Background Human cardiac progenitor cells (hCPCs) may promote myocardial regeneration in adult ischemic myocardium. The regenerative capacity of hCPCs in young patients with nonischemic congenital heart defects for potential use in congenital heart defect repair warrants exploration. Methods and Results Human right atrial specimens were obtained during routine congenital cardiac surgery across 3 groups: neonates (age, <30 days), infants (age, 1 month to 2 years), and children (age, >2 to ≤13 years). C-kit+ hCPCs were 3-fold higher in neonates than in children >2 years of age. hCPC proliferation was greatest during the neonatal period as evidenced by c-kit+ Ki67+ expression but decreased with age. hCPC differentiation capacity was also greatest in neonatal right atrium as evidenced by c-kit+, NKX2–5+, NOTCH1+, and NUMB+ expression. Despite the age-dependent decline in resident hCPCs, we isolated and expanded right atrium–derived CPCs from all patients (n = 103) across all ages and diagnoses using the cardiosphere method. Intact cardiospheres contained a mix of heart-derived cell subpopulations that included cardiac progenitor cells expressing c-kit+, Islet-1, and supporting cells. The number of c-kit+–expressing cells was highest in human cardiosphere-derived cells (hCDCs) grown from neonatal and infant right atrium. Furthermore, hCDCs could differentiate into diverse cardiovascular lineages by in vitro differentiation assays. Transplanted hCDCs promoted greater myocardial regeneration and functional improvement in infarcted myocardium than transplanted cardiac fibroblasts. Conclusions Resident hCPCs are most abundant in the neonatal period and rapidly decrease over time. hCDCs can be reproducibly isolated and expanded from young human myocardial samples regardless of age or diagnosis. hCPCs are functional and have potential in congenital cardiac repair.

Spacelab Life Sciences-1 and -2 provided skeletal muscles from rats dissected in flight for the first time and 2 h to 14 days postflight. The muscles permitted the distinguishing of primary adaptations to microgravity from secondary reloading-induced alterations. In microgravity, rats adopted bipedal forelimb locomotion with the hindlimbs relegated to grasping activities. On landing day, body posture was abnormally low and walking was stilted at a rate one-third of normal. The adductor longus (AL) and soleus muscles exhibited decreased myofiber areas that did not recover 14 days postflight. Doubling of the nonmyofiber area indicated interstitial edema in AL muscles 2.3 h postflight. Solei did not manifest edema postflight, and neither muscle showed edema in flight. Sarcomere eccentric contraction-like lesions were detected in 2.6% of AL myofibers 4.5 h postflight; lesions were absent earlier postflight and in flight. At 9 days postflight, these lesions were repaired but regenerating AL myofibers were present, which suggests that myofiber necrosis occurred 1-2 days postflight. These studies demonstrate that muscle atrophy occurs in microgravity, whereas interstitial edema and sarcomere lesions are postflight phenomena.

Slow oxidative (SO) fibers of the adductor longus (AL) were predominantly damaged during voluntary reloading of hindlimb unloaded (HU) rats and appeared explainable by preferential SO fiber recruitment. The present study assessed damage after eliminating the variable of voluntary recruitment by tetanically activating all fibers in situ through the motor nerve while applying eccentric (lengthening) or isometric contractions. Muscles were aldehyde fixed and resin embedded, and semithin sections were cut. Sarcomere lesions were quantified in toluidine blue-stained sections. Fibers were typed in serial sections immunostained with antifast myosin and antitotal myosin (which highlights slow fibers). Both isometric and eccentric paradigms caused fatigue. Lesions occurred only in eccentrically contracted control and HU muscles. Fatigue did not cause lesions. HU increased damage because lesioned- fiber percentages within fiber types and lesion sizes were greater than control. Fast oxidative glycolytic (FOG) fibers were predominantly damaged. In no case did damaged SO fibers predominate. Thus, when FOG, SO, and hybrid fibers are actively lengthened in chronically unloaded muscle, FOG fibers are intrinsically more susceptible to damage than SO fibers. Damaged hybrid-fiber proportions ranged between these extremes.

The nonreceptor protein tyrosine kinase (PTK) proline-rich tyrosine kinase 2 (PYK2) has been implicated in cell signaling pathways involved in left ventricular hypertrophy and heart failure, but its exact role has not been elucidated. In this study, replication-defective adenoviruses (Adv) encoding green fluorescent protein (GFP)-tagged, wild-type (WT), and mutant forms of PYK2 were used to determine whether PYK2 overexpression activates MAPKs, and downregulates SERCA2 mRNA levels in neonatal rat ventricular myocytes (NRVM). PYK2 overexpression significantly decreased SERCA2 mRNA (as determined by Northern blot analysis and real-time RT-PCR) to 54 +/- 4% of Adv-GFP-infected cells 48 h after Adv infection. Adv-encoding kinase-deficient (KD) and Y(402)F phosphorylation-deficient mutants of PYK2 also significantly reduced SERCA2 mRNA (WT>KD>Y(402)F). Conversely, the PTK inhibitor PP2 (which blocks PYK2 phosphorylation by Src-family PTKs) significantly increased SERCA2 mRNA levels. PYK2 overexpression had no effect on ERK1/2, but increased JNK1/2 and p38(MAPK) phosphorylation from fourfold to eightfold compared with GFP overexpression. Activation of both "stress-activated" protein kinase cascades appeared necessary to reduce SERCA2 mRNA levels. Adv-mediated overexpression of constitutively active (ca)MKK6 or caMKK7, which activated only p38(MAPK) or JNKs, respectively, was not sufficient, whereas combined infection with both Adv reduced SERCA2 mRNA levels to 45 +/- 12% of control. WTPYK2 overexpression also significantly reduced SERCA2 promoter activity, as determined by transient transfection of a 3.8-kb SERCA2 promoter-luciferase construct. Thus a PYK2-dependent signaling cascade may have a role in abnormal cardiac Ca(2+) handling in left ventricular hypertrophy and heart failure via downregulation of SERCA2 gene transcription.

Sarcomere lesions were previously observed with reloading of rat adductor longus muscles after spaceflight and hindlimb unloading (HU). Spaceflown rats displayed more lesioned fibers in the "slow-fiber" region, suggesting a damage-susceptible fiber type. Unloading induces fast myosin expression in some slow fibers, generating hybrid fibers. We examined whether lesion damage differed among slow-, hybrid-, and fast-fiber types in HU-reloaded adductor longus muscles. Temporal HU for 5, 8, 11, 14, and 17 days revealed that hybrid fiber percent, detected by antimyosin immunostaining, peaked at 29 +/- 12% by 14 days. A 14-day HU followed by 12-14 h of voluntary reloading was performed to induce lesions. chi2 analysis showed that slow fibers were preferentially damaged, accounting for 92 +/- 5% of lesioned fibers; hybrid and fast fibers accounted for 7 +/- 4 and <0.5%, respectively. Atrophy did not explain differential lesion damage across fiber types, as slow and hybrid fibers atrophied to a similar extent. Because active myofiber contractions are requisite for lesion formation, selective recruitment of slow fibers most likely explains their damage susceptibility.

Acidosis in cardiac muscle is associated with a decrease in developed force. We hypothesized that slow skeletal troponin I (ssTnI), which is expressed in neonatal hearts, is responsible for the observed decreased response to acidic conditions. To test this hypothesis directly, we used adult transgenic (TG) mice that express ssTnI in the heart. Cardiac TnI (cTnI) was completely replaced by ssTnI either with a FLAG epitope introduced into the N‐terminus (TG‐ssTnI*) or without the epitope (TG‐ssTnI) in these mice. TG mice that express cTnI were also generated as a control TG line (TG‐cTnI). Non‐transgenic (NTG) littermates were used as controls. We measured the force‐calcium relationship in all four groups at pH 7.0 and pH 6.5 in detergent‐extracted fibre bundles prepared from left ventricular papillary muscles. The force‐calcium relationship was identical in fibre bundles from NTG and TG‐cTnI mouse hearts, therefore NTG mice served as controls for TG‐ssTnI* and TG‐ssTnI mice. Compared to NTG controls, the force generated by fibre bundles from TG mice expressing ssTnI was more sensitive to Ca2+. The shift in EC50 (the concentration of Ca2+ at which half‐maximal force is generated) caused by acidic pH was significantly smaller in fibre bundles isolated from TG hearts compared to those from NTG hearts. However, there was no difference in the force‐calcium relationship between hearts from the TG‐ssTnI* and TG‐ssTnI groups. We also isolated papillary muscles from the right ventricle of NTG and TG mouse hearts expressing ssTnI and measured isometric force at extracellular pH 7.33 and pH 6.75. At acidic pH, after an initial decline, twitch force recovered to 60 ± 3 % (n= 7) in NTG papillary muscles, 98 ± 2 % (n= 5) in muscles from TG‐ssTnI* and 96 ± 3 % (n= 7) in muscles from TG‐ssTnI hearts. Our results indicate that TnI isoform composition plays a crucial role in the determination of myocardial force sensitivity to acidosis.

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. Protein kinase C-␣-induced hypertrophy of neonatal rat ventricular myocytes.