Co-immunoprecipitation of mitochondrial proteins with P-JNK and P-p38.

<p>Representative immunoblots showing interaction between P-JNK, P-p38, the MPTP components ANT, VDAC, CyP-D, and components of mitochondrial oxidative phosphorylation. (<b><i>A</i></b>) Cardiac mitochondria from each group were immunoprecipitated (IP) with P-JNK and P-p38. The complexes were subjected to SDS-PAGE followed by immunoblotting (IB) with indicated antibodies. Bands that underwent densitometry analysis are indicated by arrows (a,b). (<b><i>B</i></b>) Densitometric data for UQCRC2 (a component of mitochondrial complex III), were normalized to P-JNK. CS, non-ischemic hearts perfused for 60 min with 10 µM SU3327 added at 20 min after beginning of perfusion (n = 4). *, #, &: significantly different from the other indicators (<i>P</i><0.05). (<b><i>C</i></b>) Densitometric data for UQCRC2 (a component of mitochondrial complex III), normalized to P-p38. n = 3 per group.</p

Mitochondrial respiratory control index, MPTP opening and carbonylation of mitochondrial proteins.

<p>(<b><i>A</i></b>) Mitochondrial respiratory control index (RCI) at complex I measured in the presence of 2.5 mM 2-oxoglutarate and 1 mM L-malate substrates. (<b><i>B</i></b>) RCI at complex II measured in the presence of a 2.5 mM succinate substrate. (<b><i>C</i></b>) RCI at complex IV measured in the presence of 10 mM ascorbate and 0.3 mM TMPD. *, #, &, @, +: significantly different from the other indicators (<i>P</i><0.05). (<b><i>D</i></b>) Increment of the rate of mitochondrial swelling by addition of 100 mM CaCl₂. *: significantly different from group C (<i>P</i><0.05), (<b><i>E</i></b>) Carbonylation levels of mitochondrial proteins. The data were represented as the ratio of intensities from DNPH-treated samples to non-treated samples compared to control for each group. *, #, &, @: significantly different from the other indicators (<i>P</i><0.05). n = 5 for C, n = 8 for IR, n = 8 for IRS, n = 7 for IRSP, and n = 6 for IRSR groups.</p

MAPK activation in H9C2 cells.

<p>Representative immunoblots showing activation of JNK, p38, and ERK (<b><i>A</i></b>). Cardiomyocytes were pretreated with JNK inhibitor SU3327 (SU), then treated with 75 µM H₂O₂ (<b><i>B,D</i></b>). Cardiomyocytes were pretreated with SU and p38 inhibitor BIRB796 (BIRB), then treated with 75 µM H₂O₂ (<b><i>C,E,F</i></b>). *, #, &: significantly different from the other indicators (<i>P</i><0.05). n = 6 per group.</p

Heart function.

<p>(<b><i>A</i></b>) Left ventricular (LV) developed pressure (LVDP) calculated as the difference between LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP). Data are expressed as a percentage of pre-ischemic values. *<i>P</i><0.0001 C <i>vs</i> other groups, ^#<i>P</i><0.05 IR <i>vs</i> IRS and IRSP. (<b><i>B</i></b>) LVEDP at the end of reperfusion (for IR, IRS, IRSP and IRSR) or perfusion (C). (<b><i>C</i></b>) The rate-pressure product (RPP) calculated as RPP = LVDP×HR. RPP recovery during the reperfusion period is shown as percent of pre-ischemia values. (<b><i>D</i></b>) Lactate dehydrogenase (LDH) activity in the coronary effluent. The dotted line represents 25 min of ischemia for IR, IRS, IRSP, and IRSR groups. Time in parentheses represents perfusion time for the C group (without IR). LDH activity is shown as decrement of absorbance at 340 nm per min for liter of perfusate per gram heart. *<i>P</i><0.001 C <i>vs</i> other groups, #, &: significantly different from the other indicators (<i>P</i><0.05). n = 5 for C, n = 8 for IR, n = 8 for IRS, n = 7 for IRSP, and n = 6 for IRSR groups.</p

Effect of SU3327 on H9C2 cells exposed to H₂O₂.

<p>Cardiomyocytes were pretreated with JNK inhibitor SU3327 (SU), then treated with 75 µM H₂O₂ (<b><i>A,B,C</i></b>). Cardiomyocytes were treated with SU only (<b><i>D,E,F</i></b>). Cell viability (<b><i>A,B</i></b>), total ROS (<b><i>B,E</i></b>), mitochondrial membrane potential (<b><i>C,F</i></b>). *, #, &, @: significantly different from the other indicators (<i>P</i><0.05). n = 6 per group.</p

Proposed mechanism of cardiac dysfunction induced by inhibition of JNK.

<p>Details of the mechanism are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113526#s4" target="_blank"><i>Discussion</i></a>. MPTP, mitochondrial permeability transition pore, p38_m and P-p38_m, mitochondrial p38 and P-p38; JNK_m and P-JNK_m, mitochondrial JNK and P-JNK.</p

Table_4.xlsx

<p>Aim: Tafazzin knockdown (TazKD) in mice is widely used to create an experimental model of Barth syndrome (BTHS) that exhibits dilated cardiomyopathy and impaired exercise capacity. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that play essential roles as transcription factors in the regulation of carbohydrate, lipid, and protein metabolism. We hypothesized that the activation of PPAR signaling with PPAR agonist bezafibrate (BF) may ameliorate impaired cardiac and skeletal muscle function in TazKD mice. This study examined the effects of BF on cardiac function, exercise capacity, and metabolic status in the heart of TazKD mice. Additionally, we elucidated the impact of PPAR activation on molecular pathways in TazKD hearts.</p><p>Methods: BF (0.05% w/w) was given to TazKD mice with rodent chow. Cardiac function in wild type-, TazKD-, and BF-treated TazKD mice was evaluated by echocardiography. Exercise capacity was evaluated by exercising mice on the treadmill until exhaustion. The impact of BF on metabolic pathways was evaluated by analyzing the total transcriptome of the heart by RNA sequencing.</p><p>Results: The uptake of BF during a 4-month period at a clinically relevant dose effectively protected the cardiac left ventricular systolic function in TazKD mice. BF alone did not improve the exercise capacity however, in combination with everyday voluntary running on the running wheel BF significantly ameliorated the impaired exercise capacity in TazKD mice. Analysis of cardiac transcriptome revealed that BF upregulated PPAR downstream target genes involved in a wide spectrum of metabolic (energy and protein) pathways as well as chromatin modification and RNA processing. In addition, the Ostn gene, which encodes the metabolic hormone musclin, is highly induced in TazKD myocardium and human failing hearts, likely as a compensatory response to diminished bioenergetic homeostasis in cardiomyocytes.</p><p>Conclusion: The PPAR agonist BF at a clinically relevant dose has the therapeutic potential to attenuate cardiac dysfunction, and possibly exercise intolerance in BTHS. The role of musclin in the failing heart should be further investigated.</p

Image_7.pdf

<p>Aim: Tafazzin knockdown (TazKD) in mice is widely used to create an experimental model of Barth syndrome (BTHS) that exhibits dilated cardiomyopathy and impaired exercise capacity. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that play essential roles as transcription factors in the regulation of carbohydrate, lipid, and protein metabolism. We hypothesized that the activation of PPAR signaling with PPAR agonist bezafibrate (BF) may ameliorate impaired cardiac and skeletal muscle function in TazKD mice. This study examined the effects of BF on cardiac function, exercise capacity, and metabolic status in the heart of TazKD mice. Additionally, we elucidated the impact of PPAR activation on molecular pathways in TazKD hearts.</p><p>Methods: BF (0.05% w/w) was given to TazKD mice with rodent chow. Cardiac function in wild type-, TazKD-, and BF-treated TazKD mice was evaluated by echocardiography. Exercise capacity was evaluated by exercising mice on the treadmill until exhaustion. The impact of BF on metabolic pathways was evaluated by analyzing the total transcriptome of the heart by RNA sequencing.</p><p>Results: The uptake of BF during a 4-month period at a clinically relevant dose effectively protected the cardiac left ventricular systolic function in TazKD mice. BF alone did not improve the exercise capacity however, in combination with everyday voluntary running on the running wheel BF significantly ameliorated the impaired exercise capacity in TazKD mice. Analysis of cardiac transcriptome revealed that BF upregulated PPAR downstream target genes involved in a wide spectrum of metabolic (energy and protein) pathways as well as chromatin modification and RNA processing. In addition, the Ostn gene, which encodes the metabolic hormone musclin, is highly induced in TazKD myocardium and human failing hearts, likely as a compensatory response to diminished bioenergetic homeostasis in cardiomyocytes.</p><p>Conclusion: The PPAR agonist BF at a clinically relevant dose has the therapeutic potential to attenuate cardiac dysfunction, and possibly exercise intolerance in BTHS. The role of musclin in the failing heart should be further investigated.</p

Table_2.xlsx

<p>Aim: Tafazzin knockdown (TazKD) in mice is widely used to create an experimental model of Barth syndrome (BTHS) that exhibits dilated cardiomyopathy and impaired exercise capacity. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that play essential roles as transcription factors in the regulation of carbohydrate, lipid, and protein metabolism. We hypothesized that the activation of PPAR signaling with PPAR agonist bezafibrate (BF) may ameliorate impaired cardiac and skeletal muscle function in TazKD mice. This study examined the effects of BF on cardiac function, exercise capacity, and metabolic status in the heart of TazKD mice. Additionally, we elucidated the impact of PPAR activation on molecular pathways in TazKD hearts.</p><p>Methods: BF (0.05% w/w) was given to TazKD mice with rodent chow. Cardiac function in wild type-, TazKD-, and BF-treated TazKD mice was evaluated by echocardiography. Exercise capacity was evaluated by exercising mice on the treadmill until exhaustion. The impact of BF on metabolic pathways was evaluated by analyzing the total transcriptome of the heart by RNA sequencing.</p><p>Results: The uptake of BF during a 4-month period at a clinically relevant dose effectively protected the cardiac left ventricular systolic function in TazKD mice. BF alone did not improve the exercise capacity however, in combination with everyday voluntary running on the running wheel BF significantly ameliorated the impaired exercise capacity in TazKD mice. Analysis of cardiac transcriptome revealed that BF upregulated PPAR downstream target genes involved in a wide spectrum of metabolic (energy and protein) pathways as well as chromatin modification and RNA processing. In addition, the Ostn gene, which encodes the metabolic hormone musclin, is highly induced in TazKD myocardium and human failing hearts, likely as a compensatory response to diminished bioenergetic homeostasis in cardiomyocytes.</p><p>Conclusion: The PPAR agonist BF at a clinically relevant dose has the therapeutic potential to attenuate cardiac dysfunction, and possibly exercise intolerance in BTHS. The role of musclin in the failing heart should be further investigated.</p

Image_3.pdf

<p>Aim: Tafazzin knockdown (TazKD) in mice is widely used to create an experimental model of Barth syndrome (BTHS) that exhibits dilated cardiomyopathy and impaired exercise capacity. Peroxisome proliferator-activated receptors (PPARs) are a group of nuclear receptor proteins that play essential roles as transcription factors in the regulation of carbohydrate, lipid, and protein metabolism. We hypothesized that the activation of PPAR signaling with PPAR agonist bezafibrate (BF) may ameliorate impaired cardiac and skeletal muscle function in TazKD mice. This study examined the effects of BF on cardiac function, exercise capacity, and metabolic status in the heart of TazKD mice. Additionally, we elucidated the impact of PPAR activation on molecular pathways in TazKD hearts.</p><p>Methods: BF (0.05% w/w) was given to TazKD mice with rodent chow. Cardiac function in wild type-, TazKD-, and BF-treated TazKD mice was evaluated by echocardiography. Exercise capacity was evaluated by exercising mice on the treadmill until exhaustion. The impact of BF on metabolic pathways was evaluated by analyzing the total transcriptome of the heart by RNA sequencing.</p><p>Results: The uptake of BF during a 4-month period at a clinically relevant dose effectively protected the cardiac left ventricular systolic function in TazKD mice. BF alone did not improve the exercise capacity however, in combination with everyday voluntary running on the running wheel BF significantly ameliorated the impaired exercise capacity in TazKD mice. Analysis of cardiac transcriptome revealed that BF upregulated PPAR downstream target genes involved in a wide spectrum of metabolic (energy and protein) pathways as well as chromatin modification and RNA processing. In addition, the Ostn gene, which encodes the metabolic hormone musclin, is highly induced in TazKD myocardium and human failing hearts, likely as a compensatory response to diminished bioenergetic homeostasis in cardiomyocytes.</p><p>Conclusion: The PPAR agonist BF at a clinically relevant dose has the therapeutic potential to attenuate cardiac dysfunction, and possibly exercise intolerance in BTHS. The role of musclin in the failing heart should be further investigated.</p