytes. *<i>P<0</i>.<i>05</i>.</p