Differences Between UCn2 and UCn3
(A) Amino acid sequences of murine UCn2 and UCn3 show 42% homology. (B) Three-dimensional structure of human UCn2 and UCn3. (C) Murine UCn2 and UCn3 show small differences in receptor affinity, with UCn2 binding with somewhat higher affinity than UCn3 to CRHR2 receptor subtypes. (D) Vector maps for AAV8.CBA.mUCn2 and AAV8.CBAmUCn3. Single-strand vectors were constructed. (E) Plasma UCn2 and UCn3 before and after gene transfer. IV delivery of AAV8.UCn2 (UCn2), AAV8.UCn3 (UCn3) both at 1.9x10¹³ gc/kg, or saline was performed. Eight to 10 weeks later, blood was obtained and plasma UCn2 and UCn3 was measured. Data from individual mice are shown (mean ± SE). p Values from Student t test (2-tailed, unpaired). βGpA = β globin polyadenylation signal; CBA = chicken β-actin promoter; CMV.en = human cytomegalovirus enhancer; gc = genome copy; ITR = inverted terminal repeat; IV = intraventricular; UCn2 = urocortin-2; UCn3 = urocortin-3.

In Vivo Assessment of LV Function
Eight to 10 weeks after IV delivery of AAV8.UCn2 or AAV8.UCn3 (1.9 × 10¹³ gc/kg) or saline, mice underwent physiological studies to assess LV function. (A and B) Peak rate of LV pressure development (A) (+dP/dt) and the slope of the end-systolic pressure-volume relationship (ESPVR) (B), a relatively load-independent measurement of LV contractility, were both increased by UCn2 and UCn3 gene transfer, with no between-group differences. (C and D) Peak rate of LV pressure decline (−dP/dt) (C) and the time constant of LV pressure decline (Tau) (D), measures of LV diastolic function, were both increased by UCn2 and UCn3 gene transfer, with no between-group differences. (E) Cardiac output (CO) was increased by UCn2 and UCn3 gene transfer, with no difference between UCn2 and UCn3. (F) LV developed pressure (LVP) showed no group differences. (G) Heart rate (HR) in anesthetized animals was increased by UCn2 and UCn3 gene transfer, with no between-group differences. Individual data are shown (mean ± SE); p values are from 1-way analysis of variance (ANOVA). LV = left ventricle; LVP = left ventricular developed pressure; other abbreviations as in .

Cytosolic Ca²⁺ Transients in Cardiac Myocytes
Cytosolic Ca²⁺ transients were measured in cardiac myocytes isolated from mice 9.0 ± 0.2 weeks after IV delivery of AAV8.UCn2 or AAV8.UCn3 (both at 1.9x10¹³ gc/kg) or saline (control). (A) Representative Indo-1 Ca²⁺ transient recordings from cardiac myocytes from 1 heart in each group. (B) Mean peak Ca²⁺ transients from multiple cardiac myocytes from each group, showing increased peak Ca²⁺ transients in cardiac myocytes isolated form mice following UCn2 and UCn3 gene transfer. There was no group difference between UCn2 and UCn3. (C) Time constant of cytosolic Ca²⁺ decline (Tau) showing reduced Tau (more rapid decline) in cardiac myocytes isolated from mice following UCn2 and UCn3 gene transfer. There was no group difference between UCn2 and UCn3. (D) Time-to-peak cytosolic Ca²⁺ concentration showed no group differences. In B to D, summary data are from cardiac myocytes isolated from 4 mice per group; numbers in bars denote the number of cardiac myocytes per group; p values are from 1-way ANOVA. Bars denote mean and SE. Abbreviations as in and .

Biochemical and Molecular Effects of UCn2 and UCn3 Gene Transfer on LV Samples
LV transmural samples obtained 9.0 ± 0.2 weeks after IV delivery of AAV8.UCn2 or AAV8.UCn3 (both at 1.9 × 10¹³ gc/kg), or saline (control). (A) Basal cAMP production showed no group differences. (B) Basal PKA activity showed no group differences. (C) Phosphorylated CaMKIIα-Thr286 protein levels showed group differences: UCn2 and UCn3 gene transfer reduced CaMKIIα phosphorylation to similar degrees. (D) Phosphorylated ryanodine receptor 2 protein levels showed group differences, solely attributable to reduction conferred by UCn2 gene transfer (p = 0.01 vs. UCn3, Dunn’s multiple comparison test with correction). (E) LV SERCA2a protein levels were increased similarly by UCn2 and UCn3 gene transfer. Individual data are shown (mean ± SE); p values are from Kruskal-Wallis test. Abbreviations as in .

Glucose Disposal
Normal mice received IV saline (n = 12), AAV8.UCn2 (1.9 × 10¹³ gc/kg, n = 10), or AAV8.UCn3 (1.9 × 10¹³ gc/kg, n = 12). Eight to 10 weeks later, fasting (12-h) glucose was determined, and glucose tolerance testing was performed. (A) A group difference in fasting blood glucose was detected (ANOVA, p = 0.0002). Mice that had received UCn2 gene transfer showed reduced fasting glucose (p < 0.001 vs. control; p < 0.001 vs. UCn3). Individual mouse data are shown, and mean ± SE are indicated. (B) Glucose tolerance testing showed a difference in glucose clearance (ANOVA, p = 0.015). The area under the glucose time curve was reduced in animals that had received UCn2 gene transfer (p = 0.02 vs. control; p < 0.02 vs. UCn3). Between-group comparisons are from Sidak multiple comparison test with correction. Abbreviations as in .

Histological Analysis of LV and Liver
(A) Hematoxylin and eosin and Masson's trichrome staining were performed on liver and transmural sections of LV (original magnification 20×; presented image magnification 7×) 9.0 ± 0.2 weeks after intravenous injection of saline, AAV8.UCn2 (1.9 × 10¹³ gc/kg) and AAV8.UCn3 (2 × 10¹³ gc/kg). (B) Evaluation of fibrosis was performed using ImageJ software. AAV8.UCn2 and AAV8.UCn3 delivery were not associated with histological abnormalities. Standard is the control for Masson's trichrome. Con = control; other abbreviations as in and .

Cardiac Myocyte Volume
Cardiac myocytes were isolated 9.0 ± 0.2 weeks after intravenous delivery of saline, AAV8.UCn2 (1.9 × 10¹³ gc/kg) or AAV8.UCn3 (1.9 × 10¹³ gc/kg). Viable isolated cardiac myocytes were fixed in 10% formalin, stained with eosin and their size was measured using ImageJ software. (A) Each symbol represents the mean volume of 120 ± 11 cardiac myocytes measured per mouse. Measures were made blinded to group identity. (B) Representative isolated cardiac myocytes, indicating cardiac myocyte length (red) and width (green). There was an overall group difference in cardiac myocyte volume (p < 0.02, Kruskal-Wallis) due to a larger volume in cardiac myocytes from control mice. Between-group comparisons are from the Dunn multiple comparison test. Abbreviations as in , , and .