Muscle RING Finger-1 Promotes a Maladaptive Phenotype in Chronic Hypoxia-Induced Right Ventricular Remodeling

Exposure to chronic hypoxia (CH) induces elevated pulmonary artery pressure/resistance, leading to an eventual maladaptive right ventricular hypertrophy (RVH). Muscle RING finger-1 (MuRF1) is a muscle-specific ubiquitin ligase that mediates myocyte atrophy and has been shown to play a role in left ventricular hypertrophy and altered cardiac bioenergetics in pressure overloaded hearts. However, little is known about the contribution of MuRF1 impacting RVH in the setting of CH. Therefore, we hypothesized that MuRF1 deletion would enhance RVH compared to their wild-type littermates, while cardiac-specific overexpression would reduce hypertrophy following CH-induced pulmonary hypertension. We assessed right ventricular systolic pressure (RVSP), right ventricle to left ventricle plus septal weight ratio (RV/LV+S) and hematocrit (Hct) following a 3-wk isobaric CH exposure. Additionally, we conducted dual-isotope SPECT/CT imaging with cardiac function agent 201Tl-chloride and cell death agent 99mTc-annexin V. Predictably, CH induced pulmonary hypertension, measured by increased RVSP, RV/LV+S and Hct in WT mice compared to normoxic WT mice. Normoxic WT and MuRF1-null mice exhibited no significant differences in RVSP, RV/LV+S or Hct. CH-induced increases in RVSP were also similar between WT and MuRF1-null mice; however, RV/LV+S and Hct were significantly elevated in CH-exposed MuRF1-null mice compared to WT. In cardiac-specific MuRF1 overexpressing mice, RV/LV+S increased significantly due to CH exposure, even greater than in WT mice. This remodeling appeared eccentric, maladaptive and led to reduced systemic perfusion. In conclusion, these results are consistent with an atrophic role for MuRF1 regulating the magnitude of right ventricular hypertrophy following CH-induction of pulmonary hypertension

Improved safety and efficacy of 213Bi-DOTATATE-targeted alpha therapy of somatostatin receptor-expressing neuroendocrine tumors in mice pre-treated with l-lysine

textabstractBackground: Targeted alpha therapy (TAT) offers advantages over current β-emitting conjugates for peptide receptor radionuclide therapy (PRRT) of neuroendocrine tumors. PRRT with 177Lu-DOTATATE or 90Y-DOTATOC has shown dose-limiting nephrotoxicity due to radiopeptide retention in the proximal tubules. Pharmacological protection can reduce renal uptake of radiopeptides, e.g., positively charged amino acids, to saturate in the proximal tubules, thereby enabling higher radioactivity to be safely administered. The aim of this preclinical study was to evaluate the therapeutic effect of 213Bi-DOTATATE with and without renal protection using L-lysine in mice. Tumor uptake and kinetics as a function of injected mass of peptide (range 0.03–3 nmol) were investigated using 111In-DOTATATE. These results allowed estimation of the mean radiation absorbed tumor dose for 213Bi-DOTATATE. Pharmacokinetics and dosimetry of 213Bi-DOTATATE was determined in mice, in combination with renal protection. A dose escalation study with 213Bi-DOTATATE was performed to determine the maximum tolerated dose (MTD) with and without pre-administration of l-lysine as for renal protection. Neutrophil gelatinase-associated lipocalin (NGAL) served as renal biomarker to determine kidney injury. Results: The maximum mean radiation absorbed tumor dose occurred at 0.03 nmol and the minimum at 3 nmol. Similar mean radiation absorbed tumor doses were determined for 0.1 and 0.3 nmol with a mean radiation absorbed dose of approximately 0.5 Gy/MBq 213Bi-DOTATATE. The optimal mass of injected peptide was found to be 0.3 nmol. Tumor uptake was similar for 111In-DOTATATE and 213Bi-DOTATATE at 0.3 nmol peptide. Lysine reduced the renal uptake of 213Bi-DOTATATE by 50% with no effect on the tumor uptake. The MTD was <13.0 ± 1.6 MBq in absence of l-lysine and 21.7 ± 1.9 MBq with l-lysine renal protection, both imparting an LD50 mean renal radiation absorbed dose of 20 Gy. A correlation was found between the amount of injected radioactivity and NGAL levels. Conclusions: The therapeutic potential of 213Bi-DOTATATE was illustrated by significantly decreased tumor burden and improved overall survival. Renal protection with l-lysine immediately prior to TAT with 213Bi-DOTATATE prolonged survival providing substantial evidence for pharmacological nephron blockade to mitigate nephrotoxicity

Muscle RING finger-1 promotes a maladaptive phenotype in chronic hypoxia-induced right ventricular remodeling.

Exposure to chronic hypoxia (CH) induces elevated pulmonary artery pressure/resistance, leading to an eventual maladaptive right ventricular hypertrophy (RVH). Muscle RING finger-1 (MuRF1) is a muscle-specific ubiquitin ligase that mediates myocyte atrophy and has been shown to play a role in left ventricular hypertrophy and altered cardiac bioenergetics in pressure overloaded hearts. However, little is known about the contribution of MuRF1 impacting RVH in the setting of CH. Therefore, we hypothesized that MuRF1 deletion would enhance RVH compared to their wild-type littermates, while cardiac-specific overexpression would reduce hypertrophy following CH-induced pulmonary hypertension. We assessed right ventricular systolic pressure (RVSP), right ventricle to left ventricle plus septal weight ratio (RV/LV+S) and hematocrit (Hct) following a 3-wk isobaric CH exposure. Additionally, we conducted dual-isotope SPECT/CT imaging with cardiac function agent 201Tl-chloride and cell death agent 99mTc-annexin V. Predictably, CH induced pulmonary hypertension, measured by increased RVSP, RV/LV+S and Hct in WT mice compared to normoxic WT mice. Normoxic WT and MuRF1-null mice exhibited no significant differences in RVSP, RV/LV+S or Hct. CH-induced increases in RVSP were also similar between WT and MuRF1-null mice; however, RV/LV+S and Hct were significantly elevated in CH-exposed MuRF1-null mice compared to WT. In cardiac-specific MuRF1 overexpressing mice, RV/LV+S increased significantly due to CH exposure, even greater than in WT mice. This remodeling appeared eccentric, maladaptive and led to reduced systemic perfusion. In conclusion, these results are consistent with an atrophic role for MuRF1 regulating the magnitude of right ventricular hypertrophy following CH-induction of pulmonary hypertension

Additional file 1: of Improved safety and efficacy of 213Bi-DOTATATE-targeted alpha therapy of somatostatin receptor-expressing neuroendocrine tumors in mice pre-treated with l-lysine

Improved safety and efficacy of 213Bi-DOTATATE targeted alpha therapy of somatostatin receptor-expressing neuroendrocrine tumors in mice pre-treated with L-lysine. (DOCX 31 kb

LFA-1 antagonist (BIRT377) similarly reverses peripheral neuropathic pain in male and female mice with underlying sex divergent peripheral immune proinflammatory phenotypes

Aim: The majority of preclinical studies investigating aberrant glial-neuroimmune actions underlying neuropathic pain have focused on male rodent models. Recently, studies have shown peripheral immune cells play a more prominent role than glial cells in mediating pathological pain in females. Here, we compared the onset and duration of allodynia in males and females, and the anti-allodynic action of a potentially novel therapeutic drug (BIRT377) that not only antagonizes the action of lymphocyte function-associated antigen-1 (LFA-1) to reduce cell migration in the periphery, but may also directly alter the cellular inflammatory bias.Methods: Male and female mice were subjected to peripheral nerve injury chronic constriction injury (CCI) applying two methods, using either 4-0 or 5-0 chromic gut suture material, to examine potential sex differences in the onset, magnitude and duration of allodynia. Hindpaw sensitivity before and after CCI and application of intravenous BIRT377 was assessed. Peripheral and spinal tissues were analyzed for protein (multiplex electrochemiluminescence technology) and mRNA expression (quantitative real-time PCR). The phenotype of peripheral T cells was determined using flow cytometry.Results: Sex differences in proinflammatory CCL2 and IL-1β and the anti-inflammatory IL-10 were observed from a set of cytokines analyzed. A profound proinflammatory T cell (Th17) response in the periphery and spinal cord was also observed in neuropathic females. BIRT377 reversed pain, reduced IL-1β and TNF, and increased IL-10 and transforming growth factor (TGF)-β1, also an anti-inflammatory cytokine, in both sexes. However, female-derived T cell cytokines are transcriptionally regulated by BIRT377, as demonstrated by reducing proinflammatory IL-17A production with concurrent increases in IL-10, TGF-β1 and the anti-inflammatory regulatory T cell-related factor, FOXP3.Conclusion: This study supports that divergent peripheral immune and neuroimmune responses during neuropathy exists between males and females. Moreover, the modulatory actions of BIRT377 on T cells during neuropathy are predominantly specific to females. These data highlight the necessity of including both sexes for studying drug efficacy and mechanisms of action in preclinical studies and clinical trials

Differential Induction of Cardiac Hypertrophy/Failure Transcripts in MuRF^-/- & <i>MuRF1 Tg+</i> Mice Following CH.

<p>Effects of CH-induced PH on relative RV transcript expression of <i>(A)</i> SM a-actin, <i>(B)</i> MyHC-b, <i>(C)</i> BNP and <i>(D)</i> mtND2 DNA content from <i>MuRF1 -/-</i> and MuRF1 Tg+ mice. BNP, MyHC-b and SM a-actin expression normalized to TATA binding protein. MtND2 DNA content normalized to 18S DNA. N = 5 per group, *p<0.05 vs respective normoxic control; #p<0.05 vs chronic hypoxia Tg⁺; **p<0.05 vs respective WT.</p

Relative perfusion of right ventricular myocardium, total heart, and skeletal muscle as a reference, as determined by SPECT/CT using ²⁰¹TlCl uptake.

<p>Region of interest phantoms are depicted in (A). Hypoxia caused a significant increase in perfusion in the RV (B) of both <i>MuRF1 -/-</i> and <i>MuRF1 Tg+</i> mice relative to RV mass (N = 5–8 per group; ** denotes <i>p</i><0.01 compared to normoxia mice by ANOVA). The RV perfusion was not consistently observable in controls, so results are pooled for all mouse models. The total heart uptake also showed an effect of hypoxia that was predominant in the <i>MuRF1 -/-</i> and <i>MuRF1 Tg+</i> mice relative to WT (C). Skeletal muscle perfusion (C) was elevated in hypoxia-exposed MuRF1 -/-mice but reduced in hypoxia-exposed <i>MuRF1 Tg+</i> mice. (B,C: N = 5–8 per group; * indicates <i>p</i><0.05 compared to normoxic conditions for the same strain; † indicates P<0.05 compared to WT).</p

WT C57BL/6 mouse response to hypoxia in terms of right ventricular systolic pressure (A), right ventricular hypertrophy (B), right ventricular expression of B-type natriuretic peptide mRNA (C) and right MuRF1 mRNA (D).

<p>Asterisks denote significant difference from normoxic controls (p<0.05) by two-tailed t-test (N = 7-8 per group).</p

Hypoxia-induced right ventricular remodeling is enhanced by MuRF1 deletion, while cardiac-specific MuRF1 overexpression leads to a maladaptive dilated phenotype.

<p>(A) Chronic hypoxia induced predictable increases in RV/LVS, more so in the <i>MuRF1 -/-</i> mice than in WT. Interestingly, cardiac-specific overexpression of this atrophy-mediating ubiquitin ligase had no protective effect in terms of net hypertrophy of the right ventricle and in fact led to exacerbation of hypoxia-induced RVH relative to WT (*significantly greater than all control groups, <i>p</i><0.01; **significantly greater than all control groups and WT hypoxia, <i>p</i><0.01; ***significantly greater than all other groups, <i>p</i><0.01). (B) Axial SPECT/CT cross-section images obtained at diastole reveal marked hypertrophy of the RV in hypoxia-exposed mice, with the appearance of dilation in the MuRF1 Tg+ mice. (C) Right ventricular weight to chamber volume ratio (D) ejection fraction and (E) RV systolic and diastolic volumes determined from ECG-gated SPECT/CT images are shown. As RV wall motion was difficult to image in normoxia mice, all genotypes are pooled. (N = 3-6 per group; *significantly lower than KO, Hypoxia mice, <i>p</i><0.05).</p