Interaction between age and obesity on cardiomyocyte contractile function: role of leptin and stress signaling

Abstract

Objectives: This study was designed to evaluate the interaction between aging and obesity on cardiac contractile and intracellular Ca2+ properties.

Methods: Cardiomyocytes from young (4-mo) and aging (12- and 18-mo) male lean and the leptin deficient ob/ob obese mice were treated with leptin (0.5, 1.0 and 50 nM) for 4 hrs in vitro. High fat diet (45% calorie from fat) and the leptin receptor mutant db/db obesity models at young and older age were used for comparison. Cardiomyocyte contractile and intracellular Ca2+ properties were evaluated including peak shortening (PS), maximal velocity of shortening/relengthening (+/- dL/dt), time-to-PS (TPS), time-to-90% relengthening (TR(90)), intracellular Ca2+ levels and decay. O2(-) levels were measured by dihydroethidium fluorescence.

Results: Our results revealed reduced survival in ob/ob mice. Aging and obesity reduced PS, +/- dL/dt, intracellular Ca2+ rise, prolonged TR(90) and intracellular Ca2+ decay, enhanced O2(-) production and p(47phox) expression without an additive effect of the two, with the exception of intracellular Ca2+ rise. Western blot analysis exhibited reduced Ob-R expression and STAT-3 phosphorylation in both young and aging ob/ob mice, which was restored by leptin. Aging and obesity reduced phosphorylation of Akt, eNOS and p38 while promoting pJNK and pIkappaB. Low levels of leptin reconciled contractile, intracellular Ca2+ and cell signaling defects as well as O2(-) production and p(47phox) upregulation in young but not aging ob/ob mice. High level of leptin (50 nM) compromised contractile and intracellular Ca2+ response as well as O2(-) production and stress signaling in all groups. High fat diet-induced and db/db obesity displayed somewhat comparable aging-induced mechanical but not leptin response.

Conclusions: Taken together, our data suggest that aging and obesity compromise cardiac contractile function possibly via phosphorylation of Akt, eNOS and stress signaling-associated O2(-) release.

Figure 1. Cumulative survival curve (Kaplan-Meier survival plot) of male C57 lean and ob/ob obese mice.

The cumulative survival rate was plotted against age in months. The Log rank test was performed to compare the two mouse groups (p = 0.0007). n = 26 and 16 mice for C57 and ob/ob mice, respectively.

Figure 2. Light microscopic images of cardiomyocytes freshly isolated from young (4-month-old) and aging (12- or 18-month-old) lean (C57) and ob/ob mice.

200x, scale bar  = 100 µm.

Figure 3. Contractile properties of cardiomyocytes freshly isolated from young (4-month-old) and aging (12- or 18-month-old) lean (C57) and ob/ob mice treated with or without leptin (0.5, 1.0 and 50 nM) for 4 hrs.

A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening (TPS); F: Time-to-90% relengthening (TR₉₀); Mean ± SEM, n = 50–53 cells from 3 mice per group, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Figure 4. Intracellular Ca²⁺ transient properties of cardiomyocytes freshly isolated from young (4-month-old) and aging (12- or 18-month-old) lean (C57) and ob/ob mice treated with or without leptin (0.5, 1.0 and 50 nM) for 4 hrs.

A: Resting intracellular Ca²⁺ fluorescence intensity; B: Rise in intracellular Ca²⁺ fluorescence intensity in response to electrical stimuli; C: Single-exponential Ca²⁺ transient decay rate and D: Bi-exponential Ca²⁺ transient decay rate. Mean ± SEM, n = 36–38 cells from 3 mice per group, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Figure 5. O₂ ⁻ production (Panel A) and p ^{47 phox} NADPH oxidase subunit expression (Panel B) measured by DHE fluorescence and immunoblotting, respectively, in cardiomyocytes freshly isolated from young (4-month-old) or aging (12-month-old) lean (C57) and ob/ob mice treated with or without leptin (0.5, 1.0 and 50 nM) for 4 hrs.

Insets: Representative gel blots of p ^47phox NADPH oxidase subunit using specific anti-p ^47phox antibody. GAPDH was used as the loading control. Mean ± SEM, n = 12–14 (Panel A) and 9–11 (Panel B) per group, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Figure 6. The leptin receptor Ob-R expression (Panel A) and phosphorylation of the leptin receptor downstream signaling molecule STAT-3 (pSTAT3, Panel B) in cardiomyocytes freshly isolated from young (4-month-old) or aging (12-month-old) lean (C57) and ob/ob mice treated with or without leptin (1.0 and 50 nM) for 4 hrs.

Protein expression of Ob-R and pSTAT-3 was normalized to the loading control GAPDH or total STAT-3, respectively. Insets: Representative gel blots of Ob-R, pSTAT-3 and STAT-3 proteins using specific antibodies. Mean ± SEM, n = 3 – 6 isolations, * p<0.05 vs. respective C57 group, # p<0.05 vs. respective ob/ob group.

Figure 7. Panel A: Representative gel blots of total and phosphorylated Akt, eNOS and AMPK in cardiomyocytes freshly isolated from young (4-month-old) or aging (12-month-old) lean (C57) and ob/ob mice treated with or without leptin (1.0 and 50 nM) for 4 hrs using specific antibodies; Panel B: Phosphorylation of Akt expressed as pAkt-to-Akt ratio; Panel C: Phosphorylation of eNOS expressed as peNOS-to-eNOS ratio; and Panel D: Phosphorylation of AMPK expressed as pAMPK-to-AMPK ratio.

Mean ± SEM, n = 4 – 6 isolations, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Figure 8. Total and phosphorylated protein expression of p38 MAP kinase, JNK, ERK and IκB in cardiomyocytes isolated from young (4-month-old) or aging (12-month-old) lean (C57) and ob/ob mice treated with or without leptin (1.0 and 50 nM) for 4 hrs.

Panel A: Phosphorylation of p38 expressed as pp38-to-p38 ratio; Panel B: Phosphorylation of JNK expressed as pJNK-to-JNK ratio; Panel C: Phosphorylation of ERK expressed as pERK-to-ERK ratio; and Panel D: Phosphorylation of IκB expressed as p IκB-to-IκB ratio. Insets: Representative gel blots of total and phosphorylated p38, JNK, ERK and IκB proteins using specific antibodies. Mean ± SEM, n = 4–8 isolations, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 group, # p<0.05 vs. respective ob/ob group.

Figure 9. Contractile properties of cardiomyocytes isolated from young (4-month-old) and aging (12-month-old) male C57 mice fed a low (10%) or high (45%) fat diet for 16 weeks.

Cohorts of cardiomyocytes were treated with or without leptin (1.0 nM) for 4 hrs prior to mechanical study. A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening (TPS); F: Time-to-90% relengthening (TR₉₀); Mean ± SEM, n = 50–51 cells from 3 mice per group, * p<0.05 vs. respective low fat group, ** p<0.05 vs. young low fat group.

Figure 10. Contractile properties of cardiomyocytes isolated from young (4-month-old) and aging (12- or 18-month-old) male C57 lean and the leptin receptor-deficient db/db obese mice.

A: Resting cell length; B: Peak shortening (PS, normalized to cell length); C: Maximal velocity of shortening (+ dL/dt); D: Maximal velocity of relengthening (- dL/dt); E: Time-to-peak shortening (TPS); F: Time-to-90% relengthening (TR₉₀); Mean ± SEM, n = 102–103 cells from 3 mice per group, * p<0.05 vs. respective C57 group, ** p<0.05 vs. young C57 (4-month) group, # p<0.05 vs. young db/db (4-month) group.