MCUB Regulates the Molecular Composition of the Mitochondrial Calcium Uniporter Channel to Limit Mitochondrial Calcium Overload During Stress.

We created a stable MCUB knockout cell line ( MCUB −/− ) utilizing CRISPR-Cas9n technology and generated a cardiac-specific, tamoxifen-inducible MCUB mutant mouse (CAG-CAT-MCUB x MCM; MCUB-Tg) for in vivo assessment of cardiac physiology and response to ischemia-reperfusion (IR) injury. Live cell imaging and high-resolution spectrofluorometery were employed to determine intracellular Ca 2+ exchange and size-exclusion chromatography, blue native page and immunoprecipitation studies were utilized to determine the molecular function and impact of MCUB on the high-molecular weight mtCU complex.

Main Methods

For detailed materials and methods please refer to the online-only Data Supplement . All data presented here are freely available upon any reasonable request. Animal Studies: All animal experiments were formally approved and in accordance with institutional guidelines established by the Temple University Institutional Animal Care and Use Committee, which follows the Association for Assessment and Accreditation of Laboratory Animal Care International guidelines. All mice were on the C57/BL6N background strain. Female and male mice were used for all studies unless otherwise noted in a specific experimental assay. For this study, approximately 20 C57BL6N wild-type, 150 αMHC-MCM and 150 MCUB-Tg x αMHC-MCM mice were utilized. Whenever possible experiments were performed in a blinded fashion with randomization. For example, a lab technician ear-tagged and coded mice to mask the genotype from the experimenter. Surgical procedures and anesthetic agents utilized for each procedure are detailed in the description of each experimental procedure. Generation of a stable MCUB −/− HeLa cell line We generated a stable MCUB knockout HeLa cell line using CRISPR/Cas9n technology to target exon 1 of MCUB for genomic deletion following the protocol developed by the Zhang lab 22 . Detailed methods along with plasmids, guide RNAs, and qPCR primers/antibodies utilized to confirm gene deletion and loss of protein are listed in the online-only Data Supplement .

Generation of a cardiac-specific MCUB gain-of-function transgenic model MCUB transgenic

(MCUB-Tg) mutant mice were generated using a flox-stop strategy where mouse Mcub cDNA (CCDS 17839.1) was cloned into a custom CAG-loxP-CAT-loxP plasmid. Detailed methods about molecular cloning, selection of a transgenic lines and the tamoxifen protocol for Cre-dependent expression can be found in the Data Supplement . MCUB-Tg mice were crossed to cardiomyocyte-restricted Cre expressing mouse models (αMHC-Cre or αMHC-MerCreMer) for all experiments. Size exclusion chromatographic analysis of the high molecular weight mtCU complex 2,500 μgrams of whole cell protein lysates or pure cardiac mitochondrial protein lysates prepared in RIPA lysis buffer were fractionated by gel filtration using fast protein liquid chromatography. All procedures were carried out as previously reported 20 and detailed methods can be found in the Data Supplement .

Blue Native Page Gel Electrophoresis

Purified cardiac mitochondria were incubated on ice for 20 minutes with Invitrogen 4x NativePAGE sample buffer with digitonin at a final concentration of 2%. Samples were then centrifuged at 18,000 x g for 30 minutes at 4 degrees Celsius. The supernatant was supplemented with G-250 sample additive at a final concentration of 0.25% and samples were then loaded for gel electrophoresis runs on an Invitrogen NativePAGE Novex Bis-Tris gel system. Gels were transferred for 18 hours at 4 degrees Celsius at 20 volts. Then immunoblotted with specified antibody. Echocardiography Transthoracic echocardiography of the left ventricle was performed and analyzed on a Vevo 2100 imaging system (VisualSonics) as previously reported 7 and detailed methodology can be found in the online-only Data Supplement .

Isolation of ACMs

ACMs were isolated from ventricular tissue as described previously 23 . All cells were used within 4 h of isolation. Evaluation of m Ca 2+ Uptake, Content, and m Ca 2+ retention capacity experiments All procedures were carried out as previously reported 7 . For detailed methods please refer to the online-only Data Supplement .

Mitochondria Isolation and Swelling Assay

Hearts were excised from mice and mitochondria were isolated as reported 24 . For the swelling assay, mitochondria were diluted in assay buffer, and absorbance (abs) was recorded at 540 nm every 5 s. 500 μM CaCl 2 was injected to induce swelling 25 . Adult mouse cardiomyocyte i Ca 2+ and m Ca 2+ transient recordings Intracellular calcium dynamics in isolated ACMs were examined as previously described 7 and detailed methodology can be found in the online-only Data Supplement . Mitoplast Patch-Clamp Analysis of MCU Current Following mitochondrial isolation, mitoplasts were prepared for patch-clamp studies. I MCU was recorded as previously described in detail 26 .

Oxygen Consumption Assays

Detailed methodology has been previously reported 7 and can be found in the online-only Data Supplement .

Invasive hemodynamic measurements

All details are previously described 5 and detailed methodology can be found in the online-only Data Supplement . Permanent ligation of the LCA and ischemia-reperfusion injury LCA ligation and reperfusion was performed as previously described in detail 27 . Detailed methodology can be found in the online-only Data Supplement . c Ca 2+ and m Ca 2+ flux in HeLa cells Cells were transduced with AAV6-mito-R-GECO1 for 72 hours and live-cell imaging with a Zeiss microscope was performed to measure m Ca 2+ exchange following histamine stimulation (100 μM). Cells were also loaded with the i Ca 2+ indicator, Fluo4-AM, and imaged to monitor c Ca 2 flux following histamine stimulation. Data were collected every 0.5 s and analyzed using Zen software. Co-Immunoprecipitations Plasmids encoding MCU, MCUB, MICU1, and MICU2 tagged with HA or FLAG were generated and transfected into COS7 cells for co-immunoprecipitations. Detailed methodology can be found in the online-only Data Supplement where methods about endogenous immunoprecipitations can also be found.

Statistical Analysis

All results are presented as mean ± SEM. Statistical analysis was performed using Prism 6.0 software (GraphPad). For comparisons between two groups, an unpaired, two-tailed t test was used. For groups of three or more a one-way ANOVA with Bonferroni correction was used. For grouped analyses, either multiple unpaired t-tests with correction for multiple comparisons using the Holm-Sidak method or two-way ANOVA with Tukey post-hoc analysis were performed. For all echocardiographic analysis two-way repeated-measures ANOVA was used with Sidak’s post hoc analysis. Log-rank test was used for comparison of Kaplan-Meier survival curves.