Induction of autophagy markers is associated with attenuation of miR-133a in diabetic heart failure patients undergoing mechanical unloading
Authors: Geoffrey M Thiele
Shyam Sundar Nandi
Michael J Duryee
Hamid R Shahshahan
Paras K Mishra
Daniel R Anderson
Journal: American journal of translational research (2015)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: AMPK; Beclin1; Hypertrophy; LC3B; LVAD
Affiliations: Department of Cellular, Integrative Physiology, University of Nebraska Medical Center Omaha, NE 68198, USA., Department of Medicine, Division of Rheumatology, University of Nebraska Medical Center Omaha, NE 68198, USA ; Veterans Affair Nebraska-Western Iowa Health Care System, Research Services 151 4101 Woolworth Avenue, Omaha NE 68105, USA., Department of Medicine, Division of Cardiology, University of Nebraska Medical center Omaha, NE 68198, USA., Department of Cellular, Integrative Physiology, University of Nebraska Medical Center Omaha, NE 68198, USA ; Department of Anesthesiology, University of Nebraska Medical Center Omaha, NE 68198, USA.

Abstract:  Autophagy is ubiquitous in all forms of heart failure and cardioprotective miR-133a is attenuated in human heart failure. Previous reports from heart failure patients undergoing left ventricular assist device (LVAD) implantation demonstrated that autophagy is upregulated in the LV of the failing human heart. Studies in the murine model show that diabetes downregulates miR-133a. However, the role of miR-133a in the regulation of autophagy in diabetic hearts is unclear. We tested the hypothesis that diabetes exacerbates cardiac autophagy by inhibiting miR-133a in heart failure patients undergoing LVAD implantation. The miRNA assay was performed on the LV of 15 diabetic (D) and 6 non-diabetic (ND) heart failure patients undergoing LVAD implantation. Four ND with highly upregulated and 5 D with highly downregulated miR-133a were analyzed for autophagy markers (Beclin1, LC3B, ATG3) and their upstream regulators (mTOR and AMPK), and hypertrophy marker (beta-myosin heavy chain) by RT-qPCR, Western blotting and immunofluorescence. Our results demonstrate that attenuation of miR-133a in diabetic hearts is associated with the induction of autophagy and hypertrophy, and suppression of mTOR without appreciable difference in AMPK activity. In conclusion, attenuation of miR-133a contributes to the exacerbation of diabetes mediated cardiac autophagy and hypertrophy in heart failure patients undergoing LVAD implantation. 

Retracted: No

PubPeer comments:
 Fig 3 ![file] Fig 4 ![file] The Actin band in Fig 3Di and Fig 4 A are duplicated. Furthermore, phosphorylation of mTOR (p-mTOR) in Fig 4A raised concern due to significant unequal loading of Actin. Also, total mTOR is in question because of unequal pattern across the samples. Also, a similar observation is noted in Fig 4B for p-AMPK alpha. Can authors explain this anomaly? , 
 Comment #1: The Actin band in Fig 3Di and Fig 4 A are duplicated. Response: In this article, we used small number of human heart tissue sample to determine association of miR-133a with autophagy markers in the diabetic heart at end-stage heart failure. As this human tissue samples are precious and limited, we utilized the best possible way to get maximum outcome from these tissues. We measured ATG3 in Fig 3Di as a marker for autophagy. To evaluate upstream regulator mTOR, we reprobed the membrane with mTOR. Thus, we stripped and re-probed the Western blot membrane that was used for ATG3 (molecular weight 40kD) in Figure 3Di for mTOR (molecular weight 287-288kD) in Fig 4A. Thus, the beta-actin bands are same. Reprobing same Western blot membrane after stripping several times are not good because stripping causes loss of protein, which limits detection of protein. The detection of protein also depends on amount of loaded protein in SDS-PAGE gel. One to two stripping of membrane can be permissible if the detection of desired protein is not compromised. We observed prominent band of mTOR. Thus, it does not compromise the detection of protein. Comment # 2: Furthermore, phosphorylation of mTOR (p-mTOR) in Fig 4A raised concern due to significant unequal loading of Actin. Also, total mTOR is in question because of unequal pattern across the samples. Also, a similar observation is noted in Fig 4B for p-AMPK alpha. Response: Human samples demonstrate huge variability due to different demography, pathophysiological conditions, and treatments. Thus, there are variations in beta-actin, total mTOR and total AMPK. We presented these variabilities as they existed. To keep the integrity of the data and show the variability in the patient samples, we used one point for each sample and kept all the values of p-mTOR t-mTOR and p-AMPK t-AMPK in the bar graph in Figure 4A and 4B, respectively. For the same reason, we also kept the variability in beta-actin bands. It is different from the mouse tissue sample where variability is less and conditions are controlled. Here, I would like to clarify that we presented the results in fold change of ND (value 1). In brief, we divided the densitometric value of ATG3 with their corresponding value of beta-actin in all samples. We get the mean value of all the ND and D group. Then, we divided both ND and D group with ND value. The ND value becomes 1 and the D group was higher. Similarly, in Figure 4A, we divided pmTOR value with their corresponding t-mTOR value. Here, we presented each sample data with one point. The fold change is presented after dividing the entire sample with a particular ND sample, which is presented with the value of 1. The same applies for AMPK. It is germane to mention here that they did not achieve statistical significant. Several loading proteins have been used in literature including beta-actin. However, it is not clear which one is the best universal loading control. Thus, we did not invested our limited human tissue protein for standardizing equal loading of proteins. Moreover, we were not sure that beta-actin was the best loading control because there were huge variation in the loading even with the standard protein estimation. The recent guidelines on Western blotting suggest that using total lane protein is a better approach for normalizing protein . ,