An intrinsic mechanism of metabolic tuning promotes cardiac resilience to stress

Abstract

Defining the molecular mechanisms underlying cardiac resilience is crucial to find effective approaches to protect the heart. A physiologic level of ROS is produced in the heart by fatty acid oxidation, but stressful events can boost ROS and cause mitochondrial dysfunction and cardiac functional impairment. Melusin is a muscle specific chaperone required for myocardial compensatory remodeling during stress. Here we report that Melusin localizes in mitochondria where it binds the mitochondrial trifunctional protein, a key enzyme in fatty acid oxidation, and decreases it activity. Studying both mice and human induced pluripotent stem cell-derived cardiomyocytes, we found that Melusin reduces lipid oxidation in the myocardium and limits ROS generation in steady state and during pressure overload and doxorubicin treatment, preventing mitochondrial dysfunction. Accordingly, the treatment with the lipid oxidation inhibitor Trimetazidine concomitantly with stressful stimuli limits ROS accumulation and prevents long-term heart dysfunction. These findings disclose a physiologic mechanism of metabolic regulation in the heart and demonstrate that a timely restriction of lipid metabolism represents a potential therapeutic strategy to improve cardiac resilience to stress.

Keywords: Cardiac Metabolism; Chaperone Proteins; Doxorubicin; Pressure Overload; ROS.

Figure 1. Melusin regulates FAO by inhibiting the mitochondrial trifunctional protein.

(A) Modulation of α-MTP activity in wild-type and Mel null mice detected as NADH reduction in time in cardiac homogenates supplemented with acetoacetyl-CoA. (n = 9 per group; mean ± SD; p value by unpaired t-test; Data normalized on wt129/Sv media set to 1). (B) Palmitate oxidation capacity of cardiac isolated mitochondria from Mel null, Mel over and wild-type mice evaluated as generation of radioactive metabolites of [1−14C]-palmitate (n = 5 per group; mean ± SD; p value by unpaired t-test); (C) Trimetazidine treatment protocol: Mel null mice were treated orally from day 0 to day 4 with 30 mg/kg TMZ (TMZ+) or saline (TMZ−) as control. Wild-type mice were treated similarly with saline only. Mice were sacrificed at the end of the protocol to collect the hearts. (D) Palmitate oxidation capacity of cardiac isolated mitochondria from Mel null and wild-type mice treated as described in (C). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction). (E) The ratio of the quantity (µg/mg of tissue) of lipids in Mel null and Mel over mice compared to wild-type counterparts. Lipids have been quantified by lipidomic analysis from cardiac tissues. (n = 3 per group; mean ± SD; p value by unpaired t-test). (F) hiPSC cell models: wild-type hiPSCs were differentiated in wild-type hiPSC-CMs. hiPSCs knockout for Melusin were generated through CRISPR Cas9 technology and differentiated in Mel null hiPSC-CMs. hiPSCs were differentiated in hiPSC-CMs and infected with AAV6 and AAV9 carrying myc-Melusin, to over-express Melusin, or control AAV6 and AAV9. (G) Representative Immunofluorescence (IF) and PLA signal of Myc-Melusin (Myc-tag antibody) and α-MTP (HADHA antibody) in Mel null hiPSC-CMs infected with an AAV6-empty or carrying the construct for Myc-Melusin. DAPI was used to stain nuclei. Images were captured by confocal microscopy. Scale bare = 20/30 μm. Representative of n = 3 independent experiments (H) Palmitate oxidation capacity of isolated mitochondria from wild-type hiPSCs, wild-type hiPSC-CMs, and clone 1 and clone 2 Mel null hiPSC-CMs (left) and from AAV GFP hiPSC-CMs, AAV6 Mel hiPSC-CMs and AAV9 Mel hiPSC-CMs (right). (n = 3 per group; mean ± SD; p value by one-way ANOVA with Bonferroni correction). (I) Glucose flux through TCA cycle detected as marked CO₂ produced from 6[¹⁴C]-glucose via the tricarboxylic acid cycle in wild-type hiPSCs, wild-type hiPSC-CMs and clone 1 and clone 2 Mel null hiPSC-CMs (left) and in AAV GFP hiPSC-CMs, AAV6 Mel hiPSC-CMs and AAV9 Mel hiPSC-CMs (right). (n = 3 per group; mean ± SD; p value by one-way ANOVA with Bonferroni correction). A representative immunostaining for Melusin and vinculin levels in the cells used in (H) and (I) is reported below. (J) Representative curves (left) of the OCR modulation in wild-type (blue) and Mel null (red) hiPSC-CMs cultured in a media added with palmitate-BSA (azure arrow). Wild-type (yellow) and Mel null (purple) hiPSC-CMs cultured in the presence of the carnitine palmitoyltransferase-1 inhibitor etomoxir were used as negative controls. Maximal OCR increase (right) expressed as a percentage of Mel null hiPSC-CMs compared to the 100% of wt hiPSC-CMs. The maximal OCR increase is calculated as the OCR difference between t = 1 (empty arrow) and t = 0 (full arrow). (curves represent the media of 4–6 technical triplicates for each condition, biological n = 3 per group; mean ± SD; p value by one sample t-test). (K) AC16 cell model: AC16 cells were infected with a lentivirus carrying Myc-Melusin construct to over-express Melusin or an empty lentivirus as control. (L) Palmitate oxidation capacity of isolated mitochondria (left) and glucose flux through TCA cycle (right) in AC16 cells empty or expressing Melusin. A representative immunostaining for Melusin and vinculin level is reported below. (left: n = 3 mitochondrial preparations from independent experiments per group; right: n = 3 cell wells per group in independent experiments; mean ± SD; p value by unpaired t-test; the representative blot is common for left and right graph). (M) Representative curves (left) of the OCR modulation in empty (light green) and Mel (dark green) AC16 cells cultured in a media added with palmitate-BSA (azure arrow). Empty (violet) and Mel (yellow) AC16 cells cultured in the presence of etomoxir were used as negative controls. Maximal OCR increase (right) expressed as a percentage of empty AC16 cells compared to 100% of Mel AC16 cells. The maximal OCR increase is calculated as the OCR difference between t = 1 (empty arrow) and t = 0 (full arrow). (curves represent the media of 4–6 technical triplicates for each condition, biological n = 3 per group; mean ± SD; p value by one sample t-test). TMZ trimetazidine, FAs fatty acids, Cas9 CRISPR Cas9 technology, PLA proximity ligation assay, TCA tricarboxylic acid; OCR oxygen consumption rate, palm palmitate-BSA, etom etomoxir. Source data are available online for this figure.

Figure 2. Melusin regulates ROS generation and mitochondrial function in the myocardium.

(A) ROS content in Mel null, Mel over, and wild-type mice evaluated as DCFDA-AM fluorescence in isolated mitochondria from cardiac tissue. (n = 5 per group; mean ± SD; p value by unpaired t-test; Data normalized on wt129/Sv media set to 1). (B) ROS content in subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria isolated from Mel null, Mel over, and wild-type hearts. (n = 3 per group; mean ± SD; p value by unpaired t-test; Data normalized on wt129/Sv media set to 1). (C) ROS content in isolated mitochondria from cardiac tissue of wild-type and Mel null mice treated as described in 1C. (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction; Data normalized on wt129/Sv TMZ - media set to 1). (D) Modulation of palmitate oxidation estimated as radioactive metabolites of [1−¹⁴C]-palmitate generated in cardiac isolated mitochondria from wild-type and Mel over mice fed with high-fat diet (HFD +) or normal diet (HFD−) for 5 weeks. (n = 3 per HFD−; n = 4 per HFD +; mean ± SD; p value by two-way ANOVA with Bonferroni correction). (E) ROS content evaluated as DCFDA-AM fluorescence in cardiac isolated mitochondria from wild-type and Mel over mice feeded as in (D). (n = 3 per HFD −; n = 4 per HFD +; mean ± SD; p value by two-way ANOVA with Bonferroni correction; Data normalized on wt FVB HFD - media set to 1). (F) ROS content in mitochondria from wild-type hiPSCs, wild-type hiPSC-CMs, and clone 1 and clone 2 Mel null hiPSC-CMs. (n = 3 per group; mean ± SD; p value by one-way ANOVA with Bonferroni correction; Data normalized on wt hiPSC-CMs media set to 1). (G) ROS content in mitochondria from AAV GFP hiPSC-CMs, AAV6 Mel hiPSC-CMs, and AAV9 Mel hiPSC-CMs. (n = 3 mitochondrial preparations per group from hiPSC-CMs independently differentiated and infected; mean ± SD; p value by one-way ANOVA with Bonferroni correction; Data normalized on AAV GFP media set to 1). (H) ROS content in mitochondria from AC16 empty and mel. (n = 3 mitochondrial preparations from independent experiments; mean ± SD; p value by paired t-test; Data normalized on mel cells media set to 1). (I) Superoxide anion content was evaluated as MitoSOX fluorescence intensity per cell by confocal live imaging. Data expressed as a percentage of Mel null hiPSC-CMs compared to the 100% wt hiPSC-CMs. (technical n = 3 cell wells per condition, biological n = 4 independent experiments per group using independently differentiated hiPSC-CMs; mean ± SD; p value by one sample t-test). (J) Superoxide anion content expressed as a percentage of empty AC16 cells compared to the 100% of Mel AC16 cells. (technical n = 3 per condition, biological n = 4 per group; mean ± SD; p value by one sample t-test). (K) Modulation of the electron flux in mitochondria isolated from wild-type, Mel null and Mel over hearts, estimated as reduction rate of cytochrome C between complexes I and III. (n = 5 per group; mean ± SD; p value by unpaired t-test). (L), Level of ATP in mitochondria isolated from wild-type, Mel null and Mel over hearts, detected by luciferin-luciferase assay. (n = 5 per group; mean ± SD; p value by unpaired t-test). (M) Comparison of electron flux modulation and ATP levels between subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria isolated from Mel null, Mel over, and wild-type hearts. (n = 3 per group; mean ± SD; p value by unpaired t-test). ROS reactive oxygen species, DCFDA-AM 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein diacetate-acetoxymethyl ester, SS subsarcolemmal mitochondria, IMF intermyofibrillar mitochondria, TMZ trimetazidine, HFD high-fat diet, cit cytochrome C, EF electron flux. Source data are available online for this figure.

Figure 3. Melusin protects the heart from ROS production and mitochondrial dysfunction during stress.

(A) TAC treatment protocol: Mel null, Mel over, and wild-type mice were anesthetized and subjected to transverse aortic banding (TAC +) to induce pressure overload in the heart. Control mice were subjected to sham surgery but without aortic banding (TAC −). After 4 days, mice were sacrificed to collect the hearts. (B) Modulation of palmitate oxidation in cardiac isolated mitochondria from Mel null, Mel over, and wild-type mice treated as described in (A). (n = 5 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). (C) ROS content in cardiac isolated mitochondria from Mel null, Mel over, and wild-type mice treated as described in (A). (n = 5 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains); Data normalized on wt129/Sv TAC - media set to 1). (D) Modulation of the electron flux, estimated as reduction rate of cytochrome C between complexes I and III, in mitochondria isolated from hearts of wild-type, Mel null and Mel over mice treated as described in (A). (n = 5 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). (E) Level of ATP, detected by luciferin-luciferase assay, in mitochondria isolated from hearts of wild-type, Mel null and Mel over mice treated as described in (A). (n = 5 for each condition; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). (F) Doxorubicin single dose protocol in mice: Mel null, Mel over, and wild-type mice were injected intraperitoneally with a single dose of doxorubicin 4 mg/kg (DOX +) or saline (DOX −) as control. Mice were sacrificed 6 h later to collect the hearts. (G) Modulation of palmitate oxidation evaluated as radioactive metabolites of [1−14C]-palmitate generated in cardiac isolated mitochondria from Mel null, Mel over and wild-type mice treated as described in (F). (n = 5 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). (H) ROS content was measured as DCFDA-AM fluorescence in cardiac isolated mitochondria from Mel null, Mel over, and wild-type mice treated as described in (F). (n = 5 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains); Data normalized on wt129/Sv DOX - media = 1). (I) Modulation of the electron flux, estimated as reduction rate of cytochrome C between complexes I and III, in mitochondria isolated from hearts of wild-type, Mel null and Mel over mice treated as described in (F). (n = 5 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). (J) Level of ATP, detected by luciferin-luciferase assay, in mitochondria isolated from hearts of wild-type, Mel null and Mel over mice treated as described in (F). (n = 5 for each condition; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). DCFDA-AM 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein diacetate-acetoxymethyl ester, TAC transverse aortic constriction, cit cytochrome C, DOX doxorubicin; Source data are available online for this figure.

Figure 4. Melusin protects the heart from doxorubicin cardiotoxicity restraining ROS production and mitochondrial dysfunction, preserving the cardiac function, and sustaining the overall survival.

(A) Doxorubicin protocol in cells: AC16 cells empty e mel and hiPSC-CMs wild-type and null for Melusin were treated with 250 nM doxorubicin (DOX +) or normal media (DOX −) for 24 h before cell collection for analyses. (B) Modulation of palmitate oxidation (left) and ROS content (right) in mitochondria from AC16 cells empty and mel, treated as described in (A). (n = 3 mitochondrial preparations from independent experiments; mean ± SD; p value by two-way ANOVA with Tukey correction; ROS data normalized on AC16 mel DOX - media = 1). (C) Modulation of palmitate oxidation (left) and ROS content (right) in mitochondria from hiPSC-CMs wild-type and null for Melusin expression, treated as described in (A). (n = 3 mitochondrial preparations per group from hiPSC-CMs independently differentiated ; mean ± SD; p value by two-way ANOVA with Tukey correction; ROS data normalized on hiPSC-CMs wt DOX - media = 1). (D) Percentage of mitochondria with normal membrane potential, evaluated as red fluorescence of JC-1 probe by FACS analysis, in AC16 cells empty and expressing Melusin, treated as described in (A). A representative histogram is reported on the left, and the percentage of change graph is reported on the right. Empty AC16 cells are reported as a percentage of Mel AC16 settled as 100%. (technical n = 2/3 per condition, biological n = 3 per group; mean ± SD; p value by one sample t-test). (E) Percentage of mitochondria with normal membrane potential in hiPSC-CMs wild-type and null for Melusin, treated as described in (A). A representative histogram is reported on the left, and the percentage of change graph is reported on the right. Mel null hiPSC-CMs are reported as a percentage of wild-type hiPSC-CMs settled as 100%. (technical n = 2/3 per condition, biological n = 3 per group; mean ± SD; p value by one sample t-test). (F) Doxorubicin multiple dose protocol in mice: Mel over and wild-type mice were injected intraperitoneally with three doses of doxorubicin 4 mg/kg at t = 0, t = 1 week, and t = 2 weeks. Echocardiographic analyses were performed at 4 and 7 weeks. Mice were followed for 14 weeks for survival. (G) Percentage of fractional shortening detected by echo analysis at 4 and 7 weeks in Mel over and wild-type mice treated as described in (F). (nt0 = 12 Mel over, 12 wt FVB; nt4w = 9 Mel over, 9 wt FVB; nt7w = 9 Mel over, 9 wt FVB; mean ± SEM; p value by two-way ANOVA with Sidak correction). (H) Percentage of ejection fraction detected by echo analysis at 4 and 7 weeks in Mel over and wild-type mice treated as described in (F). (nt0 = 12 Mel over, 12 wt FVB; nt4w = 9 Mel over, 9 wt; nt7w = 9 Mel over, 9 wt FVB; mean ± SEM; p value by two-way ANOVA with Sidak correction). (I) Percentage of survival until 14 weeks in Mel over and wild-type mice treated as described in (F). (n = 13 Mel over, n = 15 wt FVB; analysis by Mantel–Cox test). DOX doxorubicin, DCFDA-AM 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein diacetate-acetoxymethyl ester, cit cytochrome C, %FS, percentage of fractional shortening, %EF percentage of ejection fraction. Source data are available online for this figure.

Figure 5. The level of FAO and ROS in steady state impacts on the cardiac response to stress.

(A) Trimetazidine and TAC treatment protocol: Mel null mice were treated orally for 5 days with 30 mg/kg TMZ (TMZ +) or saline (TMZ −) as control. At day 0 mice were subjected to TAC (TAC +) or sham (TAC −) surgery. After 4 days, mice were sacrificed to collect the hearts. (B) Modulation of palmitate oxidation estimated as radioactive metabolites of [1−14C]-palmitate generated in cardiac isolated mitochondria from Mel null mice treated as described in (A). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction). (C) ROS content evaluated as DCFDA-AM fluorescence in cardiac isolated mitochondria from Mel null mice treated as described in (A). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction; Data normalized on Mel null TAC-/TMZ- media = 1). (D) Trimetazidine and doxorubicin treatment protocol: Mel null mice were treated orally for 5 days with 30 mg/kg TMZ (TMZ +) or saline (TMZ −) as control. At day 0 mice were injected intraperitoneally with a single dose of doxorubicin 4 mg/kg (DOX +) or saline (DOX −) as control. Mice were sacrificed 6 h later to collect the hearts. (E) Modulation of palmitate oxidation in cardiac isolated mitochondria from Mel null mice treated as described in (D). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction). (F) ROS content in cardiac isolated mitochondria from Mel null mice treated as described in (D). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction; Data normalized on Mel null DOX-/TMZ- media = 1). (G) MitoQ and TAC treatment protocol: at day 0 Mel null mice were subjected to TAC (TAC +) or sham (TAC −) surgery. Starting from day 0, mice were treated daily with an oral administration of 2 µmol MitoQ (MitoQ +) or the same volume of saline (MitoQ −) as control. Mice were finally sacrificed on day 4 to collect the hearts. (H) ROS content in cardiac isolated mitochondria from Mel null mice treated as described in (G). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction; Data normalized on Mel null TAC-/MitoQ- media = 1). (I) Modulation of palmitate oxidation in cardiac isolated mitochondria from Mel null mice treated as described in (G). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction). (J) MitoQ and doxorubicin treatment protocol: Mel null mice were administered orally with 2 µmol MitoQ (MitoQ +), or saline as control (MitoQ −), 5 min before being injected with a single dose of doxorubicin 4 mg/kg (DOX +) or saline (DOX −) as control. Mice were sacrificed 6 h later to collect the hearts. (K) ROS content in cardiac isolated mitochondria from Mel null mice treated as described in (J). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction; Data normalized on Mel null DOX-/MitoQ- media = 1). (L) Modulation of palmitate oxidation in cardiac isolated mitochondria from Mel null mice treated as described in (J). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Bonferroni correction). TMZ trimetazidine, TAC transverse aortic constriction, DCFDA-AM 5-(and-6)-chloromethyl-2’,7’-dichlorodihydro-fluorescein diacetate-acetoxymethyl ester, DOX doxorubicin, MitoQ mitoquinone mesylate. Source data are available online for this figure.

Figure 6. Tuning FAO before or concomitantly to cardiac challenge sustains myocardial contractility.

(A) Trimetazidine and long TAC protocol: wild-type mice were subjected to TAC surgery and treated with 0.7 g/L TMZ dissolved in drinking water, or normal water as control. Mice were monitored by echo analyses every 3 weeks, from week 13th until week 28th. (B) Percentage of fractional shortening detected by echo analysis in wild-type mice treated as described in (A). (n = 3 ctr, n = 4 TMZ; mean ± SEM; p value by two-way ANOVA with Sidak correction). (C) Percentage of ejection fraction detected by echo analysis in wild-type mice treated as described in (A). (n = 3 ctr, n = 4 TMZ; mean ± SEM; p value by two-way ANOVA with Sidak correction). (D) Trimetazidine and doxorubicin multiple dose protocol: wild-type mice were administered orally for 5 days with 30 mg/kg TMZ, or saline as control, before each treatment with doxorubicin. Mice received three doses of 4 mg/kg doxorubicin at t = 0, t = 1 week, and t = 2 weeks. Mice were monitored by echo analyses weekly, from week 2 to week 7. (E) Percentage of fractional shortening detected by echo analysis in wild-type mice treated as described in (D). (n = 5 ctr, n = 5 TMZ; mean ± SEM; p value by one-way ANOVA with Sidak correction). (F) Percentage of ejection fraction detected by echo analysis in wild-type mice treated as described in (D). (n = 5 ctr, n = 5 TMZ; mean ± SEM; p value by one-way ANOVA with Bonferroni correction). TMZ trimetazidine, TAC transverse aortic constriction, DOX doxorubicin, %FS percentage of fractional shortening, %EF percentage of ejection fraction. Source data are available online for this figure.

Figure 7. Working model for Melusin promotion of cardiac resilience in mice.

Top: in Mel wt mice, Melusin binds and inhibits the MTP, restraining the FAO rate. This tunes the electron flux through the respiratory chain, allows an adequate ATP synthesis, and limits the generation of ROS. In Mel null mice, the absence of Melusin causes an excessive FAO rate that, in turn, enhances the electron flux through the respiratory complexes, generates more ATP but increases the risk of electron leakage and ROS generation. In Mel over mice, more Melusin binds and inhibits the MTP, reducing the FAO rate. This limits the electron flux and the ATP production, still at adequate levels for cardiac function, and significantly decreases ROS level. Bottom: stress conditions, as doxorubicin treatment or aortic constriction, generate ROS per se that, added to the basal level induced by FAO, boost the generation of ROS from multiple sites. In Mel null mice, showing a high basal level of ROS, stressful stimuli activate the RIRR process and the generation of ROS from mitochondrial and cytosolic sources. This causes mitochondrial dysfunction with a consequent severe drop in FAO together with the mitochondrial electron flux and the ATP synthesis. Conversely, stress conditions fail in boosting the generation of ROS in Mel over mice, since their very low basal level of ROS. This protects mitochondria from damage and preserves their FAO level, electron flux, and energetic production. MTP mitochondrial trifunctional protein, OM outer membrane, IM inner membrane, FAO fatty acid oxidation, FAs fatty acids, TAC transverse aortic constriction, DOX doxorubicin.

Figure EV1. Melusin binds the mitochondrial trifunctional protein.

(A) Coomassie Blue staining of Melusin immunoprecipitation (IP) from cardiac total extracts of Mel over mice and Mel null mice as controls. The indicated bands were cut out and identified by mass spectrometry. (B) Melusin immunoprecipitation from cardiac total extracts of wild-type mice and Mel null mice, as controls. The immunoprecipitated Melusin and the co-immunoprecipitated α-MTP and β-MTP were detected by immunostaining. Vinculin was stained to verify the complete removal of the total extract. Representative of n = 3 independent experiments. (C) Mitochondrial (Mito) and cytosolic (Cyto) fractions are differentially isolated from wild-type hearts. Melusin presence was evaluated by immunostaining. Vinculin and actin were stained as cytosolic markers, α-MTP and Vdac1 as mitochondrial markers. Representative of n = 4 independent experiments. (D) Mitochondria isolated from wild-type hearts treated with Proteinase K (PK), osmotic shock (OS), and Triton X-100 (TX100), or a combination of them, in order to digest proteins of different mitochondrial compartments. Melusin presence was evaluated by immunostaining. Pdh and α-MTP were stained as markers of the matrix compartment, Vdac1 as markers of the outer mitochondrial membrane. Representative of n = 3 independent experiments. (E) Fractions of cytosol (Cyto), subsarcolemmal (SS), and intermyofibrillar (IMF) mitochondria differentially isolated from wild-type hearts. Melusin presence was evaluated by immunostaining. α-MTP and Vdac1 were stained as mitochondrial markers. Representative of n = 3 independent experiments. (F) Complexes of different molecular weights were obtained as gel filtration fractions from the cardiac total extract of wild-type, Mel null and Mel over mice. Fractions were immunostained for α-MTP, β-MTP, and Melusin. Red arrows indicate the corresponding molecular weights. Representative of n = 3 independent experiments. IP immunoprecipitation, PK proteinase K, OS osmotic shock, Vinc vinculin, Vdac1 voltage-dependent anion-selective channel 1, Pdh pyruvate dehydrogenase, SS subsarcolemmal mitochondria, IMF intermyofibrillar mitochondria.

Figure EV2. Characterization of MTP in mice with different expressions of Melusin and of Melusin-null hiPSCs.

(A) Modulation of α-MTP activity in wild-type and Mel over mice detected as NADH reduction in time in cardiac homogenates supplemented with acetoacetyl-CoA. (n = 3 per group; mean ± SD; p value by unpaired t-test; Data normalized on wt129/Sv media = 1). (B) Expression of α-MTP and β-MTP mRNA evaluated by real-time qPCR in hearts from Mel null, Mel over, and wild-type mice. (n = 3 per group; mean ± SD; p value by unpaired t-test). (C) Protein level of α-MTP, β-MTP, Vdac1, and Tom20 in cardiac extracts from Mel null, Mel over, and wild-type mice. Melusin was stained as a marker of the different genotypes. Vinculin was stained as a loading control. (n = 3 per group). (D) Malonyl-CoA level detected by ELISA assay in Mel null, Mel over, and wild-type hearts. (n = 3 per group; mean ± SD; p value by unpaired t-test). (E) Expression of Glut1 and Glut4 mRNA evaluated by real-time qPCR in hearts from Mel null, Mel over, and wild-type mice. (n = 3 per group; mean ± SD; p value by unpaired t-test). (F) Protein level of pyruvate kinase and phosphorylation ratio of Pdh in cardiac extracts from Mel null, Mel over, and wild-type mice. (n = 6 per group; mean ± SD; p value by unpaired t-test). (G) NADH/NAD+ ratio estimated by HPLC analysis in cardiac extracts from Mel null, Mel over, and wild-type mice. (n = 4 per group; mean ± SD; p value by unpaired t-test). (H) RNA-seq and ATAC-seq data of WT hESCs and hESC-CMs. NONO expression is constant (RNA-seq data) and the promoter region is accessible in both cell types (ATAC-seq data). ITGB1BP2 promoter is accessible for transcription only in hiPSC-CMs. The sgRNA guides for Melusin knocking out were designed to bind the promoter region up- and down-stream the ATAC-seq peak. Primers for genotyping were designed against outside of the predicted cut sites. (I) Genotyping of Mel null hiPSC clones. The 853 bp band is the product of WT locus amplification, and the lower band (416 bp) is the result of promoter deletion. WT hiPSCs were used as control. All genome-edited clones show either the WT or promoter deletion band since Melusin is X-linked and the experiment involved male hiPSCs. (J) Karyotypes of Mel null cl.1 and cl.2 hiPSCs. Metaphase chromosomes were stained by G-banding. The karyotypes of Mel null hiPSCs are normal without clonal abnormalities and belong to a male individual. (K) RT-qPCR of WT, Mel null cl.1 and cl.2 hiPSC-CMs. ITGB1BP2 mRNA was normalized on the muscle-specific marker TNNT2, so as to better account for variations in hiPSC-CM purity in samples analyzed prior to lactate selection. Two RefSeq isoforms for the ITGB1BP2 gene were probed: the full-length canonical Melusin isoform (NM_012278.4) and a shorter isoform proved to be expressed at ~2% of Melusin (NM_001303277.3). The short isoform is predicted to share the same promoter as Melusin. Both isoforms were undetectable in both Mel null clones, validating the knockout strategy. To rule out a negative effect of the ITGB1BP2 promoter deletion on the housekeeping NONO gene, located very close upstream of ITGB1BP2, NONO mRNA was analyzed and normalized on the housekeeping gene HPRT. (n = 3 total RNA isolation per group from hiPSC-CMs independently differentiated; mean ± SD; p value by one-way ANOVA with Bonferroni correction). (L), cTnT + cells by flow cytometry of WT, Mel null cl.1 and cl.2 hiPSC-CMs post lactate selection (histograms are gated based on isotype control stains), and relative graph. (n = 3 per group; each experiment is performed by using hiPSC-CMs independently differentiated; mean ± SD; p value by one-way ANOVA with Bonferroni correction). (M) Representative Immunofluorescence (IF) and PLA signal of Myc-Melusin (Myc-tag antibody) and α-MTP (HADHA antibody) in AC16 cells empty (empty) or expressing Myc-Melusin (Mel). DAPI was used to stain nuclei. Images were captured by confocal microscopy. Scale bare = 20 μm. Representative of n = 3 independent experiments. Vinc vinculin, Vdac1 voltage-dependent anion-selective channel 1, Tom20 translocase of outer membrane 20, Pyr Kin pyruvate kinase, P-Pdh phosphorylated pyruvate dehydrogenase, Pdh pyruvate dehydrogenase, sgRNAs single guide RNAs, ITGB1BP2 Integrin β1 binding protein 2, bp base pair, TNNT2 troponin type2, cTNT cardiac troponin T, HPRT hypoxanthine phosphoribosyltransferase 1, PLA proximity ligation assay.

Figure EV3. Determination of parameters impacting mitochondrial function and ROS generation and evaluation of high-fat diet effect.

(A) Modulation of lipid peroxides detected as colorimetric reaction with malonaldehyde and 4-hydroxyalkenal in cardiac extracts of wild-type, Mel null, and Mel over mice. (n = 3 per group. mean ± SD; p value by unpaired t-test). (B) NADPH/NADP+ ratio estimated by HLPC analysis in cardiac extracts from Mel null, Mel over, and wild-type mice. (n = 4 per group; mean ± SD; p value by unpaired t-test). (C) Protein level of Nox4 in cardiac extracts from Mel null, Mel over, and wild-type mice in basal conditions (graphs of relative quantification (left) and immunostaining (right)). Vinculin was stained as a loading control. (n = 3 per group; mean ± SD; p value by unpaired t-test). (D) Heat map (left) and representative immunostaining (right) of the antioxidant enzymes catalase (Cat), glutathione peroxidase 1 (Gpx1), glutathione peroxidase 4 (Gpx4), peroxiredoxin 1 (Prdx 1), peroxiredoxin 3 (Prdx 3), superoxide dismutase 1 (Sod 1), superoxide dismutase 2 (Sod 2) in cardiac extracts from Mel null, Mel over, and wild-type mice. Vinculin was stained as a loading control. (n = 6 per group; mean ± SD; p value by unpaired t-test). (E) Left: Immunostaining for the five mitochondrial complexes in cardiac extracts from Mel null, Mel over, and wild-type mice. Vdac1 was stained as a marker of mitochondrial quantity. Vinculin was stained as a loading control. Right: relative quantifications. (n = 3 per group. mean ± SD; p value by unpaired t-test). (F) Level of ATP in total cardiac extracts of wild-type, Mel null, and Mel over mice, detected by luciferin-luciferase assay. (n = 3 per group; mean ± SD; p value by unpaired t-test). (G) Isoproterenol treatment protocol: hearts from Mel null, Mel over, and wild-type mice were excised and perfused ex-vivo with 1 μM isoproterenol (ISO +) or saline (ISO −), as control. (H) Modulation of palmitate oxidation, evaluated as generation of radioactive metabolites of [1−¹⁴C]-palmitate, in cardiac isolated mitochondria from Mel null, Mel over, and wild-type mice treated as described in (F). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). (I) ROS content, measured as DCFDA-AM fluorescence, in cardiac isolated mitochondria from Mel null, Mel over, and wild-type mice treated as described in (A). Data normalized on wt129/Sv (wt129/Sv media = 1). (n = 3 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). Cat catalase, Gpx1 glutathione peroxidase 1, Gpx4 glutathione peroxidase 4, Prdx 1 peroxiredoxin 1, Prdx 3 peroxiredoxin 3, Sod 1 superoxide dismutase 1, Sod 2 Superoxide dismutase 2, Vinc vinculin, Vdac1 voltage-dependent anion-selective channel 1, ISO isoproterenol.

Figure EV4. Evaluation of protein expression and structural morphology of mitochondria from mice subjected to pressure overload.

(A) Protein level of Nox4 in cardiac extracts from Mel null, Mel over, and wild-type mice subjected to TAC surgery for 4 days (graphs of relative quantification (left) and immunostaining (right)). Vinculin was stained as a loading control. (n = 3 per group; mean ± SD; p value by unpaired t-test). (B) Protein level of α-MTP, β-MTP, Vdac1, and Tom20 in cardiac extracts from Mel null, Mel over, and wild-type mice subjected to TAC (TAC +) or sham (TAC −) surgery for 4 days, as described in 3D. Melusin was stained as a marker of the different genotypes. Vinculin was stained as a loading control. (n = 3 per group). (C) Relative mtDNA content compared to genomic DNA evaluated by quantitative real-time PCR analysis in hearts of wild-type, Mel null and Mel over mice subjected to TAC (TAC +) or sham (TAC −) surgery for 4 days, as described in 3D. (n = 4 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). (D) Representative TEM images of cardiac sections of wild-type and Mel null hearts subjected to TAC (TAC +) or sham (TAC −) surgery for 4 days, as described in 3D. Scale bare = 1 μm. (E) Relative quantification of cristae distance in nm. Data were means ± SD. (n = 25 cristae from mitochondria from three different animals per group. mean ± SD; p value by two-way ANOVA with Bonferroni correction). (F) Level of ATP, detected by luciferin-luciferase assay, in total cardiac extracts of wild-type, Mel null and Mel over mice subjected to TAC (TAC +) or sham (TAC −) surgery for 4 days, as described in 3D. (n = 3 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). Vinc vinculin; Vdac1 voltage-dependent anion-selective channel 1, Tom20 translocase of outer membrane 20, TAC transverse aortic constriction, TEM transmission electron microscopy.

Figure EV5. Evaluation of protein expression and structural morphology of mitochondria from mice subjected to doxorubicin cardiotoxicity.

(A) Protein level of Nox4 in cardiac extracts from Mel null, Mel over, and wild-type mice treated with 4 mg/kg doxorubicin for 6 h (graphs of relative quantification (left) and immunostaining (right)). Vinculin was stained as a loading control. (n = 3 per group; mean ± SD; p value by unpaired t-test). (B) Protein level of α-MTP, β-MTP, Vdac1, and Tom20 in cardiac extracts from Mel null, Mel over, and wild-type mice treated with 4 mg/kg doxorubicin (DOX +) or saline (DOX −) for 6 h, as described in 3F. Melusin was stained as a marker of the different genotypes. Vinculin was stained as a loading control. (n = 3 per group). (C) Relative mtDNA content compared to genomic DNA evaluated by quantitative real-time PCR analysis in hearts of wild-type, Mel null, and Mel over mice treated with 4 mg/kg doxorubicin (DOX +) or saline (DOX −) for 6 h, as described in 3F. (n = 4 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). (D) Representative TEM images of cardiac sections of wild-type and Mel null mice treated with 4 mg/kg doxorubicin (DOX +) or saline (DOX −) for 6 h, as described in 3F. Scale bar = 1 μm. (E) Relative quantification of cristae distance in nm. Data were means ± SD. (n = 25 cristae from mitochondria from three different animals per group. mean ± SD; p value by two-way ANOVA with Bonferroni correction). (F) Integrity of isolated mitochondria from wild-type 129S/v and Mel null hearts, untreated or treated with doxorubicin, estimated as immunostaining of specific markers of different mitochondrial compartments (Complex Va, Complex III for the inner membrane, Vdac for the outer membrane, cyclophilin D for the matrix, cytochrome C for the intermembrane space. (n = 3 per group). (G) Level of ATP, detected by luciferin-luciferase assay, in total cardiac extracts of wild-type, Mel null and Mel over mice treated with 4 mg/kg doxorubicin (DOX +) or saline (DOX −) for 6 h, as described in 3F. (n = 3 per group; mean ± SD; p value by two-way ANOVA with Tukey correction (performed separately for the two mice strains)). Vinc vinculin, Vdac voltage-dependent anion-selective channel, Tom20 translocase of outer membrane 20, DOX doxorubicin, TEM transmission electron microscopy, CypD cyclophilin D, Cyt C cytochrome C.