Depletion of cardiac cardiolipin synthase alters systolic and diastolic function

Figures

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  Figure 1. Lower abundance of cardiomyocytic CLs and CLS in catecholamine-induced cardiomyopathy (A) Schematic overview of experimental setup: 8-week-old 129SV mice were subcutaneously injected with either NaCl (VEH) or isoproterenol (ISO) 25 mg/kg/day for 4 consecutive days; 2 h after last ISO injection, primary cardiomyocytes (PCMs) were isolated and used for indicated experiments. (B) Scatterplot displays mean estimated log2-transformed fold change (ISO versus VEH PCMs) on the y axis and mean mole percent of lipid species on the x axis. Each bubble represents a single lipid species. VEH n = 4, ISO n = 5, t test, bubble size indicates -log10 p value. (C) For each lipid class median, lower/upper quartile, min-max of total mole percent values are shown on a logarithmic scale; black dots indicate outliers. VEH n = 4, ISO n = 5, Wilcoxon test. (D) Bar graphs showing the estimated log2-fold change (PCMs ISO versus VEH) ± regression standard error of differentially and significantly changed lipid species. VEH n = 4, ISO n = 5. Colors represent -log10 p values as indicated. (E) mRNA expression of cardiolipin synthase (CLS) in PCMs from vehicle-treated (VEH) versus ISO-treated mice. n = 9–11…

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  Figure 2. Lipid peroxidation and alterations of mitochondrial respiration under catecholamine-induced cardiac stress (A) Serum levels of malondialdehyde (MDA) after 4 days ISO application assessed by the TBARS assay. n = 10–12 mice/group; data are shown as mean ± SEM, t test. (B) Mitochondrial stress test using the Seahorse XFe96 Analyzer was performed with PCMs from ISO- and VEH-treated mice. Shown is the oxygen consumption rate (OCR) over time with application of oligomycin A (OLG), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A (R/A). (C) OCR-based quantification (pmol/min/protein content in μg) of basal respiration, maximal respiration, proton leak, non-mitochondrial respiration, and spare respiratory capacity analyzed with the Seahorse XFe96 Analyzer in PCMs from ISO- and VEH-treated mice. (D) Respiratory control rate (RCR = maximal respiration/proton leak) in PCMs from ISO- and VEH-treated mice. (E) mRNA expression of uncoupling protein (UCP) 2 and 3 in PCMs from ISO- and VEH-treated mice. (F) Extracellular acidification rate (ECAR) during mitochondrial stress test (application of OLG, FCCP, R/A) using the Seahorse XFe96 Analyzer. PCMs for…

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  Figure 3. Cardiac performance in CLS mutants (A) Representative images (upper part) of heart wall movement in control wild-type flies (Ctrlw1118) using a fluorescence-based technique in unanesthetized whole flies carrying the red fluorophore tdTomato under control of a cardiomyocyte-specific enhancer (R94C02). Outline of analyzed cardiac performance parameters after digitization of fluorescence images. (B) Lipid samples from whole flies (wild-type: Ctrlw1118 and CLS mutant: CLS−/+) were analyzed by liquid chromatography-mass spectrometry (MS). Abundance of cardiolipin species is shown as MS signal intensity (n = 17–18, each sample contains a pool of three flies; data are shown as median, lower/upper quartile, and min-max; unpaired t test). (C) Representative images of heart wall movement in control wild-type flies (Ctrlw1118) and CLS mutants (CLS−/+). (D–N) Parameters of cardiac performance calculated from heart wall movement imaging as outlined in Figure 3A. AI: arrhythmia index. N = 3, n = 40/49 flies. Data are shown as mean, unpaired t test. (N) Representative images of arrhythmic events in control wild-type flies (Ctrlw1118) and CLS mutants (CLS−/+).

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  Figure 4. Cardiac performance in cardiac specific CLS knockdown flies (A) Representative images of heart wall movement in control wild-type flies (Hand/+KK and CLS RNAiKK/+) and cardiomyocyte-specific CLS-RNAi knockdown flies (Hand > CLS RNAiKK). (B–K) Parameters of cardiac performance in control wild-type flies (Hand/+KK and CLS RNAiKK/+) and cardiomyocyte-specific CLS-RNAi knockdown flies (Hand > CLS RNAiKK) calculated from heart wall movement imaging as outlined in Figure 3A. AI: arrhythmia index. N = 4, n = 57/45/38 flies. Data are shown as mean, one-way ANOVA with Bonferroni multiple comparison test. (L) Representative images of arrhythmic events in control wild-type flies (Hand/+KK and CLS RNAiKK/+) (left panels) and cardiomyocyte-specific CLS-RNAi knockdown flies (Hand > CLS RNAiKK) (right panels).

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  Figure 5. Mitochondrial respiration in hearts of CLS mutant flies (A and B) Analysis of the oxygen consumption rate (OCR) over time with application of oligomycin A (OLG), carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP), and rotenone/antimycin A (R/A) and (B) quantification of basal, maximal, and ATP-linked respiration, proton leak, non-mitochondrial (mt) oxygen consumption, spare respiratory capacity, and coupling efficiency from beating heart tubes of control (Ctrlw1118) and heterozygous CLS mutant flies (CLS−/+). (N = 2, n = 30 Ctrlw1118, n = 31 CLS−/+, mean ± SEM, unpaired t test.)

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  Figure 6. Cardiac CL levels are reduced in patients with HFrEF Cardiolipin (CL) species concentration in left ventricular myocardial samples normalized to total lipid content Cardiac samples from HFrEF (n = 10) and control subjects (Ctrl) (n = 5) were subjected to MS-based shotgun analysis. Data are shown as median, lower/upper quartile, min-max; Mann-Whitney U test.