Deficiency Of Methionine Sulfoxide Reductase A (MsrA) Causes Cardiac Dysfunction And Protective Effects Of Taurine In The Heart

Objective:To elucidate the function of methionine sulfoxide reductase A (MsrA), an enzyme that catalyzes the reduction of free and protein-bound methionine sulfoxide [Met(O)] to methionine, in intact hearts and in isolated cardiomyocytes using MsrA knock out (MsrA-/-) mouse model. To explore the beneficial effects of taurine in normal hearts and the hearts lacking MsrA. To set up the experimental protocols and criteria for cardiac function measurements in intact experimental animals using high-frequency and high-resolution echocardiography.Materials and methods:C57BL/6 wild type (WT) mice and MsrA-/- mice at various ages were used for cardiac function measurement. WT embryonic mice (E9.5 to E18.5) were used to monitor heart development using high-resolution echocardiography (Vevo 770, VisuualSonics, Toronto, ON, Canada) with a high-frequency (40MHz) transducer and a digital ultrasonic system. Cardiac function was determined in intact animal using high-frequency echocardiography, in isolated cardiac myocytes using an IonOptix system. Cell-based measurements include myocardial sarcomere contractility, myocardial cytoplasm Ca2+ transient and protein oxidative levels. Studies were performed as well in WT and MsrA-/- mice with or without treatment of taurine to evaluate the effects of taurine in the heart.Results:1. Establishing B mode, M mode, pulsed Doppler and tissue Doppler measurements in adult and embryonic mice with high-frequency echocardiography.(1) Two-dimensional B-mode imaging was taken from the cardiac long-axis view and short-axis papillary view at left parasternal position. In addition, B-mode images were obtained from a four-chamber view and a five-chamber view at apical position to evaluate cardiac morphology.(2) Anatomical M-mode imaging of left ventricle with maximal internal dimension was taken from the left parasternal short-axis papillary view.(3) Pulsed Doppler spectrum of mitral flow and tissue Doppler spectrum of mitral annulus were obtained from the apical four-chamber view.(4) Pulsed Doppler spectrum of aortic flow was obtained from the apical five-chamber view.(5) Pulsed Doppler spectrum of proximal left coronary artery flow was obtained from the"modified"left parasternal long-axis view. (6) The indexes used for evaluating cardiac systolic function included EF, FS and IVCT. The indexes used for evaluating cardiac diastolic function included IVRT, DT, E/A, E'/A'and E/E'. Among them, IVRT was the most sensitive index for diagnosing cardiac diastolic dysfunction. In addition, the indexes of aortic and proximal left coronary flow peak velocity and velocity time internal (VTI) were used for determining the aortic and coronary blood flows.(7) Determining cardiac morphology and function in embryonic mice. E9.5 was the earliest time point when embryonic hearts can be detected with echocardiography. M mode imaging of both ventricles can be obtained from E14.5 since the internal ventricular septum (IVS) is formed by then. E15.5 was the best developmental time point for observing a four-chamber embryonic heart.2. Deficiency of MsrA causes cardiac dysfunction.(1) The data from echocardiography showed an impaired systolic function in MsrA-/- mice at age of one month or elder compared to WT controls at the same age manifested by a decreased ejection fraction (EF) and fractional shortening (FS) as well as an increased left ventricular end systolic dimension (LVESD). In addition, elder MsrA-/- mice showed a further increased left ventricular end volume compared to WT controls at the same age. The systolic dysfunction was deteriorated in elder MsrA-/- mice compared to young MsrA-/- mice (1-month-old). (2) Impaired cardiac sarcomere contractility manifested as a decreased sarcomere shortening velocity and amplitude were observed in cardiac myocytes isolated from MsrA-/- mice. The impaired cardiac sarcomere contractility was worsen in aged MsrA-/- mice and in MsrA-/- cardiac myocytes under physical (a higher stimulation frequency) or oxidative (H2O2 treatment) stresses.(3) Abnormal Ca2+ transients (determined by a Fura2 ratio) were observed in cardiac myocytes isolated from MsrA-/- mice, which was worsen in aged MsrA-/- mice and in MsrA-/- cardiac myocytes under physical (a higher stimulation frequency) or oxidative (H2O2 treatment) stresses.(4) Mitochondria were more vulnerable to oxidative stress, and an increased mitochondrial protein oxidation level was observed in MsrA-/- mice under normal or oxidative conditions.3. Cardiac dysfunction was reversed in MsrA-/- mice treated with taurine in drinking water for 5 months.(1) Cardiac function measured in MsrA-/- mice treated with taurine was similar to that observed in WT controls with or without taurine treatment.(2) Taurine improved impaired cardiac sarcomere contractility in MsrA-/- cardiac myocytes manifested as an increased sarcomere shortening velocity in MsrA-/- mice treated with taurine compared to the same mice without taurine treatment (P<0.05). However, the damaged sarcomere shortening amplitude was not changed significantly in MsrA-/- mice treated with taurine. Additionally, taurine increased basic and contracted sarcomere length in WT and MsrA-/- mice. Furthermore, taurine increased basic and contracted sarcomere length in bothWT and MsrA-/- mice. Taurine treatment also improved anti-oxidant ability in both WT and MsrA-/- mice.(3) The abnormal Ca2+ transient observed in MsrA-/- mice was reversed by taurine in MsrA-/- mice treated with taurine. The Ca2+ transient measured in cardiac myocytes from MsrA-/- mice treated with taurine was similar to that measured in WT cardiac myocytes (P>0.05). Even under H2O2 stimulation, the Ca2+ transients did not change significantly in cardiac myocytes from MsrA-/- mice treated with taurine, suggesting that taurine increases the anti-oxidant ability in cardiac mycoytes.(4) Taurine increased the anti-oxidant ability in cardiac myocytes by decreasing cytoplasm and mitochondrial protein oxidation levels in both WT and MsrA-/- mice treated with taurine.Conclusion:1. High-resolution echocardiography provides us with a powerful, non-invasive, effective and reproducible way to serially assess and quantitatively analyze cardiac function in living adult and embryonic mice. 2. Deficiency of MsrA results in cardiac dysfunction in vivo. MsrA deficiency causes an impaired sarcomere contractility and abnormal Ca2+ transient at cellular level. MsrA-/- cardiac myocytes are more vulnerable to oxidative stress manifested as an enhanced mitochondrial protein oxidative level.3. Taurine can protect hearts by reversing MsrA deficiency caused cardiac dysfunction and by increasing anti-oxidant ability in cardiac myocytes.4. Mitochondria are most vulnerable to oxidative stress in the heart. Taurine can reduce the mitochondrial and cytoplasma protein oxidation levels in cardiac myocytes, suggesting that this may be one of the mechanisms underlying taurine's protection effect in the heart.