In isolated cardiomyocytes, we found that simulated cardioplegia limited the development of intracellular Ca
2+ overload. However, diastolic Ca
2+ levels still rose above baseline values by the end of the 45 minute ischemic period. In cells arrested with cardioplegia containing SEA0400, Ca
2+ overload was further reduced supporting the concept that reverse mode NCX exchange does play a role in Ca
2+ homeostasis in the cardioplegia-arrested cell. This was associated with evidence of improved myocardial protection in the whole heart including lower troponin release during reperfusion, preserved mitochondrial morphology, and better functional recovery. We hypothesize that in the whole heart, the addition of SEA0400 to cardioplegia prevents death of a proportion of cardiomyocytyes resulting in the lower troponin release observed. Furthermore, limitation of Ca
2+ overload protects the mitochondria in surviving cells as evidenced by preserved mitochondrial morphology. These two features may both contribute to improved functional recovery in hearts arrested with cardioplegia containing SEA0400. In isolated cardiomyocytes we also found that while diastolic Ca
2+ levels returned quickly to baseline values during the reperfusion period, systolic Ca
2+ levels were somewhat higher in the cells protected with cardioplegia containing SEA0400. The cause for this is not clear but one possibility includes a persistent blockade of NCX during reperfusion. While SEA0400 preferentially blocks the reverse mode of NCX, it also blocks the forward mode to some degree [
8,
25] which, during reperfusion, may impede NCX’s normal physiologic function of removing Ca
2+ from the cell after each contraction cycle. This could favor reuptake of Ca
2+ by the sarcoplasmic reticulum resulting in larger amplitude Ca
2+ induced Ca
2+ release and higher systolic Ca
2+ levels seen. While very high intracellular Ca
2+ levels during reperfusion can lead to damaging hypercontracture, we did not see this in these cells. The higher systolic Ca
2+ levels seen during reperfusion in cells protected with cardiolpegia containing SEA0400 resulted in larger contractions but no evidence of reduced diastolic cell length (data not shown).
We examined only one dose level of SEA0400 (1 uM), which was chosen based on previous studies that demonstrated relatively specific targeting of the reverse mode NCX activity (Ca
2+ influx) at or below this concentration [
9,
10,
25]. While a higher concentration of the drug might have resulted in further reductions of in Ca
2+ influx during ischemia, potential blockade of forward mode NCX or unwanted effects on other ion channels (L-type Ca
2+) during reperfusion may be problematic.