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Isolation and Physiological Analysis of Mouse Cardiomyocytes
Published on: September 7, 2014
Cardiomyocytes from hearts with left ventricular dysfunction after ischemia-reperfusion do not manifest contractile
Y Chandrashekhar1, A J Prahash, S Sen
1Division of Cardiology, Veterans Affairs Medical Center and the University of Minnesota Medical School, Minneapolis 55417, USA.
Insights
Isolated heart cells from ischemia-reperfusion injury show normal function, suggesting non-cellular factors cause cardiac stunning. This research explores the mechanisms behind heart dysfunction after reperfusion.
Area of Science:
- Cardiovascular Physiology
- Cellular Biology
- Cardiac Pathophysiology
Background:
- Ischemia-reperfusion injury causes transient cardiac contractile dysfunction, known as stunning.
- The precise cause of stunning, whether intrinsic cardiomyocyte dysfunction or other factors, remains unclear.
Purpose of the Study:
- To evaluate the contractile function of cardiomyocytes isolated from hearts exhibiting global left ventricular dysfunction post-ischemia-reperfusion.
- To determine if the observed cardiac stunning is primarily due to intrinsic cellular dysfunction.
Main Methods:
- Induction of global contractile dysfunction in isolated perfused rat hearts via transient global ischemia (20 min) and reperfusion (20 min).
- Isolation of cardiomyocytes using enzymatic digestion for functional assessment under varying inotropy (extracellular calcium) and loading conditions (perfusate viscosity).
- Measurement of mechanical function using video edge detection and intracellular calcium ([Ca2+]i) kinetics with fura-2 AM.
Main Results:
- Ischemia-reperfusion significantly impaired overall heart function (increased LV end-diastolic pressure, reduced developed pressure and dP/dt).
- Isolated cardiomyocytes did not exhibit contractile dysfunction; instead, they showed enhanced cell shortening and faster contraction rates.
- No differences were observed in calcium dose-response or the relationship between intracellular calcium and cell length between groups.
Conclusions:
- Cardiomyocytes from stunned hearts maintain normal contractile function and intracellular calcium ([Ca2+]i) transients.
- The findings suggest that non-myocyte factors, such as extracellular matrix abnormalities or altered myocyte-interstitial coupling, likely contribute to cardiac contractile failure in stunned hearts.
Objectives:
This study evaluated contractile function in cardiomyocytes isolated from hearts with global left ventricular dysfunction following ischemia-reperfusion.
Background:
Ischemia followed by reperfusion is associated with transient contractile dysfunction, termed "stunning." It is not clear whether this phenomenon is primarily due to intrinsic cardiomyocyte contractile dysfunction.
Methods:
Global contractile dysfunction was induced in isolated perfused rat hearts (n = 8) using a model of transient global ischemia (20 min) followed by reperfusion (20 min). Hearts perfused uninterrupted for 40 min were used as controls (n = 8). Cardiomyocytes were isolated using enzymatic digestion and were studied under varying degrees of inotropy (using increasing extracellular calcium [Ca2+]o) and loading conditions (varying extracellular perfusate viscosity). Mechanical function was studied with video edge detection and intracellular calcium ([Ca2+]i) kinetics using fura-2 AM.
Results:
Global ischemia-reperfusion increased left ventricle (LV) end diastolic pressure (450% vs. 33%, p < 0.01) and reduced LV developed pressure (9% vs. 33%, p < 0.01), LV positive (3% vs. 26%, p < 0.01) and negative (5% vs. 33%, p < 0.01) dP/dt. However, cells isolated from these hearts did not manifest contractile dysfunction. In fact, cell shortening (p < 0.0001) and peak rate of cell shortening (p < 0.05) and increase in [Ca2+]i with each contraction (p < 0.024) were higher in these cells during stimulation with [Ca2+]o of 1 to 10 mmol/liter. The EC50 values for calcium dose response and the slope of the relation between change in [Ca2+]i and change in cell length were no different between the groups. Cell loading (with increasing superfusate viscosity from 1 cp to 300 cp) also did not reveal any abnormalities in cells from the hearts subjected to ischemia-reperfusion.
Conclusions:
Cardiomyocytes isolated from hearts with ischemia-reperfusion-induced LV dysfunction or "stunning" have normal contractile function and normal [Ca2+]i transients, when studied both in the unloaded and loaded state. Our data suggest that nonmyocyte factors such as abnormalities in extracellular matrix or abnormal myocyte-interstitial tissue coupling may be important for the genesis of cardiac contractile failure in the stunned heart.
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