Protective action of doxycycline against diabetic cardiomyopathy in rats

Abstract

Background and purpose: Reactive oxygen and nitrogen species play an important role in the development of diabetic cardiomyopathy. They can activate matrix metalloproteinases (MMPs), and MMP-2 in particular is known to mediate early consequences of oxidative stress injury in the heart. Therefore, we investigated the role of MMP-2 and the effect of the MMP inhibitor doxycycline on the changes of heart function caused by diabetes.

Experimental approach: Using streptozotocin-induced diabetic rats, we evaluated the effect of doxycycline on both mechanical and electrical function of isolated hearts, papillary muscle and cardiomyocytes.

Key results: Doxycycline abolished the diabetes-induced depression in left ventricular developed pressure and the rates of changes in developed pressure in isolated hearts and normalized the prolongation of the action potential in papillary muscles. In cardiomyocytes isolated from doxycycline-treated diabetic rats, the altered kinetic parameters of Ca(2+) transients, depressed Ca(2+) loading of sarcoplasmic reticulum and basal intracellular Ca(2+) level, and the spatio-temporal properties of Ca(2+) sparks were significantly restored. Gelatin zymography and western blot data indicated that the diabetes-induced alterations in MMP-2 activity and protein level, level of tissue inhibitor of matrix metalloproteinase-4 and loss of troponin I were restored to control levels with doxycycline.

Conclusions and implications: Our data suggest that these beneficial effects of doxycycline on the mechanical, electrical and biochemical properties of the diabetic rat heart appear, at least in part, to be related to inhibition of MMP activity, implying a role for MMPs in the development of diabetic cardiomyopathy.

Figure 1

Electron micrographs of left ventricular heart muscle sections from control, diabetic and doxycycline-treated diabetic rats. (a) Regularly arranged myofilaments and Z-lines (Z) in myofibres and mitochondria (M), rich in cristae, were observed in control hearts. (b) Diabetes-induced changes in myofilaments and the Z-lines of myofibres, degeneration of myofibrils and destruction and loss of myofibrils over sarcomere units. Mitochondria showed loss of cristae and a granular matrix. There were also increased numbers of lipid droplets (L). (c) Doxycycline treatment normalized alterations in myofilaments, Z-lines and mitochondria. Scale bar: 2 μm.

Figure 2

Effect of doxycycline on mechanical function of hearts and action potential kinetics of papillary muscle strips isolated from diabetic rats. Despite no changes in LVEDP, both the reductions in LVDP (a) and ±dP/dt (b) induced by diabetes were abolished by doxycycline. The diabetes-induced increase in action potential duration (APD) was also restored by doxycycline (c). Bar graphs represent mean±s.e.mean values from control (C; n_heart=12, n_papillary=8); diabetic (DM; n_heart=16, n_papillary=8); doxycycline-treated diabetic (Doxy; n_heart=10, n_papillary=9) groups. ^*P<0.01 vs control, ^†P<0.01 vs diabetic, analysis of variance.

Figure 3

Effect of doxycycline on K⁺ currents in ventricular cardiomyocytes isolated from diabetic rat hearts. (a) Current–voltage (I–V) relationships of peak current densities (I_to and I_K1) in cardiomyocytes isolated from control (C; n=22 cells from five rats), diabetic (DM; n=30 cells from six rats) and doxycycline-treated diabetic groups (Doxy; n=24 cells from five rats). Voltage pulses were applied from a holding potential of 80 mV to between 120 and +70 mV, with 10 mV steps. The current densities were estimated for I_K1 at −120 mV, and for I_to, which was estimated as the difference between peak and steady state currents (Nagase et al.) elicited at +70 mV. Doxycyline significantly recovered I_K1 and I_to density. (b) I–V characteristics of I_ss were also improved in doxycycline-treated diabetic rats. Data points on the graphs represent mean±s.e.mean values.

Figure 4

Doxycycline restored the depressed intracellular Ca²⁺ homoeostasis transients in diabetic rat ventricular cardiomyocytes. (a) Intracellular Ca²⁺ transients measured through Fura-2 under voltage-clamp conditions (upper part) and I–V relationships of I_CaL density (lower part) were obtained from control (C; n=35 myocytes from 11 hearts), diabetic (DM; n=33 myocytes from seven hearts) and doxycycline-treated diabetic (Doxy; n=37 myocytes from seven hearts) groups (between −50 and +60 mV with 10 mV increments). (b) Representative I_CaL traces (upper part) and Ca²⁺ transients (lower part) recorded at 0 mV depolarization potential from the experimental groups. (c) Changes of basal fluorescence intensity indicating intracellular basal Ca²⁺ levels of the groups. Data points on the graphs and bar graphs represent mean± s.e.mean values. ^*P<0.01 vs control, ^†P<0.01 vs diabetic, analysis of variance.

Figure 5

Doxycycline restored diabetes-induced alterations of Ca²⁺ sparks parameters in cardiomyocytes. (a) Line-scan images with spontaneously arising Ca²⁺ sparks recorded using Fluo-3AM in representative cardiomyocytes from control (C; n=30 myocytes from six hearts), diabetic (DM; n=27 myocytes from five hearts) and doxycycline-treated diabetic rats (Doxy; n=28 myocytes from five hearts). (b) Averaged spatial and temporal values of Ca²⁺ sparks. Time-to-peak amplitude (TP; left-hand axis) and half-decay time (DT_50; right-hand axis), of Ca²⁺ sparks were improved by doxycycline. Diabetes itself did not affect peak amplitude (Max; right-hand graph). (c) Left: Comparison of Ca²⁺ sparks frequency. Right: Spatial distribution of the sparks represented as Ca²⁺ sparks width at half maximal amplitude (FWHM). Bar graphs represent mean±s.e.mean values. ^*P<0.01 vs control, ^†P<0.01 vs diabetic, analysis of variance.

Figure 6

Effect of doxycycline on MMP-2 and troponin I in diabetic hearts. Heart homogenates were assayed for MMP-2 activity by gelatin zymography (a), or MMP-2 (b), TIMP-4 (c) and TnI (d) protein levels by Western blotting. Upper panels show representative gelatin zymogram (a) and Western blots (b–d) bottom panels show the summary of the densitometric analysis. C: control; DM: diabetic; Doxy: doxycycline-treated diabetic. Bar graphs represent mean±s.e.mean values. ^*P<0.05 vs control (n=3–6), analysis of variance.