Progressive mitochondrial protein lysine acetylation and heart failure in a model of Friedreich's ataxia cardiomyopathy

Abstract

Introduction: The childhood heart disease of Friedreich's Ataxia (FRDA) is characterized by hypertrophy and failure. It is caused by loss of frataxin (FXN), a mitochondrial protein involved in energy homeostasis. FRDA model hearts have increased mitochondrial protein acetylation and impaired sirtuin 3 (SIRT3) deacetylase activity. Protein acetylation is an important regulator of cardiac metabolism and loss of SIRT3 increases susceptibility of the heart to stress-induced cardiac hypertrophy and ischemic injury. The underlying pathophysiology of heart failure in FRDA is unclear. The purpose of this study was to examine in detail the physiologic and acetylation changes of the heart that occur over time in a model of FRDA heart failure. We predicted that increased mitochondrial protein acetylation would be associated with a decrease in heart function in a model of FRDA.

Methods: A conditional mouse model of FRDA cardiomyopathy with ablation of FXN (FXN KO) in the heart was compared to healthy controls at postnatal days 30, 45 and 65. We evaluated hearts using echocardiography, cardiac catheterization, histology, protein acetylation and expression.

Results: Acetylation was temporally progressive and paralleled evolution of heart failure in the FXN KO model. Increased acetylation preceded detectable abnormalities in cardiac function and progressed rapidly with age in the FXN KO mouse. Acetylation was also associated with cardiac fibrosis, mitochondrial damage, impaired fat metabolism, and diastolic and systolic dysfunction leading to heart failure. There was a strong inverse correlation between level of protein acetylation and heart function.

Conclusion: These results demonstrate a close relationship between mitochondrial protein acetylation, physiologic dysfunction and metabolic disruption in FRDA hypertrophic cardiomyopathy and suggest that abnormal acetylation contributes to the pathophysiology of heart disease in FRDA. Mitochondrial protein acetylation may represent a therapeutic target for early intervention.

Fig 1. FXN KO mice exhibit diastolic dysfunction followed by dilated cardiomyopathy and heart failure.

(a) Representative mitral valve Doppler flow patterns (ratio of the early (E) to late (A) ventricular filling velocity, E/A) demonstrate restrictive cardiomyopathy in FXN KO at days of age 45 and 65. (b) Echocardiographic parasternal short axis M-mode images demonstrate progressive impairment in left ventricular wall movement with decreased fractional shortening (FS, %) in FXN KO. (c) Parasternal short axial images illustrate the transition to dilated cardiomyopathy with increased left ventricular internal diameter in diastole (LVIDd) in FXN KO at day 65. (d) FXN KO pressure volume loops represent increased end-diastolic volume (EDV) at day 65 compared to controls (p<0.001) with notable rightward shift of pressure-volume curves. Values indicated in (a), (b) and (c) are averages per group.

Fig 2. Acetylation is increased and progresses with age in FXN KO, and correlates with worse heart function.

Whole cell preparations or mitochondrial isolates from ventricular tissue were probed for proteins of interest as indicated. (a) Acetylation of lysine residues is increased early and progresses with age, and expression of electron transport chain complex subunits decreases in FXN KO hearts. (b) Plot of correlation between acetylation and systolic indices of heart function shows negative correlation between level of acetylation and ventricular function. (c) FXN hearts at day 65 had significantly increased total mitochondrial protein lysine acetylation (p = 0.0339), increased acetylation of SOD2 at Lys-68 (AcK-SOD2) (p = 0.0016), and (d) increased acetylation of LCAD compared to controls (p = 0.0291). EF = ejection fraction, FS = fractional shortening.

Fig 3. Abnormal cardiac mitochondria ultrastructure is accompanied by respiratory inhibition in FXN KO hearts.

(a) Representative images from electron micrographs of cardiomyocytes viewed at 11,000x. Mitochondrial ultrastructure abnormalities were apparent in FXN KO sections and progressed from days 30 to 65. Findings included matrix density loss, mitochondrial-to-sarcomere disarrangement, and accumulation and clumping of mitochondria. FXN KO day 65 mitochondria demonstrate cristae collapse and dissolution, and hyperdense inclusions. Arrowhead = collapsed cristae; arrow = electron-dense intramitochondrial inclusions. (b) Myofibril:mitochondria area ratios were significantly decreased at day 65 in FXN KO compared to controls (n = 3–8 micrographs per strain at each age). (c) Mitochondrial functional assays demonstrated significantly decreased respiratory control ratios in FXN KO compared to controls. There were 2–4 assay runs on pooled mitochondria from FXN KO (n = 8–12 hearts), or pooled mitochondria from controls (n = 4–6 hearts), with 2–3 pooled hearts per run.

Fig 4. FXN KO hearts exhibit features of maladaptive ventricular remodeling.

(a) Cardiac fibrosis is progressive in the FXN KO heart. Ventricular tissue was stained using Masson’s Trichrome to detect blue-staining fibrous tissue. (b) Percent area collagen was significantly increased in FXN KO hearts at postnatal day 65 compared to controls. (c) Evidence of cardiomyocyte degeneration in the FXN KO at day 65 is demonstrated by vacuolation on H&E staining.

Fig 5. Loss of FXN in the heart leads to cardiac steatosis and cold intolerance.

(a) FXN KO hearts demonstrate lipid accumulation in their hearts on oil-red-O staining. (b) Mice were subjected to a 6-hour fast after which they were placed in 4°C room for 3 hours with core body temperature monitoring. (c) FXN KO mice were unable to maintain core body temperature upon cold exposure and suffered significantly increased cold-related mortality rates compared to controls.