SMYD1a protects the heart from ischemic injury by regulating OPA1-mediated cristae remodeling and supercomplex formation

Figures

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  Fig. 1. SMYD1 is downregulated in failing heart. A Transcript variants of SMYD1 in mouse and human hearts. Two splice variants of Smyd1 in mouse heart differ by 13 amino acids inserted in Smyd1a (orange bar), which is conserved in the human transcript. Location of primers specific to either Smyd1 isoform is indicated by underlined F (red and blue, below mouse Smyd1a and Smyd1b sequences, respectively) and R (purple). B–D Evaluation of SMYD1 expression in human hearts that underwent LVAD treatment by B qRT-PCR and C, D western blot (WB). Non-resp. indicates non-responders and Resp. indicates responders. Asterisk * indicates p < 0.05, n = 5/Control, n = 10/Failure (qRT-PCR), and n = 5/group (WB). E–G qRT-PCR (E) and western blot evaluation (F) with quantification (G) of SMYD1 levels in mouse hearts 5 weeks after permanent occlusion (PO) of the LAD or Sham surgery. Asterisk * indicates p < 0.05, n = 6–7

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  Fig. 2. Generation of inducible, cardiomyocyte-specific SMYD1a overexpression in mice. A Transgenic mice were generated by crossing animals expressing a reverse tetracycline-controlled transactivator (rtTA) under the αMHC promoter with mice carrying the Smyd1a-FLAG gene under the modified αMHC promoter containing a TRE element. These mice express Smyd1a-FLAG only upon doxycycline (D) administration (80 mg/kg/day). B Genotyping of mice carrying the Smyd1a-FLAG and rtTA alleles. Doxycycline (DOX) induced expression of mouse SMYD1 isoforms was confirmed by RT-qPCR in C cardiac and D skeletal muscle showing overexpression specific to the mouse heart. Asterisk * indicates p < 0.05, n = 3–4. Western blotting (E) and quantification (F) of SMYD1 in mouse cardiac tissue 3 weeks after doxycycline treatment. Asterisk * indicates p < 0.05, n = 3. Long-term doxycycline treatment maintains SMYD1 expression at ~ twofold increase in transgenic (TG) mice as compared to wild type (WT) mice, as shown by G representative western blotting (quantified in H), and I LC/MS–MS, n = 3–4

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  Fig. 3. SMYD1a overexpression protects the heart from ischemic injury. A Mice were fed doxycycline chow for 2 weeks, subjected to permanent occlusion (PO) of the LAD, monitored weekly by echocardiography and hearts were harvested at 5 weeks after PO surgery. Biological replicates for phenotyping studies are presented in the table. B Heart weight: body weight and C heart weight: tibia length ratios show no significant changes between experimental groups. D Cardiac function was measured by echocardiography and represented by D echocardiographs and measured E at the view of the middle point of the short or long axis from M-mode to determine F ejection fraction and G fractional shortening. Echo data show preserved cardiac function in transgenic (TG) mice when compared to littermate controls (WT) that underwent PO surgery. Asterisk * indicates p < 0.05. H Five serial sections of the hearts were acquired at 5 weeks from the base (I) to apex (V) and these representative images show that SMYD1a mitigates ischemic injury in TG mice compared to WT control which lack the rtTA transgene and are also maintained on DOX chow. Bar = 100 mm. I The ischemic injury from this surgical model was quantified…

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  Fig. 4. SMYD1a overexpression enhances mitochondrial respiration and lowers ROS production. A Model of the mitochondrial electron transport chain (ETC) and ATP synthesis. IMS intermembrane space, IM inner mitochondrial membrane, MM mitochondrial matrix. B Western blotting showing expression levels of SMYD1a in H9c2 cardiomyoblasts that were transduced with Ad-SMYD1a-FLAG. C–F Cell Mito Stress Test was conducted using a Seahorse Bioscience XFe96 analyzer by sequentially injecting 1 mM oligomycin, 5 mM FCCP, and 1 mM rotenone + antymycin A inhibitors. H9c2 cardiomyoblasts were transduced with Ad-SMYD1a and oxygen consumption rates (OCR) were recorded and quantified in the presence of C, D pyruvate or E, F palmitate as substrates. Quantitative analysis (D, F) of mitochondrial OCR from H9c2 cells overexpressing SMYD1a indicates significant increases in respiration in response to various inhibitors for both substrates. Asterisk * indicates p < 0.05, † indicates p = 0.08, n = 5. G Representative respiration traces in the Complex I/II-linked OXPHOS state detected by Oroboros O2k oxygraph and quantitative analysis (H) of mitochondrial OCR from TG and WT control groups showing increased oxygen…

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  Fig. 5. SMYD1a overexpression prevents a decline in metabolic enzymes downstream of PGC-1a. A Expression of PGC-1a was quantified via A qPCR and B, C western blotting in cardiac tissue from TG and WT mice 48 h after permanent occlusion of the LAD or Sham surgeries. These data show that PGC-1α expression is significantly decreased in response to ischemic stress (PO), however, overexpression of SMYD1a is capable of maintaining PGC-1α at basal levels. Asterisk * indicates p < 0.05, n = 3–6. D Publicly available ChIP-Seq data for histone H3K4me3 at the Ppargc1α promoter was used to design primers to use in ChIP-qPCR for SMYD1a-FLAG, H3K4me3 and H3K9me3 in cardiac tissue from TG and WT mice showing E SMYD1a binding at the Ppargc1α promoter which F increases trimethylation of histone H3K4 under basal conditions. G H3K9me3 ChIP was used as a negative control. Asterisk * indicates p < 0.05, n = 4–6. H Overexpression of SMYD1a rescues expression of genes involved in OXPHOS and fatty acid oxidation which are regulated by ERRα or PPARα transcription factors, respectively, and downregulated in response to ischemic injury. Asterisk * indicates p < 0.05, † indicates p < 0.08, n = 4–6. I Under basal…

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  Fig. 6. SMYD1a overexpression regulates mitochondrial respiration by increasing the formation of supercomplexes and cristae. A Diagram showing factors influencing mitochondrial respiration. B Western blotting evaluation of the electron transport chain (ETC) complex subunits using an antibody cocktail showing no significant changes in abundance of ETC complexes in TG mice or after PO, n = 3. C Immunoblotting for additional ETC subunits showed D modest changes in a few subunits (NDUFS2, NDUFV1), but no change in most of those examined, n = 3. E–F Mitochondrial biogenesis is absent in transgenic SMYD1a mice as confirmed by three markers: E mitochondrial DNA quantified by ND1 and 16S rRNA via qPCR, n = 6, and F citrate synthase activity in TG and WT mice which showed no change after 2 weeks of DOX-induced SMYD1a overexpression, n = 3–4. G–I SMYD1a overexpression increases supercomplex formation as determined by blue-native PAGE gel stained with G Coomassie, evaluated for H Complex I activity and I quantified in WT and TG mice 48 h after permanent occlusion of the LAD or Sham surgery. Asterisk * indicates p < 0.05, n = 3–4. J Electron micrographs of cardiac tissue from WT, SMYD1a transgenic…

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  Fig. 7. SMYD1a regulates OPA1 expression in the cardiomyocyte. A OPA1 oligomers bind the inner mitochondrial membrane (IMM) at cristae junctions and regulate cristae structure. Overexpression of OPA1 has been shown to alter cristae morphology leading to reduced cristae width, increased cristae number and enhanced respiration by stabilizing supercomplex formation (right). Conversely, loss of OPA1 leads to reduced respiration and ATP, loss of cristae structure and cell death (left). B–D Western blotting analysis showing changes in OPA1 expression which is B, C increased in SMYD1a TG mice and B, D decreased in Smyd1 knockout (KO) mice at the D protein and E transcript level. Asterisk *indicates p < 0.05, n = 2–3. F–I Cultured H9c2 cardiomyoblasts were transfected with F either scr-siRNA (control) or Smyd1-siRNA for 72 h or G transduced with either empty virus (Control) or SMYD1a-FLAG adenovirus (Ad-SMYD1a) for 72 h. Both H wester blotting and I qPCR analysis show that siRNA-mediated Smyd1 knockdown in H9c2 cells led to significant downregulation of OPA1 (F, H, I) whereas overexpression of SMYD1a increased OPA1 expression (G, H, I). Asterisk * indicates p < 0.05, n = 3. J Previous ChIP-Seq…

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  Fig. 8. Schematic of SMYD1a’s role in the heart. Top panel: under basal conditions SMYD1a maintains metabolic homeostasis by regulating the expression of Ppargc1a through methylation of histone H3K4 at the promoter and its downstream targets including electron transport chain (ETC) subunits. Bottom panel: overexpression of SMYD1a regulates OPA1 expression which mediates cristae remodeling and supercomplex formation of respiratory chain complexes. This leads to enhanced mitochondrial respiration and ATP production to protect from ischemic injury and pathological remodeling in the heart