Human ventricular unloading induces cardiomyocyte proliferation

Abstract

Background: The adult mammalian heart is incapable of meaningful regeneration after substantial cardiomyocyte loss, primarily due to the inability of adult cardiomyocytes to divide. Our group recently showed that mitochondria-mediated oxidative DNA damage is an important regulator of postnatal cardiomyocyte cell cycle arrest. However, it is not known whether mechanical load also plays a role in this process. We reasoned that the postnatal physiological increase in mechanical load contributes to the increase in mitochondrial content, with subsequent activation of DNA damage response (DDR) and permanent cell cycle arrest of cardiomyocytes.

Objectives: The purpose of this study was to test the effect of mechanical unloading on mitochondrial mass, DDR, and cardiomyocyte proliferation.

Methods: We examined the effect of human ventricular unloading after implantation of left ventricular assist devices (LVADs) on mitochondrial content, DDR, and cardiomyocyte proliferation in 10 matched left ventricular samples collected at the time of LVAD implantation (pre-LVAD) and at the time of explantation (post-LVAD).

Results: We found that post-LVAD hearts showed up to a 60% decrease in mitochondrial content and up to a 45% decrease in cardiomyocyte size compared with pre-LVAD hearts. Moreover, we quantified cardiomyocyte nuclear foci of phosphorylated ataxia telangiectasia mutated protein, an upstream regulator of the DDR pathway, and we found a significant decrease in the number of nuclear phosphorylated ataxia telangiectasia mutated foci in the post-LVAD hearts. Finally, we examined cardiomyocyte mitosis and cytokinesis and found a statistically significant increase in both phosphorylated histone H3-positive, and Aurora B-positive cardiomyocytes in the post-LVAD hearts. Importantly, these results were driven by statistical significance in hearts exposed to longer durations of mechanical unloading.

Conclusions: Prolonged mechanical unloading induces adult human cardiomyocyte proliferation, possibly through prevention of mitochondria-mediated activation of DDR.

Keywords: DNA damage response; heart regeneration; heart failure; mechanical unloading; ventricular assist device.

Figure 1. LVAD Support for Heart Failure Patients Leads to Decreased Mitochondrial DNA Copy Number and Cardiomyocyte Cell Size

(A) Samples from various heart failure patients with different durations of LVAD support were used for the study. (B) Quantitative polymerase chain reaction analysis showed that in both groups 1 (short LVAD duration; 6 months or less) and 2 (long LVAD duration; longer than 6 months), the mitochondrial DNA copy number was significantly decreased compared with the nuclear DNA copy number in post-LVAD supported hearts versus pre-LVAD supported hearts. (C) Cardiomyocyte cell size analyzed by immunostaining using anti-WGA and anti-cardiac TnT antibodies showed a marked decrease in cardiomyocyte size in the overall population as well as in groups 1 and 2. AA = African American; CABG = coronary artery bypass grafting; CM = cardiomyopathy; LVAD = left ventricular assist device; TnT = troponin T; W = white; WGA = wheat germ agglutinin.

Figure 2. LVAD-Mediated Pressure Unloading Leads to Reduced DDR in Adult Human Cardiomyocytes

High-resolution confocal microscopy of immunostaining with pATM antibody shows a significant decrease in nuclear pATM foci and the DDR only in longer duration group 2 post-LVAD cardiomyocytes. DAPI = 2-(4-amidinophenyl)-1H-indole-6-carboxamidine; DDR = DNA damage response; LVAD = left ventricular assist device; pATM = phosphorylated ataxia telangiectasia mutated protein.

Figure 3. LVAD-Mediated Pressure Unloading Induces Cardiomyocyte Proliferation in Adult Human Heart

(A) Confocal z-stack imaging after pH3 antibody staining showed a significant increase in cardiomyocyte mitosis in the longer duration group 2 LVAD hearts. Scale bar=5 μm. Note that stringent criteria were used for localization of pH3 staining to cardiomyocytes. True- and false-positive examples are provided. (B) Confocal z-stack imaging of Aurora B kinase showed a marked increase in cardiomyocyte cytokinesis in the longer duration group 2 LVAD hearts. Scale bar = 100 μm. Note the localization of Aurora B kinase to the cleavage following between 2 cardiomyocytes (right inset). cTnT = cardiac troponin T, PH3 = phosphorylated histone H3; other abbreviations as in Figure 2.

Central Illustration. Cardiomyocyte Proliferation in LVAD Patients: Prolonged Mechanical Unloading Results in a Switch From Hypertrophic to Hyperplastic Cardiomyocyte Growth

Both mtDNA content (A) and cell size (B) markedly decreased post-LVAD in the combined samples. The DNA damage response was not significantly decreased in the combined samples post-LVAD, as shown by measurements of pixels/area (C) and by the number of pATM protein foci per myocyte in the combined samples. Post-LVAD, cardiomyocyte mitosis, shown by increased pH3-positive cardiomyocytes (D), and cardiomyocyte cytokinesis, shown by increased Aurora B localization to cytokinetic furrows (E), were both significantly increased in the combined samples. Collectively, these results suggest that mechanical unloading results in cardiomyocyte cell cycle re-entry. LVAD = left ventricular assist device; mtDNA = mitochondrial DNA; pATM = phosphorylated ataxia telangiectasia mutated protein; PH3 = phosphorylated histone H3.