Regulation of cardiac contractile function.

One reason why the trout heart is functional at temperatures low enough to stop a human heart is that it is 10 fold more sensitive to Ca2+. Dr. Gillis, during his PhD studies, demonstrated that this is due, in part, to trout cardiac troponin C (TnC). To examine how the regulation of contractile function has evolved in vertebrates, we have completed studies that link protein function with muscle function and then put these data into a phylogenetic context. These experiments exploited our abilities to clone genes, produce recombinant proteins, and measure the Ca2+ activation of cardiac muscle. MSc student K. Kirkpatrick found that trout troponin I (TnI) increases the Ca2+ sensitivity of the troponin (Tn) complex but reduces the influence of protein kinase A (PKA) phosphorylation on complex function. PKA is activated following adrenergic stimulation. Using skinned cardiac trabeculae we then demonstrated that trout cardiac contractile function is only subtly regulated by PKA treatment.

                                                                                                                          Gillis et al. 2011

Images of a skinned trout cardiac trabeculae preparation mounted to the muscle mechanics system. (A) Trabeculae preparation attached to force transducer and servomotor using aluminium foil clips. (B) Image of sarcomere pattern through 40× objective.


                                                                              Gillis et al. 2011

PKA treatment reduces the force generated by skinned trout cardiac trabeculae during maximum activation but it does not change the Ca2+ sensitivity of force generation. (A) Actual force generated. (B) Relative force generated. Measurements made at 15°C and a SL of 2.2 μm.

A phylogenetic study completed with PhD student J. Shaffer (UC Davis) suggests that the role of PKA and PKC in regulating cardiac contractile function has increased over evolutionary time. This is reflected in the incremental increase in the number of phosphorylation targets in TnI and myosin binding protein C (MyBP-C) as physiological temperatures increased. We proposed that such changes provided greater control of cardiac function.

                                                                                                         Shaffer & Gillis 2010

A vertebrate phylogeny onto which critical changes in the sequence of cTnI and cMyBP-C have been mapped.

One species that will provide insight into this phenomenon is the African clawed frog, Xenopus laevis. The physiological temperatures (15-30oC) of this tropical ectotherm are higher than trout (4-20oC), and as an amphibian, its heart is a morphological and functional intermediate between that of fish and endothermic vertebrates. Examination of the sequence of Xenopus contractile proteins reveals a mix of motifs from both ectotherms and endotherms. For example, Xenopus TnC contains two of the four residues identified in trout TnC as being responsible for it’s high Ca2+ affinity. The two remaining residues are the same as in human TnC. MSc student Elizabeth Sears is now working to characterize the function of Xenopus troponin and to compare this to that of trout and rat troponin.

Cardiac remodeling in trout.

PhD student J. Klaiman has demonstrated that cold and warm acclimation of trout causes distinct cardiac phenotypes. This work examined cardiac contractile function, tissue morphology as well as changes in the expression and phosphorylation of the contractile proteins. To determine how thermal acclimation influences the cardiac proteome, we utilized 2-D difference gel electrophoresis. This analysis indicated that the increased rate of actin-myosin ATPase caused by cold acclimation was due to subtle changes in the phosphorylation state of troponin T and MyBP-C.

                                                                                                                                                                          Klaiman et al., 2011

2D DIGE image of contractile proteins isolated from warm and cold acclimated trout

We also found that cardiac connective tissue increased with cold acclimation but decreased with warm acclimation. This resulted in a ~12-fold difference in the amount of connective tissue in the myocardium of cold and warm acclimated male fish.

                                                                                                                                                                      Klaiman et al.,  2011

Masson’s trichrome stained sections of ventricular compact layer from thermally acclimated rainbow trout. (A) cold, 4C, (B) control, 12C, (C) warm, 17C where pink/ purple is muscle, blue is connective tissue and white or very pale pink is ‘‘extra bundular’’ space.


                                                                                                                                                                                      Klaiman et al., 2011

Quantification of Masson’s trichrome stained sections of ventricular myocardium from thermally acclimated rainbow trout.

How this change influences the passive properties of the heart is not known. This is also the first time that cardiac connective tissue has been found to decrease in response to a physiological stressor. The next step in this project is to identify the molecular mechanisms responsible for this change and characterize the physiological consequences.

Influence of hypoxia exposure on the ontogeny of cardiac regulation in trout.

                                                                                                                          Miller et al., 2011

Chronic hypoxia (30% O2 saturation) incubation from fertilization results in delayed development and growth in trout embryos. These embryos are the result of the same fertilization event.


As an undergraduate, Silvana Miller demonstrated that hypoxia exposure reduced the metabolic rate of trout embryos and caused premature hatching. For her MSc research Silvana focused on the ontogeny of cardiac regulation in trout early life stages and how it is influenced by chronic hypoxia. Her results demonstrated that in normoxic conditions cardiac β-adrenergic receptors are functional at early life stages, while cholinergic receptors are not responsive until after hatch. Chronic hypoxia exposure triggered bradycardia, increased the response to adrenergic stimulation, and delayed the onset of cholinergic control in larvae. In non-motile stages, therefore, survival in chronic low oxygen may depend on the ability to alter the cardiac ontogenetic program to meet the physiological requirements of the developing fish. These studies were completed in collaboration with Dr. P.A. Wright (Guelph). 

                                                                                                                           Miller et al., 2011

Adrenergic regulation of heart rate in trout embryos occurs early in development and cholinergic regulation of heart rate is present after hatching. Timeline of the onset of cardiac regulatory mechanisms relative to developmental stage during the early life of rainbow trout (embryonic stages 22, 26 and 29 and larval stages 30 and 32).

Currently, undergraduate thesis student Elizabeth Johnston is examining how hypoxia exposure during embryonic development influences the aerobic capacity and development of older life stages as well as the expression of critical cardiac genes.