Hypothermia/rewarming disrupts excitation-contraction coupling in cardiomyocytes

Abstract

Hypothermia/rewarming (H/R) is poorly tolerated by the myocardium; however, the underlying intracellular basis of H/R-induced cardiac dysfunction remains elusive. We hypothesized that in cardiomyocytes, H/R disrupts excitation-contraction coupling by reducing myofilament Ca(2+) sensitivity due to an increase in cardiac troponin I (cTnI) phosphorylation. To test this hypothesis, isolated rat cardiomyocytes (13-15 cells from 6 rats per group) were electrically stimulated to evoke both cytosolic Ca(2+) ([Ca(2+)]cyto) and contractile (sarcomere shortening) responses that were simultaneously measured using an IonOptix system. Cardiomyocytes were divided into two groups: 1) those exposed to hypothermia (15°C for 2 h) followed by rewarming (35°C; H/R); or 2) time-matched normothermic (35°C) controls (CTL). Contractile dysfunction after H/R was indicated by reduced velocity and extent of sarcomere length (SL) shortening compared with time-matched controls. Throughout hypothermia, basal [Ca(2+)]cyto increased and the duration of evoked [Ca(2+)]cyto transients was prolonged. Phase-loop plots of [Ca(2+)]cyto vs. contraction were shifted rightward in cardiomyocytes during hypothermia compared with CTL, indicating a decrease in Ca(2+) sensitivity. Using Western blot, we found that H/R increases cTnI phosphorylation. These results support our overall hypothesis and suggest that H/R disrupts excitation-contraction coupling of cardiomyocytes due to increased cTnI phosphorylation and reduced Ca(2+) sensitivity.

Discontinued stimulation of cardiomyocytes provides protection against hypothermia–rewarming‐induced disruption of excitation–contraction coupling

“…The measurements were previously described in detail (Schaible et al., ). Briefly, isolated cardiomyocytes were loaded with 1 μ m fura‐2 AM in 0.1% DMSO for 5–10 min at 35°C.…”

“…After this loading, the cardiomyocytes were washed two times and then perfused with Ca 2+ Tyrode solution at 35°C aerated with 95% O 2 and 5% CO 2 while undergoing continuous stimulation. To measure simultaneously the cytosolic Ca 2+ concentration ([Ca 2+ ] i ) and SL shortening as contractile responses using an IonOptix system (Mellor et al., ; Schaible et al., ), [Ca 2+ ] i (fura‐2 fluorescence ratio; 340/380 nm) and SL shortening of isolated cardiomyocytes were elicited by continuous electrical stimulation at a frequency of 0.5 Hz. Figure illustrates an experimental protocol showing the three experimental steps; prehypothermia, 2 h of hypothermia, post‐hypothermia and time‐matched control.…”

“…Protein samples were solubilized on ice by sonication for three 15 s intervals. Samples were then spun in a 4°C centrifuge at 12,000 g for 15 min (Schaible et al., ). The Lowry assay was used to assay protein concentration.…”

“…Previous preclinical studies, in which intact animal models were exposed to experimental H/R, have shown significantly impaired myocardial contractile function after rewarming (Han, Tveita, Prakash, & Sieck, ; Kondratiev, Wold, Aasum, & Tveita, ; Tveita, Skandfer, Refsum, & Ytrehus, ). Our recent study demonstrated that H/R‐induced contractile dysfunction in cardiomyocytes is attributable to a decline in Ca 2+ sensitivity of contraction, which is associated with increased cardiac troponin I (cTnI) phosphorylation (p‐cTnI; Schaible et al., ). The intracellular mechanism responsible for this alteration appears to be closely related to an increase in protein kinase A (PKA) activation, in addition to an increase in basal [Ca 2+ ] i levels (Ca 2+ overload) and a prolongation of [Ca 2+ ] i responses to stimulation that occur during hypothermia (Schaible et al., ).…”

“…Our recent study demonstrated that H/R‐induced contractile dysfunction in cardiomyocytes is attributable to a decline in Ca 2+ sensitivity of contraction, which is associated with increased cardiac troponin I (cTnI) phosphorylation (p‐cTnI; Schaible et al., ). The intracellular mechanism responsible for this alteration appears to be closely related to an increase in protein kinase A (PKA) activation, in addition to an increase in basal [Ca 2+ ] i levels (Ca 2+ overload) and a prolongation of [Ca 2+ ] i responses to stimulation that occur during hypothermia (Schaible et al., ).…”