Genetically determined mitochondrial preservation and cardioprotection against myocardial ischemia-reperfusion injury in a consomic rat model

Abstract

Cardioprotection may be genome dependent. One example is the increased tolerance to cardiac ischemia-reperfusion (IR) in Brown Norway (BN) compared with Dahl salt-sensitive (SS) rats. By narrowing the genetic difference to chromosome 6 only, we found the consomic SS(6BN) to be similarly IR tolerant as BN. We hypothesized that better preserved mitochondrial structure and function are genetically determined and therefore critically linked to myocardial IR tolerance associated with BN chromosome 6. Langendorff-prepared BN, SS, and SS(6BN) rat hearts were subjected to IR, while corresponding controls were continuously perfused. Though largely equal in nonischemic controls, assessment of functional data and ventricular infarct size in IR experiments confirmed that BN and SS(6BN) have an equally higher tolerance to IR than SS hearts. This was complemented by equally better preserved mitochondrial structure, oxidative phosphorylation, and calcium retention capacity in BN and SS(6BN) vs. SS hearts. For the first time, our data indicate that SS(6BN) are as resistant to IR injury as BN hearts in mitochondrial and myocardial function and viability compared with SS hearts. These findings not only link myocardial and mitochondrial protection in a genetic model but also suggest that genetic information on rat chromosome 6 is critical for mitochondrial preservation and IR tolerance.

Keywords: Brown Norway; Dahl salt sensitive; SS6BN; myocardium.

Fig. 1.

Experimental protocols. Isolated, perfused hearts (n = 90) from 8 wk old male Brown Norway (BN), Dahl salt-sensitive (SS), and consomic (SS^6BN) rats were stabilized for 20 min. After baseline (bl) readings nonischemic control hearts were perfused for 185 min, whereas ischemia-reperfusion (IR) hearts were perfused for 35 min before they underwent 30 min global no-flow ischemia followed by 120 min reperfusion (RP). Ventricular infarct size (IS) was determined by TTC staining and cumulative planimetry (n = 6 per group). When Western blots (n = 3 per group) or functional experiments with isolated mitochondria (n = 6 per group) were performed, hearts were perfused for only 95 min or harvested after only 30 min reperfusion.

Fig. 2.

Functional data expressed as % of their baseline (bl) values (unless otherwise indicated) and ventricular infarct size in % after 35 min perfusion, 30 min ischemia, and 120 min reperfusion (IR, right half of each panel), or 185 min of perfusion (Con, left half of each panel) following stabilization. A: systolic left ventricular pressures (LVP); B: diastolic LVP (in mmHg); C: developed, i.e., systolic-diastolic, LVP; D: heart rate (HR); E and F: dLVP/dt_max and dLVP/dt_min, as indexes of contractility and relaxation, respectively; G: coronary flow; H: ventricular infarct size in the 3 strains BN (white), SS (black), and SS^6BN (striped). All values are means ± SE; *vs. Con, †vs. BN, ‡vs. SS^6BN; n = 6 per group.

Fig. 3.

Original Western blot data for complex I subunit NDUFA9 and complex IV subunit 1 as a loading control are shown in A. The summary of NDUFA9 levels in % BN Con is shown in B for BN (white), SS (black), and consomic SS^6BN rats (striped) after 35 min perfusion, 30 min ischemia, and 120 min reperfusion (IR, right half of B) or 185 min of perfusion (Con, left half of B) following stabilization. Complex IV subunit 1 was not different among the 6 groups (data not shown). All values are means ± SE; *vs. Con, †vs. BN, ‡vs. SS^6BN; n = 3 per group.

Fig. 4.

Mitochondrial respiration and respiratory control index (RCI). A–C: representative traces of polarographically measured chamber [O₂] concentrations (in μM) of mitochondria isolated from a nonischemic control (Con) vs. mitochondria isolated after IR from BN (A), SS^6BN (B), and SS hearts (C). Following 1 min stabilization, the complex I substrates pyruvate and malate (10 mM each) were added to initiate state 2 respiration. State 3 respiration was determined after addition of adenosine diphosphate (ADP, 250 μM) 60 s later, and state 4 respiration after complete ADP phosphorylation to ATP. RCI was calculated as the state 3-to-state 4 ratio. Results from 3 replicates per heart were averaged. D: summary of RCIs in Con (left) and IR mitochondria (right) from BN (white), SS (black), and the consomic SS^6BN strain (striped). All values are means ± SE; *vs. Con, †vs. BN, ‡vs. SS^6BN; n = 6 per group.

Fig. 5.

Mitochondrial calcium retention capacity. A: representative traces of rhodamine 123 fluorescence (in arbitrary fluorescence units, afu), indicative of membrane potential (ΔΨ_m), in mitochondria isolated from BN, SS^6BN, and SS hearts following IR in the presence of 10 mM succinate. Arrows indicate when CaCl₂ pulses (to yield 25 μM increases) were added every 60 s until ΔΨ_m depolarization. After CaCl₂-induced depolarization, 4 μM carbonyl cyanide-m-chlorophenylhydrazenone (CCCP), a mitochondrial uncoupler, was added for maximal depolarization. B: summary of the time until depolarization (in s) in Con (left) and IR mitochondria (right) from BN (white), SS (black), and the consomic SS^6BN strain (striped). All values are means ± SE; *vs. Con, †vs. BN, ‡vs. SS^6BN; n = 6 per group.