Enrichment of neonatal rat cardiomyocytes in primary culture facilitates long-term maintenance of contractility in vitro

Long-term culture of primary neonatal rat cardiomyocytes is limited by the loss of spontaneous contractile phenotype within weeks in culture. This may be due to loss of contractile cardiomyocytes from the culture or overgrowth of the non-cardiomyocyte population. Using the mitochondria specific fluorescent dye, tetramethylrhodamine methyl ester perchlorate (TMRM), we showed that neonatal rat cardiomyocytes enriched by fluorescence-activated cell sorting can be maintained as contractile cultures for long periods (24-wk culture vs. 2 wk for unsorted cardiomyocytes). Long-term culture of this purified cardiomyocyte (TMRM high) population retained the expression of cardiomyocyte markers, continued calcium cycling, and displayed cyclic electrical activity that could be regulated pharmacologically. These findings suggest that non-cardiomyocyte populations can negatively influence contractility of cardiomyocytes in culture and that by purifying cardiomyocytes, the cultures retain potential as an experimental model for longitudinal studies of cardiomyocyte biology in vitro.

Tissue-Engineered Myocardial Patch Derived From Extracellular Matrix Provides Regional Mechanical Function

Background— Extracellular matrix (ECM), a tissue-engineered scaffold, recently demonstrated cardiomyocyte population after myocardial implantation. Surgical restoration of myocardium frequently uses Dacron as a myocardial patch. We hypothesized that an ECM-derived myocardial patch would provide a mechanical benefit not seen with Dacron. Methods and Results— Using a canine model, a full thickness defect in the right ventricle was repaired with either Dacron or ECM. A third group had no surgery and determined baseline RV function. Eight weeks later, global systolic function was assessed by the preload recruitable stroke work relationship. Regional systolic function was measured by systolic area contraction (SAC), calculated by high density mechanical mapping. Tau was used to assess global diastolic function. Recoil rate and diastolic shear were used as measures of regional diastolic function. After functional data acquisition, tissue was fixed for histological evaluation. Global systolic and diastolic functions were similar at baseline and after ECM and Dacron implantation. Regional systolic function was greater in the ECM group compared with the Dacron group (SAC: 4.1±0.9% versus −1.8±1.1, P <0.05). Regional diastolic function was also greater in the ECM group (recoil rate (° sec −1 ): −44±7 versus −17±2, ECM versus Dacron; P <0.05). Immunohistochemical analysis revealed cardiomyocytes in the ECM implant region, a finding not seen with Dacron. Conclusion— At 8 weeks, an ECM-derived tissue-engineered myocardial patch provides regional mechanical function, likely related to cardiomyocyte population. These results are in sharp contrast to Dacron, a commonly used myocardial patch.