Cardiac-Specific Expression of the Tetracycline Transactivator Confers Increased Heart Function and Survival Following Ischemia Reperfusion Injury

Mice expressing the tetracycline transactivator (tTA) transcription factor driven by the rat α-myosin heavy chain promoter (α-MHC-tTA) are widely used to dissect the molecular mechanisms involved in cardiac development and disease. However, these α-MHC-tTA mice exhibit a gain-of-function phenotype consisting of robust protection against ischemia/reperfusion injury in both in vitro and in vivo models in the absence of associated cardiac hypertrophy or remodeling. Cardiac function, as assessed by echocardiography, did not differ between α-MHC-tTA and control animals, and there were no noticeable differences observed between the two groups in HW/TL ratio or LV end-diastolic and end-systolic dimensions. Protection against ischemia/reperfusion injury was assessed using isolated perfused hearts where α-MHC-tTA mice had robust protection against ischemia/reperfusion injury which was not blocked by pharmacological inhibition of PI3Ks with LY294002. Furthermore, α-MHC-tTA mice subjected to coronary artery ligation exhibited significantly reduced infarct size compared to control animals. Our findings reveal that α-MHC-tTA transgenic mice exhibit a gain-of-function phenotype consisting of robust protection against ischemia/reperfusion injury similar to cardiac pre- and post-conditioning effects. However, in contrast to classical pre- and post-conditioning, the α-MHC-tTA phenotype is not inhibited by the classic preconditioning inhibitor LY294002 suggesting involvement of a non-PI3K-AKT signaling pathway in this phenotype. Thus, further study of the α-MHC-tTA model may reveal novel molecular targets for therapeutic intervention during ischemic injury

α-MHC-tTA hearts were protected against I/R injury <i>in vitro</i> using the Langendorff-perfused heart.

<p>(A–B) LVDP was similar between α-MHC-tTA and control hearts at baseline; however, after 30 min of ischemia, control hearts recovered to 35% of baseline LVDP values whereas α-MHC-tTA had 90% recovery. (C–D) LV systolic and diastolic functions assessed by dP/dt_max and dP/dt_min respectively were significantly higher in α-MHC-tTA compared to control after 30 min of ischemia. (N = 3 for control and N = 4 for αMHC-tTA, p<0.05).</p

Echocardiographic Measurements and Heart weight/tibia length.

<p>Values are mean ± SEM. <i>P</i> values are based on 1-tail Student's <i>t</i>-test assuming unequal variance.</p

Reduced cardiac muscle damage in α-MHC-tTA hearts subjected to I/R injury.

<p>Lack of cardiac muscle cell damage was apparent in α-MHC-tTA compared to control where abundant creatine kinase levels were observed within 15 min of start of reperfusion. (N = 4, p<0.05).</p

Effect of <i>in vivo</i> I/R injury on α-MHC-tTA hearts.

<p>Significantly smaller infarct sizes were observed in α-MHC-tTA hearts subjected to 45 min of left coronary artery occlusion followed by 120 min of reperfusion. (A) Representative cross sections from control and α-MHC-tTA hearts. (B) Average infarct size in control and α-MHC-tTA hearts. (N = 5 for control and N = 7 for α-MHC-tTA, p<0.05).</p

Effect of PI3K inhibition on the protection against I/R injur in α-MHC-tTA hearts.

<p>Administration of LY294002 did not abolish the protective effect seen in α-MHC-tTA hearts however it did abolish protection imparted by IPC. (N = 5 for α-MHC-tTA and N = 3 for α-MHC-tTA+8 µM LY294002, p<0.05).</p

Myocardial Infarction in Mice Alters Sarcomeric Function Via Post-Translational Protein Modification

Myocardial physiology in the aftermath of myocardial infarction (MI) before remodeling is an under-explored area of investigation. Here, we describe the effects of MI on the cardiac sarcomere with focus on the possible contributions of reactive oxygen species. We surgically induced MI in 6-7-month-old female CD1 mice by ligation of the left anterior descending coronary artery. Data were collected 3-4 days after MI or sham (SH) surgery. MI hearts demonstrated ventricular dilatation and systolic dysfunction upon echo cardiographic analysis. Sub-maximum Ca-activated tension in detergent-extracted fiber bundles from papillary muscles increased significantly in the preparations from MI hearts. Ca(2+) sensitivity increased after MI, whereas cooperativity of activation decreased. To assess myosin enzymatic integrity we measured splitting of Ca-ATP in myofibrillar preparations, which demonstrated a decline in Ca-ATPase activity of myofilament myosin. Biochemical analysis demonstrated post-translational modification of sarcomeric proteins. Phosphorylation of cardiac troponin I and myosin light chain 2 was reduced after MI in papillary samples, as measured using a phospho-specific stain. Tropomyosin was oxidized after MI, forming disulfide products detectable by diagonal non-reducing-reducing SDS-PAGE. Our analysis of myocardial protein oxidation post-MI also demonstrated increased S-glutathionylation. We functionally linked protein oxidation with sarcomere function by treating skinned fibers with the sulfhydryl reducing agent dithiothreitol, which reduced Ca(2+) sensitivity in MI, but not SH, samples. Our data indicate important structural and functional alterations to the cardiac sarcomere after MI, and the contribution of protein oxidation to this process