High-frequency ultrasound detection of the temporal evolution of protein cross linking in myocardial tissue

The progressive increase in stiffening of the myocardium associated with the aging process and abetted by comorbid conditions such as diabetes may be linked to an excessive number of collagen cross links within the myocardial extra-cellular matrix. To determine whether ultrasound can delineate changes in the physical properties of heart tissue undergoing cross linking, the authors employed a model in which increased cross linking was induced by treating rat myocardial tissue with specific chemical fixatives. Rat hearts (n=5 each group) were arrested at end-diastole, insonified (30 to 50 MHz) fresh within a few minutes of excision in a phosphate buffered solution, placed in a fixative (10% formalin or 2.5% glutaraldehyde) and insonified at 30-minute intervals thereafter for 24 hours. Ultrasonic attenuation increased in tissues cross linked with formalin (maximal change: 27.2+/-3.4 dB/cm) and glutaraldehyde (maximal change: 40.2+/-5.6 dB/cm) over a 24-hour period. The frequency dependence of the attenuation coefficient increased as a function of the extent of collagen cross links in formalin (maximal change: 0.8+/-0.3 dB/cm-MHz) and glutaraldehyde (maximal change: 0.9+/-0.6 dB/cm-MHz). This study represents the first time that the precise time course of myocardial protein cross linking in situ has been characterized by using real time monitoring, and the physiologic effect has been delineated on microscopic material properties.