Low molecular weight fibroblast growth factor-2 signals via protein kinase C and myofibrillar proteins to protect against postischemic cardiac dysfunction.

Among its many biological roles, fibroblast growth factor-2 (FGF2) acutely protects the heart from dysfunction associated with ischemia/reperfusion (I/R) injury. Our laboratory has demonstrated that this is due to the activity of the low molecular weight (LMW) isoform of FGF2 and that FGF2-mediated cardioprotection relies on the activity of protein kinase C (PKC); however, which PKC isoforms are responsible for LMW FGF2-mediated cardioprotection, and their downstream targets, remain to be elucidated. To identify the PKC pathway(s) that contributes to postischemic cardiac recovery by LMW FGF2, mouse hearts expressing only LMW FGF2 (HMWKO) were bred to mouse hearts not expressing PKCα (PKCαKO) or subjected to a selective PKCε inhibitor (εV(1-2)) before and during I/R. Hearts only expressing LMW FGF2 showed significantly improved postischemic recovery of cardiac function following I/R (P < 0.05), which was significantly abrogated in the absence of PKCα (P < 0.05) or presence of PKCε inhibition (P < 0.05). Hearts only expressing LMW FGF2 demonstrated differences in actomyosin ATPase activity as well as increases in the phosphorylation of troponin I and T during I/R compared with wild-type hearts; several of these effects were dependent on PKCα activity. This evidence indicates that both PKCα and PKCε play a role in LMW FGF2-mediated protection from cardiac dysfunction and that PKCα signaling to the contractile apparatus is a key step in the mechanism of LMW FGF2-mediated protection against myocardial dysfunction.

Tropomyosin dephosphorylation results in compensated cardiac hypertrophy.

Phosphorylation of tropomyosin (Tm) has been shown to vary in mouse models of cardiac hypertrophy. Little is known about the in vivo role of Tm phosphorylation. This study examines the consequences of Tm dephosphorylation in the murine heart. Transgenic (TG) mice were generated with cardiac specific expression of α-Tm with serine 283, the phosphorylation site of Tm, mutated to alanine. Echocardiographic analysis and cardiomyocyte cross-sectional area measurements show that α-Tm S283A TG mice exhibit a hypertrophic phenotype at basal levels. Interestingly, there are no alterations in cardiac function, myofilament calcium (Ca(2+)) sensitivity, cooperativity, or response to ß-adrenergic stimulus. Studies of Ca(2+) handling proteins show significant increases in sarcoplasmic reticulum ATPase (SERCA2a) protein expression and an increase in phospholamban phosphorylation at serine 16, similar to hearts under exercise training. Compared with controls, the decrease in phosphorylation of α-Tm results in greater functional defects in TG animals stressed by transaortic constriction to induce pressure overload-hypertrophy. This is the first study to investigate the in vivo role of Tm dephosphorylation under both normal and cardiac stress conditions, documenting a role for Tm dephosphorylation in the maintenance of a compensated or physiological phenotype. Collectively, these results suggest that modification of the Tm phosphorylation status in the heart, depending upon the cardiac state/condition, may modulate the development of cardiac hypertrophy.

The influence of FGF2 high molecular weight (HMW) isoforms in the development of cardiac ischemia-reperfusion injury.

Fibroblast growth factor 2 (FGF2) consists of multiple protein isoforms (low [LMW] and high molecular weight [HMW]), which are localized to different cellular compartments, indicating unique biological activity. We previously showed that the LMW isoform is important in protecting the heart from myocardial dysfunction associated with ischemia-reperfusion (I/R) injury, but the roles of the HMW isoforms remain unknown. To elucidate the role of HMW isoforms in I/R and cardioprotection, hearts from novel mouse models, in which the murine FGF2 HMWs are knocked out (HMWKO) or the human FGF2 24 kDa HMW isoform is overexpressed (HMW Tg) and their wildtype (Wt) or non-transgenic (NTg) cohorts were subjected to an ex vivo work-performing heart model of I/R. There was a significant improvement in post-ischemic recovery of cardiac function in HMWKO hearts (76+/-5%, p<0.05) compared to Wt hearts (55+/-5%), with a corresponding decrease in HMW Tg function (line 20: 38+/-6% and line 28: 33+/-4%, p<0.05) compared to non-transgenic hearts (68+/-9%). FGF2 LMW isoform was secreted from Wt and HMWKO hearts during I/R, and a FGF receptor (FGFR) inhibitor, PD173074 caused a decrease in cardiac function when administered in I/R in Wt and FGF2 HMWKO hearts (p<0.05), indicating that FGFR is involved in FGF2 LMW isoform's biological effect in ischemia-reperfusion injury. Moreover, overexpression of HMW isoform reduced FGFR1 phosphorylation/activation with no further decrease in the phosphorylation state in the presence of the FGFR inhibitor. Overall, our data indicate that HMW isoforms have a detrimental role in the development of post-ischemic myocardial dysfunction.

Kappa and delta opioid receptor signaling is augmented in the failing heart.

The opioidergic system, an endogenous stress pathway, modulates cardiac function. Furthermore, opioid peptide and receptor expression is altered in a number of cardiac pathologies. However, whether the response of myocardial opioid receptor signaling is altered in heart failure progression is currently unknown. Elucidating possible alterations in and effects of opioidergic signaling in the failing myocardium is of critical importance as opioids are commonly used for pain management, including in patients at risk for cardiovascular disease. A hamster model of cardiomyopathy and heart failure (Bio14.6) was used to investigate cardiac opioidergic signaling in heart failure development. This study found an augmented negative inotropic and lusitropic response to administration of agonists selective for the kappa opioid receptor and delta opioid receptor in the failing heart that was mediated by a pertussis toxin-sensitive G-protein. The augmented decrease in cardiac function was manifested by increased inhibition of cAMP accumulation and the amplitude of the systolic Ca(2+) transient. Furthermore, increased depression of cardiac function and of two important second messengers, cAMP and intracellular Ca(2+), were independent of changes in cardiac opioid peptide or receptor expression. Thus, the cardiomyopathy-induced failing heart experiences increased cardiac depressant effects following opioid receptor stimulation which could exacerbate diminished cardiac function in end-stage heart failure. As cardiac function is already depressed in heart failure patients, administration of opioids could exacerbate the degree of cardiac dysfunction and worsen disease progression.

Hypertensive state, independent of hypertrophy, exhibits an attenuated decrease in systolic function on cardiac kappa-opioid receptor stimulation.

Opioids/opiates are commonly administered to alleviate pain, unload the heart, or decrease breathlessness in patients with advanced heart failure. As such, it is important to evaluate whether the myocardial opioidergic system is altered in cardiac disease. A hamster model of spontaneous hypertension was investigated before the development of hypertension (1 mo of age) and in the hypertensive state (10 mo of age) to evaluate the effect of prolonged hypertension on myocardial opioidergic activity. Plasma beta-endorphin was decreased before the development of hypertension and in the hypertensive state (P < 0.05). There was no change in cardiac beta-endorphin content at either time point. No differences were detected in cardiac or plasma dynorphin A, Met-enkephalin, or Leu-enkephalin, or in cardiac peptide expression of kappa- or delta-opioid receptors. mu-Opioid receptor was not detected in either model. To determine how hypertension affects myocardial opioid signaling, the ex vivo work-performing heart was used to assess the cardiac response to opioid administration in healthy hearts and those subjected to chronic hypertension. Agonists selective for the kappa- and delta-opioid receptors, but not mu-opioid receptors, induced a concentration-dependent decrease in cardiac function. The decrease in left ventricular systolic pressure on administration of the kappa-opioid receptor-selective agonist, U50488H, was attenuated in hearts from hamsters subjected to chronic, untreated hypertension (P < 0.05) compared with control. These results show that peripheral and myocardial opioid expression and signaling are altered in hypertension.

The protein kinase C pathway mediates cardioprotection induced by cardiac-specific overexpression of fibroblast growth factor-2.

Elucidation of protective mechanisms against ischemia-reperfusion injury is vital to the advancement of therapeutics for ischemic heart disease. Our laboratory has previously shown that cardiac-specific overexpression of fibroblast growth factor-2 (FGF2) results in increased recovery of contractile function and decreased infarct size following ischemia-reperfusion injury and has established a role for the mitogen-activated protein kinase (MAPK) signaling cascade in the cardioprotective effect of FGF2. We now show an additional role for the protein kinase C (PKC) signaling cascade in the mediation of FGF2-induced cardioprotection. Overexpression of FGF2 (FGF2 Tg) in the heart resulted in decreased translocation of PKC-delta but had no effect on PKC-alpha, -epsilon, or -zeta. In addition, multiple alterations in PKC isoform translocation occur during ischemia-reperfusion injury in FGF2 Tg hearts as assessed by Western blot analysis and confocal immunofluorescent microscopy. Treatment of FGF2 Tg and nontransgenic (NTg) hearts with the PKC inhibitor bisindolylmaleimide (1 micromol/l) revealed the necessity of PKC signaling for FGF2-induced reduction of contractile dysfunction and myocardial infarct size following ischemia-reperfusion injury. Western blot analysis of FGF2 Tg and NTg hearts subjected to ischemia-reperfusion injury in the presence of a PKC pathway inhibitor (bisindolylmaleimide, 1 micromol/l), an mitogen/extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) pathway inhibitor (U-0126, 2.5 micromol/l), or a p38 pathway inhibitor (SB-203580, 2 micromol/l) revealed a complicated signaling network between the PKC and MAPK signaling cascades that may participate in FGF2-induced cardioprotection. Together, these data suggest that FGF2-induced cardioprotection is mediated via a PKC-dependent pathway and that the PKC and MAPK signaling cascades are integrally connected downstream of FGF2.

The cardioprotective effect of the low molecular weight isoform of fibroblast growth factor-2: the role of JNK signaling.

UNLABELLED: Our laboratory showed that overexpression of fibroblast growth factor-2 (FGF2) protected the heart against ischemia-reperfusion injury. FGF2 has different protein isoforms (low [LMW] and high [HMW] molecular weight isoforms) produced from alternative translation start sites. However, which FGF2 isoform(s) mediates this cardioprotection, and which signaling pathway (i.e., mitogen-activated protein kinase (MAPK)) elicits FGF2 isoform-induced cardioprotection remains to be elucidated. METHODS AND RESULTS: Wildtype, Fgf2 KO (absence of all FGF2 isoforms) and FGF2 LMWKO (absence of LMW isoform) hearts were subjected to an ex vivo work-performing heart ischemic model of 60 min ischemia and 120 min reperfusion. There was a significant decrease in the recovery of post-ischemic contractile function (p<0.05) in Fgf2 KO and FGF2 LMWKO mouse hearts compared to wildtype hearts. Following ischemia-reperfusion injury, MKK4/7, JNK, and c-Jun were significantly phosphorylated (i.e., activated), and the levels of TUNEL-positive nuclei and caspase 3 cleavage were significantly increased in vehicle-treated Fgf2 KO and FGF2 LMWKO compared to wildtype hearts (p<0.05). A novel JNK pathway inhibitor, CEP11004 (50 nM), significantly restored the post-ischemic contractile function and reduced myocardial cell death, as measured by CK release and apoptotic markers, compared to DMSO-treated cohorts (p<0.05). Overall, our data indicate that the LMW isoform has an important role in restoring cardiac function after ischemia-reperfusion (I/R) injury. These results provide unequivocal evidence that inhibition of JNK signaling is involved in FGF2 LMW isoform-mediated cardioprotection and that the potential mechanism may be through inhibition of the apoptotic process.

Cardioprotection induced by cardiac-specific overexpression of fibroblast growth factor-2 is mediated by the MAPK cascade.

Our laboratory showed previously that cardiac-specific overexpression of FGF-2 [FGF-2 transgenic (Tg)] results in increased recovery of contractile function and decreased infarct size after ischemia-reperfusion injury. MAPK signaling is downstream of FGF-2 and has been implicated in other models of cardioprotection. Treatment of FGF-2 Tg and wild-type hearts with U-0126, a MEK-ERK pathway inhibitor, significantly reduced recovery of contractile function after global low-flow ischemia-reperfusion injury in FGF-2 Tg (86 +/- 2% vehicle vs. 66 +/- 4% U-0126; P < 0.05) but not wild-type (61 +/- 7% vehicle vs. 67 +/- 7% U-0126) hearts. Similarly, MEK-ERK inhibition significantly increased myocardial infarct size in FGF-2 Tg (12 +/- 3% vehicle vs. 31 +/- 2% U-0126; P < 0.05) but not wild-type (30 +/- 4% vehicle vs. 36 +/- 7% U-0126) hearts. In contrast, treatment of FGF-2 Tg and wild-type hearts with SB-203580, a p38 inhibitor, did not abrogate FGF-2-induced cardioprotection from postischemic contractile dysfunction. Instead, inhibition of p38 resulted in decreased infarct size in wild-type hearts (30 +/- 4% vehicle vs. 11 +/- 2% SB-203580; P < 0.05) but did not alter infarct size in FGF-2 Tg hearts (12 +/- 3% vehicle vs. 14 +/- 1% SB-203580). Western blot analysis of ERK and p38 activation revealed signaling alterations in FGF-2 Tg and wild-type hearts during early ischemia or reperfusion injury. In addition, MEK-independent ERK inhibition by p38 was observed during early ischemic injury. Together these data suggest that activation of ERK and inhibition of p38 by FGF-2 is cardioprotective during ischemia-reperfusion injury.

Cardiac-specific overexpression of fibroblast growth factor-2 protects against myocardial dysfunction and infarction in a murine model of low-flow ischemia.

BACKGROUND: Preconditioning the heart before an ischemic insult has been shown to protect against contractile dysfunction, arrhythmias, and infarction. Pharmacological studies have suggested that fibroblast growth factor-2 (FGF2) is involved in cardioprotection. However, because of the number of FGFs expressed in the heart and the promiscuity of FGF ligand-receptor interactions, the specific role of FGF2 during ischemia-reperfusion injury remains unclear. METHODS AND RESULTS: FGF2-deficient (Fgf2 knockout) mice and mice with a cardiac-specific overexpression of all 4 isoforms of human FGF2 (FGF2 transgenic [Tg]) were compared with wild-type mice to test whether endogenous FGF2 elicits cardioprotection. An ex vivo work-performing heart model of ischemia was developed in which murine hearts were subjected to 60 minutes of low-flow ischemia and 120 minutes of reperfusion. Preischemic contractile function was similar among the 3 groups. After ischemia-reperfusion, contractile function of Fgf2 knockout hearts recovered to 27% of its baseline value compared with a 63% recovery in wild-type hearts (P<0.05). In FGF2 Tg hearts, an 88% recovery of postischemic function occurred (P<0.05). Myocardial infarct size was also reduced in FGF2 Tg hearts compared with wild-type hearts (13% versus 30%, P<0.05). There was a 2-fold increase in FGF2 release from Tg hearts compared with wild-type hearts (P<0.05). No significant alterations in coronary flow or capillary density were detected in any of the groups, implying that the protective effect of FGF2 is not mediated by coronary perfusion changes. CONCLUSIONS: These results provide evidence that endogenous FGF2 plays a significant role in the cardioprotective effect against ischemia-reperfusion injury.