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Cardiomyocyte-specific deficiency of ketone body metabolism promotes accelerated pathological remodeling.

Schugar RC, Moll AR, André d'Avignon D, Weinheimer CJ, Kovacs A, Crawford PA - Mol Metab (2014)

Bottom Line: While germline SCOT-knockout (KO) mice die in the early postnatal period, adult mice with cardiomyocyte-specific loss of SCOT (SCOT-Heart-KO) remarkably exhibit no overt metabolic abnormalities, and no differences in left ventricular mass or impairments of systolic function during periods of ketosis, including fasting and adherence to a ketogenic diet.While TAC increased left ventricular mass equally in both groups, at four weeks post-TAC, myocardial ROS abundance was increased in myocardium of SCOT-Heart-KO mice, and mitochondria and myofilaments were ultrastructurally disordered.Eight weeks post-TAC, left ventricular volume was markedly increased and ejection fraction was decreased in SCOT-Heart-KO mice, while these parameters remained normal in hearts of control animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for Cardiovascular Research, Washington University, St. Louis, MO, USA.

ABSTRACT

Objective: Exploitation of protective metabolic pathways within injured myocardium still remains an unclarified therapeutic target in heart disease. Moreover, while the roles of altered fatty acid and glucose metabolism in the failing heart have been explored, the influence of highly dynamic and nutritionally modifiable ketone body metabolism in the regulation of myocardial substrate utilization, mitochondrial bioenergetics, reactive oxygen species (ROS) generation, and hemodynamic response to injury remains undefined.

Methods: Here we use mice that lack the enzyme required for terminal oxidation of ketone bodies, succinyl-CoA:3-oxoacid CoA transferase (SCOT) to determine the role of ketone body oxidation in the myocardial injury response. Tracer delivery in ex vivo perfused hearts coupled to NMR spectroscopy, in vivo high-resolution echocardiographic quantification of cardiac hemodynamics in nutritionally and surgically modified mice, and cellular and molecular measurements of energetic and oxidative stress responses are performed.

Results: While germline SCOT-knockout (KO) mice die in the early postnatal period, adult mice with cardiomyocyte-specific loss of SCOT (SCOT-Heart-KO) remarkably exhibit no overt metabolic abnormalities, and no differences in left ventricular mass or impairments of systolic function during periods of ketosis, including fasting and adherence to a ketogenic diet. Myocardial fatty acid oxidation is increased when ketones are delivered but cannot be oxidized. To determine the role of ketone body oxidation in the remodeling ventricle, we induced pressure overload injury by performing transverse aortic constriction (TAC) surgery in SCOT-Heart-KO and αMHC-Cre control mice. While TAC increased left ventricular mass equally in both groups, at four weeks post-TAC, myocardial ROS abundance was increased in myocardium of SCOT-Heart-KO mice, and mitochondria and myofilaments were ultrastructurally disordered. Eight weeks post-TAC, left ventricular volume was markedly increased and ejection fraction was decreased in SCOT-Heart-KO mice, while these parameters remained normal in hearts of control animals.

Conclusions: These studies demonstrate the ability of myocardial ketone metabolism to coordinate the myocardial response to pressure overload, and suggest that the oxidation of ketone bodies may be an important contributor to free radical homeostasis and hemodynamic preservation in the injured heart.

No MeSH data available.


Related in: MedlinePlus

Relative genome content and respiration studies of cardiac mitochondria. (A, B) Quantification of mitochondrial genome copy number (relative abundance) by qPCR using purified heart gDNA from SCOT-Heart-KO and αMHC-Cre mice at baseline, or after 4 wk or 8 wk TAC. Data are presented as means ± SEM; n = 4–5/group, *p ≤ 0.05 by 1-way ANOVA with Tukey's post hoc analysis. (C) Increased myocardial expression of mitochondrial transcription factor A (Tfam). (D, E) Respiration rates in the basal leak condition (state 2), ADP-stimulated condition (state 3), F1F0-ATPase independent condition (state 4, oligomycin), and uncoupled condition (FCCP) in mitochondria isolated from left ventricles of SCOT-Heart-KO and αMHC-Cre mice using palmitoyl-l-carnitine and malate or succinate and rotenone as substrates at (D) baseline or (E) following 4 wk TAC. Data are presented as means ± SEM; n = 4–7 mice/group, *p ≤ 0.05, ***p ≤ 0.001 by 2-way ANOVA with Bonferroni post hoc analysis. (F, G) No differences in NAD+, NADH, NADt or NAD+/NADH ratios were detected when comparing whole myocardial lysates of SCOT-Heart-KO and αMHC-Cre control mice at (F) baseline or (G) after 4 wk TAC. n = 5–7 mice/group.
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fig5: Relative genome content and respiration studies of cardiac mitochondria. (A, B) Quantification of mitochondrial genome copy number (relative abundance) by qPCR using purified heart gDNA from SCOT-Heart-KO and αMHC-Cre mice at baseline, or after 4 wk or 8 wk TAC. Data are presented as means ± SEM; n = 4–5/group, *p ≤ 0.05 by 1-way ANOVA with Tukey's post hoc analysis. (C) Increased myocardial expression of mitochondrial transcription factor A (Tfam). (D, E) Respiration rates in the basal leak condition (state 2), ADP-stimulated condition (state 3), F1F0-ATPase independent condition (state 4, oligomycin), and uncoupled condition (FCCP) in mitochondria isolated from left ventricles of SCOT-Heart-KO and αMHC-Cre mice using palmitoyl-l-carnitine and malate or succinate and rotenone as substrates at (D) baseline or (E) following 4 wk TAC. Data are presented as means ± SEM; n = 4–7 mice/group, *p ≤ 0.05, ***p ≤ 0.001 by 2-way ANOVA with Bonferroni post hoc analysis. (F, G) No differences in NAD+, NADH, NADt or NAD+/NADH ratios were detected when comparing whole myocardial lysates of SCOT-Heart-KO and αMHC-Cre control mice at (F) baseline or (G) after 4 wk TAC. n = 5–7 mice/group.

Mentions: A common injurious myocardial stress is sustained hypertension, which in mice is experimentally mimicked by provoking cardiac pressure overload through surgical transverse aortic constriction (TAC). This procedure provokes metabolic abnormalities that precede and drive adverse ventricular remodeling [5,45]. To determine the effect of SCOT deficiency on pathological remodeling in response to pressure overload, TAC (and sham) surgeries were performed in SCOT-Heart-KO and αMHC-Cre control mice, and echocardiographic assessments were performed following 4 wk and 8 wk TAC. Myocardial abundance of SCOT did not significantly change in control animals at 4 wk or 8 wk following TAC surgery (Supplemental Figure 1). Moreover, serum ketone body concentrations did not differ between SCOT-Heart-KO and αMHC-Cre mice after 4 wk TAC, nor did they increase from their respective baseline values; however, serum glucose concentration was modestly elevated after 4 wk TAC in SCOT-Heart-KO mice (Supplemental Table 2). While TAC-induced increases in left ventricular mass were comparable between SCOT-Heart-KO mice and αMHC-Cre control mice at both time points (Figure 3A), mean pressure gradient across the aortic arch was decreased after 8 wk TAC in SCOT-Heart-KO mice, suggesting decreased contractility and abnormal emptying of the left ventricle (Figure 3B). Furthermore, while αMHC-Cre mice maintained preserved systolic function with increased myocardial wall thickness relative to LV chamber size, SCOT-Heart-KO mice exhibited left ventricular dilation with increased end diastolic volume (2.9 ± 0.4 μL/mm tibia length and 5.3 ± 1.0 μL/mm tibia length, respectively; p = 0.04; n = 7–10/group) and decreased relative wall thickness (Figure 3C, D). In addition, systolic function was markedly decreased in hearts of pressure-overloaded SCOT-Heart-KO mice (ejection fractions of 35.0  ±  7.0%, versus 54.9  ±  6.0% in αMHC-Cre control mice; p = 0.03; n = 7–10/group) (Figure 3D). No statistically significant differences in cardiac output or evidence of worsened pulmonary edema were detected in the SCOT-Heart-KO mice after 8 wk TAC (Supplemental Table 3). Light microscopic analysis of hearts post-TAC revealed no differences in inflammatory infiltrate at either time point (Figure 3E), or in extent of collagen deposition (data not shown). To determine if the accelerated pressure overload-induced ventricular remodeling phenotype could be linked to ultrastructural abnormalities, transmission electron micrographs from SCOT-Heart-KO and αMHC-Cre hearts were compared. Mitochondrial number, organization, and ultrastructural integrity were normal in sham-operated SCOT-Heart-KO mice (Figure 4A–F). Conversely, extensive myofibrilar disarray and Z-line thickening were observed after 4 wk TAC, uniquely within cardiomyocytes from SCOT-Heart-KO mice (Figure 4H, K). High power images revealed no overt differences in mitochondrial ultrastructure relative to αMHC-Cre control mice (Figure 4I, L). Lower power electron micrographs suggest a modest increase in mitochondrial number, with more disordered packing, among myofibrils of SCOT-Heart-KO cardiomyocytes following 4 wk TAC (Figure 4G, J). Quantification of mitochondrial genome copy number indicated a trend towards increased mitochondrial number in hearts of SCOT-Heart-KO mice following 4 wk TAC (Figure 5A, B). Additionally, increased myocardial abundance of the mRNA encoding mitochondrial transcription factor A (TFAM, a mitochondrial transcription factor that coordinates mitochondrial genome replication) was observed in hearts of SCOT-Heart-KO mice following 4 wk TAC (Figure 5C). While phosphorylation of the energy sensor AMPK was normal at baseline and following 4 wk TAC in hearts of SCOT-Heart-KO mice, after 8 wk TAC relative abundance of phosphorylated AMPK was moderately increased in extracts from (Supplemental Figure 2), suggesting delayed energy deficit that was not primarily caused by the absence of ketone body oxidation.


Cardiomyocyte-specific deficiency of ketone body metabolism promotes accelerated pathological remodeling.

Schugar RC, Moll AR, André d'Avignon D, Weinheimer CJ, Kovacs A, Crawford PA - Mol Metab (2014)

Relative genome content and respiration studies of cardiac mitochondria. (A, B) Quantification of mitochondrial genome copy number (relative abundance) by qPCR using purified heart gDNA from SCOT-Heart-KO and αMHC-Cre mice at baseline, or after 4 wk or 8 wk TAC. Data are presented as means ± SEM; n = 4–5/group, *p ≤ 0.05 by 1-way ANOVA with Tukey's post hoc analysis. (C) Increased myocardial expression of mitochondrial transcription factor A (Tfam). (D, E) Respiration rates in the basal leak condition (state 2), ADP-stimulated condition (state 3), F1F0-ATPase independent condition (state 4, oligomycin), and uncoupled condition (FCCP) in mitochondria isolated from left ventricles of SCOT-Heart-KO and αMHC-Cre mice using palmitoyl-l-carnitine and malate or succinate and rotenone as substrates at (D) baseline or (E) following 4 wk TAC. Data are presented as means ± SEM; n = 4–7 mice/group, *p ≤ 0.05, ***p ≤ 0.001 by 2-way ANOVA with Bonferroni post hoc analysis. (F, G) No differences in NAD+, NADH, NADt or NAD+/NADH ratios were detected when comparing whole myocardial lysates of SCOT-Heart-KO and αMHC-Cre control mice at (F) baseline or (G) after 4 wk TAC. n = 5–7 mice/group.
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fig5: Relative genome content and respiration studies of cardiac mitochondria. (A, B) Quantification of mitochondrial genome copy number (relative abundance) by qPCR using purified heart gDNA from SCOT-Heart-KO and αMHC-Cre mice at baseline, or after 4 wk or 8 wk TAC. Data are presented as means ± SEM; n = 4–5/group, *p ≤ 0.05 by 1-way ANOVA with Tukey's post hoc analysis. (C) Increased myocardial expression of mitochondrial transcription factor A (Tfam). (D, E) Respiration rates in the basal leak condition (state 2), ADP-stimulated condition (state 3), F1F0-ATPase independent condition (state 4, oligomycin), and uncoupled condition (FCCP) in mitochondria isolated from left ventricles of SCOT-Heart-KO and αMHC-Cre mice using palmitoyl-l-carnitine and malate or succinate and rotenone as substrates at (D) baseline or (E) following 4 wk TAC. Data are presented as means ± SEM; n = 4–7 mice/group, *p ≤ 0.05, ***p ≤ 0.001 by 2-way ANOVA with Bonferroni post hoc analysis. (F, G) No differences in NAD+, NADH, NADt or NAD+/NADH ratios were detected when comparing whole myocardial lysates of SCOT-Heart-KO and αMHC-Cre control mice at (F) baseline or (G) after 4 wk TAC. n = 5–7 mice/group.
Mentions: A common injurious myocardial stress is sustained hypertension, which in mice is experimentally mimicked by provoking cardiac pressure overload through surgical transverse aortic constriction (TAC). This procedure provokes metabolic abnormalities that precede and drive adverse ventricular remodeling [5,45]. To determine the effect of SCOT deficiency on pathological remodeling in response to pressure overload, TAC (and sham) surgeries were performed in SCOT-Heart-KO and αMHC-Cre control mice, and echocardiographic assessments were performed following 4 wk and 8 wk TAC. Myocardial abundance of SCOT did not significantly change in control animals at 4 wk or 8 wk following TAC surgery (Supplemental Figure 1). Moreover, serum ketone body concentrations did not differ between SCOT-Heart-KO and αMHC-Cre mice after 4 wk TAC, nor did they increase from their respective baseline values; however, serum glucose concentration was modestly elevated after 4 wk TAC in SCOT-Heart-KO mice (Supplemental Table 2). While TAC-induced increases in left ventricular mass were comparable between SCOT-Heart-KO mice and αMHC-Cre control mice at both time points (Figure 3A), mean pressure gradient across the aortic arch was decreased after 8 wk TAC in SCOT-Heart-KO mice, suggesting decreased contractility and abnormal emptying of the left ventricle (Figure 3B). Furthermore, while αMHC-Cre mice maintained preserved systolic function with increased myocardial wall thickness relative to LV chamber size, SCOT-Heart-KO mice exhibited left ventricular dilation with increased end diastolic volume (2.9 ± 0.4 μL/mm tibia length and 5.3 ± 1.0 μL/mm tibia length, respectively; p = 0.04; n = 7–10/group) and decreased relative wall thickness (Figure 3C, D). In addition, systolic function was markedly decreased in hearts of pressure-overloaded SCOT-Heart-KO mice (ejection fractions of 35.0  ±  7.0%, versus 54.9  ±  6.0% in αMHC-Cre control mice; p = 0.03; n = 7–10/group) (Figure 3D). No statistically significant differences in cardiac output or evidence of worsened pulmonary edema were detected in the SCOT-Heart-KO mice after 8 wk TAC (Supplemental Table 3). Light microscopic analysis of hearts post-TAC revealed no differences in inflammatory infiltrate at either time point (Figure 3E), or in extent of collagen deposition (data not shown). To determine if the accelerated pressure overload-induced ventricular remodeling phenotype could be linked to ultrastructural abnormalities, transmission electron micrographs from SCOT-Heart-KO and αMHC-Cre hearts were compared. Mitochondrial number, organization, and ultrastructural integrity were normal in sham-operated SCOT-Heart-KO mice (Figure 4A–F). Conversely, extensive myofibrilar disarray and Z-line thickening were observed after 4 wk TAC, uniquely within cardiomyocytes from SCOT-Heart-KO mice (Figure 4H, K). High power images revealed no overt differences in mitochondrial ultrastructure relative to αMHC-Cre control mice (Figure 4I, L). Lower power electron micrographs suggest a modest increase in mitochondrial number, with more disordered packing, among myofibrils of SCOT-Heart-KO cardiomyocytes following 4 wk TAC (Figure 4G, J). Quantification of mitochondrial genome copy number indicated a trend towards increased mitochondrial number in hearts of SCOT-Heart-KO mice following 4 wk TAC (Figure 5A, B). Additionally, increased myocardial abundance of the mRNA encoding mitochondrial transcription factor A (TFAM, a mitochondrial transcription factor that coordinates mitochondrial genome replication) was observed in hearts of SCOT-Heart-KO mice following 4 wk TAC (Figure 5C). While phosphorylation of the energy sensor AMPK was normal at baseline and following 4 wk TAC in hearts of SCOT-Heart-KO mice, after 8 wk TAC relative abundance of phosphorylated AMPK was moderately increased in extracts from (Supplemental Figure 2), suggesting delayed energy deficit that was not primarily caused by the absence of ketone body oxidation.

Bottom Line: While germline SCOT-knockout (KO) mice die in the early postnatal period, adult mice with cardiomyocyte-specific loss of SCOT (SCOT-Heart-KO) remarkably exhibit no overt metabolic abnormalities, and no differences in left ventricular mass or impairments of systolic function during periods of ketosis, including fasting and adherence to a ketogenic diet.While TAC increased left ventricular mass equally in both groups, at four weeks post-TAC, myocardial ROS abundance was increased in myocardium of SCOT-Heart-KO mice, and mitochondria and myofilaments were ultrastructurally disordered.Eight weeks post-TAC, left ventricular volume was markedly increased and ejection fraction was decreased in SCOT-Heart-KO mice, while these parameters remained normal in hearts of control animals.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for Cardiovascular Research, Washington University, St. Louis, MO, USA.

ABSTRACT

Objective: Exploitation of protective metabolic pathways within injured myocardium still remains an unclarified therapeutic target in heart disease. Moreover, while the roles of altered fatty acid and glucose metabolism in the failing heart have been explored, the influence of highly dynamic and nutritionally modifiable ketone body metabolism in the regulation of myocardial substrate utilization, mitochondrial bioenergetics, reactive oxygen species (ROS) generation, and hemodynamic response to injury remains undefined.

Methods: Here we use mice that lack the enzyme required for terminal oxidation of ketone bodies, succinyl-CoA:3-oxoacid CoA transferase (SCOT) to determine the role of ketone body oxidation in the myocardial injury response. Tracer delivery in ex vivo perfused hearts coupled to NMR spectroscopy, in vivo high-resolution echocardiographic quantification of cardiac hemodynamics in nutritionally and surgically modified mice, and cellular and molecular measurements of energetic and oxidative stress responses are performed.

Results: While germline SCOT-knockout (KO) mice die in the early postnatal period, adult mice with cardiomyocyte-specific loss of SCOT (SCOT-Heart-KO) remarkably exhibit no overt metabolic abnormalities, and no differences in left ventricular mass or impairments of systolic function during periods of ketosis, including fasting and adherence to a ketogenic diet. Myocardial fatty acid oxidation is increased when ketones are delivered but cannot be oxidized. To determine the role of ketone body oxidation in the remodeling ventricle, we induced pressure overload injury by performing transverse aortic constriction (TAC) surgery in SCOT-Heart-KO and αMHC-Cre control mice. While TAC increased left ventricular mass equally in both groups, at four weeks post-TAC, myocardial ROS abundance was increased in myocardium of SCOT-Heart-KO mice, and mitochondria and myofilaments were ultrastructurally disordered. Eight weeks post-TAC, left ventricular volume was markedly increased and ejection fraction was decreased in SCOT-Heart-KO mice, while these parameters remained normal in hearts of control animals.

Conclusions: These studies demonstrate the ability of myocardial ketone metabolism to coordinate the myocardial response to pressure overload, and suggest that the oxidation of ketone bodies may be an important contributor to free radical homeostasis and hemodynamic preservation in the injured heart.

No MeSH data available.


Related in: MedlinePlus