High hydrostatic pressure induces slow contraction in mouse cardiomyocytes

Abstract

Cardiomyocytes are contractile cells that regulate heart contraction. Ca²⁺ flux via Ca²⁺ channels activates actomyosin interactions, leading to cardiomyocyte contraction, which is modulated by physical factors (e.g., stretch, shear stress, and hydrostatic pressure). We evaluated the mechanism triggering slow contractions using a high-pressure microscope to characterize changes in cell morphology and intracellular Ca²⁺ concentration ([Ca²⁺]_i) in mouse cardiomyocytes exposed to high hydrostatic pressures. We found that cardiomyocytes contracted slowly without an acute transient increase in [Ca²⁺]_i, while a myosin ATPase inhibitor interrupted pressure-induced slow contractions. Furthermore, transmission electron microscopy showed that, although the sarcomere length was shortened upon the application of 20 MPa, this pressure did not collapse cellular structures such as the sarcolemma and sarcomeres. Our results suggest that pressure-induced slow contractions in cardiomyocytes are driven by the activation of actomyosin interactions without an acute transient increase in [Ca²⁺]_i.

Figure 1

High hydrostatic pressure induced slow contraction in mouse cardiomyocytes. (A) Cardiomyocyte images and their computed FFT spectrum of sarcomere during the application of the pressure at 20 MPa. The gradual shift of the peak position in the FFT spectrum to higher frequency corresponds to a decrease in the SL. The black inset is magnified in the lower right corner of the image. Scale bar, 20 μm. (B) The time-dependent SL alteration under ambient pressure (0.1 MPa) and high pressure (5, 10, and 20 MPa). The SL gradually declined under pressures of 5, 10, and 20 MPa, while it remained constant under ambient pressure. Each different data point represents the different cells. (C) Comparison of SL under each pressure condition (5, 10, and 20 MPa). Pressures of 5, 10, and 20 MPa (n = 11, 8, and 12 cells, respectively) led to a decrease in SL compared with the ambient pressure of 0.1 MPa (n = 12 cells). ^∗∗p < 0.01. Images recorded at 1 frame s⁻¹. Refer to Video S1. Pressure at 0.1 MPa, Video S2. Pressure at 5 MPa, Video S3. Pressure at 10 MPa, Video S4. Pressure at 20 MPa. (D) TEM images of a single cardiomyocyte before and after the application of 20 MPa pressure. The images show that the I band was clear at a pressure of 0.1 MPa, whereas it was undetectable at a pressure of 20 MPa. No collapse of sarcomere structures (A band and Z line) was observed. Quantification of intensities derived from the white line on each image. The lowest intensity peak (darker line) corresponds to the Z line, and the second-lowest intensity peak (lighter line) corresponds to the M line. Although the I band was detected at 0.1 MPa pressure, it disappeared after the application of high pressure at 20 MPa, indicating cardiomyocyte contraction. Scale bar, 500 nm.

Figure 2

Effects of myosin ATPase inhibitor (BDM) on the pressure-induced slow contraction. (A) The pressure-induced slow contraction in live cardiomyocytes is observed in normal Tyrode solution during the application of pressure (upper panel), while its slow contraction is not observed in BDM-containing Tyrode solution (lower panel). Images were recorded at 1 frame s⁻¹. Refer to Videos S4 and S6. (B) BDM significantly inhibits the pressure-induced shortening in cells chemically fixed after the pressure treatment (n = 30 cells), while the shortening is still observed in the absence of BDM (n = 30 cells). ^∗∗∗p < 0.001.

Figure 3

Alteration in intracellular Ca²⁺ of cardiomyocytes under high-hydrostatic pressure. (A) Sequential grayscale images of Ca²⁺ fluorescence intensity in cardiomyocytes at 20 MPa pressure; Ca²⁺ intensity remains constant with time. Scale bars, 10 μm. (B) Time-dependent change of normalized Ca²⁺ intensity to maximal Ca²⁺ intensity in cardiomyocytes under each pressure condition (0.1 MPa [n = 4 cells]; 20 MPa [n = 6 cells]). (C) Comparison of Ca²⁺ intensity between 0.1 and 20 MPa (n = 4 and 6 cells, respectively) at the initial (t = 0 s) and end (t = 30 s) states. N.S., not significant. Images were recorded at 10 frame s⁻¹. Refer to Videos S8 and S9.