Abstract 10729: Freshly Isolated Fibroblasts From Normal Hearts Affect Cardiomyocyte Structure and Function Via Paracrine Communication, and These Effects are Altered in Fibroblasts From Pressure Overloaded Hearts
α-smooth muscle actin (α-SMA)-positive cardiac myofibroblasts (CMF) release paracrine mediators that affect cardiomyocyte (CM) structure and function. However, studies have used cardiac fibroblasts (CF) isolated from healthy hearts and activated to CMFs by culture. Whether freshly isolated SMA-negative CFs from healthy hearts affect CM structure and function and whether these effects are altered in CFs after pathological stimulation is unknown. We studied rat LV CFs freshly isolated from normal hearts (sham operated - S CF) and from hearts undergoing thoracic aortic constriction for 10 weeks (T CF). Within 24 hours of isolation CFs were cultured on Transwells above isolated adult rat LV myocytes to allow only paracrine communication (S CF+CM or T CF+CM). CMs co-cultured with other CMs were used as control (CM+CM). After 20 hours we measured adult CM size, contractility and Ca2+ handling. CM volume, assessed by Di-8-ANEPPS staining, was increased in S CF+CM and T CF+CM vs. CM+CM (µm3: CM+CM 40207±2171 (n=46), S CF+CM 47080±1895 (n=33), T CF+CM 48014±2049 (n=58); p<0.05). Contractility was assessed using stimulated sarcomere shortening. Baseline sarcomere length was reduced in S CF+CM and T CF+CM. Sarcomere shortening amplitude was unchanged but time to 90% peak was reduced in S CF+CM vs. CM+CM and T CF+CM (ms: CM+CM 87.8±2.4 (n=63), S CF+CM 75.9±1.5 (n=51), T CF+CM 91.0±3.1 (n=64) p<0.01). S CF+CM and T CF+CM reduce time to 50% and 90% return to baseline, with a greater effect in T CF+CM (50% decay ms: CM+CM 70.6±3.3 (n=65), S CF+CM 52.8±2.4 (n=51), T CF+CM 45.1±2.7 (n=64); p<0.05). The Ca2+ transient was assessed using Fluo-4-AM. Transient amplitude was not different between S CF+CM or T CF+CM vs. CM+CM, although was increased in S CF+CM vs. T CF+CM. Time to peak and time to 50% decay were unaffected but time to 90% decay were reduced in S CM+CF and T CM+CF (90% decay: CM+CM 373.4±15.4 (n=45), S CF+CM 328.6±10.17 (n=56), T CF+CM 339.5±16.83 (n=27); p<0.05). ELISA of the culture medium showed that TGF-β and IL-6 were increased in S CF+CM and T CF+CM compared to CM+CM. This is the first study to show that α-SMA negative CFs from normal hearts can actively modulate CM structure and function via paracrine mechanisms, with modified effects in CFs from pressure overloaded hearts.
- © 2012 by American Heart Association, Inc.